Mycophenolic acid-induced GTP depletion also affects ATP and pyrimidine synthesis in mitogen-stimulated primary human T-lymphocytes

Background. Mycophenolate mofetil (MMF) is an effective immunosuppressant d eveloped for use in organ transplantation. It specifically targets lymphocy te purine biosynthesis, However, side effects do occur. Understanding how t he active metabolite of MMF, mycophenolic acid (MPA) affects the normally i ntegrated interaction between intracellular purine and pyrimidine pathways might aid the development of improved therapeutic regimes. Methods, We used a primary human T-lymphocyte model to study how preincubat ion with MPA (0.1-50 mu M) affected normal ribonucleotide pool responses to phytohemagglutinin using radiolabeled precursors. Results, 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 ef fects were concentration dependent. By contrast, uridine pools expanded whe reas CTP pools remained at resting levels, These changes were confirmed by the altered incorporation of [C-14]-bicarbonate and [C-14]-glycine into nuc leotides. Restriction of [C-14]-hypoxanthine incorporation and reduction of [C-14]-uridine uptake comparable to that of unstimulated cells indicated t hat MPA also inhibited both salvage routes of nucleotide synthesis. Conclusion. MPA affects pyrimidine as well as purine responses to mitogens in T-lymphocytes, but not in an integrated way. The molecular mechanisms un derlying these disproportionate changes can best be explained by MPA-relate d inhibition of amidophosphoribosyltransferase, catalysing the first step i n purine biosynthesis. This would increase phosphoribosylpyrophosphate avai lability, thereby stimulating UTP biosynthesis, Such imbalances, coupled wi th ATP-depletion, could underlie reported side effects and might be overcom e by appropriate combination therapies.