Oncogenic Ras Mutations Research Articles

Emerging Issues in Mouse Liver Carcinogenesis

The mouse liver is the primary target site for carcinogenesis of more than 200 chemicals (including pesticides, food additives, pharmaceuticals, and industrial intermediates) tested in long-term toxicity safety assessment assays. Mouse liver tumors develop through defined morphological stages (similar to those found in other species) whether their origin is of undetermined etiology (spontaneous) or induced by chemicals. The morphologic type of hepatocytes in the various stages of hepatocarcinogenesis is sometimes associated with the specific inducing agent. Liver tumors developing in toxic livers often have more benign appearances and may progress to carcinomas at a slower rate than tumors developing in histologically normal livers. Specific tumors, dependent on the inducing chemical, may regress under defined protocols. Genotoxic and nongenotoxic mouse hepatocarcinogens each may induce tumors of either high malignant or low malignant potential. Liver tumors with specific H-ras oncogene mutations may appear morphologically and biologically similar to those without proven ras mutations. Thus, distinguishing mechanism of carcinogenesis by liver tumor morphology and mutation spectra may be difficult. Additionally, the presence of liver tumors with a morphology and a ras oncogene mutation spectrum characteristic of spontaneous tumors in histologically normal livers of mice exposed to a nongenotoxic test chemical may indicate promotion of spontaneous hepatocarcinogenesis by one of several potential mechanisms. Further research into the mechanisms responsible for the increased incidences of liver tumors in mice exposed to test chemicals could enhance human cancer risk assessments.

Massive programmed cell death in intestinal epithelial cells induced by three-dimensional growth conditions: suppression by mutant c-H-ras oncogene expression.

Deregulation of molecular pathways controlling cell survival and death, including programmed cell death, are thought to be important factors in tumor formation, disease progression, and response to therapy. Studies devoted to analyzing the role of programmed cell death in cancer have been carried out primarily using conventional monolayer cell culture systems. However the majority of cancers grow as three-dimensional solid tumors. Because gene expression, and possibly function, can be significantly altered under such conditions, we decided to analyze the control and characteristics of cell death using a compatible three-dimensional tissue culture system (multicellular spheroids) and compare the results obtained to those using two-dimensional monolayer cell culture. To do so we selected for study an immortalized, but nontumorigenic line of rat intestinal epithelial cells, called IEC-18, and several tumorigenic variants of IEC-18 obtained by transfection with a mutant (activated) c-H-ras oncogene. The rationale for choosing these cell lines was based in part on the fact that intestinal epithelial cells grow in vivo in a monolayer-like manner and form solid tumors only after sustaining certain genetic mutations, including those involving the ras gene family. We found that the IEC-18 cells, which grow readily and survive in monolayer cell culture, undergo massive cell death within 48-72 h when cultured as multicellular spheroids on a nonadhesive surface. This process was accompanied by a number of features associated with programmed cell death including chromatin condensation (Hoechst 33258 staining) apoptotic morphology, DNA degradation, and a virtual complete loss of colony forming (clonogenic) ability in the absence of apparent membrane damage as well as accumulation of lipid containing vacuoles in the cytoplasm. Moreover, enforced over-expression of a transfected bcl-2 gene could prevent this cell death process from taking place. In marked contrast, three different stably transfected ras clones of IEC-18 survived when grown as multicellular spheroids. In addition, an IEC cell line (called clone 25) carrying its mutant transfected ras under a glucocorticoid inducible promoter survived in three-dimensional culture only when the cells were exposed to dexamethasone. If exposure to dexamethasone was delayed for as long as 48 h the cells nevertheless survived, whereas the cells became irreversibly committed to programmed cell death (PCD) if exposed to dexamethasone after 72 h. These results suggest that intestinal epithelial cells may be programmed to activate a PCD pathway upon detachment from a physiologic two-dimensional monolayer configuration, and that this process of adhesion regulated programmed cell death (ARPCD) can be substantially suppressed by expression of a mutant ras oncogene.(ABSTRACT TRUNCATED AT 400 WORDS)

Absence of ras Mutations and Low Incidence of p53 Mutations in Renal Cell Carcinomas Induced by Ferric Nitrilotriacetate

Renal cell carcinomas induced in male Wistar rats by iron chelate of nitrilotriacetate (Fe‐NTA) were examined for mutations in ras oncogenes and p53 tumor suppressor gene. Fourteen primary tumors and two metastatic tumors from 11 animals were evaluated. Exons 1 and 2 of the H‐, K‐, and N‐ras genes were amplified by polymerase chain reaction (PCR), and the presence of mutations was examined by direct sequencing. Exon 5 through exon 7 of p53 gene, including the 3’half of the conserved region II and the entire conserved region III through V, were surveyed for point mutations by PCR‐single stranded conformation polymorphism (SSCP) analysis. Direct sequencing of the ras genes showed no mutations in codon 12, 13, or 61 among the tumors evaluated. SSCP analysis of p53 gene exon 6 indicated conformational changes in two primary tumors. One tumor had a CCG‐to‐CTG transition at codon 199, and the other had an ATC‐to‐ATT transition at codon 229 and two nonsense C‐to‐T transitions. These results suggest that neither ras genes nor p53 gene play a major role in the development of renal cell carcinomas induced by Fe‐NTA.

Relating aromatic hydrocarbon-induced DNA adducts and c-H-ras mutations in mouse skin papillomas: the role of apurinic sites.

Mouse skin tumors contain activated c-H-ras oncogenes, often caused by point mutations at codons 12 and 13 in exon 1 and codons 59 and 61 in exon 2. Mutagenesis by the noncoding apurinic sites can produce G-->T and A-->T transversions by DNA misreplication with more frequent insertion of deoxyadenosine opposite the apurinic site. Papillomas were induced in mouse skin by several aromatic hydrocarbons, and mutations in the c-H-ras gene were determined to elucidate the relationship among DNA adducts, apurinic sites, and ras oncogene mutations. Dibenzo[a,l]pyrene (DB[a,l]P), DB[a,l]P-11,12-dihydrodiol, anti-DB[a,l]P-11,12-diol-13,14-epoxide, DB[a,l]P-8,9-dihydrodiol, 7,12-dimethylbenz[a]anthracene (DMBA), and 1,2,3,4-tetrahydro-DMBA consistently induced a CAA-->CTA mutation in codon 61 of the c-H-ras oncogene. Benzo[a]pyrene induced a GGC-->GTC mutation in codon 13 in 54% of tumors and a CAA-->CTA mutation in codon 61 in 15%. The pattern of mutations induced by each hydrocarbon correlated with its profile of DNA adducts. For example, both DB[a,l]P and DMBA primarily form DNA adducts at the N-3 and/or N-7 of deoxyadenosine that are lost from the DNA by depurination, generating apurinic sites. Thus, these results support the hypothesis that misreplication of unrepaired apurinic sites generated by loss of hydrocarbon-DNA adducts is responsible for transforming mutations leading to papillomas in mouse skin.

Biochemical and growth-modulatory effects of the new S-adenosylmethionine decarboxylase inhibitor CGP 48664 in malignant and immortalized normal human breast epithelial cells in culture.

CGP 48664 [4-aminoindanon-1-(2'-amidino)hydrazone dihydrochloride monohydrate] is a newly introduced inhibitor of S-adenosylmethionine decarboxylase (SAMDC) with increased selectivity of action and reduced toxicity. We analyzed the biochemical and antiproliferative effects of this compound in a panel of hormone-dependent (3 clones of MCF-7, T47D) and -independent (MDA-MB-231, BT-20) human breast cancer cell lines in culture. For comparison, we also tested its effects in the spontaneously immortalized human breast epithelial cell line MCF-10A. All cell lines were highly sensitive to the growth-inhibitor effect of CGP 48664 with an IC50 between 0.1 and 0.5 microM. A dose-dependent bell-shaped increase in SAMDC was observed in normal and malignant breast cells resulting from enzyme stabilization by the inhibitor as supported by Western blot analysis. While ornithine decarboxylase (ODC) activity consistently increased, the effect of CGP 48664 on spermidine/spermine N'acetyltransferase (SSAT) was variable in the breast cancer cell lines. In contrast, the inhibitor consistently reduced SSAT activity level in the MCF-10A cell line and its derivative partially transformed by a mutated ras oncogene. As expected cellular putrescine levels were markedly increased by CGP 48664 administration, whereas spermidine and spermine contents were reduced. However, the degree of reduction was usually only moderate. Furthermore, exogenous polyamine administration was relatively ineffective in rescuing the antiproliferative effect of CGP 48664 in MCF-7 cells, while exerting a more complete rescue in the MDA-MB-231 cell line. We conclude that CGP 48664 exerts a potent growth-inhibitory effect on mammary cells in culture. However, its action may not always be entirely mediated through the polyamine pathway.

Advances in pathobiological research on lung carcinoma: Noguchi M. Department of Pathology, National Cancer Center Res. Inst., 5-1-1 Tsukiji, Chuo-ku, Tokyo 104. Jpn J Cancer Chemother 1994;21:2549–2554

Histological examination revealed that many peripheral type papillary adenocarcinomas appear to develop from atypical adenomatous hyperplasia (AAH), which can be called adenoma or in situ adenocarcinoma, and progress stepwise. Molecular-biologically, loss of heterozygosities of 3, 11 and 17 chromosomes, point mutation of ras oncogene and p53 anti-oncogene, amplification of myc oncogene, and overexpression of erbB2 oncogene are related to lung cancer development. Especially, ras and p53 gene abnormalities are closely associated with poor prognosis of lung adenocarcinoma. Future molecular-biological examinations should focus on AAH and/or early stage adenocarcinoma of the lung, in order to clarify the gene abnormality at the early stage of lung carcinogenesis.

Vinorelbine (Navelbine) in the adjuvant and neoadjuvant treatment of non-small cell lung cancer: Viallet J, Ayoub J, Rousseau P, Souhami L, Hohneker J, Shepherd F. Department of Oncology, Montreal General Hospital, 1650 Cedar Ave, Montreal, Que. H3G 1A4. Semin Oncol 1994;21:Suppl 10:64–72

Several Canadian centers are studying the favorable activity and toxicity profile of vinorelbine (Navelbine; Burroughs Wellcome Co, Research Triangle Park, NC; Pierre Fabre Médicament, Paris, France) in current and future trials of adjuvant and neoadjuvant treatment of non-small cell lung cancer. In locally advanced, unresectable disease, the 10-week regimen in cisplatin 100 mg/m2 during weeks 1 and 5, vinorelbine 30 mg/m2 weekly for 5 weeks with a 50% dose reduction planned for week 2 only, and accelerated fractionation thoracic irradiation during weeks 7 to 10 (30 fractions of 2 Gy in 4 weeks, once daily during weeks 7 and 8, and twice daily during weeks 9 and 10). Preliminary data on 17 patients who have completed treatment to date show it has been well tolerated, with only four cases of grade 3 nonhematologic toxicities. Favorable results from combined therapy with cisplatin and vinorelbine in advanced disease have led the National Cancer Institute of Canada Clinical Trials Group to consider testing adjuvant cisplatin and vinorelbine in completely resected non-small cell lung cancer. Surgically staged patients with T2, N0 and T1-2N1 tumors will be stratified according to nodal status and presence or absence of ras oncogene mutations in resected tumor DNA. Patients will be randomized to observation or a 16-week trial of adjuvant chemotherapy with cisplatin 50 mg/m2 days 1 and 8 every 4 weeks during the 16 weeks, and vinorelbine 30 mg/m2 weekly for 16 weeks. All resected tumors will be banked for further correlative studies to identify a clinically meaningful panel of molecular prognostic markers.

Ras oncogene and p53 gene hotspot mutations in colorectal cancers.

Ras oncogene and p53 gene mutations are frequently observed in colorectal cancers. The role of co-operation between these two genes in the tumorigenesis of colorectal cancer was evaluated. Point mutations in K-ras oncogene and hotspot codons of p53 gene of colorectal cancers were evaluated by naturally created or amplified created restriction site method. Nine of 42 cases (21.4%) of colorectal cancer showed K-ras oncogene mutations. Six of 42 cases (14.3%) of colorectal cancer showed p53 gene hotspot point mutations. The low frequency of p53 gene mutation in this series may be due to racial difference or different hotspot codons. When six cases with mutated p53 gene were examined, only one (16.7%) showed concurrent K-ras oncogene codon 12 and p53 gene codon 248 mutations. We concluded that the co-operation between ras oncogene and p53 gene hotspot point mutations in the tumorigenesis of colorectal cancer in Chinese was not common. Other factors such as adenomatous polyposis coli gene mutations, oncogene activation or tumour suppression gene inactivation may be involved.

Radiation studies on B cell differentiation marker CD24/SCLC Cluster-4 antigen expressing and non-expressing lung cancer cell lines and mouse fibroblasts.

A correlation between CD24 expression and higher intrinsic radiation sensitivity has been described in B-lineage acute lymphoblastic leukaemia (B-ALL). We recently identified the SCLC surface antigen Cluster-4 (CL-4) to be identical to the B cell differentiation marker CD24, except for one amino acid residue. The CD24/CL-4 antigen is highly expressed on SCLC, but rarely on NSCLC cells. In order to investigate the influence of the expression of CD24/CL-4 on the radiation sensitivity in a non-leukaemic cell system, sublines of the human SCLC H249 cell line transfected with mutated ras oncogene, and differing in their CD24/CL-4 expression, were studied. In addition, we stably transfected the NSCLC A125 cell line and the mouse fibroblast NIH3T3 cell line with the CL-4 cDNA. The differential expression of CD24/CL-4 on the cells had no influence on morphology, proliferation and cloning efficiency. Radiation studies were done with cells in exponential growth phase. In the highly resistant NSCLC A125 cells no difference in radioresponsiveness was observed between CD24/CL-4 expressing and non-expressing cells. In the rather radiosensitive cells, similar responses to radiation were observed between CD24/CL-4 expressing and non-expressing SCLC H249-ras cells, whereas the CL-4 transfected NIH3T3 mouse fibroblasts showed a substantially higher radioresistance than the CD24/CL-4 non-expressing control cells. In conclusion, the correlation between CD24/CL-4 expression and radiation sensitivity is controversial and depends on the cell type.

Cell transformation by c-Ha-rasVal12 oncogene is accompanied by a decrease in histone H1 zero and an increase in nucleosomal repeat length.

The activated c-Ha-rasVal12 oncogene is often involved in the genesis of human malignancies. We show here that in c-Ha-rasVal12 oncogene-transformed mouse NIH 3T3 fibroblasts the copy number and expression level of the mutant ras oncogene correlates with the degree of chromatin decondensation, as assessed by micrococcal nuclease (MNase) and DNase I digestion. MNase and DNase I analyses further revealed that the nucleosomal repeat lengths were different in the normal and ras oncogene-transformed cells, 162.3 bp and 178.1 bp, respectively. These chromatin changes were accompanied by alterations in the content of histone H1 zero. Furthermore, using DNase I as a probe, we discovered that serum stimulation of normal and transformed cells, synchronized by serum starvation, induces rapid reversible changes in the structure of bulk chromatin that may be linked to transcriptional activation. Our data thus indicate that cell transformation by ras is associated with specific changes in chromatin structure that make it more vulnerable, and prone to additional mutations characteristic of cancer development in vivo.

The minimal fragments of c-Raf-1 and NF1 that can suppress v-Ha-Ras-induced malignant phenotype.

v-Ha-Ras, an oncogenic Ras mutant, causes malignant transformation of mammalian cells by recruiting c-Raf-1, a cytosolic Ser/Thr kinase, to the plasma membranes/cytoskeleton. The kinase activity of c-Raf-1 resides in the C-terminal half, which activates mitogen-activated protein (MAP) kinase kinase, while it is the N-terminal half of c-Raf-1 (Raf257, residues 1-257) that binds the Ras-GTP complex and can compete Ras GTPase-activating proteins such as NF1 for binding to Ras. However, it still remains to be clarified whether overexpression of Raf257 or its minimal Ras-binding fragment alone is sufficient to suppress Ras-induced malignancy. In this paper we demonstrate for the first time that the 81-amino acid fragment (Raf81, residues 51-131), the minimal Ras-binding fragment of Raf, indeed can suppress v-Ha-Ras-induced malignant phenotype. A further deletion of the first 6 amino acids causes 65% reduction in the Ras binding of Raf81. The resultant 75 amino acid fragment (Raf75, residues 57-131) consists of a single alpha-helix, five anti-paralleled beta-sheets and five loops. We have found that a further deletion of either the first beta-sheet/loop or the last two beta-sheets/loops completely abolishes Ras binding. In addition we have found that the removal of the C-terminal 35 amino acids from a Ras-binding 91-amino acid fragment of NF1 (NF91, residues 1441-1531) does not abolish its ability to suppress the Ras-induced malignancy.

The genetics of human cancer: implications for ecotoxicology.

The study of human cancers has provided evidence that malignant progression is associated with genetic change. It has been suggested that some genetic alterations in tumors may be the result of direct or indirect processes related to environmental chemical exposure. This hypothesis has been supported by genetic evidence in liver tumors which has associated aflatoxin B1 exposure with the detection of inactivating DNA mutations within the human p53 tumor suppressor gene. The detection of activating ras oncogene mutations at high frequency in liver tumors of feral fish suggest that the survey of mutations in genes, such as p53 or other genes, might provide a genetic signature for specific chemical exposure in tissues of aquatic animals derived from environmentally damaged sites.

Ras oncogene activation does not induce sensitivity to natural killer cell-mediated lysis in human melanoma.

An important phenomenon in tumor immunology that has come under recent attention is the impact of oncogene activation in tumor cells on the sensitivity to lysis by immune effector cells. Several studies suggested that transfer of an activated ras oncogene into cultured rodent fibroblasts induces susceptibility to natural killer cell (NK)-mediated lysis. Experiments using human tumor cells, however, have produced conflicting data on the effect of ras activation in this respect. In studying the activation of the oncogene c-myc, which is often overexpressed in human melanoma, we have found that in cell lines expressing high levels of Myc protein, the sensitivity to lysis by NK cells was dramatically increased due to reduced expression of Human Leukocyte Antigen B locus products. Since the N-ras oncogene was found to be activated in 15% of human melanomas, we examined the possibility that in melanoma, in analogy to the murine systems, the mutated ras oncogene may influence NK susceptibility of human melanoma cells. Two N-ras genes harboring frequently found mutations were cloned into an expression vector. Transfection of the IGR39D melanoma cell line with wildtype and mutant N-ras constructs yielded several ras-expressing clones that were tested for NK sensitivity. Neither high expression of the wildtype N-ras protein, nor expression of two mutant proteins (N61-arg, N61-lys) was shown to result in enhanced NK-mediated lysis. We conclude that activation of ras oncogenes does not lead to the induction of an NK-sensitive phenotype in human melanoma cells.

State of adenomatous polyposis coli gene and ras oncogenes in Japanese prostate cancer.

Genetic alterations of ras oncogenes (K‐, H‐ and N‐ras) and adenomatous polyposis coli (APC) gene in tissues of prostate cancer from Japanese patients were examined using PCR‐SSCP (polymerase chain reaction‐single strand conformation polymorphism) analysis and direct sequencing. Tissues from 8 cases of untreated stage B prostate cancer surgically removed and from 10 cases of endocrine therapy‐resistant metastatic disease obtained at autopsy were used in the present study. In four out of 18 cases (22%), ras point mutations were found, two in either codon 12 or 61 of K‐ras and two in either 13 or 61 of H‐ras. These point mutations were detected in one of the stage B cases (13%) and in three of the autopsy cases (30%). All these cases were poorly differentiated adenocarcinoma. In autopsy cases showing ras mutation in cancerous prostate, the same alteration was observed in metastatic tissues. No APC gene mutation was detected in any sample, although polymorphism was found in some cases. These results indicate that ras oncogene mutations are related to the progression of prostate cancer, whereas APC gene alteration is not involved in tumorigenesis and development of this cancer.

Mutations in ras genes in cells cultured from mouse skin tumors induced by ultraviolet irradiation.

Mutations in ras oncogenes were detected in cultured cells of mouse skin tumors induced by near-UV irradiation. DNA extracted from the UV-induced tumor cells was transfected to golden hamster embryo cells, and focus-forming ability was confirmed in 22 of 26 cell strains, 15 of which had the repetitive mouse sequence. Mouse ras genes were detected in 10 of these 22 cell strains. Point mutations in the ras genes were at Ha-ras codon 13 (GGC-->GTC in two strains, GGC-->AGC in one strain), Ki-ras codon 61 (CAA-->GAA in two strains), and N-ras codon 61 (CAA-->CAT in two strains, CAA-->AAA in two strains). In one tumor cell strain no base change was directed. Most mutations occurred at dipyrimidine sites. Pyrimidine dimers or pyrimidine(6-4)pyrimidone photoproducts are the likely cause of the skin cancers. The base change occurred preferentially at G.C base pairs, and transversions predominated.

The clinical significance of ras oncogene mutations in non-small cell lung cancer : Sjoerd Rodenhuis, M.D. and Bert Top, Ph.D., The Netherlands Cancer Institute, Amsterdam, The Netherlands

The clinical significance of ras oncogene mutations in non-small cell lung cancer : Sjoerd Rodenhuis, M.D. and Bert Top, Ph.D., The Netherlands Cancer Institute, Amsterdam, The Netherlands

Mutant c-Ki-ras p21 protein in chemical carcinogenesis in humans exposed to vinyl chloride.

Mutations in ras oncogenes and expression of their encoded p21 protein products are believed to play an important role in carcinogenesis in humans. Detection of mutant p21 proteins in serum may be a useful molecular epidemiologic biomarker with which to study this process, and workers with heavy exposure to vinyl chloride (VC) represent a model population for such study. We studied the occurrence of a specific ras mutation (Asp 13 c-Ki-ras) by oligonucleotide hybridization and the expression of the corresponding p21 protein in tumor tissue and serum by immunohistochemistry and immunoblotting with monoclonal antibodies in five individuals with heavy exposure to VC and resultant angiosarcomas of the liver (ASL). Four of five (80 percent) of the cases of ASL were found to contain the mutation and to express the corresponding mutant protein in their tumor tissue and serum. Serum expression of the mutant protein also was examined in nine VC-exposed workers with liver angiomas and 45 VC-exposed workers with no evidence of liver neoplasia; eight of nine (89 percent) of the former and 22 of 45 (49 percent) of the latter were also positive for the mutant p21 in their serum. However, serum immunoblotting results for 28 age-gender-race matched, unexposed controls were all negative. Stratification by years of VC exposure showed a significant linear trend (P < 10(-5)) for the occurrence of the serum mutant p21 protein with increasing duration of exposure. These results suggest that detection of serum mutant p21 protein can be a valid surrogate for ras gene expression at the tissue level.(ABSTRACT TRUNCATED AT 250 WORDS)

DETECTION OF K-RAS MUTATION IN NORMAL AND MALIGNANT COLONIC TISSUES BY AN ENRICHED PCR METHOD

ras oncogene mutations appear in over 50% of colon tumors in humans. Studies in animal systems have revealed that ras mutations are also present in preneoplastic lesions, suggesting the possibility of early detection of ras mutation in morphologically normal colon tissues for diagnostic purposes. An Enriched PCR, developed by us, eliminates most of the normal ras alleles prior to amplification; subsequent analysis via RFLP enables the detection of one mutant allele within 10(4) normal alleles. Using the Enriched PCR, we have determined the frequency of mutant ras alleles in normal mucosae and in adenomatous polyps of patients with or without adenocarcinoma. Of the 42 patients who had colon tumors, 15 were found to harbor K-ras oncogene mutation (35%). In two of the 14 cases with mutant K-ras in the tumor tissue we were able to identify mutations in tissues that had been obtained from a site at considerable distance from the tumor (13%); Analysis of 7 adenomas identified one as a carrier of the mutant ras allele (14%). Of 11 normal colonic mucosa obtained from patients without neoplasia, one specimen contained K-ras mutation. Thus, mutated alleles of K-ras may be present, at low frequency, throughout the 'normal appearing' tissue. Cells of normal appearance that harbor such mutation, have the potential to undergo further changes and to develop into the transformed phenotype. Overall, our findings suggest that mutant ras alleles can be detected in preneoplastic mucosa that is morphologically normal, and in adenomas, suggesting the occurrence of an initiation event, and possibly enabling the identification of patients who may be at high risk for developing malignant tumors.

The rat N-ras gene; interference of pseudogenes with the detection of activating point mutations.

The PCR technique in combination with selective hybridization to mutation specific oligonucleotides, is a widely used methodology for the detection of activating point mutations in ras oncogenes. In the present paper we demonstrate for the N-ras gene of the rat that processed pseudogenes do interfere with this method. A first indication for this interference came from the sequence analysis of cloned PCR fragments of exon 1, amplified with primers derived from previously reported exon sequences of the mouse N-ras gene. Between different clones originating from one PCR reaction, a marked sequence heterogeneity is observed and this is shown to be the result of the presence of at least two different processed pseudogenes of the rat N-ras gene. These two pseudogenes, together with the wildtype N-ras gene and a small 3' part of the unr gene, were eventually cloned and their genomic organization and nucleotide sequences determined. Furthermore, representative examples of the confounding effects of these pseudogenes on the screening for activating point mutations are presented. Taken together, our results demonstrate that intron-specific amplification is a prerequisite for the unambiguous detection of activating point mutations in the N-ras gene of the rat.

Selective activation of ras oncogenes in follicular and undifferentiated thyroid carcinomas

A total of 96 tumour samples (88 primary tumours and 8 nodal metastases) from 88 patients with thyroid adenomas and carcinomas were investigated for ras gene mutations using polymerase chain reaction, oligonucleotide probing and sequencing. Neither the 19 adenomas nor the 31 papillary carcinomasan alysed harboured point mutations. In our cases, mutations in all three ras oncogenes were found in follicular carcinomas (five out of 21) and in the less differentiated thyroid tumour: poorly differentiated carcinomas (three out of 11) and undifferentiated carcinomas (one out of five). Finally, mutated ras oncogenes had a significant association with the appearance of haematogenous (particularly bone) metastases, suggesting a role of ras genes activation in the metastatic capability of these tumours.