COMMITMENT TO CELL-DEATH MEASURED BY LOSS OF CLONOGENICITY IS SEPARABLE FROM THE APPEARANCE OF APOPTOTIC MARKERS

Kinetic analysis of dexamethasone-induced apoptosis in the human lymph oblastoid cell line CCRF CEM C7A has revealed a point when cells, morp hologically indistinguishable from untreated cells, have irreversibly engaged a program leading to death, measured by a loss of clonogenicit y. Since all cells that fail to clone eventually died through apoptosi s, measurements of clonogenicity in this system provide an accurate me asure of commitment to apoptotic death. Inhibition of caspases by pept ide inhibitors blocked proteolysis of endogenous substrates and reduce d nuclear condensation yet did not alter either dexamethasone-induced changes in clonogenicity or mitochondrial membrane potential. In contr ast to the results with caspase inhibitors, expression of BCL-2 in CCR F CEM C7A cells proved sufficient to block all changes associated with apoptosis, including loss of both clonogenicity and changes in mitoch ondrial membrane potential. These results demonstrate that commitment to cell death can precede the key biochemical or morphological feature s of apoptosis by several hours and indicate that separate regulators govern cellular commitment to clonogenic death and the subsequent exec ution phase characterised as apoptosis.