| [Background] The development of malignancy is believed to be a multi-step process,involving multiple genetic changes.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 tumors..DOC-2 appears to be a potential tumor suppressor gene which is expressed a new phosphophoryn with signal transduction function.lt is widespreaded in a variety of normal tissues,and is highly expressed in mammary and ovarian epithelial tissue,but the aberrant expression of DOC-2 is often in tumors such as ovarian, choriocarcinoma,prostate,and mammary tumors.DOC-2 re-pression evidently inhibites the growth and proliferation of several tumor cell lines. It appears to play important roles on the growth suppression of carcimoma.In previous studies we cloned the cDNA of PID domain of DOC-2 by RT-PCR strategy. The expressed protein was used as immunogen for making antibody. A rabbit polyclonal anti-DOC2 protein serum was successfully obtained, we also constructed the recombinant eukaryotic expression vectors pcDNA3.1-p93 of DOC-2.On this basment,we designed this experiment.[ Purpose J The current studies were designed to detect the expression of DOC-2 in ovarian neoplasms and observe effects on proliferation of human ovarian cancer cell line TC-1 into which the DOC-2 was introduced, then to explore its role in generation and development of ovarian carcinoma and the feasibility of the gene therapy in human ovarian cancer.[Methods] 1.Detecting the expression of DOC-2 in epithelial ovarian neoplasms and human ovarian cancer cell line TC-1 by immunohistochemical ABC method; 2. Recombinant eukaryotic expression vector pcDNA3.1-p93 containing exogenous human DOC-2 cDNA were introduced by lipofectamine-mediated gene transfection into TC-1 cell line which does not express DOC-2 endogenously. Empty pcDNA3.1 vectors with neomycin resistance gene only were transfected TC-1 cells as controls; 3. The clones obtained were detected for their efficiency of transfection by immunocytochemical method; 4. We studied the cell growth characteristics of TC-1 and TC-pcDNA3.1 and TC-p93 cells by comparison of cell growth curves; 5. By the test of clone formation rate, we investigate the oncogenesis character of DOC-2 gene transducted cells in vitro; 6. FCM was applied to detect the effect of DOC-2 on the cell cycle.[Results] 1. DOC-2 was expressed in all benign ovarian neoplasms. In borderline ovarian neoplasms and ovarian carcinomas DOC-2 expression, which were 38.5%and 40%,were significiantly lower than benign ovarian neoplasms,which was 100 % (P <0.01), borderline ovarian neoplasms and ovarian carcinomas were not significiantly difference (P >0.05). The expression rate of DOC-2 was disassociated with histological type and differentiation and clinical stage(P >0.05). 2. DOC-2 wasn't expressed in TC-1 cell line. 3. 10 clones named TC-p93 and 6 clones named TC-pcDNA3.1 were obtained. 4. By immunocytochemical method it was proved that TC-p93 expressed DOC-2, but TC-pcDNA3.1 didn't. 5. Morphologyof cells either from TC-p93 or TC-pcDNA3.1 did not change significantly with respect to their parental TC-1. 6. The growth rate of TC-p93 cells was inhibited,while the cell growth curve of TC-pcDNA3.1 was similar to that of TC-1. 7.The average clone formation rate of TC-p93 was 8.7 3.1%,which was significantly lower than TC-1 cell' 44 3.6% or TC-pcDNA3.1 cell' 54.3 3.2%. 8. As shown by FCM, The percentage of phase Gl of TC-p93 cells increased and that of phase S cells decreased. Cells were blocked at Gl phase. The cell cycles of the TC-p93 were prolonged, and cells proliferated more slowly as comparing to their control cells.[Conclusion] DOC-2 was a tumor suppressor gene of ovarian cancer and played a crucial role in the growth and differentiation of the ovarian epithelium. The loss of DOC-2 expression was an early step in ovarian tumorigenicity. DOC-2 had growth inhibitory activity... |
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