Proceedings of the National Academy of SciencesVol. 67, No. 4, pp. 1741-1747, December 1970

Cell Density-Dependent Changes of GlycolipidConcentrations in Fibroblasts, and Loss ofThis Response in Virus-Transformed Cells

Sen-itiroh Hakomori

BIOCHEMICAL LABORATORY, DEPARTMENT OF PATHOBIOLOGY, UNIVERSITY OF WASHINGTON,

SCHOOL OF PUBLIC HEALTH AND COMMUNITY MEDICINE, SEATTLE, WASH. 98105

Communicated by Herman M. Kalckar, September 8, 1970

Abstract. The glycolipids of normal fibroblastic cells at different stages ofgrowth and with differing degrees of susceptibility to contact inhibition havebeen analyzed, as well as the glycolipids of virally transformed cells. The con-centrations (per mg protein) of certain glycolipids, including galactosylgalactosyl-glucosyl-, N-acetylneuraminosylgalactosylglucosyl-, and N-acetylneuraminosyl-(N-acetylneuraminosyl)galactosylglucosyl-ceramide, increase on cell-to-cell con-tact of susceptible cells. The concentrations of these glycolipids decrease whensusceptibility to contact inhibition is lost as a result of extensive passage in vitroor transformation by simian virus 40 or polyoma virus, and in all the malignantcells examined, the concentrations of these glycolipids were independent of celldensity.

The loss of contact inhibition and subsequent uncontrolled cell division byvirus-transformed cells has been correlated with the process of tumor growth.1'2An increase of surface negative charge' 4 and uncovering of "cryptic" carbohy-drate haptens5-8 have been correlated with the loss of contact inhibition.

In previous papers8'9 the incomplete synthesis of glycolipid carbohydratechains in transformed cells was suggested as an explanation of the absence ofcertain nonreducing carbohydrate residues in some glycolipids and the concomi-tant increase of precursor glycolipids. A similar phenomenon was described'0'1'in the membrane glycoproteins of mouse fibroblastic cells, and more recently,the absence of higher gangliosides in malignant cells has been reported. 12"3These data have suggested that altered membrane heteroglycans may be

linked to uncontrolled cell division. It should be noted, however, that the totalamount of cellular heteroglycan was higher in cells grown on monolayer than inthose grown in suspension culture, where cell-to-cell contact did not take place. 14A related observation1' is that one of the membrane glycopeptides of mouse fibro-blasts (3T3) increases on cell confluence. In the present investigation, theglycolipids of "normal" cultured cells with different degrees of contact inhibitionwere compared at different cell densities and with the glycolipids of virallytransformed cells. The results indicate that the concentration (per mg protein)of certain glycolipids increases at high cell densities. Malignant cells do notexhibit these density-dependent changes of glycolipids.

1741

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0

1742 MEDICAL SCIENCES: S. HAKOMORI PROC. N. A. S.

i 2 3 5

Iacofunt@phse 2,1pt hgceldniy1, cel lin I at. cofunhs. To sot

of samples 1-4 were glucosylceramide, overlapped with some contaminants. 5-7: "upperphase glycolipid" of human fibroblasts. 5, cell line IV at growing phase; 6, cell line IV atconfluent phase; 7, cell line V at high density. The amounts of glycolipid applied to theseplates were from identical amounts of cells (2 mg of protein).

a-e, f-k standard compounds; a, cerebroside (Gal-Cer); b, lactosylceraide; c, ceramidetrihexoside; d, globoside; e and f, N-acetylhematoside; g, N-glycolylhematoside; h, disialo-hematoside (human stomach); i, Tay-Sachs ganglioside; j, disialohematoside (cat erythro-cytes); k, monosialoganglioside (brain).

a-e, 1-4 were developed on borate-silica gel H with chloroform-methanol-water 65:30:8(lower phase); f-k, 5-7 were developed on silica gel H with chloroform-methanol-water60:35:8.

Materials and Methods. Glycolipid Samples: Pure reference samples, whosethin-layer behavior is shown in Fig. 1, were prepared in this laboratory as follows:1-galactosyl(1-.4)glucosylceramide ("lactosylceramide," spot b); galactosyl(1-4)-galactosyl(1-4)glucosylceramide ("ceramide trihexoside," CTH, spot c); ,3-N-acetyl-galactosaminosyl(1~*3)galactosyl(1 - 4)galactosyl(1--4)glucosylceramide ("globoside,"spot d) from human erythrocytes'6; N-acetylneuraminosyl(2-3)galactosyl (1-'4)glucos-ylceramide ("N-acetylhematoside," spots e and f) from human spleen'8; N-glycolyl-neuraminosyl(2-83)galactosyl(1--4)glucosylceramide ("N-glycolylhematoside," spot g)from horse erythrocytes'7; N-acetylneuraminosyl(2-~8?) [N-acetylneuraminosyl(2-.*3)]gal-actosyl(1-*4)glucosylceramide ("disialohematoside," spot h) from human gastric mucosa(unpublished results); N-glycolylneuraminosyl(2--8) [(N-glycolyl)neuraminosyl(2-3) ]-galactosyl(l14)glucosylceramide (another "disialohematoside," spot j) from cat eryth-rocytes'6; and "monosialoganglioside," /3-galactosyl(1-*3)N-acetylgalactosaminosyl-(1-94)-[N-acetylneuraminosyl(2-->3) ]galactosyl(1-4)glucosylceramide (spot k), fromhuman brain'9. Tay-Sachs ganglioside, N-acetylgalactosaminosyl(1-*4) [N-acetyl-neuraminosyl(2- 3) ]galactosyl(1-*4)glucosylceramide (spot i), was a gift from Dr. A.Makita, Tohoku University, Sendai.

Cells: The following cell lines were used: (1) Cell line I: hamster kidneyfibroblasts (BHK-21/C13, ref. 20) at extremely low passage level, kindly donated byDr. I. MacPherson, Imperial Cancer Research Fund, London, England. This cellline showed parallel arrangement on confluence, with no tendency toward overlapping.Saturation density was about 8 X 104/cm2 on "Falcon" brand plastic Petri dishes. Sub-cutaneous injection of 10)6 cells into 20-day-old hamsters did not result in tumor formation.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0

VOL. 67, 1970 GLYCOLIPIDS AND CONTACT INHIBITION 1743

(2) Cell line II: cell line I after subecultivation. for over 35 passages. (3) Cell line III:a subelone of BHK-21/C13, which originated from Dr. Stoker's laboratory and was usedin the previous study, cultured for 50 additional passages. Both cell lines II and III hadparallel arrangement on confluence but showed a lpartial overlapping figure. Saturationdensity of these cell lines was about 1.8 X 105/cm2. Upon injection of 106 cells intohamster, both cell lines II and III yielded palpable tumors after 6 weeks. (4) Cell lineI-py: cell line I transformed by polyoma virus and a clone was isolated by the agarselection method21. (5) Cell line III-py: the polyoma-transformed cell line used in theprevious study,9 cultured for 60 additional passages. These transformed cells showed aremarkable overlapping figure and injection of 106 cells into hamster gave rise to a pal-pable tumor after 1 week. The above cells were grown in Eagle's medium (2X aminoacids and vitamins) supplemented with 10% fetal calf serum. (6) Cell line IV: humandiploid fibroblast 8166 (ref. 22), isolated and donated by Dr. Pious, Department of Pedi-atrics, University of Washington. These cells showed good contact inhibition and havebeen used before cessation of growth. Cell line IV was grown in medium 199 (GrandIsland Co., New York) supplemented with 20% fetal calf serum. (7) Cell line V: ahuman heteroploid cell28 transformed by simian virus 40 (SV40). The cells showed nocontact inhibition. They were grown in Dulbecco's modified Eagle medium (GrandIsland Co.) supplemented with 10% fetal calf serum. All cell lines were grown in glassbottles or in "Falcon" brand plastic Petri dishes of various sizes.Growth conditions for different cell densities: An inoculum of 106 cells per 5-cm

Petri dish (Falcon, plastic) resulted in completely confluent cells after 48 hr, whereasthe cells from the same amount of inoculum on a 14-cm Petri dish were still in theirlogarithmic growth phase after 48 hr. An inoculum of 0.5 X 106 cells of transformedcells was grown under the same conditions in the two sizes of Petri dish for "high" and"low" densities. In some experiments 2 X 106 cells (for confluency), or 2 X 105 cells(for growing phase), were inoculated per glass bottle (growing area 16 X 7 cm) and thecells were harvested after 72-96 hr. The medium was changed every 24 or 48 hr.

Isolation and characterization of glycolipids: Cells were harvested, and the lipidswere extracted and subsequently fractionated to obtain "upper phase" glycolipid(Folch) by partitioning ten times as described previously (Fraction 1).9 The glycolipidpresent in "lower phase" was separated quantitatively as acetylated compounds bychromatography on a Florisil column. The fraction eluted with 1,2-dichloroethane-methanol 9:1 or 1,2-dichloroethane-acetone 1:1 contained all of the scteylated glyco-lipid, which was then deacetylated in chloroform-methanol 2: 1 containiag 0.1% sodiummethoxide.24 In this way the glycolipids present in the "lower phase" were quantita-tively separated from phospholipids, cholesterol, and other lipids. Glycolipids werecharacterized by: comparison of their migration rates on thin-layer chromatographywith those of standard glycolipids, determination of the molar ratio of sugar by gas-liquid chromatography [Sweeley and Walker25, and Yang and Hakomori (unpublished)],and comparison of the products of partial hydrolysis with those from known glycolipids.

Quantitation of glycolipids: Glycolipids were quantitatively determined on thin-layer chromatography by comparison of the intensities of spots developed withresorcinol reagent or with orcinol-sulfuric acid with known quantities (3-30 ,g) of stan-dard glycolipids. The comparison was made by end-point reading on serial dilutions ofthe unknown samples and standard samples. The error of this method was within 10%,if carefully controlled; the method was found to be better than the photoelectric densi-tometry or colorimetry in vitro of the extracted spots, in which the standard error oftenexceeded over 20%. In some experiments, cells were grown for 48 hr under the conditionsdescribed under Growth conditions in the presence of [U-I4C]serine plus [C6-'H]glucosamine,or plus [C6-3HIglucose. The specific activities of the labeled compounds were aboutthe same as described by Shen and Ginsburg14, or Warren and Glick26, namely 1-2 MC' of[8H]glucose or [8H]glucosamine and 0.3-0.5 MCi of [14C]serine per ml of culture medium.Serine was used to label sphingosine and proteins. Cells were harvested and extractedwith chloroform-methanol and the cell residue was collected by filtration. The radio-

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0

1744 MEDICAL SCIENCES: S. HAKOMORI PROC. N. A. S.~~~~~~~~~~~~~~~~~~~~~I'Cc,)o

0-~~~~~~~~~a-

nC e'c t c;

b4 C O O O O O O O O O o Ov04 VV 00 C 0

CZ bebD co>6ob E; wa

COPa

ad mk C)) 0 0 0 0

ek) em ~ ~ ~ ~ ~ ~ ~ .)~

00 00000 0 00 :co CD

Cs &Os 4: ) =

C -

7) :c -C)o N N L Loe ~~~~~~~~~~~~~~xo cq r- N "04 Lo T-- t O

4. 44e ci 00 00 0 Lf( X 0> O OO.<

4.) Cv 0 0 C 0 O

. B8U i3:2B 3, E

°V ~~~~~ ~~~~~~~~~~~vv &JJ

4)~~~~~~~~~~~,eO~~~~~~~~~~~e~~~~~~o~~OD~~oC

- ~ ~ . °4Q 6

X~~~~~~~~~~0 LO 00 0 cq O + n

H~~~~~~~~~c t- 0 00 t- t- *<

~4f 0 C4

.1.4%C)be to bo 93 ~ ~ ' C)~

00 ~ 00's 005

4) -

4,47)0 o)V. - 0 000 0

I..t5 0 ( - -4 4a 10,

ad-~4~-lC0

4)~~~~~~~

-~~~~4)C~~~~~~~~ -~~~~~ ~ ~~qC ~ ~ 4 44

4.)~~~~~~~~~-:3z'4 ~

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0

VOL. 67, 1970 GLYCOLIPIDS AND CONTACT INHIBITION 1745

activity (14C and 'H) of the cell residue was counted in a scintillation counter (cell proteincount). Glycolipids were isolated by acetylation24 followed by thin-layer chromatog-raphy. The amount of glycolipid fraction applied to the thin-layer plate was variedaccording to the size of the "cell protein count." Glycolipids were dissolved in 100 pl ofchloroform-methanol per 106 cpm of "cell protein count" for 14C, and 10-20 Al wasapplied onto a plate. The thin-layer plate was scanned by the Packard radioactivityscanning instrument. The radioactive spots were scraped and counted more exactly in ascintillation counter for 'H and 14C.

Results. Glycolipids of BHK Cells: BHK-21/13 cells with a low number ofpassages (cell line I) contained a ceramide trihexoside (CTH; galactosylgalac-tosylglucosylceramide) in addition to hematoside and lactosylceramide (Table1). The concentration (per mg protein) of this glycolipid decreased during 35passages of successive culture, and it was not found at all in cell line II or III,which showed lesser degree of contact inhibition. This glycolipid was not foundeither in polyoma transformants (I-py). However, there was a concomitantincrease in lactosylceramide in the freshly transformed cells (Table 1 and lanes2-4 of Fig. 1). A considerable variation was found in the concentration oflactosylceramide of the transformed BHK cells; it seemed to decrease on suc-cessive passage (Table 1).The CTH of the contact-sensitive cells (cell line I) increased at high cell density

(lanes 3 and 4 of Fig. 1 and Table 1). Higher labeling of CTH in cells at highercell density was also demonstrated by higher incorporation of isotope from [I'HIglucose and [14C]serine into this glycolipid at higher cell density (see Fig. 2)

I-G it-G d IV-GC

2 1 0e4 ~~~~~~~~~~~~~~b

2 3 4

I C~~~~~~~~~qd

Aj 5 tI-c tv-cA a~~~~~

5 ~~~~b

I-PY-Low II-PYLow d V-Lowj II-PY-High i it-PY-High V-High

3 45 ~ 23 45

d

a

B

FIG. 2. Radioactivity scan of thin-layer chromatography of glycolipids extracted fromisotope-labeled cells grown at different cell densities. I, II, IV, and V denote each cell line(G, growing phase; C, confluent phase); I-py and II-py denote the polyoma virus-transformedcell lines. Low or High denote low or high cell densities of these transformed cells (see the text).

Ordinate: total activity count (3H + 14C), 103 cpm for the scale A. Abscissa: length ofscan, 20 cm for the scale B.

Peaks: 1, hematosides; 2, ceramide trihexoside (CTH); 3, lactosylceramide; 4, glucosyl-ceramide; 5, unidentified but includes ceramide; a, monosialoganglioside; b, Tay-Sachsganglioside; c, disialohematoside; d, hematoside; e, glucosylceramide.

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0

1746 MEDICAL SCIENCES: S. HAKOMORI PROC. N. A. S.

In the cell line II or III, concentrations of all the glycolipids increased at higherdensity (as judged by chemical analysis), although only slightly. Increasedincorporation of isotope into the glycolipids (especially hematoside) at highercell density was clearly demonstrated and this was more obvious than the re-sults of chemical analysis (Table 1, Fig. 2). In contrast, the concentrationsof glycolipids in two polyoma-transformed cells (I-py and Il-py) did notchange with cell density, as was shown by chemical analysis as well as by radio-chemical analysis (Table 1 and Fig. 2).

Glycolipids of human fibroblasts: Glycolipids of human diploid fibroblasts22were identified as glucosylceramide, monosialohematoside, disialohematoside,and monosialoganglioside (Table 1). The concentrations of the last two glyco-lipids changed markedly (1- to 2-fold increase) with cell confluence (Table 1,Fig. 1).The glycolipids that responded to cell confluence disappeared or were very

greatly reduced in concentration after SV40 transformation. The SV40-trans-formed cells had ganglioside like that characteristic of Tay-Sachs disease asthe major glycolipid, and showed no variation with cell-density changes (Fig. 2).

Discussion. The results of this study suggest that the concentrations of cer-tain glycolipids increase on cell-to-cell contact of normal cells. This is presum-ably due to the attachment of sugar residue to precursor lipid. The most typicaland sensitive extension response was demonstrated by the terminal galactosylresidue of CTH in a contact-sensitive "BHK-cell line I" and by the terminalsialosyl residue of disialohematoside of human diploid cells. The observations,discussed in the Introduction, of Shen and Ginsburgl4 and of 1\eezan et al. I' maybe related to this response. Warren and Glick26 observed that the turnoverrates of lipid and of proteins in nongrowing cells are higher than those in growingcells. It is possible that a particular component of the cell surface could bemetabolically more active on cell-to-cell contact. The cell density-dependentincrease of glycolipids appeared more striking in isotope-labeling experimentsthan by chemical analysis. This tendency is particularly obvious in cell lineII, in which both synthesis and degradation of glycolipid could be enhanced bycell contact. It is conceivable that some cellular glycolipid could be metabol-ically more active on cell-to-cell contact than in the absence of contact. Itis also possible that each glycolipid showed different turnover rates, with dif-ferent responses on cell-to-cell contact. Kinetic studies are planned.

Interestingly, the glycolipids that show cell density-dependent positive re-sponses decrease in concentration on successive passages or after malignanttransformation of the cells. The "incomplete" synthesis of the carbohydratechain9 apparently observed in the transformed cells may be related to a lack of"extension response," i.e., lack of addition of terminal sugar residue in the trans-formed cells on cell-to-cell contact.The density-dependent changes of membrane glycolipids could be one of the

surface-mediated mechanisms for controlling cell division, closely linked to con-tact inhibition. Those carbohydrate chains that have shown density-dependentresponse may play some role in cell-sociologic recognition between cells. Regu-lation of synthesis and degradation of these carbohydrate chains, possibly

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0

VOL. 67, 1970 GLYCOLIPIDS AND CONTACT INHIBITION 1747

through a "contact-sensitive" enzyme system, wvill be of crucial imlortance forunderstanding various phases of cell-sociologic phenomena, including malig-nancy. The impaired activity (due to an intense negative feedback inhibition)of UDP-galactose-epimerase27 in some tumor cells as compared with progenictissue could be related to the repressed "galactosyl extension" suggested by thisinvestigation.Note added in proof. The terminal galactosyl linkage of the CTH, showing

a sensitive response, has recently been identified as a-galactosidic, in contrastto other glycosidic linkages, which are all ,3 (S. Hakomori, C. G. Hellerqvist,Y. T. Li, and B. Siddiqui, in preparation).

The author thanks Drs. Hiroshi Nikaido (University of California), Victor Ginsburg (Na-tional Institutes of Health), and Herman M. Kalckar (Harvard Medical School) for readingthis manuscript, Drs. Ian MacPherson (Imperial Cancer Res. Fund, London, England) andDonald Pious (University of Washington) for donation of cells, and Dr. Terunobu Saito, Mrs.Carol Teather, Mrs. Judy Bruce for valuable technical assistance. The work was supportedby the American Cancer Society, grant no. T475, and National Institutes of Health, grantno. CA-10909. This is the 5th Report on "Glycolipids and Malignancy."

Abbreviations: CTH, ceramide trihexoside; -py, polyoma-transformed.1 Abercrombie, M., and E. J. Ambrose, Cancer Res., 22, 525 (1962).2 Vogt, P. K., Cancer Res., 23, 1519 (1963).

<w Ambrose, E. J., A. M. James, and J. H. B. Lowick, Nature, 177, 576 (1956).4 Defendi, V., and C. Gasic, J. Cell. Comp. Physiol., 62, 23 (1963).6 Burger, M. M., Proc. Nat. Acad. Sci. USA, 62, 994 (1969).6 Inbar, M., and L. Sachs, Nature, 223, 710 (1969).7 Hiyry, P., and V. Defendi, Virology, 41, 22 (1970).8 Hakomori, S., C. Teather, and H. Andrews, Biochem. Biophys. Res. Commun., 33, 563

(1968).9 Hakomori, S., and W. T. Murakami, Proc. Nat. Acad. Sci. USA, 59, 254 (1968).

1 Wu, H. C., E. Meezan, P. H. Black, and P. W. Robbins, Biochemistry, 8, 2509 (1969)." Meezan, E., H. C. Wu, P. H. Black, and P. W. Robbins, Biochemistry, 8, 2518 (1969).12 Mora, P. T., R. 0. Brady, R. M. Bradley, and V. M. McFarland, Proc. Nat. Acad. Sci.

USA, 63,1290 (1969).13 Brady, R. O., C. Borek, and R. M. Bradley, J. Biol. Chem., 244, 6552 (1969).14 Shen, L., and V. Ginsburg, in Biological "Properties of Mammalian Surface Membrane,"

ed. L. A. Mason (Wistar Institute Symposium Monograph no. 8, 1968), p. 67.16 Handa, N., and S. Handa, Jap. J. Exp. Med., 35, 332 (1965).16 Hakomori, S., and G. D. Strycharz, Biochemistry, 7, 1279 (1968).17 Yamakawa, T., R. Irie, and M. Iwanaga, J. Biochem., 48, 490 (1960).18 Svennerholm, L., Acta Chem. Scand., 17, 860 (1963)."I Penick, R. T., M. H. Meisler, and R. H. McCluer, Biochim. Biophys. Acta, 116, 279 (1966)20 Stoker, M., and I. MacPherson, Virology, 14, 359 (1961).21 MacPherson, I., and L. Montagnier, Virology, 23, 292 (1964).22 Pious, D. A., R. N. Hamburger, and S. E. Mills, Exp. Cell Res., 33, 495 (1964).23 Todaro, G., S. R. Wolman, and H. Green, J. Cell. Comp. Physiol., 62, 257 (1963).24 Saito, T., and S. Hakomori, submitted to J. Lipid Res.25 Sweeley, C. C., and B. Walker, Anal. Chem., 36, 1461 (1964).26 Warren, L., and M. C. Glick, J. Cell Biol., 37, 729 (1968).27 Robinson, E. A., H. M. Kalckar, H. Throedsson, and K. Sanford, J. Biol. Chem., 241, 2737

(1966).

Dow

nloa

ded

by g

uest

on

Nov

embe

r 28

, 202

0