Recombinant proteins are gaining wide use as pharmaceuticals to treat patients with cancer and its sequelae. One problem with the use of these agents is the frequency with which injections must be made in order to maintain a therapeutic level in the circulation. It has been shown that erythropoietin dimers produced by chemical crosslinking or in the form of a fusion protein have enhanced potency and efficacy in vitro and in vivo. It is proposed that the molecular, cellular and pharmacological mechanisms underlying these observations be studied in detail in vivo studies in rodents and monkeys. The interaction of erythropoietin with soluble erythropoietin receptor and the demonstration that a stoichiometry of two receptors per erythropoietin molecule exists will be documented. The principal investigator will also show that an inactive mutant erythropoietin binds only one receptor. The principal investigator will study the interaction of erythropoietin with the membrane receptor and provide direct proof of membrane receptor dimerization by erythropoietin. They will study the affinity and binding stoichiometry of crosslinked erythropoietin dimmers and the fusion protein for the soluble erythropoietin receptor and will compare them with these values for both Site 1 and Site 2 mutants. The interaction of crosslinked dimers and fusion protein with the membrane bound receptor including affinity, internalization and degradation, and demonstration of receptor tetramer formation will be studied. Enhanced signaling from these tetramers will be studied using reporter constructs induced by the Ras/MAPK and JAK/STAT pathways, respectively. The pharmacokinetics and pharmacodynamic characteristics of dimmer and fusion protein will be compared with those of the monomer in rats and monkeys. The potential immunogenicity, structure and stability of these compounds will be studied. The results of these experiments will lay the foundation for the design of novel recombinant proteins for cancer therapy.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA089204-02
Application #
6489407
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Program Officer
Forry, Suzanne L
Project Start
2001-01-05
Project End
2004-12-31
Budget Start
2002-01-01
Budget End
2002-12-31
Support Year
2
Fiscal Year
2002
Total Cost
$359,125
Indirect Cost
Name
Beth Israel Deaconess Medical Center
Department
Type
DUNS #
076593722
City
Boston
State
MA
Country
United States
Zip Code
02215
Solár, Peter; Sytkowski, Arthur J (2011) Differentially expressed genes associated with cisplatin resistance in human ovarian adenocarcinoma cell line A2780. Cancer Lett 309:11-8
Jeong, Jee-Yeong; Hoxhaj, Gerta; Socha, Amanda L et al. (2009) An erythropoietin autocrine/paracrine axis modulates the growth and survival of human prostate cancer cells. Mol Cancer Res 7:1150-7
Solar, Peter; Koval, Jan; Mikes, Jaromir et al. (2008) Erythropoietin inhibits apoptosis induced by photodynamic therapy in ovarian cancer cells. Mol Cancer Ther 7:2263-71
Solar, Peter; Feldman, Laurie; Jeong, Jee-Yeong et al. (2008) Erythropoietin treatment of human ovarian cancer cells results in enhanced signaling and a paclitaxel-resistant phenotype. Int J Cancer 122:281-8
Jeong, Jee-Yeong; Feldman, Laurie; Solar, Peter et al. (2008) Characterization of erythropoietin receptor and erythropoietin expression and function in human ovarian cancer cells. Int J Cancer 122:274-80
Feldman, Laurie; Wang, Yuxun; Rhim, Johng S et al. (2006) Erythropoietin stimulates growth and STAT5 phosphorylation in human prostate epithelial and prostate cancer cells. Prostate 66:135-45
Debeljak, Natasa; Feldman, Laurie; Davis, Kerry L et al. (2006) Variability in the immunodetection of His-tagged recombinant proteins. Anal Biochem 359:216-23
Chen, Changmin; Sytkowski, Arthur J (2005) Apoptosis-linked gene-2 connects the Raf-1 and ASK1 signalings. Biochem Biophys Res Commun 333:51-7
Chen, Changmin; Sytkowski, Arthur J (2004) Erythropoietin regulation of Raf-1 and MEK: evidence for a Ras-independent mechanism. Blood 104:73-80
Chen, C; Sytkowski, A J (2001) Erythropoietin activates two distinct signaling pathways required for the initiation and the elongation of c-myc. J Biol Chem 276:38518-26