Overexpression of cyclin D1 moderately increases ploidy in megakaryocytes.

BACKGROUND AND OBJECTIVES: Megakaryocytes undergo a unique cell cycle by which they replicate their complete genome many times in the absence of cytokinesis. In the search for regulators of the endomitotic cell cycle, we previously produced mice transgenic for cyclin D3 to identify this cyclin as able to enhance ploidy and to increase the number of differentiated cells in the megakaryocytic lineage. Of the D-type cyclins, cyclin D3 and to a much lesser extent cyclin D1, are present in megakaryocytes undergoing endomitosis and these cyclins are, respectively, markedly and moderately upregulated following exposure to the ploidy-promoting factor, Mpl-ligand. Our objective was to explore whether cyclin D1 can mimic the effect of cyclin D3 on ploidy in megakaryocytes. DESIGN AND METHODS: We generated transgenic mice overexpressing cyclin D1 in megakaryocytes and analyzed megakaryocyte ploidy, number and platelet levels in these mice and control mice. RESULTS: We show that transgenic mice in which cyclin D1 is overexpressed in megakaryocytes display higher ploidy level than the control mice, with no change in the number of differentiated cells of the megakaryocytic series, or in platelet level. INTERPRETATION AND CONCLUSIONS: Our models support a key role for D-type cyclins in the endomitotic cell cycle, and also indicate that although cyclin D3, from among the D cyclins, is unique in its high levels of expression in megakaryocytes, it is not unique in its ability to promote polyploidization.

Polyploidy: occurrence in nature, mechanisms, and significance for the megakaryocyte-platelet system.

OBJECTIVE: Polyploidy, the state of having greater than the diploid content of DNA, has been recognized in a variety cells. Among these cell types, the megakaryocytes are classified as obligate polyploid cells, developing a polyploid DNA content regularly during the normal life cycle of the organism, while other cells may become polyploid only in response to certain stimuli. The objective of this review is to briefly describe the different cell cycle alterations that may lead to high ploidy, while focusing on the megakaryocyte and the importance of high ploidy to platelet level and function. MATERIALS AND METHODS: Relevant articles appearing in scientific journals and books published in the United States and in Europe during the years 1910-1999 were used as resources for this review. We selected fundamental studies related to cell cycle regulation as well as studies relevant to the regulation of the endomitotic cell cycle in megakaryocytes. Also surveyed were publications describing the relevance of high ploidy to high platelet count and to platelet reactivity, in normal situations and in a disease state. RESULTS: Different cells may achieve polyploidy through different alterations in the cell cycle machinery. CONCLUSIONS: While upregulation of cyclin D3 further augments ploidy in polyploidizing megakaryocytes in vivo, future investigation should aim to explore how normal megakaryocytes may initiate the processes of skipping late anaphase and cytokinesis associated with high ploidy. In humans, under normal conditions, megakaryocyte ploidy correlates with platelet volume, and large platelets are highly reactive. This may not apply, however, to the disease state.

Cyclin D3 and megakaryocyte development: exploration of a transgenic phenotype.

The roles of cell cycle regulatory proteins in megakaryocyte development are poorly understood. We have previously demonstrated that cyclin D3 is expressed in megakaryocytes and is induced upon treatment with Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the megakaryocytic lineage show features similar to in vivo Mpl ligand treatment, including increased megakaryocyte number and ploidy. Terminal maturation and platelet production are not enhanced, however, and transgenic megakaryocytes show a defect in demarcation membrane development. We have examined expression of the transcription factor nuclear factor (NF)-E2, known to be involved in cytoplasmic maturation and platelet fragmentation, in these transgenic mice and controls treated with Mpl ligand. Our findings demonstrate marked induction of NF-E2 mRNA in control megakaryocytes in response to Mpl ligand, but no NF-E2 increase in transgenic cells, potentially explaining the lack of platelet increase in these transgenic mice. Transgenic megakaryocytes treated with Mpl ligand display a limited increase in NF-E2. In response to literature reports of Mpl ligand-induced transient increases in p21Cip1/WAF1 mRNA in polyploidizing megakaryocytic cell lines, we have examined p21 transcript levels in both normal and transgenic megakaryocytes. In normal mouse spleen, only a small percentage of megakaryocytes express detectable levels of p21 mRNA, with the majority of these cells expressing at high intensity. p21 levels are not affected by treatment with Mpl ligand, while the frequency of expressing cells increases transiently. Transgenic megakaryocytes exposed to Mpl ligand also show an increased frequency of p21-positive cells, and stimulation with Mpl ligand resulted in a further increase in this frequency. The nature of this effect will require further investigation.

A role for cyclin D3 in the endomitotic cell cycle.

Platelets, essential for thrombosis and hemostasis, develop from polyploid megakaryocytes which undergo endomitosis. During this cell cycle, cells experience abrogated mitosis and reenter a phase of DNA synthesis, thus leading to endomitosis. In the search for regulators of the endomitotic cell cycle, we have identified cyclin D3 as an important regulatory factor. Of the D-type cyclins, cyclin D3 is present at high levels in megakaryocytes undergoing endomitosis and is markedly upregulated following exposure to the proliferation-, maturation-, and ploidy-promoting factor, Mpl ligand. Transgenic mice in which cyclin D3 is overexpressed in the platelet lineage display a striking increase in endomitosis, similar to changes seen following Mpl ligand administration to normal mice. Electron microscopy analysis revealed that unlike such treated mice, however, D3 transgenic mice show a poor development of demarcation membranes, from which platelets are believed to fragment, and no increase in platelets. Thus, while our model supports a key role for cyclin D3 in the endomitotic cell cycle, it also points to the unique role of Mpl ligand in priming megakaryocytes towards platelet fragmentation. The role of cyclin D3 in promoting endomitosis in other lineages programmed to abrogate mitosis will need further exploration.

Delayed tyrosine phosphorylation and nuclear expression of STAT1 following antigen receptor stimulation of B lymphocytes.

The regulation of the STAT1 alpha transcription factor was assessed during B cell activation induced by cross-linking the surface IgM Ag receptor. Surface Ig ligation or pharmacologic stimulation with PMA and ionomycin resulted in the delayed (2-3 h after stimulation) nuclear appearance of tyrosine-phosphorylated STAT1 alpha, in contrast to the rapid induction that follows cytokine treatment. Nuclear expression of phosphorylated STAT1 alpha was abrogated by co-incubation of anti-Ig-treated B cells with the protein synthesis inhibitor cycloheximide (CHX), with the protein kinase inhibitor H-7, or with the immunosuppressive drug rapamycin. Tyrosine-phosphorylated STAT1 alpha was found to be recruited to the STAT binding site of the IFN regulatory factor-1 (IRF-1) gene promoter only after 2 to 3 h, and this association was also inhibitable by CHX and rapamycin. The arrival of STAT1 alpha coincided with attenuation of anti-Ig-induced STAT-binding activity specific for the IRF-1 promoter site, and both rapamycin and CHX treatment counteracted the loss of this activity. Furthermore, basal transcription of the endogenous IRF-1 gene was decreased as a result of anti-Ig treatment, and this effect of anti-Ig was blocked by co-incubation with rapamycin. Thus, STAT1 alpha plays a dynamic role in the composition of IRF-1 promoter-specific DNA binding complexes stimulated by B cell Ag receptor ligation, and nuclear expression of phosphorylated STAT1 alpha is regulated in a unique fashion by Ag receptor engagement. In addition, surface Ig cross-linking imparts negative regulatory control of IRF-1 gene expression, possibly through activation of STAT1 alpha.

A new transgenic mouse model for the study of cell cycle control in megakaryocytes.

During the development of the megakaryocytic lineage, the megakaryoblasts give rise to megakaryocytes which undergo repeated S phases in the absence of cytokinesis (endomitosis). The cellular oncogene myc plays a central role in the proliferation and differentiation of several cell types. In a previous study, we generated transgenic mice carrying c-myc fused to the estrogen receptor under the control of the platelet factor four (PF4) megakaryocyte-specific promoter. The bone marrow of female transgenic mice, but not of male mice, displayed increased megakaryopoiesis. Here we report that beta-estradiol-induced activation of c-myc in cultured bone marrow cells derived from male or female transgenic mice resulted in prolonged survival of the cells in vitro. Addition of a cocktail of hemopoietic growth factors to beta-estradiol-treated cells, including interleukin 6 (IL-6), IL-3 and stem cell factor further improved the survival time in culture and increased the percentage of large mature cells, but did not result in immortalization. The majority of these PF4-expressing cells, however, did not reach the differentiation stage at which acetylcholinesterase is expressed and did not appear as large megakaryocytes. We conclude that cultured megakaryocytes overexpressing myc are induced to proliferate, but have a limited potential to fully differentiate. Under these conditions, cyclin D3 was downregulated while the level of cyclin A was slightly upregulated.

Identification of potent, selective P2Y-purinoceptor agonists: structure-activity relationships for 2-thioether derivatives of adenosine 5'-triphosphate.

Study of P2-purinoceptor subtypes has been difficult due to the lack of potent and selective ligands. With the goal of developing high affinity P2-purinoceptor-selective agonist, we have synthesized a series of analogues of adenine nucleotides modified on the purine ring as chain-extended 2-thioethers or as N6-methyl-substituted compounds. Chemical functionality incorporated in the thioether moiety included cyanoalkyl, nitroaromatic, amino, thiol, cycloalkyl, n-alkyl, and olefinic groups. Apparent affinity of the compounds for P2Y-purinoceptors was established by measurement of P2Y-purinoceptor-promoted phospholipase C activity in turkey erythrocyte membranes and relaxation of carbachol-contracted smooth muscle in three different preparations (guinea pig taenia coli, rabbit aorta, and rabbit mesenteric artery). Activity at P2X-purinoceptors was established by measurement of contraction of rabbit saphenous artery and of the guinea pig vas deferens and urinary bladder. All 11 of the 2-thioethers of ATP stimulated the production of inositol phosphates with K0.5 values of 1.5-770 nM, with an (aminophenyl)ethyl derivative being most potent. Two adenosine diphosphate analogues were equipotent to the corresponding ATP analogues. Adenosine monophosphate analogues were full agonists, although generally 4 orders of magnitude less potent. ATP 2-thioethers displayed pD2 values in the range of 6-8 in smooth muscle assay systems for activity at P2Y-receptors. There was a significant correlation for the 2-thioether compounds between the pK0.5 values for inositol phosphate production and the pD2 values for relaxation mediated via the P2Y-purinoceptors in the guinea pig taenia coli, but not for the vascular P2Y-receptors or for the P2X-receptors. At P2X-receptors, no activity was observed in the rabbit saphenous artery, but variable degrees of activity were observed in the guinea pig vas deferens and bladder depending on distal substituents of the thioether moiety. N6-Methyl-ATP was inactive at P2X-receptors, and approximately equipotent to ATP at taenia coil P2Y-receptors. This suggested that hybrid N6-methyl and 2-thioether ATP derivatives might be potent and selective for certain P2Y-receptors, as was shown for one such derivative, N6-methyl-2-(5-hexenylthio)-ATP.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF NOVEL 2-THIO DERIVATIVES OF ATP.

2-Alkylthio analogues of adenosine 5'-triphosphate were synthesized and evaluated as P2y purinoceptor agonists. ATP and analogues transiently increased intracellular Ca2+ levels in C6 glioma cells and in skeletal muscle derived myotubes in culture. Most derivatives were resistant to stepwise dephosphorylation by ecto-ATPases.

Muscarinic receptor binding and activation of second messengers by substituted N-methyl-N-[4-(1-azacycloalkyl)-2-butynyl]acetamides.

A series of substituted azacycloalkyl analogues of the muscarinic agonist UH 5 (N-methyl-N-[4-(1-pyrrolidinyl)-2-butynyl]acetamide, 1a) were synthesized and evaluated pharmacologically. These compounds were developed as intermediates for further derivatization leading to functionalized congeners of 1a. The compounds were synthesized by using a Mannich-type condensation of N-acetyl-N-methylpropargylamine to various substituted saturated azaheterocycles. The compounds were screened at a single concentration in competitive binding assays in rat cerebral cortical membranes against either [3H]N-methylscopolamine (at 100 microM) or [3H]oxotremorine-M (at 1 microM) labels. Candidates were then selected for further evaluation of their effect on phosphoinositide (PI) turnover in membranes from A9L cells transfected with cDNA of either m1-muscarinic cholinergic receptors (m1AChRs) or m3AChRs. The analogues were also tested for the inhibition of adenylate cyclase in NG108-15 cells expressing m4AChRs. The azetidine analogue of 1a had a Ki value of 12 nM for the inhibition of [3H]oxotremorine-M binding in rat brain and had an agonist potency at m1-,m3-, and m4AChRs comparable to 1a. The substituted 5- and 6-member ring analogues generally had lower binding affinities and were less potent than 1a in stimulating PI turnover. Several compounds were moderately effective in inhibiting cyclic AMP production in NG108-15 cells.

Trifunctional agents as a design strategy for tailoring ligand properties: irreversible inhibitors of A1 adenosine receptors.

The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]- oxy]phenyl]-1,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H] CGS21680 (2-[2-[4-carboxyethyl)phenyl]ethyl]amino] adenosine-5'-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversible binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.

Adenosine analogs with covalently attached lipids have enhanced potency at A1-adenosine receptors.

Chemically functionalized congeners of N6-phenyladenosine and 1,3-dipropyl-8-phenylxanthine have been covalently coupled to fatty acids, diglycerides, and a phospholipid. The lipid-drug conjugates inhibit R-[3H]-phenylisopropyladenosine binding to A1-adenosine receptors in rat cerebral cortex membranes. A xanthine-phosphatidylethanolamine conjugate bound with a Ki value of 19 nM. Various xanthine esters of low potency are potential prodrugs. Amides of an adenosine amine congener (ADAC) with 18-carbon fatty acids exhibited Ki values at A1-adenosine receptors of 70 pM, representing a 130-fold enhancement over the affinity of the corresponding acetyl amide. The very high affinity of adenosine-lipid conjugates may be due to stabilization of these adducts in the phospholipid microenvironment of the receptor protein.

An historical look at a contemporary question: the cervical cap.

PIP: The cervical cap was most likely invented during the 19th century and was rediscovered in 1908 by a Viennese physician. The cap was always more popular in Europe than in the US, and the introduction of oral contraceptives and the IUD in the 1960s led to a declining interest in barrier methods. In 1977, the US Food and Drug Administration banned distribution of the cervical cap, presumably in reaction to outbreaks of toxic shock syndrome and despite rising interest in the device on the part of the woman's health movement. It is important for health educators to be informed about empirical research about the cervical cap so that they can counsel consumers in the event that the device is reclassified for general use. Acceptor studies have identified convenience, safety, spontaneity, and comfort as reasons for selecting the cervical cap, while difficult insertion and removal, odor, partner discomfort, and uncertainty about contraceptive effectiveness are cited as reasons for disliking this device. Dislodgement has been a major problem, experienced by almost half of cap acceptors at some point. Discontinuation rates after 6 months of use have been in the 25-40% range. No cases of pelvic inflammatory disease or significant cervical pathology have been recorded. The unplanned pregnancy rate associated with the cervical cap has been estimated to be about 8%. Omission of spermicide, dislodgement, faulty technique, and irregular usage account for most of these failures. There is a need for additional research addressing the issues and documenting the limits of safe cervical cap use.^ieng