BMC Cancer 2006, 6:132 http://www.biomedcentral.com/1471-2407/6/132 script encoding both the mitochondrial and cytosolic iso- forms of the enzyme, while the second start site generates a shorter transcript encoding only the cytosolic protein. Under our conditions, these two major transcripts were detected only in Bp-ALL (NALM6 and REH) whereas in T- ALL (CCRF-CEM) cells only the longer transcript was detected. NALM6 cells which exhibited higher level of FPGS mRNA expressed both transcripts, while in REH and RCH-ACV which expressed intermediate levels of FPGS mRNA the longer transcript (mitochondrial/cytosolic) was either faint or absent, and in CCRF-CEM cells express- ing the lowest level of FPGS mRNA the shorter transcript (cytosolic) was absent. This finding is consistent with and underscores regulatory differences in the expression of the FPGS gene between Bp- and T-lineage ALL cells. It has also led us to hypothesize that the additional expression of the shorter transcript encoding for additional cytosolic pro- tein in NALM6 cells could contribute to higher FPGS mRNA expression leading to higher cytosolic protein expression in these cells. Although speculative at this time, this hypothesis which is currently being tested in our laboratory is consistent with the higher protein and enzy- matic activity observed in Bp-ALL vs. T-ALL. If confirmed, the responsible regulatory elements could be used as tar- gets for molecular or pharmacological interventions aimed at increasing FPGS expression and overcome de novo or acquired resistance in selected leukemic pheno- types were low FPGS expression mediates unresponsive- ness to MTX. An alternative, although unlikely explanation is that failure from our 5'-RACE conditions to amplify the short transcript or enriched amplification of the longer form in CCRF-CEM cells resulted in these find- ings. The 5'-flanking region of the downstream FPGS promoter contains eight forward/one reverse Sp1, two inverted NFY- boxes, and one E-box putative binding sites (see Figure 2) in which one NFY, one E-box, and three Sp1 binding sites are present within the FPGS minimal promoter region [17]. Functional activity of these Sp1 sites was previously demonstrated [17]. Herein, we determined that the puta- tive transcription factor binding site NFY, but not E-box, plays a positive role in FPGS transcription in both Bp- and T-lineages. Several studies have demonstrated that the CCAAT box plays a role in gene transcription by binding specific transcription factors such as C/EBP, NF-1, and NFY [24-26]. EMSA experiments confirmed that the NFY transcription factor is part of the complex that binds the CCAAT sequence within the FPGS promoter. The NFY transcription factor is involved in the regulation of many TATA-less promoter genes such as RAG-1, [22,27], EPHX1 [25], and Gfi-1B [28]. Therefore, it is not unexpected that NFY plays a role in the regulation of the TATA-less FPGS promoter. In addition, it has been shown that NFY coop- erates with many adjacent transcription factors such as GATA-1 [28,29], C/EBPα [25], and Sp1 [30], through pro- tein-protein interactions to mediate gene transcription. Ongoing studies are investigating the interactions of NFY and some of these proteins as regulators of FPGS gene expression. Within the FPGS promoter region we identified one CpG island at position -330 to +294 encompassing Sp1 and NFY sites. We found no role of promoter specific methyl- ation in differential FPGS gene expression between CCRF- CEM and NALM6 cells. Promoter regions with adjacent CpG islands were found to initiate multiple transcripts similar to those identified in FPGS transcription start sites [31]. Therefore, nuclear factor(s) such as Sp1 that binds to GC-rich motifs within the CpG island and NFY could operate in conjunction to regulate human FPGS gene tran- scription. Conclusion We demonstrated that the minimal FPGS promoter region previously described in CCRF-CEM is not sufficient to effectively drive FPGS transcription in NALM6 cells, sug- gesting that different regulatory elements are required for FPGS gene expression in Bp-cells. Our 5'-RACE analysis showed that two major transcripts encoding the mito- chondrial/cytosolic and cytosolic isoforms were expressed in Bp-ALL (NALM6 and REH) whereas T-ALL (CCRF- CEM) cells expressed only the mitochondrial/cytosolic transcript. We determined that the putative transcription factors binding site NFY, but not E-box, plays a role in FPGS transcription in both Bp- and T-lineages. Taken together, our data indicate that the control of FPGS expression in human hematopoietic cells is complex and involves lineage-specific differences in regulatory ele- ments, transcription initiation rates, and mRNA process- ing. Understanding the lineage-specific mechanisms of FPGS expression should lead to improved therapeutic strategies aimed at overcoming MTX resistance in leuke- mic cells by upregulating FPGS and increasing accumula- tion of MTX-PGs in those phenotypes with low FPGS expression. Physiologically FPGS is also known to be essential for eukaryotic cell survival although its targeted inhibition as a novel anticancer strategy has been elusive [32]. Further understanding of the genetic mechanisms that control FPGS expression could also lead to the devel- opment of novel molecular strategies capable of inducing apoptosis in leukemic cells by selectively turning off FPGS expression and could translate to better treatment out- comes for children with high-risk or refractory ALL. Competing interests The author(s) declare that they have no competing inter- ests. Page 10 of 12 (page number not for citation purposes)