(2003) 22, 7661–7666 & 2003 Nature Publishing Group All rights reserved 0950-9232/03 $25.00 www.nature.com/onc

Transient expression of E1A and Ras causes downregulation of c-fos gene transcription in nontransformed REF52 cells

Tatiana Usenko1, Alexander Kukushkin1, Tatiana Pospelova1 and Valery Pospelov*,1

1Institute of Cytology, Russian Academy of Sciences, 194064 St-Petersburg, Russia

Stable transformation of rat embryo fibroblast (REF) 2002). Analysing mechanisms of the negative control for cells with E1A and cHa-ras oncogenes leads to down- c-fos gene transcription, we found that although both modulation of c-fos gene transcription. This repression is ERK and p38 kinase cascades were constitutively provided in part by the association of Elk-1 transcription activated in these transformed cells, only the MEK/ factor with deacetylases mediated through effects ERK kinase inhibitor PD98059 was able to suppress of Ras on MAP-kinase cascades. Here, we focus on the serum-stimulated c-fos transcription (Kukushkin et al., primary effects of E1A and Ras displayed in transient 2002). The Elk-1 , a target for MEK/ transfection assay on the transactivating capability of ERK cascade and a key factor involved in the regulation Elk-1, which is a key transcription factor of c-fos gene of c-fos promoter activity, was also permanently regulation. Our data show that E1A is able to suppress phosphorylated in E1A þ cHa-ras cells irrespective of serum- and Ras-induced stimulation of Gal-luc reporter serum starvation or serum stimulation. In spite of the activity by a full-length Gal-Elk1-428 fusion as prior permanent phosphorylation of both ERK kinases well as the expression of c-fos promoter-driven luciferase and the Elk-1 factor, these steps were insufficient to constructs (fos-luc). The repression can be relieved by maintain the c-fos promoter activity at the elevated level trichostatin A, a histone deacetylase (HDAC) inhibitor, in E1A þ cHa-ras transformants. Downregulation of the implying the involvement of HDACs and an inactive c-fos gene in E1A þ cHa-ras cells is supposed to occur chromatin structure formed due to underacetylation of due to maintenance of a refractory state that arises in nucleosomal . Thus, upon transient transfection of normal REF cells after prolonged serum stimulation E1A and Ras oncogenes in REF52 cells or their stable (Zinck et al., 1993). Therefore, we concluded that the expression in E1A þ cHa-ras cells, E1A contributes to low level of c-fos promoter activity in these cells is likely the formation of inactive chromatin structure through to be caused by the formation of an inactive chromatin association with p300/CBP histone acetyltransferases at structure at the promoter-bound nucleosome (Herrera c-fos promoters, whereas Ras mediates its effect through et al., 1997). Consistent with this, sodium butyrate (Na constitutive activation of the MAP/ERK kinase cascade, B), an inhibitor of histone deacetylase activity (HDAC), thereby promoting the recruitment of HDAC1 to the Elk- increased the amount of c-fos transcripts both in serum- 1 transcription factor. As a result, downregulation of c-fos starved and serum-stimulated E1A þ cHa-ras cells gene transcription revealed in established E1A þ Ras (Kukushkin et al., 2002). transformants is unlikely to be a consequence of cell In this work, we have checked whether the negative transformation itself, but follows from primary effects of regulation of c-fos promoter activity observed in E1A and Ras on chromatin remodeling factors. established E1A þ cHa-ras cells can be reproduced in a Oncogene (2003) 22, 7661–7666. doi:10.1038/sj.onc.1206975 transient transfection assay when nontransformed REF52 cells are being transiently transfected by either Keywords: E1A and Ras; c-fos transcription; chromatin; E1A or Ras oncogenes. For this, we have compared the HDAC inhibitors transactivating potential of various Gal-Elk-1 con- structs comprising either N- or C-terminal parts of Elk-1 protein as well as the full-length Elk-1 fused with the DNA-binding domain (DBD) of yeast Gal4 protein Introduction on the Gal-luc reporter expression upon transient cotransfections into REF52 and E1A þ cHa-ras cells. We have recently shown that transformation of primary Further, we examined whether the negative regulation rat embryo fibroblast cells (REF) by E1A and cHa-ras could be reversed by HDAC inhibitors in transient oncogenes leads to downregulation of c-fos gene transfection assay when either the full-length Gal-Elk1- transcription (Pospelova et al., 1999; Kukushkin et al., 428 fusion constructs, together with Gal-luc or fos-luc reporters, were transiently cotransfected along with E1A- and Ras-expressing vectors into REF52 cells. It *Correspondence: V Pospelov, Institute of Cytology, Russian Acad- emy of Sciences, 194064 St-Petersburg, Tikhoretzky ave., 4, Russia; was shown that the Elk1-mediated repression revealed in E-mail: [email protected] these experiments is likely to be provided in part by Received 5 November 2002; revised 3 July 2003; accepted 10 July 2003 association of HDAC activity with the N-terminal and E1A and Ras modulate c-fos chromatin structure T Usenko et al 7662 central part of Elk-1, and formation of inactive To observe the role of the chromatin structure, we chromatin structure. Thus, Ras mediates its effect treated serum-starved E1A þ cHa-ras cells with NaB, an through constitutive activation of MAP/ERK kinase inhibitor of HDAC activity. Figure 1c shows that NaB cascade that promotes the recruitment of HDAC1 to stimulated Gal-luc reporter activity in serum-starved Elk-1 transcription factor (Kukushkin et al., 2002). The E1A þ cHa-ras cells transfected with both Gal-Elk205- E1A provides its own contribution to the downregula- 428 and full-length Gal-Elk1-428 constructs (lane 2). tion of c-fos in E1A þ cHa-ras cells by sequestering The stimulatory effect was more significant in the case of p300/CBP that have intrinsic histone acetyl- Gal-Elk1-428 construct implying that the chromatin transferase (HAT) activity, thereby shifting additionally structure at the promoter of the Gal-luc reporter was a balance of HAT/HDAC activity at various promoter- bound transcription factors.

Results

Firstly, we compared to which extent serum can stimulate the transactivating potential of Gal-Elk-1 constructs comprising different parts of Elk-1 protein transiently transfected into REF52 and E1A þ cHa-ras cells: Gal-Elk1-206, Gal-Elk205-428 and full-length Gal- Elk1-428. The data presented in Figure 1a show that serum stimulated both C-terminal Gal-Elk205-428 and full-length fusion Gal-Elk1-428 constructs, when they were cotransfected into nontransformed REF52 cells, whereas in transformed E1A þ cHa-ras cells only Gal- Elk205-428 construct was found to be stimulated by serum (Figure 1b). This suggests that the effects of negative factors interacting with the central part of Elk-1 protein are more significant in E1A þ cHa-ras transfor- mants than in nontransformed REF52 cells. Control experiments showed that the levels of RNA transcripts of Gal4 gene were practically the same when various Gal-Elk1 fusion constructs were used. This follows from RT–PCR data obtained with primers specific to the DBD-Gal4 region and presented in Figure 1a and b (panels underlying the corresponding bars).

Figure 1 Downmodulation of Elk-1 activity in E1A þ cHa-ras cells is mediated through HDAC-dependent mechanisms. REF52 and E1A þ cHa-ras cells were transiently transfected using a ratio of 1 ml Lipofectamine reagent (Life Technologies, Inc.) per 0.5 mgof plasmid DNA. The transfection mixes contained 1 mg of luciferase reporter Gal4-Luc and 0.1 mg of Gal-Elk1-206, Gal-Elk205-428 or Gal-Elk1-428 constructs. At 24 h after transfection, cells were stimulated by 20% FCS and 6 h after stimulation cell extracts were prepared, and luciferase activity was determined following a protocol supplied with the Dual luciferase kit (Promega). Data are presented as relative fold luciferase activity compared with serum-starved cells. All experiments were performed with duplicate plates of cells for each time point. Each experiment was performed in triplicate. (a) Serum does stimulate the activity of full-length Gal-Elk1-428 in REF52 cells: 1 – serum starvation, 2 – serum stimulation. (b) Serum does not activate Gal-Elk1-428 expression in E1A þ cHa-ras cells: 1 – serum starvation, 2 – serum stimulation. For parts a and b, the bottom panels display the expression of transfected Gal-Elk plasmids after control RT–PCR experiments with the primers to Gal4 gene cDNA. (c) MEK/ERK inhibitor PD98059 suppresses NaB-mediated activation of Gal-Elk1-428 in serum-starved E1A þ cHa-ras cells: 1 – controls, 2 – NaB (10 mm, 24 h), 3 – effect of NaB in the presence of PD98059 (50 mm), 4 – effect of NaB in the presence of p38 inhibitor SB203580 (10 mm)

Oncogene E1A and Ras modulate c-fos chromatin structure T Usenko et al 7663 under-regulated by HDAC/Elk-1 complexes. The level of NaB-induced Gal-luc activity was higher in the case of Gal-Elk1-428, which corroborated our data that serum activated only the Gal-Elk205-428 construct comprising C-terminal Elk-1 in E1A þ cHa-ras cells in contrast to full-length Gal-Elk1-428 (Figure1b and see Yang S-H et al., 2001). We examined the kinase cascades which could be involved in NaB-dependent transactivation of Gal-Elk1- 428 in E1A þ cHa-ras cells. For this, the MEK/ERK inhibitor PD98059 and the p38 kinase inhibitor SB203580 were added 1 h prior to serum stimulation of starved E1A þ cHa-ras cells (Figure 1c, lanes 3 and 4, respectively). The treatment of E1A þ cHa-ras cells with PD98059 caused inhibition of NaB-induced activation of Gal-luc reporter transfected with the C-terminal part of Elk-1 and full-length Elk-1 (lanes 3), whereas the p38 kinase inhibitor did not reveal such a suppressive effect (lanes 4). Therefore, we suggest that preliminarily activated (phosphorylated) ERK kinase and Elk-1 transcription factor can be potentially utilized for the activation of Gal-Elk1-428-mediated Gal-luc transcrip- tion even in serum-starved E1A þ cHa-ras cells, but at first the inactive chromatin structure must be relaxed by inhibitors of HDAC activity. We have further checked whether NaB-mediated activation of full-length Gal-Elk1-428 is realized through an indirect effect of NaB on ERK phosphory- lation and subsequent increase of Elk-1 phosphorylation Figure 2 Western blot analysis of pERKs, pElk-1 and acetylated (Yang J et al., 2001). H3 histones in serum-stimulated and NaB-treated E1A þ cHa-ras The data presented in Figure 2a show that this was cells. REF52 or E1A þ cHa-ras cells were starved in 0.1% FCS for not the case, since NaB did not stimulate ERK 24 h, then were pretreated for 1 h with 50 mm PD98059 or 20 mm SB203580 or with DMSO as solvent (control) and then stimulated phosphorylation in serum-starved and serum-stimulated by 10% FCS for 20 min until cell harvesting. For NaB treatment (a E1A þ cHa-ras cells. In turn, an inhibitory effect of and d), E1A þ cHa-ras and REF52 cells were starved for 18 h, then PD98059 on NaB-stimulated Gal-Elk1-428 activity is treated with 10 mm NaB (Calbiochem) for 6 h and stimulated by evidently mediated through downphosphorylation of 10% FCS for 30 min (a), 45 and 120 min (d) or left unstimulated. Proteins were extracted in RIPA buffer containing 1% NP-40, ERKs (Figure 2b, pERK), followed by dephosphoryla- 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitors in tion of endogenous Elk-1 (Figure 2c, pElk-1). The concentration 10 mg/ml and phosphatase inhibitors 1 mm sodium SB203580 inhibitor for p38 kinase did not suppress vanadate, 5 mm EGTA, 10 mm NaF and processed as described in ERK phosphorylation and even slightly stimulated it Bulavin et al. (1999). After electrophoresis and blotting, the (Figure 2b). The treatment of REF52 and E1A þ cHa- proteins were visualized by antibodies to phosphorylated ERK1 kinase (sc-7383), to nonphosphorylated and Ser383-phosphory- ras cells with NaB led to accumulation of acetylated H3 lated Elk-1 (sc-355 and sc-8406), to acetylated H3 histone (Lys histones under conditions of serum starvation and 9,14) (sc-8655). All antibodies were obtained from Santa Cruz stimulation with serum for 45 and 120 min (Figure 2d). Biotech. (a) NaB does not stimulate phosphorylation of ERK However, in E1A þ cHa-ras cells, this effect of NaB kinase in serum-starved and serum-stimulated E1A þ cHa-ras cells. (b, c) MEK/ERK inhibitor PD98059 suppresses serum-induced action appears less significant, implying an elevated phosphorylation of ERK kinase (b) and Elk-1 factor (c) in REF52 background level of HDAC activity in these cells. The and E1A þ cHa-ras cells. (d) NaB induces accumulation of kinetics of serum-stimulated accumulation of acetylated acetylated H3 histones both in serum-starved REF52 and H3 histones (lanes without NaB) is different in E1A þ cHa-ras cells, and in the cells stimulated by serum for 45 E1A þ cHa-ras and REF52 cells, seemingly reflecting and 120 min. Total H3 histone is visualized on the same membranes in bottom panels. Total histones (60 mg) were loaded onto gel lanes higher levels of HDAC activity in the transformed cells. We have then studied whether E1A and Ras could exert a direct primary effect on Elk1-mediated tran- wanted to observe if cotransfection of E1A12S expres- scription in a transient transfection assay. In other sion vector would abolish the serum-induced stimula- words, we were interested to know whether the down- tion. REF52 cells were transfected with the full-length modulation of c-fos observed in the established fusion construct Gal-Elk1-428 either with the E1A12S E1A þ cHa-ras cells (Kukushkin et al., 2002) could be vector or with an empty CMV vector (control). Cells reproduced upon transient transfection of E1A or Ras were then serum-stimulated for 6 h and harvested to oncogenes into nontransformed REF52 cells. As serum measure luciferase activity (Figure 3a). The results show caused the stimulation of the Gal-luc reporter in REF52 that wild-type (wt)-E1A12S suppressed serum-stimu- cells transfected with Gal-Elk1-428 (Figure 1a), we lated reporter luciferase activity, whereas the E1A12S

Oncogene E1A and Ras modulate c-fos chromatin structure T Usenko et al 7664 N-terminus RG2 point mutant that was unable to bind Then, we checked whether wt-E1A12S is able to p300 coactivator (Wang et al., 1993) did not inhibit Gal- abolish the Ras oncogene-induced stimulation of Gal- Elk-mediated Gal-luc activity. Transfection of E1A12S luc luciferase activity induced by the full-length Gal- YH47point mutant on Rb binding caused an inter- Elk1-428 construct in serum-starved REF52 cells. As mediate effect as compared with wt-E1A and RG2 can be seen from Figure 3a (right, black bars), the Ras mutants (Figure 3a). Control experiments performed to oncogene augmented Gal-luc reporter activity in serum- estimate transcription levels of the used E1A-expressing starved REF52 cells, but cotransfection of wt-E1A12S plasmids by the RT–PCR technique showed that the had a slight but reproducible inhibitory effect on the inhibitory effect of the wt E1A was not due to higher Ras-induced activation of full-length Gal-Elk1-428 at expression of this plasmid (Figure 3a, bottom panels the used ratio of E1A to Ras vectors. under the corresponding bars). E1A was known to target the p300/CBP coactivators exhibiting intrinsic HAT activity. Therefore, we sug- gested that the inhibitory effect of wt-E1A12S in the transient transfection of REF52 cells as well as in established E1A þ cHa-ras cells is likely due to a change of acetylation/deacetylation balance eventually giving rise to accumulation of hypoacetylated nucleosomal histones. As a matter of fact, cotransfection of expres- sion vectors encoding for p300 and CBP coactivator proteins caused stimulatory effect on Gal-Elk1-428- mediated activation of Gal-luc reporter in serum- stimulated E1A þ cHa-ras cells (Figure 3b). It followed from the results with trichostatin A (TSA) that the E1A effect was caused in part by the formation of inactive chromatin structure due to underacetylation of histones. This HDAC inhibitor relieved a suppressive effect imposed in serum-stimulated REF52 cells by wt- E1A12S and Ras oncogenes (Figure 3c). In these experiments, Gal-Elk1-428 was cotransfected with wt- E1A12S alone or with cHa-ras or both plasmids into REF52 cells that were then serum-stimulated. TSA was added in all transfection plates except for the control one. The obtained results show that TSA reversed both wt-E1A12S- and Ras-mediated inhibition of Gal-Elk1- 428 activity in serum-stimulated cells, although to a different extent. The TSA effect was higher in the case of E1A12S transfection and less significant in the case of Ras transfection. The latter fact is, however, very interesting because it demonstrates that Ras alone can downmodulate Elk1-mediated transcription in serum- stimulated cells through a chromatin remodeling mechanism. This corroborates early data indicating that c-fos is downmodulated in cells trans- formed by Ras in the absence of E1A (Mechta et al., 1997; Kessler et al., 1999; Abramova et al., 2003). Figure 3 (a) Effects of wt-E1A12S, its mutants and cHa-ras on Gal-Elk1-428 activity in REF52 cells. Transfection of E1A12S does Interestingly, it has recently been shown that the suppress serum-stimulated transactivation of Gal-Elk1-428 in overexpression of oncogenic Ras can increase both REF52 cells. Inhibition of Gal-Elk1-428 expression by wt-E1A phosphorylation and nuclear localization of HDAC4, depends on the N-terminal region as RG2 mutation on p300/CBP thereby linking the Ras-MAPK signal transduction binding relieves the suppression (open bars). The panel below represents transcripts of transfected E1A12S plasmids, which are pathway to the mechanism for chromatin remodeling revealed by RT–PCR with the primers to E1A cDNA. Transfection (Zhou et al., 2000). of wt-E1A weakens a stimulatory effect of Ras on Gal-Elk1-428 To compare these data obtained on the Gal-Elk-1/ expression (Ras þ wt-E1A, right, filled black bars). (b) Transfection Gal-luc reporter system with natural promoter-driven of p300/CBP expression vectors stimulates the activity of complete luciferase constructs, we used a mouse c-fos promoter Gal-Elk1-428 in serum-stimulated E1A þ cHa-ras cells. (c) HDAC inhibitor (TSA) relieves the repression of Gal-Elk1-428 activity (À503/ þ 101)-luciferase construct in the transient trans- imposed by wt-E1A in REF52 cells. Gal-luc DNA (1 mg) was fection assay (Figure 4). The overall scheme of the cotransfected with either 1 mg of expression plasmids wt-E1A12S experiments was the same as that utilized in the or/and cHa-ras per plate. TSA was added 4 h after transfection at experiments described in Figure 3. The main conclusion the concentrations of 300 nm for 24 h. The fold luciferase activity is presented relative to that in serum-starved (a, b) or to that in made from the analysis of c-fos-driven luciferase serum-stimulated nontransformed REF52 cells (c). Bars represent construct is quite similar to that arising from s.d. values from three independent transfections Figure 3a, that is, wt-E1A12S suppressed the serum-

Oncogene E1A and Ras modulate c-fos chromatin structure T Usenko et al 7665 stimulate fos-luc activity in serum-stimulated E1A þ cHa-ras cells (Figure 4b). Thus, E1A12S onco- protein is able to abolish the stimulatory effect of Ras on the c-fos-luc activity in serum-starved REF52 cells, thereby reproducing a repressive effect of E1A and Ras oncogenes on transcription of the endogenous c-fos gene in the established E1A þ cHa-ras cells (Kukushkin et al., 2002). Correspondingly, TSA abolished the repressive effect of wt-E1A and Ras on fos-luc activity in serum- stimulated REF52 cells (Figure 4c). These data indicate that chromatin-remodeling factors can be considered as one of the primary targets mediating downregulation of cell cycle-controlling genes in E1A þ Ras oncogene- expressing cells.

Discussion

Transformation of REF cells with a combination of complementing oncogenes E1A and cHa-ras leads to a selective downregulation of c-fos gene transcription, although these cells have a very high level of AP-1 DNA-binding activity (Pospelova et al., 1999; Abramova et al., 2003). We showed that ERK and p38 kinases, which were constitutively activated in E1A þ cHa-ras cells, contributed to a permanent phosphorylation of E1k-1 transcription factor. Thus, the transcription machinery is potentially capable of transcribing the c-fos gene even in serum-starved E1A þ cHa-ras cells. However, the transcription cannot start as the chroma- tin structure at the c-fos promoter is noncompetent for transcription due to accumulation of underacetylated histones. Therefore, inhibitors of HDAC activity, NaB and TSA stimulated the transcription of c-fos gene in serum-starved and serum-stimulated E1A þ cHa-ras Figure 4 (a) Effects of wt-E1A12S, its mutants and cHa-Ras on cells (Kukushkin et al., 2002). It was not clear whether fos-luc promoter activity in REF52 cells. Activity of the natural promoter in the context of the c-fos-luc construct is suppressed by downregulation of c-fos gene transcription revealed in wt-E1A12S (lane 2) and relieved by E1A mutant RG2 (lane 3) that E1A þ cHa-ras-transformed cells resulted from the are unable to bind with the p300 coactivator. The control RT–PCR events occurring during cell transformation and sub- analysis of E1A12S transcripts was performed and shown at the sequent clonal selection, or this phenomenon is due to a bottom panel under the bars. The suppressive effect of wt-E1A on fos-promoter activity is dominant over the Ras upon its cotransfec- direct effect of E1A and Ras oncogenes. tion with the Ras oncogene (Ras þ wt-E1A, right black bars). We used transient transfection of nontransformed (b) Exogenous p300 and CBP proteins stimulate fos-luc expression REF52 cells to reproduce effects of E1A and Ras on in serum-stimulated E1A þ cHa-ras cells. (c) TSA abolishes the c-fos promoter activity, which were known to be repression of fos-luc activity imposed by wt-E1A in REF52 cells. primarily regulated through the Elk-1 transcription The control plate was not treated with TSA. The fold luciferase activity is given relative to the control. Bars represent s.d. values factor in fibroblast cells. The experiments showed that from three independent transfections REF52 and E1A þ cHa-ras cells differently supported transactivating potential of full-length Gal-Elk1-428. In particular, serum stimulated Elk205-428 but not com- and Ras-stimulated activity of c-fos promoter (Figure 4a, plete Gal-Elk1-428 constructs in E1A þ cHa-ras cells, lane 2 and black bars). Cotransfection RG2 E1A12S suggesting an association of negative factors with mutant (lack of p300 binding) instead of wt-E1A12S N-terminal and central parts of Elk-1. Since HDAC produced a relatively minor suppression on c-fos-luc inhibitors make possible transactivation by Gal-Elk1- activity (lane 3), while the Rb-binding mutant YH47 428 in serum-starved E1A þ cHa-ras cells, we proposed caused an effect intermediate of wt-E1A and p300- that in the established transformants the inactive binding mutant RG2 (lane 4). All the E1A-expressing chromatin structure could be fixed due to the permanent plasmids used were shown to be expressed at similar expression of E1A and/or Ras oncogenes. levels upon cotransfection with the fos-luc plasmid as To make this point clear, we studied how E1A12S revealed by RT–PCR (bottom panel under the bars). affects serum stimulation of Gal-Elk1-428 in REF52 Consistent with data in Figure 3b, expression of cells. The following results were obtained: (i) E1A exogenous p300 and CBP proteins was shown to suppressed the activation induced by both serum and

Oncogene E1A and Ras modulate c-fos chromatin structure T Usenko et al 7666 oncogenic Ras, (ii) RG2 mutant incapable of binding kin et al., 2002), and also via activation of HDACs with p300/HAT protein coactivator restored the effect (Zhou et al., 2000). In case of E1A þ cHa-ras cells of serum, (iii) HDAC inhibitor TSA relieved the permanently expressing E1A and Ras oncogenes, the suppressive action of E1A. Altogether evidences prove E1A oncoproteins may contribute to fixation of the an important role imposed by the chromatin structure refractory state of target genes (e.g. c-fos) due to on transcription of Elk-1-regulated genes such as c-fos. interference with the function of p300/CBP proteins, The chromatin remodeling is likely to be conducted which have HAT activity. In this manner, E1A can through the association of Elk-1 with HDACs, as shown additionally shift the balance of H3/H4 histone acetyla- for some other transcription factors (Alberts et al., tion towards accumulation of hypoacetylated forms. 1998). Since the transiently transfected E1A is able to bind In normal cells, c-fos is transiently activated in cellular HAT and HDAC activities as well as pRb response to growth factor stimulation, and then protein (see Gallimore and Turnell, 2001) the balance of transcription returns to very low basal levels (Zinck these regulatory proteins at the promoter determines the et al., 1993). Within this time interval, c-fos cannot be final level of target gene expression. As the c-fos gene reactivated by growth factor stimulation (a refractory expression was found to be suppressed in some human state). Our data presented in the previous paper showed tumors (Lee et al., 1998), downregulation of c-fos gene that downregulation of c-fos gene in E1A þ cHa-ras cells transcription upon short-term transfection of E1A and is supposed to occur due to the maintenance of a Ras oncogenes mediated through a chromatin remodel- HDAC-mediated refractory state that arises in normal ing can be a primary event in the process of REF cells after prolonged serum stimulation. In carcinogenesis. E1A þ cHa-ras cells, the refractory state is likely to be constitutive due to MAP kinase activation, Elk-1 phosphorylation and HDAC1 recruitment to c-fos Acknowledgements We thank Andy Sharrocks for Gal-Elk-1 fusion constructs and promoter (Kukushkin et al., 2002). Recent data indicate Alex van der Eb for wt-E1A12S and E1A mutants. We also that Ras expression can also increase HDAC4 phos- thank Dr Svetlana Svetlikova for assistance in cell culture phorylation and cause its nuclear relocalization through work. This work was supported by grants of the Russian activation of the ERK cascade (Zhou et al., 2000). Thus, Foundation for Basic Research (RFBR) 03-04-49377 (VAP), oncogenic Ras can participate in the recruitment of 01-04-49422 (TVP), INTAS Grant 99-1036. The research was HDAC activity to Elk-1 through permanent phosphor- partially supported by the CRDF Award. No. ST-012-0. ylation of Elk-1 factor by MEK/ERK kinases (Kukush-

References

Abramova MV, Kukushkin AN, Svetlikova SB, Pospelova TV Mechta F, Lallemand D, Pfarr CM and Yaniv M. (1997). and Pospelov VA. (2003). Biochem. Biophys. Res. Commun., Oncogene, 14, 837–847. 306, 483–487. Pospelova TV, Medvedev AV, Kukushkin AN, Svetlikova SB, Alberts AS, Geneste O and Treisman R. (1998). Cell, 92, 475–487. van der Eb AJ, Dorsman JC and Pospelov VA. (1999). Gene Bulavin D, Tararova ND, Aksenov ND, Pospelov VA and Expression, 8, 19–32. Pospelova TV. (1999). Oncogene, 18, 5611–5619. Wang H-G, Rikitake Y, Carter MC, Yaciuk P, Abraham SE, Gallimore PH and Turnell AS. (2001). Oncogene, 20, 7824– Zerler B and Moran E. (1993). J. Virol., 76, 476–488. 7835. Yang J, Kawai Y, Hanson RW and Arinze IJ. (2001). J. Biol. Herrera RE, Nordheim A and Stewart AF. (1997). Chromo- Chem., 276, 25742–25752. soma, 106, 284–292. Yang S-H, Vickers E, Brehm A, Kouzarides T and Sharrocks Kessler R, Zacharova-Albinger A, Laursen NB, Kalousek M AD. (2001). Mol. Cell. Biol., 21, 2802–2814. and Klemenz R. (1999). Oncogene, 18, 1733–1744. Zhou X, Richon VM, Wang AM, Yang X-J, Rifkind RA and Kukushkin AN, Abramova MV, Svetlikova SB, Darieva ZA, Marks PA. (2000). Proc. Natl. Acad. Sci. USA, 97, Pospelova TV and Pospelov VA. (2002). Oncogene, 21, 719–730. 14329–14333. Lee HY, Chaudhary J, Walsh GL, Hong WK and Kurie JM. Zinck R, Hipskind RA, Pingoud V and Nordheim A. (1993). (1998). Oncogene, 16, 3039–3046. EMBO J., 12, 2377–2387.

Oncogene