Accumulation of 5-phosphoribosyl-1-pyrophosphate in human CCRF-CEM leukaemia cells treated with antifolates

Abstract

Amido phosphoribosyltransferase (APRT) catalyzes the first step of the de novo biosynthesis of purine nucleotides, the conversion of 5-phosphoribosyl-1-pyrophosphate (PRPP) into 5-phosphoribosylamine (PRA). APRT is a valid target for development of inhibitors as anticancer drugs. We have developed a thin layer chromatographic assay for PRPP extracted from cells. Using coupling enzymes, PRPP with excess [2- 14 C ]orotate (OA) is quantitatively converted to [2- 14 C ]OMP and then [2- 14 C ]UMP with hydrolysis of the PPi. The reaction products are isolated on poly(ethyleneimine)-cellulose (PEI-C) chromatograms. Human CCRF-CEM leukaemia cells growing in culture have been exposed to a number of antifolates and their effects upon cellular levels of PRPP determined. The steady-state level of PRPP measured in CCRF-CEM cells was 102±11 μM. Following addition of an antifolate to a culture, accumulation of PRPP in cells indicates the degree of inhibition of APRT. In human CCRF-CEM leukaemia cells, lometrexol (LTX), 2,4-diamino-6-(3,4,5-trimethoxybenzyl)-5,6,7,8-tetrahydro-quinazoline (PY899), methotrexate (MTX), N α(4-amino-4-deoxypteroyl)- N δ-hemiphthaloyl- l-ornithine (PT523), piritrexim (PTX), metoprine, 2,4-diamino-6-(3,4,5-trimethoxyanilino)-methylpyrido[3,2- d]pyrimidine (PY873) and multitargeted antifolate, N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3- d]pyrimidin-5-yl)ethyl]benzoyl]- l-glutamic acid (MTA) directly or indirectly induce inhibition of APRT indicated by time-courses for accumulation of PRPP to maximum values of 3–12-fold. These data indicate that LTX induces the most potent inhibition of APRT.