Raf-1 protein expression in human breast cancer cells

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Abstract
Background: TheRaf-1 kinase, a 72-kDa cytoplasmic serine-threonine kinase, plays a central role as a second messenger in signal transduction. After ligand binding to a variety of transmembrane tyrosine kinase growth factor receptors including epidermal growth factor (EGF) receptor, the 72-kDa kinase is activated through phosphorylation to a 74-kDa phosphoprotein. TheRaf-1 kinase is constitutively activated in many transformed cells either directly, by mutations within its amino-terminus regulatory region, or indirectly, due to overstimulation by autocrine growth factors or activated proximal oncogenes. The role ofRaf-1 kinase in breast cancer has not been studied. Methods: To investigate the role ofRaf-1 kinase expression and its activation in breast cancer, we studied three human breast cancer cell lines expressing varying amounts of EGF receptor to determine the level ofRaf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on theRaf-1 protein were studied in T47D, BT474, and MDA-MB231 cells by precipitation of cell lysates with an anti-Raf-1 antibody followed by immunoblotting. [3H]Thymidine incorporation by these cells after EGF stimulation was also determined as a measure of DNA synthesis. Results: In all three breast cancer cell lines studied, theRaf-1 protein was identified in a 70- and a 74-kDa form. The level ofRaf-1 was similar in all three cell lines and appeared unrelated to EGF receptor expression on the cell surface. The majority of the protein was found in the 74-kDa form even after serum starvation. A minor shift from the lower to higher molecular weight form ofRaf-1 was apparent in cells treated with EGF, and increased [3H]thymidine incorporation could be demonstrated in two of the cell lines after EGF stimulation. Conclusion: Baseline expression of the 74-kDa or activated form of theRaf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role ofRaf-1 protein in the pathogenesis of breast cancer is indicated.