Induction of apoptosis in IL-3-dependent hematopoietic cell lines by guanine nucleotide depletion

Inosine 5'-monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes the conversion of IMP to xanthosine monophosphate (XMP) at the branch point of purine nucleotide biosynthesis, leading to the generation of guanine nucleotides. Inhibition of IMPDH results in the depletion of guanine nucleotides, prevents cell growth by G1 arrest, and induces cell differentiation in a cell-type-specific manner. The molecular and sensing mechanisms underlying these effects are not clear. We have examined the induction of apoptosis by mycophenolic acid (MPA), a specific IMPDH inhibitor, in interleukin-3 (IL-3)-dependent murine hematopoietic cell lines. MPA treatment, at clinically relevant doses, caused apoptosis in 32D myeloid cells and in FL5.12 and BaF3 pre-B cells in the ongoing presence of IL-3. Apoptosis was completely prevented by the addition of guanosine at time points up to 12 hours, after which caspase 3 activity increased and apoptosis was not reversible. MPA treatment caused marked down-regulation of the MAP kinase kinase/extracellular regulatory kinase (MEK/Erk) pathway at 3 hours while simultaneously increasing the phosphorylation of c-Jun kinase. In addition, MPA strongly down-regulated the mammalian target of rapamcyin (mTOR) pathway, as indicated by the decreased phosphorylation of p70 S6 kinase and of 4EBP1. Inhibition of either the mitogen-activated protein kinase (MAPK) or the mTOR pathway alone by standard pharmacologic inhibitors did not induce apoptosis in IL-3-dependent cells, whereas inhibition of both pathways simulated the effects of MPA treatment. These results indicate that IMPDH inhibitors may be effective in modulating signal transduction pathways in hematopoietic cells, suggesting their usefulness in chemotherapeutic regimens for hematologic malignancies.