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Molecular Biology and Genetics |
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, D-55131 Mainz, Germany [K. O., B. K.]; and Laboratory of Structural Biology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709-2233 [R. W. S., S. H. W.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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N-Alkylation lesions, which represent the majority of alkylation-induced DNA damage (9) , are removed from DNA by BER, a mechanism that involves five sequential reactions: removal of the modified base by a specific glycosylase, 5'-cleavage of the resulting apurinic site by apurinic endonuclease, 3'-cleavage to excise the residual dRP, filling in the resulting gap by DNA polymerase, and sealing of the nick by DNA ligase (10) . A key enzyme in this repair sequence is ß-pol, one of the four known nuclear DNA polymerases in mammalian cells. It is ubiquitously distributed and highly conserved among eukaryotes, is expressed in all tissues and invariably throughout the cell cycle, fills in nucleotides in small gapped duplexes, does not exert proofreading activity, and exhibits an associated 5' dRP lyase activity (11, 12, 13, 14, 15, 16) . Furthermore, ß-pol was shown to be inducible by alkylating agents (17) . These properties make ß-pol a prime enzyme in the BER pathway. Whether ß-pol can be replaced by any one of the other mammalian DNA polymerases in vivo to carry out BER is not known, but such complementation may be unlikely because it occurs in vitro to only a limited extent (18, 19, 20) .
Germ-line knockouts, resulting in a deficiency in any one of the BER proteins ß-pol, apurinic endonuclease, or DNA ligase I, are all embryonically lethal (19, 20, 21) , which suggests that correctly performed BER is highly important for maintaining development. Although ß-pol-deficient null mice are not viable (19) , the corresponding embryonic cells survive in culture (22) , indicating that ß-pol is not essential for cell viability. ß-pol is, however, likely required in responding to genotoxic stress, as indicated by the fact that ß-pol-deficient cells are hypersensitive to the cytotoxic effect of methylating agents (22) . To address the question of the role of ß-pol in cellular defense upon the induction of DNA damage, we analyzed cells lacking ß-pol and compared their frequency of genomic changes and cell death due, notably, to apoptosis with those of cells expressing the enzyme. Here, we report that ß-pol knockout cells are dramatically more sensitive than wild-type cells with regard to chromosomal breakage induced by various mono- and bifunctional alkylating agents, and moreover, they display a higher frequency of apoptosis and necrosis. The data provide evidence that ß-pol is decisive for protection of cells against alkylation-induced apoptosis and for maintaining genomic stability upon DNA damage. They also show that unrepaired DNA lesions, notably DNA repair intermediates, may act as a primary trigger of apoptosis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Transfection Experiments.
To complement the ß-pol deficiency, ß-pol knockout cells were stably transfected with the ß-pol expression vector pRS729 harboring the human ß-pol under the control of the cytomegalovirus promoter (22)
. Transfection was performed using Lipofectamine (Life Technologies, Inc.). Cells were selected with G418, and individual clones were analyzed for ß-pol expression.
Mutagen Treatments.
MMS, MNNG, and MMC were from Sigma Chemical Co. (St. Louis, MO). HeCNU, mafosfamide, and BPDE were kindly provided by Dr. G. Eisenbrand (Kaiserslautern, Germany), Dr. J. Pohl (ASTA Medica, Frankfurt, Germany), and Dr. A. Seidel (Institute of Toxicology, University of Mainz, Germany), respectively. Preparation of mutagen stock solutions and its handling were as described (23)
. The mutagens were added directly from the stock to the medium to give the desired final concentration. If not otherwise stated, treatment was for 60 min. Thereafter, the medium was removed, and fresh medium was added to the plates. For UV (UV-C) light irradiation, the medium was aspirated, cells were irradiated with a 254-nm germicidal lamp, and fresh medium was added.
Survival Experiments.
Reproductive cell death was assayed by measuring colony formation with and without mutagen treatment. Cells (800 up to 4000 cells per plate, depending on the dose level) were seeded per 10-cm dish and treated 6 h later with the mutagen for 60 min. Colonies that appeared after 8 days were fixed with methanol, stained with crystal violet-Giemsa, and counted. Relative colony formation (%) was expressed as colonies per treatment level/colonies that appeared in the control. Colonies that were composed of more than 
50 cells were counted. The plating efficiency was 40% for the wild-type and 25% for ß-pol -/- cells. Necrotic cells were defined here as cells with disintegrated outer membrane and, thus, unable to exclude dye. The yield of necrotic cells was measured either by staining trypsinized cells with trypan blue and counting in a counting chamber (data not shown) or by the annexin V method (see below). In the non-mutagen-treated populations, the frequency of necrotic and apoptotic cells did not exceed 5%.
Apoptosis.
To detect and to quantify apoptosis, we used three different methods. (a) For detection of DNA fragmentation, cells (5 x 105 per 10-cm dish) were seeded and grown for 2 days. They were treated with the mutagens for 60 min and, after a posttreatment incubation time of 72 h, harvested by trypsinization. They were counted, and 107 cells per treatment level were lysed in a hypotonic solution as described (24)
. For selective precipitation of high molecular weight genomic DNA, a concentration of 2.5% polyethylene glycol and 1 M NaCl was used. After phenol-chloroform extraction and ethanol precipitation, the DNA was separated by gel electrophoresis on 1.5% agarose gels. (b) Mutagen-induced apoptosis was quantified by FACS analysis. Cells were seeded (5 x 105 per 10-cm dish), allowed to grow for 2 days, and then treated with the mutagens (60 min). They were further incubated at 37°C for 72 h, trypsinized, fixed, and processed for flow cytometry using FACSort (Becton Dickinson, San Jose, CA), as described previously(25)
. The fraction of cells exhibiting a DNA content lower than G1, which is an accepted marker of apoptosis (26)
, was quantified using a computer-based program (CellQuest, Becton Dickinson). (c) Alkylating agent-induced apoptosis and necrosis were quantified in the same cell population by flow cytometry using annexin V (27)
. In this assay, cells were double-stained with annexin V and propidium iodide, which allows the quantitation of different cell populations: living cells that were not labeled by either annexin V or propidium iodide, necrotic cells that were stained by both agents, and apoptotic cells that were labeled only by annexin V. Exponentially growing cells were treated as described above, harvested at various time points after mutagen treatment by trypsinization, washed with cold PBS, and subjected to annexin V staining according to the manufacturer’s protocol (ApoScreen, Annexin V apoptosis kit, Dianova, Hamburg, Germany).
Western Blotting.
Nuclear extracts were prepared from exponentially growing cells harvested by scraping off from the plates as described previously (28)
. Protein (60 µg per sample) was electrophoretically separated on a 10% polyacrylamide gel (2 h, 30 mA) and transferred to nitrocellulose membrane using a buffer containing 25 mM Tris, 100 mM glycine, and 25% methanol. Filters were preincubated with 5% nonfat dry milk-0.1% Tween 20 in PBS for 4 h. Then, rabbit polyclonal antibody raised against the mammalian ß-pol protein was added to the solution and incubated for 2 h. The filter was extensively rinsed and incubated afterwards with peroxidase-conjugated antirabbit antibody from donkey (Amersham) for 1 h. After washing in 0.1% Tween 20, antibody binding was visualized by ECL Plus (Amersham), according to the manufacturer’s protocol.
Chromosomal Aberrations.
Cells were seeded (2 x 105 per 5-cm dishes) and allowed to grow for 1–2 days. They were then treated during exponential growth with the genotoxic agents (60 min). After recovery, colcemid was added (50 ng/ml final concentration), and after further 2 h, cells were harvested by trypsinization as indicated. Slides were made as described (6)
and stained by Giemsa. At least 100 metaphases were evaluated per treatment level. The following aberration types were scored: chromatid breaks and isochromatid breaks, triradials, quadriradials, and all other types of non-, partially, and fully reunited translocations. Cells exhibiting more than seven aberrations were scored as having multiple aberrations. Gaps were scored but not included in the final evaluation. Statistical comparison of aberration frequencies was made using Fisher’s test.
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RESULTS |
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, where reproductive cell death was quantified by measuring the colony-forming ability of wild-type and ß-pol -/- fibroblasts, which was examined as a function of dose of various agents inducing either monoadducts or cross-links in DNA. Interestingly, in addition to an increased sensitivity to the methylating agents MMS and MNNG, ß-pol -/- cells are hypersensitive to the cross-linking antineoplastic drugs mafosfamide and MMC. Hypersensitivity was not observed for the chloroethylating agent HeCNU, UV-C light (Fig. 1)
, or treatment with cisplatin, melphalan, or the polycyclic hydrocarbon BPDE (data not shown).
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. Because nucleosomal degradation is difficult to measure quantitatively, comparison of apoptotic cell fraction was further conducted by determining the sub-G1 DNA content of the population (Fig. 2B)
. Those agents giving rise to elevated reproductive cell death in ß-pol -/- cells also induced a significant level of apoptosis (Fig. 2C)
. There was no difference in the apoptotic frequency between ß-pol -/- and wild-type cells after treatment with HeCNU and UV-C light.
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. The yield of apoptosis was higher than the level of necrosis (40 versus 15% for 1 mM MMS and 72-h postincubation time), indicating that apoptosis is a major pathway in methylation-induced reproductive death of ß-pol -/- cells. This study (and data not shown) also revealed that both necrosis and apoptosis are rather late effects; they were detected at a significant extent in mice fibroblasts only at time points later than 24 h after treatment with a DNA-damaging agent (Fig. 3)
. Basically similar results were obtained by quantitating necrotic cells with trypan blue staining confirming that ß-pol -/- cells are clearly more sensitive than the wild-type cells (data not shown).
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and data not shown). These lines were compared in their apoptotic response upon treatment with MMS. As demonstrated in Fig. 4B
, apoptosis increased in a dose-dependent manner in cells expressing lower amounts of ß-pol than the wild-type cells. The yield of apoptotic cells induced as a function of dose of MMS gradually increased as less ß-pol was expressed. Relating apoptosis frequency to the ß-pol expression level, a nonlinear relationship was obtained (Fig. 5)
. Thus, even a 75% reduction in ß-pol level only slightly sensitized cells to MMS-induced apoptosis. Apoptosis was dramatically enhanced, however, if ß-pol expression level decreased below 20% of the control level (of ß-pol +/+ cells). From this, we conclude that cells are able to tolerate, to some extent, a reduction in the amount of ß-pol without significant loss of protection against alkylation-induced apoptosis.
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, the frequency of chromosomal aberrations observed in ß-pol -/- cells was dramatically enhanced following treatment with MMS, as compared to wild-type cells. Thus, a dose of 0.5 mM MMS induced chromosomal aberrations in 24% of the wild-type cells, whereas 100% of the ß-pol -/- cells were affected (Fig. 6A)
. Also, the average number of aberrations per cell was clearly enhanced in ß-pol -/- cells (Fig. 6B)
. Hypersensitivity of ß-pol deficient cells to MMS was observed with each recovery time at which mitotic cells were analyzed (Fig. 6, C and D)
indicating that possible differences in cell cycle delay cannot account for the different responses of the cell types.
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, ß-pol -/- cells were significantly more sensitive to MNNG, mafosfamide, and MMC but not to HeCNU, cisplatin, melphalan, BPDE, and UV-C. These results suggest that preclastogenic lesions induced by MMS, MNNG, mafosfamide, and MMC but not by the other agents used in this study are processed by ß-pol, yielding chromosomal aberrations.
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, confirming the results obtained with the immortalized lines.
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. These cells that were partially complemented regarding ß-pol expression were clearly more resistant than ß-pol-deficient cells but still less resistant than the wild type as to their clastogenic response after treatment with MMS (Fig. 6)
. A similar complementation of chromosomal aberration frequency was observed on exposure to MNNG, mafosfamide, and MMC. The transfectants behaved similarly, however, to wild-type and ß-pol -/- cells in chromosomal sensitivity towards HeCNU, cisplatin, melphalan, BPDE, and UV-C light (Table 1)
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To elucidate the quantitative relationship between ß-pol expression and protection against induced chromosomal changes, we extended the study to various other ß-pol-transfected cell clones derived from ß-pol -/- cells (shown in Fig. 4
). Chromosomal aberration frequencies induced by MMS in the various clones depended on the level of ß-pol expression (Fig. 7A)
. As observed for the end point apoptosis, the induced aberration frequency was a nonlinear function of ß-pol level (Fig. 7B)
. Exceptionally strong increases in aberration frequency were observed in cells that expressed ß-pol at a level of <20% of the wild type. Thus, ß-pol is clearly required for defense against the clastogenic effect of methylating agents, although some variation in the amount of ß-pol can be tolerated by the cells, presumably because of saturation of ß-pol expression in the wild type.
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DISCUSSION |
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An interesting observation reported here was the high frequency of mutagen-induced apoptosis in ß-pol -/- cells. Apoptosis appears to account largely for the hypersensitivity in reproductive cell death (as measured by loss of colony-forming ability) observed in these cells. The doses of MMS and MNNG used to yield a high frequency of apoptosis in ß-pol -/- cells did not induce significant apoptosis in wild-type cells. Also, the antineoplastic drugs mafosfamide and MMC but not HeCNU induced increased yields of apoptosis in ß-pol -/- cells, which is in line with the observed hypersensitivity to reproductive cell death of ß-pol -/- cells exhibited to these agents. The results provide evidence that a deficiency in DNA repair may cause cells to become vulnerable to undergo apoptosis and that incompletely repaired DNA damage or accumulating DNA repair intermediates can act as a primary trigger in the cellular response activating the apoptotic pathway. According to our knowledge, this is the first report showing that BER acts protectively against the induction of apoptosis by various alkylating DNA damaging agents. Recently, it has been shown that repair of O6-methylguanine mediated by the repair protein MGMT as well as reduced mismatch repair exerts protection against apoptosis induced by O6-alkylguanine-generating agents, indicating that O6-alkylguanine is an apoptosis-inducing lesion (29 , 30) . Thus, together with the data presented here, accumulating evidence is available that nonrepaired or incompletely repaired DNA damage may act as a trigger of apoptosis.
Our finding that both established cells and primary fibroblasts lacking ß-pol are highly sensitive to induced chromosomal breakage and that chromosomal hypersensitivity can be complemented by ß-pol transfection strongly indicates that ß-pol is involved in the cellular defense not only against cell killing but also the clastogenic effects of methylating agents. BER is the major pathway by which MMS- and MNNG-induced DNA lesions are removed because the majority of adducts produced in DNA by both compounds are N-methylpurines. Thus, 95 and 79% of total DNA methylations produced in DNA by MMS and MNNG, respectively, are N7-methylguanine, N3-methyladenine, and N3-methylguanine (9) , all of which are removed by BER (31) . The first two enzymes involved in BER are N-methylpurine-DNA glycosylase and apurinic endonuclease. Lack of N-methylpurine-DNA glycosylase increases the toxicity of MMS both in bacteria (32) and mammalian cells (7) , indicating that N-methylpurines are potentially cytotoxic lesions. Also, down-modulation of apurinic endonuclease by antisense cDNA transfection increased cellular sensitivity to the killing effect of methylating agents (33) , and overexpression of yeast apurinic endonuclease in CHO cells rendered them more resistant to the toxic and clastogenic effect of alkylating compounds (34) . The finding that a deficiency in ß-pol makes cells dramatically hypersensitive to the chromosome breakage-inducing effect of MMS and MNNG suggests that apurinic sites cleaved by apurinic endonuclease cannot be processed efficiently by one of the other DNA polymerases still expressed in ß-pol -/- cells. Therefore, ß-pol appears to be highly important for BER of methylation lesions and the protection against methylation-induced chromosomal breakage. Interestingly, transfection of ß-pol into ß-pol-deficient cells complemented the hypersensitive phenotype in a nonlinear dose-dependent manner, which was shown both for the end point apoptosis and chromosomal aberrations. Complementation to nearly wild-type sensitivity was already observed with low ß-pol expression level (>25% of that of the wild type), which indicates that ß-pol is abundantly expressed in ß-pol +/+ mouse fibroblasts. In line with this is that ß-pol heterozygous primary fibroblasts exhibited similar aberration frequencies than the corresponding wild-type cells.4
Apurinic sites cleaved by apurinic endonuclease give rise to 5'-terminal dRPs that can be removed by the dRP lyase activity of ß-pol itself (16) . It might be interesting to speculate that the gap with 5'-dRP residue prevents other DNA polymerases from participating in an efficient repair of a methylated DNA template. The unrepaired single-stranded, gapped DNA structures may be substrates for nucleases, ultimately giving rise to DNA double-strand breaks. These breaks may illegitimately religate, leading to translocations (which amount, depending on the agent, to 30–70% of all aberrations observed both in wild-type and ß-pol -/- cells) or, if religation does not occur, may give rise to chromatid breaks. These chromosomal changes, which occur at high frequency in ß-pol -/- cells, may act as a trigger of apoptosis. This pathway of DNA damage-induced apoptosis has been proposed previously for c-Fos-deficient cells (28) . Alternatively, we hypothesize that DNA repair intermediates arising from BER that are not completely removed due to ß-pol deficiency (e.g., single-strand gaps) and/or resulting DNA breaks directly activate the apoptotic pathway. In line with the first hypothesis is that apoptosis induced by mutagens in ß-pol -/- cells is not an immediate but rather a late response after the induction of DNA damage, which occurs more than 24 h after mutagen treatment when aberrations have been formed.
There is ample evidence for ß-pol to play a major role in BER of methylation lesions but not in the repair of bulky DNA adducts. Therefore, the observation that ß-pol mediates protection against the cytotoxic and clastogenic effects of mafosfamide and MMC but not to various other agents tested inducing bulky lesions such as HeCNU, melphalan, BPDE, and UV-C attracts attention. Mafosfamide is an analogue of cyclophosphamide that forms 4-hydroxycyclophosphamide spontaneously; 4-hydroxycyclophosphamide is thought to be genotoxic due to N7-guanine interstrand cross-link formation (35 , 36) . Likewise, MMC induces monoadducts and interstrand cross-links, the latter being considered as major genotoxic lesions (37) . The hypersensitivity of ß-pol -/- cells to these agents indicates that some of the lesions induced by mafosfamide and MMC are repaired by a pathway in which ß-pol participates. Whether or not these ß-pol-repaired lesions are cross-links or monoadducts remains to be seen. It should be noted that HeCNU (which is representative of antineoplastic chloroethylnitrosoureas) and melphalan also induce interstrand cross-links. Thus, most of the genotoxic effects of HeCNU are due to O6-chloroethylguanine, which subsequently undergoes intramolecular rearrangement to form N1-guanine-N3-cytosine interstrand cross-links (38) . Assuming that cross-links are involved in hypersensitivity of ß-pol-deficient cells, one can consider the possibility that different cross-links are repaired via different DNA repair pathways, one of which requires ß-pol. Another possible explanation for the protection mediated by ß-pol against MMC and mafosfamide but not melphalan and HeCNU rests on the finding that MMC and mafosfamide induce, in addition to DNA cross-links, other kinds of DNA damage via intermediary products that could be subject to BER. Thus, MMC is able to induce oxidative DNA damage (39) and mafosfamide, which reacts in a similar way as cyclophosphamide, may induce base monoadducts via acrolein formation (40 , 41) . These lesions are likely not the major toxic one, but they may contribute to some extent to the overall genotoxicity of the agents. Mafosfamide and MMC as well as various methylating agents are being used as potent cytostatic drugs in cancer therapy. The protection mediated by ß-pol against this class of agents might be of importance in designing strategies of alleviating alkylating drug resistance of tumor cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by the Deutsche Forschungsgemeinschaft Grant SFB 519, B4. 
2 To whom requests for reprints should be addressed, at Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Strasse 67, D-55131 Mainz, Germany. Phone: 49-6131-17-3246; Fax: 49-6131-17-3421; E-mail: kaina{at}mail.uni-mainz.de
3 The abbreviations used are: MGMT, O6-methylguanine-DNA methyltransferase; BER, base excision repair; dRP, deoxyribose phosphate; ß -pol, DNA polymerase ß; MMS, methyl methanesulfonate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; MMC, mitomycin C; HeCNU, N-(2-chloroethyl)-N-nitroso-N' -(2-hydroxyethyl)urea; BPDE, benzo(a)pyrene diol epoxide.
Received 5/28/98. Accepted 1/27/99.
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, necrotic cells; 
, apoptotic cells.
