Tumorigenesis by slow-transforming retroviruses--an update.
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Animals↗
Biomedical subjects
A Berns
Publications and source records attributed to A Berns.
Transgenic mice demonstrate that epithelial homing of gamma/delta T cells is determined by cell lineages independent of T cell receptor specificity.
gamma/delta T cells with different TCR repertoires are compartmentalized in different epithelia. This raises the possibility that the TCR-gamma/delta directs homing of T cells to these epithelia. Alternatively, the signals that induce TCR-gamma/delta expression in developing T cells may also induce homing properties in such cells, presumably in the form of cell surface receptors. We have examined this issue by studying the homing of gamma/delta T cells in transgenic mice constructed with specific pairs of rearranged gamma and delta genes. In such mice, most gamma/delta T cells express the transgene-encoded TCR. We find that homing to both skin and gut epithelia is a property of T cells and is not determined by the type of gamma and delta genes used to encode their TCR. We also studied the effect of TCR replacement on the expression of Thy-1 and CD8 proteins on the gamma/delta T cells associated with gut epithelia. Our results show that the expression of the appropriate type of TCR-gamma/delta is not required for the Thy-1 expression by these T cells, suggesting that Thy-1 is not an activation marker. In contrast, CD8 expression by gut gamma/delta T cells seems to depend on the expression of the appropriate type of TCR.
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Self-tolerance to transgenic gamma delta T cells by intrathymic inactivation.
During their intrathymic differentiation, T lymphocytes expressing alpha beta T-cell receptors (TCR) are negatively and positively selected. This selection contributes to the establishment of self-tolerance and ensures that mature CD4+ and CD8+ cell populations are restricted by the self major histocompatibility complex. Little is known, however, about gamma delta T-cell development. To investigate whether selection operates in the establishment of the gamma delta T-cell class, we have generated transgenic mice using gamma- and delta-transgenes encoding a TCR that is specific for a product of a gene in the TL-region of the TLb haplotype. Similar numbers of thymocytes expressing the transgenic TCR were generated in mice of TLb and TLd haplotypes. But gamma delta thymocytes from TLb and TLd transgenic mice differed in cell size, TCR density and in their capacity to respond to TLb stimulator cells or interleukin-2 (IL-2). In contrast to gamma delta T cells from TLd transgenic mice, gamma delta T cells from TLb transgenic mice did not produce IL-2 and did not proliferate in response to TLb stimulator cells, but they did proliferate in the presence of exogenous IL-2. These results indicate that functional inactivation of self-antigen-specific T cells could contribute to the establishment of self-tolerance to thymic determinants.
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Mutations in the major histocompatibility complex class I antigen-presenting groove affect both negative and positive selection of T cells.
In several transgenic mouse models T cell development was shown to be controlled by the binding of the alpha/beta T cell receptor (TcR) to ligands in the thymus. In transgenic mice expressing a male-specific TcR alpha/beta, the presence of the restricting D major histocompatibility complex (MHC) molecule plus the male specific peptide deleted thymocytes at an early stage of development. On the other hand, maturation of T cells required an interaction of the TcR with the thymic D MHC molecules in the absence of specific peptides. This could imply that negative and positive selection of this receptor are affected differently by mutations in the HY peptide-binding groove of the D MHC molecule. Such mutants have been isolated and were shown to affect the response to HY antigen in that both the bm14 (residue Glu70----Asp) and the bm13 (residue Leu114----Glu, Phe116----Tyr and Glu119----Asp) strains do not normally mount cytotoxic responses to male cells. Here we show that these mutations affect antigenicity of male cells, as well as negative and positive selection of T cells in TcR alpha/beta transgenic mice.
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T-cell receptor gamma delta and gamma transgenic mice suggest a role of a gamma gene silencer in the generation of alpha beta T cells.
A T lymphocyte expresses on its surface one of two types of antigen receptor, T-cell receptor alpha beta or T-cell receptor gamma delta, encoded by a pair of somatically rearranged alpha and beta or gamma and delta genes. It has been suggested that alpha beta T cells are generated only from precursor T cells that failed to rearrange gamma and delta genes in a functional form. However, we found that transgenic mice constructed with functionally rearranged gamma and delta genes produce a normal number of alpha beta T cells. The transgene gamma present in these alpha beta T cells is repressed apparently through an associated cis DNA element (silencer). We propose that some T-cell precursors are committed to generate alpha beta T cells independent of the rearrangement status of their gamma gene and that this commitment involves activation of a factor(s) that interacts with the gamma gene-associated silencer.
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Identification of two genes in the unique short region of pseudorabies virus; comparison with herpes simplex virus and varicella-zoster virus.
We have determined the nucleotide sequence of two genes in the unique short region of the genome of pseudorabies virus (PRV). Near the internal repeat, upstream of the gene encoding glycoprotein gX, we identified an open reading frame (ORF) encoding a protein of 390 amino acids. We designated this gene PK because the predicted protein contains most of the conserved motifs of a eukaryotic protein kinase. The protein shares amino acid homology with the protein kinases encoded by gene US3 of herpes simplex virus type 1 (HSV-1) and gene 66 of varicella-zoster virus. Near the terminal repeat, downstream of a gene encoding an 11K protein, we identified an ORF encoding a protein of 256 amino acids. We designated this gene 28K, the Mr of the predicted protein. Part of the amino acid sequence of 28K is homologous to the predicted US2 protein of HSV-1. Northern blot analysis revealed a 2.7 kb mRNA encoding the putative protein kinase and a 1.2 kb mRNA encoding the 28K protein in PRV-infected cells. The 5' ends of the mRNAs were mapped by primer extension. Two transcriptional start sites were identified for the PK mRNA: a minor start site immediately upstream of the ORF and a major start site (greater than 95% of the mRNA) within the ORF, 64 nucleotides upstream of an internal ATG codon. A single transcriptional start site was identified for the 28K mRNA immediately upstream of the ORF. Immunoblot analysis with anti-peptide sera revealed that, in cells infected with PRV, the PK gene was translated into two proteins with Mrs of 53K and 41K, and the 28K gene into a single protein with an Mr of 28K.
Amino Acid Sequence↗
Linker insertion mutagenesis of herpesviruses: inactivation of single genes within the Us region of pseudorabies virus.
We describe a technique for the systematic inactivation of nonessential genes within the genome of a herpesvirus without the requirement for phenotypic selection. This technique is based on the insertion of an oligonucleotide containing translational stop codons at a random site within a large cloned viral DNA fragment. Mutant virus is then reconstituted by cotransfection with overlapping viral clones, together comprising the entire viral genome, as described previously (M. van Zijl, W. Quint, J. Briaire, T. de Rover, A. Gielkens, and A. Berns, J. Virol. 62:2191-2195, 1988). This technique was used to construct, in a single experiment, a set of 13 viable pseudorabies virus strains with oligonucleotide insertions within all known genes of the Us region except for the gp50 gene, which proved essential for virus growth in cell culture. The growth rate in porcine kidney cells of mutants of all nonessential Us genes was similar to that of the parental virus, with the exception of a mutant of the recently identified protein kinase gene.
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Human-mouse interspecies collagen I heterotrimer is functional during embryonic development of Mov13 mutant mouse embryos.
To investigate whether the human pro alpha 1(I) collagen chain could form an in vivo functional interspecies heterotrimer with the mouse pro alpha 2(I) collagen chain, we introduced the human COL1A1 gene into Mov13 mice which have a functional deletion of the endogenous COL1A1 gene. Transgenic mouse strains (HucI and HucII) carrying the human COL1A1 gene were first generated by microinjecting the COL1A1 gene into wild-type mouse embryos. Genetic evidence indicated that the transgene in the HucI strain was closely linked to the endogenous mouse COL1A1 gene and was X linked in the HucII transgenic strain. Northern (RNA) blot and S1 protection analyses showed that the transgene was expressed in the appropriate tissue-specific manner and as efficiently as the endogenous COL1A1 gene. HucII mice were crossed with Mov13 mice to transfer the human transgene into the mutant strain. Whereas homozygous Mov13 embryos die between days 13 and 14 of gestation, the presence of the transgene permitted apparently normal development of the mutant embryos to birth. This indicated that the mouse-human interspecies collagen I heterotrimer was functional in the animal. The rescue was, however, only partial, as all homozygotes died within 36 h after delivery, with signs of internal bleeding. This could have been due to a functional defect in the interspecies hybrid collagen. Extensive analysis failed to reveal any biochemical or morphological abnormalities of the collagen I molecules in Mov13-HucII embryos. This may indicate that there was a subtle functional defect of the interspecies hybrid protein which was not revealed by our analysis or that another gene has been mutated by the retroviral insertion in the Mov13 mutant strain.
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T cell depletion in transgenic mice carrying a mutant gene for TCR-beta.
Classical T lymphocytes recognize foreign antigens in the context of self major histocompatibility complex (MHC) molecules by means of the T-cell receptor (TCR)alpha beta heterodimer. The genes for TCR beta-chains, like immunoglobulin genes, are subject to allelic exclusion. The introduction of a functional TCR-beta gene into the germline of mice prevents rearrangement of endogenous TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta genes. Here we report that the introduction of a non-functional TCR-beta gene with a deletion of the major part of the variable region (delta V-TCR-beta), also inhibits endogenous TCR-beta gene rearrangement. This inhibition is mediated via the encoded protein because impairment of endogenous TCR-beta gene rearrangement is not found if a frameshift mutation is introduced into the DJ region of the delta V-TCR-beta transgene. The delta V-TCR-beta transgene can lead to two phenotypes, in which lymphoid development is perturbed. Phenotype A is characterized by a severe impairment of both T and B cell development as reflected by the complete absence of certain lymphoid organs. In phenotype B, lymphoid organs are macroscopically normal, but T cell differentiation is impeded. Virtually all thymocytes lack membrane expression of TCR-alpha beta, but nevertheless carry the CD4 and CD8 antigens (CD4+CD8+ phenotype); they do not, however, mature further. The defect in mice of phenotype B but not of phenotype A can be corrected by the introduction of a functional TCR-beta gene.
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Very high frequency of lymphoma induction by a chemical carcinogen in pim-1 transgenic mice.
Infection of mice with Moloney murine leukaemia virus (MuLV) induces T-cell lymphomas after an average latency period of 150 days. In these lymphomas the MuLV DNA is frequently integrated into the mouse chromosomal DNA in the vicinity of the pim-1 oncogene. Transgenic mice overexpressing the pim-1 oncogene are predisposed to develop T-cell lymphomas, but only to the extent that approximately 10% of the mice develop a lymphoma within 240 days. When these mice are infected with MuLV, lymphomas develop in all mice in only 50-60 days. In these lymphomas MuLV DNA is integrated near either the c-myc or N-myc gene, suggesting that pim-1 and myc synergize in lymphomagenesis. To determine whether this system has a more general application, we have now tested the susceptibility of pim-1 transgenic mice to N-ethyl-N-nitrosourea (ENU), a chemical carcinogen. With a single low dose of ENU, nearly all pim-1 transgenic mice, but only 15% of non-transgenic mice, develop T-cell lymphomas within 200 days. All ENU-induced lymphomas in both pim-1 transgenic and non-transgenic mice express high levels of c-myc messenger RNA, supporting the notion that pim-1 and c-myc synergize in lymphoma induction. We propose that pim-1 transgenic mice could be used to test the oncogenic potential of other chemical compounds.
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Predisposition to lymphomagenesis in pim-1 transgenic mice: cooperation with c-myc and N-myc in murine leukemia virus-induced tumors.
Transgenic mice bearing the pim-1 gene supplemented with an upstream immunoglobulin enhancer and a downstream murine leukemia virus long terminal repeat express pim-1 mRNA at high levels in both B and T cells. Between 5% and 10% of the pim-1 transgenic mice develop clonal T cell lymphomas before 7 months of age, whereas none of the age-matched control mice do, providing direct evidence for the oncogenic potential of pim-1. Histological examination and FACS analysis revealed no abnormalities in hematopoietic tissues of disease-free pim-1 transgenic mice. When newborn pim-1 transgenic mice are infected with MuLV, T cell lymphomas develop much faster (latency 7-8 weeks) than in nontransgenic mice (latency 22 weeks). In all these T cell lymphomas either c-myc or N-myc was activated by proviral insertion, suggesting strong cooperation between pim-1 and myc in lymphomagenesis.
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Transgenic mice as a means to study synergism between oncogenes.
Transgenic mice present a useful model to study the mechanisms underlying malignant transformation. (i) They can provide information on the oncogenic potential of genes as a function of tissue context. (ii) They allow the analysis of the primary effects of an oncogene on proliferation and differentiation before secondary mutations have occurred. (iii) Crossings between transgenic mice carrying different oncogenes can reveal their capacity to cooperate in transformation. (iv) Transgenic mice bearing a particular oncogene can be used to search for (new) (anti)oncogenes that synergize with the transgene. The non-acute transforming murine leukemia viruses (MuLV) appear very useful for this purpose. This has become clear from our studies with pim-I and c-myc transgenic mice. MuLV dramatically accelerates T-cell lymphomagenesis in transgenic mice overexpressing the pim-I oncogene in their lymphoid compartment. In all tumors induced by MuLV in pim-I transgenic mice, either the c-myc or the N-myc gene was activated by proviral insertion. Similarly, MuLV infection of transgenic mice overexpressing the c-myc gene in their B-cell compartment resulted in the acceleration of pre-B-cell lymphomagenesis. A significant fraction of the resulting pre-B-cell tumors showed proviral activation of pim-I. This shows that pim-I and myc synergize efficiently in both B- and T-cell lymphomagenesis. pim-I transgenic mice are also highly sensitive to tumor induction by N-ethyl-N-nitrosourea (ENU) and therefore represent an excellent in vivo model system to test the oncogenic potential of chemical compounds.
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N-myc is frequently activated by proviral insertion in MuLV-induced T cell lymphomas.
We report a new common proviral insertion site in murine leukemia virus-induced T cell lymphomas to be N-myc. Proviral activation of N-myc was found in 35% of independently induced primary tumors. The vast majority of the proviral insertions occur within a small segment of the 3'-untranslated region of the N-myc gene, directly downstream of the protein-encoding domain. This results in an increased level of expression of a truncated N-myc mRNA. Together with the previously shown c-myc activation we now find involvement of myc genes in greater than 75% of the primary T cell lymphomas induced by Moloney murine leukemia virus in C57BL10 and BALB/c mice, and show for the first time that N-myc can be over-expressed by a mechanism other than gene amplification.
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Evidence for the involvement of pim-2, a new common proviral insertion site, in progression of lymphomas.
We have compared proviral integrations near (putative) proto-oncogenes in Moloney murine leukemia virus-induced primary and transplanted T cell lymphomas. We previously found proviruses integrated near c-myc, pim-1, and N-myc in primary tumors (Selten et al., 1984; Van Lohuizen et al., 1989a; Van Lohuizen et al., 1989b). We have now identified an additional common proviral integration site, called pim-2, that carries somatically acquired proviruses in the majority of transplanted tumors. In primary tumors integration near pim-2 is usually undetectable or present in only a minor fraction of the tumor cells. This subpopulation selectively grows out upon transplantation. Insertion near pim-2 is a relatively late event in tumorigenesis and is often preceded by proviral insertions in other common insertion sites, yielding tumor clones which carry proviruses in up to three different common insertion sites within the same cell (c-myc, pim-1 and pim-2). The data suggest that pim-2 plays an important role in tumor progression.
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Transgenic expression of the muscle-specific intermediate filament protein desmin in nonmuscle cells.
The coding region of the hamster desmin gene was fused to the 5' flanking sequences of the hamster vimentin gene and introduced into the germ line of mice. The expression of this intermediate filament gene construct (pVDes) was analyzed at the RNA and protein level in transgenic mice as well as in fibroblast cell lines and primary hepatocyte cultures derived from these mice. In all transgenic mice, the pVDes-encoded protein was coexpressed with mouse vimentin in a tissue-specific fashion and was indistinguishable from normal hamster desmin. Culturing of transgenic hepatocytes induced desmin expression indicating that 3.2 kbp of the vimentin gene 5' region regulates both tissue-specific and tissue culture-induced intermediate filament protein expression. Immunohistochemical staining and double-label immunoelectron microscopy of cultured transgenic fibroblasts showed that the pVDes protein assembled into intermediate filaments which colocalized with the mouse vimentin filaments. Endogenous vimentin RNA levels were not influenced by high-level pVDes expression. The coexpression of desmin and vimentin in nonmuscle cells did not result in detectable developmental, morphological, or physiological abnormalities.
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Diversity, development, ligands, and probable functions of gamma delta T cells.
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Animals↗
T-cell-specific deletion of T-cell receptor transgenes allows functional rearrangement of endogenous alpha- and beta-genes.
In B cells the loci encoding immunoglobulin chains usually show allelic exclusion; a given B cell transcribes and translates only one productively rearranged allele of the heavy and light chain loci. This ensures that each B cell expresses only one antigen receptor. The loci encoding T-cell receptor (TCR) alpha- and beta-genes may behave similarly. We have previously reported that the expression of a transgenic TCR beta-chain prevents functional and nonfunctional V beta rearrangements in the endogenous beta-chain loci but not D beta J beta rearrangements. We have also been unable to detect the expression of the TCR gamma-chain locus in thymocytes of these mice (unpublished observations). To study the mechanisms involved in forming a mature T-cell repertoire further, we have constructed mice expressing alpha- and beta-TCR transgenes derived from a cytotoxic T-cell clone that is specific for the male antigen H-Y in the context of H-2Db MHC molecules. Here we show that in these mice rearrangement of endogenous alpha-chain loci is also suppressed, although to a lesser extent than rearrangement of beta-chain loci. In addition, in male alpha beta TCR transgenic mice we observed T-cell clones which had deleted both transgenic alpha- and beta-chain genes and expressed endogenous alpha- and beta-chain TCR genes. These cells are presumably derived from rare thymocytes that leave the male thymus because their TCR no longer recognizes self antigen. The vast majority of CD4+8+ nonmature thymocytes expressing alpha- and beta-transgenes are deleted in the male thymus.
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In transgenic mice the introduced functional T cell receptor beta gene prevents expression of endogenous beta genes.
Transgenic mice were constructed with a functional T cell receptor beta gene. Transcription of the introduced gene is largely confined to T cells, but low levels of transcripts are also seen in B cells and in other tissues. Serological analyses show that most, if not all, of the T lymphocytes express the transgenic beta chain on the cell surface and lack beta chains encoded by endogenous beta genes. Molecular genetic analyses of uncloned and cloned T lymphocytes demonstrate that rearrangement of endogenous beta genes is incomplete. Partial D beta 1-J beta 1 rearrangements are found preferentially, while complete VDJ rearrangements are not seen. These findings show that expression of the transgene regulates the rearrangement of endogenous beta genes. Although the alpha beta T cell receptors of the transgenic mice are homogeneous with respect to the beta chain, they are fully functional, at least in a variety of allogeneic responses.
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