Remarks to opening session.

A panel of 21 small cell lung cancer cell (SCLC) lines were examined for the presence of Transforming growth factor P receptors (TGF3-r) and the expression of TGFP mRNAs. By the radioreceptor assay we found high affinity receptors to be expressed in six cell lines. scatchard analysis of the binding data demonstrated that the cells bound between 4.5 and 27.5 fmol mg' protein with a KD ranging from 16 to 40 pM. TGFP, binding to the receptors was confirmed by cross-linking TGFP, to the TGFP-r. Three classes of TGFI-r were demonstrated, type I and type II receptors with M, = 65,000 and 90,000 and the betaglycan (type III) with Mr= 280,000. Northern blotting showed expression of TGFP, mRNA in ten, TGFP2 mRNA in two and TGFP3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGFP-receptors and also produce TGFI mRNAs.

III) with Mr= 280,000. Northern blotting showed expression of TGFP, mRNA in ten, TGFP2 mRNA in two and TGFP3 mRNA in seven cell lines. Our results provide, for the first time, evidence that a large proportion of a broad panel of SCLC cell lines express TGFP-receptors and also produce TGFI mRNAs.
The TGFP family consists of several members of structurally related proteins. The first member of this family to be cloned was TGFPi1 (Derynck et al., 1985). To date three other members have been cloned and described TGFP2 (Miller et al., 1989b;Madisen et al., 1988;de Martin et al., 1987) from murine and human source. TGFP3 (Miller et al., 1989a) from murine source and TGFPI4 from chicken embryonic tissue (Jakowlew et al., 1988). These members form a complex network of interacting ligands. The role for each of these has not been clearly elucidated but the expression pattern in the mouse embryo suggest a role in differentiated role in embryogenesis (Pelton et al., 1991). The TGFI3 family of peptides exerts both stimulatory and inhibitory effects depending on cell type examined (Barnard et al., 1990).
Receptors for TGFP have been demonstrated in a variety of normal cells of both epithelial and mesenchymal origin as well as in several malignancies (Frolik et al., 1984;Tucker et al., 1984;Massague & Like, 1985;Wakefield, 1987). At present five distinct TGFI3-r have been identified, type I (Mr = 60-70,000), type II (85-110,000), type III (200-400,000), type IV (60,000) and type V (40,000). The type II and III receptors have recently been cloned (Lin et al., 1992. In addition a TGFP binding protein (150,000 and 180,000) has been described, which binds TGFPi3 but not TGFP2 (MacKay & Danielpour, 1991). The type I and II receptors are the most probable candidates as the mediator of the signal induced by TGFP (Boyd & Massague, 1989;Laiho et al., 1990). The type III receptor is believed to be a surface associated proteoglycan, which binds TGFP and ultimately releases it (Andres et al., 1989) or is internalised with TGFP (Massague, 1990). The type IV receptor has been identified in pituitary cells, but its function has not been established (Cheifetz et al., 1988). The function of the type V receptor, which has been purified from bovine liver, is unclear at present (O'Grady et al., 1991). Several malignancies have been screened for the presence of TGFP-r, but in human lung cancer the data is very sparse. A few studies have demonstrated that TGFI3 mRNA was expressed in only non-SCLC (NSCLC) cell lines (Soderdahl et al., 1988;Derynck et al., 1987;Bergh, 1988). In another study all of ten SCLC cell lines examined were found to be TGF,B mRNA negative (Lagadec et al., 1991). In these studies the TGFP isoform investigated was not specified, but most probably it was TGFPI3 mRNA.
These data are the basis for the concept that only NSCLC cell lines can produce TGFP (for review, see Pelton & Moses, 1990).
In the present study we have examined the presence of TGFf-r and the production of TGFP mRNA in a panel of 21 SCLC cell lines established in five different laboratories.
The results showed that a relatively high proportion of SCLC cell lines carried high affinity TGFI-r and expressed TGF3 mRNA. Coexpression of TGFI-r and TGFP was found in six SCLC cell lines.

Materials and methods
Cell lines SCLC cell lines were cultured in 150 cm2 flasks at 37°C under standard conditions in medium containing 10% foetal calf serum (Flow Laboratories, Irvine, Scotland) without antibiotics. We have previously reported in detail the growth morphology and tissue culture media for these cell lines (Damstrup et al., 1992). Twenty-one SCLC cell lines established from 17 patients in five different laboratories were examined. Eight cell lines were established at Dartmouth Medical School, Hanover, NH, USA (DMS), seven cell lines were established at Gronningen Lung Cancer Center, Groningen, the Netherlands (GLC), two cell lines were established at the National Cancer Institute, Bethesda, MD, USA (NCI), two cell lines were established in Marburg, Germany (24H and 86M1), and two cell lines were established in our own laboratory Copenhagen, Denmark (CPH). The origin and establishment of the cell lines has been described elsewhere (Pettengill et al., 1980;Carney et al., 1985;de Leij et al., 1985;Bepler et al., 1987;Berendsen et al., 1988;Engelholm et al., 1986). AKR-2B, a mouse fibroblast cell line, which previously has been reported TGFJ-r positive (Tucker et al., 1984) was cultured in Eagle's minimal essential medium (Flow laboratory) supplemented with 10% foetal calf serum, and used as a positive control for TGFP binding. This cell line was kindly provided by Professor H.L. Moses, Vanderbilt, University, Tennessee. All cell lines were routinely checked for, and found free of, mycoplasma infection. Cells growing as monolayer cultures were assayed in 35 mm 6-well tissue dishes for radioreceptor assays. Cells were subcultured and used within 24 h of plating. Cells growing as floating aggregates were subcultured and assayed in microfuge tubes within 24 h of subculturing.

Growth factors
Porcine TGFPi1 was purchased from British Biotechnology Ltd, Oxford, England and/or was a gift from Bristol-Meyers-Squibb, Pharamaceutical Research Institute, Seattle, USA. Human recombinant EGF and TGFa was purchased from Bissendorf Biochemicals, Hannover, Germany.
'25I-labelled TGF,I with a specific activity of 100-180pCi pg`(2.5-4.5Cimol%), was purchased from New England Nuclear, Boston, USA. The binding activity of 125I-labelled TGFPI was checked at regular intervals using the positive control cell line AKR-2B. The '25I-labelled TGFP1 was used within 4 weeks of fresh lot date.
Radioreceptor assay The procedure has been described previously (Massague & Like, 1985;Massague, 1987). Cells growing as monolayer culture were plated in 35 mm 6-well dishes, usually at 2-5 x 105 cells per well, the day before experiments were performed. The cells were washed for 60 min with binding buffer (128 mM NaCl, 5 mM KCI, 5 mM MgSo4, 1.2 mM CaCl2, 50 mM HEPES, pH 7.5 and 2% -BSA). After washing, the cells were incubated with 5-1O pM 125I-labelled TGF,1 and increasing levels of native unlabelled TGFPI ranging from 0 to 200 pM, the volume of incubation being adjusted to 1 ml. After 2 h incubation at 20°C the reaction was stopped by washing the plates three times with ice cold binding buffer without albumin. After the final wash, the cells were solubilised in solubilisation buffer (128 mM NaCl, 0.25 mM EDTA, 0.5 mM Tris, pH 7.5 and 1% v/v Triton X-100). An aliquot of the supernatant was counted in a Beckmann II gamma counter (70% efficiency). Protein concentration was detennined with the BCA protein kit (Pierce Europe, B.V., Oud Beujerland, The Netherlands) (Smith et al., 1985). Cells growing as floating aggregates or cells easily detectable were assayed, as single cell suspensions, in 1.5 ml sigmacote (Pierce) treated microcentrifuge tubes. Viability after obtaining a single cell suspension, assessed by trypan blue exclusion test, was 90-95%. After incubation the reaction was stopped by centrifuging at 5,500 g for 3 min and the cell pellet was resuspended three times in ice cold binding buffer without albumin. After the final wash, the cell pellet was solubilised as above. Maximal binding (Bm.) was calculated as femtomol mg-' protein by Scatchard analysis of the binding data (Scatchard, 1949). Specificity of the binding was determined in specificity experiments with TGFPI, EGF and TGFa as the displacing agents. The displacing agents were added at the same time as the '25I-labelled TGFPI.
Cross-linking Washed single cell (2-5 x 106) suspensions were incubated with 40 pM '25I-labelled TGFPI in the presence or absence of a 100-fold excess of unlabelled TGFP1. The incubation proceeded for 4 h at 4°C. After the final wash, the cell pellet was resuspended in 950 p1 binding buffer without BSA before 50 p1 of 5 mM cross-linking agent disucinimidyl (DSS) (Pierce, France), freshly dissolved in DMSO, was added. The cross-linking reaction proceeded for 15 min at 4°C and was stopped by centrifuging and washing the pellet in a Triscontaining buffer. Finally the cell pellet was resuspending in 80 p1 solubilisation buffer, 10 p1l cocktail 1 and 10 p1l cocktail 2 as described earlier (Massague, 1987). The resulting supernatant was boiled for 5 min in sample buffer with 50 mM dithiothreitol (Pierce). One hundred fig protein/lane was run on a 5, 7 or 10%, 8 x 16 cm SDS-PAGE gel. After staining with Coomassie brilliant blue and destaining, the dried gel was exposed to an X-ray film (Amersham) with an intensifying screen at -80°C.
Northern blotting RNA was extracted by the single-step acid guanidinium thiocyanate-phenol-chloroform method (Chomczynski & Sac-chi, 1987). Ten pg total RNA samples were electrophoresed through denaturing agarose gels containing 2.2 M formaldehyde, and transferred to nylon membranes (GeneScreen Plus, NEN DuPont) as recommended by the supplier. Radiolabelled probes were prepared by the random priming method (Feinberg & Vogelstein, 1983) using [a-32P]dCTP and a commerical kit (both from Amersham). The blots were sequentially hybridised with human probes for TGFII and for P-actin. The probes for TGFI31 were a 2.0 kb full length cDNA (Kasid et al., 1988) obtained from the American Type Culture Collection (No. 59954) and a 267 bp fragment spanning nucleotides 1773-2040. The probe for TGFP2 mRNA was a 442 bp murine fragment of the plasmid pmTGFb2-9a (Miller et al., 1989b). The TGFI33 probe was a 609 bp murine fragment of the plasmid pmTGFP3-IIb (Miller et al., 1989a). The TGFP2 and TGFP3 probes were obtained from Professor H.L. Moses, Vanderbilt University. RNA extracted from murine heart and lung was used as positive controls. The P-actin probe was a 2.1 kb BamHI fragment of the plasmid pHFj3A-1 (Gunning et al., 1983). The membranes were prehybridised, hybridised and washed as recommended by the supplier, and exposed to an X-ray film at -80C with an intensifying screen.

Receptor binding studies
Saturation of the receptors were reached with a TGFPI3 concentration in the range of 50 to 100 pM (exemplified in Figure 1). Non-specific binding, defined as the cell associated radioactivity in the presence of a large excess of unlabelled TGFPI, was relatively high but was an inverse function of binding capacity -23% in AKR-2B with a Bm., of 70 femtomolmg-' protein and 70% in GLC 19, assayed as a single cell suspension, with a Bm., of 4.5 femtomolmg-' protein. This relationship has also been described in other cell types (Massague, 1987). As about half the cell lines grew as floating aggregates and half as monolayer cultures, we chose to relate all binding data to protein concentration. Scatchard analysis of the binding data showed that cells bound between 5 and 27 fmol mg-' protein with a KD of 16-40 pM ( Table I).
The specificity of the TGFI-r/ligand binding was determined using different displacing agents. The specificity of 1251I-labelled TGF,I binding to GLC 3 is shown in Figure 2.
It was found that EGF and TGFa, which both binds to the EGF-receptor (Carpenter et al., 1983) did not displace 125ilabelled TGFPI. For all cells tested and found positive in the radioreceptor assay, binding was in all cases specific and saturable.
Cross-linking studies TGFP, binding to the receptors was further visualised by cross-linking the ligand-receptor complex with DSS. Figure 3 illustrates the affinity labelling results of eight SCLC-cell lines. Following electrophoresis on a SDS-PAGE gel, specific TGFiI binding was seen as bands with calculated Mr of 65,000, 90,000 and 280,000; these bands correspond to the type I, II and III TGFI-r. The presence of excess unlabelled TGFPI resulted in the disappearance of these bands, demonstrating that the binding was specific. Seven SCLC-cell lines were TGF3-r positive in the affinity labelling experiments (Table I).
Northern blotting studies The cell lines were examined for the production of TGFI3 mRNA. Figure 4a  for TGFPi2 and TGFPI3. In two cell lines (Figure 4b) TGFP2 mRNA was detected as a faint band of 3.9 kb. TGFPi2 mRNa, size approximately 3.5 kb, was found in seven cell lines (Figure 4c). Blots were rehybridised with the 1B-actin probe to demonstrate equal loading in all lanes. The intensity of staining with the TGFP probe therefore semiquantifies the TGFJ3 mRNA content. The results for all binding data, affinity labelling and Northern blot analysis are summarised in Table I. Six of the TGF3-r positive cell lines also expressed TGFP (Table I).   elsewhere (Frolik et al., 1984;Tucker et al., 1984), and found that the binding was stable for a prolonged period of time at 20°C. Furthermore we found that the receptors could not be demonstrated if the protein concentration was lower than 150 jLl ml-'. We have previously demonstrated, in the same cell lines, studying the EGF-receptor that this critical protein level was also required to detect the EGF-receptor (Damstrup et al., 1992). Studies on other receptor systems such as the estrogen receptor has also demonstrated this critical protein limit (Skovgaard Poulsen, 1981). Therefore, to avoid underestimating binding capacity or falsely classify a SCLC cell line as TGFP-r negative, we only drew conclusions on the TGF3-receptor state in a cell line if the protein concentration was in the range of 200-600pgml-'.
Analysis of the binding data demonstrated that Scatchard plots in some cell lines were curved near saturation of the receptors. However, it was not possible, with the ligand program developed by Munson and Rodbard (Munson & Rodbard, 1980), to resolve the Scatchard plots into two or more compartments. Other investigators have, in normal rat L. DAMSTRUP et al. C] az C a (D (   Figure 4 Northern blot analysis of TGFI expression in SCLC. a, Probed with the TGFI, cDNA, b, with TGFP2 and c, with TGFP3. The resulting 7 day autoradiography is shown. The 2.5 kb TGFP, mRNA, the 3.9 kb TGFP2 mRNA and the 3.5 kb TGFP3 mRNAs are indicated. Transcript size for TGFJI was determined with reference to the 18 and 28S bands. Transcript sizes for TGFP2 and TGFP3 were determined with reference to mRNA molecular weight markers with band sizes 1.4, 2.4, 4.4, 7.5 and 9.5 kb (Life Technologies). The P-actin probing indicate that the lanes were loaded equally. The band seen in all cell lines probed with TGFPI represents non-specific binding the 28S (4.8 kb) ribosomal band. *Murine lung mRNA is included as a positive control for the TGFPi2 and TGFP3 probes. kidney cell (NRK), also only demonstrated one class of TGFP receptors, despite the fact that cross-linking studies with NRK cells have demonstrated that these cells express type I, II and III receptors (Wakefield, 1987;Massague & Like, 1985;Segarini et al., 1987). Resolving the data with a single class receptor from the linear part of the Scatchard plot demonstrated high affinity receptors in six SCLC cell lines (Figure 1, Table I). The dissociation constant was in all cases characteristic for TGFP binding (Massague, 1987;Wakefield, 1987). Maximal binding varied from 4.5 to 27.5 fmol mg-' protein. Binding of 251I-labelled TGFI,, to the positive cells was specific as only TGFP could displace the labelled TGFBI. EGF and TGFa did not influence TGFP binding ( Figure 2). The results obtained from the radioreceptor assay and the displacement studies demonstrated that a large proportion of the SCLC cell lines examined carried specific high affinity TGFI-r. Our results are in part corroborated as one of these cell lines, GLC 19, has previously been reported to be growth inhibited by TGFP (Lagadec et al., 1991). However, in the cited study the cells were not examined for the presence of TGFI-r.
To verify that the binding of TGFPI was in fact to the TGF3-r, the cell lines were tested by cross-linking. After size fractionation on SDS-PAGE gels, all cell lines found to be TGF3-r positive in the radioreceptor assay also displayed one or more specific bands with calculated Mr = 65,000, 90,000 and 280,000 (Figure 3). These sizes include reduced TGFP with a Mr of approximately 12,000. The Mr of the corresponding receptors is therefore 53,000, 78,000 and 270,000. The TGFPI receptors have previously been reported as having these calcualted molecular weight (Massague, 1987;Massague, 1990). This provides further evidence that TGFP1 binding was to the TGFI-r. One cell line, GLC 16, expressed the type I and II TGF,B-r in this assay. We could, however not demonstrate the receptor in the radioreceptor assay, even using a very high protein concentration (>800 fig ml-'). The binding capacity in this cell line could be so low that it was below the detection limit in the radioreceptor assay. In the same cell line, we have found that the receptor was functional in that exogenously added TGFP, acted as a growth inhibitor (N0rgard, P., unpubished observation).
We also examined the expression of TGF,B mRNAs in the panel. In 10/20 SCLC cell lines TGFI,B mRNA could be detected (Table I, Figure 4a). In GLC 3 and faintly in DMS 153 an additional band of 1.7 kb was found, the nature of this band is unclear. A mRNA with this approximate size has also been found in male mice germ cells (Watrin et al., 1991).
We examined 19 of the SCLC cell lines with a probe for TGFP2 mRNA (Figure 4b), and in two cell lines (DMS 53 and DMS 114) a single transcript of 3.9 kb was demonstrated. This is in accordance with one of the TGFP2 transcripts reported in other human cell lines (Mori et al., 1990), whereas none of the additional TGFI2, mRNAs reported (Jakowlew et al., 1991;Mori et al., 1990;Miller et al., 1989b) were detected in the investigated cell lines.
We have previously examined 15 of the cell lines in our panel for expression of phosphorylated retinoblastoma gene product (pRb) (Rygaard et al., 1990 detected, were found to express pRb, and immunocytochemistry demonstrated nuclear localisation of pRb (Rygaard, K., unpublished observation). Other studies (Templeton et al., 1991) have suggested that pRb is functional only when phosporylated and located in the nucleus. It has recently been reported that the pRb activates the expression of TGFI32 (Kim et al., 1992). Provided that the characteristics of pRb in DMS 53 and DMS 114 indicates that the protein is functional, our finding that TGFP2 was detected exclusively in the two cell lines also expressing a 'functional' pRb, agrees with the concept that expression of TGFP2 is activated by pRb.
Seven cell lines expressed the 3.5 kb TGFP3 mRNA ( Figure   4c), corresponding to the reported size in other human malignant tissue (Dijke et al., 1988). Two of these cell lines (GLC 2 and 24H) also expressed a transcript with a size of approximately 2.5 kb, which is the transcript size of TGFPI mRNA, however, these two cell lines did not express TGFPi mRNA.
The probing for TGFP2 and TGFP3 was performed with a murine probe, and there may not be perfect homology to the human mRNA. This implies that additional cell lines could be positive following probing with a human probe. Taken together a total of 12 of the 20 examined SCLC cell lines expressed TGFP mRNA. This finding is in contrast to earlier studies, where a few cell lines have been examined and found to be TGFP mRNA negative (Soderdahl et al., 1988;Derynck et al., 1987;Lagadec et al., 1991). In one of these studies (Lagadec et al., 1991) the examined cell lines included GLC 14, 16 and 19, NCI H69 and N417, all these were TGFP mRNA negative. These cell lines were also included in our panel, but we found expression of TGFPI mRNA in GLC 19 and NCI N417. This transcript was detected as a 2.5 kb band using both the full length cDNA and the 267 bp TGFPI fragment. The difference betwen our results and the previous reported study (Lagadec et al., 1991), is not apparent, but could be due to a difference in sensitivity.
Our results based on a panel of 21 SCLC cell lines have demonstrated that TGFI3 receptors were present in seven of 21 SCLC cell lines and more than half of the cells examined expressed TGF,B mRNA. About half of the examined cell lines grew as monolayer cultures and half as floating aggregates, but we could not detect any statistical difference between the growth morphology and the expression of TGFI-r or TGFP mRNAs (Chi-square test with Yates correction and Fisher's exact test, P < 0.2). Coexpression of TGF3-r and the ligand was found in six cell lines. These cell lines therefore have the possibility of an autocrine growth regulation. The question whether the SCLC cell lines produce TGF, protein and if this is biologically active is currently being investigated.