The long term objectives of this proposal are (1) to improve the effectiveness of currently useful anticancer compounds such as AraC and hydroxyurea (HU) by studying the detailed mechanisms of action and resistance, (2) to identify new targets for the development of new protocols or new anticancer agents by studying the properties of enzymes involved in DNA synthesis. Three areas of investigation are proposed: The first part, it to investigate the possible new targets of deoxynucleoside analog action. In addition to providing new knowledge for understanding enzymes involved in DNA metabolism, such as """"""""AraC-DNase"""""""", DNA polymerase Alpha activators, DNA primase, and DNA topoisomerase, this study will also aid in studying the biochemical determinants and the mechanism of cell resistance to araC, which is the second part of the proposal. One of the biochemical determinants of deoxynucleoside analogs, such as araC, is the intracellular content of deoxynucleoside triphosphates (dNTP). Ribonucleotide reductase (RR), the target of HU action, is the key enzyme for dNTP de novo synthesis. In order to study the regulation of deoxynucleotides and their relationship to the action of deoxynucleoside analogs, human cell lines with different activity or regulation of RR will be useful. HU was therefore used to select those cell lines. The characterization of those cell lines constitutes the third part of this proposal. This study will provide not only the basic knowledge of the regulation of deoxynucleotides which is important in understanding deoxynucleoside analog action, but also the mechanism of the resistance to HU. Through a clear understanding of the mechanism of resistance to drugs, collaterally sensitive compounds could be chosen based on these unique mechanisms of drug resistance. Furthermore, we will continue to interact with medicinal chemistry laboratories and to serve as a resource for the study of new nucleoside analogs synthesized by them, and to participate in clinical protocol design based on our understanding of drug action.
