The DNA unwinding effects of some 9-aminoacridine derivatives were compared under reaction conditions that could be used to study drug-induced topoisomerase II inhibition. An assay was designed to determine drug-induced DNA unwinding by using L1210 topoisomerase I. 9-aminoacridines could be ranked by decreasing unwinding potency: compound C greater than or equal to 9-aminoacridine greater than o-AMSA greater than or equal to compound A greater than compound B greater than m-AMSA. Ethidium bromide was more potent than any of the 9-aminoacridines. This assay is a fast and simple method to compare DNA unwinding effects of intercalators. It led to the definition of a drug intrinsic unwinding constant (k). An additional finding was that all 9-aminoacridines and ethidium bromide inhibited L1210 topoisomerase I. Enzyme inhibition was detectable at low enzyme concentrations (less than or equal to 1 unit) and when the kinetics of topoisomerase I-mediated DNA relaxation was studied. Topoisomerase I inhibition was not associated with DNA swivelling or cleavage.

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DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA-protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m-AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit antiserum elicited against calf thymus DNA modified to 4.4% (Pt drug/nucleotide ratio = 0.044) with the antitumor drug cis-diamminedichloroplatinum(ll) (cis-DDP) contains antibodies specific for the Pt-modified DNA immunogen as well as for Pt-DNA adducts formed in both cultured mouse leukemia L1210 cells and in L1210 cells from the ascites fluid of tumor-bearing mice exposed to cis-DDP. Pt-modified DNA was electrostatically complexed to methylated bovine serum albumin and injected into rabbits. Early bleedings ofthe derived antiserum were used to establish a competitive enzyme-linked immunosorbent assay (ELISA), which demonstrated specificity for the Ptmodified DNA but not for DNA or the Pt drug alone. In the ELISA, 50% inhibition occurred at a concentration of 0.5 nM Pt (on DNA) as determined by atomic absorption spectroscopy. This value corresponds to a lower limit of detectability of one adduct in 107 nucleotides, with 50 ,ug ofsample DNA added per microtiter well. DNA isolated from cultured mouse L1210 cells exposed to increasing doses of the Pt drug was found by ELISA to contain from 0.2 to 10.0 fmol of Pt adduct per ,ug of DNA. These levels remained stable for up to 4 hr after a 1-hr drug treatment, during which time DNA interstrand crosslinks developed. Thus, the antiserum appears not to be specific for DNA interstrand crosslinks. DNAs from L1210 cells exposed to trans-diamminedichloroplatinum(II) and L-phenylalanine mustard were not recognized in the ELISA. DNA prepared from the ascites cells of mice bearing the L1210 tumor 5 hr after injection of cis-DDP was found to contain about 2 fmol of Pt per jig of DNA. This work establishes that cis-DDP-DNA adducts prepared in vitro are relevant to the in vivo binding ofthe Pt drug to its biological target, DNA, and opens new avenues for studying the mechanism of action of the Pt anticancer drugs.cis-Diamminedichloroplatinum(II), cis-DDP, is currently used in the treatment of human malignancies (1, 2). Its cytocidal properties are believed to derive from covalent binding to the DNA bases, resulting in the formation ofadducts that interfere with normal DNA function (3). Because the trans isomer, trans-DDP, is not chemotherapeutically active, DNA adducts formed preferentially by cis-DDP may be responsible for its antineoplastic activity.Substantial information is now available concerning the stereochemistry of cis-DDP-DNA adducts prepared in vitro (4-8).In particular, cis-DDP has been shown to perturb the guanine-cytidine base pairs leading to unwinding and shortening of DNA (6-8) and to exhibit sequence-specific binding effects attributed to the formation of bidentate linkages between adjacent or nearby bases on the same DNA strand (4, 9, 10, 11). cis-DDP cytotoxicity in mouse and human cultured cells has been correlated with DNA interstrand crosslinks, whereas equimolar doses of trans-DDP were nontoxic and did not induce these lesions (12-15); By atomic absorption spectroscopy it has been possible to determine total Pt in DNA samples and to monito...