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.     

Identification by transfection of transforming sequences in DNA of human T-cell leukemias.

DNA from human T-cell leukemia cell lines was tested for focus-inducing activity on cultures of NIH 3T3 cells. Three leukemias yielded DNA active in this assay; restriction enzyme sensitivity of this activity indicated that similar, relatively large DNA sequences were involved. Southern blot analysis revealed conserved size classes of restriction fragments containing human repetitive (Alu) sequences in serially transfected foci derived from the active DNAs. Similar blot hybridizations with a probe specific for the human N-ras oncogene detected a 9-kilobase EcoRI fragment in all cases. DNA containing this fragment from one of the leukemias, molecularly cloned in bacteriophage lambda, displayed highly amplified focus-inducing activity in transfection assays. Thus, the N-ras oncogene appears to be active in these three human leukemias of T-cell origin.     

Amplification of the proto-neu oncogene facilitates oncogenic activation by a single point mutation.

To determine whether the amplification of the proto-neu oncogene (also called c-erbB-2) plays a role in tumorigenicity, we previously generated an NIH 3T3 transfectant (DHFR/G-8) that carried the amplified proto-neu gene. The DHFR/G-8 cells exhibited normal morphology. Their growth curve was similar to that of NIH 3T3 cells but was different from that of the B104-1 cell, and NIH 3T3 transfectant that carries the activated neu oncogene. When injected into nude mice, B104-1 cells produced tumors within 2 weeks, whereas the DHFR/G-8 cells did not produce tumors until 3 months after injection, and the NIH 3T3 cells did not produce any tumors even after 3 months. The tumors produced by the injection of the DHFR/G-8 cells were excised and grown in culture. The cells derived from the tumors were of transformed morphology and highly tumorigenic. The DNAs from the tumor cells were transfected into NIH 3T3 cells. The transfection resulted in foci on the NIH 3T3 monolayer. Southern analysis indicated that the foci derived from the transfection contained the neu gene. Using oligonucleotides as probes, the neu gene in the foci was found to carry a single-point mutation identical to the one previously found in the rat neuroblastoma and glioblastoma induced by the ethylnitrosourea. We conclude that the DNA region encoding the transmembrane domain of neu is a hot spot for converting the proto-neu gene into an activated oncogene and that amplification of the proto-neu gene facilitates mutation of the hot spot.     

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.     

High frequency of N-ras activation in acute myelogenous leukemia

Using the NIH/3T3 cell transfection assay, activated cellular oncogenes have been detected in around 10% to 20% of human tumors. From a series of DNA preparations from tissues infiltrated with acute myelogenous leukemia (AML), 50% (3/6) caused transformation of NIH/3T3 cells. Thus AML appears to be the human tumor with the highest frequency of oncogenes detected by DNA transfection. In each case the oncogene involved was N-ras, a member of the ras gene family. Biologic and clinical parameters of AML patients with and without N-ras oncogenes in their tumors are discussed.     

Activation of ras oncogene in aflatoxin-induced rat liver carcinogenesis.

The presence of activated transforming genes was investigated in four primary aflatoxin-induced rat liver tumors in male Fischer rats, in two cell lines generated from such tumors, in an epithelial liver-derived nontransformed cell line, and in the latter cell line after transformation by aflatoxin B1 in vitro. When DNA extracted from these sources was transfected into NIH 3T3 cells, negative results were obtained from focus assays. Cotransfection of these DNA samples with a gene for resistance to G418, followed by selection for resistance to that antibiotic, and tumorigenicity testing in nude mice demonstrated DNA-mediated transfer of the neoplastic phenotype in all cases except for DNA from the nontransformed cell line. DNA extracted from these primary nude mouse tumors used in a secondary round of transfection with NIH 3T3 cells gave positive results in focus assays, which were conserved through succeeding rounds of transfection. By use of appropriate radiolabeled probes, activated ras oncogenes were detected in all samples. N-ras activation was detected in three of the primary rat liver tumors and both hepatoma cell lines. Ki-ras activation was detected in one primary rat liver tumor, and Ha-ras activation was detected in the cell line transformed in vitro with activated aflatoxin B1. The activated Ki-ras oncogene was further characterized by use of synthetic oligonucleotide probes and was shown to contain a G----A transition at the second nucleotide in codon 12.     

Detection of a raf-related and two other transforming DNA sequences in human tumors maintained in nude mice.

High molecular weight DNAs prepared from a variety of human tumors maintained in nude mice were assayed for their ability to transform NIH 3T3 cells. DNAs from 4 of 21 tumors tested induced transformed foci in cultures of NIH 3T3 cells. They were from a Ewing sarcoma line, a glioblastoma line, a leiomyosarcoma line, and a lung carcinoma line. Hybridization analyses of the NIH 3T3 transformant DNAs with a human repetitive sequence as probe revealed that four distinct transforming DNA sequences were transferred to NIH 3T3 cells from the four tumor lines. The transforming DNA in a lung carcinoma line was a human homologue of the oncogene of Kirsten murine sarcoma virus (Ki-ras). On the other hand, the three other transforming DNAs showed no similarity to any known human transforming gene detected by the NIH 3T3 transformation assay. Further analyses with a series of cloned oncogenes as probes revealed that the transforming DNA in a glioblastoma line was a human homologue of the oncogene of 3611-murine sarcoma virus (raf). However, the two transforming DNAs in a Ewing sarcoma line and a leiomyosarcoma line had no sequence homology to any of the cloned oncogenes.     

Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen.

The neu oncogene encodes a 185-kDa transmembrane glycoprotein tumor antigen, termed p185. We have recently described a monoclonal antibody reactive with a cell surface domain of the p185 molecule. In vivo treatment with this anti-p185 monoclonal antibody was able to significantly inhibit the tumorigenic growth of neu-transformed NIH 3T3 cells implanted into nude mice. Such treatment had no effect on the tumorigenic growth of Ha-ras-transformed NIH 3T3 cells. Furthermore, anti-p185 antibody treatment was able to inhibit the growth of the rat neuroblastoma cells from which the neu oncogene was initially isolated. These results demonstrate that a monoclonal antibody reactive with the extracellular domain of an oncogene-encoded protein can exert a significant antitumor effect; such antibodies may prove useful in the therapy of certain malignancies.     

Isolation of transforming sequences of two human lung carcinomas: structural and functional analysis of the activated c-K-ras oncogenes.

Human lung tumors PR310 and PR371 maintained in nude mice contain activated c-K-ras oncogenes detectable by the ability of their DNAs to induce the morphological transformation of NIH 3T3 mouse fibroblasts. Using phage libraries constructed with DNA from NIH 3T3 mouse fibroblast transformants, we have isolated human sequences that span greater than 40 kilobase pairs of the c-K-ras oncogene. Based on the conservation of these human sequences in mouse fibroblast transformants, we conclude that the transforming ability of the oncogene activated in these tumors resides within a 43- to 46-kilobase-pair DNA region. No clear differences were observed between the structures of the PR310 and PR371 cloned oncogene sequences. Nucleotide sequence analysis in concert with DNA transfection experiments suggests that the PR371 oncogene has been activated by a single base change in the first exon, which results in the substitution of cysteine for glycine in position 12 of the predicted amino acid sequence. The genetic alteration responsible for the transforming activity of the PR310 oncogene, however, does not reside in the first exon. These results indicate that the activation of the c-K-ras oncogene in human lung cancer can occur by different mutational events.     

Effective tumor immunotherapy directed against an oncogene-encoded product using a vaccinia virus vector.

We have constructed a vaccinia virus recombinant that expresses the extracellular domain of the rat neu oncogene-encoded protein, a 185-kDa transmembrane glycoprotein termed p185. Strain NFS mice immunized with this recombinant virus developed a strong antibody response against the neu oncogene product and were fully protected against subsequent tumor challenge with neu-transformed NIH 3T3 cells. No tumor immunoprotection was found when recombinant virus-immunized mice were challenged with Ha-ras-transformed NIH 3T3 cells. These data indicate that immunization with a single oncogene-encoded antigen can fully and specifically protect animals against tumor cells bearing this antigen.     

Identification and expression of human c-Ha-ras and c-sis sequences in NIH3T3 transformants

High-molecular-weight DNAs from 5 bladder carcinomas were used in transfection of mouse NIH3T3 cells. The manifestation of heterologous oncogene(s) expression in NIH3T3 cells was morphological transformation very often accompanied by changes in growth characteristics of recipient cells. In DNA samples from secondary NIH3T3 transformants human c-Ha-ras and c-sis sequences were identified. In some secondary transformants these sequences were expressed. On the basis of change of the growth characteristics of some secondary transformants we could expect the integration and expression of another human gene(s) for growth factor or growth factor receptor or even activation of mouse genes. We did not manage to identify any Alu sequences in some secondary transformants carrying human c-Ha-ras sequences. On the other hand, it has not been revealed yet that BamHI DNA fragments carrying c-Ha-ras gene contained any Alu sequence. So, the identification of Alu sequences does not have to be the first step in investigation of DNA samples from NIH3T3 transformants.     

Characterization of mutagen-activated cellular oncogenes that confer anchorage independence to human fibroblasts and tumorigenicity to NIH 3T3 cells: sequence analysis of an enzymatically amplified mutant HRAS allele.

Treatment of diploid human fibroblasts with an alkylating mutagen has been shown to induce stable, anchorage-independent cell populations at frequencies (11 X 10(-4) consistent with an activating mutation. After treatment of human foreskin fibroblasts with the mutagen benzo[a]pyrene (+/-)anti- 7,8-dihydrodiol 9,10-epoxide and selection in soft agar, 17 anchorage-independent clones were isolated and expanded, and their cellular DNA was used to cotransfect NIH 3T3 cells along with pSV2neo. DNA from 11 of the 17 clones induced multiple NIH 3T3 cell tumors in recipient nude mice. Southern blot analyses showed the presence of human Alu repetitive sequences in all of the NIH 3T3 tumor cell DNAs. Intact, human HRAS sequences were observed in 2 of the 11 tumor groups, whereas no hybridization was detected when human KRAS or NRAS probes were used. Slow-migrating ras p21 proteins, consistent with codon 12 mutations, were observed i in the same two NIH 3T3 tumor cell groups that contained the human HRAS bands. Genomic DNA from one of these two human anchorage-independent cell populations (clone 21A) was used to enzymatically amplify a portion of exon 1 of the HRAS gene. Direct sequence analysis of the amplified DNA indicated equal presence of a wild-type (GGC) and mutant (GTC) allele of the HRAS gene. The results demonstrate that exposure of normal human cells to a common environmental mutagen yields HRAS GC----TA codon 12 transversions that have been commonly observed in human tumors. This oncogene as well as yet to be identified oncogene are also shown to stably confer anchorage-independence to human cells.     

Early mutation of the neu (erbB-2) gene during ethylnitrosourea-induced oncogenesis in the rat Schwann cell lineage.

The development of malignant tumors of the peripheral nervous system (schwannomas) within a defined intracranial section of the rat trigeminal nerve ("trigeminal box") was used as a model to identify genetic alterations typically associated with the process of cell-lineage-specific oncogenesis induced by exposure to N-ethyl-N-nitrosourea on postnatal day 1. All 47 trigeminal schwannomas (and 12 extracranial neurinomas) investigated carried a T.A----A.T transversion mutation at nucleotide 2012 of the neu (erbB-2) gene sequence encoding the transmembrane domain of pg185neu. This mutation was absent in all 18 tumors in the brain and spinal cord (central nervous system) isolated from the same animals. Identical observations were made in cell lines derived from N-ethyl-N-nitrosourea-induced rat schwannomas vs. brain tumors. By asymmetric PCR and mutant-specific Mnl I restriction fragment length analyses, cells carrying the mutant neu allele became detectable and could be localized within the trigeminal box as early as 7 days after the carcinogen pulse. The proliferation rate of the mutant cells strongly exceeded that of the wild-type cells up to the time of maturation of the trigeminal nerve around postnatal day 30 and thereafter to a lesser extent until the appearance of schwannomas. A specific mutation of the neu gene thus represents a very early, probably the first, step in the malignant conversion of immature rat Schwann cells exposed to N-ethyl-N-nitrosourea in vivo and is diagnostic for a subset of proliferative cells at high risk of progressing toward the expression of fully malignant phenotypes. Loss of heterozygosity for the mutant neu allele is a candidate event for a critical second step in the process.     

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.     

Molecular cloning of the neu gene: absence of gross structural alteration in oncogenic alleles.

The neu gene is distantly related to the erbB gene and encodes a cell surface protein that appears to function as a growth factor receptor. To study the mechanisms that caused the conversion of the normal neu gene to an oncogenic allele, we have isolated molecular clones of the neu oncogene as well as a clone of the corresponding protooncogene. The transforming neu oncogene and the proto-neu gene clones exhibit identical restriction enzyme patterns. Amplification of the proto-neu gene in NIH 3T3 cells by means of cotransfection with a dihydrofolate reductase gene resulted in methotrexate-resistant colonies that produce high levels of normal neu-encoded p185 protein. In contrast to cells carrying low levels of the oncogene-encoded protein, these cells appeared normal. The results suggest that the lesion that led to activation of the neu gene is a minor change in DNA sequence and is apparently located in the protein-encoding region of the gene.     

Transfection of BLV containing DNA into NIH 3T3 cells

Cell DNA isolated from bovine leukosis virus (BLV) productive cell clones was transfected into the NIH3T3 cells. DNA from some cell clones was able to transform NIH3T3 cells. The transformed cells were cloned, and in 4 cell clones out of 33 bovine leukosis virus specific sequences were detected by hybridization with labeled BLV probe. According to the restriction analysis the BLV sequences were incomplete, they were rearranged, deleted, or both. The DNA from NIH3T3 transformants with BLV sequences was able to transform in the second round transfection experiments NIH3T3 cells again, but in these transformants BLV specific sequences were not detected. Cell DNA from sheep tumors induced by BLV was able to transform the NIH3T3 cells too, but BLV specific sequences were not present in the transformants. It appears that BLV specific sequences are not required for NIH3T3 cell transformation.     

Oncogenes in human tumor cell lines: molecular cloning of a transforming gene from human bladder carcinoma cells.

The presence of dominant transforming genes in human tumor cell lines has been investigated. High molecular weight DNAs isolated from cell lines established from carcinomas and sarcomas of various organs as well as from a glioblastoma and two melanomas were utilized to transfect NIH/3T3 mouse fibroblasts. The DNAs of T24 and A2182, two cell lines derived from a bladder and a lung carcinoma, respectively, and of HT-1080, a cell line established from a fibrosarcoma, were able to transform recipient NIH/3T3 cells. First-cycle transformants exhibited anchorage-independent growth and were tumorigenic in athymic and immunocompetent mice. Moreover, they contained human DNA sequences and were able to transmit their malignant phenotype in additional cycles of transfection. Southern blot analysis of T24-derived transformants showed that a single fragment of human DNA specifically cosegregated with the malignant phenotype, suggesting that it contained the T24 oncogene. Therefore, these human sequences were molecularly cloned with lambda Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated lambda T24-15A, was shown to contain a 15-kilobase-pair EcoRI insert of human cellular DNA. lambda T24-15A DNA (either intact or EcoRI digested) transformed NIH/3T3 fibroblasts with a specific activity of 20,000 focus-forming units per pmol of cloned DNA. Our results indicate that we have molecularly cloned a biologically active oncogene present in T24 human bladder carcinoma cells.     

Expression of the metastatic phenotype in cells transfected with human metastatic tumor DNA.

NIH 3T3 mouse fibroblasts form nonmetastasizing fibrosarcomas upon transformation by the Ha-ras oncogene isolated from the EJ human bladder carcinoma cell line and subcutaneous inoculation into immunocompetent NFS/NCr mice. DNA from a human metastatic tumor was transfected into these Ha-ras transformants, and one of the resulting colonies yielded a lung metastasis after subcutaneous inoculation. DNA was isolated from this metastasis and subjected to a second round of transfer into Ha-ras-transformed NIH 3T3 cells. Inoculation of these transfected cultures into mice led once again to formation of metastases, this time at a higher frequency. Examination of four of the resulting metastases revealed discrete human DNA fragments that were common to all four. These findings demonstrate that the metastatic phenotype can be transferred via DNA from cell to cell and is associated with the presence of a discrete DNA segment. This segment is not identical to the myc oncogene or to any of the frequently detected ras tumor oncogenes.