Effects Of Human Papillomavirus Types 16 E6 And P53 Genotype On The Proliferation, Apoptosis And Chemotherapy Sensitivity Of Cervical Carcinoma Cell Lines

Objective: To investigate the effects of human papillomavirus types 16 E6 and p53 genotype on the proliferation,apoptosis and chemotherapy sensitivity of cervical carcinoma cell lines.Methods:Part one. HPV16E6 gene was stably transfected into C33A cervical carcinoma cell line by liposome, and the expression of HPV16E6 mRNA and protein in transfected cell line were identified by RT-PCR and Western blotting analysis.The effects of growth, proliferation and cell cycles of cervical carcinoma cell lines C33A by MTT colorimetry and Flow cytometry analysis.Part two: The effects of cervical carcinoma cell proliferation were examined by MTT colorimetry after the treatment of DDP,BLM,VCR,DDP+BLM+VCR.The apoptosis rates of each group were detected by AO/EB, immunofluorescence and Annexin V/PI stained methods.The expressions of HPV 16E6 and p53^mt protein after the treatment of different concentration of DDP were detected by western blotting;The amounts of HPV16E6 mRNA in each kinds of cell lines were detected by RT-PCR.Results:Part one. C33A-E6 cell expressing pcDNA3-HPV16E6 plasmid and C33A-P expressing pcDNA3 plasmid were transfected stably by liposome.The growth of C33A-E6 was more rapid than C33A-P and C33A(P＜0.05). It indicated that HPV16E6 can promote the growth and proliferation of C33A.In C33A-E6 cell lines,the percent of stage DNA pre-compound G1 boundary of the cell cycles and stage still G₀/G₁(39.27%) were significantly less than control groups C33A-P(53.73%)and C33A(55.38%)(P＜0.01). But the percent of stage S(38.83%) and the stage DNA after-compound G2 and the splitted stage M(G2/M:21.9%) were distinctly higher than control groups C33A-P(S:29.36;G2/M:16.91%)and C33A(S:28.04%; 2/M:16.58%)(P＜0.01).However, there was no significantly difference between C33A-P and C33A cell lines(P＞0.05).Part two: MTT colorimetry showed that the livability of each cell line were decreased when the chemical medicine concentration increased in the same effecting time especially in groups treated with 10 and 5 multiple PPC(peak plasma concentration,PPC).There was no statistically significant difference in groups treated with 10%, 50%PPC chemical medicine and with DMSO.The livability of groups treated with 200 %, 100 % PPC chemical medicine were significantly less than groups of 10 % PPC chemical medicine and DMSO.The livability of united application of chemical medicine more less than applicated alone.And the livability of group treated with DDP more less than groups treated with BLM and VCR. There was no significant difference between BLM and VCR. In the same concentration of chemical medicine and treated times.The livability of C33A-E6 was higher little than C33A and C33A-P.But there was significant difference only in a few groups.There was no significant difference between C33A-E6 and CaSki cell(P＞0.05).AO/EB and Annexin V/PI stained tests showed that the apoptosis rates of C33A,C33A-E6,C33A-P and CaSki cells were increased significantly when the DDP concentration increased.There were statistic- ally significant difference in apoptosis rates between groups treated with 80umol/L,8umol/L and 0.8umol/L DDP(P＜0.01).By treated with DDP in different time,there was no significant difference in apoptosis rates in groups of 80umol/L DDP,DMSO respectively after 24hr,48hr and 72hr.The apoptosis rates were increased in groups treated with 0.8umol/L DDP in 48hr to 72hr(P＜0.01).And the apoptosis rate treated with 8umol/L DDP were significantly increased after 48hr,But that was no more increased after 72hr. There was no significant difference in groups treated with 0.8umol/L DDP and DMSO.The Western Blotting showed that the protein HPV16E6 expressed weakly in C33A-E6 and CaSki cell lines. The expression of protein HPV16E6 in two groups were decreased gradually even to detect hardly following with the increased DDP and the prolonged treating time. And they were increased slightly in C33A-E6 and CaSki cell lines with treated nothing,But there was no significant difference(P＞0.05).It can't be detected HPV16E6 protein in C33A and C33A-P. The p53^mt expressed and no change in C33A-E6,C33A and C33A-P cells following the increased DDP and the prolonged treating time(P＞0.05).That can't be tested in CaSki cell.The RT-PCR showed that the amounts of HPV 16E6 mRNA were no significant difference in C33A-E6 and CaSki cells treated with nothing within 24hr.The HPV16E6 mRNA of C33A-E6 cell line was distinctly higher than CaSki(P＜0.05).And that was higher within 48hr than 24hr (P＜0.05).The expression of HPV16E6 mRNA in two groups were decreased gradually following the increased DDP and the prolonged treating time(P＜0.01).But there was no significant difference between C33A-E6 and CaSki cells under the same DDP concentration(P＞0.05).Conclusion: HPV16E6 gene can promote the proliferation and apoptosis of cervical carcinoma cells.The p53^mt was no significant effect on the proliferation and chemosensitivity of C33A. HPV16E6 gene was not cooperate with p53^mt in promoting the proliferation and apoptosis of C33A cervical carcinoma cells.The effects of HPV16E6 gene on proliferation and chemosensitivity of cervical carcinoma cell lines were not markedly relative to the p53 status(p53^mt/p53^wt).As for HPV16E6 there maybe have else mechanisms of the proliferation and chemosensitivity on C33A.