Stress Defense in Murine Embryonic Stem Cells Is Superior to That of Various Differentiated Murine Cells
http://www.100md.com
《干细胞学杂志》
a Henry Wellcome Laboratory for Biogerontology,
b Institute of Human Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom;
c Department of Biological Sciences, University of Durham, Durham, United Kingdom
Key Words. Embryonic stem cells ? Differentiation ? Antioxidant defense ? Peroxides DNA strand break ? DNA repair ? P-glycoprotein
Correspondence: Prof. Thomas von Zglinicki, Henry Wellcome Laboratory for Biogerontology, Newcastle General Hospital, University of Newcastle upon Tyne, Newcastle upon Tyne NE4 6BE, U.K. Telephone: 44-191-256-3310; Fax: 44-191-256-3445; e-mail: t.vonzglinicki@ncl.ac.uk
ABSTRACT
Murine embryonic stem (mES) cells are pluripotent cells derived from the inner cell mass of blastocysts. There is only a very small number of ES cells that give rise to all tissues of the embryo proper. This small size of the population means that ES cells should be equipped with highly efficient mechanisms to prevent DNA damage, to repair it when it happens, and to counteract any propagation of mutations arising from it. Apoptosis is one efficient way to prevent proliferation of mutations, and, in fact, mES cells are known to be much more proficient in apoptosis than differentiated cells after UV-induced DNA damage . Ionizing irradiation seems to be a less potent trigger of apoptosis in mES cells . Not much is known about the quality of primary stress defenses, specifically antioxidant defenses in mES cells, so far.
Reactive oxygen species (ROS) are a major source of DNA damage because they are continuously produced as a byproduct of normal metabolism. Most differentiated cells are sensitive to an increase in ambient oxygen pressure, because this accelerates the production of ROS from the mitochondrial respiration chain. For instance, by increasing the ambient oxygen partial pressure from 21%–40%, levels of intracellular peroxides and protein carbonyls increased, and lipofuscin accumulated at a faster rate in normal human fibroblasts . At the same time, the replicative lifespan decreased to very few population doublings (PDs), and the rate of telomere shortening increased 4- to 10-fold . Differentiated murine somatic cells are even more sensitive to oxidative stress than human ones . As a result, murine fibroblasts experience replicative senescence early under standard in vitro conditions despite the fact that their telomeres are protected by being much longer than in human cells and by expression of telomerase. If oxidative stress is minimized, these cells can grow without obvious limits, similar to telomerase-expressing human fibroblasts under standard conditions . However, immortality is not equivalent to genomic stability. In fact, mutation frequencies and genomic instability in differentiated murine cells increase with time and with increasing oxidative stress .
We speculated that mES cells should have more efficient stress defenses than differentiated ones to maintain high levels of genomic stability at the single-cell level. We believe that insight into how ES cells are able to maintain the stability of their genomes over many cell divisions will help to understand aging of somatic stem cells in vivo and its impact on age-related disease in the adult organism. mES cells can be grown in culture without apparent limit on gelatin-coated culture flasks in media containing leukemia inhibitory factor (LIF). Growth in media from which LIF is absent permits differentiation into cell types that have lost the characteristic of infinite growth, form embryoid bodies (EBs), and will eventually senesce. We and others have used this system to demonstrate that downregulation of telomerase reverse transcriptase (Tert), the catalytic subunit of telomerase, is one of the key events occurring early in ES cell differentiation . We now analyze the resistance of mES cells to oxidative stress and their capacity to repair -induced DNA strand breaks. mES cells are highly proficient in antioxidant defense, an ability that decreases during early steps of differentiation into EBs. ROS levels also remain high in mouse embryonic fibroblasts and 3T3 fibroblasts. Furthermore, mES cells perform more efficient DNA strand break repair than differentiated mouse 3T3 fibroblasts. Using microarrays and confirmation by reverse transcription–polymerase chain reaction (RT-PCR), we have identified several candidate antioxidant and stress-resistance genes that become downregulated during differentiation of ES cells into EBs.
MATERIALS AND METHODS
Murine embryonic fibroblasts (MEFs) are sensitive to ambient oxygen. Although they are able to grow essentially unrestricted in culture under physiological ambient oxygen concentration (3%), they enter a telomere length–independent senescence-like state after 10 to 20 PDs under 21% oxygen . An additional increase of the ambient oxygen partial pressure to 40% arrested MEF growth after less than 3 PDs (Fig. 1). Prolonged culture of MEFs under 21% oxygen, for instance in a 3T3 protocol, leads to spontaneous immortalization. This is accompanied by major genomic changes. Such immortalized 3T3 murine fibroblasts died by apoptosis within a few days under 40% hyperoxia (data not shown). In marked contrast, mES cells did grow fast and without limits under 21% oxygen. They even proliferated with nearly the same rate under 40% hyperoxia, and this ability to withstand chronically increased oxidative stress did not decline with age in culture (Fig. 1, data not shown). Frequencies of apoptosis did not increase when mES cells were grown under 40% oxygen (data not shown).
Figure 1. ES cells grow well under high ambient oxygen concentration. Murine ES cells at starting PD were grown under either normoxia (21% oxygen, , solid line) or hyperoxia (40% oxygen, , dotted line). Differences toward PD at the start of the experiments (,PD) are given versus time. HES cells grew nearly as fast under hyperoxia as under normoxia, and there was no significant change in growth rates of ES cells with time. However, freshly prepared murine embryonic fibroblasts ceased growth after approximately eight PDs under normoxia (, solid line) and after fewer than three PDs under 40% oxygen (, dotted line). Abbreviations: ES, embryonic stem; PD, population doubling.
These data suggested that mES cells might have especially high antioxidant defense capacities, which might be lost during differentiation. To test this suggestion, mES cells were differentiated into EBs. Differentiation toward hematopoietic lineages was determined by progenitor colony assays (CFU-GEMM), in which different cytokines and growth factors are used to promote differentiation of early hematopoietic stem/progenitor cells from ES cells. The percentage of hematopoietic CFUs increased markedly on day 4 of the differentiation protocol (24.3%), and 46.2% of EBs were committed toward the hematopoietic lineage at day 6 (Fig. 2A). To investigate whether mES cells have a better capability to maintain low intracellular levels of oxygen free radicals, we stained ES cells, EB cells, and differentiated murine cells with DCF-DA. DCF-DA is trapped within cells by esterification and converted in its fluorescent form by reaction with peroxides. DCF is a substrate for P-glycoprotein, the multidrug efflux pump that is known to be active in a wide variety of committed stem cells . In mES cells, this activity leads to a loss of DCF fluorescence, which is nearly complete within 2 hours. However, this loss is completely abrogated by verapamil, a known inhibitor of P-glycoprotein (Fig. 2B). As Figure 2C shows, the activity of this verapamil-sensitive efflux pump decreases with differentiation and is essentially lost at day 6 of the differentiation protocol. To measure the steady-state levels of peroxides irrespective of P-glycoprotein activity, 5 μM verapamil was used in all subsequent DCF staining experiments. A significant increase of cellular DCF fluorescence with time in the differentiation protocol was found. Differentiation into EBs (day 6) resulted in an approximately fourfold increase in cellular DCF fluorescence, which was similar to values found in 3T3 murine fibroblasts and MEFs (Fig. 2D). DCF fluorescence of mES cells did not increase under hyperoxic culture conditions, whereas that of differentiated cells did (data not shown).
Figure 2. Cellular peroxide content increases with differentiation. (A): EBs were allowed to differentiate in the absence of leukemia inhibitory factor, and hematopoietic commitment was assessed by counting the proportion of EBs producing mixed colonies of myeloid lineage in the colony-forming unit–granulocyte-erythroid-macrophage-megakaryocyte. The data are mean ± SEM from three experiments with triplicate measurements each. (B): Retention of DCF fluorescence in ES cells was measured over a 2-hour period after staining in the presence or absence of verapamil. Data are mean ± SEM from triplicate measurements. (C): Retention of DCF fluorescence by 5 μM verapamil in ES cells and EB cells at days 0 (d0), 2 (d2), 4 (d4), and 6 (d6) of the differentiation protocol. Measurements were performed at 1 hour after staining. (D): Peroxide content was measured by DCF staining of ES cells, EB cells at days 0, 2, 4, and 6 of the differentiation protocol, 3T3 fibroblasts, and MEFs under normoxic culture. Fluorescence values measured in ES cells were set as 100% in every experiment. Data are mean ± SEM from three experiments with triplicate measurements each. Values are significantly different with p = .016 (one-way analysis of variance). Abbreviations: CFC, colony-forming cell; DCF, 2',7-dichloro-3',5-dihydrofluorescein; EB, embryoid body; ES, embryonic stem; MEF, murine embryonic fibroblast; SEM, standard error of the mean.
To establish whether the low peroxide content in mES cells was attributable to their improved antioxidant capacity, we measured the capability of ES and EB cell lysates to delay metmyoglobin/H2O2-mediated free radical generation (TAS). As Figure 3 shows, lysates from ES cells are in fact more proficient in antioxidant defense than those from EB cells.
Figure 3. Antioxidant defense capacity of cell lysates decreases with differentiation of ES cells. TAS per milligram of protein was measured in triplicates, each in three independent experiments, and ES cell averages for each experiment were set as 100%. Data are significantly different with p = .001 (one-way analysis of variance). Abbreviations: ES, embryonic stem; TAS, total antioxidant status.
Next, we examined whether the improved stress protection of ES cells would also include their DNA damage repair capabilities. As expected, initial DNA damage induced by ionizing radiation did not differ between ES cells and differentiated immortal mouse 3T3 fibroblasts with the sole exception of the highest radiation dose (Fig. 4). The capacity of 3T3 cells to repair DNA strand breaks decreased drastically with increasing radiation dose. In contrast, ES cells performed efficient DNA repair even after doses as high as 20 Gy (Fig. 4).
Figure 4. DNA strand-break induction and repair in 3T3 mouse fibroblasts and murine ES cells after different doses of ionizing radiation. Initial damage (open columns) and damage remaining after 1-hour repair at 37°C (black columns) are calculated as indicated in Material and Methods. Data are mean ± standard error of the mean from 6 and 10 independent experiments with 3T3 cells and ES cells, respectively. Significant differences between 3T3 and ES cells under the same treatment are indicated with one (p < .05) or two (p < .01) asterisks. Abbreviation: ES, embryonic stem.
To identify candidate genes that might be involved in the differential regulation of stress defense and repair, we performed a comparative gene expression analysis of ES and day-4 EB cells using the Affymetrix Mouse 430A target array, which analyzes the expression level of approximately 14,000 well-characterized mouse genes. Eight hundred twenty-one genes were identified using twofold change in expression between the samples as cut-off criterion. Affymetrix Gene Ontology Mining tool was used to select 15 candidate genes already known to be involved in cellular response to stress and DNA repair (Table 2). Differential expression was confirmed for all of them by semiquantitative RT-PCR (Fig. 5). With the exception of only two genes (Prdx2 and Hif3a), all candidate genes were downregulated to varying degrees upon differentiation of ES cells to the day-4 EB stage. Seven of the identified genes are known to be directly involved in the regulation of the cellular redox state. In addition, four heat shock genes and one DNA repair gene were found to be downregulated during differentiation. This suggests overall a decreasing antioxidant and stress defense capacity with ongoing differentiation in accordance with the functional data shown above.
Table 2. Genes whose expression levels change significantly upon differentiation of embryonic stem cells to 4-day embryoid bodies with particular relevance to oxidative stress resistance
Figure 5. RT-PCR analysis of 15 candidate genes identified from the microarray analysis in ES cells and day-4 EBs. Total RNA was prepared, and RT-PCR was performed as described in Experimental Procedures. Aliquots of each reaction were equalized for the internal control Gapdh and were run on 2% agarose gels containing ethidium bromide. To confirm differentiation toward mesoderm and hematopoietic lineages, RT-PCR for the mesodermal marker Brachyury was carried out. Abbreviations: EB, embryoid body; ES, embryonic stem; RT-PCR, reverse transcription–polymerase chain reaction.
DISCUSSION
Work presented in this paper was supported by the Regional Developmental Agency (One North East), Life Knowledge Park, and a program grant (252) from Research into Ageing, U.K. We would like to thanks Ilka Wappler for performing the microarray analysis and Dr. Heiko Peters for useful discussions.
Gabriele Saretzki and Lyle Armstrong contributed equally to this work.
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b Institute of Human Genetics, University of Newcastle upon Tyne, Newcastle upon Tyne, United Kingdom;
c Department of Biological Sciences, University of Durham, Durham, United Kingdom
Key Words. Embryonic stem cells ? Differentiation ? Antioxidant defense ? Peroxides DNA strand break ? DNA repair ? P-glycoprotein
Correspondence: Prof. Thomas von Zglinicki, Henry Wellcome Laboratory for Biogerontology, Newcastle General Hospital, University of Newcastle upon Tyne, Newcastle upon Tyne NE4 6BE, U.K. Telephone: 44-191-256-3310; Fax: 44-191-256-3445; e-mail: t.vonzglinicki@ncl.ac.uk
ABSTRACT
Murine embryonic stem (mES) cells are pluripotent cells derived from the inner cell mass of blastocysts. There is only a very small number of ES cells that give rise to all tissues of the embryo proper. This small size of the population means that ES cells should be equipped with highly efficient mechanisms to prevent DNA damage, to repair it when it happens, and to counteract any propagation of mutations arising from it. Apoptosis is one efficient way to prevent proliferation of mutations, and, in fact, mES cells are known to be much more proficient in apoptosis than differentiated cells after UV-induced DNA damage . Ionizing irradiation seems to be a less potent trigger of apoptosis in mES cells . Not much is known about the quality of primary stress defenses, specifically antioxidant defenses in mES cells, so far.
Reactive oxygen species (ROS) are a major source of DNA damage because they are continuously produced as a byproduct of normal metabolism. Most differentiated cells are sensitive to an increase in ambient oxygen pressure, because this accelerates the production of ROS from the mitochondrial respiration chain. For instance, by increasing the ambient oxygen partial pressure from 21%–40%, levels of intracellular peroxides and protein carbonyls increased, and lipofuscin accumulated at a faster rate in normal human fibroblasts . At the same time, the replicative lifespan decreased to very few population doublings (PDs), and the rate of telomere shortening increased 4- to 10-fold . Differentiated murine somatic cells are even more sensitive to oxidative stress than human ones . As a result, murine fibroblasts experience replicative senescence early under standard in vitro conditions despite the fact that their telomeres are protected by being much longer than in human cells and by expression of telomerase. If oxidative stress is minimized, these cells can grow without obvious limits, similar to telomerase-expressing human fibroblasts under standard conditions . However, immortality is not equivalent to genomic stability. In fact, mutation frequencies and genomic instability in differentiated murine cells increase with time and with increasing oxidative stress .
We speculated that mES cells should have more efficient stress defenses than differentiated ones to maintain high levels of genomic stability at the single-cell level. We believe that insight into how ES cells are able to maintain the stability of their genomes over many cell divisions will help to understand aging of somatic stem cells in vivo and its impact on age-related disease in the adult organism. mES cells can be grown in culture without apparent limit on gelatin-coated culture flasks in media containing leukemia inhibitory factor (LIF). Growth in media from which LIF is absent permits differentiation into cell types that have lost the characteristic of infinite growth, form embryoid bodies (EBs), and will eventually senesce. We and others have used this system to demonstrate that downregulation of telomerase reverse transcriptase (Tert), the catalytic subunit of telomerase, is one of the key events occurring early in ES cell differentiation . We now analyze the resistance of mES cells to oxidative stress and their capacity to repair -induced DNA strand breaks. mES cells are highly proficient in antioxidant defense, an ability that decreases during early steps of differentiation into EBs. ROS levels also remain high in mouse embryonic fibroblasts and 3T3 fibroblasts. Furthermore, mES cells perform more efficient DNA strand break repair than differentiated mouse 3T3 fibroblasts. Using microarrays and confirmation by reverse transcription–polymerase chain reaction (RT-PCR), we have identified several candidate antioxidant and stress-resistance genes that become downregulated during differentiation of ES cells into EBs.
MATERIALS AND METHODS
Murine embryonic fibroblasts (MEFs) are sensitive to ambient oxygen. Although they are able to grow essentially unrestricted in culture under physiological ambient oxygen concentration (3%), they enter a telomere length–independent senescence-like state after 10 to 20 PDs under 21% oxygen . An additional increase of the ambient oxygen partial pressure to 40% arrested MEF growth after less than 3 PDs (Fig. 1). Prolonged culture of MEFs under 21% oxygen, for instance in a 3T3 protocol, leads to spontaneous immortalization. This is accompanied by major genomic changes. Such immortalized 3T3 murine fibroblasts died by apoptosis within a few days under 40% hyperoxia (data not shown). In marked contrast, mES cells did grow fast and without limits under 21% oxygen. They even proliferated with nearly the same rate under 40% hyperoxia, and this ability to withstand chronically increased oxidative stress did not decline with age in culture (Fig. 1, data not shown). Frequencies of apoptosis did not increase when mES cells were grown under 40% oxygen (data not shown).
Figure 1. ES cells grow well under high ambient oxygen concentration. Murine ES cells at starting PD were grown under either normoxia (21% oxygen, , solid line) or hyperoxia (40% oxygen, , dotted line). Differences toward PD at the start of the experiments (,PD) are given versus time. HES cells grew nearly as fast under hyperoxia as under normoxia, and there was no significant change in growth rates of ES cells with time. However, freshly prepared murine embryonic fibroblasts ceased growth after approximately eight PDs under normoxia (, solid line) and after fewer than three PDs under 40% oxygen (, dotted line). Abbreviations: ES, embryonic stem; PD, population doubling.
These data suggested that mES cells might have especially high antioxidant defense capacities, which might be lost during differentiation. To test this suggestion, mES cells were differentiated into EBs. Differentiation toward hematopoietic lineages was determined by progenitor colony assays (CFU-GEMM), in which different cytokines and growth factors are used to promote differentiation of early hematopoietic stem/progenitor cells from ES cells. The percentage of hematopoietic CFUs increased markedly on day 4 of the differentiation protocol (24.3%), and 46.2% of EBs were committed toward the hematopoietic lineage at day 6 (Fig. 2A). To investigate whether mES cells have a better capability to maintain low intracellular levels of oxygen free radicals, we stained ES cells, EB cells, and differentiated murine cells with DCF-DA. DCF-DA is trapped within cells by esterification and converted in its fluorescent form by reaction with peroxides. DCF is a substrate for P-glycoprotein, the multidrug efflux pump that is known to be active in a wide variety of committed stem cells . In mES cells, this activity leads to a loss of DCF fluorescence, which is nearly complete within 2 hours. However, this loss is completely abrogated by verapamil, a known inhibitor of P-glycoprotein (Fig. 2B). As Figure 2C shows, the activity of this verapamil-sensitive efflux pump decreases with differentiation and is essentially lost at day 6 of the differentiation protocol. To measure the steady-state levels of peroxides irrespective of P-glycoprotein activity, 5 μM verapamil was used in all subsequent DCF staining experiments. A significant increase of cellular DCF fluorescence with time in the differentiation protocol was found. Differentiation into EBs (day 6) resulted in an approximately fourfold increase in cellular DCF fluorescence, which was similar to values found in 3T3 murine fibroblasts and MEFs (Fig. 2D). DCF fluorescence of mES cells did not increase under hyperoxic culture conditions, whereas that of differentiated cells did (data not shown).
Figure 2. Cellular peroxide content increases with differentiation. (A): EBs were allowed to differentiate in the absence of leukemia inhibitory factor, and hematopoietic commitment was assessed by counting the proportion of EBs producing mixed colonies of myeloid lineage in the colony-forming unit–granulocyte-erythroid-macrophage-megakaryocyte. The data are mean ± SEM from three experiments with triplicate measurements each. (B): Retention of DCF fluorescence in ES cells was measured over a 2-hour period after staining in the presence or absence of verapamil. Data are mean ± SEM from triplicate measurements. (C): Retention of DCF fluorescence by 5 μM verapamil in ES cells and EB cells at days 0 (d0), 2 (d2), 4 (d4), and 6 (d6) of the differentiation protocol. Measurements were performed at 1 hour after staining. (D): Peroxide content was measured by DCF staining of ES cells, EB cells at days 0, 2, 4, and 6 of the differentiation protocol, 3T3 fibroblasts, and MEFs under normoxic culture. Fluorescence values measured in ES cells were set as 100% in every experiment. Data are mean ± SEM from three experiments with triplicate measurements each. Values are significantly different with p = .016 (one-way analysis of variance). Abbreviations: CFC, colony-forming cell; DCF, 2',7-dichloro-3',5-dihydrofluorescein; EB, embryoid body; ES, embryonic stem; MEF, murine embryonic fibroblast; SEM, standard error of the mean.
To establish whether the low peroxide content in mES cells was attributable to their improved antioxidant capacity, we measured the capability of ES and EB cell lysates to delay metmyoglobin/H2O2-mediated free radical generation (TAS). As Figure 3 shows, lysates from ES cells are in fact more proficient in antioxidant defense than those from EB cells.
Figure 3. Antioxidant defense capacity of cell lysates decreases with differentiation of ES cells. TAS per milligram of protein was measured in triplicates, each in three independent experiments, and ES cell averages for each experiment were set as 100%. Data are significantly different with p = .001 (one-way analysis of variance). Abbreviations: ES, embryonic stem; TAS, total antioxidant status.
Next, we examined whether the improved stress protection of ES cells would also include their DNA damage repair capabilities. As expected, initial DNA damage induced by ionizing radiation did not differ between ES cells and differentiated immortal mouse 3T3 fibroblasts with the sole exception of the highest radiation dose (Fig. 4). The capacity of 3T3 cells to repair DNA strand breaks decreased drastically with increasing radiation dose. In contrast, ES cells performed efficient DNA repair even after doses as high as 20 Gy (Fig. 4).
Figure 4. DNA strand-break induction and repair in 3T3 mouse fibroblasts and murine ES cells after different doses of ionizing radiation. Initial damage (open columns) and damage remaining after 1-hour repair at 37°C (black columns) are calculated as indicated in Material and Methods. Data are mean ± standard error of the mean from 6 and 10 independent experiments with 3T3 cells and ES cells, respectively. Significant differences between 3T3 and ES cells under the same treatment are indicated with one (p < .05) or two (p < .01) asterisks. Abbreviation: ES, embryonic stem.
To identify candidate genes that might be involved in the differential regulation of stress defense and repair, we performed a comparative gene expression analysis of ES and day-4 EB cells using the Affymetrix Mouse 430A target array, which analyzes the expression level of approximately 14,000 well-characterized mouse genes. Eight hundred twenty-one genes were identified using twofold change in expression between the samples as cut-off criterion. Affymetrix Gene Ontology Mining tool was used to select 15 candidate genes already known to be involved in cellular response to stress and DNA repair (Table 2). Differential expression was confirmed for all of them by semiquantitative RT-PCR (Fig. 5). With the exception of only two genes (Prdx2 and Hif3a), all candidate genes were downregulated to varying degrees upon differentiation of ES cells to the day-4 EB stage. Seven of the identified genes are known to be directly involved in the regulation of the cellular redox state. In addition, four heat shock genes and one DNA repair gene were found to be downregulated during differentiation. This suggests overall a decreasing antioxidant and stress defense capacity with ongoing differentiation in accordance with the functional data shown above.
Table 2. Genes whose expression levels change significantly upon differentiation of embryonic stem cells to 4-day embryoid bodies with particular relevance to oxidative stress resistance
Figure 5. RT-PCR analysis of 15 candidate genes identified from the microarray analysis in ES cells and day-4 EBs. Total RNA was prepared, and RT-PCR was performed as described in Experimental Procedures. Aliquots of each reaction were equalized for the internal control Gapdh and were run on 2% agarose gels containing ethidium bromide. To confirm differentiation toward mesoderm and hematopoietic lineages, RT-PCR for the mesodermal marker Brachyury was carried out. Abbreviations: EB, embryoid body; ES, embryonic stem; RT-PCR, reverse transcription–polymerase chain reaction.
DISCUSSION
Work presented in this paper was supported by the Regional Developmental Agency (One North East), Life Knowledge Park, and a program grant (252) from Research into Ageing, U.K. We would like to thanks Ilka Wappler for performing the microarray analysis and Dr. Heiko Peters for useful discussions.
Gabriele Saretzki and Lyle Armstrong contributed equally to this work.
REFERENCES
Aladjem MI, Spike BT, Rodewald LW et al. ES cells do not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage. Curr Biol 1998;8:145–155.
Van Sloun PP, Jansen JG, Weeda G et al. The role of nucleotide excision repair in protecting embryonic stem cells from genotoxic effects of UV-induced DNA damage. Nucleic Acids Res 1999;27:3276–3282.
Corbet SW, Clarke AR, Gledhill S et al. P53-dependent and -independent links between DNA-damage, apoptosis and mutation frequency in ES cells. Oncogene 1999;18:1537–1544.
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