Effects Of Recombinant Adnovirus Carrying Wild Type P53 Gene On Endometrial Carcinoma Cells: An In Vitro Study

Objective:To detect the expression of exogenous p53 gene in human endometrial carcinoma cell line RL95-2 after infected with rAd-p53 in vitro and investigate the effect of rAd-p53 on the growth and chemosensitivity of RL95-2 cells and explore the feasibility of clinical application of rAd-p53 in the treatment of endometrial carcinoma.Methods:Recombinant adenovirus carrying wild-type p53 gene (rAd-p53) was used. The efficiency of adenovirus mediated gene transfer of rAd-p53 was determined by GFP. The expression of exogenous p53 gene in RL95-2 cells was detected by immunocytochemistry(ICC). The inhibitive effect of rAd-p53 on RL95-2 cells proliferation was determined by cell growth analysis and MTT assay. The changes of morphologic of RL95-2 cells transfected with rAd-p53 were observed by the inverted microscopy. Flow cytometry and Annexin-FITC/PI was used to study the cell cycle and apoptosis. Cell proliferation was measured by MTT and colony formatting cell culture test were used to study the chemosensitivity of RL95-2 cell transfected with rAd-p53 to cisplatin (DDP) and adriamycin (ADM).Results:1. Exogenous p53 gene was successfully transfected into RL95-2 cells and the efficiency of adenovirus mediated rAd-p53 gene transfer was more than 90% when the multiplicities of infection(MOI) was 100. 2. Analysis by ICC on the expression of p53 protein in RL95-2 cells showed that the intensity of p53 expression in rAd-p53 group was remarkably strong compared with that of the adenovirus with GFP gene (Ad-GFP) infected cells and control cells(P﹤0.05). 3. rAd-p53 significantly suppressed the growth of RL95-2 cells. The growth curve showed that the speed ofproliferation of rAd-p53 group was significantly slower than that of Ad-GFP group and control group. The growth inhibition rate of rAd-p53 group in different times were higher than those of Ad-GFP group (P﹤0.01), and there was a dose-dependent inhibition of cell proliferation by rAd-p53 after infection for 72h. The cells infected rAd-p53 showed time-dependent typical morphologic changes. 4.rAd-p53 could also induce the apoptosis and arrest cell cycle in G1. There was a significant difference(P﹤0.01)between rAd-p53 group and Ad-GFP group and control group in apoptosis rate and G1/S rate. 5. The IC50 values for DDP and ADM were reduced to approximate 1/10 and 1/18 in rAd-p53 cells respectively, while the change was not observed in Ad-GFP cells and control cells. The colony formation of rAd-p53 cells was significantly inhibited compared with Ad-GFP cells and control cells(P﹤0.01).Conclusion:1. Recombinant adenovirus rAd-p53 transfection system is highly efficient in introducing wild type p53 gene into human endometrial carcinoma RL95-2 cells which bear mutant p53. 2. rAd-p53 can strongly inhibit cell proliferation and induce apoptosis and arrest cell cycle in G1 in RL95-2 cells. 3. rAd-p53 is capable of enhancing the sensitivity of human endometrial carcinoma cell line RL95-2 to DDP and ADM, and rAd-p53 may be one of the combined modality therapies for the patients in advanced stage or being too weak to tolerate surgery and large dose radiotherapy and chemotherapeutic drug or recurrent endometrial carcinoma.