BMC Cancer 2006, 6:132 http://www.biomedcentral.com/1471-2407/6/132 using the Oligotex Direct mRNA mini kit (Qiagen) were reverse transcribed using the oligonucleotide Exon5R-F8 (5'-CTTGGTGAAGAGCTCAGGACTG), a gene-specific primer in exon 5. The poly dC-tailed cDNA products were amplified using a nested primer in exon 2, Exon2R-F4 (5'- ACTCCGTGCCAGGTACAGTTCCATG), and the abridged anchor primer. Primary PCR products were re-amplified using an exon 2 upstream nested primer, 2exonR-2381 (5'-CAGGTAGCCGGCATTGGTCTG), and the abridged universal amplification primer. 5'-RACE products were separated on a 2.5% agarose gel, purified and cloned into the pCR2.1-TOPO vector (Invitrogen). Identity of the 5'- RACE products was determined by nucleotide sequence. Construction of FPGS-luciferase reporter gene fusions Regions of the FPGS gene promoter were generated by PCR, cloned into pCR2.1-TOPO vector and sub-cloned into pGL3-basic vector (Promega) or pGL2628-ATGm/c. Plasmids and primers used in this study are listed in Table 1. The BAC clone RCPI-11 465E22 contains 188,098 bp of human chromosome 9 including 104,600 bp upstream of FPGS exon 1 (obtained from Dr. P.J. de Jong, Children's Hospital Oakland). PCR conditions were optimized for each of these fragments. Nucleofection and luciferase reporter gene assays The CCRF-CEM, NALM6 and REH cell lines were trans- fected by nucleofection (Amaxa Biosystems) following manufacturer's protocol. Briefly, 5 × 106 cells were resus- pended in 100 µl of solution V® (CCRF-CEM and NALM6) or R® (REH) and mixed with 2.5 µg of plasmid pGL1374 (FPGS promoter::luc) or equimolar concentration of other FPGS promoter::luc plasmids, and 3 µg of pCMVβ. Cells were incubated at 37°C/5% CO2 for 24 hrs, har- vested, washed twice with cold PBS 1X and resuspended in dual-light lysis buffer to yield cellular extracts. Luci- ferase and β-galactosidase activities were assayed using the dual-light reporter gene assay system (Tropix, Inc.). Trans- fection efficiencies and cell viability were monitored by flow cytometry. Cell viability was 70–75% and 78–85% for CCRF-CEM and NALM6 cells, respectively. Statistical tests were achieved using one-tailed, Student paired t-test (GraphPad Prism, version 2.01). Methylation-specific PCR analysis Genomic DNA was isolated from CCRF-CEM and NALM6 cells using the DNeasy kit (Qiagen, Inc.) and treated with sodium bisulfite to convert unmethylated cytosine to uracil residues while 5-methylcytosines remain unaltered (CpGenome DNA modification kit [Chemicon Interna- tional]). Identification of CpG islands and design primer sets were determined using the MethPrimer program []. Chemically converted genomic DNA was PCR amplified using specific primer sets for methylated and unmethyl- ated forms (Table 1). Methyl-specific PCR (MSP) products were resolved on a 3% agarose gel. Site-directed mutagenesis The QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene) was used to generate substitution mutants of the human FPGS mitochondrial and cytosolic initiation codons (ATG) from the pGL2628 plasmid using oligonu- cleotides QC19934F/QC19934R and QC20060F/ QC20060R, respectively (Table 1). Similarly, FPGS puta- tive NFY868 and E-box952 binding sites were muta- genized using the plasmid pGL2628-ATGm/c plasmid DNA template. Primers NFY-868F/NFY-868R and E47- 952F/E47-952R were used for the NFY and E-box muta- genesis, respectively (Table 1). Mutations were confirmed by nucleotide sequencing. Nuclear extracts and electrophoretic mobility shift assay (EMSA) Nuclear extracts were prepared from CCRF-CEM and NALM6 cells using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce, Biotechnology, Inc.). DNA- protein interactions were carried out and detected using the LightShift Chemiluminescent EMSA kit (Pierce, Bio- technology, Inc.). Each EMSA reactions contained 25 nM labeled FPGS -32/-14 (5'-CTGCGCTGATTGGCTGGGG) oligonucleotides, 50 ng Poly (dI-dC) and 10 µg of nuclear protein. When required, competitive unlabeled NFY DNA oligomer, unlabeled Epstein-Barr nuclear antigen (EBNA) DNA, and NFY antibody (CBF-A (C20) or CBF-B (H209) (Santa Cruz, Biotechnology, Inc.)) were added to EMSA. The DNA-protein complexes were resolved on non-dena- turing 5% TBE polyacrylamide gels (Bio-Rad). Results FPGS transcription rate and mRNA transcription start sites in Bp- and T-ALL To investigate whether differences in FPGS mRNA expres- sion in Bp-ALL and T-ALL resulted from differences in FPGS promoter transcription rate, we determined the fre- quency of transcription initiation in CCRF-CEM and NALM6 cells using nuclear run-on assays. As shown in Figure 1A, ratio of FPGS/18S mRNA transcription rate was 1.64-fold higher in NALM6 (2.26 ± 0.768) vs. CCRF-CEM (1.37 ± 0.416) cells (p < 0.05). These results are consistent with the observed two- to three-fold higher levels of FPGS mRNA, protein expression and activity in NALM6 vs. CCRF-CEM [9,11,15]. To uncover potential regulatory regions involved in the lineage differences in expression of the FPGS gene, we localized the FPGS promoter in Bp-ALL vs. T-ALL cell lines by mapping FPGS transcription initiation sites using 5'- RACE. These experiments detected a long (~280 bp) and a short (~180 bp) fragment, which were individually char- Page 4 of 12 (page number not for citation purposes)