[CANCER RESEARCH 37, 1307-1312, May 1977]

SUMMARY

Lymphocytes isolated from the peripheral blood of patients with nonmalignant and malignant disorders werestudied for fluidity of membrane lipids and lateral mobilityof concanavalin A (Con A) receptors. The degree of fluidityof the surface membrane lipid core was monitored quantitatively by fluorescence polarization analysis using the probe1,6-diphenyl-1 ,3,5-hexatriene embedded in lipid regions ofthe surface membrane of intact cells. Mobility of Con Asurface receptors was determined by the cap-forming abilityafter binding of fluorescent Con A. The present studies wereperformed on lymphocytes from 28 patients with malignantlymphomas, 22 patients with leukemia, 28 individuals whoeither were healthy or had nonmalignant disorders, and 5patients with carcinoma. The results showed that lymphocytes and mononuclear cells from patients with malignantlymphomas and leukemias have a more fluid lipid layer intheir surface membrane than do lymphocytes obtained fromhealthy individuals or from patients with other malignantand nonmalignant disorders. This increase in membranefluidity was less pronounced in lymphocytes isolated fromleukemic patients in clinical remission and from leukemicpatients receiving treatment with steroids. The results alsoshow a marked difference in the cap-forming ability of lymphocytes from patients with malignant lymphomas or leukemia as compared with lymphocytes from patients with nonmalignant disorders or carcinoma. Lymphocytes isolatedfrom lymphoma and chronic lymphatic leukemia patientsduring remission stages of the disease exhibited a highercap-forming ability. The cap-forming ability of cells frompatients with chronic lymphocytic leukemia was unaffectedby treatment with steroids. The present results, which are inline with previous observations, have shown that normallymphocytes can be characterized by a low degree of lipidfluidity but a high degree of mobility of Con A receptors,whereas leukemic lymphocytes are characterized by a high

degree of lipid fluidity but a low degree of mobility of Con Areceptors. These results confirmed our general hypothesison the dynamic interrelation between membrane lipids andmembrane protein receptors, and they indicate that thewidely accepted term ‘‘membranefluidity―requires betterconsideration for different membrane components.

INTRODUCTION

Development of methods for distinguishing between normal and malignant cells may prove to be valuable in thedetection of malignant disorders of the human hematopoietic system (15). Among the methods currently available,the analysis of differences in the structure, function, anddynamics (10, 12, 27) of normal and malignant cell membranes (8, 11, 24) is of primary importance in the study ofleukemogenesis and carcinogenesis (16—18,20, 21). Theconcept of the fluid nature of biological membranes (27) isnow well documented and widely accepted . However, theterm “membranefluidity,' â€w̃hich is commonly used in thisrespect, is more complex and embraces different sites andregions of the membrane. The most prominent of these aremobility of membrane protein receptors and fluidity of themembrane lipid core. These dynamic features, which areinterrelated to some extent, (26) are believed to play a majorrole in the cellular control mechanism that determines normal and abnormal growth differentiation. The degree ofmobility of membrane protein receptors, as well as thedegree of fluidity of the membrane lipid core, can be quantitatively evaluated by fluorescence methods (22, 25). Fluorescence probes, which are embedded in the hydrophobiccore of the surface membrane, were used to monitor thedegree of lipid fluidity of cellular membranes which is determined, to a large extent, by its lipid composition. Suchstudies were carried out using the fluorescence hydrocarbon probe DPH2 (14, 17-19, 21, 22, 25, 26). In earlier studies, the Con A probe was used to study changes in the

I This work was supported by Contract 1-CP-3342 from the Virus Cancer

Program of the National Institute, USPHS.Received September 7, 1976; accepted January 26, 1977.

2 The abbreviations used are: DPH, 1 ,6-diphenyl-1 ,3,5-hexatriene; Con A,

concanavalin A; F-Con A, fluorescein isothiocyanate-treated Con A; PBS,Dulbecco's phosphate-buffered saline (pH 7.2).

MAY 1977 1307

Fluidity of Membrane Lipids and Lateral Mobility ofConcanavalin A Receptors in the Cell Surface of

Normal Lymphocytes and Lymphocytes fromPatients with Malignant Lymphomas and

Leukemias1

Hannah Ben-Bassat, Aaron Polliak, Stella Mitrani Rosenbaum, Elizabeth Naparstek, Daniel Shouval, andMichael Inbar

The Chanock Center for Virology, Department of Hematology and Medicine A, The Hebrew University-Hadassah Medical School, Jerusalem (H. B-B. , A. P., S.M. R. , E. N. , 0. SI, and Department of Membrane Research, The Weizmann Institute of Science, Rehovot fM. I.), Israel

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H. Ben-Bassat et a!.

mobility of specific receptors on the cell membrane (1-5, 6,20). Alterations in the distribution of Con A sites, the formation of Con A site complexes, and the lateral mobility ofthese complexes result in the formation of a cap. With theDPH and the Con A probes, the lipid fluidity and the lateralmobility of membrane receptors on the cell surface can bedetermined. Studies with these probes have shown differences in the structure and function of the surface membrane of normal and malignant cells. Recently (14, 18), ithas been shown that the development of leukemia in bothexperimental animals and humans is accompanied by amarked increase in the fluidity of the membrane lipid core ofthe leukemic cells. In earlier studies, we have also showndifferences in the lateral mobility of Con A receptors on thesurface membrane of normal lymphocytes and lymphocytesisolated from patients with chronic lymphocytic leukemia,Hodgkin's and non-Hodgkin's lymphomas, and AfricanBurkitt's lymphoma (1-4). The difference in the mobility ofCon A receptors, as measured by the cap-forming ability,was also observed in cells obtained from biopsy materialfrom the patients with the above cancers. The ability of cellsfrom a normal donor or a lymphoma patient to form capsappeared to be independent of the source from which thelymphocytes were derived (1).

In this study we investigate the interrelationship betweenlipid fluidity and lateral mobility of receptors in membranes

of lymphocytes isolated from patients with various cancersof the hematopoietic system as compared to patients withnonmalignant disorders by using the DPH and the F-Con Aprobes.

MATERIALSAND METHODS

Patients. Blood samples for these studies were obtainedfrom hospitalized and ambulatory patients with leukemiaand malignant lymphoma at the Hadassah University Hospital, Jerusalem, Israel. Blood samples were obtained frompatients with leukemia, Hodgkin's lymphomas, carcinoma,nonmalignant disorders, and normals, including treatedand untreated cases.

Separation of Lymphocytes. Lymphocytes were isolatedfrom peripheral blood by Ficoll-Hypaque gradient centrifugation (6), washed twice with PBS (Grand Island BiologicalCo., Grand Island, N. Y.), and diluted in PBS to the appropriate concentration. Viability of the cells used in the experiments was 95 to 100%, as determined by the trypan blueexclusion method, and only freshly isolated cells were examined. Ficoll 400 was obtained from Pharmacia, Uppsala,Sweden, and Hypaque sodium (50%) was from WinthropLaboratories, New York, N. Y.

Labeling of Cells with DPH. The fluorescence hydrocarbon DPH (Koch-Light Laboratories, Colnbrook, England)was used in the present study as a fluorescence probe formonitoring the degree of fluidity in the cell surface membrane lipid core. Labeling of cells was performed with a 2 x106 M DPH dispersion in PBS obtained by injection of 0.1ml of 2 x 10-s M DPH in tetrahydrofuran (Fluka, Buchs,Switzerland) into 100 ml PBS that has been stirred vigor

ously. This DPH dispersion is practically clear and void offluorescence (21). A volume of 2 ml cell suspension in PBSat a concentration of 106 cells/mI was incubated with 2 mlDPH dispersion for 60 mm at 25°.The labeled cells werethen washed twice with PBS, resuspended in PBS at aconcentration of 0.5 x 106 cells/mI, and immediately usedfor the fluorescence studies.

Fluorescence Polarization Analysis. The degree of lipidfluidity in the surface membrane was quantitatively determined by fluorescence polarization (P) analysis of the DPHlabeled cells. Experiments were carried out with the ElscintModel MV-i microviscosimeter (Elscint Ltd., Haifa, Israel).Excitation was performed with a polarized 365 nm bandgenerated from a 200-watt mercury arc, and the emittedlight was detected in 2 independent cross-polarized channels equipped with cutoff filters for wavelengths below 390nm. Fluorescence polarization was obtained by simultaneous measurements of I/I@ where I,,and I@are the fluorescence intensities polarized parallel and perpendicular to thedirection of polarization of the excitation beam, respectively. These values relate to the degree of fluorescencepolarization (P) and to the fluorescence anisotropy (r) bythe following equations:

p=@LL@I, + l@

— I— l@

r —l@+ 2l@

(A)

(B)

All fluorescence measurements with DPH-labeled cells inthis study were carried out at 25°.The accuracy of the Pvalues obtained with the microviscosimeter was P =±0.005.The method used in the present study for the evaluation of membrane microviscosities is based on the fluorescence polarization properties of a fluorescence probe asdescribed by the Perrin equation:

= 1 + C(r)

r(C)

where r and r1 are the measured and the limiting fluorescence anisotropies, T is the absolute temperature, r is theexcited state life-time of DPH, C(r) is a parameter thatrelates to the molecular shape of the fluorophore and has aspecific value for each r value, and@ is the microviscosity ofthe medium where the DPH molecules are embedded (25).High P values correspond to high@ values and indicate alow lipid fluidity and vice versa.

Binding of F-Con A. F-Con A was obtained from MilesYeda, Rehovot, Israel, at a fluorescein to protein ratio of1.68. For the experiment, cells (2 x 10@cells/mi) were incubated with F-Con A (200 pg/mI) for 15 mm at 37°;thecells were washed with PBS and fluorescence was determined on 1 drop of cells in a Zeiss microscope withtransmitted UV. Five hundred cells were counted for eachpoint, and only single cells and those in very small clumps(2 to 5 cells) were counted for percentage of caps.

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Degree of fluorescence polarization (P) of DPH-labeled cells and membrane microviscosity (@)oflymphocytesisolated from patients with nonmalignant disorders, carcinoma, lymphoma,andleukemiasDegree

of fluorescence Microviscosity (ti)atpolarization(P) at 25° 25°(poise)

No. of paGroup Disorder tients Range Av. RangeAv.I

Nonmalignant 28 0.280-0.305 0.295 3.55-4.594.14IICarcinoma 5 0.282-0.311 0.296 3.62-4.884.16IllMalignant lymphoma 20 0.265-0.287 0.280 3.05-3.813.55IVChronic lymphatic 5 0.260-0.275 0.269 2.90-3.373.18leukemiaV

Acute leukemia 3 0.242-0.266 0.254 2.42-3.08 2.72

Increase in membrane microviscosity (@)(decrease in lipid fluidity) of lymphocytesisolatedfromlymphoma and leukemia patients duringremissionUntreated

patients Patients in remission ortreated withsteroidsFluores-

Microvis- Fluores- Microviscence polar- cosity (i@) cence polar- cosity(@)ization

(P) at at 25° ization (P) at at25°Disorder25° (poise) 25°(poise)Malignant

lymphoma 0.280 3.55 0.2924.03Chroniclymphatic 0.269 3.18 0.2873.83leukemiaAcute

leukemia 0.254 2.72 0.275 3.37

Lipid Fluidity and Receptor Mobility

RESULTS

The degree of fluidity in the surface membrane lipid coreof lymphocytes from patients from malignant lymphomas,leukemias, carcinomas, and nonmalignant disorders andfrom normals was quantitatively determined by monitoringthe degree of fluorescence polarization (P) of DPH embedded in the surface membrane of intact cells. The presentstudies were carried out with lymphocytes isolated frompatients with malignant lymphomas (Hodgkin's disease andnon-Hodgkin's lymphoma) and patients with leukemias(chronic Iymphocytic leukemia, acute lymphocytic leukemia, and acute myeloid leukemia). The control group included patients with nonmalignant disorders (infectiousmononucleosis, autoimmune d iseases, hypolipidemia, hyperlipidemia, and normal donors). Patients with carcinomawere also included in this study. The results indicate (Table1) that lymphocytes from patients with malignant lymphomas and leukemias exhibit lower P and @jvalues (high lipidfluidity) than do lymphocytes from patients with nonmalignant disorders or carcinoma. All patients with malignantlymphomas and leukemias had lymphocytes with P valueslower than 0.280. No difference was observed in the Pvalues of lymphocytes from patients with Hodgkin's andnon-Hodgkin's lymphomas. Lymphocytes from patientswith chronic lymphatic leukemia and acute leukemia exhibited very low P values (0.269 and 0.254, respectively) ascompared with lymphocytes isolated from patients withnonmalignant disorders or carcinoma and normal donors(0.296) (Table 1). Lymphocytes isolated from patients withlymphomas and leukemias in clinical remission usually hadhigher P and@ values (Table 2). A similar increase in the P

and@ values was also obtained with chronic lymphaticleukemic patients receiving corticosteroid therapy.

Some of the patients in this series were followed up forperiods of up to 2 months, and their lymphocytes wereexamined on several occasions with the use of DPH. Theresults summarized in Table 3 indicate that the degree offluorescence polarization of DPH obtained from differentsamples of lymphocytes from the same patient was generally in good agreement and corresponded well with thesteady state of the disease. However, in a case of acutelymphocytic leukemia that was followed from the onset ofthe disease, during treatment, and in remission, the lymphocytes exhibited a remarkable and steady increase in theP and i@values, which corresponded with the clinical condition of the patient (Table 4).

The lateral mobility of specific receptors for Con A on thelymphocyte surface membrane was studied on the aliquotsof lymphocyte samples studied for membrane fluidity withDPH. Lymphocytes were isolated from blood by the FicollHypaque technique, and the cap-forming ability of the cellswas determined after binding of F-Con A, as reported inearlier studies (1, 2). Table 5 illustrates the results of theseexperiments. As in our previous studies (1, 3, 4), lymphocytes from patients with malignant lymphomas and chroniclymphocytic leukemia exhibited a significant reduction intheir ability to form caps with F-Con A. Most of the patientswith malignant lymphoma and chronic leukemia werewithin the range of 4 to 15% cap-forming ability. The vastmajority of patients with nonmalignant disorders and patients with carcinoma exhibited a pattern of cap formationsimilar to that of normal lymphocytes (25%). The decreasein cap-forming ability of leukemic lymphocytes from pa

Table 1

Table 2

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Degreeof fluaorescence

polarization of DPH and Con A formation of lymphocytes isolatedt different periods (Experiments 1 and 2) from the samepatientDegree

of fluores- Cells withcapscencepolarization after bindingwith(P)

at 25° F-ConA(%)Experi-

Experi- Experi- ExperiPatientDisorder ment 1 ment 2 ment 1 ment2M.

S.Chronic lymphatic Ieu- 0.266 0.264 11.0 10.8kemia,untreatedS.

A.Hodgkin's lymphoma 0.264 0.267 9.5 11.5duringtreatmentL.

I.Maligant lymphoma, 0.288 0.288 20.0 13.0early stage (Al)

Increasein membranemicroviscosity(ij) (decreasein lipidfluidity)inan acute lymphatic leukemic patient from onset of diseasetocomplete

remissionDegreeoffluores

cence polar- Microvisization (P) at cosity (‘i)atClinical

status 25° 25°(poise)Onset

of disease 0.2382.32Duringtreatment 0.2422.44Duringtreatment 0.2582.83Incomplete

remission 0.2673.12Remission0.290 3.93

Increase in the cap-forming ability of Con A receptors onthesurfacemembrane of lymphocytes isolated from lymphomaandleukemia

patientsCells

withcaps(%)afterbind

ingofF-ConANo.of pa

Group Disorder tients RangeAv.INonmalignant 28 17-3623IICarcinoma 5 20-3127IIIMalignant lymphoma 20 4-2613IVChronic lymphatic leu- 5 9-1912kemiaV

Acute leukemia 5 7-20 13

H. Ben-Bassat et a!.

Table 3

Table 4 donors. The results also indicate that these 2 dynamic behaviors of membrane lipids and receptors usually change tonormal in lymphocytes isolated from lymphoma and leukemia patients undergoing clinical remission.

DISCUSSION

The results of this study clearly indicate that lymphocytesisolated from peripheral blood of patients with malignantlymphomas and leukemias can be characterized by an increase in the degree of fluidity of the membrane lipid coreand by a reduced lateral mobilit@iof Con A receptor sites onthe cell surface. Previous studies with normal and leukemiccell populations obtained from experimental animals andhumans have shown that the surface membranes of leukemic cells have a lipid core less viscous (more fluid) thanthatof normal lymphocytes (14, 15, 17, 18, 21). The increasein fluidity in the leukemic cells is apparently mainly due to adecrease in a mole ratio of cholesterol to phospholipids inthe cell membrane (18). The results of the present and ofprevious studies in human leukemia indicate that the increase in membrane fluidity correlates with the activity ofthe disease. Patients with malignant lymphomas and Ieukemias in stages of clinical remission exhibit an increase inthe rigidity of the membrane lipid core as measured by theincreased values of fluorescence polarization obtained withthe DPH probe and the apparent microviscosity (@)values.Similar behavior was also observed in lymphocytes frompatients with chronic lymphocytic leukemia treated withcorticosteroids and was striking in acute lymphocytic leukemia. Moreover, studies attempting to elucidate the biological significance of membrane fluidity and of the cholesterolphospholipid ratio showed that the reduction of membranefluidity induced by the introduction of exogeneous cholesterol into the surface membrane of intact lymphoma cellsfrom mice resulted in a marked inhibition of their tumorigenicity (17). Similar effects of membrane cholesterol onthe malignancy of Ehrlich ascites carcinoma cells were alsoobtained by others (9). On the other hand, a controlledincrease in membrane fluidity of normal lymphocytes induced by a reduction of membrane cholesterol resulted in asignificant increase in the activation of the normal lymphocytes by plant mitogens (19).

Present and previous studies indicate that lymphocytesfrom patients with chronic lymphocytic leukemia, Hodg

Table5

tients with chronic lymphocytic leukemia was unaffectedduring treatment with corticosteroids. Patients with chroniclymphocytic leukemia and malignant Iymphoma in cIinical@remission exhibited cap-forming ability like that of normallymphocytes. Some of the patients were followed up forperiods of a few months and were examined several times.The results in Table 3 indicate that lymphocyte cap formation abilities with F-Con A recorded at different times for thesame patients are generally in very good agreement andusually correspond with the steady state of the disease.

Our present results have shown that lymphocytes isolatedfrom lymphoma and leukemia patients are characterized byan increase in the degree of fluidity of membrane lipids (lowP and@ values) and by a decrease in the lateral mobility ofCon A receptors (low percentage of cap-forming ability) ascompared to lymphocytes isolated from patients with nonmalignant disorders, patients with carcinoma, and normal

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Lipid Fluidity and Receptor Mobility

kin's and non-Hodgkin's lymphoma, and Burkitt's lymphoma have a reduced mobility of Con A receptor sites (1-4). This is true for lymphocytes isolated from biopsy specimens in these patients as well as for lymphocytes isolatedfrom peripheral blood. Lymphocytes isolated from peripheral blood of patients with carcinoma exhibited a mobility ofCon A sites similar to that recorded for normal lymphocytes.The present study also confirms the results of earlier studiesthat showed that lymphocytes isolated from the peripheralblood of nonlymphomatous patients (infectious mononucleosis, fever of undetermined etiology, rheumatoid arthritis, multiple sclerosis, polycythemia vera, and pancytopenia) exhibited Con A receptor mobility similar to that ofnormal lymphocytes (1). It appears that the reduced mobilityof Con A receptors is unrelated to the source of the lymphocytes in these patients, since both B- and T-Iymphocytesgive similar number of cap-forming cells (2).

The reduced mobility of Con A receptors has been interpreted (16, 20) to be characteristic of cancer. This explanation may be correct for the malignant tissue, such as theaffected lymph node or spleen, as well as for peripheralblood lymphocytes from patients with chronic lymphocyticleukemia. However, it cannot account for the behavior oflymphocytes isolated from blood of patients with malignantlymphomas, which, according to current knowledge, arenot necessarily malignant. An alternative explanation forthis phenomenon may be sought in the immunological stateof the lymphocyte. In many patients with Hodgkin's andnon-Hodgkin's lymphoma, impairment of cellular immunityhas been repeatedly demonstrated (7). The nature of thisimmunological defect has not yet been precisely established, but it is often characterized by the loss of severalmanifestations of cell-mediated immunity, without any apparent defect in the synthesis of human antibody (6, 23, 28).Whether there is an association between the abnormal behavior of the lymphocytes from patients with lymphoma andthe changes in their cellular surface membrane observedwith both the DPH and the Con A probe is not easy toestablish. In general, in the lymphoid system the mobility ofCon A receptors increases upon decreasing of the lipidfluidity and decreases with increasing fluidity of the lipidcore. These dynamic features are in accordance with thehypothesis that Con A receptors are more exposed to theaqueous surrounding when the cell surface lipid layer ismore rigid and, therefore, more mobile and vice versa (26).A similar correlation between the lipid fluidity and the mobility of Con A receptors was also observed in normal and

malignant transformed fibroblasts (26). However, it is important that this opposite interrelationship in the dynamicsof membrane lipids and proteins as determined by the DPHand the F-Con A probes may not represent a general behavior of biological membranes.

In the future, it will be of interest to determine the role ofDPH and Con A probes in clinical practice, as an earlydiagnostic aid in recognizing the presence of hematopoieticcancers. The possibility of using both probes in clinicalpractice as an aid in the differential diagnosis of malignantlymphomas and other diseases (15) and the possibility ofusing such methods for the prognosis of lymphoma and

leukemia patients (see Tables 2 and 4) are currently underinvestigation in our laboratory.

ACKNOWLEDGMENTS

The excellent technical assistance of L. Muznick is greatly appreciated.

REFERENCES

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2. Ben-Basset, H., Goldblum, N., Manny, N., and Sachs, L. Mobility ofConcanavalin A Receptors on the Surface Membrane of Lymphocytesfrom Normal Persons and Patients with Chronic Lymphocytic Leukemia.Intem. J. Cancer, 14: 367-371 , 1974.

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17. Inbar, M., and Shinitzky, M. Increase of Cholesterol Level in the SurfaceMembrane of Lymphoma Cells and Its Inhibitory Effect on Ascites TumorDevelopment. Proc. NatI. Acad. Sci. U. S.. 71: 2128-2130, 1974.

18. Inbar, M., and Shinitzky, M. Cholesterol as a Bioregulator in the Development and Inhibition of Leukemia. Proc. NatI. Aced. Sci. U. S., 71:4229-4231 , 1974.

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20. Inbar, M., Shinitzky, M. , and Sachs, L. Rotational Relaxation Time ofConcanavalin Bound to the Surface Membrane of Normal and MalignantTransformed Cells. J. Mol. Biol., 81: 245-253, 1973.

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22. Inbar, M., Yuli, I., and Raz, A. Contact-Mediated Changes in the Fluidityof Membrane Lipids in Normal and Malignant Transformed Fibroblasts.Exptl. Cell Res., in press.

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23. Jackson, S. M., Garrett, J. v., and Gaij, A. W. Lymphocytes Transforma- 26. Shinitzky, M., and Inbar, M. Microviscosity Parameters and Proteintion Changes during the Clinical Course of Hodgkin's Disease. Cancer, Mobility in Biological Membranes. Biochim. Biophys. Acta, 433: 133-25:843-848,1970. 149,1976.

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25. Shinitzky, M., and Inbar, M. Difference in Microviscosity Induced by 28. Young, A. C., Corder, M. D., Haynes, H. A., and De Vita, T. V. DelayedDifferent Cholesterol Levels in the Surface Membrane Lipid Layer of Hypersensitivity in Hodgkin's Disease: A Study on 103 Untreated PaNormal Lymphocytes and Malignant Lymphoma Cells. J. Mol. Biol., 85: tients. Am. J. Med., 52: 63-70, 1972.603-615, 1974.

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1977;37:1307-1312. Cancer Res   Hannah Ben-Bassat, Aaron Polliak, Stella Mitrani Rosenbaum, et al.   Lymphomas and LeukemiasLymphocytes and Lymphocytes from Patients with MalignantConcanavalin A Receptors in the Cell Surface of Normal Fluidity of Membrane Lipids and Lateral Mobility of

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