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Effect of Methotrexate on 5-Phosphoribosyl 1-Pyrophosphate Levels in L1210 Leukemia Cells In vitro¹

Section of Experimental Chemotherapy, Antoni van Leeuwenhoek Laboratory, Netherlands Cancer Institute, and Department of Oncology, Free University Hospital, Amsterdam, The Netherlands

The cellular content of 5-phosphoribosyl 1-pyrophosphate (PRPP) and PRPP synthetase activity levels was examined in cultured L1210 mouse leukemia cells after exposure to methotrexate (MTX). PRPP was determined in cold perchloric acid extracts of cells using a radiochemical method measuring the conversion of [¹⁴C]adenine to [¹⁴C]adenosine 5'-monophosphate in the presence of added adenine phosphoribosyltransferase activity. PRPP synthetase activity was measured in dialyzed cell-free extracts by a coupled enzyme assay determining PRPP production using the adenine phosphoribosyltransferase reaction. The intracellular levels of PRPP in untreated cells during logarithmic growth varied over 24 hr with a mean of 145 ± 65 (S.D.) pmol/10⁶ cells. Treatment of cells with 1 µM MTX caused an increase in PRPP levels by 1.5 to 3 hr with maximal levels of 5- to 10-fold higher than controls. Elevated PRPP levels persisted for up to 12 hr and declined to control values after 24 hr. Similar results were obtained with 0.1 µM MTX, while exposure of cells to 0.01 µM MTX caused a more gradual increase in cellular PRPP content which was maximal at 12 hr and was maintained for at least an additional 12 hr. The addition of 0.1 mM hypoxanthine led to recovery of PRPP to elevated levels in cells exposed to 0.1 µM MTX for 1.5 hr but not for 10 hr. No large difference in PRPP synthetase activity levels between MTX-treated cells and untreated cells was observed.

The results indicate that in L1210 cells exposure to MTX leads to increased levels of PRPP, more probably due to decreased utilization of PRPP in de novo purine synthesis than as a result of increased PRPP synthesis. Enhanced levels of PRPP are available for the reutilization of preformed purines; however, PRPP levels are afterwards only partially replenished with prolonged drug exposure because of decreased cellular capacity for PRPP synthesis despite the continued presence of PRPP synthetase activity.

¹ Supported in part by the Queen Wilhelmina Fund (Project UUKC 77-3).

² Recipient of research fellowship from the General Hospital of Asturias, Oviedo, Spain.

³ To whom requests for reprints should be addressed, at Section of Experimental Chemotherapy, Antoni van Leeuwenhoek Institute, Amsterdam, The Netherlands.

Received 5/ 9/79. Accepted 10/ 5/79.