The Role of the Protein Kinase C (PKC) Family in Glucocorticoid-Induced Apoptosis of Human Leukemic Cells.

Glucocorticoids (GC’s) are among the most important clinical agents for the treatment of human leukemias. Cells exist, however, that have a functional glucocorticoid (GC) receptor, but are resistance to GC-induced apoptosis—suggesting that major signal transduction pathways (ie PKC family) may influence cellular response to GC. In order to determine whether glucocorticoid-induced apoptosis is mediated by members of the PKC family, we first screened the CEM acute T-cell lymphoblastic leukemic cell line for the presence or absence of all known PKC isoforms. We used two clonal cell lines that were derived from the same parent, CEM-CCRF, but which subsequently developed a pronounced difference in their sensitivity to glucocorticoids. One of these cell lines, CEM C7-14 is sensitive to dexamethasone-induced apoptosis, while the second cell line, CEM C1-15, is resistant. Of the 12 known PKC isoforms, 5 were detected at the basal level, and furthermore that specific members (PKC iota) was up-regulated during resistance, while PKC theta was up-regulated during apoptosis. Selectively inhibiting the entire PKC family caused increased cellular susceptibility to GC-induced apoptosis. Selectively targeting specific isoforms (PKC iota) caused resistant cells to become sensitive to apoptosis. Using immunoflouresence imaging, we showed the selective translocation of PKC’s iota, theta, and alpha after treatment with glucocorticoids. In addition, we showed specific phosphorylation of the isoforms iota, theta, and alpha in cells undergoing DEX-induced apoptosis. Two-dimensional proteomic analysis indicated a large number of proteins in CEM cells that show significant change after DEX treatment. We successfully identified several proteins which were regulated both after treatment with DEX alone, and after treatment with DEX + the PKC specific inhibitor Safingol. These proteins may represent the potential cross talk between the glucocorticoid and PKC apoptosis pathways. Taken together, these studies show that the PKC family is directly involved in glucocorticoid-induced apoptosis.