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.     

Transforming genes of human hematopoietic tumors: frequent detection of ras-related oncogenes whose activation appears to be independent of tumor phenotype.

We surveyed 22 human hematopoietic tumors and tumor cell lines for sequences capable of transforming NIH 3T3 cells by DNA transfection. A primary human acute myelogenous leukemia, a chronic myelogenous leukemia cell line, and cell lines derived from three independent acute lymphocytic leukemias demonstrated oncogenes capable of conferring the transformed phenotype to NIH 3T3 cells through serial cycles of transfection. One of three transforming genes associated with acute lymphocytic leukemia cells (classified as thymocyte developmental stage II) was identified as the activated cellular homologue of the Kirsten murine sarcoma virus onc gene, kis, a member of the ras family of onc genes. A transforming gene, which was demonstrated to be common to several human myeloid and lymphoid tumor cells, was shown to be a distantly related member of the ras gene family. Thus, the NIH 3T3 transfection assay commonly detects related oncogenes in human hematopoietic tumor cells. Moreover, the activation of these oncogenes appears to be independent of the specific stage of cell differentiation or tumor phenotype.     

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.     

Activated protooncogenes in human lung tumors from smokers.

Fourteen primary human lung tumor DNAs from smokers were analyzed for transforming activity by two DNA transfection assays. Activated protooncogenes were detected in 3 of 11 tumor DNAs by the NIH 3T3 focus assay, whereas activated protooncogenes were detected in 11 of 13 tumor DNAs by the NIH 3T3 cotransfection-nude mouse tumorigenicity assay. K- or NRAS genes activated by point mutation at codons 12 or 61 were detected in a large cell carcinoma, a squamous cell carcinoma, and 5 adenocarcinomas. An HRAS oncogene activated by a different mechanism was detected in an epidermoid carcinoma. One adenocarcinoma was found to contain an activated RAF gene. Two unidentified transforming genes were detected in a squamous cell carcinoma DNA and two adenocarcinoma DNAs. Eight of 10 lung adenocarcinomas that had formed metastases at the time of surgery were found to contain RAS oncogenes. No significant increase in metastasis was observed in the lung adenocarcinomas that contained one or more 6-kilobase EcoRI alleles of the LMYC gene. Overall, 12 of 14 (86%) of the lung tumor DNAs from smokers were found to contain activated protooncogenes. RAS oncogenes appear to play a role in the development of metastases in lung adenocarcinomas.     

Activation of related transforming genes in mouse and human mammary carcinomas.

High molecular weight DNAs of five tumors induced by mouse mammary tumor virus (MMTV), two mouse mammary tumors induced by a chemical carcinogen, and one human mammary tumor cell line (MCF-7) were assayed for the presence of transmissible activated transforming genes by transfection of NIH 3T3 mouse cells. DNAs of all five MMTV-induced tumors, one chemical carcinogen-induced tumor, and the human tumor cell line induced transformation with high efficiencies (approximately 0.2 transformant per micrograms of DNA). NIH cells transformed by DNAs of MMTV-induced tumors did not contain exogenous MMTV DNA sequences, indicating that MMTV-induced mammary carcinomas contained activated cellular transforming genes that were not linked to viral DNA. The transforming activities of DNAs of all five MMTV-induced tumors, the chemical carcinogen-induced mouse tumor, and the human tumor cell line were inactivated by digestion with the restriction endonucleases Pvu II and Sac I, but not by BamHI, EcoRI, HindIII, Kpn I, or Xho I. These results indicate that the same or closely related transforming genes were activated in six different mouse mammary carcinomas, induced by either MMTV or a chemical carcinogen, and in a human mammary carcinoma cell line.     

Resistance of human cells to tumorigenesis induced by cloned transforming genes.

The transformation of human cells was examined by transfection of cloned oncogenic DNAs derived from the tumor virus simian virus 40 and from the human bladder carcinoma cell line EJ into diploid fibroblasts derived from foreskin (FS-2 cells). The simian virus 40 DNA was found to induce a morphologically transformed phenotype, leading to easily detectable focus formation. Tumor antigen was produced, but the transformed cells were not tumorigenic in the nude mouse. The EJ gene, a mutant form of the cellular c-Ha-ras gene, actively transforms NIH/3T3 mouse cells and CHEF/18 hamster cells but is inactive in FS-2 cells. Morphological transformation, focus formation, and tumorigenicity in nude mice were not induced when EJ DNA was transfected into FS-2 cells by using the selectable vector pSVgptEJ. The intactness of the transfected EJ DNA was established by restriction fragment analysis. This result raises the question of what role, if any, the mutated gene derived from the EJ cells played in the origin of the EJ bladder carcinoma.     

Transformation of human cells by DNAs ineffective in transformation of NIH 3T3 cells.

Neonatal human foreskin fibroblasts can be transformed to anchorage-independent growth by transfection with DNAs inefficient in transforming NIH 3T3 cells. Human cells transfected with DNA from GM 1312, a multiple myeloma cell line, or MOLT-4, a permanent lymphoblast line, grow without anchorage at a much higher frequency than do the parental cells and their DNAs can transform human cell recipients to anchorage-independent growth; they have extended but not indefinite life spans and are nontumorigenic. Human fibroblasts are also transformed by DNAs from two multiple myeloma lines that also transform 3T3 cells; however, restriction analysis suggests that different transforming genes in this DNA are acting in the human and murine systems. These results indicate that the human cell transfection system allows detection of transforming genes not effective in the 3T3 system and points out the possibility of detection of additional transforming sequences even in DNAs that do transform murine cells.     

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.     

Transformation of rodent immortalized and embryo cells by oncogenes. I. Mouse (NIH 3T3) and rat (FR 3T3) immortalized fibroblasts

Immortalized mouse NIH 3T3 cells were transformed by gene transfer of DNA isolated from a human bladder tumor cell line and plasmids containing an activated human Ha-ras oncogene insert. For gene transfer the calcium-phosphate co-precipitation method was used. Transformation was evaluated by morphological focus formation, growth in soft agar and tumor development in nude mice. In addition, immortalized rat FR 3T3 cells were transformed by Ha-ras, too. The co-transfer of ras and myc oncogenes did not enhance focus formation in FR 3T3 cells.     

Transforming genes of human bladder and lung carcinoma cell lines are homologous to the ras genes of Harvey and Kirsten sarcoma viruses.

Blot hybridization analysis indicated that NIH 3T3 mouse bladder transformed by high molecular weight DNAs of a human bladder and a human lung carcinoma cell line contained new sequences homologous, respectively, to the transforming genes of Harvey (rasH) and Kirsten (rasK) sarcoma viruses. The unique ras sequences were present in multiple independent NIH cell lines transformed in both primary and secondary transfection assays and corresponded to ras sequences normally present in human DNAs. The ras gene product was expressed in NIH cells transformed by bladder carcinoma DNAs and in the human bladder carcinoma cell lines at levels 2- to 4-fold greater than the level observed in nontransformed NIH 3T3 cells. These results indicate that the transforming genes of these human tumor cell lines are the cellular homologs of two retroviral transforming genes.     

Transforming genes in human leukemia cells

High-molecular weight DNAs of fresh bone marrow cells from 32 patients with fresh leukemia were assayed for the presence of transmissible activated transforming genes by a DNA-mediated gene transfer technique using NIH/3T3 cells. DNAs of bone marrow cells from four of the 32 patients induced transformation of NIH/3T3 cells. Two of the four cases, a chronic myelogenous leukemia and an acute lymphocytic leukemia, contained activated N-ras oncogenes. Molecular cloning and nucleotide sequence analysis revealed that the lesion responsible for the transforming activity was localized to a single nucleotide transition from guanine to thymine in codon 12 of the predicted protein in each of the two cases. These observations indicate that activation of N-ras oncogenes is independent of the specific stage of cell differentiation or the leukemia phenotype. The other two transforming genes associated with an acute myelogenous leukemia and an acute lymphocytic leukemia showed homology neither with members of the ras gene family nor with the human Blym-1 gene. Thus, the NIH/3T3 transfection assay frequently detects activated N-ras oncogenes in human leukemias, while other transforming genes, distinct from the ras gene family, can be detected in some leukemias by the transfection assay.     

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.     

Tumorigenic and metastatic properties of "normal" and ras-transfected NIH/3T3 cells.

To investigate the role of oncogene activation in the pathogenesis of malignant tumors, we have studied the tumorigenic and metastatic properties of NIH/3T3 secondary transfectants (designated A51) containing an activated c-Ha-ras-1 gene derived from the human T24 bladder carcinoma cell line and compared them with untransfected NIH/3T3 cells. Whereas subcutaneous implantation of NIH/3T3 cells in the supraclavicular region produced palpable tumors that failed to metastasize, NIH/3T3 cells inoculated in the footpad gave rise to malignant tumors that metastasized to the lung. Under identical conditions and irrespective of the site of implantation, A51 cells formed rapidly growing primary tumors that produced pulmonary metastases. In an assay for experimental metastasis, intravenously injected NIH/3T3 cells gave rise to pulmonary nodules only at high cell inocula and in long-term survivors (90 days after injection). In contrast, A51 cells formed multiple lung tumor colonies detectable 14 days after injection. These results indicate that "normal" untransfected NIH/3T3 cultures contain subpopulations of cells that express malignant properties and that transfection of NIH/3T3 cells with activated c-Ha-ras-1 accelerates formation of metastases.     

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.     

Transforming activity of human tumor DNAs.

High molecular weight DNAs of 26 human tumors and tumor cell lines were assayed for the presence of transmissible activated transforming genes by transfection of NIH 3T3 mouse cells. DNAs of two bladder carcinoma cell lines induced transformation with high efficiencies (approximately 0.2 transformant per microgram of DNA), whereas DNAs of the other tumors studied lacked detectable transforming activity. These findings suggest that dominant mutations or gene rearrangements can result in the activation of cellular transforming genes in some human tumors.     

Transforming gene in human atherosclerotic plaque DNA.

The monoclonal hypothesis equates atherosclerotic plaques with benign smooth muscle cell tumors and proposes that plaques can arise via mutational or viral events. Here, we provide direct evidence that molecular events, heretofore associated only with tumor cells, are common to plaque cells as well. Three distinct groups of human coronary artery plaque (hCAP) DNA samples transfected into NIH 3T3 cells gave rise to transformed foci. DNA samples from a panel of normal noncancerous human tissues, including coronary artery, were negative in the assay. Southern-blotted focus DNA yielded positive signals when hybridized to the 32P-labeled nick-translated repetitive human "Alu" DNA sequence. The DNA from cloned foci was used successfully in a second round of transfection. Focus DNA hybridized to nick-translated v-Ki-ras, v-Ha-ras, or N-ras probes failed to detect human fragments of these genes. Primary focus cells from each of five clones elicited tumors after injection into nude mice (6/42). Several distinct high molecular weight (greater than 6.6 kilobases) bands were detected after BamHI-digested tumor DNA was hybridized to Alu. Preliminary characterization of these hCAP DNA-associated tumors indicates that they are similar to the fibrosarcomas that arise after injection of ras-transformed cells into nude mice. We propose that transforming genes in plaque cells behave in a manner analogous to the way in which oncogenes behave in cancer cells.     

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.     

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.