Influence Of Mutant^T34A And Deleted^N-8aa Survivin Genes On The Growth And Chemotherapy Sensitivity Of Epithelial Ovarian Cancer Cells

Subjective:To study the cell cycle distribution of HO-8910 human ovarian cancer cells transfected with wild-type,mutant ^T34Aand N-end 8 amino acids deleted(deleted^N-8aa) survivin genes and the influences of CDDP,Taxol and PI3K inhibitor LY294002 on the growth inhibitions of the transfected cells,exploring their roles of the differently modified survivin genes for apoptosis induction by different chemotherapeutic drugs and possible signaling pathways involved.Methods:(1)HO-8910 cells transfected with or without wild-type,mutant ^T34A and deleted ^N-8aa survivin genes were passaged for 10 generations culture, and then co-cultured with G418 for two weeks.The total RNA were extracted from the transfected or non-transfected cells.The PCR products were prepared by using RT-PCR and agarose gel electrophoresis for sequence-specific identification.(2)The cell cycles were determined by flow cytometer(FCM); (3)After the transfected and no-transfected HO-8910 cells were treated with cisplatin,taxol and PI3K inhibitor LY294002 respectively for 48h,the growth inhibitions were determined by using MTT assay,the IC50's and sensitivity index(IC50_survivin/IC50_pcDNA) were calculated as a curvilinear regression equation.Results:(1)HO-8910 cells transfected with wild-type,mutant^T34A, deleted^N-8aa survivin genes exhibited stably expressions of survivin mRNAs,but not HO-8910 cells transfected with pcDNA and HO-8910 cells as determined by RT-PCR.Sequence analysis showed that the base sequences of PCR products(cDNAs for survivin) are fully consistent with the transfected survivin genes.(2)The cell cycle distribution of the transfected cells changed significantly. 44.72%of the cells transfected with wild-type survivin gene were found in G₁ phase,and 54.22%and 1.06%were in S and G₂ phases respectively;63.51% of the cells transfected with mutant^T34A survivin gene Were found in G₁ phase, and 36.33%and 0.16%were in S and G₂ phases respectively;54.46%of the cells transfected with deleted^N-8aa survivin gene were found in G₁ phase,and 44.92%and 0.62%in S and were G₂ phases respectively,and 49.64%of the cells transfected with pcDNA found in G₁ phase,and 49.80%and 0.55%in S and G₂ phases respectively. The cell cycles of the cells transfeced with wild-type Survivin gene and with mutant^T34A or deleted^N-8aa survivin genes were transited to S/G2 phase and G1 phase respectively,as compared to the cells transfected with pcDNA.(3)The IC_50's(μmol/L) of DDP,Taxol and LY294002·for the HO-8910 cells transfected with wild-type survivin gene were 20.381±6.132,36.702±3.974 and 44.693±14.322 respectively;for the HO-8910 cells transfected with mutant^T34A survivin gene,9.854±1.166;28.645±2.761 and 66.892±4.777 respectively;for the HO-8910 cells transfected with deleted ^N-8aasurvivin gene,7.55±1.028, 37.864±4.785 and 13.161±4.043 respectively;for the HO-8910 cells transfected with pcDNA,14.384±3.861,28.569±3.581 and 41.048±7.861 respectively. Defining drug sensitive ID of the cells transfected with pcDNA as 1.0,the sensitivity of the cells transfected with wild survivin gene to DDP,Taxol,and LY294002 decreased by 42%,28%and 9%respectively(P＜0.01);the sensitivity of the cells transfected with deleted^N-8aa survivin gene to DDP and LY294002 increased by 48%and 68%respectively(P＜0.01),but to Taxol decreased by 33%(P＜0.01);the sensitivity of the cells transfected with mutant^T34A survivin gene to Taxol showed no change,but to LY294002 decreased by 63%(P＜0.01).Conclusions:Survivin^wild could prompt the cell cycle of HO-8910 cells to transit to S/G2 phase,however,mutant^T34A,deleted^N-8aa survivins could lead the cell cycle of HO-8910 cells to transit to the,G1 phase,suggesting that survivin exist in G2 phase of cell cycle.Survivin^wild resulted in a significant,cell cycle-independent reduction in sensitivity of HO-8910 cells to DDP,Taxol,and LY294002,but mutant^T34A,deleted^N-8aa survivins showed an obvious differences between cell cycle-dependent and independent drugs.This may be related to the molecular structures of mutant^T34A and deleted^N-8aa survivins for trigger distinct apoptotic signaling pathways by different drugs,and requires further studies.