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.