PMC

Abrogation of Siva-1 expression significantly impairs cisplatin-mediated apoptosis. (A) HCT116 p53+/+ or (C) Jurkat cells were transduced with lentiviruses encoding either Siva siRNA (siSiva) or scrambled control (Scr siRNA). Transduced cells were analyzed for Siva-1 expression (A). Percentage apoptosis in HCT116 p53+/+ (B) and Jurkat cells (C) are shown (D) HCT116 p53-/- cells were transfected with plasmids expressing GST, GST-Siva-1, GST-Siva-1ΔSAH or GST-Siva-1ΔC (residues 130-149) as described earlier.[] The cell lysates were used to determine active caspase-9

Siva-1 plays a necessary but partial role in genotoxicinduced apoptosis; p53 is a central player in DNA damage-induced apoptosis and it triggers transcription of several pro-apoptotic genes such as Siva-1, Puma, Noxa, Bax etc. Siva-1, though principally regulated by p53 transcriptional activity, can function independent of p53 (unlike Puma), whereas Noxa appears to be important for mediation of ROS-induced cell death. Finally, the apoptosis pathways are kept in check by cell survival pathways, wherein Siva-1 is known to inhibit NF-kappa B.[]

Siva-1 promotes Bax activation in response to cisplatin treatment. MDA-MB-231 cells transiently expressing GST or GST-Siva-1 were treated with DMSO or cisplatin (50 μM) for the indicated time points (upper panels). Cells were iterated with BMH cross-linker (Pierce) to observe Bax multimers; arrows indicate monomeric, dimeric, and oligomeric forms (upper panels). In a parallel experiment, appropriate expression levels and protein loading were confirmed using GST and and #946; actin antibodies, respectively (lower panels)

Siva-1 and cytochrome C release. (A) HCT116 p53-/- cells were infected with either Siva-1 (Adeno-Siva-1) or GFP (Adeno-GFP) and treated as shown. Heavy membrane fraction lysates were analyzed for Siva-1 and COX IV expression (upper panels); corresponding whole cell lysate samples were analyzed for Siva-1 and GFP expression levels (lower panel). (B) Heavy membrane fractions (30 μg) from MCF-7 cells stably expressing either GFP or GFP-Bcl-xL[] were incubated with either 5 or 10 μg of SAH or control peptides. At 30 minutes, the fractions were analyzed for cytochrome C

Cisplatin induces p53 dependent Siva-1 expression. HCT116 colon cancer p53+/+ and -/- cell lines (gift of Bert Vogelstein, Johns Hopkins University) were treated with DMSO (0, Control) or 100 μM cisplatin (CDDP, Sigma), incubated for 16 hours. (A) Endogenous Siva-1 expression by SDS-PAGE and immunoblotting of lysates with Siva-1 polyclonal antibody. beta-actin served as a loading control. (B) Apoptosis by propidium iodide staining. Percentages indicate sub-G1 apoptotic DNA analyzed by flow cytometry (FACScaliber, BD Biosciences)