title: 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 [2-14C]orotate (OA) is quantitatively converted to [2-14C]OMP and then [2-14C]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 microM. 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(alpha)(4-amino-4-deoxypteroyl)-N(delta)-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.
authors.affiliation: School of Molecular and Microbial Biosciences, University of Sydney, NSW 2006, Australia.
authors.forename: M A
authors.initials: MA
authors.lastname: Kamal
authors.forename: R I
authors.initials: RI
authors.lastname: Christopherson
mesh_terms: D000582:Amidophosphoribosyltransferase / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005493:Folic Acid Antagonists / Q000494:pharmacology*; D006801:Humans; D007938:Leukemia; D015394:Molecular Structure; D010754:Phosphoribosyl Pyrophosphate / Q000032:analysis / Q000378:metabolism*; D011739:Pyrimethamine / Q000031:analogs & derivatives* / Q000494:pharmacology*
chemical_list: D000970:Antineoplastic Agents; D004791:Enzyme Inhibitors; D005493:Folic Acid Antagonists; C006308:metoprine; D010754:Phosphoribosyl Pyrophosphate; D000582:Amidophosphoribosyltransferase; D011739:Pyrimethamine