title: Reptin regulates DNA double strand breaks repair in human hepatocellular carcinoma.
abstract: 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.
authors: INSERM, U1053, F-33076 Bordeaux, France; Université de Bordeaux, F 33076, Bordeaux, France.
authors: Anne-Aurélie
authors: AA
authors: Raymond
authors: INSERM, U1053, F-33076 Bordeaux, France; Université de Bordeaux, F 33076, Bordeaux, France.
authors: Samira
authors: S
authors: Benhamouche
authors: INSERM, U1053, F-33076 Bordeaux, France; Université de Bordeaux, F 33076, Bordeaux, France.
authors: Véronique
authors: V
authors: Neaud
authors: INSERM, U1053, F-33076 Bordeaux, France; Université de Bordeaux, F 33076, Bordeaux, France.
authors: Julie
authors: J
authors: Di Martino
authors: INSERM, U1053, F-33076 Bordeaux, France; Université de Bordeaux, F 33076, Bordeaux, France.
authors: Joaquim
authors: J
authors: Javary
authors: INSERM, U1053, F-33076 Bordeaux, France; Université de Bordeaux, F 33076, Bordeaux, France.
authors: Jean
authors: J
authors: Rosenbaum
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded; D004265:DNA Helicases / Q000235:genetics* / Q000378:metabolism; D004260:DNA Repair / Q000235:genetics*; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D005720:Gamma Rays; D006657:Histones / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D011485:Protein Binding; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D000972:Antineoplastic Agents, Phytogenic; D002352:Carrier Proteins; C468783:H2AX protein, human; D006657:Histones; D009687:Nuclear Proteins; D005047:Etoposide; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C122079:RUVBL2 protein, human