BMC Cancer 2006, 6:132 http://www.biomedcentral.com/1471-2407/6/132 Authors' contributions GJL conceived of the study, participated in its design, car- ried out the molecular cloning, 5'-RACE, site-directed mutagenesis, EMSA, methylation-specific PCR, transfec- tion and luciferase/β-galactosidase assays, and drafted the manuscript. GML participated in the primers design, site- directed mutagenesis studies, EMSA, and statistical analy- sis. TTHS participated in the nuclear run-on assays. JCB conceived of the study, participated in its design and coor- dination, and drafted the manuscript. All authors read and approved the final manuscript. Acknowledgements We thank Dr. Dennis K. Watson for helpful discussions, Sanja Altman- Hamamdzic for technical assistance with run-on assays, Drs. Jacqueline Kraveka and Tapas Sengupta for critical reading of the manuscript, and the Medical University South Carolina, Charleston, SC, USA, for their assist- ance with DNA sequence analysis. References 1. McGuire JJ, Bertino JR: Enzymatic synthesis and function of folylpolyglutamates. Mol Cell Biochem 1981, 38 Spec No(Pt 1):19-48. 2. McGuire JJ, Hsieh P, Coward JK, Bertino JR: Enzymatic synthesis of folylpolyglutamates. Characterization of the reaction and its products. J Biol Chem 1980, 255(12):5776-5788. 3. Moran RG, Colman PD, Rosowsky A, Forsch RA, Chan KK: Struc- tural features of 4-amino antifolates required for substrate activity with mammalian folylpolyglutamate synthetase. Mol Pharmacol 1985, 27(1):156-166. 4. Synold TW, Willits EM, Barredo JC: Role of folylpolygutamate synthetase (FPGS) in antifolate chemotherapy; a biochemi- cal and clinical update. Leuk Lymphoma 1996, 21(1-2):9-15. 5. Baldwin SW, Tse A, Gossett LS, Taylor EC, Rosowsky A, Shih C, Moran RG: Structural features of 5,10-dideaza-5,6,7,8-tet- rahydrofolate that determine inhibition of mammalian glyci- namide ribonucleotide formyltransferase. Biochemistry 1991, 30(7):1997-2006. 6. Jackman AL, Taylor GA, Gibson W, Kimbell R, Brown M, Calvert AH, Judson IR, Hughes LR: ICI D1694, a quinazoline antifolate thymidylate synthase inhibitor that is a potent inhibitor of L1210 tumor cell growth in vitro and in vivo: a new agent for clinical study. Cancer Res 1991, 51(20):5579-5586. 7. Rots MG, Willey JC, Jansen G, Van Zantwijk CH, Noordhuis P, DeMuth JP, Kuiper E, Veerman AJ, Pieters R, Peters GJ: mRNA expression levels of methotrexate resistance-related pro- teins in childhood leukemia as determined by a standardized competitive template-based RT-PCR method. Leukemia 2000, 14(12):2166-2175. 8. Barredo J, Moran RG: Determinants of antifolate cytotoxicity: folylpolyglutamate synthetase activity during cellular prolif- eration and development. Mol Pharmacol 1992, 42(4):687-694. 9. Barredo JC, Synold TW, Laver J, Relling MV, Pui CH, Priest DG, Evans WE: Differences in constitutive and post-methotrexate folyl- polyglutamate synthetase activity in B-lineage and T-lineage leukemia. Blood 1994, 84(2):564-569. 10. Egan MG, Sirlin S, Rumberger BG, Garrow TA, Shane B, Sirotnak FM: Rapid decline in folylpolyglutamate synthetase activity and gene expression during maturation of HL-60 cells. Nature of the effect, impact on folate compound polyglutamate pools, and evidence for programmed down-regulation during mat- uration. J Biol Chem 1995, 270(10):5462-5468. 11. Galpin AJ, Schuetz JD, Masson E, Yanishevski Y, Synold TW, Barredo JC, Pui CH, Relling MV, Evans WE: Differences in folylpolygluta- mate synthetase and dihydrofolate reductase expression in human B-lineage versus T-lineage leukemic lymphoblasts: mechanisms for lineage differences in methotrexate poly- glutamylation and cytotoxicity. Mol Pharmacol 1997, 52(1):155-163. 12. Chen L, Qi H, Korenberg J, Garrow TA, Choi YJ, Shane B: Purifica- tion and properties of human cytosolic folylpoly-gamma- glutamate synthetase and organization, localization, and dif- ferential splicing of its gene. J Biol Chem 1996, 271(22):13077-13087. 13. Freemantle SJ, Taylor SM, Krystal G, Moran RG: Upstream organ- ization of and multiple transcripts from the human folylpoly- gamma-glutamate synthetase gene. J Biol Chem 1995, 270(16):9579-9584. 14. Turner FB, Taylor SM, Moran RG: Expression patterns of the multiple transcripts from the folylpolyglutamate synthetase gene in human leukemias and normal differentiated tissues. J Biol Chem 2000, 275(46):35960-35968. 15. Leclerc GJ, Barredo JC: Folylpoly-gamma-glutamate synthetase gene mRNA splice variants and protein expression in pri- mary human leukemia cells, cell lines, and normal human tis- sues. Clin Cancer Res 2001, 7(4):942-951. 16. Turner FB, Andreassi 2nd JL, Ferguson J, Titus S, Tse A, Taylor SM, Moran RG: Tissue-specific expression of functional isoforms of mouse folypoly-gamma-glutamae synthetase: a basis for tar- geting folate antimetabolites. Cancer Res 1999, 59(24):6074-6079. 17. Freemantle SJ, Moran RG: Transcription of the human folylpoly- gamma-glutamate synthetase gene. J Biol Chem 1997, 272(40):25373-25379. 18. Leclerc GJ, Leclerc GM, Barredo JC: Real-time RT-PCR analysis of mRNA decay: half-life of Beta-actin mRNA in human leukemia CCRF-CEM and Nalm-6 cell lines. Cancer Cell Int 2002, 2(1):1. 19. Hanson RD, Connolly NL, Burnett D, Campbell EJ, Senior RM, Ley TJ: Developmental regulation of the human cathepsin G gene in myelomonocytic cells. J Biol Chem 1990, 265(3):1524-1530. 20. Antequera F, Boyes J, Bird A: High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell 1990, 62(3):503-514. 21. Razin A, Cedar H: DNA methylation and gene expression. Microbiol Rev 1991, 55(3):451-458. 22. Brown ST, Miranda GA, Galic Z, Hartman IZ, Lyon CJ, Aguilera RJ: Regulation of the RAG-1 promoter by the NF-Y transcrip- tion factor. J Immunol 1997, 158(11):5071-5074. 23. Nomura M, Bartsch S, Nawata H, Omura T, Morohashi K: An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development. J Biol Chem 1995, 270(13):7453-7461. 24. Chodosh LA, Baldwin AS, Carthew RW, Sharp PA: Human CCAAT-binding proteins have heterologous subunits. Cell 1988, 53(1):11-24. 25. Zhu QS, Qian B, Levy D: CCAAT/enhancer-binding protein alpha (C/EBPalpha) activates transcription of the human microsomal epoxide hydrolase gene (EPHX1) through the interaction with DNA-bound NF-Y. J Biol Chem 2004, 279(29):29902-29910. 26. Mantovani R: The molecular biology of the CCAAT-binding factor NF-Y. Gene 1999, 239(1):15-27. 27. Kurioka H, Kishi H, Isshiki H, Tagoh H, Mori K, Kitagawa T, Nagata T, Dohi K, Muraguchi A: Isolation and characterization of a TATA-less promoter for the human RAG-1 gene. Mol Immu- nol 1996, 33(13):1059-1066. 28. Huang DY, Kuo YY, Lai JS, Suzuki Y, Sugano S, Chang ZF: GATA-1 and NF-Y cooperate to mediate erythroid-specific transcrip- tion of Gfi-1B gene. Nucleic Acids Res 2004, 32(13):3935-3946. 29. Cassel DL, Subudhi SK, Surrey S, McKenzie SE: GATA and NF-Y participate in transcriptional regulation of FcgammaRIIA in megakaryocytic cells. Blood Cells Mol Dis 2000, 26(6):587-597. 30. Liang F, Schaufele F, Gardner DG: Functional interaction of NF- Y and Sp1 is required for type a natriuretic peptide receptor gene transcription. J Biol Chem 2001, 276(2):1516-1522. 31. Cross SH, Bird AP: CpG islands and genes. Curr Opin Genet Dev 1995, 5(3):309-314. 32. Rosowsky A: Chemistry and biological activity of antifolates. Prog Med Chem 1989, 26:1-252. 33. MethPrimer - Design Primers for Methylation PCRs [http:// urogene.org/methprimer/index1.html] 34. Genomatix, MatInspector program [http://www.genomatix.de/ cgi-bin/tools/tools.pl] Page 11 of 12 (page number not for citation purposes)