CELL-CYCLE SPECIFIC INDUCTION OF HL-60 CELL-DIFFERENTIATION AND APOPTOSIS BY MYCOPHENOLIC-ACID

HL-60 cells undergo terminal differentiation and apoptosis in response to different types of sub-toxic and toxic perturbations respectively. The mechanism by which cells sense different amounts of perturbation to activate pathways that lead to the engagement of a relevant biologi cal response is not known, The response of HL-60 cells to treatment wi th the immunosuppressant mycophenolic acid (MPA), a specific inhibitor of dGTP/GTP-synthesis, allowed quantitation of a metabolic perturbati on which triggered a cellular response, 1.5 mu M MPA induced 38% termi nal differentiation to CD14 positive, early monocyte-like cells and 22 % cell death by apoptosis, whereas 3 mu M MPA induced 70% apoptosis bu t no differentiation, Despite the difference in biological outcomes, 7 2 h exposure to both 1.5 mu M and 3 mu M MPA caused a similar (similar to 75%) depletion of total GTP levels, Cells synchronized by centrifu gal elutriation were treated with MPA, Elutriated cells were overall l ess sensitive to the effects of MPA but 3 mu M MPA induced significant ly less apoptosis and more differentiation in an elutriation-enriched G1-population than in a population normally distributed in the cell cy cle, suggesting that the effects of MPA in S-phase may subsequently le ad to cell death, However, analysis of apoptosis by using a terminal d eoxynucleotidyltransferase assay and measurement of bromodeoxyuridine incorporation showed that apoptosis was engaged in G1. Analysis of the phosphorylation status of the retinoblastoma protein demonstrated tha t Rb was hypophosphorylated prior to apoptosis and that in apoptotic c ells, separated by flow cytometry, Rb protein was absent, presumably d ue to proteolysis, The loss of Rb protein did not appear to permit tra nsit to S-phase, and was not accompanied by an expression of c-Myc, Su rprisingly, therefore, an antimetabolite inducing a loss of GTP brough t about cell death by apoptosis in the G1 phase of the cell cycle.