Characterization of Protein Kinase chk1 Essential for the Cell Cycle Checkpoint after Exposure of Human Head and Neck Carcinoma A253 Cells to a Novel Topoisomerase I Inhibitor BNP1350

Abstract

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 μM concentrations of BNP1350, the IC₅₀ and IC₉₀ values, respectively. After exposure to a low concentration of BNP1350 (IC₅₀), A253 cells accumulated primarily in G₂phase. In contrast, treatment with a high concentration of BNP1350 (IC₉₀) 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 G₂ 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 G₂ checkpoint kinase chk1 are not p53-dependent.