Int. J. Cancer: 50,968-973 (1992) 0 1992 Wiley-Liss, Inc.
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Pub1car on of tne Internal onal Un on Against Cancer PLbl cat on ae I’Union internal onale Conlre le Cancer
REGULATION OF HUMAN COLON-CARCINOMA CELL ADHESION TO EXTRACELLULAR MATRIX BY TRANSFORMING GROWTH FACTOR p l Subhas CHAKRABARTY’ Division of Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA The regulatory effect of transforming growth factor P I (TGF-p I) on the adhesion of human colon-carcinoma cells t o the extracellular matrix (ECM) was investigated. ECMs used in this study included tissue-culturewells coated with fibronectin, laminin, collagen and BSA, as well as plastic wells. Three phenotypically different human colon-carcinoma cell lines (Moser, HCTl16, and KM I2SM) were used. The Moser cell line is moderately differentiated and, in terms of the diversity of responses elicited by TGF-P I, is the human colon-carcinoma cell line most responsive t o TGF-PI as reported to date. By comparison, the undifferentiated HCTl 16 and the highly metastatic KMl2SM cells are unresponsive t o this growth factor. We showed that TGF-P I regulated the adhesion responses of all 3 cell lines. However, the response profiles as well as the endogenous adhesive properties of each cell line were quite different from those of the others. Endogenous Arg-Gly-Asp(RGD)related receptors were present on the HCTl16 but not on the other cells. The observed regulatory effect of TGF-PI was contingent on the cell line, the type of ECM, and the growthfactor treatment protocol used. When cells were treated with TGF-PI for 16 hr prior t o exposure t o ECM in a 4-hr adhesion assay, a significant increase in adhesion t o fibronectin and collagen was observed for the Moser cells. For the identical experimental protocol, the KM I2SM cells respondedby increasing adhesion t o fibronectin, while the HCTl16 cells responded by decreasing adhesion t o collagen. Kinetic analyses of TGF-PI treatment showed that increased adhesion response to laminin was induced in the Moser cells after 2 hr of growth-factor treatment. This response declined rapidly upon further exposure of the cells to TGF-p I. Simultaneous exposure of cells to both TGF-PI and ECM negated the adhesion responses described above. The up-modulation of adhesion t o fibronectin, laminin and collagen by TGF-PI was mediated through RGDrelated integrin receptors. RGD-containingpeptides effectively blocked the enhanced adhesion responses induced by TGF-P I.
The extracellular matrix (ECM) regulates cellular behavior and physiology through interaction with specific cell-surface receptors for ECM-adhesion glycoproteins (Edelman, 1983; Yamada, 1983). Abnormal ECM synthesis or cell-ECM interaction, or both, underlie many pathophysiologic processes including cellular transformation (Chakrabarty et al., 1987; Varani and Chakrabarty, 1990). Altered cell-ECM interaction may be responsible for extravasation of tumor cells and their subsequent attachment to secondary sites (Humphries et al., 1986;Juliano, 1987). Transforming growth factor P l (TGF-P1) regulates the synthesis and expression of ECM-adhesion molecules and receptors for these molecules (Ignotz et al., 1987; Penttinen et al., 1988; Heino et al., 1989). Thus, TGF-P1 may play a role in matrix synthesis and cell-ECM interaction in both normal and pathophysiologic processess via autocrine or paracrine pathways (Piez and Sporn, 1990). TGF-P1 induces differentiation and synthesis of ECM glycoproteins in the human colon-carcinoma Moser cell line (Chakrabarty et al., 1988, 1989, 1990). TGF-P1 also enhances the differentiation-inducing effect of 3-dimensional collagen gel on SW1222 colorectal tumor cells (Pignatelli and Bodmer, 1989). The regulatory effect of TGF-P1 on cellular adhesion of human colon-carcinoma cells to different types of ECM sub-strata has not been reported. In this study, we investigated the regulatory effect of TGF-PI on cellular adhesion to different ECM substrata in 3 phenotypically different human colon-carcinoma cell lines: the moderately differentiated Moser (Chakrabarty et al., 1988,1989,1990), the relatively undifferen-
tiated HCT116, and the highly metastatic KM12SM (Brattain et al., 1984; Fan et al., 1989). Moser cells are the human colon-carcinoma cells most responsive to TGF-P1 as reported to date, while HCTl16 and KM12SM cells are unresponsive by comparison (Chakrabarty et al., 1990). Both HCTl16 and KM12SM cells showed a higher degree of endogenous adhesion to different type of ECM sub-strata than did the Moser cells. TGF-P1 elicited adhesion responses from all 3 cell lines. However, the Moser cells exhibited a greater degree of responses to TGF-P1. Contingent on the coloncarcinoma cell line, the type of ECM sub-stratum, and the conditions employed in the exposure of cells to growth factor and ECM, both up-modulation and down-modulation of adhesion by TGF-P1 were observed. Up-modulation of cellular adhesion to fibronectin, laminin and collagen by TGF-P1 was found to be mediated through Arg-Gly-Asp(RGD)-related integrin-like receptors. Endogenous RGD-related receptors were found to be present on HCTl16 cells but not on Moser and KM12SM cells. MATERIAL AND METHODS
Material Porcine TGF-P1 was purchased from R&D Systems (Minneapolis, MN). Human fibronectin ( f i b r o n e h ) , mouse laminin (laminin), rat-tail collagen type I (collagen), and BSA were purchased from Sigma (St. Louis, MO). 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were also purchased from Sigma, as were Arg-Gly-Asp-Ser (RGDS), Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Pro-Arg-Pro (GPRP) peptides. Cell lines and treatment with TGF-PI Working cultures of human colon-carcinoma Moser, HCTll6 and KM12SM cells were maintained in 25-cm2culture flasks at 37°C in a humidified atmosphere of 5% CO, in McCoy’s 5A medium supplemented with 5% FBS. Cells were detached from the culture flasks by trypsinization, washed with serumfree media, and seeded into sterile glass Erlenmeyer flasks for the purpose of treatment with TGF-01. The cells were finely suspended in serum-free media containing 200 pglml BSA and 5 ng/ml TGF-P1 and incubated at 37°C in the humidified CO, incubator for the period of time indicated in “Results”. Control cells were similarly treated, but without the incorporation of TGF-P1 in the serum-free medium. In these conditions, no attachment of cells to the glass culture flasks was observed over a 16-hr incubation period. Coating of tissue-culture wells with ECM glycoproteins ECM glycoproteins were coated onto 96-well tissue-culture plates as described by Pignatelli and Bodmer (1989), with slight modification. Briefly, 40 pl each of fibronectin (25 kg/ml in PBS), laminin (25 pg/ml in PBS), collagen (0.1 mg/ml in 0.2 ‘To whom correspondence and reprint requests should be addressed, at Division of Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, T X 77030, USA. Received: September 19, 1991 and in revised form November 11, 1991.
TGF-El AND COLON-CARCINOMA CELL ADHESION
N acetic acid), and BSA (200 pg/ml in serum-free media) were used to coat each tissue-culture well. The tissue-culture plates were left partially covered in a laminar-flow hood, and the solvent was allowed to evaporate completely, leaving a dried film of ECM materials in the culture wells. The tissue-culture wells were then rinsed several times with serum-free media containing 200 pg/ml BSA (binding buffer) before seeding of cells into ECM-coated wells.
Adhesion assay Adhesion assays were performed according to procedures described by Varani and Chakrabarty (1990), with some modification. Forty thousand TGF-p1 treated or untreated control cells were seeded into ECM-coated or untreated plastic culture wells. Cell number was initially determined by cell counting with the aid of a hemocytometer. The cells were allowed to attach at 37°C in a humidified CO, incubator for 4 hr unless stated otherwise. The culture wells were then washed several times with serum-free media to remove unattached cells. The number of attached cells were estimated indirectly by the tetrazolium-based colorimetric MTT assay (Carmichael et al., 1987). This assay is based on the ability of live cells to reduce a tetrazolium-based compound to a blue formazan product which can be measured spectrophotometrically. Initial experiments had shown that the amount of colored compound formed was directly and linearly correlated with cell number (1,000 to 40,000) as determined by cell counting. The average values of 4 replicates were determined in each experiment. The results presented here represent the average values of 4 experiments with their standard error as shown in the figures.
RESULTS
TGF-pl and cellular adhesion The adhesive properties of the 3 colon-carcinoma cell lines (TGF-p1 treated and untreated) on various ECM sub-strata were directly analyzed and compared (Fig. 1). In this series of experiments, TGF-P1 treatment was performed for 16 hr, after which 40,000 TGF-pl-treated or untreated control cells were seeded into previously prepared tissue-culture wells and allowed to attach at 37°C for 4 hr. Following washings to remove unattached cells, the number of attached cells was estimated by the M l T assay, as described in “Material and Methods”. Both the undifferentiated HCTll6 and the metastatic KM12SM cells showed an enhanced capacity to attach to the various ECM sub-strata, including plastic, when compared with the moderately differentiated Moser cells (Fig. 1). However, the Moser cells exhibited a significant increase in adhesion to plastic, fibronectin-, collagen- and BSA-coated sub-strata following 16 hr of TGF-P1 treatment (Fig. 1). By comparison with untreated control cells, approximately 40, 50, 30 and 40% increase in adhesion was observed on plastic and fibronectin-, collagen- and BSA-coated sub-strata respectively (Fig. Id). In these conditions, TGF-p1 did not modulate adhesion of the Moser cells to laminin-coated sub-strata (Fig. la). Though the HCT116 and the KM12SM cells possessed higher endogenous adhesive capability, the adhesive properties of these cells were less responsive to modulation by TGF-P1. In the HCT116 cells, an approximately 30% increase in adhesion to plastic and a decrease in adhesion to collagen and BSA by 20 and 25% respectively were observed following treatment with TGF-p1 (Fig. 1, b, e). No change in adhesion to either fibronectin or laminin was observed (Fig. lb). In the KM12SM cells, up-modulation of adhesion to fibronectin (30% increase) and down-modulation of adhesion to BSA (12% decrease) were observed following treatment of these cells with TGF-p1 (Fig. 1,c, f ).
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Inhibition of adhesion by RGD-containingpeptides Since integrins play a central role in cell adhesion (Ruoslahti and Pierschbacher, 1987) and human colon-carcinoma cells have been reported to possess integrins (Pignatelli and Bodmer, 1988, 1989), a series of experiments were performed to determine whether these cells possessed RGD-related integrin receptors by using RGD-containing peptides to inhibit cellular adhesion in the adhesion assay. For this purpose, cells were first incubated with RGDS, GRGDS or GPRP (control) peptides for 15 min, then seeded in the presence of these peptides into plastic and fibronectin-, laminin-, collagen- or BSA-coated tissue-culture wells; the adhesion assay was then performed as described above. In the HCT116 cells, adhesion to fibronectin- and laminin-coated sub-strata was reduced by 40 and 30% respectively, when RGD-containing peptides in the concentration of 100 kg/ml were incorporated into the assay (Table I). Control GPRP peptides had no inhibitory effect in the assay. The results of these adhesion-inhibition experiments suggested that the HCT116 cells possessed endogenous integrin-related fibronectin and laminin receptors and that the binding of these receptors to fibronectin and laminin was amenable to inhibition by RGD sequences. By comparison, in identical experimental conditions, no inhibitory effect was observed in the Moser and KM12SM cells (Table I). None of the peptides used was able to inhibit adhesion to plastic or to collagen- or BSA-coated sub-strata (Table I). Modulation of RGD-related receptors by TGF-p l TGF-pl has been reported to up-regulate the expression of integrin-related ECM receptors in human colon-carcinoma cells (Pignatelli and Bodmer, 1989). Since TGF-p1 upmodulated the adhesion of Moser cells to fibronectin and collagen, and up-modulated the adhesion of KM12SM cells to fibronectin (Fig. l), I addressed the issue of whether upmodulation of adhesion to these ECMs was due to enhanced expression of integrin-related receptors resulting from treatment with TGF-P1. RGD-containing and control peptides were used to block the adhesion of TGF-pl-treated Moser and KM12SM cells. The increase in adhesion observed in both cell types upon TGF-PI treatment was totally abolished by RGDcontaining peptides (Table 11). In the presence of RGDcontaining peptides, no differences in adhesion were observed between control and TGF-pl-treated cells. Effect of short-term treatment with TGF-p l and cellular adhesion The effect of short-term treatment with TGF-PI on cellular adhesion was investigated. Cells were treated with TGF-p1 for 0.5, 1, 2, 4 or 6 hr and the effect of treatment on cellular adhesion determined. In the Moser cells, 2 hr of treatment with growth factor resulted in a 35% increase in adhesion to laminin followed by a progressive decline in adhesion upon longer exposure to TGF-01 (Fig. Za). A progressive increase in adhesion to the other ECM as a function of exposure time was observed (Fig. 2a). In the HCTl16 cells, a 10 to 15% increase in adhesion to fibronectin, laminin, collagen, and BSA was observed with a 6-hr treatment protocol (Fig. 2b).However, a 25% increase in adhesion to plastic was observed following 1 hr of TGF-p1 treatment (Fig. 2b).This increase in adhesion to plastic was abrograted upon longer exposure of the cells to growth factor (Fig. 2b). In the KM12SM cells, an increase (10 to 25%) in adhesion to various ECMs was observed after 1 hr of treatment with growth factor (Fig. 2 ) .A slight decrease in adhesion was then observed after 4 hr of TGF-P1 treatment followed by a 30% increase in adhesion to fibronectin (Fig. 2). Effects of simultaneous exposure to ECM and TGF-p l All the above experiments were performed with cells pretreated with TGF-61 prior to determination of cellular adhesion. The following experiments were performed to assess the
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Pub1car on of tne Internal onal Un on Against Cancer PLbl cat on ae I’Union internal onale Conlre le Cancer
REGULATION OF HUMAN COLON-CARCINOMA CELL ADHESION TO EXTRACELLULAR MATRIX BY TRANSFORMING GROWTH FACTOR p l Subhas CHAKRABARTY’ Division of Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA The regulatory effect of transforming growth factor P I (TGF-p I) on the adhesion of human colon-carcinoma cells t o the extracellular matrix (ECM) was investigated. ECMs used in this study included tissue-culturewells coated with fibronectin, laminin, collagen and BSA, as well as plastic wells. Three phenotypically different human colon-carcinoma cell lines (Moser, HCTl16, and KM I2SM) were used. The Moser cell line is moderately differentiated and, in terms of the diversity of responses elicited by TGF-P I, is the human colon-carcinoma cell line most responsive t o TGF-PI as reported to date. By comparison, the undifferentiated HCTl 16 and the highly metastatic KMl2SM cells are unresponsive t o this growth factor. We showed that TGF-P I regulated the adhesion responses of all 3 cell lines. However, the response profiles as well as the endogenous adhesive properties of each cell line were quite different from those of the others. Endogenous Arg-Gly-Asp(RGD)related receptors were present on the HCTl16 but not on the other cells. The observed regulatory effect of TGF-PI was contingent on the cell line, the type of ECM, and the growthfactor treatment protocol used. When cells were treated with TGF-PI for 16 hr prior t o exposure t o ECM in a 4-hr adhesion assay, a significant increase in adhesion t o fibronectin and collagen was observed for the Moser cells. For the identical experimental protocol, the KM I2SM cells respondedby increasing adhesion t o fibronectin, while the HCTl16 cells responded by decreasing adhesion t o collagen. Kinetic analyses of TGF-PI treatment showed that increased adhesion response to laminin was induced in the Moser cells after 2 hr of growth-factor treatment. This response declined rapidly upon further exposure of the cells to TGF-p I. Simultaneous exposure of cells to both TGF-PI and ECM negated the adhesion responses described above. The up-modulation of adhesion t o fibronectin, laminin and collagen by TGF-PI was mediated through RGDrelated integrin receptors. RGD-containingpeptides effectively blocked the enhanced adhesion responses induced by TGF-P I.
The extracellular matrix (ECM) regulates cellular behavior and physiology through interaction with specific cell-surface receptors for ECM-adhesion glycoproteins (Edelman, 1983; Yamada, 1983). Abnormal ECM synthesis or cell-ECM interaction, or both, underlie many pathophysiologic processes including cellular transformation (Chakrabarty et al., 1987; Varani and Chakrabarty, 1990). Altered cell-ECM interaction may be responsible for extravasation of tumor cells and their subsequent attachment to secondary sites (Humphries et al., 1986;Juliano, 1987). Transforming growth factor P l (TGF-P1) regulates the synthesis and expression of ECM-adhesion molecules and receptors for these molecules (Ignotz et al., 1987; Penttinen et al., 1988; Heino et al., 1989). Thus, TGF-P1 may play a role in matrix synthesis and cell-ECM interaction in both normal and pathophysiologic processess via autocrine or paracrine pathways (Piez and Sporn, 1990). TGF-P1 induces differentiation and synthesis of ECM glycoproteins in the human colon-carcinoma Moser cell line (Chakrabarty et al., 1988, 1989, 1990). TGF-P1 also enhances the differentiation-inducing effect of 3-dimensional collagen gel on SW1222 colorectal tumor cells (Pignatelli and Bodmer, 1989). The regulatory effect of TGF-P1 on cellular adhesion of human colon-carcinoma cells to different types of ECM sub-strata has not been reported. In this study, we investigated the regulatory effect of TGF-PI on cellular adhesion to different ECM substrata in 3 phenotypically different human colon-carcinoma cell lines: the moderately differentiated Moser (Chakrabarty et al., 1988,1989,1990), the relatively undifferen-
tiated HCT116, and the highly metastatic KM12SM (Brattain et al., 1984; Fan et al., 1989). Moser cells are the human colon-carcinoma cells most responsive to TGF-P1 as reported to date, while HCTl16 and KM12SM cells are unresponsive by comparison (Chakrabarty et al., 1990). Both HCTl16 and KM12SM cells showed a higher degree of endogenous adhesion to different type of ECM sub-strata than did the Moser cells. TGF-P1 elicited adhesion responses from all 3 cell lines. However, the Moser cells exhibited a greater degree of responses to TGF-P1. Contingent on the coloncarcinoma cell line, the type of ECM sub-stratum, and the conditions employed in the exposure of cells to growth factor and ECM, both up-modulation and down-modulation of adhesion by TGF-P1 were observed. Up-modulation of cellular adhesion to fibronectin, laminin and collagen by TGF-P1 was found to be mediated through Arg-Gly-Asp(RGD)-related integrin-like receptors. Endogenous RGD-related receptors were found to be present on HCTl16 cells but not on Moser and KM12SM cells. MATERIAL AND METHODS
Material Porcine TGF-P1 was purchased from R&D Systems (Minneapolis, MN). Human fibronectin ( f i b r o n e h ) , mouse laminin (laminin), rat-tail collagen type I (collagen), and BSA were purchased from Sigma (St. Louis, MO). 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) and dimethyl sulfoxide (DMSO) were also purchased from Sigma, as were Arg-Gly-Asp-Ser (RGDS), Gly-Arg-Gly-Asp-Ser (GRGDS) and Gly-Pro-Arg-Pro (GPRP) peptides. Cell lines and treatment with TGF-PI Working cultures of human colon-carcinoma Moser, HCTll6 and KM12SM cells were maintained in 25-cm2culture flasks at 37°C in a humidified atmosphere of 5% CO, in McCoy’s 5A medium supplemented with 5% FBS. Cells were detached from the culture flasks by trypsinization, washed with serumfree media, and seeded into sterile glass Erlenmeyer flasks for the purpose of treatment with TGF-01. The cells were finely suspended in serum-free media containing 200 pglml BSA and 5 ng/ml TGF-P1 and incubated at 37°C in the humidified CO, incubator for the period of time indicated in “Results”. Control cells were similarly treated, but without the incorporation of TGF-P1 in the serum-free medium. In these conditions, no attachment of cells to the glass culture flasks was observed over a 16-hr incubation period. Coating of tissue-culture wells with ECM glycoproteins ECM glycoproteins were coated onto 96-well tissue-culture plates as described by Pignatelli and Bodmer (1989), with slight modification. Briefly, 40 pl each of fibronectin (25 kg/ml in PBS), laminin (25 pg/ml in PBS), collagen (0.1 mg/ml in 0.2 ‘To whom correspondence and reprint requests should be addressed, at Division of Laboratory Medicine, The University of Texas, M.D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, T X 77030, USA. Received: September 19, 1991 and in revised form November 11, 1991.
TGF-El AND COLON-CARCINOMA CELL ADHESION
N acetic acid), and BSA (200 pg/ml in serum-free media) were used to coat each tissue-culture well. The tissue-culture plates were left partially covered in a laminar-flow hood, and the solvent was allowed to evaporate completely, leaving a dried film of ECM materials in the culture wells. The tissue-culture wells were then rinsed several times with serum-free media containing 200 pg/ml BSA (binding buffer) before seeding of cells into ECM-coated wells.
Adhesion assay Adhesion assays were performed according to procedures described by Varani and Chakrabarty (1990), with some modification. Forty thousand TGF-p1 treated or untreated control cells were seeded into ECM-coated or untreated plastic culture wells. Cell number was initially determined by cell counting with the aid of a hemocytometer. The cells were allowed to attach at 37°C in a humidified CO, incubator for 4 hr unless stated otherwise. The culture wells were then washed several times with serum-free media to remove unattached cells. The number of attached cells were estimated indirectly by the tetrazolium-based colorimetric MTT assay (Carmichael et al., 1987). This assay is based on the ability of live cells to reduce a tetrazolium-based compound to a blue formazan product which can be measured spectrophotometrically. Initial experiments had shown that the amount of colored compound formed was directly and linearly correlated with cell number (1,000 to 40,000) as determined by cell counting. The average values of 4 replicates were determined in each experiment. The results presented here represent the average values of 4 experiments with their standard error as shown in the figures.
RESULTS
TGF-pl and cellular adhesion The adhesive properties of the 3 colon-carcinoma cell lines (TGF-p1 treated and untreated) on various ECM sub-strata were directly analyzed and compared (Fig. 1). In this series of experiments, TGF-P1 treatment was performed for 16 hr, after which 40,000 TGF-pl-treated or untreated control cells were seeded into previously prepared tissue-culture wells and allowed to attach at 37°C for 4 hr. Following washings to remove unattached cells, the number of attached cells was estimated by the M l T assay, as described in “Material and Methods”. Both the undifferentiated HCTll6 and the metastatic KM12SM cells showed an enhanced capacity to attach to the various ECM sub-strata, including plastic, when compared with the moderately differentiated Moser cells (Fig. 1). However, the Moser cells exhibited a significant increase in adhesion to plastic, fibronectin-, collagen- and BSA-coated sub-strata following 16 hr of TGF-P1 treatment (Fig. 1). By comparison with untreated control cells, approximately 40, 50, 30 and 40% increase in adhesion was observed on plastic and fibronectin-, collagen- and BSA-coated sub-strata respectively (Fig. Id). In these conditions, TGF-p1 did not modulate adhesion of the Moser cells to laminin-coated sub-strata (Fig. la). Though the HCT116 and the KM12SM cells possessed higher endogenous adhesive capability, the adhesive properties of these cells were less responsive to modulation by TGF-P1. In the HCT116 cells, an approximately 30% increase in adhesion to plastic and a decrease in adhesion to collagen and BSA by 20 and 25% respectively were observed following treatment with TGF-p1 (Fig. 1, b, e). No change in adhesion to either fibronectin or laminin was observed (Fig. lb). In the KM12SM cells, up-modulation of adhesion to fibronectin (30% increase) and down-modulation of adhesion to BSA (12% decrease) were observed following treatment of these cells with TGF-p1 (Fig. 1,c, f ).
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Inhibition of adhesion by RGD-containingpeptides Since integrins play a central role in cell adhesion (Ruoslahti and Pierschbacher, 1987) and human colon-carcinoma cells have been reported to possess integrins (Pignatelli and Bodmer, 1988, 1989), a series of experiments were performed to determine whether these cells possessed RGD-related integrin receptors by using RGD-containing peptides to inhibit cellular adhesion in the adhesion assay. For this purpose, cells were first incubated with RGDS, GRGDS or GPRP (control) peptides for 15 min, then seeded in the presence of these peptides into plastic and fibronectin-, laminin-, collagen- or BSA-coated tissue-culture wells; the adhesion assay was then performed as described above. In the HCT116 cells, adhesion to fibronectin- and laminin-coated sub-strata was reduced by 40 and 30% respectively, when RGD-containing peptides in the concentration of 100 kg/ml were incorporated into the assay (Table I). Control GPRP peptides had no inhibitory effect in the assay. The results of these adhesion-inhibition experiments suggested that the HCT116 cells possessed endogenous integrin-related fibronectin and laminin receptors and that the binding of these receptors to fibronectin and laminin was amenable to inhibition by RGD sequences. By comparison, in identical experimental conditions, no inhibitory effect was observed in the Moser and KM12SM cells (Table I). None of the peptides used was able to inhibit adhesion to plastic or to collagen- or BSA-coated sub-strata (Table I). Modulation of RGD-related receptors by TGF-p l TGF-pl has been reported to up-regulate the expression of integrin-related ECM receptors in human colon-carcinoma cells (Pignatelli and Bodmer, 1989). Since TGF-p1 upmodulated the adhesion of Moser cells to fibronectin and collagen, and up-modulated the adhesion of KM12SM cells to fibronectin (Fig. l), I addressed the issue of whether upmodulation of adhesion to these ECMs was due to enhanced expression of integrin-related receptors resulting from treatment with TGF-P1. RGD-containing and control peptides were used to block the adhesion of TGF-pl-treated Moser and KM12SM cells. The increase in adhesion observed in both cell types upon TGF-PI treatment was totally abolished by RGDcontaining peptides (Table 11). In the presence of RGDcontaining peptides, no differences in adhesion were observed between control and TGF-pl-treated cells. Effect of short-term treatment with TGF-p l and cellular adhesion The effect of short-term treatment with TGF-PI on cellular adhesion was investigated. Cells were treated with TGF-p1 for 0.5, 1, 2, 4 or 6 hr and the effect of treatment on cellular adhesion determined. In the Moser cells, 2 hr of treatment with growth factor resulted in a 35% increase in adhesion to laminin followed by a progressive decline in adhesion upon longer exposure to TGF-01 (Fig. Za). A progressive increase in adhesion to the other ECM as a function of exposure time was observed (Fig. 2a). In the HCTl16 cells, a 10 to 15% increase in adhesion to fibronectin, laminin, collagen, and BSA was observed with a 6-hr treatment protocol (Fig. 2b).However, a 25% increase in adhesion to plastic was observed following 1 hr of TGF-p1 treatment (Fig. 2b).This increase in adhesion to plastic was abrograted upon longer exposure of the cells to growth factor (Fig. 2b). In the KM12SM cells, an increase (10 to 25%) in adhesion to various ECMs was observed after 1 hr of treatment with growth factor (Fig. 2 ) .A slight decrease in adhesion was then observed after 4 hr of TGF-P1 treatment followed by a 30% increase in adhesion to fibronectin (Fig. 2). Effects of simultaneous exposure to ECM and TGF-p l All the above experiments were performed with cells pretreated with TGF-61 prior to determination of cellular adhesion. The following experiments were performed to assess the
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