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.     

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 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.     

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.     

ras gene mutations in human prostate cancer

Point mutations at codons 12, 13, or 61 of the Ha-, Ki-, and N-ras genes are able to convert these normal cellular genes into activated oncogenes. Previous studies have shown that ras gene mutations occur in a variety of human solid tumors and may be important in the pathogenesis of some of these tumors. In order to test the hypothesis that ras gene mutations may be associated with prostate cancer, we have used an oligodeoxynucleotide hybridization assay to detect wild-type and mutant alleles in genomic DNA from prostate tumors and prostate tumor cell lines amplified using the polymerase chain reaction. Twenty-four primary prostate tumors (23 acinar tumors and one ductal tumor) and five prostate tumor cell lines were examined for mutations at codons 12, 13, and 61 of the Ki-ras, Ha-ras, and N-ras genes. Two mutations were detected: an A----G transition causing a glutamine to arginine amino acid substitution at codon 61 of the Ha-ras gene in a primary prostatic duct adenocarcinoma and a G----T transversion causing a glycine to valine amino acid substitution at codon 12 of the Ha-ras gene in a prostate tumor cell line (TSU-PR1) derived from a lymph node metastasis. While the overall frequency of ras gene mutations in prostate tumors is low, when these mutations do occur they may have a role in the progression of disease or the development of the unusual ductal variant of prostatic adenocarcinoma.     

ras gene mutations as a prognostic marker in adenocarcinoma of the human lung without lymph node metastasis.

Adenocarcinoma of the lung obtained at surgical resection was examined for mutation at codons 12, 13, and 61 of the oncogenes K-ras, H-ras, and N-ras, using polymerase chain reaction and oligonucleotide hybridization techniques. The mutation was detected in 18 of the 115 cases (15.7%), and 15 of 18 were at codon 12, 2 were at codon 13 of K-ras, and 1 was at codon 61 of N-ras. G to T transversions were most common. The ras gene mutations were more frequent in the male patients (P = 0.0048). No significant differences were found to be related to stage of the disease or tumor-nodes-metastases classification between positive and negative groups of the ras gene mutations. A history of tobacco use was not always a factor contributing to mutation. Of the completely resected group without lymph node metastasis, the 5-year survival rate in the ras-positive group was 53.3%, which was significantly poorer than the 83.6% survival rate in the ras-negative group (P less than 0.05). Our findings suggest that ras gene mutations may be prognostic, especially in the early stage adenocarcinoma of the lung.     

Lymphomagenesis in E mu-myc transgenic mice can involve ras mutations

Transgenic mice bearing c-myc driven by the immunoglobulin heavy chain enhancer (E mu) initially exhibit a preneoplastic lymphoproliferative syndrome from which clonal pre-B or B lymphomas develop at random. To investigate whether this transition involves the activation of oncogenes capable of transforming fibroblasts, we transfected DNA from 14 E mu-myc lymphomas into NIH3T3 cells and tested the tumorigenicity of the transfectants in nude mice. By this assay and subsequent direct analysis of the lymphoma mRNA by cDNA cloning or the polymerase chain reaction, two independent E mu-myc lymphomas were shown to contain an N-ras or a K-ras oncogene mutated at codon 61. When incorporated into a recombinant retrovirus, the mutant N-ras allele could collaborate with myc to transform preneoplastic E mu-myc bone marrow pre-B cells. These results indicate that spontaneous mutation of ras genes is one pathway to lymphomagenesis in E mu-myc mice but that many of the lymphomas arise in response to changes that do not register in fibroblasts.     

ras oncogene activation in human ovarian carcinoma

Samples of 37 fresh human ovarian tumor biopsies were screened to detect proto-oncogene amplification and ras mutations. Three samples showed c-K-ras2 amplification; none of the other oncogenes tested revealed any gene amplification. 5-, 25-, and 120-fold amplifications were assessed by dilution experiments and soft laser densitometry. Corresponding elevated levels of c-K-ras2 mRNA and p21 ras protein were found in the three tumors. Analysis by the polymerase chain reaction method to detect point mutations of codon 12 or codon 61 of Harvey-, Kirsten-, or N-ras showed only the wildtype sequence in all specimens. No correlation was found between ras activation and degree of tumor progression or histological subtype. DNA from one of the tumors with c-K-ras2 amplification proved to have high transforming activity in the NIH 3T3 tumorigenicity assay, but the transforming gene was not c-K-ras2.     

N-ras mutation in ultraviolet radiation-induced murine skin cancers.

UV radiation is a potent DNA-damaging agent and a known inducer of skin cancer in experimental animals. To elucidate the role of oncogenes in UV carcinogenesis, we analyzed UV-induced murine skin tumors for mutations in codon 12, 13, or 61 of Ha-ras, Ki-ras, and N-ras oncogenes by amplification of genomic tumor DNAs by the polymerase chain reaction followed by dot-blot hybridization to synthetic oligonucleotide probes designed to detect single base-pair mutations. In addition to UV-induced C3H mouse skin tumors, we also analyzed skin tumors induced in the same strain of mice by other carcinogenic agents such as 8-methoxypsoralen + UVA, angelicin + UVA, dimethylbenz-[a]anthracene + UV + croton oil, and 4-nitroquinoline-1-oxide. We found that 4 of 20 UV-induced skin tumors contained either C----A or A----G base substitutions at N-ras codon 61. In addition, 2 of 5 melanomas possessed a G----A transition in N-ras codon 13 and an A----T transversion in N-ras codon 61, respectively. Interestingly, none of the 8-methoxypsoralen + UVA- or angelicin + UVA-induced tumors we analyzed contained mutations in any of the ras genes. However, 1 of 4 4-nitroquinoline-1-oxide-induced tumors exhibited a G----T transversion at Ki-ras codon 12, a potential site for formation of a 4-nitroquinoline-1-oxide adduct with a guanine residue. We also found that 2 nonmelanoma tumors induced by dimethylbenz[a]anthracene + UV + croton oil contained an A----T transversion at Ha-ras codon 61 position 2, which is characteristic of most dimethylbenz[a]anthracene-induced tumors. These results suggest that UV-induced C3H mouse tumors display mutations preferentially in the N-ras oncogene. Since most N-ras mutations in UV-induced tumors occurred opposite dipyrimidine sequences (T-T or C-C), one can infer that these sites are the targets for UV-induced mutation and transformation.     

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.     

Absence of ras gene mutations in UV-induced malignant melanomas correlates with a dermal origin of melanocytes in Monodelphis domestica

The South American opossum, Monodelphis domestica, has been used as a model system to study ultraviolet (UV)-induced genetic alterations that lead to the development of melanoma. Suckling young of Monodelphis exposed to multiple doses of UVB radiation can develop benign or malignant melanomas later as adults. Point mutations predominantly at codon 61 of the N-ras gene have been found in melanomas from sun-exposed body sites in humans. To determine if similar mutations are associated with UV-induced melanoma in Monodelphis, the nucleotide sequence of a Monodelphis N-ras cDNA was determined, and the occurrence of ras mutations in melanomas from UV-irradiated opossums was investigated. Single-strand conformation polymorphism analysis revealed no mutations in either the Monodelphis N-ras or H-ras genes in any of 24 primary malignant melanoma samples analyzed in this study. The disparate association of ras mutations with melanoma in humans and Monodelphis may be explained by differences in nucleotide sequence at codon 61 of the N-ras gene as well as differences in skin architecture between the two species. These results support the contention that a mutationally activated N-ras gene contributes to the vertical growth phase, which is specific to the progression of malignant melanoma in humans.     

ras gene activation in a minor proportion of the blast population in acute myeloid leukemia

DNAs from 22 acute myeloid leukaemia (AML) patients were screened for activated transforming genes using NIH3T3 transfection followed by assay for tumor formation in Nude mice. In four samples an activated N-ras oncogene, and in two samples an activated Ha-ras oncogene were detected in transfectants. Synthetic oligonucleotide probes were used to characterise the mutations in the ras genes. Three samples were found to be mutated to N-ras codon 12 ASP, one to N-ras codon 13 ASP and two to Ha-ras codon 12 VAL. When the corresponding AML DNAs were screened using direct gel hybridisation, the mutant ras genes were detectable in only one case. In two AML samples (82 and 84) with very low percentage blasts (3% and 19%), the absence of mutant ras signal from direct gel hybridisation may be due to the lack of sensitivity of this technique in detecting activated ras in such small fractions of the total DNA. These results illustrate the sensitivity of the in vivo tumour assay in detecting activated ras. When DNA from one of the remaining three AML DNAs was amplified using the polymerase chain reaction method, the mutation present in the transfectant was detected. These findings suggest that even in AMLs with high percentage blasts (40%, 70% and 90%), cells containing mutant ras may comprise only a minor proportion of the major leukaemic clone.     

Low incidence of point mutation of c-Ki-ras and N-ras oncogenes in human hepatocellular carcinoma

We examined the incidence of point mutation in codons 12, 13 and 61 of c-Ki-ras and N-ras genes in human hepatocellular carcinoma (HCC) using the polymerase chain reaction and oligonucleotide hybridization techniques. Among 34 tissues specimens surgically resected from 30 patients and 5 cell lines of human HCC, only two had ras point mutations; in one case, codon 12 of c-Ki-ras was altered from GGT, coding glycine, to GTT, coding valine; in the other case, codon 61 of N-ras was altered from CAA, coding glutamine, to AAA, coding lysine. Thus, point-mutational activation of ras oncogenes is an uncommon event in human HCC.     

A novel cell-based assay for the evaluation of anti-ras compounds.

In order to identify drugs active against mutated ras oncogenes we have developed an in vitro assay employing two clones of the human fibrosarcoma cell-line, HT1080 which carries an N-ras gene mutated at codon 61. Clone, HT1080scc2, retains the transformed phenotype of the parental line, whilst the other, HT1081c, is a morphologically flat, non-tumourigenic, revertant with under-representation of the chromosome carrying the transforming N-ras allele. The clear implication of mutant ras in maintaining the transformed nature of HT1080scc2 was confirmed when these cells were microinjected with the pan ras neutralising antibody Y13-259, which resulted in the morphological detransformation of these cells to a phenotype resembling that of the HT10801c clone. A number of known anti-cancer drugs with modes of action unrelated to ras function were found to be equipotent against both clones. However, when compounds chosen on the grounds of their potential selective cytotoxic or differentiating activity were tested some interesting results were obtained. Thus 8-bromo cAMP affected some morphological detransformation of HT1080scc2 cells and reduced their colony forming potential. The IMP-dehydrogenase inhibitors, tiazafurin and mycophenolic acid also flattened the morphology of the transformed clone. Fumagillin, an antibiotic reported to exhibit selective activity against ras transformed cells showed very marked and selective cytostatic effects against HT1080scc2 cells with IC50 values as low as 1 x 10(-11) M.     

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.     

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.     

Infrequent mutation of the ras genes in skin tumors of xeroderma pigmentosum patients in Japan.

By using PCR amplification and oligonucleotide mismatch hybridization, base-substitution mutations of the ras genes in 26 skin tumors of Japanese xeroderma pigmentosum (XP) patients were studied. Thin sections of tumor tissues which were fixed and embedded in paraffin blocks were used in this study. After analyzing codons 12, 13 and 61 of the H-, K- and N-ras genes by using 66 oligomer probes, we detected only one mutation of the K-ras gene at codon 61 in one tumor sample. All the other tumors were therefore considered not to have a mutation in the ras genes. These results suggest that mutations of the ras genes are not particularly associated with skin tumors of Japanese XP patients.     

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.     

Analysis of ras gene mutations in biliary and pancreatic tumors by polymerase chain reaction and direct sequencing

Ras gene is one of the oncogenes most commonly detected in human cancers and consists of three families (H-ras, K-ras, N-ras) that are converted to active oncogenes by point mutations occurring in codon 12, 13, or 61. The authors analyzed mutations of these codons in 12 extrahepatic bile duct carcinomas, nine gallbladder carcinomas, and 20 pancreatic tumors (18 pancreatic adenocarcinomas and two islet cell tumors) by a method to directly sequence nucleotides, using polymerase chain reaction and a direct sequencing method. Point mutations at K-ras codon 12 were found in all of 18 pancreatic adenocarcinomas and in one bile duct carcinoma, but there were no mutations in the remaining 11 bile duct carcinomas, in all of 9 gallbladder carcinomas, or in two islet cell tumors. A very high incidence of ras gene mutations may be used clinically for the diagnosis of debatable cases of pancreatic adenocarcinoma.