Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry 英文参考文献.docVIP

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Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry 英文参考文献.doc

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Rapid Detection of an ABT-737-Sensitive Primed for Death State in Cells Using Microplate-Based Respirometry 英文参考文献

RapidDetectionofanABT-737-SensitivePrimedfor DeathStateinCellsUsingMicroplate-Based Respirometry PascalineClerc1,GregoryB.Carey2,ZaraMehrabian1,MichaelWei1,HyehyunHwang1, GeoffreyD.Girnun3,HegangChen4,StuartS.Martin5,BrianM.Polster1,3* 1DepartmentofAnesthesiologyandtheShock,TraumaandAnesthesiologyResearchCenter,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,United States of America, 2Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 3Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America, 4Department of EpidemiologyandPublicHealth,UniversityofMarylandSchoolofMedicine,Baltimore,Maryland,UnitedStatesofAmerica, 5DepartmentofPhysiology,Universityof MarylandSchoolofMedicine,Baltimore,Maryland,UnitedStatesofAmerica Abstract Cellsthatexhibitanabsolutedependenceontheanti-apoptoticBCL-2proteinforsurvivalaretermed‘‘primedfordeath’’ andarekilledbytheBCL-2antagonistABT-737.Manycancersexhibitaprimedphenotype,includingsomethatareresistant toconventionalchemotherapyduetohighBCL-2expression.Weshowherethat1)stableBCL-2overexpressionalonecan induceaprimedfordeathstateand2)thatanABT-737-inducedlossoffunctionalcytochromecfromtheelectrontransport chaincausesareductioninmaximalrespirationthatisreadilydetectablebymicroplate-basedrespirometry.StableBCL-2 overexpression sensitized non-tumorigenic MCF10A mammary epithelial cells to ABT-737-induced caspase-dependent apoptosis.MitochondriawithinpermeabilizedBCL-2overexpressing cellswereselectivelyvulnerabletoABT-737-induced cytochromecreleasecomparedtothosefromcontrol-transfectedcells,consistentwithaprimedstate.ABT-737treatment causedadose-dependentimpairmentofmaximalO2consumptioninMCF10ABCL-2overexpressingcellsbutnotincontrol- transfectedcellsorinimmortalizedmouseembryonicfibroblastslackingbothBAXandBAK.Thisimpairmentwasrescued bydeliveringexogenouscytochromec