Modulation of 4'-(9-acridinylamino)methanesulfon-m-anisidide-induced, topoisomerase II-mediated DNA cleavage by...

Our earlier studies have shown that gossypol [1,1',6,6',7,7'-hexahydroxy-5,5-diisopropyl - 3,3'-dimethyl - (2,2'- binaphthalene)-8,8'-dicarboxyaldehyde], a male contraceptive, inhibits DNA synthesis by decreasing the activities of DNA polymerase alpha and beta, resulting in the arrest of cells in mid-S phase [L.J. Rosenberg, R.C. Adlakha, D.M. Desai, and P.N. Rao, Biochim. Biophys. Acta, 866: 258-267, 1986]. Now we have examined the effects of gossypol on another enzyme of importance to cellular functions, topoisomerase II (topo II). We have determined the consequences of gossypol treatment on 4'-(9-acridinylamino)methane-sulfon-m anisidide (m-AMSA)-induced topoisomerase II-mediated, protein-associated DNA cleavage using the alkaline elution technique. In HeLa cells pretreated with gossypol (3.4-17.5 microM) for 8-16 h we observed a dose- and time-dependent decrease (50-75%) in DNA cleavage compared to that quantified in cells treated with m-AMSA alone. Gossypol by itself did not induce more than 25 rad-equivalents of DNA single-strand breaks even at the highest dose tested (26 microM). [14C]m-AMSA uptake was identical in treated and untreated cells. Pretreatment of cells with another inhibitor of DNA synthesis, thymidine, which blocks cells at G1/S boundary increased the m-AMSA-induced DNA cleavage by 25%, suggesting that the effect of gossypol might be due to the arrest of cells in mid-S phase. In contrast to gossypol's effects on m-AMSA-induced DNA cleavage, m-AMSA-induced cytotoxicity was actually increased in gossypol pretreated cells. Gossypol blocked topo II strand passing activity (decatenation of kinetoplast DNA) of cellular extracts from HeLa cells. The inhibition of this activity by gossypol was synergistic with the inhibition produced by m-AMSA or etoposide. These data suggest that gossypol can both inhibit topo II catalytic activity and interfere with the stabilization of topo II-DNA complex formation by m-AMSA. These data indicate that the magnitude of m-AMSA-induced DNA cleavage may not necessarily parallel the magnitude of m-AMSA-induced cytotoxicity. The cytotoxicity data may rather be explained by an action of gossypol and m-AMSA to block topo II catalytic activity at a point in the enzyme's strand passing cycle prior to cleavage complex formation that might be particularly toxic to cells in S phase. Gossypol should therefore be useful in improving our understanding of the cellular role of topo II and the consequences of interference with topo II activity by active antineoplastic agents.