Differential gene expression, GATA1 target genes, and the chemotherapy sensitivity of Down syndrome megakaryocytic leukemia

Abstract

Children with Down syndrome (DS) with acute megakaryocytic leukemia (AMkL) have very high survival rates compared with non-DS AMkL patients. Somatic mutations identified in the X-linked transcription factor gene, GATA1, in essentially all DS AMkL cases result in the synthesis of a shorter (40 kDa) protein (GATA1s) with altered transactivation activity and may lead to altered expression of GATA1 target genes. Using the Affymetrix U133A microarray chip, we identified 551 differentially expressed genes between DS and non-DS AMkL samples. Transcripts for the bone marrow stromal-cell antigen 2 (BST2) gene, encoding a transmembrane glycoprotein potentially involved in interactions between leukemia cells and bone marrow stromal cells, were 7.3-fold higher (validated by real-time polymerase chain reaction) in the non-DS compared with the DS group. Additional studies confirmed GATA1 protein binding and transactivation of the BST2 promoter; however, stimulation of BST2 promoter activity by GATA1s was substantially reduced compared with the full-length GATA1. CMK sublines, transfected with the BST2 cDNA and incubated with HS-5 bone marrow stromal cells, exhibited up to 1.7-fold reduced cytosine arabinoside (ara-C)-induced apoptosis, compared with mock-transfected cells. Our results demonstrate that genes that account for differences in survival between DS and non-DS AMkL cases may be identified by microarray analysis and that differential gene expression may reflect relative transactivation capacities of the GATA1s and full-length GATA1 proteins.

Figure 1.

Drug sensitivities of megakaryoblasts from pediatric patients with AMkL. In vitro sensitivities to ara-C (A) and daunorubicin (B) of megakaryoblasts, obtained from newly diagnosed Down syndrome (DS) and non-DS children with AMkL, were measured by the MTT assay. Drug sensitivity results of the DS AMkL samples have been reported previously.

Figure 2.

Cluster analysis of differentially expressed genes between DS and non-DS megakaryoblasts; chromosomal localization of genes in cluster analysis. (A) Microarray analysis using the Affymetrix U133A genechip was performed with DS (n = 5) and non-DS (n = 5) AMkL samples and clinically relevant AMkL cell lines (n = 3). Following selection of genes that were present or marginal in either group and filtered for those transcripts with a minimum 2-fold change in expression between DS AMkL and non-DS AMkL, one-way analysis of variance (ANOVA) was used with the Benjamini and Hochberg multiple test correction to control the FDR at 10%, and identified 551 genes as differentially expressed between DS AMkL and non-DS AMkL. Clustering was performed using the median normalized expression data with a Euclidean distance metric and identified 105 genes overexpressed in the DS group and 447 genes overexpressed in the non-DS group. (B) Chromosome localization of the 551 genes analyzed in the cluster analysis.

Figure 3.

Schematic of the promoter region of the bone marrow stromal-cell antigen 2 gene. Putative promoter region, spanning 759 bp upstream of the translation start site ATG including 211 bp of exon 1 of the BST2 gene. Numbering is relative to the translation start site (+1). Potential cis-regulatory elements on the plus (+) and minus (-) DNA strands (determined by Transfac 3.2, MatInspector V2.2) are underlined and shown in bold.

Figure 4.

BST2 transcripts and promoter activity in AMkL cell lines; gel shifts with the +39/+65 BST2 probe. (A) Transcript levels of BST2 in the DS AMkL cell line, CMK, and non-DS AMkL cell line, Meg-01 (top panel), measured by real-time PCR, and BST2 promoter activities in CMK and Meg-01 cells (bottom panel), measured by relative luciferase activity following transient transfection of the cell lines with the BST2 reporter gene construct, pGL3B-BST2pro. Error bars indicate standard error of measurement of 3 independent experiments. (B) Gel shift assays were performed with CMK and Meg-01 nuclear extracts and the ³²P-labeled +39/+65 BST2 oligonucleotide probe in the absence and presence of 100-fold molar excess commercial consensus GATA1 oligonucleotide. The specific DNA/protein complexes are indicated by lowercase letters. For the supershifts, GATA1 antibodies were added to the reaction mixtures and incubated for 30 minutes prior to separating the DNA/protein complexes. (C) In vivo binding of the long- and short-form GATA1 proteins to the BST2 promoter was confirmed by ChIP assays, as described in “Materials and methods.”

Figure 5.

Differential activation of the BST2 promoter by long- and short-form GATA1. Activation of the BST2 promoter by the long- and short-form GATA1 in Drosophila Mel-2 cells. Drosophila Mel-2 cells were cotransfected with 1 μg of the BST2 reporter gene construct (pGL3B-BST2pro) and 125 to 500 ng pPacGATA1-L or pPacGATA1-S. For all transfections, constant plasmid was maintained (at 500 ng of the pPac series). The results represent data from 3 experiments as the mean (± SD) fold increases in luciferase activity relative to a control for which pGL3B-BST2pro was cotransfected with 500 ng pPacO vector.

Figure 6.

Correlation between BST2 and GATA1 transcripts in non-DS blast cells. Correlation between BST2 and GATA1 transcripts determined by real-time RT-PCR in 17 non-DS AML and 14 non-DS AMkL patient samples. The nonparametric Spearman rank correlation coefficient was used to analyze the relationship between BST2 and GATA1 transcripts.

Figure 7.

Functional activity of the BST2 gene in transfected CMK sublines. (A) The coding cDNA of BST2 gene was amplified by PCR and subcloned into a mammalian expression vector, pcDNA3. The BST2 construct was stably transfected into the DS AMkL cell line, CMK. Overexpression of BST2 in 2 of the stable clones was confirmed by real-time RT-PCR. (B) Protection of BST2 stable clones from ara-C-induced apoptosis by bone marrow stromal cells, HS-5, determined with annexin-FITC on an Epics-XL MCL flow cytometer, as described in “Materials and methods.” Error bars indicate standard error of 3 independent experiments. ^*Statistically significant difference (P < .005).