Human T-cell leukemia virus type I Tax transactivates the matrix metalloproteinase-9 gene: potential role in mediating adult T-cell leukemia invasiveness

Human T-cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T-cell leukemia (ATL) and of tropical spastic paraparesis/HTLV-I-associated myelopathy. Infiltration of various tissues by circulating leukemic cells is a characteristic of ATL. Matrix metalloproteinases (MMPs), which mediate the degradation of the basement membrane and extracellular matrix, play an important role in metastasis and tumor cell dissemination. The aim of this study was to explore whether expression of MMP-2 and MMP-9 was deregulated by HTLV-I infection. The data showed that HTLV-I-infected T-cell lines expressed high levels of MMP-9 compared with uninfected T-cell lines. In contrast, the levels of the related MMP-2 were not significantly altered by HTLV-I infection. In addition, the elevated expression of MMP-9 in HTLV-I-infected cells was attributable to the action of the viral transactivator protein Tax. The results show that Tax can activate the MMP-9 promoter and induce MMP-9 expression in T cells, indicating that the constitutive expression of MMP-9 in virus-infected cell lines is at least in part mediated by Tax. Activation of the MMP-9 promoter by Tax occurs mainly through the action of NF-kappaB and SP-1. The biologic significance of these observations was validated by the following 2 findings: MMP-9 expression was increased in primary ATL cells, and plasma MMP-9 levels were elevated in ATL patients. In addition, plasma levels of MMP-9 correlated with organ involvement in ATL patients. Together these data suggest that overexpression of MMP-9 in HTLV-I- infected cells may be in part responsible for the invasiveness of ATL cells.     

Virion-associated trans-regulatory protein of human T-cell leukemia virus type I.

Western blot analysis of HTLV-I virus particles from HUT-102 cells revealed a 40-kD protein strongly reactive with Tax-specific rabbit antisera. This protein subsequently was isolated from density gradient purified virions by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), purified from comigrating Gag and human cellular proteins by reversed-phase high-performance liquid chromatography (HPLC) and identified as the tax-encoded gene product by amino acid composition analysis. Among extracellular virions from five HTLV-I producing cell lines, only those from HUT-102 and C10MJ cells contained a detectable Tax protein, although all cells expressed Tax mRNA and protein intracellularly. To investigate the diagnostic implications of virion-associated Tax protein, sera from HTLV-I-infected individuals were compared on HUT-102 and MT-2 virus Western blots. The seroprevalence of antibodies to Tax, but not Gag or Env proteins, was substantially higher among adult T-cell leukemia and tropical spastic paraparesis patients using HUT-102 viral proteins. Thus, immunoassays utilizing HUT-102 virus are most sensitive for detection of Tax-reactive antibodies.     

Existence of escape mutant in HTLV-I tax during the development of adult T-cell leukemia

Although Tax protein is the main target of cytotoxic T lymphocyte (CTL) on human T-cell lymphotropic virus type I (HTLV-I)-infected cells, and Tax peptide 11 through 19 binding to HLA-A*02 has been shown to elicit a strong CTL response, there are patients with adult T-cell leukemia (ATL) bearing HLA-A*02. To explore whether there is genetic variation in HTLV-I tax that can escape CTL recognition during the development of ATL, the HTLV-I tax gene was sequenced in 55 patients with ATL, 61 patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), and 62 healthy carriers, and it was correlated with the presence of HLA-A*02. First, a premature stop codon in the 5' half of the tax gene that looses transactivation activity on the viral enhancer was observed in 3 patients with acute and 1 patient with chronic ATL. This stop codon was revealed to emerge after the viral transmission to the patient from sequence analysis in family members with ATL. Second, amino acid change in Tax peptide 11-19 was observed in 3 patients with ATL. CTL assays demonstrated that this altered Tax 11-19 peptide, observed in ATL patients with HLA-A*02, was not recognized by Tax 11-19-specific CTL. Two patients with ATL had large deletions in tax by sequencing, and 5 patients with ATL had deletions in HTLV-I by Southern blotting. These findings suggest that at some stage of ATL development, HTLV-I-infected cells that can escape the host immune system are selected and have a chance to accumulate genetic alterations for further malignant transformation, leading to acute ATL.     

NF-kappaB involvement in the activation of primary adult T-cell leukemia cells and its clinical implications.

The HTLV-I provirus-encoded Tax protein induces NF-kappaB in Tax-transfected Jurkat T cells or HTLVL-I- infected T cells in vitro. Tax induction of NF-kappaB is presumed to be involved in proliferation and activation of primary leukemia cells in vivo. Recent studies have demonstrated that NF-kappaB activities in human T cells are mediated by at least four c-Rel-related DNA binding proteins - p50, p55, p75 and p85. We examined the significance of NF-kappaB induction in primary adult T cell leukemia cells and the induction kinetics of each of the four NF-kappaB species. Marked NF-kappaB activity was detected using an electrophoretic mobility shift assay (EMSA) in the primary cells of patients with acute disease, but little activity was noted in the cells of chronic patients. NF-kappaB activity was enhanced in a time-dependent manner in acute type cells cultured with mitogen-free medium; there was no induction of activity in chronic type cells. UV crosslinking demonstrated all four species of NFkappaB complex - high levels of p50 and lower levels of p55 and p75, in acute type cells; chronic type cells showed only the p50. As a control, normal resting T cells similarly showed only p50; control cells showed little change in activity when cultured without mitogenic stimulation, analogous to chronic type ATL. Northern blotting revealed enhancement of c-rel (encoding p85) and KBFI (encoding p50 and p55) expression in acute type cells during culture, while there was no significant enhancement of mRNAs in chronic type ATL cells or unstimulated normal T cells. Northern blotting also revealed that Tax is upregulated at the mRNA level in acute- but not chronic-type cells during culture. Expression of c-rel and KBF1 mRNAs in acute type cells appeared to be related to Tax mRNA expression. These results suggest that Tax is capable of inducing nuclear expression of all four NF-kappaB species in primary ATL cells of acute type patients, with marked effects on p55, p75, and p85. Tax induction of NF-kappaB species is regulated, at least in part, at a pretranslational level involving increases in c-rel and KBF1 mRNA.     

Establishment of human T-cell leukemia virus type I T-cell lymphomas in severe combined immunodeficient mice

Human T-cell leukemia virus type I (HTLV-I) is recognized as the etiologic agent of adult T-cell leukemia (ATL), a disease endemic in certain regions of southeastern Japan, Africa, and the Caribbean basin. Although HTLV-I can immortalize T lymphocytes in culture, factors leading to tumor progression after HTLV-I infection remain elusive. Previous attempts to propagate the ATL tumor cells in animals have been unsuccessful. Severe combined immunodeficient (SCID) mice have previously been used to support the survival of human lymphoid cell populations when inoculated with human peripheral blood lymphocytes (PBL). SCID mice were injected intraperitoneally with PBL from patients diagnosed with ATL, HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), or from asymptomatic HTLV-I-seropositive patients. Many of these mice become persistently infected with HTLV-I. Furthermore, after human reconstitution was established in these mice, HTLV-I-infected cells displayed a proliferative advantage over uninfected human cells. Lymphoblastic lymphomas of human origin developed in animals injected with PBL from two ATL patients. The tumor cells represented outgrowth of the original ATL leukemic clone in that they had monoclonal or oligoclonal integrations of the HTLV-I provirus identical to the leukemic clone and predominantly expressed the cell surface markers, CD4 and CD25. In contrast, cell lines derived by HTLV immortalization of T cells in vitro did not persist or form tumors when inoculated into SCID mice, indicating differences between in vitro immortalized cells and ATL leukemic cells. This system represents the first small animal model to study HTLV-I tumorigenesis in vivo.     

Expression of terminal deoxynucleotidyltransferase gene in a case of adult T-cell leukemia

A 37-year-old male from Kagoshima Prefecture was admitted with adult T-cell leukemia (ATL). Monoclonal integration of HTLV-1 proviral DNA was found, but the integration site was different from that of terminal deoxynucleotidyltransferase (TdT)-positive MT-1 cells (an ATL cell line). The ATL cells expressed enzymatically active TdT and exhibited 2100 b TdT mRNA, which corresponds to the thymus type of TdT mRNA. The same size of TdT mRNA was also detected in MT-1. Southern blot analyses revealed no differences in the gene structure of the promoter region of TdT genes between this ATL case and TdT-positive lymphoblastic leukemia cells. There is little possibility that cis-acting viral elements promote TdT gene expression by proviral integration. The activation of TdT gene in ATL may be mediated by other trans-acting factors.     

Cell-type-specific transactivation of the parathyroid hormone-related protein gene promoter by the human T-cell leukemia virus type I (HTLV- I) tax and HTLV-II tax proteins

The human T-cell leukemia virus type I (HTLV-I) and HTLV-II Tax proteins are potent transactivators of viral and cellular gene expression. Using deletion mutants, the downstream parathyroid hormone- related protein (PTHrP) promoter is shown to be responsive to both HTLV- I and HTLV-II Tax as well as the AP1/c-jun proto-oncogene. Transactivation of PTHrP by Tax was seen in T cells but not in B-cell lines or fibroblasts. A carboxy terminal Tax deletion mutant was deficient in transactivation of both the PTHrP and IL2R alpha promoters but not the HTLV-I long terminal repeat (LTR). Exogenous provision of NFkB rescued IL2R alpha expression but not the PTHrP promoter. Thus, HTLV-I Tax, HTLV-II Tax, and c-jun transactivate PTHrP and may contribute to the pathogenesis of hypercalcemia in adult T-cell leukemia.     

Frequent lack of antibodies against human T-cell leukemia virus type I gag proteins p24 or p19 in sera of patients with adult T-cell leukemia

Summary Specificities of antibodies against human T-cell leukemia virus type I (HTLV-I) in sera of 27 patients with adult T-cell leukemia (ATL) were studied. All sera were positive for HTLV-I antigens by immunofluorescence assay using cells expressing HTLV-I antigens; sera from five ATL patients, however, did not react or hardly reacted with p19 or p24 gag proteins of HTLV-I upon immunoprecipitation assay. Therefore, the relationships among antibody specificities against HTLV-I, the proviral structures of HTLV-I genomes in leukemic cells, and the expression of viral antigens by leukemic cells after cultivation in vitro for a few days were examined. Analyses of the genomic structures of the proviruses revealed deletions in at least seven cases. However, we could not detect deletions in the proviral genomes of four out of the five ATL patients who lacked antibodies against gag proteins. Furthermore, expression of p19 and p24 was detected in these patients' peripheral blood lymphocytes (PBL) cultured in vitro for a few days. Thus, some ATL patients could not or could hardly raise antibodies against gag proteins, although they harbored complete HTLV-I genomes and their PBL expressed gag proteins in vitro. All patients harboring deleted proviruses, so far tested, raised antibodies not only against viral proteins that should be encoded by the integrated proviruses, but also against viral proteins that should be encoded by the deleted regions. Antibodies against viral proteins were detected also in sera of ATL patients whose PBL did not express viral proteins after in vitro cultivation. Specificities of antibodies against viral proteins in ATL patients could not be predicted by the structures of proviruses in leukemic cells or by expression of viral proteins in vitro. Immune responses to HTLV-I antigens were weak or lost in some ATL patients.Abbreviations used HTLV-I human T-cell leukemia virus type I - ATL adult T-cell leukemia - PBL peripheral blood lymphocytes - PAGE Polyacrylamide gel electrophoresis - kb kilobases Supported in part by a grant-in-aid from the Ministry of Health and Welfare of Japan for a comprehensive 10-year strategy for cancer control, and a grant-in-aid from the Ministry of Education, Science and Culture of Japan     

Adult T-cell leukemia

Adult T-cell leukemia (ATL) is a leukemia caused by a monoclonal expansion of HTLV-I-infected T-cells expressing a CD4 antigen. The clinical features of ATL include lymphadenopathy, hepatosplenomegaly, frequent skin lesions, hypercalcemia and a rapidly fatal course. The cell surface phenotype, cytogenetics and functions of leukemic cells are described in association with various clinical manifestations and HTLV-I infection. Leukemic cells constitutively express the p55 (Tac antigen) subunit of the interleukin-2 (IL-2) receptor. Its association with the function of HTLV-I gene products and its possible role in the leukemogenesis of ATL are discussed. Finally, the potential of some therapeutic agents which may selectively eliminate the Tac-expressing leukemic cells in vitro are described, and these may provide an improvement over currently ineffective combination chemotherapy.     

Selective inhibition of human T-lymphotropic virus type I-transformed human T-cell growth by a tax-targeted conditionally cytotoxic recombinant retrovirus

Adult T-cell leukemia (ATL), a disorder associated with high mortality rates, arises from human T-lymphotropic virus type I (HTLV-I)-infected CD4+ T cells. We designed a retroviral vector-based gene therapy approach to ATL. The long terminal repeat (LTR) of HTLV-I is transactivated by the viral tax protein. We constructed a hybrid gene consisting of herpes simplex virus thymidine kinase (HSV TK) under the control of the HTLV-I LTR and inserted it into a retroviral vector. When HTLV-I-transformed and tax-expressing human T-cell lines were infected with this recombinant retrovirus (LNLTK alpha virus), they expressed high levels of HSV TK and exhibited increased sensitivity to acyclovir, a nucleoside analog that is converted to the toxic anabolite after phosphorylation by the HSV TK. On the other hand, the retroviral infection had little effect on acyclovir-induced cytotoxicity in HTLV-I- negative human hematopoietic cell lines. Our data may provide the prospect of the gene therapy for ATL by tax-targeted selective elimination of leukemic cells.