Françoise Thierry

Expression of the papillomavirus E2 protein in HeLa cells leads to apoptosis.

The papillomavirus E2 protein plays a central role in the viral life cycle as it regulates both transcription and replication of the viral genome. In this study, we showed that transient expression of bovine papillomavirus type 1 or human papillomavirus type 18 (HPV18) E2 proteins in HeLa cells activated the transcriptional activity of p53 through at least two pathways. The first one involved the binding of E2 to its recognition elements located in the integrated viral P105 promoter. E2 binding consequently repressed transcription of the endogenous HPV18 E6 oncogene, whose product has been shown previously to promote p53 degradation. The second pathway did not require specific DNA binding by E2. Expression of E2 induced drastic physiological changes, as evidenced by a high level of cell death by apoptosis and G1 arrest. Overexpression of a p53 trans‐dominant‐negative mutant abolished both E2‐induced p53 transcriptional activation and E2‐mediated G1 growth arrest, but showed no effect on E2‐triggered apoptosis. These results suggest that the effects of E2 on cell cycle progression and cell death follow distinct pathways involving two different functions of p53.

Papillomavirus E2 induces p53-independent apoptosis in HeLa cells

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 E2-induced apoptosis is an early event, independent of p53 accumulation and unrelated to downstream p53-dependent transcriptional events.

Transcription-independent triggering of the extrinsic pathway of apoptosis by human papillomavirus 18 E2 protein.

Cervical carcinomas are most frequently associated with human papillomaviruses (HPV), whose E6 and E7 oncogenes products induce cellular immortalization. The papillomavirus E2 protein is a transcription factor, which represses the expression of the viral oncogenes, and activates viral DNA replication during the vegetative viral cycle. This protein is specifically inactivated in HPV18-associated carcinoma cells, suggesting that E2 functions prevent carcinogenic progression. Indeed, ectopic expression of E2 in cervical carcinoma cells strongly inhibits cell proliferation. Here we show that above a threshold level of expression, the E2 protein induces apoptosis, independently of other viral functions. The amino-terminal domain is responsible for this apoptotic activity, but surprisingly with no involvement of its transcriptional functions. The death pathway triggered by E2 relies on activation of the initiator caspase 8, specific of the extrinsic pathway of apoptosis. E2 itself is cleaved by caspases during cell death, providing an example of an apoptotic inducer that is itself a target for caspase processing. The autonomous proapoptotic activity of HPV18 E2 described here may counteract the proliferative functions of viral oncogenes, and renders the inactivation of E2 crucial for carcinogenic progression.

An enhanceosome containing the Jun B/Fra-2 heterodimer and the HMG-I(Y) architectural protein controls HPV18 transcription

Recent studies have reported new mechanisms that mediate the transcriptional synergy of strong tissue‐specific enhancers, involving the cooperative assembly of higher‐order nucleoprotein complexes called enhanceosomes. Here we show that the HPV18 enhancer, which controls the epithelial‐specific transcription of the E6 and E7 transforming genes, exhibits characteristic features of these structures. We used deletion experiments to show that a core enhancer element cooperates, in a specific helical phasing, with distant essential factors binding to the ends of the enhancer. This core sequence, binding a Jun B/Fra‐2 heterodimer, cooperatively recruits the architectural protein HMG‐I(Y) in a nucleoprotein complex, where they interact with each other. Therefore, in HeLa cells, HPV18 transcription seems to depend upon the assembly of an enhanceosome containing multiple cellular factors recruited by a core sequence interacting with AP1 and HMG‐I(Y).

Frequent Alterations of the β-Catenin Protein in Cancer of the Uterine Cervix

Cancer of the uterine cervix is still the leading cause of death among women with cancer in developing countries. Although infections with human papillomavirus are necessary, other molecular alterations that are needed at the cellular level for development of these tumors remain largely unknown. β-Catenin is a key regulator located within the Wnt signaling cascade whose alterations constitute an important event in colon carcinogenesis. In many malignancies increased levels of the β-catenin protein have been found, associated with its nuclear and/or cytoplasmic accumulation. To search for possible alterations of this pathway we examined the expression and localization of the β-catenin protein in tumors from the uterine cervix and cell lines derived from them. β-Catenin was found accumulated in the cytoplasm and/or nuclei of 12 out of 32 samples. In accordance, increased levels of this protein were observed in 9 out of 20 tumors analyzed. Importantly, PCR-SSCP and sequence analysis showed no mutations in exons 3, 4 and 6 of the β-catenin gene. Our findings indicate that alterations of β-catenin are frequent in these tumors and suggest that they may play an important role in the development of cancer of the uterine cervix. They also indicate that higher protein levels and abnormal localization may result from several different mechanisms.

Localization of the ts defects of ts mutants of influenza A virus using complementation analysis and gel analysis of the RNA segments of recombinants

Abstract Two sets of temperature-sensitive ( ts ) mutants of influenza A virus (derived by mutagenesis of the Hong Kong (HK) and WSN strains, respectively) were analyzed for shared lesions using a complementation-recombination assay which was performed directly on tissue culture monolayers. Each of these sets had previously been shown to consist of seven complementation groups. By this assay three of the HK groups were shown to correspond to WSN complementation groups: HK group 1 and WSN I shared a common ts lesion as did HK group 5 and WSN group III; finally, HK group 6 and WSN group II were shown to be related. The relationship of the WSN groups IV, V, VI, and VII and HK groups 2, 3, 4, and 7 could not be interpreted from the complementation-recombination studies. We therefore sought to localize the lesions of the HK mutants by preparing ts + recombinants between the HK groups 6 and 7 mutants and PR8 wildtype or WSN ts mutant 51. In this way we have localized the lesions in these mutants to RNA segments 3 (P2) and 5 (NP), respectively. The findings with HK group 6 confirm the genetic analysis while those with HK group 7 suggest the occurrence of three independent groups of lesions on the NP segment of HK (groups 2, 3, and 7). The cotransfer of the NS segment with the NP and P2 genes is discussed, as are the factors which make genetic analysis by biological methods difficult, but perhaps useful in understanding the functional interactions of the influenza polypeptides.