Mechanistic studies of amsacrine-resistant derivatives of DNA topoisomerase II. Implications in resistance to multiple antitumor drugs targeting the enzyme.

Wild-type yeast DNA topoisomerase II and three of its amsacrine-resistant derivatives L475A/L480P, L475A/R476G, and A642G, named according to amino acid changes at the codons specified, were overexpressed and purified. Because cells expressing several mutant enzymes missing portions of the carboxyl-terminal domain of the wild-type enzyme were previously found to exhibit amsacrine resistance, a carboxyl-terminal truncation protein Top2(1-1166), which lacks the last 263 amino acids of the wild-type enzyme, was also overexpressed and purified. These purified enzymes were used in the measurement of the turnover numbers of the DNA-dependent hydrolysis of ATP, the transport of one DNA segment through another, and the effects of amsacrine, teniposide or Ca(II) on the formation of the enzyme-DNA covalent intermediate. The results of these studies indicate that mutations leading to cellular resistance to amsacrine may occur by several different mechanisms, including reduction of the nuclear concentration and attenuation of the intrinsic catalytic steps of the enzyme. The significance of this underpinning mechanistic diversity of drug resistance and its relation to the simultaneous development of cellular resistance to chemically distinct drugs that target DNA topoisomerase II are discussed.