bcl-2 Protects SK-N-SH Cells From 6-Hydroxydopamine Induced Apoptosis by Inhibition of Cytochrome c Redistribution
Neurotoxicity induced by 6-hydroxydopamine (6-OHDA) is, in part, due to the production of reactive oxygen/nitrogen species (RNOS) and/or an inhibition of mitochondrial function. However, little is known about the ensuing intracellular events which ultimately result in cell death. Here we show that exposure to relatively low concentrations of 6-OHDA induce apoptosis of SK-N-SH cells. Western blots of cytosolic extracts from 6-OHDA treated cells reveal a translocation of cytochrome c from mitochondria into the cytosol. To further delineate the pathway by which 6-OHDA causes apoptosis, we investigated the effects on cell survival in bcl-2 overexpressing SK-N-SH cells and the relation to translocation of cytochrome c and free radical production. Overexpressing bcl-2 in SK-N-SH cells shifted the toxicity as well as the release of cytochrome c to higher concentrations of 6-OHDA. At concentrations of 6-OHDA in SK-N-SH-bcl-2 cells where a release of cytochrome c and subsequent cell death occurs, an increase of the oxidation of the fluorescent dye dihydrorhodamine-123 was detectable. This RNOS production paralleled the release of cytochrome c and was inhibited by bcl-2 as well. Prom these experiments, we hypothesize that cytochrome c might interact with 6-OHDA to promote an oxidative burst, which can be inhibited by bcl-2.
KeywordsCell Death Cerebellar Granule Neuron Fluorescein Diacetate Subsequent Cell Death Initiate Lipid Peroxidation
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- Cassarino, D.S., Parks, J.K., Parker, W.D., Jr., and Bennett, J.P., Jr., 1999, The parkinsonian neurotoxin MPP+ opens the mitochondrial permeability transition pore and releases cytochrome c in isolated mitochondria via an oxidative mechanism, Biochim. Biophys. Acta 1453: 49.PubMedCrossRefGoogle Scholar
- Gleichmann, M., Beinroth, S., Reed, J.C., Krajewski, S., Schulz, J.B., Wullner, U., Klockgether, T., and Weller, M., 1998, Potassium deprivation-induced apoptosis of cerebellar granule neurons: cytochrome c release in the absence of altered expression of Bcl-2 family proteins, Cell Physiol Biochem. 8: 194.PubMedCrossRefGoogle Scholar
- Korsmeyer, S.J., 1999, BCL-2 gene family and the regulation of programmed cell death, Cancer Res. 59: 1693.Google Scholar
- Kostrzewa, R.M., and Jacobowitz, D.M., 1973, Pharmacological actions of 6-hydroxydopamine,Pharmacol. Rev. 26: 199.Google Scholar
- Miller, T.M., Moulder, K.L., Knudson, C.M., Creedon, D.J., Deshmukh, M., Korsmeyer, S.J., and Johnson, E.M., 1997, Bax deletion further orders the cell death pathway in cerebellar granule cells and suggests a caspase-independent pathway to cell death, J. Cell. Biol. 139:205.PubMedCrossRefGoogle Scholar
- Pezzella, A., d’Ischia, M., Napolitano, A., Misuraca, G., and Prota, G., 1997, Iron-mediated generation of the neurotoxin 6-hydroxydopamine quinone by reaction of fatty acid hydroperoxides with dopamine: a possible contributory mechanism for neuronal degeneration in Parkinson’s disease, J. Med. Chem. 40: 2211.PubMedCrossRefGoogle Scholar
- Susin, S.A., Lorenzo, H.K., Zamzami, N., Marzo, I., Snow, B.E., Brothers, G.M., Mangion, J., Jacotot, E., Costantini, P., Loeffler, M., Larochette, N., Goodlett, D.R., Aebersold, R., Siderovski, D.P., Penninger, J.M., and Kroemer, G., 1999, Molecular characterization of mitochondrial apoptosis-inducing factor, Nature 397: 441.PubMedCrossRefGoogle Scholar
- Thomberry, N.A., Rano, T.A., Peterson, E.P., Rasper, D.M., Timkey, T., Garcia-Calvo, M., Houtzager, V.M., Nordstrom, P.A., Roy, S., Vaillancourt, J.P., Chapman, K.T., and Nicholson, D.W., 1997, A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis, J. Biol. Chem. 272: 17907.CrossRefGoogle Scholar
- Zamzami, N., Marchetti, P., Castedo, M., Decaudin, D., Macho, A., Hirsch, T., Susin, S.A., Petit, P.X., Mignotte, B., and Kroemer, G., 1995, Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death, J. Exp. Med. 182: 367.PubMedCrossRefGoogle Scholar