Regulation of apoptosis and in vitro model for studies thereof

Details Regulation of apoptosis and in vitro model for studies thereof Regulation of apoptosis and in vitro model for studies thereof

C - Chemistry, Metallurgy

12

Q

C12Q 1/37 (2006.01) C12Q 1/68 (2006.01) G01N 33/53 (2006.01)

Patent

CA 2260766

A cell-free system based on the cytosol of normally growing cells, which reproduces measurable aspects of the apoptotic program, is provided. The apoptotic program is initiated by addition of dATP in the specific exemplification of the HeLa 100,000 x g supernatant. Fractionation of the cytosol yielded a 15 kDa protein, identified by absorption spectrum and protein sequence as cytochrome c, that is required for in vitro apoptosis. Elimination of cytochrome c from cytosol by immunodepletion or inclusion of sucrose to stabilize mitochondria during cytosol preparation, diminished the apoptotic activity. Addition of exogenous cytochrome c to cytochrome c- depleted extracts restored apoptotic activity. Cells undergoing apoptosis in vivo showed increased release of cytochrome c to their cytosol, suggesting that mitochondria may function in apoptosis by releasing cytochrome c.

L'invention propose un système acellulaire fondé sur le cytosol de cellules à croissance normale qui reproduit les aspects mesurables du programme d'apoptose. Le programme d'apoptose est déclenché par l'addition de dATP dans un échantillon approprié de surnageant de HeLa à 100 000 g. Le fractionnement du cytosol permet d'obtenir une protéine de 15 kDa, que l'on identifie par son spectre d'absorption et par sa séquence protéique comme le cytochrome c, nécessaire à l'apoptose in vitro. L'élimination du cytochrome c du cytosol par déplétion immunitaire ou par inclusion de saccharose dans le but de stabiliser la mitochondrie lors de la préparation du cytosol diminue l'activité d'apoptose. L'adjonction d'un cytochrome c exogène à des extraits appauvris en cytochrome c relance l'activité d'apoptose. Les cellules où se produit l'apoptose in vivo manifestent une libération accrue de cytochrome c vers leur cytosol, ce qui laisse à supposer que la mitochondrie peut fonctionner en apoptose en libérant le cytochrome c.

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