Takayuki Miyanishi

A Novel Alternative Splicing Isoform of Human T-Cell Leukemia Virus Type 1 bZIP Factor (HBZ-SI) Targets Distinct Subnuclear Localization

ABSTRACT 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.

Resveratrol Induces Downregulation in Survivin Expression and Apoptosis in HTLV-1-Infected Cell Lines: A Prospective Agent for Adult T Cell Leukemia Chemotherapy

Abstract: 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.

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.

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

Summary. 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.

Structure and function of muscle myosin

We have studied the primary structures of myosins from chicken muscles in order to clarify the relationship between structure and function of muscle myosin. The primary structures of the various kinds of light chains from chicken muscle myosins have been determined. We also report the primary structure of the 23K fragment of subfragment-1 (S-1) component from the heavy chain of chicken fast skeletal muscle myosin. In addition, antibody was prepared against the 23K fragment. The antibody was found to inhibit the Mg2+-ATPase activity and the initial Pi burst of the ATPase in the S-1 component. The antibody suppressed the ATP-induced fluorescence enhancement of S-1, though it did not suppress the binding of ATP to S-1. These results are also discussed.