- Email: [email protected]
Isolation and characterization of [3H]fucose-labeled glycoproteins from the serum of normal rats and rats bearing Zajdela hepatoma
Cancer Letters 125 (1998) 227–231
Isolation and characterization of [3H]fucose-labeled glycoproteins from the serum of normal rats and rats bearing Zajdela hepatoma D. Ivanov, M. Antonova, J. Stoilov, S. Zacharieva, E. Gavazova, S. Ivanov* Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Received 15 May 1997; received in revised form 3 December 1997; accepted 3 December 1997
Abstract Specific [3H]fucose-labeled glycoproteins were found in the serum of rats bearing Zajdela hepatoma, which possess different isoelectric points in comparison with serum glycoproteins from normal rats. The electrophoretic profile of the serum glycoproteins is significantly altered. There is approximately a 2.5 times increase of [3H]fucose incorporation into serum glycoproteins from rats with an ascitic form of hepatoma, compared with normal rats and to animals bearing a solid form of the tumor (which have a considerably greater survival). Serum fucoproteins identical for the two forms of hepatoma were isolated, as well as glycoproteins strongly specific for each of the forms. 1998 Elsevier Science Ireland Ltd. Keywords: Serum; Glycoproteins; Hepatoma; Fucosylation; Preparative isoelectrofocusing
1. Introduction Alterations of cell surface glycosylation are closely related to tumorogenicity and the presence of newly formed tumor specific antigens, having differences in the structure of their oligosaccharide chains. It is considered that a significant part of tumor-specific antigens possess an increased incorporation of fucose and sialic acid residues into certain oligosaccharide sequences. The studies of Plotwin and Stanley [12] of the fucosylation of virus-transformed cells, as well as the earlier investigations of some authors [8, 14], conclude that the malignant phenotype of certain tumor cells is closely associated with the specific changes in the fucose-containing glycoproteins both on the cell surface and in the serum. * Corresponding author.
An increase of fucose-labeled sialoglycoproteins with high molecular weights is typical of many tumor cells [15,19,20]. It has been found that the degree of radioactively labeled fucose incorporation into the plasma membrane glycoproteins of chicken hepatoma Mc-29 is significantly higher for the high molecular glycoproteins, i.e. 70–200 kDa [1]. The much higher amount of fucose and mannose in the plasma membrane fractions of hepatoma Mc-29 verifies the assumption of the presence of glycoproteins with more branched carbohydrate chains in them [3]. There are many recent reports emphasizing the potential role and significance of these high molecular glycoproteins with branched oligosaccharide chains in the development of normal (differentiation) and pathological (neoplastic) processes [9–11]. The aim of this study was to examine the differences between the processes of serum glycoprotein
0304-3835/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0524-7
228
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231
fucosylation of normal rats and of rats bearing Zajdela hepatoma (ascites and solid forms) after the in vivo administration of [3H]fucose.
2. Materials and methods The ascites form of Zajdela hepatoma was obtained by injecting intraperitoneally a suspension of 8– 9 × 106 tumor cells into Wistar rats, weighing 150– 200 g. The ascites hepatoma (approx. 1.2 × 109 cells) was collected from the peritoneal cavity 7–8 days after inoculation. The solid form of the tumor was obtained by subcutaneous injection of 15 × 106 ascites cells and 15–20 days later the hepatoma had grown as a solid mass. 3 [H]Fucose (SA = 15.3 GBq/mmol, Amersham, UK), a radioactive precursor in the biosynthesis of glycoproteins, was injected in a dose of 100 mCi/100 g body weight. The animals were killed by decapitation 2 h later (those with the ascites form of hepatoma on the 8th day, and those with the solid form of the tumor on the 20th day after tumor inoculation). The
Fig. 1. Preparative isoelectrofocusing of [3H]fucose-labeled glycoproteins from normal rat-serum. Relative radioactivity: the activity of each fraction expressed as a percentage of the total radioactivity. Each data point represents the mean value from 4 experiments. Error bars, SEM.
Fig. 2. Preparative isoelectrofocusing of 3[H]fucose-labeled glycoproteins from the serum of rats bearing ascites form (W) and solid form (X) of Zajdela hepatoma. Relative radioactivity (see Fig. 1). Error bars, SEM.
blood was collected without anticoagulant and serum was obtained by centrifugation at 3000 × g for 15 min. The protein content was determined by the method of Bradford [2]. The procedure of Vesterberg [17] for preparative isoelectric focusing of serum glycoproteins was performed with 300 ml of serum of normal and hepatoma bearing rats (approximately 20 mg protein = 300 000 c.p.m.) on a 110 ml column (LKB 8101) using ampholytes at pH 3–10 at 500 V and 16 mA for 48 h. Fractions of 2 ml were collected at a pump speed of 120 ml/h and the radioactivity and pH of each of them were determined as described below. Electrophoretic analysis of serum proteins was performed according to the method of Laemnli [6] with 150 mg of protein. The gels were cut into 3-mm thick slices and the proteins were eluted with 1 ml of 1%sodium dodecylsulphate at 37°C overnight [16]. The radioactivity of the sample was measured in scintillation mixtures containing 4 g 2,5-Diphenyloxazol scintillation grade (Merck, Germany) (PPO) + 0.15 g 1,4Di/2-(5-phenyloxazolyl)/-benzene scintillation grade (Koch Light, UK) (POPOP) in 1 l toluene in a LKB 1110 scintillation spectrometer.
229
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231 Table 1 3
Isoelectric points of [ H]fucose-labeled glycoproteins after their separation by preparative isoelectrofocusing from the serum of normal rats and rats bearing Zajdela hepatoma pI-values Rats with hepatoma (n = 3)a Ascites form
Solid form
4.60 5.00 5.40 – – 6.25 – – 6.90 7.50 – 8.30 – –
4.60 5.00 5.40 5.90 – – 6.40 6.70 – – – 8.30 8.90 –
Normal rats (n = 3)a
4.60 5.00 – – 6.05 – – – 6.95 – 7.70 – – 9.30
a
The values in parenthesis are equal to the number of experimental animals.
3. Results and discussion The distribution of [3H]fucose-labeled serum glycoproteins according to their isoelectric points and relative radioactivity is presented in Figs. 1 and 2. The results show considerable differences between the profiles of serum glycoproteins from normal rats and rats bearing Zajdela hepatoma. Glycoproteins specific for the serum of rats bearing ascites hepatoma with pI 5.4, 6.25, 7.5 and 8.3 (Table 1), which are not presented in the profile of serum glycoproteins from normal animals were found. At the same time the serum fucoproteins from healthy rats (pI 6.05, 7.7 and 9.3) which are not detected in the sera of animals with tumor are also present. These results indicate the appearance in the serum of rats bearing hepatoma of specific glycoproteins, which differ in the higher extent of [3H]fucose incorporation, as well as in structural changes of the oligosaccharide chains, stipulating the differences in their isoelectric points. After preparative isoelectrofocusing, an elevation of the total [3H]fucose incorporation into the serum glycoproteins from normal rats and from animals with ascites and solid forms of Zajdela hepatoma was
established (Table 2). It is important to emphasize that the extent of radioactive precursor incorporation into the serum of asites hepatoma bearing rats is approximately 2.5-fold higher than that of normal serum. The experimental animals (n = 30) have a survival greater than 60 days after the subcutaneous application (rats with solid hepatoma), while the lethal phase for the rats bearing ascites tumor begins on the 8th and 9th days. The data obtained by preparative isoelectrofocusing of serum glycoproteins show the presence of specific glycoproteins which may be considered typical forms for both kinds of hepatoma (pI 5.4 and 8.3), as well as glycoproteins strongly specific for the solid form only (pI 5.9, 6.7 and 8.9). Furthermore, a correlation between the oncogenic capacity (the survival of animals bearing both types of tumor) and the degree of fucosylation was observed. The latter was about three times higher for the serum glycoproteins from rats with ascites of Zajdela hepatoma compared with that from normal rats. The extent of fucose incorporation into serum proteins from rats bearing the solid form of hepatoma was close to normal (Table 2). A correlation between the oncogenic and metastatic potential and the fucosylation of plasma membrane glycoproteins has been observed also [7]. These authors characterized the type of fucose linkage to the cell surface oligosaccharide structures from two cell lines of human colorectal tumor with differences in their metastatic capacity. The cell surface components released from the cell with a higher metastatic potential were more fucosylated than those from the other kind of tumor cells.
Table 2 Total incorporation of [3H]fucose into glycoproteins from the serum of normal rats and rats with Zajdela hepatoma Serum (300 ml)
[3H]Fucose incorporation (counts/min)a
Normal Acites form Solid form
13190 ± 1080 31517 ± 2892* 14100 ± 1150
a
Total incorporation is the sum of the radioactivity of all fractions after the isoelectric focusing of serum glycoproteins, obtained from three experimental animals for each group. *P , 0.05.
230
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231
It has also been established that the sialoglycoprotein ASPGI is specific for the cell surface of rat ascites adenocarcinoma breast cells [13]. This sialomucin is absent in solid tumors [5]. According to some authors ASPGI is closely related to the enhanced oncogenic potential of the ascites cells. On the basis of these suggestions our results can be considered a corroboration of the presence of glycoprotein with pI 7.50 specific for the ascites hepatoma cells and its absence in the serum of rats with solid tumor, as well the appearance of fucoproteins, typical only of the serum of rats bearing the solid form Zajdela hepatoma. Data of [3H]fucose incorporation into serum glycoproteins separated by slab sodium dodecylsulphate polyacrylamide gel electrophoresis are presented in Figs. 3 and 4. The extent of radioactive precursor incorporation into serum glycoproteins from rats with the ascitic form of hepatoma is greater than that of normal rats. This is evident by the 4 peaks in the zone of slice numbers 13–25 and also by the peak between slices 2 and 5. Considerable alterations of the number and localization of the peaks were observed, showing the presence of high fucose-labeled glycoproteins in the serum of rats with ascites hepatoma, with a molecular weight, different from that of the glycoproteins in the serum of normal animals. Earlier investigations [4,18] show a characteristic feature of Morris hepatoma, i.e. that there is not a significant difference between the protein profile of
Fig. 4. Incorporation of [3H]fucose into serum glycoproteins from rats bearing ascites of Zajdela hepatoma, measured by SDS-polyacrylamide gel electrophoresis. The data points present the mean from 4 individual experiments. Error bars, SEM.
malignant cells and that of normal ones, but there is a considerable difference in the number of plasma membrane glycoproteins, which are more numerous in the tumor cells than in the normal ones. Such a difference in plasma membrane glycoproteins from hepatoma Mc-29 and chicken liver was clearly demonstrated in the studies of the two precursor ([14C]glucosamine and [14C]fucose) incorporation into membrane components after polyacrylamide gel electrophoresis and gel filtration [1,3]. A higher extent of fucosylation of plasma membrane glycoproteins from hepatoma Mc-29 and especially those localized in the region of high-molecular glycoproteins was established. In the present work a similar distribution of high-labeled fucoproteins in the serum of rats with ascites of Zajdela hepatoma was also observed. The results obtained in this study can serve as an explanation for the typical alterations in the processes of fucosylation, accounting for the appearance of tumor specific fucoproteins in the serum of animals bearing Zajdela hepatoma.
References
Fig. 3. [3H]Fucose incorporation into normal rat serum glycoproteins, determined by SDS-polyacrylamide gel electrophoresis. The results are given as the mean from 4 separate experiments. Error bars, SEM.
[1] M. Antonova, S. Ivanov, E. Gavazova, H. ChelibonovaLorer, Comparative study on fucosylation of chicken liver and virus-induced hepatoma Mc-29, Int. J. Biochem. 16 (1984) 1313–1319. [2] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analyt. Biochem. 72 (1976) 248– 254.
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231 [3] H. Chelibonova-Lorer, M. Antonova, S. Ivanov, Glycopeptide profile of microsomal and plasma membrane glycoproteins from chicken liver and hepatoma Mc-29, Gen. Comp. Pathol. 24 (1988) 52–58. [4] A. Dubin, E. Paczoska-Eliasiewicz, Comparison of protein components of the plasma membrane fraction from rat liver and Morris hepatoma 5123D and 7777, Folia Histochem. Cytochem. 14 (1976) 113–122. [5] J.W. Huggins, T.P. Trenbeath, A.P. Sherblom, S.C. Howard, S.R. Hull, R.A. Laine, T. Kaizu, I. Rodriguez, K.L. Carraway, Glycoprotein differences in solid and ascites forms of the 1376 rat mammary adenocarcinoma, Cancer Res. 40 (1980) 1873–1878. [6] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [7] C.L. Lean, W.B. Mackinnon, E.J. Delikatny, R.H. Whitehead, C.E. Mountford, Cell-surface fucosylation and magnetic resonance spectroscopy characterization of human malignant colorectal cells, Biochemistry 31 (1992) 11095–11105. [8] R. Madiyalakan, S. Yazawa, K. Matta, Characterization of plasma alfa-(1,3)-l-fucosyltransferase from an ovarian cancer patient with the aid of synthetic substrates, Ind. J. Biochem. Biophys. 25 (1988) 32–35. [9] A. Nishikawa, S. Fujii, T. Sugiyama, N. Hayashi, N. Taniguchi, High expression of an N-acetylglucosaminyltransferase III in 3i-methyl DAB-induced hepatoma and ascites hepatoma, Biochem. Biophys. Res. Commun. 152 (1988) 107– 112. [10] A. Nishikarra, J. Gu, S. Bujii, N. Taniguchi, Determination of N-acetylglucosaminyltransferase III, IV and V in normal and hepatoma tissues of rats, Biochim. Biophys. Acta 1035 (1990) 313–331. [11] R. Pascale, S. Narashinhan, S. Rajalakshmi, Expression of N-
[12]
[13]
[14]
[15]
[16]
[17] [18]
[19]
[20]
231
acetylglucosaminyltransferase III in hepatic nodules, generated by different models of rat liver carcinogenesis, Carcinogenesis 10 (1989) 961–964. B. Plotwin, P. Stanley, Activation of two new alfa-(1,3)-fucosyltransferase activities in Chinese hamster ovary cells by 5azacytidine, Cell Regul. 2 (1991) 989–1000. A.P. Sherblom, R.L. Buck, K.L. Carraway, Purification of the major sialoglycoprotein of 13762 MAT-B1 and MAT-C1 rat ascites mammary adenocarcinoma cells by density gradient centrifugation in cesium chloride and guanidine hydrochloride, J. Biol. Chem. 265 (1980) 783–790. L.A. Smets, Cell transformation as a model for tumor induction and neoplastic growth, Biochim. Biophys. Acta 605 (1980) 93–111. W. Van Beek, P. Ernmelot, C. Homburg, Comparison of cellsurface glycoproteins of rat hepatomas and embryonic rat liver, Br. J. Cancer 36 (1977) 157–165. G.A. Van Nest, W.J. Grimes, A comparison of membrane components of normal and transformed BALB/c cells, Biochemistry 16 (1977) 2902–2908. O. Vesterberg, Isoelectric focusing of proteins, Methods Enzymol. 22 (1981) 389–412. P. Vischer, W. Reutter, Specific alterations of fucoprotein biosynthesis in the plasma membrane of Morris hepatoma 7777, Eur. J. Biochem. 84 (1978) 363–368. L. Warren, C.A. Buck, G.P. Tuszynski, Glycopeptide changes and malignant transformation: a possible role for carbohydrate in malignant behaviour, Biochim. Biophys. Acta 516 (1978) 97–127. C. Yogeeswaran, B. Stein, H. Sebastian, Altered cell surface organisation of gangliosides and sialoglycoproteins of mouse metastatic melanoma variant lines selected in vivo for enhanced lung implantation, Cancer Res. 38 (1978) 1336– 1344.
Isolation and characterization of [3H]fucose-labeled glycoproteins from the serum of normal rats and rats bearing Zajdela hepatoma D. Ivanov, M. Antonova, J. Stoilov, S. Zacharieva, E. Gavazova, S. Ivanov* Institute of Experimental Pathology and Parasitology, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Received 15 May 1997; received in revised form 3 December 1997; accepted 3 December 1997
Abstract Specific [3H]fucose-labeled glycoproteins were found in the serum of rats bearing Zajdela hepatoma, which possess different isoelectric points in comparison with serum glycoproteins from normal rats. The electrophoretic profile of the serum glycoproteins is significantly altered. There is approximately a 2.5 times increase of [3H]fucose incorporation into serum glycoproteins from rats with an ascitic form of hepatoma, compared with normal rats and to animals bearing a solid form of the tumor (which have a considerably greater survival). Serum fucoproteins identical for the two forms of hepatoma were isolated, as well as glycoproteins strongly specific for each of the forms. 1998 Elsevier Science Ireland Ltd. Keywords: Serum; Glycoproteins; Hepatoma; Fucosylation; Preparative isoelectrofocusing
1. Introduction Alterations of cell surface glycosylation are closely related to tumorogenicity and the presence of newly formed tumor specific antigens, having differences in the structure of their oligosaccharide chains. It is considered that a significant part of tumor-specific antigens possess an increased incorporation of fucose and sialic acid residues into certain oligosaccharide sequences. The studies of Plotwin and Stanley [12] of the fucosylation of virus-transformed cells, as well as the earlier investigations of some authors [8, 14], conclude that the malignant phenotype of certain tumor cells is closely associated with the specific changes in the fucose-containing glycoproteins both on the cell surface and in the serum. * Corresponding author.
An increase of fucose-labeled sialoglycoproteins with high molecular weights is typical of many tumor cells [15,19,20]. It has been found that the degree of radioactively labeled fucose incorporation into the plasma membrane glycoproteins of chicken hepatoma Mc-29 is significantly higher for the high molecular glycoproteins, i.e. 70–200 kDa [1]. The much higher amount of fucose and mannose in the plasma membrane fractions of hepatoma Mc-29 verifies the assumption of the presence of glycoproteins with more branched carbohydrate chains in them [3]. There are many recent reports emphasizing the potential role and significance of these high molecular glycoproteins with branched oligosaccharide chains in the development of normal (differentiation) and pathological (neoplastic) processes [9–11]. The aim of this study was to examine the differences between the processes of serum glycoprotein
0304-3835/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0524-7
228
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231
fucosylation of normal rats and of rats bearing Zajdela hepatoma (ascites and solid forms) after the in vivo administration of [3H]fucose.
2. Materials and methods The ascites form of Zajdela hepatoma was obtained by injecting intraperitoneally a suspension of 8– 9 × 106 tumor cells into Wistar rats, weighing 150– 200 g. The ascites hepatoma (approx. 1.2 × 109 cells) was collected from the peritoneal cavity 7–8 days after inoculation. The solid form of the tumor was obtained by subcutaneous injection of 15 × 106 ascites cells and 15–20 days later the hepatoma had grown as a solid mass. 3 [H]Fucose (SA = 15.3 GBq/mmol, Amersham, UK), a radioactive precursor in the biosynthesis of glycoproteins, was injected in a dose of 100 mCi/100 g body weight. The animals were killed by decapitation 2 h later (those with the ascites form of hepatoma on the 8th day, and those with the solid form of the tumor on the 20th day after tumor inoculation). The
Fig. 1. Preparative isoelectrofocusing of [3H]fucose-labeled glycoproteins from normal rat-serum. Relative radioactivity: the activity of each fraction expressed as a percentage of the total radioactivity. Each data point represents the mean value from 4 experiments. Error bars, SEM.
Fig. 2. Preparative isoelectrofocusing of 3[H]fucose-labeled glycoproteins from the serum of rats bearing ascites form (W) and solid form (X) of Zajdela hepatoma. Relative radioactivity (see Fig. 1). Error bars, SEM.
blood was collected without anticoagulant and serum was obtained by centrifugation at 3000 × g for 15 min. The protein content was determined by the method of Bradford [2]. The procedure of Vesterberg [17] for preparative isoelectric focusing of serum glycoproteins was performed with 300 ml of serum of normal and hepatoma bearing rats (approximately 20 mg protein = 300 000 c.p.m.) on a 110 ml column (LKB 8101) using ampholytes at pH 3–10 at 500 V and 16 mA for 48 h. Fractions of 2 ml were collected at a pump speed of 120 ml/h and the radioactivity and pH of each of them were determined as described below. Electrophoretic analysis of serum proteins was performed according to the method of Laemnli [6] with 150 mg of protein. The gels were cut into 3-mm thick slices and the proteins were eluted with 1 ml of 1%sodium dodecylsulphate at 37°C overnight [16]. The radioactivity of the sample was measured in scintillation mixtures containing 4 g 2,5-Diphenyloxazol scintillation grade (Merck, Germany) (PPO) + 0.15 g 1,4Di/2-(5-phenyloxazolyl)/-benzene scintillation grade (Koch Light, UK) (POPOP) in 1 l toluene in a LKB 1110 scintillation spectrometer.
229
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231 Table 1 3
Isoelectric points of [ H]fucose-labeled glycoproteins after their separation by preparative isoelectrofocusing from the serum of normal rats and rats bearing Zajdela hepatoma pI-values Rats with hepatoma (n = 3)a Ascites form
Solid form
4.60 5.00 5.40 – – 6.25 – – 6.90 7.50 – 8.30 – –
4.60 5.00 5.40 5.90 – – 6.40 6.70 – – – 8.30 8.90 –
Normal rats (n = 3)a
4.60 5.00 – – 6.05 – – – 6.95 – 7.70 – – 9.30
a
The values in parenthesis are equal to the number of experimental animals.
3. Results and discussion The distribution of [3H]fucose-labeled serum glycoproteins according to their isoelectric points and relative radioactivity is presented in Figs. 1 and 2. The results show considerable differences between the profiles of serum glycoproteins from normal rats and rats bearing Zajdela hepatoma. Glycoproteins specific for the serum of rats bearing ascites hepatoma with pI 5.4, 6.25, 7.5 and 8.3 (Table 1), which are not presented in the profile of serum glycoproteins from normal animals were found. At the same time the serum fucoproteins from healthy rats (pI 6.05, 7.7 and 9.3) which are not detected in the sera of animals with tumor are also present. These results indicate the appearance in the serum of rats bearing hepatoma of specific glycoproteins, which differ in the higher extent of [3H]fucose incorporation, as well as in structural changes of the oligosaccharide chains, stipulating the differences in their isoelectric points. After preparative isoelectrofocusing, an elevation of the total [3H]fucose incorporation into the serum glycoproteins from normal rats and from animals with ascites and solid forms of Zajdela hepatoma was
established (Table 2). It is important to emphasize that the extent of radioactive precursor incorporation into the serum of asites hepatoma bearing rats is approximately 2.5-fold higher than that of normal serum. The experimental animals (n = 30) have a survival greater than 60 days after the subcutaneous application (rats with solid hepatoma), while the lethal phase for the rats bearing ascites tumor begins on the 8th and 9th days. The data obtained by preparative isoelectrofocusing of serum glycoproteins show the presence of specific glycoproteins which may be considered typical forms for both kinds of hepatoma (pI 5.4 and 8.3), as well as glycoproteins strongly specific for the solid form only (pI 5.9, 6.7 and 8.9). Furthermore, a correlation between the oncogenic capacity (the survival of animals bearing both types of tumor) and the degree of fucosylation was observed. The latter was about three times higher for the serum glycoproteins from rats with ascites of Zajdela hepatoma compared with that from normal rats. The extent of fucose incorporation into serum proteins from rats bearing the solid form of hepatoma was close to normal (Table 2). A correlation between the oncogenic and metastatic potential and the fucosylation of plasma membrane glycoproteins has been observed also [7]. These authors characterized the type of fucose linkage to the cell surface oligosaccharide structures from two cell lines of human colorectal tumor with differences in their metastatic capacity. The cell surface components released from the cell with a higher metastatic potential were more fucosylated than those from the other kind of tumor cells.
Table 2 Total incorporation of [3H]fucose into glycoproteins from the serum of normal rats and rats with Zajdela hepatoma Serum (300 ml)
[3H]Fucose incorporation (counts/min)a
Normal Acites form Solid form
13190 ± 1080 31517 ± 2892* 14100 ± 1150
a
Total incorporation is the sum of the radioactivity of all fractions after the isoelectric focusing of serum glycoproteins, obtained from three experimental animals for each group. *P , 0.05.
230
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231
It has also been established that the sialoglycoprotein ASPGI is specific for the cell surface of rat ascites adenocarcinoma breast cells [13]. This sialomucin is absent in solid tumors [5]. According to some authors ASPGI is closely related to the enhanced oncogenic potential of the ascites cells. On the basis of these suggestions our results can be considered a corroboration of the presence of glycoprotein with pI 7.50 specific for the ascites hepatoma cells and its absence in the serum of rats with solid tumor, as well the appearance of fucoproteins, typical only of the serum of rats bearing the solid form Zajdela hepatoma. Data of [3H]fucose incorporation into serum glycoproteins separated by slab sodium dodecylsulphate polyacrylamide gel electrophoresis are presented in Figs. 3 and 4. The extent of radioactive precursor incorporation into serum glycoproteins from rats with the ascitic form of hepatoma is greater than that of normal rats. This is evident by the 4 peaks in the zone of slice numbers 13–25 and also by the peak between slices 2 and 5. Considerable alterations of the number and localization of the peaks were observed, showing the presence of high fucose-labeled glycoproteins in the serum of rats with ascites hepatoma, with a molecular weight, different from that of the glycoproteins in the serum of normal animals. Earlier investigations [4,18] show a characteristic feature of Morris hepatoma, i.e. that there is not a significant difference between the protein profile of
Fig. 4. Incorporation of [3H]fucose into serum glycoproteins from rats bearing ascites of Zajdela hepatoma, measured by SDS-polyacrylamide gel electrophoresis. The data points present the mean from 4 individual experiments. Error bars, SEM.
malignant cells and that of normal ones, but there is a considerable difference in the number of plasma membrane glycoproteins, which are more numerous in the tumor cells than in the normal ones. Such a difference in plasma membrane glycoproteins from hepatoma Mc-29 and chicken liver was clearly demonstrated in the studies of the two precursor ([14C]glucosamine and [14C]fucose) incorporation into membrane components after polyacrylamide gel electrophoresis and gel filtration [1,3]. A higher extent of fucosylation of plasma membrane glycoproteins from hepatoma Mc-29 and especially those localized in the region of high-molecular glycoproteins was established. In the present work a similar distribution of high-labeled fucoproteins in the serum of rats with ascites of Zajdela hepatoma was also observed. The results obtained in this study can serve as an explanation for the typical alterations in the processes of fucosylation, accounting for the appearance of tumor specific fucoproteins in the serum of animals bearing Zajdela hepatoma.
References
Fig. 3. [3H]Fucose incorporation into normal rat serum glycoproteins, determined by SDS-polyacrylamide gel electrophoresis. The results are given as the mean from 4 separate experiments. Error bars, SEM.
[1] M. Antonova, S. Ivanov, E. Gavazova, H. ChelibonovaLorer, Comparative study on fucosylation of chicken liver and virus-induced hepatoma Mc-29, Int. J. Biochem. 16 (1984) 1313–1319. [2] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Analyt. Biochem. 72 (1976) 248– 254.
D. Ivanov et al. / Cancer Letters 125 (1998) 227–231 [3] H. Chelibonova-Lorer, M. Antonova, S. Ivanov, Glycopeptide profile of microsomal and plasma membrane glycoproteins from chicken liver and hepatoma Mc-29, Gen. Comp. Pathol. 24 (1988) 52–58. [4] A. Dubin, E. Paczoska-Eliasiewicz, Comparison of protein components of the plasma membrane fraction from rat liver and Morris hepatoma 5123D and 7777, Folia Histochem. Cytochem. 14 (1976) 113–122. [5] J.W. Huggins, T.P. Trenbeath, A.P. Sherblom, S.C. Howard, S.R. Hull, R.A. Laine, T. Kaizu, I. Rodriguez, K.L. Carraway, Glycoprotein differences in solid and ascites forms of the 1376 rat mammary adenocarcinoma, Cancer Res. 40 (1980) 1873–1878. [6] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680–685. [7] C.L. Lean, W.B. Mackinnon, E.J. Delikatny, R.H. Whitehead, C.E. Mountford, Cell-surface fucosylation and magnetic resonance spectroscopy characterization of human malignant colorectal cells, Biochemistry 31 (1992) 11095–11105. [8] R. Madiyalakan, S. Yazawa, K. Matta, Characterization of plasma alfa-(1,3)-l-fucosyltransferase from an ovarian cancer patient with the aid of synthetic substrates, Ind. J. Biochem. Biophys. 25 (1988) 32–35. [9] A. Nishikawa, S. Fujii, T. Sugiyama, N. Hayashi, N. Taniguchi, High expression of an N-acetylglucosaminyltransferase III in 3i-methyl DAB-induced hepatoma and ascites hepatoma, Biochem. Biophys. Res. Commun. 152 (1988) 107– 112. [10] A. Nishikarra, J. Gu, S. Bujii, N. Taniguchi, Determination of N-acetylglucosaminyltransferase III, IV and V in normal and hepatoma tissues of rats, Biochim. Biophys. Acta 1035 (1990) 313–331. [11] R. Pascale, S. Narashinhan, S. Rajalakshmi, Expression of N-
[12]
[13]
[14]
[15]
[16]
[17] [18]
[19]
[20]
231
acetylglucosaminyltransferase III in hepatic nodules, generated by different models of rat liver carcinogenesis, Carcinogenesis 10 (1989) 961–964. B. Plotwin, P. Stanley, Activation of two new alfa-(1,3)-fucosyltransferase activities in Chinese hamster ovary cells by 5azacytidine, Cell Regul. 2 (1991) 989–1000. A.P. Sherblom, R.L. Buck, K.L. Carraway, Purification of the major sialoglycoprotein of 13762 MAT-B1 and MAT-C1 rat ascites mammary adenocarcinoma cells by density gradient centrifugation in cesium chloride and guanidine hydrochloride, J. Biol. Chem. 265 (1980) 783–790. L.A. Smets, Cell transformation as a model for tumor induction and neoplastic growth, Biochim. Biophys. Acta 605 (1980) 93–111. W. Van Beek, P. Ernmelot, C. Homburg, Comparison of cellsurface glycoproteins of rat hepatomas and embryonic rat liver, Br. J. Cancer 36 (1977) 157–165. G.A. Van Nest, W.J. Grimes, A comparison of membrane components of normal and transformed BALB/c cells, Biochemistry 16 (1977) 2902–2908. O. Vesterberg, Isoelectric focusing of proteins, Methods Enzymol. 22 (1981) 389–412. P. Vischer, W. Reutter, Specific alterations of fucoprotein biosynthesis in the plasma membrane of Morris hepatoma 7777, Eur. J. Biochem. 84 (1978) 363–368. L. Warren, C.A. Buck, G.P. Tuszynski, Glycopeptide changes and malignant transformation: a possible role for carbohydrate in malignant behaviour, Biochim. Biophys. Acta 516 (1978) 97–127. C. Yogeeswaran, B. Stein, H. Sebastian, Altered cell surface organisation of gangliosides and sialoglycoproteins of mouse metastatic melanoma variant lines selected in vivo for enhanced lung implantation, Cancer Res. 38 (1978) 1336– 1344.