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

Home , Adenine, Adenosine

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, nucleosides, 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.â€”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 3Â°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 38Â°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.â€”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 3Â°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 /Â¿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.â€”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 chromatography 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.â€”Incorporation of 2.0 AmÃ³lesadenine-8-CH (3) for the separation of uracil or cytosine from its (a X IO6counts/niin//imole) into C(Ã¬Lleukocytes. Patient 1. nucleoside and nucleotide. Sources of radioactive compounds.â€”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.â€”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 INCORPORATIONOF1.0Â¿iMoLEADENINECOMPOUNDSINTOCGLLEUKOCYTES

GUANDÃ•E(counts/mm//imole)14,700ADENINEDNA ADENINERNA GUANINEDNA CÂ»-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 1,500Â± 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. t Variation from the mean of duplicate flasks.

TABLE 2 Table 2. With the adenine precursor, 1.65 /Â¿moles ACID-SOLUBLEEXTRACTSFROMi-HoimINCORPORATIONS(of the original 2.00 /Â¿moles/2flasks) of adenine OFADENINECOMPOUNDSINTABLEi* and only 0.05 /tmole hypoxanthine were recovered at 1 hour. The low specific activity of hypoxan MEDIUM(counts/mm/Minole)1,790,000155,000302,000245,000202,00054,30010,0001,740,000468,000370,000608,0001,050,0002,040,000410,000928,000864AND thine compared with that of adenine indicates that most of the hypoxanthine was formed from an CÂ»-PRECCKSORtAdenine-8-C14Adenosine-8-C"AMP-8-C"COMPOUNDISOLATEDAdenineAdenosineAMPADPATPHypoxanthineGMPAdenosineAMPADPATPInosineHypoxanthineGMPAdenosineAMPADPATPHypoxanthineGMPCELLSintracellular adenine compound (s). On the other hand, only 0.11 /miÃ³leadenosine was recovered at 1 hour from the incorporation of adenosine-8- C14, plus 0.89 /Â¿moleof hypoxanthine with the original precursor specific activity and 0.23 //mole inosine with half the specific activity of the hy poxanthine. Adenosine was degraded much more rapidly than AMP, since with the latter precursor only 0.18 /Â¿molehypoxanthine was recovered. The AMP precursor was converted to adenosine, but, interestingly, inosine was not present in sufficient amounts to be isolated. The greater degradation occurring when adenosine was the precursor may represent a response of catabolic systems to an initially large amount of exogenous compound, ,000391 with a gradual production of smaller amounts of ,000745,000760,00013,200Girnole)1.65.08.21.0512.05.05.11.39.06.25.23.89.05.45.43.30.37.18.05adenosine from AMP not resulting in the same extent of degradation. Specific activities of ADP and ATP were sig nificantly higher with nucleotide and nucleoside * Two flasks, 1.0 /umole/flask, pooled for analyses, precursors than with the adenine precursor. The t 2 X IO6counts/rain//jmole. specific activity of acid-soluble GMP with the

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1962 American Association for Cancer Research. WILLIAMSâ€”NucleicAcidMetabolism in LeukemiaLeukocytes 317 adenosine precursor was 31X and 41X higher soluble AMP was much more highly labeled by the than with AMP and adenine precursors, which guanine precursor at 1 hour than was acid-soluble corresponds to the higher labeling of RNA and GMP by the adenine precursor. DNA guanine at 1 hour by the nucleoside. The Guanosine and xanthosine + xanthine were iso conversion of adenosine to inosine (and through lated from the incorporation of guanosine-8-C14 IMP to GMP) might be the reason for this more by the leukocytes from Patient 2. The specific rapid labeling of acid-soluble GMP. activity of guanosine (0.76 gniole) was 820,000 In Table 3 are compared the incorporations of counts/min//unole and that of xanthine + xan 1.0 /Â¿moleguanine, guanosine, GMP, and adenine thosine (0.27 /Â¿mole)was 1,780,000 counts/min/ into leukocytes from Patients 2 and 3. In contrast /Â¿mole.Aswith adenine compounds, the nucleoside to results for adenine compounds, guanine was was degraded more rapidly than the base. incorporated to a greater extent than were nucleo Incorporation of pyrimidine compounds.â€”Data side and nucleotide into RNA bases and to the from four 1- and 2-hour incubations of pyrimidine same extent into DNA adenine. Guanosine gave compounds with leukocytes from Patients 3-6 are the highest labeling of DNA guanine. In another given in Table 4. Thymine and uracil were poorly experiment specific activities at 1 hour were found incorporated, whereas cytosine was incorporated

TABLE 3 INCORPORATIONOFi.o /Â¿MOLEGUANINECOMPOUNDSINTOCGL LEUKOCYTES

ADEXIXE(counts/min/Vmole)5,110+GUANINEDNA GUANINERNA ADENINEDNA CJ1-PREOTHSOR*Patient

2: Guanine-8-C14Guanosine-8-C14 220f 4 + 220 + 18 120 10,300+ 240 684+ 14 9,200 + 320 224 + 10 60 2,140+ 90 1,080+ 50 896+ 68 122+ 10 Adenine-8-C14Patient 6060 764 +2624,400 208+282,920 11,400Â±1905,540+ 636+44414 3: Guanine-8-C14 + 1300 + 260 70 Â±2 Guanosine-C14 60 7,740+ 310 3,700 + 350 1,440 + 110 425 GMP-CÂ» 60 3,000+ 40 980+ 85 1,300+ 60 500 + 60 Adenine-8-C14Mm.60 60RNA 2,010480+ 3825,430 22,900120 2,610

* 2 X IO6counts/min/jumole. t Variation from the mean of duplicate flasks.

to be essentially the same with 0.5, 1.0, and 2.0 to a greater extent than were the other two bases AmÃ³lesguanine. With cells from Patient 3, guanine â€”although to a lesser extent than its nucleoside and adenine labeled their corresponding RNA and and nucleotide. If labeling by adenine-8-C14 is used DNA bases to about the same extent, but incorpo as a standard to compare different incubations, it ration of guanine into RNA adenine was almost can be calculated that cytidine was better incor 3 X that of adenine into RNA guanine. With cells porated than GMP into all parameters but DNA from Patient 2, adenine was better incorporated cytosine. In the 2-hour incubation with cells from into the corresponding RNA and DNA base, but Patient 4, uridine was better incorporated than incorporation of guanine into RNA adenine was UMP into all parameters but DNA thymine. In 7X that of adenine into RNA guanine. Acid- both 1- and 2-hour incubations, GMP labeled all soluble AMP (0.05 /Â¿mole)from the duplicate nucleic acid pyrimidines but thymine to a greater guanine-8-C14 flasks with cells from Patient 2 had extent than did UMP. Orotic acid was incorpo a specific activity of 124,000 counts/min//tmole, rated better than uracil at 1 and 2 hours into RNA compared with 1,750,000 counts/min//umole for bases, but not into DNA bases. Thymidine was guanine (1.09 jumÃ³le)and 444,000 counts/min/ much better incorporated than its base into DNA /Â¿moleforxanthine (0.05 /Â¿mole).Ifthese data are cytosine. The uridine precursor in the same experi compared with those in Table 2 for the incorpora ment gave a much higher labeling of DNA cyto tion of adenine-8-C14 by leukocytes from Patient sine. 2 in a different incubation, it can be seen that acid- Pyrimidine compounds present in significant

Cu-precursor*Patient cytosine053+ cytosine10447Â± mine240114

8:Thvmine-4-C14UrÃ cil-2-C14UMP-CÂ»CMP-C14Adenine-8-C14Patient

11,190Â± 5 96002,5704,860Â§2,7809701,880819Â§245018Â±183,150+ Â±402,0801,130350||1,7805,8401,3601Â«||1221601,760 1012,60026,600t4.7302,3004,64022 6011,2004,010#27,4007,48015,3001,460#000021,400

4:Uridine-C14UMP-C"CMP-C"Adenine-8-Ã‡14Patient

,100t00002,400+

5:Thymine-2-C14Thymidine-2-C14I'ridine-C14Patient

350+ +704,630 1318Â± +10852 905,46011079666604,170+2580912666060250+ +1,00028,4001,5902,010126114608Â±481.6301470168204430

6:Orotic acid-2-C14Uracil-2-C14Cytosine-2-C"Cytidine-4-C14Adenine-8-C14Min.6060606060HO1201201206014060140601206014060120601206014060RNA

3504,500+ 30314 +103,140Â± +24412 14039,40013,00041,400 Â±28824 5030,800+ Â±10544 +341,780 70044,500+ +102, +3,20017,000tDNA Â±110992 4001,770#Thy 420Â±50neu

Â§Uracil0112+

* 2 X 10Â«counts/min/Mmole. Â§DNAadenine. t Variation from the mean of duplicate flasks. # RNA guanine. t RNA adenine. || DNA guanine.

TABLE 5 ACID-SOLUBLEEXTRACTSFROM1-HouR INCORPORATIONS OFPYRIMIDINE COMPOUNDSINTTABLE4*

Cu-PHECUH8ORtPatient

Pmole)1,254,00015,900683,000398,000142,000(/Â¿mole).07.10.05.03.06CELLS+MEDIÃœM(counts/min//Â¿mole)1,590,0001,480,0001,460,000(/Â¿mole).97.18.52

6:Cytosine-2-C14Cytidine-4-C14Patient

pmole)1,290,000111,00047,200(itmole)1.24.05.34MEDIUM(counts/min/

3:CMP-C14COMPOUNDISOLATEDCytosineIMPCytidineUridineCMPUMPCMPLridineUMPCELLS(counts/mm/

* Two flasks, 1.0 ^mole/flask, pooled for analyses, t 2 X IO6 counts/min/jutnole.

amounts in acid-soluble extracts from the 1-hour of the pyrimidine-ribosyl linkage. Cellular acid- incubations of cytosine, cytidine, and CMP are soluble UMP and nucleic acid UMP had higher shown in Table 5. As noted in "Materials and percentages of their total radioactivities in the Methods," elution of columns was carried through sugar moiety than were present in the precursor UMP but not through UDP and UTP. Cytidine uridine, in contrast to the increased base radio was extensively deaminated to uridine; at 1 hour activities resulting when purine compounds were there was almost 3X as much uridine as cytidine precursors. present in the medium, and no cytidine was re Incorporation of glycine and AIC.â€”Table 7 covered from the acid-soluble extract of the cells, compares precursors of de novo purine biosynthesis although CMP was isolated from this fraction. with adenine-8-C14. Labeling by glycine-2-C14 was The CMP precursor was also converted to UMP measurable but low, whereas a compound entering and uridine but was not deaminated to the same at a later point in the de novo pathway, 4-amino-5- extent as the nucleoside, paralleling results with imidazole carboxamide-2-C14, was relatively well AMP and adenosine. No uracil or uridine was iso incorporated. AIC-2-C14 was incorporated some lated from the acid-soluble extract of the 1-hour what better than adenine-8-C14 into RX1A guanine, cytosine incorporation. Conversion of cytidine to but adenine labeled the other nucleic acid bases to uridine could explain the greater difference be tween cytidine-2-C14 and cytosine-2-C14 in labeling a greater extent. RXA uracil (51X higher with the cytidine precur TABLE 6 sor) than in labeling the other pyrimidine bases 1-HouRINCORPORATIONSOFCOMPOUNDS (2-9X higher with the cytidine precursor). TOTALLYLABELEDWITHC14 Measurements with totally labeled compounds.â€” ^To determine the extent of breakage of the base- inbase/C" in ribosyl linkage during these short incorporations C14-prerursorPatient isolatedAS-GMP*GMPAMPAS-Guanosine*GMPAMPAS-AMP*AMPGMPAS-UMP*UMPCMPCÂ»base/CÂ» by leukocytes, the specific activities of both RNA in sugar.19.58.40.77Compound insugar1.971.181.913.4*5.921.531.772.662.50.50.66.76 nucleotides and their bases were measured after 3:GMP-CÂ»Guanosine-C"Patient 1-hour incubations with nucleotides and nucleosides totally labeled with C14. Incorporation into DNA was not high enough to warrant similar de terminations for DXA nucleotides. Xo free purine or pyrimidine bases were found in the acid-soluble extracts of medium or cells. As shown in Table 6, 4:AMP-CÂ»Patient both nucleic acid and cellular acid-soluble nucleo tides from purine incorporations had higher per centages of their total radioactivities in the purine moiety than were present in the exogenous pre 5:Uridine-CuCÂ» cursors. Thus, considerable breakage of the purine-ribosyl linkage occurred before and/or during incorporation into acid-soluble compounds. Data for uridine-C14, however, indicated little breakage * Acid-soluble extract of cells.

TABLE 7 2-HouR INCORPORATIONOFi.o MMOLEFURINEPRECURSORS

ADEXIXERNAcrAxixE(counts/min//imole)5SADENINEDNA ADENINERNA GUANINEDNA

Patienta:Glycine-2-C"AIC-2-C"Adenine-8-C14AS +8t83,00016+ 23,76021,400 24001,220 81,7001,370 + 1,00011+ + 8039+ + 3018+2290444Â±46

*a X IO6counts/min/jumole. t Variation from the mean of duplicate flasks.

DISCUSSION greater extent than adenine labeled RNA guanine. Various other workers (4, 10; reviewed in [15]) Incorporation of glycine was barely measurable, have studied the in vitro incorporation of nucleic but AIC was not too inferior to the preformed acid hases into human leukocytes. However, ex purine bases as a precursor. cept for autoradiographic studies with tritiated Uracil and cytosine nucleosides and nucleotides thymidine and uridine, an investigation of the in were incorporated to a greater extent than were corporation of various nucleosides and nucleotides their bases, and nucleosides gave a higher labeling by human leukocytes has not been reported. In of RNA. Cytosine compounds were incorporated the present work, no study was made of permeabil better than the corresponding uracil compounds, ity to the various compounds, so that relative and orotic acid was incorporated better than uracil rates of incorporation could represent a combina into RNA bases. Thymidine gave a much higher tion of permeability and metabolism by the cells. labeling of DNA thymine than did thymine, but Although it was shown that a portion of the nu- both were incorporated to about the same extent cleotide precursor was dephosphorylated, the into DNA cytosine. Pyrimidine bases were not in fraction which entered the cell as nucleoside in corporated as well as purine bases. Labeling by stead of nucleotide was not determined. It is uracil and cytosine nucleosides and nucleotides known that alkaline phosphatase levels are low was greater than by guanosine and GMP but less in chronic granulocytic leukemic leukocytes, but than by adenosine and AMP. that acid phosphatase levels are higher than in The differences in incorporation of adenine and normal blood cells (1). guanine compounds were similar to those found Data from short incubations with totally la with Ehrlich ascites cells in vivo (14), where ade beled compounds indicated little breakage of the nine was incorporated less rapidly than were its pyrimidine-ribosyl linkage, but significant separa nucleoside and nucleotide but guanine was incor tion of purine base and sugar before incorporation porated as well as or better than guanosine and into nucleic acids. Extensive breakage of the GMP. Cytosine has been stated to be inferior to purine-ribosyl linkage was found during in vivo uracil as a precursor of nucleic acid pyrimidines incorporations by Ehrlich ascites tumor cells (14) and has not been found to react with ribose-1- and has also been reported in other systems (re phosphate, deoxyribose-1-phosphate, or with viewed in [14]). Reichard (7) found that a mince of PRPP in certain systems which are active with chick embryo cells incorporated uniformly labeled uracil (6). It should be interesting to study these cytidine-C14 into RNA cytidine without rupture reactions in extracts of the leukemic blood cells, of the ribosyl linkage and listed other references since cytosine was a considerably better precursor which indicated that pyrimidine compounds were than uracil with these cells. The better utilization incorporated relatively intact. However, Harbers of cytidine than of uridine for nucleic acid synthe and Heidelberger (5) reported that totally labeled sis has been reported for other tissues (6) and can uridine and deoxyuridine were rapidly cleaved to be partly explained by the finding of Reichard and uracil by Ehrlich ascites cells. Neither free purine Skold (8) that pyrimidine nucleoside kinase needs nor pyrimidine bases were found in significant a higher concentration of uridine than of cytidine amounts in acid-soluble extracts of the blood cells for optimal synthesis of nucleotides. when the exogenous precursor was nucleoside or Reaction in the de novosynthesis of pyrimidines nucleotide. preceding orotic acid were not investigated in the Here it would be of interest to summarize re present study. However, Smith and Baker (11) sults and compare the utilization of purines and have demonstrated aspartate carbamyl transfer- pyrimidines. In the adenine series, nucleotide and ase, dihydroorotase, and dihydroorotic dehydro- nucleoside were incorporated to a greater extent genase in sonicates of normal and leukemic human than was the base. Nucleotide gave the highest leukocytes. It has been shown in the present inves labeling of RNA and DNA adenine; nucleoside, of tigation that the leukemic leukocytes can carry RNA and DNA guanine. In the guanine series, out the final reactions in the de novo synthesis of the base was incorporated to a greater extent than purines, although a compound involved at an were nucleoside and nucleotide into RNA bases early stage (glycine) was only poorly utilized. and to the same extent into DNA adenine; nucleo These studies should be helpful in choosing pre side gave the highest labeling of DNA guanine. cursors to determine in vitro effects of agents, such Adenosine and AMP were incorporated much bet as drugs or sera, on leukocyte nucleic acid metabo ter than were guanosine and GMP, whereas dif lism. It would be necessary, of course, to weigh ferences between the two bases were not marked, both relative amounts of incorporation and rela except that guanine labeled RNA adenine to a tive rates of degradation in making such choices,

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1962 American Association for Cancer Research. WILLIAMSâ€”Nucleic Acid Metabolism in Leukemic Leukocytes 321 and to consider the metabolic products (particu 7. REICHARD,P. The Biosynthesis of Deoxyribonucleic Acid by the Chick Embryo. I. Utilization of Cytidine-C14. J. larly deaminated compounds) present in the medi Biol. Chem., 234:1244-48, 1959. um in evaluating the results. In addition, it is 8. REICHARD,P.,and SKOLD,O. Enzymes of Uracil Metabo hoped that the information obtained in this survey lism in the Ehrlich Ascites Tumour and Mammalian Liver. with whole cells with be useful in selecting isolated Biochim. et Biophys. Acta, 28:376-85, 1958. enzyme systems from these leukocytes for inves 9. ROBINSON,J. R. Some Effects of Glucose and Calcium tigation. upon the Metabolism of Kidney Slices from Adult and Newborn Rats. Biochem. J., 45:68-74, 1949. ACKNOWLEDGMENTS 10. SHAPIRA,J.; BORNSTEIN,L; WELLS,W.; and WINZLF.R, The author expresses her appreciation to Miss Elaine R. J. Metabolism of Human Leukocytes in Vitro. IV. In Joranger and Miss Frances Morris for technical help, and to corporation and Interconversion of Adenine and Guanine. Dr. R. F. Schilling, in whose laboratory this work was done. Cancer Research, 21:265-70, 1961. 11. SMITH,L.H., JR.; BAKER,F. A.; and SULLIVAN,M.Pyrim- REFERENCES idine Metabolism in Man. II. Studies of Leukemic Cells. 1. BECK,W. S., and VALENTINE,W.N. Biochemical Studies Blood, 16:360-69, 1960. on Leucocytes. II. Phosphatase Activity in Chronic Lym 12. WILLIAMS,A.M., and LEPAGE,G. A. Furine Metabolism phatic Leukemia, Acute Leukemia, and Miscellaneous in Mouse Ascites Tumor Cells. I. Effects of Preformed HÃ©matologieConditions.J. Lab. & Clin. Med., 38:245-53, Purines on in Vitro Incorporation of Glycine-2-C14. Can 1951. cer Research, 18:548-53, 1958. 2. BLAIR,D. G. R.; STONE,J. E.; and POTTER,V. R. Forma tion of Orotidine-5'-Phosphate by Enzymes from Rat 13. . Furine Metabolism in Mouse Ascites Tumor Cells. Liver. J. Biol. Chem., 236:2379-86, 1960. II. In Vitro Incorporation of Preformed Purines into Nu- cleotides and Polynucleotides. Ibid., pp. 554-61. 3. BLOCK,R. J.; DURRUM,E. L.; and ZWEIG,G. A Manual of Paper Chromatography and Paper Electrophoresis. 3d ed. 14. . Furine Metabolism in Mouse Ascites Tumor Cells. New York: Academic Press, 1958. III. In Vivo Incorporation of Preformed Purines into Nu- cleotides and Polynucleotides. Ibid., pp. 562-68. 4. GAVOSTO,F.;MARAINI,G.; and PII/ERI,A. Nucleic Acids and Protein Metabolism in Acute Leukemia Cells. Blood, 15. WILLIAMS,A.M., and SCHILLING,R.F. The in Vitro In 16:1555-63, 1960. corporation of Nucleic Acid Precursors into Leukemic Hu 5. HARBERS,E., and HEIDELBERGER,C.Studies on Nucleic man Leukocytes. I. Methodology and Effects of Therapy Acid Biosynthesis in Ehrlich Ascites Cells Suspended in a on Incorporation in Vitro. J. Lab. & Clin. Med., 58:76-85, Medium Permitting Growth. J. Biol. Chem., 234:1249-54, 1961. 1959. 16. WYATT,G. R. The Furine and Pyrimidine Composition of 6. POTTER,V. R. Nucleic Acid Outlines, pp. 168-69. Min Deoxypentose Nucleic Acids. Biochem. J., 48:584-90, neapolis: Burgess Publishing Company, 1960. 1951.

Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1962 American Association for Cancer Research. Nucleic Acid Metabolism in Leukemic Human Leukocytes: I. In Vitro Incorporation by Leukocytes from Chronic Granulocytic Leukemia

Anna Maria Williams

Cancer Res 1962;22:314-321.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/22/3/314

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/22/3/314. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.