Activation Of Ras Proto-oncogenes Research Articles

A rare point mutation in the Ras oncogene in hepatocellular carcinoma

The Ras gene is one of the oncogenes most frequently detected in human cancers, and codes for three proteins (K-, N-, and H-Ras). The aim of this study was to examine the mutations in codons 12, 13 and 61 of the three Ras genes in cases of human hepatocellular carcinoma (HCC). Paired samples of HCC and corresponding non-malignant liver tissue were collected from 61 patients who underwent hepatectomy. A dot-blot analysis was used to analyze the products of the polymerase chain reaction (PCR) amplification of codons 12, 13, and 61 of K-, N- and H-Ras for mutations. Only one mutation (K-Ras codon 13; Gly to Asp) was detected among the 61 patients. Interestingly, this patient had a medical history of surgery for both gastric cancer and right lung cancer. No mutations were found in codons 12 and 61 of K-Ras or codons 12, 13 and 61 of the N-Ras and H-Ras genes in any of the HCCs or corresponding non-malignant tissues. These findings indicated that the activation of Ras proto-oncogenes by mutations in codons 12, 13, and 61 does not play a major role in hepatocellular carcinogenesis.

Aumento da expressão do proto-oncogene ras no bócio multinodular: possível envolvimento na patogênese

A transformação neoplásica resulta de uma série de alterações genéticas, envolvendo ativação de proto-oncogenes e inativação de genes supressores tumorais. Ativação do proto-oncogene ras por mutações em ponto é a alteração genética mais freqüente em tumores espontâneos da tireóide. Avaliamos a expressão do gene ras no bócio nodular. Fragmentos de tecido tireoidiano normal e neoplásico foram coletados durante o ato cirúrgico, sendo que 79 pacientes tiveram diagnóstico histopatológico de bócio colóide e foram incluídos no estudo. O RNA total foi extraído pelo método de Trizol e o cDNA sintetizado através do Reverse Trancriptidase. Os genes H-ras e K-ras foram amplificados através de PCR com primers específicos. Do total da amostra, 62% apresentaram aumento da expressão de um dos genes ras estudados. Evidenciou-se aumento da expressão do H-ras em 9 dos 29 (31%) casos e do K-ras em 12 dos 32 (37,5%) tumores estudados. Os resultados demonstraram aumento da expressão do ras na doença nodular da tireóide e sugerem um papel importante desses genes na transformação neoplásica da tireóide.

Ras mutations are rare in solitary cold and toxic thyroid nodules.

Activation of ras proto-oncogenes as a result of point mutations is detectable in a significant percentage of most types of tumour. Similar to neoplasms of other organs, mutations of all three ras genes can be found in thyroid tumours. H-, K- and N-ras mutations have been detected in up to 20% of follicular adenomas and adenomatous nodules which were not functionally characterized. This raises the question as to whether ras mutations are specific for hypofunctional nodules and TSH receptor mutations for hyperfunctioning nodules. To investigate ras and TSH receptor mutations with respect to functional differentiation we studied 41 scintigraphically cold nodules and 47 toxic thyroid nodules. To address the likelihood of a somatic mutation we also studied the clonal origin of these tumours. Genomic DNA was extracted from nodular and surrounding tissue. Mutational hot spots in exons 1 and 2 of the H- and K-ras gene were PCR amplified and sequenced using big dye terminator chemistry. Denaturing gradient gel electrophoresis (DGGE) was used to verify sequencing results for the H-ras gene and to analyse the N-ras gene because its greater sensitivity in detecting somatic mutations. Clonality of nodular thyroid tissue was evaluated using X-Chromosome inactivation based on PCR amplification of the human androgen receptor locus. Monoclonal origin was detectable in 14 of 23 informative samples from cold thyroid nodules. In toxic thyroid nodules the frequency of clonal tissue was 20 in 30 informative cases. Only one point mutation could be found in the N-ras gene codon 61 (Gly to Arg) in a cold adenomatous nodule which was monoclonal. In toxic thyroid nodules no ras mutation was detectable. Our study suggests that ras mutations are rare in solitary cold and toxic thyroid nodules and that the frequent monoclonal origin of these tumours implies somatic mutations in genes other than H-, K- and N-ras.

Down-regulation of Lysyl Oxidase-induced Tumorigenic Transformation in NRK-49F Cells Characterized by Constitutive Activation of Ras Proto-oncogene

Several investigations have suggested a putative tumor suppressor role for lysyl oxidase because it is down-regulated in many human and oncogene-induced tumors. To address this issue we down-regulated the enzyme in normal rat kidney fibroblasts by stable transfection of its cDNA in an antisense orientation. The selected clones revealed an absence of lysyl oxidase and dramatic phenotypic changes, interpretable as signs of transformation. The antisense lysyl oxidase clones showed, indeed, loose attachment to the plate and anchorage-independent growth and were highly tumorigenic in nude mice. Moreover, we found an impaired response of the PDGF and IGF-1 receptors to their ligands. In particular, the transformed cells showed a down-regulation of both PDGF receptors and expressed the 105-kDa isoform of the IGF-1 beta receptor, which was not present in the normal control cells. The lack of response to PDGF-BB has been described as a feature of many ras-transformed phenotypes. Therefore, we looked at the status of the p21(ras). Indeed, we found a significantly higher level of active p21(ras) both during steady-state growth and prolonged starvation. Our data reveal new evidence for a tumor suppressor activity of lysyl oxidase, highlighting its particular role in controlling Ras activation and growth factor dependence.

Genetic analysis of Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes in 2′,3′-dideoxycytidine- and 1,3-butadiene-induced lymphomas in B6C3F1 mice

We have previously identified activation of ras proto-oncogenes and inactivation of tumor suppressor genes including p53, p16 INK4a and p15 INK4b in 2′,3′-dideoxycytidine (ddC)- and/or 1,3-butadiene (BD)-induced lymphomas derived from B6C3F1 (C57BL/6xC3H/He) mice, indicating that alterations of ras signaling pathway, p53 and pRb growth control pathways are important in the development of these chemically induced lymphomas. However, there is still a subset of tumors that displayed no changes in these genes. Thus, we investigated whether the Raf1, Mdm2, c-Myc, Cdc25a and Cdc25b proto-oncogenes, which are implicated in the ras or p53 or pRb pathways, are alternative oncogenic target genes. Analyses of gross genomic alterations by Southern blots failed to reveal rearrangement or amplification in any of the tumors examined. Frequent point mutations on the substrate binding domain of the Raf1 gene has been reported in 1-ethyl-1-nitrosourea (ENU)-induced murine lymphomas and lung tumors, along with a conspicuous lack of ras mutations [U. Naumann, I. Eisenmann-Tappe, U.R. Rapp, The role of raf kinases in development and growth of tumors, Recent Results Cancer Res., 143 (1997) 237–244]. To investigate whether Raf1 mutation is involved in our set of tumor especially those without ras mutations, the PCR-based single-strand conformation analyses (SSCA) and direct DNA sequencing were employed. No mutations but four genetic polymorphisms between C57BL/6 and C3H/He were found, with two of them reported as point mutations previously (op. cit.). The polymorphisms were utilized for allelic loss study of Raf1 locus. Losses of heterozygosity were found in six of 31 BD-induced lymphomas. These results indicate that genetic alterations of c-Myc, Cdc25, Raf1 and Mdm2 proto-oncogenes may not be involved in the development of ddC- and BD-induced lymphomas and the inactivation of tumor suppressor gene(s) located close to Raf1 gene might be important in the development of a subset of BD-induced lymphomas.

URINARY BLADDER TRANSITIONAL CELL CARCINOGENESIS IS ASSOCIATED WITH DOWN-REGULATION OF NF1 TUMOR SUPPRESSOR GENE IN VIVO AND IN VITRO

The NF1 gene product (neurofibromin) is known to act as a tumor suppressor protein by inactivating ras. The best documented factors involved in urinary bladder transitional cell carcinoma (TCC) are ras proto-oncogene activation and p53 suppressor gene mutations. This is the first study reporting alterations in NF1 gene expression in TCC. We examined NF1 gene expression in a total of 29 surgical urinary bladder TCC specimens representing grades 1 to 3 and in three cell lines, RT4, 5637, and T24 (representing grades 1 to 3, respectively). Decreased NF1 gene expression was observed in 23 of 29 (83%) TCC specimens as estimated by immunohistochemistry, the decrease being more pronounced in high-grade tumors. NF1 mRNA levels were markedly lower in TCC tissue compared with adjacent non-neoplastic urothelium, as studied by in situ hybridization for grade 3 TCC. Immunohistochemistry and Western blotting demonstrated that TCC cell lines expressed NF1 protein at different levels, expression being almost undetectable in T24 (grade 3) cells. Northern blotting for cell lines demonstrated reduced NF1 mRNA levels in grade 3 TCC cells. Reverse transcription polymerase chain reaction for cell lines and selected grade 2 and grade 3 tissue samples demonstrated NF1 type II mRNA isoform predominance in all samples studied. Our results show that both NF1 mRNA and protein levels are decreased in high-grade TCC, suggesting that alterations of NF1 gene expression may be involved in bladder TCC carcinogenesis.

DNA damage by mycotoxins

Mycotoxins are toxic fungal metabolites which are structurally diverse, common contaminants of the ingredients of animal feed and human food. To date, mycotoxins with carcinogenic potency in experimental animal models include aflatoxins, sterigmatocystin, ochratoxin, fumonisins, zearalenone, and some Penicillium toxins. Most of these carcinogenic mycotoxins are genotoxic agents with the exception of fumonisins, which is currently believed to act by disrupting the signal transduction pathways of the target cells. Aflatoxin B 1 (AFB 1), a category I known human carcinogen and the most potent genotoxic agent, is mutagenic in many model systems and produces chromosomal aberrations, micronuclei, sister chromatid exchange, unscheduled DNA synthesis, and chromosomal strand breaks, as well as forms adducts in rodent and human cells. The predominant AFB 1–DNA adduct was identified as 8,9-dihydro-8-( N 7-guanyl)-9-hydroxy–AFB 1 (AFB 1– N 7-Gua), which derives from covalent bond formation between C8 of AFB 1–8,9-epoxides and N 7 of guanine bases in DNA. Initial AFB 1– N 7-guanine adduct can convert to a ring-opened formamidopyrimidine derivative, AFB 1–FAPY. The formation of AFB 1– N 7-guanine adduct was linear over the low-dose range in all species examined, and liver, the primary target organ, had the highest level of the adduct. Formation of initial AFB 1– N 7-guanine adduct was correlated with the incidence of hepatic tumor in trout and rats. The AFB 1– N 7-guanine adduct was removed from DNA rapidly and was excreted exclusively in urine of exposed rats. Several human studies have validated the similar correlation between dietary exposure to AFB 1 and excretion of AFB 1– N 7-guanine in urine. Replication of DNA containing AFB 1– N 7-guanine adduct-induced G→T mutations in an experimental model. Activation of ras protooncogene has been found in AFB 1-induced tumors in mouse, rat, and fish. More strikingly, the relationship between aflatoxin exposure and development of human hepatocellular carcinoma (HHC) was demonstrated by the studies on the p53 tumor suppressor gene. High frequency of p53 mutations (G→T transversion at codon 249) was found to occur in HHC collected from populations exposed to high levels of dietary aflatoxin in China and Southern Africa. Furthermore, AFB 1-induced DNA damage and hepatocarcinogenesis in experimental models can be modulated by a variety of factors including nutrients, chemopreventive agents, and other factors such as food restriction and viral infection, as well as genetic polymorphisms.

Urinary Bladder Transitional Cell Carcinogenesis Is Associated with Down-Regulation of NF1 Tumor Suppressor Gene in Vivo and in Vitro

The NF1 gene product (neurofibromin) is known to act as a tumor suppressor protein by inactivating ras. The best documented factors involved in urinary bladder transitional cell carcinoma (TCC) are ras proto-oncogene activation and p53 suppressor gene mutations. This is the first study reporting alterations in NF1 gene expression in TCC. We examined NF1 gene expression in a total of 29 surgical urinary bladder TCC specimens representing grades 1 to 3 and in three cell lines, RT4, 5637, and T24 (representing grades 1 to 3, respectively). Decreased NF1 gene expression was observed in 23 of 29 (83%) TCC specimens as estimated by immunohistochemistry, the decrease being more pronounced in high-grade tumors. NF1 mRNA levels were markedly lower in TCC tissue compared with adjacent non-neoplastic urothelium, as studied by in situ hybridization for grade 3 TCC. Immunohistochemistry and Western blotting demonstrated that TCC cell lines expressed NF1 protein at different levels, expression being almost undetectable in T24 (grade 3) cells. Northern blotting for cell lines demonstrated reduced NF1 mRNA levels in grade 3 TCC cells. Reverse transcription polymerase chain reaction for cell lines and selected grade 2 and grade 3 tissue samples demonstrated NF1 type II mRNA isoform predominance in all samples studied. Our results show that both NF1 mRNA and protein levels are decreased in high-grade TCC, suggesting that alterations of NF1 gene expression may be involved in bladder TCC carcinogenesis.

In vitro activation of the human Harvey-ras proto-oncogene by aflatoxin B1.

Activation of ras proto-oncogenes occurs frequently in vivo in chemically induced rodent tumours, including rat hepatomas induced by aflatoxin B1. This study examines the in vitro activation of a human ras gene by this mycotoxin. A plasmid containing the human Ha-ras proto-oncogene, together with a neomycin resistance gene (pECneo), was incubated in vitro with a microsomal system generating aflatoxin B1 8,9-epoxide. Subsequent transfection of the plasmid into mouse NIH 3T3 fibroblasts, followed by G418 selection and s.c. injection of surviving cells into immunodeficient mice demonstrated that the proto-oncogene had acquired transforming capacity. Although a single tumour resulted from similar treatment of incubated unconjugated plasmid, no tumours were produced by a secondary round of transfections using DNA from this tumour. Selective PCR amplification of the human Ha-ras gene in extracted tumour DNA followed by sequencing demonstrated the presence of G-->T transversions either at the first or middle base of codon 12 in tumours resulting from transfection with the aflatoxin-B1-modified pECneo plasmid, but this was not detected in the single tumour resulting from transfection with the unmodified plasmid. Thus, although a mutation in the Ha-ras gene has not been reported for human primary hepatomas occurring in aflatoxin-exposed populations, metabolically activated aflatoxin B1 is capable of mutating this proto-oncogene to its oncogenic form in vitro. No mutations were observed in codon 61. It appears that, in contrast to the frequently reported G-->T transversions in codon 249 of the p53 gene in primary hepatomas in aflatoxin-exposed humans, the failure to detect Ha-ras mutations in these tumours is not due to an inability of aflatoxin B1 to activate this proto-oncogene. The G-->T transversions observed in this study contrast with the most frequent aflatoxin B1 in vivo induced mutations, G-->A transitions in the rat Ki-ras gene. Possible mechanisms for these differences are discussed.

Both K-ras and H-ras protooncogene mutations are associated with Harderian gland tumorigenesis in B6C3F1 mice exposed to isoprene for 26 weeks.

Isoprene is the 2-methyl analog of 1,3-butadiene, a genotoxic and carcinogenic compound in rats and mice. Male B6C3F1 mice were exposed to 0, 2200 or 7000 ppm isoprene by inhalation (6 h/day; 5 days/week) for 26 weeks. Following a 26-week recovery period, an increased incidence of Harderian gland (HG) neoplasms was observed at both concentrations. The present study was designed to characterize genetic alterations in the K-ras and H-ras protooncogenes in HG neoplasms. Mutations in K-ras and H-ras were identified by single-strand conformational analysis and direct sequencing of polymerase chain reaction (PCR) amplified DNA, isolated from paraffin-embedded sections of HG neoplasms. A higher frequency of ras mutations, in particular K-ras mutations, was detected in isoprene-induced neoplasms than in 1,3-butadiene-induced or control HG neoplasms. All of the isoprene-induced HG neoplasms exhibited activated K-ras (60%) or H-ras (40%) mutations. In contrast, ras mutations were detected in 69% of HG neoplasms from 1,3-butadiene exposed mice (14% K-ras and 55% H-ras) and in 56% of HG neoplasms obtained from control B6C3F1 mice (8% K-ras and 48% H-ras). The predominant mutations in isoprene-induced HG neoplasms, but not in previously or newly analysed 1,3-butadiene-induced HG neoplasms, consisted of A-->T transversions (CAA-->CTA) at K-ras codon 61 (15/30) and C-->A transversions (CAA-->AAA) at H-ras codon 61 (8/30). Two-thirds of the K-ras CTA mutations were detected in HG neoplasms from the 2200 ppm exposure group while one-third was present in the 7000 ppm group. Isoprene-induced HG neoplasms with K-ras or H-ras mutations had an elevated proliferating cell nuclear antigen (PCNA) index, compared to spontaneous HG neoplasms without ras mutations. The high frequency and specificity of the ras mutation profile suggest that ras protooncogene activation contributes to isoprene-induced HG tumorigenesis.

Modulation of alterations in p53 tumor suppressor gene and its association with activation of ras proto-oncogenes during chemoprevention of colon cancer

Previously, we reported (Carcinogenesis 15: 1317-1323, 1994) a high rate of activating point mutations in I ns proto-oncogenes in azoxymethane (AOM)-induced colon tumors, and a significant suppression of these mutations by dietary administration of chemopreventive agents, D,L-alpha-difluoromethylornithine (DFMO) and piroxicam. To understand the role of p53 tumor suppressor gene in chemoprevention of colon cancer and to study the association of p53 gene alterations with activation of ras genes, we determined point mutations in conserved regions (exons 5-9) of p53 gene and analyzed the occurrence of double event of ms activation acid p53 mutation. Groups of male F344 rats were fed the modified AIN-76A diet containing 0, 4000 ppm DFMO, or 150 ppm piroxicam and administered s.c. AOM at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle controls received s.c. equal volume of normal saline. Animals were sacrificed 32 weeks after the last AOM or saline injection and their grossly visible colon tumors were analyzed to determine p53 mutations by PCR amplification based single strand conformation polymorphism (SSCP) and direct DNA sequencing. Our results demonstrate that about 57% tumors from animals fed the control diet contained predominantly missense but also nonsense mutations, whereas only 30% tumors from animals on piroxicam diet, and none (0%) from animals fed the DFMO diet had similar mutations. Analysis of data revealed that about half of the tumors from animals on control diet possessed both ms and p53 mutations together, only 27% of colon tumors from animals on piroxicam diet and none of the tumors from animals on DFMO diet exhibited both ms and p53 mutations. These results indicate that the administration of piroxicam, a non-steroidal anti-inflammatory drug, and DFMO, a irreversible inhibitor of ornithine decarboxylase, may inhibit selective proliferation of initiated cells containing activated las and/or mutant p53. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing mutated ras and/or p53.

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, chi square 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.

MOLECULAR MARKERS AS INTERMEDIATE END-POINTS IN CHEMOPREVENTION OF COLON-CANCER - MODULATION OF RAS ACTIVATION BY SULINDAC AND PHENYLHEXYLISOTHIOCYANATE DURING COLON CARCINOGENESIS

Recent evidence suggests that activation of ras proto-oncogenes and inactivation of suppressor genes induce malignant phenotype in colonic cells. Thus the identification of clonal population of cells expressing activated ras may lead to a valuable intermediate biomarker to detect premalignant lesions amenable to chemoprevention. Previously, we demonstrated that sulindac inhibited the carcinogen-induced colon tumor development whereas phenylhexylisothiocyanate (PHITC) promoted the tumor outcome. The present study was conducted to investigate the effect of sulindac and PHITC on azoxymethane (AOM)induced activation of ras proto-oncogenes in order to explore the plausibility of using ras as an intermediate biomarker in chemoprevention of colon cancer. Male F344 rats were fed the AIN-76A diet containing 0, 320 ppm sulindac or 640 ppm PHITC and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt/week for 2 weeks. Vehicle control groups received s.c. equal volume of normal saline. Animals were sacrificed 52 weeks after AOM or saline treatment and their colonic mucosa and tumors were analyzed for mutations in codon 12 and 13 of K-ras and the expression of ras p21. As an alternative non-invasive approach, we developed a simple and sensitive one-step mutant-enriched PCR method to detect these genetic lesions in stools collected at 16, and again at 24 weeks after AOM treatment. AOM-induced G to A transitions were observed at the second nucleotide of 12th codon of K-ras substituting amino acid asp with wild-type gly. Sulindac not only suppressed the selective amplification of initiated cells possessing AOM-induced mutated K-ras codon 12, but significantly inhibited the AOM-induced expression of total and mutant ras-p21. PHITC did not exert any inhibitory effect on AOM-induced ras activation. Results indicated a strong correlation between ras activation and tumor outcome. Data suggest that ras activation may be a useful intermediate molecular marker in chemoprevention of colon cancer.

Dose-Dependent Carcinogenicity and Frequent Ki-ras Proto-oncogene Activation by Dietary N-Nitrosodiethylamine in Rainbow Trout

Dose-Dependent Carcinogenicity and Frequent Ki-ras Proto-oncogene Activation by Dietary N-Nitrosodiethylamine in Rainbow Trout. Hendricks, J. D., Cheng, R., Shelton, D. W., Pereira, C. B., and Bailey, G. S. (1994). Fundam. Appl. Toxicol. 23, 53-62.While the experimental data upon which current concepts in mechanistically based risk assessment and molecular epidemiology are grounded derive almost entirely from rodent models, fish models have several attributes (e.g., low background incidence, extremely low cost tumor studies, nonmammalian comparative status for extrapolation of mechanisms to humans) that make them valuable adjuncts for addressing these concepts. This report provides an initial characterization of the dose dependency of dietary N -nitrosodiethylamine (DEN) hepatocarcinogenicity in Shasta strain rainbow trout (Oncorhynchus mykiss) and the potential of DEN to elicit ras proto-oncogene activation in this species. Carcinogen was administered in the diet at five concentrations for 12 months. Necropsies were performed at 9, 12, and 18 months, the latter on fish maintained on control diet for 6 months after cessation of DEN exposure. The incidence of hepatic neoplasms at the lower dietary concentrations (⩽70 ppm) did not consistently exceed that for control groups, which were higher in this particular study (2%) than expected (historically 0.1%). For the higher DEN concentrations, a linear relationship between the hepatic tumor incidence (expressed as log odds, log [p/(1-p)], where p = proportion of fish bearing tumors), and the logarithm of total cumulative dose was observed, with response being independent of the length of time (9 or 12 months) during which the dose was accumulated. The dose-response curve for fish maintained an additional 6 months postexposure was shifted toward higher incidence but was parallel to the curve for fish killed at cessation of exposure. The model predicts that doubling the dose will produce somewhat more than a doubling of the odds (p/(100 - p) for tumor incidence and that the odds for lesions 6 months postexposure will be approximately double those at cessation of exposure. Comparison of these results with previous studies using rats suggests an overall similarity in dose-response curves, with trout being somewhat less sensitive than rats to DEN hepatocarcinogenesis. To examine the molecular basis for DEN carcinogenesis in this species, seven liver tumors induced separately by short-term DEN treatment were probed by 3′-mismatch primer polymerase chain reaction analysis for evidence of Ki-ras proto-oncogene activiting point mutations. A very high proportion (6/7) of tumors was found to carry codon 12 GGA → AGA mutations, whereas no codon 61 mutants were detected in this sample. These initial results differ from those reported using hepatic tumors from DEN-treated mice, which exhibit frequent Ha-ras codon 61 mutations [Richardson et al., Carcinogenesis 13, 1277-1279 (1992)] and rats, which appear not to carry DEN-activated ras alleles [Bauer-Hoffman et al., Carcinogenesis 11, 1875-1877 (1990)]. Thus the available oncogene data for the common carcinogen DEN do not suggest a simple, consistent oncogenic pathway or mutational spectrum useful in the molecular epidemiology of human cancers. Thus the available oncogene data for the common carcinoge DEN do not suggest a simple, consistent oncogenic pathway or mutational spectrum useful in the molecular epidemiology of human cancers.

Modulation of azoxymethane-induced mutational activation of ras protooncogenes by chemopreventive agents in colon carcinogenesis.

In our previous study, we demonstrated that azoxymethane (AOM) treatment significantly enhanced the expression of ras p21, the protein product of ras genes, and that the dietary administration of chemopreventive agents such as D,L-alpha-difluoromethylornithine (DFMO), a irreversible inhibitor of ornithine decarboxylase, and piroxicam, a non-steroidal anti-inflammatory drug (NSAID), exerted a significant inhibitory effect on AOM-induced ras p21 expression. In the present study, which is an extension of our earlier investigation, we have determined the effect of DFMO and piroxicam on mutational activation of ras protooncogenes during AOM-induced colon carcinogenesis. Groups of male F344 rats were fed the modified AIN-76A diet containing 0 or 150 p.p.m. piroxicam, or 4000 p.p.m. DFMO and administered s.c. AOM dissolved in normal saline at a dose rate of 15 mg/kg body wt, once weekly, for 4 weeks. Vehicle control groups received s.c. equal volumes of normal saline. Groups of animals were then killed at 0, 4, 16, 24 or 32 weeks after last AOM or saline injection. AOM-induced colon tumors and colonic mucosa from AOM treated as well as saline treated animals were analyzed for point mutations in K- and H-ras protooncogenes by a combination of polymerase chain reaction mediated restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing. Our results demonstrate that of the total 65 AOM-induced colon tumors analyzed, 45/50 (90%) obtained from AOM-treated animals fed the control diet, 4/11 (36%) from AOM-treated animals fed piroxicam diet, and 1/4 (25%) from AOM-treated animals fed the DFMO diet, contained single-point mutations occurring specifically at the second nucleotide of codon 12 which were identified exclusively as G to A transitions in case of K-ras, and G to A transitions and also G to T transversions in H-ras. Similar point mutations were identified in colonic mucosa of 21/30 (70%) of AOM-treated animals fed the control diet, 10/30 (33%) of AOM-treated animals fed piroxicam diet, and none of 30 (0%) of AOM-treated animals fed DFMO diet. These results indicate that the administration of piroxicam and DFMO may inhibit the selective amplification of AOM-induced initiated cells carrying mutated ras genes. Dietary DFMO exerted more pronounced inhibition of selective amplification of initiated cells containing AOM-induced mutant ras. Data suggest that determination of ras activation may be a useful marker for chemoprevention of colon cancer.

Dose-Dependent Carcinogenicity and Frequent Ki-ras Proto-oncogene Activation by Dietary N-Nitrosodiethylamine in Rainbow Trout

While the experimental data upon which current concepts in mechanistically based risk assessment and molecular epidemiology are grounded derive almost entirely from rodent models, fish models have several attributes (e.g., low background incidence, extremely low cost tumor studies, nonmammalian comparative status for extrapolation of mechanisms to humans) that make them valuable adjuncts for addressing these concepts. This report provides an initial characterization of the dose dependency of dietary N-nitrosodiethylamine (DEN) hepatocarcinogenicity in Shasta strain rainbow trout (Oncorhynchus mykiss) and the potential of DEN to elicit ras proto-oncogene activation in this species. Carcinogen was administered in the diet at five concentrations for 12 months. Necropsies were per formed at 9, 12, and 18 months, the latter on fish maintained on control diet for 6 months after cessation of DEN exposure. The incidence of hepatic neoplasms at the lower dietary concentrations (≤70 ppm) did not consistently exceed that for control groups, which were higher in this particular study (2%) than expected (historically 0.1%). For the higher DEN concentrations, a linear relationship between the hepatic tumor incidence (expressed as log odds, log [p/(1-p)1, where p = proportion of fish bearing tumors), and the logarithm of total cumulative dose was observed, with response being independent of the length of time (9 or 12 months) during which the dose was accumulated. The dose-response curve for fish maintained an additional 6 months postexposure was shifted toward higher incidence but was parallel to the curve for fish killed at cessation of exposure. The model predicts that doubling the dose will produce some what more than a doubling of the odds (pl(100 - p) for tumor incidence and that the odds for lesions 6 months postexposure will be approximately double those at cessation of exposure. Comparison of these results with previous studies using rats suggests an overall similarity in dose-response curves, with trout being somewhat less sensitive than rats to DEN hepato carcinogenesis. To examine the molecular basis for DEN carci nogenesis in this species, seven liver tumors induced separately by short-term DEN treatment were probed by 3'-mismatch primer polymerase chain reaction analysis for evidence of Ki-ras proto-oncogene activating point mutations. A very high proportion (6/7) of tumors was found to carry codon 12 GGA - AGA mutations, whereas no codon 61 mutants were detected in this sample. These initial results differ from those reported using hepatic tumors from DEN-treated mice, which exhibit frequent Ha-ras codon 61 mutations [Richardson et al., Carcinogenesis 13, 1277–1279 (1992)] and rats, which appear not to carry DEN-activated ras alleles [Bauer-Hoffman et al., Carcinogenesis 11, 1875–1877 (1990)]. Thus the available oncogene data for the common carcinogen DEN do not suggest a simple, consistent oncogenic pathway or mutational spectrum useful in the molecular epidemiology of human cancers.

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 peroxisome proliferator ciprofibrate, a hypolipidemic drug, were evaluated for the presence of transforming genes by the nude mouse tumorigenicity assay. As reported earlier, the tumors were not activated by a point mutation in codon 61 of H-ras. Two of the eight tumors examined contained a mutation in codon 13 or an H-ras gene mutated in codon 117. Screening of another 23 ciprofibrate-induced liver tumors by oligonucleotide hybridization analysis and direct DNA sequencing resulted in the identification of three tumor DNA samples with point mutations in codon 117 of the H-ras gene. In addition, another tumor sample contained a K-ras gene with a mutation in codon 61. Mutations in these codons have been seen only rarely in chemically induced liver tumors from this mouse strain. Of 15 spontaneous B6C3F1 liver tumors screened in the same manner, one exhibited a K-ras gene activated by a mutation in codon 13 and a second contained an H-ras gene activated by a mutation in codon 117. These ras gene mutations have not been reported previously from spontaneous liver tumors. The frequency and spectrum of ras oncogene mutations characterized in ciprofibrate-induced liver tumors differ significantly from the frequency and pattern identified in spontaneously occurring liver tumors. The results of this study with a limited number of samples suggest that ras protooncogene activation or activation of other protooncogenes that can be detected by the nude mouse tumorigenicity assay are not frequent events in the mechanism of carcinogenicity of the peroxisome proliferator ciprofibrate. However, the lower frequency and distinct pattern of H-ras mutations observed in these tumors disprove the assumption of promotion of spontaneous hepatocarcinogenesis by ciprofibrate.

Role of ras protooncogene activation in the formation of spsontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse : Devereux TR, Anderson MW, Belinsky SA. Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. Carcinogenesis 1991;12:299–303

Role of ras protooncogene activation in the formation of spsontaneous and nitrosamine-induced lung tumors in the resistant C3H mouse : Devereux TR, Anderson MW, Belinsky SA. Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709. Carcinogenesis 1991;12:299–303

GTP-binding proteins as oncogenes in human tumors.

Ras oncogenes are the most commonly known oncogenes in human tumors; their prevalence in different types of tumors and their role in the development of these tumors have been discussed in recent reviews. The authors have investigated the biochemical mechanism that accounts for the activation of ras proto-oncogenes to become oncogenes. In normal cells, the ras p21 cycles between the active guanosine triphosphate (GTP)-bound form and the inactive form, bound to guanosine diphosphate (GDP). The conversion of the GTP- to the GDP-bound form is catalyzed by a cellular protein referred to as GAP (GTPase activating protein). The mechanism by which the GTP-bound state is generated is not known; however, this mechanism must be much slower than the GAP-catalyzed reaction, so that most of the ras p21 in normal cells is in the GDP-bound state. Oncogenic mutants can be thought of as mutants that escape from this aspect of GAP function. The authors have used polymerase chain reaction (PCR) to amplify genomic DNA from over 300 tumor samples and have analyzed the frequency of mutations in Gs-alpha in codons 201 and 227.

Brief Report: GTP-Binding Proteins As Oncogenes in Human Tumors

Ras oncogenes are the most commonly known oncogenes in human tumors; their prevalence in different types of tumors and their role in the development of these tumors have been discussed in recent reviews (1,2). We have investigated the biochemical mechanism that accounts for the activation of ras proto-oncogenes to become oncogenes. In normal cells, the ras p21 cycles between the active guanosine triphosphate (GTP)bound form and the inactive form, bound to guanosine diphosphate (GDP). The conversion of the GTPto the GDP-bound form is catalyzed by a cellular protein referred to as GAP (GTPase activating protein) (3). The mechanism by which the GTP-bound state is generated is not known; however, this mechanism must be much slower than the GAP-catalyzed reaction, so that most of the ras p21 in normal cells is in the GDP-bound state. In ras-transformed cells, the oncogenic mutant ras protein exists in its GTP-bound state constitutively. This is because GAP fails to catalyze hydrolysis of GTP bound to these proteins. Oncogenic mutants can therefore be thought of as mutants that escape from this aspect of GAP function. Two types of GAP cDNA have been cloned from human placenta (4). Figure 1 shows the structure of the proteins encoded by these cDNAs. Type I GAP is a 116,000 Da protein, with a hydrophobic amino terminus, two SH2 regions (src-homology region 2), one SH3 region, and a catalytic domain comprising the C-terminal one-third of the protein (5). All cells express type I GAP, whereas type II, which lacks the first 180 amino acids of type I GAP and is generated by an alternative splicing mechanism, has only been detected in human placenta and in certain human cell lines, such as MRC5 diploid fibroblasts. Interaction of GAP with ras p21 may have consequences additional to conversion of the GTP-bound to the GDP-bound form. This possibility was raised by the