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.     

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.     

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.     

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.     

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.     

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.     

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.     

Transforming gene from human stomach cancers and a noncancerous portion of stomach mucosa.

DNAs from 21 human stomach cancers, 16 metastatic stomach cancers to lymph nodes, and 21 apparently noncancerous specimens of stomach mucosae from a total of 26 patients with stomach cancer were tested for their ability to induce neoplastic transformation of NIH 3T3 cells on transfection by the calcium phosphate precipitation technique. Three samples of DNA were shown to have transforming activity; one was from a primary stomach cancer of one patient, the second was from a noncancerous portion of stomach mucosa of the same patient, and the third was from a lymph node metastasis of stomach cancer from another patient. These transformants were tumorigenic in nude mice, and DNAs from the cells could induce secondary transformants. A portion of the transforming gene from the stomach cancer of one patient, which contained the sequences expressed in the NIH 3T3 transformants, was cloned. The transforming gene did not have any homology with the transforming sequences reported previously. We have applied the term hst to this novel human transforming gene. The transforming gene, hst, was found to be present in all the primary and secondary transformants induced by the other two samples of DNA.     

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.     

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.     

Abelson murine leukemia virus: molecular cloning of infectious integrated proviral DNA.

The integrated proviral genome of Abelson murine leukemia virus (A-MuLV) was cloned in lambda gtWES . lambda B bacteriophage after EcoRI endonuclease digestion and enrichment of proviral sequences by sequential RPC-5 column chromatography and agarose gel electrophoresis. Recombinant DNA clones containing a 7.8-kilobase-pair EcoRI insert were shown to have the entire integrated A-MuLV genome with both 5' and 3' ends flanked by mink cellular DNA sequences. This DNA fragment was shown to induce focus transformation upon transfection of NIH/3T3 mouse cells. Moreover, focus-forming virus could be rescued from transformed nonproducer cells upon superinfection with a type C helper virus. A polyprotein of molecular weight 120,000 (p120) containing murine leukemia virus gag gene determinants was invariably deteced by immunoprecipitation analysis of individual transformants induced by the 7.8-kilobase-pair DNA. Molecularly cloned integrated A-MuLV in its infectious form should be of use in elucidating the mechanisms involved in transformation by this virus.     

Expression of the human monocyte membrane antigen gp55 by murine fibroblasts after DNA-mediated gene transfer

Human DNA sequences that contain the gene encoding gp55, a cell surface glycoprotein expressed exclusively on mature human monocytes and monocytic leukemia cells, were isolated in a mouse genetic background. DNA from mature human monocytes was cotransfected with DNA from a molecularly cloned feline sarcoma virus containing the v-fms oncogene into NIH-3T3 cells. Transformed mouse fibroblasts that expressed gp55, based on their reactivity with the MY4, B44.1, or LeuM3 monoclonal antibodies, were selected by fluorescence-activated cell sorting. Regardless of which antibody was used for selection, equivalent binding of all three antibodies was observed for positive transformants. Secondary and tertiary mouse cell transformants were obtained after additional rounds of transfection and cell sorting with the use of DNA from primary and then secondary transformants. Southern blot analysis of the cellular DNA from two independently derived tertiary subclones revealed a limited complement of human sequences, thus indicating that the gene encoding gp55 is included in fewer than 50 kilobases of human DNA. Independently derived tertiary subclones displayed concordant patterns of reactivity with 13 monocyte-specific monoclonal antibodies, thus indicating that each recognized an epitope on the product (gp55) of a single human gene. The 55-kilodalton cell surface polypeptide was specifically immunoprecipitated with a representative monoclonal antibody, 26if, from lysates of enzymatically radioiodinated peripheral blood monocytes and tertiary transformants. We conclude that gp55 is highly immunogenic and that a large number of independently derived monoclonal antibodies specific for human monocytes react with epitopes on this one molecule.     

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.     

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.     

Molecular cloning of an oncogene from a human hepatocellular carcinoma.

A transforming DNA, named lca (for liver cancer), was obtained from a primary human hepatocellular carcinoma (HCC) in transformation assays using NIH 3T3 cells and a calcium phosphate coprecipitation method. High molecular weight DNA obtained from the HCC tissue was employed for this purpose. This transforming DNA had a linkage to the Alu sequence and was cloned in lambda phage for further studies. Restriction enzyme analyses showed that the minimal size of the lca transforming DNA is about 10 kilobase pairs and that its cleavage profiles are different from those of any one of the previously reported human transforming genes or retroviral oncogenes. No cross-hybridization was observed between these genes and the lca DNA. Southern blot analyses of DNAs from flow-sorted human chromosomes and human-mouse somatic cell hybrids indicated that the lca DNA is located on human chromosome 2. An independently obtained transforming DNA from another HCC exhibited identical restriction enzyme cleavage profiles. Thus, lca DNA is likely to represent a commonly encountered transforming DNA in HCC.     

Identification of an antigen associated with transforming genes of human and mouse mammary carcinomas.

Sera from tumor-bearing mice immunoprecipitated a 86,000-dalton glycoprotein from extracts of NIH cells transformed by human mammary carcinoma DNA. This antigen was not immunoprecipitated from extracts of NIH 3T3 cells, spontaneously transformed NIH cells, NIH cells transformed by normal human DNA, NIH cells transformed by human bladder carcinoma DNA, or NIH cells transformed by Rous sarcoma virus DNA. In addition, sera from mice bearing tumors induced by NIH cells transformed by either normal human DNA or human bladder carcinoma DNA did not immunoprecipitate this antigen from extracts of NIH cells transformed by human mammary carcinoma DNA. However, this antigen was immunoprecipitated by sera from mice bearing tumors induced by NIH cells transformed by mouse mammary carcinoma DNAs and from mice bearing primary mammary carcinomas. These results indicate that this glycoprotein represents an antigen that is specifically associated with expression of the transmissible transforming genes of human and mouse mammary carcinomas.     

Isolation of novel human genomic DNA clones related to human interferon-beta 1 cDNA.

Southern blot-hybridization analyses of human DNA (from Namalwa lymphoblastoid cells) digested with the restriction endonuclease EcoRI were carried out under optimal conditions with two human fibroblast interferon (IFN-beta 1) cDNA probes, pD19 and pD24, which contain IFN-beta 1 inserts 0.8 and 0.7 kilobase (kb) long, respectively. The analyses revealed the presence of several hybridizable DNA fragments, including two of lengths 6.8 and 5.5 kb, in addition to the classical IFN-beta 1 genomic DNA fragment of length approximately equal to 2.0 kb. We have screened a human DNA library in lambda bacteriophage Charon 4A by using a 32P-labeled IFN-beta 1 insert cDNA (pD24) and thereby isolated six strongly positive human genomic DNA clones. One of these (lambda B37) represents the classical human IFN-beta 1 gene; another (lambda B37) contains a 6.8-kb EcoRI DNA fragment(s) which cross-hybridizes with the IFN-beta 1 cDNA insert probes pD19 and pD24; and the remaining four (which are identical to each other and are exemplified by lambda B4) contain two EcoRI DNA fragments approximately 5.5 and 9 kb long which also cross-hybridize the IFN-beta 1 cDNA probes. A mRNA 0.9 kb long derived from the classical IFN-beta1 gene is expressed in poly(I) . poly(C)-induced human diploid fibroblasts (FS-4 strain). Induced FS-4 cells also contain polyadenylylated RNA 1.8, 3, 5, and approximately equal to 8 kb long derived from the lambda B3 gene, all of which appear to code for biologically active human IFN-beta as tested by using the Xenopus laevis oocyte translation assay. These data strongly indicate that lambda B3 represents a novel functional IFN-beta gene. A 12-kb polyadenylylated RNA, derived from lambda B4, is expressed constitutively at a low level in FS-4 cells, but the amount of this RNA increases 5-7 hr after exposure of the cells to poly(I) . poly(C).     

Passage of phenotypes of chemically transformed cells via transfection of DNA and chromatin.

DNA was prepared from 15 different mouse and rat cell lines transformed by chemical carcinogens in vitro and in vivo. These DNAs were applied to NIH3T3 mouse fibroblast cultures by using the calcium phosphate transfection technique. DNAs of five donor lines were able to induce foci on the recipient monolayers. Ten other donor DNAs yielded few or no foci. DNAs from control, nontransformed parental cell lines induced few or no foci. Chromosomes were transfected from one donor whose naked DNA was unable to induce foci, and morphologic transformation of recipients was observed. These experiments prove that in five of these cell lines the chemically induced phenotype is encoded in DNA, and the sequences specifying the transformed phenotype behave as a dominant allele in the NIH3T3 recipient cells. The sequences encoding the transformation are likely found on a single fragment of DNA.