Mechanism by which caffeine potentiates lethality of nitrogen mustard.

Caffeine is synergistic with many DNA-damaging agents in increasing lethality to mammalian cells. The mechanism is not well understood. Our results show that caffeine potentiates the lethality of the nitrogen mustard 2-chloro-N-(2-chloroethyl)-N-methylethanamine (HN2) by inducing damaged cells to undergo mitosis before properly repairing lesions in their DNA. Treatment with low doses of HN2 (0.5 microM for 1 hr) caused little lethality in baby hamster kidney cells (90% survival). These cells were arrested in G2 shortly after treatment with HN2 as shown by flow microfluorimetry and autoradiography. After an arrest of 6 hr, HN2-treated cells began to move into mitosis and from then on behaved like normal cells. Repair synthesis was shown to continue during the G2 arrest by using synchronized cells pulse labeled with [3H]thymidine after HN2 treatment and autoradiography. Caffeine (2mM) increased the lethality of HN2 by 5- to 10-fold. It prevented the G2 arrest. Caffeine did not prevent these HN2-treated cells from entering or completing S phase but rather allowed them to divide without finishing the repair processes and as a consequence caused nuclear fragmentation after mitosis. Caffeine-induced nuclear fragmentation and enhanced lethality were proportional, as shown with dose--response curves and time dependence. In addition, both lethality and nuclear fragmentation were abolished by low doses of cycloheximide, an inhibitor of protein synthesis.