Experimental Study On The Function Of Wild VHL Gene To Sensitize TRAIL-resistant RCC786-O To TRAIL

Part One:Experimental study on the function of wild VHL gene to sensitize TRAIL-resistant RCC786-O to TRAILObjective:Alterations in the von Hippel-Lindau (VHL) tumor suppressor gene have been reported in up to91%of case tumors in sporadic in renal cell carcinoma (RCC). VHL gene and its translational product regulate, directly or indirectly, the expression of numerous genes with varied functional consequences at multiple levels ranging from transcription to post translation in RCC. Although much is known about the effects of the VHL gene in the tumor cell, comparatively little is understood about the mechanism by which VHL proteins control the sensitivity of renal cancer cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis. Here, our goal was to determine the function of wild VHL in TRAIL-induced apoptosis in RCC786-0cells.Methods:To determine whether wide VHL gene has potential ability to sensitize RCC786-0cells to TRAIL-induced apoptosis, Human renal carcinoma cell786-0,786-O/VHL (subclone expressing wild-type VHL),786-O/LIK (subclone expressing VHL type1mutation),786-O/L2K (subclone expressing VHL type2A mutation),786-O/L3K (subclone expressing VHL type2B mutation) and786-O/L4K (subclone expressing VHL type2C mutation) were, were treated with various concentrations of TRAIL for24hours or treated with40ug/ml TRAIL for various hours and were measured by MTT and flow cytometry. RT-PCR and Western blot assay were performed to analyze the expressing of DR4, DR5and c-FLIP(L). To better define the correlation between c-FLIP(L) expression and sensitization to TRAIL-induced apoptosis in786-O/VHL cells, c-FLIP(L) was over expressed in TRAIL-sensitive786-O/VHL cells using a FLAG-tagged vector and apoptosis was measured by flow cytometry. We used the DR4and DR5block antibody to observe the correlation between the two death receptors and wild-type VHL gene. Results:MTT apoptosis detection monoclonal carried the full-length DNA fragments of wild-type VHL gene786-O/VHL cells sensitive to TRAIL-mediated apoptosis by dose-dependence and time-dependence. Flow cytometry indicated, after786-O and786-O/VHL cells treated by TRAIL (40ng/ml), nearly30%and50%of786-O/VHL cells were apoptotic when treated for24and72h, whereas the786-0cells showed significantly lower percentages of cell death after treatment with TRAIL. RT-PCR and Western blot assay showed that786-O/VHL cells displayed a significant up-regulation of DR4and down-regulation of DR5and c-FLIP(L) in both mRNA and protein levels compared to786-Ocells. Transient overexpression of c-FLIP(L) changed the phenotype of786-O/VHL cells from TRAIL sensitive to TRAIL resistant. Used DR4and DR5antagonist to detect the correlation between the wild VHL gene and sensitization to TRAIL-induced apoptosis in786-0cells, the results showed786-O/VHL cells were still sensitive to TRAIL-mediated apoptosis even though DR5was blocked. When DR4was blocked, we observed the phenotype of786-O/VHL cells from TRAIL sensitive to TRAIL resistant. Used the combined DR4and DR5antagonist antibody, the results show that TRAIL-mediated apoptosis was similar to the blocking of DR4alone.Conclusion:In this study, we demonstrate that TRAIL-sensitive786-O/VHL cells expressed higher levels of DR4and lower levels of c-FLIP(L) and DR5at both the transcription and translation levels than TRAIL-resistant786-0cells. We observed that DR4block antibody and transient overexpression of c-FLIP(L) can transform the phenotype of786-O/VHL cell from TRAIL sensitive to TRAIL resistant. In conclusion, we herein show for the first time that wide-type VHL, not VHL gene mutant-types, can sensitize TRAIL-resistant786-O/VHL cells to undergo apoptosis. Elevation of DR4and down-regulation of c-FLIP(L) expression caused by wild VHL gene play important roles in sensitization to TRAIL-induced apoptosis in RCC. Part Two:VHL-HIF-VEGF Signaling Pathway and Renal Cell CarcinomaThe von Hippel-Lindau (VHL) gene is located on human chromosome3p25-26. The VHL tumour suppressor gene product, pVHL, was relevant with a number of signaling pathways, hypoxia inducible factor and regulation of oxidative phosphorylation. Inactivation of the VHL gene can lead to abnormal VHL-HIF-VEGF signalling pathway, which was closely related to the oncogenesis and development of renal cell carcinoma. The inactivation mechanism of VHL gene included gene mutations, loss of heterozygosity and methylation. Target therapy to VHL-HIF-VEGF signalling pathway is now widely applied to clinical practice. Novel molecular markers of VHL-HIF-VEGF signalling pathway contribute to screening for early diagnosis and prognosis of renal cell carcinoma.