Primary Function Study Of DOC-2

Ovarian cancer is a fatal tumor in Gynaecology. The lack of effective diagnosis and treatment ways brings its mortality to the head. With the advance in molecular techniques,the study on the pathogenetic mechanism and the relationship among tumor, oncogene and tumor suppressor at molecular level has become one of the main developing directions in gene therapy on tumor. Using RNA fingerprinting (RAP) strategy and Northern blot analysis, Mok et al identified a differentially expressed sequence DOC-2(Differentially expressed in ovarian cancer 2) which is detectable in all normal human ovarian surface epithelial (HOSE) cell cultures but not in ovarian cancer cell lines and tissues. The DOC-2 gene is located on chromosome 5pl3 and its full length cDNA is 3268bp. Translation of the DOC-2 cDNA predicts a hydrophobic protein of 770 amino acid residues with a molecular weight of 82.5 kDa. DOC-2 has a phosphotyrosine interactingdomain (PID) on in its N-terminus and multiple proline-rich SH3 binding motifs in its C- terminus. DOC-2 is a novel phosphoprotein with signal-transducing capability. It appears to be a potential tumor suppressor gene with a growth inhibitory effect on several cancer types. However, its mechanism of action is not understood completely. The primary structure of DOC-2 reveals that DOC-2 is a putative signaling molecule with protein-protein interaction and protein phosphorylation as two possible mechanisms modulating its activity.In order to address the function of DOC-2, we cloned the cDNA of PID domain of DOC-2 by RT-PCR strategy. Then we constructed the expression vector of this cDNA and expressed protein in ?Coli.The expressed protein was used as immunogen for making antibody. A rabbit polyclonal anti-D0C2 protein serum was successfully obtained.To investigate the feasibility of DOC-2 in gene therapy on ovarian cancer, we constructed the mammalian expression vectors (pcDNAS. l-p93 and pIRES2-EGFP-p93) of DOC-2. Then we transfected them into human ovarian cancer cells HO-8910 and proved its expression by immunocytochemical method. We studied the cell growth characteristics of HO-8910, 8910-pcDNA3.1 and 8910-p93 cells by comparison of cell growth curves. By the test of clonal growth in soft agar medium , we compared the ability of cloning and proliferating among these three kinds of cells. The cell cycle was analyzed by FCM(Flow cytometry) and morphosis of cells was observed under transmission eletron microscope. To observe the cells growth in vivo, we established the model of transplant tumor in nude mice. The stable transfectants showed significantly reduced growth rate andability to form tumors in nude mice. FCM analysis showed that the ratio of S-phase cell decreased and the ratio of G1-phase cell increased. The results observed under electron microscope showed cell growth inhibition in 8910-p93 cell ultra structure. These data suggest that DOC-2 has growth inhibitory activity for ovarian cancer cells and down-regulation of DOC-2 may play an important role in ovarian carcinogenesis.To further understand the function of DOC-2, DOC-2 and nDOC-2 were used as "bait" sequence in the yeast two-hybrid system to search for protein(s) that interacts with DOC-2 and the N-terminal domain of DOC-2. Firstly we transformed the Bait vector into yeast strain AH-109. The self-activation of bait protein nDOC-2 was excluded by phenotype selection and β-galactosidase assay, and DOC-2 was proved to be self-activated. By screening the library, 21 positive clones were selected, and three positive clones were further analyzed. Further analysis of these candidated genes can give some clues for the further studies of DOC-2.