Survivin interacts with Smac/DIABLO in ovarian carcinoma cells but is redundant in Smac-mediated apoptosis.
Abnormalities in the control and execution of apoptosis are seen in many malignancies, including ovarian carcinoma. Many of these abnormalities involve the mitochondrial pathway of apoptosis, including overexpression of BIR-containing inhibitor of apoptosis protein (IAP) family proteins as well as dysregulated apoptosome function. We sought to stimulate the mitochondrial pathway of apoptosis by constructing a recombinant adenovirus encoding mature, processed Smac/DIABLO (Ad CMV tSmac), the second mitochondrial activator of caspases. Transfection of ovarian carcinoma cells with Ad CMV tSmac leads to increasing apoptosis in a dose-dependent manner. By contrast, transfection of IOSE397 immortalized normal ovarian surface epithelial cells does not cause apoptosis. We also show that the processed form of Smac is primarily expressed in the cytosol of ovarian carcinoma cells. Smac co-immunoprecipitates with both survivin and XIAP and stimulates survivin, but not XIAP, down-regulation. This down-regulation does not result from transcriptional changes, as determined by quantitative real-time PCR, but cycloheximide treatment indicates that survivin half-life is reduced from 6 to 2 h, which is secondary to ubiquitination and proteasomal degradation. RNA interference, however, suggests that survivin does not act to inhibit Smac-mediated apoptosis, which is confirmed by cotransfection with the phosphorylation mutant, survivin T34A. Finally, intraperitoneal delivery of Ad CMV tSmac increases median survival of mice bearing human ovarian carcinoma xenografts. We believe that expression of Smac/DIABLO can stimulate the intrinsic pathway of apoptosis in ovarian carcinoma without damaging normal ovarian tissue and therefore has therapeutic potential.
McNeish
I A
IA
Cancer Research UK Molecular Oncology Unit, Barts and the London School of Medicine, London EC1M 6BQ, United Kingdom. iain.mcneish@cancer.org.uk
Lopes
R
R
Bell
S J
SJ
McKay
T R
TR
Fernandez
M
M
Lockley
M
M
Wheatley
S P
SP
Lemoine
N R
NR
D017209:Apoptosis / Q000235:genetics*; D051017:Apoptosis Regulatory Proteins; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D015536:Down-Regulation / Q000235:genetics; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D005260:Female; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics / Q000494:pharmacology; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D007274:Injections, Intraperitoneal; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D009363:Neoplasm Proteins; D010051:Ovarian Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000628:therapy; D046988:Proteasome Endopeptidase Complex / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism; D015996:Survival Rate; D000077022:Survivin; D015854:Up-Regulation / Q000235:genetics; D051636:X-Linked Inhibitor of Apoptosis Protein
D051017:Apoptosis Regulatory Proteins; C107489:BIRC5 protein, human; D002352:Carrier Proteins; C412403:DIABLO protein, human; D051034:Inhibitor of Apoptosis Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D009363:Neoplasm Proteins; D011500:Protein Synthesis Inhibitors; D011506:Proteins; D000077022:Survivin; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D020169:Caspases; D046988:Proteasome Endopeptidase Complex