Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.6.1.44 (AGT)
 770 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe a sensitive, rapid, and simple assay for mammalian O6-alkylguanine DNA alkyltransferase (O6-AGT) utilizing solid-phase DNA as the substrate and a monoclonal antibody (Mab)-based immuno-slotblot (ISB) for quantitation of O6-ethylguanine (O6-EtG). lambda-phage DNA was treated with N-ethyl-N-nitrosourea and immobilized on newly developed hydrophilic latex beads. After incubation with cell extracts to be assayed for O6-AGT activity, the substrate DNA could be isolated easily by a brief centrifugation through 50% glycerol. The amount of O6-EtG retained in the substrate DNA was determined by ISB using the anti-(O6-ethyl-2'-deoxyguanosine) Mab ER-6. As little as 2 fmol of O6-AGT per reaction tube can be reproducibly measured by this procedure, which is suitable for handling large numbers of samples within a short time (e.g., 80 samples within 2 days). In normal and malignant cells, respectively, O6-AGT activity protects against O6-alkylguanine-mediated mutagenesis and oncogenesis following exposure to N-nitroso carcinogens or confers resistance against cytocidal anti-cancer drugs such as chloroethylnitrosoureas and related compounds. The analysis of cellular O6-AGT activity by a highly sensitive, routinely applicable method is, therefore, of particular interest in studies related to carcinogenesis, molecular epidemiology, and clinical oncology.
 ...
PMID:Monoclonal antibody-mediated solid-phase assay for mammalian O6-alkylguanine DNA alkyltransferase activity. 177 91

A simplified and highly sensitive assay for the determination of O6-alkylguanine-DNA-alkyltransferase has been developed and validated by the analysis of extracts of human urinary bladder mucosa. The new assay involves the use of a synthetic dodecanucleotide containing a single O6-methylguanine residue as substrate for the enzyme. This substrate is 5'-end-labelled with [35S]PO3 and separation of repaired and unrepaired oligonucleotide is achived by immuno-precipitation with polyclonal antibodies specific for O6-methyldeoxyguanosine. Kinetic analysis of the repair of the oligonucleotide by extracts of Escherichia coli and rat liver showed that the reaction is first-order in substrate and enzyme and gave the molecular rate constants 7.5 x 10(6) mol-1 1-1 sec-1 and 8.0 x 10(6) mol-1 1-1 sec-1, respectively. The rate constants for the repair of the corresponding O6-ethylguanine-containing oligonucleotide were 3.0 x 10(5) mol-1 l-1 sec-1 and 3.6 x 10(6) mol-1 l-1 sec-1, respectively. Analysis of extracts of 48 samples of normal or neoplastic human urinary bladder mucosa obtained by transurethral biopsy or at surgery, by the new method or by a method involving use of [3H]-methylated DNA as substrate and HPLC, indicated excellent agreement between the two methods. The mean AGT content of normal urinary bladder mucosa obtained from individuals without diagnosed bladder cancer was 0.181 +/- 0.081 (mean +/- SD) fmol/microgram protein, that of neoplastic samples 0.323 +/- 0.177 fmol/microgram protein and that of normal tissue obtained from patients with bladder cancer 0.183 +/- 0.068 fmol/microgram protein. The new method is convenient, rapid and extremely sensitive (it can readily measure femtomole quantities of enzyme) and should prove useful for studies of the biochemical epidemiology of DNA repair.
Carcinogenesis 1989 Jul
PMID:Development and validation of a new assay for O6-alkylguanine-DNA-alkyltransferase based on the use of an oligonucleotide substrate, and its application to the measurement of DNA repair activity in extracts of biopsy samples of human urinary bladder mucosa. 273 14

Inactivation of O6-alkylguanine-DNA alkyltransferase (O6-AGT) in HeLa CCL2 cells by cisplatin was studied. HeLa CCL2 cells treated with cisplatin showed a dose-dependent decline in O6-AGT activity. After cisplatin was removed and replaced with fresh medium, the transferase level began to rise slowly. By 72 h slightly more than 80% of the activity was recovered. It seems that the activity of the alkyltransferase can be inactivated by platinated DNA adducts. The data suggest that the O6-platinum-guanine formation and the O6-alkyltransferase depletion are not responsible for cytotoxicity but may result in a base substitution mutation in mammalian cells.
Carcinogenesis 1989 Sep
PMID:Inactivation of O6-alkylguanine-DNA alkyltransferase in HeLa cells by cisplatin. 276 59

Previous studies have demonstrated that approximately one-third of human lymphoblastoid cell lines (LCLs) are deficient in removing O6-methylguanine residues because of the lack of O6-alkylguanine-DNA alkyltransferase (O6-AGT) activity. Such LCLs have been designated Mex-, while the proficient LCLs are Mex+. Our determinations of O6-AGT activity as a function of cellular protein concentration on 37 previously-established LCLs disclosed that the expression of the enzyme was high in 14 (Mex+) and barely detectable in 16 (Mex-). The other seven LCLs showed intermediate activity of the enzyme. By contrast, all of the 28 LCLs that we newly established contained high enzyme activity, implying that they consisted of mainly Mex+ cells. Since the conventional O6-AGT assay on Mex+ cell populations was not capable of detecting the co-existence of Mex- cells as a minor component, we attempted to determine the proportion of Mex- phenotype in newly-immortalized lymphoblastoid cell clones which had been established directly on semisolid agar. All of the 15 independent clones derived from a single blood sample also showed high O6-AGT activity, rendering it unlikely that Epstein-Barr virus transformation per se was responsible for the generation of Mex- LCLs. These results collectively indicate that Mex+ cells predominate in LCLs shortly after establishment and also suggest that the possible growth advantage for Mex- cells should play an important role in the subsequent development of Mex- LCLs during the long-term culture in vitro.
Carcinogenesis 1989 Nov
PMID:Predominance of Mex+ cells in newly-established human lymphoblastoid cell lines. 280 28

The sequence selectivity of methylation at the O6 and N7 position of guanine by N-methyl-N'-nitrosourea (MNU) and the rate of removal of O6-methylguanine by O6alkylguanine-DNA alkyltransferase (AGT) was determined using dodecadeoxynucleotides of defined structure. The extent of guanine adduct formed in self-complementary dodecamers, 5'-TATACGCGTATA-3', 5'-TATACCGGTATA-3' and 5'-TATAGGCCTATA-3', after methylation with [3H]MNU in a representative experiment were, respectively, 10, 19 and 30 pmol O6-methylguanine/mumol guanine and 97, 189 and 217 pmol N7-methylguanine/mumol guanine. The O6-methylguanine/N7-methylguanine ratio remained relatively constant for each dodecamer. A direct comparison between the methylation at guanine with adenine or thymine as the 5'-flanking base was made with two dodecamers, 5'-TATACATGTATA-3' and 5'-TATACTAGTATA-3'. When the guanine residue was preceded 5' by an adenine, the level of O6 and N7-alkylation was, respectively, 2.1-fold and 1.5-fold greater than when guanine was preceded 5' by a thymine. These date are consistent with a regioselective mechanism for alkylnitrosourea alkylation of guanine. The methylated dodecamer, 5'-TATACGCGTATA-3' was repaired faster than 5'-TATACCGGTATA-3' by HT29 extract containing AGT with a loss in 10 min of 0.052 pmol and 0.025 pmol O6-methylguanine, respectively. Dodecamers of the structure 5'-dCGCGAATTCm6GCG-3' and 5'-dCGCCAATTGm6GCG-3' were labeled at the 5' end with 32P by the reaction with polynucleotide kinase and after incubation with AGT, the methylated and demethylated dodecamers were separated by reversed-phase HPLC. The amount of demethylated product formed was greater for the dodecamer containing cytosine as the 5'-flanking base to O6-methylguanine compared to guanine in that same position. A higher extent of alkylation by MNU and a slower rate of repair by AGT for sites in which a guanine or modified guanine is preceded by a purine rather than a pyrimidine may explain, at least in part, mutational hot spots.
Carcinogenesis 1988 Nov
PMID:Sequence specificity of guanine alkylation and repair. 318 Mar 51

O6-Alkylguanine-DNA-alkyltransferase is a DNA repair protein known to carry out the transfer of alkyl groups from the O6-position of guanine in alkylated DNA to a cysteine acceptor site contained within its own protein sequence. We have examined the ability of this protein isolated from either E. coli or mammalian cells to perform this repair reaction in short oligodeoxynucleotides. Dodecadeoxynucleotides of the sequence 5'-dCGNGAATTCm6GCG-3' where N is any one of the normal four bases were all repaired very rapidly by the protein with 50% repair in less than 15 s at 0 degree C. The hexadeoxynucleotide 5'-dCGCm6GCG-3' was repaired slightly more slowly with 50% removal taking 7 min at 0 degree C and 1.5 min at 37 degrees C. The tetradeoxynucleotide 5'-dTm6GCA-3' was also a substrate but was repaired much more slowly requiring 45 min for 50% repair at 37 degrees C. These results indicate that (a) the AGT has a strong but not absolute preference for double-stranded DNA substrates; (b) the repair of O6-methylguanine is independent of the base opposite the lesion; and (c) that oligodeoxynucleotides as short as tetramers are substrates for repair by this protein.
Carcinogenesis 1986 Aug
PMID:Repair of oligodeoxynucleotides containing O6-methylguanine by O6-alkylguanine-DNA-alkyltransferase. 373 92

The kinetics of accumulation of the premutagenic DNA adduct O6-methylguanine (O6-meG) in the liver, blood leukocytes, lymph nodes and bone marrow of rats was examined and compared after single or multiple doses of procarbazine, a methylating cytostatic drug employed in the treatment of Hodgkin's lymphoma patients, and methylnitrosourea (MNU), an experimental methylating agent and carcinogen. Maximal O6-meG levels occurred 1-2 h after administration of single doses of procarbazine (10 mg/kg) or MNU (1 mg/kg), thereafter decreasing with half-lives of approximately 20-45 h, depending on the tissue. A relatively uniform tissue distribution was observed with both agents, with the liver generally showing highest adduct levels, followed by the lymph nodes, bone marrow and blood leukocytes which contained broadly similar amounts of O6-meG. During daily, oral administration to rats of procarbazine for 10 days at dose rates of 2.5, 5, 10 or 20 mg/kg/day (treatment analogous to that of the MOOP chemotherapy protocol for Hodgkin's lymphoma) followed by animal death on different days (in each case 24 h after the last treatment), a biphasic mode of O6-meG induction was observed: an initially steep build-up during the first 3-4 days was followed by a transient decline in the rate of accumulation, in turn followed by a second wave of accumulation and then a further slow-down. During the same treatment, liver O6-methylguanine-DNA alkyltransferase (AGT) declined in a dose-related manner. AGT recovery after the end of treatment was slow, taking nearly 20 days after the end of the high-dose treatment to return to control levels, despite the fact that all detectable adducts had been lost from DNA within 3 days after the end of treatment. A similar depletion and slow recovery of AGT in the liver, blood lymphocytes, bone marrow and lymph nodes was observed after treatment with a single dose of 100 mg/kg procarbazine. In contrast to these observations, O6-meG accumulated smoothly during a 10 day administration of MNU (1 or 10 mg/kg/day) to reach a steady-state within 5-6 days, while liver AGT was partially depleted after the high dose and recovered fully within 72 h of cessation of treatment. Similarly, a single dose of MNU (35 mg/kg) resulted in AGT depletion followed by rapid recovery in all four tissues examined. It is concluded that procarbazine (but not MNU) causes a decrease in cellular AGT concentrations by a mechanism additional to suicide repair of O6-meG.(ABSTRACT TRUNCATED AT 400 WORDS)
Carcinogenesis 1994 Aug
PMID:Differential effects of procarbazine and methylnitrosourea on the accumulation of O6-methylguanine and the depletion and recovery of O6-alkylguanine-DNA alkyltransferase in rat tissues. 751 72

Experiments were done to show whether a G to T mis-sense mutation at the third base of codon 249 of the p53 tumour suppressor gene is a 'hot spot' of aflatoxin attack as suggested by the results of epidemiological studies. Liver tissue from liver cancer patients in Taiwan and Japan was analysed for the presence of aflatoxin-DNA adducts (ADA) as a marker for aflatoxin exposure and an AGG to AGT transversion at codon 249 of the p53 gene. Ten per cent of samples containing ADA, indicating definite exposure of the subjects to aflatoxin, was found to harbour the codon 249 mutation, whereas 18% of the samples with no detectable adducts also contained the mutation. Our data do not support the hypothesis that codon 249 of the p53 gene DNA is a hot spot for aflatoxin mutagenesis as a 'late stage event' in human hepatocellular carcinogenesis.
 ...
PMID:Recent aflatoxin exposure and mutation at codon 249 of the human p53 gene: lack of association. 766 37

The authors previously reported a significant frequency of activating point mutations in codon 12 and 13 of the K-ras gene in endometrial carcinoma and endometrial atypical hyperplasia from Osaka, Japan. They also showed that alterations of the p53 gene are found frequently in those tumors. This study was designed to reveal possible demographic differences in the prevalence of K-ras and p53 mutations in endometrial carcinoma. Tumor-enriched areas of paraffin-embedded histologic sections obtained through the Colorado Central Cancer Registry were isolated and extracted for DNA. Fragments amplified by polymerase chain reaction (PCR) were screened for transforming mutations in codon 12, 13, or 59-63 of K-ras by direct sequencing. Of 38 endometrial adenocarcinomas that were analyzed, K-ras activation was detected in 4 cases (11%), three in codon 12 (a single case with a GGT-->AGT transition, a single case with a GGT-->GAT transition, and a single case with a GGT-->TGT transversion) and one in codon 13 (a GGC-->GAC mutation). The prevalence of K-ras mutations was significantly lower in endometrial carcinomas from Colorado (4 of 38, 11%) than in those from Osaka, Japan (17 of 57, 31%; P = .02). Mutations in exons 5-8 of p53 were screened by PCR-SSCP analysis, and subsequently confirmed by direct sequencing. Mutations in the p53 gene were detected in 5 of 38 endometrial carcinomas from Colorado (13%), including a single base substitution mutation in 3 cases (60%) and a deletion mutation in 2 cases (40%). Mutations in the p53 gene were significantly more frequently found in G3 cancers (3 of 7, 43%) than G1-G2 cancers combined (2 of 31, 6%; P = .025). Although the prevalence of p53 mutations in endometrial carcinomas from Colorado was not significantly different compared to that from Osaka, Japan (9 of 40, 23%), a G:C-->A:T transition at a CpG site, which was the most common base substitution mutation among Japanese, was not included in any tumors from Colorado. A rare polymorphism in codon 213 (CGA-->CGG) was observed in three cases. These observations may indicate that genetic or environmental factors may significantly influence the pathway of endometrial carcinogenesis.
 ...
PMID:Alteration of the p53 tumor suppressor gene and activation of c-K-ras-2 protooncogene in endometrial adenocarcinoma from Colorado. 785 67

To evaluate the role of ras activation and human papillomavirus (HPV) infection in laryngeal carcinoma, we analyzed tumor DNA from 43 cases, including 25 primary laryngeal tumors, 12 lymph-node and one skin metastases, and 5 recurrent laryngeal carcinomas. Thirteen normal laryngeal tissues and 7 benign laryngeal nodule biopsy specimens along with normal tissue surrounding laryngeal carcinoma in 2 cases were also included. The polymerase-chain-reaction technique was used to amplify DNA fragments containing codon 12 and 61 of H-, K- and N-ras, also HPV 16, 18 and 33 DNA, subsequently hybridized with sequence-specific oligonucleotides. DNA samples from 22 patients with laryngeal carcinoma revealed ras mutations (18 in N-ras codon 12, 6 in H-ras codon 61, and 3 in K-ras codon 61). Likewise, HPV DNA was found in 16 cases (HPV 16, 18 and 33 in 3 cases, 14 cases and 1 case respectively). ras mutations were significantly higher in metastatic tumors (10 of 13 cases) than in primary (11 of 25 cases) and recurrent laryngeal carcinomas (1 of 5 cases). HPV DNA was detected in 60% of recurrent, 44% of primary and 15% of metastatic tumors. Only 2 of the 13 normal laryngeal tissues and 1 out of 7 laryngeal nodule specimens were found to contain HPV DNA. These results suggest that ras activation, especially in N-ras codon 12.1 (GGT-->AGT) and HPV infection are 2 important factors in (multistage) laryngeal carcinogenesis. The ras mutation may be associated with metastatic ability of the tumor.
 ...
PMID:ras gene mutations and HPV infection are common in human laryngeal carcinoma. 838 55


1 2 3 Next >>