title: Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells.
abstract: BACKGROUND
  Gene amplification is a frequent manifestation of genomic instability that plays a role in tumour progression and development of drug resistance. It is manifested cytogenetically as extrachromosomal double minutes (DMs) or intrachromosomal homogeneously staining regions (HSRs). To better understand the molecular mechanism by which HSRs and DMs are formed and how they relate to the development of methotrexate (MTX) resistance, we used two model systems of MTX-resistant HT-29 colon cancer cell lines harbouring amplified DHFR primarily in (i) HSRs and (ii) DMs.
  
  
  RESULTS
  In DM-containing cells, we found increased expression of non-homologous end joining (NHEJ) proteins. Depletion or inhibition of DNA-PKcs, a key NHEJ protein, caused decreased DHFR amplification, disappearance of DMs, increased formation of micronuclei or nuclear buds, which correlated with the elimination of DHFR, and increased sensitivity to MTX. These findings indicate for the first time that NHEJ plays a specific role in DM formation, and that increased MTX sensitivity of DM-containing cells depleted of DNA-PKcs results from DHFR elimination. Conversely, in HSR-containing cells, we found no significant change in the expression of NHEJ proteins. Depletion of DNA-PKcs had no effect on DHFR amplification and resulted in only a modest increase in sensitivity to MTX. Interestingly, both DM-containing and HSR-containing cells exhibited decreased proliferation upon DNA-PKcs depletion.
  
  
  CONCLUSIONS
  We demonstrate a novel specific role for NHEJ in the formation of DMs, but not HSRs, in MTX-resistant cells, and that NHEJ may be targeted for the treatment of MTX-resistant colon cancer.
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Xiangning
authors.initials: X
authors.lastname: Meng
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Xiuying
authors.initials: X
authors.lastname: Qi
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Huanhuan
authors.initials: H
authors.lastname: Guo
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Mengdi
authors.initials: M
authors.lastname: Cai
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Chunxiang
authors.initials: C
authors.lastname: Li
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Feng
authors.initials: F
authors.lastname: Chen
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Huan
authors.initials: H
authors.lastname: Guo
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Li
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Yuzhen
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Liu
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Xueyuan
authors.initials: X
authors.lastname: Jia
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Jingcui
authors.initials: J
authors.lastname: Yu
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Chunyu
authors.initials: C
authors.lastname: Zhang
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Wenjing
authors.initials: W
authors.lastname: Sun
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Yu
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China Key Laboratory of Medical Genetics (Harbin Medical University), Heilongjiang Higher Education Institutions, Harbin, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Jin
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Bai
authors.affiliation: State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China.
authors.forename: Mingrong
authors.initials: M
authors.lastname: Wang
authors.affiliation: Departments of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Jesusa
authors.initials: J
authors.lastname: Rosales
authors.affiliation: Cell Biology & Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Ki-Young
authors.initials: KY
authors.lastname: Lee
authors.affiliation: Laboratory of Medical Genetics, Harbin Medical University, Harbin, China Key Laboratory of Medical Genetics (Harbin Medical University), Heilongjiang Higher Education Institutions, Harbin, China.
authors.forename: Songbin
authors.initials: S
authors.lastname: Fu
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D003110:Colonic Neoplasms / Q000188:drug therapy* / Q000235:genetics* / Q000473:pathology; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D059766:DNA End-Joining Repair / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000187:drug effects / Q000235:genetics*; D005784:Gene Amplification / Q000187:drug effects; D019073:HT29 Cells; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology* / Q000627:therapeutic use*; D013194:Staining and Labeling
chemical_list: D008727:Methotrexate
keywords: Cancer: colon; Genetics; Molecular genetics; Oncology