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     

Variant mutational activation of the K-ras oncogene in renal mesenchymal tumors induced in newborn F344 rats by methyl(methoxymethyl)nitrosamine

Renal mesenchymal tumors were induced at high incidence in F344rats by a single intraperitoneal injection of methyl(methoxymethyl)nitrosamine(DMN-OMe) within 48 h after birth. DNAs from 18 of 35 mesenchymaltumors contained transforming ras sequences in NIH3T3 transfectionassays: K-ras (17/18) or N-ras (1/18). Single-stranded conformationalpolymorphism analysis or dideoxy sequencing of polymerase chainreaction-amplified K-ras gene fragments revealed that theseneoplasms contained a variety of activating mutations in theK-ras oncogene. Alterations in codon 12 predominated and includedGGT GAT transitions, GGT GTT or TGT transversions, and previouslyreported insertion mutations, although some tumors expressedmore than one mutation and the pattern of mutations even variedwithin tumors. Mutations were also found in exons 2 and 3. Inaddition, tumor trans-plantability into syngeneic hosts correlatedpositively and significantly with K-ras activation. Renal mesenchymaltumors with transforming mutations in exon 1 were often successfullypassaged (10/12) while tumors which lacked mutations in exon1 were infrequently transplantable (2/14). While the observedbase substitutions in K-ras are consistent with adduct formation,the presence of insertion mutations and intratumor heterogeneityof alterations suggest that ras activation in DMN-OMe-inducedtumors is not necessarily an early event in tumorigenesis.     

Separation of transforming amino acid-substituting mutations in codons 12, 13 and 61 of the N-ras gene by constant denaturant capillary electrophoresis (CDCE)

We used high fidelity PCR and constant denaturant capillaryelectrophoresis (CDCE) [Khrapko et al. (1994) Nucleic AcidsRes., 22, 364–369] to separate wild type and differentmutant N-ras exon 1 and 2 sequences. The set of plasmids containingN-ras cDNA, wild type or mutant sequences representing all transformingamino acid-substituting single base pair changes in codons 12,13 (exon 1) and 61 (exon 2), were amplified using Pfu polymerasein a limited cycle polymerase chain reaction. One of the primersused for the amplification of each exon included a 40 nucleotideGC rich sequence that created high and low melting domains.The amplified fragments 151 bp (exon 1) and 150 bp (exon 2)were run on the CDCE with the ‘denaturant zone’temperature of the capillary corresponding to the melting temperatureof 111 bp (exon 1) and 110 bp (exon 2) low melting domains.The separation was achieved between wild type and mutant sequencesas homoduplexes in 15 out of 19 cases, as a single base substitutionalters the electrophoretic mobility of a partially melted doublestranded fragment. The denaturation and reannealing of wildtype and mutant fragments together created wild type/ mutantheteroduplexes. All the heteroduplexes were well resolved fromwild type homoduplex. In the current form mutant sequences weredetected at a frequency of 10^–3 in the presence of wildtype. This study has resulted in obtaining electrophoretic spectrumof different N-ras mutants on CDCE as homoduplexes as well asheteroduplexes.     

Ras proto-oncogene activation in liver and lung tumors from B6C3F1 mice exposed chronically to methylene chloride

Methylene chloride has been the subject of recent toxicologicaland carcinogenesis studies because of significant human exposureand widespread use in industrial processing, food preparationand agriculture. In this study, liver and lung tumors, inducedin female B6C3F1 mice by inhalation of 2000 p.p.m. methylenechloride (6 h/day, 5 days/week continuous exposure), were examinedfor the presence of activated rasproto-oncogenes. DNA was isolatedfrom 49 spontaneous and 50 methylene chloride-induced livertumors and screened by oligonucleotide hybridization of PCRamplified H-ras gene fragments for codon 61 mutations. In thechemically induced tumors, 38 mutations were detected, 16 Cto A transversions in base 1, 16 A to G transitions in base2 and 6 A to T transversions in base 2. This mutation profilewas similar to that identified for the H-ras gene in the spontaneousliver tumors and suggests that methylene chloride acts in liverby promoting cells with spontaneous lesions. Tumors in whichH-ras codon 61 mutations were not detected were examined forthe presence of transforming genes by the nude mouse tumorigenicityassay. Except for activated K-ras genes detected in DNA fromtwo methylene chloride induced tumors and one spontaneous tumor,no other transforming genes were identified. DNA from 54 lungtumors was screened by direct sequencing of PCR amplified DNAfragments of the K-ras gene for first and second exon mutations,and 12 mutations were identified, 5 in exon one and 7 in exon2. The low number of spontaneous tumors available in this studylimits the interpretation of the data, and thus the frequencyand spectrum of K-ras activation in the methylene chloride inducedtumors was not significantly different from that in the sevenspontaneous tumors analyzed. Since K-ras activation was notdetected in 80% of the tumors, the nude mouse tumorigenicityassay was used to examine the lung tumors for the presence ofother transforming genes. At present no transforming genes otherthan ras genes were identified in either liver or lung tumors.     

International comparison on ras gene mutations in latent prostate carcinoma

Latent carcinomas of the prostate, discovered at autopsy in men with no prior treatment for prostatic disease, were studied for ras proto-oncogene mutations. Subjects included 21 Japanese, 15 U.S. whites, 15 U.S. blacks, 20 Hawaiian Japanese and 10 Colombians. PCR and sequence-specific oligonucleotide hybridization identified mutations in 5 Japanese, in 1 Hawaiian Japanese, in 1 U.S. black, in 1 U.S. white and in 3 Colombians. The 5 Japanese tumor samples contained 3 point mutations in codon 12 of K-ras and 2 mutations in codon 12 of N-ras respectively. One tumor in a Hawaiian Japanese man also showed a K-ras point mutation at codon 12. Two Colombians and one U.S. black man had tumors with mutations at codon 61 of H-ras, while 1 Colombian showed an N-ras mutation at this codon. The overall frequency of ras gene mutations was low, but point mutations in codon 12 were most common in latent tumors of Japanese, who experienced the lowest incidence and mortality from this tumor. Latent tumors in men from ethnic groups with high mortality and incidence rates showed fewer ras mutations than the Japanese, and these were more likely to involve codon 61. This finding is consistent with prior studies of more aggressive clinical cancers in Japanese men that indicated a higher frequency of mutations at codon 61.     

Transformation of human epidermal keratinocytes with fission neutrons

The biological effects of exposures to high LET radiations haveparticular relevance to radiation protection and risk assessmentSince most cancers are of epithelial origin, it is importantto obtain a better understanding of radiationinduced oncogenictransformation in this cell type. Accordingly we have initiatedstudies to determine whether immortalized human epidermal keratinocytes(RHEK) can be transformed with high LET radiations. Exponentiallygrowing RHEK cells were treated with single doses (1, 10, 25,50 and 100 cGy) of 0.85 MeV fission neutrons from the Janusreactor. Neutron exposure led to the development of morphologicallyaltered cells and foci formation after 6 weeks at confluence.These transformed cultures grew with an increased saturationdensity, exhibited anchorageindependent growth and formed tumorshi athymlc mice. Single-strand conformatlonal polymorphism analysisand DNA sequencing demonstrated the absence of point mutationsin codons 12/13 and 61 in the Ha-ras, Ki-ras, or N-ras genesand exons 4–9 of the pS3 tumor suppressor gene. Thesestudies demonstrate that high LET radiations (fission neutrons)can transform immortalized human epithelial cells to a malignantphenotype that does notappear to involve mutations in eitherthe cellular p53 or ras genes.     

A Very Large Villous Adenoma with an Adjacent Cancer of the Rectum: An Informative Case for Testing the Proposed Molecular Basis of Colorectal Tumorigenesis

It is currently accepted that colorectal tumorigenesis resultsfrom accumulation of multiple mutations in certain genes. Thisconcept prompted us to search for possible mutations in theAPC, k-ras, and p53 genes in an advanced cancer coexisting witha large villous adenoma of the rectum in a 54-year-old patientwith no family history of colorectal cancer. Genomic DNA extractedfrom multiple subregions of the tumor and surrounding normalmucosa was studied by polymerase chain reaction (PCR) followedby single-strand conformation polymorphism (SSCP) analysis anddirect sequencing. Both the adenoma and carcinoma had abnormalPCR-SSCP for APC (exon 11) and k-ras, irrespective of the locationwithin the tumors. However, p53 abnormality (exon 7) was detectedonly in samples taken from the carcinoma. Subsequent sequencingrevealed a TTC to TAG mutation at codon 479 of APC, a GGT toGAT mutation at codon 12 of k-ras in both the adenoma and carcinoma,and a CGG to TGG mutation at codon 248 of p53 (exon 7) in thecarcinoma. These findings were in accord with the current conceptof colorectal tumor progression whereby genetic alteration ofAPC and k-ras occurs relatively early while that of p53 is ratherlate and is possibly a decisive event in relation to malignancy.     

Low incidence of H-, K- and N-ras oncogene mutations in cytological specimens of laryngeal tumours

Laryngeal cancer is a rare type of neoplasia, constituting approximately 2% of all human cancers. Mutations of the ras gene family is one of the main activating mechanisms in human cancer. Their involvement in head and neck cancer has been mainly demonstrated at the level of the overexpression whereas ras mutations in these cancers are rare in the Western world. In the present study we explored the incidence of codon 12-point mutation in the H-, K- and N-ras genes, in 41 laryngeal cytological specimens. These specimens corresponded to 19 benign and 22 malignant lesions of the larynx. Only two specimens carried a codon 12-point mutation in the K-ras gene (4.8%) while no mutation was detected in the H- and N-ras genes. K-ras mutations were detected in one benign and one malignant specimen. These results indicate low incidence of ras oncogene mutations in laryngeal cytological specimens.     

Activated N-ras oncogene in a transformant derived from a rat small intestinal adenocarcinoma induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole

DNAs from five intestinal adenocarcinomas induced by 2-aminodipyrido[1,2-a:3',2'-d]imidazole,which is present in broiled fish, were subjected to transfectionassay using NIH3T3 cells as recipients. The DNA from only oneadenocarcinoma induced a morphologically transformed focus.Rat N-ras sequences were detected in the primary transformantand in three tested secondary transformants. In the activatedN-ras oncogene, a G—T transversion at the first letterof codon 12 was detected. The original tumor DNA did not hybridizewith the oligonucleotide representing the mutated allele, butdid hybridize with the one representing the normal allele. Fromthese data we concluded either that the activation of the N-rasoncogene had occurred during the transfection or that the activatedN-ras oncogene had been present in a minor population of cellsin the original tumor.     

Low frequency of H-ras mutations in hepatocellular adenomas and carcinomas and in hepatoblastomas from B6C3F1 mice exposed to oxazepam in the diet

Oxazepam has been the subject of recent toxicological and carcinogenesisstudies because it is a commonly prescribed tranquilizer andhas been shown to cause tumors in rodents. In this study, maleand female B6C3F1 mice receIved 0,125, 2500 or 5000 p.p.m. oxazepamin the diet for up to 2 years. Hepatocellular adenomas and carcinomas,as well as hepatoblastomas, which developed in these mice, wereexamined for the presence of activated ms proto-oncogenes. DNAwas Isolated from 20 or more tumors from each exposure groupand analyzed by oligonudeotide hybridiza tion, single-strandedconformation polymorphism analysis and direct sequencing ofPCR-ampllfied H-ms gene fragments for codon 61 mutations. Thirteenof 37 (35%) hepatocellular adenomas and carcinomas from the125 p.p.m. exposure group had mutations in codon 61, while mutationswere detected in only 2 of 25 or 8% of the liver tumors fromthe 2500 p.p.m. exposure group and none of the 22 tumors fromthe 5000 p.p.m. group. This compares to 63% of 126 historIcalcontrol liver tumors and 55% of 20 liver tumors from unexposedB6C3F1 mice in this study. In addition, 12 hepatoblastomas fromthe two high dose groups were examined for H-ras mutations atcodon 61, but none were detected. No tumor DNAs from any ofthe exposure groups tested had mutations In codons 12, 13 or117 of the H-ras gene or codons 12 or 13 of the K-ras gene,the other known hotspots for ras activation in mouse liver tumors.These results, together with those from the National ToxicologyProgram study showing no evidence of cytotoxicity or genotoxicityby oxazepam, suggest that oxazepam preferentially promotes cellsthat have activating lesions other than ras.     

ACCELERATED PAPER: Cloning, genetic mapping and expression studies of the rat Brca1 gene

The breast cancer gene BRCA1 has previously been cloned fromboth human and mouse. We cloned a fragment of the rat Brca1homologue in order to map it and explore its biological function.Partial cDNA fragments of the rat Brca1 homologue were isolatedby RT-PCR. Sequence analysis revealed that the RING-finger domainis well conserved among rat, mouse and human. Rat Brcal mRNAwas expressed in most tissues studied with the highest levelin testis, consistent with studies in human and mouse. Next,intron 6-containing DNA fragments were amplified by PCR fromWKY and WF strains. The splicing sites between exon 6 and exon7 are conserved between rat and human. Partial sequencing ofthe rat Brca1 intron 6 revealed a polymorphism of a pentanucleotideTTTTG repeat between the WKY and WF strains. With this intragenicmicrosatellite marker, we were able to map precisely the ratBrcal gene to chromosome 10 using a genetic linkage study of(WKYxWF)F1xWF baccross rats. Brca 1 cosegregates with markerBAND3A, and is flanked by R5123 and R5842. Using this polymorphicmarker, we also investigated the loss of heterozygosity (LOH)of the Brcal microsatellite marker in carcinogen- or radiation-inducedmammary carcinomas in (WFxF344)F1 female rats. No LOH or somaticmicrosatellite instability was detected in 18 DMBA-induced tumorsstudied. Only one LOH of the F344 allele was observed in 26radiation-induced tumors tested. Ribon-uclease protection assaysdemonstrated that Brca1 mRNA levels are similar in normal ratmammary glands and mammary carcinomas of various etiologies,including those induced by DMBA, NMU, activated-neu and activated-rasoncogenes.     

Frequent p53 alterations but low incidence of ras mutations in UV-B-induced skin tumors of hairless mice

We have investigated UV-B-induced skin tumors of hairless SKH-HRAmice for alterations in the p53 gene and for mutations in eitherof the three ras genes. Out of 32 tumors screened, only onecontained a ras mutation, i.e. in codon 12 of the K-ras gene.Alterations in the p53 gene were much more abundant, as illustratedimmunohistochemically by the accumulation of p53 protein in75% of the tumor sections examimed. Immunoreactivity was observedprimarily in the proliferative cell compartment, but no clearcorrelation between p53 staining in tumor cells and histologicalparameters for malignancy was observed. Subsequent sequenceanalysis showed that point mutations in the p53 gene are detectablein 30% (nine out of 30) of the skin tumors examined. The majorityof the mutations are located in codons 267 and 272, most likelyoriginating from UV-B-induced photo-adducts at dipyrimidinesites in the non-transcribed strand. Codon 272 corresponds tothe human codon 278, which is also a hotspot for p53 mutationsin human codon 273, which does not contain a dipyrimidine site,but represents a CpG hotspot for p53 mutations in internal malignancies.Our results demonstrate that this hairless mouse model for UV-inducedskin cancer corresponds closely to human non-melanoma skin cancerswith respect to mutations in the p53 gene.     

N-ras oncogene expression changes the growth characteristics of human melanoma in two independent SCID-hu mouse models

Fifteen percent of all human melanomas carry mutations in ras genes, the majority of which are located in codon 61 of the N-ras gene. However, the biological significance of these mutations is as yet unknown. In this study, we investigated the influence of N-ras oncogene products mutated in codon 61 on the growth characteristics of human melanoma in vivo by establishing 2 SCID-hu mouse xenotransplantation models. Tumors grown in SCID mice injected with human melanoma carrying activated N-ras genes were significantly larger (p < 0.004) than tumors grown in animals injected with the appropriate control transfectants. Additionally, tumors with N-ras point mutations clearly showed a more pleomorphic phenotype than the control groups. Our results, obtained in 2 independent SCID-hu xenotransplantation models, suggest that mutated N-ras oncogene expression may be an important factor influencing growth characteristics of human melanoma without altering metastatic potential. These novel in vivo model systems provide a tool for further study of the biology of mutated ras in melanoma and should also prove useful for testing new and improved treatment strategies for human melanoma carrying mutated ras genes. © 1996 Wiley-Liss, Inc.     

Ras oncogene activation during hepatocarcinogenesis in B6C3F1 male mice by dichloroacetic and trichloroacetic acids

Dichloroacetic (DCA) and trichloroacetic (TCA) acids, two majorby-products formed during chlorine disinfection of drinkingwater, increase the incidence of tumors in B6C3F1 mice by 6-and 3-fold respectively. In order to understand better the mechanismby which these two compounds induce liver tumors, the incidenceand spectrum of mutations in the K- and H-ras proto-oncogenesin these tumors were analyzed. DNA from spontaneous, DCA- andTCA-induced liver tumors from B6C3F1 male mice was evaluatedfor point mutations in exons 1, 2 and 3 of the two genes bysingle-stranded conformation polymorphism. Results demonstrateda similar incidence of mutations for exon 2 of H-ras in spontaneouscarcinomas (58%), and in carcinomas induced by DCA 3.5 g/l (50%),1.0 g/1(48%) and TCA 4.5 g/l (45%). Only four samples showedmutations in the other exons of H-ras or in K-ras. Sequenceanalysis of spontaneous tumor samples with second exon H-rasmutations 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-rasmutations from DCA-treated mice revealed a H-61 change fromCAA to AAA in 21 % at 3.5 g/l and 16% at 1.0 g/l. CAA to CGAwas observed in 50% of tumors from mice given DCA 3.5 or 1.0g/l, and CAA to CTA was present in 29% and 34% of the two dosagegroups respectively. Interestingly, TCA showed the same mutationalspectrum as the spontaneous liver tumors. The data indicatesthat induction of liver carcinoma by DCA and TCA involves activationof the H-ras proto-oncogene at a frequency similar to that observedin spontaneous tumors. However, the mechanism(s) for inducinghepatocellular carcinoma does not appear to be identical forDCA and TCA.     

Analysis of activated protooncogenes in B6C3F1 mouse liver tumors induced by ciprofibrate, a potent peroxisome proliferator

Liver tumors from B6C3F1 mice induced by the potent peroxisomeproliferator ciprofibrate, a hypolipidemic drug, were evaluatedfor the presence of transforming genes by the nude mouse tumorigenicityassay. As reported earlier, the tumors were not activated bya point mutation in codon 61 of H-ras. Two of the eight tumorsexamined contained a mutation in codon 13 or an H-ras gene mutatedin codon 117. Screening of another 23 ciprofibrate-induced livertumors by oligonucleotide hybridization analysis and directDNA sequencing resulted in the identification of three tumorDNA samples with point mutations in codon 117 of the H-ras gene.In addition, another tumor sample contained a K-ras gene witha mutation in codon 61. Mutations in these codons have beenseen only rarely in chemically induced liver tumors from thismouse strain. Of 15 spontaneous B6C3F1 liver tumors screenedin the same manner, one exhibited a K-ras gene activated bya mutation in codon 13 and a second contained an H-ras geneactivated by a mutation in codon 117. These ras gene mutationshave not been reported previously from spontaneous liver tumors.The frequency and spectrum of ras oncogene mutations characterizedin ciprofibrate-induced liver tumors differ significantly fromthe frequency and pattern identified in spontaneously occurringliver tumors. The results of this study with a limited numberof samples suggest that ras protooncogene activation or activationof other protooncogenes that can be detected by the nude mousetumorigenicity assay are not frequent events in the mechanismof carcinogenicity of the peroxisome proliferator ciprofibrate.However, the lower frequency and distinct pattern of H-ras mutationsobserved in these tumors disprove the assumption of promotionof spontaneous hepatocarcinogenesis by ciprofibrate.     

Detection of activated proto-oncogenes in N-nitrosodiethylamine-induced liver tumors: a comparison between B6C3F1 mice and Fischer 344 rats

DNA from B6C3F₁ mouse and Fischer 344 rat liver tumors inducedby N-nitrosodiethylamine (DEN) were examined for the abilityto induce morphological transformation of NIH3T3 cells. DNAsfrom 14 of 33 of the mouse liver tumors induced by a singleinjection of DEN at 12 or 15 days of age were positive in thisassay while DNA from only one of 28 DEN-induced rat liver tumorswas active. Southern blot analysis of the NIH3T3 transformantsderived from the mouse liver tumors revealed amplified and/orrearranged restriction fragments homologous to the H-ras proto-oncogene.DNA from two independent foci induced by the rat tumor DNA didnot hybridize to probes for members of the ras gene family orc-raf. Activating mutations in the H-ras genes from the DEN-inducedmouse liver tumors were characterized by selective oligonucleotidehybridization and the detection of a new XbaI restriction siteby Southern blot analysis. In activated H-ras genes from theDEN-induced mouse liver tumor DNA, seven of 14 had a CGAT transversionat the first base of the 61st codon, three of 14 had an ATGCtransition and four of 14 had the ATTA transversion at the secondbase of codon 61. This spectrum of mutations is very similarto that recently observed in activated H-ras genes found inspontaneously occurring B6C3F₁ mouse liver tumors. Taken together,the data suggest that the DEN-induced rat and mouse liver carcinogenesismay involve genetic targets other than or in addition to theH-ras gene.     

Mutations in ras oncogenes: Rare events in ultraviolet B radiation-induced mouse skin tumorigenesis

The activation of ras proto-oncogenes by point mutation in a broad spectrum of clinical malignancies and experimentally induced tumors suggests their critical role in cancer induction. To determine whether the activation of ras proto-oncogenes by point mutation also contributes to ultraviolet B radiation (UVB)-induced skin tumorigenesis and whether this event is responsible for the different tumorigenic potentials of UVB radiation in different mouse strains, we analyzed the skin tumors induced by UVB in SKH-1 hairless and C3H mice for specific mutations in the Ha-, Ki-, and N-ras oncogenes. With the same UVB irradiation protocol, the latency period for tumor appearance was longer in C3H mice than in SKH-1 hairless mice. In addition, tumor incidence and multiplicity were also significantly higher (P < 0.001, χ² and Wilcoxon rank sum tests) in SKH-1 hairless mice compared with C3H mice. None of the 30 skin tumor specimens (15 from each mouse strain) analyzed by polymerase chain reaction (PCR) amplification of specific codons followed by dot-blot hybridization with specific probes contained mutation in codons 13 of Ha-ras; 12, 13, and 61 of Ki-ras; or 12 and 13 of N-ras. However, three of the 15 tumors in SKH-1 hairless mice showed either a G³⁵ → A or G³⁵ → T transition at second position of Ha-ras codon 12. Interestingly, one of these tumors (with a G³⁵ → A transition) also harbored an A¹⁸² → G mutation at second position of Ha-ras codon 61. None of the tumors from C3H mice showed mutations in codons 12 or 61 of the Ha-ras oncogene. With regard to codon 61 of the N-ras oncogene, six tumors from SKH-1 hairless mice and 10 tumors from C3H mice showed an A¹⁸³ → T transversion. While G³⁵ → A or G³⁵ → T transition detected by PCR and dot-blot hybridization was confirmed by sequencing, the mutations identified similarly at codon 61 in either the Ha- or N-ras oncogenes could not be verified by sequencing of PCR-amplified products subcloned into plasmid vectors. With the exception of the low incidence of Ha-ras oncogene mutations at codon 12 in SKH-1 hairless mouse skin tumors induced by UVB, the striking absence of mutations in the Ha-, Ki-, and N-ras oncogenes in UVB-induced mouse skin tumors suggests that ras oncogene mutations are rare and thus are not an initiating event in photocarcinogenesis. © 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.     

Absence of p53 mutations and various frequencies of ki-ras exon 1 mutations in rat hepatic tumors induced by different carcinogens

Mutations of p53 and Ki-ras exon 1 were investigated in rat hepatic lesions induced by four kinds of hepatocarcinogenic protocols: continuous feeding of 3′-methyl-4-dimethylaminoazobenzene (3′-Me-DAB), daily intraperitoneal injection of aflatoxin B, (AFB,), and the Soft and Farber regimen (Nature 236:701–703, 1976), in which diethylnitrosamine (DEN) or nitrosomethylurea (NMU) was used as initiating agents. DNA from microdissected tissue sections was amplified by the polymerase chain reaction (PCR) directly using primers for p53 exons 5–8 and Ki-ras exon 1 and analyzed for mutations by denaturing gradient gel electrophoresis (DGGE) or constant denaturant gel electrophoresis (CDGE). One or both of the p53 PCR primers were located within introns to prevent amplifying the p53 processed pseudogenes. In a total of 59 hepatocellular carcinomas (HCCs), no p53 aberrations were detected, indicating that p53 mutations are not very important in rat hepatic carcinogenesis. On the other hand, Ki-ras codon 12 mutations were found at low frequency in HCCs, hyperplastic foci, and cholangiof ibroses induced by 3′-Me-DAB and by AFB! but not in the lesions induced by the Solt and Farber regimen. Although Ki-ras codon 12 mutations are generally infrequent in rat hepatic tumors, their incidence thus appears to vary depending on the carcinogen used for their induction. © 1994 Wiley-Liss, Inc.