Cytosolic Raf-1 exists in a high molecular weight complex with the heat shock protein Hsp90, the purpose of which is unknown. The benzoquinone ansamycin, geldanamycin, specifically binds to Hsp90 and disrupts certain multimolecular complexes containing this protein.Using this drug, we are able to demonstrate rapid dissociation of both Raf-1-Hsp90 and Raf-1-Ras multimolecular complexes, concomitant with a markedly decreased halflife of the Raf-1 protein. Continued disruption of the Raf-1-Hsp90 complex results in apparent loss of Raf-1 protein from the cell, although Raf-1 synthesis is actually increased. Prevention of Raf-1-Hsp90 complex formation interferes with trafficking of newly synthesized Raf-1 from cytosol to plasma membrane. These data indicate that association with Hsp90 is essential for both Raf-1 protein stability and its proper localization in the cell.

The isoflavinoid genistein is a protein-tyrosine kinase inhibitor which has been identified as a putative cancer prevention agent. Its consumption is associated with a low incidence of clinical metastatic prostate cancer in the face of a sustained high incidence of organ-confined prostate cancer. We therefore undertook studies to examine genistein's effect upon cell adhesion as one possible mechanism by which it could be acting as an antimetastatic agent. A morphogenic analysis revealed that genistein caused cell flattening in a variety of cell lines: PC3-M, PC3, and DU-145 prostate carcinoma cells, as well as MCF-7 breast carcinoma cells. Mechanistic studies focused on the highly metastatic PC3-M cell line, and revealed that cell flattening was accompanied by an increase in cell adhesion. Further investigations demonstrated that focal adhesion kinase (FAK) accumulated in areas of focal cell attachment, and that this accumulation occurred only when cells were actively undergoing genistein-mediated morphologic change. Concurrent formation of a complex between the cell attachment molecule, beta-1-integrin, and FAK was shown to occur, and to correlate with transient activation of FAK activity. Genistein is presented as a novel investigative tool for use in the study of molecular events involved in the process of cell adhesion.