APPLICATION OF AN IN-VITRO SYSTEM IN THE STUDY OF CHEMOTHERAPEUTIC DRUG EFFECTS ON DNA-REPLICATION

DNA replication machinery is an important target for chemotherapeutic drugs. We have used an in vitro system to study the effect of drugs on mammalian DNA replication, either by direct interaction with the DNA structure or with replication proteins and machinery. The anthracyclin e doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA r eplication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibiti on of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside anal og, had no significant effect on DNA synthesis. In contrast, araCTP wa s able to inhibit DNA replication in vitro. Since metabolism is dimini shed in this in vitro system, the degree of phosphorylation of araC wa s apparently low. Progesterone showed an increase in nucleotide incorp oration (sensitive to BuPdGTP inhibition of replication-specific polym erases alpha and delta) after preincubation with HeLa cell extracts, a lthough progesterone receptors were not detectable in the HeLa cell ex tracts. In addition, we observed an inhibition in DNA replication when progesterone was preincubated with DNA and nucleotides. These results suggest that progesterone may have a mechanism of action that is diff erent from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro repli cation system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication. (C) 1996 Wi ley-Liss, Inc.