CONTROLLABLE GENETIC MANIPULATION OF APOPTOSIS OF CELLS IN CULTURE

Apoptosis of mammalian cell is under the control of a wide range of in tracellular and extracellular factors - amongst them proteases, protei n kinases, cytokines and the protein products of oncogenes and tumour suppressor genes. The c-myc proto-oncogene encodes an essential compon ent of the cell's proliferative machinery and its deregulated expressi on is implicated in many cancers. Under certain conditions, c-Myc also acts as a potent inducer of apoptosis. We have developed a 'switchabl e' chimaeric c-Myc protein whose activity is dependent on the syntheti c ligand, 4-hydroxytamoxifen. In cells expressing this switchable c-My c, proliferation and apoptosis in cultured fibroblasts can be regulate d by addition of 4-hydroxytamoxifen. We have further demonstrated the utility of a switchable gene transcription system for the induction of proteins with pro-apoptotic effect. Myc-induced apoptosis is inhibite d by the action of certain cytokines or by expresson of exogenous prot eins with anti-apoptotic potential such as Bcl-2. We show that inhibit ion of p53 using dominant negative molecules inhibits apoptosis induce d by DNA damage but has little effect on Myc-induced apoptosis. Finall y, we have also been able to modulate a relatively late stage in apopt osis using inhibitors of cysteine proteases. Our data suggest a model in which the integrated activities of several proteins with diverse mo lecular functions may determine whether a particular cell undergoes ap optosis but that, once the actual catalytic machinery is engaged, the apoptotic process is irreversible.