H2O2 inhibits BCR-dependent immediate early induction of EBV genes in Burkitt's lymphoma cells.

The critical step in the Epstein-Barr virus (EBV) transition from latency to lytic replication is activation of the viral immediate early (IE) genes, BZLF1 and BRLF1. Their induction in Burkitt's lymphoma Akata cells is directly targeted by B cell receptor (BCR) signaling. On the other hand, BCR stimulation causes an outwardly directed superoxide (O(2)(*-)) burst leading to massive generation of reactive oxygen species in the cell environment. Our goal was to investigate the role of BCR-related redox changes in the IE reactivation of EBV. Production of O(2)(*-) by stimulated Akata cells was characterized using chemiluminescent dyes, lucigenin, MCLA, and coelenterazine. Expression of the EBV IE genes was analyzed by real-time PCR and Western blot assays. Catalase activity and H(2)O(2) concentration were evaluated using Amplex Red assays and by measuring light absorption at 240 nm. We show here that elevation of H(2)O(2) concentration in Akata cell suspensions inhibits the induction of the virus IE mRNA and BZLF1 protein. It was further found that Akata cells exhibit catalase-like activity that is stimulated by BCR cross-linking. The results reveal that H(2)O(2) is instrumental in the maintenance of EBV latency. Altogether they provide new evidence demonstrating the essential role of H(2)O(2) in BCR signaling.

Resveratrol-induced modification of polyamine metabolism is accompanied by induction of c-Fos.

The objective of the current study was to investigate the effect of resveratrol, a naturally occurring polyphenol with cancer chemopreventive properties, on polyamine metabolism in the human colonic adenocarcinoma cell line Caco-2. We demonstrated that inhibition of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine biosynthesis, was due to attenuated ODC protein and mRNA levels (50-200 microM). The naturally occurring resveratrol analog piceatannol (100 microM) also diminished ODC activity, protein and mRNA levels, whereas the green tea polyphenol (-)-epigallocatechin gallate (EGCG; 100 microM) exerted only weak effects on ODC. The transcription factor c-Myc, a positive regulator of the odc gene was attenuated by resveratrol treatment and to a lesser extent by piceatannol and EGCG. S-Adenosylmethionine decarboxylase, an enzyme that synthesizes higher polyamines, was concomitantly inhibited by resveratrol and piceatannol treatment, whereas EGCG did not affect its activity. In addition resveratrol, piceatannol and EGCG enhanced spermidine/spermine N(1)-acetyltransferase activity, an enzyme that degrades polyamines in cooperation with polyamine oxidase. Intracellular levels of spermine and spermidine were not affected, whereas putrescine and N(8)-acetylspermidine concentrations increased after incubation with resveratrol. These events were paralleled by an increase of the activator protein-1 constituents c-Fos and c-Jun. Whereas DNA-binding activity of c-Jun remained unchanged, DNA-binding activity of c-Fos was significantly enhanced by resveratrol and piceatannol, but inhibited by EGCG. The data suggest that growth arrest by resveratrol is accompanied by inhibition of polyamine synthesis and increased polyamine catabolism. C-Fos seems to play a role in this context. Effects of piceatannol on polyamine synthesis were similar, but not as potent as those exerted by resveratrol.

Insufficiently charged isosteric analogue of spermine: interaction with polyamine uptake, and effect on Caco-2 cell growth.

We characterised a novel, charge-insufficient isosteric analogue of spermine, 11-[(amino)oxy]-4,9-diaza-1-aminoundecane (AOSPM). This analogue was synthesised by displacing aminopropyl group by aminooxyethyl group, the latter having pK(a) of about 5. Charge deficiency of the AOSPM molecule was fixed at a definite atom, while pK(a) of the rest nitrogen was similar to the parent polyamine. AOSPM competed with putrescine, spermidine and spermine for the uptake into the cell, and was accumulated in the cells in high amounts when exogenous polyamine synthesis was impaired. It was not recognised by the cells as growth-promoting polyamine, since it was unable to restore growth arrest due to polyamine deprivation. Like natural spermine, this polyamine analogue prevented oxidative DNA damage. AOSPM could be used not only as a tool to study polyamine homeostasis in the cell, but may have distinct applications either as radiation protector, a stable and non-toxic inhibitor of polyamine uptake or, as an appropriate vector, to enhance the uptake of impermeable compounds into the cell.