[CANCER RESEARCH 47, 4345-4350, August 15, 1987]

Antiproliferative Response of Human Leukemic Cells: Lectin Induced Inhibition ofDNA Synthesis and Cellular Metabolism1

Carl A. K. Borrebaeck2 and Arne Schön

Department of Biotechnology [C. A. K. B.J and Department of Thermochemistry ¡A.SJ, University of Lund, P.O. Box ¡24,S-221 00 Lund, Sweden

ABSTRACTProliferation and DNA synthesis of human acute (CCRF-CEM,

MOLT-4, JM, 1-45) and chronic (SKW-3) lymphocytic leukemia celllines of I -cell type were inhibited in a dose dependent fashion by theisolectins of phytohemagglutinin (PHA). PHA isolectin with four Lsubunits (PHA-Lj) induced a significant antiproliferative response ofCCRF-CEM and MOLT-4 tumor cells at a concentration of only 0.05Mii/nil.A 50% inhibition of DNA synthesis of these two cell lines wasobtained at 0.4 and 0.5 nv, IMI \-l j/nil, respectively. The effect wascytostatic rather than cytotoxic. Considerably higher (>10) concentrations of PHA isolectin with four E subunits were needed to induce similargrowth inhibition of the tumor cells, as compared to PHA-L*.

The effect of PHA isolectins on cellular metabolism of the leukemiccell lines was measured using microcalorimetry. The rate of heat production (thermal power), which is the net effect of all metabolic pathways,was decreased already after a 30-min culture of tumor cells in the presenceof isolectin. PHA-L* showed a significant inhibitory effect at a concentration of 0.1 ¿iti/'in1, whereas approximately 200 times more of PHA

isolectin with four E subunits was needed to obtain the same metabolicinhibition. Measurements of glycolytic láclaleand oxygen consumptionduring isolectin treatments supported the fact that the IMIA-l 4 inducedantiproliferative response of human leukemic cell lines was specific andenergy dependent. Six and three membrane components involved in theantiproliferative response of CCRF-CEM (ALL) and SKW-3 (CLL) cells,respectively, were isolated by affinity chromatography and characterizedby electrophoresis.

INTRODUCTION

Mitogen induced activation of resting lymphocytes, progression through the cell cycle to proliferation, and the subsequentdifferentiation is accompanied by the expression of a variety ofcell surface molecules (1-8). Several of these molecules areinvolved in the induction and regulation of cellular events (6,7,9-12), and the importance of such receptors and their ligandshas been emphasized by the discovery of homologies betweenoncogene encoded products and normal cell growth factors(13). In comparison with normal lymphocytes, the surface oflymphoid tumor cell lines most often expresses a characteristicreceptor profile, representing a particular stage of normal cellular differentiation (14).

The lectin from Phaseolus vulgaris (PHA),3 a tetrameric

glycoprotein composed of two different subunits (E and L) (15),has been extensively used to probe the repertoir of cell surfacereceptors on normal lymphoid cells (16). Lymphoid tumorshave to some degree also been investigated and human chroniclymphocytic leukemic cells exhibited a decreased number ofreceptor sites (17) and an impaired and delayed blastogenicresponse to PHA (18-20). Furthermore, an inhibitory effect ofPHA on the cell growth of continuously cultured mouse lym-phoma and myeloma cell lines has been reported (21-23).

Received 10/20/86; revised 1/28/87, 5/6/87; accepted 5/14/87.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This investigation was supported by a grant from the Swedish Cancer Society.2To whom requests for reprints should be addressed.3The abbreviations used are: PHA, phytohemagglutinin; PHA-L«,PHA iso

lectin with four L subunits; I'! IA I „.PHA isolectin with four E subunits; T-ALL,acute lymphoblastic leukemia of T-cell type; 1(11 chronic lymphocytic leukemia of T-cell type.

Detection and characterization of physiologically significantsurface antigens, such as antiproliferative associated antigens,on human tumor cells is of great interest. For this reason thePHA isolectins consisting of homologous subunits (PHA-L4and -E4) exhibiting different carbohydrate specificity (24) havebeen used to detect and characterize an antiproliferative response of human acute and chronic lymphocytic leukemia celllines of the T-cell type (T-ALL/CLL). The lectin receptors ofthe leukemic cells were identified, and the effect of the isolectinson the overall cellular metabolism was also studied using thenovel approach of microcalorimetry (25) as well as specificmeasurements of metabolic pathways.

MATERIALS AND METHODS

Cell Lines. CCRF-CEM (26), MOLT-4 (27), JM (14), T-45 (28) (T-ALL), and SKW-3 (29) (T-CLL) were generously provided by Dr.Kenneth Nilsson, University of Uppsala, Sweden, and cultivated inRPMI 1640 supplemented with 4 HIM i glmam inc. 1% (v/v) 100 xnonessential amino acids, and 10% fetal calf serum (Flow Laboratories,Inc., Rockville, MD). Streptomycin (50 /ig/ml) and penicillin (50 IU/ml) were added to the cells during the short-term antiproliferativeassay. The cell lines were tested weekly for Mycoplasma contaminationusing one enzymatic and one DNA fluorescent staining assay (30, 31).Cell lines were considered free of Mycoplasma only if both tests werenegative in two consecutive tests.

Isolation and Radiolabeling of PHA Isolectins. The isolectins IMIAL4 and PHA-E4 were isolated from red kidney beans (P. vulgaris) usingfast protein liquid chromatography as recently described.4 The isolectinswere iodinated by the chloramine-T method (32). Ninety and 70% ofthe added 125Iwas incorporated into the isolectins, corresponding to aspecific activity of 1.95 x 10* cpm///g and 0.62 x 10* cpm/ug for E4

and 1,4, respectively. The binding capacity of the isolectins was notsignificantly changed by the iodination procedure, as determined bybinding inhibition studies using unlabeled PHA.

Lectin Binding Assay. Determination of the number of receptor sitesand affinity constants was performed in serum-free medium supplemented with 1% bovine serum albumin using a binding assay recentlydescribed (24). Data from each experiment were analyzed according toSteck and Wallach (33) using a regression analysis computer programbased on the least squares method.

Antiproliferative Response Assay. Tumor cells (50,000 cells/well) insupplemented growth medium were cultured in microtiter plates for 48h in the presence of PHA-L4 or -E4. The final volume was 200 jil/well,and 4 h before harvest the cells were pulsed with 1 //Ci [methyl-3H]

thymidine (5.0 Ci/mmol; Amersham International, Ltd., Amersham,United Kingdom)/well. The cells were then transferred to 0.45-ittnMillipore filters and were washed with distilled water. The filters weredried and the radioactivity of the insoluble material was determined byliquid scintillation counting in a toluene-based scintillation cocktail.Samples were run in triplicates. The percentage of tritiated thymidineuptake was expressed as

filini

X 100

where the reference was cells cultured without PHA.

4C. A. K. Borrebaeck, J. Bristulf, and B. Jergil. Antiproliferative response of

human leukemic cells. PHA modulation of cytosolic protein kinase C activity,submitted for publication.

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ANTIPROLIFERATIVE RESPONSE OF HUMAN LEUKEMIC CELLS

Calorimetry. The cellular metabolism was assessed using a microca-lorimetric technique recently described (34). Briefly, the lectin treatedleukemia cells were transferred directly to a stirred calorimetrie vesselof a 4-channel calorimeter, the BioActivity Monitor (LKB Produkter,Bromma, Sweden) (35). Three samples together with untreated reference cells were run simultaneously. These four vessels reached themeasuring point at the same time and the rate of heat production wasregistered. The volume of the cell suspension was 2.7 ml in each vesseland the cell concentration was 0.6-1.0 x 10*cells/ml. The calorimetriemeasurements were performed at 37.00 ±0.05'C.

Lactate Production. A series of test tubes containing cell suspension(0.6-1.0 x 10" cells/ml) was incubated at 37°Con an orbital shaker.

Aliquots were taken at 1-h intervals and mixed with equal volumes of0.6 M perchloric acid. The supernatant was then analyzed for thepresence of lactate according to Noll (36). Untreated cells were used asreference.

Oxygen Consumption. The oxygen consumption was measured at37*C using a polarographic oxygen sensor (Hansatech, Ltd., Norfolk,

United Kingdom). The thermostated chamber was filled with 1 ml ofcell suspension (1.2-2.0 x 10* cells/ml), and the signal was monitored

using a potentiometric recorder for approximately 20 min. The solubility of oxygen in the medium was 200 ¿imol/ml(37).

Radiolabeling and Solubilization of Cells. Tumor cells were surfacelabeled by lactoperoxidase-catalyzed iodination (38). The iodinationwas performed as follows: 2 x 10* washed tumor cells in 1 ml sodium

phosphate buffer, pH 7.2, containing 0.15 M sodium chloride weremixed with 60 MKlactoperoxidase and 2 mCi Na'2SI (Amersham Inter

national, Ltd.). Freshly prepared 0.03% H:O: was added to a finalconcentration of 0.0013% at 0 and 4 min. The reaction was terminatedafter 8 min by adding KJ to a final concentration of 0.1 mM and 7-8ml of 0.2% human serum albumin in phosphate buffer. The cells werethen washed five times in sodium phosphate buffer, pH 7.2, containing0.15 M sodium chloride and 0.2% human serum albumin.

The surface-labeled cells were solubilized in 0.05 M Tris-HCl buffer,pH 7.5, containing 25 mM KC1, 5 mM MgCl2, 5 mM CaCl2, 0.5%Nonidet I' 40. and protease inhibitors (50 kallikrein inhibitor units/mlaprotinin-5 ¿ig/mlsoya bean trypsin inhibitor I mM phenylmethylsulfonyl fluoride) for 30 min at 4°C'. The lysate was then centrifuged at

6000 x g for 20 min and dialyzed overnight against the solubilizationbuffer.

Isolation of PHA-binding Glycoproteins. Lysate (200 /il) corresponding to 30 x Ml'1cells (approximately, 700,000 cpm) was incubated with

200 n\ PHA-L4- or PHA-E4-Sepharose gel (1.5 mg isolectin/ml swollengel) at room temperature for 1 h. The gel was then washed three timesin solubilization buffer to remove unbound material. Bound materialwas eluted with 400 n\ 0.16 M Tris-sulfate buffer, pH 2.5, containing2% sodium dodecyl sulfate. The eluted material was precipitated withacetone (1:5) and the precipitate was dissolved in electrophoresis samplebuffer (39) and denatured at 100'C for 2 min.

Electrophoresis and Autoradiography. The isolation and denaturedeluate was applied to a linear polyacrylamide slab gel gradient, 7.5-15%, according to Laemmli (39). The fixed and dried gel was thencovered with Kodak X-ray film and exposed for 24 h at -70°C.

RESULTS

Inhibitory Effect of PHA Isold-tins on DNA Synthesis and

Growth of Tumor Cells. To examine the effect of PHA isolectinon cell proliferation, L4 or E4 was added to cell cultures of I -ALL or T-CLL cells and examined for their inhibitory effecton DNA synthesis and growth of leukemia cells (Fig. 1). PHA-L4 inhibited cellular DNA synthesis of CCRF-CEM, JM,MOLT-4, T-45, and SKW-3 in a dose-dependent fashion.About 50% inhibition of DNA synthesis of CCRF-CEM andMOLT-4 tumor cells was observed with PHA-L4 at a concentration of 0.4 and 0.5 pg/m\, respectively, whereas 2 ng PHA-L4/ml were needed to achieve similar inhibition of JM andSKW-3 tumor cells. T-45 was significantly more resistant toinhibition of DNA synthesis and needed a 10 times higher

concentration of PHA-L4, as compared to JM and SKW-3 cells,to induce a 50% inhibition. Considerably higher concentrations(10-30 times) of PHA-E4 was needed to obtain 50% inhibition.

The found effect of PHA-L4/E4 on the human leukemic celllines was not cytotoxic but rather cytostatic, since the viabilityof the tumor cells did not decrease during the treatment. Theantiproliferative response to the leukemic cell was also not anunspecific lectin binding effect since other lectins, e.g., Dolichosbiflorus agglutinin, did not significantly inhibit the DNA synthesis even at concentrations of more than 50 ng/m\.

The number of lectin binding sites and association constantsfor each isolectin was determined using the T-ALL/CLL cells(Table 1). There was no correlation between growth inhibition,number of binding sites, and affinity constants.

Effects of PHA Isolectins on Tumor Cell Metabolism. Therate of heat production (thermal power) as measured by themicrocalorimeter is the net effect of all metabolic pathwayseither consuming or evolving heat. All calorimetrie measurements on untreated leukemia cells showed a time-dependentlinear increase in thermal power due to cellular growth. Thethermal power at time zero for each tumor cell line was obtainedby extrapolating to this point of the thermal power curve. Thethermal power values for untreated tumor cell lines are givenin Table 2.

The kinetics of the effect of PHA-L4/E4 on tumor cell metabolism was determined using 25 /ig/ml of each isolectin. Thetumor cell lines were cultured in the presence of isolectin for0.5, 1, 2, 3, 4, and 6 h, after which times the cells were testedusing calorimetry. An effect on the cellular metabolism wasdetected after 30 min when a significant decrease (approximately, 15%) in the rate of heat production was obtained. After3 h a maximal decrease of approximately 40% was obtainedand no further inhibition of the thermal power was detectedafter this point. The kinetics was similar for all T-ALL/CLLcells.

The dose-dependent isolectin effect on tumor cell metabolismwas then determined treating each tumor cell line for 3 h withdifferent isolectin concentrations. Dose-response curves wereobtained and the effect of PHA-L4 and PHA-E4 on CCRF-CEM cells is shown (Fig. 2). PHA-L4 had a more pronouncedinhibitory effect than PHA-E4 on the cellular metabolism atlower concentrations (<5 Mg/ml). The latter isolectin had, however, a strong inhibitory effect on metabolism if concentrationsabove 30-40 ¿¿g/mlwere used. The inhibitory effect of the twoisolectins on the cellular metabolism of MOLT-4 and T-45cells was similar to that of CCRF-CEM cells (data not shown).JM and SKW-3 tumor cells differed since the PHA-E4 induceddecrease of thermal power was in the range of 35-40% using25 fig isolectin/ml, which was somewhat more than in CCRF-CEM cells. We chose CCRF-CEM tumor cells for studies onanaerobic and oxidative metabolism since these cells showedthe strongest antiproliferative response and since its overallmetabolic decrease was similar to the other leukemic cell lines.

Effect of PHA Isolectins on Lactate Production and OxygenConsumption of CCRF-CEM Tumor Cells. Exposure of CCRF-CEM cells to 2.5 ng PHA-L4/ml or 40 ng PHA-E4/ml resultedin a change in the rate of lactate production (Table 3). Theisolectin concentrations were derived from Fig. 2, as the amountneeded to obtain a significant inhibitory effect. PHA-L4 treatedCCRF-CEM tumor cells exhibited a maximal increase of lactateproduction of 3.6 times the reference value, whereas the lactateproduction for PHA-E4 treated CCRF-CEM tumor cells was4-5 times lower than the reference value.

Exposure of CCRF-CEM cells to 2.5 ng PHA-L4/ml or 40

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ANTIPROLIFERATIVE RESPONSE OF HUMAN LEUKEM1C CELLS

100

«0

BO

TO

00

go

40

30

150.15 OS

Conc.otr.lloo

~55S OÃŽ5 55 ÃŽS 5 15 25 50

Concentrât loo I pg PMA/ml) Coricanti at ton I |jg PHA /ml I

-005—555 05«a 50

Concent ral,on IpgPHA/mllConcanlrat.on IpgpHA/ml)

Fig. 1. In vitro effect of PHA-L4 and 1< isolectins on the DNA synthesis of human leukemic cells. Tumor cells (50,000) were cultured in medium containingserially diluted isolectin for 48 h. Ordinate, percentage of control (radioactivity of samples cultured with isolectin per radioactivity of samples cultured withoutisolectins). A, CCRF-CEM; B, MOLT-4; C, JM; D, T-45; £,SKW-3. PHA-L4 (•);PHA-E4 (•);TV/A,thymidine; bars, SD.

Table 1 Association constants (K„Jand total number oflectin binding sites percell (n) on human leukemic cells for /'//(/, and PHA-Et

CelltypeT-ALL

CCRF-CEMMOLT-4JMT-45T-CLLSKW-3K..XPHA-L41.7

±0.5'

5.8 ±0.43.1 ±0.11.3±0.11.7

±0.110-«

(M-')PHA-E.2.8

±0.42.8 ±0.31.6 ±0.11.4±01.8

±0.1uxPHA-L41.4

±0.10.8 ±01.0 ±0.12.2 ±0.41.7

±0.2io-5PHA-E41.5

±0.11.6 ±0.13.9 ±0.13.9 ±0.92.6

±0.4•Mean ±SD.

Table 2 Rate of heat production of untreated leukemia cell lines as determinedby microcalorimetry

The variation of CCRF-CEM is given as the mean ±2 x SD (n = 10), whilethe range is given for the others.

CelltypeT-ALL

CCRF-CEMMOLT-4JMT-45T-CLLSKW-3Rate

of heatproduction(pW/cell)14.8

±1.012.9 ±0.515.6 ±0.825.9 ±(notdetermined)29.8

±0.7

UK PI IA I a/ml also resulted in significant changes of oxygenconsumption. The mean value of oxygen consumption for untreated cells was 2.4 x IO"17 mol/(cell x s), whereas PHA-L4and PHA-E4 treated cells gave 3.4 x 1(T17and 2.1 x 10~17mol/

(cell x s), respectively.Characterization of PHA Isolectin-defined Cell Surface Anti

gens. To identify the cell surface glycoproteins that PHA isolectins bound to, one T-ALL and one T-CLL tumor cell lineswere surface labeled, solubili/ed, and subjected to affinity chro-matography on immobilized PHA-L4 or PHA-E4 followed bypolyacrylamide electrophoresis and autoradiography (Fig. 3).

Under reducing conditions, six major components of apparentmolecular weights of 150,000, 140,000, 133,000, 74,000,15,000, and 14,000 were identified by PHA-L4 from solubilizedCCRF-CEM cells. One additional component of apparent molecular weights of 98,000 was identified by PHA-E4 from theCCRF-CEM cells (Fig. 3/Õ).

PHA-L4 identified three major membrane components fromSKW-3 tumor cells with apparent molecular weights of150,000, 76,000, and 71,000; PHA-E4 identified two morecomponents of apparent molecular weights 100,000 and 88,000from the SKW-3 cells (Fig. 3B). It was evident from the auto-

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100

60

50

ANTIPROLIFERATIVE RESPONSE OF HUMAN LEUKEMIC CELLS

A

1234

116-

92.5 —

66.2

45-

31-

21.5-

14.4—

1234

U-116

- 92.5

- 66.2 *

0.1 0.5 1 2 5 10 ?5 50 10O

Concentration l ug PHA/ml )

-45

--31

-21.5

-14.4

JES

100

90

80

70

60

50

0.1 0.5 1 10 25 50 100

Concentration ( ug PHA/ml )

Fig. 2. In vitro effects of PHA-L. (A) and PHA-E«(B) isolectins on tumor cellmetabolism. CCRF-CEM leukemic cells (1 x lO'/ml) were cultured in medium

containing different amounts of isolectin for 3 h. The effect on cell metabolismwas then tested by microcalorimetry. Points, percentage of thermal power ascompared with untreated reference cells. Ban, SD.

Table 3 Lucíateproduction of isolectin treated CCRF-CEM tumor cells ascompared to untreated cells

Rate of lactate production is expressed as mol per (cell x s). PHA-L4 and -E<were used at 2.5 and 40 Mg/ml, respectively. Samples were taken each hour fortesting. The different isolectin treatments were not performed in parallel.

Lactate production [mol/(cell x s)] x IO17

Time(h)12

34Untreated

cells4.2

7.59.05.2L<

treatedcells5.2

7.120.518.5Ratio1.20.9

2.33.61

4 treatedcells12

37.97.59.81.6

2.92.40.2

0.40.2

radiograms that PHA-E4 bound to more membrane components on both tumor cell lines as compared to PHA-L4, whichis due to the broader carbohydrate specificity exhibited by PHA-

E4 (24).

DISCUSSION

This investigation clearly showed that PHA-L, isolectin defined functionally associated membrane-bound glycoproteins ofhuman leukemic cell lines and that the cellular interaction ofthis isolectin inhibited both the cellular DNA synthesis andtumor cell growth. PHA-L4 inhibited the cell growth in anoncytotoxic manner also at very low concentrations, since e.g.,0.05 /xg PHA-L4/ml caused a 15% inhibition of growth of

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Fig. 3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradi-ographic analysis of 125I-labeledcell surface proteins from CCRF-CEM and SKW-

3 tumor cells. A: lane A, labeled components in whole cell lysate; lane 2,components binding to PHA-E<; lane 3, components binding PHA-L«;lane 4,components unspecifically bound to the affinity column. B: lane I, componentsunspecifically bound to the affinity column; lanes 2 and 3, same as in A; lane 4,labeled components in whole cell lysate. The electrophoresis was run underreducing conditions, and the acrylamide gel concentration was 7.5-15%. Mw,molecular weight.

CCRF-CEM tumor cells. This is approximately 100 timeslower than the optimal isolectin dose needed to induce DNAsynthesis in normal peripheral blood lymphocytes (24). Theinhibitory effect of the isolectin is therefore not the result of anexcessive receptor cross-linking, which would change the physical state of the membrane and consequently the ability of thecells to respond, but rather a binding to proliferation associatedglycoproteins; no evidence was found for involvement of solublefactors (data not shown). The different antiproliferative response of the various human leukemic cell lines was, furthermore, not due to differences in association constants and number of binding sites for the isolectins since no obvious correlation between these parameters was found. The inability of PHA-F, to induce a strong antiproliferative response might, however,be due to its broader carbohydrate specificity which resulted ina higher number of available membrane receptors and subsequently to a higher degree of receptor cross-linking. Our bindingstudies showed that approximately 2 /¿gPHA-E4 saturated allbinding sites on 2 x IO6CCRF-CEM cells, and greater amounts

of isolectin might exert its inhibitory effects by altering physicalproperties of the cell membrane. Furthermore, the inhibitoryeffect of PHA-E4 did not increase when the experiments wereperformed under serum-free conditions (data not shown). Thisdemonstrated that an interaction of this isolectin with tumorcell membrane components was not merely inhibited by serumglycoproteins (24).

Normal human peripheral T-lymphocytes exhibit an increased glycolysis and oxygen consumption when stimulatedwith PHA (40). Furthermore, the production of glycolytic lactate in mitogen stimulated lymphocytes has also been substantiated (41) and has been proposed to be due to the rate-limitingreaction of pyruvate dehydrogenase (42,43). Therefore, it seemsto be a close kinetical relationship between the maximum ratesof glycolysis and DNA synthesis, although there is no causalassociation between these two events (16). PHA isolectins alsoaffected aspects of cellular metabolism of the human leukemiccell lines, as illustrated in more detail by the CCRF-CEM tumorcells. The net effect of all metabolic pathways either consumingor evolving heat as measured by microcalorimetry showed atime-dependent decrease in the rate of heat production when

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ANTIPROLIFERATIVE RESPONSE OF HUMAN LEUKEM1C CELLS

either of the PHA isolectins were present in the tumor cellcultures. This, together with the inhibiting effect of the isolectins on DNA synthesis, clearly demonstrates that the tumorcells respond in an antiproliferative manner, turning off thenormal cellular reactions necessary for cell growth.

Measurements of the metabolic pathway involving lactateproduction and oxygen consumption, which is the major pathway evolving heat, showed that PHA-L4 and PHA-E4 inducedopposite responses of the tumor cells. The specific antiproliferative response induced by PHA-L4 seems to be energy dependent since the increase in oxygen consumption suggests astimulation of ATP-utilizing reactions, and the increased lactateproduction indicates a higher rate of glycolysis and a rate-limiting role of pyruvate dehydrogenase similar to what hasbeen found in normal lymphocytes. However, the effects ofPHA-E4 on cellular growth and metabolic pathways of thetumor cells differ significantly from the effects we have registered with PHA-L4. The interaction of PHA-E4 with CCRF-CEM cells resulted in an overall shutdown of DNA synthesisand cellular metabolism, but only at very high lectin concentration. The data seem to indicate the possibility that a decreaseof cell proliferation at high levels of PHA-E4 is due to a lessspecific interaction compared to PHA-L4.

Identification of plasma membrane bound lectin receptorsinvolved in the antiproliferative response was performed usingradiolabeled CCRF-CEM (T-ALL) and SKW-3 (T-CLL) cells.It was evident that PHA-E4 bound to several more surfacecomponents as did PHA-L4, and the latter isolectin did notseem to identify any unique components. The membrane components identified by PHA-L4 on CCRF-CEM or SKW-3 tumorcells did not directly coincide with any of the major bandsidentified by the same isolectin on normal T-cells (44). Twoglycoproteins on normal T-cells, with apparent molecularweights of 175,000 and 125,000, are the glycoproteins mostclosely related to components identified by PHA-L4 on CCRF-CEM and SKW-3 tumor cells (44). The transferrin receptor, ahomodimer of 85,000-90,000 subunit molecular weight, wasrecently shown to be an important antiproliferative associatedantigen (2). However, PHA-L4 seems not to bind to the transferrin receptor on either CCRF-CEM cells or SKW-3 tumorcells (Fig. 2B). We are presently preparing monoclonal antibodies against cell surface components to further characterize thefunctionally associated structures on the human leukemia celllines. Monoclonal antibodies were also recently shown to bevaluable in identifying proliferation-associated antigens on arat bladder cancer cell line (45) and on human 8866 B-lym-phoma cells (46).

ACKNOWLEDGMENTS

We thank Ann-Charlott Musson for skillful technical assistance andDr. Ingemar Wadsöfor fruitful discussions on microcalorimetry.

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1987;47:4345-4350. Cancer Res   Carl A. K. Borrebaeck and Arne Schön  Induced Inhibition of DNA Synthesis and Cellular MetabolismAntiproliferative Response of Human Leukemic Cells: Lectin

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