Interaction of antifolates with enzymes of the de novo purine pathway in human CCRF-CEM leukaemia cells: Monitored using HPLC linked with UV and radioactive detectors

ABSTRACT

Estimation of the precursors of nucleic acids in the presence of anti-leukaemic developing agents is a valid approach towards seeking a cure for leukaemia. Therefore, in this study the focus was on primary inhibition of the early de novo purine pathway. A radiolabel protocol for optimum hot intermediates of the de novo purine biosynthesis produced in human leukaemia cells CCRF-CEM was developed in this study. According to recent earlier finding in this lab, methotrexate [MTX] and lome-trexol [LTX] showed blockage potency for the de novo synthesis ofpurines due to an accumulation of intracellular 5-phospho-ribosyl-1-pyrophosphate [PRPP]. Therefore, in the current study, the mechanism of action of these two antifolates on de novo purine biosynthesis in human CCRF-CEM leukaemia cells [labelled various metabolites with 14C-glycine] was elucidated by using high-pressure liquid chromatographic [HPLC] technique for separation of acid soluble metabolites and quantified using an ultraviolet detector along with a radioactivity monitor. Total level of nucleotides indicated that both MTX and LTX induced depletion ofATP, ADP, AMP, GTP, GMP, sAMP, IMP and NAD, while GDP was decreased by 63% of the control level only in the MTX [0.1 microM] treated sample. A level of nucleotides synthesized in the de novo purine biosynthesis indicated that both antifolates [0.1 microM] also induced depletion ofATP, ADP, AMP, GTP, GDP, IMP and NAD in treated human leukaemia cells. All mono-, di- and triphosphates of N-formylglycinamide ribotide [FGAR] were also decreased by MTX and LTX in the presence ofazaserine [25 microM]. These results support the finding that two tested antifolates in the present study induced primary inhibition of the early de novo purine pathway in human leukaemia cells CCRF-CEM, i.e. the inhibition of amido phosphoribosyl-transfemse [APRT] catalyses the first committed step of the de novo purine pathway