Home About us Contact
|
Home About us Contact | ||
|
|
||
Methylguanine DNA Methyltransferase (methylguanine + dna_methyltransferase)
Selected AbstractsProtection of hematopoietic cells from O6 -alkylation damage by O6 -methylguanine DNA methyltransferase gene transfer: studies with different O6 -alkylating agents and retroviral backbonesEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 1 2001Michael Jansen Abstract: Overexpression of O6 -methylguanine DNA methyltransferase (MGMT) can protect hematopoietic cells from O6 -alkylation damage. To identify possible clinical applications of this technology we compared the effect of MGMT gene transfer on the hematotoxicity induced by different O6 -alkylating agents in clinical use: the chloroethylnitrosoureas ACNU, BCNU, CCNU and the tetrazine derivative temozolomide. In addition, various retroviral vectors expressing the MGMT-cDNA were investigated to identify optimal viral backbones for hematoprotection by MGMT expression. Protection from ACNU, BCNU, CCNU or temozolomide toxicity was evaluated utilizing a Moloney murine leukemia virus-based retroviral vector (N2/Zip-PGK-MGMT) to transduce primary murine bone marrow cells. Increased resistance in murine colony-forming units (CFU) was demonstrated for all four drugs. In comparison to mock-transduced controls, after transduction with N2/Zip-PGK-MGMT the IC50 for CFU increased on average 4.7-fold for ACNU, 2.5-fold for BCNU, 6.3-fold for CCNU and 1.5-fold for temozolomide. To study the effect of the retroviral backbone on hematoprotection various vectors expressing the human MGMT-cDNA from a murine embryonic sarcoma virus LTR (MSCV-MGMT) or a hybrid spleen focus-forming/murine embryonic sarcoma virus LTR (SF1-MGMT) were compared with the N2/Zip-PGK-MGMT vector. While all vectors increased resistance of transduced human CFU to ACNU, the SF1-MGMT construct was most efficient especially at high ACNU concentrations (8,12 µg/ml). Similar results were obtained for protection of murine high-proliferative-potential colony-forming cells. These data may help to optimize treatment design and retroviral constructs in future clinical studies aiming at hematoprotection by MGMT gene transfer. [source] Frequent promoter hypermethylation and low expression of the MGMT gene in oligodendroglial tumorsINTERNATIONAL JOURNAL OF CANCER, Issue 3 2005Maria Möllemann Abstract Allelic losses on the chromosome arms 1p and 19q have been associated with favorable response to chemotherapy and good prognosis in anaplastic oligodendroglioma patients, but the molecular mechanisms responsible for this relationship are as yet unknown. The DNA repair enzyme O6 -methylguanine DNA methyltransferase (MGMT) may cause resistance to DNA-alkylating drugs commonly used in the treatment of anaplastic oligodendrogliomas and other malignant gliomas. We report on the analysis of 52 oligodendroglial tumors for MGMT promoter methylation, as well as mRNA and protein expression. Using sequencing of sodium bisulfite-modified DNA, we determined the methylation status of 25 CpG sites within the MGMT promoter. In 46 of 52 tumors (88%), we detected MGMT promoter hypermethylation as defined by methylation of more than 50% of the sequenced CpG sites. Real-time reverse transcription-PCR showed reduced MGMT mRNA levels relative to non-neoplastic brain tissue in the majority of tumors with hypermethylation. Similarly, immunohistochemical analysis showed either no or only small fractions of MGMT positive tumor cells. MGMT promoter hypermethylation was significantly more frequent and the percentage of methylated CpG sites in the investigated MGMT promoter fragment was significantly higher in tumors with loss of heterozygosity on chromosome arms 1p and 19q as compared to tumors without allelic losses on these chromosomes arms. Taken together, our data suggest that MGMT hypermethylation and low or absent expression are frequent in oligodendroglial tumors and likely contribute to the chemosensitivity of these tumors. [source] A bicistronic SIN-lentiviral vector containing G156A MGMT allows selection and metabolic correction of hematopoietic protoporphyric cell linesTHE JOURNAL OF GENE MEDICINE, Issue 9 2003Emmanuel Richard Abstract Background Erythropoietic protoporphyria (EPP) is an inherited disease characterised by a ferrochelatase (FECH) deficiency, the latest enzyme of the heme biosynthetic pathway, leading to the accumulation of toxic protoporphyrin in the liver, bone marrow and spleen. We have previously shown that a successful gene therapy of a murine model of the disease was possible with lentiviral vectors even in the absence of preselection of corrected cells, but lethal irradiation of the recipient was necessary to obtain an efficient bone marrow engraftment. To overcome a preconditioning regimen, a selective growth advantage has to be conferred to the corrected cells. Methods We have developed a novel bicistronic lentiviral vector that contains the human alkylating drug resistance mutant O6 -methylguanine DNA methyltransferase (MGMT G156A) and FECH cDNAs. We tested their capacity to protect hematopoietic cell lines efficiently from alkylating drug toxicity and correct enzymatic deficiency. Results EPP lymphoblastoid (LB) cell lines, K562 and cord-blood-derived CD34+ cells were transduced at a low multiplicity of infection (MOI) with the bicistronic constructs. Resistance to O6 -benzylguanine (BG)/N,N,-bis(2-chloroethyl)- N -nitrosourea (BCNU) was clearly shown in transduced cells, leading to the survival and expansion of provirus-containing cells. Corrected EPP LB cells were selectively amplified, leading to complete restoration of enzymatic activity and the absence of protoporphyrin accumulation. Conclusions This study demonstrates that a lentiviral vector including therapeutic and G156A MGMT genes followed by BG/BCNU exposure can lead to a full metabolic correction of deficient cells. This vector might form the basis of new EPP mouse gene therapy protocols without a preconditioning regimen followed by in vivo selection of corrected hematopoietic stem cells. Copyright © 2003 John Wiley & Sons, Ltd. [source] Prognostic significance of O6 -methylguanine DNA methyltransferase and p57 methylation in patients with diffuse large B-cell lymphomasAPMIS, Issue 2 2009SUN MI LEE To evaluate whether promoter methylation is related to responsiveness for chemotherapy or clinical outcome, we performed an association analysis between methylation and clinical outcomes. Patients with nodal diffuse large B-cell lymphomas (DLBCL) at a single institute (n=44) were studied for methylation of tumor-related genes, MGMT, p15INK4B, p16INK4A, p16INK4A, Mad2, TMS1/ASC, CASP8, and GSTP1. The clinical behavior of DLBCL after chemotherapy was followed up and analyzed. Hypermethylation of promoters of MGMT, p15INK4B, p16INK4A, p16INK4A, Mad2, and TMS1/ASC genes was observed in 52.3%, 31.8%, 54.5%, 47.7%, 50%, and 2.3% of the cases, respectively. Methylation of CASP8 and GSTP1 genes was not observed. Promoter methylation was not related to chemo-responsiveness, disease-free survival, and progress of disease after chemotherapy. However, in overall survival analyses, MGMT methylation (p<0.05) and responsiveness to chemotherapy (p<0.01) were significant prognostic factors in patients with DLBCL. In the low-risk group, patients with p57 methylation showed longer overall survival than patients without p57 methylation (p=0.02) and all patients with p57 methylation were alive during follow-up. Our results demonstrate that aberrant promoter methylation of MGMT and p57 is an additional biological marker for predicting increased overall survival in patients with DLBCL. [source] | ||||||||||