Colocalization Of Human Papillomavirus Type 16 E6 Protein And HDaxx In HeLa Cells And Their Effect On Apoptosis

Objective: To provide the experimental basis for studying hDaxx's effects on oncogenic mechanism of human papillomavirus type 16 ( HPV16 ) E6 protein, the subcellular localization of HPV 16 E6 protein and hDaxx in HeLa cells are observed, and the effect of HPV16 E6 and hDaxx on TNF-αinduced apoptosis is studied.Methods:(1) The plasmid of pDsRed-Monomer-C1/HPV16 E6 or pEGFP-C1/hDaxx was transfected into HeLa cells by LipofectamineTM 2000, respectively. The cellular protein extracts were prepared for detecting the expression of E6 protein or hDaxx using Western blot.(2) The plasmids of pEGFP-C1, pDsRed-Monomer-C1, pDsRed-Monomer-C1/HPV16 E6 or/and pEGFP-C1/ hDaxx were transfected into HeLa cells, respectively. The subcellular localization of hDaxx or HPV16 E6 were observed under confocal microscopy. Their colocalization was analyzed.(3) The plasmids of pcDNA3.1(-)/HPV16 E6 and pcDNA3.1(-)/hDaxx, which were divided into 4 groups, were cotransfected into HeLa cells. The cells'proliferation activity was examined using MTT assay.(4) HeLa cells were stained by Hoechst 33258 and were observed using fluorescent microscopy. The number of variable cells and apoptotic cells were counted for estimating apoptotic rate. pcDNA3.1(-)/HPV16 E6 ( 2.0μg ) were respectively cotransfected into HeLa cells with pcDNA3.1(-)/hDaxx which were divided into 4 groups ( 0μg, 0.5μg, 1.0μg, 2.0μg ). The control group was only transfected with pcDNA3.1(-) or pcDNA3.1(-)/hDaxx. All groups of HeLa cells were cultured in RMPI-1640 containing TNF-αfor 12h, then the cells were respectively collected, and fixed in 70% ethanol at 4℃for overnight. After the cells were stained by propidium iodide (PI), the apoptosis of every group cells was detected using Flow cytometry (FCM).(5) The groups and treatment were shown as (4). Caspase-8 or caspase-3 activity was measured using caspase-8 or caspase-3 colorimetric assay kit according to the manufacturer.Results:(1) The results of Western-blot showed that fusion proteins DsRed-HPV16 E6 or EGFP-hDaxx respectively expressed in HeLa cells.(2) Under confocal microscopy, the red fluorescence of HPV16 E6 distributed mainly in nuclei of HeLa cells, and the green fluorescence of hDaxx only distributed in the nuclei; in the cells cotransfected, red or green fluorescence were weakly uniformly distributed in cytoplasm, strongly adjacent to the nuclear membrane in nuclei. It displayed yellow fluorescent when they were merged.(3) The results of MTT assay showed the proliferation of the groups cotransfected with pcDNA3.1(-)/E6 and pcDNA3.1(-)/hDaxx was lower than the group only transfected with pcDNA3.1(-)/E6 (P<0.05).(4) The nuclei that showed patches of compact chromatin were considered as being apoptotic. It was shown that some nuclei were highly fluorescent, condensed, fragmented in all groups treated by TNF-α. The apoptotic rate of HeLa cells transfected with pcDNA3.1(-)/E6 was lower than that of vector control (P<0.05). When cotransfected with pcDNA3.1(-)/hDaxx, the apoptotic rate of HeLa cells increased in a dose-dependant manner of pcDNA3.1(-)/hDaxx (P<0.01).(5) The caspase-8 or caspase-3 relative activity of the group transfected with pcDNA3.1(-)/E6 was lower than that of vector control (P<0.01). Their activity increased when cotransfected with pcDNA3.1(-)/hDaxx in a dose-dependent manner. The activity of caspase-8 or caspase-3 of the group cotransfected with pcDNA3.1(-)/E6 and 2.0μg pcDNA3.1(-)/hDaxx was significantly higher than that transfected with pcDNA3.1(-)/E6 (P<0.01).Conclusion:(1) The fusion protein of DsRed-HPV16 E6 or EGFP-hDaxx can expresses in HeLa cells. HPV16 E6 induce the translocation of hDaxx partly from the nuclei into the cytoplasm. HPV16 E6 and hDaxx co-localize in the cells.(2) HPV16 E6 protein partly inhibits the apoptosis of HeLa cells induced by TNF-α. Over-expression of hDaxx enhance the sensitivity of HeLa cells expressing E6 protein to apoptosis induced by TNF-α. (3) HPV16 E6 may inhibit the activity of caspase-8 or caspase-3, and over-expression of hDaxx may down-regulate the inhibition.