Abstract
The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent functions. The corepressor C-terminal binding protein (CtBP) interacts with ARF, resulting in proteasome-mediated degradation of CtBP. ARF can induce apoptosis in p53-null colon cancer cells, in a manner dependent on ARF interaction with CtBP. Bik was uniquely identified in an apoptotic gene array as coordinately upregulated in colon cancer cells after either CtBP2 knockdown or ARF overexpression. Validating the array findings, ARF induced Bik mRNA and protein expression, and this activity required an intact CtBP binding domain. Apoptosis induced by CtBP deficiency was substantially impaired when Bik expression was simultaneously silenced. An analysis of the Bik promoter revealed binding sites for the CtBP-interacting basic Kruppel-like factor (BKLF). A Bik promoter luciferase reporter was repressed by BKLF and CtBP2, and ARF reversed CtBP-associated repression. Chromatin immunoprecipitation analyses showed that CtBP was recruited to the Bik promoter largely by BKLF. Expression profiling of BH3-only gene expression in ARF-expressing or CtBP-deficient cells revealed that Bik was uniquely regulated by ARF/CtBP in colon cancer cells, whereas additional BH3-only proteins (Bim, Bmf) showed CtBP-dependent repression in osteosarcoma cells. ARF antagonism of CtBP repression of Bik and other BH3-only genes may have a critical role in ARF-induced p53-independent apoptosis and tumor suppression.
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Abbreviations
- ARF:
-
alternative reading frame
- CtBP:
-
C-terminal binding protein
- BH3:
-
Bcl-2 homology domain 3
- ChIP:
-
chromatin immunoprecipitation
- siRNA:
-
small interfering RNA
- shRNA:
-
short hairpin RNA
- PCR:
-
polymerase chain reaction
- BKLF:
-
basic Kruppel-like factor
- MEF:
-
mouse embryo fibroblast
- PARP:
-
poly (ADP-ribose) polymerase
- SDS:
-
sodium dodecyl sulfate
- 7-AAD:
-
7-aminoactinomycin D
- UV:
-
ultraviolet
- PBS:
-
phosphate buffered saline
- EDTA:
-
ethylenediamine tetra-acetic acid
- NADH:
-
nicotinamide adenine dinucleotide
- NP-40:
-
Nonidet P-40
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Acknowledgements
We thank Drs. T Kowalik for high-titer Ad-hARF and Ad-lacZ, E Campeau for lentiviral shRNA vectors, and G Chinnadurai for pBLCAT2 reporter construct. SRG was supported by an American Cancer Society Research Scholar Award and the Worcester Foundation for Biomedical Research. SP was supported by Our Danny Cancer Fund.
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Kovi, R., Paliwal, S., Pande, S. et al. An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis. Cell Death Differ 17, 513–521 (2010). https://doi.org/10.1038/cdd.2009.140
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DOI: https://doi.org/10.1038/cdd.2009.140
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