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Original article
Novel role for non-homologous end joining in the formation of double minutes in methotrexate-resistant colon cancer cells
  1. Xiangning Meng1,
  2. Xiuying Qi1,
  3. Huanhuan Guo1,
  4. Mengdi Cai1,
  5. Chunxiang Li1,
  6. Jing Zhu1,
  7. Feng Chen1,
  8. Huan Guo1,
  9. Jie Li1,
  10. Yuzhen Zhao1,
  11. Peng Liu1,
  12. Xueyuan Jia1,
  13. Jingcui Yu1,
  14. Chunyu Zhang1,
  15. Wenjing Sun1,
  16. Yang Yu1,
  17. Yan Jin1,2,
  18. Jing Bai1,
  19. Mingrong Wang3,
  20. Jesusa Rosales4,
  21. Ki-Young Lee5,
  22. Songbin Fu1,2
  1. 1Laboratory of Medical Genetics, Harbin Medical University, Harbin, China
  2. 2Key Laboratory of Medical Genetics (Harbin Medical University), Heilongjiang Higher Education Institutions, Harbin, China
  3. 3State Key Laboratory of Molecular Oncology, Cancer Institute (Hospital), Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing, China
  4. 4Departments of Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta, Canada
  5. 5Cell Biology & Anatomy, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada
  1. Correspondence to Dr Songbin Fu, 157 Baojian Road, Nangang District, Harbin 150081, China; fusb{at}ems.hrbmu.edu.cn, fusongbin{at}yahoo.com

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.

  • Cancer: colon
  • Genetics
  • Molecular genetics
  • Oncology

This is an Open Access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/

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