The identification of the most suitable molecular targets for gene and drug therapy is the crucial first step in the development of new disease treatments. The rational identification of such targets depends on a detailed understanding of the pathological changes occuring at the molecular level. We have applied forward genetics approaches to the identification of the critical genes involved in the control of apoptosis in mammalian cells, since defective control of apoptosis underlies many diseases, including cancer and neurodegenerative diseases. We have identified two groups of genes by their effects on cell survival using retroviral cDNA functional expression cloning and retroviral insertional mutagenesis. The identification of these novel genes opens up new areas for apoptosis research and subsequently for the development of new gene and drug therapies.

Human promyelocytic leukemia (HL-60) cells can be induced to differentiate into macrophage-like cells by the tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Addition of this agent to HL-60 cells causes a rapid internalization of surface transferrin receptor, followed by long-term receptor down-regulation at the level of gene expression. These effects precede the inhibition of proliferation and the acquisition of differentiation markers, and it has been suggested that transferrin receptor down-regulation may play a mediating role in these later events. Here we show that HL-60 cells will grow indefinitely in serum-free medium supplemented with either 5 micrograms ml-1 transferrin or 300 microM ferric citrate and that TPA inhibits cell proliferation (assayed by cell density and rate of thymidine incorporation) and induces macrophage-like differentiation (assayed by induction of cell adhesion and increased nonspecific esterase activity) with identical dose curves in both media. Furthermore, a neutralizing anti-transferrin antibody completely inhibits transferrin-dependent cell proliferation but has no effect on differentiation in the presence or absence of transferrin. We conclude that TPA-induced down-regulation of transferrin binding and internalization does not mediate the subsequent growth arrest and differentiation of HL-60 cells.