Induction of the DNA repair gene O6-methylguanine-DNA methyltransferase by dexamethasone in glioblastomas

OBJECT: The DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT) inhibits the cytotoxic effect of alkylating agents on tumor cells. The presence of two nonconsensus glucocorticoid-responsive elements in the human MGMT promoter region indicates the potential regulation of MGMT expression by glucocorticoid agents. This study was performed to elucidate whether dexamethasone affects the expression of MGMT in glioblastoma multiforme (GBM) cells, thereby limiting the benefit of chemotherapeutic alkylating agents. METHODS: Four GBM cell lines (A172, T98G, U138MG, and U87MG) were exposed to the alkylating agent 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) with or without dexamethasone. The expression levels of MGMT were correlated with the cytotoxic effects of ACNU in GBM cells. In the presence of ACNU alone, dexamethasone alone, and the combination of both agents, messenger RNA expression of MGMT was induced to varying degrees with the highest increases seen in the later conditions. This dexamethasone-dependent induction of the MGMT gene was even observed in U87MG cells in which the promoter is methylated, although the absolute expression of MGMT mRNA was the lowest in that cell line. The induction of MGMT by dexamethasone was associated with an increased resistance of these cells to ACNU. CONCLUSIONS: These results indicate that dexamethasone-mediated upregulation of MGMT limits the efficiency of alkylating agents in the treatment of malignant gliomas.     

Prognostic significance of loss of O6-methylguanine-DNA methyltransferase expression in supratentorial diffuse low-grade astrocytoma

BACKGROUND: O(6)-Methylguanine-DNA methyltransferase is a DNA repair protein. Epigenetic silencing of MGMT function by its promoter hypermethylation is considered to contribute to carcinogenesis. If loss of function in MGMT is related to tumor progression, the immunohistochemical method may predict the malignant change of gliomas. METHOD: We investigated the expression of MGMT by immunohistochemical method in 28 supratentorial hemispheric diffuse astrocytomas. The prognostic significance of MGMT expression, proliferation index (MIB-1), and various clinical factors was evaluated. RESULTS: There were 19 MGMT-positive and 9 MGMT-negative astrocytomas. Their rates of malignant transformation at 5 years were 12.3% and 51.4%, respectively. The difference was significant in the univariate (P = .004) and multivariate analyses (P = .044). Age, sex, extent of surgery, MIB-1 value, and radiation therapy at initial treatment did not correlate with the malignant progression. The 10-year overall survival rates were 71.8% and 58.3% in the patients with MGMT-positive and MGMT-negative tumors, respectively, and were not significantly different between these 2 groups (P = .079). Two long-term survivors with MGMT-negative tumor responded well to nitrosourea-based chemotherapy and lived more than 8 years after malignant transformation. The patients' age (P = .0047) and the degree of surgical removal (P = .0082) affected the overall survival in the univariate analysis. In the multivariate analysis, none of these factors reached significance. CONCLUSION: Although the status of MGMT did not affect the overall survival, immunohistochemical evaluation of MGMT expression may be a good marker for tumor progression.     

Deficient expression ofO 6-Methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and hMSH2 is related to poor prognosis in human biliary tract carcinoma

Background O ⁶-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups fromO ⁶-methylguanine to itself. Alkylation of DNA at theO ⁶ position of guanine is an important step in the induction of mutations in the organism by alkylating agents. TheO ⁶-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway. The biliary duct is highly exposed to alkylating agents because of its anatomical location. Methods We examined 39 surgically resected gallbladder carcinomas and 35 extrahepatic bile duct carcinomas and evaluated the expression of MGMT and MMR protein (hMLH1 and hMSH2) by immunohistochemical staining. Results MGMT-negative staining was detected in 59.0% of gallbladder carcinoma specimens and 60.0% of extrahepatic bile duct carcinoma specimens. In gallbladder carcinoma, hMLH1- and hMSH2-negative staining was observed in 51.3% and 59.0%, respectively, whereas in extrahepatic bile duct carcinoma, the respective values were 57.1% and 65.7%. MGMT-negative staining correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, a combined MGMT and MMR status was shown to be a more significant prognostic biomarker in both tumor types. Conclusions Combined MGMT and MMR is a possible prognostic marker that probably reflects an accumulation of genetic mutations.     

DNA修复基因MGMT启动子区过甲基化在胆管癌中的作用

目的探讨胆管癌组织中6-氧-甲基鸟嘌呤-DNA甲基转移酶(MGMT)基因的甲基化状态及其在胆管癌中的临床意义。方法采用甲基化特异性PCR(MSP)和免疫组织化学法分析胆管癌组织中MGMT基因启动子甲基化状态及MGMT蛋白表达情况,并分析MGMT基因甲基化状态与临床病理特征的关系。结果 36例胆管癌组织中MGMT基因17例(47.2%)为甲基化阳性;MGMT蛋白阳性表达15例,阴性表达21例,21例MGMT蛋白阴性表达的胆管癌中MGMT甲基化阳性14例;15例MGMT蛋白阳性表达中MGMT甲基化阳性仅3例。MGMT蛋白表达与MGMT甲基化状态呈负相关(rs=-0.816,P<0.05)。MGMT基因的甲基化状态与胆管癌患者的肿瘤浸润深度、分化程度及TNM分期有关(P<0.05),但与患者的年龄、性别、病理学类型、淋巴结转移均无关(P>0.05)。结论 MGMT基因启动子过甲基化是胆管癌组织中常见的分子事件,并可能与胆管癌发生有关,MGMT甲基化状态可以作为评价胆管癌恶性程度的一项指标。     

Correlation of basic fibroblast growth factor expression levels with the degree of malignancy and vascularity in human gliomas.

Basic fibroblast growth factor (FGF) is a mitogen, a differentiation factor for neuroectoderm-derived cells, and a potent angiogenic factor. The authors have previously demonstrated that the messenger ribonucleic acid of basic FGF is expressed in more than 90% of human gliomas. In the present study, they examined the expression of basic FGF in human glioma tissues using immunohistochemical techniques with a mouse monoclonal antibody against human basic FGF. They also correlated the basic FGF level with the histological grades of malignancy assessed by the number of nucleolar organizer regions (NOR's). Basic FGF was detected in 18 of 19 gliomas, whereas it was undetectable in two normal brains. The expression level of basic FGF peptide increased proportionally with the degree of malignancy. There was also a tendency for the number of NOR's in glioma cells to increase in glioma samples with a high level of basic FGF expression. Furthermore, most of the cases with increased vascularity demonstrated on cerebral angiograms showed a relatively high level of basic FGF expression of tumor cells and a large number of NOR's in endothelial cells in tumor tissues. These results suggest that basic FGF is actually produced in most gliomas and is involved in tumorigenesis and malignant progression as an autocrine growth factor. Moreover, basic FGF may play an important role in tumor neovascularization as a paracrine angiogenic factor.     

Neural stem cell markers, nestin and musashi proteins, in the progression of human glioma: correlation of nestin with prognosis of patient survival

BACKGROUND: The IF protein nestin and the RNA-binding protein musashi are expressed by neural progenitor cells during CNS development. Their expression in glial tumors was evaluated by immunohistochemistry, and the histopathological scores correlated with levels of cysteine cathepsins that are known prognostic markers in several tumors. METHODS: The levels of nestin, musashi, and cathepsins B and L were assessed by immunohistochemical analysis of biopsies from 87 patients with primary CNS tumors. To confirm the immunohistochemical data, nestin expression was analyzed by real-time PCR in 12 brain tumor biopsies. The exact location of nestin-positive cells was determined by mapping the distribution of nestin in a highly invasive human glioma xenograft model. RESULTS: Immunostaining revealed nestin to be expressed in 95.8% and musashi in 80% of the patient biopsies. The total IHC score for nestin was significantly higher in high- than in low-grade tumors (P < .0001). No difference was observed for musashi (P = .11). Real-time PCR of nestin expression confirmed the immunohistochemical data. Nestin expression was shown to be a strong prognostic marker for decreased overall survival (P = .0001), whereas musashi expression has no prognostic significance. Moreover, nestin was shown by Cox regression analysis to be a stronger prognostic marker than cathepsins B and L. IHC staining of nestin in a xenograft model showed that its expression is localized mainly in the invasive tumor cells at the tumor periphery. CONCLUSIONS: Nestin is shown to be a strong prognostic marker for glioma malignancy. The presented data links the invasive glioma cells to CNS precursor cells, indicating that the most malignant cells in the gliomas may well be closely related to the glioma stem cells.     

Characterization of lymphoid cells isolated from human gliomas

To analyze the phenotypic profile of lymphoid cells freshly isolated from surgically resected human gliomas, a double-immunostaining technique was developed which permitted the investigators simultaneously to distinguish between hematogenous and tumor cell populations and to detect expression of lymphocyte-monocyte subset-specific antigens on hematogenous cells. With this technique, the profiles of tumor-infiltrating lymphocytes (TIL's) derived from high- and low-grade gliomas were compared with phenotypes of lymphocytes concurrently isolated from peripheral blood. The total leukocyte cell yield from high-grade glioma cases exceeded that of low-grade cases. In nine high-grade glioma cases the proportion of CD8-positive cells was increased within the TIL population (41.2% +/- 1.9%, mean +/- standard error of the mean) as compared to the corresponding peripheral blood lymphocyte (PBL) population (30.8% +/- 4.1%, p less than 0.05). The proportion of natural killer HNK-positive cells, some of which bear the CD8 antigen (although not necessarily the pan T cell antigens CD2 and CD3), was also increased in the TIL's (41.9% +/- 4.2%) compared to that found in PBL's (32.1 +/- 5.6%, p less than 0.05) of high-grade glioma cases. The observed phenotypic pattern of high-grade glioma TIL's is similar to that reported based on immunohistochemical analysis of tumor tissue sections, suggesting that the techniques described here resulted in isolation of lymphoid cells representative of TIL's.     

Cerebral glioblastoma with cerebrospinal fluid dissemination: a clinicopathological study of 14 cases examined by complete autopsy

During the last 17 years, complete autopsies were performed on 51 patients who died of cerebral glioblastoma, and 14 were found to have dissemination by cerebrospinal fluid (CSF). In these 14 cases of glioma, the extent of intraparenchymal invasion by the primary tumor and the degree of seeding were studied in connection with histological findings and immunohistochemical staining for glial fibrillary acidic protein (GFAP) as the most reliable marker of astrocytic differentiation. From the findings obtained, the cases were divided into two groups. In one group, consisting of 7 gliomas, autopsy revealed intense seeding, despite only slight invasion by the primary tumor. Among these 7 extensively disseminated gliomas, 4 expressed almost no GFAP, 2 contained only a few GFAP-positive cells, and only 1 displayed an immunohistochemically high degree of astrocytic differentiation. Clinically, 6 of the 7 affected patients developed symptoms attributable to CSF seeding. In the other group consisting of the remaining 7 gliomas, only slight dissemination was seen, despite extensive infiltration of the primary tumor. Each of these 7 gliomas contained many GFAP-positive cells. None of the affected patients developed symptomatic seeding. This study shows the existence of two clinicopathologically distinct groups of disseminated cerebral glioblastomas and suggests that, regardless of morphological features, glioblastomas showing immunohistochemically poor astrocytic differentiation tend to shed tumor cells more vigorously but are less invasive at the primary site than those with many GFAP-positive cells. It is also suggested that, as a consequence, the former glioma type produces symptomatic seeding more frequently than the latter type.     

EphB4在人胶质瘤过表达及意义

目的探讨人脑胶质瘤中EphB4表达情况及临床病理学意义。方法收集正常脑组织和胶质瘤组织，行逆转录-聚合酶链式反应（RT—PCR）和免疫组织化学染色（IHC），分析与病理级别、年龄、性别的相关性。结果EphB4在胶质瘤中呈过表达，表达于肿瘤细胞和血管上，与病理级别正相关，与年龄、性别无明显相关。结论EphB4／ephnnB2信号可能通过直接影响肿瘤细胞和间接影响瘤性血管的双重作用促进肿瘤发生发展。     

人胃癌组织中肝素酶基因启动子甲基化状态与肿瘤侵袭转移的关系

目的 探讨人胃癌组织中肝素酶(HPA)基因启动子的甲基化状态及其与HPA表达、肿瘤侵袭转移的关系.方法 应用免疫组织化学、逆转录-聚合酶链反应(RT-PCR)方法检测48例胃癌组织标本及相应癌旁组织中HPA蛋白和mRNA表达;采用亚硫酸氢钠修饰、甲基化PCR(MSP)、甲基化克降测序(BSP)法检测胃癌组织和癌旁组织中HPA基因启动子区域CpG岛甲基化状态,并探讨与胃癌临床病理特征的关系.结果 胃癌组织中HPA mRNA和蛋白的阳性表达率分别为79.20%(38/48)、93.75%(45/48),显著高于癌旁组织(P<0.05).MSP、BSP检测发现13例胃癌组织HPA启动子CpG岛呈低甲基化状态,且其甲基化程度与HPA的表达及胃癌的分化程度、TNM分期、淋巴结转移、远处转移明显相关(P<0.05).结论 HPA基因启动子区域CpG岛甲基化程度与HPA的表达及胃癌的发生、发展有关,在肿瘤侵袭、转移过程中起重要调节作用.     

人脑胶质瘤血管生成素-2表达及其与管生成和脑水肿的关系

目的 研究血管生成素.2(Ang-2)基因在人脑胶质瘤表达及其与胶质瘤血管生成及瘤周水肿的关系。方法 用半定量逆转录-聚合酶链反应(RT-PCR)、免疫组织化学方法测定42例人脑胶质瘤和8例正常脑组织中Ang-2 mRNA及其蛋白表达情况。用免疫组织化学方法检测肿瘤微血管密度(MVD)。结果 正常脑组织中无或弱表达Ang-2。42例胶质瘤组织中均有Ang-2 mRNA表达,不同级别间Ang-2 mRNA的表达差异有显著性(P<0.05)。随着脑胶质瘤恶性程度的增加,Ang-2 mRNA的表达增高(r=0.894,P<0.01)。免疫组织化学结果显示,胶质瘤细胞及肿瘤血管内皮细胞中均有Ang-2蛋白表达。Ang-2 mRNA表达与MVD、脑水肿指数(EI)显著相关(分别为r=0.853,P<0.01;r=0.784,P<0.01)。结论 Ang-2可能参与胶质瘤血管生成,对胶质瘤瘤周脑水肿及恶性进展有促进作用。     

Correlation between promoter hypermethylation of the O6-methylguanine-deoxyribonucleic acid methyltransferase gene and prognosis in patients with high-grade astrocytic tumors treated with surgery, radiotherapy, and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea-based chemotherapy

OBJECTIVE: O(6)-Methylguanine-deoxyribonucleic acid methyltransferase (MGMT) is a deoxyribonucleic acid repair protein associated with the chemoresistance of chloroethylnitrosoureas. We investigated whether MGMT promoter hypermethylation is associated with prognosis in patients with high-grade astrocytic tumors treated uniformly with surgery, radiotherapy, and 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-based chemotherapy. METHODS: Using the methylation-specific polymerase chain reaction, we assayed promoter hypermethylation of the MGMT gene in tumor deoxyribonucleic acid from 116 adult patients with supratentorial high-grade astrocytic tumors (42 anaplastic astrocytomas [AAs] and 74 glioblastomas multiforme [GBMs]). The Cox proportional hazards model was used in forward stepwise regression to assess the relative role of prognostic factors (i.e., age at surgery, sex, Karnofsky Performance Scale score, extent of surgical resection, methylation status of the MGMT promoter, and association between MGMT promoter methylation and survival). RESULTS: MGMT promoter hypermethylation was confirmed in 19 (45.2%) of 42 AA patients and 33 (44.6%) of 74 GBM patients. It was significantly associated with both longer overall and progression-free survival time in AA but not GBM patients. CONCLUSION: Our results demonstrate that MGMT promoter hypermethylation is associated with longer survival time in patients with AA who were treated with surgery, radiotherapy, and ACNU-based chemotherapy but not in patients with GBM.     

血管内皮细胞生长因子对胶质瘤中水孔蛋白4表达的影响

目的探讨血管内皮细胞生长因子（VEGF）对水孔蛋白4在胶质瘤中表达的影响。方法利用已鉴定的经抗性筛选的稳定表达正义／反义VEGF cDNA的C6细胞进行裸鼠皮下接种形成胶质瘤的模型，免疫组织化学方法检测VEGF和水孔蛋白4在不同种植瘤内的表达情况。结果正义VEGF组瘤内VEGF及水孔蛋白4的表达与对照组无明显差异，而反义VEGF组瘤内的VEGF及水孔蛋白4的表达均下调（P〈0．05）。结论VEGF可能通过对水孔蛋白4表达的调控而共同参与胶质瘤水肿的病理生理过程。     

Reduced expression of the transporter associated with antigen processing 1 molecule in malignant glioma cells, and its restoration by interferon-gamma and -beta

OBJECT: It remains unclear whether malignant glioma cells can deliver tumor antigens efficiently to major histocompatibility complex (MHC) Class I molecules. To elucidate the mechanism of antigen presentation in malignant gliomas, the authors examined the expression of the transporter associated with antigen processing 1 (TAP1), which transports antigens to MHC Class I molecules, and low-molecular-mass polypeptide 2 (LMP2), which is a subunit of a proteasome. They also analyzed the effects of interferon (IFN)-gamma and IFN-beta on the expression of these molecules. METHODS: Five glioma cells expressed undetectable or very low levels of TAP1 protein and did not express TAP1 messenger (m)RNA. Normal brain tissue and glioma tissue specimens also showed undetectable levels of TAP1 protein and the same levels of LMP2 protein as lymphoblastoid B cells. Treatments of the tumor cells with IFN-gamma, or -beta enhanced the expression of both TAP1 protein and mRNA as well as the expression of MHC Class I molecules. The expression of LMP2 protein was not altered by the IFN treatments. The authors analyzed structural alterations in the TAP1 promoter region in eight malignant glioma cell lines. Single nucleotide polymorphism was found in 446 bp up-stream of the translation start site of the TAP1 gene, and a point mutation was found in 34 bp upstream of the TAP1 gene. CONCLUSIONS: Malignant glioma cells may be less immunogenic due to low levels of TAP1 expression. Upregulated expression of TAP1 and MHC Class I molecules by IFN-gamma and -beta may enhance antigen presentation in malignant glioma cells.     

Delayed repletion of O6-methylguanine-DNA methyltransferase resulting in failure to protect the human glioblastoma cell line SF767 from temozolomide-induced cytotoxicity

OBJECT: Temozolomide (TMZ)-induced O6-methylguanine (MG) DNA lesions, if not removed by MG-DNA methyltransferase (MGMT), mispair with thymine, trigger rounds of futile mismatch repair (MMR), and in glioma cells lead to prolonged G2-M arrest and ultimately cell death. Depletion of MGMT by O6-benzylguanine (BG) sensitizes tumor cells to TMZ, and this combination is currently used in clinical trials. The use of the TMZ+BG combination in gliomas, however, is complicated by the prolonged TMZ-induced G2-M arrest, which may delay activation of poorly defined cell death pathways and allow for MGMT repletion and reversal of toxicity. METHODS: To address these issues, the actions of TMZ were monitored in DNA MMR-proficient SF767 glioma cells depleted of MGMT by BG, and in cells in which BG was removed at various times after TMZ exposure. In MGMT-depleted cells, TMZ exposure led to DNA single-strand breaks and phosphorylation of cdc2, followed by G2-M arrest, induction of p53/p21, and DNA double-strand breaks. Although DNA single-strand breaks, phosphorylation of cdc2, and G2-M arrest could be reversed by repletion of MGMT up to 5 days after TMZ exposure, TMZ-induced cytotoxicity could only be prevented if MGMT was replenished within 24 hours of the onset of G2-M arrest, and before the creation of DNA double-strand breaks. CONCLUSIONS: These results indicate that although SF767 glioma cells undergo a prolonged G2-M arrest in response to TMZ, their ability to escape TMZ-induced cytotoxicity by MGMT repletion is limited to an approximately 24-hour period after the onset of G2-M arrest.     

Tumor Progression Through Epigenetic Gene Silencing of O<Superscript>6</Superscript>−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers

Background We previously demonstrated in an immunohistochemical study that reduced expression of O⁶–methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer.Methods We examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry.Results Frequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, -catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations.Conclusions Reduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.     

Therapeutic efficacy and prognostic factors in diffuse astrocytomas

Diffuse astrocytomas are slowly growing tumors with a relatively long overall survival. Considerable controversy exists as to the best therapeutic management for patients with such tumors. In the present study, we retrospectively analyzed a series of 64 patients with WHO grade II astrocytomas of the cerebral hemispheres. Gross total resection and interferon-beta therapy were significantly associated with both longer progression free survival (PFS) and overall survival (OS). Immediate postoperative radiation therapy did not prolong either the PFS or OS. The presence of promoter hypermethylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene was an independent predictor of a shorter PFS. Our data suggest that radical surgery plus interferon-beta therapy may offer the best chance for long survival. Since the presence of MGMT methylation is a probable indication of an increased sensitivity to alkylating chemotherapeutic agents, determining the methylation status of MGMT could provide a potential basis for logical therapeutic intervention in identifying a subgroup of patients who could be candidates for early chemotherapy.     

Efficacy of temozolomide is correlated with 1p loss and methylation of the deoxyribonucleic acid repair gene MGMT in malignant gliomas

Promoter methylation of the deoxyribonucleic acid (DNA) repair gene, O(6)-methylguanine-DNA methyltransferase (MGMT), is associated with improved outcome of patients with glioblastoma multiforme and anaplastic astrocytoma treated with temozolomide (TMZ). Molecular genetic analysis of loss of heterozygosity (LOH) of 1p, 19q, or 10q, p53 mutation, and MGMT promoter methylation was performed in 44 assessable tumor specimens obtained from 46 patients with recurrent malignant gliomas, including 21 with glioblastoma multiforme, 17 with anaplastic astrocytoma, and eight with anaplastic oligoastrocytoma, which have heterogeneous features and variable histological diagnosis, to assess the correlation with the response to TMZ. LOHs of 1p and 19q, and MGMT promoter methylation showed positive correlations with the clinical response to TMZ therapy (p < 0.005, 0.05, and 0.05, respectively; Fisher's exact test). In addition, LOH of 1p and MGMT promoter methylation were associated with longer progression-free survival (p < 0.05 and 0.05, respectively; Cox regression analysis). LOH of 1p in the heterogeneous population of malignant gliomas may be one of the important factors besides MGMT methylation that predict better outcome in patients treated with TMZ.