Downregulation of c-myc protein by siRNA-mediated silencing of DNA-PKcs in HeLa cells

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/m2 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.