Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tumor cells resistant to chloroethylnitrosourea (CENU) therapy contain high levels of O6-alkylguanine DNA-alkyltransferase (GATase), a DNA repair enzyme that aborts DNA interstrand cross-linking by removing CENU-induced O6-alkylguanine adducts. Because the transferase binds covalently to CENU-treated oligonucleotides, we reacted partially purified GATase from cultured human lymphoblasts with a BCNU-treated, 35S-5'-end-labeled, synthetic oligonucleotide designed to have a polyadenylated 3' terminus. Immunoprobing Western blots of this reaction mixture with GATase-specific monoclonal antibody indicated that 25-30% of the transferase became complexed. We purified this complex by affinity chromatography with oligo(dT) cellulose, recovering homogenous material that appeared as a discrete 35-kDa Coomassie blue or silver-stained band after SDS-polyacrylamide gel electrophoresis. Autoradiography and Western immunoblotting confirmed that this band contained both the radiolabeled oligonucleotide and the GATase protein.
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PMID:Affinity purification and characterization of human O6-alkylguanine-DNA alkyltransferase complexed with BCNU-treated, synthetic oligonucleotide. 278 Feb 88

The chemotherapy of malignant brain tumors has been, only partially successful yet. Recently major concern is drug resistance, one of possible mechanisms of such drug resistance stems from inducible repair enzyme, especially in case of chloroethylnitrosoureas as ACNU or BCNU. We examined the changes of acquired resistance to ACNU in rat glioma cells by pretreatment with O6-methylguanine, which is a substrate for O6-methylguanine methyltransferase. ACNU-resistant (9L/AC) cells had established after 10 times treatments of ACNU. 9L/AC cells were pretreated with 2 mM O6-methylguanine for 2 hours, and subsequently challenged with increasing doses of ACNU for 2 hours. In vitro colony formation assay the survival fraction of 9L and 9L/AC cells ranged from 0.39 to 0.63 by 2-hour reaction of 1-3 mM O6-methylguanine. Based on the dose-response curve for ACNU in 9L/AC cells, by O6-methylguanine pretreatment (2 mM), ACNU-resistance decreased markedly to one-third, one-fifth, and one-two hundredth at 12, 24, 36 microM ACNU, respectively. In contrast, the survival of 9L cells against ACNU was similar under O6-methylguanine pretreatment or nontreatment condition. Therefore, ACNU-resistance is considerably related to DNA repair enzyme induction, and the substrates may potentiate the cell-killing effect of ACNU in the resistant glioma cells.
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PMID:[Circumvention of ACNU-resistance in rat glioma cells by pretreatment with O6-methylguanine]. 291 93

The mammalian DNA repair enzyme O6-alkylguanine-DNA alkyltransferase (AT) is inactivated during the repair process and its activity can only be restored by de novo synthesis. We have made use of this property to determine whether and to what extent various chemotherapeutic agents alkylate DNA in the O6-position of guanine, ie. produce lesions susceptible to AT repair. Adult female Fischer rats received a single i.p. injection of a high dose (LD50) of the respective agent and, 5 hr later, a chasing dose of N-nitroso-[14C]dimethylamine (0.2 mg/kg; 4 hr survival). The amount of 7-[14C]methylguanine formed was approximately 95 mumol/mol guanine and not significantly altered by pretreatment with any of the drugs. The ratio of O6-[14C]methylguanine/7-[14C]methylguanine was 0.019 for control animals, indicating that during the observation period of 4 hr, 83% of the O6-[14C]methylguanine produced had been removed by the hepatic AT. Little or no effect was found in rats that received spirohydantoin mustard, hexamethylmelamine, cis-platinum or mitomycin C. A significant increase in the O6-/7-[14C]methylguanine ratio was found after pretreatment with AZQ (0.026) and cyclophosphamide (0.028), agents for which lesions involving the O6-position of guanine have not yet been identified. N-(2-Hydroxyethyl)-N-nitrosourea and the cytostatic haloethylinitrosoureas, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl)-1-nitrosourea (PCNU), and N-chloroethyl-N-hydroxyethylnitrosourea (HECNU) inhibited the hepatic AT, producing a ratio of 0.025-0.035. Considerably higher ratios of 0.059 and 0.101 were observed after administration of the methylating agents procarbazine and 5-(3,3-dimethyl-1-triazeno)imidazole-4-carboxamide (DTIC), respectively. Complete saturation of the repair system (O6-/7-[14C]methylguanine ratio, 0.11) was only achieved with N-methyl-N-nitrosourea.
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PMID:Inhibition of the hepatic O6-alkylguanine-DNA alkyltransferase in vivo by pretreatment with antineoplastic agents. 293 May 93

Human DNA ligase was purified from both normal and leukemic peripheral lymphocytes and normal thymocytes. The activity of the purified enzymes was assayed in the presence of several widely used antileukemic drugs. Melphalan and prednisone at 5 mM had no effect. Carmustine, chlorambucil, and cyclophosphamide were more effective at inhibiting the enzyme from leukemic cells, whereas Adriamycin and vinblastine and their derivatives were stronger inhibitors of the enzyme from normal cells. Vincristine and etoposide inhibited DNA ligase from thymocytes and normal lymphocytes with a low Ki but were totally ineffective on the enzyme from leukemic cells. The three classes of intercalating anthracyclines, Vicia alkaloids, and podophyllotoxin derivatives, were the only drugs found to markedly inhibit DNA ligases from normal cells. Less substituted molecules of the Vicia alkaloids and podophyllotoxin classes were the more active inhibitors, whereas in the intercalating anthracycline group, it was the more substituted compounds. The clinical consequences of these observations are discussed with respect to the role of DNA ligase in DNA replication and repair.
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PMID:Inhibition of DNA ligase from human thymocytes and normal or leukemic lymphocytes by antileukemic drugs. 398 68

Anumber of DNA-damaging chemotherapeutic agents attack the O(6) position on guanine, forming the most potent cytotoxic DNA adducts known. The DNA repair enzyme O(6)-alkylguanine DNA alkyltransferase (AGT), encoded by the gene MGMT, repairs alkylation at this site and is responsible for protecting both tumor and normal cells from these agents. Cells and tissues vary greatly in AGT expression, not only between tissues but also between individuals. AGT activity correlates inversely with sensitivity to agents that form O(6)-alkylguanine DNA adducts, such as carmustine (BCNU), temozolomide, streptozotocin, and dacarbazine. The one exception is those tumors lacking mismatch repair, which renders them resistant to methylating agents. A recent study in patients with gliomas confirmed the correlation between low-level expression of the MGMT gene and response and survival after BCNU. An inhibitor to AGT, O(6)-benzylguanine (BG), depletes AGT in human tumors without associated toxicity and is now in phase II clinical trials. Finally, mutations within the active site region of the MGMT gene render the AGT protein resistant to BG inactivation. As a result, mutant MGMT gene transfer into hematopoietic stem cells has been shown to selectively protect the marrow from the combination of an alkylating agent and BG, while at the same time sensitizing tumor cells. MGMT remains a paradigm for development of new agents that modulate known mechanisms of drug resistance in cancer cells and raise the spectra of combinatorial therapies that encompass known drug resistance mechanisms.
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PMID:Clinical relevance of MGMT in the treatment of cancer. 1198 Oct 13

The prognosis of advanced melanoma is generally poor, because this tumor commonly exhibits intrinsic or acquired resistance to chemotherapy. In an attempt to identify the underlying causes of this resistance, we studied the roles played by the DNA repair enzyme O(6)-alkylguanine-DNA alkyltransferase (OGAT) and the mismatch repair (MMR) system in the sensitivity of melanoma cells to temozolomide (TMZ), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), or cis-diamminedichloroplatinum(II) (CDDP). To this end, OGAT levels and MMR efficiency of extracts of nine melanoma cell lines and selected clones derived from four of these lines were determined and correlated with the sensitivity of the respective cells to these drugs. The effectiveness of O(6)-benzylguanine (BG), a specific OGAT inhibitor, in potentiating TMZ- or BCNU-mediated cytotoxicity was also evaluated. Our results demonstrate that MMR efficiency and OGAT levels strongly affect melanoma cell sensitivity to TMZ. In MMR-proficient cells, a direct correlation between OGAT levels and TMZ IC(50) values was found. When OGAT activity was inhibited with BG, the sensitivity of these cells to TMZ increased and was then dictated largely by their MMR efficiency. MMR-deficient cells were highly resistant to the drug irrespective of their OGAT levels. Although OGAT activity and MMR status seemed to be the major determinants of melanoma sensitivity to TMZ, this was not the case for BCNU and CDDP; resistance to the latter drugs clearly involves processes other than the two DNA repair pathways analyzed in this study.
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PMID:The effect of O6-alkylguanine-DNA alkyltransferase and mismatch repair activities on the sensitivity of human melanoma cells to temozolomide, 1,3-bis(2-chloroethyl)1-nitrosourea, and cisplatin. 1253 19

O6-Benzylguanine and its metabolite, 8-oxo-O6-benzylguanine, are equally potent inhibitors of the DNA repair enzyme, O6-alkylguanine-DNA alkyltransferase. Pharmacokinetic values are derived from cancer patients participating in a phase I trial (10 or 20 mg/m2 of O6-benzylguanine in a single bolus dose or 10 to 120 mg/m2 as a 60-min constant infusion). A two-compartment model fits the plasma concentration versus time profile of O6-benzylguanine. O6-Benzylguanine is eliminated rapidly from the plasma compartment in humans (t1/2 alpha and t1/2 beta are 2 +/- 2 min and 26 +/- 15 min [mean +/- SD, n = 7], respectively), and its plasma clearance (513 +/- 148 mL/min/m2) is not dose dependent. Metabolite kinetics are evaluated using both a novel approach describing the relationship between O6-benzylguanine and 8-oxo-O6-benzylguanine and classical metabolite kinetics methods. With increasing doses of O6-benzylguanine, the plasma clearance of 8-oxo-O6-benzylguanine, decreases, prolonging elimination of the metabolite. This effect is not altered by coadministration of BCNU. The urinary excretion of drug and metabolites is minimal.
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PMID:Pharmacokinetics of O6-benzylguanine (NSC637037) and its metabolite, 8-oxo-O6-benzylguanine. 1295 45

We have previously demonstrated successful in vivo selection, chemoprotection, and modulation of donor chimerism in dogs that received myeloablative allogeneic stem cell transplantation with cells expressing the P140K mutant of the DNA repair enzyme methylguanine methyltransferase (MGMTP140K). Here, we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation, which could allow for less toxic transplantation regimens for patients with malignant and genetic diseases. Three dogs received a nonmyeloablative conditioning regimen and infusion of allogeneic stem cells transduced with MGMTP140K. All three dogs had stable gene marking and donor chimerism before receiving a course of O(6) -benzylguanine (O(6) BG)/1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) between days 210 and 589 after transplantation. One to four doses led to a marked increase in gene marking in all dogs. Furthermore, the transduced cells conferred chemoprotection and prevented severe neutropenia. Our results suggest that drug resistance gene therapy is feasible and safe in the nonmyeloablative transplantation setting.
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PMID:In vivo selection and chemoprotection after drug resistance gene therapy in a nonmyeloablative allogeneic transplantation setting in dogs. 1753 75

Current approaches for hematopoietic stem cell (HSC) and organ transplantation are limited by donor and host-mediated immune responses to allo-antigens. Application of these therapies is limited by the toxicity of preparative and post-transplant immunosuppressive regimens and a shortage of appropriate HLA-matched donors. We have been exploring two complementary approaches for genetically modifying donor cells that achieve long-term suppression of cellular proteins that elicit host immune responses to mismatched donor antigens, and provide a selective advantage to genetically engineered donor cells after transplantation. The first approach is based on recent advances that make feasible targeted down-regulation of HLA expression. Suppression of HLA expression could help to overcome limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors. Accordingly, we have recently investigated whether knockdown of HLA by RNA interference (RNAi) enables allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors that integrate into genomic DNA, thereby permanently modifying transduced donor cells. Lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA achieved efficient and dose-dependent reduction in surface expression of HLA in human cells, and enhanced resistance to allo-reactive T lymphocyte-mediated cytotoxicity, while avoiding non-MHC restricted killing. Complementary strategies for genetic engineering of HSC that would provide a selective advantage for transplanted donor cells and enable successful engraftment with less toxic preparative and immunosuppressive regimens would increase the numbers of individuals to whom HLA suppression therapy could be offered. Our second strategy is to provide a mechanism for in vivo selection of genetically modified HSC and other donor cells. We have uniquely combined transplantation during the neonatal period, when tolerance may be more readily achieved, with a positive selection strategy for in vivo amplification of drug-resistant donor HSC. This model system enables the evaluation of mechanisms of tolerance induction to neo-antigens, and allogeneic stem cells during immune ontogeny. HSC are transduced ex vivo by lentivirus-mediated gene transfer of P140K-O(6)-methylguanine-methyltransferase (MGMT(P140K)). The MGMT(P140K) DNA repair enzyme confers resistance to benzylguanine, an inhibitor of endogenous MGMT, and to chloroethylating agents such as BCNU. In vivo chemoselection enables enrichment of donor cells at the stem cell level. Using complementary approaches of in vivo chemoselection and RNAi-induced silencing of HLA expression may enable the generation of histocompatibility-enhanced, and eventually, perhaps "universally" compatible cellular grafts.
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PMID:Suppression of HLA expression by lentivirus-mediated gene transfer of siRNA cassettes and in vivo chemoselection to enhance hematopoietic stem cell transplantation. 1904 10

Major limitations to gene therapy using HSCs are low gene transfer efficiency and the inability of most therapeutic genes to confer a selective advantage on the gene-corrected cells. One approach to enrich for gene-modified cells in vivo is to include in the retroviral vector a drug resistance gene, such as the P140K mutant of the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT*). We transplanted 5 rhesus macaques with CD34+ cells transduced with lentiviral vectors encoding MGMT* and a fluorescent marker, with or without homeobox B4 (HOXB4), a potent stem cell self-renewal gene. Transgene expression and common integration sites in lymphoid and myeloid lineages several months after transplantation confirmed transduction of long-term repopulating HSCs. However, all animals showed only a transient increase in gene-marked lymphoid and myeloid cells after O6-benzylguanine (BG) and temozolomide (TMZ) administration. In 1 animal, cells transduced with MGMT* lentiviral vectors were protected and expanded after multiple courses of BG/TMZ, providing a substantial increase in the maximum tolerated dose of TMZ. Additional cycles of chemotherapy using 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) resulted in similar increases in gene marking levels, but caused high levels of nonhematopoietic toxicity. Inclusion of HOXB4 in the MGMT* vectors resulted in no substantial increase in gene marking or HSC amplification after chemotherapy treatment. Our data therefore suggest that lentivirally mediated gene transfer in transplanted HSCs can provide in vivo chemoprotection of progenitor cells, although selection of long-term repopulating HSCs was not seen.
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PMID:In vivo selection of hematopoietic progenitor cells and temozolomide dose intensification in rhesus macaques through lentiviral transduction with a drug resistance gene. 1950 70


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