| ObjectiveThe aim of this study was to evaluate the expression and the relativity of Aktl, P-Akt S473and PMS2protein in human cancer cell lines, such as, A2780, Caov3, C13K, ES2, HO8910, OV2008, A875, A549, ACHN, Lovo, SW480and MCF-7.Methods1. The expression of Aktl, P-Akt S473and PMS2protein in human cancer cell lines, such as, A2780, Caov3, C13K, ES2, HO8910, OV2008, A875, A549, ACHN, Lovo, SW480and MCF-7, was detected by Western Blot.2. A875, ES2and Caov3cell lines were collected to evaluate the protein level of Aktl, P-Akt S473and PMS2by Western Blot after treated with Aktl activity IGF-1.3. After treated with specific Aktl inhibitor API-2and Akt Inhibitor I, OV2008and A2780cell lines were collected to evaluate the protein level of Aktl, P-Akt S473and PMS2by Western Blot.4. Interfering Aktl gene by siRNA, we detected the mRNA level of Aktl and PMS2in A2780cell by Quantitative Real Time PCR; we measured the protein level of Aktl, P-Akt S473and PMS2in A2780cell line by Western Blot.Results1. Protein expression of Aktl, P-Akt S473and PMS2could be detected in all the thirteen human cancer cell lines with varied expression levels. There was an approximately equal level of P-Akt S473and PMS2protein in Hela and MCF-7cell lines. Higher PMS2expression with a simultaneous lower P-Akt S473level was observed in C13K, Caov3, ES2, HO8910, Lovo, ACHN and A875cell lines. While lower PMS2and higher activated Akt1levels were seen in A2780, OV2008, SW480, and A549cell lines. Above all, Aktl activity is inversely correlated with PMS2expression in various human cancer cell lines.2. After exposed to IGF-1, an Akt kinase stimulator, A875, ES2and Caov3cell lines were detected with a dramatically PMS2expression decreasing. Also, there was a time-dependent relationship between IGF-1and PMS2expression.3. After treated with Akt kinase specific inhibitor API-2and Akt Inhibitor I, A2780and OV2008cell lines were detected with a dramatically PMS2expression increasing, and there was a time-dependent relationship between the inhibitors and PMS2expression.4. When interfering to the mRNA expression level of Aktl, we detected that the protein expression level of Akt1was dramatically decreased, while the PMS2protein expression level was increased without changes of its mRNA level.ConclusionAktl activity is inversely correlated with PMS2expression in various human cancer cell lines, and the expression of PMS2protein may be regulated by activated Akt1. ObjectiveTo investigate whether Aktl could regulate the expression of PMS2protein or the stabilization of PMS2in human cancer cell lines.Methods1. The binding of Aktl and PMS2was detected by co-immunoprecipitation (CO-IP) and immunoblotting in parent cancer cells.2. Establishing the Aktl-wt+PMS2-wt and Aktl-wt+PMS2-T156A stable cotransfected cell lines, the binding of exogenous Aktl and PMS2was examed by CO-IP and immunoblotting in those cells.3. After exposed to Aktl specific inhibitor API-2, cells were treated with CHX and collected to detect the half-life of PMS2by Western Blot.4. Inhibited the synthesis of protein by CHX, a translational inhibitor, the half-life of PMS2was evaluated by Western Blot in PMS2-wt and PMS2-T156A stable transfected cells.5. Inhibiting the activity of Aktl by API-2and siRNA, the compartmentalization of PMS2was detected by immunofluorescence assay.Results1. In Hela, A875and A2780cells, Aktl could be precipitated by anti-PMS2antibody, and anti-Aktl antibody could precipitate the endogenous PMS2.2. In Aktl-wt and PMS2-wt cotransfected SW480cell, anti-PMS2antibody could precipitate the exogenous Aktl protein, and exogenous PMS2protein could also be precipitated by anti-Aktl antibody. In Aktl-wt and PMS2-T156A cotransfected SW480 cell, the protein of Aktl precipitated by anti-PMS2antibody and PMS2protein precipitated by anti-Aktl antibody were decreased significantly.3. In Hela, A875and A2780cells, after exposed to Aktl specific inhibitor API-2, the half-life of PMS2was prolonged.4. Compared with PMS2-wt stable transfected cells, the half-life of PMS2in PMS2-T156A stable transfected cells was prolonged in Hela, A875and A2780, respectively.5. When Aktl activity was inhibited by API-2or siRNA, the endogenous PMS2protein relocalized to nucleus from cytoplasm in Hela and A875cell lines.ConclusionAktl binds to PMS2directly to decrease the stabilization of PMS2protein, and the deficient activity of Aktl was required for the nuclear localization of PMS2. ObjectiveTo investigate whether PMS2protein could mediate the platinum analogs-induced apoptosis following DNA mismatch repair processing in ovarian cancer chemotherapy resistant.Methods1. A2780and ES2cells, with different PMS2expression levels, were treated with cisplatin, and then lysates were collected to detect apoptosis-related protein (P53, P63, P73, Caspase-3and Caspase-9) by Western Blot.2. Cells with different PMS2expression levels were treated with cisplatin, and then collected to observe the condensation and fragmentation situation in the nucleus by immunofluorescence.3. Under the same cisplatin treatment, cells with different PMS2expression levels were gathered to compare the apoptosis rate by FACS.4. Under the same cisplatin treatment, cells with different PMS2expression levels were collected to detect the cell cycle by FACS.Results1. In A2780and ES2cells, PMS2could induce the expression of P63protein by cisplatin significantly, and there was a dose-dependent correlation.2. It was verified by immunofluorescence that cells with higher PMS2protein expression appeared serious nuclear condensation and fragmentation with cisplatin treatment. 3. PMS2could promote the cisplatin-induced apoptosis following DNA mismatch repair processing in ovarian cancer cell lines.4. Treated with cisplatin, ovarian cancer cells were leaded to G2/M phase arrest and apoptosis.ConclusionPMS2protein could promote the platinum analogs-induced apoptosis following DNA mismatch repair processing in ovarian cancer, and make G2/M phase arrest, which might affect the sensitivity of cisplatin to cause chemotherapy resistant. |
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