Transforming genes in human tumors

DNAs isolated from a variety of human tumor cell lines as well as from naturally occurring human carcinomas and sarcomas were shown to induce morphologic transformation upon transfection into NIH/3T3 cells. All tested transformants contained human DNA sequences, some of which specifically cosegregated with the malignant phenotype in additional cycles of transfection. Southern blot analysis of second cycle transformants derived from T24 human bladder carcinoma cells showed the presence of a single 15 kbp EcoRI fragment of human DNA. These sequences were molecularly cloned utilizing λ Charon 9A as the cloning vector. The resulting recombinant DNA molecule, designated λ T24-15A, was shown to contain an internal 6.6 kbp Bam HI fragment of human DNA that transformed NIH/3T3 fibroblasts with a specific activity of 5 × 10⁴ focus forming units per picomol. These results indicate that we have moleculary cloned an oncogene present in T24 bladder carcinoma cells. Comparison of molecular clones containing the T24 oncogene and its normal homologue did not reveal biochemical differences that helped to explain the malignant properties of this oncogene. Finally, we report preliminary results indicating that the T24 bladder carcimoma oncogene is highly related to the transforming gene of BALB-MSV, an acute transforming retrovirus.     

Recombinant BALB and Harvey sarcoma viruses with normal proto-ras-coding regions transform embryo cells in culture and cause tumors in mice.

The ras genes of BALB and Harvey sarcoma viruses contain point mutations in codon 12 or codons 12 and 59, relative to proto-ras from normal animal and human cells. By in vitro recombination between cloned rat proto-ras and cloned BALB and Harvey sarcoma proviruses, we constructed recombinant proviruses with normal proto-ras-coding regions. These recombinant proviruses transformed mouse 3T3 cells upon transfection. However, when the transforming efficiencies of proviral DNAs were compared after transfection with helper provirus, recombinant proviruses were 2 to 30 times less efficient than the corresponding wild-type proviruses. Recombinant sarcoma viruses isolated from cells transformed by cloned proviral DNA contained the expected normal ras-coding region. They transformed rat embryo cells and induced erythroblastosis and sarcomas in newborn mice as efficiently as wild-type viruses did. We conclude that conversion of normal proto-ras genes to viral ras genes depends on truncation of normal proto-ras regulatory elements and substitution by retroviral (long terminal repeat) promoters and that the transforming function of long terminal repeat-ras genes is enhanced by point mutations.     

McDonough feline sarcoma virus: characterization of the molecularly cloned provirus and its feline oncogene (v-fms).

The genetic structure of the McDonough strain of feline sarcoma virus (SM-FeSV) was deduced by analysis of molecularly cloned, transforming proviral DNA. The 8.2-kilobase pair SM-FeSV provirus is longer than those of other feline sarcoma viruses and contains a transforming gene (v-fms) flanked by sequences derived from feline leukemia virus. The order of genes with respect to viral RNA is 5'-gag-fms-env-3', in which the entire feline leukemia virus env gene and an almost complete gag sequence are represented. Transfection of NIH/3T3 cells with cloned SM-FeSV proviral DNA induced foci of morphologically transformed cells which expressed SM-FeSV gene products and contained rescuable sarcoma viral genomes. Cells transformed by viral infection or after transfection with cloned proviral DNA expressed the polyprotein (P170gag-fms) characteristic of the SM-FeSV strain. Two proteolytic cleavage products (P120fms and pp55gag) were also found in immunoprecipitates from metabolically labeled, transformed cells. An additional polypeptide, detected at comparatively low levels in SM-FeSV transformants, was indistinguishable in size and antigenicity from the envelope precursor (gPr85env) of feline leukemia virus. The complexity of the v-fms gene (3.1 +/- 0.3 kilobase pairs) is approximately twofold greater than the viral oncogene sequences (v-fes) of Snyder-Theilen and Gardner-Arnstein FeSV. By heteroduplex, restriction enzyme, and nucleic acid hybridization analyses, v-fms and v-fes sequences showed no detectable homology to one another. Radiolabeled DNA fragments representing portions of the two viral oncogenes hybridized to different EcoRI and HindIII fragments of normal cat cellular DNA. Cellular sequences related to v-fms (designated c-fms) were much more complex than c-fes and were distributed segmentally over more than 40 kilobase pairs in cat DNA. Comparative structural studies of the molecularly cloned proviruses of Synder-Theilen, Gardner-Arnstein, and SM-FeSV showed that a region of the feline-leukemia virus genome derived from the pol-env junction is represented adjacent to v-onc sequences in each FeSV strain and may have provided sequences preferred for recombination with cellular genes.     

Isolation of a transforming sequence from a human bladder carcinoma cell line

We have isolated the component of human bladder carcinoma cell DNA that is able to transform mouse fibroblasts. The oncogenic sequence was isolated initially from a lambda phage genomic library made from DNA of a transfected mouse cell carrying the human oncogene. A subcloned insert of 6.6 kb that carried transforming activity was amplified in the plasmid vector pBR322. The subcloned oncogene has been used as a sequence probe in Southern blot analyses. The oncogene appears to derive from sequences present in normal cellular DNA. Structural analysis has failed so far to reveal differences between the oncogene and its normal cellular homolog. The oncogene is unrelated to transforming sequences detected in a variety of other types of human tumor cell lines derived from colonic and lung carcinoma and from neuroblastoma. In contrast, the EJ bladder oncogene appears closely related to one that is active in the human T24 bladder carcinoma cell line. The oncogene appears to have undergone little, if any, amplification in several bladder carcinoma cell lines.     

Regional chromosomal localization of N-ras, K-ras-1, K-ras-2 and myb oncogenes in human cells

The identification of transforming genes in human tumor cells has been made possible by DNA mediated gene transfer techniques. To date, it has been possible to show that most of these transforming genes are activated cellular analogues of the ras oncogene family. To better understand the relationship between these oncogenes and other human genes, we have determined their chromosomal localization by analyzing human rodent somatic cell hybrids with molecularly cloned human proto-oncogene probes. It was possible to assign N-ras to chromosome 1 and regionally localize c-K-ras-1 and c-K-ras-2 to human chromosomes 6pter-q13 and 12q, respectively. These results along with previous studies demonstrate the highly dispersed nature of ras genes in the human genome. Previous reports indicated that the c-myb gene also resides on chromosome 6. It has been possible to sublocalize c-myb to the long arm of chromosome 6 (q15-q21). The non-random aberrations in chromosomes 1, 6 and 12 that occur in certain human tumors suggest possible etiologic involvement of ras and/or myb oncogenes in such tumors.     

Use of gene transfer and a novel cosmid rescue strategy to isolate transforming sequences.

Mouse Lewis Lung tumor DNA was ligated to a cosmid containing a geneticin (G418)/kanamycin resistance gene and transferred into NIH3T3 cells. Recipient cells were first selected for geneticin resistance and subsequently for their ability to grow as a tumour when injected into nude mice. By repeating this transfection procedure with DNA from resultant tumours, geneticin-resistant NIH3T3 cells were obtained which were tumorigenic and contained approximately 1-5 copies of the transferred cosmid. The functional oncogene was cloned by preparing cosmid libraries of third round tumour DNAs, using a cosmid which does not contain a kanamycin resistance gene. Due to the original linkage of the oncogene with the cosmid containing the kanamycin resistance gene, a series of kanamycin-resistant cosmids were isolated, five of which contained an active oncogene. Subsequent analysis showed that the oncogene present was highly related to the human N-ras gene. Using a DNA probe from the MLL N-ras gene, a non-transforming counterpart was isolated from mouse liver DNA. A comparison between the two N-ras genes showed that a mutation at the amino acid position corresponding to 61 in the human gene is responsible for transforming activity of the rescued gene.     

Co-transfection of normal NIH/3T3 DNA and retroval LTR sequences: a novel strategy for the detection of potential c-onc genes.

Morphologically transformed, tumorigenic cell lines were obtained after co-transfecting normal NIH/3T3 DNA and cloned 3'-long terminal repeat sequences of Moloney leukemia virus (Mo-LTR) onto NIH/3T3 recipient cells. In four such cell lines the malignant phenotype was found to be associated with single and specific Mo-LTR integration sites that were retained after serial passages through NIH/3T3 and rat 208F cells, indicating that Mo-LTR sequences are linked to the activated oncogenes. In one of these clones the activated transforming gene was identified as c-raf, the cellular homologue of a recently described retroviral oncogene. This finding not only demonstrates that the mouse c-raf gene can be activated to exhibit an oncogenic potential but also that the approach chosen in this study is suitable for the detection of potential c-onc genes. In contrast to this clone, the activated transforming genes in other cell lines appear to be different from 19 previously isolated v-onc and c-onc genes. These results demonstrate the potential of the established transformation system for the detection and isolation of previously unidentified c-onc genes.     

The HL-60 transforming sequence: a ras oncogene coexisting with altered myc genes in hematopoietic tumors

The oncogene of the HL-60 human promyelocytic leukemia cell line has been passed serially through NIH/3T3 mouse fibroblasts. Oncogene-specific probes prepared from the resulting tertiary transfectants by molecular cloning have been used to show that loss of the transfected oncogene from NIH/3T3 cells correlates with reversion to nontransformed morphology. Analysis of cells transfected by the oncogenes of other tumors and tumor cell lines indicates that the transforming gene of the HL-60 leukemia cell line is closely related to oncogenes of a Burkitt's lymphoma, an acute myelogenous leukemia, an adenocarcinoma of the colon, a neuroblastoma, and two sarcomas. This oncogene is distantly related to the viral oncogenes of Kirsten and Harvey sarcoma viruses. It has been termed N-ras. The active N-ras oncogene coexists with altered versions of the myc oncogene in the HL-60 and AW Ramos human tumors. This suggests a multistep mechanism involving both ras and myc genes in the creation of these tumors.     

Molecular cloning of the Harvey sarcoma virus circular DNA intermediates. II. Further structural analyses.

Three species of unintegrated supercoiled Harvey sarcoma virus DNA (6.6, 6.0, and 5.4 kilobase pairs) have been molecularly cloned from Harvey sarcoma virus-infected cells. On the basis of restriction enzyme analyses, the 6.6- and 6.0-kilobase pair viral DNAs contain two and one copies, respectively, of a 650-base pair DNA segment which contains sequences present at the 3' and 5' termini of the viral genome. R-loop structures formed between Moloney leukemia virus RNA and the cloned Harvey sarcoma virus DNA indicated that about 500 base pairs of the 650-base pair repeating segment was complementary to the 3' end of the viral RNA. During amplification in the Escherichia coli host, some recombinants containing the 6.6- or the 6.0-kilobase pair Harvey sarcoma virus DNA insert acquired or lost the complete 650-base pair DNA segment. These changes occurred in both recA+ and recA- E. coli.     

Structure and activation of the human N-ras gene

The normal human N-ras gene has been cloned. In structure and sequence it closely resembles the human H-ras and K-ras genes. The three genes share regions of nucleotide homology and nucleotide divergence within coding sequences and have a common intron/exon structure, indicating that they have evolved from a similarly spliced ancestral gene. The N-ras gene of SK-N-SH neuroblastoma cells has transforming activity, while the normal N-ras gene does not, the result of a single nucleotide change substituting lysine for glutamine in position 61 of the N-ras gene product. From previous studies we conclude that amino acid substitutions in two distinct regions can activate the transforming potential of ras gene products.     

Transformation of primary human embryonic kidney cells to anchorage independence by a combination of BK virus DNA and the Harvey-ras oncogene.

Primary human embryonic kidney (HEK) cells were transformed by a focus assay with BK virus (BKV) DNA molecularly cloned at its unique EcoRI site. Both viral DNA sequences and viral tumor antigens were present and expressed in all the foci that we examined. However, cells isolated from foci were incapable of growth in soft agar. We then examined the transformation of HEK cells after their transfection with a combination of BKV DNA and either the normal or the activated form of the human Ha-ras oncogene (EJ c-Ha-ras-1). Only the cells transfected with a combination of BKV DNA and the activated form of Ha-ras were capable of growth in soft agar. Both BKV and Ha-ras DNAs were present in the transformed colonies. BKV tumor antigens and the Ha-ras p21 protein were also expressed.     

Absolute values of ras p21 defined by direct binding liquid competition radioimmunoassays

Several distinct and high-conserved genes comprise the ras gene family of rodents and humans, i.e., rodent Harvey and Kirsten, and human Harvey, Kirsten and neuroblastoma. Transformation, either by a point-mutation resulting in a change in one amino acid of the 21 kDa ras gene product (p21), or by increased expression of ras p21, has been demonstrated to be mediated by members of this gene family. We report here the development of direct binding liquid competition radioimmunoassays for the detection and quantitation of the ras oncogene and proto-oncogene products. Using these radioimmunoassays and ras p21 purified from Escherichia coli containing the full-length T24 mutant human Harvey ras gene protein product as a standard, we have defined the actual amount of ras p21 per micrograms of total cellular protein, or per cell, in various ras transformed and 'normal' mammalian cell lines. One of the radioimmunoassays developed is group-specific, since the antigenic determinant recognized is shared by both the point-mutated and proto-forms of Harvey, Kirsten and neuroblastoma members of the ras gene family, while the second may be termed type-selective, since it recognizes an antigenic determinant localized primarily on the Harvey ras p21. Both radioimmunoassays are interspecies, since they detect a ras p21 antigenic determinant common to cells of human and rodent origin. These studies thus describe the first means for defining absolute values of any oncogene or proto-oncogene protein product. The assays described, when used in combination with specific c-DNA probes to define specific ras proto-oncogenes or point-mutated oncogenes being expressed, will now permit truly quantitative analyses of ras p21 expression in experimental cell culture systems, animal models and human biopsy material.     

An avian transforming retrovirus isolated from a nephroblastoma that carries the fos gene as the oncogene.

A new avian transforming retrovirus, NK24, was isolated from a chicken with a nephroblastoma. This transforming virus induced fibrosarcomas with osteogenic cell proliferation and nephroblastomas in vivo and transformed fibroblast cells in vitro. From extracts of NK24-transformed cells, anti-gag serum immunoprecipitated a 100-kilodalton nonglycosylated protein with no detectable protein kinase activity. An NK24 provirus present in infected quail cells was molecularly cloned and subjected to nucleotide sequence analysis. The genome of NK24 was 5.3 kilobases long and had a 1,126-base-pair sequence of cellular origin in place of a viral sequence of avian leukosis virus containing the 3' half of the gag gene and the 5' half of the pol gene. Although the entire env gene was retained, it appeared to be inactive, possibly owing to the loss of function of its splice acceptor site as a result of a second deletion of 1,598 bases in the 3' half of the pol gene that extended to the acceptor site. Nucleotide sequence analysis revealed that the NK24 virus contained the fos gene, previously identified as the oncogene of FBJ and FBR murine osteosarcoma viruses. Unlike the v-fos gene products of FBJ and FBR, which suffer a structural alteration at their carboxyl termini, the NK24 v-fos gene product seemed to have the same carboxyl-terminal structure as the chicken c-fos gene product. A comparison of the structures of the products of the NK24 v-fos and mouse c-fos genes suggested that the fos gene product consists of highly conserved regions and relatively divergent regions.     

Molecular cloning of the 25 kbp region upstream of exon 0 of the human Ki-ras oncogene and its conservation in transformed mouse NIH 3T3 cells

Human sequences associated with the Ki-ras oncogene of the mammary tumour cell line, H-466B have been cloned from a tertiary NIH3T3 mouse transfectant. These sequences are located 5' upstream of exon 0 of the Ki-ras oncogene, span over 25 kbp of DNA and are conserved in half of the primary transfectants obtained with the Ki-ras gene of different types of tumours. No gross alterations were observed in the sequences upstream of the Ki-ras gene. The partial or total deletion of these sequences in the other half of primary transformants argues that they are not absolutely required for the transforming activity of the Ki-ras oncogene. The even distribution of the human-mouse junction points in primary transformed mouse cells suggests the absence of a specific region of recombination in the 5' flanking region of Ki-ras.     

Molecular cloning, genomic analysis, and biological properties of rat leukemia virus and the onc sequences of Rasheed rat sarcoma virus.

Rasheed rat sarcoma virus (RaSV) has been shown to code for a protein of 29,000 Mr not present in replication-competent rat type C helper virus (RaLV)-infected cells. This protein is a fused gene product consisting of a portion of the RaLV p15 gag protein and the transformation-specific 21,000 Mr (p21) ras protein, which is also found in Harvey murine sarcoma virus. We now report the molecular cloning of both the SD-1 (Sprague-Dawley) strain of RaLV and the transforming ras sequences of RaSV. Heteroduplex analysis of these cloned DNAs demonstrated that the RaSV ras gene (v-Ra-ras) was inserted into the rat type C viral genome with a small deletion of RaLV genetic information in the 5' region of the gag gene and that the v-Ra-ras gene (0.72 kilobase pair) is homologous to and colinear with the p21 ras gene of Harvey murine sarcoma virus (v-Ha-ras). Restriction enzyme mapping confirmed the homology demonstrated by heteroduplex mapping, showing strong site conservation of restriction endonucleases known to cleave v-Ha-ras. Cloned v-Ra-ras DNA transformed NIH 3T3 cells, inducing the synthesis of the p29 RaSVgag-ras protein.     

Mechanism of activation of an N-ras gene in the human fibrosarcoma cell line HT1080.

A full length N-ras gene has been cloned from both the human fibrosarcoma cell line HT1080 and from normal human DNA. N-ras isolated from HT1080 will efficiently induce morphological transformation of NIH/3T3 cells in a transfection assay, whereas N-ras isolated from normal human DNA has no effect on NIH/3T3 cells. The coding regions of the normal N-ras gene have been sequenced and the predicted amino acid sequence of the N-ras product is very similar to that of the c-Ha-ras1 and c-Ki-ras2 products. By making chimeric molecules between the two cloned genes the activating alteration in the HT1080 N-ras gene has been localised to a single base change that results in an amino acid alteration at position 61 of the p21 N-ras product.     

Transformation of quail embryo fibroblasts by a retrovirus carrying a normal human c-myc gene.

We have constructed avian retroviruses expressing the human c-myc oncogene. These viruses morphologically transformed primary quail embryo fibroblasts upon transfection and infection. Transformed cells produced viruses harboring a spliced c-myc gene and contained high levels of p64-67c-myc protein. One of these infectious viruses, vSX-AHM, was molecularly cloned and the nucleotide sequence of the spliced c-myc insert determined. No mutation was found within the c-myc coding sequence of this transforming clone when compared to the normal genomic progenitor. Thus, we concluded that no mutation within the human c-myc gene is required to induce primary avian embryo fibroblast transformation.