An ex vivo screening using mouse brain mitochondria identified seco-cycline D as an inhibitor of mitochondrial permeability transition pore.

Mitochondrial dysfunction is implicated in neuropsychiatric disorders. Inhibition of mitochondrial permeability transition pore (mPTP) and thereby enhancement of mitochondrial Ca2+ retention capacity (CRC) is a promising treatment strategy. Here, we screened 1718 compounds to search for drug candidates inhibiting mPTP by measuring their effects on CRC in mitochondria isolated from mouse brains. We identified seco-cycline D (SCD) as an active compound. SCD and its derivative were more potent than a known mPTP inhibitor, cyclosporine A (CsA). The mechanism of action of SCD was suggested likely to be different from CsA that acts on cyclophilin D. Repeated administration of SCD decreased ischemic area in a middle cerebral artery occlusion model in mice. These results suggest that SCD is a useful probe to explore mPTP function.

Increased PDGFRB and NF-κB signaling caused by highly prevalent somatic mutations in intracranial aneurysms.

Intracranial aneurysms (IAs) are a high-risk factor for life-threatening subarachnoid hemorrhage. Their etiology, however, remains mostly unknown at present. We conducted screening for sporadic somatic mutations in 65 IA tissues (54 saccular and 11 fusiform aneurysms) and paired blood samples by whole-exome and targeted deep sequencing. We identified sporadic mutations in multiple signaling genes and examined their impact on downstream signaling pathways and gene expression in vitro and an arterial dilatation model in mice in vivo. We identified 16 genes that were mutated in at least one IA case and found that these mutations were highly prevalent (92%: 60 of 65 IAs) among all IA cases examined. In particular, mutations in six genes (PDGFRB, AHNAK, OBSCN, RBM10, CACNA1E, and OR5P3), many of which are linked to NF-κB signaling, were found in both fusiform and saccular IAs at a high prevalence (43% of all IA cases examined). We found that mutant PDGFRBs constitutively activated ERK and NF-κB signaling, enhanced cell motility, and induced inflammation-related gene expression in vitro. Spatial transcriptomics also detected similar changes in vessels from patients with IA. Furthermore, virus-mediated overexpression of a mutant PDGFRB induced a fusiform-like dilatation of the basilar artery in mice, which was blocked by systemic administration of the tyrosine kinase inhibitor sunitinib. Collectively, this study reveals a high prevalence of somatic mutations in NF-κB signaling pathway-related genes in both fusiform and saccular IAs and opens a new avenue of research for developing pharmacological interventions.

Design and synthesis of curcumin derivatives as tau and amyloid ß dual aggregation inhibitors.

Alzheimer's disease (AD) is the most common form of dementia. In an AD patient's brain, senile plaques and neurofibrillary tangles, the abnormal aggregates of amyloid ß (Aß) peptide and tau protein, are observed as the two major hallmarks of this disease. To develop a new drug for treatment of AD, we have designed and synthesized a series of curcumin derivatives and evaluated their inhibitory activities against both tau and Aß aggregation. In this study, we describe the development of the more potent aggregation inhibitor 3-[(1E)-2-(1H-indol-6-yl)ethenyl]-5-[(1E)-2-[2-methoxy-4-(2-pyridylmethoxy) phenyl] ethenyl]-1H-pyrazole (compound 4, PE859). This compound has a better pharmacokinetic profile and pharmacological efficacy in vivo than curcumin, making it suitable as a drug for AD.

CD69 overexpression by human T-cell leukemia virus type 1 Tax transactivation.

Human T-cell leukemia virus type 1 (HTLV-1) infection is associated with the development of adult T-cell leukemia (ATL) and various inflammatory diseases. CD69 is a marker of early activation of lymphocytes. We investigated the effects of HTLV-1 infection on the expression of CD69. The CD69 gene was upregulated in all viral protein Tax-expressing HTLV-1-transformed T-cell lines, except MT-2 and peripheral blood mononuclear cells from patients with ATL compared with uninfected T-cell line, Tax-negative ATL-derived T-cell lines and normal peripheral blood mononuclear cells. Flow cytometric analysis and immunohistochemical analysis confirmed the enhanced expression of CD69 in HTLV-1-transformed T-cell lines and in ATL cells in lymph nodes and skin lesions, and its absence in MT-2 and peripheral blood mononuclear cells. CD69 expression was induced following infection of human T-cell line with HTLV-1, and specifically by Tax. Tax transcriptionally activated CD69 gene through both nuclear factor-κB and cyclic adenosine 3',5'-monophosphate response element-binding protein signaling pathways. Detailed analysis of the CD69 promoter indicated that the Tax-induced expression of CD69 was regulated by multiple cis-acting elements and by the interplay of transcription factors of the nuclear factor-κB, early growth response and cyclic adenosine 3',5'-monophosphate response element-binding protein families. The lack of CD69 expression in MT-2 is due to epigenetic mechanism involving deacetylation, but not methylation. We conclude that CD69 is a Tax-regulated gene, and its regulation by Tax may play a role in cellular activation and HTLV-1-induced disease pathogenesis.

Japanese encephalitis virus structural and nonstructural proteins expressed in Escherichia coli induce protective immunity in mice.

Ectodomain of Japanese encephalitis virus (JEV) E protein [domains I through III (D1-3), domains I and II (D1-2) and domain III (D3)] and the nonstructural protein 1 (NS1) were expressed in Escherichia coli, and administered to BALB/c mice via the intranasal (i.n.) route. The E protein, but not the NS1, induced JEV-specific serum IgG with virus-neutralization capacity in vitro. When mice were lethally challenged with JEV, i.n. immunization with D1-3, D1-2, D3, or a mouse brain-derived formalin-inactivated JE vaccine conferred complete protection, while an 80% protection rate was observed in the NS1 immunized mice. Cytokine analysis of the cervical lymph nodes of mice i.n. immunized with D1-3 or NS1 revealed antigen-specific IL-2 and IL-17 responses, but no IFN-γ T cell response, were observed. This study demonstrates for the first time the i.n. vaccine efficacy of the E. coli-expressed recombinant JEV proteins.

NF-kappaB activation by Helicobacter pylori requires Akt-mediated phosphorylation of p65.

BACKGROUND: The inflammatory response in Helicobacter pylori-infected gastric tissue is mediated by cag pathogenicity island (PAI)-dependent activation of nuclear factor-kappaB (NF-kappaB). Phosphatidylinositol 3-kinase (PI3K)/Akt signaling is known to play a role in NF-kappaB activation, but little information is available on the relationship between H. pylori and PI3K/Akt signaling in gastric epithelial cells. We examined whether H. pylori activates Akt in gastric epithelial cells, the role of cag PAI in this process and the role of Akt in regulating H. pylori-induced NF-kappaB activation. RESULTS: Phosphorylated Akt was detected in epithelial cells of H. pylori-positive gastric tissues. Although Akt was activated in MKN45 and AGS cells by coculture with cag PAI-positive H. pylori strains, a cag PAI-negative mutant showed no activation of Akt. H. pylori also induced p65 phosphorylation. PI3K inhibitor suppressed H. pylori-induced p65 phosphorylation and NF-kappaB transactivation, as well as interleukin-8 expression. Furthermore, transfection with a dominant-negative Akt inhibited H. pylori-induced NF-kappaB transactivation. Transfection with small interference RNAs for p65 and Akt also inhibited H. pylori-induced interleukin-8 expression. CONCLUSION: The results suggest that cag PAI-positive H. pylori activates Akt in gastric epithelial cells and this may contribute to H. pylori-mediated NF-kappaB activation associated with mucosal inflammation and carcinogenesis.

A modified version of galectin-9 suppresses cell growth and induces apoptosis of human T-cell leukemia virus type I-infected T-cell lines.

ATL is a fatal malignancy of T lymphocytes caused by HTLV-I infection and remains incurable. Galectins are a family of animal lectins that function both extracellularly (by interacting with cell surface and extracellular matrix glycoproteins and glycolipids) and intracellularly (by interacting with cytoplasmic and nuclear proteins) to modulate signaling pathways. We found that protease-resistant galectin-9 by modification of its linker peptide, hG9NC(null), prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells. The suppression of cell growth was inhibited by lactose, but not by sucrose, indicating that beta-galactoside binding is essential for hG9NC(null)-induced cell growth suppression. hG9NC(null) induced cell cycle arrest by reducing the expression of cyclin D1, cyclin D2, cyclin B1, Cdk1, Cdk4, Cdk6, Cdc25C and c-Myc, and apoptosis by reducing the expression of XIAP, c-IAP2 and survivin. Most of these genes are regulated by NF-kappaB, which plays a critical role in oncogenesis by HTLV-I. hG9NC(null) suppressed IkappaBalpha phosphorylation, resulting in suppression of NF-kappaB. Most importantly, treatment with hG9NC(null) (6.7 mg/kg injected intraperitoneally every day) reduced tumor formation from an HTLV-I-infected T-cell line when these cells were inoculated subcutaneously into SCID mice. Our results suggest that hG9NC(null) could be a suitable agent for the management of ATL.

Inhibition of heat shock protein-90 modulates multiple functions required for survival of human T-cell leukemia virus type I-infected T-cell lines and adult T-cell leukemia cells.

The molecular chaperone Hsp90 is involved in the stabilization and conformational maturation of many signaling proteins that are deregulated in cancers. The geldanamycin derivative 17-AAG is currently tested in clinical trials and known to inhibit the function of Hsp90 and promote the proteasomal degradation of its misfolded client proteins. ATL is a fatal malignancy of T lymphocytes caused by HTLV-I infection and remains incurable. Since Hsp90 is overexpressed in HTLV-I-infected T-cell lines and primary ATL cells, we analyzed the effects of 17-AAG on cell survival, apoptosis and expression of signal transduction proteins. HTLV-I-infected T-cell lines and primary ATL cells were significantly more sensitive to 17-AAG in cell survival assays than normal PBMCs. 17-AAG induced the inhibition of cell cycle and apoptosis. These effects could be mediated by inactivation of NF-kappaB, AP-1 and PI3K/Akt pathways, as well as reduction of expression of proteins involved in the G1-S cell cycle transition and apoptosis. Proteasome inhibition interfered with 17-AAG-mediated signaling proteins depletion. Collectively, our results indicate that 17-AAG suppresses ATL cell survival through, at least in part, destabilization of several client proteins and suggest that 17-AAG is a potentially useful chemotherapeutic agent for ATL.

Bisphosphonate incadronate inhibits growth of human T-cell leukaemia virus type I-infected T-cell lines and primary adult T-cell leukaemia cells by interfering with the mevalonate pathway.

Anti-resorptive bisphosphonates are used for the treatment of hypercalcaemia and bone complications associated with malignancies and osteoporosis, but also have been shown to have anti-tumour effects in various cancers. Adult T-cell leukaemia (ATL) is a fatal T-cell malignancy caused by infection with human T-cell leukaemia virus type I (HTLV-I), and remains incurable. ATL is associated with osteolytic bone lesions and hypercalcaemia, both of which are major factors in the morbidity of ATL. Thus, the search for anti-ATL agents that have both anti-tumour and anti-resorptive activity is warranted. The bisphosphonate agent, incadronate, prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells, but not of non-infected T-cell lines or normal peripheral blood mononuclear cells. Incadronate induced S-phase cell cycle arrest and apoptosis in HTLV-I-infected T-cell lines, and treatment of these cells with substrates of the mevalonate pathway blocked the incadronate-mediated growth suppression. Incadronate also prevented the prenylation of Rap1A protein. These results demonstrated that incadronate-induced growth suppression occurs by interfering with the mevalonate pathway. Importantly, treatment with incadronate reduced tumour formation from an HTLV-I-infected T-cell line when these cells were inoculated subcutaneously into severe combined immunodeficient mice. These findings suggest that incadronate could be potentially useful for the treatment of 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.

BACKGROUND: Human 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. RESULTS: Constitutive 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. CONCLUSION: Our 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.

Curcumin targets Akt cell survival signaling pathway in HTLV-I-infected T-cell lines.

The Akt signaling pathway is important for survival and growth of cancer cells. In the present paper we show that the Akt signaling pathway is constitutively activated in human T-cell leukemia virus type I (HTLV-I)-infected T-cell lines and in primary adult T-cell leukemia (ATL) cells. Curcumin, a natural compound present in turmeric, has been studied vigorously as a potent chemopreventive agent for cancer therapy because of its inhibitory effect on proliferation and induction of apoptosis in several tumor cell lines. We investigated the effect of curcumin on Akt activity in HTLV-I-infected T-cell lines and primary ATL cells. Phosphorylated PDK1 is an activator of Akt by phosphorylating Akt. Curcumin reduced phosphorylation of PDK1 and inhibited constitutive activation of Akt. Curcumin activated glycogen synthase kinase (GSK)-3beta, a downstream target of Akt kinase, by inhibiting phosphorylation of this protein. Curcumin reduced the expression of cell cycle regulators, cyclin D1 and c-Myc proteins, which are both degraded by activated GSK-3beta. Our results suggest that activation of the Akt signaling pathway plays an important role in ATL cell survival, and that curcumin may have anti-ATL properties mediated, at least in part, by inhibiting Akt activity. We propose that Akt-targeting agents could be useful for the treatment of ATL. In this regard, curcumin is a potentially promising compound for the treatment of ATL.

NIK-333 inhibits growth of human T-cell leukemia virus type I-infected T-cell lines and adult T-cell leukemia cells in association with blockade of nuclear factor-kappaB signal pathway.

Adult T-cell leukemia (ATL) is caused by human T-cell leukemia virus type I (HTLV-I) and remains incurable. NIK-333, a novel synthetic retinoid, prevents the recurrence of human hepatoma after surgical resection of primary tumors. We explored the effects of NIK-333 on HTLV-I-infected T-cell lines and ATL cells. NIK-333 inhibited cell proliferation, induced G1 arrest, and resulted in massive apoptosis in all tested HTLV-I-infected T-cell lines and ATL cells, whereas little effect was observed on normal peripheral blood mononuclear cells. NIK-333 treatment decreases the levels of cyclin D1, cyclin D2, cIAP2, and XIAP proteins. Further analysis showed that NIK-333 inactivated nuclear factor-kappaB in HTLV-I-infected T-cell lines. In animal studies, treatment with NIK-333 (100 mg/kg given orally every other day) produced partial inhibition of growth of tumors of a HTLV-I-infected T-cell line transplanted s.c. in severe combined immunodeficient mice. Our results indicate that NIK-333 is a potentially useful therapeutic agent for patients with ATL.

Transactivation of CCL20 gene by Epstein-Barr virus latent membrane protein 1.

CCL20 is expected to play a crucial role in the initiation of immune responses and tumour growth. However, expression of CCL20 in Epstein-Barr virus (EBV)-associated diseases has not been studied. We examined the contribution of EBV infection and EBV-encoded latent membrane protein (LMP)-1 to CCL20 expression. EBV infection and LMP-1 induced CCL20 mRNA expression in the EBV-negative Burkitt lymphoma (BL) cell lines and the embryonic kidney cell line. Histone deacetylase inhibitor-stimulated endogenous LMP-1 also induced CCL20 expression in an EBV-positive BL cell line. Analysis of the CCL20 promoter showed that it was activated by LMP-1 C-terminal activation region (CTAR)-1 and CTAR-2. Co-expression of IkappaB alpha, IkappaB beta, IkappaB kinase (IKK)alpha, IKKbeta, IKKgamma, nuclear factor (NF)-kappaB-inducing kinase and tumour necrosis factor receptor-associated factor 2 dominant-negative constructs with LMP-1 inhibited the activation of the CCL20 promoter by LMP-1, suggesting that LMP-1 induces CCL20 via NF-kappaB signalling. The requirement for the NF-kappaB-binding site in the CCL20 promoter in LMP-1 responsiveness was established. Our results indicate that activation of the NF-kappaB pathway by LMP-1 is required for the activation of CCL20 expression.

Curcumin (diferuloylmethane) inhibits constitutive active NF-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.

Adult T-cell leukemia (ATL) is a fatal malignancy of T lymphocytes caused by infection with human T-cell leukemia virus type I (HTLV-I) and remains incurable. Curcumin (diferuloylmethane), the major pigment of the spice turmeric, can be potentially effective by promoting cell apoptosis. Here we examined whether curcumin is effective in the treatment of ATL. Curcumin prevented cell growth of HTLV-I-infected T-cell lines and primary ATL cells but not of normal peripheral blood mononuclear cells. Curcumin induced cell cycle arrest by reducing the expression of cyclin D1, Cdk1 and Cdc25C and apoptosis by reducing the expression of XIAP and survivin. Most of these genes are known to be regulated by NF-kappaB, which plays a critical role in oncogenesis by HTLV-I. Curcumin suppressed constitutive active NF-kappaB of HTLV-I-infected T-cell lines and primary ATL cells by inhibiting phosphorylation of IkappaBalpha. Curcumin also inhibited Tax-induced NF-kappaB transcriptional activity. However, curcumin-induced suppression of cell growth did not correlate with Tax expression level. Curcumin inhibited the growth of HTLV-I-infected T-cell tumors implanted subcutaneously in SCID mice. Our results indicate that curcumin has tumor-suppressive activity against ATL.

Curcumin suppresses constitutive activation of AP-1 by downregulation of JunD protein in HTLV-1-infected T-cell lines.

Activation of the activator protein 1 (AP-1) plays a critical role in oncogenesis by human T-cell leukemia virus type 1 (HTLV-1), the etiologic agent of adult T-cell leukemia (ATL), and is required for maintenance of the malignant phenotype. Curcumin (diferuloylmethane), the major pigment of the spice turmeric, has anti-tumor activity; however, the effect of curcumin against ATL has not been elucidated. In this study, we examined the effects of curcumin on AP-1 activity in HTLV-1-infected T-cell lines. Curcumin suppressed the constitutive AP-1 DNA-binding and transcriptional activity in HTLV-1-infected T-cell line. Curcumin also inhibited HTLV-1 Tax-induced AP-1 transcriptional activity. JunD was detectable as a major component of the AP-1-DNA complex in HTLV-1-infected T-cell lines using the supershift assay. The expression of JunD was suppressed by curcumin treatment. Curcumin inhibited the growth of HTLV-1-infected T-cell lines by inducing cell cycle arrest followed by apoptosis. Our results suggest that suppression of the constitutively active AP-1 by curcumin is due to, at least in-part, reducing the expression of JunD by curcumin. Inhibition of AP-1 activity by curcumin may be one of the mechanisms responsible for the anti-ATL effect of curcumin. We propose that curcumin is a potentially promising compound for the treatment of ATL.

Potential mechanism of resistance to TRAIL-induced apoptosis in Burkitt's lymphoma.

OBJECTIVES: Members of the tumor necrosis factor family are potent inducers of apoptosis in sensitive cells and may be suitable for novel anti-cancer therapies aimed at inducing apoptosis via the activation of receptors with the death domain on malignant cells. We characterized the sensitivity of Burkitt's lymphoma (BL) cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and anti-Fas agonist, and investigated the mechanism of resistance of BL cell lines to TRAIL and Fas apoptotic pathways. METHODS: Epstein-Barr virus (EBV) status in BL cell lines was determined by PCR. The extent of apoptosis following exposure to TRAIL and anti-Fas agonist was measured by 7A6 antigen staining. Expression of TRAIL receptors and Fas was determined by flow cytometry and reverse transcriptase-PCR. Western blot analyses were used to determine the expression of proapoptotic and antiapoptotic proteins. NF-kappaB activity was evaluated by electrophoretic mobility shift assay. RESULTS: The sensitivity of BL cell lines to anti-Fas agonist depended on the expression of Fas. In contrast, the expression of TRAIL receptors did not correlate with the sensitivity to TRAIL-induced apoptosis. Interestingly, EBV-infected BL cell lines which showed constitutive levels of NF-kappaB activation, were TRAIL-resistant. NF-kappaB inhibitors reversed the resistance to TRAIL-induced apoptosis. CONCLUSIONS: Our results suggest that activation of NF-kappaB by EBV infection plays an important role in resistance of BL cell lines to TRAIL-induced apoptosis, and that NF-kappaB inhibitors may be useful adjuncts in clinical use of TRAIL against BL.

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.

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-kappaB and activator protein-1 and inhibited NF-kappaB-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.

Transcriptional activation of survivin through the NF-kappaB 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-kappaB 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-kappaB. Pharmacological NF-kappaB inhibition resulted in suppression of survivin expression and caused apoptosis of Tax-expressing CTLL-2 cells. Our findings suggest that activated NF-kappaB signaling contributes directly to malignant progression of ATL by preventing apoptosis, acting through the prosurvival protein survivin.

Transactivation of the CCL5/RANTES gene by Epstein-Barr virus latent membrane protein 1.

Chemokines and chemokine receptors mediate lymphocyte migration and tissue localization. To analyze CCL5 (RANTES) expression by EBV-infected cells, we examined the expression of CCL5 in BL cell lines. Among 4 BL cell lines, those infected with EBV selectively expressed the CCL5 gene and secreted CCL5. Four cell lines also expressed CCR5, a receptor for CCL5. EBV-encoded LMP-1, a pleiotropic protein that effects gene expression, cell transformation, growth and death, induces expression of CCL5 mRNA and secretion of CCL5 in the EBV-negative BL cell line BJAB and the embryonic kidney cell line 293T. HDACI-stimulated endogenous LMP-1 also induced CCL5 expression in an EBV-positive BL cell line. Analysis of the CCL5 promoter revealed that it is activated by both LMP-1 C-terminal activation domains, CTAR-1 and CTAR-2, which can activate NF-kappaB signaling. Coexpression of IkappaBalpha, IkappaBbeta, IKKalpha, IKKbeta, NIK and TRAF2 dominant-negative constructs, with LMP-1 inhibited the activation of the CCL5 promoter by LMP-1, suggesting that LMP-1 induces CCL5 via NF-kappaB signaling. The NF-kappaB binding sites, R(A/B), located at positions -71 to -43 relative to the putative transcription start site in the CCL5 promoter, were essential for the activation of CCL5 gene expression by LMP-1. These results indicate that the activation of the NF-kappaB pathway by LMP-1 is required for the activation of CCL5 expression.