Inhibition of topoisomerase IIalpha and G2 cell cycle arrest by NK314, a novel benzo[c]phenanthridine currently in clinical trials.
NK314 is a novel synthetic benzo[c]phenanthridine alkaloid that has recently entered clinical trials as an antitumor compound, based on impressive activities in preclinical models. The present investigations were directed at determining the mechanism of action of this agent. NK314 induced significant G(2) cell cycle arrest in several cell lines, independent of p53 status, suggesting the existence of a common mechanism of checkpoint activation. The Chk1-Cdc25C-Cdk1 G(2) checkpoint pathway was activated in response to 100 nmol/L NK314 in ML-1 human acute myeloid leukemia cells. This was associated with the phosphorylation of the histone variant H2AX, an action that was predominant in the G(2) population, suggesting that double-strand DNA breaks caused cells to activate the checkpoint pathway. Double-strand DNA breaks were visualized as chromosomal aberrations when the G(2) checkpoint was abrogated by 7-hydroxystaurosporine. In vitro assays showed that NK314 inhibited the ability of topoisomerase IIalpha to relax supercoiled DNA and trapped topoisomerase IIalpha in its cleavage complex intermediate. CEM/VM1 cells, which are resistant to etoposide due to mutations in topoisomerase IIalpha, were cross-resistant to NK314. However, CEM/C2 cells, which are resistant to camptothecin due to mutations in topoisomerase I, retained sensitivity. These findings support the conclusion that the major mechanism of NK314 is to inhibit topoisomerase IIalpha, an action that leads to the generation of double-strand DNA breaks, which activate the G(2) DNA damage checkpoint pathway.
Guo
Lei
L
Department of Experimental Therapeutics, Box 71, The University of Texas M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Liu
Xiaojun
X
Nishikawa
Kiyohiro
K
Plunkett
William
W
D000951:Antigens, Neoplasm / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D002869:Chromosome Aberrations; D002986:Clinical Trials as Topic; D004250:DNA Topoisomerases, Type II / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004354:Drug Screening Assays, Antitumor / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016195:G2 Phase / Q000187:drug effects*; D006657:Histones / Q000378:metabolism; D006801:Humans; D015470:Leukemia, Myeloid, Acute / Q000378:metabolism; D010616:Phenanthrenes; D010617:Phenanthridines / Q000494:pharmacology*; D010766:Phosphorylation; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D059005:Topoisomerase II Inhibitors
D000951:Antigens, Neoplasm; D000970:Antineoplastic Agents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; C522060:NK314; D010616:Phenanthrenes; D010617:Phenanthridines; D059005:Topoisomerase II Inhibitors; C054852:7-hydroxystaurosporine; D004250:DNA Topoisomerases, Type II; D019311:Staurosporine