Document Detail

Downregulation of c-myc protein by siRNA-mediated silencing of DNA-PKcs in HeLa cells.
MedLine Citation:
PMID:  15929110     Owner:  NLM     Status:  MEDLINE    
DNA-dependent protein kinase (DNA-PK) has been intensively investigated for its roles in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break repair and maintenance of genomic stability. Its catalytic subunit, DNA-PKcs, a serine/threonine protein kinase, has recently been reported to be overexpressed in various human cancers, but its significance is unclear. In our study, we synthesized 3 small interfering RNA (siRNA) oligonucleotides, which separately target the translation initiation region, catalytic motif and a sequence between the scid-mutation region and the FATC motif of DNA-PKcs; 3 stable cell lines were generated from HeLa cells transfected with these siRNA constructs, respectively. All 3 siRNAs resulted in remarkable depression on DNA-PKcs expression in HeLa cells, and led to an increased sensitivity to 2 or 4 Gy of gamma-ray as well as 5 or 10 J/m(2) of ultraviolet (UV) irradiation. The siRNA targeting the catalytic motif of DNA-PKcs exhibited the greatest efficiency of radiosensitization. We demonstrated that c-myc protein level was suppressed more than 80% by siRNA-mediated silencing of DNA-PKcs. Using an E-box enhancer (c-myc binding element) driving a secreted alkaline phosphatase (SEAP) reporter strategy, we further found that the transcriptional activity of c-myc was extremely suppressed by silencing DNA-PKcs. The highest suppression effect on c-myc expression was observed in the cells transfected with the siRNA targeting the catalytic motif of DNA-PKcs. Moreover, a similar suppression on c-myc expression and activity was also detected in HeLa cells treated with wortmannin, a phosphatidylinositol (PI)-3 kinase inhibitor. However, silencing DNA-PKcs did not change the level of c-myc mRNA. We have further identified the interaction between DNA-PKcs and c-myc protein. Together, our results imply that DNA-PKcs activity is necessary or contributory to the expression of c-myc protein. Targeting DNA-PKcs is an attractive anticancer strategy, which can achieve through at least two mechanistic pathways: (i) sensitizing cancer cells to radiotherapy or chemotherapy of DNA-damaging agents and (ii) downregulation of c-myc protein.
Jing An; Qing-Zhi Xu; Jian-Li Sui; Bei Bai; Ping-Kun Zhou
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  International journal of cancer. Journal international du cancer     Volume:  117     ISSN:  0020-7136     ISO Abbreviation:  Int. J. Cancer     Publication Date:  2005 Nov 
Date Detail:
Created Date:  2005-09-21     Completed Date:  2005-11-15     Revised Date:  2012-06-25    
Medline Journal Info:
Nlm Unique ID:  0042124     Medline TA:  Int J Cancer     Country:  United States    
Other Details:
Languages:  eng     Pagination:  531-7     Citation Subset:  IM    
Copyright Information:
Copyright 2005 Wiley-Liss, Inc.
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Peoples Republic of China.
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MeSH Terms
Androstadienes / pharmacology
Base Sequence
Blotting, Northern
DNA Primers
DNA-Activated Protein Kinase
DNA-Binding Proteins / genetics*,  metabolism
Gene Silencing / physiology*
HeLa Cells
Nuclear Proteins
Protein-Serine-Threonine Kinases / genetics*,  metabolism
Proto-Oncogene Proteins c-myc / antagonists & inhibitors,  metabolism*
RNA, Small Interfering / physiology*
Reg. No./Substance:
0/Androstadienes; 0/DNA Primers; 0/DNA-Binding Proteins; 0/Nuclear Proteins; 0/Proto-Oncogene Proteins c-myc; 0/RNA, Small Interfering; 19545-26-7/wortmannin; EC Protein Kinase; EC protein, human; EC Kinases

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