A micropipette technique was adopted to investigate the effect of blockade of integrin betal on adhesion of hepatocellular carcinoma (HCC) cells onto type IV collagen (Col IV) coated surfaces and pseudopod protrusion of HCC cells in response to Col IV stimulation. Adhesion strength was expressed as an adhesion force, which was defined as the product of the cross sectional area and critical negative pressure needed to detach single cell away from the substrate. Chemotactic pseudopod protrusion of an HCC cell was evaluated using a dual-pipette set-up, in which two pipettes filled with Col IV solution were positional in close contact with the same cell and pseudopod protrusion into each pipette was viewed dynamically and recorded with a tape recorder. The lengths of pseudopods were measured and plotted against time to obtain a pseudopod growth curve. The integrin beta1 subunit on the surfaces of HCC cells was analyzed by flow cytometry. The results showed that the adhesion forces for HCC cells adhering on 5 microg/ml Col IV coated surfaces were 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of HCC cells with Anti-CD29 in a protein concentration of 5 microg/ml and 10 microg/ml, the value decreased significantly to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 55), respectively. In dual pipette chemotaxis experiment, when the two pipettes were filled with Col IV in an identical concentration of 600 microg/ml, pseudopods extended from the HCC cell into each of the pipettes nearly symmetrically, i.e., with nearly identical maximum pseudopod length and similar pseudopod growth curves. Upon addition of Anti-CD29 to one of the pipettes in a protein concentration of 20 microg/ml, pseudopod protrusion was blocked nearly completely while protrusion into the opposite pipette became more evidently, with larger maximum length. Expression of integrin beta1 was up to 95.78% to cells chosen in the experiment. These results suggested that integrin beta1 subunit was important constituent receptor subunit for mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.
Antibodies, Monoclonal ; pharmacology ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Adhesion ; Chemotaxis ; immunology ; Collagen Type IV ; metabolism ; Humans ; Integrin beta1 ; biosynthesis ; immunology ; Liver Neoplasms ; metabolism ; pathology ; Neoplasm Invasiveness ; Receptors, Very Late Antigen ; biosynthesis ; immunology ; Tumor Cells, Cultured