Abstract
Epigenetic silencing of regulatory genes by aberrant methylation contributes to tumorigenesis. DNA methyltransferase inhibitors (DNMTI) represent promising new drugs for anti-cancer therapies. The DNMTI 5-Azacytidine is effective against myelodysplastic syndrome, but induces switching of latent to lytic Epstein-Barr virus (EBV) in vitro and results in EBV DNA demethylation with the potential of induction of lytic EBV in vivo. This is of considerable concern given that recurrent lytic EBV has been linked with an increased incidence of EBV-associated lymphomas. Based on the distinct properties of action we hypothesized that the newer DNMTI Zebularine might differ from 5-Azacytidine in its potential to induce switching from latent to lytic EBV. Here we show that both 5-Azacytidine and Zebularine are able to induce expression of E-cadherin, a cellular gene frequently silenced by hypermethylation in cancers, and thus demonstrate that both DNMTI are active in our experimental setting consisting of EBV-harboring Burkitt's lymphoma Akata cells. Quantification of mRNA expression of EBV genes revealed that 5-Azacytidine induces switching from latent to lytic EBV and, in addition, that the immediate-early lytic infection progresses to early and late lytic infection. Furthermore, 5-Azacytidine induced upregulation of the latent EBV genes LMP2A, LMP2B, and EBNA2 in a similar fashion as observed following switching of latent to lytic EBV upon cross-linking of the B-cell receptor. In striking contrast, Zebularine did not exhibit any effect neither on lytic nor on latent EBV gene expression. Thus, Zebularine might be safer than 5-Azacytidine for the treatment of cancers in EBV carriers and could also be applied against EBV-harboring tumors, since it does not induce switching from latent to lytic EBV which may result in secondary EBV-associated malignancies.
Highlights
Abnormal hypermethylation of the promoters of cancerrelated or tumor suppressor genes is commonly found in primary neoplasms and tumor cell lines [1]
Different types of cancers including Burkitt's lymphoma (BL) and nasopharyngeal carcinoma (NPC) harbor latent Epstein Barr virus (EBV) [7] and maintenance of latent Epstein-Barr virus (EBV) is partially mediated by hypermethylation of the EBV genome
It is not surprising that 5-Azacytidine induces switching of latent to lytic EBV in vitro [8,9,10,11] and results in EBV DNA demethylation in NPC patients with the potential of induction of lytic EBV [12]
Summary
Abnormal hypermethylation of the promoters of cancerrelated or tumor suppressor genes is commonly found in primary neoplasms and tumor cell lines [1]. To determine the effects of 5-Azacytidine and Zebularine on switching latent to lytic EBV in Akata cells, we measured the mRNA expression of BZLF1 by qRT-PCR in Akata cells treated with increasing concentrations of 5-Azacytidine at 48 h and 72 h and for Zebularine at up to 10 days. (e, f) BZLF1 expression indicating initiation of lytic EBV was observed after 1 μM 5-Azacytidine treatment (e), while 30 μM or 100 μM Zebularine do not activate BZLF1 expression, even after 8 days (f).
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