The objective of this project is to characterize the clinical mechanism(s) of resistance to the anti-cancer agents etoposide (VP-16) and to implement a strategy to overcome resistance. The applicant hypothesizes that acquired resistance to VP-16 in human K562 leukemia cells is due in part to hypophosphorylation of DNA topoisomerase II alpha (topo II). The major goal is to elucidate the protein kinase signalling pathway responsible for topo II hypophosphorylation and to establish that posttranslational modification of this enzyme target is seminal to clinical VP-16 resistance. Bryostatin 1, a protein kinase C (PKC) activator, will be examined for its ability to translocate/activate nuclear PKC and subsequently upregulate topo II phosphorylation leading to increased VP-16-induced cytotoxicity in resistant K562 cells and in marrow blasts from leukemia patients who have relapsed after receiving therapy with topo II inhibitors. In preliminary results using bryostatin 1, nuclear translocation/activation of beta II PKC has been implicated in the regulation of topo II phosphorylation in resistant K562 cells. In addition, bryostatin 1 potentiates VP-16 cytotoxicity in resistant K562 cells using a clonogenic assay. Therefore, the specific aims are: 1. To further establish the role of beta II PKC and/or other protein kinases in phosphorylation of topo II in resistant K562 cells and to demonstrate that bryostatin 1 stimulates topo II phosphorylation through a beta II PKC signalling pathway; 2. To determine the ability of bryostatin 1 to upregulate topo II phosphorylation, potentiate VP-16-induced topo II/DNA complexes and increase VP-16-induced cytotoxicity in freshly obtained marrow blasts from relapse leukemia patients. Results obtained will better define posttranslational modifications of topo II responsible for VP-16 resistance and may translate to use of VP-16/bryostatin 1 combinations as an effective clinical strategy to treat drug-resistant malignancies.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
1R01CA074972-01
Application #
2372189
Study Section
Experimental Therapeutics Subcommittee 1 (ET)
Project Start
1997-09-01
Project End
2000-08-31
Budget Start
1997-09-01
Budget End
1998-08-31
Support Year
1
Fiscal Year
1997
Total Cost
Indirect Cost
Name
University of Pittsburgh
Department
Pharmacology
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Kagan, V E; Kuzmenko, A I; Shvedova, A A et al. (2003) Direct evidence for recycling of myeloperoxidase-catalyzed phenoxyl radicals of a vitamin E homologue, 2,2,5,7,8-pentamethyl-6-hydroxy chromane, by ascorbate/dihydrolipoate in living HL-60 cells. Biochim Biophys Acta 1620:72-84
Kagan, V E; Kozlov, A V; Tyurina, Y Y et al. (2001) Antioxidant mechanisms of nitric oxide against iron-catalyzed oxidative stress in cells. Antioxid Redox Signal 3:189-202
Hasinoff, B B; Abram, M E; Barnabe, N et al. (2001) The catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces differentiation and apoptosis in human leukemia K562 cells. Mol Pharmacol 59:453-61
Kagan, V E; Kuzmenko, A I; Tyurina, Y Y et al. (2001) Pro-oxidant and antioxidant mechanisms of etoposide in HL-60 cells: role of myeloperoxidase. Cancer Res 61:7777-84
Kurz, E U; Wilson, S E; Leader, K B et al. (2001) The histone deacetylase inhibitor sodium butyrate induces DNA topoisomerase II alpha expression and confers hypersensitivity to etoposide in human leukemic cell lines. Mol Cancer Ther 1:121-31
Kagan, V E; Kuzmenko, A I; Shvedova, A A et al. (2000) Myeloperoxidase-catalyzed phenoxyl radicals of vitamin E homologue, 2,2,5,7,8-pentamethyl- 6-hydroxychromane, do not induce oxidative stress in live HL-60 cells. Biochem Biophys Res Commun 270:1086-92
Hasinoff, B B; Abram, M E; Chee, G L et al. (2000) The catalytic DNA topoisomerase II inhibitor dexrazoxane (ICRF-187) induces endopolyploidy in Chinese hamster ovary cells. J Pharmacol Exp Ther 295:474-83
Yalowich, J C; Gorbunov, N V; Kozlov, A V et al. (1999) Mechanisms of nitric oxide protection against tert-butyl hydroperoxide-induced cytotoxicity in iNOS-transduced human erythroleukemia cells. Biochemistry 38:10691-8
Kagan, V E; Yalowich, J C; Borisenko, G G et al. (1999) Mechanism-based chemopreventive strategies against etoposide-induced acute myeloid leukemia: free radical/antioxidant approach. Mol Pharmacol 56:494-506
Hasinoff, B B; Chee, G L; Thampatty, P et al. (1999) The cardioprotective and DNA topoisomerase II inhibitory agent dexrazoxane (ICRF-187) antagonizes camptothecin-mediated growth inhibition of Chinese hamster ovary cells by inhibition of DNA synthesis. Anticancer Drugs 10:47-54