Abstract
The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA. Treatment of human cells with low levels of adozelesin results in potent inhibition of both cellular and simian virus 40 (SV40) DNA replication. Extracts were prepared from adozelesin-treated cells and shown to be deficient in their ability to support SV40 DNA replication in vitro. This effect on in vitro DNA replication was dependent on both the concentration of adozelesin used and the time of treatment but was not due to the presence of adozelesin in the in vitro assay. Adozelesin treatment of cells was shown to result in the following: induction of p53 protein levels, hyperphosphorylation of replication protein A (RPA), and disruption of the p53-RPA complex (but not disruption of the RPA-cdc2 complex), indicating that adozelesin treatment triggers cellular DNA damage response pathways. Interestingly, in vitro DNA replication could be rescued in extracts from adozelesin-treated cells by the addition of exogenous RPA. Therefore, whereas adozelesin and other anti-cancer therapeutics trigger common DNA damage response markers, adozelesin causes DNA replication arrest through a unique mechanism. The S phase checkpoint response triggered by adozelesin acts by inactivating RPA in some function essential for SV40 DNA replication.
Highlights
The cyclopropylpyrroloindole anti-cancer drug, adozelesin, binds to and alkylates DNA
Cell-free simian virus 40 (SV40) DNA Replication Is Reduced in Extracts from Adozelesin-treated Cells—Extracts from adozelesin-treated cells were tested for the ability to support SV40 DNA replication in vitro
Cell extracts were prepared as described under “Experimental Procedures” and used in cell-free SV40 DNA replication assays with exogenous SV40 origin-containing plasmid template and SV40 large T antigen
Summary
Tothecin or gamma radiation can be either completely or partially blocked by the addition of the DNA polymerase inhibitor, aphidicolin This suggests that replication fork passage through sites of damaged DNA may be required to induce RPA32 hyperphosphorylation [31]. Our previous studies have shown that a trans-acting inhibitor of SV40 DNA replication is induced in cell extracts upon treatment of HeLa cells with bizelesin [11] These results are similar to effects reported for camptothecin treatment and gamma radiation [24, 25]. In this study we show that adozelesin-induced DNA replication inhibition occurs through RPA inactivation, similar to results previously reported for UV radiation and hyperthermia [23, 44] This adozelesin response is clearly different from those induced by bizelesin treatment, camptothecin treatment, or gamma radiation
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