Expression Of Wild-type P53-induced Phosphatase1in Gliomas And It Confers The Proliferation Of The Glioma Cell Lines

Gliomas are the most common primary tumors of the central nervoussystem affecting humans. Despite new improvement to the standard of caretherapy which including operation, radiation therapy and chemotherapy forhigh-grade primary malignant brain tumors, the prognosis of patients with thisdisease remains poor. According to the reports, the median survival time ofpatients with glioma III (WHO grades) is about3~5years and glioblastomaless than1year. Therefore, effective therapeutic approaches that based on abetter understanding of the pathophysiologic and molecular properties ofmalignant glioma are urgently needed. Wip1(wild-type p53-inducedphosphatase or PPM1D) is a member of the PP2C family of evolutionarilyconserved protein phosphatases maps to17q23/q24, encoded by proteinphosphatase magnesium-dependent1delta. Originally described as anoncogene, Wip1has been implicated as a negative regulator of p53via itsability to attenuate ATM, chk1/chk2and p38MAPK-p53activity. Theimportance of this negative feedback loop has been further illustrated by thefindings that Wip1cooperates with known oncogenes, such as Ras, Myc andNeu1, to transform rodent wild-type primary fibroblasts and induceanchorage-independent growth in soft agar by Bulavin in2002. In the nearyears, studies have demonstrated that Wip1is overexpressed in a wide rangeof tumor tissues including breast cancer, neuroblastoma, ovarian clear cellcarcinoma and medulloblastomas. Overexpression of Wip1is significantlyassociated with poor clinical outcome in some patients with breast cancer,gastric cancer and well-differentiated pancreatic neuroendocrine tumors.Currently, It has been successfully carried out the experiments in vivo and invitro on Wip1as a gene therapy target, and get significant results. However,previous studies have not investigated whether Wip1is expressed and its level in different type and grade gliomas, and the relationship between Wip1andoverall survival of the patients. Furthermore, there are still no reports to dateon whether Wip1is expressed in the gliomas cell lines and its roles in cellproliferation.Based on the above background, the expression of Wip1mRNA as wellas the protein levels were examined using RT-PCR, Western blot andimmunohistochemistry in different grades of primary human gliomas andnormal brain tissues. Moreover, we investigated the association of patientprognosis with Wip1expression. It is reports that Wip1transcript was inducedby ultraviolet and IR in a p53-dependent manner. Lymphoma cell lines withwild-type p53consistently showed IR-induced increases in Wip1mRNAwhile p53-deficient Burkitt lymphoma cells showed little or no induction ofWip1expression following radiation treatment. p53was initially identified asa tumor suppressor, overexpression of Wip1was associated with p53negativeexpression in breast cancer. p53phenotype was closely related to Wip1expression level that contact with poor prognosis for breast cancer patients.Therefore, it is important to explore the relationship of Wip1and p53. It isimportant to elaborate the relationship of Wip1expression and p53phenotypein glioblastoma cell lines for further understanding the maligant biologicalcharacteristics of gliomas. We intended to investigated Wip1expression indifferent p53phenotype glioblastoma cell lines, and screening Wip1advantagecell lines for lay the foundation of the next experiment.Oncogene activation or high expressed contributed to tumor developmentand the therapeutic strategies to antagonize its expression, and the mostcommonly means of gene therapy is RNA interference. We would exploreWip1role in the glioblastoma cells proliferation using RNA interferencemethods to silencing its expression. These provided the theoretical foundationfor the glioma gene therapy.The content of present thesis consists of the following3parts:Part one: The Expression of Wip1in the gliomas and its clinicalsignificance Objective: This study aimed to detect the expression of Wip1in differentgrades of primary human gliomas and to analyze its prognostic value in thepatients.Methods:1Tumor specimens and patients clinicopathological characteristicsA total of81cases with glioma were admitted for surgical treatment atthe Second Hospital of Hebei Medical University between2003and2005.Patient characteristics, including KPS score, tumor sizes and location werecollected before initial surgery. No patients received any treatment such asradiation or chemotherapy before the operation. Fifteen normal brain tissuesamples were obtained by collecting donations from consenting individualswho died in traffic accidents. These samples were confirmed as normal usinglight microcopy and special tissue staining. These tissues were divided intotwo parallel parts: one part was frozen and stored at-80°C until RNA andprotein were extracted and the other part was formalin-fixed. Histologicaltumor typing was performed using resected specimens in the department ofpathology of the same hospital by two or more experienced neuropathologists.Detailed clinical and demographic information, prognostic factors, and diseaseprogression were retrospectively collected post-operation6to12months inthis study. Clinical information was obtained by reviewing the medical records,telephone information and death certificates.2The expression of Wip1were detected by immunohistochemistry.The expression of Wip1were examined using immunohistochemistry indifferent grades of primary human gliomas and normal brain tissues.3The relationships between Wip1-positive cases and variousclinicopathological characteristics.The relationships between Wip1-positive cases and variousclinicopathological characteristics including patient's ages, sex, gliomapathological grade, tumor size, location, PCNA expression, p53expressionand Karnofsky performance scale (KPS) score at the time of surgery arerecorded. For survival analysis, we used the Kaplan-Meier method. The statistical significance between survival curves was assessed using thelog–rank test. Overall survival (OS) was determined from the date of surgeryto the time of death. Significant variables from the univariate analysis wereentered into the Cox hazard model analysis. The Cox proportional hazardsmodel was used to examine the association between the OS and potentialprognostic factors.4The expression of Wip1mRNA in gliomas and normal brain tissues.The expression of Wip1mRNA were examined using RT-PCR in differentgrades of primary human gliomas and normal brain tissues.5The expression of Wip1protein in gliomas.The expression of Wip1protein were examined using Western blot in differentgrades of primary human gliomas.Results:1Immunostaining analysis showed that Wip1protein was localized to thecytoplasm and nucleus of gliomas and highly expressed. It is also expressed inthe blood vessels of high grade gliomas. In15normal brain tissues, theexpression of Wip1was not detected or was weakly expressed. The frequencyof Wip1-stained samples was55%(45/81) of all glioma samples that wereexamined. The expression of Wip1in glioma samples were higher than innormal brain tissues(P＜0.01). In addition, gliomas with different gradesexhibited mild to strong Wip1expression. Indeed,35/43grade III-IV gliomasexhibited high levels of Wip1, whereas28/38grade II gliomas exhibitedundetectable or weak levels of the protein. The expression of Wip1in gradeIII-IV gliomas were higher than that in grade II gliomas tissues (P<0.05).2Wip1mRNA in different grade gliomas tissues and normal brain tissues wasexamined by RT-PCR. The mRNA level of Wip1expression in gliomas wasdisplayed. The normalized mRNA expression of Wip1was0.08±0.01,0.25±0.04, and0.28±0.03for normal brain tissues, low-and high-grade gliomas,respectively. Wip1mRNA expression was significantly increased in thegliomas compared to that in the normal brain tissues (P<0.05). However, theexpression of Wip1mRNA was not significantly different between the high- and low-grade gliomas (P>0.05).3Wip1protein in different grade gliomas tissues was examined by westernblot. Wip1was overexpressed in glioma tissue specimens, especially in highgrade glioma specimens normalized to GAPDH protein control. Theexpression of Wip1in grade IV gliomas tissues (3.05±0.18) and grade IIIgliomas tissues (2.53±0.11) was stronger than that in low grade gliomas(1.71±0.10)(P<0.01). Moreover, there was also a notable difference betweengrade III and grade IV glioma tissues (P<0.05).4p53protein was localized in the tumor nucleus by immunostaining. Thefrequency of p53-positive specimens was72%(31/43) in the high gradegliomas, while50%(19/38) in the low grade gliomas. Additionally, we did notfound p53positive expression in the normal brain tissues. Immunostaininganalysis showed that the proliferating cell nuclear antigen (PCNA) protein waslocalized in the nucleus. The frequency of PCNA-positive specimens was89%(72/81) of all the glioma samples that were examined. However, noPCNA-positive cells were observed in normal brain tissues. The percentage ofPCNA-positive cells was positively associated with increasing glioma grades(P<0.05).5Wip1expression did not significantly correlate with age, gender and tumorlocation (P>0.05). Increased expression of Wip1was significantly associatedwith the glioma pathological grade, PCNA expression, tumor size, p53expression and KPS score (P<0.05). Spearman's rank correlation analysisshowed a positive correlation between Wip1and PCNA (r=0.639, P<0.001).6A univariate analysis using log-rank analysis indicated that among theclinicopathological factors, the pathological grade, tumor size, KPS score andWip1-positive expression correlated with the outcome(P＜0.05). Furtherassessment using the Cox's multivariate analysis indicated that thepathological grade (grade Ⅲ-IV), KPS score (<80) and increased Wip1expression were statistically significant predictors for overall survival(P＜0.05). Conclusions:1We demonstrated that Wip1overexpressed in gliomas and increased withtumor grades. The Wip1mRNA was amplified in different grades of gliomatissues with no statistically significant differences among Wip1expressingsamples. However, the expression of Wip1was highly expressed in themajority of malignant gliomas and that Wip1protein levels correlated withincreasing tumor grades. Discrepancies in Wip1mRNA and proteinexpression profiles were observed. It is suggests that additional mechanismsfor Wip1protein expression exist, such as mechanisms that regulatepost-translational modifications and the half-life of Wip1protein.2Increased Wip1expression was significantly associated with poorer overallsurvival.3High expression of p53were detected by immunohistochemistry using p53antibody in Wip1-positive glioma tissues. The PCNA index were higher in theWip1-positive group than in the Wip1-negative group. Positive correlationbetween Wip1and PCNA expression was shown in the glioma tissues.4Increased expression of Wip1was significantly associated with the gliomapathological grade, PCNA expression, tumor size, p53expression andKarnofsky performance scale score. Wip1expression did not significantlycorrelate with age, gender and tumor location.Part two: Expression and Significance of Wip1in glioblastoma Cell LinesObjective: To explore the expression of proto-oncogene Wip1in U87and U251cell lines which related to different p53phenotypic and to screeenhigh expression cell lines. Co-immunofluorescence detection of Wip1, PCNAand p53in U87, U251and MCF-7cell lines was performed.Methods:1The expression of Wip1were detected in U87and U251cell lines whichrepresented different p53phenotypic by immunofluorescence.2Co-immunofluorescence detection of Wip1/PCNA and p53/Wip1in U87and U251cell lines were performed.3The expression of Wip1were detected in U87and U251cell lines by flowcytometry. 4The expression of Wip1mRNA were detected in U87and U251cell lines byRT-PCR.5The expression of Wip1protein were detected in U87and U251cell lines byWestern blot.Results:1The expressions of Wip1presented strong immunoreactivty in U87cells(wild-type p53phenotypic), which were higher than those in U251(mutant-type p53phenotypic) cells by immunofluorescence. There was astatistically significant difference between two cell lines(P＜0.05).2Co-immunofluorescence detection of Wip1, PCNA and p53in U87, U251and MCF-7cell lines was performed. All of them showed a large number ofpositive cells for both PCNA and Wip1. Furthermore, expression of p53andWip1were co-localized in the cell lines.3X-mode value of Wip1expression in U87cells were higher than those inU251cells by flow cytometry. There was a statistically significant differencebetween two cell lines(P＜0.05).4Both protein and mRNA expression of Wip1in U87cells were higher thanthose in U251cells by RT-PCR and Western blot. There was a significantdifference between two cell lines(P＜0.05).Conclusions:1U87cell lines (wild-type p53phenotypic) as a Wip1advantage cell lines,which may be used an object for the Wip1gene treatment experiment.2Co-expression of Wip1/PCNA and p53/Wip1by co-immunofluorescence inU87and U251cell lines were observed, demonstrating that Wip1expressingcells have a high rate of proliferation. These revealed that p53, Wip1andPCNA may play an important role in the glioma cell proliferation.Part three: Study on the roles of Wip1in malignant proliferation ofhuman glioblastoma cell lines U87Objective: To explore the roles that Wip1plays in the glioblastoma U87cell lines. Methods:1To construct the lentiviral expression vector for RNA interference (RNAi) ofWip1gene, and get stable Wip1-/-U87cell lines.2Wip1mRNA of U87cell were detected respectively by RT-PCR after Wip1silencing1d,2d,3d,4d,5d and7d.3Wip1protein of U87cell were detected respectively by western blot afterWip1silencing1d,2d,3d,4d,5d and7d.4The proliferation activities of cells were detected by3-(4,5-Dimethylthiazol,Z, yl)-3,5-di-Phenyltetrazoliumbromide(MTT)after Wip1silencing1d,2d,3d,4d,5d and7d.5Cell cyele distributions were analyzed after Wip1silencing1d,2d,3d,4d,5d and7d by flow cytometry PI staining.Results:1Three recombinant lentiviral vector expressing shRNAs against Wip1genewere obtained and confirmed by DNA sequencing. The expressions of Wip1mRNA and protein in U87cells were decreased effectively after Wip1silencing compared with mock cells, especially at3d. There was a significancedifference between them(P＜0.05).2Cells with Wip1silencing,especially at3d, had reduced proliferation abilitycompared with mock cells. The number of proliferation rate was20.15±6.22%,43.56±5.56%and48.37±6.13%for silencing group (KD), negative control(NC) and blank control one. There was a notable difference between silencinggroup and mock groups (P<0.05). However, there was not a differencebetween NC group and blank control one (P>0.05).3No significant changes of cell cycle distributions analyzed by flowcytometry were detected in U87cells after Wip1silencing1d to4d. Butincreased S phase and decreased G2phase after infection5d were detected inU87cells. There was a notable difference between silencing group and mockgroups (P<0.05). However, there was not a difference between NC group andblank control one (P>0.05).Conclusions:1Construct the lentiviral expression vector successfully for RNA interference (RNAi) of Wip1gene, and get stable Wip1-/-U87cell lines. The expressions ofWip1mRNA and protein in U87cells were decreased effectively after Wip1silencing.2Proliferation ability was inhibited after Wip1silencing. Wip1Plays animportant role in U87cell proliferation.