Aberrant activation of the interleukin-2 autocrine loop through the nuclear factor of activated T cells by nonleukemogenic human T-cell leukemia virus type 2 but not by leukemogenic type 1 virus

Human T-cell leukemia virus type 1 (HTLV-1) but not HTLV-2 is associated with adult T-cell leukemia. We found that HTLV-2 Tax2 protein stimulated reporter gene expression regulated by the interleukin (IL)-2 promoter through the nuclear factor of activated T cells (NFAT) in a human T-cell line (Jurkat). However, the activity of HTLV-1 Tax1 was minimal in this system. T-cell lines immortalized by HTLV-2 but not HTLV-1 constitutively exhibited activated NFAT in the nucleus and constitutively expressed IL-2 mRNA. Cyclosporine A, an inhibitor of NFAT activation, abrogated the induction of IL-2 mRNA in HTLV-2-immortalized T-cell lines and concomitantly inhibited cell growth. This growth inhibition was rescued by the addition of IL-2 to the culture. Furthermore, anti-IL-2 receptor antibodies significantly reduced the proliferation of HTLV-2-infected T-cell lines but not that of HTLV-1-infected cells. Our results suggest that Tax2 activates an IL-2 autocrine loop mediated through NFAT that supports the growth of HTLV-2-infected cells under low-IL-2 conditions. This mechanism would be especially important in vivo, where this autocrine mechanism establishes a nonleukemogenic life-long HTLV-2 infection. The results also suggest that differences in long-term cytokine production between HTLV-1 and HTLV-2 infection are another factor for the differences in pathogenesis.     

Altered expression of tyrosine kinases of the Src and Syk families in human T-cell leukemia virus type 1-infected T-cell lines

During the late phase of adult T-cell leukemia/lymphoma, a severe lymphoproliferative disorder caused by human T-cell leukemia virus type 1 (HTLV-1), leukemic cells no longer produce interleukin-2. Several studies have reported the lack of the Src-like protein tyrosine kinase Lck and overexpression of Lyn and Fyn in these cells. In this report we demonstrate that, in addition to the downregulation of TCR, CD45, and Lck (which are key components of T-cell activation), HTLV-1-infected cell lines demonstrate a large increase of FynB, a Fyn isoform usually poorly expressed in T cells. Furthermore, similar to anergic T cells, Fyn is hyperactive in one of these HTLV-1-infected T-cell lines, probably as a consequence of Csk downregulation. A second family of two proteins, Zap-70 and Syk, relay the signal of T-cell activation. We demonstrate that in contrast to uninfected T cells, Zap-70 is absent in HTLV-1-infected T cells, whereas Syk is overexpressed. In searching for the mechanism responsible for FynB overexpression and Zap-70 downregulation, we have investigated the ability of the Tax and Rex proteins to modulate Zap-70 expression and the alternative splicing mechanism which gives rise to either FynB or FynT. By using Jurkat T cells stably transfected with the tax and rex genes or inducibly expressing the tax gene, we found that the expression of Rex was necessary to increase fynB expression, suggesting that Rex controls fyn gene splicing. Conversely, with the same Jurkat clones, we found that the expression of Tax but not Rex could downregulate Zap-70 expression. These results suggest that the effect of Tax and Rex must cooperate to deregulate the pathway of T-cell activation in HTLV-1-infected T cells.     

Resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis and constitutive expression of Apo2L/TRAIL in human T-cell leukemia virus type 1-infected T-cell lines

Adult T-cell leukemia (ATL), a CD4+-T-cell malignancy caused by human T-cell leukemia virus type 1 (HTLV-1), is difficult to cure, and novel treatments are urgently needed. Apo2 ligand (Apo2L; also tumor necrosis factor-related apoptosis-inducing ligand [TRAIL]) has been implicated in antitumor therapy. We found that HTLV-1-infected T-cell lines and primary ATL cells were more resistant to Apo2L-induced apoptosis than uninfected cells. Interestingly, HTLV-1-infected T-cell lines and primary ATL cells constitutively expressed Apo2L mRNA. Inducible expression of the viral oncoprotein Tax in a T-cell line up-regulated Apo2L mRNA. Analysis of the Apo2L promoter revealed that this gene is activated by Tax via the activation of NF-kappaB. The sensitivity to Apo2L was not correlated with expression levels of Apo2L receptors, intracellular regulators of apoptosis (FLICE-inhibitory protein and active Akt). NF-kappaB plays a crucial role in the pathogenesis and survival of ATL cells. The resistance to Apo2L-induced apoptosis was reversed by N-acetyl-L-leucinyl-L-leucinyl-lLnorleucinal (LLnL), an NF-kappaB inhibitor. LLnL significantly induced the Apo2L receptors DR4 and DR5. Our results suggest that the constitutive activation of NF-kappaB is essential for Apo2L gene induction and protection against Apo2L-induced apoptosis and that suppression of NF-kappaB may be a useful adjunct in clinical use of Apo2L against ATL.     

Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells

Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.     

Human T-cell leukemia virus type 1 tax transactivates the matrix metalloproteinase 7 gene via JunD/AP-1 signaling

Adult T-cell leukemia (ATL) is a T-cell malignancy associated with human T-cell leukemia virus type 1 (HTLV-1) and characterized by visceral invasion. Degradation of the extracellular matrix by matrix metalloproteinases (MMPs) is a crucial process in invasion of tumors and metastasis. MMP-7 (or matrilysin), is a "minimal domain MMP" with proteolytic activity against components of the extracellular matrix. To determine the involvement of MMP-7 in visceral spread in ATL, this study investigated MMP-7 expression in ATL. MMP-7 expression was identified in HTLV-1-infected T-cell lines, peripheral blood ATL cells and ATL cells in lymph nodes, but not in uninfected T-cell lines or normal peripheral blood mononuclear cells. MMP-7 expression was induced following infection of a human T-cell line with HTLV-1, and specifically by the viral protein Tax. Functionally, MMP-7 promoted cell migration of HTLV-1-infected T cells. The MMP-7 promoter activity was increased by Tax and reduced by deletion of the activator protein-1 (AP-1) binding site. Electrophoretic mobility shift assay showed high levels of AP-1 binding proteins, including JunD, in HTLV-1-infected T-cell lines and ATL cells, and Tax elicited JunD binding to the MMP-7 AP-1 element. Tax-induced MMP-7 activation was inhibited by dominant negative JunD and augmented by JunD/JunD homodimers. Short interfering RNA against JunD inhibited MMP-7 mRNA expression in HTLV-1-infected T-cell lines. These results suggest that the induction of MMP-7 by Tax is regulated by JunD and that MMP-7 could facilitate visceral invasion in ATL. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Apoptosis induced by the histone deacetylase inhibitor FR901228 in human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

Inhibition of histone deacetylase (HDAC) activity induces growth arrest, differentiation, and, in certain cell types, apoptosis. FR901228, FK228, or depsipeptide, is an HDAC inhibitor effective in T-cell lymphomas. Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. We examined whether FR901228 is effective for treatment of ATL by assessing its ability to induce apoptosis of HTLV-1-infected T-cell lines and primary leukemic cells from ATL patients. FR901228 induced apoptosis of Tax-expressing and -unexpressing HTLV-1-infected T-cell lines and selective apoptosis of primary ATL cells, especially those of patients with acute ATL. FR901228 also efficiently reduced the DNA binding of NF-kappaB and AP-1 in HTLV-1-infected T-cell lines and primary ATL cells and down-regulated the expression of Bcl-x(L) and cyclin D2, regulated by NF-kappaB. Although the viral protein Tax is an activator of NF-kappaB and AP-1, FR901228-induced apoptosis was not associated with reduced expression of Tax. In vivo use of FR901228 partly inhibited the growth of tumors of HTLV-1-infected T cells transplanted subcutaneously in SCID mice. Our results indicated that FR901228 could induce apoptosis of these cells and suppress the expression of NF-kappaB and AP-1 and suggest that FR901228 could be therapeutically effective in ATL.