The Study On Mechanism Of Small T Antigen Of Simian Virus 40 Endues The Benign Immortalized Prostatic Epithelial Cells With TRAIL-sensitive Phenotype

PART 1:Sensitivity and mechanism of the benign prostatic immortalized cells to TRAIL-induced apoptosisObjective Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it induces apoptosis in cancer cells but spares most normal cells. To determine whether benign prostatic immortalized cells have variable sensitivity to TRAIL-induced apoptosis and explore the possible mechanism.Methods Normal prostatic epithelial cells immortalized by combination of the early region of simian virus 40 large T antigen and a catalytic subunit of telomerase (hTERT) and then followed by the early region of simian virus 40 small T antigen (LE) were treated with different doses of TRAIL. MTT assay was used to detect the cell viability. Flow cytometry and cell morphology were performed to examine apoptotic percentages of treated cells. Western blot were used to detect the expression of PP2A and c-Fos. The PP2A activity of LH and LE cells were determined by phosphatase assays after treatment with TRAIL.Statistic analyses were performed using SPSS software version 13.0.Results After treated with increasing doses of TRAIL, LH cells were resistant to TRAIL, whereas LE cells were sensitive to TRAIL. Nearly 45% of LE cells had undergone apoptosis after administrated with TRAIL, whereas the LH cells showed much lower percentages of cell death. Western blot results revealed that PP2 A protein expressions showed no significant differences between LH cells and LE cells with/without TRAIL treatment; the protein expression of c-Fos/AP-1 significantly increased in TRAIL-sensitive LE cells.There is decreased PP2A activity in LE cells compared to LH cells. And the PP2A activity in both LH and LE cells did not change when treated with TRAIL.Conclusion SV40 ST endues the immortalized benign prostatic epithelial cells LE cells with TRAIL-sensitive phenotype. PART 2:Okadaic acid Endues the TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells LH with TRAIL-sensitive PhenotypeObjective To determine whether okadaic acid changes LH cells from TRAIL-resistant to TRAIL-sensitive and its possible mechanism.Methods The TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells (LH) were treated with different doses of TRAIL after treated by Okadaic acid, an inhibitor of PP2A. MTT assay was used to detect the cell viability. Flow cytometry and cell morphology were performed to examine apoptotic percentages of treated cells. Western blot were used to detect the expression of PP2A and c-Fos. The PP2A activity of LH and LE cells were determined by phosphatase assays after treatment with TRAIL. Statistic analyses were performed using SPSS software version 13.0.Results OA combined with TRAIL significantly increased apoptosis in the TRAIL-resistant LH cells as shown by Annexin V and cell viability assays. Western blot results revealed that OA alone or OA combined with TRAIL not only decreased the protein expression of PP2A, but also increased the protein expression of c-Fos. In accordance with our immunoblot findings, OA alone or OA combined with TRAIL decreased PP2A activity.Conclusion Okadaic acid endued the TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells LH with TRAIL-sensitive Phenotype. PART 3:A-Fos converts the LH cells from a relatively TRAIL-sensitive to a TRAIL-resistant phenotype after treatment by OA combined with TRAILObjective To explore the effect of c-Fos on the benign prostatic immortalized cells sensitive to TRAIL and its possible mechanism.Methods The TRAIL-resistant Benign Immortalized Prostatic Epithelial Cells (LH) were treated with OA, TRAIL or in combination with OA and TRAIL after transfection with/without the A-Fos vector. MTT assay was used to detect the cell viability. Flow cytometry and cell morphology were performed to examine apoptotic percentages of treated cells. Western blot were used to detect the expression of PP2A and c-Fos. Statistic analyses were performed using SPSS software version 13.0.Results OA did not enhance the ability of TRAIL to promote apoptosis like before.Western blot results revealed that the activity of c-Fos/AP-1 was inhibited by the dominant negative form A-Fos.Conclusion ectopic expression of the dominant-negative form of c-Fos, A-Fos, converted the LH cells from a relatively TRAIL-sensitive to a TRAIL-resistant phenotype after treatment by OA combined with TRAIL.