Identification of apoptotic tyrosine‐phosphorylated proteins after etoposide or retinoic acid treatment

A main shortcoming of using HL‐60 cells as a model of granulocyte‐macrophage differentiation is that some cells in the differentiating population undergo apoptosis. To address this issue, we have identified which tyrosine‐phosphorylated proteins are involved in apoptosis and differentiation, respectively. HL‐60 cells were induced specifically to undergo apoptosis with 68 μ M etoposide, and to undergo granulocytic differentiation with 1 μ M retinoic acid (RA). The corresponding two‐dimensional electrophoretic maps of tyrosine‐phosphorylated proteins from treated cells were compared. In the 8 h etoposide‐treated HL‐60 cell population, 83% of the cells were apoptotic. In the 120 h RA‐treated cells, 50% of the cells were apoptotic. Eighteen cytosolic and nuclear tyrosine‐phosphorylated proteins were found in both the 8 h etoposide‐ and the 120 h RA‐treated cells, but not in the proliferating HL‐60 cell population. Matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry analyses suggested that some of the proteins may be involved in signal transduction pathways (NFκB, GTP‐binding protein, protein disulfide isomerase, Cyclophilin A), others in cell transcriptional and translational control (hnRNP H, hnRNP L, Hsp60, Hp1, Hcc‐1, 26S proteasome beta‐subunit, ATP synthase beta‐chain), and a third group in cell cytoskeleton organization and receptor cycling (profilin, caveolin‐1). An understanding of signal transduction in apoptosis initiation by screening for tyrosine‐phosphorylated proteins associated with apoptosis may provide new targets for the treatment of leukemia.