Controllable genetic manipulation of apoptosis of cells in culture.

Apoptosis of mammalian cell is under the control of a wide range of intracellular and extracellular factors-amongst them proteases, protein kinases, cytokines and the protein products of oncogenes and tumour suppressor genes. The c-myc proto-oncogene encodes an essential component of the cell's proliferative machinery and its deregulated expression is implicated in many cancers. Under certain conditions, c-Myc also acts as a potent inducer of apoptosis. We have developed a 'switchable' chimaeric c-Myc protein whose activity is dependent on the synthetic ligand, 4-hydroxytamoxifen. In cells expressing this switchable c-Myc, proliferation and apoptosis in cultured fibroblasts can be regulated 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 inhibited by the action of certain cytokines or by expresson of exogenous proteins with anti-apoptotic potential such as Bcl-2. We show that inhibition of p53 using dominant negative molecules inhibits apoptosis induced by DNA damage but has little effect on Myc-induced apoptosis. Finally, we have also been able to modulate a relatively late stage in apoptosis using inhibitors of cysteine proteases. Our data suggest a model in which the integrated activities of several proteins with diverse molecular functions may determine whether a particular cell undergoes apoptosis but that, once the actual catalytic machinery is engaged, the apoptotic process is irreversible.