Recurrent breast cancer and an adenocarcinoma of the lung occurring in one patient: c-myc oncogene amplification and K-ras codon 12 point mutation as tumour markers.
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Adenocarcinoma↗
Biomedical subjects
J L Bos
Publications and source records attributed to J L Bos.
E1A products of adenoviruses reduce the expression of cellular proliferation-associated genes.
Products of the adenovirus (Ad) E1A region are capable of modulating the expression of a variety of genes, both of viral and cellular origin. We have investigated the expression of two proliferation-associated genes, c-myc and JE, in various adenovirus-transformed cell lines. Rodent and human cell lines, transformed as primary cultures by the E1 region of Ad5 or of Ad12, exhibit a markedly reduced expression of both genes. Ad5 E1-transformed cells show a more pronounced reduction of c-myc RNA than Ad12 E1-transformed cells. Even the very low levels of E1A products in Ad5 E1A-immortalized cells are sufficient for this reduction. Analysis of c-myc and JE expression in cell lines, transformed by other viral or cellular oncogenes, indicated that this reduction is specific for adenoviruses. These observations show that E1A does not contribute to cellular transformation by activating the proliferation-associated genes c-myc and JE, but instead suggest an alternative mechanism of E1A-mediated transformation.
Adenovirus Early Proteins↗
Ras oncogenes in hematopoietic malignancies.
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Genes, ras↗
Activation of N-ras induced by ultraviolet irradiation in vitro.
Ultraviolet (UV) light is an important environmental mutagen and one of the major factors in the etiology of skin cancer in man. In this study we have addressed the question whether UV light can activate in vitro the N-ras proto-oncogene into an active oncogene, and whether pyrimidine cyclobutane dimers or (6-4) photoproducts are the premutagenic lesion. To that end, the cloned human N-ras proto-oncogene (pN-ras) was irradiated with UV light (254 nm) which results in the formation of cyclobutane dimers and (6-4) photoproducts and the irradiated DNA was transfected into Rat-2 cells. Whereas untreated pN-ras never gave foci upon transfection into Rat-2 cells, UV-irradiated pN-ras (100-300 J.m-2) yielded 1-2 foci/micrograms DNA. The DNAs of 49 primary foci were subjected to a second round of transfection. 13 of these were positive. The DNAs that did not give rise to secondary foci invariably contained high copy numbers of pN-ras. The DNAs from the secondary foci had only a low copy number of pN-ras. Using synthetic oligonucleotide probes we have found that all N-ras genes that gave rise to secondary foci contained a mutation at positions in codon 12 or 61. Only one primary transformant, negative in the secondary transformation assay, was shown to harbor a mutated N-ras gene. From these results we conclude that UV light can activate N-ras genes. Mutations were preferentially found at the 61st codon (11), harboring a TT doublet, while only 3 mutations were found at the 12th codon and none at the 13th codon, both harboring CC doublets. Of the 14 point mutations analysed 6 represented transitions and 8 transversions. No specific base-substitution could be regarded as predominant. Furthermore, treatment of the irradiated N-ras plasmids with photoreactivating enzyme prior to transfection, which specifically monomerizes cyclobutane dimers but not other photoproducts, reduced the transformation frequency several fold. None of the 36 DNAs from the primary foci obtained after transfection of photoreactivating enzyme-treated N-ras gave rise to secondary foci. These DNAs all contained high copy numbers of unmutated pN-ras. From this result we conclude that cyclobutane dimers, and not (6-4) photoproducts, are the major premutagenic lesions, responsible for the activation of N-ras. The possible role of ras genes as targets for UV-induced carcinogenesis is discussed.
Animals↗
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.
Carcinoma↗
Mutational activation of the K-ras oncogene. A possible pathogenetic factor in adenocarcinoma of the lung.
To define the role of cellular oncogenes in human cancers, we studied the prevalence of mutational activation of ras oncogenes in untreated non-small-cell lung cancer. Genomic DNA was extracted from 39 tumor specimens obtained by thoracotomy and was examined for activating point mutations in codons 12, 13, and 61 of the H-ras, K-ras, and N-ras genes. A novel, highly sensitive assay based on oligonucleotide hybridization following an in vitro amplification step was employed. The K-ras gene was found to be activated by point mutations in codon 12 in 5 of 10 adenocarcinomas. Two of these tumors were less than 2 cm in size and had not metastasized. No ras gene mutations were observed in 15 squamous-cell carcinomas, 10 large-cell carcinomas, 1 carcinoid, 2 metastatic adenocarcinomas from primary tumors outside the lung, and 1 small-cell carcinoma. An approximately 20-fold amplification of the unmutated K-ras gene was observed in a tumor that proved to be a solitary lung metastasis of a rectal carcinoma. We conclude that mutational K-ras activation may be an important early event in the pathogenesis of adenocarcinoma of the lung but that amplification of ras genes or mutational activation of H-ras or N-ras does not play a major part in non-small-cell lung cancer.
Adenocarcinoma↗
The human c-Kirsten ras gene is activated by a novel mutation in codon 13 in the breast carcinoma cell line MDA-MB231.
We have detected amplified human Ki-ras sequences in tumorigenic NIH 3T3 cells transfected with genomic DNA from the human breast carcinoma cell line MDA-MB231. Hybridization of synthetic oligonucleotides specific for human Ki-ras sequences showed a mutation at codon 13. The polymerase chain reaction with Ki-ras specific amplimers revealed a guanosine to adenosine transition at the second position of codon 13, resulting in a substitution of glycine by aspartic acid. The codon 13 mutation is also detected in one Ki-ras allele of the MDA-MB231 cell line.
Animals↗
Activation of adenovirus 5 E1A transcription by region E1B in transformed primary rat cells.
The human adenovirus 5 E1A region can immortalize primary cultures of baby rat kidney cells, but requires the presence of the E1B region for complete oncogenic transformation. One of the effects of the E1B region in the transformation process is the activation of E1A expression. We have investigated the mechanism of this stimulation of E1A expression using nuclear run-on assays with nuclei from Ad5 E1A- and Ad5 E1-transformed cells. It was found that E1B enhances E1A at the level of transcription-initiation. This activation is mainly observed when the E1A and E1B regions are integrated simultaneously into the cellular genome and only minimally when these genes are integrated separately, strongly suggesting that a close physical linkage of these regions is essential for the observed effect.
Adenovirus Early Proteins↗
Detection of alpha-1-antitrypsin deficiency variants by synthetic oligonucleotide hybridization.
Oligonucleotide probes, specific for the two most common deficiency variants, Z and S, of alpha-1-antitrypsin have been successfully applied for the diagnosis at the DNA-level. The possible presence of silent alleles necessitates a careful study of the parents both at the protein- and DNA-level in prenatal diagnostic cases.
Amino Acid Sequence↗
RAS gene mutations in acute and chronic myelocytic leukemias, chronic myeloproliferative disorders, and myelodysplastic syndromes.
We report on investigations aimed at detecting mutated RAS genes in a variety of preleukemic disorders and leukemias of myeloid origin. DNA transfection analyses (tumorigenicity assay) and hybridization to mutation-specific oligonucleotide probes established NRAS mutations in codon 12 or 61 of 4/9 acute myelocytic leukemias (AML) and three AML lines. Leukemic cells of another AML patient showed HRAS gene activation. By using a rapid and sensitive dot-blot screening procedure based on the combination of in vitro amplification of RAS-specific sequences and oligonucleotide hybridization we additionally screened 15 myelodysplastic syndromes, 26 Philadelphia chromosome-positive chronic myelocytic leukemias in chronic or acute phase, and 19 other chronic myeloproliferative disorders. A mutation within NRAS codon 12 could thus be demonstrated in a patient with idiopathic myelofibrosis and in another with chronic myelomonocytic leukemia. Moreover, mutated NRAS sequences were detected in lymphocytes, in granulocytes, as well as in monocytes/macrophages of the latter case.
DNA, Neoplasm↗
Mutations in N-ras predominate in acute myeloid leukemia.
Using synthetic oligomers we investigated fresh samples of acute myeloid leukemia (AML) for the presence of mutated ras oncogenes. Our original results showed that five of eight samples contained a mutation in codon 13 of the N-ras gene. In a subsequent study involving 37 samples, we found only one N-ras-13 mutation, and, in addition, mutations in codon 61 of the N-ras gene in four cases and a mutation in codon 12 of the Ki-ras gene in two cases. Amplification of ras genes was not observed. We conclude that in approximately 20% to 25% of AML cases, a mutated ras oncogene is present, predominantly the N-ras gene. The occurrence of mutations does not correlate with the cytological features of the leukemia.
Base Sequence↗
Transforming properties of a 15-kDa truncated Ad12 E1A gene product.
A mutant Ad12 E1A region (Ad12 R11E1A) was constructed, which directs the synthesis of only a 15-kDa N-terminal E1A product. When controlled by the SV40 early promoter plus enhancer region (SVR11E1A) this mutant E1A region is capable of immortalizing primary baby rat kidney (BRK) cells, showing that the information essential for immortalization is located in the N-terminal part of region E1A and is shared by the 13 S and 12 S mRNA gene products. This immortalization is thought to be an essential step in the process of oncogenic transformation. Primary BRK cells transformed by SVR11E1A in the presence of Ad12 E1B are nononcogenic. This implies that the E1A region also codes for activities required for oncogenicity. However, in the presence of an activated c-Ha-ras oncogene the SVR11E1A region can oncogenically transform primary BRK cells, showing that the c-Ha-ras oncogene not only can complement for the Ad12 E1B region, but also for the E1A function lost by the R11 deletion.
Animals↗
A human gastric carcinoma contains a single mutated and an amplified normal allele of the Ki-ras oncogene.
The DNA from various human tumors and tumor cell lines was screened for the presence of mutated ras oncogenes with synthetic oligonucleotide probes, as well as with the NIH/3T3 cell transfection assay. Among the various mutations found we discovered two novel Ki-ras mutations in codon 12: gly to ala and gly to ser. A gastric carcinoma was found to possess a single mutated Ki-ras allele (gly-12 to ser), as well as a 30-50 fold amplified normal allele. This implies that two activating steps must have occurred in this malignancy.
Alleles↗
A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides.
To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155-9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [Science 230 (1985) 1350-1353] and results in a more than 10(4)-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 microgram of tumor DNA.
Cell Line↗
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.
Acetoxyacetylaminofluorene↗
Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene.
We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene, transformed the primary cells with a low frequency. Ad5 E1A plus Ha-ras-transformed cells differed in phenotype from cells transformed by Ad12 E1A plus Ha-ras. The cells expressing Ad5 E1A appeared highly transformed and practically failed to adhere to plastic. This phenotype may be due to the virtually complete absence of fibronectin gene expression in these cells. In contrast, the cells expressing Ad12 E1A were flatter and adhered to plastic, whereas fibronectin gene expression was reduced but not absent. The oncogenic potential of the two types of E1A plus ras-transformed cells was tested by their injection into both athymic nude mice and weanling syngeneic rats. The Ad5 E1A plus ras-transformed cells were found to be highly oncogenic in both animal species, whereas the Ad12 E1A plus ras-transformed cells were only weakly oncogenic in both syngeneic rats and nude mice. The difference in oncogenic potential of the Ad5 E1A plus ras- and the Ad12 E1A plus ras-transformed cells is discussed in terms of the different capacities of the Ad5 and Ad12 E1A-encoded proteins to modulate cellular gene expression.
Adenoviruses, Human↗
Absence of oncogene amplifications and occasional activation of N-ras in lymphoblastic leukemia of childhood.
To examine whether determination of (1) the copynumber or restriction pattern of certain oncogenes or (2) the mutational activation of the N-ras gene might contribute to the risk classification of acute lymphoblastic leukemia of childhood (ALL), we investigated DNA isolated from lymphoblasts of untreated patients. Restriction enzyme analysis of cellular oncogenes was performed on DNA of 25 patients. No rearrangements could be demonstrated within or near the genes c-myc, c-myb, c-abl, bcr, c-Ki-ras, and N-ras. No amplifications of these genes nor of N-myc or c-Ha-ras were present. Eight of 21 patients were heterozygote for "rare" Ha-ras allelic restriction fragments that have been associated with an increased risk of developing a malignancy. These patients were clinically indistinguishable from patients lacking these fragments. The breakpoint cluster region (bcr) that is rearranged in all patients with Philadelphia chromosome positive chronic myeloid leukemia, was normal in all cases, including at least one patient with Philadelphia chromosome positive ALL. A 2.8 kb HindIII fragment of a hitherto unknown gene or pseudogene related to v-myb probably derives from the Y chromosome. Nineteen patients were examined for point mutations in the N-ras gene, using a novel synthetic oligonucleotide hybridization assay. In two patients activating point mutations were present, both in positions 1 of the 12th codon. Both patients were somewhat older than the others (16 and 11 years), had L2 morphology, and were shown to have high growth fractions of tumor in their bone marrow.
Adolescent↗
Three different mutations in codon 61 of the human N-ras gene detected by synthetic oligonucleotide hybridization.
The activation of ras genes in naturally occurring tumors has, thus far, been found to be due to mutations in codon 12 or 61 resulting in single amino acid substitutions. We have used highly labeled synthetic oligonucleotides to detect mutations in these codons and to determine the exact position of the mutation. Using this approach we have found three different mutations in codon 61 of the N-ras gene of various human tumor cell lines. In the fibrosarcoma line HT1080 the first nucleotide of the codon is mutated; in the promyelocytic line HL60 the second and in the rhabdomyosarcoma line RD301 the third nucleotide. For RD301 this implies that the normal glutamine residue at position 61 is replaced by histidine. In addition to the mutated N-ras gene the three cell lines have a normal N-ras gene which is indicative of the dominant character of the mutations.
Base Sequence↗