Downregulation of c-myc protein by siRNA-mediated silencing of DNA-PKcs in HeLa cells.
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.
An
Jing
J
Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, Peoples Republic of China.
Xu
Qing-Zhi
QZ
Sui
Jian-Li
JL
Bai
Bei
B
Zhou
Ping-Kun
PK
D000730:Androstadienes / Q000494:pharmacology; D001483:Base Sequence; D015152:Blotting, Northern; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D020868:Gene Silencing / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D047468:Immunoprecipitation; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000235:genetics* / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000037:antagonists & inhibitors / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000502:physiology*; D000077191:Wortmannin
D000730:Androstadienes; D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D016271:Proto-Oncogene Proteins c-myc; D034741:RNA, Small Interfering; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin