Concentration-dependent differences in the mechanisms by which caffeine potentiates etoposide cytotoxicity in HeLa cells.

Morphological examination of HeLa cells exposed to etoposide for 1 h revealed two distinct modes of death: (a) within 6 h of drug removal, shrunken cells appeared which contained vacuolated cytoplasm and regions of intense chromatin staining, consistent with apoptosis; and (b) concomitant with release from G2 arrest, enlarged cells appeared which contained evenly staining nuclear fragments, consistent with mitotic death. The methylxanthine, caffeine, enhanced cytotoxicity in a concentration-dependent manner when applied for 24 h following etoposide exposure. One mM caffeine alleviated etoposide-induced G2 arrest and increased the incidence of mitotic death, accounting for the potentiation of cytotoxicity. Brief caffeine exposures (5 or 10 mM for 1-2 h) caused specific tyrosine dephosphorylation and activation of p34cdc2 kinase, and mitotic progression to a limited extent, in cells which were arrested in G2 following etoposide treatment. However, longer exposure times at a high caffeine concentration (10 mM) caused inhibition of both cell cycle progression and mitotic death, and the enhancement of etoposide cytotoxicity could be accounted for by up to a 3-fold increase in the proportion of morphologically apoptotic cells. Thus, caffeine potentiates etoposide cytotoxicity by two morphologically distinct mechanisms depending on its concentration.