Role Of PTEN In The Malignant Development Of Glioma Cells With Different P53 Genotypes

Background:Glioma is one of the most lethal human tumors,which originate within brain.The prognosis for patients with high-grade gliomas is very poor owing to the invasiveness,rapid progression and recurrence of the tumor.Therefore it will significantly improve the patient's prognosis if we are able to inhibit or delay the progression of gliomas somehow.PTEN(phosphatase and tensin homolog deleted on chromosome 10) and p53 (also known as tumor protein p53,TP53) acting as the two tumor suppressor genes are involved in cell cycle arrest,cell differentiation and apoptosis.The interaction between PTEN and p53 in glioma progression remains obscure.Glioblastoma(GBM) is the most malignant glioma presenting as one of two subtypes with distinct clinical histories and molecular profiles.The primary GBM subtype presents acutely as a high-grade disease that typically harbours EGFR amplification(36%) and the mutation rates in PTEN(25%) and p53(28%),and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses EGFR amplification(8%) and mutation rates in PTEN(4%) and p53(65%).We pay much attention to the phenomenon of low PTEN mutation and high p53 mutation in secondary GBM.The reason is that PTEN has tumor-promoting properties in the setting of gain-of-function p53 mutations,which possibly indicate that low-grade gliomas with wild type PTEN are more likely to develop into secondary GBM. Objective:In this study,we try to elucidate how PTEN is inactivated and the role of PTEN during the malignant gliomas development via investigating the distinct function of PTEN between glioma cells harbouring wild type and mutant p53,and the effect of PTEN phosphorylation on its function as well.Methods:1.To identify the mutational site of p53 in SWO-38 cell,the exons 5,6,8 were amplified by PCR and sequenced.Using U251 glioma cell(mut-PTEN,mut-p53) as the control of SWO-38 cell,p53 and nestin were detected by immunocytochemistry(ICC) and immunofluorescence(IF);GFAP,MMP-2 and MTA1 were detected by Western blot;cell proliferation was evaluated by WST-8 assay;cell migration and invasion were evaluated by Transwell assay.2.PTEN expression in SWO-38 cell was knocked down by target-specific siRNAs of wild type PTEN.The changes of PTEN and p53 expression in SWO-38 cell were detected by Western blot;cell proliferation was evaluated by WST-8 assay; cell cycle was analyzed by flow cytometry(FCM);cell migration was evaluated by Transwell assay.Adenoviruses encoding wild type PTEN(Ad-PTEN) were constructed to infect U87 cell(mut-PTEN,wt-p53).Methods of above-mentioned were used to detect the changes of cell proliferation,cell cycle and cell migration in U87 cell.3.The expression of total PTEN,phosphorylated PTEN,MMP-2 and MTA1 in SWO-38 cell and its sublines Z1,Z2 were detected by Western blot,as well as the change of phosphorylated PTEN in SWO-38 cell treated with DRB(CK2 inhibitor).The effect of DRB on SWO-38 cell proliferation was evaluated by WST-8 assay. Results:1 Biological feature of SWO-38 cell1.1 The sequences of p53 exons 5 and 6 in SWO-38 cell were identical to wild type p53.Exons 8 harbouring a missense mutation which was designated as R280T.The ICC result showed that p53 was observed in the nucleus of SWO-38 cell.1.2 By WST-8 assay,the growth curves of SWO-38 and U251 cell were distinct. The proliferation ability of SWO-38 was higher than U251 cell.At day 5, SWO-38 reached the death phase while U251 stayed at stationary phase.1.3 In migration assay,SWO-38 cells migrated below the Transwell chamber was much fewer than U251.The cells were quantified by WST-8 assay,and the values of absorbance were 0.316±0.029(SWO-38) and 1.297±0.035(U251) with statistical significance(P＜0.05),which indicated that the migration ability of SWO-38 cell was much lower than that of U251 cell.In invasion assay,the values of absorbance were 0.478±0.027(SWO-38) and 0.481±0.034 (U251) with no statistical significance(P＞0.05).1.4 By immunofluorescence staining,nestin was filamentous in the cytoplasm of SWO-38 cell,but in U251 cell,nestin appeared as a perinuclear distribution and extending to the cytoplasm.The results in Western blot revealed that two cell lines had different molecular weights of nestin.1.5 The results in Western blot revealed that PTEN was positive in SWO-38 cell while negative in U251 cell,and the GFAP expression level of SWO-38 cells was lower and MMP-2,MTA1 were higher compared with those of U251.2 The distinct function of PTEN between glioma cells harbouring wild type and mutant p532.1 The results in Western blot revealed that among three PTEN-target siRNAs, si-h-PTEN₀03 significantly knocked down PTEN expression level in SWO-38 cell.2.2 Ad-PTEN were confirmed by PCR and sequencing.PTEN was expressed in U87 cell infected with Ad-PTEN.2.3 By WST-8 assay,no significant difference of proliferation ability was found between SWO-38 cells in siRNA-Control and siRNA-PTEN group(P＞0.05); the proliferation ability of U87 cell in Ad-PTEN group was significantly inhibit compared with that of Ad-GFP group(P＜0.05).2.4 Cell cycle analyses by FCM showed that G₀/G₁,S,G₂/M distributions of SWO-38 cells in siRNA-Control group were 53.4±4.5%,34.5±3.4%and 10.5±1.0%,respectively;while 54.6±5.5%,36.1±3.7%and 11.2±1.1%in siRNA-PTEN group,respectively.No statistical significance was found between two groups(P＞0.05).G₁/G₁,S,G₂/M distributions of U87 cells in Ad-GFP group were 71.2±4.6%,19.3±3.4%and 10.4±1.0%,respectively; whereas 87.5±5.3%,10.7±2.3%and 2.5±0.8%in Ad-PTEN group, respectively.Statistical significance was found between two groups(P＜0.05), which indicated that Ad-PTEN caused the cell cycle arrest in U87 cells.2.5 In migration assay,SWO-38 cells in siRNA-Control group migrated below the Transwell chamber was much fewer than those in siRNA-PTEN group.The cells were quantified by WST-8 assay,and the values of absorbance were 0.482±0.064(siRNA-Control) and 0.633±0.065(siRNA-PTEN) with statistical significance(P＜0.05).As for U87 cells,the values of absorbance were 0.842±0.054(Ad-GFP) and 0.653±0.063(Ad-PTEN) also with statistical significance(P＜0.05).The results indicated that PTEN inhibit migration in both cells.2.6 The results in Western blot revealed that no change of p53 expression was found in siRNA-PTEN group SWO-38 cells,compared with that of siRNA-Control group. 3 Expression of phosphorylated PTEN in SWO-38 cell and its sublines Z1,Z23.1 The results in Western blot showed that no difference of total PTEN expression was found in SWO-38 cell and its sublines Z1,Z2,while the expression level of PTEN phosphorylated at Ser380/Thr382/Thr383 is the highest in Z1 cells.3.2 The results in Western blot showed that the expression levels of MMP-2 and MTA1 is the highest in Z1 cells compared with that of SWO-38 and Z2 cells.3.3 The results in Western blot showed that in SWO-38 cell,no change of total PTEN expression was found,while phosphorylated PTEN was down-regulated after treated with DRB.3.4 DRB significantly inhibit cell proliferation of SWO-38 cell,compared with control group(P＜0.05).3.5 The effect of DRB on SWO-38 cell proliferation was different between siRNA-Control group and siRNA-PTEN group.By WST-8 assay,the values of absorbance were 0.379±0.032 and 0.657±0.019 with statistical significance (P＜0.05) after treated with DRB.Conclusions:1.The p53 mutation site of SWO-38 cell was R280T,which is a gain-of-function mutant.It is because of the biological characteristic of SWO-38 cell line that the cell line could be a useful model in the studies of glioma progression and the mechanisms of interaction between PTEN and p53.2.PTEN possess distinct effect on cell proliferation between glioma cells harbouring wild type and mutant p53.PTEN did not affect cell proliferation and cell cycle in mut-p53 SWO-38 cell,while PTEN inhibit cell proliferation and caused cell cycle arrest in wt-p53 U87 cell.The results suggested that cell proliferation regulated by PTEN was p53-dependent.However the inhibitive ??role of PTEN on cell migration in both cell lines was p53-independent.It is obvious that PTEN regulate cell proliferation and cell migration independently, which the combination determined the role of PTEN in glioma progression.3.The expression level of PTEN phosphorylated at Ser380/Thr382/Thr383 is the highest in Z1 cells which was the most malignant.Downregulation of PTEN phosphorylation inhibited cell proliferation,which suggested that PTEN phosphorylation resulted in functional inactivation and caused glioma progression.PTEN phosphorylation is the important pattern of posttranslational inactivation.