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Neuron-specific enolase in relation to differentiation in human neuroblastoma
69
Brain Research, 224 (1981) 69-82 Elsevier/North-Holland Biomedical Press
N E U R O N - S P E C I F I C ENOLASE IN R E L A T I O N TO D I F F E R E N T I A T I O N IN HUMAN NEUROBLASTOMA
L E N A O D E L S T A D , S V E N P A H L M A N * , K E N N E T H N I L S S O N , ER1K L A R S S O N , G O R A N L ~ C K G R E N , K A R L - E R I K J O H A N S S O N , S T E L L A N HJERTI~N and G U N N A R G R O T T E
Institute of Biochemistry, Box 576 and (K.N., E.L.) The Wallenberg Laboratory and Department ~1" Pathology, University of Uppsala, and (G.L., G.G.) Department of Pediatric Surgery, University Hospital, lack, Uppsala (Sweden) (Accepted March 12th, 1981)
Key words: h u m a n - - n e u r o b l a s t o m a - - g a n g l i o n e u r o b l a s t o m a
-- g a n g l i o n e u r o m a - -
neuron-
specific enolase (14-3-2) - - differentiation
SUMMARY
The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCI, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultured cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.
INTRODUCT[ON
During the last decade cultured neuroblastoma cells have been used for in vitro studies of differentiationa, 2s. Most of the results have been obtained using the mouse * To whom correspondence should be addressed at: Institute of Biochemistry, Biomedical Center, Box 576, S-751 23 Uppsala, Sweden.
0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
70 neuroblastoma C1300 cell line or its subclones 1.23. The substances used to induce differentiation include nerve growth factor (NGF), dibutyryl cyclic AMP (db-cAMP) and dimethylsulfoxide (DMSO) (~,9,11,2°,'-'z. The most widely used criteria for in vitro differentiation have been morphological changes, i.e. formation of cell processes. These changes have also been correlated to alterations in nerve cell functions, e.g. an increase in the concentration of enzymes for the neurotransmittor synthesisZ3, stimulation of sodium uptake 2° or increased number of acetylcholine receptor ~:'. Neuron-specific enolase (NSE), also called 14-3-25,9,l~ is the major enolase isoenzyme in differentiated neurons z,7,'1, while a non-neuronal enolase (NN E), found in glial cells of the mature brain, is the major enolase form in non-differentiated neurons 5,7,17. It has been suggested that NNE compensates for the low levels in nondifferentiated neuronal cells and is replaced by NSE during differentiation TM 18,24. NSE is thus a possible differentiation marker also in neuroblastoma. Cultured human and mouse neuroblastoma cells have low activities of NSE t°,~=~, but a small increase in NSE levels was noted after exposure to db-cAMP. NSE and NNE are dimers of different subunits, and tissues containing both enolases also have a hybrid enolase. The two enolase forms and the hybrid can be separated by ion exchange chromatographyV,lv,ot. In this work we show that biopsy cells from human neuroblastoma have a much higher relative NSE content than cells of three cultured human neuroblastoma cell lines, but lower relative levels than in biopsy cells of ganglioneuroblastoma which morphologically represents a form of neuroblastoma of intermediate differentiation. Furthermore, treatment of cultured human neuroblastoma cells with nerve growth factor (NGF) or db-cAMP gave rise to a small increase in relative NSE levels. MATERIALS AND METHODS Preparation of brain and tumor extracts Fresh tumor tissues were obtained from the Department of Pediatric Surgery, Necrotic parts of the tumors were avoided when material was taken for enolase fractionation. Part of this material was taken for preparation of histological sections. These sections were used to estimate the degree of microscopic necrosis and the proportion of tumor cells in the tissue taken for NSE determination. Human brain material was taken out within 12 h after death. The tissues were disintegrated and homogenized in a teflon-glass homogenizer with 1 part (w/v) 10 mM Tris-HCl buffer (pH 7.4) 1 mM MgSO4. The homogenate was then centrifuged at 130,000 g for 60 min at 4 °C. All tissues and tissue extracts were stored at --70 °C until use. Cell cultures The human neuroblastoma cell line I MR-32 was obtained from American Tissue Culture Association, Rockville, MD. The three human neuroblastoma cell lines SKN-MC, SK-N-SH and SH-SYSY 3 were a kind gift from Dr. June Biedler of the Sloan Kettering Institute, New York. The human glial cell line U-787 CG has been described previously from our laboratory z2. All cells were grown in t00 mM tissue culture plates
71 (Nunclone, Roskilde, Denmark) in Eagles MEM supplemented with 15 ~ fetal calf serum, penicillin (100 l.U./ml), streptomycin (50 #g/ml) and amphotericin B (I.25 l~g/ml) and incubated at 37 °C in a 95% air-5°g] CO,, humidified incubator. The medium was changed twice a week. The confluent cultures were transferred after trypsinization (0.25 '.!/ootrypsin in phosphate-buffered saline (PBS)). Cells for NSE tests were harvested after treatment for 5 rain with EDTA (0.02°//o in PBS), washed with PBS and stored at --20 °C until use.
Treatment with db-cA MP and NGF Cells were grown in the above medium supplemented with 1 mM Na,O`,'-dibuty ryl-adenosine-Y:5'-cyclic monophosphoric acid (db-cAMP) (Sigma) or 5 units of NGF/ml medium. N G F prepared from mice was a generous gift from Dr. Ted Ebendahl, Department of Zoology, Uppsala. The substances were included in the culture medium directly after subcultivation of the cells and was added again at the same concentration at each medium removal. After 7-11 days the cells were confluent and harvested as described above. Preparation of cell extracts Cells were homogenized with a Dounce glass homogenizer and then centrifuged at 100,000 g for 40 rain at 4 °C. Approximately 10s cells were used for each chromatographic experiment. Protein was measured by the method of Lowry et al. 12 with human serum albumin as standard. Ion exchange chromatograph), The sample containing 3-29 mg of protein was desalted on a Sephadex G-25 column (PD 10, prepacked, Pharmacia, Uppsala, Sweden). The different enolase isoenzymes were then separated by ion exchange chromatography on preswoIlen DEAE-Sephacel (Pharmacia) in 1.5 x 10 cm columns. The ion exchanger was equilibrated with 10 mM Tris-HCl buffer (pH 7.4) containing [ mM MgSO4. The samples were applied to the ion exchanger, which was then washed with two column volumes of the buffer. Adsorbed material was eluted by I00 ml of a linear gradient of 0-1.5 M KCI in the buffer. The flow rate was 25 ml/h. All experiments were run at 4 °C. Fractions of about 2 ml were collected and analysed for enolase activity immediately after the run. The KCI concentrations in the fractions were determined by measuring the conductivity and comparing with the conductivity of KC1 solutions of known molarity. Enolase assay Enolase activity was, with some modifications, determined as described by Baranowski and Wolna 2. The assay implies direct initial rate measurement by following the change in optical density at 240 nm. The total assay volume was 1 ml containing 50 mM imidazole-HCl buffer (pH 6.8), 1.5 mM MgSO4, 50 mM KCI, 1 mM 2-phospho-D-glycerate (sodium salt, Sigma) and 0.5 ml of sample. The reaction was carried out at 25 °C. The activity is expressed in enzyme units per ml sample
72 solution, where one enzyme unit corresponds to the conversion of one /zmole of substrate per minute. Since the molar absorption coefficient of phosphoenolpyruvate is 1300 M - l c m -1 under the conditions of the assay, a linear absorbancc increase of 0.100 at 240 nm was taken to be equivalent to the conversion of 77/tM of substrate'-'. The totally recovered enolase activity after the chromatography was determined and the relative amounts of the different isoerlzymes were expressed as percentage of the total activity.
Inactivation by heat, KBr and NaCI Pooled fractions from the ion exchange chromatography of ganglioneuroblastoma isoenzymes corresponding to peaks 1, 11 and 1II in Fig. 4 were used. The three fractions were first dialyzed against 10 mM Tris-HCl buffer (pH 7.4) containing 1 mM MgSO4. The enolase activity was then adjusted to be about the same in the different fractions by dilution with the buffer. The temperature inactivation experiment was performed at 50 :C. Aliquots were withdrawn for direct activity measurement at different incubation times. KBr (analytical grade) was added to the enolase fractions to a final concentration of 0.5 M and inactivation was performed at 37 ~C. Samples were withdrawn at different incubation times and the enolase activity was measured immediately. The three enolase fractions from the ion exchange chromatography were also incubated at 4 °C with different concentrations of KCI and NaCI. After 2 h the samples were thermostated at 25 °C and 500 ktl of each sample were analysed for enolase activity.
Double imrnunodiffusion Samples were analysed by double immunodiffusion according to Ouchterlony's method 19 in 1 3/o agarose C (Pharmacia Fine Chemicals) at 30 °C for 48 h. The gel was prepared in 80 mM Tris-acetic acid buffer (pH 8.6). Bovine NSE and antiserum against this protein 4 was a generous gift from Dr. Manfred Braun, Max Planck Institute, Cologne, F.R.G. RESULTS
Histopathological.findings The analyzed neuroblastomas were all of the classical type, consisting of small, dark basophilic cells with little cytoplasm (Fig. 1). Within the group of neuroblastoma a considerable heterogeneity with respect to the relative amounts of stromal and necrotic tumor cells was found (Table 1). Ganglioneuroblastoma, accounting for 5-10 % of all neuroblastomas, is a partially differentiated form, consisting of immature small neuroblasts, larger, more differentiated neuroblasts with prominent nuclei and scattered ganglia cells (Fig. 2). The admixture of stromal cells was low. In the benigngangtioneuroma, a terminally differentiated variant of neuroblastoma found at an even lower frequencey than ganglioneuroblastoma, sparse ganglie cells were found within a background of fibrious and neurofibrillary stroma (Fig. 3 and Table I).
73
Fig. 1. Neuroblastoma with rather small, relatively uniform cells as the major diagnostic hallmark. Typical pseudorosettes are frequent (arrow). Hematoxylin eosin, 150.
Fig. 2. Ganglioneuroblastoma. The figure shows a more differentiated part of the tumor with a mixture of mature ganglia-like cells (G) and larger neuroblasts (N). Hematoxylin-eosin, ,-~ 150.
I
male female male female male
4 yr 8 mo 4 yr 5 yr 22 m o
*
female right adrenal NB male intrathoracal NB
NB NB NB GNB GN
-------
-cytostatics irradiation
cytostatics irradiation
---
7,~,
C
~,
Necrosis* Cytostatics or irradiation
( )
Stroma *
**
m
n
_ 4
Presence * * Presence* * o f mature o f partly ganglia cells dtfferentiated nem'oblasts
GN, ganglioneuroma.
* (3, p r e s e n c e of s t r o m a o r n e c r o s i s ; ( , l a r g e r a r e a s o f s t r o m a or n e c r o s i s . ** -~, p r e s e n c e o f t h e cell t y p e ; 5 - " , m a j o r cell t y p e ; i : , e n t i r e cell p o p u l a t i o n . d a t a t a k e n f r o m T a b l e 11.
right adrenal left a d r e n a l left a d r e n a l right adrenal intrathoracal
NB
10 m o 8 mo
para-aortic
male
Histopathological diagnosis
3 yr
Anatomical site
Sex
Age
Abbreviations: NB, neuroblastoma; GNB, ganglioneuroblastoma;
Characteristics o f investigated tumors
TABLE
-
....
-
•
- ~ : : +
31 36.5 48 36 40 28 62.5 48
No No Yes No No No
Calculated N S E * * *, % o f total activity
No Yes
Presence * * Rosettes o f dark small undifferentiated neuroblasts
",,-a 4~
75
Fig. 3. Ganglioneuroma. Built up of clusters of mature ganglia-like cells (G). Hematoxylin-eosin, 150.
hm exchange chromatography: identification of NSE Fractionation of neuroblastoma or brain tissue extracts always resulted in three
fractions of enolase activity (I-111) with reproducable elution positions of fi'action II and II1 with respect to the salt gradient (Fig. 4). By analogy with the literature we
0.4Z
~0.4
(,...)
¢-.
~0.3
0.3~
](19%)
o ¢-
i~ 0.2 r~ e-
0.1 0
19 r" O ¢,D
LLI
20
40 60 80 100 E[ution vo[urne (ml.)
120
Fig. 4. Separation of enolase isoenzymes of human ganglioneuroblastoma by ion exchange chromatography on DEAE-Sephacel. One ml of the tissue extract containing 14 mg of protein was chromatographed as described in Materials and methods. Enolase activity ( 0 ) was determined and expressed as enzyme units per ml sample solution. ~ , KC[ concentration. The enolase activity in fractions I, II and Ill are expressed as percentage of totally recovered activity. The yield of enolase activity was in all experiments more than 8 0 %
76 A
B
100 8O 60
20
O0
1;
2?
3'0
1'0
2~0
30
Incubation timQ (rain)
Fig. 5. Inactivation of human ganglioneuroblastoma enolases. Fractions I, II and 111 from the ion exchange chromatography shown in Fig. 4 were used. A: incubation in the presence of 0.5 M KBr at 7-37 °C. B: incubation at t 50 °C in the absence of KBr. ~, fraction I: ~,, fraction I1; ~3, fraction llI. assumed that the enolase activities in fractions I - I I I c o r r e s p o n d e d to N N E , hybrid form a n d NSE, respectively. This was confirmed by the d e m o n s t r a t i o n that the enolase activity in fraction I was more labile in 0.5 M K B r or at 50 °C, which is in agreement with the greater heat a n d K B r lability of N N E" c o m p a r e d to N SE. F r a c t i o n II showed
Fig. 6. Immunodiffusionof the enolase forms found in human ganglioneuroblastoma. Antiserum against bovine NSE was placed in the central wells. The antigen loads in peripheral wells labeled 1 were : O, 5 fll of bovine NSE (0.4 mg/ml); A, 5 t*1of fraction I (6.1 units/ml); B, 5 pl of fraction II (6.2 units/ml); C, 5 #1 of fraction III (12.8 units/ml). In the peripheral wells 2-5 the antigens were diluted 1:2, 1:4, 1:8 and 1:16, respectively.
77 either intermediate lability or the same lability as the enolase in fraction I, depending on the inactivation conditions. It has been shown that anti-bovine NSE cross-reacts with NSE from other species 14. Fig. 6 shows an immunodiffusion experiment, with the three enolase fractions from the ion exchange chromatography, analyzed against anti-bovine NSE. The same number of enolase units from fractions I and II and approximately twice as much of fraction 11I were applied giving a strong immunoprecipitate with fraction III, still present at a 1:16 dilution of the antigen. The second fraction gave a weaker immunoprecipitate visible down to a 1:2 dilution, while no immunoprecipitate could be detected with the first fraction. The reproducability of the NSE measurements was controlled by running a series of samples from the brain of a 37-week-old fetus. The calculated NSE levels were 60 ± 4'!~,;(n - 6), but in experiments where the same sample batch was used and all the chromatographical and assay procedures were done in parallel, the variations in calculated NSE levels were less than ~ 1 o//o. Effect o f KCI and NaCI on enolase activity Initially, NaC1 was used to form the salt gradient during the ion exchange chromatography. However, NaC1 inhibited the enolase activity and was therefore replaced by KCI. As shown in Fig. 7, KC1 had much less influence on the enolase activity than NaC1 at similar concentrations. For example, the KCI concentration in fraction 1II (0.28 0.33 M KC1) had only a small (8 ~o increase in activity) effect on the enolase activity while NaC1 at the same concentrations decreased the activity to approximately 35 ~ of the activity in the absence of NaCI (Fig. 7). N S E in tissue extracts The relative calculated NSE levels in 6 tumor biopsies, diagnosed as neuroA
C
B
100' ~80
Sz, O C
2O
0
01s
0.'zs
0'.s
0'2s
0'.5
Satt concentration (M)
Fig. 7. Effect of NaCI (;5~)and KCI (×) on the enolase activity. Fractions from the ion exchange chromatography shown in Fig. 4 were used. The fractions were incubated in the presence of different concentrations of NaCI and KCI respectively at ~ 4 °C for 2 h before activity measurement. A: fraction I. B: Fraction 11. C: fraction III.
78 TABLE II
Levels of enolase isoenzymes in human neuroblastoma and]etal brain Soluble extract of the tissues were chromatographed on DEAE-Sephaeel and analyzed for enolase activity. The relative amount of enolase activity in each fraction is given as ~ of totally recovered enolase activity (cf. Fig. I). The calculated NSE value is given in 70 of total enolase activity and is the sum of the amount of enolase in fraction III and half of the amount in fraction II.
Tissue
Enolase activity (units/mg protein)
Neuroblastoma
1 I1 III IV V V] Ganglioneuroblastoma Ganglioneuroma Foetal brain 17 weeks 20 weeks 37 weeks
0.65 1.8 0.84 0.49 0.48 1.4 1.2 1.2 0.78 1.0 1.2
% of total activity Fraction l
11
IH
53 43 32 48 42 58 19 37 43 30 27
32 41 40 32 36 28 37 30 32 31 28
15 16 28 20 22 14 44 33 25 39 45
Calculated NSE % of total activity
31 36.5 48 36 40 28 62.5 48 41 54.5 59
blastoma, ranged from 28 to 48 °/o of total enolase activity with a mean value of 37 °/ /o • The corresponding value for the more differentiated form of the tumor, i.e. ganglioneuroblastoma, was found to be 62 ~o (Table II). Histological examination revealed that, in all 7 biopsies, tumor cells constituted more than 80 ~o of the tissue, the rest being stromal fibroblasts, collagen or necrotic parts (Figs. 1 and 2). The biopsy of the completely differentiated and benign form of the tumor, ganglioneuroma, however, contained mainly fibrous stroma. Less than 20 % of the tissue contained ganglia cells ,
II
0,10
III
57%
E
O.Lg c ,,r"
0.3 "6 ~ 0.05 u ¢tl
8
17%
0.2~ 0.1 ~=
o
c)
UJ
20
60 80 100 E[ution vol.ume (rot)
/~0
120
Fig. 8. Rechromatography of the second fraction from the DEA,E-Sephacel chromatography of human fetal brain. The fraction was dialyzed against 10 m M Tris-HC1 buffer (pH 7.4) containing l m M MgSO4 and frozen at --20 °C before the experiment, which was performed in the same way as the first chromatography.
79 TABLE llI Levels of enolase isoenzymes in cultured human neuroblastoma cells and human glial cells
Soluble extract of the cultured cells, chromatographed on DEAE-Sephacel and analyzed for enolase activity. Enolase and calculated NSE levels are expressed as in Table II. Cells
SK-N-SH* SK-N-SH ! NGF* SK-N-SH ~ db-cAMP* SH-SY5Y** SH-SY5Y ~ NGF** SH-SY5Y + db-cAMP** SK-N-MC IMR-32 787 CG (human gila)
Enolase activity (un#s/mg protein)
3.0 2.8 4.1 2.1 1.8 2.5 3.9 1.9 0.92
%, of total activity Fraction 1
11
111
83 76 78 81 79 71 95 76 89
17 24 22 18 18 29 5 20 11
---1 3 --4 --
CalculatedNSE, % of total activity
8.5 12 11 10 12 14.5 2.5 14 5.5
* Measured in parallel experiments. ** Measured in parallel experiments. (Fig. 3). The relative calculated N S E level in this biopsy was, despite this sparsity of ganglie ceils, 48 ° o (Table I1). The relative NSE activity in normal fetal brains at different stages of development increased with age (Table I1), as could be expected 7. F o r each biopsy the total level ofenolase per mg protein was determined. The activities varied between 0.5 and 1.8 units/rag protein, but there seemed to be no correlation between the total activity of enolase and the relative NSE activity. R e c h r o m a t o g r a p h y o f the hybrid form (fraction II) on D E A E - S e p h a c e l after dialysis and homogenization in the same way as the tissue extracts were treated gave three fractions of enolase activity at positions in the c h r o m a t o g r a m corresponding to N N E , hybrid and NSE (Fig. 8). It seems as if the level o f hybrid is affected by the treatment of the sample, and it has been suggested that the hybrid is an artefact due to the storage conditions and preparation procedures 17. Hybrid and thus NSE-levels might then be dependent on how the tissue extracts have been prepared. I f instead a calculated NSE value, i.e. the a m o u n t of NSE and half of the a m o u n t o f hybrid given as per cent o f total enolase activity, is used, the comparison o f N S E levels in tissues are not dependent on how the tissues were handled assuming the same specific activity for NSE and the hybrid. The calculated N S E levels for the analyzed tissues are given in Table 1I. N S E in cultured cells
Cultured h u m a n neuroblastoma cells also showed varying relative NSE activities (Table IIl) but they were always lower than in the neuroblastoma biopsies (Table II). The lowest level o f NSE was found in the cholinergic neuroblastoma S K - N - M C cell line. Small amounts o f NSE were also found in cultured h u m a n glial cells. The NSE in
80 the cultured cells were mainly found in fraction II (hybrid) and not in fraction Ill (NSE). After treatment with N G F or db-cAMP, the adrenergic neuroblastoma cell line SK-N-SH and the clone SH-SY5Y changed their growth pattern from growth in dense colonies to growth as single cells. The growth was not markedly affected by N G F while db-cAMP caused some cell death. Only a small proportion of the cells showed morphological changes, suggestive of induced differentiation. These cells had thin plasma membrane projections with a length 2-5 times that of the control cells. N G F and db-cAMP induced a small (20-45 °/o) but significant increase in the relative amount of NSE in both SK-N-SH and SH-SY5Y cells detected as increased enolase activity mainly in the hybrid fraction (Table 1ll). DISCUSSION The hybrid enolase can apparently dissociate and reassociate into all three forms under the same conditions as we use for preparation of the tissue and cell extracts. Fletcher et al. 7 showed that maturation of the rat brain and thus expression of NSE initially was reflected as an increase in the hybrid level and later also in the NSE level. Evidently the amount of hybrid must also be taken into account when the total relative NSE levels of different tissues are compared. The calculated NSE values given in Tables II and III are based on the assumption that the specific activity of NSE, N N E and the hybrid is the same. This has not been shown for the human system but Marangos et al. la have shown that the rat brain enolase forms have similar kinetic parameters. Furthermore, Hullin et al. s found good agreement between enolase activity and immunoreactive material of fractions II and III assuming that half of the enolase activity from the hybrid was contributed by NSE. In animal systems as well as in man, there is a correlation between the stage of maturation of the brain and the level of NSE 5,7. The present results suggest that the same correlation holds also for the various forms of human neuroblastoma, In tumors, histopathologically diagnosed as partly or fully differentiated (ganglioneuroblastoma and ganglioneuroma, respectively) the relative NSE levels were higher, if the low content of gangliecells in ganglioneuroma are taken into account, than in the nondifferentiated ('true' neuroblastoma) form of the tumor. However, the number of analyzed ganglioneuroblastoma and ganglioneuroma is still limited and as the possibility exists that these cases are not representative for differentiated forms of neuroblastoma in general this conclusion should merely be considered as a strong suggestion. Absolute enolase levels are not a proper basis for comparison since the biopsies are heterogeneous with respect to the extent of necrosis and infiltration of non-neoplastic cells (Table I). We have instead compared the relative enolase levels which probably are not seriously effected by a variation in necrosis but still leave us with a varying contribution from infiltrating cells. The variation in NSE levels between the different neuroblastoma biopsies might be explained by differences in necrosis or presence of non-neoplastic cells, but could also reflect an existing heterogeneity within the histopathological entity neuroblastoma not detectable by current morphological
8l methods. Quantitation of enolases would, if this assumption is true, be of possible clinical use as a mean to further subclassify neuroblastomas. To further investigate the correlation between NSE and differentiation of neuroblastoma cells we performed controlled studies with cells of human neuroblastoma in vitro cell lines. Compared to the biopsies, the cultured cells had very low NSE levels. A question that arises is therefore if the established cell lines might originate from neuroblastoma cells with low NSE levels or if the cells have diminished their capacity for NSE production as a consequence of prolonged in vitro culture. The latter possibility is favored by the fact that a number of other enzymes have lower levels of activities in cultured than in freshly isolated neuroblastoma cells z3. The NSE level in the cultured neuroblastoma cells increased significantly when the cells were treated with known inducers of differentiation like N G F and db-cAMP. Since only a small fraction of the cells showed morphological changes suggestive of differentiation, the result could be explained by either a small increase in the amount of NSE in a majority of the cells or a large increase in the few differentiated cells. It has been reported that the NSE level in cultured mouse neuroblastoma cells is lower in logarithmically growing than in confluent cell monolayers 10. We have not found this difference in the human cell lines SK-N-SH and SH-SY5Y (unpublished data). ACKNOWLEDGEMENTS
We wish to thank Mrs. Aino Ruusala and Mrs. Karin Elenbring for skilful technical assistance. The work was supported by the Swedish Natural Science Research Council, 'H.K.H. kronprinsessan Lovisas f6rening f6r barnasjukvS.rd', 'Ollie och Elof Ericssons stiftelse' and the Swedish Cancer Society.
REFERENCES 1 Augusti-Tocco, G., Neuroblastoma culture: an experimental system for the study of cellular differentiation, Trends Bioehem. Sci., 1 (1976) 151 154. 2 Baranowski, T. and Wolna, E., Enolase from human muscle, Meth. Enzymol., 42 (1975) 335-338. 3 Biedler, J. L., Helson, L. and Spengler, B. A., Morphology and growth tumorigenicity, and cytogenetics of human neuroblastoma cells in continuous culture, Cancer Res., 33 0973) 26432652. 4 Braun, M., Grasso, A. and Wechsler, W., 14-3-2 protein in rat primary and transplanted gliomas and neurinomas and in clonal cell lines, Exp. Brain Res., 25 (1976) 93-97. 5 Cicero, T..I., Cowan, W. M. and Moore, B. W., Changes in the concentrations of the two brain specific proteins, S-100 and 14-3-2, during the development of the avian optic tectum, Brain Research, 24 (1970) 1 10. 6 Ehrlich, Y. H., Prasad, K. N., Sinha, P. K., Davis, L. G. and Brunngraber, E. G., Selective changes in the phosphorylation of endogenous proteins in subcellular fractions from cyclic AMPinduced differentiated neuroblastoma cells, Neurochem. Res., 3 (1978) 803 813. 7 Fletcher, k., Rider, C. C. and Taylor, C. B., Enolase isoenzymes. Ill. Chromatographic and immunological characteristics of rat brain enolase, Bioehem. biophys. Acta, 452 (1976)245-252. 8 Hullin, D. A., Brown, K., Kynoch, P. A. M., Smith, C. and Thompson, R. J., Purification, radioimmunoassay, and distribution of human brain 14-3-2 protein (nervous-system specific enolase) in human tissues, Biochim. biophys. Acta, 628 (1980) 98 108.
82 9 Kolber, A. R., Goldstein, M. N. and Moore, B. W., Effect of nerve growth factor on the expression of colchicine-binding activity and 14-3-2 protein in an established line of human neuroblastoma, Proc. nat. Acad. Sci. USA, 71 (1974) 4203-4207. 10 Legault-Demare, L., Zeitoun, Y., Lando, D., Lamande, N., Grasso, A. and Gros, F., Expression of a specific neuronal protein, 14-3-2, during in vitro differentiation of neuroblastoma cells, Exp. Cell Res., 125 (1980) 233-239. 11 Littauer, U. Z., Giovanni, M. Y. and Glick, M. C., Differentiation of human neuroblastoma cells in culture, Biochem. biophys. Res. Commun., 88 (1979) 933-939. 12 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randell, R. J., Protein measurement with the Folic phenol reagent, J. biol. Chem., 193 (1951) 265-275. 13 Marangos, P. J., Zomzely-Neurath, C. and York, C., Determination and characterization of neuron specific protein (NSP) associated enolase activity, Biochem. biophys. Res. Commun,. 68 (1976) 1309-1316. 14 Marangos, P. J., Zomzely-Neurath, C. and Goodwin, F. K., Structural and immunological properties of neuron specific protein (NSP) from rat, cat and human brain: comparison to bovine 14-3-2, J. Neurochem., 28 (1977) 1097-1107. 15 Marangos, P. J., Goodwin, F. K., Parma, A., Lauter, C. and Trams, E., Neuron specific protein (NSP) in neuroblastoma cells: relation to differentiation, Brain Research, 145 (1978) 49-58. 16 Marangos, P. J., Zis, A. P., Clark, R. L. and Goodwin, F. K., Neuronal, non-neuronal and hybrid forms of enolase in brain: structural, immunological and functional comparisons, Brain Research, 150 (1978) 117-133. 17 Marangos, P. J., Parma, A. M. and Goodwin, F. K., Functional properties of neuronal and glial isoenzymes of brain enolase, J. Neurochem., 31 (1978) 727-732. 18 Marangos, P. J., Schrnechel, D. E., Parma, A. M: and Goodwin, F. K:, Developmental profile of neuron-specific (NSE) and non-neuronal (NNE) enolase. Brain Research, 190 (1980) 185-193. 19 Ouchterlony, O. and Nilsson, L. A., lmunodiffusion and immunoelectrophoresis. In D. M. Weir (Ed.), Handbook of Experimental Immunology, Ifol. 1, Blackwell Scientific Publ., Oxford, 1967, pp. 655-706. 20 Perez-Polo, J. R., Werrback-Perez, K. and Tiffany-Castiglioni, E., A human ctonal cell line model of differentiating neurons, Develop. Biol., 71 (1979) 341-355. 21 Pickel, V. M., Reis, D. J., Marangos, P. J. and Zomzely-Neurath, C., lmmunocytochemical localization of nervous system specific protein (NSP-R) in rat brain, Brain Research, 105 (1976) 184-187. 22 Pont6n, J . , Westermark, B. and Hugosson, R., Regulation of proliferation and movement of human glia-like cells in culture. Exp. Cell Res., 58 (1969) 393-400. 23 Prasad, K. N., Differentiation of neuroblastoma cells in culture, Biol. Rev., 50 (1975) 129-265. 24 Schmechel, D. E., Brightman, M. W. and Marangos, P. J., Neurons switch from non-neuronal enolase to neuron-specific enolase during differentiation, Brain Research, 190 (1980) 195--214. 25 Sinator, R. and Sachs, L., Regulation of acetylcholine receptors in relation to acetylcholinesterase, Proc. nat. Acad. Sci. (U.S.A.), 70 (1973) 2902-2905.
Brain Research, 224 (1981) 69-82 Elsevier/North-Holland Biomedical Press
N E U R O N - S P E C I F I C ENOLASE IN R E L A T I O N TO D I F F E R E N T I A T I O N IN HUMAN NEUROBLASTOMA
L E N A O D E L S T A D , S V E N P A H L M A N * , K E N N E T H N I L S S O N , ER1K L A R S S O N , G O R A N L ~ C K G R E N , K A R L - E R I K J O H A N S S O N , S T E L L A N HJERTI~N and G U N N A R G R O T T E
Institute of Biochemistry, Box 576 and (K.N., E.L.) The Wallenberg Laboratory and Department ~1" Pathology, University of Uppsala, and (G.L., G.G.) Department of Pediatric Surgery, University Hospital, lack, Uppsala (Sweden) (Accepted March 12th, 1981)
Key words: h u m a n - - n e u r o b l a s t o m a - - g a n g l i o n e u r o b l a s t o m a
-- g a n g l i o n e u r o m a - -
neuron-
specific enolase (14-3-2) - - differentiation
SUMMARY
The presence of the two forms of enolase, neuron-specific enolase (NSE) and non-neuronal enolase (NNE), have been examined in biopsy material of human neuroblastoma, ganglioneuroblastoma, ganglioneuroma and cultured neuroblastoma cells, after separation with ion exchange chromatography. The enolase activities were inhibited in the presence of NaCl but remained active in KCI, which were used in the chromatographic step. The relative NSE levels in the neuroblastoma tissues were found to be lower than in the histopathologically more differentiated forms of the tumour, i.e. ganglioneuroblastoma and ganglioneuroma. The human neuroblastoma in vitro cell lines SK-N-SH, SH-SY5Y, SK-N-MC and IMR-32 contained considerably lower relative levels of NSE compared to the levels in the neuroblastoma biopsies. After treatment of the cultured cells with nerve growth factor or dibutyryl-cAMP some cells showed morphological differentiation and concomitantly an increase in the NSE levels. The results indicate that NSE might be useful as a marker for differentiation in human neuroblastoma.
INTRODUCT[ON
During the last decade cultured neuroblastoma cells have been used for in vitro studies of differentiationa, 2s. Most of the results have been obtained using the mouse * To whom correspondence should be addressed at: Institute of Biochemistry, Biomedical Center, Box 576, S-751 23 Uppsala, Sweden.
0006-8993/81/0000-0000/$02.50 © Elsevier/North-Holland Biomedical Press
70 neuroblastoma C1300 cell line or its subclones 1.23. The substances used to induce differentiation include nerve growth factor (NGF), dibutyryl cyclic AMP (db-cAMP) and dimethylsulfoxide (DMSO) (~,9,11,2°,'-'z. The most widely used criteria for in vitro differentiation have been morphological changes, i.e. formation of cell processes. These changes have also been correlated to alterations in nerve cell functions, e.g. an increase in the concentration of enzymes for the neurotransmittor synthesisZ3, stimulation of sodium uptake 2° or increased number of acetylcholine receptor ~:'. Neuron-specific enolase (NSE), also called 14-3-25,9,l~ is the major enolase isoenzyme in differentiated neurons z,7,'1, while a non-neuronal enolase (NN E), found in glial cells of the mature brain, is the major enolase form in non-differentiated neurons 5,7,17. It has been suggested that NNE compensates for the low levels in nondifferentiated neuronal cells and is replaced by NSE during differentiation TM 18,24. NSE is thus a possible differentiation marker also in neuroblastoma. Cultured human and mouse neuroblastoma cells have low activities of NSE t°,~=~, but a small increase in NSE levels was noted after exposure to db-cAMP. NSE and NNE are dimers of different subunits, and tissues containing both enolases also have a hybrid enolase. The two enolase forms and the hybrid can be separated by ion exchange chromatographyV,lv,ot. In this work we show that biopsy cells from human neuroblastoma have a much higher relative NSE content than cells of three cultured human neuroblastoma cell lines, but lower relative levels than in biopsy cells of ganglioneuroblastoma which morphologically represents a form of neuroblastoma of intermediate differentiation. Furthermore, treatment of cultured human neuroblastoma cells with nerve growth factor (NGF) or db-cAMP gave rise to a small increase in relative NSE levels. MATERIALS AND METHODS Preparation of brain and tumor extracts Fresh tumor tissues were obtained from the Department of Pediatric Surgery, Necrotic parts of the tumors were avoided when material was taken for enolase fractionation. Part of this material was taken for preparation of histological sections. These sections were used to estimate the degree of microscopic necrosis and the proportion of tumor cells in the tissue taken for NSE determination. Human brain material was taken out within 12 h after death. The tissues were disintegrated and homogenized in a teflon-glass homogenizer with 1 part (w/v) 10 mM Tris-HCl buffer (pH 7.4) 1 mM MgSO4. The homogenate was then centrifuged at 130,000 g for 60 min at 4 °C. All tissues and tissue extracts were stored at --70 °C until use. Cell cultures The human neuroblastoma cell line I MR-32 was obtained from American Tissue Culture Association, Rockville, MD. The three human neuroblastoma cell lines SKN-MC, SK-N-SH and SH-SYSY 3 were a kind gift from Dr. June Biedler of the Sloan Kettering Institute, New York. The human glial cell line U-787 CG has been described previously from our laboratory z2. All cells were grown in t00 mM tissue culture plates
71 (Nunclone, Roskilde, Denmark) in Eagles MEM supplemented with 15 ~ fetal calf serum, penicillin (100 l.U./ml), streptomycin (50 #g/ml) and amphotericin B (I.25 l~g/ml) and incubated at 37 °C in a 95% air-5°g] CO,, humidified incubator. The medium was changed twice a week. The confluent cultures were transferred after trypsinization (0.25 '.!/ootrypsin in phosphate-buffered saline (PBS)). Cells for NSE tests were harvested after treatment for 5 rain with EDTA (0.02°//o in PBS), washed with PBS and stored at --20 °C until use.
Treatment with db-cA MP and NGF Cells were grown in the above medium supplemented with 1 mM Na,O`,'-dibuty ryl-adenosine-Y:5'-cyclic monophosphoric acid (db-cAMP) (Sigma) or 5 units of NGF/ml medium. N G F prepared from mice was a generous gift from Dr. Ted Ebendahl, Department of Zoology, Uppsala. The substances were included in the culture medium directly after subcultivation of the cells and was added again at the same concentration at each medium removal. After 7-11 days the cells were confluent and harvested as described above. Preparation of cell extracts Cells were homogenized with a Dounce glass homogenizer and then centrifuged at 100,000 g for 40 rain at 4 °C. Approximately 10s cells were used for each chromatographic experiment. Protein was measured by the method of Lowry et al. 12 with human serum albumin as standard. Ion exchange chromatograph), The sample containing 3-29 mg of protein was desalted on a Sephadex G-25 column (PD 10, prepacked, Pharmacia, Uppsala, Sweden). The different enolase isoenzymes were then separated by ion exchange chromatography on preswoIlen DEAE-Sephacel (Pharmacia) in 1.5 x 10 cm columns. The ion exchanger was equilibrated with 10 mM Tris-HCl buffer (pH 7.4) containing [ mM MgSO4. The samples were applied to the ion exchanger, which was then washed with two column volumes of the buffer. Adsorbed material was eluted by I00 ml of a linear gradient of 0-1.5 M KCI in the buffer. The flow rate was 25 ml/h. All experiments were run at 4 °C. Fractions of about 2 ml were collected and analysed for enolase activity immediately after the run. The KCI concentrations in the fractions were determined by measuring the conductivity and comparing with the conductivity of KC1 solutions of known molarity. Enolase assay Enolase activity was, with some modifications, determined as described by Baranowski and Wolna 2. The assay implies direct initial rate measurement by following the change in optical density at 240 nm. The total assay volume was 1 ml containing 50 mM imidazole-HCl buffer (pH 6.8), 1.5 mM MgSO4, 50 mM KCI, 1 mM 2-phospho-D-glycerate (sodium salt, Sigma) and 0.5 ml of sample. The reaction was carried out at 25 °C. The activity is expressed in enzyme units per ml sample
72 solution, where one enzyme unit corresponds to the conversion of one /zmole of substrate per minute. Since the molar absorption coefficient of phosphoenolpyruvate is 1300 M - l c m -1 under the conditions of the assay, a linear absorbancc increase of 0.100 at 240 nm was taken to be equivalent to the conversion of 77/tM of substrate'-'. The totally recovered enolase activity after the chromatography was determined and the relative amounts of the different isoerlzymes were expressed as percentage of the total activity.
Inactivation by heat, KBr and NaCI Pooled fractions from the ion exchange chromatography of ganglioneuroblastoma isoenzymes corresponding to peaks 1, 11 and 1II in Fig. 4 were used. The three fractions were first dialyzed against 10 mM Tris-HCl buffer (pH 7.4) containing 1 mM MgSO4. The enolase activity was then adjusted to be about the same in the different fractions by dilution with the buffer. The temperature inactivation experiment was performed at 50 :C. Aliquots were withdrawn for direct activity measurement at different incubation times. KBr (analytical grade) was added to the enolase fractions to a final concentration of 0.5 M and inactivation was performed at 37 ~C. Samples were withdrawn at different incubation times and the enolase activity was measured immediately. The three enolase fractions from the ion exchange chromatography were also incubated at 4 °C with different concentrations of KCI and NaCI. After 2 h the samples were thermostated at 25 °C and 500 ktl of each sample were analysed for enolase activity.
Double imrnunodiffusion Samples were analysed by double immunodiffusion according to Ouchterlony's method 19 in 1 3/o agarose C (Pharmacia Fine Chemicals) at 30 °C for 48 h. The gel was prepared in 80 mM Tris-acetic acid buffer (pH 8.6). Bovine NSE and antiserum against this protein 4 was a generous gift from Dr. Manfred Braun, Max Planck Institute, Cologne, F.R.G. RESULTS
Histopathological.findings The analyzed neuroblastomas were all of the classical type, consisting of small, dark basophilic cells with little cytoplasm (Fig. 1). Within the group of neuroblastoma a considerable heterogeneity with respect to the relative amounts of stromal and necrotic tumor cells was found (Table 1). Ganglioneuroblastoma, accounting for 5-10 % of all neuroblastomas, is a partially differentiated form, consisting of immature small neuroblasts, larger, more differentiated neuroblasts with prominent nuclei and scattered ganglia cells (Fig. 2). The admixture of stromal cells was low. In the benigngangtioneuroma, a terminally differentiated variant of neuroblastoma found at an even lower frequencey than ganglioneuroblastoma, sparse ganglie cells were found within a background of fibrious and neurofibrillary stroma (Fig. 3 and Table I).
73
Fig. 1. Neuroblastoma with rather small, relatively uniform cells as the major diagnostic hallmark. Typical pseudorosettes are frequent (arrow). Hematoxylin eosin, 150.
Fig. 2. Ganglioneuroblastoma. The figure shows a more differentiated part of the tumor with a mixture of mature ganglia-like cells (G) and larger neuroblasts (N). Hematoxylin-eosin, ,-~ 150.
I
male female male female male
4 yr 8 mo 4 yr 5 yr 22 m o
*
female right adrenal NB male intrathoracal NB
NB NB NB GNB GN
-------
-cytostatics irradiation
cytostatics irradiation
---
7,~,
C
~,
Necrosis* Cytostatics or irradiation
( )
Stroma *
**
m
n
_ 4
Presence * * Presence* * o f mature o f partly ganglia cells dtfferentiated nem'oblasts
GN, ganglioneuroma.
* (3, p r e s e n c e of s t r o m a o r n e c r o s i s ; ( , l a r g e r a r e a s o f s t r o m a or n e c r o s i s . ** -~, p r e s e n c e o f t h e cell t y p e ; 5 - " , m a j o r cell t y p e ; i : , e n t i r e cell p o p u l a t i o n . d a t a t a k e n f r o m T a b l e 11.
right adrenal left a d r e n a l left a d r e n a l right adrenal intrathoracal
NB
10 m o 8 mo
para-aortic
male
Histopathological diagnosis
3 yr
Anatomical site
Sex
Age
Abbreviations: NB, neuroblastoma; GNB, ganglioneuroblastoma;
Characteristics o f investigated tumors
TABLE
-
....
-
•
- ~ : : +
31 36.5 48 36 40 28 62.5 48
No No Yes No No No
Calculated N S E * * *, % o f total activity
No Yes
Presence * * Rosettes o f dark small undifferentiated neuroblasts
",,-a 4~
75
Fig. 3. Ganglioneuroma. Built up of clusters of mature ganglia-like cells (G). Hematoxylin-eosin, 150.
hm exchange chromatography: identification of NSE Fractionation of neuroblastoma or brain tissue extracts always resulted in three
fractions of enolase activity (I-111) with reproducable elution positions of fi'action II and II1 with respect to the salt gradient (Fig. 4). By analogy with the literature we
0.4Z
~0.4
(,...)
¢-.
~0.3
0.3~
](19%)
o ¢-
i~ 0.2 r~ e-
0.1 0
19 r" O ¢,D
LLI
20
40 60 80 100 E[ution vo[urne (ml.)
120
Fig. 4. Separation of enolase isoenzymes of human ganglioneuroblastoma by ion exchange chromatography on DEAE-Sephacel. One ml of the tissue extract containing 14 mg of protein was chromatographed as described in Materials and methods. Enolase activity ( 0 ) was determined and expressed as enzyme units per ml sample solution. ~ , KC[ concentration. The enolase activity in fractions I, II and Ill are expressed as percentage of totally recovered activity. The yield of enolase activity was in all experiments more than 8 0 %
76 A
B
100 8O 60
20
O0
1;
2?
3'0
1'0
2~0
30
Incubation timQ (rain)
Fig. 5. Inactivation of human ganglioneuroblastoma enolases. Fractions I, II and 111 from the ion exchange chromatography shown in Fig. 4 were used. A: incubation in the presence of 0.5 M KBr at 7-37 °C. B: incubation at t 50 °C in the absence of KBr. ~, fraction I: ~,, fraction I1; ~3, fraction llI. assumed that the enolase activities in fractions I - I I I c o r r e s p o n d e d to N N E , hybrid form a n d NSE, respectively. This was confirmed by the d e m o n s t r a t i o n that the enolase activity in fraction I was more labile in 0.5 M K B r or at 50 °C, which is in agreement with the greater heat a n d K B r lability of N N E" c o m p a r e d to N SE. F r a c t i o n II showed
Fig. 6. Immunodiffusionof the enolase forms found in human ganglioneuroblastoma. Antiserum against bovine NSE was placed in the central wells. The antigen loads in peripheral wells labeled 1 were : O, 5 fll of bovine NSE (0.4 mg/ml); A, 5 t*1of fraction I (6.1 units/ml); B, 5 pl of fraction II (6.2 units/ml); C, 5 #1 of fraction III (12.8 units/ml). In the peripheral wells 2-5 the antigens were diluted 1:2, 1:4, 1:8 and 1:16, respectively.
77 either intermediate lability or the same lability as the enolase in fraction I, depending on the inactivation conditions. It has been shown that anti-bovine NSE cross-reacts with NSE from other species 14. Fig. 6 shows an immunodiffusion experiment, with the three enolase fractions from the ion exchange chromatography, analyzed against anti-bovine NSE. The same number of enolase units from fractions I and II and approximately twice as much of fraction 11I were applied giving a strong immunoprecipitate with fraction III, still present at a 1:16 dilution of the antigen. The second fraction gave a weaker immunoprecipitate visible down to a 1:2 dilution, while no immunoprecipitate could be detected with the first fraction. The reproducability of the NSE measurements was controlled by running a series of samples from the brain of a 37-week-old fetus. The calculated NSE levels were 60 ± 4'!~,;(n - 6), but in experiments where the same sample batch was used and all the chromatographical and assay procedures were done in parallel, the variations in calculated NSE levels were less than ~ 1 o//o. Effect o f KCI and NaCI on enolase activity Initially, NaC1 was used to form the salt gradient during the ion exchange chromatography. However, NaC1 inhibited the enolase activity and was therefore replaced by KCI. As shown in Fig. 7, KC1 had much less influence on the enolase activity than NaC1 at similar concentrations. For example, the KCI concentration in fraction 1II (0.28 0.33 M KC1) had only a small (8 ~o increase in activity) effect on the enolase activity while NaC1 at the same concentrations decreased the activity to approximately 35 ~ of the activity in the absence of NaCI (Fig. 7). N S E in tissue extracts The relative calculated NSE levels in 6 tumor biopsies, diagnosed as neuroA
C
B
100' ~80
Sz, O C
2O
0
01s
0.'zs
0'.s
0'2s
0'.5
Satt concentration (M)
Fig. 7. Effect of NaCI (;5~)and KCI (×) on the enolase activity. Fractions from the ion exchange chromatography shown in Fig. 4 were used. The fractions were incubated in the presence of different concentrations of NaCI and KCI respectively at ~ 4 °C for 2 h before activity measurement. A: fraction I. B: Fraction 11. C: fraction III.
78 TABLE II
Levels of enolase isoenzymes in human neuroblastoma and]etal brain Soluble extract of the tissues were chromatographed on DEAE-Sephaeel and analyzed for enolase activity. The relative amount of enolase activity in each fraction is given as ~ of totally recovered enolase activity (cf. Fig. I). The calculated NSE value is given in 70 of total enolase activity and is the sum of the amount of enolase in fraction III and half of the amount in fraction II.
Tissue
Enolase activity (units/mg protein)
Neuroblastoma
1 I1 III IV V V] Ganglioneuroblastoma Ganglioneuroma Foetal brain 17 weeks 20 weeks 37 weeks
0.65 1.8 0.84 0.49 0.48 1.4 1.2 1.2 0.78 1.0 1.2
% of total activity Fraction l
11
IH
53 43 32 48 42 58 19 37 43 30 27
32 41 40 32 36 28 37 30 32 31 28
15 16 28 20 22 14 44 33 25 39 45
Calculated NSE % of total activity
31 36.5 48 36 40 28 62.5 48 41 54.5 59
blastoma, ranged from 28 to 48 °/o of total enolase activity with a mean value of 37 °/ /o • The corresponding value for the more differentiated form of the tumor, i.e. ganglioneuroblastoma, was found to be 62 ~o (Table II). Histological examination revealed that, in all 7 biopsies, tumor cells constituted more than 80 ~o of the tissue, the rest being stromal fibroblasts, collagen or necrotic parts (Figs. 1 and 2). The biopsy of the completely differentiated and benign form of the tumor, ganglioneuroma, however, contained mainly fibrous stroma. Less than 20 % of the tissue contained ganglia cells ,
II
0,10
III
57%
E
O.Lg c ,,r"
0.3 "6 ~ 0.05 u ¢tl
8
17%
0.2~ 0.1 ~=
o
c)
UJ
20
60 80 100 E[ution vol.ume (rot)
/~0
120
Fig. 8. Rechromatography of the second fraction from the DEA,E-Sephacel chromatography of human fetal brain. The fraction was dialyzed against 10 m M Tris-HC1 buffer (pH 7.4) containing l m M MgSO4 and frozen at --20 °C before the experiment, which was performed in the same way as the first chromatography.
79 TABLE llI Levels of enolase isoenzymes in cultured human neuroblastoma cells and human glial cells
Soluble extract of the cultured cells, chromatographed on DEAE-Sephacel and analyzed for enolase activity. Enolase and calculated NSE levels are expressed as in Table II. Cells
SK-N-SH* SK-N-SH ! NGF* SK-N-SH ~ db-cAMP* SH-SY5Y** SH-SY5Y ~ NGF** SH-SY5Y + db-cAMP** SK-N-MC IMR-32 787 CG (human gila)
Enolase activity (un#s/mg protein)
3.0 2.8 4.1 2.1 1.8 2.5 3.9 1.9 0.92
%, of total activity Fraction 1
11
111
83 76 78 81 79 71 95 76 89
17 24 22 18 18 29 5 20 11
---1 3 --4 --
CalculatedNSE, % of total activity
8.5 12 11 10 12 14.5 2.5 14 5.5
* Measured in parallel experiments. ** Measured in parallel experiments. (Fig. 3). The relative calculated N S E level in this biopsy was, despite this sparsity of ganglie ceils, 48 ° o (Table I1). The relative NSE activity in normal fetal brains at different stages of development increased with age (Table I1), as could be expected 7. F o r each biopsy the total level ofenolase per mg protein was determined. The activities varied between 0.5 and 1.8 units/rag protein, but there seemed to be no correlation between the total activity of enolase and the relative NSE activity. R e c h r o m a t o g r a p h y o f the hybrid form (fraction II) on D E A E - S e p h a c e l after dialysis and homogenization in the same way as the tissue extracts were treated gave three fractions of enolase activity at positions in the c h r o m a t o g r a m corresponding to N N E , hybrid and NSE (Fig. 8). It seems as if the level o f hybrid is affected by the treatment of the sample, and it has been suggested that the hybrid is an artefact due to the storage conditions and preparation procedures 17. Hybrid and thus NSE-levels might then be dependent on how the tissue extracts have been prepared. I f instead a calculated NSE value, i.e. the a m o u n t of NSE and half of the a m o u n t o f hybrid given as per cent o f total enolase activity, is used, the comparison o f N S E levels in tissues are not dependent on how the tissues were handled assuming the same specific activity for NSE and the hybrid. The calculated N S E levels for the analyzed tissues are given in Table 1I. N S E in cultured cells
Cultured h u m a n neuroblastoma cells also showed varying relative NSE activities (Table IIl) but they were always lower than in the neuroblastoma biopsies (Table II). The lowest level o f NSE was found in the cholinergic neuroblastoma S K - N - M C cell line. Small amounts o f NSE were also found in cultured h u m a n glial cells. The NSE in
80 the cultured cells were mainly found in fraction II (hybrid) and not in fraction Ill (NSE). After treatment with N G F or db-cAMP, the adrenergic neuroblastoma cell line SK-N-SH and the clone SH-SY5Y changed their growth pattern from growth in dense colonies to growth as single cells. The growth was not markedly affected by N G F while db-cAMP caused some cell death. Only a small proportion of the cells showed morphological changes, suggestive of induced differentiation. These cells had thin plasma membrane projections with a length 2-5 times that of the control cells. N G F and db-cAMP induced a small (20-45 °/o) but significant increase in the relative amount of NSE in both SK-N-SH and SH-SY5Y cells detected as increased enolase activity mainly in the hybrid fraction (Table 1ll). DISCUSSION The hybrid enolase can apparently dissociate and reassociate into all three forms under the same conditions as we use for preparation of the tissue and cell extracts. Fletcher et al. 7 showed that maturation of the rat brain and thus expression of NSE initially was reflected as an increase in the hybrid level and later also in the NSE level. Evidently the amount of hybrid must also be taken into account when the total relative NSE levels of different tissues are compared. The calculated NSE values given in Tables II and III are based on the assumption that the specific activity of NSE, N N E and the hybrid is the same. This has not been shown for the human system but Marangos et al. la have shown that the rat brain enolase forms have similar kinetic parameters. Furthermore, Hullin et al. s found good agreement between enolase activity and immunoreactive material of fractions II and III assuming that half of the enolase activity from the hybrid was contributed by NSE. In animal systems as well as in man, there is a correlation between the stage of maturation of the brain and the level of NSE 5,7. The present results suggest that the same correlation holds also for the various forms of human neuroblastoma, In tumors, histopathologically diagnosed as partly or fully differentiated (ganglioneuroblastoma and ganglioneuroma, respectively) the relative NSE levels were higher, if the low content of gangliecells in ganglioneuroma are taken into account, than in the nondifferentiated ('true' neuroblastoma) form of the tumor. However, the number of analyzed ganglioneuroblastoma and ganglioneuroma is still limited and as the possibility exists that these cases are not representative for differentiated forms of neuroblastoma in general this conclusion should merely be considered as a strong suggestion. Absolute enolase levels are not a proper basis for comparison since the biopsies are heterogeneous with respect to the extent of necrosis and infiltration of non-neoplastic cells (Table I). We have instead compared the relative enolase levels which probably are not seriously effected by a variation in necrosis but still leave us with a varying contribution from infiltrating cells. The variation in NSE levels between the different neuroblastoma biopsies might be explained by differences in necrosis or presence of non-neoplastic cells, but could also reflect an existing heterogeneity within the histopathological entity neuroblastoma not detectable by current morphological
8l methods. Quantitation of enolases would, if this assumption is true, be of possible clinical use as a mean to further subclassify neuroblastomas. To further investigate the correlation between NSE and differentiation of neuroblastoma cells we performed controlled studies with cells of human neuroblastoma in vitro cell lines. Compared to the biopsies, the cultured cells had very low NSE levels. A question that arises is therefore if the established cell lines might originate from neuroblastoma cells with low NSE levels or if the cells have diminished their capacity for NSE production as a consequence of prolonged in vitro culture. The latter possibility is favored by the fact that a number of other enzymes have lower levels of activities in cultured than in freshly isolated neuroblastoma cells z3. The NSE level in the cultured neuroblastoma cells increased significantly when the cells were treated with known inducers of differentiation like N G F and db-cAMP. Since only a small fraction of the cells showed morphological changes suggestive of differentiation, the result could be explained by either a small increase in the amount of NSE in a majority of the cells or a large increase in the few differentiated cells. It has been reported that the NSE level in cultured mouse neuroblastoma cells is lower in logarithmically growing than in confluent cell monolayers 10. We have not found this difference in the human cell lines SK-N-SH and SH-SY5Y (unpublished data). ACKNOWLEDGEMENTS
We wish to thank Mrs. Aino Ruusala and Mrs. Karin Elenbring for skilful technical assistance. The work was supported by the Swedish Natural Science Research Council, 'H.K.H. kronprinsessan Lovisas f6rening f6r barnasjukvS.rd', 'Ollie och Elof Ericssons stiftelse' and the Swedish Cancer Society.
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