JOURNAL OF CELLULAR PHYSIOLOGY 146:451459 119911
The RGD Containing Site of the Mouse Laminin A Chain is Active for Cell Attachment, Spreading, Migration and Neurite Outgrowth KEN-ICHIRO TASHIRO, GREGORY C. SEPHEL, DAVE GREATOREX, MAKOTO SASAKI, NORIO SHIRASHI, GEORGE R. MARTIN, HYNDA K. KLEINMAN, AND YOSHlHlKO YAMADA* Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National lnstitutes of Health, Bethesda, Maryland 20892 The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.
Laminin (Mr = 900,000) is a large glycoprotein specific to basement membrane (Timpl et al., 1979; Chung et al., 1979). Laminin binds to other basement membrane components, such as collagen IV, heparan sulfate proteoglycan, and nidogedentactin, and mediates cellular interactions with this matrix (Timpl and Martin, 1982; Timpl et al., 1984; Kleinman et al., 1986; Laurie et al., 1986, Martin et al., 1989). Current models suggest that laminin contains one A chain (Mr = 440,000), one B1 chain (Mr = 225,0001, and one B2 chain (Mr = 205,000) (Barlow et al., 1984; Paulsson et al., 1985; Palm et al., 19851, arranged as shown in Figure 1 to form a cross-shaped molecule. Laminin exhibits a number of biological activities, including promotion of attachment, migration, growth, differentiation, tumor cell metastasis, collagenase IV activity, and neurite outgrowth (Baron Van Evercooren et al., 1982;Aumailley et al., 1983; Grover et al., 1983; Kleinman et al., 1985; Hadley et al., 1985; Greenberg and Hay, 1986; Edgar et al., 1984; Engvall et al., 1986). Based on studies using proteolytic fragments, cell binding has been attributed to the portion of laminin containing the intersecting chains and the end of the long arm. A pentapeptide, YIGSR-NH, (Tyr-Ile-GlySer-Arg) from the cysteine-rich domain (domain 111) of the B1 chain, was found to support cell attachment, 67 Kd receptor binding, and migration. It also prevents tumor cell metastasis in experimental animals (Graf et al., 1987; Iwamoto et al., 1987; 1988). This peptide, however, did not mimic all the activities of laminin and was unable to promote neurite outgrowth or cell growth, or stimulate collagenase IV production. Neurite outgrowth activity has been localized to an another region of the molecule (Edgar et al., 1984). C 1991 WILEY-LISS, INC.
We have recently cloned and sequenced the A chain of laminin. It is comprised of 3084 amino acids, which form at least 8 distinct structural domains, (Sasaki et al., 1988). Particular attention was directed to the potential biological role of domain IIIb due to the presence of an RGD sequence which is known to be a versatile cell attachment site in fibronectin and in various other proteins (Ruoslahti and Pierschbacher, 19861, including the basement membrane glycoproteins entactin (Chakravarti et al., 1990) and laminin (Grant et al., 1989;Aumailley et al., 1990).One study suggested that the RGD sequence of the laminin-entactin type IV collagen mixture matrigel was active for endothelial cells (Grant et al., 1989) and another study demonstrated that the RGD site on laminin was latent and required proteolysis (Aumailley et al., 1990). Also, some of the integrin family of cell matrix receptors are known to recognize the RGD sequence. We, therefore, sought to extend these findings and to determine the biological importance of the RGD site in laminin using synthetic peptides to the region containing the RGD sequence and to determine the cellular receptor inReceived July 20, 1990; accepted December 7, 1990. *To whom reprint requestsicorrespondence should be addressed. Gregory C. Sephel’s present address is Vanderbilt University School of Medicine, Laboratory Service, 1310 24th Avenue South, Nashville, TN 37203 George R. Martin’s present address is National Institute on Aging, Gerontology Research Center, 4940 Eastern Avenue, Baltimore, MD 21224 Ken-ichiro Tashiro’s present address is Department of Psychiatry, Saga Medical School, Nabeshima, Saga 840-01, Japan.
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Fig. 1. Structural model of laminin. The Roman numerals designate the domains in each chain. G designates the A chain terminal domain. The location of the peptides used in this study are indicated by arrows. The peptides and their respective residue numbers in laminin are: peptide PA21, residues 1115-1129; PA26, residues 1120-1127; PA36, residues 1113-1123; PAlO, residues 2109-2128.
volved. Our data show that laminin-derived RGDcontaining peptides were active for cell attachment, migration, and neurite outgrowth via an integrin. These data identify the RGD sequence in laminin as a potential cell interaction site and indicate that laminin has multiple sites with biological activity. MATERIALS AND METHODS Synthesis of peptides a n d preparation of laminin Peptides with a terminal amide group were synthesized using an automated Model 430A synthesizer (Applied Biosystems, Inc., Foster City, Calif.). Deprotection and release of the peptides from the solid-phase support matrix were accomplished by treating the protected peptide on the resin with anhydrous HF containing 10% thioanisole or 10% anisole for 1-2 hours at 0°C. Following deprotection and release from the resin, the peptides were extracted with either ethyl acetate or diethyl ether. The peptides were then dissolved in 20-50 ml of 10% aqueous acetic acid, filtered to remove the resin and lyophilized. Peptides were screened for biological activity and were further urified by preparative HPLC when analytical HPL indicated that the products were heterogeneous. The purity and composition of the peptides were verified by HPLC and amino acid analysis. Mouse laminin was extracted and purified from the Engelbreth-Holm-Swarm (EHS) tumor, using methods previously described ("imp1 et al., 1979). Laminin as isolated by this procedure contains approximately 5% entactin.
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Conjugation of peptides Prior to conjugation of peptides to Keyhold Lim et Hemocyanin (KLH), the KLH (30 mg) was dissolvecfin 5 ml of 0.1 M NaHC03 with stirring for 15 minutes and then m-Maleimidobenzoylsulfosuccinimideester (MBS, 10 mg) was added to the KLH solution and stirred for 15 minutes, For the conjugation reaction, the peptide (50 mg) was combined with the KLH/MBS solution, and the conjugation reaction was allowed to proceed for 2 hours at 20°C. Following the conjugation process, the conjugates were dialyzed against 0.1 M NH,C03 for 18 hours at 4°C and lyophilized. Cells and culture Various cells were studied, including M2 mouse melanoma, HT-1080 human fibrosarcoma cells (Rasheed et al., 1974). B16F10 mouse melanoma (Fidler, 19731, NG108-15 a mouse neuroblastoma-rat glioma hybrid (Nirenberg et al., 1983), calf bovine Dulmonarv endothelial cells (CPAE:ATCC. CCL 209), ind a human embryonal rhabdomyosarcoma cell line (RD;ATCC,CCL136).M2 cells (a gift of I.J. Fidler, M.D. Anderson) were maintained in Dulbecco's Modified Eagle's Medium (DMEM)supplemented with 10%fetal calf serum (FCS) and grown to 80% confluence. For adhesion assays, cells were washed with PBS (pH 7.4) lacking Ca2+ and Mg2+ and then detached with 0.025% trypsin, 0.025% EDTA (GIBCO).The cells were then sedimented by low sFeed centrifugation, suspended in serum-free Eagle s Minimal Essential Medium (EMEM) containing 0.02% BSA, and then used immediately in the adhesion assay. HT-1080 cells were maintained in DMEM supplemented with 10% FCS. These cells were grown and prepared for adhesion assays as described for M2 cells. B16F10 murine melanoma cells (a gift from I.J. Fidler, Houston, TX), were maintained in EMEM supplemented with 5% FCS, non-essential amino acids, and vitamins. Cells were grown to 80% confluency in Falcon tissue culture dish. For chemotaxis assays, cells were harvested as described above, and washed twice, and then the final pellet was resuspended in serum-free EMEM containing 0.1% BSA. These cells were immediately added to the upper compartment of the Boyden Chamber. NG108-15 neuroblastoma x glioma cells attach and extend out long neuronal processes in the presence of laminin (Luckenbill-Edds et al., 1986). These cells were grown to confluence in DMEM containing NaHC03 and supplemented with 0.1 mM hypoxanthine, 0.4 p.M aminopterin, 16 pM thymidine, and 10% FCS. Dishes were rinsed with serum-free EMEM containing 0.02% BSA, and the cells were detached by pipetting and sedimented by low-speed centrifugation. The pellet was resuspended in serum-free EMEM containing 0.02% BSA and then used immediately in the adhesion assay and in the neurite outgrowth assay. CPAE cells were maintained in EMEM supplemented with 20% FCS. NIH 3T3 cells were maintained in DMEM supplemented with 10%calf serum. RD cells were maintained in EMEM supplemented with 10% FCS and twice the standard concentration of amino acids and vitamins. These cells were grown and prepared for adhesion assays as described for M2 cells.
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Cell adhesion Various concentrations of either laminin or peptide were added to 96 well tissue culture dishes in a total volume of 0.1 ml of serum-free EMEM and incubated for 2 hours at 37°C in 5% COz, 95% air. Unbound surfaces were subsequently blocked with 0.1 ml of EMEM containing 3% BSA for 30 minutes at 37°C. At the end of this period, the wells were rinsed with PBS to remove unattached cells and attached cells were trypsinized and electronically counted. Each assay was carried out in duplicate, and the du licates did not vary by more than 10%. Each pepti c fe was tested in 5 separate experiments and a t least 3 concentrations. Peptide inhibition of cell attachment was assayed using 96 well dishes coated with 2 pg/ml of either laminin or fibronectin as described above. Prior to addition of the cells, varying amounts of peptide solubilized in serum-free EMEM containing 0.02% BSA were added to each well. Cells were added and incubated for 30 minutes at 37°C and the number of attached cells was determined as described above. The effect of polyclonal antisera against the fibronectin (a5pl complex)receptor (integrin) (gift of Drs. Steve Akiyama and Ken Yamada, NCI; Akiyama et al., 1990) on cell attachment was assayed. Each well of a 96 well dish was coated with either 2.5 pg of laminin or fibronectin or 55 pg of various peptides as described above. HT-1080 cells (5000/well)were incubated with 2 different dilutions (150 and 1:lOO) of antisera for 15 minutes, sedimented by low speed centrifugation, resuspended in 100 pl of serum-free MEM plus 0.02% BSA, and then added into the substrate-coated wells. Cells were incubated at 37°C for 20 minutes, unattached cells were rinsed from the dish and attached cells were stained with hematoxylin and eosin and counted by microscope. Monoclonal antibody, GoH3 (gift of Dr. Sonnenberg, Central Laboratory of the Netherlands, Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, University of Amsterdam, The Netherlands) to the integrin, VLA-6, was also used in the inhibition assay for cell attachment. The assay was same as described above except dilutions of 1:5 and 1:20 were employed. Each concentration was tested in duplicate and the duplicates did not differ by more than 10%. The experiment was repeated two times. Immunoprecipitationof integrins HT-1080 cells were cell surface labeled with lZ5Iiodine using the lactoperoxidase technique. The antibodies to the integrins described and preimmune controls mentioned above were used in a standard immunoprecipition assay. Cell migration Chemotaxis assays were carried out as previously described (Mensin et al., 1984; McCarthy and Furcht, 1984). In brief, PO ycarbonate filters (8 pm pore size; Neuroprobe) were coated with type IV collagen (5 pg per filter). A 1 pm pore size uncoated filters were placed in a modified Boyden Chamber and 8 km pore size type IV collagen-coated filters were laced on top. B16F10 melanoma cells (3.0 x 105/0.9m ) were placed
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TABLE 1. Synthetic peptides Peptide PA21 PA26 PA36 PA10
Sequence
Hesidues’
CQAGTFALRGDNPQG C’FALRGDNP C~SKCQAGTFALR DCIRAYOPQTSSTNYNTLII,
11 15-1 129 1120-1127 1113- 1I23 2109-2128
:Residues are from Sasaki et al. (1988) C(Cyskine) i s artificially attached.
in the upper compartment of the Boyden Chamber. The chemoattractant was placed in the lower compartment including either test peptide (200-300 pgiml), or laminin (200-300 pg/ml) in serum-free DMEM with 0.1% BSA (0.22 ml). The chambers were sealed and then incubated for 5 hours at 37°C. The cells that had attached to the upper side of the 8 pm pore size filter were mechanically removed. The cells that had migrated to the lower side of the 8 pm pore size filter were fixed in methanol and then stained with hematoxylin and eosin. The cells were counted by using an Optomax V HR. Each sample was assayed in duplicate and the duplicates did not differ by more than 10%.Each assay was repeated 5 times. The ability of the peptides to block migration to laminin was tested. The chemoattractant in the lower chamber was laminin (20 pg/ml). The cells were placed in the upper compartment of the Boyden Chamber in the presence of the peptide (10 to 300 pg/ml) being tested. The inhibition assay was carried out in duplicate and the assays were repeated 5 times.
RESULTS Cell adhesion Peptides including the laminin A chain RGD sequence or neighboring sequences were tested for their ability to induce attachment and spreading. A 15-mer designated PA21 (Table 11, corresponding to an RGD containing sequence in domain IIIb, was found to promote the attachment and spreading of M2, mouse melanoma cells (Fig. 2). Although more peptide was required on a molar basis, the level of attachment and spreading on the peptide was comparable to that observed on a laminin substrate (Figs. 2 and 3a). Although quite s read on the peptide, however, the cells were not as e ongated as on the laminin substrate. Smaller peptides comprising the carboxyl terminal half (PA261or the amino terminal half (PA361of PA21 were tested (Table 1 and Fig. 3). RGD containing peptides PA21 and PA26, coated at 5-25 pg/well, stimulated M2 cell attachment in a dose-dependent fashion (Fig. 3a). An adjacent but non RGD-containing peptide, PA36, showed some low activity and a control peptide from another region of the A chain (PA101 lacked activity. We also assessed the ability of these peptides to inhibit laminin- and fibronectin-mediated cell attachment (Fig. 3b,c). Peptides PA21 and PA26 blocked M2 cell attachment to both substrates in a dose-de endent manner. PA21 and PA26 were more potent inhi itors of the laminin-mediated adhesion than of the fibronectinmediated adhesion and more potent on laminin than the fibronectin peptide GRGDS. The GRGDS was equally as active as the PA21 and PA26 on the fi-
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Fig. 2. The adhesion and spreading of cells on laminin and on peptide PA21. M2 murine melanoma cells were allowed to attach for 60 minutes to laminin (a,b)and to peptide PA21 (c,d) coated dishes and photographed without removing unbound cells. M2 cells were spread on laminin and on peptide PA21. Cells were photographed at 2 different magnifications ( 1 0 a~ and c, and 320X b and a).
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The RGD Containing Site of the Mouse Laminin A Chain is Active for Cell Attachment, Spreading, Migration and Neurite Outgrowth KEN-ICHIRO TASHIRO, GREGORY C. SEPHEL, DAVE GREATOREX, MAKOTO SASAKI, NORIO SHIRASHI, GEORGE R. MARTIN, HYNDA K. KLEINMAN, AND YOSHlHlKO YAMADA* Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, National lnstitutes of Health, Bethesda, Maryland 20892 The laminin A chain has been sequenced by cDNA cloning and was found to contain an RGD sequence. Synthetic peptides containing the RGD sequence and flanking amino acids were active in mediating cell adhesion, spreading, migration, and neurite outgrowth. Furthermore, endothelial cell attachment to a laminin substrate was inhibited by an RGD-containing synthetic peptide. Antisera against the integrin (fibronectin) receptor, and monoclonal antibody to the integrin, VLA-6, inhibited cell interaction with laminin, as well as with peptides containing an RGD sequence. These results suggest that the RGD containing site of laminin is active and interacts with the integrin family of receptors in certain cells.
Laminin (Mr = 900,000) is a large glycoprotein specific to basement membrane (Timpl et al., 1979; Chung et al., 1979). Laminin binds to other basement membrane components, such as collagen IV, heparan sulfate proteoglycan, and nidogedentactin, and mediates cellular interactions with this matrix (Timpl and Martin, 1982; Timpl et al., 1984; Kleinman et al., 1986; Laurie et al., 1986, Martin et al., 1989). Current models suggest that laminin contains one A chain (Mr = 440,000), one B1 chain (Mr = 225,0001, and one B2 chain (Mr = 205,000) (Barlow et al., 1984; Paulsson et al., 1985; Palm et al., 19851, arranged as shown in Figure 1 to form a cross-shaped molecule. Laminin exhibits a number of biological activities, including promotion of attachment, migration, growth, differentiation, tumor cell metastasis, collagenase IV activity, and neurite outgrowth (Baron Van Evercooren et al., 1982;Aumailley et al., 1983; Grover et al., 1983; Kleinman et al., 1985; Hadley et al., 1985; Greenberg and Hay, 1986; Edgar et al., 1984; Engvall et al., 1986). Based on studies using proteolytic fragments, cell binding has been attributed to the portion of laminin containing the intersecting chains and the end of the long arm. A pentapeptide, YIGSR-NH, (Tyr-Ile-GlySer-Arg) from the cysteine-rich domain (domain 111) of the B1 chain, was found to support cell attachment, 67 Kd receptor binding, and migration. It also prevents tumor cell metastasis in experimental animals (Graf et al., 1987; Iwamoto et al., 1987; 1988). This peptide, however, did not mimic all the activities of laminin and was unable to promote neurite outgrowth or cell growth, or stimulate collagenase IV production. Neurite outgrowth activity has been localized to an another region of the molecule (Edgar et al., 1984). C 1991 WILEY-LISS, INC.
We have recently cloned and sequenced the A chain of laminin. It is comprised of 3084 amino acids, which form at least 8 distinct structural domains, (Sasaki et al., 1988). Particular attention was directed to the potential biological role of domain IIIb due to the presence of an RGD sequence which is known to be a versatile cell attachment site in fibronectin and in various other proteins (Ruoslahti and Pierschbacher, 19861, including the basement membrane glycoproteins entactin (Chakravarti et al., 1990) and laminin (Grant et al., 1989;Aumailley et al., 1990).One study suggested that the RGD sequence of the laminin-entactin type IV collagen mixture matrigel was active for endothelial cells (Grant et al., 1989) and another study demonstrated that the RGD site on laminin was latent and required proteolysis (Aumailley et al., 1990). Also, some of the integrin family of cell matrix receptors are known to recognize the RGD sequence. We, therefore, sought to extend these findings and to determine the biological importance of the RGD site in laminin using synthetic peptides to the region containing the RGD sequence and to determine the cellular receptor inReceived July 20, 1990; accepted December 7, 1990. *To whom reprint requestsicorrespondence should be addressed. Gregory C. Sephel’s present address is Vanderbilt University School of Medicine, Laboratory Service, 1310 24th Avenue South, Nashville, TN 37203 George R. Martin’s present address is National Institute on Aging, Gerontology Research Center, 4940 Eastern Avenue, Baltimore, MD 21224 Ken-ichiro Tashiro’s present address is Department of Psychiatry, Saga Medical School, Nabeshima, Saga 840-01, Japan.
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TASHIRO ET AL.
Fig. 1. Structural model of laminin. The Roman numerals designate the domains in each chain. G designates the A chain terminal domain. The location of the peptides used in this study are indicated by arrows. The peptides and their respective residue numbers in laminin are: peptide PA21, residues 1115-1129; PA26, residues 1120-1127; PA36, residues 1113-1123; PAlO, residues 2109-2128.
volved. Our data show that laminin-derived RGDcontaining peptides were active for cell attachment, migration, and neurite outgrowth via an integrin. These data identify the RGD sequence in laminin as a potential cell interaction site and indicate that laminin has multiple sites with biological activity. MATERIALS AND METHODS Synthesis of peptides a n d preparation of laminin Peptides with a terminal amide group were synthesized using an automated Model 430A synthesizer (Applied Biosystems, Inc., Foster City, Calif.). Deprotection and release of the peptides from the solid-phase support matrix were accomplished by treating the protected peptide on the resin with anhydrous HF containing 10% thioanisole or 10% anisole for 1-2 hours at 0°C. Following deprotection and release from the resin, the peptides were extracted with either ethyl acetate or diethyl ether. The peptides were then dissolved in 20-50 ml of 10% aqueous acetic acid, filtered to remove the resin and lyophilized. Peptides were screened for biological activity and were further urified by preparative HPLC when analytical HPL indicated that the products were heterogeneous. The purity and composition of the peptides were verified by HPLC and amino acid analysis. Mouse laminin was extracted and purified from the Engelbreth-Holm-Swarm (EHS) tumor, using methods previously described ("imp1 et al., 1979). Laminin as isolated by this procedure contains approximately 5% entactin.
e
Conjugation of peptides Prior to conjugation of peptides to Keyhold Lim et Hemocyanin (KLH), the KLH (30 mg) was dissolvecfin 5 ml of 0.1 M NaHC03 with stirring for 15 minutes and then m-Maleimidobenzoylsulfosuccinimideester (MBS, 10 mg) was added to the KLH solution and stirred for 15 minutes, For the conjugation reaction, the peptide (50 mg) was combined with the KLH/MBS solution, and the conjugation reaction was allowed to proceed for 2 hours at 20°C. Following the conjugation process, the conjugates were dialyzed against 0.1 M NH,C03 for 18 hours at 4°C and lyophilized. Cells and culture Various cells were studied, including M2 mouse melanoma, HT-1080 human fibrosarcoma cells (Rasheed et al., 1974). B16F10 mouse melanoma (Fidler, 19731, NG108-15 a mouse neuroblastoma-rat glioma hybrid (Nirenberg et al., 1983), calf bovine Dulmonarv endothelial cells (CPAE:ATCC. CCL 209), ind a human embryonal rhabdomyosarcoma cell line (RD;ATCC,CCL136).M2 cells (a gift of I.J. Fidler, M.D. Anderson) were maintained in Dulbecco's Modified Eagle's Medium (DMEM)supplemented with 10%fetal calf serum (FCS) and grown to 80% confluence. For adhesion assays, cells were washed with PBS (pH 7.4) lacking Ca2+ and Mg2+ and then detached with 0.025% trypsin, 0.025% EDTA (GIBCO).The cells were then sedimented by low sFeed centrifugation, suspended in serum-free Eagle s Minimal Essential Medium (EMEM) containing 0.02% BSA, and then used immediately in the adhesion assay. HT-1080 cells were maintained in DMEM supplemented with 10% FCS. These cells were grown and prepared for adhesion assays as described for M2 cells. B16F10 murine melanoma cells (a gift from I.J. Fidler, Houston, TX), were maintained in EMEM supplemented with 5% FCS, non-essential amino acids, and vitamins. Cells were grown to 80% confluency in Falcon tissue culture dish. For chemotaxis assays, cells were harvested as described above, and washed twice, and then the final pellet was resuspended in serum-free EMEM containing 0.1% BSA. These cells were immediately added to the upper compartment of the Boyden Chamber. NG108-15 neuroblastoma x glioma cells attach and extend out long neuronal processes in the presence of laminin (Luckenbill-Edds et al., 1986). These cells were grown to confluence in DMEM containing NaHC03 and supplemented with 0.1 mM hypoxanthine, 0.4 p.M aminopterin, 16 pM thymidine, and 10% FCS. Dishes were rinsed with serum-free EMEM containing 0.02% BSA, and the cells were detached by pipetting and sedimented by low-speed centrifugation. The pellet was resuspended in serum-free EMEM containing 0.02% BSA and then used immediately in the adhesion assay and in the neurite outgrowth assay. CPAE cells were maintained in EMEM supplemented with 20% FCS. NIH 3T3 cells were maintained in DMEM supplemented with 10%calf serum. RD cells were maintained in EMEM supplemented with 10% FCS and twice the standard concentration of amino acids and vitamins. These cells were grown and prepared for adhesion assays as described for M2 cells.
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BIOLOGICALLY ACTIVE RGD IN LAMININ
Cell adhesion Various concentrations of either laminin or peptide were added to 96 well tissue culture dishes in a total volume of 0.1 ml of serum-free EMEM and incubated for 2 hours at 37°C in 5% COz, 95% air. Unbound surfaces were subsequently blocked with 0.1 ml of EMEM containing 3% BSA for 30 minutes at 37°C. At the end of this period, the wells were rinsed with PBS to remove unattached cells and attached cells were trypsinized and electronically counted. Each assay was carried out in duplicate, and the du licates did not vary by more than 10%. Each pepti c fe was tested in 5 separate experiments and a t least 3 concentrations. Peptide inhibition of cell attachment was assayed using 96 well dishes coated with 2 pg/ml of either laminin or fibronectin as described above. Prior to addition of the cells, varying amounts of peptide solubilized in serum-free EMEM containing 0.02% BSA were added to each well. Cells were added and incubated for 30 minutes at 37°C and the number of attached cells was determined as described above. The effect of polyclonal antisera against the fibronectin (a5pl complex)receptor (integrin) (gift of Drs. Steve Akiyama and Ken Yamada, NCI; Akiyama et al., 1990) on cell attachment was assayed. Each well of a 96 well dish was coated with either 2.5 pg of laminin or fibronectin or 55 pg of various peptides as described above. HT-1080 cells (5000/well)were incubated with 2 different dilutions (150 and 1:lOO) of antisera for 15 minutes, sedimented by low speed centrifugation, resuspended in 100 pl of serum-free MEM plus 0.02% BSA, and then added into the substrate-coated wells. Cells were incubated at 37°C for 20 minutes, unattached cells were rinsed from the dish and attached cells were stained with hematoxylin and eosin and counted by microscope. Monoclonal antibody, GoH3 (gift of Dr. Sonnenberg, Central Laboratory of the Netherlands, Red Cross Blood Transfusion Service and Laboratory of Experimental and Clinical Immunology, University of Amsterdam, The Netherlands) to the integrin, VLA-6, was also used in the inhibition assay for cell attachment. The assay was same as described above except dilutions of 1:5 and 1:20 were employed. Each concentration was tested in duplicate and the duplicates did not differ by more than 10%. The experiment was repeated two times. Immunoprecipitationof integrins HT-1080 cells were cell surface labeled with lZ5Iiodine using the lactoperoxidase technique. The antibodies to the integrins described and preimmune controls mentioned above were used in a standard immunoprecipition assay. Cell migration Chemotaxis assays were carried out as previously described (Mensin et al., 1984; McCarthy and Furcht, 1984). In brief, PO ycarbonate filters (8 pm pore size; Neuroprobe) were coated with type IV collagen (5 pg per filter). A 1 pm pore size uncoated filters were placed in a modified Boyden Chamber and 8 km pore size type IV collagen-coated filters were laced on top. B16F10 melanoma cells (3.0 x 105/0.9m ) were placed
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TABLE 1. Synthetic peptides Peptide PA21 PA26 PA36 PA10
Sequence
Hesidues’
CQAGTFALRGDNPQG C’FALRGDNP C~SKCQAGTFALR DCIRAYOPQTSSTNYNTLII,
11 15-1 129 1120-1127 1113- 1I23 2109-2128
:Residues are from Sasaki et al. (1988) C(Cyskine) i s artificially attached.
in the upper compartment of the Boyden Chamber. The chemoattractant was placed in the lower compartment including either test peptide (200-300 pgiml), or laminin (200-300 pg/ml) in serum-free DMEM with 0.1% BSA (0.22 ml). The chambers were sealed and then incubated for 5 hours at 37°C. The cells that had attached to the upper side of the 8 pm pore size filter were mechanically removed. The cells that had migrated to the lower side of the 8 pm pore size filter were fixed in methanol and then stained with hematoxylin and eosin. The cells were counted by using an Optomax V HR. Each sample was assayed in duplicate and the duplicates did not differ by more than 10%.Each assay was repeated 5 times. The ability of the peptides to block migration to laminin was tested. The chemoattractant in the lower chamber was laminin (20 pg/ml). The cells were placed in the upper compartment of the Boyden Chamber in the presence of the peptide (10 to 300 pg/ml) being tested. The inhibition assay was carried out in duplicate and the assays were repeated 5 times.
RESULTS Cell adhesion Peptides including the laminin A chain RGD sequence or neighboring sequences were tested for their ability to induce attachment and spreading. A 15-mer designated PA21 (Table 11, corresponding to an RGD containing sequence in domain IIIb, was found to promote the attachment and spreading of M2, mouse melanoma cells (Fig. 2). Although more peptide was required on a molar basis, the level of attachment and spreading on the peptide was comparable to that observed on a laminin substrate (Figs. 2 and 3a). Although quite s read on the peptide, however, the cells were not as e ongated as on the laminin substrate. Smaller peptides comprising the carboxyl terminal half (PA261or the amino terminal half (PA361of PA21 were tested (Table 1 and Fig. 3). RGD containing peptides PA21 and PA26, coated at 5-25 pg/well, stimulated M2 cell attachment in a dose-dependent fashion (Fig. 3a). An adjacent but non RGD-containing peptide, PA36, showed some low activity and a control peptide from another region of the A chain (PA101 lacked activity. We also assessed the ability of these peptides to inhibit laminin- and fibronectin-mediated cell attachment (Fig. 3b,c). Peptides PA21 and PA26 blocked M2 cell attachment to both substrates in a dose-de endent manner. PA21 and PA26 were more potent inhi itors of the laminin-mediated adhesion than of the fibronectinmediated adhesion and more potent on laminin than the fibronectin peptide GRGDS. The GRGDS was equally as active as the PA21 and PA26 on the fi-
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Fig. 2. The adhesion and spreading of cells on laminin and on peptide PA21. M2 murine melanoma cells were allowed to attach for 60 minutes to laminin (a,b)and to peptide PA21 (c,d) coated dishes and photographed without removing unbound cells. M2 cells were spread on laminin and on peptide PA21. Cells were photographed at 2 different magnifications ( 1 0 a~ and c, and 320X b and a).
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