Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis.

BACKGROUND: Osteoarthritis (OA) is a common joint disease that causes disabilities in elderly. However, few agents with high efficacy and low side effects have been developed to treat OA. In this study, we evaluated the effects of the alginate extract named CTX in OA cell and rabbit models. RESULTS: CTX was formulated by hydrolyzing sodium alginate polymers with alginate lyase and then mixing with pectin. HPLC was used to analyze the CTX content. Human chondrosarcoma SW1353 cells treated with interleukin-1ß were used as OA model cells to investigate the effects of CTX on chondrocyte inflammation and anabolism. CTX at concentrations up to 1000 µg/ml exerted low cytotoxicity. It inhibited the gene expression of proinflammatory matrix metalloproteinases (MMPs) including MMP1, MMP3 and MMP13 in a dose-dependent manner and increased the mRNA level of aggrecan, the major proteoglycan in articular cartilage, at 1000 µg/ml. Thirteen-week-old New Zealand White rabbits underwent a surgical anterior cruciate ligament transection and were orally treated with normal saline, glucosamine or CTX for up to 7 weeks. Examinations of the rabbit femur and tibia samples demonstrated that the rabbits taking oral CTX at a dosage of 30 mg/kg/day suffered lesser degrees of articular stiffness and histological cartilage damage than the control rabbits. CONCLUSIONS: The gene expression profiles in the cell and the examinations done on the rabbit cartilage suggest that the alginate extract CTX is a pharmaco-therapeutic agent applicable for OA therapy.

Epstein-Barr virus Rta-mediated transactivation of p21 and 14-3-3σ arrests cells at the G1/S transition by reducing cyclin E/CDK2 activity.

Many herpesviral immediate-early proteins promote their robust lytic phase replications by hijacking the cell cycle machinery. Previously, lytic replication of Epstein-Barr virus (EBV) was found to be concurrent with host cell cycle arrest. In this study, we showed that ectopic expression of EBV immediate-early protein Rta in HEp-2 cells resulted in increased G1/S population, hypophosphorylation of pRb and decreased incorporation of 5-bromo-2'-deoxyuridine. In addition, EBV Rta transcriptionally upregulates the expressions of p21 and 14-3-3σ in HEp-2 cells, 293 cells and nasopharyngeal carcinoma TW01 cells. Although p21 and 14-3-3σ are known targets for p53, Rta-mediated p21 and 14-3-3σ transactivation can be detected in the absence of p53. In addition, results from luciferase reporter assays indicated that direct binding of Rta to either promoter sequences is not required for activation. On the other hand, a special class of Sp1-responsive elements was involved in Rta-mediated transcriptional activation on both promoters. Finally, Rta-induced p21 expression diminished the activity of CDK2/cyclin E complex, and, Rta-induced 14-3-3σ expression sequestered CDK1 and CDK2 in the cytoplasm. Based on these results, we hypothesize that through the disruption of CDK1 and CDK2 activities, EBV Rta might contribute to cell cycle arrest in EBV-infected epithelial cells during viral reactivation.

N-methyl-N'-nitro-N-nitrosoguanidine induces and cooperates with 12-O-tetradecanoylphorbol-1,3-acetate/sodium butyrate to enhance Epstein-Barr virus reactivation and genome instability in nasopharyngeal carcinoma cells.

Seroepidemiological studies implicate a correlation between Epstein-Barr virus (EBV) reactivation and the development of nasopharyngeal carcinoma (NPC). Moreover, N-nitroso compounds are known chemical carcinogens in preserved foodstuffs and cigarettes and have been implicated as risk factors contributing to the development of NPC. Here, NPC cell lines latently infected with EBV, NA and HA, and the corresponding EBV-negative NPC cell lines, NPC-TW01 and HONE-1, were used as the model system in this study. We demonstrate that the reactivation of EBV increases with increasing concentrations of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG at a single non-toxic concentration (0.1µg/ml) did not induce discernible reactivation of EBV, but repeated treatment with this concentration of MNNG significantly induced viral reactivation. Furthermore, low dose MNNG (0.1µg/ml) had a synergistic effect with 12-O-tetradecanoylphorbol-1,3-acetate (TPA)/sodium butyrate (SB) (10ng/ml and 0.75mM, respectively) on EBV reactivation. Through promoter activity assay, MNNG was found to enhance the transcriptional activity of Rta on Rta and Zta promoters. Using siZta to block EBV reactivation, the concomitant induction of genome instability was diminished indicating that reactivation is critical for enhancing genome instability. Co-treatment with TPA/SB and MNNG markedly increased the levels of γH2AX and ROS formation in NPC cells, which may be responsible for the increase of genome instability. Our findings offer a possible mechanism by which N-nitroso compounds induce reactivation of EBV and contribute to malignant progression by enhancing genome instability in NPC cells.

Role of the TSG101 gene in Epstein-Barr virus late gene transcription.

Rta, an Epstein-Barr virus (EBV)-encoded immediate-early protein, governs the reactivation of the viral lytic program by transactivating a cascade of lytic gene expression. Cellular transcription factors such as Sp1, ATF2, E2F, and Akt have been demonstrated to mediate Rta transactivation of lytic genes. We report herein that Rta associates with another potent transcription factor, tumor susceptibility gene 101 (TSG101), to promote the activation of EBV late genes. Results from an EBV cDNA array reveal that depletion of TSG101 by siRNA potently inhibits the transcription of five Rta-responsive EBV late genes, BcLF1, BDLF3, BILF2, BLLF1, and BLRF2. Depletion of TSG101 impairs the Rta transactivation of these late promoters severely. Moreover, a concordant augmentation of Rta transactivating activity is observed when TSG101 is overexpressed following ectopic transfection. Mechanistically, Rta interaction with TSG101 causes the latter to accumulate principally in the nuclei, wherein the proteins colocalize and are recruited to the viral promoters. Of note, TSG101 is crucial for the efficient binding of Rta to these late promoters. As a result, cells with defective TSG101 fail to express late viral proteins, leading to a decrease in the yield of virus particles. Thus, the contribution of TSG101 to Rta-mediated late gene activation is of great importance for completion of the EBV productive lytic cycle. These observations consolidate a role for TSG101 in the replication of EBV, a DNA virus, that differs from what is observed for RNA viruses, where TSG101 aids mainly in the endosomal sorting of enveloped late viral proteins for assembly at the plasma membrane.

Genome-wide transcription program and expression of the Rta responsive gene of Epstein-Barr virus.

Infection with Epstein-Barr virus (EBV) usually leads to a latent state in B lymphocytes. The virus can be reactivated through two viral transactivators, Zta and Rta, leading to a cascade of gene expression. An EBV DNA array was generated to analyze the pattern of transcription of the entire EBV genome under various conditions. Firstly, a complete set of temporal expression clusters of EBV genes was displayed by analyzing the array data of anti-IgG-induced Akata cells. In addition to assigning genes of unknown function to the various clusters, increasing expression of latent genes, including EBNA2, EBNA3A and EBNA 3C, was observed during virus replication. Secondly, gene expression independent of viral DNA replication was analyzed in PAA blocked Akata cells and in chemically induced Raji cells. Several genes with presumed late functions were found to be expressed with early kinetics and independent of viral DNA replication, suggesting possible novel functions for these genes. Finally, the EBV array was used to identify Rta responsive gene expression in Raji cells, and in the EBV-positive epithelial cells NA, using a Zta siRNA strategy. The array data were confirmed by Northern blotting, RT-PCR and reporter assays. All the information here thus provides a better understanding of the control of EBV lytic gene expression.

Characterization of three essential residues in the conserved ATP-binding region of Epstein-Barr virus thymidine kinase.

The thymidine kinase encoded by Epstein-Barr virus (EBV TK) is an important target for antiviral therapy and the treatment of EBV-associated malignancies. Through computer-assisted alignment with other human herpesviral TK proteins, EBV TK was shown to contain a conserved ATP-binding motif as for the other TK enzymes. To investigate functional roles of three highly conserved residues (G294, K297, T298) within this region, site-directed mutagenesis was employed to generate various mutants. The TK enzyme activity and ATP-binding ability of these mutant TK enzymes were determined and compared with EBV wild-type TK (wtTK). Mutant G294V lost its ATP-binding ability and was inactive in enzyme activity assay. As the enzyme activity of G294A was reduced to 20% of that of wtTK, the K(m) for ATP binding of G294A was 48.7 microM as compared with 30.0 microM of EBV wtTK. These results suggested that G294 participates in ATP binding and contributes to maintenance of structure. EBV TK mutants K297E, K297Q, and K297R lost their ATP-binding ability and enzyme activity. However, K297R was shown to have a preference for usage of GTP (K(m): 43.0 microM) instead of ATP (K(m): 87.6 microM) as the phosphate donor. This implies that, in addition to nucleotide binding, K297 was involved in the selection of phosphate donor. While EBV TK mutant T298S retained approximately 80% of wtTK enzyme activity, T298A lost its enzyme activity, suggesting that a hydroxyl group at this position is important for the enzyme activity. Interestingly, T298A retained its ATP-binding ability, suggesting a role of T298 in the catalytic process but not in the coordination of ATP. This study demonstrated that amino acid residues G294, K297, and T298 in the ATP-binding motif of EBV TK enzyme are essential for the enzymatic activity but are involved in different aspects of its action.

Reactivation of Epstein-Barr virus can be triggered by an Rta protein mutated at the nuclear localization signal.

Rta, an immediate-early protein of Epstein-Barr virus (EBV), is a transcriptional activator that induces lytic gene expression and triggers virus reactivation. Being located predominantly in the nucleus, Rta can exert its transactivation function through either direct DNA binding or certain indirect mechanisms mediated by cellular signalling and other transcriptional factors. This study examined whether the subcellular localization of Rta was critical for the induction of target genes. First, 410KRKK413 was identified as a nuclear localization signal (NLS) of Rta. An Rta mutant with the NLS converted to 410AAAA413 showed cytoplasmic localization and failed to activate the promoter of BGLF5. Interestingly, ectopic expression of the Rta mutant still disrupted EBV latency in an epithelial cell line. Reporter gene assays revealed that the NLS-mutated Rta retained the ability to activate two lytic promoters, Zp and Rp, at a considerable level. Thus, the cytoplasmic Rta mutant could induce expression of endogenous Zta and Rta, triggering reactivation of EBV.

Induction of Epstein-Barr virus latent membrane protein 1 by a lytic transactivator Rta.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a transforming protein that affects multiple cell signaling pathways and contributes to EBV-associated oncogenesis. LMP1 can be expressed in some states of EBV latency, and significant induction of full-length LMP1 is also observed frequently during virus reactivation into the lytic cycle. It is still unknown how LMP1 expression is regulated during the lytic stage and whether any EBV lytic protein is involved in the induction of LMP1. In this study, we first identified that LMP1 expression is associated with the spontaneous virus reactivation in EBV-infected 293 cells and that its expression is a downstream event of the lytic cycle. We further found that LMP1 can be induced by ectopic expression of Rta, an EBV immediate-early lytic protein. The Rta-mediated LMP1 induction is independent of another immediate-early protein, Zta. Northern blotting and reverse transcription-PCR analysis revealed that Rta upregulates LMP1 at the RNA level. Reporter gene assays further demonstrated that Rta activates both the proximal and distal promoters of the LMP1 gene in EBV-negative cells. Both the amino and carboxyl termini of the Rta protein are required for the induction of LMP1. In addition, Rta transactivates LMP1 not only in epithelial cells but also in B-lymphoid cells. This study reveals a new mechanism to upregulate LMP1 expression, expanding the knowledge of LMP1 regulation in the EBV life cycle. Considering an equivalent case of Kaposi's sarcoma-associated herpesvirus, induction of a transforming membrane protein by a key lytic transactivator during virus reactivation is likely to be a conserved event for gammaherpesviruses.

Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase.

Thymidine kinase (TK), encoded by EBV (Epstein-Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure-function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392-->Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3'-deoxy-2',3'-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3'-azido-3'-deoxythymidine, iododeoxyuridine and beta-L-5-iododioxolane uracil (L-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

Site-directed mutagenesis in a conserved motif of Epstein-Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases.

Sequence alignment of human herpesvirus DNases revealed that they share several conserved regions. One of these, the conserved motif D203...E225XK227 (D.EXK) in the sequence of Epstein-Barr virus (EBV) DNase, has a striking similarity to the catalytic sites of some other nucleases, including type II restriction endonucleases, lambda exonuclease and MutH. The predicted secondary structures of these three residues were shown to resemble the three catalytic residues of type II restriction endonucleases. Site-directed mutagenesis was carried out to replace each of the acidic residues near the motif by residues with different properties. All substitutions of D203, E225 and K227 were shown to cause significant reductions in nuclease activity. Six other acidic residues, within the conserved regions, were also replaced by Asn or Gln. Five of these six variants retained nuclease activity and mutant D195N alone lost nuclease activity. The four charged residues, D195, D203, E225 and K227, of EBV DNase were found to be important for nuclease activity. Biochemical analysis indicated that the preference for divalent cations was altered from Mg2+ to Mn2+ for mutant E225D. The DNA-binding abilities of D203E, E225D and E225Q were shown to be similar to that of wild-type. However, K227 mutants were found to have variable DNA-binding abilities: K227G and K227N mutants retained, K227E and K227D had reduced and K227R lost DNA-binding ability. Comparison of the biochemical properties of the corresponding substitutions among EBV DNase and type II restriction enzymes indicated that the D...EXK motif is most likely the putative catalytic centre of EBV DNase.

Characterization of integration patterns and flanking cellular sequences of hepatitis B virus in childhood hepatocellular carcinomas.

Hepatitis B virus (HBV) DNA integration into host chromosomes is detected in more than 80% of HBV-related hepatocellular carcinomas (HCC), yet its significance in tumor development remains obscure. In this study, we re-examined the integration pattern of HBV in childhood HCC tissues, which has less environmental confounding factors than adult HCC. The HBV junctions and flanking cellular sequences were amplified from five childhood HCC patients by the inverse polymerase chain reaction (IPCR) method using primers located near HBV direct repeats (DR) 1 and 2. The viral junctions in nine of the ten obtained IPCR clones were demonstrated to be located near HBV DR1, and their patterns were classified to type I integrants. Southern blot analyses demonstrate that the cellular junctions derived from two of the five HCC tissues were male specific and contained sequences homologous to human long interspersed DNA elements (LINE-1). HBV integrant of one HCC tissue (1217T) was integrated into a RNA binding motif Y chromosome (RBMY) gene. The expression of RBMY, which is normally found only in male germ cells, was detected in HCC tissue 1217T by RT-PCR but not in the corresponding non-tumor liver tissue. The prevalence of RBMY expression in liver tissues from the tumor and non-tumor parts of ten other HCC children and seven biliary atresia (BA) children was studied by RT-PCR. No RBMY transcripts were detected in the non-tumor parts of HCC patients or the cirrhotic livers of BA children, whereas 30% (three of ten) of HCC tissues specifically expressed RBMY. The results indicate that HBV integration and activation of RBMY gene expression in liver cells may be associated with the development of childhood HCC.

Expression of Epstein-Barr virus latent membrane protein 1 and B-cell leukemia-lymphoma 2 gene in nasopharyngeal carcinoma tissues.

Co-expression of B-cell leukemia-lymphoma 2 gene (Bcl-2) and Epstein-Barr virus latent membrane protein 1 (LMP-1) in nasopharyngeal carcinoma tissues were tested using immunohistochemical methods. Results showed that there were 32% (14/44) and 68% (30/44) LMP-1 and Bcl-2-positive cases, respectively. Among the LMP-1-positive tissues, 8 (57%) of 14 specimens were also Bcl-2-positive. The level of LMP-1 and Bcl-2 expression was associated with the clinical stages of nasopharyngeal carcinoma. Furthermore, when LMP-1- and Bcl-2-positive cases were combined, the highest positive score was found in clinical stage II as well as in the early stage (stages I and II) of nasopharyngeal carcinoma. While further studies with more cases are needed, this study suggests that co-expression of Bcl-2 and LMP-1 may be involved in the process of nasopharyngeal carcinoma aggravation.

Resistance to tumor necrosis factor-alpha-induced apoptosis in human T-lymphotropic virus type I-infected T cell lines.

Induction of apoptosis of virus-infected cells is an important host cell defense mechanism. It is well documented that T cells may undergo apoptosis due to interactions between Fas and Fas ligand (FasL). In addition, signals that induce apoptosis in T cells can result from interaction of tumor necrosis factor (TNF)-alpha with TNF receptors (TNFRs). It has been shown that human T cell lines expressing HTLV-I have decreased sensitivity to Fas-mediated apoptosis. The susceptibility of HTLV-I-infected cells to TNF-alpha-induced apoptosis remains to be elucidated. In the present study, we examined the expression of TNFRs on HTLV-I-infected T cell lines that expressed T-cell activation markers and thus phenotypically resemble activated T cells. Different from primary activated T cells that expressed both TNFRs, none of the five HTLV-I-infected T cell lines studied had detectable TNFR1 and only three had TNFR2 on their cell surfaces, although, the RNA transcripts of both TNFR genes could be detected via reverse transcription-polymerase chain reaction in these cell lines. The T cell blasts, which we activated in vitro, were sensitive to apoptosis induced by TNF-alpha and by antibodies to TNFR1 and/or TNFR2. However, all of the HTLV-I-infected cell lines expressing TNFR2 were resistant to TNF-alpha-mediated apoptosis. These findings suggest that HTLV-I infection may interfere with the autonomous suicide programs of T cells, not only Fas/FasL but also TNFRs/TNF-alpha pathways, to prolong the life of the infected cells. This may contribute to viral persistence and favor survival and subsequent expansion of dysregulated infected T cells with the potential to produce HTLV-I-associated autoimmune-like diseases or malignancies.