Topoisomerase II (topo II) is required to maintain DNA topology during various DNA metabolic processes and is also an important molecular target in cancer therapy. Drugs acting on topo II can be divided in two groups; “poisons” like the anthracyclines daunorubicin and doxorubicin which lead to DNA double strand breaks, and “catalytic inhibitors” such as dexrazoxane (ICRF-187) that inhibit topo II without creating DNA strand breaks at pharmacologically relevant concentrations. Clinically dexrazoxane is used to prevent anthracyline-mediated cardiotoxicity, and has also shown activity as an antidote in accidental anthracycline extravasation. Furthermore, dexrazoxane antagonizes etoposide-induced myelosuppression in the mouse allowing for increased etoposide dosing and improved targeting of brain tumors. Although it is well established that dexrazoxane targets topo II in vitro, the role of topo II in mediating these in vivo effects of dexrazoxane remains elusive. We have introduced a point mutation (Y165S) in the topo II α gene in mice predicted to abrogate dexrazoxane binding in vivo. The Y165S mutation was chosen on the basis of biochemical studies with recombinant Y165S protein which was previously shown to lack dexrazoxane binding. This mutant protein exhibits wild-type levels of DNA strand passage activity at saturating ATP levels, but has reduced ATP hydrolysis. By homologous recombination a targeting vector containing the Y165S mutation was integrated in embryonic stem cells (ES cells) at the topo II α loci. Correctly targeted ES cell clones were isolated, used for blastocyst injection, and mice with germline transmission obtained. When crossing heterozygous mice, no homozygous mutants were born. The homozygous mutant embryos die after day 7 of the embryonic development, but they are smaller in size than wild type and heterozygous embryos, indicating that the Y165S mutant allele is lethal during the late part of embryonic development when homozygous. On the other hand, no differences in size, mortality and life span were observed between heterozygous mutant mice and their wild type siblings. Clonogenic assays using heterozygous ES cells and bone marrow cells isolated from heterozygous mice showed that these cells are highly resistant to dexrazoxane. No resistance to other topoisomerase inhibitors such as daunorubicin, etoposide and camptothecin were seen. We are currently testing dexrazoxane-mediated effects on extravasation, myelosuppression and cardiotoxicity in Y165S/wt mice to elucidate the functional role of topo II α.
[Proc Amer Assoc Cancer Res, Volume 47, 2006]