Investigation of roles of hur and VPS39 on cell surface expression of DR5

Objective: TRAIL (TNF-Related Apoptosis Inducing Ligand) is a member of TNF superfamily cytokines, which can induce apoptosis when bound to its cognate receptors. It has two apoptosis-initiating receptors. Although much about TRAIL signaling pathway has been elucidated, details of regulation of its receptors remain highly elusive. First step to TRAIL-induced apoptosis in cancer cells is the requirement that they express TRAIL receptors DR4 and DR5 on their surface. Unlike DR4, cell surface expression mechanism of which is relatively well understood, DR5 receptor's translocation to plasma membrane is still unknown. Methods: In the context of this thesis study, LNCaP and HeLa cell lines were transfected with anti-HuR and anti-Vps39 siRNA. Cytoplasmic, nuclear, and membrane fractions were isolated from these transfected cells. Effects of HuR and Vps39 knockdown on DR5 expression in different subcellular fractions were evaluated by Western Blot and ELISA. Moreover, TRAIL-induced apoptosis and necroptosis levels of above-mentioned transfected cells were determined by a commercial ELISA based kit. Results: HuR knockdown in LNCaP cells were found to decrease DR5 surface expression as shown by both Western Blot and ELISA (p=0,036). Similar results were obtained from experiments performed on HeLa cell line. HuR knockdown-related decrease in surface expression of DR5 diminished TRAIL-induced apoptosis only in HeLa cells. On the other hand, Vps39 was failed to be relevant with DR5 surface expression, but was shown to alter nuclear localization of DR5 in both cell lines. Conclusion: In sum, HuR protein influences plasma membrane expression of DR5. In order to fully reveal this mechanism however, further research is necessary.