Yin Yang 1 is a potent activator of human T lymphotropic virus type 1 LTR-driven gene expression via RNA binding.

Yin Yang 1 (YY1) is a DNA-binding transcription factor that either activates or represses gene expression. YY1 has previously been implicated in the transcriptional silencing of many retroviruses by binding to DNA sequences in the U3 region of the viral long terminal repeat (LTR). We here show that YY1 overexpression leads to profound activation, rather than repression, of human T lymphotropic virus type 1 (HTLV-1) expression, while YY1 down-regulation reduces HTLV-1 expression. The YY1 responsive element mapped not to YY1 DNA-binding sites in the HTLV-1 LTR but to the R region. The HTLV-1 R sequence alone is sufficient to provide YY1 responsiveness to a nonresponsive promoter, but only in the sense orientation and only when included as part of the mRNA. YY1 binds to the R region of HTLV-1 RNA in vitro and in vivo, leading to increased transcription initiation and elongation. The findings indicate that YY1 is a potent transactivator of HTLV-1 gene expression acting via binding viral RNA, rather than DNA.

The effect of alcoholic extract from Eucalyptus camaldulensis leaves on HTLV-1 Tax activities.

HTLV-1 is a human retrovirus responsible for adult T-cell leukemia (ATL) and certain other clinical disorders. The viral Tax oncoprotein plays a central role in HTLV-1 pathogenicity, mainly due to its capacity of inducing the transcriptional activity of various transcriptional factors like NFқB. Eucalyptus camaldulensis (Ec) is considered as a traditional medicinal plant with valuable therapeutic effects. Here we evaluated the activity of its ethanolic leave extract on different Tax activities by testing its influence on Tax-induced activity of NFқB and HTLV-1 LTR in Jurkat cells. Our results showed that Ec inhibited Tax induced activation of NFқB -, SRF- dependent promoters and HTLV-1 LTR. Ec extract has no effect on the binding of Tax to NFқB while it strongly prevented the degradation of IҝBα induced by Tax probably as a result of preventing the link between Tax and IKKγ. In addition, increasing the cellular level of P-TEFb-cyclinT1 significantly reduced the inhibitory effect of Ec on Tax activities, probably by preventing the interaction between Tax and P-TEFb-cyclin T1. The 40%-MeOH fraction of this extract, which is rich with polyphenols, offered the highest inhibitory effect against Tax activities. Further studies are required for the isolation and identification of active component/s in this extract which may be developed in the future as preventive/curing drugs for HTLV-1 related diseases.

Bovine leukemia virus nucleocapsid protein is an efficient nucleic acid chaperone.

Nucleocapsid proteins (NCs) direct the rearrangement of nucleic acids to form the most thermodynamically stable structure, and facilitate many steps throughout the life cycle of retroviruses. NCs bind strongly to nucleic acids (NAs) and promote NA aggregation by virtue of their cationic nature; they also destabilize the NA duplex via highly structured zinc-binding motifs. Thus, they are considered to be NA chaperones. While most retroviral NCs are structurally similar, differences are observed both within and between retroviral genera. In this work, we compare the NA binding and chaperone activity of bovine leukemia virus (BLV) NC to that of two other retroviral NCs: human immunodeficiency virus type 1 (HIV-1) NC, which is structurally similar to BLV NC but from a different retrovirus genus, and human T-cell leukemia virus type 1 (HTLV-1) NC, which possesses several key structural differences from BLV NC but is from the same genus. Our data show that BLV and HIV-1 NCs bind to NAs with stronger affinity in relation to HTLV-1 NC, and that they also accelerate the annealing of complementary stem-loop structures to a greater extent. Analysis of kinetic parameters derived from the annealing data suggests that while all three NCs stimulate annealing by a two-step mechanism as previously reported, the relative contributions of each step to the overall annealing equilibrium are conserved between BLV and HIV-1 NCs but are different for HTLV-1 NC. It is concluded that while BLV and HTLV-1 belong to the same genus of retroviruses, processes that rely on NC may not be directly comparable.

Novel cytotoxic isolated from Jamaican Hyptis verticillata jacq induces apoptosis and overcomes multidrug resistance.

BACKGROUND: Adult T-cell leukemia is an aggressive hematological malignancy with a poor clinical prognosis, and a rapid resistance to chemotherapy is rapid. MATERIALS AND METHODS: Cytotoxicity assay-directed fractionation identified a novel lignan-related agent, 4-methoxy-9-(3,4,5-trimethoxyphenyl)-8, 9 - dihydrofuro[3',4':6,7]naphtho[2,3-d][1,3]dioxol-6(5H)-one (4-MTDND) from the Jamaican plant Hyptis verticillata jacq, and its effects on apoptosis, cell cycle and drug resistance were elucidated. RESULTS: The novel agent, 4-MTDND, exhibited cytotoxicity against myriad cancer types, with a wide therapeutic index of 30- to 40-fold, promoted G(2)/M arrest and up-regulated expression of pro-apoptotic proteins p53 and BAX, as well as enhanced activation of caspase-3, caspase-9 and poly (ADP ribose) polymerase, consistent with apoptosis induction. Multidrug-resistant cancer cells were as susceptible to 4-MTDND as their non-resistant control counterparts, with 4-MTDND having greater efficacy compared to standard chemotherapy agents etoposide and mitoxantrone. CONCLUSION: The novel cytotoxic agent 4-MTDND induces G(2)/M arrest and apoptosis in cancer cells possibly due to direct DNA damage or interference with topoisomerase II.

Oridonin, a diterpenoid purified from Rabdosia rubescens, inhibits the proliferation of cells from lymphoid malignancies in association with blockade of the NF-kappa B signal pathways.

This study found that oridonin, a natural diterpenoid purified from Rabdosia rubescens, inhibited growth of multiple myeloma (MM; U266, RPMI8226), acute lymphoblastic T-cell leukemia (Jurkat), and adult T-cell leukemia (MT-1) cells with an effective dose that inhibited 50% of target cells (ED50) ranging from 0.75 to 2.7 microg/mL. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining showed that oridonin caused apoptosis of MT-1 cells in a time-dependent manner. We explored effects of oridonin on antiapoptotic Bcl-2 family members and found that it down-regulated levels of Mcl-1 and BCL-x(L), but not Bcl-2 protein, in both MT-1 and RPMI8226 cells. Further studies found that oridonin inhibited nuclear factor-kappa B (NF-kappa B) DNA-binding activity in these cells as measured by luciferase reporter gene, ELISA-based, and electrophoretic mobility shift assays. Oridonin also blocked tumor necrosis factor-alpha- and lipopolysaccharide-stimulated NF-kappa B activity in Jurkat cells as well as RAW264.7 murine macrophages. Of note, oridonin decreased survival of freshly isolated adult T-cell leukemia (three samples), acute lymphoblastic leukemia (one sample), chronic lymphocytic leukemia (one sample), non-Hodgkin's lymphoma (three samples), and MM (four samples) cells from patients in association with inhibition of NF-kappa B DNA-binding activity. On the other hand, oridonin did not affect survival of normal lymphoid cells from healthy volunteers. Taken together, oridonin might be useful as adjunctive therapy for individuals with lymphoid malignancies, including the lethal disease adult T-cell leukemia.

Role of accessory proteins of HTLV-1 in viral replication, T cell activation, and cellular gene expression.

Human T-cell lymphotropic virus type 1 (HTLV-1), causes adult T cell leukemia/lymphoma (ATLL), and initiates a variety of immune mediated disorders. The viral genome encodes common structural and enzymatic proteins characteristic of all retroviruses and utilizes alternative splicing and alternate codon usage to make several regulatory and accessory proteins encoded in the pX region (pX ORF I to IV). Recent studies indicate that the accessory proteins p12I, p27I, p13II, and p30II, encoded by pX ORF I and II, contribute to viral replication and the ability of the virus to maintain typical in vivo expression levels. Proviral clones that are mutated in either pX ORF I or II, while fully competent in cell culture, are severely limited in their replicative capacity in a rabbit model. These HTLV-1 accessory proteins are critical for establishment of viral infectivity, enhance T-lymphocyte activation and potentially alter gene transcription and mitochondrial function. HTLV-1 pX ORF I expression is critical to the viral infectivity in resting primary lymphocytes suggesting a role for the calcineurin-binding protein p12I in lymphocyte activation. The endoplasmic reticulum and cis-Golgi localizing p12I activates NFAT, a key T cell transcription factor, through calcium-mediated signaling pathways and may lower the threshold of lymphocyte activation via the JAK/STAT pathway. In contrast p30II localizes to the nucleus and represses viral promoter activity, but may regulate cellular gene expression through p300/CBP or related co-activators of transcription. The mitochondrial localizing p13II induces morphologic changes in the organelle and may influence energy metabolism infected cells. Future studies of the molecular details HTLV-1 "accessory" proteins interactions will provide important new directions for investigations of HTLV-1 and related viruses associated with lymphoproliferative diseases. Thus, the accessory proteins of HTLV-1, once thought to be dispensable for viral replication, have proven to be directly involved in viral spread in vivo and represent potential targets for therapeutic intervention against HTLV-1 infection and disease.

The HBZ factor of human T-cell leukemia virus type I dimerizes with transcription factors JunB and c-Jun and modulates their transcriptional activity.

The human T-cell leukemia virus type I (HTLV-I)-encoded Tax protein activates transcription from the viral promoter via association with the cellular basic leucine zipper factor cAMP-response element-binding protein-2. Tax is also able to induce cellular transformation of T lymphocytes probably by modulating transcriptional activity of cellular factors, including nuclear factor-kappaB, E2F, activator protein-1 (AP-1), and p53. Recently, we characterized in HTLV-I-infected cells the presence of a novel viral protein, HBZ, encoded by the complementary strand of the HTLV-I RNA genome (Gaudray, G., Gachon, F., Basbous, J., Biard-Piechaczyk, M., Devaux, C., and Mesnard, J.-M. (2002) J. Virol. 76, 12813-12822). HBZ is a nuclear basic leucine zipper protein that down-regulates Tax-dependent viral transcription by inhibiting the binding of cAMP-response element-binding protein-2 to the HTLV-I promoter. In searching for other cellular targets of HBZ, we identified two members of the Jun family, JunB and c-Jun. Co-immunoprecipitation and cellular colocalization confirmed that HBZ interacts in vivo with JunB and c-Jun. When transiently introduced into CEM cells with a reporter gene containing the AP-1 site from the collagenase promoter, HBZ suppressed transactivation by c-Jun. On the other hand, the combination of HBZ with Jun-B had higher transcriptional activity than JunB alone. Consistent with the structure of its basic domain, we demonstrate that HBZ decreases the DNA-binding activity of c-Jun and JunB. Last, we show that c-Jun is no longer capable of activating the basal expression of the HTLV-I promoter in the presence of HBZ in vivo. Our results support the hypothesis that HBZ could be a negative modulator of the Tax effect by controlling Tax expression at the transcriptional level and by attenuating activation of AP-1 by Tax.

Green tea polyphenols induce apoptosis in vitro in peripheral blood T lymphocytes of adult T-cell leukemia patients.

Green tea polyphenols (TEA) are known to exhibit antioxidative activity as well as tumor-suppressing activity. In order to examine the tumor-suppressing activity of TEA against adult T-cell leukemia (ATL), we cultivated peripheral blood T lymphocytes of ATL patients (ATL PBLs), an HTLV-I-infected T-cell line (KODV) and healthy controls (normal PBLs) for 3 days in the presence of TEA and its main constituent, epigallocatechin-3-gallate (EGCg), to measure cell proliferation and apoptosis, and to quantitate mRNAs of HTLV-I pX and beta-actin genes of the cultured cells. Growth of ATL PBLs was significantly inhibited by 9-27 microg/ml of TEA and EGCg, in contrast to minimal growth inhibition of T cells of normal PBLs. Inhibition of KODV was intermediate between ATL PBLs and normal PBLs. The ATL PBLs and KODV treated with 27 microg/ml of either TEA or EGCg induced apoptotic DNA fragmentation, producing terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive cells, while the normal PBLs treated with the same concentration of TEA or EGCg produced a negligibly small number of TUNEL-positive cells, in which apoptotic DNA fragmentation was not detectable. Expression of HTLV-I pX mRNA was suppressed more than 90% in ATL PBLs by treatment with 3-27 microg/ml of either TEA or EGCg, while expression of beta-actin mRNA was much less suppressed by treatment with the same concentration of TEA or EGCg. These results indicate that TEA and EGCg inhibit growth of ATL PBLs, as well as HTLV-I-infected T-cells, by suppressing HTLV-I pX gene expression and inducing apoptotic cell death.

In vitro anti-human immunodeficiency virus activity of polysaccharide from Rhizophora mucronata Poir.

A polysaccharide was extracted with 1% sodium carbonate from the bark of Rhizophora mucronata and its antiviral activities against human immunodeficiency virus (HIV) were assessed by an in vitro cell culture system. The anti-HIV activity of the alkaline extract was mainly recovered in the 25-75% ethanol-precipitated fraction. Rhizophora mucronata polysaccharide (RMP) protected MT-4 cells from the HIV-induced cytopathogenicity and blocked the expression of HIV antigens. RMP completely inhibited the viral binding to the cell and the formation of syncytium upon cocultivation of MOLT-4/HIV-1IIIB cells and MOLT-4 cells. These results suggest that RMP inhibited early steps of the virus life cycle especially virus adsorption to the cell.

Delayed tumor onset in transgenic mice fed an amino acid-based diet supplemented with red wine solids.

Increased consumption of vegetable foods (cereals, legumes, fruits) and some beverages (tea, cider, wine) is associated with reduced risk of cancer. Polyphenols in these foods and beverages are thought to be responsible, based on data from in vitro assays and from in vivo studies that used animals pretreated with carcinogen and given tea or polyphenol-spiked water to drink. We tested the hypothesis that dehydrated-dealcoholized red wine (wine solids), when consumed as part of a precisely defined complete diet, would delay tumor onset in transgenic mice that spontaneously develop externally visible tumors without carcinogen pretreatment. Sibling transgenic mice were weaned onto an amino acid-based diet alone or supplemented with red wine solids. Mice were examined daily; the age at which a first tumor appeared was recorded as the age of tumor onset. The concentration of the major polyphenol of red wine (catechin) in blood serum was also measured at the end of the study. The supplemented diet was fed continuously for three generations to ensure that it supported normal growth and reproduction. We discovered that the wine solid supplement delayed tumor onset, that intact catechin was absorbed, and that the supplemented diet supported normal growth and reproduction for three generations. Also, our simple experimental protocol offers an alternate and/or complementary way to identify foods, beverages, and their constituents that delay tumor onset and to investigate possible mechanisms involved.