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