Both N-ras and c-myc are activated in the SHAC human stomach fibrosarcoma cell line

A transforming N-ras gene was isolated from the SHAC human stomach fibrosarcoma cell line. A single-point mutation resulting in the substitution of histidine for glutamine at codon 61 was found in the SHAC transforming allele. The N-ras gene is overexpressed in the tumor cells and transformant cells. The N-ras p21 product was studied by immunoprecipitation and showed no alteration in mobility as compared to the normal p21 protein. The c-myc gene is amplified and overexpressed in these cells. This report gives evidence that an amplified c-myc and a mutated N-ras gene are both present in this tumor cell line and provides support for the idea that co-operation of at least 2 activated cellular oncogenes is required for carcinogenesis.     

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.     

Ras gene mutation and amplification in human nonmelanoma skin cancers

Our previous studies have shown that human skin cancers occurring on sun-exposed body sites frequently contain activated Ha-ras oncogenes capable of inducing morphologic and tumorigenic transformation of NIH 3T3 cells. In this study, we analyzed human primary squamous cell carcinomas (SCCs) and basal cell carcinomas (BCCs) occurring on sun-exposed body sites for mutations in codons 12, 13, and 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 the primary human skin cancers, we also analyzed Ha-ras-positive NIH 3T3 transformants for mutations in the Ha-ras oncogene. The results indicated that all three NIH 3T3 transformants, 11 of 24 (46%) SCCs, and 5 of 16 (31%) BCCs contained mutations at the second position of Ha-ras codon 12 (GGC----GTC), predicting a glycine-to-valine amino acid substitution, whereas only 1 of 40 skin cancers (an SCC) displayed a mutation in the first position of Ki-ras codon 12 (GGT----AGT), predicting a glycine-to-serine amino acid change. In addition, three of the SCCs contained highly amplified copies of the N-ras oncogene in their genomic DNA. Interestingly, two of the SCCs containing amplified N-ras sequences also had G----T mutations in codon 12 of the Ha-ras oncogene. These studies demonstrate that mutations in codon 12 of the Ha-ras oncogene occurred at a high frequency in human skin cancers originating on sun-exposed body sites, whereas mutation in codon 12 of Ki-ras or amplification of N-ras occurred at a low frequency. Since the mutations in the Ha-ras and Ki-ras oncogenes were located opposite potential pyrimidine dimer sites (C-C), it is likely that these mutations were induced by ultraviolet radiation present in sunlight.     

Screening of N-ras Gene Mutations in Urothelial Cell Carcinomas of the Urinary Bladder in the Kashmiri Population

Background and Aims: The objective of this study was to assess the frequency of specific-point mutations inN-ras of the RAS gene family in a group of Kashmiri patients with bladder cancer and to observe any associationwith clinicopathological parameters. Methods: Paired tumor and normal tissue specimens of 55 consecutivepatients with urothelial cell carcinoma were screened and DNA was extracted for detection of N-ras activatingmutations in exons 1 and 2. In addition, blood was also collected from all the cases to rule out any germ linemutation. Results: Specific point mutations of activated N-ras were detected in 9% (5 of 55) of the bladdercancer patients, all being missense. The base substitutions identified included three transversions (two G toTand one A to T) and two transitions ( A-G). Sixty % of the mutations were detected in codon 61 and 40% incodon 12. No significant correlations were found between the mutations and clinical features. Conclusion:Although N-ras gene mutation might be one of the mechanisms underlying oncogenesis of urothelial cancer, itseems to be a relatively rare event in Kasmiris, pointing to involvement of different etiological factors in theinduction of bladder tumor in this population     

Activation of the Ha-, Ki-, and N-ras genes in chemically induced liver tumors from CD-1 mice.

We compared the profile of ras gene mutations in spontaneous CD-1 mouse liver tumors with that found in liver tumors that were induced by a single i.p. injection of either 7,12-dimethylbenz(a)anthracene (DMBA), 4-aminoazobenzene, N-hydroxy-2-acetylaminofluorene, or N-nitrosodiethylamine. By direct sequencing of polymerase chain reaction-amplified tumor DNA, the carcinogen-induced tumors were found to have much higher frequencies of ras gene activation than spontaneous tumors. Furthermore, each carcinogen caused specific types of ras mutations not detected in spontaneous tumors, including several novel mutations not previously associated with either the carcinogen or mouse hepatocarcinogenesis. For example, the model compound DMBA is known to cause predominantly A to T transversions in Ha-ras codon 61 in mouse skin and mammary tumors, consistent with the ability of DMBA to form bulky adducts with adenosine. Our results demonstrate that the predominant mutation caused by DMBA in mouse liver tumors is a G to C transversion in Ki-ras codon 13 (DMBA is also known to form guanosine adducts), illustrating the influence of both chemical- and tissue-specific factors in determining the type of ras gene mutations in a tumor. 4-Aminoazobenzene and N-hydroxy-2-acetylaminofluorene also caused the Ki-ras codon 13 mutation. In addition, we found that N-nitrosodiethylamine, 4-aminoazobenzene, and N-hydroxy-2-acetylaminofluorene all caused G to T transversions in the N-ras gene (codons 12 or 13). This is the first demonstration of N-ras mutations in mouse liver tumors, establishing a role for the N-ras gene in mouse liver carcinogenesis. Finally, comparison of the ras mutations detected in the direct tumor analysis with those detected after NIH3T3 cell transfection indicates that spontaneous ras mutations (in Ha-ras codon 61) are often present in only a small fraction of the tumor cells, raising the possibility that they may sometimes occur as a late event in CD-1 mouse hepatocarcinogenesis.     

Activation of the c-Ha-ras-1 proto-oncogene by methylation in vitro with alpha-acetoxy-N-nitrosodimethylamine

alpha-Acetoxy-N-nitrosodimethylamine, an activated derivative of the carcinogen N-nitrosodimethylamine, methylated in vitro a plasmid containing the human c-Ha-ras-1 proto-oncogene, resulting in the generation of a transforming oncogene, assayed by transfection into NIH 3T3 cells. The resulting transformed cells were tumorigenic and metastatic in immune-deprived mice. Further transfection using tumor DNA led to the formation of three secondary NIH 3T3 transformants. DNA from these secondary transformants contained human ras gene sequences. Two of the three secondary transformants contained G----A mutations at guanine 35 in codon 12, and the third secondary transformant retained the wild-type sequence at codons 12, and 61. For the latter, the activating mutation was not determined. These results demonstrate that a simple methylating agent can activate a normal human ras proto-oncogene to a transforming oncogene.     

Oncogenes in multiple myeloma: point mutation of N-ras

Alterations of ras, c-myc and bcl-1 have been described in hematologic malignancies of lymphoid origin. We investigated the structure of these genes and evaluated the frequency of point mutations involving H-, K- or N-ras in bone marrow samples from patients with multiple myeloma. No abnormalities were detected in the c-myc and bcl-1 genes, but two of 17 patients were found to have N-ras mutations by differential oligonucleotide hybridization and dideoxynucleotide sequencing following amplification by polymerase chain reaction. Bone marrow DNA from both patients had identical missense mutations of N-ras codon 61 changing CAA to AAA, resulting in a substitution of lysine for glutamine in the encoded protein. Multiple myeloma is the first mature B cell neoplasm found to harbor ras mutations.     

Glycine-cysteine substitution at codon 13 of the N-ras proto-oncogene in a human T cell non-Hodgkin's lymphoma

Tumor-derived DNA from a non-Hodgkin's (T cell) lymphoma patient, assayed by NIH3T3 transfection followed by inoculation of cells into nude mice, was found to contain an activated N-ras proto-oncogene. The mode of activation was determined by hybridization with N-ras-specific oligonucleotide probes detecting mutations at codons 12, 13 and 61. A transversion in codon 13 (GGT----TGT) resulting in replacement of glycine13 by cysteine13 in ras p21 protein was found. The mutation was detected in DNA from mouse tumors induced by transfected NIH3T3 cells and in DNA from patient tumor lymphoblasts. The patient was heterozygous for this mutation. These data identify the first base of codon 13 as a novel mutation site in ras genes and indicate that cysteine at position 13 of the ras p21 is a transforming substitution.     

Activated N-ras gene was found in human hepatoma tissue but only in a small fraction of the tumor cells

Activated N-ras gene was isolated from human hepatoma tissue by DNA transfection assay coupled with the neomycin selection method and molecular cloning and a point mutation in the codon 61 (CAA----AAA) was noted. However, examination of the proportion of the mutated N-ras gene in the tumor part by molecular cloning and by hybridization using synthetic oligonucleotide probes indicated that the mutated gene occurred with very low frequency. The activated N-ras gene appears located only in a small fraction of the tumor cells. The experimental data indicate activation of this gene as possibly not the major cause of carcinoma, but rather a manifestation of tumor heterogeneity.     

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.     

Analysis of ras DNA sequences in rat renal cell carcinoma

The DNA sequences for Ha-, Ki-, and N-ras were determined in six cell lines derived from independent rat hereditary renal cell carcinomas (RCC). Genomic regions encompassing codons 12, 13, and 61 of Ha-ras, Ki-ras, and N-ras, and codon 117 of Ha-ras were PCR amplified and directly sequenced. The DNA sequences of Ha-ras and Ki-ras were normal in all lines tested, as were the codon 12 and 61 sequences of N-ras. However, DNA sequence variations that could code for amino acid substitutions were observed in codons 13, 14, and 18 of N-ras in all the lines. The codon 13 Gly----Val alteration observed was consistent with activating N-ras mutations previously reported. When normal kidney DNA from rats with the hereditary tumor syndrome was sequenced, the same N-ras sequence variations observed in the tumor lines were found. DNA from outbred Long-Evans and inbred Fischer rats also had the altered N-ras sequences. The variant N-ras sequence was not observed in PCR-amplified N-ras cDNA from the RCC lines. Thus, tumor-associated activation of ras oncogene appears to be an infrequent event in spontaneous rat RCC. In addition, these data indicate that rats contain an N-ras DNA polymorphism that appears to be a species-specific anomaly.     

Rapid Screening of Mutant N-ras Alleles by Analysis of PCR-Induced Restriction Sites: Allele Specific Restriction Analysis (ASRA)

We have developed a rapid screening method for analysis of codon 12, 13 and 61 N-ras gene mutations, since these mutations have been observed in approximately 25% of patients with acute myeloid leukemia and myelodysplastic syndromes. The method, termed allele specific restriction analysis (ASRA), involves polymerase chain reaction amplification of DNA or RNA using a mismatched primer which introduces appropriately positioned base substitutions in N-ras and creates a restriction site provided the adjacent sequence is normal. Simultaneous analysis of codons 12 and 61 is also possible by the use of a multiprimer reaction mixture. Resistance of the amplified product to digestion indicates the presence of a mutation in the original template. Since ASRA allows simultaneous analysis of mutant and wild type sequences in DNA and RNA, an estimate of the ratio of gene copies and relative expression of N-ras alleles can be obtained for heterozygous individuals.     

Spontaneous Ha-ras gene activation in cultured primary murine keratinocytes: consequences of Ha-ras gene activation in malignant conversion and malignant progression

The activation of the c-Ha-ras gene and its contribution to the tumorigenic phenotype were examined in cultured mouse keratinocytes and squamous tumors using transfection into NIH 3T3 cells and nucleic acid hybridization. When normal keratinocytes were cultured in medium with 0.05 mM Ca2+ (low Ca2+ medium), many cells died within 2-3 wk, while others formed rapidly growing foci that could be subcultured. These rapidly growing cells produced benign tumors when grafted to nude mice and possessed a heterozygous mutation in the c-Ha-ras gene with an A----T transversion in codon 61. Fibroblast-conditioned low Ca2+ medium prevented cell death, focus formation, c-Ha-ras gene mutation, and tumorigenicity. Thus, suboptimal culture conditions favored a spontaneous mutation in codon 61 of the c-Ha-ras gene of keratinocytes. When a v-Ha-ras gene was introduced into normal keratinocytes by a replication-defective retrovirus, the recipient cells produced papillomas in vivo, and after 2 mo, 60% of the tumors converted to squamos cell carcinomas. None of the 22 converted tumors had an endogenous c-Ha-ras gene mutation at codon 61. However, the A----T transversion mutation developed when these carcinoma cells were cultured in low Ca2+ medium but not in fibroblast-conditioned medium. Cells with both an exogenous v-Ha-ras and an activated c-Ha61-ras gene produced undifferentiated, rapidly lethal carcinomas, while cells with only v-Ha-ras maintained the squamous carcinoma phenotype. Undifferentiated carcinomas also developed when the v-Ha-ras gene was introduced into papilloma cells with a chemically induced endogenous c-Ha61-ras gene mutation. These results suggest that mutation in the c-Ha-ras gene can contribute to initiation, malignant conversion, and malignant progression in skin carcinogenesis, and gene dosage may determine the phenotype expressed.     

Mechanism of activation of human ras genes cloned from a gastric adenocarcinoma and a pancreatic carcinoma cell line

We have analyzed the mechanism of activation of two human ras oncogenes. We have also identified a rasN gene from a human gastric adenocarcinoma which efficiently induced both morphological transformation and tumorigenicity of NIH3T3 cells in a transfection assay. The rasN gene in tumor tissue DNA did not appear to be rearranged or amplified. A molecular clone, which contained an EcoRI fragment spanning the first and second rasN exons, was molecularly cloned directly from the human tumor DNA. Chimeric constructions and DNA sequencing defined the mechanism of activation of the gene as a mutation in the 61st amino acid codon substituting arginine for glutamine. Normal DNA isolated from Epstein-Barr virus immortalized lymphocytes derived from the same patient did not induce morphological transformation or tumorigenicity in NIH3T3 cells. A cloned cell line isolated from the human pancreatic carcinoma cell line Panc1 had previously been shown to contain an activated rasK-2. Sequence analysis of the cloned transfected gene reveals a G to A change within codon 12, which is presumably responsible for its biological activity. This represents the first identification of a codon 12 aspartic acid substitution of a c-rask oncogene from a human tumor-derived cell line.     

Activation of protooncogenes in spontaneously occurring non-liver tumors from C57BL/6 x C3H F1 mice

The C57BL/6 x C3H F1 (hereafter called B6C3F1) mouse is an important animal model for long-term carcinogenesis studies. Maintained under normal laboratory conditions, these mice develop various types of spontaneous tumors during their lifetime. Activated Ha-ras genes have been detected in 66% of spontaneous hepatocellular tumors in the B6C3F1 mouse [Reynolds et al., Science (Washington DC), 237:1309, 1988]. In this study 49 spontaneous non-liver tumors were investigated for oncogene activation by DNA transfection techniques. Of the 49 tumor DNAs analyzed, only 5 yielded multiple foci in the NIH 3T3 focus assay: 2 of 10 pulmonary adenocarcinomas; 0 of 25 lymphomas; 2 of 2 Harderian gland adenomas; 0 of 1 adenocarcinoma of the small intestine; 1 of 6 malignant skin tumors; 0 of 4 hemangiosarcomas; and 0 of 1 lung metastasis of a hepatocellular carcinoma. DNA from six lymphomas which were negative in the NIH 3T3 focus assay were further analyzed for transforming genes by the nude mouse tumorigenicity assay. One of the five lymphomas tested positive with this assay. Southern blot analysis identified five activated ras genes: H-ras in two Harderian gland adenomas; K-ras in one pulmonary adenocarcinoma and in one s.c. adenocarcinoma; and N-ras in one lymphoma. The mutations involved were CG to AT and AT to TA in codon 61 of the Ha-ras genes, GC to AT or TA in codon 12 of the K-ras genes, and a GC to AT mutation in codon 12 of the N-ras gene. Transformant DNA from a pulmonary adenocarcinoma which yielded multiple foci in the transfection assay did not hybridize to DNA probes specific for the K-, H-, and N-ras, raf, neu, and met genes. Thirteen additional tumor DNAs yielded a single focus in the NIH 3T3 transfection assay. The transformant DNAs retransmitted in a second cycle transfection assay. Rearranged and/or amplified raf genes were detected in six of the transformant DNAs. At present we do not know whether these activated raf genes were present in the original tumor DNA. The other seven transformant DNAs did not hybridize with any of the above mentioned specific DNA probes utilized in Southern blot analysis. Unlike liver tumors, the activation of ras protooncogenes is not a frequent event in the development of spontaneous non-liver tumors of the B6C3F1 mouse. The results from this study should aid in understanding the neoplastic development associated with exposure to chemical carcinogens in the B6C3F1 mouse.     

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.     

Analysis of oncogene alterations in human endometrial carcinoma: prevalence of ras mutations

The molecular genetics of human endometrial carcinoma have yet to be defined to any significant extent. Cell lines from 11 endometrial carcinomas were examined for alterations in proto-oncogenes that might predictably be present, based on existing data from the better-characterized human carcinomas of the uterine cervix, ovary, and breast. Codons 12, 13, and 61 of the Ha-ras, Ki-ras, and N-ras genes were examined for possible point mutations, and the c-erbB2/neu, c-myc, and epidermal growth factor receptor (EGFR) genes were examined for amplification or overexpression. Ras mutations were found in seven of 11 (64%) tumors, including three in codon 61 of Ha-ras (CAG----CAT) and four in codon 12 of Ki-ras (GGT----GAT in two and GGT----GTT in two). No evidence was found for amplification or overexpression of the c-erbB2 or EGFR genes in any tumor. One tumor contained amplified c-myc sequences and exhibited relative overexpression of c-myc. These data suggest that the amplification or overexpression of several proto-oncogenes frequently observed in other human gynecologic and breast tumors are not prevalent in endometrial carcinoma and that ras gene mutations are relatively common in this tumor type.     

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.