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Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.

Volk A, Li J, Xin J, You D, Zhang J, Liu X, Xiao Y, Breslin P, Li Z, Wei W, Schmidt R, Li X, Zhang Z, Kuo PC, Nand S, Zhang J, Chen J, Zhang J - J. Exp. Med. (2014)

Bottom Line: We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC.We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway.Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, Department of Biology, Loyola University Chicago, Chicago, IL 60660.

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Tnf receptor endocytosis and Mkps regulate Jnk activity in LC and HSPC. (A) HSPC and LC were stimulated with TNF for the indicated times (in minutes). Jnk activity was examined by Western blot and the normalized ratios of p-Jnk levels were quantified in bar graph to the right. Arbitrary units are normalized to vehicle-treated lane. (B) LCs endocytose Tnfr1 in response to Tnf stimulation as determined by mean fluorescence intensity by flow cytometry. (C) HSPC and LC were pretreated with Dynasore followed by Tnf for the indicated times. Jnk activity was examined by Western blot. (D) LCs were treated with Tnf and/or Dynasore overnight, and assayed the next day for cell death by Annexin V/PI analysis. (E) Basal expression of Mkps was compared between HSPC and LC by qRT-PCR. (F) HSPC and LC were treated with Tnf for the indicated times. Expression of Jnk-specific Mkps were examined by qRT-PCR. Transcript levels were normalized to the corresponding basal levels. (G) shRNA knockdown of Mkps 1, 3, and 5 in LC was evaluated by qRT-PCR. (H) LCs were transduced with shRNA-specific Mkp1, Mkp3, or Mkp5, respectively. The transduced cells were treated with the indicated dosages of Tnf, and Jnk activity was examined by Western blotting of p-Jnk. Quantification of p-Jnk Western blot analysis is shown to the right. Arbitrary units are normalized to vehicle treated lane. (I) The CFU of Mkp1, Mkp3, and Mkp5 shRNA-transduced LCs were examined after Tnf treatment. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 when compared with vehicle-treated/shSCR control as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with the indicated conditions. A, B, C, and H are representative results from three independent trials.
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fig5: Tnf receptor endocytosis and Mkps regulate Jnk activity in LC and HSPC. (A) HSPC and LC were stimulated with TNF for the indicated times (in minutes). Jnk activity was examined by Western blot and the normalized ratios of p-Jnk levels were quantified in bar graph to the right. Arbitrary units are normalized to vehicle-treated lane. (B) LCs endocytose Tnfr1 in response to Tnf stimulation as determined by mean fluorescence intensity by flow cytometry. (C) HSPC and LC were pretreated with Dynasore followed by Tnf for the indicated times. Jnk activity was examined by Western blot. (D) LCs were treated with Tnf and/or Dynasore overnight, and assayed the next day for cell death by Annexin V/PI analysis. (E) Basal expression of Mkps was compared between HSPC and LC by qRT-PCR. (F) HSPC and LC were treated with Tnf for the indicated times. Expression of Jnk-specific Mkps were examined by qRT-PCR. Transcript levels were normalized to the corresponding basal levels. (G) shRNA knockdown of Mkps 1, 3, and 5 in LC was evaluated by qRT-PCR. (H) LCs were transduced with shRNA-specific Mkp1, Mkp3, or Mkp5, respectively. The transduced cells were treated with the indicated dosages of Tnf, and Jnk activity was examined by Western blotting of p-Jnk. Quantification of p-Jnk Western blot analysis is shown to the right. Arbitrary units are normalized to vehicle treated lane. (I) The CFU of Mkp1, Mkp3, and Mkp5 shRNA-transduced LCs were examined after Tnf treatment. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 when compared with vehicle-treated/shSCR control as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with the indicated conditions. A, B, C, and H are representative results from three independent trials.

Mentions: Studies show that the contradictory functions of the Jnk signal stimulated by Tnf are in part determined by the duration of Jnk activity. A short duration (<2 h) promotes survival/proliferation, whereas longer duration (>2 h) promotes cell death (Sakon et al., 2003; Liu and Lin, 2005). We found that Jnk signal duration is limited in LC compared with HSPC in response to TNF stimulation (Fig. 5 A). We predicted that extending the duration of Tnf–induced Jnk activation might convert the survival signal into death signal in LC.


Co-inhibition of NF-κB and JNK is synergistic in TNF-expressing human AML.

Volk A, Li J, Xin J, You D, Zhang J, Liu X, Xiao Y, Breslin P, Li Z, Wei W, Schmidt R, Li X, Zhang Z, Kuo PC, Nand S, Zhang J, Chen J, Zhang J - J. Exp. Med. (2014)

Tnf receptor endocytosis and Mkps regulate Jnk activity in LC and HSPC. (A) HSPC and LC were stimulated with TNF for the indicated times (in minutes). Jnk activity was examined by Western blot and the normalized ratios of p-Jnk levels were quantified in bar graph to the right. Arbitrary units are normalized to vehicle-treated lane. (B) LCs endocytose Tnfr1 in response to Tnf stimulation as determined by mean fluorescence intensity by flow cytometry. (C) HSPC and LC were pretreated with Dynasore followed by Tnf for the indicated times. Jnk activity was examined by Western blot. (D) LCs were treated with Tnf and/or Dynasore overnight, and assayed the next day for cell death by Annexin V/PI analysis. (E) Basal expression of Mkps was compared between HSPC and LC by qRT-PCR. (F) HSPC and LC were treated with Tnf for the indicated times. Expression of Jnk-specific Mkps were examined by qRT-PCR. Transcript levels were normalized to the corresponding basal levels. (G) shRNA knockdown of Mkps 1, 3, and 5 in LC was evaluated by qRT-PCR. (H) LCs were transduced with shRNA-specific Mkp1, Mkp3, or Mkp5, respectively. The transduced cells were treated with the indicated dosages of Tnf, and Jnk activity was examined by Western blotting of p-Jnk. Quantification of p-Jnk Western blot analysis is shown to the right. Arbitrary units are normalized to vehicle treated lane. (I) The CFU of Mkp1, Mkp3, and Mkp5 shRNA-transduced LCs were examined after Tnf treatment. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 when compared with vehicle-treated/shSCR control as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with the indicated conditions. A, B, C, and H are representative results from three independent trials.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4042653&req=5

fig5: Tnf receptor endocytosis and Mkps regulate Jnk activity in LC and HSPC. (A) HSPC and LC were stimulated with TNF for the indicated times (in minutes). Jnk activity was examined by Western blot and the normalized ratios of p-Jnk levels were quantified in bar graph to the right. Arbitrary units are normalized to vehicle-treated lane. (B) LCs endocytose Tnfr1 in response to Tnf stimulation as determined by mean fluorescence intensity by flow cytometry. (C) HSPC and LC were pretreated with Dynasore followed by Tnf for the indicated times. Jnk activity was examined by Western blot. (D) LCs were treated with Tnf and/or Dynasore overnight, and assayed the next day for cell death by Annexin V/PI analysis. (E) Basal expression of Mkps was compared between HSPC and LC by qRT-PCR. (F) HSPC and LC were treated with Tnf for the indicated times. Expression of Jnk-specific Mkps were examined by qRT-PCR. Transcript levels were normalized to the corresponding basal levels. (G) shRNA knockdown of Mkps 1, 3, and 5 in LC was evaluated by qRT-PCR. (H) LCs were transduced with shRNA-specific Mkp1, Mkp3, or Mkp5, respectively. The transduced cells were treated with the indicated dosages of Tnf, and Jnk activity was examined by Western blotting of p-Jnk. Quantification of p-Jnk Western blot analysis is shown to the right. Arbitrary units are normalized to vehicle treated lane. (I) The CFU of Mkp1, Mkp3, and Mkp5 shRNA-transduced LCs were examined after Tnf treatment. Results shown are mean values ± 1 SD from three independent trials. * indicates P < 0.05 when compared with vehicle-treated/shSCR control as determined by Student’s t test two-tailed analysis. # indicates P < 0.05 significant difference when compared with the indicated conditions. A, B, C, and H are representative results from three independent trials.
Mentions: Studies show that the contradictory functions of the Jnk signal stimulated by Tnf are in part determined by the duration of Jnk activity. A short duration (<2 h) promotes survival/proliferation, whereas longer duration (>2 h) promotes cell death (Sakon et al., 2003; Liu and Lin, 2005). We found that Jnk signal duration is limited in LC compared with HSPC in response to TNF stimulation (Fig. 5 A). We predicted that extending the duration of Tnf–induced Jnk activation might convert the survival signal into death signal in LC.

Bottom Line: We determined that TNF stimulation drives the JNK-AP1 pathway in a manner parallel to NF-κB, leading to the up-regulation of anti-apoptotic genes in LC.We found that we can significantly sensitize LC to NF-κB inhibitor treatment by blocking the TNF-JNK-AP1 signaling pathway.Our data suggest that co-inhibition of both TNF-JNK-AP1 and NF-κB signals may provide a more comprehensive treatment paradigm for AML patients with TNF-expressing LC.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Biology Program, Department of Biology, Loyola University Chicago, Chicago, IL 60660.

Show MeSH
Related in: MedlinePlus