Doctoral Dissertations

Date of Award

12-1984

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Biomedical Sciences

Major Professor

James D. Regan

Committee Members

Julian Preston, Rhoda F. Grell, Robert Fujimura

Abstract

The eight arabinofuranosyl(ara)nucleosides and one ara-nucleotide: ara-adenine 5'-monophosphate (ara-AMP), ara-adenine (ara-A), ara-2-fluoroadenine (FAA), ara-guanine (ara-G), ara-hypoxanthine (ara-H), ara-thymine (ara-T), ara-2'-fluorocytosine (FAG), ara-2'- fluoro-5-iodo-cytosine (FIAC) and ara-2'-fluoro-5-methyl-uraci1 (FMAU) were compared to ara-cytosine (ara-C) in their ability to inhibit ultraviolet (UV) light-induced DNA repair in log phase and confluent human diploid fibroblasts. In UV-irradiated growth arrested human fibroblast cultures, treatment with any of the ara-nucleosides leads to accumulation of single-strand breaks, the effective dose for this inhibition varied greatly: ara-C and its derivatives > ara-A, FAA > ara-G, ara-H > ara-T. In rapidly cycling cells sensitivity to repair inhibition was exhibited only in response to ara-C and FAC, and if 2 mM hydroxyurea (HU) was coadministered with ara-A, FAA or FMAU. Few or no DNA single-strand breaks were observed in UV-irradiated log phase cells streated with FIAC, ara-H, ara-G or ara-T even in the presence of HU. All four deoxynucleosides were found to reverse ara-A, ara-H, ara-G, and ara-AMP inhibition of DNA repair, while only (dCd) was able to reverse ara-C induced inhibition. The efficiencies of inhibition of unscheduled DNA synthesis (UDS) and semi conservative DNA synthesis by the ara-nucleosides is consistent with their relative efficiencies at producing strand breaks. Using ara-C the initial rates of UV-induced DNA repair in growth-arrested confluent monolayers VI of normal diploid human fibroblasts were examined by assaying the accumulation of DNA breaks due to DNA incisions. The accumulation of DNA breaks followed first-order kinetics, and was analyzed via Henri-Michealis-Menten kinetics. Based on the initial rates of DNA repair the expected times for the removal of pyrimidine dimers were calculated and found to be in good agreement with other measurements of pyrimidine dimer excision. The reversal of ara-C accumulated UV-induced DNA breaks with deoxycytidine was found to follow noncompetitive type inhibition. A kinetic model for ara-C inhibition of DNA repair is discussed.

The number of sites undergoing DNA excision repair in human fibroblasts following treatment with the highly carcinogenic anti- and weakly carcinogenic syn- diastereomers of BPDE was assayed by both the bromodeoxyuridine photolysis technique and ara-C accumulated DNA incisions. During a four hour DNA repair period (including a one hour BPDE treatment), the number of sites undergoing repair were approximately 2-4 fold higher for the syn-isomer than the anti-isomer when calculated on a per adduct basis. Bromodeoxyuridine photolysis measurements indicated an average patch size for both diastereomers between 40 to 45 nucleotides. BPDE treatment induced DNA breaks in normal fibroblasts and to a much lesser extent in XP12BE cells. The UVR ABC endonuclease from Escherichia coli was used in a modified endonuclease sensitive assay (ESS) to examine the kinetics of repair of anti- and syn-BPDE DNA adducts and was found to be biphasic in normal human diploid fibroblasts (HSBP). The repair defective XP12BE VI1 cells exhibited only a fast repair component, losing 30-40% of the BPDE-DNA adducts in the first 12 hours following treatment, with no apparent BPDE-DNA adduct removal at later times. Small differences in the kinetics of repair for anti- and syn-BPDE were found to be significant, but cannot solely explain the marked differences in the biological activities of these two compounds. It was found that in HSBP cells ara-C/HU treatment inhibited the repair of BPDE adducts by the slow repair component. This inhibition of adduct removal in normal cells resembles the situation in XP12BE cells in the absence of the inhibitors. Experiments correlating the number of adducts re moved with the number of repair sites indicated a more rapid loss of adducts than sites undergoing repair, which may reflect a two-step repair process with the removal of an alkylated base by the action of a DNA glycosylase, followed by an apurinic/apyrimidinic endonuclease incision, for a specific class of DNA adducts.

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