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1.
Oncogene Res ; 4(4): 311-8, 1989.
Article in English | MEDLINE | ID: mdl-2475843

ABSTRACT

Expression of eleven oncogenes was analyzed in thymomas induced by X-rays in the BALB/c strain of mice. H-, K-, N-ras, c-myc, c-myb, and c-abl genes were consistently expressed in thymomas, as well as in normal, age-matched thymus. Only c-myc transcription appeared to be altered in thymomas: a 1.5-3-fold elevated expression of c-myc was found in 42% of the thymomas tested. An altered ratio of two normal promoters, P1 and P2, of the c-myc gene has also been observed in 6 samples out of 15 tested. In one sample, expression from only the P2 promoter was found. A change in DNA sequence to the 5' side of this promoter was detected in an RNAase cleavage assay; this could have disrupted transcription from the P1 promoter. No other structural alteration has been detected within approximately 1800 bases upstream or 700 bases downstream from the 1st exon including exon 1 of the c-myc gene. With the exception of the one sample described, the results of RNAase cleavage assays, as well as Southern blotting of the c-myc gene, show that the structural alteration of this region of c-myc is not generally associated with radiation-induced thymomas in this strain of mice.


Subject(s)
Gene Expression Regulation , Neoplasms, Radiation-Induced/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogenes , Thymoma/genetics , Thymus Neoplasms/genetics , Animals , Blotting, Northern , DNA Restriction Enzymes , Gene Amplification , Mice , Mice, Inbred BALB C , Proto-Oncogene Proteins c-myc , RNA/analysis , Thymoma/etiology , Thymus Neoplasms/etiology , Transcription, Genetic
2.
J Virol ; 62(5): 1808-9, 1988 May.
Article in English | MEDLINE | ID: mdl-3357211

ABSTRACT

We propose a revised standardized nomenclature for the proteins common to all retroviruses on the basis of biological function, enzymatic activity, and/or virion location data. (We do not discuss proteins specific for subfamilies or only some retroviruses.)


Subject(s)
Retroviridae Proteins/classification , Terminology as Topic
3.
Proc Natl Acad Sci U S A ; 85(7): 2191-5, 1988 Apr.
Article in English | MEDLINE | ID: mdl-2965388

ABSTRACT

We report that a 2.4-kilobase (kb) pim-1 transcript is expressed in the germ cells of mouse testis. Analysis of purified populations of spermatogenic cell types indicates that the 2.4-kb transcript is selectively expressed in haploid postmeiotic early spermatids. The evidence for a developmentally regulated expression of pim-1 in haploid spermatids suggests a possible developmental role for this protooncogene product. The 2.4-kb pim-1 transcript present in postmeiotic cells differs in size from the 2.8-kb transcript usually detected in somatic tissues. Similar testis-specific transcripts have been seen for mos and abl genes. These data suggest specificity in transcription or processing of certain genes in haploid male germ cells. We have also analyzed other representative protooncogenes, including examples of protein kinases, the ras family, and the "nuclear" protooncogenes. The results indicate that additional protooncogenes are preferentially expressed in either meiotic pachytene cells or postmeiotic early spermatids. These findings suggest a differential regulation of gene expression in these two developmental stages of germ cells. In particular, analysis of expression of the three members of the ras gene family indicates a distinct temporal differential regulation in the expression of the Harvey, Kirsten, and N-ras genes in these germ cells.


Subject(s)
Gene Expression Regulation , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogenes , RNA, Messenger/biosynthesis , Spermatids/metabolism , Animals , Haploidy , Infertility, Male/genetics , Male , Mice , Mice, Mutant Strains/genetics , Mice, Quaking/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-abl , Proto-Oncogene Proteins c-fos , Proto-Oncogene Proteins c-mos , Proto-Oncogene Proteins c-pim-1 , Proto-Oncogene Proteins p21(ras) , Spermatogenesis
4.
Oncogene ; 2(3): 223-6, 1988 Mar.
Article in English | MEDLINE | ID: mdl-3281092

ABSTRACT

An interleukin-3 (IL-3) dependent mast cell line (MC) was infected with a recombinant retrovirus expressing the proto-oncogene c-myc and the drug selectable marker neo. Cells containing the transcriptionally activated c-myc gene displayed an increased growth rate in liquid culture and a higher cloning efficiency in soft agar when compared to control virus infected cells. All infected cells remained absolutely dependent on IL-3 for growth and were not tumorigenic in nude mice. Similar results were obtained with two additional IL-3 dependent cell lines, the mast cell 32D and the pre-B-cell Ea3. Thus, while constitutive expression of c-myc potentiates the response of mast cells to IL-3, it is not sufficient to eliminate their requirement for growth factors.


Subject(s)
Growth Substances/pharmacology , Interleukin-3/pharmacology , Mast Cells/cytology , Proto-Oncogene Proteins/genetics , Animals , Cell Division , Gene Expression Regulation , Mast Cells/drug effects , Mice , RNA, Messenger/genetics
5.
Oncogene Res ; 2(2): 189-95, 1988.
Article in English | MEDLINE | ID: mdl-3064027

ABSTRACT

Mouse C3H10T1/2 cells were co-transfected with a plasmid containing the KiSV DNA and a plasmid carrying sequences coding for resistance to mycophenolic acid. Only 30% of the transfected colonies had a transformed phenotype, i.e. highly refractile rounded cells. The remaining colonies had varied morphologies with flat or slightly elongated cells. Analysis of p21 ras protein indicated that higher levels of the protein were expressed in cells with the more transformed phenotype. Tumors formed by a poorly tumorigenic clone were found to have undergone in vivo amplification of the transfected KiSV sequence. Transformed variants of this clone were also isolated in vitro. Treatment with 5-azacytidine resulted in an increase of about 10 fold in the formation of transformed variants. All transformed cells isolated, either spontaneous or 5-azacytidine induced, were tumorigenic in nude mice. The neoplastic conversion of these cells was accompanied by amplification of the transfected K-ras sequences. The data reported here indicate that a chemical agent that modifies DNA structure can enhance the frequency of amplification events in cellular DNA and, by affecting ras copy number, promote cell transformation.


Subject(s)
Carcinogens , Cell Transformation, Neoplastic , Gene Amplification , Genes, ras , Mutation , Animals , Cell Line , Cell Transformation, Neoplastic/drug effects , DNA, Viral/genetics , Kirsten murine sarcoma virus/genetics , Mice , Mice, Inbred C3H , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins p21(ras) , Transfection
6.
J Natl Cancer Inst ; 79(1): 189-95, 1987 Jul.
Article in English | MEDLINE | ID: mdl-3110476

ABSTRACT

For the investigation of whether Abelson murine leukemia virus (A-MuLV) is able to transform in vivo lymphocytes other than those of the B-cell lineage, newborn BALB/c and C57BL/6 mice were given an injection of A-MuLV directly into the thymus. Thymic lymphomas appeared with a short latent period of 4-5 weeks in BALB/c mice and 8 weeks in C57BL/6 mice. Cell lines derived from some thymic lymphomas presented a very immature phenotype and did not express cellular markers of either T-cells (Thy 1.2, Lyt 1.2, and Lyt 2.2) or B-cells (cytoplasmic IgM) even after treatment with several differentiation inducers. Molecular analysis showed that T-cell receptor (TCR) beta chain genes were never rearranged; in one case only, rearrangement of TCR gamma chain genes could be demonstrated, confirming the immaturity of the presumptive T-cell lines studied. Furthermore, the cell lines consistently carried diversity (D)-joining (J) but not variable (V)-D-J rearrangements of the immunoglobulin heavy chain genes. On the whole, these findings suggest that following intrathymic A-MuLV injection neoplastic transformation does involve lymphocytes possibly of T-cell lineage, at a very early stage of differentiation.


Subject(s)
Cell Transformation, Neoplastic/etiology , Lymphocytes/ultrastructure , Lymphoma/etiology , Thymus Neoplasms/etiology , Tumor Virus Infections/etiology , Abelson murine leukemia virus , Animals , Animals, Newborn , Cell Line , DNA, Neoplasm , Genes, MHC Class II , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Receptors, Antigen, T-Cell
7.
Proc Natl Acad Sci U S A ; 84(12): 4131-4, 1987 Jun.
Article in English | MEDLINE | ID: mdl-3473497

ABSTRACT

C3H/10T1/2 cells were infected with a retroviral vector expressing a mouse c-myc oncogene and a drug-selection marker. The resulting cells, morphologically indistinguishable from C3H/10T1/2, displayed a greatly enhanced sensitivity to neoplastic transformation by ionizing radiation or by a chemical carcinogen. Constitutive expression of myc therefore appears to synergize with an initial carcinogenic event, providing a function analogous to a subsequent event that apparently is required for the neoplastic transformation of these cells. This cell system should prove useful in exploring early stages in radiation-induced transformation.


Subject(s)
Cell Transformation, Neoplastic , Neoplasms, Radiation-Induced/pathology , Proto-Oncogenes , Animals , Cells, Cultured , Genes, Viral , Genetic Vectors , Mice , Mice, Inbred C3H , Neoplasms, Radiation-Induced/genetics , Retroviridae/genetics
8.
Virology ; 158(1): 69-78, 1987 May.
Article in English | MEDLINE | ID: mdl-3576974

ABSTRACT

We have introduced a genomic DNA clone of a mutated human N-ras gene from a T-cell leukemia cell line into a retroviral vector equipped with a neo resistance gene and with SV40 and pBR322 origins of replication. The helper free N-ras virus, which was recovered after transfection of the construction in the psi 2 packaging cell line, contained a correctly spliced N-ras gene. Proviral DNA was amplified in cos cells and subsequently cloned in bacteria. Nucleic acid sequence analysis of the activated N-ras gene revealed a point mutation at codon 12 resulting in a glycine to aspartic acid substitution. The N-ras virus was able to transform mouse fibroblastic cell lines, but failed to fully transform mouse primary embryo fibroblasts. MoMuLV or amphotropic 4070A pseudotypes of the virus were injected intraperitoneally into newborn mice. The MoMuLV pseudotype produced only helper-virus-induced leukemias. The amphotropic pseudotype caused fibrosarcomas after a long latent period. The results of these and other in vivo experiments are discussed in relation to known pathogenic effects of other retroviruses carrying H-ras or K-ras genes.


Subject(s)
Cell Transformation, Neoplastic , Moloney murine leukemia virus/genetics , Oncogenes , Animals , Cell Line , Cell Transformation, Viral , Cloning, Molecular , DNA, Recombinant , Fibrosarcoma/microbiology , Genes, Viral , Genetic Vectors , Humans , Leukemia, Experimental/microbiology , Mice , Mice, Inbred AKR , Mice, Inbred BALB C , Transfection
10.
Proc Natl Acad Sci U S A ; 83(21): 8167-71, 1986 Nov.
Article in English | MEDLINE | ID: mdl-3490663

ABSTRACT

The c-myc oncogene has been implicated in deregulation of cell growth in neoplastic cells and response to "competence-inducing" growth factors in normal cells. In the latter case, expression of c-myc has been shown to be associated with the transition from the G0 to the G1 phase of the cell cycle induced by platelet-derived growth factor (PDGF). In the work reported here, we have introduced the c-myc coding region, in a retroviral vector, into mouse and rat cells. We show that under conditions of anchorage-independent growth, constitutive c-myc expression increases the response of rodent cells to PDGF, as well as to other growth factors of both the competence-inducing and "progression" classes. These effects of the myc product are observed whether or not an exogenous ras gene has also been introduced into the same cells. Possible models for the influence of myc on growth responses are discussed.


Subject(s)
Growth Substances/pharmacology , Proto-Oncogenes , Cell Line , Cell Transformation, Neoplastic , Epidermal Growth Factor/pharmacology , Models, Biological , Peptide Biosynthesis , Platelet-Derived Growth Factor/pharmacology , Transforming Growth Factors
11.
Mol Cell Biol ; 6(11): 4088-92, 1986 Nov.
Article in English | MEDLINE | ID: mdl-3025631

ABSTRACT

A high frequency (greater than or equal to 65%) of thymomas induced by mink cell focus-forming virus 69L1 in AKR/J mice contain proviral integrations which are clustered 0.7-kilobase upstream of the c-myc oncogene predominantly in the opposite transcriptional orientation. Blot hybridization experiments showed that on the average there was only a twofold elevation of steady-state c-myc RNA in the thymomas as compared with levels in normal AKR/J thymocytes. Such an increase would not appear to be sufficient as a mechanism of oncogene activation in this system. In contrast, S1 nuclease analysis of transcripts initiated from the two known c-myc promoters indicated a strong shift in promoter usage in virtually all thymomas tested. In normal thymus the ratio of transcripts initiated from the proximal promoter P1 to the distal promoter P2 was 0.2 to 0.3. In contrast, most of the thymomas tested (18 of 23) showed an average P1/P2 ratio of 1.2 regardless of whether or not proviral integrations could be detected within a 21-kilobase EcoRI fragment containing the three c-myc exons. We conclude that alterations in P1/P2 ratios are good indicators of c-myc deregulation in thymic lymphomas.


Subject(s)
Leukemia Virus, Murine/pathogenicity , Lymphoma/microbiology , Mink Cell Focus-Inducing Viruses/pathogenicity , Oncogenes , Thymoma/microbiology , Thymus Neoplasms/microbiology , Animals , Genes, Viral , Lymphoma/genetics , Mice , Mice, Inbred AKR , Nucleic Acid Hybridization , Thymoma/genetics , Thymus Neoplasms/genetics
12.
Immunogenetics ; 24(2): 103-14, 1986.
Article in English | MEDLINE | ID: mdl-2427440

ABSTRACT

TL antigens are class I glycoproteins which are expressed on thymocytes and which are coded by the Tla region of the major histocompatibility complex of the mouse. Biochemical analysis of TL molecules from different strains of mice revealed structural variation determined by the Tla region which is detectable by peptide mapping, isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two-dimensional gels, and by differential reactivity of allelic forms of TL molecules with a panel of anti-TL reagents. The quantity of TL expressed on thymocytes is also influenced by the Tla region; three quantitative phenotypes were identified: high (Tlaa, Tlad, Tlae), intermediate (Tlac, Tlaf), and low (Tlab). (Relative amounts: 1000: 100: 1.) Some thymic leukemias arising in (Tlab, Tlac) mice with genetically determined reduced levels of thymic TL were found to express TL molecules which were structurally indistinguishable from TL isolated from thymocytes but were present in larger amounts. This suggests that TL structural genes are intrinsically capable of full expression in all mice but that the Tla region of mice expressing an intermediate or low quantity of TL is marked by some feature which causes the thymocyte to express less than the full amount of TL possible.


Subject(s)
Antigens, Neoplasm/genetics , Major Histocompatibility Complex , Membrane Glycoproteins , T-Lymphocytes/immunology , Age Factors , Animals , Antigens, Neoplasm/immunology , Autoantibodies/immunology , Epitopes , Gene Expression Regulation , Genotype , Heterozygote , Isoelectric Point , Leukemia, Experimental/immunology , Macromolecular Substances , Mice , Molecular Weight , Polymorphism, Genetic , Thymus Gland/immunology
13.
J Virol ; 55(2): 500-3, 1985 Aug.
Article in English | MEDLINE | ID: mdl-2991574

ABSTRACT

Blot hybridization of thymocyte DNA from AKR/J mice was used to detect new proviral junction fragments as markers of clonality at different stages of viral leukemogenesis and to detect DNA rearrangements at the c-myc locus due to proviral insertion. Clonal populations of thymocytes were observed in mink cell focus-forming virus-injected mice as early as 35 days postinjection, at a stage distinguishable from frank leukemia by flow cytometric analysis and transplantation bioassay. Specific proviral integrations in the c-myc locus were detected in 15% of these early clones and in up to 65% of late-developing thymomas and frank leukemias. Thus, in this system c-myc activation appears to be a common mechanism in T-cell leukemogenesis.


Subject(s)
Leukemia Virus, Murine/genetics , Leukemia, Experimental/microbiology , Oncogenes , Recombination, Genetic , Animals , Cell Transformation, Neoplastic , Cell Transformation, Viral , Clone Cells , Genes, Viral , Leukemia, Experimental/genetics , Leukemia, Experimental/pathology , Mice , Mice, Inbred AKR , T-Lymphocytes/microbiology , T-Lymphocytes/pathology , Thymoma/genetics , Thymoma/microbiology , Thymoma/pathology , Thymus Neoplasms/genetics , Thymus Neoplasms/microbiology , Thymus Neoplasms/pathology
14.
Virology ; 140(1): 102-12, 1985 Jan 15.
Article in English | MEDLINE | ID: mdl-2981445

ABSTRACT

Endogenous murine leukemia virus (MuLV) proviral copies were analyzed in thymomas induced in normal BALB/c (Fv-1b) and in Fv-1n congenic mice by X-irradiation. Both strains of mice developed leukemia with similar kinetics, indicating that N-tropism of endogenous MuLV was not a rate-limiting factor in development of disease. Southern blot analysis, using a probe specific for ecotropic virus and for ecotropic-specific sequences retained in pathogenic, env-recombinant viruses, showed that the majority of radiation leukemias lacked newly acquired, clonally integrated, proviruses. This was in contrast to virus-induced leukemias, which routinely exhibited several new proviral integration sites. When an internal proviral DNA restriction fragment was monitored, some radiation leukemias showed evidence of nonclonal infection, accounting for more frequent isolation of infectious virus from such leukemias. Differences in expression of T-cell surface antigens were found in X-ray-induced and virus-induced leukemias. All radiation leukemias were TL positive, whereas virus-induced leukemias were primarily negative for TL. Some differences were also found in Lyt-1 and Lyt-2 expression. The data as a whole suggest that, in the majority of cases, radiation leukemogenesis is not initiated by a viral route--that is, the sort of viral mechanism for which exogenous infection by known pathogenic MuLV is the paradigm.


Subject(s)
Leukemia, Experimental/microbiology , Leukemia, Radiation-Induced/physiopathology , Thymoma/physiopathology , Thymus Neoplasms/physiopathology , Animals , Antigens, Surface/analysis , DNA, Neoplasm/analysis , Leukemia Virus, Murine/pathogenicity , Leukemia, Experimental/physiopathology , Mice , Mice, Inbred BALB C , T-Lymphocytes/immunology , Thymoma/microbiology , Thymus Neoplasms/microbiology , X-Rays
16.
Proc Natl Acad Sci U S A ; 80(21): 6676-9, 1983 Nov.
Article in English | MEDLINE | ID: mdl-6605534

ABSTRACT

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.


Subject(s)
Leukemia/genetics , Oncogenes , Animals , Cell Line , Cell Transformation, Neoplastic , Cloning, Molecular , Humans , Mice , T-Lymphocytes/physiology , Transfection , Transformation, Genetic
17.
J Virol ; 48(1): 102-9, 1983 Oct.
Article in English | MEDLINE | ID: mdl-6193284

ABSTRACT

Murine leukemia virus-related RNA species were examined in a set of radiation-induced T-cell leukemias from BALB/c mice. No evidence was found for linkage of viral long terminal repeat-derived (U5) sequences to information of host origin. A novel class of 2-kilobase (kb) env-related transcripts, about 1kb shorter than normal viral env messenger, was found in all the leukemias. All of the 2-kb transcripts contained sequences homologous to the xenotropic virus-related env sequences in the Friend spleen focus-forming virus, representing the N-terminal portion of gp70. In two of the leukemias, these transcripts were found to contain both ecotropic p15E and U3 sequences in addition to the xenotropic gp70-related sequence. These two leukemias, but not others in which ecotropic sequences were absent from the 2-kb RNA, harbored several copies of a specific class of env recombinant proviruses. These proviruses possessed full-size env genes and were submethylated, as shown by SmaI and XmaI digests of proviral DNA. Low levels of 2-kb RNA were found in normal thymocytes from strains BALB/c, AKR, and 129 but not from congenic 129 GIX- mice. It is possible that the 2-kb RNA may originate by a novel splicing step that removes portions of the gp70 and p15E sequences from full-length env transcripts.


Subject(s)
Leukemia Virus, Murine/genetics , Leukemia, Radiation-Induced/microbiology , RNA, Viral/genetics , Transcription, Genetic , Viral Proteins/genetics , Animals , Cell Line , Mice , Mice, Inbred AKR , Mice, Inbred BALB C , Nucleic Acid Hybridization , Poly A/analysis , Poly A/genetics , RNA/analysis , RNA/genetics , RNA Splicing , RNA, Messenger , RNA, Viral/analysis , Recombination, Genetic , Repetitive Sequences, Nucleic Acid , T-Lymphocytes/analysis , Viral Envelope Proteins
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