Definition of the human raf amino-terminal regulatory region by deletion mutagenesis.

Activation of transforming potential of the cellular raf gene has uniformly been associated with the deletion of amino-terminal coding sequences. In order to determine whether 5' truncation alone could activate cellular raf, we constructed 21 human c-raf-1 cDNAs with variable BAL 31-generated deletions distal to a Moloney murine sarcoma virus long terminal repeat and a consensus translation initiation sequence. The deletions ranged from 136 to 1,399 nucleotides of coding sequence and shortened the 648-amino-acid raf protein by 44 to 465 amino acids. The full-length c-raf-1 cDNA was nontransforming upon transfection of NIH 3T3 cells, as were four mutants with deletions of 142 or fewer amino acids. Seven of nine mutants with deletions of 154 to 273 amino acids induced transformation with efficiencies ranging from 0.25 to 70 foci per micrograms of DNA. Mutants with deletions of 303 to 324 amino acids displayed high transforming activities (comparable with that of v-raf), with a peak activity of 2,400 foci per microgram of DNA when 305 amino acids were deleted. Deletions of greater than 383 amino acids, extending into the raf kinase domain, lacked transforming activity. Northern (RNA) blotting and immunoprecipitation assays indicated that transfected NIH cells expressed raf RNAs and proteins of the expected sizes. Thus, 5' truncation alone can activate raf transforming potential, with a sharp peak of activation around amino acid 300. Analysis of three raf genes previously detected by transfection of tumor DNAs indicated that these genes were activated by recombination in raf intron 7 and encoded fusion proteins containing amino-terminal non-raf sequences. The extend of deletion of raf sequences in these recombinant genes corresponded to BAL 31 mutants which did not display high transforming activity, suggesting that the fused non-raf coding sequences may also contribute to biological activity.     

Activation of human c-raf-1 by replacing the N-terminal region with different sequences.

Two transformants of NIH 3T3 cells, obtained by the transfection of human colon cancer and normal colon DNAs, contained activated c-raf-1. In both the activated c-raf-1, the 5' half of the c-raf-1 sequence was replaced by sequences other than c-raf-1 as a result of recombinations which occurred at the intron between exons 7 and 8. It was suggested, however, that these recombinations, which conferred the transforming activity on the c-raf-1, occurred during the transfection. In one case analyzed, characteristic sequences were found near the breakpoint and these may be involved in the recombination. It was found, upon analysing the structure of the cDNA derived from one of the activated c-raf-1, that fused mRNA had been transcribed from the recombined gene comprising the non-raf gene and c-raf-1. The mRNA possibly encodes a fused protein. One cDNA clone was derived from alternatively spliced mRNA, although its physiological role is unclear. On comparing the structure of the two human activated c-raf-1 and the rat activated c-raf which we have reported previously, it was revealed that, in these three cases, the sequences joined to the truncated c-raf(-1)1 were different. It was suggested from data which we and others have previously reported that various sequences could be capable of activating c-raf(-1) by replacing its 5' half.     

Activation of a novel human transforming gene, ret, by DNA rearrangement

A novel transforming gene was detected by transfection of NIH 3T3 cells with human lymphoma DNA. The tumor DNA induced a single focus in primary transfections, whereas DNAs of transformed NIH cells induced transformation with high efficiencies in secondary and tertiary assays. Molecular clones spanning about 37 kb of human sequence were isolated from tertiary transformant DNA. Blot hybridization indicated that the transforming gene consisted of two segments that were unlinked in both normal human and primary lymphoma DNAs. The two segments of human DNA were cotranscribed in transformed NIH cells but not in any human cells examined. The transforming gene thus appeared to be activated by recombination between two unlinked human DNA segments, possibly by cointegration during transfection.     

Stage-specific transforming genes of human and mouse B- and T-lymphocyte neoplasms

DNAs of 20 B- and T-lymphocyte neoplasms of human and mouse origin induced transformation of NIH/3T3 cells with high efficiencies, indicating that these neoplasms contained activated transforming genes that were detectable by transfection. Analysis of the susceptibility of the transforming activities of lymphocyte-neoplasm DNAs to digestion with restriction endonucleases indicated that the same or closely related transforming genes were activated in independent neoplasms representative of the same stage of normal cell differentiation. However, different transforming genes were activated in neoplasms representative of different stages of normal B- and T-lymphocyte differentiation. These results indicate that specific transforming genes are activated in neoplasms of discrete stages of differentiation within these cell lineages.     

H-ras activation in benign and self-regressing skin tumors (keratoacanthomas) in both humans and an animal model system.

The involvement of the ras oncogenes in tumorigenesis was investigated in keratoacanthomas, which are benign and self-regressing skin tumors, both in humans and in a corresponding animal model system. Keratoacanthomas were induced on rabbit ears by repeated applications of 7,12-dimethylbenz(a)anthracene. About 60% of the tumor DNAs produced transformed foci after transfection into NIH 3T3 cells, and in all of them the transforming gene was identified as H-ras by Southern and Northern (RNA) hybridization. Immunoprecipitation experiments suggested that the transforming rabbit H-ras protein carried a mutation in codon 61. In addition, an activated H-ras gene was detected in a human keratoacanthoma by using a nude mouse tumorigenesis assay after transfection of tumor DNA into NIH 3T3 cells. This is the first report of ras activation in a benign human tumor. The transforming human H-ras gene showed a point mutation in codon 61 that would result in leucine instead of the glutamine present in the normal gene product. The finding of ras activation in tumors that are not only benign but also self-regressing indicates that activated ras genes are not sufficient to maintain a neoplastic phenotype, although they likely play a role in early stages of tumorigenesis.     

Human cell transformation in the study of sunlight-induced cancers in the skin of man

Human cell transformation provides a powerful approach to understanding--at the cellular and molecular levels--induction of cancers in the skin of man. A principal approach to this problem is the direct transformation of human skin cells by exposure to ultraviolet and/or near-UV radiation. The frequency of human cells transformed to anchorage independence increases with radiation exposure; the relative transforming efficiencies of different wavelengths implies that direct absorption by nucleic acids is a primary initial event. Partial reversal of potential transforming lesions by photoreactivation suggests that pyrimidine dimers, as well as other lesions, are important in UV transformation of human cells. Human cells can also be transformed by transfection with cloned oncogenes, or with DNAs from tumors or tumor cell lines. Cells treated by the transfection procedure (but without DNA) or cells transfected with DNAs from normal mammalian cells or tissues show only background levels of transformation. Human cells can be transformed to anchorage-independent growth by DNAs ineffective in transformation of NIH 3T3 cells (including most human skin cancers), permitting the analysis of oncogenic molecular changes even in tumor DNAs difficult or impossible to analyze in rodent cell systems.     

Malignant cell transformation and oncogenes

In genome of all transforming retroviruses special genes (oncogenes) have been identified which play a key role in malignant conversion of the cells, infected with these viruses. The homologues of these genes (protooncogenes) are persist in all normal cells. During transformation protooncogenes can be activated as a result of one of following processes: insertion of promotor-like elements, mutations, translocations, amplifications or rearrangements. Using transfection technique the transforming genes were isolated from different human tumors. The activation of one of the cellular oncogenes may switch on the other genes and malignant cell transformation may be characterized as a multifactor and multistage process.     

Novel human endogenous sequences related to human immunodeficiency virus type 1.

Endogenous retrovirus-related sequences exist within the normal genomic DNA of all eukaryotes, and these endogenous sequences have been shown to be important to the nature and biology of related exogenous retroviruses and may also play a role in cellular functions. To date, no endogenous sequences related to human immunodeficiency virus type 1 (HIV-1) have been reported. Herein we describe the first report of the presence of nucleotide sequences related to HIV-1 in human, chimpanzee, and rhesus monkey DNAs from normal uninfected individuals. We also present the isolation and characterization of two of these endogenous HIV-1-related sequences, EHS-1 and EHS-2. With use of low-stringency Southern blot hybridization, complex banding patterns were detected in human DNA with 5' and 3' HIV-1-derived probes. When an HIV-1 env region probe was used, we detected a less complex, conserved banding pattern in human DNA as well as a related but distinct banding pattern in chimpanzee and rhesus monkey DNAs. EHS-1 and -2 were cloned from normal human genomic DNA libraries by using the env region probe. Clone EHS-1 shows sequence similarity with the domain of the envelope cellular protease cleavage site of HIV-1, while EHS-2 has sequence similarity to the overlapping reading frame for Rev and gp41. Stringent hybridization of EHS-1 back to primate genomic DNA indicates two distinct EHS-1 loci in normal human DNA, an identical band pattern in chimpanzee DNA, and a single locus in rhesus monkey DNA. Likewise, EHS-2 is present as a single highly conserved locus in all three species. An oligonucleotide derived from EHS-2 across a region of near identity to HIV-1 detects a complex banding pattern in all primates tested similar to that seen with the 3' HIV-1 probe. These data suggest that most of the HIV-1-related sequences identified in primate DNA share a common core of nucleic acid sequence found in both EHS-2 and rev and that some of these HIV-1-related sequences have additional larger regions of sequence similarity to HIV-1.     

Altered gene products are associated with activation of cellular rasK genes in human lung and colon carcinomas

Two lung and two colon carcinoma cell lines of human origin, which contained the same activated ras^K transforming gene, expressed abnormal species of p21 that were distinct from the p21 proteins expressed in normal human cells and other human carcinomas. The abnormal species of p21 expressed by three of these cell lines were indistinguishable from each other, but differed from the abnormal p21 expressed by one lung carcinoma cell line. NIH cells transformed by DNAs of these carcinomas expressed the same abnormal p21 species, indicating that these abnormal proteins were encoded by the activated ras^K genes detected by transfection. These results indicate that transforming activity of ras^K genes in human lung and colon carcinoma cell lines is activated by mutations which alter the structure of their gene products, and that activation of ras^K genes can result from different molecular alterations in different individual neoplasms.     

Transforming activity of DNA fragments from normal human lymphocytes results from spontaneous activation of a c-Ha-ras1 gene.

An activated human Ha-ras gene was present in a secondary NIH 3T3 transformant isolated after serial transfection of originally low-molecular-weight DNA fragments from normal human cells. This gene appeared to have acquired its transforming properties by a spontaneous mutation in codon 12 by substitution of a deoxythymidine residue for a deoxyguanosine residue. DNA rearrangements in the flanking sequences of the transferred Ha-ras gene were not involved in the activation of the protooncogene.     

Susceptibility of Xeroderma pigmentosum cells to transformation with oncogenes

Oncogenes capable of transforming 3T3-Vill cells were not detected in 'normal' Xeroderma pigmentosum (XP) fibroblasts but were detected in two out of six XP epitheliomas. Preliminary results concerning the transfection of 'normal' XP fibroblasts with activated ras genes seem to indicate that these cells are as resistant as the healthy controls to the transforming action of the group II oncogenes. However, after transfection with v-myb oncogene in XP fibroblasts several cellular clones have been isolated showing some new phenotypic characteristics.     

Highly frequent detection of transforming genes in acute leukemias by transfection using in vivo selection assays

DNAs from nine out of ten acute leukemia cases that were negative by in vitro focus forming assays exhibited transforming activity tested by in vivo selection assays in nude mice using transfected NIH3T3 cells. Of the nine cases, six cases contained activated N-ras genes, and one case exhibited activation of the c-K-ras gene. None of the ras gene family showed homology with the transforming genes derived from the other two cases. Our observations indicate that in vivo selection assays detect transforming genes including ras oncogenes at high frequency, and that activated N-ras genes are frequently detected in human acute leukemias.     

Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas

DNAs from nitrosoethylurea-induced rat neuroblastomas transform NIH/3T3 mouse fibroblasts in a transfection assay. DNAs of such transformed cells can be used in a subsequent cycle of transfection to generate secondary foci that contain virtually no foreign genetic material besides the sequences carrying the rat neuroblastoma transforming function. These secondary neuroblastoma transfectants were injected into young mice and grew out into fibrosarcomas. Sera from these mice were examined for reactivity with any proteins which were induced specifically by the neuroblastoma transforming sequence. These sera precipitate a polypeptide of about 185,000 daltons from ³⁵S-methionine-labeled cell lysates of the rat neuroblastoma cells that served as DNA donors and in all transfection-derived primary and secondary foci. This protein is present in high levels in all neuroblastoma transfectant clones, but was not detectable in a variety of other transformed cells. Antisera were prepared from mice bearing tumors induced by transformed cells derived by transfection of DNAs from various tumor cell types unrelated to rat neuroblastoma. These antisera failed to immunoprecipitate the 185,000 dalton protein. These data indicate that the synthesis of the 185,000 dalton protein is specifically induced by the neuroblastoma transforming sequence. The protein may be encoded by the transforming sequence and may mediate transformation in this chemically induced tumor.     

Effect of human papillomavirus type 16 oncogenes on MAP kinase activity.

The mitogen-activated protein (MAP) kinase signal transduction pathway is an intracellular signaling cascade which mediates cellular responses to growth and differentiation factors. The MAP kinase pathway can be activated by a wide range of stimuli dependent on the cell types, and this is normally a transient response. Oncogenes such as ras, src, raf, and mos have been proposed to transform cells in part by prolonging the activated stage of components within this signaling pathway. The human papillomavirus (HPV) oncogenes E6 and E7 play an essential role in the in vitro transformation of primary human keratinocytes and rodent cells. The HPV type 16 E5 gene has also been shown to have weak transforming activity and may enhance the epidermal growth factor (EGF)-mediated signal transduction to the nucleus. In the present study, we have investigated the effects of the oncogenic HPV type 16 E5, E6, and E7 genes on the induction of the MAP kinase signaling pathway. The E5 gene induced an increase in the MAP kinase activity both in the absence and in the presence of EGF. In comparison, the E6 and E7 oncoproteins do not alter the MAP kinase activity or prolong the MAP kinase activity induced with EGF. These findings suggest that E5 may function, at least in part, to enhance the cell response through the MAP kinase pathway. However, the transforming activity of E6 and E7 is not associated with alterations in the MAP kinase pathway. These findings are consistent with E5 enhancing the response to growth factor stimulation.     

Conserved sequence related to the 3'-terminal region of retrovirus RNA'S In normal cellular DNAs.

The nucleotide sequences related to the 3'-terminal protion of retrovirus genomic RNA have been detected in the DNA of animals, including humans. The DNA complementary to the 400 to 700 nucleotides from the 3'-terminal end of retrovirus RNA (cDNA3'), which contains the enriched conserved region, was hybridized with DNA from a variety of animal cells. Under the conditions of annealing in 0.72 M NaCl at 67 degrees C and hydroxyapatite chromatography at 55 degrees C, 20 to 50% of the radioactivity of the cDNA3' prepared from two retroviruses, a murine Rauscher virus (RLV) and a baboon virus (M7), annealed with normal cellular DNA of animals, including human tissue. The thermal denaturation profile revealed considerable mismatching between the duplex of the cDNA3' and human DNA, cDNA3' of retroviruses is most homologous to cellular DNA of the host species of origin and is less homologous to cellular DNA of species that are distant in the phylogeny of the host species. The conservation and evolution of nucleotide sequences related to the 3' end of retrovirus genomes in animal DNAs, including humans, suggest that the sequences may have important functions.     

Structural organization and biological activity of molecular clones of the integrated genome of a BALB/c mouse sarcoma virus.

BALB/c mouse sarcoma virus (BALB-MSV) is a spontaneously occurring transforming retrovirus of mouse origin. The integrated form of the viral genome was cloned from the DNA of a BALB-MSV-transformed nonproducer NRK cell line in the Charon 9 strain of bacteriophage lambda. In transfection assays, the 19-kilobase-pair (kbp) recombinant DNA clone transformed NIH/3T3 mouse cells with an efficiency of 3 X 10(4) focus-forming units per pmol. Such transformants possessed typical BALB-MSV morphology and released BALB-MSV after helper virus superinfection. A 6.8-kbp DNA segment within the 19-kbp DNA possessed restriction enzyme sites identical to those of the linear BALB-MSV genome. Long terminal repeats of approximately 0.6 kbp were localized at either end of the viral genome by the presence of a repeated constellation of restriction sites and by hybridization of segments containing these sites with nick-translated Moloney murine leukemia virus long terminal repeat DNA. A continuous segment of at least 0.6 and no more than 0.9 kbp of helper virus-unrelated sequences was localized toward the 3' end of the viral genome in relation to viral RNA. A probe composed of these sequences detected six EcoRI-generated DNA bands in normal mouse cell DNA as well as a smaller number of bands in rat and human DNAs. These studies demonstrate that BALB-MSV, like previously characterized avian and mammalian transforming retroviruses, arose by recombination of a type C helper virus with a well-conserved cellular gene.