The use of a combinatorial library method to isolate human tumor cell adhesion peptides.
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 degrees C. The peptide bead coated with a monolayer of cells was then isolated for microsequencing. 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 alpha 6 beta 1 integrin on the cell surface. The use of a function-blocking antibody (GoH3) allows for the detection of peptides which are alpha 6-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 alpha 6 integrin candidate peptide beads (five in total) were recovered and two of the beads were microsequenced. These two peptides, RU-1 (LNIVS-VNGRHX) 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.
Pennington
M E
ME
Department of Radiation Oncology, University of Arizona, Tucson 85824, USA.
Lam
K S
KS
Cress
A E
AE
D015703:Antigens, CD / Q000378:metabolism; D002277:Carcinoma; D002448:Cell Adhesion; D015815:Cell Adhesion Molecules / Q000302:isolation & purification* / Q000502:physiology; D005434:Flow Cytometry; D006801:Humans; D039503:Integrin alpha6; D016023:Integrins / Q000378:metabolism; D008297:Male; D008863:Microspheres; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000502:physiology; D011471:Prostatic Neoplasms; D011485:Protein Binding; D013499:Surface Properties; D014407:Tumor Cells, Cultured
D015703:Antigens, CD; D015815:Cell Adhesion Molecules; D039503:Integrin alpha6; D016023:Integrins; D019151:Peptide Library; D010455:Peptides