Tumor suppressor protein p53 regulates megakaryocytic polyploidization and apoptosis.

The molecular mechanisms underlying differentiation of hematopoietic stem cells into megakaryocytes are poorly understood. Tumor suppressor protein p53 can act as a transcription factor affecting both cell cycle control and apoptosis, and we have previously shown that p53 is activated during terminal megakaryocytic (Mk) differentiation of the CHRF-288-11 (CHRF) cell line. Here, we use RNA interference to reduce p53 expression in CHRF cells and show that reduced p53 activity leads to a greater fraction of polyploid cells, higher mean and maximum ploidy, accelerated DNA synthesis, and delayed apoptosis and cell death upon phorbol 12-myristate 13-acetate-induced Mk differentiation. In contrast, reduced p53 expression did not affect the ploidy or DNA synthesis of CHRF cells in the absence of phorbol 12-myristate 13-acetate stimulation. Furthermore, primary Mk cells from cultures initiated with p53-null mouse bone marrow mononuclear cells displayed higher ploidy compared with wild-type controls. Quantitative reverse transcription-PCR analysis of p53-knockdown CHRF cells, compared with the "scrambled" control CHRF cells, revealed that six known transcriptional targets of p53 (BBC3, BAX, TP53I3, TP53INP1, MDM2, and P21) were down-regulated, whereas BCL2 expression, which is known to be negatively affected by p53, was up-regulated. These studies show that the functional role of the intrinsic activation of p53 during Mk differentiation is to control polyploidization and the transition to endomitosis by impeding cell cycling and promoting apoptosis.

Gene Ontology-driven transcriptional analysis of CD34+ cell-initiated megakaryocytic cultures identifies new transcriptional regulators of megakaryopoiesis.

Differentiation of hematopoietic stem and progenitor cells is an intricate process controlled in large part at the level of transcription. While some key megakaryocytic transcription factors have been identified, the complete network of megakaryocytic transcriptional control is poorly understood. Using global gene expression microarray analysis, Gene Ontology-based functional annotations, and a novel interlineage comparison with parallel, isogenic granulocytic cultures as a negative control, we closely examined the mRNA level of transcriptional regulators in megakaryocytes derived from human mobilized peripheral blood CD34(+) hematopoietic cells. This approach identified 199 differentially expressed transcription factors or transcriptional regulators. We identified and detailed the transcriptional kinetics of most known megakaryocytic transcription factors including GATA1, FLI1, and MAFG. Furthermore, many genes with transcription factor activity or transcription factor binding activity were identified in megakaryocytes that had not previously been associated with that lineage, including BTEB1, NR4A2, FOXO1A, MEF2C, HDAC5, VDR, and several genes associated with the tumor suppressor p53 (HIPK2, FHL2, and TADA3L). Protein expression and nuclear localization were confirmed in megakaryocytic cells for four of the novel candidate megakaryocytic transcription factors: FHL2, MXD1, E2F3, and RFX5. In light of the hypothesis that transcription factors expressed in a particular differentiation program are important contributors to such a program, these data substantially expand our understanding of transcriptional regulation in megakaryocytic differentiation of stem and progenitor cells.

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-kappaB signaling, which was explored using phosphorylation assays for IkappaBalpha and p65 (RELA). Up-regulation was noted among several pro-apoptotic 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.

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

OBJECTIVES: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Deregulated CDC25A expression promotes mammary tumorigenesis with genomic instability.

Checkpoint pathways help cells maintain genomic integrity, delaying cell cycle progression in response to various risks of fidelity, such as genotoxic stresses, compromised DNA replication, and impaired spindle control. Cancer cells frequently exhibit genomic instability, and recent studies showed that checkpoint pathways are likely to serve as a tumor-suppressive barrier in vivo. The cell cycle-promoting phosphatase CDC25A is an activator of cyclin-dependent kinases and one of the downstream targets for the CHK1-mediated checkpoint pathway. Whereas CDC25A overexpression is observed in various human cancer tissues, it has not been determined whether deregulated CDC25A expression triggers or promotes tumorigenesis in vivo. Here, we show that transgenic expression of CDC25A cooperates markedly with oncogenic ras or neu in murine mammary tumorigenesis. MMTV-CDC25A transgenic mice exhibit alveolar hyperplasia in the mammary tissue but do not develop spontaneous mammary tumors. The MMTV-CDC25A transgene markedly shortens latency of tumorigenesis in MMTV-ras mice. The MMTV-CDC25A transgene also accelerates tumor growth in MMTV-neu mice with apparent cell cycle miscoordination. CDC25A-overexpressing tumors, which invade more aggressively, exhibit various chromosomal aberrations on fragile regions, including the mouse counterpart of human 1p31-36, according to array-based comparative genomic hybridization and karyotyping. The chromosomal aberrations account for substantial changes in gene expression profile rendered by transgenic expression of CDC25A, including down-regulation of Trp73. These data indicate that deregulated control of cellular CDC25A levels leads to in vivo genomic instability, which cooperates with the neu-ras oncogenic pathway in mammary tumorigenesis.

Nicotinamide (vitamin B3) increases the polyploidisation and proplatelet formation of cultured primary human megakaryocytes.

Megakaryocytic (Mk) cell maturation involves polyploidisation, and the number of platelets produced increases with Mk DNA content. Ploidy levels in cultured human MK cells are much lower than those observed in vivo. This study demonstrated that adding the water-soluble vitamin nicotinamide (NIC) to mobilised peripheral blood CD34+ cells cultured with thrombopoietin (Tpo) more than doubled the percentage of high-ploidy (> or = 8N) MK cells. This was observed regardless of donor-dependent differences in Mk differentiation. Furthermore, MK cells in cultures with NIC were larger, had more highly lobated nuclei, reached a maximum DNA content of 64N (vs. 16N with Tpo alone), and exhibited more frequent and more elaborate cytoplasmic extensions. NIC also increased the ploidy of cultured primary murine MK cells and a cell line model (CHRF-288) of Mk differentiation. However, NIC did not alter Mk commitment, apoptosis, or the time at which endomitosis was initiated. Despite the dramatic phenotypic differences observed with NIC addition, gene expression microarray analysis revealed similar overall transcriptional patterns in primary human Mk cultures with or without NIC, indicating that NIC did not disrupt the normal Mk transcriptional program. Elucidating the mechanisms by which NIC increases Mk maturation could lead to advances in the treatment of Mk and platelet disorders.