Abstract
Abstract Most human cervical cancers are caused by human papilloma virus (HPVs) infection. The HPV-E7 protein of HPV can bind to retinoblastoma protein (Rb) then inhibits the cell cycle regulation activity of Rb protein. Previous study of this lab showed that sodium arsenite (SA) could restore p53 tumor suppressor pathway in human cervical carcinoma SiHa cells, but not mutant-p53-SiHa (SiHa-p53m) cells. In present study showed that cisplatin could restore p53 expression in HPV-16 E6 containing SiHa cells after 10 mM cisplatin treatment for more than 4 hours (4 h) and enhance the radiosensitivity (in sub-G1 apoptosis) in HPV16 E6 containing SiHa cells, but not SiHa-p53m cells. Whether E7 involving in SA or cisplatin restored p53 function or cisplatin enhanced radiosensitivity (X-ray) in E6/E7 containing SiHa cells was not yet known. In order to determine E7 function in SiHa cells after treat with SA, cisplatin or X-ray, SiHa and human fibroblast (HF) cells were transfected with the retrovirus-derived vector pLXSN containing both the neo and HPV-16 E7 gene. Four individual clones (SiHa-E7#4, SiHa-E7#8, SiHa-E7#14 and HF-E7#8) were selected because of their high level of E7 protein and hypo-phosphorylated Rb protein (pRb) among a total of 30 (15 SiHa-E7; 15 HF-E7) G418-resistant transfected clones. Most E7 protein were located in the nucleus of SiHa and SiHa-E7#14 cells after immuno-fluorescence stain, but some E7 protein were found in cytoplasm surrounding the nucleus. Compared to parental SiHa cells, the SiHa-E7#14 cells have 1-3 fold more pRb protein, but less p53 and p21 protein. In HF-E7#8 cells, the E7 protein increased 3-6 fold of p53 expression than parental HF cells. The E7 effects on sub-G1 apoptosis and cytotoxicity were assayed by means of Flow-Cytometry (A) and SRB assay (B) in E7-transfected cells and parental cells. SiHa-E7#14 and HF-E7#8 cells both induced 1-3 fold sub-G1 apoptosis than parental cells after 0-8 Gy X-ray treatments. They also have 1-3 fold more sub-G1 apoptosis than parental cells after 0-5 μM cisplatin treatment for 24 h. SiHa-E7 #14 and HF-E7 #8 cells both appeared to have a 1.5-2 fold SRB-cytotoxicity than parental cells after the same dose of X-ray or cisplatin treatment. After 0-32 μM SA treatment for 24 h, SiHa and SiHa-E7#14 cells showed no significant difference in cytotoxic results from protocol (A) and (B). These results confirmed the previous study of this lab: SA induced SiHa cells’ apoptosis via decreasing E6 gene expression and then restoring p53 protein function. E7 protein had no significant effects on SA-induced apoptosis. In contrast HF-E7#8 cells induced more sub-G1 apoptosis than parental HF cells after the same dose of SA for 24 h treatment. Western blotting indicated that an increase in dose-dependent p53 and pRb protein expression (according to X-ray 0-8 Gy or cisplatin 0-10 μM for 24 h) was found in SiHa and SiHa-E7 #14 cells. In the other hand p21, p53 down stream protein, expression was a decreased in dose-dependent. The results from the above treatment showed that, active-caspase-3 protein in two kinds of SiHa cells were no different before or after treatment. P53 protein increased in both cells with agent doses in HF cells, but p53 expression level showed no difference in HF-E7#8 cells. In this study, E7 gene was successfully transfected into SiHa and HF cells stably. Flow-cytometry and SRB assay showed that, E7 protein had no significant effect on SA-induced apoptosis, but increased the sensitivity of SiHa cells to cisplatin and X-ray. The E7 protein may through binding pRb protein and decreasing p21 protein expression to increase cells undergoing apoptosis.