Specific sequences of fibronectin activate the protein kinase C signal transduction pathway in invasive bladder cancer.

The mechanism of human bladder cancer cell invasion is not clear, but it appears that extracellular matrix components, such as fibronectin, may be involved. To investigate the role of fibronectin in tumor cell invasion and progression, we used an in vitro invasion assay to define the motility stimulating fragment of fibronectin for invasive human bladder cancer T24 cells. Using a modified Boyden chamber assay and purified fragments of fibronectin, we demonstrated that both the 120 kDa chymotrypsin generated fragment of fibronectin (containing the cell attachment RGD motif and additional sequences towards the carboxyl-terminal heparin binding domain), as well as the trypsin generated 60 kDa fragment of fibronectin (containing the carboxyl-terminal heparin binding domain and additional sequences towards the cell attachment RGD motif), were able to stimulate the migration of invasive human bladder cancer T24 cells. Control fragments containing only the amino-terminal gelatin binding region of fibronectin did not stimulate the motility of the human bladder cancer T24 cells. To determine the molecular mechanism in which these fragments may stimulate the migration of the T24 cells, we assayed for intracellular signal transduction pathway protein kinase C (PKC). We demonstrated that both the 120 kDa and the 60 kDa fragments were able to stimulate the activation of protein kinase C. Non-motility stimulating fragments of fibronectin were not able to activate protein kinase C. We conclude that the PKC signal transduction pathway may be involved in matrix mediated motility, and suggest that the inhibition of such pathway(s) may alter the malignant phenotype of human bladder cancer.

Bacillus Calmette-Guérin interacts with the carboxyl-terminal heparin binding domain of fibronectin: implications for BCG-mediated antitumor activity.

Intravesical bacillus Calmette-Guérin (BCG) has been shown to be an effective treatment for superficial transitional cell carcinoma of the bladder. The mechanisms by which BCG achieves this effect remain unclear. Reports have attributed an important role to fibronectin both in the initial attachment of BCG to bladder surfaces and in the limitation of tumor cell motility. In the present study, using limited protease cathepsin B degradation followed by Western blot analyses with antibodies to various domains of the fibronectin molecule, we showed that BCG appears to bind to fibronectin near the carboxyl terminal and adjacent to the heparin binding domain. Furthermore a 51-chromium release assay with human bladder cancer cell line T24 as target cells and lymphokine activated killer (LAK) cells as effector cells showed that fibronectin was needed for tumor cytotoxicity by the LAK cells. By using antibodies and peptides to various domains of the fibronectin molecule, the heparin binding domain, but not the cell binding domain, carboxyl terminal region, or the amino terminal region of the fibronectin molecule, was identified as essential to tumor cell lysis by the LAK cells. Flow cytometric analysis showed that both peripheral blood lymphocytes and the LAK cells express fibronectin receptors VLA-3, VLA-4 and VLA-5 on their surfaces. However, the numbers of receptors are not significantly different in the two cell populations. We conclude that, by binding near the carboxyl terminal region and adjacent to the heparin-binding domain of the fibronectin molecule, BCG may protect this region of the molecule from tumor proteases, and may thus allow the antitumor activity of the host immune cells to take place.