| As one of the most important tumor suppressor genes,the products of p53 expression known as p53 protein(P53),is posttranslationally modified. In response to various cellular stresses, P53 expression is increased and resulting in either arrest of the cell cycle at G1 and G2/M,and then DNA damage repair,or commitment to death through apoptosis. The p53 is also one of the most frequently mutated genes. Mutations in this gene have been found in about 50% of human cancers. The other 50% of human tumours retain wild-type p53,but it is not known how the function of p53 protein is suppressed and, moreover, by what and how it can be reactivated in these tumors? These questions remain unclear recently, a new family of protein, apoptosis-stimulating protein of p53 (ASPPs) was discovered. This discovery may shed some light on above conundrum.Objective:To observe in p53 wild-type tumor cell lines A549 and A375p whether the status of ASPP proteins expression might be important in controlling chemoresensitivity, especially, in DNA-damaging chemotherapeutic agents induced apoptosis.Methods:Human non-small cell lung cancer A549 and melanoma A375p which remain wild-type p53 were treated with cisplatin (CDDP), Pirarubicin (THP) orβ-elemene (β-Ele). The smallest efficacious concentrations of different DNA-damaging agents and the growth arrest of A549 and A375p cell lines were detected by MTT assay. Morphological features of apoptotic A549 and A375p cells induced by CDDP,THP andβ-Ele were observed with electronmicroscopy, meanwhile, apoptotic rates(AR) were analysed by the flow cytometry(FCM). Immunofluorescence was used to show the expression and localization of iASPP in A549 and A375p cells. To examine the role of ASPP2 in these tumour cells, we analyzed the changes of ASPP2 mRNA expression in cells treated with CDDP,THP andβ-Ele by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR).finally all the data were processed in SPSS software . Means was denoted with means±SD, and analyzed by ANOVA or Wilcoxon statistical methods according to the features of different data.Results:The results of MTT assay showed that a time-dependent and concentration-dependent inhibition of both A549 and A375p cells proliferation and viability were observed upon treatment with CDDP andβ-Ele, but there are little concentration-dependent in THP-treated group. Exponentially growing A549 and A375p cells were treated with CDDP (3.0μg/ml), THP(1.25μmol/l) andβ-Ele(50.0μg/ml), and harvested for flow cytometric analysis of DNA content by propidium iodide(PI)staining. The results indicated that rates of apoptotic cells of A549 and A375p treated after 24 hour were significantly higher(P<0.01) than that of untreated cells. A549 cells are more sensitive to CDDP-induced apoptosis(P<0.01) and less sensitive to THP andβ-Ele-induced apoptosis than A375p(P<0.01, P<0.05 respectively). Condensation and crack of nucleus and apoptotic bodies appeared in apoptotic cells of A549 and A375p cell lines in all treated groups and necrocytosis were to be seen in some groups. Fluorescence imaging experiments demonstrated that accumulation of iASPP in cytoplasm but little distribution in nucleus in all groups. untreated groups in two cell lines presented a bright-green colour,followed by CDDP-treated groups,and THP-treated groups is the darkest one in all groups.But there was no significant difference betweenβ-Ele treated groups and control groups. The analysis of RT-PCR products indicated that the mRNA expression of ASPP2 in A549 cell line treated with CDDP and THP,and in A375p cell line treated with CDDP,THP andβ-Ele were higher than that of their control groups(P=0.029,P=0.001;P=0.047,P< 0.001, P= 0.01 respectively) . However, there was no significant difference betweenβ-Ele treated group and control group in A549 cell line(P=0.185).Conclusion:Our study suggested that the cell growth of A549 and A375p with wild-type p53 can be inhibit markedly by CDDP,THP andβ-Ele . Apoptosis may be an important mechanism of A549 and A375p cell lines killed by these three drugs, and ASPPs may play a role in the process of apoptosis induced by chemotherapeutic agents. In both A549 and A375p cell lines after treated with CDDP or THP, The expression of ASPP2 is upregulated significantly, expression of iASPP is downregulated. These results, together with the research of others proved that the apoptotic function of p53 is stimulated by ASPP2 and inhibited by iASPP in tumors with wild-type p53. |
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