Raf-1 protein expression in human breast cancer cells.
BACKGROUND
The Raf-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. The Raf-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 of Raf-1 kinase in breast cancer has not been studied.


METHODS
To investigate the role of Raf-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 of Raf-1 protein and the proportion expressed in the higher molecular weight form. Effects of serum starvation and stimulation with EGF on the Raf-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, the Raf-1 protein was identified in a 70- and a 74-kDa form. The level of Raf-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 of Raf-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.


CONCLUSIONS
Baseline expression of the 74-kDa or activated form of the Raf-1 kinase appeared to be elevated in the breast cancer cells studied, indicating constitutive activation. Further investigation into the role of Raf-1 protein in the pathogenesis of breast cancer is indicated.
Department of Surgery, Hospital of the University of Pennsylvania, Philadelphia 19104.
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Callans
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Naama
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Khandelwal
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Plotkin
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Jardines
D001943:Breast Neoplasms / Q000235:genetics* / Q000473:pathology; D003470:Culture Media; D004273:DNA, Neoplasm / Q000235:genetics; D004789:Enzyme Activation / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D015151:Immunoblotting; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D019908:Proto-Oncogene Proteins c-raf; D015290:Second Messenger Systems / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D013936:Thymidine / Q000378:metabolism; D014316:Tritium; D014407:Tumor Cells, Cultured
D003470:Culture Media; D004273:DNA, Neoplasm; D011518:Proto-Oncogene Proteins; D014316:Tritium; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D013936:Thymidine