Neuropathology and Applied Neurobiology 199 I , 17,201-2 17

Expression of alternative isoforms of the neural cell adhesion molecule (NCAM) on normal brain and a variety of brain tumours G. FROST*, K. PATEL*, S. BOURNE?, H. B. C O A K H A M * t A N D J. T. K E M S H E A D * *The Imperial Cancer Research Fund, Paediatric and Neuro-oncology Group and Brain Tumour Research Fund, ?Department of Neurosurgery, Frenchay Hospital, Bristol

FROST G., PATELK., BOURNES., COAKHAM H. B. & KEMSHEAD J. T. (1991) Neuropathology and Applied Neurobiology 17,207-2 I 7 Expression of alternative isoforms of the neural cell adhesion molecule (NCAM) on normal brain and a variety of brain tumours

A panel of monoclonal antibodies, including a reagent designated ERIC-1, have been characterized as binding to the human neural cell adhesion molecule (NCAM). These monoclonal antibodies bind in a relatively uniform manner to a variety of normal and neoplastic tissues arising from the neuroectoderm. However, multiple forms of the protein are known to arise from the differential splicing of exons within the NCAM gene located on chromosome 1 1 at q23. On human adult brain, four isoforms of 180, 170, 145 and 120 kDa have been identified. Here, we report the identification of another NCAM isoform of 95 kDa that is apparent on tissues following either N-glycanase or neuraminidase treatment to remove carbohydrate and sialic acid residues from the molecule respectively. NCAM expression is further complicated by differential post-translational modification of the molecule which is developmentally regulated. In general, fetal NCAM is more heavily polysialylated than the adult forms of the molecule. Human fetal brain has been shown to express the heavily sialylated embryonic form of NCAM, but following neuraminidase digestion, a similar pattern of NCAM expression is seen to that in adult brain. A variety of human brain tumours examined also show different patterns of NCAM expression, despite their uniform staining with monoclonal antibodies. The significance of these observations for designing new molecular and immunological approaches to the diagnosis of a variety of primary tumours is reviewed. Keywords: monoclonal antibodies, NCAM, brain tumours, neuroectoderm

INTRODUCTION Over the last decade, monoclonal antibodies have found a role in assisting in the diagnosis of a variety of tumour types. This is of prime importance when conventional morphological studies indicate a particularly anaplastic phenotype. However, monoclonal antibodies present the user Correspondence to: J. T. Kernshead, The Imperial Cancer Research Fund, Paediatric and Neuro-oncology Group, Frenchay Hospital, Bristol BS16 ILE.

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with a unique set of problems not encountered when using conventional polyclonal antisera. Due to their unique specificity, monoclonal antibodies recognize either a single or set of very closely related epitopes. Both quantitative and qualitative heterogeneity in antigen expression can lead to a lack of binding of individual monoclonal antibodies to individual tumour cells. When no binding is observed with a monoclonal antibody, it is often assumed that the antigen detected by the reagent is absent from the cell. This may not be the case, as it may be that theepitope for a particular monoclonal antibody is either missing or masked. It is now clear that individual genes consist of coding regions (exons) interspersed by non-coding areas of DNA (introns). Several different protein products can result from the differential splicing of exons within a single gene and these can subsequently be modified by glycosylation and phosphorylation in different ways. This naturally generates a diverse array of epitopes on a particular protein. If the binding of monoclonal antibodies to such gene products is to be fully understood, it is necessary to obtain a detailed biochemical knowledge of their antigens. Recently, we have characterized a group of monoclonal antibodies raised against neuroectodermally derived cells as binding to NCAM. The expression of this particular protein is highly complex, with several different gene products resulting from the phenomenon of differential splicing (Cunningham er af., 1987). Four major forms of NCAM have been identified in mouse brain. The two largest forms of 180 and 140 kDa are transmembrane proteins which differ primarily in the length of their cytoplasmic tail (Murray ef af., 1986). The 120 kDa isoform is linked into the membrane via a glycosylphosphoinositol(GPI) anchor and the smallest isoform is believed to be a secretory protein (He, Finne & Goridis, 1987). In human brain, an NCAM isoform of 170 kDa has also been identified, but it remains unclear as to how this links into the membrane (Bhat & Silberberg, 1988). NCAM isoforms have also been studied in detail in human skeletal muscle. In this instance, a transmembrane protein of 140 kDa and two GPI linked isoforms of 125and 155 kDa are present. A 115 kDa secretory protein has been identified (Moore er af., 1987; Gower et al., 1988). Whilst NCAM expression has been extensively studied in animals such as the mouse and chicken, less work has been undertaken on the human homologues of the protein. As indicated above, the isoforms of NCAM found on human adult brain and muscle have been characterized but not to the same degree as in the mouse. Furthermore, human fetal tissues have not been examined in detail and no work has been reported on the expression of NCAM on primary brain neoplasms. Here, we report such a study and demonstrate that, underlying a uniform staining pattern with anti-NCAM monoclonal antibodies, a marked diversity in the expression of different isoforms of the molecule exists. The possible biological significance of these findings, along with their relevance for further diagnostic studies, is discussed in detail. MATERIALS A N D METHODS Tissues The samples of normal and tumour tissue were snap frozen in liquid nitrogen and stored at - 70°C. Monoclonal antibodies Monoclonal antibody ERIC- 1 was raised following immunization of mice with a membrane preparation obtained from human retinoblastoma tissue (Bourne et al., 1990). The monoclonal antibody has been shown to bind selectively to the human NCAM using a variety of biochemical and molecular techniques. Briefly, ERIC-1 binds to a pattern of proteins synonymous with that

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seen with purified NCAM. Transfection of 3T3 cells with a full length cDNA clone coding for the 120 kDa isoform of human muscle NCAM results in the expression of the human protein in the murine cells (Gower et al., 1988). ERIC-1 binds specifically to the transfected cells, but not the control 3T3 cells. Western blot analysis of extracts of transfected cells shows that ERIC-1 binds to a 120 kDa protein identical to that seen with a NCAM monoclonal antibody and a polyclonal antiserum to human NCAM (Bourne et al., 1990). Indirect immunofluorescence For indirect immunofluorescence, 5 pm cryostat tissue sections were incubated with monoclonal antibody under conditions of antibody excess. Binding of monoclonal antibody (MAb) was visualized using a fluorescein-conjugated F(ab)2 fragment of goat anti-mouse immunoglobulin (Ig). Samples were analysed using a Zeiss fluorescence microscope with epi-illumination optics. Western blot analysis Tissues were gently disrupted and homogenized in 62.5 mM Tris/HCI pH 6.8 containing 2.5 mM PMSF, 12.5% glycerol, 1.25 mM EDTA, 12.5 pg/ml leupeptin and 2% NP40. The resulting homogenate was centrifuged for 5 rnin at 8OOOg at 4°C and the protein concentration of the supernatant determined using the Bio-Rad Protein Assay (Bio-Rad Laboratories, Munich). Proteins from the lysate were size-separated under reducing conditions by polyacrylamide gel electrophoresis (7%) at 200 V for 3 to 4 h. Transfer of proteins to 0.2 pm nitrocellulose filters was carried out at 134 mA for I t h using the LKB Novablot transfer apparatus. Non-specific protein binding sites on the filter were blocked by overnight incubation in PBS containing 5% Marcel (Cadbury Schweppes, London, UK). Incubation with MAbs was performed for 30 min at room temperature. This was followed by two washes for 5 rnin each in phosphate buffered saline (PBS). The filters were subsequently incubated with alkalinephosphatase-conjugated rabbit anti-mouse Ig for 30 rnin at room temperature. After washing twice with PBS and once with TBS (25 mM Tris, 135 mM NaCI, 5 mM KCI, 1 mM levamisole), binding of the MAbs was visualized by incubating the filter for 15-30 min in the dark with the substrate consisting of 0.2 mg/ml naphthol AS-MX phosphate, 2% N,N-dimethylformamide, 1 mg/ml Fast Red T R in 0.1 M Tris-HC1 pH 8.2 and 1 mM levamisole. The filters were finally washed twice in TBS and air-dried. Enzyme treatment Neuraminidase Appropriate amounts of 1.5 M NaCl and 0.5 M sodium acetate were added to the tissue extracts to give final concentration of 150 mM NaCl and 50 mM sodium acetate. The p H was adjusted to 5.0 usingeither glacial acetic acid or sodium hydroxide. Neuraminidase type X (50 U/ml in PBS) was added to the extracts to give a final concentration of 5 U/ml and the mixture was incubated at 37°C for 5 h. N-glycanase Extracts for treatment with N-glycanase were boiled for 2 min in the presence of 1% SDS. A buffer consisting of 20 mM sodium phosphate pH 7.2, 10 mM NaN,, 10 mM EDTA, 0.5% n-octylglucoside was added to the extract (1 vol. extract:4 buffer) and boiled for a further 2 min. After cooling, 3 U of N-glycanase were added and the solution incubated at 37°C for 18 h. Extracts were subsequently size separated on SDS polyacrylamide gels.

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180 kDa

--

145 kDa

-

120 kDa 95 kDa

-

- 200 kDa

-200 kDa 180 kDa=

-116 kDa -97 kDa

1-1 (+I a

b

c

145 kDa

-

120 kDa

-

-116 kDa -97 kDa

95 kDa-

(-i

(it

d

e

f

Figure 1. NCAM isoforms expressed in human adult brain (grey and white matter). Tissue samples were cut into small pieces and gently homogenized in extraction buffer at 4°C (materials and methods). The resulting extracts were centrifuged to remove debris and subjected to Western blot analysis. NCAM proteins were visualized using the MAb ERIC-I. The data presented here is representative of that seen using at least three different adult brain samples. Lanes: a, NP-40 extract + MAb ERIC-I. b, NP-40 extract neuraminidase treatment + MAb ERIC- I . c, NP-40 extract + irrelevant monoclonal antibody M340. d, NP-40 extract + monoclonal anti-NCAM antibody ERIC- I .e, NP-40 extract + N-glycanase treatment + MAb ERIC- I . f, NP-40 extract + irrelevant MAb M340.

+

RESULTS Indirect immunofluorescence studies

Monoclonal antibody ERIC-I binds to all areas of human fetal and adult brain examined, as determined by indirect immunofluorescence studies on frozen tissue sections. In addition, the MAb reacts with astrocytomas (8/8), ependymomas (5/5), oligodendrogliomas (6/6),medulloblastomas (12/12) and meningiomas (2/3). All tissues subjected to Western blot analysis were checked for binding to ERIC- I prior to the preparation of cell extracts and all shown to bind the MAb. NCAM expression on human brain Western blots of extracts of human brain, containing both grey and white matter, reveal four major bands on staining with the anti-NCAM MAb ERIC-I. These are of 180, 170, 145 and 120 kDa and correspond to the known isoforms of NCAM found on normal human neural tissue. In addition, some weaker bands of 160, 130 and 95 kDa are observed. This microheterogeneity presumably reflects differential post-translational modification of the main NCAM isoforms, which can be both glycosylated and phosphorylated. Neuraminidase treatment to remove sialic acid residues (Figure 1, lane b) and N-glycanase digestion to strip N-linked carbohydrates (Figure I , lane e) reduces this heterogeneity. The 180, 170, 145 and 120 kDa isoforms of NCAM all appear slightly smaller on Western blot anlysis, following removal of carbohydrate residues (Figure 1, Table 1). In addition, another major band of 95 kDa is visualized following either neuraminidase or N-glycanase treatment. This presumably represents another major NCAM isoform, the presence of which is only revealed after carbohydrate moieties are stripped from the molecule. Isolation of both grey and white matter from adult brain and Western blot analysis reveals a broadly similar pattern of NCAM expression in the two extracts. The only difference in the

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Table 1. The differential expression of NCAM isoforms in normal neural tissue and malignant brain tumours Tissue

Normal Adult brain Fetal brain Meninges Malignant Astrocytoma Medullo blastoma Meningioma Oligodendroglioma Schwannoma

Major NCAM isoforms detected (kDa)

NP-40 extracls

+ Neuraminidase

180-170--145-120

175-1 65-1 40- 120-95 175-1 65-140-1 20-95 140-120-95

Smear* 165-135-125

145-12s

Smear 165-1 35-1 25 180-145-125

Smear

140-120-95 175-165-140-120-95 140-120-95 175-140-120-95 140-120-95

~

*This represents the embryonic form of NCAM which is heavily glycosylated causing the proteins to smear on the blots

NCAM isoforms noted is a diminution in the intensity of the 180 and 170 kDa proteins observed in white matter, along with a concomitant increase in the 120 and 140 kDa isoforms (data not presented). Sixteen week human fetal brain, however, shows a different pattern of NCAM expression to that seen in adult brain. A smear ranging from 120-> 300 kDa is observed on Western blots of NP-40 extracts of human fetal brain (Figure 2, lane a). This reflects the high degree of polysialylation of embryonic NCAM in comparison to the adult forms of the molecule. Following either neuraminidase or N-glycanase treatment, the pattern of NCAM expression observed broadly parallels that seen in human adult brain (Figure2, lanes b, c). In fetal brain, the intensity of the 95 kDa band appears less than that seen in adult brain. In addition, a weaker band of 80 kDa is observed. Whether this represents a further isoform of NCAM or degradation product is as yet unclear. The bands observed in human fetal and adult brain are specific to monoclonal antibody ERIC-] as no binding to similar bands is noted with an irrelevant MAb [M340]. NCAM expression on human brain tumours Prior to preparation of cell extracts, frozen tumour biopsies were checked to ensure that they predominantly consisted of malignant tissue and the diagnosis of tumour type was confirmed by reference to Dr T. Moss, Consultant Neuropathologist, Frenchay Hospital, Bristol. Several tumours of each type were examined and the Western blots presented are representative of the pattern of NCAM expression observed in the different tumours. Medulloblastoma extracts demonstrated a pattern of NCAM expression analogous to that seen in normal human fetal brain. In three childhood medulloblastomas examined, a smear ranging between 140 and 250 kDa was observed following Western blot analysis and visualization of the NCAM isoforms with MAb ERIC-1 in NP-40 tissue extracts. Neuraminidase

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180 kDa

-

145 kDa

-

120 kDa 95 kDa

-

200 kDa

116 kDa 97 kDa

Figure 2. NCAM isoforms expressed in human 16 week fetal brain. See Figure for methodology. Lanes: a, NP-40 extract + MAb ERIC- 1. b, NP-40 extract + neuraminidase treatment + MAb ERIC-1. c, NP-40 extract+N-glycanase treatment+ MAb ERIC-1. d, NP-40 extract + irrelevant MAb M340.

digestion of the extracts revealed major bands of 175,165,140,120 and 95 kDa (Figure 3), along with a weak band of approximately 80 kDa. The only other primary brain tumour found to express the embryonic, highly polysialylated form of the NCAM molecule were Schwannomas as extracts of this tissue also revealed a smear of 140-250 kDa on Western blot analysis with MAb ERIC-1 (Figure 4). However, following neuraminidase treatment, bands of 140,130,120 and 95 kDa are observed. The 130 kDa band was found to resolve to either a 120 or 95 kDa species if neuraminidase treatment was prolonged (overnight) suggesting that this results from post-translational modification of one of the other NCAM isoforms. Oligodendrogliomas reveal a pattern of NCAM expression somewhat analogous to that seen in human adult brain. The molecule is less glycosylated as distinct NCAM isoforms can be identified following Western blot analysis of NP-40 tumour extracts. However, only the 180, 145 and 120 kDa isoforms of the molecule are clearly identifiable along with a very weak band at 95 kDa neuraminidase treatment reduces the intensity of the 180-145 kDa bands with a concomitant increase in the 120 and 95 kDa isoforms (Figure 5). Astrocytomas express an even more restricted pattern of NCAM isoforms to those seen in oligodendrogliomas. Major bands of 145 and 120 kDa, along with a minor 95 kDa band are observed following Western blot analysis of extracts of astrocytomas and identification of NCAM isoforms with MAb ERIC-1. Neuraminidase treated extracts present a similar pattern of NCAM expression, although the intensity of the 120 and 95 kDa isoforms is markedly increased (Figure 6 ) . Meningiomas reveal a different pattern of NCAM expression to other tumours. Bands of 165, 135 and 125 kDa are observed following Western blot analysis of tumour extracts (Figure 7). These bands resolve to the 140,120 and 95 kDa isoforms ofNCAM following neuraminidase treatment. This suggests that the difference in size of the NCAM proteins isolated from meningiomas are due to differential post-translational modification of the molecule. Normal meninges also exhibit the same isoforms of NCAM as meningiomas, and neuraminidase treatment again results in the generation of 140,120 and 95 kDa proteins (Table 1).

Alternative isoforms of N C A M on normal and neoplastic tissue

-

200 kDa

--

116 kDa 97 kDa

2 13

180

140

120 kDa

-

(-1 a

(+I b

C

Figure 3. NCAM isoforms expressed in medulloblastoma. See Figure 1 for methodology. Lanes: a, NP-40 extract + MAb ERIC-I. b, NP-40 extract + neurarninidase treatment + MAb ERIC- I . c, NP-40 extract firrelevant MAb M340. ~

..

DISCUSSION A11 of the tissues and tumours investigated in this study bind to anti-NCAM MAb such as ERIC-I and UJ13A, as determined by indirect binding assays (Pate1er al., 1989a,b). Although it is difficult to determine accurately, the intensity of staining appears to be similar for all the tissues examined. However, biochemical studies reveal that, underlying the apparent uniformity in antigen expression, there exists a marked divergence in the forms of NCAM found on both tumours and normal brain at different developmental stages. This is explicable, as the alternative isoforms of NCAM differ predominantly in their C-terminal regions. Thus, the 180 and 140 kDa transmembrane isoforms differ in the length of the protein tail found within the cell. It was originally assumed that the extracellular regions of the different NCAM isoforms were identical in their primary sequence and this is the region where the ERIC- 1 and UJ13A epitopes are to be found. Recent work on the expression of NCAM at the molecular level indicates that the premise concerning the similarity of the extracellular sequences of different NCAM isoforms is incorrect. In both animal and human muscle NCAM, a region of 35 amino acids, the Muscle Specific Domain-1 (MSD-I), is spliced into the protein in between the sequences coded for by exons 12 and 13. This region is coded for by three mini exons of 15,48 and 42 nucleotides; termed a, b and c. These are spliced into the 120 and 155 kDa isoforms of muscle NCAM and are thought not to be present in human neural NCAM (Dickson et al., 1987). In the rat and mouse, a 30 base pair sequence, designated x, codes for a 10 amino acid sequence that is spliced selectively into neural NCAM (Small, Haines & Akeson, 1988). The

200 kDa

-

180 kDa

116 kDa

-

66 kDa

-

-

(-1 a

(+I b

-

.

145 kDa t

-

200 kDa

-

116 kDa 97 kDa

-

68 kDa

120 kDa

C

Figure 4.

Figure 5.

(-)

a

(+)

b

Figure 6.

c

Figure 7.

68 kDa

Alternative isoforms of NCAM on normal and neoplastic tissue

21 5

status of the expression of the JI and MSD mini exons in human brain tumours remains unclear. However, recent studies indicate that mini exons 12a, b and c are expressed both independently and in different combinations in NCAMs isolated from human tumour cell lines (unpublished observations). The incorporation of these exons into NCAM is, therefore, not restricted to muscle and it is plausible that at least some of the MSD exons (a, b or c) will be expressed in primary brain tumours. The status of the JI region within human neural NCAM is also unresolved. Recent studies on mice indicate that the MSD and JI regions are not the only sequences alternatively spliced into NCAM (Santoni et al., 1989). With the advent of the polymerase chain reaction technology, it is relatively simple to obtain such information as long as tissues are rapidly frozen to preserve RNA. Whilst the pattern of NCAM expression in primary brain tumours differs, insufficient diversity exists to identify each individual tumour type by this criteria. In addition, it is as yet unclear if tumours such as medulloblastoma and astrocytomas express the same isoforms of NCAM when found in children and adults. The characterization of sequences coded for by mini exons, diffentially expressed in a range of primary brain tumours, may prove to be a way of generating highly specific reagents that recognize particular malignant types. Once either a unique or selectively expressed region is identified, it is possible to artificially synthesize the sequence in the laboratory. The resulting peptide can be coupled to a carrier and used to immunize animals to raise either highly specific monoclonal or polyclonal antibodies. The function(s) of the NCAM family of proteins remains unclear. Originally, NCAM was described as only being present on neural and muscle tissue. As such, it was described as having a role in neural/neural and neural/muscle interaction. However, recently NCAM has been identified on human fetal kidney, fetal liver, some haematopoietic cells and a sub-set of bone marrow stromal cells. This makes its role less clear, but it could still function in some way to influence cell adhesion. NCAM is known to contain homophilic binding sites. This region on one NCAM molecule is capable of recognizing a similar region on another NCAM protein. In addition, NCAM is known to have a heparin binding site, but the significance of this observation remains unclear (Edelman, 1988). The reason underlying the expression of different isoforms of the NCAM on a variety of cell types is also totally unknown. It is possible that the combination of alternative isoforms within the membrane may affect cell-cell interactions in different ways. Alternatively, it has recently been demonstrated that NCAM can function in co-association with another adhesion molecule, namely L1.It is possible that the expression of different isoforms of NCAM affect the way in which the molecule co-associates with other proteins in the membrane. The degree of polysialylation of NCAM proteins also influences function. In the mouse and chick embryo NCAM is highly polysialylated. Expression of this form of the molecule coincides with a high degree of cell migration. In contrast, adult NCAM is less sialylated, this phenotype

Figure 4. NCAM isoforms expressed in human Schwannomas. See Figure 1 for methodology. Lanes: a, NP-40 extract + MAb ERIC-I. b, NP-40 extract +neuraminidase treatment MAb ERIC-I. e, NP-40 extract irrelevant MAb M340. Figure 5. NCAM isoforms expressed in oligodendrogliomas. See Figure 1 for methodology. Lanes: a, NP-40 extract+ MAb ERIC-I. b, NP-40 extract neuraminidase treatment+ MAb ERIC-I. c, NP-40 extract irrelevant MAb M340. Figure 6. NCAM isoforms expressed in astrocytomas. See Figure 1 for methodology. Lanes: a, NP-40 extract + MAb ERIC- I. b, NP-40 extract neuraminidase treatment MAb ERIC- I. c, NP-40 extract irrelevant MAb M340. Figure 7. NCAM isoforms expressed in rneningiornas. See Figure 1 for methodology. Lanes: a, NP-40 rneningioma extract+ MAb ERIC-I. b, NP-40 meningioma extract + neuraminidase treatment + MAb ERIC-I. c, NP-40 adult brain extract + MAb ERIC-I. d, NP-40 meningiorna extract + irrelevant MAb M340.

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being associated with a less motile cell. Medulloblastomas from paediatric patients and Schwannomas express the embryonic form of NCAM, but the significance of this observation is unclear. All of the other tumours express the adult forms of the molecule as Western blot analysis with the antibody ERIC-1 reveals distinct bands rather than a smear characteristic of embryonic NCAM. As a generalization, brain tumours do not readily metastasize. If NCAM were solely responsible for cell adhesion, the protein would have to be functionally active within the cell membrane. However, there are a variety of molecules.associated with both the cell membrane and the extracellular matrix that are involved in cell adhesion. Ultimately, it is probably the quantitative expression and spatial arrangements of these molecules that controls metastatic potential. However, unless we determine the distribution of the different isoforms of these molecules, it will be impossible to ultimately understand how they function. ACKNOWLEDGEMENTS We wish to thank the Imperial Cancer Research Fund, the Bristol Brain Tumour Fund and the Neuroblastoma Society for Funding this research. We are grateful to Dr T. Moss for his help in characterizing the tumours investigated and also thank Miss S. Murphy for typing the manuscript. REFERENCES Bhat S. & Silberberg D.H. (1988) Adult human brain expresses four different molecular forms of neural cell adhesion molecules. Neurochemistry Inrernational13.487-49 1 Bourne S.P., Patel K., Walsh F.S., Popham C.J., Coakham H.B. & Kemshead J.T. (1991) A monoclonal antibody (ERIC- I), raised against retinoblastoma, that recognizes the neural cell adhesion molecule (NCAM) expressed on brain and tumours arising from the neuroectoderm. Journa/of Neuro-Oncology, in press Cunningham B.A., Hemperly J.J., Murray B.A., Prediger E.A., Brackenbury R. & Edelman G.M. (1987) Neural cell adhesion molecule: structure, immunoglobulin like domains, cell surface modulation and alternative RNA splicing. Science 236.799-806 Dickson G., Gower H.J., Barton C.H., Prentice H.M., Elsom V.L., Moore S.E., Cox R.D., Quinn C., Putt W. & Walsh F.S. (1987) Human muscle neural cell adhesion molecule (N-CAM): identification of a muscle-specific sequence in the extracellular domain. Cell50, I 119-1 130 Edelman G.M. (1988) Morphoregulatory molecules. Biochemisrry 27,3533-3543 Gower H.J., Barton C.H., Elsom V.L., Thompson J., Moore S.E.,Dickson G. & Walsh F.S. (1 988) Alternative splicing generates a secreted form of N-CAM in muscle and brain. Cell 55,955-964 He H.-T., Finne J. & Goridis C. (1987) Biosynthesis, membrane association, and release of N-CAM-120, a phosphdtidylinositol-linked form of the neural cell adhesion molecule. Journal of Cell Biology 105,2489-2500 Kemshead J.T. & Coakham H. (1983) The use of monoclonal antibodies for the diagnosis of intracranial malignancies and small round cell tumours of childhood. Journal of Pathology 141,249-257 Moore S.E., Thompson J., Kirkness V., Dickson J.G. & Walsh F.S.(1987) Skeletal muscle neural cell adhesion molecule (N-CAM): changes in profein and mRNA species during myogenesis of muscle cell lines. Journal of CellBio/ogy 105, 1377-1386 Murray B.A., Hemperly J.J., Prediger E.A., Edelman G.M. & Cunningham B.A. (1986) Alternatively spliced mRNAs code for different polypeptide chains of the chicken neural cell adhesion molecule (N-CAM). Journal of Cell Biology 102, 189-193 Patel K., Moore S.E.,Dickson G., Rossell R.J., Beverley P.C., Kemshead J.T.& Walsh F.S.(1989a) Neural cell adhesion molecule (NCAM) is the antigen recognized by monoclonal antibodies of similar specificity in small cell lung carcinoma and neuroblastoma. Inrernarional Journal of Cancer 44,573578 Patel K., Rossell R.J., Bourne S., Moore S.E..Walsh F.S. & Kemshead J.T.(1989b) Monoclonal antibody UJ13A recognizes the neural cell adhesion molecule NCAM. Inrernarional Journal of Cancer 44,1062-1069

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Santoni M.J., Barthels D., Vopper G.. Boned A., Goridis C. & Wille W. (1989) Differential exon usage involving an unusual splicing mechanism generates at least eight types of NCAM cDNA in mouse brain. EMBO Journal 8, 385-392 Small S.J., Haines S.L.& Akeson R.A. (1988) Polypeptide variation in an N-CAM extracellular imunoglobulin-like fold is developmentally regulated through alternative splicing. Neuron I, 1007-1017

Received 14 September 1990 Accepted 14 December 1990