Cellular
topoisomerase I is an important target in
cancer chemotherapy. A novel
karenitecin,
BNP1350, is a
topoisomerase I-targeting
anticancer agent with significant antitumor activity against human head and neck
carcinoma A253 cells in vitro. As a basis for future clinical trials of
BNP1350 in human head and neck
carcinoma, in vitro studies were carried out to investigate its effect on DNA damage and cell cycle checkpoint response. The treatment of A253 cells with
BNP1350 caused biphasic profiles of DNA fragmentation displayed from 0 to 48 h after 2-h exposure. Pulsed-field gel electrophoresis demonstrated that the first wave of DNA damage was mainly megabase DNA fragmentation, but the second wave of DNA damage was 50- to 300-kb DNA fragmentation in addition to megabase DNA damage. The cell cycle checkpoint response was characterized after exposure to 0.07 and 0.7 microM concentrations of
BNP1350, the IC(50) and IC(90) values, respectively. After exposure to a low concentration of
BNP1350 (IC(50)), A253 cells accumulated primarily in G(2) phase. In contrast, treatment with a high concentration of
BNP1350 (IC(90)) resulted in S phase accumulation. The concentration-associated cell cycle perturbation by
BNP1350 was correlated with different profiles of cell cycle-regulatory
protein expression. When treated with the low concentration of
BNP1350,
cyclin B/cdc2
protein expression was up-regulated, whereas with the high concentration, no significant change was observed at 24 and 48 h. In addition, increased phosphorylation of a G(2) checkpoint
kinase chk1 was observed 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 appears to be associated with specific phases of cell cycle arrest induced by
BNP1350. Because A253 cells do not express the p53
protein, the
drug-induced alterations of the G(2) checkpoint
kinase chk1 are not p53-dependent.