. The availability of recombinant colony-stimulating factors (CSFs) for clinical use has presented medical science with its first opportunity to stimulate blood cell formation and host defense in man. Potential benefits of CSF administration include treating primary hematologic diseases, enhancing host defense against infection, and preventing chemotherapy-induced cytopenias. Elucidating the role colony-stimulating factors play in normal hematopoiesis, inflammation and leukemia cell growth requires a thorough understanding of the mechanisms by which cells control their production of CSFs. Studies on the inducible expression of GM-CSF in T cells and the constitutive expression of GM-CSF in HTLV infected T cell lines have identified a critical region of the GM-CSF promoter required for its mitogen-inducible activity and its responsiveness to the HTLV-I and -II tax proteins. In this grant we propose to investigate the DNA-protein interactions in this region to understand the role that expression of sequence-specific DNA-binding proteins play in the normal and abnormal production of hematopoietic growth factors. We will identify the precise nucleotide sequence recognized by GM-CSF promoter DNA-binding proteins by mutating specific nucleotides in the promoter and analyzing their effect on the binding of transcription factors using gel shift and DNase I footprinting assays. The proteins binding specifically to the GM-CSF promoter sequences will be characterized using Southwestern blotting. UV cross-linking and biochemical manipulations designed to identify post-transcriptional protein modifications. We will use these techniques to determine the presence and activity of these factors in nuclear extracts prepared from stimulated and unstimulated mature T cell lines and from a variety of immature or transformed cell lines. T cell DNA-binding proteins will be purified using DNA affinity chromatography and amino acid sequencing will enable us to design oligonucleotides for cloning DNA-binding factors from a T cell cDNA library. We will obtain polyclonal antisera for screening our lambda gtll T cell cDNA expression library and will also use radiolabelled DNA recognition site probes to isolate transcription factor cDNA clones. The studies proposed will provide valuable insights into the transcriptional mechanisms controlling hematopoiesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK043025-05
Application #
3244279
Study Section
Hematology Subcommittee 2 (HEM)
Project Start
1990-09-01
Project End
1995-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
5
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Sloan-Kettering Institute for Cancer Research
Department
Type
DUNS #
064931884
City
New York
State
NY
Country
United States
Zip Code
10065
Wang, Lan; Huang, Gang; Zhao, Xinyang et al. (2009) Post-translational modifications of Runx1 regulate its activity in the cell. Blood Cells Mol Dis 43:30-4
Moldenhauer, Anja; Frank, Richard C; Pinilla-Ibarz, Javier et al. (2004) Histone deacetylase inhibition improves dendritic cell differentiation of leukemic blasts with AML1-containing fusion proteins. J Leukoc Biol 76:623-33
Burns, Caroline Erter; DeBlasio, Tony; Zhou, Yi et al. (2002) Isolation and characterization of runxa and runxb, zebrafish members of the runt family of transcriptional regulators. Exp Hematol 30:1381-9
Mulloy, James C; Cammenga, Jorg; MacKenzie, Karen L et al. (2002) The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells. Blood 99:15-23
MacGrogan, D; Alvarez, S; DeBlasio, T et al. (2001) Identification of candidate genes on chromosome band 20q12 by physical mapping of translocation breakpoints found in myeloid leukemia cell lines. Oncogene 20:4150-60
Alvarez, S; MacGrogan, D; Calasanz, M J et al. (2001) Frequent gain of chromosome 19 in megakaryoblastic leukemias detected by comparative genomic hybridization. Genes Chromosomes Cancer 32:285-93
Jakubowiak, A; Pouponnot, C; Berguido, F et al. (2000) Inhibition of the transforming growth factor beta 1 signaling pathway by the AML1/ETO leukemia-associated fusion protein. J Biol Chem 275:40282-7
Klampfer, L; Cammenga, J; Wisniewski, H G et al. (1999) Sodium salicylate activates caspases and induces apoptosis of myeloid leukemia cell lines. Blood 93:2386-94
Frank, R C; Sun, X; Berguido, F J et al. (1999) The t(8;21) fusion protein, AML1/ETO, transforms NIH3T3 cells and activates AP-1. Oncogene 18:1701-10
Mao, S; Frank, R C; Zhang, J et al. (1999) Functional and physical interactions between AML1 proteins and an ETS protein, MEF: implications for the pathogenesis of t(8;21)-positive leukemias. Mol Cell Biol 19:3635-44

Showing the most recent 10 out of 25 publications