We next explored the methylation status of the PAX5 promoter by using MSP (Fig. 2A). Full or partial methylation was detected in HCC cell lines (Hep3B, huH4, huH6, Mahlavu, SNU398, and PLC5), which showed silenced or down-regulated PAX5 expression, whereas methylation was not detected in the cell lines with PAX5 expression (hUH7, SNU475, RAD001 and SNU449) (Fig. 2A). The methylation density within the PAX5

promoter region was then characterized and validated by BGS (Fig. 2B). The BGS results were consistent with those of MSP in which dense methylation was found in methylated cell lines by MSP, but not in normal liver tissues (Fig. 2B). To confirm whether the promoter methylation is involved

in the silencing of PAX5, five cell lines with silenced PAX5 expression including Hep3B, HepG2, SNU387, SNU398, and PLC-5 were treated with 5-Aza combined with or without trichostatin A. This treatment resulted in the restoration of PAX5 expression in all cell lines examined (Fig. MG 132 2C), further implicating that the transcriptional silence of PAX5 was mediated by promoter methylation. The frequent inactivation of PAX5 in HCC cell lines but not in normal liver tissue suggested that PAX5 may play a role in tumor growth. We thus examined the growth-suppressive effect through ectopic expression of PAX5 in HepG2 and Hep3B, which showed no PAX5 expression. Reexpression of PAX5 in the stable transfected HepG2 and Hep3B cells was confirmed by RT-PCR (Fig. 3A1) and western blot (Fig. 3A2). Ectopic expression of PAX5 in these HCC cell lines caused a significant decrease in cell viability (Fig. 3B). The inhibitory effect on HCC cell growth was further confirmed by colony formation assay. The colonies formed in PAX5-transfected cells were significantly fewer in number and smaller in size than in empty vector-transfected

cells (down to 44%-54% of vector control, P < 0.01) (Fig. 3C). We examined the contribution of apoptosis to the observed growth inhibition in HCC cells derived by PAX5. The number of HepG2 cells with sub-G1 DNA content after PAX5 transfection was substantially increased compared with the control vector transfection learn more (24.75% ± 2.09% versus 33.11% ± 2.06%; P < 0.05). Apoptosis was further assessed by immunoblot detection of the active form of caspase-7, caspase-8, caspase-9, and poly (ADP-ribose) polymerase (PARP). As shown in Fig. 3D, overexpression of PAX5 enhanced the levels of active caspase-7, -8, -9, and PARP. The subcutaneous tumor growth curve of Hep3B stably transfected with PAX5 or empty vector in vivo is shown in Fig. 4A. The tumor volume was significantly lower in PAX5-transfected nude mice as compared to the vector control mice (P < 0.0001). At the end of experiments tumors were isolated and weighed.