| The role of ARC (A&barbelow;poptosis R&barbelow;epressor with C&barbelow;aspase Recruitment Domain) in PC12 cell serum withdrawal driven apoptosis was studied. A progressive and massive increase in ARC protein occurred during serum withdrawal that correlated with declining survival and processing of caspase-2, previously shown to associate with ARC. The accumulation of ARC protein was significantly reduced in the presence of the survival agent, nerve growth factor, added at the time of serum withdrawal. This suggested that the ARC protein accumulation correlated with death. The transfection of PC12 cells with exogenous ARC (hARC-Flag) failed to protect the cells from apoptosis by serum withdrawal. Moreover, 48 h post-transfection hARC-Flag promoted death in PC12 cells. These results called into question the role of ARC as a repressor of apoptosis in this model.; The mechanism mediating the ARC accumulation was also investigated. The ARC protein accumulation during serum withdrawal occurred independent of transcription and translation. In addition, ARC was found localized exclusively in the nucleus of PC12 cells where several other CARD/homophilic-domain containing proteins have been found. During apoptosis, the nucleus undergoes condensation and provides an excellent environment for the aggregation of homophilic-domain containing proteins. Aberrant ARC migration patterns were observed on immunoblots, especially at 24–48 h serum withdrawal, and provided indirect evidence that ARC may be aggregating with itself or other proteins. The aggregation of ARC may also protect it from degradation and account for its accumulation during serum withdrawal driven apoptosis.; To better understand the role of ARC during apoptosis, co-transfection experiments were carried out with hARC-Flag and caspase-2 or caspase-8. The over-expression of caspase-2 alone elicited ∼40% total caspase. activation and 20% death. Co-expression of hARC-Flag with caspase-2 did not alter caspase activation or prevent death. Whereas, caspase-8 alone elicited ∼35–40% total caspase activation and death. Expression of hARC-Flag with caspase-8 significantly reduced (by about 50%) caspase activation and death. The results indicated that ARC acts differentially in apoptosis depending on the stimuli and the caspase employed. |
