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Published on: March 5, 2018
Apoptosis induced by the nuclear death domain protein p84N5 is associated with caspase-6 and NF-kappa B activation
J Doostzadeh-Cizeron1, S Yin, D W Goodrich
1Department of Cancer Biology, University of Texas M.D. Anderson Cancer Center, Houston 77030, USA.
Abstract:
Although the mechanisms involved in responses to extracellular or mitochondrial apoptotic signals have received considerable attention, the mechanisms utilized within the nucleus to transduce apoptotic signals are not well understood. We have characterized apoptosis induced by the nuclear death domain-containing protein p84N5. Adenovirus-mediated N5 gene transfer or transfection of p84N5 expression vectors induces apoptosis in tumor cell lines with nearly 100% efficiency as indicated by cellular morphology, DNA fragmentation, and annexin V staining. Using peptide substrates and Western blotting, we have determined that N5-induced apoptosis is initially accompanied by activation of caspase-6. Activation of caspases-3 and -9 does not peak until 3 days after the peak of caspase-6 activity. Expression of p84N5 also leads to activation of NF-kappaB as indicated by nuclear translocation of p65RelA and transcriptional activation of a NF-kappaB-dependent reporter promoter. Changes in the relative expression level of Bcl-2 family proteins, including Bak and Bcl-Xs, are also observed during p84N5-induced apoptosis. Finally, we demonstrate that p84N5-induced apoptosis does not require p53 and is not inhibited by p53 coexpression. We propose that p84N5 is involved in an apoptotic pathway distinct from those triggered by death domain-containing receptors or by p53.
Insights
The nuclear protein p84N5 effectively induces apoptosis in tumor cells via a novel pathway. This process involves caspase-6 activation and is independent of the p53 tumor suppressor.
Area of Science:
- Molecular Biology
- Cell Biology
- Cancer Research
Background:
- Mechanisms of nuclear apoptosis signaling remain poorly understood.
- Extracellular and mitochondrial apoptotic pathways are well-characterized.
- The role of nuclear proteins in apoptosis requires further investigation.
Purpose of the Study:
- To characterize the apoptotic pathway induced by the nuclear protein p84N5.
- To elucidate the molecular mechanisms underlying p84N5-mediated apoptosis.
- To determine if p84N5-induced apoptosis is p53-dependent.
Main Methods:
- Adenovirus-mediated gene transfer and transfection of p84N5 expression vectors.
- Assessment of apoptosis using cellular morphology, DNA fragmentation, and annexin V staining.
- Analysis of caspase activation (caspase-6, -3, -9) via peptide substrates and Western blotting.
- Evaluation of NF-kappaB activation through p65RelA nuclear translocation and reporter assays.
- Monitoring changes in Bcl-2 family protein expression (Bak, Bcl-Xs).
Main Results:
- p84N5 expression induced apoptosis in tumor cell lines with high efficiency.
- N5-induced apoptosis was characterized by initial caspase-6 activation, followed by caspases-3 and -9.
- p84N5 expression led to NF-kappaB activation and altered Bcl-2 family protein levels.
- p84N5-induced apoptosis was independent of p53 and unaffected by p53 coexpression.
Conclusions:
- p84N5 is a potent inducer of apoptosis in tumor cells.
- p84N5 activates a distinct apoptotic pathway involving early caspase-6 activation and NF-kappaB signaling.
- This p84N5-mediated apoptotic pathway is independent of the p53 tumor suppressor.
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