Gangliosides of Active and Inactive Neuroblastoma Clones J. CIESIELSKI-TRESKA, J. ROBERT, G. REBEL’, and P. MANDEL Centre de Neurochimie du CNRS 1 1, rue Humann, F-67085 Strasbourg-Cedex, France

Received April 1976lRevised December I976

It is possible to divide neuroblastoma cells into clones able to synthesize neurotransmitters (active clones) or not (inactive clones). The analysis of gangliosides of active and inactive clones shows that their total lipid sialic acids is markedly lower than that of neuron-enriched fractions prepared from brain. The ganglioside pattern of the cultured cells also differs notably from those obtained with neuronal fractions from brain. The absence of tri- and tetrasialogangliosides and the presence of appreciable amounts of the simplest monosialogangliosides are particularly noticeable in the neuroblastoma. Morphological differentiation obtained by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine does not restore a true neuronal pattern. Gangliosides could not therefore be used as a marker of neuronal differentiation in this type of cell. No correlations can be found between the ganglioside pattern and the ability of cells to synthesize neurotransmitters.

Introduction

Gangliosides are a class of lipid present in a higher concentration in the nervous tissue compared to tissue of non-nervous origin. The low concentration of gangliosides found in the myelin 111 and in cultured astroblasts [2l suggests that these lipids are chiefly localized in neuronal structure and as such could be considered as a neuronal marker. The high level of gangliosides found in synaptosomal plasma membranes [3-81 further supports this hypothesis. Another characteristic is the presence in nervous tissues of appreciable levels of tri- and tetrasialogangliosides, compounds which are absent from practically all the non-nervous organs analyzed [91. The biological functions of gangliosides in the nervous system is not known. Related to their localization in synaptosomal plasma membrane, it has been suggested that these lipids could be associated with the membrane receptors of some neurotransmitters [4. 5, 10, 111. Studies of neuronal cells have been impeded by the fact that the classical methods for cell fractionation on sucrose gradient give cross-contaminated fractions [12-141. Many workers have looked for a better model Abbreviations and Nomenclature. Gangliosides are named according to Svennerholm [651 or Wiegandt [661. Dibutyryl cyclic AMP = DBcAMP; bromodeoxyuridine = BRDU To whom reprints must be requested

Differentiation 8, 31-37 (1977) - 0 by Springer-Verlag 1977

of neuronal cells and have used neuroblastoma, a tumoural cell line originating from the mouse neuronal crest. These cells, in a medium supplemented with serum, grow with a round morphology (proliferating cells). When serum is withdrawn [151 or when agents such as bromodeoxyuridine I161 or dibutyryl cyclic AMP [17, 181 are added, cell growth stops and cells undergo morphological and biochemical differentiation. Neuroblastoma clones can be divided into two groups according to their ability to produce neurotransmitters: those which cannot synthesize catecholamine and/or acetylcholine represent the “inactive clones”, and the others which make these neurotransmitters are the “active clones”. In these active clones, a high increase in the activity of enzymes involved in neurotransmitter metabolism is observed after differentiation [ 191. An increase of cellular respiration [201 and of excitabdity [21-231 are also found after differentiation. Although disputed by some workers [24,251, gangliosides are generally considered to be mainly localized in cell plasma membranes, particularly in the case of nervous tissues [5--8, 26-301. These molecules could therefore be good markers for following the changes of these membranes during the differentiation of neuroblastoma cells. Few studies have been devoted to the analy-

32

J. Ciesielski-Treska et al.:

sis of gangliosides in neuroblastoma. Dawson et al. [3 11, then Yogeeswaran et al. [321 have studied the glycolipid pattern of some clones in proliferation. The distribution of the gangliosides in these clones was very different from those reported for nerve cells. Working with the inactive Neuro 2 A clone, Yogeeswaran et al. [321 found that no qualitative or quantitative changes occur in the gangliosides when the cells were grown in the same medium either in spinner culture (cells in proliferation) or in static culture flasks (cells in differentiation). In contrast, using serum deprivation to differentiate the Neuro 2 A clone, we have observed an increase of the monosialoganglioside level in the neuron-like cells [331. We now complete this observation and report the ganglioside composition of some inactive and active clones and the changes observed when they are differentiated by serum deprivation, dibutyryl cyclic AMP or bromodeoxyuridine.

Methods Five clones of neuroblastoma cells were used: NIE 115, NIE 18 and NS 20 were kindly given by Dr. Nirenberg; Neuro 2 A, isolated by Klebe and Ruddle [341, was provided by Jackson Laboratories; M1 was cloned in our laboratory. The main characteristics of the different clones used are summarized in Table 1. The cells were grown in Falcon flasks on Eagle-Dulbecco’s medium supplemented with 10% foetal calf serum, in an atmosphere of 5% C0,-95% air. Differentiation was obtained either by withdrawing the serum from the medium or by adding to the medium the following inducers: DBcAMP: 49 mg/100 ml or BRDU: 0.3 mg/100 ml. Cells were used between the 30th and the 65th replication after clonage. During this time no appreciable changes were detected in the ganglioside level and distrbution of the studied clones.

Gangliosides Determination The cells were recovered by scraping with a rubber policeman, washed with physiological saline then lyophilyzed. After the extraction of lyophilyzed cells with phosphate buffer pH 7.0, the lipids were obtained according to the technique of Suzuki [351. The lipid extract was washed with 0.2 vol 0.8% aq KCl. The resulting lower phase

was washed again twice with the theoretical Folch’s upper phase [361. The pooled upper phases were concentrated under vacuum then dialyzed against water. Gangliosides were purified by mild alkaline hydrolysis (modified from Schneider and Kennedy [371). Lipid sialic acid was measured according to Miettinen and Luukkainen [381. Ganglioside pattern was obtained by thin layer chromatography according to Van Den Eijnden [391. After detection with orcinol-HC1 [401 the quantitation was carried out by densitometry [411. Dawson et al. [31l and Yogeeswaran et al. [32] reported that the neuroblastoma gangliosides were structurally similar to brain gangliosides. We have therefore only characterized the spots detected after chromatography by comparison of their migration in different solvent systems with known samples from pig brain. The markers of G,, and GM3were obtained by Dr. L. Sarlieve from autopsy pieces of Tay-Sachs and from bovine spleen.

Results

Some neuroblastoma clones contained high levels of the G,, ganglioside. It is known that this ganglioside partitions between the upper and the lower phase during the aqueous washings of the chloroform-methanol extract 1421. Analysis of the lower-phase glycolipids, obtained from the different clones, showed that when 50 mg (dry weight) cells are extracted, two washings are needed to bring more than 95% of the total G,, to the upper phase. With 100 mg or more, three washings are necessary. With additional washings we extract a yellowish non-dialyzable, alkalistable compound which migrates on thin layer plates in the tetrasialoganglioside region. Kanfer and Spielvogel [431 have reported a loss of gangliosides during the dialysis of low concentrations of gangliosides. Comparison of extracts with high or low ganglioside concentration shows that under our conditions the dialysis steps remove not more than 8- 10% of the total lipid sialic acid. In accord with Kanfer and Spielvogel no changes were observed in the ganglioside pattern. However, this purification step must be maintained in order to remove the free sialic acid and some glycosidic material extracted from the culture medium which interferes in the ganglioside analysis.

Table 1. Adrenergic and cholinergic activities of the studied clones Clones

Neuro 2 A

NS 20

NIE 18

NIE 115

Acetylcholine transferasen Catecholamine synthesisa Lactic0 dehydrogenase isoenzyme (Tholey et al. [671)

Negative Negative Not tested

Negative

Negative

Negative

a

Ebel, A., Ciesielski-Treska, J., Mandel, P.: Unpublished results

* Not tested

* Muscle type

+++

Muscle type

M1

++

+++

Muscle + 2 brain types

33

Gangliosides of Neuroblastoma Cells

Analyzing the NB 41 A and Neuro 2 A clones, Dawson and Yogeeswaran [31, 321 found, in agreement with our results, a predominance of monosialogangliosides with G,, or G,, as the principal compound and an absence of tri- and tetrasialogangliosides. However, the pattern that was found for the Neuro 2 A differs in the relative amount of each ganglioside to that reported for the same clone by Yogeeswaran et al. 1323. This could be the result of an evolution of the clone during the numerous replications following its derivation from the original tumour. A similar discrepancy was also observed in the ganglioside patterns obtained for the clone NB 41 A by Dawson et al. or Yogeeswaran et al. [31, 321.

Gangliosides of Proliferating Cells The total lipid sialic acid of all analyzed clones (Table 2) is rather low compared to the value obtained for the neuronal cells prepared by density-gradient centrifugation. The ganglioside level cannot be correlated with the ability of the clone to synthesize neurotransmitters: the value found for NIE 115 (a highly active clone) lies in the same range as the Neuro 2 A (inactive clone) value and is notably lower than the NS 20 (slightly active) one. Comparisons of NS 20 and MI (highly active) confirms this view. Each clone has a different ganglioside pattern (Table 2). Differences in the glycolipid pattern between sub-clones derived from the same tissue or same cell has already been observed by other workers [32, 44, 451. All the clones show a large amount of monosialogangliosides. In contrast to normal nervous tissue, a high level of G,, is also observed in these cells. A second major ganglioside in all clones is the GDla.Most important is perhaps the total absence of tri- and tetrasialogangliosides. Comparison of NS 20, NIE 115 and neuronal fractions obtained by gradient centrifugation shows that there is also no parallelism between the activity of the clone and the ganglioside pattern. M1 is the clone which most ressembles the neuronal pattern; it is the only clone containing some GDlb.

Gangliosides of Serum Deprived Neuroblastoma Cells Differentiation of neuroblastoma cells by withdrawing the serum from the culture medium results in an increase of total lipid sialic acid. When the gangliosides changes are considered, three types of clones are found. In the Neuro 2 A clone the differentiation is accompanied by an increase in the monosialogangliosides (Table 2). In the NS 20 or M1 (Table 3) no changes are observed. Finally the NIE 18 and NIE 115 show a notable increase in the disialogangliosides. Yogeeswaran et al.

Table 2. Gangliosides of proliferating and serum deprived clones Clone

NIE 18

NIE 115

NS 20

Neuro 2 A [31

MI

Prol.

Diff.

Prol.

Diff.

Prol.

Diff.

Prol.

Diff.

Prol.

Diff.

Total lipid sialic acids pg/g dry weight

301 520

396 f 2 2

282 f 1 8

413 k25

459 + I 7

510 f 2 5

498 f 1 8

630 f 2 1

240 +20

303 f 1 9

% of increase

+ 32%

+ 46%

between proli. and differ.

+

+ 26%

11%

+ 26%

96 of total gangliosides'

G,,/G

Lac 1

15.9f 2.0

4.72 0.9

4.75 0.5

G,,/G

GNTr 1

47.1+ 3.2

46.5f 2.7

63.9+ 4.1

1 G M ~ GNT G G,,/G Lac 2

10.6f 3.5 -

-

-

-

-

-

-

-

-

-

-

-

-

a

22.7+ 2.3

14.2+ 3.7 15.9f 2.2

GJGGNT

G,,/GGNT4

39.5+ 2.5

8 . 7 5 2.5 Traces

23.6k 3.8

G D d G GNT 2a G,,,/G GNT 2b 3

26.45 2.4

9.3k 3.0 -

-

46.3f 2.6

Each value is the mean of three experiments f standard deviation

14.15 3.1

17.92 2.5

5.7-t 0.9

1.1+ 0.3

21.35 4.8

33.6f 3.9

25.9k 2.6

27.0k 2.3

23.9f 3.1

24.7f 3.9

44.9f 5.1

39.9-t 3.9

12.6f 4.1

17.3f 2.9

15.05 2.2 -

45.0f 5.0

11.5f 2.9 -

43.6f 5.3

IS+ 0.5

~

53.1f 3.9

51.7k 4.2

4.7+ 0.3

3.7-t 0.5

-

Traces -

33.82 3.7

5.3f 1.1 21.2f 3.8

J. Ciesielski-Treska et al.:

34

Table 3. Gangliosides of clones neuro 2 A and M I differentiated by dibutyryl cyclic AMP or bromodeoxyuridine

Neuro 2 A

Clone

Prol. Total lipid sialic acid pg/g dry weight

240

& 20

M1

DBcAMP

BRDU

Prol.

399

-b

498

f 26

k 18

+ 66%

% of increase between

BRDU

DBcAMP 736

-I 21

564

k 23

+ 13%

+ 47%

prolif. and differ. % of total gangliosidesa

G,,/G

Lac 1

G,,*/G GNTr 1 G,,/G

GNT 1

G,,/G

Lac 2

GDIa/GGNT 2a

G,,,/G

GNT 2b

21.3

+

4.8

44.9 k 5.1

24.4 f

Traces

4.9 f 1.0

33.8 k 3.7

45.7

3.8

13.2 k 3.2

16.5 -

-

k 6.1

34.0 k 3.2 -

1.9

7.6 & -

4.4

49.9 k 3.7

*

0.9

20.5 -I 2.1

23.9 k 3.1

26.3 k 1.9

17.3 & 2.0

13.4 f 3.2

13.7 & 3.6

5.7

12.6 f 4.1

k 2.0

+

2.8

+

3.9

39.1

+

2.8

0.5 f 0.3

4.7 f 0.3

0.7

2.8 k 1.0

Traces 53.1

1.0

57.4

+

5.4

7.8 f 2.2

-

G,,/G GNT 3 G,,/G GNT 4

Each result is the mean of three different experiments f standard deviation In all the experiments the measurement was perturbed by the presence of a brown compound. The estimated value is 790 pg sialic acid/g dry weight a

[321 did not observe any change in the Neuro’2 A gangliosides when the cells were grown on the same culture medium in Falcon flasks (differentiated cells) and in spinner (undifferentiated cells).

Gangliosides of Neuro 2 A and M1 Clones Differentiated with Bromodeoxyuridine and Dibutyryl CAMP

the notable decrease of the disialoganglioside GDla.The results are similar to those we found previously when differentiation was induced by serum deprivation [331. This decrease was, in the case of DBcAMP, compensated by a similar increase of all the monosialogangliosides and the GD3.In contrast, with BRDU only the G,, increased.

Discussion

Since the M 1 clone is more similar to the neuronal cells than the other active clones studied, the effect on this clone of some other compounds known to induce a good differentiation of neuroblastoma cells was also studied. For comparison, the effect of the same inducers on the inactive Neuro 2 A clone was studied. DBcAMP produced (Table 3) a notable increase of the total gangliosides, due mainly to a striking rise of the G, level. In contrast, the differentiation induced by BRDU involves only a slight change in the total sialic acid, but this is accompanied by a net increase of the disialogangliosides. In spite of the higher amount of GDlb,the known direct precursor of tri- and tetrasialogangliosides [461, no compound of these groups was detected in the cells. When the Neuro 2 A cells were differentiated by DBcAMP or BRDU the most important change lay in

The ganglioside level and pattern of all the analyzed clones are very different from those reported for neuronal cells [2, 6, 47, 481. Even the MI clone which is the most “active” of the studied clones, has a ganglioside composition which remains far from the neuronal one. The principal differences between neurones and neuroblastoma cells lies first in the high percentage of the two monosialogangliosides G,, and GM2,and secondly in the absence of tri- and tetrasialogangliosides. The decreased amount of total gangliosides and the simplification of the pattern is probably a consequence of the malignant transformation of the cell. Indeed, this phenomena had been largely observed in a great number of non-nervous cells [49, 501. These results are similar to those found by Dawson et al. 1311 and Yogeeswaran et

35

Gangliosides of Neuroblastoma Cells

al. [321. They contrast with the activity of the sialyltransfereases measured with different acceptors in the NIE 115 cells by Moskal et al. [511. These authors principally found that the NIE 115 clone has an appreciable trisialoganglioside-synthetizingactivity. Although never detected in neuroblastoma clones, trace amount of trisialogangliosides have been seen in a human neuroblastoma sample by Barton and Rosenberg [521. Therefore it is possible that some clones have retained the ability to synthetize trisialogangliosides but that this lipid cannot be incorporated in cell membranes being rapidly catabolized. Indeed Schengrund et al. [531 found that in fibroblasts, tumourigenic transformation is accompanied by the appearance of a notable sialidase activity. Differentiation of the neuroblastoma cells induces in active clones a great increase in the synthesis of neurotransmitters [19, 541, in cellular respiration [201 and in exitability (21-231. We could therefore expect that the gangliosides of such clones change to a more neuronal distribution. This is not the case whatever be the kind of differentiating agent used. Neuroblastoma cells contain virus particles I55-571. The cell differentiation involves an arrest of the viral production 1551 but only a decrease in the ability to produce tumours [55, 571. The conservation of some tumourigenic properties could explain the inability of the cells to restore a normal ganglioside pattern. However, one cannot forget that even virustransformed cell revertants do not recover a normal ganglioside pattern [ 5 8 , 591. This inability to recover a neuronal pattern for the gangliosides after differentiation, even in the most active clones, leads us to suggest that this class of glycolipid, in contrast to other neuronal characteristics such as neurotransmitter synthesis, cannot be used as a marker of differentiation. Although neuroblastoma cells, when they differentiate, grow neurite-like processes, they never develop nerve endings or establish synaptic connections [55, 56, 601. As these structures are rich in gangliosides 15-81, we could hypothesize that the results obtained on differentiated neuroblastoma cells could be the consequence of the absence of synapses. However observations on the gangliosides of new-born rat cerebellum, which does not contain any synaptic material, show the presence of tri- and tetrasialogangliosides [6 1, 621. This suggests that the absence of polysialogangliosides in neuroblastoma cells is not related to the absence of synapses. Another conclusion which could be made from our results is the absence of correlation between the ganglioside changes and the capacity of cells to synthetize the neurotransmitters. Comparing the gangliosides pattern obtained with the Neuro 2 A, grown in the same medium either in

spinner or in static flasks, Yogeeswaran et al. [321 did not find any modifications, concluding that differentiation in itself does not require variations in the gangliosides. Using other methods which give better differentiation of the cells, we found variations which differ from one clone to the other. Following the conclusions of Yogeeswaran et al., we could hypothesize that the observed changes are the consequence of a specific action of the differentiating agent of the ganglioside metabolism and are independent of the morphological changes. However we cannot discard the hypothesis that the morphological changes could be obtained by numerous biochemical means, some needing a variation in the ganglioside metabolism, others not. Supporting this hypothesis are the results of Furmanski [631 and Steinbach and Schubert [64] showing that there exist multiple modes for the formation of processes in such cells. Acknowledgements: The authors wish to thank Mrs P. Guerin for her excellent technical assistance. This work was supported by a grant to G. R. (ATP 1852, Differenciation cellulaire from the C.N.R.S.).

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Gangliosides of Neuroblastoma Cells 51. Moskal, J., Gardner, D. A,, Basu, S.: Changes in glycolipid gly-

52.

53.

54.

55.

56.

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Gangliosides of active and inactive neuroblastoma clones.

Gangliosides of Active and Inactive Neuroblastoma Clones J. CIESIELSKI-TRESKA, J. ROBERT, G. REBEL’, and P. MANDEL Centre de Neurochimie du CNRS 1 1,...
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