PMC

Nontumorigenic ovarian surface epithelial cells are transformed by Daxx overexpression. A, Western blot results for DAXX overexpression in immortalized OSE cells. B and C, mOSE and mOSE-Daxx cells (10⁶ cells for each) were implanted subcutaneously into different nude mice, respectively. After 60 days, tumors were removed and weighed (n = 10). D, immunohistochemical staining for the DAXX and PML expression levels in mOSE-Daxx tumor tissues. Scale bar, 200 μm. E, immunohistochemical staining for pHistone H3 (pHH3) and collagen IV showing active cell proliferation and angiogenesis in mOSE-Daxx tumor tissues. Scale bar, 200 μm; **, p < 0.001.

DAXX protects ovarian cancer cells against DNA damage insults. A, immunofluorescence results showing increased pH2AX and pCHK2 in ES-2 cells after x-ray irradiation. Scale bar, 10 μm for all images. B, percentages of Daxx-overexpressing or Daxx-depleted cells that were positive for p-H2AX and p-CHK2. **, p < 0.001. C and D, immunofluorescence results for the levels of pH2AX and pCHK2 in Daxx-overexpressing or Daxx-depleted ES-2 cells, with or without x-ray treatment (8 gray). Green, GFP-labeled Daxx overexpressing or Daxx-depleted cells; red, p-H2AX and p-CHK2; blue, DNA stained with DAPI. E, Western blot results for the dose-dependent induction of H2AX phosphorylation by x-ray irradiation (0, 5, and 8 gray) in ES-2 cells, with or without overexpressed GFP-DAXX. F and G, Western blot results for x-ray irradiation-induced phosphorylation of H2AX and CHK1 in ES-2 cells and their derivatives (ES-2-GFP-Daxx and ES-2-siDaxx). Cells were harvested at the indicated times (hours) after x-ray irradiation (8 gray).

DAXX cooperates with PML in ovarian cancer cells in response to DNA damage insults. A and B, immunofluorescence results for nuclear pH2AX, pCHK2, and PML foci formation after bleomycin treatment. ES-2 cells that stably expressed GFP-DAXX fusion proteins were treated with bleomycin (20 μg/ml) for 0, 5, 9, and 12 h. Scale bar, 10 μm. C, quantification of PML foci in nuclei after bleomycin treatment at the indicated times. D, Western blot results showing increased DAXX and PML protein levels after BLM treatment. E, PML expressions in normal ovary and ovarian cancer tissues were determined by immunohistochemistry. Scale bar, 200 μm. F and G, immunofluorescence staining (F) and Western blotting (G) for PML and DAXX in cells transfected with siDaxx and/or siPml. Phospho-H2AX was detected by immunofluorescence before and after x-ray irradiation. Scale bar, 10 μm. H, Western blot results for PML RNAi-induced changes in DAXX and pH2AX. I, quantification of p-H2AX-positive cells in siPml and siDaxx-treated cells, with or without x-ray irradiation. Shown is mean ± S.E. (n = 3): #, p < 0.05; *, p < 0.01; **, p < 0.001.

DAXX expression patterns in human ovarian cancer samples and cell lines. A and B, immunohistochemistry results for DAXX expression in normal human ovary and ovarian cancer tissues. Sections were counterstained with hematoxylin. Scale bar, 200 μm. C, DAXX immunofluorescent staining in cultured ovarian cancer cells (ES-2, OV2008, SKOV3, and A2780), immortalized mOSE cells, and HeLa cells. Cells were seeded on glass coverslips in 24-well plates overnight before staining. Nuclei were visualized by 4′,6′-diamidino-2-phenylindole (DAPI) staining (blue). Scale bar, 10 μm. D, quantitative RT-PCR for Daxx mRNA expression levels in cultured ovarian cancer cells, mouse oocyte, and granular cells. E, Western blot analysis for DAXX protein expression levels in mouse tissues and cultured cell lines. β-Actin was used as the loading control. F, quantitative RT-PCR and Western blot results for the effects of Daxx silencing with RNAi. ES-2 cells were transfected with either control shRNA (shCON), Daxx-shRNA2, or Daxx-shRNA3; total mRNAs and proteins were isolated 48 h after transfection. G, Western blot results showing stable overexpression of DAXX protein in ovarian cancer cells. Ovarian cancer cells (A2780, ES-2, and SKOV3) were transfected with pLEGFP-Daxx and selected with G418 for more than 2 weeks. H, fluorescence microscopy results for the localization of overexpressed DAXX (green) in ovarian cancer cells (ES-2, SKOV3, and A2780) and mOSE cells that were stably transfected with GFP-Daxx. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.

DAXX promotes ovarian cancer cell colony formation and migration. A, DAXX-induced ovarian cancer cell proliferation. Cells were seeded into 96-well plates (3,000 cells/well) overnight, treated with 5 μg/ml bleomycin for 24 h, and then assessed by MTT assay. B and C, colony formation assay for the growth of ES-2 and SKOV3 cells, with or without Daxx overexpression. Cells were cultured in Matrigel. Colonies that formed at days 40 are shown. Colony numbers were counted at day 40. Scale bar, 100 μm. D, immunofluorescent staining of cleaved caspase 3 after RNAi depletion of Daxx or Pml. Scale bar, 100 μm. E, quantification of cleaved caspase 3-positive cells in D, and Western blotting results showing the levels of cleaved caspase 3 with or without Daxx/Pml RNAi. F, wound-healing assay for the migration capability of ES-2 cells and their derivatives (GFP-Daxx overexpression and siDaxx stable cell lines) cultured in serum-free medium. Scale bar, 100 μm. G, transwell experiment for the migration capability of ES-2 cells and their derivatives (GFP-Daxx overexpression and siDaxx stable cell lines). Cells were added to transwells and allowed to migrate for 12 h. Cells at the upper surface of the membrane were removed with cotton swabs, and the cells on the bottom surface were stained with hematoxylin and eosin. Numbers of cells (Daxx-overexpressing group or Daxx-depleted group) on the bottom surface were counted and were compared with the control group (set as 1). *, p < 0.01; **, p < 0.001. Scale bar, 50 μm.

DAXX promotes ovarian cancer cell proliferation and metastasis in vivo. A, ES-2 cells and their derivatives (GFP-Daxx overexpression and siDaxx stable cell lines) were injected intraperitoneally into nude mice (10⁶ cells/mouse). After 30 days, mice that received Daxx-overexpressing ES-2 cells had significant ascites accumulation. Tumors were removed and weighed (n = 18). Results are the means ± S.E. of six independent experiments. B, ascites fluid cells were harvested from nude mice and cultured in vitro. Abundant GFP signals were observed by fluorescent microscopy. Left panel, GFP. Right panel, merge (GFP and DAPI). Scale bar, 50 μm. C, ES-2 cells and their derivatives (GFP-Daxx overexpression and siDaxx stable cell lines) were injected underneath the abdomen membrane of nude mice (10⁶ cells/mouse). In situ colonization of tumor cells and metastasis to the intestine and liver were examined 3 weeks later. D, ovarian cancer cells (SKOV3 and ES-2) with or without DAXX overexpression (10⁶ cells for each) were implanted subcutaneously into different nude mice, respectively. After 30 days, tumors were removed and weighed (n = 10). E, cryosections were prepared from tumor tissues derived from ES-2 and DAXX-overexpressing ES-2 cells. Prominent angiogenesis was shown by immunofluorescent staining for collagen IV (red). DAXX overexpression was shown by GFP fluorescence (green). Tissues were counterstained with DAPI (blue). Scale bar, 100 μm. F, immunohistochemistry staining for DAXX, PML, cleaved caspase3, and pHistone H3 in representative ES-2-derived tumor tissues. Scale bar, 50 μm.