4255

MGMT

-;O-6-methylguanine-DNA methyltransferase;MGMT;O-6-methylguanine-DNA methyltransferase

6-O-methylguanine-DNA methyltransferase|O-6-methylguanine-DNA-alkyltransferase|O6-methylguanine-DNA methyltransferase|methylated-DNA--protein-cysteine methyltransferase|methylguanine-DNA methyltransferase

10q26

protein-coding

Human tumor cell lines that do not express O6-methylguanine-DNA methyltransferase (MGMT) in detectable quantities (Mer-) are hypersensitive to the effects of O6-guanine-alkylating agents. Because the Mer- phenotype enhances tumor responseto such agents, we investigated possible mechanisms involved in regulation of MGMT expression in a panel of Mer+ and Mer- pediatric rhabdomyosarcoma xenograftand cell lines. ALL Mer- cell and xenograft lines lacked not only MGMT activity but also the protein and mRNA as well, suggesting that its expression is transcriptionally regulated. Transfection of Mer+ and Mer- rhabdomyosarcoma celllines with MGMT gene promoter-CAT constructs yielded similar levels of CAT expression, indicating that Mer- cells possessed the necessary factors to support transcription. Methylation in the 5'-untranslated region of the MGMT gene was assayed by Southern analysis using methylation-sensitive restriction enzymes. Digestion with HpaII and its methylation-insensitive isoschizomer, MspI, revealed little overall correlation between methylation and MGMT expression. However, methylation in a single SmaI site at position-69 was observed in ALL MGMT deficient lines but not in any MGMT expressing lines. These results suggest thatmethylation of specific cytosines in the MGMT promoter may play a role in suppressing its expression, as well as being a potentially useful marker for theMer- phenotype.

DNA methylation and epigenetic inactivation of the O6-methylguanine methyltransferase (MGMT) gene induces MGMT deficiency, reducing the tumor cell'sDNA repair capacity and increasing its susceptibility to alkylating chemotherapeutic agents. Consequently, adult patients whose tumors are deficientin MGMT have better outcomes with alkylator chemotherapy, and MGMT methylation has been proposed as a screening marker of deficient tumors. In order to test the feasibility of this approach for medulloblastoma, a common brain tumor in children, we determined the methylation status, mRNA expression pattern, and protein expression of MGMT in a panel of clinical specimens. Methylation-specific polymerase chain reaction analysis revealed methylation of MGMT in 28 of 37 tumor samples. Quantitative real-time reverse transcriptase-polymerase chain reaction showed a range of expression of MGMT mRNA varying more than 20-fold. However, there was no correlation found between MGMT methylation and mRNA expression. Immunohistochemistry demonstrated that ALL tumors were immunoreactive for MGMT in the nucleus of the medulloblastoma cells in a heterogeneous pattern. The intercell variability of MGMT complement explained the discordance between methylation and expression. Therefore, MGMT methylation as determined by methylation-specific polymerase chain reaction cannot be used as a marker for MGMT deficiency in medulloblastoma. Further, these findings support the use of pharmacological MGMT depletion as a rational approach for intensification of alkylator chemotherapy in the treatment of medulloblastoma.

BACKGROUND: Osteosarcoma is the most common solid malignant diseases of childhood, occurring in approximately 6 children per million annually; however, to the authors' knowledge to date, the cause of osteosarcoma has remained mostlyunknown. Genetic alterations of genes that are specific for osteosarcoma have not been identified. Genetic alternations in the status of DNA methylation, known asepigenetic alterations, are the most common molecular alterations in human neoplasia. Aberrant methylation in the promoter region of tumor-related genes isassociated closely with epigenetically mediated gene silencing, which is a common feature in human tumors. METHODS: The authors analyzed CpG islands of 5 different gene loci for aberrant methylation profiles in 30 pairs of osteosarcoma and corresponding normal tissues by using the quantitative methylation-specific polymerase chain reaction method. The objectives of this study were to characterize the methylation changes in osteosarcoma more extensively and to identify epigenetic biomarkers that may be useful in the diagnosis and prevention of osteosarcoma. RESULTS: For the Ras effector homologue (RASSF1A), tissue inhibitor of metalloproteinase 3 (TIMP3), O-6-methylguanine DNA methyltransferase (MGMT), and death-associated protein kinase 1 (DAPK1) genes, significant differences were observed in the degree of hypermethylation between tumors and normal tissues (P < 0.01 and P < 0.001, respectively). Measurement of the cumulative multiple promoter hypermethylation revealed striking differences between tumor specimens and normal tissues (t = 7.31; P < .001). There also was a significant difference in the levels of DNA methylation between the metastatic and nonmetastatic high-grade osteosarcomas (t = 4.57; P < .01). In addition, themethylation levels were associated closely with gender (t = 6.44; P < .001). CONCLUSIONS: The results indicated that tumor tissues from patients with osteosarcoma had a significantly higher incidence of hypermethylation for several genes compared with corresponding normal tissues. The epigenetic changes observed in this study may have prognostic importance for patients with osteosarcoma.#CI- Copyright 2006 American cancer Society.

BACKGROUND: Recently, 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 thedrug resistance property of CD133 positive cancer stem cells, however, are stillunknown. RESULTS: In 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. CONCLUSION: Our 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.

Insufficient response to oral temozolomide (TMZ) in children with brain tumor may depend on the repair-action of inducible O6-methylguanine-DNA methyltransferase (MGMT). To investigate the clinical relevance of MGMT expression, we analyzed MGMT levels by qRT-PCR and immunohistochemistry, and the methylation of gene promoter in patients with relapsed or refractory brain tumor, enrolled in an off-label trial with oral temozolomide. The drug was administered at the dose of200 mg/m(2)/day in patients with no prior cranio-spinal irradiation, and 180 mg/m(2)/day in those with previous radiotherapy and/or high-dose chemotherapy followed by autologous hematopoietic stem cell rescue. Nine patients with recurrent ependymoma (n=3), low grade glioma (n=3), glioblastoma (n=1), relapsedmedulloblastoma (n=2) were enrolled in the study. Median absolute MGMT mRNA expression level standardized for GAPDH was 1.06 (range -0.453 to 3.932). The median relative expression level (RQ=2(-ddC)) was 4.29 (range 1.585 to 12.228). By immunohistochemistry, the score was 2+ in 6 of the 9 tumor samples, and 1+ in3, while none was MGMT negative. Methylation of MGMT promoter was detected in only one ependymoma sample. The heterogeneous PFS in our patients treated with second line TMZ, indicates that MGMT expression alone is insufficient to predictthe response to TMZ, presumably because of the DNA repair mechanisms involved.