Doxorubicin induces prolonged DNA damage signal in cells overexpressing DEK isoform-2

Abstract

DEK has a short isoform (DEK isoform-2; DEK2) that lacks amino acid residues between 49-82. The full-length DEK (DEK isoform-1; DEK1) is ubiquitously expressed and plays a role in different cellular processes but whether DEK2 is involved in these processes remains elusive. We stably overexpressed DEK2 in human bone marrow stromal cell line HS-27A, in which endogenous DEKs were intact or suppressed via short hairpin RNA (sh-RNA). We have found that contrary to ectopic DEK1, DEK2 locates in the nucleus and nucleolus, causes persistent γH2AX signal upon doxorubicin treatment, and couldn't functionally compensate for the loss of DEK1. In addition, DEK2 overexpressing cells were more sensitive to doxorubicin than DEK1-cells. Expressions of DEK1 and DEK2 in cell lines and primary tumors exhibit tissue specificity. DEK1 is upregulated in cancers of the colon, liver, and lung compared to normal tissues while both DEK1 and DEK2 are downregulated in subsets of kidney, prostate, and thyroid carcinomas. Interestingly, only DEK2 was downregulated in a subset of breast tumors suggesting that DEK2 can be modulated differently than DEK1 in specific cancers. In summary, our findings show distinct expression patterns and subcellular location and suggest non-overlapping functions between the two DEK isoforms.

Fig 1. Expression level of DEK isoforms.

(A) RT-PCR results show endogenous mRNA expression levels of each DEK isoform in human cell lines. The same amount of RNA and cDNA was analyzed for each sample by using primers specific to either DEK1 or DEK2 mRNA. GAPDH analysis was performed for the normalization. (B) RT-qPCR analyses of the same cell lines that were shown in the upper panel. HepG2 cells were used as a calibrator in these analyses. DEK1 and DEK2 expression that was normalized with GAPDH (left and middle panel) or DEK2 expression that was normalized with DEK1 (right panel) were shown. (C) The expression level of DEK1 and DEK2 in HS-27A cell lines that overexpress Flag (left panel) or Myc (right panel) tagged DEK isoforms was analyzed by RT-qPCR. The expression level of each isoform was normalized with GAPDH expression. Fold change was calculated by using the expression level of the DEK1 isoform in the control cells (GFP-only) as a calibrator. "Target" indicates the primer pairs that were specific to each isoform, recognizing both ectopic and endogenous mRNAs. (D) Western blot analyses show ectopic expression of Flag (left panel) or Myc (right panel) epitope-tagged DEK1 and DEK2 in HS-27A cells. Anti-GAPDH antibody was used to show equal loading.

Fig 2. Cellular localization of DEK isoforms.

(A, B) HS-27A cells stably expressing Flag-tagged (A) or Myc-tagged (B) DEK isoforms were labeled with anti-Flag or anti-Myc antibodies (red) and anti-NPM1 antibody (green in panel B) using immunofluorescence staining. GFP (green) shows transduction with the retrovirus carrying DEK1 or DEK2 cDNA. DAPI (blue) labels DNA in the nucleus. (C) Immunofluorescence analysis shows the expression and cellular location of ectopic DEK1GFP or DEK2GFP fusion proteins (green) in transiently transfected HeLa cells. Nucleolar marker (labeled with anti-NPM1 antibody (red)) co-localizes with DEK2GFP fusion protein. (D) Co-immunoprecipitation (Co-IP) assays using anti-Flag and anti-Myc antibodies showed that despite the interaction between the large number of FlagDEK1 and MycDEK1 molecules, only a small number of FlagDEK2 and MycDEK1 molecules interact. (E) Co-IP assay in the cells that transiently co-expressed FlagDEK2 and MycDEK2 indicated that DEK2 doesn’t form homodimers.

Fig 3. Stable overexpression of DEK isoforms slows the proliferation and affects the response to doxorubicin.

(A) The growth curve of HS-27A cells stably expressing the FlagDEK1 or FlagDEK2 was generated by following the cell proliferation for 5 consecutive days using WST-1 assay. Cells transduced with an empty vector (GFP-only) were used as a control. The growth rate on each day was calculated by dividing each day’s absorbance by the absorbance of Day-0. The graphic shows mean values (± SEM) obtained from three independent experiments, each performed as triplicates (Two-way ANOVA Tukey’s multiple comparisons test: **P = 0.005; ***P<0.001, ns: not significant). (B) Parallel to growth curve analysis, the cell cycle was also analyzed using the same cells on days 1, 3, 4, and 5. Graphics reveal the percentage of cells at each stage of the cycle on the corresponding days. (C) Doxorubicin dose-response (IC50 values) of HS-27A-MycDEK1 or MycDEK2 cells were analyzed by using WST-1 assay. The graphic indicates the mean of three independent experiments (± SD, Paired t-test: *P = 0.0420 (MycDEK1 vs. MycDEK2); P = 0.0132 (GFP-Only vs. MycDEK2), **P = 0.0027).

Fig 4. DEK2 does not compensate lack of DEK1.

(A) RT-qPCR analysis showing DEK knockdown efficiency. Primer sets specific to DEK1 or DEK2 (X-axis, target) were used to determine the expression level of each isoform. Relative expression was calculated using the DEK1 expression level in the control HS-27A cells (GFP only) as a calibrator. (B) Confocal pictures of immunofluorescence staining with anti-Flag antibody showing ectopic FlagDEK2 expression in the shDEK+FlagDEK2 cells (mid-low panel, red). DAPI labels the nucleus (blue) (40X, with oil). (C) Representative confocal pictures of double immunocytochemistry analysis showing staining with anti-γH2AX (red) and anti-Flag (green) antibodies in the vehicle (untreated) or doxorubicin (24 hr doxo.) treated cells. Cells recovered for 24 hr after doxorubicin treatment depicted as "24 hr recovery". DAPI staining (blue) labels the nucleus (40X, with oil). (D) Representative graphic showing anti-γH2AX staining intensity per nucleus of each cell (Y-axis of the graph; au: arbitrary units) that was determined by using Image-J software. Each dot in the graph represents a cell and between 50 and 100 cells were analyzed per each corresponding cell line (X-axis) (bar indicates mean value. Mann Whitney test: ****P<0.0001, ns: not significant). (E) Reduction in the γH2AX signal intensity after the 24 hr of recovery from the doxorubicin treatment was determined by dividing the mean intensity of the recovery cells (24 hr rec.) by the mean intensity of the doxorubicin-treated (24 hr doxo.) cells in each group. The graph indicates the mean of two independent experiments (error bar indicates ± SD. Two-way ANOVA Tukey’s multiple comparisons test: *P = 0.0325, **P = 0.0087).

Fig 5. Effect of DEK1 or DEK2 overexpression on DNA repair efficiency.

(A) MycDEK1 (anti-Myc antibody, green in middle panels), MycDEK2 (anti-Myc antibody, green in lower panels), and γH2AX expression (anti-γH2AX antibody, red) were determined by immunocytochemistry and representative pictures captured by confocal microscope were shown in the vehicle (untreated), doxorubicin (24 hr doxo.) and recovery (24 hr recovery) groups (40X, with oil). DAPI (blue) labels the nucleus. (B) A graphic of one representative experiment shows the anti-γH2AX staining intensity per nucleus (Y-axis) in each group that was determined by using Image-J software. Each dot represents a cell and between 50 and 100 cells were analyzed in each group (bar indicates mean value. Mann Whitney test: **P = 0.0010 (GFP-only vs. MycDEK2), **P = 0.0011 (MycDEK1 vs. MycDEK2), ****P<0.0001, ns: not significant). (C) Reduction in the γH2AX signal intensity after the 24 hr of recovery from the doxorubicin treatment was determined by dividing the mean intensity of the recovery cells (24 hr rec.) by the mean intensity of the doxorubicin-treated (24 hr doxo.) cells in each group. The graph indicates the mean of three independent experiments (error bar indicates ± SD. Two-way ANOVA Tukey’s multiple comparisons test: *P = 0.0384). (D) Annexin-V analysis after the 24 hr doxorubicin (50 nM) treatment (0 hr) and following 72 hr or 96 hr recovery of control (GFP only), MycDEK1 or MycDEK2-cells. (E) Western blot analysis of the same cells shown in D exhibited an increased level of cleaved p89-PARP in the MycDEK2 cells. Anti-GAPDH was used to show equal protein loading. Quantification of band intensity was calculated using Image-J software (PARP/GAPDH). Fold change was determined by using GFP-only (24 hr doxo) sample as a calibrator and indicated between the Western blot images of PARP and GAPDH. (F) RT-qPCR analysis showing the endogenous DEK1 and DEK2 expression levels in HS-27A cells that were treated with 50nM doxorubicin for the indicated time points (X-axis). Twenty-four hr of recovery (24 (rec)) was also applied after the 24 hr of doxorubicin treatment. Vehicle-treated cells for each time point were used as a calibrator in the RT-qPCR analyses.

Fig 6. The mRNA expression profiles of DEK1 and DEK2.

A) CCLE dataset; B) Tissue array panel; and C, D) TCGA datasets. DEK1 and DEK2 Ct values were normalized to ACTB (tissue array) and reported as -ΔCt while CCLE and TCGA dataset transcript values were transformed as log2(RSEM+1) and log2(RSEM TPM+1), respectively. The Mann-Whitney U Test was used for statistical analysis. Asterisks represent the statistical significance.