Mariko Tomita

Transcriptional activation of survivin through the NF‐κB pathway by human T‐cell leukemia virus type I tax

Survivin, a unique member of the inhibitor of apoptosis protein family, is overexpressed in many cancers and considered to play an important role in oncogenesis. We previously reported the survivin expression profile in ATL, a CD4‐positive T‐cell malignancy caused by HTLV‐I. HTLV‐I Tax is thought to play an important role in immortalization of T cells. We have shown also that the expression of Tax protected the mouse T‐cell line CTLL‐2 against apoptosis induced by deprivation of IL‐2 and converted its growth from being IL‐2 dependent to being IL‐2 independent through the NF‐κB pathway. In our study, we demonstrate that constitutive expression of survivin was associated with resistance to apoptosis after IL‐2 deprivation in Tax‐expressing CTLL‐2 cells. Transient transfection assays showed that survivin promoter was transactivated by Tax, via the activation of NF‐κB. Pharmacological NF‐κB inhibition resulted in suppression of survivin expression and caused apoptosis of Tax‐expressing CTLL‐2 cells. Our findings suggest that activated NF‐κB signaling contributes directly to malignant progression of ATL by preventing apoptosis, acting through the prosurvival protein survivin.

Fucoidan extracted from Cladosiphon okamuranus Tokida induces apoptosis of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells.

Abstract: Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type 1 (HTLV-1) and remains incurable. The highest endemic area of HTLV-1 carriers in Japan is located in Okinawa, and novel treatments are urgently needed in this area. We extracted fucoidan, a sulfated polysaccharide, from the brown seaweed Cladosiphon okamuranus Tokida cultivated in Okinawa, Japan and examined its tumor-suppression activity against ATL. Fucoidan significantly inhibited the growth of peripheral blood mononuclear cells of ATL patients and HTLV-1-infected T-cell lines but not that of normal peripheral blood mononuclear cells. Fucoidan induced apoptosis of HTLV-1-infected T-cell lines mediated through downregulation of cellular inhibitor of apoptosis protein-2 and survivin and G1 phase accumulation through the downregulation of cyclin D2, c-myc, and hyperphosphorylated form of the retinoblastoma tumor suppressor protein. Further analysis showed that fucoidan inactivated NF-κB and activator protein-1 and inhibited NF-κB-inducible chemokine, C-C chemokine ligand 5 (regulated on activation, normal T expressed and secreted) production, and homotypic cell-cell adhesion of HTLV-1-infected T-cell lines. In vivo use of fucoidan resulted in partial inhibition of growth of tumors of an HTLV-1-infected T-cell line transplanted subcutaneously in severe combined immune deficient mice. Our results indicate that fucoidan is a potentially useful therapeutic agent for patients with ATL.

Retracted: Curcumin (diferuloylmethane) inhibits constitutive active NF-κB, leading to suppression of cell growth of human T-cell leukemia virus type I-infected T-cell lines and primary adult T-cell leukemia cells

Retraction: The following article has been retracted through agreement between the first author and several coauthors, the journal Editor‐in‐Chief, Peter Lichter, and John Wiley & Sons, Ltd.: Tomita M, Kawakami H, Uchihara JN, Okudaira T, Masuda M, Takasu N, Matsuda T, Ohta T, Tanaka Y, Ohshiro K, Mori N. (2006). Curcumin (diferuloylmethane) inhibits constitutive activeNF‐kappaB, leading to suppression of cell growth of human T‐cell leukemia virus type I‐infected T‐cell lines and primary adult T‐cell leukemia cells. Int J Cancer; 118 (February (3)): 765‐772, published online on 16 AUG 2005 in Wiley Online Library (www.onlinelibrary.wiley.com). After an investigation the retraction has been agreed due to inappropriate duplication of images and overlap with other published work

Antiadult T‐cell leukemia effects of brown algae fucoxanthin and its deacetylated product, fucoxanthinol

Adult T‐cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by human T‐cell leukemia virus type 1 (HTLV‐1) infection and remains incurable. Carotenoids are a family of natural pigments and have several biological functions. Among carotenoids, fucoxanthin is known to have antitumorigenic activity, but the precise mechanism of action is not elucidated. We evaluated the anti‐ATL effects of fucoxanthin and its metabolite, fucoxanthinol. Both carotenoids inhibited cell viability of HTLV‐1‐infected T‐cell lines and ATL cells, and fucoxanthinol was approximately twice more potent than fucoxanthin. In contrast, other carotenoids, β‐carotene and astaxanthin, had mild inhibitory effects on HTLV‐1‐infected T‐cell lines. Importantly, uninfected cell lines and normal peripheral blood mononuclear cells were resistant to fucoxanthin and fucoxanthinol. Both carotenoids induced cell cycle arrest during G1 phase by reducing the expression of cyclin D1, cyclin D2, CDK4 and CDK6, and inducing the expression of GADD45α, and induced apoptosis by reducing the expression of Bcl‐2, XIAP, cIAP2 and survivin. The induced apoptosis was associated with activation of caspase‐3, ‐8 and ‐9. Fucoxanthin and fucoxanthinol also suppressed IκBα phosphorylation and JunD expression, resulting in inactivation of nuclear factor‐κB and activator protein‐1. Mice with severe combined immunodeficiency harboring tumors induced by inoculation of HTLV‐1‐infected T cells responded to treatment with fucoxanthinol with suppression of tumor growth, showed extensive tissue distribution of fucoxanthinol, and the presence of therapeutically effective serum concentrations of fucoxanthinol. Our preclinical data suggest that fucoxanthin and fucoxanthinol could be potentially useful therapeutic agents for patients with ATL.

Inhibition of constitutively active Jak-Stat pathway suppresses cell growth of human T-cell leukemia virus type 1-infected T-cell lines and primary adult T-cell leukemia cells

BackgroundHuman T-cell leukemia virus type 1 (HTLV-1), the etiologic agent for adult T-cell leukemia (ATL), induces cytokine-independent proliferation of T-cells, associated with the acquisition of constitutive activation of Janus kinases (Jak) and signal transducers and activators of transcription (Stat) proteins. Our purposes in this study were to determine whether activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells, and to explore mechanisms by which inhibition of Jak-Stat pathway kills ATL cells.ResultsConstitutive activation of Stat3 and Stat5 was observed in HTLV-1-infected T-cell lines and primary ATL cells, but not in HTLV-1-negative T-cell lines. Using AG490, a Jak-specific inhibitor, we demonstrated that the activation of Stat3 and Stat5 was mediated by the constitutive phosphorylation of Jak proteins. AG490 inhibited the growth of HTLV-1-infected T-cell lines and primary ATL cells by inducing G1 cell-cycle arrest mediated by altering the expression of cyclin D2, Cdk4, p53, p21, Pim-1 and c-Myc, and by apoptosis mediated by the reduced expression of c-IAP2, XIAP, survivin and Bcl-2. Importantly, AG490 did not inhibit the growth of normal peripheral blood mononuclear cells.ConclusionOur results indicate that activation of Jak-Stat pathway is responsible for the proliferation and survival of ATL cells. Inhibition of this pathway may provide a new approach for the treatment of ATL.

MicroRNA miR‐146a is induced by HTLV‐1 tax and increases the growth of HTLV‐1‐infected T‐cells

Human T‐cell leukemia virus type 1 (HTLV‐1) is the causative agent of adult T‐cell leukemia (ATL), which is an aggressive and fatal CD4+ T cell malignancy. MicroRNA (miRNA), a novel class of RNA that regulates gene expression, is involved in many cellular processes such as growth, development and apoptosis. It has recently been linked to several cancer phenotypes. However, aberrant miRNA expression and its pathologic significance in ATL are not well documented. Here, we investigated the role of miRNAs in HTLV‐1‐related leukemogenesis. The results showed that miR‐146a was upregulated in HTLV‐1‐infected T‐cell lines compared to uninfected T‐cell lines. Tax‐induced miR‐146a expression in a NF‐κB‐dependent manner and inhibited the expression of gene harboring the target sequence of miR‐146a on its 3′UTR. Inhibition of miR‐146a function by anti‐miRNA inhibitor reduced the proliferation of HTLV‐1‐infected T‐cell lines but not that of uninfected T‐cell lines. Moreover, overexpression of miR‐146a enhanced the growth of an HTLV‐1‐infected T‐cell line. Our findings suggest that miR‐146a is a potentially suitable therapeutic target of ATL.

Activation of hypoxia-inducible factor 1 in human T-cell leukaemia virus type 1-infected cell lines and primary adult T-cell leukaemia cells.

HTLV-1 (human T-cell leukaemia virus type 1) is the causative agent for ATL (adult T-cell leukaemia). HTLV-1 Tax can activate the PI3K (phosphoinositide 3-kinase)/Akt signalling pathway, which is responsible for survival of HTLV-1-infected T-cells. HIFs (hypoxia-inducible factors) are transcriptional regulators that play a central role in the response to hypoxia. Overexpression of HIF-1alpha in many cancers is associated with a poor response to treatment and increased patient mortality. Our objectives in the present study were to investigate whether HIF-1 was activated in HTLV-1-infected T-cells and to elucidate the molecular mechanisms of HIF-1 activation by focusing on the PI3K/Akt signalling pathway. We detected a potent pathway that activated HIF-1 in the HTLV-1-infected T-cells under a normal oxygen concentration. Enhanced HIF-1alpha protein expression and HIF-1 DNA-binding activity were exhibited in HTLV-1-infected T-cell lines. Knockdown of HIF-1alpha by siRNA (small interfering RNA) suppressed the growth and VEGF (vascular endothelial growth factor) expression of the HTLV-1-infected T-cell line. HIF-1 protein accumulation and transcriptional activity were enhanced by Tax, which was inhibited by dominant-negative Akt. Importantly, mutant forms of Tax that are defective in activation of the PI3K/Akt pathway failed to induce HIF-1 transcriptional activity. The PI3K inhibitor LY294002 suppressed HIF-1alpha protein expression, HIF-1 DNA-binding and HIF-1 transcriptional activity in HTLV-1-infected T-cell lines. In primary ATL cells, HIF-1alpha protein levels were strongly correlated with levels of phosphorylated Akt. The results of the present study suggest that PI3K/Akt activation induced by Tax leads to activation of HIF-1. As HIF-1 plays a major role in tumour progression, it may represent a molecular target for the development of novel ATL therapeutics.

Aurora A selective inhibitor MLN8237 suppresses the growth and survival of HTLV-1-infected T-cells in vitro

Aurora A kinase plays an essential role in the proper assembly and function of the mitotic spindle. We have shown previously that Aurora A expression is increased aberrantly in human T‐cell leukemia virus type 1 (HTLV‐1)‐infected T‐cell lines and primary adult T‐cell leukemia cells, and a pan‐Aurora kinase inhibitor, which inhibits both Aurora A and Aurora B kinases, reduces viability and induces apoptosis in these cells. However, the specific effects of Aurora A inhibition on HTLV‐1‐infected T‐cells are poorly understood. In this study, we addressed this question by comparing the effects of MLN8237, a selective inhibitor of Aurora A, on cell viability, cell cycle progression, and induction of apoptosis in HTLV‐1‐infected and ‐uninfected T‐cell lines. MLN8237 reduced the viability of HTLV‐1‐infected T‐cell lines within 24 h, but its effects on that of HTLV‐1‐uninfected T‐cell lines were moderate. MLN8237 induced early apoptosis of HTLV‐1‐infected T‐cell lines without induction of polyploidy. It induced p53 and p21 expression in HTLV‐1‐infected but not in ‐uninfected T‐cell lines, suggesting that MLN8237‐treated HTLV‐1‐infected T‐cell lines exit from mitosis and activate a p53‐dependent postmitotic G1 checkpoint, leading to G1 arrest followed by the induction of apoptosis. Our results suggest that specific inhibition of Aurora A kinase is a potentially useful therapeutic strategy in the treatment of adult T‐cell leukemia and that further in vivo exploration is warranted.

The Kaposi's sarcoma-associated herpesvirus K-bZIP protein represses transforming growth factor β signaling through interaction with CREB-binding protein

Kaposis sarcoma (KS)-associated herpesvirus (KSHV) is involved in the pathogenesis of KS, primary effusion lymphoma, and multicentric Castlemans disease. K-bZIP, the protein encoded by the open reading frame K8 of KSHV, is a member of the basic region-leucine zipper family of transcription factors. We studied the mechanisms that underlie KSHV-induced oncogenesis by investigating whether K-bZIP perturbs signaling through transforming growth factor β (TGF-β), which inhibits proliferation of a wide range of cell types. K-bZIP repressed TGF-β-induced, Smad-mediated transcriptional activity and antagonized the growth-inhibitory effects of TGF-β. Since both K-bZIP and Smad are known to interact with CREB-binding protein (CBP), the effect of CBP on inhibition of Smad-mediated transcriptional activation by K-bZIP was examined. K-bZIP mutants, which lacked the CBP-binding site, could not repress TGF-β-induced or Smad3-mediated transcriptional activity. Overexpression of CBP restored K-bZIP-induced inhibition of Smad3-mediated transcriptional activity. Competitive interaction studies showed that K-bZIP inhibited the interaction of Smad3 with CBP. These results suggest that K-bZIP, through its binding to CBP, disrupts TGF-β signaling by interfering with the recruitment of CBP into transcription initiation complexes on TGF-β-responsive elements. We propose a possibility that K-bZIP may contribute to oncogenesis through its ability to promote cell survival by repressing TGF-β signaling.

Elevated expression of CCL5/RANTES in adult T‐cell leukemia cells: Possible transactivation of the CCL5 gene by human T‐cell leukemia virus type I tax

HTLV‐I is the etiologic agent of ATL and of tropical spastic paraparesis/HTLV‐I‐associated myelopathy. Infiltration of various tissues by circulating leukemic cells and HTLV‐I‐infected T cells is a characteristic of ATL and HTLV‐I‐associated inflammatory diseases. Chemokines play important roles in migration and tissue localization of various lymphocyte subsets. Here, we report the highly frequent expression of CCL5 (RANTES) in ATL and HTLV‐I‐infected T‐cell lines. Among various human T‐cell lines, those infected with HTLV‐I selectively expressed the CCL5 gene and secreted CCL5. Furthermore, CCL5 was expressed by leukemic cells in peripheral blood and lymph nodes from patients with ATL. Inducible expression of HTLV‐I transcriptional activator Tax in a human T‐cell line Jurkat, up‐regulated CCL5 mRNA and induced CCL5 secretion. Analysis of the CCL5 promoter revealed that this gene is activated by Tax, via the activation of NF‐κB, whose responsive element, R(A/B), is located at positions −71 to −43 relative to the putative transcription start site. Aberrant expression of CCL5 by HTLV‐I‐infected T cells may impact on the pathophysiology of HTLV‐I‐associated diseases.