Growth and death of hela cells in the presence of caffeine

Proliferation and death were measured in synchronously growing cultures of HeLa S3 cells during treatment with up to 30 mM caffeine. Changes in the number of colony‐forming cells were determined by single‐cell plating, while changes in the total number of cells were measured both by electronic counting and by monitoring cell division and physiological death cinemicrographically. At concentrations between 2 and 5 mM, cell killing occurs over several days during which the cells traverse the generation cycle once or a few times before losing colony‐forming ability, with consequent proliferation of non‐colony‐forming cells. This indicates that lethal damage is accumulated with time. Death occurs more rapidly at higher concentrations, without proliferation, the kinetics of inactivation being strongly dependent on the phase of the cycle (cell age) at which treatment is initiated. G 1 cells are killed more slowly in 10 mM caffeine than are S cells, but G 1 cells respond rapidly to 20 mM caffeine, suggesting the inception of an additional mode of killing. The incidence of sister‐cell fusion increases with increasing caffeine concentration above 1 mM. On addition of 10 mM caffeine to a culture prepared from collected mitotic cells, the cells undergo a transient rounding and then respread after several hours; with 20 mM, they never respread. The generation cycle is prolonged in a concentration‐dependent fashion, as is the duration of G 1 ; the generation time is doubled in 5–6 mM caffeine. G 2 and M are also prolonged at concentrations above 3 mM, but S is not prolonged even by 10 mM caffeine.