BMC Cancer 2006, 6:132 http://www.biomedcentral.com/1471-2407/6/132 FFPigGuSrepr3omoter-luciferase constructs and activity in CCRF-CEM and NALM6 cells FPGS promoter-luciferase constructs and activity in CCRF-CEM and NALM6 cells. Genetic organization of the FPGS promoter-luciferase constructs is shown with their normalized luciferase/β-galactosidase activity in CCRF-CEM and NALM6 cells. Horizontal lines represent fragments cloned into pGL3 (luc) vector. Size of the fragments is indicated. Pointed boxes represent exons, and arrows direction of gene expression. Exponential cultures of CCRF-CEM and NALM6 cells (5 × 106) were transfected by nucleofection with equimolar concentration of pGL1374/pCMVβ and other FPGS promoter::luc con- structs as described in Materials and Methods. Luciferase activity (RLU; relative light units) was initially subtracted from the level of luciferase activity detected with pGL3 (empty vector) and normalized with respect to the level of β-galactosidase activ- ity (RLU). Each experiment was performed at least two times in triplicate. Bars represent mean ± SD. See details in Materials and methods. of luciferase/β-galactosidase activity in CCRF-CEM com- pared to NALM6 cells (Figure 3). To validate our reporter gene assay and rule out any technical differences in luci- ferase activity in NALM6 vs. CCRF-CEM cells, we assayed a CMV promoter-luciferase driven vector (pCMV-luc) and found no significant differences in both cell lines (data not shown). Therefore, these data indicate that DNA frag- ments containing the previously described minimal pro- moter region are not sufficient to effectively drive FPGS transcription in Bp-ALL (NALM6) compared to T-ALL (CCRF-CEM). Similar experiments with other Bp-ALL cell lines such as RCH-ACV (t(1:19)) and REH (t(12;21)) were performed and yielded same results (data not shown). To exclude that differences in nucleotide sequence were responsible for low promoter activity observed in Bp-ALL, we PCR amplified and sequenced the 1374 bp fragment containing the minimal promoter from normal bone marrow (BM), CCRF-CEM and NALM6 cells. No differ- ence in nucleotide sequence was detected. The inability of the minimal promoter region to effi- ciently drive FPGS transcription in NALM6 cells implied that additional unidentified regulatory regions may be required in Bp-ALL cells. To test this hypothesis we then investigated the presence of enhancers or transcription factors required for FPGS transcription in NALM6 vs. CCRF-CEM cells, by analyzing the effect in cis of DNA fragments located within the exons A1b-1 and in the 3'- UTR of the FPGS gene on the FPGS-luc expression of pGL2628-ATGm/c. As shown in Figure 3, plasmids pGL2299-2628, pGL2158-2628, pGL1163-2628, and pGL2881-2628 constructs yielded 1.8- to 4.2-fold lower level of luciferase activity in NALM6 vs. CCRF-CEM cells. Therefore, the regulatory elements required for FPGS gene expression in Bp-ALL appear not to be localized within a region encompassing 12 kb upstream of exon 1 nor within the 3'-untranslated region. Methylation status of the FPGS promoter in Bp- and T- lineage DNA methylation has been associated with transcrip- tional inactivation and gene silencing [20,21]. Nucleotide sequence analysis of the 1374 bp fragment (-791 to +582) containing the FPGS minimal promoter predicted one CpG island (54% GC content) at position -330 to +294. We determined whether DNA methylation could contrib- ute to the lineage-specific differences in FPGS expression in ALL cell lines by methylation-specific PCR (MSP) anal- ysis using bisulfite-treated DNA. Primers sets were designed to anneal to unmethylated DNA (U) and meth- ylated templates (M). As shown in Figure 4A, amplifica- tion products (142 bp) were detected in both CCRF-CEM Page 7 of 12 (page number not for citation purposes)