Summary 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 (≥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.

HoxA10 is a homeodomain transcription factor that is frequently overexpressed in human acute myeloid leukemia. In murine bone marrow transplantation studies, HoxA10 overexpression induces a myeloproliferative disorder with accumulation of mature phagocytes in the peripheral blood and tissues. Over time, differentiation block develops in these animals, resulting in acute myeloid leukemia. In immature myeloid cells, HoxA10 represses transcription of some genes that confer the mature phagocyte phenotype. Therefore, overexpressed HoxA10 blocks differentiation by repressing myeloid-specific gene transcription in differentiating myeloid cells. In contrast, target genes involved in myeloproliferation due to HoxA10 overexpression have not been identified. To identify such genes, we screened a CpG island microarray with HoxA10 co-immunoprecipitating chromatin. We identified the DUSP4 gene, which encodes mitogen-activated protein kinase phosphatase 2 (Mkp2), as a HoxA10 target gene. We analyzed the DUSP4 5-flank and identified two proximal-promoter cis elements that are activated by HoxA10. We find that DUSP4 transcription and Mkp2 expression decrease during normal myelopoiesis. However, this down-regulation is impaired in myeloid cells overexpressing HoxA10. In hematopoietic cells, c-Jun N-terminal kinases (Jnk) are the preferred substrates for Mkp2. Therefore, Mkp2 inhibits apoptosis by dephosphorylating (inactivating) Jnk. Consistent with this, HoxA10 overexpression decreases apoptosis in differentiating myeloid cells. Therefore, our studies identify a mechanism by which overexpressed HoxA10 contributes to inappropriate cell survival during myelopoiesis.The 39 human and murine HOX genes are arranged in four paralog groups on four different chromosomes These genes encode homeodomain transcription factors that are highly conserved from Drosophila to humans. During embryogenesis, HOX gene transcription is activated 3Ј to 5Ј with the 3Ј-most genes regulating cephlad development and the 5Ј-most genes regulating caudal development (1). Transcription of the HOX genes is also tightly regulated during definitive hematopoiesis.Genes 3Ј in the locus (HOX1 to -4) are actively transcribed in hematopoietic stem cells, and more 5Ј genes (HOX5 to -11) are actively transcribed in lineage-committed progenitors (2). Therefore, HOX1 to -4 genes are transcribed in CD34 ϩ CD38 Ϫ cells. In contrast, HOX5 to -11 genes (also referred to as ABD HOX genes) are transcribed throughout the CD34 ϩ compartment and down-regulated in CD34 Ϫ cells. In poor prognosis AML, the normal decrease in HoxA7, A9, and A10 expression in CD34Ϫ cells does not occur (3, 4). Consistent with this, mice transplanted with bone marrow overexpressing either HoxA9 or HoxA10 rapidly develop a myeloproliferative disorder, which evolves to acute myeloid leukemia over time. This myeloproliferative disorder is characterized by an increase in mature phagocytes in the peripheral blood and tissues. Murine studies also indicate that forced overexpression of HoxA9 or 10 has a common ...

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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.

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. [Cancer Res 2007;67(3):984-91]