Erythropoietin Jerry L. Spivak* Department of Medicine, Johns Hopkins University School of Medicine, Traylor 924 720 Rutland Avenue, Baltimore, MD 21205, USA * corresponding author tel: 410-955-5454, fax: 410-955-0185, e-mail:
[email protected] DOI: 10.1006/rwcy.2000.09007.
SUMMARY This chapter reviews current knowledge about erythropoietin from the level of its gene to the application of its recombinant version for the correction of anemia with special emphasis on the molecular biology, physiology and pharmacology of the protein, its tissue-specific expression and regulation of that expression in health and disease.
BACKGROUND
Discovery A relationship between hypoxia and increased erythropoiesis was first recognized in 1863 and by the end of the nineteenth century not only were the kinetics of this relationship well established, but a hypothesis linking enhanced erythropoiesis with marrow hypoxia had been developed (Erslev, 1953). In 1906, Carnot and Deflandre proposed an alternative hypothesis that hypoxia enhanced erythropoiesis not by a direct effect on the bone marrow but rather indirectly through a humoral mechanism. This hypothesis was attractive but could not be substantiated by other investigators using the Carnot and Deflandre protocol and lay dormant until 1950. At this time, Kurt Reissmann, using parabiotic rats, conclusively demonstrated that hypoxic induction of erythropoiesis was under humoral control (Reissmann, 1950). For technical reasons, Reissmann's demonstration was indirect and it remained for Erslev to demonstrate directly and conclusively using large quantities of anemic rabbit plasma that Carnot and Deflandre were indeed correct in their intuition if not their experimental methods (Erslev, 1953). The
putative humoral mediator was named erythropoietin, as suggested previously (Grant and Root, 1947). With scientific interest now rekindled, it was soon established that the kidneys were a major site of erythropoietin production (Jacobson et al., 1957). An in vivo bioassay was developed and efforts were initiated to purify erythropoietin, an accomplishment which proved exceedingly difficult owing to the minute quantities of the hormone normally present in the plasma and urine. Indeed, the initial biochemical purification of erythropoietin required 2550 L of urine from extremely anemic individuals (Miyake et al., 1977). Once purified, it was possible to raise antibodies to erythropoietin and thereby develop an immunoassay sufficiently sensitive and specific, in contrast to the existing bioassays, to define the physiology of the hormone (Egrie et al., 1987). With development of recombinant DNA technology, knowledge of the amino acid sequence of erythropoietin permitted cloning of its gene (Jacobs et al., 1985; Lin et al., 1985).
Alternative names EPO. The two commercially available erythropoietin preparations have been designated Epoietin and Epoietin .
Structure This is a globular protein (predicted radius 20.2 AÊ) with an observed Stokes radius of 32 AÊ due to its carbohydrate residues (Davis and Arikawa, 1987), constituted as a four antiparallel helical bundle with one short and two long intervening loops and several stretches of sheets (Elliott et al., 1997), a structure
942 Jerry L. Spivak which is characteristic of hematopoietic growth factors in general, even though they share little amino acid sequence homology (Bazan, 1990a). The protein has two independent binding sites for its receptor, which differ in their binding affinity (Philo et al., 1996; Elliott et al., 1997).
et al., 1986; Powell et al., 1986). There are no documented relevant linkages, mutations or related genes.
Main activities and pathophysiological roles
A GATA sequence is present 50 to the cap site, but the putative promoter region of the erythropoietin gene does not contain the TATA or CAAT sequences typical of a classical promoter (Shoemaker and Mitsock, 1986). Rather, the erythropoietin gene contains a 50 nucleotide enhancer sequence in its immediate 30 flanking sequence, a liver inducible element mapping between 0.4 kb 50 to the gene and 0.7 kb 30 , a negative regulatory element between 0.4 and 6 kb 50 to the gene which represses expression in most tissues, and a region between 6 and 14 kb 50 which controls renal expression (Semenza et al., 1991; Semenza and Wang, 1992). Adding to this level of complexity is the existence of multiple transcription initiation sites which appear to be differentially utilized in a tissuespecific fashion. In addition to hypoxia-induced transactivating factors that upregulate erythropoietin gene transcription, factors that can negatively regulate erythropoietin production have also been described. These include an as yet unidentified 47 kDa nuclear protein and a ribonucleoprotein (Beru et al., 1990) and the transcription factors GATA-1, -2, and -3 (Imagawa et al., 1997). The von Hippel-Lindau (VHL) gene product may also be a negative regulator of erythropoietin production under normoxic conditions (Krieg et al., 1998). Finally, in contrast to the genes for other hematopoietic growth factors such as IL-3, GM-CSF, and G-CSF, the erythropoietin gene lacks the 30 ATTA sequences that enhance mRNA degradation (Shaw and Kamen, 1986). This may be a consequence of its oxygen-dependent expression.
Erythropoietin is a member of the hematopoietic growth factor family and is one of the few hematopoietic growth factors which behaves like a hormone. The principal function of erythropoietin is to couple oxygen delivery by circulating red cells to long-term tissue oxygen needs. Produced primarily in the kidneys and to a small extent in the liver in adults, erythropoietin interacts in the bone marrow with specific receptors on the surface of erythroid progenitor cells to initiate their entry into cell cycle if dormant (Spivak et al., 1991, 1996) or to maintain their viability while differentiating, if they are already in active cell cycle (Koury and Bondurant, 1990). Erythropoietin achieves its effects by causing homodimerization of its receptor with the resultant autophosphorylation of the tyrosine kinase JAK2 (Witthuhn et al., 1993) and phosphorylation of the receptor itself, as well as various substrate proteins leading to the upregulation of a number of signaling pathways and the activation of gene transcription (Damen and Krystal, 1996). In keeping with its high level of evolutionary conservation and its vital role in maintaining an adequate supply of red cells for oxygen transport, no mutant erythropoietin molecule has ever been described and antibody production to endogenous erythropoietin or its exogenous recombinant counterpart is exceedingly rare (Casadevall et al., 1996).
GENE AND GENE REGULATION
Accession numbers Gene: M11319, X02158 cDNA: X02157
Chromosome location The human erythropoietin gene is present as a single copy on chromosome 7(q11-q22) and is composed of five exons (582 bp) and four introns (1562 bp) (Law
Regulatory sites and corresponding transcription factors
Cells and tissues that express the gene Cloning of the erythropoietin gene provided an opportunity to define its tissue-specific expression formally. In the normoxic state, erythropoietin mRNA was detected in the kidneys, liver, lung, testes, and brain (both infra- and supratentorial) (Tan et al., 1991; Digicaylioglu et al., 1995). In the setting of hypoxia, erythropoietin mRNA was upregulated in kidney, liver, testes, and brain and become detectable in the spleen. Using RT-PCR analysis, it has also been possible to study the ontogeny of erythropoietin
Erythropoietin 943 gene expression. Erythropoietin mRNA was detectable in embryonic stem cells before their differentiation together with erythropoietin receptor mRNA (Schmitt et al., 1991; Keller et al., 1993). Thus, erythropoietin gene expression occurs before the formation of the kidneys or liver and even before the expression of multipotent growth factors such as IL-1, IL-3, and GM-CSF (Schmitt et al., 1991). To date, only two cell lines, HepG2 (HB-8065) and Hep3B (HB-8064), derived from human hepatomas, have been documented to produce erythropoietin in vitro (Goldberg et al., 1987). Recently, data have been obtained suggesting that erythropoietin is also produced by erythroid burst-forming units (BFU-E) and might be upregulated by erythropoietin itself (Stopka et al., 1998b).
PROTEIN
Accession numbers PO1588
Sequence See Figure 1.
Description of protein The erythropoietin gene codes for a 193 amino acid protein, of which 27 serve as the signal peptide, while the remaining 166 constitute the native protein. However, based on studies employing fast atom bombardment mass spectrometry and peptide mapping, the terminal amino acid, arginine 166, is absent not only from recombinant erythropoietin produced by Chinese hamster ovary cells but also from human urine erythropoietin, the only native form of erythropoietin purified to date (Recny et al., 1987). The site for this posttranslational modification has not been identified, nor is its biologic significance understood. Physicochemical studies indicate that erythropoietin has a high helix content, a calculated Stokes
radius of 20.2 AÊ with an observed Stokes radius of 32 AÊ, presumably due to its carbohydrate side chains, heterogeneity with respect to its pI (4.2±4.6) owing to variable degrees of sialation, and a net negative charge at physiologic pH also due to its sialic acid residues (Davis and Arakawa, 1987). The pI of the aglycone is 9.2 and the protein becomes destabilized below its isoelectric point, presumably as a result of the loss of sialic acid residues. In spite of its acidic pI, fluorescent quenching studies indicate the protein interacts better with negatively charged ions, suggesting that the net positive charge of the protein as opposed to the net negative charge of the whole molecule is dominant in ionic interactions (Davis and Arakawa, 1987), a behavior which is consonant with respect to the interaction of erythropoietin with its receptor. Erythropoietin is a hydrophobic protein, a characteristic which is also important with regard to its ligand±receptor interactions. While human erythropoietin contains four cysteine residues (at positions 7, 29, 33, and 161), which form two internal disulfide bonds, cysteine 33 is replaced by proline in murine erythropoietin (Wen et al., 1993). The disulfide bridge between cysteine 7 and 161 is, however, necessary for biological activity since alkylation of these residues or their replacement through site-directed mutagenesis inactivated the protein under physiologic circumstances. Normally this internal disulfide bond is not exposed, since erythropoietin in solution is not inactivated by reducing agents. Internal disulfide bonds are also a feature of GM-CSF, G-CSF, and IL-3. Early studies indicated that iodination of more than one of erythropoietin's four tyrosine residues, occupation of its lysine residues, or alteration of their charge also inactivated erythropoietin. With respect to tyrosine iodination, this might have been due to steric hindrance since site-directed mutagenesis at tyrosine residues 49 and 145 did not abolish biologic activity, while only nonaromatic substitutions for tyrosine 15 or 156 affected biologic activity (Elliott et al., 1997). Futhermore, erythropoietin can be iodinated without loss of biological activity. The net charge of erythropoietin is positive and neutralization of certain basic amino acids such as lysine impairs receptor-ligand binding (Elliott et al., 1997). Indeed,
Figure 1 Amino acid sequence for erythropoietin.
MGVHECPAWL NITTGCAEHC LRGQALLVNS ASAAPLRTIT
WLLLSLLSLP SLNENITVPD SQPWEPLQLH ADTFRKLFRV
LGLPVLGAPP TKVNFYAWKR VDKAVAGLRS YSNFLRGKLK
RLICDSRVLE MEVGQQAVEV LTTLLRALGA LYTGEACRTG
RYLLEAKEAE WQGLALLEAV QKEAISPPDA DR
944 Jerry L. Spivak electrostatic interactions between hematopoietic growth factors and their receptors appear to be an essential factor in ligand binding (Wlodawer et al., 1993, Demchuk et al., 1994). Mapping studies using site-directed mutagenesis and monoclonal antibodies have confirmed secondary and tertiary structure predictions based on computer modeling as well as X-ray diffraction and NMR analysis of other hematopoietic growth factors (De Vos et al., 1992; Boissel et al., 1993; Elliott et al., 1996, 1997). Indeed, in spite of their different target cells and cognate receptors, hematopoietic growth factors as well as growth hormone and prolactin share in common a four helical antiparallel bundle structure with long crossover loops between the first two and last two helices. Erythropoietin follows this pattern, containing long crossover loops between the A and B and C and D helices, and a short loop between B and C. There are also two short antiparallel strands formed by the long AB and CD crossover loops (Syed et al., 1998). Epitope mapping and site-directed mutagenesis revealed four regions of the protein which are important for biological activity: amino acid residues 11±15 in helix A; 44±51 in the A±B connecting loop; 100±108 in helix C and 147±151 in helix D (Matthews et al., 1996; Elliott et al., 1997). These four regions map to two sites on erythropoietin based on their effects on receptor binding and biological activity. Mutations of the amino acids comprising site 1 impair both receptor binding and bioactivity, while mutations in site 2 have lesser effects on receptor binding but greater effects on bioactivity. These observations support a model of ligand± receptor binding where erythropoietin initially binds to one erythropoietin receptor with high affinity (Kd=1 nM) and subsequently to a second receptor molecule with a lower affinity (Kd 1 mM), causing receptor dimerization. Site 1, the high-affinity site, is composed of portions of the A, B, and D helices and the AB loop while site 2, the low-affinity site, involves portions of the A and C helices (Elliott et al., 1996, 1997). Physiochemical analysis using soluble erythropoietin receptors containing only its extracellular domain (Philo et al., 1996) support this model, as have chemical crosslinking studies using soluble fulllength erythropoietin receptors (Avedissian et al., 1995) and studies using activating monoclonal receptor antibodies (Elliott et al., 1996; Schneider et al., 1997).
Discussion of crystal structure Although the native erythropoietin molecule has not been crystallized nor studied by NMR,
crystallography at 1.90 AÊ of an erythropoietin aglycone±erythropoietin receptor extracellular ligandbinding domain complex has recently been reported (Syed et al., 1998). The crystallized ligand±receptor complex confirmed studies employing mutational analysis, epitope mapping and ligand±receptor crosslinking, indicating that one erythropoietin molecule binds to two molecules of its cognate receptor. Thus, erythropoietin was found to interact with its receptor at two sites with both hydrophobic and hydrophilic interactions. Phenylalanine 93 of the erythropoietin receptor was the dominant residue with respect to hydrophobic interactions and mutations of this residue resulted in loss of erythropoietin binding (Middleton et al., 1996). As predicated by epitope mapping, the charge interactions between erythropoietin and its receptor involve lysine and arginine resides of the former and aspartate and glutamate residues of the latter (Syed et al., 1998). The most important erythropoietin residues with respect to its interaction with its receptor appear to be glycine 151, which dictates the appropriate conformation of the D helix and the arginine residues 14 and 103.
Important homologies Erythropoietin is highly active across mammalian species, a behavior which correlates well with the high degree of homology amongst the cloned human, simian, and rodent genes (McDonald et al., 1986; Shoemaker and Mitsock, 1986). Whether analyzed at the level of the gene or its protein, with one exception erythropoietin is highly conserved amongst all mammalian species studied to date (Wen et al., 1993). For example, the human erythropoietin gene is 91% identical to monkey erythropoietin, 85% identical to cat and dog erythropoietin, and 80±82% identical to pig, sheep, and rat erythropoietin. Interestingly, while mouse and human erythropoietin stimulate guinea pig erythroid progenitor cells, guinea pig erythropoietin did not stimulate mouse or human erythropoietin progentor cells in vitro, suggesting loss of epitope homology between guinea pig erythropoietin and other mammalian erythropoietins (Stopka et al., 1998a). With respect to gene conservation, there are extensive regions of sequence homology between human and mouse erythropoietin genes upstream of the cap site and in the 30 UTR as well as in the first intron, together with a high frequency of Alu sequences (or their murine homologs) in the 50 region and within the transcription unit (Shoemaker and Mitsock, 1986; Galson et al., 1993). Erythropoietin has no significant homology to any other known protein except for thrombopoietin,
Erythropoietin 945 the hematopoietic growth factor which regulates megakaryocyte and platelet production as well as stimulation of primitive, uncommitted hematopoietic progenitor cells (Bartley et al., 1994). Thrombopoietin can be divided into two domains on the basis of its amino acid sequence; the Nterminal domain of 155 resides has 21% sequence identity and overall a 46% sequence similarity to erythropoietin. It is this domain which binds to the thrombopoietin receptor.
Posttranslational modifications Based on its amino acid composition, the estimated molecular weight of erythropoietin is 18,398, while based on its behavior during sedimentation equilibrium, its apparent molecular weight is 30,400 (Davis and Arakawa, 1987). The difference between the estimated and apparent molecular weights is due to glycosylation and, as might be expected from the extent of glycosylation, this is the most important posttranslational modification of the protein. Erythropoietin contains one O-linked (serine 126) and three N-linked (asparagine 24, 38, and 83) glycosylation sites and the latter are invariant amongst mammalian erythropoietins. The N-linked carbohydrates consist primarily of tetra-antennary saccharides with or without one or more N-acetyl lactosaminyl repeats and lesser quantities of biantennary and triantennary saccharides, the latter of which may also contain N-acetyl lactosaminyl repeats. All the N-linked saccharides are sialated, as is the O-linked saccharide. On a mole/mole basis, the carbohydrate composition of human urinary erythropoietin consists of fucose (2.9), mannose (9.2), galactose (12.9), N-acetylglucosamine (16.3), Nacetylgalactosamine (0.9), and N-acetylneuraminic acid (10.4) (Sasaki et al., 1987). With the exception of the type of sialic acid linkage, the glycosylation of human recombinant erythropoietin is similar to human urinary erythropoietin. The erythropoietin aglycone, produced in Escherichia coli or by enzymatic deglycosylation, has a conformation similar to fully glycosylated erythropoietin as defined by circular dichroism (Narhi et al., 1991). Thus, glycosylation is not required for proper folding of the protein or even refolding after denaturation by a chaotrophic agent. However, glycosylation does appear important for stabilizing the tertiary structure of the protein under a variety of denaturing conditions. The sialic acid residues of erythropoietin, in keeping with their abundance, are essential for maintaining erythropoietin in the circulation. Desialation of
erythropoietin exposes the penultimate galactose residues which are recognized by hepatic galactosyl receptors and desialated erythroporetin is rapidly cleared from the plasma and degraded in hepatocytes. Oxidation of the exposed galactose residues restored the ability of the hormone to remain in the circulation (Spivak and Hogans, 1989). There is no evidence that the sialic acid residues serve another purpose. To date, no role has been defined for the single O-glycosylation site of erythropoietin. Murine erythropoietin has a proline at amino acid 126 rather than serine, suggesting that O-glycosylation per se does not have a physiologic role (Wen et al., 1993). Supporting this contention is the observation that erythropoietin produced in cells defective in O-glycosylation behaved identically in vivo to O-glycosylated erythropoietin (Wasley et al., 1991). Studies employing enzymatic O-deglycosylation have yielded similar results (Higuchi et al., 1992). Although one study employing site-directed mutagenesis suggested that O-glycosylation was essential for biosynthesis and secretion of the hormone (Dube et al., 1988), other studies also employing site-directed mutagenesis indicated that this result was an artifact due to the amino acid (glycine) chosen to replace serine at site 126. A similar result was obtained with alanine but not with valine, threonine, histidine, or glutamic acid (Delorme et al., 1992). Since serine 126 occurs in the long loop between the C and D helices of erythropoietin which is not near its active sites, it is likely that the alanine or glycine substitutions altered protein conformation and were not truly neutral substitutions. At the same time, it is possible that the O-glycosylation site serves to stabilize the conformation of the protein, a role served by proline in murine erythropoietin. In contrast to O-glycosylation, the highly conserved N-linked saccharides of erythropoietin are required for its secretion and in vivo biologic activity. With respect to secretion, glycosylation at either site 38 or 83 was sufficient but glycosylation at site 24 was not (Delorme et al., 1992), while complete loss of Nlinked glycosylation markedly impaired secretion of erythropoietin but incomplete glycosylation did not (Yamaguchi et al., 1991). Thus, erythropoietin produced in insect cells or certain mammalian cells which lack the capacity for complex glycosylation secreted erythropoietin in a comparable fashion to mammalian cells which have this capacity (Wojchowski et al., 1987; Wasley et al., 1991). Glycosylation of erythropoietin was also required for biologic activity in vivo and, in contrast to the differential effects on secretion, each N-linked site appeared to contribute equally to the in vivo activity of the hormone. This is presumably because their sialation prevents rapid degradation of
946 Jerry L. Spivak circulating erythropoietin since elimination or oxidation of the penultimate galactose residues also stabilized the hormone in the circulation (Spivak and Hogans, 1989). The erythropoietin aglycone produced in E. coli is said to have in vitro biologic activity but this has been incompletely quantified, while studies employing enzymatically deglycosylated erythropoietin appear to be confusing in this regard because of the definition of the extent of deglycosylation. Based on molecular weight determinations, fully deglycosylated but otherwise intact eukaryotic erythropoietin has very little in vitro biologic activity (Takeuchi et al., 1990). This is probably a consequence of a loss of stability or a change in conformation of the protein since the Nlinked glycosylation sites are not near its putative active sites. Thus, the saccharides of erythropoietin appear to be necessary to sustain the physical half-life of the hormone in the circulation and to maintain it in a biologically active conformation. Specific carbohydrate structural motifs on erythropoietin may also have roles in determining its circulatory residence time and rate of elimination. For example, it has been demonstrated that erythropoietin molecules rich in biantennary oligosaccharides not only have lower in vivo biologic activity, but are also preferentially cleared by the kidney (Misaizu et al., 1995). Furthermore, erythropoietin molecules containing three N-acetyl lactosamine repeats are preferentially cleared from the circulation by hepatocyte galactose receptors (Fukuda et al., 1989). Thus, there appear to be mechanisms operative in vivo which control the specific forms of erythropoietin that are physically available to the bone marrow.
CELLULAR SOURCES AND TISSUE EXPRESSION
Cellular sources that produce Early studies of erythropoietin production identified the kidneys and the liver as major sites for this. In the kidneys, erythropoietin is produced in peritubular fibroblastoid cells in the inner cortex and outer medulla (Koury et al., 1988; Lacombe et al., 1988). In the liver, erythropoietin is produced by hepatocytes and interstitial fibroblasts (Koury et al., 1991). With cloning of the erythropoietin gene, it was finally possible to establish definitively the sites of erythropoietin production using more sensitive and specific techniques. Under normoxic conditions, expression of erythropoietin mRNA was identified in the lung, testes, and brain, in addition to the liver and kidneys.
With hypoxia, erythropoietin mRNA was not only upregulated in these organs but became detectable in the spleen as well (Tan et al., 1992). Erythropoietin mRNA synthesis in an oxygen-dependent manner has also been demonstrated in murine, primate, and human brain in a broad distribution (Digicaylioglu et al., 1995) and erythropoietin has been identified in the cerebrospinal fluid (Marti et al., 1997). In mice, astrocytes were identified as a site of erythropoietin production (Masuda et al., 1994). Since neurons express erythropoietin receptors (Morishita et al., 1997) erythropoietin may have a role in maintaining neuronal viability under conditions of hypoxic stress (Sakanaka et al., 1998).
Eliciting and inhibitory stimuli, including exogenous and endogenous modulators Hypoxia is the only physiological stimulus for erythropoietin production (Reissmann, 1950). In the kidneys, erythropoietin production is constitutive and maximal in each cell. With hypoxia, additional renal interstitial fibroblasts are recruited to produce it in a watershed fashion (Koury et al., 1989). By contrast, in the liver, erythropoietin production occurs in every hepatocyte, is most marked in hepatocytes closest to central veins and with increasing hypoxia can be upregulated in individual hepatocytes (Koury et al., 1991). The characteristics of erythropoietin production in hepatic interstitial fibroblasts have not yet been investigated. During gestation, the liver is the major site of erythropoietin production and the switch to renal dominance appears to occur after birth (Zanjani et al., 1981; Dame et al., 1998). In adults, under both normoxic and hypoxic conditions, the kidneys are the major site of erythropoietin production. This is in part due to a differential sensitivity to hypoxia in this organ as compared to the liver. Thus, with mild hypoxia, the increase in erythropoietin mRNA is much greater in the kidneys than liver and only when hypoxia becomes severe is hepatic synthesis of the hormone substantial. Indeed, with severe hypoxia, the liver can account for over 35% of erythropoietin mRNA production (Tan et al., 1991). With renal parenchymal damage, hepatic erythropoietin production becomes even more dominant. However, it is never sufficient to sustain erythropoiesis at an adequate level when renal function is severely impaired or in the anephric state. This appears to be a consequence of the relative insensitivity of hepatocytes to hypoxia, possibly because of the dual blood supply
Erythropoietin 947 that the liver enjoys. Alternatively, it could be a consequence of a difference between fetal and adult hepatocytes. In this regard, it is well established that in patients with chronic renal failure amelioration of anemia could occur in the setting of acute hepatitis. Indeed, in one such anephric patient with acute viral hepatitis, plasma erythropoietin rose and the hematocrit was restored to normal transiently but subsequently both fell to their previous levels, even though chemical evidence of hepatocellular injury was still present. (Klassen and Spivak, 1990). This suggested that either erythropoietin production occurred as part of a recapitulation of ontogeny associated with hepatocyte regeneration or, less likely, that hepatocyte injury was associated with a greater reduction in erythropoietin catabolism than production. Chronic inflammatory or infectious disorders and neoplasms are commonly associated with anemia. With the development of a sensitive assay for erythropoietin and the identification of the cells and cell lines which produce the hormone, it has been possible to evaluate the mechanisms involved in the suppression of erythropoiesis by infection, inflammation, or neoplasia. The inflammatory cytokines, IL-1, TNF , and TGF inhibited erythropoietin production by Hep3B and Hep2G cells while IL-6 and IFN did not (Faquin et al., 1992; Jelkmann et al., 1992). IL-1 also inhibited erythropoietin production by an isolated perfused kidney (Jelkmann et al., 1992). IL-6, however, actually potentiated the effect of hypoxia on Hep3B cell erythropoietin production. Endotoxin (Schade and Fried, 1976), IL-1, TNF , TGF , and IFN also inhibited the proliferation of erythroid progenitor cells in response to erythropoietin (Means and Krantz, 1992), while IL-6 and its soluble receptor were capable of stimulating erythroid progenitor cell proliferation in the absence of erythropoietin (Sui et al., 1996). Renal erythropoietin production can be stimulated in vivo or in vitro by exposure to cobalt or by intrarenal injections of nickel but the mechanisms involved remained undefined because of lack of a model system to study the molecular regulation of erythropoietin production. With cloning of the erythropoietin gene it was finally possible to identify cells that produced erythropoietin in vitro in response to hypoxia. It is of interest that, to date, no renal cell line has been identified that displays this property, but several hepatoma cell lines have been useful in defining the mechanism for hypoxia-induced erythropoietin production (Goldberg et al., 1987). Oxygen-sensitive hepatoma cell lines respond to cobalt as well as hypoxia and also produce erythropoietin after exposure to nickel and manganese but
not zinc, iron, calcium, or tin (Goldberg et al., 1988). Cycloheximide inhibited cobalt, nickel and manganese-stimulated erythropoietin production, indicating that protein synthesis was required for transcriptional activation of the erythropoietin gene. Because these elements are interchangeable with iron in the porphyrin ring of heme and because their effects were not additive with respect to themselves or hypoxia, it appeared that a heme protein served as the oxygen sensor in erythropoietin-producing cells. Support for this conclusion was provided by the observation that carbon monoxide inhibited erythropoietin production by hypoxic hepatoma cells but not by those exposed to cobalt or nickel, to which carbon monoxide cannot bind. Additionally, desferrioxamine, which inhibits heme synthesis, also inhibited hypoxia-, cobalt- or nickel-stimulated erythropoietin synthesis (Goldberg et al., 1988). In this regard, it is of interest that iron deficiency can potentiate the production of erythropoietin out of proportion to the degree of tissue hypoxia (Kling et al., 1996a), which is consonant with the observation that desferrioxamine induced erythropoietin gene expression (Wang and Semenza, 1993). The process involved in the induction of erythropoietin production by hypoxia or cobalt appears to be a universal mechanism for oxygen-regulated gene expression. To date, genes that are regulated in this fashion include vascular endothelial growth factor (VEGF), heme oxygenase I, nitric oxide synthase, and a variety of glycolytic enzymes (Ratcliffe et al., 1995). The oxygen-sensing heme protein has not been identified to date, but a cytochrome b with NAD(P)H oxidase activity is considered to be a putative candidate (Bunn and Poyton, 1996). Identification of a cis-acting enhancer sequence in the 30 UTR of the erythropoietin gene provided an opportunity to define the activators involved in oxygen-regulated gene transcription. When this cisacting enhancer sequence was transfected into cells, it responded in an identical fashion in the presence of cobalt or hypoxia to its endogenous counterpart in the intact erythropoietin gene (Wang and Semenza, 1993). With this enhancer sequence, it was possible to identify the transactivating factor (hypoxia-inducible factor 1, HIF-1) that was responsible for activation of erythropoietin gene transcription (Wang et al., 1995a). As might be expected, renal innervation has no influence on oxygenmediated erythropoietin gene expression (Eckardt et al., 1992). Among the antioxidant vitamins, vitamin A (Jelkmann et al., 1997) and its derivative, retinoic acid (Okano et al., 1994) upregulate both renal and hepatic erythropoietin expression in vitro and in vivo.
948 Jerry L. Spivak HIF-1 is a heterodimeric DNA binding complex involving two basic helix-loop-helix PAS proteins (HIF-1 and HIF-1 ) (Wang et al., 1995a). HIF-1 is a novel, 120 kDa, nonheme iron-containing protein (Srinivas et al., 1998), while HIF-1 is the 91±90 kDa product of the aryl hydrocarbon nuclear translocator (ARNT) gene (Semenza et al., 1997). The HIF-1 heterodimer binds to a specific (TACGTGCT) 50 sequence in the 30 erythropoietin gene enhancer element and to p300, a non-DNA binding transcriptional activator through its subunit. HNF-4, an orphan nuclear steroid receptor, that binds to two random hormone-responsive elements in the 30 end of the enhancer, also binds to HIF-1 , completing the transcriptional complex (Huang et al., 1997). The expression of HIF-1 and mRNAs is ubiquitous and their protein products are upregulated by hypoxia, consistent with the universal role of this complex in modulating oxygen-regulated gene expression. Since the HIF-1 mRNAs are constitutively expressed, it appears that regulation of HIF-1 levels does not simply involve protein synthesis but also posttranslational stabilization of these proteins (Wenger et al., 1997). HIF-1 protein is present in excess of HIF-1 which is constitutively synthesized and rapidly degraded by the ubiquitin±proteasome pathway (Huang et al., 1998). HIF-1 is stabilized by hypoxia or iron chelation and dimerizes with HIF-1 (Kallio et al., 1997). Sulfhydryl reduction is also required for HIF-1 and dimerization. Importantly, dimerization is required not only for high-affinity DNA binding but also for the resistance to proteolysis which permits HIF-1 accumulation. Posttranslational modifications such as protein phosphorylation (both serine/threonine and tyrosine) that appear to be required not only for DNA binding but also protein synthesis confer another level of complexity on transcription factor complex behavior (Wang et al., 1995b). Finally, induction of both HIF1 activity and hypoxia-induced expression of erythropoietin is blocked by inhibitors of RNA or protein synthesis. Under normoxic conditions there is a low level of constitutive erythropoietin gene expression in hepatoma cell lines that is not suppressible by increasing tissue oxygenation. The half-life of erythropoietin mRNA is normally approximately 2 hours but is increased 3-fold in the presence of actinomycin D or cycloheximide, suggesting that a short-lived protein was involved in its degradation (Goldberg et al., 1991). With hypoxia, erythropoietin mRNA transcription was initiated within 30 minutes and increased exponentially, reached a maximal level by 6 hours and eventually leveled off or declined to approximately 50% of the maximum level achieved
(Schuster et al., 1987). While erythropoietin mRNA levels increase 50- to 100-fold in response to hypoxia, nuclear run-off studies indicated only a 5- to 10-fold increase in the actual rate of transcription (Goldberg et al., 1991). Resolution of this discrepancy was provided by the observation that with hypoxia not only was erythropoietin gene transcription increased but there was also an increase in the stability of its mRNA which was similar to that achieved under normoxic conditions with actinomycin or cycloheximide (Goldberg et al., 1991). The hypoxia-mediated stability of erythropoietin mRNA appears to be due to the upregulation of an erythropoietin mRNAbinding protein (Rondon et al., 1991; McGary et al., 1997) in association with a heat shock protein (Scandurro et al., 1997). Therefore, posttranscriptional events involving erythropoietin mRNA parallel those of HIF-1 and have an important role in the regulation of erythropoietin production.
RECEPTOR UTILIZATION Erythropoietin interacts with its target cells through a specific receptor that is a member of the hematopoietic growth factor receptor superfamily (Bazan, 1989, 1990b). The human erythropoietin receptor gene, located on chromosome 19, consists of eight exons and seven introns and codes for a 508 residue glycoprotein (Winkelmann et al., 1990) (507 in the mouse: D'Andrea et al., 1989). The promoter region of the gene contains CACC, GATA-1 and SP-1binding sites and one inhibitory and two enhancer regions 50 to the ATG intiation site. Erythroidspecific expression of the gene appears to be provided by elements both 50 and 30 to the promoter which exert a negative effect on nonerythroid tissues and elements within the first intron which appear to provide erythroid specificity (Youssoufian et al., 1993). The erythropoietin receptor is expressed not only by erythroid cells but also by embryonic stem cells (Schmitt et al., 1991), endothelial cells (Anagnostou et al., 1990), and neural cells (Morishita et al., 1997). The receptor has a 24 residue signal peptide, a 224 amino acid extracellular domain, a single 24 amino acid membrane-spanning domain, and a 236 amino acid cytoplasmic domain (D'Andrea et al., 1989). There are 11 cysteine residues but no disulfide bridges and only one N-linked glycosylation site. Based on its amino acid composition, the molecular weight of the erythropoietin receptor is 55,000 but after posttranslational processing apparent molecular weights as high as 78 kDa have been observed for
Erythropoietin 949 membrane-bound receptor (Sawyer and Hankins, 1993). The posttranslational modifications include glycosylation and tyrosine and serine-threonine phosphorylation. In general, the form of the receptor present in the plasma membrane has a higher molecular weight than its cytoplasmic counterpart, presumably due to phosphorylation. The isoelectric point of a purified baculovirus-expressed human erythropoietin receptor is 5.6 (Avedissian and Spivak, 1995). The erythropoietin receptor is a member of the hematopoietic growth factor receptor superfamily whose other members include the receptors for IL-2 ( chain), IL-3, IL-4, IL-5, IL-6, IL-7, GM-CSF, GCSF, thrombopoietin, leukemia-inhibitory factor (LIF), ciliary neutrotropic factor (CNTF), oncostatin M, growth hormone, and prolactin (Bazan, 1989, 1990b). They share in common four positionally conserved cysteines in their extracellular domain as well as a tryptophan-serine-X-tryptophan-serine (WSXWS) motif or its homolog located near the transmembrane region, and each lacks kinase motifs in their intracellular domain.
IN VITRO ACTIVITIES
In vitro findings Erythropoietin promotes the proliferation of its target cells and maintains their viability as they differentiate. The effect of the hormone varies with the particular target cell. For primitive erythroid progenitor cells (BFU-E) which express a low number of erythropoietin receptors (Sawada et al., 1990), erythropoietin acts as a mitogen, promoting their proliferation (Spivak et al., 1991). For late erythroid progenitor cells, which express a high number of erythropoietin receptors and are actively cycling, erythropoietin acts as a survival factor (Koury and Bondurant, 1990). The hormone is not required for erythroid cell differentiation since erythroid progenitor cells overexpressing Bcl-xL can differentiate in the absence of erythropoietin (Silva et al., 1996). In the absence of erythropoietin, erythroid cells become quiescent and enter a G0ÿG1 state (Spivak et al., 1996). Other growth factors also participate in stimulating the proliferation of erythroid progenitor cells. They include insulin-like growth factor type 1 (IGF-1) (Sawada et al., 1989), IL-3 (Goldwasser et al., 1983), stem cell factor (Dai et al., 1991), and thrombopoietin (Kobayashi et al., 1995). Other agents which can enhance the effects of erythropoietin include tyrosine kinase inhibitors such as zinc chloride and sodium vanadate and activators of protein kinase C such as bryostatin (Spivak et al., 1992).
Bioassays used The standard in vivo assay for erythropoietin is the exhypoxic polycythemic mouse assay which is relatively insensitive (lower limit of detection 0.05 U/mL), cumbersome, time-consuming, and expensive. In vitro assays for the hormone are more sensitive and specific and there are a variety of techniques from which to choose that differ with respect to their end points. The mouse spleen bioassay is based on the incorporation of labeled thymidine into the DNA of mouse splenic erythroblasts after exposure to erythropoietin (Krystal, 1983). In vitro clonal assays using freshly explanted marrow or peripheral blood mononuclear cells are based on the number of erythroid colonies which form in the presence of erythropoietin. These colony-forming assays which can employ either human or animal cells require strict attention to the components of the culture medium to ensure reproducibility (Spivak and Seiber, 1983). Erythropoietindependent cell lines are also useful for bioassay purposes and are, perhaps, the simplest, most direct method for the in vitro assay of erythropoietin. With the development of recombinant reagents, in vitro immunoassays that are specific, sensitive, and highly reproducible have replaced the bioassay procedures for clinical purposes (Egrie et al., 1987).
IN VIVO BIOLOGICAL ACTIVITIES OF LIGANDS IN ANIMAL MODELS
Normal physiological roles The major physiologic role of erythropoietin is to couple blood oxygen transport with long-term tissue oxygen requirements. Erythropoietin production is controlled at the level of its gene. Hypoxia upregulates erythropoietin production and hyperoxia suppresses erythropoietin production, although never completely. Because erythropoietin is a survival factor as well as a mitogen, it is produced constitutively and is always present in the plasma (Spivak and Hogans, 1987).
Species differences As mentioned previously, erythropoietin is highly conserved and, with the exception of guinea pig erythropoietin (Stopka et al., 1998a), species differences are negligible with respect to molecular homology: this is also true for the erythropoietin receptor.
950 Jerry L. Spivak
Knockout mouse phenotypes Both knockout and transgenic mouse studies have provided additional insight into the behavior of this highly conserved ligand±receptor pair. Mice heterozygous for erythropoietin or its receptor appear phenotypically normal. However, homozygosity for loss of either was embryonically lethal due to failure of definitive erythropoiesis (Wu et al., 1995). Importantly, however, committed erythroid progenitor cells were present in homozygous knockout mice, indicating that neither erythropoietin nor its receptor are required for lineage-specific commitment. This should not be a surprising observation given the overlapping functions of many of the growth factors which influence primitive hematopoietic progenitor cells (Walker et al., 1985), and also the stochastic nature of lineage±specific commitment (Nakahata et al., 1982). Indeed, immortalized, factor-independent multipotent hematopoietic progenitor cells are capable of lineage-specific commitment in the absence of either hematopoietic growth factors or serum (Fairbairn et al., 1993). Interestingly, thrombopoietin was capable of rescuing a fraction of the erythroid progenitor cells which lacked the receptor for erythropoietin in homozygous knockout mice (Kieran et al., 1996).
Transgenic overexpression Transgenic mice that overexpress erythropoietin behave in concordance with the lineage-restricted behavior of the hormone. Thus, these mice develop erythrocytosis but not leukocytosis or thrombocytosis (Semenza et al., 1989).
Pharmacological effects The major pharmacologic effect of erythropoietin is to increase the red blood cell mass, an effect which comes at the expense of the plasma volume (Lim et al., 1989). Other effects attributed to erythropoietin, such as hypertension or improved hemostasis, are a consequence of the increased red blood cell mass.
Interactions with cytokine network The flu-like syndrome experienced by patients receiving bolus injections of large quantities of recombinant erythropoietin in vivo may be a cytokine storm triggered by the hormone. In this regard, it is of interest that, while recombinant erythropoietin only
causes an increase in circulating red blood cells, it is associated with increases in the number of various types of hematopoietic progenitor cells in active cell cycle within the bone marrow without a change in their number in the blood (Dessypris et al., 1988). Myeloid growth factors also appear to have overlapping effects with erythropoietin. For example, GCSF can potentiate the effects of erythropoietin in vivo (Miles et al., 1991). Since most growth factors, in contrast to erythropoietin, are produced and act locally, it is likely that a number of synergistic interactions are occurring with respect to the growth factor network in the bone marrow, and we are as yet unaware of the details of this.
PATHOPHYSIOLOGICAL ROLES IN NORMAL HUMANS AND DISEASE STATES AND DIAGNOSTIC UTILITY
Normal levels and effects Cloning of the human erythropoietin gene provided an opportunity to develop the reagents necessary to measure accurately the quantity of erythropoietin in the circulation. The immunoassay for erythropoietin has proved to be useful because: 1. There is only one form of erythropoietin in the circulation. 2. Immunoreactive erythropoietin is equivalent to biologically active erythropoietin. 3. Erythropoietin is biochemically unique. 4. There are no preformed stores of the protein. 5. Production of erythropoietin is independent of its plasma level. 6. The plasma clearance of erythropoietin is independent of its plasma level. 7. Production of erythropoietin is controlled at the level of its gene. 8. There is no effect of age or gender on plasma erythropoietin. 9. Erythropoietin is always present in the plasma. 10. Its plasma level is constant in a given individual (Spivak, 1993). Erythroid progenitor cells metabolize erythropoietin and, therefore, the extent of bone marrow erythroid activity influences the plasma clearance of erythropoietin (Cazzola et al., 1998). Generally, marrow metabolism of erythropoietin is influenced by the same environmental factors which modify erythropoietin production and in the same direction.
Erythropoietin 951 For example, situations such as inflammation, infection, or neoplasia which downregulate erythropoietin production also impair erythroid progenitor cell proliferation, while the relative increase in plasma erythropoietin due to an increase in its production caused by tissue hypoxia depends on the extent of the marrow committed erythroid precursor pool. This is because erythropoietin is metabolized by its targets cells (Cazzola et al., 1998). Thus, in anemic patients the plasma erythropoietin level should be high if the kidneys are normal and marrow function is impaired, while a low plasma erythropoietin level in an anemic patient suggests impaired erythropoietin production alone. However, it is not possible in the latter situation to predict whether the marrow will respond to recombinant erythropoietin without a clinical trial; in the former situation, if the marrow is cellular and the erythropoietin level is greater than 1000 mU/mL, a response to exogenous erythropoietin is unlikely. There is a small diurnal variation in erythropoietin production with the highest levels occurring in the morning and this needs to be considered when comparative or serial measurements are made (Wide et al., 1989).
Role in experiments of nature and disease states Since tissue hypoxia is the only physiologic stimulus for erythropoietin production and production of the protein is regulated at the level of its gene, there is an inverse, log-linear relationship between plasma erythropoietin and tissue hypoxia whether measured by the hemoglobin level or arterial oxygen saturation. Unfortunately, however, plasma erythropoietin cannot simply be used as a surrogate measure of tissue oxygenation because of a central tendency for downregulation of erythropoietin production normally, and because of factors which positively or negatively regulate erythropoietin production. Thus, within the normal range for hemoglobin or hematocrit for both men and women, there is no linear correlation between plasma erythropoietin and the hemoglobin or hematocrit level (Spivak, 1993). Importantly, when anemia develops, even if it is mild, plasma erythropoietin will increase but because the normal range is wide (4±26 mU/mL), the level will not rise outside the normal range until the hemoglobin level falls below 10.5 mg% (hematocrit 30%) (Spivak, 1993). In states of compensated hemolytic anemia, the erythropoietin level generally does not rise indefinitely or even outside the normal range. This is also true in patients with tissue hypoxia due to cardiac shunts or obstructive lung disease (Wedzicha et al.,
1985; Haga et al., 1987). In part, this is due to the wide range of normal values for plasma erythropoietin and also to the increased metabolism of the hormone by the expanded marrow as well as by the suppressive effect on erythropoietin production by the increase in blood viscosity associated with the elevated red cell mass (Singh et al., 1993). Thus, a `normal' plasma erythropoietin level in a patient with erythrocytosis does not exclude hypoxia as a cause for the erythrocytosis. Other situations can elevate plasma erythropoietin, although only transiently, including chemotherapy which reduces erythroid progenitor cell proliferation and parenchymal liver damage when hepatocyte regeneration results in upregulation of erythropoietin production. For unknown reasons, zidovudine also causes an elevation in plasma erythropoietin (Spivak et al., 1989). Initially chemotherapy can increase erythropoietin levels (Birgegard et al., 1989), presumably by reducing the number of erythroid progenitors but prolonged treatment with certain chemotherapeutic drugs can also reduce erythropoietin production (Miller et al., 1990). A number of conditions are associated with impaired erythropoietin production. Intrinsic renal disease is the most common and once the serum creatinine rises above 1.5 mg%, the inverse linear correlation between hemoglobin and plasma erythropoietin is lost. However, it is important to note that patients with renal failure are capable of increasing erythropoietin production in response to hypoxia but the threshold for this is higher than normal and the response is not sustained (Walle et al., 1987; Chandra et al., 1988). In patients with diabetes mellitus, the effect of the disease on renal endocrine function is usually out of proportion to its effect on renal exocrine function and there can be impaired erythropoietin production and anemia without clinical evidence of a significant decrease in renal excretory function. Other situations with impaired erythropoietin production include inflammatory disorders, infections, cancer, pregnancy, surgery, and prematurity (Spivak, 1993), liver disease (Siciliano et al., 1995) and post bone marrow transplantation (Miller et al., 1992).
IN THERAPY
Preclinical ± How does it affect disease models in animals? Recombinant human erythropoietin is the most successful recombinant protein currently in clinical
952 Jerry L. Spivak use in terms of safety and efficacy. It can truly be considered a medical miracle since it provides most of the benefits of blood transfusion without transfusing blood. Its development was remarkably timely, occurring when the nation's blood supply was under duress, not only with respect to the adequacy of its inventory but also with respect to its safety. Indeed, with the focus of transfusion medicine on blood conservation rather than blood transfusion, recombinant erythropoietin was a welcome therapeutic addition. In comparison to the other hematopoietic growth factors, erythropoietin not only had the advantage of being the first to be discovered but also being one of the few such growth factors to behave like a hormone. As a consequence, its physiology was easier to define and erythropoietin deficiency could be directly identified by bioassay or immunoassay. Furthermore, since decades of study of erythropoietin physiology preceded the development of recombinant erythropoietin, a solid base of information was also available to delineate those situations in which the hormone would be most effective and to guide clinical trials. For example, the ability of exogenous erythropoietin to increase the red blood cell mass in both small and large animal models of renal insufficiency (Anagnostou et al., 1977; Mladenovic et al., 1985), as well as the documented impairment of erythropoietin production in anemic patients with end-stage renal disease (Gallagher et al., 1960), strongly supported the contention that recombinant erythropoietin would prove effective in correcting anemia in this group of patients. Additionally, both in vivo and in vitro models (DeGowin and Gibson, 1979; Dainiak et al., 1983) indicated that erythroid progenitor cells were responsive to exogenous erythropoietin in the presence of cancer or chemotherapeutic agents. In vitro studies (Shannon et al., 1987) also supported the contention that erythropoietin might be useful in the treatment of the anemia of prematurity while studies of serum erythropoietin using a more sensitive and specific immunoassay (Egrie et al., 1987) further defined specific groups of anemic patients in whom serum erythropoietin was inappropriately low and who might, therefore, be appropriate candidates for receiving the hormone. These included patients with infectious (Spivak et al., 1989) or inflammatory disorders (Hochberg et al., 1988) or neoplasia (Miller et al., 1990), premature infants (Kling et al., 1996b) and patients undergoing elective surgery (Clemens and Spivak, 1994) or bone marrow transplantation (Miller et al., 1992). Of course, before recombinant erythropoietin therapy is used all correctable causes for anemia must be excluded, and the patient should be symptomatic from the anemia or transfusiondependent.
Pharmacokinetics The plasma clearance of erythropoietin is complex and best explained by a two-compartment model with exponential clearance (Emmanouel et al., 1984; Steinberg et al., 1986; Spivak and Hogans, 1989). The volume of distribution of the hormone is slightly larger than the plasma volume and the initial phase of clearance reflects the distribution of the protein between the intravascular and extravascular spaces. Thereafter, there is monoexponential elimination of erythropoietin from the plasma. The half-time of elimination for plasma erythropoietin in humans is between 6 and 10 hours and is not appreciably affected by its plasma level, absence of the kidneys (McMahon et al., 1990), or cirrhosis of the liver (Jensen et al., 1995). Under normal circumstances, less than 10% of erythropoietin is eliminated by the kidneys (Emmanouel et al., 1984). In humans, erythropoietin does not cross the placenta (Widness et al., 1991). Administration of recombinant erythropoietin by subcutaneous or intraperitoneal injection results in remarkably different clearance kinetics than intravenous injection (MacDougall et al., 1989). In contrast to the exponential clearance from the plasma following intravenous injection, following subcutaneous or intraperitoneal administration there is a depot-like gradual increase in plasma erythropoietin followed by decline. Subcutaneous injection results in higher plasma levels than intraperitoneal injection. Importantly, although a higher plasma concentration of erythropoietin is achieved by intravenous administration, from a dose-response perspective, subcutaneous administration is approximately 30% more effective (Albitar et al., 1995; Kaufman et al., 1998). This may be due to its more sustained plasma residence time. Furthermore, the peak plasma levels of erythropoietin achieved by intravenous administration probably far exceed the available number of erythropoietin receptors, resulting in elimination of much of the hormone before it has any biological effect. The metabolic fate of erythropoietin over and above that utilized by erythroid progenitor cells is unknown. Following infusion of labeled recombinant human erythropoietin into rats, initial accumulation in the liver, spleen, kidneys, or bone marrow was insufficient to account for its elimination from the plasma, indicating equilibration with the extracellular space (Spivak and Hogans, 1989). No accumulation of acid-soluble, labeled metabolites occurred over the 4-hour period of observation, indicating that catabolism of the hormone did not contribute to its plasma clearance. Native erythropoietin eventually accumulated in the bone marrow but only to a
Erythropoietin 953 slightly greater extent than in the kidneys and spleen. Importantly, the plasma clearance kinetics of desialated, oxidized, biologically inactive erythropoietin were identical with native erythropoietin, indicating determinants other than its carbohydrate moieties or its biological activity could be involved in its catabolism under certain circumstances. Of course, repeated infusions of recombinant erythropoietin result in a shortening of its plasma half-life (McMahon et al., 1990), presumably due to an increase in the erythroid progenitor cell proliferation and metabolism of the hormone (Cazzola et al., 1998).
Toxicity In the decade since recombinant erythropoietin was first introduced into clinical practice, it has had a remarkable safety record, particularly in patients with normal renal function. The flu-like syndrome observed when erythropoietin was administered intravenously by bolus injection is rarely encountered when it is administered subcutaneously. Improvements in the excipient have also reduced the discomfort and local skin reactions associated with subcutaneous injection. Allergic reactions are rare, as is the development of antibodies to the recombinant protein. Hypertension or exacerbation of pre-existing hypertension occurred frequently in the initial clinical trials involving anemic hemodialysis patients (Eschbach et al., 1989). Although the frequency of hypertension is much lower in patients with normal renal function, they are not immune to this complication, which generally occurs in the first several months of therapy. Therefore, it is worth monitoring the blood pressure periodically during the early phase of therapy, particularly if there is pre-existing hypertension. Vascular thrombosis is the most serious side-effect of recombinant erythropoietin and, while hemostasis is improved with successful erythropoietin therapy (Moia et al., 1987), therapy-associated thrombosis is purely a function of the extent to which the red cell mass is increased. This should not be surprising since there is a direct linear correlation between the hematocrit and whole-blood viscosity. Indeed, a recent study indicated that elevating the hematocrit to 42% in hemodialysis patients was associated with an increased cardiovascular mortality (Besarab et al., 1998). While the mechanism for hypertension is still undefined, the reduction in the plasma volume (Lim et al., 1989) that accompanies the increase in red cell mass is undoubtedly involved. Thus, it is always prudent, particularly in patients with renal disease or preexisting hypertension, to initiate erythropoietin
therapy with a low dose (50 U/kg) of the hormone and to avoid excessive elevation of the hematocrit. There is no evidence from either in vitro studies or clinical trials that recombinant erythropoietin can promote the growth of tumor cells (Berdel et al., 1991). Erythropoietin can, however, stimulate extramedullary erthropoiesis and the development of splenomegaly and even splenic infarcts in patients with myelodysplasia or myeloproliferative disorders has been described. In this regard, recombinant erythropoietin has no significant influence on platelet production. Iron deficiency was a significant problem in renal dialysis patients receiving recombinant erythropoietin and emphasized the importance of an adequate supply of iron to sustain a response to the hormone. However, hemodialysis patients represent a unique population who are at constant risk of negative iron balance due to blood loss incurred through diagnostic phlebotomies, gastrointestinal hemorrhage or in dialyzer dead space. It is well established, however, that individuals with normal body iron stores can sustain a weekly blood loss of 500 mL while taking oral iron without developing significant anemia (Coleman et al., 1953). As a corollary, they can also increase their red cell mass in response to erythropoietin without receiving iron. As a general rule, if the serum ferritin is greater than 100 ng/mL in normal individuals or 200 ng/mL in patients with renal disease, body iron stores are sufficient for a maximal response to recombinant erythropoietin (Rutherford et al., 1994). Certainly, in the absence of iron deficiency, there is no evidence that parenteral iron as opposed to oral iron is a necessary adjunct to erythropoietin therapy.
Clinical results Anemia Associated with Renal Disease Since erythropoietin is produced primarily in the kidneys in adults and renal disease is associated with impaired erythropoietin production, anemic patients with end-stage renal disease were the first to participate in clinical trials of human recombinant erythropoietin (Winearls et al., 1986; Eschbach et al., 1987). As anticipated, recombinant erythropoietin proved to be remarkably effective in correcting anemia and alleviating transfusion requirements in 97% of patients (Eschbach et al., 1989). Importantly, correction of anemia was associated with improvement in the quality of life as a consequence of improved tissue oxygenation, cardiac function, muscle strength, and cognitive function (Evans et al., 1990). Recombinant erythropoietin therapy also proved to be effective in anemic patients with
954 Jerry L. Spivak predialysis renal failure (Watson et al., 1990). Contrary to initial concerns, recombinant erythropoietin did not accelerate renal failure in predialysis patients, was not associated with an increased incidence of seizures, dialyzer heparin requirements, hypercalcemia, or access thrombosis, at least with respect to native fistulas. Elevation of the hematocrit was also associated with an improvement in hemostasis (Moia et al., 1987). Subcutaneous administration of erythropoietin has proved to be more effective than intravenous administration (Albitar et al., 1995; Kaufman et al., 1998) and, most recently, administration of the total weekly dose of erythropoietin as a single injection has proved to be as effective as its administration in divided doses (Goldberg et al., 1996). In patients with end-stage renal disease, it is best to initiate erythropoietin therapy at a low dose (30±50 U/kg s.c.) and escalate gradually if necessary. Resistance to recombinant erythropoietin in patients with chronic renal failure can occur due to a variety of causes other than inadequate dosing. They include iron deficiency, infection, inflammation (surgery, connective tissue disorder), bleeding, aluminum toxicity, metabolic bone disease, folate deficiency, hemolysis, a hemoglobinopathy, or splenomegaly. Iron deficiency is the most common cause of erythropoietin resistance and may require the administration of intravenous iron if oral supplementation is not effective. The extent to which anemia needs to be corrected in patients with end-stage renal disease has been the subject of substantial debate not only from the perspective of cost but also because this particular patient population has a high incidence of cardiovascular disease. Furthermore, although erythropoietin can alleviate anemia and prevent iron overload, it does not change the need for dialysis. Thus, while it appears that a target hematocrit of 33% is too low, the extent to which it should be raised above 36% is unclear. A recent study of anemic hemodialysis patients with ischemic heart disease or congestive heart failure indicated that elevation of the hematocrit to 42% was associated with an increased risk of cardiacrelated morbidity and mortality (Besarab et al., 1998). Anemia Associated with Cancer Anemia is a common complication of cancer and, although there are multiple reasons for this, the most common cause is impairment of erythropoietin production presumably secondary to the elaboration of inflammatory cytokines (Means and Krantz, 1992) or chemotherapy (Miller et al., 1990). At the same time, erythroid progenitor cells from cancer patients were found to be responsive to erythropoietin in vitro, although the response was somewhat blunted
(Dainiak et al., 1983). Importantly, however, chemotherapeutic agents did not abrogate erythropoietin-responsiveness (Reissmann and Udupa, 1972). To date, four randomized, double-blind, placebo-controlled studies have established that recombinant human erythropoietin can alleviate the anemia associated with cancer and its chemotherapy and reduce transfusion requirements (Abels et al., 1991; Cascinu et al., 1994; Heiss et al., 1996; Wurnig et al., 1996). The overall response rate irrespective of the type or malignancy or treatment regimen was approximately 60%. In the absence of chemotherapy, the response rate appeared to be lower for hematologic malignancies than solid tumors. Failure to respond to erythropoietin had significant prognostic implications because such patients usually had a shorter survival (Ludwig et al., 1994). A reduction in transfusion requirements was obtained in approximately 50% of patients but is rarely absolute, since it takes 4±8 weeks for recombinant erythropoietin to elevate the hemoglobin level (Glaspy et al., 1997). Importantly, erythropoietin therapy has been shown to improve the quality of life in responders (Demetri et al., 1998). Unfortunately, no study to date has demonstrated that the use of erythropoietin perioperatively either alone or in conjunction with autologous blood donation results in a reduction in allogeneic blood exposure in cancer patients. Since there is no evidence that allogeneic blood exposure promotes tumor metastases, recombinant erythropoietin cannot be recommended for perioperative use in cancer patients (Busch et al., 1993). With respect to dose, it has been established that 300 U/kg is not better than 150 U/kg. Approximately 30,000±40,000 U/week should be an effective dose in most patients and this can be administered once a week subcutaneously (Goldberg et al., 1998). Given the importance of cost considerations, algorithms have been developed to predict which patients are most likely to respond. An erythropoietin level greater than 400 mU/mL in the absence of chemotherapy suggested that a response was unlikely (Osterborg et al., 1996). Failure to achieve an increase in hemoglobin of at least 0.5 g after 2 weeks of therapy (or > 1 g after 4 weeks), together with a serum erythropoietin level of > 100 mU/mL, or a serum ferritin level of > 400 ng/mL, were also highly predictive for nonresponders (Ludwig et al., 1994). The reticulocyte count is, unfortunately, a sensitive indicator of responsiveness (Henry et al., 1995). Although recombinant erythropoietin is only approved for use in preventing symptomatic anemia in patients with nonmyeloid malignancies undergoing chemotherapy, it has been widely used in patients with anemia due to myelodysplasia (Mittleman and
Erythropoietin 955 Lessin, 1994). In this situation the hormone has proved to be most effective in patients with refractory anemia or refractory anemia with ringed sideroblasts. However, responses have been obtained in patients with all types of myelodysplasia regardless of cytogenetic status unless the serum erythropoietin level was greater than 1000 mU/mL. In many patients, the addition of G-CSF may be necessary to obtain a response to erythropoietin (Negrin et al., 1993, 1996; Hellstrom-Lindberg et al., 1998). Recombinant erythropoietin therapy does not seen to accelerate the underlying myelodyplasia but can cause splenomegaly and splenic infarction. Anemia Associated with HIV Infection Anemia is a common complication of HIV infection and is multifarious with respect to its etiology (Holland and Spivak, 1990). Serum erythropoietin levels are depressed in HIV-infected patients (Spivak et al., 1989) and HIV is capable of suppressing erythropoietin production by Hep3B cells in vitro (Wang et al., 1993). Although HIV does not directly infect erythroid progenitor cells (Potts et al., 1992), the immunosuppressed state it creates permits other pathogens such as MAI, parvovirus B19, or cytomegalovirus to attack the marrow and inhibit erythropoiesis. Anemia is a poor prognostic sign in HIV-infected patients (Moore et al., 1998; Sullivan et al., 1998) and blood transfusions can activate HIV expression in infected individuals (Mudido et al., 1996). Furthermore, the antiretroviral agent zidovudine suppresses erythropoiesis. These observations led to clinical trials of recombinant erythropoietin in anemic HIV-infected patients (Henry et al., 1992). These trials demonstrated that erythropoietin therapy could alleviate anemia and reduce tranfusion requirements in patients receiving zidovudine whose endogenous erythropoietin level was less than 500 mU/mL. In a large open-label trial, using doses of 40,000 U s.c. 6 days per week, transfusion requirements were reduced by 50%, and 44% of the patients archived a 6% increase in hematocrit (Phair et al., 1993). Combined therapy with erythropoietin and G-CSF was also effective in 70% of patients receiving zidovudine who were unable to tolerate this drug because of marrow toxicity (Miles et al., 1991). Correction of anemia by erythropoietin in HIV-infected patients was also associated with an improvement in quality of life (Revicki et al., 1994). HIV infection does not appear to impair erythropoietin-responsiveness in patients with end-stage renal disease (Ifudu et al., 1997). Although high does of zidovudine are no longer used in treating HIV infection, the recognition that anemia has an adverse
impact on survival in HIV-infected patients and that a response to erythropoietin is associated with improved survival suggests that recombinant erythropoietin may still have a role in the management of anemic HIV-infected patients. Perioperative Use Most blood use occurs with surgical procedures and, given public concern about blood safety, the perioperative use of recombinant erythropoietin has received intense scrutiny. There is a certain irony to this since the blood supply is safer now then ever before due to the development of sensitive tests for the detection of known viral pathogens. Indeed, the risk of acquiring HIV by transfusion is equivalent to the risk of a fatal hemolytic transfusion reaction (Popovski, 1998). The impetus to employ recombinant erythropoietin perioperatively in part stemmed from the observation that erythropoietin production did not increase substantially unless the hemoglobin fell below 10.5 g% (Spivak, 1993), and that autologous blood donors bled weekly did not increase erythropoietin production substantially and become progressively anemic (Kickler and Spivak, 1988). Furthermore, the time available before surgery is sometimes insufficient to collect the quantity of blood needed to avoid allogeneic blood exposure. Thus, it appeared that recombinant erythropoietin might have a useful role in the perioperative period. Unfortunately, this has proved not to be the case. Rather, it was observed that, with a more aggressive blood donation schedule, nonanemic, iron-replete individuals with an adequate blood volume (> 5000 mL) could donate the quantity of blood desired (Biesma et al., 1994). However, estimated blood needs and actual blood requirements proved not to be equal since surgical blood loss is not predictable. Thus, neither erythropoietin (Canadian Perioperative Orthopedic Erythropoietin Study Group, 1993) nor erythropoietin coupled with autologous blood donation prevented exposure to allogeneic blood in either nonanemic or anemic patients (Goodnough et al., 1989, 1994). Given the current safety of the blood supply, autologous blood donation is not cost-effective (Etchason et al., 1995) and obviously erythropoietin therapy does not improve the situation. While recombinant erythropoietin is now approved clinically for use in mildly anemic patients (hemoglobin < 13.5 g% > 11.5 g%) undergoing noncardiac surgical procedures, it must be remembered that it may not abrogate allogeneic blood exposure under these conditions. Whether erythropoietin together with acute normovolemic hemodilution will be superior to
956 Jerry L. Spivak autologous blood donation remains to be determined but recent results are encouraging (Ness et al., 1992) and may be applicable to patients with cardiac disease who cannot donate autologously (Sowade et al., 1997) and also to adherents of the Watchtower Society who refuse blood transfusion on a religious basis. Individuals with rare blood groups, severe alloimmunization, a small blood volume, or severe IgA deficiency are other candidates for perioperative use of recombinant erythropoietin.
may be unavoidable; in part due to more conservative transfusion and phlebotomy usage as well as improved transfusion safety; and finally, in part to unavoidable medical complications which affect the need for transfusion independently of erythropoietin therapy. When recombinant erythropoietin is used for the anemia of prematurity, its use should be restricted to very low birth weight infants and at a dose of not more than 750 U/kg week (Maier et al., 1998) in combination with iron supplementation.
Anemia of Prematurity
Miscellaneous Uses of Recombinant Erythropoietin
After birth, there is a reduction in hemoglobin which is most marked in premature infants, often resulting in the need for transfusions (Keyes et al., 1989). Studies of serum erythropoietin postpartum indicate that, while these infants are capable of producing erythropoietin in response to hypoxia, their response is blunted when compared to the responses of adults to a similar degree of anemia (Brown et al., 1984). Since the number of circulating erythroid progenitor cells is not reduced in premature infants and their sensitivity to erythropoietin is normal, it was concluded that inadequate erythropoietin production was a major factor in the anemia of prematurity (Shannon et al., 1987). A recent evaluation of erythropoietin pharmacokinetics challenges that assumption in part, since it demonstrated that the plasma clearance of erythropoietin was accelerated in premature infants (Widness et al., 1996). Given the evidence of increased circulating erythroid progenitor cells in premature infants, this should not be a surprising observation (Shannon et al., 1987). Thus, although there appears to be a defect in erythropoietin production in infants, it is apparently magnified by an increase in erythropoietin metabolism. Because of the documented adverse effects of blood transfusion, erythropoietin therapy was widely embraced for the treatment of the anemia of prematurity (Maier et al., 1994; Ohls et al., 1997; Kumar et al., 1998; Shannon et al., 1998). The results of these trials indicate that recombinant erythropoietin therapy is safe in premature infants but that the dose required to enhance erythropoiesis was larger than the effective dose in adults, that erythropoietin therapy was not beneficial in infants weighing more than 1000 g and that, despite erythropoietin therapy, exposure to allogeneic blood could not be avoided (Strauss, 1994). Furthermore, the cost±benefit ratio of employing erythropoietin was not favorable (Fain et al., 1995). This was in part due to the fact that it takes several weeks for erythropoietin therapy to be effective and within this time interval transfusions
Recombinant erythropoietin has been demonstrated to ameliorate anemia in patients with rheumatoid arthritis (Pincus et al., 1990) and inflammatory bowel disease (Schreiber et al., 1996), to accelerate erythropoiesis after allogenic but not autologous marrow transplantation (Miller et al., 1992), to improve blood pressure control in patients with orthostatic hypotension (Hoelktke and Streeten, 1993), and to improve erythropoiesis in patients with aplastic anemia (Bessho et al., 1990) or thalassemia major (Rachmilewitz et al., 1995). However, the extent to which its use can be justified in these situations on a cost±benefit basis has not been established. Nevertheless, given patient perceptions about blood safety as well as the demonstrated improvement in quality of life associated with alleviation of anemia, it is difficult to fault the appropriate use of the hormone in `off-label' situations. Indeed, whenever it can be shown that recombinant erythropoietin alleviates the need for blood transfusions, an argument can be made for its use.
References Abels, R. I., Larholt, K. M., Krantz, K. D., and Bryand, E. C. (1991). In ``Blood Cell Growth Factors'' (ed M. J. Murphy), Recombinant human erythropoietin (r-HuEPO) for the treatment of the anemia of cancer pp. 122±140. AlphaMed Press, Dayton, OH. Albitar, S., Meulders, Q., Hammoud, H., Soutif, C., Bouvier, P., and Pollini, J. (1995). Subcutaneous versus intravenous administration of erythropoietin improves its efficiency for the treatment of anaemia in haemodialysis patients. Nephrol. Dial. Transplant. 6, 40±43. Anagnostou, A., Barone, J., Kedo, A., and Fried, W. (1977). Effect of erythropoietin therapy on the red cell volume of uraemic and non-uraemic rats. Br. J. Hematol. 37, 85±91. Anagnostou, A., Lee, E. S., Kessimian, N., Levinson, R., and Steiner, M. (1990). Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells. Proc. Natl Acad. Sci. USA 87, 5978±5982. Avedissian, L. S., and Spivak, J. L. (1995). In ``Human Protein Data'' (ed A. Haebeck), Erythropoietin receptor. VCH Verlagsgesellschaft, Weinheim.
Erythropoietin 957 Avedissian, L. S., Poola, I., and Spivak, J. L. (1995). Ligand binding kinetics of a soluble full-length murine erythropoietin receptor. Biochem. Biophys. Res. Commun. 216, 62±68. Bartley, T. D., Bogenberger, J., Hunt, P., Li, Y.-S., Lu, H. S., Martin, F., Chang, M. S., Samal, B., Nichol, J. L., Switft, S., Johnson, J., Hsu, R. Y., Parker, V. P., Suggs, S., Skrine, J. D., Metewether, L. A., Clogston, C., Hsu, E., Hokom, M. M., Hornkohl, A., Choie, E., Pangelinan, M., Sun, Y., Mar, V., McNinch, J., Simonet, L., Jacbsen, F., Xie, C., Shutter, J., Chute, H., Basu, R., Selander, L., Trollinger, D., Sieu, L., Padilla, D., Trail, G., Xu, W., Del Castillo, K., Biron, J., Cole, S., Hu, M. C. T., Pacific, R., Ponting, I., Saris, C., Wen, D., Yung, Y. P., Lin, H., and Bosselman, R. A. (1994). Identification and cloning of a megakaryocyte growth and development factor that is a ligand for the cytokine receptor Mpl. Cell 77, 1117±1124. Bazan, J. F. (1989). A novel family of growth factor receptors. Biochem. Biophys. Res. Commun. 164, 788±796. Bazan, J. F. (1990a). Haemopoietic receptors and helical cytokines. Immunol. Today 11, 350±354. Bazan, J. F. (1990b). Structural design and molecular evolution of a cytokine receptor superfamily. Proc. Natl Acad. Sci. USA 87, 6934±6938. Berdel, W. E., Oberberg, D., Reufi, B., and Thiel, E. (1991: Studies on the role of recombinant human erythropoietin in the growth regulation of human nonhematopoietic tumor cells in vitro. Ann. Hematol. 63, 5±8. Beru, N., Smith, D., and Goldwasser, E. (1990). Evidence suggesting negative regulation of the erythropoietin receptor by ribonucleoprotein. J. Biol. Chem. 265, 14100±14104. Besarab, A., Bolton, W. K., Browne, J. K., Egrie, J. C., Nissenson, A. R., Okamoto, D. M., Schwab, S. J., and Goodkin, D. A. (1998). The effects of normal as compared with low hematocrit values in patients with cardiac disease who are receiving hemodialysis and epoietin. N. Engl. J. Med. 339, 584±901. Bessho, M., Hirashima, K., Asano, S., Ikeda, Y., Ogawa, N., Tomonaga, M., Toyama, K., Nakahata, T., Nmura, T., Mizoguchi, H., Yoshida, Y., Niitsu, Y., and Kohogo, Y. (1997). Treatment of the anemia of aplastic anemia patients with recombinant human erythropoietin in combination with granulocyte colony-stimulating factor: a multicenter randomized controlled study. Eur. J. Hematol. 58, 265±272. Biesma, D. H., Marx, J. J. M., Karaaijenhagen, R. J., Franke, W., Messinger, D., and van de Wiel, A. (1994). Lower homologous blood requirment in autologous blood donors after treatment with recombinant human erythropoietin. Lancet 334, 367±370. Birgegard, G., Wide, L., and Simonsson, B. (1989). Marked erythropoietin increase before fall in Hb after treatment with cytostatic drugs suggests mechanism other than anaemia for stimulation. Br. J. Haematol. 72, 462±466. Boissel, J. P., Lee, W.-R., Presnell, S. R., Cohen, F., and Bunn, H. F. (1993). Erythropoietin structure-function relationships. J. Biol. Chem. 268, 15983±15993. Brown, M. S., Garcia, J. F., Phibbs, R. H., and Dallman, P. R. (1984: Decreased response of plasma immunoreactive erythropoietin to ``available oxygen'' in anemia of prematurity. J. Pediatr. 105, 793±798. Bunn, J. F., and Poyton, R. O. (1996). Oxygen sensing and molecular adaptation to hypoxia. Physiol. Rev. 76, 839. Busch, O. R. C., Hop, W. C. J., Hoynck van Papendrecht, M. A. W., Marquet, R. L., and Jeekel, J. (1993). Blood transfusions and prognosis in colorectal cancer. N. Engl. J. Med. 32, 1372±1376.
Canadian Orthopedic Perioperative Erythropoietin Study Group (1993). Effectivness of perioperative recombinant human erythropoietin in elective hip replacement. Lancet 341, 1227± 1232. Casadevall, N., Dupuy, E., Molho-Sabatier, P., Tobelem, G., Varet, B., and Mayeux, P. (1996). Autoantibodies against erythropoietin in a patient with pure red-cell aplasia. N. Engl. J. Med. 334, 630±633. Cascinu, S., Fedeli, A., Del Ferro, E., Luzi Fedeli, S., and Catalano, G. (1994). Recombinant human erythropoietin treatment in cisplatin-associated anemia: a randomized, doubleblind trial with placebo. J. Clin. Oncol. 12, 1058±1062. Cazzola, M., Gurnone, R., Cerani, P., Centenara, E., Rovati, A., and Beguin, Y. (1998). Red blood cell precursor mass as an independent determinant of serum erythropoietin level. Blood 91, 2139±2145. Chandra, M., Clemons, G. K., and McVicar, M. I. (1988). Relation of serum erythropoietin levels to renal excretory functions: evidence for lowered set point for erythropoietin production in chronic renal failure. J. Pediatr. 113, 1015±1021. Clemens, J., and Spivak, J. L. (1994). Serum immunoreactive erythropoietin during the perioperative period. Surgery 115, 510±515. Coleman, D. H., Stevens, A. R., Dodge, H. T., and Finch, C. A (1953). A rate of blood regeneration after blood loss. Arch. Intern. Med. 92, 341±349. Dai, C. H., Krantz, S. B., and Zsebo, K. M. (1991). Human burstforming units-erythroid need direct interaction with stem cell factor for further development. Blood 78, 2493±2524. Dainiak, N., Kulkarni, V., Howard, D., and Kalamanti, M. (1983). Mechanisms of abnormal erythropoiesis in malignancy. Cancer 51, 1101±1106. Dame, C., Fahnenstich, H., Freitag, P., Hofmann, D., AbdulNour, T., Bartmann, P., and Fandrey, J. (1998). Erythropoietin mRNA expression in human fetal and neonatal tissue. Blood 92, 3218±3225. Damen, J. E., and Krystal, G. (1996). Early events in erythropoietin-induced signaling. Exp. Hematol. 24, 1455±1459. D'Andrea, A. D., Lodish, H. F., and Wong, G. G. (1989). Expression cloning of the murine erythropoietin receptor. Cell 57, 277±285. Davis, J. M., and Arakawa, T. (1987). Characterization of recombinant human erythropoietin produced in Chinese hamster ovary cells. Biochemistry 26, 2633±2638. DeGowin, R. L., and Gibson, D. P. (1979). Erythropoietin and the anemia of extramedullary tumor. J. Lab. Clin. Med. 94, 303±311. Delorme, E., Lorenzini, T., Giffin, J., Martin, F., Jacobsen, F., Boone, T., and Elliott, S. (1992). Role of glycosylation on the secretion and biological activity of erythropoietin. Biochemistry 31, 9871±9876. Demchuk, E., Mueller, T., Oschkinat, H., Sebald, W., and Wade, R. C. (1994). Receptor binding properties of fourhelix-bundle growth factors deduced from electrostatic analysis. Protein Sci. 3, 920±935. Demetri, G. D., Kris, M., Wade, J., Degos, L., and Cella, D. (1998). Quality-of-life benefit in chemotherapy patients treated with epoetin alfa is independent of disease response. J. Clin. Oncol. 16, 3412±3425. Dessypris, E., Graber, S. E., Krantz, S. B., and Stone, W. J. (1988). Effects of recombinant erythropoietin on the concentration and cycling status of human marrow hematopoietin progentior cells in vivo. Blood 72, 2060±2062. De Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992). Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science 255, 306±312.
958 Jerry L. Spivak Digicaylioglu, M., Bichet, S., Marti, H. H., Wenger, R. H., Rivas, L. A., Bauer, C., and Gassmann, M. (1995). Localization of specific erythropoietin binding sites in defined areas of the mouse brain. Proc. Natl Acad. Sci. USA 92, 3717± 3720. Dube, S., Fisher, J. W., and Powell, J. S. S. (1988). Glycosylation at specific sites of erythropoietin is essential for biosynthesis, secretion and biological function. J. Biol. Chem. 263, 17516± 17521. Eckardt, K.-U., LeHir, M., Tan, C. C., Ratcliffe, P. J., Kaissling, B., and Kurtz, A. (1992). Renal innervation plays no role in oxygen-dependent control of erythropoietin mRNA levels. Am. J. Physiol. 263, F925±F930. Egrie, J. C., Cotes, P. M., Lane, J., Gaines Das, R. E., and Tam, R. C. (1987). Development of radioimmunoassays for human erythropoietin as tracer and immunogen. J. Immunol. Methods 99, 235±241. Elliott, S., Lorenzini, T., Chang, D., Brazilay, J., Delorme, E., Griffin, J., and Hesterberg, L. (1996). Fine-structure epitope mapping of antierythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure. Blood 87, 2702±2713. Elliott, S., Lorenzini, T., Chang, D., Barzilay, J., and Delorme, E. (1997). Mapping of the active site of recombinant human erythropoietin. Blood 89, 493±502. Emmanouel, D. S., Goldwasser, E., and Katz, A. I. (1984). Metabolism of pure human erythropoietin in the rat. Am. J. Physiol. 247, F168±F176. Erslev, A. (1953). Humoral regulation of red cell production. Blood 8, 349±357. Eschbach, J. W., Egrie, J. C., Dowining, M. R., Browne, J. K., and Adamson, J. W. (1987). Correction of the anemia of endstage renal disease with recombinant human erythropoietin. N. Engl. J. Med. 321, 73±78. Eschbach, J. W., Abdulhardi, M. H., Browne, J. K., Delano, B. C., Downing, M. R., Egrie, J. C., Evans, R. W., Friedman, E. A., Graber, S. E., and Haley, N. R. (1989). Recombinant human erythropoietin in anemic patients with end-stage renal disease. Results of a phase III multicenter clinical trial. Ann. Intern. Med. 111, 992±1000. Etchason, J., Petz, L., Keeler, E., Calhoun, L., Kleinman, S., Snider, C. Y., Fink, A., and Brook, R. (1995). The cost effectiveness of preoperative autologous blood donations. N. Engl. J. Med. 332, 719±724. Evans, R. W., Rader, B., and Mannine, D. L. (1990). The quality of life of hemodialysis recipients treated with recombinant human erythropoietin. J. Am. Med. Assoc. 263, 825±830. Fain, J., Hilsenrath, P., Widness, J. A., Strauss, R. G., and Mutnick, A. H. (1995). A cost analysis comparing erythropoietin and red cell transfusions in the treatment of anemia of prematurity. Transfusion 35, 936±943. Fairbairn, L. J., Cowling, G. J., Reipert, B. M., and Dexter, T. M. (1993). Suppression of apoptosis allows differentiation and development of a multipotent hemopoietic cell line in the absence of added growth factors. Cell 74, 823±832. Faquin, W. C., Schneider, T. J., and Goldberg, M. A. (1992). Effect of inflammatory cytokines on hypoxia-induced erythropoietin production. Blood 79, 1987±1994. Fukuda, M. N., Sasaki, H., Lopez, L., and Fukuda, M. (1989). Survival of recombinant erythropoietin in the circulation: the role of carbohydrates. Blood 73, 84±89. Gallagher, N. J., McCarthy, J. M., and Lange, R. D. (1960). Observations on erythropoietic stimulating factor (ESF) in the plasma of uremic and non-uremic anemia patients. Ann. Intern. Med. 52, 1201±1212.
Galson, D. L., Tan, C. C., Ratcliffe, P. J., and Bunn, H. F. (1993). Comparison of the human and mouse erythropoietin genes shows extensive homology in the flanking regions. Blood 82, 3321±3326. Glaspy, J., Bukowski, R., Steinberg, T. C., Taylor, C., Tchekmedyian, S., and Vadhan-Raj, S. (1997). Impact of therapy with epotein alfa on clinical outcomes in patients with nonmyeloid malignancies during cancer chemotherapy in community oncology practice. J. Clin. Oncol. 15, 1218±1234. Goldberg, M. A., Glass, G. A., Cunningham, J. H., and Bunn, H. F. (1987). The regulated expression of erythropoietin by two human hepatoma cell lines. Proc. Natl Acad. Sci. USA 84, 7972±7976. Goldberg, M. A., Dunning, S. P., and Bunn, H. F. (1988). Regulation of the erythropoietin gene: evidence that the oxygen sensor is a heme protein. Science 242, 1412±1414. Goldberg, M. A., Gaut, C. C., and Bunn, H. F. (1991). Erythropoietin mRNA levels are governed by both the rate of gene transcription and posttranscriptional events. Blood 77, 271±277. Goldberg, M. A., McGutchen, J. W., Jove, M., Di Cesare, P., Friedman, R. J., Pos, R., Guilfoyle, M., Frei, D., and Young, D. (1996). A safety and efficacy comparison study of two dosing regimens of epoetin alfa in patients undergoing major orthopedic surgery. Am. J. Orthop. 25, 544±556. Goodnough, L. T., Rudnick, S., Price, T. H., Ballas, S. K., Collins, M. L., Crowley, J. P., Kosman, M., Drushall, M. S., Lenes, B. A., Menitove, J. E., Silberstein, L. E., Smithe, K. J., Wallas, C. H., Abels, R., and Von Tress, M. (1989). Increased preoperative collection of autologous blood with recombinant human erythropoietin therapy. N. Engl. J. Med. 321, 1163± 1168. Goodnough, L. T., Price, T. H., Friedman, K. D., Johnson, M., Ciavarella, D., Khan, N., Sacher, R., Vogler, W. R., Wissel, M., and Abels, R. I. (1994). A phase III trial of recombinant human erythropoietin therapy in nonanemic orthopedic patients subjected to aggressive removal of blood for autologous use: dose, response, toxicity, and efficacy. Transfusion 34, 66±71. Grant, W. E., and Root, W. S. (1947). The relation of O2 in bone marrow blood to posthemorrhagic erythropoiesis. Am. J. Physiol. 150, 618±627. Haga, P., Cotes, P. M., Till, J. A., Minty, B. D., and Shinebourne, E. A. (1987). Serum immunoreactive erythropoietin in children with cyanotic and acyanotic congenital heart disease. Blood 70, 822±826. Heiss, M. M., Tarabichi, A., Delanoff, C., Allgayer, H., Jauch, K. W., Hernandez-Richter, T., Mempel, W., Beck, K. G., Schildberg, F. W., and Messmer, K. (1996). Perisurgical erythropoietin application in anemic patients with colorectal cancer: a double-blind randomized study. Surgery 119, 523±527. Hellstrom-Lindberg, E., Ahlgren, T., Beguin, Y., Carlsson, M., Carneskog, J., Dahl, I. M., Dybedal, I., Grimfors, G., KanterLewensohn, L., Linder, O., Luthman, M., Lofvenberg, E., Nilsson-Ehle, H., Samuelsson, J., Tangen, J. M., Winqvist, I., Oberg, G., Osterborg, A., and Ost, A. (1998). Treatment of anemia in myelodysplastic syndromes with granulocyte colonystimulating factor plus erythropoietin: results from a randomized phase II study and long-term follow-up of 71 patients. Blood 92, 68±75. Henry, D. H., Beall, G. N., Benson, C. A., Carey, J., Cone, L. A., Eron, L. J., Fiala, M., Fischl, M. A., Gabin, S. F., Gottlieb, M. S., Galpin, J. E., Groopman, J. E., Hooton, T. H., Memsek, J. G., Levin, R. L., Miles, S. A., Riehart, J. J., Rios, A., Robbins, W. J., Ruckdeschel, J. C., Smith, J. A., Spruance, S. L., Starrett, B.,
Erythropoietin 959 Toney, J., Zalusky, R., Abels, R. I., Bryand, E. C., Larholt, K. M., Sampson, A. R., and Rudnick, S. A. (1992). Recombinant human erythropoietin in the treatment of anemia associated with human immunodeficiency virus (HIV) infection and zidovudine therapy. Ann. Intern. Med. 117, 739±747. Henry, D., Abels, R., and Larholt, K. (1995). Prediction of response to recombinant human erythropoietin (r-HuEPO/ epoetin) therapy in cancer patients. Blood 85, 1676±1678. Higuchi, M., Oh-eda, M., Kuboniwa, H., Tomonoh, K., Shimonaka, Y., and Ochi, N. (1992). Role of sugar chains in the expression of the biological activity of human erythropoietin. J. Biol. Chem. 267, 7703±7709. Hochberg, M. C., Arnold, C. M., Hogans, B. B., and Spivak, J. L. (1988). Serum immunoreactive erythropoietin in rheumatoid arthritis: impaired response to anemia. Arthritis Rheum. 31, 1318±1321. Hoelktke, R. D., and Streeten, D. H. P. (1993). Treatment of orthostatic hypotension with erythropoietin. N. Engl. J. Med. 329, 611±615. Holland, K., and Spivak, J. L. (1990). Haematological manifestations of the acquired immunodeficiency syndrome. Clin. Haematol. 3, 103±109. Huang, L. E., Ho, V., Arany, Z., Krainc, D., Galson, D., Tendler, D., Livingston, D. M., and Bunn, H. F. (1997). Erythropoietin gene regulation depends on heme-dependent oxygen sensing and assembly of interacting transcription factors. Kidney Int. 51, 548±552. Huang, L. E., Gu, J., Schau, M., and Bunn, H. F. (1998). Regulation of hypoxia-inducible factor 1 is mediated by an O2-dependent degradation domain via the ubiquitin-proteasome pathway. Proc. Natl Acad. Sci. USA 95, 7987±7992. Ifudu, O., Matthew, J. J., Mayers, J. D., Macey, L. J., Brezsnyak, W., Reydel, C., McLendon, E., Surgrue, T., Rao, S., and Friedman, E. A. (1997). Severity of AIDS and the response to EPO in uremia. Am. J. Kidney Dis. 30, 28±35. Imagawa, S., Yamaoto, M., and Miura, Y. (1997). Negative regulation of the erythropoietin gene expression by the GATA transcription factors. Blood 89, 1430±1439. Jacobs, K., Shoemaker, C., Rudersdorf, R., Neill, S. D., Kaufman, R. J., Mufson, A., Seebra, J., Jones, S. S., Hewick. R., Fritsch, E. F., Kawakita, M., Shimizu, T., and Miyake, T. (1985). Isolation and characterization of genomic and cDNA clones of human erythropoietin. Nature 313, 806±810. Jacobson, L. E., Goldwasser, E., Fried, W., and Plzak, L. (1957). Role of the kidney in erythropoiesis. Nature 179, 633±634. Jelkmann, W., Pagel, H., Wolff, M., and Fandrey, J. (1992). Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys. Life Sci. 30, 301±308. Jelkmann, W., Pagel, H., Hellwig, T., and Fandrey, J. (1997). Effects of antioxidant vitamins on renal and hepatic erythropoietin production. Kidney Int. 51, 497±501. Jensen, J. D., Jensen, L. W., Madsen, J. K., and Poulsen, L. (1995). The metabolism of erythropoietin in liver cirrhosis patients compared with healthy volunteers. Eur. J. Haematol. 54, 111±116. Kallio, P. J., Pongratz, I., Gradin, K., McGuire, J., and Poellinger, L. (1997). Activation of hypoxia-inducible factor 1 : posttranscriptional regulation and conformational change by recruitment of the Arnt transcription factor. Proc. Natl Acad. Sci. USA 94, 5667±5672. Kaufman, J. S., Reda, D. J., Fye, C. L., Goldfarb, D. S., Henderson, W. G., Kleinman, J. S., and Vaamonde, C. A. (1998). Subcutaneous compared with intravenous epoetin in patients receiving hemodialysis. N. Engl. J. Med. 399, 578±583.
Keller, G., Kennedy, M., Papayannopoulou, T., and Wiles, M. V. (1993). Hematopoietic commitment during embryonic stem cell differentiation in culture. Mol. Cell. Biol. 13, 473±486. Keyes, W. G., Donohue, P. K., Spivak, J. L., Jones, M. D., and Oski, F. A. (1989). Assessing the need for transfusion of premature infants and role of hematocrit clinical signs and erythropoietin level. Pediatrics 84, 412±417. Kickler, T., and Spivak, J. L. (1988). Effect of repeated phlebotomy on serum immunoreactive erythropoietin levels in autologous blood donors. J. Am. Med. Assoc. 260, 65±67. Kieran, M. W., Perkins, A. C., Orkin, S. H., and Zon, L. I. (1996). Thrombopoietin rescues in vitro erythroid colony formation from mouse embryos lacking the erythropoietin receptor. Proc. Natl Acad. Sci. USA 93, 9126±9131. Klassen, D. K., and Spivak, J. L. (1990). Hepatitis-related hepatic erythropoietin production. Am. J. Med. 89, 684±686. Kling, P. J., Dragsten, P. R., Roberts, R. A., Dos Santos, B., Brooks, D. J., Hedlung, B. E., and Taetle, R. (1996a). Iron deprivation increases erythropoietin production in vitro, in normal subjects and patients with malignancy. Br. J. Haematol. 95, 241±248. Kling, P. J., Schmidt, R. L., Roberts, R. A., and Widness, J. A. (1996b). Serum erythropoietin levels during infancy; associations with erythropoiesis. J. Pediatr. 128, 791±796. Kobayashi, M., Laver, J. H., Kato, T., Miyazaki, H., and Ogawa, M. (1995). Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors. Blood 86, 2494±2499. Koury, M. J., and Bondurant, M. C. (1990). A survival model of erythropoietin action. Science 248, 378±381. Koury, S. T., Bondurant, M. C., and Koury, M. J. (1988). Localization of erythropoietin synthesizing cells in murine kidneys by in situ hybridization. Blood 71, 524±527. Koury, S. T., Koury, M. J., Bondurant, M. C., Caro, J., and Graber, S. E. (1989). Quantitation of erythropoietin-producing cells in kidneys of mice by in situ hybridization: correlation with hematocrit, renal erythropoietin mRNA and serum erythropoietin concentration. Blood 74, 645±651. Koury, S. T., Bondurant, M. C., Koury, M. J., and Semenza, G. L. (1991). Localization of cells producing erythropoietin in murine liver by in situ hybridization. Blood 77, 2469±2471. Krieg, M., Marti, H. H., and Plate, K. H. (1998). Coexpression of erythropoietin and vascular endothelial growth factor in nervous system tumors associated with von Hippel-Lindau tumor suppressor gene loss of function. Blood 92, 3322±3389. Krystal, G. (1983). A simple microassay for erythropoietin based on 3H-thymidine incorporation into spleen cells for phynylhydrazine treated mice. Exp. Hematol. 11, 649±660. Kumar, P., Shankaran, S., and Krishnan, R. G. (1998). Recombinant human erythropoietin therapy for treatment of anemia of prematurity in very low birth weight infants; a randomized, double-blind, placebo-controlled trial. J. Perinatol. 18, 173±177. Lacombe, C., Da Silva, J. L., Bruneval, P., Fournier, J. G., Wending, F., Casadevall, N., Camilleri, J. P., Bariety, J., Varet, B., and Tambourin, P. (1988). Peritubular cells are the site of erythropoietin synthesis in the murine hypoxic kidney. J. Clin. Invest. 81, 620±623. Law, M. L., Cai, G.-Y., Lin, K., Wei, Q., Huang, S. Z., Hartz, J. H., Morse, H., Lin, C. H., Jones, C., and Kao, F. T. (1986). Chromosomal assignment of the human erythropoietin gene and its DNA polymorphism. Proc. Natl Acad. Sci. USA 83, 6920±6924. Lim, V. F., Degrown, R. L., Zavala, D., Kirchner, P. T., Abels, R., Perry, P., and Fangman, J. (1989). Recombinant human
960 Jerry L. Spivak erythropoietin treatment in pre-dialysis patients. Ann. Intern. Med. 110, 104±108. Lin, F.-K., Suggs, S., Lin, C.-H., Browne, J. K., Smalling, R., Egrie, J. C., Chen. K. K., Fox, G. M., Martin, F., Stabinsky, Z., Badrawi, S. M., Lai, P.-H., and Goldwasser, E. (1985). Cloning and expression of the human erythropoietin gene. Proc. Natl Acad. Sci. USA 82, 7580±7584. Ludwig, H., Fritz, E., Leitgeb, C., Perchestorfer, M., Samonigg, H., and Schuster, J. (1994). Prediction of response to erythropoietin treatment in chronic anemia of cancer. Blood 84, 1056±1063. McDonald, J. D., Lin, F.-K., and Goldwasser, E. (1986). Cloning, sequencing and evolutionary analysis of the mouse erythropoietin gene. Mol. Cell. Biol. 6, 842±848. MacDougall, I. C., Neubert, P., Coles, G. A., Roberts, D. E., Dharmasena, A. D., and Williams, J. D. (1989). Pharmacokinetics of recombinant human erythropoietin in patients on continuous ambulatory peritoneal dialysis. Lancet 1, 425±427. McGary, E. C., Rondon, I. J., and Beckman, B. S. (1997). Posttranscriptional regulation of erythropoietin mRNA stability by erythropoietin mRNA-binding protein. J. Biol. Chem. 272, 8628±8634. McMahon, F. G., Vargas, R., Ryan, M., Jain, A. K., Abels, R. I., Perry, B., and Smith, I. L. (1990). Pharmacokinetics and effects of recombinant human erythropoietin after intravenous and subcutaneous injections in healthy volunteers. Blood 76, 1718±1722. Maier, R. F., Obladen, M., Kattner, E., Natzschka, J., Messer, J., Regazzoni, B. M., Speer, C. P., Fellman, V., Grauel, E. L., Groneck, P., Wagner, M., Moriette, G., Salle, B. L., Verellen, G., and Scigalla, P. (1998). High-versus low-dose erythropoietin in extremely low birth weight infants. J. Perinatol. 132, 866±870. Maier, R. F., Obladen, M., Scigalla, P., Linderkamp, O., Duc, G., Hieronimi, G., Halliday, H. L., Versmold, H. T., Moriette, G., and Jorch, G. (1994). The effect of epoietin (recombinant human erythropoietin) on the need for transfusion in very low-birth-weight infants. N. Engl. J. Med. 330, 1173±1178. Marti, H. H., Gassmann, M., Wenger, R. H., Kvietikova, I., Morganti-Kossmann, M. C., and Kossmann, T. (1997). Detection of erythropoietin in human liquor: intrinsic erythropoietin production in the brain. Kidney Int. 51, 416±418. Masuda, S., Okano, M., Yamgishi, K., Nago, M., Ueda, M., and Saski, R. (1994). A novel site of erythropoietin production. Oxygen-dependent production in cultured rat astrocytes. J. Biol. Chem. 269, 19488±19493. Matthews, D. J., Topping, R. S., Cass, R. T., and Giebel, L. B. (1996). A sequential dimerization mechanism of erythropoietin receptor activation. Proc. Natl Acad. Sci. USA 93, 9471±9476. Means Jr, R. T., and Krantz, S. B. (1992). Progress in understanding the pathogenesis of the anemia of chronic disease. Blood 80, 1639±1647. Middleton, S. A., Johnson, D. L., Jin, R., McMahon, F. J., Collins, A., Tullai, J., Gruninger, R. H., Jolliffe, L. K., and Mulcahy, L. S. (1996). Identification of a critical ligand binding determinant of the human erythropoietin receptor. Evidence for common ligand binding motifs in the cytokine receptor family. J. Biol. Chem. 271, 14045±14054. Miles, S. A., Mitsuyasu, R. T., Moreno, J., Baldwin, G., Alton, N. K., Souza, L., and Glaspy, J. A. (1991). Combined therapy with recombinant granulocyte colony-stimulating factory and erythropoietin decreases hematologic toxicity from zidovudine. Blood 77, 2109±2117. Miller, C. B., Jones, R. J., Piantadosi, S., Abeloff, M. D., and Spivak, J. L. (1990). Decreased erythropoietin response in
patients with the anemia of cancer. N. Engl. J. Med. 322, 1689±1692. Miller, C. B., Jones, R. J., and Zahurak, M. L. (1992). Impaired erythropoietin response to anemia after bone marrow transplantation. Blood 80, 2667±2682. Misaizu, T., Matsuki, S., Strickland, T. W., Takeuchi, M., Kobata, A., and Takasakai, S. (1995). Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin. Blood 86, 4097±4104. Mittleman, M., and Lessin, L. S. (1994). Clinical application of recombinant erythropoietin in myelodysplasia. Hematol./Oncol. Clin. N. Am. 8, 993±1009. Miyake, T., Kung, C. K.-H., and Goldwasser, E. (1977). Purification of human erythropoietin. J. Biol. Chem. 252, 5558±5564. Mladenovic, J., Eschback, J. W., Koup, J. R., Garcia, J. F., and Adamson, J. W. (1985). Erythropoietin kinetics in normal and uremic sheep. J. Lab. Clin. Med. 105, 659±663. Moia, M., Mannucci, P. M., Vizzotto, L., Masati, S., Cattaneo, M., and Ponticelli, C. (1987). Improvement in the haemostatic defect of uraemia after treatment with recombinant human erythropoietin. Lancet 2, 1227±1229. Moore, R. D., Keruly, J. C., and Chaisson, R. E. (1998). Anemia and survival in HIV infection. J. AIDS Hum. Retrov. 19, 29±33. Morishita, E., Masuda, S., Nagao, M., Yasuda, Y., and Sasaki, R. (1997). Erythropoietin receptor is expressed in rat hippocampal and cerebral cortical neurons, and erythropoietin prevents in vitro glutamate-induced neuronal death. Neuroscience 76, 105±116. Mudido, P. M., Georges, D., Dorazio, D., Yen-Lieberman, B., Base, S., O'Brien, W. A., Spritzler, J., and Lederman, M. M. (1996). Human immunodeficiency virus type 1 activation after blood transfusion. Transfusion 36, 860±865. Nakahata, T., Gross, A. J., and Ogawa. M. (1982). A stochastic model of self-renewal and commitment to differentiation of the primitive hemopoietic stem cells in culture. J. Cell. Physiol. 113, 455±458. Narhi, L. O., Arakawa, T., Aoki, K. H., Elmore, R., Rohde, M. F., Boone, T., and Strickland, T. W. (1991). The effect of carbohydrate on the structure and stability of erythropoietin. J. Biol. Chem. 266, 23022±23026. Negrin, R. S., Stein, R., Vardiman, J., Doherty, K., Cornwell, J., Krantz, S., and Greenberg, P. L. (1993). Treatment of the anemia of myelodysplastic syndromes using recombinant human granulocyte colony-stimulating factor in combination with erythropoietin. Blood 82, 737±743. Negrin, R. S., Stein, R., Doherty, K., Cornwell, J., Vardimann, J., Krantz, S., and Greenberg, P. L. (1996). Maintenance treatment of the anemia of myelodysplastic syndromes with recombinant human granulocyte colony-stimulating factor and erythropoietin: evidence for in vivo synergy. Blood 87, 4076±4081. Ness, P. M., Bourke, D. L., and Walsh, P. C. (1992). A randomized trial of perioperative hemodilution versus transfusion of preoperatively deposited autologous blood in elective surgery. Transfusion 32, 226±230. Ohls, R. K., Harcum, J., Schibler, K. R., and Christensen, R. D. (1997). The effects of erythropoietin on the transfusion requirements of preterm infants weighing 750 grams or less; a randomized, double-blind, placebo-controlled study. J. Pediatr. 131, 653±655. Okano, M., Masuda, S., Narita, H., Masushige, S., Kato, S., Imagawa, S., and Saski, R. (1994). Retinoic acid up-regulates erythropoietin production in hepatoma cells and in vitamin Adepleted rats. FEBS Lett. 349, 229±233. Osterborg, A., Boogaerts, M. A., Cimino, R., Essers, U., Holowiecki, J., Juliusson, G., Jager, G., Najman, A., and
Erythropoietin 961 Peest, D. (1996). Recombinant human erythropoietin in transfusion-dependent anemic patients with multiple myeloma and non-Hodgkin's lymphoma ± a randomized multicenter study. The European study group of erythropoietin (epoetin ) treatment in multiple myeloma and non-Hodgkin's lymphoma. Blood 87, 2675±2682. Phair, J. P., Abels, R. I., McNeill, M. V., Pharm, D., and Sullivan, D. J. (1993). Recombinant human erythropoietin treatment; investigational new drug protocol for the anemia of the acquired immunodeficiency syndrome. Arch. Intern. Med. 153, 2669±2675. Philo, J. S., Aoki, K. H., Arakawa, T., Narhi, L. O., and Wen, J. (1996). Dimerization of the extracellular domain of the erythropoietin (EPO) receptor by EPO; one high-affinity and one lowaffinity interaction. Biochemistry 35, 1681±1691. Potts, B., Hogan, M. D., Lamperth, L., and Spivak, J. L. (1992). Replication of HIV-1 and HIV-2 in human bone marrow cultures. Virology 188, 840±849. Popovski, M. A. (1998). Infection and America's blood supply: a 1998 status report. Am. J. Clin. Pathol. 109, 659±661. Powell, J. S., Berkner, K. L., Lebo, R. V., and Adamson, J. W. (1986). Human erythropoietin gene: high level expression in stably transfected mammalian cells and chromosome localization. Proc. Natl Acad. Sci. USA 83, 6465±6469. Rachmilewitz, E. A., Aker, M., Perry, D., and Dover, G. (1995). Sustained increase in haemoglobin and RBC following longterm administration of recombinant human erythropoietin to patients with homozygous -thalassaemia. Br. J. Haematol. 90, 341±345. Ratcliffe, P. J., Ebert, B. L., Ferguson, D. J., Firth, J. D., Gleadle, J. M., Maxwell, P. H., and Pugh, C. W. (1995). Regulation of the erythropoietin gene. Nephrol. Dial. Transplant. 10, 18±27. Recny, M. A., Scoble, H. A., and Kim, Y. (1987). Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin, identification of des-arginine 166 erythropoietin. J. Biol. Chem. 262, 17156±17163. Reissmann, K. R. (1950). Studies on the mechanism of erythropoietic stimulation in parabiotic rats during hypoxia. Blood 5, 372±380. Reissmann, K. R., and Udupa, K. B. (1972). Effect of erythropoietin on proliferation of erythropoietin-responsive cells. Cell Tissue Kinet. 5, 481±489. Revicki, D. A., Brown, R. E., Henry, D. H., McNeill, M. V., Rios, A., and Watson, T. (1994). Recombinant human erythropoietin and health-related quality of life of AIDS patients with anemia. J. AIDS 7, 474±484. Rondon, I. J., MacMillan, L. A., Beckman, B. S., Goldberg, M. A., Schneider, T., Bunn, H. F., and Malter, J. S. (1991). Hypoxia up-regulates the activity of a novel erythropoietin mRNA binding protein. J. Biol. Chem. 25, 16594±16598. Rutherford, C. J., Schneider, T. J., Dempsy, H., Kirn, D. H., Brugnara, C., and Goldberg, M. A. (1994). Efficacy of different dosing regimens for recombinant human erythropoietin in a simulated perisurgical setting: the importance of iron availability in optimizing response. Am. J. Med. 96, 139±145. Sakanaka, M., Wen, T. C., Matsuda, S., Mauda, S., Morishita, E., Nagao, M., and Sasaki, R. (1998). In vivo evidence that erythropoietin protects neurons from ischemic damage. Proc. Natl Acad. Sci. USA 95, 4635±4640. Sasaki, H., Bothner, B., Dell, A., and Fukuda, M. (1987). Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem. 262, 12059±12076.
Sawada, K., Krantz, S. B., Dessypris, E. N., Koury, S. T., and Sawyer, S. T. (1989). Human colony-forming units-erythroid do not require accessory cells, but do require direct interaction with insulin-like growth factor I and/or insulin for erythroid development. J. Clin. Invest. 83, 1701±1709. Sawada, K., Krantz, S. B., Dai, C.-H., Koury, S. T., Horn, S. T., Glick, A. D., and Civin, C. I. (1990). Purification of human blood burst-forming units-erythroid and demonstration of the evolution of erythropoietin receptors. J. Cell. Physiol. 142, 219±230. Sawyer, S. T., and Hankins, W. D. (1993). The functional form of the erythropoietin receptor is a 78-kDa protein: correlation with cell surface expression, endocytosis, and phosphorylation. Proc. Natl Acad. Sci. USA 90, 6849±6853. Scandurro, A. B., Rondon, I. J., Wilson, R. B., Tenenbaum, S. A., Garry, R. F., and Beckman, B. S. (1997). Interaction of erythropoietin RNA requires an association with heat shock protein 70. Kidney Int. 2, 579±584. Schade, S. G., and Fried, W. (1976). Suppressive effect of endotoxin on erythropoietin-responsive cells in mice. Am. J. Physiol. 231, 73±76. Schmitt, R. M., Bruyns, E., and Snodgrass, H. R. (1991). Hematopoietic development of embryonic stem cells in vitro: cytokine and receptor gene expression. Genes Dev. 5, 728±740. Schneider, H., Chaovapong, W., Matthews, D. J., Karkaria, C., Cass, R. T., Zhan, H., Boyle, M., Lorenzini, T., Elliott, S. G., and Giebel, L. B. (1997). Homodimerization of erythropoietin receptor by a bivalent monoclonal antibody triggers cell proliferation and differentiation of erythroid precursors. Blood 89, 473±482. Schreiber, S., Howaldt, S., Schnoor, M., Nikolaus, S., Bauditz, J., Gasche, C., Lochs, H., and Raedler, A. (1996). Recombinant erythropoietin for the treatment of anemia in inflammatory bowel disease. N. Engl. J. Med. 334, 619±623. Schuster, S. J., Wilson, J. H., Erslev, A. J., and Caro, J. (1987). Physiologic regulation and tissue localization of renal erythropoietin messenger RNA. Blood 70, 316±318. Semenza, G. L., and Wang, G. L. (1992). A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol. Cell. Biol. 12, 5447±5454. Semenza, G. L., Traystman, M. D., Gearheart, J. D., and Antonarakis. S. E. (1989). Polycythemia in transgenic mice expressing the human erythropoietin gene. Proc. Natl Acad. Sci. USA 86, 2301±2305. Semenza, G. L., Koury, S. T., Nejfelt, M. K., Gearhart, J. D., and Antonarakis, S. E. (1991). Cell-type-specific and hypoxia-inducible expression of the human erythropoietin gene in transgenic mice. Proc. Natl Acad. Sci. USA 88, 8725±8729. Semenza, G. L., Agani, F., Booth, G., Forsythe, J., Iyer, N., Jiang, B. H., Leung, S., Roe, R., Wiener, C., and Yu, A. (1997). Structural and functional analysis of hypoxia-inducible factor 1. Kidney Int. 51, 553±555. Shannon, K. M., Naylor, G. S., Torkildson, J. C., Clemons, G. K., Schaffner, V., Goldman, S. L., Lewis, K., Bryant, P., and Phibbs, R. (1987). Circulating erythroid progenitors in the anemia of prematurity. N. Engl. J. Med. 317, 728±733. Shannon, K. M., Keith, J. F., 3rd Mentzer, W. C., Ehrenkranz, R. A., Brown, M. S., Widness, J. A., Gleason, C. A., Bifano, E. M., Millard, D. D., and Davis, C. B. (1998). Recombinant human erythropoietin stimulated erythropoiesis and reduces erythrocyte transfusions in very low birth weight preterms infants. Pediatrics 95, 1±8. Shaw, G., and Kamen, R. (1986). A conserved AU sequence from the 30 untranslated region of GM-CSF mRNA mediates selective mRNA degradation. Cell 46, 659±667.
962 Jerry L. Spivak Shoemaker, C. B., and Mitsock, L. D. (1986). Murine erythropoietin gene: cloning, expression and human gene homology. Mol. Cell. Biol. 6, 849±858. Siciliano, M., Tomasello, D., Milani, A., Ricerca, B. M., Storti, S., and Rossi, L. (1995). Reduced serum levels of immunoreactive erythropoietin in patients with liver cirrhosis and chronic anemia. Hepatology 22, 1132±1137. Silva, M., Grillot, D., Benito, A., Richard, C., Nunez, G., and Fernandez-Luna, J. L. (1996). Erythropoietin can promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2. Blood 88, 1576±1582. Singh, A., Eckardt, K. U., Zimmerman, A., Gotz, K. H., Hamann, M., Ratcliffe, P. J., Kurtz, A., and Reinhart, W. H. (1993). Increased plasma viscosity as a reason for inappropriate erythropoietin formation. J. Clin. Invest. 91, 251±256. Sowade, O., Warnke, H., Scigall, P., Sowade, B., Franke, W., Messinger, D., and Gross, J. (1997). Avoidance of allogeneic blood transfusions by treatment with epoetin (recombinant human erythropoietin) in patients undergoing open-heart surgery. Blood 89, 411±418. Spivak, J. L. (1993). The clinical physiology of erythropoietin. Semin. Hematol. 30, 2±11. Spivak, J. L., and Hogans, B. B. (1987). Clinical evaluation of a radioimmunoassay for serum erythropoietin using reagents derived from recombinant erythropoietin. Blood 70, 143a. Spivak, J. L., and Hogans, B. B. (1989). The in vivo metabolism of recombinant human erythropoietin in the rat. Blood 73, 90±99. Spivak, J. L., and Seiber, F. (1983). Erythropoietin. Horm. Norm. Abnorm. Hum. Tissues 3, 63±96. Spivak, J. L., Barnes, D. C., Fuchs, E., and Quinn, T. C. (1989). Serum immunoreactive erythropoietin in HIV-infected patients. J. Am. Med. Assoc. 261, 3104±3107. Spivak, J. L., Pham, T., Isaacs, M., and Hankins, W. D. (1991). Erythropoietin is both a mitogen and a survival factor. Blood 77, 1228±1233. Spivak, J. L., Fisher, J., Isaacs, M. A., and Hankins, D. (1992). Protein kinases and phosphatases are involved in erythropoietin mediated signal transduction. Exp. Hematol. 20, 500± 504. Spivak, J. L., Ferris, D. K., Fisher, J., Noga, S. J., Isaacs, M., Connor, E., and Hankins, W. D. (1996). Cell cycle-specific behavior of erythropoietin. Exp. Hematol. 24, 141±150. Srinivas, V., Zhu, X., Salceda, S., Nakamura, R., and Caro, J. (1998). Hypoxia-inducible factor 1 (HIF-1) is a non-heme iron protein. J. Biol. Chem. 273, 18019±18022. Steinberg, S. E., Garcia, J. F., Matzke, G. R., and Mladenovic, J. (1986). Erythropoietin kinetics in rats: generation and clearance. Blood 67, 646±649. Stopka, T., Zivny, J. H., Goldwasser, E., Prchal, J. F., Nescas, E., and Prchal, J. T. (1998a). Guinea pig serum erythropoietin (EPO) selectively stimulates guinea pig erythroid progenitors: human or mouse erythroid progenitors do not form erythroid burst-forming unit colonies in response to guinea pig serum EPO. Exp. Hematol. 26, 910±914. Stopka, T., Zivny, J. H., Goldwasser, E., Prchal, J. F., Nescas, E., and Prchal, J. T. (1998b). Human hematopoietic progenitors express erythropoietin. Blood 91, 3766±3772. Strauss, R. (1994). Erythropoietin and neonatal anemia. N. Engl. J. Med. 330, 1227±1228. Sui, X., Tsuji, K., Tajima, S., Tanaka, R., Muraoka, K., Ebihara, Y., Ikebuchi, K., Yasukawa, K., Taga, T., Kishimoto, T., and Nakahata, T. (1996). Erythropoietinindependent erythrocyte production; signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34 cells. J. Exp. Med. 183, 837±845.
Sullivan, P. S., Hanson, D. L., Chu, S. Y., Jones, J. L., and Ward, J. W. (1998). Epidemiology of anemia in human immunodeficiency virus (HIV)-infected persons: results from the multistate adult and adolescent specturm of HIV disease surveillance project. Blood 91, 301±308. Syed, R. S., Reid, S. W., Li, C., Cheetham, J. C., Aoki, K. H., Liu, B., Zhan, H., Osslund, T. D., Chirino, A. J., Zhang, J., FinerMoore, J., Elliott, S., Sitney, K., Katz, B. A., Matthews, D. J., Wendoloski, J. J., Egrie, J., and Stroud, R. M. (1998). Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature 395, 511±516. Takeuchi, M., Takasaki, S., Shimada, M., and Kobata, A. (1990). Role of sugar chains in the in vitro biological activity of human erythropoietin produced in recombinant Chinese hamster ovary cells. J. Biol. Chem. 265, 12127±12130. Tan, C. C., Eckardt, K.-U., and Ratcliffe, P. J. (1991). Organ distribution of erythropoietin messenger RNA in normal and uremic rats. Kidney Int. 40, 69±76. Tan, C. C., Eckardt, K.-U., and Firth, J. D. (1992). Feedback modulation of renal and hepatic erythropoietin mRNA in response to graded anemia and hypoxia. Am. J. Physiol. 263, F474±F481. Walker, F., Nicola, N. A., Metcalf, D., and Burgess, A. W. (1985). Hierarchical down-modulation of hemopoietic growth factor receptors. Cell 43, 269±276. Walle, A. J., Wong, G. Y., Clemons, G. K., Garcia, J. F., and Niedermayer, W. (1987). Erythropoietin-feedback circuit in the anemia of end-stage renal disease. Kidney Int. 31, 1205± 1209. Wang, G. L., and Semenza, G. L. (1993). Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction. Blood 82, 3610±3615. Wang, Z., Goldberg, M. A., and Scadden, D. T. (1993). HIV-1 suppresses erythropoietin production in vitro. Exp. Hematol. 21, 683±688. Wang, G. L., Jiang, B.-H., Rue, E. A., and Semenza, G. L. (1995a). Hypoxia-inducible factor 1 is a basic-helix-loop-helixPAS heterodimer regulated by cellular O2 tension. Proc. Natl Acad. Sci. USA 92, 5510±5514. Wang, G. L., Jiang, B. H., and Semenza, G. L. (1995b). Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1. Biochem. Biophys. Res. Commun. 216, 669±675. Wasley, L. C., Timony, G., Murtha, P., Stoudemire, J., Dorner, A. J., Caro, J., Krieger, M., and Kaufman, R. J. (1991). The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin. Blood 77, 2624±2632. Watson, A. J., Gimenez, L. F., Cotton, S., Walser, M., and Spivak, J. L. (1990). Treatment of the anemia of chronic renal failure with subcutaneous recombinant human erythropoietin. Am. J. Med. 89, 432±435. Wedzicha, J. A., Cotes, P. M., Empey, D. W., Newland, A. C., Royston, J. P., and Tam, R. C. (1985). Serum immunoreactive erythropoietin in hypoxic lung disease with and without polycythemia. Clin. Sci. 69, 413±422. Wen, D., Boissel, J.-P. R., Tracy, T. E., Gruninger, R. H., Mulcahy, L. S., Czelusniak, J., Goodman, M., and Bunn, H. F. (1993). Erythropoietin structure±function relationships: high degree of sequence homology among mammals. Blood 82, 1507±1516. Wenger, R. H., Kvietikova, I., Rolfs, A., Gassmann, M., and Marti, H. H. (1997). Hypoxia-inducible factor-1 is regulated at the post-mRNA level. Kidney Int. 51, 560±563.
Erythropoietin 963 Wide, L., Bengtsson, C., and Birgegard, G. (1989). Circadian rhythm of erythropoietin in human serum. Br. J. Haematol. 72, 85±90. Widness, J. A., Sawyer, S. T., Schmnidt, R. L., and Chestnut, D. H. (1991). Lack of maternal to fetal transfer of 125 I-labelled erythropoietin in sheep. J. Dev. Physiol. 15, 139±143. Widness, J. A., Veng-Pedersen, P., Peters, C., Pereira, L. M., Schmidt, R. L., and Lowe, L. S. (1996). Erythropoietin pharmacokinetics in premature infants:developmental, nonlinearity, and treatment effects. J. Appl. Physiol. 80, 140±148. Winearls, C. G., Pippard, M. J., Dowinging, M. R., Oliver, D. O., Reid, C., and Cotes, P. M. (1986). Effect of human erythropoietin derived from recombinant DNA on the anaemia of patients maintained by chronic haemodialysis. Lancet 2, 1175±1178. Winkelmann, J. C., Penn, L. A., Deaven, L. L., Forget, B. G., and Jenkins, R. B. (1990). The gene for the human erythropoietin receptor: analysis of the coding sequence and assignment of chromosome 19p. Blood 76, 24±30. Witthuhn, B. A., Quelle, F. W., Silvennoinen, O., Yi, T., Tang, B., Miura, O., and Ihle, J. N. (1993). JAK2 associates with the erythropoietin receptor and is tyrosine phosphorylated and activated following stimulation with erythropoietin. Cell 74, 227±236. Wlodawer, A., Pavlovsky, A., and Gustchina, A. (1993). Hematopoietic cytokines: similarities and differences in the
structures, with implications for receptor binding. Protein Sci. 2, 1373±1382. Wojchowski, D. M., Orkin, S. H., and Sytkowski, A. J. (1987). Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide. Biochim. Biophys. Acta 910, 224±232. Wu, J., Liu, X., and Jaenisch, R. (1995). Generation of committed erythroid BFU-E and CFU-E progenitors does not require erythropoietin or the erythropoietin receptor. Cell 83, 59±67. Wurnig, C., Windhager, R., Schwameis. E., Kotz, R., Zoubek, A., Stockenhuber, F., and Kurz, R. W. (1996). Prevention of chemotherapy-induced anemia by the use of erythropoietin in patients with primary malignant bone tumors (a double-blind, randomized, phase III study). Transfusion 36, 155±159. Yamaguchi, K., Akai, K., Kawanishi, G., Ueda, M., Masuda, S., and Sasaki, R. (1991). Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties. J. Biol. Chem. 266, 20434± 20439. Youssoufian, H., Longmore, G., Neumann, D., Yoshimura, A., and Lodish, H. F. (1993). Structure, function and activation of the erythropoietin receptor. Blood 81, 2223±2236. Zanjani, E. D., Ascensao, J. L., McGlave, P. B., Banisadre, M., and Ash, R. C. (1981). Studies on the liver to kidney switch of erythropoietin production. J. Clin. Invest. 67, 1183±1188.
