Ploidy could be the key to understanding megakaryocyte (MK) biology and platelet production. Human CD34 ؉ cells purified from umbilical cord blood (CB) and peripheral blood (PB) were investigated on their capability to give rise, in a serumfree medium containing thrombopoietin, to MKs and platelets. CB-MKs showed reduced polyploidization and platelet number compared with PB-MKs, but a similar membrane phenotype. Most CBMKs showed a 2N content of DNA (ϳ80%) and only 2.6% had 8N, whereas 40% of the PB cells had 8N or more. Platelets were substantially released in PB culture from day 12; at day 14 the CB-derived MKs were able to release platelets although at a reduced level (ϳ35%), correlating with their reduced size. A direct correlation was demonstrated by sorting polyploid cells from PB-MKs and evaluating the platelets released in the supernatant. Furthermore, the study analyzed the expression and distribution of cyclin D3 and cyclin B1. Cyclin D3 protein was increased in PB in comparison to CB-MKs; in PB culture most cells rapidly became positive, whereas in CB-derived cells cyclin D3 expression was evident only from day 9 and in a reduced percentage. Cyclin B1 was essentially localized at the nuclear level in the CB and was expressed during the whole culture. In PB-MKs, at day 9, a reduction was observed, correlating with an advanced ploidy state. The data indicate the inability of the CB-MKs to progress in the endomitotic process and a direct correlation between DNA content and platelet production. IntroductionThe main feature of megakaryocyte (MK) maturation is the development of a single, large, lobulated, polyploid nucleus; the mature MKs cease to proliferate but continue to increase their DNA content without undergoing late stages of mitosis. [1][2][3][4] Increase in megakaryocytic ploidy is associated with increase in megakaryocytic volume; the large size and abundant cytoplasm allow MKs to produce several thousand platelets per cell. 3 It was presumed that higher-ploidy cells could produce more platelets than lower-ploidy cells or that production and release is more efficient from a single large cell than from several smaller ones, but none of these suppositions has been proven. 5 Peripheral blood (PB)-mobilized CD34 ϩ cells induced to differentiate into megakaryocytic lineage gave rise to 3-fold augmentation of platelets compared with bone marrow (BM) CD34 ϩ cells, although the proportion of proplateletdisplaying MKs were similar. 6 Choi et al 7 reported the functionality of the platelets released in vitro from CD34 ϩ cells derived from PB stimulated to form MKs.The generation of large numbers of megakaryocytes became possible by the identification and cloning of thrombopoietin (TPO), the key regulatory cytokine of megakaryocytopoiesis. [8][9][10][11] Then several culture systems have been developed permitting all stages of megakaryocytopoiesis until platelet formation. 6,[12][13][14][15] TPO was shown to induce endomitosis and consequently to increase the polyploidy state of MKs in a significant meas...

The homoeobox is a 183 base-pair (bp) DNA sequence conserved in several Drosophila genes controlling segmentation and segment identity. Homoeobox sequences have been detected in the genome of species ranging from insects and anellids to vertebrates and homoeobox containing genes have been cloned from Xenopus, mouse and man. We recently isolated human homoeobox containing complementary DNA clones, that represent transcripts from four different human genes. One clone (HHO.c10) is selectively expressed in a 2.1 kilobase (kb) polyadenylated transcript in the spinal cord of human embryos and fetuses 5-10 weeks after fertilization. We report the characterization of a second cDNA clone, termed HHO.c13, that represents a new homoeobox gene. This clone encodes a protein of 255 amino-acid residues, which includes a pentapeptide, upstream of the homoeo domain, conserved in other Drosophila, Xenopus, murine and human homoeobox genes. By Northern analysis HHO.c13 detects multiple embryonic transcripts, which are differentially expressed in spinal cord, brain, backbone rudiments, limb buds and heart in 5-9-week-old human embryos and fetuses, in a striking organ- and stage-specific pattern. These observations suggest that in early mammalian development homoeobox genes may exert a wide spectrum of control functions in a variety of organs and body parts, in addition to the spinal cord.

The globin chain synthetic pattern and the extent of DNA methylation within embryonic, fetal, and adult f-like globin gene domains were evaluated in 290% purified human erythroblasts from yolk sacs and fetal livers in the 6-to 12-wk gestational period as well as from adult marrows. The 6-wk erythroblasts produce essentially embryonic E chains, whereas the 12-wk erythroblasts synthesize largely fetal gamma globin and the adult marrow erythroblasts synthesize almost exclusively adult /3 chains. In all phases of ontogenic development, a strong correlation exists between DNA hypomethylation in the close flanking sequences of globin genes and their expression. These results suggest that modulation of the methylation pattern may represent a key mechanism for regulating expression of human globin genes during embryonic --fetal and fetal --adult Hb switches in humans. In ontogenic development this mechanism might in turn correlate with a gradual modification of chromatin structure in the non-a gene cluster, thus leading to a 5' -* 3' activation of globin genes in a balanced fashion.In mammalian DNA =2-7% of cytosine residues are converted to 5-methylcytosine shortly after DNA replication (1). More than 90% of 5-methylcytosine residues occur in the dinucleotide CpG with a nonrandom distribution, which is both species and tissue specific (1, 2). The DNA methylation pattern in proliferating cells is preserved by "maintenance" methylase(s), which recognize CpG sequences in the newly synthesized unmethylated DNA strand (3,4).Growing evidence indicates that in higher eukaryotes DNA methylation and gene expression are inversely correlated (reviewed in refs. 1, 2, and 5). DNA in germline cells is heavily methylated (1,2). In differentiated cells, =70% of CpG sequences are methylated in the overall DNA vs. only =20-30% in chromatin regions containing active gene sequences (6). A variety of protein encoding genes is undermethylated or methylated when expressed or not expressed, respectively (1,2,5). Similarly, endogenous proviral genomes are commonly heavily methylated when inactive but unmethylated when expressed (7-9). However, inactive genes may be undermethylated in both cell lines (10, 11) and primary tissues (e.g., placenta and carcinomas) (12, 13). In rare instances, active genes are totally methylated (14).A cause-effect relationship between DNA undermethylation and gene expression has been suggested by a number of experiments. In particular, (i) expression of cloned genes inserted in eukaryotic cells is dependent upon their methylation state prior to insertion (15, 16) and (ii) inhibition of DNA methylation by 5-azacytidine leads to hypomethylation and activation of endogenous viral DNAs (7, 17) and cellular genes (18)(19)(20).The methylation pattern of human globin genes also relates to the debated issue of mechanisms underlying the Hb switches in humans. In this regard, the embryonic --fetal (i.e., a, e--y) globin chain switch has been scarcely investigated so far, whereas the perinatal y -+ ( switch has been ...

BackgroundThe identification of signaling pathways that affect the cancer stem-like phenotype may provide insights into therapeutic targets for combating embryonal rhabdomyosarcoma. The aim of this study was to investigate the role of the MEK/ERK pathway in controlling the cancer stem-like phenotype using a model of rhabdospheres derived from the embryonal rhabdomyosarcoma cell line (RD).MethodsRhabdospheres enriched in cancer stem like cells were obtained growing RD cells in non adherent condition in stem cell medium. Stem cell markers were evaluated by FACS analysis and immunoblotting. ERK1/2, myogenic markers, proteins of DNA repair and bone marrow X-linked kinase (BMX) expression were evaluated by immunoblotting analysis. Radiation was delivered using an x-6 MV photon linear accelerator. Xenografts were obtained in NOD/SCID mice by subcutaneously injection of rhabdosphere cells or cells pretreated with U0126 in stem cell medium.ResultsMEK/ERK inhibitor U0126 dramatically prevented rhabdosphere formation and down-regulated stem cell markers CD133, CXCR4 and Nanog expression, but enhanced ALDH, MAPK phospho-active p38 and differentiative myogenic markers. By contrast, MAPK p38 inhibition accelerated rhabdosphere formation and enhanced phospho-active ERK1/2 and Nanog expression. RD cells, chronically treated with U0126 and then xeno-transplanted in NOD/SCID mice, delayed tumor development and reduced tumor mass when compared with tumor induced by rhabdosphere cells. U0126 intraperitoneal administration to mice bearing rhabdosphere-derived tumors inhibited tumor growth . The MEK/ERK pathway role in rhabdosphere radiosensitivity was investigated in vitro. Disassembly of rhabdospheres was induced by both radiation or U0126, and further enhanced by combined treatment. In U0126-treated rhabdospheres, the expression of the stem cell markers CD133 and CXCR4 decreased and dropped even more markedly following combined treatment. The expression of BMX, a negative regulator of apoptosis, also decreased following combined treatment, which suggests an increase in radiosensitivity of rhabdosphere cells.ConclusionsOur results indicate that the MEK/ERK pathway plays a prominent role in maintaining the stem-like phenotype of RD cells, their survival and their innate radioresistance.Thus, therapeutic strategies that target cancer stem cells, which are resistant to traditional cancer therapies, may benefit from MEK/ERK inhibition combined with traditional radiotherapy, thereby providing a promising therapy for embryonal rhabdomyosarcoma.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0501-y) contains supplementary material, which is available to authorized users.

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