Abstract
The high risk human papillomavirus type 16 (HPV-16) have been found to be strongly associated with the carcinoma of the uterine cervix. E6 and E7 proteins of HPV-16 viruses are thought to underlie the development of cervical cancer. Recent reports indicated that E7 would suppress apoptosis (Lee et al., 1998; Thompson et al., 2001). Interestingly, E7 have also been reported to mediate pro-apoptotic effects (Basile et al., 2001; Hickman et al., 1997; Puthenveettil et al., 1996; Stoppler et al., 1998). In this study, E7-mediated apoptosis was determined in negative HF cells and in HPV16 positive SiHa cells (cervical carcinoma cells). The mechanism for E7 modulated apoptosis in these cells was investigated. pLXSN-E7/neo and pCDNA-neo was stably transfected into HPV-16 negative human fibroblast (HF). HF-E7#8 clone which had higher level of E7 and pRb protein and lower level of p53 protein was selected as the model cell line. On the other hand, SiHa-E7 cell line, constructed by Ms. Chen Ting Ting, and SiHa-neo cell line was also used in this study. The biological effects of E7 protein in both HF and SiHa cells were assayed by Sulforhodamine B (SRB) survival assay and Sub-G1 apoptotic analysis (Flow cytometry). E7 transfection in HF or SiHa cells led to increased Sub-G1 apoptosis. Furthermore, in response to DNA damage agents (0-8 Gy X-irradiation or 0-1μM cisplatin), both HF -E7 and SiHa -E7 cells were more sensitive than their parental cells because that these two cells had more sub-G1 apoptosis and less survival fraction. That is, E7 had synergistic apoptotic effects in SiHa or HF cells after X-ray or cisplatin treatment. However, no synergistic apoptotic effect of E7 was observed in E7 transfected SiHa cells after SA treatment. In conclusion, E7 protein sensitized HF and SiHa cells to X-irradiation and cisplatin but not SA. In both HF-E7 and SiHa-E7 cells, E7 protein had been proved its functionality by increasing phosphorylated Rb. E7 protein up-regulated the apoptosis-related protein, p53, ser15-phospho p53, and p21, in HF cells. In contrast, it down-regulated these three proteins expression in SiHa cells. Moreover, all of these proteins increased in X-irradiated HF/HF-E7 cells but p21 decreased in SiHa/SiHa-E7 cells after X-ray or cisplatin. Despite the differences, active form of caspase 3 increased in both HF-E7 and SiHa-E7 cell indicating that E7 mediated caspase 3-dependent apoptosis. All these results indicated that E7 transfection both in HPV16 positive SiHa and HPV16 negative HF cells would increase X-irradiated sub-G1 apoptosis and cell killing, but the molecular mechanisms for pro-apoptosis were not exactly the same in these two cell lines. Therefore, the pro-apoptotic function of E7 in human cells (HF and SiHa) had been investigated in this study.