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Downregulation of c‐myc protein by siRNA‐mediated silencing of DNA‐PKcs in HeLa cells
Author(s) -
An Jing,
Xu QingZhi,
Sui JianLi,
Bai Bei,
Zhou PingKun
Publication year - 2005
Publication title -
international journal of cancer
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.475
H-Index - 234
eISSN - 1097-0215
pISSN - 0020-7136
DOI - 10.1002/ijc.21093
Subject(s) - microbiology and biotechnology , gene silencing , dna pkcs , small interfering rna , transfection , hela , biology , dna , oligonucleotide , kinase , chemistry , protein kinase a , cell culture , gene , biochemistry , genetics
Abstract 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 γ‐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. © 2005 Wiley‐Liss, Inc.