Effect Of Interaction Of Human Papillomavirus Type 16 E2 Protein And Daxx On Apoptosis

Objective: To provide the experimental basis for further elucidating the effects and their mechanisms of human papillomavirus(HPV) type 16(HPV16) E2 on the HPV16-induced carcinogenesis, interactions of HPV16 E2 and Daxx in human acute monocytic leukemia cells(THP-1) and human cervical cancer cells(C-33A) were studied, and the effects of their interaction on apoptosis were also analyzed.Methods:(1) The recombinant plasmid pc DNA3.1(+) / HPV16 E2 were transiently transfected into C-33 A or THP-1 cells. Indirect immunofluorescence assay was used to analyze the disposition and locations of HPV16 E2 and Daxx in THP-1 or C-33 A cells.(2) The recombinant plasmid pc DNA3.1(+) / HPV16 E2 were transiently transfected into C-33 A cells, Co-immunoprecipitation and Western blot were used to detect the interaction of HPV16 E2 protein and Daxx protein.(3) The recombinant plasmid pc DNA3.1(+)/HPV16 E2 were transiently transfected or co-transfected with the recombinant plasmid pc DNA3.1(-)/Daxx into C-33 A or THP-1 cells. Then, q RT-PCR or Western blot was respectively used to detect the expression of Daxx gene or Daxx protein in C-33 A or THP-1 cells.(4) The recombinant plasmid pc DNA3.1(+) / HPV16 E2 were transiently transfected into C-33 A cells. At 12 h, 24 h, 36 h and 48 h after the cells transfected with the plasmids, OD490 value for C-33 A cells and OD570 value for THP-1 cells was respectively detected. The ratios of cellular inhibition were calculated. Flow cytometry was used to detect the percentages of cell cycle phases and the apoptotic rates.Results:(1) Under inverted fluorescence microscope, HPV16 E2 proteins were red signal and Daxx proteins were green signal, both located in nuclei of C-33 A or THP-1 cells. it showed yellow signal when two signals were imerged.(2) It was found the interaction between HPV16 E2 proteins and Daxx proteins in C-33 A cells by using Co-immunoprecipitation assay and Western blot.(3) The expression of Daxx gene or Daxx proteins were both ehanced in C-33 A or THP-1 cells with over-expressed immediately HPV16 E2 proteins. Both HPV16 E2 proteins and Daxx proteins showed over-expressions in C-33 A or THP-1 cells after the plasmids were transfected into the cells.(4) When immediate over-expression of HPV16 E2 proteins were in C-33 A or THP-1 cells, the relative inhibition rates were obviously higher than that in the control(P <0.05); the phases of G1 was extended(P <0.05) and apoptotic rates were incraesed significantly compared to the control(P <0.05).Conclusions:(1) HPV16 E2 could interact with Daxx in C-33 A cells.(2) Over-expression of HPV16 E2 proteins could enhance the Daxx expression, and inhibit the cellular proliferation, and extend the G1 phase and increase the apoptotic rate in C-33 A or THP-1 cells.