Effect of separase depletion on ionizing radiation-induced cell cycle checkpoints and survival in human lung cancer cell lines.

OBJECTIVES

This study is to evaluate the effect of separase depletion on cell cycle progression of irradiated and non-irradiated cells through the G(2)/M phases and consecutive cell survival.

MATERIALS AND METHODS

Separase was depleted with siRNA in two human non-small cell lung carcinoma (NSCLC) cell lines. Cell cycle progression, mitotic fraction, DNA repair, apoptotic and clonogenic cell death were determined.

RESULTS

By depletion of endogenous separase with siRNA in NSCLCs, we showed that separase affects progression through the G(2) phase. In non-irradiated exponentially growing cells, separase depletion led to an increased G(2) accumulation from 17.2% to 29.1% in H460 and from 15.7% to 30.9% in A549 cells and a decrease in mitotic cells. Depletion of separase significantly (P < 0.01) increased the fraction of radiation-induced G(2) arrested cells 30-56 h after irradiation and led to decrease in the mitotic fraction. This was associated with increased double-strand break repair as measured by gamma-H2AX foci kinetics in H460 cells and to a lesser extent in A549 cells. In addition, a decrease in the expression of mitotic linked cell death after irradiation was found.

CONCLUSIONS

These results indicate that separase has additional targets involved in regulation of G(2) to M progression after DNA damage. Prolonged G(2) phase arrest in the absence of separase has consequences on repair of damaged DNA and cell death.