Int J Cuncer 52,455-460 (1992) 0 1992 Wiley-Lm, Inc.
I
Publicationof the International Union Against Cancer Publicationde I Union Internationale Contre le Cancer
SPONTANEOUSLY TRANSFORMED RAT-LIVER EPITHELIAL-CELL LINE PRODUCING AUTOCRINE AND PARACRINE GROWTH FACTORS Takeo NARITA Department of Pathology, Hirosaki University School of Medicine, Hirosaki (036),Japan. A spontaneously transformed rat-liver epithelial-cellline that could proliferate in unsupplemented. serum-free medium and be passaged with trypsinization has been established. At the time of writing, the line has undergone more than 100 passages in serum-free culture. This cell line produced an autocrine growth-stimulatory factor (AGSF) and a paracrine growthstimulatory factor (PGSF). AGSF had a remarkable growthstimulatory effect on transformed rat-liver epithelial-cells, but had no such effect on non-transformed rat-liver epithelial-cells that could not proliferate in serum-free medium. AGSF did not show a growth-inhibitory or stimulatory effect on normal rat kidney (NRK) cells in monolayer culture, and did not induce anchorage-independent growth in soft-agar culture even with epidermal growth factor (EGF) or transforming growth factor (TGF)-p. AGSF was protease-sensitive, but heat- and acidstable. The molecular weight was about 700 Dalton (Da) on size-exclusion chromatography. PGSF showed a growthstimulatory effect on NRK cells and induced anchorageindependent growth in soft-agar culture without EGF or TGF-p. On the other hand, PGSF slightly inhibited the growth of the spontaneously transformed rat-liver epithelial cells. PGSF was heat-, protease- and acid-sensitive. The molecular weight was 30 kDa on size-exclusionchromatography. 8 1992
Wilq-Liss, lnc.
Following the work of Sporn and Todaro (1980), the serum-free culture system has come into use throughout the world to examine whether or not tumor cells can produce an autocrine growth factor, o r to determine what kind of growth factors can be produced by tumor cells. But almost all serum-free culture systems contain growth factors such as transferrin, insulin, glucocorticoid, sex hormone and others (Kurokawa et al., 1989). In other systems, after cells have been cultured in serum-containing medium, the medium is replaced by serum-free medium, and the cells are recultured to analyze the production of one o r more growth factors for a short time (Anzano et al., 1989; Shapiro and Wagner, 1989). In such systems, cells cannot be passaged. We have established a spontaneously transformed, rat-liver epithelial-cell line that can proliferate in unsupplemented, serum-free medium and be passaged with trypsinization. This cell line produces an autocrine growth-stirnulatory factor that is different from reported growth factors in molecular weight and biological behavior, as well as a paracrine growth-stimulatory factor which shows a growth-stirnulatory effect on NRK cells in both monolayer and soft-agar culture. MATERIAL AND METHODS
Preparation of rat-liver epithelial cells The liver of a young Donryu rat (purchased from Charles River, Tokyo, Japan) was finely minced and suspended in a solution of Ca2+-and Mgz+-free Hanks’ BSS containing 0.1% trypsin and 0.1% collagenase (Sigma, St Louis, MO, type IV). The suspension was shaken at 37°C for 30 min, and centrifuged at 800 rpm (g = 114). Cells were washed and suspended in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) and plated in Corning plastic culture dishes (6 cm in diameter). Then they were incubated at 37°C in a humidified atmosphere containing 5% COz. Twenty-four hours later, the original medium was replaced by fresh medium. In about 10 days, small colonies of epithelial cells were observed with a phase-contrast microscope. These colonies were circled with
silicone-coated glass cylinders, and the cells in the cylinder were trypsinized, transferred to 6-cm dishes, cultured, and passaged at split ratios of 1:s using 0.5% trypsin. No antibiotics were included in the medium throughout the experimental procedures.
Histochemical study Adenosine-triphosphatase (ATP), and glucose-6-phosphatase (G6P) were stained according to the method of Wachstein and Meisel (1959), and y-glutamyltranspeptidase (GTP) was stained according to the method of Rutenburg etal. (1969). Immunohistochemical identification of the rat-liver epithelial cells Rat albumin (fraction V) was purchased from Sigma. Albumin solution (1 mg/ml PBS), emulsified in an equal volume of Freund’s complete adjuvant, was injected S.C. at various sites of the back of a rabbit, 3 times on alternate weeks. The rabbit was bled I week after the last injection and the antisera were purified with ammonium sulfate. Monospecificity was examined using Ouchterlony’s test. Immunohistochemically, the presence of the rat albumin was investigated. In brief, cells in a semi-confluent phase were fixed with methanol and rehydrated in PBS. Rabbit anti-rat albumin antibody ( ~ 4 0 0 was ) applied to the cells for 45 min at 37°C after blocking the endogenous peroxidase and applying normal swine serum. After washing in PBS, swine anti-rabbit antibody ( x 100) was applied, and then rabbit peroxidase anti-peroxidase antibody (PAP) ( ~ 9 0 was ) applied. Further, to visualize the localization of albumin, a peroxidase reaction with 3,3’diaminobenzidine (DAB)as a substrate was performed. As a positive control, thin sections of rat liver, after formalinfixation and paraffin-embedding, were used. Serum-free culture Cells grown semi-confluently in serum-containing medium were harvested with trypsinization and washed twice in PBS. The cells were cultured in unsupplemented RPMI 1640 medium. Every 4th day, the medium was changed. Cells Normal rat kidney (NRK) cells were provided from the Japanese Cancer Research Resources Bank (JCRB, Tokyo) and maintained in RPMI 1640 medium with 10% FBS. Effect of conditioned medium The conditioned medium in serum-free culture was collected, filtered (Millipore, Bedford, MA) to sterilize, and examined to define the growth-stimulatory effect on SFRLE or NRK cells. SFRLE cells (lo4cells/6-cm dish) were cultured in serum-free RPMI 1640 medium (4 ml) with 1ml of RPMI 1640 medium o r the conditioned medium. NRK cells (2 x lo4 cells/6-cm dish) were also cultured in RPMI 1640 medium (4 ml) containing 0.1% FBS with 1 ml of RPMI 1640 medium or the conditioned medium. The medium was not changed. Cells were photographed with a phase-contrast microscope, or the culture dishes were subjected to Giemsa staining after about 1 week. Received: March 24,1992 and in revised form May 25,1992.
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Concentration of conditioned medium The conditioned medium (10 1) in serum-free culture was collected, filtered to sterilize (0.22 km, Millipore), and concentrated to 100 ml with Amicon (Tokyo, Japan) Hollow Fiber System (molecular weight cut-off, 10 kDa). Saturated ammonium sulfate was added to the concentrated conditioned medium to give a concentration of 80%. The precipitate was collected and resolved in 10 ml of distilled water, and the solution was dialyzed against PBS. This sample was applied to a Sephadex (3-200 column. The filtered, conditioned medium (MW below 10 kDa) in Amicon Hollow Fiber System was collected, lyophilized, and resuspended in 200 ml of distilled water. This sample was applied to a Sephadex G-15 column. Gel-filtration chromatograp\iy A Sephadex G-15 column (130-ml bed volume) and a Sephadex (3-200 column (130-ml bed volume) were equilibrated with PBS. PBS was used as an eluent. The column was calibrated using albumin (M, 67,000), chymotrypsinogen A (M, 25,700), bacitracin (M, 1,450), and bradykinin (M, 1,060). SFRLE cells (lo4 cells/6-cm dish) were cultured in serum-free medium with 100 PI of PBS or a fraction of the Sephadex (3-15 column. NRK cells (2 x lo4 cellsi6-cm dish) were cultured in 0.1% FBS-containing medium with 100 PI of PBS or a fraction of the Sephadex (3-200 column. As a control, 100 PI of PBS was used. Cell counts were performed in one week in triplicate. Soft-agar cultirre NRK cells free of aggregates werc cultured in 0.3% agar over a 0.5% agar underlayer containing RPMI 1640 medium with 5% FBS in plastic dishes. Colonies were observed with a phase-contrast microscope. Cell aggregates composed of 2-5 cells were not counted as colonies. Protease, thermal, and acid treatment Insoluble protease (Sigma) was used at 37°C for 60 rnin at a concentration of 50 units per ml. Thermal treatment was carried out for 30 rnin at 90°C. Acid treatment was performed at pH 2.5 for 5 hr at 4"C, after neutralization with 1 N NaOH. Growth factors and anti-growlh-factor antibodies Epidermal growth factor (EGF) was purchased from Biomedical Technologies (Stoughton, MA), transforming growth factor (TGF)-a from Bachem (Torrance, CA), TGF-p from Biotope (Redmond, WA), platelet-derived growth factor (PDGF) from R & D Systems (Minneapolis, MN), fibroblast growth factor (FGF) from Collaborative Research (Bedford, MA), and insulin-like growth factor (IGF-I) from A m m o (Nagoya, Japan). Rabbit anti-EGF antibody was purchased from Biomedical Technologies, anti-TGF-a from Biotope and anti-TGF-P, anti-PDGF and anti-EGF from R & D Systems. Irnmirnoblotting Nitrocellulose sheets were imnicrsed in PBS containing 0.05% Tween 20 (TPBS), and 100 kl of a sample were applied to the sheets. Then the sheets were immersed in rabbit anti-growth-factor antibody solution for 60 min at room tcmperature. After washing with TPBS. the sheets werc immersed in swine anti-rabbit antibody solution for 60 min at room temperature. After another wash with TPBS, the sheets were immersed in rabbit peroxidase anti-peroxidase antibody (PAP) solution for 60 min at room temperature. After a final wash with TPBS, the sheets were immersed in DAB-HzO? solution for visualization. Tunzorigenicit?,test Cells (105 cells in 0.1 ml of PBS) were inoculated S.C. into the backs of BALB/c athymic mice. The mice were observed for 2 months. The tumor was formalin-bed, paraffin-embedded, thin-sectioned, and stained with hematoxylin-eosin.
FIGURE1 -Primary RLE cells ( u ) were polygonal or triangular and proliferated in a paved pattern. RLE cells at passage 70 were partly fusiform and proliferated in a fasciculating pattern (b). Scale bars = 40 krn.
Chromosome study Logarithmic-phase cells were treated for 2 hr with Colcemid and harvestcd by ordinary 0.2% trypsin treatmcnt. They were then treated hypotonically with 0.07.5 M KCI for 20 min at 37°C and fixed with methanol acetic acid (3:l in vol) for 30 min, and ordinary air-dried chromosome spreads were made on clean slide glasses. Chromosome number counts were carried out on enlarged photographs of metaphase plates. RESULTS
Growth characteristics of rat-liver epithelial (RLE) cells in senim-containing medium Primary RLE cells wcre triangular or polygonal, had 2 or more nucleoli and proliferated in a sheet-like pattern forming small colonies (Fig. 1). At passage 10. the cells were still triangular or polygonal and proliferated forming small colonies. These characteristics were maintained during passages 1 to 30. R L E cells at passages 3.540 became spindle-shaped, and some of the cells proliferated in a spindle o r fasciculating pattern. RLE cells at passage 80 showed the same pattern. During passages 2 to 10, the population doubling time was about 40 to 44 hr. During passages 30 to 40, the population doubling time was about 15 hr. After that, the growth rate was not changed (Fig. 1). Charactelistics of rat-liver epithelial cells Rat-liver epithelial (RLE) cells at passage 2 in serumcontaining medium were ATP-positive at the cell periphery, but 7-GTP- and G6P-negative. lmmunohistochemically, the cytoplasm was positive for rat albumin (Fig. 2). The cells were confirmed to be of rat-liver epithelial origin. Establishment of a spontaneously transfonned rat-liver epithelial cell line propagating in iinsupplernented, senim-free medium RLE cells were serially cultured for a long time in serumcontaining medium. At intervals, i.e. every 3rd or 4th passage, cells were examined for their ability to proliferate in unsupplemented, serum-free medium. After passages 50-55, cells that might have been spontaneously transformed proliferated in scrum-free medium (Fig. 3). These cells (SFRLE cells) were picked up and serially cultured in serum-free medium with no supplement at split ratios of 1 5 , using trypsin. At the time of writing, the line has gone through more than 100 passages. Primary SFRLE cells were polygonal or fusiform and prolifer-
TRANSFORMED RAT LIVER EPITHELIAL CELLS AND GROWTH FACTORS
457
FIGURE2 - ATP was demonstrated at the periphery of the cells (a). and rat albumin was diffusely positive in the cytoplasm (b). Scale bars = 40 pm.
FIGURE4 - Effect of SFRLE-cell-conditioning medium on SFRLE cells and NRK cells. SFRLE cells (loJ cellsi6-cm-dish) were cultured in 4 ml of RPMI 1640 medium with 1 ml of RPMI 1640 medium (a) or the conditioned medium (b). NRK cells (2 x loJ cellsidish) were cultured in 4 ml of 0.1% FBS containing RPMI 1640 medium with 1 ml of RPMI 1640 medium (c) or the conditioned medium (d). One week later, cells were photographed or dishes were subjected to Giemsa staining. Conditioned medium showed a remarkable growth-stimulatory effect on SFRLE cells and NRK cells. Scale bars = 100 km.
FIGURE3 -Primary SFRLE cells ( a ) and SFRLE cells at passage YO (h) in serum-free culture. Cells were polygonal or fusiform. and proliferated in a paved or fasciculating pattern. Scale bars = 40 km.
ated in sniall colonies, in which they formed a partly paved and partly fasciculating pattern (Fig. 3). Cell population doubling time was about 85 hr at passage 5 and 72 hr at passage 20. After that, doubling time was stable. Morphological and proliferation characteristics were not so changed at passages 5 , 20, 50, 70 and 100.
Effect of SFRLE cell-conditioriit~gmedium SFRLE cell-conditioning medium showed a remarkable growth-stimulatory effect on SFRLE cells in serum-free culture. Compared with a control, SFRLE cells formed numerous and larger colonies in serum-free culture (Fig. 4). The conditioncd medium also showed a remarkable growth-stiniulatory cffect on NRK cells in 0.1% FBS-containing medium. NRK cells did not proliferate in the 0.1% FBS-containing medium without the conditioned medium, but administration of the conditioned medium caused NRK cells to proliferate rapidly (Fig. 4). Gel filtrution profile On Sephadex (3-15 column (130-ml bed volume) analysis, there was one pcak which showed a growth-stimulatory effcct
on SFRLE cells (Fig. 5 ) . The molecular weight was cstimated to be about 700 Da. The autocrine growth factor of this peak fraction was designated AGSF (autocrine growth-stimulatory factor). On Sephadex (3-200 column (130-nil bed volume), analysis, there was also one pcak which showed a growthstimulatory effect on NRK cells (Fig. 6). The molecular weight was 30 kDa. The growth factor of this peak fraction was designated PGSF (paracrine growth-stimulatory factor).
Characteristics of A G S F SFRLE cells were cultured in serum-free medium with 100 *I of PBS or AGSF. Cell counts were performed every 4th day in triplicate. AGSF showed a growth-stimulatory effect on transformed self-cells that could proliferate in serum-free medium (Fig. 7). but showed no such effect on nontransformcd self-cells that could not proliferate in serum-free medium. Furthermore, AGSF did not show a growthstirnulatory or inhibitory effect on NRK cells, and did riot induce anchorage-independent growth of NRK cells even with E G F or TGF-P. No colonies were found. AGSF was proteasesensitive, but heat- and acid-stable (Table I). Charnctmristics of PGSF NRK cells were cultured in 0.1% FBS containing medium with 100 p1 of PBS or PGSF. Cells were counted every other day in triplicate. PGSF showed a remarkable growthstimulatory effect on NRK cells (Fig. 8). When SFRLE cells were cultured with 100 p1 of PGSF, the growth of SFRLE cells was slightly inhibited (data not shown). Additionally, PGSF induced anchorage-independent growth of NRK cells without E G F or TGF-P. Compared with controls, many colonies
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FIGURE5 - Gel filtration rofile on Sephadex G-15. SFRLE-cellconditioning medium (10 !I was concentrated in Amicon Hollow Fiber System (MW cut-off, 10 kDa). The filtered, conditioned medium (MW < 10 kDa) was collected, lyophilized, and resolved in distilled water, then 2 ml of this solution were applied to a Sephadex G-15 column. PBS was used as an eluent and the fraction volume was 5 ml. From each fraction 100 pl were examined, and 100 pl of PBS were used as a control. The column was calibrated using bacitracin (M, 1,450) and bradykinin (M, 1,060). SFRLE cells (lo4 cells/dish) were cultured in serum-free RPMl 1640 medium. Cells were counted after 7 days. There was one peak corresponding to MW 700 Da, which showed a remarkable growth-stimulatory effect on SFRLE cells in serum-free culture.
IU'
i
8
i2
i6
Days in culture FIGURE7 - Effect of AGSF on self-cells. Self SFRLE cells ( lo4 cells/dish) were cultured in serum-free medium with PBS ( 0 )or 100 pl of AGSF (0).Cell counts were performed every 4th day in triplicate.
106'
3D tl
P
G
2 105. 3
c
2
1
L
50
100 Elution volume
150
ml
FIGURE6 - Gel filtration profile on Sephadex G-200. SFRLE-cellconditioning medium (10 I) was concentrated using Amicon Hollow Fiber System (MW cut-off, 10 kDa), and saturated ammonium was added. The precipitate was collected, resolved in distilled water and dialyzed against PBS, then 2 ml of this sample were applied to a Sephadex G-200 column. PBS was used as an eluent. Fraction volume was 5 ml. From each fraction 100 pl were examined, and 100 pl of PBS were used as a control. The column was calibrated using albumin (M, 67,000) and chymotrypsinogen A (M, 25,700). NRK cells (2 x lo4 cells/dish) were cultured in RPMI 1640 medium with 0.1% FBS. Cells were counted after 7 days. There was one peak corresponding to MW 30 kDa showing a growth-stimulatory effect on NRK cells. composed of 10-20 cells were found in 7 days (Fig. 9). PGSF was heat-, protease- and acid-sensitive (Table I).
Effect of growth factors on SFRLE cells SFRLE cells were cultured in RPMI 1640 medium supplemented with 20 ng of EGF, TGF-a, TGF-P, PDGF, FGF or
4
6
8
Days in culture
FIGURE 8 - Effect of PGSF on NRK cells. NRK cells (2 x lo4 cellsidish) were cultured in 0.1% FBS containing medium with 100 pl of PBS ( 0 )or PGSF (0).Cell counts were performed every other day in triplicate. TABLE 1 -TREATMENT OF AGSF AND PGSF Treatment
AGSF Cdl numbers ( ~ 1 0 ' )
Crll numbers ( x 10')
PGSF
Protease Heat Acid N o treatment
95 237 228 242
83 79 104 243
SFRLE cells ( lo4 cellsidish) were cultured in serum-free medium (RPMI 1640) with 100 pl of treated or non-treated AGSF. NRK cells ( 2 x lo4 cells/dish) were cultured in 0.1% FBS-containing medium with 100 pI of treated or non-treated PGSF. Data are mean cells counted in triplicate about 1 week later. IGF-I. Seven days later, cells were counted. None of these growth factors promoted the growth of SFRLE cells (Table 11). Biologically, AGSF was thought to be different from these growth factors.
459
TRANSFORMED RAT LIVER EPITHELIAL CELLS AND GROWTH FACTORS
Immunoblottinganalysis PGSF was examined using anti-EGF, TGF-a, TGF-P, PDGF and F G F antibodies, but did not cross-react with any of them. Immunologically, PGSF was thought to be different from EGF, TGF-a. TGF-P, P D G F and FGF. Titmorigenicip SFRLE cells at passages 3, 10,50 and 100 showed tumorigenicity (incidence, loo%, n = 5 ) . In about 20 days, a small nodule was found on the back of each athymic mouse. Histologically, SFRLE cells proliferated in a trabecular or alveolar pattern with thin fibrous stroma. Neither glandular nor bile pigment was observed (Fig. 10). Chromosomal nnalysis One hundred RLE cells in metaphase at passages 4 and 45 and 100 SFRLE cells in metaphase at passage 4 were examined with Giemsa stain. The chromosome number of R L E cells at passage 4 was 42 (99.9%), and that of RLE cells at passage 45 was 42 (92.5%). The chromosome number of SFRLE cells at passage 4 varied from 57 to 118 with a peak of 65 (Table 111).
DISCUSSION
A spontaneously transformed, rat-liver epithelial-cell line propagating in unsupplemented, serum-free medium produced an autocrine growth-stimulatory factor (AGSF), and a paracrine growth-stimulatory factor (PGSF) which showed a growth-stimulatory effect on NRK cells. AGSF had a remarkable growth-stimulatory effect on transformed self-cells (SFRLE cells), but not on NRK cells or on non-transformed self-cells ( R L E cells) that could not proliferate in serum-free medium. In soft-agar culture of NRK cells, AGSF did not induce anchorage-independent growth even with E G F or TGF-P. E G F stimulates the growth of NRK cells in monolayer culture, and induces anchorage-independent growth of NRKcells with TGF-P. Other growth factors such as EGF, TGF-a, TGF-P, PDGF, F G F and IGF-I did not stimulate the growth of SFRLE cells. Protease treatment reduced the effect of AGSF. AGSF, which is thought to be an oligopeptide, has a molecular weight of 700. T o the best of our knowledge, there is no report on an autocrine growth factor with such a low molecular weight. AGSF may be a novel autocrine growth factor derived from epithelial cells. Its purification is now under way. PGSF showed a growth-stimulatory effect on NRK cells in monolayer culture, induced anchorage-independent growth of NRK cells in soft-agar culture without EGF or TGF-a, and slightly inhibited the growth of SFRLE cells. TGF-P did not induce anchorage-independent growth of NRK cells without E G F or TGF-a, and inhibited the growth of NRK cells in monolayer culture only after acid treatment. In a neutral state PGSF is active on NRK cells. TGF-y is also active in a neutral state, but does not induce anchorage-independent growth of NRK cells. P D G F and F G F are active in a neutral state, but in general they are not active on epithelial cells. The molecular
FIGURE 9 - Anchorage-independent growth: NRK cells free of aggregates were cultured in 0.3% soft agar over a 0.5% agar underlayer, then 100 )LI of PBS (a) or PGSF (b) were added to the 0.37%soft-agar layer. After about 7 days, many colonies composed of 10-20 cells were found, but in a control there were hardly any colonies. Scale bars = 20 km. TABLE 11 - EFFECT OF GROWTH FACTORS ON SFRLE CELLS Cell numbers ( x 10’)
Growth factor
57 234
None AGSF EGF TGF-a
55
48
TGF-R -__
52_
PDG; FGF IGF-I
45 46 60
SFRLE cells ( lo4 cells/dish) were cultured in serum-free medium with 100 )LI of AGSF, or with 20 ng of EGF, TGF-a, TGF-P, PDGF, FGF or IGF-I. Data are mean values from cells counted in triplicate 1 week later.
FIGURE10-Tumorigenicity of SFRLE cells. SFRLE cells at passages 3, 10, 50 and 100 were injected S.C.into the backs of athymic BALB/c mice. After about 20 days, small nodules were observed, showing SFRLE cells to be tumorigenic in athymic mice. Cells with prominent nucleoli proliferated in a trabecular pattern with thin, fibrous stroma (Hematoxylin-eosin stain). Scale bar = 40 km.
TABLE 111-CHROMOSOMAL ANALYSIS Cells -
RLE cells at passage 4 RLE cells at passage 45 SFRLE cells at passage 4
Chromosomal distribution 41
42
1 3
99
85
43
5040
60-70
7W8U
9&100
100-
11
1 6
48
21
14
11
After cells were treated with Colcemid, metaphase preparation were achieved by hypotonic treatment. Details are found in the text.
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weight of PGSF was 30 kDa on size-exclusion chromatography, but that of EGF, TGF-P, TGF-y, PDGF and F G F was about 6 kDa (Holladay et al., 1976), 23-25 kDa (Massague, 1984), 12 kDa (Hirai et al., 1983), 23 kDa (Deuel et a/., 1981) and 13 kDa (Gospodarowicz, 1975) respectively. Furthermore, immunoblotting analysis showed that PGSF did not cross-react with anti-EGF, TGF-a, TGF-P, P D G F or FGF. These findings suggest that PGSF may be a new type of T G F inasmuch as it induces anchorage-independent growth of NRK cells. Molecular similarities between PDGF and oncogene sisproducts have been reported (Doolittle et al., 1983; Waterfield et al., 1983). At present, there is no information as to what oncogene products are related to AGSF or PGSF. Hill et al. (1990), who established a cell line from Chinese hamster fibroblasts infected with Rous sarcoma virus, which could proliferate in serum-free medium, report that virus oncogene v-src is not related to cell proliferation in serum-free culture.
But, unlike SFRLE cells, their cell line is of non-epithelial origin. Carcinomas can be divided into 2 types, depending upon the stromal component. One is medullary carcinoma with a scanty fibrous component, and the other is scirrhous carcinoma with an abundant fibrous component. AGSF stimulated the growth of self epithelial cells, and PGSF stimulated the growth of fibroblasts. If malignant epithelial cells generally produce 2 growth factors such as AGSF and PGSF, higher production of AGSF may result in medullary carcinoma and higher production of PGSF may lead to scirrhous carcinoma. SFRLE cells formed a tumor in athymic mice in which the cells proliferated in an alveolar or trabecular pattern with thin, fibrous stroma. ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Ministry of Education (04807025).
REFERENCES ANZANO.M.A., RIEMAN, D.. PRICHFIT, W., BOWEN-POPE, D.F. and GKEIG,R., Growth factor production by human colon carcinoma cell lines. Cancer Res., 49,2898-2904 (1989). DLLIEL, T.F., HUANG,J.S., PROFFIIT, R.T., BAENZIGEK, J.U., CHANG, D. and KENNEDY, B.B., Human platelet-derived growth factor. J. biol. Chem., 256,88968899 (1981). DOOLITTLE,R.F., HUNKAPILLER, M.W., Hooo, L.E., DEVARE,S.G.. S.A. and ANTONIADES, H.N., Simian ROBBINS,K.C., AAKONSON, sarcoma virus oncogene, v-sis derived from the gene (or genes) encoding a platelet-derived growth factor. Scierice. 221, 275-277 ( 1983). GOSPODAROWICZ, D., Purification of a fibroblast growth factor from bovine pituitary. J. biol. Cliern., 250, 2515-2520 (1975). HILL,M., HILLOVA, J. and MARIAGE-SAMSON, R., Autocrine-factorindependent growth of mammalian fibroblasts established in fully synthetic medium. Nov-oric requirement in establishment. Vitro Cell Dew/. Biol.,25,44-50 (1990). H I K A I ,R., YAMAOKA,K. and MI'ISUI. H., Isolation and partial purification of a new class of transforming growth factor from an avian sarcoma virus-transformed rat cell line. Cancer Rex, 43, 5742-5746 (1983). HOLLADAY, L.A., SAVAGE,C.R., JR., C O H ~ NS., and PUEI-I.,D., Conformation and unfolding thermodynamics of epidermal growth factor and derivatives. Biochemistiy. 15,26242633 (1976).
KUKOKAWA, R., KYAKUMOTO, S. and OTA, M., Autocrine growth factor in defined serum-free medium of human salivary gland adenocarcinoma cell line HSG. CuncerRes., 49,5163-5142 (1989). MASSAGUE, J., Type p transforming growth factor from feline sarcoma virus-transformed rat ce1ls.J. bid. Chern., 259,9756-9761 (1984). RUTENBURG, A.M., K I M . H . , FISHBEIN,J.W.. HANhER, J.S., WASSERKRUNG, H.L. and SELIGMAN, A.M., Histochemical and ultrastructural demonstration of y-glutamyl transpeptidase activity. J. Hisrochem. Cyfochern.,17,517-526 (1969). SHAPIRO, L.E. and WAGNER,N., Transferrin is an autocrine growth factor secreted by Reuber H-35 cells in serum-free culture. In Vitro Cell Devel. Biol., 25, 650-654 (1989). SPORN,M.B. and TODAKO,G.J., Autocrine secretion and malignant transformation of cells. Nen Engl. J. Med.. 303,878-880 (1980). WACHSTEIN, M. and MEISEL,E., Enzymic histochemistry of ethionin induced liver cirrhosis and hepatoma. J. Hisrochem. Cytochem., 7, 189-201 (1959). WATERFIELD, M.D., SCARACE, G.T., WHITTLE,N., STROOBANT, P., JOHNSON, A,, WASTERSON, A.. WESTERMARK, B.J., HELDIN,C-H., HUANG,J.S. and DEUEL,T.F., Platelet-derived growth factor is structurally related to the putative transforming protein p28"' of simian sarcoma virus. Nutitre (Lond.), 304,35-39 (1983).
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Publicationof the International Union Against Cancer Publicationde I Union Internationale Contre le Cancer
SPONTANEOUSLY TRANSFORMED RAT-LIVER EPITHELIAL-CELL LINE PRODUCING AUTOCRINE AND PARACRINE GROWTH FACTORS Takeo NARITA Department of Pathology, Hirosaki University School of Medicine, Hirosaki (036),Japan. A spontaneously transformed rat-liver epithelial-cellline that could proliferate in unsupplemented. serum-free medium and be passaged with trypsinization has been established. At the time of writing, the line has undergone more than 100 passages in serum-free culture. This cell line produced an autocrine growth-stimulatory factor (AGSF) and a paracrine growthstimulatory factor (PGSF). AGSF had a remarkable growthstimulatory effect on transformed rat-liver epithelial-cells, but had no such effect on non-transformed rat-liver epithelial-cells that could not proliferate in serum-free medium. AGSF did not show a growth-inhibitory or stimulatory effect on normal rat kidney (NRK) cells in monolayer culture, and did not induce anchorage-independent growth in soft-agar culture even with epidermal growth factor (EGF) or transforming growth factor (TGF)-p. AGSF was protease-sensitive, but heat- and acidstable. The molecular weight was about 700 Dalton (Da) on size-exclusion chromatography. PGSF showed a growthstimulatory effect on NRK cells and induced anchorageindependent growth in soft-agar culture without EGF or TGF-p. On the other hand, PGSF slightly inhibited the growth of the spontaneously transformed rat-liver epithelial cells. PGSF was heat-, protease- and acid-sensitive. The molecular weight was 30 kDa on size-exclusionchromatography. 8 1992
Wilq-Liss, lnc.
Following the work of Sporn and Todaro (1980), the serum-free culture system has come into use throughout the world to examine whether or not tumor cells can produce an autocrine growth factor, o r to determine what kind of growth factors can be produced by tumor cells. But almost all serum-free culture systems contain growth factors such as transferrin, insulin, glucocorticoid, sex hormone and others (Kurokawa et al., 1989). In other systems, after cells have been cultured in serum-containing medium, the medium is replaced by serum-free medium, and the cells are recultured to analyze the production of one o r more growth factors for a short time (Anzano et al., 1989; Shapiro and Wagner, 1989). In such systems, cells cannot be passaged. We have established a spontaneously transformed, rat-liver epithelial-cell line that can proliferate in unsupplemented, serum-free medium and be passaged with trypsinization. This cell line produces an autocrine growth-stirnulatory factor that is different from reported growth factors in molecular weight and biological behavior, as well as a paracrine growth-stimulatory factor which shows a growth-stirnulatory effect on NRK cells in both monolayer and soft-agar culture. MATERIAL AND METHODS
Preparation of rat-liver epithelial cells The liver of a young Donryu rat (purchased from Charles River, Tokyo, Japan) was finely minced and suspended in a solution of Ca2+-and Mgz+-free Hanks’ BSS containing 0.1% trypsin and 0.1% collagenase (Sigma, St Louis, MO, type IV). The suspension was shaken at 37°C for 30 min, and centrifuged at 800 rpm (g = 114). Cells were washed and suspended in RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS) and plated in Corning plastic culture dishes (6 cm in diameter). Then they were incubated at 37°C in a humidified atmosphere containing 5% COz. Twenty-four hours later, the original medium was replaced by fresh medium. In about 10 days, small colonies of epithelial cells were observed with a phase-contrast microscope. These colonies were circled with
silicone-coated glass cylinders, and the cells in the cylinder were trypsinized, transferred to 6-cm dishes, cultured, and passaged at split ratios of 1:s using 0.5% trypsin. No antibiotics were included in the medium throughout the experimental procedures.
Histochemical study Adenosine-triphosphatase (ATP), and glucose-6-phosphatase (G6P) were stained according to the method of Wachstein and Meisel (1959), and y-glutamyltranspeptidase (GTP) was stained according to the method of Rutenburg etal. (1969). Immunohistochemical identification of the rat-liver epithelial cells Rat albumin (fraction V) was purchased from Sigma. Albumin solution (1 mg/ml PBS), emulsified in an equal volume of Freund’s complete adjuvant, was injected S.C. at various sites of the back of a rabbit, 3 times on alternate weeks. The rabbit was bled I week after the last injection and the antisera were purified with ammonium sulfate. Monospecificity was examined using Ouchterlony’s test. Immunohistochemically, the presence of the rat albumin was investigated. In brief, cells in a semi-confluent phase were fixed with methanol and rehydrated in PBS. Rabbit anti-rat albumin antibody ( ~ 4 0 0 was ) applied to the cells for 45 min at 37°C after blocking the endogenous peroxidase and applying normal swine serum. After washing in PBS, swine anti-rabbit antibody ( x 100) was applied, and then rabbit peroxidase anti-peroxidase antibody (PAP) ( ~ 9 0 was ) applied. Further, to visualize the localization of albumin, a peroxidase reaction with 3,3’diaminobenzidine (DAB)as a substrate was performed. As a positive control, thin sections of rat liver, after formalinfixation and paraffin-embedding, were used. Serum-free culture Cells grown semi-confluently in serum-containing medium were harvested with trypsinization and washed twice in PBS. The cells were cultured in unsupplemented RPMI 1640 medium. Every 4th day, the medium was changed. Cells Normal rat kidney (NRK) cells were provided from the Japanese Cancer Research Resources Bank (JCRB, Tokyo) and maintained in RPMI 1640 medium with 10% FBS. Effect of conditioned medium The conditioned medium in serum-free culture was collected, filtered (Millipore, Bedford, MA) to sterilize, and examined to define the growth-stimulatory effect on SFRLE or NRK cells. SFRLE cells (lo4cells/6-cm dish) were cultured in serum-free RPMI 1640 medium (4 ml) with 1ml of RPMI 1640 medium o r the conditioned medium. NRK cells (2 x lo4 cells/6-cm dish) were also cultured in RPMI 1640 medium (4 ml) containing 0.1% FBS with 1 ml of RPMI 1640 medium or the conditioned medium. The medium was not changed. Cells were photographed with a phase-contrast microscope, or the culture dishes were subjected to Giemsa staining after about 1 week. Received: March 24,1992 and in revised form May 25,1992.
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Concentration of conditioned medium The conditioned medium (10 1) in serum-free culture was collected, filtered to sterilize (0.22 km, Millipore), and concentrated to 100 ml with Amicon (Tokyo, Japan) Hollow Fiber System (molecular weight cut-off, 10 kDa). Saturated ammonium sulfate was added to the concentrated conditioned medium to give a concentration of 80%. The precipitate was collected and resolved in 10 ml of distilled water, and the solution was dialyzed against PBS. This sample was applied to a Sephadex (3-200 column. The filtered, conditioned medium (MW below 10 kDa) in Amicon Hollow Fiber System was collected, lyophilized, and resuspended in 200 ml of distilled water. This sample was applied to a Sephadex G-15 column. Gel-filtration chromatograp\iy A Sephadex G-15 column (130-ml bed volume) and a Sephadex (3-200 column (130-ml bed volume) were equilibrated with PBS. PBS was used as an eluent. The column was calibrated using albumin (M, 67,000), chymotrypsinogen A (M, 25,700), bacitracin (M, 1,450), and bradykinin (M, 1,060). SFRLE cells (lo4 cells/6-cm dish) were cultured in serum-free medium with 100 PI of PBS or a fraction of the Sephadex (3-15 column. NRK cells (2 x lo4 cellsi6-cm dish) were cultured in 0.1% FBS-containing medium with 100 PI of PBS or a fraction of the Sephadex (3-200 column. As a control, 100 PI of PBS was used. Cell counts were performed in one week in triplicate. Soft-agar cultirre NRK cells free of aggregates werc cultured in 0.3% agar over a 0.5% agar underlayer containing RPMI 1640 medium with 5% FBS in plastic dishes. Colonies were observed with a phase-contrast microscope. Cell aggregates composed of 2-5 cells were not counted as colonies. Protease, thermal, and acid treatment Insoluble protease (Sigma) was used at 37°C for 60 rnin at a concentration of 50 units per ml. Thermal treatment was carried out for 30 rnin at 90°C. Acid treatment was performed at pH 2.5 for 5 hr at 4"C, after neutralization with 1 N NaOH. Growth factors and anti-growlh-factor antibodies Epidermal growth factor (EGF) was purchased from Biomedical Technologies (Stoughton, MA), transforming growth factor (TGF)-a from Bachem (Torrance, CA), TGF-p from Biotope (Redmond, WA), platelet-derived growth factor (PDGF) from R & D Systems (Minneapolis, MN), fibroblast growth factor (FGF) from Collaborative Research (Bedford, MA), and insulin-like growth factor (IGF-I) from A m m o (Nagoya, Japan). Rabbit anti-EGF antibody was purchased from Biomedical Technologies, anti-TGF-a from Biotope and anti-TGF-P, anti-PDGF and anti-EGF from R & D Systems. Irnmirnoblotting Nitrocellulose sheets were imnicrsed in PBS containing 0.05% Tween 20 (TPBS), and 100 kl of a sample were applied to the sheets. Then the sheets were immersed in rabbit anti-growth-factor antibody solution for 60 min at room tcmperature. After washing with TPBS. the sheets werc immersed in swine anti-rabbit antibody solution for 60 min at room temperature. After another wash with TPBS, the sheets were immersed in rabbit peroxidase anti-peroxidase antibody (PAP) solution for 60 min at room temperature. After a final wash with TPBS, the sheets were immersed in DAB-HzO? solution for visualization. Tunzorigenicit?,test Cells (105 cells in 0.1 ml of PBS) were inoculated S.C. into the backs of BALB/c athymic mice. The mice were observed for 2 months. The tumor was formalin-bed, paraffin-embedded, thin-sectioned, and stained with hematoxylin-eosin.
FIGURE1 -Primary RLE cells ( u ) were polygonal or triangular and proliferated in a paved pattern. RLE cells at passage 70 were partly fusiform and proliferated in a fasciculating pattern (b). Scale bars = 40 krn.
Chromosome study Logarithmic-phase cells were treated for 2 hr with Colcemid and harvestcd by ordinary 0.2% trypsin treatmcnt. They were then treated hypotonically with 0.07.5 M KCI for 20 min at 37°C and fixed with methanol acetic acid (3:l in vol) for 30 min, and ordinary air-dried chromosome spreads were made on clean slide glasses. Chromosome number counts were carried out on enlarged photographs of metaphase plates. RESULTS
Growth characteristics of rat-liver epithelial (RLE) cells in senim-containing medium Primary RLE cells wcre triangular or polygonal, had 2 or more nucleoli and proliferated in a sheet-like pattern forming small colonies (Fig. 1). At passage 10. the cells were still triangular or polygonal and proliferated forming small colonies. These characteristics were maintained during passages 1 to 30. R L E cells at passages 3.540 became spindle-shaped, and some of the cells proliferated in a spindle o r fasciculating pattern. RLE cells at passage 80 showed the same pattern. During passages 2 to 10, the population doubling time was about 40 to 44 hr. During passages 30 to 40, the population doubling time was about 15 hr. After that, the growth rate was not changed (Fig. 1). Charactelistics of rat-liver epithelial cells Rat-liver epithelial (RLE) cells at passage 2 in serumcontaining medium were ATP-positive at the cell periphery, but 7-GTP- and G6P-negative. lmmunohistochemically, the cytoplasm was positive for rat albumin (Fig. 2). The cells were confirmed to be of rat-liver epithelial origin. Establishment of a spontaneously transfonned rat-liver epithelial cell line propagating in iinsupplernented, senim-free medium RLE cells were serially cultured for a long time in serumcontaining medium. At intervals, i.e. every 3rd or 4th passage, cells were examined for their ability to proliferate in unsupplemented, serum-free medium. After passages 50-55, cells that might have been spontaneously transformed proliferated in scrum-free medium (Fig. 3). These cells (SFRLE cells) were picked up and serially cultured in serum-free medium with no supplement at split ratios of 1 5 , using trypsin. At the time of writing, the line has gone through more than 100 passages. Primary SFRLE cells were polygonal or fusiform and prolifer-
TRANSFORMED RAT LIVER EPITHELIAL CELLS AND GROWTH FACTORS
457
FIGURE2 - ATP was demonstrated at the periphery of the cells (a). and rat albumin was diffusely positive in the cytoplasm (b). Scale bars = 40 pm.
FIGURE4 - Effect of SFRLE-cell-conditioning medium on SFRLE cells and NRK cells. SFRLE cells (loJ cellsi6-cm-dish) were cultured in 4 ml of RPMI 1640 medium with 1 ml of RPMI 1640 medium (a) or the conditioned medium (b). NRK cells (2 x loJ cellsidish) were cultured in 4 ml of 0.1% FBS containing RPMI 1640 medium with 1 ml of RPMI 1640 medium (c) or the conditioned medium (d). One week later, cells were photographed or dishes were subjected to Giemsa staining. Conditioned medium showed a remarkable growth-stimulatory effect on SFRLE cells and NRK cells. Scale bars = 100 km.
FIGURE3 -Primary SFRLE cells ( a ) and SFRLE cells at passage YO (h) in serum-free culture. Cells were polygonal or fusiform. and proliferated in a paved or fasciculating pattern. Scale bars = 40 km.
ated in sniall colonies, in which they formed a partly paved and partly fasciculating pattern (Fig. 3). Cell population doubling time was about 85 hr at passage 5 and 72 hr at passage 20. After that, doubling time was stable. Morphological and proliferation characteristics were not so changed at passages 5 , 20, 50, 70 and 100.
Effect of SFRLE cell-conditioriit~gmedium SFRLE cell-conditioning medium showed a remarkable growth-stimulatory effect on SFRLE cells in serum-free culture. Compared with a control, SFRLE cells formed numerous and larger colonies in serum-free culture (Fig. 4). The conditioncd medium also showed a remarkable growth-stiniulatory cffect on NRK cells in 0.1% FBS-containing medium. NRK cells did not proliferate in the 0.1% FBS-containing medium without the conditioned medium, but administration of the conditioned medium caused NRK cells to proliferate rapidly (Fig. 4). Gel filtrution profile On Sephadex (3-15 column (130-ml bed volume) analysis, there was one pcak which showed a growth-stimulatory effcct
on SFRLE cells (Fig. 5 ) . The molecular weight was cstimated to be about 700 Da. The autocrine growth factor of this peak fraction was designated AGSF (autocrine growth-stimulatory factor). On Sephadex (3-200 column (130-nil bed volume), analysis, there was also one pcak which showed a growthstimulatory effect on NRK cells (Fig. 6). The molecular weight was 30 kDa. The growth factor of this peak fraction was designated PGSF (paracrine growth-stimulatory factor).
Characteristics of A G S F SFRLE cells were cultured in serum-free medium with 100 *I of PBS or AGSF. Cell counts were performed every 4th day in triplicate. AGSF showed a growth-stimulatory effect on transformed self-cells that could proliferate in serum-free medium (Fig. 7). but showed no such effect on nontransformcd self-cells that could not proliferate in serum-free medium. Furthermore, AGSF did not show a growthstirnulatory or inhibitory effect on NRK cells, and did riot induce anchorage-independent growth of NRK cells even with E G F or TGF-P. No colonies were found. AGSF was proteasesensitive, but heat- and acid-stable (Table I). Charnctmristics of PGSF NRK cells were cultured in 0.1% FBS containing medium with 100 p1 of PBS or PGSF. Cells were counted every other day in triplicate. PGSF showed a remarkable growthstimulatory effect on NRK cells (Fig. 8). When SFRLE cells were cultured with 100 p1 of PGSF, the growth of SFRLE cells was slightly inhibited (data not shown). Additionally, PGSF induced anchorage-independent growth of NRK cells without E G F or TGF-P. Compared with controls, many colonies
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FIGURE5 - Gel filtration rofile on Sephadex G-15. SFRLE-cellconditioning medium (10 !I was concentrated in Amicon Hollow Fiber System (MW cut-off, 10 kDa). The filtered, conditioned medium (MW < 10 kDa) was collected, lyophilized, and resolved in distilled water, then 2 ml of this solution were applied to a Sephadex G-15 column. PBS was used as an eluent and the fraction volume was 5 ml. From each fraction 100 pl were examined, and 100 pl of PBS were used as a control. The column was calibrated using bacitracin (M, 1,450) and bradykinin (M, 1,060). SFRLE cells (lo4 cells/dish) were cultured in serum-free RPMl 1640 medium. Cells were counted after 7 days. There was one peak corresponding to MW 700 Da, which showed a remarkable growth-stimulatory effect on SFRLE cells in serum-free culture.
IU'
i
8
i2
i6
Days in culture FIGURE7 - Effect of AGSF on self-cells. Self SFRLE cells ( lo4 cells/dish) were cultured in serum-free medium with PBS ( 0 )or 100 pl of AGSF (0).Cell counts were performed every 4th day in triplicate.
106'
3D tl
P
G
2 105. 3
c
2
1
L
50
100 Elution volume
150
ml
FIGURE6 - Gel filtration profile on Sephadex G-200. SFRLE-cellconditioning medium (10 I) was concentrated using Amicon Hollow Fiber System (MW cut-off, 10 kDa), and saturated ammonium was added. The precipitate was collected, resolved in distilled water and dialyzed against PBS, then 2 ml of this sample were applied to a Sephadex G-200 column. PBS was used as an eluent. Fraction volume was 5 ml. From each fraction 100 pl were examined, and 100 pl of PBS were used as a control. The column was calibrated using albumin (M, 67,000) and chymotrypsinogen A (M, 25,700). NRK cells (2 x lo4 cells/dish) were cultured in RPMI 1640 medium with 0.1% FBS. Cells were counted after 7 days. There was one peak corresponding to MW 30 kDa showing a growth-stimulatory effect on NRK cells. composed of 10-20 cells were found in 7 days (Fig. 9). PGSF was heat-, protease- and acid-sensitive (Table I).
Effect of growth factors on SFRLE cells SFRLE cells were cultured in RPMI 1640 medium supplemented with 20 ng of EGF, TGF-a, TGF-P, PDGF, FGF or
4
6
8
Days in culture
FIGURE 8 - Effect of PGSF on NRK cells. NRK cells (2 x lo4 cellsidish) were cultured in 0.1% FBS containing medium with 100 pl of PBS ( 0 )or PGSF (0).Cell counts were performed every other day in triplicate. TABLE 1 -TREATMENT OF AGSF AND PGSF Treatment
AGSF Cdl numbers ( ~ 1 0 ' )
Crll numbers ( x 10')
PGSF
Protease Heat Acid N o treatment
95 237 228 242
83 79 104 243
SFRLE cells ( lo4 cellsidish) were cultured in serum-free medium (RPMI 1640) with 100 pl of treated or non-treated AGSF. NRK cells ( 2 x lo4 cells/dish) were cultured in 0.1% FBS-containing medium with 100 pI of treated or non-treated PGSF. Data are mean cells counted in triplicate about 1 week later. IGF-I. Seven days later, cells were counted. None of these growth factors promoted the growth of SFRLE cells (Table 11). Biologically, AGSF was thought to be different from these growth factors.
459
TRANSFORMED RAT LIVER EPITHELIAL CELLS AND GROWTH FACTORS
Immunoblottinganalysis PGSF was examined using anti-EGF, TGF-a, TGF-P, PDGF and F G F antibodies, but did not cross-react with any of them. Immunologically, PGSF was thought to be different from EGF, TGF-a. TGF-P, P D G F and FGF. Titmorigenicip SFRLE cells at passages 3, 10,50 and 100 showed tumorigenicity (incidence, loo%, n = 5 ) . In about 20 days, a small nodule was found on the back of each athymic mouse. Histologically, SFRLE cells proliferated in a trabecular or alveolar pattern with thin fibrous stroma. Neither glandular nor bile pigment was observed (Fig. 10). Chromosomal nnalysis One hundred RLE cells in metaphase at passages 4 and 45 and 100 SFRLE cells in metaphase at passage 4 were examined with Giemsa stain. The chromosome number of R L E cells at passage 4 was 42 (99.9%), and that of RLE cells at passage 45 was 42 (92.5%). The chromosome number of SFRLE cells at passage 4 varied from 57 to 118 with a peak of 65 (Table 111).
DISCUSSION
A spontaneously transformed, rat-liver epithelial-cell line propagating in unsupplemented, serum-free medium produced an autocrine growth-stimulatory factor (AGSF), and a paracrine growth-stimulatory factor (PGSF) which showed a growth-stimulatory effect on NRK cells. AGSF had a remarkable growth-stimulatory effect on transformed self-cells (SFRLE cells), but not on NRK cells or on non-transformed self-cells ( R L E cells) that could not proliferate in serum-free medium. In soft-agar culture of NRK cells, AGSF did not induce anchorage-independent growth even with E G F or TGF-P. E G F stimulates the growth of NRK cells in monolayer culture, and induces anchorage-independent growth of NRKcells with TGF-P. Other growth factors such as EGF, TGF-a, TGF-P, PDGF, F G F and IGF-I did not stimulate the growth of SFRLE cells. Protease treatment reduced the effect of AGSF. AGSF, which is thought to be an oligopeptide, has a molecular weight of 700. T o the best of our knowledge, there is no report on an autocrine growth factor with such a low molecular weight. AGSF may be a novel autocrine growth factor derived from epithelial cells. Its purification is now under way. PGSF showed a growth-stimulatory effect on NRK cells in monolayer culture, induced anchorage-independent growth of NRK cells in soft-agar culture without EGF or TGF-a, and slightly inhibited the growth of SFRLE cells. TGF-P did not induce anchorage-independent growth of NRK cells without E G F or TGF-a, and inhibited the growth of NRK cells in monolayer culture only after acid treatment. In a neutral state PGSF is active on NRK cells. TGF-y is also active in a neutral state, but does not induce anchorage-independent growth of NRK cells. P D G F and F G F are active in a neutral state, but in general they are not active on epithelial cells. The molecular
FIGURE 9 - Anchorage-independent growth: NRK cells free of aggregates were cultured in 0.3% soft agar over a 0.5% agar underlayer, then 100 )LI of PBS (a) or PGSF (b) were added to the 0.37%soft-agar layer. After about 7 days, many colonies composed of 10-20 cells were found, but in a control there were hardly any colonies. Scale bars = 20 km. TABLE 11 - EFFECT OF GROWTH FACTORS ON SFRLE CELLS Cell numbers ( x 10’)
Growth factor
57 234
None AGSF EGF TGF-a
55
48
TGF-R -__
52_
PDG; FGF IGF-I
45 46 60
SFRLE cells ( lo4 cells/dish) were cultured in serum-free medium with 100 )LI of AGSF, or with 20 ng of EGF, TGF-a, TGF-P, PDGF, FGF or IGF-I. Data are mean values from cells counted in triplicate 1 week later.
FIGURE10-Tumorigenicity of SFRLE cells. SFRLE cells at passages 3, 10, 50 and 100 were injected S.C.into the backs of athymic BALB/c mice. After about 20 days, small nodules were observed, showing SFRLE cells to be tumorigenic in athymic mice. Cells with prominent nucleoli proliferated in a trabecular pattern with thin, fibrous stroma (Hematoxylin-eosin stain). Scale bar = 40 km.
TABLE 111-CHROMOSOMAL ANALYSIS Cells -
RLE cells at passage 4 RLE cells at passage 45 SFRLE cells at passage 4
Chromosomal distribution 41
42
1 3
99
85
43
5040
60-70
7W8U
9&100
100-
11
1 6
48
21
14
11
After cells were treated with Colcemid, metaphase preparation were achieved by hypotonic treatment. Details are found in the text.
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weight of PGSF was 30 kDa on size-exclusion chromatography, but that of EGF, TGF-P, TGF-y, PDGF and F G F was about 6 kDa (Holladay et al., 1976), 23-25 kDa (Massague, 1984), 12 kDa (Hirai et al., 1983), 23 kDa (Deuel et a/., 1981) and 13 kDa (Gospodarowicz, 1975) respectively. Furthermore, immunoblotting analysis showed that PGSF did not cross-react with anti-EGF, TGF-a, TGF-P, P D G F or FGF. These findings suggest that PGSF may be a new type of T G F inasmuch as it induces anchorage-independent growth of NRK cells. Molecular similarities between PDGF and oncogene sisproducts have been reported (Doolittle et al., 1983; Waterfield et al., 1983). At present, there is no information as to what oncogene products are related to AGSF or PGSF. Hill et al. (1990), who established a cell line from Chinese hamster fibroblasts infected with Rous sarcoma virus, which could proliferate in serum-free medium, report that virus oncogene v-src is not related to cell proliferation in serum-free culture.
But, unlike SFRLE cells, their cell line is of non-epithelial origin. Carcinomas can be divided into 2 types, depending upon the stromal component. One is medullary carcinoma with a scanty fibrous component, and the other is scirrhous carcinoma with an abundant fibrous component. AGSF stimulated the growth of self epithelial cells, and PGSF stimulated the growth of fibroblasts. If malignant epithelial cells generally produce 2 growth factors such as AGSF and PGSF, higher production of AGSF may result in medullary carcinoma and higher production of PGSF may lead to scirrhous carcinoma. SFRLE cells formed a tumor in athymic mice in which the cells proliferated in an alveolar or trabecular pattern with thin, fibrous stroma. ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Ministry of Education (04807025).
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KUKOKAWA, R., KYAKUMOTO, S. and OTA, M., Autocrine growth factor in defined serum-free medium of human salivary gland adenocarcinoma cell line HSG. CuncerRes., 49,5163-5142 (1989). MASSAGUE, J., Type p transforming growth factor from feline sarcoma virus-transformed rat ce1ls.J. bid. Chern., 259,9756-9761 (1984). RUTENBURG, A.M., K I M . H . , FISHBEIN,J.W.. HANhER, J.S., WASSERKRUNG, H.L. and SELIGMAN, A.M., Histochemical and ultrastructural demonstration of y-glutamyl transpeptidase activity. J. Hisrochem. Cyfochern.,17,517-526 (1969). SHAPIRO, L.E. and WAGNER,N., Transferrin is an autocrine growth factor secreted by Reuber H-35 cells in serum-free culture. In Vitro Cell Devel. Biol., 25, 650-654 (1989). SPORN,M.B. and TODAKO,G.J., Autocrine secretion and malignant transformation of cells. Nen Engl. J. Med.. 303,878-880 (1980). WACHSTEIN, M. and MEISEL,E., Enzymic histochemistry of ethionin induced liver cirrhosis and hepatoma. J. Hisrochem. Cytochem., 7, 189-201 (1959). WATERFIELD, M.D., SCARACE, G.T., WHITTLE,N., STROOBANT, P., JOHNSON, A,, WASTERSON, A.. WESTERMARK, B.J., HELDIN,C-H., HUANG,J.S. and DEUEL,T.F., Platelet-derived growth factor is structurally related to the putative transforming protein p28"' of simian sarcoma virus. Nutitre (Lond.), 304,35-39 (1983).
