Toshihisa Hayashibara

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.

Clinical significance of serum thymidine kinase in adult T‐cell leukaemia and acute myeloid leukaemia

To clarify the clinical and biological significance of serum thymidine kinase (TK) in adult T‐cell leukaemia (ATL) associated with human lymphotropic virus type‐I (HTLV‐I) and in acute myeloid leukaemia (AML), TK was measured in 52 patients with ATL (acute ATL, 35 patients; lymphoma ATL, two patients; chronic ATL, 12 patients; smouldering ATL, three patients), and in 27 patients with AML (one FAB MO, one Ml, 10 M2, seven M3, five M4, one M5, one M6, one MU). In ATL patients, statistical analysis disclosed a close correlation between TK level and the leucocyte count (P<0–01), and absolute number of abnormal lymphocytes (P<0–01). However, no correlation was observed between serum lactic dehydrogenase (LDH) level and these items. Concerning the therapeutic response, a statistical difference was present in TK between complete remission and no response (P<005), but not in LDH. We also investigated a significant inverse correlation between TK level as well as LDH level and the length of survival after the initial diagnosis (P<001). In AML patients a close correlation of TK level with the count of leucocytes (P<001), percentage of blasts in the blood (P<005), therapeutic response (P<0–01) and the length of survival after the initial diagnosis (P< 005) was present.

Lactacystin activates FLICE (caspase 8) protease and induces apoptosis in Fas- resistant adult T-cell leukemia cell lines

Abstract: Lactacystin (LC) is a specific inhibitor of the proteasome, and has recently been shown to induce apoptosis in certain cell lines. In the present study, we established Fas‐resistant adult T‐cell leukemia (ATL) cell subclones RSO4 and RST1 from their parental Fas‐sensitive cell lines SO4 and ST1, and examined whether LC can overcome Fas resistance. LC completely inhibited proteasome function as determined by a peptidyl‐MCA substrate (LLVY‐MCA and LLE‐MCA), and induced apoptosis in these cell lines irrespective of Fas sensitivity at low concentrations (∼10μM). LC induced the activation of caspase 3 (CPP32/Yama) and caspase 6 proteases in an identical manner to Fas‐mediated apoptosis. Moreover, LC induced the activation of caspase 8 (FLICE) protease, which is the initiator of the Fas‐mediated apoptotic cascade. Synthesized proteasome inhibitory peptide MG‐115 (ZLLnV‐CHO) also induced apoptosis in these cell lines. These results indicated that proteasome inhibitors overcome Fas‐resistance by bypassing the proximal part of the Fas signal. Inhibition of the proteasome function may be a new strategy for the treatment of ATL.

De novo acute myeloid leukemia in the elderly; a consistent fraction of long-term survivors by standard-dose chemotherapy

To clarify the characteristics of de novo acute myeloid leukemia (AML) among the elderly, we reviewed 112 patients over 60 years old (median age 72 years) who were treated at hospitals in Nagasaki Prefecture with a population of 1.5 million between 1987 and 1994. Reclassification of morphological diagnosis revealed that the proportion of M3 was lower but that of M6 and the incidence of cases with trilineage dysplasia (TLD), known as poor prognostic features, were higher in the elderly than in patients less than 60 years old. Similarly, chromosomal data showed a lower frequency of favorable karyotypes such as t(8;21) and t(15;17) in the elderly. The overall survival of all 112 patients was 10.3% at 5 years. Multivariate analysis indicated that good performance status (PS), low WBC at diagnosis, standard dose multi-drug chemotherapy and all-trans retinoic acid (ATRA) treatment for M3 patients, and morphological findings without TLD were significantly correlated with longer survival. Most of the long-term survivors were found among those who received standard dose therapy in this series, although no consensus has been established how to treat elderly AML patients. We propose that a prospective controlled trial is necessary to confirm the role of standard dose chemotherapy for elderly patients with de novo AML.

Fas-resistance in ATL cell lines not associated with HTLV-I or FAP-1 production

A preventive role for human T-cell leukemia virus type-I (HTLV-I) and Fas-associated phosphatase-1 (FAP-1) in Fas-mediated apoptosis has been reported in HTLV-I-infected cells. In the present study, we examined whether these molecules increased during the acquisition of Fas-resistance in adult T-cell leukemia (ATL) cell lines. SO4, ST1 and KK1 are Fas-sensitive ATL cell lines, and produce small amounts of HTLV-I in vitro. Although their subclones RSO4 and RST1 are completely Fas-resistant, they produced an equivalent amount of HTLV-I to SO4 and ST1. Moreover, FAP-1 mRNA was not detected in these cell lines irrespective of Fas sensitivity. Thus, Fas resistance in ATL cells was not directly associated with the increased production of HTLV-I or FAP-1.

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

Abstract:  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 γ‐chain (γc)‐receptor family and shares γ 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‐4Rα (CD124) and IL‐7Rα (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‐4Rα 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 γc‐receptors except IL‐9R. Overlapping and switching of cytokine receptors supported the idea that ATL cells can rapidly select the appropriate γc‐receptor according to conditions.

Therapeutic and Prognostic Value of Modal Number of Chromosomes at the Blastic Phase of Philadelphia-Chromosome-Positive Chronic Myeloid Leukemia: Comparison Based on the Same Criteria between Nagasaki University and Roswell Park Memorial Institute

In a comparison of 47 patients with Philadelphia-chromosome (Ph)-positive chronic myeloid leukemia (CML) in the Nagasaki University School of Medicine and 64 patients with the same disease in the Roswell Park Memorial Institute, the correlation between the modal number of chromosomes and the therapeutic response and/or survival after the onset of the blastic phase (BP) was evaluated. The patients were divided into four groups on the basis of the modal number of chromosomes of the cells in the bone marrow: those with hypodiploidy (group 1), those with pseudodiploidy carrying a Ph chromosome (group 2), those with 47 chromosomes (group 3), and those with 48 or more chromosomes (group 4). The results revealed similar trends in the two institutes. Namely, the therapeutic response and the survival after the onset of the BP in groups 1 and 4 were more unfavorable and shorter than those in groups 2 and 3, although the former (group 2) had a better prognosis than the latter (group 3). Thus, the statistical analysis revealed that the numerical chromosome findings at the BP are useful parameters for assessing the therapeutic response and survival after the onset of the BP of CML.