Methylguanine DNA Methyltransferase Levels Research Articles

TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT.

BackgroundOur previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.MethodsThe distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.ResultsOverexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.ConclusionsOur results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.

Predictive MGMT status in a homogeneous cohort of IDH wildtype glioblastoma patients

Methylation of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter is predictive for treatment response in glioblastoma patients. However, precise predictive cutoff values to distinguish “MGMT methylated” from “MGMT unmethylated” patients remain highly debated in terms of pyrosequencing (PSQ) analysis. We retrospectively analyzed a clinically and molecularly very well-characterized cohort of 111 IDH wildtype glioblastoma patients, who underwent gross total tumor resection and received standard Stupp treatment. Detailed clinical parameters were obtained. Predictive cutoff values for MGMT promoter methylation were determined using ROC curve analysis and survival curve comparison using Log-rank (Mantel-Cox) test. MGMT status was analyzed using pyrosequencing (PSQ), semi-quantitative methylation specific PCR (sqMSP) and direct bisulfite sequencing (dBiSeq). Highly methylated (> 20%) MGMT correlated with significantly improved progression-free survival (PFS) and overall survival (OS) in our cohort. Median PFS was 7.2 months in the unmethylated group (UM, < 10% mean methylation), 10.4 months in the low methylated group (LM, 10-20% mean methylation) and 19.83 months in the highly methylated group (HM, > 20% mean methylation). Median OS was 13.4 months for UM, 17.9 months for LM and 29.93 months for HM. Within the LM group, correlation of PSQ and sqMSP or dBiSeq was only conclusive in 51.5% of our cases. ROC curve analysis revealed superior test precision for survival if additional sqMSP results were considered (AUC = 0.76) compared to PSQ (cutoff 10%) alone (AUC = 0.67). We therefore challenge the widely used, strict PSQ cutoff at 10% which might not fully reflect the clinical response to alkylating agents and suggest applying a second method for MGMT testing (e.g. MSP) to confirm PSQ results for patients with LM MGMT levels if therapeutically relevant.

Transcriptional control of O6 -methylguanine DNA methyltransferase expression and temozolomide resistance in glioblastoma.

O6 -methylguanine DNA methyltransferase (MGMT) promoter methylation is a predictive biomarker for benefit from alkylating chemotherapy, specifically temozolomide (TMZ), in glioblastoma, the most common malignant intrinsic brain tumor. Glioma-initiating cells (GIC) with stem-like properties have been associated with resistance to therapy and progression. We assessed the levels of MGMT mRNA and MGMT protein by real-time PCR and immunoblot and evaluated the impact of MGMT on TMZ sensitivity in clonogenicity assays in GIC sphere cultures (S) or differentiated adherent monolayer cultures (M). Nuclear factor kappa B (NF-κB) signaling was assessed by reporter assay and immunoblot. Compared to M cells, S cells expressed higher levels of MGMT. Differentiation of GIC induced by S-to-M transition resulted in a gradual loss of MGMT expression and increased TMZ sensitivity. This transcriptional regulation of MGMT was restricted to cell lines without MGMT promoter methylation and was not coupled to any specific neurobasal (NB) stem cell medium supplement or loss of cell adhesion. Expression levels of p50/p65 subunits of NF-κB, a transcriptional regulator of MGMT, were increased in S cells. Inhibition of NF-κB by the small molecule inhibitor, BAY 11-7082, or siRNA-mediated gene silencing, reduced MGMT levels. In summary, alkylator resistance of S cells is mainly promoted by over-expression of MGMT which results from increased activity of the NF-κB pathway in this cell culture model of glioma stem-like cells. Read the Editorial Highlight for this article on page 688.

Abstract 3493: Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane

Abstract Background: O6 methylguanine DNA methyltransferase (MGMT) is overexpressed in a majority of cancers, including pancreatic cancer. MGMT has been the focus of significant research for its role in the repair of DNA damage caused by chemotherapeutic agents. Aldehyde Dehydrogenase as a progenitor/stem cell marker has been linked to chemotherapy and radiation resistance. Methods: We tested the administration of Antabuse (disulfiram, DSF) at nontoxic doses in combination with gemcitabine (GEM) and Abraxane (ABX) on pancreatic cancer cells. Results: DSF at very low doses (achievable in human serum with standard DSF clinical dosing) decreases pancreatic cancer cell growth in a dose-dependent manner. DSF further sensitizes pancreatic cancer cells to GEM and ABX and significantly inhibits pancreatic cancer growth without causing unwanted side effects to the normal pancreatic cells. Dose effect and isobologram studies confirm synergistic activity of DSF and ABX combination and moderate synergism for DSF and GEM combination. DSF, either alone or in combination with GEM (DSF ± GEM) and/or ABX (DSF ± ABX), significantly inhibits expression of MGMT, aldehyde dehydrogenase, antigen Ki-67 and BIRC5 gene (survivin) - all involved in pancreatic cancer tumorigenesis. DSF+/-GEM+/-ABX induces the expression of p21Cip1, a cell cycle inhibitor. We show an inverse correlation between aldehyde dehydrogenase activity and MGMT expression, where MGMT expression is high, aldehyde dehydrogenase activity is low and where aldehyde dehydrogenase activity is high, MGMT levels were low. The ALDH3A1 expression is significantly higher in highly metastatic cancer cells (L3.6pl) compared to other metastatic cancer cells (PANC1, Mia PaCa2, ASPC1). We further report that in our model DSF at very low doses (achievable in human serum with standard DSF clinical dosing) effectively inhibits both aldehyde dehydrogenase and MGMT in pancreatic cancer cells. DSF also induces caspase activation indicative of mitochondrial induced apoptosis in these cells. Conclusions: Our findings suggest that DSF as dual inhibitor of MGMT and aldehyde dehydrogenase potentially provides a novel therapeutic approach to inhibiting pancreatic cancer growth/metastasis and synergistically enhances GEM and ABX activity. Citation Format: George C. Bobustuc, Alisher Holmuhamedov, Kalkunte S. Srivenugopal, Jacob C. Frick, JAMES L. WEESE, Santhi D. Konduri. Disulfiram a dual mgmt and aldehyde dehydrogenase inhibitor sensitizes pancreatic cancer to gemcitabine and abraxane. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3493. doi:10.1158/1538-7445.AM2015-3493

NEO212, temozolomide conjugated to perillyl alcohol, is a novel drug for effective treatment of a broad range of temozolomide-resistant gliomas.

Patients with glioblastoma multiforme (GBM), a malignant primary brain tumor, inevitably develop resistance to standard-of-care chemotherapy, temozolomide. This study explores the effects of the novel agent NEO212, a conjugate of temozolomide to perillyl alcohol, on temozolomide-resistant gliomas. NEO212 was tested for cytotoxic activity on three human temozolomide-resistant glioma cell lines, which were resistant to temozolomide based on overexpression of the base excision repair (BER) pathway, mismatch repair (MMR) deficiency, or overexpression of O(6) methyl-guanine-DNA methyltransferase (MGMT). BER expression was evaluated by Western blotting and PARP activity. MMR deficiency was determined by Western blotting and microsatellite instability. MGMT overexpression was evaluated by Western blotting and O(6)-benzylguanine (O(6)BG) inhibition. For in vivo evaluation of NEO212, temozolomide-resistant glioma cells were implanted into immune-incompetent mice, and NEO212 was administered. NEO212, at equimolar concentrations of temozolomide, was more cytotoxic for temozolomide-resistant cells than temozolomide and not toxic to normal cells. NEO212-induced cell death in temozolomide-resistant glioma cells was independent of such mechanisms of resistance as high levels of MGMT, MMR deficiencies, or overexpression of BER proteins. NEO212 functions as a DNA alkylating agent, similar to temozolomide; however, this novel conjugate is unique for it may induce endoplasmic reticulum (ER) stress and inhibits autophagy. In vivo studies show that NEO212 reduces intracranial tumor growth and increases animal survival without significant toxicity. These results demonstrate that NEO212 is an effective drug against malignant gliomas that can be used for a broad range of newly diagnosed and temozolomide-resistant gliomas.

Abstract C2: Cross-talk between the O(6)-Methylguanine-DNA methyltransferase (MGMT) and p53 in glioblastoma multiforme.

Abstract Background. Glioblastoma multiforme (GBM) is a fatal malignant primary brain tumor in adults with a median survival time of approximately 14.6 months, despite treatment with surgery, concurrent radiation therapy (RT) and the alkylating agent temozolomide (TMZ). O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein involved in resistance to TMZ in GBM. Our group showed that MGMT is a critical regulator of GBM angiogenesis, invasion and response to antiangiogenic treatment (sunitinib). While sunitinib treatment significantly decreased invasion in MGMT-positive cell lines, it led to a significant increase of invasion in MGMT-negative cell lines. Our group launched the first phase II clinical trial of concurrent sunitinib with RT and TMZ for selected patients with MGMT-positive tumors. The p53 tumor suppressor protein is involved in proliferation, angiogenesis and response to RT. Previous studies showed the effect of p53 in regulating MGMT levels. The role of MGMT and p53 in response to RT and sunitinib is unknown. We hypothesize that MGMT may affect levels of p53 and response to RT and sunitinib treatment. Results. We used isogenic human GBM cell lines isogenic for MGMT and assessed p53 levels by Western blotting and immunofluorescence. We showed for the first time that stable transfection of GBM cell line negative for MGMT (U87MG) and harboring wild type TP53 with a vector encoding for MGMT (U87MGMT) led to significant increase of p53 expression and its nuclear localization. Strikingly, increased p53 in U87MGMT cells did not lead to increased p21 protein, one of the main targets of p53. As shown by western blotting analysis, ionizing radiation (6 and 10 Gy) did not increase p21 levels compared to sham control. Interestingly, it was previously shown that irradiation does not lead to p21 activation in GBM cells with basal overexpression of p21 mRNA and can even lead to its repression as a mechanism of resistance to RT. We suggest that MGMT could be an upstream regulator of this resistance. Conclusion. Our study revealed a novel mechanism for regulation of p53 through MGMT, which may affect response to RT in GBM. We will further establish the relationship between MGMT/p53 status and response to combined RT and sunitinib in GBM cell lines and samples from patients enrolled in our clinical trial. This will lead to more efficient strategies of treatment for patients with tumors unresponsive to combined RT and sunitinib. Grant acknowledgments. This work has been funded by the Cedar Cancer Foundation, Research Institute of McGill University Health Center and the McGill-CIHR Drug Development Training Program (DDTP). Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C2. Citation Format: Mariia Patyka, Bassam Abdulkarim, Yaoxian Xu, Siham Sabri. Cross-talk between the O(6)-Methylguanine-DNA methyltransferase (MGMT) and p53 in glioblastoma multiforme. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C2.

Triazene Compounds in the Treatment of Acute Myeloid Leukemia: A Short Review and a Case Report

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 O(6)-position of guanine producing O(6)-methylguanine (O(6)-MeG). If not repaired, O(6)-MeG frequently mispairs with thymine during DNA duplication. O(6)-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 O(6)-MeG is achieved by the DNA repair protein, O(6)-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.

Diethylnitrosamine-induced hepatocarcinogenesis is suppressed in lecithin:retinol acyltransferase-deficient mice primarily through retinoid actions immediately after carcinogen administration

Loss of retinoid-containing lipid droplets upon hepatic stellate cell (HSC) activation is one of the first events in the development of liver disease leading to hepatocellular carcinoma. Although retinoid stores are progressively lost from HSCs during the development of hepatic disease, how this affects hepatocarcinogenesis is unclear. To investigate this, we used diethylnitrosamine (DEN) to induce hepatic tumorigenesis in matched wild-type (WT) and lecithin:retinol acyltransferase (LRAT) knockout (KO) mice, which lack stored retinoid and HSC lipid droplets. Male 15-day-old WT or Lrat KO mice were given intraperitoneal injections of DEN (25 mg/kg body wt). Eight months later, Lrat KO mice showed significantly less liver tumor development compared with WT mice, characterized by less liver tumor incidence and smaller tumor size. Two days after DEN injection, lower serum levels of alanine aminotransferase and decreased hepatic levels of cyclin D1 were observed in Lrat KO mice. Lrat KO mice also exhibited increased levels of retinoic acid-responsive genes, including p21, lower levels of cytochrome P450 enzymes required for DEN bioactivation and higher levels of the DNA repair enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), both before and after DEN treatment. Our results indicate that Lrat KO mice are less susceptible to DEN-induced hepatocarcinogenesis due to increased retinoid signaling and higher expression of p21, which is accompanied by altered hepatic levels of DEN-activating enzymes and MGMT in Lrat KO mice also contribute to decreased cancer initiation and suppressed liver tumor development.

Significance of Phosphatase and Tensin Homologue (PTEN), O6-Methylguanine-DNA Methyltransferase (MGMT), and DNA-dependent Protein Kinase Catalytic Subunit (DNA-PKcs) Protein Expression in Gynaecomastia

This study was designed to investigate the pathogenesis of gynaecomastia by measuring phosphatase and tensin homologue (PTEN), O(6)-methylguanine-DNA methyltransferase (MGMT) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein in breast tissue specimens from 68 patients with gynaecomastia and 24 normal male controls using immunohistochemical staining. The gynaecomastia cases were divided into three different histological types: florid, intermediate and fibrous. The PTEN, MGMT and DNA-PKcs proteins were detected in both gynaecomastia and normal breast tissue, but the levels of immunohistochemical staining of each protein were significantly lower in gynaecomastia breast tissue than in normal breast tissue. There were also significant differences in the levels of immunohistochemical staining for the three proteins according to gynaecomastia histological type. These results suggest that abnormally low levels of PTEN, MGMT and DNA-PKcs protein in gynaecomastia breast tissue may play a role in the development of gynaecomastia. Further research is required to elucidate fully their individual roles in the pathophysiology of gynaecomastia.

Low susceptibility to N-ethyl-N-nitrosourea-induced transplacental carcinogenesis in Long-Evans Cinnamon (LEC) rats

The Long-Evans Cinnamon (LEC) rat, an animal model of Wilson's disease, is resistant to a variety of chemical carcinogenesis except liver and colon. In the present study, N-ethyl-N-nitrosourea (ENU)-induced transplacental carcinogenesis was examined in male and female LEC, Long-Evans Agouti (LEA), a sibling line of the LEC rat, and F344 rats (n=21). ENU was administered to pregnant rats as a single s.c. injection at a dose of 60 mg/kg body weight on the 17th day after conception. Cerebral/spinal gliomas and trigeminal/spinal nerve schwannomas developed in both LEA and F344 rats at 30 weeks of age, but no nervous system tumors developed in LEC rats, the difference being statistically significant. Lung adenomas also developed in LEA and F344 rats, but not in LEC rats. Semiquantitative RT-PCR demonstrated that metallothionein (MT)1a, MT2 and O(6)-methylguanine-DNA methyltransferase (MGMT) mRNA levels in the liver of LEC rats were higher than those in F344 and LEA rats. In addition, Western blot analysis showed that MT (MT1 plus MT2) in the liver of LEC rats was also higher than that in other strains. Present results suggest that high levels of MT and/or MGMT contribute to the resistance to nitrosamine-induced carcinogenesis in LEC rats.

Inhibition of O6-methylguanine DNA methyltransferase (MGMT) in solid tumors by lomeguatrib

3597 Background: MGMT is implicated in tumor resistance to O6-alkylating agents, since the protein repairs DNA damage before replication results in cell death. Lomeguatrib (LM) is an orally bioavailable pseudosubstrate for MGMT that inactivates the protein through transfer of a boromthenyl group to the active site cysteine. De novo synthesis is required to restore cellular MGMT levels. We determined the dose of LM needed reliably to deplete MGMT in 3 tumor types, to inform future combination studies with methylating agents. Methods: Patients due to undergo surgery for prostate, colorectal or primary CNS cancers were invited to take part. They were dosed with LM PO 6 to 18 hours before surgery, and a portion of the tumor specimen retained for assessment of total and active MGMT. Active MGMT was assessed by transfer of tritium from methylated DNA to the protein fraction, and total MGMT by ELISA. 3 patient cohort dose escalations were used, with expansion to 6 patients where total depletion was encountered. Dose levels were 20, 40, 80, 120, and 160 mg. Results: LM was well tolerated and depleted MGMT in all 3 tumor types (table 1). Prostate cancers had the highest levels of MGMT (550 Fmol/mg protein) but were the most readily depleted tumour type. CNS tumours required more LM to achieve 100% depletion. Conclusions: Lomeguatrib depletes MGMT in prostate, colorectal and CNS cancers after single oral doses of 120 mg, 120 mg, and 160 mg respectively. Mean MGMT depletion (%) with a single LM dose Dose Level (mg) Prostate Cancer Colorectal Cancer Glioblastoma 20 81.7 44.7 40 96.8 61 46 80 98.3 96.5 57 120 99.5 97.7 73.4 160 100 Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Cancer Research UK

Mgmt deficiency alters the in vivo mutational spectrum of tissues exposed to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

It has been proposed that O(6)-methylguanine DNA methyltransferase (MGMT) gene silencing in premalignant lesions and cancers of the lung might result in the acquisition of a 'mutator' phenotype. Previously, however, we found that Mgmt(-/-) mouse DNA failed to show an increase in spontaneous mutations. We thus hypothesized that only during exposure to specific environmental carcinogens would the consequences of MGMT deficiency become evident. Metabolism of the tobacco-derived nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) generates alkylating species that can react with the O(6) position of deoxyguanine, thereby yielding substrates for MGMT-mediated repair. To investigate how MGMT might regulate the mutational effects of NNK, Mgmt(-/-) mice were crossed with a lacI-based transgenic reporter line (Big Blue) thus enabling an assessment of the in vivo mutagenic effects of this agent. We observed the induction of a complex spectrum of NNK-dependent lacI mutations in both control and Mgmt(-/-) tissues, but only a trend in the mutant frequency increases that could be attributed to MGMT deficiency. The mutational spectra of NNK-treated Mgmt(-/-) lungs revealed an increase in the absolute number of G:C to A:T changes accompanied by a shift in these from CpG to GpG sites, consistent with an S(N)1 alkylation mechanism. In keeping with the high levels of MGMT expressed in the liver, more pronounced mutagenic effects and greater differences in O(6) position of deoxyguanosine adduct levels following NNK were observed in Mgmt(-/-) versus wild-type mice. Extrapolating to humans, MGMT-deficient cells would likely exhibit an increased mutational burden, but only following exposures to specific environmental mutagens such as NNK.

Temozolomide induces senescence but not apoptosis in human melanoma cells

Temozolomide (TMZ), a DNA alkylating agent used in the treatment of melanoma, is believed to mediate its effect by addition of a methyl group to the O6 position of guanine in DNA. Resistance to the agent may be in part due to the activity of O6-methylguanine-DNA methyl transferase (MGMT). In the present study, we show that sensitivity of melanoma cells to TMZ was dependent on their p53 status and levels of MGMT. Analysis of the mechanisms underlying reduced viability showed no evidence for induction of apoptosis even though marked levels of apoptosis was seen in TK6 lymphoma cells. Sensitivity of melanoma cells was associated with p53-dependent G2/M cell cycle arrest and induction of senescence. To verify the role of p53, the assays were repeated in presence of pifithrin-α, an inhibitor of p53. This resulted in increased viability of melanoma cells with wild-type p53 and reversed G2/M cell cycle arrest. Paradoxically, apoptosis was increased in melanoma but decreased as expected in TK6 lymphoma cells. These results are consistent with the view that TMZ is relatively ineffective against melanoma due to defective apoptotic signalling resulting from activation of p53. The nature of the defects in apoptotic signalling remains to be explored.

Increased expression of the MGMT repair protein mediated by cysteine prodrugs and chemopreventative natural products in human lymphocytes and tumor cell lines

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein which protects the cellular genome and critical oncogenic genes from the mutagenic action of endogenous and exogenous alkylating agents. An expedited elimination of O6-alkylguanines by increasing MGMT activity levels is likely to be a successful chemoprevention strategy. Here, we report for the first time that cysteine/glutathione enhancing drugs and certain plant antioxidants possess the ability to increase human MGMT expression beyond its steady-state levels that may afford protection. The non-toxic cysteine prodrugs, 2-oxothiazolidine-4-carboxylic acid (OTC) and N-acetyl-L-cysteine (NAC), metabolized, respectively by 5-oxoprolinase and acylases, increased the MGMT protein and its repair activity levels in a dose- and time-dependent manner in several cancer cell lines and peripheral blood lymphocytes with a maximum of 3-fold increase by 72 h. The natural antioxidants, namely, curcumin, silymarin, sulforaphane and resveratrol were also effective in raising the MGMT levels to different extents. Among the synthetic agents, oltipraz and N-(4-hydroxyphenyl) retinamide (4-HPR) also increased MGMT expression, albeit to a lesser extent. Augmented mRNA levels accounted at least, in part, for the increased activity of MGMT in this setting. However, evidence from cysteine/methionine deprivation, acivicin treatment, and protein synthesis measurements in OTC-treated cells suggested that an increased cysteine flux also contributed significantly to enhanced MGMT expression. Many of these treatments increased the glutathione S-transferase-P1 (GSTP1) levels as well. These findings raise the possibility of MGMT-targeted chemoprevention strategies through dietary supplementation of OTC and herbal antioxidants. Further, the studies reveal the antioxidant responsiveness of the human MGMT gene.

Inverse Correlation Between the Expression of O6-Methylguanine-DNA Methyl Transferase (MGMT) and p53 in Breast Cancer

Expression of O(6)-methylguanine-DNA methyl transferase (MGMT), a DNA repair protein, has been associated with tumor resistance to alkylating agents such as cyclophosphamide. Promoter hypermethylation of MGMT is a strong predictor of survival, and expression of mutant p53 protein may be associated with downregulated MGMT expression in brain tumors. In order to clarify further the correlation between MGMT and p53 expression, we investigated the expression levels of both MGMT and p53 in breast cancer. MGMT and p53 expression was examined in tissues from 48 consecutive cases of primary breast cancer patients using immunohistochemical staining with antibodies specific for MGMT and p53. The prognosis for survival was analyzed based on MGMT and p53 immunoreactivity and clinicopathological characteristics. Expression of either MGMT or p53 was classified as negative or positive based on immunohistochemical staining. The expression of MGMT showed a significant inverse correlation with three clinicopathological characteristics, the status of the estrogen receptor, local recurrence and distant metastasis. There was a significant correlation between the expression level of p53 and the lymphatic involvement, estrogen receptor status and distant metastasis. MGMT and p53 were correlated with distant metastasis and local recurrence, but Kaplan-Meier curves did not show a significant difference. MGMT immuno-negative specimens showed a significantly higher expression of p53 (P = 0.026, chi(2) test). p53 may be associated with the regulation of MGMT expression in breast cancers.

Human osteosarcoma xenografts and their sensitivity to chemotherapy.

Despite the increased survival rates of osteosarcoma patients attributed to adjuvant chemotherapy, at least one third of the patients still die due to their disease. Further improvements in the management of osteosarcoma may rely on a more individualised treatment strategy, as well as on the introduction of new drugs. To aid in the preclinical evaluation of new candidate substances against osteosarcoma, we have established 11 human osteosarcoma xenograft lines and characterised them with regard to response to five different reference drugs. Doxorubicin, cisplatin methotrexate, ifosfamide and lomustine were effective in 3/11, 3/11, 1/10, 5/11 and 4/11 of the xenografts, respectively. Five xenografts were resistant to all compounds tested. We also assessed the mRNA expression levels of the xenografts for the O(6)-Methylguanine DNA Methyltransferase (MGMT), DNA topoisomerase II- (Topo II)-alpha, Gluthathione-S-transferase (GST)-pi, Multidrug-resistance related protein (MRP) 1 and Multidrug-resistance (MDR) 1 genes. There was an inverse correlation between the transcript levels of GST-pi and doxorubicin growth inhibition (r=-0.66; p<0.05), and between the transcript levels of MGMT and the effect of lomustine (r=-0.72; p<0.01), whereas the expression of MRP1 and cisplatin growth inhibition was positively correlated (r=0.82; p<0.005). This panel of xenografts should constitute a good tool for pharmacological and molecular studies in osteosarcoma.

Hammerhead ribozyme-mediated sensitization of human tumor cells after treatment with 1,3-bis(2-chloroethyl)-1-nitrosourea.

O(6)-Methylguanine DNA methyltransferase (MGMT) protects tumor cells from the cytotoxic effects of DNA-alkylating agents such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU). To improve the therapeutic index of BCNU, biochemical strategies to inhibit MGMT temporarily by systemic administration of small molecules, such as O(6)-benzylguanine, have been developed and are showing promise in clinical trials. In this study, an alternative molecular strategy for modulating BCNU resistance was explored using hammerhead ribozymes (Rz) designed to degrade the long-lived MGMT mRNA. We had previously identified several ribozymes capable of decreasing MGMT levels in HeLa cells. Using colony formation assays, the BCNU-induced cell kill was shown to be increased by 1 to 3 logs in the HeLa/Rz clones compared with wild-type HeLa cells at a BCNU dose of 100 microM. In the current study, 10 randomly selected clones of Rz161, 212, and a reconstructed Rz178/212 were assayed for MGMT activity, MGMT mRNA, and sensitivity to BCNU. The 30 clones exhibited almost identical results in the three assays, i.e., nearly undetectable MGMT activity, greatly diminished MGMT mRNA, and comparable sensitivity to BCNU using the 4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate (WST-1) viability assay. The effects of catalytically inactive ribozymes carrying a single point mutation were compared with their active counterparts in vitro and in stably transfected clones to determine whether antisense inhibition was a contributor to the inhibition of MGMT activity we observed. Collectively, these results suggest that the hammerhead ribozymes characterized in this study will be excellent candidates for future gene therapy approaches targeting MGMT.

DNA repair protein MGMT protects against N-methyl-N-nitrosourea-induced conversion of benign into malignant tumors.

Tumor formation is a multi-step process that can be divided into the stages of tumor initiation, promotion and progression. Previously, we showed that overexpression in skin of mice of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) protects against N-methyl-N-nitrosourea (MNU)-induced tumor initiation without affecting tumor promotion. This indicated that O(6)-methylguanine, which is specifically repaired by MGMT, is a major tumor-initiating lesion. Here we extended this transgenic approach to the study of tumor progression. Benign papillomas that arose on the skin of CkMGMT transgenic mice upon initiation with 7,12-dimethylbenz[a]anthracene (DMBA) and promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA) expressed higher levels of MGMT than papillomas that appeared on DMBA/TPA treated non-transgenic NMRI mice. Treatment of papillomas with MNU resulted in the formation of malignant carcinomas to a significantly lower frequency in CkMGMT mice as compared with the non-transgenic control. The data provide evidence that increased DNA repair protects against the conversion of benign into malignant tumors. They show at the same time that a particular type of damage induced in DNA, namely O(6)-methylguanine, is decisively involved in triggering tumor progression. This supports the concept that the major cause of both tumor initiation and tumor progression is mutation. Data also indicate that alkylating anti-neoplastic drugs may provoke tumor progression in case of failure of tumor therapy, which is attenuated by DNA repair.

Correlation of tumor O6 methylguanine-DNA methyltransferase levels with survival of malignant astrocytoma patients treated with bis-chloroethylnitrosourea: a Southwest Oncology Group study.

Prior studies show that increased levels of the DNA repair protein O6 methylguanine-DNA methyltransferase (MGMT), also referred to as O6-alkylguanine-DNA alkyltransferase (AGT) correlate with the resistance of glioma cell lines to nitrosoureas. The observed nitrosourea sensitivity of MGMT-deficient lines (methyl excision repair negative [MER-]) and those repair-proficient lines pretreated with MGMT-specific inhibitors (eg, O6 benzylguanine) has raised the possibility that tumor MGMT levels may be an important predictor of survival in patients with gliomas. We correlated the MGMT level in malignant astrocytoma tissues, obtained from patients treated with radiotherapy and bis-chloroethylnitrosourea (BCNU) on a prior prospective trial (Southwest Oncology Group [SWOG] 8737), with overall and failure-free survival. Of 64 assessable patients with malignant astrocytoma (63% glioblastoma, 37% anaplastic astrocytoma), 64% had high (> 60,000 molecules/nucleus) MGMT levels. The overall median survival for patients with high versus low MGMT levels was 8 and 29 months, respectively (P=.0002), and median failure-free survival 3 and 6 months, respectively (P=.008). Subset analysis by histology (high v low MGMT levels) for anaplastic astrocytoma was 14 versus 62 months (n=24) and for glioblastoma was 7 versus 12 months (n=40). The overall hazards ratio (risk for death) for high versus low MGMT levels was 3.41; in young patients, the hazards ratio was higher (age 18 to 40 years, 4.19; age 41 to 60 years, 3.08) but became equal by MGMT level at age older than 60 years (1.11). Multivariate analysis showed that MGMT was independent of other known prognostic factors (age, performance status, histology). The MGMT level in tumor tissue specimens may be a predictive marker of survival in patients with malignant astrocytoma that is independent of other previously described prognostic variables.

Intracellular Localization and intercellular heterogeneity of the human DNA repair protein O(6)-methylguanine-DNA methyltransferase.

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair protein that removes alkyl adducts from DNA and may be important in tumor resistance to alkylation chemotherapy. MGMT was visualized in human cells and tumor tissues with monoclonal antibodies against MGMT and immunofluorescence microscopy, and fluorescent signals were quantified by digital image analysis. MGMT was found both in the cytoplasm and the nucleus, and in either locale the protein reacts with alkylated DNA bases and becomes inactivated and lost from the cell. Cell lines in culture and xenografts showed a broad normal distribution of nuclear MGMT levels, but human brain tumors often showed a skewed distribution, with a significant fraction of cells with high levels of MGMT. O(6)-Benzylguanine, a suicide substrate inactivator for MGMT activity, reduced MGMT in human cells and in a mouse xenograft to levels undetectable by antibody assay 1 h post-treatment. In melanoma specimens taken from a patient 3 h post-treatment with temozolomide, MGMT levels were reduced by 70%. This quantitative immunofluorescence assay can be used to monitor MGMT and it depletion in human tumors to improve the use of alkylating agents in cancer chemotherapy.