The abnormal cells from patients with myelodysplastic syndromes and acute myelogenous leukemias fail to differentiate normally. A better understanding of the biochemical events regulating myeloid cell differentiation could potentially lead to more specific therapies of these disorders. The human promyelocytic cell line HL-60 provides the opportunity to study myeloid cell differentiation in vitro as it differentiates into granulocytes in response to several inducers, including dimethylsulfoxide (DMSO), and cAMP- and cGMP-elevating agents. Since cAMP and cGMP exert their effects almost exclusively through their respective protein kinases, it appears that differentiation of HL-60 cells can be induced by phosphorylation of key regulatory protein(s). The PI has recently isolated stable mutant HL-60 sublines which are resistant to the differentiating effects of elevated cGMP concentrations but differentiate normally in re- sponse to other inducing agents including DMSO and 8-Br-cAMP. Preliminary characterization of these mutants indicates a defect in phosphorylation of a cGMP-dependent protein kinase substrate (or substrates). The three major goals of this proposal are: (i) to identify the proteins phosphorylated in wild type HL-60 cells during cGMP-induced differentiation that are not phosphorylated in the mutant HL-60 cells; (ii) to purify and partially sequence one or several of these phosphoproteins; and (iii) to isolate a cDNA clone of one of the phosphoproteins. The proteins will be identified by 2-D PAGE/autoradiography and selected ones will be purified using a combination of radio-HPLC and PAGE/autoradiography. The partial amino acid sequence of the proteins will provide information about their physiological function and will allow synthesis of degenerative oligonucleotides and peptide specific antibodies. A cDNA clone and peptide-specific antibodies will allow study of the regulation of the phosphoprotein's synthesis during differentiation of HL-60 cells and of the molecular defect in the mutant HL-60 cells. The cloned gene will be transfected into the mutant cells to determine if it will correct their defect and the phosphoprotein will be microinjected into wild type HL-60 cells to determine if it is sufficient to induce differentiation. These studies will provide the PI with training in protein biochemistry and molecular biology and lay the foundation for future studies in patients with myelodysplastic syndromes and acute myelogenous leukemias.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Clinical Investigator Award (CIA) (K08)
Project #
1K08CA001548-01
Application #
3080018
Study Section
Special Emphasis Panel (SRC (B3))
Project Start
1990-08-16
Project End
1995-07-31
Budget Start
1990-08-16
Budget End
1991-07-31
Support Year
1
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of California San Diego
Department
Type
Schools of Medicine
DUNS #
077758407
City
La Jolla
State
CA
Country
United States
Zip Code
92093
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Pilz, R B; Huvar, I; Scheele, J S et al. (1997) A decrease in the intracellular guanosine 5'-triphosphate concentration is necessary for granulocytic differentiation of HL-60 cells, but growth cessation and differentiation are not associated with a change in the activation state of Ras, the transformin Cell Growth Differ 8:53-9
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Pilz, R B; Berjis, M; Idriss, S D et al. (1994) Isolation and characterization of HL-60 cells resistant to nitroprusside-induced differentiation. J Biol Chem 269:32155-61
Scheele, J S; Pilz, R B; Quilliam, L A et al. (1994) Identification of a ras-related protein in murine erythroleukemia cells that is a cAMP-dependent protein kinase substrate and is phosphorylated during chemically induced differentiation. J Biol Chem 269:18599-606
Pilz, R B (1993) Impaired erythroid-specific gene expression in cAMP-dependent protein kinase-deficient murine erythroleukemia cells. J Biol Chem 268:20252-8
Pilz, R B; Eigenthaler, M; Boss, G R (1992) Chemically induced murine erythroleukemia cell differentiation is severely impaired when cAMP-dependent protein kinase activity is repressed by transfected genes. J Biol Chem 267:16161-7
Idriss, S D; Pilz, R B; Sharma, V S et al. (1992) Studies on cytosolic guanylate cyclase from human placenta. Biochem Biophys Res Commun 183:312-20