Abstract

FdUMP[N] compounds constitute a novel class of fluoropyrimidine (FP) chemotherapeutic that may be useful for the treatment of human malignacies that are refractory to current chemotherapy. The goal of this research project is to understand the unique cytotoxic mechanism of FdUMP[N] compounds. Human prostate cancer (PC) cells will be used as a model system to investigate FdUMP[N] cytotoxicity because of the relative sensitivity of PC cells to FdUMP[10], and because of the need for new and more effective drugs to treat late-stage PC. PC results in nearly 30,000 deaths in the U.S. each year.
Aim 1 focuses on evaluating the extent that FdUMP[N] multimers enter PC cells via active transport and identifying proteins expressed by PC cells that are involved in receptor-mediated endocytosis. Cellular uptake kinetics will be determined from the time- and concentration-dependence of radioactivity in PC cellular lysates following exposure to 32P-labeled FdUMP[N] compounds. The contribution of active transport to cellular uptake will be evaluated using metabolic inhibitors. Structure/function analyses will be performed to identify structural features of FdUMP[N] compounds that promote intracellular internalization of FdUMP[N] compounds via active transport. Protein(s) involved in the active transport of FdUMP[N] compounds will be identified by UV cross-linking, and sequences for these protein(s) will be determined using mass spectrometry.
In Aim 2, the intracellular metabolism of [6-3H]FdUMP[N] compounds to monomeric FP metabolites will be evaluated, and thymidylate synthase inhibition and nucleotide pool imbalances will be quantified.
In Aim 3, the misincorporation of FdUTP into DNA and the extent and type of DMA damage, including DNA damage resulting from topoisomerase I cleavage complex formation, will be quantified using alkaline elution, pulsed-field gel electrophoresis, and an in vivo complex of enzyme bioassay. These studies greatly enrich our understanding of the unique cytotoxic mechanism for FdUMP[N] compounds towards PC cells. ? ?

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01CA102532-04
Application #
7479667
Study Section
Drug Discovery and Molecular Pharmacology Study Section (DMP)
Program Officer
Lees, Robert G
Project Start
2005-08-10
Project End
2010-07-31
Budget Start
2008-08-01
Budget End
2009-07-31
Support Year
4
Fiscal Year
2008
Total Cost
$300,200
Indirect Cost

  Institution

Name
Wake Forest University Health Sciences
Department
Biology
Type
Schools of Medicine
DUNS #
937727907
City
Winston-Salem
State
NC
Country
United States
Zip Code
27157

  Publications

Gmeiner, William H; Boyacioglu, Olcay; Stuart, Christopher H et al. (2015) The cytotoxic and pro-apoptotic activities of the novel fluoropyrimidine F10 towards prostate cancer cells are enhanced by Zn(2+) -chelation and inhibiting the serine protease Omi/HtrA2. Prostate 75:360-9
Gmeiner, William H; Jennings-Gee, Jamie; Stuart, Christopher H et al. (2015) Thymineless death in F10-treated AML cells occurs via lipid raft depletion and Fas/FasL co-localization in the plasma membrane with activation of the extrinsic apoptotic pathway. Leuk Res 39:229-35
Gmeiner, William H; Willingham, Mark C; Bourland, J Daniel et al. (2014) F10 Inhibits Growth of PC3 Xenografts and Enhances the Effects of Radiation Therapy. J Clin Oncol Res 2:
Pardee, Timothy S; Stadelman, Kristin; Jennings-Gee, Jamie et al. (2014) The poison oligonucleotide F10 is highly effective against acute lymphoblastic leukemia while sparing normal hematopoietic cells. Oncotarget 5:4170-9
Jennings-Gee, Jamie; Pardee, Timothy S; Gmeiner, William H (2013) Replication-dependent irreversible topoisomerase 1 poisoning is responsible for FdUMP[10] anti-leukemic activity. Exp Hematol 41:180-188.e4
Huang, Shar-yin N; Murai, Junko; Dalla Rosa, Ilaria et al. (2013) TDP1 repairs nuclear and mitochondrial DNA damage induced by chain-terminating anticancer and antiviral nucleoside analogs. Nucleic Acids Res 41:7793-803
Ghosh, Supratim; Salsbury Jr, Freddie R; Horita, David A et al. (2013) Cooperative stabilization of Zn(2+):DNA complexes through netropsin binding in the minor groove of FdU-substituted DNA. J Biomol Struct Dyn 31:1301-10
Pardee, Timothy S; Gomes, Evan; Jennings-Gee, Jamie et al. (2012) Unique dual targeting of thymidylate synthase and topoisomerase1 by FdUMP[10] results in high efficacy against AML and low toxicity. Blood 119:3561-70
Gmeiner, William H; Salsbury Jr, Freddie; Olsen, Chris M et al. (2011) The stability of a model substrate for topoisomerase 1-mediated DNA religation depends on the presence of mismatched base pairs. J Nucleic Acids 2011:631372
Ghosh, Supratim; Salsbury Jr, Freddie R; Horita, David A et al. (2011) Zn2+ selectively stabilizes FdU-substituted DNA through a unique major groove binding motif. Nucleic Acids Res 39:4490-8

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