Int. J. Cancer: 50,118-123 (1992) 0 1992 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
Publication de I Union Internationale Contre le Cancer
SPLICING OF THE VASE EXON OF NEURAL CELL ADHESION MOLECULE (NCAM) IN HUMAN SMALL-CELL LUNG CARCINOMA (SCLC) H.L.P. VAN DUIJNHOVEN’,w . HELFRICH’,L. DE LEU2,A.J.M. ROEBROEK3,W.J.M. VAN DE VEN’’3,K. HEALEY4, A. CLJLVERWELL~,R.J. ROssELL4, J.T. u M S H E A D 4 and K. PATEL4” ‘Molecular Oncology Section, Department of Biochemistry, University of Nijmegen, Adelbertusplein 1, 6525 EK Nijmegen, The Netherlands; ’UniversityHospital Groningen, Dept of Clinical Immunology, Oostersingel59, 9713 EZ Groningen, The Netherlands; ’Molecular Oncology Section, Center of Human Genetics, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium; and 4The Imperial Cancer Research Fund, Paediatnc and Neuro-Oncology Group, Frenchay Hospital, Bristol BS16 ILE, UK. Expression of the neural cell adhesion molecule (NCAM) on small-cell Lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.
Carcinoma of the lung is one of the most common cancers affecting adults. This tumour expresses a wide variety of phenotypes. According to the World Health Organization Classification (WHO, 1982), lung carcinoma can be divided into a number of major types; squamous carcinoma, adenocarcinoma and large-cell carcinoma, which are collectively referred to as non-small-cell lung carcinoma (NSCLC), carcinoid tumour, and finally small-cell lung carcinoma (SCLC). SCLCs and carcinoids are characterized by the presence of neuroendocrine phenotypes. For the study of lung carcinoma biology, cell lines derived from the different histological types of tumours have been established. These cell lines manifest many features of the tumour of origin and thus have found use in in-vitro studies. SCLC cell lines can be subdivided into 2 major categories, namely classic and variant (Carney et a/., 1985). The classic type exhibits the most pronounced neuro-endocrine phenotype whereas this is lost to some extent in the variant cell lines. A wide variety of markers have been described which can be used for human lung carcinoma classification (Minna et a/., 1989). In addition, a number of monoclonal antibodies (MAbs) have been developed for use both in diagnosis of various sub-divisions of lung carcinoma and in therapy. The First and Second International Workshops on Small-Cell Lung Carcinoma (SCLC) (Beverley et aZ., 1988) brought together numerous MAbs in an attempt to better understand the reactivities of so-called “SCLC-specific MAbs” and to identify the antigens recognized by such MAbs. Groups of MAbs were identified which appear to recognize similar antigens. The Cluster 1 MAbs (Beverley et al., 1988) recognize a molecule involved in cell-cell interactions during normal growth and development, namely, the neural cell adhesion molecule (NCAM) (Patel et al., 1989). Since a feature of lung carcinoma, and cancers in general, is to metastasize to sites distant from the primary tumour, it is tempting to speculate that adhesion molecules may have a role in the biology of the tumour. Many families of adhesion
molecules have now been described, some of which appear to be either under-expressed, aberrantly expressed or overexpressed in tumours (Erickson, 1989; Natali et al., 1990; Zimmerman et al., 1988). Furthermore, in some tumours, there is a switch from expression of one form to another (Matsumura and Hakomori, 1985; Borsi et a/., 1987). Finally, a tumour suppressor gene has been shown to code for a molecule with homology to NCAM (Fearon et a/., 1990). As a prerequisite to understanding the biology of NCAM expression on SCLC tumours, we have characterized different isoforms of the molecule at the biochemical and molecular levels. NCAMs are a family of cell-surface glycoproteins that are transcribed from a single gene located on chromosome l l q 2 3 in humans (Nguyen et al., 1986). Post-transcriptional and translational modifications give rise to a series of products that are expressed in tissues in a developmentally regulated manner (Cunningham et al., 1987; Edelman, 1988; Walsh and Dickson, 1989). Alternative splicing of the large RNA segments, and their subsequent translation, give rise to 6 proteins in brain. Two large isoforms of 180 and 140 kDa are wellcharacterized transmembrane products and have similar extracellular domains, but differing lengths of cytoplasmic tails (Cunningham et a/., 1987). The 120-kDa isoform is a glycosylphosphatidylinositol (GP1)-anchored protein (Cunningham et a/., 1987) and the 115-kDa product is believed to be a secreted molecule (Walsh and Dickson, 1989). Further isoforms of 170 kDa and 95 kDa have been identified in human brain, but these remain poorly characterized (Bhat and Silberberg, 1988; Frost et a/., 1991). Different forms of NCAM are associated with skeletal muscle. In this instance, one transmembrane (140 kDa), one secretory (115 kDa) and 2 GPI-linked (155 and 125 kDa) proteins have been described (Walsh and Dickson, 1989). In addition to these biochemically distinct forms of NCAM, further heterogeneity occurs which can only be detected by isolation and sequencing of individual cDNA clones or by PCR. One such region, designated VASE, is a 30-bp sequence which is differentially inserted between exons 7 and 8 (Santoni et al., 1989; Small et a/., 1988). The VASE sequence, originally described in mice and rats, was thought to be spliced only in neural NCAM (Santoni et al., 1989; Small et a/., 1988). However, Reyes et al. (1991) suggest that VASE can be spliced into rat heart muscle. The insertion of VASE into the 4th Ig-like domain changes its conformation from a C2 type to one that resembles the variable domain of immunoglobulins. The functional consequences of this insertion are unknown. In addition to isoforms resulting from alternative splicing, post-translational modifications such as phosphorylation. sulphation and glycosylation, in particular polysialylation, can also increase the complexity of NCAMs. The degree of polysialylation is substantially altered during development. ’To whom correspondence and reprint requests should be sent.
Received: May 30,1991 and in revised form July 24, 1991.
MINI-EXON VASE OF NCAM Here, we describe the characterization of NCAMs expressed in SCLC cell lines and tissues at the biochemical and molecular levels. In addition, we describe both the presence and sequence of the VASE exon from human SCLC, which has not been previously reported. MATERIAL AND METHODS
Cell lines The human SCLC classic cell lines, GLC-14, -16, -19 (Berendsen et al., 1988), and GLC-28 and the variant line, GLC-1 (De Leij etal., 1985) were grown in a 5% CO, incubator in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 IUiml penicillin and 100 Fgirnl streptomycin. All lines grow as aggregates. The GH1 line (Minowada et al., 1972), a haemopoietic cell line, was also grown under similar conditions. Tissues Lung carcinoma specimens from patients were selected from the files of the Pathology Department of St. Antonius Hospital, Nieuwegein, The Netherlands. Tumours were characterized by routine microscopical and histopathological techniques and classified according to W H O criteria. Two specimens of control lung tissue were also obtained. Monoclonal antibodies (MAbs) MAb ERIC-1, raised against human retinoblastoma, recognizes all isoforms of human NCAM (Bourne et al., 1990). In addition, MAb M340 was used as irrelevant MAb. Western blotting Tissues or cell lines 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 pgiml leupeptin and 2% NP40. The resulting homogenate was centrifuged for 5 rnin at 8,OOOg at 4°C and the protein concentration of the supernatant determined using the Bio-Rad Protein Assay (Munich, Germany). Proteins from the lysate, either treated with neuraminidase (Clostridium perfnngens, Sigma X; 5 U for 5 hr) or left untreated (controls) were size-separated under reducing conditions by polyacrylamide gel electrophoresis (7%) at 200 V for 3 4 hr. Transfer of proteins to 0.2-ym nitrocellulose filters was carried out at 134 mA for 90 min using the LKB (Bromma, Sweden) Novablot transfer apparatus. Non-specific protein binding sites on the filter were blocked by overnight incubation in PBS containing 5% Marvel (Cadbury Schweppes, London, UK). Incubation with MAbs was performed for 30 rnin at room temperature. This was followed by 2 washes for 5 min each in PBS. The filters were subsequently incubated with alkaline-phosphatase-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 rnin in the dark with the substrate consisting of 0.2 mgiml napthol AS-MX phosphate, 2% N, N-dimethylformamide, 1 mgiml Fast red T R in 0.1 M Tris-HCI pH 8.2 and 1 mM levamisole. The filters were finally washed twice in TBS and air-dried. RNA isolation and Northern blot analysis Total cellular R N A was isolated using the lithium-urea procedure described by Auffray and Rougeon (1980). Total RNA (15 Fg) was glyoxylated and size-fractionated on 1.0% agarose gels and transferred to Hybond-N as recommended by the manufacturers (Amersham, Aylesbury, UK). OD,,,, and ethidium bromide staining were used to estimate RNA amounts loaded per well.
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To study NCAM expression, the 1.1-kbp NCAM-specific human cDNA NC7 was used (data not shown). This was labelled by random priming and hybridized by the method of Church and Gilbert (1984). Blots were washed at high stringency and exposed to autoradiographic films for an appropriate period. For re-hybridization, the probes were stripped by incubation in 5 mM Tris-HCI (PH S), 2 mM EDTA and X O . l Denhardt’s solution ( x 1 Denhardt’s solution contains 0.02% (w/v) BSA, 0.02% (wiv) polyvinylpyrrolidone and 0.02% (wiv) Ficoll) at 65°C for 2 hr. Polymerase chain reaction (PCR) Total R N A was extracted by the method of Cathala et al. (1983); 1.5 p g was reverse-transcribed in a total volume of 20 yI consisting of 50 mM Tris-HCI pH 8.5, 40 mM KCI, 10 mM MgCI,, 0.4 mM P-mercaptoethanol, 1 FM 3’-prjmer (oligo 2), 20 U AMV-reverse transcriptase and 0.5 mM each dNTP at 42°C for 45 min. The resulting cDNA was amplified using PCR in a total volume of 100 p*.1containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCI,, 0.02% gelatin, 200 p M each dNTP, 0.2 FM S’-primer (oligo l ) , 0.2 p M 3’-primer (oligo 2) and 2.5 U Taq polymerase. Samples were overlaid with 50 p1 mineral oil and subjected to denaturation at 94°C for 4 min. This was followed by 30 cycles of denaturation at 94°C for 2 min, annealing at 60°C for 2 min and extension at 72°C for 90 sec. The last cycle had an extension period of 7 min. As a positive control, the murine NCAM clone N1 which contains the VASE sequence was used (Santoni et al., 1989). In some experiments, poly (A)’ mRNA was extracted using a commercial kit (British Bio-tech, Oxford, UK) and used instead of total RNA. Analysis of PCR products Mineral oil was removed using 100 ~l ch1oroform:isoamylalcoho1 (24:l). 10 pl of PCR product was run on a 2.5% agarose gel in ~1 T A E buffer. The bands were blotted onto GeneScreen Plus (NEN, Boston, MA) membrane. Oligonucleotides 3 and 4 were end-labelled by T4 polynucleotide kinase and used at 106cpmiml for hybridization in a solution consisting of 10% dextran sulphate, 1 % SDS, 1 M NaCl and 100 Fgirnl salmon-sperm-denatured DNA. The blots were washed to a final stringency of 0.1 x SSC, 1% SDS at 60°C and exposed to Fuji X-ray films for an appropriate period. Oligonucleotides Oligonucleotides were numbered according to Barton et al. (1988). Oligonucleotide 1 AGC AGG TCA CTC TTA CCT GT (967-987) Oligonucleotide 2 GGC GGT GCA CAT GTA CTC (1044-1062) Oligonucleotide 3 (complementary to exon 7) GAT GTT CCG GGT AGA AGT CCT CCA (1003-1045) GGT GAT GGA GGG AAT GGG Oligonucleotide 4: (Complementary to mouse VASE) CTC TTG CTT CTC TGG TCG AGT CCA CGA TGC
DNA sequencing Separated bands were excised from the agarose gel, DNApurified and subjected to asymmetric PCR as described above, but with the following modification: (0.5 )J.M 3’ primer: 0.01 p~ 5‘ primer); 0.2 pmole of the asymmetric PCR product was sequenced by the dideoxynucleotide chain termination method as described by Winship (1989).
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RESULTS
Western blotting Five well-characterized SCLC cell lines were examined for expression of NCAM by Western blot analysis using the anti-NCAM MAb ERIC-1. GLC-14, -16, -19 and -28 are “classic” SCLC lines, whereas GLC-1 has characteristics associated with the “variant” type. In the absence of neuraminidase treatment, a smear ranging from 140 to 200 kDa was identified in all lines (Fig. 1) although in some instances the smear was weak (e.g., Fig. 1, lane B). In addition, some distinct bands within this smear could be observed (Fig. 1). The smear is indicative that NCAM expressed on these cell lines is the highly polysialylated form containing high levels of a2-8 linked polysialic acid (PSA) residues (Frost et al., 1991). After neuraminidase treatment to remove the PSA, distinct bands of 140 and 120 kDa could be seen. Furthermore, GLC-1, -19 and -28 also contained an additional band of 95 kDa. The expression of NCAM in freshly frozen SCLC tumour tissue obtained from surgically excised specimens was also compared to that in the cell lines. The data obtained were essentially similar; i.e., a smear was detected in the absence of neuraminidase treatment, whereas distinct bands of 140, 120 and 95 kDa were present after removal of PSA by neuraminidase treatment (Fig. 1; lane A). No such bands were detected in any samples using an irrelevant antibody M340. For the sake of brevity, only data for line GLC-1 are presented (Fig. 1; lane G). Northern blotting T o confirm the Western blot data, the NCAM mRNA species present in SCLC cell linesltissues was examined. Total R N A was separated on agarose gels, blotted and hybridized with the human NCAM cDNA probe NC7. A high degree of concordance was observed between the Western and Northern blot data. Cell lines expressing the 140- and 120-kDa isoform of NCAM contained NCAM mRNA transcripts of 6.7 and 5.4 kb. For the sake of brevity, we present only data on the GLC-1
cell line which expresses a major mRNA species of 6.7 kb and weak bands of 5.5 and 5.0 kb which were not easy to reproduce photographically (Fig. 2; lane A). In occasional tumour samples, a weak band of 7.4 kb was also identified (Fig. 2; lane C). This NCAM mRNA codes for the 180-kDa form of the molecule, suggesting that this isoform can also be expressed in SCLC tumours. Unfortunately, due to lack of material, these results could not be confirmed biochemically. No NCAM transcripts were detected in normal lung, squamous carcinoma or adenocarcinoma (Fig. 2). Similar data were obtained from tissue samples. Screening a large panel of cell lines and tissues showed that only SCLC and carcinoids contained mRNA for NCAM (Table I). The 5.0-kb band (mentioned above) present in some of the cell lines and tumours was not due to non-specific hybridization of NCAM probe with rRNA, as identical blots were obtained using poly (A)’ RNA. Furthermore, this transcript has also been identified in other tumour cell lines expressing NCAM, namely neuroblastoma and rhabdomyosarcoma (data not presented).
PCR analysis of the splicing of the VASE mini-exons The splicing of the mini-exon, VASE, was examined in the SCLC cell lines by PCR and Southern blotting. Total RNA was reverse transcribed and the resulting cDNA amplified using the PCR primers oligos 1 and 2. The samples were separated on 2.5% agarose gels, blotted and hybridized with appropriate oligonucleotides. Using an oligonucleotide complementary to the murine VASE sequence, a major band of 224 b p and a minor band of 260 bp were detected in SCLC lines. Furthermore, similar data were obtained with human adult brain (Fig. 3; lane A). The position of the 224-bp fragment corresponds to the position of the PCR product amplified from a murine cDNA clone containing the VASE exon (Fig. 3, lane H). In contrast, no bands were detected in GHI, a haemopoietic cell line known not to express NCAM (Fig. 3; lane G).
FIGURE 1 - NCAM expression in human small-cell lung carcinoma cell lines and tissues. NP-40 cell or tissue lysates were electrophoresed on 7% SDS-PAGE gels and electroblotted. Blots were subsequently incubated with MAb ERIC-1 or an irrelevant MAb (M340). After washing, the blots were further incubated with sheep anti-mouse Ig conjugated to alkaline phosphatase and colour developed as described in “Methods”. Amersham International Rainbow Markers were used for estimation of molecular weight. (200 kDa myosin, 116 kDa P-galactosidase, 95 kDa phosphorylase P, 69 kDa bovine serum albumin, 46 kDa ovalbumin, 30 kDa carbonic anhydrase). -, no neuraminidase; +, neuraminidase-treated [Closfridiumpe~~ingen~ ( 5 U for 5 hr)J. A = SCLC tumour tissue, incubated with ERIC-1; B = GLC-1 cell line incubated with ERIC-1; C = GLC-14 cell line incubated with ERIC-1; D = GLC-16 cell line incubated with ERIC-1; E = GLC-19 cell line incubated with ERIC-1; F = GLC-28 cell line incubated with ERIC- 1; G = GLC-1 cell line incubated with irrelevant antibody M340.
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MINI-EXON VASE OF NCAM IN LUNG CARCINOMA
FIGURE 2-NCAM mRNA species detected in tumour and normal lung tissue. Total RNA was glyoxylated, separated on 1% agarose gel and transferred to Hybond N membrane. The membrane was hybridized with radiolabelled NC7 probe and washed to high stringency. Glyoxylated Hind111 digest markers were used to estimate the size of the mRNA bands. A = GLC-1 cell line; B = normal lung; C = SCLC tumour tissue 1; D = SCLC tumour tissue 2; E = carcinoid tumour; F = squamous carcinoma; G = adenocarcinoma. The 5.5- and 5.0-kb bands in track A were too weak to be identified on the photograph, but are clearly visible on the autoradiogram. TABLE I - NCAM mRNA EXPRESSION IN CARCINOMA OF THE LUNG Cell line?
Classic SCLC Variant SCLC NSCLC
NCAM mRNA
515' 818 012
Tumourr
SCLC Carcinoid Adenocarcinoma Sauamous-cell carcinoma
516 415 017 019
'Number positive out of total tested.
The weak band of 260 b p appears to be an artifact since separation of the PCR products under denaturing conditions and subsequent hybridization did not reveal its presence (data not shown). Using an oligonucleotide lying outside the VASE exon, i.e., oligonucleotide 3, 3 products of 194, 224 and 260 bp (weak) were identified. The 194-bp fragment is derived from NCAM mRNA lacking the VASE exon.
DNA sequencing To confirm the identity of the 194- and 224-bp fragments, asymmetric PCR was performed on separated bands from the first round of PCR. The product from asymmetric PCR reaction was sequenced by the dideoxy-nucleotide chain termination method. Figure 4 shows the comparison of the published murine VASE sequence and the sequence obtained for human VASE. It can be seen that both sequences are identical except for one base change in the 3rd nucleotide of the sequence where a
FIGURE3 - Identification and characterization of the cDNA amplified from SCLC mRNA. Total RNA was reverse transcribed and the resulting cDNA amplified by polymerase chain reaction. Ten microlitres of the PCR product were run on a 2.5% agarose gel, transferred to Genescreen plus membrane and hybridized either with radiolabelled oligonucleotide complementary to murine VASE (panel a ) or an oligonucleotide complementary to exon 7 (panelb). A = GLC-1; B = GLC-14; C = GLC-16; D = GLC-19; E = GLC-28; F = adult human brain; G = GH1; H = Clone N1.
thymidine replaces an adenosine residue. This substitution does not result in a change in the amino-acid coded for by the triplet. In addition, sequencing of the regions flanking the VASE mini-exon indicates that no other new mini-exons could be identified in the 194- and 224-bp PCR fragments. DISCUSSION
Many families of adhesion molecules have been described which are believed to play an important role in a variety of developmental processes. These include the immunoglobulin gene superfamily (Edelman, 1985, 1988),cadherins (Takeichi, 1988), integrins (Ruoslahti and Pierschbacher, 1987), extracelM a r matrix molecules (Ruoslahti, 1988) and selectins (Springer and Lasky, 1991). Since many of the processes that occur during normal development, e.g. cell-cell interactions and migration, are also relevant t o tumour biology and metastasis, it is likely that some of these molecules will be important in carcinogenesis. We have previously shown that a number of MAbs reactive with SCLC (Cluster 1) recognize the neural cell adhesion molecule (NCAM) (Pate1 et al., 1989) which is involved in cell-cell interactions (Cunningham et al., 1987). The expression of this molecule is complex, with multiple forms of the protein being described. These various forms of the molecule arise from a single gene through post-transcriptional control, such as alternative splicing and use of multiple polyadenylation sites, and through post-translational modifications, such as phosphorylation, sulphation and glycosylation. Although the precise functions of these various isoforms of NCAM remain unclear, their expression appears to be strictly regulated during development. This suggests that they play important, but subtly different roles. We have investigated the expression of NCAM in SCLC since it appears to be a major antigen that is expressed in this
122 EXON 7
VAN DUIJNHOVEN ET AL.
5' EXON 8 5' -------------.....--------------VASE-EXON----......-----------------3'
3'
AAC ATC AGC AGT GAA GAA AAG GCA TCG TGG ACT CGA CCA GAG AAG CAA GAG ACT CTG GAT GGG CAC ATG Mouse AAC ATC AGC AGC GAA GAA AAG GCT TCG TGG ACT CGA CCA GAG AAG CAA GAG ACT CTG GAT GGG CAC ATG Human N I S S E E K A S W T R P E K Q E T L D G H M
FIGURE 4 - Comparison of the human and murine VASE sequence. Asymmetrically amplified PCR fragment (224 bp) was sequenced by the dideoxy chain termination method.
tumour. Our present data show that the NCAM protein isoforms and mRNA species are differentially expressed in the SCLC cell lines and tumour tissues we have investigated. The 140- and 120-kDa isoforms and their corresponding mRNAs, 6.7 and 5.5 kb respectively, were present in all lines/tissues, whereas the 7.4-kb transcript coding for 180-kDa isoform was found in occasional samples. A 95-kDa protein was also identified in some SCLC samples. The nature of this NCAM product is currently unclear, although a similar protein has been found in adult brain (Frost eta[., 1991; Patel et a[., 1991). In some respects, our data are at variance with those published previously. Firstly, at the protein level we have observed highly sialylated NCAM, whereas previously discrete bands of 180 and 140 kDa have been described (Kibbelaar et a/., 1989; Aletsce-Ufrecht et al., 1990) which suggests that, although these forms were reported to be highly sialylated by Kibbelaar et al. (1989), these are less sialylated NCAMs. Secondly, we show that the 140 kDa band is not specific for SCLC, as previously believed (Aletsce-Ufrecht et al., 1990) and finally, we describe the presence of a 95-kDa band. The reasons for these discrepancies could be manifold. For example, they may b e due to different methods of investigation, e.g. immunoprecipitation vs. Western blotting or use of different MAbs. Moreover, the polysialic acid on NCAM is very labile. The presence of highly sialylated NCAM has been demonstrated in other round-cell tumours e.g. neuroblastoma and rhabdomyosarcoma. In neuroblastoma, only a proportion of the NCAM isoforms described in human brain are present (Phimister et al., 1991). This is also the case with SCLC. Because of the differences between the data described here and those reported previously, we have attempted to confirm the Western blotting data by characterizing NCAM mRNA species present in SCLC linesitissues. Transcripts of 6.7, 5.5 and 5.0 kb were present with differential expression of the 7.4 kb. The presence of the 5.0 kb has not been described previously in normal tissues, but its presence on neuroblastoma and rhabdomyosarcoma cell lines has been documented (Patel et al., 1991). The detection of this transcript is not due to non-specific hybridization to rRNA since use of poly (A)' RNA gives identical results to those documented above.
The mini-exon, VASE, spliced between exons 7 and 8, has been studied in mice and rats (Santoni et al., 1989; Small t't al., 1988). It has been shown to consist of a 30-bp sequence whose insertion is developmentally regulated. Initially, it was thought to be neuron-specific, but its presence in rat heart muscle has been reported (Reyes et al., 1991). In this report, PCR fragments lacking or containing VASE mini-exons have been shown to be present in SCLC lines. D N A sequencing has confirmed the presence of VASE in the 224-bp fragment. W e have shown biochemically that multiple forms of NCAM are associated with SCLC. Furthermore, through PCR analysis, it appears that VASE-containing, as well as VASE-lacking, NCAMs are present. However, we have been unable to correlate the expression of various NCAMs in SCLC with general adhesiveness of the cells or with disease states. We found similar levels of NCAM expression in one variant (GLC-1) vs. classic (GLC-14, -16, -19 and -28) lines by Western blotting and also no difference in mRNA expression in a larger panel of classic SCLCs vs. variant SCLCs by Northern blotting (Table I). In addition, there was no discernible difference in NCAM expression between cell lines established from the same patient prior to chemotherapy (GLC-14) and those established after first (GLC-16) and second rounds (GLC-19) of therapy. GLC-14 grows as tight aggregates, whereas GLC-16 and -19 grow as loose aggregates. Adhesion is almost certainly a multifactorial process, with interactions occurring between a number of different adhesion molecules, of which NCAM is only one. Before the process can be fully understood, a detailed knowledge of the different forms of the molecules expressed on cells is critical. Once this has been achieved, it may also lead to opportunities to develop highly selective diagnostic tools in the future. ACKNOWLEDGEMENTS
We are grateful to the Imperial Cancer Research Fund for financial support. In addition, we thank Dr. Barthels (University of Cologne, Germany) for the generous gift of clone N1, Ms E. Moors, Ms E. Timmer and Mr A. Groeneveld for technical assistance and Ms S. Murphy for typing the manuscript.
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BRACKENBURY, R. and EDELMAN, G.M., Neural cell adhesion mole- PATEL,K., MOORE,S.E., DICKSON, G., ROSSELL, R.J., BEVERLEY, P.C., cule: structure, immunoglobulin-like domains, cell surface modulaKEMSHEAD,J.T. and WALSH, F.S., Neural cell adhesion molecule tion, and alternative RNA splicing. Science, 236,799-806 (1987). (NCAM) is the antigen recognized by monoclonal antibodies of similar DE LEIJ,L., POSTMUS, P.E., BUYS,C.H., ELEMA,J.D., RAMAEKERS, F., specificity in small-cell lung carcinoma and neuroblastoma. Int. J. POPPEMA, S., BRALWER, M., VAN DER VEEN,A,, MESANDER, G. and Cancer, 44,573-578 (1989). THE,T.H., Characterisation of three new variant type cell lines derived PATEL.K.. KIELY.F.. WALSH.F.. PHIMISTER. E. and KEMSHEAD. J.T.. from small cell carcinoma of the lung. Cancer Res., 45, 6024-6033 Potential diagnostic usefulness of tissue-specific NCAMs in differen: (1985). tial identification of small, round-cell tumours. Advanc. Neuroblast. EDELMAN, G.M., Cell adhesion molecule expression and the regula- Res., 3,275-281 (1991). tion of morphogenesis. Cold Spr. Harb. Symp. quant. Biol., 50,877-889 PHIMISTER, E., KIELY,F., KEMSHEAD, J.T. and PATEL,K., Expression (1985). of neural cell adhesion molecule (NCAM) isoforms in neuroblastoma. EDELMAN, G.M., Morphoregulatory molecules. Biochemistry, 27,3533- J. din. Pathol., 44,58&585 (1991). 3543 (1988). REYES,A.A., SMALL,S.J. and AKESON,R., At least 27 alternatively spliced forms of the neural cell adhesion molecule mRNA are ERICKSON, H.P., Tenascin: an extracellular matrix protein prominent in specialized embryonic tissues and tumours. Ann. Rev. Cell Biol., 5, expressed during rat heart development. Mol. cell. Bid., 11,1654-1661 (1991). 71-92 (1989). E., Fibronectin and its receptors. Ann. Rev. Biochem., 57, FEARON,E.R., CHO, K.R., NIGRO,J.M., KERN,S.E., SIMON,J.W., RUOSLAHTI, RUPPERT,J.M., HAMILTON, S.R., PREISINGER, A.C., THOMAS,G., 3 7 5 4 1 3 (1988). KINZLER, K.W. and VOGELSTEIN, B., Identification of a chromosome RUOSLAHTI, E. and PIERSCHBACHER, M.D., New perspectives in cell 18q gene that is altered by colorectal cancers. Science, 247, 49-56 adhesion: RGD and integrins. Science, 238,491-497 (1987). (1990). SANTONI, M.J., BARTHELS, D., VOPPER,G., BONED,A,, GORIDIS, C. FROST,G., PATEL,K., BOURNE,S., COAKHAM, H.B. and KEMSHEAD, and WILLE,W., Differential exon usage involving an unusual splicing J.T., Expression of alternative isoforms of the neural cell adhesion mechanism generates at least eight types of NCAM cDNA in mouse molecule (NCAM) on normal brain and a variety of brain tumours. brain. EMBOJ., 8,385-392 (1989). Neuropath. Neurobiol., 17,207-219 (1991). SMALL,S.J., HAINES, S.L. and AKESON,R.A., Polypeptide variation in KIBBELAAR,R.E.,MOOLENAAR,C.E.C.,MICHALIDES,R.J.A.M.,BI~ERan N-CAM extracellular immunoglobulin-like fold is developmentally SUERMANN, D., ADDIS, B.J. and MOOI, W.J., Expression of the regulated through alternative splicing. Neuron, 1,1007-1017 (1988). embryonal neural cell adhesion molecule N-CAM in lung carcinoma. SPRINGER, T.A. and LASKY,L.A., Sticky sugars for selections. Nature Diagnostic usefulness of monoclonal antibody 735 for the distinction (Land.), 349,196-197 (1991). between small-cell lung carcinoma and non-small-cell lung carcinoma. TAKEICHI, M., The cadherins: cell-cell adhesion molecules controlling J. Pathol., 159,23-28 (1989). animal morphogenesis. Development, 102,639-655 (1988). MATSUMURA, H. and HAKOMORI, S.I., The oncofoetal domain of fibronectin defined bv monoclonal antibodv FDC-6: its uresence in WALSH,F.S. and DICKSON,G., Generation of multiple N-CAM fibronectins from foeial and tumour tissues and its abseice in those polypeptides from a single gene. Bioessays, 11,83-88 (1989). from normal adult tissues and plasma. Proc. nat. Acad. Sci. (Wash.), 82, WINSHIP,P.R., An improved method for directly sequencing PCR 6517-6521 (1985). amplified material using dimethyl sulphoxide. Nucl. Acids Res., 17, 1266 (1989). MINNA,J.D., PASS,H., GLATSTEIN, E. and IHDE,D.C., Carcinoma of the lung. In: V.T. Devita, S. Hellman and S.V. Rosenberg (eds.), WHO. The World Health Organization histological typing of lung Cancer: principles and practice in oncology, Lippincott, Philadelphia tumours, 2nd ed.Amer. J. clin. Path., 77,123-136 (1982). (1989). ZIMMERMAN, W., WEBER,B., ORTLIEB, B., RUDENT, F., SCHEMPP, W., MINOWADA, J., DNUMA, T. and MOORE,G.E., Brief communication: FIEBIG,M., SHIRELY,J.E., VON KLEIST, S. and THOMPSON, J.A., rosette-forming human lymphoid cell lines. I. Establishment and Chromosomal localisation of the carcinoembryonic antigen gene evidence for origin of thymus-derived lymphocytes. J. nat. Cancer Znst., family and differential expression in various tumours. Cancer Res., 48, 49,891-895 (1972). 2443-2550 (1988).
Publication of the International Union Against Cancer
Publication de I Union Internationale Contre le Cancer
SPLICING OF THE VASE EXON OF NEURAL CELL ADHESION MOLECULE (NCAM) IN HUMAN SMALL-CELL LUNG CARCINOMA (SCLC) H.L.P. VAN DUIJNHOVEN’,w . HELFRICH’,L. DE LEU2,A.J.M. ROEBROEK3,W.J.M. VAN DE VEN’’3,K. HEALEY4, A. CLJLVERWELL~,R.J. ROssELL4, J.T. u M S H E A D 4 and K. PATEL4” ‘Molecular Oncology Section, Department of Biochemistry, University of Nijmegen, Adelbertusplein 1, 6525 EK Nijmegen, The Netherlands; ’UniversityHospital Groningen, Dept of Clinical Immunology, Oostersingel59, 9713 EZ Groningen, The Netherlands; ’Molecular Oncology Section, Center of Human Genetics, University of Leuven, Herestraat 49, B-3000 Leuven, Belgium; and 4The Imperial Cancer Research Fund, Paediatnc and Neuro-Oncology Group, Frenchay Hospital, Bristol BS16 ILE, UK. Expression of the neural cell adhesion molecule (NCAM) on small-cell Lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.
Carcinoma of the lung is one of the most common cancers affecting adults. This tumour expresses a wide variety of phenotypes. According to the World Health Organization Classification (WHO, 1982), lung carcinoma can be divided into a number of major types; squamous carcinoma, adenocarcinoma and large-cell carcinoma, which are collectively referred to as non-small-cell lung carcinoma (NSCLC), carcinoid tumour, and finally small-cell lung carcinoma (SCLC). SCLCs and carcinoids are characterized by the presence of neuroendocrine phenotypes. For the study of lung carcinoma biology, cell lines derived from the different histological types of tumours have been established. These cell lines manifest many features of the tumour of origin and thus have found use in in-vitro studies. SCLC cell lines can be subdivided into 2 major categories, namely classic and variant (Carney et a/., 1985). The classic type exhibits the most pronounced neuro-endocrine phenotype whereas this is lost to some extent in the variant cell lines. A wide variety of markers have been described which can be used for human lung carcinoma classification (Minna et a/., 1989). In addition, a number of monoclonal antibodies (MAbs) have been developed for use both in diagnosis of various sub-divisions of lung carcinoma and in therapy. The First and Second International Workshops on Small-Cell Lung Carcinoma (SCLC) (Beverley et aZ., 1988) brought together numerous MAbs in an attempt to better understand the reactivities of so-called “SCLC-specific MAbs” and to identify the antigens recognized by such MAbs. Groups of MAbs were identified which appear to recognize similar antigens. The Cluster 1 MAbs (Beverley et al., 1988) recognize a molecule involved in cell-cell interactions during normal growth and development, namely, the neural cell adhesion molecule (NCAM) (Patel et al., 1989). Since a feature of lung carcinoma, and cancers in general, is to metastasize to sites distant from the primary tumour, it is tempting to speculate that adhesion molecules may have a role in the biology of the tumour. Many families of adhesion
molecules have now been described, some of which appear to be either under-expressed, aberrantly expressed or overexpressed in tumours (Erickson, 1989; Natali et al., 1990; Zimmerman et al., 1988). Furthermore, in some tumours, there is a switch from expression of one form to another (Matsumura and Hakomori, 1985; Borsi et a/., 1987). Finally, a tumour suppressor gene has been shown to code for a molecule with homology to NCAM (Fearon et a/., 1990). As a prerequisite to understanding the biology of NCAM expression on SCLC tumours, we have characterized different isoforms of the molecule at the biochemical and molecular levels. NCAMs are a family of cell-surface glycoproteins that are transcribed from a single gene located on chromosome l l q 2 3 in humans (Nguyen et al., 1986). Post-transcriptional and translational modifications give rise to a series of products that are expressed in tissues in a developmentally regulated manner (Cunningham et al., 1987; Edelman, 1988; Walsh and Dickson, 1989). Alternative splicing of the large RNA segments, and their subsequent translation, give rise to 6 proteins in brain. Two large isoforms of 180 and 140 kDa are wellcharacterized transmembrane products and have similar extracellular domains, but differing lengths of cytoplasmic tails (Cunningham et a/., 1987). The 120-kDa isoform is a glycosylphosphatidylinositol (GP1)-anchored protein (Cunningham et a/., 1987) and the 115-kDa product is believed to be a secreted molecule (Walsh and Dickson, 1989). Further isoforms of 170 kDa and 95 kDa have been identified in human brain, but these remain poorly characterized (Bhat and Silberberg, 1988; Frost et a/., 1991). Different forms of NCAM are associated with skeletal muscle. In this instance, one transmembrane (140 kDa), one secretory (115 kDa) and 2 GPI-linked (155 and 125 kDa) proteins have been described (Walsh and Dickson, 1989). In addition to these biochemically distinct forms of NCAM, further heterogeneity occurs which can only be detected by isolation and sequencing of individual cDNA clones or by PCR. One such region, designated VASE, is a 30-bp sequence which is differentially inserted between exons 7 and 8 (Santoni et al., 1989; Small et a/., 1988). The VASE sequence, originally described in mice and rats, was thought to be spliced only in neural NCAM (Santoni et al., 1989; Small et a/., 1988). However, Reyes et al. (1991) suggest that VASE can be spliced into rat heart muscle. The insertion of VASE into the 4th Ig-like domain changes its conformation from a C2 type to one that resembles the variable domain of immunoglobulins. The functional consequences of this insertion are unknown. In addition to isoforms resulting from alternative splicing, post-translational modifications such as phosphorylation. sulphation and glycosylation, in particular polysialylation, can also increase the complexity of NCAMs. The degree of polysialylation is substantially altered during development. ’To whom correspondence and reprint requests should be sent.
Received: May 30,1991 and in revised form July 24, 1991.
MINI-EXON VASE OF NCAM Here, we describe the characterization of NCAMs expressed in SCLC cell lines and tissues at the biochemical and molecular levels. In addition, we describe both the presence and sequence of the VASE exon from human SCLC, which has not been previously reported. MATERIAL AND METHODS
Cell lines The human SCLC classic cell lines, GLC-14, -16, -19 (Berendsen et al., 1988), and GLC-28 and the variant line, GLC-1 (De Leij etal., 1985) were grown in a 5% CO, incubator in RPMI 1640 medium supplemented with 10% FCS, 2 mM glutamine, 100 IUiml penicillin and 100 Fgirnl streptomycin. All lines grow as aggregates. The GH1 line (Minowada et al., 1972), a haemopoietic cell line, was also grown under similar conditions. Tissues Lung carcinoma specimens from patients were selected from the files of the Pathology Department of St. Antonius Hospital, Nieuwegein, The Netherlands. Tumours were characterized by routine microscopical and histopathological techniques and classified according to W H O criteria. Two specimens of control lung tissue were also obtained. Monoclonal antibodies (MAbs) MAb ERIC-1, raised against human retinoblastoma, recognizes all isoforms of human NCAM (Bourne et al., 1990). In addition, MAb M340 was used as irrelevant MAb. Western blotting Tissues or cell lines 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 pgiml leupeptin and 2% NP40. The resulting homogenate was centrifuged for 5 rnin at 8,OOOg at 4°C and the protein concentration of the supernatant determined using the Bio-Rad Protein Assay (Munich, Germany). Proteins from the lysate, either treated with neuraminidase (Clostridium perfnngens, Sigma X; 5 U for 5 hr) or left untreated (controls) were size-separated under reducing conditions by polyacrylamide gel electrophoresis (7%) at 200 V for 3 4 hr. Transfer of proteins to 0.2-ym nitrocellulose filters was carried out at 134 mA for 90 min using the LKB (Bromma, Sweden) Novablot transfer apparatus. Non-specific protein binding sites on the filter were blocked by overnight incubation in PBS containing 5% Marvel (Cadbury Schweppes, London, UK). Incubation with MAbs was performed for 30 rnin at room temperature. This was followed by 2 washes for 5 min each in PBS. The filters were subsequently incubated with alkaline-phosphatase-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 rnin in the dark with the substrate consisting of 0.2 mgiml napthol AS-MX phosphate, 2% N, N-dimethylformamide, 1 mgiml Fast red T R in 0.1 M Tris-HCI pH 8.2 and 1 mM levamisole. The filters were finally washed twice in TBS and air-dried. RNA isolation and Northern blot analysis Total cellular R N A was isolated using the lithium-urea procedure described by Auffray and Rougeon (1980). Total RNA (15 Fg) was glyoxylated and size-fractionated on 1.0% agarose gels and transferred to Hybond-N as recommended by the manufacturers (Amersham, Aylesbury, UK). OD,,,, and ethidium bromide staining were used to estimate RNA amounts loaded per well.
IN LUNG CARCINOMA
119
To study NCAM expression, the 1.1-kbp NCAM-specific human cDNA NC7 was used (data not shown). This was labelled by random priming and hybridized by the method of Church and Gilbert (1984). Blots were washed at high stringency and exposed to autoradiographic films for an appropriate period. For re-hybridization, the probes were stripped by incubation in 5 mM Tris-HCI (PH S), 2 mM EDTA and X O . l Denhardt’s solution ( x 1 Denhardt’s solution contains 0.02% (w/v) BSA, 0.02% (wiv) polyvinylpyrrolidone and 0.02% (wiv) Ficoll) at 65°C for 2 hr. Polymerase chain reaction (PCR) Total R N A was extracted by the method of Cathala et al. (1983); 1.5 p g was reverse-transcribed in a total volume of 20 yI consisting of 50 mM Tris-HCI pH 8.5, 40 mM KCI, 10 mM MgCI,, 0.4 mM P-mercaptoethanol, 1 FM 3’-prjmer (oligo 2), 20 U AMV-reverse transcriptase and 0.5 mM each dNTP at 42°C for 45 min. The resulting cDNA was amplified using PCR in a total volume of 100 p*.1containing 10 mM Tris-HCI pH 8.3, 50 mM KCI, 1.5 mM MgCI,, 0.02% gelatin, 200 p M each dNTP, 0.2 FM S’-primer (oligo l ) , 0.2 p M 3’-primer (oligo 2) and 2.5 U Taq polymerase. Samples were overlaid with 50 p1 mineral oil and subjected to denaturation at 94°C for 4 min. This was followed by 30 cycles of denaturation at 94°C for 2 min, annealing at 60°C for 2 min and extension at 72°C for 90 sec. The last cycle had an extension period of 7 min. As a positive control, the murine NCAM clone N1 which contains the VASE sequence was used (Santoni et al., 1989). In some experiments, poly (A)’ mRNA was extracted using a commercial kit (British Bio-tech, Oxford, UK) and used instead of total RNA. Analysis of PCR products Mineral oil was removed using 100 ~l ch1oroform:isoamylalcoho1 (24:l). 10 pl of PCR product was run on a 2.5% agarose gel in ~1 T A E buffer. The bands were blotted onto GeneScreen Plus (NEN, Boston, MA) membrane. Oligonucleotides 3 and 4 were end-labelled by T4 polynucleotide kinase and used at 106cpmiml for hybridization in a solution consisting of 10% dextran sulphate, 1 % SDS, 1 M NaCl and 100 Fgirnl salmon-sperm-denatured DNA. The blots were washed to a final stringency of 0.1 x SSC, 1% SDS at 60°C and exposed to Fuji X-ray films for an appropriate period. Oligonucleotides Oligonucleotides were numbered according to Barton et al. (1988). Oligonucleotide 1 AGC AGG TCA CTC TTA CCT GT (967-987) Oligonucleotide 2 GGC GGT GCA CAT GTA CTC (1044-1062) Oligonucleotide 3 (complementary to exon 7) GAT GTT CCG GGT AGA AGT CCT CCA (1003-1045) GGT GAT GGA GGG AAT GGG Oligonucleotide 4: (Complementary to mouse VASE) CTC TTG CTT CTC TGG TCG AGT CCA CGA TGC
DNA sequencing Separated bands were excised from the agarose gel, DNApurified and subjected to asymmetric PCR as described above, but with the following modification: (0.5 )J.M 3’ primer: 0.01 p~ 5‘ primer); 0.2 pmole of the asymmetric PCR product was sequenced by the dideoxynucleotide chain termination method as described by Winship (1989).
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VAN DUIJNHOVEN ET AL.
RESULTS
Western blotting Five well-characterized SCLC cell lines were examined for expression of NCAM by Western blot analysis using the anti-NCAM MAb ERIC-1. GLC-14, -16, -19 and -28 are “classic” SCLC lines, whereas GLC-1 has characteristics associated with the “variant” type. In the absence of neuraminidase treatment, a smear ranging from 140 to 200 kDa was identified in all lines (Fig. 1) although in some instances the smear was weak (e.g., Fig. 1, lane B). In addition, some distinct bands within this smear could be observed (Fig. 1). The smear is indicative that NCAM expressed on these cell lines is the highly polysialylated form containing high levels of a2-8 linked polysialic acid (PSA) residues (Frost et al., 1991). After neuraminidase treatment to remove the PSA, distinct bands of 140 and 120 kDa could be seen. Furthermore, GLC-1, -19 and -28 also contained an additional band of 95 kDa. The expression of NCAM in freshly frozen SCLC tumour tissue obtained from surgically excised specimens was also compared to that in the cell lines. The data obtained were essentially similar; i.e., a smear was detected in the absence of neuraminidase treatment, whereas distinct bands of 140, 120 and 95 kDa were present after removal of PSA by neuraminidase treatment (Fig. 1; lane A). No such bands were detected in any samples using an irrelevant antibody M340. For the sake of brevity, only data for line GLC-1 are presented (Fig. 1; lane G). Northern blotting T o confirm the Western blot data, the NCAM mRNA species present in SCLC cell linesltissues was examined. Total R N A was separated on agarose gels, blotted and hybridized with the human NCAM cDNA probe NC7. A high degree of concordance was observed between the Western and Northern blot data. Cell lines expressing the 140- and 120-kDa isoform of NCAM contained NCAM mRNA transcripts of 6.7 and 5.4 kb. For the sake of brevity, we present only data on the GLC-1
cell line which expresses a major mRNA species of 6.7 kb and weak bands of 5.5 and 5.0 kb which were not easy to reproduce photographically (Fig. 2; lane A). In occasional tumour samples, a weak band of 7.4 kb was also identified (Fig. 2; lane C). This NCAM mRNA codes for the 180-kDa form of the molecule, suggesting that this isoform can also be expressed in SCLC tumours. Unfortunately, due to lack of material, these results could not be confirmed biochemically. No NCAM transcripts were detected in normal lung, squamous carcinoma or adenocarcinoma (Fig. 2). Similar data were obtained from tissue samples. Screening a large panel of cell lines and tissues showed that only SCLC and carcinoids contained mRNA for NCAM (Table I). The 5.0-kb band (mentioned above) present in some of the cell lines and tumours was not due to non-specific hybridization of NCAM probe with rRNA, as identical blots were obtained using poly (A)’ RNA. Furthermore, this transcript has also been identified in other tumour cell lines expressing NCAM, namely neuroblastoma and rhabdomyosarcoma (data not presented).
PCR analysis of the splicing of the VASE mini-exons The splicing of the mini-exon, VASE, was examined in the SCLC cell lines by PCR and Southern blotting. Total RNA was reverse transcribed and the resulting cDNA amplified using the PCR primers oligos 1 and 2. The samples were separated on 2.5% agarose gels, blotted and hybridized with appropriate oligonucleotides. Using an oligonucleotide complementary to the murine VASE sequence, a major band of 224 b p and a minor band of 260 bp were detected in SCLC lines. Furthermore, similar data were obtained with human adult brain (Fig. 3; lane A). The position of the 224-bp fragment corresponds to the position of the PCR product amplified from a murine cDNA clone containing the VASE exon (Fig. 3, lane H). In contrast, no bands were detected in GHI, a haemopoietic cell line known not to express NCAM (Fig. 3; lane G).
FIGURE 1 - NCAM expression in human small-cell lung carcinoma cell lines and tissues. NP-40 cell or tissue lysates were electrophoresed on 7% SDS-PAGE gels and electroblotted. Blots were subsequently incubated with MAb ERIC-1 or an irrelevant MAb (M340). After washing, the blots were further incubated with sheep anti-mouse Ig conjugated to alkaline phosphatase and colour developed as described in “Methods”. Amersham International Rainbow Markers were used for estimation of molecular weight. (200 kDa myosin, 116 kDa P-galactosidase, 95 kDa phosphorylase P, 69 kDa bovine serum albumin, 46 kDa ovalbumin, 30 kDa carbonic anhydrase). -, no neuraminidase; +, neuraminidase-treated [Closfridiumpe~~ingen~ ( 5 U for 5 hr)J. A = SCLC tumour tissue, incubated with ERIC-1; B = GLC-1 cell line incubated with ERIC-1; C = GLC-14 cell line incubated with ERIC-1; D = GLC-16 cell line incubated with ERIC-1; E = GLC-19 cell line incubated with ERIC-1; F = GLC-28 cell line incubated with ERIC- 1; G = GLC-1 cell line incubated with irrelevant antibody M340.
121
MINI-EXON VASE OF NCAM IN LUNG CARCINOMA
FIGURE 2-NCAM mRNA species detected in tumour and normal lung tissue. Total RNA was glyoxylated, separated on 1% agarose gel and transferred to Hybond N membrane. The membrane was hybridized with radiolabelled NC7 probe and washed to high stringency. Glyoxylated Hind111 digest markers were used to estimate the size of the mRNA bands. A = GLC-1 cell line; B = normal lung; C = SCLC tumour tissue 1; D = SCLC tumour tissue 2; E = carcinoid tumour; F = squamous carcinoma; G = adenocarcinoma. The 5.5- and 5.0-kb bands in track A were too weak to be identified on the photograph, but are clearly visible on the autoradiogram. TABLE I - NCAM mRNA EXPRESSION IN CARCINOMA OF THE LUNG Cell line?
Classic SCLC Variant SCLC NSCLC
NCAM mRNA
515' 818 012
Tumourr
SCLC Carcinoid Adenocarcinoma Sauamous-cell carcinoma
516 415 017 019
'Number positive out of total tested.
The weak band of 260 b p appears to be an artifact since separation of the PCR products under denaturing conditions and subsequent hybridization did not reveal its presence (data not shown). Using an oligonucleotide lying outside the VASE exon, i.e., oligonucleotide 3, 3 products of 194, 224 and 260 bp (weak) were identified. The 194-bp fragment is derived from NCAM mRNA lacking the VASE exon.
DNA sequencing To confirm the identity of the 194- and 224-bp fragments, asymmetric PCR was performed on separated bands from the first round of PCR. The product from asymmetric PCR reaction was sequenced by the dideoxy-nucleotide chain termination method. Figure 4 shows the comparison of the published murine VASE sequence and the sequence obtained for human VASE. It can be seen that both sequences are identical except for one base change in the 3rd nucleotide of the sequence where a
FIGURE3 - Identification and characterization of the cDNA amplified from SCLC mRNA. Total RNA was reverse transcribed and the resulting cDNA amplified by polymerase chain reaction. Ten microlitres of the PCR product were run on a 2.5% agarose gel, transferred to Genescreen plus membrane and hybridized either with radiolabelled oligonucleotide complementary to murine VASE (panel a ) or an oligonucleotide complementary to exon 7 (panelb). A = GLC-1; B = GLC-14; C = GLC-16; D = GLC-19; E = GLC-28; F = adult human brain; G = GH1; H = Clone N1.
thymidine replaces an adenosine residue. This substitution does not result in a change in the amino-acid coded for by the triplet. In addition, sequencing of the regions flanking the VASE mini-exon indicates that no other new mini-exons could be identified in the 194- and 224-bp PCR fragments. DISCUSSION
Many families of adhesion molecules have been described which are believed to play an important role in a variety of developmental processes. These include the immunoglobulin gene superfamily (Edelman, 1985, 1988),cadherins (Takeichi, 1988), integrins (Ruoslahti and Pierschbacher, 1987), extracelM a r matrix molecules (Ruoslahti, 1988) and selectins (Springer and Lasky, 1991). Since many of the processes that occur during normal development, e.g. cell-cell interactions and migration, are also relevant t o tumour biology and metastasis, it is likely that some of these molecules will be important in carcinogenesis. We have previously shown that a number of MAbs reactive with SCLC (Cluster 1) recognize the neural cell adhesion molecule (NCAM) (Pate1 et al., 1989) which is involved in cell-cell interactions (Cunningham et al., 1987). The expression of this molecule is complex, with multiple forms of the protein being described. These various forms of the molecule arise from a single gene through post-transcriptional control, such as alternative splicing and use of multiple polyadenylation sites, and through post-translational modifications, such as phosphorylation, sulphation and glycosylation. Although the precise functions of these various isoforms of NCAM remain unclear, their expression appears to be strictly regulated during development. This suggests that they play important, but subtly different roles. We have investigated the expression of NCAM in SCLC since it appears to be a major antigen that is expressed in this
122 EXON 7
VAN DUIJNHOVEN ET AL.
5' EXON 8 5' -------------.....--------------VASE-EXON----......-----------------3'
3'
AAC ATC AGC AGT GAA GAA AAG GCA TCG TGG ACT CGA CCA GAG AAG CAA GAG ACT CTG GAT GGG CAC ATG Mouse AAC ATC AGC AGC GAA GAA AAG GCT TCG TGG ACT CGA CCA GAG AAG CAA GAG ACT CTG GAT GGG CAC ATG Human N I S S E E K A S W T R P E K Q E T L D G H M
FIGURE 4 - Comparison of the human and murine VASE sequence. Asymmetrically amplified PCR fragment (224 bp) was sequenced by the dideoxy chain termination method.
tumour. Our present data show that the NCAM protein isoforms and mRNA species are differentially expressed in the SCLC cell lines and tumour tissues we have investigated. The 140- and 120-kDa isoforms and their corresponding mRNAs, 6.7 and 5.5 kb respectively, were present in all lines/tissues, whereas the 7.4-kb transcript coding for 180-kDa isoform was found in occasional samples. A 95-kDa protein was also identified in some SCLC samples. The nature of this NCAM product is currently unclear, although a similar protein has been found in adult brain (Frost eta[., 1991; Patel et a[., 1991). In some respects, our data are at variance with those published previously. Firstly, at the protein level we have observed highly sialylated NCAM, whereas previously discrete bands of 180 and 140 kDa have been described (Kibbelaar et a/., 1989; Aletsce-Ufrecht et al., 1990) which suggests that, although these forms were reported to be highly sialylated by Kibbelaar et al. (1989), these are less sialylated NCAMs. Secondly, we show that the 140 kDa band is not specific for SCLC, as previously believed (Aletsce-Ufrecht et al., 1990) and finally, we describe the presence of a 95-kDa band. The reasons for these discrepancies could be manifold. For example, they may b e due to different methods of investigation, e.g. immunoprecipitation vs. Western blotting or use of different MAbs. Moreover, the polysialic acid on NCAM is very labile. The presence of highly sialylated NCAM has been demonstrated in other round-cell tumours e.g. neuroblastoma and rhabdomyosarcoma. In neuroblastoma, only a proportion of the NCAM isoforms described in human brain are present (Phimister et al., 1991). This is also the case with SCLC. Because of the differences between the data described here and those reported previously, we have attempted to confirm the Western blotting data by characterizing NCAM mRNA species present in SCLC linesitissues. Transcripts of 6.7, 5.5 and 5.0 kb were present with differential expression of the 7.4 kb. The presence of the 5.0 kb has not been described previously in normal tissues, but its presence on neuroblastoma and rhabdomyosarcoma cell lines has been documented (Patel et al., 1991). The detection of this transcript is not due to non-specific hybridization to rRNA since use of poly (A)' RNA gives identical results to those documented above.
The mini-exon, VASE, spliced between exons 7 and 8, has been studied in mice and rats (Santoni et al., 1989; Small t't al., 1988). It has been shown to consist of a 30-bp sequence whose insertion is developmentally regulated. Initially, it was thought to be neuron-specific, but its presence in rat heart muscle has been reported (Reyes et al., 1991). In this report, PCR fragments lacking or containing VASE mini-exons have been shown to be present in SCLC lines. D N A sequencing has confirmed the presence of VASE in the 224-bp fragment. W e have shown biochemically that multiple forms of NCAM are associated with SCLC. Furthermore, through PCR analysis, it appears that VASE-containing, as well as VASE-lacking, NCAMs are present. However, we have been unable to correlate the expression of various NCAMs in SCLC with general adhesiveness of the cells or with disease states. We found similar levels of NCAM expression in one variant (GLC-1) vs. classic (GLC-14, -16, -19 and -28) lines by Western blotting and also no difference in mRNA expression in a larger panel of classic SCLCs vs. variant SCLCs by Northern blotting (Table I). In addition, there was no discernible difference in NCAM expression between cell lines established from the same patient prior to chemotherapy (GLC-14) and those established after first (GLC-16) and second rounds (GLC-19) of therapy. GLC-14 grows as tight aggregates, whereas GLC-16 and -19 grow as loose aggregates. Adhesion is almost certainly a multifactorial process, with interactions occurring between a number of different adhesion molecules, of which NCAM is only one. Before the process can be fully understood, a detailed knowledge of the different forms of the molecules expressed on cells is critical. Once this has been achieved, it may also lead to opportunities to develop highly selective diagnostic tools in the future. ACKNOWLEDGEMENTS
We are grateful to the Imperial Cancer Research Fund for financial support. In addition, we thank Dr. Barthels (University of Cologne, Germany) for the generous gift of clone N1, Ms E. Moors, Ms E. Timmer and Mr A. Groeneveld for technical assistance and Ms S. Murphy for typing the manuscript.
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