A chimeric protein induces tumor cell apoptosis by delivering the human Bcl-2 family BH3-only protein Bad.
Deregulation of PI3K/Akt and Raf/Mek/Erk signal transduction cascades is one of the principal causes of neoplastic transformation. The inactivation of the proapoptotic protein Bad, upon phosphorylation by different kinases of these two pathways, may play an important role in different human malignancies. Therefore, we have expressed and purified a new chimeric protein, hGM-CSF-Bad, linking the human granulocyte-macrophage colony-stimulating factor to the N-terminus of the proapoptotic protein human Bad, to deliver Bad into tumor cells and induce apoptosis. Indeed, the human GM-CSF receptor is a good target because it is overexpressed on many leukemias and solid tumors and is not detectable on stem cells. We found that the chimeric protein binds the human GM-CSF receptor, is endocytosed, and appears to reach the cytosol via retrograde ER transport. After entering cells, the protein is able to induce apoptosis of human leukemia cells and human colon and gastric carcinoma cell lines (IC(50) values as low as 1 muM). We conclude that GM-CSF-Bad can overcome the inappropriate survival stimuli in transformed cells and restore the apoptotic pathway. The completely human sequence and the elevated selectivity for cancer cells could prevent immunogenicity and the nonspecific toxicity of targeted toxins in future clinical application of this fusion protein.
Antignani
Antonella
A
Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892-3704, USA.
Youle
Richard J
RJ
D000409:Alanine / Q000235:genetics; D017209:Apoptosis / Q000235:genetics*; D051023:BH3 Interacting Domain Death Agonist Protein; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000502:physiology / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000235:genetics; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D006131:Growth Inhibitors / Q000235:genetics / Q000378:metabolism / Q000633:toxicity; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism*; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism* / Q000502:physiology / Q000633:toxicity; D012694:Serine / Q000235:genetics; D020298:U937 Cells; D051025:bcl-Associated Death Protein
C494819:BAD protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002352:Carrier Proteins; D006131:Growth Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011993:Recombinant Fusion Proteins; D051025:bcl-Associated Death Protein; D012694:Serine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000409:Alanine