Skip to main content
SpringerLink
Log in

The use of a combinatorial library method to isolate human tumor cell adhesion peptides

  • Research Papers
  • Published:
Molecular Diversity Aims and scope Submit manuscript

Summary

Tumor cell progression is dependent in part on the successful adhesive interactions of the cells with the extracellular matrix. In this study, a new approach is described to isolate linear peptide ligand candidates involved in cellular adhesion. A synthetic combinatorial peptide library based on the ‘one-bead-one-peptide’ concept was incubated with live human prostate cancer cells for 90 min at 37 °C. The peptide bead coated with a monolayer of cells was then isolated for micro sequencing. The DU145 (DU-H) cells were chosen since they have been previously characterized as containing elevated levels of a laminin receptor for cell adhesion, the α6β1 integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are α6-specific ligand candidates. From two different libraries (linear 9-mer and 11-mer) of a total of 1 500 000 beads, 68 peptide beads containing attached cells were isolated. These positive beads were then retested to determine the ability of the GoH3 antibody to block binding of the cells to the peptide beads. The α6 integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVSVNGRHX) and RX-1 (DNRIRLQAKXX), resemble the previously reported active peptide sequences (GD-2 and AG-73) from native laminin. The RU-1, RX-1 and AG-73 peptides were tested for their ability to support cell attachment and to bind the cell surface of DU-H prostate carcinoma cells in suspension using fluorescence-activated cell-sorting (FACS) analysis. Both RU-1 and AG-73 peptides supported cellular attachment within 1 h. In contrast, after 1 h, EHS laminin supported both cellular attachment and spreading. The RX-1 peptide exhibited only weak binding to the DU-H prostate carcinoma cells. FACS analysis indicated that AG-73 peptide attached to tumor cell surfaces over a range of concentrations, whereas the RU-1 peptide showed a homogeneous concentration required for attachment. The described strategy for screening a random peptide library offers three advantages: (i) ligands for conformationally sensitive receptors of adhesion can be isolated using live cells; (ii) specific binding can be selected for using function-blocking antibodies; and (iii) peptides supporting adhesion independent of spreading properties can be distinguished. In principle, specific adhesive peptides without prior knowledge of the sequence could be isolated for any epithelial cell surface receptor for which a function-blocking reagent is available.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Van Waes, C.,Cell adhesion and regulatory molecules involved in tumor formation, hemostasis, and wound healing, Head Neck, 17 (1995) 140–147.

    Google Scholar 

  2. Yoneda, T., Sasaki, A. and Mundy, G.R.,Osteolytic bone metastasis in breast cancer, Breast Cancer Res. Treat., 32 (1994) 73–84.

    Google Scholar 

  3. Kemperman, H., Driessens, M., La Riviere, G., Meijne, A.M. and Roos, E.,The role of integrins and integrin activation in liver metastasis, Invasion Metastasis, 14 (1994) 98–108.

    Google Scholar 

  4. Hynes, R.O.,Integrins: Versatility, modulation, and signaling in cell adhesion, Cell, 69 (1992) 11–25.

    Google Scholar 

  5. Danen, E.H., Van Muijen, G.N., Van de Wiel-Van Kemenade, E., Jansen, K.F., Ruiter, D.J. and Figdor, C.G.,Regulation of integrin-mediated adhesion to laminin and collagen in human melanocytes and in non-metastatic and highly metastatic human melanoma cells, Int. J. Cancer, 54 (1993) 315–321.

    Google Scholar 

  6. Ramos, D.M., Cheng, Y.F. and Kramer, R.H.,Role of laminin-binding integrin in the invasion of basement membrane matrices by fibrosarcoma cells, Invasion Metastasis, 11 (1991) 125–138.

    Google Scholar 

  7. Castronovo, V.,Laminin receptors and laminin-binding proteins during tumor invasion and metastasis, Invasion Metastasis, 13 (1993) 1–30.

    Google Scholar 

  8. Coopman, P., Verhasselt, B., Bracke, M., De Bruyne, G., Castronovo, V., Sobel, M., Foidart, J.M., Van Roy, F. and Mareel, M.,Arrest of MCF-7 cell migration by laminin in vitro: Possible mechanisms, Clin. Exp. Metastasis, 9 (1991) 469–484.

    Google Scholar 

  9. Rabinovitz, I., Cress, A.E. and Nagle, R.B.,Biosynthesis and secretion of laminin and S-laminin by human prostate carcinoma cell lines, Prostate, 25 (1994) 97–107.

    Google Scholar 

  10. Kosir, M.A. and Quinn, C.C.,Sorting of heparan sulfate proteoglycan into matrix compartments of prostate adenocarcinoma cells, J. Surg. Res., 58 (1995) 46–52.

    Google Scholar 

  11. Nomizu, M., Weeks, B.S., Weston, C.A., Kim, W.H., Kleinman, H.K. and Yamada, Y.,Structure-activity study of a laminin alpha-1 chain active peptide segment Ile-Lys-Val-Ala-Val (IKVAV), FEBS Lett., 365 (1995) 227–231.

    Google Scholar 

  12. Kawahara, E., Imai, K., Kumagai, S., Yamamoto, E. and Nakanishi, I.,Inhibitory effects of adhesion oligopeptides on the invasion of squamous carcinoma cells with special reference to implication of alpha v integrins, J. Cancer Res. Clin. Onc., 121 (1995) 133–140.

    Google Scholar 

  13. Grant, D.S., Kinsella, J.L., Fridman, R., Auerbach, R., Piasecki, B.A., Yamada, Y., Zain, M. and Kleinman, H.K.,Interaction of endothelial cells with a laminin A chain peptide (SIKVAV) in vitro and induction of angiogenic behavior in vivo, J. Cell. Physiol., 153 (1992) 614–625.

    Google Scholar 

  14. Gehlsen, K.R., Sriramarao, P., Furcht, L.T. and Skubitz, A.P.,A synthetic peptide derived from the carboxy terminus of the laminin A chain represents a binding site for the alpha-3 beta-1 integrin, J. Cell Biol., 117 (1992) 449–459.

    Google Scholar 

  15. Nomizu, M., Kim, W.H., Yamamura, K., Utani, A., Song, S.Y., Otaka, A., Roller, P.P., Kleinman, H.K. and Yamada, Y.,Identification of cell binding sites in the laminin alpha-1 chain carboxylterminal globular domain by systematic screening of synthetic peptides, J. Biol. Chem., 270 (1995) 20583–20590.

    Google Scholar 

  16. Skubitz, A.P., Letourneau, P.C., Wayner, E. and Furcht, L.T.,Synthetic peptides from the carboxy-terminal globular domain of the A chain of laminin: Their ability to promote cell adhesion and neurite outgrowth, and interact with heparin and the beta-1 integrin subunit, J. Cell Biol., 115 (1991) 1137–1148.

    Google Scholar 

  17. Lee, E.C., Lotz, M.M., Steele Jr., G. and Mercurio, A.M.,The integrin alpha-6 beta-4 is a laminin receptor, J. Cell Biol., 117 (1992) 671–678.

    Google Scholar 

  18. Mercurio, A.M. and Shaw, L.M.,Laminin-binding proteins, Bioassays, 13 (1991) 469–473.

    Google Scholar 

  19. Niessen, C.M., Hogervorst, F., Jaspars, L.H., de Melker, A.A., Delwel, G.O., Hulsman, E.H., Kuikman, I. and Sonnenberg, A.,The alpha-6 beta-4 integrin is a receptor for both laminin and kalinin, Exp. Cell Res., 211 (1994) 360–367.

    Google Scholar 

  20. Brown, J.C. and Goodman, S.L.,Different cellular receptors for human placental laminin and murine EHS laminin, FEBS Lett., 282 (1991) 5–8.

    Google Scholar 

  21. Weinel, R.J., Rosendahl, A., Neumann, K., Chaloupka, B., Erb, D., Rothmund, M. and Santoso, S.,Expression and function of VLA-alpha-2, -alpha-3, -alpha-5 and -alpha-6-integrin receptors in pancreatic carcinoma, Int. J. Cancer, 52 (1992) 827–833.

    Google Scholar 

  22. Bonkhoff, H., Stein, U. and Remberger, K.,Differential expression of alpha-6 and alpha-2 very late antigen integrins in the normal, hyperplastic, and neoplastic prostate: Simultaneous demonstration of cell surface receptors and their extracellular ligands, Hum. Pathol., 24 (1993) 243–248.

    Google Scholar 

  23. Cress, A.E., Rabinovitz, I., Knox, J.D. and Nagle, R.B.,The α 6 β 1 and α 6 β 4 integrins in human prostate cancer progression, Cancer Metastasis Rev., 14 (1995) 219–228.

    Google Scholar 

  24. Knox, J.D., Cress, A.E., Clark, V., Manriquez, L., Affinito, K.S., Dalkin, B.L. and Nagle, R.B.,Differential expression of extracellular matrix molecules and the alpha-6-integrins in the normal and neoplastic prostate, Am. J. Pathol., 145 (1994) 167–174.

    Google Scholar 

  25. Nagle, R.B., Brawer, M.K., Kittelson, J. and Clark, V.,Phenotypic relationships of prostatic intraepithelial neoplasia to invasive prostatic carcinoma, Am. J. Pathol., 138 (1991) 119–128.

    Google Scholar 

  26. Nagle, R.B., Knox, J.D., Wolf, C., Bowden, G.T. and Cress, A.E.,Adhesion molecules, extracellular matrix, and proteases in prostate carcinoma, J. Cell Biochem., 19 (1994) 232–237.

    Google Scholar 

  27. Nagle, R.B., Hao, J., Knox, J.D., Dalkin, B.L., Clark, V. and Cress, A.E.,Expression of hemidesmosomal and extracellular matrix proteins by normal and malignant human prostate tissue, Am. J. Pathol., 146 (1995) 1498–1507.

    Google Scholar 

  28. Friedrichs, K., Ruiz, P., Franke, F., Gille, I., Terpe, I. and Imhof, B.A.,High expression level of α 6 integrin in human breast carcinoma is correlated with reduced survival, Cancer Res., 55 (1995) 901–906.

    Google Scholar 

  29. Dedhar, S., Saulnier, R., Nagle, R. and Overall, C.M.,Specific alterations in the expression of alpha-3 beta-1 and alpha-6 beta-4 integrins in highly invasive and metastatic variants of human prostate carcinoma cells selected by in vitro invasion through reconstituted basement membrane, Clin. Exp. Metastasis, 11 (1993) 391–400.

    Google Scholar 

  30. Weinel, R.J., Rosendahl, A., Pinschmidt, E., Kisker, O., Simon, B. and Santoso, S.,The alpha-6-integrin receptor in pancreatic carcinoma, Gastroenterology, 108 (1995) 523–532.

    Google Scholar 

  31. Wolf, G.T. and Carey, T.E.,Tumor antigen phenotype, biologic staging, and prognosis in head and neck squamous carcinoma, Monographs, National Cancer Institute, 1992, pp. 67–74.

  32. Danen, E.H., Van Muijen, G.N., Ten Berge, P.J. and Ruiter, D.J.,Integrins and melanoma progression, Rec. Results Cancer Res., 128 (1993) 119–132.

    Google Scholar 

  33. Ruiz, P., Dunon, D., Sonnenberg, A. and Imhof, B.A.,Suppression of mouse melanoma metastasis by EA-1, a monoclonal antibody specific for alpha-6 integrins [published erratum appears in Cell. Adhes. Commun. 1993 Sep;1(2):following 190],

  34. Lam, K.S., Salmon, S.E., Hersh, E.M., Hruby, V.J., Kazmierski, W.M. and Knapp, R.J.,A new type of synthetic peptide library for identifying ligand-binding activity, Nature, 354 (1991) 82–84.

    Google Scholar 

  35. Lam, K.S. and Salmon, S.E., Method of Screening a Peptide Library, United States Patent No. 5510240, 1996.

  36. Lebl, M., Krchñák, V., Sepetov, N.F., Seligmann, B., Strop, P., Felder, S. and Lam, K.S.,One-bead-one-structure combinatorial libraries, Biopolymers, 37 (1995) 177–198.

    Google Scholar 

  37. Furka, A., Sebestyen, F., Asgedom, M. and Dibo, G.,General method for rapid synthesis of multicomponent peptide mixtures, Int. J. Pept. Protein Res., 37 (1991) 487–493.

    Google Scholar 

  38. Houghten, R.A., Pinilla, C., Blondelle, S.E., April, J.R., Dooley, C.T. and Cuerro, J.H.,Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery, Nature, 354 (1991) 84–86.

    Google Scholar 

  39. Lam, K.S., Lebl, M., Krchñák, V., Wade, S., Abdul-Latif, F., Ferguson, R., Cuzzocrea, C. and Wertman, K.,Discovery of D-amino-acid-containing ligands with Selectide technology, Gene, 137 (1993) 13–16.

    Google Scholar 

  40. Lam, K.S. and Lebl, M.,Streptavidin and avidin recognize peptide ligands with different motif, ImmunoMeth., 1 (1992) 11–15.

    Google Scholar 

  41. Salmon, S.E., Lam, K.S., Lebl, M., Kandola, A., Khattri, P.S., Wade, S., Patek, M., Kocis, P., Krchñák, V., Thorpe, D. and Felder, S.,Discovery of biologically active peptides in random libraries: Solution-phase testing after staged orthogonal release from resin beads, Proc. Natl. Acad. Sci. USA, 90 (1993) 11708–11712.

    Google Scholar 

  42. Smith, M.H., Lam, K.S., Hersh, E.M. and Grimes, W.,Mapping the peptide binding groove of MHC class I proteins using a synthetic peptide library approach, Mol. Immunol., 31 (1994) 1431–1437.

    Google Scholar 

  43. Lam, K.S., Wu, J. and Lou, Q.,Identification and characterization of a novel synthetic peptide substrate specific for Src-family protein tyrosine kinases, Int. J. Pept. Protein Res., 45 (1995) 587–592.

    Google Scholar 

  44. Wu, J., Ma, Q.N. and Lam, K.S.,Identifying substrate motifs of protein kinases by a random library approach, Biochemistry, 33 (1994) 4825–4833.

    Google Scholar 

  45. Lam, K.S., Lou, Q., Zhao, Z., Smith, J., Chen, M., Pleshko, E. and Salmon, S.E.,Idiotype specific peptides bind the surface immunoglobulins of two murine B-cell lymphoma lines, inducing signal transduction, Biomed. Pept. Proteins Nucleic Acids, 1 (1995) 205–210.

    Google Scholar 

  46. Rabinovitz, I., Nagle, R.B. and Cress, A.E.,Integrin alpha-6 expression in human prostate carcinoma cells is associated with a migratory and invasive phenotype in vitro and in vivo, Clin. Exp. Metastasis, 13 (1995) 481–491.

    Google Scholar 

  47. Lam, K.S. and Lebl, M.,Selectide technology: Bead-binding screening, Methods: A companion to Methods Enzymol., 6 (1994) 372–380.

    Google Scholar 

  48. Lam, K.S. and Lebl, M.,Combinatorial library based on the one-bead-one-chemical concept, In Jung, G. (Ed.), Peptide and Nonpeptide Libraries — A Handbook, VCH, New York, NY, U.S.A., 1996, pp. 173–201.

    Google Scholar 

  49. Rabinovitz, I., Nagle, R.B. and Cress, A.E.,Integrin α 6 influences the migratory and invasive phenotype of human prostate carcinoma cells in vitro and in vivo, Clin. Exp. Metastasis, 13 (1995) 481–491.

    Google Scholar 

  50. Juliano, R.L.,The role of beta-1 integrins in tumors, Semin. Cancer Biol., 4 (1993) 277–283.

    Google Scholar 

  51. Chorev, M., Dresner-Pollak, R., Eshel, Y. and Rosenblatt, M.,Approach to discovering novel therapeutic agents for osteoporosis basedon integrin receptor blockade, Biopolymers, 37 (1995) 367–375.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Radiation Oncology, University of Arizona, 1501 North Campbell Avenue, 85824, Tucson, AZ, USA

    Michael E. Pennington & Anne E. Cress

  2. Department of Medicine, University of Arizona, 1501 North Campbell Avenue, 85824, Tucson, AZ, USA

    Kit S. Lam

  3. Department of Microbiology and Immunology, University of Arizona, 1501 North Campbell Avenue, 85824, Tucson, AZ, USA

    Kit S. Lam

Authors
  1. Michael E. Pennington
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kit S. Lam
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Anne E. Cress
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Pennington, M.E., Lam, K.S. & Cress, A.E. The use of a combinatorial library method to isolate human tumor cell adhesion peptides. Mol Divers 2, 19–28 (1996). https://doi.org/10.1007/BF01718696

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01718696

Keywords

Search

Navigation