The long term objective of this application is to develop methods that enable the rapid identification of the mechanistic type and frequency of antineoplastic drug resistant cells within populations of blast cells obtained from patients with acute nonlymphocytic leukemia (ANLL). The importance of such methodologies cannot be overstated, since they will not only aid in the immediate selection of therapy, but will also give direction to future research in the design of new therapeutic approaches, aimed at overcoming those mechanisms of drug resistance identified. These studies will focus on the two most effective agents currently available for treatment of ANLL, daunorubicin (DNR) and 1-B-D-arabinofuranosylcytosine (ara-C). We will utilize state-of-the-art flow cytometric and cell sorting technology to enhance detection of drug resistant cells.
Our specific aims propose: 1) to perfect methods for detecting DNR resistance by studying factors influencing the intracellular biochemistry or pharmacology of DNR. These studies will focus on detection of markers of multidrug resistance such as P-glycoprotein and intracellular DNR concentration, as well as other biochemical parameters which may serve as markers for other forms of DNR resistance; 2) to perfect methods for detecting ara-C resistance by studying biochemical events related to ara-C metabolism and mechanism of action. To this end, we will attempt to produce a monoclonal antibody that recognizes ara-C incorporated into DNA. Nascent DNA containing ara-C, obtained by the pH-step alkaline elution method, will be used as antigen. We will also utilize the pH-step alkaline elution method to study the effects of ara-C on DNA synthesis; 3) to detect resistance to ara-C or DNR on the basis of their cytotoxicity to leukemic cells. These studies will utilize a sensitive flow cytometric viability assay we have developed that quantifies leukemic cell survival following exposure to cytotoxic drug.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
2R01CA040188-04
Application #
3179812
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1985-07-01
Project End
1991-06-30
Budget Start
1988-07-01
Budget End
1989-06-30
Support Year
4
Fiscal Year
1988
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Type
Schools of Medicine
DUNS #
003255213
City
Baltimore
State
MD
Country
United States
Zip Code
21201
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Wills, P; Hickey, R; Ross, D et al. (1996) A novel in vitro model system for studying the action of ara-C. Cancer Chemother Pharmacol 38:366-72
Lenehan, P F; Gutierrez, P L; Wagner, J L et al. (1995) Resistance to oxidants associated with elevated catalase activity in HL-60 leukemia cells that overexpress multidrug-resistance protein does not contribute to the resistance to daunorubicin manifested by these cells. Cancer Chemother Pharmacol 35:377-86
Ross, D D; Cuddy, D P (1995) Consequences of 2',2'-difluorodeoxycytidine (gemcitabine) on replicative DNA synthesis in intact HL-60 cells. Semin Oncol 22:26-34
Ross, D D; Wooten, P J; Tong, Y et al. (1994) Synergistic reversal of multidrug-resistance phenotype in acute myeloid leukemia cells by cyclosporin A and cremophor EL. Blood 83:1337-47
Ross, D D; Cuddy, D P (1994) Molecular effects of 2',2'-difluorodeoxycytidine (Gemcitabine) on DNA replication in intact HL-60 cells. Biochem Pharmacol 48:1619-30
Ross, D D; Wooten, P J; Sridhara, R et al. (1993) Enhancement of daunorubicin accumulation, retention, and cytotoxicity by verapamil or cyclosporin A in blast cells from patients with previously untreated acute myeloid leukemia. Blood 82:1288-99
Ross, D D; Cuddy, D P; Cohen, N et al. (1992) Mechanistic implications of alterations in HL-60 cell nascent DNA after exposure to 1-beta-D-arabinofuranosylcytosine. Cancer Chemother Pharmacol 31:61-70
Gervasoni Jr, J E; Fields, S Z; Krishna, S et al. (1991) Subcellular distribution of daunorubicin in P-glycoprotein-positive and -negative drug-resistant cell lines using laser-assisted confocal microscopy. Cancer Res 51:4955-63
de Valeriola, D L; Ross, D D; Forrest, A et al. (1991) Use of plasma cytotoxic activity to model cytotoxic pharmacodynamics of anticancer drugs. Cancer Chemother Pharmacol 29:133-40

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