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IASPP in MCF7 cells and C57 in mouse thymocytes apoptosis resistance

Author: WangLin
Tutor: PangTianXiang
School: Beijing Union Medical College
Course: Pathology and Pathophysiology
Keywords: ASPP family iASPPsv MCF7 apoptosis Real time PCR H2AXASPP family iASPP transgenic mouse thymocytes H2AX
CLC: R363
Type: PhD thesis
Year: 2010
Downloads: 16
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Abstract

Objective:iASPP is an inhibitory member of ASPP (apoptosis stimulating protein of p53) and can specifically inhibit the apoptotic function of p53. In previous study, we identified a novel isoform of human iASPP, which encodes a407aa protein and highly matches with the C terminus of iASPP(828aa). It was found to be able to inhibit the transcriptional activity of p53on the promoters of Bax in our previous study. Here, we investigate the effects of overexpression of iASPPsv on the biological function and sensitivity to chemotherapeutic drug of MCF7(human breast cancer cell line).Methods:The lentiviral vectors pCDH containing iASPPsv was constructed to transduce MCF7-cells. The iASPPsv-GFP+clones were screened following limiting dilution and the GFP+clone only containing empty vector pCDH was used as control. After treated with Etoposide (VP-16), the proliferation and apoptosis of the cells were analyzed by MTT and FACS, respectively. The expression of apoptosis-associated genes Bax, PUMA and Noxa were detected using Real-time PCR. Moreover, H2AX was analyzed following Western blot.Results:1. Three pCDH-iASPPsv clones showed obvious up-regulation of iASPPsv protein than that of pCDH clone.2. The inhibition rate of proliferation decreased in pCDH-iASPPsv clones compared with that in pCDH clone.3. After treated with VP-16at200μg/mL for24h,48h, the apoptotic rate and the mRNA levels of Bax, PUMA and Noxa were lower in pCDH-iASPPsv clones than those in pCDH clone, while the expression of H2AX protein was higher at24h treatment.Conclusion:iASPPsv gene could reduce the chemosensitivity of MCF-7cells to cytotoxic drugs VP-16and exhibit the inhibitory effect on cell apoptosis, and that can promote the accumulation of the cells harboring DNA damages. Perhaps, this is the mechanism that iASPPsv involve in the tumorgenesis. Objective:As an inhibitory member of ASPP (apoptosis stimulating protein of p53) family, iASPP can inhibit the p53induced transcription of proapoptosis genes, therefore inhibit the p53induced apoptosis. We have demonstrated that iASPPsv can relieve the inhibitory proliferation of MCF7cells from VP16treatment. iASPPsv also decreased the expression of proapoptosis genes Bax, PUMA and Noxa in these VP-16treated cells. As a result, iASPPsv could reduce the VP-16induced apoptosis in MCF7cells, whereas the expression of H2AX protein increased, which represented more DNA damage accumulated in cells. To investigate the role of iASPP/iASPPsv in normal cells, we have already established an transgenic mouse model with iASPP/iASPPsv expression.In this study, we isolate the thymocytes of these transgenic mouse and expose them to Dex or VP16, and to explore if iASPP/iASPPsv can antagonise the effect on apoptosis induced by the chemotherapeutic agents.Methods:To isolate the thymocytes from thymus of wild type C57mouse and the iASPP/iASPPsv transgenic mouse using lymphocyte separation medium. The thymocytes were treated with Dex or VP16, then the inhibition rate of proliferation was masured by MTT, apoptosis of the cells was analyzed by FACS and the expression of H2AX protein was detected by Western blot.Results:(1) The cluster of differentiation antigen of thymocytes from the wild type mouse C57and the iASPP/iASPPsv transgenic mouse is measured by FACS, there are no significent differences between different groups.(2) The two groups of thymocytes were treated with Dex or VP-16at various concentrations for48h, we found that the inhibition rate on proliferation of the thymocytes from transgenic mouse is lower than that of the wild type mouse.(3) After treated with Dex or VP-16for12h,24h and48h, the thymocytes from the transgenic mouse showed lower apoptosis rate than the control group.(4) The expression of H2AX protein is higher in thymocytes of the transgenic mouse than that of the wild type mouse after treated with1nM Dex or0.1μg/ml VP-16for48h.Conclusion:iASPP/iASPPsv could antagonise the inhibition rate of proliferation and the apoptosis caused by Dex or VP-16in mouse thymocytes, meanwhile, iASPP/iASPPsv increased the proportion of the thymocytes containing more DNA damages.

IASPP in MCF7 cells and C57 in mouse thymocytes apoptosis resistance

Abstract

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