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 anthracycline doxorubicin (Dox) showed a dose-dependent inhibitory effect on DNA replication, whether incubated with HeLa cell extracts or with DNA and nucleotides. Earliest-labeled fragment analysis revealed that inhibition of replication began within the origin-containing fragment in both control and Dox-containing reactions in vitro. AraC, a nucleoside analog, had no significant effect on DNA synthesis. In contrast, araCTP was able to inhibit DNA replication in vitro. Since metabolism is diminished in this in vitro system, the degree of phosphorylation of araC was apparently low. Progesterone showed an increase in nucleotide incorporation (sensitive to BuPdGTP inhibition of replication-specific polymerases alpha and delta) after preincubation with HeLa cell extracts, although progesterone receptors were not detectable in the HeLa cell extracts. 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 different from any known to be mediated through progesterone receptors. In conclusion, these results indicate that this mammalian in vitro replication system will be useful for the study of mechanisms and design of therapeutic drugs that inhibit mammalian DNA replication.
Diaz-Perez
M J
MJ
Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada.
Wainer
I W
IW
Zannis-Hadjopoulos
M
M
Price
G B
GB
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D001085:Arabinofuranosylcytosine Triphosphate / Q000494:pharmacology*; D003561:Cytarabine / Q000494:pharmacology*; D004261:DNA Replication / Q000187:drug effects*; D004317:Doxorubicin / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation; D010957:Plasmids; D011374:Progesterone / Q000378:metabolism; D011980:Receptors, Progesterone / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D011980:Receptors, Progesterone; D003561:Cytarabine; D001085:Arabinofuranosylcytosine Triphosphate; D011374:Progesterone; D004317:Doxorubicin