Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Activated c-Ki-ras with a point mutation (GGT to CGT) at codon 12, resulting in the substitution of arginine for glycine, was found in DNA from metastatic pancreatic adenocarcinoma in a lymph node. By means of restriction endonuclease length polymorphism with SacI digestion, we were able to demonstrate that the same point mutation of c-Ki-ras was present in the primary tumor and in metastases in lymph nodes. DNA from the normal spleen of the patient did not have this type of point mutation. Moreover, amplifications of 3- to 6-fold of the activated c-Ki-ras and 50-fold of c-myc were found in the primary tumor and the metastases in the two lymph nodes, indicating that point mutation had occurred at a relatively early stage of the tumor development, before amplification of the gene. This is the first clear demonstration of amplification of activated c-Ki-ras accompanied by amplification of c-myc in both primary and metastatic human tumors in vivo.
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PMID:Amplifications of both c-Ki-ras with a point mutation and c-myc in a primary pancreatic cancer and its metastatic tumors in lymph nodes. 300 77

The clonal composition of cancers of the female reproductive tract was evaluated by analysis of patterns of X-chromosome inactivation. Using DNA extracted from frozen tissues or paraffin-embedded archival specimens as template, polymerase chain reaction (PCR) was performed to generate amplified DNA fragments of exon 1 of the X-linked androgen receptor gene, which contains a highly polymorphic trinucleotide repeat. Predigestion of tumor DNA with methylation-sensitive restriction endonuclease Hha I or Hpa II permitted selective PCR amplification from the methylated (uncleaved) allele. Of a total of 54 tumors analyzed, 50 cases showed heterozygosity (93%) and were therefore informative for clonal analysis. Monoclonal composition of the tumors was suggested in a total of 49 of 50 cases, including 12 adenocarcinomas of the uterine endometrium, 13 squamous cell carcinomas of the uterine cervix, 6 adenocarcinomas of the uterine endocervix, and 18 epithelial tumors of the ovary. However, polyclonal composition was observed in one mucinous carcinoma of the ovary, in which we previously showed that both GGT-->GAT and GGT-->GTT mutations are present in > 20% of total K-ras copies in the tissue. Our studies demonstrate the utility of PCR amplification of highly polymorphic repetitive sequences for analysis of patterns of X-chromosome inactivation. This approach is practical for the analysis of clonal cell composition in a high proportion of both formalin-fixed and frozen archival tissues.
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PMID:Analysis of clonality by amplification of short tandem repeats. Carcinomas of the female reproductive tract. 786 41

Hepatic angiosarcoma (HA) is an uncommon neoplasm associated with known etiologic factors in 25% to 42% of cases. It is, however, one of the most common sarcomas found in the liver. The aim of this study was to find was to find mutations in the K-ras-2 oncogene in sporadic and Thorotrast (TT)-induced HA. Point mutations in K-ras-2 were sought in archival, formalin-fixed tissue blocks from 24 patients with angiosarcoma. Of these, 19 cases were sporadic and 5 were TT-induced. Mutational analysis was performed by topographic microdissection with PCR amplification followed by genotyping. Specific mutations were determined by two independent methods: (a) direct sequencing of the PCR product confirmed by rePCR and by using a different sequencing primer, and (b) PCR-based selective enrichment of mutant DNA by endonuclease digestion followed by heteroduplex DNA analysis using denaturing gradient gel electrophoresis. Eleven K-ras-2 point mutations were detected in 7 of 24 (29%) tumors, including 5 of 19 (26%) sporadic HA and 2 of 5 (40%) TT-induced HA. There were seven G:C > A:T and four G:C > T:A mutations. All seven mutated tumors contained a codon 12-aspartate amino acid substitution. In addition, a second codon 12-cysteine mutant cell population was present in one of two codon 12-aspartate mutated TT-induced HA and in three of five codon 12-aspartate sporadic tumors. Of these four tumors, three contained both aspartate and cysteine mutations and were composed of multiple nodules; the fourth was a single mass. Seventeen tumors had multiple nodules; whereas 5 had a K-ras-2 mutation, 12 were wild-type. The molecular pathology of both sporadic and TT-induced HA is characterized by a high rate of K-ras-2 mutations characteristic of oxidative damage (ie, G:C > A:T and G:C > T:A mutations) resulting in two mutated population sets: codon 12 GGT > GAT and GGT > TGT (glycine to aspartic acid and cysteine). This is, to date, the first study to characterize the K-ras-2 gene mutations within human sporadic and TT-induced HA by direct sequence analysis and denaturing gradient gel electrophoresis. These data further support the hypothesis linking adduct-forming vinyl chloride exposure to HA containing a much higher frequency of K-ras-2 mutations and a mutational spectrum characteristic of chloroethylene oxide, a carcinogenic metabolite of vinyl chloride.
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PMID:Sporadic and Thorotrast-induced angiosarcomas of the liver manifest frequent and multiple point mutations in K-ras-2. 901 Apr 58

We report 8 newly established gastric-carcinoma cell lines (SNU-216, 484, 520, 601, 620, 638, 668, 719) from Korean patients. Morphologic study was carried out using light and electron microscopes. CEA, alpha FP, and CA 19-9 and TPA in supernatant and in cell lysate were measured by radioimmunoassay. p53 and c-Ki-ras gene mutations were screened and confirmed by sequencing. The cell lines, derived from tumors with moderate differentiation, grew as a diffuse monolayer, and those from tumors with poor differentiation and minimal desmoplasia grew exclusively as non-adherent. Out of the 8 gastric-cancer cell lines, 5 had detectable levels of CEA both in supernatant and in cell lysate; there was no expression or secretion of alpha FP in these cells; 4 cell lines showed high levels of CA 19-9 in cell pellets. All cell lines except SNU-484 had high concentrations of TPA both in cell lysate and in supernatants. p53 mutation was found in 6 cell lines (75%): 2 (SNU-216 and SNU-668) had mutations in exon 6, and other 3 in exon 8. The c-Ki-ras mutation was found in 2 cell lines (25%), SNU-601 and SNU-668. The former showed GGT-to-GAT transition mutation at codon 12, while the latter showed CAA-to-AAA transversion mutation at codon 61. DNA profiles using restriction endonuclease HinfI and polymorphic DNA probes ChdTC-15 and ChdTC-114 showed different unique patterns; which suggests that these cell lines are unique and not cross-contaminated. We believe that the newly characterized gastric-cancer cell lines presented in this paper will provide a useful in vitro model for studies related to human gastric cancer.
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PMID:Establishment and characterization of human gastric carcinoma cell lines. 903 53

The adeno-associated virus (AAV) Rep78 and Rep68 proteins are required for site-specific integration of the AAV genome into the AAVS1 locus (19q13.3-qter) as well as for viral DNA replication. Rep78 and Rep68 bind to the GAGC motif on the inverted terminal repeat (ITR) and cut at the trs (terminal resolution site). A similar reaction is believed to occur in AAVS1 harboring an analogous GAGC motif and a trs homolog, followed by integration of the AAV genome. To elucidate the functional domains of Rep proteins at the amino acid level, we performed charged-to-alanine scanning mutagenesis of the N terminus (residues 1 to 240) of Rep78, where DNA binding and nicking domains are thought to exist. Mutants were analyzed for their abilities to bind the GAGC motif, nick at the trs homolog, and integrate an ITR-containing plasmid into AAVS1 by electrophoretic mobility shift assay, trs endonuclease assay, and PCR-based integration assay. We identified the residues responsible for DNA binding: R107A, K136A, and R138A mutations completely abolished the binding activity. The H90A or H92A mutant, carrying a mutation in a putative metal binding site, lost nicking activity while retaining binding activity. Mutations affecting DNA binding or trs nicking also impaired the site-specific integration, except for E66A and E239A. These results provide important information on the structure-function relationship of Rep proteins. We also describe an aberrant nicking of Rep78. We found that Rep78 cuts predominantly at the trs homolog not only between the T residues (GGT/TGG), but also between the G and T residues (GG/TTGG), which may be influenced by the sequence surrounding the GAGC motif.
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PMID:Charged-to-alanine scanning mutagenesis of the N-terminal half of adeno-associated virus type 2 Rep78 protein. 1007 14

The mutagenicity of a prominent tobacco carcinogen, benzo[a]pyrene (B[a]P), is believed to result from chemical reactions between its diol epoxide metabolite, (+)-anti-7r,8t-dihydroxy-c9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), and DNA, producing promutagenic lesions, e.g., (+)-trans-anti-7R,8S,9S-trihydroxy-10S-(N(2)-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[a]pyrene (N(2)-BPDE-dG). Previous studies used the DNA repair enzyme UvrABC endonuclease in combination with ligation-mediated PCR (LMPCR) to demonstrate an increased reactivity of BPDE toward guanine nucleobases within codons 157, 248, and 273 of the p53 tumor suppressor gene (Denissenko, M. F., Pao, A., Tang, M., and Pfeifer, G. P. Science 274, 430-432). These sites are also "hot spots" for mutations observed in lung tumors of smokers, suggesting an involvement of B[a]P in the initiation of lung cancer. However, the LMPCR approach relies on the ability of the repair enzyme to excise BPDE-induced lesions, and thus the slowly repaired lesions may escape detection. Furthermore, BPDE-DNA adduct structure and stereochemistry cannot be determined. In the present work, we performed a direct quantitative analysis of N(2)-BPDE-dG originating from specific guanine nucleobases within p53- and K-ras-derived DNA sequences by using a stable isotope labeling-mass spectrometry approach recently developed in our laboratory. (15)N-labeled dG was placed at defined positions within DNA sequences derived from the K-ras proto-oncogene and p53 tumor suppressor gene, the two genes most frequently mutated in smoking-induced lung cancer. (15)N-labeled DNA was annealed to the complementary strands, followed by BPDE treatment and liquid chromatography-electrospray ionization tandem mass spectrometry analysis (HPLC-ESI-MS/MS) of N(2)-BPDE-dG lesions. The extent of adduct formation at (15)N-labeled guanine was determined directly from the HPLC-ESI-MS/MS peak area ratios of (15)N-N(2)-BPDE-dG and N(2)-BPDE-dG. BPDE-induced guanine adducts were produced nonrandomly along K-ras and p53 gene-derived DNA sequences, with over 5-fold differences in adduct formation depending on sequence context. N(2)-BPDE-dG yield was enhanced by the presence of 5-Me substituent at the cytosine base-paired with the target guanine nucleobase, an endogenous DNA modification characteristic for CpG dinucleotides within the p53 gene. In the K-ras-derived DNA sequence, the majority of N(2)-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G --> T transversions observed at this nucleotide in smoking-induced lung cancer. On the contrary, the pattern of N(2)-BPDE-dG formation within the p53 exon 5 sequences did not correlate with the mutational spectrum in lung cancer, suggesting that factors other than N(2)-BPDE-dG formation are responsible for these mutations. The stable isotope labeling HPLC-ESI-MS/MS approach described in this work is universally applicable to studies of modifications to isolated DNA by other carcinogens and alkylating drugs.
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PMID:Formation of benzo[a]pyrene diol epoxide-DNA adducts at specific guanines within K-ras and p53 gene sequences: stable isotope-labeling mass spectrometry approach. 1213 76

Apolipoprotein CIII (apo-CIII) participates in the regulation of triglyceride-rich lipoprotein metabolism. Several polymorphic sites have been detected within and around the apo-CIII gene. Here, we examined the relationship between apo-CIII SstI polymorphism (CC, CG, GG genotypes) and plasma triglyceride (TG) levels in a group of 159 Japanese individuals living in Southern Brazil. The sample was divided into a group of Japanese descendants (N = 51) with high TG (HTG; >200 mg/dL) and a group of Japanese descendants (N = 108) with normal TG (NTG; <200 mg/dL). TG and total cholesterol levels were analyzed by an enzymatic method using the Labtest-Diagnostic kit and high- and low-density lipoproteins by a direct method using the Labtest-Diagnostic kit and DiaSys Diagnostic System International kit, respectively. A 428-bp sequence of apo-CIII gene was amplified using oligonucleotide primers 5' GGT GAC CGA TGG CTT CAG TTC CCT GA 3' and 5' CAG AAG GTG GAT AGA GCG CTG GCC T 3'. The PCR products were digested with a restriction endonuclease SstI. Rare G allele was highly prevalent in our study population (0.416) compared to Caucasians (0.00-0.11). G allele was almost two times more prevalent in the HTG group compared to the NTG group (P < 0.001). The genotype distribution was consistent with the Hardy-Weinberg equilibrium. There was a significant association between rare G allele and HTG in Japanese individuals living in Southern Brazil as indicated by one-way ANOVA, P < 0.05.
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PMID:Apolipoprotein CIII polymorphism and triglyceride levels of a Japanese population living in Southern Brazil. 1856 Jun 72

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1-/-Neil1-/- mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1-/- and Neil1-/-. The pulmonary tumors contained, exclusively, activating GGT-->GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT-->GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1-/- and Neil1-/-Nth1-/- mice. The high incidence of tumors in our Nth1-/-Neil1-/- mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua.
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PMID:Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA. 1934 69