CASPASE ACTIVATION IN MCF7 CELLS RESPONDING TO ETOPOSIDE TREATMENT

Studies of the biochemical mechanisms evoked by conventional treatment s for neoplastic diseases point to apoptosis as a key process for elim ination of unwanted cells. Although the pathways through which chemoth erapeutics promote cell death remain largely unknown, caspase protease s play a central role in the induction of apoptosis in response to a v ariety of stimuli including tumor necrosis factor, fas ligand, and gro wth factor deprivation. In this article, we demonstrate the induction of caspase protease activity in MCF7 human breast carcinoma cells expo sed to the topoisomerase inhibitor, etoposide. Caspase protease activi ty was assessed by incubating cell lysates with the known caspase subs trates, acetyl-L-aspartic-L-glutamic-L-valyl-L-aspartic acid 4-methyl- 7-aminocoumarin or acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7 -aminocoumarin. We observed maximal cleavage of acetyl-L-aspartic-L-gl utamic-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin within 6 hr fo llowing etoposide addition, a time that precedes cell death. In contra st, acetyl-L-tyrosyl-L-valyl-L-aspartic acid 4-methyl-7-aminocoumarin was resistant to cleavage activity. This substrate cleavage specificit y implies that a caspase-3-like protease is activated in response to D NA damage. Consistent with the lysate protease activity, an intracellu lar marker of caspase activation, poly-ADP ribose polymerase (PARP), w as cleaved in a concentration-and time-dependent manner after etoposid e-treatment. PARR cleavage followed caspase activation and reached max imum cleavage between 12 and 16 hr. Incubation of the cells with the p eptidic caspase inhibitor z-valine-alanine-asparagine-CH2F prevented c aspase activation, inhibited PARP cleavage, and inhibited cell death. Thus, etoposide killing of MCF7 cells requires a caspase-3-like protea se.