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Glycosaminoglycans and electrokinetic behavior of rat ascites hepatoma cells
Experimental
GLYCOSAMINOGLYCANS
Cell Research 67 (1971) 142-146
AND
RAT ASCITES K. KOJIMA Laboratory of Cell Biology, Department of Chemistry, Faculty
Aichi
ELECTROKINETIC HEPATOMA
BEHAVIOR
OF
CELLS
and T. YAMAGATA Cancer
of Science,
Center Research Nagoya University,
Institute, Nagoya
and 464, Japan
SUMMARY Cellular electrophoretic mobility of AH-130, an island-forming strain of rat ascites hepatomas, was reduced by chondroitinase-ABC treatment of cells but not affected by neuraminase. Assay of released sugars demonstrated the presence of chondroitin sulfates at the cell surface of AH-130, indicating that acidic residues of chondroitin sulfates were one of the factors responsible for negative surface charge of these cells and sialic acid was not. Surface-located chondroitin sulfates in AH-130 cells were abundant in chondroitin sulfate A. The mobility of freecell-type subline cells was also lowered by the chondroitinase as well as by the neuraminidase, indicating the presence of chondroitin sulfates on the cell surface. The mobility of rat erythrocytes, however, was not affected by the chondroitinase.
Negative surface charge in many cell types is largely dependent on a carbohydrate-rich protein layer on the cell surface. A major contributor to the negative surface potential of mammalian cells is sialic acid (N-acetylneuraminic acid), but charged side chains of membrane proteins, membrane RNA and presumably membrane lipids also contribute to the net cell-surface charge so far reported [l]. Enzymic removal of sialic acid from the cell surface results in reduction in electrophoretic mobility of many cell types [2]. In some cell types, however, it has been reported that no reduction in the mobility was found by the enzymic treatment of cells in spite of removal of sialic acid from their surface [3, 4, 51. Recently, we have suggestedthe possibility that sialic acid is deeply located and charged groups other than sialic acid major factors responsible for negative surface charge in Exptl
Cell Res 67
the cells of island-forming strains in rat ascites hepatomas [5]. In the present study, this possibility on the cell surface was examined by electrophoresis of cells and chemical analysis of released sugars from the surface following the enzymic treatments of cells for chondroitin sulfates.
MATERIALS
AND METHODS
Cell materials Ascites hepatoma AH-130 and its subline cells [6], maintained in rats of Moriyama inbred strain, and rat erythrocytes were used in the present study. In AH-130. tumor cells make cell association to form ‘hepatoma islands’ and individually isolated tumor cells are also present. The sublines of AH-130 consist of almost entirely single isolated tumor cells.
Enzymic treatments of cells The ourified bacterial chondroitinase-ABC and -AC [7] were used in a concentration of 1 unit/ml of the Veronal-buffered saline (0.05 M sodium acetate, 0.05 M sodium barbiturate, HCl and 0.85 % NaCI, pH
Surface charge of cells due to chondroitin sulfates 8.0), supplemented 1 mg of egg albumin/ml of the buffer. Neuraminidase from vibrio cholerae was obtained from General Biochemicals, Inc. Chagrin Falls, Ohio. The neuraminidase solution used in this study was prepared by addition of 20 units of the enzyme to 1 ml of Krebs-Ringer phosphate (pH 7.0). Excess quantities of the enzymes used herein were free of proteolytic enzyme activity. Tumor ascites were harvested from the abdominal cavity of rats, and erythrocytes from the heart. Cells were washed twice with a large amount of the appropriate buffer solutions. About 5 x 10’ cells were suspended in 5 ml of each enzyme solution and incubated at 37°C for 30 min. The enzyme-treated cells were washed 3 times with the electrophoretic medium used. Their electrophoretic mobility was then compared with that of each control similarly treated inthe appropriate buffer without the enzyme. No difference in viability between the enzyme- and sham-treated cells was revealed by the trypan blue exclusion test.
Electrophoresis of cells The electrophoretic medium used was a l/15 M phosphate buffer supplemented 5.4 % glucose (pH 7.3, ionic strength 0.167). Measurement of electrophoretic mobility of cells was made at 25+0.5”C by a cytopherometer according to the method described previously 151.
Assay for chondroitin sulfates Assay of chondroitin sulfates released from the cell surface by the enzymic treatment was performed in AH-130 cells. The tumor ascites was harvested from 5-dav-old tumor-bearing animal which received intraperitoneal injection of
143
distilled water, and the saccharides were then reprecipitated with the ethanol. After washing 5 times by repeat of this procedure, the test sample was exhaustively digested with the enzymes. Products were examined by paper chromatogram.
Assay for sialic acid The thiobarbituric acid method [9] was used for determination of sialic acid. For sialic acid of bound form, the assay was also performed on the samples after acid hydrolysis.
RESULTS Changes in electrophoretic mobility of cells after enzymic treatments were summarized in table 1. The mobility of AH-130 cells was reduced by chondroitinase-ABC treatment, while no significant change in the mobility was detectable in neuraminidase treatment as compared with that of each control. In the free-cell-type subline cells, AH-130FN and FG cells, their mobility was remarkably reduced by the chondroitinase treatment as well as by the neuraminidase. The reduction rate of the mobility in the chondroitinasetreated cells was similar to that in the neuraminidase. On the contrary, erythrocytes were not detected by any changes in the mobility by the chondroitinase treatment. Assay of sialic acid revealed that no sialic acid was released from the cell surface in any forms, free or bound, by the chondroitinase treatment. When sulfate-labeled AH-130 cells were treated with chondroitinase-ABC or -AC, disaccharides released from the cell surface were examined on the supernatant by paper chromatography. Radioactivity was shown at the area corresponding to unlabeled ADi4S, as internal marker, by radioautography of the resulting chromatogram equally well in either enzymic system (fig. 1). Significant radioactivity was also detectable at the area corresponding to ADi-6S by liquid scintillation spectrometry. Radioactive ratio of Exptl
Cell Res 67
144 K. Kojima & T. Yamagata Table 1. Effects of chondroitinase-ABC
Cell type
Treatment
AH-1 30
CHase
40 40 30 30
cant { exp 1 cant exp
40 40 30 30
NAase
1 cant exp cant exp
40 40 30 30
- 1.387kO.112 - 1.116+0.070 + 0.057 - 1.263 1.240+0.047 - 1.323 i-O.096 - 0.806 & 0.079 - 1.160+-0.066 -0.741 kO.051 - 1.238iO.103 - 0.808 k 0.046 - 1.174+-0.068 - 0.772 & 0.049
CHase
cant exp
40 40
- 1.097 io.033 - 1.097 * 0.044
CHase i
NAase CHase
AH-l 30FG Erythrocyte
Mobility of cells meankS.D. (pm/set/V/cm)
cant exp ( cant exp
NAase AI’ 1-13urn ’ qApX’
No. of cells measured
and neuraminidase on electrophoretic mobility
cant-exp
Difference ( %I
0.271
19.5
co.01
0.023
1.8
> 0.05
0.517
39.1
< 0.01
0.419
36.1
< 0.01
0.430
34.7
co.01
0.402
34.2
< 0.01
of cells
P
0
Abbreviations: CHase, chondroitinase-ABC; NAase, neuraminidase; cant, control; exp, experimental. Chondroitinase-ABC was used in a concentration of 1 unit/ml of Veronal-buffered saline supplemented 0.1 % albumin (pH 8.0) and neuraminidase 20 units/ml of Krebs-Ringer phosphate (pH 7.0), respectively. Cells were incubated at 37°C for 30 min in each enzymic solution.
products was about 73 of ADi-4S to 1 of ADi-6s. The chondroitinase-treated cells and cellfree ascites were also analysed for labeled chondroitin sulfates. No significant radioactivity was detectable in disaccharides from the chondroitinase-treated cells and cellfree ascites by radioautography and liquid scintillation spectrometry.
DISCUSSION The presence of sialic acid at the surface of most animal cells so far examined has been demonstrated and shown to be responsible for electrophoretic mobility of many cell types. Our recent results, however, have demonstrated that electrophoretic mobility of the cells in island-forming hepatoma lines was independent of sialic acid in spite of its presence on the surface, and suggestedthat charged groups other than sialic acid were ExptI
CeN Res 67
factors responsiblefor negative surface charge in them [5]. The present results have shown that the cellular electrophoretic mobility of AH-l 30, an island-forming line, is lowered by the chondroitinase-ABC treatment of cells but not affected by the neuraminidase. Chemical analysis has also shown that products from chondroitin sulfates are released from the cell surface by the chondroitinase treatments. These results demonstrate the presence of chondroitin sulfates at the cell surface of AH-130, and indicate that acidic residues of chondroitin sulfates, sulfate and/or carboxyl groups, are at least one of the factors responsible for negative surface charge of these cells but sialic acid is not. It has been established previously that ADi4S is produced from dermatan sulfate and chondroitin sulfate A, and ADi-6S from chondroitin sulfate C in the chondroitinase-ABC system, while ADi-4S is produced only from chondroitin sulfate A in the
Surface
charge of cells due to chondroitin sulfates 145
Fig. 1. Paperchromatographic separationof disaccharides released from thecellsurfaceof YGlabeledAH-130. UV-photography(lefr) and radioautography(right, 10 daysexposure)are shown.Radioactivity is found at the area corresponding to aDi-4S by eitherenzymicdigestionalike.a and b indicatethe areacorresponding to ADi-4S and aDi-6S, respectively.CHase-ABC:chondroitinase-ABCdigest.CHase-AC:chondroitinaseAC digest.
chondroitinase-AC system [7]. Since radioactive ratio of ADi-4S and ADi-6S is about 73 :1, and ADi-4S is similarly detectable in the chondroitinase-AC system, it implies that sulfated polysaccharides of the surface of AH-130 cells is abundant in chondroitin sulfate A. The electrophoretic mobility of free-celltype subline cells is also reduced by chondroitinase treatment. Its reduction rate by the treatment is similar to that by the neuraminidase. Since the chondroitinase used is free of proteolytic enzyme activity and no sialic acid is released by the chondroitinase, this result may also indicate the presence of lo-
711811
chondroitin sulfates on the surface of these cell types. On the contrary, erythrocyte membrane may be lacking in the substancessusceptible to chondroitinase-ABC. Recently, we have suggested the presence of sulfated mucopolysaccharides on the cell surface of other tumors [lo] and cultured cell lines [l 11, indicating that pattern of the macromolecules synthesized was dependent on cell lines. As shown in the present results, islandforming and free-cell-type lines in the ascites hepatomas differ from each other with respect to their electrokinetic behavior after enzymic treatment. This finding implies that differences in the surface structure exist Exptl
Cell Res 67
146 K. Kojima & T. Yamagata between two cell types and are related to the surface property of each cell type. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education in Japan.
REFERENCES 1. Wallach, D F H, New Engl j med 280 (1969) 761. 2. Doljanski, F & Eisenberg, S, Cell electrophoresis (ed E J Ambrose) p. 78. Churchill, London (1965). 3. Cook, G M W, Seaman, G V F & Weiss, L, Cancer res 23 (1963) 1813. 4. Wallach, D F H & Esandi, M V P, Biochim biophys acta 83 (1964) 363.
Exptl
Cell Res 67
5. Kojima, K & Maekawa, A, Cancer res 30 (1970) 2858. 6. Hirono, I, Kojima, K, Kachi, H, Ohashi, A & Sasaoka, I, Gann 55 (1964) 363. 7. Yamagata, T, Saito, H, Habuchi, 0 & Suzuki, S, J biol them 243 (1968) 1523. 8. Saito, H, Yamagata, T & Suzuki, S, J biol them 243 (1968) 1536. 9. Aminoff, D, Biochem j 81 (1961) 384. 10. Kojima, K, Takeichi, N, Kobayashi, H & Maekawa, A, Nagoya med j 16 (1970) 7. 11. Suzuki, S, Kojima, K & Utsumi, K R, Biochim biophys acta 222 (1970) 240. Received December 7, 1970 Revised version received March 15, 1971
GLYCOSAMINOGLYCANS
Cell Research 67 (1971) 142-146
AND
RAT ASCITES K. KOJIMA Laboratory of Cell Biology, Department of Chemistry, Faculty
Aichi
ELECTROKINETIC HEPATOMA
BEHAVIOR
OF
CELLS
and T. YAMAGATA Cancer
of Science,
Center Research Nagoya University,
Institute, Nagoya
and 464, Japan
SUMMARY Cellular electrophoretic mobility of AH-130, an island-forming strain of rat ascites hepatomas, was reduced by chondroitinase-ABC treatment of cells but not affected by neuraminase. Assay of released sugars demonstrated the presence of chondroitin sulfates at the cell surface of AH-130, indicating that acidic residues of chondroitin sulfates were one of the factors responsible for negative surface charge of these cells and sialic acid was not. Surface-located chondroitin sulfates in AH-130 cells were abundant in chondroitin sulfate A. The mobility of freecell-type subline cells was also lowered by the chondroitinase as well as by the neuraminidase, indicating the presence of chondroitin sulfates on the cell surface. The mobility of rat erythrocytes, however, was not affected by the chondroitinase.
Negative surface charge in many cell types is largely dependent on a carbohydrate-rich protein layer on the cell surface. A major contributor to the negative surface potential of mammalian cells is sialic acid (N-acetylneuraminic acid), but charged side chains of membrane proteins, membrane RNA and presumably membrane lipids also contribute to the net cell-surface charge so far reported [l]. Enzymic removal of sialic acid from the cell surface results in reduction in electrophoretic mobility of many cell types [2]. In some cell types, however, it has been reported that no reduction in the mobility was found by the enzymic treatment of cells in spite of removal of sialic acid from their surface [3, 4, 51. Recently, we have suggestedthe possibility that sialic acid is deeply located and charged groups other than sialic acid major factors responsible for negative surface charge in Exptl
Cell Res 67
the cells of island-forming strains in rat ascites hepatomas [5]. In the present study, this possibility on the cell surface was examined by electrophoresis of cells and chemical analysis of released sugars from the surface following the enzymic treatments of cells for chondroitin sulfates.
MATERIALS
AND METHODS
Cell materials Ascites hepatoma AH-130 and its subline cells [6], maintained in rats of Moriyama inbred strain, and rat erythrocytes were used in the present study. In AH-130. tumor cells make cell association to form ‘hepatoma islands’ and individually isolated tumor cells are also present. The sublines of AH-130 consist of almost entirely single isolated tumor cells.
Enzymic treatments of cells The ourified bacterial chondroitinase-ABC and -AC [7] were used in a concentration of 1 unit/ml of the Veronal-buffered saline (0.05 M sodium acetate, 0.05 M sodium barbiturate, HCl and 0.85 % NaCI, pH
Surface charge of cells due to chondroitin sulfates 8.0), supplemented 1 mg of egg albumin/ml of the buffer. Neuraminidase from vibrio cholerae was obtained from General Biochemicals, Inc. Chagrin Falls, Ohio. The neuraminidase solution used in this study was prepared by addition of 20 units of the enzyme to 1 ml of Krebs-Ringer phosphate (pH 7.0). Excess quantities of the enzymes used herein were free of proteolytic enzyme activity. Tumor ascites were harvested from the abdominal cavity of rats, and erythrocytes from the heart. Cells were washed twice with a large amount of the appropriate buffer solutions. About 5 x 10’ cells were suspended in 5 ml of each enzyme solution and incubated at 37°C for 30 min. The enzyme-treated cells were washed 3 times with the electrophoretic medium used. Their electrophoretic mobility was then compared with that of each control similarly treated inthe appropriate buffer without the enzyme. No difference in viability between the enzyme- and sham-treated cells was revealed by the trypan blue exclusion test.
Electrophoresis of cells The electrophoretic medium used was a l/15 M phosphate buffer supplemented 5.4 % glucose (pH 7.3, ionic strength 0.167). Measurement of electrophoretic mobility of cells was made at 25+0.5”C by a cytopherometer according to the method described previously 151.
Assay for chondroitin sulfates Assay of chondroitin sulfates released from the cell surface by the enzymic treatment was performed in AH-130 cells. The tumor ascites was harvested from 5-dav-old tumor-bearing animal which received intraperitoneal injection of
143
distilled water, and the saccharides were then reprecipitated with the ethanol. After washing 5 times by repeat of this procedure, the test sample was exhaustively digested with the enzymes. Products were examined by paper chromatogram.
Assay for sialic acid The thiobarbituric acid method [9] was used for determination of sialic acid. For sialic acid of bound form, the assay was also performed on the samples after acid hydrolysis.
RESULTS Changes in electrophoretic mobility of cells after enzymic treatments were summarized in table 1. The mobility of AH-130 cells was reduced by chondroitinase-ABC treatment, while no significant change in the mobility was detectable in neuraminidase treatment as compared with that of each control. In the free-cell-type subline cells, AH-130FN and FG cells, their mobility was remarkably reduced by the chondroitinase treatment as well as by the neuraminidase. The reduction rate of the mobility in the chondroitinasetreated cells was similar to that in the neuraminidase. On the contrary, erythrocytes were not detected by any changes in the mobility by the chondroitinase treatment. Assay of sialic acid revealed that no sialic acid was released from the cell surface in any forms, free or bound, by the chondroitinase treatment. When sulfate-labeled AH-130 cells were treated with chondroitinase-ABC or -AC, disaccharides released from the cell surface were examined on the supernatant by paper chromatography. Radioactivity was shown at the area corresponding to unlabeled ADi4S, as internal marker, by radioautography of the resulting chromatogram equally well in either enzymic system (fig. 1). Significant radioactivity was also detectable at the area corresponding to ADi-6S by liquid scintillation spectrometry. Radioactive ratio of Exptl
Cell Res 67
144 K. Kojima & T. Yamagata Table 1. Effects of chondroitinase-ABC
Cell type
Treatment
AH-1 30
CHase
40 40 30 30
cant { exp 1 cant exp
40 40 30 30
NAase
1 cant exp cant exp
40 40 30 30
- 1.387kO.112 - 1.116+0.070 + 0.057 - 1.263 1.240+0.047 - 1.323 i-O.096 - 0.806 & 0.079 - 1.160+-0.066 -0.741 kO.051 - 1.238iO.103 - 0.808 k 0.046 - 1.174+-0.068 - 0.772 & 0.049
CHase
cant exp
40 40
- 1.097 io.033 - 1.097 * 0.044
CHase i
NAase CHase
AH-l 30FG Erythrocyte
Mobility of cells meankS.D. (pm/set/V/cm)
cant exp ( cant exp
NAase AI’ 1-13urn ’ qApX’
No. of cells measured
and neuraminidase on electrophoretic mobility
cant-exp
Difference ( %I
0.271
19.5
co.01
0.023
1.8
> 0.05
0.517
39.1
< 0.01
0.419
36.1
< 0.01
0.430
34.7
co.01
0.402
34.2
< 0.01
of cells
P
0
Abbreviations: CHase, chondroitinase-ABC; NAase, neuraminidase; cant, control; exp, experimental. Chondroitinase-ABC was used in a concentration of 1 unit/ml of Veronal-buffered saline supplemented 0.1 % albumin (pH 8.0) and neuraminidase 20 units/ml of Krebs-Ringer phosphate (pH 7.0), respectively. Cells were incubated at 37°C for 30 min in each enzymic solution.
products was about 73 of ADi-4S to 1 of ADi-6s. The chondroitinase-treated cells and cellfree ascites were also analysed for labeled chondroitin sulfates. No significant radioactivity was detectable in disaccharides from the chondroitinase-treated cells and cellfree ascites by radioautography and liquid scintillation spectrometry.
DISCUSSION The presence of sialic acid at the surface of most animal cells so far examined has been demonstrated and shown to be responsible for electrophoretic mobility of many cell types. Our recent results, however, have demonstrated that electrophoretic mobility of the cells in island-forming hepatoma lines was independent of sialic acid in spite of its presence on the surface, and suggestedthat charged groups other than sialic acid were ExptI
CeN Res 67
factors responsiblefor negative surface charge in them [5]. The present results have shown that the cellular electrophoretic mobility of AH-l 30, an island-forming line, is lowered by the chondroitinase-ABC treatment of cells but not affected by the neuraminidase. Chemical analysis has also shown that products from chondroitin sulfates are released from the cell surface by the chondroitinase treatments. These results demonstrate the presence of chondroitin sulfates at the cell surface of AH-130, and indicate that acidic residues of chondroitin sulfates, sulfate and/or carboxyl groups, are at least one of the factors responsible for negative surface charge of these cells but sialic acid is not. It has been established previously that ADi4S is produced from dermatan sulfate and chondroitin sulfate A, and ADi-6S from chondroitin sulfate C in the chondroitinase-ABC system, while ADi-4S is produced only from chondroitin sulfate A in the
Surface
charge of cells due to chondroitin sulfates 145
Fig. 1. Paperchromatographic separationof disaccharides released from thecellsurfaceof YGlabeledAH-130. UV-photography(lefr) and radioautography(right, 10 daysexposure)are shown.Radioactivity is found at the area corresponding to aDi-4S by eitherenzymicdigestionalike.a and b indicatethe areacorresponding to ADi-4S and aDi-6S, respectively.CHase-ABC:chondroitinase-ABCdigest.CHase-AC:chondroitinaseAC digest.
chondroitinase-AC system [7]. Since radioactive ratio of ADi-4S and ADi-6S is about 73 :1, and ADi-4S is similarly detectable in the chondroitinase-AC system, it implies that sulfated polysaccharides of the surface of AH-130 cells is abundant in chondroitin sulfate A. The electrophoretic mobility of free-celltype subline cells is also reduced by chondroitinase treatment. Its reduction rate by the treatment is similar to that by the neuraminidase. Since the chondroitinase used is free of proteolytic enzyme activity and no sialic acid is released by the chondroitinase, this result may also indicate the presence of lo-
711811
chondroitin sulfates on the surface of these cell types. On the contrary, erythrocyte membrane may be lacking in the substancessusceptible to chondroitinase-ABC. Recently, we have suggested the presence of sulfated mucopolysaccharides on the cell surface of other tumors [lo] and cultured cell lines [l 11, indicating that pattern of the macromolecules synthesized was dependent on cell lines. As shown in the present results, islandforming and free-cell-type lines in the ascites hepatomas differ from each other with respect to their electrokinetic behavior after enzymic treatment. This finding implies that differences in the surface structure exist Exptl
Cell Res 67
146 K. Kojima & T. Yamagata between two cell types and are related to the surface property of each cell type. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education in Japan.
REFERENCES 1. Wallach, D F H, New Engl j med 280 (1969) 761. 2. Doljanski, F & Eisenberg, S, Cell electrophoresis (ed E J Ambrose) p. 78. Churchill, London (1965). 3. Cook, G M W, Seaman, G V F & Weiss, L, Cancer res 23 (1963) 1813. 4. Wallach, D F H & Esandi, M V P, Biochim biophys acta 83 (1964) 363.
Exptl
Cell Res 67
5. Kojima, K & Maekawa, A, Cancer res 30 (1970) 2858. 6. Hirono, I, Kojima, K, Kachi, H, Ohashi, A & Sasaoka, I, Gann 55 (1964) 363. 7. Yamagata, T, Saito, H, Habuchi, 0 & Suzuki, S, J biol them 243 (1968) 1523. 8. Saito, H, Yamagata, T & Suzuki, S, J biol them 243 (1968) 1536. 9. Aminoff, D, Biochem j 81 (1961) 384. 10. Kojima, K, Takeichi, N, Kobayashi, H & Maekawa, A, Nagoya med j 16 (1970) 7. 11. Suzuki, S, Kojima, K & Utsumi, K R, Biochim biophys acta 222 (1970) 240. Received December 7, 1970 Revised version received March 15, 1971