A novel alternative splicing isoform of human T-cell leukemia virus type 1 bZIP factor (HBZ-SI) targets distinct subnuclear localization.

Adult T-cell leukemia (ATL) is associated with prior infection with human T-cell leukemia virus type 1 (HTLV-1); however, the mechanism by which HTLV-1 causes adult T-cell leukemia has not been fully elucidated. Recently, a functional basic leucine zipper (bZIP) protein coded in the minus strand of HTLV-1 genome (HBZ) was identified. We report here a novel isoform of the HTLV-1 bZIP factor (HBZ), HBZ-SI, identified by means of reverse transcription-PCR (RT-PCR) in conjunction with 5' and 3' rapid amplification of cDNA ends (RACE). HBZ-SI is a 206-amino-acid-long protein and is generated by alternative splicing between part of the HBZ gene and a novel exon located in the 3' long terminal repeat of the HTLV-1 genome. Consequently, these isoforms share >95% amino acid sequence identity, and differ only at their N termini, indicating that HBZ-SI is also a functional protein. Duplex RT-PCR and real-time quantitative RT-PCR analyses showed that the mRNAs of these isoforms were expressed at equivalent levels in all ATL cell samples examined. Nonetheless, we found by Western blotting that the HBZ-SI protein was preferentially expressed in some ATL cell lines examined. A key finding was obtained from the subcellular localization analyses of these isoforms. Despite their high sequence similarity, each isoform was targeted to distinguishable subnuclear structures. These data show the presence of a novel isoform of HBZ in ATL cells, and in addition, shed new light on the possibility that each isoform may play a unique role in distinct regions in the cell nucleus.

Helicobacter pylori induces matrix metalloproteinase-9 through activation of nuclear factor kappaB.

BACKGROUND & AIMS: Matrix metalloproteinases (MMPs), enzymes capable of degrading extracellular matrix components, are believed to be active in connective tissue remodeling associated with various physiologic processes and in pathologic conditions. The aim of this study was to analyze the molecular mechanism responsible for Helicobacter pylori-mediated MMP expression. METHODS: Expression of MMP messenger RNA and MMP activity were assessed by reverse-transcription polymerase chain reaction and zymography, respectively. Chloramphenicol acetyltransferase assay was used to monitor activation of the MMP-9 gene promoter, and electrophoretic mobility shift assay was used to explore the binding of transcription factors to this promoter. Gastric tissue samples were immunohistochemically stained for MMP-9. RESULTS: H. pylori induced MMP-9 expression in 2 gastric epithelial cell lines but had no effect on MMP-2. Induction of MMP-9 was dependent on an intact cag pathogenicity island. Activation of the MMP-9 promoter by H. pylori occurred through the action of nuclear factor kappaB. Transfection of kinase-deficient mutants of IkappaB kinase and nuclear factor kappaB-inducing kinase inhibited H. pylori-mediated activation of MMP-9. MMP-9 expression was higher in epithelial cells of H. pylori-positive tissue compared with those of H. pylori-negative tissue. CONCLUSIONS: H. pylori induced activation of nuclear factor kappaB through an intracellular signaling pathway that involved IkappaB kinase and nuclear factor kappaB-inducing kinase, leading to MMP-9 gene transcription. MMP-9 induction by H. pylori may play an important role in gastric inflammation, ulcer formation, and carcinogenesis.

Possible involvement of aryl hydrocarbon receptor (AhR) in adult T-cell leukemia (ATL) leukemogenesis: constitutive activation of AhR in ATL.

Human T-cell leukemia virus type 1 is the etiologic agent of adult T-cell leukemia (ATL), although the precise mechanism involved in the transformation process has not yet been defined. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that can influence cell proliferation and differentiation. We investigated the expression and activation of AhR in ATL. RT-PCR and Western blot analyses showed high expression levels of AhR in ATL cell lines. The elevated expression of AhR was in part attributable to the action of the viral transactivator protein, Tax. Interestingly, activation of the AhR was found in ATL cell lines in the absence of apparent exogenous ligands. Importantly, the increased expression and activation of AhR were also observed in some primary ATL cells. To our best knowledge, this is the first report to show the lymphoid malignancy having constitutive activation of AhR. A possible link between increased AhR expression and leukemogenesis in ATL is discussed.

Multiple gammac-receptor expression in adult T-cell leukemia.

Constitutive expression of the IL-2 receptor (IL-2R) on adult T-cell leukemia (ATL) cells and the presence of permanent IL-2-dependent ATL cell lines indicate that the signal transduction system via IL-2R is a key element for the development of this disease. IL-2R is a member of the common gamma-chain (gammac)-receptor family and shares gamma with IL-4R, IL-7R, IL-9R, and IL-15R. In addition to IL-2R, ATL cells express IL-15R and respond to IL-15. In the present study, we examined other members of this receptor family. ATL cells showed various levels of IL-4Ralpha (CD124) and IL-7Ralpha (CD127) expression, and responded to these cytokines. In contrast, ATL cells hardly responded to IL-9. As primary samples were a mixed population and the results may have been modified by contaminating normal cells, we used ATL cell lines as pure ATL cell populations. Here, we report that IL-2-dependent ATL cell lines also express IL-4Ralpha and respond to IL-4, which was verified by the activation of cytoplasmic transcriptional activator Stat6 protein. Moreover, a novel ATL cell line that grows stably in an IL-7-dependent manner was established from one of the cell lines, and IL-7 induced Stat5 activation in this cell line. These results indicated that ATL cells have the potential to express all gammac-receptors except IL-9R. Overlapping and switching of cytokine receptors supported the idea that ATL cells can rapidly select the appropriate gammac-receptor according to conditions.

The two actin-binding regions on the myosin heads of cardiac muscle.

In the presence of myosin S1 or myosin heads, actin filaments tend to form bundles. The biological meaning of the bundling of actin filaments has been unclear. In this study, we found that the cardiac myosin heads can form the bundles of actin filaments more rapidly than can skeletal S1, as monitored by light scattering and electron microscopy. Moreover, the actin bundles formed by cardiac S1 were found to be more stable against mechanical agitation. The distance between actin filaments in the bundles was approximately 20 nm, which is comparable to the length of a myosin head and two actin molecules. This suggests the direct binding of S1 tails to the adjacent actin filament. The "essential" light chain of cardiac myosin could be cross-linked to the actin molecule in the bundle. When monomeric actin molecules were added to the bundle, the bundles could be dispersed into individual filaments. The three-dimensional structure of the dispersed actin filaments was reconstructed from electron cryo-microscopic images of the single actin filaments dispersed by monomer actin. We were able to demonstrate that cardiac myosin heads bind to two actin molecules: one actin molecule at the conventional actin-binding region and the other at the essential light-chain-binding region. This capability of cardiac myosin heads to bind two actin molecules is discussed in view of lower ATPase activity and slower shortening velocity than those of skeletal ones.

Matrix metalloproteinase-9 and vascular endothelial growth factor: a possible link in adult T-cell leukaemia cell invasion.

Plasma from a total of 57 patients with adult T-cell leukaemia (ATL) (acute ATL, 39 patients; lymphoma ATL, one patient; chronic ATL, 15 patients; smouldering ATL, two patients) and 20 healthy controls was analysed for the presence of type IV gelatinase activity with clinical features. A significant elevation of plasma matrix metalloproteinase-9 (MMP-9) was observed in some ATL patients, particularly in the patients with malignant cell infiltration. MMP-9 was found to be secreted into the conditioned medium from all ATL cell lines examined. Moreover, the corresponding mRNA was detectable both in all ATL cell lines examined and in the majority of primary acute ATL cells, indicating that ATL cells are capable of synthesizing and secreting MMP-9. We previously demonstrated that a high incidence of ATL cell infiltration was closely related to a high plasma level of vascular endothelial growth factor (VEGF) produced by ATL cells themselves. This present study showed that the presence of increased plasma MMP-9 was closely associated with elevated plasma VEGF in ATL patients. Furthermore, we showed that both increased plasma MMP-9 and VEGF were significantly related to high ATL cell infiltration. All these findings strongly suggest that MMP-9 and VEGF act co-operatively in the process of ATL cell invasion.

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.

Resveratrol induces downregulation in survivin expression and apoptosis in HTLV-1-infected cell lines: a prospective agent for adult T cell leukemia chemotherapy.

Resveratrol, a phytoalexin found in grapes and wine, has been shown to exhibit a wide range of pharmacological properties and is believed to play a role in the chemoprevention of human cancer. Resveratrol has also been shown to induce antiproliferation and apoptosis of several leukemia cell lines. In the present study, we investigated the effect of resveratrol in adult T cell leukemia. Our present observations showed that resveratrol induced growth inhibition in all five human T cell lymphotrophic virus-1-infected cell lines examined, with 50% effective dose of 10.4-85.6 mM. In the resveratrol-treated cells, induction of apoptosis was confirmed by annexin V-based analyses and morphological changes. The most surprising observation was that resveratrol treatment resulted in a gradual decrease in the expression of survivin, an antiapoptotic protein, during cell apoptosis. These findings indicate that resveratrol inhibits the growth of human T cell lymphotrophic virus-1-infected cell lines, at least in part, by inducing apoptosis mediated by downregulation in survivin expression. In view of the accumulating evidence that survivin may be an important determinant of a clinical response in adult T cell leukemia, our present findings have led to the suggestion that resveratrol, a common constituent of the human diet, merits further investigation as a potential therapeutic agent for this incurable disease.