title: Analysis of CDK Inhibitor Action on Mitochondria-Mediated Apoptosis.
abstract: The role of cyclin-dependent kinase inhibitors (CDKIs) is to negatively regulate cyclin-dependent kinases as a mechanism of control of cell proliferation. As such, CDKIs are being used to induce apoptosis in cancer cells to prevent their excessive reproduction. This chapter describes procedures to study apoptosis induction upon treatment with any CDKI through the evaluation of morphological and functional mitochondrial alterations, in particular, how to measure the mitochondrial membrane potential (ΔΨm) using TMRE dye, determine the content of intracellular ATP, observe mitochondrial network morphology using HeLa cells stably expressing fluorescent reporter DsRed targeting mitochondrial matrix, observe ultrastructure of the organelle using transmission electron microscopy, and, finally, assure that mitochondrial outer membrane permeabilization takes place by assessing the subcellular localization of cyt C in HeLa cells stably expressing fluorescent cyt C-GFP.
authors.affiliation: Department of Anatomical Pathology, Pharmacology and Microbiology, University of Barcelona, Casanova 143, Barcelona, 08036, Spain, gortat@o2.pl.
authors.forename: Anna
authors.initials: A
authors.lastname: Gortat
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry; D017209:Apoptosis; D002460:Cell Line; D049109:Cell Proliferation; D050756:Cyclin-Dependent Kinase Inhibitor Proteins / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors*; D045304:Cytochromes c / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry; D053078:Membrane Potential, Mitochondrial; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*
chemical_list: D050756:Cyclin-Dependent Kinase Inhibitor Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C413663:fluorescent protein 583; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; D045304:Cytochromes c; D018844:Cyclin-Dependent Kinases