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A new method to quantify mitochondrial cytochrome c release during cell death: immunodetection of cytochrome c following selective permeabilization of the plasma membrane

Grant number: 02/09210-3
Support type:Research Grants - Support for Intellectual Property Rights and Licensing (PAPI/Nuplitec)
Duration: December 01, 2002 - November 30, 2005
Field of knowledge:Biological Sciences - Biochemistry
Principal Investigator:Aníbal Eugênio Vercesi
Grantee:Aníbal Eugênio Vercesi
Home Institution: Faculdade de Ciências Médicas (FCM). Universidade Estadual de Campinas (UNICAMP). Campinas , SP, Brazil

Abstract

This invention proposes an easy, fast, quantitative and reliable technique to assay mitochondrial cytochrome c release during apoptotic cell death by immunodetection of cytochrome c following selective permeabilization of plasma membrane using fluorescence, chemoluminescence or colorimetric methodology. Cytochrome c is released from mitochondria to the cytosol after the triggering of apoptosis in many different cell types in response to a variety of cellular insults (Martinou and Green, 2001; Zamzami and Kroemer, 2001), been its release one of the most used feature to characterize this type of cell death. The techniques currently available to measure cytochrome c release are Western blot and fluorescence microscopy. The first one is time-consuming, usually taking at least two days to get the results. Fluorescence microscopy is faster and easier then a Western blot, but it can also be time-consuming. More important, accuracy and sampling are always the main concern when using this techniques. The technique described here greatly decreases the time spent for the determination of cytochrome c release, while increases the accuracy of the measurement of this protein and the number of cells analyzed during apoptosis. The method is based on the differences among the lipids that constitute plasma and mitochondrial membranes. The cholesterol/phospholipid ratio is very high in the plasma membrane, while outer and inner mitochondria membranes are very poor or are virtually free of cholesterol, respectively. Using low concentrations of cholesterol-directed detergents, such as digitonin, it is possible to selectively disrupt the plasma membrane without affecting intracellular organelle membranes, such as mitochondrial membrane (Granger and Lehninger, 1982; Harris et al, 1981; Moreadith and Fiskum, 1984). Once the plasma membrane is permeabilized, it is possible to wash out any cytosolic component able to pass over digitonin-forming holes, including cytochrome c released from mitochondria of apoptotic cells. A further fixation prevents loss of unreleased cytochrome c during the following steps. The cells are then labeled with an anti-cytochrome c antibody and developed using a secondary antibody conjugated with a marker suitable for fluorescence, chemoluminescence or color detection. Non-apoptotic cells will be positive for cytochrome c since all cytochrome c remains retained inside mitochondria. In this case, flow cytometric analysis (judged to be the most reliable method to be used for determination of cytochrome c release) would detect a population of cells with high fluorescence. In apoptotic cells, the cytochrome c released to the cytoplasm will be further released from the cells to the washing media during digitonin treatment and washes, and the cells will turn negative for cytochrome c. The fluorescence of the apoptotic cells population detected by flow cytometry would be around background fluorescence. Flow cytometers, plate readers, spectrophotometers, fluorometers, or any instrument that detects light, fluorescence or color can analyze the result of this procedure, although flow cytometry is the most suitable technique in this case. Any described compound, or a compound yet to be described, that would selectively permeabilize plasma membrane based on cholesterol/phospholipid ratio is included in this invention to detect cytochrome c release. ReferencesGranger, D.L., Lehninger, A. L. (1982) Sites of inhibition of mitochondrial electron transport in macrophage-injured neoplastic cells. J.Cell.Biol. 95:527-535.Harris, S.I., Balaban, R.S., Barrett, L., Mandel, L.J. (1981) Mitochondrial respiratory capacity and Na+- and K+-dependent adenosine triphosphate-mediated ion transport in the intact renal cells. J.Biol.Chem. 256:10341-10328.Martinou, J.C., Green, D.R. (2001) Breaking the mitochondrial barrier. Nat. Rev. Mol. Cell. Biol. 2:63-67.Moreadith, R.W., Fiskum, G. (1984) Isolation of mitochondria from ascitis tumor cells permeabilized with digitonin. Anal. Biochem. 137:360-367.Zamzami, N., Kroemer, G. (2001) The mitochondrion in apoptosis: how Pandora's box opens. Nat. Rev. Mol. Cell. Biol. 2:67-71. (AU)

Filed patent(s) as a result of this research project

DETECTION, QUANTIFICATION AND COMPOSITION FOR CYTOCHROME C RELEASED FROM MITOCHONDRIA PCT/BR2004/000022 - Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) . Cláudia Barbosa Ladeira de Campos; Ricardo G. Cosso; Roger F. Castilho; Hagai Rottenberg; Aníbal E. Vercesi - February 2004, 16

MÉTODO PARA QUANTIFICAR LIBERAÇÃO MITOCONDRIAL DE CITOCROMO c, COMPOSIÇÃO PARA DETECTAR LIBERAÇÃO MITOCONDRIAL DE CITOCROMO c, KIT PARA DETECTAR LIBERAÇÃO MITOCONDRIAL DE CITOCROMO c, USO DE COMPOSIÇÃO PARA LIBERAÇÃO MITOCONDRIAL DE CITOCROMO c PI0300660-3 - Universidade Estadual de Campinas (UNICAMP) . Roger F. Castilho; Ricardo G. Cosso; Claudia Barbosa Ladeira Campos; Aníbal E. Vercesi; Hagai Rottenberg - February 2003, 28