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-nega tive and Bcl-2-positive MDA-MB-468 breast cancer cells. A comparison was ma de 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 i n Bcl-2-negative cells treated with the kinase inhibitor staurosporine or t he calcium-ATPase inhibitor thapsigargin. No release of endogenous cytochro me 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 c ells 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 endogen ous cytochrome c, due to accumulation of both cyt-c.EGFP and free EGFP in t he cytoplasm of both treated and untreated cells. In summary, cyt-c.EGFP ma y 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 r elease in cells that overexpress Bcl-2.