Comparative, genome-scale transcriptional analysis of CHRF-288-11 and primary human megakaryocytic cell cultures provides novel insights into lineage-specific differentiation

Abstract

Little is known about the transcriptional events underlying megakaryocytic (Mk) differentiation. We sought to identify genes and pathways previously unassociated with megakaryopoiesis and to evaluate the CHRF-288-11 (CHRF) megakaryoblastic cell line as a model system for investigating megakaryopoiesis. Using DNA microarrays, Q-RT-PCR, and protein-level assays, we compared the dynamic gene expression pattern of phorbol ester-induced differentiation of CHRF cells to cytokine-induced Mk differentiation of human mobilized peripheral blood CD34(+) cells. Transcriptional patterns of well-known Mk genes were similar between the two systems. CHRF cells constitutively express some early Mk genes including GATA-1. Expression patterns of apoptosis-related genes suggested that increased p53 activity is involved in Mk apoptosis, and this was confirmed by p53-DNA-binding activity data and flow-cytometric analysis of the p53 target gene BBC3. Certain Rho and G-protein-coupled-receptor signaling pathway components were upregulated, including genes not previously associated with Mk cells. Ontological analysis revealed upregulation of defense-response genes, including both known and candidate platelet-derived contributors to inflammation. Upregulation of interferon-responsive genes occurred in the cell line, but not in the primary cells, likely due to a known genetic mutation in the JAK2/STAT5 signaling pathway. This analysis of megakaryopoiesis, which integrates dynamic gene expression data with protein abundance and activity assays, has identified a number of genes and pathways that may help govern megakaryopoiesis. Furthermore, the transcriptional data support the hypothesis that CHRF cells resemble an early Mk phenotype and, with certain limitations, exhibit genuine transcriptional features of Mk differentiation upon treatment with phorbol esters.