Advances in Cancer Research Volume 63

ADVANCES IN CANCER RESEARCH VOLUME 63

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ADVANCES IN CANCERRESEARCH Edited by

GEORGE F. VANDE WOUDE ABL - Basic Research Program NCI - Frederick Cancer Research and Development Center Frederick, Maryland

GEORGE KLEIN Department of Tumor Biology Karolinska lnstitutet Stockholm, Sweden

Volume 63

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9 8 7 6 5 4 3 2 1

CONTENTS

CONTRIBUTORS TO VOLUME 63 .......................................

ix

FOUNDATIONS IN CANCER RESEARCH

Alone on the Heart of the Earth: An Immunogeneticist’s Journey into the Past

JAN KLEIN I. Part One: The Uncertainty Principle ............................... 11. Part Two: Interrogating the Sphinx ................................ 111. Part Three: Points of View ........................................ References .......................................................

2 10 32 37

FOUNDATIONS IN CANCER RESEARCH

Hemopoietic Regulators and Leukemia Development: A Personal Retrospective

DONALDMETCALF 1. 11. 111. IV. V. VI. VII.

Introduction ...... ............................................ Background and Early in V i m Work . , ................ Hemopoietic Clonal Cultures ...................................... Colony-Stimulating Factors ........................................ .......... Biological Actions of the Colony-Stimulating Factors Myeloid Leukemic Cells in Culture ................................. Role of the Colony-Stimulating Factors in Initiation of Myeloid Leukemia .............................................

41 42 47 49

52 59 62

vi

CONTENTS

VIII.

Membrane Receptors for (:oloiiy-Stimulating Factors . . . . . . . . . . . . . . . .

71

IX. Heinopoietic Regulators in the Context of Known X. XI. XlI.

Inducers of Leukemia ............................. Role of Heniopoietic in Suppressing Myeloid Leukemia . . . Recapitulation . . . . . . .................................... T h e Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... References ..........................................

72 76 82

85 86

MAP Kinases ERKl and ERK2: Pleiotropic Enzymes in a Ubiquitous Signaling Network

DAVID J. ROBBINS,ERZHEN ZHEN, MANGENGCHENG, SHUICHAN Xu, DOUGLAS EBERT,AND MELANIEH. COBB I. 11. 111.

I v. V. VI.

VII. VIII. IX.

.................... In trod uction . . . . . . . . . . . . . . Honiology of MAP Kinases w ................................ in the Yeast Mating Pathway Regulation o f MAP Kinases ............................ Identification and Purification of MEK ............................. Kas and Heterotrimeric G Proteins Regulate the ERK Network ... Protein-Protein Interactions That Regulate the MAP Kinase Cascade ....................... MAP Kinases Phosphorylate Upstream Compo Substrates of ERKl and ERKZ . . . . . . . . . . . . . . . .......... ERKl and ERKP Are Essential Regulators of C References . . . . . . . . . . . . . . . . . . ................

93 97 99 I02 I05

I06 107 1 on 111

112

How DNA Viruses Perturb Functional MHC Expression to Alter Immune Recognition

GRANTMCFADDEN AND KEVINKANE 1. 11.

MHC Expression and Ininiurie Recognition of Viral Antigens by ?’ Cells ....................................... Poxviruses . . . . . . . . . . . ................................

111. IV. Herpesviruses: T h e Cytomegalovirus Model ........................ V. Hepatitis B Virus ..................... ........................................ VI. ........... VII. Conclusions .................... References ...............................................

118 145 155

I65 173 180 18.5

I90

CONTENTS

vii

Viral Transformation of Human T Lymphocytes RALPH GRASSMANN. BERNHARD FLECKENSTEIN. AND RONALDC . DESROSIERS I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1 . Transformation of Human T Helper Lymphocytes by Human T Cell Leukemia Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ill . Developing a T-Lymphotropic Herpesvirus Vector . . . . . . . . . . . . . . . . . . IV . Immortalization by Herpesvirtu saimiri-HTLV Recombinants V. Transformation of Human T-Lymphocytes with Wild-Type Herpe.svim saimiri . . ........................... VI Growth Regulation a Rhadinovirus-Transformed T Cell Lines and Clones ................. al stp Oncogene . . . . . . . . . . . . . VII . Transforming Potential of the ............................ VIII . Concluding Remarks . . . . . . . . References .....................................................

211 214 219 22 1 223 228 231 236 237

Lymphomagenesis in AKR Mice: B Cell Lymphomas as a Model of Tumor Dormancy NECHAMA HARAN-GHERA I . Introduction ..................................................... I 1 . Identification of Potential Lymphoma Cells in AKR Mice ............ Ill . Enhanced T Cell Lymphoma Development Pathways . . . . . . . . . . . . . . . . IV . T h e Level of Dormant PLCs Following Prevention of Spontaneous T Cell Lymphoma Development .................... V. Maintenance and Termination of the B-PLC Dormant State . . . . . . . . . . VI . Ly-I+ (CD5+) B Cell Lymphoma Characteristics ..................... VII Concluding Remarks .............................................. References .......................................................

.

245 249 253

262 270 277 286 289

The Tumor Biology of Gastrin and Cholecystokinin JENS

I. I1 . Ill . IV . V.

F . REHFELDAND WOUTERW . VAN SOLINCE

Introduction ..................................................... Definition of the Gastrin-Cholecystokinin Family .................... Normal Biology .................................................. Tumor Biology ................................................... Requirement of Gastrin and Cholecystokinin Measurements in Oncology ........................................

295 296 301 316 331

...

Vlll

CONTENTS

VI . Methods for Measurement of Gastrin and Cholecystokinin . . . . . . . . . . . VII . Perspectives ...................................................... References .......................................................

INDEX ..................................................................

333 336 337

349

CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors' contributions begin.

MANGENG CHENG,University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235 (93) MELANIE H . COBB,University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235 (93) RONALDC. DESROSIERS, New England Regional Primate Research Center, Hamard Medical School, Southborough, Massachusetts 0 1 772 (211 ) DOUGLASEBERT,University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235 (93) BERNHARD FLECKENSTEIN, Institut f u r Klinische und Molekulare Virologie der Universitat Erlangen-Nurnberg, D-91054 Erlangen, Germany (211) RALPHGRASSMANN, Institut f u r Klinische und Molekulare Virologie der Universitat Erlangen-Niirnberg, D-91054 Erlangen, Germany (211 ) NECHAMA HARAN-GHERA, Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel (245) KEVIN KANE,Department of Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2 H 7 (117) JAN KLEIN, Max-Planck-Institut f u r Biologie, Abteilung Immungenetik, 0 - 7 4 0 0 Tubingen, Germany; and Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33136 (1) GRANTMCFADDEN, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2 H 7 (117) DONALDMETCALF,The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia (41) JENS F. REHFELD, Department of Clinical Biochemistry, University of Copenhagen, Rigshospitalet, DK-2100 Copenhagen, Denmark (295) DAVIDJ . ROBBINS,University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235 (93) WOUTERW. VAN SOLINGE, Department of Clinical Biochemistry, University of Copenhagen, Rigshospitalet, DK-2100 Copenhagen, Denmark, and Department of Clinical Chemistry, Ziskenhuis Eemland, Amersfort, The Netherlands (295)

X

SHUICHAN Xu, University of of Pharmacology, Dallas, E R Z H E N Z H E N , Uniuersity of of Phamnacology, Dallas,

CONTRIBUTORS

Texas Southwestern Medical Center, Departrnent Texas 75235 ( 9 3 ) Texas Southwestern Medical Center, Department Texas 75235 ( 9 3 )

FOUNDATIONS IN CANCER RESEARCH ALONE ON THE HEART OF THE EARTH: AN IMMUNOGENETICIST’S JOURNEY INTO THE PAST Jan Klein Max-Planck-lnstitut fur Biologie, Abteilung Immungenetik, D-7400 Tiibingen, Germany; and Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, Florida 33136

Ognuno sta solo sul cuor della terra traffito da un ragio di sole: ed P subito sera.

Each alone on the heart of the earth impaled upon a ray of sun: and suddenly, it’s evening.

Salvatore Quasimodo: Ed L sub& sera

Salvatore Quasimodo: And Suddenly, It’s Evening (translated by Allen Mandelbaum)

I. Part One: T h e Uncertainty Principle A. Window with Grandfather’s Herbarium B. T h e Violet Window C. T h e Orwellian Window D. T h e “Oops, Wrong Profession” Window E. T h e Sweikian Window 11. Part Two: Interrogating the Sphinx A. Joining the Club B. What a Stool Pigeon Caused C. Fateful Decision in Tokyo D. T h e Chase Is On E. Ockham’s Razor F. Playing the Accordion G. Trapper in Michigan H. T h e First Sequences-End of‘ an Era 111. Part Three: Points of View A. Where O u r Judgements Err B. Muskrats and Little Beavers C. T h e Calling D. T h e Kingfisher References

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1. Part One: The Uncertainty Principle We who pass through a world Changeful as the dews of evening, Uncertain as the skies of spring, We that are as foam ujon the &earn,can anybody be our foe? Zenchiku Ujinobu: 7 h e Hijka Priests

Weird is the life of a photon. When nobody is looking, it exists in a superposition of all possible states and spreads throughout space with no definitive position. But the moment a curious observer offers it a choice between two routes-two narrow slits o r windows in a plate-a photon may pass through one of these, but never through both. The windows compel the photon to choose one from an infinity of possible paths. Weird is the life of a photon, but weirder still is the life of a person. Life, like an unobserved subatomic particle, teems with boundless potentials and unfathomable possibilities. When observed, it springs into existence and takes a specific course determined by the encountered window. And because it is observed all the time, life zigzags from one window to another. A window here deflects it in one direction, a window there steers it in an entirely different direction. It is a disconcerting thought that at any place, at any time, a different window, or simply the different slanting of a window, could have deflected life onto a different path and that an entirely different set of potentials could have been realized. Here, then, is a selection of windows that determined one person’s life-a humbling reminder, as if I needed one, that life is but foam upon the stream. A. WINDOWWITH GRANDFATHER’S HERBARIUM He m a n e m , H e ~ O B Y ,H e nnaqy, Bce npOkllineT, KBK C 6enbrx s6nosb IIblM. YBFInaHbB 3OJlOTOM OXBaqeHHbIk, He

6yny 60nbme

MOJlORbIM.

Sergei Yesenin

I hazte no regrets, velreals, or weepings, Smoke front while apple lrees: all will go. Gripped as I am by the gold id w ilhering, I will no1 be young again, I know. Sergei Yesenin (translated by V. Markov and M . Sparks)

Rummaging through bric-A-brac in the attic, an 8-year-old boy discovers a ledger of folios with pressed plants identified in his grandfather’s

A N IMMUNOGENETICIST’S JOURNEY INTO THE PAST

3

meticulous handwriting. Next to it, he finds a book from which his grandfather and father learned botany in the Gymnasium,’ an oldfashioned text aspiring to nothing more than a mere characterization of the main plant families and their representatives. From the treatment it had suffered at the hands of its owners, the boy can tell how much his grandfather and father must have hated memorizing dry descriptions of species after species. But curiously, the herbarium and the book hold a wondrously strange attraction and they are to become the boy’s most treasured possessions. Whenever he can steal a moment between attending school and helping on the farm, the boy retreats with ledger and book to the meadow, the ravine, or the woods, and tries to match the pictures and the pressed plants with those he finds growing around the village. He fails most of the time, or he misidentifies some of the plants, but the frustration he experiences only serves to enhance his curiosity. Where did the curiosity come from? I have often thought about this question, but have not been able to come up with a satisfactory answer. There had been no external stimulus to the boy’s interest in nature and, in the beginning at least, there was no one to nurture it. It appeared suddenly and spontaneously-a cultural mutation. It had, however, from the onset, been linked to a strong, esthetic feeling-a love of nature fused with a love of beauty. The tiniest flower, the commonest bird, the lowliest beetle, the plainest butterfly evoked in the child a sensation of enchantment and delight. It was as if he had been born with a mysterious chord that resonated with pleasing vibrations on contact with nature. T h e pleasure intensified with his growing knowledge of this garden of earthly delights. Discovering the tiny secrets of each species, discovering the bewildering ties that connected the communities of creatures, or just learning the identities of these creatures heightened and deepened the resonations of that chord. Each season brought with it a unique splendor, but spring was always the most glorious. For the boy, the greatest adventure, unmatched by any others later in his life, was to record spring’s progress, from the first appearance of the firebugs at the base of an old linden tree to the first kee-wi cry of the lapwing over the marshes, from an attempt to dig out toothwort growing out of the roots of an elm tree to sketching the heterostyly of cowslips carpeting an old garden, from the discovery of frog spawn to the thrill of an all-night frog concert on a local pond. One day, the boy, by then a junior-high-school student, was surprised to find in the school’s window a display of plants, each with a tag identifying the family and the species. The display was the work of a young I A secondary school, the last 4 years of which might be compared to the senior high school in the American educational system.

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J A N KLEIN

teacher, an enthusiastic naturalist, who in this manner tried to familiarize his students with the local flora. It was not long before the teacher became aware of the boy’s interest and the two of them began to explore nature together. They identified new species, undertook joint excursions to places where rare species had been reported, studied from the same books, and influenced each other culturally as well. Eventually they developed a friendship that has endured to this day. Under the teacher’s guidance the boy began to think of becoming a professional botanist, and it was the teacher who convinced the boy’s mother that he should be sent to college, no matter what hardships it might bring upon the family. Looking back on those times, I cannot help but wonder what would have happened had the boy not found his grandfather’s herbarium and had he not met the teacher. Would he have become a farmer like his kinsfolk? A carpenter? A musicologist? A writer? Idle thoughts! Considerable time has passed since then, and I, too, know I cannot be young again. But as I close my eyes and see once more the tender corydalis sprouting between last year’s leaves, the inconspicuous yellow star of Bethlehem quivering in May’s gentle sun, and, with each stirring of the wind, clouds of petals, the smoke from the white apple trees rising from the orchard, the mysterious chord vibrates in me now as it did then. B. THEVIOLETWINDOW Nunc de vana et stub vitu vneu vobk seribere cupio uc dr exordio transitus mei mundnni, u! cedere vobis vuleunt in examplum. Gruciam uutem michi a deo ir+.sarn et amortrn studii, quod mei pectoris habuit tenucitzcs. . . Vila karidi Quarti

Now I want to write about my vain and unwise l f e and about the beginning of my cureer so that it can serue as an example to you. I will not keep silent about the Grace that God infused in me, nor about my love of study, which inhabited the eSfort of my bosom. . . The Life if Char1e.i IV

Entrance examination for the University. The boy, now 18 years old, is sweating. Though the other subjects went well, he is doing poorly in physics. T h e examiner has discovered a weakness and is pursuing it mercilessly. Could it be that he resents the lad’s resolve to study in Prague rather than Olomouc or Brno, which are closer to Opava, the province of the boy’s origin? Whatever the reason, it does not look good for the youth, as the examiner seems dead set on not letting him pass.

A N IMMUNOGENETICIST’S JOURNEY INTO THE PAST

5

Then it is time for the last subject, biology. The young assistant professor, who witnessed the slaughter by his colleague and apparently feels compassion, wants to put the student at ease. “In your application,” he begins, “you state that you were involved in the survey of the flora in the Opava province. Which plant families did you focus on?” The boy enumerates them. “Also Violaceae?”is the next question. Yes, Violaceae, too. “In that case, how do you tell Viola hirta from Viola canina?” asks the examiner. But that’s easy: By the hairs on the leaves, and by the shape as well as the color of the spur. “Good. Which species, besides the common ones, did you find in your province?” And as the boy lists them, he is stopped in the middle. “Viola bgora? Where did you find Viola bijlora? That’s a mountain species!” Indeed, the boy had found it on the PradZSd mountain. . . . And so the examination gradually evolves into an exchange of information between two enthusiastic plant collectors. T h e young assistant, an expert on violets, about which he is collecting data for a monograph, and the student who knows all the plant species in his province. An hour passes. The assistant professor and the candidate fail to notice. In the meantime, exams over, they have been surrounded by the other examiners who are watching the exchange incredulously and with amusement. Time to quit. When the boy is called into the room a little later, he is granted admittance to the University on the condition that he catch up on his physics during the remainder of his vacation. T h e physicist has apparently been prevailed upon by the violet expert. Blessed be violets, for they helped to fulfill one boy’s dream! They became a window that enabled the realization of one potential from an infinity of possibilities. Prague, the city of Charles IV, the city the boy has learned to love even before he has set foot on her pavements for the first time, welcomes him. For the next few years, the love of study and the love of the city merge. C. THEORWELLIAN WINDOW Andreu: Ungliicklich das Land, das keine Helden hat! Galilei: Nein. Ungliicklich das Land, rim Helden notig hat.

Andrea: Unfortunate the country that has no heroes! Galilei: No. Unfortunate the country thal need heroes.

Bertolt Brecht: Leben des Galilei

Bertolt Brecht: Lye of Galilei

At the Charles University in Prague there are several old traditions that go back all the way to Charles IV, the University’s founder, Emperor

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of the Holy Roman Empire, and king of Bohemia. One of them is the Mujules Pm'vilepum, which grants the students the right to march through the city on the first Sunday in May and poke fun at anybody they please-the rector, the professors, the merchants, the burghers, even the Emperor. For that one day, the students need not fear punishment for their deeds. The communist regime, of course, revoked the Privilepum. The last thing needed was students running wild through the city insulting university officials, not to mention the government. But even communist functionaries apparently felt the need to release built-up pressure occasionally and so, from time to time, they loosened the screws and opened the valve ever so slightly, only to close it quickly again, lest the citizens became intoxicated by the taste of freedom. In 1956 the opening of the valve was signaled by the permission to celebrate Mujules Privilegzum for the first time since the communist takeover. It was also to be the last time! T h e boy, a young man by then, actually did not know what was brewing, but he happened to come upon the parade and so joined it and walked its entire length. Everything appeared to be set in medieval time-the masks, the costumes, the historic figures-but in reality nobody was fooled by the thin disguises. Everybody knew to whom the slogans, the inscriptions, the chantings referred, and those careless enough to do so were laughing and applauding. T h e more cautious individuals stayed home. The students were having a tremendous time but prudent onlookers knew that their fun would be brief and the consequences harsh. When he returned to his apartment, the young man fired off an enthusiastic letter to his mother, describing the events. A few days later, as he was studying in his room, the doorbell rang, and when he answered it he was confronted by two men in leather coats. They flashed an STB badge (STB was the Czech equivalent of the Russian KGB) and ordered him to come with them. A black Moskvitch with darkened windows waited on the street. They all climbed in and sped off. Where were they taking him? What had he done? What would happen to him? He drew some comfort from the fact that they did not stop at BartolomEjska, the secret police headquarters, and that they were not heading for Packrac, the notorious jail for political prisoners. Eventually, they ended up at Letna, at the Ministry of Internal Affairs. T h e interrogation lasted the whole afternoon and until late into the night. Who had organized the provocations at the Mujules? Were foreign provocateurs involved? What was his role in the affair? Who had prepared the reactionary slogans? From the more specific questions, it

A N IMMUNOGENETICIST’S JOURNEY INTO THE PAST

7

gradually dawned on him that the interrogators had intercepted the letter to his mother and that they had also taped discussions at philosophical seminars, where students were encouraged to express themselves freely, which he, stupidly, often did. At some point after midnight he was allowed to leave but was told that he would be expelled from the University. A week or so later the expulsion order arrived at the Dean’s office. What transpired at the faculty meeting, which was expected to result in a rubber stamp for the expulsion, he was never able to learn. He was later told, however, that had it not been for his botany professor, who happened to be the Dean at that time, he would never have completed his studies. I do not believe that anybody born and raised in western democracy can ever really appreciate the courage the Dean must have mustered to defend a student against the STB. It could only be comprehended by someone who has been driven in a black limousine by two thugs in leather coats to an unknown destination for an unknown purpose. Unfortunate the country that needs heroes-indeed! WINDOW D. THE“OOPS,WRONGPROFESSION” VtGina uziletnjch lidskjch povoldni j e mofnci jen z nevtdomosti!

Most useful human professions are possible out of ignorance only!

Karel capek: Vtc Makropulos

Karel capek: The Thing Makropulos

To a true researcher, science is a hobby, and the young man wrestled for some time with the question: Should a person be paid for working on his hobby? “I will be running around the countryside,” he reasoned, “collecting plants, studying their distribution, describing new variants, recording which species are disappearing and why, and all the time enjoying myself immensely. Isn’t it immoral to draw a salary for it?” He resolved the quandary by deciding to become a biology teacher at a Gymnasium. In this decision he drew on some illustrious examples of famous taxonomists who were also teachers and who carried out research as a secondary occupation. So, at the University he enrolled in courses that would prepare him for a teaching career, and he was reaffirmed in his decision when he discovered he actually enjoyed teaching. In Orwellian Czechoslovakia, Big Brother was watching closely over His herd of souls, and the decision as to where University graduates should be allowed to teach was, of course, His alone. For the young man,

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the decision, undoubtedly prompted by his dubious political past, was: As far away from Prague as possible! In the westward direction you could go no further than AS, a little town that then had the reputation of being a Dodge City of the Czech Wild West. T h e young man was told that in AS he could start and, if the authorities could help it, also end, his teaching career. Had he gone there, his life would undoubtedly have unfolded in an entirely different way. Once again, the window that deflected his life’s course was opened not by a guardian angel but by a man whose only concern was for high professional standards. He was the professor at the University in charge of the model school program providing students with their first teaching experiences. The professor thought that the young man possessed a talent f o r teaching and decided he wanted him for the model school. The kind of cunning dialectic the professor must have used to convince the authorities that the teacher was not going to debase politically the young souls under his guidance will never be known. Somehow, however, he succeeded, and the young man’s “marching orders” were changed: From AS to Prague! At the model school, the young man quickly learned that the education system had changed since Jean-Henri Fabre’s time and that the possibilities open to a biology professor at a Gymnasium 100 years ago no longer existed in the new system. He realized that he had been naively uninformed when he had opted for teaching as a means of pursuing a research career. T h e teaching load (and he had to teach not only biology and chemistry, for which he was trained, but sometimes also physics, astronomy, and mathematics) and his other duties were so heavy that botanical excursions or other research activities were simply not possible. After three years at the model school he attempted to change his profession and to become a full-time researcher. T h e only possibility open to him was to enroll in a Ph.D. program either at the University o r at the Czechoslovak Academy of Sciences. He applied at the latter. His admission was a small miracle. There were over 30 candidates and all were better qualified, having had research experience at the University. During his teaching years, however, the young man had continued to study and had discovered a new love-molecular genetics. He had read everything that he could lay his hands on (mostly Russian translations of English books, for books in English were largely unavailable in Czechoslovakia at that time) and he excelled at the interview. So, in 1961, at the age of 26, he abandoned the useful profession that he had chosen out of ignorance and was finally ready to embark on a research careerin plant genetics.

A N IMMUNOGENETICIST’S JOURNEY INTO THE PAST

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E. THESWEIKIAN WINDOW Kdyi bozr scihnou k ironii Elovt‘k Je tupi sutirou.

When gods reach for irony hurnans insult each other with sutirr.

Vladimir Holan: Cnta ntmku

Vladimir Holan: The Way of a Cloud

Because he had lost 3 years already, the young man was eager to get on with research. Unfortunately, his thesis advisor was in no hurry to let him work on his own project. The advisor still lacked the necessary qualifications for supervising Ph.D. students and would need another 2 to 3 years to obtain the required approbation. T h e young man was not willing to wait that long and so he quit. This angered the advisor, who swore to see to it that the young man would not be accepted in any other department of the Academy. Once more, the future looked bleak until someone advised the young man: “Go downstairs and see Milan HaSek. Milan is the only person in the whole Academy who could care less what your former advisor says about you.” Nationally and internationally known for his research on immunological tolerance and for his charismatic personality, Professor Milan HaSek was head of the Institute of Experimental Biology and Genetics, in which a group of enthusiastic young people strived to blend immunology with genetics. Joining this group should have been the dream of every aspiring young scientist in Czechoslovakia, nonetheless the young man hesitated. If accepted, it would mean switching from plants to animals, and for as long as he could remember he had always wanted to be a botanist. In the end he realized, however, that if he wanted to become a researcher he had no other choice than to knock on HaSek’s door. The interview went well (see Klein, 1989). HaSek was indeed unconcerned about the incident “upstairs.” The only thing he seemed to be probing was intelligence and enthusiasm. When it was over and they stood up to shake hands, HaSek, himself a tall man, said: “I need people at the Institute whom I can look straight in the eye. You can start next week.” Thus, the gods reached for irony. After years of believing that no career other than one in botany would satisfy him, the young man was suddenly confronted with the fact that he might become either an immunologist or an animal geneticist, and perhaps even both. When the time came to choose the topic of the young man’s thesis, HaSek, who, as far as I know, was not related to the author of “The Good Soldier Sweik,” decided to perpetrate a Sweikian prank. To the two Kleins already work-

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ing on antigenic variation in tumors, he decided to add a third: In his own words, to “confuse people a bit.” So, there I was, totally ignorant of immunology and not very familiar with animal genetics, but surrounded by people who were as enthusiastic as I about research. And I was prepared to work on something I had never come across before-antigenic variation in tumors. It was then that I first heard mention of the puzzling acronym “H-2.” What was H-2?

II. Part Two: Interrogating the Sphinx

A.

JOINING THE

CLUB

E douremo dunque negarti, Dio dei tumori, Dio del fiore uiuo, e cominciare con un no all’oscura pietru Cio sono-, e consentiere alla morle P su ogni tomba scriuere h sola nostru cerlezza: ethanalos athanatos- ?

And shall we have to deny thee, then, God of tumors, God of the living flower, begin with a no to the obscure rock “ I am”, consenl to death and on each tomb inscribe our only certaanty: “thanutos athanatos”?

Salvatore Quasirnodo: T h a t u s Athanntus

Salvatore Quasirnodo: Thh7lUkJS A ~ / L ~ M J ~ U S

(translated by Allen Mandelbauni)

To those brought up on the notion that, if you want to characterize a gene, why, you just pick it up from your genomic library, it may be difficult to understand the frustrations of mammalian geneticists before the DNA cloning era. At the time when microbial geneticists were able to take their analyses down to the level of operons, cistrons, and even mutons, mammalian geneticists were still using the same methods Gregor Mendel had pioneered, the counting of progenies from a cross between disparate individuals. And they were tremendously proud of themselves when they managed to screen 500 offspring to obtain a 1-cM resolution of their maps. While searching for new methods, they placed high hopes on somatic cell genetics, which became a catchword of the 1960s. T h e fact that somatic cells could be fused (“hybridized”)had been established by then and the occurrence of mitotic recombination could also no longer be doubted (Pontecorvo, 1958). But could the two processes be fashioned into a method of fine gene mapping? One proposal, which originated with Joshua Lederberg (1956), was to induce tumors in

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F, hybrids of two mouse strains differing at the H - 2 complex only and then transplant the tumor tissue to either of the parental strains. T h e heterozygous tumor will, of course, express H-2 antigens of both parents and so the P, parent will reject it because it responds immunologically to P2-derived antigens, and the P2 parent will do the same because of anti-PI-directed response. But what if rare variants arose in the tumor cell population, which, because of recombination or some other mechanism, had lost some or all of the antigens of one parent? Such variants would sprout from the inoculum under attack by the host immune system and would be recognized as tumors developing in the incompatible host. In this manner, millions of cells could easily be screened in each experiment, variants could be found even if they occurred at very low frequencies, and the system could be used to increase the resolution of genetic analysis. Testing of the proposal was undertaken by George and Eva Klein, as well as their students Erna and Goran Moller, Karl Erik, and Ingegerd Hellstrom, and others at the Institute for Tumor Biology, Karolinska Institutet, Stockholm, Sweden (reviewed in Klein, 1975). It seemed to work, and there was a great deal of excitement about the results flowing in from Stockholm. My task was to map the H-2 complex by this method. In the 1960s, there was only a handful of true insiders working with the H - 2 system. There were the two patriarchs, Peter A. Gorer, the discoverer of the system, who was then already losing his battle with cancer, and George D. Snell, the cofounder of the field, and surely one of the gentlest scientists who ever walked this earth. There was Corer’s student, Bernard D. Amos, who was then already in the process of switching camps and establishing himself as one of the founders of the HLA studies. Then there was Jack H. Stimpfling, the H - 2 guru, who, in the serene Montana wilderness, sought and found refuge from the quickening pace of research. There were Gustavo (“Pancho”) Hoecker and Olga Pizarro, who sequestered themselves beyond the Andes and continued H - 2 studies at their leisure. And finally, there was Donald C. Shreffler, a relative newcomer, who landed in the middle of the H - 2 complex while chasing the gene of the “Serum serological” (Ss, alias C4) protein discovered by him. And that was it. The H - 2 had then the reputation of being a system that was important but difficult to understand. One reason behind the fear of H - 2 was serology, the method used in its description. To detect H-2 antigens, you would transplant tissue of one mouse strain into the abdomen of a mouse of another strain (everybody had their own magic formula as to how many cells to use, how often, and at what intervals) and then utilize

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the antiserum thus produced to clump red blood cells of the donor. But herein lies the first difficulty: mouse erythrocytes are, for some reason, difficult to agglutinate by H-2 antibodies. In fact, most of the time they would not agglutinate at all, unless you made the mouse serum more viscous by adding to it in the right proportion (magic formula number two) concoctions such as serum from patients with myeloma, umbilical cord extract, dextran, or polyvinyl pyrrolidone. Of course, by using such a mixture you always walked a thin line between too much clumping (nonspecific reaction) and too little clumping (false negative reaction), and it was only after spending many months developing your own magic recipes that you would begin getting reproducible results. T h e other reason the H - 2 system appeared so intimidating to outsiders was in the reaction patterns the serological analysis generated. Each antiserum you produced would react not only with the donor cells, but often with cells of most of the other strains (except the recipient, of course) that you had room for in your animal colony. You would then resort to absorption analysis, trying to remove some of the antibodies from the mixture by exposing the antiserum to third-party cells. If you were really determined you could end up with an operationally nionospecific antiserum, a euphemism expressing, in effect, “I have gone to considerable pains to make the antiserum react with only one H-2 antigen.” Through the combined efforts of Gorer, Snell, Amos, Stimpfling, Hoecker, Pizarro, Shreffler, and those of us who followed in their footsteps, a magnificently formidable “H-2 chart” was produced, something like a telephone book in which individual numbers could consist of 15 digits or more (Klein, 1975). To insiders, this was the most exciting document in existence, and they never seemed to tire of discussing individual digits. To outsiders it appeared as exciting as-well, as a telephone book. Matters were not made easier by the continuous evolution of the H-2 chart and by occasional radical revisions of H - 2 nomenclature. An outsider might spend months memorizing the directory, only to wake up one morning and find that letters had been changed to numbers, for instance. For all these reasons, the H - 2 fraternity was regarded by outsiders as an exclusive club, the membership of which was limited to those who knew the H-2 chart. After months of repeatedly immunizing, bleeding, agglutinating, and absorbing, I realized one day that I had completed the rites of passage and become a fully accredited member of the club. Familiarity with the H-2 chart and the rest of the H-2 lore allowed me to play little combinatorial games with the H-2 determinants. By choosing the right third-party hosts, for example, I could design experiments in which the immune response was directed against some, but not

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against other, determinants derived from one parent, and ask the question: If determinants selected against are lost, what will happen to determinants not under selection pressure? In this way, I was hoping to be able to decide which determinants were controlled by which loci and t o map the loci. But the Dio dei tumori refused to cooperate with me and the answers I was getting were different from those I expected. I observed that whenever one determinant was lost under the selection pressure, other determinants not under selection pressure were also lost (Klein, 1966). It seemed either that there were only two H-2 molecules, each carrying a plethora of determinants, o r that the H-2 complex was divided into two blocks that behaved as units in whatever process was responsible for generating the antigenic variants. These and similar findings were deemed sufficient by Milan HaSek for a Ph.D. thesis, for which I took my degree in 1964. B. WHATA STOOLPIGEONCAUSED Tyltyl: Que c b l bmu! . , Qu’il fail beaa! . . . O n se croirait en plein 616.

..

Maurice Maeterlinck: L’oiscau blvu

Tyltyl: How beautiful il is! . . Arul what lovely weather! . . . It i s jwll like midyummer . . . Maurice Maeterlinck: The Blue Bard (translated by A. T. d e Mattas)

In 1965, the 100th anniversary of the publication of Johann Gregor Mendel’s seminal work on heredity was to be celebrated by a series of symposia in Brno and Prague. One of the symposia was to be devoted to somatic cell genetics, and I was involved in its organization (Klein et al., 1966). I was delighted, because it gave me the opportunity to meet personally some of the researchers with whose work I was well acquainted. Among those who attended the conference were Leonard and Leonore A. Herzenberg from the Department of Genetics, Stanford University Medical Center, Stanford, California. T h e Herzenbergs and their colleagues were trying to achieve in vitro what the rest of us working in the field were striving to accomplish in vizm, namely, the selection of antigenic tumor variants by the treatment of cultured cells with H-2-specific antibodies and complement (Cann and Herzenberg, 1963a,b; Papermaster and Herzenberg, 1966). I was anxious to discuss their work and my own results with them, and as there was little time for this during the meeting, we decided to d o so in the stimulating atmosphere of a nightclub. The discussion, of course, wandered from science to other topics, although it was some-

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what hampered by my poor command of English. As we were leaving the bar, a commotion took place which led me to make disparaging remarks about the Prague police. I knew that there were probably informers among the customers, but I did not think that they would understand English. How stupid of me to think that the secret police would plant stool pigeons who did not understand English in a nightclub frequented by foreigners! We were stopped at the exit. T h e Herzenbergs were sent to their hotel and I was taken for interrogation in one of the rooms adjacent to the bar. How convenient for the police: you capture the bird and pluck it right on the spot! After that I had to report to the police, and another black mark was entered into the dossier the STB kept on me. But the incident had consequences that, I am sure, the informer had not foreseen. Whether out of compassion o r because, on account of the incident, they felt I would make an interesting addition to the collection of characters in their laboratory, the Herzenbergs invited me to Stanford to work there as a postdoctoral fellow. It was something short of a miracle when, some time later, the government issued to me a permit to travel to the United States, in spite of all the black marks on my record. Today, I know that it was more of a testimony to HaSek’s political standing than anything else. I am sure he must have vouched personally for my return, and one of the reasons I did return was my suspicion that it was so. HaSek was a pronounced communist, but he used his political power to provide shelter for persons like myself, who without his protection would not have been allowed to work, not to mention to travel abroad. A few months later I was on a plane to California. When I stepped out in San Francisco, I felt like Tyltyl and Mytyl entering the enchanted palace of Fairy Berylune in search of the blue bird. The feeling of unreality persisted during my entire stay at Stanford and sometimes returns to me whenever I visit California. Once settled in Palo Alto, I discovered to my dismay that there was nobody in Herzenberg’s laboratory working on anything remotely related to antigenic variation or the H-2; the only thing they all wanted to talk about were immunoglobulin allotypes. Eventually, I worked out a compromise and focused on a project that was halfway between Len’s interests and my interests (Klein and Herzenberg, 1967). In the meantime, however, I discovered that there was a person at Stanford who had a keen interest in H-2 and who was willing to discuss the system with me ad nauseam. He was Hugh 0. McDevitt. Hugh had just returned from a stay abroad, where he had made an

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exciting discovery. While studying the formation of antibodies specific for synthetic antigens, he serendipitously turned up evidence for genetic control of the immune response (McDevitt and Sela, 1965). What was most remarkable about this response was that it seemed to be controlled by a single gene, the immune response-1 o r Ir-1, gene. I used to argue with Hugh, as he never tires of reminding me, that this surely must be because of the artificiality of the situation, the combination of synthetic antigens and of inbred strains. A quantitative trait such as the height of antibody response, I reasoned, ought to be under polygenic control. I should have known better! It should already have been clear to me that Hugh’s keen common sense, combined with a rare instinct for correct interpretation, were two characteristics that would have made any opponent think twice before challenging him. Hugh was, of course, right: Ir-I was not just one of many immune response genes, it was the Zr gene. Although many other Ir genes were described later, Ir-1 retained a special position among them. What made me gravitate to Hugh’s laboratory, however, was not the Ir-Z gene (I was not too crazy about immunology then), but the evidence just emerging on my arrival at Stanford that the gene was closely linked to H-2 (McDevitt and Chinitz, 1969). Hugh was eager to map the gene as precisely as possible, and was collecting all available H-2 recombinants. I had some, too, which I had produced in Prague at the time that I was losing confidence in the power of somatic cell genetics and had turned to the old-fashioned but time-honored procedures of gene mapping. One of these recombinant strains, derived from H - 2 haplotypes a and q and hence dubbed AQR, together with Shreffler’s A.TL strain, proved to be crucial for mapping the Ir-1 gene. To everybody’s surprise, the gene mapped within the H - 2 complex (McDevitt et al., 1972)! This finding was a jolt registering 8.6 on the immunological Richter scale, and the waves emanating from the epicentrum at Stanford were soon reverberating through the entire immunological world. In retrospect, I feel somewhat guilty about not having contributed much to the line of investigation Len was then pursuing, but allotypes just left me cold. On the other hand, I got a great deal out of my stay at Stanford. I recall with particular fondness the evening seminars at the Herzenbergs’ home: to witness Len’s sharp intellect at work was a real treat. Henry H. Wortis, another postdoctoral fellow in Len’s laboratory at that time, and I confided to each other much later that we keep trying to recreate the atmosphere of these meetings with our students, but continue to fail miserably.

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C. FATEFUL DECISION I N TOKYO Et par 1~ pouvoir d’un mol J P recommence ma vie Je suu ni pozii te connuttre Pour Ie noinmer Lzberli

Arid by the power 01a word I stail my lzfe again I w m b o w to know you To name you LibPrty

Paul Eluard: Liber/P

Paul Eluard: Lzberty

Humans might be the only animals that return to their cages voluntarily. In 1967, after tasting a few months of freedom, I returned of my own free will to Czechoslovakia. I did so for several reasons, one of which I have already mentioned. Another was the ferment that was then brewing in my country and that would soon become the “Prague Spring.” I was sceptical about the long-term prospects of the movement (“communism with a human face” is a contradiction in terms), but I expected the valve to remain open for a few years and thus give me time to arrange my personal affairs and to decide about my own future. It was not to be, however, and I was forced to make a decision sooner than I had planned. In August 1968 I attended the International Congress of Genetics in Tokyo. On the third day of the meeting I returned late at night to my hotel, and as I was approaching my room I could hear the telephone ringing persistently. It was Len Herzenberg, who had also been at the meeting. “Look,” he said, “I can imagine how you feel. If you prefer not to go back to Prague under these circumstances, I will go with you tomorrow to the U.S. Embassy and arrange a visa for you to return to my lab. You can stay with me until you find a suitable position elsewhere.” I was nonplussed. I had no idea what he was talking about. “Haven’t you heard?,” he explained. “the Russians have invaded Czechoslovakia!” In the morning we did as he suggested and a few days later I was back at Stanford, to start my life again. My possessions consisted of one suitcase. T h e only thing I was worried about was what was going t o happen to my relatives and my friends, in particular because, the next day, many newspapers across the country carried a story about a “young Czech scientist” who had defected. How the reporters learned about me, I have never found out. I n any case, once my future wife was able to join me and we began to settle down in what was to become our new country, 1 became almost intoxicated by the sudden realization that there was no cage to have to return to any more. Soon afterward, I accepted an offer from Donald C. Shreffler, whom I had visited briefly during my first stay in the United States, to join his

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laboratory as a Research Associate. When I arrived in Ann Arbor, at the Department of Human Genetics at the University of Michigan School of Medicine, Chella S. David was already there, as was Jane S. Schultz and Howard C . Passmore; Dona1 B. Murphy and Ted H. Hansen joined the group shortly afterward. We were a convivial bunch, Chella being the Till Eulenspiegel of the group. His pranks were legendary and we all look back on those times as being among the happiest of our lives. Chella and Jane have remained among niy most trusted and most respected friends. I spent most of the time characterizing my recombinants, while Chella worked with Don’s. The characterization seemed to suggest that several H-2 antigens were encoded in different loci or “regions.” The trend to divide the H-2 complex into regions started with Gorer and Amos and was then continued by Stinipfling and Shreffler (Shreffler, 1970). At the time the trend was peaking, there were eight regions in the H-2 complex, K, I , A, S, E , V, C , and D (the letters were derived from the original designations of the antigens believed to be encoded by the regions). There was a problem with this interpretation, however: several of the antigens mapped to different positions depending on the H-2 recombinant analyzed. Antigen “3,” for example, was believed to be controlled by the C region, which most recombinants mapped to the D end of the H-2 complex, but some mapped it to the K end. This was a puzzling finding, which we tried to accommodate by various explanations. At one point, we even went so far as to speculate that the antigens might be sugar based and resemble the complicated system of Salmonellu lipopolysaccharide antigens (Shreffler and Klein, 1970). Another possibility we considered was that the H-2 complex was symmetrically duplicated around the S region, so that each region was represented twice (Shreffler et al., 1971). T h e explanation we gradually began to favor, however, was that the mapping of the same antigen to different ends of the H - 2 complex was the result of serological cross-reactivity (Klein and Shreffler, 1971). We imagined it to work like this: You might produce antibodies such as those against the D end-encoded antigen 3, but in some mouse strains there might also be K-encoded molecules that would bear antigenic determinants resembling the D-encoded 3, and these would cross-react with the antibodies. If you then obtained a recombinant inheriting this K-end 3, it would appear that the C region mapped to two different ends of the H-2 complex. This interpretation squared well with the results of my thesis work, which seemed to point to the existence of two H-2 molecules only, K and D, both bearing a multitude of H-2 determinants. T h e serological analysis now suggested that the

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two molecules were so similar to each other that antibodies produced against one often cross-reacted with the other. When we carefully analyzed all the serological typings of the H-2 recombinants, Don and I concluded that all the data were consistent with the existence of only two regions, K and D, with the Ss protein gene residing between them. This “two-locus model” (Klein and Shreffler, 1971), as it came to be known, was also consistent with the notion of two segregant series in the human homologue of the H-2 complex, later to be termed the HLA complex. A similar model was also put forward by George D. Snell and Peter Demant (Snell et al., 1971) as well as by Erik Thorsby (197 l), although Erik got the H-2 chart wrong and we wasted no time pointing this out to him (Klein and Shreffler, 1972). I described the two-locus model at the Midwinter Conference on Immunology at Asilomar, California in 1970, and I remember very well the reaction to my presentation. Before this meeting, attempts to explain the H-2 complex at seminars or symposia were usually met with indifference. Half of the audience dozed off shortly after you flashed the H-2 chart on the screen, while the other half spent the time suppressing their yawns. At Asilomar the atmosphere was different. There was a peculiar tension in the air, a sense of expectation, which kept everybody in the room tuned attentively to what I was saying, from beginning to end. Later, I would witness similar charged atmospheres on other occasions, and it would always remind me of a pack of hounds, quivering and alert, on the brink of a chase. D. THECHASEIs ON En cette confusion uenteuse de bruits de ruporh el opinions vulgaires qui nous powsent, il ne se peut Ctablir aucune route qui vaille. Ne now proposons point une fin si flotante el uagabonde; allons constammant aprt?s la ration. Montaigne: Essuis

In that windy confwion of rumors, reports, and popular opinions that push us about, no worthwhile road can be charted. Let us not set ourselves a goal so ,fluctuating and wauering: let u.s steadfastly follow reason. Montaigne: E,ssr?ys (tmnslutrd by D. M . Frumu)

What excited the pack at Asilomar was this: T h e Zr-1 gene of McDevitt and similar genes discovered by Baruj Benacerraf and his colleagues in the guinea pig (see Benacerraf, 1973) seemed to be controlling some important step in the immune response. Their association with the H-2 and homologous complexes in other species could no longer be doubted.

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By sweeping away all the regions from the middle part of the H-2 complex and piling them up at the ends of the complex, Don and I cleared the ground for the product of the Ir-1 gene. If the known H-2 antigens were not the Ir-1 gene products, what was? Did the Ir-1 gene have a product? If so, could it be recognized serologically? And if so, on which cells was it expressed? These and similar thoughts must have raced through the minds of the Asilomar audience. They were certainly racing through Don’s and mine. Two things were clear to us. One was that we had to learn new methods. Up until then we had been satisfied with hemagglutination as our main source of serological information. It had drawbacks, but once mastered properly, it served us well. Now, however, it was obvious that the Ir-1 gene product was not to be found on red blood cells and that we had to develop a means of typing lymphocytes instead. The second thing we realized was that two-strain combinations were the best bets for producing antibodies against the Ir-1 gene product, if such antibodies could indeed be obtained. One combination was A.TL-A.TH, two of Don’s recombinants, the other was AQR-B 1O.T(6R), mine and Stimpfling’s recombinants, respectively. Chella and Don began immunizing in the former combination; Vera Hauptfeld, who had recently arrived from Czechoslovakia via Yugoslavia, and I began immunizing in the latter. Just around then I left Don’s laboratory and established my own across the central campus of the University of Michigan, at the Dental Research Institute. The choice of affiliation may seem unusual, but in reality it was a practical move. I agreed to devote part of my research effort to the genetic factors controlling the outcome of tooth bud transplantation, which was not an uninteresting project at the time, and had the rest of my time free to pursue my main interests. James V. Neel, the Chairman of the Department of Human Genetics, and Don were the prime movers behind this arrangement, finding it a feasible way of keeping me in Ann Arbor. My secondary appointment in the Human Genetics Department formalized the ties with Don’s laboratory. We planned to have a free exchange of information between our two groups through joint lab meetings and collaborations on some projects. Things, however, turned out differently. Shortly after the hunt for the Zr-1 gene product began in earnest, a rift began to develop between our laboratories. T h e fact that I no longer remember how it started indicates that it must have been something trivial, some windy confusion of rumors, perhaps. Gradually, however, an element of mistrust began creeping into our relationship, each side apparently believing the other was hiding something. It was not true on our part and, in retrospect, I d o not think it was true on Don’s and

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Chella’s part, but in the emotionally charged atmosphere of the chase, trivial accidents tended to produce exaggerated consequences. The rift eventually widened to the extent that the two groups stopped comniunicating altogether. Although later we managed to clear up all misunderstandings, reestablish normal relationships, and, with Chella at least, fully renew our former friendship, my relationship with Don has never been the same since. I regret this very much, because I hold Don in high esteem for his personal integrity, his scientific accomplishment, and for everything he has done for me. The method we used initially to detect AQR-B lO.T(6R) antibodies was indirect ininiunofluorescence, mainly because Dagmar BednPfovA, who by then had become my wife, had worked with it in Donald J . Merchant’s laboratory. In retrospect, it was not a good choice. We had great difficulties with nonspecific staining and it took us some time to find an antiimmunoglobulin that gave a background that was not too high and that, in combination with the BlO.T(GR) anti-AQR serum, stained AQR lymphocytes specifically. T h e gene controlling the determinant detected by this combination of antibodies mapped to the Ir-1 region and we therefore decided to call it Zr-1. (This decision was then criticized as inferring too much, but in fact it was quite proper; the two genes were inseparable by recombination and the determinant WUJ the Ir-1 gene product.) Vera, Dagmar, and I published the findings in the July 1973 issue of Science (Hauptfeld et al., 1973). It was the first published description of what would later become known as Ia, or class 11, antigens. The report from Don’s laboratory describing the results obtained with the A.TH anti-A.TL serum appeared in the September issue of the Proceedings of the National Academy of Sciences U.S.A. in the same year (David et al., 1973). The following year several other laboratories reported similar results. T h e fox had been hunted down.

E. OCKHAM’S RAZOR 9 nOMd

7 C O l l O i S F&l

dl&@O@Og

PQOT63V.

Euripides: Mrdea

Not as the world thinks /hink I oftentimes. Eutipides: MdePa

Nature operates on the priqciple of parsimony, which, plainly stated, means that if there is a simple way and a complex way of doing things, nature will always choose the former. Immunologists more often than not seem to be unaware of this. Again and again they offer complicated hypotheses and explanations, which almost always turn out to be too confounding. T h e developments that followed the discovery of class I1 antigens were a good example of this.

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Having established the existence of a new class of H-2 antigens, Don’s laboratory and mine focused on different aspects of these antigens. Chella, Don, and Jeffrey A. Frelinger continued their serological characterization and eventually wound up with an Ia chart akin to the H-2 chart of the class I antigens. My co-workers and I concentrated on the involvement of the Ir region in cellular responses such as the mixed lymphocyte reaction (MLR), graft-versus-host reaction (GVHR), cellmediated lymphocytotoxicity (CML), and graft rejection. For the initial work on the MLR, we teamed u p with Fritz H. Bach, then at the Immunobiology Research Center, University of Wisconsin, Madison, Wisconsin (Bach et al., 1972a,b). Fritz and his co-workers did most of the work, while our role was primarily to provide appropriate strains and an intimate knowledge of the H-2 system. As an example of the latter, Fritz likes to tell the story about how a discussion at a seminar in his home once got bogged down because nobody could remember the genetic difference between a particular pair of H-2 congenic strains. Fritz resolved the problem by calling me in Ann Arbor, forgetting how late it was. The call woke me up at one o’clock in the morning. Only half awake, I recited the requested information and then fell asleep again. That I could churn out H-2 differences while half in the arms of Queen Mab impressed Fritz very tnuch. For “H-2 workers” like us, however, this was nothing unusual: knowledge of the H-2 chart penetrated even to our subconscious! Later, we continued the MLR studies on our own and, simultaneously with this work, carried out an analysis of the influence the individual H-2 regions had on the GVHR, one phase of which was then considered to be an in uiuo analog of the MLR (Klein and Park, 1973; Livnat et al., 1973). Both the MLR and GVHR data revealed that, contrary to what might have been expected, major determinants stimulating these reactions were controlled by the Ir, and not by the K and D regions. This was surprising because the MLR and GVHR were then believed to reflect phases of graft rejection, and graft rejection was the hallmark of the K and D antigens. After all, it was through graft rejection that Gorer first perceived the existence of the H-2 complex (Gorer, 1937), and the antigens he originally described were later shown to be controlled by the K and D regions. Yet, here we were, with data on our hands demonstrating unambiguously that the MLR/GVHR stimulus emanating from the K and D regions was considerably weaker than that from the Ir region. Later, many immunologists, Fritz among them, went to the other extreme by denying any stimulatory role of the K and D antigens, and I fought a long, protracted battle trying to convince them that they were mistaken. They attributed the weak stimulation obtained across K or D disparities to unrevealed “Zr genes.” No such genes, however, were ever

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discovered, and H-2 mutants demonstrated very clearly that K and D antigens alone could stimulate the MLR and GVHR (Klein and Egorov, 1973). Fritz and I parted on the issue of how the results should be interpreted. He championed the view that there were two kinds of principally different determinants, serologically defined (SD) and lymphocyte defined (LD). I argued that the t w o methods simply revealed two different facets of similar molecules. It was, in fact, in an effort to neutralize Fritz’s terminology, which was catching on rapidly among immunologists, that I introduced the designations “class I” and “class 11” antigens (Klein, 1977). Fortunately, the class terminology prevailed over the LD-SD sobriquets. Another bone of contention was the identity of the CML-stimulating determinants. According to the then prevailing view, only K - and D-encoded determinants were responsible for CML responses, but in our experiments we were also getting reproducible CML in strain combinations that differed only in the Zr region (Klein et al., 1977). I remember that when I first presented them at a meeting of immunologists at Cold Spring Harbor in 1976, these data were given the big freeze. After I had finished my talk, Harvey Cantor, who was then considered to be the expert on in nitro lymphocyte responses, stood up and said in essence: “We used the same strain combinations and we never got any response. You must be measuring some nonspecific reaction.” After that I could not convince anybody that there was such a thing as Ir region-controlled CML determinants. Yet we were right, and the truth eventually prevailed. Today most immunologists do not even realize that class I1 region CML was once a contentious issue. Consistent with the CML data was our observation that Zr region disparities were sufficient to activate rejection of skin and other tissue grafts (Klein et al., 1974). Here, too, however, we had to fend off arguments that the strains we used were not truly congenic, that the rejections were caused by “contaminating” minor histocompatibility loci, or that there were as yet undiscovered “SD” loci in the Ir region. Although we were able to demonstrate that the rejections were accompanied by the production of Ir-specific antibodies, immunologists were difficult to convince. This was becoming a pattern that would repeat itself’ again and again. Immunologists eagerly embraced the flimsiest evidence that fitted their preconceived notions, but were extremely critical of data contradicting a fashionable hypothesis, if they paid any attention to such data at all. I do not believe that much has changed since. By about 1973 or 1974, we knew that products of the IT region controlled immune response, stimulated the production of serologically detectable antibodies, stimulated the MLR and GVHR, were the targets of

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CML, and caused graft rejection. All these functions, with the exception of the control of immune response, the Zr region genes shared with the K and D region genes. To me, these observations indicated two things: first, that there was no principal difference between the KID (class I) and Zr region (class 11) gene products, and second, that the various functions were carried out by the same molecules. Very few immunologists agreed with me then, however, and from that time on I would mostly be swimming against the current, and a strong current, too. There was nevertheless one function that even I had difficulty in bringing under the one umbrella, the control of immune response. I had no trouble visualizing how the same molecule could stimulate antibody production, MLR, GVHR, CML, and graft rejection, but I could not rationalize how such a molecule could also decide whether an animal would produce high or low levels of antibodies when immunized, say, against a synthetic polypeptide. T h e various propositions made at that time and invoking “interaction structures,” a plethora of “factors,” and the baroquely complex “suppressor circuits” simply did not make sense. Little wonder, for much of the data on which the hypotheses were based later proved to be irreproducible. A future historian will have a hard time explaining how so much absurdity could have been produced in such a short time-and taken seriously. All that changed in 1974 with the publication in Nature of a brief communication by Rolf M. Zinkernagel and Peter C. Doherty. This article flung open the door to a rational interpretation of the Zr genes. Suddenly it became evident that the Zr genes, too, could be integrated with the rest of the knowledge into a unified concept of the H-2 complex. Zinkernagel and Doherty (1974) had discovered that T lymphocytes do not recognize viral antigens alone but in conjunction with H-2 molecules. This “dual recognition,” which was subsequently quickly extended to nonviral antigens as well, was an entirely new concept, not deducible from any of the previous discoveries. In particular, it could not have been gleaned from the existence of Ir genes, and indeed, none of the fanciful speculations inspired by Zr gene-related phenomena contained even a hint of what the Zinkernagel-Doherty discovery revealed. In fact, for some time after the Zinkernagel-Doherty discovery, immunologists failed to add two and two together and continued to regard Zr genes as having nothing to do with dual recognition. After all, they reasoned, dual recognition concerned class I genes, from which Zr genes were clearly separable by recombination. Immunologists continued to revere the Zr region as if the Holy Grail lay hidden in it, and to look down on class I genes as something much more mundane and even profane. This schizophrenic penchant for regarding KID and Zr regions as two unrelated worlds persisted unabated for an unreasonably long time af-

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ter the discovery of dual recognition, and when the first sequences of the class I and class I1 proteins became known, immunologists were surprised by how similar to each other they were. Up to this point, the principle of parsimony had been lost on them. Yet, as Doherty and Zinkernagel (1975) realized, their discovery provided the key to the true function of not only class I but also class I1 (Zr) genes. If class I1 molecules were also capable of providing a context for the recognition of foreign antigens, then the failure to perceive a particular antigen-class I1 molecule complex, either because of a lack of association between the two components o r because of missing T cells with appropriate receptors, would result in low-level or nonresponsiveness, the very phenomenon used to identify the Ir genes. By the end of the 1970s, all the pieces necessary for formulating a unified concept of the H-2 complex and, more generally, of the major histocompatibility complexes of all other species, were in place. We put together the pieces and published just such a concept in Nature in 1981 (Klein et al., 1981). We argued that both class I and class I1 molecules functioned by providing a context for the recognition of foreign antigens and that Ir genes had no existence of their own: They were the class I1 genes. We surmised that recognition of allogeneic histocompatibility antigens, which was the basis for MLR, GVHR, CML, and graft rejection, was a perverted form of dual recognition and that all these “functions” were therefore performed by the same molecules that provided the context for the recognition of foreign antigens. It took immunologists some time to come around to accepting this concept, and I suspect that even today many of them perceive the class I1 region as being something special when compared to the class I region. Why else would they continue referring to it as the Zr or Z region, when it is now obvious that both the class I and class I1 regions operate on the Zr principle? F. PLAYING THE ACCORDION Accordhn, cheual de &acre Le dernier Foupir arrarhe‘, Tu meurs, en riont de la naue Sur les genow de ton cocher Jean Cocteau: Arcordeon (LP Mirlilon D’ItinP)

Accordion, like a coach-horse Wheezing your last sigh, memy, in mother-vf-peorl, on your coachman5 knees, you die. Jean Cocteau: Accordzon (Ireiidr fennyw/iist~e) (translated by Alastair Reid)

After Don Shreffler and I removed the A, E, V, and C regions from the H-2 map (Klein and Shreffler, 1971), it remained simple for a short

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while only, consisting of the K and D regions at the ends and the Ir and Ss regions in the middle. But as immunologists began to supplement the serological methods of characterizing the H-2 complex by histogenetic methods, such as MLR, GVHR, CML, and graft rejection, the H-2 map began to expand again. Suddenly there were, in addition to the K and D loci, IT, Is, la, Lad, and H loci, not to mention some 30 or so other genes believed to be in the H-2 complex but not mapped with any degree of precision and hence not assigned to particular regions (Klein, 1978). We contracted the expanding H-2 map by our unified hypothesis in which we proposed that the “new” genes were in reality identical with the “old” serologically defined genes (Klein et al., 1981). However, ours turned out to be a Sisyphian undertaking, for no sooner had we simplified the H-2 map than it began to grow again. The new expansion was the result of the serological analysis of class I1 determinants on the one hand, and the mapping of Ir genes on the other. The serological studies seemed to suggest the existence of at least five regions encoding class I1 determinants-A, C , E , J , and V. The results of the Ir (Lad) gene mapping studies were interpreted in terms of at least eight regions, A, B , C, F, H, N , R , and T (see “Madman’s Alphabet” in Klein et al., 1983). We did our best to contract the map again. The C, F , H, N , and R regions dissolved by themselves when the determinants they were supposed to encode either proved to be irreproducible or the mapping data turned out to be in error. The B region had to be scrapped when we found an alternative explanation for the phenomenon that led to its proposal (Baxevanis et al., 1981). T h e T region was shown to be identical with the Qa-1 locus (Klein and Chiang, 1978). The most resistant to removal proved to be the J region. It was originally defined by the chimeric combination of a serological method and cellular reaction: the antibodies were produced by immunization in a particular combination of recipient and donor strains, but their presence was demonstrated by the inhibition of immune suppression (Murphy et al., 1976; Tada et al., 1976). We tried to produce J-specific antibodies many times and always failed. We spent much time attempting to demonstrate J-specific antibodies in sera o r culture fluids provided by other laboratories, but were unsuccessful both in making such reagents stain cells specifically or in making them kill cells in the presence of complement. On one occasion only did w e obtain an inhibitory effect with the reagents sent to us, which, however, was probably not specific. Don Murphy continues in his attempts to rationalize the J locus, but, in the face of the molecular genetics data now available, it is hard to escape the conclusion that the locus was a phantom created by a combination of artifacts, nonspecific reactions, and wishful thinking.

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Over the years, the H-2 map resembled the bellows of an accordion, expanding and contracting to the tune in the player’s mind. Others were good at expanding the bellows, I specialized in contracting them. I never added a region to the H-2 map, but I removed quite a few, until the accordion wheezed its last and died on the coachman’s knees. G. TRAPPER IN MICHIGAN Palmstrom hat nicht Speck im Haw, dahingegen eine Maus.

Palm lacks bacon in the house, but is troubled by a mouse.

KO$, bewegt von seinem Jammer, baut ihm eine Gitterkammer.

KO$ aroused by Palmstroem’s gloom, builds for him a wire room.

Und mil einer Geige fein setzt er seinen Freund hinein.

And he puts his buddy in, next to him a violin.

Nacht ists, und die Sterne funkeln. Palmstriim musizier! im Dunkeln.

Night arrives with stars aspark; Palmstroem fiddles in the dark.

Und denueil er konzertiert, kommt die M a w hereinspaziert.

As the artist hits his stride, comes the mouse and steps inside.

Hinter ihr, geheimer Weise, fullt die Pforte leicht und Leise.

Back of it, like mystery, drops the trapdoor quietly.

Vor ihr sinkt in Schlaf alsbald Palmstroms schweigende Gestalt.

Shortly, Palmstroem falls in deep, undisturbed, and quiet sleep.

Chrislkan Morgenstern:

Christian Morgenstern: The Mouse Trap (Gallow Songs) (translated by Max Knight)

Die Mausrfalle (Gnlgenlieder)

The discovery of the Ir genes started a goldrush, and H - 2 immunogenetics was soon overrun with the usual mob of gold diggers. I am uncomfortable in crowds, and so began to look for a secluded niche in which I could work undisturbed by mass hysteria. I thought I had found it in H-2 polymorphism. Until then, H - 2 immunogeneticists had focused their efforts on inbred strains exclusively, among which they found plenty of haplotypes to keep them busy. But questions such as “How many alleles were there per locus? How many haplotypes? What was the average heterozygosity at the H-2 loci? How was the polymorphism related to population structure? Did the H - 2 polymorphism have a biological meaning and if so,

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which?” could not be answered by studying inbred strains; they would have to be addressed by surveying wild mice. I realized that H-2 serology of wild mice was going to be tough. It was difficult enough with inbred strains, which provided unlimited material for repeated immunizations; a situation in which every donor could be expected to be genetically unique would dramatically magnify these difficulties. On the other hand, I reasoned that these obstacles were likely to scare away the goldminers and I would have the niche to myself. To make the task somewhat easier, I decided to start by producing a series of congenic strains that would carry H-2 haplotypes of wild mice (each strain a different haplotype) but would receive the rest of their genetic makeup from the inbred strain C57BLlO/Sn. So, one brisk morning in the summer of 1969, Howard Passmore (who had already had some experience trapping wild mice) took me out and introduced me to several farmers in the vicinity of Ann Arbor. Later, I would make additional contacts on my own and thus more o r less cover the territory all the way to Salinas. The farmers were generally cooperative. Nowadays one would have to worry that the farmer might shoot first and ask questions afterward, but at that time it was still possible to approach a house unannounced. I believe that for the farmers, a thickly accented guy chasing wild mice might not have seemed quite right in the head, but probably appeared harmless. For several months that summer and fall I spent the mornings driving around the environs of Ann Arbor, checking the traps that I had set the day before, and learning that catching wild mice is not as easy as it may seem. Like fishing, hunting, and mushroom collecting, wild mouse trapping is an art that has its own lore, and when mouse trappers get together over a drink, they might spend the entire evening swapping stories and experiences. Eventually I, too, developed my own ideas about the best traps, the best places to set them, and how to recognize before entering a house whether there were mice in it. But I stopped short of the extreme to which Korf and Palmstrom went. I brought the captured mice to an abandoned building on the University of Michigan campus and attempted to breed them. “Attempted” is the right word, for, their notorious reproductive prowess notwithstanding, wild mice, especially females, are difficult to breed in captivity. Here, too, however, I eventually acquired the necessary experience, and after a few years could begin typing my first “B10.W” strains (Klein, 1972). Altogether, I produced over 30 BIO.W strains, which proved to be invaluable, not only for my own line of work but for the work of others as well. Over the years, they have been used in a variety of studies from cancer research to the study of susceptibility to diabetes mellitus, and

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from the search for new H-2 genes to the mapping of loci on chromosome 17. The entire project, spread over a number of years, was funded by the National Institutes of Health at Bethesda, Maryland. Fortunately, at that time Study Section members were still wise enough to recognize the merits of long-term projects. More recently, the same Study Section group turned down an application because it would have taken three years to obtain the first results. The Section members thought that was too long to wait. From the springboard of the B10.W strains, I jumped into the murky waters of wild mouse serology. With my students, postdoctoral fellows. and collaborators, who included Vera Hauptfeld, Zofia ZaleskaRutczynska, Edward K. Wakeland, Joseph H. Nadeau, William R. Duncan, Dietrich Giitze, Felipe Figueroa, and Herbert Tichy, we were able t o identify the H-2 genes and haplotypes of the BlO.W strains (Klein and Zaleska-Rutczynska, 1977; Zaleska-Rutczynska and Klein, 1977; Wakeland and Klein, 1979; Duncan and Klein, 1980; Zaleska-Rutczynska ~t al., 1983). Aided by these strains, we generated antisera (and later also monoclonal antibodies) that enabled us to determine gene frequencies in populations from different parts of the world and thus to obtain the first glimpse of the real extent of the H-2 polymorphism. I t turned out to be enormous. We discovered that close to 100% of wild mice are heterozygous at some of the H-2 loci (Duncan et al., 1979b) and that mice from different farms, buildings, or even different floors in the same building (“upstairs/downstairs” populations) often have different combinations of 11-2 alleles (Klein, 1970, 1971; Duncan et al., 1979a; Gijtze el al., 1980; Figueroa rt nl., 1986). By using the H-2 polymorphism, we were able to find out about the way wild mice live (Klein and Bailey, 1971), about their origin and spreading (Figueroa and Klein, 1987), including their colonization of North America, about the origin of t haplotypes (Hammerberg and Klein, 1975; NitetiC et al., 1984; Figueroa et al., 1985; Neufeld et al., 1986), about the relationship between 1-1-2 and chromosomal variation (Figueroa ~t al., 1982), and many other interesting things. One finding in particular was important to me personally, because it was instrumental in switching my interests to a different track. By typing mice from different regions and even mice of different subspecies and species, we observed repeated occurrences of what appeared to be identical alleles (Wakeland and Klein, 1983; Figueroa and Klein, 1987). We were able to confirm the serological identity by protein analysis (Arden and Klein, 1982) and by DNA sequencing (Figueroa et al., 1988). The presence of identical H-2 alleles in different populations was, of course, to be expected. More surprising was the finding that some of the populations may have been separated for more than one

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million years. This observation suggested that, contrary to the general belief at the time, the H - 2 genes were changing slowly, perhaps no faster than ordinary genes. A logical extension of this conclusion was the transspecies theory of MHC polymorphism (Klein, 1980, 1987), the notion that much of the H-2 polymorphism now present in the population was generated long before the last speciation took place. The efforts to test the theory and take advantage of its implications led me away from the turf I had started on and took me to entirely new fields. But that is another story.

H. THEFIRSTSEQUENCES-END OF A N ERA Hu nat bych o.clidu mad sve'mu lal, ie zahrcivul s i mnou zde jako mitem, ie hned mne hladil, hned mne .flehal bitem, ie ze mne ledacos ui udllal od skromnc' oluzky a i hrde' ku Vdlle't P, i e padl jsevn a ustal zcls nastokrdtjci ledafim ui by1 11 tom boZim svlttp a tim jsem byl, t h j s e m by1 rcid. Jan Neruda: VFimj s t m by1 rad! (Knahy verfh)

Then why should I bemoan my fate, That it kicked me about like u bull, That it first caressed me, then lashed me with a whip, That out of me il fashioned all manner of things, From a humble query to u proud response, That I fell and rose again a hundred timesOh, I was many things in this world of Our Lord And knewpy in all thot I did. Jan Neruda:

I Knew Joy

in All that I Dad!

(Books of Venes)

When I summarized the H - 2 system in 1975, I needed 620 pages to d o it, and that was before the explosion of knowledge triggered by the Zinkernagel-Doherty discovery! One of the shortest chapters in the book was on biochemistry, and I concluded it with these words: After almost 2 0 years of intensive work and several hundred publications, the accomplishments of H-2 biochemistry are not very impressive. The facts established during these 20 years can be summarized in three sentences: ( 1 ) the H-2 molecules are membrane-bound glycoproteins; (2) the H-2 antigenicity most likely resides in the protein moiety of the molecule; and (3) one haplotype controls at least two H-2 molecules carrying the K and D region antigens, respectively. The rest of the biochemical knowledge of the H-2 antigens is controversial. (Klein, 1975, pp. 379-380).

The biochemists did not like what I had to say about them, but it was true: the uncertainty about the nature of the H-2 product was becoming

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an embarrassment. On the other hand, it was also true that the isolation of pure H-2 molecules was a tough nut to crack. Never before had biochemists faced the task of separating a glycoprotein from the embedding lipids. New methods had to be developed and that took time. All kinds of approaches had been tried, from sonication to leaving cells to rot, but the ones that ultimately worked included dissolution of the lipids with detergent and pulling out the glycoprotein by immunoprecipitation. In 1976, 1 year after the publication of my book, five laboratories reported simultaneously the first amino-terminal sequences of class I molecules (Ewenstein et al., 1976; Henning et al., 1976; Silver and Hood, 1976; Terhorst et al., 1976; Vitetta et al., 1976). I remember very well the meeting at Brook Lodge, Augusta, Michigan, in November 1975, where at one point representatives of the five groups agreed to lay aside the strict secrecy that until then had prevented them from sharing their data, and one after another wrote down their results on the blackboard. As the sequence fragments, many riddled with errors, emerged, I could not help feeling that I was witnessing an historical event, like the signing of the Declaration of Independence or the ratification of the Constitution. Here they were at last, for all of us to see, the first sequences of the product that had eluded us for such a long time. After so many years of guessing, speculation, and conjecturing, we were all finally stepping onto solid ground: the H-2 was a protein! The big question that remained was the nature of the class I1 molecules. Were they also proteins? If so, how were they related to class I molecules? But no sooner had the gates of the Brook Lodge estate closed behind the last conference participant than the secrecy returned. T h e competing laboratories again began to guard their data closely and imposed a total ban on the circulation of any preliminary results. T h e grapevine, of course, remained open. Rumors abounded and intelligence reports were leaked. There was tremendous excitement, and the atmosphere was once more highly charged with expectations. One of the competing groups was that of Jonathan W. Uhr, Ellen S. Vitetta, and J. Donald Capra, from the Department of Microbiology at the University of Texas Southwestern Medical School at Dallas. T h e group needed a serologist to provide them with antisera and the necessary expertise, and Jonathan approached me with the offer of a position in his department. The offer was decent, the prospect of collaborating with biochemists exciting, and the open space alluring, and so I moved down to Dallas with all my mice, for which I almost had to charter a plane. Texas was an interesting experience, both socially and scientifically.

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T h e five years I stayed in Dallas, before I made the big move to Tiibingen to my present position, were productive and, in a special way, unforgettable. As well as with Ellen, Don, and Jonathan, I particularly enjoyed my contacts with James Forman and Wayne J. Streilein, with whom I also collaborated scientifically. Both Jim and Wayne have a keen and very individual sense of humor, so interacting with them was great fun. Wayne would later become the prime mover behind my returning on a seasonal basis to the United States and the obtaining of my current position in the Department of Microbiology and Immunology at the University of Miami School of Medicine. I also nurtured several external collaborations, particularly with Rolf Zinkernagel, then at the Scripps Clinic and Research Foundation, La Jolla, California. Rolf was in the midst of making his second big scoop by discovering that T cells learn how to recognize antigens and H-2 in the thymus, and seemed continually to be in need of all kinds of weird mouse strains, most of which I was able to supply. T h e collaboration with Ellen, Don, and Jon was almost overwhelming and also, at times, amusing, especially because, as a mere provider of antisera and mice, I could afford to remain above the priority and authorship squabbles. Once again, four or five groups came out with the class I1 protein sequences at about the same time, and our group was one of them (Cook et al., 1977; McMillan et al., 1977; Silver et al., 1977; Springer et al., 1977). For me, it was gratifying to see how similar class I and class I1 turned out to be, and thus to have my firm belief in the parsimony principle once more vindicated. The isolation and sequencing of the H-2 proteins brought to an end an era in the history of the H-2 complex. By this time Corer was dead, Amos fully on the HLA track, Snell retired, Stimpfling semiretired, and Hoecker swamped in administrative duties. T h e second generation, Don, Chella, Hugh, myself, and others, had taken over, and the third generation was clambering onto the stage. This generational changeover is a good point at which to cut the thread of this narrative. As I look back on events in the stretch of time from the day I discovered my grandfather’s herbarium to the moment of writing this very personal and idiosyncratic memoir, of all the coincidences that shaped my life and career, I judge the encounter with H-2 my luckiest. If it were not terribly presumptuous, I would say that the H-2 system has been tailored to my needs. I have a wide range of interests, both cultural and scientific, and the H-2 has enabled me to fulfill many of them. When I became excited about somatic cell genetics, the H-2 provided the entrance ticket into the arena. When I felt like being an immunogeneticist for a while, the H-2 was a perfect model with which to dally. Did I feel

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like dabbling in immunology? The H - 2 system led me right into it. Did I develop a taste for evolution? Why, there was hardly a more interesting system to woo than the H - 2 . And on the side, I could flirt with molecular genetics, taxonomy, anthropology, fish biology, and even history. And I had a tremendous time doing each of these things. With Jan Neruda, I can truthfully say: I w a s many things i n this world of Our Lord, and knew j o y i n all that I did. Ill. Part Three: Points of View

A. WHERE

O U R JUDGEMENTS

Em

Time! the cowector where our judgemenk err; The test of truth, love-sole philosopher, For a11 beside are sophisls-from thy thrift, Which never loses though it doth deferTime, the avenger! unto thee I lift My hands and eyes, and heart, and crave of thee u g$. Lord Byron: Childe Huroldj. Pilp'vnage

To avoid giving the impression of claiming that I was always right, I can provide an example wherein Time, the corrector upended my judgment. Several years ago, during my digression into immunology, I became involved in a controversy over antigen processing, the notion that foreign proteins must first be degraded inside a cell before they can be presented to T lymphocytes. One could say that I created the controversy by questioning whether the evidence available then warranted the universal acceptance of the antigen-processing hypothesis and by claiming that our own data went against the hypothesis. On the latter point I was wrong. Antigen processing is now a well-established concept and Emil R. Unanue, Paul M. Allen, Howard M. Grey, Alain Townsend, and others deserve all the credit for developing it. My attempt to deny antigen processing was perhaps my most spectacular, but not my only, blunder. Nonetheless, I do not regret having challenged the antigenprocessing hypothesis, and if we had at hand today only that knowledge that was available then, I would do so again for two reasons. T h e first is my belief that in science it is wrong to focus on one explanation and to ignore others. A few years ago, antigen processing was accepted by the overwhelming majority of immunologists, although the evidence on which it was based was open to alternative interpretations. Immunologists either ignored the alternatives completely or dismissed them offhandedly. Whenever this kind of thing happens, I automatically take the opposite side and try to stir up the lacking discussion.

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The second reason for arguing against antigen processing was that the hypothesis quickly became fashionable. I have a low regard for any fashion, but for fads in science I have the profoundest contempt. Fashion leads to the negation of the very principle on which science is based: the principle of objectivity. In science, a dictate-any dictate-is unacceptable. Yes, I have often erred in my judgment. But I firmly believe that it is better to have a wrong opinion than to have no opinion at all. A wrong opinion can help resolve a problem by stimulating a debate. N o opinion, however, contributes nothing.

B. MUSKRATSA N D LITTLEBEAVERS So much the better!-I may stand alone, But would not change my free thoughts for a throne. Lord Byron: Don Juan

Miroslav Holub, the Czech immunologist and poet, recounts in one of his short pieces an event he witnessed in a movie theater. In those days, the theaters used to show short movies before the main feature, and the one shown on that particular occasion was about muskrats, which the narrator mistakenly referred to as “little beavers.” At the first mention of “little beavers,” a man sitting in front of Holub said quietly but distinctly: “They are not beavers, they are muskrats!” The narrator, of course, went on with the text and his little beavers, and the man grew more and more agitated, correcting him continually. Eventually, the audience’s initial “shushing” evolved into shouts of “Quiet!” and “Shut up!,” but the man would not stop. A fierce argument erupted, and when it became clear that he would be thrown out, the man stood up and left. He preferred to miss the main feature than to remain silent in the face of a falsehood. The story is not about me, but it could have been. I do not know whether I would have argued with the narrator of a movie, but I might have written to the movie producers, suggesting they straighten out their zoology. I would almost certainly have spoken about the error to somebody somewhere. For, like Holub’s moviegoer, I suffer from the same urge to put wrong right. This urge is a highly disadvantageous trait, which, if genetically controlled, should have been eliminated by natural selection. It is a trait whose expression may bring one into physical peril, endanger one’s career, alienate one’s friends, and win one enemies left, right, and center. Although not spurred by a malicious intent, the tendency to speak up is often interpreted that way. It is overpowering, and

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at least as strong as other powerful human drives, sex included. One is punished for it continuously, yet one never learns. One can make thousands of resolutions to keep one’s mouth shut, yet as sure as the sun rises, one will break them next time around. It is an impulse that has driven many a man and woman before inquisition tribunals, into dungeons, or even onto the executioner’s block. Yet, men and women continue to voice their beliefs and would not change their free thoughts f o r a throne. We may be muzzled trying to speak up, we may even be frightened into silence, we may be forced to recant our beliefs, but, on leaving the tribunal, we must mutter, at least to ourselves, Eppur si muove, “and yet it does move.” C. THE CALLING Time can but make it easier 10 be wise Though now it .seem impossible, and so All that you need is patience W. B. Yeats: The Folly of Being Comforted

As a person who knew, from the age of eight, that he would be a scientist, it is sometimes difficult for me to understand young people who choose a research career for want of anything better to do. For me, being a scientist is not an occupation, but a vocation. Science is a calling without an alternative, a summons one is compelled to obey, and no sacrifice is too great in its service. Evenings, weekends, and holidays are among the first things to be relinquished, but ultimately science becomes a person’s whole life. I have been impatient with students who believe that putting in 8 hours of leisurely work is already too much, and who forget about science the moment they step outside of the laboratory. This intolerance has sometimes thrown me on a collision course with such students, and those who left in anger have made sure others would hear only their side of the story. Thus, I have acquired the reputation of being a slave driver. Time has made it easier for me to be wise, and I have learned to accept that not everybody is as zealous as I am. Still, in my own laboratory, I d o not want to be surrounded by people who do not feel the calling. I would rather live with the reputation of being an autocrat than be in charge of young people who retire at the beginning of their careers.

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D. THEKINGFISHER

/

Gustav Mahler: Das Lied uon der Erde (The Song of the Earth)

Through the memories of my childhood flows a creek. It meanders gently through the meadows, runs swiftly past the ravine, skirts the village, and then makes a sharp turn right under the windows of my attic room. I sometimes hear it murmuring in my dreams, the only sound in the stillness of a moonlit night. I remember the creek as a place of awe-inspiring beauty, an inexhaustible source of wonder. When it froze in winter, you could read it like an open book. You could see where the pheasant searched for food, where a hare crossed it, pursued by a fox, and where the neighbor’s cat stalked a sparrow. You could tell spring was coming by the symphony of sounds created by the surging water beneath the cracking ice. Soon afterward, the first wood anemones, lesser celandines, and primroses would appear under the alder trees. And later, when dense foliage clothed the banks, the orioles would fill the valley with their sonorous whistling cries. In the heat of summer, the waters would teem with fish and crayfish, and the banks with muskrats and water shrews. Each bend of the stream had its own secrets, each cluster of reddened tree roots was an invitation to exploration and adventure. Fall would first announce itself by a few yellowing leaves on age-old willows,and then one morning you would wake up to a riot of colors, a fire-breathing dragon winding under your window. And so the seasons passed, each bringing new delights to us kids. One thrill, however, always seemed to elude me: I could never catch sight of the kingfisher! A few lucky villagers reported seeing the bird, but, no matter how hard I tried, I could not track it down. I longed to see that marvel of the avian world, a creature that nested in burrows, dived after fish, and, by all accounts, sported brilliant colors, but the bird was by then already extremely rare. One day, however, as I was daydreaming under a natural overhang of foliage, out of the corner of my eye I caught

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a blue sparkle in the air above the quiet water. And then, right in front of me, so near that if I had stretched out my hand I could have touched it, was the kingfisher! Through the shimmering light, it looked like a patch of blue sky that had descended onto the branch, its image mirrored in the water beneath it. A moment later it was off, and were it not for the swinging branch, I would have thought I had dreamed it. I visited the village of my childhood recently. T h e creek was still there, but it was dead. Years earlier, the authorities had decided to build a distillery in an upstream village and had allowed the waste to be dumped into the creek. It took only a few months for the pollution, like a spreading cancer, to reach our village. The crayfish were the first to go, followed by all the fish, and then by all the other water animals. T h e bankinhabiting mammals were next, and finally the birds. That once-magical stream had become a sluggish, lifeless, mud-filled, stinking sewage. The world now is very different from that of my childhood. T h e death of a creek is just one of many brutal metamorphoses the world has undergone in these 50-odd years. Others are apparent everywhere I look. Everything has changed, the environment, the social system, the mores, the philosophical outlook. Science, too. I try desperately to cling to the romantic notion of science as a source of wonder, an enriching activity that lifts mortals above the mere struggle for existence. But science is changing from adventure to venture, from endeavor to enterprise. Instead of “Why?” and “By what mechanism?” I hear dishearteningly more often “How much?” and “In what currency?” It is not for me to judge which of these changes are for the better and which for the worse; only future generations will be in a position to do so competently. And I do realize that Homo supiens is the most adaptable of all animal species: a visit to Mexico City or a coal-mining town in Rumania should convince anybody that we can adapt to almost anything, perhaps even to Hell itself. Children growing u p along the banks of my creek today do not know what it was once like. They will never realize what joys they have been deprived of. But they have adapted to the circumstances and have found other sources of entertainment: the television set and the computer. And so, on the surface at least, all seems well. Half a century ago, the creek spawned a scientist, and some day it may do so again. But it will be a different kind of scientist, not one haunted by the fleeting image of a kingfisher, but one better adapted to the business-like character science is rapidly assuming. Better adapted he may be, but I would not want to be in his place. I am almost glad that for me suddenly it’s evening.

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AN APPRECIATION If I have dwelled on some early moments in my life and career longer than was perhaps called for, it was to express, for the first time publicly, my gratitude to Mr. Karel Kousal, the high-school teacher who delicately nurtured my interest in natural sciences and helped me to form my own philosophy; to the examiner in Prague, whom I never met again, but who convinced the committee that I should be admitted to the University; to Professor Zdenek cernohorskf, who courageously interceded on my behalf and thus enabled me to complete my studies at the University; to Professor Alfons Jungert, whose intervention saved me from banishment to the Czech equivalent of Dodge City; to Professor Milan HaSek, who helped an unknown candidate, at a critical moment, to find his niche and who introduced him to the H-2 complex; to Professor Leonard A. Herzenberg, who brought me to the United States and taught me how to think scientifically; and to Professor Donald C. Shreffler, who helped me to get on my feet. Without these men, I would not be where I am: T h e Principle of Uncertainty would undoubtedly have swept me in an entirely different direction. I thank Ms. Lynne Yakes and Ms. Donna Devine for editorial help in the preparation of this manuscript. REFERENCES

LITERARY WORKS Byron, Lord, “The Coniplete Poetical Works of Byron.” Houghton Mifflin, Boston, 1933. capek, K., “Hry.” ceskoslovenskf spisovatel, Prague, 1956. Holan, V., “Sebrane spisy. VII. Pfibehy,” Odeon, Prague, 1970. Maeterlinck, M., “The Blue Bird. A Fairy Play in Six Acts” (A. T. d e Mattos, trans.). Dodd, Mead, New York, 1958. Maeterlinck, M., “L‘Oiseau Bleu.” Libraire Charpentier et Fasquelle, Paris, 1912. Markov, V., and Sparks, M., “Modern Russian Poetry.” Bobbs-Merill, Indianapolis, IN, 1966. Montaigne, “The Complete Essays of Montaigne” (D. M. Frame, trans.). Stanford University Press, Stanford, CA, 1943. Montaigne, “Essais. Oeuvres Completes.” Editions Galliniard, Paris, 1962. Morgenstern, C., “Galgenlieder und andere Gedichte. Gallowsongs and Other Poems” (M. Knight, trans.). Piper, Munich, 1990. Neruda, J., “Knihy b9sni.” Orbis, Prague, 1951. Quasimodo, S., “The Selected Writings of Salvatore Quasimodo” (A. Mandelbaum, ed. and trans.). Farrar, Straus 8t Cudahy, New York, 1960. “Vita Karoli Quarti. Karel IV. Vlastni iivotopis.” Odeon, Prague, 1978. Yeats, W. B., “The Collected Works of W. B. Yeats. Vol. I: T h e Poems” (R. J. Finneran, ed.). Macmillan, New York, 1983. Yesenin, S., “Sotchinenia. V dvuch tomach. Tom pervyj. Stichotvorenia.” Gosudarstvennoe Izdatelstvo Chudozhestvennoj Literatury, Moscow, 1956.

SCIENTIFIC WORKS Arden, B., and Klein, J. (1982). Proc. Nntl. Acad. Sci. U . S . A . 79, 2342-2346. Bach, F. H., Widmer, M. B., Segall, M., Bach, M. L., and Klein, J. (1972a). Science 176, 1024- 1037.

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Bach, F. H., Widnier, M. B., Bach, M. L., and Klein, J. (1972b).J. Exp. Med. 136, 14201444. Baxevanis, C. N., Nagy, Z. A., and Klein, J. (1981).P ~ / J Natl. c . Acud. Sci. U.S.A. 78, 38093813. Benacerraf, B. (1973). Harvey Lect. 67, 109-141. Cann, H. M., and Herzenberg, L. A. (1963a).J. Exp. Med. 117,259-265. Cann, H. M., and Herzenberg, L. A. (1963b).J. Exp. Med. 117, 267-283. Cook, R., Vitetta, E. S., Capra, D., a n d Uhr, J. W. (1977). Immunogmetic.~5, 437-443. David, C. S.. Shreffler, D. C., and Frelinger, J. A. (1973). Proc. Nutl. A d . Sci. U.S.A. 70, 2509-25 14. Doherty, P. C., and Zinkernagel, R. M. (1975). Lancet 1, 1406-1409. Duncan, W. R., and Klein, J. (1980).Immunogenetics, 10, 45-65. Duncan, W. R., Wakeland, E. K., and Klein, J. (1979a). Immunogenetics 9, 261-272. Duncan, W. R., Wakeland, E. K., and Klein, J. (1979b). Nature (London) 281, 603-605. Ewenstein, B. M., Freed, J. H., Mole, L. E., and Nathenson, S. C . (1976). Proc. Nutl. Acad. Sci. U.S.A. 73, 915-918. Figueroa, F., and Klein, J. (1987). Irr “H-2 Antigens: Genes, Molecules, Function” (C. S. David, ed.), pp. 61-76. Plenum, New York. Figueroa, F., Zaleska-Rutczynska, Z., Adolph, S., Nadeau, J. H., and Klein J. (1982). C h i d Re.$.41, 135-144. Figueroa, F., Golubii., M., NiZetii., D., and Klein, J. (1985). Proc. Nutl. Acud. Sci. U.S.A. 82, 28 19-2823. Figueroa, F., Tichy, H., McKenzie, I., Hammerling, U., and Klein, J. (1986). C u m To#. Microbial. Immunol. 127, 229-235. Figueroa, F., Gunther, E., and Klein, J. (1988). Nature (London) 335, 265-267. Gorer, P. A. (1937).J. Pathol. Bacteriol. 44,69 1-697. Gotze, D., Nadeau, J.. Wakeland, E. K., Berry, R. J., Bonhomme, F., Egorov, I. K., Hjorth, J. P., Hoogstrall, H., Vives, J., Winking, H., and Klein, J. (1980).J. Immunol. 124,2675268 1. Hammerberg, C., and Klein, J. (1975). Nature (London) 258, 296-299. Hauptfeld, V., Klein, D., and Klein, J. (1973). Science 181, 167-169. Henning, R., Milner, R. J., Reske, K., Cunningham, B. A., and Edelman, G. M. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 1 18-1 22. Klein, J. (1966).“ T h e Use of Tissue Incompatibility in the Genetics of the Somatic Cell” (in Czech). Academia, Prague. Klein, J. (1970). Science 168, 1362-1364. Klein, J. (1971). Nature (London) 229, 635-637. Klein, J. (1972). Tramplantation 13, 291-299. Klein, J. (1975). “Biology of the Mouse Histocompatibility Complex: Principles of Immunogenetics Applied to a Single System.” Springer-Verlag, New York. Klein, J. (1977). In “ T h e Major Histocompatibility System in Man and Animals” (D. Gotze, ed.), pp. 339-378. Springer-Verlag. New York. Klein, J. ( 1 978). A h . Immunol.26, 55- 146. Klein, J. (1980). In ”Immunology 80” (M. Fougereau and J. Dausset, eds.), pp. 239-253. Academic Press, London. Klein, J. (1987). Hum. Immunol. 19, 155-162. Klein, J. (1989). In “Realm of Tolerance” (P. Ivanyi, ed.), pp. 73-79. Springer-Verlag, Berlin. Klein, J.. and Bailey, D. W. (1971). Genetics 68, 287-297. Klein, J., and Chiang, C. L. (1978). Immunugenetic.s 6, 235-243.

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Klein, J., and Egorov, 1. K. (1973).J. Immunol. 111, 976-979. Klein, J., and Herzenberg, L. A. (1 967). Tramplantation 5, 1484-1495. Klein, J., and Park, J. M. (1973).j. Exp. Med. 137, 1213-1255. Klein, J., and ShrefHer, D. C. (1971). Tramplant. Rev. 6, 3-29. Klein, J., and Shreffler, D. C. (1972). Tissue Anligenr 2, 78-83. Klein, J., and Zaleska-Rutczynska, 2. (1977).J. Immunol. 119, 1912-1915. Klein, J., VojtiSkova, M., and Zeleny, V., eds. (1966). “Genetic Variations in Somatic Cells.” Academia, Prague. Klein, J., Hauptfeld, M., and Hauptfeld, V. (1974). Immunogenetics 1, 45-56. Klein, J,, Chiang, C. L., and Hauptfeld, V. (1977).J. Exp. Med. 145, 450-454. Klein, J., JuretiC, A., Baxevanis, C. N., and Nagy, Z. A. (1981). Nature (London) 291, 455460. Klein, J., Figueroa, F., and David, C. S. (1983). Immunogenetics 17, 553-596. Lederberg, J. (1956). Ann. N.Y. Acad. Sci. 63, 662-665. Livnat, S., Klein, J., and Bach, F. H. (1973). Nature (London) New B i d . 243, 42-49. McDevitt, H. O., and Chinitz, A. (1969). Science 163, 1207-1208. McDevitt, H. O., and Sela, M . (1965).J. Exp. Med. 122, 517-531. McDevitt, H. O., Deak, B. D., Shreffler, D. C., Klein, J., StimpHing, J. H., and Snell, G . D. (1972).J. Exp. Med. 135, 1259-1278. McMillan, M., Cecka, J. M., Murphy, D. B., and McDevitt, H. 0. (1977). Proc. Natl. Acad. S C ~U.S.A. . 74, 5135-5139. Murphy, D. B., Herzenberg, L. A,, Okumura, K., Herzenberg, L. A., and McDevitt, H. 0. (1976).J . Exp. Med. 144, 699-712. Neufeld, E., Ritte, U., Figueroa, F., and Klein, J. (1986). Immunogenetics 24, 374-380. Niietit, D., Figueroa, F., and Klein, J. (1984). Immunogenetics 19, 31 1-320. Papermaster, B. W., and Herzenherg, L. A. (1966).J. Cell. Physiol. 67, 407-420. Pontecorvo, G. (1958). “Trends in Genetic Analysis.” Columbia Univ. Press, New York. Shreffler, D. C. (1970). In “Blood and Tissue Antigens” (D. Aminoff, ed.), pp. 85-99. Academic Press, New York. ShrefHer, D. C., and Klein, J. (1970). Tramfllant. Proc. 2, 5-14. Shreffler, D. C., David, C. S., Passmore, H. C., and Klein, J. (1971). Transplant. Proc. 3, 175179. Silver, J., and Hood, L. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 599-603. Silver, J., Russell, W. A., Reis, B. L., and Frelinger, J. A. (1977). Proc. Natl. Acad. Sci. U.S.A. 74,5131-5134. Snell, G . D., Cherry, M., and Demant, P. (1971). Transplant. Proc. 3, 1183-1186. Springer, T. A., Kaufman, J. F., Terhorst, C., and Strominger J. L. (1977). Nature (London) 268, 213-218. Tada, T., Taniguchi, M., and David, C. S. (1976).J. Exp. Med. 144, 713-725. Terhorst, C., Parham, P., Mann, D. L., and Strominger, J. L. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 910-914. Thorsby, E. (1971). Eur. J. Immunol. 1, 57-59. Vitetta, E. S., Capra, J. D., Klapper, D. G., Klein, J., and Uhr, J. W. (1976). Proc. Natl. Acad. Sci. U.S.A. 73, 905-909. Wakeland, E. K., and Klein, J. (1979). lmmunogenetics 8, 27-39. Wakeland, E. K., and Klein, J. (1983).J. Immunol. 130, 1498-1499. Zaleska-Rutczynska, Z., and Klein, J. (1977).J. Immunol. 119, 1903-191 1. Zaleska-Rutczynska, Z., Figueroa, F., and Klein, J. (1983). Immunogenetics 18, 189-203. Zinkernagel, R. M., and Doherty, P. C. (1974). Nature (London) 248, 701-702.

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FOUNDATIONS IN CANCER RESEARCH HEMOPOIETIC REGULATORS AND LEUKEMIA DEVELOPMENT: A PERSONAL RETROSPECTIVE Donald Metcalf The Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Victoria 3050, Australia

I. 11. 111. IV. V. VI. VII. VIII. IX. X.

Introduction Background and Early in Vivo work Hemopoietic Clonal Cultures Colony-Stimulating Factors Biological Actions of Colony-Stimulating Factors Myeloid Leukemic Cells in Culture Role o f Colony-Stimulating Factors in Initiation of Myeloid Leukemia Membrane Receptors for Colony-Stimulating Factors Hemopoietic Regulators in the Context of Known Inducers of Leukemia Role of Hemopoietic Regulators in Suppressing Myeloid Leukemia XI. Recapitulation XII. The Future References

I. Introduction

The editors of this series have made a somewhat unusual request that I write a personalized analysis of the work my colleagues and I have been doing in the past 40 years, on the possible involvement of specific hemopoietic regulators in the development of leukemia. Why were such regulators postulated as being involved in leukemia development? How was the question approached experimentally? How were the regulators in question discovered and developed? Does excessive or inappropriate stimulation by regulators lead to leukemia development? How have ideas on these questions evolved over this period and in what direction does current work appear to be heading? In the spirit of what was requested of me, the present review will not attempt an exhaustive analysis of the literature in the field but will approach the subject in the form of a narrative account of what led me into the field and what transpired when technical advances allowed us to dig

41 ADVANCES IN CAN(:ER RESEARCH. VOL. G3

C:opyrighI 0 1904 by Academic Press, Inc. All rights of reproduction in any Iorm reserved.

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more deeply into the subject. I am wary of this approach. The account may well appear egocentric, may seem to pass lightly over major developments due to others, and may arrive at personal views of the data that are quite erroneous. I hope that this has not happened. There is, I suppose, a partially effective safeguard against extreme o r untenable views. When one works on communal projects with successive groups of colleagues over the years, extreme views tend not to be sustainable and a group consensus emerges that, in turn, becomes modified by competitive o r collaborative interactions with other groups. Ideally, the group may retain a distinctive approach, but in general its ideas need to conform with what evolves internationally. Short of being distinctly odd, one’s views tend therefore to be tempered to a large degree by what is acceptable to others in the field. II. Background and Early in Vivo Work

When I was in medical school in the late 1940s, it became apparent to me that lectures on cancer were no more than descriptions of the pathological appearances of the tissues and that very little seemed to be known about the biology of cancer. This absence of answers to questions that seemed reasonable to pose led me to choose a career in cancer research. Leukemia, as a possibly neoplastic version of normal hemopoiesis, became the most fascinating subject of all-an interest reinforced by my inability as a young intern to offer any sort of treatment for this bizarre and then uniformly fatal disease. In 1954, if one accepted published work on chickens and mice as possibly also being valid for humans, human leukemia could be regarded either as a virus-induced disease of some type (the virus yet to be isolated) or, alternatively, based on work with inbred mice, as a genuine transplantable cancer. In patients with acute leukemia, it was still rare in 1954, except in childhood acute lymphoid leukemia, to witness a temporary complete remission, but when this event occurred, the transient return to apparent normality dramatically set the disease apart from any other type of cancer and raised serious doubts whether acute leukemia was in fact a cancer at all. The most dramatic hematological success of the generation preceding mine had been the understanding and control of pernicious anemia, a disease that in its untreated state exhibits an accumulation of abnormal immature cells in the marrow, not unlike leukemia. If pernicious anemia was merely a profound aberration of hemopoiesis correctable by vitamin treatment, was myeloid leukemia essentially similar in nature? Did myeloid leukemia arise because of some deficiency o r imbalance in vitamins

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or specific regulators (Whitby, 195 1; Israels, 1954)? Conversely, were there natural agents whose administration could convert the blast cells of acute leukemia into normal-enough maturing hemopoietic cells? Although these notions were not particularly popular at the time, the phenomenon of remissions raised the exciting possibility that leukemia-whatever the name denoted-might be a potentially reversible disease. Such a possibility appeared to become progressively more fanciful in the following decades with accumulating evidence of the clonality of leukemic populations and of specific chromosomal translocations, the hallmarks of cancer. However, recent events, with the dramatic induction of remissions in acute promyelocytic leukemia by the administration of retinoic acid (Daenen et al., 1986; Huang et al., 1988; Castaigne et al., 1990), have shown that the possibility was far from fanciful. Certainly, in the early 1950s, such a possibility was reasonable enough to at least entertain. The major formative influence on my own ideas on the actual process of leukemogenesis was the work of Jacob Furth on the development of tumors in target tissues of hormones (Furth, 1953, 1954). Essentially, what he had documented in an elegant series of studies was that experimental manipulations resulting in a sustained hormonal imbalance favoring proliferation first induced hyperplasia in the target tissue, then the development of tumors that were transplantable and behaved as cancers, but only in the continuing presence of the initiating hormonal imbalance. Such tumors were termed dependent tumors. With time, mutations occurred in dependent tumors that conferred autonomy on the affected cells and a capacity to grow progressively even in normal recipients (autonomous tumors). It remains unclear what mechanisms, under these circumstances, lead to neoplastic transformation in organs such as the pituitary, thyroid, o r gonadal tissues. These are not favorite tissues for today s molecular biologists. Nevertheless, the conclusion emerging at the time was clear enough-prolonged growth factor imbalance can somehow lead to tumor development in a target tissue, usually with a definable sequence of dependent tumor formation followed by autonomy. It seemed reasonable to anticipate that hormonelike agents might control the proliferation of hemopoietic tissues, because this would be a feasible method for regulating and coordinating the proliferation of the dispersed populations of hemopoietic cells in the body. Indeed, one such humoral regulator, erythropoietin, had been discovered almost 50 years previously (Carnot and Deflandre, 1906) and was the subject of active investigation in the 1950s. It was possible, therefore, that comparable regulatory factors might control myeloid and lymphoid populations and

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DONALD METCALF

11954 HYPOTHESIS1

Growth Excess Growth Excess Growth Factors Factors Factors

1 1

1

,----

0 0

Normal

Hyperplasia

Dependent Leukemia

Autonomous Leukemia

Ftc. 1. The starting hypothesis in 3954. Excessive growth factor stimulation causes hyperpbasia of hernopoietic cells, with the eventual emergence of a leukemic population with a continuing dependency on excess growth factor stimulation. With time, emerging intitant cells generate an autonomous leukemic population. Such autonomous populations could be clonal. as shown.

were likely to be present in the circulation. If there was a similarity between endocrine target tissues and hemopoietic populations, a possible sequence of events leading to leukemia development might be that persons at risk of leukemia development would exhibit an imbalance of humoral regulatory factors, forcing the hemopoietic cells to become hyperplastic, then to transform to dependent or autonomous leukemias (Fig. 1). Furth was well aware of this possibility. In 1954 he wrote “On the basis of events with other regulated cells it can be postulated that a permanent disturbance of the homeostatic balance might result in leukaemias in which the proliferating cells are essentially unaltered, and which could be controlled at their inception by restoration of the deranged equilibrium of the regulatory forces.” He concluded his very perceptive 1954 essay with this paragraph: Years ago, when I came to the conclusion that the mammalian leukaemias then studied were composed of permanently altered cells, I was depressed by the consequence of this conclusion, for control of leukaemia, like that ofcommon cancer, would call for an agent whirh selectively destroys all leukaemic cells without harming their normal prototype. T h e hypothesis that some leukaemias are conditioned neoplasms has at least one virtue: it raises the hope that some leukaemias might be controlled by restoring the normal balance, and may lead to renewal of much-needed research on forces regulating normal haemopoiesis.

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T h e sequence of these last phrases is illuminating. As a true scientist, he placed understanding on a higher plane than that of merely developing a treatment for a human disease. It seemed to me that two methods to explore this question were, first, to attempt to detect abnormal levels of hemopoietic regulators in the serum of preleukemic or leukemic subjects and, second, to seek evidence of a continuing dependency of at least some leukemic cells on proliferative stimulation by such regulators. These considerations led me to investigate the effects of injected serum from leukemic patients on white cell levels in the blood of mice (Metcalf, 1956a,b). T h e protocol chosen was somewhat curious-to inject neonatal mice intracerebrally with leukemic serum. The neonatal animal was chosen because its small size allowed the use of small volumes of test material, and the intracerebral route was used because it was a standard procedure in the virus laboratory in which I was working. In neonatal mice, this mode of injection is without apparent ill effects and allows larger volumes to be injected compared to the intraperitoneal o r subcutaneous routes. T h e parameter measured initially was absolute white cell numbers in the blood but, with time, this was modified, because of interanimal variation, to a simpler estimation of the lymphocyte-to-neutrophil ratio in blood films. A survey of leukemic sera indicated that the injection of sera from patients with chronic lymphoid leukemia or lymphomas often had the novel ability to elevate the lymphocyte-to-neutrophil ratio in recipient mice. Of various organ extracts tested, only thymus extracts produced a similar response, with higher levels of activity in the thymus from humans or mice with lymphoid leukemia (Metcalf, 1956~).T h e active factor was termed the thymic lymphocytosis-stimulating factor. Regrettably, these observations on the effects of thymus extracts were never confirmed by others. However, because only one negative report was actually published, it remains unclear what techniques had been used by other workers and what the exact negative data had been. There were two useful outcomes of this unpromising beginning. First, my attention was directed to the thymus-then an organ of unknown function other than that it was an obvious site of lymphocyte formation. Second, it caused me to seek a postdoctoral position with Jacob Furth (Fig. 2), who among his many discoveries had made the intriguing observation that thymectomy prevented the development of lymphoid leukemia in the high-leukemia AKR mouse strain developed by him (McEndy et al., 1944). Although he had demonstrated that the first leukemic cells arose in the thymus (Furth and Boon, 1945) and that there was there-

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FIG. 2. Jacob Furth and the author in 1958 working in the Children’s Cancer Research Foundation, Boston, now the Dana-Farber Center.

fore a semimechanical explanation for the effects of thymectomy, several questions remained. Why d o cells transform for preference in the thymus, and might the thymus be influencing the behavior of lymphoid tissues elsewhere in the body in some manner relevant to leukemogenesis? In AKR mice, the thymic lymphomas are now recognized to be virally induced and it has been demonstrated that the tumors in these mice often exhibit retroviral insertion that activates the c-my protooncogene (Graham et al., 1985). Why thymic lymphoid cells are particularly susceptible to transformation remains an unresolved question. In experiments in Furth’s laboratory, I was able with some difficulty to show that thymectomy led to lymphocyte depletion in specific regions of the spleen and lymph nodes, now termed T-dependent regions (Metcalf, 1960). Subsequent work by Jacques Miller (1961) using the more favorable model of neonatal thymectomy documented the dramatic suppressive effects of neonatal thymectomy on immune responsiveness. Ultimately, through the work of Warner and Szenberg (1964),the existence

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of T lymphocytes and B lymphocytes was documented, leading to a great number of studies on the special functions of these two populations and their interactions during immune responses. Most of this work regarded antigenic stimulation as the dominant stimulating event for cell proliferation in lymphoid populations, with a consequent rather slow subsequent recognition of the existence and importance of regulatory factors such as IL-2, IL-4, IL-6, and IL-7. None of these regulatory factors is uniquely thymic in origin and although agents such as thymosin and thymopoietin have been described, the nature of the lymphopoietic regulatory factors exclusively produced by the thymus remains rather obscure. I n the absence at that time of any known lymphocyte regulatory factors other than foreign antigens, I did undertake one long-term study attempting chronic overstimulation of lymphoid tissues. T h e procedure used was to inject C3H mice weekly for life with the foreign antigens bovine serum albumin or Salmonella flagellin. This procedure proved to be a good method for inducing amyloid disease and the mice did develop an increased incidence of a miscellany of tumors of lymphoid tissues (Metcalf, 1961). However, the results were far from convincing. My own studies on the thymus in the period 1960-1966 became focused on the puzzling autonomous behavior of the organ. T h e data indicated that the enormous proliferation of T lymphocytes occurring within the organ was essentially being regulated locally by thymic stroma1 and/or epithelial cells (Metcalf, 1963, 1966). However, how this was achieved could not be established and remains a fascinating problem. During this period, some evidence was produced that lymphoid leukemias developing in AKR mice pass through a thymus-dependent phase (Metcalf, 1962). 111. Hemopoietic Clonal Cultures These often frustrating experiences with the thymus and lymphoid populations made it evident to me that the questions being posed were not likely to be answered by in vivo studies. The hemopoietic and lymphoid systems appeared far too complex to be perturbed significantly by simple experimental manipulation or by the injection of crude test materials containing probably only low concentrations of the regulatory molecules being sought. Until these regulators could be identified and massproduced it seemed unlikely that the question of their possible involvement in leukemogenesis could be approached. It would be satisfying to claim that this impasse directed my efforts to the use of tissue culture to detect the sought-for regulators. However,

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this was not the case. In the early 1960s, no technology existed for the satisfactory culture of hemopoietic cells. Indeed, the only monograph to come to my attention on the subject made depressing reading (Woodliff, 1964). Some blood cell formation seemed to occur in the cultures being used, but it seemed impossible to quantify or manipulate reproducibility, and tissue culture did not seem to offer much prospect as a method for identifying regulators. However, what the preceding decade of mounting frustration had produced was my firm conviction that specific regulatory factors did exist, that they had to be of importance in understanding hemopoiesis, and were likely to be involved in leukemogenesis, despite the growing evidence that leukemias were genuine neoplasms and not simply dysplasias. I was therefore in a thoroughly primed state, waiting to exploit whatever opportunity presented itself. That opportunity came in a quite unforeseen manner and led me virtually overnight to abandon the thymus and lymphoid cells in favor of two cells, the granulocyte and the macrophage, which had become the neglected stepchildren of hematology. Despite the abundant evidence from the turn of the century that these were crucial cells for resistance to infections, their importance had become downgraded largely due to the dominance of cellular immunologists and their proper fascination by the specific antigenic responsiveness of T and B lymphocytes. It also happens that the occurrence of myeloid leukemia is unusual in mice and the major groups then working on leukemogenesis rarely attempted studies on myeloid leukemia. The experiments leading to the accidental discovery of clonal hemopoietic cultures were carried out in Melbourne for a quite different purpose. Attempts were being made by my colleague in the University of Melbourne, Ray Bradley, to culture murine lymphoid leukemic cells. He used semisolid agar cultures because current folklore, based on experiments with fibroblasts, held that the ability of cells to grow in agar was a specific property of transformed cells (Macpherson, 1970). My role in these experiments was initially a very minor one-merely to supply AKR thymic lymphomas for culture. T h e lymphoma cells did not grow in the agar medium and in an attempt to assist their growth, Ray Bradley followed the lead of Puck and Marcus (1955), who had demonstrated a feeder or conditioning effect of cocultured irradiated cells that permitted single fibroblasts to proliferate effectively in culture. He therefore included in a second agar layer a variety of tissues or cells in the hope that the lymphoma cells might be induced to proliferate. To his astonishment, in cultures in which marrow cells had been used as the “feeder cells,” large colonies developed, but, disconcertingly, these were in the marrow layer, not the layer containing the lymphoma cells. What had

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appeared to have happened was that the lymphoma cells had acted as a feeder layer, allowing some of the marrow cells to proliferate and form colonies. I t was soon found that inclusion of other tissues in the feeder layer could induce bone marrow cells to form similar colonies (Bradley and Metcalf, 1966). Investigation of the cells in these colonies revealed them to be granulocytes and/or mononuclear macrophages. T h e folklore stating that only transformed cells could grow in agar caused us much concern, but the colony cells appeared morphologically normal and did not form further colonies on reculture. Nevertheless, for 1 or 2 years, we had very much in mind the possibility that these marrowderived colonies might really be composed of cells transformed by some possible viral agent in the cocultures. Unknown to us, the same phenomenon of granulocyte-macrophage colony formation was being encountered independently in the laboratory of Dov Pluznik and Leo Sachs in the Weizmann Institute, in experiments initially involving the attempted culture of Rauscher virusinduced leukemic cells (Pluznik and Sachs, 1965, 1966).The phenomenon of simultaneous independent discovery is more familiar today than it was a few decades ago, and our colleagues were moved to declare in print on more than one occasion that we had copied their techniques. However, this was not the case. Indeed, it would have been almost impossible not to discover granulocyte and macrophage colony formation if agar cultures had been prepared containing mouse hemopoietic cells, so vigorous is the growth of these colonies and so widespread the sources of cells able to produce at least a little of the required specific growth factors. In retrospect, it is odd that tissue culture should have made such an unpromising start in exploring hemopoiesis. I suspect that a combination of circumstances permitted its sudden emergence as a feasible and useful technique. These included the commercial introduction of adequate culture mediutn, the recognition that care needs to be taken in selecting batches of fetal calf serum, improvements in incubator design, and eventually the general availability of suitable plastic culture dishes and bottles. T h e single most important event, however, was the use of solid-state cultures in which individual precursor cells divided to produce colonies of maturing progeny. This made the events occurring in culture suddenly comprehensible and so dramatic as to be believable. IV. Colony-Stimulating Factors The morphological appearance of granulocyte-macro phage colonies was visually so striking that the system demanded extensive exploration.

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However, the fact that no such colonies developed unless feeder layers or tissues were added was to me the most important aspect of the phenomenon. T h e implication was that hemopoietic cells, at least in this type of culture, could not proliferate spontaneously. They required stimulation for cell division and it followed that the stimulus being added might be the long sought-for hemopoietic regulator, albeit not for lymphoid cells but for granulocytes and macrophages. My initial concern was to build a reasonable case from indirect evidence that this particular factor-soon termed colony-stimulating factor (CSF)-might be a genuine specific regulator of granulocytes and macrophages. Questions addressed were whether the factor was detectable in the serum or urine, was it present in tissues, and, if so, which tissues. Did the levels of CSF fluctuate in situations, such as infections o r following the injection of endotoxin, where perturbations occur in the production of granulocytes and macrophages? The answers to these questions were affirmative. CSF was detectable in some mouse sera and, later, in human urine and in some human sera. Intriguingly, levels of CSF appeared to be elevated in the serum of mice with lymphoid leukemia (Robinson et al., 1967)-a not particularly puzzling phenomenon because most mice with lymphoid leukemia also have elevated levels of neutrophils in the blood, a still not well-recognized fact. CSF was also detectable in extracts of all tissues (Sheridan and Stanley, 197 1) and CSF levels were elevated in the serum in at least some animals and humans with infections (Metcalf and Foster, 1967; Foster et al., 1968a,b). Based on these initial observations, we considered it to be a reasonable hypothesis that CSF was in fact a genuine regulator worth characterizing, and this led us to a 15-year program in which CSF was purified and characterized. T h e project proved formidable because of the low concentrations of CSF produced by tissues or cell lines and because the initial assumption of the existence of a single CSF was incorrect. There are in fact four CSFs, now designated on the basis of their differing bias of granulocyte (G) and macrophage/monocyte (M) stimulation as GMCSF, G-CSF, M-CSF, and multi-CSF (or IL-3) (Fig. 3). Of these, M-CSF was the first to be purified from human urine and mouse fibroblast conditioned medium (Stanley et al., 1975; Stanley and Heard, 1977), then GM-CSF from mouse lung conditioned medium (Burgess et al., 1977), multi-CSF (IL-3) from medium conditioned by the murine WEHI-3B myelomonocytic leukemic cell line (Ihle et al., 1982), and finally G-CSF, again from mouse lung conditioned medium (Nicola et al., 1983). T h e existence of other human CSFs, analogous to the murine

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51

Macrophages

Cranulocyte-

Granulocytes

FIG. 3. The formation of granulocytes and macrophages from committed progenitor cells is controlled by the action and interaction of four specific glycoprotein colonystimulating factors. Stimulation by G-CSF results mainly in granulocyte formation and stimulation by M-CSF results mainly in monocyte-macrophage formation.

molecules, was recognized, but work on their purification lagged behind that on the murine CSFs. T h e introduction of clonal cultures for human cells (Pike and Robinson, 1970) allowed exploration of the possibility implied in Fig. 1 that CSF levels might be elevated in the serum of patients with myeloid leukemia or the potentially preleukemic myelodysplasias. Assays using cultures of human or murine marrow cells showed that CSF levels in the serum o r urine were sometimes elevated but certainly not uniformly so in patients with leukemia (Chan et al., 1971; Metcalf, 1974, 1977, 1984; Lind et al., 1974). Furthermore, the elevations seemed more related to the occurrence of infections in these patients (Metcalf etal., 1971). These data placed in considerable doubt the original simple hypothesis implied from Fig. 1 that myeloid leukemia might be the consequence of readily demonstrable sustained excess stimulation by CSFs-or at least circulating CSFs. The CSFs proved to be glycoproteins in the MW range 18,00090,000 that were highly active biologically in subnanngram per milliliter concentrations. For the few laboratories possessing purified native CSFs,

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the late 1970s and early 1980s were an exciting time spent in determining the biological actions of these new regulators. However, the minute amounts available also made this period one of near despair for those of us wishing to establish effects in vivo o r hoping to establish possible roles in leukemogenesis. Noone had been able to produce enough CSF to test in vivo and the low amounts of CSF in even the richest tissue source offered no real prospect for mass-producing CSF. The situation was altered dramatically by the entry of molecular biologists into the field. In the period 1983-1987, cDNAs encoding all four murine and human CSFs were cloned and biologically active recombinant CSFs were mass-produced in bacterial, yeast, or mammalian expression systems. The history of how these developments led to the widespread clinical use of the CSFs in stimulating granulocyte and macrophage formation has been detailed elsewhere (Metcalf, 199 lb). From the viewpoint of the present discussion, there were a number o f important consequences of the success of molecular biologists in cloning the CSF genes. Recombinant CSFs became available for use in a much wider range of laboratories. Above all, it was now possible to look for CSF and, later, CSF-receptor gene rearrangements in leukemic cells, to monitor at least the transcription of individual CSF genes in leukemic cells, to develop immunoassays for the CSFs, and, most important, to induce the autocrine production of CSF by myeloid cells by CSF gene insertion into appropriate hemopoietic cells. However, before discussing the role played by these approaches in our present ideas on the development of myeloid leukemia, it is necessary to summarize briefly what is now known about the biological actions of the CSFs. V. Biological Actions of Colony-Stimulating Factors

The CSFs proved to be not simply growth factors but to have multiple actions on responding grdnulocyte-macrophage populations (Fig. 4). T h e action leading to their discovery was the mandatory role they play in controlling cell division in these populations. Acting alone, or in combination, they control entry of Go cells into the cell cycle, the transit of the cells through G , , and the length of the G , period. T h e CSF concentration thus effectively controls how many cells are dividing, how rapidly they complete the cell cycle, and the total number of progeny produced by individual progenitor cells in a fixed time interval (Metcalf, 1991b). Production of progeny by progenitor cells is accompanied by matura-

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53

@ PROLIFERATIVE STIMULATION

0

,

@ DIFFERENTIATION COMMITMENT @@Neutrophils

6

Macrophages

@

MATURATION INDUCTION I

@

SUPPRESSION OF APOPTOSIS I

@ FUNCTIONAL

STIMULATION Phagocytosis Superoxide

Monocyte

@-'&

Phagocytosis Lysozyme 11-1, IFN-7, TNF, P.A., etc.

FIG. 4. The colony-stimulating factors not only control cell proliferation but also differentiation commitment in progenitor cells, the initiation of maturation changes, membrane transport integrity with suppression of apoptotic death, and the stimulation of various functions of mature granulocytes and macrophages.

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tion, a process that eventually terminates further cell division when the progeny reach the postmitotic stage. It is common to describe proliferation and maturation as being “tightly linked” in normal cells but uncoupled in leukemic populations where little or no maturation may occur (Sachs, 1980, 1987). This is a broadly valid distinction, but the “tight linkage” in normal cells should not be misinterpreted as indicating that a fixed number of divisions necessarily occurs between each maturation step. Studies using different CSF concentrations on paired progeny of progenitor cells showed that the number of progeny able to be generated by CSF stimulation before maturation terminates the process is not fixed: the higher the CSF concentration used, the larger the number of progeny produced (Metcalf, 1980). Because maturation terminates further cell division, this relationship between CSF concentration and progeny number could be interpreted as indicating that CSF action may delay o r prevent maturation. It has proved difficult to design experiments to determine whether the CSFs can directly influence maturation, because cell viability in uitro depends on CSF action and control cells studied in the absence of CSF are in fact dying cells. Despite this difficulty, studies with appropriate cell lines have produced some evidence indicating that the CSFs can initiate maturation, the converse of what might have been deduced from the above data (Valtieri et al., 1987; Heyworth et al., 1990). This apparent conflict in the evidence concerning the effects of CSFs on maturation induction has yet to be resolved fully, but the question potentially is a matter of some importance for a proper understanding of the leukemic state because a defect in maturation is evident in most acute myeloid leukemic populations. An important action of the CSFs is their ability to influence the process of differentiation commitment in hemopoietic cells. Differentiation commitment and maturation induction are almost certainly different cellular processes. Differentiation commitment is an event that precedes maturation induction and is not associated with morphological change in the affected cells. It appears to be a process linked with the cell cycle and, where analyzed, to occur late in G I or early S (Boyd and Metcalf, 1984; von Melchner and Hoffken, 1985). Commitment appears to be irreversible and commonly is asymmetrical in nature with only one of the daughter cells being affected (Metcalf, 1982b). Commitment probably requires a series of linked transcriptional activations or suppressions of relevant genes that limit previous options available to a cell o r make possible certain future events. Commitment may well involve the expression of new receptors on the cell membrane, cessation of expression of

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other receptors, and major alterations in available signaling pathways within the cell. In its simplest form, differentiation commitment in a bipotential granulocyte-macrophage progenitor cell results in one or more of the progeny becoming restricted to the future formation only of granulocytes or macrophages. Thus M-CSF can induce differentiation commitment in responding cells such that they will thereafter form only macrophage progeny, regardless of the type of CSF then used to stimulate subsequent cell divisions (Metcalf and Burgess, 1982). G-CSF has the opposite effect of inducing commitment to granulocyte formation and thus M-CSF can compete with G-CSF in inducing commitment into opposing lineages. Although this type of differentiation commitment action addresses a question of fundamental importance in understanding the biological processes of hemopoiesis, it might be regarded as a trivial side issue for an understanding of leukemogenesis. However, the exact opposite is the situation, because differentiation commitment is probably the key process becoming perturbed during leukemogenesis. Here the reader needs some assistance in interpreting the terminology used in some publications. T h e expression “uncoupling of proliferation from maturation” is potentially a little misleading. Maturation is of course an important process without which no functional mature cells will be produced-a failure very evident in untreated acute myeloid leukemia, leading to death from either infections o r uncontrolled bleeding. However, a more fundamental abnormality exists in leukemia, which leads to progressive expansion of the leukemic clone. In normal hemopoiesis, immature stem cells self-generate but also produce equal numbers of cells that undergo differentiation commitment, leading eventually to the formation by such cells of maturing progeny. This arrangement ensures maintenance of stable numbers of stem cells but at the same time the continuous formation of mature cells. Perturbation of this differentiation commitment process, such that the level of self-generation of stem cells rises above the 50% level, results in a progressive expansion of the stem cell compartment in a Gompertzian manner. This is the behavior of an emerging leukemic clone. In the process of abnormal self-renewal, it matters little what the actual maturation stage is of the affected cells. Thus a leukemic “stem cell” may indeed be at the maturation stage of a normal hemopoietic stem cell o r may, by marker analysis, be identifiable as an aberrant committed progenitor cell in a particular lineage. T h e principles remain the same, and if such a cell displays an aberrant capacity for self-renewal, a progressively expanding population will result-the hallmark of a leu-

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kemic population. For those leukemic cells that do undergo differentiation commitment, what capacity they retain for producing maturing cells is irrelevant for the dynamics of the leukemic population. This may be an initially confusing concept if, as in chronic myeloid leukemia, the maturing cells grossly outnumber the stem cells. However, the key issue in such a population remains the self-renewal behavior of the minority stem cell population. Thus the term “uncoupling of proliferation from maturation” really refers to the uncoupling of proliferation from differentiation commitment, an event resulting in an abnormally high level of self-renewal. T h e ability of the CSFs to induce differentiation commitment (suppression of self-renewal) in clonogenic leukemic stem cells has been well documented. T h e consequent ability of the CSFs to suppress appropriate leukemic cell lines is a highly important process but one more logically reviewed in a later section. At this stage in the discussion, it is more useful to continue the general description of the biological actions of the CSFs by considering how their proliferative and differentiation commitment actions can in fact influence all members of a leukemic clone even if some members of the clone are in differentiation stages or lineages outside the committed granulocyte-macrophage lineage. The CSFs d o not act solely on committed granulocyte-macrophage populations. Each, particularly when acting in association with one or more other hemopoietic growth factors, can stimulate cell division in at least some of the more ancestral hemopoietic stem cells with the formation by them of committed progenitor cells. Thus, whereas stem cell factor (SCF, kitligand) can stimulate stem cells to form small blast colonies composed of progeny progenitor cells (Metcalf and Nicola, 1991), costimulation by a CSF expands the formation of progenitor cells and can also influence the relative frequency of the particular types of committed progenitor cells being formed. Thus SCF plus G-CSF enhances the formation of macrophage progenitor cells whereas SCF plus multi-CSF enhances the formation of granulocytic and eosinophil progenitor cells (Metcalf, 1991a). This process of progenitor cell formation demonstrates nicely the dual actions of CSFs on cell division and differentiation commitment, although there are some intriguing paradoxes evident. For example G-CSF, when acting on progenitor cells, is a selective stimulus for granulocyte formation, whereas, when acting with SCF on stem cells, G-CSF action is broader and in fact results in the biased formation of committed macrophage progenitor cells. T h e CSFs can also influence cells outside the granulocyte-

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macrophage lineage. This is particularly evident for GM-CSF, which is also a proliferative stimulus for eosinophil progenitors and at higher concentrations for megakaryocyte and some erythroid progenitors (Metcalf et al., 1986). multi-CSF has the broadest action ofthe four CSFs, being a proliferative stimulus for granulocytic, macrophage, eosinophil, megakaryocyte, erythroid, and mast cell precursors (Metcalf et al., 1987). It can be seen from these considerations that the CSFs can potentially regulate the biology of the entire myeloid leukemic population, regardless of whether the leukemic clone only involves granulocytic and macrophage populations, as may be the case in some acute myeloid leukemias (AMLs), o r whether the clone includes eosinophil, erythroid, and megakaryocytic cells, as is clearly the case in chronic myeloid leukemia (CML) and probably the case in most AMLs. To complete the discussion of the multiple actions of the CSFs on responding cells, experiments have shown that the CSFs also control a variety of functional activities in these cells. These include maintenance of membrane transport integrity, which is essential for preserving the viability of the cells and for preventing death from apoptosis. T h e CSFs can also influence the level of functional activity of mature granulocytes and macrophages, an action involving functions such as phagocytosis, superoxide production, killing of microorganisms or tumor cells, and the production of other agents, such as interferon-y, tumor necrosis factor, interleukin-1, and other CSFs (Metcalf, 1991b; Demetri and Griffin, 1991; Gasson, 1991). These latter actions of the CSFs on the functions of mature cells at present seem unlikely to have much relevance for leukemogenesis. However, the action of CSFs in preventing death from apoptosis requires further comment because it may well have some relevance, if only in an indirect manner, in the leukomogenesis process. The protooncogene bcl-2 has been linked to the development of follicular lymphoma in man, becoming activated by the 14: 18 translocation (Tsujimoto et al., 1984). T h e most obvious function of the bcl-2 product so far characterized is its ability to prolong the life span of cells by preventing death from apoptosis (Vaux et al., 1988; Nunez et al., 1990). This naturally occurring death process is an important facet of the biology of short-lived hemopoietic cells and most often involves perfectly normal aging cells. T h e mechanism might well have an additional important role in eliminating cells that have undergone mutations of one type or another that are potentially involved in leukemogenesis. It is on this basis that bcl-2 is postulated to play a significant role in the emergence of follicular lymphomas. T h e apoptotic process involves the action

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of the nuclear transcription factor p53, and overexpression of p53 accelerates apoptotic death (Yonish-Rouach et al., 1991). In this function, p53 can be viewed as a censor system continually eliminating preneoplastic cells from the body. Loss of p53 is a common event in the complex sequences of events in tumor evolution in both solid tumors and leukemias-possibly because of the consequent loss of this censoring function (Lane and Benchimol, 1990). The experiments documenting that the CSFs prevent death from apoptosis (Williams et al., 1990) have been carried out exclusively in vitro and it remains to be shown that they exhibit a similar function in vivo. If they do, it could be postulated that CSF action is a potential antagonist of the censory action of p53 and, if so, the action of CSFs on a myeloid population carrying mutations of relevance for leukemogenesis would tend to protect such cells from elimination. Thus, in essence, CSF action might be seen to favor the emergence of mutant preleukemic cells, not by direct mutagenic action but by protecting these cells from elimination by apoptosis. As a general aside, recognition of the obvious polyfunctionality of the CSFs highlighted the principle that growth factors are usually not simply proliferative factors. Subsequent to the work on the CSFs, it has become common to read accounts describing similar polyfunctionality for growth factors acting on other cell types. I have encountered one referee who vehemently disputed the claim that this concept originated with the hemopoietic growth factors. Not being an assiduous reader of the scientific literature, I am not well placed to argue the case. It may be, as claimed, that this principle was well known to workers studying other cell types prior to the early 1980s. All I can say is that this was not the impression I gained at the time when giving general lectures on the subject, for the response of the audience was usually somewhat skeptical. In defense of my hematological colleagues, I would say, however, that recognition of the key role played by specific regulators in the biology of myeloid leukemic cells, because of their twin ability to control cell division and differentiation commitment, was firmly established in the 1970s, before such issues were actively explored for other cancers. This discussion of the action of the CSFs on normal granulocytic and macrophage cells raises the expectation that their actions on myeloid leukemic cells might be quite complex and at times be opposing, because the CSFs might simultaneously be able to stimulate leukemic cell proliferation yet abort proliferation by actions on differentiation commitment o r maturation induction. As shall be discussed below, both types of action have indeed been documented.

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VI. Myeloid Leukemic Cells in Culture

Adaptation in 1970 of the agar culture technique to permit the growth of human granulocytic and macrophage progenitor cells (Pike and Robinson, 1970) revealed characteristics of cultured human marrow cells that had not been obvious with murine cells. In particular, it became evident that, in crowded cultures, some “spontaneous” colony formation could occur in cultures to which no source of CSF had been added. T h e phenomenon was clearly dependent on the concentration of cells cultured, and cell separation studies showed that adherent cells in the cultured population were providing an endogenous source of CSF (Moore and Williams, 1972). It was established that in order to assess genuine autonomy in such cultures, the cultures needed to be prepared using fewer than 50,000 cells/ml and ideally required a preliminary fractionation step, such as removal of adherent cells, to reduce the number of CSF-producing cells (Moore et al., 1973a). Failure to recognize these technical points led to some initial confusion in reports on the possible autonomy of human myeloid leukemic cells in culture. The leukemic cells from most myeloid leukemias in man can proliferate in clonal cultures, and the clones generated have been verified by karyotypic analysis as belonging to the leukemic clone (Metcalf, 1984). There are some distinctive features of these leukemic clones that have raised some unresolved problems. In cultures of marrow or blood cells from patients with chronic myeloid leukemia, the leukemic colonies that develop are essentially identical to those grown from normal marrow cells in terms of colony shape, size, and content of maturing cells. There are, however, certain abnormalities: ( 1) an unusually high frequency of clonogenic cells, (2)an abnormally light density and low cycling status of the colony-forming cells, and (3)certain anomalies in the responsiveness of colony formation to inhibitory agents such as prostaglandin E (Moore et al., 1973b; Metcalf, 1984). Analysis has shown that normal progenitor cells persist in these patients in near-normal absolute numbers but are greatly outnumbered by additional Ph-positive leukemic progenitor cells (Eaves and Eaves, 1987). In sharp contrast, although leukemic clones can be grown from the marrow and blood from most patients with acute myeloid leukemia, the frequency of such clones is highly variable from one patient to another and in most cases the clones are of abnormally small size and contain cells exhibiting little o r no maturation (Moore et al., 1974; Lowenberg and Touw, 1993). Typically, no surviving normal progenitor cells are detectable, although such cells reappear following induction of a remission.

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There is a persisting unease that agar cultures of this type do not detect the true clonogenic cells in either chronic or acute myeloid leukemia, but rather are detecting more mature members of the leukemic clone. This disquiet is based on an inability to detect clonogenic cells in clones grown from either type of leukemia, because an ability to selfgenerate is a reasonable property to expect of a genuine stem cell in a leukemic clone. There remain two general possibilities: ( 1) different culture systems may be required to detect the true stem cells o r (2) the cells in leukemic clones may have had their ability to self-renew suppressed by the factors used to stimulate their proliferation. T h e growth of less numerous and less mature blast cell colonies was subsequently described in cultures of both chronic and acute myeloid leukemic cells (Buick et al., 1976; Griffin and Liiwenberg, 1986). T h e cells in such colonies do have some clonogenic potential but the degree of selfrenewal demonstrable in such cells is strictly limited and somewhat unconvincing. These reservations unfortunately do raise doubts concerning the information obtained from clonal cultures of primary human myeloid leukemic cells, and it seems wise to accept the possibility that we may not yet possess adequate information on the nature of the most ancestral members of the leukemic clone in either disease. Placing these reservations to one side, the culture studies in the early 1970s revealed somewhat surprising information on the responsiveness and dependency of the clonogenic cells with respect to the CSFs and other possible growth factors. T h e clonogenic cells from all patients with chronic myeloid leukemia and most with acute myeloid leukemia exhibited a complete inability to proliferate autonomously in semisolid cultures. Proliferation of these cells required stimulation by CSFcontaining material and the quantitative responsiveness of the leukemic cells was similar to that of normal cells (Metcalf, 1984; Begley et al., 1987, 1988). These basic conclusions remained unchanged when purified human CSFs subsequently became available in recombinant form and when purified populations of clonogenic myeloid leukemic cells were obtained by cell sorting. All four CSFs are active in stimulating leukemic cell proliferation. With individual cases, one CSF may have more activity than another, but there is no fixed pattern of reactivity correlating with the subtype of leukemia (Liiwenberg and Touw, 1993). There can be wide differences in the quantitative responsiveness of cells from different patients with acute myeloid leukemia. However, it needs to be recalled that these populations are clonal and that individual normal progenitor cells also differ widely in their quantitative responsiveness to

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stimulation by CSFs. Because of this, the variations observed between different AML populations remain within normal limits of variation. T h e continuing dependency of human myeloid leukemic cells on stimulation by normal regulators was initially interpreted as indicating that these cells could not be producing their own growth factors, otherwise this should have resulted in autonomous growth in vitro. This conclusion has needed to be reviewed because subsequent studies have shown that some AML cells appear to have a capacity to transcribe and produce one or more of the CSFs (Young and Griffin, 1986; Oster et al., 1988; Murohashi et al., 1989), although this property does not necessarily correlate with an ability to proliferate autonomously in vitro. However, it has been recognized that CSF transcription can be induced by the handling of leukemic cells prior to testing and can be strongly induced by interleukin- 1 (IL- I ) (Delwel et al., 1989). These technical problems have made it a little uncertain how often human acute myeloid leukemic cells actually produce CSF in vivo. The likely situation is that some probably do while the majority probably may not unless significant amounts of IL-1 are available for priming. With populations of chronic myeloid leukemic cells it is much easier to document CSF production by members of the leukemic clone. T h e purified progenitor cells do not appear to be CSF producing, but the mature monocytes are consistent CSF producers (Metcalf, 1984). These leukemic monocytes resemble normal monocytes in this regard. For both cell types, this property raises a puzzle. If these cells can produce CSF for which the cells express receptors, why do they not exhibit autonomous proliferation in vitro? T h e most likely explanation is that the cells have matured to an essentially postmitotic state similar to that of polymorphs. Even if mitotic signaling is initiated from an occupied receptor, reprogramming of the genome of the cells during maturation has probably made it impossible for them to undergo cell division, although other types of CSF-initiated responses may remain possible. The phenomenon of mature cells in a CML leukemic clone producing CSF but possibly not the clonogenic cells does require careful assessment of the data obtained on CSF production by AML blast cells. It may be that only a subset of these cells is producing CSF and that autocrine CSF production may not be of genuine relevance for the behavior of the clonogenic cells in the population. There has not been extensive experience with the behavior in clonal culture of primary myeloid leukemias induced by agents such as viruses o r irradiation. In general, however, the clonogenic cells are usually CSF dependent or responsive and perform relatively poorly in primary clo-

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nal cultures (Radke et al., 1982; Klein et al., 1982; Metcalf, 1984). This in sharp contrast to the behavior of long-established murine leukemic cell lines, such as M 1 or WEHI-SB, wherein the leukemic cells are autonomous and form very large colonies. T h e selective and other changes undergone during establishment of these leukemic cell lines are likely to have made the cell lines unrepresentative and potentially misleading for studies on the regulatory control of emerging primary leukemic populations. To bring the situation up to date, there are still no firm data for the existence of leukemia-specific growth factors with no action on normal hemopoietic cells. One o r two other hemopoietic factors have some ability to stimulate the proliferation of both normal and leukemic cells. These include stem cell factor (Ikeda et al., 1991; Kuriu et al., 1991) and IL-6 (Hoang et al., 1988), but the concentrations required are higher and the resulting proliferation less than with the CSFs. At present, these other factors seem less likely to be of importance as proliferative stimuli for myeloid leukemic cells in vivo than the CSFs, but their possible role does require further investigation. By 1980, therefore, analysis of clonal cultures of human myeloid leukemic cells had shown them to have an unexpected dependency on extrinsic CSFs for proliferation. By this time, the documented clonality of myeloid leukemic populations and the increasing examples of specific translocations in various leukemic cells seemed to have eliminated the original simple hypothesis that myeloid leukemia might merely be an aberrant regulator-driven unrestrained proliferation of otherwise normal-enough granulocytic and macrophage cells. However, a modified proposal could be advanced regarding the role of regulators in myeloid leukemia development. Regardless of the mechanism leading to the emergence of the first clonogenic myeloid leukemic cells, stimulation by the CSFs, whether in normal or elevated concentrations, could be proposed as being mandatory for the expansion of the clone and the development of clinically overt leukemia (Fig. 5 ) .

VII. Role of Colony-Stimulating Factors in Initiation of Myeloid Leukemia Granted that a case had been established for an important role of the CSFs in the clonal expansion of transformed myeloid leukemic cells, was it also possible that the CSFs might actually be involved in the transformation events leading to the development of the first fully leukemic cell? Because none of the common chromosomal translocations in human myeloid leukemia appeared to involve CSF genes, it seemed, at least for

HEMOPOIETIC REGULATORS AND LEUKEMIA DEVELOPMENT

11980 HYPOTHESIS

CSF

1

Normal

63

1 CSF

1

First

Leukemic Cell

CSF

1

CSF

1

Expansion of Leukemic Clone

FIG. 5. T h e 1980 minimal hypothesis for the role o f the CSFs in myeloid leukemogenesis. Regardless of what processes lead to the appearance of the first leukemic cell, such cells remain dependent for proliferation on stimulation by CSF, and expansion of the leukemic clone is therefore CSF dependent.

these leukemias, that autocrine production of CSF would be unlikely to be a relevant event for initiating leukemic transformation. Moreover, as noted above, excessive levels of circulating CSFs had not been observed consistently in preleukemic or leukemic patients, so this possible inducing event seemed also to have been eliminated. This conclusion has been in general reinforced by subsequent assays on GM-CSF and G-CSF levels in the circulation using immunoassays (Watari et al., 1989; Sallerfors and Olofsson, 1991; Omori et al., 1992; Verhoef etal., 1992), although, in one study, a high frequency of elevated M-CSF levels was noted (JanowskaWieczorek et al., 1991). It could be argued, however, that the transformation process in all types of myeloid leukemias might require the existence of multiple abnormalities. The initiation and progressive growth of leukemic cells might require, as one component, excessive o r aberrant proliferative stimulation, but excessive exposure to excess circulating levels of CSF is only one possible method. The same outcome might be achieved by autocrine production of cell-associated CSF or the development of a surrogate system mimicking CSF signaling in the cell. Despite the fact that survey data had failed to document consistently high circulating CSF levels in preleukemic or leukemic patients, it was felt to be worthwhile to attempt experiments using model systems to establish more directly the possible role of growth factor stimulation in the induction of leukemia. Accordingly, in our laboratories, an experimental program was initiated in mice to establish whether acquisition of an autocrine capacity to produce CSF or exposure to excess levels of CSF could initiate the development of myeloid leukemia. A three-pronged

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approach was eventually developed: ( 1) the generation of transgenic mice with constitutively elevated CSF levels, (2) insertion of CSF cDNAs into hemopoietic cells, which were then used to repopulate irradiated recipients, and (3) insertion of CSF cDNAs into immortalized nonleukemic hemopoietic cell lines. For convenience, the results will be reviewed slightly out of chronological sequence. Two lines of GM-CSF transgenic mice were developed by injecting genomic DNA under the control of the Moloney virus long terminal repeat (LTR) into fertilized oocytes (Lang et al., 1987). In both lines, the only cells so far demonstrated to express the transgene are macrophages-representing a potentially autostimulatory system for these cells. Serum concentrations of GM-CSF are constitutively elevated 80- to 100-fold compared with littermate control mice and comparable elevations are present in the resident peritoneal and pleural fluid. These mice develop a massive increase in the numbers of peritoneal and pleural macrophages in a proliferative response that declines when the animals reach adult age (Metcalf et al., 1992). Despite the elevated GMCSF levels, the hemopoietic populations in the marrow and spleen are essentially normal and so in this model the proliferative stimulation achieved is restricted to macrophage populations. Several thousand transgenic mice have been closely monitored but no cases of myeloid leukemia have developed, the mice dying in middle age from tissuedamaging effects of overstimulation of macrophage production of toxic products (Metcalf and Moore, 1988; Lang et al., 1992). Even administration of 3.5 Gy whole-body irradiation, the optimal dose for inducing myeloid leukemia in suitable mouse strains, failed to result in leukemias of granulocytic o r macrophage populations. A similar negative outcome was observed in transgenic mice expressing high levels of IL-6. In the original model, massive plasma cell hyperplasia was observed but not development of plasma cell tumors (Suematsu et al., 1989). A much more powerful model system was then developed using highefficiency retroviruses to insert into marrow cells CSF cDNA linked to the stronger myeloproliferative sarcoma virus LTR promoter. These cells were then used to repopulate the hemopoietic tissues of lethally irradiated recipients. In experiments using cDNA for GM-CSF (Johnson et al., 1989), multi-CSF (Chang et al., 1989b), or G-CSF (Chang et al., 19894, the various recipient animals developed 1000-fold elevations of the respective CSFs and extreme hyperplasia of granulocytic and macrophage populations. Like the transgenic model, the repopulation models again contained an element of autocrine stimulation because the hemopoietic cells were both the source and potential targets of the CSF. T h e animals developed spleen enlargement and, particularly in

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the GM-CSF model, massive invasion of the liver and lungs by proliferating populations of granulocytes and macrophages. Although a lethal disease resulted in mice with excess levels of GM-CSF and multi-CSF and the affected tissues exhibited the histological appearance of leukemia, the cells from such tissues in fact failed to produce transplanted tumors in normal syngeneic recipients, showing that the tissues were not leukemic but merely hyperplastic. Similar negative results were obtained with engraftment models involving excess levels of erythropoietin (Villeva1 et al., 1992) or IL-5 (Vaux et al., 1990) and the negative results with multi-CSF have also been observed by others (Wong et al., 1989). T h e apparent conclusion from these studies is that overstimulation of otherwise normal hemopoietic cells leads to hyperplasia but not to leukemia development, even if, for many of the cells, the excessive amounts of growth factor are being produced by cells that are able to respond to the same factor. These results seemed to have unequivocally eliminated the 1954 hypothesis (Fig. 1). However, a potentially important caveat to this conclusion is that the life span of the animals in the various models was severely restricted and possibly if they had lived beyond middle age, leukemia might have developed. Given the very large numbers of transgenic mice monitored, this argument loses much of its force for the transgenic model, because leukemia development tends to show a skewed age of onset and at least a few cases should have developed by early middle age. However, the limited life span of the repopulated animals does provide grounds for questioning the validity of the conclusions. Moreover, the system in which sustained hyperplasia was induced in erythroid cells by insertion of a mutant, activated, erythropoietin receptor did result eventually in the development of some erythroleukemias (Longmore and Lodish, 1991), and transgenic IL-7 mice with excess levels of IL-7 transcription develop hyperplastic lymphoid tissues with the later emergence of clonal T and B cell tumors (Rich et al., 1993). Therefore, sustained overstimulation of proliferation on occasion may have some relevance for leukemogenesis, a matter that will be returned to later. A quite different outcome was observed in studies using immortalized, but nonleukemic, hemopoietic cell lines. These cell lines remain wholly dependent on CSF stimulation for survival and cell division, but the clonogenic cells exhibit an abnormally high ability to self-renew and little capacity for maturation. FDC-P1 cells are useful in such studies because they respond to stimulation either by GM-CSF or multi-CSF (Dexter et al., 1980; Hapel et al., 1984). When GM-CSF cDNA was inserted into FDC-P1 cells using a retrovirus, some cells exhibited immediate transformation to autonomous cells and these were able to generate

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NORMAL CELL

-@ -

Q

CSF Gene

CSF

Hyperplasia

CSF

Leukemic Transformation

CSF

Leukemic Transformation

Self-Renewal

IMMORTALIZED CELL

NORMAL CELL

Self-Renewal

CSF Gene Hox 2.4 Gene

FIG. 6. T h e consequences of insertion of independently regulated CSF cDNA into hemopoietic cells. If normal cells acquire an autocrine capacity to produce CSF, hyperplasia but not leukemia results. If CSF cDNA is inserted into an immortalized hemopoietic cell with an abnormal capacity for self-generation, leukemia results. T h e active CSF operates in a cell-associated (intracellular?) manner. Normal hemopoietic cells can be transformed to leukemic cells by the insertion of the Hox 2.4cDNA plus a CSF cDNA, the Hox 2.4 gene conferring on the cells the required anomalous self-generation.

rapidly growing transplanted tumors on injection into normal syngeneic recipients (Lang et al., 1985). Although the transformed cells secreted GM-CSF, antibodies to GM-CSF did not block the autonomous growth of our transformed cells, leading us to conclude that the autocrine GMCSF was cell associated-either activating receptors within the cytoplasm or on the cell membrane in some manner that was not blockable by extrinsic antibody (Fig. 6). Transformation of FDC-P1 cells by insertion of GM-CSF cDNA was reproduced in another laboratory (Laker et al., 1987), but in this study the cells did not always, o r did not immediately, behave as autonomous cells in clonal culture. This was somewhat surprising because acquisition of the autocrine capacity to produce GM-CSF was the only transformation event likely to have occurred and such cells should not have continued to be dependent in clonal culture on extrinsic stimulation by GMCSF. This curious combination has now been observed in two other

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variants of this model. When multi-CSF cDNA was inserted into FDC-P1 or other cloned lines, some cells also promptly transformed to transplantable leukemic cells (Hapel et al., 1986; Metcalf, 1988; Suda et al., 1988; Browder et al., 1989). Although the cells secreted multi-CSF, the evidence from various laboratories was conflicting as to whether cell proliferation was being stimulated by secreted, as distinct from cellassociated, multi-CSF. In our studies, some transformed FDC-P1 cell lines behaved as completely autonomous cells but others exhibited a partial dependency on added extrinsic multi-CSF, while some exhibited continuing complete dependency on added CSF (D. Metcalf, unpublished data). We have also observed that leukemic transformation of FDC-PI cells can occur spontaneously if the cells are injected intravenously into irradiated syngeneic DBA mice. After a latent interval of several months, these animals develop leukemia, and karyotypic analysis of the leukemic cells confirmed that they are derived from the injected FDC-P1 cells (Duhrsen and Metcalf, 1988, 1989). Many of these leukemic populations can be shown to have acquired the autocrine capacity to produce either GM-CSF o r multi-CSF, and in many populations a rearrangement of the respective genes was demonstrated, due to insertion of an intracisternal A particle (IAP) or an IAP LTR upstream of the gene. This insertion seems to have been responsible for transcriptional activation of the CSF gene (Duhrsen et al., 1990). As in the case of the retrovirally inserted multi-CSF model, only some of the leukemias developing in engrafted animals exhibited autonomous proliferation in clonal culture. Others, although shown to be producing CSF, exhibited partial or complete dependency on growth stimulation by added CSF (Duhrsen, 1988). T h e anomalous behavior of cells, certifiable as being leukemic and known to be producing CSF as the likely transforming event, but continuing to exhibit complete dependency on extrinsic CSF for proliferative stimulation in vitro, represents a bizarre combination. T h e phenomenon reemphasizes the need to reconsider the conclusions reached earlier regarding human myeloid leukemic cells on the basis of their dependency on extrinsic stimulation by CSF. This dependency can no longer be assumed to exclude their autocrine production of CSF or the possibility that such autocrine CSF production was of relevance for their leukemic transformation. What these studies on immortalized cell lines documented was that the CSF genes can function as typical protooncogenes able to transform cells from a nonleukemic to a leukemic state. The failure of activated CSF genes to transform normal hemopoietic cells indicates, however, that autocrine CSF production is only one of the necessary transforming

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steps. T h e immortalized cell lines are highly abnormal with their high level of self-generation and inability to undergo full maturation changes and these abnormalities appeared to be crucial if leukemic transformation was to occur following acquisition by the cells of a capacity to produce their own growth factors. In important parallel studies in this Institute, a method was devised for achieving immediate leukemic transformation of normal hemopoietic cells. It had been noted by others (Ymer et al., 1985) that in the murine myelomonocytic leukemia WEHI-3B there is an IAP insertion that activates transcription of the multi-CSF gene. Extension of this analysis revealed that these cells also exhibit an IAP-activated transcription of the homeobox gene Hox 2.4 (Blatt et al., 1988; Kongsuwan et al., 1989). When a retroviral construct was made containing the cDNAs for both multi-CSF and Hox 2.4, infection of normal hemopoietic cells resulted in rapid transformation of these cells to WEHI 3B-type leukemias (Perkins et al., 1990) (Fig. 6). Subsequent analysis of the action of the Hox 2.4 gene has indicated that it encodes a nuclear transcription factor whose overexpression appears to interfere with the normal process of differentiation commitment and to confer on some normal hemopoietic precursors a sustained capacity for self-generation if the cells are maintained in the presence of high concentrations of multi-CSF (A. Perkins, personal communication). Taken together, these studies have therefore demonstrated a mechanism for myeloid leukemia initiation that involves two essential changes: (1) an acquired abnormal capacity for self-generation and (2) an acquired capacity for autocrine proliferative stimulation by an appropriate CSF. T h e two changes may not need to occur in the sequence listed, but have done so in the models so far analyzed. This “formula” for leukemogenesis is best regarded as documenting a principle rather than being exclusive of other comparable leukemogenic mechanisms. For example, the Hox2.4 product is not likely to be the only nuclear transcription factor able to perturb the pattern of selfgeneration in early hemopoietic cells. It might be that products of other protooncogenes o r nuclear transcription factor genes are more usually the agents inducing a comparable perturbed state of self-generation. Similarly, autocrine growth factor production is only one of several mechanisms whereby a cell can achieve autonomy of cell proliferation. As shall be discussed below, mutations of growth factor receptors can result in constitutive activation of the receptor, leading to signaling in the absence of ligand. It is also probable that agents such as the products of the src group of genes can achieve comparable cellular proliferation

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a

b

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C

a b C FIG. 7. T h e twin events of autocrine stimulation and aberrant self-renewal can be achieved by a variety of mechanisms. Autocrine stimulation is achieved (a) by CSF production, (b) by a constitutively activated mutated receptor, signaling without ligand binding, and (c) by an oncogene product that can enter the signaling pathway normally activated by ligand-receptor binding. Aberrant self-renewal requires the aberrant operation of nuclea r transcription factors but a variety of gene products, e.g., Hox 2.4, Myc, or MyURARa may achieve this final outcome. by impinging on the signaling pathway normally activated by ligandreceptor binding (Fig. 7). This cellular formula for leukemogenesis can account for the development of an expanding population of leukemic cells, but the full pathological picture in AML, with its suppression of normal hemopoietic cells, probably requires the operation of additional mechanisms with selective suppressive actions on normal stromal or hemopoietic cells. Our experiments on leukemogenesis repeatedly identified autocrine growth factor production as a common change occurring during transformation. This was curious because it is easy enough to envisage other mechanisms for achieving autocrine growth stimulation and because the amounts of growth factor produced were often very low, implying that cell-associated factors may be peculiarly efficient in achieving the type of proliferative stimulation relevant during leukemic transformation. These observations seemed to downgrade the possibility that excessive stimulation by extrinsic growth factors might be able to play a significant role in leukemogenesis-the starting point for my own studies on leukemogenesis.

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However, we have recently reinvestigated the possible role of excess extrinsic regulatory stimulation in leukemogenesis by examining the behavior of FDC-P 1 cells after engraftment in GM-CSF transgenic mice with high circulating levels of GM-CSF. T h e results proved dramatic and readily reproducible. Within 100 days of injection of certified nonleukemic FDC-P 1 cells, all GM-CSF transgenic recipients developed advanced leukemia, but no disease developed in the injected littermate control mice. T h e leukemias were of FDC-P1 origin, were commonly polyclonal, and were readily transplantable to normal recipients. In culture, some leukemias were autonomous, others dependent on extrinsic CSF stimulation. More than half were able to produce either GM-CSF o r multi-CSF and, in many, variable rearrangements of the CSF genes were demonstrable. Analysis showed that the leukemias developed independently in vivo and were not the consequence of accidental injection of pretransformed FDC-PI cells (Metcalf and Rasko, 1993). T h e mechanism responsible for transformation in this model has yet to be determined but it could be quite indirect. T h e simplest, but not necessarily the full, explanation is that excessive stimulation by GM-CSF merely expanded the size of the engrafted population, which then allowed randomly occurring transformation events to occur more often. It is again intriguing how often the transformed populations in this model exhibited autocrine CSF production despite the excess concentrations of CSF available in the animal. Equally intriguing was the fact that those leukemias demonstrable as producing CSF did not arise earlier than those with no evidence of autocrine CSF production. As in the other models, a residue of leukemias could not be accounted for by acquired autocrine growth factor production, leaving open the possible development of quite different autocrine mechanisms. These observations indicate that excess levels of circulating GM-CSF can accelerate leukemic transformation, at least in immortalized cells responsive to GM-CSF. If the model at all resembles the situation in some myelodysplastic patients, then the prolonged administration of CSF, which is a useful clinical treatment to improve hemopoiesis, might entail some risk of increased transformation to acute leukemia. The foregoing experiments have relied heavily on one useful cell line to establish likely mechanisms of leukemogenesis and are open to the criticism that the results may be relevant only to this particular cell line. However, what the experiments have sought to establish is the general nature of the basic cellular changes required for leukemogenesis. Comparable basic changes may well be able to be achieved by alternative mechanisms not necessarily involving the CSFs.

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VIII. Membrane Receptors for Colony-Stimulating Factors

To induce changes in responding cells, the CSFs need to interact with specific membrane receptors but the polyfunctionality and shared actions of the CSFs raised a number of questions. Are there specific receptors for each CSF or are receptors shared? Is there a single type of receptor for each CSF or do different receptors initiate the different actions of the CSFs? If multiple signaling cascades can be initiated from a single class of receptor, how is this achieved? Information on the physical structure of CSF receptors has accumulated rapidly in the past few years with the successful cloning and expression of cDNAs encoding the receptors. Each CSF has a specific membrane receptor that is unable to bind other CSFs. T h e numbers of CSF receptors on responding cells are typically small-only a few hundred per cell-and signaling is achieved with low levels of receptor occupancy. Most granulocyte-macrophage progenitors coexpress receptors for all four CSFs, an arrangement allowing a variety of interactions between the CSFs on individual cells (Nicola, 1989). The membrane receptor for M-CSF was recognized to be the product of the protooncogene c-fms and is a transmembrane glycoprotein with a tyrosine kinase signaling domain in the cytoplasmic region (Sherr, 1990). Binding of M-CSF to its receptor induces homodimerization of two receptor chains with transphosphorylation at or near the tyrosine kinase domain as the initial event in cell signaling. T h e membrane receptors for the other three CSFs belong to a newly defined class of growth factor (hemopoietin) receptors characterized by shared regions of homology in their extracellular domains and an absence of a tyrosine kinase or other known signaling motif in their cytoplasmic regions (Bazan, 1990). These transmembrane receptors (a chains) bind CSF with low affinity due to fast off-rate kinetic (Nicola and Metcalf, 1991). However, in the case of the GM-CSF and multi-CSF receptors, following ligand binding the complex undergoes heterodimer formation by association with a second transmembrane chain (the p chain). T h e resulting heterodimer is of high affinity and is able to initiate signaling (Miyajima, 1992). It remains possible that additional subunits may exist for the CSF receptors. T h e presence of at least two chains in an activated CSF receptor allows the possibility that differing signaling cascades may be initiated by different portions of the complex. Preliminary mapping data using mutagenized receptors have supported this possibility by showing that differ-

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ent regions of the p chain are associated with an ability to initiate the transcription of different sets of nuclear transcription factor genes following binding of GM-CSF (A. Miyajima, personal communication). Data obtained from an analysis of a truncated form of the erythropoietin receptor suggested that different regions of the receptor might initiate signals preventing apoptotic death on the one hand, and stimulating mitosis on the other (Nakamura et al., 1992). These observations require extension but seem to offer a possible mechanism for uncoupling proliferative from differentiative responses in a cell if it expresses suitably abnormal receptors. Studies with the M-CSF receptor have shown that mutated receptors can initiate signaling in the absence of ligand binding, and aberrant receptors of this type can function as a surrogate autocrine system for stimulating cell division and transformation (Wheeler et al., 1986). Similarly, a mutation in the extracellular domain of the erythropoietin receptor leads to constitutive activation of the receptor in the absence of erythropoietin and, in erythroid cells expressing this mutated receptor, sustained hyperplasia develops with the eventual emergence of genuine erythroleukemias (Yoshimura et al., 1990; Longmore and Lodish, 1991). Thus the work on growth factor receptors has identified two phenomena of relevance for leukemogenesis: ( 1) possible receptor-based mechanisms for uncoupling proliferation from differentiation and (2)the ability of mutated receptors to induce proliferation in the absence of ligand binding. In this context, we have been interested recently by the recognition that the genes encoding the human GM-CSF and multi-CSF receptor 01 chains are both located in the pseudoautosomal regions of the X and Y chromosomes (Gough et al., 1990; M. Vadas, unpublished data), a finding of potential relevance in AML, particularly M2 AML, where loss of a sex chromosome is unusually common. If the remaining allele was mutated, could it then exhibit constitutive activation? So far, our analysis of the GM-CSF receptor gene in AML has revealed no obvious rearrangements (Brown et d . , 1993), but some of the point mutations observed are still in need of further functional analysis. IX. Hemopoietic Regulators in the Context of Known Inducers of Leukemia

There were times during the work on the colony-stimulating factors and their role in the development of human myeloid leukemia when our colleagues seemed to regard the role of hemopoietic regulators as being little more than the lunatic fringe of mainstream work on leuke-

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mogenesis. These attitudes have changed but there does remain a necessity to link together the various streams of information of leukemogenesis. It is accepted wisdom that the major human leukemias exhibit characteristic chromosomal abnormalities, the most dramatic of which are translocations that have activated protooncogenes or have resulted in the production of abnormal fusion proteins of clear relevance to leukemogenesis. It is also accepted wisdom that human or animal leukemias or lymphomas are induced by one or the other of the “big four”irradiation, chemical carcinogens, leukemia viruses, or gross deficiencies in immune responsiveness. However, a critical appraisal of our present knowledge of the causes of leukemia in man reveals some serious deficiencies. Chromosomal abnormalities exist in most leukemias and are clearly of major relevance. However, there is a notable deficiency: we completely lack information on what provokes the development of such specific rearrangements or abnormalities. As for the “big four” being causes of leukemia in humans, even the most enthusiastic have to admit to some problems. Only two viruses have been established as being leukemogenic in man-HTLV- 1 for adult T cell leukemia and possibly a variety of leukemias and lymphomas in certain geographical regions, and Epstein-Barr virus (EBV) for Burkitt’s lymphoma and probably at least some Hodgkin’s disease and lymphomas. No human viruses have been described for the myeloid leukemias. Irradiation is certainly leukemogenic in humans but cannot account for more than a small fraction of cases. According to one’s scientific beliefs, extrinsic chemical carcinogens are either the cause of most cancers and leukemias, or may merely be responsible for a few: the problem is so complex and the likely dose rate so low that the questions do not lend themselves to experimental analysis. Gross immunological deficiencies certainly allow the emergence of B lymphomas, but this is an uncommon situation with little obvious relevance for the average case of human leukemia. However, the work on hemopoietic regulators should not be viewed as some sort of competitive enterprise to establish the “true” or usual causes of leukemia in man. T h e work on hemopoietic regulators has merely sought to characterize the nature of the cellular changes that appear to be necessary for leukemic transformation, not the initiating causes of these changes. Nevertheless, there is clearly an intellectual gap to be bridged between oncogenes and leukemia viruses on the one hand, and dysplastic hemopoietic mechanisms on the other. This gap may be bridged by considering whether the products of known oncogenes or tumor viruses can be fitted into the hypothesis that induction of leuke-

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mia requires two major abnormalities to develop-abnormal selfgeneration and autocrine growth stimulation (Fig. 7). There are some oncogenes or protooncogenes whose protein products are, or interact with, nuclear transcription factors potentially able to interfere with the self-generative process. These would include products of myc (Adams and Cory, 1992), myb (Slamon et al., 1986), v-erb A (Sap et al., 1986; Weinberger et al., 1986), scl (Green et al., 1991), and Evi-1 (Morishita et al., 1988), and the aberrant retinoic acid receptor-mylencoded fusion protein (de T h e et al., 1990). Conversely, there are other oncogenes whose products are either mutated growth factor receptors or cause receptor activation, such as products of v-fm (Sherr, 1990), v-erbB (Ullrich et al., 1984), and the Friend virus gp55 (Li et al., 1990), o r are likely signaling intermediates, such as the products of raf (Carroll et al., 1991), lck (Hatakeyama et al., 1991), ras (Greenberger, 1989), abl (Mathey-PrCvot et al., 1986), and bcrlabl (Daley and Ben-Neriah, 1991). In the latter context, infection of FDC-P1 cells by the Abelson virus results in leukemic transformation without evidence for autocrine CSF production (Cook et al., 1985; Pierce et al., 1985), the abl product possibly achieving surrogate signaling that substitutes for normal CSFinitiated signaling. Similar results have been obtained by inserting bcrlabl into a CSF-dependent cell line, transformation again being achieved without autocrine growth factor production (Daley and Baltimore, 1988). However, oncogenes may also act indirectly by inducing autocrine production of colony-stimulating factors. This has been reported in the Abelson virus transformation of mast cell lines, wherein the induced production of GM-CSF and multi-CSF was noted (Humphries et al., 1988), and induced production of multi-CSF was also observed by FDCP1 cells transformed by the bcrlabl oncogene (Hariharan et al., 1988). Similarly, in the development of myeloid leukemia in chickens, infection by v-mil confers autonomy on macrophages by inducing them to produce a CSF (Metz et al., 1991). Where oncogenes of this type are involved and offer other possible mechanisms for transformation, it becomes necessary to question the significance of the observed autocrine growth factor production. Is any level of production, however small, of relevance, or must the level of production exceed a certain threshold? Some assessment may be possible in suitable systems by the use of antibodies, but the use of antisense nucleotides probably offers the best approach to resolving the question. It is well recognized that pairs of oncogenes can cooperate to induce the accelerated development or an increased incidence of leukemia in murine models (Adams and Cory, 1991, 1992). This has also been noted

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in avian models in which complementation is evident between an oncogene product that is nuclear in location (candidate transcription factor) and an oncogene product that is cytoplasmic in location (candidate signaling intermediate) (Graf, 1988). Is it absolutely necessary to have the combined action of an oncogene of each class? Could the combined action of two nuclear transcription factors achieve transformation? There are increasing examples of enhanced transformation resulting from combination of two nuclearacting factors and if signaling remains intact, normal levels of growth factor might well provide sufficient mitotic signaling. Conversely, can aberrant growth stimulation alone induce transformation? Is it absolutely necessary to have an abnormality in self-renewal induced independently by a nuclear-acting factor? The data on the CSFs reviewed above indicated that excess CSF stimulation alone was only able to induce hyperplasia. A similar answer was obtained for sustained excess mitotic stimulation by erythropoietin (Villeval et al., 1992), IL-5 (Vaux et al., 1990), and IL-6 (Suematsu et al., 1989). There are some experiments, however, that suggest that growth factor stimulation might be able to achieve immortalization o r sustained self-generation. Studies using chicken erythroid precursor cells and TGFa, acting through the c-erb B receptor, have documented a capacity of TGFa to induce the long-term proliferation of undifferentiated erythroid precursors, a process described as being substantially self-renewal (Pain et al., 1991). Similarly, under the action of multi-CSF, normal mast cell lines can be generated that exhibit a sustained capacity for self-renewal. However, this latter proliferation is finite and eventually such lines terminate unless they become transformed to genuine immortalized lines (Moore, 1988). These various examples suggest that growth factor stimulation alone can sometimes achieve in suitable normal cells a sustained level of what approximates abnormal self-renewal. However, in each case, the evidence suggests that genuine immortalization is not achieved, indicating the probable need for a separate perturbation of the self-renewal process to achieve the genuinely aberrant state of self-renewal needed for leukemic transformation. Where leukemogenic viruses lack a formal transforming oncogene, transformation is achieved by insertional mutagenesis. It remains intriguing in the model systems analyzed in this laboratory how often transformation under such circumstances has involved activation of either the GM-CSF or multi-CSF genes. For example, there are IAP insertions in WEHI-3B leukemic cells upstream of the multi-CSF gene and IAP insertions upstream of the GM-CSF or multi-CSF genes in FDC-P1 cells, transforming either in irradiated or transgenic recipients. With the

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in vitro transformation of FDC-PI cells by the Moloney virus it has also been of interest that there is a similar activation of these two CSF genes (J. Rasko, personal communication). It has also been intriguing that, when FDC-P1 cells were engineered in our laboratory to exhibit the M-CSF receptor (c-fms), multiple autonomous cell lines emerged independently, all of which had acquired an autocrine capacity to produce M-CSF (D. Metcalf, unpublished data). These may be an unrepresentative collection because, as commented above, an insertional abnormality might well influence nuclear transcription genes and achieve transformation by perturbing the process of self-generation. The biology of oncogenes remains largely outside my field of competence, but I am acutely conscious of the gap that needs to be bridged between the two general fields and have therefore raised the above possibilities to stimulate further efforts to achieve this bridging by those better fitted to explore these questions.

X. Role of Hemopoietic Regulators in Suppressing Myeloid Leukemia Ichikawa (1969, 1970) noted a curious phenomenon in cultures of the established leukemic cell line, M I . These cells formed large, tight colonies of undifferentiated cells in semisolid cultures, but, if various conditioned media were added to the cultures, many could induce a striking differentiation of the cells to relatively mature granulocytes and macrophages. When the WEHI-3B myelomonocytic leukemic cell line (Warner et al., 1969) was eventually adapted to more satisfactory growth in agar culture, this line also exhibited a comparable phenomenon of differentiation induction (Metcalf, 1979). At the time, many of the types of conditioned media used in these studies were also being documented as containing CSF. Considerable confusion arose in the literature during the following decade regarding the identity or nonidentity of the growth factors (CSFs)being detected using normal cells, with the differentiationinducing factors detected using leukemic cell lines. The confusion was ultimately resolved by purification of the regulatory factors involved. Using the WEHI-3B cell line model, it was demonstrated that G-CSF was highly active in inducing differentiation (Nicola et al., 1983) in a complex action in which G-CSF initially stimulated leukemic cell division, but at the same time induced irreversible asymmetrical differentiation commitment in the cells that then proceeded to differentiate (Metcalf, 198213). With time, differentiation commitment dominated the response, with progressive suppression of the further potential for proliferation in this leukemic cell line. Cells exposed to

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G-CSF

FIG. 8. Depending on the niyeloid leukemic cell line used, one or other of a number of regulators can partially or completely suppress the population by blocking self-renewal and enforcing differentiation commitment. The committed progeny produced lose their proliferative capacity and eventually die. These cells may or may not exhibit morphological maturation changes.

G-CSF exhibited a reduced ability to produce transplanted leukemias (Metcalf, 1982a) and, using a comparable cell line, it was shown that injection of transplanted animals with G-CSF could suppress or delay leukemia development (Tamura et al., 1987). GM-CSF was also shown to have some ability to induce differentiation in WEHI-3B cells but its action was clearly weaker than that of G-CSF (Metcalf, 1979) (Fig. 8). These findings did not agree with the data on the molecular weight and properties of the active agents inducing differentiation in M1 leukemic cells. Hozumi and his colleagues in Sapporo described a differentiation factor (DF) of MW 58,000 as the active factor inducing differentiation in M1 cells (Tomida et al., 1984); Sachs and his colleagues in Rehovot, also working with M 1 cells, described an MGI-2 with differing properties (Lipton and Sachs, 1981). To resolve these discrepancies we obtained M1 cells from Hozumi and the Krebs I1 ascites tumor used as one source of his DF. Purification of Krebs I1 ascites tumor conditioned medium revealed that it contained G-CSF and GM-CSF but also a more active molecule, of MW 58,000, with an ability to induce profound dif-

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ferentiation in M 1 colonies with suppression of most colonies. We termed this factor leukemia inhibitory factor (LIF) (Hilton et al., 1988a,b; Metcalf et al., 1988) and isolated cDNAs encoding the murine and human LIF molecules (Gearing et al., 1987; Cough et al., 1988). LIF has no direct colony-stimulating activity for normal hemopoietic cells but can stimulate the proliferation of several hemopoietic cell lines (Moreau et al., 1988; Cory et al., 1991). Subsequent sequencing and cloning of DF confirmed that it was LIF (Lowe et al., 1989). Further resolution of the discrepancies resulted when sequencing of purified MGI-2 revealed it to be IL-6 (Shabo et al., 1988) and when a comparative study revealed that the Rehovot sublines of M1 were highly responsive to IL-6 but unresponsive to LIF, whereas the Sapporo/Melbourne lines were highly responsive to LIF and only weakly responsive to IL-6 (Lotem et al., 1989) (Fig. 8). From the limited perspective of work on M1 and WEHI-3B cells, certain conclusions were apparent. A number of hemopoietic regulators had the ability to induce differentiation in leukemic cell lines, and which agent was more active depended on the cell population under study. Extension of this work to human leukemic cell lines resulted in similar conclusions. For example, the U937 cell line responded strongly to GMCSF, but HL-60 cells, only weakly (Maekawa and Metcalf, 1989). Furthermore, combinations of CSFs, IL-6, and LIF all resulted in enhancement of differentiation induction as measured by the key parameter of reduced clonogenicity (Maekawa et al., 1990). Interestingly, the differentiating macrophages derived from autonomous leukemic cell lines appeared to develop dependency on growth factors for survival and growth (Fibach and Sachs, 1976) and, as a consequence, inclusion of M-CSF in such cultures could actually enhance the number of maturing macrophages being generated (Metcalf, 1989). To place these observations on hemopoietic regulators in proper context, it needs to be stated that a bewildering variety of chemical agents, ranging from dimethyl sulfoxide to sodium butyrate, have been shown to have a capacity to induce differentiation in these same and other leukemic cell lines, often with much more rapid and complete cellular maturation (Abraham and Rovera, 1981). Indeed, much of the suppression of leukemic cells by the CSFs or LIF is not associated with obvious maturation. Rather, the cells retain their blast morphology but exhibit a reduced or no capacity for further self-generation. Maturation is therefore not a necessary accompaniment of differentiation induction (clonogenic suppression), although often the two can be associated. There may well be nothing special in terms of cell biology about the actions of hemopoietic regulators in inducing differentiation commit-

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ment in leukemic cells. However, the important fact remains that these regulators are normal products of the body and are therefore available to influence the biology of emerging myeloid leukemic populations. T h e action of agents like the CSFs on leukemic cells is the basis for the speculation raised earlier that the anomalous growth of AML cells in culture may have been the result of the culture system used to obtain clonal proliferation. AML cells require stimulation by CSFs to proliferate, but the same CSFs may possibly be inducing differentiation commitment (without maturation) in the cells, causing them to be incapable of further proliferation on recloning. I n this context, a CSF-stimulated culture of AML cells can resemble exactly a clonal culture of M1 cells after suppression by LIF, i.e., scattered collections of small clusters of cells. T h e preceding discussion on the role of the CSFs in leukemogenesis has largely been concerned with their action in stimulating cell division -an obviously necessary component for the emergence and expansion of a leukemic clone. However, the differentiation commitment (inhibitory) actions of the CSFs on myeloid leukemic cells raise an additional possibility. Is it possible that a failure to deliver an effective CSF-initiated signal for differentiation commitment can represent o r influence the required abnormality in self-generation needed by transforming cells? (See Fig. 9.) The anomalous behavior of the CSFs in being both proliferative stimuli and inducers of differentiation commitment and maturation represents a curious combination. If there is a design purpose in this arrangement it may well be to ensure, through the action of a single regulator, that a limited and orderly generation of mature cells is accomplished from immature precursors. Some suggestive evidence has been obtained that the CSFs may exhibit a skewing of their actions on hemopoietic cells, with GM-CSF and multi-CSF having more evident proliferative actions, whereas G-CSF and M-CSF have relatively stronger actions in differentiation commitment (Nicola and Metcalf, 1985). Could an imbalance in the relative concentrations of these factors lead to a significant imbalance between proliferation and differentiation commitment? It has even been proposed that the regulators are classifiable into two distinct subsetsproliferative factors and differentiation-inducing factors (Sachs, 1987) -but I believe that this proposal goes beyond the known facts. On analysis, each factor possesses both types of action. T h e known interactions between differing occupied CSF receptors (Walker et al., 1985), the existence of receptor subunit sharing for at least two regulator subgroups (GM-CSF, multi-CSF, and IL-5 on the one

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CM-CSF Multi-CSF

Proliferation

Differentiation

CSF

Proliferation

C-CSF M-CSF

CM-CSF Multi-CSF

C-CSF M-CSF

Proliferation

CSF

Differentiation Proliferation

Differentiation

FIG. 9. In normal cells, stimulation by the CSFs elicit a balanced proliferative and tlifferentiative response. G-CSF and M-CSF may have stronger differeritiative than proliferative actions and loss of their receptors could result in an unbalanced proliferative response to GM-CSF o r multi-CSF. A mutation in a CSF receptor could result in proliferative but not differentiative signals following CSF binding. More likely, aberrations in the genome could allow a cell to exhibit a proliferative response to CSF stimulation, but not a ditt'erentiative response.

hand, and LIF, IL-6, IL-11, and Oncostatin M (OSM) on the other), and the emerging information that differing regions of the receptor may initiate proliferative versus differentiative signaling all provide possible mechanisms whereby proliferative and differentiation induction signaling might become uncoupled or imbalanced in response to stimulation by even normal concentrations of CSF. T h e situation in many AMLs,

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where there is loss of one sex chromosome and the possibility of a mutation in the remaining allele for either the GM-CSF or multi-CSF a-chain receptor, has already been noted, but again could have significance if any abnormality led to a dissociation of normally linked signaling. In the context of imbalanced signaling in a system whose design allows multiple regulators to act on individual cells, if there is substance in the possibility that some regulators have stronger differentiationinducing actions than others, then failure to express particular receptors becomes a possibly important cause of signaling imbalance It is of interest that two main sublines of the WEHI-3B leukemia exist, one (D+)able to exhibit differentiation, the other not (D-). WEHI-3B D- cells lack receptors for G-CSF (Nicola and Metcalf, 1984), certainly providing an explanation for their failure to respond to G-CSF-initiated differentiation. Similarly, in a survey of primary human AML populations, the situation in most was that a significant subset of cells in each population appeared to lack receptors for G-CSF, except in the case of M3 promyelocytic leukemias (Begley et d., 1987). Loss of receptors for G-CSF might therefore allow some imbalance in the proliferative responses elicited by other CSFs (Fig. 9). However, there are likely to be a multiplicity of abnormalities that might result in a failure of a leukemic cell to respond by coupled differentiation induction to CSF signaling even with an intact receptor system (Sachs, 1987; Hoffman-Liebermann and Liebermann, 1991). CSF signaling would then provide a fatal proliferative stimulus uncoupled from differentiation commitment. The peculiar frequency or effectiveness of autocrine, cell-associated CSF in murine leukemogenesis suggests that signaling of this type may be potentially more prone to uncoupling than conventionally delivered signaling by extrinsic CSF. The availability of agents such as the CSFs, LIF, or IL-6 for clinical use raises the practical question of whether they can usefully be incorporated in treatment regimens for the suppression of myeloid leukemic populations. In this context, the dramatic ability of retinoic acid treatment to correct the maturation blockage in acute promyelocytic leukemic cells and to lead to a complete remission validates the general utility of this approach. The CSFs and IL-6 have the apparent disadvantage that, being proliferative stimuli, their administration will enhance the expansion of the leukemic clone. However, this property can be exploited clinically when S-phase-specific chemotherapeutic agents are in use. Myeloid leukemic cells are commonly out of cell cycle o r have longer G, periods than normal cells and the CSFs do have an ability to force cells into cycle and to shorten the G , period. Their use, combined with cycle-specific drugs,

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does result in heightened cytotoxicity in vitro and possibly improved induction of remissions in vzvo (Lowenberg and Touw, 1993). There has not yet been a serious attempt to make use of the differentiation commitment action of these regulators in enhancing remission induction or in sustaining remissions where some leukemic cells survive. This remains a possibility worthy of clinical trial, particularly if the specific leukemic population exhibits some responsiveness in vitro. An agent such as LIF, which has little proliferative action on myeloid leukemic cells, seems a promising candidate for use in this manner, and because combinations of regulators are more active in nitro than are single agents, this principle is also worthy of clinical exploration.

XI. Recapitulation The work of our group over the past 40 years began with studies on lymphoid leukemia but evolved through circumstance to be concerned mainly with myeloid leukemia. T h e four colony-stimulating factors were characterized and identified as the major regulators of granulocytemacrophage populations. From studies on murine models, two major intrinsic changes appear to be required for myeloid cells to transform to leukemic cells: (1) a perturbation of the manner in which they self-renew and (2)acquisition of a mechanism for autocrine growth stimulation. Extrinsic or autocrine CSF is likely to be necessary to stimulate the subsequent clonal expansion of a transformed myeloid cell, whether murine or human. These concepts represent our current basic hypothesis of leukemogenesis (Fig. 10). Perturbation in self-renewal appears to involve the aberrant action of one or more nuclear transcription factors controlling the gene(s) responsible for the self-renewal versus differentiation commitment decision in dividing immature cells. Hox 2.4 has been identified as one such nuclear transcription gene and various protooncogenes can also operate singly or in collaboration to achieve this outcome. The acquired mechanism for autocrine growth stimulation can involve activation of a CSF gene by viral or IAP insertion and the CSF produced then acts preferentially in a cell-associated manner. Other methods for achieving autocrine growth stimulation appear to be aberrant, constitutively activated growth factor receptors or the development of dysregulated concentrations of a signaling intermediate in the growth factor-receptor signaling pathway. It is now possible to propose several accessory hypotheses (Fig. 11). Excess extrinsic CSF levels can accelerate leukemic transformation, pos-

HEMOPOIETIC REGULATORS AND LEUKEMIA DEVELOPMENT

a3

I 1993 HYPOTHESIS I CSF

CSF

CSF

CSF

Aberrant

Aberrant

Dominant

Self-Renewal

Self -Renewal

Leukemic

Autocrine Stimulation

Clone

1

0

ooo + 0 0

Normal

+

FIG. 10. The 1993 hypothesis of the most likely role played by the CSFs in myeloid leukemogenesis. Normal, preleukemic, and leukemic populations remain CSF dependent throughout. The leukemogenic process initiates by a cell acquiring an aberrant capacity for self-generation, leading to the emergence of a dominant preleukemic clone. Leukemic transformation occurs when one of these cells acquires the autocrine capacity for selfstimulation, often by CSF production. The leukemic clone then expands and suppresses preexisting preleukernic or normal populations.

sibly by increasing the population size of responding cells, so allowing one or the other of the above intrinsic abnormalities to be more likely to develop. CSF action may also protect abnormal cells from apoptotic death, encouraging the persistence of such preleukemic or leukemic cells. Conversely, because the CSFs and other hemopoietic growth factors, such as IL-6 and LIF, can enforce differentiation commitment and reduce self-generation, these regulators can suppress some emerging myeloid leukemic cells with o r without associated maturation of the cells. Hemopoietic growth factors such as the CSFs therefore play a key role in the development and emergence of myeloid leukemic populations, being often involved in the autocrine growth stimulation component of leukemogenesis and probably always in the subsequent expansion of the leukemic clone. T h e studies in our laboratory have not identified causative agents of leukemia and have merely addressed the cellular processes subsequently becoming abnormal. Although they have offered some new approaches in therapy, they have not provided any insight into the prevention of these diseases.

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I

1993 ACCESSORY HYPOTHESES

Hyperplasia

Preleukemic

Dominant

+Aberrant

Clone

leukemic

Cell

CSF

CSF

B

Clone

1

t

CSF

0

%X

ooo .--, 00

0

I

Normal

0 -

Apoptosist

c

f

Apoptosir

CSF

CSF

CSF

1

- -

00

00

000 0 Normal

1

00 000 0

I

Persistence of

t

1

000 Normal

+

Suppression of Leukemic Cell

FIG. 1 1 . Accessory hypotheses for the involvement of the CSFs in myeloid leukemia development. (A) A variant of the events in Fig. 1 0 in which excess stimulation by extrinsic CSF expands the population at risk, making the emergence of cells with abnormalities more likely to occur. (B) T h e possibility that CSF-induced suppression o f the apoptotic proress may allow the persistence and subsequent expansion of abnormal cells of the above types. (C) T h e contrary role that the CSFs may play in suppressing emerging preleukemic or leukemic cells by enforcing differentiation commitment with or without maturation. (In both this figure and in Fig. 10, aberrant self-renewal is shown as preceding the acquisition of autocrine stimulation. This may not need to be so, but has been the situation in the models analyzed in the author's laboratory.)

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XII. The Future

T h e “two-abnormality” formula discussed above should not be misinterpreted as suggesting that leukemogenesis is necessarily a simpler process than the multistep process of cancer development in epithelial tissues. Such a view has been expressed but is probably not correct. Some of the “steps” proposed in the development of carcinomas are merely progression changes in established tumors, and similar progression is common in leukemias. I suspect that there are additional subtle changes required during the actual developmental process in leukemogenesis. For example, in our current model in which leukemic transformation occurs in FDC-P1 cells engrafted in GM-CSF transgenic mice, there is an obvious “preleukemic” change wherein the cells remain CSF dependent but exhibit abnormal clonal proliferation in nitro. This change invariably precedes the emergence of transformed leukemic cells and its nature is under investigation. It is obvious that this work has at best merely identified the general nature of the major changes required for transformation but has not documented a unique abnormality responsible for each. An immediate task for the future is to try to establish whether o r not the known agents implicated in leukemogenesis achieve one or the other of these general abnormalities. Although data have been discussed that implicate the CSFs in both processes, the CSFs may not in fact necessarily be involved and have merely been a convenience for characterizing the nature of the changes occurring. Surrogate signaling by oncogene products that makes use of the CSF signaling pathway can clearly substitute for the actual requirement for CSF-initiated proliferation, and the CSF-driven differentiation commitment/maturation sequence influencing selfgeneration is likely to be perturbed in many ways. It can be anticipated that future studies will succeed in identifying the actual abnormalities in the more common forms of myeloid leukemia. It seems timely to apply the principles deduced from the studies on the myeloid leukemias to a more extensive analysis of the role of lymphoid regulatory factors in the transformation of lymphoid cells. It is also reasonable to expect that the same principles will apply in the transformation of other cell types, although the corresponding tissue-specific regulators are only just being uncovered. It would be rewarding at some stage to identify the specific initiating causes of the common leukemias-if such exist. If we are to remain with nothing more than a random mutation hypothesis as the origin of the required genetic abnormalities, we will have achieved nothing of value in preventive medicine and will always be faced with the prospect of treat-

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ing an already-established disease state. I hope that this will not prove to be the case. Understanding may be rewarding in itself, but to be able to use knowledge to prevent the initiation of a disease, I believe, takes understanding to a higher plane in human affairs. In retrospect, the hypothesis I began with in 1954 has taken a battering but was not entirely ill-founded. Such is the nature of hypotheses that the present set can be assumed to be fated for a similar battering in the next few decades. Despite this, I believe that considerable progress has been made in understanding, if not the initiating causes, at least the nature of the cellular changes involved in leukemogenesis and along the way agents have been developed of clinical value for a wide variety of other disease states. I am therefore pleased in retrospect to have taken the tortuous course I did. If setting out in 1993 to undertake a career in cancer research, I think that I would remain unrepentant and again enter the world of biological regulators-perhaps not of hemopoietic cells, for these are now becoming well defined, but of some other cell type, offering the prospect of future adventures in the unknown. ACKNOWLEDGMENTS I thank the Anti-Cancer Council of Victoria for generous support of my laboratory throughout the whole period described in this review, the many creative co-workers it has been my good fortune to have had over the years, and, finally, Suzanne Cory and Nick Nicola for their kindness in critically reviewing this manuscript. REFERENCES

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ribosomal protein S6 (Gressner and Wool, 1974). Extracellular cues, including insulin and growth factors, cause the phosphorylation of S6 on serine residues, although most of the receptors for these extracellular ligands have intrinsic tyrosine kinase activity o r signal by activating an associated tyrosine kinase. Enhanced S6 phosphorylation may come about by the activation of two different S6 protein kinase families (Jones et al., 1988; Kozma et al., 1990; Bannerjee et al., 1990; Novak-Hofer and Thomas, 1985). Both are regulated by phosphorylation on serine/ threonine residues. It has been suggested that the 70K, single-catalyticdomain S6 kinases may be the enzymes responsible for S6 phosphorylation in intact cells (Chung et al., 1992), whereas the two-catalytic-domain, 90K S6 kinases, also called Rsks, phosphorylate S6 fortuitously. S6 phosphorylation is believed to contribute to discrimination among messenger RNAs for translation. However, the issue of whether both, neither, o r only one of the known types of S6 kinase phosphorylate S6 in intact cells awaits a detailed understanding of the function of the event. Experiments from several laboratories suggest that the 90K and 70K S6 kinases lie on separate signaling pathways (Ballou et al., 1991; Mukhopadhyay et al., 1992). The 90K, two-catalytic-domain S6 kinases are now known to be controlled by a protein kinase cascade that involves the sequential activation of at least four serine/threonine, or dual-specificity, protein kinases. Receptors activate this cascade via GTP binding proteins, either the protooncogene product Ras or certain heterotrimeric G proteins, including the Gi class (Anderson et al., 1991a; Robbins et al., 1992; Wood et al., 1992; Thomas et al., 1992; Gupta et al., 1992; Kahan et al., 1992; Levers and Marshall, 1992; Gallego et al., 1992). It became clear in 1988 that S6 protein kinases were controlled through phosphorylation cascades; Sturgill and Maller showed that a partially purified insulin-stimulated MAP kinase was able to phosphorylate and activate the Rsk-type S6 kinase I1 (Sturgill et al., 1988). Other groups confirmed this observation (Gregory et ad., 1989; Ahn and Krebs, 1990; Kyriakis and Avruch, 1990; Chung et al., 1991). The term MAP kinase is presently used to refer to at least two distinct proteins, known as the extracellular signal-regulated protein kinases ERK 1 (p44 MAP kinase) (Boulton et al., 1990) and ERK2 (p42 MAP kinase) (Boulton et al., 1991b), and has also been used to refer to other protein kinases [e.g., p54 MAP kinase (Kyriakis and Avruch, 1990)l. Following activation by phosphorylation on threonine and tyrosine, ERK 1 and ERK2 phosphorylate the sequence S/TP within a protein or peptide substrate. Other kinases, such as p54 MAP kinase, also undergo dual phosphorylation (Kyriakis et al., 1991) and have been shown to phosphorylate similar sites (Kyriakis and Avruch, 1990). Because these kinases are tyrosine phos-

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MAP KINASES ERKI AND ERK2: PLEIOTROPIC ENZYMES IN A UBIQ UITOUS SIGNALING NETWORK David J. Robbins, Erzhen Zhen, Mangeng Cheng, Shuichan Xu, Douglas Ebert, and Melanie H. Cobb University of Texas Southwestern Medical Center, Department of Pharmacology, Dallas, Texas 75235

I. Introduction A. Distribution of MAP Kinases B. Purification and Cloning of ERKs 11. Homology of MAP Kinases with Proteins in the Yeast Mating Pathway 111. Regulation of MAP Kinases A. Regulation of ERKl and ERK2 by Phosphorylation B. Regulation of Recombinant MAP Kinases in Vztro IV. Identification and Purification of MEK A. Isolation of cDNAs Encoding MEKs B. Regulation of MEK C. Raf, Mos, and MEK Kinase Phosphorylate and Activate MEK V. Ras and Heterotrimeric C Proteins Regulate the ERK Network VI. Protein-Protein Interactions That Regulate the MAP Kinase Cascade A. Ras-Raf B. Connection of Ras to Receptors VII. MAP Kinases Phosphorylate Upstream Components of the Cascade VIII. Substrates of ERKl and ERK2 A. Primary Sequence Specificity B. Sites of Possible Physiological Consequence IX. ERKl and ERK:! Are Essential Regulators of Cell Function References

I. Introduction Receptor-mediated signal transduction can be divided into four discrete steps: (1) transmission of the signal across the plasma membrane, (2) amplification of the signal on the cytoplasmic side of the membrane, (3) activation of downstream effector molecules, and (4) production of the biological response. One approach to determine mechanisms by which cells respond to environmental cues is to identify an assayable biochemical event activated by the signal of interest and then detect, purify, and reconstitute in vitro the molecules responsible for the activation (Krebs, 1993). One such biochemical event, whose study has led to the identification of a protein kinase cascade, is the phosphorylation of 93 ADVANCES IN CAN(:ER RESEARCH. VOL. 63

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ribosomal protein S6 (Gressner and Wool, 1974). Extracellular cues, including insulin and growth factors, cause the phosphorylation of S6 on serine residues, although most of the receptors for these extracellular ligands have intrinsic tyrosine kinase activity o r signal by activating an associated tyrosine kinase. Enhanced S6 phosphorylation may come about by the activation of two different S6 protein kinase families (Jones et al., 1988; Kozma et al., 1990; Bannerjee et al., 1990; Novak-Hofer and Thomas, 1985). Both are regulated by phosphorylation on serine/ threonine residues. It has been suggested that the 70K, single-catalyticdomain S6 kinases may be the enzymes responsible for S6 phosphorylation in intact cells (Chung et al., 1992), whereas the two-catalytic-domain, 90K S6 kinases, also called Rsks, phosphorylate S6 fortuitously. S6 phosphorylation is believed to contribute to discrimination among messenger RNAs for translation. However, the issue of whether both, neither, o r only one of the known types of S6 kinase phosphorylate S6 in intact cells awaits a detailed understanding of the function of the event. Experiments from several laboratories suggest that the 90K and 70K S6 kinases lie on separate signaling pathways (Ballou et al., 1991; Mukhopadhyay et al., 1992). The 90K, two-catalytic-domain S6 kinases are now known to be controlled by a protein kinase cascade that involves the sequential activation of at least four serine/threonine, or dual-specificity, protein kinases. Receptors activate this cascade via GTP binding proteins, either the protooncogene product Ras or certain heterotrimeric G proteins, including the Gi class (Anderson et al., 1991a; Robbins et al., 1992; Wood et al., 1992; Thomas et al., 1992; Gupta et al., 1992; Kahan et al., 1992; Levers and Marshall, 1992; Gallego et al., 1992). It became clear in 1988 that S6 protein kinases were controlled through phosphorylation cascades; Sturgill and Maller showed that a partially purified insulin-stimulated MAP kinase was able to phosphorylate and activate the Rsk-type S6 kinase I1 (Sturgill et al., 1988). Other groups confirmed this observation (Gregory et ad., 1989; Ahn and Krebs, 1990; Kyriakis and Avruch, 1990; Chung et al., 1991). The term MAP kinase is presently used to refer to at least two distinct proteins, known as the extracellular signal-regulated protein kinases ERK 1 (p44 MAP kinase) (Boulton et al., 1990) and ERK2 (p42 MAP kinase) (Boulton et al., 1991b), and has also been used to refer to other protein kinases [e.g., p54 MAP kinase (Kyriakis and Avruch, 1990)l. Following activation by phosphorylation on threonine and tyrosine, ERK 1 and ERK2 phosphorylate the sequence S/TP within a protein or peptide substrate. Other kinases, such as p54 MAP kinase, also undergo dual phosphorylation (Kyriakis et al., 1991) and have been shown to phosphorylate similar sites (Kyriakis and Avruch, 1990). Because these kinases are tyrosine phos-

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Pheromone

FIG. 1 . Components of the pheromone response pathway.

phorylated, they have been recognized widely as stimulus-dependent tyrosine phosphoproteins. Our present understanding of the participants of the network leading to ERK activation is presented in Fig. 1. This review will focus on the two MAP kinases, ERKl and ERK2. T h e MAP kinases (sometimes called MAP2 kinases) were originally named for their ability to phosphorylate the exogenous substrate microtuble-associated protein-2 (Ray and Sturgill, 1987). They were subsequently renamed mitogen-activated protein kinases (Rossomando et al., 1989), although they are also activated by numerous nonmitogenic stimuli. T h e MAP kinase ERK2 was the first to be described as a rapidly stimulated insulin-sensitive serine/threonine protein kinase activity in 3T3-LI preadipocytes by Ray and Sturgill (1987). This was recognized independently in the laboratories of Krebs (Cicirelli et al., 1988; Pelech et al., 1988) and of Nishida (Hoshi et al., 1988). Krebs’ group discovered ERK2 as a myeline basic protein (MBP) kinase activity in both Xenopus oocytes undergoing maturation and in the sea star following germinal vesicle breakdown. Hoshi et al. (1988) found it as a MAP2 kinase activity in extracts from mammalian cells stimulated with phorbol esters and epidermal, fibroblast, and platelet-derived growth factors. Factors that promote differentiation as well as those that lead to proliferation activate MAP kinases and have been discussed in previous reviews (Cobb et al., 1991). A. DISTRIBUTION OF MAP KINASES Based on Northern and Western analyses, either ERKl or ERK2, o r both, are found in all tissues and cell lines (Boulton et al., 1991b). Their

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subcellular distributions are less certain and vary with cell type and system. Both are found in the cytoplasm, in nuclei, and associated with the cytoskeleton (Boulton and Cobb, 1991; Loeb et al., 1992; Chen eta!., 1992; Seth et al., 1992). Both have been reported to translocate to nuclei following cellular stimulation. In PC 12 cells, ERK2 moves to the nucleus in response to nerve growth factor (NGF) but not epidermal growth factor (EGF) (Traverse et al., 1992). Extracellular signals may also cause one or both to become associated with plasma membranes (Loeb et al., 1992). ERKl but not ERK2 binds to the activated NGF receptor (Loeb et al., 1992). As yet uncharacterized MAP kinase isoforms are likely to account for activity and immunoreactivity found in some of these compartments.

B. PURIFICATION A N D CLONING OF ERKs Several groups independently purified MAP kinases to homogeneity and obtained sequence. ERKl was purified as a MAP2 kinase from insulin-stimulated Rat 1 HIRc B cells (Boulton et al., 1991a),and was also purified from vanadate-treated KB cells as an EGF receptor peptide kinase activity (Northwood et al., 1991) shortly thereafter. An enzyme with similar properties was isolated from sea star (Sangherd et al., 1990). T h e Xenopzls ERK2 homologue was first purified from oocytes (Gotoh et al., 1991b); ERKP had been partially purified earlier from 3T3 cells (Ray and Sturgill, 1988b). Using degenerate oligonucleotides designed to tryptic peptides from highly purified ERK1, Boulton et al. succeeded in cloning the rat forms of ERKl (1990), ERK2, and a related enzyme, ERKS (199 1b). The cloned enzymes were called extracellular signalregulated protein kinases to reflect the diversity of signals known to activate them and because Southern analysis indicated that the first three cloned were from a larger family of related protein kinases. Others have subsequently been identified: ERK4, a 45K protein, was recognized by its differential reactivity with a panel of ERK-peptide antibodies (Boulton and Cobb, 1991), and p57 was identified as an enzyme tyrosine phosphorylated in response to fibroblast growth factor (Lee et al., 1993). MAP kinase clones have now been isolated from many organisms, including mice, humans, cows, frogs, fruit flies, and plants (Her et al., 1991; Gonzalez et al., 1992; Gotoh et al., 1991a; Biggs and Zipursky, 1992; Duerr et al., 1993). Within their catalytic domains, ERKl and ERKS are 90% identical to each other, with ERK3 approximately 50% identical to either ERKl o r ERK2. ERKS is categorized as a member of the ERK subfamily based on characteristic features of its sequence. Among these three protein kina-

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ses and their yeast relatives (see below), there are clusters of similarity that distinguish the ERK family from other types of protein kinases (Boulton et al., 199lb). In particular, subdomains [as defined by alignment of the available protein kinase sequences (Hanks et al., 1988)l V, VII, IX, and XI are highly conserved between ERKl and ERK3. For example, ERK3 subdomain V is 83% identical and subdomain IX is 72% identical to ERK1. cdc2, the mammalian protein kinase most similar to ERKs, is only 40% identical to ERKl in subdomain IX, whereas protein kinase A is only 25% identical. Second, the inserts between conserved subdomains are of similar lengths among the ERKs. Other kinase groups d o not share this property. Very little is known about the biochemical properties and functions of ERK3. We find that it has protein kinase activity with very narrow specificity and that it is found predominantly in the nucleus. Its manner of regulation appears to be similar to that of ERKl and ERK2.

II. Homology of MAP Kinases with Proteins in the Yeast Mating Pathway Yeast protein kinases, including KSSl (Courchesne et al., 1989), and FUSS (Elion et al., 1990) from Saccharomyes cerevisiae and spkl (Toda et al., 1991) from Schizosaccharomyces pombe, are the enzymes with greatest sequence identity to the MAP kinases, with nearly 55% sequence identity to ERKl and ERK2. ERK2 partially complements an spkl gene disruption, as can KSSl and FUSS (Neiman et al., 1993). These enzymes are essential for mating in yeast. The finding that ERKs were related in sequence to yeast kinases accelerated progress in discovering how these enzymes are regulated by extracellular cues, because information about yeast signaling pathways could be exploited. Genetic techniques have been used to identify protein kinases and other components required for this and related signaling pathways in yeast. Knowledge of these pathways has led to the identification of components of the mammalian system as well. Therefore, we include a brief discussion of the pheromone response pathway here (Sprague and Thorner, 1993; Marsh et al., 1991; Kurjan, 1992). Binding of pheromone to receptors on haploid yeast causes cells of the opposite mating type to stick to each other, arrest in G, of the cell cycle, and fuse. T h e newly formed diploid cells can proceed through mitosis or undergo meiosis to form four spores. Hartwell (1980) isolated a series of mutants, called sterile (STE) mutants, defective in this pathway. Based on genetic criteria some of the components have been ordered (Fig. 1). T h e pheromone receptors, STE2 (for the pheromone

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a-factor) and STE3 (for a-factor), are believed to span the membrane seven times and couple to heterotrimeric G proteins. STE4 and STE18 encode the G protein p and y subunits, whereas GPAl encodes the a subunit. Genetic evidence indicates that the p and y subunits are the signal transducers, and the a subunit mediates pheromone desensitization. T h e effector for the G protein remains unknown. It has been speculated that STE20, a newly identified serine/threonine protein kinase, may fill this role, because genetically it acts upstream of other characterized components of this yeast signaling pathway (Leberer et al., 1992; Ramer and Davis, 1993). Other proteins whose contributions to this pathway have been ordered genetically are STE5, STEl 1 and STE7 (protein kinases), FUS3 (an ERK homologue), and STE 12 (a transcription factor). STE5 may form complexes with other proteins in this cascade, including STE 11, STE7, FUS3, and STE12 (Elion et al., 1993). Its interaction with FUS3 and STE 12 has been demonstrated using the two-hybrid system. Therefore, it has been proposed that it serves as a docking protein for a signal transduction complex. Interestingly, this protein has limited sequence similarity to FAR1, particularly in a Cys-rich region common to the proteins. FARl is a pheromone-inducible protein that inhibits the function of G, cyclins and is phosphorylated by FUS3 (Peter et al., 1993). Overexpression of FARl bypasses the requirement for the kinase FUSS in GI arrest, suggesting that FUS3 may contribute to G, arrest by phosphorylating FARl to increase its binding to cyclins. Like STE5, FARl associates with FUS3 using the two-hybrid system (Elion et al., 1993). Several proteins upstream in this cascade may be substrates for FUSS, including STE5, STEl 1, and STE7. Mammalian homologues of STE5 and STE20 have not been identified. Mammalian homologues of the protein kinases STE 1 1 and STE7 have been found, as discussed further below. An analogous pathway in the fission yeast S. pombe has many similar elements. The mating pheromones are also believed to transduce signals through G-protein-coupled receptors to a series of three protein kinases, byr2 (an S T E l l homologue), byrl (an STE7 homologue), and spkl (a MAP kinase homologue) (Neiman et al., 1993). One unique feature of the S. pombe pathway is that the small G protein Ras is believed to lie upstream of byr2 but downstream of the heterotrimeric G protein. Thus, S. cerevisiae has a heterotrimeric G-protein-dependent, Ras-independent signaling pathway, whereas S. pombe has an analogous heterotrimeric G-protein-dependent and Ras-dependent pathway, suggesting that multiple mechanisms have evolved to regulate this type of protein kinase

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cascade. Additional analogous cascades have also been found in yeast (Irie et al., 1993; Brewster et al., 1993).

111. Regulation of MAP Kinases A. REGULATION OF ERKl AND ERK2 BY PHOSPHORYLATION MAP kinases require phosphorylation on both tyrosine and threonine for maximum enzymatic activity (Fig. 2). Serinehhreonine-selective phosphatases, such as phosphatase 2A, tyrosine-specific phosphatases, such as CD45, and dual-specificity phosphatases dephosphorylate and inactivate ERKl and ERK2 (Anderson et al., 1990; Ahn et al., 1991; Boulton and Cobb, 1991; Charles et al., 1993; Alessi et al., 1993). Following exhaustive dephosphorylation by any one of these phosphatases, kinase activity is reduced to 2% or less of the original specific activity. Thus, there are three low-activity forms of each kinase: enzymes containing no phosphate, only a tyrosine phosphate, or only a threonine phosphate (see below). There is only one high-activity form of ERKl and ERK2, the doubly phosphorylated form. In cells labeled with 32P, phosphorylation of ERKl and ERK2 occurs on tyrosine, threonine, and serine (Ray and Sturgill, 1988a; Robbins and

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Autop hosphorylation Serine/Threonine

lnactlvation by Phosphatases FIG. 2. Regulation of the MAP kinases ERKl and ERK2 by phosphorylation.

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Cobb, 1992). Serine phosphorylation is insensitive to stimulation of cells. Tyrosine phosphorylation in response to stimuli precedes threonine phosphorylation (Robbins and Cobb, 1992; Haystead et al., 1992), but the latter best correlates with increased kinase activity. Two-dimensional maps of tryptic phosphopeptides from ERKl and ERK2 isolated from labeled cells reveal the same three phosphopeptides in both enzymes regardless of cellular stimulus (Robbins and Cobb, 1992). Two of these contain only phosphotyrosine, whereas the third contains both phosphotyrosine and phosphothreonine. These same phosphopeptides are detected in ERKs phosphorylated in vitro (see below). Analysis by mass spectroscopy indicated that one of the peptides containing only tyrosine phosphate had the same sequence as the doubly phosphorylated peptide (R. Seger, D. Robbins, N. Ahn, K. Walsh, M. Cobb, and E. Krebs, unpublished). Payne et al. (1991) identified two sites, T183 and Y185, phosphorylated in active ERK2 isolated from mouse T cells stimulated with phorbol ester. These t w o sites lie between subdomains VII and VIII in ERKs and account for the tyrosine and threonine phosphorylated peptide found in peptide maps. These sites are highly conserved between ERK homologues and are the sites of activating phosphorylation in ERKl and ERK2 from all species examined. B. REGULATION OF RECOMBINANT MAP KINASESIN VITRO

Mutations of ERKl and ERK2 have been made that remove the two activating phosphorylation sites (Posada and Cooper, 1992; Robbins et al., 1993; L'Allemain et al., 1992). Wild-type and mutant proteins produced in bacteria have been phosphorylated in vitro with MAP kinase kinase, which will be referred to here as MEK (MAP kinase/ERK kinase). Properties of MEK are discussed below. Mutants of ERKl and ERK2 lacking either of the activating phosphorylation sites are not activated by MEK in vitro. Mutations lacking both phosphorylation sites, Y185 and T183 of ERK2 (and the comparable sites, Y204 and T202 of ERKl), are not substrates of MEK. Phosphorylation of the wild-type kinases by MEK causes a 1000-fold increase in activity of the two ERKs u p to the specific activity of ERK 1 purified from insulin-stimulated cells (Robbins et al., 1993). MEK will only phosphorylate native ERKs; neither denatured ERKs nor peptides from ERKs containing the MEK phospho_ are recognized. T h e rylation sites (e.g., IADPDHDHTGFLTEYVATR) peptides are neither substrates nor inhibitors of MEK. Thus, the tertiary structures of ERKs are important for recognition by MEK. Wild-type ERKl and ERK2 expressed in Escherichia coli react with

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antibodies to phosphotyrosine (Seger et al., 1991; Wu et al., 1991; Crews et al., 1991). The purified recombinant proteins autophosphorylated in uitro contain phosphate predominantly on tyrosine, and also on threonine and serine. Peptide mapping and mutagenesis showed that the activating sites of tyrosine phosphorylation (ERK1 Y204 and ERK2 Y185) were the major sites of autophosphorylation (Wu et al., 1991; Rossomando et al., 1992). A small fraction of autophosphorylated ERK2 molecules also contain phosphate on the site of activating threonine phosphorylation. The self-catalyzed phosphorylation reaction is intramolecular (unimolecular) at least for the predominant phosphoamino acid (Wu et al., 1991; Rossomando et al., 1992). However, autophosphorylation is inefficient; only a small fraction of the molecules are phosphorylated. It was believed that autophosphorylation could have regulatory significance based primarily on four lines of evidence: (1) recombinant ERK2 had low MBP kinase activity that increased with time of autophosphorylation, implying a low rate of autoactivation (Seger et al., 1991); (2) autophosphorylation occurred on the physiologically relevant tyrosine and threonine (Robbins and Cobb, 1992); (3) ERKl purified in the active form from stimulated cells and then dephosphorylated with CD45 became partly reactivated on incubation with MgATP (Ahn et a!., 1991); and (4) MEK required the native ERK structure to activate ERKs. All of these observations suggest that ERKs might contribute catalytically to their own activation. Various laboratories tested the ability of catalytically impaired ERKs to become phosphorylated by MEKs to resolve this question (LAllemain et al., 1992; Posada and Cooper, 1992; Robbins et al., 1993; Seger et al., 1992a; Nakielny et al., 1992). T h e lysine in protein kinase subdomain I1 has been mutated. In ERKl this results in over a 100-fold loss of catalytic activity, whereas in ERK2 this mutation only reduces activity to -5% of the wild-type enzyme (Robbins et al., 1993). Nevertheless, whether such mutant proteins were incubated with MEK in uitro o r were placed into oocytes or mammalian cells, they became phosphorylated on threonine and tyrosine following stimulation of cells. For example, Cooper’s laboratory demonstrated that Xp42, a Xenopus ERK homologue, was phosphorylated on threonine and tyrosine following progesterone-induced meiotic maturation in Xenopus oocytes (Posada and Cooper, 1992). Based on a protein band shift assay, the phosphorylation states of wild-type Xp42 and the inactive I1 mutant were similar. Additionally, they showed that the regulatory tyrosine or threonine could be phosphorylated independently of each other. The aggregate of these in vitro and intact cell studies indicates that (1) MEK is the physiological regulator of MAP kinases, (2) phosphorylation on two sites by MEK is sufficient to activate

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ERKs to their maximum specific activities, thus, no other protein kinases are required, and (3) autophosphorylation is not required for activation of ERKs. MAP kinases in the pheromone response pathway are regulated like their mammalian counterparts. Ballard et al. (1991) have described a 40-kDa protein that becomes tyrosine phosphorylated in response to pheromone. This is probably FUSS, because a FUSS knockout strain that is mating competent is devoid of this pheromone-induced tyrosinephosphorylated band. Gartner et al., (1992) have shown more directly that FUSS (and KSS 1) become rapidly phosphorylated after pheromone treatment on T I 8 0 and Y 182, the analogous sites phosphorylated in mammalian ERKs. Mutants of either FUS3 o r KSSl lacking the phosphorylation sites resulted in loss of function in viva STE7 (a MEK homologue) phosphorylates FUSS on the physiologically relevant tyrosine and threonine (Errede et al., 1993). STE7 has also been shown to be multiply phosphorylated in response to either pheromone or constitutively active S T E l l (Stevenson et al., 1992; Cairns et al., 1992). Signal propagation requires an active protein kinase catalytic domain, because an inactive STE7 mutant is defective in mating. Pheromone-induced phosphorylation of STE7 requires FUSS/KSSl, as well as S T E l l . T h e reason for this interdependence of STE7 and FUS3/KSS1 is unknown.

IV. Identification and Purification of MEK The kinase that activates MAP kinases was discovered by Ahn and Krebs by fractionating extracts from EGF-stimulated and untreated Swiss 3T3 cells on Mono Q to identify kinases that would activate other kinases (Ahn et al., 1988, 1991). They divided the fractions into pools to create an 8 x 8 mixing matrix, incubated the mixed fractions under phosphorylating conditions, and rechromatographed the products, assaying for increased kinase activity. Using this strategy, they identified two fractions that activated ERKl and ERK2. Activation of ERKs was reversible by either tyrosine-specific or serine/threonine-specific phosphatases; activation correlated with tyrosine and threonine phosphorylation of ERKs 1 and 2 on the physiologically relevant sites. They originally called these activities MAP kinase activator and subsequently MAP kinase kinase (or MKK). Other terms in the literature include MAP kinase/ERK kinase, or MEK, which will be used in this review. Krebs’ laboratory purified two differentially migrating forms of MEK activity (Ahn et al., 1991; Seger el al., 1992a). T h e relationship between the two pools of activity is unknown, but they are likely to be the products of closely related genes (see below). The two preparations each

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contained one major silver-stainable protein band, one of 45 kDa and the other of 46 kDa, on SDS-PAGE. ERKl and ERK2 were excellent substrates, but few other proteins were phosphorylated. The next best substrate for these enzymes, out of a screen of 21 potential kinase substrates, was MBP, for which the specific activity was less than 1% of that for ERK2. T h e enzymes were shown to be dual-specificity protein kinases by kinase assays performed on the purified proteins eluted from denaturing gels. Several other groups also characterized and purified MEKs (Gomez and Cohen, 1991; L'Allemain et al., 1992; Nakielny et al., 1992; Adams and Parker, 1992; Kosako et al., 1992; Matsuda et al., 1992; Crews and Erikson, 1992; Alessandrini et al., 1992; Wu et al., 1992). Nishida's group purified a 45-kDa Xenopw MEK approximately 1300-fold from progesterone-treated oocytes. They demonstrated that microinjection of purified MEK into oocytes was sufficient to stimulate ERK activity (Matsuda et al., 1992). Using an antibody, they showed that MEK was ubiquitously expressed but enriched in brain and spleen. Crews and Erickson (1992) partially purified two differentially migrating forms of MEK from TPA treated T cell hybridomas. Cohen and co-workers, in conjunction with the groups of Sturgill (Nakielny et al., 1992) and of Haystead (Wu et al., 1992), isolated MEK from rabbit muscle and demonstrated that it was similar to enzyme from stimulated cells. T h e muscle preparation phosphorylated ERK2 on T183 and Y 185, as determined by Edman degradation. They also showed that phosphorylation by MEK increases MAP kinase activity comparable to that purified from an insulin-stimulated source. OF cDNAs ENCODING MEKs A. ISOLATION

A number of groups were able to sequence peptides from preparations of MEK (Kosako et al., 1992; Crews and Erikson, 1992; Wu et al., 1992; Seger et al., 1992b), which led to the isolation of cDNA clones. Eriksons' group isolated their clone based on a polymerase chain reaction (PCR) strategy using degenerate oligonucleotides made to tryptic peptide sequence (Crews et al., 1992). Their murine transcript was most highly expressed in the brain and had an open reading frame of 393 amino acids (43.5 kDa). To verify that their clone had MEK activity, they expressed it in E. coli as a GST fusion protein and demonstrated tyrosine and threonine phosphorylation of ERK2. Seger et al. (1992b) cloned two cDNAs from a human T cell cDNA library, MKKla, which was 43.4 kDa, and MKKlb, which was 40.7 kDa. MKKlb is believed to be an alternatively spliced form of MKKla. T h e

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MKKl transcript was detectable in all human tissues and cell lines exaniined, and enriched in skeletal muscle. Surprisingly, there was little of the transcript in brain, although the MEK activity in neuronal tissue is high (Ahn el al., 1992). Immunoblotting using the Xenopus MEK antibody detects large amounts of MEK in neuronal tissue as well (Kosako et al., 1992). Transient expression of MKKla in COS cells was associated with an increase in TPA-stimulated activity of 1.5- to 3-fold over that in mocktransfected cells. MEK has also been cloned by other groups (Wu et al., 1993; Ashworth el al., 1992). An additional mammalian MEK clone has been isolated that is -85% identical to the MEK clone isolated earlier (Zheng and Guan, 1993). These enzymes are now referred to as MEKl and MEK2. Based on the number of MEK-like proteins in yeast and on MEK activities found in mammalian systems, additional MEK clones will undoubtedly be found that may have a range of substrate specificities. MEK sequences, whether from Xenopus oocytes (Kosako et al., 1992), a human cell line (Crews and Erikson, 1992), or rabbit muscle (Wu et al., 1992), are similar to STE7 and byrl, kinases from the pheromone response pathway. In some regions the identity with the yeast enzymes is 60-70%, although overall identity is closer to 40% (Seth et al., 1992).

B. REGULATION OF MEK Shortly after its initial characterization, MEK was shown to be inactivated by serinelthreonine-specific but not tyrosine-specific phosphatases (N. G. Ahn, unpublished data; Gbmez and Cohen, 1991). A Xenopus MEK immunoprecipitated from "P-labeled oocytes following progesterone-induced maturation was phosphorylated primarily on threonine but to a smaller extent on serine residues. MEKs are capable of autophosphorylating, although to relatively low molar ratios of phosphate. This is true of the enzyme purified from Xenopus (Kosako et al., 1992), human (Seger et al., 1992a), and murine sources (Crews and Erikson, 1992; Crews et d., 1992), as well as recombinant protein. Autophosphorylation occurs on serine, threonine, and tyrosine residues. C. RAF,Mos, A N D MEK KINASE PHOSPHORYLATE A N D ACTIVATE MEK

Because MEK is regulated by phosphorylation, MEK kinases have been sought. Several groups have found that the protooncogene product Raf functions as a MEK kinase (Howe et al., 1992; Kyriakis et al.,

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1992; Dent et al., 1992). Raf is a serine/threonine protein kinase, with an N-terminal regulatory domain that is truncated in certain constitutively active oncogenic forms. Kyriakis and Avruch first demonstrated that both ERK and MEK activity were constitutively activated in v-Raftransformed NIH-3T3 cells (Kyriakis et al., 1992). Partially purified MEK deactivated with phosphatase 2A was reactivated, more than 30fold, by an immunoprecipitated, constitutively activated c-Raf-1 mutant. Dent et al. (1992) showed that active Raf mutants partially purified over one gel filtration column reactivated partially purified dephosphorylated MEK to approximately 70% of its initial activity. Immunoprecipitates of Raf also reactivate dephosphorylated MEK. Experiments with highly purified enzyme preparations have not yet been performed, but the data are sufficient to conclude that Raf is an important MEK kinase. Johnson and co-workers have suggested that mechanisms of ERK activation may be yet more complex. They showed that the effects of four different oncogene products, v-Raf, v-Ras, v-Src, and gip2, on activation of both MEK and ERK activity were cell type dependent (Gupta et al., 1992; Gallego et al., 1992). For instance, v-Src and gip2 activated ERK and MEK activity in Rat 1A cells but not in NIH-3T3 cells, whereas v-Raf and v-Ras activated these enzymes in NIH-3T3 cells but not in Rat 1A cells. Wood et al. (1992) also gathered data of this type. Deducing that there is a higher eukaryotic homologue of the yeast protein kinases STEl 1 and byr2, Lange-Carter et al. (1993) used PCR with degenerate oligonucleotides designed from the yeast kinases to isolate the mammalian enzyme, which they called MEK kinase (MEKK). The catalytic core o f MEKK is -60% identical to that of STE7 or byr2. When transiently expressed in Cos cells, it activates MEKl or MEK2. Finally, work from Posada et al. (1993) and Nebreda and Hunt (1993) has demonstrated that Mos may also, like Raf and MEKK, act as a MEK kinase. Thus, three distinct protein kinases appear to activate MEKs. Perhaps this complexity contributes to differences in the abilities of various extracellular stimuli to activate the MAP kinase cascade and to elicit distinct responses.

V. Ras and Heterotrimeric G Proteins Regulate the ERK Network Ras has been implicated in the actions of tyrosine kinases based on work from a number of laboratories. Feig and Cooper (1988) described a Ras mutation (S 17N) that had the ability, when overexpressed, to act with a dominant-negative phenotype. When this Ras mutant was overex-

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pressed in PC 12 cells it prevented NGF-dependent neurite outgrowth, presumably by competing with endogenous Ras for an upstream guanine nucleotide exchange protein (SzeberCnyi et al., 1990). Such cell lines have now been used to demonstrate that NGF-stimulated ERK activation is Ras dependent. Robbins et al. (1992) showed that three sequential steps in a NGF-stimulated protein kinase cascade-MEK activity, ERK activity, and S6 kinase activity-were blocked in this dominant-negative Ras-containing cell line. ERKs could be activated in these cell lines by aluminum fluoride. T h e mechanism of action of aluminum fluoride is most likely through activation of a heterotrimeric G-protein-dependent pathway, apparently in a Ras-independent fashion. Nori et al. (1992) also provide evidence for this bifurcated model of ERK activation. They showed that overexpression of GAP in 3T3 cells blocked TPA-induced ERK activation, presumably by maintaining Ras in its GDP-bound inactive form. GAP overexpression, however, did not block ERK activation by aluminum fluoride, once again presumably because of activation of a heterotrimeric G-protein-dependent pathway. Both Wood et al. (1992) and Thomas et al. (1992) showed that a similar dominant-negative Ras mutant, overexpressed from a dexamethasone-inducible promoter, blocked NGF- and TPA-stimulated ERK activation in PC12 cells. They also demonstrated that constitutively active oncogenic Ras was able to activate ERKs. Wood et al. further demonstrated that activation of 90K S6 kinases, and kinases causing Raf phosphorylation by NGF, were also blocked by overexpression of the S17N Ras mutant (Wood et al., 1992). Xenopus ERK homologues have also been shown to be activated by oncogenic mutants of Ras. Microinjection of oncogenic Ras into Xenopus oocytes activated ERKs, as well as S6 kinase I1 (Pomerance et al., 1993). Shibuya et al. (1992) and others have shown that addition of oncogenic Ras to cell-free extracts leads to ERK activation. Farnesylated forms seem to be more effective in these assays (Shibuya et al., 1992; Itoh et al., 1993).

VI. Protein-Protein Interactions That Regulate the MAP Kinase Cascade A. RAS-RAF

Ras appears to link to this cascade via direct interaction with Raf (X. Zhang et al., 1993; Warne et al., 1993; Vojtek et at., 1993). The interaction is believed to be dependent on GTP and may involve a large complex composed of several protein kinases, including Raf and MEK (Moodie et al., 1993). The two-hybrid system has also been used by Wigler and coworkers (Van Aelst et al., 1993), Avruch and colleagues (X. Zhang et al.,

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1993), and Cooper and associates (Vojtek et al., 1993) to demonstrate Ras-Raf association. The N-terminal regulatory domain of Raf alone is sufficient for the interaction. Mutations of Ras that reduce its transforming potential also impair its interaction with Raf. Activation of purified Raf by Ras-GTP has not yet been demonstrated, suggesting that additional components or modifications may be required. B. CONNECTION OF RASTO RECEPTORS

SH2 domains, stretches of about 100 amino acids found in many signaling molecules, bind to tyrosine phosphate-containing proteins with varying specificities (Pawson and Gish, 1992). SH3 domains, roughly 50 residues, are another protein interaction domain. Some molecules with SH2 or SH3 domains have intrinsic enzymatic activity. Others, often with multiple SH2 and/or SH3 domains, serve as bivalent or multivalent adapters or docking proteins that mediate interactions among signaling molecules. One such adapter protein, GRB2, contains one SH2 and two SH3 domains. It links tyrosine-phosphorylated receptors via its SH2 domain with the Ras-GTP/GDP exchange factor SOS, which binds to its SH3 domains (Rozakis-Adcock et al., 1992). Another SH2-containing protein, SHC, has been cloned based on conserved sequences within SH2 domains, which may also contribute to these interactions (RozakisAdcock et al., 1992). A variation on this theme exists for the insulin receptor. One of the earliest consequences of insulin receptor activation is tyrosine phosphorylation of a protein known as insulin receptor substrate 1 (IRS-1). Phosphorylated tyrosines on IRS-1, rather than the insulin receptor itself, form the majority of recognition sites for proteins containing SH2 domains (Sun et al., 1991; Skolnik et al., 1993).

VII. MAP Kinases Phosphorylate Upstream Components of the Cascade Like FUS3 in the pheromone response pathway, MAP kinases may exert negative feedback or some other type of regulatory impact on upstream components of their cascade. ERKl and ERK2 have been reported to phosphorylate Raf- 1 on physiologically relevant sites (Anderson et al., 1991b; Lee et al., 1992). However, no enzymatic activation of Raf was detected. MAP kinases phosphorylated MEK (Matsuda et al., 1993). T h e major peaks of soluble MEK phosphorylating activity that can be measured in fractionated cell extracts are ERKl and ERK2 (D. J. Robbins, unpublished data). Phosphorylation by ERKs does not stimulate MEK activity; other functional effects have not been measured.

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ERKs also phosphorylate certain receptors, such as the EGF receptor (see below). How these phosphorylations of upstream components influence their regulatory properties is not known. VIII. Substrates of ERKI and ERK2

A. PRIMARY SEQUENCE SPECIFICITY A comparison of the primary sequences of ERK substrates has led to a proposed consensus sequence of PXS/TP, where X is a neutral or basic amino acid (Gonzalez e l al., 1991). Peptide substrates may also contain two residues between the P at n - 2 and the phosphorylation site (PXXS/TP). In known protein substrates, however, no sites have yet been found of the PXXS/TP type. Further, some sites do not contain P at the n - 2 site (Haycock et al., 1992). Thus, P at the n + 1 residue (S/TP) is the minimum primary sequence determinant for ERK phosphorylation (Table I). Conformational constraints clearly play a major part in interaction of potential substrates with ERKs. Major insights into this specificity come from the crystal structure of ERK2, which has recently been solved (F. Zhang et al., 1993). Large side chains in the peptide binding pocket decrease space that can be occupied by substrates. It is of interest that the consensus phosphorylation site of cdc2 protein kinase is similar to that of ERKs in that both can phosphorylate proteins with S/TP in the right environment (Shenoy et ul., 1989). Although a few common substrates have been identified (e.g., tau), not all proteins with this sequence are substrates for both o r either (Kamijo et al., 1992). TABLE I SEQUENCES OF MAP KINASE PHOSPHORYLATION Srres Substrate

Sequence

Residue

Ref.

MBP EGF receptor PLA, Rsk, rabbit MAPKAP-2 Tyrosine hydroxylase, rat Tyrosine hydroxylase, human c-Myc c-Jim c-Elk Tall

RRN IVTPRTPPPSQCKGR

(T97)

SYPLSPLSD-

(S.505)

Erickson rt (11. (1990) Northwood el n1. (1991) Lin rt (11. ( 1 993) Sutherland et (11. (19%) Stokoe rt r d . (1992)

(T669) RRELVEPLTPSGEA

ENGLLMTPCYTANF VPQTPLH'TSR -

-

KQAEAVTSPR

Haycock el nl. (1!W2)

EAIMSPRFK LPPT~PSRR TPPL~PIDME

(S3 1 ) (S62) (S243)

IAPRSPAKL MVQLSPPAL -

(SSSU) (5122)

Haycock et nl. (1992) Alvarez et (11. (1991) Alvarez el nl. (1991) H. Gille (unpublished) Cheng et (11. (1993)

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B. SITESOF POSSIBLE PHYSIOLOGICAL CONSEQUENCE The first MAP kinase phosphorylation site to be sequenced was from MBP (Erickson et al., 1990) (Table 1). High molecular weight MAPS,such as MAP2, are also thought to be ERK substrates in vivo (Tsao et al., 1990; Gotoh et al., 1991b; Sat0 et al., 1988; Shiina et al., 1992). Although no phosphorylation site sequences have been published, MAP2 is phosphorylated on both threonine and serine (Hoshi et al., 1988). Another, low molecular weight, MAP that is phosphorylated by ERKs is tau (Drewes et al., 1992; Drechsel et al., 1992). Tau is phosphorylated by ERKs on multiple sites. Following phosphorylation tau has one-tenth the ability to bind to microtubules, and has a decreased ability to promote microtubule assembly (Drechsel et al., 1992). Additionally, Drewes et al. (1992) have found that phosphorylation of tau by ERKs, on multiple sites, transforms tau into an Alzheimer-like state. ERKs have been reported to phosphorylate at least six other protein kinases on physiologically relevant sites, activating two of them (Sturgill et al., 1988; Chunget al., 1991; Gregory et al., 1989; Stokoeet al., 1992),inhibiting one of them (Northwood and Davis, 1990; Northwood et al., 199 1 ; Takishima et al., 1991),and having unknown effects on the last three (Lee et al., 1992; Anderson et al., 1991b; Mukhopadhyay et al., 1992; Robbins et al., unpublished observations). Rsk-type S6 kinases have been shown to be activated by ERKs, accompanied by phosphorylation of some physiologically relevant sites, both serine and threonine (Sturgill et al., 1988; Chung et al., 1991; Gregory et al., 1989; Sutherland et al., 1993). Additionally, ERKs have been shown to phosphorylate recombinant 70K S6 kinase on serine/threonine residues within its putative autoinhibitory domain. Some tryptic phosphopeptides phosphorylated by ERK 1 and ERK2 comigrated with tryptic phosphopeptides of the 70K kinase isolated from insulin-treated cells. However, ERKs were unable to activate the 70K S6 kinase (Mukhopadhyay et al., 1992). Stokoe et al. (1992) have recently purified a novel insulin-stimulated rabbit muscle protein kinase they termed MAPKAP kinase-2. MAPKAP kinase-2, when active, phosphorylates glycogen synthase. Once dephosphorylated, MAPKAP kinase-2 can be rephosphorylated and activated by ERKs. This phosphorylation occurs on the first threonine of the peptide VPQIPLHTSR (Stokes et al., 1992). As noted above two groups have demonstrated that Raf-1 and MEK are phosphorylated by ERKs (Anderson et al., 1991b; Lee et al., 1992). ERKl and ERK2 phosphorylate T669 of the EGF receptor (Northwood and Davis, 1990; Northwood et al., 1991; Takishima et al., 1991); a peptide based on this site is a very good substrate in vitro. T h e physiological consequences of this phosphorylation remain controver-

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sial, although, like the phosphorylation of Raf- 1, the phosphorylation of T699 by ERKs has been suggested to involve a feedback loop. Several DNA-binding proteins are phosphorylated by ERK 1 and ERK2 (Gille et al., 1992; Seth et al., 1992; Alvarez et al., 1991; Pulverer et al., 1991; Cheng et al., 1993). Phosphorylation of p62-ternary complex factor, which appears to be identical to c-Elk, by ERKl and ERK2 leads to tight binding of a ternary complex factor/serum response factor complex and subsequent transcriptional activation of thefos promoter (Gille et al., 1992). Phosphorylation occurs on four or five sites in vztro, several of which have been sequenced (H. Gille, unpublished data). c-Myc, c-Jun, Tal-1, and El2 are also phosphorylated by ERKs on physiologically relevant sites. c-Myc is phosphorylated on a serine, in the peptide TPPLSPS, important for transcriptional activation (Alvarez et al., 1991; Seth-et al., 1992). In vitro c-Jun is phosphorylated by ERKs on two to five sites with preferential phosphorylation of two to three residues on the C terminus followed by phosphorylation of two residues within the N terminus. The sites phosphorylated in vivo by these enzymes remain controversial (Alvarez et al., 1991; Pulverer et al., 1991). Cytosolic phospholipase A, (PLA,), which causes hormone-dependent release of arachidonic acid in many cell types, is regulated by growth factors (Lin et al., 1989). This is believed to occur via serine phosphorylation, and has been shown to correlate with enzymatic activation. Serine 505 of PLA, is phosphorylated by ERKs in vitro (Drewes et al., 1992; Lin et al., 1993) within the consensus site PLSP. The identification of cPLA, as a potential in vivo substrate implies that ERKs may lie at a pivotal point in a pathway to control numerous potential second messengers derived from arachidonic acid. This discussion of proteins that may be regulated by ERKs points to the pleiotropic nature of signaling pathways regulated by this family of protein kinases (Fig. 3). Relatively few data are yet in hand to prove that any of these proteins are substrates for these enzymes in vivo. Nevertheless, the impact of these enzymes on cell function can be deduced from recent experiments using mutated enzymes. The ERKl phosphorylation site and lysine mutants have proved useful dominant inhibitors. In Jurkat cells (C. Whitehurst and T. Geppert, unpublished data) the mutants inhibit induction of the cytokine IL-2. Frost et al. (1993) find that ERKl and ERK2 mutants block the ability of Ras, serum, and phorbol ester to induce transcription from a TPA response element. Further, Sontag et al. (1993) find that ERK2 mutants prevent proliferation caused by activated Raf EGF or small tau antigen.

ERKl

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KINASES

J. Protein kinases

:gt* MAPKAP Kinase-2

EGF R

J.

Cytoskeletai Elements

-

90 Kinases K S6

/j

Tau

111

Transcription Factors

PiA2

. y c ps2TCF T u n

HighMW

MAPS

FIG.3. ERKl and ERK2 are pleiotropic regulators of cell function.

IX. ERKl and ERK2 Are Essential Regulators of Cell Function I n summary, the ubiquitous MAP kinases are activated by a remarkable variety of hormones in differentiated cells and growth factors in dividing cells. Their activation has been linked to the transition from Go to G, in the cell cycle and to the induction of differentiated phenotypes. These enzymes are essential components of a universal protein kinase cascade implicated in the control of many cellular processes. Subversion of this cascade can lead to and is critical for cellular transformation and oncogenesis. Among enzymes in this cascade, ERKl and ERK2 have the broadest known specificity and the most diverse substrates, phosphorylating numerous transcription factors, membrane proteins, cytoskeletal elements, as well as other protein kinases. Thus, the MAP kinases may be considered the primary disseminators of the regulatory functions of this pathway. ACKNOWLEDGMENTS We thank Tom Geppert ( U T Southwestern), Natalie Ahn (University of Colorado at Boulder), Michael Wigler (Cold Spring Harbor), Jim Feramisco (University of California at San Diego), and Gary Johnson (National Jewish Hospital) for valuable discussions, and Jo

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Hicks f o r preparation of the manuscript. Work from the authors’ laboratory was supported by a grant from the Texas Advanced Research Program, a grant from theJuvenile Diabetes Foundation, National Institutes of Health Research Grant DK34128 and Kesearch Career Development Award DK01918 (to MHC), National Institutes of Health Training Grant GM07062 (to DJR), and Merck training fellowship (to DE).

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HOW DNA VIRUSES PERTURB FUNCTIONAL MHC EXPRESSION TO ALTER IMMUNE RECOGNITION Grant McFadden* and Kevin Kanet ‘Department of Biochemistry and tlmmunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7

I. MHC Expression and Immune Recognition of Viral Antigens by T Cells A. Introduction B. Development of the T Cell Repertoire C. Roles of T Cell Subsets D. MHC Restriction of Viral Antigen Recognition E. Class I and I1 MHC Molecules F. Structure and Diversity of T Cell Antigens That Bind MHC Molecules G. T Cell Recognition of Viral Peptide-MHC Complexes H. Viral Antigen Processing and Presentation: Class I Pathway 1. Assembly of MHC-I Heavy Chain-P,M-Peptide Complexes J. ABC Transporters and MHC-I Assembly with Endogenous Peptide K. Exceptions to the Rule: “Exogenous” Viral Protein Entry into the MHC-I Pathway L. Viral Antigen Processing and Presentation: Class I1 Pathway M. Biosynthesis and Endosomal Targeting of Class 11 Molecules N. Binding of Peptide Antigens to Class I1 Molecules 0. Other Candidate Molecules That May Affect MHC-11-Restricted Antigen Processing and Peptide Binding P. Exceptions to the Rule: “Endogenous” Viral Protein Entry into the MHC-I1 Pathway Q. Regulation of MHC Transcription R. Cytokine Regulation of MHC Gene Expression S. DNA Viruses That Modulate MHC Expression 11. Poxviruses A. Introduction B. Cellular Immune Response to Poxvirus Infection C. Poxvirus Regulation of MHC-I D. Poxvirus Regulation of MHC-I1 E. Possible Mechanisms of MHC Regulation by Poxviruses 111. Adenoviruses A. Introduction B. Cellular Immune Response to Adenovirus Infection C . Adenovirus Regulation of MHC-I D. Adenovirus Regulation of MHC-I1 E. Significance of MHC Regulation by Adenoviruses IV. Herpesviruses: T h e Cytomegalovirus Model A. Introduction B. Cellular Immune Response to CMV Infection

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C. CMV Regulation o f MHC-I D. CMV Regulation of MHC-I1 E. Possible Mechanisms of MHC Regulation by CMV V. Hepatitis B Virus A. Introduction B. Cellular Immune Response to HBV Infection C. HBV Regulation of MHC-I D. HBV Regulation of MHC-I1 E. Possible Mechanisnis of MHC Regulation by HBV VI. Papillomavirus A. Introduction B. Cellular Immune Response to PV Infection C. PV Regulation of MHC-I D. PV Regulation of MHC-11 E. Possible Mechanisms of MHC Regulation by PV VII. Conclusions References

I. MHC Expression and Immune Recognition of Viral Antigens by T Cells A. INTRODUCTION T h e response to infection by viruses involves the participation of multiple components of the vertebrate immune system. T h e acquired immune system includes both humoral and cell-mediated responses, in the form of antibody production and the activation of lymphocytes that recognize particular viral antigens. Cellular immunity is especially effective against cells harboring active virus replication, and is critical for the elimination of ongoing infections, regression of virus-specific tumors, and reducing or preventing the reactivation of persistent viruses (for more detailed reviews, see McChesney and Oldstone, 198’7; Bangham and McMichael, 1989; McMichael et al., 1989; Oldstone, 1989, 1991; Melief and Kast, 1990, 1992; Whitton and Oldstone, 1990; Randall and Souberbielle, 1990; Maudsley and Pound, 1991; McMichael, 1992a; Murray and McMichael, 1992; Yewdell and Bennink, 1992). The cellular immune system becomes sensitized to a virus infection only after viral proteins are degraded to short linear peptide epitopes that become complexed with class I or I1 major histocompatibility complex (MHC) proteins, and the resulting complexes are transported to the cell surface, where they are presented as “nonself” entities to T lymphocytes. If the viral antigen has not been previously seen by the T cell repertoire of the host, the initial antigen-specific activation event is generally believed to require the appearance of MHC-peptide complexes on professional antigen-presenting cells (APCs), but if activated T

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cells that have been previously sensitized to the viral epitope(s) are available, then a broader class of somatic cells, especially for class I-restricted responses, becomes competent for presentation of viral antigens and can be targeted for lysis or clearance by cytotoxic T cells (CTLs). In either event, the ability to discriminate self molecules from foreign viral epitopes is dependent on the presentation of the nonself peptide to T cells within specific peptide-binding grooves of the presenting MHC molecules. In this review we focus on how gene products encoded by DNA viruses can alter MHC expression and function so as to perturb antigen recognition and thus potentially provide selective advantage for the virus. In general, viruses can affect MHC expression by either elevating surface levels to generate inappropriate immune response, especially in cases where class I1 levels are normally low, or lower surface levels to reduce T cell recognition. Reduction of the number of functional class I molecules at the surface can be accomplished by (1) decreasing constitutive expression levels of MHC or related genes required for antigen presentation, (2) blocking some aspect of the intracellular processing/presentation pathway, o r (3) inhibiting the extent of MHC upregulation by inducing signals such as cytokines. Because the subject of immune subversion by proteins encoded by DNA viruses is relatively new (Gooding, 1992), this review will also summarize recent developments in MHC expression and antigen presentation to suggest other potential targets for viral intervention that might prove to be fruitful avenues of inquiry for future studies in virus-induced immune dysfunction. OF THE T CELLREPERTOIRE B. DEVELOPMENT

Thymus-derived (T) cells are intimately involved in self/nonself discrimination. This distinction is critical for elimination of foreign invading pathogens, including viruses, bacteria, and protozoa, while at the same time sparing host tissues from destruction. The identity of molecules that are recognized as self is largely determined in the thymus through positive and negative selection events (von Boehmer, 1986, 1988). Immature thymocytes undergo germ-line gene rearrangements of variable gene segments of the T cell receptor (TCR), resulting in the expression of a/@or y/6 heterodimers in association with several additional subunits responsible for transmembrane signaling by this TCR complex (Weiss, 1993). Thymocytes bearing TCRs with low affinity for self proteins encountered in the thymic environment are apparently positively selected for survival, whereas those with high affinity for self are eliminated by apoptotic mechanisms (von Boehmer et al., 1989;

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Jenkinson et al., 1989). Only a very small percentage of thymocytes that enter the thymus survive selection events to exit the thymus as mature T cells for destinations in peripheral lymphoid organs (von Boehmer, 1986). MHC-encoded class I and class I1 molecules expressed on thymic epithelium and stromal elements are likely to be responsible for the presentation of self proteins to developing thymocytes (Sprent et al., 1988). Peptide fragments of self proteins derived from intracellular proteolytic events are bound and presented by the unique collection of MHC molecules expressed by each individual and constitute the self repertoire of epitopes for recognition by these particular thymocytes. Not only does this process eliminate unwanted and potentially deleterious T cells with high affinity for self, it also skews the T cell repertoire toward preferential responsiveness to antigens presented by that individual’s (self) MHC molecules (Janeway, 1988; NikoliC-ZugiC and Bevan, 1990). At an intermediate stage of the thymic maturation process the thymocytes express two cell surface glycoprotein coreceptor molecules, termed CD4 and CD8, simultaneously, in addition to the TCR. T h e CD4 and CD8 molecules can bind the same MHC-peptide complex as the TCR, albeit at distinct sites, and coreceptor engagement by CD4 o r CD8 can be essential for maturation of thymocytes (Ingold et al., 1991; Killeen et al., 1992), as well as responsiveness of mature peripheral T cells (Connolly et al., 1990).Double-positive thymocytes (expressing both CD4 and CD8) subsequently mature into single-positive thymocytes expressing CD4 or CD8. Whether positive selection occurs at the doublepositive o r the later single-positive stage has not been firmly established (von Boehmer and Kisielow, 1993). It is understood, however, that single-positive cells are end-stage thymocytes, of which at least some will exit the thymus to take u p positions in peripheral lymphoid tissues and the circulatory system. C. ROLESOF T CELLSUBSETS

Peripheral T cells are readily divided into two subsets by the mutually exclusive expression of CD4 or CD8 (Julius et al., 1993; O’Rourke and Mescher, 1993). Historically, CD4 and CD8 subsets were identified by the original functional activities attributed to each of them. In general, CD4+ T cells were found to augment antibody production by B lymphocytes or expansion of CD8+ cells, whereas CD8+ T cells were shown to function directly as cytotoxic T lymphocytes. However, more recently it has been demonstrated that at least some CD4+ T cells are just as efficient as CD8+ cells at lysing antigen-bearing cells, and the effector func-

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tions (i.e., helping versus killing) of these two classes of ‘rcells may well be even more overlapping in viva This blurring of functional operations of the two kinds of T cell, however, should not obscure the fact that each T cell class uniquely recognizes only one type of presented antigen: CD4+ T cells recognize only antigens expressed on class I1 MHC (MHC11) molecules and CD8+ T cells recognize only antigens expressed on MHC-I molecules (Swain, 1983). The primary effector function of CD4+ T cells remains the recruitment and expansion of antibody-producing B cells and other T cells through secretion of antigen-induced cytokines, which have responsive cell targets bearing cognate cytokine receptors, mostly within the immune system. It has only recently been appreciated how important CD4+ T cells are in orchestrating the type and degree of immune response, particularly to what extent humoral or cell-mediated responses are propagated and balanced in response to a particular pathogen challenge (Mosmann and Coffman, 1989). Antigen-specific CD4+ T helper (TH) cells can be subdivided into at least two subsets that secrete largely exclusive sets of cytokines. T h e T H l subset usually secretes interleukin-2 (IL-2), interferon-? (IFN-?), and tumor necrosis factor alpha (TNFa), cytokines capable of propagating a strong cell-mediated response (Mosmann and Coffman, 1989). In contrast, the T H 2 subset secretes cytokines capable of sustaining inflammatory humoral responses, including IL-3, -4,- 5 , -6, and -10 (Mosmann and Coffman, 1989; Moore et al., 1993). Development of CD4+ T helper cell patterns in response to viral antigen, either in the TH1 or T H 2 direction, is poorly understood and may be determined, at least in part, by the invading virus (Moore et al., 1993; Clerici and Shearer, 1993). The CD8+ T cell subset as a group is quite efficient at cell contactdependent destruction of antigen-expressing target cells (McMichael, 1992b). For instance, antigen-specific CD8+ T cells, primed by a previous virus challenge, can detect and lyse infected cells at early stages in a virus infection, often before expression of complete virions. This has apparent benefits for the elimination of infected cells that may act as reservoirs for intracellular pathogens and is likely to be the reason that CD8+ T cells are essential for the clearance of certain viral infections (Wells et ul., 1981; Cerny et al., 1986; Moskophides et al., 1987). Some CD8+ T cells also secrete a number of cytokines on engagement with infected antigen-presenting cells, including IL-2, IFN-y, and TNFa: i.e., a cytokine profile similar to CD4+ T H 1 T cells. T h e activities of CD8+ T cells are therefore not likely to be limited to cytotoxicity but to a constellation of responses collectively aimed toward the elimination of intracellular pathogens (Martz and Howell, 1989).

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It is important to again emphasize at this point the fundamental functional differences between CD4+ and CD8+ T cells. Although the effector functions of CD4+ and CD8+ T cells overlap to some degree, the primary role of CD8+ T cells is to curtail the replication of intracellular parasites, including viruses, during an ongoing infection. It is likely for this reason that self class I MHC molecules responsible for presentation of peptide fragments of viral proteins to CD8+ T cells are expressed constitutively on most somatic cell types. In this way, intracellular viral pathogens would, at least in the absence of viral countermeasures, be subject to immune detection, because almost all infected cell types, in theory, could be identified and eliminated. In contrast, the major function of CD4+ T cells is to enhance humoral immune responses and to amplify CD8+ responses indirectly through production of T H l-type cytokines. Consistent with this function, class I1 MHC molecules that present antigen CD4+ T cells are expressed constitutively on a limited number of cell types, largely restricted to the immune system, particularly macrophages, dendritic cells, and antibody-producing B cells. Finally, while monitoring the intracellular environment for invading pathogens is the razson d’2tre of CD8+ T cells, surveillance of foreign antigens found in the extracellular environment is the purview of CD4+ T cells, through recognition of such antigens after antigen uptake and presentation by class II-bearing cells of the immune system.

D. MHC RESTRICTIONOF VIRAL RECOGNITION ANTIGEN T cell recognition of antigen is fundamentally different from that by B cells. Whereas B cells are capable of binding soluble intact virus antigens through surface immunoglobulin (Ig) receptors, T cells must recognize fragments of viral proteins displayed on the surface of APCs. Zinkernagel and Doherty (1974) made the critical observation that antigen-specific recognition and lysis of virally infected cells by CD8+ T cells requires that the target cells and killer T cells be of the same MHC haplotype. Similar requirements were also found for CD4+ T cell responsiveness to viral antigens (Rosenthal and Shevach, 1973; Shevach and Rosenthal, 1973; Katz et al., 1973). This phenomenon is termed MHC restriction and is a consequence of thymic maturation and selection events that result in the emergence of a T cell population that collectively has the potential to be responsive to a wide spectrum of nonself antigenic epitopes presented in conjunction with self MHC molecules. The MHC maps to a region of chromosome 17 in mice and chromo-

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some 6 in humans, termed the H-2 and HLA regions, respectively (Milner and Campbell, 1992; DeMars and Spies, 1992). T h e MHC-encoded genes are highly polymorphic and were originally characterized by their influence on tissue transplant survival or rejection (Clark, 1983). T h e mouse H-2 encodes two o r three class I genes, designated K, D, and L (in some cases), whereas the human HLA contains three class I loci, designated A, B, and C (MHC-I). Genes encoding subunits of mouse class 11, o r Ia molecules (MHC-11), are clustered in the I region between the K and D regions, whereas genes for human DR, DQ, and DS MHC-I1 molecules are dispersed in a region upstream of the MHC-I loci (Trowsdale et al., 199 1). Genes encoding additional MHC-like molecules exhibiting limited polymorphism are encoded in the MHC regions; however, such “nonclassical” MHC molecules are likely to play a limited role, if any, in viral antigen presentation due to their limited polymorphism (Stroynowski, 1990). Given the existence of multiple alleles at each class I and class I1 locus, and significant recombination having occurred in the MHC, an outbred population can offer diverse combinations of MHC alleles that may therefore optimize antigen presentation, perhaps serving to preserve the species, although not necessarily the individual, during selective pressure by a pathogen.

E. CLASSI

AND

I1 MHC MOLECULES

Class I MHC molecules consist of two common subunits; a polymorphic 45-kDa heavy chain glycoprotein that is noncovalently associated with a conserved 12-kDa P,-microglobulin (P2M) light chain (Bjorkman and Parham, 1990; Pease et al., 1991). T h e class I heterodimer is expressed as a transmembrane complex at the cell surface with three N-terminal heavy chain domains called a-1, a-2, and 01-3, extending outward from the membrane. The heavy chain-P2M complex bound with its antigenic peptide is anchored by a single transmembrane segment on the heavy chain that is followed by a short cytoplasmic sequence of variable length. The membrane proximal external domain, a-3, folds in a manner similar to that of an immunoglobulin domain and has extensive contact with the P2M light chain. T h e structures of several class I molecules as determined by X-ray crystallography reveal that the two most N-terminal domains fold as a unit to form a prominent groove on the top face of the molecule (Bjorkman et al., 1987; Garrett et ad., 1989; Madden et al., 1991). Two parallel a helices and eight antiparallel P sheets comprise the walls and base of the groove, respectively. T h e groove was found to be of dimensions appropriate to accommodate

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short peptides. Recent structural data obtained from class I molecules crystallized bound with single defined viral peptides have provided a clear view of each viral peptide in the groove (Fremont et al., 1992). These studies revealed the importance of allele-specific pockets within the groove in accommodating tightly bound peptides and suggest how polymorphic residues in class I molecules and viral peptide secondary structure may influence the specificity of peptide binding (Gairin and Oldstone, 1993). Polymorphic residues of the heavy chain are clustered in or near the groove and some appear to alter the size, shape, and predicted biochemical properties of pockets within the groove (Garrett et al., 1989). Class I1 molecules are expressed at the cell surface as transmembrane heterodimers composed of a 29- to 33-kDa a chain and a 24- to 29-kDa p chain (Robinson and Kindt, 1989; Gorga, 1992). Each MHC-I1 chain has two external domains, a-I,a-2 for the a chain and p-1, p-2 for the p chain. As determined by X-ray crystallography, the N-terminal a-1 and p-1 domains of the class I1 subunits are predicted to fold in a manner analogous to the a-1 and a-2 domains of class I and form a groove similar in overall structure to that observed on class I, with the notable exception that unlike class I, the ends of the class I1 groove are open, i.e., not occluded by amino acid side chains (Brown et al., 1993).This distinction may influence the type of peptides bound in the class I1 groove (discussed later). F. STRUCTURE A N D DIVEHSITY OF T CELLANTIGENS THAT BINDMHC MOLECULES

It has been known for some time that T cells do not recognize whole protein antigens, but rather peptide fragments derived from protein antigens. This was first demonstrated for CD4+ T helper cells when trypsin- o r cyanogen bromide (CNBR)-generated peptide fragments, but not intact protein antigen, incubated with metabolically inactivated or giutaraldehyde-fixed MHC-matched APCs, could stimulate T cells (Unanue, 1989). Townsend and co-workers demonstrated that this principle was also relevant to CD8+ T cell recognition because short synthetic peptides corresponding to segments of viral antigen sensitized targets for antigen-specific class I MHC-restricted CTL lysis (Townsend et al., 1986; reviewed in Townsend and Bodmer, 1989). Furthermore, expression of transfected minigenes encoding short segments of viral proteins also served to sensitize target cells for lysis, indicating that expression of full-length viral protein is not necessary for expression of viral epitopes for T cell recognition (Sweetser et al., 1988, 1989).

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Analysis of peptides generated from processing events within the cell and presented by MHC molecules was aided by the discovery that naturally processed MHC-bound peptides could be eluted away from purified MHC molecules or live cells under acidic conditions and separated on the basis of size (<5 kDa) (Buus et al., 1988; Falk et al., 1990). T h e isolated peptides can be purified and, when incubated with appropriate APCs, will trigger T cell responses (Fdlk et al., 1990; Rotzschke et al., 1991). Biochemical analysis of class I MHC-eluted peptides has revealed that they are of remarkably uniform lengths, usually consisting of either eight or nine residues, depending on the class I allele product or allomorph from which they were isolated (Rotzschke and Falk, 1991). Peptides isolated from a given MHC-I allomorph often share residues at one or more of the same positions within the peptide sequence. These “consensus motifs” appear to reflect allele specificity of peptide binding. Peptides eluted from class I1 MHC proteins are more heterogeneous in both length, usually 12- 17 residues, and composition compared to class I-bound peptides (Rudensky et al., 1991). These differences may be due in part to the open groove ends that may allow for the observed accommodation of larger peptides (Rudensky et al., 1991). Also, the proteolytic machinery that generates peptides for the class I and I1 pathways functions in distinct cellular compartments and may operate with different specificity constraints. For any particular class I or class I1 allele product, it has been found that 10-20 peptides derived from constitutively expressed “housekeeping” proteins, including histones and ribosomal protein subunits, for instance, can occupy 50% or more of the available peptide-binding capacity of the expressed MHC molecules (Jardetsky et ul., 1991; Rudensky et al., 1991). Diversity within the peptide pool bound to a given allele product, however, may include hundreds to low thousands of unique peptides as estimated by tandem mass spectrometry analysis of class 11 IAd-bound peptides (Hunt et al., 1992). It is not readily apparent how processed peptides from foreign proteins can compete for MHC binding in the presence of the constitutively expressed self peptides described above. Recent studies of the intracellular processing and transport events controlling preparation and association of peptides with MHC molecules suggest that the availability of self and foreign peptides for MHC binding is usually limiting (Germain and Hendrix, 1991; Lie et al., 1991). Consistent with this conclusion is the observation that a percentage of MHC molecules expressed at the cell surface arrive there unoccupied with bound peptide (Schumacher et al., 1990). Viral protein fragments may therefore associate with MHC molecules under the prevailing circumstances in vivo, where MHC occupancy does not

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appear to be saturated. Furthermore, the available pool of nascent peptides in infected cells may be skewed in favor of viral epitopes, because many viruses successfully outcompete cellular needs for the translational machinery. Van Bleek and Nathenson (1990) took advantage ofjust such a situation to identify the immunodominant vesicular stomatitis virus (VSV) peptide bound and presented by the murine MHC-I K b molecule to VSV-specific cytotoxic T cells. The Kb molecules were immunoprecipitated, and the bound peptides were subsequently acid eluted, during a period in the VSV infection when the synthesis of most cellular proteins other than the virus and MHC products was shut down, thus enriching the immunoprecipitated Ktl for the desired peptide.

G. T CELLRECOGNITION OF VIRAL PEPTIDE-MHC COMPLEXES

T cells are very responsive to virally infected cells, yet exquisitely selective in this process (Murray and McMichael, 1992; Yewdell and Bennink, 1992). The efficiency of presentation of viral peptides to T cells is determined by the number of peptide antigen-MHC complexes expressed on APCs. T h e minimum threshold density of specific MHCpeptide antigen complexes that trigger T cell responses is estimated to be perhaps less than 300 (Harding and Unanue, 1990; Demotz et al., 1990; Christinck et al., 1991). This suggests that specific peptide-MHC complexes represented at even very low density on the cell surface, e.g., 1 in 500, may potentially stimulate a T cell response. Activation then leads to up-regulation of cytokine receptors on the responding T cells, secretion of cytokines, and clonotypic expansion of antigen-specific T cells. Although activation of T cells by virally infected cells is absolutely dependent on an antigen-specific interaction between the TCR and peptide-MHC complexes, it is also dependent on the participation of a number of additional receptor-ligand interactions between the T cell and the APC. As mentioned previously, simultaneous coengagement of class I or class I1 by the TCR and CD8 or CD4 coreceptors, respectively, can be essential for induction of responsiveness. In addition to coreceptor function, CD8 and CD4 molecules can engage nonantigen MHC molecules independent of the TCR to augment antigen-specific T cell activation (Goldstein and Mescher, 1986; Doyle and Strominger, 1987; Kane et al., 1989). Furthermore, a growing number of additional accessory molecule interactions, including lymphocyte function-associated antigen (LFA-I) and its cognate intercellular adhesion molecule ligands ICAM-1, -2, and -3, and CD28 and its B7 ligand on APCs, can positively influence T cell responsiveness to antigen (reviewed in van Seventer et

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al., 1991; Schwartz, 1992). Recent studies indicate that the functional activity of a number of participating accessory molecules are rapidly stimulated by minimal TCR engagement, resulting in amplification of the overall avidity of the cell-cell interaction and subsequent T cell transmembrane signaling (Dustin and Springer, 1989; O’Rourke et al., 1990; Schweighoffer and Shaw, 1992). The TCR-dependent amplification of receptor-ligand interactions provides a likely explanation for the surprisingly small number of specific viral peptide-MHC complexes needed to trigger T cell activation.

H. VIRALANTIGEN PROCESSING AND PRESENTATION: CLASSI PATHWAY Both infectious and noninfectious forms of viral antigens can enter the endocytic pathway of a professional APC to be processed and presented, in the context of MHC-I1 molecules, to CD4+ T cells. In contrast, infection with live virus is, except in rare cases, a requirement for induction of class I-restricted CD8+ T cell responses (Morrison et al., 1986; Braciale et al., 1987). This difference results from the distinct intracellular location of processing activities for MHC-I and MHC-I1 antigen presentation. Indeed, the MHC-I and MHC-I1 molecules can be viewed as peptide scavengers that target two different cellular compartments. Viral epitopes presented by class I molecules are almost exclusively derived from the viral proteins synthesized de novo in an infected cell, whereas viral antigens that enter the endosomal/lysosomal compartments have been specifically routed to these compartments, usually after capture from an extracellular location. T h e importance of cytoplasmic localization for entry into the class I antigen-processing and presentation pathway was elegantly approached by Bevan and colleagues (see Moore et al., 1988). They first established a CD8+ T cell line specific for a soluble protein, bovine serum albumin (BSA), by DNA-mediated transfection (normally CD8+ T cells would not be stimulated by a soluble protein present in the extracellular environment). They found that the BSA-specific CD8+ T cells were not responsive to cells incubated with soluble BSA in isotonic medium, but if they manipulated the osmolarity of the medium to allow cytosolic entry of the BSA via osmotic lysis of endosomal membranes, then the intact BSA could be presented by the class I pathway (Moore et al., 1988). Additional agents or manipulations that allow entry into the cytosol provide concomitant entry to the class I antigen presentation pathway (Harding et al., 1991). The cytosolic proteolytic processing enzymes required for the genera-

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tion of class I-presented viral peptide fragments have not been positively identified. However, large 1300-kDa (26s) or 580- to 650-kDa (20s) ATP-dependent cytosolic multicatalytic proteinase complexes having broad proteolytic specificity are likely candidates for this function, because these multicatalytic complexes are the major proteinases responsible for the normal turnover and degradation of cytosolic proteins (Rechsteiner, 1987; Hershko, 1991; Goldberg and Rock, 1992). A large portion of the 26s protease complex is composed of the smaller 20s complex, which is also known as the proteasome. Proteasomes consist of 15-20 proteins ranging from 2 1 to 35 kDa, which, through noncovalent subunit associations, form a cylinder-shaped structure composed of-four stacked rings (Goldberg and Rock, 1992). Proteasomes can exist as free entities in the cytosol and the nucleus and are capable of degrading proteins independent of ubiquitinylation (Goldberg and Rock, 1992; Yang et ul., 1992). Proteasomes can also associate with two additional regulatory subunits (600 and 250 kDa) to form the 26s complex, which can carry out ubiquitin-dependent proteolysis. Proteasomes and the larger ubiquitin-dependent complex are attractive candidates for generating peptides from larger proteins for class I antigen presentation because of their broad specificity, their ability to cleave on the carboxyl side of hydrophobic, basic, o r acidic residues, and the demonstration that proteasomes can process proteins into oligomers within an appropriate length range for class I binding without further degradation to single amino acids (reviewed in Tanaka et al., 1992). The relative contributions of ubiquitin-dependent and independent processing for generation of viral epitopes for class I antigen presentation have not been determined and are currently under investigation. A recent report has demonstrated that ubiquitinylation is essential for the processing of at least one antigen to yield a peptide appropriate for presentation to class I-restricted T cells (Michalek et al., 1993). A connection between the MHC and proteasomes was established by Monaco and McDevitt, when they discovered that alloantisera generated across class I1 region differences immunoprecipitated a subset of 20s proteasomes that they termed low molecular mass polypeptide (LMP) complexes (Monaco and McDevitt, 1982, 1984). It has since been determined that two polymorphic subunits of the LMP complex, LMP-2 and LMP-7, are encoded in the class I1 region of the MHC (Monaco, 1992). Intriguingly, the genes encoding LMP-2 and LMP-7 are up-regulated by IFN-y, similar to the genes encoding class I and class I1 (Monaco, 1992). The coordinated IFN-y up-regulation of class I, class 11, and LMPs has suggested, albeit indirectly, that LMPs have a role in antigen processing or presentation, particularly under circumstances associated with viral

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infections wherein significant levels of IFN-y are produced (Driscoll and Finley, 1992). The MHC-encoded proteasome subunits are presumed not to have catalytic activity, because they d o not have obvious homology to any known proteases. However, Peterson and co-workers have demonstrated that up-regulated expression of MHC LMP-2 and LMP-7 by IFN-y can influence the association of several other subunits within proteasome complexes, resulting in the differential gain or loss of certain subunits distinct from LMP-2 and LMP-7 (Yang et al., 1992; Brown et al., 1993). These studies suggested that MHC-encoded proteasome subunits may influence the composition of proteasome complexes and by this means perhaps alter the proteolytic pattern to favor production of peptides appropriate for class I-restricted antigen presentation. An alternative possibility is that the MHC-encoded subunits may mediate interaction of LMP complexes with other cytosolic or membrane components to facilitate efficient delivery of generated peptides into appropriate compartments for association with class I MHC molecules. Despite teleological arguments for the involvement of MHC-encoded proteasome subunits in the class I antigen-processing pathway, the expression of these subunits has been found not to be essential for the generation of the majority of peptides normally bound to class I molecules, as well as a limited number of peptides presented by class I molecules for T cell recognition (Arnold et al., 1992; Momburg et al., 1992). T h e LMP subunits may have more subtle effects on antigen processing or presentation, effects that could easily have gone undetected in these preliminary analyses. In fact, it has recently been demonstrated that MHC-encoded LMP gene products specifically alter the peptidase activities of the proteasome to favor cleavages that result in peptides possessing basic residues on their C-termini (Driscoll et al., 1993; Gaczynska et al., 1993). Such an alteration may favor the generation of peptides suitable for class I binding since basic C-termini are often necessary to anchor peptide binding in class I grooves. The precise role, if any, for MHC-encoded subunits of proteasomes in antigen processing and presentation must therefore await further investigation, but viral regulation of proteasome function remains a possible target for the manipulation of functional MHC expression. I. ASSEMBLY OF MHC-I HEAVY CHAIN-&MPEPTIDECOMPLEXES

The intracellular association of appropriate octamer or nonamer peptides with class I heavy chains is essential for stable assembly and transport of peptide-loaded class I complexes to the cell surface

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(Tsomides and Eisen, 1991; Brodsky and Guagliardi, 1991; van Bleek and Nathenson, 1992; Braciale, 1992). The principle that peptide is required for stable assembly of class I molecules was deduced from an elegant series of experiments utilizing a number of mutant murine and human cell lines defective in class I surface expression, despite the appropriate intracellular expression of normal levels of class I heavy and light chains (reviewed by Elliott, 1991). It was determined that exogenously applied synthetic peptides corresponding to viral T cell epitopes could induce/stabilize assembly of MHC complexes on the cell surface and in cell extracts of the class I-deficient cell lines (Townsend et al., 1989). Peptides of eight to nine residues in length were found to be essential for high-affinity peptide binding and stabilization of class I heavy chain-P,M association (Cerundolo et al., 1991). Peptides are believed to bind newly synthesized and translocated class I molecules in the endoplasmic reticulum. This conclusion is supported by studies using Brefeldin A, a potent fungal inhibitor of egress from the endoplasmic reticulum (ER) (Lippincott-Schwartz et al., 1989). Brefeldin A is able to block presentation of intracellularly expressed and naturally processed viral antigens but not exogenous peptide antigen presentation, implying that naturally processed peptide does not associate with class I molecules at later stages of class I export through the Golgi apparatus o r at the cell surface (Yewdell and Bennink, 1989; Cox et al., 1990). As would be predicted from this model, newly synthesized class I molecules, lacking extensive glycosylation modifications, and therefore likely to be in the ER o r cis-Golgi compartment, readily bind exogenously supplied peptides in cell extracts as compared to their more mature counterparts, as determined by pulse-chase studies (Townsend el d., 1990). The proper assembly of protein complexes for secretion or cell surface expression can require the involvement of ER-resident chaperonelike molecules (Gething and Sambrook, 1992). T h e p88 molecule, also termed calnexin, is a Ca*+-binding ER-retained protein that rapidly binds free class I heavy chains as they are translocated into the ER (Degen and Williams, 1991). The function of p88 with respect to heavy chain-P*M-peptide trimolecular assembly appears to be to retain the heavy chain in the ER and prevent its denaturation until P2M and peptide have properly associated with the heavy chain (Degen et al., 1992). Once peptide and P2M have stably associated with the heavy chain, the complexes are exported rapidly through the medial and trans-Golgi to the cell surface, while p88 remains in the ER (Degen et al., 1992). It should be noted that numerous other unrelated proteins are also bound

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and transiently retained by p88 in the ER (Wada et al., 1991; Ahluwalia et al., 1992). J. ABC TRANSPORTERS AND MHC-I ASSEMBLY WITH ENDOGENOUS PEPTIDE

If, as it appears, peptide fragments of viral proteins are generated in the cytosol, and peptide binding to class I occurs in the ER, how do such peptides gain access to the lumen of the ER? Again, studies with mutant cell lines deficient in class I surface expression due to posttranslational defects have provided clues as to how peptides may be transported across the ER membrane. The murine RMA-S cell line and a series of human cell lines have a mutated gene or lack this gene, and the human hybrid cell line T 2 has deleted two linked genes; these genes all map close to but are distinct from the LMP-encoding genes in the MHC. Transfection of wild-type genes corresponding to the altered o r deleted genes restores peptide loading of class I molecules in the ER and cell surface expression of class I molecules (Spies et al., 1990, 1992; Powis et al., 1991; Kelly et al., 1992). T h e two genes encode separate highly homologous (77% at the amino acid level) subunits of an ER-localized transmembrane heterodimer with significant homology to the ATPbinding cassette (ABC) family of transmembrane transporters (Deverson et al., 1990; Juranka et al., 1989). A common feature of this family, as reflected in its name, is an ATP-binding site on the carboxy-terminal domain of each subunit of the heterodimer. T h e ABC family contains at least 30 members distributed between prokaryotes and eukaryotes that function in the transport of a variety of solutes, including various ions and sugars, amino acids, peptides, and even proteins larger than 100 kDa (reviewed in Juranka et al., 1989). The genes encoding the two MCH-encoded ABC homologues have been termed Ham-1 and Ham-2 in the mouse, RING4, R I N G l l , or PSF-1 in the human, and mtp-1 and mtp-2 in the rat. The nomenclature for these genes has been simplified by applying the acronyms Tap-1 and Tap-2 (for transporter for antigen presentation) to represent the corresponding genes. T h e predicted lengths of individual Tap-1 and Tap-2 products described to date range from 577 to 808 residues, corresponding to approximately 63-88 kDa (reviewed in Yewdell and Bennink, 1992). T h e putative transporter subunits are also predicted to have multiple membrane-spanning domains (Deverson et al., 1990; Juranka et al., 1989). Genetic reconstitution studies suggest that the putative peptide transporter is likely to function as a heterodimer, because neither wild-

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type subunit on its own is able to facilitate proper loading of class I molecules, yet expression of both wild-type genes restores class I expression. T h e coimmunoprecipitation of Tap-1 and Tap-2 products from cell extracts using antibodies that recognize only one subunit provides strong additional evidence that these two gene products form a heterodimer (Kelly et al., 1992). It must be emphasized at this point that the evidence for peptide transport by the MHC-encoded Tap proteins is indirect. The heterodimer has the ability to provide efficient loading of class I molecules, but it has not been directly demonstrated, however, that the complex actually transports peptides. Levy et al. (1991) have provided evidence using microsomal membranes that peptides can traverse ER-derived membranes independent of Tap-1 and Tap-2. It is not clear to what extent these studies reflect in uiuo peptide translocation and therefore the issue of whether Tap-1 and Tap-2 transport peptides must await further study.

K. EXCEPTIONS TO THE RULE:“EXOGENoUS” V I R A L PROTEINENTRYINTO T H E MHC-1 PATHWAY Our discussion thus far has involved a model in which class I molecules present endogenously synthesized viral proteins available in the cytosol only during active virus replication. There exist exceptions to this basic tenet, however, in which some exogenous protein antigens of certain viruses and as well as certain bacteria (Jin et al., 1988; Barnaba et al., 1990; Pfeifer et al., 1993) can enter the class I pathway and be presented to CD8+ T cells. The class I presentation of DNA viral products falling into this category include certain cytomegalovirus proteins and hepatitis B virus surface antigen, which will be discussed in later sections of this review. L. VIRALANTIGEN PROCESSING A N D PRESENTATION: CLASSI1 PATHWAY

MHC-I1 molecules present peptide fragments derived from exogenous protein ant.igens, including structural components of virus particles or secreted viral proteins, to CD4+ T cells. Exogenous viral antigens are taken u p into endosomal compartments, cleaved into short peptides that associate with class I1 molecules targeted to this compartment, and then the peptide-MHC-I1 complexes are routed to the cell surface for T cell recognition (Unanue, 1992; Brodsky, 1992; Long, 1992). A brief description of the details of this process follows.

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M. BIOSYNTHESIS A N D ENDOSOMAL TARGETING OF CLASSI1 MOLECULES On translocation into the ER, the MHC-11 ci and p chains rapidly associate with one another together with a third, nonpolymorphic o r invariant (Ii) chain (Jones et al., 1979). The Ii chain is a type I1 transmembrane protein, with the amino terminus extending into the cytosol and the C terminus residing in the lumen o f t h e ER (Teyton and Peterson, 1992). The Ii chain has at least four forms that differ in the N-terminal region due to differential translation initiation and/or alternative exon usage (Germain and Margulies, 1993). The MHC-I1 a/@ heterodimer associated with the invariant chain in the ER is relatively unstable and contains an empty peptide-binding site. The Ii chain has been demonstrated to prevent exogenous peptides from binding the associated MHC-I1 (Roche and Cresswell, 1990; Teyton et al., 1990). the ci/p-Ii trimeric complex is thought to play an important role in class I1 biosynthesis by preventing peptides that may be generated in, or transported to, the lumen of the ER from binding MHC-I1 molecules before their transit from the ER. The Ii-MHC-I1 complexes transit through the cis- and niedial-Golgi normally; however, at the trans-Golgi the complexes are routed to endosomal compartments rather than follow the default exocytosis pathway to the cell surface. The Ii chain cytoplasmic domain is responsible for the targeting of MHC-I1 heterodimers to the endocytic compartments (Roche et al., 1992). The MHC-I1 complexes that contain Ii appear to route through early endosomal compartments but apparently accumulate in late endosomes that are distinct from lysosonies, and MHC-I1 molecules can remain there for several hours (Neefjes rt a/., 1990; Romagnoli et al., 1993). N. BINDINGOF PEPTIDEANTIGENS TO CLASSI1 MOLECULES I t was established early on that agents such as chloroquine, which raises the pH of endosomal compartments, and leupeptin, which affects resident acid proteases such as cathepsin B, could inhibit MHC-I1 but not MHC-I antigen processing and presentation (reviewed by Unanue, 1989). Despite intensive investigation, the precise eridocytic compartment in which peptides derived from exogenous proteins bind to MHCI1 molecules has not been determined. What is now evident, however, is that Compartments of reduced pH are required not only for the proteolytic activities needed for the unfolding and partial degradation of

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exogenous antigens but also for the efficient association of peptide antigens with MHC-I1 molecules (Sadegh-Nasseri and Germain, 1991; Reay et al., 1992). Proteolytic cleavage of the associated Ii chain is also necessary to reveal the previously occluded peptide binding site (Roche and Cresswell, 1991). It has been observed that mature MHC-I1 molecules with tightly bound peptide remain associated as noncovalent dimers if the samples are not boiled prior to polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) (Springer et al., 1977). Heating o r low pH results in the loss of bound peptide and an unstable MHC-I1 heterodimer that readily dissociates when analyzed by SDS-PAGE. Using pulse-chase methodologies, Germain and Hendrix ( 1991) demonstrated that newly associated MHC-I1 dimers were SDS-PAGE unstable, and it was not until the Ii chain had dissociated that the MHC-I1 dimer could assume the SDS stable phenotype. Class I1 dimers that dissociate from the Ii chain in endosomal compartments and have not bound peptide may rapidly aggregate and be degraded (Germain and Margulies, 1993). However, increases in the concentration of exogenous antigen result in increases in the percentage of mature class I1 molecules that assume the stable conformation, suggesting that binding of peptides derived from the antigen is responsible for increased dimer stabilization. Binding of peptide to class I1 molecules in endosomal compartments appears, therefore, to have a profound effect on the conformational stability of the MHC-I1 heterodimer. One role of bound peptide may be to ensure that cell surface expression of empty class I1 molecules is minimized, and in this respect peptide pools may regulate class I and class I1 cell surface expression in a similar manner (Rothbard and Gefter, 1991; Sette and Grey, 1992). 0. OTHERCANDIDATE MOLECULESTHATMAY AFFECTMHC-I I-RESTRICTEDANTIGEN PROCESSING A N D PEPTIDE BINDING

A pair of constitutively expressed molecules termed p72/74, which are members of the heat-shock protein (hsp70) family may play a role in antigen processing or loading of processed peptide on MHC-I1 molecules (Lakey et al., 1987; Pierce and De Nagel, 1992). This is an attractive possibility because members of the heat-shock family have been found to have the capacity to bind peptides (Rippmann et al., 1991). One possibility would be that ~ 7 2 1 7 4molecules protect partially degraded proteins in the endosomal compartments from rapid and complete degradation for association with MHC-11. Another possibility is that ~ 7 2 1 7 4

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may scavenge peptides generated in lysosomal compartments and shuttle them to endosomal compartments, where class I1 has been found to accumulate (Romagnoli et al., 1993). Riberdy and Cresswell (1992) have found that the human T 2 cell line that has a deletion in the class I1 region of MHC is not only defective in class I antigen presentation due to loss of Tap-1 and Tap-2, but it is also defective in exogenous antigen presentation by transfected MHC-I1 molecules. T h e implication from these studies is that genes may be encoded in this region of the MHC, which may also be required for MHCI1 antigen processing or presentation. P.

EXCEPTIONS TO THE RULE: “ENDOGENOUS” VIRAL PROTEINENTRYINTO THE MHC-I1 PATHWAY

Not all class II-restricted peptide antigens are derived from exogenous proteins. In fact, there are reports describing the MHC-I1 presentation of cytosolically expressed proteins to CD4+ T cells, for example, measles matrix protein (Jacobson et al., 1989; Malnati et al., 1992). It has not been firmly established how and where any of these antigens associate with MHC-I1 molecules. A Tap dependence of presentation of a cytoplasmic hemagglutinin minigene product suggests that in this case transport into the secretory pathway may be required. This could suggest that the peptide is transported to the cell surface bound to MHC-I and only later becomes accessible to MHC-I1 molecules in the endosome on endocytosis and degradation of MHC-I-peptide complexes, or perhaps the peptide associates directly with MHC-I1 molecules in the ER. Considering the latter possibility, it is unclear at present to what extent MHC-11 molecules, usually associated with the Ii chain, are receptive to peptide binding in the ER. Some cytosolic proteins o r peptides may enter endosomal compartments, either nonspecifically by cytoplasmic vesicle formation and autophagy, or more specifically through the participation of chaperones such as Hsc 70, which has been documented to transport proteins or peptides into lysosomes (Chiang et al., 1989). Once in endosomal compartments, the cytosolically derived antigens would presumably be subject to processing and MHC-I1 peptide loading similar to that of internalized exogenous antigens.

Q.

REGULATION

OF

MHC TRANSCRIPTION

Because MHC levels are believed to be tightly regulated at the level of mRNA synthesis, the cis-acting DNA elements responsible for regulating MHC gene transcription are possible targets for perturbation by

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viruses attempting to evade detection by the immune system. T h e following brief review addresses the known upstream regulatory elements for MHC-I and MHC-I1 genes and pertinent factors that may bind these elements to regulate MHC expression. More detailed information and further references can be found in several recent reviews o n MHC-I gene regulation (David-Watine t t al., 1990; Singer a n d McCuire, 1990; Ting and Baldwin, 1993) and on MHC-I1 gene regulation (Benoit and Mathis, 1990; Glimcher and Kara, 1992; Ting and Baldwin, 1993). 1. MHC-I

MHC-I gene expression is developmentally regulated; MHC-I antigens a r e only significantly detectable during and after the midsomite stage of embryogenesis (Ting an d Baldwin, 1993). Class 1 molecules a re found on nearly all somatic cells of the adult; however, the level of expression is quite variahle in d rent cell types (Daar et al., 1984; Singer and McGuire, 1990). T h e highest expression levels of MH(:-I a r e found on lyniphoid cells (David-Watine 01 al., 1990). In contrast, neurons normally lack class I expression, as d o sperm cells, certain placental cells, and early embryo cells (Daar et al., 1984; Singer and McCuire, 1990). Essentially all adult cells express the &M light chain coordinately with the heavy chain. Class I MHC expression is regulated by cis-acting DNA sequences (promoters and enhancers) upstream from the KNA polymerase 11 start site. Mouse and human MHC-I promoters contain several regions that regulate the expression of these genes through the specific binding of proteins (Shirayoshi et d., 1987; Baldwin and Sharp, 1988; Singh el al., 1988; Kanno et nl., 1989; Israel et al., 1989a,b). Two highly conserved enhancer-like sequences found within promoters of several classical MHC-I genes, termed enhancer A and enhancer B, reside at -209 t o - 160 and -85 to -60 (-121 to - 107, in humans) respectively, from the RN A polymerase start site. Furthermore, a n interferon response sequence (IKS)is immediately adjacent and continues downstream from the beginning o f enhancer A (Ting and Baldwin, 1993). Recent experiments employing in uizio footprinting have confirmed that the original upstream regulatory elements identified in uitro are indeed occupied in actively transcribing cells and are not occupied in brain tissues, for instance, that are negative for MHC-I expression (Dey et al., 1992). T h e enhancer A region is composed of a series of three overlapping palindromes (Israel el al., 1989a). A perfect palindrome (ah) constitutes the core of the A enhancer and an imperfect copy of it (ab’) is slightly upstream. A third (perfect) palindrome (b’d’) partially overlaps the imperfect palindrome and includes the spanning sequence (d‘) that

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connects the first two palindromes. A nuclear factor, termed KBFI, consisting of a dimer composed of two 48-kDa subunits, binds independently to the core perfect palindrome with high affinity and to the imperfect palindrome with reduced affinity. This factor also binds a similar sequence within the promoter of the mouse P2M gene (Israel et al., 1987). Enhancer A binding studies done with cells expressing I class heavy chain and P2M compared to cells that do not express either show a correlation between KBF 1 binding and constitutive expression of both chains (Burke et al., 1989; Zakow and Orr, 1989). KBFl is therefore thought to function as a positive factor in the expression of MHC-I. At least three other nuclear factors, KBF2, H2TF1, and NF-KB are known to bind the enhancer A ab and ab' palindromes (Israel et al., 1989b; Baldwin and Sharp, 1988). KBF2 is a 58-kDa monomer that requires only a half palindrome for binding (Israel et al., 1989b). T h e functional relationship of KBFl and KBF2 has not been established. H2TF1 binds the same nucleotide sequence in enhancer A that KBFl binds, but H2TFl is a distinct molecule of larger molecular mass (1 10 kDa) (Baldwin and Sharp, 1988; Singer and McGuire, 1990). It and KBFl appear to function in a similar fashion, serving as positive factors for MHC-I transcription. T h e nuclear factor KB (NF-KB)binds the enhancer A palindromes with the same affinities as exhibited by KBFI. NF-KB is not bound to enhancer A in all cells expressing MHC-I; instead, its binding is often up-regulated in response to a variety of stimulators, including TNFa and phorbol esters (Lenardo and Baltimore, 1989). If KBFl is lacking in a cell or is significantly reduced, NF-KBcan substitute for KBFl to promote MHC-I transcription (Ting and Baldwin, 1993). A fourth nuclear factor, termed AP2, is a ubiquitous DNAbinding protein that binds the overlapping/connecting b'd' palindrome, and in so doing, is likely to prevent KBFl from binding to the imperfect palindrome, ab'. T h e simultaneous binding of AP2 and KBFl is believed to be responsible for maintaining the basal transcription activity of enhancer A (Israel et al., 1989a). Further upstream, toward the 5' boundary of enhancer A, is a binding site for a factor termed H-2 region I1 binding protein (H-2RIIBP), or RXRb (Hamada et al., 1989). T h e H-2RIIBP is a member o f a nuclear hormone receptor family that heterodimerizes with thyroid hormone and retinoic acid receptors (Marks et al., 1992). T h e heterodimerization results in enhanced H-2RIIBP DNA binding to the upstream enhancer A element (region II), which in turn enhances the effects of positive downstream enhancer A interactions, leading to increases in MHC-I gene expression (Shirayoshi et al., 1987; Israel et al., 1989a).

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The MHC-I genes can also be transcriptionally regulated by cyclic AMP (CAMP),usually in a positive manner (Israel et al., 1989a), although it has been reported to regulate MHC-I expression negatively in some cell types (Saji et al., 1992). There are two DNA-binding sites for CREBATF-like factors, one located within enhancer B and one at the 5’ border of enhancer A (Roesler et al., 1988).Both of these sites are responsive to cAMP and up-regulate MHC expression (Roesler et al., 1988; Israel et al., 1989a). A third mechanism of positive regulation by cAMP that is observed can be accounted for by the increased binding of AP2 to b’d’ (Israel et al., 1989a). It has been reported recently by McGuire and co-workers (1992) that gene silencer as well as additional enhancerlsilencer elements may exist much further upstream than the enhancer A elements, mapping at -489 to -395 and -769 to -690. In addition, the authors found evidence for factors binding to the silencer and enhancerlsilencer sequences (McGuire et al., 1992). The detailed functions of these elements and their binding factors must await further investigation. It is apparent from these studies that MHC-I expression is probably regulated by a diverse array of positive and negative signals resulting largely from multiple DNA-protein interactions in the promoter region, and possibly additional regulatory protein-protein interactions not discussed here that may, for instance, restrict the availability of nuclear binding factors such as NF-KB(Beg et al., 1992). It could be argued that the system offers several potential targets for viral intervention and manipulation of MHC-I expression; however, it should be noted that the redundant and overlapping nature of MHC-I gene regulation may in fact provide sufficient flexibility to avoid simple strategies for viral manipulation at the level of individual transcription factors. 2. MHC-I1

In contrast to the ubiquitous expression patterns of MHC-I, class I1 molecules are constitutively expressed only on specialized APCs, including B cells, macrophages, dendritic cells, and thymic epithelium (Benoit and Mathis, 1990), at levels that are dependent on the cell’s differentiation and activation states (Glimcher and Kara, 1992). Class I1 molecules can also be found on a variety of other cell types as well (Klareskog and Forsum, 1986); however, such expression is mostly cytokine dependent (Momburg et al., 1986a,b; Pober et al., 1983). The level of MHC-I1 expression is generally controlled at the level of MHC-I1 gene transcription (Glimcher and Kara, 1992). T h e MHC-I1 promoter-proximal region is located between - 180 and the RNA poly-

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merase I1 start site and is intimately involved in regulating class I1 gene expression. Several conserved cis-acting regulatory elements have been identified in this region (reviewed in Saito et al., 1983; Kelly and Trowsdale, 1985; Schopfer et al., 1987; Glimcher and Kara, 1992). Two of these are short sequences of 14 and 10 base pairs (bp), and are termed X and Y boxes, respectively. The X and Y boxes are essential for promoter activity as determined by deletional and segment replacement analyses (Koch et al., 1988; Dedrick and Jones, 1990; Tsang et al., 1990). Analyses of human and mouse MHC-I1 promoters indicate that the highly conserved X and Y boxes are also essential sequences for IFN and T N F up-regulated expression (Glimcher and Kara, 1992). The X and Y boxes are separated from one another by a stretch of 19-20 bp. This linker segment is variable in sequence, but conserved in length, between class I1 alleles. T h e activities of the X and Y boxes appear to be linked (Benoit and Mathis, 1990) and this may be responsible for the conserved spacing of the X and Y boxes. T h e X box has been further subdivided since the discovery of an additional regulatory site termed X2 (Liou et al., 1990). T h e X2 consists of a sequence that partially overlaps with the 3' end of the X box and includes the 5' end of a spacer between the X and Y boxes (Glimcher and Kara, 1992). T h e X2 sequence has homology to CAMPand TPA regulatory elements (Liou et al., 1988, 1990; Kara et al., 1990; Ivashkiv et al., 1990). T h e core of the X box is now referred to as X1 (Kara and Glimcher, 1991). The Y box is composed of a 10-bp sequence that includes a reverse CCAAT sequence (Glimcher and Kara, 1992). Another sequence of approximately 7 bp in length that resides 15-17 bp upstream of the X box, termed the S box, is also thought to play an important role in positively regulating MHC-I1 gene expression. Mutations altering the S box result in a significant reduction in promoter activity (Koch et al., 1988; Dedrick and Jones, 1990; Tsang et al., 1990). T h e following transcriptional regulators have been implicated in class I1 expression: 1 . X I binding factors. A human protein named R-FX that has been identified interacts with the X1 site of the X box of MHC-I1 promoters (Reith et al., 1989). T h e RF-X can bind to the X box as either a single protein o r as a homodimer. A murine factor with similar properties has been identified and named NF-X. NF-X will bind to both the MHC-I1 Ea (mouse) and DRa (human) promoters (Koch et al., 1988; Kouskoff et al., 1991). A human homologue of NF-X has been identified and is a protein distinct from RF-X (Kouskoff et al., 1991). 2. X2 binding factors. A protein designated hXBP-1 has been cloned

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from human cells based on the ability to bind to the X2 site of the DR-a promoter (Liou et al., 1990). T h e XBPl is a 250-residue protein that has a leucine zipper dimerization domain and shows homology with c-fos and c-jun (Glimcher and Kara, 1992). The hXBP-1 can form a heterodimer with c-fos (On0 et al. 1991a). A mouse homologue to XBPl has also been identified (Boothby et al., 1989). Structural features of XBPl are consistent with the predicted function of X2 determined from its nucleotide sequence. T h e palindromic sequence of X2 is identical to the CAMP-responsiveelement (CRE) that is bound by the CREB and fos/jun family members (Vogt and Bos, 1989) and may be associated with protein kinase C-linked activation pathways (Benoit and Mathis, 1990). Transfection of antisense DNA to the hXBP gene has been found to inhibit IFN-y-induced, as well as constitutive expression of MHC-11 genes (On0 et al., 1991a,b). 3. Y box binding factors. NF-Y/YEBP is a factor that binds to the Y box (Dorn et al., 1987a,b; Miwa et at., 1987; Zeleznick-Le et al., 1991). NF-Y is a ubiquitous heterodimeric factor that is also involved in the transcription of a number of genes that are unrelated to MHC genes (Miwa et al., 1987; Koch et al., 1988; Benoit and Mathis, 1990). T h e binding of NF-Y is dependent on the CCAAT motif, which constitutes the core sequence of the Y box (Dorn et al., 1987a). NF-Y seems to play a very important role in the formation of stable transcriptional initiation complexes for classical MHC-I1 genes (Mantovani et al., 1992; Milos and Zaret, 1992). NF-Y is identical to CP1, a CCAAT-binding factor described in a HeLa cell system with identical properties (Chodosh et al., 1988; van Huijsduijnen et al., 1990). The Y box is also bound by a factor distinct from NFY/YEBP, termed YB1. This factor consists of a single-chain, 36-kDa protein whose mRNA expression is generally inversely correlated with MHC-I1 gene expression (Didier et al., 1988). This observation has lead to speculation that YB1 may be a repressor of Y box activity (Benoit and Mathis, 1990). 4. S box binding factors. The S box has been reported to bind proteins (Tsang et al., 1990; Cogswell et al., 1990; Dedrick and Jones, 1990); however, the identity of these factors has yet to be determined. As with MHC-I genes, transcriptional regulation of MHC-I1 genes is dependent on the interplay of multiple cis-acting regulatory elements and their respective binding factors. The regulation of MHC-I1 gene expression can become particularly complicated when the cells are responsive to two or more (sometimes opposing) cytokines. Some of these situations will be discussed in the following section.

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REGULATION OF MHC R. CYTOKINE GENEEXPRESSION Several cytokines have the capacity to influence MHC-I and I1 expression, including interleukin-4, interleukin-10, type I interferons (IFN-a, IFN-P), type I1 interferon (IFN-y),TNFa, granulocyte/monocyte-colony stimulating factor (GM-CSF), transforming growth factors (Y and P- 1 (TGF-a, TGF-Pl), and epidermal growth factor (EGF) (Maudsley et al., 1989; Todd et al., 1990; Glimcher and Kara, 1992; Ting and Baldwin, 1993). It is important to emphasize at this point that some cytokines can directly affect MHC-I o r MHC-I1 gene expression, whereas other cytokines can act indirectly by regulating the expression of other cytokines o r extracellular mediators, such as prostaglandins. Second, it is useful to bear in mind that several cytokines can have differential effects depending on the responding cell type. It can therefore be difficult to attribute a specific regulatory activity (enhancing o r suppressing) for MHC expression to a particular cytokine without careful consideration of the cell type involved and the status of any other activating signals. A generalized summary of cytokine effects on MHC expression follows, with certain specific situations included as examples. More detailed discussions in this area are reviewed elsewhere (Glimcher and Kara, 1992; Moore et al., 1993). The cytokines that are most responsible for up-regulating MHC expression are IFN-a, IFN-@,and IFN-y (Maudsley et al., 1989). Of these, IFN-y is the most potent (Revel and Chebath, 1986; Farrar and Schreiber, 1993). This difference in potency may be due to the distinct cell surface receptors that type I and type I1 IFNs bind to transmit signals (Revel and Chebath, 1986; Langer and Pestka, 1988; Aguet, 1990; Bazan, 1990). Type I IFNs are produced by fibroblasts, leukocytes, as well as other cell types during a viral infection. In contrast, IFN-.)Iis a T cellderived cytokine (lymphokine, more specifically) that is released by T cells in response to activation by APCs bearing MHC and antigen (Klein et al., 1985; Taniguchi, 1988; Farrar and Schreiber, 1993). The interferons can significantly enhance MHC-I expression on a wide variety of cell types of diverse embryologic origin (David-Watine et al., 1990), particularly cell types with a low basal MHC-I expression level (Momburg et al., 1986a,b). The differences in basal class I expression commonly observed among normal cell types are generally due to differences in gene transcription rather than to posttranslational regulation (Ting and Baldwin, 1993; Drezen et al., 1993). The mRNA levels for genes encoding MHC-I heavy chain and P2M increase rapidly, usually

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within 3 hours following IFN treatment (Rosa et al., 1983), suggesting that the primary effect of IFNs is to up-regulate transcriptional activity of heavy chain and P2M genes. Post-transcriptional up-regulation of MHC-I surface expression has been observed in tumor cells deficient in MHC-I surface expression (Klar and Hammerling, 1989), suggesting that additional IFN-responsive genes, possibly Tap- 1, Tap-2, or others, may also play a role in IFN-enhanced MHC-I expression at the plasma membrane. Induction of MHC-I gene transcription by interferons is regulated by IFN-inducible trans-acting nuclear factors that bind the conserved upstream IRS motifs located in classical MHC-I gene promoters. Shirayoshi et al. (1988) have found that the H-2Ld IRS is occupied constitutively with a nuclear binding factor in lymphocytes and fibroblasts, but on treatment of the cells with IFN-a/P two additional IRS binding activities are detectable. T h e constitutive and two IFN-inducible binding factors bind the same site within the IRS, extending from - 152 to - 142 (Shirayoshi et al., 1988). The existence of indistinguishable binding sites suggests the possibility that the induced binding factors may displace the constitutively bound factor to allow enhanced transcription. An IFN-inducible MHC-I IRS binding factor termed IRF-1 (IFN response factor-1), also known as IBFl (IRS binding factor-1) has been cloned and sequenced (C.-H. Chang et al., 1992). This gene encodes a 349-residue protein that binds the IRS of the MHC-I promoter and this interaction is sufficient to trans-activate the MHC-I promoter (C.-H. Chang el al., 1992). Furthermore, mutant cells lacking expression of IRF-1 are apparently defective in the inducibility of MHC-I expression by IFN-y treatment (C.-H. Chang et al., 1992). It is likely that one of the IFN-inducible factors described by Shirayoshi and co-workers (1988) is IRF-1. Results discussed here and additional data found elsewhere (Harada et ul., 1990) support the conclusion that IRF-1 is the major IRS binding factor responsible for the positive regulation of MHC-I gene expression induced by the IFNs. A second IFN-inducible IRS binding protein, called IRF-2, has been cloned and sequenced (Harada et al., 1989). T h e N-terminal regions of IRF-2 and IRF-1 exhibit over 60% homology (Harada et ul., 1989), whereas the C-terminal regions possess only 25% homology. IRF-2 binds the identical IRS nucleotide sequence that is recognized by IRF-1 (Harada et al., 1989), presumably through the similar N-terminal domain(s). I n contrast to IFN-1, binding of IRF-2 to the IRS sequence does not upregulate MHC-I gene expression (Harada et al., 1990). In fact, coexpression of IRF-2 inhibits the induction of MHC-I gene expression observed when IRF-1 is expressed in the absence of IRF-2. IRF-2 has a similar

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effect on IRF-1 promoter activities in other IFN-inducible genes (Harada et ul., 1990). Although IRF-1 and IRF-2 are IFN inducible, there is low level expression of both IRFs in differentiated cells (Harada et al., 1990). Harada et al. (1990) have proposed a model in which IRF-2 remains bound constitutively to the IRS, repressing induction under normal circumstances in differentiated somatic cells. On IFN exposure o r viral infection, however, IRF-1 and IRF-2 are up-regulated. The IRF-1, by a mechanism that is not clear, would be able to compete effectively with IRF-2 for binding to the IRS, leading to the induction of MHC-1 expression. Subsequently, IRF-2 would replace IRF- 1 to repress the induction of MHC-I expression and return transcription to basal levels. Further studies will be necessary to clarify the relationship of IRF-1 and IRF-2 in IFN-induced up-regulation of MI-IC-I expression. TNFa is a 17-kDa protein produced by monocytes and T H 1 T cells. It was first identified by its cytotoxic activity on a variety of tumor cells (Beutler and Cerami, 1989; Fiers, 1991). TNF also has the ability to increase the cell surface expression of MHC-I proteins on several cell types (Collins et al., 1986; Pfizenmaier et al., 1987). T h e MHC-I upregulation by TNFa appears to be due to a severalfold increase in the steady-state levels of MHC-I mRNA (David-Watine et al., 1990). Whereas IFNs induce both MHC-I heavy chain and P2M gene expression, TNFa increases heavy chain gene transcription only. The IRS is not involved in TNFa-dependent MHC-I upregulation; rather, TNFa up-regulates class I by inducing an NF-KB-like enhancer binding activity that displaces both constitutively bound AP-2 and KBFlIHPTFl factors in the MHC-I promoter (Israel et al., 1989a). Two NF-KB-like factors must be bound simultaneously to stimulate MHC-I gene transcription over the basal rates observed when AP-2 and KBFlIH2TFl are bound to the promoter (Israel et al., 1989a). A concomitant up-regulation of P2M gene expression may not be observed because the P2M promoter contains one but not two binding sites for a NF-KB-like DNA-binding factor. A large number of cytokines act as MHC-I1 inducers or repressors and in many instances the same cytokine can serve both functions depending on the type and differentiation state of the responding cell. In the B cell lineage, MHC-I1 expression is believed to be mainly induced physiologically by IL-4 but not IFN-y. In fact, IFN-y can inhibit IL-4mediated MHC-I1 induction, consistent with the antagonistic relationship between these two cytokines (Mond et al., 1986). In contrast, MHC-I1 expression in monocytes/macrophages and some nonhemopoietic cell lineages is induced by IFN-y and IL-4. It is notable that IL-4 and IFN-y are antagonists for MHC-I1 expression in B cells but not normally in other cell types. Up-regulation of MHC-I1 on macrophages and non-

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hemopoietic lineage cells is also mediated by additional cytokines, including GM-CSF (Fischer et al., 1988; Falk et al., 1988; Willman et al., 1989), and TNFa (Zimmer and Jones, 1990). TNFa can synergize with IFN-y to induce high levels of MHC-I1 on monocytes and macrophages (Chang and Lee, 1986; Pfizenmaier et al., 1987). MHC-I1 expression on B cells is induced by IL-10 in addition to IL-4, consistent with a T H 2 cytokine response pattern for this cell lineage (Go et al., 1990). Expression of MHC-I1 on macrophages and/or nonhemopoietic lineages can be expressed by cytokines, including IFN-a and IFN-P (Ling et al., 1985; Inaba et al., 1986), as well as TGF-P1 (Czarniecki et al., 1988), whereas IFN-y remains the principal repressor of MHC-I1 expression in B cells. With only a few exceptions regulation of MHC-I1 expression by cytokines is at the transcriptional level. T h e mechanisms by which MHC-I1 transcriptional activity is regulated by cytokines are poorly understood. In the most thoroughly exaniined systems involving interferon induction, IRS motifs similar to those in MHC-I promoters have not been found in the upstream promoter region of MHC-I1 genes (Glimcher and Kara, 1992). T h e activity of IFN-y seems to be exerted through the same promoter sequences that are responsible for constitutive MHC-I1 expression, namely the S, X1, and Y elements (Moses et al., 1992; Sloan et al., 1992). Recently, an X box binding protein has been found that is induced by IFN-y but does not bind the IRS found in MHC-I promoters (Moses et al., 1992). Furthermore, in vivo footprinting analyses have found that a HLA-DR promoter X box binding protein is up-regulated by IFN-y treatment (Wright and Ting, 1992). It is worth mentioning that the gene for the nonpolymorphic Ii chain that associates with the MHC-I1 heterodimer intracellularly is also inducible by IFN-y (Rahmsdorf et al., 1986; Pessara et al., 1988) and T N F (Pessara and Koch, 1990). In most instances the up-regulation of MHCI1 genes and the Ii chain genes is coordinate, and the Ii and MHC-I1 promoters share similar upstream S/H/W, X, and Y enhancer elements (Zhu and Jones, 1990). However, a set of additional promoter elements is found downstream of the human and mouse Ii Y boxes, including KB and SP1 sites, and a CCAAT box. T h e KB site is thought to mediate the response of Ii to TNFa by binding NF-KB(Pessara and Koch, 1990). An IFN response element has been identified in the mouse Ii promoter region and a protein from IFN-?-treated fibroblasts has been found to bind this sequence (Eades et al., 1990). A better understanding of MHC gene regulation by cytokines will likely follow further identification and characterization of transcription

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factors that bind to, or are released from, DNA promoter/enhancer elements in response to cytokine treatment. For the moment, however, the list of known transcription factors must be considered as potential targets for regulation by DNA viruses. S. DNA VIRUSES THAT MODULATE MHC EXPRESSION

T h e preceding sections have outlined the basic processes involved in MHC expression and immune recognition. These complicated activities offer a number of regulatable sites for viral perturbation, and several DNA viruses have evolved clever and sophisticated means to manipulate the expression of MHC products in attempts to evade or subvert the immune system to their obvious advantage. Figure 1 illustrates the potential intracellular events that could be potentially manipulated, at stages common to MHC-I and MHC-I1 pathways (steps 1-3), or that are specific to the class I pathway (steps 4-10) or the class I1 pathway (steps 4’-10’). Not all of these steps have been shown to be regulated by viral intervention, but it is worthwhile to consider that the study of virus gene products that perturb MHC expression and antigen presentation is still in its early stages, and that it is likely that as more specific examples are discovered and analyzed, other sites of viral intervention are likely to be uncovered. The following sections provide descriptions of the interplay between a variety of specific DNA viruses and their hosts as each competes for the control of MHC expression and the presentation pathways. As will become clear, the outcomes of individual DNA virus infections are sometimes far from certain, often dependent on the tropism of the virus, the permissiveness of host tissues to viral activities, the allelic composition of the immune repertoire of the host, and the state of activation of the various responsive immune cells, as well as other parameters that remain to be better characterized.

I I. Poxviruses A, INTRODUCTION Poxviruses are among the largest of the eukaryotic viruses, and are the only virus group to replicate exclusively in the cytoplasm of infected cells (Traktman, 1990; Moss, 1990a, 1992). Unlike most other DNA viruses, poxviruses execute only a productive replication cycle and generally do not enter into states of latency o r persistence, although cases of

FIG. 1. Class I and I1 pathways of MHC-restricted presentation of viral antigens. The processing and presentation of intracellular viral antigens by MHC-I molecules and the uptake, processing, and presentation of extracellular viral antigens by MHC-I1 molecules are shown. Steps 1 to 3 indicate stages of transcription, translation, and transport to cellular compartments; these stages are similar in the class I and I1 pathways (see Table I for examples of viral intervention). Steps 4 to 10 are unique to MHC-I presentation to CD8+ T cells (see Table I1 for examples of virus regulation) and steps 4’ to 10’are unique to MHCI1 presentation to CD4+ T cells (very few known examples of regulation by DNA viruses).

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abortive infections do arise in certain cell types (Dales, 1990). Instead, poxviruses generally express the full spectrum of encoded proteins (frequently in excess of 200) in successive waves of gene expression, denoted early, intermediate, and late (Moss, 1990b). Because many of these viral proteins are excellent antigens, poxviruses have evolved a multiplicity of strategies to evade or down-regulate immune recognition, and the final outcome of a particular infection in a vertebrate host reflects a balance between virus cytotoxicity/cellular tropism/tissue spread and the functions of the various arms of the immune system, both native (e.g., N K cells and macrophages) and acquired (T cells and B cells). By and large, the cellular immune response is critical for virus clearance whereas humoral immunity plays a major role in resistance to secondary challenges (Turner and Moyer, 1990; Buller and Palumbo, 1991). In poxviruses with pronounced virulence characteristics, such as variola in humans, ectromelia (mousepox) in mice, and myxoma in rabbits, the virus first replicates in cells at the portal of entry (frequently the skin) and then spreads as free virions or within infected lymphoid cells to lymph nodes, where infection of professional APCs leads to MHCI-restricted presentation of viral epitopes and stimulation of CD8+ T cells (Buller and Palumbo, 1991; Fenner, 1992). Class I presentation may also occur via dendritic cells of the skin, such as Langerhans cells (Sprecher and Becker, 1993), although the extent of the contribution of these populations to overall T cell priming is still unclear. At the same time, virus particles and extracellular viral antigens are taken up by MHC-11+ presenting cells, such as macrophage and B cells, and serve to stimulate CD4+ T cells that help B cells secrete neutralizing antibody and help CD8+ T cell function. The severity of poxviral diseases and their individual pathogenic characteristics reflect a balance between the viral propensity for invasive spread and the vigor with which the cellular immune repertoire is allowed to develop. For infections by the less virulent poxviruses, such as vaccinia, Molluscum contugiosum, the benign orthopoxviruses, and rabbit fibroma virus, the immune response is sufficiently vigorous to permit containment of the virus at the portal of entry, and systemic infections are uncommon unless the host is immunocompromised (McFadden, 1988; Fenner et ul., 1989). Thus, the discussion of mechanisms by which poxviruses perturb MHC expression to alter cellular immune responses must reflect the fact that there is a large spectrum in the ability of different poxviruses to evade o r depress cellular immunity. For example, vaccinia virus was until recently used as a live vaccine against smallpox (variola) and is now utilized extensively as a recombinant vector for novel vaccines and as a delivery vehicle for het-

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erologous antigens (Moss, 1991; Cox et al., 1992; Fenner, 1992; Smith and Mackett, 1992). However, the origins of vaccinia virus are obscure and many of the poxvirus virulence genes derived from the original cowpox-like inoculum used by Jenner, which gave rise to the modernday strains of vaccinia, have been altered by two centuries of passage and propagation outside a native host (Baxby, 1981). Thus, the extensive attenuation of vaccinia and lack of a native host complicate the formal analysis of its immunosuppressive characteristics during infections of test animals. Despite this caveat, vaccinia has turned out to be an important model system to study the mechanics of MHC-restricted antigen presentation (Bennink and Yewdell, 1990; Andrew et ul., 1992; Yewdell et al., 1993; Cox et al., 1993). Vaccinia can be inoculated into test animals by a variety of routes and in mice elicits a specific CTL response within about 6 days (Buller and Palumbo, 1991). When the virus has been genetically altered to express foreign antigens, CTLs to the encoded antigen often can be readily isolated, and cells infected in vitro with recombinant vaccinia viruses are usually good targets for cytotoxicity assays by antigen-specific CTLs. T h e use of vaccinia-based vectors, for example, established that internal proteins can be inimunodominant and that recognition of nonself antigens depends on MHC haplotypes (Bennink and Yewdell, 1990). There is now a long list of vaccinia constructs that have been shown to induce high levels of protective antibody and CTL response (for example, see Tartaglia et al., 1990; Smith, 1990; Mahr and Payne, 1992; Cox et al., 1992), and immunization with vaccinia vectors that express tumorspecific antigens (e.g., neu, T antigen, p97 melanoma antigen) has also been shown to prevent induction of syngeneic tumors o r cause clearance of existing tumors in animal models, further attesting to a vigorous CTL response (Smith, 1990). Vaccinia viruses that are engineered to encode MHC molecules can also present antigens bound to the expressed MHC (Coupar et al., 1986b; Bennink and Yewdell, 1990; Lobigs and Mullbacher, 1993). For example, in a recent variation on this theme, Restifo et al. (1993) used a vaccinia vector that expresses H-2Kd of mouse to study antigen presentation properties in 26 human tumor lines and showed that the ability to present viral antigens to Kd-restrict.ed vacciniaspecific CTLs was blocked in small cell lung carcinomas, apparently because MHC transport from the ER was specifically abrogated in these cells. Clearly, poxvirus-based vectors can be a powerful tool to analyze the mechanisms of antigen presentation, and their optimal usage requires consideration of the potential MHC-modulatory capabilities of the parent poxviral vectors.

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B. CELLULAR IMMUNE RESPONSE TO POXVIRUS INFECTION There is considerable evidence indicating the importance of CD8+ T cells in clearance of poxviruses from infected hosts (Buller and Palumbo, 1991; Fenner, 1992). T h e presence of CD8+ CTLs in vaccinia-infected animals was demonstrated some years ago, but CTL generation in humans has been difficult to prove (e.g., Graham et al., 1991) and was only recently convincingly shown in vaccinated humans (Demkowicz and Ennis, 1993; Erickson and Walker, 1993). In ectromelia-infected mice, specific CTLs are detected 2-4 days postinfection and peak at day 6, before antibody appears at day 8 (Buller and Palumbo, 1991). Buller et al. (1987) showed that CD8+ CTLs are specifically critical for clearance of ectromelia from C57BL/6 mice and that depletion of CD4+ T cells did not impede recovery. Ramshaw’s group has demonstrated that during vaccinia infection of mice, CD8+ T cells are major producers of IFN-y at the height of infection and that IFN-y is one of the critical effector molecules of the virus-specific CTLs, whereas CD4+ T cells preferentially elaborate TNF (Ramshaw et al., 1992; Ramsay et al., 1993). Because both IFN-y and TNF are powerful inducers of MHC antigens, local upregulation of class I and I1 molecules by infiltrating lymphocytes is a major determinant of functional MHC levels at the site of virus replication (Maudsley et al., 1989; Sibille et al., 1992). Cytokine regulation of MHC is particularly relevant in light of the observation that some, but not all, poxviruses elaborate proteins that mimic cellular receptors and function as antagonists for TNFa/P and IFN-y. These “viroceptors” are expressed from infected cells as secreted glycoproteins and are believed to bind and inactivate their target cytokines in the local environment of the infected tissue. The first example of this was the T2 protein encoded by Shope fibroma virus, which was shown to possess sequence homology with the ligand-binding domain of one of the t w o known cellular TNF receptors, p75 (Smith et al., 1990). Subsequently, the expressed T 2 protein was demonstrated to be secreted as an extracellular glycoprotein capable of binding both TNFa and TNFP (Smith et al., 199 1). When the closely related T 2 gene of myxoma was disrupted by insertional mutagenesis, the resulting recombinant virus was unaffected for replication in cultured cells but was dramatically attenuated in rabbits, indicating that T 2 was a bonaJide poxvirus virulence gene and that virus-specific TNF inactivation is an important component of viral pathogenesis (Upton et al., 1991). Notably, the genomes of other poxviruses, including vaccinia, variola, and cowpox, also contain

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open reading frames with homology to T2, but, at least in the case of vaccinia, the two homologues (A53R and C22L) are interrupted by stop codons and frameshifts, suggesting that this anticytokine defense mechanism has been lost from vaccinia during protracted passage outside of a vertebrate host (Howard e l al., 1991; Upton at al., 1991; Shchelkunov et al., 1993). A second poxvirus viroceptor, the T 7 protein expressed from myxoma virus, has been shown to be a secreted glycoprotein, with homology to the cellular IFN-y receptor, that can functionally bind and inactivate rabbit IFN-y, suggesting that this cytokine also plays a major role in the cellular immune response to poxviruses (Upton et al., 1992). In this particular case, the homologous genes in vaccinia (B8R) and variola (B9R) bear intact homologous open reading frames and may very well express active proteins with anti-IFN-y properties (Upton et al., 1992; Upton and McFadden, 1993; Shchelkunov et al., 1993). Work with poxviruses in test animals has confirmed the importance of cell-mediated immunity in the antiviral response. For example, although normal mice undergo only a localized and relatively benign infection by vaccinia, nude mice, o r sublethally irradiated euthymic mice, undergo a lethal systemic disease (Buller and Palumbo, 1991; Ramshaw et al., 1992). Importantly, when the inoculating vaccinia has been engineered to express certain critical cytokines, such as IL-2, TNF, or IFN-y, the resulting infection is cleared even in immunodeficient mice, confirming the relevance of these ligands in the immune response to poxvirus infection (Flexner et al., 1987; Kohonen-Corish et al., 1989, 1990; Ruby and Ramshaw, 1991; Giavedoni et al., 1992). One curious observation is that homozygous P2M- mice successfully respond to vaccinia challenge and clear the infection (Spriggs et al., 1992). Although it is known that such mice are depleted of CD8+ T cells, recent results indicate that P2Mmice can in fact generate CD8+ peritoneal exudate leukocytes, indicating that at least a limited class I-restricted immune response may still be possible in the absence of P2M (Apasov and Sitkovsky, 1993).This latter explanation is particularly appealing in the light of the recent demonstration that mice carrying a genetic knockout for the receptor to IFN-y, and hence are nonresponsive to the biological activity of IFN-y, undergo systemic infection when challenged with vaccinia (Huang el al., 1993). Finally, it has been well known for several years that patients with compromised cellular immunity, but not humoral immunity, are at high risk for poxvirus infections (Buller and Palumbo, 1991, 1992). For example, generalized vaccinia infections have been noted in AIDS patients exposed to live vaccinia virus vaccines (e.g., Guillaume et al., 1991), and these individuals are also at risk for progressive infection by the usually benign poxvirus, Molluscum contagiosum (Koopman et al., 1992; Charles

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and Friedberg, 1992), which does not tend to regress in states of T cell depletion (Robinson et al., 1992). Similarly, mice that have been treated with anti-T antisera, thymectomized, or carry nulnu alleles are susceptible to increased virulence by both vaccinia and ectromelia (Buller and Palumbo, 1991). Thus it is reasonable to expect that the more efficient poxviral pathogens have evolved mechanisms to perturb MHC expression and the presentation of viral antigens. C. POXVIRUS REGULATIONOF MHC-I Vaccinia vectors have been used extensively to deliver intact foreign proteins and minipeptides into the class I pathway of antigen presentation, and in some cases, such as the influenza matrix protein in B cells, the class I1 pathway (Yewdell and Bennink, 1990). For example, to study the role of MHC-I transport in antigen presentation, Cox et at. (1990) expressed the gp19/E3 gene product of adenovirus type 5 from an early promoter in vaccinia and showed that presentation of viral antigens to Ld- and Kd-restricted CTLs was interrupted, without affecting total surface levels of Ld and Kd molecules (at least in the first 5 hours of infection). Thus, the prevention of nascent class I egress to the surface will functionally inhibit MHC-I presentation of viral antigens and determination of gross surface class I levels may not necessarily reveal a virusspecific transport blockade, especially at relatively early times during the poxvirus replication cycle. Nevertheless, evidence exists that several poxviruses can induce major down-regulation of surface class I molecules at later stages of the lytic cycle. Koszinowski and Ertl(l975) showed by immunofluorescence analysis that within 19 hours after infection of L929 cells at high multiplicity with vaccinia there was a substantial reduction of cell surface H-2k antigens and that this loss corresponded to a decrease in the ability of antiH-2k CTLs to kill virus-infected cells. A similar observation was made for ectromelia-infected cells (Gardner et al., 1975). Later, Lakdhar and Senik (1982) demonstrated that both H-2Kk and H-2Dk levels at the surface of vaccinia-infected mouse cells dropped to near baseline levels following high multiplicities of infection. When a vaccinia vector that expressed human IFN-y was assessed in similar experiments, an increase in MHC-I levels of nearby uninfected cells was observed at low multiplicities, but only decreases comparable to the control virus were detected at higher multiplicities (Kohonen-Corish et al., 1989). Importantly, a vaccinia vector that expressed H-2Kd induced an increase in H-2Kd levels but a decrease in native H-2Kk levels at the surface of infected L929 cells, indicating that the down-regulation effect was tar-

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geted to cellular class I expression. More recently, Brutkiewicz et al. (1992) demonstrated that H-2Kd and Dk reductions at the surface of vaccinia-infected L929 cells correlated with decreases in CTL lysis but increases in MHC-unrestricted NK cell killing. This is likely to be a biologically relevant observation because NK cell response may be critical for virus clearance under conditions where MHC-restricted viral antigen presentation is blocked (Reiter and Rappaport, 1993). For example, immunodeficient mice infected with vaccinia recombinants that express 11-2 induce a strong NK response during recovery and mice depleted of NK cells generate higher titers of vaccinia (Flexner et al., 1987; Ramshaw et al., 1992). T h e loss of killing by CTLs during the vaccinia infection was greater for H-2-specific CTLs than for vaccinia-specific CTLs, suggesting that decreases in MHC-I levels cannot alone explain the loss of allospecific reactivity. One reasonable hypothesis is that viral peptides rapidly outcompete host peptides for the available class I molecules that reach the ER/cis-Golgi. Surface MHC-I down-regulation has also been observed during the infections of cultured cells with myxoma virus (Boshkov et al., 1992). Cellular immune functions are dramatically depressed in rabbits during the development of myxomatosis (Fenner and Ratcliffe, 1965; McFadden, 1988; Strayer, 1989), and surface analyses of class I molecules on myxoma-infected cells indicate drastic reductions of MHC-I epitopes to near background levels within 24 hours of infection (Boshkov et al., 1992). Comparable infections with vaccinia or Shope fibroma virus also induced detectable, but less drastic, reductions. Importantly, blockade of de nova cellular MHC-I expression by nonspecific inhibitors of translation resulted in only partial surface MHC-I reductions within 24 hours, probably due to recycling and turnover, indicating that the myxoma down-regulation effect included class-I molecules that existed at the cell surface prior to the virus infection (Boshkov et al., 1992). Further experiments using DNA synthesis inhibitors to block late myxoma gene expression indicate the one or more late gene products contribute to the MHC-I decline, but the relevant gene(s) remain to be mapped and charac terized.

D. POXVIRUS REGULATIONOF MHC-I1 T h e class I1 peptide-processing pathway can be shown to be different from the class I pathway in that it is relatively less efficient, sensitive to chloroquine, resistant to Brefeldin A, and usually relies on exogenous antigen. The effects of poxviruses, which can replicate in MHC-11+ cells on class II-restricted presentation, vary considerably and are relatively

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cell and epitope specific (e.g., Littaua et al., 1992; Freer and Senesi, 1993). Morrison et al. (1986) and Sweetser et al. (1989) demonstrated that an influenza hemagglutinin (HA) epitope minigene expressed from vaccinia could present HA to class II-restricted CTLs only after ultraviolet (UV) light inactivation of the virus, suggesting direct entry of HA into the endosome/lysosome compartment during abortive infection. It was further shown by Domanico and Pierce (1992) that productive vaccinia infection of B cells caused a decline in the ability to present certain epitopes (such as intact exogenous cytochrome c) to class II-restricted CTLs. Only certain cells, such as LK35-2 (a B cell hybridoma), manifested the block, and only some low-affinity epitopes were susceptible when introduced as intact protein. However, the antigenic epitope was not excluded from presentation when exogenously added in the fully processed peptide form, suggesting that the viral block was manifested at the level of protein uptake and/or degradative release of the antigenic peptide epitope. On the other hand, Jaraquemada et al. (1990) showed that endogenously synthesized influenza M 1 protein expressed from a vaccinia vector could be presented to class II-restricted CTLs, suggesting that intracellular antigens synthesized from the virus genome can still enter into effective complexes with class I1 molecules and can outcompete at least some exogenous antigens for class I1 loading. To date there is no information on the capacity of any poxvirus to modulate MHC-I1 expression directly, other than by the overall reduction of cellular gene transcription that normally accompanies most productive poxvirus infections. E. POSSIBLE MECHANISMSOF MHC REGULATION BY POXVIRUSES

Perhaps fortuitously, the first promoter used extensively to express foreign antigens in recombinant vaccinia virus vectors was the 7.5K promoter, which is active at both early and late stages during the virus infection (Smith, 1990; Moss, 1991). Many of these early recombinant viruses were capable of vigorous antigen-specific CTL induction and proved to be highly useful as vaccines. However, the possibility that vaccinia might be capable of altering MHC-restricted presentation was suggested by the finding that many antigens under the control of late viral promoters were capable of inducing humoral but not cellular immunity (reviewed by Andrew et al., 1992). Coupar et al. (1986a) first showed that the major influenza HA epitope expressed from a late viral promoter in a vaccinia recombinant failed to prime CTLs in CBA/H mice, could not restimulate primed splenocytes, and did not make target

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cells sensitive to HA-specific CTLs. This late restriction was haplotype specific (i.e., not observed in Balb/c mice) and not all epitopes were equally affected. Townsend et al. (1988) later demonstrated that the defect in late class-I restricted presentation in vaccinia infected cells is selective: for HA, the block was detected only at late times, but for peptide 366-379 of the influenza nucleoprotein antigen, a presentation deficit was observed at both early and late times, while there was relatively little effect on a second epitope (peptide 50-63) from the same polypeptide. Furthermore, for those epitopes that exhibited the block, the defect could be circumvented by enhanced degradation of the protein, for example, by deletion of the HA signal sequence or fusion of the nucleoprotein antigen with a ubiquitin sequence. Because the late blockade did not interfere with the presenting function of Db molecules, at least for exogenous peptides exchanged at the cell surface, it is reasonable to conclude that the virus effect must be exerted at the level of antigen trafficking, rate of protein degradation, peptide loading, and/or efficiency of transport to the surface. Because the viral gene product(s) that presumably mediate this blockade have not yet been identified, the molecular basis for the defect remains unknown. One suggested explanation is that a virus-encoded serine protease inhibitor (serpin) might perturb antigen processing into peptides, but this remains to be experimentally demonstrated (Turner and Moyer, 1990; Smith, 1990;J. Zhou et al., 1990). Virus-induced shutdown of cellular genes, a nonspecific phenomenon that presumably includes heavy chain and P2M, may very well contribute to the overall competition for MHC-I loading by viral versus host peptides, but simple blockade of heavy chain/P,M synthesis by nonspecific inhibitors of protein synthesis does not mimic the epitope-specific late blockade observed in virus-infected cells (Townsend et al., 1988). IFN and TNF are known inducers of MHC expression, and many viruses have evolved mechanisms to circumvent the activity of these cytokines (Staeheli, 1990; Joklik, 1990; Samuel, 1991; Kerr and Stark, 1992a; McNair and Kerr, 1993). As previously mentioned, several poxviruses encode and elaborate secreted viroceptors (homologues of the cellular receptors) for TNFa/P and IFN-y that are believed to neutralize the target ligands before they come into contact with their cognate cellular receptor. Vaccinia virus, additionally, exhibits variable sensitivity to the inhibitory effects of type I interferon (Rodriguezet al., 1991; McNair and Kerr, 1993) by virtue of several distinct gene products that modulate IFN responsiveness. The first of these (E3L) possesses sequence homology with DAI, a cellular dsRNA-dependent protein kinase that inhibits protein synthesis by phosphorylating and inactivatingeIF2 (H. W. Chang et al., 1992; Davies et al., 1993; Chang and Jacobs, 1993).Another

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vaccinia gene that inhibits IFN signal transduction is the K3L gene product, which is a homologue of eIF2a and inhibits activation of DAI by acting as an inappropriate substrate (Davies et al., 1992, 1993). Vaccinia mutants deleted for K3L exhibit greater susceptibility to IFN inhibition (Beattie et al., 1991), but whether this lesion has a direct effect on MHC-I inducibility by IFN has not been investigated. Another gene implicated in the vaccinia antiinterferon response is A18R, which has been implicated in the cellular ribonucleolytic 2'-5' adenylate pathway of the interferon response (Pacha and Condit, 1985; Cohrs et al., 1989; Bayliss and Condit, 1993). Taken together, there is circumstantial evidence that specific poxviral gene products might directly mediate at least some of the late blockade to MHC-restricted antigen presentation. Virus genes so far identified that perturb MHC expression have been restricted to those that inhibit cytokine function (TNFa/P and IFN-y) or signal transduction (IFN-alp), but the impressive extent of MHC-I surface-level downregulation at late times following infection by variety of poxviruses and the apparently specific class 11-peptide-processing defects in vacciniainfected phagocytic cells suggest that more poxviral proteins that can directly perturb functional MHC expression may very well exist but remain to be identified. 111. Adenoviruses A. INTRODUCTION

Adenoviruses are nonenveloped, double-stranded DNA viruses that infect a broad range of species (Ishibashi and Yasue, 1984; Nermut, 1984; Wigand and Adrian, 1986).The best characterized are the human adenoviruses, which can cause persistent infections in humans and animals (reviewed in Wadell, 1984; Horwitz, 1990). Human adenoviruses are associated with a variety of respiratory, gastrointestinal, and urinary tract diseases and some ocular diseases, including keratoconjunctivitis. It is estimated that adenoviruses cause about 30% of viral respiratory diseases (Horwitz, 1990). Infants and young children usually exhibit the most frequent clinical manifestations from adenovirus infections, especially in the respiratory tract, although certain adenovirus infections cause infantile diarrhea (Uhnoo et al., 1983; Horwitz, 1990). Adenoviruses can be readily cleared from primary sites of infection; however, subsequent persistent infection in lymphoid tissue is a characteristic feature of human adenoviruses. On rare occasions, systemic adenovirus infections are found, usually with immunocompromised in-

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dividuals (Zahradnik et al., 1980; Siegal et al., 1981; Hierholzer et al., 1988). There are over 50 subtypes of adenoviruses that have been subdivided into six groups, A through F, based on serology, DNA homology, and tumorigenicity (Green et al., 1979; Wadell, 1984). T h e pathogenicity of a given adenovirus varies with the group and type (reviewed in Gooding and Wold, 1990; Braithwaite et al., 1993). T h e adenovirus group A, including Ad 12, Ad 18, and Ad3 1, are associated with upper respiratory tract infections and may be associated with childhood diarrhea (Gooding and Wold, 1990). Group B, including Ad types 3, 7, 11, 14, 16, 21, 34, and 35, are relatively common and are associated with a variety of upper respiratory tract, ocular, and gastrointestinal diseases (Wigand and Adrian, 1986; Horwitz, 1990). T h e most common group, C, includes types 1, 2, 5, and 6, which mostly induce mild to severe upper respiratory diseases. Group D includes the highest number of distinct adenovirus types, at least 19, and they are all associated with epidemic keratoconjunctivitis (Gooding and Wold, 1990). At the DNA level, the group E Ad4 is quite divergent from the other groups and is believed to be responsible for acute respiratory disease epidemics in military recruits (Wadell, 1984). Little is known about the genome of group F, Ad40 and Ad4 1; however, a direct association with infantile gastroenteritis has been implicated (Uhnoo et al., 1983; Horwitz, 1990). Human cell lines can be productively infected with adenoviruses in vitro, yielding infectious virions (reviewed in Gooding and Wold, 1990). In contrast, rodent cells are nonpermissive or semipermissive for adenovirus replication in vitro. Infection by any type of human adenovirus of established or primary rodent cells, however, results in cellular transformation at low frequency (van der Eb and Zantema, 1992). Although all human adenoviruses transform rodent cells in vitro with equivalent efficiency, only group A, e.g., Ad12, and to some extent group B viruses generate tumors in rodent animals (reviewed by Horwitz, 1990; Braithwaite et al., 1993). Consistent with these observations are the findings that rodent cells transformed in culture by group A or B viruses are tumorigenic when transferred to animals, but this is not the case for cells transformed by group C viruses, such as Ad5 (Trentin et al., 1962; van der Eb and Zantema, 1992). T h e adenovirus life cycle and the basic organization of the virus genome are similar to a variety of animal viruses (reviewed by Horwitz, 1990; Gooding and Wold, 1990) in that early, intermediate, and late genes are expressed in a highly sequential and ordered fashion. A major function of the early genes is devoted to the subversion of the cellular

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macromolecular synthesis machinery to initiate the process that produces mature virions. A number of the intermediate and late genes encode structural components and proteins directly required for virus assembly. However, in addition to basic replicative functions, the adenovirus genome encodes a number of genes that have roles in evasion from cell-mediated immune detection and function (Wold and Gooding, 1991; Miillbacher, 1992). This review will focus on the early genes that may affect cell-mediated immunity. A very brief description of adenovirus early gene expression and function follows to provide context for the discussion of early gene viral activities affecting MHC-restricted immune detection and function (reviewed in Paabo et al., 1989; Braithwaite et al., 1993). Adenovirus early genes are transcribed from both DNA strands by RNA polymerase I1 in the following order: E l a , E4, E3, E l b , and E2. Each transcript can yield multiple mRNAs due to differential splicing and multiple polyadenylation sites. The existence of several open reading frames on the mRNAs allows for expression of different proteins from the same transcript. The E l a mRNAs code for two proteins with identical sequences except at the C terminus, where one product has 46 more residues (289R) than the other (243R). Both proteins are involved in the down-regulation of cellular transcription and the activation of other adenovirus early genes. T h e E l a products can affect transcription of both viral and cellular genes through multiple mechanisms and are reviewed in detail elsewhere (Shenk and Flint, 1991; Nevins, 1993). T h e E l a gene is responsible for the down-regulation of MHC-I transcription observed in cells acutely infected with adenovirus (Friedman and Ricciardi, 1988; Lassam and Jay, 1989; Meiljer et al., 1989). T h e EZa gene is also required for the induction of viral DNA replication, perhaps through the binding of cellular proteins thought to be negative regulators of DNA replication, such as the retinoblastoma susceptibility gene product (Harlow et al., 1986; Bellett et al., 1989; Howe et al., 1990; Braithwaite et al., 1991). The diverse regulatory activities of E l a contribute to the immortalization of primary cells that are nonpermissive or semipermissive hosts for adenoviruses (van der Eb and Zantema, 1992; Nevins, 1993). T h e Elb region immediately adjacent to E l a encodes two proteins, 19K and 55K, respectively, that are translated in different reading frames. T h e El b 19K product serves to prevent viral and cellular DNA degradation that is induced as a result of virus infection (Pilder et al., 1984; White et al., 1984). The principal function of the E l b 55K product, in contrast, is to inhibit host cell protein synthesis (Babiss and Gins-

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berg, 1984; Babiss et al., 1985). The combined activities of E l a and E l 6 region products can induce full cell transformation (Ruley, 1983; Moran and Matthews, 1987). Adenovirus E 2 and E 4 region products are involved in DNA replication and late viral gene expression; descriptions of their functions can be found elsewhere (Shenk and Williams, 1984; Halbert et al., 1985; Horwitz, 1990). In contrast to E 2 and E4, none of the nine E3 regionencoded proteins is required for virus replication, yet E3 region genes are retained in all natural adenovirus isolates from humans (Morin et al., 1987; Adrian et al., 1989a,b). As will be discussed later, E3 products are devoted to functions involved in protecting adenovirus-infected cells from a variety of cellular immune responses. Finally, all adenoviruses encode genes for virus-associated (VA) RNAs, VA RNAl and RNA2 (Akusjarvi et al., 1980). The VA region genes are distinct from other adenovirus genes in that they are transcribed by RNA polymerase 111 (Shenk and Williams, 1984). Efficient synthesis of late viral proteins requires VA RNAl, but a role for VA RNA2 has not been clearly defined (Schneider et al., 1984). Although present early in adenovirus infection, expression of VA RNAs increases dramatically toward the end of the infectious cycle (Soderlund et al., 1976).

B. CELLULAR IMMUNE RESPONSE TO ADENOVIRUS INFECTION The importance of cellular immune mechanisms in controlling adenovirus infections is indicated by the fact that immunosuppressed patients lacking functional cell-mediated immunity but retaining normal humoral immune function can often suffer fatal systemic adenovirus infections (Zahradnik et al., 1980; Burns and Saral, 1985; Shields et al., 1985; Horwitz, 1990). A role for T cell-mediated immune function in controlling adenovirus-induced tumorigenicity has been demonstrated in rodents (Harwood and Gallimore, 1975; Bernards and van der Eb, 1984). The depletion of T cells in rats with specific antisera allows normally nononcogenic adenovirus group C-transformed cells to form tumors in vivo (Harwood and Gallimore, 1975). Furthermore, adenovirus group C-transformed cells that are also not oncogenic in normal mice are oncogenic in athymic (nude) animals (Bernards and van der Eb, 1984), suggesting that T cells are required to control group C virus tumors. It was demonstrated early on that adenovirus-specific CTL activity could be generated in adenovirus-infected mice and rats (Inada and

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Uetake, 1978; Gallimore and Williams, 1982). Proteins encoded in the E l a gene are the predominant adenovirus CTL antigens (Bellgrau et al., 1988). The immunodominant epitope for Ad5-specific CTLs in the rat has been localized by the use of a series of E l a mutants in the C-terminal region of Ela (Urbanelli et al., 1989). In the mouse, the E l a determinant recognized is dependent on the strain of the infected cells. The H-2Db molecule has been found to present an immunodominant T cell determinant contained within a 12-amino acid stretch of the E l a second exon to an Ad5-specific CTL clone. This was determined by using a series of overlapping 12-residue synthetic peptides encompassing the entire amino acid sequences of Ela-encoded proteins for CTL lytic sensitization of noninfected H-2Db-expressing cells (Kast et al., 1989). Evidence for antigen-specific CTL elimination of Ad5 tumors in vivo was provided by Kast et al. (1989), who injected Ad5-specific CTLs and recombinant IL-2 into nude mice carrying Ad5 tumors and caused the elimination of even very large solid tumors within 2 weeks. These results suggested that Ad5-induced tumors may be nontumorigenic because of their susceptibility to antigen-specific CTLs. A number of reports suggest that, in addition to CTLs, natural killer (NK) cells may play a significant role in determining the tumorigenicity of adenovirus-transformed cells (reviewed in van der Eb and Zantema, 1992). Consistent with this proposal is the observation that adenovirustransformed cells that are NK resistant, (e.g., Ad12-transformed cells) are also likely to be tumorigenic, whereas NK-sensitive Ad-transformed cells (e.g., Ad5 transformed) are usually nontumorigenic. It has been reported that the non-MHC-restricted lysis mediated by NK cells can nevertheless be modulated by MHC molecules on the target in a manner that is the reciprocal of T cell recognition (Reiter and Rappaport, 1993). In other words, cells that express low levels of MHC-I molecules are generally more susceptible to NK lysis than are cells expressing higher MHC-I at the cell surface. The regulation of MHC-I expression on adenovirus-transformed cells by the infecting virus cannot readily explain the observed NK susceptibility pattern for adenovirus tumor cells, because in the case of the highly tumorigenic Ad12 virus, for instance, infection greatly reduces cellular MHC-I expression, yet Ad 12 infection renders the cells resistant to NK lysis (Raska and Gallimore, 1982; Cook et al., 1987). Furthermore, no correlation between MHC-I levels and sensitivity to NK cell lysis has yet been established for adenovirusinfected cells (Bosse and Ades, 1991; Routes, 1992). Viral infections generally result in production and release of cytokines, including IFN-y and TNFa, by T lymphocytes, NK cells, and macrophages. Human adenovirus infections are no exception, but

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adenovirus-infected cells are relatively insensitive to IFN and TNF because adenovirus gene products can actively interfere with the antiviral activities of these cytokines. For instance, the adenovirus VA R N A l inhibits the activity of the IFN-inducible double-stranded RNA protein kinase responsible for shutting down cellular translation processes in cells undergoing viral infection (Katze et al., 1987; O’Malley et al., 1989). In addition, the E l a from Ad5 has been shown to repress the signal transduction pathways of the type I and I1 IFNs (Reich et al., 1988).The E l a product has been reported to carry out this inhibition by preventing formation of appropriate transcription initiation complexes for initiation of interferon-responsive gene transcription (Kalvakolanu et al., 1991; Gutch and Reich, 1991; Ackrill et al., 1991). Based on experiments in transfected cells, it has been observed that the expression of E l a renders infected cells susceptible to TNFamediated lysis (Chen et al., 1987; Duerksen-Hughes et al., 1989) in the absence of other adenovirus gene products. To counteract such potential vulnerability, at least three adenovirus gene products are committed wholly or partially to conferring resistance to TNF-mediated cytolysis of the infected cell, including the E l b 19K protein and three E 3 regionencoded proteins, E l 3 14.7K, and a heterodimer of the 10.4K/14.5K products (Wold and Gooding, 1991). The efficacy of the adenovirus products in preventing TNF cytolysis is dependent on the infected cell type. However, the fact that 4 of the 25-30 adenovirus genes are dedicated in some way to TNF resistance suggests that TNF may normally be a potent effector of host viral resistance.

REGULATIONOF MHC-I C. ADENOVIRUS 1. Transcriptional Control Early observations that adenovirus infections persist for extended periods in humans and that certain adenovirus-transformed cells are highly tumorigenic in animals suggested that these viruses have learned to cope with normal antiviral activities of cellular immune responses. The role of adenovirus gene products in disrupting some of the cytokine-dependent effector phases of cell-mediated immunity has been discussed above. However, the possibility that adenoviruses also interfere with the inductive and cell-cell contact-dependent cytotoxic effector phases of antiviral T cell-mediated immunity was supported initially by the correlation of adenovirus tumorigenicity with the ability of specific adenovirus types, e.g., Ad 12, to down-regulate class I MHC gene expression (Schrier et al., 1983; Bernards et al., 1983; Vasavada et al., 1986).Although nontumori-

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genic adenoviruses, such as Ad5, do not down-regulate MHC-I gene expression, they are effective at interfering with MHC-I expression, but at the post-translational level (Wold and Gooding, 1991). Therefore, all adenoviruses have mechanisms of modulating MHC-I expression, presumably with the intent of evading CD8+ T cell-mediated immune surveillance in an effort to sustain viral persistence in viva The down-regulation of MHC-I gene expression by Ad12 has been observed in human (Vasavada et al., 1986) and rodent cells (Eager et al., 1985) and has been ascribed to the 289R E l a product of the Ad12 13s RNA transcript (Bernards et al., 1983), although the 243R may also down-regulate MHC-I gene expression, albeit less effectively (Braithwaite el al., 1993).The corresponding E l a proteins from Ad5 are unable to down-regulate MHC-I gene expression and, in fact, counteract the down-regulation mediated by Ad12 E l a when Ad12 and Ad5 E l a genes are coexpressed in the same cell (Vaessen et al., 1986).The regulation of MHC-I expression therefore is complex, and is further complicated by the observation that Ad12 as well as Ad5 E l a can induce MHC-I transcription in primary mouse embryo cells, results that clearly differ from those obtained with Ad 12-transformed cells. Finally, IFN-y can induce expression of MHC-I gene expression in Ad 12-infected cells, suggesting that IFN can overcome the transcriptional repression of Ad12 E l a (Eager et al., 1985; Hayashi et al., 1985). It is therefore likely that the effects of Ad12 E l a in MHC-I transcription are dependent on several factors, including the combination of trans-activating and repressing transcriptional factors present in the cell at the time of infection, which in turn may depend on the developmental stage of the infected cells and the status of cellular activation (Nielsch et al., 1991). There is also some evidence that adenoviruses can also exert some control over MHC-I mRNA stability (Shemesh et al., 1991). In Ad 12-infected cells that have reduced MHC-I expression, the steady-state level of MHG-I mRNA is lowered and it is at the stage of transcription initiation that E l a has its negative effects (Friedman and Ricciardi, 1988; Lassam and Jay, 1989; Katoh et al., 1990). The mechanism(s) through which Ad12 E l a exerts its negative effect on MHC-I transcription initiation has not been fully resolved, but several other cellular genes, such asjunB, are coregulated in a similar manner (Meijer et al., 1991). Recent studies from a number of groups have described different mechanisms for Ad12 E l a down-regulation of MHC-I promoter activity, but we will only briefly describe some of the recent developments in this area (Friedman and Ricciardi, 1988; Katoh et al., 1990; Meijer et al., 1992; Ge et al., 1992; Kralli et al., 1992).

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One study has suggested that Ad12 E l a reduces MHC-I promoter activity by reducing the extent of KBFl and NF-KBbinding to the MHCI promoter enhancer A element (Meijer et al., 1992). Another group (Kralli et al., 1992) has found that the region I1 element RXRlH2RIIBP binding region of MHC-I enhancer A is necessary for the Ad12 reduction of MHC-I promoter activity, and that it must be juxtaposed to the positive enhancer element (KBFl binding region) to mediate the transcriptional repression. Furthermore, a region I1 binding factor, termed R2BF (55-60 kDa), was found to be at higher levels in extracts from Ad 12-transformed cells than from Ad5-transformed cells (Ge et al., 1992; Kralli et al., 1992). The R2BF was found to bind the same site as HBRIIBP, which is a member of the steroid hormone receptor superfamily (Hamada el al., 1989). Kralli et al. (1992) argue that it is unlikely that region I1 binding protein blocks the binding of proteins to the positive enhancer A site, because their in vitro DNA binding assays showed that the presence of the region I1 nucleotide sequence does not interfere with binding of nuclear factors to the positive enhancer A element, which contrasts with the results of Meijer et al. (1992). It is suggested by Kralli et al. (1992) that the repression may be a consequence of masking of activation domains on positive regulators by the region I1 binding protein due to its juxtaposition, similar to the yeast a2 repressor system (Kelleher et al., 1988). The regulatory effects of Ad12 E l a are further complicated by the identification of a far upstream regulatory element required for repression of H-2Kh’ (a murine MHCI) gene transcriptional activity in rat embryonal fibroblasts (Katoh et al., 1990). Further studies, using a larger spectrum of Ad12-infected or -transformed cells, may be needed to clarify the effect@)of Ad12 E l a on MHC-I promoter activities. 2. Post-translational Control The 19-kDa protein product of the E3 region (gpl9/E3) is able to regulate negatively the expression of MHC-I molecules at the posttranslational level (reviewed in Wold and Gooding, 1991).The first indication of how gp19/E3 function was from the demonstration by Kvist et al. (1978) that gp19/E3 binds to MHC-I molecules in adenovirustransformed rat cells. It was later determined that gp19/E3 bound human as well as rodent MHC-I molecules, although the affinity of gp19/E3 for MHC-I allomorphs is variable and binding is not detectable with certain MHC-I products (reviewed in Wold and Gooding, 1991). The association between gp19/E3 and MHC-I is noncovalent, does not require the participation of other adenovirus products, and occurs when

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P2M is complexed to the MHC-I heavy chain. The gp19/E3 proteins from adenovirus groups B, C, (Ad2 and Ad5), D, and E, but notably not A (Ad12), bind MHC-I molecules (Paabo et al., 1986a; Wold and Gooding, 1989). The gp19/E3 molecule is expressed as a resident ER protein with a typical type I transmembrane orientation; the N-terminal domain extends into the lumen of the ER, followed by a short transmembrane segment and a cytoplasmic domain of 15 residues. The signal that retains gp19/E3 in the ER resides in the cytoplasmic domain; however, the composition of the retention "motif" remains controversial. One group has reported that a pair of lysines within a linear sequence of the cytoplasmic domain retains gp19/E3 in the ER (Nilsson et al., 1989;Jackson et al., 1990), whereas another group claims that a noncontiguous sequence, folded in the proper orientation, is what serves as the retention signal (Gabathuler and Kvist, 1990). Pulse-chase studies have indicated that gp19/E3 binds newly synthesized MHC-I molecules at a step prior to the acquisition of mature forms of glycosylation, which are typically found on proteins that have transited through the medial and trans-Golgi (Severinsson and Peterson, 1985; Burgert and Kvist, 1987). The intracellular transit of MHC-I molecules is severely compromised by gp19/E3. The newly synthesized MHC-I are retained for extended periods in the ER/cis-Golgi and are eventually degraded (Gooding and Wold, 1990). The overall effect of gpl9/E3 is to block MHC-I egress from the ER/cis-Golgi compartments, which presumably is the reason that multiple post-translational events, such as phosphorylation of the MHC-I chains, are also blocked (LippC et al., 1991). Several groups are now in the process of identifying sites on gp19/E3 and MHC-I molecules that mediate their interaction. It has been demonstrated that the a 1 and a 2 domains of the MHC-I heavy chain are necessary for gp19/E3 binding (Burgert and Kvist, 1987). The N-terminal lumenal domain of gp19/E3 has been found to be required for binding to MHC-I molecules, although the transmembrane segment and cytosolic tail may influence MHC-I binding (Paabo et al., 1986b, 1987; Flomenberg et al., 1992; Hermiston et al., 1993). Recently, two novel proteins (100 and 110 kDa) were detected in immunoprecipitates of class I-gp19/E3 complexes from cells expressing the adenovirus gp19/E3 protein (Feuerbach and Burgert, 1993). The association of the novel proteins was induced with glucose starvation and the 100- to 110-kDa proteins were displaced by class I binding peptides. Such properties suggest that the 100- to 110-kDa proteins could be chaperones that

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promote class I folding o r possibly peptide binding to class I molecules. T h e adenovirus gp19/E3 product may serve as a valuable tool in determining the mechanism(s) of peptide loading on class I in viva

D. ADENOVIRUS REGULATIONOF MHC-I1 A very large body of studies have documented the often profound effects adenovirus can have on MHC-I expression at the transcriptional and post-translational levels. Very little is known about how adenoviruses may affect MHC-I1 expression. However, it has been reported that Ad5 E l a gene expression can inhibit the transcriptional up-regulation by IFN-y of several IFN-y-responsive promoters, including an HLA class I1 promoter (Ackrill et al., 1991). Results from this study indicate that E l a in some way blocks the association of interferon-inducible DNA-binding elements from associating with DNA sequences in the interferon response sequence of these promoters. To our knowledge, no evidence exists for post-transcriptional regulation of MHC-I1 expression by adenoviruses. It appears then, that adenoviruses may indeed down-regulate MHC-I1 expression; however, it is perhaps only in the context of a generalized effect on all IFN-y-responsive genes. OF MHC REGULATION E. SIGNIFICANCE BY ADENOVIRUSES

The group A adenoviruses, e.g., Ad12, down-regulate MHC-1 gene transcription by E l a , whereas other adenovirus groups, including B, C, D, and E, which lack such a capability, down-regulate MHC-I expression by a gp 19/E3-mediated post-translational mechanism. Thus, all adenoviruses are capable of down-regulating MHC-I expression by one means or another. T h e down-regulation of host cell MHC-I expression by adenoviruses, using two independent mechanisms, underscores the likelihood that this function is very important for some aspect of viral propagation or persistence in the native host. T h e fact that the gpl9/E3 molecule is not required for any viral replicative functions suggests that the purpose of gp19/E3 down-regulation of MHC-I is to evade immune detection by CD8+ T cells. Consistent with that interpretation are the observations that cells expressing gp19/E3 are less sensitive to alloreactive SV40 virus and adenovirus-specific CTL lysis (Anderson et al., 1987; Burgert et al., 1987; Rawle et al., 1989; Cox et al., 1991). Furthermore, it has been demonstrated that Ad 12 E l a transformation renders cells refractory to allospecific CTL-mediated lysis (Bernards et al., 1983). It is quite clear,

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then, that the down-regulation of MHC-I expression induced by adenoviruses can significantly affect the ability of CTLs to recognize adenovirus-infected cells in vitro. On the other hand, not all cells are equally sensitive to gp19/E3 and it has been reported that endogenous expression of adenovirus E l a potentiates the gp19/E3 effect even in normally resistant cells, suggesting that the status of the infected cell plays a major role in determining MHC levels (Routes and Cook, 1990; Routes et al., 1993). It remains to be determined what effect(s) adenovirus regulation of MHC-I expression has on the virulence and immunopathology of adenovirus infections in vivo. There are cases, for example, in which increases of MHC-I levels actually increase the tumorigenicity for Ad 12-transformed cells (Soddu and Lewis, 1992), further suggesting that immune clearance can be a complicated process. Progress in this area may now be possible with the observation that cotton rats infected with Ad5 develop pneumonia-like symptoms similar to human symptoms (Pacini et al., 1984). An Ad5 mutant that has a deletion in the E 3 region produces a dramatic increase in pulmonary infiltration compared to the wild-type virus (Ginsberg et al., 1987), consistent with the hypothesis that the E3encoded products alter immune recognition of the virus in order to avoid immune signaling that would lead to subsequent lymphocyte infiltration. Further studies using additional adenovirus deletion mutants in the cotton rat model, and potentially other model animal systems, may clarify the role of adenovirus modulation of MHC-I expression in virus virulence and persistence as well as shed light on the nature of immunopathologies that sometimes accompany adenovirus infection. IV. Herpesvirus: The Cytomegalovirus Model

A. INTRODUCTION Most human herpesviruses are kept in check by cell-mediated immune mechanisms, and impaired 'T cell function frequently leads to fulminant infections (Doymaz and Rouse, 1992; Moss et al., 1992; Rickinson et al., 1992). Herpesvirus strategies to combat cellular immunity include a variety of mechanisms designed to prevent or alter MHCrestricted presentation of viral antigens (Rinaldo, 1990; Banks and Rouse, 1992). For example, virus-specific down-regulation of MHC antigens has been reported for herpes simplex, types 1 and 2 (Jennings et al., 1985; Kuzushima et al., 1990), Epstein-Barr virus (Masucci et al., 1987, 1989; Anderson et al., 1991; Gavioli et al., 1992), and cytomegalovirus (CMV). Because relatively more is known about the role of

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CMV with respect to MHC expression than is known for the other herpesviruses, this section will focus on the CMV model. Cytomegalovirus is ubiquitous in the human population, but in the majority of cases the virus remains in a latent state or causes only subclinical infections. However, in situations involving immunocompromise, particularly defects in cellular immunity, the virus can enter into productive replication in a variety of tissues and cell types (Griffiths and Grundy, 1987; Alford and Britt, 1990). Although CMV latency has been associated with lymphocytes and cells of the macrophage/monocyte lineage, a variety of nonhemopoietic cells, such as ductal epithelia, renal tubules, NK cells, and fibroblasts, can support viral replication to various degrees (Grundy, 1990). The virus has a propensity to penetrate the placental barrier and induce congenital birth defects, and reactivated CMV is a major pathogen in transplant recipients undergoing immunosuppressive therapy. The replication of the virus within some cell types, such as those in the retina, is associated with direct virus-induced cytopathology; in other tissues, such as lung tissue, damage is the primary consequence of responsive immune cells (Waner, 1989; Landini and La Placa, 1991). CMV has particular affinity for major APCs such as monocytes/macrophages and can down-regulate cytokine release from these cells, thus contributing to overall immune dysfunction (Pasternack et al., 1990). The fully productive CMV replication cycle involves successive waves of viral gene expression, denoted immediate-early, early, and late (Mocarski, 1988; Stinksi, 1990), but some target cells, such as monocytes, are only semipermissive, i.e., immediate-early and early gene expression is relatively low, whereas other cell types, such as T and B lymphocytes, are relatively nonpermissive but do permit some transcription of immediate-early genes. Thus, the extent to which CMV can enter into persistence within a given immunocompetent host is critically dependent on (1) the extent of virus permissivity of the susceptible cell populations, (2) the ability of the CMV strain to depress lymphocyte and APC function, especially cytokine release and responsiveness, and (3) the load of infecting virus (Blanden, 1988; Alford and Britt, 1990; Koszinowski et al., 1992). In humans, CMV disease can become a clinical problem in various clinical situations, including leukemias, lymphomas, AIDS, organ transplants, and bone marrow allografts. Patients with clinical CMV syndromes frequently exhibit highly impaired test scores for cellular immunity, such as T cell proliferation in response to antigen or mitogens, elaboration of IFNy, antibody-independent NK cell killing activity [although not antibody-dependent cell-mediated cytotoxicity (ADCC)], IFN induction by heterologous viruses, the ability to mount an antibody

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response to foreign antigens, and the generation of virus-specific CTLs (Pasternack et al., 1990; Grundy, 1990; Alford and Britt, 1990; Koszinowski et al., 1993). Furthermore, CMV is a positive risk factor for graft rejection, particularly in renal transplant patients and graftversus-host disease in bone marrow transplants (von Willebrand et al., 1986; Griffiths and Grundy, 1987; Grundy, 1990).In contrast to the risks of CMV recrudescence in patients with compromised cellular immunity, CMV is not a major pathogen in patients with exclusive defects in humoral immunity (Alford and Britt, 1990; Landini and La Placa, 1991). A critical factor in the clinical path to either persistence or systemic CMV infection lies in the ability of CMV to induce interferon. Both classes of IFN (alp and y) play a major role in mediating CMV clearance and also are important inducers of MHC-I and MHC-I1 expression in infected tissues. Not only is MHC up-regulation critical for viral antigen presentation, but there have been some claims that cellular MHC molecules could function directly as receptors for CMV entry (Grundy et al., 1987b). This latter claim, however, remains controversial, and Beersma et at. (1991, 1992) have reported the absence of a correlation between human CMV infectivity and HLA-I expression levels at the cell surface. On the other hand, MHC-I expression levels can be correlated with susceptibility to infection by murine CMV, and recent data indicate that infectivity is dependent on surface class I conformations (Wykes et al., 1993). Although the virus encodes a homologue of the MHC-I heavy chain (Beck and Barrell, 1988), there is no evidence that either this viral gene product or cellular class I heavy chain is associated with CMV virions (Rose and Grundy, 1992). Nevertheless, virions have been reported to bind host P2M and CMV infection induces the release of P2M from the surface of infected cells (Grundy et al., 1987a; McKeating et al., 1987; Yamashita et al., 1992). RESPONSE B. CELLULAR IMMUNE TO CMV INFECTION

In humans, primed T cells specific for CMV immediate-early gene products have been detected in peripheral blood and CD8+ CTLs in particular are believed to play a major role in protection and clearance of CMV (reviewed by Grundy, 1990; Koszinowski et al., 1993).In the case of bone marrowlrenal transplant recipients, HLA-restricted CTLs and NK cells are both believed to contribute to clearance of virus infected cells and recovery (Alford and Britt, 1990; Koszinowski et al., 1992). In the murine model, CTLs that recognize the pp89 immediate-early protein are the predominant species and a dominant nonapeptide epitope has

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been mapped (del Val et al., 1988). I n adoptive transfer experiments, pp89-specific CD8+ CTLs can limit virus spread and protect immunosuppressed mice from lethal infections (Reddehase et al., 1987), although clearance from the salivary gland uniquely requires CD4+ T cells and is likely mediated by IFN--y (Lucin et al., 1992). Vaccinia virus vectors have been used to express individual murine CMV epitopes derived from pp89, and live recombinant virus vaccination has been shown to provide protection to CMV challenge in mice (JonjiC et al., 1988; del Val et al., 1991b). When the immunodominant pp89 nonapeptide was linked to a foreign protein, the hepatitis B core antigen, the sequence context of the epitope within the fusion construct was critical for functional presentation of the CMV determinant, suggesting that the number of viral determinants that can be presented to T cells is directly related to the proteolytic machinery that cleaves out the appropriate peptide for entry into the MHC-I assembly pathway (del Val et al., 1991a). Interestingly, the presentation of the dominant pp89 epitope to T cells by murine CMV-infected fibroblasts in vitro is inhibited during early gene expression under conditions in which MHC-I surface expression (Ld) is unaffected. This has led to the hypothesis by Koszinowski’s group that productively infected cells do not present pp89 epitopes during most of early phase in vitro, but that in vivo functional presentation is by APCs harboring viral replication that is arrested in the immediate-early phase and thus these APCs d o not express early or late gene products (del Val et al., 1989). T h e nature of the early phase inhibition of pp89 presentation is unknown but could reflect either an inhibitory protein that directly perturbs some aspect of peptide/MHC assembly o r else the overloading of the system with an early viral peptide that is nonimmunogenic but that effectively outcompetes the pp89 nonapeptide for binding to the available MHC molecules. A similar early phase block to the presentation of heterologous antigen, namely, epitopes from SV40, has been noted in CMV-infected mice that were immune to SV40 and in CMV-infected macrophage and fibroblasts that had been transformed by SV40 (Campbell et al., 1989, 1992; Slater et al., 1991). This blockade to the class I-restricted presentation of SV40 epitopes was also detected at early stages of CMV replication (Campbell et al., 1992), suggesting that the interference was indeed derived from murine CMV early gene product($. In healthy seropositive humans, CD8+ CTL response is principally against a 72-kDa irnmediate-early gene product and a virion glycoprotein (gB) that can be directly introduced into the class I peptideprocessing pathway after viral penetration (Borysiewicz et al., 1988; Riddell et al., 1991). Note that in the mouse CMV model, the type of CTL

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response measured is to acute high-multiplicity virus challenge in an immunologically naive host, whereas in the human population that bears latent CMV, the CTL response may reflect epitopes important for the prevention of virus reactivation from reservoir cells, probably lymphocytes and macrophages, that harbor quiescent viral genomes. The importance of CD8+ CTL for limiting CMV-induced pathology in humans was recently highlighted by the observation by Riddell et al. (1992) that adoptive transfer of CTL clones that recognize CMV structural proteins would provide protection against CMV disease in human bone marrow transplant recipients. The propensity of CMV to subvert the cell-mediated immune response is typified by the loss of CMV-infected peripheral monocyte function and responsiveness to IFN-y during acute disease (Schrier and Oldstone, 1986; Buchmeier and Cooper, 1989). Infection of primary monocytes by human CMV can result in impaired presentation of heterologous antigens and loss of MHC class I1 induction by IFN-y but not the ability to elaborate interleukin-1 (Buchmeier and Cooper, 1989). C. CMV REGULATION OF MHC-I The ability of CMV to either up-regulate or down-regulate surface MHC-I molecules is controversial, and the literature is filled with seemingly contradictory observations. In some cases, the differences can be explained by variations in the multiplicity of infection in nitro, because at low multiplicities infected cells release IFN-P, which is a potent MHC inducer on neighboring uninfected cells. Although this paracrine phenomenon may be extremely important in the development of latency or the progression to more severe clinical syndromes, especially as it relates to graft rejection, the interpretation of the molecular basis for CMValtered MHC expression remains a challenge. Further complicating the biological relevance of MHC modulations is the fact that NK cells are also important for CMV clearance, and loss of MHC-I molecules from infected cell surfaces may in fact potentiate NK recognition and killing. CMV infection, for example, has been associated with increases in expression of adhesion molecules, such as LFA-3 and ICAM-1, which are required for both HLA-restricted and nonrestricted killing of infected cells (Hutchinson et al., 1991; Grundy and Downes, 1993; Grundy et al., 1993; van Dorp et al., 1993). As an example of how MHC-I levels can be subject to experimental conditions, infection of human endothelial cells with CMV was initially believed to cause a large increase in MHC-I expression (van Dorp et al., 1989; Tuder et al., 1991), but later studies by Scholz et al. (1992) sug-

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gested that class I level increases were only marginal, although surface ICAM- 1 levels were significantly augmented. Similarly, Grundy et al. (1988) originally observed large increases in MHC-I levels in CMVinfected fibroblasts, but later experiments showed the increases could be explained by the secondary action of IFN-P released from infected cells on neighboring uninfected cells (Barnes and Grundy, 1992). Large increases in surface MHC-I levels observed in CMV-infected aortic smooth muscle cells (Hosenpud et al., 1991) and primary lung epithelial cells (Ibrahim et al., 1993) can probably also be explained by the paracrine effects of induced IFN-P. Although there is evidence that CMV immediate-early gene products can positively transactivate the MHC-I promoter in transfected cells (Burns et al., 1993), there is as yet no evidence that this up-regulation plays a role in viva In recent experiments to address the question of MHC-I expression in CMV-infected human embryonic lung,fibroblasts, Yamashita et al. (1993) showed that at a high multiplicity of infection there was a marked loss of surface class I molecules to almost background levels by 72 hours postinfection, but no obvious alteration in the cytoplasmic expression of class I heavy chain or P2M. They also demonstrated that late viral gene expression was not needed for surface MHC-I down-regulation and, importantly, infection with UV-inactivated CMV caused an enhancement of MHC-I surface levels. This has led to the hypothesis that semior nonpermissive conditions for viral replication can lead to upregulation of class I molecules while permissive conditions lead to downregulation (Yamashita et al., 1993). In related experiments, Barnes and Grundy (1992) showed in fibroblasts infected with human CMV at high multiplicities that nascent MHC-I molecules became trapped in a perinuclear compartment, most likely the Golgi complex. As a result, surface class I molecules dropped to very low levels, while several surface adhesion molecules (ICAM-1, LFA-3) actually increased (Grundy and Downes, 1993). The nature of the apparent blockade to surface expression was investigated by del Val et al. (1992),who showed that transport of MHC-I molecules through the Golgi apparatus was arrested in mouse embryo fibroblasts infected at high multiplicity with murine CMV. Importantly, although MHC-I molecules were no longer transported to the surface, intracellular class I heavy chain and P2M levels were unaffected and the dominant pp89 nonapeptide was still generated. Despite the presence of all three components required for the class I trimolecular complex, infected cells were unable to present the pp89 epitope to MHC-I (Ld)-restrictedCTLs. To test whether there was a generalized presentation defect, del Val et al.

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(1992) inserted a lac2 gene under the control of an immediate-early promoter into a recombinant CMV and showed that, at immediate-early times, presentation of either lacZ or pp89 was unaffected, whereas at early times functional presentation of both epitopes was inhibited. Although the peptide epitopes were properly liberated and associated with MHC-I (Ld), the glycosylation profile of Ld was altered (del Val et al., 1992). MHC-I normally acquires a high mannose core of N-linked oligosaccharides in the ER, but during CMV early phase the majority of MHC-I molecules remained sensitive to endoglycosidase H, suggesting that the trimolecular MHC-I complexes did not transit into the medial Golgi compartment. This blockage was observed for a variety of class I molecules (Dd, Kd, Ls, Kb, Db, Kk, and Dk), whereas other surface molecules such as transferrin receptor (CD71) and CD44 were unaffected. Thus, in some respects, the CMV blockade is reminiscent of the gp19/E3 retention of MHC-I by Ad2, although no CMV counterpart to gp19 has yet been identified. One appealing model to explain these results is that the function of a chaperone molecule such as p88 (Degen and Williams, 1991; Degen et al., 1992) might be directly affected by CMV early gene product(s). A related but apparently distinct blockade of the presentation of the major immediate-early 72-kDa protein of human CMV to CTLs was reported by Gilbert et al. (1993),who provided evidence that anti-72-kDa CTLs were underrepresented and that 72-kDa epitopes were poor stimulators of T cell responses. Furthermore, the anti-72-kDa CTLs that were isolated recognized CMV-infected cells poorly at all times during the lytic cycle, unlike CTLs against other CMV antigens, such as glycoprotein B, suggesting a specific defect in 72-kDa peptide processing or an inability of 72-kDa peptide epitopes to compete for MHC-I binding (Gilbert et al., 1993). D. CMV

REGULATION

OF

MHC-I1

In contrast to the case of MHC-I, there is little evidence to suggest that CMV directly affects MHC-I1 expression. However, CMV-infected human endothelial cells become refractory to the ability of IFN-y to induce class I1 antigens (Scholz et al., 1992), which would thus functionally preclude recognition by class II-restricted T cells. Sedmak et al. (1990) detected no induction of MHC-I1 in human umbilical vein endothelial cells infected with CMV, and speculated that IFN-P induction might suppress any effects of IFNy on CMV-infected monocytes. In contrast, van Dorp et al. (1989) observed no impairment of IFN-y induc-

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tion of class I1 molecules in CMV-infected human endothelial cells, but the lower multiplicity of infection used in these experiments makes the interpretation of these results difficult. Unlike the above cases, Arnold et al. (1991) observed an increase in HLA-DR in the bile duct epithelium of liver transplant recipients and Ustinov et al. (1993) reported that cultured rat heart endothelial cells exposed to rat CMV exhibited increased MHC-I1 expression. Although the direct cause of these inductions is not established, indirect effects of secondary cytokines is very likely, especially during episodes of allograft rejections (von Willebrand et al., 1986). On the other hand, the apparent induction by CMV of a state that is refractory to class-I1 induction by IFN-y (Scholz et al., 1992) is reminiscent of the blockade of IFN upregulation of MHC-I and MHC-I1 by the hepatitis B virus polymerase gene in hepatocytes (see later), and suggests that CMV gene product(s) may play a direct role in this process as well.

E. POSSIBLE MECHANISMSOF MHC REGULATIONBY CMV The first suggestion that CMV proteins might directly perturb MHC expression came from the observation that human CMV encodes a homologue of the MHC-I heavy chain (Beck and Barrell, 1988). T h e 67kDa protein product of the UL18 open reading frame was coexpressed from vaccinia virus vectors with P2M and shown to migrate to the surface as a heterodimer, leading to the prediction that UL 18 might interfere with proper MHC-I assembly and transport to the surface (Browne et al., 1990). However, Barnes and Grundy (1992) and Yamashita et al. (1993) could find no evidence for UL18 binding to P2M in infected cells, and when Browne et al. (1992) created a UL18 deletion in human CMV the resulting recombinant virus was unaffected in its ability to downregulate MHC-I expression in vitro. Indeed, these latter authors were unable to detect the translated UL18 protein product at all in wild-type CMV-infected cells by Western analysis. Taken together, these results suggest that UL18 probably does not play a role in class I downregulation in fibroblasts in vitro, but it remains to be shown whether a comparable deletion in murine CMV might have an effect on antigen presentation from an APC subset in infected animals. At present, the explanation for the observed down-regulation of class I molecules in cells infected at high multiplicity by CMV is unknown, but there is reason to suspect that specific viral gene products either cause trapping of heavy chain&M/peptide complexes in a cis-Golgi/ER com-

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partment or else interfere with the transport machinery that carries trimolecular complexes through the Golgi apparatus and to the cell surface (Barnes and Grundy, 1992; del Val et al., 1992; Yamashita et al., 1993). Given the relative paucity of information regarding the role of chaperone proteins such as p88 in MHC-I transport, it is premature to speculate how CMV proteins might interfere with this process. However, identification of CMV gene products that bind or colocalize with host proteins other than class I molecules in the ER/Golgi might very well provide clues as to the molecular nature of this blockade. The recent evidence that the p88 chaperone protein that transiently binds MHC-I molecules in the ER is the product of the calnexin gene (Calvin et al., 1992) and that p88 participates in the assembly of class I molecules (Krishna et al., 1992; Degen and Williams, 1991; Degen et al., 1992; DeNagel and Pierce, 1993) suggests that this aspect of the transport pathway is an attractive target for putative CMV inhibitory gene product(s).

V. Hepatitis 6 Virus A. INTRODUCTION Hepatitis B virus (HBV) can latently infect lymphocytes but replicates productively only in hepatocytes and is a major causative agent for chronic and acute liver disease, including hepatic necrosis, cirrhosis, and hepatocarcinoma (reviewed in Hollinger, 1990; Schroder and Zentgraf, 1990; Sherker and Marion, 1991; Koshy, 1992; Slagle et al., 1992; Yoffe and Noonan, 1993). The human HBV DNA genome is 3.2 kb in length and encodes four open reading frames: surface antigen, or env, which is expressed as three overlapping proteins; core antigen, or nucleocapsid, which is expressed as a cell-associated c-antigen and a secreted e-antigen; polymerase/reverse transcriptase; and X, a transactivator of gene expression (Robinson, 1990). In addition to serving direct functions for the virus, several of these gene products have the capacity to regulate viral and cellular gene expression, either directly or indirectly (Robinson, 1990; Barnaba and Balsano, 1992). The majority of neonates, and about 4-5% of adults, that become infected with HBV are unable to clear the virus infection and become chronic carriers. In the initial carrier state, high level9 of virus replication occur in infected hepatocytes but the virus is not cytopathic per se and there is relatively little liver damage. Later, as viral replication recedes, liver damage caused by inflammation and necrosis becomes more

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prominent and at this point the disease can either resolve, become persistent, or progress to cirrhosis or hepatocarcinoma (Escobar, 1988; Rogler et al., 1989; Desmet, 1991). When HBV develops into persistence, partial immune dysfunction in the host is frequently observed, characterized by reduced levels of interferon production or responsiveness and altered lymphocyte function (Thomas, 1989; Alexander, 1990; Stremmel et al., 1991; Sylvan, 1991). Chronic hepatitis is frequently associated with reduced levels of induced IFN-y and loss of sensitivity of hepatocytes to IFN is common (Finter et al., 1991; Baron et al., 1991; Martin and Friedman, 1992; Braken et al., 1992). Recovery from HBV is dependent on the interplay of the host IFN system with cellular immunity, and failure to clear virus is frequently associated with low interferon production and/or T cell deficits, especially as found in the very young or elderly, immunosuppressed patients, subjects of chemotherapy, Down’s syndrome, and patients with AIDS or certain malignancies. In some cases of depressed cellular immunity, active virus replication may proceed in the absence of liver disease and the status of such asymptomatic carriers can be revealed by the persistence of circulating surface antigen and e-antigen (Thomas, 1989; Alexander, 1990; Sylvan, 199 1; Desmet, 199 1). Frequently in these cases, a strong nonresponsiveness (tolerance) to env epitopes results in a failure to elicit antibody to surface antigen (Milich et al., 1990). Chronic HBV that is characterized by continuous synthesis of viral DNA, e-antigen and polymerase becomes clinically important when liver damage caused by necrotic fibrosis, persistence of inflammatory cells/infiltrating CTLs, and progressive cycles of liver cell regeneration leads to hepatic dysfunction and, often, neoplasia (Sherker and Marion, 1991; Slagle et al., 1992; Koshy, 1992). Considerable data exist to support the notion that the cell-mediated immune response to HBV antigens, or in some cases self-antigens, is a principal mediator of the hepatic destruction associated with HBVinduced hepatitis rather than direct viral cytopathology (Robinson, 1990; Alexander, 1990; Desmet, 1991). For example, in transgenic mice that express the HBV surface antigens (Moriyama et al., 1990), adoptive transfer of virus-specific class I-restricted CTLs can cause hepatocyte destruction reminiscent of HBV-associated hepatitis (reviewed in Barnaba and Balsano, 1992). Class I- and 11-restricted CTLs against surface and nucleocapsid antigens have been detected in the circulation of HBV-infected pqtients (but not asymptomatic carriers) and are believed to mediate at least a component of the liver damage (Sylvan, 1991). Furthermore, the major infiltrating cells in necrotizing liver sections are CTLs, frequently in contact with hepatocytes in the portal and periportal areas (Robinson, 1990).

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B. CELLULAR IMMUNE RESPONSETO HBV INFECTION MHC-I- and MHC-11-restricted epitopes have been mapped on a variety of HBV proteins, particularly the nucleocapsid and surface antigens (Penna et al., 1991, 1992; Bertoletti et al., 1991, 1993; Kamogawa et al., 1992; Missale et al., 1993; Yamauchi et al., 1993; Nayersina et al., 1993). HBV-infected hepatocytes frequently express both MHC-I and MHC-I1 antigens and thus both CD4+ and CD8+ CTLs can be of importance in causing liver pathology (MiIich, 1988; van den Oord et al., 1990). Because class I MHC can be induced by both IFN-odP and IFN-y, whereas class I1 MHC is inducible by only IFN-y, IFN-P production from infected hepatocytes and IFN-y elaborated by infiltrating CTLs and NK cells profoundly affect cytotoxicity levels in infected livers. Chronic HBV carriers frequently show a defect in both IFN elaboration and responsiveness (Kerr and Stark, 1992), and IFN-a therapy has been used clinically to up-regulate virus-specific CTL function and down-regulate viral replication (Bonnem, 1991; Muller, 1991; Martin and Friedman, 1992; Braken et al., 1992; Perrillo, 1993). There is some evidence that HBV can directly reduce hepatocyte responsiveness to IFN, and transfected viral DNA has been shown to mimic this loss of sensitivity (Onji et al., 1989). Two of the major consequences of IFN action are the induction of the antiviral state, which limits virus spread, and up-regulation of cellular MHC antigens, which facilitates clearance of infected hepatocytes by CTLs. The HBV nucleocapsid antigens are particularly important targets for class I-restricted CTLs in areas of spotty necrosis, whereas the surface antigen is an important epitope for both cellular and humoral immunity. The surface antigen in particular is of interest because, unlike most viral proteins, the extracellular form can directly access the ER/Golgi processing pathway after internalization by uninfected APCs and can be presented by class I molecules to CD8+-restricted T cells (Jin et al., 1988). C. HBV REGULATION OF MHC-I Uninfected hepatocytes have been reported to have low or nondetectable levels of MHC-I antigens, but some reports have suggested that class I molecules are present but difficult to detect by some methods (reviewed by van den Oord et al., 1990). Multiple lines of evidence, however, suggests that a variety of liver diseases, including those caused by HBV, markedly up-regulate surface class I molecules, especially on the basolateral membrane of hepatocytes and on bile duct cells

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(Nagafuchi and Scheuer, 1986; Pignatelli et al., 1986; Chu et al., 1987; Franco et al., 1988; Paul et al., 1991; Krawitt et al., 1991; Nonomura et al., 1992). In the case of the HBV-infected chimpanzee model, a rise in interferon levels in the serum is correlated with a concomitant rise in MHC-I expression throughout the liver lobule (van den Oord et al., 1990). I n cases of human patients with chronic HBV infection, MHC-I induction is frequently observed but levels in posthepatic cirrhosis are variable. Patients with high levels of viral replication, however, frequently display low levels of HLA-A, -B, and -C antigens when levels of HBV surface antigen on liver cell membranes and core antigen in nuclei are high, suggesting an inverse correlation between MHC-I and viral gene expression (van den Oord et al., 1990; Lau el al., 1993). Such conclusions are not uniformly accepted, however, and reports of variable MHC-I levels in livers of infected children suggest caution in overinterpreting these results (Lobo-Yeo et al., 1990; Senaldi et al., 1991). Both type I and I1 interferons are believed to play a role in MHC-I induction in HBV-infected livers. In acute and chronic hepatitis, treatment with IFN-ol frequently causes MHC-I levels in hepatocytes to increase in patients that respond to treatment by resolving the infection (Paul el al., 1991; Caselmann et al., 1992). Because induced levels of MHC-I on peripheral blood lymphocytes tend to rise more acutely than do those of hepatocytes in response to interferon treatment, it has been suggested that HBV replication in hepatocytes specifically switches off the MHC-I induction pathway by interferon (Ikeda et al., 1986; Onji et al., 1989). However, normal carriers with antibody to e-antigen do not show reduced levels of MHC-I on hepatocytes and elimination of surface antigen is unrelated to class I levels (van den Oord et al., 1990). These cases are difficult to interpret because actual levels of HBV transactivator gene products in diseased livers (see later) are not known. There are several examples wherein HLA-A, HLA-B, and HLA-C are shown to become elevated only after seroconversion when viral replication drops and core/e-antigens disappear (Thomas, 1989). At late stages of HBV-induced hepatic damage, when viral replication is fully suppressed, MHC-I expression is frequent and almost all HBV-associated hepatic carcinomas express class I heavy chain and &M, in dramatic contrast to adjacent normal liver tissue (Paterson et al., 1988). In an important experiment, Takehara et al. (1992) showed that in the HB611 cell line, which contains multiple integrated HBV genomes, replication of HBV inhibited expression of MHC-I antigens and treatment with acyclovir o r IFN-y caused a rise in MHC-I levels concomitant with a decrease in HBV expression. Thus, it seems reasonable to postulate that MHC-levels in HBV-infected liver tissues is a consequence of a tug-of-

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war between the up-regulation effects of IFN-f3 from infected hepatocyteslIFN-y from infiltrating lymphocytes, and a competing downregulation effect of viral gene products that dampen MHC-I expression directly and/or reduce cellular responsiveness to the local interferons. D. HBV REGULATIONOF MHC-I1 There is generally uniform agreement that MHC-I1 levels in uninfected hepatocytes are either very low or absent, but can be induced in chronic liver disease (van den Oord et al., 1990). Class 11+ hepatocytes can present viral antigens to T cells and CD4+ CTLs may play a role in the hepatocyte lysis frequently associated with viral hepatitis. Penna et al. ( 1992) have described surface antigen-specific class II-restricted CTLs in HBV-infected patients. The majority of macrophage-like Kuppfer cells in the liver are HLA-DR, DQ, DP+ and have the capacity to prime class II-restricted T cells with processed viral antigens. Normal HLA-DRhepatocytes express DR antigen in response to IFN-y, but not IFN-a, and thus infiltrating lymphocytes or N K cells may be responsible for the HLA-DR induction frequently observed in HBV liver disease, especially in areas of piecemeal and spotty necrosis (van den Oord et al., 1986; Lobo-Yeo et al., 1990; Senaldi et al., 1991).By and large, MHC-I1 antigen is found only on hepatocytes in which MHC-I levels are also enhanced, again consistent with a role for IFN-y, which is a potent inducer of both MHC-I and MHC-11. E. POSSIBLE MECHANISMSOF MHC REGULATION BY HBV

Several HBV gene products have been described to have transactivator/transrepressor activities that could possibly affect MHC gene induction or interfere with IFN expression and/or signal transduction. 1 . Polymerase/Termina Protein

To study the mechanism whereby transfected HBV DNA reduces the responsiveness of transfected cells to interferon, Foster e? al. (1991) expressed each HBV gene independently and showed that only the polymerase gene could induce this inhibitory effect. Furthermore, the polymerase N-terminal 150-amino acid domain (called terminal protein), which lacks the reverse transcriptase and RNaseH activities, could both down-regulate the response of transfected cells to IFN-(r and IFN-y and also abrogate the ability of dsRNA to induce IFN-P. Analysis showed that the production and/or activation of the a subunit of TF-E (ISGFS),

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a transcriptional factor that mediates IFN signal transduction, was affected and that MHC-I or MHC-I1 levels could not be induced under these conditions. More recently, expression of terminal protein in liver biopsy specimens of patients chronically infected with HBV was found to be correlated with an inability of interferon to induce P2M, suggesting that this viral protein may be a good clinical marker for interferon responsiveness (Foster et al., 1993).

2. Nucleocapsid (Corele) Protein Whitten el al. (1991) reported that in cells carrying an expression vector that overexpressed core/e-antigen, IFN-P induction was inhibited and that comparable HBV polymerase gene constructs had no effect. Direct trans-repression of core/e-antigen on the IFN-P promoter (Twu et al., 1988; Twu and Schloemer, 1989) would be of important biological significance and should be confirmed with constructs in which the terminal protein open reading frame domain has been disrupted. It should be noted that the N-terminal region of the polymerase gene overlaps in another reading frame with the C terminus of the nucleocapsid gene in HBV and the transfected core constructs tested could, in theory, express remnants of the terminal protein domain. Thus the question of whether polymerase and/or core protein are the sole mediators for the repression of IFN inducibility remains to be resolved. 3. X Protein The HBV-X protein bears some similarities to other virus-encoded transcriptional activators, such as HIV-1 tat, which have the potential to up-regulate a variety of viral and cellular promoters (Gilman, 1993).The cellular promoters known to be up-regulated by X protein of relevance to this discussion include those for MHC-I, MHC-I1 (HLA-DR), and IFN-P (Twu and Schloemer, 1987; D. X. Zhou et al., 1990; Hu et al., 1990). These inductions are believed to occur by the activation of multiple transcription factors, such as NF-KB,CREB, ATF-2, AP1, and AP2 (Twu et al., 1989; Seto et al., 1990; Unger and Shaul, 1990; Maguire etal., 1991; Lucito and Schneider, 1992; Gross et al., 1993). There is one, as yet unconfirmed, report of serinehhreonine kinase activity for X protein (Wu et al., 1990).More recently, KekulC et al. (1993) provide evidence that the X protein triggers a phospholipase to generate diacylglycerol and facilitate the translocation of protein kinase C to the membrane. In the case of the MHC-I promoter, D. X. Zhou et al. (1990) have shown that promoter targets for X transactivation include the interferon response sequence and a motif responsive to H ~ T F ~ / N F - KIn B .the case of the

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HLA-DR promoter, there are no NF-KBor AP2 domains, but X is a potent trans-activator of DR expression, suggesting that levels of X expression may also play a role in regulating the magnitude of class IIrestricted responses against infected hepatocytes (Hu et al., 1990). Another potential biological effect of class I1 inductiov is that upregulation of HLA-DR on hepatocytes, which normally do not present class 11-restricted antigens, might induce T cell anergy because of the absence of the costimulatory signals required to prime T cells. 4 . Truncated Suface Antigen

Intact HBV surface antigen is not a trans-activator, but many integrated versions of the HBV genome express a 3’-truncated version of middle surface antigen that possesses novel trans-activation activities. Although the truncated surface antigen is synthesized as an integral membrane component associated with the HBV envelope, the modified protein can trans-activate a variety of promoters (e.g., c-myc and c-fos) and up-regulate genes controlled by NF-KB,including a number of cytokines and cytokine receptors (Caselmann et al., 1990; KekulC et al., 1990; Meyer et al., 1992; Lauer et al., 1992). Although not yet formally demonstrated, the possibility exists that positive up-regulation of NF-KBcould also induce the MHC-I promoter in cells bearing truncated surface antigens.

5 . HBV-DNA Integrations During progression from HBV-induced hepatitis to hepatocarcinoma, viral DNA becomes integrated into the cellular DNA of the majority of tumor cells (Sherker and Marion, 1991; Slagle et al., 1992). Although the insertional mutagenesis model of HBV transformation has been difficult to establish, in the woodchuck hepatitis model activations of c-myc and N-myc are observed in the majority of tumor cells. In human hepatocarcinomas, insertion sites near a variety of regulatory genes, such as retinoic acid receptor and cyclin A, have been reported (reviewed by Koshy, 1992; Buendia, 1992). In theory, two divergent pathways could independently alter MHC expression: ( 1) direct repression of the MHC-I promotor by activated N-myc (van’t Veer et al., 1993) and (2) up-regulation of the MHC-I promoter by the action of truncated surface antigen. Given the observation that almost all hepatocarcinomas overexpress class I heavy chain and P2M (Paterson et al., 1988), the latter pathway seems more likely, but a variety of explanations for MHC-I overexpression that do not involve HBV gene products are possible. In summary, there is evidence to suggest that HBV can both upregulate and down-regulate MHC expression through the concerted

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action of several viral proteins. Because HBV pathogenesis follows a complicated and variable series of MHC antigen variations, and the virus has a documented ability to regulate the response of infected cells to the major MHC-inducing cytokines (IFN-a/g and IFN-.I), it is likely that the eventual response reflects a summation of viral host and microenvironment influences, and the level of CTL damage in an individual case will reflect the unique balance of these parameters in the affected liver tissues.

VI. Papillomavirus A. INTRODUCTION Papillomaviruses (PVs) are highly restricted to the squamous epithelial cells of skin and mucosa, and replicate within differentiating keratinocytes as the infected cells migrate from basal layers of the epidermis to the surface (reviewed by Howley, 1990; Shah and Howley, 1990). These ubiquitous viruses are frequently present in latent form within normal epithelium but under complex and poorly understood conditions, involving both environmental and genetic factors, become activated to induce hyperplastic papillomatory lesions that can either regress, persist in relatively stable state, or else progress onto more malignant neoplasia, notably cervical carcinomas (zur Hausen, 1989; Howley, 1991; Gissmann, 1992; Richart and Wright, 1992). During this progression to increased malignancy, viral DNA is frequently found integrated into the DNA of tumor cells and only a subset of viral genes, especially E6 and E7, are found to be expressed (zur Hausen, 1991; Campo, 1992). There is no viremia associated with viral replication in lesions and the infected cells are relatively inaccessible to the elements of the immune system that are not associated with the skin. Thus, the principal mediators of the immune reactions to PV infection are the keratinocytes, intraepithelial lymphocytes, and the dendritic Langerhans cells (Vardy et al., 1990; Jenson et d.,1991; Roche and Crum, 1991). Because cervical cancer is the second most common female cancer, and certain human papillomavirus (HPV) types (especially T16/ 18) are highly associated with these tumors, the mechanism(s) of how the immune system responds to HPV infection is an important medical issue (Arends et al., 1990). There are over 70 types of HPV, based on host range and DNA homologies, and several animal model systems, especially cottontail rabbit PV and bovine PV, have been useful in defining how these viruses interact with the immune system. For example, the progression of rabbit

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PV lesions to squamous carcinoma is linked to the genetic background of the individual rabbit, in particular to markers that map within the MHC (Han et al., 1992). This is an important model system in view of the fact that only known correlation in humans between cancer progression and MHC is an increased frequency of cervical carcinomas associated with HLA-DQw3 (Wank and Thomssen, 1991; Wank et al., 1992). In patients with nonprogressing warts, anti-HPV antibodies are generally of low titer, suggesting that there is limited antigenic stimulation by the virus unless skin trauma induces the liberation of viral antigens (Barbanti-Brodano, 1989). In the case of the rabbit PV model, carcinogenic progression is accompanied by variable levels of antibody response to the viral proteins, but the antibodies have little or no ability to induce regression (Lin et al., 1993). In fact, much evidence exists to indicate that the capacity to clear HPV infection lies at the level of cellular immunity (Vardy et al., 1990; Jenson et al., 1991).

B. CELLULAR IMMUNE RESPONSETO PV INFECTION When HPV infections fail to resolve, depressed cellular immune function is frequently observed, as measured by phytohemagglutin activation of T cells, CD4/CD8 ratios, total T cell count, leukocyte migration inhibition tests, and NK activity (Vardy et al., 1990; Woodworth et al., 1992). During lesion regression, enhancement of both in uivo and in uitro tests is observed and a strong positive correlation exists between increased T cell activation and virudtumor clearance. Some of this balance between regression/progression is determined by the genetic background of the virus. For example, tests to measure cell-mediated immunity are generally unaffected by HPV-1, -4,and -7, but are altered by HPV-5 and HPV-8, and surgery for the latter group often results in improvement of cellular immune function (Acs et al., 1988). The activity of sentinal APCs, in particular Langerhans cells, is also relevant to the clinical outcome (Viac et al., 1990; Roche and Crum, 1991). In some regressing HPV-associated warts, Langerhans cells become morphologically activated, whereas in other wart conditions, their total number can either rise or fall dramatically (Acs et al., 1988; Vardy et al., 1990; Viac et al., 1992). Patients with depressed or compromised cellular immunity are at greater risk for HPV-associated complications. For example, Hodgkin’s patients, chronic lymphocytic leukemia patients, HIV-infected patients, organ transplant recipients, and patients undergoing immunosuppressive chemotherapy are at greater risk for HPV warts and progression to

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carcinomas than are patients with humoral immune deficiencies (Vardy et al., 1990; Howley, 1990; Jenson et al., 1991). A variety of T cell epitopes have been mapped within HPV proteins (e.g., Strang et al., 1990; Meneguzzi et al., 1991; Chen et al., 1991, 1992b; Zhou et al., 1991; Altmann et al., 1992). Many of these T cell epitopes function as tumor rejection antigens and mediate regression of HPVassociated tumors. It was shown that costimulatory signals provided by B7 on cells that present HPV-16 E7 antigen to T cells are required to induce HPV tumor regression, and the absence of B7 resulted in T cell clonal anergy (Chen et al., 1992a; Travis, 1993).The ability to respond to HPV antigens therefore revolves around the capacity of infectedhransformed cells to effectively present viral epitopes to T cells, and HPV regulation of surface MHC-I and MHC-IT levels is an important parameter in the overall cellular immune response (see also Bangham and McMichael, 1989; Melief 'and Kast, 1990, 1992; Hoglund et al., 1990; McMichael, 1992a, for more detailed discussion of HPV antigens during tumor regression). C. PV

REGULATION

OF

MHC-I

Reductions in MHC-I levels, as assessed by surface staining of class I heavy chain or the associated P2M light chain, have been reported for a variety of HPV-associated lesions and tumors (Ruiz-Cabelloet al., 1991; Browning and Bodmer, 1992; Kaklamanis and Hill, 1992; Ljunggren, 1992). Given the extent to which MHC-I levels can be affected by indirect means, such as availability of stimulatory or inhibitory cytokines, care must be taken in assigning modulations of surface class I levels to direct virus intervention (Maudsley et al., 1989). Some human patients with recalcitrant wart lesions, for example, have no altered deficits in cellular immunity but respond well to interferon treatment, which is known to up-regulate MHC expression, and manifest complete regression (Trofatter, 1991). When tested on HPV-16-transformed human cervical cells, IFN-y and leukoregulin synergystically induced class I transcription and IFN-y alone induced class I1 (Woodworth et al., 1992). Normal cervical epithelial cells are sensitive to the inhibitory effects of leukoregulin and IFN-y, but the HPV- 16-transformed cells acquired partial resistance, suggesting that HPV-16 may contribute to loss of responsiveness of malignant cells to potent MHC-inducing cytokines (Woodworth et al., 1992). The mechanism for this effect remains unknown. Another documented inducer of MHC-I is TNF (Fiers, 1991; Vilcek and Lee, 1991; Vassalli, 1992); keratinocytes transformed with HPV-16 are known to overproduce TNFa, which may affect progression

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of neoplasia by multiple mechanisms, including the modulation of viral antigen presentation (Malejczyk et al., 1992). Thus, the relationship between HPV infection and MHC-I regulation is complex and variable. Cases of &M/heavy chain loss or reductions have been described in HPV-induced hyperproliferative lesions (Mahrle et al., 1982; Viac et al., 1987; Markey et al., 1990). Connor and Stern (1990) have reported that over 30% of cervical carcinomas show alterations of surface MHC-I levels, but no direct correlation was observed with the presence of T16/18 DNA, tumor type, extent of differentiation, or disease state. Similarly, Viac et al. (.1990) have reported that cervical intraepithelial neoplasia patients (grade II/III) frequently exhibited reductions of surface P2M in epithelial cells, but no linkage with the presence of HPV-6/11 or HPV-16/18 could be documented. Torres et al. (1993), using a more sensitive PCR strategy to identify HPV-16 sequences in a variety of HPV-associated lesions, concluded that MHC-1 reductions were most prominent in invasive cervical carcinomas, especially those of the more aggressive malignant phenotype. To date, the class I down-regulation in these cases cannot as yet be directly ascribed to the direct function of specific viral gene products, but may reflect local alterations in synthesis of, or responsiveness to, MHC-inducing cytokines such as IFN-y and TNF. OF MHC-I1 D. PV REGULATION

Because keratinocytes can functionally express class I1 antigens, MHC-II-restricted responses may be critical for clearance of HPVinfected and -transformed cells (Glew et al., 1992). T h e fact that the HLA-DQw3 allele in Caucasian women is associated with a sevenfold increased risk of squamous cell carcinoma of the cervix (Wank et al., 1992) suggests the possibility that class I1 recognition of HPV antigens may influence disease progression, although other explanations are also possible. In the rabbit PV model of neoplastic progression, Han et al. ( 1992) examined restriction fragment length polymorphisms (RFLPs) for MHC-I, MHC-11, and TCR genes in regressor versus progressor rabbits and found a positive linkage for regression with the class I1 DRaB marker and for progression with a DQa allele. It remains to be shown if the assigned markers relate to MHC-linked presentation or to functional expression of other closely linked genes (such as TNF), but the direct association of disease outcome with elements of the class I1 locus is clearly provocative. Both CD4+ and CD8+ T cells have been observed to infiltrate HPV tumors, and it is presumed that local levels of secreted IFN-y will have a

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major affect on overall class 1 and I1 levels (Arends et al., 1990; Vardy et al., 1990). Because keratinocytes express MHC-I constitutively and MHC-I1 inducibly, presentation of viral epitopes by the two classes of molecules can present targets for both class I- and II-restricted CTLs. Indeed, in rabbit PV regressor rabbits a predominance of class 11+ T cells are observed in regressing lesions, whereas progressors are excessively depleted of class 11+ T cells (Okabayashi et al., 1991, 1993). In cervical intraepithelial neoplasia patients (grade lI/III), decreases in HLA-DR expression have been observed at the surface of transformed cells, but variations in levels of class I1 expression have been similarly noted in many other carcinomas as well, such as malignant melanomas and carcinomas of hepatocellular, gastric, colorectal, or laryngeal origin (Ruiz-Cabello et d., 1991; Glew et d., 1992; Kaklamanis and Hill, 1992). The majority of cervical squamous tumors are class 11+ (unlike normal cervical epithelium), but there is no direct correlation of this up-regulation with the presence of HPV-16 DNA or the presence of tumor-infiltrating lymphocytes (Markey et al., 1990; Glew et al., 1992). It is still unknown if any specific HPV gene products can be correlated with class I1 inductions, and the causes of this up-regulation may be indirect.

E. POSSIBLE MECHANISMS OF MHC REGULATION BY P v A useful paradigm for HPV progression is epidermodysplasia verriciformis (EV), a genetically predisposed disease characterized by innate defects in several measurable parameters of cell-mediated immune competence, including reduced numbers of Langerhans cells and T cells, lowered responsiveness to phytohemagglutinin, and increased N K activity (Cooper et al., 1990; Majewski and Jablonska, 1992). EV patients have high risks for HPV infection and progression to malignancy, especially in conjunction with sunlight, and this risk is directly associated with impaired T cell responsiveness in the epidermis (Majewski and Jablonska, 1992). There is no known linkage with any particular MHC allele, and the nature of the genetic defect(s) is unknown, .but HPV lesions associated with the EV syndrome exhibit local overproduction of TNFa, which may modulate MHC expression during the course of the disease (Majewski et al., 1991). One parameter that could contribute to variable MHC perturbations by HPV might be at the level of viral DNA integration sites (Howley, 1990). In several cases of HPV- 16118-associated genital cancers, HPV DNA was found integrated near c-my and N - m y genes such that the expressed protein was altered and/or unregulated (Couturier et al.,

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1991). Such events have been more closely examined with reference to transformation mechanisms, because in intraepithelial neoplasia HPV is usually detected in episomes whereas in invasive carcinomas viral DNA is frequently found integrated into the tumor cell genome (Shah and Howley, 1990; Howley, 1991; Campo, 1992). Such alterations of myc expression might also be relevant to MHC expression, in view of the inverse relationship between myc activation and MHC-I expression (Schrier and Peltenburg, 1993). One potential candidate for a viral gene product that might interact directly with MHC surface antigens is the E5 protein, which is found associated with several growth factor receptors (Martin et al., 1989; Petti et al., 1991; Leechanachai et al., 1992; Pim et al., 1992). In the case of bovine PV-1, the E5 protein has been shown to activate several cytokine receptors and to associate with the 16K subunit of the proton-ATPase found in vacuoleslclathrin-coated vesicleslendosomeslGolgi vesicles, and thus could potentially modify such diverse processes as protein sorting, processing, or endocytosis (Goldstein et al., 1992). No physical association with E5 and MHC proteins were detected by cross-linking studies, but formal analysis of E5 and functional antigen presentation remains to be done. More recently, the product of the newly identified E5B open reading frame of bovine PV, which has analogous counterparts in HPV-6 and HPV-16, was shown to alter the processing pathways of a variety of cellular proteins such as calreticulin (O’Banion et al., 1993). T h e potential role of E5B in MHC processing remains to be investigated, but until such data are in hand we must conclude that there is relatively little firm evidence to suggest that the observed upregulations of MHC-I1 and down-regulations of MHC-I can be directly linked with the expression of specific PV gene products. More likely, cellular alterations that are induced by viral DNA integrations or selected for during tumorigenic progression and alterations of the cytokine networks (particularly the type I and I1 interferons and TNF) play the predominant roles in MHC antigen regulations observed during PV infections.

VII. Conclusions As can be readily surmised, the regulation of functional MHC expression by viruses is complex, and many important questions remain, both in terms of the molecular nature of observed modulations and the functional significance of how the virus-specific alterations of MHC function actually provide a selective advantage for the virus. In this review we have focused on the interaction between DNA viruses and the MHC

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because a number of specific DNA virus-encoded gene products have already been identified as directly affecting MHC expression o r antigen presentation (Maudsley and Pound, 199 l), whereas RNA viruses generally interact with the MHC by indirect mechanisms, although the transrepression of the class I promoter by HIV is a notable exception (Howcroft et al., 1993). Most DNA viruses seem to be capable of modulating cellular immunity at least to some degree, and, encoded within the larger viral genomes, the large number of viral open reading frames that still have no known function make it likely that more viral gene products that can perturb MHC function remain to be identified. For example, in vaccinia at least 55 open reading frames have been shown to be dispensible for virus viability in cultured cells (Perkus et al., 1991) and it is presumed that some of the functions of the encoded proteins are to secure an advantage for virus propagation in the face of a host capable of a mature cellular immune response. Furthermore, given the fact that most of the viruses considered in this review have evolved in outbred populations of immunocompetent hosts that possess a variable array of polymorphic alleles for both class I and I1 loci, it should be taken as a given that virus defense mechanisms are the product of selective pressure from immune mechanisms that have likely been subject to selection by virus pathogens (McMichael, 1993). For example, EBV strains from HLA-A1 1 populations in New Guinea carry a single point mutation in the dominant EBNA-4 T cell epitope that precludes binding and recognition of the processed viral peptide with the A1 1 allomorph, suggesting that virus evolution can indeed be shaped by the MHC repertoire in the resident host population (de Campos-Lima et al., 1993). Aside from the relatively trivial explanation of hiding in cells that simply lack MHC surface molecules, such as neurons (Joly and Oldstone, 1992; Drew et al., 1993), viruses can utilize strategies that modify MHC expression either by direct mechanisms within the infected cells, or by indirect strategies that interfere with MHC induction, particularly at the level of cytokine regulation. Although a number of cytokines have been implicated in MHC up-regulation, such as 11-4, M-CSF, GM-CSF, TGF-P, and EGF/TGF-a, the two dominant families of cytokine inducers of MHC are T N F and IFN. Viruses have evolved a multiplicity of strategies against these latter ligands, in part because TNF and IFN possess direct antiviral properties and are potent inducers of class I and I1 MHC expression (for reviews, see Vilcek and Lee, 1991; Loetscher et al., 1991; Samuel, 1991; Ruddle, 1992; Vassalli, 1992; Kerr and Stark, 1992; McNair and Kerr, 1993). As can be seen in Table I, the number of different strategies directed at TNF and IFN is very impressive, and +

TABLE I DNA VIRUSESA N D MHC: CONTROL SITESCOMMON TO CLASSI MHC induction/expression MHC induction by cytokines Expression of cytokines

Potential control sites IFN-dP or IFN-y synthesis blocked

Ligand function

TNFa/P receptor homologue

Ligand function

IFN-y receptor homologue

Signal transduction

Lack of responsiveness to TNFdP

Signal transduction

Lack of responsiveness to IFN-a/P or IFN-y

Expression of MHC-I and MHC-I1 genes Transcription Inhibit host protein synthesis de novo (H chain, P2M, a, b) Alter transcription factor synthesis or function (e.g., NF-KB, interferon response sequence binding proteins) Outcompetition of host Translation messages by viral mRNAs Perturb posttranslation Transport modifications or transit to ER/Golgi or to endosonie Expression of other Ag presentation-related genes

h

Examples include B7, ICAM-I, LFA-3, ABC transporter, LMPs, HSPs, p88, chaperones, ubiquitinylation enzymes, peptide chain release factors, proteases (cathepsins), p72/74, invariant (Ii) chain

AND

I1 PATHWAYS

Virus Regulation HBV (core antigen) Ad5 (Ela) EBV (BCRFlp SFV/myxorna (T2) Cowpox (crniB) Variola (G4R) SFV/myxoma (T7) Vaccinia (B8R) Variola (B9R) Ad (E3- 14.7K) Ad (E3- 10.4/14.5K) Ad (Elb-19K) Vaccinia (K3L/E3L/A18R) ? EBV (EBER RNA)” Ad (VAl RNA)b EBV (EBNA 2)6 Ad5 (Ela) HBV (pol/terminal protein) CMV (unmapped) Many DNA viruses Ad12 (Ela/Elb) HBV (truncated S) HBV (X protein) PV (DNA integration) Many DNA viruses

CMV (unmapped) ? PV (e.g., E5B?)

?

Moore rl al. (1991, 1993). See McNair and Kerr (1993) (other references are found in text).

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more examples of cytokine blockade targeted against other MHCinducing ligands may well exist (discussed in McFadden, 1993). Because the ability of CTLs to clear virus-infected cells is directly proportional to the quantity of surface MHC molecules loaded with a given viral epitope, and entry into the presentation pathway is a function of nascent MHC chain synthesis and viral peptide generation, it is also not surprising that the expression of MHC genes can be subject to direct viral regulation by transacting transcriptional regulators (Brown et al., 1988). Many DNA viruses shut down host transcriptional/translational machineries to favor the selective expression of viral genes, which would reduce de nova MHC chain availability for the presentation pathway, but other viruses have more subtle effects on host macromolecular synthesis and some cell types permit only partial or abortive viral replication cycles. Therefore, appreciation of the roles that specific virus gene products play in MHC regulation requires not only functional characterization of the viral proteins, but also a detailed consideration of the types of cells that come into contact with the virus in an infected host and have the capacity to function as APCs in the context of the normal portal of entry for the specific virus in question. A topical example of this is the UL18 gene product of CMV, which has been conjectured to be an MHC down-regulator because of its ability, in certain circumstances in vztro, to form complexes with P2M at the cell surface (Browne et al., 1990). Despite the intriguing homology between UL18 and host class I heavy chain (Beck and Barrell, 1988), directed knockout of the UL18 gene in CMV surprisingly had no effect on surface MHC-I down-regulation, at least in cultured fibroblasts (Browne et al., 1992). However, the tentative conclusion that UL18 might not affect MHC-restricted antigen presentation is premature, and it is entirely possible that UL18 may play a role in a particular subset of key presenting cells that would be manifested only in an infected host, perhaps restricted by the route of inoculation o r circumstances of initial infection. Another important biological issue is the consequence of upregulating MHC, particularly class I1 levels, in cells that normally express low levels of MHC. If the induced cells do not also express appropriate costimulating signaling molecules, such as B’7, the increased level of viral antigen presentation may result in abortive T cell stimulation (i.e., anergy) and tolerance to the viral epitope. Another variation of this theme is T cell exhaustion (Moskophidis e l al., 1993), in which responsive T lymphocytes become overstimulated by excessive MHC-restricted presentation of antigen to the point where they literally disappear as a

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clonal population. In either event, a selective advantage for the virus can actually result from the up-regulation of MHC-restricted presentation. Relatively little is known about the effect of viruses on the expression of the growing number of genes that participate in MHC-restricted presentation, some of which are listed at the bottom of Table I. When to this list are added the host gene products necessary for proper MHC/peptide signaling to T cells, such as B7 and adhesion molecules such as ICAM-1 and LFA-3, it is readily apparent that there is a large array of control points that could serve as the targets for virus intervention to effectively subvert the class I- or 11-restricted presentation of viral antigen. Table I1 provides a limited picture of what is known about how DNA viruses interfere with the class I processing pathway, but it is fair to say that even the function of the best studied viral protein in this class, gp 19/E3 of adenovirus, is still incompletely understood with respect to the cellular immune response in the infected host. To date, other than the cytokine blockade strategy, very little is known about how viruses affect MHC class I1 antigen presentation, but given the complexity of this pathway (see Fig. 1) it would be surprising if viruses that TABLE I1 DNA VIRUSESAND MHC: SPECIFIC CLASSI CONTROL SITES FOR ANTIGENPRESENTATION IN VIRUS-INFECTED CELLS Pathway Degradation of viral antigen

Peptide transport to ER

Assembly of H chain/P,M/peptide complex Transport of complex to Golgi Trans-Golgi processing

Transport to surface Recycling/stability at surface ~~~

Potential control sites Antiproteases Homologues of ubiquitinylation enzymes Inhibit ABC transporters Poor recognition of viral peptides Inhibit p88 or other chaperones Homologues of H chain ER-anchoring mechanism Transport defect Inhibit glycosylationl phosphorylation of trimolecular complex Trans-Golgi defect Disruption of surface MHC-I complex ~~

~~

Rodriguez et al. (1992; other references are found in text).

Virus example ? Poxviruses (? serpins) African swine fever virus (1215L)o ? ?

? CMV (unmapped) ? CMV (ULIS?) Ad (gp19/E3) ? PV (e.g., E5B?) ? Ad (gp19/E3)

? ? Myxoma (unmapped)

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can infect class 11+ cells have left this side of cellular immunity out of their antiimmune defense repertoire. T h e challenge for the future, then, is the daunting task of deciphering how viruses have evolved to accommodate the cellular immune recognition process at the level of individual infected cells in an animal host. Gene products encoded by the larger DNA viruses that apparently have been acquired to modulate the MHC-restricted antigen presentation pathway provide useful in vim probes for MHC function and should facilitate the longer term objective of understanding how the diverse viral strategies to elude immune destruction can provide clues as to how the nonself recognition process actually functions in the living host. ACKNOWLEDGMENTS G.M. a n d K.K. are Medical Scientist and Scholar, respectively, of the Alberta Heritage Foundation for Medical Research. K.K. is also a Cancer Research lnstitute Investigator a n d an MRC Scholar. This work was supported by the MRC and NCI of Canada. We thank Shari Kasinec for help with the preparation of the manuscript.

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VIRAL TRANSFORMATION OF HUMAN T LYMPHOCYTES Ralph Grassmann," Bernhard Fleckenstein,"and Ronald C. Desrosierst 'Institut fur Klinische and Molekulare Virologie der UniversitP Erlangen-Nurnberg, D-91054 Erlangen, Germany; and *New England Regional Primate Research Center, Harvard Medical School, Southborough, Massachusetts 0177'2

I. Introduction 11. Transformation of Human T Helper Lymphocytes by Human T Cell

Leukemia Virus 111. Developing a T-Lymphotropic Herpesvirus Vector IV. Immortalization by Herpesvirus saimarilHTLV Recombinants V. Transformation of Human T Lymphocytes with Wild-Type Herpesvirus saimiri VI. Growth Regulation and Antigen Specificity of Rhadinovirus-Transformed T Cell Lines and Clones VII. Transforming Potential of the Herpesviral stp Oncogene VIII. Concluding Remarks References

I. Introduction

T cell activation induces both differentiation and amplification of T lymphocytes and is thus pivotal for eliciting effective and specific immune responses. This has focused the attention of cell biology and molecular immunology on the signaling cascades that control growth and differentiation of T lymphocytes. Intense study of these activation pathways has provided models for signaling, starting from receptor-ligand interactions at the cell membrane to nuclear events, including transcription of activation-specific genes, DNA synthesis, and mitosis (Janeway, 1992, Herschman, 1991; Ullman et al., 1990). Aberrations in T cell activation pathways have important pathological implications. T h e nonspecific activation of T lymphocytes by prokaryotic superantigens is a critical feature of the pathogenesis of some bacterial diseases (Zumla, 1992), Early steps of leukemogenesis may include a chronic state of T cell activation that may predispose cells to somatic mutations associated with tumor progression. Various viruses from different taxonomic groups have selected lymphocytes for their site of permanent residence. Persistence of human 21 1 ADVANCES IN CANCER RESEARCH. VOL. 63

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retroviruses in these cells is believed to be crucial for their pathogenesis. Long-term persistence of the human T cell leukemia virus (HTLV), for example, can cause malignant transformation of the host cell, resulting in the development of leukemia (Uchiyama et al., 1977; Poiesz et al., 1980; Hinuma et al., 1981; Yoshida et al., 1982). Two types of genetically distinct HTLVs have been identified to date. HTLV-1, which is endemic in southern Japan, the Caribbean, and regions of Africa, is a causative, initiating agent of adult T cell leukemia/lymphoma (ATL). T h e incidence of ATL is highest in those regions where HTLV-1 is endemic, and ATL generally develops only many years after initial infection with HTLV-1 (reviewed in Cann and Chen, 1990). Current models suggest that the infection of T lymphocytes by HTLV-1 causes an early growth stimulation, resulting in an amplified population of virus-harboring T lymphocytes, from which further steps of malignant conversion are likely (Franchini et al., 1984). Infection of primary cultures of human peripheral blood mononuclear cells (PBMCs) with HTLV-1 results in continuous growth and transformation of T lymphocytes (Yamamoto et al., 1982; PopoviC et al., 1983). T h e closely related but genetically distinct HTLV-2 is less prevalent than HTLV-1 and may be associated with different pathologic manifestations, e.g., atypical hairy cell leukemia (Kalyanaraman et al., 1982; Saxon et al., 1978; Rosenblatt et al., 1986, 1988). HTLV- 1, HTLV-2, and the more distantly related bovine leukemia virus (BLV) are generally classified as a distinct group within the oncovirus subfamily because of their unusual genome organization (see below). The capacity for growth transformation and persistence in lymphocytes is also characteristic of some herpesviruses. The gamma- 1 herpesviruses include Epstein-Barr virus (EBV) and closely related B lymphotropic herpesviruses from Old World primates and great apes. Infection with EBV is usually restricted to B lymphocytes by its membrane receptor, complement receptor 2 (CR2) (Fingeroth et al., 1984). Primary infection of humans with EBV causes polyclonal B cell proliferation, which is usually limited by an active cytotoxic T lymphocyte (CTL) response (Moss et al., 1978; Rickinson et al., 1979). Fatal lymphoproliferative syndromes involving B lymphocytes may result from inherited o r acquired deficiencies in CTL responsiveness. EBV is able to immortalize B lymphocytes in cell culture to continuous growth (reviewed in Middelton et al., 1991). T h e growth-promoting effects of EBV seem to be mediated, at least in part, by virally encoded EBV nuclear antigens (EBNAs) and lymphocyte membrane proteins (LMPs). Human herpesvirus type 6 (HHVG) is a member of the beta or cytomegalolike subfamily of herpesviruses (Roizman, 1982; Roizman et al., 1992). HHVG is widespread in the human population and has a pro-

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nounced tropism for T lymphocytes. Viral replication in cell culture seems to be confined to primary cultures of mononuclear blood cells and T lymphoma cell lines. HHV6 has been linked to certain forms of human lymphoproliferative syndromes. It has been difficult to determine, however, whether these result from autonomous growth or reactive proliferation; monoclonal T lymphocyte amplification has been found in at least one case (Borisch et al., 1991). Rhadinoviruses, or gamma-2 herpesviruses, represent a distinct subgroup of lymphotropic viruses in the herpesvirus family (Roizman, 1982; Roizman et al., 1992). T h e rhadinoviruses typically replicate and persist in T lymphocytes. Herpesvirus saimiri, the prototype of the rhadinoviruses, is highly prevalent in squirrel monkeys (Saimiri sciureus). T h e virus can easily be isolated from the peripheral blood by cocultivation of mononuclear blood cells with permissive kidney cell cultures (Falk et al., 1972). Herpesvirus saimiri does not seem to cause overt illness in its natural host, but it does induce fulminant T cell leukemia and lymphoma in a wide spectrum of other New World primates (for review, see Fleckenstein and Desrosiers, 1982; Fleckenstein, 1979). Herpesvirus ateles, a related virus of spider monkeys (Ateles sp.), likewise is the cause of various forms of T-proliferative syndromes in South American monkeys (Melendez et al., 1972). It is still an open question why T cells in heterologous hosts respond to rhadinovirus infection with uncontrolled constitutive proliferation, whereas the growth of lymphocytes in the naturally infected animals seems to be unaffected. Possible explanations include inherent, evolved species differences in the extent to which lymphocytes become activated by the virus, differences in the ability to respond immunologically, or differences in the MHC or the T cell receptor repertoire. T h e capacity of herpesviruses and retroviruses to induce lymphoproliferation has quite naturally led to their use for transformation of lymphocytes in cell culture. Lymphocyte immortalization can be a useful tool for immunologists who want to expand certain cell types of the hematopoietic system. Human B lymphocytes are readily transformed in vitro by Epstein-Barr virus, and human T lymphocytes are growth transformed by HTLV- 1. However, use of HTLV- 1 for transformation of T lymphocytes suffers from several limitations. In addition to the inefficiency of transformation with cell-free virus, difficulties in working with infectious cloned DNAs in primary lymphocyte cultures have limited molecular and genetic studies (De Rossi et al., 1985). HTLV-1immortalized T cell lines usually lose T cell functions within a few months of culturing (Mitsuya et al., 1984a, 1986; PopoviC et al., 1984; Suciu-Foca et al., 1986; Yarchoan et al., 1986; Inatsuki et al., 1989). In

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addition, HTLV- l-carrying tumor cells and in vztro-transformed T cell lines usually have a helper (CD4+/CD8-) phenotype, confining experiments to this cell population only. More recent studies from our laboratories have shown that certain strains of H. saimiri and H . saimirilHTLV-1 recombinant viruses can transform T lymphocytes to continuous growth. In this review, we have attempted to summarize some of the salient features of viral immortalization of human T lymphocytes and the status of knowledge on the herpesvirus and retrovirus oncogenes functional in these cells.

II. Transformation of Human T Helper Lymphocytes by Human T Cell Leukemia Virus Infection with HTLV-1 may result in the development of the fatal lymphoproliferative disease ATL (Uchiyama et al., 1977; Poiesz et al., 1980; Hinuma et al., 1981) only after decades of inapparent persistence. T h e extended latency period (Kawano et al., 1985), the relatively low incidence (Kondo et al., 1987a,b),and specific properties of the leukemic cells suggest that T cell leukemia is the result of a multistep process that begins with effects of virus on the growth control of CD4+ T lymphocytes. Tumor cells have a characteristic phenotype, including the appearance of highly lobulated nuclei, the presence of the CD4 antigen in the cell membrane (Hattori et al., 1981; Sugamura et al., 1984), and the expression of high levels of IL-2 receptors (IL-2Ra) at the cell surface (Uchiyama et al., 1981). Cells of CD4- CD8+ phenotype are rare exceptions (Amagasaki et al., 1985). Other T cell subset markers are heterogeneous (Morimoto et al., 1985; Yamada et al., 1985). Tumor cells derived from a single patient represent dominant clones; this was demonstrated by the analysis of T cell receptor P-chain rearrangements (Jarrett et al., 1986; Matsuoka et al., 1988) and proviral integration sites (Hoshino et al., 1983; Seiki et al., 1984). Chromosomal abnormalities are frequent in these cells (Fukuhara et al., 1983; Miyamoto et al., 1983; Whang-Peng et al., 1985) and are most frequently observed in highly malignant ATL cases (Sanada et al., 1985). HTLV- 1 T cell lines can be established from the peripheral blood cells from leukemic patients, from nonleukemia patients with tropical spastic paraparesis/HTLV-associated myelopathy (TSPIHAM) and from asymptomatic individuals (Gootenberg et al., 1981; Depper et al., 1984; Jacobson et al., 1988). Cultured cells often produce HTLV-1 particles. Both HTLV-1 and HTLV-2 are capable of immortalizing primary human lymphocytes in culture (Chen et al., 1983; Miyoshi el al., 1981, PopoviC et al., 1983; Yamamoto et al., 1982). However, immortalization is +

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not specific to human cells, because T cells from monkeys, rabbits, cats, and rats can also be transformed to permanent growth in culture (Miyoshi et al., 1982, 1983; Hoshino et al., 1984; Tateno et al., 1984). Infection of normal T cells is most effectively achieved by cocultivation with lethally irradiated HTLV-producing cell lines, but was found to be also possible with cell-free virus (De Rossi et al., 1985). Immediately after infection, polyclonal stimulation of T cell growth with IL-2 production can be observed (Gazzolo and Duc Dodon, 1987; Kimata and Ratner, 1991; Wucherpfennig et al., 1992). This stimulation can be inhibited by monoclonal antibodies recognizing the surface molecules CD2 and LFAIII (Wucherpfennig et al., 1992; Duc Dodon et al., 1989),which have important functions in T cell activation. Following this initial stimulation, lymphocyte proliferation ceases for a 2- to 4-week period; subsequent expansion of infected cells indicates the establishment of permanent lines. These cells do not produce detectable IL-2 activity or mRNA, but do express high levels of IL-2Ra and viral RNA (Kimata and Ratner, 1991). T h e cells usually depend on exogenous IL-2 for their growth, are oligoclonal or monoclonal, and can be kept in culture for very long times without antigenic stimulation (Popovik et al., 1983; Grassmann et al., 1989). Clonal lines can emerge from cultures that have lost the requirement for exogenous IL-2. Functional properties of T cells immortalized by HTLV- 1 have been investigated using antigen-specific T cell clones. Several reports have described loss of specific T cell functions, such as impairment of cytotoxicity (Mitsuya et al., 1984a,b, 1986; Popovik et al., 1984; Suciu-Foca et al., 1986; Yarchoan et al., 1986; Inatsuki et al., 1989). The T helper reaction of CD4+ T cell clones infected with HTLV-1 is affected to varying extents. During the initial IL-2-dependent phase, it is only partially impaired; this function is often completely lost in cells growing independently of IL-2 (Inatsuki et al., 1989). This may be explained by a concomitant reduction of the CD3-TCR complex (Yarchoan et al., 1986; Inatsuki et al., 1989). T h e ability to transform T lymphocytes in vitro and the absence of specific integration sites in HTLV-infected lymphocytes suggest the involvement of viral gene products in the immortalization of T cells. Like other retroviruses, HTLV and BLV contain the genes gag, pol, and env. However, unlike other members of the type C oncoviruses, HTLV and BLV contain additional open reading frames in the X region near the 3' ends of their genomes (Seiki et al., 1983). Long terminal repeats (LTRs) at both ends of the proviral genome contain transcriptional control elements (Sodroski et al., 1984). Three major transcripts are synthesized from the promoter in the 5' LTR (Fig. 1). The unspliced mRNA repre-

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FIG. 1. Gene expression of human T cell leukemia virus HTLV-1. The proviral genome contains the structural genes (gag, pol, e m ) , the pX region, and the long terminal repeats (LTRs). Starting from the promoter in the 5' LTR, three types of mRNA are synthesized (bold lines). The unspliced RNA, which equals the viral genome, and the singly spliced RNA are translated into structural proteins. The doubly spliced transcript is needed for the expression of the pX region genes (trans-activator, Tax; regulator of virion synthesis, Rex). The gag-derived proteins are assembled into the nucleocapsid and matrix of the virus; pol encodes proteins of the reverse transcriptase complex and env encodes envelope proteins.

sents the viral genome and is translated into Gag and Pol proteins (Seiki et d.,1985). The gag-derived polypeptides form the matrix and nucleocapsid of the virion; pol encodes the reverse transcriptase and integrase functions. A single-spliced mRNA is translated into the envelope glycoproteins. A double-spliced RNA species is used for the expression of the regulatory genes encoded by the X region. This genomic region between the 3' end of the enu reading frame and the 3' LTR is unique to the members of the HTLV-BLV viruses. The X region encompasses two overlapping reading frames encoding the transactivator (Tax),a protein

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of 40 kDa, and the regulator of virion protein expression (Rex), a protein of 27 kDa. A truncated Rex protein of 21 kDa can be translated initiating at an internal start codon (Kiyokawa et al., 1985). The protein p27rex is required for cytoplasmatic expression of the unspliced and the singly spliced mRNAs (Inoue et al., 1987). Rex activity is mediated by a cis-acting signal sequence located between the poly(A) signal and poly(A) addition site. This stretch of RNA forms a complex, highly stable secondary structure, which is bound specifically by p27rex (Grassmann et al., 1991; Toyoshima et al., 1990; Ballaun et al., 1991; Bogert et al., 1991). T h e same structure mediates the functional arrangement of the polyadenylation signals and is therefore essential for this process (Ahmed et al., 1991). T h e p40& trans-activator, a nuclear protein, stimulates viral transcription by acting on three imperfect repeats of 21 bp located in the viral promoter (for review, see Cullen, 1992). Additionally, this protein activates transcription of a significant number of cellular and viral genes. These include genes for cytokines, a cytokine receptor, several transcription factors (Table I), HIV-1, and the human cytomegalovirus (HCMV) immediate-early promoters (Bijhnlein et al., 1989; Moch et al., 1992). Tax does not bind directly to DNA; however, the protein can function as a transcriptional activator if it is artificially attached to promoter sequences (Fujisawa et al., 1991). Deletion analysis of various Tax-inducible promoters revealed that binding sites of cellular transcription factors mediate the stimulatory effect. These sites include the CAMPreponsive elements (CRE) in the HTLV (Yoshimura et al., 1990; Jeang et al., 1988) and HCMV (Moch et al., 1992) promoters, the CArG boxes in the c-fos and egrV2 promoters (Alexandre et al., 1991), and binding sites of the nuclear factor KB (NF-KB),which are found in cytokine, cytokine receptor, and HIV promoters (Leung and Nabel, 1988; Ruben et al., 1988; Bohnlein et al., 1989; Paul et al., 1990). The notion that Tax can act indirectly by complexing to inducible promoters is supported by the observation that it can bind CREB protein and p67Nin vitro (Fujii et al., 1992; Zhao and Giam, 1992; Suzuki et al., 1993). This reaction may promote transcription either by increasing the binding affinity of the cellular transcription factor to DNA or by the inherent activation domain of Tax. T h e activation of various cytokine promoters by Tax is mediated by NF-KB.This factor, a heterodimer, is normally bound by the inhibitor IKBand is located in the cell cytoplasm. Activation requires dissociation of the cytoplasmatic inhibitor and this results in transport of the NF-KB heterodimer into the nucleus. NF-KBcan be activated not only by intracellularly expressed Tax, but also by addition of Tax protein to the culture medium, which can be taken up into the cells (Marriot et al.,

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TABLE I CELLULAR GENESTRANS-ACTIVATED BY HTLV TAX Gene

Function of product

Interleu kin-2 Interleukin-3 Interleu kin-4 Tumor necrosis factor a

Cytokine C ytokine Cytokine Cytokine

Tumor necrosis factor p (lymphotoxin) Granulocyte-macrophage colon y-stimulating factor Transforming growth factor p1 Interleukin-2 receptor a chain Class I major histocompatibility complex antigen c-fos

Cytokine

Transcription factor

Fra-I c-jun junB junD Egr-I (Krox24)

Transcription Transcription Transcription Transcription Transcription

Egr-2 (Krox20, 225)

Transcription factor

p105 NF-KB

Transcription factor progenitor Transcription factor Transcript ion factor Cytoskeleton Neurotransmitter progenitor Tyrosine kinase

c-re1 c-my (mouse) Vimentin Proenkephalin c-lyn

Ref. Miyatake et al. (1988) Miyatake et al. (1988) Miyatake et al. (1988) Tschachler et al. (1989); H. Albrecht et al. (1992) Tschachler et al. (1989); Paul et al. (1990) Miyatake et al. (1988); Nimer et al. (1989)

Cytokine

Cytokine

Kim et al. (1990)

Cytokine receptor

Cross et al. (1987); Inoue el al. (1986) Sawade et al, (1990)

Surface receptor

factor factor factor factor factor

Fujii el a/. (1988); Nagata el al. (1989) Fujii et al. (1991) Fujii et al. (1991) Fujii et al. (1991) Fujii et al. (1991) Fujii et al. (1991); Alexandre et al. (1991) Fujii et al. (1991); Alexandre et al. (1991) Arima et al. (1991) Arima rt al. (1991) Duyao el al. (1992) Lilienbaum et al. (1990) Joshi and Dave (1992) Uchiumi et al. (1992)

1992). Similar to other NF-KB-inducing compounds, Tax-induced NF-KBstimulation seems to be mediated by oxygen radicals (Schreck et al., 1991, 1992). T h e capacity to stimulate CREB-mediated and NF-KBmediated transcription seems to depend on different domains of the Tax protein, because mutants have been described that have lost only one of these functions (Semmes and Jeang, 1992; Smith and Greene, 1990). T h e ability of HTLV to transform cells in vitro implicates the X region as the responsible factor. However, difficulty in working with

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gene and proviral DNA transfections in primary lymphocytes renders direct demonstration difficult. Such demonstration has been facilitated by development and use of H. saimiri as a T cell viral vector system.

Ill. Developing a T-Lyrnphotropic Herpesvirus Vector Several unique features of the rhadinovirus H. saimiri led to its use as a eukaryotic vector. The virus is capable of infecting T cells, in which it can persist over very long time periods as multicopy episomes without signs of virus production. It can be easily propagated in a fully replication permissive epithelial cell line derived from owl monkey kidneys (OMKs). Specific features of the rhadinovirus genome (J.-C. Albrecht et al., 199213) seem to favor the generation of recombinants. T h e viral genome (M-DNA) is a linear DNA molecule of 150-170 kb consisting of a coding region of 113 kb (L-DNA) that is flanked by variable numbers of directly repeated noncoding sequence elements of 1.4 kb (H-DNA) (Bornkamm et al., 1976; Bankier et al., 1985). The high content of redundant, potentially replaceable sequence information allows the insertion of long stretches of foreign DNA. T h e replication and persistence of herpesviruses in the nucleus allow the use of heterologous promoters. Furthermore, the insertion of genetic information interrupted by intron sequences should result in mRNA that is processed like cellular transcripts. T h e generation of recombinant H. saimiri is most efficiently achieved by cotransfection of infectious virion DNA and suitable plasmid DNA into permissive OMK cells. The plasmid contains the transcription unit to be inserted flanked by stretches of herpesviral sequences. Homologous recombination of these sequences with virion DNA results in the generation of replication-competent recombinants that, together with wild-type virus, destroy the transfected monolayer. Different regions of the H. saimiri genome have been used for the insertion of heterologous sequences into replication-competent viruses. These regions include nontranscribed DNA at the right junction of H/L-DNA, H-DNA, and nonessential genes in the left-terminal L-DNA (Desrosiers et al., 1985b; Grassmann and Fleckenstein 1989; Aepinus et al., 1990; Medveczky et al., 1989). Whereas insertion of foreign sequences into L-DNA and in H/L junctions results in stable recombinants, integration of foreign sequences into H-DNA does not yield stable recombinant virus. T h e presence of a single contiguous stretch of H. saimiri DNA at one flank of the heterologous DNA to be inserted is sufficient for cloning into the H/L junction. L-DNA sequences as small as 2.0 kb are sufficient to direct foreign sequences into the right H/Ljunction (Alt etal., 1991).This

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FIG. 2. Generation of Herpavirus saimin' recombinants (model). The H. saimin' virion DNA (top) consists of a coding, light component (L-DNA; 34.5% GC), which is flanked by noncoding, tandemly repeated heavy components (H-DNA; 70.8% GC). The junctions between H-DNA and L-DNA were chosen as insertion sites of heterologous sequences. To allow homologous recombination, infectious virion DNA is cotransfected with linearized plasmid DNA. A single cross-over event between these molecules could result in a linear intermediate, which is assumed to circularize. Replication according to the rolling circle mechanism results in the formation of concatemers that were cut to recombinant monomers and packaged into infectious particles.

strongly suggests that recombinants can arise. from a single cross-over event within L-DNA. This may be followed by circularization and DNA replication according to the model of the rolling circle, resulting in replication-competent recombinant viruses (Fig. 2). T h e content of recombinant virus within viral stocks resulting from contransfection can vary over several orders of magnitude. Values of up to 10% recombinants are possible (Desrosiers et al., 1985b) and allow direct purification from the supernatant of cotransfected OMK cells. However, more frequently, less than 1% recombinants are observed (Alt et al., 1991). This led to the application of dominant selectable markers for enrichment of recombinant virus (Grassmann and Fleckenstein, 1989; Alt et al., 1991; Medveczky et al., 1989). T h e genes used include the gene for neomycinlkanamycin phosphotransferase (neor), which confers resistance to geneticin (G4 18), and hygromycin B phosphotransferase ( h y g ) .The corresponding open reading frames were fused to eukaryotic transcription signals derived from SV40 and HCMV before they were introduced into the H. saimim' genome. Viral stocks resulting from cotransfection experiments with a content of 0.5-1% recombinants were used to investigate the utility of these markers for the purification of recombinants. Two

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successive cultivations in the presence of hygromycin B resulted in virus stocks contaihing 50%hyg’ recombinants (Ah et al., 1991). Similar results were obtained using the neor gene as selectable marker (Grassmann and Fleckenstein, 1989). Thus, inoculation of permissive O M K cells in the presence of sublethal concentrations of hygromycin B or geneticin allows significant enrichment of recombinants. Selectable recombinants containing the neor o r hygr genes were used to analyze the potential of H. saimiri recombinants to infect and to persist in various human cell lines (Simmer et al., 1991). T h e lines used represent cells from epithelium and connective tissue as well as from all hematopoetic lineages. After infection, most of the lines converted to resistance against geneticin or hygromycin B. All resistant lines harbored recombinant viral episomes. These cell lines include cells of B and T-lymphoid origin as well as myeloid, fibroblast, and carcinoma-derived cells. Infection with both of the selectable recombinants resulted in double resistance and simultaneous persistence of both recombinants in a T cell line (Ah et al., 1991). This may be of considerable value for the analysis of cooperating regulatory genes. Lines derived from the Burkitt lymphoma B cell line Raji contain simultaneously persisting episomes of another member of the herpesvirus family, the Epstein-Barr virus. Most of the persistently infected human cell cultures, except for a pancreatic carcinoma line and foreskin fibroblasts, did not synthesize infectious virus. T h e absence of virus production in the lymphoid human cell lines may be due to a block in viral gene expression, including the two known immediate-early transcripts; these RNAs could not be detected in persistently infected cells. With these procedures, several cellular and viral genes were introduced and successfully expressed using the H. saimiri vector. These include the bovine growth hormone (BGH) gene (Desrosiers et al., 1985b),the X region of human T cell leukemia virus (Grassmann et al., 1992), the c-fos protein (Alt and Grassmann, 1993), and the env-encoded glycoprotein of HIV (Aepinus et al., 1990). Using a semipermissive monkey cell line it was possible to demonstrate release of up to 500 ng BGH per lo6 cells in 24 hours (Desrosiers et al., 1985b). Experimentally infected owl monkeys made BGH that could be detected in plasma, and the animals developed high-titered antibodies to the bovine growth hormone (Desrosiers et al., 1985b). IV. Immortalization by Herpesvirus saimirilHTLV Recombinants

T h e X region of the human T cell leukemia virus was cloned into the S4 deletion variant of H. saimiri strain A l l (Grassmann et al., 1989,

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1992). T h e wild-type strain A1 1 is capable of immortalizing CD8+ lymphocytes of common marmosets, but not human cells. The deletion in H . saimiri A l l S4 removes a genetic segment that is required for the immortalization of New World primate lymphocytes and tumorigenesis (Desrosiers et al., 1984, 1985a). T h e removed sequences include those that encode the H. saimiri transformation-associated protein (STP-A) (Jung et al., 1991; Murthy et al., 1989). The X-region sequences inserted included the 3' end of the provirus, starting with the 3' end of the env gene as well as a short stretch of DNA overlapping the 3' end of the pol gene and the 5' end of the env gene. T h e latter region contains the first codons of the tax and rex open reading frames and the 5' splice site, which is used for the synthesis of the authentic X-region mRNA. This first coding exon is spliced to an acceptor that is located at the 5' end of the Tax- and Rex-encoding exon. T h e promoter of the murine retrovirus SL3-3 was used for the expression of these sequences. Termination can take place at the homologous sites located in the 3' LTR. This expression cassette synthesized a 40-kDa Tax protein and a 27-kDa Rex protein, but no Env protein. Variants of this expression cassette that were generated were selectively suppressed in the synthesis of Tax, Rex, or both Tax and Rex. Wild-type and mutagenized X regions were inserted into the H. saimiri genome, yielding virus with the genotypes taX+lrex+, tax+lrex-, tax-lrex+, and tax-lrex-. The immortalizing potential of the HTLV- 1/herpesvirus recombinants was investigated by infection of primary phytohemagglutinin (PHA)-stimulated human lymphocytes obtained from cord blood or thymus. Recombinant viruses containing an intact tax open reading frame resulted in permanent growth of cell cultures. These were in culture for a minimum of 6 months and can be referred to as immortalized. T h e Rex protein was not required for the immortalization of T cells in this system, because a recombinant of the phenotype Tax+ Rex- could still transform T cells to permanent growth. Cultures infected with recombinants lacking the potential to synthesize functional Tax behaved like uninfected controls or cord blood cells infected with the vector strain alone. In summary, these experiments indicate that the presence of a functional tax gene is necessary and sufficient for T cell immortalization in this system. The human cell lines established by the recombinant virus harbor episomally persisting recombinant herpesvirus DNA and synthesize functional Tax protein. T h e p27r.x protein was made in T cells immortalized by the tux+/rex+ recombinant, but not in cord blood cells infected with the rex- strain. T h e p21rex protein was not detectable in any of these cell lines. The phenotype of the cells immortalized by these recombi-

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nants resemble HTLV- l-immortalized T cells (CD3+). They resemble T lymphocytes of the helper subtype (CD4+ CD8-), which express major histocompatibility complex (MHC) class I1 molecules (HLA-DR) and large amounts of IL-2Ra at their surface. These cells and HTLV-1immortalized cultures, which were established at the same time by cocultivation with a HTLV-producing cell line, are dependent on exogenous IL-2 for growth (Grassmann et al., 1989, 1992). In summary, these results indicate that the HTLV- l-X-region gene tax is a key factor responsible for T cell immortalization caused by HTLV- 1. T h e capacity of the Tax protein to interfere with the control of cell proliferation was also demonstrated in other experimental systems. Expression of tax can alter the growth properties of rodent fibroblasts. Cell lines derived from rat and mouse grew to increased saturation densities and formed small colonies in soft agar (Tanaka et al., 1990). Tax can cooperate with the ras oncogene to transform rat embryo fibroblasts to focus formation, permanent growth in culture, and oncogenicity in nude mice (Pozzatti et al., 1990). Transgenic mice expressing tax but not rex developed neurofibromas (Nerenberg et al., 1987), but not lymphoproliferative disease (Nerenberg et al., 1991). Cell lines established by infection of primary T cells with taxexpressing H. saimiri as well as cells transformed by HTLV alone were analyzed for the expression of early response genes for T cell activation (Kelly et al., 1992; Fujii et al., 1991). The transcription of this group of genes represents the immediate genetic response to a specific interaction with an antigen-presenting cell. These genes are strictly regulated in normal T cells and corresponding RNAs can be observed only for very short time intervals spanning the transition from the Go to the GI phase of the cell cycle (Zipfel et al., 1989). Genes of the early response group were overexpressed to a very high level in tux- and HTLV-transformed lymphocytes (Kelly et al., 1992; Fujii et al., 1991).T h e normal function of this group of genes includes the initiation of proliferation and differentiation following antigenic stimulation. Trans-activation by the Tax protein of the same group of genes therefore resembles permanent T cell activation and may result in lymphoproliferation. According to this model (Fig. 3) it is likely that Tax contributes directly to the proliferation of HTLV-carrying T cells in infected patients.

V. Transformation of Human T Lymphocytes with Wild-Type Herpesvirus saimiri Herpesvirus ateles was the first rhadinovirus shown to transform T lymphocytes in culture. Falk et a!. (1974) found that peripheral and

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FIG. 3. Hypothetical model of Tax-mediated growth stimulation. The normal activation o f T cells is induced by the interaction of molecules of'the T cell receptor complex and membrane proteins with ligands localized at the surface of antigen-presenting cells. Signals (white arrows) were passed into the nucleus, resulting in the transient expression of the group of early activation genes. T h e corresponding gene products mediate a signal for cellular proliferation. A high percentage of these genes is also activated by the Tax protein, bypassing the natural signal pathways.

splenic lymphocytes from marmoset monkeys are immortalized by cocultivation with X-ray-inactivated virus-producing lymphoid cells. Later it became apparent that cell-free H. ateles also immortalizes marmoset lymphocytes (Falk et al., 1978). Lymphoid cell lines were obtained from tamarin (Saguinus oedaw, Saguinusfuscicollis, and Saguinus labiatus) and common marmosets (Callithrzxjacchus). 'The first signs of virus-induced proliferation usually became discernible some 3-4 weeks after infection of peripheral blood mononuclear cells with H. ateles. The transformed cells expressed CD2, which was demonstrated by rosetting with sheep erythrocytes; they did not express B cell-specific antigens such as membrane-bound immunoglobulin G or complement receptor 2 (Falk et al., 1978). All in vitro-transformed cell lines produced infectious virus on

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cocultivation with permissive epithelial monolayer cells, at least during the first months of growth in suspension culture. T h e number of cellfree virus particles in culture fluids of producing in vitro-transformed cell lines is always low (approximately 10 PFU/ml or less). Parallel attempts to immortalize tamarin marmoset lymphocytes by H. saimiri were surprisingly unsuccessful. Several laboratories experienced difficulties in trying to achieve immortalization of peripheral leukocytes from tamarin monkeys of the genus Sapinus by H. saimiri in culture. In one of numerous experiments, a cottontop marmoset cell line (termed H1591) was obtained from mononuclear blood cells infected with a H. saimiri strain of the subgroup A (see below), employing squirrel monkey fibroblasts as a feeder layer (Schirm et al., 1984). Subsequently, Desrosiers et al. (1986) found that T lymphocytes of common marmosets are readily immortalized in vitro by H . saimiri, establishing an easy standard system to analyze the transforming potential of wild-type virus and deletion variants of H. saimiri. A genomic segment required for growth transformation of lymphocytes and oncogenicity was localized to the left-terminal region of the 113 kilobase pair (kb) L-DNA in the H. saimiri genome (Koomey etal., 1984; Desrosiers et al., 1986; Murthy et al., 1989). Based on extreme sequence variation at the same left terminus of L-DNA, the known H. saimim' strains have been classified into three subgroups (A, B, and C) (Desrosiers and Falk, 1982; Medveczky et al., 1984). These subgroups appear to differ in their transforming and oncogenic potential; group A and C strains are efficiently transforming and highly oncogenic, whereas group B strains are less so (Medveczky et al., 1989). All lymphoid cell lines obtained by immortalization of monkey lymphocytes with H. saimiri o r H . ateles carry the viral genomes as nonintegrated, covalently closed circular DNA molecules at high multiplicity (KaschkaDierich et al., 1982; Gardella et al., 1984). Long-term culture of transformed cells can result in a stable nonproducer status of the cells, which is frequently accompanied by deletion of large regions of coding L-DNA sequences (Schirm et al., 1984). Early attempts to immortalize cells from human peripheral blood using H. saimim' were unsuccessful. However, in these early experiments virus strains from subgroups A and B were used. The more potent transforming potential of the oncogene encoded by the group C strains (Jung et al., 1991) prompted a reevaluation of the transforming potential for human lymphocytes. Wild-type strains of subgroup C were indeed found to be capable of transforming human T lymphocytes. Cultures of mononuclear cells from cord blood, from the peripheral blood of adults, from bone marrow, and from thymus yielded continuously growing lymphoblastoid cell lines following infection with the group C

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strains 484-77 o r 488-77 (Biesinger et al., 1992). All cells infected with strains 11 (group A) or SMHI (group B) and uninfected cells ceased to proliferate within about 4 weeks. Numerous lymphoid cell lines were established in the presence of IL-2 by infection with the subgroup C strain 488, some of them following stimulation by phytohemagglutinin, anti-CD3, or mixed lymphocyte reaction. All cell lines appeared to grow continuously; some cell lines have now been in culture for more than 20 months. Generally, these lines had a normal diploid phenotype (Troidl et al., 1993). Genomic H. sazmiri DNA sequences were found in all cell lines except one at 30-60 genome equivalents per diploid cellular genome (Biesinger et al., 1992). T h e lymphoid cell lines contained the multicopy viral genomes as episomes, as has generally been observed in lymphomaderived monkey lymphocytes (Kaschka-Dierich et al., 1982) and in human tumor cell lines persistently infected with selectable H. saimim' (Grassmann and Fleckenstein, 1989; Simmer et al., 1991) or HTLV-1IH. saimiri recombinants (Grassmann et al., 1989, 1992). The immortalized human lymphoblastoid cells, in contrast to monkey cell lines, did not secrete detectable amounts of infectious virus into the culture supernatant. Transcripts from the stp-C oncogene (see below) and its respective protein product, a polypeptide of 102 amino acids, have been detected in the transformed lymphocyte lines. Whereas several transcripts of this genomic L-region are found in RNA from lytically infected and semipermissive cultures, human H. saimiri-transformed cell lines have a single transcript of 1.7 kb (H. Fickenscher, B. Biesinger, S. Wittmann, and B. Fleckenstein, unpublished). Surface phenotyping for a variety of membrane antigens consistently indicated that all continuous human cell lines expressed the characteristic profile of mature, activated T cells. All lines, except one thymusderived culture (Lucas), were either CD4+ CD8- o r CD4- CD8+. T h e thymus-derived cell line Lucas displayed a mixed phenotype with a small subpopulation of CD4/8 double-positive cells. Other characteristic T cell surface antigens, such as CD2, CD3, TCR-a/P, and CD7, were expressed in high amounts on all cell lines; CD5 was nearly always present. The interleukin-2 receptor (Y chain CD25 was found to be expressed to varying degrees in all cell lines tested to date; most, but not all, continuous lines were dependent on exogenous IL-2. All cell lines were negative for the nonspecific killer (NK) cell marker CD57 (HNK-1). A second marker for lymphocytes with NK activity, CD56 (NKH-I), was found on nearly all of 13 cell lines (Biesinger et ad., 1992). Strong cytotoxic activity on K562 cells was observed for all CD8+ lines. T h e CD4+ CD8- human lines showed low-level nonspecific killing. Likewise, marmoset lymphocytes transformed by strains of H. saimiri subgroup A were CD8+, had

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NK activity on sensitive cell targets, and expressed the surface marker CD56 (Kiyotaki et al., 1986). Thus, the human T cell lines immortalized by H. saimiri were not typical NK populations, as they always expressed T cell receptors and the CD3 complex, and the markers CD16 and CD57 were missing. Marker analysis for isoforms of CD45, a transmembrane phosphatase, indicated that the H. saimiri-immortalized T cells always had CD45 RO and CD45 RA, as is characteristic for memory T cells. Remarkably, the CD8+ human T cell lines, regardless of the organ from which they originated, expressed the CD45 RA isoform in addition; this marker is typically present on the surface of naive T cells and their CD3negative percursors. The early differentiation marker CD 1 was not detected, but the activation marker CD30 was present in various amounts, and MHC class I1 antigens were expressed consistently in high quantities (B. Simmer and E. Platzer, unpublished). Transformed cells of T helper subgroup 1 and T helper subgroup 2 spontaneously secreted the subgroup-specific cytokines (De Carli et al., 1993). In summary, the H. saimiri-immortalized cell lines obtained from mixed primary cultures of peripheral blood, thymus, o r bone marrow always had the phenotype of activated and mature CD4+ or CD8+ T lymphocytes. Mature T lymphocyte cell lines also arose when bone marrow cultures from patients with acute lymphocytic o r chronic myelogenic leukemia were infected. This could be explained by a growth advantage for the rhadinovirus-transformed mature T cells relative to other cell populations that may be transformable by the virus. It is also possible that growth transformation may be strictly confined to lymphocytes that have undergone differentiation to mature T cells. Alternatively, undifferentiated T lymphocyte precursors could be the primary target for rhadinovirus transformation, but regularly undergo differentiation in culture to result in mature cell populations. It is not yet possible to differentiate among these possibilities. T h e repertoire of human T lymphocytes amenable to immortalization by H. saimiri has not yet been fully determined. In this context it is noteworthy that transformation trials with preselected T cells, such as CD4+ T cell clones of known antigen specificity and cytokine secretion profile, have usually been successful (De Carli et al., 1993). This suggests that transformability is not restricted to a limited set of T cell subpopulations, but that mature T lymphocytes expressing T cell receptors of the a/p type in general are susceptible to the H. saimiri growth-stimulating effects. This was corroborated by studies on the clonality of T lymphoblasts growing out from mononuclear blood cell cultures after virus-induced transformation. Fluorescence-activated cell sorting (FACS) analysis was performed with monoclonal antibodies with specificity for particular TCR VP chain fam-

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ilies. There was no preferential expansion of T cells expressing selected TCR Vp elements during the initial phase of proliferation. Biased expression of TCR V genes was observed only after long-term culture (>3 months), as would be expected following clonal outgrowth of the most rapidly proliferating cells. (H. Fickenscher, R. Sekaly, B. Simmer, and B. Broker, unpublished observation). Furthermore, long-term cultures established from different donors at differing times displayed widely varying patterns of TCR Vp gene expression. Based on these results, it seems unlikely that viral products stimulate T cells expressing selected Vp elements in a superantigen-like fashion. The transformation process seems to be independent of superantigens. These observations are relevant because H. saimiri has been found to encode a protein product with sequence similarity to the MMTV superantigen (Thomson and Nicholas, 1991; J.-C. Albrecht et al., 199213). VI. Growth Regulation and Antigen Specificity of Rhadinovirus-Transformed T Cell Lines and Clones

An initial study on the growth regulation of virus-transformants was performed with three cell lines-two T cell lines with CD4+ phenotype derived from cord blood cells and a CD8+ line from thymocytes (Mittrucker et al., 1992). A common characteristic of the cells was their response to allogeneic stimulator cells, regardless of their MHC class I or I I expression. T h e stimulatory signals seemed to be mediated by binding of CD2 molecules to their natural ligand, LFA-3 (CD58). This was demonstrated by the selective inhibition of growth stimulation by antLCD2 and anti-CD58 monoclonal antibodies. Furthermore, this notion is supported by the potent stimulatory activity of cross-linked monoclonal antibodies directed against the ligand-binding site of CD2, and by the response to sheep erythrocytes and to CD58-expressing transfected cells. Thus, in the virus-transformed human cell lines, binding of CD2 to its natural ligand seems sufficient for activation in the absence of other ligdnds for T cell surface structures. In contrast to resting T lymphocytes, herpesvirus-transformed cells may have an independent activation pathway through CD2 that is uncoupled from TCR/CD3 signaling, or, alternatively, an activation pathway may be constitutively switched on that otherwise depends on the TCR-mediated signaling cascade. Following stimulating signals, the virus-transformed cells react, like normal lymphocytes, with a proliferative response, with IL-2 secretion, and with induction of the IL-2 receptor (Y chain. T h e interaction of CD2 with LFA-3 by cell-to-cell contact seems to be the signal leading to autocrine growth. Permanent activation by these signals seems essential for

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growth; cyclosporin A and the compound FK506 suppressed IL-2 production and proliferation in parallel (Mittriicker et al., 1992). Likewise, monoclonal antibodies directed against Tac antigen (IL-2 receptor (Y chain) inhibited growth further. This suggests that H. saimiritransformed T cell clones grow via an IL-2/IL-2 receptor autocrine mechanism in response to activation signals given by their CD2 and CD58 molecules during mutual cell-to-cell contact, providing the first known example of activation-dependent permanent autocrine growth. Further studies on the proliferation control were performed using T cell clones of known Vp subtype that were transformed to antigenindependent growth by H. saimiri (Broker et al., 1993). In general, the virus-transformed cells and their nontransformed progenitors were strikingly similar in parameters of early signal transduction, namely tyrosine phosphorylation and mobilization of calcium. Tyrosine phosphorylation is believed to be one of the first steps in signal transduction in T lymphocytes. The tyrosine phosphorylation patterns elicited by monoclonal antibodies against CD4 and/or the TCR/CD3 complex appeared essentially identical in the H . saimiri-transformed clones and their nontransformed parent clones. In both cases there were only low levels of constitutive phosphorylation; the hierarchy of general phosphorylation intensity in response to the various signals was also preserved in the transformants. In contrast, the tumor cell line Jurkat had pronounced constitutive tyrosine phosphorylation in the absence of external signals, whereas phosphorylation of p561ck was diminished (Broker et at., 1993). Increases of the cytoplasmic free calcium result from signal-induced phosphoinositol turnover after activation of phosphoinositol-phospholipase C and are linked to T cell activation. T h e characteristics of calcium mobilization remained qualitatively unchanged in the virus-transformed lymphoblasts as compared to the antigen-dependent precursor clones (Broker et al., 1993a). Long-term (>24 hours) incubation with antibodies against CD4 suppressed dramatically the basal proliferation of H. saimiri-immortalized T cells; the same growth suppression was obtained by anti-CD4 F(ab), fragments, although 100- to 1000-fold higher doses were required in the case of the monovalent Fab-fragments or the gp120 surface molecule of HIV, a ligand for CD4. T h e growth inhibition was overcome by high concentrations of exogenous IL-2. There was no apoptosis, but a rapid and longlasting down-regulation of the IL-2 receptor a-chain expression was characteristic for the incubation with anti-CD4. The growth inhibition through CD4 ligands was accompanied by a parallel reduction of CD4-bound p56"k protein, whereas the unbound fraction remained unchanged. Because IL-2 binding activated only the CD4-bound fraction of p56kk,

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ligation of CD4 followed by reduction of CD4-bound p561ck probably rendered signaling through the IL-2 receptor less effective. Thus, the data provided evidence for a cross-talk between CD4 and the IL-2 receptor in T lymphocytes mediated through p56"k (Broker et al., 1994). Whether rhadinovirus-transformed cell lines will help immunologists analyze T cell function will depend to a large extent on the continued expression of the TCR and conservation of its function. Mittriicker et al. (1993) showed that 1L-2 secretion, cytotoxicity, and exocytosis of granule esterase can be triggered by recognition of the TCR through a bacterial superantigen, suggesting that the cells are functionally normal in their response to TCR-mediated signals. Antigen recognition through the TCR of immortalized cells was demonstrated first with CD4+ human T cell lines specific for myelin basic protein (MBP) (Pette et al., 1990). T h e transformed T cells conserved their membrane phenotype (CD3+,CD4+, CD8-) and continued to express the original T cell receptor as demonstrated by VP-specific monoclonal antibodies and TCR gene sequencing. MHC class II-restricted antigen recognition and signal transduction by the TCR were demonstrated by MBP-induced secretion of interferon-y and induction of proliferation (Weber et al., 1993). Broker and colleagues (1993a) also found that a CD4+ T cell clone, directed against MHC class II-presented tetanus toxoid, preserved its specific activation pattern as measured by growth response and interferon-y secretion following immortalization with H. saimiri strain C488. This indicates that rhadinovirus transformation will allow production of large quantities of T cells with functional TCRs and stable membrane phenotype. T h e availability of T cell lines with membrane phenotype and signaling pathways most closely resembling primary cultures suggests their use as permissive culture systems for viruses that optimally grow in primary lymphocyte cultures. CD4+ human lymphoid cell lines transformed by H. saimiri were found susceptible to infection with the human immunodeficiency virus (HIV) prototype strains HIV-1 IIIB and HIV-2 ROD. The cell lines were also suitable for the propagation of an HIV-2 isolate with restricted cell tropism originating from an asymptomatic individual. Rhadinovirus-transformed human T cells thus represent a new permissive system for HIV and may provide a useful alternative means for isolating, culturing, or quantitating HIV. The cells may be especially useful for large-scale production and biological analyses of primary HIV isolates that are not able to grow in tumor cell lines (Nick et al., 1993). Likewise, immortalization of rhesus monkey peripheral blood CD4+ T lymphocytes by H. saimiri C488 has generated a convenient autologous production system for simian immunodeficiency virus (SIVn,ac) (Desrosiers et al., 1994).

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VII. Transforming Potential of the Herpesviral stp Oncogene T h e complete nucleotide sequence of the H. saimiri group A strain 11 (A1 1) was determined by the cooperation of two laboratories (J.-C. Albrecht et al., 1992b). The detailed analysis of this sequence has greatly facilitated the search for viral T cell-transforming oncogenes and puta-

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tive virus-encoded cofactors. The L-DNA region has a total of 112,930 bp, including a short GC-rich transition at the left junction of H - and L-DNA (Murthy et al., 1989). The entire L-region encompasses 76 major open reading frames and a set of seven U-RNA genes for a total of 83 potential genes (Fig. 4).The GC content of L-DNA is 34.5%. It is markedly depleted in CpG, whereas this dinucleotide occurs at near-random frequencies in a- and P-herpesviruses. This may be related to methylation of the episomal viral DNA persisting in T lymphocytes, their precursors, or derivatives in the natural hosts (Honess et al., 1989). The nucleotide 5-methylcytosine is a hot spot for mutations, because spontaneous desamination of 5-methylcytosine results in a change to thymidine (Coulondre et ul., 1978). The dinucleotide CpC may have been specifically lost, because methylation activity is particularly high in lymphoid cells containing H . ~aimiri(Desrosiers, 1982; Desrosiers et al., 1979). Open reading frame (ORF) 27 has the characteristic signature of a cytosine-specific methylase (J.-C. Albrecht et al., 1992b), suggesting that its expression during the state of persistence may be related to the de novo methylation detected in H . saimiri episomes; none of the other herpesviruses that have been sequenced contains a similar sequence motif. Herpesvirus saimiri in addition, encodes two enzymes involved in dTMP synthesis, i.e., thymidylate synthase (ORF70) (Honess et al., 1986; Bodemer et al., 1986) and dihydrofolate reductase (ORF2) (Trimble et al., 1988), which may contribute to the high AT content of L-DNA. The left-end L-DNA of H . saimiri A l l has seven U-RNA genes (HSUR genes), including promoters with typical transcription regulatory elements of cellular U-RNAs (Albrecht and Fleckenstein, 1992b; Biesinger et al., 1990; Murthy et al., 1986; Lee et al., 1988; Wassarman et al., 1989). The HSUR genes were not found to be expressed in productively infected cells, but were abundant in transformed cells and H . saimiri-induced tumors (Murthy et d., 1986). Myer et al. (1992) hypothesized that the HSURs may contribute to lymphocyte transformation by sequestering a protein factor involved in the degradation of cellular mRNAs relating to growth regulation. Besides HSURs, at least three other virus genes can be considered as candidates possibly contributing to stimulation of T cell proliferation. ORF14, a gene expressed during the immediate-early phase of viral replication (Nicholas et al., 1990), has sequence similarity to an ORF in the long terminal repeat of the niouse mammary tumor proviral DNA and mls genes of mice that function as superantigens (Thomson and Nicholas, 1991). ORF72 is homologous to members of the cyclin family (Nicholas et al., 1992) and is most closely related to human cyclin DI (Xiong et al., 1991), whose derivative PRAD-1 is a candidate oncogene that may play an important role in the develop-

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ment of centrocytic lymphoma and parathyroid tumors (Motokura et al., 1991; Rosenberg et al., 1991). ORF74 is related to G-protein-coupled receptors (Nicholas et al., 1992), which have been shown to be involved in malignant cell transformation (Julius et al., 1989). Also worth noting is the presence of two genes (ORF4 and ORF15) encoding glycoproteins related to complement control proteins, which down-regulate complement activity at two distinct steps of complement activation (Albrecht and Fleckenstein, 1992a; J.-C. Albrecht et al., 1992a; Rother et al., 1993). Herpesvirus saimiri could possibly use these products for avoidance of' host immune defenses. It remains to be seen, however, the extent to which these products contribute to the ability of the virus to persist. With an exceptionally high number of genes with pronounced homology to cellular genes, H . saimiri seems to be particularly prone to capturing cellular genes that, like retroviral oncogenes, could be involved in growth stimulation of persistently infected cells. Analysis of spontaneously attenuated deleted variants of H . saimiri strain A1 1 indicated that the left-most L-DNA region may be involved in oncogenic transformation. The attenuated variant 1latt was obtained by serial propagation of H . saimiri A l l (Schaffer et al., 1975). Cottontop marmosets inoculated with strain 1 latt did not develop lymphoma even though the virus was infectious. Persistent infection was confirmed by antibody response and by the recovery of virus from circulating blood cells over time. Persistent infection of common marmosets with strain A1 latt was able to protect common marmosets from challenge by wildtype oncogenic virus (Wright et al., 1980). Herpesvirus saimiri strain 1latt has a deletion of 2.3 kb that spans its left junction between the repetitive H- and L-DNA (Koomey et al., 1984). Complementation of the deleted sequences restored the oncogenic capacity (Desrosiers et al., 1985a). Desrosiers et al. (1984, 1985a, 1986) constructed a series of mutants of H . saimiri strain A1 1 that were deleted in the same genomic region. These mutants failed to immortalize marmoset lymphocytes in nitro, when the mutation affected the left-most reading frame (ORF1) of the A l l genome. These experiments provide strong evidence that the corresponding gene product, a predicted membrane-spanning polypeptide of 164 amino acids, is required for transformation; the gene product was termed Stp-A ( H . saimiri transformation-associated protein of group A virus) (Murthy et al., 1989). Pronounced nucleotide sequence divergence in the left-end L-DNA forms the basis of subclassification of known H . saimiri strains into three subgroups, A, B, and C. The virus strains of subgroups A and C are highly oncogenic for a wide spectrum of New World primates, whereas subgroup B strains appear to have a limited tumor-inducing capacity

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(Medveczky et al., 1984). Subgroup C viruses are highly oncogenic in New Zealand white rabbits; group B viruses are not. However, recombinants of a subgroup B virus with a fragment from the H- to L-DNA junction of subgroup C strain 484 were as pathogenic as strain 484 (Medveczky et al., 1989). This indicates that sequences collinear to the stp region of subgroup A determine the transforming properties of these group C viruses as well. The nucleotide sequence of group C strain 488 left-end L-DNA indicated a genomic organization related to but distinct from the previously sequenced group A strain 11. Strain 488 contains two open reading frames with the potential to code polypeptides of 102 and 256 amino acids at the position equivalent to stp-A (Biesinger et al., 1990). Both polypeptides share minimal sequence homology with Stp-A and the corresponding peptide of subgroup B virus, Stp-B, but do contain a carboxy-terminal hydrophobic domain in the absence of aminoterminal signal sequences. A similar structure is found in the polyoma virus middle T antigen; it lacks a hydrophobic leader and its membranespanning domain is important for transformation (Markland and Smith, 1987). There were no similarities between these H. saimiri ORFs and the conserved patterns found in membrane-bound protein kinases (Hanks et al., 1988), including the pim-1 and lck oncogenes, which are implicated in T cell lymphomagenesis (Selten et al., 1986; Koga et al., 1988). Likewise, Stp-A and the corresponding peptides of group B and C viruses did not have the transformation-relevant sequence motifs found in ras oncoproteins, and no homology was detected to any known retrovirus o r DNA virus oncogene. There was also no compelling evidence for zinc finger o r leucine zipper secondary structures, which would indicate the potential of the protein to bind nucleic acids in a sequence-specific manner akin to many transcription factors. Sequencing of the left-terminal 6.5 kb o r L-DNA from subgroup B virus SMHI revealed an open reading frame (stp-S) for a polypeptide of 171 amino acids at an equivalent position and orientation to Stp-A. They have in common a hydrophobic carboxy-terminal putative transmembrane domain and a weakly acidic amino terminus. They show common amino-terminal putative phosphorylation sites, but no consistently conserved protein or nucleotide sequences motifs (Ensser et al,, 1994). A most remarkable structural feature of all stp gene products are characteristic collagen-like repeats (Fig. 5 ) . T h e primary amino acid sequence of Stp-A11 has nine repeated 3-amino acid motifs, each of the structure Gly-X-Y, where X and/or Y are proline. The collagen motifs are directly repeated 18 times in the central region of the 102-amino acid gene product from ORF2 of strain C488; the nine collagen motifs of Stp-A1 1 are similarly concentrated in the central portion of the protein, but not directly repeated as in Stp-C

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FIG. 5. Schematic representation of sfp gene products from H . saimiri A l l and H . sairniri C488. Collagen-like repeats, hydrophobic regions, acidic segments, and putative phosphorylation sites are indicated.

(Jung et al., 1991). Stp-B of one H. sairniri B strain was found to be more like Stp-A (Ensser el al., 1993). Transfer of expression cassettes for stp genes onto rodent monolayer cells indicated transforming and tumor-inducing capabilities. Specifically, ORF2 of strain C488 (spC488)can transform the established cell line rat-1 to loss of contact inhibition, formation of cellular foci, growth under reduced serum concentration, and tumorgenicity in nude mice (Jung et al., 1991). Stp-C is found associated with cytoplasmic membranes and is primarily located in perinuclear compartments of transformed cells (Jung and Desrosiers, 1991). Stp-C488 is phosphorylated to about 15% in transformed cells. A serine residue near the amino terminus was shown to be the site of phosphorylation (Jung and Desrosiers, 1992). Stp-A1 1 is only weakly transforming for rat-1 cells. The rat-1 cells transformed by Stp-C488 formed invasive tumors in nude mice. Experiments to analyze the growth-transforming capabilities of Stp-C were performed by generating transgenic mice. To obtain s1p-C transcription in a wide variety of tissues, the reading frame was cloned under the control of a mouse H-2 promoter (Murphy el al., 1994). Seventeen transgenic mice were obtained, the majority of which died within the first month. Histopathological evaluation revealed hyperplasia of the epithelial cell compartment of salivary glands, liver, pancreas, and thymus, which occurred as early as day 16 postnatally and progressed into adenomas and thymomas between days 28 and 41. All transgenic mice exhibited characteristic alterations in the salivary glands, frequently developing into adenomas. Some animals developed bile duct tumors characteristic of cholangiofibromas. Of the 17 transgenic mice, 15 had lesions, for example, medullary thymomas with excessive epithelial

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hyperplasia. T h e stp-C transgene was expressed at low levels in most organs. Indirect immunofluorescence tests with salivary gland tissue revealed a distinct cytoplasmic staining pattern for Stp; the protein was uniformly expressed throughout the section. Western blots confirmed the presence of Stp-C488 protein and its phosphorylated form, of the expected sizes. Stp-C488 was also expressed in thymocytes, but remarkably there was no indication of increased proliferation of T lymphocytes o r their progenitors. Thus, T cell transformation may require additional virus sequences, a higher gene dosage, or specific regulation of stp expression. The absence of proliferative stimulation could also reflect a species variation in the susceptibility of T lymphocytes. T h e induction of epithelial tumors in the transgenic mice clearly documents that stp-C has oncogenic activity in the absence of the remainder of the viral genome. T h e study of Murphy et al. (1994) defines stp-C as a herpesviral oncogene sufficient for induction of tumors in animals. A number of transformation trials have been attempted with human and common marmoset T lymphocytes using a retroviral expression cassette of stp-C488. Although a number of slowly growing IL-2dependent human cell cultures were obtained that replicated for several months or nearly 1 year, it is not yet possible to decide if stp-C alone is sufficient to sustain continuous growth of lymphocytes in the absence of other H . saimiri genes. Common marmoset lymphocyte cultures receiving stp-C survived much longer than control cultures, but continuously growing cell lines were not obtained (J.Jung, personal communication). These results suggest that other viral gene products may contribute to the full transforming potential of H . saimiri for T lymphocytes or that quantitative or regulatory aspects of stp expression are critical for growth stimulation. VIII. Concluding Remarks

Two unrelated viruses, the retrovirus HTLV and herpesvirus saimiri, have developed strategies to stimulate the proliferation of T lymphocytes, the cellular target of their persistence. In this article we have summarized the present state of knowledge on in vitro growth transformation of human T lymphocytes by HTLV-1, H . saimiri, and H . saimidHTLV-1 recombinants. T h e permanent T cell replication that these viruses can induce is accompanied by and probably caused by induction and/or maintenance of an activated state of the infected cell. This type of T cell activation is likely to bypass components of the normal activation pathway, which normally initiates with ligand binding to surface receptors. T h e capacity for immortalization of human T cells in

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vitro has proved useful for the investigation of cellular immunity, T cell signaling, lymphoproliferation, and leukemogenesis. Whereas HTLVtransformed T cells frequently undergo functional changes, human T lymphocytes infected with the H. saimiri C strains seem to be unaltered with respect to T cell function and early T cell signaling. Herpesvirus saimiri C strains allow direct functional comparison of transformed and nontransformed cells of the same origin. This is not possible with the commonly used leukemic T cell lines, because their normal progenitors are not known. Use of immortalized T cell lines for studies of T cell signaling avoids the problems caused by contaminating feeder cells or by accessory cells present in preparations of fresh lymphocytes. The H. saimiri-infected T cell lines can also serve as a source of unlimited amounts of a specific T cell receptor, which could be used for the investigation of antigen-specific immunoregulation in uitro and potentially in experimental animals, e.g., SCID mice. The ability of H . saimiri to immortalize T cells depends on the expression of the herpesviral oncogene stp; the HTLV-mediated growth stimulation requires the expression of the regulatory gene tax. The transforming potential of both proteins is not restricted to T cells, Whereas Tax is needed for viral replication, Stp is nonessential with respect to multiplication of virus in cell culture. T h e capacity of Tax, a nuclear protein, to transactivate the promoters of various activation genes, at least in part explains the activated phenotype and the proliferative stimulation of HTLV-transformed lymphocytes. T h e localization of Stp-C in the cytoplasm suggests a different mechanism for influencing T cell activation and growth control. Additional research will be needed to identify the cellular targets of Stp action and the contribution of the other viral factors to the full transforming potential of the virus. Studies in both H. saimiri and HTLV systems can help define the signaling pathway used in normal and transformed cells for the induction of T cell proliferation.

ACKNOWLEDGMENTS Original work reported in this review was supported by Deutsche Forschungsgemeinschaft, Forschergruppe “DNA-Viren des hamatopoetischen Systems,” Ria Freifrau von Fritsch-Stiftung (to R.G.), Johannes und Frieda Marohn-Stiftung, and from grants from the US. Department of Health and Human Services. We are grateful to Barbara Broker, Bryan Cullen, and Helmut Fickenscher for critically reading the text and thank Irene Stubrach for preparing the manuscript.

REFERENCES Aepinus, C., Grassniann, R., Voll, R., and Fleckenstein, B. (1990).In “Progress in AIDS Research in the Federal Republic of Germany” (M. Schauzu, ed.), pp. 41-46. MMV Medizin Verlag, Miinchen.

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LYMPHOMAGENESIS IN AKR MICE: B CELL LYMPHOMAS AS A MODEL OF TUMOR DORMANCY Nechama Haran-Ghera Department of Chemical immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

I. Introduction 11. Identification of Potential Lymphoma Cells in AKR Mice 111. Enhanced T Cell Lymphoma Development Pathways

1V.

V. VI.

A. Acceleration by Recombinant Dual Tropic Viruses B. Induction by the Ecotropic Retrovirus SL3-3 C. Induction by the Radiation Leukemia Virus Variant A-RadLV D. Lymphomagenic Effect of Fractionated Irradiation E. N-Methyl-N-Nitrosourea-Induced T Cell Lymphomas F. Conclusions The Level of Dormant PLCs Following Prevention of Spontaneous T Cell Lymphoma Development A. PLCs in Thymectoniized AKR Mice B. PLC Level Following Lymphoma Prevention by Viral Interference C. PLCs in AKR-Fv-I” Congenic Mice D. Elimination of PLCs in AKR/J Mice Following Passive Inimunotherapy E. Conclusions Maintenance and Termination of the B-PLC Dormant State Ly-l+ (CD5+) B Cell Lymphoma Characteristics A. Ly-l B Cells B. Analysis of the Heavy and Light Chain Loci of Ly-I+ B Cell Lymphomas C. T h e Pattern of IgH Rearrangements in Spleens of Grossly Normal Old AKR-Fv-lh and Thymectomized AKR Mice D. Lack of Class I MCF Virus Involven~entin Ly-l+ B Cell Lymphomas Concluding Remarks References +

VII.

1. Introduction

The A K R inbred mouse strain, established by Furth some 60 years ago (Furth et al., 1933), displays a high incidence of spontaneous T cell lymphomas that arise predominantly in the thymus of 6- to 12-month old mice. Beyond this age, sporadic B cell lymphomas arise mainly in the peripheral lymphoid organs of old mice (Greenberg et al., 1977). Besides the central role of the thymus in A K R lymphomagenesis, a group of heterogeneous nonacute transforming retroviruses are associated with 245 ADVANCES IN CANCER RESEARCH, VOL. 63

Copyright 8 1994 by Academic Press, Inc. All rights of 1-eproduction in any form reserved.

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NECHAMA HARAN-GHERA

the etiology of the disease: the endogenous ecotropic viruses inherited in AKR mice at two nonlinked chromosomal loci, Akv-1 and Akv-2 (Rowe, 1972; Chattopadhyay et al., 1980): the xenotropic virus (Levy, 1973); and a new class of recombinant viruses. This de novo-arising class of dual tropic recombinant murine leukemia viruses (DTVs) can be identified by its ability to form cytopathic foci in mink lung cell cultures, and members are accordingly called mink cytopathic focus-forming (MCF) viruses. This class of viruses has been established through a series of genetic recombinations between endogenous ecotropic virus and endogenous xenotropic o r MCF-like proviral gene sequences (Fischinger et al., 1975; Hartley et al., 1977). MCF viruses, detected in the spleen and thymus, are subdivided into two groups, Class I and Class 11, based on their oncogenicity and genomic structure. High titers of ecotropic viruses are expressed at all ages in AKR mice, whereas low levels of xenotropic virus are expressed in young mice (<5 months), but the xenotropic virus titer increases with age, particularly in the thymus of preleukemic mice. The ecotropic murine leukemia virus (MuLV) is not lymphomagenic: insertion of the AKR viral genes (Akv-1 and Akv-2) on NFS background (lacking endogenous virus), resulting in NFS-V mice congenic for ecotropic MuLV induction loci, did not enhance T cell lymphoma development but rendered these mice more susceptible to the development of nonthymic lymphomas of the B cell phenotype (Fredrickson et al., 1984). The xenotropic virus was also shown to lack lymphomagenic activity (Yanagihara et al., 1982). Class I MCF viruses formed in the thymus of 4- to 5-month-old AKR mice (during the preleukemic period) are generated by recombination events within the env gene and 3' region of ecotropic and nonecotropic viral genomes (Herr and Gilbert, 1983). These viruses preferentially infect cells of the thymic cortex and accelerate T cell lymphoma development when injected into young AKR mice. Class I MCF viruses have therefore been considered as the proximal vector of transformation in AKR mice (O'Donnell et al., 1981). T h e mechanism by which MCF viruses induce lymphomas is not defined; however, insertional activation of cellular oncogenes by provirus integration is considered to be responsible for this acceleration phenomenon. MCF Class I1 viruses [present in spleens of 3-week-old mice (Cloyd, 1983)],which contain one substituted region, usually encompassing the entire env gene (Holland et al., 1989), are neither thymotropic nor lymphomagenic. T h e development of spontaneous T cell lymphomas in AKR mice can be prevented by thymus removal at the age of 1-3 months (McEndy et al., 1944). As a consequence of the prolonged life span of these thymectomized AKR mice, they develop a low incidence (up to 30% at a mean

LYMPHOMAGENESIS I N AKR MICE

247

latency of 600 days) of B cell lymphomas. The susceptibility to lymphoma development in thymectomized AKR/J mice can be restored by subcutaneous thymus grafts (Law and Miller, 1950; Miller, 1960). I n thymectomized F, hybrid mice, made by crossing high-incidence and low-incidence lymphoma strains of mice, susceptibility to T cell lymphoma development was restored by thymus grafts (into young recipients) from the high-incidence leukemia strain but not by the grafts from the low-incidence leukemia strain parent (Miller, 1960). T h e thymus graft is composed of donor epithelial and reticulum cells and host lymphoid cells. Because the lymphomas that develop in the graft are mostly derived of host cells that have become neoplastic in the environment of the thymus graft (Law and Potter, 1956), the thymic epithelial and reticulum cells seem to exert a proliferative stimulus to the lymphoid cells that seed the thymus, probably through the expression of the leukemogenic retrovirus. A stromal cell, the macrophage, was actually found to be the first thymic element to produce detectable levels of recombinant retrovirus, approximately 12 weeks before thymocytes (Kim et al, 1991). Thus, the preventive effect of thymectomy stresses the role of the recombinant retroviruses and the thymus microenvironment in lymphomagenesis. Their role is also indicated in the athymic mutant genotype of AKR mice (referred to as streaker mice). I n these mice both ecotropic and xenotropic viruses are produced, but in conjunction with the absence of a thymus there is a low-level recombinant virus expression that leads to refractoriness of these mice to spontaneous T cell lymphoma development (Bedigian et al., 1979); some of these mice develop B cell lymphomas and granulocytic leukemias. The thymus grafting experiments in thymectomized mice suggest that the development of the disease involves a multiphase process. T h e initial phase of this sequence may occur in cells located elsewhere in the body (in the bone marrow), which later seed in the thymus (the site of the recombinant virus establishment) and there finally acquire full characteristics of neoplastic cells. Experimental data support this suggestion. Establishment of long-lived allogeneic bone marrow chimerism between DBA/2 and AKR mice (Pollard and Truitt, 1973) or between BL/6 and AKR/J mice (Wustrow and Good, 1985) prevented lymphoma development. In contrast, AKR mice reconstituted with syngeneic marrow cells did not afford any protection (Legrand et al., 198 1). It should be stressed that in the chimeric mice, similar viral gene expressions and viral gene amplification (preceding overt lymphoma development in normal AKR mice) occurred in the thymus and spleen of these lymphoma-resistant chimeric mice. Thus, viral amplification, although necessary for T cell lymphoma development, is not sufficient. The presence of potential

248

NECHAMA HARAN-GHERA

lymphoma cells (PLCs) among bone marrow cells of young intact or thymectomized AKR mice, causing a high T cell lymphoma incidence on transplantation into appropriate recipients (Haran-Ghera, 1980), could explain the preventive effect of allogeneic bone marrow transplantation. Following lethal irradiation the AKR bone marrow containing PLCs is replaced by the DBA/2 and BL/6 bone marrow (lacking PLCs) used in the above-cited experiments, thereby eliminating PLC seeding in the AKR thymus. In the syngeneic chimera, PLCs present in the reconstituting bone marrow of donor AKR mice replace the PLCs eradicated by lethal irradiation. Prevention of T cell lymphoma development in AKR mice was also achieved by restricting MuLV replication and spread of its endogenous N-tropic MuLV. Relative susceptibility to infection of mouse cells by ecotropic MuLV is regulated by alleles at the cellular Fu-1 locus (Pincus et al., 1971). There are two primary alleles at the Fv-I locus: Fu-Z” and Fv-lb (Hartley et al., 1970). The Fu-In allele is nonpermissive for the replication of the B type ecotropic viruses and Fv-16 is nonpermissive for replication of N-type ecotropic viruses. AKR/J mice carry the Fv-l)~ allele and in the AKR-Fv-lb congenic strain the Fu-ln allele was substituted with the Fu-lb allele of the C57BL/6 mice (Stockert et al., 1979). This Fv-lb substitution was shown to limit viral replication and spread of the endogenous N-tropic MuLV in thymocytes, splenocytes, and bone marrow cells of AKR-Fu-lb mice (Rowe and Hartley, 1972). As a result of this genetic viral restriction, these AKR-Fu-16 mice develop a very low incidence (7%)of spontaneous T cell lymphomas and about 28% B cell lymphomas, observed in old mice (Haran-Ghera et al., 1993). Several studies (Huebner et al., 1976; Schafer et al., 1977; Schwarz et al., 1979; Fischinger et al., 1982) have shown that passive administration of antibody directed against viral gp71 to neonatal mice had a strong inhibitory effect on T cell lymphoma development in AKR mice. This therapy must be initiated during a narrow “window,” comprising the first 3 days of life, in order to be effective. The success of the treatment correlated with elimination of detectable ecotropic or recombinant MuLV, elimination of somatic proviral DNA amplification, and appearance of host antiviral humoral reactivity (Weinhold et al., 1984). More recent studies correlated the leukemosuppressive immunotherapy with suppression of the early development of MuLV infectious cell centers (ICCs) in the bone marrow, spleen, and thymus populations (Buckheit et al., 1989) as well as elimination of PLCs from bone marrow of AKR mice (Haran-Ghera and Peled, 1991). Such treated mice developed a low incidence of T cell lymphomas (7%) and about 20% B cell lymphomas (mean latency of 650 days).

LYMPHOMAGENESIS IN AKR MICE

249

Another method to reduce T cell lymphoma development in intact AKR mice was through interference with the generation and spread of the dual tropic viruses that are formed de novo in the AKR thymus during the late preleukemic phase. Intrathymic injection of a dual tropic MuLV originally derived from a Moloney MuLV stock was found to protect AKR mice from developing MuLV-accelerated lymphoma and spontaneous lymphoma (Stockert et al., 1980). Similarly, injection of a nonlymphomagenic ecotropic virus (designated 24-666), isolated from a B cell lymphoma of AKR origin, into young AKR mice was also shown to prevent spontaneous T cell lymphoma development (Peled and HaranGhera, 1991). T h e virus seems to act through interference with the establishment of the recombinant dual tropic virus in the thymus. Beyond the age of 300 days, sporadic B cell lymphomas were observed and their cumulative incidence increased with age, reaching 34% at the age of 600 days. Thus, prevention of spontaneous T cell lymphoma development in AKR mice, using different experimental methods that interfere with DTV formation or PLC availability, was accompanied with a moderate increase in spontaneous B cell lymphomagenesis. These experimental manipulations provided means to study the role of PLC and DTV in the pathogenesis of T and B cell lymphomagenesis in AKR mice-the subject of the present article. II. Identification of Potential Lymphoma Cells in AKR Mice

Restoration of the potential to develop spontaneous T cell lymphomas following thymus removal at young age, by subcutaneous thymus grafting, was shown to involve host cells that seed and repopulate the thymus graft. We have demonstrated that potential lymphoma cells that develop predominantly into overt T cell lymphomas are present in fetal liver of 16-day-old embryos, and mostly among bone marrow cells rather than in the thymus of young (1-4 months old) AKR mice (Haran-Ghera, 1980; Haran-Ghera et al., 1987). The presence of PLCs was established using the transplantation assay, which is based on the capacity of lymphoid cells to give rise to lymphomas after being transferred into histocompatible recipients, including F, hybrids in which one parent is AKR. T h e use of hybrid mice in this analysis made possible the identification of the origin of the transformed cells, whether from donor or recipient origin. Genotype analysis, based on H-2 differences, was done serologically or by transplantation (into F, and parental strains of mice), thereby providing data on the lymphoma incidence of AKR origin. Our

250

NECHAMA HARAN-GHERA

results are in accordance with similar observations by other investigators (Legrand et al., 1975; Hays, 1982), indicating the presence of potential lymphoma cells among bone marrow cells and their absence in thymus or spleen during the first few months of life (Metcalf, 1963, 1966). These findings suggest that PLCs do not originate from the thymus, but rather the thymus provides a suitable environment for the progression and differentiation into T cell lymphomas of PLCs that have the characteristics of prothymocytes (Haran-Ghera, 1980). The results of a survey of the age-related spread of PLCs among thymus, bone marrow, and spleen cells of intact AKR mice aged 1, 6, 9, and 12 months are summarized in Table I. Lymphoid cells (107) were injected into the thymus of 2-month-old (AKR X DBA/2)F, recipients that were exposed to 4 Gy of whole-body irradiation 2-3 hours before cell transfer (cells from one donor were transferred to one recipient to prevent possible “pool contamination” by high-PLC carriers). Transplantation of bone marrow, spleen, or thymus cells from l-month-old donor mice indicated the presence of PLCs only among bone marrow cells (resulting in 81% T cell lymphoma of AKR origin); at the age of 6 months PLCs were identified among all tested lymphoid organs. With age increase (mostly among lymphoid cells from 9- to 12-month-old AKR donors) a reduction in PLCs developing into T cell lymphomas was observed. In AKR survivors that did not succumb to T cell lymphoma development at the age of 12 months, the development of B cell lymphoma (37% following bone marrow transfer, 25% in spleen cell recipients, and 57% after transfer of thymocytes) was observed (Table I). It should be pointed out that transplantation of DBA/2 or (AKR X DBA/2)F, bone marrow cells into F, irradiated recipients, used in this survey, did not enhance lymphoma development beyond the spontaneous incidence (Haran-Ghera, 1980). These survey studies indicate an age-related transition from T to B cell-oriented PLCs. In spite of the optimal conditions for PLC progression to T cell lymphoma (by intrathymic PLC transfer), the disease developed mostly in spleen and lymph nodes (though sometimes there was also thymus involvement) and was characterized as Ly- 1 pre-B or B cell lymphomas (Peled and Haran-Ghera, 1985). Establishment of PLCs in young AKR mice was shown to be thymus independent (PLCs were present in mice thymectomized when 3 days old; their bone marrow was tested for PLC presence 80 days later). However, progression of PLCs from young intact AKR donors (1-4 months old) into overt T cell lymphoma, following transplantation into F, recipients, was shown to depend on specific host conditions, including intact thymus, exposure of cell recipients to sublethal doses of wholebody irradiation (3-4 Gy) before cell transfer, and a homologous Fv-ln” +

AGE-RELATED IDENTIFICATION OF PLCS

IN

TABLE I BONEMARROW,SPLEEN, A N D THYMUS OF INTACT A N D

THYMECTOMIZED

AKR/J MICE ~~

T cell lymphoma Age (months)

Cells tested

Incidence

Null cell lymphoma

Latency (days)

Incidence

Latency

Bcell lymphoma Incidence

Latency

Intact AKR Donors (Lymphoma development of AKR origin following lymphoid cell transfer)u 220 c 196 2 120 2 85?

1 6 9 12

Bone Bone Bone Bone

1 6 9 12

Spleen Spleen Spleen Spleen

0113 8/15 (53%) 11/16 (70%) 318 (37%)

180 2 46 98 2 40 62 ? 12

0113 0115 01 16 218-25%

1

6 9 12

Thymus Thymus Thymus Thymus

0115 11/15(73%) 14/16 (87%) 217 (28%)

98 2 56 102 2 24 (45; 52)

0/15 0115 0116 117-14%

6 12

Bone marrow Bone marrow

4/14 (28%) 2/22 (8%)

197 2 39 (225; 260)

0/14 0122

6 12

Spleen Spleen

6/20 (30%) 012 1

148 2 48

2120-10% 012 1

marrow marrow marrow marrow

13/16 (81%) 10115 (66%) 14/16 (87%) 2/8 (25%)

48 31 36 12

0/16 3/15-20% 1/16-6% 218-25%

260 2 33 140 90 c 8

0/16 0115 0/16 318 (37%)

149 2 36

(88; 100)

0113 2/15 (13%) 1/16 (6%) 2/8 (25%)

(210; 350) (285) (160; 216)

(65)

0/15 0115 O / 16 417 (57%)

125 2 17

7/14 (50%) 19/22 (86%)

248 2 32 162 -t 24

9/20 (45%) 20/21 (95%)

198 2 28 140 2 21

Thx AKR Donorsb

(145; 114)

a The tested lymphoid cells (107) were injected into the thymus of 2-month-old (AKR X DBA12)Fl recipient mice. 3 hours after their exposure to 4Gy whole body irradiation;cell transfer was done from one donor to one recipient. The lymphomas genotype was tested by transplantation and only tumors of AKR origin were included in the table. h Thymectomy was done at the age of 55 days.

TABLE I1 HOST EFFECTSON BONEMARROW-DERIVED PLC PROLIFERATION (AKRxDBA/2)FI + 4Gy Oricrin of tested cells

Number

%

Latency (days)

(AKR x DBAI2) Number

5%

Tx(AKRXDBAIB)F,

Latency (days)

Number

%

+ 4Gy

Latency (days)

(AKR

X

Number

B1/6)F,

+ 4Gy

%

Latency (days)

Lymphoma development of AKR origin in cell recipients0

T cell lymphoma (lo6) B cell lymphoma (lo7) Intact AKR ( 1 month old) Intact AKR (3-month-old) Intact AKR (8-month-old)

5/5 5/5 12/16 8/10b 12/14

100 100 75 80 85

20 55 220

616 515 0112

100 100

190 110

0110

-

10114

71

145

517

T h x AKR (6-month-old)

7/14<

50

248

317

43

285

T h x AKR (12-month-old)

12/15c

80

160

14/16

87

154

24 40

515 515

26 45 380

13/15

275

4/23

50

275

145

718

87

168

515 515 1/10

71

120

317

43

9/10

90

0110 0110

-

100 100 10 86

20 35

100 100 -

0110

120

fl Two-month-old recipient mice were used; (AKR x DBA/2)F, have the Fv-l*’~‘ allele; (AKR X BI/6)Fl have the nlb allele. Cells (2 X 107) were injected intravenously; cell transfer to irradiated recipients was done 24 hours following exposure 10 4Cy. Thymectomy was done at the age of 55 days. b All characterized as T cell lymphomas. Characteri~edas B cell lymphomas.

LYMPHOMAGENESIS IN AKR MICE

253

allele when using F, hybrids as cell recipients (Haran-Ghera et al., 1987). T h e provided host factors seem to contribute to the transition of PLCs to autonomous lymphoma cells expressed in the thymus. The age-related dependence of bone niarrow-derived PLCs on specific host conditions, in comparison to the autonomous growth capacity of fully transformed T or B cell lymphomas, was demonstrated by the following transplantation studies (results summarized in Table 11). Bone marrow cells from intact AKR mice aged 1,3, or 8 months were transferred (i.v.) into (AKR X DBA/2)F, recipients with or without previous exposure to 4 Gy as well as into similar thymectomized F, irradiated recipients. T h e possible dependence of PLCs on the Fv-1 allele of the recipient [thereby indicating viral involvement in PLC proliferation, because the Fv-1 allele affects replication and spread of the endogenous N-tropic virus (Pincus et al., 1971)] was tested by bone marrow transfer into (AKR X BL/6)F, (Fv-l’lb) irradiated recipients. PLCs among bone marrow cells of 1- and 3-monthold intact AKR mice required for their proliferation the presence of a thymus in the cell recipients and their exposure to radiation. T h e requirement of a thymus for PLC proliferation coincides with our observations suggesting that PLCs among bone marrow cells have the characteristics of prothymocytes and need the thymus microenvironment for their progression into T cell lymphoma (Haran-Ghera et al., 1978; Haran-Ghera, 1980). T h e contribution of host irradiation to PLC proliferation may be related to radiation-triggered cytokine production (Tartakovsky et al., 1993). T h e required Fv-1”” combination of the hybrid F, recipients suggest that viruses have an important role in the transition and progression of PLCs to overt lymphoma development, similar to previous observations showing the effect of the Fv-1 allele on sensitivity of mice to lymphoma induction (Cloyd et al., 1980; Cloyd, 1983). However, bone marrow cells from 8-month-old intact female mice yielded a similar lymphoma incidence and latent period irrespective of the recipients’ pretreatments (Table 11). These results indicate the presence of autonomously growing lymphoma cells in female intact grossly normal 8-month-old AKR mice, versus “dependent” PLCs in younger mice (1-6 moths old).

I l l . Enhanced T Cell Lymphoma Development Pathways

We have suggested that in intact AKR/J mice the development of spontaneous T cell lymphomas is a stepwise process. Our working hypothesis has been that the dual tropic recombinant virus, produced in the thymus of 4- to 5-month-old mice, may act as a promoter on preex-

254

NECHAMA HARAN-GHERA

isting potential lymphoma cells (present mostly among bone marrow cells), enhancing their ability to progress to fully transformed cells following their seeding in the thymus (Haran-Ghera, 1985; Haran Ghera et al., 1987). T h e validity of this hypothesis was elucidated by studying the requisite of PLCs and/or DTVs for enhanced T cell lymphomagenesis (induced by viruses, chemical carcinogen, or radiation). Thus, intact AKR mice as well as AKR mice lacking PLCs since birth (due to suppressive immunotherapy) and AKR-Fu-I b mice having virus replication restrictions were similarly treated with different lymphomagenic agents (including Class I MCF virus, important in AKR T cell lymphomagenesis). T h e susceptibility to T cell lymphoma development in these different tested groups indicated that distinct developmental pathways are involved. Acceleration of T cell lymphomagenesis in AKR mice (treatments yielding 100% lymphomas at the age of 3-4 months) has been demonstrated using different experimental methods: infection with Class I MCF recombinant virus isolates (Cloyd et al., 1980), ecotropic retrovirus (Kato and Hays, 1985), exposure to fractionated irradiation (Legrand and Duplan, 1971), or administration of the chemical carcinogen methylnitrosourea (MNU) (Frei, 1980). Do all these treatments indeed accelerate spontaneous AKR lymphomagenesis (affecting promotion of PLCs) or d o some of them induce de novo T cell lymphomas (acting directly on thymocytes)? The analysis of mechanisms underlying accelerated lymphoma development in AKR mice (following different “enhancing” treatments) will be described in this section. A. ACCELERATION BY RECOMBINANT DUALTROPIC VIRUSES Lymphoma development in AKR mice is usually delayed until the age of 6-12 months. T h e enigma of this long latent period has been attributed to the delayed formation of the recombinant dual tropic MuLV established in the thymus of 5- to 6-month-old mice. The recombinant dual tropic (MCF) viruses are produced spontaneously in high endogenous ecotropic MuLV mouse strains, and concurrent replication of ecotropic MuLV greatly enhances MCF MuLV replication and lymphomagenicity. Several studies have indicated that the lymphomagenic activity of Class I AKR MCFs depends on the expression of endogenous Akv MuLVs [shown in NFS and NIH Swiss mice lacking the endogenous ecotropic virus (Fredrickson et al., 1984)l. One exception of a highly lymphomagenic helper-independent MCF virus, isolated from a thymic T cell lymphoma associated with infection with Cas-Br-M wild mouse ecotropic MuLV, that does not require ecotropic helper MuLV to pro-

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duce short-latency T cell lymphoma was described (Chattopadhyay et al., 1989). Class I MCF viruses injected intrathymically in newborn o r young adult AKR mice were shown (1) to be thymotropic, replicating selectively in the major thymus population of immature cells; (2) to be responsible for amplified expression of MuLV gp70 on thymocytes, (3) to trigger increased expression of H-2 alloantigens and decreased Thy- 1 expression, and (4) to amplify expression of xenotropic MuLV antigens on thymocytes (Kawashima el al., 1976; O’Donnell et al., 198 1).The lymphomagenic potential of these viruses has been primarily defined by their ability to accelerate lymphoma development when inoculated in newborn or young adult AKR mice, in contrast to ecotropic and xenotropic viruses that did not exert an acceleration effect. O’Donnell et al. (1984) studied sequential cellular changes in the thymus of AKR mice during Class I MCF-accelerated lymphoma development and defined three stages in lymphoma development: Stage I (lasting 28-40 days postvirus injection), involving steady-state infection of immature thymocytes by the virus; Stage I1 (50-60 days), representing emergence of a clonal population of cells with restricted transplantation properties (can be abrogated by sublethal irradiation); and Stage 111 (from 70 days onward), concerned with outgrowth of fully transformed cells. We studied the effect of the recombinant dual tropic virus isolate DTV-70, which accelerates lymphoma development (100% T cell lymphoma observed at the age of 90-120 days following virus injection into the thymus of 14-day-old AKR mice), on the distribution and proliferation of PLCs among bone marrow, thymus, and spleen cells of virusinfected mice and matching controls (Haran-Ghera et al., 1987). In 30and 75-day-old control AKR mice, PLCs were predominantly identified (by transplantation studies) among bone marrow cells (yielding 80% lymphoma of AKR origin); they occurred less in spleen (30-50% lymphomas of AKR origin) and were practically nonexistent in the thymus (7- 11% incidence). Similar transplantation studies done 15 days after intrathymic DTV inoculation showed no substantial changes in PLC distribution or in length of the latent period beyond the control level. In contrast, assays done 30 days after DTV infection indicated shortened latency for lymphoma development by bone marrow-derived PLCs (although PLC proliferation was still dependent on specific host conditions) and occurrence of a comparative lower level of PLCs among thymocytes (causing 40% lymphoma development of AKR origin versus 80% by PLCs from bone marrow). At 60 days after DTV infection the distribution of autonomous lymphoma cells was observed in all three tested organs (independent of specific host conditions for their growth),

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thus markedly differing from PLCs in uninfected control mice (75 days old) in relation to organ distribution and specific requirements for their growth and proliferation. These studies confirm that in DTV-infected mice, PLCs are also initially detected among bone marrow cells rather than in the thymus of young mice. The occurrence of PLCs in the thymus 30 days post-DTV infection might be due to DTV-accelerated migration of PLCs from the bone marrow to the thymus. The thymus was found to be essential for PLC proliferation for at least 30 days after DTV infection. The PLC “dependence” on specific host conditions for the transition to autonomous lymphoma-developing cells was observed for both spontaneous (from 75 day-old mice) and DTV-accelerated PLCs shortly (within 30 days) after virus infection (Haran-Ghera et al., 1987). Thus, DTV-70 inoculation into 14-day-old mice did not change the spontaneous PLC distribution pattern in the tested host organs within 30 days postinfection, nor did it change PLC specific host requirements for further progression into lymphomas; however, it enhanced PLC transition to autonomous lymphoma cells at about 60 days after infection. We conclude that DTV may act as a promoter on preexisting PLCs present mostly among bone marrow cells of young AKR mice. This suggestion was further confirmed using another experimental model in which the accelerating effect of MCF-247 was tested in AKR mice treated since birth (for 10 days) by passive immunotherapy, namely, injected with a monoclonal antibody (18-5) to specific gp71 determinants; this treatment was shown to eradicate PLC occurrence and ultimately prevented T cell lymphoma development (Haran-Ghera and Peled, 1991). Intrathymic inoculation of MCF-247 to mice 40 days after terminating the treatment with mAb 18-5 (the mice were 50 days old) resulted in 27% T cell lymphoma at the “regular” nonaccelerated mean latency of 256 days, in contrast to 100% T cell lymphoma (with a mean latency of 130 days) when 50-day-old AKR mice were injected intrathymically with MCF-247. These results clearly indicate that PLC is the required target cell for the induction of DTV acceleration. €3. INDUCTIONBY

THE

ECOTROPIC RETROVIRUS SL3-3

The SL3-3 highly lymphomagenic ecotropic retrovirus, isolated from a cell line derived from a spontaneous AKR lymphoma (Hays el al., 1982), induces 100% T cell lymphoma when injected into newborn AKR mice; there is a latency period of 60-90 days. T h e nucleotide sequences of the structural genes of SL3-3 virus are essentially similar to those of the nononcogenic endogenous Akv virus of A K R mice (Pederson et al., 1981). T h e differences lie in the nonencoding long terminal repeat

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(LTR) region; the LTR of SL3-3 has been shown to be responsible for the viral properties of tropism for thymocytes and oncogenicity (Lenz et al., 1984; Rosen et al., 1985). The initial transformation occurs in the thymus irrespective to the presence or absence of PLC. Eradication of PLC in AKR/Cu mice by passive immunotherdpy, namely by neonatal injection of mAb 18-5 and further inoculation of SL3-3 into the thymus of these mice when 30 days old, yielded a high T cell lymphoma incidence (E. Hays, personal communication). Bone marrow-transformed cells were observed only after thymic disease was overt. Virus infection of the thymus per se is the step that precedes lymphomagenic transformation of cortical thymocytes in this model. Virus must reach certain levels in the thymus before transformation can occur. The lymphomagenic pathway of SL3-3 involves virus infection of thymic stroma, which allows infection of thymocyte progenitors. The maturation of these cells in the thymus is delayed and these thymocytes have prolonged intrathymus survival; these thymus-dependent immature self-renewing cells become targets for virus integration and reintegration, permitting genetic changes necessary for malignant transformation (Hays et al., 1990). Two stages of lymphomagenesis were observed: thymus-"dependent" PLCs, which produce donor-type lymphomas when inoculated into the thymus of cell recipients, but not when injected subcutaneously; and thymus-independent transformed cells, which yield donor-type lymphomas 3-4 weeks after inoculation to cell recipients via any route. Although this virus was used to study the mechanism underlying AKR spontaneous lymphomagenesis (Hays el al., 1990), it seems to represent a direct virus-thymocyte transformation interaction, differing from the spontaneous T cell lymphoma model that consists of Thy-lnegative PLCs present in the bone marrow of very young AKR mice (Haran-Ghera, 1980); these PLCs have to migrate to the thymus, which contains DTV, for their promotion to overt T cell lymphoma (HardnGhera et al., 1987). C. INDUCTION BY THE RADIATION LEUKEMIA VIRUS VARIANTA-RADLV

The radiation leukemia virus variant A-RadLV, originally isolated from an irradiated BL/6 mouse (Haran-Ghera et al., 1977), induces a high T cell lymphoma incidence (70-100% at 90-120 days latency) when injected into the thymus of young adult BL/6 mice. PLCs were initially observed among thymocytes (cortisone-resistant Thy+Cd4+ and/or CD8+ cells) of injected mice (Gokhman et al., 1990). Because A-RadLV viruses exhibit both N and B tropism (Yefenof et al., 1984), we

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tested their lymphomagenic activity in three different groups of AKR mice; AKR/J mice highly susceptible to T cell lymphomagenesis; AKR/J mice treated since birth with mAb 18-5, thereby eliminating PLCs among bone marrow cells (Haran-Ghera and Peled, 1991); and AKR-FV-lbmice resistant to spontaneous T cell lymphoma development (Haran-Ghera et al., 1993). A-RadLV was injected into the thymus of 30day-old mice; in all three experimental groups tested, we observed a high T cell lymphoma incidence (90-100% at a mean latency of 75-80 days) (Haran-Ghera et al., 1993). Because A-RadLV contained both N and B tropic MuLVs, permissive infection with one of these components was presumably responsible for the accelerated disease observed in these treated AKR mice. We assume that A-RadLV interacts and transforms thymocytes, rather than promoting the spontaneous endogenous PLCs present in AKR mice, because it acted similarly in AKR/J mice carrying or lacking PLCs among their bone marrow cells. D. LYMPHOMAGENIC EFFECTOF FRACTIONATED IRRADIATION Extensive studies by Legrand and Duplan showed that young AKR mice exposed to fractionated irradiation (1.75 Gy once a week for 4 consecutive weeks) were highly sensitive to T cell lymphoma development, and injection of syngeneic bone marrow shortly after the last radiation exposure [a procedure shown to prevent radiation-induced lymphomas in BL/6 mice (Kaplan et al., 1953)] did not reduce lymphoma incidence in AKR mice (Legrand and Duplan, 1971; Legrand et aL., 1981). Thymectomized AKR mice similarly treated developed only null and B cell lymphomas (in 50% of irradiated mice beyond the age of 450 days versus 5% in thymectomized nonirradiated control mice) (Legrand et al., 1981). To gain an insight into the role of endogenous MuLV and of spontaneously occurring PLCs in young AKR mice and their susceptibility to radiation lymphomagenesis, 30-day-old AKR/J mice, AKR/J mice lacking spontaneously occurring PLCs (following treatment since birth for 10 consecutive days with mAb 18-5), and AKR-Fv-Zb mice (having N-tropic viral replication restriction and a variable level of PLCs) were exposed to 1.7 Gy whole-body irradiation once a week for four consecutive weeks. AKR/J and AKR-FV-lbmice were found to be highly sensitive to radiation-induced T cell lymphomas (92- 100% incidence at a mean latency of 120 days) although AKR-FV-lb mice developed a low (7%) incidence of spontaneous T cell lymphomas (Haran-Ghera et al., 1993). In contrast, AKR/J mice treated with mAb 18-5 (thereby eradicating

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259

PLCs) were found to be resistant to radiation lymphomagenesis: 9% developed T cell lymphoma (197 days latency), 9% developed null tumors (177 days), and 2 1% developed B cell lymphomas (220 days mean latency; N. Haran-Ghera and A. Peled, unpublished observation).The spontaneous lymphoma incidence in nonirradiated mAb 18-5-treated mice was similar: 10% T cell lymphoma (350 days latency) and 10% B cell lymphoma (480 days latency). Thus, radiation did not increase lymphoma incidence beyond the spontaneous level, but perhaps shortened the latent period. Studies concerned with the virological status of T lymphomas arising in AKR-Fv-I6 mice indicated that most spontaneously developing T lymphomas in untreated or thymectomized mice showed Class I MCFs, as did all those infected with MCF-247 or A-RadLV (Haran-Ghera et al., 1993). Thus, as for normal AKR/J mice, in AKR-Fv-Zb mice Class I MCFs appear to play a role in T cell lymphomagenesis. The low frequency and long latency of T lymphomas in these mice might be explained at least in part by the inefficiency of N-tropic virus spread (Akv and AKR MCFs) in Fv-lb mice. I n contrast to the other T lymphomas, the high incidence of T lymphomas induced by fractionated irradiation in AKR-Fv-16 mice was not associated with Class I MCF viruses (HaranGhera et al., 1993). Thus, these T lymphomas appear to arise without the involvement of Class I MCF viruses. These results may indicate that in the AKR genetic background, potentially leukemic cells of the T-lymphoid lineage also exist, but their expression is not dependent on the presence of Class I MCF viruses; in addition, eradication of PLCs before exposure to radiation does indeed prevent susceptibility to radiation lymphomagenesis.

E. N-METHYL-N-NITROSOUREA-INDUCED T CELLLYMPHOMAS AKR mice are highly sensitive to accelerated T cell lymphoma development by the chemical carcinogen MNU; a single injection of 50-75 mg/kg in young AKR mice results in 80- 100% lymphomas that arise 812 weeks later, prior to the onset of spontaneous T cell lymphomas (Frei, 1980). It was suggested that the early appearance of lymphomas in AKR mice is the result of their endogenous viruses (Frei, 1980). Studies on the etiological role of viruses in MNU lymphomagenesis indicated that Class I MCF proviruses were not integrated into tumor DNA and therefore were not likely to play a significant role in MNU-induced lymphomas in AKR mice (Richie et al., 1985, 1988). Further studies on the role of oncogene activation and the involvement of MuLV in the development of MNU-induced lymphomas in AKR mice showed that a high propor-

260

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tion of MNU-induced tumors contained activated rus, not detected in spontaneously developing T cell lymphomas in AKR mice (Warren et al., 1987). T h e alterations of the c-my and Pim-1 genes found in a small proportion of spontaneous lymphomas were not changed following MNU treatment. There seems to be a cooperation between the chemical carcinogen MNU and endogenous MuLV. The majority of MNUinduced lymphomas contain newly acquired murine lymphoma proviral sequences. In contrast to spontaneous lymphomas, which contain multiple integrations of MCF recombinant proviruses, MNU-induced lymphomas contain only ecotropic-related proviruses that have genomic structures distinct from Class I or Class I1 viruses (Richie et ul., 1988). The new ecotropic-related proviral integrations may contribute to the shortened latency observed in T cell lymphoma in MNU-treated AKR mice. Further attempts were made to determine the potential role of AKR ecotropic MuLV loci in MNU lymphomagenesis, by testing MNUinduced incidence and latency in AKR/J mice in comparison to resistant NFS/N and congenic NFS/N mice containing either the Akv-l or Akv-2 endogenous ecotropic locus (Becker, 1990). T h e NFS-Akv-1 mice had a significantly higher lymphoma incidence than did NFS/N or NFS-Akz)-2 strains. It was proposed that the Akv-1 locus confers enhanced susceptibility to MNU-induced lymphomas by increasing the number of target cells susceptible to MNU activity. To further determine the role of provirus integrations of endogenous MuLV in enhanced susceptibility of AKR mice to MNU, AKR-Fv-16 and AKR/J mice were treated similarly with MNU; both incidence and latency were similar in both treated groups. Thus, the Fv-1 viral restriction did not alter tumor incidence in AKR-Fw16 mice (Richie et d.,1991). Southern blot analysis confirmed that lymphoma DNA from AKR-Fv-lh mice did not contain somatically acquired provirus integration, thereby confirming that virus integration is not an essential element in MNU-induced lymphomagenesis. MNU treatment was found to stimulate high-level expression of newly acquired ecotropic-like proviruses in both AKR-Fv-1" and AKR/J mice treated with MNU, suggesting that somatic integrations of novel recombinant ecotropic-like MuLV contribute to the high incidence of lymphoma development in a relatively short latent period. We tested the susceptibility of AKR/J mice lacking PLCs (following mAb 18-5 treatment) to MNU in comparison to AKR/J mice (single i.p. injection of 75 mg/kg into 35-day-old mice) and observed a 100% T cell lymphoma incidence in both treated groups at a similar mean latency of 100 days (N. HaranGhera and A. Peled, unpublished results). Thus, the target population o f MNU-induced lymphomas does not seem to reside among the bone marrow PLC compartment. In accordance with our results, it was pre-

LYMPHOMAGENESIS IN AKR MICE

26 1

viously shown that thymectomy prevented the appearance of MNUinduced T cell lymphomas, and implants of neonatal syngeneic thymus grafts into thymectomized recipients failed to restore thymic lymphoma development unless the grafted thymus was exposed to MNU (Baines et al., 1979). It has been suggested that MNU acts on thymocytes at the CD4- CD8+ immature stage of differentiation (Richie et al., 1991). F. CONCLUSIONS

Enhanced T cell lymphoma development in AKR mice occurs in 80100% of treated mice prior to the onset of spontaneous lymphomas, following treatment with different lymphomagenic agents. Introduction of interstrain variabilities, changing the PLC level, or restricting viral replication before the onset of the different lymphomagenic treatments suggest that distinct mechanisms in lymphoma development are involved. Similar high incidences of lymphomas following inoculation of SL3-3, A-RadLV, o r MNU to AKR mice, in spite of viral or PLC presence or absence in the treated mice, suggest a direct action of the lymphomagenic agents on thymocytes. In contrast, the accelerated effect of MCF-247 seems to depend on the presence of PLCs among bone marrow cells, because AKR/J mice treated with 18-5 mAb (thereby eradicating the PLCs) did not respond to the acceleration effect of MCF-247 infection. T h e lack of MCF-247 acceleration effect in AKR-Fv-I" mice is obvious, due to the Fv-lb-induced restriction to N-tropic virus replication and spread. T h e susceptibility to radiation-induced T cell lymphoma seems also to be PLC dependent because AKR/J mice depleted of PLCs (following treatment with 18-5 mAb) prior to exposure to X-rays were shown to be resistant to lymphomagenesis by fractionated irradiation. The sensitivity of AKR-Fu-16 mice to radiation lymphomagenesis might be related to the radiation-triggered high-level expression of ecotropic proviruses. The variable level of PLCs observed among bone marrow cells of AKR-Fv-Ib mice (20-50%) might be increased following radiation-induced elevated levels of ecotropic virus. Treatment of AKR/J mice with mAb 18-5 (with specificity for ecotropic and MCF virus envelope glycoproteins) suppresses the early development of MuLV infectious centers, which express high levels of gp7 1 (these ICCs represent about 1% of bone marrow cells). Following treatment with mAb 18-5, the thymus remains devoid of detectable ICCs throughout life, and PLCs, being susceptible to antiviral antibody therapy, are eliminated. These observations may suggest a role of ecotropic proviruses in the initiation of PLC in AKR mice.

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NECHAMA HARAN-CHERA

IV. The Level of Dormant PLCs Following Prevention of Spontaneous T Cell Lymphoma Development Following the prevention of overt spontaneous T cell lymphoma development, a moderate increase of Ly-l+ B cell lymphomas was observed. T h e question is whether these preventive treatments (administered to young AKR mice) eliminate PLC occurrence, or whether the high level of PLCs is maintained in a dormant state, because only about 30% of these mice will develop overt T or B cell lymphomas. Is the pathway of PLC progression toward B cell rather than T cell lymphoma development enhanced with the increased age of the PLC carriers? PLCs may represent precursor pre-T and/or pre-B potential lymphoma cells. Overt T cell lymphoma will develop provided that PLCs reach the thymus by the time that the recombinant virus has already emerged in this organ. Lack of the appropriate thymus microenvironment for T cell differentiation and progression to T cell lymphoma results in progression to B cell lymphomas in older mice (beyond the age of 1 year). T h e level of PLCs, their dormant state, and their developmental pathways to Ly- 1 B cell lymphoma, following different experimental manipulations, will be addressed in the following discussion. +

A. PLCs

IN

THYMECTOMIZED AKR MICE

Beyond the age of 1 year, AKR mice thymectomized at the age of 55 days developed 6% T cell lymphomas and about 20-30% B cell lymphomas (mean latency of 600 days) (Rosner et al., 1993). Analysis of the lymphomagenic potential of bone marrow and spleen cells of 6- and 12month-old thymectomized AKR mice demonstrated the presence of PLCs in most thymectomized mice (see Table I). PLCs in 6-month-old thymectomized mice were present at a similar level among bone marrow and spleen cells. About 28-30% of the lymphomas of AKR origin developing in the F, irradiated recipients were characterized as T cell lymphomas and 4 5 5 0 % as Ly-l+ pre-B o r B cell lymphomas. A few null lymphomas (from spleen PLCs) were also observed. With age, a reduction in T cell lymphoma incidence and an increase in B cell lymphomagenesis, with shortened latency, were observed ( 0 4 % T cell lymphoma and 86-95% incidence of Ly-l+ B cell lymphomas, following transfer of bone marrow or spleen cells from 12-month-old thymectomized AKR donors). T h e route of cell transfer did not affect tumor characteristics: bone marrow o r spleen cells (107) from individual 12month-old thymectomized AKR mice were injected into F, irradiated recipients via three different routes-intravenous, intrathymic, or intra-

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LYMPHOMAGENESIS I N AKR MICE

splenic. T h e incidence (80-90%) and latency of Ly- 1 B cell lymphoma development were similar in all the tested groups (including the intrathymic group). I n contrast to “host-dependent” progression of PLCs from young intact AKR mice to T cell lymphoma development, PLCs from thymectomized mice progressed into B cell lymphomas following transplantation into histocompatible recipients, irrespective of the presence or absence of thymus, of the recipients’ exposure to radiation, o r of the Fv-1 homologous allele of the PLC recipients (see Table 11). Thus, different host conditions are prerequisites for the progression of PLCs to T or B cell lymphomas. T h e differences in lymphoma characteristics, regardless of whether PLCs originate from intact or thymectomized AKR mice, is remarkable. A high incidence of T cell lymphomas developed from PLCs taken from 1- to 6-month-old intact AKR mice through reduced T cell lymphomas and increased B cell lymphomas reaching a peak of 70-95% Ly- 1 B cell lymphomas following transfer of lymphoid tissues from 12-month-old thymectomized donors (Table I). Regulatory signals provided by the intact thymus of AKR mice may influence the direction of differentiation of the PLCs (probably representing pluripotent stem cells serving as precursors) toward T or B cell neoplasms of AKR origin. T h e agerelated effects on the characteristics of lymphomas resulting from lymphoid cell transfer were stressed when thymus, bone marrow, and spleen cells from 12-month-old intact AKR mice were transplanted into young recipients. In these transfer experiments, null, T, and B cell lymphomas were observed. This age effect on the pathway of PLC progression suggests that age-related changes (including age-dependent thymus atrophy) in hemapoietic regulators affect the orientation of PLCs toward the B or T lymphoid cell pathway. These observations coincide with similar findings on presence of null and B cells among thymocytes of SJL/J mice from 7-9 months onward (Ben-Yaakov and Haran-Ghera, 1975). These SJL/J mice are subject to a neoplastic disorder of the lymphoreticular system and their growth was shown to depend on specific T cell functions (Bonavida, 1983). The obvious unanswered problem is whether PLCs in intact and thymectomized AKR mice are the same dual potential lymphoid precursor stem cells or alternatively if two different lymphoid progenitors restricted to T or B cell pathways are involved in T and Ly-l+ B cell lymphoma development in AKR mice. In intact AKR mice the progression of PLCs to overt T cell lymphoma is dependent on the delayed formation of the dual tropic virus in the thymus of 5- to 6-month old mice. DTVs induce thymus injury, accelerate changes in thymus subpopulations, and amplify the expression of xenotropic MuLV antigens on thymoctyes (Kawashima et al., 1976). These DTV +

+

264

NECHAMA HARAN-GHERA

effects could trigger PLC migration into the “injured” thymus and promote PLC progression to autonomous T cell lymphomas (Haran-Ghera et al., 1987; Peled et al., 1987). In thymectomized A K R mice [carriers of dormant PLCs (Peled and Hardn-Ghera, 1985)], DTV formation does not seem to occur, and, indeed, Class I MCF-type recombinant viruses are not involved in the generation of Ly-l+ B cell lymphomas (HaranGhera et al., 1992). Thus, a state of PLC growth arrest seems to be maintained throughout life in about 70-80% of thymectomized A K R mice (a follow-up of thymectomized A K R mice in several experiments indicated an ultimate B cell lymphoma incidence in 10-30% far beyond the age of 1 year), without the age-related interference of DTV that promotes lymphoma development in intact A K R mice. It is plausible to assume that prolonged thymectomy in A K R mice causes changes in regulatory mediators, including deficiency of T cell factors necessary for B cell growth turnover (Mosmann and Coffman, 1989; Swain et al., 1988). Lack of such factors may play a role in the transition of pre-T to pre-B PLCs. B. PLC LEVELFOLLOWING T CELLLYMPHOMA PREVENTION BY VIRALINTERFERENCE Spontaneous T cell lymphoma development can be prevented in A K R mice without surgical removal of the thymus. Injection of a nonlymphomagenic ecotropic virus (designated 24-666), isolated from a B cell lymphoma of A K R origin, into young A K R mice (1 to 60 days old) inhibited spontaneous T cell lymphoma development. A reduction in T cell lymphoma incidence (to 15%) was accompanied by the appearance of B cell lymphomas (34%) in older mice (500 days mean latency) (Peled and Haran-Ghera, 1991). Infection of newborn to 60-day-old A K R mice with 24-666 prevented changes in thymus subpopulations and expression of MuLV-related cell surface antigens, normally observed in the thymus of 5- to 6-month-old A K R mice, prior to lymphoma development (Kawashima et al., 1976). Thymuses of 24-666-infected 3- to 12month-old mice lacked recombinant dual tropic virus expression and retained the thymus subpopulation pattern of 2-month-old A K R mice. At 12 months after 24-666 administration, a striking decrease in Thy-1.1 level and in CD4+ CD8+ populations and an increase in CD4- CD8cells and in p+ B cells, predominantly Ly-1+, were observed in the thymus. The presence of B cells in these thymuses was also reflected in the high response of thymocytes to lipopolysaccharide blastogenesis accompanied by a decreased response to phytohemagglutinin (Peled and Haran-Ghera, 1991). These observations imply that prevention of dual

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265

tropic virus generation and replication in the thymus is related to the observed prevention of spontaneous T cell lymphoma development in these 24-666-infected mice. We showed that PLC establishment is thymus independent (not dependent on DTV formation in the thymus) and that the initial site of PLC occurrence is among bone marrow cells, thus it seemed obvious that mice infected with 24-666, though free of T cell lymphomas, should be PLC carriers. T h e age-related levels of PLCs among bone marrow, spleen, and thymus cells of 3-, 6-, or 12-month-old AKR mice injected with 24-666 at the age of 14 days (intrathymic virus inoculation) and their potential of PLCs to develop into T or B cell lymphomas were tested using the transplantation method (results are summarized in Table 111). T h e possible dependence of PLC proliferation on host pretreatment (exposure to radiation) was evaluated by injecting lymphoid cells of one donor, divided into two equal doses of (1-2) x 107 cells, into irradiated and nonirradiated F, recipients. Bone marrow cells were injected intravenously whereas spleen and thymus cells were injected intrathymically. T h e results obtained indicated that the inhibitory effect of 24-666 infection on spontaneous T cell lymphoma development did not prevent PLC occurrence; most tested mice were PLC carriers (see Table 111). The route of cell transfer and pretreatment of the cell recipients did not change markedly lymphoma incidence and latency. Transplantation of lymphoid cells from 3-month-old infected mice yielded a similar low level of T and B cell lymphomas (about 20% of each type at a prolonged latent period above 1 year, independent of host conditions), in contrast to 80% T cell lymphoma of AKR origin developing in irradiated F, recipients injected with bone marrow cells of 3-month-old intact AKR mice (Table 11). Transplantation of bone marrow, spleen, or thymus from 6- and 12-month-old virus-infected donors resulted in a high incidence (60-80%) of Ly- 1+ B cell lymphomas and a low incidence (720%) of T cell lymphomas (Table 111).Thus, the ratio of T:B lymphoma development following PLC transfer from normal or 24-666-infected donors was reverted. T h e prevalent B-PLC pathway in 24-666-infected mice, following T cell lymphoma prevention, coincides with our observations on the presence of B-PLCs in lymphoid organs of 8- to 12-monthold AKR mice that were thymectomized at the age of 6-8 weeks. It seems that prevention of DTV formation by surgical removal of the thymus of young mice, o r by inoculation of another nonlymphomagenic virus into young mice, results in diverting the pathway of PLC progression toward B cell rather than T cell lymphoma development (with age increase of the PLC carriers).

TABLE 111 AGE-RELATED PLC LEVELS IN 24-666

~NFECTED MICE

Age of cell donors (months)a

Cells tested

Route of cell transfer

3 3 3

Bone marrow Spleen Thymus

Intravenous Intrathymic Intrathymic

4/10 (40%) (396 2 23)c 2/10 (20%) (240; 268) 2/10 (20%) (274; 300)

2/10 (20%) (355; 452) 2/10 (20%) (410; 417) 2/10 (20%) (314; 450)

2/10 (20%) (133; 440) 2/10 (20%) (350; 390) 2/10 (20%) (300; 340)

3/10 (30%) (452 2 50) 2/10 (20%) (345; 450) 2/10 (20%) (395; 410)

6 6 6

Bone marrow Spleen Thymus

Intravenous Intrathymic Intrathymic

2/9 (22%) (156; 305) 118 (12%) (368) 2/10 (20%) (149; 300)

719 (77%) (340 2 36) 618 (75%)(325 2 48) 8/10 (80%) (329 2 76)

2/10 (20%) (310; 360) 1/10 (20%) (202) 411 1 (36%) (266 34)

5/10 (50%) (330 2 37) 9/10 (90%) (376 2 62) 6/11 (55%) (380 48)

12 12 12

Bone marrow Spleen Thymus

Intravenous Intrathymic Intrathymic

01 12

9/12 (75%) (109 2 20) 10113 (77%) (92 2 14) 9/14 (64%) (99 2 23)

1/14 (7%) (78) 1/12 (8%)(124) 0/10

11/14 (78%) (80 20) 9/12 (75%) (123 2 24) 7/10 (70%) (100 2 12)

(AKR/J x DBA/2)Fl T cell lymphoma

+ 4Gy

B cell lymphoma

(AKR

X

T cell lymphoma

DBA12)Fl B cell lymphoma

T and B cell lymphoma development of AKR origin in cell recipients”

1/13 (7%) (134) 2/14 (14%) (217; 248)

*

*

The virus isolate 24-666 was injected into the thymus of 14-day-old AKR mice that served as cell donors. Two-month-old (AKR/J X DBA/2)Fl cell recipients were used; lo7 lymphoid cells were injected intravenously or intrathymically into irradiated recipients; The cells were transferred 3 hours after exposure to 4Gy; Cell transfer‘was done from one donor to one recipient; The lymphomas genotype was tested serologically (H-2 and Thy-I differences) and only tumors of AKR origin (H-2k; Thy-I. 1) were included in the Table. c Mean latency days. b

LYMPHOMAGENESIS IN AKR MICE

C. PLCs

IN

267

AKR-FV-lb CONGENIC MICE

In the AKR-Fv-lb congenic strain, the Fv-1" allele of the AKR/J mice was substituted with.the F v - l b allele of BL/6 mice (Stockert et al., 1979). T h e F v - l b gene product causes limited viral replication and spread of the endogenous N-tropic MuLV in thymocytes, splenocytes, and bone marrow of AKR-Fv.lb mice (Rowe and Hartley, 1972), which is also reflected in the reduced infectious virus-producing cells in all hemopoietic organs of AKR-Fv-16 mice (Buckheit et al., 1988). As a result of this genetic viral restriction, AKR-Fwlb mice develop a very low (7%) incidence of spontaneous T cell lymphomas at a mean latency of 470 days, and 28% Ly-l+ B cell lymphomas at a mean latency of 680 days. T h e low frequency and long latency of T cell lymphomas in AKR-Fv-lb mice might be explained at least in part by the inefficiency of N-tropic virus spread (Akv and MCF) in these mice (Haran-Ghera et al., 1993). T h e characteristic changes in thymus subpopulations of AKR/J mice preceding overt T cell lymphoma development, associated with the formation of DTV in the thymus of 5- to 6-month-old mice, were not observed in thymuses of 2- to 18-month-old AKR-Fv-lb mice. Thymuses of l-year-old mice maintained the population pattern of 2-month-old AKR/J mice; in 24-month-old thymuses a drop in the double-positive population and an increase in the CD4+ CD8- population were observed (Haran-Ghera et al., 1993). T h e search for PLCs among bone marrow and spleen cells of old AKR-Fv-lb mice within the age range of 16-24 months revealed a variable level of PLCs. In most tested groups, transfer of bone marrow or spleen cells caused a 20-40% B cell lymphoma development of AKR-Fv-lb origin, though in one group 50-70% incidence was observed (Haran-Ghera et al., 1993). This wide variable range of B-PLCs demonstrated in AKR-Fv-Ib mice (in contrast to the regular high B-PLC incidence of 80-100% in 10- to 12-month-old thymectomized AKR/J mice) might be related to lower levels of, or lack of, infectious virus-producing cells in the bone marrow of AKR-Fv-lb mice (Buckheit et al., 1988). D. ELIMINATION OF PLCs I N AKR/J MICE FOLLOWING PASSIVEIMMUNOTHERAPY

AKR/J mice were protected from developing spontaneous T cell lymphomas by a course of daily treatment with antibody 18-5 to gp7 1 determinants, administered from birth to 10 days (Haran-Ghera and Peled, 1991). T h e characteristic age-related DTV-associated changes in thymus subpopulations of AKR/J mice were not observed in thymuses of AKR/J mice treated with mAb 18-5. Transfer of thymocytes from 250-day-old

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AKR/J mice, treated at birth with mAb 18-5, into irradiated (AKR X DBA/2)F, recipients (107 cells intrdthymically) caused only 7% T cell lymphomas of AKR origin in comparison to 93% following transfer of thymoctyes from 250-day-old untreated controls, within a short latent period of 38 days. Intravenous transfer of (2-4) x 107 bone marrow cells into F, irradiated recipients yielded 13% T cell lymphoma incidence at a mean latency of 213 days, in comparison to 86% (42 days latency) following marrow transfer of untreated donor mice (the F, recipients that did not develop lymphomas were followed until they were 400 days old). These results indicate elimination of PLCs following anti-18-5 mAb treatment. E. CONCLUSIONS Prevention of spontaneous T cell lymphoma development in AKR mice by surgical removal of the thymus (thereby removing the site of DTV formation and the microenvironment for the promotion of PLC) or by hindering the dual tropic virus formation in the thymus (through viral interference or through genetic restriction of virus replication) did not affect the spontaneous PLC occurrence characteristic to AKR mice. Prevention of T cell lymphoma development by mAb 18-5 treatment seems to act on a different level, by eliminating the occurrence of PLCs, probably by reacting with gp7 1 determinants present on their surface. Eradication of PLCs by mAb 18-5, demonstrated in transplantation studies, was also indicated based on the following observations: the inoculation of MCF-247 into the thymus of young 18-5-treated AKR mice did not accelerate T cell lymphomagenesis, in contrast to its T-PLCpromoting effect in untreated control AKR/J mice; transfer of lymphoid cells from 24-666-infected AKR/J mice yielded a high incidence of Ly-l+ B cell lymphomas rather than T cell lymphomas, whereas transfer of bone marrow or thymus cells from mAb 18-5-pretreated AKR/J mice when 250 days old did not result in Ly-l+ B cell lymphoma development. These results may suggest that 18-5 treatment eliminated PLCs that have the dual capacity to progress to T or B cell lymphomas. In intact AKR mice, changes in thymus subpopulations coincide with DTV formation in the thymus. Prevention of T cell lymphomagenesis by viral interference with DTV formation in the thymus delayed or prevented the thymus subpopulation changes and involved occurrence of p,+ B cells in the thymus (see Table IV). These changes were probably triggered by B-PLCs, which have been shown to be present among thymocytes of these virus-treated mice. Lack of changes in thymus subpopulations (including absence of B cells) in 12-month-old 18-5 mAb-

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treated A K R mice (Haran-Ghera and Peled, 1991) may also be due to the absence of PLCs in these mice.

V. Maintenance and Termination of the B-PLC Dormant State AKR/J mice thymectomized at young age develop a low incidence of spontaneous T and/or B cell lymphomas. But transplantation of lymphoid cells from 8- to 12-month-old AKR mice, thymectomized at the age of 6 to 8 weeks, into intact or thymectomized recipients yielded 80100% Ly-l+ (CD5+) pre-B or B cell lymphomas of AKR origin (Peled and Haran-Ghera, 1985). Thus, these thymectomized mice are carriers of dormant PLCs that can be triggered to develop into overt lymphomas only after their removal from their natural environment to appropriate compatible young recipients. The mechanism controlling PLC dormancy in old thymectomized mice remains elusive. Host anti-PLC activity as well as immunological impairment due to endogenous factors (viruses, hemapoietic regulatory factors) may affect PLC dormancy and eventual lymphoma recurrence. In intact AKR mice, during the first few months of life, PLCs (present since birth) were identified among bone marrow cells rather than in the thymus. Several observations have suggested the presence of anti-PLC immune responses in AKR mice within the age range of 2-5 months (Haran-Ghera, 1980; Haran-Ghera et al., 1987). DTV formation in the thymus of 5-month-old AKR mice may contribute to PLC progression by interfering with antiprelymphoma immune reactivity present in 2- to 5-month-old mice and thereby permit PLC transition to overt T cell lymphoma. Removal of the thymus at a young age prevents DTV formation, which is important for PLC promotion in intact mice (Haran-Ghera et al., 1987). In thymectomized AKR mice (lacking DTV), PLC-induced immunostimulation may suppress PLC proliferation (without complete PLC destruction, perhaps by cytostatis) and thereby establish the PLC dormant state; only by removing these cells from the inhibitory environment (following cell transfer) do they proliferate and give rise to overt lymphoma. This host-PLC immune interaction has been favored because bone marrow cells from 12-monthold thymectomized mice did not cause B cell lymphoma development when transplanted into mice of similar age, but induced a high B cell lymphoma incidence of donor origin when injected into young recipients (N. Haran-Ghera, unpublished observations). Thymectomy causes changes in the balance of T cell populations involved in the immune responses and in secretion of regulatory factors, which are important for differentiation, proliferation, and progression of other cells of the im-

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mune system. Prolonged absence of thymus was shown to affect T cells involved in development of cytotoxic T cell responses (Galli and Driige, 1980), to deplete short-lived T helper (TH) cells (Swain et al., 1988), to diminish generation of suppressor T cells that affect either T or B cell responses (Bore1 et al., 1980), and to abrogate the ability of surviving T cells to produce IL-2 (Mosmann and Coffman, 1989). It is therefore plausible to suggest that thymectomy causes changes in the regulatory mediators that play a role in PLC dormancy o r in PLC progression to the overt disease. T h e possible involvement of humoral immune reactivity in maintenance of PLC dormancy also seems plausible. Most of the B cell lymphomas of AKR origin express the Ly- 1 antigen. Because Ly- 1+ B cells were shown to secrete IgM autoantibodies (Hayakawa et al., 1984), some of the autoantibodies might be antiidiotypic antibodies or antibodies to regulatory factors that are necessary for the proliferation of PLCs to autonomous tumor cells. Removal of PLC from the thymectomized host into another young recipient may prevent the interaction of the autoantibodies with PLCs and thereby facilitate their proliferation (following transplantation into the appropriate recipients) to Ly-l+ B cell lymphomas. One obvious problem has been whether it is possible to terminate tumor dormancy in the PLC carriers, resulting in a high incidence of lymphoma in old thymectomized AKR mice, instead of transferring the lymphoid cells to young compatible recipients. The outgrowth of tumor cells at the end of the dormant state could be due to changes in the antigenic properties of the transformed cells, to decreased host-tumor suppressive activity, to regulatory influences affected by interaction between T H and suppressor cells, and/or to deficiency of hemopoietic regulatory mediators, including deficiency of T cell factors necessary for B cell growth. The effects due to interference with T cell functions or administration of T cell growth factors on termination of PLC dormancy, resulting in a high incidence of B cell lymphomas, have been analyzed. Replenishment of deficiencies caused by thymectomy, thereby preventing DTV formation, was tested. Thymectomized AKR mice received a subcutaneous thymus graft (thymus taken from newborn or 3-month-old AKR mice) at the age of 12 months; this replenishment of the T cell reservoir and humoral factors (produced by the thymus epithelial reticulum of 3-month-old thymus graft, which is not repopulated by lymphoid cells) by syngeneic thymus grafting terminated the dormant state. Irrespective of the age of the grafted thymus, 80% of these treated mice developed lymphomas within a long mean latent period of about 200 days after thymus grafting, versus 36-40% in the control groups (untreated o r grafted subcutaneously with newborn spleen) (Haran-Ghera et al.,

27 1

LYMPHOMAGENESIS IN AKR MICE 100

1

Thymex Thymex + n.b Thymus Thymex + rlL-2 --C

Thymex + MCF-247 Thymex + AntlCD8

"

300

A

400

500

Days

600

700

800

FIG. 1. Incidence of Ly-l+ B cell lymphoma following termination of tumor dormancy by treating 12-month-old AKR mice thymectomized at the age of 55 days. Thymex, control group; thymex + n.b. thymus, syngeneic newborn thymus transplanted subcutaneously; thymex + rIL-2, administration of rIL-2, 2000 U intraperitoneally, twice daily for 10 days; thymex + MCF-247, 1 ml intravenously; thymex + anti-CD8 mAb, 0.1 mg antibody injected intraperitoneally every second day within 30 days.

1992). All the lymphomas developed extrathymically, predominantly in the spleen, and in many cases also involved lymph nodes and liver dissemination, in contrast to thymus graft involvement in restoring T cell lymphoma development in young AKR-thymectomized regrafted mice (Metcalf, 1966). T h e cell surface phenotypes of these lymphomas examined by flow microfluorometry indicated that the lymphomas were Ly-l+ pre-B o r B cell lymphomas. Replenishing the deficiency of the recombinant virus (due to thymus removal at young age) was tested by injecting the virus isolate MCF-247 or DTV-71 (1 rnl, intravenously) into 12-month-old thymectomized AKR mice. Virus administration triggered the development of Ly-l+ B cell lymphomas in 90-97% of the infected mice within a short latency period of 75 days (Haran-Ghera et al., 1992)-(see Fig. 1). This efficient termination of PLC dormancy can be attributed to the capacity of DTV to impair T cell functions in AKR mice [reduction of Lyt-2+ and mac-l+ cells; decreased delayed-type hypersensitivity response and reduction in response to mitogens (Peled et al., 1987)] and thereby abrogate the immune control of PLC proliferation. However, an alternative explanation for the accelerated lympho-

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magenesis is direct DTV infection of PLCs, e.g., leading to insertional activation of protooncogenes (Hayward et al., 1981; Li et al., 1989). One approach to distinguish between these two possibilities was to test the resulting tumors for DTV/MCF infection: if the mechanism was direct virus infection, all tumors should be MCF infected; if the mechanism was suppression of immune response, this would not necessarily be the case. Therefore, a series of independent Ly- 1 B cell lymphomas arising in thymectomized AKR mice injected with MCF-247 (when 12 months old) were tested for MCF-247 viral DNA by Southern blot hybridization. The fact that 60% of these tested lymphomas did not have MCF-247 DNA (Haran-Ghera et al., 1992) strongly argued against lymphomagenesis resulting from direct viral infection of PLCs and rather was consistent with an immunologic mechanism. T h e function of T and B cell subsets in spleens of old thymectomized mice infected with MCF-247, in comparison to noninfected thymectomized mice of matched age, was evaluated by testing cellular responses to the mitogens (phytohemagglutin, concanavalin A, and lipopolysaccharide (PHA, Con A, and LPS) and by their capacity to produce cytokines (IL-1, IL-2, IL-4, and IL-6). Splenocytes from untreated thymectomized AKR/J mice showed a decreased response only to Con A, in contrast to MCF-247infected mice, which exhibited significantly decreased responses to PHA (reflecting reduced levels of mature T cells) and Con A, and less to LPS. The possible effect of MCF-247 associated with the coinduction of some cytokines was also tested. Splenocytes of virus-infected or control mice were incubated for 24 hours with medium alone, in the presence of LPS o r Con A. The supernatants were collected and tested for the presence of IL-1 and IL-6 (in the LPS-activated cells) and IL-2 and IL-4 (in the Con A-activated cells). T h e virus infection was found to reduce markedly the level of IL-4; a milder reduction of IL-2 production was also observed. In contrast, IL-1 and IL-6 production, following activation of splenocytes with LPS, reached levels four to six times higher in the virusinfected mice versus the uninfected control mice; similar results were obtained with peritoneal exudate cells from the same mice (A. Peled, unpublished observations). It is interesting to point out that cytokine production by splenocytes from AKR mice infected with MCF-247 when 14 days old (thereby accelerating T cell lymphoma development), and tested up to 90 days after virus infection, showed an IL-I production level similar to that of the age-matched controls and less IL-6 production than in the controls. Thus, infection of young AKR mice with MCF-247, leading to accelerated T cell lymphomagenesis, did not involve increased cytokine production, in contrast to termination of B-PLC dormancy by MCF-247 administration to thymectomized 12-month-old mice, coincid+

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ing with increased production of IL-1 and IL-6. IL-6 is a pleiotropic cytokine that has been cloned from T cell-lines but is also produced by other peripheral blood mononuclear cell subsets, such as monocytes and B lymphocytes (Kishimoto and Hirdno, 1988; Van Snick, 1990). IL-6 has a wide variety of biologic effects supporting the growth of T and B cells; it has been shown to play an important role in the immunopathology of various diseases. IL-6 is an autocrine growth factor for multiple myeloma (Kawano et al., 1988), and continuous overproduction of IL-6 contributes to B cell hyperactivity in SLE patients and seems to play an important role in SLE pathogenesis (Linker-Israeli et al., 1991). Infection with HIV is associated with increased production of IL-6 in vivo (by stimulating monocytes to produce IL-6), suggesting that IL-6 overproduction may contribute to the polyclonal B cell activation and Ig secretion characteristically seen in HIV infection (Breen el al., 1990). T h e enhanced production of IL-6 by splenocytes of old thymectomized AKR mice after MCF-247 administration may affect B-PLCs directly by stimulating their growth and proliferation and may thereby contribute to the termination of the dormant state. The possible effect of IL-6 on PLC proliferation was also suggested from preliminary studies (A. Peled, unpublished observations) in which MCF-247-mediated termination of PLC dormancy was partially inhibited (in about 50% of such treated mice) by the administration of monoclonal antibodies to IL-6. Because administration of DTV was found to impair T cell functions in intact AKR mice and triggered efficiently the termination of the dormant state, it seemed of interest to test whether different treatments affecting T cell functions could stimulate the progression of dormant PLCs toward B cell lymphoma development. In vivo elimination of T cell subsets was performed by the administration of cyclosporin A, which is an immunosuppressant with a high degree of specificity for T cells (Shevach, 1985) or by monoclonal antibodies to T cell subsets, which provided substantial and long-term depletion of T cell subsets and impaired immunological responses after administration to adult thymectomized mice (Cobbold et al., 1984). The mAb to mouse T cell antigen L3T4 (CD4) inhibits the T cell response to MHC class I1 restriction, and the T cell antigen Lyt-2 (CD8) displays MHC class I restriction and corresponds chiefly to suppressor cytolytic T cell lineages. The most striking results concerning termination of PLC dormancy were observed after in vivo depletion of suppressor/cytotoxic T cells by the administration of antLCD8 mAb: 94% of these treated mice developed Ly- 1 B cell lymphomas within a mean latency of 80 days after onset of treatment, in contrast to 40% in the control group at 130 days mean latency (HaranGhera el al., 1992). Treatment with anti-CD4 mAb yielded only a 50% +

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tumor incidence and cyclosporin A had a mild enhancing effect, resulting in 64% B cell lymphoma development within a longer latent period. T h e efficient termination of PLC dormancy after elimination of Lyt-2+ cells by antLCD8 mAb may suggest that this population mediates an anti-PLC immune response. Elimination of Lyt-2+ cells may activate L3T4+ cells and N K cells (Grau et al., 1986), which play a role in the modulation of dormancy. The observations that a cytokine produced by one class of mouse T H cells can inhibit synthesis of cytokines by other clones (Fiorentino et al., 1989; Moore et al., 1990) suggests some crossinhibitory cytokine effects between CD8+ and CD4+ subpopulations. Complete elimination of CD8+ T cells may modify the function of CD4+ cells, producing cytokines more effective to B cell proliferation. Tests were done to elucidate whether the anti-CD8 mAb effect was associated with coinduction of cytokines. Splenocytes from 12-month-old thymectomized AKR mice treated in vivo with anti-CD8 mAb were incubated for 24 hours with medium alone, in the presence of LPS or Con A. T h e supernatants were collected and tested for the presence of IL-1 o r IL-6 (in LPS-activated splenocytes) and IL-2 and IL-4 (in Con A-activated cells) using the proper indicator cells, dependent for their growth on one of the cytokines. T h e impaired responses of splenocytes to PHA and Con A were particularly reflected in the reduced production of IL-4 and to a lesser extent in the IL-2 production by Con A stimulation. The nonactivated splenocytes secreted only marginal amounts of IL-2 and IL-4. In contrast, IL-1 and IL-6 production, following activation with LPS, reached levels two- to threefold higher in the anti-CD8-treated mice versus the controls (A. Peled, unpublished observations). Thus, the increased level of IL-6, following treatment with anti-CD8 mAb, might trigger activation and proliferation of B-PLCs and thereby contribute to the termination of their dormant state. Elimination of T cell subsets may interfere with hemopoietic regulatory mediators, triggering lymphokine production that is important for B cell proliferation. IL-2 produced by T lymphocytes acts on T cells, B cells, and N K cells, causing these cells to proliferate or manifest different cell functions. It also activates subsets of non-T, non-B lymphocytes that can lyse tumor cells, a phenomenon referred to as lymphokine-activated killing. IL-2 also induces the release of several other lymphokines, including interferon-y (IFN-y) and tumor necrosis factor (TNF). Because of these properties, IL-2 has been used in recent therapeutic trials for a variety of malignant diseases and was shown to exhibit antitumor effects in both experimental animals and cancer patients (Rosenberg, 1988). Interference with tumor growth in mice was observed after administration of high doses of rIL-2 (that are often quite toxic to the animals), i.e.,

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100,000 U/injection, three times daily, for several days (Slavin et al., 1988). We tested the possible effect of low doses of IL-2 (2000 U/injection, twice daily for 10 days) on tumor dormancy. Its administration to 12-month-old thymectomized AKR mice triggered a burst of Ly-l+ B cell lymphoma development in almost all treated mice within a short mean latency of 60 days (Haran-Ghera et al., 1992) (see Fig. 1). Thus, administration of IL-2 at low doses might be hazardous because of its capacity to enhance proliferation of dormant tumor cells. Interestingly, the injection of rIL-2 (similar schedule) to intact AKR mice, starting when 14 days or 3 months old, did not enhance T cell lymphoma development (A. Peled, unpublished observation). T h e mode of action of IL-2 is unresolved. Because the Ly- 1 B cell lymphomas lack receptors to IL-2, we assume that IL-2 might act indirectly by affecting host-mediated antitumor immune responses. IL-2 administration in vivo (250 U/mouse) was shown to induce proliferation of Thy-l+ L3T4- Lyt-2- cells with macrophage cytolytic activity in vivo (Kamitani et al., 1990); IL-2 was also shown to be capable of replacing P-B cell growth factor activity and to act as a maturation-inducing factor on primary B cells (Lernhardt et al., 1987). Enhanced Ly- 1 B cell lymphoma development was also observed in AKR-Fv-Zb mice treated with anti-CD8 mAb at the age of 18 months (Haran-Ghera et al., 1993); 77% of such treated mice developed tumors at a mean latency period of 140 days (versus 30% in the untreated controls). The enhancing effect of anti-CD8 mAb on B cell lymphoma development may be due to clonal expansion of Ly-l+ B cells, shown to be increased in spleens of old AKR-Fv-lb mice (up to 35%) (Rosner et al., 1993). Because Ly- 1 B cells are long lived and have self-renewal properties, they may have an increased tendency to become malignant through a time-dependent accumulation of mutations (Stall et al., 1988). Enhancement of Ly-l+ B cell lymphoma development in thymectomized AKR-Fv-26 mice was also observed following treatment of 18month-old mice with IL-4 (Haran-Ghera et al., 1993), which has been shown to modulate B cell responses, trigger resting B cells, and cause proliferation of activated B cells. T h e different experimental manipulations that were found to terminate efficiently the dormant B-PLC state at variable latent periods (see Fig. 1) suggest that T cell subsets and/or their products are involved in the proliferation arrest of PLCs present in thymectomized AKR mice o r in old intact AKR-Fv-Zb mice. T h e state of tumor dormancy has been described in several experimental models, all involving exogenous tumor cell challenge in normal pretreated mice. Extensive studies on tumor dormancy have been car+

+

+

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ried out by Wheelock and colleagues (1981) studying the fate of the L5 1784 lymphoma cells injected into DBA/2 mice that had previously received viable tumor cells that were excised 10 days later. Such animals were shown to display immunity to the second challenge of tumor cells, which remain in a dormant state. A strong peritoneal cytolytic T lymphocyte generation was shown to be responsible for establishing tumor dormancy, and peritoneal macrophage-mediated immunosuppressive activity contributed to the termination of the dormant state (Liu et al., 1986). These investigations have more recently suggested that IFN-y and tumor necrosis factor secreted by activated cells act on macrophages to induce the release of arginase, which affects tumor cell growth (Suzuki et al., 1991). Another model of tumor dormancy involves the BCL, Ly- 1 B cell lymphoma that arose spontaneously in an aged BALB/c mouse (Slavin and Strober, 1978). T h e dormant state has been induced by different experimental methods, including induction of allogeneic chimeric mice (Weiss et ul., 1983; Siu et ul., 1986) or immunization with a BCL, purified surface immunoglobulin idiotype (George et al., 1987; Uhr et ul., 1991). Studies by George et d.(1987) showed that the growth of BCL, cells in mice immunized with idiotypic IgM, rescued from BCL, lymphomas, was suppressed by the modulation of the tumor cell idiotype determinants; the presence of cytotoxic antiidiotypic antibodies was detected in the sera of the immunized mice. Another model of tumor dormancy established by idiotypic IgM vaccination against Ly-l+ B cell lymphoma A31, developing in CBAlH mice treated with YOSr, was described by Dyke Pt al. (1991). Antiidiotypic antibody appeared to have a major role in protection against further tumor cell proliferation in the immunized mice, but the dormant state could be disrupted by transferring spleen cells carrying dormant tumor cells. The expression of idiotypic IgM on the emergent tumor was indistinguishable from that of the parental A3 1 cells. T h e mechanism of protection in both BCLl and A3 1 tumor models remains obscure. Ly-l+ B cell lymphomagenesis in aged thymectomized AKR mice or in congenic AKR-Fu-1" mice involves a phase of spontaneously occurring endogenous PLCs in a dormant state, thereby providing a unique model for studying host factors that contribute to the long-lasting PLC dormant state and PLC growth recurrence. +

VI. Ly-1 (CD5+) B Cell Lymphoma Characteristics +

A variety of spontaneous and induced lymphomas and leukemias, in different strains of mice, are derived from the B cell lineage. They

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represent all the major stages of normal B cell differentiation. T h e CD5 B cells (which coexpress a 67-kDa pan T lymphocyte surface glycoprotein) (Ledbetter et al., 1980) are defined by their reactivity with mAb Ly-1 in mice or Leu-1 in humans. Both murine and human CD5 cells express roughly 20% of the level of CD5 expressed by most T lymphocytes (Manohar et al., 1982); however, these cells lack expression of other T cell-associated differentiation antigens but coexpress other B cell surface antigens. Several animal models that involve Ly-l+ (CD5+)B cell lymphoma development have been described. Spontaneous murine Ly-l+ B cell lymphomas have been observed in old BALB/c mice, including the BCL, tumor (Slavin and Strober, 1978), in old NZB mice (East, 1970), and in NFS mice congenic for ecotropic MuLV loci from AKR and C58 mice (Davidson et al., 1984). CD5 lymphomas also arose following chronic antigenic stimulation associated with aging. The CH series of sIgM and Ly- 1 B cell lymphomas in B 10-H-2aH-46 p/wts mice developed in aging recipients of adoptively transferred syngeneic spleen cells after hyperimmunization with sheep red blood cells (Lanier et al., 1978, 1982). Aged NZB mice were shown to have clonal expansions of long-lived, slow-growing hyperdiploid cells characterized as Ly- 1 IgM (CD5+) B cells; continued serial passage of these cells in compatible recipients resulting in Ly- 1+ (CD5+)B cell lymphoma development (Seldin et al., 1987). Our studies have shown the development of Ly-l+ (CD5+) B cell lymphomas of AKR origin, following treatments that prevent spontaneous T cell lymphoma development in this strain of mice (Peled and Haran-Ghera, 1985, 1991; Haran-Ghera and Peled, 1991; Haran-Ghera et al., 1993). The cell surface phenotypes of the B cell lymphomas (examined by flow cytometry) developing spontaneously in old intact or thymectomized AKR/J mice, o r in old AKR-Fu-lh congenic mice, were similar. All primary tumors were Thy-1 negative, IgM+, and Ly-l+ (CD5+), stained with antisera specific for pre-B o r B cells, and expressed p. and k antigens on their surface (Peled and Haran-Ghera, 1985). During in zdro maintenance of lymphoma cell lines established from primary tumors, these cell lines lost their surface p. and k expression, but retained cytoplasmic p, B-220, and Ly-1. Lymphomas obtained by retransplantation of the cells from tissue culture back into animals regained their surface p. and k. I n contrast, the B cell lymphoma cells developing in AKR/J mice treated with mAb 18-5 (thereby preventing T cell lymphoma development) differed morphologically (smaller cells) and phenotypically, having the characteristics of late pre-B cells; only about 30% of the tested tumor cells expressed surface IgM and Ia and all of them lacked the CD5, thus being Ly-l- (A. Peled, unpublished observations). It is inter+

+

+

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esting to point out that cell lines are easily established from these primary Ly-l- B cell lymphomas, in contrast to difficulties in maintaining Ly-l+ B cell lymphomas as permanent tumor cell lines. Cytogenetic studies of primary Ly-1 B cell lymphomas and several established cell lines of these tumors by G-banding analysis revealed normal diploid karyotypes in 70% of the analyzed tumors. T h e rest of the lymphomas with the model number of 39-41 chromosomes had different chromosome markers specific for each tumor (Trakhtenbrot et al., 1987). The development of Ly-I+ B cell lymphomas in AKR/J and AKR-Fv.Zb mice and the identification of PLCs in spleens of old grossly normal mice raised the question whether the number of Ly- 1 B cells was increased in the spleens of older mice, whether Ly- 1 B cell populations were clonal, and whether the pattern of IgH rearrangements in those clones was similar to those observed in the Ly- 1 B cell lymphomas developing in thymectomized AKR/J mice and AKR-Fv-lb congenic mice. T h e following studies were carried out in order to answer these questions. +

+

+

+

A . Ly-l+ B CELLS The Ly-l+ B cells (CD5+) that have a high concentration of IgM (Hayakawa et al., 1984) are a minor subset of the mouse B cells and have been considered as a separate lineage with special organ distribution (Herzenberg et a/., 1986). Unlike other lymphocytes, the absolute number of Ly-l+ B cells rapidly reaches adult levels early in lymphocyte ontogeny and decreases with age (Dexter and Corley, 1987). In adult mice Ly-l+ B cells constitute a major lymphoid subpopulation in the murine peritoneal cavity (30-60% of total B cells (Hayakawa et al., 1986). I n old mice an increase in the level of Ly-1 B cells has been observed in peritoneal cavity, lymph nodes, and spleen (Stall et al., 1988). Several mouse strains that develop autoimmune pathology (e.g., NZB mice) have elevated numbers of splenic and peritoneal Ly-l+ B cells (Hayakawa et al., 1983). Comparison of inbred mouse strains indicates that the level of Ly-l+ B cells is under genetic control (Kipps, 1989). These cells do not respond like normal B cells to exogenous antigens, but appear to contribute to the production of autoantibodies, especially the IgM class (Herzenberg et al., 1986). It has been suggested that these cells appear early in the ontogeny, developing from precursors present in fetal or newborn liver and bone marrow (rather than from precursors in adult spleen o r bone marrow (Hayakawa et al., 1986). Thus, the adult Ly-l+ B cell population consists predominantly of long-lived self-renewing cells

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and their recruitment requires antigenic stimulation (Lalor et al., 1989; Hardy et al., 1991). This suggestion implies that if these Ly-l+ B cells are depleted during neonatal life, there is no self-renewal, because adult bone marrow cells lack precursors of Ly- 1 B cells; in contrast, conventional B cells can be reconstituted because their precursors can be continually supplied by bone marrow (Lalor et al., 1989). Other recent studies have suggested that spleen and Ig- bone marrow cells from adult mice can persist, expand, and reconstitute both Ly-l- and Ly-l+ B cells on transfer to sublethally irradiated hosts (Thomas-Vaslin et al., 1992). These observations also coincide with findings in humans; adult bone marrow was shown to contain cells that may reconstitute the CD5+ B cell population (Ault et al., 1985). Ly-l+ B cells have been shown to be present in the thymus of normal mice (Miyama-Inaba et al., 1988). Using radiation chimeras, these investigators demonstrated that adult thymic Ly- 1 B cells could be reconstituted with fetal liver cells o r bone marrow cells from both young and old mice, whereas peritoneal Ly-l+ B cells could not be reconstituted with bone marrow cells from old mice. These studies have suggested that thymic and peritoneal Ly- 1 B cells differ in ontogeny and function (Than et al., 1992). Ly-l+ B cells have unique characteristics, intrinsically linked to autoimmune diseases and autoantibody production (Herzenberg et al., 1986; Casali et al., 1987). In humans, a large fraction of CD5 B cells produce autoantibodies (Casali et al., 1987). These antibodies are frequently noted to have reactivity to immunoglobulins, to proteolytically processed erythrocyte membranes, and to denaturated DNAs. Polyreactive (mainly IgM+) Ly-l+ B cells (and their Leu-1 human counterparts) were found to produce antibodies directed against self antigens and bacterial antigens (Hayakawa et al., 1984; Forster and Rajewsky, 1987) and as such were directly involved in autoimmunity and in natural defense (Herzenberg et al., 1986; Rajewski et al., 1987). On the basis of the V gene usage, the Ly-l+ B cells appear to use germ-line unmutated V genes and may select V, genes (Painter et al., 1986). There are functional subsets of Ly-1 B cells with apparent immunoregulatory activity (Akamura et al., 1982; Sherr et al., 1987). The immunoregulatory potential of Ly-1 B cells may be mediated by cytokines and secreted antibodies with anti-Ig reactivity. It was shown that Ly-1 B cells may synthesize IL-1 (Pistoia et al., 1986) and produce IL-10 (Moore et al., 1990), that a high proportion of peritoneal B cells bear functional IL-5R (poorly expressed in splenic B cells), and that IL-5 can regulate proliferation and maturation of Ig secretion of mouse Ly-1 B cells (Wetzel, 1989). I n old thymectomized AKR/J mice and AKR-Fv-lb mice we demonstrated that the Ly-1 B cell population increased as the animal became +

+

+

+

280

NECHAMA

HAKAN-GHERA

older (not seen in old AKR/J mice treated, when newborn, with mAb 18-5, thereby eliminating PLCs and preventing T cell lymphoma development; N. Haran-Ghera and A. Peled, unpublished observation). The analysis of a Ly- 1 IgM+ B cell population of spleen cells (determined by FACS analysis on cells double labeled with anti-Ly-1 and anti+ antibodies) showed that in 6-month-old thymectomized AKR/J mice, 9% of B cells were Ly-l+ versus 30-60% in 15- to 18-month-old mice. In spleens of 18-month-old grossly normal AKR-Fv-lb congenic mice, 22-45% of the B cells were of the Ly-1 B type versus 5% in spleens of 6-month-old mice. T h e size and number of these populations varied among individual mice and between batches of animals. Similar findings have been described for peritoneal cells of NZB and (N2B X NZW)F, females (Hakayawa et al., 1983; Tarlintonetal., 1988)andCB.20 mice(Forsteretal., 1988).Thelarge Ly-l+ B population in normal spleens of old AKR mice might already represent the preleukemic B-PLC clone that would expend following antigenic stimulation. We speculate that the different experimental manipulations used by us to enhance progression of B-PLCs to overt B cell lymphoma development involve breakdown of immunological surveillance, which normally arrests the proliferation of the PLCs. This increased proliferation would increase the risk of additional genetic alterations, culminating in the malignant transition to autonomous tumors. +

OF THE HEAVY A N D LIGHTCHAIN LOCI B. ANALYSIS OF Lv-l+ B CELLLYMPHOMAS

High molecular weight DNAs prepared from 23 Ly-l+ B cell lymphomas derived from different individual mice were digested with EcoRI and analyzed by Southern blot technique using probes that recognize all possible rearrangements of the IgH locus. Similar analysis was performed to examine the loci coding for the light chains of the imniunoglobulins (Rosner et al., 1993). None of the tumors showed rearrangement of the lambda locus, but most tumors tested contained rearranged kappa alleles. Southern blot analysis of the IgH locus revealed that the germ-line fragment of 6.2 kb was detected in some tumors, and indicated common utilization of two specific sets of IgH VDJ segments (a 2.6-kb EcoRI fragment and a 5.5-kb EcoRI DNA). The same species showed a common restriction pattern with three enzymes in several tumors and therefore represented a unique set of VDJ segments. T h e analyzed tumors could be divided into three categories: in group 1, about 40% of the analyzed tumors exhibited rearranged fragments of 5.5 and 2.6 kb; in group 2, 17% of tumors showed a 5.5-kb fragment; and in group 3, tumors had randomly sized IgH rearranged fragments

LYMPHOMAGENESIS IN AKR MICE

28 1

(Rosner et d., 1993). Thus, in many tumors, both IgH alleles were rearranged. Southern blot analysis of the IgH rearrangement pattern in 16 Ly- 1 B cell lymphomas of AKR-Fu-Ib origin demonstrated rearrangement patterns similar to those found in Ly-l+ B lymphomas of AKR/J mice. To study further commonly sized rearranged fragments, the 5.5- and 2.6-kb species were molecularly cloned from several independently derived Ly-l+ B cell lymphomas (Rosner et al., 1993). Comparison of the physical maps of these cloned fragments to the map of the cloned germline DNA indicated deletion of sequences upstream of -J2 and 53 in the 5.5- and 2.6-kb fragments, respectively. T h e sequences of DNAs derived from two lymphomas that differed in their karyotype [one had a normal karyotype and the other had the (17:18) translocation marker (Trakhtenbrot et al., 1987)] were identical. This indicates the absence of somatic mutations, and no insertion of N-region nucleotides (Alt and Baltimore, 1982). Joining of VDJ segments retained the open reading frames in phase. The sequences of the 2.6-kb DNA fragments cloned from three Ly- 1 B lymphomas were identical to one another (Rosner et al., 1993). Whereas the common 5.5-kb DNA detected in tumors contained an open reading frame, this was not the case with the 2.6-kb DNA species. It is possible that the latter rearrangement becomes productive through posttranscriptional or translational modifications such as those found in other systems (Powell et al., 1987;Jacks et al., 1988). An alternative possibility is that the 2.6-kb DNA rearrangement is giving rise to a truncated Ig, containing predominantly the V region, but not the Cp. T h e reproducible emergence and properties of clones with particular IgH rearrangements is most likely due to the characteristics of the Ly- 1 B cell population from which they are derived. Several recent publications have shown repetitive utilization of certain VH and/or D segments in hybridomas and Ly-1 B lymphomas. Tarlinton et al. (1988) showed that many Ly-1 B cells isolated form peritoneal cavities of different (NZB x NZW)F, mice had the same size rearrangements at both IgH and IgK loci. In one mouse, the productive rearrangements had identical unmutated V, and D elements joined to different J H elements. Forster et al. (1988) described repetitive utilization of V, segments in hybridomas derived from unrelated clones. Two extreme examples, antibody produced in cell lines obtained as spontaneous outgrowth cultures of spleen cells (Braun and King, 1989), and autoantibodies against bromelain-treated red blood cells (Pennel et al., 1988), exhibit the same properties of IgH molecules described in our work: (1) usage of identical V, D, and J elements, (2) lack of somatic mutations, and (3) absence of an N-region. The sequence within the 5.5+

+

282

NECHAMA HARAN-GHERA

kb EcoRI fragment shows 88% homology to the IgH expressed in two Ly-l+ B hybridomas (Forster et al., 1988). Moreover, the sequence within the 2.6-kb DNA species exhibits 94% homology to that of IgH expressed in the CHlO and CH13 tumors (Pennel et al., 1988) and in 12 hybridomas (Forster el al., 1988) and 89% homology to the sequence of IgH expressed in Ly-1 B cells found in B/W mice (Tarlinton et al., 1988). Repetitive utilization of Ig gene segments could be due to favorable rearrangements during the recombination process, or to selection of the cells through their antibody receptors. The first possibility is usually based on the assumption that the VDJ elements proximal to each other are easiest to recombine. The VH region within the 5.5-kb fragment most commonly used in our tumors is a member of the V, Q52 family, which is indeed positioned as the second most proximal to the D region (Paige and Wu, 1989). I n contrast, The V, region within the 2.6-kb species belongs to the V, J558 family positioned away from the D-J-C segment. Another possibility is that the prominent Ly-1 B oligoclones that eventually give rise to Ly-1B lymphomas in AKR mice are antigen driven. T h e antigen could be an AKR self-antigen, an endogenous virus such as 24-666, isolated from a B cell lymphoma (Peled and HaranGhera, 1991),or an antiidiotypic antibody. Interestingly, the sequence of the V region within the 2.6-kb AKR rearranged DNA fragment is 90% homologous to V, sequences expressed in lupus-prone mice (Eilat et al., 1989). Thus, it is possible that the AKR Ly-I+ B clones, by virtue of specific IgH molecules that they express, are stimulated to proliferate by repeated exposure and interaction with a self antigen. Continuous proliferation of these clones would increase their risk for additional genetic alterations, leading to malignant transition. In humans the most common malignancy of the CD5 (Leu-1) B cell is chronic B lymphocytic leukemia (B-CLL) (Martin et al., 1980; Royston et al., 1980). Similar to the Ly-l+ B cell lymphomas, the CLL cells express sIg that uses a restricted set of conserved IgV genes (which have been highly associated with IgM autoantibodies) with specific VDJH rearrangements and minimal somatic mutations. For example, Humpries et al. (1988) reported that a new human V, family was rearranged in about 30% of B-CLL patients. Similar observations were reported by Kipps et al. (1989). In contrast to B-CLL, malignant B cell NHLs of folicular center cell origin may permute their expressed IgV genes through somatic hypermutations (Kipps, 1989). We have suggested that Ly-l+ B oligoclones that give rise to Ly- 1+ B lymphomas in thymectomized AKR mice o r old AKR-Fz,-lb mice are antigen driven (the antigen could be an endogenous virus, an AKR self antigen, or an antiidiotypic antigen). Similar suggestions of intense antigenic stimulation (which could also be

LYMPHOMAGENESIS I N AKR MICE

283

generated by infectious agents such as malaria o r HIV infection associated with B cell proliferation) playing a role in human B cell lymphoproliferation have been delineated by several investigations (Kipps, 1989; Potter, 1990). Other similar observations characteristic of our murine model and human CLL involve its association with aging, immunodeficiency, and genetic predisposition; the developmental pattern is one of bone marrow being the initial site of transformation and further spleen and lymph node involvement and often also liver infiltration. T h e occurrence of the dormant B-PLC state in AKR mice also coincides with the observation that CLL can remain asymptomatic for a long time (Boggs et al., 1966). Thus, the AKR Ly-l+ B cell lymphomagenesis model has the potential to yield valuable information on the pathogenesis of B-CLL. OF IGH REARRANGEMENTS IN C. THEPATTERN SPLEENS OF GROSSLY NORMAL OLDAKR-Fu-Ib AND THYMECTOMIZED AKR/J MICE

An age-related increase in Ly-1 B cells was observed in spleens of grossly normal mice, reaching a level of 30-60% of the total B cell population in the spleens of mice beyond the age of 1 year. T h e IgH rearrangement pattern of spleens from AKR/J and AKR-Fu-Ib mice of different ages was studied. Spleen DNAs were digested with EcoRI and analyzed by Southern blotting for hybridization to the probe. This probe contains the genomic segments J3 and J 4 of the heavy chain locus and could be used to detect all rearrangements of this locus. When normal spleens from 2-month-old mice were analyzed, only the germline EcoRI fragment of 6.2 kb was detected. Spleens from 6-month-old mice showed the germ-line band as well as many very faint bands corresponding to rearranged IgH. Southern blot analysis of DNA samples derived from individual spleens of 12-month-old thymectomized AKR/J mice and 24-month-old AKR-FV-Ibmice indicated IgH EcoRI bands of 6.2 (germ line), 5.0, and 2.6 kb, and others (Rosner et al., 1993). T h e analysis of about 80 “normal” spleens of old mice indicated several IgH EcoRI-rearranged fragments of common size in different spleens. Fragments of 2.6,2.9, or 5 kb were most common and were detected alone o r in combinations in about 50% of spleens analyzed. Thus, common utilization of two specific sets of IgH VDJ segments were indicated. These observations imply that the Ly-l+ B cell population in spleens of these mice is mono- o r oligoclonal. T h e clonal rearrangements observed in spleen cells of old mice were shown to occur in Ly- 1 B FACS-sorted cells (Rosner et al., 1993). DNAs prepared from Ly-l+ B cells of individual

284

NECHAMA HARAN-CHERA

spleens were analyzed by Southern blotting and showed rearranged IgH similar to that observed for the tumors. Molecular cloning of the 2.6-kbp EcoRI fragment of the prominent rearranged species in normal spleens was performed. Several clones with identical restriction maps were obtained, thus ensuring that they represented the species observed by Southern blot analysis. T h e 2.6-kb EcoRI fragments obtained from two different spleens of 2-year-old AKR-Fv-1 (J mice were sequenced. T h e sequences were found identical, suggesting germ-line configurations as well as the absence of an N-region. Three 5-kb EcoRI fragments were cloned from three individual spleens of 2-year-old AKR-Fv-16 mice; sequencing analysis indicated that the fragments represented DJ2 rearrangements, where the D segment was different in each spleen. Thus, while the 2.6-kb EcoRI-rearranged fragments detected in individual spleens appear to represent a unique DNA species, the 5-kb EcoRIrearranged bands correspond to at least three different types of rearrangements. In summary, analysis of individual spleens of old mice and individual primary Ly- 1+ B cell lymphomas indicated common rearranged IgH fragments and utilization of specific sets of IgH VDJ segments. A 2.6-kb EcoRI fragment detected in many “normal” spleens and tumors was analyzed on the sequence level in two spleens and three tumors, and was found to represent an identical VDJH joining. D. LACKOF CLASSI MCF VIRUS INVOLVEMENT I N L Y - ~B + CELLLYMPHOMAS Following experimental manipulations that accelerated lymphomagenesis (Haran-Ghera et al., 1992),a series of Ly-I+ B cell lymphomas developing in thymectomized A K R mice was tested for the presence of MCF recombinant viruses by Southern blot hybridization. Two different combinations of restriction enzymes and probes were used to detect Class I o r Class I1 MCF in the analyzed tumors. A high percentage of tumors lacked Class 1 MCF [which are pathogenic for T cell lymphomagenesis in A K R mice, in contrast to Class I1 MCF, which are not pathogenic (Thomas and Coffin, 1982)l. Thus, the Ly-I+ B cell lymphomas developing in thymectomized A K R mice did not result from the development of pathogenic Class I MCF. The virological characterization of normal tissues and Ly-l+ B lymphomas arising in AKR-FzI-I” mice was also examined (Haran-Ghera et al., 1993). When normal spleens from 1l-month-old mice were examined, none showed evidence of Class I MCF infection. Spleens from old AKR-Fu-l* mice showing high percentages of Ly-I+ €3 cells also showed

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285

no evidence of Class I MCFs. Likewise, when Ly- 1 B lymphoid tumors arising spontaneously or following “acceleration” treatments were examined, Class I MCFs were infrequently detected. Thus, Ly-l+ B cell lymphomas arising in AKR-Fu-lb mice appear to result from a mechanism independent of Class I MCF formation. Presumably, other genetic features of these mice are responsible for the propensity to develop B lymphomas. In contrast to these observations, many of the T lymphomas arising in AKR-Fu-1 mice showed evidence of Class I MCF infection, thereby suggesting a causal role in T lymphoma development (Haran-Ghera et al., 1993). T h e virological studies concerned with B cell lymphomagenesis were focused on Class I MCF viruses, because they are the most strongly implicated pathogenic agents in the AKR mouse. However, the Southern blot hybridizations were not used to test for infection of tumors with endogenous Akv MuLV. In fact, it is formally possible that the other genetic factor in AKR mice responsible for establishing potentially leukemic B and T cell lymphomas is the endogenous Akv MuLV itself. In NFS-V-congenic mice there is a clear relationship between the levels of ecotropic MuLV expressed early in life and subsequent risk for B cell tumor development (Davidson et al., 1984). In addition, MCF viruses can be recovered from these lymphomas, but these agents do not accelerate B cell lymphoma development in NFS-V-congenic mice. In this regard, Class I MCF pathogenicity is typically measured by the ability of these viruses to accelerate spontaneous leukemia in AKR mice. If Class I AKR MCFs are injected into mice that do not express the endogenous Akv MuLVs (e.g., NIH Swiss), then they do not induce disease (Cloyd et al., 1980). +

VI I. Concluding Remarks

T h e high susceptibility of AKR mice to spontaneous T cell lymphomagenesis is dependent on the presence of an intact thymus and is associated with two classes of endogenous retroviruses: the ecotropic virus and the dual tropic recombinant virus class expressed in the thymus prior to the development of the lymphomas. The dual tropic recombinant Class I MCF viruses (DTVs) are the most strongly implicated pathogenic agents in T cell lymphomagenesis in AKR mice, but are not involved in Ly- 1 B cell lymphomagenesis. We have demonstrated (by in vivo transplantation bioassay studies) the presence of potential lymphoma cells that have the capacity to develop into T cell lymphomas, among fetal liver cells and mostly among bone marrow cells of young intact or thymectomized AKR mice (thereby sug+

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gesting that they do not originate from the thymus). T h e importance of the recombinant dual tropic viruses (formed in the thymus of 5- to 6-month-old mice) and PLCs (preexisting, since birth, among bone marrow cells) in spontaneous and induced T cell lymphomagenesis was delineated by analyzing mechanisms underlaying their enhancement o r prevention. Enhanced T cell lymphomagenesis (yielding 80- 100% T cell lymphoma development in 3- to 4-month-old mice) was either PLC dependent (required for tumorigenesis by Class I MCF viruses or exposure to fractionated irradiation) or independent of the presence of both PLCs and DTVs (induction by ecotropic viruses of MNU), thus involving a direct transformation of thymocytes. In contrast, prevention of spontaneous T cell lymphomagenesis in AKR mice was attained either by preventing DTV formation in the thymus or by PLC eradication. Thus, besides the multiphase event in spontaneous T cell lymphoma development in AKR mice, whereby DTV acts as a promoter on preexisting PLCs, enhancing their ability to migrate into the thymus and further progress to fully transformed autonomous tumor cells, other pathways underlie induced T cell lymphomagenesis in AKR mice (summarized in Table IV). Although PLC establishment in young A K R mice is thymus independent, progression of PLCs from young intact (1-4 months old) AKR mice toward overt T cell lymphoma development (demonstrated by transplantation studies) was dependent on host conditions, including intact thymus, exposure of PLC recipients to radiation, and an Fu-1 nn allele when using F, PLC recipients; with age increase (beyond 6-8 moths) PLCs were autonomously growing tumor cells. These observations suggest that both viruses and regulatory growth factors contribute to the transition and progression of “dependent” PLCs toward overt T cell lymphoma development. Prevention of spontaneous T cell lymphoma development in all the experimental models studied was accompanied by a moderate increase (up to 30%) in Ly-l+ B cell lymphoma development. One interesting problem was whether T cell prevention would affect the PLC level in these treated mice. Transplantation studies actually demonstrated the presence of dormant PLCs in SO-100% of these grossly normal old mice. T h e association between overt T / B cell lymphoma incidence and PLC levels in intact versus pretreated AKR mice (affording prevention of the disease) is delineated in Table IV. The effect of DTV formation and PLC presence on changes in thymus subpopulations (occurring in normal 5- to 6-month-old AKR mice) and their contribution to accelerated T cell lymphomagenesis is also indicated in this table. The level of T-PLCs in AKR mice, developing ultimately to T cell

TABLE IV BETWEEN T/B-PLC LEVEL AND T/B LYMPHOMAGENESIS ASSOCIATION Accelerated T cell lymphoma development by Spontaneous T cell lymphoma incidence

Host treatment

Spontaneous B cell lymphoma incidence

T-PLC

B-PLC

Thymus subpopulation changes(6- 12 months old)

+A-RadLV (30 days old, intrathymic)

+MCF (14-30 days old, intrathymic)

+170r x 4 (30 days old)

~~

AKRIJ

80-95% 5-10% (630 2 45) (260 5 30)a

5% 80% (in 30-day-old mice)

++++

100% (115

2

24)

90% (75

92% f

10)

(140

* 25)

65% 20-28% (in 12-month-old mice) AKR/J thymectomized

5-10% (460 2 58)

20-30% (600 f 50)

0-5% 90-100% (in 12-month-old mice)

-

AKR/J + intrathymic 24-666, to 14-30 days old

14%

(485 2 65)

35% (570 t 48)

0-1476 64-80% (in 12-month-old mice)

AKR/J + antibody 18-5 (newborn to 10 days old)

7% (245 t 35)

20% (650 f 45)

AKR-Fv- I' congenic

7%

30% (680 2 55)

0

b

Mean latency days. n.d., not determined.

(470 2 85)

-

-

-

No changes

Resistant

n.d.0

n.d.

7-13% 10-20% (in 10-month-old mice)

N o changes

Resistant

85% (74

10% 40-70% (in 10-month-old mice)

No changes

Resistant

* 8)

100% (75 2 18)

Resistant

100 (125 f 24)

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NECHAMA HAKAN-GHEKA

lymphoma, was shown to be age related; with age increase (from 10 months onward) a reduction in T-PLCs and occurrence of B-PLCs, developing ultimately to B cell lymphoma, was observed (summarized in Table IV). The increased level of B-PLCs was observed following experimental manipulations that prevented T cell lymphoma development (thymectomy, viral interference with DTV formation, genetic restriction of viral replication using the congenic AKR-Fv-lb mice). In contrast to T-PLC dependency on host conditions for their further transition to T cell lymphomas, B-YLCs from older mice were autonomously growing cells (when transplanted), independent of host conditions. This age effect on the pathway of PLC characteristics and growth conditions suggests that age-related changes in hemopoietic regulators affect the orientation of lymphoid progenitor cells toward the B or T lymphoid cell pathway. T h e dormant PLCs could be triggered to develop into Ly- 1 B cell lymphomas following their removal from their “restrictive environment” to appropriate compatible young recipients. Host-PLC immune interactions seem to control this PLC dormant state. Interference with T cell functions was found to trigger termination of B-PLC dormancy. Elimination of T cell subsets (by administration of MCF-247 or mAb to CD8 cells) or administration of T cell growth factors (IL-2 or IL-4) resulted in a high incidence of Ly-l+ IgM+ pre-B or B cell lymphoma development with a short latency. Infection with MCF-247 o r elimination of CD8+ T cells was actually found to be associated with the overproduction of IL-6 and IL-1. The increased level of IL-6 may trigger activation and proliferation of B-PLCs and thereby contribute to the termination of their dormant state. Different experimental models of tumor dormancy, involving exogenous tumor cell challenge in normal pretreated mice, have been described. T h e present AKR model involves occurrence of spontaneous endogenous dormant PLCs, thereby providing a unique model for studying host factors that contribute to the long-lasting PLC dormant state and PLC growth recurrence. Studies of the characteristics of the Ly- 1 B cell lymphomas indicated that rearrangements of the IgH locus involved rearranged kappa alleles. Southern blot analysis of the IgH locus in individual tumors indicated common utilization of one or two specific sets of IgH VDJ segments (the 2.6-kb EcoRI fragment and 5.5-kb EcoRI DNA) in about 50% of the analyzed tumors. T h e 2.6- and 5.5-kb species from several independently derived Ly- 1 B lymphomas were molecularly cloned and their DNA sequences were found to be identical, indicating the absence of somatic mutations and no insertion of N-region nucleotides. Similar +

+

+

LYMPHOMAGENESIS IN AKR MICE

289

analysis of IgH rearranged patterns in spleens of grossly normal old mice (having an increased Ly- 1 B cell population) indicated rearranged fragments, including common utilization of two specific sets of IgH VDJ segments, in about 50% of spleens, similar to the IgH rearrangements in the Ly-1 B lymphomas. T h e large Ly- 1 B cell population observed in spleens of old normal mice might already represent the enlarged B-PLC clones. We speculate that different experimental manipulations used to enhance progression of B-PLCs to overt lymphomas involve breakdown of immunological surveillance, which normally arrests the proliferation of B-PLCs. The increased proliferation would increase the risk for additional genetic alterations, culminating in the malignant transition to autonomous Ly- 1 lymphoma cells. There is much similarity between Ly-l+ B cell lymphoma development in AKR mice and the human CD5+ B-CLLs that express sIg and use a restricted set of conserved IgV genes with specific VDJH rearrangements and minimal somatic mutations. Other similarities include the role of antigenic stimulation in triggering CD5+ B cell expansion, association with aging, immunodeficiency, genetic predisposition, and asymptomatic long latency before overt expression of the disease. B cell lymphomagenesis in AKR mice may therefore provide valuable information on the ontogeny and pathogenesis of CD5+ B-CLL. +

+

+

+

ACKNOWLEDGMENTS Work cited from the author’s laboratory was supported by the Israel Science Foundation (Grant 147191). I am indebted to Drs. A. Peled and Jay A. Levy for valuable discussions, suggestions, and critical comments on the manuscript. Special thanks to E. Majerowich for careful and dedicated attention to the preparation of this review.

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THE TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ Jens F. Rehfeld and Wouter W. van Solingel Department of. Clinical Biochemistry, University of Copenhagen, Rigshospitalet, DK-2100 Copenhagen, Denmark

I. Introduction 11. Definition of the Gastrin-Cholecystokinin Family 111. Normal Biology A. Genes and mRNAs of Gastrin and Cholecystokinin B. Post-translational Processing of Progastrin C. Post-translational Processing of Procholecystokinin D. Cellular Release of Gastrin and Cholecystokinin E. Receptors for Gastrin and Cholecystokinin IV. Tumor Biology A. Gastrin Expression in Tumors B. Cholecystokinin Expression in Tumors C. Gastrin as Tumor Growth Factor D. Cholecystokinin as Tumor Growth Factor V. Requirement for Gastrin and Cholecystokinin Measurements in Oncology VI. Methods for Measurement of Gastrin and Cholecystokinin VII. Perspectives References

1. Introduction Most growth factors of interest in tumor biology have been discovered in the past few decades (for reviews, see Aaronson, 1991; Deuel, 1987). Gastrin and cholecystokinin (CCK), however, were discovered in the beginning of this century-as hormones. Gastrin was discovered as an acidstimulating factor in extracts of the antral part of the stomach (Edkins, 1905). CCK was found as a gallbladder-emptying factor in extracts of the small intestine (Ivy and Oldberg, 1928). Purification from gut extracts subsequently revealed that CCK is identical to pancreozymin, the intestinal hormone that regulates enzyme secretion from the pancreas (Harper and Raper, 1943; Jorpes and Mutt, 1966). Simultaneously, determination of the primary structures of gastrin and CCK disclosed that the active site of the two hormones is identical (Gregory et al., 1964; Mutt and Jorpes, 1968). Thus, biologically active gastrin and CCK peptides have the same C-terminal pentapeptide amide sequence, and this strucPresent address: Department of Clinical Chemistry, Ziekenhuis Eemland, Amersfort, The Netherlands.

295 ADVANCES IN CANCER RESEARCH. VOL. 63

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ture has been exceedingly well-preserved during evolution (Anastasi et al., 1967; Larsson and Rehfeld, 1977; Johnsen and Rehfeld, 1990). During the past two decades, the concept of gastrin and CCK as simple gut hormones has changed dramatically (for reviews, see Dockray and Gregory, 1989; Rehfeld, 1989a). By now both gastrin and CCK are known to occur in multiple hormonal forms, and they are both expressed in a tissue-specific manner in a wide variety of cells outside the gastrointestinal tract. For instance, CCK is the most abundant peptide system in the brain, and, vice versa, the brain is the main production site of CCK (Rehfeld, 1978~).Accordingly, CCK and gastrin are not only hormones. They are also potent transmitters in the central and peripheral nervous systems. Moreover, gastrin and CCK have been shown to be growth factors in the gastrointestinal tract, and perhaps elsewhere (for review, see Johnson, 1989). Today, the view is even favored that growth stimulation is the most important function of gastrin and CCK (Johnson, 1976, 1989). In synchrony with the increased focus on growth effects, the clinical interest in gastrin and CCK has gradually expanded from roles in duodenal ulcer, gallbladder, and pancreatic diseases to their significance for tumor development. So far, particular interest has been given to carcinoid tumors in the stomach and adenocarcinomas in the pancreas and colorectal mucosa. But, as shown in the following discussion, gastrin and CCK may, in accordance with their widespread expression, also be involved in tumor development elsewhere. The new trend suggests that time is ripe for a broader consideration of the tumor biology of gastrin and CCK. The present review intends to serve this purpose by giving a description of the normal biology of the gastrin and CCK systems followed by a discussion of the expression and growth stimulation of gastrin and CCK peptides in carcinomas, sarcomas, and benign tumors. Finally, we will mention novel methods for measurement of gastrin and CCK expression in experimental and clinical oncology. Specific aspects of the association between gastrin or CCK and cancer have been reviewed briefly elsewhere (Axelson et al., 1992; Creutzfeldt, 1988; Laniers and Jansen, 1988; Lamers et al., 1990; Rehfeld and Hilsted, 1992; Waldum et al., 1992). However, because the emphasis has been on clinical correlations, a more comprehensive review including basic aspects is needed. II. Definition of the Gastrin-Cholecystokinin Family Most biologically active peptides occur in families whose members display significant structural homology. T h e occurrence of peptide fami-

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lies is supposed to reflect evolution from a single ancestral peptide gene by gene duplication, and subsequent mutations (for reviews, see Acher, 1981; Adelson, 1971; Barrington, 1982; Young, 1992). Also, gastrin and CCK are members of a family (for review, see Rehfeld, 1981). As mentioned, CCK and gastrin have an identical active site that has been extremely well-preserved during evolution (Larsson and Rehfeld, 1977; Johnsen and Rehfeld, 1990, 1992; Bjornskov et al., 1992). It is possible that the putative common ancestor resembles a dityrosyl-sulfated peptide, cionin, which recently was isolated from protochordean neurons in our laboratory (Johnsen and Rehfeld, 1990). The frog skin peptides, caerulein and phyllocaerulein (Anastasi et al., 1967, 1969), are also members of the gastrin-CCK family (Fig. 1). Our present state of knowledge recognizes only CCK and gastrin as members of the gastrin family in mammals. All known biological effects of gastrin and CCK peptides reside in the conserved common C-terminal tetrapeptide amide [-Trp-Met-AspPhe-NH, (Fig. l.)].Any modification of this sequence grossly reduces o r abolishes its biological effects (Morley et al., 1965), although binding to the receptors may still occur (Galleyrand et al., 1992). The different N-terminal extensions of the tetrapeptide amide increase the biological potency and the specificity for the CCK and gastrin receptor binding, respectively. Of particular importance in this respect is the tyrosyl residue in position six of gastrin and in position seven of CCK peptides, as counted from the C-terminal phenylalanyl amide (Fig. 1). Structurally, the gastrins are consequently defined as peptides that stimulate gastric acid secretion and have the C-terminal sequence Tyr-X-Trp-MetAsp-Phe-NH, (where X in most mammalian species is a glycyl residue). CCKs are defined as gallbladder-emptying peptides having the C-termi(where X in most nal sequence Tyr-X ,-X,-Trp-Met-Asp-Phe-NH, mammals is a methionyl residue and X, is a glycyl residue). CCK peptides normally occur with the tyrosyl residue O-sulfated, whereas antral gastrin may occur with its tyrosyl residue either sulfated or nonsulfated (Andersen, 1985; Gregory et al., 1964). It has been suggested that sulfakinins, i.e., carboxyamidated and tyrosyl-sulfated peptides isolated from the cockroach, Leucophaea maderue (Nachman et al., 1986), or deduced from cDNA cloned from Drosophila melanoguster (Nichols et al., 1988) might belong to the family. However, as shown in Fig. 1, the difference in the C-terminal active site comprises substitutions of two decisive residues (Trp + His and Asp + Arg). Such marked substitutions render a phylogenetic relationship less likely. T h e active-site homology of gastrin with CCK has posed large problems in the study of these peptides, because the active site is strongly

,

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JENS F. REHFELD A N D WOUTER W. VAN SOLINGE

S?i

: -Ser-Asp-Arg-A~p-Tyr-Met-Gly!Trp-Met-Arp-Phe-NH2 Cholecyrtoklnln

I S?i

I

-G1~Glu-01u-GIu-Ala-Tyr-Gly-Trp-M~ t-Asp-Phe-NH

Gartrln

I

Caeruleln

59;

pQlu-Glu-Tyr-Thr-Gly-Trp~e~-Asp-~e-NH

Phyllocamruleln

s?; sp; Clonln I

Leucosulfaklnln I

:

LeucomulfaklnlnII :

Olu-Gln-Phe-Glu-Arp-T yrGly-Hir-Met-Arg-Phe-NH

sp;

Drosulfaklnln I

:

Drorulfaklnln II

(S?J : -Arp-GIn-Phe-Asp-Arp-Tyr-Gly-Hlr-Mmt-Arg-Phe-NH2

I

FIG. 1 . The C-terminal amino acid sequences of members of the gastrincholecystokinin family (top) and of the sulfakinins (bottom).

immunogenic (Rehfeld, 1984) and because both CCK and gastrin receptors bind peptides having the common sequence (Wank et al., 1992; Kopin et al., 1992). Consequently, evaluation of antibody and receptor binding of gastrin and CCK as well as studies of the carcinogenic effects of gastrin and CCK require due consideration of functional interactions and immunochemical cross-reactivity between the two systems of peptides. Human gastrin and CCK peptides display, not unexpectedly, extensive homology with corresponding peptides from other species. T h e extent of the species differences has been elucidated by cDNA deduction of the preprogastrin and preproCCK structures for various mammals (Boel et al., 1983; Deschenes et al., 1984; Fuller et al., 1987; Gantz et al., 1990; Gubler et al., 1984; Kang et al., 1989; Lund el al., 1989; Takahashi et al., 1985; Yo0 et al., 1982). Comparison of the sequences is quite

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I

SIGNAL -2 1

1

5

36 37

19

.2!

53 54

73 74 80

-10

MAN : Met -Qln-Arg-Leu-Cys-Val -Tyr -Val -Leu- Ile -Phe-AIa-Leu-Ala PIG : -Ala -His -Val cow : - Ala - His -Leu- Val -Cye-Met-Val -Leu-Val RAT : -proDOG : -Leu -

-Leu-Ala-Ala-P~-Ser-Olu -cy8-Cp-

- Thr -Thr-

10

PIG : COW: RAT : DOG :

-

- His -Leu - Gln - Leu- Gln -

-Ser -Ser -Pro - Val Ala -Pro Arp -Ser-Ser-Pro-Arg-Thr-

-

-Arg-Leu-Qln

~

-Ser-

-Pro

-

Qly -Lyr Gly -Qh -Gly-Ak-

-

u c y .-

30

-

40

MAN : Glu -Leu - Pro- Trp -Leu- Qh - Qln-Qln Gly - Pro- Ala - Ser- His -His PIG : -Pro- His-Glu-AspArg-Leu-Am- Pro cow: -Pro - Leu- Arp - Met- Aan- Arg- LeuRAT : -Gln-Hia-(Yn- Lys -Leu DOG : pro - Hi8 -my -Alp-Leu~

-Leu -Leu-

0

50

MAN : Gly-Pro - P r o - ~ i s - ~ e u - V a-Ala-Asp-Pro-Ser l PIG : - Leu- Ala COW: Aep-Met-LeuRAT : -an-Phe-Ib -LeuDOG : -Qln -Leu -

70

MAN : PIG :

cow: RAT : DOG :

D

an-Gly-Pro-Trp -Leu-Qb-Glu-Glu-Glu-Met -Val -Arg-Pro-Met-Met

-

-

w

Glu-Ala -Tyr -Gly -Trp -Met-Asp-Phe-Qly Arg -Arg Ser-Ala -QIu -Asp-QIu -Am -Glu -Qly -Alp-Oln -Arg-Pro -Glu-Gly-Asp-Gln-ma-Pro Ala -0111 - -Aap-Qln - Tyr - A m -0Iu -Gly -Asp-Qln -Arg-Pro Ala -

FIG. 2. The cDNA-deduced amino acid sequences of preprogastrins from five mammalian species. Boxes indicate established proteolytic processing points-mainly at di- and monobasic sites. Note the preserved primary structures around the active site (residues 68-7 1 ) and processing sites.

300

JENS F. REHFELD A N D WOUTEK W. VAN SOLINGE

Arg Arg Arp2 Arg Arg

Arg Arg-Lys Lys Arg Arg

25 35

Arg2

56

-20

-10

MAN : Met - A m - Ser-Gly-Val -Cys-Leu-Cys-Val -Leu-Met-Ala-Val -Leu-Ala -Ala-Gly - A l a - L e u Thr PIG : -Gly-Leu-Thr-LYs-CYS-Val RAT : 1

20

10

MAN : Gln -Pro-Val -Pro - Pro- Ala-Asp- Pro- Ale-Gly- Ser-Gly -Leu-Glu -Arg -Ala -Glu -Glu -Ala -Pro PIG : -Val-Pro-Ala-Gln Glu -HisRAT : -Val -Val-Glu-Ala-Asp-Pro-Met-Glu -Gln-

-

40

Ala- His -Leu-Gly -Ala -Arg

1 -

PIG : RAT : DOG :

MAN PIG RAT DOG

60

Ale -Pro - Ser- GI) Arg Met-Ser- Ile -Val-Met- Ile Val -Val -Leu-Val - 118 -

0

s

80

Lys Asn-Leu-Gln -Asn-Leu-Asp-Pro-Ser-His Arg Ile -Ser-Asp Arg Asp-Tyr -Met-Gly-Trp : -Ser: -Gly:

:

' 80

O

n

-

MAN : Met-Asp-Phe-Gly Arg-Arg Ser-Ala-Glu -Glu-Tyr-Glu-Tyr-Pro.Ser -Thr PIG : RAT : -Asp-

[II

FIG. 3. The cDNA-deduced amino acid sequences of proprocholecystokinins from three mammalian species. The known canine sequence is also shown. Boxes indicate established proteolytic processing points-mainly at di- and monobasic sites. Note the preserved primary structures around the active site (residues 80-83) and processing sites.

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

30 1

instructive. It shows how well the active site and structures around major processing sites are preserved (Figs. 2 and 3). Ill. Normal Biology

A. GENESAND MRNAs OF GASTRIN AND CHOLECYSTOKININ The single-copy genes for human gastrin and CCK are located on chromosome 17q and chromosome 3 in the region 3q12-3pter, respectively (Lund et al., 1986; Takahashi et al., 1986). The cloning of the human gastrin gene (Ito et al., 1984; Kato et ul., 1983; Wiborg et ul., 1984) and CCK gene (Takahashi et al., 1985, 1986), showed that the genes are structurally similar, both in the overall exon-intron organization and in certain peptide coding sequences. The gastrin gene spans 4.1 kb of chromosomal DNA and contains two introns of 3041 and 130 bp, respectively. Antral G-cells generate a single mRNA of 0.7 kb, which encodes the 101-amino acid preprogastrin (Boel et al., 1983; Wiborg et al., 1984). The first exon encodes the 5’-untranslated region (5’-UTR) (Fig. 4). T h e CCK gene spans 7 kb and contains, like the gastrin gene,

FIG. 4. Structure of the human gastrin gene, rnRNA, and prepropeptide. Exons are shown as boxes, introns as straight lines. The shaded area of the mRNA indicates the coding region. Numbers indicate number of base pairs (bp) or kilobase pairs (kb) in each section of the gene. In preprogastrin the position of gastrin-34 is shown.

302

JENS F. REHFELD A N D WOUTER W. VAN SOLINGE

FIG. 5. Structure of the human cholecystokinin gene, mRNA, and prepropeptide. Exons are shown as boxes, introns as straight lines. The shaded area of the mRNA indicates the coding region. Numbers indicate number of base pairs (bp) or kilobase pairs (kb) in each section of the gene. In preprocholecystokinin (preproCCK)the position of CCK-33 is shown.

two introns, although of -1.1 and -5 kb. T h e I cells in the small intestine express a single mRNA of 0.8 kb, coding for the 115-amino acid preproCCK (Takahashi et al., 1986). Also for CCK, the first exon encodes the 5’-UTR of the transcript (Fig. 5 ) . Gastrin and CCK cDNAs derived from tumor tissues are identical to those isolated from normal tissues (Brand and Wang, 1988; Kariya et al., 1986; Kuwano et al., 1984). Studies on the promoters of the gastrin and CCK genes are of importance, because the differences in expression between normal tissues and tumor tissues is likely to be caused entirely or partly by differences in transcriptional activation of the genes. Haun and Dixon (1990) found several cis-acting elements that are important for the expression of the rat CCK gene in vitro. They also defined the interacting trans-acting factors. Similar studies on the human CCK promoter have not yet been published. Although the regulation of the human gastrin promoter in a human system remains to be reported, several studies have identified important regulatory domains in the human promoter using rodent cell systems. A cell-specific regulatory element has been located in the cap exon I region

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

303

of the human gastrin gene (Theill et al., 1987). Wang and Brand (1990) later identified a pancreatic islet cell-specific regulatory domain in the gastrin promoter, containing adjacent positive and negative DNA elements. T h e authors suggested that the regulatory domain is a switch controlling the transient transcription of the gastrin gene during fetal islet development. Godley and Brand (1989) reported that gastrin gene transcription was stimulated by epidermal growth factor (EGF) and inhibited by somatostatin. Later the EGF-responsive element was identified (Merchant et al., 1991). This element is of particular interest in the tumor biology of gastrin, because EGF/TGF-a is expressed in a variety of carcinoma cell lines derived from the colon (Karnes et al., 1992; Huang et al., 1991; Coffey et al., 1986), ovary (Morishige et al., 1991; Kurachi et al., 1991; Stromberg et al., 1992; Kohler et al., 1992), lung (Imanishi et al., 1988), pancreas (Smith et al., 1987), and stomach (Yasui et al., 1988). Therefore, synthesis of gastrin in tumors may be due to transcriptional activation of the gastrin gene by EGF and TGF-a. T h e transcriptional inhibition by somatostatin is probably mediated by a specific cis regulatory element, located closely to the EGF-responsive element (Brand et al., 1991). I n addition to transcriptional inhibition by somatostatin, Karnik et ul. (1989) have suggested that somatostatin may affect also the posttranscriptional processing of gastrin RNA. So far, no tissue-specific splicing or use of alternative promoters has been described. Therefore, the cell-specificmolecular patterns of gastrin and CCK peptides in different tissues are probably due to differences in the post-translational processing, rather than differential processing o r transcription of the gene (Brand and Fuller, 1988; Friedman et al., 1985; Gubler et al., 1984). However, in addition to the 0.7-kb gastrin mRNA, other transcripts have been found. Northern analysis of colorectal and testicular tissue extracts showed a transcript of -3.3 kb hybridizing with a gastrin probe (Hoosein et al., 1990; Schalling et al., 1990; van Solinge et al., 1993a). It probably represents unprocessed gastrin RNA, including the first intron of the gastrin gene. A minor band of 0.5 kb has also been observed (Schalling et ad., 1990; van Solinge et al., 1993a). The nature of the latter mRNA is uncertain. It may encode a different but homologous peptide. T h e gastrin transcript containing the second 130-bp intron, detected by reverse transcription polymerase chain reaction in colonic cell lines (Baldwin et al., 1990), has now been found to be genomic DNA (Baldwin and Zhang, 1992; van Solinge et al., 1993a). Evidence of translational regulation of CCK and gene expression has been reported by several authors. Gubler et al. (1987) observed a major discrepancy between the levels of CCK mRNA and peptides in the cerebellum. It was later shown that the discrepancy is partly due also to

304

.JENS F. REHFELD A N D

WOUrER W. VAN

SOLINCE

incomplete processing of proCCK (Rehfeld et al., 1992). In other words, expression is in this case regulated both at the translational level and at the post-translational level. Recently, we found two additional cases of translational regulation. Thus, a human gastric carcinoma cell line (AGS) contained CCK mRNA but neither proCCK nor amidated CCK (van Solinge and Rehfeld, 1992). The second observation was that the CCK mRNA concentrations in the rat colon increased after birth, without concomitant peptide synthesis (Luttichau et al., 1993). In the developing rat colon, gastrin mRNA concentrations also increase from birth to adult life apparently without a corresponding increase of peptide synthesis (Luttichau et al., 1993). Therefore, the expression of the gastrin gene also appears to be regulated at the translational level. Comparison of rat, pig, and human cDNA sequences showed remarkably conserved base sequences in the 5‘- and 3’-untranslated regions (Fuller et al., 1987). It is therefore possible that such sequences in the 5’-UTR may determine the translational efficiency of gene expression. The 3’-UTR may be involved in regulation of polyadenylation, termination of transcription, or message stability. Further studies on gastrin and CCK mRNA are necessary to explain these observations. The expression of both the gastrin and the CCK gene are ontogenetically regulated (Bardram et al., 1990; Brand and Fuller, 1988; Friedman et al., 1985; Luttichau et al., 1993; Rehfeld et al., 1992). Consequently, expression of gastrin and CCK genes in tumors may involve regulatory factors that normally are expressed only in fetal life. B. POST-TRANSLATIONAL PROCESSING OF PROGASTRIN I . Antral Progastrin

In normal mammals the antral G cells are the main site of gastrin synthesis (for reviews, see Larsson, 1980; Rehfeld, 1981). The G cells are present also in the proximal duodenum. After antrectomy, the duodenal G cells “antralize” and increase their synthesis considerably (Brodin et al., 1979; Nilsson and Brodin, 1977). Gastrin biosynthesis studies have so far focused on antral tissue (Brand et al., 1984a; Hilsted and Rehfeld, 1987; S. Jensen et al., 1989; Sugano et al., 1985). Combination of the results of these studies with general knowledge about peptide hormone synthesis (for reviews, see Kelly, 1985; Schwartz, 1990) provides a clear picture of the biosynthetic pathway of antral gastrin (Fig. 6): After translation of gastrin mRNA in the rough endoplasmic reticulum (RER) and cotranslational removal of the N-terminal signal peptide from preprogastrin, intact progastrin is transported to the Golgi appara-

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

SIGNAL -2 1

Arg

Arg

Arg-Arg

I

I

I

1 ' 1

5

Lys-Lys

Arg-Arg

I

I

1 I

I

19

36 37

53 54

7374

305

80

ROUGH ENDOPLASMIC RETICULUM: SIQNALASE

-

TRANS-GOLGI APPARATUS AND

TYROSYL-PROTEIN SULFOTRANSFERASE

I

I

I

IMMATURE SECRETORY VESICLES:

TRYPSIN-LIKE ENDOPEPTIDASE

SECRETORY GRANULES:

TRYPSIN-LIKE ENOOPEPTIDASES

1. PROGASTRINS

CARBOXYPEPTDASE E-LIKE EXoPEPTIDASE

,

2. GLYCINE-EXTENDED INTERMEDIATES

-

TRYPSIN-LIKE E + , S E S

T L G L Y C I N E (YAMIDATING MONO'y OXYGENASE

7 Gly Gly

t

3. BlOACTlVE

CONH,

Gastrin.71

7CONH,

Gastrin-34

GASTRINS k

CONH,

Gastrin-17

CONH2

Gastrin-14

CONH?

Gastrin-6

FIG.6. Schematic illustration of the posttranslational processing of preprogastrin in the antral G cell.

tus. In the trans-Golgi the first post-translational modifications occur. These are 0-sulfation of the tyrosyl-66 residue neighboring the active site, and the first of the trypsinlike endoproteolytic cleavages at two monobasic and three dibasic processing sites. From the trans-Golgi network vesicles carry the processing intermediates of progastrin toward

306

JENS F. REHFELD AND WOUTER W. VAN SOLINGE

the basal part of the G cells, where the gastrin peptides are stored in characteristic secretory granules (Hlkanson et al., 1982; Larsson and Rehfeld, 1979a). We assume that the endoproteolytic trypsinlike and exoproteolytic carboxypeptidase E-like processings as well as the subsequent glutamyl cyclization-corresponding to the N-termini of gastrin-34 and gastrin-17 (Figs. 2 and 5)-continue during the transport from the Golgi to the early secretory granules. The last and decisive processing step in the synthesis of gastrin then occurs during storage and maturation in the secretory granules. The secretory granules contain the amidation enzymes (Eipper et ul., 1987; Murthy et at., 1986), which remove glyoxylate from the immediate precursors, the glycineextended gastrins, to complete the synthesis of bioactive a-carboxyamidated peptides (Fig. 6). Amidation of gastrin is a crucial all-or-none activation process, which is carefully controlled (Hilsted et al., 1986, 1988; Hilsted and Rehfeld, 1987; S.Jensen et al., 1989).Activation of the enzymatic amidation process requires copper, oxygen, ascorbic acid, and a pH around 5 (Eipper et al., 1987; for reviews, see Hilsted, 1991; Eipper et al., 1992). Recently, a-amidation of peptides was shown to require two sequentially acting enzymes: a copper- and ascorbate-dependent peptidylglycine a-hydroxylating monooxygenase (PHM), derived from the N-terminal part of the PAM precursor, and a separate peptidyl-ahydroxyglycine a-amidating lyase (PAL), derived from the remaining intragranular region of the PAM precursor (Katopodis et al., 1990; Perkins et al., 1990; Suzuki et al., 1990; Takahashi et al., 1990). Partial phosphorylation of serine in the C-terminal flanking fragment of progastrin also occurs. The significance of the phosphorylation is not yet known and the phosphokinase is unknown (Dockray et al., 1987). As a result of the elaborate biosynthetic pathway, the normal human antral G cells release a heterogeneous mixture of progastrin products from the mature secretory granules. A small percentage are nonamidated precursors, mainly glycine-extended gastrins. But in humans more than 95% are a-amidated bioactive gastrins, of which the longest molecular form is gastrin-7 1. Thus, of the amidated gastrins, 90% are gastrin-17, 5% are gastrin-34, and the rest is a mixture of gastrin-71 and -52, gastrin- 14, and short C-terminal hepta- to tetrapeptide amide fragments (Gregory et al., 1983; Hilsted and Hansen, 1988; S. Jensen et al., 1989; Malmstom et al., 1976; Rehfeld and Larsson, 1979; Rehfeld et al., 1974; Rehfeld and Uvnas-Wallensten, 1978; Yalow and Berson, 1970). Approximately half of the amidated gastrins are tyrosyl-sulfated (Andersen et al., 1983; Andersen, 1985; Brand et al., 1984b; Gregory and Tracy, 1964; Hilsted and Rehfeld, 1987). Due to gross differences in metabolic clearance rates, the distribution of gastrins in peripheral plas-

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

307

ma changes so that larger gastrins with their long half-lives predominate over gastrin-17 and shorter gastrins (S. Jensen et al., 1989). Increased gastrin synthesis changes the molecular pattern further. Abnormally increased antral synthesis occurs in man by achlorhydria, as seen in pernicious anemia. In antrum-sparing pernicious anemia the translational activity of gastrin mRNA in the G cells seems to be so high that the enzymes responsible for the processing of progastrin cannot keep u p with the maturation, i.e., the carboxyamidation process (S. Jensen et al., 1989). Consequently, G cells release more unprocessed and incompletely processed nonamidated progastrin products, when the synthesis is increased. Also, the carboxyamidated gastrins are less sulfated (Borch et al., 1986) and the N-terminus of progastrin is less cleaved (S. Jensen et al., 1989). Precursors, processing intermediates, and longchained carboxyamidated gastrins, such as gastrin-7 1, gastrin-52, and gastrin-34, are, as mentioned, cleared at a relatively slow rate from the circulation and therefore accumulate in plasma when synthesis and release are increased. Therefore, assays that measure progastrin and its products irrespective of the degree of processing provide a better measure of increased gastrin synthesis than do conventional assays, which recognize only the fully processed amidated gastrins. 2. Extraantral Progastrin The gastrin gene is expressed at the peptide level in several cell types other than the antroduodenal G cells. Quantitatively, these other cells contribute very little gastrin in the blood of normal organisms. Partly because the secretion seems to serve local purposes rather than a general endocrine purpose, and partly because the biosynthetic processing is cell specific, i.e., so different from that of the antral G cells, the bioactive amidated gastrin may not even be synthesized. So far we have encountered expression of progastrin and its products outside the antroduodenal mucosa in the distal small intestine, perhaps originating from the socalled tetragastrin (TG) cells (Buchan et ul., 1979; Larsson and Rehfeld, 1979a), in unidentified cells in the colon (Luttichau et al., 1993; van Solinge et al., 1993a),in endocrine cells in the fetal and neonatal pancreas (Bardram etal., 1990; Brand et al., 1984a; Larssonetal., 1976),in pituitary corticotrophs and melanotrophs (Larsson and Rehfeld, 1981; Rehfeld, 1978a, 1986), in oxytocinergic hypothalamo-pituitary neurons (Rehfeld, 1978a, 1986; Rehfeld et al., 1984), in a few cerebellar (Rehfeld, 1991) and vagal neurons (Uvnas-Wallensten et al., 1977), in the adrenal medulla of some species (Bardram and Rehfeld, unpublished results), in the bronchial mucosa (Rehfeld et al., 1989),in postmenopausal ovaria (van Solinge et al., 1993b), and in spermatogenic cells (Schalling et al., 1990). As shown

308

JENS F. REHFELD A N D WOUTER W. VAN SOLINGE

TABLE I EXPRESSION OF THE GASTRIN GENEAT F’EPTIDE LEVEL IN NORMAL ADULT MAMMALIAN TISSUE^' Tissue

Total translation product (pironioles per gram of tissue)

Precursor percentage

Gastrointestinal tract Antral niucosa Duodenal mucosa Jejunal mucosa Ileal niucosa Colonic mucosa

10,000 400 40 20 0.2

5 20 30 85 100

5 8 200 30 2 2

20 10 98 5 100 95

Neuroendocrine tissue Cerebellum Vagal nerve Adenohypophysis Neuroh ypophysis Adrenal medulla Pancreas Genital tract Ovaries Testicles Spermatozoa

0.5 6 2

100 100 55

Respiratory tract Bronchial mucosa

0.3

100

u

Orders of magnitude based on examination of diffcrent inamnialian species

in Table I , the concentrations and presumably also the synthesis in the extraantral tissues are far below those of the antral “main factory.” The precise function of gastrin synthesized outside the antroduodenal mucosa is largely unknown. Several suggestions can be offered. First, an obvious possibility is paracrine or autocrine regulation of growth. Second, it is possible that the low concentration of peptides is without significant function in the adult, but is a relic of a more comprehensive fetal synthesis for local stimulation of growth (Bardram et al., 1990; Luttichau et al., 1993). A third possibility is that the low cellular concentration is due to constitutive rather than regulated secretion (Kelly, 1985). By constitutive secretion there is no storage of peptides in the cells in spite of a considerable release per time unit. Although it is possible that the extraantral synthesis of gastrin is without function in the normal adult organism, recognition of the phenomenon has considerable clinical relevance. Hence, tumors originating from

T U M O R BIOLOGY OF GASTRIN A N D CHOLECYSTOKININ

309

tissues and cells that normally express the gastrin gene even at low level may produce gastrin in lethal amounts.

C. POST-TRANSLATIONAL PROCESSING OF PROCHOLECYSTOKININ 1. Intestinal proCCK

Although normal mammals synthesize most of their cholecystokinin in the central nervous system, an essential portion is synthesized in the I cells o f t h e small intestine (Rehfeld, 1978~). Moreover, almost all CCK in plasma originates from the endocrine I cells of the small intestine. T h e I cells are, however, so disseminated in the intestinal mucosa, and the intensity of the biosynthesis per gram of small intestine therefore so incomprehensive, that dynamic biosynthesis studies have so far been impossible to perform. However, the dynamics of cerebral CCK synthesis have been studied in detail (Goltermann et al., 1980; StengaardPedersen et al., 1984). I n addition, a variety of proCCK-derived peptides of different chain lengths have been identified from extracts of both the small intestine and the brain (Blanke et al., 1993; Dockray et al., 1978; Eberlein et al., 1992; Engetal., 1983, 1984; Mutt, 1976; Mutt and Jorpes, 1968, 1971; Reeve et al., 1986, 1991; Rehfeld and Hansen, 1986). Combination of these results with general knowledge about peptide hormone synthesis (for reviews, see Kelly, 1985; Schwartz, 1990) has provided a picture of the biosynthetic pathway of CCK in intestinal I cells (Fig. 7). After translation of CCK mRNA in the RER and cotranslational removal of the N-terminal pre- o r signal peptide from preproCCK, intact proCCK is transported to the Golgi apparatus. As for other peptide hormones, the first posttranslational modifications occur in the Golgi apparatus, where proCCK is completely tyrosyl O-sulfated in three positions (Tyr-77, -92, and -95). T h e trypsinlike endoproteolytic cleavages at multiple monobasic and one dibasic processing site also begin in the trans-Golgi apparatus, and continue in small vesicles carrying the processing intermediates toward the basal parts of the I cells, where the processing continues in the secretory granules. The last and decisive processing step in the synthesis of bioactive CCK peptides then, as for the gastrins, occurs during storage and maturation in the secretory granules. T h e secretory granules contain the precursor for the two enzymes necessary for amidation (Eipper et al., 1987; Katopodis et al., 1990; Murthy et al., 1986; Perkins et al., 1990), which removes glyoxylate from the immediate precursors, the glycine-extended CCKs, to com-

310

JENS F. REHFELD A N D WOUTER W. VAN SOLINGE Arg-Arg

-20

1

Arg

Arg-Lys

Arg Arg Arg

Arg

Arg-Arg

Arg Lys

Arg Arg

ROUGH ENDOPLASMIC RETICULUM:

TRANS-GOLGI APPARATUS: AND

TYROSYL-PROTEINSULFOTRANSFERASE

:

IMMATURE SECRETORY VESICLES: TRYPSIN-LIKE ENDOPEPTIOASE

SECRETORY GRANULES: TRY PSIN-LIKE ENDOPEPTIDASES

CARBOXYPEPTIDASE

1. PROCCKs PEPTIDYLQLYCINE (ILAMlDATlNQ

I

'Iy

c

'Iy QIY

I

2. GLYCINE-EXTENDED

INTERMEDIATES

7

Q

I

MONOOXYQENASE

y

C-Qly

c I

3. BlOACTlVE CCKs

-

CONHp

CCK-83

CONHp

CCK-58

CONHp

CCK-3Q

7 CONHp

CCK-33

L CON&

CCK-22

+ CONHp

CCK-8

C CONHp

CCK-5

FIG. 7. Schematic illustration of the post-translational processing of preprocholecystokinin in the I cells of the small intestine.

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

31 1

plete the synthesis of bioactive a-carboxyamidated peptides (vide supra and Fig. 7). As a result of the elaborate biosynthetic pathway, the normal intestinal I cells in humans release a heterogeneous mixture of proCCK products from the mature secretory granules. A small percentage are nonamidated precursors. The amidated CCKs constitute a mixture of the longest possible bioactive product of proCCK, i.e., CCK-83 (Eberlein et al., 1992), in addition to the medium-sized CCK-58, -39, -33, and -22 (Eng et al., 1984; Mutt and Jorpes, 1968; Mutt, 1976; Reeve et al., 1986; Rehfeld, 1978b) and the short CCK-8 and CCK-5 (Dockray et al., 1978; Rehfeld and Hansen, 1986; Shively et al., 1987). The distribution and release patterns vary grossly among species (Cantor and Rehfeld, 1989; Eberlein et al., 1988; Rehfeld, 1993).

2. Extraintestinal proCCK The CCK gene is expressed at peptide level in several cell types other than the small intestinal I cells. Entirely predominating are CCK neurons in the brain. Neurons in all regions of the central nervous system synthesize CCK peptides, though cerebellar neurons do so only in the fetal state (Larsson and Rehfeld, 1979b; Mogensen et al., 1990; Rehfeld, 1978c; Rehfeld et al., 1992). The highest expression occurs in neocortical regions, which explains why CCK is the most abundant peptide system in the human brain (Crawley, 1985; Rehfeld, 1978~).CCK peptides are also widely expressed in peripheral neurons, primarily in the intestinal tract, but also in the genitourinary tract and elsewhere (Larsson and Rehfeld, 1979b). Low-level expression has been found also in pituitary corticotrophs (Rehfeld, 1986, 1987), in thyroid C cells (Rehfeld et al,, 1990b), in the adrenal medulla (Bardram et al., 1989), in the bronchial mucosa (Ghatei et al., 1982),and in spermatogenic cells of certain species (Persson et al., 1989). Quantitative measures of the tissue-specific expression are presented in Table 11. D. CELLULAR RELEASEOF GASTRIN AND CHOLECYSTOKININ In order to understand the mitogenic effect of gastrin and CCK peptides, it is necessary to realize that the different types of cells, in which the gastrin and CCK genes are expressed, also release the peptides in different ways (Fig. 8). As mentioned in Section I, secretion of gastrin and CCK were until 15 years ago supposed to be endocrine only, i.e., secretory granules from the endocrine I and G cells in the gastroin-

312

J E N S F. REHFELD A N D WOUTER W. VAN SOLINCE

TABLE I 1 EXPHESSION OF THE CHOLECYSTOKININ GENEAr P W ~ I D E LEVEL I N NORMAL ADuLr MAMMALIAN TISSUE^' Tissue

Total translation product (picomoles per gram of tissue)

Precursor percentage

Gastrointestinal tract Duodenal niucosa Jejunal mucosa Ileal mucosa Colonic mucosa

200 250 20 5

5 20

Neuroendocrine tissue Adenohypophysis Neurohypophysis Thyroid gland Adrenal medulla

25 20 2 1

100 10 20 50

Genital tract Testicles Spermatozoa*

5 -

80

400 350 200 2

2 2

Central nervous system Cerebral cortex Hippocampus Hypothalamus Cerebellum

50 50

-

2 80

Orders of niagnitude based on examination of different mammalian species. Cliolecystokinin peptides are present in spcrniatozoa of nonhuman ntarnmals. The conccntration, however, has not Ixcn qtramitateti. (1

testinal mucosa emptied their peptides into surrounding capillaries after appropriate stimulation. In contrast to the systemic release of gastrin and CCK peptides from classical endocrine cells (Fig. 8A), four alternative routes of secretion to neighboring cells o r to the secretory cell have been discovered during the past 15 years. First, the peptides synthesized in neurons are released, from synaptosomal vesicles in the nerve terminals, to the receptors of adjacent target cells, i.e., neurotransmitter or neurocrine release (Fig. 8B). I t is possible that a spillover of CCK and gastrin released from peripheral neurons may be transported via blood, in analogy with other neuropeptides. It is also possible that some CCK and gastrinergic neurons, for instance the hypothalamo-pituitary neurons, release the peptides directly to blood vessels. Second, in the wake of the discovery of the morphological substrate for paracrine secretion, i.e., short cytoplasmatic processes to neighboring cells (Larsson et al., 1978a), it has been shown that there are gastrin-producing paracrine cells in the small intestinal

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

313

FIG.8. Five types of cellular release of gastrin and cholecystokinin peptides. (A) Endocrine release from hormonal cells to capillaries. (B) Neurocrine release from neurons to the synaptic cleft. (C) Paracrine release to a neighboring cell through short cellular processes. The paracrine cells are often of the closed type, which do not reach the lumen. (D) Autocrine release to receptors on the membrane of the same cell that synthesizes the peptides. (E) Spermiocrine release from the acrosomal granule of a spermatozoon to receptors in the egg cell membrane.

mucosa (Larsson, 1980). These cells carry gastrin granules through cytoplasmatic extensions to specific target cells in the neighborhood. Paracrine cells can be conceived as hybrids of classical endocrine cells and neurons (Fig. 8C). It is consequently possible that also a local spillover of gastrin from paracrine cells may reach the circulation. Self-stimulation, or autocrine secretion, was described first by Sporn and Todaro (1980; see also Cuttita et al., 1985; Hoosein et al., 1990; Sporn and Roberts, 1985). By autocrine secretion, cells stimulate their own growth. Trophic peptides bind to specific receptors in the membranes of cells in which they are also synthesized (Fig. 8D). Autocrine secretion is supposed to play a decisive role in tumor and cancer development (Sporn and Roberts, 1985). T h e first example was small cell bronchogenic cancers, which were stimulated by autocrine secretion of gastrin-releasing polypeptide (GRP) (Cuttita et al., 1985; Layton et al.,

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1988). Recent evidence suggests that the growth of certain cultured bronchial carcinoma cells (Sethi and Rozengurt, 199l), colon carcinoma cells (Hoosein et al., 1988, 1990), gastric carcinoma cells (Weinstock and Baldwin, 1988), and pancreatic tumor cells (Blackmore and Hirst, 1992; Seva et al., 1990; Watson et al., 1992) is stimulated by autocrine secretion of gastrin, and that growth of certain human pancreatic cancers and cell lines is stimulated by CCK peptides (Smith et al., 1991; Townsend et al., 1981). Cellular release of CCK and gastrin peptides also occurs in a fifth way that cannot be ascribed to any of the four above-mentioned ways (Fig. 8E). As already mentioned, spermatogenic cells in mammals other than man express the CCK gene (Persson et al., 1989), whereas human spermatogenic cells express the gastrin gene (Schalling et al., 1990). In spermatozoa, CCK o r gastrin peptides are fully carboxyamidated, and are concentrated in the acrosome. I n accordance with the acrosomal reaction, the peptides are released from the spermatozoon by contact with the jelly coat of the egg, and are subsequently bound to receptors in the egg membrane. So far, CCK or gastrin receptors have not been demonstrated in mammalian egg cell membranes, but availability of the recently cloned CCK and gastrin receptors (Kopin et al., 1992; Wank et al., 1992) makes such demonstration feasible in a foreseeable future. Acrosoma1 release may prove to be the single most important and fundamental mechanism of secretion of gastrin and CCK peptides. We suggest that the release of bioactive peptides from acrosomal granules be designated “spermiocrine release” (Fig. 8E).

E.

RECEPTORS

FOR

GASTRINAND CHOLECYSTOKININ

The development of a procedure for nonoxidative isotope labeling of CCK peptides (Rehfeld, 1978b) paved the way for the first characterization of the CCK receptors (Jensen et al., 1980; Miller et al., 1981; Sankaran et al., 1979; for review, see also Gardner and Jensen, 1989). T h e biological and structural characterizations were greatly advanced by the subsequent design of receptor antagonists, of which the potent and selective benzodiazepine derivative L-364,7 18 and its derivatives were the first available (Chang and Lotti, 1986; Evans et al., 1986). Using these tools, two subtypes of CCK receptors (CCKA and CCK,) have been classified (R. T. Jensen et al., 1989). The CCKA receptors mediate the physiologic gallbladder contraction, pancreatic growth and enzyme secretion, delay of gastric emptying, and relaxation of the sphincter of Oddi (for review, see R. T. Jensen et al., 1989). CCK, receptors have also been found in the anterior pituitary,

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the myenteric plexus, and important areas of the midbrain in which CCK-containing dopaminergic neurons have been implicated in the pathogenesis of schizophrenia and certain other neuropsychiatric diseases (Hokfelt et al., 1980; for review, see Crawley, 1991). T h e CCK, receptor binds with high affinity CCK peptides that are both carboxyamidated and tyrosyl-sulfated. In contrast, nonsulfated CCK peptides and gastrin peptides are bound with low affinity. The CCKA receptor from rat pancreas has been purified and the corresponding cDNA cloned (Wank et al., 1992). The cDNA encodes a 444-amino acid protein containing seven putative transmembrane domains, and thus belongs to the G-protein-coupled receptor superfamily. The CCKA receptor G protein activates phospholipase C, breakdown of inositol phospholipids, mobilization of intracellular calcium, and activation of protein kinase C. Interestingly, the CCKA receptor is also expressed in some pancreatic carcinoma cell lines (Wank et al., 1992) and is even overexpressed in azaserine-induced pancreatic carcinomas in the rat (Bell et al., 1992). T h e CCKB receptor is the predominant CCK receptor of the central nervous system, the “brain receptor”. It is expressed with particularly high density in the cerebral cortex (Saito et al., 1980). T h e CCKB receptor is less selective than the CCKA receptor, as it binds both tyrosylsulfated and nonsulfated CCK peptides as well as gastrins and short C-terminal fragments of CCK and gastrin, with almost similar affinity (Saito et al., 1980; Williams et al., 1986). The CCKB receptor has not yet been purified as a protein, but cDNA encoding the human CCKB receptor was recently cloned and characterized by Lee et al. (1993). Observations on the recently identified gastrin receptor from canine parietal cells (Kopin et al., 1992) strongly suggest that the CCKB receptor is either identical to or highly homologous with the gastrin receptor. Procedures for mild labeling of gastrin peptides of high specific radioactivity (Rehfeld, 1980; Stadil and Rehfeld, 1972) paved the way also for the initial characterization of the gastrin receptor (Soll et al., 1984; for review, see Soll, 1989). The gastrin receptor appears to be only moderately selective, as it binds carboxyamidated gastrin and CCK peptides with essentially similar affinities (Soll, 1989). As mentioned above, gastrin receptor cDNA from a canine parietal cDNA expression library was cloned (Kopin et al., 1992). By Northern analysis it showed a high degree of similarity with the CCK, receptor. Thus, apparently the gastrin/CCK, receptor is abundantly expressed both in the brain and in the stomach. At present we d o not know whether this receptor is expressed in other tissues. T h e canine gastrin receptor is a 453-amino acid protein that also contains seven putative transmembrane domains, and hence presumably belongs to the G-protein-coupled receptor superfamily. Like

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the CCKA receptor it has a number of N-terminal glycosylation sites, several serine and threonine residues for phosphorylation, but no tyrosine phosphorylation site. The gastrin receptor coupled G protein also activates phospholipase C, degrades inositol phospholipids, mobilizes intracellular calcium, and activates protein kinase C (Kopin et al., 1992). Recently, Beinborn et al. (1993) showed that valine-319 in the sixth transmembrane domain of the human gastrin/CCK, receptor determines the specificity for binding of the nonpeptide receptor antagonists. This receptor site for antagonist binding differs from that of agonist binding, as shown also for other peptide receptors (Fong et al., 1993; Gether et al., 1993; Sachais et al., 1993). Hence, allosteric binding of nonpeptide antagonists seems to be a common feature of the G-protein-coupled, seventransmembrane-spanning family of peptide receptors. The identification of the CCKA and gastrin/CCK, receptors has opened new avenues for gastrin and CCK receptor studies. It should soon be possible to answer a number of pertinent questions. For instance, is the low-affinity binding protein characterized by Baldwin et al. (1986) another gastrin receptor? Does a CCK, receptor exist in certain endocrine tissues (Rehfeld, 1992)? Can the CCKA and gastrin receptors account for the binding of CCK and gastrin peptides as described in colorectal, gastric, and bronchogenic carcinomas (Frucht et al., 1992; Ishizuka et al., 1992; Sethi and Rozengurt, 1991; Singh et al., 1985,1986; Staley et al., 1989; Weinstock and Baldwin, 1988)? Do other cancers express CCK, and gastrin receptors? Are there oncoproteins homologous with the CCK and/or gastrin receptors? IV. Tumor Biology

A. GASTRIN EXPRESSION IN TUMORS 1 . Pancreatic Endocrine Tumors

In 1955 Zollinger and Ellison described a syndrome consisting of severe duodenal ulcer disease, massive gastric acid hypersecretion, and endocrine tumors in the pancreas. The syndrome was subsequently shown to be due to hypersecretion of gastrin from pancreatic tumors (Gregory et al., 1960). These tumors, now called gastrinomas, are generally malignant. Although most gastrinomas originate in the pancreas, small gastrin-producing tumors associated with the Zollinger-Ellison syndrome are now being recognized in the duodenum with increasing frequency (Pipeleers-Marichal et al., 1990). A few gastrinomas have been found in the antrum (Larsson et al., 1973; Royston et al., 1972;

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Thompson et al., 1989) and the ovaries (Bardram, 1990a; Boixeda et al., 1990; Bollen et al., 1981; Cocco and Conway, 1975; Heyd et al., 1989; Julkunen et al., 1983; Long et al., 1980; Maton at al., 1989; Morgan et al., 1985; Primrose et al., 1988). Some pancreatic gastrinomas are mixed and contain, in addition to G cells, cells that produce pancreatic hormones such as insulin, glucagon, pancreatic polypeptide (PP), vasoactive intestinal polypeptide (VIP), and somatostatin (Larsson et al., 1978b). Furthermore, neoplastic G cells may additionally express peptides encoded by genes for nonpancreatic hormones, especially the POMC gene (Maton et al., 1986). T h e Zollinger-Ellison (gastrinoma) syndrome occurs with a frequency of one per million inhabitants per year (Jacobsen et al., 1986). However, because gastrinomas should be suspected among the relatively frequent patients with common duodenal ulcer disease andfor diarrhea, the requirement for gastrin measurements is significant; i.e., measurement in approximately 400 patients per million inhabitants per year (Jacobsen et al., 1986). It is important to diagnose and localize gastrinomas at an early stage, even when the symptoms are relatively mild and transient, because most gastrinomas-although often slow growingare malignant (Zollinger and Coleman, 1974). Thus, if not removed they will eventually metastatize and the gastrin synthesis will increase, leading to a fulminant and lethal Zollinger-Ellison disease. The diagnostic problems are considerable, because symptoms and gastrin levels in most early gastrinomas are difficult to distinguish from those of the common chronic duodenal ulcer disease. Fortunately, fulminant ZollingerEllison syndromes are rarely seen today. In Denmark we have found gastrinomas to be the most frequent endocrine tumor in the pancreas (Jacobsen et al., 1986). Insulinomas are slightly less frequent. I n contrast, only a few cases of pancreatic glucagonomas, somatostatinomas, VIPomas, and PPomas have been encountered during the last decade in spite of intensive search. Insulin, glucagon, somatostatin, PP, and VIP are, however, synthesized in the normal human pancreas, where gastrin hitherto could not be detected. This so-called gastrinoma enigma has now been solved by the recent demonstration of low-level expression of progastrin, which is not processed to bioactive gastrin in the normal adult human pancreas (Bardram et al., 1990). Therefore, pancreatic gastrinomas cannot be considered ectopic. Interestingly, however, mature carboxyamidated gastrins were expressed in significant quantities in the fetal pancreas (Bardram et al., 1990; Brand et al., 1984b; Brand and Fuller, 1988; Larsson et al., 1976). T h e reappearance of mature gastrin in pancreatic tumors and in

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a pancreatic cell line that grows under the influence of gastrin (Blackmore and Hirst, 1992) indicates that gastrin may have local oncofetal importance as a pancreatic growth factor. As mentioned above, prospective clinical-biochemical studies in a small country (Denmark) showed gastrinomas and insulinomas to be the most frequent endocrine tumors in the pancreas. Retrospective immunohistochemical examinations of tumors collected by surgery o r autopsy often reveal a different pattern of distribution, where “nonfunctioning” tumors predominate and insulinomas occur more frequently than gastrinomas (Heitz et al., 1982; Klijppel and Heitz, 1988; Larsson et al., 1975; Mukai et al., 1982). It remains to be shown whether the clinically and functionally “silent” tumors secrete either new hormones of unknown function or inactive precursors of known hormones. Per definition such tumors are nearly impossible to detect in prospective clinical studies. Their true incidence shall therefore remain obscure until the secretory product(s) become known.

2. Pancreatic Exocrine Tumors Apart from pancreatic G cell carcinomas (gastrinomas) and mixed endocrine pancreatic tumors, little is known about gastrin or CCK gene expression in pancreatic tumors (for example, the common pancreatic adenocarcinomas), which generally have been supposed to be without hormone production. Recently, however, nonneoplastic pancreatic tissue from patients with adenocarcinomas of papilla vateri was found to contain a significant concentration of both progastrin and bioactive carboxyamidated gastrins (Bardram, 1990b). These data suggest that paracrine or autocrine synthesis and secretion of gastrin (and/or perhaps CCK) may occur in pancreatic adenocarcinomas. This was supported by the recent finding of bioactive gastrin in pancreatic cell lines that were susceptible to gastrin (Blackmore and Hirst, 1992), and by the demonstration of significant expression of gastrin/CCK, receptors in azaserine-induced adenocarcinomas in the rat pancreas (Zhou et al., 1992). Thus, evidence for an essential local role of gastrin in the development and growth of exocrine pancreatic cancers is rapidly accumulating. Further examination of this problem is certainly worthwhile, particularly in the light of the high frequency of occurrence of these cancers (they are the fifth most common cancer in the western world) and in view of their sombre prognosis. It is not surprising that pancreatic cancer is called “the greatest challenge in oncology” (Williamson, 1988). 3 . Colorectal Tumors Although gastrin synthesis in the relatively rare pancreatic and duodenal gastrinomas is well established, expression of the gastrin gene in

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319

common colorectal carcinomas has been a topic of much debate. The significance of gastrin synthesis in colon cancers is unsettled. The high frequency and poor prognosis of colorectal carcinomas have, however, drawn attention to the possibility that gastrin might be an autocrine/paracrine growth factor in the normal colonic mucosa and in colorectal malignancies. The interest began 20 years ago, when Johnson et al. (1969; Johnson and Guthrie, 1974a,b, 1978)demonstrated that pentagastrin (a synthetic analog of gastrin and CCK) stimulates growth of colonic mucosal cells in rats. Later it was shown that pentagastrin and gastrin stimulate the growth of colorectal cell lines and experimental tumors (Elwyn et al., 1985; Hoosein et al., 1990; Imdahl et al., 1989; Karlin et al., 1985; Kusyk et al., 1986; Lamote and Willems, 1988; McGregor et al., 1982; Singh et al., 1986; Sirinek et al., 1985; Sumiyoshi et al., 1984; Watson et al., 1989; Winsett et al., 1986). Unfortunately the issue became confused due to lack of distinction between local paracrine/autocrine secretion and the massive secretion of gastrin from the antral mucosa. In other words, several groups have studied the correlation between gastrin concentrations in plasma (of mainly antral origin), growth of the colorectal mucosa, and the occurrence of colorectal cancer without taking local colorectal gastrin synthesis into account (Creutzfeldt and Lamberts, 1991; Elsborg and Mosbech, 1979; Graffner et al., 1992; Hikanson et al., 1986, 1988; Seitz et al., 1991; Smith et al., 1989; Suzuki et al., 1988; Talley et al., 1989; Wong et al., 1991; Yapp et al., 1992). So far, the results have been conflicting. Some groups have found a positive correlation with hypergastrinemia in both benign and-more pronounced-malignant tumors (Seitz et al., 1991; Smith et al., 1989; Wong et al., 1991). In a few cases the hypergastrinemia was reduced after tumor-resection (Wong et al., 1991). Others have not been able to demonstrate changes of the circulating gastrins in clinical or experimental colorectal cancers (Graffner et al., 1992; Kikendall et al., 1992; Suzuki et al., 1988; Yapp et al., 199,Z).Obviously, examination of large groups of patients before and after surgery is required. Moreover, it is necessary to evaluate the origin of the hypergastrinemia that may occur in patients with colorectal cancer. The hypergastrinaemia might be due to coincidental hypo- or achlorhydria in the stomach, because it is unlikely that colorectal cancers and their metastases can release gastrin in amounts sufficient to explain the hypergastrinemia. But perhaps some carcinomas secrete one or more factors that stimulate antral gastrin secretion. The role of gastrin as a local growth factor for the colonic mucosa and in particular for colonic carcinomas has recently attracted further interest. Because autocrine mechanisms require synthesis of the functional

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SOLINGE

gene product by the cells of interest (Sporn and Roberts, 1985), reports have focused on detection of gastrin gene expression in colonic cell lines. Hoosein et al. (1990) found gastrin/CCK immunoreactivity in growth media of colon cell lines, but unfortunately unspecific antibodies were used. Gastrin mRNA has been demonstrated by polymerase chain reaction in a number of colonic cell lines (Baldwin et al., 1990; Baldwin and Zhang, 1992), but no transcripts could be detected using Northern analysis, and no peptide data were reported. Expression of the gastrin gene in normal colonic tissue and in solid human adenocarcinomas and carcinoma cell lines was recently examined in more detail (van Solinge et al., 1993a). The results were unambiguous. Both normal adult tissue and cancer cells synthesized progastrin-although at a low level (Tables I and 11). The posttranslational maturation of progastrin, however, was incomplete. Hence, bioactive, carboxyamidated gastrin was undetectable (van Solinge et al., 1993a) (Fig. 9). Similar results have been published also by Kochman et ad. (1992) and Nemeth et al. (1993). Interestingly, the fetal rat colon synthesizes considerable amounts of bioactive, amidated gastrin (Luttichau et ul., 1993). Thus, it is possible that gastrin is expressed as a local growth factor in colonic mucosa early in development. In the adult colon (normal as well as neoplastic), the synthesis of bioactive, amidated gastrins appears to be almost completely switched off. Consequently, colorectal adenocarcinomas hardly contribute to the hypergastrinemia occasionally encountered in these patients. The results, however, do not

Human Ovarian Bronchogenic Colon antrum carcinomas carcinomas carcinomas FIG.9. Fractions of carboxyamidated, bioactive gastrins (hatched areas) versus biologically inactive processing intermediates and progastrins (open areas) in extracts of common human cancers. For comparison, similar fractions of the total translation product in extracts of the normal human antral mucosa are also shown (first column).

TUMOR BIOLOGY OF GASTRIN AND CHOLECYSTOKININ

32 1

exclude the possibility of more comprehensive colonic gastrin synthesis in a subpopulation of colon cancer patients. Taken together, gastrin gene expression in colorectal mucosa and the mitogenic effect of gastrin suggest that locally released gastrin in some instances may contribute to the development of colorectal cancer. At which stage gastrin may play a role in the “multiple hit” hypothesis of colonic cancer development (for reviews, see Fearon and Vogelstein, 1990; Powell et al., 1992; Weinberg, 1989) is so far unknown. A tempting suggestion is in the hyperproliferative epithelium, stage I. T h e human gastrin gene is, as earlier mentioned, located on chromosome 17q (Lund et al., 1986), the same chromosome that harbors the p53 gene, which so often is mutated and deleted in colorectal tumors (Baker et al., 1989). Although p53 is located on 17p, it may be rewarding to look for possible abnormalities in the gastrin gene in colorectal carcinomas, especially because the number of Alu repeat sequences (nine) in the gastrin gene and its surrounding region is unusually high. Gene rearrangement can be expected to occur because of the high density of the repetitive DNA sequences, which can be used as “hot spots” of recombinations (Kariya et al., 1986). However, no mutations or other genetic alterations in the gastrin gene have been reported thus far. 4. Bronchial Tumors

A few years ago we found progastrin in each tumor in a series of 17 bronchogenic carcinomas, including all histological classifications (Rehfeld et al., 1989). T h e degree of progastrin processing varied considerably from tumor to tumor without relationship to any particular class of carcinoma (small cell, large cell, squamous cell, etc.). Generally, the level of synthesis was low in comparison with that of the antral mucosa (Tables I and 111). In analogy with colorectal carcinomas, it is possible, however, that locally synthesized gastrin in some bronchial carcinomas may have influenced tumor growth. Hence, Staley et al. (1989) and Sethi and Rozengurt ( 1991) have found receptors resembling the CCK,/gastrin receptor associated with growth in some small cell bronchogenic carcinoma cell lines. Gastrin may therefore play a role in a subset of lung cancers. Examination of larger samples is now necessary to determine the extent to which gastrin is of significance in pulmonary carcinogenesis. 5 . Ovarian Tumors

A few cases of the Zollinger-Ellison syndrome have been reported to be due to excessive gastrin synthesis from ovarian mucinous adenocarcinomas (case reports by Bardram, 1990a; Boixeda et al., 1990; Bollen et

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TABLE I11 EXPRESSION OF THE GASTRIN GENEAT PEPTIDELEVELIN TUMORS (RANGE) Total translation product (picomoles per gram of tissue)

Tumor”

Precursor percentage

Acoustic neurornash (14119)

0.2-10

10-100

Pituitary adenomas (12187)

0.5-200

20-90

Bronchogenic carcinomasb (17117)

0.2-20

25- 100

Pancreatic gastrinornas (27127)

400-2,500,000

15-90

Pheochrornocytomasb (13113)

0.2-400

90- 100

Colorectal carcinomas ( I21 12)

0.5-2.0

85- 100

Ovarian carcinomas* (16116)

0.8-25

10-50

~~~~

~~

~

~

~

Numbers in parentheses indicate the number of tumors with detectable gastrin expression in relation to total number of tumors examined. * Tumor type in which carhoxyamidated, bioactive gastrin occurs only as gastrin-17. 0

al., 1981; Cocco and Conway, 1975; Heyd et al., 1989; Julkunen et al., 1983; Long et al., 1980; Maton et al., 1989; Morgan et al., 1985; Primrose et al., 1988). In order to see how common the expression of gastrin is, we recently examined a series of ovarian cancers and found significant amounts of gastrin in all tumors, whereas normal adult ovarian tissue contained only trace concentrations of gastrin peptides (van Solinge et al., 1993b). In contrast to the bronchogenic and colorectal carcinomas, ovarian progastrin was to a high degree processed to bioactive, carboxyamidated gastrins in the malignant tumors (Table I11 and Fig. 9). Hence, the malignant ovarian tumors appear-like the pancreatic tissue-to be well equipped with the processing enzymes necessary for maturation of progastrin (Figs. 6 and 9). Lack of appropriate processing enzymes and/or cofactors may explain why Zollinger-Ellison syndromes have so far never been reported in association with bronchogenic and colorectal carcinomas.

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6. Gastric Tumors In a few cases gastrinomas have been found in the antral mucosa, occasionally associated with other endocrine disturbances such as nesidioblastosis (Larsson et al., 1973; Royston et al., 1972). It is an enigma why antral gastrinomas are so exceedingly rare in comparison with pancreatic and duodenal gastrinomas, because the antral mucosa contains far more G cells than do the duodenum and pancreas. Hypergastrinemia occurs, however, also in association with common gastric carcinomas (den Hartog et al., 1988; Pawlikowski et al., 1989; RakiC and MiliEeviC, 1991).The degree of hypergastrinemia is moderate, although higher in patients with the intestinal type of gastric carcinoma than in patients with the diffuse type (Rakid and MiliteviC, 1991). It is likely that the hypergastrinemia mainly is of antral origin, and that it is a consequence of the achlorhydria often found in patients with gastric cancers. However, some human gastric cancer cell lines do in fact express the gastrin gene at both the mRNA and peptide levels (van Solinge and Rehfeld, 1992),and growth effects of gastrin have been reported on gastric cell lines (Ishizuka et al., 1992; Watson et al., 1992). Therefore, it is possible that neoplastic cells in some gastric cancers release gastrin and that gastrin acts as an autocrine growth factor, because gastric epithelial cells have been assumed to be well equipped with gastrin receptors (however, see also Mezey and Palkovits, 1992).

7. Neuroendocrine Tumors In addition to the common carcinomas mentioned above, the gastrin gene is also expressed at the peptide level in an array of other tumors (Table 111), but these tumors are without the symptoms of the ZollingerEllison syndrome and hypergastrinemia. So far we have found low-level expression of gastrin peptides in some brain tumors [acoustic neuromas (Rehfeld el al., 1990a)], in the pituitary Nelson and Cushing tumors (Bardram et al., 1987), and in pheochromocytomas (Bardram et al., 1989). There are several factors that explain the lack of hypergastrinemia and Zollinger-Ellison symptoms in these tumor types. First, the level of expression is often so low (Table 111) that gastrins released from the tumors do not contribute significantly to the concentration of gastrin in plasma. Second, often only a small fraction of the progastrin is processed to bioactive gastrins, although as shown in Table 111, the range of processing varies considerably with each individual tumor. Third, the tumors may synthesize other peptides or substances that interfere with and inhibit gastric acid secretion. However, even though the secretion of

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SOLINGE

gastrin from these neuroendocrine tumors is too small to increase the concentration of bioactive, amidated gastrins in plasma significantly, the expression of gastrin may have tumorigenic significance. Local autocrine secretion may stimulate growth of tumor cells equipped with receptors for gastrin (Beauchamp et al., 1985; Hoosein et al., 1988, 1990; Imdahl et al., 1989; Singh et al., 1986; Weinstock and Baldwin, 1988). Moreover, it is possible-in some instances even likely-that the release of inactive precursors may contribute to the concentrations of progastrin in plasma. Hence progastrin may serve as a tumor marker (Bardram, 1990a; Hilsted and Rehfeld, 1986; Rehfeld et al., 1989; van Solinge and Rehfeld, 1990). Progastrin concentrations in plasma also show promise as a prognostic indicator of the degree of malignancy of gastrinomas (Bardram, 1990a).

B. CHOLECYSTOKININ EXPRESSION I N TUMORS In contrast to gastrinomas and the Zollinger-Ellison syndrome, so far no CCK-producing tumors (“CCKomas”) have been found that synthesize CCK peptides in amounts sufficient to cause clinical syndromes. If CCKomas accompanied by hyperCCKemia exist, they should, based on the actions of CCK, be associated with symptoms such as pancreatic hypertrophia, persistent gallbladder contraction, diarrhea, gastric acid hypersecretion with ensuing duodenal ulcers, anorexia, and perhaps anxiety. ‘The unsuccessful search for CCKomas has been hampered by the worldwide scarcity of reliable assays for measurement of CCK in plasma (for review, see Rehfeld, 1984). Unfortunately, development of sufficiently sensitive and specific CCK assays has proved extremely difficult. Although the existence of proper CCKomas thus remains an open question, low-level expression of the CCK gene has been detected in a number of different carcinomas and benign tumors (Table IV). None of these have, however, been accompanied by hyperCCKemia.

I. Gastrointestinal Tumors A cell line (AGS) from a human gastric adenocarcinoma, which contained significant amounts of gastrin mRNA and progastrin peptides, also expressed the CCK gene at the mRNA but not at the peptide level (van Solinge and Rehfeld, 1992). The observation is interesting, because simultaneous expression of gastrin and CCK peptides in the same cell has thus far never been found, and because such lack of coexpression has been assumed to be regulated at the transcriptional level. Consequently, the AGS cell line shows that differentiation of expression in homologous peptide systems may be regulated at the translational level.

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TABLE IV EXPRESSION OF THE CHOLECYSTOKININ GENEAT F'EPTIDE LEVELI N TUMORS (RANGE) ~

Tumor"

Total translation product (picomoles per gram of tissue)

Precursor percentage

Acoustic neuromasD ( 1 4/ 19)

0.5- 10

30- 100

Gliomash ( 1 2/ 12)

0.2-10

70- 100

Pituitary adenomash ( 1 7/87) Medullary thyroid carcinomasb (16/ 16) Pheochromocytomas'J ( 1 2/13)

5-15,000

10-40

0.5-30

15-80

2-50

10-70

u Numbers in parentheses indicate the number of tumors with detectable cholecystokinin expression in relation to total number of tumors examined. h Tumors showing reduced tyrosine sulfation of cholecystokinin peptides (see also Fig. 10).

In contrast to the AGS cell line, CCK peptides have been detected by specific immunocytochemistry in a few cells in a duodenal carcinoid tumor (C. Bordi, personal communication), but, otherwise, there have been no reports of CCK synthesis in gastrointestinal tumors. 2 . Bronchial Tumors Whereas solid human bronchogenic carcinomas, including small cell carcinomas, expressed the gastrin gene without traces of CCK (Rehfeld et al., 1989), a human small cell carcinoma cell line, U1690, was recently found to express the CCK gene without traces of gastrin mRNA or gastrin peptides (Geijer et al., 1990). The cell line even processed proCCK to bioactive amidated CCK peptides. The discrepancy may reflect that bronchogenic carcinomas are mixed, so that some express gastrin and a few others only CCK, and that the number of tumors and cell lines examined so far is small. Alternatively, the cell line result is unique, because the environment of cell lines differs greatly from that of solid cancers in vivo. Some cell lines from human small cell lung carcinomas apparently are equipped with CCK, receptors (Staley et al., 1989; Sethi and Rozengurt, 1991), thus it is possible that CCK peptides occasionally contribute to the development of bronchogenic cancer by autocrine mechanisms.

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3. Neuroendocrine Tumors

There is a widespread expression of CCK in a large number of neuroendocrine tumors. I n accordance with the normal pituitary corticotrophic expression of proCCK, corticotrophic Cushing and, in particular, Nelson tumors contain substantial amounts of CCK peptides (Rehfeld et al., 1987, and Table IV). The corticotrophic processing of proCCK is, however, incomplete, with a grossly reduced degree of tyrosyl-0-sulfation (Rehfeld, 1987) (Fig. 10). Because tyrosyl-sulfation is essential for the specific biological activity of CCK, corticotrophic Nelson tumors are, in spite of substantial synthesis, functionally silent with respect to CCK (Rehfeld, 1987; Rehfeld et al., 1987, and Fig. 10). Medullary thyroid carcinomas of both human and rat origin also express the CCK gene (Deschenes et al., 1984; Rehfeld et al., 1990b; @durn and Rehfeld, 1990). Although the rat carcinoma cell line contains abundant tyrosyl sulfotransferase activity (@durn and Rehfeld, 1990), CCK peptides in human medullary thyroid carcinomas are poorly sulfated (Rehfeld et al., 1989). Medullary thyroid carcinomas in humans are therefore also silent with respect to CCK (Fig. 10). Pheochromocytomas synthesize a wide range of neuropeptides, including CCK (Bardram et al., 1989). In contrast to the Nelson tumors and medullary thyroid carcinomas, pheochromocytomas are so well equipped with tyrosyl sulfotransferase that proCCK and its products are more completely sulfated (Bardram et al., 1989), and consequently bio-

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$f

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0.6

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530 3%

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0.4-

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Human Acoustic cerebral neuromas adenomas cytomas thyroid cortex carcinomas FIG.10. Fractions of tyrosyl 0-sulfated cholecystokinins (hatched areas) in extracts o f neuroendocrine tumors. For comparison, similar fractions of carboxyamidated cholecystokinins in extracts of normal human cerebral cortex are also shown (first column).

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logically active in relation to CCKAreceptor-containing tissues. The level of expression is, however, so modest that hyperCCKemia has never been observed in patients with pheochromocytomas (Table IV). There is a single report about CCK gene transcription in a solid human pancreatic glucagonoma (Philippe et al., 1988), but CCK peptide secretion has so far never been found in solid pancreas tumors (Rehfeld et al., 1988). In cell lines derived from an experimentally induced rat islet cell tumor there was, however, a substantial CCK peptide synthesis (Madsen et al., 1986). Moreover, transplantable glucagonomas cloned after further passages of this islet cell tumor released a mixture of glucagon and CCK peptides, and subsequent implantation into nude mice induced lethal anorexia (Madsen et al., 1993). Because CCK induces satiety in most mammals, it is likely that CCK contributes to the cachectic anorexia in the mice (Madsen et al., 1993). Perhaps the combination of CCK and glucagon is particularly anorectic. Recently, expression of both CCK mRNA and proCCK was found in cell lines derived from pediatric tumors such as neuroepitheliomas, Ewing sarcomas, and rhabdomyosarcomas (Friedman et al., 1992). Only one neuroepithelioma cell line matured proCCK to bioactive amidated CCK-8. The findings suggests that measurement of the CCK expression may serve as a marker in classification of pediatric tumors. Moreover, measurement of CCK peptides may also be of use in the control or treatment of these malignant tumors.

4 . Cerebral Tumors Because the brain is the main production site of CCK, expression of CCK in brain tumors is an obvious possibility. Examination of common gliomas and astrocytomas has so far revealed only low-level expression of CCK peptides (C. Kruse-Larsen and J. F. Rehfeld, unpublished data). It has not been possible to attribute symptoms of these tumors to CCK release. AS TUMOR GROWTH FACTOR C. GASTRIN

Gastrin has trophic effects on the normal gastric and colorectal mucosae (Johnson, 1976; Johnson and Guthrie, 1974a,b, 1978), and several normal gastric and colorectal mucosal cells apparently express gastrin receptors (Baldwin et al., 1986; Kopin et al., 1992; Sol1 et al., 1984). It is consequently possible that the growth of some gastric and colorectal carcinomas is influenced by gastrin. Accordingly, recent in vitro studies have indicated that gastrin may promote growth of several gastric and

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W. VAN

SOLINGE

colorectal carcinomas (Beauchamp et al., 1985; Borch et al., 1987; Imdahl et al., 1989; Kobori et al., 1982; McGregor et al., 1982; Singh et al., 1986; Sirinek et al., 1985; Watson et al., 1989). Different types of studies have been performed to elucidate the effect of gastrin on tumor growth. First, the effect of exogenously administered gastrin has been examined by evaluating the i n vitro effect either on tumor cell lines, on the growth of tumor cells transplanted into suitable animal models, or on the development of malignant changes induced i n vitro by carcinogens (Beauchamp et al., 1985; Imdahl et al., 1989; Kobori et al., 1982; Moyer et al., 1986; Singh et al., 1986; Sirinek et al., 1985; Sumiyoshi et al., 1984; Tahara and Haizuka, 1978; Tatsuta et al., 1985; Watson et al., 1989; Weinstock and Baldwin, 1988). Second, some studies have examined the effect of manipulation of endogenous gastrin secretion on the growth of transplanted tumors or the development of neoplastic changes by carcinogens (Karlin et al., 1985; McGregor et al., 1982; Tahara et al., 1982; Tatsuta et al., 1977, 1985). Changes of endogenous gastrin secretion and plasma concentration have been achieved by feeding of specific nutrients or by surgical interventions on the stomach and/or the small bowel. Third, the effect of growth-inhibiting hormones such as somatostatin, secretin, and CCK on tumor development in the models mentioned above has also been studied (Elwyn et al., 1985; Kobori et al., 1982; Moyer et al., 1986; Tahara and Haizuka, 1978; Yasui et al., 1986). Fourth, the effect of specific gastrin receptor antagonists has been examined in the various models (Beauchamp et al., 1985; Hoosein et al., 1988, 1990; Imdahl et al., 1989). Finally, the occurrence and biochemical nature of gastrin binding to cancer cell membranes have been characterized (Chicone et al., 1989; Hoosein et al., 1988; Singh et al., 1986; Upp et al., 1989; Weinstock and Baldwin, 1988). With the recently cloned gastrin receptor cDNA at hand (Kopin et al., 1992), more definite characterization of the gastrin binding to cancer cell membranes is now feasible. In other words, is the binding caused by a receptor (or binding protein) identical to, homologous to, or different from the gastrin receptor in normal stomach cell membranes? Several studies have shown that gastrin stimulates the growth of rat and human gastric cancer cells i n vitro and promotes the growth of rat and human gastric cancer transplanted into the nude mouse (Kobori et al., 1982; Moyer et al., 1986; Sumiyoshi et al., 1984; Yasui et al., 1986). Less convincing are the reports on the effect of gastrin on gastric carcinogenesis induced by carcinogens. It has been reported (Tahara and Haizuka, 1978) that administration of gastrin increased the growth of scirrhous gastric cancer in rats produced by N-methyl-N’-nitro-Nnitrosoguanidine (MNNG). Others, however, were unable to demon-

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strate such an effect of gastrin in the same model (Deveny et al., 1980; Tatsuta et al., 1977). It is, however, possible that the timing of gastrin administration is of importance and that differences in the administration can explain some of the earlier discrepancies (Tahara et al., 1982). It has recently been shown that long-term hypergastrinemia induces development of specific carcinoid tumors in the stomach of some rats (Carlsson et al., 1986; Mattsson et al., 1991; for review, see Waldum et al., 1992). Because these tumors arise from entero-chromaffin-like (ECL) cells in the gastric body, they are named ECLomas. The marked hypergastrinemia in these experiments was secondary to achlorhydria induced by long-term administration of high doses of the antisecretory drug, omeprazole. The development of gastric carcinoids was preceded and accompanied by ECL cell hyperplasia. When hypergastrinemia was prevented by antrectomy, omeprazole induced neither ECL cell hyperplasia nor development of carcinoid tumors (Sundler et al., 1986). In this respect it is interesting to note that the occurrence of ECL cell hyperplasia and gastric carcinoids is extremely rare in normogastrinemic subjects, but occurs more regularly in hypergastrinemic patients with pernicious anemia (Borch et al., 1987; Larsson et al., 1978b). Moreover, the degree of hypergastrinemia correlates with the degree of ECL cell proliferation (Borch et al., 1986). Gastrinoma-induced hypergastrinemia also stimulates ECL cell growth. Interestingly, however, malignant transformation of ECLomas occurs only in patients in which the gastrinoma is part of a multiple endocrine neoplasia type 1 (MEN-1) syndrome. Thus, malignant ECLomas require at least both hypergastrinemia and deletions or mutations of the MEN- 1 gene on chromosome 11 (C. Larsson et al., 1988; Bystrom et al., 1990; Pipeleers-Marichal et al., 1990). T h e conception of the MEN-1 gene as a tumor suppressor gene is in full agreement with the observation on the occurrence of malignant ECLomas. Gastrin seems to promote the in vitro growth not only of gastric cancers, but also of colorectal cancers in humans and rats. Furthermore, exogenous gastrin stimulates growth of transplanted mouse and rat colon cancer in mice (Beauchamp et al., 1985; McGregor et al., 1982; Sirinek et al., 1985; Watson et al., 1989; Winsett et al., 1986). T h e stimulation of colon carcinomas is accompanied by decreased survival due to accelerated tumor growth (Winsett et al., 1986). However, studies on the effect of gastrin on the growth of colon cancer induced by carcinogens are conflicting. One study found increased tumor concentration of DNA, RNA, and protein in gastrin-treated rats after previous tumor induction with methylazoxymethanol (McGregor et al., 1982), whereas another group (Tatsuta et al., 1985) could not show that gastrin potentiates the growth of colon cancer induced by rectal instillation of MNNG.

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Endogenous hypergastrinemia after antral exclusion or small bowel resection in rats has been shown to increase DNA synthesis in colon tumors or increase the incidence of tumors in carcinogen-treated rats (Elwyn et al., 1985; Karlin et al., 1985). The rat pancreatic acinar cell-line, AR4-2J, possesses both CCKA and gastrin/CCKB receptors (Lambert et al., 1991; Scemama et al., 1987, 1989). Recent studies showed that gastrin stimulates the growth of the AR4-2J cell line (Blackmore and Hirst, 1992; Watson et al., 1992; Seva et al., 1990). Because the AR4-2J cells also synthesize and release amidated gastrins (Blackmore and Hirst, 1992), gastrin seems to act as an autocrine growth factor in that particular cell system. The molecular mechanisms by which gastrin acts as growth factor in tumor cells are unclear. Moreover, it is unknown whether gastrin expression is associated with cell transformation. Thus, it remains to be settled whether the gastrin gene, the receptor gene, or genes encoding proteins of the gastrin signal transduction pathway are oncogenes-or perhaps tumor suppressor genes.

D. CHOLECYSTOKININ AS TUMOR GROWTH FACTOR CCK peptides are well established trophic factors in pancreatic growth (for reviews, see Johnson, 1976, 1989). It is therefore less surprising that CCK peptides also stimulate growth of pancreatic cancer cell lines and experimental pancreatic cancers (Andrkn-Sandberg et al., 1984; Chu et al., 1993; Heald et al., 1992; Howatson and Carter, 1985; Smith et al., 1990a,b, 1991; Townsend et al., 1981). Whether the result of exogenous administered CCK peptides can be extrapolated to the in vivo development of human pancreatic carcinomas is unknown. In other words, we do not know if CCK peptides are expressed in pancreatic cancers to act as autocrine/paracrine growth factors. Elevated plasma CCK concentrations in patients with pancreatic carcinomas have not been reported. With specific and sensitive CCK assays at hand, examination of the CCK concentrations in human pancreatic carcinomas and corresponding plasma is an obvious task. In a preliminary study, we have in fact found normal CCK concentrations in plasma from patients with pancreatic cancer (J. P. Giitze and J. F. Rehfeld, unpublished observations). In the rat and hamster, experimental hyperCCKemia is induced by pancreaticobiliary diversion, and the hyperCCKemia stimulates pancreatic growth (Chu et al., 1992; Gasslander et al., 1990). Interestingly, the growth of experimental pancreatic cancers is grossly exaggerated by hyperCCKemia (Chu et al., 1993). On the other hand, administration of a specific CCKA receptor antagonist (L-364,718) reduced the growth of

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33 1

transplantable pancreatic adenocarcinomas in hamsters only moderately (Shivaram et al., 1992). However, Shivaram et al. did not exclude the possibility that their pancreatic carcinomas expressed CCK, receptors in addition to or rather than C C K A receptors (Zhou et al., 1992). The pancreas is the only organ in which CCK peptides are known to regulate growth. However, because pentagastrin is not only an analog of gastrin, but also of CCK, it is possible that many tissues and organs stimulated by pentagastrin are stimulated also by CCK. Thus, it is possible that CCK peptides may contribute to the carcinogenesis of gastric, colorectal, and, for instance, medullary thyroid carcinomas. Although such suggestions are hypothetical, the tools are now at hand for proper examination of the ideas. In view of the frequency and depressive prognosis of pancreatic and gastrointestinal carcinomas, this CCK hypothesis deserves evaluation.

V. Requirement for Gastrin and Cholecystokinin Measurements in Oncology As already mentioned, measurement of gastrin in plasma has so far had a minor but well-defined place in the diagnosis, localization, and control of the therapy of gastrinomas associated with the ZollingerEllison syndrome. In order to comply with this purpose, measurements in plasma from approximately 400 patients per million people per year have been necessary (Jacobsen et al., 1986). In addition, gastrin measurements are useful in screening for pancreatic and/or duodenal gastrinomas associated with the multiple endocrine neoplasia type 1 syndrome (Oberg et al., 1982; Shepherd, 1991). In this disease, an autosomal dominant disorder characterized by tumors of the parathyroids, the endocrine pancreas, and the anterior pituitary (Wermer, 1954), the involvement of pancreatic tumors is considered most life-threatening (Oberg et al., 1982). Since Lamers et al. (1988) showed that gastrinomas are the only disorder in MEN-1 accounting for abnormal gastrin secretion, measurement of serum gastrin concentrations is important in the screening, treatment, follow-up, and counseling of the members of families with MEN-1 (Shepherd, 1991). It has since been shown that the gastrinomas are not necessarily localized to the pancreas, but in the MEN-1 syndrome often occur in the duodenum. These tumors may be so small that they easily escape detection (Pipeleers-Marichal et al., 1990). A relatively new oncological indication for plasma gastrin measurements is in monitoring the treatment of duodenal ulcer patients receiving omeprazole or H, receptor antagonists (Carlsson et al., 1986; Sun-

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dler et a/., 1986). Hypergastrinemia induced by long-term treatment with these drugs-or by nature’s own experiment, chronic achlorhydria-may involve a risk of developing gastric carcinoid tumors. Until the size of this risk has been precisely evaluated, control of gastrin concentrations in plasma is considered advisable. Therefore, there has been a substantial demand for gastrin measurements in trials of antiulcer drugs. Early results of such trials, however, suggest that the risk of neoplastic proliferations of ECL cells is negligible (Creutzfeldt and Lamberts, 1991; Lundell et al., 1991). In the future, however, the oncological requirement for gastrin measurements may increase further. As already discussed, gastrin peptides may be involved in the development and growth of bronchial, gastric, pancreatic, colorectal, ovarian, and other common carcinomas. Colorectal and lung cancers are the most frequently occurring malignant tumors in the western world. A significant number of them apparently express gastrin receptors, and hence may be regulated by autocrine gastrin secretion (Hoosein et al., 1988, 1990). Consequently, it is possible that subclassification and selection of treatment of colorectal, bronchial, gastric, and ovarian carcinomas require measurements of gastrin sensitivity. In other words, we have to decide which tumors synthesize gastrin and/or have gastrin receptors by measurement of extracts of tumor biopsies. It is unlikely that plasma or serum gastrin concentrations provide guidance (except possibly for certain malignant ovarian carcinomas), in spite of the increased concentrations encountered in some patients with colorectal cancers (Seitz et d., 1991; Smith et al., 1989; Wong et al., 1991). Even if it holds true that plasma gastrin concentrations are increased in patients with carcinomas in the colon, the increase is likely to be due mainly to secondary antroduodenal disturbances. Thus, direct measurements of the concentrations of gastrins, progastrins, or gastrin receptors in extracts of tumor biopsies are probably the most useful parameters. Proper oncological diagnosis and treatment may therefore require refined measurement of the gastrin system of peptides and/or receptors in the near future. I n view of the apparent low frequency of CCK-producing tumors, the requirements for CCK measurements are probably limited. On the other hand, the rare occurrence of these tumors may also reflect the fact that there are few specific CCK assays available. Thus, it may well be argued that there is a substantial need for CCK measurements until more knowledge about the type and frequency of CCK-producing tumors is available. This point is illustrated by the recent discovery of CCK expression in pediatric tumor cell lines. Although it is still unsettled whether expression also occurs in solid tumors, measurement of CCK

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may be useful to distinguish between different types of small round-cell tumors (Friedman et al., 1992). VI. Methods for Measurement of Gastrin and Cholecystokinin

If gastrin and CCK measurements in plasma or tissue extracts are to gain wider use as tumor markers, conventional assays must be improved. Conventional assays include radioimmunoassays developed against the principal antral form of gastrin (carboxyamidated gastrin- 17) o r against CCK-33 or CCK-8. Assays for gastrin-17 have been commercially available for many years. Moreover, it has long been easy to develop sensitive and specific radioimmunoassays for amidated gastrins (Rehfeld, 1988b; Rehfeld et al., 1972; Stadil and Rehfeld, 1972), whereas specific CCK assays have been difficult to develop (Rehfeld, 1984). The problem is, however, that the delicate posttranslational maturation process easily is disturbed so that abnormal gastrin- or CCK-producing cells process progastrin or proCCK less well, irrespective of whether the cells are endocrine G or I cells in duodenal ulcer patients or tumor cells (Bardram, 1990a; Rehfeld and Bardram, 1991). In other words, neoplastic CCK- and gastrin-producing cells release more unprocessed o r poorly processed precursor products. These products are frequently not carboxyamidated (vide supra). Consequently they are not detected in conventional assays. Therefore, in order to use measurements of gastrin o r CCK as markers of CCK- or gastrin-producing carcinomas, it is necessary to develop assays that measure the entire translation product of gastrin or CCK mRNA independent of the degree of posttranslational processing (Rehfeld and Bardram, 199 1). We have developed a library of new assays that detect glycineextended processing intermediates (Hilsted and Rehfeld, 1986), unprocessed precursors in the form of glycine-extended gastrins after in uitro trypsin and carboxypeptidse B cleavage (Hilsted and Rehfeld, 1986), o r the total translation product irrespective of the degree of processing (Bardram and Rehfeld, 1988; von Solinge and Rehfeld, 1990). Preliminary results indicate that the latter types of assays can be performed in a simple manner and at the same time improve the diagnostic specificity for tumors considerably (Bardram, 1990a; Rehfeld and Bardram, 1991; van Solinge and Rehfeld, 1990). The general principle of the assay is illustrated in Fig. 11. T h e processing-independent assays (PIAs) have been developed by production of high-avidity antibodies, which are monospecific for a suitable progastrin or proCCK sequence (L. Paloheimo and J. F. Rehfeld, unpublished data; Rehfeld and Bardram,

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FIG.11. General scheme of the posttranslational maturation of secretory proteins and peptides. T h e scheme also shows the principle of the processing-independent analysis. T h e shaded bar illustrates a peptide sequence that does not undergo posttranslational modifications, but is present in precursors, processing intermediates, and mature, bioactive products. The “processing-independent” sequence has to be localized between suitable cleavage sites. Thus, by in vitro treatment with appropriate endoproteases, one processingindependent peptide fragment is released per molecule of translated proprotein, irrespective of the degree of processing. After inactivation of the endoprotease added in vitro, quantitation of the processing-independent peptide can be performed by a suitable assay (radioimmunoassay, other immunoassays, or HPLC, but not bioassays).

1991; van Solinge and Rehfeld, 1990). The sequence has to be located immediately C terminal to a trypsin-sensitive cleavage site (Figs. 6 and 7). Monospecificity of the antibodies is ensured by immunization with a short synthetic deca- to pentadecapeptide hapten directionally coupled at its C terminus to a carrier (Bardram and Rehfeld, 1988, 1989; van Solinge and Rehfeld, 1990). A monoiodinated tracer is prepared by labeling naturally occurring or a synthetically coupled tyrosyl residue in a position C terminal to the antibody-bound sequence. By incubation of the samples with trypsin (or another suitable endoprotease) prior to measurement, the selected epitope on the precursor is invariably exposed to binding of the antibodies. Moreover, the tryptic cleavage ensures that the peptide fragment to be measured always has the same size, i.e., the minimal tryptic fragment. Using such a fragment as standard,

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the trypsin treatment therefore ensures optimal accuracy, because the substance to be measured always corresponds to the standard. As illustrated in Fig. 11, the described approach ensures that the entire translation product is quantitated accurately irrespective of the degree of processing. In other words, stoichiometrically one peptide fragment is measured per translated propeptide molecule. We believe that processing-independent assays may substantially improve the diagnostic sensitivity for CCK- and gastrin-producing tumors (Rehfeld and Bardram, 1991). For the classic pancreatic gastrinomas, evidence of the diagnostic value of the PIA is already accumulating (Tables V and VI) (Bardram, 1990a).The ability to diagnose a pancreatic or duodenal gastrinoma in an early, clinically silent stage of the disease is also of importance in screening members of families with MEN-1 disease (vide supra). In addition, the recent observation of incompletely processed CCK in pediatric tumors (Friedman et al., 1992)illustrates the potential diagnostic importance of a PIA for CCK. Therefore, PIAs for gastrin and CCK may prove valuable in the diagnosis and treatment of a variety of tumors. It is important to note that the peptide assays we mention above for use in clinical oncology are based on radioimmunochemical technology. Such assays combine high sensitivity with the necessary selective sequence specificity, in particular for biologically inactive precursors. Additionally, they are fast, simple, and relatively inexpensive. Receptor assays and the few sensitive bioassays developed for CCK in the 1980s (see, for instance, Liddle et al., 1985; Sjodin e l al., 1989) are interesting and have considerable potential in experimental pharmacology and physiology. However, because bioassays and receptor assays, by definition, TABLE V SERUM CONCENTRATIONS OF CARBOXYAMIDATED GASTRIN AND OF T H E TOTAL TRANSLATION IN A GASTRINOMA PATIENTY Disease progression

Carboxyarnidated gastrin (picomoles per liter)

Gastrinoma suspected

Total translation product (picomoles per liter) 276

Three years later-no tumor found

60

642

Four years later-after tumor resection

10

75

1500

7200

Six years later-hepatic metastases a

From Bardram (1990a).

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TABLE VI S E R U M CONCENI'RATION OF CARBOXYAMIDATED G A S T R I N IN PERCENTAGE OF T H E

TOTAL TRANSLATION PRODUCT

Patient status Patients with hepatic metastases Patients with lymph node metastases Patients without metastases n

IN G A s T R I N O M A PATIENTS"

n

Median (76)

10

23

16-49

5

39

25-66

33

54

17-100

Range (%)

From Bardram (199Oa).

measure only bioactive peptides (and neither prohormones nor processing intermediates), their usefulness in clinical oncology is limited. Moreover, they are generally complicated and expensive (for further discussion, see Rehfeld, 1989b; Nielsen and Rehfeld, 1994). VII. Perspectives

This review has substantiated the large body of evidence showing gastrin and CCK gene expression in a wide variety of tumors. For years to come, the task will be to delineate the biological significance and clinical implications of the tumor expression of both peptide and receptor genes. The single most important aspect in gastrin and CCK tumor biology is the possible involvement of gastrin in the development of several common carcinomas. It has been surprising to find expression of the gastrin gene not only in colorectal carcinomas, but also in bronchial, ovarian, pancreatic, and apparently some gastric carcinomas. T h e findings raise three immediate questions: Does the expression of gastrin peptides have carcinogenetic significance? If so, what is the mechanism? Why is the gastrin gene so widely expressed, whereas the homologous CCK gene is expressed only in rare neuroendocrine tumors and in specific pediatric sarcomas? These questions and others have to be addressed by careful studies of factors that control the expression cascade for the genes of both gastrin and CCK, as well as for their receptors. I n the end, such studies may show whether the gastrin or CCK genes are oncogenes or tumor suppressor genes. Both possibilities are open. Another avenue of research comprises examination of other common tumor and cancer forms about which we still are ignorant with regard to CCK and gastrin expression. Such studies may also give clues to the mechanisms of the differentiated occurrence of the two hormones in tumors.

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Finally, we also have to examine in comprehensive clinical studies whether the degree of expression of hormone andlor the corresponding receptor genes may have significance for subclassification and diagnosis of the common cancers with respect to treatment, prognosis, and prevention. If measurement of elements of the gastrin or CCK system turns out to have diagnostic and/or prognostic significance, application of analytical principles, such as the described processing-independent analysis, may prove particularly useful. ACKNOWLEDGMENTS The skillful and patient secretarial assistance of Charlotte Vienberg is gratefully acknowledged. We also thank Drs. Linda Hilsted and Finn Cilius Nielsen for helpful suggestions for improvement of the manuscript. The review is to a significant extent based on studies of gastrin, CCK, and cancer undertaken in the Department of Clinical Biochemistry, Rigshospitalet. These studies have been supported by grants from the Danish Medical Research Council, the Danish Cancer Union, and the Alfred Benzon, Gangsted, and Vissing Foundations.

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INDEX

A Acute myeloid leukemia clones from blood of patients, 59 and colony-stimulating factors production, 61 proliferation stimulation, 79 regulation of cells, 57 imbalanced signaling effects, 80-81 loss of sex chromosome, 72 suppression of hemopoietic cells, 69 Adenoviruses, effect on MHC expression cellular immune response, 158-160 characteristics, 155- 158 MHC regulation MHC-I post-translational control, 162- 164 transcriptional control, 160-162 MHC-11, 164 significance, 164- 165 AKR mouse lymphomas, see Lymphomagenesis, in AKR mouse in thymic leukemia study, 46-47 Antigens processing hypothesis, 32-33 specificity of rhadinovirus-transformed T cell lines, 228-231 truncated surface, MHC regulation by, 179 Antigens, and DNA viruses MHC class I expression effects adenoviruses, 160- 164 cytomegalovirus, 169- 171 hepatitis B virus, 175-177 herpesviruses, 169- 17 1 immune recognition by T cells mechanism, 123-124

349

MHC-I assembly with endogenous peptide, 131-132 pathway, 127-129, 132 peptide complexes, 129-131 transcription regulation, 136138 papillomavirus, 182- I83 poxviruses, 151-152 MHC class 11 expression effects adenoviruses, 164 binding factors S box, 140 XI, 139 X2, 139 Y box, 139-140 cytomegalovirus, 171-172 hepatitis B virus, 177 herpesviruses, 171- 172 immune recognition by T cells binding of peptide antigens, 133134 biosynthesis, 133 endosomal targeting, 133 pathway, 132, 135 restriction, 134-135 transcription regulation, 138-140 papillomavirus, 183-184 poxviruses, 152-153 MHC expression effects adenoviruses, 155- 165 control sites, 187, 189 cytomegalovirus, 165- 173 hepatitis B virus, 173-180 herpesviruses, 165-173 immune recognition by T cells antigen diversity, 124- 126 antigen structure, 124-126

350

INDEX

cytokine regulation of gene expression, 141-145 mechanisms, 185- 190 modulation, 145-146 presentation, 118-1 19 restriction, 122-123 subset roles, 120-122 T cell repertoire development, 119-120

future research, 336-337 gene expression at peptide level, 312, 325

identification, 295 measurement methods, 333-336 requirements, 33 1-333 as tumor growth factor, 330331

transcription regulation, 135- 140 viral peptide-MHC complexes, 126- 127

papillomavirus, I8 1- 185 poxviruses, 145, 147-155

B B cell lymphomas, see also Lymphomagenesis, in AKR mouse Ly-I+ heavy and light chain loci, analysis, 281-284

role of class I MCF, 285-286 as model of tumor dormancy, 245 B cells, Ly-I+, characteristics, 279-281 Bronchial tumors cholecystokinin expression, 325 gastrin expression, 321

Chromosomes, abnormalities in leukemia, 73

Colony-stimulating factor genes cloning, 52 granulocyte-macrophage induced production, 74 insertional mutagenesis, 75 multi-colony-stimulating factors induced production, 74 insertional mutagenesis, 75 Colony-stimulating factors, see also Granulocyte colony-stimulating factors; Granulocyte-macrophage colonystimulating factors and leukemia development biological actions, 52-58 characterization, 50-5 1, 8283

detection, 50 differentiation commitment, 54-56, 83

C Cerebral tumors, cholecystokinin expression, 327 Cholecystokinin biology, normal cellular release, 31 1-314 genes, 301-304 mRNAs, 301-304 procholecystokinin, post-translational processing, 309-312 receptors, 3 14-316 biology, tumor bronchial tumors, 325 cerebral tumors, 327 gastrointestinal tumors, 324-325 neuroendocrine tumors, 326-327 definition, 296-296,300-301 function, 296

excess extrinsic levels, 82-84 gene cloning, 52 maturation induction, 54-55 membrane receptors, 71-72 myeloid leukemia initiation, 6270

myeloid leukemic cell proliferation, 60-62

production by macrophages, 74 study, 49-50 multi, see Multi-colony-stimulating factors protection of abnormal cells from apoptotic death, 83 Colorectal tumors, gastrin expression, 3 18-32 1

Cytokines, regulation of MHC gene expression, 141- 145 Cytomegalovirus model, for herpesviruses effect on MHC expression, 165-173

35 I

INDEX

Cytotoxic T lymphocytes Epstein-Barr virus infection response, 212 and MHC antigens adenovirus infection cellular immune response, 158-159 regulation, significance, 164-165 poxvirus infection mechanism of regulation, 153-154 MHC-I1 regulation, 153 MHC-I regulation, 151-152 response to, 149 vaccinia virus infection response, 148

D DNA, complementary, MEKs, isolation, 103-104 DNA viruses, effects on MHC expression adenoviruses cellular immune response, 158- 160 characteristics, 155- 158 MHC regulation MHC-11, 164 MHC-I post-translational control, 162- 164 MHC-I transcriptional control, 160-162 significance of, 164-165 control sites, 187, 189 hepatitis B virus cellular immune response, 175 characteristics, 173-174 MHC regulation mechanisms, 177-180 MHC-I, 175-177 MHC-11, 177 herpesviruses cellular immune response, 167- 169 characteristics, 165-167 MHC regulation mechanisms, 172- 173 MHC-I, 169-171 MHC-11, 171-172 immune recognition of viral antigens by T cells class I molecules assembly with peptide, 131132

peptide complexes, 129-131 structure, 123-124 class I1 molecules binding of peptide antigens to, 133-1 34 biosynthesis, 133 endosomal targeting, 133 restriction, 134-135 structure, 123-124 cytokine regulation of gene expresssion, 141-145 diversity of antigens, 124-126 mechanisms, 118-1 19, 185-190 modulation, 145-146 pathway class I, 127-129.132 class 11, 132, 135 restriction, 122-123 structure of antigens, 124-126 T cells repertoire development, 1 19- 120 subset roles, 120-122 transcription regulation, 135- 140 viral peptide-MHC complexes, 126127 papillomavirus cellular immune response, 181-182 characteristics, 180-181 MHC regulation mechanism, 184-185 MHC-I, 182-183 MHC-11, 183-184 poxviruses cellular immune response, 149-151 characteristics, 145, 147- 148 MHC regulation mechanisms, 153- 155 MHC-I, 151-152 MHC-11, 152-153 Dormancy, tumor, B cell lymphoma as model, 245 Dual tropic viruses, lymphomagenesis role in AKR mouse, 246, 249, 254-256, 286-287

E Epstein-Barr virus induction of lymphomas, 73

352

INDEX

T lymphocyte transformation by, 212 ERK, see Extracellular signal-regulated protein kinases Extracellular signal-regulated protein kinases cell function regulation, 11 1 cloning, 96-97 distribution, 95-96 MEK activation, 104-1 05 cDNAs, isolation, 103-104 identification, 102-103 purification, 102- 103 regulation, 104 network, regulation by G proteins, 105106 phosphorylation, 94-95, 99-100, 107 properties, 100-102 purification, 96-97 signal transduction mechanisms, 93-94 phosphorylation role, 94-95 substrates primary sequence specificity, 108 sites of possible physiological consequence, 109- 1 1 I

F FDC-PI cells, transformation by CSFs, 66-67, 70, 75 by oncogenes, 74 by viruses, 74, 76

G Gastric tumors, gastrin expression, 323 Gastrin biology, normal cellular release, 3 11-3 14 genes, 301-304 mRNAs, 30 1-304 progastrin, post-translational processing, 304-309 receptors, 3 14-3 16 biology, tumor bronchial tumors, 321

colorectal tumors, 3 18-321 gastric tumors, 323 neuroendocrine tumors, 323-324 ovarian tumors, 320-322 pancreatic endocrine tumors, 3 16318 pancreatic exocrine tumors, 318 definition, 296-299, 301 function, 296 future research, 336-337 gene expression at peptide level, 308, 322 identification, 295 measurement methods, 333-336 requirements, 33 1-333 as tumor growth factor, 327-330 Gastrointestinal tumors, cholecystokinin expression, 324-325 G-CSF, see Granulocyte colony-stimulating factors Genes bcl-2, follicular lymphoma development role, 57 cholecystokinin, 30 1-304, 308, 325 colony-stimulating factor, see Colonystimulating factor genes cytokine regulation of MHC gene expression, 141-145 gastrin, 301-304, 312, 322 H-2 complex, see H-2 complex Ir-I, research, 15, 18-26 major histocompatibility complex, expression, see Antigens, and DNA viruses oncogenes, see Oncogenes stp, T lymphocyte transformation by, 231-236 GM-CSF, see Granulocyte-macrophage colony-stimulating factors, and leukemia development Granulocyte colony-stimulating factors, and leukemia development characterization, 50 differentiation commitment, 55 myeloid leukemia initiation, 63-64, 70 suppression, 76-77, 81 progenitor cell formation, 56

353

INDEX

Granulocyte-macrophage colonystimulating factors, and leukemia development characterization, 50 membrane receptors, 7 1-72 myeloid leukemia initiation, 63-66, 70 suppression, 77, 79, 81 progenitor cell formation, 57 Growth factors myeloid leukemia initiation role, 68-69 autocrine growth factors, 6 9 extrinsic growth factors, 69 self-renewal of cell lines by, 75 tumor cholecystokinin as, 330-33 1 gastrin as, 327-330

H H-2 complex, history of research early studies, 11-15, 17-18 haplotype study, 26-27 heterozygosity, 28 Ir-1 gene research integration, 23-26 protein identification, 29-30 Hemopoietic regulators, and leukemia development colony-stimulating factors, see Colonystimulating factors early in vivo work, 42-47 future studies, 85-86 inducers, 72-76 myeloid leukemia cells in culture, 59-62 suppression, 76-82 research perspective, 4 1-42 Hepatitis B virus, effect on MHC expression cellular immune response, 175 characteristics, 173-174 MHC regulation HBV-DNA integrations, 179-1 80 mechanism, 177- 180 MHC-I, 175-177 MHC-11, 177 nucleocapsid protein, 178 polymerase/termini protein, 177178

truncated surface antigen, 179 X protein, 178-179 Herpesviruses effect on MHC expression cellular immune response, 167-169 characteristics, 165-167 cytomegalovirus model, 165-1 73 MHC regulation mechanism, 172-173 MHC-I, 169-171 MHC-11, 171-172 human herpesvirus type 6, T lymphocyte transformation by, 212-213 stp oncogene, T lymphocyte transformation by, 231-236 T-lymphotropic vector, development of, 2 19-22 1 Herpesvirus saimiri human T lymphocyte transformation by, 223-228 immortalization. 22 1-223 HTLV, see Human T cell leukemia virus Human herpesvirus type 6, T lymphocyte transformation by, 212-213 Human T cell leukemia virus human T helper lymphocyte transformation, 2 13-22 1 immortalization, 22 1-223 induction of adult T cell leukemia, 73

I Immunoglobulin H, in Ly-I+ (CD5+) B cell lymphoma heavy and light chain loci, analysis, 28 1-284 in spleen of grossly normal old and thymectomized mice, rearrangements, 284-285 Immunotherapy, passive, elimination of PLCs in AKR/J mouse following, 267-269 Interferon, and MHC regulation gene expression, 14 1- 144 poxvirus infection, 154-155 Interferon-y, and MHC regulation gene expression, 144 poxvirus infection, 149-15 I

354

INDEX

Interferon response factors, regulation of MHC gene expression, 142-143 Interleukin-3, see Multi-colony-stimulating factors, and leukemia development Interleukin-6, myeloid leukemia suppression, 78, 81 Irradiation, fractionated, lymphomagenic effect, 258-259

K Klein, Jan, retrospective of life and studies, 1-36

L Leukemia, see also Acute myeloid leukemia; Myeloid leukemia chromosomal abnormalities in, 73 development, hemopoietic regulators and, see Hemopoietic regulators, and leukemia development radiation leukemia virus A-RadLV, lymphomagenesis in AKR mice by, 257-258 Leukemia inhibitory factor, myeloid leukemia suppression, 78, 81-82 Ly-I (CD5+) B cell lymphoma characteristics, 277-2&287-289 heavy chain loci analysis, 281284 IgH rearrangements, 284-285 lack of class I MCF involvement, 285286 light chain loci analysis, 281-284 Lymphocytes, see B cells; Cytotoxic T lymphocytes; T cells Lymphomagenesis, in AKR mouse B cell lymphoma, see Ly-I+ (CD5+) B cell lymphoma characteristics, 245-249 potential lymphoma cells age-related levels, 266 bone marrow-derived, proliferation, host effects, 252-253 B-PLC dormant state maintenance and termination, 270-277 +

dormant, levels after prevention of spontaneous T lymphoma development, 262-270 identification, 247-253 prevention, 248-249 T cell lymphoma development mechanisms, 253-254, 26 1 development pathways, enhanced, 253-261 dual tropic viruses in, 247, 286287 tumorigenesis acceleration by recombinant viruses, 254-256 fractionated irradiation effect, 258259 N-meth yl-N-nitrosourea-induced, 259-261 radiation leukemia virus A-RadLVinduced, 257-258 SL3-3 ecotropic retrovirus-induced, 256-257 transplantation studies, 287

M Macrophage colony-stimulating factors, and leukemia development characterization, 50 differentiation commitment, 55 membrane receptors, 71-72 myeloid leukemia suppression, 79 production by FDC-P1 cells, 76 Major histocompatibility complex, expression, effect of DNA viruses, see Antigens, and DNA viruses MCF, see Mink cytopathic focus-forming viruses M-CSF, see Macrophage colonystimulating factors N-Methyl-N-nitrosourea, lymphomagenesis in AKR mouse by, 259-261 MHC antigens, expression, effect of DNA viruses, see Antigens, and DNA viruses Microtubule-associated protein kinases characterization, 95 distribution, 95-96

355

INDEX

extracellular signal-regulated, see Extracellular signal-regulated protein kinases homology with proteins in yeast mating pathway, 97-99 phosphorylation of upstream components of cascade, 107-108 recombinant, in vitro regulation, 100102 regulation, protein-protein interactions in, 106-107 Mink cytopathic focus-forming viruses, class I, role in Ly-I+ B cell lymphomas, 285-286 Multi-colony-stimulating factors, and leukemia development characterization, 50 membrane receptors, 71-72 myeloid leukemia initiation, 65, 67, 70 suppression, 79, 81 progenitor cell formation, 57 Myeloid leukemia, see ah0 Acute myeloid leukemia cells in culture, 59-62 chronic cell culture, 59 and colony-stimulating factor regulation of cells, 57 and colony-stimulating factors cell proliferation, 60-62 initiation role, 62-70 production by macrophages, 74 suppression, 76-8 1 initiation by growth factors, 68-69 suppression G-CSF, 76-77,81 GM-CSF, 77, 79, 81 hemopoietic regulators, 76-82 interleukin-6, 78, 81 leukemia inhibitory factor, 78, 81-82 M-CSF, 79 multi-CSF, 79, 81

N Natural killer cells, response to adenovirus infection, 159

Neuroendocrine tumors cholecystokinin expression, 326-327 gastrin expression, 323-324

0 Oncogenes leukemia development role FDC-PI cell transformation, 74 induction of CSF production, 74 stp, T lymphocyte transformation by, 231-236 Ovarian tumors, gastrin expression, 320322

P Pancreatic tumors, gastrin expression endocrine tumors, 316-318 exocrine tumors, 3 18 Papillomavirus, effect on MHC expression cellular immune response, 181-182 characteristics, 180-1 8 1 MHC regulation mechanism, 184-185 MHC-I, 182-183 MHC-11, 183-184 Potential lymphoma cells, in AKR mouse, see Lymphomagenesis, in AKR mouse Poxviruses, effect on MHC expression cellular immune response, 149-151 characteristics, 145, 147- 148 MHC regulation mechanism, 153-155 MHC-I, 151-152 MHC-11, 152-153 Procholecystokinin, post-translational processing extraintestinal, 31 1-312 intestinal, 309-31 1 Progastrin, post-translational processing antral, 304-307 extraantral, 307-309 Protein kinases, see Extracellular signalregulated protein kinases; Microtubule-associated protein kinases

356

INDEX

Proteins exogenous viral, entry into MHC pathways, 132, 135 C proteins ERK network regulation, 105-106 microtubule-associated protein kinase homology, 98 KSSI, microtubule-associated protein kinase homology, 97 MHC regulation by nucleocapsid, 178 polymerase/termini, 177- 178 X protein, 178-179 Mos, MEK activation, 104 Raf, in MAP kinase casade regulation. 106- I07 Ras ERK network regulation, 105-106 in MAP kinase casade regulation, 106-107 MEK activation, 103-104 spk I , microtubule-associated protein kinase homology, 97-98 in yeast mating pathway, microtubuleassociated protein kinase homology, 97-99

R Radiation leukemia virus A-RadLV, lymphomagenesis in AKR mouse by, 257-258 Retrovirus SL3-3, ecotropic, lymphomagenesis in AKR mouse by, 256-257 Rhadinovirus, T lymphocyte transformation by, 2 13, 228-230 RNA, messenger cholecystokinin, 30 1-304 gastrin, 301-304

S Saccharomyces cereuisiae, microtubuleassociated protein kinase homology, 97-98 Saccharomyces pombe, microtubuleassociated protein kinase homology, 97-98

Somatic cell genetics, history of research. 10 Spleen, grossly normal old and thymectoniized mice, IgH rearrangements. 284-285 Stem cell factor, progenitor cell formation, 56

T T cell lymphomas, in AKR mouse, see Lymphomagenesis, in AKR mouse T cells cytotoxic, see Cytotoxic T lymphocytes human, viral transformation characteristics of virus, 212-2 13 herpesviral stp oncogene, 23 1-236 Herpesvirus saimiri immortalization with HTLV recombinants, 221-223 mechanisms, 223-228 human T cell leukemia virus, 214219 mechanisms, 2 1 1-2 13, 236-237 rhadinovirus, 228-230 T-lymphotropic herpesvirus vector, 2 19-22 1 immune recognition of viral antigens by, and MHC expression, see DNA viruses, effects on MHC expression Thymectomy, AKR mouse. effects on potential lymphoma cell levels, 262-264 Transcription factors, leukemic transformation role, 74-75 Transplantation, studies of lyniphomagenesis in AKR mouse, 287 Tumor growth factors cholecystokinin as, 330-33 I gastrin as, 327-330 Turnor necrosis factor, and MHC antigens poxvirus infection mechanism of regulation, 154- 155 response to, 149- 150 regulation of MHC gene expression, 143- I44 Tumors cholecystokinin expression bronchial tumors, 325 cerebral tumors. 327

INDEX

gastrointestinal tumors, 324325 neuroendocrine tumors, 326327 dormancy, B cell lymphomas as model, 245 gastrin expression in bronchial tumors, 321 gastric tumors, 323 neuroendocrine tumors, 323324 ovarian tumors, 320-322 pancreatic endocrine tumors, 316318 pancreatic exocrine tumors, 318 rectal tumors, 3 18-32 1

V Vaccinia virus, effect on MHC expression, 147-148

Viral interference, T cell lymphoma prevention by, effect on PLC level, 264265 Viruses, see also specific viruses DNA, effect on MHC expression, see DNA viruses, effects on MHC expression FDC-PI cell transformation, 74, 76 transformation of human T lymphocytes by, see T cells

Y Yeast, microtubule-associated protein kinase homology, 97-99

z Zollinger-Ellison syndrome, and gastrin expression, 3 16-3 17

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