title: Targeting Rad50 sensitizes human nasopharyngeal carcinoma cells to radiotherapy.
abstract: BACKGROUND
  The Mre11-Rad50-Nbs1 (MRN) complex is well known for its crucial role in initiating DNA double strand breaks (DSBs) repair pathways to resistant irradiation (IR) injury and thus facilitating radioresistance which severely reduces radiocurability of nasopharyngeal cancer (NPC). Targeting native cellular MRN function would sensitize NPC cells to IR.
  
  
  METHODS
  A recombinant adenovirus containing a mutant Rad50 gene (Ad-RAD50) expressing Rad50 zinc hook domain but lacking the ATPase domain and the Mre11 interaction domain was constructed to disrupt native cellular MRN functions. The effects of Ad-RAD50 on the MRN functions were assessed in NPC cells lines using western blot, co-immunoprecipitation and confocal microscopy analyses. The increased radiosensitivity of transient Ad-RAD50 to IR was examined in NPC cells, including MTT assay, colony formation. The molecular mechanisms of radiosensitization were confirmed by neutral comet assay and western bolts. Nude mice subcutaneous injection, tumor growth curve and TUNEL assay were used to evaluate tumor regression and apoptosis in vivo.
  
  
  RESULTS
  Rad50 is remarkably upregulated in NPC cells after IR, implying the critical role of Rad50 in MRN functions. The transient expression of this mutant Rad50 decreased the levels of native cellular Rad50, Mre11 and Nbs1, weakened the interactions among these proteins, abrogated the G2/M arrest induced by DSBs and reduced the DNA repair ability in NPC cells. A combination of IR and mutant RAD50 therapy produced significant tumor cytotoxicity in vitro, with a corresponding increase in DNA damage, prevented proliferation and cell viability. Furthermore, Ad-RAD50 sensitized NPC cells to IR by causing dramatic tumor regression and inducing apoptosis in vivo.
  
  
  CONCLUSIONS
  Our findings define a novel therapeutic approach to NPC radiosensitization via targeted native cellular Rad50 disruption.
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. lillychang121719@126.com.
authors.forename: Lihong
authors.initials: L
authors.lastname: Chang
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. sallot33@hotmail.com.
authors.forename: Jiancong
authors.initials: J
authors.lastname: Huang
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. wangkai.sysu@163.com.|Department of Otorhinolaryngology-Head & Neck Surgery, The First People's Hospital of Foshan, Cancheng District, NO.81 Lingnan Bei Road, Foshan, 528000, China. wangkai.sysu@163.com.
authors.forename: Kai
authors.initials: K
authors.lastname: Wang
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. li-jingjia@hotmail.com.
authors.forename: Jingjia
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. razingyan@126.com.|Department of Otolaryngology-Head & Neck Surgery, Zengcheng District People's Hospital of Guangzhou (Boji-Affiliated Hospital of Sun Yat-sen University), Zengcheng District, NO.1 Guangming Dong Road, Guangzhou, 511300, China. razingyan@126.com.
authors.forename: Ruicheng
authors.initials: R
authors.lastname: Yan
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. ling0412@126.com.|Department of Otolaryngology-Head & Neck Surgery, Nanhai Maternity and Child Healthcare Hospital, Nanhai District, NO.6 Guiping Xi Road, Foshan, 528000, China. ling0412@126.com.
authors.forename: Ling
authors.initials: L
authors.lastname: Zhu
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. entyj@126.com.
authors.forename: Jin
authors.initials: J
authors.lastname: Ye
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. wuxifuent@163.com.
authors.forename: Xifu
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. zhuangsm05@163.com.|Department of Otolaryngology-Head & Neck Surgery, The Sixth Affiliated Hospital of Sun Yat-sen University, NO.26 Yuancun Erheng Road, Guangzhou, 510655, China. zhuangsm05@163.com.
authors.forename: Shimin
authors.initials: S
authors.lastname: Zhuang
authors.affiliation: Department of Otorhinolaryngology-Head & Neck Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. lidaqing@mail.med.upenn.edu.
authors.forename: Daqing
authors.initials: D
authors.lastname: Li
authors.affiliation: Department of Otolaryngology-Head & Neck Surgery, The Third Affiliated Hospital, Sun Yat-sen University, NO.600 Tianhe Road, Guangzhou, 510630, China. gehuazh@hotmail.com.
authors.forename: Gehua
authors.initials: G
authors.lastname: Zhang
mesh_terms: D017766:Acid Anhydride Hydrolases; D000818:Animals; D017209:Apoptosis; D002277:Carcinoma; D018797:Cell Cycle Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded / Q000528:radiation effects; D045643:DNA Repair Enzymes / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004195:Disease Models, Animal; D059565:G2 Phase Cell Cycle Checkpoints / Q000528:radiation effects; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic / Q000528:radiation effects; D006801:Humans; D000076228:MRE11 Homologue Protein; D051379:Mice; D046912:Multiprotein Complexes; D009154:Mutation; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000235:genetics* / Q000532:radiotherapy; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011836:Radiation Tolerance / Q000235:genetics*; D011839:Radiation, Ionizing; D023041:Xenograft Model Antitumor Assays
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D046912:Multiprotein Complexes; C112156:NBN protein, human; D009687:Nuclear Proteins; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes