A spontaneously transformed pulmonary embryonic rat epithelial cell line (PER) is described in terms of growth, tumorigenicity, growth factor responsiveness and biosynthetic capacity. At low-passage subcultures, PER cells grew as a monolayer and did not form colonies in soft agar. After long-term subcultivation, they lost contact inhibition, became anchorage-independent and formed tumours in nude mice. Low concentrations of foetal calf serum permit the maximum growth rate. The multiplication and metabolic activity, assessed by 2-deoxy-D-glucose uptake, was significantly stimulated by growth factors. PER cells synthesized collagen types I, III, IV and V, laminin and fibronectin, and organized a pericellular matrix made up of only basement membrane components (type IV collagen and laminin) and fibronectin. These data enabled us to define PER cells as a transformed epithelial cell line evolving towards malignancy with long-term subcultivation. These cells appeared to be a valuable tool in studies of tumour cell-matrix interactions and regulation of growth factor receptors in tumorigenesis.

The regulation and/or the maintenance of cell differentiation operated by the supporting extracellular matrix or the basement membrane might be significant in the preservation of physiological characteristics. This interaction might depend on information received by the cells through membrane receptors recognizing extracellular components such as fibronectin (Grinnell & Feld, 1979; Takashima & Grinnel, 1984), collagen type I (Klebe, 1974; Burke et al. 1983) and laminin (Terranova et al. 1980; Couchman et al. 1983).

Experimental models in vitro and in vivo provide support for the importance of this interaction in the process of tumour development, invasion and metastasis that might be significant in the expression of a transformed phenotype (Gospodarowicz, 1983). For example, Michiels et al. (1981) failed to observe malignant transformation developing in embryonic lung explants exposed to carcinogens while the dissociation of the cells from the explants resulted in the establishment of a transformed tumorigenic cell line. A similar cell line was obtained without treatment with a carcinogen just by dissociation of lung explants (Michiels et al. 1981). These cells proved to be most interesting in terms of cell-matrix relationship (Paye & Lapière, 1986) as well as in the alteration of the turnover of epidermal growth factor (EGF) receptors (Nishiyama et al. unpublished data).

We describe here the growth properties of such cells in vitro, their biosynthetic capacity in terms of collagen, basement membrane components and attachment glycoproteins, as well as their metabolic and mitotic response upon stimulation with defined growth factors.

Materials

Reagents were obtained from the following sources: 3,5-diamino-benzoic acid (DABA), fibroblast growth factor (FGF), epidermal growth factor (EGF) from Sigma; [methyl-M]thymidine (6 · 7 Ci mmol− 1), 2-deoxy-D-[l-3H]glucose (8 · 3 Ci mmol− 1), [5-3H]proline (10 Ci mmol− 1) from New England Nuclear. Culture medium was purchased from Gibco and foetal calf serum from Gibco and Flow Laboratories. Tissue culture plates were obtained from Nunc. Anti-rabbit fluorescein-conjugated goat immunoglobulin G (IgG) and anti-guinea-pig rhodamine-conjugated goat IgG were purchased from Dako.

Cell culture

Pulmonary embryonic rat (PER) epithelial cells were obtained by dissociation of lung explants from 15-day-old Wistar rat embryos after organ culture for 16 days according to the technique described by Michiels et al. (1981). These cells are known to undergo transformation spontaneously upon prolonged subculture.

The cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% (v/v) foetal calf serum (FCS), glutamine (292mgl− 1), ascorbic acid (50 μ gml− 1), penicillinstreptomycin (100 units ml− 1) at 37°C in a controlled atmosphere containing 5 % CO2-They were propagated by brief treatment with 0 · 1 % trypsin and 0 · 02% EDTA solution at pH 7 · 4 by a 1:3 split ratio every 5 days. Unless stated otherwise, the cells were used between passages 27 and 45. Normal bovine skin fibroblasts used for comparison were cultivated and propagated using the same conditions.

DNA measurement

The DNA content was measured by the fluorimetnc technique described by Johnson-Wint & Hollis (1982) using recrystallized DABA reagent. Briefly, cell layers were fixed for 24h at 4°C in 10 % formaldehyde in 0-1 M-borate buffer, pH 8, washed with 15 % isopropanol solution, dried for 30 min at 50°C. DABA reagent was added and allowed to react for 45 min at 60°C. After stabilizing the DABA-DNA reaction by adding 6M-HC1, the fluorescence of the reaction medium was measured in a spectrofluorimeter at an excitation wavelength of 420 nm and an emission wavelength of 510nm.

Measurement of the mitogenic response

The cells (7 · 5 × 104) were plated in 30 mm dishes in DMEM containing 10% FCS. Immediately after attachment, they were made quiescent by incubation in DMEM containing 0-05 % FCS. After 24 h, the quiescence medium was replaced with DMEM containing FCS at increasing concentrations or purified growth factors. Twenty-four hours later, [3H]thymidine (7 · 5 μ Ci, 1 μ M) was added for 18 h. After washing three times with cold phosphate-buffered saline (PBS), the cells were lysed in 1ml of 0 · 5M-NaOH for 4h at 37°C. DNA was precipitated by addition of cold trichloracetic acid (30 % final concentration) and harvested by filtration under reduced pressure on Whatman G FA filters and repeatedly washed. The radioactivity retained on the filters was measured by liquid scintillation.

Determination of2-deoxy-D-glucose (2-DOG) transport

Cells were plated on 24-well tissue culture plates (Nunc) and allowed to reach confluence in DMEM with 10% FCS. One day after confluence the cells were maintained overnight in a quiescence medium (DMEM containing 0 · 1% FCS) before being preincubated for 5h in the quiescence medium containing increasing concentrations of dialysed FCS or purified growth factors as indicated. The cells were washed with PBS buffer at 37°C and incubated for 15 min in 1 ml of PBS containing the same concentration of growth factors or dialysed FCS as above. The measurement of 2-DOG transport was initiated by adding 1 · 5 μ Ci of [3H]2-DOG to each well. After 20 min of incubation, cells were washed three times with 1 ml of cold PBS and lysed at room temperature in 0 · 4ml of 0 · 2% sodium dodecyl sulphate (SDS) solution in 0 · 2M-NaOH. Radioactivity of the lysate was measured by liquid scintillation.

Anchorage-independent growth assay

Anchorage-independent growth assay of the cultured cells at passages 38 and 75 was performed in soft agar according to the technique described by Courtenay (1976).

Characterization of the biosynthetic products

Four days after reaching confluence, cultures of PER cells were labelled in the maintenance medium freshly supplemented with ascorbic acid (50 μ gml− 1) and containing beta-aminopropio-nitrile (50 μ gml− 1) and [5-3H]proline (10 μ Ciml− 1). After a 24-h period of labelling, the medium was removed and the cells were washed with physiological saline, scraped in 0 · 05 M-Tris. HC1, pH 7 · 2, 0 · 15M-NaCl, and briefly sonicated. Culture medium and cell homogenate were supplemented with a mixture of protease inhibitors (final concentration 20mM-EDTA, 0 · 5 mM-A?-ethylmaleimide and 0T mM-phenylmethane sulphonyl fluoride) and precipitated by adding ammonium sulphate to 40% saturation. After 18 h at 4°C, the precipitated material was collected by centrifugation and solubilized in 0T M-acetic acid. Samples of the labelled material were digested with pepsin for 18 h at 4°C. Native and pepsin-digested material were submitted to polyacrylamide slab gel electrophoresis in 6 · 25 % separation gel under non-reducing and reducing conditions, according to the technique of Laemmli (1970) slightly modified by the addition of 4M-urea in the separation gel. The labelled bands were revealed by fluorography (Bonner & Laskey, 1974) and exposure to Royal SO-mat Kodak film.

Immunofluorescence studies

Cells were seeded on glass coverslips in bacteriological dishes and allowed to reach confluence. After washing with PBS, they were air-dried and fixed in acetone at −20°C for 10 min. Indirect immunofluorescence studies were performed according to the technique described by Foidart & Reddi (1980). Antibodies, raised in rabbit or guinea-pig, were directed against type I and pN-III collagen purified from bovine foetal skin (Pierard et al. 1984), type IV collagen extracted from human placenta (Van Cauwenberge et al. 1983), fibronectin isolated from human plasma and laminin from EHS tumour (Nusgens et al. 1986). The specificity of the antibodies was tested by ELISA as described by Rennard et al. (1980) and by immunoblotting using the technique of Towbin et al. (1979). The cytokeratin filaments were revealed by a monoclonal antibody from Sanbio (Utrecht, Holland). Control preparations were performed using non-immune serum from rabbit, guinea-pig or mouse. The preparations revealed by goat anti-rabbit fluorescein- or antiguinea-pig rhodamin-conjugated IgG were observed in a Leitz ultraviolet microscope with epiillumination and photographed.

Morphology and transformed phenotype

At low cell density (Fig. 1A), PER cells displayed a roundish and irregular shape and a large amount of cytoplasm containing inclusions that did not stain with Papanicolaou coloration. A small number of cells (7 %) had an elongated fibroblastic shape. At high cell density, the cells had a hexagonal aspect with a small amount of cytoplasm (Fig. IB). Until passage 50, cells grew as a monolayer without overlapping. At higher passages, contact inhibition at confluence was lost and multilayers formed (Fig. 1C). The cytoplasm was very small and no inclusion was visible.

Fig. 1.

Phase-contrast micrographs of PER cells: A, at low density (× 340); B, at confluency and passage 27 (× 170); and C, growing in multilayers at passage 75 (× 170).

Fig. 1.

Phase-contrast micrographs of PER cells: A, at low density (× 340); B, at confluency and passage 27 (× 170); and C, growing in multilayers at passage 75 (× 170).

At passage 38, the PER cells do not grow in soft agar (Fig. 2A) whereas after long-term subcultivation (75 passages) cells were clearly anchorage-independent and formed visible colonies in soft agar 9 days after cloning (Fig. 2B).

Fig. 2.

Phase-contrast micrographs of PER cells seeded in soft agar (104 cells) and maintained in culture for 9 days. A. PER cells at passage 38; and B, PER cells at passage 75 (× 100).

Fig. 2.

Phase-contrast micrographs of PER cells seeded in soft agar (104 cells) and maintained in culture for 9 days. A. PER cells at passage 38; and B, PER cells at passage 75 (× 100).

Responsiveness to FCS and growth factors

At a concentration of FCS of 10% in DMEM the doubling time of PER cells is 23 h. The incorporation of thymidine by sparse culture of PER cells for increasing concentrations of FCS is illustrated in Fig. 3. The stimulation of cellular multiplication was dose-dependent up to 1 % FCS. At this concentration, the uptake of thymidine was maximal and levelled off at higher FCS concentrations. The nutritional requirements of normal skin fibroblasts taken for comparative purpose were significantly higher since the plateau of maximum uptake was not reached even at 5 % FCS. EGF significantly stimulated the DNA synthesis of the PER cells while almost no response was observed upon addition of F-GF up to 100 ng ml-1 (Fig. 4) in confluent culture. The response of fibroblasts to these growth factors was the reverse of that observed in the PER cells, strong activation by FGF and mild stimulation by EGF.

Fig. 3.

[3H]thymidine incorporation (ctsmin− 1 per dish) into DNA of PER cells (O) and normal skin fibroblasts (A) seeded at low density (7 · 5 × 104 cells per 3cm dish) and made quiescent in 0 · 05% FCS for 24 h. After stimulation by increasing concentration of FCS for 24h, [methyl-3H]thymidine was added for 18 h and the radioactivity incorporated into DNA was measured as described in Materials and Methods.

Fig. 3.

[3H]thymidine incorporation (ctsmin− 1 per dish) into DNA of PER cells (O) and normal skin fibroblasts (A) seeded at low density (7 · 5 × 104 cells per 3cm dish) and made quiescent in 0 · 05% FCS for 24 h. After stimulation by increasing concentration of FCS for 24h, [methyl-3H]thymidine was added for 18 h and the radioactivity incorporated into DNA was measured as described in Materials and Methods.

Fig. 4.

[3H]thymidine incorporation (ctsmin− 1, × 104 per dish) into DNA of confluent PER cells supplemented with increasing concentrations (1—100 ngml− 1) of EGF (◼) and FGF (□).

Fig. 4.

[3H]thymidine incorporation (ctsmin− 1, × 104 per dish) into DNA of confluent PER cells supplemented with increasing concentrations (1—100 ngml− 1) of EGF (◼) and FGF (□).

The responsiveness to these growth factors in terms of metabolic activity was assessed by the uptake of 2-DOG (Table 1). 2-DOG transport was stimulated, with a dose-dependent response, by FCS. Both EGF and FGF also induced a stimulation, with the maximum response at 10 ng ml− 1.

Table 1.

Effect ofFCS, EGF and FGF on 2-DOG transport by PER cells

Effect ofFCS, EGF and FGF on 2-DOG transport by PER cells
Effect ofFCS, EGF and FGF on 2-DOG transport by PER cells

Characterization of the biosynthetic products of the PER cells

The polypeptides synthesized in vitro after radioactive labelling with [3H] proline were characterized by SDS-polyacrylamide slab gel electrophoresis (Fig. 5). The radiolabelled collagen polypeptides were found mainly in the form of soluble precursors in the culture medium. After pepsin digestion (Fig. 5, lanes b and d) they proved to be collagen type I (75 %), type III (25 %) and type V (traces). A labelled band of 220 × 103Mr co-migrating with authentic fibronectin and susceptible to pepsin was also observed in the culture medium (Fig. 5, lane c).

Fig. 5.

Fluorogram of 3H-labelled polypeptides precipitated from the culture medium of PER cells by ammonium sulphate at 40 % saturation. The electrophoresis was performed in a 6 · 25 % acrylamide gel under non-reducing (lanes a and b) and reducing (lanes c and d) conditions, before (lanes a and c) and after (lanes b and d) pepsin digestion.

Fig. 5.

Fluorogram of 3H-labelled polypeptides precipitated from the culture medium of PER cells by ammonium sulphate at 40 % saturation. The electrophoresis was performed in a 6 · 25 % acrylamide gel under non-reducing (lanes a and b) and reducing (lanes c and d) conditions, before (lanes a and c) and after (lanes b and d) pepsin digestion.

By indirect immunofluorescence performed on the cell layer, a well-developed extracellular network of collagen type IV (Fig. 6A), laminin (Fig. 6B) and fibronectin (Fig. 6C) was observed. No type I or type III collagen could be detected in the matrix although fine granular intracytoplasmic deposits of pN-I and pN-IH collagen were visible (data not shown). The cytoplasm of all cells contained a well-developed network of cytokeratin filaments (Fig. 6D).

Fig. 6.

Indirect immunofluorescent staining of the extracellular matrix of confluent PER cells using antiserum against: A, type IV collagen (× 450); B, laminin (× 450); and C, fibronectin (× 450). Subconfluent PER cells were stained with a monoclonal antikeratin antibody (D) and with non-immune mouse serum (E); × 1200.

Fig. 6.

Indirect immunofluorescent staining of the extracellular matrix of confluent PER cells using antiserum against: A, type IV collagen (× 450); B, laminin (× 450); and C, fibronectin (× 450). Subconfluent PER cells were stained with a monoclonal antikeratin antibody (D) and with non-immune mouse serum (E); × 1200.

This study was performed to define the morphological characteristics, growth properties, nutritional requirements, biosynthetic activity and metabolic response to growth factors of an established pulmonary embryonic rat (PER) epithelial cell line. These characteristics will be discussed in relation to some specific properties of these cells that might be related to their transformed phenotype and described in detail in the accompanying paper (Paye & Lapière, 1986) and elsewhere (Nishiyama et al. unpublished data).

As often reported for transformed cells (Sanford, 1965; San et al. 1979; Exilie et al. 1980; Mukherji et al. 1984), the PER cells display an irregular, roundish shape at low density. Contact inhibition of growth is observed at low passage and lost at high passage where they form foci of multilayered cells. The malignant potential is supported by the development of anchorage-independent growth and the formation of tumours after inoculation in syngeneic rats (Michiels et al. 1981, and unpublished data).

We have demonstrated the ability of PER cells to live in a synthetic medium containing a low concentration of foetal calf serum while maximum growth is reached with a foetal calf serum concentration as low as 1 %. Multiplication is stimulated by low concentrations of EGF and less significantly by FGF. The metabolic response of PER cells, estimated by 2-DOG transport, is triggered by foetal calf serum, EGF and FGF. As observed in several transformed cell lines (Oshiro & Dipaolo, 1974; San et al. 1979; Siddigi & Lype, 1975), the basal level of sugar transport is about three times higher than that expressed by normal skin fibroblasts and C3H10T 1/2 (unpublished observations).

PER cells display in vitro biosynthetic activities compatible with their epithelial origin as suggested by their content of cytokeratin filaments. A well-developed extracellular network of type IV collagen, laminin and fibronectin supports their capacity to synthesize and to assemble basement membrane components. Labelling experiments show that they also synthesize interstitial collagen type I and type III and traces of collagen type V as observed in several other lines of epithelial cells (Keski-Oja et al. 1982; Alitalo et al. 1982). The PER cells were, however, unable to polymerize these collagens in the extracellular matrix, perhaps due to a lack of procollagen peptidase activity and/or to a lack of appropriate receptors on the cell surface.

All together these data strongly suggest that PER cells are transformed epithelial cells evolving towards malignant transformation with long-term subcultivation. The absence of Papanicolaou positive granules in the cytoplasm, a feature of type II alveolar cells (Kikkawa & Yoneda, 1974) that is, however, rapidly lost in culture, prevents us from confirming that PER cells are a line of pneumocytes II.

This cell line represents an experimental model that is useful in the study of the regulatory activity of growth factors in tumorigenesis since, unlike A431, their multiplication is stimulated by EGF. Moreover, the down-regulation of the EGF receptors of the PER cells is unusual as 50% of the receptors remain at the cell surface after contact with EGF (Nishiyama et al. unpublished data). A similar feature has been recently described in normal rat kidney (NRK) fibroblasts exposed to TGF-beta (Assoian & Spom, 1984).

PER cells also demonstrate defective attachment properties to interstitial collagens that can be corrected by fibronectin modified by a transglutaminase (activated FXIII) of blood coagulation. This provides an interesting in vitro assay for investigating the requirements of tumour cells for adhesion to an extracellular matrix (Paye & Lapière, accompanying paper).

We acknowledge the excellent technical assistance of Mrs Y. Goebels and the typographical help of Mrs I. Leclercq. This work has been supported in part by a grant no. 3.4512.85 from the FRSM of Belgium and a grant from the Fonds Cancérologique de la Caisse Générale d’Epargne et de Retraite.

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