Targeting Sphingosine Kinase 1 in Carcinoma Cells Decreases Proliferation and Survival by Compromising PKC Activity and Cytokinesis

Sphingosine kinases (SK) catalyze the phosphorylation of proapoptotic sphingosine to the prosurvival factor sphingosine 1-phosphate (S1P), thereby promoting oncogenic processes. Breast (MDA-MB-231), lung (NCI-H358), and colon (HCT 116) carcinoma cells were transduced with shRNA to downregulate SK-1 expression or treated with a pharmacologic SK-1 inhibitor. The effects of SK-1 targeting were investigated by measuring the level of intracellular sphingosine, the activity of protein kinase C (PKC) and cell cycle regulators, and the mitotic index. Functional assays included measurement of cell proliferation, colony formation, apoptosis, and cell cycle analysis. Downregulation of SK-1 or its pharmacologic inhibition increased intracellular sphingosine and decreased PKC activity as shown by reduced phosphorylation of PKC substrates. In MDA-MB-231 cells this effect was most pronounced and reduced cell proliferation and colony formation, which could be mimicked using exogenous sphingosine or the PKC inhibitor RO 31-8220. SK-1 downregulation in MDA-MB-231 cells increased the number of cells with 4N and 8N DNA content, and similar effects were observed upon treatment with sphingosine or inhibitors of SK-1 or PKC. Examination of cell cycle regulators unveiled decreased cdc2 activity and expression of Chk1, which may compromise spindle checkpoint function and cytokinesis. Indeed, SK-1 kd cells entered mitosis but failed to divide, and in the presence of taxol also failed to sustain mitotic arrest, resulting in further increased endoreduplication and apoptosis. Our findings delineate an intriguing link between SK-1, PKC and components of the cell cycle machinery, which underlines the significance of SK-1 as a target for cancer therapy

Pleistocene Climate, Phylogeny, and Climate Envelope Models: An Integrative Approach to Better Understand Species' Response to Climate Change

Mean annual temperature reported by the Intergovernmental Panel on Climate Change increases at least 1.1°C to 6.4°C over the next 90 years. In context, a change in climate of 6°C is approximately the difference between the mean annual temperature of the Last Glacial Maximum (LGM) and our current warm interglacial. Species have been responding to changing climate throughout Earth's history and their previous biological responses can inform our expectations for future climate change. Here we synthesize geological evidence in the form of stable oxygen isotopes, general circulation paleoclimate models, species' evolutionary relatedness, and species' geographic distributions. We use the stable oxygen isotope record to develop a series of temporally high-resolution paleoclimate reconstructions spanning the Middle Pleistocene to Recent, which we use to map ancestral climatic envelope reconstructions for North American rattlesnakes. A simple linear interpolation between current climate and a general circulation paleoclimate model of the LGM using stable oxygen isotope ratios provides good estimates of paleoclimate at other time periods. We use geologically informed rates of change derived from these reconstructions to predict magnitudes and rates of change in species' suitable habitat over the next century. Our approach to modeling the past suitable habitat of species is general and can be adopted by others. We use multiple lines of evidence of past climate (isotopes and climate models), phylogenetic topology (to correct the models for long-term changes in the suitable habitat of a species), and the fossil record, however sparse, to cross check the models. Our models indicate the annual rate of displacement in a clade of rattlesnakes over the next century will be 2 to 3 orders of magnitude greater (430-2,420 m/yr) than it has been on average for the past 320 ky (2.3 m/yr)

Developmental Stability Covaries with Genome-Wide and Single-Locus Heterozygosity in House Sparrows

Fluctuating asymmetry (FA), a measure of developmental instability, has been hypothesized to increase with genetic stress. Despite numerous studies providing empirical evidence for associations between FA and genome-wide properties such as multi-locus heterozygosity, support for single-locus effects remains scant. Here we test if, and to what extent, FA co-varies with single- and multilocus markers of genetic diversity in house sparrow (Passer domesticus) populations along an urban gradient. In line with theoretical expectations, FA was inversely correlated with genetic diversity estimated at genome level. However, this relationship was largely driven by variation at a single key locus. Contrary to our expectations, relationships between FA and genetic diversity were not stronger in individuals from urban populations that experience higher nutritional stress. We conclude that loss of genetic diversity adversely affects developmental stability in P. domesticus, and more generally, that the molecular basis of developmental stability may involve complex interactions between local and genome-wide effects. Further study on the relative effects of single-locus and genome-wide effects on the developmental stability of populations with different genetic properties is therefore needed

BMP-2/6 Heterodimer Is More Effective than BMP-2 or BMP-6 Homodimers as Inductor of Differentiation of Human Embryonic Stem Cells

Bone Morphogenetic Protein (BMP) signaling pathways are involved in differentiation of stem cells into diverse cell types, and thus BMPs can be used as main guidance molecules for in vitro differentiation of human stem cells.We have analyzed the ability for inducing differentiation of the heterodimer BMP-2/BMP-6 (BMP-2/6) compared to the homodimers BMP-2 or BMP-6, using human embryonic stem (hES) cells H9 as model system. When incubated in a medium with high concentration of basic fibroblastic growth factor (FGF2), 100 ng/ml of human recombinant BMPs induced morphological changes and differentiation of hES cells in 24 to 48 hours. After 5 days, expression of differentiation markers was induced and quantified by quantitative PCR (qPCR) and flow cytometry. BMP-2/6 exhibited stronger activity for the induction of the expression of trophectodermal (CDX2) and endodermal (SOX17, GATA4, AFP) markers than BMP-2 or BMP-6 homodimers. BMP-2/6 also induced the expression of BMPR2 gene more effectively than BMP-2 or BMP-6 when used at the same concentration and time. Moreover, the percentage of cells expressing the surface endodermal marker CXCR4 was also increased for the heterodimer when compared to both homodimers. BMP-2/6 was a more potent activator of Smad-dependent (SMAD1/5) and Smad-independent signaling (mitogen-activated protein kinases ERK and p38) than BMP-2 and BMP-6, and the activation of these pathways might play a role in its increased potency for inducing hES cell differentiation.Therefore, we conclude that BMP-2/6 is more potent than BMP-2 or BMP-6 for inducing differentiation of hES cells, and it can be used as a more powerful substitute of these BMPs in in vitro differentiation guidance

Turtle Carapace Anomalies: The Roles of Genetic Diversity and Environment

Background: Phenotypic anomalies are common in wild populations and multiple genetic, biotic and abiotic factors might contribute to their formation. Turtles are excellent models for the study of developmental instability because anomalies are easily detected in the form of malformations, additions, or reductions in the number of scutes or scales. Methodology/Principal Findings: In this study, we integrated field observations, manipulative experiments, and climatic and genetic approaches to investigate the origin of carapace scute anomalies across Iberian populations of the European pond turtle, Emys orbicularis. The proportion of anomalous individuals varied from 3 % to 69 % in local populations, with increasing frequency of anomalies in northern regions. We found no significant effect of climatic and soil moisture, or climatic temperature on the occurrence of anomalies. However, lower genetic diversity and inbreeding were good predictors of the prevalence of scute anomalies among populations. Both decreasing genetic diversity and increasing proportion of anomalous individuals in northern parts of the Iberian distribution may be linked to recolonization events from the Southern Pleistocene refugium. Conclusions/Significance: Overall, our results suggest that developmental instability in turtle carapace formation might be caused, at least in part, by genetic factors, although the influence of environmental factors affecting the developmental stability of turtle carapace cannot be ruled out. Further studies of the effects of environmental factors, pollutants an

KA1-targeted regulatory domain mutations activate Chk1 in the absence of DNA damage

The Chk1 protein kinase is activated in response to DNA damage through ATR-mediated phosphorylation at multiple serine-glutamine (SQ) residues within the C-terminal regulatory domain, however the molecular mechanism is not understood. Modelling indicates a high probability that this region of Chk1 contains a kinase-associated 1 (KA1) domain, a small, compact protein fold found in multiple protein kinases including SOS2, AMPK and MARK3. We introduced mutations into Chk1 designed to disrupt specific structural elements of the predicted KA1 domain. Remarkably, six of seven Chk1 KA1 mutants exhibit constitutive biological activity (Chk1-CA) in the absence of DNA damage, profoundly arresting cells in G2 phase of the cell cycle. Cell cycle arrest induced by selected Chk1-CA mutants depends on kinase catalytic activity, which is increased several-fold compared to wild-type, however phosphorylation of the key ATR regulatory site serine 345 (S345) is not required. Thus, mutations targeting the putative Chk1 KA1 domain confer constitutive biological activity by circumventing the need for ATR-mediated positive regulatory phosphorylation