The effect of combinations of the anthracycline aclarubicin and the 
topoisomerase II targeting drugs 
4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucopyra noside) 
(VP-16) and 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) was 
investigated in a clonogenic assay. The cytotoxicity of VP-16 was almost 
completely antagonized by preincubating cells with nontoxic concentrations of 
aclarubicin. The inhibition of cytotoxicity was not seen when the cells were 
exposed to aclarubicin after exposure to VP-16. The inhibition was significant 
over a wide range of aclarubicin concentrations (3 nM to 0.4 microM), above 
which the toxicity of aclarubicin became apparent. A similar effect was seen on 
the toxicity of m-AMSA. In contrast to aclarubicin, preincubation with 
Adriamycin did not antagonize the effect of VP-16. With purified topoisomerase 
II and naked DNA, aclarubicin did not stimulate the formation of cleavable 
complexes between topoisomerase II and DNA. Aclarubicin concentrations above 1 
microM inhibited the baseline formation of cleavable complexes elicited with the 
enzyme alone. Low (1 to 10 nM) aclarubicin concentrations increased the 
formation of cleavable complexes obtained with VP-16 and m-AMSA; however, at 
aclarubicin concentrations above 1 microM an antagonistic effect was obtained. 
In cells, the m-AMSA- and VP-16-induced, protein-concealed DNA strand breaks 
were completely inhibitable by aclarubicin preincubation with no synergic dose 
levels. Our results suggest that aclarubicin inhibits topoisomerase II-mediated 
DNA cleavage. This inhibition could represent the mechanism of action of the 
drug and explain the lack of cross-resistance to the classical anthracyclines. 
The observed antagonism could have consequences for scheduling of aclarubicin 
with topoisomerase II-active anticancer drugs.