Relationship of Dehydroascorbic Acid Transport to Cell Lineage in Lymphocytes from Normal Subjects and Patients with Chronic Lymphocytic Leukemia1

[CANCER RESEARCH 45, 6507-6512, December 1985]

Robert L. Stahl,2 Charles M. Farber, Leonard F. Liebes,3 and Robert Silber4

Department of Medicine, New York University School of Medicine, New York, New York WO T6

ABSTRACT maintain its intracellular AA content against a gradient. Since L- gulonolactone oxidase (EC 1.1.3.8), the enzyme necessary for Dehydroascorbic acid is the principal form for the cellular AA synthesis, is not present in human tissues, these processes uptake by blood cells of vitamin C. Since previous studies from appear to regulate intracellular ascorbate levels. Previous studies this laboratory had shown a higher content of ascorbic acid and from this laboratory have shown elevated AA (8) and DHA (9) dehydroascorbic acid (DHA) in chronic lymphocytic leukemia concentrations in CLL B-lymphocytes. In view of these findings, (CLL) lymphocytes when compared to their normal counterparts, we have now investigated the transport of DHA in CLL and DHA uptake was characterized using these cells. The affinities normal lymphocyte subpopulations. of CLL and normal lymphocytes for DHA uptake were similar, as demonstrated by the Km values of 3.7 and 3.5 HIM, respectively. Differences were found in other kinetic constants of DHA uptake. MATERIALS AND METHODS The Vmaxfor normal lymphocytes, 634 ^mol/liter cell H20/min, Purification of Lymphocytes. Heparinized blood was obtained from was approximately twice that of CLL cells, 392 /Â¿mol/litercell normal subjects and from patients with untreated CLL. Informed consent H2O/min. In addition, the initial velocity and the maximal DHA was obtained in all cases. The diagnosis and staging of CLL was uptake by normal lymphocytes were greater than that of CLL established by standard criteria. Mononuclear cells were isolated by lymphocytes. These differences were not simply a reflection of Ficoll-Hypaque density centrifugation (10) and were depleted of mono- lymphocyte subsets since CLL B-cells demonstrated lower up cytes by adherence to Falcon plastic culture dishes (11). The degree of take rates than did normal B-cells whereas CLL T-cells were cellular purity was assessed by cell sizing using a Za Coulter Counter similar to their normal counterparts. The alterations appear to be (Coulter Electronics, Hialeah, FL). T- and B-lymphocyte subpopulations specific for the leukemic B-cell since they were not shared by were isolated by standard resetting techniques using EN and ETAC, neoplastic cells from two patients with T-cell CLL. When ana respectively (12, 13) with modifications as previously described (14). lyzed in light of the 3-fold greater cellular DHA and ascorbic acid These procedures yielded preparations with 90-95% B-cells and 90- content in B-cell CLL as compared to normal lymphocytes, these 97% T-cells in samples from normal subjects and from 69-84% B-cells and 76-95% T-cells in specimens from patients with CLL. kinetic parameters support the occurrence of a concentration- Preparation of Radiolabeled Dehydroascorbate. Radioactive |'4C|- dependent transport system for DHA. We conclude that the DHA ascorbic acid with a specific activity of 100 /Ã¼Ci/1.0mg (Amersham, uptake properties of CLL lymphocytes of B-cell origin serves to Arlington Heights, IL) was mixed with unlabeled AA to a final specific distinguish this lineage from T-cell CLL or normal lymphocytes. activity of 17 Â¿Â¿Ci/mg.Thismaterial was converted to DHA by the procedure of Staudinger and Weis (15). Aliquots were dried in a Savant Speedvac (Savant, Hicksville, NY) and stored at -20Â°C. Quantitative INTRODUCTION conversion to DHA was documented by HPLC (9). DHA5 is the major form of vitamin C taken up by blood cells. Measurement of DHA Transport. Transport was studied using an adaptation of the method of Segel and Lichtman (16). Packed lymphocyte Studies in human (1) and animal (2) erythrocytes as well as in aliquots of 1 x 107 cells were resuspended in 0.25 ml RPMI 1640 human leukocytes (3, 4, 5) have shown that DHA is rapidly containing 1% human serum albumin and varying concentrations of DHA. transported into these cells where a glutathione-dependent re The DHA concentration was 1 HIM except in experiments on uptake duction to AA occurs. The accumulation of DHA and AA by cells kinetics where it ranged up to 10 HIM. Since studies using [14C]AA against a concentration gradient is thought to be facilitated by confirmed previous reports (2, 4) that this form of the vitamin was not the D-glucose carrier system (6). Following reduction of DHA, taken up by blood cells, no further experiments with this compound were performed. Incubations were at 37Â°Cfor specified time intervals, usually there is a gradual transfer of AA to the medium (1, 2). The low permeability of the plasma membrane to polar AA (7) probably 15 or 30 sec. In order to measure initial rates of uptake, cytochalasin B (50 /Â¡.M)wasadded to control samples as an inhibitor of DHA transport accounts for this slow efflux as well as the ability of the cell to (6,17). In some experiments, the inhibitors sodium azide and 2-deoxy- Received 4/8/85; revised 7/9/85; accepted 8/16/85. glucose were used in 10 HIM and 5 mw concentrations, respectively. 1This work was supported by NIH Grant CA28376. Samples were layered over 0.5 ml of butyl phthalate:corn oil (10:3, v/v) 2 Recipient of a National Research Service Award (T32HL07151) from NIH. in 1.5-ml microcentrifuge tubes and spun for 1 min at 8000 x g. The Present address: Department of Medicine, Emory University School of Medicine, aqueous supernatant and the oil phase were aspirated separately and 201 Woodruff Memorial Building, Atlanta, GA 30322. 3 Scholar of the Leukemia Society of America, Inc. the wall of the tube was wiped dry with a cotton applicator after each 4To whom requests for reprints should be addressed, at Department of Medi step. The tip of the tube containing the cell pellet was severed and cine, New York University Medical Center, 550 First Avenue, New York, NY 10016. 5The abbreviations used are: DHA, dehydroascorbic acid; AA, ascorbic acid; placed overnight at room temperature in a counting vial containing 2.0 ml 0.3 M KOH. Fifteen ml of Ready-Solv HP (Beckman, Fullerton, CA) CLL, chronic lymphocytic leukemia; HPLC, high-performance liquid chromatography; Vi, initial velocity; EN, neuraminidase-treated sheep erythrocytes; ETAC, were then added to the counting vials and the radioactivity was deter complement-coated sheep erythrocytes. mined in an LS 7000 liquid scintillation counter (Beckman Instruments,

CANCER RESEARCH VOL 45 DECEMBER 1985 6507

DHA uptake per 10' cells at 30 s. The intracellular disposition of radio- Pato Alto, CA). The initial velocity of DHA uptake was calculated according to Segel labeled DHA was analyzed by preparing an extract from the cell pellet and LJchtman (18) from the formula using 60% v/v methanol as outlined above. Determination of Ascoltiate Synthesis. In view of the high ascorbic acid content of CLL B-lymphocytes (8), the remote possibility that these cells might synthesize AA was investigated using a modification of the x Ac procedure of Loewus ef al. (22). Aliquots of 5 x 10' cells were incubated dt at 37Â°C in 5 ml RPMI 1640 medium containing [1-14C]glucose (8 /iCi/ where d[Ai/Ao]/dt is measured over the initial linear portion of the uptake 134.4 /tmol) for either 3 or 20 h. Following four washings, extracts were curve. Ai and Ao represent the intracellular and extracellular radioactivity prepared using 60% v/v methanol. The residue was dissolved in elution and Ac Â¡sthe IHMDHA concentration in the incubation medium. V, is buffer and one half of the sample was immediately analyzed by HPLC ^mol/liter cell water/min. The volume of CLL lymphocytes was calculated as described above. The remainder was treated for 30 min with 3 units from a ratio determined from the median lymphocyte volume distribution ascorbate oxidase (EC 1.10.3.3) (1440 units/mg enzyme protein; Sigma profile generated by a Channelyzer attached to a ZBI Coulter Counter of Chemical Co.). One unit of ascorbate oxidase was effective in oxidizing 95 preparations from 34 patients with untreated CLL and 54 lymphocyte an AA standard to DHA at 1.0 ^mol/min. Following enzyme treatment, preparations from 38 normal donors adjusted for tonicity using a refer the sample was applied to the HPLC column. Elution fractions were ence normal lymphocyte volume of 210 fl (19). collected from both the ascorbate oxidase treated and untreated profiles at 20-s intervals with a flow rate of 1.5 ml/min. These were dissolved in Alterations in intracellular DHA content were also monitored by HPLC 3 ml Ready-Solv HP and the radioactivity was determined. using nonradiolabeled DHA. For these experiments, a modification of the above uptake method was necessary since the presence of minute quantities of oil was found to interfere with the preparation of cell RESULTS extracts. The preparation of cells and the incubation with DHA were performed as described above for radiolabeled experiments. Uptake, Uptake of Radiolabeled DHA. Chart 1 shows the time course however, was stopped by the addition of an excess volume of iced RPMI 1640 to which 50 ftu cytochalasin B had been added. The samples were of DHA uptake by CLL and normal lymphocytes. When lympho then centrifugea at 8000 x g for 1 min and the supernatant was aspirated. cytes were incubated in the presence of 1 mÂ«DHA, an initial Cell pellets were analyzed for AA and DHA content as described below. linear uptake phase was seen over the first 15s. The 15-s results In addition to the experiments with fresh lymphocyte preparations, were therefore used to calculate the initial velocity of DHA studies were also performed following cellular depletion of AA and DHA. uptake. The rate of uptake decreased thereafter, reaching a This was accomplished by a 20-h incubation at a concentration of 5 x plateau by 30-60 s. Studies using time points up to 15 min lO/Vml in ascorbate-free RPMI medium containing 10% fetal calf showed that uptake after 1 min remained at the plateau level serum: 1% penicillin, in a 5% CO2 atmosphere. Cell viability always (data not shown). The 30-s measurements were therefore used exceeded 90%. Following incubation, cells were exposed to 1 mw DHA and their AA and DHA content was determined by HPLC as described below. AA and DHA Content. Cell pellets of 1 x 10' lymphocytes were extracted with 60% (v/v) methanol, evaporated under reduced pressure using a Savant Speedvac, and the residue was stored at -70Â°C (20). This material was dissolved in 0.1 ml H2O and analyzed for AA content on a 5-/itn 4.5- x 250-mm SOTA SAX column (SOTA Chromatography, Crompond, NY) using an isocratic elution with 7 mw KCI:7 Ã•TIMKH2PO4, pH 4.0, which was modified from the method of Uebes et al. (8). QuantitÃ¤ten of DHA was performed as described by Farber ef al. (9). The DHA was separated from AA, reduced with dithiothreitd, and then measured as AA following rechromatography. Cell pellet protein was assayed according to Lowry ef al. (21). To evaluate whether lymphocyte AA is protein bound, 1x10Â° cells from two patients with CLL were incubated with [14C]DHA as described above. Cells were washed 3 times in phosphate buffered saline and homogenized by freeze-thawing. Fol lowing a 60-min centrifugation at 100,000 x g, the supernatant fluid was applied to a 1- x 50-cm Sephacryl S-200 column which had previously been calibrated with AA and marker proteins. The column was eluted with phosphate buffered saline, 1-ml fractions were collected, and the radioactivity was determined. Efflux Experiments. The extent of efflux was determined following uptake of [14C]DHA by normal and CLL lymphocytes by measuring both the decrease in cellular AA and the increase in supernatant radioactivity. Cells were labeled for 30 s with [14C]DHA as outlined above. The reaction was stopped by the addition of an excess volume of iced RPMI 1640 medium. Following four washings, the supernatant fluid was collected 0 15 45 60 and the radioactivity was determined as a base-line measurement. Cells were resuspended at a concentration of 1 x 107/ml in RPMI 1640 Time (seconds) medium and incubated at 37Â°Cfor 30 min. At 15 min intervals, aliquots Chart 1. Time course of DHA uptake by normal (O) and CLL (â€¢)lymphocytes. The effect of cytochalasinB on normal (A) and CLL (A) lymphocytes is also shown. were taken and centrifuged at 1700 x g for 4 min. The radioactivity of Data are the mean Â±SE(bars)of determinations In 10 samples at 15,12 samples the supernatant fluid was determined as the increment over baseline at 30, and 3 samples at 60 s for CLL and normal lymphocytes. Single or duplicate following incubation and was expressed as the percentage of the total determinations were performed at 10 and 12.5 s.

CANCER RESEARCH VOL. 45 DECEMBER 1985 6508

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. DMA TRANSPORT IN CLL LYMPHOCYTES to determine the maximal level of DHA uptake. Cytochalasin B, 49.5 Â± 8.6% (n = 7) in normal lymphocytes while the DHA which interferes with monosaccharide transport, was an effective content decreased to 59.8 Â±26.7% (n = 7) in CLL lymphocytes inhibitor of DHA uptake. Sodium azide, which prevents oxidative and to 70.8 Â±9.9% (n = 4) in normal lymphocytes. When these phosphorylation and hence active transport, and the analogue cells were exposed to 1 ITIM DHA, the DHA uptake by CLL 2-deoxyglucose also completely blocked DHA uptake to an lymphocytes was greater than that of normal lymphocytes (6.1 identical extent as that seen with cytochalasin B (Chart 1). versus 1.9 nmol/mg protein, respectively). The V>of DHA uptake in CLL and normal lymphocytes and in Kinetic Constants. Values for the Kmand VmaxofDHA uptake their T- and B-cell subpopulations is shown in Table 1. This by normal and CLL lymphocytes as determined by Uneweaver- comparison required correction for lymphocyte volume since CLL Burk analysis are shown in Table 2. The V,â„¢Â«forCLL lympho lymphocytes were found to be 13.8% smaller than normal lym cytes was half that for normal lymphocytes. In contrast, the phocytes (181 fl versus 210 fl, respectively) consistent with a values for Kmwere similar. These affinity constants are in agree previous report (23) that CLL lymphocyte cell protein content ment with those reported in human neutrophils and fibroblasts was lower than that of normal controls. The mean VÂ¡fornormal (6). No data have previously been published for normal or CLL lymphocytes was 1.5 times that of CLL lymphocytes. Normal B- lymphocytes. and T-cell subsets had the same VÂ¡asthe unfractionated lym AA and DHA Content. A comparison of the uptake by these phocyte preparation. The V,of CLL B-cells was equivalent to the cell types must take into account the differences in intracellular unfractionated preparation whereas the V, of CLL T-cells was ascorbate content. Table 3 lists the intracellular concentrations not different from that of normal lymphocytes. Similar results of AA and DHA in normal and CLL lymphocytes and in their T- were obtained when cell subpopulations were isolated using and B-cell subpopulations. Similar results were obtained when either ETAC or EN pelleting techniques. Two patients with T-cell cell subpopulations were isolated using either ETAC or EN CLL were studied. One had cells which were T8+ and the other pelleting techniques. A larger series than that published from this had T4+ cells. Their VÂ¡,360.9 and 551.3 /jmol/liter cell water/ laboratory (8, 9) is shown in Table 3. Also included are values min, respectively, were within 1 SD of the mean V, of normal giving the DHA contents of CLL B- and T-cells and normal T- lymphocytes. cells, which have not previously been reported. In addition, The maximal increment in cellular DHA content, determined at lymphocytes from two patients with T-cell CLL were studied. 30 s, is also shown in Table 1. The amount of DHA taken up by The AA and DHA concentrations were 2.38 and 0.91 mmol/liter CLL lymphocytes was one-half that of normal lymphocytes. As cell H2O, respectively, for the patient with T8+ cells, and 2.96 would be expected, the uptake of CLL B-cells obtained by ETAC and 0.59 mmol/liter cell H20 for the patient with T4+ cells. These or EN pelleting was similar to that of unfractionated CLL lympho values are within the range for normal lymphocytes. These cytes. The results with normal B- and T-cell subsets was within results confirm our earlier finding of a higher content of AA and 1 SD of the mean uptake by unfractionated normal lymphocytes. DHA in B-cell CLL as compared to normal controls and show CLL T-cells did not differ at 30 s from normal B- and T-cells. In that this anomaly does not extend to T-cell CLL. To evaluate the the patients with T-cell CLL, the levels of maximal DHA uptake possibility that intracellular AA and/or DHA may be protein were 146.4 (T8+) and 143.3 (T4+) ^mol/liter cell H2O, values bound, supernatant fluid prepared from an homogenate of cells close to the mean uptake by normal lymphocytes. incubated with [14C]DHA was applied to a Sephacryl S-200 Uptake of Unlabeled DHA. Cells were also incubated with column (see "Materials and Methods"). The radioactivity did not nonradioactive DHA and the increment in DHA content was elute with the protein fraction but instead was more internal in determined by HPLC. The CLL lymphocyte DHA content after a the column as indicated by the AA marker (data not shown). This 30-s incubation increased by 87.2 Â±54.6 (SD) ^mol/liter cell H20 finding provides evidence against a binding protein operating (n = 3) above baseline which was less than that noted in normal under these assay conditions. lymphocytes, 224.4 and 210.4 //mol/liter cell H2O (n = 2). These Efflux Studies. The contribution of vitamin C efflux to cellular results are comparable to the maximal DHA uptake obtained AA and DHA content was examined in normal and CLL lympho with labeled DHA as shown in Table 1. cytes following the uptake of labeled [14C]DHA (Chart 2). HPLC Effect of Cellular AA and DHA Depletion on Uptake. To test analysis of the cell pellets showed that 71 % of the label taken the hypothesis that cytosol DHA concentration affects uptake, up by the cell was AA and 29% was DHA. This finding was cells were depleted of endogenous substrate and the rate of consistent with previous studies (1,2,4,5) describing a reduced DHA uptake was determined. Following a 20-h incubation in an glutathione-dependent reduction to AA following DHA uptake. ascorbate-free medium, intracellular AA content decreased to When the labeled cells were resuspended and incubated for 15 61.6 Â±23.3% (n = 8) of baseline in CLL lymphocytes and to min, an increase in supernatant radioactivity was noted for CLL

Table 1 Initial velocity and maximal level of DHA uptake in CLL and normal lymphocytes The data are the mean Â±SD with the number of determinations shown in parentheses. Tests of significance (Student's t test) for V,: normal lymphocytes versus CLL (P < 0.01); CLL B-cells versus normal lymphocyte B-cells (P < 0.01); CLL B-cells versus CLL T-cells (P < 0.001); CLL T-cells versus normal lymphocyte T-cells (not significant); normal lymphocyte B-cells versus normal lymphocyte T-cells (not significant); for maximal uptake: CLL versus normal lymphocytes (P < 0.001); CLL B-cells versus normal lymphocyte B-cells (P < 0.01); CLL B-cells versus CLL T-cells (P < 0.001); CLL T-cells versus normal lymphocyte T-cells (not significant); normal lymphocyte B-cells versus normal lymphocyte T-cells (not significant). B- and T-cells were isolated as described in "Materials and Methods."

B-cell278.4 T-cell363.2 lymphocytes423.3 B-cell408.7 T-cell451 V, (fimol/liter cell water/min) Â±84.5 (8) Â±44.9 (6) Â±119.0 (6) Â±130.3 (10) Â±70.8 (3) .5 Â±75.7 (5) Maximal DHA uptake Â¿mol/liter 83.0 Â±20.5 (12)CLL 66.7 Â±14.2 (6)CLL 149.6 Â±22.0 (5)Normal142.1 Â±29.9(12)Normal 108.5 Â±4.9 (3)Normal148.2 Â±9.1 (5) cell water)CLL269.3

CANCER RESEARCH VOL. 45 DECEMBER 1985 6509

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1985 American Association for Cancer Research. DMA TRANSPORT IN CLL LYMPHOCYTES but not for normal lymphocytes, an effect which is associated medium when compared to their normal counterpart. The cor with a decrease in cellular ascorbate radioactivity. The efflux of responding decrease in uptake suggests the operation of a radiolabel from CLL lymphocytes accounted for 29.8 Â±10.4% concentration-dependent system. Support for this concept is (SE) (n = 5) of the total uptake of [14C]DHA by these cells. In provided by the similar carrier affinities or Kn,of DHA uptake by contrast, normal lymphocytes had minimal efflux, comprising 2.4 these cells. In addition, the finding that CLL lymphocytes de Â±2.4% of their total uptake (n = 3). These values were obtained pleted of endogenous substrate show an accelerated uptake of at equilibrium and do not represent rates. DHA provides strong support for the suggestion that it is the Absence of Ascorbate Synthesis. The possible de novo high concentration of DHA within the cell which is responsible synthesis of AA was evaluated by the HPLC analysis of extracts for the diminished DHA uptake noted under base-line conditions. prepared from cells which had been incubated with [1-14C]- Finally the lymphocytes from two patients with T-cell CLL, which glucose. Radioactivity was noted in the region corresponding to had normal AA and DHA contents, were noted to have V, and the retention time of AA (Chart 3). To determine whether this maximal DHA uptake values within the normal ranges. We there radioactivity represented newly synthesized AA, the cell extract fore conclude that the decrease in V,observed with CLL lympho was treated with ascorbic acid oxidase. Chromatography of the cytes reflects the high concentration of DHA within the cell and ascorbic acid oxidase treated aliquot showed the complete dis is specific to the leukemic B-cell lineage rather than representing appearance of the AA peak which is converted to DMA by the a trait of leukemic cells in general. enzyme. The remaining small peak represents cyclic nucleotides The elevated AA and DHA levels of CLL B-cells cannot at which comigrate with AA. DHA Ã©lÃ»tesearlierfrom the column present be explained by differences in DHA uptake. The finding along with nucleosides and bases. In contrast to the enzymatic of a high cellular content and accelerated efflux in CLL B-cells conversion of AA, no change was noted in the radioactivity suggests that the conditions for the regulation of uptake and elution profile following ascorbic acid oxidase treatment (3029 exodus may differ. Be this as it may, impaired efflux is not cpm versus 3061 cpm prior to treatment for the 20-h incubations responsible for the increased level in CLL B-cells. De novo and 2624 cpm versus 2630 cpm, respectively, for the 3-h incu synthesis appears also to have been excluded as an underlying bations). These results indicate that the radioactivity was not AA mechanism. An AA binding protein was not demonstrated in but represented other compound(s) with similar retention time(s) preliminary studies. No information is available concerning the toAA.

DISCUSSION 15MinutesNORMALiCLL4Â»3OMinutesNORMALCLLIÂ»

The present studies compare several kinetic parameters of DHA uptake in normal and CLL lymphocytes. The V,, V^, and I 0 maximal increment in DHA content for CLL lymphocytes, mainly B-cells, were approximately one-half that of normal lymphocytes, 40 consisting largely of T-cells. When selectively enrich popula tions were tested, uptake by CLL T-cells was similar to a.A of their normal counterpart. The uptake by CLL B-cells, as in the unfractionated population, was lower than that of normal B-cells. 2 30 While these findings at first suggest an impairment of DHA uptake by CLL B-cells, they must be interpreted in light of the differences in intracellular DHA content (Table 3) which are r LU oÃ2O known to affect the initial rates of uptake. Since CLL B-cells O have three times the DHA concentration of normal B-cells, an increased gradient exists between CLL cells and the incubation 10 Table2 Kinetic parameters of DHA uptake The data are the mean Â±SE. The number of lymphocyte preparations studied is shown in parentheses. CLL Normal lymphocytes Chart 2. Efflux of labeled ascorbate at 15 and 30 min in normal and CLL lymphocytes expressed as the percentage of the total DHA uptake. Data are the VmÂ».(jimol/liter cell water/min) 392 Â±8.5 634 Â±67.1 mean Â±SE (bars) of measurements in lymphocytes from 5 patients with CLL and Km(mMDHA) 3.73 Â±0.11(4) 3.48 Â±0.44(4) 3 normal subjects.

Tabte3 AA and DHA content of normal and CLL lymphocytes The data are the mean Â±SE with the number of determinations shown in parentheses. mmol/hter cell water

CLL CLL B-cell CLL T-cell Normal lymphocytes Normal B-cell Normal T-cell AA Â±0.82(22) Â±1.24(11) Â±0.28(12) Â±0.36(27) Â±0.36(11) Â±0.18(10) DHA10.85 2.11 Â±0.23(14)10.98 2.57 Â±0.18(4)2.88 1.03;0.95(2)2.78 0.59 Â±0.06(12)2.70 0.60 Â±0.04(3)2.24 0.84 Â±0.09(3)

CANCER RESEARCH VOL. 45 DECEMBER 1985 6510

possible intracellular subcompartmentalization of AA and DMA. cose, supporting the belief of the occurrence of a common carrier Since B-CLL cells appear to be arrested at an intermediate stage mechanism for glucose and DHA in human lymphocytes (6,17). of lymphocyte differentiation, the finding of their decreased up Comparison of normal and CLL lymphocytes has revealed take must be considered in light of the recently reported corre multiple abnormalities of CLL membrane composition and func lation of DHA uptake with stages of granulocyte maturation (24). tion. These include absent or decreased blood group antigens Using cultures of developing granulocyte-macrophage progeni (29), a lower sialic acid content (30, 31 ), changes in lipid ratios tors in vitro, an increase in the rate of DHA uptake was observed (32), a reduced number of catecholamine hormone receptors as a concomitant of neutrophil differentiation. In this respect, a (33), anomalous cap formation in response to lectins (34), fewer parallel may be drawn between immaturity in these two types of receptor sites for phytohemagglutinin (30) or concanavalin A cells and a decreased rate of DHA uptake. (35), diminished immunoglobulin density (36), abnormal surface protein glycosylation (37), absence of membrane 5'-nucleotidase The DHA transport system has previously been characterized in several ways. In experiments using insulin-treated diabetic in 70% of patients (38), absence of a Mr 185,000 macromolecular dogs, urinary AA excretion diminished, plasma AA levels de insoluble cold globulin found in normal B-cells (39), and a de creased, and the AA content of the buffy coat increased (25). creased L-system for amino acid transport (16,40). The maximal The concept that DHA and glucose share a carrier mechanism velocity of the CLL L-system functioned at a rate less than 10% was first suggested by Mann (26) who showed that the transport of that observed in normal blood lymphocytes, normal tonsillar of DHA in human erythrocytes was impaired by increasing glu lymphocytes, and a normal B-cell line, indicating that this system cose concentrations (27). This relationship is consistent with the is rudimentary in B-cell CLL. In the work reported above, it reported observation that patients with diabetes mellitus have seems probable that the deviation from normal is not an intrinsic increased serum DHA levels (28). Recent studies of DHA uptake membrane phenomenon but rather reflects the altered internal by human neutrophils and fibroblasts showed that DHA and milieu, i.e., AA and DHA content of the CLL B-cell. This derange glucose analogues were equally effective competitive inhibitors ment of DHA uptake is characteristic of the CLL B-cell and of cellular uptake with ratios of Kmand KÂ¡nearunity (6). This was serves to distinguish this lineage from T-cell CLL or normal taken to indicate a common transport system. In the present lymphocytes. Further studies are needed to explain the reasons study, the DHA uptake of both normal and CLL lymphocytes for the higher concentration of this important vitamin in CLL B- was inhibited not only by cytochalasin B but also by 2-deoxyglu- lymphocytes.

ACKNOWLEDGMENTS

We are grateful to Dr. Ralph Zalusky for allowing us to study his patients.

REFERENCES

1. Christine, L., Thomson, G., Iggo, B., Brownie, A. C., and Stewart, C. P. The reduction of dehydroascorbic acid by human erythrocytes. Clin. Chim. Acta, 7: 557-569,1956. 2. Hughes, R. E., and Maton, S. C. The passage of Vitamin C across the erythrocyte membrane.Br. J. Haematol., 14:247-253,1968. 3. Bigley, R., Stankova, L., Roos, D., and Loos, J. Glutathione dependent dehydroascorbate reduction: a determinant of dehydroascorbate uptake by human polymorphonuclearleukocytes. Enzyme(Basel),25: 200-204,1980. 4. Bigley, R. H , and Stankova, L. Uptakeand reduction of oxidized and reduced ascorbate by human leukocytes.J. Exp. Med., 739. 1084-1092,1974. 5. Stahl, R. L., Liebes, L. F., Farber, C. M., Kanganis, D. N., and Silber, R. Glutathioneas a regulator of lymphocyte ascorbate content (Abstract). Blood, 64: 81a, 1984. 6. Bigley, R., Wirth, M., Layman, D., Riddle, M., and Stankova, L. Interaction between glucose and dehydroascorbate transport in human neutrophils and fibroblasts. Diabetes,32: 545-548,1983. 7. Martin, G. Studies on the tissue distribution of vitamin C. Ann. NY Acad. Sci., 92: 141-147,1961. 8. Liebes, L., Krigel, R., Kuo, S., Nevrla, D., Pelle, E., and Silber, R. Increased ascorbic acid content in chronic lymphocytic leukemia B lymphocytes. Proc. Nati. Acad. Sci. USA, 78: 6481-6484,1981. 9. Fatter, C. M., Kanengiser, S., Stahl, R. L., Liebes, L. F., and Silber, R. A specific high-performanceliquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes. Anal. Biochem., 734: 355-360,1983. 10. Boyum, A. Isolationof mononudear cells and granulocytes from human blood. Scand.J. Clin. Lab. Invest., 27(Suppl97): 77-89, 1968. 11. Rabinovitch, M., and DeStefano,M. J. Useof the local anesthetic lidocainefor cell harvesting and subcultivation. In Vitro (Rockville),77: 379-381,1975. 5 10 0 5 10 12. Broome, J. D., Zucker-Franklin, D., Weiner, M. S., Bianco, C., and Nussen- Elution Time (minutes) zweig, V. Leukemia cells with membrane properties of thymus-derived (T) lymphocytes in a case of Sezary syndrome. Clin. Immunol.Immunopathol., 7: Chart 3. HPLC and radioactivity elution profiles of extracts prepared from cells 319-329,1973. incubated for 20 h with [1-'*C]glucose are shown before (left) and after (right) 13. Weiner, M. S., Bianco, C., and Nussenzweig, V. Enhanced binding of neura- treatment with ascorbic acid oxidase. HPLC analyses were performed with a SOTA minidase-treated sheep erythrocytes to human T-lymphocytes. Blood, 42: SAX column with elution conditions as described in the text. NAD. nicotinamide 939-946,1973. adenine dmucleotide. 14. Farber, C. M., Liebes, L. F., Kanganis, D. Nâ€žandSilber, R. Human B

CANCER RESEARCH VOL. 45 DECEMBER 1985 6511

lymphocytesshow greater susceptibility to HA toxicity than T lymphocytes. NY Acad. Sci., 258: 243-252,1975. J. Immunol., 732: 2543-2546,1984. 28. Chatterjee,I. B., Majumder,A. K., Nandi,B. K., and Subramanian,N. Synthesis 15. Staudmger.H., and Weis, W. Reindarstellungund Kristallisationvon Dehydro- and some major functions of vitamin C in animals. Ann. NY Acad. Sci., 258: L-ascorbinsaure.Z. Phys. Chem., 337: 284-285, 1964. 24-47,1975. 16. Segel,G. B., and Ã¼chtman,M.A. Decreasedu-systemfor aminoacid transport 29. Brody, J. I., and Beizer, L. H. Alteration of blood group antigens in leukemic in chronic lymphocyte leukemiclymphocytes.J. Btol.Chem.,257:9255-9247, lymphocytes.J. Clin. Invest., 44: 1582-1589,1965. 1982. 30. Kornfeld, S. Decreased phytohemagglutininreceptor sites in chronic lympho 17. Jung, C. Y.. and Rampai, A. L. Cytochalasin B binding sites and glucose cytes leukemia. Biochim. Biophys. Acta, 792: 542-545,1969. transport carrier in human erythrocyte ghosts. J. Btol. Chem., 252: 5456- 31. Speckart, S. F., BokJt, D. H., and MacDermott, R. P. Chronic lymphatic 5463, 1977. leukemia (CLL): cell surface changes detected by lectin binding and their 18. Segel,G. B., and Ã¼chtman,M.A. Amino acid transport in human lymphocytes: relation to altered glycosyltransferaseactivity. Blood, 52: 681-695,1978. distinctions in the enhanced uptake with PHA treatment or amino acid depri 32. Peel,W. E., and Thomson, A. E. R. The fatty acyl chain composition of human vation. J. Cell. Physiol., 106: 303-308,1981. normal and leukaemic lymphocytes and its modulation by specialized hydro gÃ©nation.Leuk.Res., 7: 193-204,1983. 19. Segel, G. B., Gokelet, G. R., and Lichtman, M. A. The measurement of lymphocyte volume: importance of reference particle deformabilHyand count 33. Sheppard, J. R., Gormus, R., and Moldow, C. F. Catecholamine hormone ing solution tonicity. Blood, 57: 894-899,1981. receptors are reducedon chronic lymphocytic leukaemiclymphocytes. Nature (Lond.),269: 693-695,1977. 20. Donofrio, J., Coleman, M. S.. Mutton, J. J., Daoud, A., Lampkin, B., and Dyminski, J. Overproduction of adenine deoxynucleosides and deoxynucleo- 34. Cohen, H. J. Human lymphocyte surface immunoglobuUncapping. Normal characteristics and anomalous behavior of chronic lymphocytic leukemic lym tides in adenosine deaminase deficiency with severe combined immunodefi phocytes. J. Clin. Invest., 55: 84-93,1975. ciency disease. J. Clin. Invest., 62: 884-887,1978. 35. Novogrodsky, A., Biniaminov, M., Ramot, B., and Katchalski, E. Binding of 21. Lowry, O. H., Rosebrough, N. J., Fair, A. L., and Randall, R. J. Protein concanavalinA to rat, normal human,and chronic lymphaticleukemialympho measurement with the Polin phenol reagent. J. Btol. Chem., Ã•93:265-275, cytes. Blood, 40: 311-316,1972. 1951. 22. Loewus, F. A., Kelly, S., and Hiatt, H. H. Ascorbic acid synthesis from o- 36. Shevach, E. M., Herberman, R., Frank, M. M., and Green, I. Receptors for glucose-2-14Cinthe liver of the intact rat. J. Btol. Chem.,235:937-939,1960. complementand immunogtobulinon human leukemiccells and lymphoblastoid cell lines.J. Clin. Invest., 51:1933-1938,1972. 23. Liebes, L. F., Pelle, E., Zucker-Franklin, D., and Silber, R. Comparisonof lipid 37. Andersson, L. C., Gahmberg,C. G., SÃ»mes,M.A., Teerenhovi,L., and Vuopio, composition and 1,6-diphenyH ,3,5-hexatrienefluorescence polarizationmea P. Cell surface glycoprotein analysis: a diagnostic tool in human leukemias. surementsof hairycellswith monocytes and lymphocytesfrom normalsubjects Int. J. Cancer, 23: 306-311,1979. and patients with CLL. Cancer Res., 41: 4050-4056,1981. 38. Lopes, J., Zucker-Franklin,D., and Silber, R. Heterogeneityof 5'-nudeotidase 24. Anderson, R., Stankova, L., Bigley, R. H., and Bagby, G. C. Dehydroascorbate activity in lymphocytes in chronic lymphocytic leukemia. J. Clin. Invest., 52: uptake as an in vitro biochemicalmarker of granulocytedifferentiation. Cancer 1297-1300,1973. Res., 43: 4696-4698,1983. 39. Simmonds, M. A., Sobczak, G., and Hauptman, S. P. Chronic lymphocytic 25. Sherry, S., and Ralli, E. P. Further studies of the effects of insulin on the leukemiacells lack the 185,000-dalton macromolecularinsolublecold globulin metabolismof vitamin C. J. Clin. Invest., 27:217-225,1948. present on normal B-lymphocytes.J. Clin. Invest., 69: 624-631,1981. 26. Mann, G. U. Hypothesis: the role of vitamin C in diabetic angiopathy. Perspect. 40. Segel, G. B., Simon, W., and Lichtman, M. A. Multicomponent analysis of Btol. Med., 17: 210-217,1975. amino acid transport in human lymphocytes. DiminishedL-systemtransport in 27. Mann, G. U., and Newton, P. The membrane transport of ascorbic acid. Ann. chronic leukemic B lymphocytes. J. Clin. Invest., 74:17-24,1984.

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Robert L. Stahl, Charles M. Farber, Leonard F. Liebes, et al.

Cancer Res 1985;45:6507-6512.

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