|
|
Blood, Vol. 113, Issue 18, 4281-4288, April 30, 2009
Myc sensitizes p53-deficient cancer cells to the DNA-damaging effects of the DNA methyltransferase inhibitor decitabine
Blood Höglund et al.
113: 4281
Supplemental materials for: Hoglund et al
Files in this Data Supplement:
- Figure S1. Puma partially mediates the apoptosis induced by Decitabine (JPG, 49.1 KB)
-
(A) Quantitative RT-PCR on RNA from Akata cells cultured in the presence or absence of 2 µM Decitabine (Dec) shows that a lentiviral shRNA can knockdown the expression of PUMA. (B) Western blot analysis on the same samples as in A confirms shRNA-mediated knockdown of the Puma protein. (C) Puma knockdown blocks some of the Decitabine-induced apoptosis. Early apoptotic cells are present in the lower right quadrant and late apoptotic/necrotic are present in the upper right quadrant.
- Figure S2. Decitabine causes a growth arrest in p53 proficient BL cells but aneuploidy and/or apoptosis in p53 deficient BL and mouse lymphoma cells (JPG, 132 KB)
-
B-cell lymphoma lines were cultured in the presence or absence of 2 µM Decitabine for 24 h (not shown) and 48 h followed by flow cytometry analysis of PI-stained cells. Data are presented in two groups based on presence of the EBV genome and their p53 status is indicated (see table for literature references). Apoptosis was measured as the amount of cells with one log less DNA content than diploid cells and is presented in red numbers. Note the presence of cells with higher than tetraploid DNA content in Akata, Raji, P3HR1, and the two mouse lymphoma lines.
- Figure S3. Decitabine treatment induces the lytic regulator ZEBRA in Akata and Raji cells (JPG, 70.8 KB)
-
(A) Akata and KemI cells were treated with Decitabine (Dec.) for the time points indicated and the expression of EBV encoded mRNAs was determined by qRT-PCR. (B) Akata and Raji cells cultured in the absence (U) or presence of Decitabine (D) for 48 h. Protein was analyzed by Western blot analysis using antibodies against EBNA-2 (Dako), LMP-1 (Dako), and ZEBRA (Santa Cruz). A Raji cell line with EBNA3C reconstituted by stable transfection was used as a control for LMP-1 expression and was a kind gift from Dr Georg Klein. Total protein from KemIII cells was used as a positive control.
- Figure S4. Decitabine treatment induces a DNA damage response (DDR) (JPG, 60.7 KB)
-
KemI (A) and Akata cells (B) were treated with either 2 µM Decitabine (Dec) or 10 Gy of γ-IR and analyzed for the presence of phosphorylated NBS1 and Chk1 by Western blot analysis using phospo-specific antibodies. (C) A representative γ-H2AX staining of a cell subjected to DNA damage.
- Figure S5. Decitabine-induced cell death in mouse lymphoma cells is partially caspase-dependent and modestly dependent on Caspase-2 (JPG, 73.1 KB)
-
(A) λ#820 cells were established from a tumor of a λ-Myc transgenic mouse and have spontaneously lost expression of p53 during tumor development (data not shown), were grown in the presence or absence of 2 µM Decitabine (Dec) and/or the pan-caspase inhibitor Q-VD-OPH. Apoptosis (=sub-G1) was measured by FACS analysis of PI-stained cells and plotted in the DNA histograms. (B) Quantitative RT-PCR of the level of caspase-2 transcript in λ#820 cells expressing the best casp2 shRNA. The vector control was the construct which elicited the smallest decrease in casp2 transcript levels. (C) DNA histogram showing that caspase-2 knockdown results in a very small decrease in the amount of apoptosis (=sub-G1). (D) A DNA histogram showing that blocking caspase-2 with Z-VDVAD-FMK results in a very small decrease in the amount of Decitabine-induced apoptosis (=sub-G1) at 48 h.
- Figure S6. Tumors arising in iMycEµ; p53+∕− mice are B-cell lymphomas (JPG, 158 KB)
-
(A) Slides of 5–6 µm sections of formalin-fixed paraffin-embedded tissues were deparaffinized and stained with hematoxylin & eosin, dehydrated, and then cover slipped. Slides were analyzed for the presence of lymphoma cells in anatomic sites such as lymph nodes, spleen, thymus, liver, and bone marrow (sternum) from three individual mice. Pathologists (Professors Roos and Lundgren, Umeå University) confirmed the presence of infiltrating lymphoma cell which replaces most normal lymphocytes and destroys normal organ architecture. (B) Lymph node-derived tumor cells from the same mice as in A were stained with the indicated R-PE–conjugated antibodies and analyzed by flow cytometry. B220-positivity confirms that the tumors are of B-cell origin.
- Figure S7. The p53 inhibitor pifithrin-α (PFTα) 46 augments the therapeutic effect of Decitabine (JPG, 89.3 KB)
-
(A) Identification of tumors from λ-Myc mice that express wildtype p53. Live cells from lymphomas that had been cryo-frozen were injected into two C57/BL6 mice per original lymphoma. When palpable tumors had developed, one of the mice was subjected to 5 Gy of γ-IR and the tumors were harvested from both mice six hours later. Western blot analysis of two different lymphomas (#1 and #2) confirms wildtype and thus inducible p53. A tumor which expresses mutant and henceforth high levels of p53 is used as a positive control (+). (B) Survival curve of 18 C57/BL6 mice transplanted with a λ-Myc;p53wt tumor and treated as indicated (six animals per treatment group). Decitabine (Dec) was injected 7 days post-transplant at a dose of 5 mg/kg 3 times during the course of 24 h. To block p53 function 2.2 mg/kg PFTα was injected daily for four days starting 6 days post-transplant. The survival curve was generated and analyzed using GraphPad Prizm and is a representative of transplantation experiments of two different tumors. (C) Tumors arising in transplanted and PFT and/or Decitabine treated mice were analyzed for the expression of c-Myc and p53 protein levels by Western blot analysis. Table inset The lymph-node derived tumors analyzed by Western blot had developed in mice which carried tumors in lymph nodes and in the thymus. In all mice treated with both Decitabine and PFTα tumors predominantly localized to the thymus.
|
|
