P16INK4A sensitizes human leukemia cells to FAS- and glucocorticoid-induced apoptosis via induction of BBC3/Puma and repression of MCL1 and BCL2
ABSTRACT Loss of CDKN2A/p16(INK4A) in hematopoietic stem cells is associated with enhanced self-renewal capacity and might facilitate progression of damaged stem cells into pre-cancerous cells that give rise to leukemia. This is also reflected by the frequent loss of the INK4A locus in acute lymphoblastic T-cell leukemia. T-cell acute lymphoblastic leukemia cells designed to conditionally express p16(INK4A) arrest in the G(0)/G(1) phase of the cell cycle and show increased sensitivity to glucocorticoid- and tumor necrosis factor receptor superfamily 6-induced apoptosis. To investigate the underlying molecular mechanism for increased death sensitivity, we interfered with specific steps of apoptosis signaling by expression of anti-apoptotic proteins. We found that alterations in cell death susceptibility resulted from changes in the composition of pro- and anti-apoptotic BCL2 proteins, i.e. repression of MCL1, BCL2, and PMAIP1/Noxa and the induction of pro-apoptotic BBC3/Puma. Interference with Puma induction by short hairpin RNA technology or retroviral expression of MCL1 or BCL2 significantly reduced both glucocorticoid- and FAS-induced cell death in p16(INK4A)-reconstituted leukemia cells. These results suggest that Puma, in concert with MCL1 and BCL2 repression, critically mediates p16(INK4A)-induced death sensitization and that in human T-cell leukemia the deletion of p16(INK4A) confers apoptosis resistance by shifting the balance of pro- and anti-apoptotic BCL2 proteins toward apoptosis protection.
- SourceAvailable from: Judith Hagenbuchner
T-Cell Leukemia, Edited by Olga Babusikova, Sinisa Dovat, Kimberly J. Payne, 01/2011; InTech., ISBN: 978-953-307-400-9
- "Retrovirally transduced Survivin compensates for the loss of the endogenous protein in CEM/p16-Survivin cells. Re-expression of p16INK4A accelerates death-receptor-induced (anti-FAS anibody, 0.1 mg/ml, for four hours) and glucocorticoidinduced apoptosis (10 nM dexamethasone, 24 hours) as shown in B und C (Obexer et al, 2009a). Ectopic expression of Survivin did not change the sensitivity to dexamethasone (C), but prevents increased sensitivity to Fas-induced apoptosis (B). "
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ABSTRACT: Neuroblastoma is the most frequent extracranial solid tumor in children. Here, we report that the proteasome inhibitor bortezomib (PS-341, Velcade) activated the pro-apoptotic BH3-only proteins PMAIP1/Noxa and BBC3/Puma and induced accumulation of anti-apoptotic MCL1 as well as repression of anti-apoptotic BCL2L1/Bcl-xL. Retroviral expression of Bcl-xL, but not of MCL1, prevented apoptosis by bortezomib. Gene knockdown of Noxa by shRNA technology significantly reduced apoptosis, whereas Puma knockdown did not affect cell death kinetics. Immunoprecipitation revealed that endogenous Noxa associated with both, Bcl-xL and MCL1, suggesting that in neuronal cells Noxa can neutralize Bcl-xL, explaining the pronounced protective effect of Bcl-xL. Tetracycline-regulated Noxa expression did not trigger cell death per se but sensitized to bortezomib treatment in a dose-dependent manner. This implies that the induction of Noxa is necessary but not sufficient for bortezomib-induced apoptosis. We conclude that MCL1 steady-state expression levels do not affect sensitivity to proteasome-inhibitor treatment in neuronal tumor cells, and that both the repression of Bcl-xL and the activation of Noxa are necessary for bortezomib-induced cell death.Journal of Biological Chemistry 03/2010; 285(10):6904-12. DOI:10.1074/jbc.M109.038331 · 4.57 Impact Factor
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ABSTRACT: The tumor suppressor p16(INK4a) has earned widespread attention in cancer studies since its discovery as an inhibitor of cyclin-dependent kinases (CDKs) 4/6. Structurally, it consists of four complete ankyrin repeats, believed to be involved in CDK4 interaction. According to the previous disparities concerning the importance of domains and inactivating mutations in p16, we aimed to search for the domain possessing the functional properties of the full length protein. Upon our in silico screening analyses followed by experimental assessments, we have identified the novel minimum functional domain of p16 to be the C-terminal half including ankyrin repeats III, IV and the C-terminal flanking region accompanied by loops 2 and 3. Transfection of this truncated form into HT-1080 human fibrosarcoma cells, lacking endogenous p16, revealed that it is able to inhibit cell growth and proliferation equivalent to p16(INK4a). The functional analysis showed that this fragment like p16 can interact with CDK4/6, block the entry into S phase of the cell cycle and suppress growth as indicated by colony formation assay. Identification of p16 minimum functional domain can be of benefit to the future peptidomimetic drug design as well as gene transfer for cancer therapy.Journal of Cellular Biochemistry 12/2010; 111(6):1598-606. DOI:10.1002/jcb.22892 · 3.37 Impact Factor