MAB16200 Sigma-AldrichAnti-MGMT Antibody, clone MT3.1

Glioma stem-like cells (GSCs) could have potential for tumorigenesis, treatment resistance, and tumor recurrence (GSC hypothesis). However, the mechanisms underlying such potential has remained elusive and few ultrastructural features of the cells have been reported in detail. We therefore undertook observations of the antigenic characteristics and ultrastructural features of GSCs isolated from human glioblastomas. Tumor spheres formed by variable numbers of cells, exhibiting a variable appearance in both their size and shape, were frequently seen in GSCs expressing the stem cell surface markers CD133 and CD15. Increased cell nucleus atypia, mitochondria, rough endoplasmic reticulum, coated vesicles, and microvilli, were noted in the GSCs. Furthermore, cells at division phases and different phases of the apoptotic process were occasionally observed. These findings could imply that GSCs have certain relations with human neural stem cells (NSCs) but are primitively different from undifferentiated NSCs. The data may provide support for the GSC hypothesis, and also facilitate the establishment of future glioblastoma treatments targeting GSCs.

Acute myeloid leukemia (AML) is a highly lethal disease, especially in old patients. Chemoresistance and the absence of host immune responses against autochthonous malignancy play a major role in the poor prognosis of AML. The triazene compounds Dacarbazine and Temozolomide are monofunctional alkylators that donate methyl groups to many sites in DNA, including the O6-position of guanine producing O6-methylguanine (O6-MeG). If not repaired, O6- MeG frequently mispairs with thymine during DNA duplication. O6-MeG:T mismatches can be recognized by the mismatch repair (MMR) system which activates a cascade of molecular events leading to cell cycle arrest and cell death. If MMR is defective, cells continue to divide and GC → AT transition mutations occur. In preclinical models, such mutations can lead to the appearance of abnormal proteins containing non-self peptides ("chemical xenogenization" CX) that can be recognized by host cell-mediated immunity. Repair of O6-MeG is achieved by the DNA repair protein, O6- methylguanine-DNA methyltransferase (MGMT), which removes the methyl adduct in an autoinactivating stoichiometric reaction. High MGMT levels attenuate the pharmacodynamic effects of triazenes. In the last few years, triazenes, alone or with MGMT inhibitors, have been tested in AML. In view of their potential activity as CX inducers, triazenes could offer the additional advantage of host anti-leukemia immune responses. The present paper describes several studies of leukemia treatment with triazenes and a case of acute refractory leukemia with massive skin infiltration by malignant cells. Treatment with Temozolomide and Lomeguatrib, a potent MGMT inhibitor, produced a huge, although transient, blastolysis and complete disappearance of all skin lesions.

Although implementation of temozolomide (TMZ) as a part of primary therapy for glioblastoma multiforme (GBM) has resulted in improved patient survival, the disease is still incurable. Previous studies have correlated various parameters to survival, although no single parameter has yet been identified. More studies and new approaches to identify the best and worst performing patients are therefore in great demand.This study examined 225 consecutive, non-selected GBM patients with performance status (PS) 0-2 receiving postoperative radiotherapy with concomitant and adjuvant TMZ as primary therapy. At relapse, patients with PS 0-2 were mostly treated by reoperation and/or combination with bevacizumab/irinotecan (BEV/IRI), while a few received TMZ therapy if the recurrence-free period was greater than 6 months.Median overall survival and time to progression were 14.3 and 8.0 months, respectively. Second-line therapy indicated that reoperation and/or BEV/IRI increased patient survival compared with untreated patients and that BEV/IRI was more effective than reoperation alone. Patient age, ECOG PS, and use of corticosteroid therapy were significantly correlated with patient survival and disease progression on univariate analysis, whereas p53, epidermal growth factor receptor, and O⁶-methylguanine-DNA methyltransferase expression (all detected by immunohistochemistry), tumor size or multifocality, and extent of primary operation were not. A model based on age, ECOG PS, and corticosteroids use was able to predict survival probability for an individual patient.The survival of RT/TMZ-treated GBM patients can be predicted based on patient age, ECOG PS, and corticosteroid therapy status.

Antiestrogen therapy resistance remains a huge stumbling block in the treatment of breast cancer. We have found significant elevation of O(6) methylguanine DNA methyl transferase (MGMT) expression in a small sample of consecutive patients who have failed tamoxifen treatment. Here, we show that tamoxifen resistance is accompanied by upregulation of MGMT. Further we show that administration of the MGMT inhibitor, O(6)-benzylguanine (BG), at nontoxic doses, leads to restoration of a favorable estrogen receptor alpha (ERα) phosphorylation phenotype (high p-ERα Ser167/low p-ERα Ser118), which has been reported to correlate with sensitivity to endocrine therapy and improved survival. We also show BG to be a dual inhibitor of MGMT and ERα. In tamoxifen-resistant breast cancer cells, BG alone or in combination with antiestrogen (tamoxifen [TAM]/ICI 182,780 [fulvestrant, Faslodex]) therapy enhances p53 upregulated modulator of apoptosis (PUMA) expression, cytochrome C release and poly (ADP-ribose) polymerase (PARP) cleavage, all indicative of apoptosis. In addition, BG increases the expression of p21(cip1/waf1). We also show that BG, alone or in combination therapy, curtails the growth of tamoxifen-resistant breast cancer in vitro and in vivo. In tamoxifen-resistant MCF7 breast cancer xenografts, BG alone or in combination treatment causes significant delay in tumor growth. Immunohistochemistry confirms that BG increases p21(cip1/waf1) and p-ERα Ser167 expression and inhibits MGMT, ERα, p-ERα Ser118 and ki-67 expression. Collectively, our results suggest that MGMT inhibition leads to growth inhibition of tamoxifen-resistant breast cancer in vitro and in vivo and resensitizes tamoxifen-resistant breast cancer cells to antiestrogen therapy. These findings suggest that MGMT inhibition may provide a novel therapeutic strategy for overcoming antiestrogen resistance.

MGMT (O⁶-methylguanine-DNA methyltransferase) promoter hypermethylation is a helpful prognostic marker for chemotherapy of gliomas, although with some controversy for low-grade tumors. The objective of this study was to retrospectively investigate MGMT promoter hypermethylation status for a series of 350 human brain tumors, including 275 gliomas of different malignancy grade, 21 glioblastoma multiforme (GBM) cell lines, and 75 non-glial tumors. The analysis was performed by methylation-specific PCR and capillary electrophoresis. MGMT expression at the protein level was also evaluated by both immunohistochemistry (IHC) and western blotting analysis. Associations of MGMT hypermethylation with IDH1/IDH2 mutations, EGFR amplification, TP53 mutations, and 1p/19q co-deletion, and the prognostic significance of these, were investigated for the gliomas. MGMT promoter hypermethylation was identified in 37.8% of gliomas, but was not present in non-glial tumors, with the exception of one primitive neuroectodermal tumor (PNET). The frequency was similar for all the astrocytic gliomas, with no correlation with histological grade. Significantly higher values were obtained for oligodendrogliomas. MGMT promoter hypermethylation was significantly associated with IDH1/IDH2 mutations (P = 0.0207) in grade II–III tumors, whereas it had a borderline association with 1p deletion (P = 0.0538) in oligodendrogliomas. No other association was found. Significant correlation of MGMT hypermethylation with MGMT protein expression was identified by IHC in GBMs and oligodendrogliomas (P = 0.0001), but not by western blotting. A positive correlation between MGMT protein expression, as detected by either IHC or western blotting, was also observed. The latter was consistent with MGMT promoter hypermethylation status in GBM cell lines. In low-grade gliomas, MGMT hypermethylation, but not MGMT protein expression, was associated with a trend, only, toward better survival, in contrast with GBMs, for which it had favorable prognostic significance.

In this study several tumor-related volumes were assessed by means of a computer-based application and a survival analysis was conducted to evaluate the prognostic significance of pre- and postoperative volumetric data in patients harboring glioblastomas. In addition, MGMT (O6-methylguanine methyltransferase) related parameters were compared with those of volumetry in order to observe possible relevance of this molecule in tumor development.We prospectively analyzed 65 patients suffering from glioblastoma (GBM) who underwent radiotherapy with concomitant adjuvant temozolomide. For the purpose of volumetry T1 and T2-weighted magnetic resonance (MR) sequences were used, acquired both pre- and postoperatively (pre-radiochemotherapy). The volumes measured on preoperative MR images were necrosis, enhancing tumor and edema (including the tumor) and on postoperative ones, net-enhancing tumor. Age, sex, performance status (PS) and type of operation were also included in the multivariate analysis. MGMT was assessed for promoter methylation with Multiplex Ligation-dependent Probe Amplification (MLPA), for RNA expression with real time PCR, and for protein expression with immunohistochemistry in a total of 44 cases with available histologic material.In the multivariate analysis a negative impact was shown for pre-radiochemotherapy net-enhancing tumor on the overall survival (OS) (p = 0.023) and for preoperative necrosis on progression-free survival (PFS) (p = 0.030). Furthermore, the multivariate analysis confirmed the importance of PS in PFS and OS of patients. MGMT promoter methylation was observed in 13/23 (43.5%) evaluable tumors; complete methylation was observed in 3/13 methylated tumors only. High rate of MGMT protein positivity (greater than 20% positive neoplastic nuclei) was inversely associated with pre-operative tumor necrosis (p = 0.021).Our findings implicate that volumetric parameters may have a significant role in the prognosis of GBM patients. Furthermore, volumetry could help not only to improve the prediction of outcome but also the outcome itself by identifying patients at high risk of treatment failure and, thus, seek alternative treatment for these patients. In this small series, MGMT protein was associated with less aggressive tumor characteristics.

Antiepileptic drugs (AEDs) are frequently used to treat seizures in glioma patients. AEDs may have an unrecognized impact in modulating O(6)-methylguanine-DNA methyltransferase (MGMT), a DNA repair protein that has an important role in tumor cell resistance to alkylating agents. We report that levetiracetam (LEV) is the most potent MGMT inhibitor among several AEDs with diverse MGMT regulatory actions. In vitro, when used at concentrations within the human therapeutic range for seizure prophylaxis, LEV decreases MGMT protein and mRNA expression levels. Chromatin immunoprecipitation analysis reveals that LEV enhances p53 binding on the MGMT promoter by recruiting the mSin3A/histone deacetylase 1 (HDAC1) corepressor complex. However, LEV does not exert any MGMT inhibitory activity when the expression of either p53, mSin3A, or HDAC1 is abrogated. LEV inhibits malignant glioma cell proliferation and increases glioma cell sensitivity to the monofunctional alkylating agent temozolomide. In 4 newly diagnosed patients who had 2 craniotomies 7-14 days apart, prior to the initiation of any tumor-specific treatment, samples obtained before and after LEV treatment showed the inhibition of MGMT expression. Our results suggest that the choice of AED in patients with malignant gliomas may have an unrecognized impact in clinical practice and research trial design.

We studied the expression of O(6)-methylguanine-DNA methyltransferase (O(6)-MGMT), P-glycoprotein (Pgp), and multidrug resistance protein-1 (MRP-1) in 23 glioblastomas using RT-PCR, methylation-specific PCR, and immunohistochemistry, and analyzed their association with overall patient survival. Univariate analysis of collected data demonstrated that the expressions of O(6)-MGMT and MRP-1 detected by immunohistochemistry, in addition to the consistent factors, including preoperative Karnofsky performance scale (KPS), radical surgery, and tumor location and extension, were significant prognostic factors for the overall survival (OS) of patients with glioblastoma, who received nimustine (ACNU)-based chemotherapy in association with surgery and radiotherapy. Among them, following multivariate analysis, preoperative KPS, radical surgery, tumor location, and the expression of O(6)-MGMT remained as significant prognostic factors. These findings suggest that immunohistochemical analysis of O(6)-MGMT in patients with glioblastoma can be a useful method to predict the effects of chemotherapy and identify alternative chemotherapeutic regimens for O(6)-MGMT-positive patients.

PURPOSE: We sought to determine whether administration of a MGMT blocker, O(6)-benzyl guanine (O(6)BG), at an optimal biological dose alone or in combination with gemcitabine inhibits human pancreatic cancer cell growth. EXPERIMENTAL DESIGN: Human pancreatic cancer L3.6pl and PANC1 cells were treated with O(6)BG, either alone or in combination with gemcitabine, and the therapeutic efficacy and biological activity of these drug combinations were investigated. RESULTS: O(6)BG sensitized pancreatic cancer cells to gemcitabine. Protein and mRNA expression of MGMT, cyclin B1, cyclin B2, cyclin A, and ki-67 were significantly decreased in the presence of O(6)BG. In sharp contrast, protein expression and mRNA message of p21(cip1) were significantly increased. Interestingly, O(6)BG increases p53-mediated p21(cip1) transcriptional activity and suppresses cyclin B1. In addition, our results indicate that p53 is recruited to p21 promoter. Furthermore, an increase in p21(cip1) and a decrease in cyclin transcription are p53 dependent. The volume of pancreatic tumors was reduced by 27% in mice treated with gemcitabine alone, by 47% in those treated with O(6)BG alone, and by 65% in those mice given combination. Immunohistochemical analysis showed that O(6)BG inhibited expression of MGMT and cyclins, and increased expression of p21(cip1). Furthermore, there was a significant decrease in tumor cell proliferation and an increase in tumor cell apoptosis. CONCLUSIONS: Collectively, our results show that decreased MGMT expression is correlated with p53 activation, and significantly reduced primary pancreatic tumor growth. These findings suggest that O(6)BG either alone or in combination with gemcitabine may provide a novel and effective approach for the treatment of human pancreatic cancer.

AIM: To evaluate MGMT promoter hypermethylation as prognostic factor in a retrospective study of 104 cases of glioblastoma multiforme (GBM). MATERIALS AND METHODS: The O(6)-methylguanine-DNA methyltransferase (MGMT) status was evaluated by methylation-specific PCR (MSP), immunohistochemistry and Western blotting analysis in formalin-fixed paraffin-embedded surgical samples. RESULTS: The MGMT gene was found to be methylated in 29 of 101 tumors (28.7%) by MSP, according to the evaluation methods employed. By immunohistochemistry, different categories were identified on the basis of reaction intensity, percentage of positive cells and homogeneous or heterogeneous distribution. MSP did not correlate with immunohistochemistry, with the exception of the category with the highest percentage of positive cells and homogeneity of immunostaining. Western blotting analysis correlated with immunohistochemical findings (Pearson's correlation coefficient r=0.268, p=0.0211), but not with MSP. By Kaplan-Meier survival analysis, radiotherapy was a significant prognostic factor (p=0.0001). When uncensored patients alone were considered, MGMT methylation status showed a significant correlation with survival (p=0.026). Temozolomide therapy correlated with survival (p=0.022), but not with MGMT methylation. After multivariate Cox regression analysis, only radiotherapy remained as an independent prognostic factor (p=0.0001). CONCLUSION: Correlation was inconclusive among MSP, immunohistochemistry and Western blotting analysis, despite the sophisticated score system for the immunohistochemical quantitative evaluation. MGMT expression is a complex event in which many factors beside epigenetic silencing are implicated.