The proto-oncogene c-fos increases the sensitivity of ... - Nature

Oncogene (1997) 14, 1555 ± 1561  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

The proto-oncogene c-fos increases the sensitivity of keratinocytes to apoptosis ValeÂrie Mils1,2, Jacques Piette2, Caroline Barette2, Jean-luc Veyrune2, Anne TesnieÁre1,2, Chantal Escot2, Jean-Jacques Guilhou1 and Nicole Basset-SeÂguin1,2 1

Laboratoire de Dermatologie MoleÂculaire. IURC. 75, rue de la cardonille. 34093 Montpellier Cedex 5. France; 2Institut de GeÂneÂtique MoleÂculaire. UMR5535 1919, route de Mende. BP5051. 34033 Montpellier Cedex. France

In human skin, most studies have suggested a role of cfos or c-fos related genes in keratinocyte dierentiation. The aim of our work was to more directly address this question by transfecting more or less dierentiated keratinocyte cell lines (A431 and HaCaT) with constitutive expression vectors for c-Fos or c-Fos+c-Jun. Our results showed that c-Fos expression decreased keratinocyte growth, yet addition of c-Jun seemed to revert this c-Fos induced growth inhibition. Whereas no obvious dierentiation program was turned on by c-Fos or c-Fos+c-Jun expression in our tissular model, apoptotic ®gures were observed and con®rmed by in situ DNA fragmentation studies. These results do not rule out a role of c-Fos in keratinocyte dierentiation but may indicate that the cell lines we used have reached an irreversible state of transformation so that they no longer respond to dierentiation signals and rather die from apoptosis. These data add further evidence in favor of a role of c-Fos in epidermal homeostasis. Keywords: c-fos; c-jun; keratinocytes; apoptosis; differentiation

Introduction Several studies have shown that normal cell growth and dierentiation depend on the tightly controlled expression of a variety of genes at both transcriptional and post-transcriptional levels. AP-1 factors are involved in the transcriptional regulation of gene expression and are composed of phosphoprotein dimers of the Fos and Jun families. AP1 factors bind to the TPA-responsive element (TRE) in the promoter of target genes (Curran and Franza, 1988; Angel and Karin, 1991). The Jun proteins (c-Jun, JunB, JunD) are able to form homodimers with low DNA binding capacity and transactivating potency. Conversely, the Fos proteins (c-Fos, Fra1, Fra2, FosB) need to be associated with a Jun member to gain DNA binding capacity. However, Fos/Jun heterodimers were shown to have higher transactivating capacity and DNA binding anity than Jun homodimers. Moreover, the AP1 activity is also modulated by phosphorylation events (Smeal et al., 1991; Pulverer et al., 1991; Adler et al., 1992) and by a cell type-speci®c inhibitor, IP1 (Auwerx and Sassone-Corsi, 1992; Briata et al., 1993). Correspondence: N Basset-SeÂguin Received 2 June 1996; revised 17 December 1996; accepted 17 December 1996

The c-Fos protein has been largely studied in recent years. It is a proline-rich phosphoprotein of 55 to 65 kD apparent molecular weight. c-Fos expression was shown to be tissue- and cell type-speci®c. Whereas it was found to be high in some tissues and cell-types like mast cells and monocytes/macrophages of bone marrow (Conscience et al., 1986; MuÈller et al., 1984; Gonda et al., 1984), extra-embryonic membranes (MuÈller et al., 1983, 1984), and skin keratinocytes (Basset-SeÂguin et al., 1990), most other tissues show faint to undetectable levels of c-Fos. In vitro, c-Fos expression depends on external stimuli and c-fos mRNA and protein are rapidly and transiently induced after stimulation of quiescent cells by growth factors (Sassone-Corsi and Verma, 1987). c-Fos expression is considered as a master switch involved in many cellular processes such as proliferation (MuÈller, 1986), dierentiation (MuÈller, 1986), and apoptosis (Colotta et al., 1992; Marti et al., 1994; Smeyne et al., 1993). Recent studies in our laboratory support a role of c-Fos in epidermal dierentiation (Basset-SeÂguin et al., 1990, 1991, 1994). Epidermis is one of the adult tissues with a spontaneous high expression of c-Fos. In cell culture, this spontaneous expression is lost but is still inducible by calcium which promotes dierentiation of keratinocytes. Furthermore, in a skin equivalent system, c-Fos expression is detected when the neo-epidermis has completed its dierentiation. Other recently published data support the hypothesis of a role of c-Fos in keratinocyte dierentiation. They include the increase of c-Fos expression in the granular layer of mouse epidermis just before corni®cation (Fisher et al., 1991), the presence of AP1 sites in the promoter regions of genes coding for various epidermal dierentiation markers (Oshima et al., 1990; Takahashi and Iizuka, 1993; Casatorres et al., 1994; Disepio et al., 1995) and v-fos transgenic animal studies (Greenhalgh et al., 1993). In order to provide direct evidence for a role in cFos in epidermal dierentiation, we investigated whether the introduction of a constitutive c-Fos expression vector in the human keratinocyte cell lines A431 and HaCaT could improve the respectively more or less undierentiated phenotype of the epidermis reconstructed by these cells in a skin equivalent system. Whereas no induction of dierentiation was observed in both types of transfected clones, apoptotic images were observed in c-Fos expressing keratinocyte cell lines. These results suggest a role of c-Fos in keratinocyte apoptosis. Additionally, because c-Fos/cJun heterodimers are more ecient DNA transactivators, we studied the eect of co-transfection of c-Jun.

Role of c-fos in keratinocyte apoptosis V Mils et al

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In clones in which both transgenes were expressed an increased tissue fragility was noticed. However no in¯uence on dierentiation neither major in¯uence on c-Fos induced apoptosis was demonstrated. We propose that c-Fos could be involved in an early and common pathway shared by dierentiation and apoptosis in keratinocytes.

Results Tissue models used A431 and HaCaT cells reconstruct a respectively more or less undierentiated tissue. (Figure 1A and C). Additionally, by opposition with epidermis reconstructed from normal skin biopsies, tissues reconstructed from both cell lines lack spontaneous c-Fos expression by immuno¯uorescence (Figure 1B and D). Establishment of stable transgene expression conditions In preliminary assays, the experimental parameters for transfection of human keratinocyte cell lines were de®ned by co-transfection of a pSVNLSLacZ expression vector and a G418 selection vector. After selection, geneticin-resistant clones were screened for LacZ expression. Positive clones were subcultured and then used for skin reconstruction either in the presence

Figure 1 A431 and HaCaT cell lines reconstructed more or less undierentiated tissues in vitro. Hematoxylin and eosin stained tissue sections (A, C). Reconstructed tissues from HaCaT cells showed an epidermal like organization without complete maturation and stratum corneum formation (A). Reconstructed tissues from A431 cells strati®ed but were tumoral like (abnormal architecture, absence of maturation, cell atypia) (C). Endogenous Fos expression was undetectable in both types of reconstructed tissues (B, D) by immuno¯uorescence. Magni®cation 6200

Figure 2 Transgene expression in transfected A431 cells was heterogeneous and was improved by the maintenance of the selection pressure during reconstruction. A431 cells transfected with pSVNLSLacZ expression vector were used in the reconstruction model. b-galactosidase expression activity was detected in situ by X-gal staining. Tissular reconstruction was performed in the absence (A) (6200) or in the presence of geneticin (500 mg/ml) (B, C) (6200 and 6400 respectively). The percentage of blue stained cells was clearly higher when the selection pressure was maintained during epidermal reconstruction. However, transgene expression was found to vary from cell to cell with some nuclei appearing less positive than others (arrows). This variability was also observed with c-fos transfected clones as shown by immuno¯uorescence (D, E) (magni®cation 6200 and 6400 respectively)


or absence of geneticin. Results showed that, if skin culture was performed in the presence of geneticin, most cells of the tissue expressed the LacZ transgene (Figure 2B and C). By contrast, tissues reconstructed in the absence of geneticin showed numerous cells lacking the transgene expression, (Figure 2A) suggesting that these cells tend to be eliminated and are rapidly replaced by non-expressing cells when cultured under non selective conditions. For that reason, skin reconstructions were performed under selection pressure in further experiments. Additionally, it should be noted that if most cells expressed the transgene after tissue-reconstruction in the presence of geneticin, a certain degree of irregularity in the levels of transgene expression was still observed (Figure 2C arrows, D and E). Characterization of selected clones Eight out of more than 100 clones isolated were selected by Northern blot according to levels of transgene expression. As shown in Figure 3, all clones expressed rather high levels of the c-fos transgene (Figure 3b). Among the ®ve co-transfected clones (Figure 3 lines 5, 6, 7, 9, 10) only two co-expressed c-fos and c-jun transgenes (Figure 3a). Growth properties of these clones are shown in Figure 4A. There is a decrease in growth ability for almost all transfected clones except the two c-Fos and c-Jun co-transfected positive clones for which growth rates were either close or even superior to that of the control (Figure 4A, clone 6). Additionally, cell release in culture supernatant showed a signi®cant increase for all clones (Figure 4B).

(Fos+/Jun+) was similar to that untransfected clones or pSVNeo controls (data not shown). However, reconstructed tissues from the transfectants were characterized by a signi®cative increase in the occurrence of fragmented nuclei that evoked apoptotic ®gures (Figure 5B, C and D), yet absent from pSVNeo A431 cell controls (Figure 5A). Mainly two patterns of c-Fos expressing cells and apoptotic ®gures could be observed in A431 c-Fos-positive clones (Figure 5B, C, F and G). Whereas two clones (Figure 5B and F) showed apoptotic ®gures in suprabasal layers that co-localized with c-Fos expression (Figure 5J), other clones demonstrated a regular distribution of c-Fos expressing cells and apoptotic-like changes in more super®cial layers (Figure 5C, G and K). Distribution of proliferative cells as analysed by Ki67 detection showed an inverse correlation with that of fragmented nuclei (Figure 5N, O and P). Both c-Fos and c-Jun co-transfected A431 positive clones showed a similar morphology with increased tissue fragility, apoptotic-like nuclei in super®cial layers and a rather regular expression of c-fos

Apoptosis is induced in c-fos transfected clones Dierentiation markers analysis (including cytokeratins no 1, 10, 5, 14, ®laggrin and involucrin) of reconstructed tissue with both clones A431 cells (Fos+) and A431 cells

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Figure 3 Transgene expression analysis by Northern blot. Eight out of more than 100 clones isolated were selected according to levels of transgenes expression. (a) c-jun transgene expression. (b) c-fos transgene expression. (c) GAPDH internal control. Lane 1: A431 control clone transfected with pSVNeo alone. Lane 2: A431 Clone 2 transfected with c-fos (pbireFosD). Lanes 3 and 4: A431 clone 7 and clone 10, transfected with c-fos (P19.1). Lanes 5, 6 and 7: A431 clone 36, clone 37 and clone 6 co-transfected with cfos (P19.1) and pSVJun. Lane 8: HaCaT control clone transfected with pSVNeo alone. Lanes 9 and 10: HaCaT clone 47 and clone 49, co-transfected with c-fos (P19.1) and pSVJun

Figure 4 A431 cells stably transfected by constitutive c-fos expression vectors showed reduced cell growth and increased cell shedding. Graph A: Cell growth was followed during 95 h after cell seeding and was evaluated by MTT conversion. Each measure was performed in triplicate and expressed as minimal/maximal/ median value of DO 570 nm. The apparent growth of all c-fos alone transfected clones was clearly lower than that of control. However, the c-fos+c-jun co-transfected clone grew above the control. Graph B: Cell shedding was measured during the same period by cell count. At each point, the cumulated number of released cells was calculated. A direct correlation was observed between increased cell release and decrease cell growth

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Figure 5 Transfection of A431 cells with constitutive c-Fos or C-Fos+c-Jun expression vectors did not improve dierentiation but correlated with the presence of apoptosis. c-fos and c-fos+c-jun transfected clones were used in the tissue reconstruction model. Histological analysis is shown in (A) to (D), detection of DNA fragmentation in (E to H) and immunohistological analysis for c-fos in (I to L) and for Ki67 detection in (M to P). The following transfectants are successively shown: panel (A, E, I, M): PSVNeotransfected A431 cell controls. No apoptosis is observed: (B, F, J, N) c-fos-transfected A431 cells clone 7. Apoptosis is seen essentially in basal layers and correlates with c-fos expression; (C, G, K, O) c-fos transfected A431 cells clone 37. Apoptosis and cfos expression are observed throughout the reconstituted tissue: (D, H, L, P) c-fos+c-jun co-transfected A431 clones. Acantholyticlike changes with intercellular enlargement is observed. c-Fos expression and DNA fragmentation are seen throughout the tissue. Magni®cation 6200

transgene expression throughout the epidermis (Figure 5D, H and L). Only c-Fos positive HaCaT transfected cells were obtained. They also revealed apoptotic ®gures in super®cial layers together with a regular distribution of c-Fos expressing cells (data not shown). Apoptosis was con®rmed by detection of DNA fragmentation in situ Using terminal deoxynucleotidyl labelling in the presence of fragmentation was con®rmed in Patterns correlated mostly with

transferase DNA end dATP aP33, DNA all transfected clones. morphological detec-

tion of apoptotic ®gures by standard microscopy but in situ DNA end labelling appeared as a much more sensitive method. Some dierences in the amounts of grains could be detected between the dierent clones (c-Fos+ or c-Fos+/c-Jun+ clones) but transfection of c-Jun did not seem to signi®cantly modify c-Fos induced apoptosis (Figure 5E to H). Discussion This study was initiated in an attempt to demonstrate a role of c-Fos in keratinocyte dierentiation. While we


could not observe any eect of constitutive c-Fos expression on dierentiation, we could show a positive correlation between c-Fos expression and the presence of apoptotic ®gures in keratinocytes. When transfected clones were cultured as cell monolayers, c-Fos positive clones grew more slowly than control clones and this correlated with an elevated rate of cell shedding. Moreover, the detached cells showed numerous morphological characteristics of apoptotic cells including membrane blebbing, perinuclear condensation of chromatin and nuclear and cytoplasmic fragmentation. Because not all c-Fos expressing cells underwent apoptosis and could be cultured and expended in vitro, we believe that c-Fos may be necessary but not sucient to induce apoptosis in keratinocytes. This could re¯ect either a dose-eect (i.e. level of c-Fos expression within the cell) or the need of another factor or both. In particular, the dierentiation of the cell could be important, since in a reconstituted tissue system, apoptosis is observed mostly in non-dividing cell layers. Co-expression of cJun did not change our observations on tissue dierentiation and apoptosis. However, it is noteworthy that c-Fos+c-Jun positive clones grew more quickly than c-Fos positive clones and/or controls. This could suggest a role of c-Jun in keratinocyte proliferation as previously proposed for ®broblasts (Chen et al., 1994). Alternatively, since c-Jun protein catalyzes proteasome-dependent c-Fos degradation (Tsurumi et al., 1995), its constitutive expression could, in part, attenuate the eects of c-Fos overexpression. In reconstituted epidermis, both c-Fos protein and apoptosis displayed the same localization that varied according to the clone used. In neo-epidermis derived from clones 7 and 10 (Figure 5B to N), c-Fos expression and apoptosis were restricted mostly to basal layers. The lack of c-Fos protein in suprabasal layers could be related to heterogeneity in transgene expression that was previously reported, even in the presence of geneticin (see Results section). However, the regular distribution of Fos expressing versus non expressing cells renders this hypothesis unlikely. By in situ hybridization, we observed the absence of c-fos mRNA in super®cial layers of these clones suggesting that it is either not produced or rapidly degradated (data not shown). The strati®cation process could titer an external factor necessary for the optimal expression of the transgene by creating a gradient within the tissue as classically observed within the normal epidermis. However, other clones behaved dierently with a more regular distribution of apoptosis and cFos expression throughout the epidermis. Therefore, it is likely that the genomic insertion site of the transgene is responsible in these observations. Whatever the distribution of c-Fos exogenous protein, all the neo-epidermis derived from transfected clones showed signi®cant levels of apoptotic ®gures and in situ DNA fragmentation. So, we conclude that the cell death that we observed in both cell monolayer culture and skin equivalents systems corresponds to apoptosis and is most probably due to c-Fos overexpression. Apoptosis is a very important phenomenon that is involved in embyronic development and in the control of adult tissue homeostasis (reviewed in Kerr and

Harmon, 1991). In normal adult epidermis, a rapidly reneweing tissue, the basal rate of apoptosis is low. The importance of apoptosis in skin homeostasis was revealed by recent studies from Haake's group (Haake and Polakowska, 1993; Polakowska and Haake, 1994). However, the role of c-Fos in apoptosis is controversed. Recent in vivo studies performed with the Fos/LacZ transgenic mice strongly suggested a role of c-Fos in developmental apoptosis (Smeyne et al., 1993). In this model, cell death in embryonal organs was preceeded by increased expression of c-Fos. The role of c-Fos in apoptosis of lymphocytes (Colotta et al., 1992) and epithelial cells (Marti et al., 1994) was also previously hypothesized by some authors. Additionally, a recent paper provides evidence in the same direction using a chimeric c-Fos-estrogen receptor fusion protein. In this work c-Fos protein-induced apoptosis of a Syrian hamster embryo cell line and a human colorectal carcinoma cell line occurred via a wild type p53 protein (Preston et al., 1996). Conversely, the role of c-Fos in mammalian u.v. response was recently studied and showed that 3T3 ®broblasts lacking the c-fos gene were hypersensitive to u.v. with increased cell death partly by apoptosis, suggesting that c-Fos was not important in u.v.-induced apoptosis (Schreiber et al., 1995). Our study does not rule out the latter hypothesis, but we think that the models used (mouse ®broblasts/human keratinocytes) are too dierent to critically be compared. Another work using c-fos -/- mice also suggested that this gene was not essential for apoptosis, however its absence could be compensated by other members of the fos/jun families (Gajate et al., 1996). The relation between the high c-Fos expression in adult epidermis and keratinocyte apoptosis has never been explored. By using immortalized keratinocyte cell lines, we were able to show a relation between c-Fos expression and apoptosis. This latter could result from the antagonism between the immortalized status of the cell lines used and the Fos delivered antiproliferative signal (cell death by signal antagonism). In that hypothesis, Fos apoptotic eect is similar to that observed upon transfection of wild type p53 in transformed cell lines (Yonish-Rouach et al., 1991). However, since A431 and HaCaT cell lines are p53 mutated, apoptosis induced by Fos expression is here p53-independent. In this work, we bring further evidence for a role of c-Fos in epidermal homeostasis. We showed that c-Fos protein expression can induce growth inhibition of keratinocyte cell lines and increases keratinocyte's sensitivity to apoptosis. We propose that expression of this gene could be involved in the transition from proliferative to post-mitotic state and thus would be requested for the further proceeding of apoptosis or epidermal dierentiation. Materials and methods Cell lines and culture The HaCaT cell line (passage 29) was kindly provided by Dr Fusenig (Heidelberg). Characteristics of A431 and HaCaT cell types were described elsewhere (Boukamp et al., 1988). These cell lines were characterized by mutations on both alleles of p53 gene. Cells were routinely cultured in

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DMEM (Gibco BRL) supplemented with 10% v/v foetal calf serum (Gibco BRL), 1% penicilline/streptomycin and Fungizone. Cultures were maintained at 378C in a 5% CO2 atmosphere and culture medium was changed every 2 ± 3 days. Vectors The pSVNLSLacZ constructed from pCH110 vector contains the cDNA of b-galactosidase gene associated with a nuclear localisation of signal from SV40. Constitutive expression of the enzymatic activity depends on the SV40 promoter. Two expression vectors encoding for c-Fos were used: P19.1 (generous gift of U RuÈther) where the murine c-fos genomic sequence was cloned downstream to the metallothionein gene promoter (RuÈther et al., 1985) and pbireFosD where the same genomic fragment containing a small deletion (3701 to 3831) was cloned downstream to the rat b-actin gene promoter (Veyrune et al., 1995). Both types of promoters allowed a constitutive expression of transgene in human cells. pSVJun, the expression vector for c-Jun used in cotransfection experiments, contained a cDNA sequence of murine c-jun placed under control of the SV40 promoter (generous gift of R Bravo). The selection vector was the vector pSVNeo that encodes for a resistance to geneticin. Transfection Transfection of A431 and HaCaT cells was performed by lipofectin. Seventy to eighty per cent con¯uent culture plates were rinsed twice with PBS then 1.5 ml of serum-free medium containing 15 mg of DNA (including expression vector and selection vector) and 50 ml of lipofectin reagent (Sigma) was distributed. After a 6 h long incubation period, 3.5 ml of serum-free medium was added to the transfection mixture. The next morning, plates were rinsed three times with PBS then fed with DMEM containing serum and antibiotics (see culture section). After a 48 h period, culture medium was supplemented with 265 mg/ml geneticin base for selection of steadily transfected cells. The selection period needed about 4 weeks. At this time, small clones were seen in the petri-dishes. When colonies were 3 ± 4 mm large, they were independently subcultured in the presence of geneticin. Analysis of transgene expression For b-galactosidase detection, cell monolayers or tissue sections were incubated in the presence of X-Gal (5Bromo-4-chloro-3-indolyl-b-D-galactopyranosidase: Boehringer Mannheim) for 4 ± 24 h at 378C. Positive cells appeared with blue stained nuclei. The expression of c-fos and c-jun transgenes was checked in each individual clone by immunohistological analysis and/ or Northern blot analysis. In order to avoid the endogenous expression of human genes, cultures were serum-deprived for 48 h before RNA and protein analysis. c-Fos and c-Jun proteins were detected by indirect immuno¯uorescence using speci®c antibodies (see immuno¯uorescence section). For Northern blot analysis, total RNA were extracted using RNAzol reagent (Bioprobe systems, France). Puri®ed RNAs were then separated on a 1.5% agarose gel containing 1% formaldehyde. After blotting on nylon membrane (Amersham), RNA were ®xed by a 3 min UVC exposure. Hybridization was performed overnight at 658C in church buer (0.5 M phosphate buer, 7% SDS and 1 mM EDTA) with 32P-labelled probes prepared according to the random priming procedure using the puri®ed murine cDNA inserts from expression vectors as DNA template.

Analysis of cell growth and cell apoptosis For cell growth analysis, 24 well plates were seeded with 46104 cells per well. Cell growth was determined every day by MTT conversion or by cell numeration using a cell counter for one week. During the same time, cell shedding was measured daily by counting the number of cells present in the culture supernatant. In graph B, the number of shedded cells reported for a given day was expressed as the cumulated number of released cells from day 1. For morphological analysis, cells shedded within 24 h were harvested by centrifugation of the culture supernatant and the pellet was included in OCT compound. After cryosectioning, stainings by hematoxylin and propidium iodinate were performed to visualize the nuclear fragmentation.

Skin reconstruction Clones that constitutively expressed one or both transgenes were used in the skin equivalent system described by PrunieÂras et al. (1983). Brie¯y, 36105 cells were seeded on a dead de-epidermized dermis and maintained at the airliquid interface by a metallic support. The culture medium was DMEM/Ham's F12 3 : 1 v/v (Gibco BRL) containing 10% fetal calf serum (Gibco BRL), 1% penicillin/ streptomycin (Gibco BRL), 1% fungizone (Gibco BRL), 0.4 mg/ml hydrocortisone (Sigma), 5 mg/ml insulin (Sigma), 0.1 nM cholera toxin (Sigma), 10 ng/ml EGF (Sigma) and 265 mg/ml geneticin. The culture medium was changed every 2 ± 4 days for 15 days. Samples were then embedded in OCT-compound (Miles, USA), frozen in liquid nitrogen and stored at 7808C until use.

Morphological and immuno¯uorescence studies Morphological examination was performed by hematoxylin/eosin staining on both cell monolayers after formaldehyde/aceton ®xation and on cryosections of cell pellets and skin-equivalents. Transgene expression in both cell monolayers and skin equivalents was performed by immuno¯uorescence analysis using antibodies speci®c for c-fos (rabbit polyclonal antibody, generous gift of C Gauthier RouvieÁre) and c-jun (PC07 AB-2, Oncogene Sciences). Proliferation and dierentiation state of the skin equivalents were determined by immuno¯uorescence analysis using Ki67 monoclonal antibody speci®c for cycling cells (Dako, Copenhagen, Denmark), monoclonal antibodies speci®c for keratins K1, K10, K5, 14, K6 (generous gift of I Leigh) and ®laggrin (Euromedex, Germany) and a polyclonal antibody speci®c for involucrin (Euromedex, Germany). For immuno¯uorescence studies, slides were saturated for 5 min in PBS containing 0.5% bovine serum albumin (BSA, Euromedex, Germany). For detection of c-Fos, c-Jun and Ki67 antigens, slides were ®xed for 5 min in 3.7% formaldehyde in PBS immediately after sectioning, treated for 30 s with cold acetone and washed before saturation. All slides were then incubated for 45 min at 378C in a moist chamber with the ®rst antibody (monoclonal or antiserum) at the appropriate dilution (see antibodies section). After three 5 min washes in PBS 0.5% BSA, slides were incubated for 30 min at 378C with either goat anti-rabbit IgG antibodies (1/ 20 in PBS 0.5% BSA, Sigma) or goat anti-mouse Igs (G,M,A) antibodies coupled to ¯uorescein or biotin (1/50 in PBS 0.5% BSA, Sigma). In the latter case, a 30 min incubation with Texas red-streptavidine (Amersham, Buckinghamshire, England) used at 1/100 dilution was then performed. After three washes in PBS alone, slides were mounted and observed with a Zeiss Axioscop microscope equipped with a camera.


In situ terminal deoxynucleotidyl transferase (TdT) assay DNA fragmentation was detected in situ using the procedure of Groczyca et al. (1993) on cell suspension combined to that of Escot et al. (1991) used for RNA in situ hybridization. Five mm thick cryosections were mounted on poly-L-lysine coated slides ®xed in 4% paraformaldehyde, dehydrated in graded series of alcohol and stored at 7708C until use. To perform the TdT assay, slides were rehydrated 2 min in PBS and prewarmed at 378C. Excess reaction mixture containing 10 mCi of 33P dATP (3000 Ci/mmole, Amersham, France), 16reaction buer pH 6 (56reaction buer is 1 M potassium : cacodylate, 0.125 M Tris-HCl and 1.25 mg/ml BSA), 2.5 mM cobalt chloride, and ®ve units of terminal transferase (Boehringer Mannheim) was applied to each slide. Sections were then covered with silicon treated coverslips and incubated in a humid chamber for 45 min at 378C. Coverslips were removed by dipping the slides in PBS at room temperature. Sections were sequentially rinsed in PBS and dipped 15 min in 1 liter/20 slides of PBS at room temperature, 10 min in 0.16SSC at 658C and

®nally in 0.1 SSC at room temperature. Slides were then dehydrated in graded series of alcohol. The slides were then dipped in K5 emulsion (Ilford) melted at 408C, drained, allowed to set horizontally and exposed at 48C for autoradiography. After exposure, the slides were developed in Kodak D19 or 2 min 30 s at 228C, rinsed in 2% acetic acid and ®xed for 5 min in Kodak L4 ®xer. Finally, the hybridized tissue sections were lightly counterstained with hematoxylin. Abbreviations BSA: Bovine serum albumin; DNA: deoxyribonucleic acid; DMEM: Dulbecco's modi®ed eagle medium; EGF: epidermal growth factor; Ig: immunoglobulin(s); MTT: (3)-(4.5dimethylthiazol-2-yl-)-2.5-diphenyl tetrazolium bromide; PBS: phosphate buered saline; RNA: ribonucleic acid; TGFb: transforming growth factor beta: u.v.: ultraviolet Acknowledgements This work was supported by MRT (ER91), GEFLUC, CHU de Montpellier, FRM grants.

References Adler V, Franklin CC and Kraft AS. (1992). Proc. Natl. Acad. Sci. USA, 89, 5341 ± 5345. Angel P and Karin M. (1991). Biochem. Biophys. Acta, 1072, 129 ± 157. Auwerx J and Sassone-Corsi P. (1992). Oncogene, 7, 2271 ± 2280. Basset-SeÂguin N, Escot C, Blanchard JM, Kerai C, Verrier B, Mion H and Guilhou JJ. (1990). J. Invest. Dermatol., 94, 418 ± 422. Basset-SeÂguin N, Escot C, MoleÁs JP, Blanchard JM, Kerai C and Guilhou JJ. (1991). J. Invest. Dermatol., 97, 672 ± 678. Basset-SeÂguin N, Demoly P, MoleÁs JP, TesnieÁre A, GauthierRouvieÁre C, Richard S, Blanchard JM and Guilhou JJ. (1994). Oncogene, 9, 765 ± 771. Boukamp P, Petrusseevska RT, Breitkreutz D, Hornung J, Markham A and Fusenig NE. (1988). J. Cell Biol., 106, 761 ± 771. Briata P, D'Anna F, Franzi AT and Gherzi R. (1993). Exp. Cell Res., 204, 136 ± 146. Casatorres J, Navarro JM, Blesing M and Jorcano JL. (1994). J. Biol. Chem., 269, 20489 ± 20496. Chen SL, Tsao LT and Tsao YP. (1994). Cancer Lett., 85, 119 ± 123. Colotta F, Polentarutti N, Sironi M and Mantovani A. (1992). J. Biol. Chem., 267, 18278 ± 18283. Conscience JF, Verrier B and Martin G. (1986). EMBO J., 5, 317 ± 323. Curran T and Franza BR. (1988). Cell, 55, 395 ± 397. Disepio D, Jones A, Longley MA, Bundman D, Rothnagel JA and Roo DR. (1995). J. Biol. Chem., 270, 10792 ± 10799. Escot C, Le Roy X, Chalbos D, Joyeux C, Simonsen E, Daures JP, Soussaline F and Rochefort H. (1991). Anal Cell. Pathol., 3, 215 ± 224. Fisher C, Byers MR, Iadarola MJ and Powers EA. (1991). Development, 11, 253 ± 258. Gajate C, Alonso MT, Schimmang T and Mollinedo F. (1996). Biochem. and Biophys. Res. Commun., 218, 267 ± 272. Gonda TJ and Metcalf D. (1984). Nature, 310, 249 ± 251. Gorczyca W, Gong J, Darzynkiewicz Z. (1993). Cancer Res., 53, 1945 ± 1951. Greenhalgh DA, Rothnagel JA, Wang XJ, Quintanilla MI, Orengo CC, Gagne TA, Bundman DS, Longley MA, Fisher C and Ropp DR. (1993). Oncogene, 8, 2145 ± 2157. Haake AR and Polakowska RR. (1993). J. Invest. Dermatol., 101, 107 ± 112.

Kerr JFK and Harmon BV. (1991). Tomei LD and Cope FO (eds.). Apoptosis: The molecular basis of cell death. Current Communications in Cell and Molecular Biology. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Marti A, Jehn B, Costello E, Keon N, Ke G, Martin F and Jaggi R. (1994). Oncogene, 9, 1213 ± 1223. MuÈller R, Slamon DJ, Tremblay JM, Cline MJ and Verma IM. (1983). Nature, 299, 640 ± 644. MuÈller R, MuÈller D and Guilbert L. (1984). EMBO J., 3, 1887 ± 1890. MuÈller R. (1986). Biochim. Biophys. Acta, 823, 207 ± 225. Oshima RG, Abrams L and Kulesh D. (1990). Genes Dev., 4, 835 ± 848. Polakowska RR and Haake AR. (1994). Cell Death and Dier., 1, 19 ± 31. Preston GA, Lyon TT, Yin Y, Lang JE, Solomon G, Annab L, Dayalan GS, Alcorta DA and Barrett JC. (1996). Mol. Cell Biol., 16, 211 ± 218. PrunieÂras M, Regnier M and Woodley D. (1983). J. Invest. Dermatol., 81, 28s ± 33s. Pulverer BJ, Kyriakis JM, Avruch J, Nikolakaki E and Woodgett JR. (1991). Nature, 353, 670 ± 674. RuÈther U, Wagner EF and MuÈller R. (1985). EMBO J., 4, 1775 ± 1781. Sassone-Corsi P and Verma IM. (1987). Nature, 326, 507 ± 510. Schreiber M, Baumann B, Cotten M, Angel P and Wagner EF. (1995). EMBO J., 14, 5338 ± 5349. Smeal T, Binetruy B, Mercola DA, Birrer M and Karin M. (1991). Nature, 354, 494 ± 496. Smeyne RJ, Vendrell M, Hayward M, Baker SJ, Miao GG, Schilling K, Robertson LM, Curran T and Morgan JI. (1993). Nature, 363, 166 ± 169. Takahashi H and Iizuka H. (1993). J. Invest. Dermatol., 100, 10 ± 15. Tsurumi C, Ishida N, Tamura T, Kakizuka A, Nishida A, Okumura E, Kishimoto T, Inagaki M, Okazaki K, Sagata N, Ichihara A and Tanaka K. (1995). Mol. Cell Biol., 15, 5682 ± 5687. Veyrune JL, Carillo S, VieÂ A, Blanchard JM. (1995). Oncogene, 11, 2127 ± 2134. Yonish-Rouach E, Resnitzky D, Lotem J, Sachs L, Kimchi A and Oren M. (1991). Nature, 352, 345 ± 352.

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