Shuvayeva G. Expression of adaptor protein Ruk/CIN85 isoforms in human tumors

The thesis is devoted to investigation of Ruk/CIN85 expression profiles in tumors of urogenital (kidney, prostate) and glial (brain) origins. To realize the main objectives of this study, polyclonal and monoclonal antibodies (Abs) against recombinant GST-fused proteins which include C- terminal and N-terminal parts, correspondingly, of adaptor protein Ruk/CIN85 have been generated. Expression profiles of Ruk/CIN85 isoforms and their oligomerization in cell lines of various tissue origins were analyzed using Western blotting and immunoprecipitation. Possibly, these features may reflect specific biological role of Ruk/CIN85 isoforms in cell lines of various species and tissue origins. Decrease as well as increase of full-length form of Ruk/CIN85 expression level was revealed in clear cell carcinoma samples in comparison with corresponding control samples (morphologically nontransformed tissue isolated from the same operated organ under radical nephrectomy) both at the level of mRNA and protein using Nothern-blot and Western-blot analysis respectively. It was shown with immunocytochemical analysis that decrease of Rukl/CIN85 expression in investigated renal carcinomas is accompanied with changes of adaptor protein subcellular distribution - translocation to the nuclei and predominant accumulation near plasma membrane zone. Polymorphism in the expression level of main Ruk/CIN85 isoforms was demonstrated in benign prostate hyperplasia (BPH) samples. In adenocarcinomas, the immunoreactive band with apparent molecular weight of 85 kDa was detected at very low level or was not detected. The data of Western-blot analysis were correlated with Nothern-blotting results. Inverse relationship between expression of Rukl/CIN85 and EGFR was revealed in the central and peripheral regions of glioblastoma samples: the high level of Rukl/CIN85 expression in the central area corresponded to low level of EGFR expression and vice versa. Low level of Rukl/CIN85 expression in investigated types of human neoplasia coincides with their high grade of tumorigenicity (G3-G4) that allow using this parameter to predict the progression potential of tumor growth. The obtained results suggest that decrease in the expression level of full-length form of Ruk/CIN85 adaptor protein can lead to the loss of coordinated control of apoptosis and proliferation in the transformed epithelium cells.