MGMT mRNA Levels Research Articles

Constitutional BRCA1 and MGMT Methylation Are Significant Risk Factors for Triple-Negative Breast Cancer and High-Grade Serous Ovarian Cancer in Saudi Women.

Breast cancer (BC) and ovarian cancer (OC) are rapidly increasing in Saudi Arabia. BRCA1 and MGMT epimutations have been linked to a higher risk of these malignancies. The present research investigated the impact of these epimutations on the prevalence of BC and OC among Saudi women. DNA methylation was evaluated using methylation-specific PCR, whereas mRNA expression levels were assessed using qRT-PCR. We evaluated white blood cell (WBC)-BRCA1 methylation in 1958 Saudi women (908 BC patients, 223 OC patients, and 827 controls). MGMT methylation was determined in 1534 of the 1958 women (700 BC patients, 223 OC patients, and 611 controls). BRCA1 methylation was detected in 8.6% of the controls and 11% of the BC patients. This epimutation was linked to 13.8% of the early-onset BC patients (p = 0.003) and 20% of the triple-negative breast cancer (TNBC) patients (p = 0.0001). BRCA1 methylation was also detected in 14% of the OC patients (p = 0.011), 19.4% of patients aged <55 years (p = 0.0007), and 23.4% of high-grade serous ovarian cancer (HGSOC) patients. In contrast, the BRCA1 mutation was detected in 24% of the OC patients, 27.4% of patients aged ≥55 years, and 26.7% of the HGSOC patients. However, MGMT methylation was detected in 10% of the controls and 17.4% of the BC patients (p = 0.0003). This epimutation was linked to 26.4% of the late-onset BC patients (p = 0.0001) and 11% of the TNBC patients. MGMT methylation was also found in 15.2% of the OC patients (p = 0.034) and 19.1% of HGSOC patients (p = 0.054). Furthermore, 36% of the BRCA1-methylated patients and 34.5% of the MGMT-methylated patients had a family history of cancer, including breast and ovarian cancer. Notably, BRCA1 and MGMT mRNA levels were greater in the WBC RNA of the BC patients and cancer-free methylation carriers than in that of the OC patients. Our data indicate that BRCA1 and MGMT epimutations significantly contribute to the development of breast cancer and ovarian cancer in Saudi cancer patients. These blood-based biomarkers could help identify female patients at high risk of developing TNBC and HGSOC at an early age.

EXTH-78. CRISPROFF EPIGENOME EDITING AND FUNCTIONAL GENOMICS DEFINE MEDIATORS OF CHEMOTHERAPY RESISTANCE IN GLIOBLASTOMA

Abstract Alkylating chemotherapies exhibit survival benefit for patients with glioblastoma (GBM). CRISPRoff is a programmable epigenetic memory writer that stably and heritably silences genes through DNA methylation. Epigenetic silencing of MGMT via promoter methylation predicts response to chemotherapy and is prognostic for overall survival. Here, we performed epigenome editing using CRISPRoff to stably silence MGMT through induced promoter methylation as a therapeutically tractable approach for potentiating GBM to chemotherapy. We then used genome-wide CRISPR interference (CRISPRi) screens to broadly define novel chemosensitizing targets of GBM cells. Epigenome editing was performed through electroporation of CRISPRoff mRNA and sgRNAs into MGMT hypomethylated GBM cell lines (LN18 and T98G) and primary GBM cells. Whole genome bisulfite sequencing, RT-qPCR, and western blot assessed gene silencing. Cell viability assays measured drug sensitivity. GBM xenografts with CRISPRoff-induced silencing of MGMT were intracranially transplanted in mice and treated with temozolomide. Genome-wide functional genomics screens were performed using CRISPR interference. CRISPRoff against MGMT reduced MGMT mRNA and protein levels by 99.97%, generating up to 150-fold sensitization to temozolomide and 9-fold sensitization to lomustine in GBM cells. Silencing remained stable and heritable in clonally isolated GBM cells at 145 days after transient delivery of CRISPRoff, and gene repression and drug sensitization were equivalent to Cas9-mediated homozygous deletion of MGMT in vitro and in vivo. CRISPRoff of MGMT in intracranial xenografts exhibited significant reduction in tumor growth with temozolomide treatment compared to temozolomide treatment with control CRISPRoff (p = 0.0047). CRISPRi screens validated MGMT and defined 266 and 238 additional modifiers of temozolomide and lomustine response, respectively, including the ATR (BRCA2), DNA repair (REV1), cell cycle (PSMD13), and Fanconi anemia pathways (FANCI, FANCD2). In summary, we integrate targeted epigenome editing with unbiased genome-wide approaches to establish a novel discovery and therapeutic platform against glioblastoma.

Mechanism of DNA methylation-mediated downregulation of O6-methylguanine-DNA methyltransferase in cartilage injury of Kashin-Beck disease.

Kashin-Beck disease (KBD) is an endemic osteoarthropathy, in which excessive apoptosis of chondrocytes occurs. O6-methylguanine-DNA methyltransferase (MGMT), a DNA damage repair gene, plays an important role in apoptosis, but the mechanism is unclear in KBD cartilage injury. This study was to investigate the expression and promoter methylation of MGMT in KBD patients and its role in DNA damage and apoptosis of chondrocytes. MGMT mRNA and protein level were detected by quantitative real-time PCR and immunohistochemistry. Demethylation of MGMT was carried out using 5-Aza-2'-deoxycytidine, and the methylation level of MGMT promoter was measured by quantitative methylation specific PCR. Next, small hairpin RNA was used to knockdown the expression of MGMT. Cell viability, apoptosis and DNA damage were determined by MTT assay, flow cytometry, Hoechst 33342 staining and alkaline comet assay following T-2 toxin and selenium treatment. MGMT protein expression and mRNA levels were decreased (P = 0.02, P = 0.007) and promoter methylation was increased (P = 0.008) in KBD patients. Meanwhile, MGMT level was upregulated by 5-Aza-2'-deoxycytidine in chondrocytes (P = 0.0002). DNA damage and apoptosis rates were increased in MGMT-silenced chondrocytes (all P < 0.0001). Furthermore, DNA damage and apoptosis were increased in chondrocytes treated with T-2 toxin (all P < 0.0001), but were decreased after selenium treatment (P < 0.0001, P = 0.01). Decreased mRNA level and increased methylation of MGMT were found in the T-2 toxin group (P = 0.005, P = 0.002), while selenium reversed it (P = 0.02, P = 0.004). MGMT might play a crucial part in the pathogenesis of KBD cartilage injury, which could provide a therapeutic target for KBD.

Genomic Space of MGMT in Human Glioma Revisited: Novel Motifs, Regulatory RNAs, NRF1, 2, and CTCF Involvement in Gene Expression.

Background: The molecular regulation of increased MGMT expression in human brain tumors, the associated regulatory elements, and linkages of these to its epigenetic silencing are not understood. Because the heightened expression or non-expression of MGMT plays a pivotal role in glioma therapeutics, we applied bioinformatics and experimental tools to identify the regulatory elements in the MGMT and neighboring EBF3 gene loci. Results: Extensive genome database analyses showed that the MGMT genomic space was rich in and harbored many undescribed RNA regulatory sequences and recognition motifs. We extended the MGMT’s exon-1 promoter to 2019 bp to include five overlapping alternate promoters. Consensus sequences in the revised promoter for (a) the transcriptional factors CTCF, NRF1/NRF2, GAF, (b) the genetic switch MYC/MAX/MAD, and (c) two well-defined p53 response elements in MGMT intron-1, were identified. A putative protein-coding or non-coding RNA sequence was located in the extended 3′ UTR of the MGMT transcript. Eleven non-coding RNA loci coding for miRNAs, antisense RNA, and lncRNAs were identified in the MGMT-EBF3 region and six of these showed validated potential for curtailing the expression of both MGMT and EBF3 genes. ChIP analysis verified the binding site in MGMT promoter for CTCF which regulates the genomic methylation and chromatin looping. CTCF depletion by a pool of specific siRNA and shRNAs led to a significant attenuation of MGMT expression in human GBM cell lines. Computational analysis of the ChIP sequence data in ENCODE showed the presence of NRF1 in the MGMT promoter and this occurred only in MGMT-proficient cell lines. Further, an enforced NRF2 expression markedly augmented the MGMT mRNA and protein levels in glioma cells. Conclusions: We provide the first evidence for several new regulatory components in the MGMT gene locus which predict complex transcriptional and posttranscriptional controls with potential for new therapeutic avenues.

Low MGMT digital expression is associated with a better outcome of IDH1 wildtype glioblastomas treated with temozolomide.

Glioblastoma (GBM) is the deadliest primary brain tumor. The standard treatment consists of surgery, radiotherapy, and temozolomide (TMZ). TMZ response is heterogeneous, and MGMT promoter (MGMTp) methylation has been the major predictive biomarker. We aimed to describe the clinical and molecular data of GBMs treated with TMZ, compare MGMT methylation with MGMT expression, and further associate with patient's outcome. We evaluate 112 FFPE adult GBM cases. IDH1 and ATRX expression was analyzed by immunohistochemistry, hotspot TERT promoter (TERTp) mutations were evaluated by Sanger or pyrosequencing, and MGMTp methylation was assessed by pyrosequencing and MGMT mRNA expression using the nCounter® Vantage 3D™ DNA damage and repair panel. Of the 112 GBMs, 96 were IDH1WT, and 16 were IDH1MUT. Positive ATRX expression was found in 91.6% (88/96) of IDHWT and 43.7% (7/16) of IDHMUT. TERTp mutations were detected in 70.4% (50/71) of IDHWT. MGMTp methylation was found in 55.5% (35/63) of IDHWT and 84.6% (11/13) of IDHMUT, and as expected, MGMTp methylation was significantly associated with a better response to TMZ. MGMT expression was inversely correlated with MGMTp methylation levels (- 0.506, p < 0.0001), and MGMT low expression were significantly associated with better patient survival. It was also observed that integrating MGMTp methylation and expression, significantly improved the prognostication value. MGMT mRNA levels evaluated by digital expression were associated with the outcome of TMZ-treated GBM patients. The combination of MGMT methylation and mRNA expression may provide a more accurate prediction of TMZ response in GBM patients.

S-phase Specific Downregulation of Human O6-Methylguanine DNA Methyltransferase (MGMT) and its Serendipitous Interactions with PCNA and p21cip1 Proteins in Glioma Cells

Whether the antimutagenic DNA repair protein MGMT works solo in human cells and if it has other cellular functions is not known. Here, we show that human MGMT associates with PCNA and in turn, with the cell cycle inhibitor, p21cip1 in glioblastoma and other cancer cell lines. MGMT protein was shown to harbor a nearly perfect PCNA-Interacting Protein (PIP box) motif. Isogenic p53-null H1299 cells were engineered to express the p21 protein by two different procedures. Reciprocal immunoprecipitation/western blotting, Far-western blotting, and confocal microscopy confirmed the specific association of MGMT with PCNA and the ability of p21 to strongly disrupt the MGMT-PCNA complexes in tumor cells. Alkylation DNA damage resulted in a greater colocalization of MGMT and PCNA proteins, particularly in HCT116 cells deficient in p21 expression. p21 expression in isogenic cell lines directly correlated with markedly higher levels of MGMT mRNA, protein, activity and greater resistance to alkylating agents. In other experiments, four glioblastoma cell lines synchronized at the G1/S phase using either double thymidine or thymidine-mimosine blocks and subsequent cycling consistently showed a loss of MGMT protein at mid- to late S-phase, irrespective of the cell line, suggesting such a downregulation is fundamental to cell cycle control. MGMT protein was also specifically degraded in extracts from S-phase cells and evidence strongly suggested the involvement of PCNA-dependent CRL4Cdt2 ubiquitin-ligase in the reaction. Overall, these data provide the first evidence for non-repair functions of MGMT in cell cycle and highlight the involvement of PCNA in MGMT downregulation, with p21 attenuating the process.

Abstract 2081: The CCCTC-binding factor (CTCF) functions to downregulate the transcription of MGMT gene in human glioblastoma cells

Abstract The O6-methylguanine-DNA methyltransferase (MGMT) gene located at chromosome 10q26 encodes a DNA repair protein that abrogates the therapeutic effects of alkylating agents by preventing the formation of mutagenic lesions or cytotoxic DNA crosslinks. MGMT is highly expressed in human gliomas and its transcriptional regulation has emerged as a major translational focal point in neuro-oncology, because the epigenetic silencing of the MGMT gene which occurs in a subset of these malignancies confers improved treatment responses to both chemo and radiation therapies. While the promoter methylation and its contribution to glioma therapy has been much studied, the regulation of MGMT transcription per se, proteins accessory to the process and the chromatin structure thereof are not well understood. The CCCTC-binding transcription factor (CTCF) is one such multifunctional protein involved not only in DNA methylation but also with transcriptional activation/repression, and induction of short or long range chromatin looping. CTCF, an 11 zinc-finger containing protein is more frequently known for its insulating effects by binding diverse nucleotide sequences and restricting the function of transcriptional enhancers. Our bioinformatic analysis of the human MGMT gene showed an unusually high (7-10 sites) congregation of CTCF-binding sites distal to the promoter in human cancer cell lines. Given these considerations, we used CTCF siRNA to eliminate the transcription factor in two MGMT-proficient GBM cell lines (SF180 and GBM10). A specific reduction of CTCF protein by individual and pooled siRNAs was consistently accompanied by increased MGMT protein and mRNA levels. Furthermore, the DNA repair activity of MGMT was increased by 50%, which correlated with a proportional reduction of the CTCF protein in siRNA-exposed cells. Currently, we are investigating the effect of CTCF on MGMT reactivation in cells with methylation, MGMT promoter activity in CTCF silenced cells and the effect of enforced CTCF expression on MGMT transcription. Our results for the first time highlight the involvement of multivalent CTCF in MGMT expression. Since CTCF is tightly linked with DNA methylation, further studies should reveal its role in silencing of MGMT in human GBMs (supported by CPRIT RP130266 and NIH RO3CA125872 grants to KSS). Citation Format: Kalkunte S. Srivenugopal, Mohammed Ibrahim Al-Obaide. The CCCTC-binding factor (CTCF) functions to downregulate the transcription of MGMT gene in human glioblastoma cells. [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 2081. doi:10.1158/1538-7445.AM2015-2081

DNA repair gene expression level in peripheral blood and tumour tissue from non-small cell lung cancer and head and neck squamous cell cancer patients

BackgroundThe nucleotide excision repair pathway is crucial for cellular DNA integrity and the ERCC1 helicase is also potentially involved in resistance to platinum-based chemotherapy, and high levels of ERCC1 mRNA in tumours have been associated with cisplatin resistance in different human cancers. The aim of this work was to investigate the correlation between DNA repair gene expression levels in tumour tissue, normal tissue and peripheral blood samples from patients with two common human cancers, non-small cell lung cancer (NSCLC) and squamous cell carcinoma of the head and neck (HNSCC), to test if blood gene expression could be a proxy for tumour tissue gene expression to predict response to platinum-based chemotherapy. MethodsUsing RT-qPCR we determined ERCC1, ERCC2, ERCC4, XPA, XPC, XRCC1, XRCC3, APEX, OGG1, MGMT mRNA levels in fresh NSCLC, normal lung and HNSCC tissue, as well as blood, from NSCLC and HNSCC patients who were treated surgically. ResultsTarget gene expression in NSCLC and HNSCC tissue was higher than in blood. A statistically significant correlation (p<0.05) was found between target gene mRNA expression in tumour tissue and blood, in particular ERCC1, MGMT, XPC, XRCC1 and XRCC3 in NSCLC and APEX, ERCC1, ERCC2, ERCC4, XRCC1 and XRCC3 in HNSCC. ConclusionsThe existence of a significant correlation between blood and tumour tissue expression of some genes of clinical interest, such as ERCC1 in NSCLC and HNSCC, could allow the introduction in clinical practice of a simple test that would measure mRNA levels of DNA repair genes in peripheral blood samples instead of tissue samples to determine prognostic and predictive factors in NSCLC and HNSCC patients.

MGMT and PTEN as potential prognostic markers in breast cancer

AimTo evaluate the prognostic importance of MGMT and PTEN concerning their correlation with other prognostic factors evaluated by immunohistochemistry (IHC) and the molecular phenotype of breast cancers. MethodsIHC for estrogen and progesterone receptors, HER2, Ki67, p53, p63, e-cadherin, EGFR, CK5, CK14, MGMT and PTEN was performed on 200 breast tumors. Basal-like and luminal breast carcinomas were defined by the IHC evaluation of these markers. Fluorescent in situ hybridization (FISH) was performed for PTEN and HER2 analysis using the Vysis PTEN and HER2 DNA probe kits (Abbott™). RT-PCR was performed to evaluate gene expressions of MGMT and PTEN in frozen tissue of 59/200 cases. Results147/200 cases were triple-negative (73.5%), 47/147 were basal-like carcinomas (31.9%). 53 cases (26.5%) were luminal-like type A or B. 56 (93.3%) and 46 samples (76.6%) expressed lower levels of MGMT and PTEN mRNA, respectively, compared with normal breast (p<0.001). There was a positive correlation between the IHC results and the RT-PCR values for MGMT and PTEN. Tumors with homozygotic deletion of PTEN expressed little or no mRNA or protein. Positive p53, high Ki67, and basal-like tumors expressed significant lower MGMT and PTEN. ConclusionsWe hypothesize that MGMT and PTEN expressions have prognostic significance in breast cancer. Also, based on their predictive value of response to therapy, evaluating MGMT and PTEN and learning to interpret their patterns of immunoexpression will undoubtedly lead to a greater understanding of breast cancer and its treatment.

Expression of glioma stem cell marker CD133 and O6-methylguanine-DNA methyltransferase is associated with resistance to radiotherapy in gliomas

In the present study, we investigated the prognostic roles of the O6-methylguanine-DNA methyltransferase (MGMT) gene methylation status, the protein profiles of MGMT, and the glioma stem cell (GSC) marker CD133 in malignant glioma resistance to radiotherapy. The proliferation of glioma cells was assessed using a clonogenic survival assay and flow cytometry. CD133 expression was assessed in SHG-44-GSCs using RT-PCR and flow cytometry. MGMT exhibited resistance to radiation in the SHG-44-GSCs using siRNA transfection. The effects of the siRNA on mRNA and protein expression of MGMT in SHG-44-GSCs were detected using semi-quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting. MGMT methylation status, MGMT and CD133 expression profiles were assessed in 59 malignant glioma patients using methylation-specific polymerase chain reaction (MSP), and immunohistochemistry. Invitro, SHG-44-GSCs exhibited a characteristic resistance to radiation that was not observed in SHG-44 cells. This resistance was attributed to the unmethylated status of the MGMT promoter and to high expression levels of MGMT mRNA in the glioma cells. In these patients, the CD133 marker, but not MGMT promoter methylation or MGMT protein level, was associated with resistance to radiotherapy (n=59; hazard ratio=2.838; 95% CI, 1.725-7.597; p=0.001). The median progression-free survival (PFS) among patients with the CD133 marker was 14 months, whereas it was 35 months in patients without CD133 (p=0.001). Notably, co-expression of the methylated MGMT promoter and the CD133 marker was associated with the poorest outcome in patients with gliomas treated by radiotherapy; in these patients, PFS was 7 months. These results suggest that assessment of GSC MGMT and CD133 levels will guide future clinical targeted therapies and stratify glioma patient treatment regimens. High expression levels of the CD133 protein could be used as a predictor for poor survival in patients treated with radiotherapy.

Effects of sodium arsenite on hypermethylation, transcription and expression of O6-methylguanine-DNA methyltransferase gene in HaCaT cells

Objective To investigate the DNA methylation feature and DNA methylation regulation to its transcription and expression of O6-methylguanine-DNA methyltransferase gene (MGMT) in NaAsO2-treated HaCaT cells. Methods HaCaT cells were treated 72 hours at intervals and repeatedly by 3.13, 6.25,12.50, and 25.00 μmol/L NaAsO2, MGMT gene promoter region was amplified in the transcription initiation site - 329 - + 93 region by bisulfate-sequencing polymerase chain reaction (BSP), the mRNA transcription and the protein expression of MGMT was detected by real-time quantitative PCR and Western blotting. NaAsO2-untreated HaCaT cell was set as a blank control, and human epidermal squamous carcinoma cell strain A431 was set as a positive control. Results Among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, the positive rates of the DNA methylation of promoter region in MGMT gene were 0.63%(l/160), 6.25% (10/160), 10.63%( 17/160) and 18.75% (30/160), respectively, and methylated CpG sites were mainly located in - 249--146 region relative to transcription start site. There was no DNA methylation in the blank control. There were significant differences between the blank control and the NaAsO2-treated cells (x2 = 76.687, P< 0.05). Average levels of MGMT mRNA were 1.518 31 ± 0.180 54, 1.425 22 ± 0.180 39, 1.014 54 ± 0.096 79 and 0.887 72 ± 0.020 00, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells(1.198 29 ± 0.159 97), there were significant differences(F = 37.359, P < 0.05). Average levels of MGMT protein were 1.174 47 ± 0.064 75, 0.848 83 ± 0.057 01, 0.471 63 ± 0.023 34 and 0.240 34 ± 0.014 43, respectively among the groups of HaCaT cells treated with 3.13, 6.25, 12.50 and 25.00 μmol/L NaAsO2, compared with the blank control cells (1.066 19 ± 0.061 24), there were significant differences(F = 20.687, P < 0.05). Conclusions Arsenic can cause CpC island hypermethylation in the promoter region of MGMT gene, which results in inhibited MGMT mRNA transcription and protein expression. It might be one of the important mechanisms of arsenic-induced skin lesion. Key words: Arsenic; DNA methylation; Transcription, genetic; Proteins; O6-methylguanineDNA methyltransferase gene

Abstract 619: O6-Benzylguanine inhibits tamoxifen-resistant breast cancer cell growth and resensitizes breast cancer cells to anti-estrogen therapy

Abstract Endocrine therapies using anti-estrogens are less toxic and very effective for breast cancers however tumor resistance to tamoxifen remains a stumbling block for successful therapy. Based on our recent study on the involvement of the DNA repair protein MGMT in pancreatic cancer (Clin Cancer Res. 15, 6087, 2009), we investigated whether MGMT overexpression mediates tamoxifen resistance. Specifically, we determined whether administration of MGMT inhibitor [O6-benzylguanine (BG)] at a non-toxic dose alone or in combination with the anti-estrogens (tamoxifen/fulvestrant) curtails human tamoxifen resistant breast cancer cell growth. Furthermore, we also determined whether BG sensitizes breast cancer cells to tamoxifen using tamoxifen resistant cells. MGMT expression was found to be increased in breast cancer cells relative to normal breast epithelial cells. Also, MGMT levels were significantly higher in tamoxifen resistant MCF-7 compared to the parent cells. Silencing of the ER-α expression using a specific siRNA resulted in augmentation of MGMT mRNA and protein levels by two fold. We also observed an inverse correlation between MGMT and p53 levels in breast cancer cell lines; moreover, p53 downregulation was accompanied by increased MGMT expression. Other experiments showed that BG alone or BG in combination with tamoxifen or fulvestrant decreased ER-α expression, whereas tamoxifen alone and fulvestrant alone increased and decreased the same respectively. All these treatments increased the p21cip1 mRNA and protein expression significantly. BG inhibited tamoxifen resistant breast cancer growth in a dose-dependent manner and it also resensitized resistant breast cancer cells to anti-estrogen therapy (TAM/ICI). These combinations also enhanced the cytochrome C release and PARP cleavage, indicative of apoptosis. In breast cancer xenografts, BG alone or a combination of BG with tamoxifen or fulvestrant caused a significant tumor growth delay and immunohistochemistry revealed that BG inhibited the expression of MGMT, ER- α, ki-67 and increased p21cip1 staining. These findings suggest that MGMT inhibition may provide a novel and effective approach for overcoming tamoxifen resistance (supported by a Florida Biomedical grant to SK). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 619. doi:10.1158/1538-7445.AM2011-619

Endocrine disrupting chemicals modulate expression of O 6-methylguanine DNA methyltransferase ( O6-MGMT) gene in the hermaphroditic fish, Kryptolebias marmoratus

O 6-methylguanine-DNA methyltransferase (O 6-MGMT; EC 2.1.1.63) is a key repair enzyme that helps to protect the cell against alkylation on DNA by removing a methyl group from the O 6-position of guanine. Here, we cloned and sequenced the full-length O 6 -MGMT cDNA from the hermaphroditic fish, Kryptolebias marmoratus. Complete Km-O 6 -MGMT cDNA was 1324 bp in length, and the open reading frame of 567 bp encoded a polypeptide of 188 amino acid residues. Phylogenetic analysis revealed that Km-O 6 -MGMT was clustered with those of other fish species. Embryo, juveniles, and aged secondary fish had low levels of Km-O 6 -MGMT mRNA than adults, indicating more susceptibility to DNA damage by alkylating agent exposure during these developmental stages. Km-O 6 -MGMT mRNA levels differed according to tissue type and was highest in the liver. Exposure to an alkylating agent, N-methyl- N-nitrosourea (MNU) exposure increased the mRNA expression of tumor suppressor gene such as p53 and oncogenes such as R-ras1, R-ras3, N-ras, c-fos as well as Km-O 6 -MGMT mRNA in a time-dependent manner. On the contrary, several (anti)estrogenic compounds (17β-estradiol 100 ng/L, tamoxifen 10 μg/L, bisphenol A 600 μg/L, and 4- tert-octylphenol 300 μg/L) suppressed mRNA expression of Km-O 6 -MGMT in most tissues, especially the liver. In juvenile fish, 17β-estradiol, bisphenol A, and 4- tert-octylphenol also decreased the expression of Km-O 6 -MGMT mRNA in a time-dependent manner. Overall, our finding shows that Km-O 6 -MGMT mRNA levels can be modulated by environmental estrogenic compounds as well as alkylating agents. This finding will be helpful to improve our knowledge of the effects of estrogenic compounds that contain the genotoxic ability to inhibit the DNA repair process in aquatic animals.

Promoter hypermethylation and expression profile of MGMT and CDH1 genes in oral cavity cancer

Background Several genetic alterations have been reported to contribute to the development of oral squamous cell carcinoma (OSCC). Methylation of CpG-islands in cancer-related genes may serve as epigenetic biomarkers for oral cancer diagnosis and prognosis. The objective of this study was to analyze methylation profile of MGMT and CDH1 genes and their link with expression activity in patients with oral cavity cancer. Methods Promoter hypermethylation status of MGMT and CDH1 genes were assayed by Methylation-specific PCR (MSP) in OSCC ( n = 76) tissues kept in paraffin and normal oral tissues ( n = 57) served as control. Also, we investigated MGMT and CDH1 mRNA levels by real-time quantities reverse transcripts PCR. Methylation and mRNA expression profiles of these genes and their association with clinical data were determined. Results Aberrant promoter hypermethylation of CDH1 and MGMT genes were detected in 61.8% (47 of 76) and 73.7% (56 of 76) of the OSCC cases, respectively, with significant difference between cases and controls for MGMT ( P = 0.027). CDH1 promoter methylation in cases and healthy controls was not significant. The mRNA expression level results showed statistically significant ( P = 0.03) differences between cases and healty controls for the MGMT gene. However, the difference for the CDH1 gene was not significant. Conclusion Our findings, for the first time, in a South-Eastern Iranian population, indicate that the two genes are aberrantly methylated in OSCC, and that MGMT methylation may be considered as a potential molecular marker for the poor survival in advanced OSCC.

Gene expression profiling predicts response to temozolomide in malignant gliomas

Malignant gliomas are highly lethal neoplasms that cannot be cured with currently available therapies. Temozolomide (TMZ) is a recently introduced alkylating agent that has yielded significant benefits and become a key agent in the treatment of high-grade gliomas. However, its survival benefit remains unsatisfactory. Understanding the molecular basis of TMZ sensitivity/resistance is necessary for improving the treatment outcome by devising strategies that are able to circumvent primary drug resistance. We therefore combined the in vitro TMZ response with microarray gene expression data to identify genes that could potentially be used to predict the response of malignant gliomas to TMZ therapy. We first obtained the individual IC50 values for TMZ in seven malignant glioma cell lines (A-172, AM-38, T98G, U-87MG, U-138MG, U-251MG and YH-13) and then identified the genes whose expression correlated most highly with TMZ sensitivity employing a cDNA microarray. We present here a list of the most highly up-regulated and down-regulated genes which may be involved in conferring TMZ sensitivity/resistance in malignant gliomas, although most of the genes have not been implicated as a causal factor in the TMZ response except MGMT. We also demonstrated and confirmed the MGMT methylation status, quantitative MGMT mRNA levels, and MGMT protein expression levels in TMZ resistant glioma cells in vitro. Our results are thus consistent with previous studies and suggest that a dominant mechanism conferring sensitivity/resistance to TMZ exists in malignant glioma cells. Although the present study dose have several limitations, our reported candidate genes could represent not only potential molecular markers for TMZ sensitivity/resistance but also chemotherapy targets. Furthermore, the present study could provide a foundation for alternative therapeutic strategies including novel combination treatments that incorporate additional reagents directed at overcoming resistance to TMZ.

Effect of IFN-β on human glioma cell lines with temozolomide resistance

Human interferon-beta (IFN-beta) is known to exhibit pleiotropic biological activities including antitumor effects. On the other hand, temozolomide (TMZ), an oral bioavailable alkylating agent with excellent tolerability, has demonstrated efficacy and has become a key therapeutic agent in patients with malignant gliomas; however, its survival benefit remains unsatisfactory. More recent studies have indicated that there might be favorable therapeutic interactions between IFN-beta and TMZ, although the therapeutic advantages of such a combination have not yet been fully explored. The main aim of the present study was to determine whether an antitumor effect could be potentiated by a combination of IFN-beta and TMZ. The antitumor effect of and cell sensitivity to IFN-beta and TMZ and the synergistic potential of IFN-beta and TMZ in combination were evaluated in six malignant glioma cell lines. Correlations among the MGMT methylation status, quantitative level of MGMT mRNA, MGMT protein expression and the antitumor effect of these agents were also evaluated, since one of the most prominent resistance mechanisms to TMZ involves the DNA repair protein MGMT. The cell growth inhibitory effects of IFN-beta and TMZ on all tumor cell lines were observed in a dose-dependent manner, and the human malignant glioma-derived cell lines differed in their sensitivity to TMZ. The MGMT status, including promoter hypermethylation, quantitative mRNA expression and protein expression, was strongly correlated with TMZ sensitivity. A synergistic cell growth inhibitory effect and down-regulated MGMT mRNA levels were significantly observed when a clinically achievable CNS dose of IFN-beta was combined with TMZ, as compared to treatment with IFN-beta or TMZ alone in TMZ-resistant T98G cells. Furthermore, significant amounts of endogenous IFN-beta protein were detected in TMZ-treated T98G cells by ELISA. These results suggest that the clinical therapeutic efficacy of TMZ might be improved by a combination with IFN-beta in malignant gliomas unmethylated at the MGMT gene. The data provide an experimental basis for future strategies in TMZ chemotherapy, although further studies are needed to determine the detailed role of combined IFN-beta and TMZ chemotherapy in increasing tumor sensitivity.

Influence of Helicobacter pylori infection on the expression of MLH1 and MGMT in patients with chronic gastritis and gastric cancer

The aim of the present study was to evaluate the influence of Helicobacter pylori on MLH1 and MGMT mRNA levels in patients with chronic gastritis and gastric cancer. The study included 217 patients, of which 26 were uninfected, 127 had chronic gastritis and were H. pylori-positive, and 64 had gastric cancer. Bacterial genotypes were evaluated by polymerase chain reaction (PCR), and the expression levels of MLH1 and MGMT were determined by quantitative real-time PCR and immunohistochemistry. There was an association between infection with cagA, vacA s1m1 strains and gastric cancer development. When the gastric epithelium and associated inflammation were examined for expression of MLH1 and MGMT, an overall increase in expression was observed. On the other hand, these levels decrease significantly among gastric cancer patients. The loss of MLH1 and MGMT expression in gastric cancer patients suggests that it is not an early event in H. pylori-associated gastric carcinogenesis.

Alterations in S-adenosylhomocysteine metabolism decrease O6-methylguanine DNA methyltransferase gene expression without affecting promoter methylation

The DNA repair enzyme O6-methylguanine DNA methyltransferase (MGMT) protects cells against the cytotoxic effects of alkylating agents. Therefore, modulation of MGMT expression in tumors is a possible strategy for improving the efficiency of cancer therapy. MGMT expression and activity is lost frequently in association with DNA hypermethylation of the MGMT promoter region. Since DNA and mRNA methylation are controlled by intracellular S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) levels, we hypothesized a role for AdoMet/AdoHcy ratio in the regulation of MGMT promoter methylation and mRNA expression.Our initial studies showed that AdoMet/AdoHcy ratios vary over a wide range (7.0–50) in different glioblastoma and hepatoma cell lines. The studied cell lines exhibit distinct MGMT promoter methylation patterns: MGMT promoter was completely unmethylated in LN-18 and Tu 132 cells, hypermethylated in LN-229, U87-MG, and Tu 113 cells, and partially methylated in HepG2 cells. Furthermore, MGMT promoter methylation patterns and global DNA methylation are not related to intracellular AdoMet/AdoHcy ratio under control conditions. To lower AdoMet/AdoHcy ratio to values <1 we used AdoHcy hydrolase inhibitor adenosine-2′,3′-dialdehyde (30μM) and found that neither short-term (24h) nor long-term changes (7 weeks) in AdoMet/AdoHcy ratio altered global or MGMT promoter methylation. However, experimentally elevated AdoHcy levels significantly decreased MGMT mRNA levels by >50% in all MGMT-expressing cell lines, which is most likely the result of impaired mRNA methylation. Thus, the present study suggests elevation of AdoHcy levels by AdoHcy hydrolase inhibition as a novel pharmacological approach to modulate MGMT expression and to increase the responsiveness to alkylating agents.

Expression silence of DNA repair gene hMGMT induced by RNA interference

ObjectiveMGMT protein expression has been associated with tumor resistance to alkylating agents. The objective of this paper is to construct the RNA interference vector which can specifically induce the expression silence of human DNA repair gene hMGMT.MethodsThe hMGMT specific siRNA expression cassette was made by two steps PCR, linked with pUC19 to get pU6-MGMTi, co-transfected with pEGFP-C1 into 16HBE and screened by G418. The MGMT mRNA and protein levels were detected by RT-PCR and Western Blot respectively.ResultshMGMT specific RNA interfere vector pU6-MGMTi was constructed successfully. In transfected 16HBE cells MGMT mRNA level could hardly be detected and the protein level was only 10% of control.ConclusionMGMT specific RNAi expression cassette can effectively inhibit MGMT expression. MGMT silence cell line was built by co-transfection technology, which offered condition for studying the gene function of MGMT.

Analysis of gene expression and chemoresistance of CD133+ cancer stem cells in glioblastoma

BackgroundRecently, a small population of cancer stem cells in adult and pediatric brain tumors has been identified. Some evidence has suggested that CD133 is a marker for a subset of leukemia and glioblastoma cancer stem cells. Especially, CD133 positive cells isolated from human glioblastoma may initiate tumors and represent novel targets for therapeutics. The gene expression and the drug resistance property of CD133 positive cancer stem cells, however, are still unknown.ResultsIn this study, by FACS analysis we determined the percentage of CD133 positive cells in three primary cultured cell lines established from glioblastoma patients 10.2%, 69.7% and 27.5%, respectively. We also determined the average mRNA levels of markers associated with neural precursors. For example, CD90, CD44, CXCR4, Nestin, Msi1 and MELK mRNA on CD133 positive cells increased to 15.6, 5.7, 337.8, 21.4, 84 and 1351 times, respectively, compared to autologous CD133 negative cells derived from cell line No. 66. Additionally, CD133 positive cells express higher levels of BCRP1 and MGMT mRNA, as well as higher mRNA levels of genes that inhibit apoptosis. Furthermore, CD133 positive cells were significantly resistant to chemotherapeutic agents including temozolomide, carboplatin, paclitaxel (Taxol) and etoposide (VP16) compared to autologous CD133 negative cells. Finally, CD133 expression was significantly higher in recurrent GBM tissue obtained from five patients as compared to their respective newly diagnosed tumors.ConclusionOur study for the first time provided evidence that CD133 positive cancer stem cells display strong capability on tumor's resistance to chemotherapy. This resistance is probably contributed by the CD133 positive cell with higher expression of on BCRP1 and MGMT, as well as the anti-apoptosis protein and inhibitors of apoptosis protein families. Future treatment should target this small population of CD133 positive cancer stem cells in tumors to improve the survival of brain tumor patients.