Matrix Vol. 12/1992, pp. 213-220 © 1992 by Gustav Fischer Verlag, Stuttgart
The Stimulation of Fibroblasts Collagen Synthesis by Neoplastic Cells is Modulated by the Extracellular Matrix A. NOEL1 , C. MUNAUT1 , B. NUSGENS2 , J. M. FOIDART 1 and CH. M. LAPIERE 2 1
2
Laboratory ofBiology and Laboratory of Experimental Dermatology, University of Liege, Sart Tilman, B4000 Liege, Belgium.
Abstract Human fibroblasts cocultured with neoplastic MCF7 cells produce increased amounts of collagen. A maximal stimulation requires direct cell-cell contacts between tumor cells and fibroblasts. However, this effect could be reproduced, although to a lesser extent, by medium conditioned by MCF7 cells, suggesting that it is mediated by a factor produced by MCF7 cells and secreted, at least partly, under a soluble form (Noel et al., 1992). This Collagen Stimulating Factor ("COSF") present in the culture medium displayed a molecular mass between 3,500 to 10,000 daltons, bound to heparin and appeared to be different from the growth factors described until now. The "COSF" can be released from the surface of MCF7 cells by treatment with heparin. The aim of the present work was to investigate the influence of various extracellular matrix components on the production and the release of "COSF". A 3- to 4-fold enhancement of collagen synthesis was observed in coculture on plastic and collagen type I substrates without significant modification of the non-collagen proteins. The increased collagen synthesis was paralleled by an elevation of specific collagen mRNAs level suggesting a regulation at a pretranslationallevel. On the opposite, in the presence of soluble or insoluble laminin, this stimulation was abolished. Similarly, coculture on "reconstituted basement membrane matrix", matrigel, did not increase collagen production. The "COSF" was found to bind to matrigel and could be released from the basement membrane matrix by treatment with heparin. The absence of collagen stimulation in the presence of laminin could not be ascribed to a lesser responsiveness of fibroblasts, an absence of "COSF" secretion by MCF7 cells or its binding to laminin. This absence of collagen stimulating effect was only observed when the two cell types cultured on laminin were in close contacts suggesting the requirement of direct intercellular contacts between tumoral cells and fibroblasts. Key words: collagen, desmoplasia, extracellular matrix, fibroblasts, neoplastic cells.
Introduction The microenvironment of malignant cells modulates the growth and the evolution of neoplasm. The major environmental factors involved in such a process are the contacts between tumoral cells and the extracellular matrix (ECM), and their interactions with host cells. Growing neoplasms
are often surrounded by abundant and excessive connective tissue. This "desmoplastic reaction" accentuated in some invasive carcinoma of the breast (Martinez-Hernandez, 1988) might result from the secretion by cancer cells of growth factors known to modulate the biosynthetic activity of mesenchymal cells as TGF~, TGF a, PDGF, FGF (Ignotz and Massague, 1986; Ross et al., 1986; Varga et al., 1987).
214
A. Noel et al.
Tumor cells in culture have been reported to produce diffusible factors able to specifically stimulate glycosaminoglycans synthesis by human skin fibroblasts (Merrilees and Finlay, 1985). Mammary carcinoma cells also secrete a 68 kDa polypeptide that enhances collagen synthesis by other cells, including fibroblasts (Bano et aI., 1983; 1985). Other biosynthetic products of tumor cells could also modulate the ECM macromolecules turnover by regulating their synthesis, degradation or both. This altered ECM can in turn influence various tumoral cell functions such as morphology (Noel et aI., 1988), differentiation, attachment, metastatic potential, biosynthetic activity (Kleinman et aI., 1985; Terranova et aI., 1984) and modulate their responsiveness to hormones and growth factors (Colige et aI., 1990; Gospodarowicz et aI., 1984). We have previously demonstrated that coculturing breast adenocarcinoma cells (MCF7 cells) and normal human fibroblasts resulted in a stimulation of collagen synthesis by fibroblasts. This effect was mediated by a factor synthesized by the carcinoma cells and released at least partly in the medium (Noel et aI., 1992). This collagen stimulating factor ("COSF") appeared different from any of the growth factors described until now. We have now investigated the influence of ECM proteins on the production and the release of "COSF" by MCF7 cells and the responsiveness of fibroblasts to this factor.
Materials and Methods Cells
Normal human breast or skin fibroblasts were obtained by outgrowth from explants and used between passages 4 and 12 as previously described by Delvoye et al. (1983). MCF7 cells, a human breast carcinoma cell line (Soule et aI., 1973) were generously provided by Dr. Leclercq (Bordet Institute, Brussels, Belgium). Cells were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Gibco), glutamine (292 mg/ml) , sodium bicarbonate (2.1 g/l), ascorbic acid (50 mg/l) and penicillin-streptomycin (100 U/ml) (standard medium). Preparation ofbiological reagents
Collagen type I was purified as described by Lapiere et al. (1977). Laminin was extracted from murine EngelbrethHolm-Swarm (EHS) tumor and purified according to Timpl et al. (1979). Fibronectin was collected from human plasma (Yamada, 1983). The purity of the preparations was monitored by SDS-polyacrylamide slab gel electrophoresis (Laemmli, 1970) and Western blot (Towbin et aI., 1979). Tissue culture plastic dishes (35 mm, Falcon) were coated with laminin (75 !.lgldish), fibronectin (100 !.lgldish) or collagen type I (100 !.lgldish) by drying overnight at room tem-
perature a solution of these proteins in 50 mM Tris-HCI (pH 7.4) or phosphate-buffered saline (PBS). After three washings with PBS, dishes were saturated with 1% solution of heat denaturated bovine serum albumin (BSA) in PBS, for 1 h at room temperature. Wells were then washed three times with PBS. Solid gels of basement membrane (BM) proteins (matrigel) were prepared from dialyzed urea extracts of EHS tumor as previously described (Kleinman et aI., 1986; Noel et aI., 1988). The major components of this BM gel were laminin (85%), entactin (10%), type IV collagen (4%) and heparan sulfate proteoglycan (1 %). One milliliter of matrigel (10 mg of protein/ml) was deposited in 35-mm plastic dishes and allowed to polymerize overnight at 37°C. Preparation ofconditioned medium (CM)
MCF7 cells (5 x 105 ) were cultured in 100-mm culture plastic dishes (Falcon) coated or not with purified laminin (100 !.lgldish) or collagen type 1(150 !.lgldish). CM was prepared by incubating cells for 48 h with 10 ml of DMEM without serum and centrifuged to remove cell debris before use. In some experiments, CM was dialyzed against serum free DMEM using membranes with a molecular weight cut off of 1,500 or 3,500 (Spectrapor, Polylab, Antwerpen, Belgium) or 10,000 (Analis, Ghent, Belgium). Metabolic labeling
Fibroblasts (2 x 105 ) and tumor cells (2 x 105 ) were plated on plastic either in monoculture or in coculture in DMEM supplemented or not with 10% dialyzed and decomplemented FCS and ascorbic acid (50 !.lglml). After one day of culture, metabolic labeling was performed for 24 h in the same but freshly replaced medium containing L-5- 3 H proline (10 !.lCi/ml) (37 Ci/mmol, NEN, Brussels, Belgium). In some experiments, 3 ml of medium conditioned by tumor cells were added to 2 ml of fresh medium at the beginning of the metabolic labeling period. Fresh medium or medium conditioned by fibroblasts was used as control. Collagen synthesis was identical in both control conditions. Soluble laminin (50 !.lglml) was added to some cultures. Collagen synthesis was measured, as previously described (Noel et aI., 1992). Measurement ofthe steady-state level ofspecific mRNAs
Total RNA was purified mainly as described by Chomczynski and Sacchi (1987). Monolayer cultures were washed with PBS at room temperature and directly solubilized in the lytic solution (RNAzoI) (Cinna/Biotecx, Texas, USA). Northern and dot-blot analysis were preformed as previously described (Noel et aI., 1991). The membranes were exposed to Kodak S-O-mat films. The extent of hybridiza-
Matrix Modulation of Collagen Production tion was quantified by LASER scanning densitometry (Ultroscan XL, LKB). For each experiment, the amount of blotted total RNA was controlled by hybridization with a 28 S rRNA specific oligonucleotide (Clontech Laboratories, USA) as previously described (Noel et aI., 1992). The cDNA probes specific for a1(1) (Hf-677) (Chu et aI., 1982) and a2(I) (Hf-32) (Myers et aI., 1981) collagen chains were kindly provided from Dr. F. Ramirez (New York) and for a1(III) (pIII-33) (Miskulin et aI., 1986) by Dr. R. Crystal (NIH, Bethesda, MD).
Release offactor from matrigel MCF7 cells (3 X 105 cells/35 mm dish) were cultured for 48 h on polymerized matrigel in serum-free DMEM. The medium was harvested and the gel was washed 3 times with serum-free medium. The matrix was then incubated with medium containing heparin (1 mg/ml) (Sigma) at room temperature, on a shaking platform, for 1 hour and the supernatant collected by centrifugation. Control experiments were performed with matrigel alone, without cells.
Statistical analysis Differences between the experimental conditions were evaluated using Student's t-test (p values lower than 0.02 were considered as significant).
Results In cocultures of fibroblasts and MCF7 cells, at a cell ratio of 1: 1, collagen synthesis by fibroblasts was stimulated by a factor of 2.5 to 5 (p < 0.01). In the presence of conditioned medium (CM) of MCF7 cells, the stimulation was only of 1.5 to 2.5 fold (p < 0.01) (Noel et aI., 1992). Preliminary observations suggest that the "Collagen Stimulating Factor" (COSF) displayed an apparent molecular size between 3,500 and 10,000 and could bind to heparin. The effect of vari~us extracellular matrix macromolecules on the production and the mode of release of this factor was investigated.
Effect ofvarious substrates on the collagen synthesis by fIbroblasts in coculture with MCF7 cells
Release offactor from cells surface Cells cultured on plastic or laminin were washed with 50 mM Tris-HCI, pH 7.4 (Tris buffer), and incubated for 2 h with a suspension of heparin-Sepharose beads (Pharmacia, LKB) (50 mg/ml) in the Tris buffer, at 4°C under agitation. The heparin-Sepharose was then harvested and extensively washed with the Tris buffer. Elution was performed successively with 1 M NaCl and 2 M NaCI in the Tris buffer. Samples were dialysed against serum-free medium using Spectrapor membranes (cut off: 3,500) and tested on fibroblasts cultures.
12.50 Fig. 1. Effect of various substrates on collagen stimulation in coculture of fibroblasts and MCF7 cells. Normal human fibroblasts were labeled with eHl-proline in monoculture or in coculture with tumoral MCF7 cells on various substrates and collagen synthesis was determined as described in Materials and Methods. The enhancement of collagen production was most significant in cocultures on plastic, fibronectin (FN) and type I collagen (COLL) coated dishes. No stimulation was observed in the presence of coated laminin (LAM), of soluble laminin (LAMsol.) added in the medium at a final concentration of 50 !!g/ml and of matrigel. In these culture conditions, MCF7 cells did not synthesize collagen.
215
As illustrated in Figure 1, subconfluent cocultures of fibroblasts and MCF7 cells on plastic, collagen or fibronectin-coated dishes, at a cellular ratio of 1: 1 produced increased amounts of collagen as compared to monocultures. ~n the contrary, this enhancement of the collagen productIOn by stromal cells was not observed in the presence of laminin either in soluble form in the medium, coated on plastic or included in a polymerized gel of basement membrane components (matrigel). W~en fibroblasts and MCF7 cells were plated at high density, at an 1: 1 ratio, on plastic or laminin-coated dishes the MCF7 cells developed a monolayer over the confluen;
r--------------------.
10.00 7.50 5.00 2.50 0.00 plastic
c::J
FN
fibroblasts
COLL
LAM _
LAMsoI MATRIGEL
cocultlr&
216
A. Noel et al.
fibroblasts and were physically separated from the laminin coat. In such culture conditions, collagen stimulation was maximal in both conditions (4 fold) (p < 0.01) (data not shown). These various substrates did not affect by themselves the synthesis of collagen by human fibroblasts since no significant modification was observed in monoculture (Fig. 1). The lack of collagen production by MCF7 cells in cultures on plastic or the various substrates was again observed (data not shown).
Measurement ofspecific mRNA levels in coculture With the aim to define at which step the collagen synthesis was regulated in coculture, the steady state level of the mRNAs specific for collagen types I and III was analyzed by slot blot hybridization. Total RNA was extracted from subconfluent cocultures of fibroblasts and MCF7 cells maintained during 48 h on piatic or laminin-coated dishes. The MCF7 cells did not expressed mRNA coding for these collagen chains (Noel et aI., 1991). As control, the two cell types were cultured separately for 48 h on both substrates and mixed just before RNA isolation. Densitometric analy-
PLASTIC
LAMININ
2.50
0.80
...
2.00
...
!J)
0.64
The Stimulation of Fibroblasts Collagen Synthesis by Neoplastic Cells is Modulated by the Extracellular Matrix A. NOEL1 , C. MUNAUT1 , B. NUSGENS2 , J. M. FOIDART 1 and CH. M. LAPIERE 2 1
2
Laboratory ofBiology and Laboratory of Experimental Dermatology, University of Liege, Sart Tilman, B4000 Liege, Belgium.
Abstract Human fibroblasts cocultured with neoplastic MCF7 cells produce increased amounts of collagen. A maximal stimulation requires direct cell-cell contacts between tumor cells and fibroblasts. However, this effect could be reproduced, although to a lesser extent, by medium conditioned by MCF7 cells, suggesting that it is mediated by a factor produced by MCF7 cells and secreted, at least partly, under a soluble form (Noel et al., 1992). This Collagen Stimulating Factor ("COSF") present in the culture medium displayed a molecular mass between 3,500 to 10,000 daltons, bound to heparin and appeared to be different from the growth factors described until now. The "COSF" can be released from the surface of MCF7 cells by treatment with heparin. The aim of the present work was to investigate the influence of various extracellular matrix components on the production and the release of "COSF". A 3- to 4-fold enhancement of collagen synthesis was observed in coculture on plastic and collagen type I substrates without significant modification of the non-collagen proteins. The increased collagen synthesis was paralleled by an elevation of specific collagen mRNAs level suggesting a regulation at a pretranslationallevel. On the opposite, in the presence of soluble or insoluble laminin, this stimulation was abolished. Similarly, coculture on "reconstituted basement membrane matrix", matrigel, did not increase collagen production. The "COSF" was found to bind to matrigel and could be released from the basement membrane matrix by treatment with heparin. The absence of collagen stimulation in the presence of laminin could not be ascribed to a lesser responsiveness of fibroblasts, an absence of "COSF" secretion by MCF7 cells or its binding to laminin. This absence of collagen stimulating effect was only observed when the two cell types cultured on laminin were in close contacts suggesting the requirement of direct intercellular contacts between tumoral cells and fibroblasts. Key words: collagen, desmoplasia, extracellular matrix, fibroblasts, neoplastic cells.
Introduction The microenvironment of malignant cells modulates the growth and the evolution of neoplasm. The major environmental factors involved in such a process are the contacts between tumoral cells and the extracellular matrix (ECM), and their interactions with host cells. Growing neoplasms
are often surrounded by abundant and excessive connective tissue. This "desmoplastic reaction" accentuated in some invasive carcinoma of the breast (Martinez-Hernandez, 1988) might result from the secretion by cancer cells of growth factors known to modulate the biosynthetic activity of mesenchymal cells as TGF~, TGF a, PDGF, FGF (Ignotz and Massague, 1986; Ross et al., 1986; Varga et al., 1987).
214
A. Noel et al.
Tumor cells in culture have been reported to produce diffusible factors able to specifically stimulate glycosaminoglycans synthesis by human skin fibroblasts (Merrilees and Finlay, 1985). Mammary carcinoma cells also secrete a 68 kDa polypeptide that enhances collagen synthesis by other cells, including fibroblasts (Bano et aI., 1983; 1985). Other biosynthetic products of tumor cells could also modulate the ECM macromolecules turnover by regulating their synthesis, degradation or both. This altered ECM can in turn influence various tumoral cell functions such as morphology (Noel et aI., 1988), differentiation, attachment, metastatic potential, biosynthetic activity (Kleinman et aI., 1985; Terranova et aI., 1984) and modulate their responsiveness to hormones and growth factors (Colige et aI., 1990; Gospodarowicz et aI., 1984). We have previously demonstrated that coculturing breast adenocarcinoma cells (MCF7 cells) and normal human fibroblasts resulted in a stimulation of collagen synthesis by fibroblasts. This effect was mediated by a factor synthesized by the carcinoma cells and released at least partly in the medium (Noel et aI., 1992). This collagen stimulating factor ("COSF") appeared different from any of the growth factors described until now. We have now investigated the influence of ECM proteins on the production and the release of "COSF" by MCF7 cells and the responsiveness of fibroblasts to this factor.
Materials and Methods Cells
Normal human breast or skin fibroblasts were obtained by outgrowth from explants and used between passages 4 and 12 as previously described by Delvoye et al. (1983). MCF7 cells, a human breast carcinoma cell line (Soule et aI., 1973) were generously provided by Dr. Leclercq (Bordet Institute, Brussels, Belgium). Cells were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 10% fetal calf serum (FCS) (Gibco), glutamine (292 mg/ml) , sodium bicarbonate (2.1 g/l), ascorbic acid (50 mg/l) and penicillin-streptomycin (100 U/ml) (standard medium). Preparation ofbiological reagents
Collagen type I was purified as described by Lapiere et al. (1977). Laminin was extracted from murine EngelbrethHolm-Swarm (EHS) tumor and purified according to Timpl et al. (1979). Fibronectin was collected from human plasma (Yamada, 1983). The purity of the preparations was monitored by SDS-polyacrylamide slab gel electrophoresis (Laemmli, 1970) and Western blot (Towbin et aI., 1979). Tissue culture plastic dishes (35 mm, Falcon) were coated with laminin (75 !.lgldish), fibronectin (100 !.lgldish) or collagen type I (100 !.lgldish) by drying overnight at room tem-
perature a solution of these proteins in 50 mM Tris-HCI (pH 7.4) or phosphate-buffered saline (PBS). After three washings with PBS, dishes were saturated with 1% solution of heat denaturated bovine serum albumin (BSA) in PBS, for 1 h at room temperature. Wells were then washed three times with PBS. Solid gels of basement membrane (BM) proteins (matrigel) were prepared from dialyzed urea extracts of EHS tumor as previously described (Kleinman et aI., 1986; Noel et aI., 1988). The major components of this BM gel were laminin (85%), entactin (10%), type IV collagen (4%) and heparan sulfate proteoglycan (1 %). One milliliter of matrigel (10 mg of protein/ml) was deposited in 35-mm plastic dishes and allowed to polymerize overnight at 37°C. Preparation ofconditioned medium (CM)
MCF7 cells (5 x 105 ) were cultured in 100-mm culture plastic dishes (Falcon) coated or not with purified laminin (100 !.lgldish) or collagen type 1(150 !.lgldish). CM was prepared by incubating cells for 48 h with 10 ml of DMEM without serum and centrifuged to remove cell debris before use. In some experiments, CM was dialyzed against serum free DMEM using membranes with a molecular weight cut off of 1,500 or 3,500 (Spectrapor, Polylab, Antwerpen, Belgium) or 10,000 (Analis, Ghent, Belgium). Metabolic labeling
Fibroblasts (2 x 105 ) and tumor cells (2 x 105 ) were plated on plastic either in monoculture or in coculture in DMEM supplemented or not with 10% dialyzed and decomplemented FCS and ascorbic acid (50 !.lglml). After one day of culture, metabolic labeling was performed for 24 h in the same but freshly replaced medium containing L-5- 3 H proline (10 !.lCi/ml) (37 Ci/mmol, NEN, Brussels, Belgium). In some experiments, 3 ml of medium conditioned by tumor cells were added to 2 ml of fresh medium at the beginning of the metabolic labeling period. Fresh medium or medium conditioned by fibroblasts was used as control. Collagen synthesis was identical in both control conditions. Soluble laminin (50 !.lglml) was added to some cultures. Collagen synthesis was measured, as previously described (Noel et aI., 1992). Measurement ofthe steady-state level ofspecific mRNAs
Total RNA was purified mainly as described by Chomczynski and Sacchi (1987). Monolayer cultures were washed with PBS at room temperature and directly solubilized in the lytic solution (RNAzoI) (Cinna/Biotecx, Texas, USA). Northern and dot-blot analysis were preformed as previously described (Noel et aI., 1991). The membranes were exposed to Kodak S-O-mat films. The extent of hybridiza-
Matrix Modulation of Collagen Production tion was quantified by LASER scanning densitometry (Ultroscan XL, LKB). For each experiment, the amount of blotted total RNA was controlled by hybridization with a 28 S rRNA specific oligonucleotide (Clontech Laboratories, USA) as previously described (Noel et aI., 1992). The cDNA probes specific for a1(1) (Hf-677) (Chu et aI., 1982) and a2(I) (Hf-32) (Myers et aI., 1981) collagen chains were kindly provided from Dr. F. Ramirez (New York) and for a1(III) (pIII-33) (Miskulin et aI., 1986) by Dr. R. Crystal (NIH, Bethesda, MD).
Release offactor from matrigel MCF7 cells (3 X 105 cells/35 mm dish) were cultured for 48 h on polymerized matrigel in serum-free DMEM. The medium was harvested and the gel was washed 3 times with serum-free medium. The matrix was then incubated with medium containing heparin (1 mg/ml) (Sigma) at room temperature, on a shaking platform, for 1 hour and the supernatant collected by centrifugation. Control experiments were performed with matrigel alone, without cells.
Statistical analysis Differences between the experimental conditions were evaluated using Student's t-test (p values lower than 0.02 were considered as significant).
Results In cocultures of fibroblasts and MCF7 cells, at a cell ratio of 1: 1, collagen synthesis by fibroblasts was stimulated by a factor of 2.5 to 5 (p < 0.01). In the presence of conditioned medium (CM) of MCF7 cells, the stimulation was only of 1.5 to 2.5 fold (p < 0.01) (Noel et aI., 1992). Preliminary observations suggest that the "Collagen Stimulating Factor" (COSF) displayed an apparent molecular size between 3,500 and 10,000 and could bind to heparin. The effect of vari~us extracellular matrix macromolecules on the production and the mode of release of this factor was investigated.
Effect ofvarious substrates on the collagen synthesis by fIbroblasts in coculture with MCF7 cells
Release offactor from cells surface Cells cultured on plastic or laminin were washed with 50 mM Tris-HCI, pH 7.4 (Tris buffer), and incubated for 2 h with a suspension of heparin-Sepharose beads (Pharmacia, LKB) (50 mg/ml) in the Tris buffer, at 4°C under agitation. The heparin-Sepharose was then harvested and extensively washed with the Tris buffer. Elution was performed successively with 1 M NaCl and 2 M NaCI in the Tris buffer. Samples were dialysed against serum-free medium using Spectrapor membranes (cut off: 3,500) and tested on fibroblasts cultures.
12.50 Fig. 1. Effect of various substrates on collagen stimulation in coculture of fibroblasts and MCF7 cells. Normal human fibroblasts were labeled with eHl-proline in monoculture or in coculture with tumoral MCF7 cells on various substrates and collagen synthesis was determined as described in Materials and Methods. The enhancement of collagen production was most significant in cocultures on plastic, fibronectin (FN) and type I collagen (COLL) coated dishes. No stimulation was observed in the presence of coated laminin (LAM), of soluble laminin (LAMsol.) added in the medium at a final concentration of 50 !!g/ml and of matrigel. In these culture conditions, MCF7 cells did not synthesize collagen.
215
As illustrated in Figure 1, subconfluent cocultures of fibroblasts and MCF7 cells on plastic, collagen or fibronectin-coated dishes, at a cellular ratio of 1: 1 produced increased amounts of collagen as compared to monocultures. ~n the contrary, this enhancement of the collagen productIOn by stromal cells was not observed in the presence of laminin either in soluble form in the medium, coated on plastic or included in a polymerized gel of basement membrane components (matrigel). W~en fibroblasts and MCF7 cells were plated at high density, at an 1: 1 ratio, on plastic or laminin-coated dishes the MCF7 cells developed a monolayer over the confluen;
r--------------------.
10.00 7.50 5.00 2.50 0.00 plastic
c::J
FN
fibroblasts
COLL
LAM _
LAMsoI MATRIGEL
cocultlr&
216
A. Noel et al.
fibroblasts and were physically separated from the laminin coat. In such culture conditions, collagen stimulation was maximal in both conditions (4 fold) (p < 0.01) (data not shown). These various substrates did not affect by themselves the synthesis of collagen by human fibroblasts since no significant modification was observed in monoculture (Fig. 1). The lack of collagen production by MCF7 cells in cultures on plastic or the various substrates was again observed (data not shown).
Measurement ofspecific mRNA levels in coculture With the aim to define at which step the collagen synthesis was regulated in coculture, the steady state level of the mRNAs specific for collagen types I and III was analyzed by slot blot hybridization. Total RNA was extracted from subconfluent cocultures of fibroblasts and MCF7 cells maintained during 48 h on piatic or laminin-coated dishes. The MCF7 cells did not expressed mRNA coding for these collagen chains (Noel et aI., 1991). As control, the two cell types were cultured separately for 48 h on both substrates and mixed just before RNA isolation. Densitometric analy-
PLASTIC
LAMININ
2.50
0.80
...
2.00
...
!J)
0.64
