1. Myc sensitizes p53-deficient cancer cells to the DNA-damaging effects of the DNA methyltransferase inhibitor decitabine
by Andreas Höglund, Lisa M. Nilsson, Linus Plym Forshell, Kirsteen H. Maclean, and Jonas A. Nilsson
Blood
Volume 113(18):
April 30, 2009
©2009 by American Society of Hematology

2. Decitabine induces aneuploidy in p53 deficient Burkitt lymphoma (BL) cells.
Decitabine induces aneuploidy in p53 deficient Burkitt lymphoma (BL) cells. (A) The BL cell lines Akata, KemI, and KemIII were treated with 2 μM decitabine (Dec) for 48 hours. The cell-cycle distribution was determined by flow cytometry of cells stained with propidium iodide. Cells regarded as aneuploid are indicated by arrows. (B) TP53 status correlates with presence of decitabine-induced aneuploidy of BL cells. Cells were irradiated with 10 Gy of ionizing radiation (γ-IR) and harvested 24 hours after treatment for analysis with Western blot using a monoclonal antibody against p53. The KemI cell line exhibits abnormally high levels of p53 irrespective of DNA damage, which is indicative of a TP53 mutant status. KemIII cells induce p53 after γ-IR treatment, as predicted by cells carrying wild-type TP53. Akata cells do not express p53, as previously shown.32
Andreas Höglund et al. Blood 2009;113:
©2009 by American Society of Hematology

3. Decitabine-induced aneuploidy and cell death is preceded by a DNA damage-induced transient G2 block.
Decitabine-induced aneuploidy and cell death is preceded by a DNA damage-induced transient G2 block. (A) Cell-cycle distribution of Akata cells 24 and 48 hours after 10 Gy of γ-IR or start of culture in the presence of 2 μM decitabine (Dec). The amount of cells with a less than diploid DNA content was regarded as apoptotic (ie, sub-G1, only number shown). (B) Decitabine and γ-IR treatment transiently arrests Akata cells in the G2 phase. To distinguish between the G2 and M phase of the cell cycle, loss of granularity due to nuclear envelope breakdown in mitosis was used during FACS analyses. To be able to set the M-phase gate correctly, Akata cells were treated with 1 μg/mL Taxol, which resulted in an accumulation of cells in gate R3. The remaining cells with the same DNA content but with higher granularity were collected in gate R2, depicted as the G2-phase. Quantifications of the cell-cycle distribution are shown in the table. (C) Decitabine treatment induces a DNA damage response (DDR). Akata cells were treated with either 5 μM decitabine or 10 Gy of γ-IR and analyzed for the presence of phosphorylated H2AX (γ-H2AX) in foci by immunofluorescence. The graph shows quantification of the percentage of cells with a signal that exceeds that of an arbitrarily chosen background staining. The results are based on quantifications of at least 100 cells counted from at least 3 different slides. A representative staining of a cell subjected to DNA damage is shown in Figure S4C. The bottom panel shows a Western blot analysis of lysates from cells treated with decitabine or γ-IR. The loading order is the same as the label on the graph. (D) Decitabine treatment leads to phosphorylation of p53 on serine 15. KemI and KemIII cells were treated with either decitabine or γ-IR and analyzed for phosphorylation of p53 serine 15. Both mutant (KemI) and wild-type (KemIII) p53 showed serine 15 phosphorylation, which is indicative of a DNA damage response.
Andreas Höglund et al. Blood 2009;113:
©2009 by American Society of Hematology

4. Myc provokes G2 checkpoint override that sensitizes p53 knockout mouse embryo fibroblasts (MEFs) to decitabine-induced cell death.
Myc provokes G2 checkpoint override that sensitizes p53 knockout mouse embryo fibroblasts (MEFs) to decitabine-induced cell death. (A) Low passage p53−/− MEFs were infected with retroviruses made either with MSCV-IRES-puro (vector control) or with MSCV-MycER-IRES-puro vectors. After selection, the cells were cultured in the presence or absence of 4-HT, to induce nuclear translocation and activation of MycER, and either treated with 10 Gy γ-IR or 2 μM decitabine (Dec) for the indicated time points. The cells were harvested and stained with propidium iodide and their cell-cycle distribution was analyzed by FACS. (B) The same cells as in panel A were also seeded into 6-well plates and cultured for 10 days after γ-IR or decitabine. Cultures with freshly added decitabine daily for 3 or 10 days produced the same results and a representative experiment of 3 independent clonogenic survival assays is shown. (C) Western blot analysis showing levels of total and phosphorylated cyclin-dependent kinase 1 (Cdc2) in p53−/− MEFs subjected to decitabine and Myc activation for the indicated times. Less phosphorylated Cdc2 suggests checkpoint override. (D) Akata cells were treated with either decitabine or γ-IR and analyzed for phosphorylation of Cdc2 by Western blot analysis.
Andreas Höglund et al. Blood 2009;113:
©2009 by American Society of Hematology

5. Decitabine-induced cell death in Akata cells is caspase-dependent but can occur in the absence of induced caspase-2 activity.
Decitabine-induced cell death in Akata cells is caspase-dependent but can occur in the absence of induced caspase-2 activity. (A) Akata cells, or Akata cells expressing an shRNA against Puma (Figure S1) and/or were pretreated with 5 μM of the pan-caspase inhibitor Q-VD-OPH, were grown in the presence or absence of 2 μM decitabine (Dec). Apoptosis (= sub-G1), G1, G2 and aneuploidy (> 4N) were measured by FACS analysis of PI-stained cells and plotted in the table (inset) or DNA histograms. (B) Caspase-2 activity of Akata cells or Akata cells expressing an shRNA against Puma treated or untreated with decitabine was measured by a colorimetric method as measured by the cleavage of the caspase-2 substrate VDVAD-pNA into the chromophore p-nitroanilide (pNA). The specificity of the assay was ascertained by pretreating (1 hour) and culturing the cells in the presence of 20 μM of the caspase-2 inhibitor Z-VDVAD-FMK. The arbitrary units (AU) were calculated by multiplying the absorbance minus background measured at 400 nm with 100. (C) DNA histogram showing that blocking Puma and/or caspase-2 (with Z-VDVAD-FMK) results in a very small decrease in the amount of apoptosis (= sub-G1).
Andreas Höglund et al. Blood 2009;113:
©2009 by American Society of Hematology

6. Decitabine treatment delays tumor formation in vivo.
Decitabine treatment delays tumor formation in vivo. (A) iMycEμ mice were bred to p53 knockout mice to produce offspring that were Myc-transgenic and heterozygous for p53. These mice exhibited an accelerated course of disease (median survival 48 days) compared with the iMycEμ mice previously born in the laboratory (dashed line, median survival 198 days). The survival curves and the statistical analyses were performed using the Survival curve function of the GraphPad Prism software (San Diego, CA). (B) Tumors arising in iMycEμ; p53+/− mice were frozen down as live cells, but pieces were also snap frozen for analyses. The snap frozen tumor pieces were analyzed for their p53 status by PCR genotyping and Western blot analysis (WB). In the PCR, intensities between the top mutant and the bottom wild-type bands were compared with assess loss of heterozygosity (LOH). High amount of p53 protein on the WB indicates mutant protein (*). (C) Vials of live cells were thawed, and the tumor cells were transplanted into the tail veins of 12 recipient C57/BL6 mice. One week after transplant, 6 mice were treated with 3 injections of 5 mg/kg decitabine with 8-hour intervals. The survival curve was generated and analyzed using GraphPad Prism and is a representative of transplantation experiments of 3 different tumors. (D) Tumors arising in transplanted mice were analyzed for the expression of c-Myc protein levels by Western blot analysis.
Andreas Höglund et al. Blood 2009;113:
©2009 by American Society of Hematology