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• News Volume 1, Issue 3 pp 275-277

  Partners in death: a role for p73 and NF-kB in promoting apoptosis

  Relevance score: 8.055986

  Karen H. Vousden

  Keywords: p73, NF-kB, NOXA, genotoxic stress

  Published in Aging on March 28, 2009

• Research Paper Volume 1, Issue 3 pp 335-349

  Activation of p73 and induction of Noxa by DNA damage requires NF-kappa B

  Relevance score: 8.05309

  Angel G. Martin, Jason Trama, Diane Crighton, Kevin M. Ryan, Howard O. Fearnhead

  Keywords: apoptosis, p73, NF-κB B, Noxa

  Published in Aging on February 18, 2009

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  Although the transcription factor NF-κB is most clearly linked to the inhibition of extrinsic apoptotic signals such as TNFα by upregulating known anti-apoptotic genes, NF-κB has also been proposed to be required for p53-induced apoptosis in transformed cells. However, the involvement of NF-κB in this process is poorly understood. Here we investigate this mechanism and show that in transformed MEFs lacking NF-κB (p65-null cells) genotoxin-induced cytochrome c release is compromised. To further address how NF-κB contributes to apoptosis, gene profiling by microarray analysis of MEFs was performed, revealing that NF-κB is required for expression of Noxa, a pro-apoptotic BH3-only protein that is induced by genotoxins and that triggers cytochrome c release. Moreover, we find that in the absence of NF-κB, genotoxin treatment cannot induce Noxa mRNA expression. Noxa expression had been shown to be regulated directly by genes of the p53 family, like p73 and p63, following genotoxin treatment. Here we show that p73 is activated after genotoxin treatment only in the presence of NF-κB and that p73 induces Noxa gene expression through the p53 element in the promoter. Together our data provides an explanation for how loss of NF-κB abrogates genotoxin-induced apoptosis.

  

  (A) retrovirus-mediated reconstitution of p65 null MEFs with p65 restores NF-κB function as measured by EMSA. Wild type (wt), p65 null (vector) and p65 null reconstituted MEFs were stimulated with 10 ng/ml TNFα for 6 hr. Nuclear proteins were extracted and equal amounts of extract incubated with a radio-labeled NF-κB consensus probe. (B) p65 null cells are resistant to genotoxin-induced apoptosis. Cells were treated with 10 μM etoposide or 5 mJ UV-irradiation for 18 hr. Floating and attached cells were then collected and stained with propidium iodide (PI). DNA content was analyzed by flow cytometry. Results are presented as percentage of cells with sub-G₁ DNA content. The data shown represent the mean and SEM of three independent experiments. **statistically significant by student t-test analysis (p<0.05). (C) S-100 extracts from p65 null (vector) and reconstituted cells (p65) treated with 10 μM etoposide were used to assess caspase activity by cleavage (arbitrary fluorescence units per minute [AFU/min]) of the fluorogenic substrate, Ac-DEVD-afc. The data shown represent the mean and SEM of three independent experiments.

  
  
  

  

  (A) Expression of several key components of the apoptotic machinery in p65 null and reconstituted cells was compared. Expression of Apaf-1, caspase-2, cytochrome c, and XIAP was detected by immuno-blot. Expression of caspase-3 and -9 was assessed by RT-PCR from total RNA extracted from the cells as indicated. (B) S-100 extracts from p65 null (vector) and reconstituted cells (p65) were incubated with 1 mM ATP and 1 μM equine cytochrome c at 37 °C for 1 hr. Caspase activity was then assessed by cleavage (arbitrary fluorescence units per minute [AFU/min]) of the fluorogenic substrate, Ac-DEVD-afc. (C) caspase-9 processing by autoradiography. S-100 extracts were incubated under the conditions described above with in vitro translated caspase 9 and subjected to SDS-PAGE.

  
  
  

  

  (A) p65 null (vector) and reconstituted cells were treated with 10 μM etoposide or 5 mJ UV-irradiation in the presence of the caspase inhibitor, zVAD-fmk (50 μM) for 18 hr and then fixed and stained with a specific antibody for native cytochrome c. An Alexa Green coupled secondary antibody was used to reveal the localization of cytochrome c. (B) Results are expressed as percentage of cells showing cytosolic cytochrome c.

  
  
  

  

  (A) RT-PCR of bcl2 family members. cDNA was prepared from total RNA from p65 null (vector) and reconstituted cells (p65). Specific oligonucleotides for each gene (and three pairs for Noxa) were used to determine expression. GAPDH expression was used as a control. Bax expression was detected by immunoblot. (B) Northern Blot for Noxa after genotoxic treatments. Total RNA was extracted from p65 null and reconstituted cells after treatment with 10 μM etoposide or 5 mJ UV-irradiation for the times indicated. Expression of Noxa, and GAPDH as control, was revealed by blotting with specific radio-labeled probes. (C) Expression of Noxa sensitizes p65 null MEFs to genotoxic agents. Cloned murine Noxa was introduced into p65 null cells by retroviral transfer and sensitivity to etoposide and UV-irradiation compared. Noxa cloned in the anti-sense orientation was used as a control. After selection cells were treated with 10 μM etoposide or 5 mJ UV-irradiation for 24 hr and apoptosis assessed by flow cytometry as described in Figure 1. Results are representative of three different viral clones for both control and Noxa. Northern blotting confirmed Noxa expression.

  
  
  

  

  (A-B) 0.5 μg of PG13-Luc p53 luciferase reporter (A) or Noxa promoter luciferase reporter (B) were co-transfected into SaOS-2 cells along with increasing amounts of the wild type or P275R mutant p53 vectors. 48 hr after transfection luciferase activity was compared. Results are expressed as fold induction above mock (empty pcDNA3 vector) control. (C) p53 P275R or wild type expression was demonstrated by immunoblotting in extracts derived from SaOS-2 p53 tet-on cells transfected as described for A-B. As a control, p53 was induced by doxocycline treatment. Endogenous p21 induction was assessed by immunoblotting from the same extracts. A non-specific band detected with the p21 antibody was used as loading control.

  
  
  

  

  (A) SaOS-2 cells were transfected with 1 μg of pcDNA3 control vector, p73α or p73β expression vectors along with the following luciferase reporter plasmids: Noxa promoter reporter, Noxa promoter p53 mutant reporter, PG13-Luc p53 reporter or NF3TK-Luc NF-κB reporter (as control). 48 hr after transfection luciferase activity was compared. Results are expressed as fold induction above mock (empty pcDNA3 vector) control. (B) p73 activation in p65 null and reconstituted cells. Cells were treated with 10μM etoposide for 24 hr and p73 levels determined by immunoblotting with a pan-p73 antibody.

  
  
  

  

  p73β wild type was introduced into p65 null cells and p73β dominant negative was introduced into p65 reconstituted cells by retroviral transfer. Then apoptosis induction and Noxa expression was assessed after treatment with 10 μM etoposide for 18 hr. (A) Floating and attached cells were then collected and stained with propidium iodide (PI). DNA content was analyzed by flow cytometry. Results are presented as percentage of cells with sub-G₁ DNA content. The data shown represent the mean and SEM of three independent experiments. (B) S-100 extracts from p65 null (vector) and reconstituted cells (p65) were used to assess caspase activity by cleavage (arbitrary fluorescence units per minute [AFU/min]) of the fluorogenic substrate, Ac-DEVD-afc. The data shown represent the mean and SEM of three independent experiments. (C) Northern Blot for Noxa expression. Total RNA was extracted from p65 null (vector) and reconstituted (p65) cells after treatment with etoposide. Expression of Noxa was revealed by blotting with a specific radio-labeled probe. As loading control the ethidium bromide stained gel previous to transfer onto membrane is shown. This blot is representative of three independent experiments.