Inhibition of melanoma cell binding to type IV collagen by analogs of cell adhesion regulator.
Integrin-mediated tumor cell adhesion to type IV collagen is believed to play a role in the invasion of basement membrane proteins and the subsequent metastatic process. The cellular protein CAR (cell adhesion regulator) has been proposed to influence integrin-mediated binding to extracellular matrix proteins, including basement membrane (type IV) collagen. Three analogs of the CAR138-142 have been tested for activity. The first contains the 138-142 sequence (CAR138-142, Val-Glu-Ile-Leu-Tyr-NH2), the second contains the 138-142 sequence with a phosphorylated Tyr [pCAR138-142, Val-Glu-Ile-Leu-Tyr(PO3H2)-NH2], and the third contains the reversed 138-142 sequence (rCAR138-142, Tyr-Leu-Ile-Glu-Val-NH2). When added extracellularly, none of the analogs had a significant affect on cell adhesion to type IV collagen. Using a novel reversible cell permeabilization method, we found that intracellular incorporation of both CAR138-142 and pCAR138-142 resulted in inhibition of cell adhesion in a dose-dependent fashion. The IC50 values were approximately 90 and approximately 10 microM for CAR138-142 and pCAR138-142, respectively. Intracellular incorporation of the rCAR138-142 peptide had no affect on cell adhesion. Fluorescence microscopy of a fluorescein-labeled CAR138-142 peptide revealed that the reversible permeabilization procedure resulted in the peptides crossing the cell membrane. Affinity chromatography of melanoma cell lysates with pCAR138-142 or rCAR138-142 attached to a solid support of magnetic beads suggested that one protein was bound uniquely by pCAR138-142. Immunoprecipitation analysis identified vinculin, a protein associated with the actin cytoskeleton, as the protein specifically bound by pCAR138-142. Immunoprecipitation with pp125FAK- or beta 1-integrin-derived mAbs gave negative results. Our study suggests that a possible therapeutic approach for inhibition of melanoma cell adhesion adhesion to extracellular matrix proteins is the use of CAR peptide analogs intracellularly.
Lauer
J L
JL
Department of Laboratory Medicine, University of Minnesota, Minneapolis 55455, USA.
Furcht
L T
LT
Fields
G B
GB
D000074183:ATPases Associated with Diverse Cellular Activities; D000595:Amino Acid Sequence; D001485:Basement Membrane; D002448:Cell Adhesion / Q000187:drug effects* / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000737:chemistry*; D002463:Cell Membrane Permeability; D003094:Collagen; D006801:Humans; D007700:Kinetics; D008545:Melanoma; D008666:Metalloendopeptidases; D009362:Neoplasm Metastasis; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D015815:Cell Adhesion Molecules; D009842:Oligopeptides; D010446:Peptide Fragments; D003094:Collagen; D008666:Metalloendopeptidases; C112889:SPG7 protein, human; D000074183:ATPases Associated with Diverse Cellular Activities