Papillomavirus E2 induces p53-independent apoptosis in HeLa cells

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Oncogene (1999) 18, 4538 ± 4545 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc

Papillomavirus E2 induces p53-independent apoptosis in HeLa cells Christian Desaintes1,2, Sylvain Goyat1, Serge Garbay1, Moshe Yaniv1 and FrancËoise Thierry*,1 1

UniteÂ des virus oncogeÁnes, deÂpartement des biotechnologies, URA 1644 du CNRS, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, cedex 15, Paris, France

We have previously shown that expression of the papillomavirus E2 protein in HeLa cells induces p53 accumulation and causes both cell cycle arrest and apoptosis. In contrast to growth arrest, onset of apoptosis was not correlated with an increase of p53 transcriptional activity. In the present study, we conducted biochemical and genetic experiments in order to determine whether E2-induced apoptosis was independent of p53 induction. We showed that E2 did not alter the transcription of Bax, a known p53-activated cell death inducer. The time course of apoptotic cell death preceded p53 induction by several hours. Overexpression of the HPV18 E6 oncogene prevented E2-mediated p53 accumulation, but did not alter the rate of cell death. Finally, point mutants of the HPV18 E2 transactivation domain induced apoptosis, although they were unable to induce high p53 accumulation or cell cycle arrest. In addition, the results obtained with these mutants indicated that both transcriptional activation and replication functions of E2 were dispensable for the induction of cell death. These observations show that E2induced apoptosis is an early event, independent of p53 accumulation and unrelated to downstream p53-dependent transcriptional events. Keywords: HPV18 E2 mutants; apoptosis; p53 HeLa cells

Introduction Papillomaviruses are small DNA viruses whose infection lead to skin or squamous epithelium lesions. A number of viral types that infect the genital epithelium are associated with the development of cervical cancer. These viruses encode two oncogenes E6 and E7 that can transform keratinocytes by interacting with cell cycle regulators. E6 binds to and degrades the p53 regulatory protein, while E7 interacts with members of the pRb family (Dyson et al., 1989; Schener et al., 1990). However, expression of the viral oncogenes is not sucient for carcinogenic progression, which requires additional events, one of them being the integration of the viral DNA into the host cell genome. This event occurs in the great majority of HPV18associated cervical carcinomas and usually only part of the viral genome is integrated in the cellular genome

*Correspondence: F Thierry 2 Current address: Radiobiology Unit, SCK-CEN 200, Boeretang, B-2400 Mol, Belgium Received 14 December 1998; revised 11 March 1999; accepted 11 March 1999

(Berumen et al., 1994). The mRNAs coding for the E6 and E7 oncogenes are actively transcribed from the integrated viral DNA under the control of the viral regulatory region (Schwarz et al., 1985). In contrast, the portion of the viral genome expressing the E1 and E2 regulatory proteins is usually disrupted. E2 regulates both viral transcription and DNA replication when bound to palindromic DNA sequences present in four copies in the regulatory region of human genital papillomaviruses. The E2 protein contains two functional domains. The amino-terminal domain, of about 200 amino acids, is required for activation of both transcription and replication. The carboxy-terminal domain, of about 80 aa, binds DNA speci®cally. It contains a dimerization interface which is composed of a b-barrel structure formed of b-sheets, while the interaction with DNA occurs through a conserved a-helix for each subunit (Hegde et al., 1992). In contrast, less is known about the structure of the amino-terminal transactivation domain (E2TD). Secondary structures predictions indicate that it might be constituted of two subdomains, the most N-terminal one containing two or three large acidic a-helices, while the domain closest to the hinge may contain a series of small b-sheets. Systematic mutational analysis of the E2TD domain by several groups has not allowed to unambiguously discriminate its functions in transcription and replication (reviewed in McBride and Myers, 1996). Although E2 proteins have initially been considered as potent transcriptional transactivators, they also act as transcriptional repressors of the E6 and E7 transforming genes in the context of the human genital papillomaviruses. This repression is caused by the binding of E2 to the HPV18 early promoter which consequently hinders the binding of two essential transcription factors, TBP and Sp1 (Demeret et al., 1994; Dong et al., 1994; Dostatni et al., 1991; Tan et al., 1992; Thierry and Yaniv, 1987). Repression mainly involves the DNA binding domain of E2, although its transactivation domain may play a role by modulating the stability of the E2/DNA complexes (Demeret et al., 1997). As mentioned above, E2 is not expressed in cervical carcinomas because the E2 gene has been disrupted due to the integration of the viral DNA into the host genome. Consequently, in cervical carcinomas, the transcription of the viral E6 and E7 oncogenes is derepressed. Furthermore, disruption of both E1 and E2 does not allow viral DNA replication to take place. We have challenged this model by expressing ectopic E2 in the HPV18-associated cervical carcinoma HeLa cell line (Desaintes et al., 1997). We have found that expression of E2 induces the accumulation of wild-type p53 partly by inhibiting the synthesis of E6, the inducer of its degradation and partly by an unknown

Papillomavirus E2-mediated apoptosis C Desaintes et al

mechanism. This results in G1 cell cycle arrest. In addition, a fraction of the cells expressing E2 undergo apoptosis. Overexpression of a p53 trans-dominant negative protein abolishes the E2-mediated p53 transcriptional activity and growth arrest but does not abolish the induction of cell death. This intriguing observation led us to postulate that, while the accumulation of p53 in E2 expressing cells was directly linked to the cell cycle arrest, induction of apoptosis may follow an alternative pathway. In an attempt to test this hypothesis, we used both biochemical and genetic approaches to con®rm that apoptosis induced by both BPV1 and HPV18 E2 proteins in HeLa cells is independent of p53. First we show that E2-mediated apoptosis preceded the onset of p53 induction by several hours. Furthermore, even though E2 expression resulted in the accumulation of transcriptionally active p53, Bax transcription was not activated, and overexpression of E6 did not block apoptosis. We also generated HPV18 E2 mutants that could not induce p53 accumulation, but eciently induced cell death, con®rming that these two functions of E2 are not linked.

p53-induced apoptotic promoting genes (Miyashita and Reed, 1995) in comparison with transcriptional activation of p21WAF1/CIP1. Full-length E2 proteins from BPV1 and HPV18 induced a massive accumulation of p53 protein in HeLa cells (Figure 1a and Desaintes et al., 1997) without altering the levels of p53 mRNA (Figure 1b). In contrast, a strong transcriptional activation of the p21WAF1/CIP1 gene was observed in cells transfected with the full-length E2, while in cells transfected with the transcriptionally inactive E2TR, p21WAF1/CIP1 mRNA accumulation was strongly attenuated (Figure 1b). The activation of p21WAF1/CIP1 transcription is probably responsible for the G1 growth arrest as previously demonstrated. In contrast, no change of Bax mRNA levels was observed in E2-expressing cells (Figure 1b). Altogether, these results show that transcriptional activation of the Bax gene is not required for E2induced apoptosis, supporting the hypothesis that E2induced apoptosis is independent of p53-responsive genes.

Results

In order to clarify further the potential role of p53 in both E2-mediated G1 growth arrest and apoptosis, we measured the levels of E2 and p53 transcriptional activities, cell death, and cell cycle arrest. Apoptosis occurred early, between 20 and 24 h after transfection (Figure 2). At this time, the transcriptional activity of E2 was 100-fold above that of non-transfected cells, while that of p53 was very low. After 24 h, p53 started to accumulate, while the fraction of apoptotic cells declined. FACS

E2-induced apoptosis is independent of Bax To further characterize the role of p53 transcriptional activation in both G1 growth arrest and apoptosis of E2-transfected HeLa cells, we examined whether two p53-inducible genes involved in these two phenomena were concomitantly induced. We tested the transcriptional activation of Bax, one of the best documented

Time course of E2-induced apoptosis precedes p53 accumulation

Figure 1 E2 activates transcription of p21WAF1/CIP1 but not Bax. (a) Western blot analysis showing the concomitant expression of E2 and p53 in HeLa cells transfected with 2 mg of wild-type BPV1 E2 expression plasmid. (b) Northern blot analysis of p53, p21WAF1/CIP1 and Bax RNA from HeLa cells transfected with BPV1 E2 or BPV1 E2TR. As negative control, cells were transfected with pCGE1TTL plasmid (7)

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analysis of surviving cells 48 h after transfection revealed that they were mostly arrested at G0/G1 (data not shown). To clarify further whether cells were still undergoing apoptosis after 24 h, or whether the dead cells observed at 40 h originated from cells which had undergone apoptosis before 24 h, we video-recorded HeLa cells cotransfected with E2 and GFP expression plasmids. Following change of the medium 14 h after transfection, cells

Figure 2 E2-induced cell death occurs before the onset of p53 transcriptional activity. One mg of HPV18 E2 (~) or HPV18 E2DBD (&) expression plasmids were cotransfected with 4 mg of the GFP expression plasmid (upper graph) or with 2 mg of the MDM2-CAT (middle graph) or TKE2-CAT (lower graph) reporter plasmids. At various times after transfection, cells were examined for cell death by counting dead ¯uorescent transfected cells (upper graph) and for CAT activities (middle and lower graphs)

were transferred to a thermostated CO2 chamber under a phase contrast microscope and were videorecorded for an additional 48 h. Video images given in Figure 3 show that E2-induced cell death occurred early after the onset of the recording between 20 and 24 h after transfection, depending on the experiment. No increase in the number of cells undergoing apoptosis was observed at later time points. Apoptosis was observed exclusively in transfected cells which were identi®ed by the green ¯uorescence signal (Figure 3b). In addition, we observed numerous cells with mitotic morphology in non-transfected cells (shown by a white arrow in Figure 3a), indicating that cells were not suering from the culture conditions during recording. Identical experiments performed with non-transfected cells or cells transfected with control plasmids did not show such a burst of cell death (not shown). In contrast, HeLa cells treated with cisplatin showed a dierent kinetics of cell death. Apoptosis occurred gradually to reach 100% of the cells around 70 h after cisplatin treatment (data not shown). In contrast, E2induced apoptosis occurred in a short interval of 2 ± 3 h (corresponding to high E2 transactivation) in only about 20% of transfected cells. However, the apoptotic process per se appeared indistinguishable in both video-recordings. It was a very abrupt event with almost no visible precursor state. Apoptotic cells became rounded very rapidly, then collapsed within a 4 ± 10 min time lapse.

Figure 3 Cell death of E2-transfected HeLa cells occurs shortly after transfection. (a) Images from the phase contrast videomicroscopy recording of HeLa cells transfected with 2 mg of BPV1 E2 expression plasmid and 4 mg of GFP expression plasmid. Black arrows show dead cells. White arrows show a mitotic cell. (b) Corresponding images taken from the ¯uorescent objective


Overexpression of E6 does not alter E2-induced apoptosis We have previously shown that inhibition of p53 transcriptional activity by a trans-dominant negative mutant had no eect on the level of E2-mediated apoptosis (Desaintes et al., 1997). In order to exclude that transcriptionally inactive p53 might still mediate E2-apoptosis, we decided to eliminate p53 by overexpressing the HPV18 E6 oncoprotein which triggers its degradation through the proteasome complex (Scheffner et al., 1990). We found that cotransfection of HeLa cells with an E2 expression plasmid and increasing doses of a plasmid expressing HPV18 E6 directed by a CMV promoter drastically reduced p53 transcriptional activity, presumably due to its degradation. However, the decrease in p53 did not correlate with a decreased

Figure 4 Eect of E6 expression on p53 transcriptional activation and apoptosis. Increasing amounts of HPV18 E6 expression plasmid (from 1 ± 4 mg) were cotransfected in HeLa cells with 1 mg of BPV1 E2 expression plasmid and with either 2 mg of MDM2-CAT reporter plasmid (black bars are the relative CAT activities) or 4 mg of GFP expression plasmid (hatched bars are the % cell death among transfected cells)

rate of cell death, which remained unchanged in the presence of excess E6 (Figure 4). These experiments indicated that E2-induced apoptosis was independent of the accumulation of the p53 protein. Mutations in the transactivation domain of E2 can discriminate between activation of p53 and cell death If the E2-induced apoptosis and the E2-mediated accumulation of p53 are separate functions, one should be able to discriminate between the two by mutagenesis. We have previously shown that both processes require the transactivation domain present in the N-terminal part of E2 (Desaintes et al., 1997). In addition, this domain functions as an activator of transcription and of viral DNA replication, therefore allowing us to check whether these functions are required for the induction of p53 and/or apoptosis in HeLa cells. To construct HPV18 E2 point mutants, we took advantage of mutational analyses performed on other papillomavirus types. Very few positions can discriminate between transcriptional activation and stimulation of viral DNA replication, (reviewed by McBride and Myers, 1996). We chose to modify two positions of the transactivation domain previously shown to discriminate between transcription and replication in the analogous HPV16 E2 protein (Sakai et al., 1996). We substituted the conserved glutamic acid residue at position 43 (E43A) or the isoleucine at position 77 (I77A) for an alanine residue. These mutated proteins expressed from the strong cytomegalovirus early promoter were nuclear and as stable as the wild-type protein, as judged by immuno¯uorescence and gel shift assays (Figure 5). Both mutants were devoid of any transcriptional activity, since they were unable to induce a CAT reporter plasmid containing six E2 binding sites upstream of the thymidine kinase promoter (Figure 5c and Table 1). This was a surprise because the E43A equivalent mutants in HPV16 or HPV11 E2 (E39A) retain a wild-type transcriptional activity. In transient replication assays, mutant E43A was unable to replicate, while I77A retained between 30 and 60% of the wild-type activity (Figure 6 and

Figure 5 Expression and transactivation of HPV18 E2 mutants. (a) Shows immuno¯uorescence of transfected HeLa cells with 2 mg of expression plasmids for the wild-type HPV18 E2 (a and d), mutant I77A (b and e) and mutant E43A (c and f). Panels a ± c are DAPI staining and d ± f speci®c detection of E2 with anti-E2 antibody. (b) Is a gel shift assay with whole cell extracts of HeLa cells transfected with 2 mg of wild-type E2 (lane 1), mutant E43A (lane 2), mutant I77A (lane 3) and E1TTL (lane 4). (c) Is a cotransfection of E2 expression plasmids with the TKE2-CAT reporter plasmid. Wild-type E2 at 1 mg and 2 mg respectively (lanes 1 and 2), mutant E43A at 1 mg and 2 mg (lanes 3 and 4), mutant I77A at 1 mg and 2 mg (lanes 5 and 6 ) and 1 mg of E2DBD (lane 7)

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Table 1 Mutants of E2 induce apoptosis as eciently as the wildtype protein in HeLa cells Transcription DNA activation replication E2 wt E43A I77A E2DBD

100 0 0 0

100 0 46 (9) 0

MDM2Relative CAT increase of % activation G1 (%) apoptosis 80 30 16 9

(8) (16) (13) (5)

22 (5) 5 (5) 2 (8) 1

14 15 15 8

(2) (4) (2) (2)

Transactivation assays are described in the legend of Figure 5. DNA replication assays were done as described in Figure 6. The amount of DpnI resistant DNA from cells transfected with the mutant E2 expression plasmids were normalized to that of wild-type E2. Activation of MDM2-CAT reporter plasmid by 1 mg of E2 expression vectors was calculated compared to an E1TTL negative control. Cell cycle analyses were performed according to Desaintes et al. (1997) with cells cotransfected with 4 mg of E2 and 2 mg of H2Kd expression vectors and subsequent FACS analyses of the H2Kd positive cells. Increase in %G1 was calculated compared to nontransfected HeLa cells. For apoptosis, cells were cotransfected with 2 mg of E2 expression vectors and 4 mg of GFP expression plasmid. Green ¯uorescent dead cells were counted in four independent transfection experiments. Cell-death of E2 expressing cells was compared to that of HeLa cells transfected with the GFP expression vector alone which gave background apoptotic levels comparable to E2DBD as previously reported (Desaintes et al., 1997). Standard deviations given in parenthesis were calculated from at least three independent experiments

monitor E2-induced accumulation of transcriptionally active p53 in our system. At relatively high amounts of mutant E2 expressing plasmids, activation of MDM2 was between three- and ®vefold lower than with the wild-type protein, although activities of both mutants were higher than that of the E2 protein lacking the transactivation domain (E2DBD) (Table 1). However, this reduced p53 activation was correlated with a decrease in cell-cycle arrest even when mutant proteins were expressed at high concentrations (Table 1). We deduced that both mutants exhibited comparable defective phenotypes with regard to p53 activation and growth arrest, although they could be clearly discriminated for their functions in activation of transcription and replication. In contrast, both mutants induced cell death in around 15% of transfected cells, which was comparable to apoptosis mediated by the wild-type protein (Table 1). In control experiments, overexpression of the truncated E2 protein induced a level of cell death of 5 ± 8% which was also observed with a null control plasmid (data not shown and Desaintes et al., 1997). In conclusion, mutants of the E2TD that do not eciently activate p53, and consequently do not arrest the cell cycle, are still able to induce cell death, showing that the two functions are genetically separable. Discussion

Figure 6 DNA replication assays of HPV18 E2 mutants. Small DNA was extracted from cells cotransfected with the HPV18 ori plasmid, E1 expression plasmid and either none, wild-type or mutant E2 expression plasmids as indicated. DpnI-resistant replicated DNA were resolved on an agarose gel. Quanti®cation was calculated with a phosphorimager relative to the wild-type set at 100%

Table 1). This mutant therefore discriminates between the two activating functions of E2. The same two mutants were assayed for transcriptional activation of a MDM2-CAT promoter in cotransfected HeLa cells. This assay has been used to

We have deciphered, at least partially, the mechanisms by which E2 activates p53 in transfected HeLa cells. This induction results partly from transcriptional repression of the endogenous E6 transcription by E2. Repression of E6, that is an inducer of p53 degradation by the ubiquitin pathway, favors accumulation of a transcriptionally active p53 (Hwang et al., 1993, 1996; Desaintes et al., 1997; Dowhanick et al., 1995). E2mediated transcriptional repression of E6 requires binding of the viral protein to speci®c recognition sites in the HPV18 early promoter contained within the viral DNA integrated in the HeLa cell genome. However, we and others have found that, in addition to an intact DNA binding domain, the transactivation domain was required for ecient induction of p53, since N-terminally truncated E2 proteins were almost inactive. The role of the transactivation domain was con®rmed here with transcriptionally inactive point mutants of E2 that did not activate p53 as eciently as the wild-type protein, although they bind DNA with anities similar to the wt protein in vitro. It has been suggested that the E2 transactivation domain is required for ecient p53 accumulation because it augments transcriptional repression of E6 (Goodwin et al., 1998). Similarly, we found that the transactivation domain in¯uences the stability of the E2/DNA complexes in vitro (Demeret et al., 1997). Since induction of p53 does not occur at the transcriptional level, the need for E2 transcriptional activity is obviously indirect. Interestingly, while E2 mutants were totally inactive for transcriptional activation, they could still stimulate p53 to a certain extent, this stimulation being greater than with the N-terminally truncated protein. One possible explanation could be that point mutants of the transactivation domain repress endogenous E6 transcription more eciently


than truncated proteins, thereby inducing a better stabilization of p53. However, these observations argue against a complete correlation between transcriptional transactivation and activation of p53. In addition, since the two mutants used in this study could be distinguished by their ability to stimulate viral DNA replication although they were very similar in their ability to induce p53, we deduce that the DNA replicating function is not required for p53 induction. We have previously shown that the BPV1 E2 protein mutated in its DNA binding domain, but containing an intact transactivation domain, could induce apoptosis (Desaintes et al., 1997). We con®rmed this observation with an equivalent mutant of the HPV18 E2 protein (data not shown). Therefore, the amino-terminal domain of E2 appears as the main inducer of apoptosis, although we have demonstrated here that apoptosis was not aected by mutations inactivating its transcription or replication activity. In addition, apoptosis does not seem to correlate with p53 accumulation. The mechanism of cell death induction by E2 is probably mediated by novel function(s) of E2. Few molecular partners, thought to play a role in E2 transcriptional activation, have been described (Rank and Lambert, 1995; Steger et al., 1995; Benson et al., 1997; Breiding et al., 1997; Yao et al., 1998). These include TFIID, TFIIB and an additional cellular protein of unknown function. However, E2TD is a large domain containing more than 200 aa that also has the capacity to bind to the E1 viral replication protein (Benson and Howley, 1995) and the RPA cellular replication protein (Li and Botchan, 1993). It might interact with additional cellular or viral proteins. One of these putative partners might be either an inducer or a repressor of apoptosis whose identi®cation would require further studies. Our video-microscopy recording experiments have been extremely informative in several aspects. Induction of cell death by E2 was clearly established as an early event that coincides with the presence of elevated E2 activity in transfected cells. This result indicates that E2 might promote cell death through direct interaction of its N-terminal domain with a cell death control protein, rather than by activating downstream eector genes. Moreover, when recordings of cisplatintreated and E2-transfected HeLa cells were compared, cell death appeared faster in the E2-transfected cells than in cisplatin-treated cells. Cisplatin treatment of cells induces DNA damage followed by nuclear p53 accumulation, which in turn may activate apoptosis (Fritsche et al., 1993). Interestingly, cisplatin-induced cell death is considered as a classical p53-dependent apoptotic pathway and our experiments indicate that the process of cell death per se was similar by videomicroscopy to that observed in the presence of E2. These experiments suggest that, although E2 and cisplatin-induced apoptosis are phenotypically similar, the E2-induced process may require fewer downstream steps. p53-dependent apoptosis is a well-documented process that occurs in many cell types in response to a variety of cellular stresses (reviewed in Levine, 1997). It is clear that p53 can activate apoptosis or arrest cell cycle as alternative responses to stress. In both cases the resulting eect is to eliminate cells that would otherwise accumulate genetic damages. Although both

cell-cycle arrest and apoptosis can be induced by p53, it is unclear whether the two phenomena are linked, even though they can occur in the same cell type. The underlying mechanism of p53-dependent growth arrest is due to the transcriptional activation of the p21WAF1/ CIP1 cyclin dependent kinase inhibitor (El-Deiry et al., 1993; Harper et al., 1995; Xiong et al., 1993). The role of p53 in the induction of apoptosis appears more complex although it can activate transcription of proapoptotic genes such as Bax (Miyashita and Reed, 1995). Apparently, p53-dependent growth arrest and apoptosis rarely occur in the same cell. Rather it seems that these two pathways are mutually exclusive. It has been suggested that induction of p21WAF1/CIP1 could protect cells against apoptosis. In addition, it has been proposed that transcriptionally dependent and independent functions of p53 contribute to cell death, the importance of each of these functions depending on the cell type. Numerous examples of apoptosis induced by transcriptionally inactive p53 have been reported (Allday et al., 1995; Bissonnette et al., 1997; Delia et al., 1997; Haupt et al., 1995; Polyak et al., 1996; Rowan et al., 1996). On the other hand, there are also many examples of p53-independent apoptosis, although the underlying mechanisms are ignored. Overexpression of E2F has been shown to activate apoptosis in a p53-independent manner (DeGregori et al., 1997; Field et al., 1996; Fueyo et al., 1998; Hsieh et al., 1997; Phillips et al., 1997). The mechanism of this induction seems to involve relief of repression of still unknown proapoptotic genes by the E2F/Rb complex, rather than direct activation of transcription (Hsieh et al., 1997). The ras oncogene also induces p53-independent apoptosis that could be suppressed by NF-kB (Mayo et al., 1997). An example particularly relevant to our model, is the p53-independent apoptosis induced by the adenovirus early region E4orf4 protein. Adenovirus carries both anti-apoptotic and pro-apoptotic functions that are used at dierent moments of the viral cycle. For instance, cell killing by E4orf4 seems to be a late event occurring at the ®nal stages of infection that may represent the ultimate death of infected cells and spread of the progeny (Marcellus et al., 1998). During control of the papillomavirus growth cycle, a similar mechanism might be used. Late in infection and independently of expression of the viral oncogenes, E2 could play a prominent role in the ®nal death of infected cells and spread of the virus. In contrast, it might modulate cell proliferation during the initial stages of infection. Materials and methods Plasmids MDM2-CAT containing the p53-responsive mdm2 promoter was kindly provided by Moshe Oren. The H2Kd expression plasmid was previously described (Desaintes et al., 1997). The following HPV18 E2 expression vectors pCGE218 and pCGE2DBD or BPV1 E2 expression vectors, pCGE2B and pCGE2TR have been described previously (Demeret et al., 1997). pCGE1.B TTL (Le Moal et al., 1994), or pGGE1.18 TTL (Desaintes et al., 1997) were used as negative controls. The point mutations were introduced into HPV18 E2 sequence by a PCR-directed mutagenesis method. The primers were designed to change the target codon to GCA

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or GCT at both E43 and I77 positions. In addition, silent mutations were introduced at position 46 disrupting a SspI site and at position 84 disrupting a StuI site. The HPV18 E6 expression plasmid which expresses the E6 protein under the control of the cytomegalovirus promoter is a kind gift of M Schener. CMVgfp expresses the green ¯uorescent protein from the cytomegalovirus promoter. The E2-responsive plasmid TKE2-CAT contains six E2 binding sites in front of the minimal TK promoter (Thierry et al., 1990). For replication experiments an E1 expression plasmid was constructed from the originally described E1/E2 expression plasmid EapCG (Demeret et al., 1995) by inserting a TTL (Translation Termination Linker) in the restriction sites SexA1 in the E2 ORF, and Blp1 in the E7 ORF. The origin-containing plasmid, poril8, was described in (Demeret et al., 1995). Cell cultures and transient transfections HeLa and 293 cells were maintained in Dulbecco's Modi®ed Eagle's Medium supplemented with 7% fetal calf serum, penicillin (500 IU/ml) and streptomycin (125 mg/ml). Twentyfour hours before transfection, 26105 or 56105 cells were plated in 6 cm dishes. Transfections were performed by the calcium phosphate co-precipitation method as described previously (Desaintes et al., 1997). DNA was prepared by the Qiagen procedure and adjusted to 6 mg per transfection with Blue Scribe plasmid. CAT assays were performed with 1/10 to 1/2 of total cell extract as described earlier (Thierry and Yaniv, 1987). Cell death assay and video-microscopy recording HeLa cells were cotransfected with various amounts of E2 expression plasmids or the E1TTL negative control and 4 mg of the GFP expression plasmid. After change of medium 18 h post-transfection, cells were observed under ¯uorescent microscope. Dead cells were counted among successfully transfected green ¯uorescent cells, various times after transfection. Cell cycle analyses were done by ¯ow cytometry as described in (Desaintes et al., 1997). Cisplatin treatment was done according to (Donaldson et al., 1994). Brie¯y, 5 mg of cisplatin per ml of medium were added to cell cultures. To increase the eect of cisplatin, cells were arrested in G1 by adding 2.5 mM of thymidine 18 h before addition of cisplatin. For video-recording, cells were transferred to a thermostatic chamber under the microscope, immediately after change of medium following transfection or drug treatment. Cells were recorded for 48 ± 72 h with images taken every 5 min. Immuno¯uorescence HeLa cells grown on coverslips were rinsed with PBS 40 h after transfection with E2 expression plasmids, then ®xed in 4% paraformaldehyde. After rehydration cells were permeabilized with 0.1% triton and incubated with a rabbit polyclonal antibody against HPV18 E2 followed by an antirabbit antibody coupled to FITC and DAPI 0.15 mg/ml.

DNA binding assays A double stranded oligonucleotide, end-labeled by Klenow ®ll-in with 32P-dATP, was used as probe in gel shifts. It contains the core sequence of the E2 binding site #2 of the HPV18 LCR 5'-AATTGTAGTATATAAAAAAGTTAGTGACCGAAAACGGTCGGG (the conserved palindrome recognized by E2 is underlined). Binding reactions were carried out in a ®nal volume of 20 ml in a buer containing 12 mM HEPES pH 7.9, 10% glycerol, 0.5 mM EDTA, 4 mM MgCl2, 60 mM KCl, 8 mM DTT. Reactions containing 4 ± 5 mg of proteins of total cell extracts were pre-incubated 15 min at 48C in the presence of 1 mg of PolydIdC and 1 mg of salmon sperm DNA. After addition of the speci®c labeled probe, they were further incubated 5 min at 48C then loaded on a polyacrylamide gel and run for 2 h at room temperature. Replication assays Replication assays were performed as previously described in (Demeret et al., 1995). Brie¯y, 2 mg of the pori18 plasmid were cotransfected with 2 mg of the E1 expression plasmid and 1 mg of plasmids expressing either wt or mutant E2 proteins in 293 cells. Seventy-two hours after transfection, low molecular weight DNA was extracted from the transfected cells by the Hirt method and was subjected to a double digestion with the BamHI and DpnI enzymes. DpnI resistant DNA was resolved on an agarose gel, blotted on a membrane and probed with a 32P-labeled pori18 plasmid. DpnI resistant DNA was quanti®ed with a phosphorimager. Western and Northern blots Western blots were performed as previously described (Desaintes et al., 1997). Northern blots were done with total RNA prepared from E2-transfected HeLa cells 48 h after transfection. Five mg of RNA were loaded on a 1% MOPS-agarose gel. After transfer, the nitrocellulose membranes were hybridized with 32P-labeled p53, p21 or Bax probes. Probes were generated by PCR ampli®cations of human cDNAs. Note added in proof After submission of this manuscript, Thomas and Banks (Thomas M and Banks L 1998, Oncogene, 17, 2943 ± 2954), have published that the HPV E6 proteins can degrade the proapoptotic protein Bak. According to this pathway, E2mediated repression of E6 would lead to p53-independent apoptosis. Acknowledgements This work was supported by the `Association pour la Recherche contre le Cancer (ARC)' and the `Ligue FrancËaise contre le Cancer'. We are very grateful to Jonathan Weitzman and Caroline Demeret for critical reading of the manuscript. We thank Martin Schener for the gift of HPV18 E6 expression plasmid and Moshe Oren for MDM2-CAT.

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