Early caspase activation in leukemic cells subject to etoposide-induced G(2)-M arrest: Evidence of commitment to apoptosis rather than mitotic cell death

After exposure to cytotoxic drugs at relatively low concentration, many cel l types undergo G(2)-M arrest and then either mitotic cell death or, in the case of hematopoietic cells, apoptosis, We have sought to examine this phe nomenon in two lymphoblastoid cell lines. After continuous or short-term ex posure to etoposide (final concentration, 0.5 mu M), up to 80% of cells acc umulated at G(2)-M by 24 h, and subsequently either underwent apoptosis or re-entered the cell cycle. In this and the other studies undertaken, the CE M and MOLT-4 lines behaved similarly. Progressive accumulation of cells at G(2)-M was accompanied by increasing levels of cyclin B1, Commitment to apo ptosis was assessed by evidence of caspase activation using a number of dif ferent criteria. A decreased amount of M-r 32,000 procaspase-3 was evident 24-48 h after drug treatment. However, cleavage of caspase substrates poly( ADP-ribose) polymerase and lamin B indicated caspase activation occurring w ithin 3-6 h of drug treatment, Protease activity in corresponding cell extr acts increased progressively from 6 h or earlier to 24 h after the addition of etoposide to the medium. Such increase was consequent on drug treatment and not attributable to cells being at G(2)-M, Treatment with 1.5 mM caffe ine abrogated etoposide-induced G(2)-M arrest, and in cells so treated, the etoposide-induced increase in protease activity was also abrogated, Howeve r, there was no impact of caffeine on cytotoxicity under these conditions, Although mitotic cell death is precipitated subsequent to prolonged G(2)-M arrest in many cell types, the present data suggest that commitment to apop tosis occurs in parallel to G(2)-M arrest in leukemic cells.