RESUMO
ABL-MYC is a recombinant retrovirus that constitutively expresses the v-abl and c-myc oncogenes. When used to infect immunized mice this virus rapidly and efficiently induces plasmacytomas of which an unusually high percentage secrete antigen (Ag)-specific monoclonal antibodies. These findings suggested that ABL-MYC targets Ag-stimulated B cells for transformation and that infection of lymphoid cells in vitro might be a useful, alternative method for generating monoclonal, Ag-specific plasmacytomas (ASPCTs). Therefore, we used helper virus-free ABL-MYC to infect suspensions of cells from spleens and other lymphoid organs from mice that had been immunized with a variety of Ags and transplanted them into naive mice. The results show that ABL-MYC preferentially transforms splenocytes that are Ag-reactive. They also demonstrate that ASPCTs can be produced by in vitro infection of cell suspensions from the spleen, lymph nodes and Peyer's patches of mice that had been immunized intraperitoneally with sheep red blood cells, Escherichia coli core RNA polymerase or Epstein-Barr virus gp340 protein or immunized orally with live Giardia lamblia parasites. The ASPCTs usually consisted of one to three colnes, secreted antibodies that were quantitatively and qualitatively similar to those obtained from hybridomas, and could continue to secrete Ag-reactive antibody over eight transplant generations.
Assuntos
Linfócitos B/microbiologia , Genes abl , Genes myc , Plasmocitoma/imunologia , Retroviridae/patogenicidade , Animais , Linfócitos B/imunologia , Transformação Celular Viral , Células Clonais , DNA de Neoplasias/genética , DNA Recombinante , Ativação Linfocitária , Linfócitos/imunologia , Tecido Linfoide/citologia , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologia , Baço/imunologiaRESUMO
ABL-MYC, a murine retrovirus that encodes the v-abl and c-myc oncogenes, was constructed from Abelson murine leukemia virus (A-MuLV) in order to assess the biological consequences of co-expression of these genes in lymphoid cells. When inoculated into mice this retrovirus induced plasmacytomas in up to 100% of infected mice and less frequently induced pre-B lymphomas. Both tumor types contained genome-length proviruses in one or a few chromosomal locations, were mono- or oligoclonal as judged by immunoglobulin gene rearrangement and had unrearranged endogenous c-myc loci. The type of tumor induced depended upon the age and strain of mouse, and whether helper virus was present in the inoculated virus pool. ABL-MYC induced plasmacytomas with or without helper virus, with or without pretreatment of the mice with pristane, and in strains of mice resistant to pristane-induced plasmacytomas. Pristane treatment prior to ABL-MYC infection shortened the latent period of plasmacytomagenesis and produced mostly IgM-secreting tumors rather than IgA-secreting tumors, which predominantly arose in the absence of pristane. Control viruses for ABL-MYC with either a deletion in v-abl or a frameshift mutation in c-myc caused predominantly monocyte/macrophage tumors and pre-B-cell lymphomas respectively. Histopathological analysis of ABL-MYC-infected mice showed foci of transformed plasma cells as early as 14 days after infection. These results indicate that v-abl and c-myc act synergistically to transform mature B cells with high efficiency.
Assuntos
Transformação Celular Neoplásica/genética , Genes abl , Genes myc , Proteínas Oncogênicas v-abl/genética , Plasmocitoma/genética , Proteínas Proto-Oncogênicas c-myc/genética , Animais , DNA de Neoplasias/genética , Expressão Gênica , Vetores Genéticos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Neoplasias Experimentais/genética , Neoplasias Experimentais/patologia , Plasmocitoma/patologia , RNA Mensageiro/genética , RetroviridaeRESUMO
ABL-MYC, a recombinant murine retrovirus that expresses v-abl and c-myc, rapidly induces transplantable mono- or oligoclonal plasmacytomas in BALB/c mice. To determine if the targets for transformation of this retrovirus are antigen-committed B lymphocytes and to explore this system as an alternative technique for producing antigen-specific monoclonal antibodies, plasmacytomas were induced in mice that had been immunized with two different types of immunogens, hen egg white lysozyme and sheep red blood cells. The majority of these plasmacytomas secreted immunogen-specific antibodies. Plasmacytomas induced in unimmunized mice did not react with hen egg white lysozyme or sheep red blood cells. The specific antibodies were comparable in concentration, specificity, and affinity to monoclonal antibodies obtained with conventional hybridoma technology, but, in addition to IgGs and IgMs, they included specific IgA antibodies, which are rare among splenic-derived hybridomas. Our results demonstrate that a principal target for ABL-MYC is an antigen-committed B lymphocyte. In addition this procedure provides an alternative method for the production of monoclonal antibodies, without a requirement for hetero-caryon formation by cell fusion techniques.
Assuntos
Anticorpos Monoclonais/imunologia , Plasmocitoma/imunologia , Animais , Especificidade de Anticorpos , Northern Blotting , Southern Blotting , Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Muramidase/imunologia , Proteínas Oncogênicas v-abl/genética , Oncogenes , Proteínas Proto-Oncogênicas c-myc/genética , Proto-Oncogenes , RNA Mensageiro/genética , Retroviridae/genéticaRESUMO
Molecular analysis has shown that the majority of Abelson murine leukemia virus (Ab-MuLV)-induced primary thymomas represent transformed gamma/delta thymocytes. Many of these thymomas are of monoclonal origin as judged by provirus integration pattern and contain rearranged genes encoding T-cell receptor (TCR) gamma and delta chains but germ-line immunoglobulin heavy-chain genes. Some of the monoclonal tumors contain multiple rearranged alleles encoding TCR gamma, delta, and beta chains. Further, one Ab-MuLV thymoma cell line contained germ-line-configuration TCR gamma- and delta-chain genes, which became rearranged after in vitro propagation. Clones of this cell line were observed to rearrange these genes after intrathymic passage. Also, some subclones of this cell line underwent rearrangement of their immunoglobulin heavy-chain genes in culture. These observations suggest that the thymic targets for Ab-MuLV transformation are early gamma/delta thymocytes, some of which continue to rearrange their TCR gamma- and delta-chain genes.
Assuntos
Vírus da Leucemia Murina de Abelson/crescimento & desenvolvimento , Rearranjo Gênico do Linfócito T , Subpopulações de Linfócitos T/microbiologia , Timoma/genética , Timo/microbiologia , Neoplasias do Timo/genética , Animais , Southern Blotting , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T gama-delta , Timoma/microbiologia , Timo/citologia , Neoplasias do Timo/microbiologiaRESUMO
The Drosophila melanogaster abl and the murine v-abl genes encode tyrosine protein kinases (TPKs) whose amino acid sequences are highly conserved. To assess functional conservation between the two gene products, we constructed Drosophila abl/v-abl-chimeric Abelson murine leukemia viruses. In these chimeric Abelson murine leukemia viruses, the TPK and carboxy-terminal regions of v-abl were replaced with the corresponding regions of D. melanogaster abl. The chimeric Abelson murine leukemia viruses were able to mediate morphological and oncogenic transformation of NIH 3T3 cells and were able to abrogate the interleukin-3 dependence of a lymphoid cell line. We also found that a virus that contained both TPK and carboxy-terminal Drosophila abl regions had no in vitro transforming activity for primary bone marrow cells and lacked the ability to induce tumors in susceptible mice. A virus that replaced only a portion of the v-abl TPK region with that of Drosophila abl had low activity in in vitro bone marrow transformation and tumorigenesis assays. These results indicate that the transforming functions of abl TPKs are only partially conserved through evolution. These results also imply that the TPK region of v-abl is a major determinant of its efficient lymphoid cell-transforming activity.
Assuntos
Vírus da Leucemia Murina de Abelson/genética , Transformação Celular Neoplásica , Drosophila melanogaster/genética , Vírus da Leucemia Murina/genética , Proteínas Tirosina Quinases/genética , Proteínas Virais/genética , Sequência de Aminoácidos , Animais , Células da Medula Óssea , Células Cultivadas , Quimera , Sondas de DNA , DNA Viral/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Mapeamento por Restrição , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
Helper virus-free Abelson murine leukemia virus (A-MuLV) was used to induce monoclonal pre-B-cell tumors in mice. The clonality, patterns of immunoglobulin heavy-chain gene rearrangement, tumorigenicity, and v-abl oncogene expression in individual preleukemic and leukemic colonies were compared. Our results indicate that A-MuLV preleukemic cells with low or undetectable tumorigenic potential give rise to leukemic cells with high tumorigenic potential by a process of subclone selection. The levels of v-abl oncogene product in preleukemic and leukemic cell populations were not significantly different. These results suggest that an additional event(s) unrelated to the level of the v-abl protein product is required for A-MuLV-transformed cells to become fully malignant.
Assuntos
Vírus da Leucemia Murina de Abelson/patogenicidade , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/microbiologia , Pré-Leucemia/genética , Vírus da Leucemia Murina de Abelson/genética , Animais , Southern Blotting , Divisão Celular , Células Clonais , DNA de Neoplasias/genética , Regulação da Expressão Gênica , Rearranjo Gênico de Cadeia Pesada de Linfócito B , Leucemia Experimental/patologia , Camundongos , Proteínas Oncogênicas Virais/genética , Pré-Leucemia/microbiologia , Pré-Leucemia/patologiaRESUMO
The long terminal repeat (LTR) of the pre-B cell tropic Abelson murine leukemia virus (A-MuLV) was replaced with the LTR of the erythrotropic Friend MuLV or with the LTR of the erythropic/fibrotropic Harvey murine sarcoma virus (Ha-MuSV) to generate the viruses F-ABL and H-ABL, respectively. The parental A-MuLV and the recombinant viruses induced pre-B cell lymphomas in susceptible mice with similar frequencies. Recombinant virus-induced tumor DNAs were analysed by nucleic acid hybridization and were shown to contain the appropriate recombinant provirus. F-ABL was 100-1000 fold less efficient than A-MuLV or H-ABL in the in vitro transformation of primary bone marrow cells, as detected by lymphoid colony formation in agarose. To compare the level of transcription initiated from the different viral LTRs, we investigated the ability of the U3 region of these retroviral LTRs to promote transcription in a battery of cell lines using the chloramphenicol acetyl-transferase (CAT) assay, and with some exceptions we found the following hierarchy of activities: Ha-MusSV greater than or equal to M-MuLV greater than A-MuLV greater than F-MuLV, regardless of the cell line transfected. These results indicate that the LTR is not a determinant of the pre-B cell disease specificity of A-MuLV, and suggest that this specificity resides in the v-abl oncogene. Also, our results suggest that a threshold amount of the v-abl protein product is necessary for in vitro transformation, and this level of expression may be different from the level selected during in vivo tumorigenesis.
Assuntos
Vírus da Leucemia Murina de Abelson/genética , Transformação Celular Viral , Vírus da Leucemia Murina/genética , Neoplasias Experimentais/genética , Animais , Sequência de Bases , Elementos Facilitadores Genéticos , Vírus da Leucemia Murina de Friend/genética , Regulação da Expressão Gênica , Vírus do Sarcoma Murino de Harvey/genética , Camundongos , Dados de Sequência Molecular , Proteínas Oncogênicas Virais/genética , Oncogenes , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Sequências Repetitivas de Ácido NucleicoRESUMO
We investigated the role of the Moloney helper virus, Moloney murine leukemia virus (Mo-MuLV), in cell transformation and tumor induction by the defective Abelson murine leukemia virus (Ab-MuLV). A molecular clone of Ab-MuLV (P160 strain) was transfected into the psi 2 packaging cell line, and helper virus-free Ab-MuLV (psi 2) was harvested from the supernatant medium. Ab-MuLV (psi 2) was as efficient as helper virus-containing Ab-MuLV (Mo-MuLV) in the transformation of primary bone marrow cells in vitro. Inoculation of weanling BALB/c mice with Ab-MuLV (psi 2) induced nonthymic pre-B-cell lymphomas with high efficiency and short latency (28 days). Adult BALB/c mice were less sensitive to tumor induction by a factor of 100. Ab-MuLV (psi 2) did not induce tumors in weanling C57BL/6 mice, unlike Ab-MuLV (Mo-MuLV). Examination of the proviral integration pattern in Ab-MuLV (psi 2)-induced tumor cell DNA revealed that each of the tumors contained a single integrated provirus. Immunoprecipitation of viral-encoded proteins in helper virus-free tumor cell lines detected the P160 Ab-MuLV-transforming protein; however, no trace of the gag, pol, and env helper virus-encoded proteins was found. Our results indicate that integration and expression of a single Ab-MuLV genome is sufficient for efficient transformation of primary bone marrow cells by Ab-MuLV in vitro and tumor induction in susceptible mice. However, the helper virus may contribute to tumor induction in weanling resistant mice.
Assuntos
Transformação Celular Neoplásica , Vírus Auxiliares , Vírus da Leucemia Murina , Leucemia Experimental/patologia , Animais , Medula Óssea/microbiologia , Células da Medula Óssea , Leucemia Experimental/microbiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Peso Molecular , Proteínas Virais/biossínteseRESUMO
We examined the clonality of tumors induced by an acutely transforming retrovirus which carries a single oncogene. Contrary to our expectation, tumors induced by the Abelson murine leukemia virus (A-MuLV) showed one to four major proviral integration events. To further investigate the process by which clonality was established, we analyzed the number of cells infected and transformed by A-MuLV at various times after in vivo infection. At the midpoint of tumor latency (14 days postinfection), we found that infection of total bone marrow cells by A-MuLV was efficient and polyclonal. However, only a minority of these infected cells were transformed as assayed in cell culture, and clonal dominance had already been established in this transformed cell population. Examination of the in vitro growth properties of transformed cells recovered from preleukemic and leukemic mice indicated that preleukemic cells had lower cloning efficiencies than primary tumor cells. Our results suggest that the rate-limiting step in this system of lymphomagenesis is the initial transformation of bone marrow target cells and that these cells undergo subsequent changes in cloning ability during the course of the disease that lead to an autonomous neoplastic state.
Assuntos
Vírus da Leucemia Murina de Abelson/fisiologia , Células Clonais/patologia , Vírus da Leucemia Murina/fisiologia , Leucemia Experimental/patologia , Vírus da Leucemia Murina de Abelson/genética , Animais , Transformação Celular Neoplásica/etiologia , Transformação Celular Neoplásica/patologia , Transformação Celular Viral , DNA de Neoplasias/análise , Células-Tronco Hematopoéticas/microbiologia , Células-Tronco Hematopoéticas/patologia , Leucemia Experimental/etiologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pré-Leucemia/patologia , Recombinação Genética , Ensaio Tumoral de Célula-TroncoAssuntos
Vírus da Leucemia Murina de Abelson/patogenicidade , Transformação Celular Neoplásica , Vírus da Leucemia Murina/patogenicidade , Leucemia Experimental/microbiologia , Linfoma/microbiologia , Vírus da Leucemia Murina de Abelson/genética , Animais , Animais Recém-Nascidos , Enzimas de Restrição do DNA , CamundongosRESUMO
The susceptibility of mice to lymphoma induction by Moloney or Abelson murine leukemia virus has been compared in BALB/c, C57BL/6, and BALB/cXC57BL/6 recombinant inbred strains. BALB/c mice were found to be susceptible to lymphoma induction by either virus, and C57BL/6 mice were found to be relatively resistant to lymphoma induction by either virus. The genes that control these patterns of susceptibility to each virus are not the same because susceptibility to each virus segregated independently in CXB recombinant inbred strains. We also found, as reported by Cook (W. Cook, Proc. Natl. Acad. Sci. U.S.A. 79:2917-2921, 1982), when injected intrathymically that Abelson murine leukemia virus rapidly induced thymomas in weanling B6 mice. Examination of the cellular phenotypes of the tumors induced by Abelson murine leukemia virus or by Moloney murine leukemia virus indicated that different lymphocyte subpopulations were the targets for tumor induction by each virus.