Topoisomerase inhibitors can selectively interfere with different stages of simian virus 40 DNA replication.

Abstract

I have found that antineoplastic drugs which are known to be inhibitors of mammalian DNA topoisomerases have pronounced and selective effects on simian virus 40 DNA replication. Ellipticine, 4'-(9-acridinylamino)methanesulfon-m-aniside, and Adriamycin blocked decatenation of newly replicated simian virus 40 daughter chromosomes in vivo. The arrested decatenation intermediates produced by these drugs contained single-strand DNA breaks. Ellipticine in particular produced these catenated dimers rapidly and efficiently. Removal of the drug resulted in rapid reversal of the block and completion of decatenation. The demonstration that these drugs interfere with decatenation suggests that they may exert their cytotoxic and antineoplastic effects by preventing the separation of newly replicated cellular chromosomes. Camptothecin rapidly breaks replication forks in growing Cairns structures. It is likely that the target of camptothecin is the "swivel" topoisomerase required for DNA replication and that it is located at or very near the replication fork in vivo. Evidence is presented that many of the broken Cairns structures are in fact half-completed sister chromatid exchanges. One pathway for the resolution of these structures is completion of the sister chromatid exchange to produce a circular head-to-tail dimer.DNA topoisomerases are now recognized as important targets for cancer chemotherapy (for reviews, see references 20 and 32). A number of cytotoxic drugs which were found to have antineoplastic properties have recently been shown to be topoisomerase inhibitors (3,12,15,25,26,31,33). These drugs typically interfere with the breakage-reunion cycles of topoisomerases to produce single-and double-strand DNA breaks. Recent studies have shown a correlation between cytotoxicity and levels of DNA strand breakage caused by these drugs (6,7,21). It is also possible that these drugs can exert their cytotoxic and antineoplastic effects through interference with reactions that require topoisomerases, such as replication fork progression, transcription, and separation (decatenation) of newly replicated daughter chromosomes. The work reported here demonstrates that topoisomerase inhibitors can selectively and reversibly interfere either with replication forks or with the decatenation process in simian virus 40 (SV40) DNA replication. Improved understanding of the modes of action of this class of drugs should aid in the rational design of a second generation of antineoplastic topoisomerase inhibitors.MATERIALS AND METHODS Cell culture and virus infection. African green monkey kidney cells (CV-1) were grown in Eagle minimal essential medium (Gibco) supplemented with 14 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.2) and 4 mM NaHCO3. Cells were infected with SV40 strain 777 at a multiplicity of 10 PFU/cell, and experiments were carried out 36 h after infection at the peak of SV40 DNA replication.Radiolabeling and preparation of viral DNA. Replicating SV40 DNA was pulse-labeled with [methyl-3H]thymidine as in...

Topoisomerase I and II activities are required for Epstein--Barr virus replication

“…The role of topoisomerases in DNA replication has been studied in a number of organisms (Maxwell & Geltert, 1985;Wang, 1985). In the case of viruses, Snapka (1986) has reported that topoisomerase II inhibitors including ellipticine block decatenation of newly replicated simian virus 40 daughter chromosomes and lead to an accumulation of catenated dimers containing single-strand DNA breaks and that camptothecin rapidly breaks the replication fork in growing Cairns structures. Recent reports have shown that the cellular topoisomerase II can induce double-strand breaks at specific locations in herpes simplex virus type 1 DNA and is involved in aspects of viral replication at late times in the infection cycle (Ebert et aL, 1990).…”

“…In the case of animal viruses, topoisomerases play important roles in viral DNA replication, transcription, packaging and integration (Foglesong & Bauer, 1984;Snapka, 1986;Nishiyama et al, 1987;Benson & Huang, 1988;Champoux, 1988; Richter & Strausfeld, 1988;Ebert et al, 1990;Priel et al, 1990Priel et al, , 1991 Schaack et al, 1990a, b;Wong & Hsu, 1990;Yamada et al, 1990;Gu & Rhode, 1991;Priel et al, 1991 ;Wang & Rogler, 1991). Topoisomerase I changes the linking numbers of topological DNA domains in integral steps of one by introducing transient singlestrand breaks.…”

Evaluation of Topoisomerase Inhibitors as Potential Antiviral Agents

“…The virus replicates in the infected cell nucleus forming structures very similar to eukaryotic chromatin (minichromosomes) (Griffeth et el., 1975;Tooze et et., 1980). In vitro studies have suggested that topoisomerase I and II enzymes are important for the replication of SV40 (Yang et aI., 1987;Snapka, 1986). Topoisomerase I acts during elongation to relax the positive supercoils which are generated as the parental DNA unwinds.…”

A virus-like agent associated with neurofibromatosis in damselfish

“…The genomes of most DNA viruses and retroviruses are detectable in the nuclei as eDNA during at least a portion of the viral life cycle (Fields et al 1996). Infected cells often contain complex patterns of different size viral eDNAs which reflect various stages in replication of the viral genome (Snapka 1987, Cotmore & Tattersall 1998. These patterns are produced by pauses in replication which result in the accumulation of discrete size fractions of DNA rather than simply a continuum of sizes of elongating DNA (Snapka 1987).…”

“…Infected cells often contain complex patterns of different size viral eDNAs which reflect various stages in replication of the viral genome (Snapka 1987, Cotmore & Tattersall 1998. These patterns are produced by pauses in replication which result in the accumulation of discrete size fractions of DNA rather than simply a continuum of sizes of elongating DNA (Snapka 1987). In addition to extrachromosomal forms, some DNA viruses and all retroviruses also exhibit integrated stages where one or more viral genomes are integrated into chromosomal DNA.…”