Mutagenesis, Vol. 16, No. 3, 233-241,
May 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Effect of ultraviolet light, methyl methanesulfonate and ionizing radiation on the genotoxic response and apoptosis of mouse fibroblasts lacking c-Fos, p53 or both
Institute of Toxicology, Division of Applied Toxicology, University of Mainz, Obere Zahlbacher Straße 67, D-55131 Mainz, Germany
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Abstract |
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c-Fos and p53 are DNA damage-inducible proteins that are involved in gene regulation, cell cycle checkpoint control and cell proliferation following exposure to genotoxic agents. To investigate comparatively the role of c-Fos and p53 in the maintenance of genomic stability and the induction of apoptosis, we generated mouse fibroblast cell lines from knockout mice deficient for either c-fos (fos –/–) or p53 (p53–/–) or for both gene products (fosp53–/–). The sensitivity of these established cell lines was compared with the corresponding wild-type cells as to the cytotoxic, clastogenic and apoptosis-inducing effects of ultraviolet (UV-C) light and methyl methanesulfonate (MMS). Additionally, we analysed the frequency of apoptosis of the cell lines after treatment with ionizing radiation (IR). We observed c-fos–/–, p53–/– and fosp53–/– cells to be more sensitive than wild-type cells with respect to cell death, as measured in a cytotoxicity (MTT) assay. Regarding apoptosis, all deficient cell lines displayed hypersensitivity to UV-C light, MMS and IR. With chromosomal aberrations as the endpoint, the sensitivity of the double-knockout cells was between wild-type and single-knockouts. The results indicate that both c-Fos and p53 play an important role in protecting fibroblasts against a broad range of genotoxic agents. The results also show that, in fibroblasts, apoptosis induced by UV-C light, MMS and IR does not require p53 and that, in this cell type, p53 rather protects against DNA damage-induced apoptotic cell death.
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Introduction |
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Exposing cells to DNA-damaging agents triggers a number of cellular defence functions (Kaina et al., 1999
). Among them are the product of the proto-oncogene c-fos and the tumor-suppressor protein p53, both of which are involved in essential cellular processes. The cellular concentration of p53 is enhanced by increasing the half-life of the protein in response to various stimuli, such as hypoxia, nucleotide deprivation, or induced DNA damage (Fritsche et al., 1993; Siegel et al., 1995). The stabilization of p53 is based on post-translational modification of the protein by phosphorylation/dephosphorylation and acetylation (Jimenez et al., 1999; Lakin and Jackson, 1999). At the same time, the DNA binding ability of p53 is enhanced (Lakin and Jackson, 1999). Because of its function as a sequence-specific transcription factor and because of direct interaction with other proteins, p53 is involved in the regulation of genes whose products play a role in cell cycle control (Janus et al., 1999; Kaufmann and Paules, 1996), DNA replication and repair (Albrechtsen et al., 1999; Grombacher et al., 1998; Smith et al., 2000), and apoptosis (Levine, 1997). One of the consequences of p53 induction is cell cycle arrest, which can occur in the G1/S or G2/M phase of the cell cycle. It is believed that the cell cycle arrest extends the period of DNA repair prior to DNA replication or entry of cells into mitosis, and thus reduces the probability of fixation of DNA damage. The DNA damage-induced G1/S arrest mediated by p53 occurs via transactivation of the inhibitory protein p21. p21 prevents cell cycle progression by interacting with cyclin–cdk (cyclin dependent kinase) complexes and proliferating cell nuclear antigen (PCNA) (Kuerbitz et al., 1992). Another pathway triggered by p53 in certain (e.g. hematopoietic) cell types is apoptosis, resulting in permanent elimination of highly damaged or mutated cells (Ko and Prives, 1996; Levine, 1997). The decision between these alternative pathways following genotoxic stress seems to depend on various circumstances, such as cell type, degree of DNA damage induced, presence of growth factors, extracellular environment and the given experimental situation.
In contrast to p53, the induction of c-Fos by genotoxic treatment is based mainly on enhanced transcription of the c-fos gene. Induction of c-fos is achieved via Ras, Raf, MAP kinase and Jun kinase (Cavigelli et al., 1995; Coso et al., 1995; Radler-Pohl et al., 1993) leading to post-translational modification, i.e. phosphorylation of the c-Fos protein. As a consequence, the activity of the heterodimeric transcription factor activator protein 1 (AP-1) that is formed by c-Fos and one of the members of the Jun family (Angel and Karin, 1991) is enhanced. AP-1 regulates genes involved in cell proliferation (Kovary and Bravo, 1991), tumorigenesis (Jenuwein et al., 1985) and differentiation (Lord et al., 1993). In addition, c-Fos was shown to play a general role in cellular protection against a broad spectrum of DNA-damaging agents (Haas and Kaina, 1995; Kaina et al., 1997). This conclusion has been drawn from the finding that c-Fos-deficient mouse fibroblasts are hypersensitive to the cytotoxic and genotoxic effects of various agents inducing different kinds of DNA damage, all of which exert their genotoxic effect in an S-phase dependent manner. Increased apoptosis induced by these agents in c-fos knockout cells and in cells deprived of c-Fos by antisense techniques indicates that c-Fos is also important for defence against DNA damage-induced programmed cell death (Kaina et al., 1997; Roffler-Tarlov et al., 1996; Schreiber et al., 1995; Smeyne et al., 1993).
As the pathways in which c-Fos and p53 are involved seem to overlap, an interaction of these regulatory factors in cellular defence appears to be possible. Thus, a number of genes have been found to harbour binding sites for both AP-1 and p53 in their promoters. Examples of such genes putatively controlled by both transcription factors are the drug resistance gene mdr1 (Chin et al., 1992; Scanlon et al., 1994), the replication and repair factor PCNA (Gillardon et al., 1995; Shivakumar et al., 1995), and the DNA repair gene O6-methylguanine-DNA methyltransferase (Boldogh et al., 1998; Grombacher et al., 1998; Rafferty et al., 1996). Furthermore, p53 has the ability to repress transcription of the c-fos gene, due to direct interaction of p53 with components of the basal transcription machinery, such as TFIIH, TBP (TATA binding protein) and TAFs (TBP-associated factors) (Horikoshi et al., 1995; Ko and Prives, 1996).
To investigate the role of c-Fos and p53 in cellular defence against genotoxic exposure, we established mouse fibroblasts deficient for either c-Fos or p53. Also, to gain insight into a possible co-operation of c-Fos and p53 in defence, we generated c-fos/p53 double-knockout cells. This kind of cell has not been described by others previously. The response of these cells was compared following treatment with ultraviolet (UV-C) light, the alkylating agent methyl methanesulfonate (MMS) and, for the endpoint apoptosis, with 
-rays. The agents used cause different kinds of DNA damage that is repaired via distinct pathways (Friedberg et al., 1995). Our results show that cells deficient for c-Fos, p53 or both gene products are hypersensitive to the clastogenic, cytotoxic and apoptosis-inducing effect of the agents we applied.
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Materials and methods |
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Cell lines and culture conditions
The cell lines +/+BK4, fos+/+1-98M, fos–/–7-98M, p53+/–BK1, p53–/–2-98M, fosp53–/–BK3 and fosp53–/–BK10 are spontaneously immortalized fibroblastoid cell lines established in our laboratory. The cell lines were derived from cross-breeding of wild-type, c-fos knockout and p53 knockout mice purchased from The Jackson Laboratory (Bar Harbor, ME) and cultivated as described (Kaina et al., 1997
). The lines +/+f20 and fos–/–f10 are immortalized, 3T3-like mouse fibroblasts provided by Dr E.F.Wagner (Research Institute of Molecular Biology, Vienna, Austria). Growth characteristics have been described previously (Brüsselbach et al., 1995; Haas and Kaina, 1995) The cell line p53–/–E was obtained from Dr A.Balmain (Glasgow). Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies) containing 10% heat inactivated fetal bovine serum at 37°C in a humidified atmosphere containing 7% CO2.
Mutagen treatment
MMS (Sigma Chemical) was stored at –20°C as stock solution in sterile distilled water and used immediately after thawing and dilution with phosphate-buffered saline (PBS, if necessary). Exponentially growing cells were incubated with the agent for 1 h in the CO2 incubator, and the medium was then replaced by fresh medium. For UV-C irradiation, a germicidal lamp (254 nm) was used. After aspiration of the medium and irradiation, the conditioned medium was returned to the cells. -irradiation was performed using a 137Cs source. Cells were exposed to radiation in an adherent state with complete culture medium at room temperature.
PCR analysis
PCR reactions were performed using Taq DNA polymerase (Amersham Pharmacia) (30 cycles; annealing temperature 65°C, synthesis temperature 72°C). Specific primer pairs were designed either for the wild-type gene or for the knocked-out gene containing a neo insert.
Western blot analysis
Nuclear extracts were prepared as described (Grombacher et al., 1998). Forty micrograms of nuclear protein per lane were separated on a 7.5 or 10% SDS–polyacrylamide gel and electroblotted onto Protran nitrocellulose transfer membrane (Schleicher & Schuell). The blotting quality was checked by staining with Ponceau S-red. After blocking with 5% non-fat dry milk in PBS/0.1% Tween-20 (at least 3 h at room temperature or overnight in the cold), the mouse anti-p53 antibody [p53 (Ab-1) monoclonal mouse IgG, Calbiochem] was applied in a dilution of 1:500 in blocking solution for 1–1.5 h. The membranes were then washed several times with PBS/0.1% Tween-20 and, afterwards, incubated with a 1:3000 dilution of secondary antibody (Antimouse Ig from sheep, Amersham Life Science) in blocking solution for 1 h. After extensive rinsing with PBS/0.1% Tween-20, the horse- radish peroxidase coupled to the secondary antibody was detected by Renaissance® (NEN)/HyperfilmTMECLTM (Amersham Life Science) according to the manufacturer's protocol. For loading control, blots were re-incubated with anti-ERK2 antibody [ERK2 (C-14) rabbit polyclonal IgG, Santa Cruz Biotechnology, 1:3000] and horseradish peroxidase-conjugated antirabbit Ig from donkey (Amersham, dilution 1:3000).
Northern blot analysis
Total RNA was isolated from untreated and treated cells 1 h after exposure to 40 J/m2 UV-C light. Twenty micrograms of each sample were denatured with 6% formaldehyde for 15 min at 56°C. After electrophoresis on a 1.4% agarose gel containing 6% formamide, the RNA was blotted to Hybond N+ membrane (Amersham) as described (Fritz and Kaina, 1992). As a hybridization probe, a PstI fragment of the plasmid pUCp/v-fos (Rahmsdorf et al., 1987) was radioactively labelled with [
-32P]dCTP using a random priming method (Prime-It Kit, Stratagene). Hybridization was performed at 65°C overnight.
Survival experiments
The metabolic activity of UV-C- or MMS-treated cells in comparison with untreated cells was determined by means of the MTT assay. Thirty-six to forty hours after treatment, MTT solution (5 mg/ml in PBS) was added to the culture medium to a final concentration of 0.5 mg/ml. After 2–6 h of incubation, the medium was aspirated. The cells were lysed and the blue redox product was dissolved by adding 0.1–1 ml ethanol. The absorption at 540 nm was determined; the 620 nm background absorption was subtracted.
Clastogenicity assays
Treatment of exponentially growing cells with different doses of UV-C or MMS was performed as described above. After 18–19 h post-incubation time, metaphase arrest was caused by incubation in the presence of 50 ng/ml colcemid (Life Technologies) for 2.5 h. Cells were trypsinized, treated with hypotonic KCl solution (75 mM) for 7 min and then fixed with ice-cold methanol/glacial acetic acid (3/1). Chromosome preparations were made by standard procedures. After conventional Giemsa staining, 100 metaphases per sample were microscopically analysed. Cells with more than eight aberrations were considered as metaphases with multiple aberrations. Gaps were scored but not included in the final evaluation. Chromosomal aberration frequencies induced in the knockout lines were statistically compared with the wild-type using the 
2 test.
Quantification of apoptosis and necrosis
The frequencies of apoptosis and necrosis were determined by the Annexin V/propidium iodide double staining method (Vermes et al., 1995) as previously described (Ochs et al., 1999) using a FACSort flow cytometer (Becton Dickinson, San Jose, CA). Before harvesting the cells, the medium was aspirated but not discarded since a large proportion of the apoptotic/necrotic cells did not adhere to the plate. After trypsinization, cells were suspended in the original medium and washed twice with cold PBS. Fifty microlitres of a suspension of the cells in binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl2, 0.1% BSA) at a concentration of 106–107 cells/ml were incubated with 2.5 µl FITC-coupled Annexin V (PharMingen) on ice in the dark for 15–20 min. Afterwards, 0.5 µg propidium iodide in 450 µl binding buffer (propidium iodide stock solution 50 µg/ml in water) was added. At least 104 cells per sample were analysed by flow cytometry using Cell Quest software (Becton Dickinson). Apoptotic cells were identified as the population of cells stained by Annexin V-FITC, but not by propidium iodide. Necrotic cells show both increased FITC- and propidium iodide-fluorescence if compared with viable cells. The frequencies of apoptosis induced in the knockout lines were compared with the wild-type using the t-test.
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Results |
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Generation and characterization of cell lines
The experiments were performed with spontaneously immortalized, established fibroblast cell lines derived from newborn mice that were phenotypically either wild-type (+/+) or knockout for c-fos or p53, or for both c-fos and p53 (double-knockout cells). Except for the cell lines +/+f20, fos–/–f10 and p53–/–E, all other lines were generated in our laboratory. The cell line +/+BK4 was obtained from wild-type mice, whereas the line fos+/+1-98M, which is also wild-type concerning c-fos, is the product of cross-breeding of c-fos heterozygous mice. The homozygous c-Fos-deficient cell line fos–/–7-98M was derived from the same litter as fos+/+1-98M (thus having the identical genetic background). Crossing of heterozygous p53+/– mice resulted in the heterozygous cell line p53+/–BK1 and the homozygous p53-deficient cell line p53–/–2-98M, respectively. From embryos resulting from cross-breeding fos+/– and p53+/– mice, we obtained two independent fos/p53 double-knockout lines, designated as fosp53–/–BK3 and fosp53–/–BK10.
The cell lines generated in our laboratory were genotypically characterized with respect to the corresponding knocked-out gene(s) by PCR before (data not shown) and after immortalization (Figure 1). The double-knockout cell lines we generated as well as the cell lines obtained from other laboratories were checked for the expression of c-fos mRNA by northern blotting. Neither in the double-knockout cell lines nor in fos–/–f10 was c-fos mRNA detectable (Figure 2A). The p53 expression was checked at the protein level by western blot analysis (Figure 2B). The high basal level and non-inducibility of p53 in the cell line fos–/–f10 indicate mutant p53 protein. All the established wild-type cell lines and the c-Fos-deficient cell line fos–/–7-98M showed UV-C-inducible p53 protein, indicating that p53 had not been mutated. The four p53–/– cell lines (including both double-knockouts) did not show any p53 protein whereas the heterozygous line p53+/–BK1 displayed an intermediate level of p53 protein as compared with the wild-type (p53+/+) lines.
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Cytotoxicity
The sensitivity of exponentially growing established mouse fibroblasts to the cytotoxic effect of UV-C light and the alkylating agent MMS was checked by means of the MTT assay (Figure 3
). All tested cell lines deficient for either p53, c-Fos or both proteins proved to be more sensitive towards UV-C light and MMS than wild-type cells. There was no significant difference in sensitivity between c-Fos-deficient, p53-deficient and double-knockout cell lines. The cell line heterozygous for p53 (p53+/–BK1) was also more sensitive than the wild-type, which is probably due to the mutant p53 protein encoded by the remaining allele (see Figure 2B
) or to a dosage effect.
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Apoptosis and necrosis UV-C light and MMS
The frequencies of apoptosis and necrosis induced in wild-type and p53, c-fos and fos/p53 knockout fibroblasts were determined by flow cytometric analysis after double-staining with FITC-coupled Annexin V and propidium iodide. The apoptotic response of exponentially growing cells 3 days after exposure to UV-C light or MMS is shown in Figure 4A and B
. It mirrors the enhanced sensitivity of c-fos knockout cell lines to these agents. p53-deficient fibroblasts are also significantly more sensitive to UV-C and MMS than wild-type cells. The cell line p53+/–BK1 showed an intermediate apoptotic response indicating a dosage effect of the remaining (possibly mutated) p53 allele. For fos/p53 double-knockout cells, the apoptosis frequencies were higher than those of wild-type and also of p53 knockout cells. However, the effects based on c-Fos and on p53 deficiency were not additive. The population of necrotic cells was generally much smaller than that of apoptotic cells and never surpassed 10%. The percentage of apoptotic cells was also determined by flow cytometric quantification of the sub-G1 population. The data obtained were comparable to the results gained with Annexin V (data not shown).
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Ionizing radiation
The data on induction of apoptosis following
-ray irradiation are shown in Figure 4C. For these experiments we chose one cell line of each knockout cell type as a representative example. Whereas c-Fos-deficient cells (fos–/–7-98M) displayed only slightly enhanced frequencies of apoptosis 72 h after treatment, apoptosis of the p53-deficient cell line (p53–/–2-98M) was significantly enhanced if compared with the wild-type cell line (+/+BK4). The double-knockout cell line fosp53–/–BK3 exhibited the highest level of sensitivity, indicating additivity of the apoptotic effects detected in c-fos–/– and p53–/– cells. This applies to both doses used, 3 and 10 Gy, respectively. The frequencies of necrosis were also enhanced in a dose-dependent manner. However, except for the double-knockout cells at 10 Gy, they remained below 5%.
Chromosomal sensitivity
The frequency of chromosomal aberrations was determined 18 h after UV-C irradiation or MMS treatment of exponentially growing cells. Data are summarized in Tables I and II for UV-C light and MMS, respectively. Cells deficient for c-Fos and p53 proved to be hypersensitive to the clastogenic effect of UV-C light and MMS. This was the case for both aberration rates (percentage of aberrant cells) and aberration yields (aberrations per cell). For UV-C light, the heterozygous p53 knockout cell line p53+/–BK1 was as sensitive as the homozygous line p53–/–E to the induction of chromosomal aberrations but less sensitive than the line p53–/–2-98M (which was the most hypersensitive of all cell lines analyzed in this study) (Table I). For MMS, the effect of p53 again seems to depend on gene dosage (Table II). The sensitivity of fos/p53 double-knockout cells (tested for UV-C irradiation only) was higher than that of wild-type cells but, surprisingly, lower compared with the cell lines deficient for c-Fos or p53. This was especially true when aberrations were expressed as aberrations per cell (aberration yield). The types of induced aberrations are also shown in Tables I and II. It becomes obvious that UV-C and MMS induce mainly chromatid-type aberrations. There was no difference in the aberration spectrum between the cell lines.
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Discussion |
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This study was performed to compare the role of c-Fos and p53 in cellular defence after treatment with DNA-damaging agents. We comparatively analyzed the sensitivity of established mouse fibroblasts derived from knockout mice deficient for c-Fos, p53 or for both. As endpoints, we analyzed cell death (by means of the MTT assay), apoptosis and the formation of chromosomal aberrations. For mutagenic treatment we used UV-C light and the alkylating agent MMS. UV-C and MMS cause DNA damage that is repaired by different repair mechanisms. Therefore we considered them as representative inducers of DNA damage, which are removed from DNA by nucleotide excision repair (NER) and base excision repair (BER) and alkyltransferase, respectively. Since p53 is a major player in apoptosis induced by ionizing radiation in various cell systems, for which contradictory results were reported (Clarke et al., 1993
; Kuerbitz et al., 1992; Lee et al., 1994; Lee and Bernstein, 1993; Lowe et al., 1993), we also included treatment with -rays in our apoptosis experiments.
p53 is known to be involved in DNA damage-induced apoptosis via two different mechanisms: (i) in the mitochondrial pathway by down-regulation of Bcl-2 and induction of Bax (Miyashita et al., 1994) and (ii) in the Fas/CD95 pathway by stimulation of the transcription of the gene encoding the Fas receptor and thus activating the pathway (Muller et al., 1998). In both models, p53 is an essential element in triggering apoptosis. Alternatively, p53 activation leads to cell cycle arrest in the G1/S-phase, which is supposed to exert protection due to pre-replicative DNA repair. In p53-deficient fibroblasts, but not in wild-type cells, we found highly increased frequencies of apoptosis following treatment with UV-C light and MMS as well as with -rays. The high sensitivity of p53–/– cells to the induction of apoptosis indicates that apoptosis in fibroblasts proceeds in a p53-independent manner. Moreover, in fibroblasts p53 even appears to act protectively, by preventing apoptotic cell death upon DNA damage.
The role of p53 in the induction of chromosomal aberrations has not been extensively studied and, notably, fibroblasts deficient in p53 have not yet been investigated as to their genotoxin-induced chromosomal sensitivity. For fibroblasts obtained from Li–Fraumeni patients spontaneous genomic instability has been demonstrated resulting in aneuploidy and accumulation of structural chromosomal aberrations (Boyle et al., 1998). It would be interesting to see whether these fibroblasts respond to UV-C or alkylating agent treatment with elevated sensitivity. For p53 mutated glioma cells it has been reported that they do not display more X-ray-induced chromosomal aberrations than cells expressing wild-type p53 (Gupta et al., 1996). For lymphoblastoid cells it has been shown that treatment with alkylating agents leads to higher chromosomal aberration rates in two p53 mutated strains when compared with p53 wild-type cells (Greenwood et al., 1998). Since p53 is essential for the induction of apoptosis in these cells, the authors concluded that heavily damaged cells not eliminated by apoptosis contribute to the increased aberration rates in the p53-mutated lymphoblastoid population. In our study, mouse fibroblasts lacking p53 display clearly higher levels of UV-C- and MMS-induced chromosomal aberrations (measured in the first post-treatment mitosis) than wild-type cells. Since, as revealed by time-course experiments (not shown), apoptotic cells appeared at a later stage after mutagen treatment than aberrant mitotic cells, we conclude that preferential elimination of aberrant cells by apoptosis did not occur in our cell system. The protective, anti-clastogenic and anti-apoptotic effect of p53 in fibroblasts is probably mediated by G1/S arrest, providing more time for the removal of preclastogenic and pretoxic DNA lesions. Consequently, the clastogenic (as well as the cytotoxic and apotosis-inducing) effect of a mutagen would be lower in wild-type than in p53-deficient cells. Alternatively, the protective effect of p53 could be due to its direct involvement in DNA repair, as it was reported to exhibit exonuclease activity (Mummenbrauer et al., 1996), binding to Rad51 (Süsse et al., 2000) and to be required for global NER (Ford and Hanawalt, 1997; Smith et al., 2000). In addition, there are findings suggesting that p53 promotes rejoining of DNA double-strand breaks (Tang et al., 1999).
The finding that all c-fos knockout cell lines we examined so far are mutagen-hypersensitive clearly indicates that c-Fos, as a component of the heterodimeric transcription factor AP-1, protects against apoptosis and chromosomal damage induced by genotoxic agents. It appears unlikely that the expression of DNA repair enzymes is largely affected because the activity of different repair proteins was found to be normal in c-fos–/– cells (Haas and Kaina, 1995; Kaina et al., 1997). Cross-sensitivity to various S-phase-dependent genotoxic agents causing damage repaired via different pathways rather indicates involvement of cell cycle regulation. The findings are consistent with the hypothesis that wild-type c-Fos controls gene(s), the product(s) of which are responsible for the release from S-phase blockage. In this case, absence of c-Fos would cause prolongation of the S-phase arrest, indirectly leading to increased DNA breakage and consequently to chromosomal aberrations (Kaina, 1998; Morgan et al., 1998) and apoptosis (Karina et al., 1997; Meng et al., 1998).
To analyze the relationship between c-Fos and p53 in cellular defence, we generated immortalized c-fos/p53 double-knockout fibroblast cell line. This type of cell has not been described before. After UV-C and MMS treatment of these cells, we did not observe clear additive or synergistic effects (compared with the single-knockout cells), which could indicate co-operation of c-Fos and p53 in cellular defence. The chromosomal sensitivity of the double-knockout cells was even lower compared with the single-knockout fibroblasts. This could be due to, in comparison with single-knockout cells, stronger cell cycle blockage, which would prevent highly damaged cells from reaching mitosis. In this case, cells with extremely severe chromosomal damage would not be included in the evaluation. Instead, these heavily damaged cells could undergo apoptosis, which would result in enhanced apoptotic sensitivity of fosp53–/– cells to UV-C and MMS, which was indeed observed. Interestingly, as to apoptosis induced by -rays the c-fos/p53 double-knockout cell line was the most sensitive one. This finding as well as the comparatively low sensitivity of c-fos–/– cells to -irradiation may be explained, as discussed previously (Kaina et al., 1997), by the mechanism of IR-induced clastogenicity, which does not require DNA replication. Although the molecular mechanism underlying the hypersensitivity of c-Fos- and p53-deficient fibroblasts awaits to be clarified, this study shows the significance of c-Fos as well as p53 in the protection of fibroblasts against the clastogenic and apoptosis-inducing effects of genotoxic agents.
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Acknowledgments |
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We are grateful to A.Piee-Staffa for technical assistance. This work was supported by a grant from the DFG to B.K. (DFG 724/8-1).
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Notes |
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1 To whom correspondence should be addressed. Tel: +49 6131 393 3246; Fax: +49 6131 393 3421; Email: kaina{at}mail.uni-mainz.de
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References |
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Received on August 4, 2000; accepted on December 20, 2000.
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