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In over 90% of cervical cancers and cancer-derived cell lines, the p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV). The HPV E6 protein promotes the degradation of p53 and thus inhibits the stabilization and activation of p53 that would normally occur in response to HPV E7 oncogene expression. Restoration of p53 function in these cells by blocking this pathway should promote a selective therapeutic affect. Here we show that treatment with the small molecule nuclear export inhibitor, leptomycin B, and actinomycin D leads to the accumulation of transcriptionally active p53 in the nucleus of HeLa, CaSki, and SiHa cells. Northern blot analyses showed that both actinomycin D and leptomycin B reduced the amount of HPV E6-E7 mRNA whereas combined treatment with the drugs showed almost complete disappearance of the viral mRNA. The combined treatment activated p53-dependant transcription, and increases in both p21 WAF1͞CIP1 and Hdm2 mRNA were seen. The combined treatment resulted in apoptotic death in the cells, as evidenced by nuclear fragmentation and PARP-cleavage indicative of caspase 3 activity. These effects were greatly reduced by expressing a dominant negative p53 protein. The present study shows that small molecules can reactivate p53 in cervical carcinoma cells, and this reactivation is associated with an extensive biological response, including the induction of the apoptotic death of the cells. Mutations in the p53 tumor suppressor gene are the most common specific genetic changes in human tumors. They occur only rarely, however, in tumors in which p53 may be inactivated by interaction with cellular or viral proteins. For instance, in cervical cancer, p53 mutation is uncommon, but human papillomavirus (HPV) is present in more than 90% of the tumors. HPV infection is the major risk factor of this disease, which, worldwide, is the second most common form of cancer in women (1). The correlation of HPV infection and p53 mutation has been closely studied in cervical cancer, and, because the HPV E6 protein inactivates p53, it appeared that p53 mutations would be confined to HPV-negative cases. This is, with a few exceptions, found to be the case (2).The HPV E6 protein complexes with cellular proteins E6-AP and p53 and facilitates p53 degradation via the ubiquitindependent proteolytic system (3). E6 proteins of both high risk and low risk HPV types bind to p53 in vitro, but only E6 proteins of oncogenic HPV types can target p53 for degradation (4). Apart from the p53 interaction, evidence is accumulating that E6 has p53-independent transforming and growth-regulating activities (5, 6).Different stress signals, such as DNA damage, hypoxia, heat shock, and oncogene activation induce an increase in the stability of the wild-type p53 protein. The accumulation of p53 is associated with the transcription of a series of p53-responsive cellular genes, including bax, p21 WAF1͞CIP1, and gadd45, that mediate p53 induction of growth arrest and apoptosis (7). The activated p53 also induces the transcription ...

The transcription factor E2F-1 directs the expression of genes that induce or regulate cell division, and a role for E2F-1 in driving cells into apoptosis is the subject of intense discussion. Recently it has been shown that E2F-1 binds and coprecipitates with the mouse double-minute chromosome 2 protein (Mdm2). A domain of E2F-1 (amino acids 390 to 406) shows striking similarity to the Mdm2 binding domain of the tumor suppressor protein p53. It is known that interaction of Mdm2 with p53 through this domain is required for Mdm2-dependent degradation of p53. We show here that E2F-1 protein is upregulated in response to DNA damage. The kinetics of induction are dependent upon the source of DNA damage, i.e., fast and transient after irradiation with X rays and delayed and stable after irradiation with UVC, and thus match the kinetics of p53 induction in response to DNA damage. We show further that E2F-1 is also upregulated by treatment with the transcription inhibitor actinomycin D and with the kinase inhibitor DRB, as well as by high concentrations of the kinase inhibitor H7, all conditions which also upregulate p53. In our experiments we were not able to see an increase in E2F-1 RNA production but did find an increase in protein stability in UVC-irradiated cells. Upregulation of E2F-1 in response to DNA damage seems to require the presence of wild-type p53, since we did not observe an increase in the level of E2F-1 protein in several cell lines which possess mutated p53. Previous experiments showed that p53 is upregulated after microinjection of an antibody which binds to a domain of Mdm2 that is required for the interaction of Mdm2 with p53. Microinjection of the same antibody also increases the expression of E2F-1 protein, while microinjection of a control antibody does not. Furthermore, microinjection of Mdm2 antisense oligonucleotides upregulates E2F-1 protein, while microinjection of an unrelated oligonucleotide does not. These data suggest that E2F-1 is upregulated in a similar way to p53 in response to DNA damage and that Mdm2 appears to play a major role in this pathway.

The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.