Real-Time Imaging of HIF-1α Stabilization and Degradation 英文参考文献.docVIP

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Real-Time Imaging of HIF-1α Stabilization and Degradation 英文参考文献.doc

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Real-Time Imaging of HIF-1α Stabilization and Degradation 英文参考文献

Real-TimeImagingofHIF-1aStabilizationand Degradation EkaterinaMoroz1,SeanCarlin2,KaterinaDyomina1,SeanBurke2,HowardT.Thaler4 ,Ronald Blasberg1,3*,InnaSerganova1 1DepartmentofNeurology,MemorialSloan-KetteringCancerCenter,NewYork,NewYork,UnitedStatesofAmerica,2DepartmentofMedicalPhysics,MemorialSloan- KetteringCancerCenter,NewYork,NewYork,UnitedStatesofAmerica,3DepartmentofRadiology,MemorialSloan-KetteringCancerCenter,NewYork,NewYork,United StatesofAmerica,4DepartmentofEpidemiologyandBiostatistics,MemorialSloan-KetteringCancerCenter,NewYork,NewYork,UnitedStatesofAmerica Abstract HIF-1a is overexpressed in many human cancers compared to normal tissues due to the interaction of a multiplicity of factorsandpathwaysthatreflectspecificgeneticalterationsandextracellularstimuli.WedevelopedtwoHIF-1achimeric reporter systems, HIF-1a/FLuc and HIF-1a(DODDD)/FLuc, to investigate the tightly controlled level of HIF-1a protein in normal (NIH3T3andHEK293) andglioma(U87) cells. Thesereporter systems provided anopportunity toinvestigate the degradationofHIF-1aindifferentcelllines,bothincultureandinxenografts.Usingimmunofluorescencemicroscopy,we observeddifferentpatternsofsubcellularlocalizationofHIF-1a/FLucfusionproteinbetweennormalcellsandcancercells; similardifferenceswereobservedforHIF-1ainnon-transduced,wild-typecells.Adynamiccytoplasmic-nuclearexchangeof the fusion protein and HIF-1a was observed in NIH3T3 and HEK293 cells under different conditions (normoxia, CoCl2 treatmentandhypoxia).Incontrast,U87cellsshowedamorepersistentnuclearlocalizationpatternthatwaslessaffected by different growing conditions. Employing a kinetic model for protein degradation, we were able to distinguish two componentsof HIF-1a/FLucproteindegradation andquantifythehalf-life ofHIF-1afusion proteins. Therapidclearance component(t1/2,4–6min)wasabolishedbythehypoxia-mimeticCoCl2,MG132treatmentanddeletionofODDdomain, andreflectstheoxygen/VHL-dependentdegradationpathway.Theslowclearancecomponent(t1/2,20