Cif (Cytochrome c Efflux-Inducing Factor) Activity Is Regulated by Bcl-2 and Caspases and Correlates with the Activation of Bid

Abstract

The cytosolic factor Cif (cytochrome c-efflux inducing factor) was activated by the apoptosis inducers staurosporine and anti-Fas antibodies and rapidly induced the efflux of cytochrome c from purified human mitochondria. HL-60 cells that stably overexpressed a bcl-2 cDNA transgene (Bcl-2:HL-60 cells) contained mitochondria and a cytosol that were resistant to exogenous Cif and that lacked detectable endogenous Cif activity, respectively. Therefore, Bcl-2 overexpression negated Cif activity and suggested that the requirement for Cif resides upstream of Bcl-2 on the apoptotic signal transduction pathway. The addition of purified caspase 3, caspase 7, or caspase 8 to the cytosolic extract from Bcl-2:HL-60 cells, however, restored Cif activity, demonstrating that the inhibition of Cif by Bcl-2 overexpression could be overcome by activated caspases. Moreover, the addition of purified caspases to cytosolic extracts prepared from parental HL-60 cells was also sufficient to cause Cif activation, suggesting that caspases might be required for Cif activation. Consistent with these observations, Fas-induced apoptosis in Jurkat cells resulted in caspase 8 activation and subsequently in activation of Cif. Finally, we demonstrate that the activation of Cif correlated with the activation of the Bcl-2 family member Bid by caspases and that Cif activity was selectively neutralized by anti-Bid antibodies. Taken together, these results indicate that Cif is identical to Bid and that it can be inhibited by Bcl-2 and activated by caspases. Thus, Cif (Bid) is an important biological regulator for the transduction of apoptotic signals.

Cleavage of BID during cytotoxic drug and UV radiation-induced apoptosis occurs downstream of the point of Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for amplification of apoptosis-associated mitochondrial cytochrome c release

“…6,7,29 Recent studies have implicated BID in the cytochrome c release that is seen during FasL and TNF-induced apoptosis. 16,17,18,19,20 However, it is unclear whether BID plays a similar role in nondeath receptor-mediated cell deaths. To address this question, we induced apoptosis in Jurkat T lymphoblastoid cells using a range of pro-apoptotic stimuli and monitored BID protein levels as cells entered apoptosis.…”

“…Active caspase-8 cleaves cytosolic BID, resulting in translocation of the C-terminal portion of BID to the mitochondria, followed by integration into the outer mitochondrial membrane. 16,17,18,19 Insertion of BID into the mitochondrial membrane was found to be a potent instigator of cytochrome c release from mitochondria, via an as yet unknown mechanism. Significantly, whereas Bcl-2 and Bcl-x did not prevent cleavage of BID in the context of death receptor-initiated apoptosis, these proteins were capable of blocking the subsequent release of cytochrome c from mitochondria, in agreement with their perceived role as regulators of mitochondrial integrity.…”

“…Significantly, whereas Bcl-2 and Bcl-x did not prevent cleavage of BID in the context of death receptor-initiated apoptosis, these proteins were capable of blocking the subsequent release of cytochrome c from mitochondria, in agreement with their perceived role as regulators of mitochondrial integrity. 16,17,18,19 Mitochondrial cytochrome c release in non-death receptor pathways is less well understood. Recent data suggests that the pro-apoptotic Bcl-2 family proteins Bax and Bak may promote cytochrome c efflux, possibly due to their ability to bind and regulate components of the multiprotein permeability transition pore complex.…”

“…9 Recent studies have presented data to argue that BID, a death promoting member of the Bcl-2 family, promotes cytochrome c release in the Fas/CD95-and TNF-initiated cell death pathways. 16,17,18,19,20 In these contexts, receptor engagement results in early activation of caspase-8 through proximity-induced transprocessing that is facilitated by the FADD and TRADD adaptor proteins. Active caspase-8 cleaves cytosolic BID, resulting in translocation of the C-terminal portion of BID to the mitochondria, followed by integration into the outer mitochondrial membrane.…”

Human Mcm10 Regulates the Catalytic Subunit of DNA Polymerase-α and Prevents DNA Damage during Replication

“…Apoptosis was induced by adding staurosporine (STS) to 5 ϫ 10 6 cells at a final concentration of 5 M for up to 8 h (Han et al, 1999). Cells were irradiated using a 137 Cs ␥-ray source at a dose rate of 2.5 Gy/min.…”