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

The protein O6-alkylguanine-DNA alkyltransferase (O6-AGT) has been implicated as a major determinant of resistance of diverse tumors to chloroethylnitrosoureas. To evaluate the contribution of O6-AGT to resistance of medulloblastomas to chloroethylnitrosoureas, we assessed the role of O6-AGT in determining (BCNU). Sensitivity to BCNU cytotoxicity, measured as dose dependent survival of soft agar colony forming ability, varied among the lines. Two lines (UW443 and UW228-3) displayed linear survival curves and comparable BCNU sensitivity (D37 ca. 140 microM). The other lines (UW228-2 and UW228-1) had biphasic survival curves indicating that each line was composed of two sub-populations that differed in BCNU sensitivity. The D37 for these sub-populations ranged from 51 microM to 253 microM. The O6-AGT activities of the cell lines, however, did not reflect their varied susceptibilities to BCNU as evidenced by a 9-fold difference in O6-AGT activity between UW443 and UW228-3. Moreover, elimination of O6-AGT activity by the inhibitor O6-benzylguanine did not appreciably increase sensitivity to BCNU compared with the response of other human tumor cells [Dolan et al. Cancer Res. 51:3367-3372, 1991]. Our results demonstrate that O6-AGT is not a major determinant of BCNU sensitivity in the four medulloblastoma lines.
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PMID:O6-alkylguanine DNA-alkyltransferase is not a major determinant of sensitivity to 1,3-bis(2-chloroethyl)-1-nitrosourea in four medulloblastoma cell lines. 142 17

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.
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PMID:Monoclonal antibody-mediated solid-phase assay for mammalian O6-alkylguanine DNA alkyltransferase activity. 177 91

The molecular mechanism of acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl)-methyl-3-(2-chloroethyl)-3-nitroso ure a hydrochloride (ACNU) was investigated using ACNU-resistant clones (ACNUr-1-4) isolated from the V79 cell line. The binding level of alkyl cyanate, a decomposition product of ACNU, to protein in ACNUr-1 cells was not less than that in the parental V79 cells, indicating that the acquired resistance was not due to a reduced intracellular concentration of ACNU. Because O6-chloroethylguanine, an intermediate in cytotoxic interstrand cross-link formation by ACNU, is known to be repaired by the same mechanism as O6-ethyldeoxyguanosine (O6-EtdGuo), we quantitated O6-EtdGuo by radioimmunoassay at various times after exposure of cells to 100 micrograms/ml N-ethyl-N-nitrosourea for 20 min. In V79 cells, elimination of O6-EtdGuo was negligible, but in all four resistant clones, 30 to 59% of the O6-EtdGuo was removed within 24 hr after exposure. This increased removal of O6-EtdGuo among the resistant clones was associated with the activity of O6-alkylguanine DNA alkyltransferase (O6-AGT) determined using cell extracts. The present results indicate that increased removal of O6-chloroethylguanine in ACNU-resistant clones by O6-AGT is mechanistically linked to the acquisition of resistance to ACNU.
Jpn J Cancer Res 1987 Oct
PMID:Acquisition of resistance to 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride in V79 cells through increased removal of O6-alkylguanine. 311 42

We demonstrated a germline p53 replication error in two generations of a Li-Fraumeni family affected with liposarcoma, adrenocortical carcinoma, and osteosarcoma. The trinucleotide repeat mutation changed 5'-AGT GTG GTG GTG-3' at codons 215-218 to 5'-AGT TGG TTG GTG GTG-3'. The predicted protein would be elongated by one amino acid (val216-->trp leu) without a change in charge. Detection of p53 in the adrenal tumor by immunostaining suggested that the mutant protein was expressed. Persistence of the mutation in the germline may suggest a defect in DNA repair in the family member first affected. This is the first report where germline transmission of replication-damaged p53 trinucleotide repeats is associated with the Li-Fraumeni syndrome.
Cancer Res 1995 Aug 01
PMID:Complex replication error causes p53 mutation in a Li-Fraumeni family. 761 54

In nine human colon tumour cell lines the relationship between O6-alkylguanine-DNA alkyltransferase (O6-AGT) activity and carmustine resistance was investigated. Three lines with O6-AGT activity below 25 fmol/mg protein had carmustine ED50 values ranging from 5.0 microM to 11.9 microM; six lines displaying an O6-AGT activity above 240 fmol/mg protein had ED50 values ranging from 28.9 microM to 69.5 microM. In lines with low O6-AGT activity, depletion of the repair protein by O6-benzylguanine resulted in a marginal increase of carmustine cytotoxicity (sensitization factors less than 2). In the majority of cell lines with high O6-AGT activity, pretreatment with O6-benzylguanine resulted in a pronounced sensitization to carmustine. In one line, an optimal time schedule for sensitization of colon tumour cells by O6-benzylguanine was assessed; continuous exposure to O6-benzylguanine > or = 16 h after carmustine exposure) was superior to short-term exposure limited to a period before carmustine treatment.
J Cancer Res Clin Oncol 1995
PMID:Sensitization of human colon tumour cell lines to carmustine by depletion of O6-alkylguanine-DNA alkyltransferase. 775 21

O6-Alkylguanine-DNA alkyltransferase (O6-AGT) activity in rat ovarian tumor lines O-342 and O 342/DDP was 103.4 +/- 18.4 and 240.9 +/- 40.2 fmol/mg protein, respectively; thus, cisplatin (DDP) resistance was paralleled by an increase in O6-AGT activity by a factor of approximately 2.3. The DDP-resistant line expressed a collateral resistance to BCNU. Both lines could be sensitized to BCNU by O6-BG, with sensitization factors of 6.0 and 2.1, respectively. In neither line did depletion of O6-AGT have any sensitizing effect towards DDP. In the human ovarian cancer lines SK-OV-3 and OAW 42, O6-AGT activity was 337.6 +/- 18.2 and 180.0 +/- 39.9 fmol/mg protein, respectively; in these lines depletion of O6-AGT activity by O6-BG treatment resulted in sensitization factors of 3.0 and 4.1, respectively. The increase in sensitivity of ovarian tumor cell lines against a chloroethylating agent by O6-AGT depletion and possible pharmacological advantages of regional (i.p.) administration of this combination might be beneficial in advanced ovarian cancer.
Cancer Chemother Pharmacol 1995
PMID:Inhibition of O6-alkylguanine-DNA alkyltransferase in animal and human ovarian tumor cell lines by O6-benzylguanine and sensitization to BCNU. 780 87

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.
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PMID:Alteration of the p53 tumor suppressor gene and activation of c-K-ras-2 protooncogene in endometrial adenocarcinoma from Colorado. 785 67

Reduced or heterogeneous expression of E-cadherin has been demonstrated immunohistochemically in poorly differentiated carcinoma, which frequently shows weak intercellular adhesiveness and marked invasiveness. In vitro, not only reduced expression but also structural abnormalities of E-cadherin have been observed in human carcinoma cell lines which grow in a loosely adhering manner. To clarify the participation of structural abnormalities of E-cadherin in cancer invasion in vivo, sequence abnormalities were examined in the cadherin domain (exons 5, 6, 7 and 8) including the region essential for E-cadherin specific binding, using the polymerase chain reaction-single-strand conformation polymorphism method and direct sequencing in invasive lobular carcinoma of the breast, in which cancer cells become detached from each other and invade the stroma in a particularly scattered pattern. In 2 (10%) of the 20 cases examined, an identical sequence abnormality was detected in E-cadherin exon 7, i.e. a point mutation of codon 315 (AAT to AGT) which resulted in a single amino acid substitution (asparagine to serine). This mutation may abolish the E-cadherin-mediated cell-cell adhesion and be at least partly responsible for the weak intercellular adhesiveness and scattered histological pattern of the tumor.
Jpn J Cancer Res 1994 Oct
PMID:Point mutation of the E-cadherin gene in invasive lobular carcinoma of the breast. 796 Nov 5

The effect of human O6-methylguanine-DNA methyltransferase (MGMT) on the cytotoxicity, the mutagenicity, and the specific kinds of base substitutions induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were examined in non-MGMT transfected Chinese hamster ovary cells (CHOM cells) and in those cells which had been transfected with human MGMT complementary DNA (AGT cells). AGT cells containing a high level of human MGMT activity were markedly more resistant to the cytotoxic and mutagenic effects of MNNG than CHOM cells which had no detectable MGMT activity. The dosages of MNNG which reduced to 50% of colony forming ability were estimated to be 0.8 microM for CHOM and 10 microM for AGT cells. The induction frequency of 6-thioguanine-resistant cells was significantly declined in AGT cells. At 4 microM MNNG, this frequency was declined from 273 mutants/10(6) viable CHOM cells to 13 mutants/10(6) viable AGT cells. The entire coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene in 37 AGT and 22 CHOM mutants was characterized by direct sequencing of the mRNA-polymerase chain reaction-amplified complementary DNA. Base changes at the intron-exon boundaries of the hprt DNA in the splicing mutants were further examined. Those results indicated that G to A transitions were significantly reduced in MNNG-treated AGT cells (chi 2 test, P < 0.001), suggesting that O6-methylguanine was repaired error free by human MGMT. In contrast, no difference arose in the frequencies of T to C transitions induced by MNNG in these two populations. All of the G to A transitions induced in AGT cells were located on the nontranscribed strand, assuming that the causative lesion was O6-methylguanine (P < 0.05). Such a strand specificity was not observed in CHOM mutants. Most of the G to A transitions observed in CHOM mutants were located at the middle guanine of 5'-GGPu sequences. Transitions observed at these sites, particularly 5'-GGG, were significantly reduced in AGT mutants (P < 0.05). Our results have suggested that human MGMT specifically repairs O6-methylguanine with a preference to remove those located on the transcribed strand and middle guanine of 5'-GGG.
Cancer Res 1994 Jul 15
PMID:Strand- and sequence-specific attenuation of N-methyl-N'-nitro-N-nitrosoguanidine-induced G.C to A.T transitions by expression of human 6-methylguanine-DNA methyltransferase in Chinese hamster ovary cells. 803 7

Three series of biopsy specimens of premalignant and malignant oral lesions, together with seven human keratinocyte cultures, previously established from oral squamous cell carcinomas, were analysed for point mutation in exons 1 and 2 of the c-Ha-ras, c-Ki-ras and N-ras genes by direct nucleotide sequencing of DNAs amplified in the polymerase chain reaction (PCR). Only one out of 12 biopsy samples (8.3%), a well-differentiated carcinoma which was the latest in a series of floor of mouth lesions from 1 of the 3 patients studied, harboured a mutant c-Ha-ras gene, being heterozygous at codon 12 for a GGA-GTA change. One cell line (H357) showed heterozygosity in both exons 1 and 2 of c-Ha-ras, harbouring a GGT to AGT mutation over codon 13 and a CAG to CAA mutation over codon 61. The remaining six oral carcinoma cell lines (85.7%) were homozygous normal at both exons 1 and 2 of c-Ha-ras. All cell lines showed normal c-Ki-ras and N-ras loci. We conclude that ras gene mutation is an infrequent occurrence in the malignant progression of oral epithelial cells, despite the probable importance of chemical carcinogens in the aetiology of the disease. We emphasise the need to search for other cellular sequences which may be targets for chemical or viral carcinogens.
Eur J Cancer B Oral Oncol 1993 Jan
PMID:Ras gene point mutation is a rare event in premalignant tissues and malignant cells and tissues from oral mucosal lesions. 818 May 79


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