Cell Surface Glycolipids - ACS Publications - American Chemical

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Fucolipids and Gangliosides of Human Colonic Cell Lines BADER SIDDIQUI and Y. S. KIM

Veterans Administration Medical Center and Department of Medicine, University of California, San Francisco, CA 94121 Carcinoma of the large bowel is a major hazard in most affluent countries. In the United States alone, 100,000 persons get colonic cancer each year and half of them die from it. Cancerous growth of tissues appears to be the result of cells not following the normal differentiation pathway towards formation and maintenance of normal functional organs. One approach to treatment of cancer is to redirect the cellular differentiation pathway toward normal growth with chemical agents. Several chemical agents have been used to modify the differentiation process in cultured tumor cell lines. A variety of chemical compounds, including cyclic AMP, sodium butyrate, dimethylformamide, dimethylsulfoxide, 5-bromodeoxyuridine, and tri-fluoro-methyl-2deoxyuridine can affect morphological and biochemical properties of cells. Some reports demonstrate that the tumorigenicity of cancer cells is markedly reduced or completely abolished by these agents. (See review by Prasad and Sinha, 1.) Butyrate treated Hela cells (2) and KB cells showed marked increases in the amounts of G gangliosides and elevated levels of the enzyme, CMP:sialic acid: lactosylceramide sialosyltransferase, required for its synthesis. Human colonic mucosa and colonic tumors are rich in glycolipids including gangliosides and several fucolipids. These lipids are important because they often determine blood group and other surface properties of cells. To understand better the effects of differentiating agents on tumor cells, we have been concentrating our efforts on the effect of agents like sodium butyrate or dimethylsulfoxide on colonic tumor cell lines. Previous studies in our laboratory have dealt with some of the effects of sodium butyrate on two colonic tumor cell lines, SW-480 and SW-620. This study describes the effect of sodium butyrate on glycolipids from four human colonic tumor cell lines, SKCO-1, HT-29, SW-480 and SW-620 and a human fetal intestinal line, FHS. M3

0-8412-0556-6/ 80/ 47-128-177$5.00/ 0 © 1980 American Chemical Society Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Materials and Methods C e l l Lines. The human fetal intestinal c e l l line (FHS) was kindly supplied to us by Dr. Walter A. Nelson-Rees at the Naval Bioscience Laboratory, Oakland, California. The human colonic c e l l line, SKCO-1 developed by Drs. G. Trempe and L.F. Olds, was obtained from Dr. Jorgen Fogh, Sloan Kettering Institute, Rye, New York. The HT-29 c e l l line was developed and obtained from Dr. J . Fogh. SW-480 and SW-620 were developed at Scott and White C l i n i c in Temple, Texas and were obtained from Col. A. Liebovitz. A l l of the c e l l lines are routinely maintained as monolayer in Dulbecco's modified Eagle's medium supplemented to 10% with fetal bovine serum, 100 units/ml of p e n i c i l l i n and 100 mg/ml of streptomycin. Labelling of Cells. For labelling experiments, Dulbecco's modified Eagle's medium was used, except i t contained only 1% glucose. Cells were seeded at 1 to 2 x 106 cells/75 cm2 flask i n medium at 37° and allowed to attach for 20-24 hours. The medium was then replaced with fresh medium containing sodium butyrate, 1.0 mM in case of SKC0-1 c e l l s or 2.5 mM for the other c e l l lines. Medium was changed every 3-4 days. After 8 days, medium containing 50uCi of Q H3-galactose (specific activity 9.1 Ci/m mole, (New England Nuclear Corporation, Boston, Massachusetts)) or £3H]fucose (specific activity 13.2 mCi/m mole, (NEN)) was added with or without butyrate. The c e l l s were further incubated for 20-24 hours. The c e l l s were harvested with 10 mM phosphate-buffered, 0.15 M saline, pH 7.4, containing 2 mM EDTA and washed three times with cold phosphate-buffered saline. Cells were collected by centrifugation. 3

Isolation of Labelled Glycolipids. Cells were sonicated in a small volume of saline and the total protein was determined on an aliquot by the method of Lowry et. a l . (_5) . Total l i p i d s were extracted with 20 volumes of chloroform; methanol (2:1) f i l t e r e d , and the residue re-extracted with 10 volumes of chloroform: methanol: water (1:2:0.15). Extracts were combined and concentrated at 40o under vacuum and dialyzed against d i s t i l l e d water for 2 days at 4°. The dialyzate was dried and applied on a 1 x 10 cms DEAE-Sephadex column (6). Labelled neutral glycolipids, along with other l i p i d s , were eluted with 50 ml chloroform: methanol: water (30:60:8) and the ganglioside fraction, also containing sulfoglycolipids, was eluted with chloroform: methanol: 0.8 M sodium acetate (30:60:8). In some experiments, total l i p i d s were separated into upper phase and lower phase. Each phase was applied separately to columns containing DEAE-Sephadex to isolate three classes of glycolipids: neutral glycolipids, sulfoglycolipids and gangliosides {!). Gangliosides and sulfoglycolipid fractions were dialyzed and lyophilized. Glycolipids were resolved by thin layer chromotography.

Sweeley; Cell Surface Glycolipids ACS Symposium Series; American Chemical Society: Washington, DC, 1980.

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Fucolipids

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Thin Layer Chromotography. Unless otherwise stated, a l l thin layer chromotography was on plates coated with s i l i c a Gel G (E. Merck, Dramstadt). A l l the solvents were mixed on a volume basis. Neutral glycolipid fractions were developed i n chloroform: methanol: water (60:35:6.5). Labelled fucolipid fractions were developed in chloroform: methanol: water (40:40:10). For separation of gangliosides, chloroform: methanol: 2.5N aqueous NH4OH (60:40:9) was used. Fluorography of TLC Plates. TLC plates were developed in the appropriate solvent system and dried at 50° for 10-15 minutes. The plates were impregnated with the s c i n t i l l a t i n g medium by dipping them into 20% 2,5,Diphenyloxazole (PPO) i n toluene, dried and exposed to X-ray film (Kodak, X-Qmat R XR ) for several days at -70°. Fluorgraphs were then developed as described (8). 2

Results Effect of Sodium Butyrate on Morphology and C e l l Growth. FHS, SKCO-1, HT-29 did not show any significant morphological changes with sodium butyrate. SW-480 and SW-620 c e l l s produce angular c e l l s rich i n cellular membranes. These processes were pronounced with SW-620 c e l l lines. Cells were seeded at 1 to 2 X 10 cells/75cm flask with sodium butyrate concentrations from 0.5 to 5.0 mM and without butyrate in growth medium. After 8 days, the c e l l s were harvested and protein was determined. Figure 1 shows total milligram protein/T-75cm flasks as plotted against sodium butyrate concentrations. The c e l l protein per flask of SKCO-1 decreased sharply with increased concentrations of butyrate when compared with control culture c e l l s . With SW-480 and SW-620 culture c e l l s , protein was decreased against butyrate concentrations, but the decrease was more pronounced with SW-620 c e l l s . C e l l protein of FHS and HT-29 cultures were unaffected (Fig. 1). 6

2

2

TLC of Ganglioside. Figure 2 TLC patterns of gangliosides obtained from fetal c e l l lines and three colonic cancer c e l l lines. The fetal c e l l lines (track 1) contained uncharacterized gangliosides, a through f; SW-480 (track 4) contained uncharacterized gangliosides, a through h; HT-29 gangliosides (track 3) have a simpler pattern; GJ43 i s the major ganglioside i n the SKCO-1 line (track 2). Labelled Fucolipids. Figure 3 shows fluorograms which were obtained from c e l l s labelled with G*lQ-fucose with and without butyrate treatment. Fucolipids were not found i n fetal c e l l s and, therefore, are not shown here. Figure 3A, track 2, shows fucolipid patterns of SW-480 cells without butyrate. Although


CELL SURFACE

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GLYCOLIPIDS

Figure 1. Total milligrams of protein/ T-75 cm flasks vs. millimolar concentrations of sodium butyrate in growth medium. Total cell protein was determined after cells were incubated in growth medium with sodium butyrate for eight daysat37°C. O, FHS; A, SKCO-1; HT-29; • , SW-480; | , SW-620.

Figure 2.

TLC chromatogram of gangliosides in chloroform:methanol:2.5N

NH, OH t

(60:40:9) 1, FHS; 2, SKCO-1; 3, HT-29; 4, SW-480 cell lines; 5, small intestine gangliosides used as standards; 6, 7 contain human brain standard gangliosides. Apparent discrepancy in mobilities among A and B is because they were obtained from different runs and conditions vary slightly. Gangliosides visualized by spraying with resorcinol, followed by heating at 130°C for 15-25 min. G , NeuAca2 -> 3Galfil -> 4GIB1 l'Cer G , NeuAca2 -> 8NeuAca2 -» 3Galf31 -> 4Glc/31 -» VCer G , GalNAcpl -» 4Gal(3 «- 2aNeuAc)(31 -* 4Glcf31 -» VCer G , Gaipi 3GalNAc/31 -» 4Gal(3 4Glc/31 -> VCer G , NeuAca2 -> 3Gal/31 -> 3GalNA pi -» 4Gal(3 4Glc(31 -> VCer G , Gal/31 3GalNAcf31 -> 4Gal(3 8NeuAca2 -» JGa/jSi -» 4GlcNacf31 -> JGa/y37 -> 4Gfcj8J -> i'Cer m

D3

M2

M1

Dla

C

Dih

Lc


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Figure 3.

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Thin-layer chromatographic autoradiograms of labeled fucolipids

A: 1, labeled standard glycolipids GL-2a through Le ; 2, labeled fucolipids from SW-480 control cells. B: 3, labeled standard glycolipids GL-lb through Gl-5a. Labeled fucolipids from cells grown in butyrate-free medium are: HT-29, track 4; SW-480, track 6; SW-620, track 8. Fucolipids from cells grown in butyrate are: HT-29, track 5; SW-480, track 7; SW-620, track 9. A was developed in chloroform:methanol:water (60:35:8); B was developed in chloroform.methanohwater (40:40:10). In B equal amounts of fucolipid activity from both control cultures and butyrate-treated cells were applied. A is the result of direct radioautography of glycolipids. TLC plate B was dipped in 20% PPO in toluene and dried prior to exposure to x-ray film for several days at —70°C. Arrows show reactions of faint bands. b

GL-lb, GL-2a, GL-3a, GL-4a, GL-5a,

Gal/31 -> VCer Galfll -» 4Gkpi -» VCer Galal 4Galf31 4Glc/31 -> VCer GalNAc/31 3Galal 4Galf31 -» 4Glc/31 -> VCer GalNAcal 3GalNA pi -> 3Galal -> 4Galpl -> 4Glcf31 -* C

VCer


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the pattern of SKCO-1 c e l l s are now shown here, there was no change i n the fucolipid patterns of these c e l l s with and without butyrate. Figure 3B shows fucolipid patterns of c e l l s with and without butyrate treatment. Tracks 4 and 5 show fucolipid patterns of HT-29 c e l l s with and without butyrate treatment. In the HT-29 c e l l line fucolipid FL-1 i s not present but there i s a decrease in FL-5 when c e l l s are grown i n butyrate. Track 6 and 8 show fucolipid patterns of SW-480 and SW-620 c e l l lines. There i s a difference i n fucolipid patterns between these two c e l l lines although they were both derived from the same patient. On treatment with sodium butyrate, FL-1 i s markedly decreased or disappears i n these two c e l l lines (Fig. 3B, Track 7 and 9) and reappearance of slow-migrating fucolipids (FL-7 through FL-9). Labelled Gangliosides. Figure 4 shows fluorograms of gangliosides labelled with £*H]\-galactose from c e l l s grown with or without butyrate. In the fetal c e l l line (FHS) there was no marked difference between treated and untreated c e l l s . There was a slight difference in the intensities between the two spots of Gj43 (Fig. 4A) . GM3 i s a major ganglioside i n SKCO-1 c e l l s and labelling appeared to be unaffected by butyrate treatment (Fig. 4B). In HT-29 c e l l lines, the amount of G appeared to remain the same; however, the distribution of GJ43 components was affected by butyrate. Minor changes in other gangliosides could be seen (Fig. 4C). Although the overall pattern of ganliosides of SW-480 c e l l s with and without butyrate i s similar, there are some changes i n G143, GM2 and G ^ regions which may be due to alterations in the l i p i d moieties(Fig. 4D). Similar results are also observed with SW-620 c e l l s , as shown in Figure 4E. M 3

M

Labelled Neutral Glycolipids. Neutral glycolipids were labelled with -galactose. As" was seen with the ganglioside, the butyrate affected the neutral glycolipid patterns but the most marked alterations appeared to be due to changes in the l i p i d moeities. Discussion In the present study, sodium butyrate had a differentiated effect on c e l l morphology. Sodium butyrate caused the SW-620 lines to become markedly angular with extension of many membraneous processes. These effects were also seen with the SW-480 c e l l lines but were less pronounced. No morphological changes were observed when SKCO-1, HT-29 and FHS c e l l lines were cultured in sodium butyrate. The concentration of sodium butyrate was observed to have a d i f f e r e n t i a l effect on c e l l growth i n colonic c e l l lines. After culturing for 8 days with 5 mM sodium butyrate, the c e l l protein per flask of the SCK0-1 line was decreased to less than 10% of the control cultures. In the SW-620 culture, c e l l protein per


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Figure 4.

Fucolipids

KIM

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Gangliosides

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Thin-layer chromatographic fluorograms of labeled gangliosides

The plates were developed in chloroform:methanol:2.5N NH OH (60:40:9). There are apparent discrepancies in the mobilities among the fluorograms because each plate was obtained from different runs and the conditions varied slightly. Standard [ H]-gangliosides G , G , and G were used in tracks 1, 4, 7, 10, and 12. A: labeled gangliosides obtained from FHS cell lines with (3) and without (2) butyrate treatment. B: gangliosides of SKCO-1 with (6) and without (5) butyrates. C: gangliosides of HT-29 with (9) and without (8) butyrate. D: gangliosides of SW-480 with (12) and without (11) butyrate. E: gangliosides of SW-620 cell lines with (15) and without (14) butyrate. Bands above G in C, D, and E are sulfoglycolipids. L

3

y3

M2

M1

M3


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was reduced to 20-25% of the control while the related line SW-480 showed a 50% reduction in c e l l protein. Cell protein of FHS and HT-29 cultures was unaffected. When c e l l s were cultured in labelled fucose or galactose i n the presence or absence of butyrate, alterations i n the labelled glycolipids were observed. Treatment of a l l of the c e l l lines with butyrate did not markedly affect the incorporation of L H J galactose in ganglioside per milligram of c e l l protein. In a l l of the lines except SW-480, butyrate caused a decrease in monoglycosylceramide compared to diglycosylceradmie; however, the changes were not as distinct as the changes in gangliosides. When SW-480 and SW-620 c e l l lines were grown i n the presence of butyrate, the fastest migrating fucolipid disappeared concomitant with the appearance of slow-migrating fucolipids. Although there were few qualitative changes i n the gangioside patterns of the SKCO-1 and FHS lines, there were marked alterations of gangliosides in the HT-29, SW-480 and SW-620 c e l l lines. The major changes were seen within the components compiling the Gj43 fraction. In HT-29, SW-480, and SW-620, there was a s h i f t i n GM3 to less polar components suggesting that the carbohydrates may be unchanged but the l i p i d moieties are altered. Alternatively, there may an acetylation of a hydroxyl group i n the carbohydrate moiety since i t has been shown that the butyrate increases the amount of acetylated histones i n Friend erythroleukemic c e l l s (9). The butyrate-induced s h i f t to less polar components i s also seen in the G fraction. The s h i f t i n GM2 and G143 components may be important in disturbing c e l l surface properties. SW-480 and SW-620 showed dramatic morphological alterations when cultured i n butyrate, and these c e l l s had obvious shifts to less polar components within the GM2 and GM3 fractions. In the SKCO-1 and FHS lines, these shifts were not observed and thus there were no morphological changes i n these two c e l l lines i n butyrate. However, since HT-29 c e l l s did not change morphology i n butyrate but also demonstrated the polarity shift in GM2 and GM3 components, the correlation between the two may be more complex, such as being dependent upon concentration or distribution of these components on the c e l l surface. We are currently exploring the effects of butyrate on ganglioside components of other c e l l lines to determine i f this glycolipid shift i s related to morphological alterations and to the malignant properties of c e l l s . 3

M 2

Summary In the present study, we examined the pattern of fucolipids and gangliosides i n cultured c e l l lines and alterations produced by a differentiating agent. A human f e t a l intestinal line (FHS), and four human colonic tumor lines (SKCO-1, HT-29, SW-480 and SW-620) were used. Cells were grown with or without sodium butyrate, (1.0 mM i n SKCO-1 and 2.5 mM in a l l other c e l l lines) i n


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growth medium. After 8 days medium containing 50yCi of C3HJgalactose or Q3Hj-f se was added with or without butyrate, followed by incubation for another 20-24 hours. Glycolipids were purified by column chromatography, characterized by thin-layer chromatography and were detected by radioautography or by conventional staining. Each tumor line revealed a distinct pattern of labelled fucolipids consisting of at least 10 components. No labelled fucolipids were detected in the FHS cell lines. The butyrate treated SKCO-1 cells did not show any change in fucolipid patterns. In HT-29 cell lines, there was a decrease of fucolipid FL-5 when the cells were grown in butyrate. There is a difference in fucolipid patterns between SW-480 and SW-620 cell lines. On treatment with sodium butyrate FL-1 (fast moving fucolipid) is markedly decreased or disappears, and there is appearance of slow migrating fucolipids (FL-7 through FL-9). Gangliosides were labelled with galactose. In the fetal cell lines (FHS) and SKCO-1 there was no marked difference between treated and untreated cells. In HT-29, SW-480, and Sw-620 cell lines, the amounts of GM3 appeared to remain the same, but the distribution of GM3 components was affected by butyrate. These changes, might be due to alterations in the lipid moieties or, alternatively, there might be an acetylation of a hydroxyl group in the carbohydrate moiety, since it has been shown that the butyrate increases the amount of acetylated histones. UCO

Acknowledgements This work was supported in part by the United States Public Health Service Grant CA-14905 from the National Cancer Institute through the National Large Bowel Cancer Project, and by the Veterans Administration Medical Research Service. We are indebted to Dr. J . S. Whitehead for his critical review and valuable discussions in the preparation of this manuscript. We also appreciate the technical assistance of Mr. James Bennett. Literature Cited 1. 2. 3. 4. 5.

Prasad, K.N., and Sinha, P.K. (1978) In "Cell Differentiation and Neoplasia" (G.F. Saunders, ed.), pp 111-141. Raven Press, New York. Fishman, P.H., Bradley, R.M., and Henneberry, R.C. (1976) Arch. Biochem. Biophys. 172, 618-626. Macher, B.A., Lockney, M., Moskal, J.R., Fung, Y.K., and Sweeley, C.C. (1978) Exptl. Cell Res. 117, 95-102. Kim, Y.S., Tsao, D., Siddiqui, B., Whitehead, J.S., Arnstein, P., Bennett, J., and Hicks, J . Cancer, In press. Lowry, O.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275.


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6. Ledeen, R.W., Yu, R.K., and Eng, L.F. (1973) J . Neurochem. 21, 829-939. 7. Siddiqui, B., Whitehead, J.S., and Kim, Y.S. (1978) J . Biol. Chem. 253, 2168-2175. 8. Mills, A.D., and Laskey, R.A. (1975) Eur. J . Biochem. 56, 335-341. 9. Reeves, R., and Cserjesi, P. (1979) J . Biol. Chem. 254, 4283-4290. RECEIVED December

10, 1979.


Cell Surface Glycolipids - ACS Publications - American Chemical

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