Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have examined the effects of the nitrosoureas, streptozotocin (STZ) and 1,3-bis(chloroethyl)-1-nitrosourea (BCNU), on a human multiple myeloma cell line, RPMI 8226, and its drug-resistant variants. Cell lines selected for doxorubicin (DOX) resistance alone displayed a STZ and BCNU cytotoxicity profile similar to that of the parent cell line. In contrast, two of the drug-resistant variants selected with DOX plus verapamil, an agent which inhibits P-glycoprotein-mediated multidrug resistance, displayed a collateral sensitivity to STZ and BCNU. Verapamil was included in the selection protocol because it has been shown to inhibit the P-glycoprotein-mediated multidrug resistance phenotype and is now in clinical trials as a chemosensitizing agent. The collateral sensitivity to these nitrosoureas seen in the DOX plus verapamil-selected cell lines is due to the functional loss of a DNA repair molecule, O6-Methylguanine DNA methyltransferase (MGMT). The functional loss of MGMT is secondary to the loss of MGMT gene expression. The loss of MGMT gene expression is not due to loss or gross rearrangement of the MGMT-coding region. If this selection pressure applied in vitro reflects the in vivo situation, then new chemotherapeutic strategies may be devised to exploit this phenomenon. These cell lines will serve as useful models for delineating mechanisms which govern MGMT expression.
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PMID:Collateral sensitivity to nitrosoureas in multidrug-resistant cells selected with verapamil. 138 86

The marginal level of clinical responses to the Chloroethylnitrosoureas (CENU, i.e. BCNU, CCNU, MeCCNU) suggests that there may exist an innate mechanism of resistance in tumors to these chemotherapeutic agents. A decade of research from many laboratories around the world has led to the identification of the mechanisms for tumor cell resistance to the CENU. The ability to prevent the formation of DNA interstrand crosslinks, thought to be the critical lethal lesion induced by these agents, is accomplished in a majority of human tumors by the unique DNA repair protein O-6 methylguanine DNA methyltransferase (MGMT). This review addresses the identification of this mechanism of resistance to therapy, and chemotherapeutic strategies to inhibit this DNA repair system, in an attempt to sensitize resistant tumors to the CENU.
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PMID:The role of O-6 methylguanine DNA methyltransferase (MGMT) in drug resistance and strategies for its inhibition. 183 90

Severe and delayed myelosuppression is a major side effect encountered with the clinical use of nitrosourea-type chemotherapeutic drugs. The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) has been shown to repair nitrosourea-induced DNA damage. We therefore investigated the effect of expressing MGMT in hematopoietic cells (via retrovirus-mediated gene transfer) on nitrosourea-induced toxicity. A retroviral vector (N2/ZipPGK-MGMT) expressing the human MGMT cDNA from the phosphoglycerate kinase promoter was constructed. Infection of murine bone marrow with the N2/ZipPGK-MGMT retrovirus significantly increased the survival of murine bone marrow-committed progenitor cells following in vitro exposure to N-N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine). MGMT gene transfer also protected murine hematopoietic cells in vivo in a murine model of BCNU-induced myelosuppression. The infusion of 4-6 x 10(6) N2/ZipPGK-MGMT-transduced bone marrow cells into mice every 2 weeks significantly increased peripheral leukocyte counts, platelet counts, and hematocrits compared to infusions of mock-infected bone marrow cells. In addition, bone marrow-committed progenitor cells from some recipient animals demonstrated increased resistance to BCNU in vitro when analyzed 2.5 months after initial treatment. The integration of the N2/ZipPGK-MGMT provirus in the spleen DNA from these animals correlated with committed progenitor cell resistance to BCNU. These data suggest that MGMT expression in hematopoietic progenitor and precursor cells protects against nitrosourea-induced toxicity and that gene transfer may prove useful in attempts to reduce nitrosourea-induced myelosuppression in the clinical setting.
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PMID:Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo. 778 Sep 76

The purpose of this study was to evaluate the anti-tumor activity of sequenced administration of O6-benzylguanine (BG), streptozotocin (STZ), and 1,3-bis(2-chloroethyl)-1- nitrosourea (BCNU) in vitro and in vivo. We measured the recovery of O6-methylguanine DNA methyltransferase (MGMT) and BCNU cytotoxicity in the human glioma SF767 cell line, and anti-tumor activity against xenografts following exposure to BG, STZ or the combination of BG + STZ combined with BCNU. In SF767 cells, the combination of BG (10 microM) + STZ (0.05 mM) produced sustained inhibition of MGMT activity for at least 24 hr, and a greater potentiation of BCNU cytotoxicity than either agent alone. The combined treatment of BG + STZ increased BCNU-induced cell kill by 0.5 to 1.0 log over BG or STZ alone. The maximally tolerated doses of the combination of BG + STZ + BCNU administered to nude mice i.p. were the following: BG (80 mg/kg), STZ (100 mg/kg), and BCNU (15 mg/kg). Utilizing these doses of BG and STZ, the depletion and repletion profile of MGMT activity in SF767 xenografts was measured. STZ at 100 mg/kg did not affect xenograft MGMT activity. Subsequent to BG treatment, xenograft MGMT activity was inactivated completely for 12 hr, and the tumors gradually recovered approximately 40% of control activity by 24 hr. The combination of BG + STZ produced sustained inhibition of MGMT activity for 24 hr in the xenografts with complete recovery of MGMT activity by 48 hr. Administration of the combination of BG + BCNU to nude mice bearing SF767 tumor resulted in significant inhibition of tumor growth for 23 days. However, the addition of STZ to this combination provided no greater anti-tumor activity than that observed with BG + BCNU. The three-drug combination of BG, STZ, and BCNU produced no more than 2.4 to 13.0% weight loss with occasional lethal toxicity. Collectively, these data suggest that prolonged depletion of MGMT might be required for optimal reversal of BCNU resistance both in vitro and in vivo.
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PMID:Anti-neoplastic activity of sequenced administration of O6-benzylguanine, streptozotocin, and 1,3-bis(2-chloroethyl)-1-nitrosourea in vitro and in vivo. 780 3

This study was undertaken to ascertain the importance of prolonged depletion of O6-methylguanine DNA methyltransferase (MGMT) activity, following O6-benzylguanine (BG) and streptozotocin (STZ) exposure, in reversing 1,3 bis(2-chloroethyl)-1-nitrosourea (BCNU) resistance in vitro. We evaluated BCNU-induced cytotoxicity and measured the temporal recovery of MGMT activity in human colon carcinoma HT-29 cells following treatment with BG, STZ, or the combination of BG and STZ. The pretreatment regimens which provided the greatest potentiation of BCNU cytotoxicity were those exhibiting the greatest temporal inhibition of MGMT activity. The combination of BG (10 microM) and STZ (1.0 mM) produced sustained inhibition of MGMT activity through 24 h and potentiated BCNU cytotoxicity by at least one log greater than either agent alone. Similarly, BG (10-100 microM) produced marked reductions in MGMT activity and increased BCNU cytotoxicity in a dose-dependent fashion. A 100-microM dose of BG inhibited MGMT activity for 48 h and potentiated BCNU induced cell kill by 3 logs greater than BCNU alone. In addition, we observed that during the period of sustained inhibition of MGMT activity, no changes in the steady-state MGMT mRNA levels occurred. We conclude that prolonged inhibition of MGMT activity is an important determinant in reversing BCNU resistance and that chemotherapeutic regimens targeting the inactivation of MGMT activity should be optimized such that MGMT activity is depleted for at least 24 h following BCNU administration.
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PMID:Prolonged depletion of O6-methylguanine DNA methyltransferase activity following exposure to O6-benzylguanine with or without streptozotocin enhances 1,3-bis(2-chloroethyl)-1-nitrosourea sensitivity in vitro. 836 24

The chloroethylnitrosourea (CNU) alkylating agents are commonly used for cancer chemotherapy, but their usefulness is limited by severe bone marrow toxicity that causes the cumulative depletion of all hematopoietic lineages (pancytopenia). Bone marrow CNU sensitivity is probably due to the inefficient repair of CNU-induced DNA damage; relative to other tissues, bone marrow cells express extremely low levels of the O6-methylguanine DNA methyltransferase (MGMT) protein that repairs cytotoxic O6-chloroethylguanine DNA lesions. Using a simplified recombinant retroviral vector expressing the human MGMT gene under control of the phosphoglycerate kinase promoter (PGK-MGMT) we increased the capacity of murine bone marrow-derived cells to repair CNU-induced DNA damage. Stable reconstitution of mouse bone marrow with genetically modified, MGMT-expressing hematopoietic stem cells conferred considerable resistance to the cytotoxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a CNU commonly used for chemotherapy. Bone marrow harvested from mice transplanted with PGK-MGMT-transduced cells showed extensive in vitro BCNU resistance. Moreover, MGMT expression in mouse bone marrow conferred in vivo resistance to BCNU-induced pancytopenia and significantly reduced BCNU-induced mortality due to bone marrow hypoplasia. These data demonstrate that increased DNA alkylation repair in primitive hematopoietic stem cells confers multilineage protection from the myelosuppressive effects of BCNU and suggest a possible approach to protecting cancer patients from CNU chemotherapy-related toxicity.
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PMID:Increasing DNA repair methyltransferase levels via bone marrow stem cell transduction rescues mice from the toxic effects of 1,3-bis(2-chloroethyl)-1-nitrosourea, a chemotherapeutic alkylating agent. 855 5

O6-Methylguanine-DNA methyltransferase (MGMT), a constitutively expressed DNA repair protein, removes alkyl groups from the O6-position of guanine in DNA. Tumor cells with high MGMT activity are resistant to nitrosoureas and other agents that form toxic O6-alkyl adducts. O6-Benzylguanine (BG) inactivates the MGMT protein and thereby enhances the sensitivity of tumor cells to alkylating drugs. However, the therapeutic potential of BG is limited by its poor solubility and its nonspecific inactivation of MGMT in normal tissues as well as in tumor tissues. Consequently, BG analogues are being developed to identify agents that have more favorable pharmacological characteristics. We evaluated O6-benzyl-2'-deoxyguanosine (dBG), the 2'-deoxyribonucleoside analogue of BG, for its ability to inhibit MGMT and to potentiate 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in a MGMT-positive human brain tumor xenograft, Daoy. When given i.p. 1 h before BCNU (25 mg/m2) to animals bearing s.c. tumors, dBG (134 mg/m2) produced a growth delay of 24.7 days, compared to 21.6 days after treatment with an equimolar dose of BG (90 mg/m2) plus BCNU and -0.6 days after treatment with BCNU alone. The combination of dBG + BCNU also increased the survival of animals bearing intracranial tumors by 65%. By increasing the dose of dBG to 300 mg/m2 (the maximum dose that could be delivered i.p. in a standard treatment volume), the growth delay of s.c. tumors increased from -0.1 days with BCNU alone to 39.3 days. dBG suppressed both tumor and liver MGMT activity to less than 1.5% of baseline, and dBG + BCNU induced extensive perivascular apoptosis. Because dBG is a 10-fold less potent MGMT inhibitor than BG in HT-29 cell extracts, these results illustrate the capacity of BG analogues to potentiate BCNU toxicity, despite less in vitro activity than the parent compound, and emphasize the importance of in vivo evaluation of BG analogues.
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PMID:Treatment of human brain tumor xenografts with O6-benzyl-2'-deoxyguanosine and BCNU. 861 53

O6-Methylguanine-DNA methyltransferase (MGMT) is an important DNA repair protein that plays a key role in cancer chemotherapy by alkylating agents such as carmustine (BCNU) and Dacarbazine (DTIC). Therapy by BCNU and DTIC is reduced by dose-limiting hematological toxicity as a result of low MGMT repair activity in bone marrow cells. In this study, we have constructed a Moloney murine leukemia virus retroviral vector containing the human mgmt gene. High-titer retrovirus producer cells lines have been generated. Retroviral-mediated transfer of the human mgmt gene into murine multi-potent hematopoietic stem cells, FDCP-1, resulted in the expression of a high level of MGMT activity. In comparison with the control cells that were transduced with the parent vector, the MGMT-expressing clones were considerably more resistant to the cytotoxicity of the methylating agents, such as N-methyl-N'-nitro-N-nitrosoguanidine, N-nitroso-N-methyl-urea, and temozolomide, as well as the chloroethylating agents 1-(2-chloroethyl)-1-nitrosourea and BCNU. The protection provided by MGMT could be eliminated by the MGMT inactivator O6-benzylguanine. Thus, the principal lethal lesions produced by these alkylating agents in the murine hematopoietic stem cells and the MGMT deficiency in these cells can be complemented by retroviral-mediated gene transduction.
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PMID:Retrovirus-mediated transfer of the human O6-methylguanine-DNA methyltransferase gene into a murine hematopoietic stem cell line and resistance to the toxic effects of certain alkylating agents. 864 46

Human CD34 cells express low levels of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) and are sensitive to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Gene transfer of the AGT gene, methylguanine DNA methyltransferase (MGMT), results in only modest BCNU resistance. Recently, an AGT inhibitor, O6-benzylguanine (BG), entered clinical trials. In preclinical studies, BG potentiated the cytotoxic effect of BCNU in tumors but increased toxicity to normal CD34 cells. We transferred a mutant MGMT containing a glycine-to-alanine mutation at position 156, resulting in marked resistance to BG, into Chinese hamster cells; the K562 cell line and human CD34 cells used the retroviral backbone MFG. In each instance, cells expressed increased AGT and were much more resistant to the combination of BG and BCNU than the parental cells or cells transduced with wild-type MGMT. Furthermore, the transduction efficiency in human CD34 cells was in excess of 70%, and the proportion of CD34 transduced cells resistant to the combination was > 30%. Thus, retroviral-mediated transduction of a mutant MGMT into CD34 cells appears to be an effective way to induce selective resistance to a drug combination designed to overcome a significant resistance mechanism to nitrosoureas in tumors.
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PMID:Retroviral transduction of a mutant methylguanine DNA methyltransferase gene into human CD34 cells confers resistance to O6-benzylguanine plus 1,3-bis(2-chloroethyl)-1-nitrosourea. 894 65

Bone marrow toxicity is a dose-limiting side effect of chloroethylnitrosourea (CNU) chemotherapeutic alkylating agents. A major determinant of CNU cytotoxicity is the methylation of guanine at the O6-position and the subsequent formation of interstrand DNA cross-links. O6-Methylguanine DNA methyltransferase (MGMT) removes alkyl groups from the O6 position of guanine and has been shown to repair CNU-induced DNA damage. We have previously demonstrated that transplantation of murine bone marrow cells transduced with a recombinant retroviral vector expressing MGMT via the human phosphoglycerate kinase promoter (PGK-MGMT) protects animals in vivo from acute myelotoxicity associated with CNU treatment. In the present study, we examined the effects of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), a commonly used CNU, on long term recovery of the lymphoid compartment, including thymus reconstitution, peripheral T and B cell populations, and lymphocyte mitogen responses in mice reconstituted with PGK-MGMT-transduced hemopoietic cells. Mice transplanted with either mock-infected control or PGK-MGMT-transduced stem cells were treated with five weekly doses of BCNU. Analysis of the lymphoid compartment demonstrated significant damage 3 mo after the last BCNU dose in control animals. In contrast, the profound deficiency in CD4+CD8+ double-positive thymocytes and mature lymphocytes observed in control mice surviving BCNU treatment was completely reversed in mice transplanted with PGK-MGMT-transduced bone marrow and was associated with molecular evidence of in vivo selection of transduced cells in the lymphoid compartment. Thus, long term immunodeficiency following CNU therapy may be prevented by genetic modification of murine hemopoietic stem cells with MGMT, leading to significant improvement in post-transplant immune function.
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PMID:Reversal of 1,3-bis(2-chloroethyl)-1-nitrosourea-induced severe immunodeficiency by transduction of murine long-lived hemopoietic progenitor cells using O6-methylguanine DNA methyltransferase complementary DNA. 899 23


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