Anti-Human SHC1 (a.a. 272-559) Polyclonal antibody

Core promoters with adjacent regions of the human genes CDC6, POLD1, CKS1B, MCM2, and PLK1 were cloned into a pGL3 vector in front of the Photinus pyrails gene Luc in order to study the tumor specificity of the promoters. The cloned promoters were compared in their ability to direct luciferase expression in different human cancer cells and in normal fibroblasts. The cancer-specific promoter BIRC5 and non-specific CMV immediately early gene promoter were used for comparison. All cloned promoters were shown to be substantially more active in cancer cells than in fibroblasts, while the PLK1 promoter was the most cancer-specific and promising one. The specificity of the promoters to cancer cells descended in the series PLK1, CKS1B, POLD1, MCM2, and CDC6. The bidirectional activity of the cloned CKS1B promoter was demonstrated. It apparently directs the expression of the SHC1 gene, which is located in a "head-to-head" position to the CKS1B gene in the human genome. This feature should be taken into account in future use of the CKS1B promoter. The cloned promoters may be used in artificial genetic constructions for cancer gene therapy.

P66shc protein is an alternative transcript product of the SHC1 gene. Whereas two other isoforms (p52shc and p46shc) have adaptor function in the RAS signaling pathway, p66shc regulates the amount of reactive oxygen species (ROS) in a cell. Previously, it was demonstrated that the p66shc genome knockout significantly extends maximal lifespan in mice. P66shc could translocate into the mitochondria and increase intracellular ROS level, although basic mechanisms of this activity remain poorly understood. P66shc seems to play a significant role in carcinogenesis since an increased expression of p66shc correlates with poor prognosis in colorectal carcinoma. In our study, we applied RNA interference using lentiviral constructions that express short hairpin RNA (shRNA) against the N-terminal CH2 domain of the p66shc isoform. As a consequence, the p66shc but not p52shc and p46shc isoforms was selectelively suppressed in the RKO colon carcinoma cell line. We found that RKO cells with p66shc knockdown have been shown to be more resistant to oxidative stress induced by hydrogen peroxide exposure or serum starvation. Moreover, mitochondrial fragmentation that depends on the mitochondrial ROS amount was significantly decreased in p66shc-deficient RKO cells. Our findings are consistent with the hypothesis that p66shc participates in mitochondrial accumulation of ROS during oxidative stress and consequently promotes induction of apoptosis. DOI: 10.1134/S0026893312010062