A neuploidy (aberrant chromosome number) is a hallmark feature of human malignancies (1, 2) and has also been proposed as a necessary event for tumorigenesis (2). Although there have been many proposed hypotheses, there is no general agreement as to why aneuploidy is so highly prevalent in cancer cells, and how it contributes to tumor progression (3, 4). Importantly, if aneuploidy forms an underlying cause of human cancer, it has not been fully substantiated. The mechanisms of aneuploidy also remain a fundamental unresolved problem in cancer biology.To understand how aneuploidy might originate in mammalian tissues, we have focused on the elements that regulate chromosomal segregation, particularly those involved in sister chromatid cohesion and separation, because chromosome missegregation, for example during mitosis, can lead to aneuploidy. A key gene in our analysis is ESPL1, which encodes an endopeptidase called Separase that separates sister chromatids by cleaving cohesin Rad21/Mcd1/Scc1 during the metaphase to anaphase transition. The hypothesis we tested is that hormonal stimulation of the p53-null mouse mammary gland results in misexpression of the ESPL1 gene, thus promoting aneuploidy and breast cancer formation. Dysregulation of the mitotic machinery that helps maintain chromosomal stability in mammary cells can result in aneuploidy and subsequently, cancer formation. We focused on Separase for the following reasons that have important implications for breast cancer: (i) Separase plays a central role in promoting faithful chromosome segregation; (ii) our previous studies strongly indicated that hormonal stimulation of p53-null mice mammary gland results in overexpression of the ESPL1 and Separase protein, which may be a direct cause of aneuploidy (5); and (iii) siRNA-mediated knockdown of Separase and Separase deficient mouse embryonic fibroblasts results in genomic instability (6-8).An evolutionarily conserved protein complex called cohesin and an endopeptidase named Separase play pivotal roles in the accurate segregation of sister chromatids into two daughter cells. Cohesion along the length of the sister chromatids is formed during DNA replication in S phase. Cohesion along the chromosomal arms is removed during prophase and from centromeric regions at the metaphase-to-anaphase transition when Separase is activated after its inhibitory chaperone securin is degraded (9, 10).To understand how aberration in sister chromatid separation may contribute to chromosomal missegregation, we investigated the role of Separase overexpression in mouse mammary cells by using a mammary epithelial transplant model (11) as well as various biochemical and functional assays. Our results indicate that conditional overexpression of Separase alone in mammary epithelial cells with a p53 mutant background is sufficient to induce aneuploidy and tumorigenesis in vitro and in vivo. Results Conditional Expression of Mouse Separase (mSeparase) Results inAneuploidy in Mouse Mammary Epithelial Cells. To examine the direct effect of ...

The mechanism by which aspirin consumption is linked to significant reductions in the incidence of multiple forms of cancer and metastatic spread to distant tissues, resulting in increased cancer patient survival is not well understood. In this study, using colon cancer as an example, we provide both in vitro (cell culture) and in vivo (chemically-induced mouse model of colon cancer) evidence that this profound anti-neoplastic action may be associated with aspirin’s ability to irreversibly inhibit COX-1 mediated platelet activation, thereby blocking platelet-cancer cell interactions, which promote cancer cell number and invasive potential. This process may be driven by platelet-induced Epithelial-Mesenchymal Transition (EMT), as assessed using confocal microscopy, based upon changes in cell morphology, growth characteristics and fibronectin expression, and biochemical/molecular analysis by measuring changes in the expression of the EMT markers; vimentin, β-catenin, and SNAIL. We also provide evidence that a novel, GI-safer phosphatidylcholine (PC)-associated aspirin, PL2200 Aspirin, possesses the same or more pronounced actions vs unmodified aspirin with regard to antiplatelet effects (in vitro-reducing platelet activation as determined by measuring the release of thromboxane and VEGF release in culture medium; in vivo-inhibiting platelet number/activation and extravasation into tumor tissue) and chemoprevention (in vitro-inhibiting colonic cell growth, and invasive activity; in vivo-inhibiting colonic dysplasia, inflammation and tumor mass). These results suggest that aspirin’s chemopreventive effects may be due, in part, to the drug blocking the pro-neoplastic action of platelets; and the potential use of Aspirin-PC/PL2200 as an effective and safer chemopreventive agent for colorectal cancer and possibly other cancers.

Integrin ␣ IIb ␤ 3 activation is critical for platelet physiology and is controlled by signal transduction through kinases and phosphatases. Compared with kinases, a role for phosphatases in platelet integrin ␣ IIb ␤ 3 signaling is less understood. We report that the catalytic subunit of protein phosphatase 2A (PP2Ac) associates constitutively with the integrin ␣ IIb ␤ 3 in resting platelets and in human embryonal kidney 293 cells expressing ␣ IIb ␤ 3 . The membrane proximal KVGFFKR sequence within the cytoplasmic domain of integrin ␣ IIb is sufficient to support a direct interaction with PP2Ac. Fibrinogen binding to ␣ IIb ␤ 3 during platelet adhesion decreased integrinassociated PP2A activity and increased the phosphorylation of a PP2A substrate, vasodilator associated phosphoprotein. Overexpression of PP2Ac ␣ in 293 cells decreased ␣ IIb ␤ 3 -mediated adhesion to immobilized fibrinogen. Conversely, small interference RNA mediated knockdown of endogenous PP2Ac ␣ expression in 293 cells, enhanced extracellular signal-regulated kinase (ERK1/2) and p38 activation, and accelerated ␣ IIb ␤ 3 adhesion to fibrinogen and von Willebrand factor. Inhibition of ERK1/2, but not p38 activation, abolished the increased adhesiveness of PP2Ac ␣ -depleted 293 cells to fibrinogen. Furthermore, knockdown of PP2A c␣ expression in bone marrow-derived murine megakaryocytes increased soluble fibrinogen binding induced by protease-activated receptor 4-activating peptide. These studies demonstrate that PP2Ac ␣ can negatively regulate integrin ␣ IIb ␤ 3 signaling by suppressing the ERK1/2 signaling pathway.Integrin cytoplasmic tails are devoid of any intrinsic catalytic activity. Nevertheless, integrins can transmit bidirectional signals across the plasma membrane of a cell and regulate several cellular processes, such as, adhesion, migration, and apoptosis. In the context of the major platelet integrin ␣ IIb ␤ 3 , emerging evidence indicates that cytoplasmic tails act as a molecular scaffold for intracellular enzymes and for both cytoskeletal and adaptor proteins and can either positively or negatively regulate signaling (1). For example, during an agonist-mediated insideout signaling process, talin interacts with the integrin ␤ 3 tail and induces integrin ␣ IIb ␤ 3 activation (2), whereas calcium and integrin-binding protein 1 binds to the ␣ IIb tail and negatively regulates ␣ IIb ␤ 3 activation (3).Subsequent binding of fibrinogen to the activated ␣ IIb ␤ 3 integrin initiates an outside-in signaling process that regulates platelet function. Outside-in signaling can be mediated by intricate interplay of a set of proteins that associate constitutively with the integrin and by others that either associate or dissociate with the integrin in response to fibrinogen binding. For instance, c-Src associates constitutively to the ␤ 3 tail (4). Fibrinogen binding to ␣ IIb ␤ 3 induces association of protein tyrosine phosphatase 1B, spleen tyrosine kinase, and protein kinase C␤ to the ␤ 3 tail (4 -6) and calcium and integrin-binding protein 1 to t...

BACKGROUND Continuous-flow left ventricular assist devices (LVADs) expose blood cells to high shear stress, potentially resulting in the production of microparticles that express phosphatidylserine (PS+) and promote coagulation and inflammation. In this prospective study, we attempted to determine whether PS+ microparticle levels correlate with clinical outcomes in LVAD-supported patients. METHODS We enrolled 20 patients undergoing implantation of the HeartMate II LVAD and 10 healthy controls who provided reference values for the microparticle assays. Plasma was collected before LVAD implantation, at discharge, at 3-month follow-up, and when an adverse clinical event occurred. We quantified PS+ microparticles in the plasma using flow cytometry. RESULTS During the study period, 8 patients developed adverse clinical events: ventricular tachycardia storm (n=1), non–ST-elevation myocardial infarction (n=2), arterial thrombosis (n=2), gastrointestinal bleeding (n=2), and stroke (n=3). Levels of PS+ microparticles were higher in patients at baseline than in healthy controls (2.11%±1.26 vs 0.69±0.46, P=0.007). After LVAD implantation, patient PS+ microparticle levels increased to 2.39%±1.22 at discharge and then leveled to 1.97%±1.25 at 3-month follow-up. Importantly, patients who developed an adverse event had significantly higher levels of PS+ microparticles than did patients with no events (3.82%±1.17 vs 1.57%±0.59, P<0.001), even though the 2 patient groups did not markedly differ in other clinical and hematologic parameters. CONCLUSIONS Our results suggest that an elevation of PS+ microparticle levels may be associated with adverse clinical events. Thus, measuring PS+ microparticle levels in LVAD-supported patients may help identify patients at increased risk for adverse events.