Inductioin and activation of p53 tumor suppressor protein by Cdt in HPV-related cancer cells

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The cytolethal distending toxin (Cdt) from Actinobacillus actinomycetemcomitans has been shown to induce cell cycle arrest in the G2 phase as well as cell death in mammalian cells. However, the precise mechanism by which the toxin induces these effects remains unknown. Human papillomavirus-16 (HPV-16) and HPV-18 are considered as causative agents of most human rectogenital cancers and part of oropharyngeal cancers. The E7 viral oncoprotein inactivates the retinoblastoma-related gene-product (Rb) and the E6 oncoprotein inhibits the p53 tumor suppressor protein by promoting its degradation.We have found that Cdt caused accumulation of p53 during the induction of G2 arrest and cell death in HPV-18 positive HeLa cells. HS-72 mouse hybridoma cells expressing wild-type p53 were stably transfected with HPV-16 E6/E7. Using E6/E7-expressing HS-72 cells, we demonstrated that these cells were also sensitive to Cdt-mediated G2 arrst and cell death. To explore the molecular mechanism of p53 accumulation, we established the the siRNA system in HPV-positive cancer cell lines (HeLa, SiHa).Using lamin A/C siRNA which had been reported to exhibit a strong RNAi activity, we maximize the induction rate of siRHA into these cells. He found that HeLa cells and SiHa cells can be introduced with siRNA with a extremely high efficiency (more than 90%). We also found synthesized HPV-16 E6 and E7 siRNAs down-regulated the expression E6 and E7 mRHA by 70% and 80%, respectively. These results showed that SiHa cells were highly susceptible to siRNA. We are now planning to study if either ATM, ATR, Chkl, Chk2, DNA-PK or ARF is involved in the Cdt-induced p53 accumulation using the siRNA method.