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[Cancer Research 62, 1524-1530, March 1, 2002]
© 2002 American Association for Cancer Research

Molecular Biology and Genetics

Deficiency in DNA Polymerase ß Provokes Replication-dependent Apoptosis via DNA Breakage, Bcl-2 Decline and Caspase-3/9 Activation1

Kirsten Ochs, Jochen Lips, Simone Profittlich and Bernd Kaina2

Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells deficient in DNA polymerase ß (ß-pol) are impaired in base excision repair (BER) and hypersensitive to various DNA damaging agents, including methylating mutagens. Hypersensitivity of ß-pol-deficient cells to methylating agents is because of induction of apoptosis (Ochs et al., Cancer Res., 59: 1544–1551, 1999), indicating incompletely repaired DNA damage to trigger the response. Here we show that defective BER in ß-pol-null cells results in an early and transient increase in the frequency of DNA single-strand breaks on treatment with methyl methanesulfonate. These breaks arising as repair intermediates are not likely to trigger apoptosis directly because they were repaired efficiently and generated both in resting and proliferating cells, whereas only proliferating cells underwent with high frequency apoptosis after methylation. Therefore, we propose that single-strand breaks are converted into another kind of critical apoptosis-triggering lesion during replication. These critical secondary DNA lesions are likely to be nonrepaired DNA double-strand breaks (DSBs), which are formed at higher frequency in ß-pol-null than in wild-type cells. Apoptosis was a late response not detectable before 24 h after methylation and was preceded by DSBs formation, extensive chromosomal breakage, and decline in Bcl-2 level and caspase-9 and caspase-3 activation. Caspase-8 was not significantly activated. Transfection of ß-pol-null cells with bcl-2 protected against methylation-induced apoptosis, indicating Bcl-2 to be causally involved. Overall, the data demonstrate that in cells lacking ß-pol, defective BER results in incompletely repaired DNA damage, which triggers apoptosis in a replication-dependent way by activating the mitochondrial death pathway. It is suggested that DSBs act as a critical ultimate apoptosis-inducing lesion.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Many, if not all, DNA repair-deficient cell types are hypersensitive to the killing effect of DNA damaging agents, supporting the view that nonrepaired DNA damage is responsible for genotoxin-induced cell death (1) . The molecular mechanism of DNA damage-induced cell death is, however, not yet understood. Reproductive cell killing effects, as measured by loss of colony-forming ability, can be the result of induced apoptosis, necrosis, or irreversible cell cycle blockage or caused by the so-called mitotic death. The main interest of research is currently directed toward apoptosis, which appears to be the main route of cell killing on treatment of cells with death receptor ligands (e.g., tumor necrosis factor {alpha}, Fas-L) and various genotoxic agents, including DNA-damaging anticancer drugs (2, 3, 4, 5) . A major question currently addressed is whether DNA-damaging agents induce apoptosis via receptor activation (6) or by activating receptor-independent apoptotic pathways (7 , 8) . A prerequisite for studies of the mechanism of apoptosis evoked by DNA damage would be a definition of the DNA damage itself, which provokes apoptosis. There are only very few examples for which clear evidence does exist that a particular type of DNA damage triggers apoptosis. Most convincing evidence was provided for O6-methylguanine induced by simple methylating agents in DNA. This lesion acts via the mediation of mismatch repair as a primary trigger of apoptosis (9, 10, 11, 12) , activating the mitochondrial damage pathway (13) .

O6-methylguanine amounts to only <=8% of total DNA methylations (14) . The majority of DNA damage induced spontaneously, on treatment with simple alkylating agents and by oxidative stress, is repaired by BER3 (15 , 16) . In this repair pathway, ß-pol plays a key role. The protein possesses two enzymatic activities: dRPase activity, which is responsible for removal of the sugar residue (5'-deoxyribose phosphate) from the incised DNA strand, and a DNA polymerase activity required for sealing the repair gap (for review, see Ref. 17 ). ß-pol appears to be the major DNA polymerizing enzyme in the short-patch BER pathway, which is characterized by singlenucleotide insertion into the repaired patch (18, 19, 20) . ß-pol-null cells are hypersensitive to various DNA-damaging agents, notably methylating compounds, which provided evidence that BER-involving ß-pol is highly important in avoiding genotoxic and cytotoxic effects on alkylation (21 , 22) . Hypersensitivity to the killing effect of methylating agents has been attributed to the lack of dRPase function (23) . ß-pol-null cells display a high frequency of methylating agentinduced apoptosis, indicating that incompletely repaired DNA alkylation lesions trigger apoptosis (22) .

To elucidate the mechanism of cell death and apoptosis induced by nonrepaired DNA damage in more detail, we investigated the mechanism of apoptotic death in ß-pol-null (ß-pol-/-) cells and compared it with the response of the corresponding wild-type fibroblasts (ß-pol+/+). In particular, we wished to address the question of how defective BER can trigger cytotoxicity and apoptosis. The data obtained show that incompletely repaired DNA damage in ß-pol-null cells gives rise to a transiently elevated level of DNA SSBs. These breaks do not provide the apoptotic signal per se, because they are rapidly removed in both dividing and nondividing cells, whereas only dividing cells undergo apoptosis. Therefore, nonrepaired SSBs are supposed to be converted by DNA replication into ultimate critical lesions, which are likely to be DNA DSBs triggering Bcl-2 decline and caspase-9 and caspase-3 activation. Overall, we conclude that incompletely and delayed repaired base damage because of defective BER induces DNA replication-dependent apoptosis via secondary lesions by activating the intrinsic mitochondrial damage pathway.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mutagens.
MMS was prepared and handled as described (22) . If not otherwise stated, treatment lasted for 60 min. Thereafter, cells were washed, and fresh medium was added.

Antibodies.
Antibodies against Bcl-2, Bax, ERK-2, and PARP were from Santa Cruz Biotechnology. The polyclonal antibody against Bcl-xL was purchased from Transduction Laboratories, Inc.

Cell Lines and Culture Conditions.
The cell lines ß-pol null (-/-; Mb19tsA, clone2B2) and the corresponding wild type (+/+; Mb16tsA, clone 1B5) used in this study were described previously (21 , 22) . They were derived from embryonal tissue of either ß-pol knockout or wild-type mice. The cells were cultivated in DMEM, as described (21) .

Transfection Experiments.
To overexpress Bcl-2, ß-pol-/- cells were stably transfected with the expression vector pcDNA3.1(-)Myc-His harboring wild-type bcl-2 under the transcriptional control of the cytomegalovirus promoter (24) . Transfection was performed using Lipofectamine (Life Technologies, Inc.). Cells were selected with G418 (0.8 mg/ml), and individual clones were checked for Bcl-2 expression by Western blot analysis.

Clonogenic Survival Assay.
Reproductive cell death was assayed by measuring colony formation as described (25) . Cell growth and cytotoxicity after treatment with MMS were measured using the MTT assay. Cells (2500/well) were treated with MMS 24 h after seeding. Their potential to metabolize MTT was quantified as described (26) , with some modifications. MTT (50 µl/500 µl medium) dissolved in PBS (5 mg/ml) was added to each well and incubated for 4 h at 37°C. After discarding the supernatants, 0.2 ml of 96% ethanol was added to dissolve formazan crystals. Absorbance was measured at 540 nm and at 690 nm for reference.

Apoptosis and Necrosis.
The frequency of apoptosis and necrosis in untreated cell populations and after treatment with MMS was determined using flow cytometry and Annexin V/propidium iodide staining (27) as described previously (13) . Evaluation of cell populations was performed using a computer-based program (Cell Quest; Becton Dickinson).

SCGE (Comet Assay).
The procedure described for the alkaline comet assay for detection of SSBs (28) was followed with modifications. Cells (3 x 105) were seeded/5-cm dish and treated 48 h later with MMS. At various times after treatment, cells were trypsinized, washed with cold PBS, and kept on ice until assayed. Cells were embedded in 0.5% low-melting point agarose, and microscope slides were immersed in ice-cold lysis solution [2.5 M NaCl, 100 mM EDTA, 10 mM Tris, and 1% sodium laurylsarcosine (pH 10); 1% Triton X-100 and 10% DMSO were added freshly] and kept at 4°C for 1 h. After lysis, the slides were placed in a horizontal electrophoresis box and exposed to alkali [300 mM NaOH and 1 mM EDTA (pH > 13)] for 40 min to allow for DNA unwinding and expression of alkali-labile sites. After electrophoresis (25 V, 300 mA, 15 min), the slides were neutralized in 0.4 M Tris buffer (pH 7.5). The fixed and ethidium bromide-stained slides were analyzed using image analysis system (Kinetic Imaging, Ltd.; Komet 4.0.2, Optilas) determining the Olive Tail Moment (percentage of DNA in the tail x tail length) of 50 cells/sample. For determination of the frequency of DSBs, the neutral comet assay was applied essentially as described previously (29) .

Chromosomal Aberrations.
Cells were seeded (3 x 105/5-cm dish) and after 1–2 days treated with MMS. Metaphase preparation and evaluation of the slides were performed as described (22) .

Cell Cycle Analysis.
Cells were seeded (5 x 105/10-cm dish), allowed to grow for 2 days, and then treated with MMS. After the indicated recovery times, cells were trypsinized, fixed, stained with propidium iodide, and processed to flow cytometry, as described (30) . The fraction of cells in G1-, S-, and G2-M-phase and the fraction of apoptotic cells (exhibiting a DNA content lower than G1) were quantified using a computer-based program (Cell Quest; Becton Dickinson).

Preparation of Cell Extracts and Western Blotting.
Nuclear cell extracts were prepared as described (31) in the presence of 1 mM phenylmethylsulfonylfluorid and 10 µg/ml aprotinin, 10 µM bestatin, 10 µM leupeptin, 1 µM pepstatin, and 0.1 mM phenylmethylsulfonyl fluoride. Whole cell extracts were prepared by lysis of PBS-washed cells in 10 µl of ice-cold sample buffer containing 25 mM Tris-HCl (pH 6.8), 1% SDS, 5% glycerol, and 2.5% 2-mercaptoethanol followed by sonification on ice. For detection of Bcl-2 family proteins, whole cell extracts were separated onto 0.1% SDS and 12% polyacrylamide gels and subjected to Western blot analysis (22) . Nuclear extracts for detection of PARP were fractionated onto 0.1% SDS and 7% polyacrylamide gels. Proteins were visualized by enhanced chemiluminescence (Amersham).

Measurement of Caspase Activity.
Caspase activity assays were performed using the CPP32/caspase-3, FLICE/caspase-8 colorimetric kit (Chemicon), or the corresponding colorimetric kits (R & D Systems) as described (13) .


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cell Killing and Apoptosis in Wild-type and ß-pol-/- Cells.
Cells lacking ß-pol are hypersensitive to the cytotoxic effect of simple alkylating agents. This is shown in Fig. 1ACitation for the end-point reproductive cell death (data are from Ref. 22 and shown here for comparison) and in Fig. 1BCitation for cell killing measured by the MTT assay as a function of time after treatment. Hypersensitivity to MMS is mainly because of the induction of apoptosis (40% apoptosis versus 15% necrosis after treatment with 1 mM MMS and 72-h recovery; Fig. 1CCitation and data not shown). Apoptosis in ß-pol-/- cells is a late response. It is detectable not earlier than 28 h after treatment and increases with postincubation time (Fig. 1C)Citation . Interestingly, loss of viability was detected already 24 h after methylation (see Fig. 1B)Citation . This early cytotoxicity is because of necrosis, which was detected at lower frequency and time points before apoptosis was appearing (data not shown).



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  		Fig. 1. Cytotoxicity and apoptosis induced in ß-pol-proficient (ß-pol+/+) and ß-pol-deficient (ß-pol-/-) cells by MMS. A, reproductive cell death, as measured by the loss of colony-forming ability, after treatment with different doses of MMS (data are from Ref. 22 ). B, cytotoxicity of MMS as determined by the MTT test at different times after treatment with 0.75 mM (closed symbols) and 1 mM (open symbols) MMS. Data were obtained from three independent experiments performed in triplicate. C, frequency of apoptosis as a function of time after treatment with 0.75 and 1 mM MMS, respectively. Apoptosis was measured as described in "Materials and Methods" using the method of Annexin V/PI double staining. Data are the mean of at least three independent experiments; bars, SD.

 
DNA SSBs in ß-pol-/- Cells.
Cells lacking ß-pol are defective in the intermediate step of BER: the removal of 5'-deoxyribose phosphate and sealing of the resulting abasic site. It is reasonable to assume that this leads to an accumulation of SSBs arising from the action of apurinic endonuclease on abasic sites. Furthermore, it may be speculated that these SSBs act as a trigger of cell death, notably apoptosis. The frequency of SSBs in wild-type and ß-pol-/- cells was measured by SCGE at various times after MMS treatment. As shown in Fig. 2ACitation , the frequency of SSBs was significantly higher in ß-pol-/- than in wild-type cells. Significantly higher yields of SSBs were observed already immediately after treatment with MMS, even if the exposure occurred for a rather short period, i.e., pulse treatment for 15 min (Fig. 2B)Citation . Interestingly, the frequency of SSBs decreased during the postexposure period with similar kinetics in ß-pol-/- and ß-pol+/+ cells. This indicates that ß-pol-deficient cells have the ability to repair SSBs that arise as intermediates during BER. However, the repair occurs delayed, as compared with the wild type, resulting in a higher yield of residual SSBs in ß-pol-/- cells at all postincubation times (Fig. 2)Citation . Similar results were obtained by the alkaline elution assay (data not shown).



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  		Fig. 2. ß-pol-deficient cells exhibit elevated levels of SSBs in response to MMS. A, formation of SSBs, as determined by alkaline SCGE (comet assay), in ß-pol+/+ and ß-pol-/- cells at various time points after treatment for 60 min with 1 mM MMS. Data are the mean of three independent experiments; bars, SD. B, frequency of SSBs in ß-pol+/+ and ß-pol-/- cells immediately after treatment with MMS (1 mM) for 60, 30, and 15 min. Data of one representative experiment are shown.

 
Apoptosis Is Related to Cell Proliferation.
To address the question of whether nonrepaired SSBs induced in ß-pol-/- cells attributable to impaired BER trigger directly apoptosis, cells were analyzed, which were exponentially growing or held in confluence. In the exponentially growing state, 43% of cells were in G1, in contrast to the confluent state where 77% were in G1 (Fig. 3A)Citation . The frequency of SSBs was the same in exponentially growing and arrested cells (Fig. 3B)Citation , indicating that BER takes place both in resting and dividing cells, resulting in nonsealed DNA breaks because of incomplete BER. If these breaks directly trigger apoptosis, one would expect apoptosis to occur immediately without the requirement of cell proliferation. This, however, is not the case. As shown in Fig. 3CCitation , the frequency of MMS-induced apoptosis is much higher in exponentially growing than in resting cells (38.2 versus 9.2% in growing and resting cells treated with 1 mM MMS, respectively). This indicates that SSBs per se do not lead to apoptosis. Therefore, we suppose that SSBs arising as BER intermediates become converted into secondary lesions and chromosomal aberrations during DNA replication, which acts as the ultimate trigger of apoptosis.



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  		Fig. 3. Only proliferating ß-pol-/- cells show induction of apoptosis in response to MMS. A, cell cycle distribution of exponentially growing and serum-starved, arrested ß-pol-/- cells. B, yield of DNA SSBs in exponentially growing and confluent, serum-starved cells on treatment with 1 mM MMS; frequency of SSBs in cells treated with MMS (1 mM) under the same experimental conditions as shown under A. exp, exponentially growing; confl, serum-starved confluent cells. C, frequency of necrotic (Nec), apoptotic (Apo), and living (Liv) cells 72 h after pulse treatment (60 min) with 1 mM MMS, respectively. To arrest cells in G1, they were, after seeding, kept in confluence for 2 days and thereafter fed with serum-free medium. After 2 days without serum, they were treated with MMS. Exponentially growing and arrested cells were harvested on day 0 (untreated) and analyzed by flow cytometry to determine cell cycle distribution, which is given in percentages above each DNA histogram. Cells were treated with MMS, or left untreated (Con), harvested on day 3 (72 h), and subjected to cytometric measurement. Determination of apoptotic, necrotic, and living cells was performed using the method or Annexin V/PI double staining, as described in "Materials and Methods." The resulting dot blot diagrams are shown from a representative experiment.

 
DNA DSBs and Cell Cycle Inhibition in ß-pol-/- Cells.
Critical secondary lesions derived from DNA replication at incompletely repaired patches in ß-pol-/- cells could be DSBs, which were demonstrated previously to be highly efficient in triggering apoptosis (29) . DSBs were indeed detected at higher levels on alkylation in ß-pol-/- cells compared with the wild type, as measured at various times after methylation (Fig. 4A)Citation . DSBs can lead to the formation of chromosomal aberrations, which become detectable when the cells harboring them enter mitosis. Indeed, under the treatment conditions applied, aberration frequency was clearly enhanced in ß-pol-/- cells, which was observed already 8 h after MMS treatment (Fig. 4B)Citation , suggesting DSBs attributable to defective BER to cause aberrations. Nonrepaired DNA damage, i.e., either SSBs or DSBs arising from them, seem also to cause cells to become transiently arrested in S phase, because in ß-pol-/- cells, the proportion of S phase increased at early times (2–5 h) after methylation. At late time points (15–30 h), a significant increase in the G2-M population was also observed. In contrast, in wild-type cells, the same dose of MMS affected cell cycle distribution of the population only very slightly (Fig. 4C)Citation .



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  		Fig. 4. Induction of DSBs, chromosomal aberrations, and S-G2 phase arrest in ß-pol-/- cells. A, relative DSB frequency in wild-type and ß-pol-/- cells nontreated (Control) and MMS treated (0.75 mM, 60 min). DSBs were quantified by neutral SCGE at the times indicated after MMS exposure. Data of three independent experiments ±SD are shown. B, induction of chromosomal aberrations in MMS-treated cells. Aberration yield (aberrations/cell) was determined at different times after pulse treatment with the mutagen (0.25 mM, 60 min) in exponentially growing cell populations. C, effect of MMS treatment on cell cycle progression of ß-pol wild-type (+/+) and ß-pol-/- cells. Cells were treated with 0.8 mM MMS for 60 min and analyzed by flow cytometry determining cell cycle distribution at the times indicated. The data shown are the mean of three independent experiments.

 
Apoptosis Induced in ß-pol-/- Cells Is Mediated by Bcl-2 Decline.
The expression level of proteins of the Bcl-2 family in wild-type and ß-pol-/- cells is demonstrated in Fig. 5ACitation . The data show that MMS treatment caused a decline in Bcl-2 level, whereas Bax and Bcl-xL remained largely unaltered. Bcl-2 decline was already observed 15 h after MMS pulse treatment (for quantification, see bottom panel of Fig. 5ACitation ) and thus clearly preceded the appearance of apoptotic cells. Interestingly, in the repair-competent ß-pol+/+ cells, MMS caused a slight but still significant increase in Bcl-2 protein level (see Fig. 5ACitation bottom panel), which was also observed previously in Chinese hamster cells on methylation (13) . Decline of Bcl-2 occurred dose dependently and was related inversely to the frequency of MMS-induced apoptosis (Fig. 5B)Citation , which may be taken to indicate that it is causally involved in the strong apoptotic response of ß-pol-/- cells.



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  		Fig. 5. Induction of apoptosis in ß-pol-deficient cells in response to MMS is accompanied by a decrease in Bcl-2 level. A, expression of Bcl-2, Bax, and Bcl-xL in wild-type (ß-pol+/+) and ß-pol-/- cells untreated (Con) and at the indicated times treated with 1 mM MMS, as determined by Western blot analysis. The filters were reincubated with ERK-2 antibody, which served as a loading control. The relative expression levels of Bcl-2 in MMS-treated cells, as compared with untreated controls, were determined by quantification of the Bcl-2 signals, which were set in relation to ERK-2. The relative expression levels of Bcl-2 in ß-pol-proficient (+/+) and -deficient (-/-) cells are shown as a function of time in the graph below. B, expression of Bcl-2 in ß-pol-/- cells 30 h after treatment with various doses of MMS, as determined by Western blot analysis. The relative Bcl-2 expression in ß-pol-deficient cells was quantified and plotted as a function of dose. The frequencies of apoptosis were calculated from flow cytometric measurements (fraction exhibiting subG1 DNA content) and shown for comparison. The frequency of apoptosis at the highest dose (1 mM) was set to 100%.

 
Involvement of Bcl-2 in DNA damage-induced apoptosis in ß-pol-/- cells was verified by transfection experiments with bcl-2 (using an vector expressing myc-tagged Bcl-2). Two stably transfected clones (designated as bcl-2wt/cl.1 and bcl-2wt/cl.6) clearly expressing the transfected Bcl-2 protein were established (Fig. 6A)Citation . Although treatment of these cells with MMS resulted in decrease of Bcl-2 protein level, which pertained to both the endogenous and the transfected Bcl-2 protein, the remaining overall Bcl-2 level was clearly higher in the Bcl-2 transfectants than in the neotransfected control (Fig. 6A)Citation . Survival assays with these cells showed that the viability of the Bcl-2 transfectants after MMS treatment was enhanced (Fig. 6B)Citation , and the frequency of apoptosis was reduced (Fig. 6CCitation and data not shown). This supports the conclusion that Bcl-2 decline is a decisive event in DNA damage-induced apoptosis in ß-pol-deficient cells.



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  		Fig. 6. Overexpression of Bcl-2 protects ß-pol-deficient cells from MMS-induced cytotoxicity and apoptosis. ß-pol-/- cells were stably transfected with an expression vector encoding human myc-tagged, wild-type Bcl-2 (enabling to distinguish between transfected and endogenous Bcl-2). A, expression of myc-tagged Bcl-2 and endogenous Bcl-2 in Bcl-2-overexpressing ß-pol-/- cell clones (designated as bcl-2wt/cl.1 and bcl-2wt/cl.6) and the corresponding neotransfected ß-pol-/- cells. Total cell extracts of cells untreated (Con) and treated with 1 mM MMS were harvested 48 h after treatment and subjected to Western blot analysis. B, cytotoxicity of the cells as determined by the MTT test 48 h after treatment with 0.75 mM MMS. Values were obtained from three independent experiments performed in triplicate. The relative viability of the Bcl-2-overexpressing clones was calculated by setting the viability of the neotransfected ß-pol-/- cell clone to 100%. C, relative apoptosis of the Bcl-2-overexpressing clones bcl-2wt/cl.1 and /cl.6. The frequency of apoptosis 48 h after treatment with 0.75 mM MMS in ß-pol-/- neo, ß-pol-/- bcl-2wt/cl.1, and /cl.6 was measured by using the method of Annexin V/PI double staining. The data shown are the mean of four independent experiments with the values of the neotransfectants set to 100%.

 
Caspase Activation and PARP Cleavage.
Next, we measured the major mitochondrial damage-related caspases, caspase-9 and caspase-3, as well as the CD95/Fas-triggered caspase-8 on alkylation. Whereas MMS treatment was without significant effect on caspase activity in wild-type cells, in ß-pol-/- cells, caspase-3 and caspase-9 became significantly activated. Activation was observed as early as 16 h after methylation and raised during further postexposure time (Fig. 7A)Citation . There was only a slight, insignificant increase in caspase-8 activity (Fig. 7A)Citation . Induction of caspase-9 and caspase-3 activity resulted in PARP cleavage, which was detected in ß-pol-/- but not in ß-pol+/+ cells (Fig. 7B)Citation . Interestingly, PARP cleavage was found to occur quite precisely between 15 and 30 h after MMS pulse treatment that corresponds to the period of maximal activity of caspase-3. We should note that caspase-3 started to get activated already at 14 h after MMS treatment, whereas PARP cleavage occurred slightly later, indicating the existence of a threshold above which a rise in caspase activity results in PARP cleavage. Overall, the data show that defective BER results in activation of the mitochondrial damage pathway.



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  		Fig. 7. Activity of caspase-3, caspase-9, and caspase-8 and cleavage of PARP in wild-type and ß-pol-/- cells on MMS treatment. A, lysates of ß-pol+/+ and ß-pol-/- cells were assayed for DEVD (caspase-3-like proteases), LEHD (caspase-9), and IETD (caspase-8) activity as a function of time after pulse treatment (for 60 min) with MMS (1 mM). The caspase activity of untreated cells was set to 100%. The values are derived from two to three independent experiments performed in duplicate; bars, SD. B, expression level and cleavage of the caspase substrate PARP. Nuclear cell extracts of ß-pol wild-type (+/+) and deficient (-/-) cells at different times after treatment with 1 mM MMS were harvested and subjected to Western blot analysis. Arrows, the uncleaved form of PARP (Mr 116,000) and the Mr 85,000 cleavage product.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
It has been shown previously that cells deficient in ß-pol are hypersensitive to the cytotoxic and clastogenic effect of simple methylating agents (21 , 22) . This study was aimed at elucidating the mechanism of cytotoxicity in more detail. We show that ß-pol-/- cells display a significantly higher frequency of apoptosis on methylation than wild-type cells. The frequency of necrosis was only slightly enhanced, indicating the induction of apoptosis to be the major route of alkylation-induced cell killing in ß-pol-/- fibroblasts. The high frequency of apoptosis in ß-pol-/- cells compared with wild-type fibroblasts provides evidence that incompletely and erroneously repaired DNA base methylation lesions trigger the apoptotic pathway.

Which is the mechanism involved? ß-pol has been shown to play a key role in SP-BER of DNA methylation lesions removed by N-methylpurine-DNA glycosylase (17) . Because N-methylpurine-DNA glycosylase is a monofunctional glycosylase without endonuclease activity (32) , apurinic endonuclease is required to cleave the apurinic site at the 5' position. This generates a sugar residue, which is removed by the dRPase function of ß-pol. This is an essential step in SP-BER because transfection of ß-pol lacking dRPase activity was unable to complement the ß-pol hypersensitive phenotype (23) . This indicates that the remaining 5'-dRP group in the BER repair patch blocks ligation during SP-BER. We initially supposed that this would result in an accumulation of SSBs in ß-pol-/- cells on methylation. Surprisingly, although the frequency of SSBs was significantly enhanced in ß-pol-/- cells compared with the wild type immediately after pulse treatment with MMS, the rate of removal was comparable with that detected in wild-type cells. This indicates that SP-BER catalyzed by ß-pol is a very fast repair step, which, if it is blocked, can be overtaken by a repair pathway using a DNA polymerase other than ß-pol. This is very likely to be LP-BER involving DNA polymerases {delta} and {epsilon} (for review, see Ref. 33 ). A similar conclusion has been reported recently by another group (34) . Because LP-BER was shown to be able to fill in apurinic endonuclease-mediated repair intermediates (35) , it is reasonable to suppose that this pathway competes for SP-BER, thus eliminating SSBs arising as BER intermediates. Interestingly, a higher frequency of SSBs was observed in ß-pol-/- cells even if the time of MMS exposure was reduced to 15 min, indicating that ß-pol/dRPase is very fast acting on SSBs generated by apurinic endonuclease. It should be noted that ß-pol presumably acts in concert with apurinic endonuclease, because both enzymes are present in the cell as a complex (36) . Irrespective of the exact mechanism of processing of nonrepaired patches in cells lacking ß-pol, it could be speculated that SSBs generated in ß-pol-/- cells account for their hypersensitive phenotype.

On the other hand, it is a generally accepted paradigm dating back to the work of Evans and Scott (37) that SSBs and base damages are not genotoxic per se but need DNA replication to be converted into ultimate clastogenic lesions. This is also true for simple alkylating agents, such as MMS, whose clastogenicity is replication dependent (38 , 39) . Indeed, we did not observe significant induction of aberrations 2 h after MMS treatment, whereas aberrations were strongly induced 8 and 14 h later when cells that had passed through S phase entered mitosis. Therefore, it appears that DNA lesions resulting from erroneous BER, such as SSBs, are converted during replication into chromosomal breaks and exchanges, thus leading to the high clastogenicity in ß-pol-/- cells. This hypothesis gained further support by the finding that the frequency of nonrepaired DSBs was significantly enhanced in ß-pol-/- cells various hours after methylation. This indicates that DSBs, which are the major ultimate clastogenic lesions (40 , 41) , derive from incompletely repaired BER patches during DNA replication. Cell cycle analysis revealed that cells accumulate at early times (<2 h) in S phase and at late times (>15 h) in G2-M, which is likely because of cell cycle blockage of heavily damaged cells in the second post-treatment cell cycle.

Regarding the induction of cell death, it is important to note that apoptosis in ß-pol-deficient cells is a late response detectable not earlier than 30 h after pulse treatment with MMS. Therefore, it is unlikely that incompletely repaired DNA lesions, such as DNA gaps or SSBs, detectable immediately after treatment trigger apoptosis directly. Apoptosis induced in ß-pol-/- cells was significantly reduced in nonproliferating cultures compared with an exponentially growing one, indicating that DNA replication is required for eliciting the apoptotic response. We should note that the yield of necrosis was similar in exponentially growing and arrested cells, suggesting necrosis to be replication independent. On the other hand, the frequency of SSBs was not affected under these culture conditions supporting the view that SSBs are not the primary trigger of apoptosis. On the basis of the results, we hypothesize that nonrepaired BER intermediates (i.e., SSBs characterized by unremoved 5'deoxyribose phosphate) are converted during DNA replication into critical secondary DNA lesions, which finally trigger apoptosis. These secondary lesions are likely to be DSBs or chromosomal aberrations derived from them, both of which were found to be induced at higher levels in ß-pol-/- cells compared with the wild type. Various other data are in line with the view that DSBs are the critical ultimate apoptosis-inducing lesions in ß-pol-/- cells: (a) they preceded caspase activation and the appearance of apoptotic cells (this work); (b) they are highly efficient in inducing apoptosis, as shown by electroporation experiments with restriction enzymes (29) ; and (c) they are critical events in ionizing radiation-induced cell death rather than base damage (42 , 43) .

A hallmark of methylation-induced apoptosis in ß-pol-deficient cells was the decline of Bcl-2, whereas the amounts of Bax and Bcl-xL remained largely unaffected. Bcl-2 decline was observed already 15 h after MMS treatment and, therefore, clearly preceded apoptosis. Causal involvement of Bcl-2 was confirmed by transfection experiments with Bcl-2 expression plasmid. Although treatment with MMS clearly reduced both the endogenous and the transfected Bcl-2 level, the transfectants displayed a lower frequency of apoptosis than the neotransfected control, which assigns to Bcl-2 a central role in DNA damage triggered apoptosis. Bcl-2 decline was also a hallmark of apoptosis induced by O6-methylguanine in DNA (13) and was shown to be provoked by DSBs (29) .

It is well known that a reduced level of Bcl-2 leads to cytochrome c release from mitochondria and caspase activation (44 , 45) . Here we showed that after Bcl-2 decline, caspase-9 and caspase-3, but not to a significant extent caspase-8, became activated, resulting in cleavage of PARP. PARP cleavage occurred between 15 and 30 h after treatment with MMS in ß-pol-/- cells, which correlates with the maximal increase of caspase-3 activity. On the basis of the data, the scenario of methylation-induced apoptosis in ß-pol-/- cells looks as follows: nonrepaired 5' incised apurinic sites are either processed by LP-BER or interfere with DNA replication, leading to critical secondary lesions. These are very likely to be DSBs, which trigger Bcl-2 decline, leading to activation of downstream caspases and DNA degradation. The model presented here is reminiscent to apoptosis triggered by O6-methylguanine, which, as a consequence of faulty mismatch repair at O6-MeG/T lesions, causes DNA and chromosomal breakage, Bcl-2 decline, and caspase-9/3 activation (13) . Although the critical primary DNA lesions were different, the execution phase of apoptosis evoked by a common type of critical ultimate DNA lesion (i.e., DSBs) appears to be identical. The experimental repair-deficient systems available will be useful to further reconstruct the chain of events leading to cell death on the induction of DNA damage by environmental carcinogens and anticancer drugs.


   ACKNOWLEDGMENTS
 
We thank Drs. S Wilson and R. Sobol for providing the ß-pol-deficient cell lines.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by Stiftung Rheinland-Pfalz and German Research Foundation (SFB 519/B4). Back

2 To whom requests for reprints should be addressed, at Institute of Toxicology, Division of Applied Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany. Phone: 0049-6131-393-3246; Fax: 0049-6131-393-3421; E-mail: Kaina{at}mail.uni-mainz.de. Back

3 The abbreviations used are: BER, base excision repair; ß-pol, polymerase ß; dRPase, deoxyribose phosphate lyase; SSB, single-strand break; DSB, double-strand break; MMS, methyl methanesulfonate; ERK, extracellular signal-regulated kinase; PARP, poly(ADP-ribose) polymerase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; SCGE, single cell gel electrophoresis; SP-BER, short-patch base excision repair; PI, propidium iodide; LP-BER, long-patch base excision repair. Back

Received 7/26/01. Accepted 12/28/01.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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