Rearranged NF-kappa B2 gene in an adult T-cell leukemia cell line

Adult T-cell leukemia (ATL) is an aggressive type of leukemia, originating from T-cells infected with human T-cell leukemia virus type 1. Accumulating evidence suggests the aberrant activation of NF-κB to be a causative factor mediating the abnormal proliferation of leukemic cells, thus resulting in the development of ATL. A rearranged NF-κB2/p100 gene was isolated from an ATL-derived cell line, which was generated by a chromosomal translocation. The isolated NF-κB2 mutant is fused with the with no (lysine) deficient protein kinase 1 gene, coding for a 58 kDa protein that retains the DNA binding Rel homology domain, but it lacks the entire ankyrin repeat inhibitory domain, thus suggesting its constitutive activation. This rearranged NF-κB2 gene product (p58) was localized in the nucleus, and formed a complex with NF-κB p65 or RelB. Moreover, a T-cell line expressing p58 increased the amount of an NF-κB2-inducible gene, NF-κB2/p100 by itself. These results suggest that such NF-κB2 gene rearrangement may therefore be a factor in the constitutive activation of NF-κB in ATL, and thereby playing a role in the ATL pathogenesis. ( Cancer Sci 2008; 99: 792–798)     

Constitutive activation of nuclear factor-κB is preferentially involved in the proliferation of basal-like subtype breast cancer cell lines

Constitutive nuclear factor (NF)-κB activation is thought to be involved in survival, invasion, and metastasis in various types of cancers. However, neither the subtypes of breast cancer cells with constitutive NF-κB activation nor the molecular mechanisms leading to its constitutive activation have been clearly defined. Here, we quantitatively analyzed basal NF-κB activity in 35 human breast cancer cell lines and found that most of the cell lines with high constitutive NF-κB activation were categorized in the estrogen receptor negative, progesterone receptor negative, ERBB2 negative basal-like subtype, which is the most malignant form of breast cancer. Inhibition of constitutive NF-κB activation by expression of IκBα super-repressor reduced proliferation of the basal-like subtype cell lines. Expression levels of mRNA encoding NF-κB-inducing kinase (NIK) were elevated in several breast cancer cell lines, and RNA interference-mediated knockdown of NIK reduced NF-κB activation in a subset of the basal-like subtype cell lines with upregulated NIK expression. Taken together, these results suggest that constitutive NF-κB activation, partially dependent on NIK, is preferentially involved in proliferation of basal-like subtype breast cancer cells and may be a useful therapeutic target for this subtype of cancer. ( Cancer Sci 2009; 100: 1668–1674)     

HTLV-1 provirus load in peripheral blood lymphocytes of HTLV-1 carriers is diminished by green tea drinking

Human T-cell lymphotropic virus type 1 (HTLV-1) is causatively associated with adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Since a high level of HTLV-1 provirus load in circulating lymphocytes is thought to be a risk for ATL and HAM/TSP, diminution of HTLV-1 provirus load in the circulation may prevent these intractable diseases. Our previous study (Jpn J Cancer Res 2000; 91: 34-40) demonstrated that green tea polyphenols inhibit in vitro growth of ATL cells, as well as HTLV-1-infected T-cells. The present study aimed to investigate the in vivo effect of green tea polyphenols on HTLV-1 provirus load in peripheral blood lymphocytes on HTLV-1 carriers. We recruited 83 asymptomatic HTLV-1 carriers to examine HTLV-1 provirus DNA with or without administration of capsulated green tea extract powder. Thirty-seven subjects were followed up for 5 months by measuring HTLV-1 provirus load after daily intake of 9 capsules of green tea extract powder per day (equivalent to 10 cups of regular green tea), and 46 subjects lived ad libitum without intake of any green tea capsule. The real-time PCR quantification of HTLV-1 DNA revealed a wide range of variation of HTLV-1 provirus load among asymptomatic HTLV-1 carriers (0.2-200.2 copies of HTLV-1 provirus load per 1000 peripheral blood lymphocytes). Daily intake of the capsulated green tea for 5 months significantly diminished the HTLV-1 provirus load as compared with the controls (P = 0.031). These results suggest that green tea drinking suppresses proliferation of HTLV-1-infected lymphocytes in vivo.     

Human T-cell leukemia virus type 1 can infect a wide variety of cells in mice.

Analysis of human T-cell leukemia virus type 1 (HTLV-1)-infected cell types and the interplay of these infected cells in vivo should provide valuable information to elucidate the pathogenesis of HTLV-1-associated diseases in humans and in animal models. In this study, HTLV-1-infected cell types were identified in HTLV-1-infected C3H/HeJ mice. Pan T, CD4+, CD8+, granulocyte and pan B cell fractions in the splenocytes of MT-2 cell-inoculated mice were sorted by use of their cell surface high-density expression of CD3e, CD4, CD8, Gr-1 and B220 antigens, respectively, with a fluorescence-activated cell sorter. The pX sequence of HTLV-1 provirus in the lysate of each fraction was amplified by polymerase chain reaction and detected by Southern hybridization. Interestingly, in addition to the CD4+ cell fraction, the pX sequence was also found in CD8+ cell, B cell and granulocyte fractions. The broad cell spectrum of HTLV-1 infection in mice is consistent with the situation in humans. Our finding indicate that HTLV-1 receptor or coreceptor is widely distributed among different cell types in mice.     

Human T-cell Leukemia Virus Type 1 Can Infect a Wide Variety of Cells in Mice

Analysis of human T-cell leukemia virus type 1 (HTLV-1)-infected cell types and the interplay of these infected cells in vivo should provide valuable information to elucidate the pathogenesis of HTLV-1-associated diseases in humans and in animal models. In this study, HTLV-1-infected cell types were identified in HTLV-1-infected C3H/HeJ mice. Pan T, CD⁺₄, CD8⁺⁸, granulocyte and pan B cell fractions in the splenocytes of MT-2 cell-inoculated mice were sorted by use of their cell surface high-density expression of CD_3e, CD₄, CD₈, Gr-1 and B₂₂₀ antigens, respectively, with a fluorescence-activated cell sorter. the pX sequence of HTLV-1 provirus in the lysate of each fraction was amplified by polymerase chain reaction and detected by Southern hybridization. Interestingly, in addition to the CD⁺₄ cell fraction, the pX sequence was also found in CD8+ cell, B cell and granulocyte fractions. The broad cell spectrum of HTLV-1 infection in mice is consistent with the situation in humans. Our findings indicate that HTLV-1 receptor or coreceptor is widely distributed among different cell types in mice.     

HTLV-I messenger RNA is expressed in vivo in adult T-cell leukemia/lymphoma patients: An in situ hybridization study

Adult T-cell leukemia/lymphoma (ATLL) is a malignancy involving peripheral blood, lymph nodes, skin and other organs. Human T-cell leukemia virus type 1 (HTLV-1) is etiologically associated with ATLL but cannot be detected by conventional methods in fresh samples of peripheral blood and skin taken from ATLL. The aim of this study was to investigate the feasibility of an in situ hybridization technique for detection of HTLV-1 mRNA in atypical lymphoid cells of peripheral blood and skin lesions of patients with ATLL. We detected variable amounts of HTLV-1 tax mRNA in the nuclei and cytoplasm of these cells in fresh peripheral blood samples and skin lesions from ATLL patients, and also in asymptomatic HTLV-1 infected donors to a lesser extent. Out of 10 patients with ATLL, 7 showed strong positive in situ hybridization whereas the other 3 were only weakly positive. However, in the last 3 cases, the reaction became strongly positive after cells had been cultured for 24 hr. Furthermore, all 3 asymptomatic HTLV-1-infected donors exhibited a weakly positive response in their apparently mature lymphoid cells.     

Synergistic inhibition of HTLV-1-infected cell proliferation by combination of cepharanthine and a tetramethylnaphthalene derivative

The tetrahydrotetramethylnaphthalene derivative TMNAA has recently been identified as a selective inhibitor of human T-lymphotropic virus type 1 (HTLV-1)-infected T-cell lines and adult T-cell leukemia (ATL) cells but not of uninfected T-cell lines and peripheral blood mononuclear cells (PBMCs). Although the target molecule of TMNAA is still unknown, it does not inhibit nuclear factor-κB (NF-κB) activity. Therefore, TMNAA was examined for its inhibitory effect on the cell proliferation in combination with the NF-κB inhibitor cepharanthine. Synergism was observed for the combination, in inhibiting the proliferation of HTLV-1-infected T-cell lines. Although TMNAA alone did not induce the apoptosis of HTLV-1-infected T-cell lines, it strongly enhanced their apoptosis induced by cepharanthine. Thus, TMNAA may have potential as a therapeutic agent against ATL either alone or in combination with cepharanthine, which is clinically used as an anti-inflammatory drug in Japan.     

Provirus Load in Patients with Human T-Cell Leukemia Virus Type 1 Uveitis Correlates with Precedent Graves' Disease and Disease Activities

We previously demonstrated the increased provirus load in the peripheral blood of patients with human T-cell leukemia virus type 1 (HTLV-1) uveitis (HU). To delineate the relevance of the increased provirus load to clinical and immunologic parameters, we studied the correlation between them. Seventy-nine HU patients (24 male and 55 female) were included in the study, with their informed consent. Plasma samples and genomic DNA of the peripheral blood mononuclear cells were isolated and the provirus load was estimated by semi-quantitative polymerase chain reaction of the gag region sequence. Serum levels of anti-HTLV-1 antibodies and soluble IL-2R were determined by electrochemiluminescence immuno assay and by ELISA, respectively. Disease activities were assessed and graded 0 to 4 according to the evaluation system. Recurrence of the disease during the follow-up period was diagnosed ophthalmologically. The provirus load was significantly higher in the HU patients after Graves' disease (GD) than in those without GD ( P <0.05). It correlated with disease activities assessed in terms of vitreous inflammation and interval to recurrence (both P <0.05). In the HU patients without GD, it correlated with the serum levels of soluble IL-2 receptor ( P <0.01), and nearly with those of HTLV-1 antibody ( P =0.063). These correlations were not found in the HU patients after GD under methimazole treatment. The results suggested a direct involvement of HTLV-1-infected cells in the pathogenesis of uveitis, and raise the possibility that hyperthyroidism may contribute to the clonal expansion of HTLV-1-infected cells.     

Interferon-gamma sensitizes hepatitis B virus-expressing hepatocarcinoma cells to 5-fluorouracil through inhibition of hepatitis B virus-mediated nuclear factor-kappaB activation

Nuclear factor (NF)-κB is important for immune responses and cell survival; however, abnormal activation of NF-κB is linked with many types of diseases, including hepatocellular carcinoma (HCC). Our previous report indicated that hepatitis B virus (HBV) induces NF-κB activation through NF-κB-inducing kinase (NIK), and this can be blocked specifically by interferon (IFN)-γ. In the present study, we report that HBV expression in HCC cell lines induces drug resistance against 5-fluorouracil (5-FU). This drug resistance was abolished by inhibition of NF-κB activation through small interfering RNA-mediated NIK 'knockdown' and IFN-γ treatment. In addition to the reduced NF-κB activation and drug resistance, the upregulated growth arrest- and DNA damage-inducible protein 45β (Gadd45β) in HBV-expressing HCC cell lines was downregulated by the small interfering RNA-mediated NIK knockdown and IFN-γ treatment. The overexpression of Gadd45β in HCC cell lines also induces drug resistance against 5-FU. Based on our data, we suggest that IFN-γ treatment might be helpful for chemotherapy in HBV-integrated HCC through inhibition of the NIK-mediated NF-κB activation and downregulation of the NF-κB target gene Gadd45β . ( Cancer Sci 2007; 98: 1758–1766)     

Selective inhibition of HTLV-1-infected cell proliferation by a novel tetramethylnaphthalene derivative

Adult T-cell leukemia (ATL) is caused by infection with human T-lymphotropic virus type 1 (HTLV-1). A novel tetramethylnaphthalene derivative, TMNAA, selectively inhibited the proliferation of various HTLV-1-infected cells, including ATL cell lines and peripheral blood mononuclear cells (PBMCs) from ATL patients. In contrast, the proliferation of uninfected cell lines and PBMCs from healthy donors was hardly affected by the compound. Cell-cycle analysis revealed that TMNAA increased the population of the G0/G1 phase and reduced that of the S phase in HTLV-1-infected cells. TMNAA was found to suppress the phosphorylation of retinoblastoma protein and the expression of cyclin-dependent kinase 4 in HTLV-1-infected cells. Furthermore, the inhibition of cell proliferation was partially annihilated by removing the compound. These results indicate that TMNAA exerts selective inhibition of HTLV-1-infected cells through a novel mechanism, presumably modulating cell cycle regulatory proteins associated with the G0/G1 phase.     

Anti-adult T-cell leukemia effects of Bidens pilosa

We evaluated the effects of Bidens pilosa, a plant found in tropical and subtropical regions, and investigated the molecular pathways responsible for the anti-adult T-cell leukemia (ATL) effect. Water extracts of B. pilosa had growth suppressive effects on human T-cell leukemia virus type 1 (HTLV-1)-infected T-cell lines and ATL cells. B.?pilosa extracts arrested cells in G1 cell cycle and induced apoptosis of HTLV-1-infected T-cell lines. B. pilosa extracts inhibited also the phosphorylation of IκB kinase β and IκBα, and NF-κB-DNA binding, in conjunction with reduction of expression of proteins involved in G1/S cell cycle transition and suppression of apoptosis. Reactive oxygen species played a role in B.?pilosa-mediated suppression of NF-κB activity. B.?pilosa extracts also inhibited the expression of JunB and JunD, resulting in suppression of AP-1-DNA binding. In animals harboring tumors of HTLV-1-infected T-cell origin, treatment with B. pilosa extracts suppressed tumor growth. Our results suggest that B. pilosa is a potentially useful medicinal plant for treatment of ATL.     

Identification of HTLV-I markers in patients with hemoblastoses

Serologic and molecular biologic techniques were used to identify association of HTLV-1 with adult T-cell leukemia/lymphoma as well as with B-cell leukemia. HTLV-1 markers (antibodies and integrated provirus) were identified in all lympho- and myeloproliferative diseases, while integrated provirus genome--in T-cell population only.     

A unique T-cell line derived from an HTLV-1-negative adult T-cell leukemia patient

A unique T-cell line, designated ATL-5T, was established from lymphoma cells in pericardial effusion of an adult T-cell leukemia (ATL) patient not carrying HTLV-1 provirus. The cell line is OKT4 and/or Leu3a+ and OKT8 and/or Leu2a+, but interleukin 2 receptor (IL2R)- and HTLV-1 provirus genome negative, and has cytogenetically abnormal karyotypes. The cell line contains rearranged T-cell receptor beta-chain gene, which was identical in rearrangement pattern to the T-cell receptor beta-chain gene in primary cells. These results suggest that factors other than HTLV-1 may sometimes be associated with HTLV-1-negative ATL. The ATL-5T cell line we describe here is unique, and should contribute to further elucidation of the mechanisms involved in the pathogenesis of HTLV-1-negative ATL and HTLV-1-positive ATL.     

The oncogenic driving force of CD30 signaling-induced chromosomal instability in adult T-cell leukemia/lymphoma

Adult T-cell leukemia/lymphoma (ATL) develops via stepwise accumulation of gene mutations and chromosome aberrations. However, the molecular mechanisms underlying this tumorigenic process are poorly understood. We previously reported the presence of a biological link between the expression of CD30, which serves as a marker for ATL progression, and the actively proliferating fraction of human T-cell leukemia virus type 1 (HTLV-1)-infected cells that display polylobulation. Here, we demonstrated that CD30 signaling induced chromosomal instability with clonal expansion through DNA double-strand breaks (DSBs) via an increase of intracellular reactive oxygen species. CD30⁺ATL cells were composed of subclones with additional genomic aberrations compared with CD30⁻ATL cells in ATL patients. Furthermore, we found an accumulation of copy number loss of DSB repair-related genes as the disease progressed. Taken together, CD30 expression on ATL cells appears to be correlated with genomic instability, suggesting that CD30 signaling is one of the oncogenic factors of ATL progression with clonal evolution. This study provides new insight into the biological roles of CD30 signaling and could improve our understanding of tumorigenic processes of HTLV-1-infected cells.     

Transient inhibition of NF-κB by DHMEQ induces cell death of primary effusion lymphoma without HHV-8 reactivation

Primary effusion lymphoma (PEL) is a refractory malignancy caused by human herpes virus 8 (HHV-8) in immunocompromised individuals. The tumor cells of PEL are characterized by constitutive NF-κB activation. Dehydroxymethylepoxyquinomicin (DHMEQ) is a new NF-κB inhibitor and is effective on various tumor cells with constitutively activated NF-κB. Thus, in search for a new therapeutic modality of PEL, we examined the effect of DHMEQ on PEL cells. We confirmed constitutive activation of NF-κB with subcomponents of p50 and p65 in PEL cell lines. DHMEQ quickly and transiently abrogated NF-κB activation and reduced the cell viability in dose- and time-dependent manners, inducing apoptosis through activation of both mitochondrial and membrane pathways. Array analysis revealed that DHMEQ down-regulated expression levels of NF-κB target genes, such as interleukin-6 (IL6), Myc , chemokine (C-C motif) receptor 5 ( CCR5 ) and NF-κB1, whereas it up-regulated expression levels of some genes involved in apoptosis, and cell cycle arrest. DHMEQ did not reactivate HHV-8 lytic genes, indicating that NF-κB inhibition by DHMEQ did not induce virus replication. DHEMQ rescued CB-17 SCID mice xenografted with PEL cells, reducing the gross appearance of effusion. Thus, DHMEQ transiently abrogated the NF-κB activation, irreversibly triggering the apoptosis cascade without HHV-8 reactivation. In addition, DHMEQ could rescue the PEL-xenograft mice. Therefore, we suggest DHMEQ as a promising candidate for molecular target therapy of the PEL. ( Cancer Sci 2009; 100: 737–746)     

Involvement of IL-2/IL-2R system activation by parasite antigen in polyclonal expansion of CD4(+)25(+) HTLV-1-infected T-cells in human carriers of both HTLV-1 and S. stercoralis

The intermediate state of HTLV-1 infection, often found in individuals dually infected with Strongyloides stercoralis (S. stercoralis) and HTLV-1, is assumed to be a preleukemic state of adult T-cell leukemia (ATL). To investigate the effects of S. stercoralis superinfection on the natural history of HTLV-1 infection, we characterized peripheral blood samples of these individuals in Okinawa, Japan, an endemic area for both HTLV-1 and S. stercoralis and we studied effects of the parasite antigen on T-cells. The dually infected individuals showed a significantly higher provirus load and an increase in CD4(+)25(+) T cell population, with a significant, positive correlation. This increase was attributable to polyclonal expansion of HTLV-1-infected cells, as demonstrated by inverse-long PCR analysis of the integration sites. S. stercoralis antigen activated the IL-2 promoter in reporter gene assays, induced production of IL-2 by PBMC in vitro, and supported growth of IL-2 dependent cell lines immortalized by HTLV-1 infection or the transduction of Tax. Taken collectively, these results indicate that S. stercoralis infection induces polyclonal expansion of HTLV-1-infected cells by activating the IL-2/IL-2R system in dually infected carriers, an event which may be a precipitating factor for ATL and inflammatory diseases.