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Nucleic Acid Metabolism in Leukemic Human Leukocytes I. in Vitro Incorporation by Leukocytes from Chronic Granulocytic Leukemia*

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Nucleic Acid Metabolism in Leukemic Human Leukocytes I. in Vitro Incorporation by Leukocytes from Chronic Granulocytic Leukemia* Nucleic Acid Metabolism in Leukemic Human Leukocytes I. In Vitro Incorporation by Leukocytes from Chronic Granulocytic Leukemia* ANNAMARIAWILLIAMS (Department of Medicine, University of Wisconsin Medical School, Madison, Wisconsin) SUMMARY A survey has been made of the incorporation of purine and pyrimidine bases, nu- cleosides, and nucleotides into nucleic acid bases and acid-soluble compounds of white blood cells from chronic granulocytic leukemia. The leukocyte suspensions were incu bated in a glucose-salts medium for periods of 1-4 hours. A comparison of the various precursors is summarized in the "Discussion." The information obtained should be useful in choosing precursors to determine in vitro effects of agents on leukocyte nucleic acid metabolism, and in studying isolated enzyme systems from these cells. White blood cells can be obtained in large num white blood cells. The studies reported in the pres bers from patients with chronic leukemias. Sus ent paper were designed to survey the incorpora pensions of these cells are convenient both for tion of various purine and pyrimidine precursors studies of leukocyte metabolism and for possible by leukocytes from a limited number of CGL pa assay systems to test activity and mechanism of tients. Such information should be useful for fur action of therapeutic agents. Under our incubation ther investigations of in vitro effects of agents on conditions, significant incorporation into both leukemic leukocytes and on isolated enzyme sys RNA1 and DNA of leukocyte suspensions from tems from these cells. chronic granulocytic leukemia occurs during time intervals of 1-4 hours. There is low or negligible MATERIALS AND METHODS incorporation into DNA of similar suspensions Preparation and incubation of leukocyte suspen from chronic lymphocytic leukemia or normal sion.—Heparinized blood was removed by veni- blood, although these show good incorporation puncture from leukemic patients having white into RNA. We have, therefore, used leukocytes blood counts of 100,000-400,000 cells per cu. mm. from CGL for the larger number of our studies. The blood was allowed to settle at 3°C. for 1 hour, In another publication (15) we described the use after which the leukocyte-plasma layer was re of adenine-8-C14 in an assay system for testing the moved by aspiration and centrifuged for 5 min action of therapeutic agents in vitro and for test utes in a clinical centrifuge at 1470 X g. The cells ing the effects of in vivo therapy on peripheral were washed in a 3-times volume of saline, centri fuged, and suspended in Robinson's salts mixture * This research was supported by grants from the National Institutes of Health, U.S.P.H.S. (CY-5936), and the K. K. (9), supplemented with 100 ing. per cent glucose Holz Fund. and 209 ing. per cent sodium bicarbonate. Con 1Abbreviations used: RNA, ribonucleic acid; DNA, deoxy- tamination with erythrocytes, as determined by ribonucleic acid; CGL, chronic granulocytic leukemia; PCA, perchloric acid; AMP, GMP, CMP, UMP, the 5'-monophos- counting stained smears of the leukocyte suspen sions, was less than 5 per cent. Duplicate 60-ml. phates of adenosine, guanosine, cytidine, and undine; ADPand GDP, the fl'-diphosphates of adenosine and guanosine; ATP Warburg vessels, each containing 4-6 X IO8 cells and GTP, the 5'-triphosphates of adenosine and guanosine; in 12 ml. medium plus radioactive precursor, were AS, acid-soluble; AIC, 4-amino-5-imidazole carboxamide; incubated at 38°C. in a 95 per cent air: 5 per cent PRPP, 5-phosphorylribose-l-pyrophosphate. carbon dioxide atmosphere. The vessels were re Received for publication September 25, 1961. moved to an ice bath after incubation for the in- 314 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1962 American Association for Cancer Research. WILLIAMS-Nucleic Acid Metabolism in Leukemic Leukocytes 315 cheated time, and 1 ml. 2.6 M PCA was added RESULTS when cells and medium were to be extracted to Incorporation of purine compounds.—In the gether. For separate extraction of cells and medi studies of assay conditions with adenine-8-C14 (15) um, contents of the vessels were centrifuged 10 it was found that incorporation for 1 hour was minutes at 3°C. at 1470 X g, and the supernatant essentially the same under 95 per cent air + 5 per medium from each vessel was poured into 1 ml. cent COj or 95 per cent N2 + 5 per cent COs, and 2.6 M PCA. Cells were washed once with 2 ml. cold with 0.5, 1.0, or 2.0 /¿molesadenine per flask. nonradioactive medium, centrifuged, and then ex Labeling of RNA adenine and guanine did not tracted with 2 ml. 0.2 M PCA. seem to depend on cell maturity, but incorporation Isolation of compounds for radioactivity measure into DNA adenine was related at least in part to ments.—Furine and pyrimidine bases were isolated, the (weighted) percentages of immature cell types. purified, and measured for radioactivity as de All cell suspensions used in the experiments de scribed (15). When specific activities were deter scribed in the present report were from patients mined for both RNA nucleotide and its base in with total leukocyte counts above 100,000 cells per experiments with totally labeled precursors, the cu. mm. and significant numbers of immature mononucleotides were isolated for radioactivity measurements as described previously (14). Spe cific activities of sugar moieties were calculated by difference. Acid-soluble compounds were isolated by the manual addition of a series of eluents of increasing formate ion concentration, described in (12) for purines. The procedure for pyrimidines was modified from the hand-column method de scribed by Blair et al. (2). Acid-soluble extracts pooled from duplicate flasks and adjusted to pH 6.8-7.2 were poured on 1.0 X 5.0 cm. Dowex-1- formate columns and eluted with 40 ml. water (uracil, uridine, cytosine, cytidine), 40 ml. 0.05 M formic acid (CMP), 40 ml. 0.5 M formic acid (AMP), 30 ml. water, 80 ml. 0.2 M ammonium for mate (UMP, orotic acid). Two paper chromatog- raphy systems were used to purify compounds from the column fractions: isopropyl.'HCl (16) for 240 the separation of uracil compounds from cytosine compounds, and isobutyric acid:NHs at pH 2.6 CHART 1.—Incorporation of 2.0 Amólesadenine-8-CH (3) for the separation of uracil or cytosine from its (a X IO6counts/niin//imole) into C(ìLleukocytes. Patient 1. nucleoside and nucleotide. Sources of radioactive compounds.—AMP-8-C14 cells. Differential leukocyte counts of Patients 2 was prepared from ascites tumor cells, GMP-8-C14 and 3 (C. Fr. and A. M.) are given in (15). Flasks from E. coli, and adenosine-8-C14, guanosine-8-C14, with adenine-8-C14 have been included in experi and guanosine-C14 from the corresponding nucleo ments with other precursors to allow comparison side monophosphates as described in (13). Totally of the various incubations. labeled C14 nucleo tides were prepared from Hy- Time curves for the incorporation of adenine- drogenomonas as described in (14). Other com 8-C14 by leukocytes from Patient 1 are shown in pounds were obtained from commercial sources. Chart 1. The rate of increase in labeling was great Presentation of data.—For direct comparisons of est during the 1st hour for acid-soluble AMP and ull experiments specific activity values have been RNA adenine, and during the 2d hour for RNA calculated for a precursor radioactivity of 2 X IO6 guanine. Incorporation into DNA adenine was counts/min/Vmole, which was the specific activity nearly linear for the 4-hour period. Data for an of the adenine-8-C14 and guanine-8-C14. The spe incubation of leukocytes from Patient 2 with 1.0 cific activity of glycine-2-C14 was 6 X IO6 counts/ yumole each of adenine, adenosine, and AMP are min/jumole, and that of other compounds varied given in Table 1. The nucleoside and nucleotide from 0.25 X IO6 to 2.5 X IO6 counts/min//umole. were incorporated to a greater extent than was the With compounds labeled in both base and sugar, base. One- and 2-hour specific activities of RNA calculations have been made for a base specific adenine were similar for nucleoside and nucleotide activity of 2 X IO6 counts/min/jumole. precursors. Labeling of RNA adenine at 4 hours Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1962 American Association for Cancer Research. 316 Cancer Research Vol. 22, April 1962 and of DNA adenine at all time periods was higher DNA guanine was most rapid during the 1st hour. with the AMP precursor, whereas labeling of RNA Similar results for the relative extent of incorpo and DNA guanine was higher with the adenosine ration of the three adenine compounds at 1 hour precursor. Incorporation of both nucleoside and were obtained with cells from another patient. nucleotide into RNA and DNA guanine was high Data for the purine compounds present in sig er with the adenosine precursor. Incorporation of nificant amounts in acid-soluble extracts from the both nucleoside and nucleotide into RNA and 1-hour incorporations of Table 1 are given in TABLE1 INCORPORATIONOF1.0¿iMoLEADENINECOMPOUNDSINTOCGLLEUKOCYTES GUANDÕE(counts/mm//imole)14,700ADENINEDNA ADENINERNA GUANINEDNA C»-PBECUBSOR*Patienta: Adenine-8-C14Adenosine-8-C14AMP-8-C"MIN.60 12060 22,100 +1200t46,300+ 801,220+ 1,500+407,540 484 +50684 500 10 + 590 + 10 120 53,200 + 1300 1,500± 60 11,100 + 100 870 + 34 24060 68,60052,100 2,8502,330 15,5003,980+ 1,740360 + 4400 + 110 30 120 63,500 + 1900 4,120 + 180 6,250 + 350 416 + 16 240RNA 109,0005321,330+ 4,610464 9,450296 1,090 * 2 X IO6counts/min/jjmole.
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