DNA Repair in Normal and Cancer Stem Cells, with Special Reference tothe Central Nervous System (Article)

Cu rrent M edicinal Chemistry, 2009, 16, 854-866

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DNA Repair in Normal and Cancer Stem Cells, with Special Reference to the Central Nervous System Guido Frosina* Molecular Mutagenesis & DNA Repair Unit, Istituto Nazionale Rice rca Cancro, Largo Rosanna Benzi n. 10, 16132 Genova, Italy Abstract: Stem cells build and maintain organisms. Accordingly, they are particularly well-protected from damage to DNA and other cellular components. T his feature becomes a serious drawback when stem cells transform and develop cancer, because they resist to radiation and chemotherapy. Various mechanisms ensure protection of stem cells. In nonnal stem cells enhanced DNA repair is often on e of them. Whether the same holds for cancer stem cells still is an open questioo.

Keyword s: Stem cells, differentiation, DNA repair, CDl33, cancer. INTRODUCTION Unlike normal somatic cells, stem cells can proliferate indefinitely in culture in an undifferentiated state where they do not appear to undergo senescence and yet remain nontran sformed. Cells maintain th eir pluripotency both in vivo and in vitro, exhibit high telomerase acti vi ty and maintain telomere length after prolonged in vitro culture [1]. Mutation frequencies at some loci in mammalian so matic cells in vivo approach 10-4 This high level of DNA damag e is clearly untenable for embryonic stem (ES) cell s that must build a whole organism. Although ES cells are genetically hyperactive [2], their mutation frequencies and frequencies of mitotic recombination are about 100-fold lower than in adult somatic cells or in isogenic mouse embryonic fibroblasts (MEFs) [3] . ES cells must therefore be equipped with highly efficient defense mechanisms against various kinds of stress including DNA damage. Adult stem cells are al so extremely important in the long-term maintenan ce of ti ssues throughout life [4]. They regenerate tissues in respon se to damage and replace senescent terminally differenti ated cells that no longer function. Oxidative stress, tox ic byproducts, reduced mitochondrial function and external expo sures all damage DNA causing base modification and mis-incorporation, fragmentation of deoxyribose, induction of pho sphotriesters, single strand breaks (SSB) and double strand breaks (DSB). As in most cell s, this damage may limit the survival of the ste m cell population affecting tissue regeneration and eventually lifespan. One established defense mechanism against toxic drugs is a high activity of a verapamil- sensitive multidrug efflux pump. A peculiarity of many stem-cell populations is their relatively high expression of ATP-binding cassette (ABC) drug transporters, which can protect cell s from cytotoxic agents [5]. For instance, haematopoietic stem cells have been isolated on the basis of their ability to efflux the fluorescent dye Hoechst 33342, which give them a unique profile, referred to as the side population, when analysed by flow cytometry. Side-popUlation cells have also been isolated from other tissues e.g. in the central nervous system (CNS).

*Address correspondence to this author at the Molecular Mutagenesis & DNA Repair Unit, Is tituto N azionale Ricerca Cancro, Largo Rosarma Benzi n. 10, 16 132 Genova, Italy; Tel : +39.0 10.5737543; Fax: +39.010. 5737237; E-mail: [email protected] 0929-8673109 $55.00+.00

Although stem cells can be identified within this side population, not all of the stem cell population is contained within this group, nor does the side population contain a pure stemcell population, therefore indicating this property as a means to enrich, but not purify, stem cells. Another way by which stem cells protect their genome may be selective elimination of tho se cell s that have acquired a mutational burden [6,7]. This would be accomplished by lack of a G 1 checkpoint and related signalling pathways. The checkpoint kinase, Chk2, which participates in signalling pathways for G 1 checkpoint, is sequestered at centro somes in stem cells and does not phosphorylate its substrates (p53 and Cdc25A) that must be modified to produce a G 1 arrest. Thus, wild type stem cell s exposed to ionizing radiation (IR) do not accumulate in G 1 but do so in S-phase and in G2 where apoptosis inevitabl y follo ws due to unsustainable mutational burden [6]. Consistentl y, keratinocyte populations enriched for stem cells from human epidermi s respond to IR by the regulation of genes functionally related to cell death and apoptosis [8] . As under many oth er aspects , mice may differ from human s with respect to selective elimination of mutated stem cells. Chambers and coworkers [9] using purified hematopo ietic stem cells from mice aged 2 to 21 months found a deficit in function yet an increase in stem cell number with advancing age. Lo ss of ep igenetic regulation with age could dri ve fu nctio nal attenuation of murine hematopoietic stem cells [9]. Apoptosis and cell cycle delay after DNA damage re main as important safeguard mechanisms of the murine ES cell geno me [7] . One further mechanism of stem cells preservation could be retention of those DNA strands with the fewest mutations acquired during DNA replication [10] . Studies of stem cell s in the mouse small intestine [ll], breast [1 2], brain [13] and muscle [14] have shown that these stem cells do ind eed keep the same parental DNA strands through successiv e divi sions and, in one case, that their non stem-cell daughters do not [14] . Thi s arrangement ensures that any errors ari sing in stem cells during gene duplication avoid being permanently fix ed because thay are passed on, at the asymmetric division, to the differentiating daughter cell and will therefore soon be discarded. The interactions of gene products underlying these properties of stem cells are not understood, but they may involve the action of p53 [15]. This "immortal strand" hypothesis has been recently questioned [1 6,17 ]. In human hematopoietic stem cell s, the ex press ion of some antioxidant proteins increases with age. The tran scrip© 2009 Bentham Science Publishers Ltd.

DNA Repair in Stem Cells

Table 1.

Current Medicinal Chemistry, 2009 Vol. 16, No.7

DNA Repair Capacity in Normal Stem vs Differentiated Cells

Stem cell system

DNA repair mechanism/enzyme

Higher

Similar

Lower

Remarks

Ref.

Strand break repair Murine Embryonic Stem Cells

Expression of antioxidant and strand break repair genes

,j

Compared to differentiated cells

[25]

Human fetal mesenchymal stem cells

DNA repair gene expression

,j

Compared to adult mesenchymal stem cells

[22]

Human CD34+ 38- hematopoietic stem cells

Removal ofENU ormelphalan-induced DNA adducts

,j

Compared to progenitor or mature cells Resistant to DNA-reactive drugs

[27]

Human CD34+ 38- hematopoietic stem cells

Resealing of strand breaks and • reparr gaps

,j

Compared to progenitor or mature cells Resistant to DNA-reactive drugs

[27]

Human mesenchymal stem cells from bone marrow transplant. patients

DSB repah

,j

Compared to lung or breast cancer cells Resistant to IR

[28]

Murine neural precursors

,j

Sensitive to IR

[32]

Rat neural precursors

,j

Sensitive to IR

[33]

Negatively correlated with donor age

[18]

Resistant to IR High telomerase activity

[34]

Human CD 34+ hematopoietic stem cells

KU70 expression

Human mesenchymal stem cells

Single and double -strand break repair

,j ,j

BER Human mesenchymal stem cells obtained from bone marrow transplant. patients

ROS-scavenging capacity

,j

Resistant to IR

[28]

Murine neural stem/progenitor cells

BER (OGGI)

,j

Compared to differentiated cells

[35]

Murine neural stem/progenitor cells

BER (NEIL3)

,j

Compared to differentiated cells

[36]

Murine fetal hematopoietic cells

BER (DNA pol P)

Compared to adult hematopoietic cells Low point mutation frequency

[38]

Murine myoblasts

BER (DNA ligase I and XRCCI)

,j

Compared to myotubes

[37]

Human embryonic stem cell lines

Expression of antioxidant and DNA repair genes

,j

Compared to differentiated cells

[26]

,j

NER Human cells of the monocytic lineage

NER

,j

Compared to macrophages

[46]

Human neural precursors

NER

,j

Compared to neurons

[43,44]

Murine ES cells

NER

,j

Strong apoptosis

[47]

Murine ES cells

NER

,j

Strong apoptosis S-phase delay

[7]

Murine keratinocytes

NER

,j

[48]

Alkylation damage repair Human cycling CD 34+ hematopoietic stem cells

MGMT MMR

Murine ES cells

MGMT MMR

,j

,j

Compared to mature CD34- cells Normally sensitive to methylation damage

[51]

Compared to differentiated cells Highly sensitive to methylation damage Strong apoptosis

[49]

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tion levels of microsomal glutathione S-transferase 1 (MGSTl), a gene protecting against oxidative stress have been found progressively increased with age [18]. Expression was lowest in newborn, 1.8-2.6-fold higher in young (mean age: 30 years) and 4.1-4.3-fold higher in old (mean age: 87.6 years) donors [18].

Guido Frosina

DNA repair may contribute to genome stability in normal stem cells [19-21]. This may particularly apply to ES cells where DNA repair genes are induced at higher levels than in adult stem cells [22,23].

[32]. In the second one, radiation injury was specifically associated with irreversible damage to the neural stem cell compartment in the sub ventricular zone and loss of oligodendrocyte precursor cells in both rodent and human brain [33]. Hence, the DNA repair machinery may process radiation damage more slowly in the neural precursors in relation to their greater radiosensitivity. Overall DNA repair has been found similar in human mesenchymal stem cells and their telomerase-immortalized derivatives [34]. The latter show higher stability at telomeric regions and resistance to IR than primary stem cells indicating that high telomerase activity is another mechanism by which sensitivity to IR may be reduced in stem cells.

Strand Break Repair

DNA Base Excision Repair

DSBs are typically repaired by the non homologous end joining (NHEJ) pathway in ES cells, but in the absence of NHEJ components, a substantial fraction of breaks can be efficiently channeled into alternative pathways such as homology-directed repair (HDR) [24]. Several strand break repair genes become downregulated during differentiation of murine ES cells [23,25,26] (Table 1; Fig. (la)). In the human Iymphohematopoietic system, maturation-dependent alterations in strand break repair have been observed as well. Bracker and coworkers [27] have correlated the expression of DNA damage response genes and the functional repair capacity of cells at distinct stages of human hematopoietic differentiation after treatment with ENU or melphalan (Fig. (lb)). Comparing fractions of mature (CD34-), progenitor (CD34+ 38+) and stem cells (CD34+3810w) isolated from umbilical cord blood, these authors observed that the removal of DNA adducts, the resealing of strand breaks and repair gaps, and the resistance to DNA-reactive drugs were clearly higher in stem cells compared with progenitor cells of the same individual [27]. Hence, the organism might protect the small number of valuable slow dividing stem cells by extensive DNA repair, whereas fast-proliferating progenitor cells, once damaged, may be rather eliminated by apoptosis. Likewise, mesenchymal stem cells obtained from bone marrow transplantation patients appear resistant to IR and possess better antioxidant ROS-scavenging capacity and DSB repair as compared to lung cancer and breast cancer cells [28]. KU70 is an important component of the NHEJ repair pathway. The expression of this protein, which further exerts a major role in immunoglobulin gene recombination, was negatively correlated with donor age in CD 34+ hematopoietic stem cells showing highest expression levels in newborn, 2.6-fold lower levels in young (mean age: 30 years) and 6.3-fold lower levels in old (mean age: 87.6 years) donors [18]. Two recent murine studies confirm that efficient DSB repair is important for hematopoietic stem cell function [29-31]. Mutations in the DSB repair pathway caused a progressive loss of hematopoietic stem cells and decrease of bone marrow cell count during ageing and severely impaired stem cell function in tissue culture and transplantation [30]. Stem cells in the CNS might behave differently. Two studies failed to observe a pronounced resistance of neural precursors to IR [32, 33]. In the first one, gamma-irradiation of the developing mouse brain induced a massive apoptosis of neural precursors but not of neurons and the different radiosensitivity was not related to variations in the numbers of IR-induced DSBs in the two cell types

In the neonatal mouse brain, expression of 8-oxoguanine DNA glycosylase (OGG 1) is detectable in a distinct layer of cells in the medial wall of the lateral ventricle and in some scattered cells in the sub ventricular zone, a brain region rich in neural stem/progenitor cells [35] (Fig. (lc)). Both expression and activity of OGG 1 are high in neurospheres derived from newborn mice and decrease in adults and upon induction of cell differentiation (Table 1; Fig. (ld)). Enhanced OGGl-mediated DNA base excision repair (BER) may be a mechanism by which neural stem/progenitor cells maintain their genome [35]. Murine Nei endonuclease VIII-like 3 (Nei13) glycosylase follows as well a discrete expression pattern in brain regions harbouring stem cell populations [36]. The levels of endogenous oxidative DNA damage and BER capacity of mouse proliferating myoblasts and their differentiated counterpart, the myotubes, have been analyzed [37]. Changes in the expression of oxidative stress marker genes during differentiation, together with an increase in 8oxoguanine (8-oxoGua) DNA levels in terminally differentiated cells, suggested that reactive oxygen species (ROS) were produced during this process. Both short and long patch BER pathways were delayed in terminally differentiated muscle cells. The defect in BER was ascribed to the nearly complete lack of DNA ligase I (Fig. (Ie)) and to the strong down-regulation of XRCCI with subsequent destabilization of DNA ligase III alpha [37] (Fig. (1f)). Likewise, during spontaneous differentiation of two human embryonic stem cell lines the expression of antioxidant and DNA repair genes was downregulated and DNA damage levels consistently increased [26].

DNA REPAIR IN NORMAL STEM CELLS

Unexpectedly, BER activity has been found significantly lower in fetal hematopoietic cells than in adult hematopietic cells, due to a lower level of DNA polymerase (pol) beta [38]. Pol beta has been suggested as the rate-limiting enzyme in repair of AP sites, while DNA glycosylases are ratelimiting in BER of uracil and oxidized bases [39-41]. In fetal hematopoietic cells, the low BER activity correlated with elevated mitotic recombination but low point mutation frequency [38]. The mutational response to IR in hematopoietic stem cells may be partly determined by developmentally regulated phenotypes other than DNA repair, e.g. mitotic index [38]. Nucleotide Excision Repair Nucleotide excision repair (NER) preferentially repairs the transcribed strand of active genes, as compared to the

DNA Repair in Stem Cells

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Current Medicinal Chemistry, 2009 Vol. 16, No.7

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Fig. (1). DNA repair is higher in stem than in differentiated cells. a. Endogenous expression levels of the HDR protein Rad51 is higher in ES cells than in MEFs. SDS-PAGE was performed using 20 flg of I 29/Sv ES cell or passage 3 MEF whole cell extracts and probed with Rad51 (top) or a-actinin (bottom) antibodies (from [23], with permission). b. Processing of DNA lesions in hematopoietic cells is impaired in progenitor compared with stem cells. Following exposure to N-ethylnitrosourea the kinetics of repair-induced DNA strand breaks were determined by the comet assay [measurement of olive tail moment (OTM)] in CD34+3810w (stem) or CD34+38+ (progenitor) cells from four individual samples. The frequency of initial DNA strand incision (OTM, to - top) and repair velocity (t.OTM/min - bottom) were consistently higher in the stem cell fraction (from [27], with permission). c. OGGI is highly expressed in the ventricular wall of the developing forebrain of newborn mice, an area known to harbour neural stem cells. In situ hybridization was performed on coronal sections from rostral regions, using an OGGI-specific probe. CC, corpus callosum; LV, lateral ventricle; S, septum; Str, striatum. Right: magnification of boxed area. Arrows indicate a defined layer of OGG I-positive cells (from [35], with permission). d. Enhanced OGG I activity in nuclear extracts from newborn derived neurospheres. 8-oxoG excision activity was measured by incubating increasing amounts of nuclear extracts derived from neurospheres from newborn (NSC nb) and juvenile (NSC juv) mice, cells differentiated from newborn neurospheres (Diff NSC nb) and MEF cells (OggJ+I+ and Oggi-I-) with a l2P -labeled 8-oxoG paired with C substrate. Strand cleavage was analyzed by denaturing PAGE followed by phosphorimaging (from [35], with permission). e. DNA ligase I protein level (top) is higher in proliferating (myoblasts) than in terminally differentiated (myotubes) muscle cells. ~-tubulin protein levels (bottom) were used as a loading control (from [37], with permission). f. XRCCI and DNA ligase III protein levels drop during differentiation. XRCCI and DNA ligase III in nuclear extracts from muscle cells at the indicated times (days) after addition of differentiation medium are shown. DNA polymerase ~ protein levels were used as a loading control (bottom) (from [37], with permission). non-transcribed strand [transcription-coupled repair (TCR)] [42]. This subpathway of NER provides cells with a mechanism removing DNA lesions from actively expressed genes. Upon differentiation NER is strongly attenuated in several human cell types (e.g. in cells of the monocytic lineage when they differentiate into macrophages or neural stem cells differentiating to neurons) [43-45] (Table 1). The attenuation of NER during differentiation results from the lack of ubiquitination of NER factors, most likely owing to differences in phosphorylation of the ubiquitin-activating enzyme E 1 [44]. However, differentiated cells possess besides TCR, a

second specialized mechanism to ensure proficient repair of active genes termed differentiation-associated repair (DAR). DAR grants transcribed genes of differentiated cells preferential access to the remaining proficient NER enzymes not engaged in TCR. As a consequence, in differentiated cells both DNA strands of active genes are eventually repaired [44]. Downregulation of selected NER genes with small interfering (si) RNA has shown that DAR is a subset of global NER, restricted to the sub-nuclear compartments or chromatin domains within which transcription occurs [44,46]. Mouse cells are less well characterized. Murine ES

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cells exhibit low levels of NER after UV irradiation, and cell cycle arrest and a strong apoptotic response eliminating damaged cells, seem major safeguard mechanisms here [7 ,47] . No DAR has been observed upon differentiation of murine keratinocytes [48]. Alkylation Damage Repair Upon treatment with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), murine ES cells undergo apoptosis at much higher frequency than differentiated cells, although 6 they express a high level of 0 methylGua DNA methyltransferase (MGMT) [49] (Table 1). The high sensitivity of 6 ES cells to 0 methylating agents is due to high expression of the mismatch repair (MMR) proteins MSH2 and MSH6 6 that trigger futile cycles of 0 methylGua repair/replication [50] . Roos and coworkers [49] propose that the high apop6 totic response of murine ES cells to 0 methylGua adducts may contribute to reduction of the mutational load in the progenitor population. In contrast, Casorelli et al. [51] find that cycling CD34+ (stem) and CD34- (mature) human hematopoietic cord blood cells are equally sensitive to methylation damage. In this study, MGMT provided significant protection against N-Methyl-N-Nitro sourea (MNU) toxicity and MGMT and MMR exerted the expected roles in the MNU sensitivity of the cells [50,51]. Whether those differences in sen sitivity could be linked to species (mouse vs human) or tissue (ES vs hematopoietic) variations (or both) is undetermined. Other Antimutagenic Mechanism s A critical role for Fanconi anemia (FA) pathway in neural stem and progenitor cells during developmental and adult neurogenesis has been recently described [52,53]. In adult FA mice, a reduced proliferation of neural progenitor cells related to apoptosis and accentuated neural stem cell exhaustion with ageing was shown. In addition, embryonic and adult FA neural stem cells showed a reduced capacity to selfrenew in vitro [52,53]. Neural stem cells distinguish them selves in their basal mitochondrial metabolism from po stmitotic neural cells by their lower ROS levels and higher express ion of key antioxidant enzymes [e.g. uncoupling protein 2 (UCP2) and glutathione peroxidase (GPX)] [54]. Following exposure to the mitochondrial toxin 3-nitropropionic acid and unlike postmitotic cells, neural stem cells react by fast upregulation of UCP2, GPX and superoxide dismuta se 2 and successfully recover from an initial deterioration . Thus, an increased "vigilance" of antioxidant mechanisms is a hallmark of neural stem cells and might help them to counteract oxidative stres s in CNS. DNA REPAIR IN CANCER STEM CELLS Ca ncer Stem Cells? A few years ago parallels were hypothes ized between ste m cells and cancer cells [55]: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells. and cancer cells may include "cancer stem

Guido Frosina

cells" - rare cells with indefinite potential for self renewal and differentiation that drive tumorigenesis [55 ,56]. Merely to quote a few observations supporting this theory, comparison of expression data of leukemia and glioma cells with those of normal hematopoietic or brain stem cells revealed that an elevated portion of the modulated genes are shared by cancer and normal stem cells [57-59]; human stem cells can undergo spontaneous transformation following long-term (45 months) in vitro culture [60,61]; down-regulation of several tumor suppre ssors (e.g. DOCK4 and SPARCLl) can be observed in CDI33+/CD34+ stem cells [62,63]; side populations with ability to efflux Hoechst 33342 dye (see above) have been shown to be enriched sources of tumor initiating cells [64]; expression of the CD133 stem cell antigen negatively correlates with glioma patient survival in several studies [65-69]. Overall, cancer stem cells may share many of the properties of normal stem cells that provide for a long lifespan including relative quiescence, resistance to drug s and toxins through the expression of ABC transporters and resistance to apoptosis [70-75]. A detailed knowledge of the biological distinctness of cancer stem cells may be crucial for the development of specific tumour therapies [76-80]. However, despite the current enthusiasm on the cancer stem cell theory, several exceptions may exist [81,82] . It has been pointed out that much of the supporting evidence for tumors arising from rare cancer stem cells is derived from xenotransplantation experiments in which human leukemia cells are grown in immunocompromised mice [83,84]. However, when lymphomas and leukemias of mou se origin are transplanted into histocompatible mice, a very high frequency (at least 1 in 10) of the tumor cells can seed tumor growth, thus suggesting that the low frequency of tumorsustaining cell s observed in xenotransplantation studies may reflect the limited ability of human tumor cell s to adapt to growth in a foreign (mouse) milieu [83,84]. Further, in some cases CD133- cells may even be more aggressive than CD133+ cells in tumor initiation and progression [85]. Kennedy and coworkers [86] have replied that the cancer stem cells hypothesis actually depends on pro spective purification of cells with tumor-initiating capacity, irrespective of frequency and that similar frequencies of leukemia-initiating cells in genetically comparable leukemias using syngeneic or xenogeneic models have been observed [86]. Anyway, the essential unresolved point is whether in a number of tumors, the cancer-derived stem cells may be not the "cause", but the "consequence" of carcinogenesis [87] . Mature cells and cancer cells may dedifferentiate or reprogram to cancer stem cells by genetic and / or epigenetic events and thu s gain selfrenewal activity and multipotentiality [88-90] . Even from a mathematical point of view, many studies on cancer stem cells have been questioned [91]. Some inconsistencies may simply derive from use of inadequate terminology, the term cancer stem cell being still poorly defined [92,93]. Vescovi and coworkers [5] have proposed five cardinal features to define brain tumor stem cells: i) cancer-initiating ability upon orthotopic implantation; ii) extensive cell renewal ability; iii) karyotypic or genetic alterations; iv) capacity to generate non-tumorigenic end cells; v) differentiation capacity. We recently found these features a valuable tool to standardize work in the field [94]. In particular, in our opinion the ability to differentiate to one or more lineages (v) is an indispensable, albeit often disregarded , feature to describe

DNA Repair in Stem Cells

"stem" cells. In the presence of such a hallmark, whether the term "cancer" might further be applied would depend on fulfilment of criteria i-iii. Cells possessing the latter three features but unable to differentiate should be better termed "cancer-initiating cells" or " tumor-propagating cells" [73, 83,92,95]. Mechanisms of resistance to drugs and radiation in cancer stem cells may resemble those in normal stem cells [96,97]. Cancer stem cells express high levels of ABC transporters although multidrug resistance reversal agents often fail to increase the therapeutic index of substrate antineoplastic agents [76,98,99]. Additional mechanisms of cancer stem cell resistance to chemo/radiotherapy may involve the telomere complex [100] and downregulation of apoptosis [101]. Like normal stem cells, cancer stem cells are further supposed to resist therapies by an increased DNA repair capacity [102]. With some cell types, however, some observations do not fit this model. For instance, it has been unexpectedly observed that myeloid progenitor bone marrow cells derived from BER-defective mice are more resistant than those from wild-type mice to the cytotoxic effects of several alkylating agents [103]. Hence, in some stem cell types, the initiation of BER may be more lethal to the cell than leaving the damaged base unrepaired. Absence of MMR has therapeutic implications as well [104]. For instance, in alkylating chemotherapy-related acute myeloid leukaemia (AML), loss of DNA MMR may promote the emergence of resistant stem clones with chromosomal instability [105]. Glioma Stem Cells As for other cancers, the existence of glioma cells endowed with features of primitive progenitor cells and tumorinitiating function has been demonstrated [78,106-108]. Most gliomas contain considerable portions of cells expressing stem cell markers such as CD133, Nestin, Sox-2 and Musashi-1 [109-110] and expression of some of them has been shown to be prognostic of decreased overall survival [69,111-113]. As mentioned above for hematological malignancies, whether the currently identified glioma stem cells may be the true cell-of-origin for tumour initiation and progression or the results of such processes still is an open question [114,115]. Further, gliomas produce factors [e.g. urokinase plasminogen activator (uPA) and its receptor (uPAR)] that selectively attract neural stem cells [116-121] and this has originated the question as to whether stem cells may be attracted by, rather than originate, the tumor [109,122-124]. Recent data showing high cellular coexpression of CD133 with Musashi-1 but not CD34 would suggest a local origin of these cells and a negative answer [69]. Anyway, gliomas do not clash with the general concept that if only a rare subset of tumor stem cells, whatever their origin, drives tumor formation, then it is important to identify this population and develop therapies that target it [59,125130]. Methods to Study DNA Repair in Glioma Stem Cells One of the most widespread in vitro models to study glioma stem cells is based on neurospheres, long-term cultures of neural cells that can be grown in suspension as clonal aggregates [131-133]. These multicellular spheroids

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may mimic heterogeneous cellular behaviour and some functional characteristics of in vivo solid tumors and thus serve as an important in vitro model to investigate response to potential therapeutic agents [133]. However, stem cells constitute only a fraction of the cell population in brain-derived neurospheres which consist predominantly of committed progenitors, presumably because aggregation induces differentiation, analogous to embryoid body differentiation by ES cell aggregates [134]. To minimize this drawback and make morphological analysis easier, adherent glioma stem cells can also be cultured [135]. The findings reported by Conti and coworkers [136-140] establish that the epidermal growth factor (EGF) and fibroblast growth factor (FGF) -2 plus insulin are sufficient to sustain robust expansion and inhibit differentiation of neural stem cells in defined adherent monolayer cultures. Even under adherent conditions, the neural stem-cell titre is very high soon after the dissociation and re-plating (which selects against cells other than neural stem cells), but tends to decline progressively with subsequent subculturing steps. This is due to the production of more mature precursors that occur spontaneously during cell proliferation. Selection of active-growing subpopulations, which lose their stem properties and differentiation capacity, can be counteracted by isolation of CD133+ precursors [141]. CD133+ is often expressed on the surface of human glioma stem cells and its expression has been recently described in murine glioma stem cells as well [142]. Further, CD133+ cells are preserved during orthotopic transplantations of human gliomas in the mouse [143]. However, purification of stem cells by CD133+ sorting pays the price of discarding significant subpopulations of CD133- stem cells [144]. In a recent study, 11115 primary glioblastomas contained a significant CD133+ subpopulation that displayed asymmetrical cell divisions yielding cells expressing markers characteristic for all three neural lineages [145]. However, four of 15 cell lines were driven by CD133- tumor cells that also fulfilled stem cell criteria (e.g. pluripotency and tumorigenicity in nude mice in vivo). Consistently, analysis of the expression profiles of nine glioma cell lines established under neural stem cell conditions yielded two distinct clusters [146]. Four cell lines were characterized by the expression of neurodevelopmental genes, a multipotent differentiation profile, expression of CD133 and formation of highly invasive tumors in vivo. The other five cell lines shared expression signatures enriched for extracellular matrix-related genes, had a more restricted differentiation capacity, contained no or fewer CD133+ cells and displayed reduced tumorigenicity and invasion in vivo. Taken together, the above data indicate that CD133+ stem cells may maintain only a subset of primary glioblastomas, the remainder deriving from CD133- tumor stem cells with distinct phenotypical features [145-149]. A number of additional cell surface markers have been used to hallmark glioma stem cells including CD 117, CD71, CD45 and A2B5, with problems similar to or deeper than those found with CD133 [150-152]. In conclusion, in vitro expansion of pure stem cell populations to obtain the relatively high numbers of cells required in most high-resolution DNA repair protocols (e.g all methods using cell extracts repairing single-lesion substrates) is currently unfeasible: purification may be possible by sorting for CD133 or other stemness markers, but after a few cell divisions, some stem cells will commit again to losing, at

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least partially, their "stemness" features. DNA repair analyses on pure cancer stem cell populations are therefore currently limited to relatively non specific and low-resolution procedures such as the single cell gel electrophoresis (SCGE) and unscheduled DNA synthesis (UDS) assays, where low numbers of freshly purified cells are sufficient. With DNA repair protocols employing cell extraction, at the moment there is no better way than doing experiments with enriched populations of glioma stem cells, that are far from being 100% pure. For some research aspects, a recently established human neural stem cell line may be of help [153]. These cells originate by v-myc-mediated immortalization of wild type human neural stem cells and demonstrate a number of properties similar to parental wild type cells including multipotentiality. Upon growth factor removal the immortalized cell line completely downregulates v-myc expression, ceases proliferation and differentiates terminally into the three major neural lineages: astrocytes, oligodendrocytes and neurons. Importantly, those cells are endowed with an extensive and rapid proliferation capacity although no features of transformed cells, such as growth factor independence or aberrant karyotypic properties, are displayed. Thus this cell line may serve as a surrogate for DNA repair studies requiring elevated numbers of neural stem cells [153]. A novel in vivo method exploiting a combination of confocal microscopy, three dimensional modelling and mathematical algorithms has recently been used to visualize and characterize the spatial distribution of neural stem cells throughout an orthotopic glioma in a mouse model [154]. In a different approach, a metabolic biomarker [1.28-parts per million (ppm)] even allows detection and quantification of neural stem and progenitor cells in the human brain in vivo [155]. Here, proton nuclear magnetic resonance spectroscopy is used to identify and characterize this biomarker in which neural progenitor cells are enriched and its use as a reference for monitoring neurogenesis has been demonstrated [155]. Further development of these methodologies might lead to investigate the role and features of brain tumor stem cells in vivo and optimize the efficacy of stem cell-targeting glioma therapies [154,156,157]. DNA Repair as a Resistance Mechanism in Glioma Stem Cells Bao and coworkers [158] have shown that glioma stem cells may resist radiation through preferential activation of the DNA damage checkpoint response. The fraction of tumour cells expressing CD133 was enriched after radiation in gliomas. In both cell culture and the brains of immunocompromized mice, CD133+ glioma cells survived IR in increased proportions relative to most tumour cells, which lack CD133. CD133+ tumour cells isolated from both human glioma xenografts and primary patient glioblastoma specimens preferentially activated the DNA damage checkpoint in response to radiation and repaired IR-induced-DNA damage more effectively than CD133- tumour cells [158,159]. In addition, the radioresistance of CD133+ glioma stem cells could be reversed with a specific inhibitor of the Chkl and Chk2 checkpoint kinases. In the mouse, G2 and G I-arrested neural stem cells rapidly increase after IR and this correlates with phosphorylation of cdc2 and p53 respectively, and

Guido Frosina

inactivation of the Notch signal [160]. Besides IR, resistance of glioma stem cells to chemotherapeutic drugs has been reported [161,162]. As aforementioned, expression signatures comprising CD133, may predict poor survival in glioma patients treated with concomitant chemo/radiotherapy [69,111-113,163]. Hence, CD133+ tumour cells may represent one (albeit probably not the only) cellular population that confers glioma resistance to chemo/radiotherapy and activation of the DNA damage checkpoint response may be a major mechanism in this regard. One further important resistance mechanism could be multidrug resistance. As for other cancers, expression of multidrug resistance-associated protein genes has been found elevated in cancer stem-like cells isolated from glioblastoma and astrocytoma [98,99,164,165]. Finally, elevated expression of antiapoptic proteins [e.g. the myeloid cell leukemia-l (Mel-i) protein] can be found in glioma stem cells [165,166] and reduced expression of major histocompatibility complex genes may also contribute to their resistance to therapies [167,168]. The relative importance of DNA repair as a resistance mechanism in glioma stem cells still is an open question. We have recently examined DNA repair in five stem and non-stem glioma cell lines [94]. The stem lines fulfilled the defining criteria proposed by Vescovi and coworkers [5]. The population doubling time was significantly increased in stem as compared to non-stem cells and enhanced activation of Chkl and Chk2 kinases was observed in untreated CD133+ as compared to CD133- cells. However, neither BER nor SSB repair nor resolution of pH2AX nuclear foci were increased in CD133+ as compared to CD133- cells. Hence, elongated cell cycle and enhanced basal activation of checkpoint proteins may be common mechanisms by which glioma stem cells resist therapies while enhanced DNA repair is not [94]. Therapies Targeting Glioma Stem Cells Bone morphogenetic proteins (BMPs), amongst which BMP4 elicits the strongest effect, activate their cognate receptors (BMPRs) and trigger the Smad signalling cascade in cells isolated from human gliomas [169]. This is followed by a reduction in proliferation and increased expression of differentiated neural markers, without affecting cell viability. The concomitant reduction in the clonogenic ability, both in the size of the CD133+ side population and in the growth kinetics of glioblastoma cells, indicates that BMP4 triggers a reduction in the in vitro cancer stem cell pool. Most important, in vivo delivery of BMP4 effectively blocks the tumour growth and associated mortality which occur in 100% of control mice in less than 12 weeks, following intracerebral grafting of human glioma cells. Thus, the BMP signalling system which controls the activity of normal brain stem cells may also act as a key inhibitory regulator of tumourinitiating, stem-like cells from gliomas and identify novel, non-cytotoxic therapeutic effectors, which may be used to prevent growth and recurrence of gliomas in humans [169,170]. Notch signaling may promote the formation of cancer stem cell-like cells in human glioma [171]. Apoptotic rates following Notch blockade are almost lO-fold higher in primitive nestin-positive cells as compared with nestinnegative ones. Notch blockade further reduces the CD133positive cell fraction almost 5-fold and totally abolishes the side population [172]. Stem-like cells in brain tumors thus

DNA Repair in Stem Cells

may be selectively vulnerable to agents inhibiting the Notch pathway [172]. Similarly, as maintenance of human brain tumor stem cells requires EGF, tyrosine kinase inhibitors of EGF signaling (e.g. AG1478 and gefitinib) potentially inhibit proliferation and induce apoptosis of these cells [173]. Activation of the phosphatidylinositol-3-0H kinase/AKT signaling pathway occurs frequently in gliomas due to inactivation of the tumor suppressor phosphatase and tensin homologue (PTEN) [174,175]. The therapeutic effect of targeting the phosphatidylinositol-3-0H kinase/AKT pathway or the efficiency of PTEN itself is certainly worth exploring. Derivatives of Cannabis may be another possibility: recent results demonstrate that these drugs target glioma stem-like cells, promote their differentiation and inhibit gliomagenesis, suggesting their potential use for the management of malignant gliomas [176]. Antagonists of hyaluronan-CD44 interaction, especially small hyaluronan oligomers, suppress glioma growth in vivo by enhancing apoptosis and possibly decreasing recruitment of cancer stem-like cells expressing ABC drug transporters [177,178]. MicroRNAs have been used to induce differentiation of adult mouse neural stem cells, mouse oligodendroglioma-derived stem cells and human glioblastoma multiforme (GBM)derived stem cells and induce GBM cell cycle arrest. Hence, targeted delivery of microRNAs to GBM tumor cells might be explored as a novel treatment [179]. Over-expression of multiple ion channel genes [e.g. chloride intracellular channell (CLICl)] may contribute to resistance of glioma stem cells to nitrosoureas promoting drug efflux [180]. Using the chloride channel blocker, 4,4'-diisothiocyanostilbene-2,2'disulfonic acid (DIDS) enhanced apoptosis and sensitization of glioma stem cells to 1,3-bis-(2- chloroethyl)-I-nitrosourea (BCNU) chemotherapy has been observed [180]. Oncolytic adenoviruses can be engineered to selectively attack brain tumor stem cells, recognized by linage-specific cell surface markers, dysfunctional stem cell-signaling pathways, or upregulated oncogenic genes [181,182]. The neuronal cell adhesion molecule LICAM is required for maintaining the growth and survival of CD133( +) glioma cells both in vitro and in vivo [183]. Targeting LlCAM using lentiviralmediated short hairpin RNA (shRNA) interference in CD133+ glioma cells potently disrupts neurosphere formation, induces apoptosis, and inhibits growth specifically in glioma stem cells. Use of LICAM as a cancer stem cellspecific therapeutic target for improving the treatment of malignant gliomas has been proposed [183]. Finally, normal neural stem cells retrovirally transduced with therapeutic genes such as the cytosine deaminase gene, endostatin or cytochrome P450 have been shown to have the capability of selectively migrating into glioma mass and delivering therapeutic agents with elimination of their transformed cousins [115,184-187]. CONCLUSIONS The DNA repair capacity of normal (embryonic or adult; human or murine) stem cells as compared to differentiated cells has been reported 22-fold (Table 1). In 14 cases (64 %), an increased DNA repair capacity was observed, suggesting that normal stem cells often protect their genome through enhanced DNA repair. On the contrary, reduced proliferation rate and activation of checkpoint proteins, rather than DNA

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repair, seem major resistance mechanisms in glioma stem cells [94]. Even the fundamental point of whether these cells may be at the origin or outcome of neoplastic transformation is currently uncertain [89,90]. That loss of differentiation in neoplastic cells may lead to acquisition of stem properties rather than neoplastic transformation preferentially occurring in normal stem cells, still is a worth exploring possibility. ACKNOWLEDGEMENTS Work partially supported by Compagnia di S. Paolo, Programma "Oncologia" ; Italian Ministry of University and Research, "Fondo Investimenti Ricerca Base (FIRE Internazionalizzazione )"; Istituto Superiore Sanita, Programma Italia-USA "Malattie Rare". ABBREVIATIONS ABC

ATP-binding cassette

AML

acute myeloid leukaemia

BCNU

1,3-bis-(2- chloroethyl)-I-nitrosourea

BER

DNA base excision repair

BMP

bone morphogenetic protein

BMPR

receptor of bone morphogenetic protein

CLICI

chloride intracellular channell

CNS

central nervous system

DAR

differentiation-associated repair

DIDS

4,4' -diisothio cyano stilbene-2,2' -disulfonic acid

DSB

double-strand break

EGF

epidermal growth factor

ES

embryonic stem

FA

Fanconi anemia

FGF

fibroblast growth factor

GBM

glioblastoma multiforme

GPX

glutathione peroxidase

HDR

homology-directed repair

IR

ionizing radiation

Mcl-l

myeloid cell leukemia-l

MEF

mouse embryonic fibroblast 6

MGMT

0 methylGua DNA methyltransferase

MGSTl

microsomal glutathione S-transferase 1

MMR

mismatch repair

MNNG

N -methyl-N' -nitro-N -nitrosoguanidine

MNU

N-methyl-N-nitrosourea

Nei13

Nei endonuclease VIII-like 3 glycosylase

NER

nucleotide excision repair

NHEJ

non homologous end joining

862

Current M edicinal Chem istry, 2009 Vol. 16, No.7

- 8-oxoGua DNA glycosylase

OGGl

8-oxoGua

8-oxoguanine

- DNA polymerase

pol ppm

parts per million

PTEN

phosphatase and tensin homologue

- reactive oxygen sp ecies

ROS SCGE

single cell gel electrophoresis

shRNA

short hairpin RNA

- small interfering RNA

SSB

single strand break

TCR

transcription-coupled repa ir

UCP2

[1 51 [1 6] [17]

[18]

[1 9] [20]

[2 1]

- uncoupling protein 2

U DS

unscheduled DNA synthesis

uPA

urokinase plasminogen activator

uPAR

[14]

olive tail moment

OTM

siRNA

Guido Frosina

[22]

- urokinase plasminogen activator receptor. [23]

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Received: November 19,:2()(6

Revised: January 31, 2009

Accepted: January 31, 2009

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