Figure 3

Analysis of Slit-Robo pathway gene expression by RT-PCR in cervical cancer cell lines and primary tumors. A. Expression of SLIT1, SLIT2, SLIT3, and ROBO1 genes. Note the high-levels of expression in normal cervix, complete loss or down-regulated expression in the cell lines (CaSki, HT-3, SiHa, SW756, ME-180, and HeLa) and primary tumors (shown by prefix "T" for tumor). B-C. Effect of demethylation and acetylation on SLIT2, ROBO1, SLIT1, and SLIT3 genes. FANCF gene is shown as a control for reactivation of expression in SiHa and ME-180 in panel B [31]. Lanes 1, untreated; 2, 5-aza-CdR-treated (5 or 10 μM for 5 days); 3, TSA treated (100 nM for the last 24 hours); 4, 5-aza-CdR and TSA treated. Note that SLIT2 promoters were methylated in all three-cell lines but only ME-180 showed reactivation, while HT-3 showed minimal reactivation only in combined 5-aza-CdR and TSA treated cells but not with other treatments. SiHa failed to reactivate. For ROBO1 gene, ME-180 had methylated promoter and showed reactivated expression with all treatments. No reactivation of SLIT1 and SLIT3 genes in promoter methylated CaSki cell line was found. Beta actin (empty arrow) used as an internal control; Filled arrows indicate specific genes used for RT-PCR. Promoter methylation status of each gene is shown below the panels.