Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells - PubMed (original) (raw)

Zebularine reactivates silenced E-cadherin but unlike 5-Azacytidine does not induce switching from latent to lytic Epstein-Barr virus infection in Burkitt's lymphoma Akata cells

Sieta P Rao et al. Mol Cancer. 2007.

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.

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Figure 1

Figure 1

Response of Burkitt's lymphoma Akata cells to 5-Azacytidine and Zebularine treatment. (a, b) Determination of non-toxic concentrations for Akata treatment in vitro with 5-Azacytidine (a) and Zebularine (b). 5 × 105 viable cells were seeded in 1 ml medium with increasing 5-Azacytidine or Zebularine concentrations. Living cells (trypan-blue exclusion) were then counted after 48 h and 72 h. The highest non-toxic-concentrations are: 1 μM 5-Azacytidine (a) and 100 μM for Zebularine (b). (c, d) Expression of E-cadherin (CDH1) normalized to HMBS. Treatment with 1.0 μM 5-Azacytidine resulted in a 30-fold induction of CDH1 expression after 48 h (c), and with slower kinetics 100 μM Zebularine induced a 60-fold activation after 120 h and was sustained after 8 days of continuous Zebularine treatment (d). (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). Data in a-f are given as means ± SD from three independent experiments (g, h) Expression of early and late lytic EBV genes. Treatment with 5-Azacytidine results in increased expression of both EBV early and late antigens (g). Treatment with Zebularine did not show any significant increase of expression of early or late lytic antigens (h). (i, j) Expression of latent EBV genes LMP2A and EBNA2 increased around 10-fold upon treatment with 1.0 μM 5-Azacytidine. EBNA2 expression increased about 60-fold by 0.5 μM and 1.0 μM 5-Azacytidine at 48 h. The lowest 5-Azacytidine concentration used (0.1 μM) was also able to induce a 50-fold increase of EBNA2 expression at 72 h (i). LMP2B did show a maximal increase when treated with 1 μM 5-Azacytidine. (i). Treatment with Zebularine concentrations between 0.03 mM and 1 mM did not have a significant effect on LMP2B and EBNA2 gene expression up to 72 h (j). Data in g-j are given as means of three independent experiments.

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