Michiko Kawanishi

Combined effect of the extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli and n-butyrate on Epstein-Barr virus expression in human lymphoblastoid P3HR-1 and Raji cells

The combined usage of n-butyrate and 12-O-tetradecanoylphorbol-13-acetate (TPA) or the oily extracts from Croton tiglium, Euphorbia lathyris or Euphorbia tirucalli exerted a marked effect on induction of Epstein-Barr virus (EBV)-associated early (EA) and viral capsid (VCA) antigens in EBV genome-carrying human lymphoblastoid cell lines. In producer P3HR-1 cells, the enhancing effect of the 2 components was additive both for EA and VCA, while in non-producer Raji cells, a synergistic increase of EA was observed. The possible implication of these findings relating to the cause of EBV-associated diseases is discussed.

Generation of hydrogen peroxide precedes loss of mitochondrial membrane potential during DNA alkylation-induced apoptosis

Pulsed field gel electrophoresis showed that the initiation time of DNA breakage induced by the DNA alkylating agent duocarmycin A, which is not a redox‐cycling agent, was almost the same in the human leukemia cell line HL‐60 and its H2O2‐resistant clone HP100. Catalase activity of HP100 cells was much higher than that of HL‐60 cells. Duocarmycin A‐mediated DNA ladder formation in HP100 cells was delayed compared with that in HL‐60 cells, suggesting the involvement of H2O2 in duocarmycin A‐induced apoptosis. Flow cytometry demonstrated that peroxide formation preceded loss of mitochondrial membrane potential (ΔΨm) in cells treated with duocarmycin A. Then, caspase‐3 was activated, followed by DNA ladder formation. These findings suggest that DNA damage by duocarmycin A induces H2O2 generation, which causes ΔΨm loss and subsequently caspase‐3 activation, resulting in apoptosis.

Reactive nitrogen species-dependent DNA damage in EBV-associated nasopharyngeal carcinoma: the relation to STAT3 activation and EGFR expression.

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein‐Barr virus (EBV) infection. Recently, reactive nitrogen and oxygen species are considered to participate in inflammation‐related carcinogenesis through DNA damage. In our study, we obtained biopsy and surgical specimens of nasopharyngeal tissues from NPC patients in southern China, and performed double immunofluorescent staining to examine the formation of 8‐nitroguanine, a nitrative DNA lesion and 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine, an oxidative DNA lesion, in these specimens. Strong DNA lesions were observed in cancer cells and inflammatory cells in stroma of NPC patients. Intensive immunoreactivity of iNOS was detected in the cytoplasm of 8‐nitroguanine‐positive cancer cells. DNA lesions and iNOS expression were also observed in epithelial cells of EBV‐positive patients with chronic nasopharyngitis, although their intensities were significantly weaker than those in NPC patients. In EBV‐negative subjects, no or little DNA lesions and iNOS expression were observed. EGFR and phosphorylated STAT3 were strongly expressed in cancer cells of NPC patients, but NF‐κB was not expressed, suggesting that STAT3‐dependent mechanism is important for NPC carcinogenesis. IL‐6 was expressed mainly in inflammatory cells of nasopharyngeal tissues of EBV‐infected patients. EBV‐encoded RNAs (EBERs) and latent membrane protein 1 (LMP1) were detected in cancer cells from all EBV‐infected patients. In vitro cell system, nuclear accumulation of EGFR was observed in LMP1‐expressing cells, and IL‐6 induced phosphorylated STAT3 and iNOS. These data suggest that nuclear accumulation of EGFR and STAT3 activation byIL‐6 play the key role in iNOS expression and resultant DNA damage, leading to EBV‐mediated NPC.

Adult T cell leukemia-like disease experimentally induced in rabbits.

An HTLV‐I‐transformed T cell line, obtained from the peripheral blood of a virus‐infected (B/J × Chbb:HM) F1 rabbit, was able to kill syngeneic newborn rabbits within 7 days, when inoculated intraperitoneally at a dose of 1 × 108 cells. Inoculation of 1 × 107 cells killed or rendered moribund 50% of inoculated animals, while surviving animals exhibited cell‐mediated cytotoxic activities against the transformed cells. The peripheral blood leukocyte counts increased in all surviving animals, in association with appearance of abnormal lymphocytes with convoluted or lobulated nuclei. Pathological examination of animals that died one week post‐inoculation revealed no tumors in the abdominal cavity, but accumulation of ascites containing abnormal lymphocytes. Histological examination showed leukemic infiltration in the liver, lungs, spleen and mesenteric lymph nodes. The same cell line was also able to kill syngeneic adult rabbits in 8–10 days when inoculated intravenously, but not intraperitoneally, at a dose of 1 × 108 cells. Leukemic infiltration was observed in the major organs of these animals. Adult animals which were already virus carriers were resistant to this lethal inoculation. This rabbit ATL‐like disease may prove to be useful as an experimental model for acute adult T cell leukemia.

Epstein–Barr virus BHRF1 functions downstream of Bid cleavage and upstream of mitochondrial dysfunction to inhibit TRAIL-induced apoptosis in BJAB cells

We have previously reported that TNF-related apoptosis inducing ligand (TRAIL) causes cleavage of Bid via activation of caspase-8 and the loss of mitochondrial membrane potential (DeltaPsim), resulting in apoptosis. Experiments with BJAB clones expressing Epstein-Barr virus (EBV) anti-apoptotic protein BHRF1 showed that BHRF1 drastically inhibited TRAIL-mediated apoptosis. Although Western blot analysis demonstrated that TRAIL-induced Bid cleavage was not inhibited by BHRF1, the decrease in DeltaPsim caused by TRAIL was effectively blocked by BHRF1. These findings suggest that in BJAB cells, BHRF1 acts downstream of Bid cleavage and upstream of mitochondrial damage, resulting in inhibition of TRAIL-induced apoptosis.

Topoisomerase I and II activities are required for Epstein—Barr virus replication

The roles of topoisomerases I and II in Epstein-Barr virus (EBV) replication were investigated using Raji cells infected with EBV. The topoisomerase II inhibitor ellipticine inhibited the synthesis of EBV polypeptides at concentrations which did not affect total protein synthesis. Slot blot analysis of total cellular DNA showed that camptothecin and ellipticine inhibited replication of progeny EBV DNA in superinfected Raji cells at concentrations which did not inhibit synthesis of EBV early polypeptides prerequisite for EBV DNA replication. Analysis of the structure of EBV DNA termini demonstrated that both inhibitors affected the replicating EBV DNA. Gardella gel electrophoresis showed that both inhibitors affected the formation of the linear form of EBV DNA. However, restriction analysis of EBV DNA in superinfected Raji cells demonstrated that both inhibitors degraded neither endogenous nor exogenous EBV DNA. Cell viability was not affected by either inhibitor at the concentrations tested. These findings suggest that topoisomerase II is required for expression of the EBV genome and that both topoisomerases I and II are involved in replication of the EBV genome during the lytic phase of the life cycle. The effects of topoisomerase inhibitors on the circular form of EBV DNA during virus replication are discussed.

Epstein-Barr virus-induced polypeptides: a comparative study with superinfected Raji, IUdR-Treated, and N-butyrate-treated P3HR-1 cells.

Abstract Three Epstein-Barr virus (EBV)-induced in vitro systems, EBV-superinfected Raji, IUdR-, and n -butyrate-treated P3HR-1 cells, were studied using a radioimmunoprecipitation method, and the EBV-specific polypeptide profiles were analyzed with anti-EBV human sera. In the three cell systems, 16, 20, and 22 polypeptides were identified, respectively. All 16 polypeptides detectable in the EBV-infected Raji cells belonged to the early products insensitive to phosphonoacetic acid (PAA) and were also shared by the other two systems. Among these common polypeptides, two with molecular weights of 140,000 (140K) and 120K were identified both in the cytoplasm and in the nucleus. However, these are not the major components of EA induced in the EBV-infected Raji cells. All other 14 polypeptides were found exclusively in the cytoplasm and were precipitated by all VCA(+)EA(+) sera, indicating that all of these polypeptides are EA components and that 90K, 54K, and 38K in particular are the major constituents of the EA. The six polypeptides (150K, 145K, 75K, 46K, 34K, 18K), which were detectable in n -butyrate-treated P3HR-1 cells, and four of which are common to those in IUdR-treated P3HR-1 cells, are the late virus-specific products sensitive to PAA. In contrast to the early polypeptides, these late products were found both exclusively in the cytoplasm and the nucleus. Among these products the 150K polypeptide is probably the main component of VCA.

Nitric Oxide Inhibits Epstein-Barr Virus DNA Replication and Activation of Latent EBV

Nitric oxide (NO), a mediator of biological functions, has antimicrobial activity against a variety of pathogens including viruses. Effects of NO donors on EBV replication in two EBV lytic systems, Raji cells infected with P3HR-1 virus and P3HR-1 cells activated with TPA plus n-butyrate, were studied. S-nitroso-N-acetylpenicillamine (SNAP), which generates NO when placed in an aqueous solution, and 3-morpholinosydnonimine (SIN-1), which liberates NO and O2-, resulting in the formation of peroxynitrite, were used as NO donors. Immunoprecipitation analysis showed that in superinfected Raji cells, SNAP inhibited EBV late protein synthesis but not EBV early protein expression. Analysis of the structure of EBV DNA termini demonstrated that SNAP suppressed the amplification of EBV DNA in superinfected Raji cells at a dose which did not affect synthesis of EBV early proteins required for EBV DNA replication. In TPA plus n-butyrate-treated P3HR-1 cells, SNAP inhibited synthesis of both early and late proteins of EBV. Northern blot analysis of RNA expressed in TPA plus n-butyrate-treated P3HR-1 cells demonstrated that expression of EBV immediate-early mRNAs coded from BZLF1 and BRLF1 genes was inhibited by SNAP. SIN-1 showed no or little effect on EBV replication in both cell systems. Cell viability and cellular protein synthesis were not affected by either NO donor under the conditions used. These findings suggest that NO prevents EBV replication by inhibiting EBV DNA amplification during the lytic phase of the life cycle as well as by blocking activation of the latent EBV genome. The mechanism for inhibiting of EBV replication by NO was discussed in relation to the role of NO in EBV latency in vivo.

Epstein-Barr virus-induced early polypeptides in Raji and NC37 cells activated by diterpene ester TPA in combination with N-butyrate

Abstract Epstein-Barr virus (EBV)-induced early polypeptides in activated nonproducer Raji and NC37 cells, and such polypeptides induced in producer P3HR-1 cells were studied for comparison. The major difference was that the two polypeptides with molecular weights of 140,000 (140K) and 120,000 (120K) which are presumably involved in viral DNA synthesis in the activated producer cells, were not detectable in the activated nonproducer cells.

Effect of short-chain fatty acids on Epstein-Barr virus early and viral capsid antigen induction in P3HR-1 cells.

The effects of short-chain fatty acids were assayed for their capacity to induce Epstein-Barr virus (EBV) from the EBV genome-carrying human lymphoblastoid P3HR-1 cells. Not only the n-butyric acid, the activity of which is now well established, but also the n-valeric acid was found to induce EBV-associated early antigen (EA) and viral capsid antigen (VCA) at an appreciable level. Similarly, i-valeric acid showed a considerably lower, but significant, level of this activity, while the i-butyric acid was inactive. All other fatty acids with either a chain shorter than butyric or longer than valeric showed only a marginal effect or none at all. Thus, the decisive factor for EBV-inducing capacity of the fatty acids concerns adequate length and configuration of the basic structure of the molecules.