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Acute myeloid leukemia induced by MLL-ENL is cured by oncogene ablation despite acquisition of complex genetic abnormalities
Blood Horton et al.
10.1182/blood-2008-07-170480
Supplemental materials for: Horton et al
Files in this Data Supplement:
- Figure S1. Immortalized cells with conditional MLL-ENL expression induce leukemia in vivo (JPG, 119 KB)
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(A) Diagrams represent the retroviral expression vectors used to generate immortalized cells expressing MLL-ENL in a constitutive (pMSCV-MLL-ENL-neo) and conditional (pMSCV-neo-TRE-MLL-ENL & pMSCV-tTA-IRES-EGFP) manner. The BamHI restriction endonuclease site and region corresponding to the probe (shown in grey) used in the southern blot analysis are indicated. (B) Relative level of MLL-ENL mRNA expression in immortalized ME4, ME5, ME7, and cME3 cells after treatment (grey bars or indicated with arrow) or not (black bars) for 48 hours with 2 µg/ml doxycycline. Values determined by quantitative RT-PCR are normalised to untreated cME3 cells. Columns represent the mean of triplicate measurements, error bars represent the standard deviations. (C) Photomicrographs of haematoxylin & eosin (H&E) stained sections of liver demonstrating infiltration with transplanted leukemic cells (original magnifications ×100). (D) Southern blot analysis of genomic DNA isolated from immortalized cells ME5 and ME7 and their leukemic progeny ME5a, ME7a, and ME7b. Blots show 5′ end-fragments produced by digestion of the integrated provirus and genomic DNA with BamHI.
- Figure S2. Reduced proliferation but unaltered growth-factor requirements of leukemic cells (JPG, 54.7 KB)
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(A) Fold accumulation (log10) in cell number of immortalized ME4 (filled squares), and leukemic ME4a (open triangles) and ME4b (open diamonds) cells. (B) Viability of cells cultured in various cytokine conditions (SCF, IL-6, IL-3, GM-CSF) measured with an MTS assay at an absorbance of 490 nm. The values plotted are normalized to the growth of each cell line in the combination of SCF, IL3, and IL6. Bars represent means of triplicate values and error bars their standard deviations.
- Figure S3. Comparative genomic hybridization of DNA samples from immortalized and leukemic cells (JPG, 79.9 KB)
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Figure illustrates the DNA copy number profile across all chromosomes and the increase (red bars) and decrease (green bars) in copy number of chromosomal regions in ME4b (A) and ME7b (B) leukemic cells when compared to ME4 (A) and ME7 (B) immortalized cells (CGH Analytics v3.4, Agilent Technologies).
- Figure S4. Relative MLL-ENL DNA copy number and mRNA expression in immortalized and leukemic cells (JPG, 56.6 KB)
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(A) Relative MLL-ENL DNA copy numbers measured by quantitative RT-PCR. Copy numbers were calculated for ME4a and ME4b relative to ME4, for ME5a relative to ME5, for ME7a and ME7b relative to ME7 and for cME3a relative to cME3. Columns represent the mean of triplicate measurements, error bars represent the standard deviations. (B) Relative level of MLL-ENL mRNA expression measured by quantitative RT-PCR shown for all cell lines normalised to ME4. Columns represent the mean of triplicate measurements, error bars represent the standard deviations.
- Figure S5. Leukemic cells require MLL-ENL expression for survival in vivo (JPG, 57.6 KB)
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Effect of doxycycline on survival of recipient mice (left panel) and elimination of leukemic cells from peripheral blood (right panel). Recipient mice were transplanted with the indicated leukemic cells and following their detection in the peripheral blood, one group was given doxycycline in their water (open diamonds; ME4b n=5, ME4c n=5, ME7a n=5, ME7b n=5) and the other left untreated (filled circles; ME4b n=5, ME4c n=5, ME7a n=5, ME7b n=4). Arrows indicate the point at which doxycycline treatment started and the bars on the survival curves indicate the length of treatment in each experiment. The five doxycycline treated mice transplanted with ME7b cells are still alive 280 days after transplantation. The graphs depict the presence of leukemic Mac1+EGFP+ cells as a percentage of total Mac1+ cells in the peripheral blood of recipient mice. Points on the graphs represent mean values and bars their standard deviations.
- Figure S6. Leukemic cells require MLL-ENL expression for survival in vivo (JPG, 46.8 KB)
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(A–B) Relative level of MLL-ENL mRNA expression in immortalized ME4 cells after treatment (open bars or indicated with arrow) or not (filled bars) for 48 hours with 2 µg/ml doxycycline. Also shown in (A) are levels of MLL-ENL mRNA expression in leukemias isolated from the spleens of secondary recipient mice 54 days (ME4cI and ME4cII) after transplantation with ME4c leukemic cells or 60 days (ME4bI) after transplantation with ME4b leukemic cells (all on doxycycline treatment). Also shown in (B) are levels of MLL-ENL mRNA expression in ME4b leukemias following culture in methylcellulose with (open bars) or without (filled bars) doxycycline. The cells were isolated from the spleens of secondary recipient mice 60 days (ME4bI, on doxycycline treatment) and 101 days (ME4bII, off doxycycline treatment) after transplantation. Values determined by quantitative RT-PCR are normalised to untreated ME4 cells. Columns represent the mean of triplicate measurements, error bars represent the standard deviations.
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