N-terminal and core-domain random mutations in human topoisomerase II alpha conferring bisdioxopiperazine resistance.
Random mutagenesis of human topoisomerase II alpha cDNA followed by functional expression in yeast cells lacking endogenous topoisomerase II activity in the presence of ICRF-187, identified five functional mutations conferring cellular bisdioxopiperazine resistance. The mutations L169F, G551S, P592L, D645N, and T996L confer > 37, 37, 18, 14, and 19 fold resistance towards ICRF-187 in a 24 h clonogenic assay, respectively. Purified recombinant L169F protein is highly resistant towards catalytic inhibition by ICRF-187 in vitro while G551S, D645N, and T996L proteins are not. This demonstrates that cellular bisdioxopiperazine resistance can result from at least two classes of mutations in topoisomerase II; one class renders the protein non-responsive to bisdioxopiperazine compounds, while an other class does not appear to affect the catalytic sensitivity towards these drugs. In addition, our results indicate that different protein domains are involved in mediating the effect of bisdioxopiperazine compounds.
Jensen
L H
LH
Department of Pathology, Laboratory Center, Righospitalet, Copenhagen, Denmark. lhjensen@rh.dk
Wessel
I
I
Møller
M
M
Nitiss
J L
JL
Sehested
M
M
Jensen
P B
PB
D000255:Adenosine Triphosphate / Q000378:metabolism; D000677:Amsacrine / Q000494:pharmacology; D000951:Antigens, Neoplasm; D004250:DNA Topoisomerases, Type II / Q000235:genetics; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D004351:Drug Resistance; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005047:Etoposide / Q000494:pharmacology; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000235:genetics; D016296:Mutagenesis; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D010879:Piperazines / Q000494:pharmacology*; D011929:Razoxane / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D054659:Diketopiperazines; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D019384:Nucleic Acid Synthesis Inhibitors; D010879:Piperazines; D059005:Topoisomerase II Inhibitors; D000677:Amsacrine; C070899:4,4'-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione; D011929:Razoxane; D005047:Etoposide; D000255:Adenosine Triphosphate; D004250:DNA Topoisomerases, Type II