DNA double-strand breaks trigger apoptosis in p53 ... - Oxford Academic

Carcinogenesis vol.22 no.4 pp.579–585, 2001

DNA double-strand breaks trigger apoptosis in p53-deficient fibroblasts

Jochen Lips and Bernd Kaina1 Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Str. 67, D-55131 Mainz, Germany 1To

whom correspondence should be addressed Email: [email protected]

DNA double-strand breaks (DSBs) are induced by ionizing radiation (IR) and various radiomimetic agents directly, or indirectly as a consequence of DNA repair, recombination and replication of damaged DNA. They are ultimately involved in the generation of chromosomal aberrations and were reported to cause genomic instability, gene amplification and reproductive cell death. To address the question of whether DSBs act as a trigger of apoptosis, we induced DSBs by means of restriction enzyme electroporation and compared the effect with IR in mouse fibroblasts that differ in p53 status [wild-type (⍣/⍣) versus p53-deficient (–/–) cells]. We show that (i) electroporation of PvuII is highly efficient in the induction of DSBs, (ii) electroporation of PvuII increases the rate of apoptosis, but not of necrosis in p53–/– cells, (iii) treatment with γ-rays induces both apoptosis and necrosis in p53–/– cells, (iv) the frequency of DSBs correlates with the yield of apoptosis and (v) both PvuII and γ-ray treatment reduce the level of anti-apoptotic Bcl-2 protein in p53–/– cells whereas the level of Bax remains unaltered. Cells expressing wild-type p53 were more resistant than p53-deficient cells as to the induction of apoptosis and did not show Bcl-2 decline upon treatment with PvuII and γ-rays. The data provide evidence that blunt-ended DSBs induced by restriction enzyme PvuII act as a highly efficient trigger of apoptosis, but not of necrosis. This process is related to Bcl-2 decline and does not require p53. Introduction DNA double-strand breaks (DSBs) are induced in DNA either directly by high-energy ionizing radiation (IR) or indirectly by many genotoxic agents. They can arise as intermediates during DNA repair and recombination and emerge as a consequence of replication of damaged DNA containing singlestrand breaks, critical minor lesions or bulky adducts (1). As a result of DSB formation, chromosomal aberrations, i.e. chromosomal breaks and translocations, are formed. In addition, mitotic homologous recombination, mutations and oncogenic transformation may result from DSBs (2–5). The role of DSBs in cytotoxicity brought about by genotoxic agents has been a matter of debate for years. For IR a correlation has been found between reproductive cell death and the induction and repair of DSBs (6–8). However, other reports failed to support these data (9,10), which indicates the complexity of the processes involved. Recently, a correlation has been reported Abbreviations: DSBs, DNA double-strand breaks; IR, ionizing radiation; SCGE, single cell gel electrophoresis. © Oxford University Press

between residual non-repaired DSBs and radiosensitivity (11). These data fit the model derived from the correlation of chromosomal breaks and reproductive cell death that nonrepaired or misrepaired DSBs result in cell killing (12). Reproductive cell death, as measured by loss of colonyforming ability, may be caused by three non-exclusive processes: irreversible blockage of cell division (non-toxic cell inactivation), necrosis and apoptosis. Although many, if not all DNA damaging agents induce apoptosis, the role of DSBs in its induction is unclear. A major cause of uncertainty is due to the fact that genotoxic agents do not only damage DNA but also activate cellular receptors and signal pathways that are shown to be involved in apoptosis (13,14). To address the question of whether DSBs act as a primary trigger of apoptosis, we made use of the approach to introduce into cells restriction enzymes that cleave DNA in situ (15,16). Electroporation of restriction enzymes into cells was found to be very efficient in inducing chromosomal aberrations (17–19). Restriction enzymes are not likely to damage other cellular constituents and therefore are an ideal instrument in elucidating the role of DSBs in apoptosis. As p53 is a critical determinant of apoptosis, we compared the effect of restriction enzyme-mediated DNA cleavage in p53 wild-type (p53⫹/⫹) and p53-deficient (p53–/–) fibroblasts, which were derived from p53 knockout mice. We compared the data obtained with those gained by IR of the same pair of cells. The results provide evidence that DSBs induced by PvuII in situ trigger apoptosis. They do not trigger necrosis whereas IR does both. Since p53-deficient cells were more responsive than p53-proficient cells, we infer that p53 is not required for the induction of apoptosis triggered by DSBs in fibroblast cells. Materials and methods Cell culture The BK4⫹/⫹ (p53⫹/⫹) cell line is an established fibroblast line derived from newborn mice in this laboratory (background 129/C57BL/6). The p53–/– E cell line is a fibroblast line that was established from p53 knockout mouse tissue with similar genetic background (20). It was kindly provided from the laboratory of Dr A.Balmain (Glasgow). Cells were grown in Dulbecco’s modified Eagle’s medium (Gibco BRL) supplemented with 10% inactivated fetal bovine serum (Greiner) at 37°C and 7% CO2. Electroporation and irradiation Electroporation was performed according to a modified, previously described procedure (21). Subconfluent cells were trypsinized and washed once with pre-warmed medium and once with ice-cold sucrose buffer (272 mM sucrose, 1 mM MgCl2, 7 mM phosphate pH 7.4). Electroporation of PvuII restriction enzyme (Amersham Pharmacia) into cells (3⫻106) was performed in a Gene Pulser Cuvette (0.4 cm electrode gap, Bio-Rad) in a total volume of 800 µl sucrose buffer (400 V, 400 W, 25 µF). After 10 min of recovery on ice, cells were seeded and incubated on 10 cm dishes for the time periods indicated. Control experiments were performed with 400 U of heat-inactivated PvuII (15 min, 95°C). For γ-ray irradiation cells were trypsinized, suspended in medium and irradiated on ice. γ-rays were generated by 137Cs (1800 Ci) in a Gammacell 2000 device (Molsgaard Medical) at 7.7 rad/s. Immediately after treatment, cells were seeded into pre-warmed fresh medium. Neutral single cell gel electrophoresis (SCGE) The neutral SCGE (‘comet assay’) was performed basically as described (22,23), with slight modifications. In brief, following electroporation of

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J.Lips and B.Kaina restriction enzyme or γ-ray irradiation and post-treatment incubation, cells were harvested by trypsinization and cooled down on ice. 1⫻104 cells (in 10 µl) were embedded in 120 µl low-melting point agarose (0.5% in dH2O at 37°C) onto agarose-coated (1.5% in PBS) and dried slides that were submersed for 1 h in pre-cooled lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 1% sodium-laurylsarcosine, pH 7.5; 1 h before use 1 ml Triton X-100 and 10 ml DMSO per 100 ml were added). Slides were electrophoresed at 25 V for 15 min at 4°C in electrophoresis buffer (90 mM Tris, 90 mM boric acid, 2 mM EDTA, pH 7.5). After ethanol fixation, drying and ethidium bromide (20 µg/ml) staining, cells were analyzed using a microscope (Olympus BX50, 100⫻ magnification) and olive tail moment was determined by measuring the fluorescence intensity automatically using Kintetic Imaging Komet 4.0.2 software (BFI Optilas). In background experiments the method proved to be suitable for specific detection of DSBs (data not shown). Relative DSB frequency is defined as olive tail moment of comets in a linear range of dose response. Apoptosis (i) After electroporation or irradiation, apoptotic and necrotic cell population was quantified after double-staining with annexin V and propidium iodide by flow cytometric analysis using FACSort (Becton Dickinson) as previously described (24). Necrotic cells were defined by staining with both agents whereas apoptotic cells were labeled by annexin V only (25). In brief, cells were harvested by trypsinization, washed once with PBS and incubated in annexin V binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 1% BSA). 1⫻106 cells suspended in binding buffer were first stained with annexin V (ApoScreen, Annexin V apoptosis kit, Dianova,) and then with propidium iodide (50 µg/ml) and measured immediately by flow cytometry. (ii) For detection of nucleosomal fragmentation, treated cells (1⫻107) were lysed in a hypotonic solution as described (26). 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, DNA was separated by gel electrophoresis on 1.5% agarose gels. Western blotting Protein extracts were prepared from adhering cells together with those from supernatant. In brief, after washing with ice-cold PBS, cells were suspended in protein extraction buffer (20 mM Tris pH 7.4, 1 mM EDTA, 50 mM NaCl, 1% SDS, 1 mM PMSF, 2 mM DTT). The suspension was sonified (3⫻ 10 pulses, Branson Sonifier) and centrifuged for 10 min at 10 000 r.p.m., 4°C. 30 µg of protein was electrophoretically separated on a 10% SDS–polyacrylamide gel and blotted onto nitrocellulose membrane (PROTRAN, Schleicher & Schuell) using a buffer containing 25 mM Tris, 100 mM glycine, 25% methanol. After preincubating the filter in 5% non-fat dry milk, 0.1% Tween-20 in PBS, Bcl-2, Bax or ERK2 antibodies (Santa Cruz Biotechnology) were added to the solution and incubated for several hours. The filter was extensively rinsed with PBS, 0.1% Tween-20 and incubated with peroxidase-conjugated secondary antibody from donkey (Amersham Life Science). After washing, antibodybinding was visualized by using Renaissance Western Blot Chemiluminescence Reagent Plus (NEN Life Science Products) and Hyperfilm ECL (Amersham Life Science). The signals onto the filters were quantified using Multianalyst image analysis system (Bio-Rad) and set in relation to the loading control ERK2.

Results DSBs induced by PvuII and IR An efficient method to induce selectively DSBs in mammalian cells is based on electroporation of restriction enzymes (15,16). To establish optimal conditions of DSB induction in our experimental system, we determined in background experiments the conditions of treatment and electroporation. As demonstrated in Figure 1A, the endonuclease PvuII induces DSBs depending on the concentration applied. Interestingly, p53–/– cells turned out to be more sensitive than p53⫹/⫹ cells. In control experiments, heat-inactivated PvuII (15 min, 95°C) was electroporated, which did not lead to any induction of DSBs (basal frequency of olive tail moment, data not shown) indicating that the effects observed are due to the enzymatic activity of the restriction enzyme inside the cell. Next we determined in time-course experiments the appearance of DSBs after PvuII electroporation (Figure 1B). Maximum induction of DSBs was measured 6 h after electro580

Fig. 1. Induction of DSBs after electroporation of PvuII restriction enzyme as a function of dose and post-exposure time. (A) Dose-dependent induction of DSBs was quantified after electroporation of p53⫹/⫹ and p53–/– mouse fibroblasts with 100 and 400 U of PvuII. In control experiments, 400 U of heat-inactivated PvuII was electroporated into cells, which gave essentially the same result as mock electroporation (olive tail moment ⬍5). After 6 h of incubation cells were subjected to neutral SCGE. (B) For time-dependent induction and repair of DSBs cells were electroporated with 400 U of PvuII and neutral SCGE was performed at the time points indicated. Data are the mean of at least three independent experiments ⫾SD.

poration in both p53⫹/⫹ and p53–/– cells. Thereafter the number of DSBs declined reaching nearly control levels 24 h after treatment. Again, the p53–/– cell line was more sensitive displaying higher peak values of DSBs (2.5-fold as compared to p53⫹/⫹ cells). However, after 24 h of incubation, the residual DSB frequency was nearly the same in both cell types. In order to compare the induction of DSBs by PvuII with breaks induced by IR, the same assay was performed with the same pair of cells exposed to γ-rays. The induction of DSBs was a linear function of dose, and there was no significant difference between p53⫹/⫹ and p53–/– cells in the DSB frequency initially induced (Figure 2A). With increasing post-exposure times, the frequency of DSBs was declining in both cell types. Interestingly, p53–/– cells displayed a higher residual level of DSBs than p53⫹/⫹ cells after 6 h of recovery (Figure 2B).

DNA breaks trigger apoptosis

Fig. 2. Induction and repair of DSBs after γ-ray irradiation as a function of dose and post-exposure time. (A) p53⫹/⫹ and p53–/– fibroblasts were trypsinized, incubated on ice, irradiated with 2–15 Gy and subjected to neutral SCGE after 1 h of incubation at 37°C, as described in Materials and methods. (B) Cells were irradiated on ice with 15 Gy and subjected to neutral SCGE at the indicated times. Data are the mean of three independent experiments, each performed in duplicate ⫾SD.

Apoptosis induced by PvuII and IR Having shown that PvuII induces DSBs in mouse fibroblasts, we analyzed the induction of apoptosis and necrosis in the same experimental system. Electroporation of PvuII yielded a high rate of apoptosis in p53–/– cells (up to 60% as quantified by annexin V staining and subsequent flow cytometry), whereas in p53⫹/⫹ cells only a slight induction of apoptosis was observed (maximally 10%) even with the highest concentration of PvuII applied (Figure 3A). Interestingly, the rate of necrosis was not significantly enhanced in both cell types. Apoptosis induced by PvuII was a late response, as shown in time-course experiments. It increased slightly 24 h after treatment in p53–/– cells with a sharp rise 3 days later (Figure 3B). Again, in the whole time period of analysis (up to 96 h after electroporation) necrosis was not significantly induced. Induction of apoptosis in p53–/– cells by PvuII was confirmed by analyzing nucleosomal fragmentation of DNA, which is a hallmark of apoptosis. Apoptotic DNA fragmentation

Fig. 3. Apoptosis and necrosis as a function of concentration and time after electroporation of PvuII into p53⫹/⫹ and p53–/– cells. Apoptosis and necrosis were quantified by double-staining of cells with annexin V and propidium iodide and subsequent flow cytometric analysis. (A) Dosedependence of the frequency of apoptosis and necrosis in p53 wild-type and p53-deficient cells as measured 72 h after electroporation. (B) Time-course of induction of apoptosis and necrosis of p53–/– and p53⫹/⫹ cells that were electroporated with PvuII (400 U). Data are the mean of at least four independent experiments. For the last measurement point (96 h) two experiments were performed. (C) Nucleosomal degradation of DNA was observed in p53–/– cells after electroporation of PvuII.

was clearly observed in p53-deficient cells. In p53⫹/⫹ cells only a very weak DNA degradation was detectable (Figure 3C), which is in line with the low level of apoptosis quantified by FACS analysis (see Figure 3B). Treatment of the same pair of cells with γ-rays also induced apoptosis as a function of dose (Figure 4A). Again, p53–/– cells were more sensitive than p53⫹/⫹ cells. Most interestingly, γ-rays induced not only apoptosis but also necrosis to nearly comparable levels. The induction of apoptosis was again a late response reaching the highest levels at the latest time points of measurement, i.e. 96 h after treatment (Figure 4B). The high sensitivity of p53–/– fibroblasts to γ-ray induced apoptosis was confirmed by gel electrophoresis, 581

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Fig. 4. Apoptosis and necrosis after γ-ray irradiation of p53⫹/⫹ and p53–/– cells. (A) Dose-dependence of the rate of apoptosis and necrosis which was measured 3 days after irradiation in p53–/– and p53⫹/⫹ cells. (B) Timecourse of induction of apoptosis and necrosis after irradiation (10 Gy) of p53 wild-type and p53-deficient cells. (C) Apoptotic DNA degradation, as visualized by gel electrophoresis, was observed after γ-ray irradiation (10 Gy) in p53–/– cells.

displaying clearly apoptotic laddering in p53–/– but not in p53⫹/⫹ cells (Figure 4C). Correlation between DSBs and apoptosis To see whether there is a correlation between DSBs and apoptosis, the yield of apoptosis was plotted as a function of DSB frequency. As shown in Figure 5, both for PvuII and γ-rays a linear relationship does exist between both endpoints. We should stress that PvuII (with the concentrations applied) was much more efficient in inducing DSBs and apoptosis than γ-rays (doses of 0–15 Gy; note the different scales in the figure), which supports the view that ‘clean’ DSBs are a highly efficient trigger of apoptosis. 582

Fig. 5. Relationship between DSBs and apoptosis for p53-deficient cells treated with PvuII (A) or γ-rays (B). Data are from Figures 1–4. The correlation coefficient for the curve shown in (A) is 0.9436, and for the curve shown in (B) 0.9400. Note that the scales of the plots in (A) and (B) are different.

Bcl-2 and Bax expression upon treatment with PvuII and IR Previously, we have shown that Bcl-2 is crucially involved in DNA alkylation damage-induced apoptosis in Chinese hamster and mouse fibroblasts (ref. 27 and data not published). To see whether Bcl-2 is also affected upon the induction of DSBs, the Bcl-2 level upon treatment with PvuII and γ-rays was determined in the mouse fibroblast lines. As shown in Figure 6A, electroporation of cells with PvuII led to a decline in Bcl-2 level 2–3 days after treatment in p53–/– cells, but not in p53⫹/⫹ cells. The expression of Bax was not significantly altered (see Figure 6A for western blots and their quantification). Similarly, treatment with γ-rays caused a strong decline in Bcl-2 level in p53–/– cells, which was not observed in p53⫹/⫹ cells. Again, there was no effect of treatment on the level of Bax (Figure 6B). Overall, the decline in Bcl-2

DNA breaks trigger apoptosis

Fig. 6. Expression levels of Bcl-2 and Bax protein in p53⫹/⫹ and p53–/– cells upon exposure to PvuII or IR. (A) Western blot analysis of p53 wildtype and p53-deficient cells electroporated with PvuII (400 U). Cells were harvested at the times indicated in the figure. (B) Western blot analysis of cells treated with γ-rays (15 Gy). Cells were harvested various times after treatment. Filters were re-incubated with ERK2, which served as a loading control. Quantification of the relative amount of Bcl-2 and Bax was performed by densitometric measurement. Expression values were set in relation to ERK2.

protein appears to be related to the induction of DSBs and apoptosis, and was most obvious in p53-deficient cells. Discussion In this study, we addressed the question of whether DSBs act as a primary trigger of apoptosis. Since IR induces a broad spectrum of DNA lesions including DNA single-strand breaks, DSBs and various kinds of base damage (28) and, moreover, targets extranuclear constituents including cellular membrane receptors (29), data obtained with genotoxic agents on apoptotic signaling are difficult to interpret. In order to induce

highly specific DSBs in DNA without damaging other potential targets, we made use of electroporation of the restriction enzyme PvuII into cells. This blunt-end cutter was previously shown to be highly efficient in inducing chromosomal aberrations upon electroporation (17–19). Since p53 plays an important role in apoptosis, we compared the effect of PvuII in mouse fibroblasts that are either wildtype (p53⫹/⫹) or knockout (p53–/–) for p53. In background experiments, we determined the optimal conditions of electroporation. Electroporation per se (mock) and with heat-inactivated PvuII did not affect the basal level of DSBs and did not induce apoptosis (data not shown). On the other hand, electroporation with native PvuII efficiently induced DSBs and, at the same time, was highly efficient in inducing apoptosis in mouse fibroblasts. Since PvuII induces DSBs without any known side-effects, we conclude that DSBs are the primary trigger of the signaling cascade resulting in apoptosis. Interestingly, the kinetics of induction of DSBs was different following PvuII and γ-ray treatment. Comparison of the timecourse of DSBs after treatment with PvuII and γ-rays revealed that the yield of DSBs increased with time after electroporation reaching a maximum at 6 h whereas for γ-rays the maximum effect was seen immediately after irradiation. This indicates that the enzymatic generation of DSBs upon PvuII electroporation lasts several hours after introducing the enzyme into the cells. After maximum induction of DSBs, their frequency declined with post-incubation time indicating their repair. We should note that the overall amount of DSBs induced by PvuII and the level of residual DSBs both after PvuII and γ-ray treatment (as detected 24 and 6 h after treatment respectively) was higher in p53–/– than in p53⫹/⫹ cells, which is probably due to inefficient repair at least of a subfraction of DSBs in p53–/– cells. PvuII induces DSBs with blunt-end morphology. There are data to indicate that the predominant process involved in the rejoining of DSBs with blunt-end morphology is nonhomologous end-joining (NHEJ) (30), which is different from homologous recombination (31). The protein complex involved in NHEJ is composed of three subunits of DNA-dependent protein kinase (DNA-PK) (32), XRCC4 (33) and DNA ligase IV (34). Interestingly, the catalytic subunit of DNA-PK recently proved to act as an upstream effector for p53 in response to IR (35). Besides, p53 interacts with RAD51, which is involved in DSB repair (36,37). Moreover, p53 exhibits a 3⬘→5⬘exonuclease activity supposed to be involved in DNA repair (38). Data are also available to show that cells lacking p53 are impaired in global genomic nucleotide excision repair (39,40) and in the rejoining of DSBs with short complementary ends of single-stranded DNA (41). Overall, the available data suggest a direct or at least supporting role of p53 in DSB repair, which might explain our findings that p53–/– cells are more sensitive than p53⫹/⫹ cells as to the induction of non-repaired DSBs and apoptosis. Hypersensitivity of p53deficient fibroblasts pertains to not only PvuII electroporation and γ-rays. It was also shown for the endpoints chromosomal breakage and apoptosis after treatment with ultraviolet (UV) light and methyl methanesulfonate both in primary diploid p53 knockout fibroblasts (42) and various established p53deficient cell lines (Lackinger and Kaina, unpublished data). Therefore, hypersensitivity to DNA damaging agents appears to be a more general property of p53-deficient fibroblasts. This seems to contradict the view that DNA 583

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damage-induced apoptosis is p53 dependent (43–45), due to p53-mediated upregulation of the Fas/CD95 receptor (46) or to upregulation of Bax protein level (47) or downregulation of Bcl-2 (48). Apoptosis mediated by activation of Fas/CD95, however, is probably the major mechanism only in some cell types, such as lymphoblastoid cells. In other cell systems, including fibroblasts, apoptosis appears to be independent of p53 (42,49,50) and seems not to involve as a central event upregulation of Fas/CD95 and Bax (27). In these cells p53 rather exerts protection against genotoxicity. Protection could be due to both p53-mediated DNA repair and G1/S blockage (43) enhancing the pre-replicative repair period. We should note that apoptosis in our experiments is a late response detectable not earlier than 2–3 days after PvuII or γ-ray treatment. Similar results have been obtained with DNA repair deficient Chinese hamster ovary (CHO) cells and mouse fibroblasts upon treatment with alkylating agents (24,27) indicating the signaling from DSBs to the endpoint apoptosis to be rather complex. An important observation we would like to note is the complete lack of necrosis in PvuII-treated cells, whereas upon γ-ray exposure necrosis amounted to nearly 50% of the cell killing response. Furthermore, DSBs (as measured by SCGE) and apoptosis were linearly related and PvuII was much more potent in inducing DSBs and apoptosis than γ-rays. Based on this it is reasonable to conclude that blunt-ended DSBs trigger exclusively the apoptotic pathway whereas other kinds of damages induced by IR, such as protein and membrane alterations, cause necrosis. In this light, the apoptotic versus the necrotic effect of different genotoxic agents gains a new interpretation. For instance, ganciclovir incorporated into DNA induces nearly exclusively apoptosis (51) whereas UV light and alkylating agents induce both apoptosis and necrosis (~60 and 40% respectively) in CHO cells. This might indicate that for the latter group of agents damages other than DSBs are additionally involved in the cell killing response, notably the induction of necrosis. A decisive factor in the apoptotic pathway are proteins of the Bcl-2 family (52). In previous work we showed that Bcl-2, but no other proteins of the family (e.g. Bax, BclXL), is reduced in expression, and that Bcl-2 transfection protects against alkylating agent-induced apoptosis (27). In a first step of elucidating apoptotic signaling upon the induction of DSBs we therefore checked the expression level of Bcl-2. In p53–/– cells, but not in the refractory p53⫹/⫹ cell type, Bcl-2 was down-modulated. This was true both after PvuII and γ-ray treatment. The mechanism of Bcl-2 decline in p53deficient cells upon exposure to a DNA damaging agent is still unclear. The finding, however, that PvuII is able to trigger this response indicates that DSBs and not unspecific membrane or protein damage are primarily involved. The finding that PvuII-induced DSBs trigger apoptosis may be taken to indicate that for many agents other than IR, which do not directly induce DNA breakage, replication or repair-mediated DSBs are critically involved in the induction of apoptosis. This hypothesis gains support by a previous report showing that the apoptotic pathway triggered by the specific DNA base damage O6-methylguanine is likely to be activated by DSBs, which can be considered as a critical ultimate lesion in the apoptotic process (27). Overall, the data show that DSBs induced by PvuII trigger Bcl-2 decline and apoptosis, but not necrosis in p53-deficient mouse fibroblasts. This supports the view that DNA damage 584

is a primary trigger of apoptotic death upon genotoxic treatments of cells. Therefore, the induction of apoptosis in fibroblasts upon mutagen treatment cannot be explained solely on the basis of receptor activation and the induction of cellular apoptotic functions independent of DNA damage. Acknowledgements We are grateful to Uta Eichhorn, Kirsten Ochs and Torsten Dunkern for FACS analysis and to Dr A.Balmain (Glasgow) for providing the p53 knockout cell line. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 519/B4 and KA724/8–1) and Stiftung Rheinland-Pfalz.

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