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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.

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Authors and Affiliations

  1. Division of Surgical Oncology, Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania, USA

    Linda S. Callans MD, Hassan Naama (FRCSI), Mamta Khandelwal, Randi Plotkin & Lori Jardines MD

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  1. Linda S. Callans MD
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  2. Hassan Naama
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  3. Mamta Khandelwal
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  4. Randi Plotkin
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Callans, L.S., Naama, H., Khandelwal, M. et al. Raf-1 protein expression in human breast cancer cells. Annals of Surgical Oncology 2, 38–42 (1995). https://doi.org/10.1007/BF02303700

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