The overall significance of this project relates to the ability to imbalance the DNA base excision repair (BER) pathway in ovarian tumor ceils, increasing their sensitivity to chemotherapeutic and ionizing radiation (IR) agents. We will attempt to accomplish this goal using mutants of the human apurinic/apyrimidinic endonuclease (APE1) enzyme, overexpression of N-methylpurine DNA glycosylase (MPG), both targeted to the nucleus and mitochondria, as well as small (short) interfering RNA (siRNA) for APE1. We will also utilize folic acid-derivatized liposomes and adenoviral targeting along with tumor specific promoter expression using the human telomerase reverste transcriptase (hTERT) promoter in both cell lines and an NOD/SCID animal model to develop the usefulness of this approach. Hypothesis: Overexpression of MPG in the nucleus and/or mitochondrial compartments, altered human APE1 proteins (dominant-negative), or siRNA for APE1 either independently, or in various combinations will enhance ovarian cancer cells to standard or decreased levels of commonly used chemotherapeutic agents (e.g. alkylators) and/or IR.
The Specific Aims are:
Specific Aim 1 :
This first aim i ncludes determining the effectiveness of overexpressing MPG or dominant-negative APE1 in multiple ovarian cancer lines and evaluating tumor cell response to chemotherapeutic and IR treatment. This includes both nuclear and mitochondrial targeting of the MPG enzyme and overexpression and the knockdown of APE1 with siRNA.
Specific Aim 2 : Determine the effects of co-overexpression of nucMPG, mitoMPG and nuclMPG+mitoMPG, nucMPG+APE1 mutant, mitoMPG+APE1 mutant and nucMPG or mitoMPG and APEI-siRNA. We will monitor whether combined expression enhances the tumor cell killing effect of chemotherapeutic agents or IR.
Specific Aim 3 : Constructs using the hTERT promoter will be used in ovarian cancer cell lines in both plasmid (folic acid-derivatized liposome) and adenoviral based delivery systems for tumor specific expression studies using best candidate APE1 mutants, nuc- or mitoMPG, or siRNA as determined by the results in Aims 1-2.
Specific Aim 4 : Determine in vivo chemo- and radiosensitivity due to the expression of the various constructs of APE1 mutants, or nuc-/mitoMPG as well as APEI-siRNA in NOD/SCID mice. Adenoviral constructs with the hTERT promoter as well as folic acid-derivatized liposomes containing selected genes from the first three aims will be used with xenograft NOD/SCID mice. If successful, we feel these studies will create very effective reagents in a therapeutic gene transfer/therapy setting in the clinic, as well as shed light on the role of both nuclear and mitochondrial BER in cancer cells.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA106298-05
Application #
7413989
Study Section
Special Emphasis Panel (ZRG1-TPM (02))
Program Officer
Arya, Suresh
Project Start
2004-05-01
Project End
2010-04-30
Budget Start
2008-05-01
Budget End
2010-04-30
Support Year
5
Fiscal Year
2008
Total Cost
$285,864
Indirect Cost
Name
Indiana University-Purdue University at Indianapolis
Department
Pediatrics
Type
Schools of Medicine
DUNS #
603007902
City
Indianapolis
State
IN
Country
United States
Zip Code
46202
Kelley, Mark R; Georgiadis, Millie M; Fishel, Melissa L (2012) APE1/Ref-1 role in redox signaling: translational applications of targeting the redox function of the DNA repair/redox protein APE1/Ref-1. Curr Mol Pharmacol 5:36-53
Jiang, Aihua; Gao, Hua; Kelley, Mark R et al. (2011) Inhibition of APE1/Ref-1 redox activity with APX3330 blocks retinal angiogenesis in vitro and in vivo. Vision Res 51:93-100
Vascotto, Carlo; Bisetto, Elena; Li, Mengxia et al. (2011) Knock-in reconstitution studies reveal an unexpected role of Cys-65 in regulating APE1/Ref-1 subcellular trafficking and function. Mol Biol Cell 22:3887-901
Onyango, David O; Naguleswaran, Arunasalam; Delaplane, Sarah et al. (2011) Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii. DNA Repair (Amst) 10:466-75
Rohrabaugh, Sara L; Hangoc, Giao; Kelley, Mark R et al. (2011) Mad2 haploinsufficiency protects hematopoietic progenitor cells subjected to cell-cycle stress in vivo and to inhibition of redox function of Ape1/Ref-1 in vitro. Exp Hematol 39:415-23
Jedinak, Andrej; Dudhgaonkar, Shailesh; Kelley, Mark R et al. (2011) Apurinic/Apyrimidinic endonuclease 1 regulates inflammatory response in macrophages. Anticancer Res 31:379-85
Kelley, Mark R; Luo, Meihua; Reed, April et al. (2011) Functional analysis of novel analogues of E3330 that block the redox signaling activity of the multifunctional AP endonuclease/redox signaling enzyme APE1/Ref-1. Antioxid Redox Signal 14:1387-401
Luo, Meihua; He, Hongzhen; Kelley, Mark R et al. (2010) Redox regulation of DNA repair: implications for human health and cancer therapeutic development. Antioxid Redox Signal 12:1247-69
Nyland, Rodney L; Luo, Meihua; Kelley, Mark R et al. (2010) Design and synthesis of novel quinone inhibitors targeted to the redox function of apurinic/apyrimidinic endonuclease 1/redox enhancing factor-1 (Ape1/ref-1). J Med Chem 53:1200-10
Fishel, Melissa L; Colvin, E Scott; Luo, Meihua et al. (2010) Inhibition of the redox function of APE1/Ref-1 in myeloid leukemia cell lines results in a hypersensitive response to retinoic acid-induced differentiation and apoptosis. Exp Hematol 38:1178-88

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