MYCOPHENOLIC ACID-INDUCED GTP DEPLETION ALSO AFFECTS ATP AND PYRIMIDINE SYNTHESIS IN MITOGEN-STIMULATED PRIMARY HUMAN T-LYMPHOCYTES1

Abstract

Mycophenolate mofetil (MMF) is an effective immunosuppressant developed for use in organ transplantation. It specifically targets lymphocyte purine biosynthesis. However, side effects do occur. Understanding how the active metabolite of MMF, mycophenolic acid (MPA) affects the normally integrated interaction between intracellular purine and pyrimidine pathways might aid the development of improved therapeutic regimes. We used a primary human T-lymphocyte model to study how preincubation with MPA (0.1-50 microM) affected normal ribonucleotide pool responses to phytohemagglutinin using radiolabeled precursors. MPA not only restricted the mitogen-induced expansion of GTP pools, but actually induced a severe drop in both GTP (10% of unstimulated cells) and GDP-sugar pools, with a concomitant fall in ATP (up to 50%). These effects were concentration dependent. By contrast, uridine pools expanded whereas CTP pools remained at resting levels. These changes were confirmed by the altered incorporation of [14C]-bicarbonate and [14C]-glycine into nucleotides. Restriction of [14C]-hypoxanthine incorporation and reduction of [14C]-uridine uptake comparable to that of unstimulated cells indicated that MPA also inhibited both salvage routes of nucleotide synthesis. MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms underlying these disproportionate changes can best be explained by MPA-related inhibition of amidophosphoribosyltransferase, catalysing the first step in purine biosynthesis. This would increase phosphoribosylpyrophosphate availability, thereby stimulating UTP biosynthesis. Such imbalances, coupled with ATP-depletion, could underlie reported side effects and might be overcome by appropriate combination therapies.