Activating mutations of the c-Ha-ras protooncogene in chemically induced hepatomas of the male B6C3 F1 mouse.

Activated c-Ha-ras protooncogenes have recently been identified in the DNA of some spontaneous hepatic tumors found in 2-year-old B6C3 F1 mice. Activation of c-Ha-ras has now been demonstrated in DNA from well-differentiated hepatomas initiated by a single dose of carcinogen given to male B6C3 F1 mice at 12 days of age. DNA from each of 25 hepatomas, induced by N-hydroxy-2-acetylaminofluorene, vinyl carbamate, or 1'-hydroxy-2',3'-dehydroestragole, containing transforming activity in the NIH 3T3 transfection assay. Southern analysis of NIH 3T3 cells transformed by DNA from 24 of these hepatomas revealed amplified and/or rear-ranged restriction fragments homologous to a Ha-ras probe. The other tumor contained an activated Ki-ras gene. Immunoprecipitation and NaDodSO4/PAGE analysis of p21 ras proteins in NIH 3T3 transformants derived from a majority of the hepatomas suggested that the activating mutations were localized in the 61st codon of the c-Ha-ras gene. Creation of a new Xba I restriction site by an AT----TA transversion at the second position of codon 61 was detected in DNA from primary tumors and NIH 3T3 cells transformed by DNA from 6 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced hepatomas. Selective oligonucleotide hybridization demonstrated a CG----AT transversion at the first position of the 61st codon in NIH 3T3 transformants derived from 7 of 7 N-hydroxy-2-acetylaminofluorene-induced hepatomas. By the same criterion, an AT----GC transition at the second position of codon 61 was the activating mutation in 1 of 7 vinyl carbamate- and 5 of 10 1'-hydroxy-2',3'-dehydroestragole-induced tumors. Thus, c-Ha-ras activation is apparently an early event in B6C3 F1 mouse hepatocarcinogenesis that results directly from reaction of ultimate chemical carcinogens with this gene in vivo.     

Characterization of c-Ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors.

Activated c-Ki-ras genes in liver tumors from rats exposed to the potent hepatocarcinogen aflatoxin B1 were analyzed to determine the nature of their activation by characterization of two c-Ki-ras alleles present in tumor-derived NIH 3T3 mouse transformants. Using selective hybridization of synthetic oligonucleotides to transformant DNA, we have determined that a single G X C to A X T base transition in either the first or second position of the 12th codon is associated with activation of the gene. Such mutations would lead to amino acid substitutions of aspartate or serine for glycine in the mutant proteins. To confirm these findings, we applied a technique for direct sequence analysis of a 90-base-pair region of the rat c-Ki-ras gene produced by primer-directed enzymatic amplification. Findings produced by this approach, which provides a convenient method to characterize mutations in multiple alleles without the necessity to clone individual genes, confirmed the presence and identity of the 12th codon mutations in the activated oncogene, as initially determined by the oligonucleotide hybridization technique.     

Mutations in c-Ki-ras oncogenes in diseased livers of winter flounder from Boston Harbor.

Livers of a natural population of winter flounder from a contaminated site in Boston Harbor were examined for the presence of oncogenes by transfection of DNA into NIH 3T3 mouse fibroblasts. Tissues analyzed contained histopathologic lesions including abnormal vacuolation, biliary proliferation, and, in many cases, hepatocellular and cholangiocellular carcinomas. Fibroblasts transfected with liver DNA samples from 7 of 13 diseased animals were effective in the induction of subcutaneous sarcomas in nude mice. Further analysis revealed the presence of flounder c-Ki-ras oncogenes in all 10 nude mouse subcutaneous tumors analyzed. Direct DNA sequencing and allele-specific oligonucleotide hybridization following amplification of the tumor DNA by the polymerase chain reaction showed mutations in the 12th codon in this gene. Analysis of DNA from all nude mouse tumors as well as the livers from which they were derived showed mutations at this codon. The mutations comprised G.C----A.T or G.C----T.A base changes resulting in substitution of serine, valine, or cysteine for glycine. Liver DNA samples from five histologically normal livers of animals from a less polluted site were ineffective in the transfection assay and showed only wild-type DNA sequences (GGT) at the 12th codon of c-Ki-ras. The prevalence of mutations in this gene region was associated with the presence of liver lesions and could signify DNA damage resulting from environmental chemical exposure.     

Activation of the c-Ki-ras oncogene in aflatoxin B1-induced hepatocellular carcinoma and adenoma in the rat: detection by denaturing gradient gel electrophoresis.

Sequence alterations in the exon 1 region of the rat c-Ki-ras gene were studied in DNA isolated from aflatoxin B1 (AFB1)-induced rat liver carcinomas and precursor lesions appearing 56 weeks after administration of the carcinogen. To detect the mutations with high sensitivity, DNA samples were analyzed by using polymerase chain reaction (PCR) amplification in conjunction with allele-specific oligonucleotide (ASO) hybridization together with a modified PCR-G+C clamp-denaturing gradient gel electrophoresis (DGGE) method. Mutations in the Ki-ras gene were present in all adenomas and carcinomas examined. The predominant mutation observed was a G.C-to-A.T base transition in codon 12 (GGT to GAT). Also present, but at low frequency, was a G.C-to-T.A base transversion in the same codon (GGT to TGT). In addition, 20% of the samples contained a G.C-to-T.A transversion in the second base position of codon 12 (GGT to GTT), a mutation not previously observed in AFB1-induced rat liver tumors. These results confirm and extend our previous findings that Ki-ras mutation is a prevalent event in hepato-cellular carcinogenesis induced in Fischer 344 rats by AFB1. The modified DGGE method described is applicable to the screening of multiple mutations in neoplastic lesions with high fidelity and sensitivity.     

Characterization of c-Ki-ras and N-ras oncogenes in aflatoxin B1-induced rat liver tumors.

c-Ki-ras and N-ras oncogenes have been characterized in aflatoxin B1-induced hepatocellular carcinomas. Detection of different protooncogene and oncogene sequences and estimation of their frequency distribution were accomplished by polymerase chain reaction, cloning, and plaque screening methods. Two c-Ki-ras oncogene sequences were identified in DNA from liver tumors that contained nucleotide changes absent in DNA from livers of untreated control rats. Sequence changes involving G.C to T.A or G.C to A.T nucleotide substitutions in codon 12 were scored in three of eight tumor-bearing animals. Distributions of c-Ki-ras sequences in tumors and normal liver DNA indicated that the observed nucleotide changes were consistent with those expected to result from direct mutagenesis of the germ-line protooncogene by aflatoxin B1. N-ras oncogene sequences were identified in DNA from two of eight tumors. Three N-ras gene regions were identified, one of which was shown to be associated with an oncogene containing a putative activating amino acid residing at codon 13. All three N-ras sequences, including the region detected in N-ras oncogenes, were present at similar frequencies in DNA samples from control livers as well as liver tumors. The presence of a potential germ-line oncogene may be related to the sensitivity of the Fischer rat strain to liver carcinogenesis by aflatoxin B1 and other chemical carcinogens.     

c-Ki-ras mutations in dysplastic fields and cancers in ulcerative colitis.

A sensitive restriction fragment length polymorphism assay and DNA sequencing were used to detect c-Ki-ras mutations in 56 specimens of colonic epithelium from 18 patients with chronic ulcerative colitis. Mutations were not detected in biopsy specimens that were negative or indefinite for dysplasia. In 4 of 8 patients with high-grade dysplasia, a c-Ki-ras codon 12 or 13 mutation was detected. In three colectomy specimens, a wide area of dysplastic cells (greater than 10 cm2) contained a specific ras mutation. In two of these specimens, an invasive cancer contained a c-Ki-ras mutation identical to that found in adjacent dysplastic epithelium. These studies indicate that mutations of c-Ki-ras may be an excellent molecular genetic marker to map dysplastic fields and invasive cancer in ulcerative colitis.     

Mutations activating human c-Ha-ras1 protooncogene (HRAS1) induced by chemical carcinogens and depurination.

In vitro modification of plasmids containing the human c-Ha-ras1 protooncogene (HRAS1) with the ultimate carcinogens N-acetoxy-2-acetylaminofluorene and r-7, t-8-dihydroxy-t-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[alpha]pyrene (anti-BPDE) generated a transforming oncogene when the modified DNA was transfected into NIH 3T3 cells. The protooncogene was also activated by heating the plasmid at 70 degrees C, pH 4, to generate apurinic/apyrimidinic sites in the DNA. DNA isolated from transformed foci was analyzed by hybridization with 20-mer oligonucleotides designed to detect single point mutations within two regions of the gene commonly found to be mutated in tumor DNA. Of 23 transformants studied, 7 contained a mutation in the region of the 12th codon, whereas the remaining 16 were mutated in the 61st codon. Of the codon-61 mutants, 6 were mutated at the first base position (C X G), 5 at the second (A X T), and 5 at the third (G X C). The point mutations induced by anti-BPDE were predominantly G X C----T X A and A X T----T X A base substitutions, whereas four N-acetoxy-2-acetylaminofluorene-induced mutations were all G X C----T X A, and a single depurination-induced activation that was analyzed contained an A X T----T X A transversion. Together, these methods provide a useful means of determining point mutations produced by DNA-damaging agents in mammalian cells.     

Relationship between O6-alkylguanine-DNA alkyltransferase activity and N-methyl-N''-nitro-N-nitrosoguanidine-induced mutation, transformation, and cytotoxicity in C3H/10T1/2 cells expressing exogenous alkyltransferase genes.

While a great deal of evidence has directly implicated the importance of O6-alkylation of guanine in the mutagenicity of alkylating agents, evidence demonstrating the oncogenic potential of this lesion has been largely indirect. We have combined a well-studied in vitro neoplastic transformation system (using C3H/10T1/2 mouse cells) with a proven method of gene transfection for expressing the bacterial O6-alkylguanine-DNA alkyltransferase (AT; EC 2.1.1.63) repair genes ada and ogt to generate subclones which possess augmented repair capability toward specific DNA lesions. The products of these genes specifically and differentially repair O6-methylguanine (O6-MeGua), O4-methylthymine (O4-MeThy), and methylphosphotriesters. We show that the level of expression of either the ada or the ogt AT gene in C3H/10T1/2 cells directly correlates with protection against mutation to ouabain resistance by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Subclones expressing 70 fmol of AT per 10(6) cells exhibited a mutation frequency approximately 1/40th of that of clones expressing 15 fmol of AT per 10(6) cells when treated with MNNG at 0.4 micrograms/ml. Protection against mutagenesis by MNNG at 0.8 micrograms/ml, however, did not exceed 12-fold even in subclones expressing greater than 100 fmol of AT per 10(6) cells. As an MNNG dose of 0.6 micrograms/ml was sufficient to saturate more than 95% of the AT activity in any of the clones, the residual mutation frequency may have been caused by unrepaired O6MeGua lesions. In contrast to mutagenesis, protection against neoplastic transformation in vitro, in cells expressing high levels of AT, was most pronounced in cells treated with the highest dose of MNNG used (1.2 micrograms/ml). Low levels of transformation caused by MNNG at 0.4 and 0.8 micrograms/ml were not consistently inhibited in those clones. These data suggest that O6-MeGua formation is of major but not unique significance in the neoplastic transformation of C3H/10T1/2 cells by MNNG.     

Identification of an activated c-Ki-ras oncogene in rat liver tumors induced by aflatoxin B1.

Weanling male Fischer rats were administered 40 intraperitoneal injections of aflatoxin B1 (25 micrograms per animal per day) over a 2-month period. This chronic dosing regimen resulted in the sequential formation of hyperplastic foci, preneoplastic nodules, and hepatocellular carcinomas in all of the animals treated. The presence of transforming DNA sequences was detected by formation of anchorage-independent foci after transfection of tumor-derived DNA in NIH 3T3 mouse fibroblasts. Transfection of genomic DNA isolated from individual tumors from eight animals resulted in specific transforming activities ranging from 0.05 to 0.2 foci per micrograms of DNA. Primary transfectant DNAs were analyzed by Southern blot hybridization with DNA probes homologous to c-Ha-ras, c-Ki-ras, and N-ras oncogenes. A highly amplified c-Ki-ras oncogene of rat origin was detected in transformants derived from tumors in two of the eight animals tested. There was no evidence to suggest the presence of c-Ha-ras or N-ras sequences in any of the transformants. Analysis of primary liver tumor DNA showed no Ki-ras DNA amplification when compared to control liver DNA samples. Increased levels of c-Ki-ras p21 proteins were detected in 3T3 transformants containing activated rat c-Ki-ras genes. The presence of c-Ki-ras sequences of rat origin capable of inducing transformed foci can be taken as evidence that the c-Ki-ras gene has been activated in the primary liver tumors.     

N-RAS activation in the terminal stage of undifferentiated chronic myeloproliferative disease.

The relationship between activation of the N-RAS gene and the leukemic progression of undifferentiated chronic myeloproliferative disease (UCMPD) was investigated in a 71-year-old male. Hematologically, it was difficult to differentiate the UCMPD from chronic myelogenous leukemia. Chromosomal analysis revealed no Philadelphia chromosome (Ph1-), and DNA analysis revealed no BCR rearrangement (BCR-) either at the beginning or in the terminal stages of the disease. We performed a tumorigenicity assay, using NIH3T3 cells, and molecular analysis, using the polymerase chain reaction (PCR) and direct sequencing. The DNA of leukemic cells at the beginning of the leukemic progression did not show any abnormalities, but at the terminal stage of the disease the DNA showed a point mutation in codon 12 (GGT----GCT) of the N-RAS gene. Interestingly, a codon 13 mutation (GGT----GTT) was also detected by tumorigenicity assay. These observations suggest that the activated N-RAS gene contributes to the hematologic progression of UCMPD.     

Effect of 1,25-(OH)2-vitamin D3 on growth, homologous receptor and c-myc regulation in C3H/10T1/2 cells

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) receptor concentration, cell proliferation, and the steady-state level of c-myc mRNA were examined in the C3H/10T1/2 mouse embryo fibroblasts, before and after exposing the cells to 1,25-(OH)2D3. The non-transformed, logarithmically growing C3H/10T1/2 Cl 8 cells contained a high concentration of 1,25-(OH)2D3 receptor (164 fmol/mg of protein). An up-regulation of the 1,25-(OH)2D3 receptor and a potent inhibition of cell growth were observed by exposing the cells to 10 nM 1,25-(OH)2D3. The concentration of 1,25-(OH)2D3 receptor in the two chemically transformed, tumorigenic cell lines. C3H/10T1/2 Cl 16 and C3H/10T1/2 TPA 482, was 218 and 63 fmol/mg of protein, respectively. In the two transformed cell lines, 10 nM 1,25-(OH)2D3 had only negligible effect on cell growth. In the Cl 16 cells, an up-regulation of the 1,25-(OH)2D3 receptor was demonstrated, but only a weak up-regulation was found in the TPA 482 cells by the 1,25-(OH)2D3 treatment. No major changes were found in c-myc mRNA levels by the 1,25-(OH)2D3 treatment. Despite inhibition of cell growth, the steady-state level of c-myc mRNA was slightly induced (35%, mean) in the Cl 8 cells compared to control cells. In the transformed cells, no consistent change of the c-myc level was found. In contrast to earlier reports, we did not find any correlation between the 1,25-(OH)2D3 receptor and c-myc level, nor did we find any decrease of c-myc mRNA by 1,25-(OH)2D3 treatment in the C3H/10T1/2 fibroblasts.     

Homozygous deletions of p16/MTS1 gene are frequent but mutations are infrequent in childhood T-cell acute lymphoblastic leukemia

Fifty-six primary childhood T-cell acute lymphoblastic leukemia (T-ALL) samples and 17 T-ALL cell lines were examined for mutations and homozygous deletions of the p16/MTS1 gene using polymerase chain reaction single-strand conformation polymorphism and Southern blot analysis. Homozygous deletions were found in 22 primary samples (39%) and in 10 cell lines (59%). In contrast, mutations including small deletions and/or insertions were identified in only 4 primary samples (7%) and in 2 cell lines (12%). Mutations included samples (7%) and in 2 cell lines (12%). Mutations included one nonsense mutation at codon 72, one missense mutation at codon 58, one deletion (29 bp from codon 52-61), one insertion (7 bp into codon 50), and two deletion/insertions (codon 63 and intron 1). Four of the six mutations caused subsequent stop codon and presumably produced truncated p16 protein. Our results suggest that p16 gene alterations are involved in the development of T- ALLs and that the inactivation of the p16 gene occurs mainly through homozygous deletions rather than mutations.     

Haplotypes linked to three rare beta-thalassemia mutations, originally reported in Tunisia

The polymorphism of the beta-globin gene haplotypes and frameworks are useful in the determination of the unicentric and multicentric origin of a mutational event. In order to improve our knowledge of the chromosomal background of the beta-globin gene in three beta-thalassemia (thal) mutations originally reported in Tunisia, namely codons 25/26 (+T), codon 30 (G-->C) and IVS-I-2 (T-->G), we have investigated 13 unrelated individuals. There were five non transfusion-dependent patients homozygous for the IVS-I-2 (T-->G) mutation, five others were homozygous for the codon 30 (G-->C) mutation, one was a homozygote for the codons 25/26 (+T) insertion mutation and one patient was a compound heterozygote for the codon 39 (C-->T) and codon 25/26 (+T) mutations; the last patient had a betaS/codon 25/26 (+T) compound heterozygous genotype. Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based methods. The framework polymorphism was established by direct sequencing. beta-Globin gene analyses demonstrated that all IVS-I-2 (T-->G) cases were associated with haplotype IX; the codon 30 (G-->C) mutation was supported by haplotype I, while the codons 25/26 (+T) mutation was linked to haplotypes I and IX.     

Mutagenesis of the Ha-ras oncogene in mouse skin tumors induced by polycyclic aromatic hydrocarbons.

The importance of mutational activation of the Ha-ras protooncogene in polycyclic aromatic hydrocarbon-induced mouse skin tumors was investigated in a complete carcinogenesis model using repetitive applications of 7,12-dimethylbenz[a]anthracene (DMBA), or in an initiation-promotion model using a single application of dibenz[c,h]acridine (DB[c,h]ACR) or benzo[a]pyrene (B[a]BP) followed by chronic treatment with phorbol 12-myristate 13-acetate. DNA isolated from carcinomas induced by DMBA or DB[c,h]ACR, but not by B[a]P, efficiently transformed NIH 3T3 cells, and a high percentage of the transformed foci had an amplified Ha-ras gene. Restriction enzyme Southern blot analysis and DNA sequencing revealed that the amplified Ha-ras genes of the transformants had an A----T transversion in the second position of the 61st codon. The same mutation was also detected in primary tumor DNA in a high percentage of the DMBA- or DB[c,h]ACR-induced carcinomas. Identification of the mutation in NIH 3T3 cells transformed with DNA from DB[c,h]ACR-induced benign skin papillomas suggests that it is an early event in skin carcinogenesis. Thus, mutation of the 61st codon of the Ha-ras-1 gene appears to be a critical step in the formation of mouse skin tumors induced in both of the two models tested. Our analyses also delineate two other classes of hydrocarbon-induced carcinomas--namely, tumors whose DNAs efficiently transform 3T3 cells but do not contain mutated ras genes and tumors whose DNAs do not transform 3T3 cells.     

c-Ki-ras mutations in chronic ulcerative colitis and sporadic colon carcinoma

Mutations in the first exon of the c-Ki-ras protooncogene were analyzed in carcinomas and dysplasias from patients with sporadic colon cancer and chronic ulcerative colitis by a combination of histological enrichment, cell sorting, polymerase catalyzed chain reaction, and direct sequencing. In contrast to sporadic colon carcinomas, where 52% (11 of 21) contained mutations in codon 12, only 1 of 28 samples of ulcerative colitis associated carcinoma or dysplasia contained a c-Ki-ras mutation, despite the presence of aneuploid cell populations. These results suggest that a different genetic pathway for tumor progression may exist between sporadic colon carcinoma and carcinomas arising in chronic ulcerative colitis.     

Haplotypes Linked to Three Rare β-Thalassemia Mutations,Originally Reported in Tunisia

The polymorphism of the β-globin gene haplotypes and frameworks are useful in the determination of the unicentric and multicentric origin of a mutational event. In order to improve our knowledge of the chromosomal background of the β-globin gene in three β-thalassemia (thal) mutations originally reported in Tunisia, namely codons 25/26 (+T), codon 30 (G→C) and IVS-I-2 (T→G), we have investigated 13 unrelated individuals. There were five non transfusion-dependent patients homozygous for the IVS-I-2 (T→G) mutation, five others were homozygous for the codon 30 (G→C) mutation, one was a homozygote for the codons 25/26 (+T) insertion mutation and one patient was a compound heterozygote for the codon 39 (C→T) and codon 25/26 (+T) mutations; the last patient had a β^S/codon 25/26 (+T) compound heterozygous genotype. Haplotype analysis was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based methods. The framework polymorphism was established by direct sequencing.

β-Globin gene analyses demonstrated that all IVS-I-2 (T→G) cases were associated with haplotype IX; the codon 30 (G→C) mutation was supported by haplotype I, while the codons 25/26 (+T) mutation was linked to haplotypes I and IX.     

Increased radiation-induced transformation in C3H/10T1/2 cells after transfer of an exogenous c-myc gene.

C3H/10T1/2 cells were infected with a retroviral vector expressing a mouse c-myc oncogene and a drug-selection marker. The resulting cells, morphologically indistinguishable from C3H/10T1/2, displayed a greatly enhanced sensitivity to neoplastic transformation by ionizing radiation or by a chemical carcinogen. Constitutive expression of myc therefore appears to synergize with an initial carcinogenic event, providing a function analogous to a subsequent event that apparently is required for the neoplastic transformation of these cells. This cell system should prove useful in exploring early stages in radiation-induced transformation.     

Novel mutations in the MEN1 gene in subjects with multiple endocrine neoplasia-1

OBJECTIVE: To identify MEN1 gene mutations and characterize clinical manifestations in Chinese kindred with multiple endocrine neoplasia type 1 (MEN1) in Taiwan. PATIENTS AND METHODS: Eight unrelated subjects (one male and seven females, age range 26-70 years) with clinical manifestations of MEN1 were analysed. In addition, 45 relatives that included 10 affected (three males and seven females, age range 32-53 years) and 35 unaffected (17 males and 18 females, age range 15-80 years) subjects were evaluated. Genomic DNA extraction, polymerase chain reaction (PCR) and DNA sequence analysis were performed according to standard procedures. RESULTS: We identified heterozygous MEN1 gene mutations in all eight probands and 10 affected subjects as well as in 13 clinically asymptomatic relatives. Novel mutations included a missense mutation in a heterozygous mutation in exon 9 (GAC --> CAC) resulting in a substitution of aspartic acid by histidine at codon 418 (family 1); a nonsense mutation at codon 556 of exon 10 (GAG --> TAG) resulting in a stop codon and termination (family 2); a missense mutation in exon 2 (GGG --> GAG) causing the substitution of glycine by glutamic acid at codon 110 (family 3); and a deletion/insertion mutation in nucleotide 1200 of exon 8 resulting in frameshift and early termination (family 4). Affected subjects in families 5-7 shared the same C insertion at nucleotide 1650 of exon 10, similar to that previously described as a hotspot for mutation, and proband 8 had a previously described mutation in intron 4 of the MEN1 gene (IVS4-9 G --> A). We also found that 18 (58%) of our 31 MEN1 mutant carriers had clinical symptoms, whereas four (13%) had biochemical abnormalities without clinical symptoms, and nine (29%) were unaffected both clinically and biochemically. CONCLUSIONS: We have identified four novel mutations in the MEN1 gene in patients with MEN1 in Taiwan.