Expression of O⁶-Methylguanine-DNA Methyltransferase Examined by Alkyl-Transfer Assays, Methylation-Specific PCR and Western Blots in Tumors and Matched Normal Tissue

The tumor selectivity of alkylating agents that produce guanine O6-chloroethyl (laromustine and carmustine) and O6-methyl (temozolomide) lesions depends upon O6-methylguanine-DNA methyltransferase (MGMT) activity being lower in tumor than in host tissue. Despite the established role of MGMT as a tumor resistance factor, consensus on how to assess MGMT expression in clinical samples is unsettled. The aim of this study is to examine the relationship between the values derived from distinctive MGMT measurements in 13, 12, 6 and 2 pairs of human tumors and matched normal adjacent tissue from the colon, kidney, lung and liver, respectively, and in human cell lines. The MGMT measurements included 1) alkyl-transfer assays using [benzene-3H]O6-benzylguanine as a substrate to assess functional MGMT activity, 2) methylation-specific PCR (MSP) to probe MGMT gene promoter CpG methylations as a measure of gene silencing, and 3) western immunoblots to analyze the MGMT protein. In human cell lines, a strict negative correlation existed between MGMT activity and the extent of promoter methylation. In tissue specimens, by contrast, the correlation between these two variables was low. Moreover, alkyl-transfer assays identified 3 pairs of tumors and normal tissue with tumor-selective reduction in MGMT activity in the absence of promoter methylation. Cell line MGMT migrated as a single band in western analyses, whereas tissue MGMT was heterogeneous around its molecular size and at much higher molecular masses, indicative of multi-layered post-translational modifications. Malignancy is occasionally associated with a mobility shift in MGMT. Contrary to the prevalent expectation that MGMT expression is governed at the level of gene silencing, these data suggest that other mechanisms that can lead to tumorselective reduction in MGMT activity exist in human tissue.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory

Background:The dysregulation of Isocitrate dehydrogenase(IDH)and the subsequent production of 2-Hydroxyglutrate(2HG)may alter the expression of epigenetic proteins in Grade 4 astrocytoma.The interplay mechanism between IDH,O-6-methylguanine-DNA methyltransferase(MGMT)-promoter methylation,and protein methyltransferase proteins-5(PRMT5)activity,with tumor progression has never been described.Methods:A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors.Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis.Inter-cohort statistical significance was evaluated.Results:Both IDH-mutant WHO grade 4 astrocytomas(n=22,64.7%)and IDH-wildtype glioblastomas(n=12,35.3%)had upregulated PRMT5 gene expression except in one case.Out of the 22 IDH-mutant tumors,10(45.5%)tumors showed MGMT-promoter methylation and 12(54.5%)tumors had unmethylated MGMT.All IDH-wildtype tumors had unmethylated MGMT.There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma(p-value=0.006).Statistically significant differences in progression-free survival(PFS)were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide(TMZ)or TMZ plus other chemotherapies,regardless of their IDH or MGMT-methylation status(p-value=0.0014).Specifically,IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation,who received only TMZ,have exhibited longer PFS.Conclusions:The relationship between PRMT5,MGMT-promoter,and IDH is not tridirectional.However,accumulation of D2-hydroxyglutarate(2-HG),which partially activates 2-OG-dependent deoxygenase,may not affect their activities.In IDH-wildtype glioblastomas,the 2HG-2OG pathway is typically inactive,leading to PRMT5 upregulation.TMZ alone,compared to TMZ-plus,can increase PFS in upregulated PRMT5 tumors.Thus,using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.

ANTITUMOR EFFECT OF SARCNU IN A 0~6-METHYLGUANINE-DNA METHYLTRANSFERASE POSITIVE HUMAN GLIOMA XENOGRAFT MODEL

Objective: To assess whether novel analogue of nitrosoureas, 2-chloroethyl-3-sarcosinamide-1-nitrosourea (SarCNU), has antitumor effect to 06-methylguanine-DNA methyltransferase (MGMT) positive tumorsin vivo. Methods: MGMT positive human glioma cell line SF-767 xenografts in nude mice were treated with SarCNU. The antitumor efficacy of SarCNU was compared with the results of 1, 3-bis(2-chloroethyl)-1-nitrosourea (BCNU) treatment with or without 06-benzylguanine (06-BG) preadministration. Results: Since the SF-767 is MGMT strongly positive, BCNU treatment alone did not result in a satisfactory anticancer effect. As expected, 06-BG by depleting MGMT activity, significantly enhanced BCNU antitumor efficacy (P<0.001). More interestingly, SarCNU treatment alone had a better antitumor effect than O6-BG plus BCNU treatment (F=51.7,P=0.00036). Conclusion: Since SarCNU enters cells via extraneuronal monoamine transporter (EMT), the enhanced antitumor activity of SarCNU in this MGMT positive human tumor xenograft model may be due to the presence of EMT in SF-767. SarCNU may be used as an alternative treatment for MGMT positive tumors, specifically for tumors expressing EMT.

GM-CSF在替莫唑胺抗高级别胶质瘤中的作用

目的探讨粒-巨噬细胞集落刺激因子(granulocyte-macrophage colony-stimulating factor,GMCSF)在替莫唑胺(temozolomide,TMZ)抗高级别胶质瘤中的作用及机制。方法选取6例高级别胶质瘤患者来源的肿瘤组织培养肿瘤细胞,待细胞状态稳定后采用MTT法进行细胞增殖毒性实验,流式细胞仪检测细胞周期变化和细胞凋亡情况,甲基化特异性PCR和免疫组织化学染色法分别检测六氧甲基鸟嘌呤DNA甲基转移酶(O6-methylguanine-DNA methyltransferase,MGMT)基因启动子甲基化状态和MGMT蛋白表达水平。结果 MTT实验显示,GM-CSF处理组的细胞存活率与对照组相比均有不同程度的增加。MGMT基因启动子甲基化的3例细胞,GM-CSF+TMZ组的细胞存活率比TMZ组显著降低(P<0.05),另3例MGMT启动子非甲基化细胞,GM-CSF+TMZ组与TMZ组相比,其存活率差异均无统计学意义(P>0.05)。6例细胞GM-CSF组的G1期细胞比例均比对照组降低,而S期细胞比例GMCSF处理组较对照组显著增加(P<0.05)。流式细胞仪凋亡检测显示,MGMT启动子甲基化的3例细胞,GM-CSF+TMZ组凋亡率与单药TMZ组凋亡率相比均显著增加(P<0.05),而MGMT非甲基化细胞GM-CSF+TMZ组与单药TMZ组凋亡率相比无统计学差异。结论 GM-CSF可通过诱导高级别胶质瘤细胞快速进入细胞周期,显著提高TMZ对MGMT基因启动子甲基化的高级别胶质瘤细胞的杀伤作用,而对MGMT基因启动子非甲基化高级别胶质瘤细胞的作用不明显。