Binding of formyl peptides to Walker 256 carcinosarcoma cells and the chemotactic response of these cells.
N-Formylmethionylleucylphenylalanine (fMLP) induces chemotaxis in leukocytes, the response being mediated by peptide binding to a receptor on the plasma membrane. In tumor cells, this peptide has been reported to induce cellular swelling and chemotaxis in vitro and to enhance the localization of circulating tumor cells in vivo. In the Boyden chamber, we evaluated the migratory responses of Walker carcinosarcoma 256 cells to varying concentrations of fMLP. Sigmoidal dose-response curves were obtained with the dose of chemotactic factor that elicits a half-maximal chemotactic response of 5.0 +/- 2.5 X 10(-8) M. Checkerboard analysis indicated that these responses were dependent upon a concentration gradient of fMLP with increases in migration of circa 2 to 2.5 times that of random movement. To examine the binding of fMLP, the tumor cells were incubated with 5 X 10(-9) M fML-[3H]P in Hanks' balanced salt solution. Specific binding (0.5 to 1% of total radioligand, to whole cells inhibited by 5 X 10(-6) M fMLP) approached equilibrium after 4 to 6 h at 4 degrees C and after 6 to 10 h at 22 degrees C. Autoradiographic studies demonstrated heterogeneous binding of the peptide by tumor cells and also showed its intracellular localization. In homogenates of Walker cells prepared in 0.1 M Tris HCl, pH 7.4, with 10 mM MgCl2 and bovine serum albumin (1 mg/ml), specific binding of approximately 0.5% of total fML-[3H]P reached equilibrium after 60 min at 4 degrees C. In whole cells and homogenates, binding was reversible by addition of unlabeled fMLP. In whole cells, displacement curves demonstrated a Kd of 1.9 +/- 0.1 X 10(-7) M, whereas in homogenates there was a background of low affinity (Kd greater than 10(-5) M) nonsaturable binding, but also a high-affinity component with Kd of 4.9 +/- 1.8 X 10(-8) M. Both chemotaxis and binding were inhibited by the oligopeptide, N-carbobenzoxy-L-phenylalanyl-L-methionine, which is a competitive inhibitor of formyl peptide-induced neutrophil chemotaxis. These data suggest that fMLP stimulates chemotaxis in tumor cells by a receptor-mediated pathway.
D000818:Animals; D002279:Carcinoma 256, Walker / Q000378:metabolism*; D002478:Cells, Cultured; D002633:Chemotaxis; D004151:Dipeptides / Q000494:pharmacology; D005260:Female; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D051381:Rats; D014316:Tritium
D004151:Dipeptides; D014316:Tritium; C035192:carbobenzoxyphenylalanylmethionine; D009240:N-Formylmethionine Leucyl-Phenylalanine