Bizelesin, a Bifunctional Cyclopropylpyrroloindole Alkylating Agent, Inhibits Simian Virus 40 Replication in Trans by Induction of an Inhibitor

Abstract

Bizelesin, a bifunctional DNA minor groove alkylating agent, inhibits both cellular and viral (SV40) DNA replication in whole cells. Bizelesin inhibition of SV40 DNA replication was analyzed in SV40-infected cells, using two-dimensional (2D) neutral agarose gel electrophoresis, and in a cell-free SV40 DNA replication assay. Within 1 h of bizelesin addition to infected cells, a similar rapid decrease in both the level of SV40 replication intermediates and replication activity was observed, indicating inhibition of initiation of SV40 DNA replication. However, prolonged bizelesin treatment (>/=2 h) was associated with a reduced extent of elongation of SV40 replicons, as well as the appearance on 2D gels of intense spots, suggestive of replication pause sites. Inhibition of elongation and induction of replication pause sites may result from the formation of bizelesin covalent bonds on replicating SV40 molecules. The level of in vitro replication of SV40 DNA also was reduced when extracts from bizelesin-treated HeLa cells were used. This effect was not dependent upon the formation of bizelesin covalent bonds with the template DNA. Mixing experiments, using extracts from control and bizelesin-treated cells, indicated that reduced DNA replication competence was due to the presence of a trans-acting DNA replication inhibitor, rather than to decreased levels or inactivation of essential replication factor(s).

Adozelesin Triggers DNA Damage Response Pathways and Arrests SV40 DNA Replication through Replication Protein A Inactivation

“…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).…”

“…If the inhibition of DNA replication in adozelesin-treated cell extracts is primarily due to a trans-acting DNA replication inhibitor, the addition of drug-treated cell extract would have a negative effect on DNA synthesis by the control cell extract, resulting in DNA synthesis levels below that of the base-line reaction (100%). This has been shown to be the case for treatment of cells with other DNA-damaging agents (11,24,25) but is clearly not the case for adozelesin treatment (Fig. 4).…”

“…Equivalent amounts of dimethylacetamide/dimethyl sulfoxide were added to control cultures. Cells were harvested, and extracts were prepared as described (11,14). Briefly, after washing once with cold phosphate-buffered saline and once with hypotonic buffer (20 mM HEPES-K ϩ (pH 7.5), 1.5 mM MgCl 2 , 0.5 mM Na 2 EDTA, 5 mM KCl, and 1 mM DTT), the cells were resuspended in 1.4ϫ cell pellet volume of hypotonic buffer for 10 min before being lysed by seven passages through a 25-gauge needle.…”

“…Conditions used for SV40 cell-free DNA replication were as described (11,14). Briefly, 30 ng of SV40 origin-containing plasmid pSV011, 500 ng of T antigen, 200 M each of UTP, CTP, and GTP, 4 mM ATP, and 100 M each of dCTP, dGTP, dTTP, 25 M dATP, creatine phosphokinase (24 units/ml), 40 mM phosphocreatine, and 2 Ci of [␣-…”

“…Lack of a trans-Acting DNA Replication Inhibitor in Adozelesin-treated Cell Extracts-Adozelesin-treated cell extracts were evaluated for the presence of a trans-acting DNA replication inhibitor, which is characteristic of DNA replication inhibition in gamma radiation-, bizelesin-, or camptothecin-treated cells (11,24,25). Extracts were prepared from 293 cells treated with 100 nM adozelesin for 2 h. The indicated amounts of adozelesin-treated cell extract were mixed with a fixed amount (40 g) of control cell extract, and the extract mixtures were used to carry out in vitro SV40 DNA replication.…”