The Molecular Biologic Study about the Role of Deubiquitination Gene in Bladder Tumor Progression

Kim, Hyung Yun; Lee, Young Goo; Lee, Sang Eun

Abstract

PURPOSE: Ubiqutin-proteasome-mediated proteolysis is an important pathway of nonlysosomal protein degradation, which controls the timed destruction of cellular regulatory proteins of the p21, p27, p53 gene (p53), Retinoblastoma gene (Rb), E2F-1, E2F-4 and cyclin etc. The deubiqutination (DUB) gene prevents ubiquitin-proteasome- mediated proteolysis by removal of ubiquitin from ubiqutinated proteins. We investigated the role of the DUB gene (DFFRY or DFFRX) in the progression of a bladder tumor associated with Rb and p53, which follows ubiquitin-proteasome-mediated proteolysis.
MATERIALS AND METHODS
RT4 (bladder pailloma cell line), 5637 (superficial bladder transitional cell carcinoma cell line), and HT1376 (invasive bladder transitional cell carcinoma cell line) were cultured. mRNA was extracted from each cell line by the Poly-A tract mRNA isolation system. Relative quantitative RT-PCR (reverse transcription-polymerase chain reaction) for the DUB gene (DFFRY or DFFRX) was performed. Northern blotting for the DUB gene, p53 and Rb were performed. Proteins were harvested at 80% confluence from each cell line. Western blotting for the p53 and Rb were performed.
RESULTS
The relative quantitative RT-PCR for the DUB gene showed it to be expressed most strongly in RT4, and most weakly in HT1376, which were identical to the findings of the Northern blotting. The p53 was expressed at similar levels on the Northern blots of all three cell lines, whereas on Western blotting, it was expressed most strongly in 5637, most weakly in RT4, and moderately in HT1376. The Rb was expressed only in RT4 on Northern and Western blotting.
CONCLUSIONS
The DUB gene might be related to the inhibition of the progression of bladder tumors associated with Rb and p53, which follows ubiquitin-proteasome- mediated proteolysis.