The major focus of this proposal is the elucidation of the mechanisms of action of cytosine arabinoside, ara C. This drug, the single most effective agent in the treatment of AML, appears to exert most of its actions through fraudulent incorporation into DNA. We will attempt to define in precise molecular terms the effects of this misincorporation on the functions of DNA; better define the nature and locus of ara C misincorporation; further explore the intermediary metabolism of the principal metabolite uracil arabinoside (ara U); and finally study which of several biochemical parameters may be predictors of clinical response to the drug. The role of DNA in controlling the life and growth of cells is so central that any derangement is likely to be damaging or lethal. The complex, interrelated functions of DNA make assessment of the impact of misincorporated ara C on any one function of DNA in living cells highly problematic. We have thus taken the approach of using synthetic DNA fragments as simplified in vitro models. We have developed methodology for the chemical synthesis of DNA oligomers containing ara C. We will utilize these oligomers for in vitro assays of DNA functions including chain elongation, replication fidelity, chemical stability, fragment ligation, and as a substrate for 3' 5' exonuclease activity. We will look for evidence of sequence specificity at the nearest neighbor and short sequence levels of DNA organization. We will also look for evidence of removal of ara C from DNA by excision repair mechanisms and for its spontaneous deamination to ara U. Although ara U is usually considered an inactive metabolite of ara C, reports indicate that it may have some activity and that it may modulate the activity of ara C. We will examine whether ara UTP cn serve as a substrate for dUTPase, and whether ara U can be excised from DNA by the uracil DNA glycosylase system. During our clinical treatment program for childhood acute myelogenous leukemia we will study the efficacy of ara C as a single agent in producing early cell kill. We will measure biochemical parameters related to the metabolism of ara C and subsequently look for correlations between these parameters in each patient and the degree of early cell kill produced in that patient.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA042300-02
Application #
3183389
Study Section
Experimental Therapeutics Subcommittee 2 (ET)
Project Start
1985-07-01
Project End
1987-02-28
Budget Start
1986-03-01
Budget End
1987-02-28
Support Year
2
Fiscal Year
1986
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Marshalko, S J; Schweitzer, B I; Beardsley, G P (1995) Chiral chemical synthesis of DNA containing (S)-9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) and effects on thermal stability, duplex structure, and thermodynamics of duplex formation. Biochemistry 34:9235-48
Mikita, T; Beardsley, G P (1994) Effects of arabinosylcytosine-substituted DNA on DNA/RNA hybrid stability and transcription by T7 RNA polymerase. Biochemistry 33:9195-208
Schweitzer, B I; Mikita, T; Kellogg, G W et al. (1994) Solution structure of a DNA dodecamer containing the anti-neoplastic agent arabinosylcytosine: combined use of NMR, restrained molecular dynamics, and full relaxation matrix refinement. Biochemistry 33:11460-75
Sokoloski, J A; Beardsley, G P; Sartorelli, A C (1989) Induction of HL-60 leukemia cell differentiation by the novel antifolate 5,10-dideazatetrahydrofolic acid. Cancer Res 49:4824-8
Mikita, T; Beardsley, G P (1988) Functional consequences of the arabinosylcytosine structural lesion in DNA. Biochemistry 27:4698-705
Spigelman, Z; Duff, R; Beardsley, G P et al. (1988) 2',3'-Dideoxyadenosine is selectively toxic for TdT-positive cells. Blood 71:1601-8
Beardsley, G P; Mikita, T; Klaus, M M et al. (1988) Chemical synthesis of DNA oligomers containing cytosine arabinoside. Nucleic Acids Res 16:9165-76
Kremer, A B; Mikita, T; Beardsley, G P (1987) Chemical consequences of incorporation of 5-fluorouracil into DNA as studied by NMR. Biochemistry 26:391-7
Clark, J M; Joyce, C M; Beardsley, G P (1987) Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. J Mol Biol 198:123-7