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.

Objective-Megakaryocytic cells (Mks) undergo endomitosis and become polyploid. Mk ploidy correlates with platelet production. We previously showed that nicotinamide (NIC) greatly increases Mk ploidy in cultures of human mobilized peripheral blood (mPB) CD34 + cells. This study aims to examine the generality of NIC effects, NIC's impact on Mk ultrastructure, and potential mechanisms for the increased ploidy.Methods-We used electron microscopy to examine Mk ultrastructure and flow cytometry to evaluate NIC effects on Mk differentiation and ploidy in mPB CD34 + cell cultures under diverse megakaryopoietic conditions. Mk ploidy and NAD(H) content were evaluated for NIC and other NAD + precursors. We tested additional inhibitors of the SIRT1 and SIRT2 histone/protein deacetylases and, after treatment with NIC, evaluated changes in the acetylation of SIRT1/2 targets.Results-NIC increased ploidy under diverse culture conditions and did not alter Mk ultrastructure. 6.25 mM NIC increased NAD + levels 5-fold. Quinolinic acid increased NAD + similar to that for 1 mM NIC, but yielded a much smaller ploidy increase. Similar increases in Mk ploidy were obtained using NIC or the SIRT1/2 inhibitor cambinol, while the SIRT2 inhibitor AGK2 moderately increased ploidy. SIRT1/2 inhibition in cells treated with NIC was evidenced by increased acetylation of nucleosomes and p53. Greater p53 acetylation with NIC was associated with increased binding of p53 to its consensus DNA binding sequence. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Conflict of Interest DisclosureNo financial interest/relationships with financial interest relating to the topic of this article have been declared. Nicotinamide has diverse functions in cells. NIC inhibits the activity of the silent information regulator 2 (Sir2) family of histone/protein deacetylases (sirtuins or SIRTs) [9][10][11][12]. Sirtuins catalyze a unique NAD + -dependent deacetylation reaction and are important for a wide variety of biological processes including transcriptional silencing, lifespan regulation, and the regulation of apoptosis [13][14][15]. NIC is also a precursor of NAD + via the salvage pathway [16,17]. NAD + regulates a variety of intracellular activities such as the DNA-binding specificity of p53 [18,19]. We have previously established that the p53-mediated Mk apoptotic program is intimately linked with terminal maturation and polyploidization, possibly through the downstream effects of p53 on MDM2 and BCL2 expression [20,21]. NIH Public AccessThe aim of this study was to examine the generality of the NIC effects, NIC's ...