Unreliability of the cytochrome c-enhanced green fluorescent fusion protein as a marker of cytochrome c release in cells that overexpress Bcl-2.
A cytochrome c-enhanced green fluorescent protein chimera (cyt-c.EGFP) was used to monitor the release of cytochrome c from mitochondria in Bcl-2-negative and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was made with the intracellular distribution of endogenous cytochrome c based on Western blotting of cell fractions and immunocytochemistry. The release of endogenous cytochrome c from mitochondria into the cytoplasm was detected in Bcl-2-negative cells treated with the kinase inhibitor staurosporine or the calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochrome c was evident in Bcl-2-positive cells, consistent with earlier evidence that Bcl-2 overexpression inhibits cytochrome c release from mitochondria. Cyt-c.EGFP appeared to be localized to the mitochondria in Bcl-2-negative cells and to be released into the cytoplasm following treatment with either staurosporine or thapsigargin. However, in Bcl-2-positive cells the pattern of distribution of cytochrome c-EGFP was inconsistent with that of endogenous cytochrome c, due to accumulation of both cyt-c.EGFP and free EGFP in the cytoplasm of both treated and untreated cells. In summary, cyt-c.EGFP may be useful for monitoring cytochrome c release in living cells that do not express high levels of Bcl-2 but is an unreliable marker of cytochrome c release in cells that overexpress Bcl-2.
Unkila
M
M
Division of Hematology/Oncology, Department of Medicine and Comprehensive Cancer Center at Case Western Reserve University and University Hospitals of Cleveland, Cleveland, Ohio 44106, USA.
McColl
K S
KS
Thomenius
M J
MJ
Heiskanen
K
K
Distelhorst
C W
CW
D017209:Apoptosis; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005819:Genetic Markers; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008928:Mitochondria / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D019311:Staurosporine / Q000494:pharmacology; D013347:Subcellular Fractions; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D003574:Cytochrome c Group; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005819:Genetic Markers; D008164:Luminescent Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D019284:Thapsigargin; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D019311:Staurosporine