title: Rac1 protein signaling is required for DNA damage response stimulated by topoisomerase II poisons.
abstract: To investigate the potency of the topoisomerase II (topo II) poisons doxorubicin and etoposide to stimulate the DNA damage response (DDR), S139 phosphorylation of histone H2AX (γH2AX) was analyzed using rat cardiomyoblast cells (H9c2). Etoposide caused a dose-dependent increase in the γH2AX level as shown by Western blotting. By contrast, the doxorubicin response was bell-shaped with high doses failing to increase H2AX phosphorylation. Identical results were obtained by immunohistochemical analysis of γH2AX focus formation, comet assay-based DNA strand break analysis, and measuring the formation of the topo II-DNA cleavable complex. At low dose, doxorubicin activated ataxia telangiectasia mutated (ATM) but not ATM and Rad3-related (ATR). Both the lipid-lowering drug lovastatin and the Rac1-specific inhibitor NSC23766 attenuated doxorubicin- and etoposide-stimulated H2AX phosphorylation, induction of DNA strand breaks, and topo II-DNA complex formation. Lovastatin and NSC23766 acted in an additive manner. They did not attenuate doxorubicin-induced increase in p-ATM and p-Chk2 levels. DDR stimulated by topo II poisons was partially blocked by inhibition of type I p21-associated kinases. DDR evoked by the topoisomerase I poison topotecan remained unaffected by lovastatin. The data show that the mechanisms involved in DDR stimulated by topo II poisons are agent-specific with anthracyclines lacking DDR-stimulating activity at high doses. Pharmacological inhibition of Rac1 signaling counteracts doxorubicin- and etoposide-stimulated DDR by disabling the formation of the topo II-DNA cleavable complex. Based on the data we suggest that Rac1-regulated mechanisms are required for DNA damage induction and subsequent activation of the DDR following treatment with topo II but not topo I poisons.
authors.affiliation: Institute of Toxicology, Heinrich-Heine-University Düsseldorf, Moorenstrasse 5, D-40225 Düsseldorf, Germany.
authors.forename: Stefanie C
authors.initials: SC
authors.lastname: Huelsenbeck
authors.forename: Anne
authors.initials: A
authors.lastname: Schorr
authors.forename: Wynand P
authors.initials: WP
authors.lastname: Roos
authors.forename: Johannes
authors.initials: J
authors.lastname: Huelsenbeck
authors.forename: Christian
authors.initials: C
authors.lastname: Henninger
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
authors.forename: Gerhard
authors.initials: G
authors.lastname: Fritz
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D016923:Cell Death; D002460:Cell Line; D004249:DNA Damage; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000187:drug effects / Q000378:metabolism*; D006657:Histones / Q000737:chemistry; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D007150:Immunohistochemistry / Q000379:methods; D009369:Neoplasms / Q000188:drug therapy; D011042:Poisons / Q000737:chemistry; D051381:Rats; D015398:Signal Transduction; D059005:Topoisomerase II Inhibitors / Q000494:pharmacology; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D000970:Antineoplastic Agents; D006657:Histones; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D011042:Poisons; D059005:Topoisomerase II Inhibitors; D020830:rac1 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II