Int. J . Cancer: 47, 742-745 (1991) Publication of the International Union Against Cancer Publication de I‘Union lnternationale Contre 1e Cancer 0 199 1 Wiley-Liss, Inc .

CHANGES IN THE GANGLIOSIDE COMPOSITION OF HUMAN NEUROBLASTOMA CELLS UNDER DIFFERENT GROWTH CONDITIONS Hiroshi SASAKI’, Toru MOMOI, Chutaro YAMANAKA, Tohru YORIFUJI, Masayuki KAJI and Haruki MIKAWA Department of Pediatrics, Kyoto University Faculty of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606, Japan.

The ganglioside composition of human neuroblastoma cells (LA-N-I and LA-N-5) was studied in samples obtained from (I) original cells in tissue cultures, (2) tumors grown in nude mice inoculated with original cells and (3) cells in tissue cul- tures re-established from the mouse tumors. The amounts of “a” pathway gangliosides (GW2, G,, and Gqi3 and those of the “b” pathway (Gq3, GDz, G,,, and GTib) differed according to the culture conditions. The “b” pathway gangliosides were markedly increased in the tumors grown in nude mice. In contrast, the “a” pathway gangliosides were abundant in cul- tures of both original and re-established cells. We also mea- sured the enzymatic activities of UDP-N-acetylgalac- tosamine: GM3, N-acetylgalactosaminyl transferase (EC 2.4. I .92) and of CMP-N-acetylneuraminic acid: G,, sialyl transferase (EC 2.4.99.8) in neuroblastoma cells cultured un- der these conditions. These enzymes are thought to be the key enzymes involved in the synthesis of the ‘‘a” and “b” pathway gangliosides. Though there was no significant differ- ence in the activity of N-acetylgalactosaminyl transferase be- tween original cells and tumors in nude mice, re-established cells showed a definitely higher activity (3.5 times higher than in the original cells). On the other hand, tumors grown in nude mice had a markedly higher activity of sialyl transferase than that of original cells or re-established cells. These find- ings suggest that the culture conditions and/or the type of cell growth play some role in the synthesis and expression of gan- gliosides in neuroblastoma cells.

Gangliosides, sialic acid-containing glycosphingolipids, are mainly found in cell membranes. They have been identified as receptors for hormones, toxins and various growth factors (Ha- komori, 1981; Alessandri et ul . , 1987; Spiegel and Fishman, 1987). A number of ganglioside fractions have been detected,

ganglioside synthesis in vertebrates (Ando and Yu, 1979). It has been suggested that accumulations of specific gangliosides may be markers for certain tumors (Hakomori and Kannagi, 1983). Furthermore, gangliosides have been recognized as functionally important molecules related to oncogenic transfor- mation, cell differentiation, and density-dependent cell growth in some human tumor cells (Hakomori and Kannagi, 1983; Schengrund and Repman, 1982). Some ganglioside species

GDla, G,,, and G,,,) play an important regulatory role in the morphologic and metabolic differentiation in cultures of neuroblastoma and pheochromocytoma (Matta et al . , 1986).

Human neuroblastoma is a tumor of the sympathetic nervous system which has a high ganglioside content. The ganglioside composition may reflect the state of neuroblastoma cell differ- entiation (Wu et al . , 1986; Schengrund et al . , 1985). Neuro- blastoma cells can be induced to undergo differentiation in vitro by various agents and/or under certain culture conditions (Sidell, 1982; Bottenstein, 1980; Rama and Prasad, 1986). However, it is not known whether the ganglioside expression of neuroblastoma cells varies with the culture conditions. Such information would be useful in evaluating the function of gan- gliosides in the differentiation of neuroblastoma cells.

In this work we employed nude mice for xenogeneic trans- plantation of cultured neuroblastoma cells and re-established the cell line from cells thus grown. We compared the gangli- oside composition in 3 groups: original cultured cells, neuro- blastoma tumors grown in nude mice inoculated with original cells and cultured cells re-established from the tumors of nude

and it is known that there are 2 pathways, “a” and “b”, of

mice. We also investigated the effect of these varied culture conditions of neuroblastoma cells on the activities of UDP- N-acetylgalactosamine: G,,, N-acetylgalactosaminyl trans- ferase (EC 2.4.1.92) (GalNAc transferase) and of CMP- N-acetylneuraminic acid: GM3, sialyl transferase (EC 2.4.99.8) (sialyl transferase) which are thought to be the key enzymes involved in ganglioside synthesis.

MATERIAL AND METHODS

Cell cultures The LA-N-1 and LA-N-5 human neuroblastoma cell lines

were kindly provided by Dr. R.C. Seeger (Department of Pe- diatrics, School of Medicine, UCLA, Los Angeles, CA) (See- ger et a l . , 1977; Sidell et d., 1984). These cells were main- tained in RPMI- 1640 medium (Nissui, Tokyo) supplemented with 10% heat-inactivated fetal calf serum, 50 IU penicillin/ ml, 50 p,g streptomycin/ml, and 1 p,g Fungizone/ml (complete medium). Cells were grown in a humidified incubator at 37°C in a 5% CO, atmosphere. The cells were fed with medium twice weekly and subcultured with 0.025% trypsin in a Versene buffer consisting of 0.13 M NaCl, 5.4 m~ EDTA-4 Na, 7 mM NaHCO,, and 1 .O% glucose. The cells were assayed when 80 to 90% confluence was attained. Inoculation of neuroblastoma cell lines into nude mice

Four-week-old female BALB/c athymic nude mice were purchased from Nihon SLC, Shizuoka, Japan; 1.5 X lo7 cells were suspended in 0.3 ml of medium and inoculated s .c . in the dorsum. The mice were monitored for tumor development ev- ery 3 or 4 days. On day 56, tumors were removed and either processed immediately or stored at - 70°C until further anal- yses. Some portions of excised tumors were mechanically dis- persed and re-established in culture under the conditions de- tailed above. Ganglioside analysis

Gangliosides were isolated and purified from 2-3 X lo7 cultured cells or 0.1-0.3 g of tumors grown in nude mice (Ladisch and Gillard, 1985). Briefly, samples were homoge- nized and extracted twice with 10 vol of chloroformimethanol (1/1, v/v). After centrifugation, the extracts were combined and dried under a stream of nitrogen. The dried total lipid extract was then partitioned twice in 10 vol of diisopropyl ether/l-butanol/50 mM aqueous NaCl (6:4:5, v/v/v). The gan- glioside-containing aqueous phase was lyophilized, dissolved in distilled water and then purified by Sephadex G-50 gel fil- tration. Gangliosides were recovered in the void volume, ly- ophilized, and redissolved in chloroformimethanol (1/1, v/v).

The distribution of individual gangliosides was analyzed with HPTLC plates (Silica gel 60; E. Merk, Darmstadt, Ger- many) together with reference mixtures of gangliosides of

‘To whom correspondence and reprint requests should be sent, at the Department of Pediatrics, Yamato-Takada City Hospital, 1 , Isono, Ya- mato-Takada, Nara, 635, Japan.

Received: August 27, 1990 and in revised form November 19, 1990.

GANGLIOSIDES OF HUMAN NEUROBLASTOMA 743

known composition according to the method of Ladisch and Gillard (1985). The plates were developed in chloroform, methanol and 0.2% aqueous calcium chloride (in a ratio of 60/40/9 by volume). Gangliosides were visualized with resor- cinol-HC1 agent. Relative proportions (in percentage) of the ganglioside fractions on HPTLC were measured by a Shimadzu CS-930 densitometer (Shimadzu Seisakusho, Kyoto). The quantity of sialic acid in the gangliosides was determined by a modified resorcinol method (Jourdian et al., 1971).

Assay of GaNAc transferase activity The assay for GalNAc transferase in human neuroblastoma

cells was performed as described by Keenan et al. (1974) and modified as follows. Twenty million cells, or 0.2-0.3 g of tumor tissue, were homogenized in 0.32 M sucrose containing 14 m~ 2-mercaptoethanol and were used as the enzyme source. The standard reaction mixture contained 50 nM of GM3 as the acceptor lipid, 0.3 mg of Triton X-100, 15 p~ of cacodylate- HCl (PH 7.3), 2.5 pmol of MnCl,, 3 X lo5 dpm of UDP-[ l-14C]N-acetylgalactosamine (55.0 m C i / m , New En- gland Nuclear, Boston, MA), and 0.4 mg of enzyme protein, in a final volume of 0.1 ml. The mixtures were incubated at 37°C for 3 hr, and reactions were terminated by boiling for 1 min in a water bath at 100°C.

A SEP-PAK C18 cartridge (Waters Associates, Milford, MA) was pre-washed with 20 ml methanol and equilibrated with 10 ml of 0.1 M KCl. Reaction mixtures were suspended in 1 ml of 0.1 M KCl and transferred to the cartridges. The assay tube was washed twice with 2 ml of 0.1 M KCl, and the washings were also applied to the cartridge. Then the cartridge was washed with 25 ml of distilled water to remove non-lipid compounds. The radioactive lipid product was eluted with 7.5 ml of methanol, collected in a scintillation vial and dried under a stream of nitrogen. The radioactivity incorporated into the lipid fraction was counted in a liquid scintillation counter. The reaction product was identified by HPTLC autoradiography with authentic gangliosides in the same solvent system.

Assay of sialyl transferase activity Sialyl transferase activity was measured by the method of

Yu et al. (1988): 2 X lo7 cells, or 0.2-0.3 g of tumor tissue, were homogenized in distilled water. The incubation mixture contained total homogenate (300 pg of protein), 10 pl of 0.1 M MnCI,, 10 p1 of 0.25 m~ cacodylate buffer (PH 6.3), 125 pg of Triton CF-54, 40 nM of GM3, and 3 X lo5 dpm of CMP-N-A~ety1[4-~~C]neuraminic acid (1.8 mCi/mM; NEN, Boston, MA). The tubes were incubated for 30 min at 37”C, and the reaction was stopped by boiling for 30 sec. Then 0.5 ml of octyl-Sepharose 4B (Pharmacia, Uppsala, Sweden) was added to each reaction tube. This mixture was vortexed and allowed to stand for 1 hr at room temperature. It was added to the octyl-Sepharose column (bed volume = 0.3 ml) and rinsed 3 times with 2 ml of distilled water. The radiolabelled gly- colipids were then eluted with 6 ml of chlorofodmethanol

(111, v/v) and dried under a stream of nitrogen. Purified GD, was added as the carrier for HPTLC and, after spotting on an HPTLC plate, the plate was developed with chloroform/ methanol (85/15, v/v) to remove interfering neutral lipids and phospholipids, and then with chloroform/methanol/aqueous 0.02% CaCl, 2H20 (55/45/10, v/v/v) to resolve the ganglio- sides. Products of enzyme reactions were scraped off the plate corresponding to the position of reference ganglioside and the radioactivity was counted in a liquid scintillation counter.

These experiments were performed in duplicate, and control assays were performed without an acceptor lipid. Proteins were determined by the method of Bradford (1976) using BSA as the standard.

RESULTS

Cell growth In both the human neuroblastoma cell lines, LA-N-1 and

LA-N-5, there were no differences in growth pattern or mor- phology between the original cell lines and the re-established cell lines after growth in nude mice. When neuroblastoma cells were inoculated into nude mice, tumors were first recognized about 3 weeks after inoculation of both cell lines. On day 56, 13 tumors were obtained from 16 inoculations of LA-N-1 cells, and 8 from 10 inoculations of LA-N-5 cells. The mean weights of these tumors were 2.63 g and 1.68 g, respectively.

Ganglioside composition The distribution of the individual gangliosides of the original

cultured cells (LA-N- 1 -OR, LA-N-5-OR), neuroblastoma tu- mors grown in nude mice (LA-N- 1 -NU, LA-N-5-NU) and cul- tured cells re-established from the tumors in nude mice (LA- N-1-RE, LA-N-5-RE) are shown in Table I. The 2 original neuroblastoma cell lines had different ganglioside composi- tions. The LA-N-1-OR cells had a high proportion of “a” pathway gangliosides ( ( 3 ~ 2 , GMl and GDl,), while the LA- N-5-OR cells had a high level of “b” pathway gangliosides (GD3, G,,, GDlb and GTlb). When inoculated into nude mice, both cell lines induced the formation of neuroblastoma tumors, and a specific trend observed in the ganglioside composition was the predominance of “b” pathway gangliosides. The quantity of these gangliosides increased from 38% to 68.4% in LA-N-1 cells and from 67.9% to 87.2% in LA-N-5 cells. On the other hand, the content of GMl decreased markedly in nude mouse tumors. However, the ganglioside composition of LA- N-1-RE cells and LA-N-5-RE cells was very similar to that of LA-N-1-OR and LA-N-5-OR cells, respectively. This was un- changed when we analyzed the ganglioside composition of re-established cells after 8-10 passages. With regard to the total amount of gangliosides, cultured cells re-established from the mouse tumors had a significantly higher level than did original cells or nude mice tumors from both human neuro- blastoma cell lines (Table I). However, after 8-10 passages, the ganglioside content of re-established cells decreased to the

TABLE I - CHANGES IN THE GANGLIOSIDE COMPOSITION OF HUMAN NEUROBLASTOMA CELLS UNDER 3 DIFFERENT SETS OF CULTURE CONDITIONS

Ganglioside composition (Yo) Total gangliosides

(nmol LBSN GM3 mg protein)

Samples (n) “a” pathway gangliosides “b” pathway gangliosides

GM2 GMI GDla GD3 GDl b + GD2 GTlb

LA-N-1-OR (5) ND 0.8 -+ 1.0 44.9 f 7.8 16.3 f 3.4 1.4 f 1.0 36.6 t 7.8 ND 7.04 f 0.92 LA-N-1-NU (5) ND ND 14.5 t 9.2 17.1 f 9.7 0.3 f 0.6 4 6 . 0 k 9.8 22.1 * 5.3 5.44 f 1.14 LA-N-1-RE (4) ND ND 44.8 f 8.1 23.2 2 3.8 2.5 k 1.7 29.5 f 3.2 ND 15.51 t 0.57 LA-N-5-OR (4) 0.6 t 0.4 3.8 t 3.2 21.6 k 5.9 6.1 f 4.8 0.2 f 0.3 48.4 f 6.3 19.3 f 5.8 8.91 k 1.09 LA-N-5-NU (5) ND 0.6 f 0.4 7.8 f 4.2 4.2 f 3.2 ND 54.7 f 7.6 32.5 f 4.6 6.33 t 0.61 LA-N-5-RE (7) 0.5 * 0.4 1.2 f 0.7 22.9 f 4.0 12.2 f 4.7 1.0 f 2.1 43.8 f 3.7 18.0 f 3.9 13.48 f 0.84 All results are shown as the mean t SD. LBSA, lipid-bound sialic acid. OR, original cells in tissue culture; NU, tumors grown in nude mice; RE, cells in tissue culture

re-established from the murine t u m ~ r ~ . ND, not detected.

744 SASAKI ET AL.

level of original cells (6.51 ? 0.50 n M LBSNmg protein in LA-N-1-RE cells and 9.49 ? 0.49 ILM LBSNmg protein in LA-N-5-RE cells).

GaWrAc tran$erase activity and sialyl trangerase activity We studied the changes in the activity of these enzymes in

LA-N- 1 human neuroblastoma cells cultured in 3 different ways as described above. A significantly higher level of N- acetylgalactosaminyl transferase activity was found in LA- N-I-RE cells (61.8 ? 4.2 pmol/hr/mg protein) than in LA- N-1-OR cells (18.4 ? 2.1 pmoYhr/mg protein). However, af- ter 8-10 passages, this high activity decreased to 27.5 ? 1.0 pmol/hr/mg protein. Individual LA-N- 1 -NU tumors showed various levels of activity of this enzyme (22.7 k 11.3 pmol/ hr/mg protein) (Fig. 1).

The highest sialyl transferase activity was found in LA- N-1-NU tumors (10.27 ? 3.65 nmol/hr/mg protein). This was significantly higher than that in LA-N- 1-OR cells (1.52 k 0.63 nmol/hr/mg protein) or LA-N-1-RE cells (3.10 ? 0.67 nmol/hr/mg protein). The enzymatic activity in LA-N-1-RE cells decreased to 1.39 2 0.74 nmol/hr/mg pro- tein after 8-10 passages (Fig. 2).

DISCUSSION

Our studies show that the ganglioside composition of neu- roblastoma cells varies when cells are cultured under different conditions. This is characterized by changes in the “a” and “b” pathway ganglioside content. The amounts of these 2 groups of gangliosides vary under different culture conditions. It is of particular interest that the quantity of “b” pathway gangliosides in neuroblastoma tumors grown in nude mice was much higher than in the original cells cultured in vitro. The ganglioside composition reverted to that of the original cells in cell lines re-established from these tumors. Disialoganglioside GD2, one of the major gangliosides of the “b” pathway, may be related to the degree of differentiation and the metastatic growth of neuroblastoma (Wu et al., 1986; Schulz et al., 1984; Cheresh et al., 1986). Unfortunately, we could not separate 2 gangliosides (G,,, and GDJ clearly on HPTLC plates. How- ever, the changes in the amounts of G,,, and GDlb + GD2 suggest that these 3 gangliosides are related to the growth and

60

C a, +J .-

40 M E

O 20

\ S 1

!i

0 1 T

L

OR NU RE E- P

FIGURE 1 - Changes in the activity of UDP-N-Acetylgalac- tosamine: G,, , N-Acetylgalactosaminyl transferase in LA-N-1 human neuroblastoma cells under different growth conditions. OR, original cells in tissue culture; NU, tumors grown in nude mice; RE, cells in tissue culture re-established from the mouse tumors; RE-P, re- established cells after 8-10 passages. Data are mean values * SD (bars) (n = 3).

15

(I

+J ‘5 10

2 a

E

- 5

M

\ r \

E C

0

T

OR NU RE RE-P

FIGURE 2 - Changes in the activity of CMP-N-acetylneuraminic acid: GM3, sialyl transferase of LA-N- 1 human neuroblastoma cells under different growth conditions. OR, original cells in tissue culture; NU, tumors grown in nude mice; RE, cells in tissue culture re- established from the mouse tumors; RE-P, re-established cells after 8-10 passages. Data are mean values ? SD (bars) (n = 4).

tumor formation of neuroblastoma under in vivo growth con- ditions.

It is not certain whether the changes in ganglioside compo- sition are due to nutritional changes and other variations in the culture environment, or to the differences in cell growth due to culture in vitro or in nude mice (Tsuchida et al., 1987). There must be some differences in the process of cell attachment between cells growing as monolayers in tissue culture flasks and cells growing as “solid tumors” in nude mice. Ganglio- sides in cell membranes play an important role in cell-to-cell interactions (Hakomori, 1981; Alessandri et al., 1987; Skipski et al., 1980). Cheresh et al. (1986) reported that the disialo- gangliosides GD, and GD, are involved in the attachment of human neuroblastoma and melanoma cells to extracellular ma- trix proteins.

The inverse correlations between ‘ ‘a’ ’ pathway gangliosides and “b” pathway gangliosides in our study led us to measure the activities of GalNAc transferase and sialyl transferase in neuroblastoma cells. The former is considered to be the starting enzyme of the “a” pathway and the latter, that of the “b” pathway. We expected a lower activity of GalNAc transferase or a higher activity of sialyl transferase in LA-N-1-NU, which had more “b” pathway gangliosides, than in LA-N-1-OR or LA-N-1-RE, which had more “a” pathway gangliosides. However, there was no significant difference in the enzymatic activities of GalNAc transferase between LA-N- 1 -OR and LA- N-1-NU, but, to our surprise, LA-N-1-RE had a significantly higher specific activity than LA-N-1-OR (3.5-fold), even though the ganglioside composition of these cells was similar. Sialyl transferase activity was highest in LA-N-1-NU tumors (6.8 times higher than in LA-N-1-OR cells), as we had ex- pected. A possible explanation for these observations might be that some enzymatic activation occurred when the cells were cultured in nude mice where they formed solid tumors. A decrease in the activity of these enzymes in LA-N-1-RE cells after several passages might support this hypothesis. Fucogan- glioside accumulates in malignant hepatomas in vivo but dis- appears when the tumor cells are grown as cell lines in tissue culture (Holms and Hakomori, 1983). This is due to the in- duction of alpha-fucosyl transferase in malignant cells in vivo and the absence of alpha-galactosyl transferase in the cultured cells. Alterations in glycosyltransferase activity in the synthe-

GANGLIOSIDES OF HUMAN NEUROBLASTOMA 745

the activity of specific enzymes in the synthesis of ganglio- sides. We believe that these results should be taken into ac- count when studying the changes in ganglioside composition accompanying the differentiation of human neuroblastoma cells in vivo or in vitro.

sis of gangliosides occur during embryonic development in vivo (Yu et al., 1988) or during treatment with various inducers of differentiation in some transformed cells in vitro (Moskal et al., 1987). Our results indicate that changes in culture condi- tions and/or differences in the type of cell growth could affect

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