Aug 14, 1995 - cells. Transcriptional induction of the c-Fos target genes collagenase I, stromelysin-1 and stromelysin-2 by UV is almost absent in cells lacking ...
The EMBO Journal vol. 14 no.21 pp.5338-5349, 1995
Fos is an essential component of the mammalian UV response
Martin Schreiber, Bernd Baumann', Matthew Cotten, Peter Angell and Erwin F.Wagner2 Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030 Vienna, Austria and 'Forschungszentrum Karlsruhe, Institut fur Genetik, PO Box 3640, D-76021 Karlsruhe, Germany
2Corresponding author
Mouse 3T3 fibroblasts lacking c-fos were employed to demonstrate an essential function of the UV-inducible transcription factor AP-1 (Fos/Jun) in the response to the cytotoxic effects of short-wavelength ultraviolet (UVC) radiation. Clonogenic survival and proliferation of cells lacking c-fos were drastically reduced following UV irradiation. This UV hypersensitivity manifests itself primarily in increased cell death, partly by apoptosis, and prolonged recovery time from UVinduced cell cycle arrest. Co-culture with wild-type cells did not ameliorate the hypersensitivity of mutant cells. Transcriptional induction of the c-Fos target genes collagenase I, stromelysin-1 and stromelysin-2 by UV is almost absent in cells lacking c-fos which correlates with a reduced UV induction of AP-1 DNAbinding and transactivation activity. The repair of UVinduced DNA lesions was not affected, as shown by unscheduled DNA synthesis and host cell reactivation assays. These data demonstrate that c-Fos is involved in a novel protective function other than DNA repair against the harmful consequences of UVC. Keywords: AP-1/c-fos/cell death/DNA repair/UV response
Introduction A major adverse effect of UV on cells is damage to DNA caused primarily by pyrimidine dimers, leading to either cell death or somatic mutations, which are thought to be one of the initial steps of neoplastic transformation. However, UV irradiation not only leads to the destruction of cellular integrity, but also induces specific cellular reactions. This induction response, known as the 'mammalian UV response', is characterized by transcriptional activation or repression of a specific set of genes, gene amplification, an increase in the rate of recombination and the induction of endogenous viruses (Ronai et al., 1990; Herrlich et al., 1992; Angel, 1995). This cellular response is not unique to UV irradiation, but is also observed following exposure to other DNA damaging agents such as ionizing radiation, mitomycin C, 4-nitroquinoline oxide, and H202. Among the first gene products that are activated via increased transcription and/or post-translational modification are the tumour suppressor protein p53 and several transcription factors encoded by the class of immediate
early genes, such as c-myc, egr-1, NF-,KB, p62TCF/elk-1, and c-fos and c-jun, components of the dimeric transcription factor AP-1 (reviewed in Herrlich et al., 1992; Rahmsdorf, 1994; Angel, 1995). UV irradiation induces both the enhanced de novo synthesis of c-Fos and c-Jun proteins and the activation of pre-existing and newly synthesized proteins by posttranslational modification, which together lead to increased AP-1 activity in irradiated cells (Rahmsdorf, 1994). Induction of c-fos transcription by UV is primarily mediated by the cis element also responsible for activation by mitogens, the serum response element (SRE) at position -320 to -300 of the c-fos promoter (Buscher et al., 1988). This element is bound by dimeric serum response factor (Treisman, 1987) complexed with the ternary complex factor p62TCF/elk-1 (Shaw et al., 1989). There is strong evidence that largely overlapping signal transduction pathways lead to the activation of TCF upon mitogen stimulation and UV irradiation (Karin, 1994; Rahmsdorf, 1994). Several of the components of this pathway such as Ras, Raf and MAP-kinases are activated upon UVC irradiation, and tyrosine kinase inhibitors or dominant negative mutants of Ras, Src, Raf, MAP-kinase and the EGFreceptor all attenuate UV-induced transcriptional activation of AP- 1-dependent reporter constructs (Devary et al., 1992, 1993; Radler-Pohl et al., 1993; Sachsenmaier et al., 1994). Whereas these and other findings provide strong evidence for common signal transduction pathways mediating cellular proliferation and the UV response, the molecular nature and subcellular localization of the primary 'receptor' of UV irradiation is still unknown. Based on studies with DNA repair-deficient Xeroderma pigmentosum cells (Schorpp et al., 1984; Stein et al., 1989) and in analogy to the bacterial SOS response (Walker, 1985), DNA damage or by-products of DNA damage have been hypothesized to provide this primary signal. However, the rapid UV activation of Ras and other factors located at or near the plasma membrane argues strongly against DNA damage as the primary signal (Devary et al., 1992). In fact, activation of NF-kB and Jun kinase by UV is not affected in enucleated cytoplasts, demonstrating that a nuclear signal is not required at least for this part of the UV response (Devary et al., 1993). As an alternative to DNA damage, the production of oxidative stress by UV and many other DNA damaging agents has been proposed as a trigger for this signalling cascade (Devary et al., 1992). In vitro studies have demonstrated redox-dependent binding of Jun/Jun and Jun/Fos to DNA (Abate et al., 1990), and there are at least two UV-inducible genes regulated by AP- 1 for which a role in counteracting the adverse effects of free radicals might be inferred: glutathione transferase and methallothioneins (Angel,
1995). Whether or not DNA damage is the actual trigger, a
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Defective UV response in fibroblasts lacking c-Fos
likely function for the UV response is to enhance the DNA-repair capacity of the exposed cell, in analogy to the bacterial SOS-response (Walker, 1985). Therefore, an obvious concept for the function of a UV-inducible transcription factor like AP-1 may be to induce the production of DNA repair enzymes. However, the repair genes known in mammalian cells are constitutively expressed, and no role for AP-1 in their regulation has been found (Herrlich et al., 1992). In addition, none of the UV-inducible genes identified in mammalian cells appears to be involved in DNA repair (Herrlich et al., 1992). Together with the fact that the initial signal does not appear to be generated in the nucleus, these results suggest that the mammalian UV response mediated by AP- 1 is involved in a protective function other than DNA repair (Devary et al., 1992; Engelberg et al., 1994). Most DNA damaging agents, including UV radiation, are known to damage other cellular constituents such as membrane lipids, proteins, RNAs and ribosomes. A protective function for the eukaryotic UV response is suggested by three lines of evidence: first, treatment with tyrosine kinase inhibitors reduces the survival rate of HeLa cells after UV irradiation by one order of magnitude (Devary et al., 1992); second, transformation of cells with activated raf-1, Ha-ras and v-src increases their radioresistance (Kasid et al., 1987; Sklar, 1988; Shimm et al., 1992); finally, analysis of mutant yeast strains revealed that Ras-dependent activation of target genes of GCN4 (one of the yeast homologues of mammalian AP-1) also has a role in protecting cells from the effects of UV irradiation (Engelberg et al., 1994). Genetic evidence indicates that although this response is responsible for increased survival after UV irradiation it is unlikely to be involved in DNA repair (D.Engelberg and M.Karin, personal communication). Using immortalized 3T3 fibroblast cell lines with a targeted disruption of the c-fos gene (Brusselbach et al., 1995), we show that the UV-inducible transcription factor c-Fos is an essential component of the cellular defence mechanism against the cytotoxic effects of UVC irradiation. The lack of c-Fos results in significantly lower UVinduced mRNA levels of the AP- 1 dependent genes collagenase I, stromelysin- 1 and stromelysin-2, which correlates with a reduction of UV-induced AP-1 DNA binding and transactivation activity. Furthermore, these cells are hypersensitive to UV irradiation, yet they are fully capable of repairing UV-damaged DNA, demonstrating that functions of the UV response other than DNA repair are indeed relevant to the survival of mammalian cells.
Results Cells lacking c-Fos are hypersensitive to UVC irradiation To analyse whether c-Fos serves any protective function in cells exposed to UV radiation, we have used immortalized 3T3 fibroblasts derived from mouse embryos lacking c-Fos due to targeted disruption of the c-fos gene by homologous recombination (Brusselbach et al., 1995). We first examined whether the absence of c-Fos affects cell survival following UV irradiation. The number of colonies formed from single cells was determined following expo-
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Fig. 1. The lack of an intact c-fos gene affects cell survival and cell proliferation after UV irradiation. (A) Clonogenic survival assays of wild-type (+/+) and c-fos4- 3T3 fibroblasts (-/-) after irradiation with increasing doses of UVC. The plating efficiency at each UV dose (colonies obtained/cells plated) is indicated. Each point represents the mean ± SD of triplicate wells of experiments performed with two independent cell lines of each genotype. (B) Increase in cumulative cell numbers of wild-type (+/+) and (-/-) 3T3 fibroblasts after UV irradiation (+UV) or mock-treatment. Irradiation with 10 J/m2 UVC was performed 8 h after plating, and cells were passaged at 3 day intervals. Each point represents the mean ± SD of experiments performed with three wild-type (NIH 3T3, 3T3f-20 and 3T3j-56) or two c-fos4- (3T3f-1 and 3T3f-10) cell lines. (C) Cells were plated at a density of 105 cells per 12.5 cm2 well, irradiated with 10 J/m2 UVC or mock-treated 6 h after plating and counted after culture for the indicated times after plating. Each time point represents the mean ± SD of triplicate wells of two independent cell lines of each genotype.
sure to increasing doses of UVC. The clonogenic survival of two independent cell lines lacking c-Fos was considerably reduced compared with wild-type cells (Figure IA). This UV hypersensitivity increased with increasing UV doses, and at a dose of 20 J/m2, five times more wildtype colonies than c-fos-'- colonies had formed. Using the
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same assay, two independent c-fos4- embryonic stem (ES) cell lines were also found to be UV-hypersensitive (data not shown). We next measured the effect of UV irradiation on the proliferation rates of these fibroblasts (Figure iB). When cell numbers were determined 3 days after irradiation, three independent wild-type cell lines had grown to 55% of the numbers obtained for non-irradiated control cells, whereas the cell numbers for two c-fos-'cell lines were only 20% of their non-irradiated control. Thereafter, wild-type as well as mutant cells resumed the same proliferation rates as non-irradiated cells, indicating that all UV-damaged cells had either repaired their damage or had been eliminated from the cell population (Figure 1B). Interestingly, the largest difference in proliferation between irradiated and control cells was not observed during the first 10 h, but from 10 to 34 h post-treatment, suggesting that a significant fraction of UV damaged cells survives for at least 10 h after the irradiation event (Figure IC). These results demonstrate that c-fos' cells are hypersensitive to the cytotoxic effects of UVC, as both the clonogenic survival and the proliferation rate following UV irradiation are reduced. Since UV-irradiated cells secrete soluble factors which are involved in the autocrine and paracrine induction of the UV response (Schorpp et al., 1984; Kramer et al., 1993), we tested whether co-culture with wild-type cells could overcome this UV hypersensitivity. Wild-type and c-fos4- cells were mixed in a 1:1 ratio, and the changes in this ratio during 4 days of co-culture after UV irradiation or mock treatment were analysed (Figure 2). Wild-type and mutant cells can be distinguished by Southern blot analysis of the c-fos locus, thus allowing determination of the relative abundance of each cell type in the mixture. When a co-culture of non-irradiated wild-type and c-fos4cells was analysed, a 1:1 ratio was maintained throughout the entire culture period of 96 h, consistent with their essentially identical proliferation rates. However, when cells were irradiated with a dose of 10 J/m2 of UVC after plating, the relative abundance of mutant cells in the coculture progressively decreased, as demonstrated by the lower intensity of the c-fos-'- band (Figure 2A). When a growth curve for the UV-irradiated wild-type and mutant cells was calculated from the intensities of the Southern blot (Figure 2B), the c-fos4- cells clearly lagged behind the wild-type cells, as has been found previously for the growth curves of UV-irradiated cells (Figure 1). Therefore, putative soluble factors secreted by wild-type cells following UV irradiation, such as IL-la and bFGF (Kramer et al., 1993), are not sufficient to overcome the UV hypersensitivity of c-fos4- cells.
Increased cell death of c-fosg-l fibroblasts following UV irradiation Since UV irradiation reduces the proliferation rates of wild-type cells, and to a larger extent of c-fos4- cells, we next analysed to what extent cell death contributes to this decrease in cell numbers. Cell death was measured by quantification of the amount of the cytosolic enzyme lactate dehydrogenase (LDH) released into the culture medium due to cell lysis. A dose-response curve of LDH release showed that both wild-type and mutant cells responded to increasing doses of UVC irradiation with a roughly linear increase in the fraction of dead cells (Figure
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Fig. 2. Effect of co-culture with wild-type cells (3T3f-20) on the proliferation rate of c-fos4- 3T3 fibroblasts (3T3f-1) after irradiation with 10 J/m2 UVC. (A) Wild-type (+/+) and (-/-) cells were trypsinized, counted and mixed in a 1:1 ratio. The mixture was plated into multiple wells, irradiated or mock-treated 6 h after plating, and cells were harvested after culture for the indicated times after plating. At each time point, cells from three identical wells were pooled for Southern blot analysis. The 1.8 kb band represents the wild-type allele of c-fos, whereas the 1.3 kb band is diagnostic for the targeted allele present in c-fos4- cells. The intensity of each band was quantified using a Phosphorlmager, and the percentage of c-fos4- cells in the mixture (% -/-) calculated from these intensities is indicated at the bottom of each lane. (B) Cells were counted at the time points of harvesting for the Southern blot in (A), and a growth curve for the UV-irradiated mixture of wild-type and c-fos4- cells was calculated from these numbers. Each time point represents the mean ± SD of triplicate cultures. The broken lines represent the increase in the number of wild-type (+/+) and c-fos4- cells (-/-) in the co-culture, which was calculated from the relative intensities of the bands in the Southern blot shown in (A).
3A). The increase was considerably larger in c-fos4- cells, resulting in almost twice as many dead cells at higher doses. A time-course analysis of cell death revealed that during the first 12 h post-irradiation a relatively small fraction of cells of both genotypes dies (Figure 3B). However, after this initial period of 'immediate' cell death, the percentage of dead wild-type cells does not increase significantly, whereas the fraction of dead cells in the mutant population continues to increase at a linear rate
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for up to 48 h (Figure 3B). As a consequence, 48 h after irradiation the fraction of dead cells is three times higher than in the wild-type population. This difference reflects a dramatic increase in cell death due to the lack of c-Fos, which significantly contributes to the UV hypersensitivity of mutant cells. To determine the role of apoptosis in this UV-induced cell death, we analysed UVC-treated wild-type and mutant cells by propidium iodide staining and flow cytometry (Huschtscha et al., 1994). The number of particles with a hypodiploid (sub-2N) DNA content was drastically elevated in both cell types following irradiation with a dose of 40 J/m2 of UVC (Figure 3D). The frequency of such apoptotic cells was -3-fold higher in c-fos4- cells than in wild-type cells, correlating with the frequency of total cell death (Figure 3B). Furthermore, the flow cytometry analysis revealed a significant reduction of cells with a 4N DNA content, i.e. G2 and M-phase cells following UV irradiation consistent with the UV-induced transient block of DNA synthesis observed in these cells (Figure 5). Agarose gel electrophoresis of DNA extracted from mutant cells 12 h after UVC irradiation revealed the characteristic fragmentation of DNA into bands with a size of multiples of .200 bp, a hallmark of apoptosis (Figure 3D). No nucleosome laddering was observed in non-irradiated cells or irradiated wild-type cells. Furthermore, the frequency
of UV-induced cell death of wild-type and c-fos4- cells was significantly reduced following transient overexpression of the anti-apoptotic gene bcl-2 (Korsmeyer, 1992), again indicating that a significant fraction of these cells dies by apoptosis (Figure 3C).
Overexpression of c-fos ameliorates the UVhypersensitivity of c-fos-1- cells To demonstrate that the UV hypersensitivity of the c-fos43T3 fibroblast lines used in this study is specific for the absence of c-Fos, we introduced a functional c-fos gene into these cells by transfection with the c-fos expression plasmid H2-c-fos-LTR (Grigoriadis et al., 1993). The plasmid was introduced into cells using the transferrinfection method which has independently been shown by LacZ staining to lead to expression of the introduced gene in >80% of the target cells (Cotten et al., 1992; Wagner et al., 1992). Ectopic expression of c-Fos in c-fos4fibroblasts resulted in a dramatic drop in the number of dead cells following UVC irradiation, making them almost as resistant to the cytotoxic effects of UVC as the corresponding wild-type cells (Figure 3C). Some 14.5% dead cells were identified by lack of trypan blue exclusion in this experiment, as opposed to 46% dead cells observed when the same mutant cell line was transfected with empty plasmid, and compared with 12.5% dead wild-type 5341
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Fig. 4. DNA repair efficiency after UV irradiation in wild-type (+/+) and c-fos fibroblasts (-I-). (A) Ability of transiently transfected cells to by DNA repair the CAT activity expressed from an in vitro UVC irradiated SV40-CAT reporter plasmid (host cell reactivation assay). CAT activity relative to the non-irradiated plasmid at 12 h after transfection (before repair) and 48 h after transfection (after repair) is indicated. The amounts of extracts used were normalized to the luciferase activity expressed from a co-transfected internal control plasmid. Values are the averages + SD of three independent wild-type and two independent c-fo.s t cell lines. Data of one representative out of three independent experiments are shown. (B) Relative [3H]thymidine incorporation by unscheduled DNA synthesis (UDS) into non-S-phase cells after irradiation with increasing doses of UVC. Each value represents the mean + SD of quadruplicate wells of two independent cell lines. (C and D) UV-induced (40 J/m2) UDS in wildtype (C) and ctfo.s-- (D) fibroblasts. The number of nuclei with the indicated mean grey levels resulting from I3Hlthymidine incorporation is shown. Thus, high levels of incorporation correlate with reduced mean grey levels. restore
cells transfected with c-fos. We also observed a small, but significant reduction of cell death of UV-treated wild-type cells overexpressing c-fos, suggesting a gene dosage effect of c-fos on the UV-resistance of fibroblasts. In contrast, expression of exogenous c-Fos had no significant effects on the viability of non-irradiated wild-type and mutant cells (Figure 3C). A parallel transfection with the antiapoptotic gene bcl-2 also reduced the frequency of cell death of wild-type and mutant cells. Whereas the protective effect of Bcl-2 was more pronounced than that of c-Fos in wild-type cells, c-fos-'- cells were protected more efficiently by transfection with c-fos.
Repair of UV-induced DNA damage is independent of c-Fos A reason for the decreased survival of c-fos-A- cells might be a defect in cellular DNA repair mechanisms. To test this possibility, DNA repair efficiency was analysed using three independent assays. First, we analysed the restoration of transcriptional activity of an in vitro UV-irradiated CAT-reporter plasmid following transient transfection into wild-type and mutant cells (Wang et al., 1995). UV irradiation introduces mutations into the plasmid DNA, which interfere with CAT activity relative to that of 5342
the same plasmid transfected in parallel without prior irradiation. Thus, the extent to which CAT expression is restored is proportional to the ability of a given cell to repair the UV-induced DNA damage. At 12 h after transfection, a reduction in CAT activity expressed from the UV-irradiated plasmid was observed in wild-type and c-fos- cells, indicating that the CAT plasmid was indeed damaged (Figure 4A). After an additional 36 h of culture, expression of CAT activity from the UV-irradiated plasmid reached levels comparable with the non-irradiated control plasmid in both cell types, indicative of efficient plasmid reactivation by the host cell DNA repair mechanisms. Since UV-induced DNA lesions are eliminated primarily by nucleotide excision repair, we next measured the efficiency of unscheduled DNA synthesis (UDS), i.e. DNA synthesis due to repair replication in non-S-phase cells. Two wild-type and two c-fos-k- cell lines were irradiated with increasing doses of UVC, and [3H]thymidine incorporation into DNA during 5 h post-irradiation was determined by scintillation counting. The background of replicative DNA synthesis was eliminated by arresting these cells with 0.5% serum and hydroxyurea before and after irradiation. UV exposure resulted in elevated [3H]thymidine incorporation levels relative to the non-
Defective UV response in fibroblasts lacking c-Fos
irradiated control, which increased with increasing UV doses (Figure 4B). Thymidine incorporation levels of mutant cells were not significantly different from those of wild-type cells at all UV doses tested, demonstrating that UDS is not impaired in the absence of c-Fos. The efficiency of UDS was also determined on a single cell level by [3H]thymidine incorporation and in situ autoradiography. The number of autoradiographic grains above the nuclei of UV-irradiated cells was determined by automated image processing (Figure 4C and D) or manual counting (not shown). The number of grains was not significantly different between two independent wild-type and mutant cell lines at a dose of 40 J/m2 (Figure 4C and D) and 10 J/m2 (data not shown), confirming that UV-induced nucleotide excision repair occurred with normal efficiency in c-fos#- cells. The observed incorporation of [3H]thymidine was specific for UV-induced UDS, as the number of autoradiographic grains was not above background in untreated cells (data not shown). These results demonstrate that although c-fos-'- cells are UV-hypersensitive, they do not appear to be defective in DNA repair.
Prolonged S-phase arrest following UV irradiation of c-fos-1- cells The initiation and elongation of DNA synthesis is blocked following UV irradiation of cells, which is assumed to be critical for cell survival, allowing for repair of UV-induced DNA damage before DNA replication (Kaufmann and Cleaver, 1981; Wang and Ellem, 1994). We analysed whether the kinetics and magnitude of this UV-induced S-phase arrest were altered in the absence of c-Fos, which might contribute to the observed reduced proliferation rates. A relatively low dose of UVC was used to avoid any specific effects being masked due to a high incidence of cell death. Wild-type and c-fos-'- cells were irradiated with a dose of 10 J/m2 and [3H]thymidine incorporation into newly synthesized DNA was measured during the first 10 h post-exposure (Figure 5A). Both cell types responded with a rapid decrease in relative DNA synthesis rates within the first 2 h, which was slightly more pronounced in the mutant cells. Wild-type cells recovered from this transient S-phase block within 6 h and even reached thymidine incorporation levels exceeding those of the non-irradiated control, presumably due to partial UV-induced cell cycle synchronization. In contrast, cfos'- cells fail to reach pre-irradiation levels of DNA synthesis as late as 10 h post-irradiation. As an internal control for cell viability, the conversion of the tetrazolium salt MTS into a formazan was quantified in parallel to [3H]thymidine incorporation. MTS is converted by constitutively active dehydrogenases of viable cells, whose activity is independent of normal cell cycle progression. MTS conversion of both wild-type and mutant cells remained at levels >90% of that of the non-irradiated control throughout the 10 h post-irradiation period (data not shown), demonstrating that the reduced thymidine incorporation levels are specific for S-phase block and not due to cell death. We next measured the levels of thymidine incorporation and MTS conversion 24 h after irradiation, a time point when the difference in [3H]thymidine incorporation of wild-type and c-fos-1- cells is not significant (Figure 5A). Exposure to increasing doses of UVC resulted in a dose-dependent inhibition of DNA synthesis, which
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reached 90% at UV doses >20 J/m2 (Figure 5B). MTS conversion dropped significantly below control levels at higher UV doses, indicative of partial cell death, and cfos4- cells were again more sensitive in this respect. The level of thymidine incorporation was significantly below the level of MTS conversion for both cell types, suggesting that both cell death and partial block of S-phase had occurred. Interestingly, we observed a small, but characteristic increase in MTS conversion at low doses of UVC compared with the non-irradiated control in both cell types. A similar, but even stronger increase was observed when a dose-response curve was performed 2 h after irradiation (data not shown). The thymidine incorporation data indicate a role for c-Fos in cell cycle re-entry of S-phase-arrested UV-irradiated cells, presumably after
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ts_tz9.,X Fig. 6. Expression of c-Jun, c-Fos and AP-l target genes in wild-type (WT) and c-fos4- 3T3-like fibroblasts. Cells were irradiated with UVC (40 J/m2) and RNA was isolated at the indicated time points (45 min and 36 h post-irradiation). Co, RNA from mock-treated cells. Probes specific for mouse type I collagenase (coll-1), mouse type I and II stromelysin (strom-i and strom-2), c-Jun and c-Fos were used for Northern blot analyses using 20 gg of total RNA. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control for equal loading.
completion of DNA repair processes. In contrast, a role for c-Fos in the UV-induced initiation of this S-phase arrest seems unlikely, since the initial inhibition of [3H]thymidine incorporation occurs essentially with the same efficiency and kinetics in normal and mutant cells. UV induction of AP-1 target gene expression and AP-1 DNA-binding and transactivation activity is reduced in cells lacking c-Fos Based on its function as a transcriptional regulator, it is likely that the differences in UV sensitivity between wildtype and c-fos'- cells are caused by alterations in the expression of Fos-regulated genes. To analyse whether UV inducibility of Fos-dependent genes is affected we determined the expression of the matrix metalloproteinases collagenase I, stromelysin-1 and stromelysin-2 (Figure 6). UV treatment resulted in transcriptional activation of collagenase I in wild-type cells reaching maximum levels at 36 h, in agreement with previous findings (Angel et al.,
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1987; Gack et al., 1994). In contrast, basal level expression of this gene is reduced and UV induction is severely affected in c-fos- cells. Similarly, UV induction of stromelysin-1 and stromelysin-2 is completely abolished in mutant cells. In contrast, expression of the immediate early genes c-fos and c-jun is normally inducible by UV, and no significant differences in the levels of endogenous c-fos RNA in wild-type cells and the 3.2 kb and 2.0 kb Fos/neo fusion transcripts in c-fos-'- cells were detected 45 min after irradiation. c-Fos mRNA levels were maximally induced 12-fold 60 min after UV irradiation (data not shown) and returned to basal levels after 36 h, which is consistent with previous results (Buscher et al., 1988). Consistent with the lack of transcriptional activation of c-Fos target genes, UV-induction of DNA binding of AP-1 dimeric complexes was very inefficient in mutant cells. When protein binding to the AP-1 site of the mouse collagenase gene was determined in vitro, low amounts of protein-DNA complexes were detected in non-irradiated wild-type cells (Figure 7), which contain rather small amounts of c-Fos (Briisselbach et al., 1995 and Figure 7B). The levels of AP-1 DNA-protein complexes in untreated mutant cells are slightly higher, which is most likely due to increased levels of c-Jun (Figure 7C). UV treatment of wild-type cells resulted in a long-lasting enhancement of DNA binding for at least 36 h. Using specific antibodies, these UV-induced complexes were found to contain considerable amounts of c-Fos (Figure 7B), c-Jun (Figure 7C) and JunD, Fra-1 and Fra-2 (data not shown). In contrast, UV-induced enhancement of DNA binding is reduced and not as long-lasting in c-fos-'- cells (Figure 7). As expected, these complexes do not contain any c-Fos (Figure 7B), which presumably is the cause for the reduced UV inducibility of AP-1 DNA binding, especially at later time points. Very similar results were obtained using the AP-1 consensus sequence from the human collagenase gene, which is also found in the mouse stromelysin-I promoter (data not shown). As an internal control for equal DNA-binding capacity of the whole cell extracts used, protein binding to an SP- 1 consensus oligonucleotide probe was analysed (Figure 7D). We next compared the UV-induced transactivation of a promoter containing multimerized AP-1 sites (5X TRECAT) in wild-type and c-fos'- cells. Whereas transactivation activity was induced almost 3-fold in two independent wild-type cell lines within 8 h, no increase in transactivation activity following UV-irradiation was observed in two c-fos-/- fibroblast lines (Figure 8), which is consistent with the observed lack of UV inducibility of Fos-regulated genes. The parental vector lacking AP- 1 sites (TATACAT) was not affected by UV irradiation. At 24 h after irradiation, some UV inducibility was also observed in c-fos-'- cells, but it was lower than in the corresponding wild-type cells (data not shown). In summary, UV-induction of AP-1 target gene expression, AP-1 DNA-binding activity and transactivation activity is considerably impaired in c-fos-'- cells, which is most likely the cause of their UV hypersensitivity.
Discussion Exposure of mammalian cells to UV irradiation induces a response that closely resembles the response to growth
Defective UV response in fibroblasts lacking c-Fos WN T
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Fig. 7. Differences in DNA binding activity of AP-I in wild-type (WT) and c-fos4- fibroblasts. Whole cell extracts were prepared from untreated cells (0) or from UV-irradiated cells (40 J/m2) at the indicated times post-irradiation. Extracts were incubated with labelled probes containing AP-1 sites from the mouse type I collagenase gene (A-C) or, as an internal control for extract quality, an SP-1 consensus oligonucleotide (D). (-), no extracts were added. The positions of the AP-1-DNA complex and the SP-1-DNA complex are indicated. For supershift analysis, extracts were preincubated with specific antiserum for c-Fos (B; supershift c-Fos, ss cFos) and c-Jun (C; ss cJun), and the positions of the resulting supershifts are indicated. The numbers at the bottom of each lane refer to the fold induction of AP-1 DNA-binding activity in (A) compared with the level of complex formation using extracts from untreated cells. The values were calculated for each cell line separately. The amounts of DNA-protein complexes were quantified using a Phosphorlmager.
factors ('pseudo-growth response'). Like growth factors, UV irradiation activates a number of cellular components involved in the regulation of cell proliferation, such as the EGF receptor, Src, Ras, Raf, and certain MAP-kinases, which ultimately results in activation of transcription factors p62TCF, ATF-2 and AP-1 (Devary et al., 1991, 1992, 1993; Radler-Pohl et al., 1993; Sachsenmaier et al., 1994; van Dam et al., 1995; Cavigelli et al., 1995). However, the functional relevance of this response, termed the UV response, is still not clear. Since the AP- 1 component c-Fos is activated rapidly and to high levels after exposure to UV (Biischer et al., 1988), we tested whether it serves a function in protecting cells against the cytotoxic effects of UV. Both clonogenic survival and proliferation were indeed reduced several-fold in UVirradiated 3T3 fibroblasts and ES cells lacking c-fos, primarily due to increased cell death, demonstrating that the lack of c-Fos considerably enhances the cytotoxic
11
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Fig. 8. UV-induction of an AP-l-regulated promoter in wild-type (WT) and c-fos4- 3T3 fibroblasts. Cells were transfected with CATreporter plasmids containing either a pentameric AP-1 site (5x TRE) preceding a TATA box, or a TATA box only. Cells were then incubated for 10 h in 10% serum and 24 h in 0.5% serum before irradiation with a dose of 40 J/m2 UVC to reduce background AP-1 activity. Cell extracts were harvested at the indicated times after UV irradiation (+; black bars) or mock-treatment (-; white bars) and normalized to the luciferase activity expressed from a co-transfected plasmid (pRK7). The percentage of chloramphenicol converted to an acetylated product (% CAT conversion) was quantified using a Phosphorlmager. Each value represents the mean + SD of two independent experiments performed with two cell lines of each genotype.
effect of UVC. Our findings provide the first genetic evidence in mammalian cells that the UV induction of growth-regulating genes serves an essential protective role. This is consistent with previous reports using tyrosine kinase inhibitors or yeast mutants, which also support a biological role for the UV response in cell survival (Devary et al., 1992; Engelberg et al., 1994). The molecular nature of the signal for the AP-1mediated UV response is not known, but it is most likely not DNA damage, at least for induction of the early events (Devary et al., 1993). Nevertheless, part of the UV response may involve DNA repair, even if the signal is generated elsewhere. However, the DNA repair capacity of c-fos4- cells is not significantly different from that of wild-type cells. Therefore, c-fos4- cell lines provide a unique example of a single-gene defect resulting in UV hypersensitivity, yet leaving DNA repair functions apparently unaffected. These data are in agreement with previous data showing that the expression of DNA repair enzymes is not increased following UV irradiation (Herrlich et al., 1992). All other known UV-hypersensitive cell lines for which the genetic cause has been identified exhibit defects in DNA repair, e.g. Xeroderma pigmentosum or Cockayne syndrome cells (Lehmann, 1982). Another example is the tumour suppressor gene p53, since interference with p53 function by viral oncoproteins results not only in reduced survival, but also in reduced nucleotide excision repair efficiency of UV-irradiated cells (Smith et al., 1995). We frequently observed a smaller increase in p53 protein levels following UV irradiation in c-fos4- cells compared 5345
M.Schreiber et aL
with wild-type cells (data not shown). However, cells lacking c-fos are fully capable of undergoing apoptosis and exhibit normal levels of nucleotide excision repair following UV irradiation, arguing against a defect in p53 function in these cells. Regardless of the exact role of p53, our results demonstrate that additional functions of the mammalian UV response distinct from DNA repair are relevant for cell survival. As DNA repair activity of c-fos4- cells is not significantly reduced, c-Fos is most likely involved in the repair or replacement of other UV-damaged cellular constituents. In addition to DNA damage, exposure to UV and most other DNA damaging agents results in damage to biomembranes, proteins and, most importantly, RNA and ribosomes, either directly or by generating oxidative stress (Angel, 1995). A simple protective mechanism against damage to such components is to replace them with newly synthesized ones. This might explain why the mammalian UV response leads to the activation of a signal transduction cascade similar to that of a growth response, since both proliferation and replacement of damaged counterparts would require enhanced macromolecule synthesis. Interestingly, we have repeatedly observed increased conversion of MTS by cellular dehydrogenases after low-dose UV irradiation of wild-type cells, which might reflect a general UV-induced acceleration of cellular metabolism. The UV hypersensitivity of cells lacking c-fos manifests as a drastic increase in cell death following UV exposure. Interestingly, this large decrease in viability compared with wild-type cells only becomes apparent at late time points after irradiation. This could be due to the efficient activation of protective mechanisms in wild-type cells by this time, and the deficiency of such defence mechanisms in the absence of c-Fos. In agreement with these findings, the largest reduction of proliferation was not observed immediately after UV irradiation, but 10-34 h later. A significant fraction of hypodiploid cells, an acknowledged measure of apoptotic activity (Huschtscha et al., 1994; Jacobsen et al., 1994), was detected in UV-irradiated wildtype and mutant populations, suggesting that apoptosis contributes to the UV-induced cell death in these cells. Support for this interpretation comes from the fact that transient transfection with the anti-apoptotic gene bcl-2 could significantly reduce this cell death. Apoptosis is increased in mutant cells by roughly the same factor as total cell death, suggesting that enhanced apoptotic activity significantly contributes to the UV hypersensitivity of c-fos-'- cells. In addition to affecting cell survival, c-Fos appears to be required for efficient cell cycle re-entry of UV-irradiated, S-phase-arrested cells, since c-fos'- cells recover to pre-irradiation levels of thymidine incorporation incompletely and with a delay of several hours, which results in a pronounced decrease in the frequency of cells in the G2 and M-phases of the cell cycle following UV irradiation. This defect is consistent with the proposed role of AP-1 in cell cycle re-entry of quiescent cells (Kovary and Bravo, 1991). On the other hand, S-phase arrest is a common protective mechanism against the harmful consequences of UV irradiation, allowing DNA repair before replication and macromolecule resynthesis. Hence, the UV-hypersensitivity of c-fos4- cells with their prolonged S-phase arrest and, more generally, the activation of a signalling pathway
5346
similar to the growth response may seem paradoxical. However, under many circumstances DNA repair presumably is highly efficient, and therefore most cells escape moderate DNA damage with an intact genome. Under such circumstances, a prolonged or even permanent cell cycle block is certainly undesirable. While the cellular components responsible for the enhanced UV sensitivity of c-fos'1 cells are presently unknown, it is very likely that alterations in gene expression resulting in enhanced or reduced synthesis of one or more gene products are instrumental. A variety of possibilities should be considered to explain how the absence of c-Fos might affect gene expression. First, lack of c-Fos expression presumably reduces or abrogates the expression of AP-1-dependent target genes. In fact, we observed an almost complete loss of UV inducibility of collagenase I, stromelysin-1 and stromelysin-2 expression in c-fos-'- fibroblasts, which are UV-inducible genes predominantly regulated by AP-1 complexes containing Fos proteins (Angel, 1995). Regulation of these genes may exemplify the loss of expression of other gene products required for cell survival following UV irradiation. Second, c-Fos has been found to positively or negatively regulate the activity of other transcription factors without contacting DNA, such as the p65 subunit of NF-KB, MyoD, steroid hormone receptors, and possibly other transcription factors of yet unknown identity (Jonat et al., 1990; Schiile et al., 1990; Yang-Yen et al., 1990; Li et al., 1992; Stein et al., 1993). Due to these activities of c-Fos, genes that are essential parts of the UV response may be altered in their transcription rate in c-fos4- cells. The lack of UV inducibility in c-fos-'- cells was only observed for genes that depend on UV-induced c-Fos expression, such as collagenase I, but not for immediate early genes such as c-fos and c-jun. The induction of c-jun and c-fos transcription by UV is mediated by changes in the phosphorylation status of promoter-associated c-Jun/ ATF-2 and SRF/TCF complexes, respectively (Herrera et al., 1989; Sachsenmaier et al., 1994; van Dam et al., 1995; Cavigelli et al., 1995). Therefore, these data demonstrate that the lack of c-Fos does not interfere with the integrity of signal transduction pathways initiated by UV, such as the presence and activation of protein kinases that regulate the transactivation function of c-Jun, ATF-2 and TCF/Elk-1. The UV induction of genes of the delayed type, such as collagenase I, is mediated, at least in part, by soluble factors released upon UV irradiation of cells (Schorpp et al., 1984; Kramer et al., 1993). One reason for the UV hypersensitivity of c-fos'1 cells could be a lack of efficient transcriptional induction of these extracellular factors following UV irradiation. However, co-culture with wild-type cells did not ameliorate this hypersensitivity, demonstrating that soluble factors secreted by normal cells following UV irradiation are not sufficient. The data on the UV response presented here provide evidence for an important contribution of c-Fos to AP-1 activity in fibroblasts. In a different study using the same cell lines, we could not detect any defect in either cell proliferation or cell cycle re-entry of quiescent cells upon serum stimulation, presumably due to redundancy with other Fos family members (Brusselbach et al., 1995). The more important role of c-Fos in cell cycle re-entry of UV-arrested cells could be based on two fundamental
Defective UV response in fibroblasts lacking c-Fos
differences. First, serum starvation arrests cells uniformly in Go, whereas UV-irradiated cells can be arrested in GI, in G2 or at the beginning and within S-phase (Wang and Ellem, 1994). Second, c-Fos is the only member of the Fos gene family that is highly inducible by UV irradiation (Devary et al., 1991; our unpublished data), whereas serum stimulation efficiently activates FosB, Fra-I and Fra-2 as well, which might functionally complement for the absence of c-Fos in this context. The latter finding may also explain why the UV-induced AP-1 DNA-binding and transactivation activity is considerably reduced in cfos4- cells, whereas no significant difference to wild-type cells was observed in unstimulated cells and growth factoror TPA-stimulated cells (Hu et al., 1994; Bruisselbach et al., 1995). On the other hand, a decreased induction of collagenase I was observed not only for UV, but also following TPA and cytokine stimulation of c-fos4fibroblasts (Hu et al., 1994; B.Baumann et al., unpublished data). It is very likely that a different set of AP-1 target genes is important in the UV response than in normal growth and cell cycle progression. Since the binding specificities of different AP-1 complexes are not absolutely identical (Ryseck and Bravo, 1991), it is conceivable that c-Fos cannot be functionally complemented in the regulation of UV-induced target genes to the same extent as in the regulation of genes involved in cell proliferation. Alternatively, c-Fos function may not be required at all under normal growth conditions and might only become critical under adverse environmental influences such as exposure to UV. Introduction of functional c-Fos into c-fos' cells almost completely restored wild-type levels of resistance to the cytotoxic effects of UVC, demonstrating that the UV hypersensitivity of these cells is specific for the absence of c-Fos and not due to any secondary mutations that might have occurred in the process of immortalization. This finding is supported by the fact that UVC exhibits an enhanced cytotoxic effect on three independently derived 3T3 fibroblast lines and two independent ES cell lines, which are expected to be genetically more stable. In the future, transient overexpression of exogenous genes in c-fos4- fibroblasts will allow us to determine whether other members of the Fos family, which are not efficiently induced by UV irradiation directly, can complement for the absence of c-Fos in the UV response. Moreover, different Fos mutants with defined functional defects could be analysed in this assay to determine which domains of c-Fos are required for an efficient UV response. The present study with c-fos'1 fibroblasts underscores the power of genetic approaches to examine the function of the mammalian UV response. Previous studies have primarily focused on unravelling the signal transduction cascades activated by UV irradiation, whereas very few investigations into the functional relevance of these activations were carried out, which used inhibitors or dominant negative mutants (e.g. Devary et al., 1992; Smith et al., 1995). Transgenic mice lacking other specific genes involved in the UV response, such as c-src or c-jun, have recently become available (Soriano et al., 1991; Hilberg et al., 1993), and it will be interesting to determine whether the absence of these gene products affects the UV response. We have recently analysed fibroblasts lacking cjun and found them to be similarly UV-hypersensitive as
the c-fos'1 cell lines described here (M.Schreiber and E.F.Wagner, unpublished data). In the future, it will be interesting to combine the mutation of c-fos with a mutation affecting DNA repair, and to determine the interplay between these two distinct functions of the mammalian UV response.
Materials and methods Culture and UV irradiation of 3T3 fibroblasts Fibroblasts from embryos derived from intercrosses of c-fos+/- mice were isolated and immortalized as described in Brilsselbach et al. (1995). The 3T3-like cell lines used in this study were NIH-3T3 (WT), 3T3j56 (WT), 3T3-f20 (WT), 3T3-fl (c-fos4-) and 3T3-flO (c-fos4-) (Brusselbach et al., 1995). The genotype at the c-fos locus of each cell line was determined by Southern blot as described (Wang et al., 1992). 3T3 fibroblasts were cultured in DMEM supplemented with 10% fetal calf serum. As a UVC source we used either a StratalinkerTm 2400 UV crosslinker (Stratagene, La Jolla, CA) with a peak emission wavelength of 254 nm and a flow rate of 2000 J/m2/min, or a 15 W mercury lamp (Vetter GmbH, Wiesloch, Germany) with a maximum output at 254 nm. Cells were typically 50-80% confluent at the time of irradiation. For irradiation, the medium was removed and retained, cells were washed once with PBS, and the PBS removed. Uncovered tissue culture plates were then irradiated with the indicated UV doses, and the retained medium was added back to the cells to avoid any serum stimulation effects of fresh medium. For mock-treated control cells, the same procedure was followed without irradiation.
Survival assays and proliferation curves For survival assays, cells were plated into multiple 12.5 cm2 plates at 500 cells per plate and irradiated 8 h later with graded doses of UVC. The medium was changed 20 h after irradiation and then at 3-day intervals. At 12 days after irradiation, the medium was removed, plates were washed with PBS, fixed with methanol and stained with 0.2% methylene blue. Colonies were counted, and the plating efficiency at each UV dose (colonies counted/500 cells plated) was calculated. To determine the proliferation rates during the first 9 days after irradiation, two cultures per cell line were initiated with 2.5X 105 cells per 25 cm2 dish, and one dish per cell line was irradiated with 10 J/m2 UVC 8 h after plating. Cells were passaged at 3-day intervals, replating the same number of cells at each passage. Cell numbers were determined at each passage, and cumulative cell numbers were calculated from these determinations. In a different experiment, cells were plated at a density of 105 cells per 12.5 cm2 well, irradiated with 10 J/m2 UVC 6 h after plating and counted after culturing for 16-96 h after plating.
Assays for cell death and apoptosis For colorimetric quantification of the amount of lactate dehydrogenase (LDH) released into the culture medium, a CytoTox 96 assay kit (Promega, Madison, WI) was used according to the manufacturer's instructions. LDH is a stable cytosolic enzyme which is released upon cell lysis. 104 cells were plated into multiple wells of a 96-well plate and irradiated with the indicated doses of UVC 8 h after plating. At the indicated times after irradiation, plates were centrifuged for 4 min at 250 g, and 50 gl of the supernatant were transferred to a fresh 96-well plate. The amount of LDH in this supematant was measured with a coupled enzymatic assay which results in the conversion of a tetrazolium salt into a red formazan product. The absorbance at 490 nm of this product was measured using an ELISA plate reader. To determine the maximum LDH release (100%), control wells were irradiated in parallel with the experimental wells, and additionally incubated in 1% Triton X-100 for 45 min before measurements. For trypan blue exclusion assays, cells were trypsinized, pooled with the floating fraction of cells of the respective culture dish, centrifuged and resuspended in 500 ,l of trypan blue solution (0.4% in PBS). The percentage of trypan bluepermeable (dead) cells was determined by counting in a haemocytometer. For DNA flow cytometry, 106 cells were harvested by trypsinization 36 h after UV irradiation (40 J/m2) or mock treatment and fixed in 70% ethanol at 4°C for 16 h. Before analysis, cells were washed twice with PBS, incubated with 1 mg/ml RNase A for 20 min at room temperature, and stained with propidium iodide solution (40 ,ug/ml in PBS) for 10 min in the dark. Data were collected and analysed with a Becton Dickinson FACScan and CellQuest software (Becton Dickinson, San
5347
M.Schreiber et aL Jose, CA). Fragmentation of cellular DNA was monitored by agarose gel electrophoresis as described (Fritsche et al., 1993; Huschtscha et al., 1994). In all cases, the floating and adherent fractions of cells of individual tissue culture plates were pooled for analysis.
Gene delivery by transferrinfection Cells were plated at 3XI04 cells/well into 24-well dishes. The cells were exposed 18-24 h later to adenovirus/polylysine/DNA transfection complexes containing the luciferase expression plasmid (pCLuc; Plank et al., 1992) plus either empty plasmid (pUC19); c-fos expression plasmid pH2-c-fos-LTR (Grigoriadis et al., 1993) or Bcl-2 expression plasmid pCMV-Bcl2. pCMV-Bcl-2 was obtained from Michael Buschle and contains the human Bcl-2 cDNA (Seto et al., 1988) cloned downstream from a CMV immediate-early promoter. Transfection complexes were prepared as previously described (Cotten et al., 1992; Wagner et al., 1992) and contained, in 500 gl of HBS (150 mM NaCl, 20 mM HEPES, pH 7.4), 6 jg of DNA (4 ,ug of pCLuc plus 2 jg of pUC19, pH2-c-fos-LTR or pCMV-Bcl-2) complexed with polylysine and 109 particles of psoralen-inactivated adenovirus. Transfection complexes were supplied to the cells (15 jl per well) in 200 gl of 2% horse serum/ DMEM. 2 h later the medium was replaced with 2 ml of 10% FCS/ DMEM. After 24 h, one set of wells was harvested for measuring luciferase gene expression to verify equal levels of gene delivery, and a second set of wells was UV-irradiated and analysed for cell death as described above.
Assays for 13Hlthymidine incorporation and MTS conversion Thymidine incorporation and MTS conversion were measured in parallel to assess the number of cells undergoing DNA synthesis (S-phase cells) and the total number of viable, metabolically active cells, respectively. 104 cells were plated into multiple wells of a 96-well plate (two identical plates each for thymidine and MTS), irradiated with the indicated doses of UVC, pulse-labelled for 1 h immediately before harvesting with either [3H]thymidine (26 Ci/mmol, Amersham, UK; 8 gCi/ml final concentration) or MTS solution (Cell Titer 96 kit, Promega, Madison, WI), and harvested at the indicated times. To determine thymidine incorporation, cells were lysed by freeze-thawing, transferred onto filter paper using a cell harvester, and subjected to scintillation counting. The conversion of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; Owen's reagent] into a coloured formazan by dehydrogenase enzymes found in metabolically active cells was quantified colorimetrically according to the manufacturer's instructions. Briefly, cells were incubated with MTS solution together with an electron-coupling agent (phenazine methosulfate; PMS) for 1 h at 37'C, and absorbance at 490 nm was recorded using an ELISA plate reader. DNA repair assays For host cell reactivation assays (Wang et al., 1995), pSV2-CAT plasmid (Promega, San Diego, CA) was damaged in vitro by exposure to 450 J/m2 UVC. 10 jg of damaged or untreated control plasmid, together with 1 jig luciferase expression plasmid (pRK7), were transiently transfected into each of three independent wild-type and two independent c-fos4- cell lines. The amount of extract used in the CAT assays was normalized to equal luciferase units expressed from the internal standard pRK7. CAT activity was analysed by thin-layer chromatography 12 h after transfection (before repair) and 48 h after transfection (after repair). CAT activity values were quantified with a PhosphorImager instrument (Molecular Dynamics, Krefeld, Germany), and expressed relative to the CAT activity obtained for the undamaged CAT plasmid in each cell line at the specified time points after transfection. Unscheduled DNA synthesis (UDS) of a population of cells in 96-well plates was measured as described by Wang et al. (1995). To eliminate background of replicative (S-phase) DNA synthesis, cells were cultured in 0.5% serum for 30 h, and 10 mM hydroxyurea was added 30 min before irradiation. Cells were then exposed to graded doses of UVC and labelled with [3H]thymidine (26 Ci/mmol, Amersham, UK; 8 jCi/ml final concentration) for 5 h, still in the presence of 10 mM hydroxyurea. Thereafter, cells were lysed by freeze-thawing, and the amount of thymidine incorporation was determined by scintillation counting. Alternatively, UDS assays were performed as described by Vermeulen et al. (1986). Cells were grown on microscope slides and irradiated with 40 J/m2 UVC or mock-treated. Cells were then labelled with [3H]thymidine (26 Ci/mmol, Amersham, UK; 10 gCi/ml final concentration) for 2 h, fixed with Bouin's solution, and exposed with Kodak photographic emulsion for 8 days. After
developing and fixing, cells were stained with haematoxylin and eosin or with Giemsa stain. Labelling due to UDS was readily distinguishable 5348
from S-phase labelling under these conditions since the high-level incorporation of [3H]thymidine as a result of DNA replication resulted in uniformly black nuclei. To determine UDS activity, the number of autoradiographic grains above 100 individual nuclei per cell line was counted, or the overall level of greyness (correlating with the number of grains) of 300 nuclei per cell line was quantified by automatic image processing system colourmorph version 4.2 (Perceptive Instruments, UK). Non-S-phase nuclei of non-irradiated cells resulted in mean grey levels which were not above background.
RNA isolation, Northern blot and Southern blot analysis Total RNA was prepared according to Chomczynski and Sacchi (1987). Northern blot analysis was performed as described previously (Stein et al., 1992). Genomic DNA from cells was isolated by Proteinase K treatment and high-salt extraction, and Southern blot analysis was performed as described (Wang et al., 1992). A 400 bp fragment 5' to exon 1 of the c-fos gene was used as a probe. The PvuII digest used allows distinction to be made between the endogenous and targeted alleles of c-fos based on different restriction fragment lengths, since a PvuII site is present in the neo R gene which was inserted into the targeted allele by homologous recombination (Wang et al., 1992).
Electrophoretic mobility shift assays Whole cell extracts from 3T3 fibroblasts were prepared as described by van Dam et al. (1993). Gel retardation assays (EMSAs) were performed as described previously (Stein et al., 1989). Briefly, 5 jig of whole cell extract was incubated in 5% glycerol; 20 mM HEPES-KOH, pH 7.9; 50 mM KCI; 1 mM EDTA; 1 mM DTT; 1 jig BSA and 1 jig poly(dIdC) with 25 fmol of radioactively labelled probe for 30 min at room temperature, followed by separation of the DNA-protein complexes on 4% Tris-glycine gels (Buratowski et al., 1989). For supershift assays, 2 jil of antiserum were added to the whole cell extract and incubated for 2 h at 4'C before addition of reaction buffer and labelled probe. The levels of DNA-protein complexes were quantified using a Phosphoimager Fujix BAS 100 (Fuji Photo Film Co., Japan). The following doublestranded oligonucleotides were used as probes: 5'-AGTGGTGACTCATCACT-3' and 5'-AGTGATGAGTCACCACA-3' [mouse (m)-Col AP- 1, position -52 to -36; Schorpp et al., 1995], and 5'-AGCTAGCATGAGTCAGACAC-3' and 5'-AGCTGTGTCTGACTCATGCT-3' [human (hu)Col AP-1, position -76 to -61, Angel et al., 1987]. The SP-l consensus oligonucleotide was obtained from Serva Feinbiochemica (Heidelberg, Germany). c-Jun-specific antiserum was provided by Curt Pfarr, c-Fosspecific antiserum was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Transfections and CAT assays SX 105 cells per 75 cm2 tissue culture plate were transfected with 10 jig CAT-reporter plasmid and 1 jig luciferase control plasmid (pRK7) by the CaPO4 co-precipitation method as described (Briisselbach et al., 1995). The promoter elements regulating CAT expression were: a TATAbox only (TATA-CAT, Jonat et al., 1990), a TATA-box preceded by a pentameric synthetic TRE (5 X TRE-TATA-CAT, Jonat et al., 1990) and a fragment of the SV40 early promoter (pSV2, Promega, San Diego, CA). Cell extracts were harvested 12-48 h after transfection and CAT assays were performed as described (Brusselbach et al., 1995). Extracts for CAT assays were normalized to equal amounts of luciferase activity, which was determined according to the protocol of de Wet et al. (1987).
Acknowledgements We are grateful to Meinrad Busslinger, Gerhard Christofori, Peter Herrlich, Hans Rahmsdorf, Laura Stingl and Zhao-Qi Wang for critical reading of the manuscript; Curt Pfarr and Moshe Yaniv for Fos- and Jun-specific antibodies; Michael Buschle for the pCMV-Bcl-2 plasmid; Christina Kelke and Mediyha Saltik for technical assistance; the members of our groups for helpful discussions; Jan Hoeijmakers for advice on UDS assays and Michael Karin for communication of unpublished results. This research was supported in part by the Austrian Industrial Research Promotion Fund and by grants from the Deutsche Forschungsgemeinschaft (An 182/6-2, He 551/8-1) and the European Economic Community Biomedicine and Health Program.
References Abate,C., Patel,L., Rauscher,F.J.,III. and Curran,T. (1990) Science, 249, 1157-1161.
Defective UV response in fibroblasts lacking c-Fos Angel,P. (1995) In Baeuerle,P. (ed.), Inducible Gene Expression and Cytoplasmic/Nuclear Signal Transduction. Birkhauser Verlag, Boston, pp. 62-92. Angel,P., Baumann,I., Stein,B., Delius,H., Rahmsdorf,H.J. and Herrlich,P. (1987) Mol. Cell. Biol., 7, 2256-2266. Brusselbach,S., MBhle-Steinlein,U., Wang,Z.-Q., Schreiber,M., Lucibello,F.C., Muller,R. and Wagner,E.F. (1995) Oncogene, 10, 79-86. Buratowski,S., Hahn,S., Guarente,L. and Sharp,P.A. (1989) Cell, 56, 549-561. Buscher,M., Rahmsdorf,H.J., Litfin,M., Karin,M. and Herrlich,P. (1988) Oncogene, 3, 301-311. Caelles,C., Helmberg,A. and Karin,M. (1994) Nature, 370, 220-223. Cavigelli,M., Dolfi,F., Claret,F.-X. and Karin,M. (1995) EMBO J., in press. Chomczynski,P. and Sacchi,N. (1987) Anal. Biochem., 162, 156-159. Cotten,M., Wagner,E., Zatloukal,K., Phillips,S., Curiel,D. and Birnstiel,M.L. (1992) Proc. Natl Acad. Sci. USA, 89, 6094-6098. Devary,Y., Gottlieb,R.A., Lau,L.F. and Karin,M. (1991) Mol. Cell. Biol., 11, 2804-2811. Devary,Y., Gottlieb,R.A., Smeal,T. and Karin,M. (1992) Cell, 71, 1081-1091. Devary,Y., Rosette,C., DiDonato,j.A. and Karin,M. (1993) Science, 261, 1442-1445. de Wet,W., Bernard,M., Benson-Chanda,V., Chu,M.L., Dickson,L., Weil,D. and Ramirey,F. (1987) J. Biol. Chem., 262, 16032-16036. Engelberg,D., Klein,C., Martinetto,H., Struhl,K. and Karin,M. (1994) Cell, 77, 381-390. Fritsche,M., Haessler,C. and Brandner,G. (1993) Oncogene, 8, 307-318. Gack,S., Vallon,R., Schaper,J., Ruther,U. and Angel,P. (1994) J. BioL Chem., 269, 10363-10369. Grigoriadis,A.E., Schellander,K., Wang,Z.Q. and Wagner,E.F. (1993) J. Cell Biol., 122, 685-701. Herrera,R.E., Shaw,P.E. and Nordheim,A. (1989) Nature, 340, 68-70. Herrlich,P., Ponta,H. and Rahmsdorf,H.J. (1992) Rev. Physiol. Biochem. Pharmacol., 119, 187-223. Hilberg,F., Aguzzi,A., Howells,N. and Wagner,E.F. (1993) Nature, 365, 179-181. Hu,E., Mueller,E., Oliviero,S., Papaioannou,V.E., Johnson,R. and Spiegelman,B.M. (1994) EMBO J., 13, 3094-3103. Huschtscha,L.I., Jeitner,T.M., Andersson,C.E., Bartier,W.A. and Tattersall,M.H. (1994) Exp. Cell Res., 212, 161-164. Jacobsen,K.A., Prasad,V.S., Sidman,C.L. and Osmond,D.G. (1994) Blood, 84, 2784-2794. Jonat,C., Rahmsdorf,H.J., Park,K.-K., Cato,A.C., Gebel,S., Ponta,H. and Herrlich,P. (1990) Cell, 62, 1189-1204. Karin,M. (1994) Curr. Biol., 6, 415-424. Kasid,U., Pfeifer,A., Weichselbaum,R.R., Dritschilo,A. and Mark,G.E. (1987) Science, 237, 1039-1041. Kaufmann,W.K. and Cleaver,J.E. (1981) J. Mol. Biol., 149, 171-187. Korsmeyer,S.J. (1992) Blood, 80, 879-886. Kovary,K. and Bravo,R. (1991) Mol. Cell. Biol., 11, 2451-2459. Kramer,M., Sachsenmaier,C., Herrlich,P. and Rahmsdorf,H.J. (1993) J. Biol. Chem., 268, 6734-6741. Lehmann,A.R. (1982) Cancer Surv., 1, 93-118. Li,L., Chambard,J.-C., Karin,M. and Olson,E.N. (1992) Genes Dev., 6, 676-689. Plank,C., Zatloukal,K., Cotten,M., Mechtler,K. and Wagner,E. (1992) Bioconjugate Chem., 3, 533-539. Radler-Pohl,A., Sachsenmaier,C., Gebel,S., Auer,H.-P., Bruder,J.T., Rapp,U., Angel,P., Rahmsdorf,H.J. and Herrlich,P. (1993) EMBO J., 12, 1005-1012. Rahmsdorf,H.J. (1994) In Angel,P.E. and Herrlich,P.A. (eds), The FOS and JUN Families of Transcription Factors. CRC Press, Boca Raton, FL, pp. 169-189. Ronai,Z.A., Lambert,M.E. and Weinstein,I.B. (1990) Cell Biol. Toxicol., 6, 105-126. Ryseck,R.-P. and Bravo,R. (1991) Oncogene, 6, 533-542. Sachsenmaier,C., Radler-Pohl,A., Zinck,R., Nordheim,A., Herrlich,P. and Rahmsdorf,H.J. (1994) Cell, 78, 963-972. Schorpp,M., Mallick,U., Rahmsdorf,H.J. and Herrlich,P. (1984) Cell, 37, 861-868. Schorpp,M., Mattei,M.G., Herr,I., Gack,S., Schaper,J. and Angel,P. (1995) Biochem. J., 308, 21 1-217. Schule,R., Rangarajan,P., Kliewer,S., Ransone,L.J., Bolado,J., Yang,N., Verma,I.M. and Evans,R.M. (1990) Cell, 62, 1217-1226.
Seto,M., Jager,U., Hockett,R.D., Graninger,W., Bennett,S., Goldman,P. and Korsmeyer,S.J. (1988) EMBO J., 7, 123-131. Shaw,P.E., Schroter,H. and Nordheim,A. (1989) Cell, 56, 563-572. Shimm,D.S., Miller,P.R., Lin,T., Moulinier,P.P. and Hill,A.B. (1992) Radiat. Res., 129, 149-156. Sklar,M.D. (1988) Science, 239, 645-647. Smith,M.L., Chen,I.-T., Zhan,Q., O'Connor,P.M. and Fornace,A.J.,Jr (1995) Oncogene, 10, 1053-1059. Soriano,P., Montgomery,C., Geske,R. and Bradley,A. (1991) Cell, 64, 693-702.
Stein,B., Rahmsdorf,H.J., Steffen,A., Litfin,M. and Herrlich,P. (1989) Mol. Cell. Biol., 9, 5169-5181. Stein,B., Angel,P., van Dam,H., Ponta,H., Herrlich,P., van der Eb,A. and Rahmsdorf,H.J. (1992) Photochem. Photobiol., 55, 409-415. Stein,B., Baldwin,A.S.,Jr, Ballard,D.W., Greene,W.C., Angel,P. and Herrlich,P. (1993) EMBO J., 12, 3879-3891. Treisman,R. (1987) EMBO J., 6, 2711-2717. van Dam,H., Duyndam,M., Rottier,R., Bosch,A., de Vries-Smits,L., Herrlich,P., Zantema,A., Angel,P. and van der Eb,A.J. (1993) EMBO J., 12, 479-487. van Dam,H., Wilhelm,D., Herr,I., Steffen,A., Herrlich,P. and Angel,P. (1995) EMBO J., 14, 1798-1811. Vermeulen,W., Osseweijer,P., de Jonge,A.J.R. and Hoeijmakers,J.H.J. (1986) Mutat. Res., 165, 199-206. Wagner,E., Zatloukal,K., Cotten,M., Kirlappos,H., Mechtler,K., Curiel,D. and Birnstiel,M.L. (1992) Proc. Natl Acad. Sci. USA, 89, 6099-6103. Walker,G.C. (1985) Annu. Rev. Biochem., 54, 425-457. Wang,X.-Q. and Ellem,K.A. (1994) Exp. Cell Res., 212, 176-189. Wang,Z.-Q., Ovitt,C., Grigoriadis,A.E., Mohle-Steinlein,U., Ruither,U. and Wagner,E.F. (1992) Nature, 360, 741-745. Wang,Z.-Q., Auer,B., Stingl,L., Berghammer,H., Haidacher,D., Schweiger,M. and Wagner,E.F. (1995) Genes Dev., 9, 509-520. Yang-Yen,H.-F., Chambard,J.-C., Sun,Y-L., Smeal,T., Schmidt,T.J., Drouin,J. and Karin,M. (1990) Cell, 62, 1205-1215. Received on June 8, 1995; revised on August 14, 1995
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