A systems-biology analysis of isogenic megakaryocytic and granulocytic cultures identifies new molecular components of megakaryocytic apoptosis

Background: The differentiation of hematopoietic stem cells into platelet-forming megakaryocytes is of fundamental importance to hemostasis. Constitutive apoptosis is an integral, yet poorly understood, facet of megakaryocytic (Mk) differentiation. Understanding Mk apoptosis could lead to advances in the treatment of Mk and platelet disorders. Results: We used a Gene-ontology-driven microarray-based transcriptional analysis coupled with protein-level and activity assays to identify genes and pathways involved in Mk apoptosis. Peripheral blood CD34(+) hematopoietic progenitor cells were induced to either Mk differentiation or, as a negative control without observable apoptosis, granulocytic differentiation. Temporal gene-expression data were analyzed by a combination of intra- and inter-culture comparisons in order to identify Mk-associated genes. This novel approach was first applied to a curated set of general Mk-related genes in order to assess their dynamic transcriptional regulation. When applied to all apoptosis associated genes, it revealed a decrease in NF-kappa B signaling, which was explored using phosphorylation assays for I kappa B alpha and p65 ( RELA). Up-regulation was noted among several proapoptotic genes not previously associated with Mk apoptosis such as components of the p53 regulon and TNF signaling. Protein-level analyses probed the involvement of the p53-regulated GADD45A, and the apoptosis signal-regulating kinase 1 (ASK1). Down-regulation of anti-apoptotic genes, including several of the Bcl-2 family, was also detected. Conclusion: Our comparative approach to analyzing dynamic large-scale transcriptional data, which was validated using a known set of Mk genes, robustly identified candidate Mk apoptosis genes. This led to novel insights into the molecular mechanisms regulating apoptosis in Mk cells.