Neuronal ‘differentiation’ of murine neuroblastoma cells induced by neocarzinostatin: neural cell adhesion molecules

Neuronal ‘differentiation’ of murine neuroblastoma cells induced by neocarzinostatin: neural cell adhesion molecules

Brain Research, 613 (1993) 123-131 123 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00 BRES 18875 Neuronal 'differ...

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Brain Research, 613 (1993) 123-131

123

© 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

BRES 18875

Neuronal 'differentiation' of murine neuroblastoma cells induced by neocarzinostatin: neural cell adhesion molecules Jeffrey A. Lowengrub

a,b a n d N i n a F e l i c e S c h o r b,c,d

Departments o f a Psychiatry, b Pediatrics, c Neurology, and d Pharmacology, University of Pittsburgh, Pittsburgh, PA 15213 (USA)

(Accepted 29 December 1992)

Key words: Neuroblastoma; Differentiation; Neural crest; Neocarzinostatin

Neural crest tumor cells which have been pharmacologically induced in culture to undergo neuronal 'differentiation' have been proposed as a model for normal neural crest cell differentiation. We have previously reported that murine neuroblastoma cells treated with the antineoplastic agent neocarzinostatin (NCS) adopt the light microscopic appearance of differentiated neurons. After undergoing morphologic change, the cells no longer divide. As part of an effort to compare the process of differentiation in these cells with what is known about normal neural crest cells, we have examined the cellular distribution and isoform complement of neural cell adhesion molecules (NCAMs) in native and NCS-treated neuroblastoma cells. Our studies show that NCS induces profound changes in NCAM distribution. Immunohistochemical staining indicates that, in contrast to native neuroblastoma cells, more than 80% of treated cells display surface NCAM by 4 days following treatment. Unlike the case for normal neurons, NCAM is uniformly distributed over the treated cell surface. Neuroblastoma cells treated with NCS are more avidly adherent to culture plates coated with NCAM than are control neuroblastoma cells, reflecting the homophilic binding characteristics of NCAM. Interestingly, Western blot analysis for NCAM demonstrates similar total cellular content of a single NCAM species in both control and treated neuroblastoma cells. Furthermore, this 120 kDa mol. wt. NCAM is an isoform of NCAM not found on normally differentiated cerebellar neurons. While the presence of NCAM on these treated murine neuroblastoma cells is evidence for 'differentiation' along neuronal lines, the isoform complement and cell surface distribution of NCAM in treated cells are not normal. The change in surface staining for NCAM without a change in the total amount of N C A M suggests a redistribution of N C A M within the cell. This is supported by our finding that, while untreated cells do not stain superficially for NCAM, permeabilization of these cells leads to marked staining, implying that, upon treatment, cytoplasmic NCAM is translocated to the membrane.

INTRODUCTION

n e o c a r z i n o s t a t i n (NCS), r e s e m b l e m o r e closely differ-

t o n ~ p r o p o s e d that d i f f e r e n t i a t i o n - i n d u c t i o n could be

e n t i a t e d sympathetic n e u r o n s t h a n do their u n t r e a t e d c o u n t e r p a r t s . T h r e e days after a 1-h exposure to NCS, the cells d o u b l e in size, exhibit m u l t i p l e p r o m i n e n t nucleoli, a n d e x t e n d at least 3 processes. Like n o r m a l n e u r o n s , in the a b s e n c e of a d d e d n e r v e growth factor, N C S - t r e a t e d n e u r o b l a s t o m a ceils die within two weeks of c u l t u r e 11. I n a n effort to d e t e r m i n e the biochemical correlates of N C S - i n d u c e d morphological change, we have c o m p a r e d N C S - t r e a t e d n e u r o b l a s t o m a ceils a n d n o r m a l m a t u r e n e u r o n s with respect to their total c o n t e n t , isoform c o m p l e m e n t , a n d cellular d i s t r i b u t i o n of n e u r a l cell a d h e s i o n molecules (NCAMs).

used as a t h e r a p e u t i c modality for the t r e a t m e n t of n e u r o b l a s t o m a . W e have r e p o r t e d that m u r i n e n e u r o b lastoma cells t r e a t e d with the a n t i n e o p l a s t i c agent,

N C A M s are a family of m o l e c u l e s involved with the m i g r a t i o n of n e u r a l a n d glial cells, a n d the establishm e n t of stable c e l l - c e l l contacts in the n e r v o u s system.

N e u r o b l a s t o m a is a t u m o r which arises from cells of the n e u r a l crest. O n e theory as to its p a t h o g e n e s i s suggests that the t u m o r cells are d e v e l o p m e n t a l l y arrested n e u r o b l a s t s 16. N e u r o b l a s t o m a cells can b e p h a r macologically i n d u c e d in c u l t u r e to u n d e r g o m o r p h o logic c h a n g e s which r e s e m b l e by light microscopy those seen in n e u r o n a l d i f f e r e n t i a t i o n 9'11'~2. This f i n d i n g has led to the suggestion that the i n d u c t i o n of these c h a n g e s in n e u r o b l a s t o m a cells could b e used to p r o d u c e a m o d e l of n o r m a l d i f f e r e n t i a t i o n 15. I n addition, P i n k e r -

Correspondence: N.F. Schor, Children's Hospital of Pittsburgh, 3705 Fifth Avenue, Pittsburgh, PA 15213, USA. Fax: (1) (412) 692-5723.

124 In mature central nervous system tissue, NCAM exists in three discrete forms with molecular weights of 120, 140, and 180 kDa. In embryonic brain, NCAM is polysialated, and can have tool. wts. in excess of 200 kDa 1°. Recent studies have suggested that a more 'mature' phenotype in both murine and human neuroblastoma cells is associated with a shift in NCAM type from the 140 kDa form to the 180 kDa form 7, and with a shift in NCAM localization from diffuse over the cell surface to restricted to cell-cell contact sites 4. We present herein evidence that murine neuroblastoma cells induced by NCS to undergo morphological alteration exhibit a change in the distribution of NCAM in and on the cell, without a change in total NCAM expression or molecular weight distribution. Further, we present evidence consistent with either a translocation of NCAM from the cytoplasm to the membrane or a change in the accessibility of membrane-associated NCAM to antibody staining in these cells. MATERIALS AND METHODS Chemicals and immunochemical reagents NCS was obtained as a sterile solution (0.5 m g / m l ) in 0.015 M sodium acetate buffer, pH 5, from Dr. William T. Bradner (Bristol Myers-Squibb, Wallingford, CT). Purified murine N C A M and polyclonal antibodies raised in rabbits to murine N C A M were gifts from Dr. Carl Lagenaur (University of Pittsburgh, Pittsburgh, PA). The characterization of these antibodies has been performed by both Western blot analysis and developmental histochemical study 2. Polyclonal antibodies to tubulin Polysciences (Warrington, PA). Monoclonal antibodies to neurofilament 200 kDa protein (clone RT97) were obtained from Boehringer-Mannheim (Indianapolis, IN). Biotinylated goat antibodies to rabbit lgG and mouse IgG ('secondary' antibody), horseradish peroxidase-streptavidin conjugate, aminoethyl carbazole (chromogen), 3% hydrogen peroxide, hematoxylin, and aqueous mounting solution for immunohistochemical staining were obtained as a Histostain-SP Immunoperoxidase Stain Kit from Zymed Laboratories, Inc. (San Francisco, CA). E D T A and the protease inhibitor PMSF were obtained from Sigma Chemical Corporation (St. Louis, MO). The non-ionic detergent, NP-40, was obtained from Calbiochem (San Diego, CA).

treated ceils were stained for the presence of N C A M at 24, 96, and 168 h after treatment with polyclonal rabbit anti-mouse N C A M antibodies using a Histostain-SP kit (Zymed Laboratories, Inc., San Francisco, CA). Briefly, cells were fixed for 10 min at room temperature with 4% paraformaldehyde, and then washed in phosphatebuffered saline (PBS). Where indicated in the text, cells were permeabilized by treatment with NP-40 (1%, w / v , for 2 min) and rewashed with PBS. The coverslips were then submerged for 10 min in peroxidase blocking solution (30% hydrogen peroxide:methanol::1:9), and washed with PBS. The cells were covered with serum blocking buffer (10% horse s e r u m / l % BSA) for 10 rain. Polyclonal rabbit antimurine N C A M antibodies (1/250 dilution)were put on each coverslip and allowed to sit at room temperature for 20 min. The coverslips were rinsed in PBS and covered with biotinylated goat anti-rabbit IgG for 10 min. Again cells were rinsed in PBS and incubated for 5 rain with the peroxidase-strepavidin conjugate. Cells were then washed with PBS and covered with substrate chromogen mixture for 15 min. Distilled water was used to wash cells prior to counterstaining with hematoxylin and mounting on glass slides. Control slides for each treatment condition at each timepoint were simultaneously prepared in the absence of primary antibody to NCAM. For each slide, three sets of 100 adjacent cells each were graded as 'positive' or 'negative' for peroxidase staining by an observer blinded to the treatment group from which the slide came. Details of the criteria for designation as 'positive' or 'negative' are given in the Results section. The results were expressed as the average percentage of cells staining positively ( + S.E.M.) in a given set of 100 cells. Adherence of native and NCS-treated C1300 cells to NCAMcoated plates: because N C A M is known to undergo homophilic binding, and to mediate cellular adhesion to matrices and other cells harboring N C A M on their surfaces, we examined the adherence to NCAM-coated tissue culture plates of native and NCS-treated C1300 cells. Plastic petri dishes (60 m m diameter) were coated with nitrocellulose and this coating was overlain in discrete spots with murine N C A M by the technique of Lagenaur and L e m m o n 5. C1300 cells (1 × 106 cells/dish) were allowed to adhere to these plates at 37°C for 24 h. The plates were gently washed free of non-adherent cells with fresh medium. The n u m b e r of adherent cells for each of three high-power fields was then counted for the area of the plate which had been coated with nitrocellulose alone and for the area of the plate coated with N C A M on nitrocellulose.

100-

80-

Z

• Control [] NCS-~eated

60"

Cell culture Murine neuroblastoma cells of the C1300 line (NB41A3 subline) were obtained from the American Type Culture Collection (Bethesda, MD), and maintained at 37°C as adherent cultures in F-10 Nutrient Mixture supplemented with 15% horse serum and 5% fetal calf serum (GIBCO, Grand Island, NY). For transfer of cells from stock flasks to six-well plates, cells were gently scraped off of the flask surface with sterile cell scrapers (Sarstedt, Newton, NC), and allowed to stabilize on the plate overnight before studies were performed. T r e a t m e n t of neuroblastoma cells with NCS: for studies involving NCS treatment, neuroblastoma cells were transferred to and stabilized on sterilized glass coverslips (12 m m diameter; Fisher Scientific, Pittsburgh, PA) which had been placed into six-well tissue culture plates. Each well contained approximately 4 x 10 s cells and 2 coverslips. Cells were treated with either NCS (0.5 p . g / m l in complete medium; 'treated cells') or an equivalent volume of complete medium ('control cells') for 1 h at 37°C. Cells were washed twice with complete medium (1 ml/wash), and then incubated with 3 ml of complete medium for the duration of the study. Immunoperoxidase staining: coverslips containing control and

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Day Fig. 1. Percent of control and NCS-treated C1300 cells which stain positively for NCAM. Cells were plated on glass coverslips and treated on day 0 with control medium or with NCS ( 0 . 5 / z g / m l ) for 1 h at 37°C. They were then fixed and stained with a polyclonal antibody to murine N C A M under non-permeabilizing conditions, as detailed in the Materials and Methods section. On days 1, 4, and 7, the percent of the cells staining positively for NCAM in each of three sets of 100 adjacent cells were determined for control and NCStreated cells. The figure shows the mean percentage for each treatment group on each day in a representative experiment. Error bars represent the S.E.M. of the three determinations performed on each slide.

125 Western blot analysis of NCAM in control and treated C1300 cells: Neuroblastoma cells for Western blot analysis of NCAM expression were maintained and treated in 75 cm 2 tissue culture flasks. Two flasks of cells at approximately half-confluence (0.5X10 7 cells/flask) were treated with 0.5 p.g/ml neocarzinostatin for 1 h at 37°C ('treated' cells). Two sister flasks were treated with an equivalent volume of medium alone ('control' cells). All cells were washed twice with complete medium and incubated at 37°C for 7 days. On day 7 after treatment, the medium was removed from each flask and all cells were washed twice with PBS and then gently scraped off of the flask surface into PBS. Cells from the two control flasks were pooled, as were cells from the two treated flasks. Cells for each treatment were counted by hemacytometry, and 1.4× 106 cells from each treatment group were used for the remainder of the experiment. All subsequent preparative steps were performed at 4°C. Cells

were pelleted by centrifugation (1,300 rpm for 5 min), and the pellet was taken up in 50 /xl of PBS made 0.2 mM in PMSF (added as a stock solution in isopropanol) and 1 mM in EDTA. Cells were homogenized and centrifuged at 14,000 rpm for 30 min to remove nuclei. The supernatant from each preparation (control and treated) was boiled in sample buffer, applied to an SDS-polyacrylamide gel (stacking ge1+7.5% lower gel), and electrophoresed along with prestained high mol. wt. standards (Bio-Rad, Redmond, CA) using standard techniques. As a positive control, the whole brain of a male 5-week-old A / J mouse was homogenized in 5 ml of PBS containing 0.2 mM PMSF and 1 mM EDTA, and 50 jzl of this homogenate were applied to the same gel and simultaneously electrophoresed. Transfer to nitrocellulose membranes was performed using a Trans-Blot system with overnight elution (Bio-Rad). Staining of the nitrocellulose membrane was performed using an Immunoblot-SP Kit (Zymed,

Fig. 2. Photomicrographs of control (A,B) and NCS-treated (C,D) C1300 cells stained for NCAM using a polyclonal antibody and the immunoperoxidase method under non-permeabilizing conditions (see Materials and Methods). Treatment was performed on day 0; fixation and staining were performed on day 7. Cells shown in panels A and C were stained in the absence of the anti-NCAM antibody, and serve as negative controls. Note that the magnification for all photomicrographs shown is 133 ×. Cells which have undergone morphologic change in response to neocarzinostatin treatment are two- to four-times as large as untreated cells, and elaborate at least three processes11.

126

Fig. 2 (continued).

San Francisco, CA) and a 1/500 dilution of the same polyclonal rabbit anti-murine NCAM antibodies used for histochemical staining. RESULTS Immunohistochemical studies of N C A M on nonpermeabilized native and NCS-treated C1300 cells: We have previously reported that C1300 cells treated with NCS increase in size, elaborate cell processes, and develop multiple nucleoli by 4 days in culture. In addition the cell culture growth rate is markedly reduced by NCS treatment 11. Because N C A M is thought to participate in cell-cell interaction, and because

N C A M expression changes over the course of neuronal differentiation, we were interested in the effects of NCS treatment upon N C A M expression and distribution in C1300 cells. Immunoperoxidase staining of C1300 cells for N C A M was performed at several timepoints in native cells and in cells which had been treated with NCS as described in Materials and Methods. Cells were not permeabilized, and staining was presumed to represent cell surface NCAM. In all cases, stained cells were either completely blue or blue containing one or two single dark red-brown granules, or covered over more than 50% of their surface with dark red-brown granular aggregates. No cells of intermedi-

127 4O rl

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NCS-Tteated

Fig. 3. Adhesion of control and NCS-treated C1300 neuroblastoma cells to nitrocellulose plates coated with murine N-CAM. The 'adherence ratio' is defined as the mean number of cells per high-power field adherent to the N-CAM spot on the nitrocellulose plate, divided by the mean number of cells per high-power field adherent to the uncoated nitrocellulose background on the same plate. Adherent cells were counted after gentle washing of the plate with serumfree F-10 Nutrient Mixture (Gibco, Grand Island, NY). The results of four separate experiments are shown.

ate staining were seen. For this reason, cells of the stain-covered type were designated as positive; cells which which were completely blue or which contained only a single granule of red-brown stain were considered negative. Minimal staining was seen in both control and treated cells 24 h after treatment (9 + 3% control vs. 3 + 5% treated). In contrast, by 96 h, significantly more treated cells than control cells stained positively (80 + 12% treated vs. 2 + 2% control, P <

0.001, Student's t-test; see Fig. 1). The staining of individual cells became more intense by 168 h (data not shown), although the percentage of cells staining positively remained the same. Fig. 2 illustrates that the staining of treated cells was evenly distributed over the cell surface, and not simply at cell-cell contact points. In addition, cells from each treatment group processed simultaneously with non-immune serum do not stain with immunoperoxidase, indicating that the staining seen in treated cells is not the result of a non-specific reaction of the stain with the cells or with adherent reagents from the staining process, or of the serum with other cellular components. Adhesion of native and NCS-treated C1300 cells to NCAM substrata: to provide a functional assessment of the level of cell surface NCAM on treated and untreated C1300 cells, these cells were plated on nitrocellulose-coated plates spotted with NCAM. It has previously been shown that NCAM undergoes homophilic binding 3, making it likely that cells with surface NCAM will adhere more readily to NCAM-coated nitrocellulose than to nitrocellulose alone. This was the case with C1300 cells treated with NCS. As is shown in Fig. 3, the preferential adherence of NCS-treated C1300 cells to NCAM-coated nitrocellulose was between 5and 15-fold higher than that of untreated cells. This corroborates the above finding that more NCAM is

Fig. 4. Western blots for N-CAM of nucleus-free homogenates of control and NCS-treated C1300 neuroblastoma cells on day 4 and day 7 after treatment. NCS treatment involved a 1-h exposure to 0.5/zg/ml NCS on day 0. The anti-murine N-CAM antibody was prepared in rabbits 2. The positions of mol. wt. markers run on the gel are indicated in the center. Whole routine brain electrophoresed and blotted with the same antibody gives three distinct bands at 180, 140, and 120 kDa mol. wt., corresponding to the three known isoforms of NCAM (data not shown).

128

129 accessible on the surface of C1300 cells after treatment with NCS. Western blot analysis of NCAM in native and NCStreated C1300 cells: the histochemical and functional studies presented above do not distinguish between an increase in the total amount of NCAM present in the cells and a change in the binding of antibody or NCAM to NCAM on the cell surface. The latter situation could result from changes in the relative orientation or amount of NCAM in the membrane or in the relative amounts of each of the NCAM isoforms present. To address this issue, Western blots were performed on nucleus-free homogenates of C1300 neuroblastoma cells 4 or 7 days after exposure to either control conditions or NCS (0.5 ~ g / m l for 1 h), using the same antibody as was used in the immunohistochemical staining. As is shown in Fig. 4, these studies show no change in the absolute amount of NCAM present per cell, or in the mol. wts. of the NCAM species present. Furthermore, in both treated and control cells, only the 120 kDa species is present. Simultaneous blotting of mouse whole brain homogenate run on the same gel gave three distinct bands at 180, 140, and 120 kDa mol. wt., as has previously been reported for this antibody 2 (data not shown). These results imply that the immunohistochemical and functional differences seen in the whole cells are the result of either redistribution of NCAM within the cell or 'unmasking' of NCAM; the latter situation could be due to changes in the configuration of the cell membrane or alterations in the relationship of other membrane macromolecules to the haptens involved in polyclonal antibody and NCAM binding to NCAM. Immunohistochemical studies of NCAM in permeabilized native and NCS-treated C1300 cells: To test the hypothesis that the change in NCAM staining characteristics after NCS treatment is the result of translocation of cytoplasmic NCAM to the membrane, we compared non-permeabilized and NP-40-permeabilized native and NCS-treated C1300 cells with respect to staining for NCAM. As is shown in Fig. 5, native cells become positive for NCAM staining after permeabilization. The staining of NCS-treated cells does not change after permeabilization (see Table I). Neither permeabilized nor non-permeabilized native or treated

TABLE I

Immunohistochernical staining of native and NCS-treated C1300 cells with antibodies to cell surface and cytoplasmic antigens under non-perrneabilizing and permeabilizing (NP-40) conditions. Details of methods for NCS treatment and staining are given in the Materials and Methods section. Fixation and staining were performed on day 7 after NCS treatment. In all cases, simultaneous processing with n o n - i m m u n e serum resulted in no immunoperoxidase staining.

NCAM Tubulin Neurofilaments

Non-permeabilized

Permeabilized

Native

NCS-treated

Native

NCS-treated

ND ND

+ ND ND

+ + -

+ + -

ND: Not done, since these are cytoplasmic antigens which do not appear on the surface of cells.

cells stain after treatment with non-immune serum (data not shown). In contrast, neither treated nor native permeabilized cells stain with a monoclonal antibody to neurofilament protein (200 kDa). Staining with antitubulin antibodies results in equivalent staining of treated and untreated permeabilized cells (see Table I). These resuits indicate that the change in NCAM staining with permeabilization of native C1300 cells, but not with permeabilization of treated cells, is not a non-specific effect of permeabilization upon stain uptake. DISCUSSION

Previous studies have shown that neural crest tumor cells can be induced in culture to Undergo morphological changes which resemble those seen during the course of normal differentiation9'ta. It has been suggested that this system could be used as a model to study normal neural crest development tS. The validity of this system as a model depends critically upon its physiological and biochemical resemblance to normal neural crest cells. The present study examines the expression of NCAMs by native neural crest tumor cells and by neural crest tumor cells which have been induced to undergo morphological 'differentiation' with the antimitotic agent, NCS. They suggest that morphological equivalence does not necessarily imply the bio-

Fig. 5. Photomicrographs of control non-permeabilized (A) and permeabilized (B) C1300 cells stained for N C A M using a polyclonal antibody and the immunoperoxidase method (see Materials and Methods). T r e a t m e n t was performed on day 0; fixation and staining were performed on day 7. The magnification for both photomicrographs shown is 200 x . Permeabilization with NP-40 results in staining of these control cells with the antibody. In contrast, both non-permeabilized and permeabilized NCS-treated cells stain avidly with this antibody (see Table I). Simultaneous processing with n o n - i m m u n e serum results in no staining under any of the experimental conditions (data not shown).

130 chemical or physiological similarity of this system to normal neural crest differentiation. In normal brain, NCAM exists in 120, 140, and 180 kDa mol. wt. forms. While 140 mol. wt. NCAM is distributed over the cell bodies of differentiating cerebellar cells, NCAM 180 is found at the growth cones of cells in contact with their targets. This differentiation at once results in an increase in the total amount of cellular NCAM, a change in the cellular prevalence of the individual isoforms of NCAM, and a change in the subcellular distribution of NCAM. This is thought to result in homophilic binding between cells ~°. The present study examines cell surface NCAM by antibody staining under non-permeabilizing and permeabilizing conditions. When treated with NCS, murine neuroblastoma cells display more cell surface NCAM as seen by immunoperoxidase staining, and show increased adhesion to NCAM spotted on nitrocellulose plates. These findings give both chemical and functional evidence of increased cell surface NCAM on C1300 cells after NCS treatment. Despite this feature which resembles superficially the changes seen during neuroblast maturation, the NCAM isoform displayed on the surface of both native and treated murine neuroblastoma cells is the 120 kDa species. Furthermore, the total amount of NCAM in the treated cells is not different from that of the untreated murine neuroblastoma cells, and the NCAM of treated cells is distributed uniformly over the surface of the cell. Thus, while the light microscopic changes seen in these cells resemble differentiation, the biochemical changes which accompany them are not the same as those seen in normal developing neurons. Previous studies have reported the appearance of the 140 kDa and 180 kDa mol. wt. NCAM isoforms in neuroblastoma cells, especially after treatment with retinoic acid or dibutyryl-cAMP6'8. These studies differ from ours in the use of different species and sublines of neuroblastoma cells and of different pharmacological agents to induce morphological change in the cells. Permeabilization of native cells leads to avid staining with the polyclonal antibody for NCAM, supporting the notion that something other than total NCAM changes after NCS treatment. Several mechanisms would explain the discrepancy between the antibody staining characteristics of NCS treated cells relative to native cells and the lack of change in the absolute amount of NCAM present after treatment. One possibility is that both treated and native cells produce NCAM, but that the NCAM produced by native cells is not inserted into the membrane. This could result from structural aberrancy of the NCAM produced, from failure to produce a signal-bearing NCAM precursor,

or from membrane aberrancy which precludes insertion of NCAM into the native C1300 cell membrane. In any case, one would have to postulate that NCS 'corrects' the abnormality or produces a compensatory one, allowing NCAM to be placed in the membrane of treated cells. Alternatively, the configuration of NCAM or of surrounding proteins in the membrane may differ between native and treated cells, such that surface NCAM is 'unmasked' by treatment. A third and more intriguing possibility is that NCS could trigger a cellular 'program' which has as one of its components the redistribution of cellular macromolecules, including NCAM. Although this latter scenario is perhaps the most interesting of the three, the present studies do not directly distinguish among these possibilities. In all three cases, membrane permeabilization with NP-40 might increase staining. However, preliminary data obtained in our laboratory indicate that treatment of C1300 cells with cycloheximide (10 /xg/ml) before, during, and after treatment with NCS does not impede the formation of processes by these cells. This would imply that new protein synthesis is not necessary for the elaboration of processes in response to NCS treatment, favoring a mechanism where NCAM is 'unmasked' or translocated. The induction of morphological changes which resemble maturation, and redistribution of macromolecules associated with such maturation, without an increase in the synthesis of such macromolecules, has also been reported for GAP-43 in neuroblastoma cells induced with dibutyryl-cAMP to undergo neuritogenesis 13. Preliminary studies in our laboratory indicate that NCAM might not be the only surface molecule which undergoes a change in surface abundance. The adhesion-related molecule L1 exhibits increased staining in permeabilized cells after NCS treatment, although it is present on the surface of native cells (Lowengrub, Belz, Lagenaur, and Schor, unpublished data). Associations between NCAM and L1 have been previously reported 8. The use of a murine cell line in our initial studies stemmed from the ready availability of a C1300-A/J mouse model in which to test the in vivo effects of NCS on neuroblastoma. We have recently begun extending our findings in murine cells to human neuroblastoma lines. Our preliminary data for the cell lines SK-N-SH, SK-N-MC, and IMR-32 indicate that, as is the case for C1300 cells, NCS does not change the quantitative expression of the various isoforms of NCAM in these cells (Hartsell and Schor, preliminary results). In contrast to this, retinoic acid and phorbol esters induce similar morphologic differentiation in human neuroblastoma cells with a concomitant increase in the

131 expression of NCAM 6 and a decline in the expression of N-myc mRNATM. The N-myc oncogene codes for a protein the level of expression of which has been shown to correlate inversely with the favorableness of prognosis in patients with neuroblastoma7. Transfection studies indicate that expression of N-myc in rat neuroblastoma cells leads to a dramatic reduction in the expression of NCAM by these cells 1. Our studies show that, at least in murine neuroblastoma cells, NCS treatment is not accompanied by changes in NCAM levels. If this is true of NCS-treated human neuroblastoma cells, as well, it would suggest that NCS induces differentiation through a proximate mechanism distinct from those of retinoic acid or phorbol esters. Additional evidence that other pharmacologically induced states which have been termed 'differentiation' differ from that induced by NCS has recently been obtained in our laboratory. The 'differentiation' induced by nerve growth factor in nerve growth factor receptor-positive neuroblastoma cell lines results in somatic and process morphology which is significantly different, by both light and electron microscopy, from that induced by NCS (Falcione, Gilbert, and Schor, preliminary results). Determination of the molecular events which underlie these differences awaits further study. Acknowledgements. Funded by grants to N.F.S. from the American Cancer Society (CH-446) and the National Institutes of Health (CA-47161). The authors wish to thank Drs. Carl Lagenaur and Aviva Abosch for many helpful scientific and technical discussions. Thanks are also due to Mark Belz and Theresa Hartsell for expert technical assistance.

REFERENCES 1 Akeson, R. and Bernards, R., N-myc down-regulates neural cell adhesion molecule expression in rat neuroblastoma, Mol. Cell. Biol., 10 (1990) 2012-2016.

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