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Metabolism of Human Leukocytes in Vitro I. Effects of A-Methopterin on Formate-C14 Incorporation' RICHARDJ. WINZLER,ALBERTD. WILLIAMS,ANDWILLIAMR. BEST WITH THETECHNICALASSISTANCE OF IRENEBOHNSTEIX,MAHTJo BURR,ANDWARRENWELLS (Departments of Biological Chemistry and of Medicine, Unirersity of Illinois, College of Medicine, Chicago, III.} A considerable amount of evidence exists to show that various types of human leukocytes may differ markedly in their content of certain en zymes and in their metabolic behavior. Much of this work has been previously reviewed (2, 3, 5, 6, 11, 13, 14, 18, 25-27). Additional biochemical studies with human leukocytes are reported here. Biochemical differ ences between normal leukocytes and those from the several types of leukemic patients are of particular importance, since such dissimilarities may be re sponsible for the variations in clinical response to chemotherapeutic agents used in the treatment of leukemia. It is likewise possible that development of resistance by leukemic patients to a given therapeutic agent may reflect metabolic changes in morphologically similar leukocytes. Our approach to this problem has been to study the incorporation of isotopie precursors into the ribonucleic acid and protein of isolated leukocytes. These studies have provided further evidence that leukocytes isolated from the blood of normal indi viduals and from the blood of patients with chronic lymphocytic (CLL), chronic granulocytic (CGL), or acute leukemia (AL) differ biochemical- and leukemic human leukocytes. In the present re port, the effects of one of these agents, A-methop terin, on the incorporation, in vitro, of formate-C14 into the gross protein fraction, into serine, and into the purine ribonucleotides of human leukocytes will be described. MATERIALS AND METHODS Separation of leukocytes.—-Types of leukemia were identified by usual clinical criteria (20). For purposes of analysis, the acute leukemias have been considered as a single group and include pre dominantly individuals over 15 years of age suf fering from acutely granulocytic or undifferentiated acute leukemia. Leukocytes from peripheral blood samples of normal individuals and leukemic patients were obtained1 by a method (Chart 1) which is essentially that reported by Beck and Valentine (3). A 60-80 per cent recovery of leukocytes was obtained by this method. The preparations frequently contained equal numbers of leukocytes and erythrocytes. This red cell con tamination affected the results to be reported here only negligibly. Preliminary studies showed that detectable amounts of formate-C14 were not in corporated by erythrocytes. However, a small error was introduced by the contribution of the red cells in the incubation mixture to the planchet weights. The error was estimated to average about 25 per cent with CLL cells and about 15 per cent with other types of cells. Usually, 300 ml. of blood was drawn for the isolation of leukocytes from normal individuals. The amount needed from leukemic patients depended upon the peripheral cell count, about 30 ml. being obtained from pa- ly. Certain chemotherapeutic agents have proved moderately effective in bringing about temporary remission of some leukemias. The clinical use of several such compounds including 4-amino-N10methyl-pteroylglutamic acid (A-methopterin) (10), 1,4-dimethanesulfonoxybutane (Myleran) (15), 6-mercaptopurine (Purinthal) (8), and Odiazo-acetyl-L-serine (azaserine) (12) has prompted a study of the effects of these drugs, and others, on certain aspects of the metabolism of normal 1Blood specimens were obtained from patients at the Illi nois Research and Educational, Cook County, West Side Veterans Administration, and Presbyterian Hospitals in Chicago, and at the Veterans Administration Hospital, Hiñes, Illinois. We wish to acknowledge and thank the many physi cians from these institutions who aided in this regard. »This work was supported by grants Cl828(C-3) and C2347(C-2) from the National Institutes of Health, Public Health Service. Received for publication September 15,1956. 108 Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. WiNZLER et al.—Metabolism of Human Leukocytes 109 tients with a white count of 100,000/mm. Sixty to 90 minutes were required to prepare the leuko cytes for incubation. Per cent inhibition Incubations.—A calculated volume of freshly drawn or once-frozen serum from normal donors, supplemented with 3 mg of glucose/ml, was added to the concentrated leuko cytes to yield a suspension containing 1 X 10* white cells/ml. One-mi, aliquots of this suspension were distributed into 10-ml. beakers for incubation. Further additions to the incubation vessels consisted of 1 ml. of normal human serum supple mented with 3 mg glucose/ml for control studies, or 1 ml. of the same serum containing A-methopterin and supplemented with 3 mg of glucose/ml.2 The concentrations of A-methopterin used are indicated in the charts and tables. The vessels were shaken at 100-120 oscillations per minute in a Dubnoff meta bolic shaking incubator at 37°C. in an atmosphere of 95 per Separation of ribonucleic acid purine nucleotides.—Ribonucleic acid (RNA) was extracted from a portion of the GPF and was hydrolyzed to the free ribonucleotides with previ ously described technics (28). Following a preliminary sepa ration of the ribonucleotides on a Dowex-1 formate anión exchange column (17), the purine ribonucleotides were further purified by filter paper ionophoresis and by filter paper chro- cent Oj:5 per cent COj. After a 15-minute preincubation period, 0.2 ml. of 0.9 per cent saline containing 2 juc. (usually 0.1 mg.) of sodium formate-C14 was added,8 and the vessels were incubated for 4 hours. In some cases Krebs-Ringerbicarbonate (24) containing 6 mg of glucose/ml (KRBG) was used instead of serum. This modification and other variations are indicated in the specific charts and tables in which results are given. A minimum of three and usually four or more repli cate incubations was carried out for each variable in a single experiment. Separation of "gross protein fraction."—After incubation = loo x ( i - cpm/mg in experimental incubations cpm/mg in control incubation BLOOD Collect in citrate-fibrinogenl?(l ml. 3.8 per cent sodium citrate and 10 mg. bovine fibrinogen for each 10 ml. of blood) andiallow[to stand for 30 minutes. ÃŒ LEUKOCYTE SUSPENSION RED CELLSEDIMENT Centrifuge at 3000 r.p.m. for 10 minutes LEUKOCYTES the cells were recovered and washed twice with 0.9 per cent saline by centrifugation. They were then resuspended in 2 ml. of 5 per cent trichloroacetic acid (TCA) at 5°C. to obtain the TCA-insoluble fraction. It was found that five washes with2-ml. portions of cold 5 per cent TCA were necessary to remove acidsoluble radioactivity from the insoluble fraction. The TCAinsoluble residue was extracted twice at 60°C. with 3 ml. of a 3:1 ethanol-ether mixture, and, after a final ether wash, the insoluble residue was dried. The resulting lipide-free residue is designated the "gross protein fraction" (GPF). It was dis solved in 1 ml. of 90 per cent formic acid and dried under an infrared lamp on tared copper planchéis for determination of radioactivity. After drying, the plancheta were reweighed to obtain the dry weight of the GPF on the planchet for selfabsorption corrections and calculation of formate incorpora tion.4 The weight of protein on each planchet was between 4 and 10 mg., depending on the type of cells used. Radioactivity was measured with a shielded mica end-window counter, and the samples were counted for a sufficient time to reduce the counting error to less than 5 per cent. The standard error of the mean of three to five replicate incubations carried out with each variable in a given experiment rarely exceeded 10 per cent. The values shown in the tables represent the mean and stand ard deviations of separate experiments carried out at different times with leukocytes from different sources. The results for the GPF were expressed as /¿molesof formate incorporated/ mg of protein and as per cent inhibition by A-methopterin, with the following relations: /¿molesformate incorporated = tic formate/mg protein pc formate 1 We wish to acknowledge X ¿¿moles formate added added generous gifts of A-methopterin, folie acid, and leucovorin from the Lederle Company, Pearl River, New York. 1 Formate-C14 was obtained from Isotopes Specialties Co., Burbank, California, and from Nuclear Instrument Co., Chicago, Illinois. \ / ' PLASMA Wash with saline Suspend at 108cells/ml in glucoseserum Agitate 4 hrs. in Oj-COs at 37°C. with isotope, centrifuge cells, and wash with saline \ SERUM AND WASH LEUKOCYTES Extract cells 5 times with cold TCA TCA-DJSOLDBLE FRACTION TCA-8OLUBLE FRACTION Extract with hot ethanol-ether GROSSPROTEINFRAOTION (GPF) CHART 1.—Isolation, incubation, cytes. LIPIDS and fractionation of leuko matography as described previously (29). Quantitative measurement of purine nucleotides was made spectrophotometrically, and the radioactivity of infinitely thin deposits of the purine nucleotides on copper planchéis was determined. The specific activities of RNA adenylic and guanylic acids were expressed as pinoles of formate incorporated/^mole of the compound. Separation of protein-bound serine.—Serine was isolated from another portion of the GPF which had been hydrolyzed 4 Although drying of formic acid alone in the copper planchéis resulted in an increase in weight, this did not occur when formic acid solutions of protein were dried. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 110 Cancer Research in 6 N HCl by being autoclaved at 115°-120° C. for Ij hours. After removal of excess HCl by evaporation to dryness in vacuo, the residue was dissolved in 0.01 N HCI, and the amino acids were absorbed on a 10 X 1 cm. Dowex-SO column (hydrogen form). The column was washed with 20 ml. of 0.01 N HCl and was then eluted with three 10-ml. fractions of l N NHjOH, the second fraction containing all the amino acids (80). Small portions of the concentrated amino acid mixture were applied as a 10-cm. band on Whatman No. 1 filter paper and developed with a water-saturated phenol solvent (9). Parallel strips from each edge of the paper were sprayed with ninhydrin solution and dried to locate the various amino acid bands. The serine band, well separated from other amino acids, was eluted with water. One aliquot of the eluate was used for quantitative amino acid assay by the ninhydrin reaction (28), while another aliquot was counted at infinite thinness for radioactivity. The specific activity of serine was expressed as jumóles of formate incorporated/iimole of serine. The major portion of the radio activity in the amino acid mixture was found in the serine band. An average of 2.5 mg of serine/100 mg of GPF was recovered from CGL and CLL cells. TABLE 1 INFLUENCEOFMEDIUMONFORMATE-C'*UPTAKEBY CHRONICGRANULOCYTIC LEUKEMIACELLS* Medium No. ezpa. Per cent of normal aerum control 9 100 Normal serum control 164+ 15ÃŽ 6 Dialy zed normal serum t Krebs-Ringer-bicarbonate-glucose 7 249± 50 Normal serum —¿ 95 per cent N2: 5 per 4 48 to 120 cent CO2 * 10»cells incubated 4 hours at 37°C. in 2.2 ml. normal serum containing 6 mg. of glucose and 1.47 Amóles formateC14 (2 ¿tc.)in an atmosphere of 95 per cent O2: 5 per cent CO2, unless otherwise indicated. t Dialyzed 24 hours at 5°C. against Krebs- Ringer-bicar bonate-glucose solution. ÃŽStandard deviation. Separation of acid-soluble adenylic acid. —¿ Most of the TCA was removed from the acid-soluble fraction by extraction with ether. The nucleotides were separated by chromatography on Dowex-1 formate ion exchange columns essentially by the method described by Hurlbert et al. (17). The ultra violet-absorbing peaks eluted from the column were located spectrophotometrically, and the tubes from the adenylic acid (AMP) peak were pooled and concentrated by lyophylization for further purification by Chromatographie tech nics described previously (29) . The specific activity of AMP was expressed as Amóles of formate incorporated/Vmole of the compound. in the KRBG solution and in various modifications thereof. Since it was desired to maintain the cells in as normal a physiological state as possible, most of the work to be reported in this paper was carried out with cells incubated in serum from normal donors, the serum being used immediately or kept in the frozen state until used. Leukocytes incu bated with formate-C14 in serum which previously had been dialyzed against KRBG buffer resulted in an increased rate of formate uptake (Table 1). The effect of anaerobiosis on formate incorpora tion has been somewhat variable. However, in only one instance has more than a 25 per cent inhibition been observed in the absence of oxygen. Erythrocytes incubated under the conditions described for leukocytes did not incorporate formate-C14 into the GPF to a significant extent. Preparations made by exposing leukocytes in KRBG buffer to sonic vibrations (Raytheon-2KW, 3 minutes), in which less than 2 per cent of the leukocytes re mained intact, just prior to incubations, did not fix formate into a protein-bound state. Variations of the numbers of cells from 0.5 X IO8 to 2.0 X IO8 per incubation vessel had little influence on the specific activity of the isolated GPF. Formate-C14 incorporation was reduced in the absence of glucose. TABLE 2 EFFECTOFA-METHOPTERIN ONFORMATE-C" INCORPORATION BYLEUKOCYTES* cent inhibition by A-mcthopterinî + 0.62§(37) 3.9 + 4.1 (33) 11.1+ 5.1 (35) 6.7±5.4(25) CLL 21.2+ 7.2 (49) CGL 80 5 ±8 . 2 (49) 60.6 + 15.8 (13)Per S5.0±4.5(1S) ALIsotope * 10a cells incubated 4 hours at 37°C. in 2.2 ml. of Type cellNormal of t4.8 incorporât ion normal serum containing 6 mg. of glucose and 1.47 Amóles (2 /ne.) formate-C" in an atmosphere of 95 per cent Oj: 5 per cent COj. Number of experiments indi cated in parentheses. t Amólesformate incorporated X 10~'/mg of protein in 4 hours. t A-methopterin §Standard concentration, 100 /jg/ml. deviation. Formate-C1*incorporation into the "gross protein fraction" (GPF) of different types of leukocytes.— RESULTS Influence of medium. —¿ Preliminary experiments Marked differences were observed in the rates of were carried out with cells from patients with formate-C14 incorporation into the GPF of leuko chronic granulocytic leukemia incubated for 4 cytes from normal individuals, from patients hours in various media to determine some of the with chronic lymphocytic leukemic (CLL) or factors influencing formate uptake. Some of these with chronic granulocytic leukemia (CGL), and results are summarized in Table 1. It is of interest from adults with acute leukemia (AL). The that formate-C14 was incorporated into the GPF incorporation rate increased in the order: normal <CLL<CGL<AL, with only slight overlapping more rapidly when cells were incubated in KRBG solution than when they were incubated in normal between groups. This is shown in Table 2 for all human serum. However, the cells usually clumped the data thus far available. Also included in this Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. WiNZLER et al.—Metabolism of Human Leukocytes table are the effects of A-methopterin on formateC14incorporation by these cells. These results will be discussed later. The uptake of formate was linear with time, as is shown in Chart 2, forCGL cells in the presence and absence of A-methopterin. It is evident that the standard deviations for formate incorporation were relatively large in the leukemic series. The relation of this variability to variations in the average maturity of cellular populations was considered. Chronic granulocytic leukemia was chosen for this comparison because of the larger number of studies which were avail able for analysis and because of the greater range of relative cellular maturity in this series. Ade quate data for analysis were available in 37 studies on twenty patients with chronic granulocytic leukemia. Cellular maturity was expressed as the percentage of myelocytes plus progranulocytes plus myeloblasts, based on differential counts of 100 cells on peripheral smears. Chart 3 shows the relation of formate uptake to cellular maturity in five patients studied serially. Consecutive tests are joined by lines char acteristic for each patient. A positive slope indi cates that immature populations tended to in corporate formate more rapidly than did mature populations during that segment of the patient's disease encompassed by the consecutive tests. On this graph there are fourteen positive slopes and three negative slopes. If there were no relation be Relotion of Leukocyte 60-.? Immoturity 111 tween cellular maturity and formate uptake in consecutive tests, there should be an equal number of positive and negative slopes. The probability of the observed differences occurring on the basis of chance alone is less than 1 in 100. Chart 4 combines the first tests on these five patients with single tests on the other fifteen patients. The scatter of values CHART2.—Timecourse of formate incorporation into CGL leukocytes in the presence and absence of A-methopterin. 10* CGL cells incubated at 37°C. in normal human serum containing Õ¡ic.(0.1 mg.) sodium formate-C14 in the presence and absence of 100 ¡igA-methopterin/ml. to Leukocyte Incorporation of Formóte tests)Therapy testo at time of NoneD Myleran Cortisone«X-rayStatus A 50ooo.en test:o six mos. after Alive 40-E\Sa.Jso-oO,t ¿a• ¿ro Dead Unknown ¿j,-'s-'s''S Ac.^,'' Exac^^•*,,»•**'„****Tests S•''•ff/"ai'' q«./\ .''jki/ 20-0 ,''''/ 0—09o /TW ¿¿W \> \ .--•""""" '"'rf^3?^ \ I ^^ <Õ/^¡ ' &' ^,»,-''' 10-0u_0(Serial sequentiallyPatients: linked testsf~ i— f AT First of serial ff n —¿o*T.R,56 J. B.,43 9T. d*O.Y., D.,39 49 9 ! 10 20 30 40 Myelocytes -I- progranulocytes 50 60 -I- myeloblasts 70 (percent) 80 90 CHAUT3.—Theeffect of cellular maturity on formate incorporation into CGL leukocytes (serial studies in five patients) Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 112 Cancer Research suggests a crude correlation, and this is confirmed by calculation of a correlation coefficient, which is 0.54 with a P value less than .02. This graph in cludes data for one patient who was in preterminal acute exacerbation of his disease. One might ob ject to inclusion of acute exacerbations on the grounds that the process has undergone some fundamental change other than just a change in relative maturity. Of the entire 37 tests, including repeats, two tests were made during acute exacer bation. A correlation coefficient on the remaining 35 tests is 0.45 with P less than 0.01. Thus, there appears to be little doubt that formate uptake is related to relative cellular maturity. Charts 3 and the concentrations of the formate-C14 used in some of the early experiments were different from those used subsequently, it was necessary to deter mine the effects of formate concentration on the extent of formate incorporation. The fraction of formate-C14 incorporation into the GPF was be tween 0.1 and 1.0 per cent of the amount added, and most of the added radioactivity remained as formate in the medium. The results of typical ex periments with the several types of leukocytes are given in Chart 5. These data show that all the types of cells achieved a maximum uptake of the isotope when the formate concentration was above about 0.5 ¿imoles/ml. All data reported here have Relotion of Leukocyte Immoturity to Leukocyte Incorporotion (Individual Patients) 60 Therapy at time of tests * of Formóte 0 None 0 Myleran * X-roy u-fe-mercaptopurine > !?50 ^* u Streptonivison ok. o. 6.40 E ^^ Status 6mos. öftertest °Alive •¿ Dead »Unknown First of serial tests \<i^^ ^ \^"** Ac. ft%*«^'-' Exac. o."1^ ^^^ V'• ^'' 30c o o o C ¡OH o D. i O IO i ! I ! i ! 20 30 40 50 60 70 Myelocytes +• progronulocytes + myeloblasts (percent) i ; 80 90 (x) CHART4.—The effect of cellular maturity on formate incorporation into CGL leukocytes (first determinations in twenty patients). and 4 indicate therapy at time of testing as well as subsequent fate of the patients. No relationship between formate uptake and concomitant therapy is evident. There is a suggestion, which cannot be statistically validated with the present data, that, as the course of the disease progresses, formate up take becomes greater, even at the same level of cellular maturity. Thus, there appears to be little doubt that a direct correlation exists between formate uptake and leukocyte maturity. The specific activity of the labeled cellular com ponents depends on the specific activity of the exogenous formate pool and the concentration of formate in the medium. Since the specific activity been expressed as /¿molesof formate incorporated/ mg of protein at this saturation level of formate, unless otherwise stated. In some cases this in volved correction from observations made at lower formate concentrations with the use of the curves in Chart 5. Formate-C1* incorporation into serine and into acid-soluble and RNA purine nucleotides.—Table 3 shows the specific activities of the gross protein fraction, protein-bound serine, RNA adenine and guanine, and acid-soluble adenylic acid (AMP) of an experiment with CGL cells incubated in KRBG medium. The results are expressed as /imoles of formate incorporated per /¿moleof the compound Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. WiNZLER et al.—Metabolism of Human Leukocytes except for the gross protein fraction. The specific activities of serine and the RNA purines were comparable, while that of the acid-soluble AMP was some tenfold higher. Seventy to 80 per cent of the total radioactivity could be accounted for as protein-bound serine. Very little radioactivity was observed in other amino acids, including methionine or histidine, into which formate may be incorporated in some systems. The radioactivity in RNA adenine and guanine accounted for part but not for all the remaining radioactivity in the GPF. 70-, 113 poration into the GPF of AL cells was intermedi ate between CGL and normal leukocytes. The per cent inhibition by A-methopterin of formate in corporation into the GPF was the same for CGL cells incubated in normal serum, KRBG medium, or serum dialyzed against KRBG medium. Table 3 shows that A-methopterin inhibited formate-C14 incorporation into the GPF, the pro tein-bound serine, the RNA purines, and acidsoluble AMP of CGL cells to approximately the same extent. The effect of A-methopterin on formate incorpo ration by CGL cells was immediate and nonpro gressive. In the experiment shown in Chart 2, CGL leukocytes were equilibrated with A-methopterin for 15 minutes prior to the addition of the radio active formate. Pre-incubation of the cells in the TABLE 3 EFFECTOFA-METHOPTERIN ONUPTAKE OFFORMATE INTOSEVERAL COMPONENTS OFCHRONIC GRANÃœLOCYTIC LEUKEMIA LEUKOCYTES Tissue component Gross protein fractionf Control43.6 X10-"0.06 terin*8.9 X10-"0. cent inhibi tion80807f. X10-» 012X10-» Protein-bound serineî 0. 065X10-* 0. 016X10-* RNA adeninel 0.05 X10-» 0. 006X10-» 88 RNA guaninef 1.47 X10-*A-Methop0.11 X10-*Per 92.5 Acid-soluble AMPJ * 100 pg A-methopterin/ml. t ¿«mole formate incorporated/mg GPF. } jumóleformate incorporated//jmole of component. presence of A-methopterin for as long as 2 hours before the addition of the labeled substrate did not increase the inhibitory effect of the folie acid an tagonist. This absence of a latent period for the A-methopterin effect suggests that neither perme ability barriers nor conversion to active deriva 0.3 0.4 0.5 0.6 0.7 0.8 02 tives is involved in A-methopterin inhibition of Formate formate uptake by CGL cells. The fact that formate uptake could be only CHART5.—Theeffect of formate concentration on formate incorporation into normal, CLL, CGL, and AL leukocytes. 10* partially inhibited (80 per cent in CGL and 50 per cells incubated in normal human serum for 4 hours at 37°C. cent in AL cells, Table 2) even at high levels of A-methopterin was of particular interest. Previous Effect of A-methopterin.—The effect of A- in vitro studies with a transplantable mouse leuke mia (29) showed that a similar A-methopterin-remethopterin on the incorporation of radioactive formate into the GPF of leukocytes from patients sistant formate uptake occurred in this tissue, and that development of A-methopterin resistance was with different types of leukemia differed conspicu paralleled by an increase in the A-methopterinously. This is shown in the second column of Table 2, in which it is evident that formate incorporation insensitive formate incorporation. Others have shown a folie acid antagonist-resistant formate up into the GPF by normal and by CLL cells was es sentially unaffected by high concentrations of take in leukemic mouse spleen (1) and rabbit bone A-methopterin, whereas uptake of formate-C14 marrow (22). Typical dose-response curves, in which formate into this fraction by CGL cells was inhibited by about 80 per cent. Inhibition of formate-C14 incor uptake is plotted as a function of A-methopterin Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. 114 Cancer Research concentration, are shown in Chart 6 for normal, CLL, CGL, and AL leukocytes. This figure shows that the maximum effect of A-methopterin on CGL and AL cells was reached at concentrations of about 2-4 jug/ml, and that higher concentrations of A-methopterin did not increase the inhibition. The low level of A-methopterin necessary for the maximum inhibitory effect is in accord with those affecting formate incorporation in vitro into purines of rabbit bone marrow suspension (22) and into the protein fraction of mouse leukemia (29). than does folie acid (21). This same relationship was found in the present experiments. Chart 7 shows that there was a marked stimulation of for mate-C14 incorporation by CGL cells incubated with leucovorin2 in the absence of A-methopterin. There was a reversal of the inhibitory effect of A-methopterin by rather high concentrations of leucovorin in a manner suggestive of a competitive relationship. Folie acid2 produced neither the stimulating nor the A-methopterin-reversing ef fects shown by leucovorin. DISCUSSION It is interesting that the rate of formate incor poration into leukocytes was faster in KrebsRinger-bicarbonate-glucose (KRBG) medium than in normal serum (Table 1). Some of this ef fect might have been due to a dilution of the spe cific activity of the exogenous formate pool by one- 150 •¿ AL 'CLL -.CGL ~°Norm 10 100 . Amethopterir\/ml. CHART6.—¿ Effect of A-methopterin concentration on for mate uptake by normal, CLL, CGL, and AL leukocytes. 10* cells incubated at 37°C. for 4 hours in normal human serum containing 2 me. (0.1 mg.) sodium fonnate-C14. The same series of patients with chronic granulocytic leukemia analyzed in Charts 3 and 4 were examined for a relationship between A-methop terin inhibition and cellular immaturity. No sig nificant correlation was found between inhibition by A-methopterin of formate incorporation and the percentages of myelocytes plus progranulocytes plus myeloblasts in 35 tests on these twenty patients. Consideration of therapy and subsequent clinical course in these patients did not reveal any apparent relationship to per cent inhibition by A-methopterin. Effect of leucovorin.—¿ It has been shown in other systems (16) that citrovorum factor more effec tively reverses the effects of folie acid antagonists o" 1 1 1 —¿i 1 10 20 30 40 50 Leucovorin (pg./flask) CHART7.—Leucovorinreversal of A-methopterin inhibition of formate incorporation into CGL leukocytes. 10* CGL cells incubated at 37°C. for 4 hours in normal human serum con taining 2 me. (0.1 mg.) sodium formate-C". carbon donors in the serum. However, dialysis of serum against KRBG solution did not result in uptake rates as high as those seen in cells incubat ed in KRBG. Therefore, components which inhibit formate uptake may be present in normal serum. The per cent inhibition by A-methopterin of for mate incorporation into CGL fractions was the same in KRBG solution as in serum, indicating that the inhibition was not governed by the abso lute rate of formate uptake. Also, the uptake of formate by AL cells was much higher than by CGL Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. WiNZLER et al.—Metabolism of Human Leukocytes cells, although the inhibitory effect of A-methopterin was considerably greater in CGL cells. The incorporation of formate by leukocytes in the absence of oxygen was studied in four experi ments with CGL cells. In these cases the per cent uptake ranged from 48 to 120 per cent of that ob served in control aerobic experiments (Table 1). These results suggest that the high glycolysis rates of leukocytes (2, 4) are sufficient to provide what ever energy is required for the incorporation reactions. The data reported in Table 2 are consonant with other work which indicates consistent differences in the metabolism and composition of various types of human leukocytes (2-6, 11, 13, 14, 18, 25-27). It is evident that human leukocytes may be as different biochemically and metabolically as they are morphologically. The available evidence is not sufficient to deter mine whether the different rates of formate incor poration by the several types of leukocytes are due to differences in net de novo synthesis or to differ ences in exchange reaction rates involving active one-carbon units. Buchanan (7) and others have demonstrated such exchange reactions by purine nucleotides in cell-free systems. The question of de novo synthesis is one of considerable significance. The formate uptake into serine and RNA-purines resulted in specific activities which corresponded to an uptake of .06 X 10~2 Amólesof formate/ jumóleat the end of a 4-hour incubation period in CGL cells (Table 3). Thus, the de novo synthesis of about 0.06 per cent of the amount of serine, RNA adenine, or guanine initially present would account for the radioactivity found in these com pounds. De novo synthesis of this slight magnitude is certainly a possibility. Measurement of the rela tive incorporation of formate into the carbon 2 and carbon 8 positions of RNA-purines should aid in distinguishing between de novo synthesis and ex change reactions, and such experiments are cur rently being carried out in our laboratory. The physiological and biochemical basis for the differences in the A-methopterin sensitivity of dif ferent types of cells is a problem of fundamental biological and clinical interest. Such differences may underlie the variations seen in the clinical ef fectiveness of this drug and also the development of resistance during a course of therapy. High concentrations of A-methopterin result in only partial inhibition of formate incorporation of CGL and AL cells. This raises the question of whether there are two populations of cells in these conditions, one sensitive and the other insensitive to the drug, or whether A-methopterin-insensitive pathways for formate incorporation exist in all 115 cells. It is not yet possible to distinguish between these possibilities, since in both situations all the components could (as is found in the data shown in Table 3) be inhibited to an equal extent. Although formate incorporation by leukocytes from CGL patients was markedly inhibited by A-methopterin in these in vitro studies, it should be noted that this drug was not used in the treat ment of these individuals. A-methopterin has been most effectively used clinically in the treatment of acute leukemia in children, but studies with this type of cell in this in vitro system are difficult to carry out owing to the leukopenia commonly pres ent in these patients. No correlation can be made at this time between the clinical effectiveness of A-methopterin and the in vitro effects of the com pound on formate incorporation by leukocytes. It would be advantageous to extend the studies de scribed here to cells from patients under treatment with A-methopterin. Biochemical situations responsible for differ ences in sensitivity to A-methopterin by the vari ous cell types may include differences in citrovorum factor synthesis, permeability to A-methop terin, affinities of the drug for certain one-carbon transferring enzymes, and alternative metabolic pathways of one-carbon metabolism (19). It is hoped that this most fundamental problem will attract continued attention. SUMMARY The effects of A-methopterin on the rate of in corporation of formate-C14, in vitro, by human leukocytes have been studied. The rate of formate incorporation by different types of human leukocytes increases in the follow ing order: normal, chronic lymphocytic, chronic granulocytic, and acute leukemic cells, with little overlapping between values obtained with the various cell types. In the granulocytic series a linear relationship existed between the relative maturity of circulating leukocytes and the rate of formate incorporation by these cells, the more im mature populations taking up more formate. Leukovorin stimulated this rate of formate uptake, whereas folie acid did not. In chronic granulocytic cells, protein-bound serine and ribonucleic acid adenine and guanine had similar specific activities, whereas the specific activity of acid-soluble adenylic acid was much higher. A-methopterin inhibited incorporation into all the components equally. 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Conversions of Glucose to Amino cytes in Health and Disease. J. Lab. & Clin. Med., 40: Acids by Brain and Liver of the New-Born Mouse. J. 825-40, 1952. Biol. Chem., 199:485-92, 1952. uptake into acute leukemic cells and little or no effect on formate incorporation into the protein fraction of chronic lymphocytic or normal leukocytes. Downloaded from cancerres.aacrjournals.org on June 18, 2017. © 1957 American Association for Cancer Research. Metabolism of Human Leukocytes in Vitro: I. Effects of A-Methopterin on Formate-C14 Incorporation Richard J. Winzler, Albert D. Williams, William R. Best, et al. Cancer Res 1957;17:108-116. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/17/2/108 Sign up to receive free email-alerts related to this article or journal. 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