Reptin Regulates DNA Double Strand Breaks Repair in Human Hepatocellular Carcinoma

<div><p>Reptin/RUVBL2 is overexpressed in most hepatocellular carcinomas and is required for the growth and viability of HCC cells. Reptin is involved in several chromatin remodeling complexes, some of which are involved in the detection and repair of DNA damage, but data on Reptin involvement in the repair of DNA damage are scarce and contradictory. Our objective was to study the effects of Reptin silencing on the repair of DNA double-strand breaks (DSB) in HCC cells. Treatment of HuH7 cells with etoposide (25 μM, 30 min) or γ irradiation (4 Gy) increased the phosphorylation of H2AX by 1.94 ± 0.13 and 2.0 ± 0.02 fold, respectively. These values were significantly reduced by 35 and 65 % after Reptin silencing with inducible shRNA. Irradiation increased the number of BRCA1 (3-fold) and 53BP1 foci (7.5 fold). Depletion of Reptin reduced these values by 62 and 48%, respectively. These defects in activation and/or recruitment of repair proteins were not due to a decreased number of DSBs as measured by the COMET assay. All these results were confirmed in the Hep3B cell line. Protein expression of ATM and DNA-PKcs, the major H2AX kinases, was significantly reduced by 52 and 61 % after Reptin depletion whereas their mRNA level remained unchanged. Phosphorylation of Chk2, another ATM target, was not significantly altered. Using co-immunoprecipitation, we showed an interaction between Reptin and DNA-PKcs. The half-life of newly-synthesized DNA-PKcs was reduced when Reptin was silenced. Finally, depletion of Reptin was synergistic with etoposide or γ irradiation to reduce cell growth and colony formation. In conclusion, Reptin is an important cofactor for the repair of DSBs. Our data, combined with those of the literature suggests that it operates at least in part by regulating the expression of DNA-PKcs by a stabilization mechanism. Overexpression of Reptin in HCC could be a factor of resistance to treatment, consistent with the observed overexpression of Reptin in subgroups of chemo-resistant breast and ovarian cancers.</p></div

Reptin depletion reduces the recruitment on chromatin of BRCA1 and 53BP1 after gamma ray irradiation.

<p>HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated with doxycycline (sh Reptin +) or left untreated (sh Reptin-) for 4 days. (A) Representative images of 53BP1 and BRCA1 foci in HuH7 cells detected using immunofluorescence 2h after gamma ray irradiation. (B) The bars represent the mean number of foci per cell from two independent experiments (>200 cells were counted per experiment). Expression levels of BRCA1 and 53BP1 were assessed by Western Blot on whole cell extracts 4 days after doxycycline treatment. A representative picture is shown in (C). The migration positions of molecular weight standards (in kDa) are indicated on the left. (D) Quantification of 3 Western blot experiments.</p

Reptin depletion and DNA damage cooperate to reduce cell viability.

<p>(A) HuH7 cells transfected with a control siRNA were either left untreated (yellow) or received etoposide (ETO, green) after 3 days. Similarly, cells transfected with an anti-Reptin siRNA (siR2) were untreated (blue) or received etoposide (red). The effect on cell number was determined at 1, 3, 4 and 5 days after seeding, using the MTS assay. The results are the mean ± SD of 3 independent experiments. After 5 days, the difference between siR2 and siControl without treatment was significant with a p value <0.001 and the difference between untreated siR2 and treated siR2 was significant with a p value <0.05 (Two-way ANOVA followed by Bonferroni test). (B) Same design as in (A) except that DNA damage was induced with γ-irradiation (IRR). The figure shows the mean of 2 independent experiments. (C) Clonogenic tests were carried out as described in Materials and Methods using HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA that were either treated with doxycycline (sh Rep +) or untreated (sh Rep-). The graph shows the mean of 3 replicates (*** p<0.001 and ** p<0.01 by One-way ANOVA followed by Bonferroni test).</p

Reptin depletion impairs H2AX phosphorylation at serine 139 (γH2AX) after DNA damage.

<p>(A) and (B) HuH7 cells were transfected with a control or a Reptin siRNA. After 3 days, they were then treated with etoposide (ETO, 25 μM) (A) or exposed to gamma radiation (IRR, 4 Gy) (B), stained for γ-H2AX and analyzed by flow cytometry. The bar graphs show the mean of 3 experiments (*** p<0.001 by One-way ANOVA followed by Bonferroni test). (C) and (D) HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated with doxycycline (sh Reptin +) or left untreated (sh Reptin-) for 4 days. Extracts from HuH7 cells treated with etoposide as in (A) were analyzed by Western blot with an anti-phospho-H2AX antibody (C). Data were normalized relative to the Sypro Ruby staining of the membrane. The graph on the right shows the mean of 9 experiments (*** p<0.001 by One-way ANOVA followed by Bonferroni test). (D) Control or Reptin depleted HuH7 cells were treated with etoposide or irradiated, then fixed at different time points and immunostained for phospho-H2AX. The graphs below show for each treatment the evolution of the number of foci per cell (left) or the fractional decrease of the number of foci, setting the initial time point as 100% (right).</p

Effect of Reptin depletion on signaling intermediates upstream of H2AX phosphorylation.

<p>(A) Protein expression of DNA-PKcs and ATM after Reptin depletion were analyzed by Western Blot on whole cell extracts 4 days after Reptin silencing with doxycycline (sh Rep +) (n = 6 and n = 3 for DNA-PKcs and ATM, respectively: * p<0.05 by Mann and Whitney test) (B) Phosphorylation of CHK2 after Reptin depletion and 2h after gamma ray irradiation (+ IRR) was detected by Western Blot and normalized on total CHK2 (n = 4: *** p<0.001 by One-way ANOVA followed by Bonferroni test) (C) RNA was extracted 4 days after Reptin silencing and expression of DNA-PKcs and ATM mRNAs was analyzed by RT-qPCR. (n = 3).</p

Reptin interacts with DNA-PKcs and regulates its stability.

<p>(A) Interaction between Reptin and DNA-PKcs was tested by immunoprecipitation. The migration positions of molecular weight standards (in kDa) are indicated on the left. The faint band seen in the IgG lane with the Reptin antibody corresponds to traces of IgG heavy chains. The picture is representative of 3 similar experiments. (B) Metabolic labeling and pulse chase. HuH7 cells stably expressing a doxycycline-inducible Reptin shRNA were treated (sh Reptin) or not (Control) with doxycycline. After 4 days, they were labeled with EXPRE³⁵S³⁵S as described in Materials and Methods. Following the indicated periods of chase, DNA-PKcs was immunoprecipitated and the eluates were separated on SDS-PAGE. The top panel shows the autoradiographic image, and the bottom one the Coomassie blue staining of the gel with DNA-PKcs. (C) The graph shows the quantitative analysis of the data following normalization of the autoradiographic signal on the amount of immunoprecipitated DNA-PKcs.</p

Reptin depletion does not reduce the number of double strand breaks.

<p>HuH7 cells were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123333#pone.0123333.g001" target="_blank">Fig 1</a>. The neutral COMET assay was used to assess DNA double-strand breaks that were quantified using calculation of the comet tail moment. Bars represent the mean ±SD from 3 independent experiments (>140 cells were counted per experiment). Left, etoposide treatment, right, γ irradiation. * p<0.05, ** p<0.01 and *** p<0.001 by One-way ANOVA followed by Bonferroni test. Representative COMET images are shown below.</p

HERP-R50H stabilizes Herp endogenous and increases cell toxicity.

<p>(<b>A</b>) Immunoblot of 1AT, HRD1, BiP and Hsp90 protein expression in cell lysates following siRNA-mediated silencing of HERP and HRD1 in Z-IB3 cells. Tunicamycin (Tun) treatment at 2 μg/ml for 24 hours is used as positive control. Quantitative analyses of the immature (black bar graph) and mature (white bar graph) forms of Z-1AT after HERP and HRD1 silencing relative to scrambled control (Scr). Data denote the -fold change in the protein expression of the indicated Z-1AT forms relative to Scr (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels, the * indicate <i>p</i> < 0.05 as determined by two-tailed t-test using Scr as reference. (<b>B</b>) Effect of HRD1 and HERP silencing on steady state levels of Z-1AT in Z-IB3 cells. Cell homogenates were separated into insoluble (left) and soluble (right) fractions and these fractions were then subjected to immunoblot analysis for 1AT (top) and GAPDH (bottom). (<b>C</b>) Percentage of live cells was estimated as described in Materials and methods upon pcDNA, HERP-WT or HERP-R50H overexpression at 48 h after transfection. Data are presented as a ratio relative to control pcDNA empty vector (mean ± SD, n = 3 independent experiments). The asterisk indicates p < 0.05 as determined by two-tailed t-test using pcDNA empty vector as the reference. (<b>D</b>) Immunoblot of Flag tag (upper) and HERPUD1 (Herp) protein expression (lower) in cell lysates following cycloheximide (CHX) treatment. Black arrowhead indicates Flag-Herp expression bands and grey arrowhead indicates endogenous (Endo)-Herp expression bands. Z-IB3 cells were transfected with plasmids encoding the empty vector (pcDNA), HERP-WT, HERP-R50H and HERP-K61R mutation. At 48 h after transfection, 50 μg/ml cycloheximide (CHX) were added and the chase was performed in the absence or presence of CHX for the indicated times. Quantitative analysis of the cycloheximide-chase experiments for the Flag tag (left) and Herp endogenous (right) upon pcDNA, HERP-WT, HERP-R50H or HERP-K61R overexpression is shown. Data shown denote the -fold change relative to <i>t</i> = 0 (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels asterisks indicate <i>p</i> < 0.05 as determined by two-way ANOVA followed by Bonferroni post-test.</p

Genotypes of Herp and HFE variants among ZZ individuals who developed or not liver symptoms.

<p>Genotypes of Herp and HFE variants among ZZ individuals who developed or not liver symptoms.</p

HRD1 silencing affects the viability, the traffic and secretion of human cells lines expressing the Z variant.

<p>(<b>A</b>) Viability is measured using GF-AFC Substrate. This compound can enter live cells where it is cleaved by the live-cell protease to release AFC. WT and Z-IB3 cells were treated as described in Material and methods. Quantitative analysis of candidate genes silencing on viability in WT or Z-IB3 cells (mean ± S.D., n = 3 independent experiments). The * indicates p < 0.05 as determined by two-tailed t-test using WT-IB3 cells as reference. (<b>B</b>) Immunoblot analysis of 1AT and Hsp90 protein expression in cell lysates and culture media following siRNA-mediated silencing of the candidate genes HDAC2 and HRD1 in WT and Z-IB3 cells. Traffic of the 1AT glycoprotein through the secretory pathway can be monitored by a change in its migration on SDS-PAGE in response to the processing of ER-acquired N-linked oligosaccharides (the immature form: I) during trafficking through the Golgi to generate the slower migrating, mature glycoform (Mat). The latter is secreted in the serum (secreted form: S) by the cell. Quantitative analyses of the immature, mature, and secreted forms of WT-1AT (black bar graph) and Z-1AT (white bar graph) after candidate genes silencing relative to scrambled control (Scr). Data denote the -fold change in the protein expression of the indicated WT or Z-1AT forms relative to Scr (mean ± S.D., <i>n</i> = 3 independent experiments). In all panels, the * indicates <i>p</i> < 0.05 as determined by two-tailed t-test using Scr as reference.</p