The human DEK oncogene regulates DNA damage response signaling and repair

Abstract

The human DEK gene is frequently overexpressed and sometimes amplified in human cancer. Consistent with oncogenic functions, Dek knockout mice are partially resistant to chemically induced papilloma formation. Additionally, DEK knockdown in vitro sensitizes cancer cells to DNA damaging agents and induces cell death via p53-dependent and -independent mechanisms. Here we report that DEK is important for DNA double-strand break repair. DEK depletion in human cancer cell lines and xenografts was sufficient to induce a DNA damage response as assessed by detection of γH2AX and FANCD2. Phosphorylation of H2AX was accompanied by contrasting activation and suppression, respectively, of the ATM and DNA-PK pathways. Similar DNA damage responses were observed in primary Dek knockout mouse embryonic fibroblasts (MEFs), along with increased levels of DNA damage and exaggerated induction of senescence in response to genotoxic stress. Importantly, Dek knockout MEFs exhibited distinct defects in non-homologous end joining (NHEJ) when compared to their wild-type counterparts. Taken together, the data demonstrate new molecular links between DEK and DNA damage response signaling pathways, and suggest that DEK contributes to DNA repair.

Figure 1.

DEK depletion increases DNA damage markers in vitro and in vivo. (A) U2OS cells were infected with Ad-GFP or AdDEKsh adenovirus and harvested 3 days post-infection, then analyzed by western blot analysis for DEK, γH2AX, FancD2 and p53^ser15, and quantified as described in the ‘Materials and Methods’ section. (B) U2OS cells were infected as in (A) and focus formation was visualized by immunofluorescence on Day 3 post-infection using either γH2AX or FANCD2 primary antibodies together with FITC- or rhodamine-conjugated secondary antibodies. At least five fields and 200 cells, from three independent experiments, were counted for quantification of the data, which is presented in the graphs on the right. Columns, mean values of 200 cells per experiment; bars, SEM; magnification = 100×; scale bar = 10 μm. (**P = 0.01, ***P = 0.0001) (C) Xenografts generated in nude mice were injected with 10⁸ infectious units Ad-GFP or AdDEKsh in the left and right flanks, respectively, three times at 3 day intervals. Tumors were removed, fixed, and two consecutive sections in each case were subjected to immunofluorescent detection of GFP and immunohistochemical detection of γH2AX. (D) SAOS-2 cells were infected as in (A) and probed with γH2AX primary and rhodamine-conjugated secondary antibody, and counterstained with DAPI on Day 4 post-infection. The graphs represent the percentage of cells positive for foci quantitated on Days 3 and 4 post-infection. Columns, mean values of 200 cells per experiment; bars, SEM; magnification = 100×; scale bar = 10 μm. (*P < 0.05).

Figure 2.

DEK knockdown leads to increased ATM and decreased DNA-PK signaling. (A) HeLa cells were infected with Ad-GFP and AdDEKsh adenoviruses. Three days post-infection, cells were collected, suspended in agarose gel, lysed and subjected to electrophoresis for comet assay analysis. Cells were then stained with SYBR green and assessed for DNA damage by determining the tail moment. Columns, mean value of tail moment from three independent experiments; bars, SEM. (***P < 0.0001) (B) HeLa cells were infected as in (A), and treated with 25 μM etoposide for the time points indicated 3 days post-infection. Whole cell lysates were subjected to western blot analysis using antibodies specific for γH2AX, pATM^Ser1981, total ATM, pSMC1^Ser957, total SMC1, pDNA-PKcs^Ser2056 and total DNA-PKcs. (C) U2OS cells were infected as in (A) and treated with 25 μm etoposide for 3 h at 3 days post-infection and analyzed for pDNA-PKcs^Ser2056 and total DNA-PKcs by western blot analysis. (D) HeLa cells were infected as in (A) and treated with 10 Gy IR 3 days post-infection, allowed to recover for 3 h, and whole cell lysates analyzed by western blot analysis. Western blots depicted in (C) and (D) are from different parts of the same immunoblot, respectively. (E) HeLa cells were infected with Ad-GFP or AdDEKsh, and nuclear extracts collected 3 days post-infection. Endogenous DNA was removed from the nuclear extracts by passage through a DEAE sepharose column, and then kinase assays were performed using a biotinylated DNA-PK specific p53 target substrate, and activity determined using a scintillation counter. Columns, mean value of activity of 3 independent experiments; bars, SEM. (*P = 0.05, **P = 0.02, ***P = 0.001).

Figure 3.

DEK depletion impairs Ku70/80 heterodimer mobilization. (A) HeLa cells were infected with Ad-GFP or AdDEKsh, treated with 25 μm etoposide for 10 min and 1 h 3 days post-infection, and treated with extraction buffer for 2 min prior to fixation in paraformaldehyde. Cells were analyzed by immunofluorescence using a Ku70/80 heterodimer specific antibody (red). (B) HeLa cells were treated with 25 μM etoposide for 3 h and analyzed for DEK (red) and γH2AX or SC35 (green) localization. Magnification = 100×; scale bars = 10 μm.

Figure 4.

Dek knockout MEFs are hyper-sensitive to genotoxic stress. (A) Asynchronous populations of primary wild-type and Dek knockout MEFs were analyzed for BrdU incorporation via flow cytometry. The percentage of cells in S-phase is indicated. (B) One hundred thousand wild-type or Dek knockout MEFs were plated in six-well plates in media only (left graph) or media containing 4 μM etoposide (right graph) and counted on Days 1, 3 and 5 post-plating for cell viability. Media +/− etoposide was replenished every 24 h. Line graphs, mean value of total number of cells in three independent experiments; bars, SEM. (**P = 0.0168) (C) Cells were plated and treated as in (B), and fixed 5 days post-plating for SA-β-galactosidase assays to determine levels of senescence. Columns, mean value of 200 cells counted in three independent experiments; bars, SEM. (*P < 0.05, ***P < 0.001) (D) Wild-type and Dek knockout cells were plated and treated as in (B). At 4 h post-plating, cells were trypsinized and counted to determine plating efficiency. Columns, mean value of attached cells from three independent experiments; bars, SEM.

Figure 5.

Dek-deficient MEFs exhibit increased levels of DNA strand breaks and decreased repair by NHEJ. (A) Primary MEFs were isolated from Dek wild-type, heterozygous, and knockout MEFs and treated with 1 mM HU for 24 h. Cells were then collected, suspended in agarose gel, lysed and subjected to electrophoresis. Cells were stained with SYBR green and assessed for DNA damage by determining the tail moment. Columns, mean value of the tail moment from three independent experiments; bars, SEM. (*P < 0.05, ***P < 0.001). (B) Primary MEFs were subjected to hydroxyurea (HU), camptothecin (C) and etoposide (E) treatment for 3 h, and protein lysates harvested and analyzed by western blot analysis for DEK, γH2AX and actin. To confirm the slight baseline increase of γH2AX in the knockout MEFs (compare lanes 1, 5 and 9), untreated samples were rerun separately (right panels). (C) Dek knockout MEFs were retrovirally infected with an empty vector (R780) or a DEK-expressing vector (R780-DEK). Following flow cytometric sorting, the cells were treated and analyzed as in (B). (D) Wild-type and Dek knockout MEFs were co-transfected with pEGFP-Pem1-Ad2 linearized with I-SceI along with DsRed-Express to control for transfection efficiency. The cells were harvested after 3 days and analyzed by flow cytometric analysis. DSB repair frequency was calculated from the percentage of EGFP positive cells divided by the percentage of transfection efficiency. DSB repair frequency of Dek wild-type MEFs were set to 100% each (absolute mean value: 80 × 10⁻²). Columns, mean value of four independent experiments; bars, SEM. (**P = 0.01) (E) Wild-type and Dek knockout MEFs were transfected with a repair reporter plasmid to detect NHEJ along with an I-SceI expressing vector. One day post-transfection, cells were collected and analyzed for EGFP expression by flow cytometry to determine repair efficiency. DSB repair frequency was calculated from the percentage of EGFP positive cells divided by the percentage of transfection efficiency in each case. DSB repair frequency of Dek wild-type MEFs were set to 100% each (absolute mean value: 30.0 × 10⁻³). Columns, mean values of 10 measurements per cell type and construct; bars, SEM (*P < 0.05).