Transgenic thymocytes are refractory to transformation by the human T-cell leukemia virus type I tax gene.

Previous transgenic work demonstrated transforming activity of the human T-cell leukemia virus type I Tax protein in fibroblasts. In the present study, a Thy-1-based vector was used to express Tax in thymocytes. These mice developed no functional or neoplastic abnormalities of T cells but developed fibroblastic tumors with a longer latency than in the previous model.     

Infection with the human T-lymphotropic virus type I. A review for clinicians.

The human T-cell lymphotropic virus type I (HTLV-I) is the first retrovirus identified in humans. It has been responsible for a number of clinical syndromes, most notably adult T-cell leukemia or lymphoma and tropical spastic paraparesis. In the United States, infection with this virus is most frequently found in specific subsets of our population, particularly in those who live in the southeastern states, have southern Japanese ancestry, or share intravenous drug paraphernalia. Understanding the epidemiology and clinical manifestations of this virus is necessary to properly diagnose and care for patients with HTLV-I infection.     

The bovine papillomavirus E5 oncogene can cooperate with ras: identification of p21 amino acids critical for transformation by c-rasH but not v-rasH.

We have previously used a series of insertion-deletion mutants of the mutationally activated v-rasH gene to identify several regions of the encoded protein that are dispensable for cellular transformation (B. M. Willumsen, A. G. Papageorge, H.-F. Kung, E. Bekesi, T. Robins, M. Johnsen, W. C. Vass, and D. R. Lowy, Mol. Cell. Biol. 6:2646-2654, 1986). To determine if some of these amino acids are more important for the biological activity of c-rasH, we have now tested many of the same insertion-deletion mutants in the c-rasH form for their ability to transform NIH 3T3 cells. Since the transforming activity of c-rasH is low, we have used cotransfection with the bovine papillomavirus (BPV) genome to develop a more sensitive transformation assay for c-rasH mutants. The increased sensitivity of the assay, which is seen both in focal transformation and in anchorage-independent growth, is mediated by cooperation between the BPV E5 gene and ras. E5-dependent cooperation was seen for v-rasH as well as for c-rasH, which suggests that the major effect of E5 was to increase the susceptibility of the cell to transformation to a given level of ras activity. The cooperation assay was used to test the potential importance, in c-rasH, of codons 93 to 108, 123 to 130, and 166 to 183, which were nonessential for v-rasH transformation. Relative to the respective transforming activity of wild-type c-rasH and v-rasH, mutants with lesions in codons 102 and 103 were significantly less active in their c-rasH forms than in their v-rasH forms. We conclude that a region including amino acids 102 and 103 encodes a function that is more critical to c-rasH than to v-rasH. Guanine nucleotide exchange is one function that is compatible with such a phenotype.     

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4(-) and CD8(-), but CD44(+), CD25(+) and cytoplasmic CD3(+). This phenotype is indicative of a thymus-derived pre-T-cell phenotype, and disease development was associated with the constitutive activation of NF-kappaB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.     

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene.

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.     

Isolation of a human T-lymphotropic virus type I strain from Australian aboriginals.

A human T-lymphotropic virus type I (HTLV-I) strain was isolated in a CD4+ T-lymphocyte culture established from a healthy seropositive Australian Aboriginal. This isolate, identified as HTLV-IMSHR-1, was detected by immunofluorescence with monoclonal antibodies, by the presence of gag-encoded protein p24 in the culture supernatant, and by cocultivation leading to infection and transformation of lymphocytes from an HTLV-I-negative donor. By using the polymerase chain reaction technique, the env gene and segments of the pol and pX regions of the proviral genome of HTLV-I(MSHR-1) were amplified and sequenced. Comparison with the envelope sequences of prototype strains revealed up to 7% divergence at the nucleotide level and 3.1 to 4.3% divergence in the predicted amino acid sequence. Phylogenetic analysis showed that the Australian and Melanesian isolates are related. Differential reactivity with monoclonal antibodies suggests that gag protein p19 of HTLV-I(MSHR-1) is also divergent. The potential for antigenic divergence between the prototype HTLV-I isolates and the Austro-Melanesian variants requires further investigation, because it would have implications for serodiagnosis and vaccine development.     

Mutation is required to activate the p53 gene for cooperation with the ras oncogene and transformation.

Previous experiments have brought into question which amino acid sequence of the p53 oncogene product should be considered wild type and whether the normal protein is capable of cooperating with the ras oncogene to transform cells in culture. To address these questions, a series of p53 cDNA-genomic hybrid clones have been compared for the ability to cooperate with the ras oncogene in transformation assays. From these experiments, it has become clear that the amino acid alanine at position 135, in either the genomic clone or the cDNA clone, failed to produce a p53 protein that cooperated with the ras oncogene and transformed cells. Replacing alanine with valine at this position in either the genomic or the cDNA clone activated for transformation in this assay. Using restriction enzyme polymorphisms in the p53 gene, it was shown that normal mouse DNA encodes alanine at position 135 in the p53 protein. Thus, mutation is required to activate the p53 protein for cooperation with the ras oncogene. After cotransfection with the activated ras gene, the genomic p53 DNA clone always produced more transformed cell foci (1.7-fold) than similar cDNA clones and these foci were more readily cloned (3.6-fold) into permanent cell lines. A series of deletion mutants of the genomic p53 clone were employed to show that the presence of intron 4 in the p53 gene was sufficient to provide much enhanced clonability of transformed foci from culture dishes. The presence of introns in the p53 gene constructions also resulted in elevated levels of p53 protein in the p53-plus-ras-transformed cell lines. Thus, qualitative changes in the p53 protein are required to activate p53 for transformation with the oncogene ras. Quantitative improvements of transformation frequencies are associated with the higher expression levels of altered p53 protein that are provided by having one of the p53 introns in the transforming plasmid.     

trans activation of granulocyte-macrophage colony-stimulating factor and the interleukin-2 receptor in transgenic mice carrying the human T-lymphotropic virus type 1 tax gene.

Three lines of transgenic mice carrying the human T-cell lymphotropic virus type 1 tax gene have previously been reported to develop neurofibromas composed of perineural fibroblasts (S. H. Hinrichs, M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1340-1343, 1987; M. Nerenberg, S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1324-1329, 1987). Tumors from these mice and tumor cell lines derived from them expressed high levels of tax RNA and protein. They also expressed high levels of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene as measured by proliferative responses of FD-CP1 target cells using conditioned media from tumor cells and by Northern (RNA) blot analysis of RNA from tumors and tumor cell lines. Although other tissues, such as salivary glands and muscles, in the transgenic mice also expressed high levels of tax, they did not express the gene for GM-CSF. This indicates that tissue-specific cellular factors, in addition to tax, are required for GM-CSF gene expression. Systemic effects of excessive GM-CSF production were demonstrated by infiltration of polymorphonuclear leukocytes into tumor tissues which are not necrotic, by peripheral granulocytosis, and by splenomegaly resulting from myeloid hyperplasia. The interleukin-2 (IL-2) receptor was also found to be expressed by the tumors and tumor cell lines as measured by IL-2-binding and cross-linking studies. This is the first demonstration that the IL-2 receptor can be activated by tax in a nonlymphoid cell type. These in vivo findings are consistent with other reports which have demonstrated in vitro cis-regulatory elements within the 5'-flanking regions of the genes for GM-CSF and the IL-2 receptor which are responsive to trans activation by the tax gene.     

Phenotypic progression of a rat lymphoid cell line immortalized by human T-lymphotropic virus type I to induce lymphoma/leukemia-like disease in rats.

Rat lymphoid cells, TARS-1, immortalized by coculture with adult T-cell leukemia cells, were intraperitoneally injected into 65 newborn, inbred WKAH/Hkm rats. In most of the rats, tumor nodules were discernible 7 to 15 days after transplantation but were completely rejected within 5 to 6 weeks. Two rats with no tumor nodules exhibited gait disturbances and paralysis of the hind legs 3 to 4 weeks after transplantation. Histological and hematological examinations revealed that a lymphoma/leukemia-like disease had developed in one of the two rats, and the T-lymphoid cell line WLeuk-1 was established from peripheral blood mononuclear cells from this rat. When the WLeuk-1 cells were transplanted into newborn WKAH/Hkm rats, the animals died of a lymphoma/leukemia-like disease within several weeks after transplantation, in contrast to their rejection of the TARS-1 cells. Southern blot and karyotype analyses revealed that WLeuk-1 cells had retained the marker chromosomes and human T-lymphotropic virus type I (HTLV-I) integration patterns of the parent cell line, TARS-1. The additional specific chromosome abnormalities 3p+,t (12;13), and Xq+ were found in the WLeuk-1 cells. Moreover, the expression of HTLV-I structural proteins was slightly depressed in WLeuk-1 cells, while that of the transacting factors p40tax and p21x, but not that of p27rex, was enhanced about fivefold compared with that in TARS-1. The transactivating function of p40tax was intact in WLeuk-1, as evidenced by enhanced interleukin-2 receptor alpha chain expression. These results suggest that aberrant expression of HTLV-I regulatory genes and alteration of cellular genes were associated with the phenotypic progression of the WLeuk-1 cell line.     

Simian virus 40 large T antigen contains two independent activities that cooperate with a ras oncogene to transform rat embryo fibroblasts.

The simian virus 40 large T antigen immortalizes growing primary cells in culture. In addition, this viral oncoprotein cooperates with an activated ras protein to produce dense foci on monolayers of rat embryo fibroblasts (REF). The relationship between independent immortalization and cooperative transformation with ras has not been defined. Previously, two regions of T antigen were shown to contain immortalization activities. An N-terminal fragment consisting of amino acids 1 to 147 immortalizes rodent cells (L. Sompayrac and K. J. Danna, Virology 181:412-415, 1991). Loss-of-function analysis indicated that immortalization depended on integrity of the T-antigen segments containing amino acids 351 to 450 and 533 to 626 (T. D. Kierstead and M. J. Tevethia, J. Virol. 67:1817-1829, 1993). The experiments described here were directed toward determining whether these same T-antigen regions were sufficient for cooperation with ras. Initially, constructs that produce T antigens containing amino acids 176 to 708 (T176-708) or 1 to 147 were tested in a ras cooperation assay. Both polypeptides cooperated with ras to produce dense foci on monolayers of primary REF. These results showed that T antigen contains two separate ras cooperation activities. In order to determine the N-terminal limit of the ras cooperation activity contained within the T176-708 polypeptide, a series of constructs designed to produce fusion proteins containing T-antigen segments beginning at residues 251, 301, 337, 351, 371, 401, 451, 501, 551, 601, and 651 was generated. Each of these constructs was tested for the capacity to cooperate with ras to produce dense foci on REF monolayers. The results indicated that a polypeptide containing T-antigen amino acids 251 to 708 (T251-708) was sufficient to cooperate with ras, whereas the more extensively truncated products were not. The abilities of the N-terminally truncated T antigens to bind p53 were examined in p53-deficient cells infected with a recombinant vaccinia virus expressing a phenotypically wild-type mouse p53. The results showed that polypeptides containing T-antigen amino acids 251 to 708, 301 to 708, 337 to 708, or 351 to 708 retained p53-binding capacity. The introduction into the T251-708 polypeptide of deletions that either prevented p53 binding (dl434-444) or did not prevent p53 binding (dl400) abrogated ras cooperation. These results indicated that although p53 binding may be necessary for ras cooperation, an additional, as-yet-undefined activity contained within the T251-708 polypeptide is needed.     

Monocyte-driven activation-induced apoptotic cell death of human T-lymphotropic virus type I-infected T cells.

We attempted apoptotic cell death induction of T cells infected with human T lymphotropic virus type I (HTLV-I) which induces HTLV-I-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. T cells acutely infected and expressing HTLV-Igag Ags were killed by cross-linking their TCR with anti-CD3 mAb. Cells in apoptotic process were found by staining with annexin V. The apoptosis was not affected by costimulation through CD28 molecules and was resistant to ligation of Fas molecules. Whereas the virus-infected T cells expressed higher levels of HLA-DR, CD25, CD80, and CD86 Ags than apoptosis-resistant PHA-blasts, the T cell apoptosis was enhanced by addition of exogenous IL-2. Furthermore, in this apoptosis, monocytes played an important role because T cells infected in the absence of monocytes were resistant to the death signals. The apoptosis-sensitive T cells responded to TCR signaling more strongly by proliferating than those apoptosis-resistant cells. Monocytes weakly affected the expression levels of viral Ags on T cells. However, HTLV-I-infected monocytes primed T cells to die by subsequent TCR signaling. T cells primed with the monocytes, subsequently infected in the absence of monocytes, were killed by TCR signaling. These observations suggest that primed and infected T cells could be killed by activation-induced cell death.     

Overexpression of transforming growth factor-beta in transgenic mice carrying the human T-cell lymphotropic virus type I tax gene.

Human T-cell lymphotropic virus type I (HTLV-I) has been associated with an adult form of T-cell leukemia as well as tropical spastic paraparesis, a neurodegenerative disease. Adult T-cell leukemia patients express high levels of the type 1 isoform of transforming growth factor-beta (TGF-beta 1), which is mediated by the effects of the HTLV-I Tax transactivator protein on the TGF-beta 1 promoter. To understand further the regulation of TGF-beta 1 expression by Tax, we examined its expression in transgenic mice carrying the HTLV-I tax gene. We show that tumors from these mice and other tissues, such as submaxillary glands and skeletal muscle, which express high levels of tax mRNA selectively express high levels of TGF-beta 1 mRNA and protein. Moreover, TGF-beta 1 significantly stimulated the incorporation of tritiated thymidine into one of three cell lines derived from neurofibromas of tax-transgenic mice, which suggests that the excessive production of TGF-beta 1 may play a role in tumorigenesis and that these mice may serve as a useful model for studying the biological effects of TGF-beta in vivo.     

Activated Ha-ras can cooperate with defective simian virus 40 in the transformation of nonestablished rat embryo fibroblasts.

We studied the ability of activated Ha-ras to cooperate with simian virus 40 (SV40) in the transformation of nonestablished rat embryo fibroblasts. Cotransfection with Ha-ras greatly accelerated the rate of focus induction by wild-type SV40. Moreover, a series of transformation-defective SV40 mutants could be partially complemented by Ha-ras. This was true not only for mutants retaining an intact N-terminal immortalization-competent domain, but also for a nonkaryophilic SV40 mutant. In the latter case, all detectable T antigen was cytoplasmic, indicating that efficient transformation can be achieved through the interaction of two nonnuclear proteins. By employing cell lines derived with various SV40 mutants, it was determined that the ability to complex with p53 depends on the integrity of a relatively large region in the C-terminal half of large T. Finally, we report that nonkaryophilic SV40 large T forms a complex with the major heat shock protein HSP70, and we discuss its possible implications.     

trans activation of nerve growth factor in transgenic mice containing the human T-cell lymphotropic virus type I tax gene.

Three lines of transgenic mice containing the human T-cell lymphotropic virus type I (HTLV-I) tax gene develop neurofibromas composed of perineural fibroblasts (S. H. Hinrichs, M. Nerenberg, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1340-1343, 1987; M. Nerenberg, S. H. Hinrichs, R. K. Reynolds, G. Khoury, and G. Jay, Science 237:1324-1327, 1987). Tumors and tumor cell lines derived from these mice produce neurite outgrowth from PC-12 cells and nerve growth factor (NGF), as determined by RNA (Northern) blot analysis and enzyme-linked immunosorbent assays. In vitro cotransfection studies demonstrate that Tax is able to trans activate the NGF promoter in NIH 3T3 fibroblast cells. The major cis-acting tax-responsive element in the NGF promoter (AGGGTGTGACGA) has 92% homology with a tax-responsive element contained within the 21-bp repeats of the HTLV-I long terminal repeat. The receptor for NGF is also expressed in the transgenic tumor cells, suggesting that Tax may activate an autocrine mechanism through the upregulation of NGF.