Comparative Study of 6-Mercaptopurine Metabolism in Human Leukemic Leukocytes and L1210 Cells

Abstract

Leukocytes from patients with leukemia and L1210 cells from mice were examined for the rate offormation and cellular concentration of phosphoribosylpyrophosphate, the rate of thioinosinic acid formation, and a number of selected enzymes involved in purine nucleotide synthesis. The amount of thioinosinic acid formed in L1210 cells was much higher than that in human leukemic leukocytes. In cell extracts, the synthesis of thioinosinic acid was similar in both cell types, and the amount of purine phosphoribosyltransferase was not rate limiting in either case. Much higher concentrations and rates of formation of phosphoribosylpyrophosphate were found in L1210 cells than in human leukemic leukocytes. The difference in response to 6-mercaptopurine between L1210 cells and human leukemic leukocytes might be attributed to their difference in supply of phosphoribosylpyrophosphate. Phosphoribosylpyrophosphate-amidotransferase was found to be high in L1210 cells, but was not detected in human leukemic leukocytes.The enzymes responsible for purine nucleotide synthesis have been only partially studied in L1210 cells (4,26). Few studies have been done on the characterization of the enzymes that are essential to the mechanism of action of 6-mercaptopurine (6-MP) in humans (5,19,20). It is well known that nucleotides can be formed by means of a de novo pathway from single metabolites or, alternatively, synthesized from preformed purine bases by means of salvage pathways. Synthesis ofpurine nucleotides from preformed purines in cells has been shown to be controlled more extensively by the endogenous supply of phosphoribosylpyrophosphate (PRPP) than by the activity of the purine phosphoribosyltransferase in experimental animal tumor cells (7), human erythrocytes (24), and lymphoblasts (6). To study the regulation of purine nucleotide synthesis and to clarify the mechanism of action of 6-MP in humans, we have undertaken comparative studies on the salvage and de novo pathways, the cellular concentration of PRPP, and the enzymes responsible for PRPP formation in human leukemic leukocytes and L1210 cells. been given 6-MP during the preceding month. Blood was drawn by venipuncture into heparinized tubes. The plasma and buffy coat were separated by centrifugation in the cold and then removed. Sedimented erythrocytes were washed twice with cold, 0.9% NaCl solution.Isolation of leukocytes. Leukocytes were isolated from peripheral blood by the dextran sedimentation procedure (23). The cells were mixed with 1 volume of 3% dextran in 0.9% NaCl solution and allowed to sediment at room temperature for 30 to 45 min until a good separation was noted. The supernatant containing leukocytes, platelets, and some erythrocytes was decanted into a test tube in which all subsequent steps were performed. The cells were sedimented at 500 x g for 10 min, washed with 0.9% NaCl solution, and sedimented at 500 x g for 10 min. The cells were suspended in 0.9% NaCl solution and sedimented as described above. The leukocyte pellet was suspended in 1 ml of 0.9% N...