Radiation response and regulation of apoptosis induced by a combination of TRAIL and CHX in cells lacking mitochondrial DNA: A role for NF-κB–STAT3-directed gene expression
Authors: Hongning Zhou
Sarah X Huang
Yunfei Chai
Vladimir N Ivanov
Tom K Hei
Sally A Amundson
Shanaz A Ghandhi
Journal: Experimental Cell Research (2011)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: Mitochondrial DNA; ρ(0); NF-κB; STAT3; oxidative phosphorylation
Affiliations: Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, USA. 

Abstract:  Mitochondrial DNA depleted (ρ(0)) human skin fibroblasts (HSF) with suppressed oxidative phosphorylation were characterized by significant changes in the expression of 2100 nuclear genes, encoding numerous protein classes, in NF-κB and STAT3 signaling pathways, and by decreased activity of mitochondrial death pathway, compared to the parental ρ(+) HSF. In contrast, the extrinsic TRAIL/TRAIL-Receptor mediated death pathway remained highly active, and exogenous TRAIL in a combination with cycloheximide (CHX) induced higher levels of apoptosis in ρ(0) cells compared to ρ(+) HSF. Global gene expression analysis using microarray and qRT-PCR demonstrated that mRNA expression levels of many growth factors and their adaptor proteins (FGF13, HGF, IGFBP4, IGFBP6, and IGFL2), cytokines (IL6, ΙL17Β, ΙL18, ΙL19, and ΙL28Β) and cytokine receptors (IL1R1, IL21R, and IL31RA) were substantially decreased after mitochondrial DNA depletion. Some of these genes were targets of NF-κB and STAT3, and their protein products could regulate the STAT3 signaling pathway. Alpha-irradiation further induced expression of several NF-κB/STAT3 target genes, including IL1A, IL1B, IL6, PTGS2/COX2 and MMP12, in ρ(+) HSF, but this response was substantially decreased in ρ(0) HSF. Suppression of the IKK-NF-κB pathway by the small molecular inhibitor BMS-345541 and of the JAK2-STAT3 pathway by AG490 dramatically increased TRAIL-induced apoptosis in the control and irradiated ρ(+) HSF. Inhibitory antibodies against IL6, the main activator of JAK2-STAT3 pathway, added into the cell media, also increased TRAIL-induced apoptosis in HSF, especially after alpha-irradiation. Collectively, our results indicated that NF-κB activation was partially lost in ρ(0) HSF resulting in downregulation of the basal or radiation-induced expression of numerous NF-κB targets, further suppressing IL6-JAK2-STAT3 that in concert with NF-κB regulated protection against TRAIL-induced apoptosis. 

Retracted: No

PubPeer comments:
 ![file] , 
 Dear Ficus Calopilina, Thank you for your attention to our paper. This is comparison of Rho(+) and Rho (0) cells before and after radiation. To make this presentation more compact and representative, some unnecessary central lanes were removed from the images of original gels. The blue arrows on the western with p53-P(S35) and AKT-P (S473) are correctly indicate the boundary between Rho(+) and Rho (0) cells. The small central arrow on the COX2 western is also correct while two side arrows are not necessary. Minimal editing of original western images without change of the final conclusion was allowed at year 2011. , 
 #2  To make this presentation more compact and representative, some unnecessary central lanes were removed from the images of original gels.  Lanes were removed from some panels, but not others. How can you compare the panels? , 
 #2 Can the authors provide said u201cimages of the original gels?u201d , 
 Could the authors confirm whether the images in Figure 8 also had lanes removed? Partial view of 8, inverted, shown below. *** ![file] , 
 #3 In our experiments we used two doses of alpha-irradiation (0.5 Gy and 1Gy). However 1 Gy irradiation resulted in higher levels of cell death and we used for further experiments only 0.5 Gy. However, some western blots contained probes after 1 Gy irradiation. Finally, we removed these lanes from this presentation, due to their variability, and focused only on data at 0.5 Gy.The additional technical problem was with XIAP, where control samples were used twice and, finally, one control lane was removed. I highlighted the area of scans where cutting was made by dotted lines. I have attaches these figures. I am quite sorry that this minimal editing initiates some questions. However, 10 years ago certain degree of proper editing was allowed and there was no question from reviewers. I would like to thank you for your reply. Please, do not hesitate ask any additional questions. I will be happy to address these questions. ![file] ![file] , 
 #4 Scans of original western , 
 #5 Only at the position indicated with large arrow in the middle of the upper western. , 
 Data in this paper looks very similar to data in a 2008 paper. Experimental conditions are different. ![file] , 
 I am afraid to disappoint, but  NO . It was different experiments and different westerns. , 
 #9 I prepared this illustration to support your concern. After adjusting contrast and sizes to better match, I agree that these may be a repurposing of the same data. *** ![file] , 
 Thank you. I will check original scans... Yes, it was error in assembly for the second paper. I will fix it by repeating this western. Thank you again. , 
 #12 Dr. Ivanov: Thank you for responding. For my education: What changed between your response in #10 (u201cNOu201d) and #12 (u201cI will fixu201d)? If it was the editing (adjustments to dimensions, contrast and brightness) I did to the images so that they looked more similar, how can the mistake be attributed to figure assembly? Is it a common practice to edit the scanned gels prior to publication? I had the impression from other PubPeer discussions that that practice was viewed as inappropriate. , 
 #12 I still have no opportunity to repeat Western for COX-2 detection in Fig. 5B 2011 Exp Cell Res, where a wrong image with COX-2 was used. Hope to resolve this problem in the future. In contrast, there are no problem with COX-2 Western in Fig. 4B from Cancer Research 2008. Vladimir Ivanov , 
 #14 I am sorry for old text of the previous comment. Correction with a new COX-2 Western data for Fig. 5B of 2001 Exp Cell Res has been done some times ago and was published as my comment on PubPeer to our paper:  Mitochondrial function and nuclear factor-kappaB-mediated signaling in radiation-induced bystander effects Cancer Research (2008) - 2 Comments (PubPeer) pubmed: 18381429 doi: 10.1158 0008-5472.can-07-5278 issn: 1538-7445 issn: 0008-5472 Hongning Zhou , Vladimir N. Ivanov , Yu-Chin Lien , Mercy Davidson , Tom K. Hei Please see this comment with updated Fig. 5B (for Exp. Cell Res. 2001) , 
 #15 Exp Cell Res 2011 , 
 2021 Correction: https: pubmed.ncbi.nlm.nih.gov 34399973 The authors regret that several errors were found in this paper. Irradiation of cell cultures of u03c1+ and u03c10 HSF (human skin fibroblasts) was performed using u03b1-irradiation at 0.5 Gy. Several individual images of Western blots contained excessive information (bystander response of cells after alpha irradiation) that was not used in this paper. These needless lanes were removed for p53-P(S15) and AKT-P(S473) images in Fig. 5B. Vertical solid lines in updated Fig. 5B indicate a localization of image cutting. Western blots for XIAP and PTGS2 (COX2) were also removed, because they mistakenly used during assembly of Fig. 5B. , 
 Figure 6C appears to have a differential splice. *** ![file] , 
 Actin is present in mammalian cells at very high concentrations and therefore tends to partially merge on electropherograms. This is a very usual situation. ,