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Regulation of De Novo Purine Synthesis in Human Bone Marrow Mononuclear Cells by Hypoxanthine

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Regulation of De Novo Purine Synthesis in Human Bone Marrow Mononuclear Cells by Hypoxanthine Regulation of de novo purine synthesis in human bone marrow mononuclear cells by hypoxanthine. M E King, … , J M Honeysett, S B Howell J Clin Invest. 1983;72(3):965-970. https://doi.org/10.1172/JCI111068. Research Article In previous studies from this laboratory, human bone marrow hypoxanthine concentrations were found to average 7.1 microM, three times higher than plasma hypoxanthine concentrations measured simultaneously. To assess the significance of this finding, the relationship between hypoxanthine concentration and the rate of purine nucleotide synthesis by the de novo pathway was studied in normal human bone marrow mononuclear cells and in the human promyelocytic cell line, HL-60, in vitro. Utilizing a [14C]formate incorporation technique, rates of total cellular de novo purine synthesis as well as rates of de novo adenine, de novo guanine, and thymine synthesis and incorporation into RNA and DNA were measured as a function of hypoxanthine concentration. In normal human marrow cells, the rate of total de novo purine synthesis declined by 81%, while the rate of de novo adenine and de novo guanine synthesis and incorporation into macromolecules declined by 89 and 75%, respectively, when media hypoxanthine was increased from 0 to 10 microM. Similar results were seen in the HL-60 cell line. In contrast, rates of thymine synthesis and incorporation into DNA as well as overall rates of RNA and DNA synthesis did not change with varying media hypoxanthine concentrations. In addition, hypoxanthine salvage and incorporation into RNA and DNA was shown to progressively increase with increasing media hypoxanthine concentrations. These results indicate that physiologic […] Find the latest version: https://jci.me/111068/pdf Regulation of De Novo Purine Synthesis in Human Bone Marrow Mononuclear Cells by Hypoxanthine MARK E. KING, J. MICHAEL HONEYSETT, and STEPHEN B. HOWELL, Department of Medicine, the Cancer Center, University of California, Sant Diego, La Jolla, California 92093 A B S T R A C T In previous studies from this laboratory, of RNA and DNA, they regulate enzymatic activity human bone marrow hypoxanthine concentrations as components of vitamins and cofactors, and they were found to average 7.1 MM, three times higher than mediate energy transfer in the cell. This remarkable plasma hypoxanthine concentrations measured simul- diversity of function emphasizes the fundamental im- taneously. To assess the significance of this finding, the portance of these compounds. Mammalian cells obtain relationship between hypoxanthine concentration and purine nucleotides through two separate metabolic the rate of purine nucleotide synthesis by the de novo pathways: by de novo synthesis utilizing 5-phosphori- pathway was studied in normal human bone marrow bosyl-l-pyrophosphate (PRPP)' and glutamine or by mononuclear cells and in the human promyelocytic salvage of extracellular purine bases and nucleosides. cell line, HL-60, in vitro. Utilizing a ['4C]formate in- The regulation of these pathways has been the subject corporation technique, rates of total cellular de novo of numerous investigations, and an early observation purine synthesis as well as rates of de novo adenine, was the strikingly low rate of de novo purine synthesis de novo guanine, and thymine synthesis and incor- in several normal tissues, including intestine and bone poration into RNA and DNA were nmeasured as a func- marrow (1-5). It has been suggested that these tissues tion of hypoxanthine concentration. In normal human have little, if any, capacity for de novo purine synithesis marrow cells, the rate of total de novo purine synthesis and are therefore dependent on the salvage of circu- declined by 81%, while the rate of de novo adenine lating purines (5, 6). Recent studies in this laboratory and de novo guanine synthesis and incorporation into have demonstrated a much higher concentration of the macromolecules declined by 89 and 75%, respectively, purine base hypoxanthine in human bone marrow than when media hypoxanthine was increased from 0 to 10 in plasma (7). This observation has been confirmed by uM. Similar results were seen in the HL-60 cell line. other investigators (8). The average hypoxanthine con- In contrast, rates of thymine synthesis and incorpo- centration in eight bone marrow samples was 7.1 ,uM ration into DNA as well as overall rates of RNA and (range 1.9 to 20 MM), while that in venous plasma DNA synthesis did not change with varying media measured simultaneously was 2.0 MM (range 0.8 to 7.9 hypoxanthine concentrations. In addition, hypoxan- MM). Hypoxanthine has been shown to inhibit the de thine salvage and incorporation into RNA and DNA novo pathway of purine biosynthesis in human and was shown to progressively increase with increasing animal cells in vitro (9-15). Therefore, the apparent media hypoxanthine concentrations. These results in- absence of de novo purine synthesis in bone marrow dicate that physiologic concentrations of hypoxanthine may have been the result of inhibition of this pathway are sufficient to regulate the rate of de novo purine by elevated concentrations of hypoxanthine. synthesis in human bone marrow in vivo. In the present study, the effect of varying media concentrations of hypoxanthine on the rate of de novo INTRODUCTION purine synthesis was investigated in normal human Purine nucleotides participate in a wide variety of bone marrow mononuclear cells and in the human cellular functions. They serve as the building blocks promyelocytic leukemia cell line, HL-60, in vitro. The results demonstrate that physiologic levels of hypo- Dr. Howell is a Clayton Foundation investigator. Dr. King's present address is Oklahoma City Clinic, Oklahoma City, OK 73104. Received for publication I November 1982 and in revised ' Abbreviations used in this paper: PRPP, 5-phosphori- form 6 May 1983. bosyl- 1-pyrophosphate. J. CliGu. Invest. ©) The American Society for Clinical Investigation, Inc. - 0021-9738/83/09/0965/05 $1.00 965 Volume 72 Septemllber 1983 965-970 xanthine markedly reduce the rate of de novo purine min. After cooling, the samples were diluted with 400 Ml of synthesis in both human marrow and HL-60 cells. This water and neutralized with 6 ml of alanine/freon as de- scribed by Khym (18). A 150-AI aliquot of each resulting finding indicates a key regulatory role for physiologic sample was then subjected to high pressure liquid chro- concentrations of hypoxanthine in purine nucleotide matographic analysis of purine and pyrimidine base content metabolism. using a method similar to that of Willis et al. (19). Chro- matography was performed using a Waters Associates liquid METHODS chromatograph at ambient temperature on a Bondapak C- 18 reverse phase column (Waters Associates, Inc., Milford, HL-60 cell line and bone marrow samples. The human MA) with a gradient developed from buffer A (10 mM po- promyelocytic cell line HL-60 (16) was obtained from Dr. tassium phosphate, pH 5.0) and buffer B (60% methanol in Steven Collins (Veterans Administration Medical Center, water). Absorbance was measured at 254 nM and 280 nM Seattle, WA). Exponentially growing cells were routinely and peak areas determined with a Waters Associates model cultured in RPMI 1640 medium (cell culture facility, Uni- 730 data module. The bases were quantitated and elution versity of California, San Diego, La Jolla, CA) supplemented times confirmed by comparison with pure standards. The with 10% fetal calf serum (Irvine Scientific, Santa Ana, CA), adenine, guanine, and thymine peaks were collected sepa- 1% glutamine, and 1% antibiotic/antimycotic mixture (both rately and quantitated in 10-ml volumes of Aquasol by liquid from Gibco Laboratories, Grand Island, NY). Before use in scintillation counting. the experiments described below, cells were washed and re- Measurement of rates of purine salvage incorporation suspended in RPMI 1640 alone to eliminate preformed pu- into RNA and DNA. In the HL-60 cell line, rates of hy- rines from the media. poxanthine salvage and incorporation into RNA and DNA Heparinized bone marrow aspirates were obtained from were determined with varying media hypoxanthine concen- either normal volunteers or patients undergoing staging bone trations. 2-ml aliquots of cells (106 cells/ml) were incubated marrow examinations in the evaluation of cancer. In all can- for 90 min in ['4C]hypoxanthine (0.1 mCi/ml, 56 mCi/mmol; cer patients reported here, concomitant histologic studies of Moravek Biochemical, City of Industry, CA). The total me- bone marrow aspirate and biopsy material were normal. The dia hypoxanthine concentration varied from 0.1 to 10 MM, bone marrow mononuclear cells were isolated by Ficoll-Hy- but serial dilutions of stock ['4C]hypoxanthine were used such paque gradient centrifugation (17) and resuspended in RPMI that the specific activity of the ['4C]hypoxanthine was the 1640 medium. The viability of both cultured and bone mar- same (56 mCi/mmol) for each sample. The incubation was row cells during the incubation periods of experiments de- terminated by centrifugation at 4°C for 5 min at 500 g and scribed below was >95% by trypan blue dye exclusion. the supernatent discarded. The cell pellet was resuspended Measurement of rates of total cellular de novo purine in 2 ml of ice-cold 10% trichloroacetic acid and maintained synthesis. Rates of total cellular de novo purine synthesis on ice for 20 min. The precipitate was collected on GF/C were determined by a modification of the method of Hersh- filter paper (Whatman Chemical Separation Inc., Clifton, field and Seegmiller (12). 2-ml samples of cells (106 cells/ml) NJ) and washed three times with 5 ml of cold 10% trichlo- were incubated in concentrations of hypoxanthine (Sigma roacetic acid. The filters were dried and then suspended in Chemical Co., St. Louis, MO) varying 0-10 mmol for 60 min 10 ml of Betaphase (West Chem Products, San Diego, CA) at 37°C under a 5% CO2 atmosphere with occasional shaking.
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