©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Peptide Model of Basement Membrane Collagen 1(IV) 531543 Binds the Integrin (*)

(Received for publication, September 25, 1995)

Andrew J. Miles (1) Jennifer R. Knutson (1) Amy P. N. Skubitz (1) (3) Leo T. Furcht (1) (3)(§) James B. McCarthy (1) (3) Gregg B. Fields (1) (3) (2)(¶)

From the  (1)Departments of Laboratory Medicine and Pathology and (2)Biochemistry, and the (3)Biomedical Engineering Center, University of Minnesota, Minneapolis, Minnesota 55455

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Tumor cell adhesion to the triple-helical domain of basement membrane (type IV) collagen occurs at several different regions. Cellular recognition of the sequence spanning alpha1(IV)531-543 has been proposed to be independent of triple-helical conformation (Miles, A. J., Skubitz, A. P. N., Furcht, L. T., and Fields, G. B.(1994) J. Biol. Chem. 269, 30939-30945). In the present study, integrin interactions with a peptide analog of the alpha1(IV)531-543 sequence have been analyzed. Tumor cell adhesion (melanoma, ovarian carcinoma) to the alpha1(IV)531-543 chemically synthesized peptide was inhibited by a monoclonal antibody against the alpha(3) integrin subunit, and to a lesser extent by monoclonal antibodies against the beta(1) and alpha(2) integrin subunits. An anti-alpha(5) monoclonal antibody and normal mouse IgG were ineffective as inhibitors of tumor cell adhesion to the peptide. Two cell surface proteins of 120 and 150 kDa bound to an alpha1(IV)531-543 peptide affinity column and were eluted with 20 mM EDTA. When the eluted proteins were incubated with monoclonal antibodies against either the alpha(3) or beta(1) integrin subunit, proteins corresponding in molecular weight to alpha(3) and beta(1) integrin subunits were precipitated. No proteins were immunoprecipated with monoclonal antibodies against the alpha(2) or alpha(5) integrin subunits. Thus, the alpha(3)beta(1) integrin from two tumor cell types has been shown to bind directly to the alpha1(IV)531-543 peptide. The alpha1(IV)531-543 peptide is the first collagen-like sequence that has been shown to bind the alpha(3)beta(1) integrin.


INTRODUCTION

Basement membranes are specialized extracellular matrices that delineate tissue boundaries. The metastatic process involves tumor cell interaction with and invasion through the basement membrane. Basement membrane (type IV) collagen can promote the adhesion and migration of diverse cell types including melanoma, corneal epithelial, endothelial, and neural crest cells (Herbst et al., 1988; Chelberg et al., 1989; Etoh et al., 1993; Olivero and Furcht, 1993; Perris et al., 1993). Normal and tumorigenic cellular interactions with type IV collagen can be mediated by integrins and/or cell surface proteoglycans (reviewed in Faassen et al., 1992). The alpha(1)beta(1) and alpha(2)beta(1) integrins can bind directly to type IV collagen (Staatz et al., 1989; Vandenberg et al., 1991; Kern et al., 1993; Eble et al., 1993; Carmeliet et al., 1994), while the alpha(3)beta(1) integrin has been implicated in cell migration on type IV collagen (Yoshinaga et al., 1993; Vink et al., 1994; Melchiori et al., 1995). Identification of specific adhesion sites within type IV collagen for the individual integrins has proven difficult, most likely due to the conformational dependence of integrin binding to the collagen triple-helix (Kühn and Eble, 1994). Type IV collagen cyanogen bromide fragment 3 (CB3(IV)), which includes alpha1(IV) residues 388-551 and alpha2(IV) residues 407-570, has been reported to contain the individual binding sites for the alpha1beta(1) and alpha2beta(1) integrins (Vandenberg et al., 1991; Kern et al., 1993). The N-terminal region of a trypsin-derived fragment of CB3(IV), incorporating residues alpha1(IV)414-452 and alpha2(IV)432-469, is believed to be a high affinity binding site for the alpha1beta(1) integrin from fibrosarcoma cells (Eble et al., 1993). The alpha2beta(1) integrin from human fibroblasts and human platelets adheres to a peptide model of alpha1(I)430-442 in a Mg-dependent fashion (Staatz et al., 1990, 1991). This peptide inhibits platelet and breast adenocarcinoma cell adhesion to type I collagen (Staatz et al., 1991) but does not inhibit alpha2beta(1)-mediated chondrosarcoma cell adhesion to type II collagen (Tuckwell et al., 1994).

Other collagen-derived sequences may also function as integrin binding sites. Peptide models of the alpha1(IV)1263-1277 region promote the adhesion, spreading, and migration of highly metastatic tumor cells (Chelberg et al., 1990; Mayo et al., 1991; Fields et al., 1993). A peptide incorporating the alpha1(IV)531-543 (^1)(Gly-Glu-Phe-Tyr-Phe-Asp-Leu-Arg-Leu-Lys-Gly-Asp-Lys-Tyr) sequence promotes keratinocyte, corneal epithelial, melanoma, ovarian carcinoma, and Jurkat cell adhesion (Wilke and Furcht, 1990; Cameron et al., 1991; Miles et al., 1994) and migration of corneal epithelial cells and keratinocytes (Cameron et al., 1991; Kim et al., 1994). Cellular recognition of the alpha1(IV)531-543 peptide is, in general, independent of substrate conformation and configuration (chirality) (Miles et al., 1994). Competition studies suggested that the L- and D-peptides incorporating alpha1(IV)531-543 are bound by the same receptor (Miles et al., 1994). Preliminary results indicated that the beta(1) integrin subunit was involved in mediating cell adhesion to this sequence (Miles et al., 1994). We have presently examined integrin binding to the alpha1(IV)531-543 sequence. Since an alpha1beta(1) integrin binding site in type IV collagen has already been identified (see above), we have focused on the alpha2beta(1) and alpha(3)beta(1) integrins as possible receptors for alpha1(IV)531-543. In addition, the alpha(5)beta(1) integrin has been considered as a potential receptor for alpha1(IV)531-543 in this study, as it can mediate cell adhesion to denatured collagen (Gullberg et al., 1992; Tuckwell et al., 1994). We have utilized a number of assays including inhibition of cell adhesion and affinity chromatography of solubilized cells to characterize the integrin(s) involved in cellular recognition of the alpha1(IV)531-543 sequence. Two different tumor cell lines have been utilized, melanoma and ovarian carcinoma, since both of these cell types adhere well to type IV collagen and the alpha1(IV)531-543 peptide (Miles et al., 1994).


EXPERIMENTAL PROCEDURES

Materials

All standard peptide synthesis chemicals were analytical reagent grade or better and purchased from Applied Biosystems, Inc. (Foster City, CA) or Fisher. The synthesis, purification, and characterization of the peptide incorporating the alpha1(IV)531-543 sequence has been described (Miles et al., 1994). Monoclonal antibody (mAb) P5D2 was prepared against the beta(1) integrin subunit using methods described previously (Wayner and Carter, 1987). mAbs prepared against the integrin subunits alpha2 (P1E6), alpha(3) (P1B5), and alpha(5) (P1D6) were purchased from Chemicon International (Temecula, CA). The anti-alpha(3) mAb was obtained as ascites fluid (2.5 mg of IgG/ml of ascites); all other mAbs were purified IgGs. Purified normal mouse IgG was purchased from Organon Teknika Corp. (Durham, NC).

Cells

A375SM melanoma cells were originally obtained from Dr. I. J. Fidler, M. D. Anderson Cancer Center, Houston, TX, and propagated as described previously (Miles et al., 1994). Briefly, melanoma cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 0.1 mg/ml gentamicin (Boehringer Mannheim), 50 units/ml penicillin, 0.05 mg/ml streptomycin, and minimum essential medium vitamin solution. SKOV3 ovarian carcinoma cells were obtained from Dr. Robert C. Bast, Jr., M. D. Anderson Hospital, Houston, TX. Ovarian carcinoma cells were cultured in modified McCoy's 5A medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 50 units/ml penicillin, and 0.05 mg/ml streptomycin.

Cells were passaged for 4-5 weeks and then replaced from frozen stocks of early passage cells to minimize phenotypic drift. All cells were maintained at 37 °C in a humidified incubator containing 5% CO(2). All media reagents were purchased from Sigma except where noted.

Cell Adhesion

Melanoma and ovarian carcinoma cell adhesion and inhibition of cell adhesion assays were performed as described previously (Chelberg et al., 1990; Wilke and Furcht, 1990; Miles et al., 1994) using the alpha1(IV)531-543 peptide at a substrate concentration of 5.7 µM. For inhibition assays, cells were preincubated for 1 h at 37 °C with various concentrations (0.005-5 µg/ml) of the anti-integrin subunit mAb; then the cells (50,000/ml), in the continued presence of the mAb, were added to the Immulon plate wells and allowed to adhere for another 1 h at 37 °C.

Affinity Chromatography

The alpha1(IV)531-543 peptide was coupled to activated CH-Sepharose according to the manufacturer's instructions (Pharmacia Biotech Inc.). In addition, a mock-coupled column was made without peptide for use as a control. Briefly, 30 mg of high performance liquid chromatography-purified peptide was dissolved in 200 µl of Me(2)SO and diluted to 5 ml with coupling buffer (15 mM sodium carbonate and 35 mM sodium bicarbonate, pH 8.6). The peptide solution was added to 3 ml of preswollen beads and mixed overnight at 4 °C. Unbound peptide was removed by washing the beads with coupling buffer, and the remaining reactive groups were hydrolyzed at pH 8.0 with 0.1 M Tris-HCl for 2 h. Cells were surface labeled with I as described (Gehlsen et al., 1992), extracted in buffer (50 mM Tris-HCl, pH 7.4, 50 mM octyl-beta-D-glucopyranoside, 15 mM NaCl, 1 mM MgCl(2), 1 mM MnCl(2), 1 mM CaCl(2), 1 mMN-ethylmaleimide, 100 µg/ml soybean trypsin inhibitor, and 1 mM phenylmethylsulfonyl fluoride) for 30 min at 4 °C, and ultracentrifuged at 36,500 times g for 1 h at 4 °C. The I-labeled cell lysates were precleared with the mock-coupled Sepharose beads as a slurry by constant mixing overnight at 4 °C. Cleared lysates were then incubated with the peptide-Sepharose beads as a slurry with constant mixing overnight at 4 °C. The peptide-Sepharose beads were packed into a column, and the column was washed with extraction buffer and eluted with 20 mM EDTA in extraction buffer lacking the cations. Eluates were incubated with anti-integrin subunit mAbs to immunoprecipitate specific integrins (Gehlsen et al., 1992). 2-Mercaptoethanol (10%) was added to some samples (see ``Results''), followed by heating at 100 °C for 5 min to reduce disulfide bonds. Elution fractions were electrophoresed by 7.5% SDS-polyacrylamide gel electrophoresis and analyzed by autoradiography (Gehlsen et al., 1992). Molecular mass standards (Sigma) were rabbit muscle myosin (205 kDa), Escherichia coli beta-galactosidase (116 kDa), rabbit muscle phosphorylase b (97.4 kDa), bovine albumin (66 kDa), and chicken egg albumin (43 kDa).


RESULTS

We had shown previously that 40-60% maximum cell adhesion is achieved with 5-10 µM amounts of a peptide analog of the alpha1(IV)531-543 sequence (Miles et al., 1994). Inhibition of cell adhesion studies were thus performed at a peptide concentration of 5.7 µM. Ovarian carcinoma cell adhesion to the peptide could be inhibited in a dose-dependent manner by mAbs against beta(1), alpha2, or alpha(3) integrin subunits, with maximum inhibition occurring at the highest mAb concentration tested (5 µg/ml) (Fig. 1A). The mAb against the alpha(3) subunit was most effective, producing >60% inhibition of adhesion at a mAb concentration as low as 2.5 µg/ml (data not shown). A similar level of inhibition (50%) was achieved by the anti-beta(1) subunit mAb at a concentration of 5 µg/ml (Fig. 1A). The anti-alpha2 subunit mAb was less effective as an inhibitor, causing only 35% inhibition at 5 µg/ml (Fig. 1A). A mAb against the alpha(5) integrin subunit and normal mouse IgG did not give dose-dependent inhibition of cell adhesion, even up to a concentration of 5 µg/ml (Fig. 1A). In a similar fashion, melanoma cell adhesion to the peptide could be inhibited in a dose-dependent manner by mAbs against the beta(1), alpha2, or alpha(3) integrin subunits, with maximum inhibition occurring at a mAb concentration of 5 µg/ml (Fig. 1B). The anti-alpha(3) subunit mAb was the most effective inhibitor, causing 60% inhibition. A mAb against the alpha(5) integrin subunit and normal mouse IgG did not give dose-dependent inhibition of melanoma cell adhesion up to a concentration of 5 µg/ml (Fig. 1B).


Figure 1: Inhibition of ovarian carcinoma (A) or melanoma cell (B) adhesion to Immulon I plate surfaces adsorbed with 5.7 µM alpha1(IV)531-543 peptide by 5 µg/ml of mAbs to the integrin subunits beta(1), alpha(2), alpha(3), or alpha(5). Normal mouse (n.m.) IgG was used as a negative control. Cells were preincubated with the mAbs for 1 h, then added to the wells in the presence of the mAbs for a 1 h incubation. All assays were repeated at least in triplicate. Conditions are given under ``Experimental Procedures.''



The alpha1(IV)531-543 peptide was immobilized to CH-Sepharose, and affinity chromatography was performed with an I-labeled extract of the ovarian carcinoma cells. Following application of cells, the column was first washed with extraction buffer, then eluted by 20 mM EDTA. Eluants were incubated with 5 µg/ml mAbs against alpha2, alpha(3), alpha(5), or beta(1) integrin subunits. Precipitated proteins were then analyzed by 7.5% SDS-polyacrylamide gel electrophoresis with detection by autoradiography. Immunoprecipitation of the EDTA eluant with the anti-beta(1) integrin subunit mAb resulted in detection of a 135-kDa protein under reducing conditions (Fig. 2). Immunoprecipitation of the same eluant with the anti-alpha(3) integrin subunit mAb, followed by reduction with 2-mercaptoethanol, resulted in detection of a 135-kDa protein (Fig. 2). In other studies, the beta(1) and alpha(3) integrin subunits have been shown to have similar apparent molecular weights under reducing conditions (Gehlsen et al., 1992; Sonnenberg, 1993). No proteins were seen following incubation of the EDTA eluant with anti-alpha2 integrin subunit mAb or normal mouse IgG (Fig. 2). When the immunoprecipitants were not reduced, two proteins of 150 and 120 kDa were immunoprecipitated from the EDTA eluant by the anti-beta(1) integrin subunit mAb (Fig. 3). The molecular weights correspond to the alpha(3) and beta(1) integrin subunits, respectively (Sonnenberg, 1993). In similar fashion, two proteins of 150 and 120 kDa were immunoprecipitated from the EDTA eluant by the anti-alpha(3) integrin subunit mAb (Fig. 3). No proteins were seen using normal mouse IgG or mAbs against the alpha2 or alpha(5) integrin subunits (Fig. 3).


Figure 2: Immunoprecipitation analysis of ovarian carcinoma cell surface proteins eluted from the alpha1(IV)531-543 peptide affinity column. The proteins eluted by EDTA from the peptide column were immunoprecipitated with anti-integrin mAbs and reduced. Immunoprecipitation of a 135-kDa protein(s) was seen using mAbs against either the beta(1) or alpha(3) integrin subunit. No proteins were immunoprecipitated when an anti-alpha2 mAb or normal mouse (n.m.) IgG were used.




Figure 3: Immunoprecipitation analysis of ovarian carcinoma cell surface proteins eluted from the alpha1(IV)531-543 peptide affinity column. The proteins eluted by EDTA from the peptide column were immunoprecipitated with either an anti-beta(1) or anti-alpha(3) integrin subunit mAb. Two proteins of 120 and 150 kDa, corresponding to the beta(1) and alpha(3) integrin subunits, respectively, were immunoprecipitated. No proteins were immunoprecipitated when an anti-alpha2 mAb, an anti-alpha(5) mAb, or normal mouse (n.m.) IgG were used.



Affinity chromatography and immunoprecipitation experiments were repeated using I-labeled melanoma cells. Incubation of the EDTA eluant with the anti-alpha(3) integrin subunit mAb resulted in immunoprecipitation of a 135-kDa protein under reducing conditions (data not shown). Similarly, incubation of the eluant with the anti-beta(1) integrin subunit mAb resulted in immunoprecipitation of a 135-kDa protein under reducing conditions (data not shown). Under non-reducing conditions, two proteins of 150 and 120 kDa were immunoprecipitated from the EDTA eluant by the anti-alpha(3) integrin subunit mAb (Fig. 4). The apparent molecular weights correspond to the alpha(3) and beta(1) integrin subunits, respectively (Sonnenberg, 1993). No proteins were seen using normal mouse IgG (Fig. 4) or mAbs against the alpha2 (Fig. 4) or alpha(5) integrin subunits (data not shown).


Figure 4: Immunoprecipitation analysis of melanoma cell surface proteins eluted from the alpha1(IV)531-543 peptide affinity column. The proteins eluted by EDTA from the peptide column were immunoprecipitated with an anti-alpha(3) integrin subunit mAb. Two proteins of 120 and 150 kDa, corresponding to the beta(1) and alpha(3) integrin subunits, respectively, were immunoprecipitated. No proteins were immunoprecipitated when an anti-alpha2 mAb or normal mouse (n.m.) IgG were used.




DISCUSSION

In an attempt to further refine our knowledge of cellular receptors for type IV collagen, melanoma and ovarian carcinoma cell adhesion to the alpha1(IV)531-543 peptide was examined in the presence of anti-integrin subunit mAbs. For both cell types, adhesion to this peptide was most effectively inhibited by the anti-alpha(3) integrin subunit mAb, followed by the anti-beta(1) and anti-alpha2 integrin subunit mAbs. Cell surface proteins with molecular masses of 120 and 150 kDa bound to a alpha1(IV)531-543 peptide affinity column in an EDTA-dependent fashion. These proteins could be immunoprecipitated with mAbs against either the alpha(3) or beta(1) integrin subunit, and the protein molecular weights corresponded to alpha(3) and beta(1) integrin subunits. Thus, it would appear that the alpha(3)beta(1) integrin binds directly to the alpha1(IV)531-543 peptide.

The alpha1(IV)531-543 peptide is the first collagen-like sequence identified as a specific alpha(3)beta(1) integrin/ligand binding site. Two different tumor cell types, melanoma and ovarian carcinoma, bind this site via the alpha(3)beta(1) integrin. At present, it is unclear as to what role this integrin plays during tumor cell invasion, where extravasating cells have contact with type IV collagen. The level of alpha(3)beta(1) expression varies amongst tumor cell types. Metastatic melanoma cells up-regulate the alpha(3)beta(1) integrin compared with primary melanoma cells (Yoshinaga et al., 1993), while highly invasive prostate carcinoma cells have decreased expression of the alpha(3)beta(1) integrin compared to the parental cell line (Dedhar et al., 1993). Transformed fibroblasts retain the same level of alpha(3)beta(1) as non-transformed cells while decreasing levels of other integrins (Plantefaber and Hynes, 1989).

The level of alpha(3)beta(1) integrin expression may correlate to the utility of this integrin for cell migration. mAbs to the alpha(3) subunit inhibit melanocyte and melanoma cell motility (Morelli et al., 1993; Yoshinaga et al., 1993; Melchiori et al., 1995) and dysplastic nevus cell spreading and migration (Vink et al., 1994) on type IV collagen. Tumorigenic cell types such as metastatic melanoma cells may have increased levels of alpha(3)beta(1), which enhances motility on the basement membrane or basement membrane molecules. Other cell types may use different receptors for migration. For example, keratinocytes use alpha2beta(1) integrins for migration on type IV collagen (Chen et al., 1993; Kim et al., 1994). There are suggestions that cellular interactions with the basement membrane via alpha(3)beta(1) integrins may also lead to basement membrane degradation. Antibodies to alpha(3)beta(1) integrin stimulate the expression of matrix metalloproteinase-9 (92 kDa type IV collagenase) (Larjava et al., 1993). Matrix metalloproteinase-9 can be induced in transformed cells (Wilhelm et al., 1989) or by direct contact with tumorigenic cells (Himelstein et al., 1994) and has been localized to the invasion front of oral squamous cell carcinoma (Kawahara et al., 1993). Matrix metalloproteinase-9 efficiently degrades type IV collagen (Morodomi et al., 1992).

The alpha2beta(1) integrin may have a role in cellular recognition of the alpha1(IV)531-543 sequence based on the inhibition of cell adhesion assays. alpha2beta(1) integrin binding to the anti-alpha2 integrin subunit mAb used in this study (P1E6) does not result in signal transduction (Kapron-Bras et al., 1993); thus, the function of the alpha(3)beta(1) integrin is probably not indirectly altered by alpha2beta(1) interaction with the mAbs used here. More likely, the alpha2beta(1) integrin has lower affinity for the alpha1(IV)531-543 peptide than alpha(3)beta(1). An alpha2beta(1) binding site is found within alpha1(IV)453-551 (Vandenberg et al., 1991; Eble et al., 1993). The overlap between the alpha2beta(1) site and the sequence examined in this study (alpha1(IV) residues 531-543) may be part of the alpha2beta(1) binding site. There is also a greater expression of the alpha(3)beta(1) integrin than the alpha2beta(1) integrin for the ovarian carcinoma (^2)and melanoma (^3)cell types studied here, possibly contributing to more alpha(3)beta(1) receptor-ligand binding events and a more avid binding overall.

Other studies indicate that both the alpha2beta(1) and alpha(3)beta(1) integrins can participate in cellular interactions with the alpha1(IV)531-543 peptide. mAbs against the alpha2, alpha(3), and beta(1) integrin subunits have been used to demonstrate alpha2beta(1) and alpha(3)beta(1) integrin involvement for corneal epithelial cell adhesion to a peptide incorporating alpha1(IV)531-543 (Maldonado and Furcht, 1995). Also, the alpha1(IV)531-543 peptide promotes the motility of keratinocytes and inhibits keratinocyte migration on type IV collagen via the alpha2beta(1) integrin (Kim et al., 1994).

In addition to type IV collagen, type I collagen, fibronectin, laminin, and epiligrin have all been reported to be ligands for the alpha(3)beta(1) integrin (see reviews by Ruoslahti(1991), Hynes(1992), and Kühn and Eble (1994)). From these various other proteins, only two specific peptide sequences have been identified as alpha(3)beta(1) integrin binding sites. A mAb against the human squamous cell carcinoma alpha(3)beta(1) integrin inhibits cell adhesion to peptide GD-2 (Lys-Glu-Gly-Tyr-Lys-Val-Arg-Leu-Asp-Leu-Asn-Ile-Thr-Leu-Glu-Phe-Arg-Thr-Thr-Ser-Lys, which corresponds to mouse laminin A chain residues 2890-2910). (^4)The human melanoma cell line C8161 alpha(3)beta(1) integrin binds to peptide GD-6 (Lys-Gln-Asn-Cys-Leu-Ser-Ser-Arg-Ala-Ser-Phe-Arg-Gly-Cys-Val-Arg-Asn-Leu-Arg-Leu-Ser-Arg, which encompasses mouse laminin A chain residues 3011-3032) (Gehlsen et al., 1992). Peptides GD-2 and GD-6 have virtually no sequence homology to the alpha1(IV)531-543 peptide (GD-6 and alpha1(IV)531-543 have a Leu-Arg-Leu overlap). In addition, GD-6 is considerably more basic than alpha1(IV)531-543. The activity of alpha1(IV)531-543 is dependent upon the acidic residues Asp and Asp (Miles et al., 1994), while peptide GD-6 contains neither Asp nor Glu. These results suggest that different sites of the alpha(3)beta(1) integrin may be used to bind different ligands. (^5)One would predict this behavior based on the variety of alpha(3)beta(1) ligands. Consistent with this notion are prior studies demonstrating that alpha(3)beta(1) binds fibronectin in an Arg-Gly-Asp-dependent fashion but that laminin binding to the alpha(3)beta(1) integrin is Arg-Gly-Asp-independent (Gehlsen et al., 1988; Elices et al., 1991; Sonnenberg et al., 1991). There are also different isoforms of alpha(3) (Tamura et al., 1991), which may result in different binding specificities and/or affinities of the alpha(3)beta(1) integrin. Further investigations are thus required to better understand the function of the alpha(3)beta(1) integrin in tumor cell invasion.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants KD 44494 and AR 01929 (to G. B. F.), CA 21463, CA 29995, and EY 09065 (to L. T. F.), CA 60658 (to A. P. N. S.), and CA 63671 (to J. B. M. and G. B. F.) and by the American Cancer Society. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Allen-Pardee Professor.

Recipient of a National Institutes of Health research career development award. To whom correspondence should be addressed: Department of Laboratory Medicine and Pathology, Box 107, 420 Delaware St. S.E., University of Minnesota, Minneapolis, MN 55455. Tel.: 612-626-2446; Fax: 612-625-1121.

(^1)
The abbreviations used are: alpha1(IV)531-543 peptide, Gly-Glu-Phe-Tyr-Phe-Asp-Leu-Arg-Leu-Lys-Gly-Asp-Lys-Tyr; mAb, monoclonal antibody.

(^2)
S. Pattaramalai, K. M. Skubitz, and A. P. N. Skubitz, unpublished results.

(^3)
A. J. Miles, J. R. Knutson, J. B. McCarthy, and G. B. Fields, unpublished results.

(^4)
S. Pattaramalai, K. M. Skubitz, and A. P. N. Skubitz, submitted for publication.

(^5)
Although the anti-alpha(3) mAb P1B5 inhibits cell adhesion to both GD-6 (Gehlsen et al., 1992) and the alpha1(IV)531-543 peptide, it should be noted that this mAb was screened and selected based on its ability to inhibit cell adhesion to multiple extracellular matrix proteins, including laminin and type IV collagen (Takada et al., 1988; Carter et al., 1990). This mAb may sterically restrict interactions of ligands with several alpha(3) binding sites.


ACKNOWLEDGEMENTS

We thank Drs. I. J. Fidler and Robert C. Bast, Jr. for providing cell lines.


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