The physicochemical and biochemical effects of anthracycline antibiotics on DNA

Abstract

The anthracycline antibiotics are effective clinical anticancer agents for the treatment of leukemias, lymphomas, and solid tumors. The main biochemical effect of these drugs is believed to be interference with template DNA function during the transcription and replication processes. A highly potent anthracycline analog, cyanomorpholinodoxorubicin (CNM-DOX), was used to study the physicochemical effects of anthracyclines on duplex DNA. CNM-DOX complexed with DNA and increased the physicochemical stability of double-stranded DNA. The nature of this stabilized DNA-CNM-DOX complex is not DNA crosslinks. The interaction between CNM-DOX and DNA phosphate backbone, however, is crucial to highly stabilized CNM-DOX-DNA complex formation. Structure-activity relationship analysis revealed a link between the ability of anthracyclines to increase physicochemical stability of double-stranded DNA and their cytotoxicity. Since the enzyme helicase separates double-stranded DNA and prepares the strands for enzymatic replication or transcription, it is a potential cellular target for anthracyclines. We examined the effect of stabilized anthracycline-DNA complex on the dissociating activities of DNA helicases using a duplex DNA helicase substrate of {dollar}\rm\sp{lcub}32{rcub}P{dollar} labeled 17mer annealed to complementary M13 circular single stranded DNA. SV40 T-antigen helicase activity is potently inhibited by the anthracycline antibiotics with {dollar}\rm IC\sb{lcub}50{rcub}{dollar} values from 2 {dollar}\times{dollar} 10{dollar}\sp{lcub}-7{rcub}{dollar}M to 6 {dollar}\times{dollar} 10{dollar}\sp{lcub}-6{rcub}{dollar}M. Partially purified helicases from HeLa cells and murine FM3A cells also are potently inhibited by doxorubicin (DOX) with {dollar}\rm IC\sb{lcub}50{rcub}{dollar} values of 4 {dollar}\times{dollar} 10{dollar}\sp{lcub}-7{rcub}{dollar}M and 9 {dollar}\times{dollar} 10{dollar}\sp{lcub}-7{rcub}{dollar}M respectively. Both the enzymatic reaction rate and the maximal level of T-antigen helicase activity were significantly reduced with DOX treatment. While the DOX-DNA complex formation did not interfere with helicase associated ATPase activity, it blocked the conformational change of T-antigen during DNA dissociation process. These findings suggested that helicase is trapped by the DOX-DNA complex as a stable ternary complex (enzyme-DNA-drug), which subsequently prohibits the conformational change of helicase that is required for its DNA dissociating activity.