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seems clear that the input virus alone provided the stimulus." The close associa-tion of foci of giant cells with sites of subsequent transformation may also be relatedto this chronic infectious process; if the giant cells are sites of maximal virus growth,the neighboring cells may be most affected. In this connection, Shein et al.3 notedthat nuclei of the early giant cells consistently showed immune fluorescence withanti-SV40 rabbit serum.Summary.-When hamster kidney monolayers are exposed to the simian vac-

uolating virus-SV40, abnormal proliferation of tissue occurs after a variable latentperiod. The length of the latent period is dependent on the type of medium, virusstrain, and dosage of virus used. The transformed cells produce mixed tumors(carcinosarcomas) when transplanted to hamsters. SV40 proliferated continuallyin the primary cultures and became progressively more difficult to detect in trans-formed cultures; in late passages of transformed cells, virus could only be recoveredwhen these were planted directly on Cercopithecus kidney monolayers.

The authors are grateful to Mr. Richard Maloof for technical assistance and to Mr. JohnMcGuire for microphotography.

1 Rabson, A. S., and R. L. Kirschstein, Proc. Soc. Exptl. Biol. Med., 111, 323 (1962).2 Black, P. H., and W. P. Rowe, Virology, 19, 107 (1963).3 Shein, H. M., J. F. Enders, J. D. Levinthal, and A. E. Burket, these PROCEEDINGS, 49, 28 (1963).4Ashkenazi, A., and J. L. Melnick, J. Nat. Cancer Inst., 30, 1227 (1963).5 Black, P. H., and W. P. Rowe, submitted to J. Nat. Cancer Inst.6 Eddy, B. E., G. E. Grubbs, and R. D. Young, Proc. Soc. Exptl. Biol. Med., 111, 718 (1962).7 "Agamma" calf serum from Hyland Laboratories, Los Angeles, California.8 Eddy, B. E., G. S. Borman, G. E. Grubbs, and R. D. Young, Virology, 17, 65 (1962).9 Gerber, P., and R. L. Kirschstein, Virology, 18, 582 (1962).

10 Girardi, A. J., B. H. Sweet, and M. R. Hilleman, Proc. Soc. Exptl. Biol. Med., 112, 662 (1963).'1 Malherbe, H., and R. Harwin, Brit. J. Exptl. Path. 38, 539 (1957).12 Sweet, B. H., and M. R. Hilleman, Proc. Soc. Exptl. Biol. Med., 105, 420 (1960).13 Cooper, H. L., and P. H. Black, J. Nat. Cancer Inst., 30, 1015 (1963).14 Sabin, A. B., and M. A. Koch, these PROCEEDINGS, 49, 304 (1963).15 Gerber, P., Science, 140, 889 (1963).16 Stoker, M., and I. MacPherson, Virology, 14, 359 (1961).17 Black, P. H., W. P. Rowe, and H. L. Cooper, these PROCEEDINGS, in press.18 Stoker, M., Virology, 20, 366 (1963).

REACTIONS OF NORMAL AND TUMOR CELL SURFACES TO ENZYMES,I. WHEAT-GERM LIPASE AND ASSOCIATED

MUCOPOLYSACCHARIDES* t

BY JOSEPH C. AUB, CAROL TIESLAU, AND ANN LANKESTER

MASSACHUSETTS GENERAL HOSPITAL, BOSTON

Communicated August 2, 1968

Within the last few years many scientists have focused their attention on thenature of the cell membrane. It is well established that cellular adhesions andinteractions are largely dependent on the surface properties of cells. With theobservation that neoplastic cells differ from normal cells in the nature of these

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reactions, the cell surface was implicated as an important factor in the assumptionof a neoplastic state. The pioneering work of such men as Coman, Abercrombie,Easty, and Ambrose, in establishing certain major differences in normal and neo-plastic cell surface structure, further underlined the importance of the cell mem-brane.1-4' 6, 8-10, 12, 13

This laboratory has recently been interested in the nature of cell surfaces andthe changes taking place in them during malignancy. The electron microscopeproved valuable in the study of the uptake of ferritin particles by tumor cells,23but provided minimal information on the chemical nature of the tumor cell mem-brane. We therefore turned our attention to the study of the reaction of cellsurfaces to enzymes, working on the theory that if isolated normal and tumor cellsof comparable derivation respond differently to the same substance under care-fully controlled conditions, the nature of the response and of the substance causingthe response might throw light on the chemical structure of the tumor cell membrane.

Materials and Methods.-Cells for study: Isolated normal and tumor cells derived from com-parable cell types were needed for these observations. Normal rat and mouse thymocytes wereselected for comparison With lymphoma tumor cells, and tissue culture fibroblasts for comparisonwith Ehrlich ascites tumor cells. Normal mouse thymocytes were harvested in phosphate buf-fered saline (PBS) and isolated by the method of Roof and Aub22 from the thymuses of five-week-old Swiss mice; normal rat thymocytes were harvested in the same manner from five-week-oldWistar rats; normal fibroblasts, L-wild strain, were grown in tissue culture "Spinners" in supple-mented18 Eagle's Minimal Essential Media and later transferred into PBS for the experiments;L-C2 lymphoma cells (malignant thymocytes) were transplanted intraperitoneally into six-week-old Swiss (or ALN and A strain) interbred white mice, and after seven days the peritoneal fluidwas collected in PBS from the freshly killed mice; Ehrlich ascites hyperdiploid malignant mousefibroblasts were transplanted by intraperitoneal injection of 0.25 cc into six-week old-Swiss miceand harvested in the same manner as the lymphoma tumor cells.Enzyme preparations: The following enzymes were used in this series of experiments: (1)

phospholipase-D PL-D from C. F. Boehringer and Soehne GmbH, Mannheim; (2) porcine pan-creatic lipase from California Corporation for Biochemical Research; and (3) wheat-germ lipase,Grade B, from California Corporation for Biochemical Research and from Sigma Corporation.

Experimental preparation: The harvested cells were suspended in PBS by aspiration with aglass pipette, centrifuged for 5 min at 900 rpm, the supernatant poured off, and the cells resuspendedin 20 ml of PBS: washing and centrifugation were repeated three times. The cells were thensuspended in 10 ml. of PBS, counted, and diluted with PBS to the required cell concentrations.An aliquot of 0.9 ml. of cells/ml PBS solution was combined in a beaker with 0.1 ml of the enzymebeing tested, and the cell-enzyme solution incubated at room temperature for 10 min. A 0.05ml drop of this solution was then placed on a cover glass and enclosed in a Sykes-Moore cellchamber (Bellco Glass Co.); the hanging drop was observed immediately with the phase contrastmicroscope, and at 15-min intervals for several hours. This simple procedure and the use of theSykes-Moore cell chamber allowed for controlled variations of the cell concentrations, the pH ofthe solution, the nature of the buffer, the amount of calcium in the media, the concentration of theenzymes, the temperature of the enzyme solution, the incubation time, etc. The effect on the cellsof these variations could be easily and promptly observed in the microscope with minimal effectsdue to evaporation.

Determination of wheat-germ lipase activity: The activity of wheat-germ lipase was determined ina Warburg apparatus according to a modification of the method described by Singer and Hofstee.25The experiment was conducted at 38°C, pH 7.4, oscillations 150/min, gas phase 95% N2 and 5%C02; 1.6 ml of a 5.0 mg/ml solution of lipase was incubated with 0.046 ml of triacetin dissolved in2.35 ml of 0.025 M NaHCOa solution plus 1% gelatin.

Results were expressed in terms of juliters of CO2 liberated by lipase from the substrate at 5-minintervals for one hour.

(1) Inactivation of wheat-germ lipase with p-chloromercuribenzoic acid (p-CMB): Lipase was in-

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activated according to the procedure of Singer:24 0.6 ml of a 3.3 mg/ml solution of lipase wasincubated for 20 min with 0.4 ml of 0.025 M Na 1C003 and 2.0 ml of 4.5 X 10-4p-CMB in thebody of a Warburg flask. 0.02 ml of 0.02 M triacetin was added to the solution, and the Mulitersof C02 liberated from the substrate during one hour were determined.

(2) Inactivation of wheat-germ lipase with heat: Two ml aliquots of 5.0 mg/ml lipase in PBS wereplaced in beakers and incubated for 15 min in a Dubnoff metabolic shaking incubator at temper-atures of 45S85oC. After incubation, the solutions were centrifuged at 2,500 rpm for 10 min, andthe precipitate discarded. A 0.6 ml of the supernatant from each aliquot was incubated with thetriacetin substrate, and its lipolytic activity was determined as described above.

Results.-When phospholipase-D and porcine pancreatic lipase were tested in con-centrations ranging from 0.01 to 7.0 mg enzyme/ml of cell solution, no effects wereobserved on the cells. However, upon the addition of wheat-germ lipase to a sus-pension of isolated cells, it was observed that tumor cells were attracted to one an-other, forming a tightly bound clump of cells which, once formed, could not be dis-sociated by agitation, the addition of proteolytic enzymes, or EDTA. Normal cellsexposed to comparable concentrations of the enzyme remained almost entirely iso-lated. (See illustrations.) The cell membranes, though closely bound, appeared toremain intact. No mucinous coagulum was observed upon the addition of proteo-lytic enzymes. The viability of the cells was unaltered, as indicated by the com-parable impermeability to nigrosin of the control and enzyme-exposed cells and bythe continued ability of the agglutinated cells to produce tumor when 0.25 cc ofagglutinated solution was injected intraperitoneally into a mouse.

Table 1 indicates the degree of clumping of normal and tumor cells when exposedto varying dilutions of supernatant from heated wheat-germ lipase. The reason

FIG. 1. Tumor cells. FIG. 2. Tumor cells with lipase.

FIG.3.-NormalcellW.FI,. 4. Normal cells wit

WN-16-~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~~~~

Fi oml elK Nra el wt iae

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TABLE 1

THE DEGREE OF CLUMPING OF NORMAL AND TUMOR CELLS SUSPENDED INDILUTIONS OF HEATED "LIPASE"*

Normal- TumorRat Mouse Mouse

Heated thymocytes thymocytes fibroblasts Mouse L#1 Mouse"lipase"* 5 X 106 (5 X 106 (1 X 106 lymphoma Ehrlich ascites

(ml/ml of cells) cells/ml) cells/ml) cells/ml) (1 X 106 cells/ml) (1 X 106 cells/ml)0.100 + + ++ +++ +++0.075 0 + ++ +++ +++0.050 0 + ++ +++ +++0.025 0 + + +++ +++0.010 0 0 + ++ ++0.0075 0 0 0 ++ +0.0050 0 0 0 + +0.0025 0 0 0 + 00.0010 0 0 0 0 00.00075 0 0 0 0 0

* 10 mgm/ml wheat germ lipase heated to 650C for 15 min; centrifuged; supernatant used.+ refers to the presence in the drop of only a few agglutinated cells, the majority of the cells remaining isolated;

+ + indicates the presence of a greater number of small clumps; ++ + indicates that all cells are clumped in smallgroups; + + + + (Table 2 only) indicates massive agglutination of all cells.

for correlating clumping with dilutions of supernatant of lipase heated to 650Crather than with mg/ml of unheated lipase solution will be discussed below.

It can be seen in the table that at all dilutions of "heated lipase supernatant"tumor cells clumped far more than normal cells. Normal cells stop clumping at adilution of 0.01-0.05 ml of lipase supernatant, while tumor cells stop at 0.001-0.005 ml, or a dilution factor of 10. In some of our experiments, fibroblasts werefound to clump slightly more than normal cells, thereby raising the averagedegree of clumping for this type of cell as indicated in the chart. It has beensuggested that "tumor-like" alterations of the cell membranes occur when somecells are grown in tissue culture.26 Transplantation of these tissue-culture-grownfibroblasts into the abdominal cavity of mice occasionally results in tumor cellgrowth. Therefore, we think that these fibroblasts were not purely benign cells, butwere a mixture of normal and occasional tumor cells which produced an intermediateclumping reaction as indicated in Table 1. Not only does this differential clumpingreaction hold true for the cells tested here, but for human normal and leukemicwhite blood cells as well.5The average surface area of the normal and tumor cells was determined, and

it was found that rat and mouse thymocytes were approximately five times smallerthan the other cells. Therefore, in most of the experiments, the cell concentrationsused were 5 X 106 cells/ml for thymocytes and 1 X 106 cells/ml for other cells.To be sure that the observed differential clumping reaction was not merely a resultof this variation in cell size or of the concentration of the cells used, the degree ofcell clumping was determined at various concentrations of normal and tumor cells.Table 2 indicates that tumor cells even in very dilute concentrations are still muchmore reactive to wheat-germ lipase than normal cells. As tumor cell concentrationincreased, massiveness of the clumping increased; but normal cells, even thoughclearly adjacent, tended to remain almost unattached.

Other factors that might have had an effect on the clumping reaction weretested. It was found that tumor cells clumped as effectively in Tyrodes bufferand Versene as in PBS, although cells remained viable longer in the PBS. Additionof calcium to the media or its removal by EDTA had no effect on the degree of

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TABLE 2EFFECT OF THE VARIATION IN CELL CONCENTRATION ON THE DEGREE OF

CLUMPING BY HEATED "LIPASE"Heated"lipase" --Mouse thymocytes (cells/ml)- , Mouse lymphoma (cells/ml)

(ml/ml of cells) 1 X 1O6 5 X 106 10 X 106 5X 105 1 X 106 5 X 10'0.100 + + + ++ +++ ++++0.075 + + + t + +++ ++++0.050 0 + + ++ +++ +++0.025 0 0 + + +++ +++0.010 0 0 + + ++ +++0.0075 0 0 0 + ++ ++0.0050 0 0 0 + + +0.0025 0 0 0 0 0 00.0010 0 0 0 0 0 00.00075 0 0 0 0 0 0

agglutination. The reaction was unaltered by incubation of the enzyme solutionin temperatures up to 750 or by alteration of the pH (within physiological limits forcell viability). The reaction appeared to be tumor cell specific, for when normaland tumor cells were mixed in the same chamber, only the tumor cells agglutinated.

Further analysis of the nature of the reaction taking place at the cell surfacenecessitated an assay of the wheat-germ lipase for lipolytic activity. Severalaliquots of two commercial preparations of wheat-germ lipase were assayed in theWarburg apparatus according to a method described by Singer and Hofstee.25It was found that lipase made by Sigma Company generated 179 1l C02/mg oflipase in one hr, while California Biochemical lipase generated only 62 1AI C02/mgin the same period. However, when the activities of these two preparations werecorrelated with their ability to agglutinate tumor cells, it was discovered that theCalifornia Biochemical preparation was the more effective. These results led us towonder whether the lipolytic portion of the lipase were responsible for the clumping.We therefore undertook to inactivate the lipolytic portion of the enzyme and test theremaining solution for clumping effectiveness. Addition of p-chloromercuribenzoicacid to the lipase resulted in 85 per cent inhibition of lipolytic activity. But this"inactivated" lipase continued to clump cells as effectively as untreated lipase.Table 3 shows the effect of heat inactivation on the degree of clumping. It canbe seen that lipase activity decreased markedly with increasing temperature andceased entirely at 60'C, while the clumping factor was not inactivated until 750C.Cell clumping and lipolytic activity appeared to have no correlation. Until thistime, cell clumping had been correlated with mg/ml lipase solution. When it wasfound that heating lipase to 650C removed most of the protein but left the clumpingfactor intact, we decided to express the degree of clumping in terms of ml of heatedlipase solution added to the cells.

TABLE 3INACTIVATION OF LIPASE BY HEAT: COMPARISON OF LIPASE ACTIVITY AND CELL CLUMPING

Preincubation oflipase for 15 min Lipase activity Ehrlicb ascites cells(temperature-0C) (% of control) (degree of clumping)

24 100.0 +++45 52.0 +++55 24.0 +++60 2.5 +++65 0 ++75 0 085 0 0

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Discussion.-We have found a substance, present in the supernatant of heatedwheat-germ lipase, which, so far, appears to be tumor cell specific. When thissubstance is added in comparable concentrations to suspensions of normal and tumorcells, it causes the agglutination of the tumor cells while the normal cells remainalmost completely isolated.The fact that the tumor cells, although agglutinated, appear to retain their

viability and tumor-producing capacities seems to indicate that the agglutinationis not a result of cellular destruction or alteration of metabolic processes but israther a surface phenomenon. Other substances usually classified as hemagglu-tinins or lectins, which cause the agglutination of cells, have been described.7Many of these substances are plant-derived and produce their agglutinating effectby reacting with specific antigenic groups on the surfaces of red blood cells.20' 21Perhaps we, too, are dealing with such an antigenic reaction, but thus far we havebeen unable to find any mention in the literature of a plant agglutinin known toreact specifically with tumor cells.

This laboratory is now undertaking a thorough chemical analysis of the clumpingfactor, in the hope that further knowledge of its chemical composition will serve as ameans toward an understanding of the nature of the agglutination reaction. Ourexperiments thus far seem to indicate that the clumping factor is not a lipase. Ofall the lipases tested, only wheat-germ lipase elicited agglutination of tumor cells,but inactivation of this wheat-germ lipase failed to inactivate the clumping factor.Coman has suggested that calcium is a major factor in cellular adhesions; however,addition of calcium to our suspending media or its removal by EDTA failed to affectthe agglutination reaction. Subsequent dialysis of the wheat-germ lipase solutionindicated that our clumping factor was a large molecule and therefore not a metal.Preliminary laboratory reports at this time indicate that our clumping factor maybe a mucopolysaccharide capable of being inactivated by periodic acid. Other in-vestigators have implicated mucopolysaccharides in cellular interactions andadhesions'419 and have suggested that a change in their nature might play a role inthe assumption of the metastatic state.,-"7 If our clumping factor is a mucopoly-saccharide, the similarity between it and plant agglutinins would be further under-lined, as would the possibility that we are dealing with an antigenic response oftumor specific antigens.We do not at this time know how our clumping factor works-whether it combines

with a reactive site on the tumor cell membrane which is absent on normal cells, orwhether it removes a portion of the membrane rendering the cell surface sticky. Wethink that we have a tumor specific substance and, at this time, we are trying to iso-late other tumor and normal cells of comparable cell derivation in an effort to deter-mine the extent of its selective effect. It is our hope that, until we fully understandthe nature of the agglutination reaction, our finding will serve as a valuable aid inthe study of the alterations of cell membranes that occur during neoplasia.

We should like to thank Dr. Mary L. Stephenson for her invaluable advice, and Dr. EmmaShelton for the gift of the ILC2 lymphoma tumor.

* Supported in part by grant C-2867 from the U.S. Public Health Service, National CancerInstitute, and the American Cancer Society, Massachusetts Division.

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VOL. 50, 1963 PHYSIOLOGY: TASAKI AND TAKENAKA 619

t Presented before the American Association for Cancer Research, Toronto, Canada, May 25,1963.

1 Abercrombie, M., and E. J. Ambrose, Cancer Research, 22, 525 (1962).2Ambrose, E. J., in Biological Interactions in Normal and Neoplastic Growth, ed. M. J. Brennan

and W. L. Simpson (Little, Brown and Company, 1962), p. 149.3Ambrose, E. J., and G. C. Easty, Proc. Roy. Phys. Soc. Edin., 28, 53 (1959).4Ambrose, E. J., J. A. Dudgeon, D. M. Easty, and G. C. Easty, Exptl. Cell Res., 24, 220

(1961).5 Aub, J. C., and G. Kury, unpublished data.6Berwick, L., and D. R. Coman, Cancer Res., 22, 982 (1962).7Boyd, W. C., Vox Sanguinis, 8, 1 (1963).8 Coman, D. R., Cancer Res., 21, 1436 (1961).9 Coman, D. R., and T. F. Anderson, Cancer Res., 15, 541 (1955).10Cormack, D. H., G. C. Easty, and E. J. Ambrose, Nature, 190, 1207 (1961).11 DeLong, R. P., D. R. Coman, and I. Zeidman, Cancer, 3, 718 (1950).12 Easty, G. C., D. M. Easty, and E. J. Ambrose, Exptl. Cell Res., 19, 539 (1960).13 Easty, G. C., and V. Mutolo, Exptl. Cell Res., 21, 374 (1960).14 Essner, E., H. Sato, and M. Belkin, Exptl. Cell Res., 6-7, 430 (1954).15Gasic, G., and T. Baydak, in Biological Interactions in Normal and Neoplastic Growth, ed.

M. J. Brennan and W. L. Simpson (Little, Brown and Company, 1962), p. 709.16 Gasic, G., and T. Gasic, these PROCEEDINGS, 48, 1172 (1962).17 Gasic, G., F. Loebel, and 0. Badinez, Nature, 185, 864 (1960).8 Littlefield, J. W., Exptl. Cell Res., 26, 318 (1962).19 Moscona, A. A., in Biological Interactions in Normal and Neoplastic Growth, ed. M. J. Brennan

and W. L. Simpson (Little, Brown and Company, 1962), p. 113.20 Nowell, P. C., Cancer Res., 20, 462 (1960).21 Rigas, D. A., and E. E. Osgood, J. Biol. Chem., 212, 607 (1955).22 Roof, B. S., and J. C. Aub, Cancer Res., 20, 1426 (1960).23 Ryser, H., J. B. Caulfield, and J. C. Aub, J. Cell Biol., 14, 255 (1962).24 Singer, T. P., J. Biol. Chem., 174, 11 (1948).2" Singer, T. P., and B. H. J. Hofstee, Arch. Biochem. Biophys., 18, 299 (1948)."6Weiler, E., in Biological Interactions in Normal and Neoplastic Growth, ed. M. J. Brennan

and W. L. Simpson (Little, Brown and Company, 1962), p. 141.

RESTING AND ACTION POTENTIAL OF SQUID GIANT AXONSINTRACELLULARLY PERFUSED WITH SODIUM-RICH SOLUTIONS

BY I. TASAKI AND T. TAKENAKA

LABORATORY OF NEUROBIOLOGY, NATIONAL INSTITUTES OF HEALTH, BETHESDA, MARYLAND, ANDTHE MARINE BIOLOGICAL LABORATORY, WOODS HOLE

Communicated by Hallowell Davis, July 31, 1963

The axoplasm of freshly excised squid giant axons contains a relatively low con-centration of sodium (see, e.g., Steinbach and Spiegelman'). It is generallybelieved that a low sodium and a high potassium concentration in the axoplasm isthe condition necessary for the maintenance of normal excitability of the axon.Until quite recently, however, there was no direct means of varying the intracellularionic composition to test whether this condition is actually necessary. Owing tothe development of the methods of intracellular perfusion in recent years,2-6 it isnow possible to examine the effects of high sodium in the interior of the axon uponthe resting and action potentials.

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