Characterization of protein kinase chk1 essential for the cell cycle checkpoint after exposure of human head and neck carcinoma A253 cells to a noveltopoisomerase I inhibitor BNP1350

Cellular topoisomerase I is an important target in cancer chemotherapy. A n ovel karenitecin, BNP1350, is a topoisomerase I-targeting anticancer agent with significant antitumor activity against human head and neck carcinoma A 253 cells in vitro. As a basis for future clinical trials of BNP1350 in hum an head and neck carcinoma, in vitro studies were carried out to investigat e its effect on DNA damage and cell cycle checkpoint response. The treatmen t of A253 cells with BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophores is demonstrated that the first wave of DNA damage was mainly megabase DNA f ragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragm entation in addition to megabase DNA damage. The cell cycle checkpoint resp onse was characterized after exposure to 0.07 and 0.7 mu M concentrations o f BNP1350, the IC50 and IC90 values, respectively. After exposure to a low concentration of BNP1350 (IC50), A253 cells accumulated primarily in G(2) p hase. In contrast, treatment with a high concentration of BNP1350 (IC90) re sulted in S phase accumulation. The concentration-associated cell cycle per turbation by BNP1350 was correlated with different profiles of cell cycle-r egulatory protein expression. When treated with the low concentration of BN P1350, cyclin B/cdc2 protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In a ddition, increased phosphorylation of a G(2) checkpoint kinase chk1 was obs erved when cells were treated with a low concentration of BNP1350, whereas only slight inhibition of chk1 activity was found in the cells treated with the higher concentration. Altered chk1 phosphorylation after DNA damage ap pears to be associated with specific phases of cell cycle arrest induced by BNP1350. Because A253 cells do not express the p53 protein, the drug-induc ed alterations of the G(2) checkpoint kinase chk1 are not p53-dependent.