Frequent mutations of Ki-ras but no mutations of Ha-ras and p53 in lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine in rats

Point mutations of the Ki-ras and p53 genes in rat lung lesions induced by N-nitrosobis(2-hydroxypropyl)amine (BHP) were investigated by polymerase chain reaction-single-strand conformation polymorphism analysis followed by direct sequencing using paraffin-embedded tissues. Male Wistar rats 6 wk old were given 2000 ppm BHP in drinking water for 15 wk. Another group was given drinking water without BHP. The rats were killed 20–27 wk after the beginning of the experiment. Lung adenomatous and squamous lesions, including carcinomas, were induced. The frequencies of Ki-ras mutations were 40% (six of 15) in alveolar hyperplasias, 36% (five of 14) in adenomas, 72% (18 of 25) in adenocarcinomas, 20% (three of 15) in squamous metaplasias, 50% (three of six) in squamous cell carcinomas, and 50% (five of 10) in adenosquamous carcinomas. The mutations were all G → A transitions at the second position of codon 12; no other mutations were detected. However, Ha-ras mutations in exons 1 and 2 and p53 mutations in exons 5, 6, and 7 were not detected in adenocarcinomas and squamous cell carcinomas. These results indicate that Ki-ras mutation is an early genetic event in some adenomatous and squamous lung carcinogenesis and that Ki-ras mutations can cause benign lesions to convert to malignant lesions. The results also show that Ha-ras and p53 mutations are not involved in rat lung carcinogenesis induced by BHP. © 1996 Wiley-Liss, Inc.     

Infrequent Ha-ras mutations and absence of Ki-ras,N-ras,and p53 mutations in 4-nitroquinoline 1-oxide-induced rat oral lesions

The alkylating agent 4-nitroquinoline 1-oxide (4-NQO) is a powerful carcinogen and induces squamous cell hyperplasia, squamous cell dysplasia, papilloma, and squamous cell carcinoma (a) in rat oral epithelia. Oral cancers induced by a single application of 4-NQO develop through a multistage process in a way similar to the development of this cancer in humans. In this study, mutations in exons 1 and 2 of Ki-ras, N-ras, and Ha-ras and exons 4–7 of p53 were examined by polymerase chain reaction (a) -single strand conformation polymorphism (a) analysis, followed by PCR-direct sequencing for the confirmation of mutations. Samples for the mutation analysis were obtained from dysplasias, papillomas, and SCCs on the tongue epithelia induced in F344 rats by adding 4-NQO (20 ppm) to their drinking water for 8 wk. The Ha-ras mutations (⁶¹A→T transversions in the second position) were found in five of 29 (17%) samples (one dysplasia and four SCCs). However, no mutations were detected in either Ki-ras, N-ras, or p53 under two different conditions of PCR-SSCP analysis. We suggest that some neoplasms in oral carcinogenesis induced by 4-NQO may involve Ha-ras mutations but not mutations in Ki-ras, N-ras, or p53. The 4-NQO-induced rat oral carcinogenesis model may provide a system for evaluation of the mechanisms of multistage oral carcinogenesis associated with Ha-ras mutation without Ki-ras, N-ras, or p53 mutation. © 1995 Wiley- Liss, Inc.     

High frequency of ki-ras amplification and p53 gene mutations in adenocarcinomas of the human esophagus

Mutated ras genes have been found to be conspicuously absent from primary tumors of the esophagus, although high expression of ras p21 oncoprotein in some esophageal squamous cell carcinomas and mutations of the Ki- and Ha-ras genes in esophageal carcinoma cell lines have been reported. In this study, we found amplification of the Ki-ras gene in four of 10 esophageal adenocarcinomas (40%). No such amplification was observed among 61 squamous cell carcinomas, one pseudosarcomatous carcinoma, and eight esophageal cell lines, nor in six adenocarcinomas of the stomach. In two samples on which immunohistochemical analysis could be performed, we found overexpression of Ki-ras proteins when compared with normal samples. This Ki-ras amplification in esophageal tumors did not correlate with any pathological feature of the tumors, with the survival of the patients, or with the presence of other genetic alterations. These findings provide the first evidence for amplification of the Ki-ras gene in human esophageal cancer, which is restricted to adenocarcinomas. We also found that six of eight adenocarcinomas had point mutations in the p53 gene; this is a considerably higher prevalence than that reported for esophageal squamous cell carcinomas. These results strongly suggest that esophageal adenocarcinomas differ from squamous cell carcinomas in their molecular genetic characteristics. © 1995 Wiley- Liss, Inc.     

Ki-ras gene mutations and absence of p53 gene mutations in spontaneous and urethane-induced early lung lesions in CBA/J mice

Ki-ras and p53 genes are involved in human lung carcinogenesis; however, the role of these genes in experimental lung tumors is not well known. In our study, the CBA/J mouse strain was used to investigate the presence of Ki-ras and p53 alterations in lung carcinogenesis of spontaneous tumors and tumors induced with high and low doses of urethane (ethyl carbamate). To study the presence of these alterations in the early stages of lung carcinogenesis and in very small lung tumors, restriction fragment length polymorphism and single-strand conformation polymorphism analyses were performed on polymerase chain reaction–amplified DNA from microdissected tumoral and normal lung samples. Ki-ras gene mutations in codons 12 and 61 were detected in all types of lung lesions, even in small and preneoplastic lesions, and their incidence increased with progression from lung hyperplasias (18%) to adenomas (75%) and to carcinomas (80%). Urethane exposure, in both high and low doses, increased the incidence of Ki-ras mutations in lung tumors, especially in adenomas. The presence of Ki-ras gene mutations in very small urethane-induced lung tumors and the absence of hyperplasias among the treated-group lesions may indicate that urethane accelerates tumoral progression. No p53 mutations were detected in exons 5–8 in any of the epithelium-derived lung tumors. Only one p53 mutation in exon 5 was found in a spontaneous lymphoma. Therefore, p53 mutations do not seem to cooperate with Ki-ras gene mutations or represent an alternative molecular pathway in murine carcinogenesis. Mol. Carcinog. 21:251–260, 1998. © 1998 Wiley-Liss, Inc.     

Assessment of mutations in ki-ras and p53 in colon cancers from azoxymethane- and dimethylhydrazine-treated rats

Mutations in the Ki-ras oncogene and the p53 tumor suppressor gene are known to occur at high frequencies in human colon cancers. We measured the frequency of mutations in these two genes in colon adenocarcinomas obtained from a widely used experimental model of human colon carcinogenesis: F344 rats treated with the carcinogens azoxymethane (AOM) or dimethylhydrazine (DMH). We detected codon 12 mutations in Ki-ras in approximately 60% of colon adenocarcinomas induced by either carcinogen. We characterized the rat p53 intron-exon junctions to construct primers for polymerase chain reaction amplification of this gene. We discovered that the rat p53 gene was structurally different from the human p53 gene, as the rat gene was missing one intron between exons 6 and 7. Both single-stranded DNA conformational polymorphism analysis and direct DNA sequencing of the highly conserved regions of rat exons 5–7 were conducted because the corresponding human regions (exons 5–8) have been reported as being mutated most frequently in human colon cancers. Using these methods, we were unable to identify any p53 mutations in the highly conserved regions of exons 5–7 in either AOM- or DMH-induced colon adenocarcinomas. These data confirm that Ki-ras was mutated in most colon cancers in AOM- or DMH-treated rats but indicate that molecular alterations in the p53 gene, if they occur in this animal model, are different from most p53 mutations in human colon cancers. Mol. Carcinog. 19:137–144, 1997. © 1997 Wiley-Liss, Inc.     

ras mutations in 2-amino-3-methylimidazo-[4,5-f]quinoline—induced tumors in the CDF1 mouse

2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a very potent mutagen that is carcinogenic in rodents and nonhuman primates. IQ-induced CDF₁ mouse lung and liver tumors were examined for activated Ki-ras and Ha-ras genes, respectively. Polymerase chain reaction (PCR)—amplified target DNAs were analyzed for mutations of codons 12, 13, and 61 by single-strand conformation polymorphism (SSCP) and direct sequencing methods. All mutations were localized to codon 61 of the ras genes. Forty-nine of 54 lung tumors induced by IQ possessed activating Ki-ras mutations, as did 20 of 26 lung tumors from the vehicle-treated animals; 80% and 75% of these mutations, respectively, were A → T transversions of the second nucleotide redundant. One lung adenoma from the IQ-treated group contained a tandem duplication of the sequence corresponding to codons 50–57 of the Ki-ras gene (unpublished observations). In addition, seven of 34 IQ-induced liver tumors harbored activating Ha-ras mutations: five were C → A (G → T) transversions at the first nucleotide, and two were A → T transversions at the second nucleotide of codon 61. None of the 15 liver tumors collected from the vehicle-treated mice possessed Ha-ras mutations in codon 12, 13, or 61. These data indicate that IQ induces Ha-ras gene activation in CDF₁ mouse liver tumors. The mechanism of lung tumor induction by IQ, however, is obscured by the high frequency of Ki-ras A → T mutations observed in both the IQ-induced and spontaneous lung tumors. The different ras mutational spectra in lung and liver tumors may suggest either that two different pathways of IQ metabolism exist in these organs or that IQ contributes to CDF₁ lung tumorigenesis by a mechanism other than its direct interaction with the Ki-ras gene.     

Far less frequent mutations in ras genes than in the p53 gene in skin tumors of xeroderma pigmentosum patients

Mutations in Ha-ras, and n-rasNras genes in squamous and basal cell carcinomas in patients with xeroderma Pigmentosum (XP) were examined by the polymerase chain reaction followed by single-strand conformation polymorphism analysis and direct base sequencing. No mutation was detected in codons 12, 13, and 61 of the ras genes in XP skin tumors. This was in contrast with previous findings of a high frequency of mutation in the p53 gene in skin tumors in XP patients. A novel mutation in codon 6 of the ki-ras gene was detected in a squamous cell carcinoma. The mutation was a C→T transtion at a dipyrimidine (5′-CT) sequence and could have been produced by solar ultraviolet light. The mutated ras gene did not have the ability to transform NIH/3T3 cells. In three tumors, multiple base substitutions were detected in exon 1 of the Ki-ras and N-ras genes. These results and our previous work on p53 gene mutations suggest that mutations in ras genes are far less frequent than in the p53 gene in the skin tumors in XP patients and that ras genes are less important in skin tumorigenesis in XP patients that is the p53 gene.     

Overexpression of MDM-2 mRNA and mutation of the p53 tumor suppressor gene in bladder carcinoma cell lines

To investigate the importance of oncogenes and tumor suppressor genes in bladder carcinogenesis, we determined the status of the expression of the MDM-2 and p53 genes and genetic alterations in the p53 gene in five bladder carcinoma cell lines and one kidney urothelial carcinoma cell line. Overexpression of MDM-2 mRNA was observed in three bladder carcinoma cell lines, J82, SCaBER, and BFTC-905. Amplification of the MDM-2 gene was not detected in any of the six cell lines by Southern analysis. The deletion in the p53 gene was observed in J82, and point mutation was detected in J82 and BFTC-909, the kidney urothelial carcinoma cell line. In contrast, no mutations were found in codons 12, 13, and 61 in the Ha-ras and Ki-ras genes in these six cell lines. These results indicate that alterations in the p53-regulated pathway are important in bladder carcinogenesis. © 1995 Wiley-Liss, Inc.     

G→A mutations in p53 and Ha-ras genes in esophageal papillomas induced by N-nitrosomethylbenzylamine in two strains of rats

In human esophageal cancers, no ras gene mutations but a relatively high prevalence of p53 gene mutations have been reported. We found a high prevalence of point mutations in Ha-ras and p53 genes in N-nitrosomethylbenzylamine (NMBA)-induced esophageal tumors in two strains of rats (BD VI and F344). Our analysis showed the point mutation GGA←GAA (expected from the known mechanisms of action of NMBA) at Ha-ras codon 12 in 22 of 46 (48%) and 22 of 38 (58%) papillomas from BD VI and F344 rats, respectively. There was no significant difference in the prevalence of ras mutations in tumors induced by high doses (5.0 mg/kg) and low doses (2.5 mg/kg) of NMBA. Eleven papillomas from each strain were analyzed for p53 mutations. The prevalent mutations found were G←A and C←T transitions. The frequency of p53 mutation was 36% (four of 11) for each strain. No apparent hot-spot codon or exon was found in the p53 gene, and two papillomas contained double mutations in this gene. The high prevalence of G←A mutations in the rat Ha-ras gene contrasts with that in the human gene, in which no ras mutations have been found in primary tumors, and suggests either that the biology of esophageal carcinogenesis differs in humans and rats or that nitrosamines are not the major etiological risk factor for human esophageal cancers. © 1994 Wiley-Liss, Inc.     

Carcinogenic alterations in murine liver,lung, and uterine tumors induced by in utero exposure to ionizing radiation

The atomic bombing of Hiroshima and Nagasaki and the nuclear accident at Chernobyl raised the question of prenatal sensitivity to ionizing radiation–induced cancer. In this study, mice were exposed to single doses of γ-radiation (0.2–2.0 Gy) at different embryonic stages. The tumor incidence increased with dose from 15% in control mice to 35% in mice irradiated with 2.0 Gy on 18 d of prenatal life. Various oncogenic events were investigated in lymphoid, liver, lung, and uterine tumors. We observed threefold to fivefold increases in myc expression in 25% of the lymphomas, and the expression of Ha-ras and p53 genes decreased in 40% and 60% of the lung tumors by twofold to fivefold. Point mutations were tissue specific: Ha-ras codon 61 mutations were found in about 40% of the liver adenocarcinomas, Ki-ras codon 12 mutations in about 17% of lung tumors, and p53 mutations in about 15% of the lymphomas. Amplification and rearrangement of the p53, myc, and Ha-, Ki- and N-ras genes were not detected. Loss of heterozygosity on chromosome 4 at the multiple tumor suppressor 1 and 2 genes was observed in all types of malignancies. Allelic losses on chromosome 11 at the p53 locus were found in lymphoid, liver, and lung tumors, but they were absent from uterine tumors. Multiple oncogenic changes were often detected. The frequency of carcinogenic alterations was similar in spontaneous and radiation-induced lymphoid, liver, and uterine tumors. In radiation-induced lung adenocarcinomas, however, the incidences of many oncogenic changes were different from those found in their spontaneous counterparts. This suggests that different oncogenic pathways are activated during spontaneous and in utero γ-radiation–induced murine lung carcinogenesis. Mol. Carcinog. 21:100–110, 1998. © 1998 Wiley-Liss, Inc.     

Regulatory elements in the first intron of the mouse ha-ras gene

The Ha-ras gene is one of the three oncogenes (Ha-ras, Ki-ras, and N-ras) of the ras superfamily of small G proteins. The p21^ras proteins encoded by the ras genes are key proteins involved in the transduction of signals from membrane receptor-tyrosine kinases to downstream targets. The ras genes seem to play a ubiquitous role in the control of cell proliferation and cell differentiation. At the same time, ras genes may perform specific differentiated functions in certain cell types. Little is known about the regulation of expression of the Ha-ras gene. The first intron of the Ha-ras gene has been reported to be highly conserved between human and rodent. We investigated the role that this intron may play in the regulation of expression of Ha-ras. The promoter region of the Ha-ras gene exhibits characteristics of a housekeeping gene. Deletion analysis shows the existence of an enhancer-type element in the 5′ region of the first intron (intron 0). DNase I footprinting experiments reveal five sites that interact with nuclear proteins from fibroblast and epithelial cell lines. Deletion and site-directed mutagenesis of three of these sites show that two are involved in a positive effect and one in a negative effect on the regulation of expression of the mouse Ha-ras gene. © 1995 Wiley-Liss Inc.     

Complete cDNA sequence of the Ki-ras proto-oncogene in the liver of wild English sole (Pleuronectes vetulus) and mutation analysis of hepatic neoplasms and other toxicopathic liver lesions

A complete copy of Ki-ras b cDNA from English sole (Pleuronectes vetulus), a benthic marine flatfish, was cloned and sequenced. The percent identity between the predicted amino acid sequence of English sole and human Ki-ras b was 97%, whereas the percent identity between the English sole gene and rainbow trout or Rivulus Ki-ras b was 98%. Areas of amino-acid sequence conservation included codons 12, 13, and 61, the positions in which mutations are observed in ras cellular oncogenes in other species. The 5′ untranslated region (UTR), consisting of 217 nt, was not highly GC rich but contained four ATG start codons upstream of the major open reading frame. The 3′ UTR, containing 26 nt, was AU rich. Analysis of Ki-ras mutations was performed on a variety of necrotic, preneoplastic, and neoplastic lesions in livers from 13 English sole collected from contaminated waterways in Puget Sound, WA. Despite reports of Ki-ras mutations in hepatic tumors from other fish, no mutations in codons 12, 13, or 61 were found in hepatic lesions from English sole by direct DNA sequencing of polymerase chain reaction–amplified genomic DNA. Although mutations could exist at levels below the detection limits of this analysis, the results suggest that Ki-ras has a role in liver carcinogenesis that varies according to the fish species or carcinogen. Furthermore, future studies of the etiology of chemically induced cancer in feral English sole should consider mutations in other cancer-related genes, such as p53, Ha-ras, and N-ras. Mol. Carcinog. 23:207–216, 1998. © 1998 Wiley-Liss, Inc.     

Ki-ras and p53 mutations are early and late events,respectively, in urethane-induced pulmonary carcinogenesis in A/J mice

In the A/J strain of mice, urethane (ethyl carbamate) induces lung hyperplasia, adenoma, and adenocarcinoma in a time-dependent manner. These distinct morphological stages may correlate with sequential molecular genetic changes in this mouse model. To test this hypothesis, we investigated the presence of mutations involving Ki-ras and p53 in urethane-induced lung lesions in A/J mice at early and late stages of tumorigenesis. We precisely microdissected 40 lung lesions from paraffin-embedded sections. Ki-ras mutations around codon 61 and p53 mutations in exons 5–8 were identified by polymerase chain reaction-single-strand conformation polymorphism and DNA sequencing techniques. In 29 early-stage lung lesions classified as hyperplasias (seven) or adenomas (22), we observed 19 Ki-ras mutations (66%), including three silent mutations and one double mutation at different codons, and one silent p53 mutation (3.5%). In 11 late-stage adenomas, we identified nine activating Ki-ras mutations (82%) and four missense p53 mutations (36%). These results indicate that Ki-ras mutations arise early, whereas p53 mutations occur relatively late during the benign stages of urethane-induced lung carcinogenesis in A/J mice. © 1996 Wiley-Liss, Inc.     

No involvement of Ki-ras or p53 gene mutations in colitis-associated rat colon tumors induced by 1-hydroxyanthraquinone and methylazoxymethanol acetate

1-Hydroxyanthraquinone (1-HA), which is present in some herbs, and methylazoxymethanol (MAM) acetate, a metabolite of azoxymethane, show synergistic carcinogenicity in rat colon, and 1-HA induces ulcerative changes with simultaneous severe inflammation of the entire colon. In this study, mutations in Ki-ras (exons 1 and 2) and p53 (exons 4–7) were studied by polymerase chain reaction (PCR)–single-strand conformation polymorphism (SSCP) analysis. Of 18 adenomas and 38 adenocarcinomas induced in male F344 rats (52 tumors induced by 1-HA plus MAM acetate, three by 1-HA alone, and one by MAM acetate alone), no mutations in Ki-ras of p53 were detected under two conditions of PCR-SSCP analysis. Because human colon carcinomas from patients with ulcerative colitis have a very low incidence of Ki-ras mutation, this experimental system would be a good animal model of human colon carcinomas with ulcerative colitis and of human colon carcinomas without Ki-ras of p53 mutations. © 1995 Wiley-Liss Inc.     

High frequency and low specificity of ras gene mutations in rat Zymbal's gland tumors induced by 2-amino-3-methylimidazo[4, 5-f]quinoline

The activating mutations of all three ras genes in rat Zymbal'sgland tumors induced by a food mutagen, 2-amino-3-methyl-imidazo[4,5-f) inoline (IQ) were analyzed. DNA fragments of the Ha-ras,Ki-ras and N-ras oncogenes were amplified from formalin-fixedand paraffin-embedded tissues by the polymerase chain reaction(PCR) and analyzed for activating mutations involving codons12, 13 and 61 by oligoncleotide differential hybridization.All nine Zymbal's gland tumors examined, including three papillomas,were found to contain either an Ha-ras or Ki-ras mutation. Thesemutations were located in either codon 13 or 61 of Ha-ras, andin either codon 12 or 13 of Ki-ras. Of the nine mutations, threewere G     

N-methylnitrosourea—lnduced Ki-ras codon 12 mutations: Early events in mouse thymic lymphomas

N-Methylnitrosourea (NMU)—induced codon 12 Ki-ras mutations were analyzed in premalignant thymic lymphomas from C57BL/6J mice by using a selective polymerase chain reaction amplification strategy. The frequency of codon 12 Ki-ras mutations was 67% (16 of 24) in NMU-treated animals with premalignant stage I disease. Previously, animals with different stages of disease had been analyzed for cytogenetic changes and for mutations in the p53 tumor suppressor gene. The genetic changes observed were early-activating codon 12 G³⁵→A transition mutations of the Ki-ras gene, followed closely by trisomy 15 and infrequent mutation of the p53 gene late in tumor development. The consistent and early detection of Ki-ras mutations in NMU-treated animals but not in untreated controls suggests that the mutations result from direct carcinogen exposure. Alternate pathways of NMU-induced thymic lymphomagenesis were implicated. One pathway involved putative NMU-induced mutations in other, non-ras oncogenes that cooperate with trisomy 15 to produce similar T-cell tumors. The frequency of p53 gene mutations in human and murine T-cell tumors is similar but low. © 1995 Wiley-Liss, Inc.     

Characterization of mutant Ha-ras gene expression in transformed murine keratinocyte lines grown under in vitro and in vivo conditions

We investigated the Ha-ras and Ki-ras gene status, tumorigenicity, pathology, line derivation, and intercellular communication of 7,12-dimethylbenz[a]anthracene-initiated papilloma-, carcinoma-, and hyperplastic skin-producing cell lines to further characterize them. Six of nine tumor cell lines grown in vitro expressed both mutant and normal Ha-ras proteins, and three lines expressed only normal Ha-ras. However, when grown subcutaneously in nude mice, seven of the nine lines expressed both mutant and normal Ha-ras, one line expressed normal Ha-ras, and one line did not grow subcutaneously. One papilloma line, P2/15, appeared to have an inducible mutant Ha-ras gene, as it was expressed only in vivo. These findings suggest that mutant Ha-ras genes may be lost in only a minor population of tumor lines during growth in culture. Finally, we found that mutant Ha-ras gene expression was strongly correlated with tumorigenicity in nude mice and that intercellular communication was strongly correlated with the derivation of the lines. © 1996 Wiley-Liss, Inc.     

Infrequent mutation of ha-ras and p53 in rat mammary carcinomas induced by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine(PhlP) is the most abundant of the heterocyclic amines, a group of potent carcinogens contained in cooked meat and fish. Female F344 rats fed a diet containing 100 or 400 ppm PhIP developed mammary carcinomas within 104 or 52 wk, respectively, at the rate of 47% for each group; these carcinomas were examined for mutations in three members of the ras gene family and in the p53 gene. Single-strand conformation polymorphism (SSCP) analysis and direct sequencing demonstrated a G←A transition at the second position of Ha-ras codon 12, with the resultant substitution of glutamic acid for glycine, in two of 10 carcinomas induced by 100 ppm PhIP and in one of seven induced by the 400 ppm dose. No mutations in Ki-ras or N-ras were detected. cDNA polymerase chain reaction-SSCP analysis and direct sequencing demonstrated a G←Ttransversion at the third position of p53 codon 130, with the resultant substitution of asparagine for lysine, in one of the 10 carcinomas induced by 100 ppm of PhIP for which freshly frozen samples were available. PhlP-induced rat mammary carcinogenesis can be regarded as a unique system in that rat mammary carcinomas are negative for ras and p53 mutations. © 1994 Wiley-Liss, Inc.