RESUMO
Oncogenic retroviruses are generated by transduction of the coding region of a protooncogene and acquire genetic changes during subsequent replication. Critical genetic events which occurred during and after transduction of rat proto-ras-1Ha into Harvey sarcoma virus were identified by evaluating the transforming activity of plausible synthetic progenitor proviruses encompassing the complete proto-ras genomic region with or without various 5' deletions. All progenitor proviruses induced phenotypic transformation of mouse NIH 3T3 cells, although with a 5- to 10-fold lower frequency than Harvey sarcoma provirus. Although no tumor formation was observed in vivo after inoculation in the absence of helper murine retrovirus, both wild-type and progenitor viruses inoculated in the presence of helper virus induced tumors in newborn BALB/c mice. No critical alterations of the p21ras coding region and no deletion of 5' genomic elements were detected in a progenitor virus encompassing the complete proto-ras genomic region that had been isolated from tumors. However, one progenitor virus that included all proto-ras exons induced tumors with a decreased latency. This virus contained a mutation in codon 12 (glycine to valine), which had apparently been selected during tumorigenesis in vivo. During the genesis of Harvey sarcoma virus, critical steps conferring transforming function are therefore transduction of coding proto-ras exons and enhancement of their transforming function by specific amino acid changes in p21ras.
Assuntos
Transformação Celular Viral/genética , Genes ras , Vírus do Sarcoma Murino de Harvey/genética , Transdução Genética , Células 3T3 , Sequência de Aminoácidos , Animais , Sequência de Bases , Transformação Celular Neoplásica/genética , Células Cultivadas , Códon/genética , DNA Viral/genética , Vírus do Sarcoma Murino de Harvey/patogenicidade , Vírus Auxiliares/genética , Camundongos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas p21(ras)/genética , Provírus/genética , Ratos , Sarcoma Experimental/etiologia , Sarcoma Experimental/genética , Homologia de Sequência de AminoácidosRESUMO
The malignant histiocytosis sarcoma virus (MHSV), in contrast to other viruses with the ras oncogene, induces acute histiocytosis in newborn and adult mice. Molecular structure and function studies were initiated to determine the basis of its unique macrophage-transforming potential. Characterization of the genomic structure showed that the virus evolved by recombination of the Harvey murine sarcoma virus (Ha-MuSV) and a virus of the Friend-mink cell focus-forming virus family. Structural analysis of MHSV showed two regions of the genome that are basically different from the Ha-MuSV: (i) the ras gene, which is altered by a point mutation in codon 181 leading to a Cys----Ser substitution of the p21 protein, and (ii) the U3 region of the long terminal repeat, which is largely derived from F-MCFV and contains a deletion of one direct repeat as well as a duplication of an altered enhancer-like region. Biological studies of Ha-MuSV, MHSV, and recombinants between the two viruses show that the U3 region of the MHSV long terminal repeat is essential for the malignancy and specificity of the disease. A contributing role of the ras point mutation in determining macrophage specificity, however, cannot be excluded.
Assuntos
Transformação Celular Neoplásica , Vírus do Sarcoma Murino de Harvey/genética , Vírus da Leucemia Murina/genética , Vírus Indutores de Focos em Células do Vison/genética , Recombinação Genética , Sequências Repetitivas de Ácido Nucleico , Vírus do Sarcoma Murino/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Códon/genética , DNA Viral/genética , Genes Virais , Genes ras , Vírus do Sarcoma Murino de Harvey/patogenicidade , Histiocitose/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Vírus Indutores de Focos em Células do Vison/patogenicidade , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , Baço/microbiologia , Transfecção , Proteínas do Envelope Viral/genéticaRESUMO
The rat-derived Harvey murine sarcoma virus (Ha-MuSV) contains a transduced ras oncogene activated by two missense mutations and flanked by rat retroviruslike VL30 sequences. Ha-MuSV induces focal transformation of mouse NIH 3T3 cells in vitro and tumors (fibrosarcomas and splenic erythroleukemias) in newborn mice. We have used these two assays to study the contribution of coding and noncoding viral sequences to the biological activity of Ha-MuSV. A good correlation was found between the in vitro and in vivo assays. In several different isogenic Ha-MuSV variants, those with a rasH gene that had one or both of the Ha-MuSV missense mutations were much more active biologically than the corresponding proto-oncogene. A Ha-MuSV variant that encoded the proto-oncogene protein induced lymphoid leukemias (with thymomas), with a relatively long latent period, rather than the fibrosarcomas and erythroleukemias characteristic of Ha-MuSV with one or both missense mutations. A VL30-derived segment with enhancer activity was identified downstream from v-rasH. A mutant Ha-MuSV from which this 3' noncoding segment was deleted expressed lower levels of the wild-type viral protein, displayed impaired transforming activity in vitro, and induced lymphoid leukemias (with thymomas). 5' noncoding rat c-rasH sequences were found to increase the biological activity of the virus when substituted for the corresponding segment of v-rasH. We conclude that (i) the biological activity of Ha-MuSV can be influence significantly by noncoding sequences located outside the long terminal repeat as well as by coding sequences, (ii) VL30 sequences positively regulate the expression of v-rasH, (iii) relatively low biological levels of ras, whether resulting from low-level expression of wild type v-rasH or high-levels of ras proto-oncogene protein, induce a type of tumor that differs from tumors induced by high biological levels of ras, and (iv) the in vivo pathogenicity of the Ha-MuSV variants correlated with their transforming activity on NIH 3T3 cells.
Assuntos
Transformação Celular Viral , Vírus do Sarcoma Murino de Harvey/genética , Leucemia Experimental/genética , Proteínas Oncogênicas Virais/genética , Oncogenes , Vírus do Sarcoma Murino/genética , Sarcoma Experimental/genética , Animais , Análise Mutacional de DNA , Elementos Facilitadores Genéticos , Vírus do Sarcoma Murino de Harvey/patogenicidade , Técnicas In Vitro , Camundongos , Proteína Oncogênica p21(ras) , Sequências Reguladoras de Ácido Nucleico , Relação Estrutura-AtividadeRESUMO
Transglutaminase (TGase) activity was reduced in intact mitogen-stimulated human peripheral blood lymphocytes (PBL) when compared to intact resting PBL. Moreover, a treatment of the same quiescent immunocompetent cells with purified liver TGase and Ca2+ completely suppressed the mitogen-induced blast transformation. A decrease in TGase activity in neoplastically transformed seminal vesicle epithelial cells with respect to their normal parent counterpart was also observed. Our data support the notion of a possible implication of TGase in cell proliferation and transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Transglutaminases/metabolismo , Animais , Ligação Competitiva , Cálcio/farmacologia , Catálise , Divisão Celular , Linhagem Celular , Concanavalina A/farmacologia , Meios de Cultura , Vírus do Sarcoma Murino de Harvey/patogenicidade , Vírus do Sarcoma Murino de Kirsten/patogenicidade , Fígado/enzimologia , Linfócitos/patologia , Mitógenos/farmacologia , Proteínas/análise , Ratos , Ratos Endogâmicos , Transglutaminases/antagonistas & inibidores , Transglutaminases/fisiologia , Células Tumorais Cultivadas/enzimologiaRESUMO
Activated Harvey murine sarcoma virus ras genes were introduced into epidermal cells in vivo by direct application of retroviruses to mouse skin. Subsequent treatment with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced benign papillomas, some of which progressed to invasive carcinomas. Initiation with virus was irreversible for at least 4 months, since TPA treatment after this latency period produced papillomas within 4 weeks. Analysis of viral integration sites showed that carcinomas are clonal in origin. Both papillomas and carcinomas express virus-specific ras mRNA and the viral form of ras P21 protein. The results show that activated ras genes can replace chemical carcinogens in initiation of mouse skin carcinogenesis. This system presents a novel approach to in vivo analysis of the biological role of oncogenes in epithelial tumorigenesis.
