engleski

The Role of p21WAF1/Cip1 Gene in Different Cell Death Responses to DNA-damage Treatment of Colon Carcinoma Cells

p21WAF1/Cip1 is a well characterized cyclin-dependent kinase inhibitor that negatively modulates the cell cycle progression by arresting G1, G2 or S phase of the cell cycle. Moreover, p21 has a role in cell differentiation, senescence and apoptosis where it can act as an inhibitor or activator of apoptosis. Because of its complex and contradictory functions, it is important to study its roles in different cell death responses to DNA-damage treatment. In this study we evaluated the function of p21 after the DNA damage stress caused by three different chemotherapeutics (cisplatin, camptothecin and adriamycin), especially in the activation of different types of cell death, such as: apoptosis, autophagy, mitotic catastrophe and cell senescence. Four different colon cancer cell lines with diverse endogenous p53 and p21 status were used for this study (SW480, SW620, HCT116 and CaCo-2). Exogenous overexpression of p21 in >50% of the cells was achieved by infection at MOI 60 with replication-incompetent adenoviruses bearing the human p21 gene. For most of the experiments, cells were treated with IC50 concentrations of the compounds that were first established with MTT proliferation assay. The influence on the cell cycle was assessed with two parameter flow cytometry (BrdU/PI) labeling. Occurrence and magnitude of programmed cell death was measured by labeling the cells with annexin V conjugated to alexa flour 488. A specific mechanism of induced cell death in treated cells was further analyzed. Emergence of autophagic vesicle organelles was examined by fluorescent microscopy and flow cytometry after staining the cells with acridine orange. As a marker of senescence, senescence-associated β -galactosidase assay was used. Cells were fixated and then stained with X-gal substrate (pH=6). Mitotic catastrophe was further examined under fluorescent microscope after staining the DNA with DAPI and appearance of aberrant mitosis. Apoptosis was additionally confirmed by western blot with appearance of cleaved PARP-1 and activation of caspase-3. The results obtained in this study show that exogenous expression of p21 markedly influences the cell cycle, shifting a significant amount of the cells from initial G2/M arrest caused by DNA damage to G1 phase of the cell cycle. The impact on the type and the number of cells affected by programmed cell death differed after expression of p21. Both cases were also intrinsically influenced with the endogenous status of the p53 and p21 in each cell line. These results give additional insights into diverse, but interconnected mechanisms of programmed cell death and determine whether and how p21 characterizes chemosensitivity of tumor cells. We hope that the discovery of precise mechanisms that p21 bears under these circumstances could lead to the design of new and more effective treatments of cancer.

p21 ; cell death ; senescence ; autophagy

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