ras Mutations in Endocrine Tumors: Mutation Detection by Polymerase Chain Reaction-Single Strand Conformation Polymorphism

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Mutations of the H-, K-, N- ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N- ras codon 61, and one contained the mutated H- ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H- ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.     

Low frequency of ras gene mutations in neuroblastomas, pheochromocytomas, and medullary thyroid cancers

Little is known about the prevalence and significance of ras gene activation in neural crest tumors such as neuroblastomas, pheochromocytomas, and medullary thyroid cancers (MTCs). Therefore, we analyzed DNA from 10 human neuroblastoma cell lines and 10 primary human pheochromocytomas for activating mutations in N-ras, H-ras, and K-ras. We also studied DNA from 24 primary neuroblastomas and 10 MTCs for N-ras mutations. ras genes were analyzed by direct sequencing of specific DNA fragments amplified by the polymerase chain reaction. With the exception of the SK-N-SH cell line, the examined ras gene sequences were normal in all the neuroblastomas, pheochromocytomas, and MTCs tested. A single point mutation was identified at codon 59 (GCT(ala)----ACT(thr)) in one N-ras allele in an SK-N-SH subline. Interestingly, this mutation is different from the activating codon 61 mutation which resulted in the initial identification of N-ras from SK-N-SH DNA. Therefore, we analyzed the sequences of earlier passages and sublines of the SK-N-SH cell line, but mutations at codon 59 or 61 were not detected, suggesting that neither mutation was present in the primary tumor. Our results indicate that N-ras mutations may occur spontaneously during in vitro passage of cell lines but rarely, if ever, occur in primary neuroblastomas, pheochromocytomas, and MTCs. In addition, we have not found H-ras or K-ras mutations in any neuroblastoma cell line or primary pheochromocytoma.     

High rates of ras codon 61 mutation in thyroid tumors in an iodide-deficient area

Using polymerase chain reaction and sequence-specific oligonucleotide hybridization, the frequency of three ras oncogene mutations (N-ras, Ha-ras, and K-ras) in thyroid tumors (25 adenomas, 16 follicular carcinomas, and 22 papillary carcinomas) was investigated in both iodide-deficient and iodide-sufficient areas. The ras oncogene mutation rate was significantly higher in the iodide-deficient area, being 85 versus 17% in the adenomas, and 50 versus 10% in the follicular carcinomas. No mutations were found in papillary carcinomas. The most common mutation site was Ha-ras codon 61 with Gln----Arg substitution. Two ras mutations at codon 61 (Gln----Lys in N-ras and Gln----Arg in Ha-ras) were found in a microfollicular adenoma specimen from Eastern Hungary. We conclude that dietary iodine may modulate ras oncogene mutations, and that in the iodide-deficient area, ras oncogene activation may play a more important role in the initiation and/or maintenance of follicular tumors. Additional factors are, however, necessary to initiate carcinogenesis.     

Detection of ras gene mutations in human lung cancers by single-strand conformation polymorphism analysis of polymerase chain reaction products

A simple, sensitive method of DNA analysis of nucleotide substitutions, namely, single-strand conformation polymorphism analysis of polymerase chain reaction products (PCR-SSCP analysis), was used for detection of mutated ras genes in surgical specimens of human lung cancer. Of a total of 129 tumors analysed, 22 contained a mutated ras gene. Of the 66 adenocarcinomas analysed, 14 contained an activated c-Ki-ras2 gene (the mutations in codon 12 in 6, in codon 13 in 4, in codon 18 in one, and in codon 61 in 3), one contained a c-Ha-ras1 gene with a mutation in codon 61 and 3 contained N-ras genes with mutations (in codon 12 in one and in codon 61 in 2). Mutated rats genes were also found in 2 of 36 squamous cell carcinomas (c-Ha-ras1 genes with mutations in codon 61) and 2 of 14 large cell carcinomas (c-Ki-ras2 genes with mutations in codon 12). No mutation of the ras gene was detected in 8 small cell carcinomas and 5 adenosquamous cell carcinomas. These results indicate that activation of the ras gene was not frequent (17%) in human lung cancers, that among these lung cancers mutation of the ras gene was most frequent in adenocarcinomas (27%) and 73% of the point mutations were in the c-Ki-ras2 gene in codon 12, 13, 18 or 61.     

ras oncogenes in human cancer: a review

Mutations in codon 12, 13, or 61 of one of the three ras genes, H-ras, K-ras, and N-ras, convert these genes into active oncogenes. Rapid assays for the detection of these point mutations have been developed recently and used to investigate the role mutated ras genes play in the pathogenesis of human tumors. It appeared that ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90%), the colon (50%), and the lung (30%); in thyroid tumors (50%); and in myeloid leukemia (30%). For some tumor types a relationship may exist between the presence of a ras mutation and clinical or histopathological features of the tumor. There is some evidence that environmental agents may be involved in the induction of the mutations.     

Activating mutations of ras family genes in prostatic-cancer

ras family genes (H-, K- and N-ras) encode for a 21 kD membrane protein which possesses GTPase activity and participates in a signal transduction pathway. Activating mutations of the ras family genes occur at codons 12, 13 and 61 and have been detected in a variety of human tumours, including colonic, bladder and pancreatic cancers. Prostatic cancer is among the most common malignancies throughout the world and a major cause of death from cancer in males. Data reported on the implication of the ras family genes in the development of the disease are conflicting. The aim of this study was to determine the incidence of mutations at codon 12 of H-ras, codon 12 of K-ras and codon 61 of N-ras proto-oncogenes, in a Greek population with prostatic cancer. Our analysis revealed that 4 out of 20 (20%) samples harboured a K-ras codon 12 point mutation, 1 out of 20 (5%) specimens contained mutations at codon 12 of the H-ras and 1 out of 20 (5%) at codon 61 of the N-ras, indicating a role for the ras genes in the development of the disease.     

Screening of N-ras codon 61 mutations in paired primary and metastatic cutaneous melanomas: mutations occur early and persist throughout tumor progression.

PURPOSE: Mutations in the ras genes often occur during tumorigenesis. In malignant melanoma, the most common ras alterations are N-ras codon 61 mutations. This study was aimed to measure the frequency of such mutations in a large series of paired primary and metastatic melanomas to determine their role in melanoma initiation and progression. EXPERIMENTAL DESIGN: Seventy-four primary melanomas and 88 metastases originating from 54 of the primary tumors were screened for N-ras codon 61 mutations using single-strand conformation polymorphism and nucleotide sequence analyses. RESULTS: Twenty-one of the 74 primary tumors (28%) had activating N-ras codon 61 mutations. From 20 of the mutated primary tumors, a total of 34 metastases were analyzed, and all but one showed the same mutation as the primary tumor from which they originated. The remaining 53 primary tumors and corresponding metastases (n = 54) were wild-type for N-ras codon 61. Analysis of the different growth phases of the mutated primary tumors showed that the mutations were already present in the radial growth phase. Mutations were also detected in tumor-associated nevi. N-ras codon 61 mutations were associated with a higher Clark level of invasion (P = 0.012) and a lower age at diagnosis (P = 0.042) but did not affect survival (P = 0.671). CONCLUSIONS: This study shows that N-ras codon 61 mutations occur early in primary melanomas rather than in the metastatic stage and that once the mutations have occurred, they persist throughout tumor progression. This suggests that activated N-ras may be an attractive target for therapy in the subset of melanoma patients carrying such mutations.     

H-ras activation and ras p21 expression in bladder tumors induced in F344/NCr rats by N-butyl-N-(4-hydroxybutyl)nitrosamine

Bladder tumors were induced in male F344/NCr rats by administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) at 500 p.p.m. in their drinking water for 12 weeks. Twenty-one bladder tumors that developed between 25 and 50 weeks after BBN administration was begun were evaluated for immunoreactivity with polyclonal or monoclonal antibodies raised against ras p21, for amplification of ras genes by Southern blotting, and for activating point mutations in ras genes by selective oligonucleotide hybridization of products from polymerase chain reaction (PCR). Increased expression of ras p21 was detected by avidin-biotin immunohistochemistry in 18/21 (85%) of the neoplastic bladder lesions. By Southern analysis, there was no significant amplification of H-ras, K-ras or N-ras in any of the tumors except one that showed a 5-fold amplification of K-ras. Point mutations in ras genes were detected by selective oligonucleotide hybridization of the products of PCR. Of the 21 bladder tumors, three tumors were shown to have mutations in codon 12 (GGA----GAA), six tumors in codon 61 (two CAA----CTA, four CAA----CGA), and one in both codon 12 (GGA----GAA) and codon 61 (CAA----CGA), all in H-ras. Thus 10 of 21 tumors has ras gene mutations in a portion of the tumor cells. The variable pattern of point mutation in H-ras suggests that these mutations may not all be a direct consequence of interaction of BBN metabolites with H-ras. Enhanced expression of ras p21 was always focal and was not necessarily associated with transforming ras mutations. It is therefore suggested that tumorigenesis in BBN-initiated bladder cells might involve H-ras activation as part of a multistep pathway; however, H-ras involvement is not obligatory for tumor development.     

Point Mutations of ras and Gsα Subunit Genes in Thyroid Tumors

We studied 43 thyroid tumors including 5 adenomatous goiters, 7 follicular adenomas, 22 papillary carcinomas, and 9 medullary carcinomas with regard to the presence of point mutations in the genes of Gs alpha subunit ( Gsα ), Gi2 alpha subunit ( Gi2α ), H- ras , K- ras , and N- ras by a polymerase chain reaction-direct sequencing method. An adenomatous goiter and a follicular adenoma showed double mutations at codon 227 and 231, and 4 papillary carcinomas showed mutation at codon 231 of the Gsα gene. An adenomatous goiter, a follicular adenoma, and a papillary carcinoma showed a missense mutation in codon 13 of the K- ras gene. There were no such missense mutations of these G-protein or ras genes in medullary carcinomas. These data indicate that the genetic events involved in the oncogenesis of parafollicular C-cells are different from those of thyroid follicular cells, in which missense mutations of Gsα and ras genes seem to play important roles in tumorigenesis.     

Analysis of ras gene mutations in human hepatic malignant tumors by polymerase chain reaction and direct sequencing

The ras gene is one of the oncogenes most commonly detected in human cancers, and it consists of three families (H-ras, K-ras, N-ras). These genes are converted to active oncogenes by point mutations occurring in either codon 12, 13, or 61. We analyzed mutations of these codons in 23 primary hepatic malignant tumors (12 hepatocellular carcinomas, nine cholangiocarcinomas, and two hepatoblastomas) by a method to directly sequence nucleotides, using polymerase chain reaction and a direct sequencing method. Of 23 hepatic malignant tumors, point mutations at K-ras codon 12 or K-ras codon 61 were found in six of nine cholangiocarcinomas. In contrast, there were no point mutations in any of 12 hepatocellular carcinomas or two hepatoblastomas around codon 12, 13, or 61 of the ras genes. These observations suggest that ras gene mutations are not related to pathogenesis of hepatocellular carcinoma, but play an important role in pathogenesis of cholangiocarcinoma.     

Thyroid targeting of the N-ras(Gln61Lys) oncogene in transgenic mice results in follicular tumors that progress to poorly differentiated carcinomas

Ras oncogenes are frequently mutated in thyroid carcinomas. To verify the role played by N-ras in thyroid carcinogenesis, we generated transgenic mice in which a human N-ras(Gln61Lys) oncogene (Tg-N-ras) was expressed in the thyroid follicular cells. Tg-N-ras mice developed thyroid follicular neoplasms; 11% developed follicular adenomas and approximately 40% developed invasive follicular carcinomas, in some cases with a mixed papillary/follicular morphology. About 25% of the Tg-N-ras carcinomas displayed large, poorly differentiated areas, featuring vascular invasion and forming lung, bone or liver distant metastases. N-ras(Gln61Lys) expression in cultured PC Cl 3 thyrocytes induced thyroid-stimulating hormone-independent proliferation and genomic instability with micronuclei formation and centrosome amplification. These findings support the notion that mutated ras oncogenes could be able to drive the formation of thyroid tumors that can progress to poorly differentiated, metastatic carcinomas.     

Activation of ras oncogenes and expression of tumor-specific transplantation antigens in methylcholanthrene-induced murine fibrosarcomas

The DNA of 22 fibrosarcomas, newly induced in BALB/c mice by subcutaneous doses of 3-methylcholanthrene (3-MCA), was tested in NIH 3T3 transformation assay. Activation of K-ras and N-ras was found in 7 and 3 cases respectively. No H-ras activation was detected. Polymerase chain reaction and oligonucleotide hybridization performed on the DNA of the 22 sarcomas revealed 5 cases of K-ras mutation at codon 12, 3 at codon 13 and 1 at both codons. One case of K13 mutation was not detectable by transfection. Three cases of mutation at codon 61 of N-ras were also found, one of which was simultaneous with a K12 mutation. Tumor-specific transplantation antigens (TSTA) were assessed in the 22 original tumors. Altogether 16 sarcomas were immunogenic, with the highest frequency of TSTA+ tumors (10/11 and 5/6) in the groups given 1.0 and 0.1 mg of 3-MCA respectively, the lowest (1/5) in that with 0.01 mg of carcinogen; ras mutations occurred in the DNAs of 11 out of the 16 TSTA+ sarcomas, but none of the DNAs of the 6 TSTA- tumors showed ras mutation. The results suggest that 3-MCA-induced transformation of subcutaneous fibroblasts can involve mutations in codons 12, 13 or 61 of K- and N- but not H-ras gene and that such mutation is accompanied by the expression of TSTA.     

Presence of mutations in all three ras genes in human thyroid tumors

Polymerase chain reaction (PCR) amplification followed by oligonucleotide probing was used to investigate the presence of ras genes mutations in human thyroid adenomas and carcinomas. The results confirm the frequent occurrence of mutations in all three ras genes in both adenomas and carcinomas, in agreement with the hypothesis that the ras mutations may constitute early steps in thyroid tumorigenesis. No evident correlation between the frequency of ras mutations, the identity of the mutated ras gene, the position affected in the ras gene or the type of mutation and the pathological features is apparent. However, definitive conclusion on this point is precluded because of the small number of tumors examined at the present time.     

Mapping of UV photoproducts within ras proto-oncogenes in UV-irradiated cells: correlation with mutations in human skin cancer.

Mutations in ras proto-oncogenes have been found in human skin cancers. Since ultraviolet light is implicated in the development of skin cancers, we have investigated the formation of UV-induced photoproducts along exons 1 and 2 of the three ras proto-oncogenes, H-ras, K-ras, and N-ras, in UV-irradiated human cells. The two major types of DNA photoproducts, cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidone photoproducts [(6-4) photoproducts], were mapped at the DNA sequence level by ligation-mediated polymerase chain reaction (LMPCR). No significant differences were seen between irradiated purified DNA and irradiated cells, implying that local chromatin structure does not influence the distribution of photoproducts along exons 1 and 2 of the three ras genes. We find that the transcribed strand near codon 61 in H-ras, K-ras and N-ras shows a high frequency of potentially mutagenic cyclobutane dimers and (6-4) photoproducts. Codon 12 of H-ras, K-ras and N-ras displays only barely detectable photoproducts at a CpC dinucleotide. In human skin cancers, mutations were most frequently detected at codon 12 of H-ras and K-ras. These results imply that the initial frequency distribution of a mutagenic DNA adduct may not correlate with mutation spectra in human tumors.     

High frequency of ras oncogene activation in all stages of human thyroid tumorigenesis

Using polymerase chain reaction amplification and oligonucleotide probing, the activation of ras oncogenes in 24 benign and 20 malignant human thyroid neoplasms was examined. The frequency of ras oncogene activation was similar at all stages of tumorigenesis in this system, being found in 33% of adenomas overall (50% of microfollicular tumours), 53% of differentiated follicular carcinomas and 60% of undifferentiated carcinomas. This supports the contention that mutation of these oncogenes occurs at an early step in tumorigenesis. The predominant amino acid substitution in the differentiated tumours was glutamine to arginine at position 61 of Ha-ras or N-ras, but this mutation was not found in any of the undifferentiated tumours. It was noted that while transition mutations predominated in differentiated tumours (both benign and malignant), transversions were more common in the undifferentiated tumours.     

Point mutation analysis of ras genes in spontaneous and chemically induced C57Bl/10J mouse liver tumours.

The high incidence and profile of ras gene mutations reported in spontaneous and chemically induced liver tumours of the B6C3F1 mouse provides a potential means of determining in vivo genotoxicity and its relevance to carcinogenicity. We analysed spontaneous and chemically induced [with 4-amino-biphenyl (ABP), 2-acetylaminofluorene (AAF) and diethylnitrosamine (DEN)] hepatocellular tumours of the C57Bl/10J mouse for H-ras, K-ras and N-ras gene mutations to see if mutational analysis of the ras genes could be useful for such a determination in this strain. Regions of DNA spanning codons 12, 13 and 61 of the ras genes were amplified from formalin fixed liver tumour sections using the polymerase chain reaction. Mutations were detected using allele specific oligonucleotide probing and confirmed by sequencing. We have found that there are few ras mutations in either spontaneous or chemically induced liver tumours in the C57Bl/10J mouse. Out of 25 spontaneous tumours two contained an A to T transversion and one contained an A to G transition in base 2 of H-ras codon 61 and two contained a G to A transition in base 2 of K-ras codon 13 (the K-ras mutations were only faintly detectable and may be present in a subpopulation of the tumour cells). In the case of the 18 ABP induced tumours one contained a C to A transversion in base 1 of H-ras codon 61, and one contained an A to T transversion in base 2 of H-ras codon 61 and one contained a G to C transversion in base 1 of K-ras codon 13. One C to A transversion in base 1 of H-ras codon 61 was detected out of eight AAF induced tumours. Of the 25 DEN induced tumours, one contained an A to G transition and one contained an A to C transversion in base 2 of H-ras codon 61. The data indicate that at least in hepatocellular tumours of the C57Bl/10J strain and using chronic dosing regimes the ras genes do not represent markers for in vivo genotoxic activity.     

Preferential DNA damage and poor repair determine ras gene mutational hotspot in human cancer

BACKGROUND: Mutations in ras genes are commonly found in human cancers and in animal models. Although mutations at codons 12, 13, and 61 of H-, N- and K-ras genes can activate their oncogenic function, mutations at codon 12 of K-ras are the most common mutations found among the three ras genes in human cancers. To investigate whether codon 12 of human K-ras is especially susceptible to carcinogens and/or whether carcinogen-DNA adducts at this codon are repaired less efficiently, we examined tobacco smoke carcinogen-induced DNA damage in normal human bronchial epithelial and fibroblast cells. METHODS: We used the UvrABC nuclease incision method in combination with ligation-mediated polymerase chain reaction to map the distribution of DNA adducts induced by benzo[a]pyrene diol epoxide (BPDE) and other bulky carcinogens within exons 1 and 2 in H-ras, N-ras, and K-ras. We also analyzed BPDE-DNA adduct repair efficiency in these three genes using the same method. RESULTS: Codons 12 and 14 of the K-ras gene were hotspots for carcinogen-DNA adduct formation, with little and no adduct formation at codons 13 and 61, respectively. The BPDE-DNA adducts formed at codon 14 were repaired almost twice as quickly as those formed at codon 12. There was some BPDE-DNA adduct formation at codons 12 of H-ras and N-ras, but this codon was not a hotspot. Furthermore, no substantial difference in repair rates between codon 12 and the other codons analyzed (codons 3 and 18) was observed in either the H-ras or N-ras genes. CONCLUSION: These findings link the human cancer mutational hotspot at codon 12 of K-ras to preferential DNA damage and poor repair.     

Detection of activated ras oncogenes in human thyroid carcinomas

Focus formation following DNA transfection of mouse 3T3-Vill cells was used to search for the presence of activated oncogenes in human thyroid tumors. Oncogenes belonging to the ras family were detected in four out of six thyroid carcinomas (Ki-ras in one anaplastic tumor and one follicular moderately differentiated tumor and Ha-ras and N-ras in two papillary tumors). Normal thyroid tissue samples obtained from two patients, one with an anaplastic tumor and one with a benign adenoma, and samples from 4 benign adenomas and from one toxic goiter of a patient with Graves' disease gave negative results. In one case, restriction enzyme analysis demonstrated the presence of a mutation in codon 12 of the activated Ha-ras oncogene. Our data show that all three ras proto-oncogenes can become activated in malignant thyroid tumors.