1. The C/EBPδ tumor suppressor is silenced by hypermethylation in acute myeloid leukemia
by Shuchi Agrawal, Wolf-Karsten Hofmann, Nicola Tidow, Mathias Ehrich, Dirk van den Boom, Steffen Koschmieder, Wolfgang E. Berdel, Hubert Serve, and Carsten Müller-Tidow
Blood
Volume 109(9):
May 1, 2007
©2007 by American Society of Hematology

2. Microarray analysis of Aza-treated U937 cells reveals multiple differentially regulated genes.
Microarray analysis of Aza-treated U937 cells reveals multiple differentially regulated genes. (A) SAM of a paired analysis between Aza-exposed or nonexposed U937 cells to identify genes that are induced by Aza treatment. The scatter plot indicates the expected as well as the observed variability. Induced and repressed genes are indicated in red and green, respectively. (B) The scatter plot depicts the induced expression of genes (2-fold) by 6 days of Aza treatment of U937 cells. Red dots indicate 274 genes that were significantly induced after 6 days of Aza treatment. (C) Pathway analysis by PANTHER Classification System indicates biochemical pathways to be regulated by Aza treatment of U937 cells for all differentially expressed genes or for genes that were 2-fold induced (n = 274). (D) Relative levels of mRNA expression of transcripts identified from microarray analysis. Real-time RT-PCR was performed using mRNA from U937 cells treated with Aza for the indicated time. Expression levels for each gene were normalized to GAPDH levels.
Shuchi Agrawal et al. Blood 2007;109:
©2007 by American Society of Hematology

3. Aza treatment induced the expression of C/EBPδ.
Aza treatment induced the expression of C/EBPδ. (A) Relative levels of mRNA expression of C/EBP family members' transcripts in Aza-cytidine–exposed and nonexposed U937 cells. Indicated are means and standard deviations. (B) Relative levels of C/EBPδ mRNA in Aza-treated or untreated U937 and NB4 cells by real-time RT-PCR. Expression levels were normalized to GAPDH levels. Results shown are mean ± SD of 3 independent experiments. (C) Western blot analysis of C/EBPδ expression in lysates from Aza-treated U937 cells. (D) Densitometry of the Western blot shown in panel C normalized to actin verified induction of C/EBPδ protein after Aza treatment of U937 cells. (E) Western blot showing expression of C/EBPδ in different leukemic cell lines. Total protein lysate was subjected to Western blotting and probed with anti-C/EBPδ and antiactin antibodies. (F) Bisulfite sequencing for C/EBPδ promoter methylation analysis in leukemic cell lines. Filled squares indicate methylated and open squares nonmethylated CpG dinucleotides. The transcriptional start site and the location of the analyzed CpGs (n = 32) are indicated. Individual clones were sequenced.
Shuchi Agrawal et al. Blood 2007;109:
©2007 by American Society of Hematology

4. C/EBPδ promoter activity is suppressed by methylation.
C/EBPδ promoter activity is suppressed by methylation. (A) Luciferase assays showing C/EBPδ promoter repression in response to methylation. Methylated (SssI) or mock-methylated C/EBPδ promoter reporter constructs were transfected into 32D cells. The fold repression was calculated as 1/activity (normalized to Renilla values) with the control set as 1. The results are shown as the mean ± SD of 3 independent experiments. (B) Luciferase assay in S2 Drosophila cells, indicating that C/EBPδ promoter repression is dependent on MeCP2 activity. Methylated or mock-methylated reporter constructs were transfected into S2 Drosophila cells along with an Sp1 expression plasmid and either an empty vector or a MeCP2 expression vector. Fold repression was calculated as 1/relative activity, with the control set as 1. The results are shown as the mean ± SD of 3 independent experiments. (C) Luciferase assay showing fold repression in promoter activity of C/EBPδ, C/EBPα, cyclin E, and c-myc promoters after SssI methylase treatment. Methylated (SssI) or mock-methylated C/EBPδ promoter reporter constructs were transfected into 32D cells. The results are shown as the mean ± SD of 3 independent experiments. (D) Chromatin modification of histone H3-K4 methylation and histone H3 acetylation. Chromatin immunoprecipitations were performed with Aza-exposed U937 cells. Antibodies against histone H3-methylated K4 (K4-Me), acetylated histone H3, or IgG isotype control were used. Real-time PCR was performed using C/EBPδ promoter–specific primers and probe. The results shown are mean ± SD of 2 independent experiments.
Shuchi Agrawal et al. Blood 2007;109:
©2007 by American Society of Hematology

5. The C/EBPδ promoter is hypermethylated in acute myeloid leukemia.
The C/EBPδ promoter is hypermethylated in acute myeloid leukemia. (A) The number of hypermethylated CpGs in the promoter region of the C/EBP family members was analyzed in AML patients and controls by MALDI-TOF. Hypermethylation was defined as methylation above the mean methylation levels in controls plus 2 times the controls' standard deviation. The number of hypermethylated CpGs was analyzed for each sample and each gene. (B) The C/EBPδ promoter was methylated at a high frequency in AML. The bars indicate the percent of AML patients' samples hypermethylated for the different C/EBP genes as assessed by MALDI-TOF. (C-D) Correlation between C/EBPδ promoter methylation and mRNA expression. C/EBPδ promoter methylation was associated with decreased C/EBPδ expression levels (P = .005). The scatter plot indicates the relative mRNA expression levels normalized to GAPDH with regard to a low (up to 5 hypermethylated CpGs), intermediate (6 to 15 hypermethylated CpGs), and high (more than 150 methylated CpGs) degree of C/EBPδ promoter methylation (P = .016, nonparametric test). The median level of expression is indicated for each group with a horizontal line.
Shuchi Agrawal et al. Blood 2007;109:
©2007 by American Society of Hematology

6. C/EBPδ suppresses growth of primary hematopoietic progenitors.
C/EBPδ suppresses growth of primary hematopoietic progenitors. (A) Colony assays showing the growth inhibitory effect of C/EBPδ on hematopoietic progenitor cells. Murine bone marrow cells were transduced with an ER-inducible C/EBPδ construct or empty vector as a control. After 48 hours of transduction, cells were puromycin selected for 2 days. Colony assays were performed in the presence of β-estradiol to activate C/EBPδ. The results are shown as the mean ± SD of 3 independent experiments. (B) Western blot analysis of the colonies obtained in panel A. Blots were probed with anti-C/EBPδ and antiactin antibodies to confirm ectopic expression of C/EBPδ in the colonies. (C) C/EBPδ-transduced primary bone marrow cells in colony assay after replating. C/EBPδ expression completely abolished the colony growth of primary cells on replating. Photograph of representative areas of the plates demonstrates the lack of colony growth after replating in C/EBPδ-overexpressing cells in the presence of β-estradiol. (D) The bars indicate the inhibitory effect of C/EBPδ on colony formation in replating experiments. Results show mean ± SD of triplicates of 2 independent experiments. (E) Western blot analysis with G-CSFR and c-myc antibodies in total protein lysate from C/EBPδ or empty vector control–transduced bone marrow cells. The blot was stripped and rehybridized with actin antibodies. (F) Wright-Giemsa–stained cytospin slides under light microscopy. C/EBPδ or the empty vector–transduced primary bone marrow cells were incubated with β-estradiol for induction of C/EBPδ. Representative morphologic changes seen at day 7 of β-estradiol treatment are shown.
Shuchi Agrawal et al. Blood 2007;109:
©2007 by American Society of Hematology

7. C/EBP inhibits the growth of Flt3-ITD–transformed cells.
C/EBP inhibits the growth of Flt3-ITD–transformed cells. (A) Western blot analysis of transfected 32D-Flt3-ITD cells. The 32D-Flt3-ITD stable cells were transfected with an ER-inducible C/EBPδ construct or empty vector. Cells were puromycin selected. Lysates were probed with anti-C/EBPδ and antiactin antibody. (B) Growth curve of C/EBPδ-expressing 32D-Flt3-ITD cells. 32D-Flt3-ITD cells with C/EBPδ or control cells were seeded in suspension cultures at the density of 2 × 105/mL in the presence of β-estradiol or ethanol. Cells were counted every 24 hours by trypan blue exclusion method. The results are shown as the mean ± SD of 2 independent experiments. (C) Colony assays were performed with 32D-Flt3-ITD cells expressing the C/EBPδ ER construct. A total of 1000 cells were seeded per dish with either β-estradiol or ethanol. Colonies were analyzed on day 8. Each bar represents the mean ± SD of a representative triplicate experiment. (D) Wright-Giemsa–stained cytospin slides under light microscopy. C/EBPδ or the empty vector–transduced 32D-Flt3-ITD cells were incubated with β-estradiol for induction of C/EBPδ. Representative morphologic changes seen at day 7 of β-estradiol treatment are shown.
Shuchi Agrawal et al. Blood 2007;109:
©2007 by American Society of Hematology