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Query: UMLS:C0162871 (abdominal aortic aneurysm)
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In order to better understand the molecular events in murine hepatocarcinogenesis, the frequency and types of mutations in the murine H-ras proto-oncogene isolated from 184 independent, spontaneously occurring hepatic lesions were determined. Hepatocellular foci, hyperplasias, adenomas and carcinomas were obtained from archival samples of control male (134 samples) and female (50 samples) B6C3F1 mice used in oncogenicity studies that were conducted at Lilly Research Laboratories from 1979 to 1986. The 61st codon region of the H-ras oncogene from these sections was amplified using the polymerase chain reaction. Mutation frequencies were determined by restriction fragment length polymorphism analysis. The types of mutations were characterized by allele-specific oligonucleotide hybridization and confirmed by DNA sequencing. Forty-two per cent of the carcinomas, 44% of the adenomas, 42% of the hyperplasias and 29% of the foci contained mutations at the 61 codon. The mutation spectra for the carcinomas, adenomas and hyperplasias consisted of mostly CAA-AAA transversions, followed by CAA-CGA transitions, followed by CAA-CTA transversions. These results demonstrate that: (i) the frequency of spontaneous mutations in the H-ras 61st codon is equivalent in murine hyperplasias, adenomas and carcinomas, and (ii) sex was not a determining factor in either the mutation frequency or mutation spectrum for the spontaneous lesions. If these lesions represent successive stages in the carcinogenic process, then these results suggest that mutations in the 61st codon of H-ras are early events in spontaneous murine hepatocarcinogenesis.
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PMID:Genetic alterations in the 61st codon of the H-ras oncogene isolated from archival sections of hepatic hyperplasias, adenomas and carcinomas in control groups of B6C3F1 mouse bioassay studies conducted from 1979 to 1986. 135 Sep 49

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.
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PMID:Polymerase chain reaction/sequencing analysis of ras mutations in paraffin-embedded tissues as compared with 3T3 transfection and polymerase chain reaction/sequencing of frozen tumor deoxyribonucleic acids. 158 89

Hepatocellular tumors were induced in 15 day old male B6C3F1 mice following a single exposure to N-nitrosodiethylamine (DEN; 5 mg/kg, i.p.). Tumors were collected at 38 and 65 weeks to compare the frequencies and types of mutations in the 61st codon of the H-ras oncogene. The 61st codon was amplified using the polymerase chain reaction (PCR). Allele-specific oligonucleotide (ASO) probes were used to determine the frequency and types of mutations present in these tumors. Forty-nine nodular hepatic lesions were obtained from seven animals at the 38 week timepoint. Five of these samples (10%) had mutations at the 61st codon with one CAA-AAA, one CAA-CGA and three CAA-CTA. Thirty-six nodular hepatic lesions were obtained from six animals at the 65 week timepoint. Ten of these samples (28%) had mutations at the 61st codon with one CAA-AAA, five CAA-CGA and four CAA-CTA. These data indicate that DEN-induced mutations at the 61st codon of the mouse H-ras oncogene (i) are an infrequent event, (ii) have different frequencies at the 38 and 65 week timepoints and (iii) are different from the types of mutations seen in spontaneous lesions.
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PMID:Temporal changes in the mutant frequency and mutation spectra of the 61st codon of the H-ras oncogene following exposure of B6C3F1 mice to N-nitrosodiethylamine (DEN). 163 98

Twenty-nine patients with acute myelocytic leukemia (AML) and 14 patients with Philadelphia chromosome-positive chronic myelocytic leukemia (CML) were analyzed to detect the presence of mutations in their ras genes by the polymerase chain reaction and oligonucleotide hybridization methods. Deoxyribonucleic acid (DNA) isolated from blood or bone marrow samples was screened for mutations in codons 12, 13 and 61 of N-ras and in codons 12 and 61 of K-ras and H-ras. We detected mutations of the ras gene in 7 patients with AML (7/29), all in N-ras. The mutations were 3 GGT- greater than GAT transitions in codon 12, 1 GGT- greater than TGT transition in codon 13, and 3 CAA- greater than AAA transitions in codon 61. No correlation has been observed between French-American-British subtypes and the incidence of N-ras mutation, nor between cytogenetic changes and the incidence of N-ras mutation. All ras gene mutations detected by the oligonucleotide hybridization method were further confirmed by direct sequencing. No mutations were detected in ras genes in samples from the 14 Philadelphia chromosome-positive CML patients (12 in chronic phase, 2 in blastic phase). These findings are in line with previous results indicating that ras gene mutations in the codons tested play only a small role in the tumorigenesis of CML.
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PMID:Mutation analysis of the ras gene in myelocytic leukemia by polymerase chain reaction and oligonucleotide probes. 168 80

The H-ras gene of the BALB murine sarcoma virus (BALB-MSV) was placed under the transcriptional control of the tightly regulated PL promoter of bacteriophage lambda in the expression vectors pEV-vrf-1 and pRC23. Upon derepression of the PL promoter, large amounts (10-20% of total cellular protein) of the H-ras gene product p21 are synthesized in Escherichia coli. We constructed three H-ras gene expression vectors, designated pJCL-H5, pJCL-E30, and pJCL-33. pJCL-H5 directs the synthesis of p21, a fusion protein whose four amino-terminal residues are replaced by eight amino acids coded for by plasmid sequences. The 13 5' coding nucleotides of the BALB-MSV H-ras gene missing in pJCL-H5 were regenerated in pJCL-E30 by inserting a pair of complementary synthetic oligodeoxynucleotides. As a result, pJCL-E30 encodes a p21 protein, p21T, of sequence identical to that of the transforming p21 protein of BALB-MSV. pJCL-33 is a derivative of pJCL-E30 in which the 12th codon, AAA, a lysine codon, was replaced by GGA, a glycine codon. Thus, pJCL-33 directs the synthesis of a p21 protein, p21N, whose sequence corresponds to that of a normal cellular p21 protein. We report the purification of H-ras p21 proteins to apparent homogeneity by a method involving solubilization with chaotropic agents followed by reverse-phase high-performance liquid chromatography.
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PMID:Expression of normal and transforming H-ras genes in Escherichia coli and purification of their encoded p21 proteins. 608 91

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two major by-products formed during chlorine disinfection of drinking water, increase the incidence of tumors in B6C3F1 mice by 6- and 3-fold respectively. In order to understand better the mechanism by which these two compounds induce liver tumors, the incidence and spectrum of mutations in the K- and H-ras proto-oncogenes in these tumors were analyzed. DNA from spontaneous, DCA- and TCA-induced liver tumor from B6C3F1 male mice was evaluated for point mutations in exons 1, 2 and 3 of the two genes by single-stranded conformation polymorphism. Results demonstrated a similar incidence of mutations for exon 2 of H-ras in spontaneous carcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%), 1.0 g/l (48%) and TCA 4.5 g/l (45%). Only four showed mutations in the other exons of Hras or in K-ras. Sequence analysis of spontaneous tumor samples with second exon H-ras mutations revealed a change in codon 61 from CAA to AAA in 80% and CAA to CGA in 20% of tumors. In contrast, tumors with H-ras mutations from DCA-treated mice revealed a H-61 change from CAA to AAA in 21% at 3.5 g/l and 16% at 1.0 g/l. CAA to CGA was observed in 50% of tumors from mice given DCA 3.5 or 1.0 g/l, and CAA to CTA was present in 29% and 34% of the two dosage groups respectively. Interestingly, TCA showed the same mutational spectrum as the spontaneous liver tumors. The data indicates that induction of liver carcinoma by DCA and TCA involves activation of the H-ras proto-oncogene at a frequency similar to that observed in spontaneous tumors. However, the mechanism(s) for including hepatocellular carcinoma does not appear to be identical for DCA and TCA.
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PMID:Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids. 769 4

The frequency and mutation spectra of proto-oncogene activation in hepatocellular neoplasms induced by tetrachloroethylene, trichloroethylene and dichloroacetic acid were examined to help define the molecular basis for their carcinogenicity. H-ras codon 61 activation was not significantly different among dichloroacetic acid- and trichloroethylene-induced and combined historical and concurrent control hepatocellular tumors (62%, 51% and 69% respectively). The mutation spectra of H-ras codon 61 mutations showed a significant decrease in AAA and increase in CTA mutations for dichloroacetic acid- and trichloroethylene-induced tumors when compared to combined controls. The H-ras codon 61 mutation frequency for tetrachloroethylene-induced tumors was significantly lower (24%) than that of combined controls and also that of the two other chemicals. Mutations at codons 13 and 117 plus a second exon insert contributed 4% to the total H-ras frequencies for trichloroethylene and tetrachloroethylene. There was also a higher incidence of K-ras activation (13%) in tetrachloroethylene-induced tumors than in the other chemically induced or control tumors. Four liver tumors were found to contain insertions of additional bases within the second exon of K- or H-ras. These findings suggest that exposure to dichloroacetic acid, trichloroethylene and tetrachloroethylene provides a selective growth advantage to spontaneously occurring mutations in codon 61 of H-ras and, at the same time, is responsible for a small number of unique molecular lesions suggestive of either a random genotoxic mode of action or a non-specific result of secondary DNA damage. However, the absence of ras activation in many of the liver neoplasms suggests that alternative mechanisms are also important in B6C3F1 mouse hepatocarcinogenesis.
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PMID:ras proto-oncogene activation in dichloroacetic acid-, trichloroethylene- and tetrachloroethylene-induced liver tumors in B6C3F1 mice. 795 63

Treatment of B6C3F1 mice with concentrations of 62.5-625 p.p.m. 1,3-butadiene by inhalation for up to 2 years causes a significantly increased incidence of Harderian gland (HG) neoplasms over untreated controls (Melnick,R., Huff,J., Chou,B.J. and Miller,R.A. Cancer Res., 50, 6592-6599, 1990). Since a specific K-ras mutation (codon 13 GGC-->CGC) had previously been described in lung and liver tumors from 1,3-butadiene-treated B6C3F1 mice, we analyzed 23 adenomas and six adenocarcinomas of the HG from mice exposed to 1,3-butadiene for this mutation and mutations in the H-ras gene. We also examined ras activation in 16 spontaneously occurring HG adenomas and one adenocarcinoma. DNA samples were prepared from paraffin-embedded tissues and analyzed by PCR followed by direct sequencing methods. Only one 1,3-butadiene-induced HG tumor contained the K-ras codon 13 mutation previously detected in lung and liver tumors. However, 16/29 HG tumors from the treated B6C3F1 mice contained H-ras codon 61 mutations. The mutations detected were: 12 CAA-->CGA transitions, two CAA-->CTA and two CAA-->AAA transversions. Eleven of 17 spontaneous HG tumors contained mutations in H-ras codon 61: five CAA-->CGA transitions, two CAA-->CTA transversions and four CAA-->AAA transversions. While the spectrum of ras mutations did not differ between the spontaneously occurring and chemically induced tumors, these data indicate that activation of H-ras contributes to the process of HG tumorigenesis in both groups of these neoplasms.
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PMID:Activation of H-ras is prevalent in 1,3-butadiene-induced and spontaneously occurring murine Harderian gland tumors. 795 23

Previous reports from several laboratories have consistently shown that approximately 30% of spontaneous hepatocellular adenomas and 70-80% of spontaneous hepatocellular carcinomas found in aged B6C3F1 [C57BL/6 (liver tumor resistant) x C3H (liver tumor susceptible)] male mice contain one of three missense point mutations in codon 61 of the H-ras oncogene, CAA-->AAA, CGA or CTA. Irrespective of subline, the C3H mouse, the paternal parent strain of the B6C3F1 hybrid, is more susceptible to spontaneous liver tumorigenesis than the B6C3F1 mouse. However, the role of H-ras in the pathogenesis of hepatocellular tumors in C3H mice is less clear, as widely different frequencies of activation of this gene, but by the same point mutations in codon 61, have been reported by various laboratories. The present study was undertaken to characterize H-ras involvement in hepatocellular tumors of aged C3H/He mice from the NCI-Frederick Cancer Research and Development Center Colony (C3H/HeNCr). Oncogene activation was evaluated in 45 C3H/HeNCr hepatocellular tumors by the NIH 3T3 transfection assays, and point mutations in the H-ras oncogene were detected and characterized in DNA fragments amplified by PCR, using dot blot hybridization analysis with mutation-specific oligonucleotide probes and direct dideoxy sequencing of PCR products. The only transforming gene detected in these tumors by NIH 3T3 transfection was H-ras. Only 17% (1/6) of spontaneous carcinomas and 8% (3/39) of spontaneous adenomas contained transforming H-ras sequences, each with a point mutation in codon 61. In all four cases with H-ras mutations, mutated sequences comprised a minor fraction of total H-ras gene copies in DNA extracted from primary tumors. H-ras mutations thus appear to have arisen relatively late in the pathogenesis of the neoplasms. For comparison, sections of formalin-fixed, paraffin-embedded hepatocellular tumors that occurred in untreated B6C3F1 hybrid mice sired by C3H/HeNCr males were assayed for the same H-ras mutations by PCR and dot blot hybridization. Nine of 13 such tumors (4/6 carcinomas, 5/7 adenomas) were positive. The overall difference in frequency of H-ras codon 61 mutations in hepatocellular tumors in C3H/HeNCr (4/45) versus B6C3F1 (9/13) was highly significant (P = 0.000035, Fisher's exact test). These data indicate that point mutations in H-ras do not generally play a major or an initiating role in spontaneous hepatocarcinogenesis of inbred C3H/HeNCr mice and contrast with the high rate of ras mutations in liver tumors of the B6C3F1 hybrid.
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PMID:Low frequency of H-ras activation in naturally occurring hepatocellular tumors of C3H/HeNCr mice. 840 22

The MutEx assay is a technique that was developed to detect and map mutations. This assay takes advantage of the Escherichia coli mismatch binding protein MutS, which binds and protects mismatched, heteroduplex DNA from subsequent exonuclease digestion. The plausibility of using the MutEx assay as part of a genotypic selection scheme was investigated. Heteroduplexes were formed between mouse H-ras gene PCR products or restriction fragments that contained wild-type sequence and sequence with a single base change at codon 61 (wild-type, CAA and mutant, AAA). The heteroduplexes were incubated with MutS and then treated with the exonuclease activity of T7 DNA polymerase. MutS-protected DNA sequences were amplified by PCR. When this method was linked to single nucleotide primer extension (SNuPE) for mutant base identification, original mutant fractions of 1 in 50000 and above were detected. Using comparable DNA template mixtures, the sensitivity of SNuPE alone was 1 in 5 or 1 in 50, depending on the direction of SNuPE priming and the particular base being incorporated. We conclude that the MutEx assay was able to enrich the mutant sequence approximately 1000-fold and, therefore, has considerable potential as a tool for mutation detection.
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PMID:Evaluation of MutS as a tool for direct measurement of point mutations in genomic DNA. 910 Aug 51

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