A mechanism of insulin-like growth factor binding protein 2-induced cell mobility in glioma

Insulin-like growth factor binding protein 2 (IGFBP2) is a protein known to be overexpressed in a majority of glioblastoma multiforme (GBM) tumors. While it is known the IGFBP2 is involved in promoting GBM tumor cell invasion, no mechanism exists for how the protein is involved in signal transduction pathways leading to enhanced cell invasion. ^ We follow up on preliminary microarray data on IGFBP2-overexpressing GBM cells and protein sequence analysis of IGFBP2 in generating the hypothesis that IGFBP2 interacts with integnn α5 in regulating cell mobility. Microarray data showing upregulation of integrin α5 by IGFBP2 is validated and evidence of protein-protein interaction between IGFBP2 and integrin α5 is found. The exact binding domain on IGFBP2 responsible for its interaction with integrin α5 is also determined, confirming our initial findings and reaffirming that the IGFBP2/integrin α5 interaction is specific. Disruption of this interaction resulted in attenuation of IGFBP2-enhanced cell mobility. Further, we found that cell mobility is only enhanced when IGFBP2 and integrin α5 are both overexpressed and able to interact with each other. ^ We also determined fibronectin to be a critical player in the activation of the IGFBP2/integrin α5 pathway. The activation of this pathway appears to be progressive and initiates once GBM cells have sufficiently established anchorage. ^

Body mass index trajectories and predictors among 3rd to 12th graders using growth curve mixture modeling the Child and Adolescent Trial for Cardiovascular Health (CATCH) study

This dissertation examined body mass index (BMI) growth trajectories and the effects of gender, ethnicity, dietary intake, and physical activity (PA) on BMI growth trajectories among 3rd to 12th graders (9-18 years of age). Growth curve model analysis was performed using data from The Child and Adolescent Trial for Cardiovascular Health (CATCH) study. The study population included 2909 students who were followed up from grades 3-12. The main outcome was BMI at grades 3, 4, 5, 8, and 12. ^ The results revealed that BMI growth differed across two distinct developmental periods of childhood and adolescence. Rate of BMI growth was faster in middle childhood (9-11 years old or 3rd - 5th grades) than in adolescence (11-18 years old or 5th - 12th grades). Students with higher BMI at 3rd grade (baseline) had faster rates of BMI growth. Three groups of students with distinct BMI growth trajectories were identified: high, average, and low. ^ Black and Hispanic children were more likely to be in the groups with higher baseline BMI and faster rates of BMI growth over time. The effects of gender or ethnicity on BMI growth differed across the three groups. The effects of ethnicity on BMI growth were weakened as the children aged. The effects of gender on BMI growth were attenuated in the groups with a large proportion of black and Hispanic children, i.e., “high” or “average” BMI trajectory group. After controlling for gender, ethnicity, and age at baseline, in the “high BMI trajectory”, rate of yearly BMI growth in middle childhood increased 0.102 for every 500 Kcals increase (p=0.049). No significant effects of percentage of energy from total fat and saturated fat on BMI growth were found. Baseline BMI increased 0.041 for every 30 minutes increased in moderate-to-vigorous PA (MVPA) in the “low BMI trajectory”, while Baseline BMI decreased 0.345 for every 30 minutes increased in vigorous PA (VPA) in the “high BMI trajectory”. ^ Childhood overweight and obesity interventions should start at the earliest possible ages, prior to 3rd grade and continue through grade school. Interventions should focus on all children, but specifically black and Hispanic children, who are more likely to be highest at-risk. Promoting VPA earlier in childhood is important for preventing overweight and obesity among children and adolescents. Interventions should target total energy intake, rather than only percentage of energy from total fat or saturated fat. ^

Activation of c -Met contributes to growth and metastasis of human colorectal carcinoma

c-Met is the protein tyrosine kinase receptor for hepatocyte growth factor/scatter factor (HGF/SF) and mediates several normal cellular functions including proliferation, survival, and migration. Overexpression of c-Met correlates with progression and metastasis of human colorectal carcinoma (CRC). The goals of this study were to determine if overexpression of c-Met directly contributes to tumorigenicity and liver metastatic potential of colon cancer, and what are the critical pathways regulated by c-Met in this process. The studies used two colon tumor cell lines, KM12SM and KM20, which express high levels of constitutively active c-Met and are highly metastatic in nude mice. To examine the effects of c-Met overexpression, subclones of theses lines with reduced c-Met expression were obtained following transfection with a c-Met specific targeting ribozyme. Reduction of c-Met in KM12SM cells abolished liver metastases when cells were injected intrasplenically in an experimental metastasis assay. However, c-Met downregulation in theses clones was unstable. Three stable KM20 clones with a 25–35% reduction in c-Met protein levels but 60–90% reduction in basal c-Met autophosphorylation and kinase activity were obtained. While HGF increased c-Met kinase activity in the clones with reduced c-Met, the activity was less than that observed in parental or control transfected cells. Correlating with the reduction in c-Met kinase activity, subclones with reduced c-Met expression had significantly reduced in vitro growth rates, soft-agar colony forming abilities, and increased apoptosis. HGF/SF treatment did not affect anchorage-dependent growth or soft-agar colony forming abilities. Further, c-Met downregulation significantly impaired the ability of HGF/SF to induce migration. To examine the effects of reduced c-Met on tumor formation, parental and c-Met reduced KM20 cells were grown subcutaneously and intrahepatically in nude mice. c-Met downregulation delayed, but did not abolish growth at the subcutaneous site. When these cells were injected intrahepatically, both tumor incidences and size were significantly reduced. To further understand the molecular basis of c-Met in promoting tumor growth, the activation of several signaling intermediates that have been implicated in c-Met mediated growth, survival and migration were compared between KM20 parental cells and subclones with reduced c-Met expression levels. The expression and activity (as determined by phosphorylation) of AKT and Erk1/2 were unaltered. In contrast, Src kinase activity, as measured by immune complex kinase assay, was reduced 2–5 fold following c-Met downregulation. As Src has been implicated in growth, survival and migration, Src activation in c-Met overexpressing lines is likely contributing to the tumorigenic and metastatic capabilities of colon tumor cell lines that overexpress c-Met. Collectively, these results suggest that c-Met overexpression plays a causal role in the development of CRC liver metastases, and that c-Src and c-Met inhibitors may be of potential therapeutic benefit for late-stage colon cancer. ^

Seawater carbonate chemistry, nutrients and growth rate of coral (Acropora intermedia) and coral-reef seaweed (Lobophora papenfussii) during experiments, 2011

Space competition between corals and seaweeds is an important ecological process underlying coral-reef dynamics. Processes promoting seaweed growth and survival, such as herbivore overfishing and eutrophication, can lead to local reef degradation. Here, we present the case that increasing concentrations of atmospheric CO2 may be an additional process driving a shift from corals to seaweeds on reefs. Coral (Acropora intermedia) mortality in contact with a common coral-reef seaweed (Lobophora papenfussii) increased two- to threefold between background CO2 (400 ppm) and highest level projected for late 21st century (1140 ppm). The strong interaction between CO2 and seaweeds on coral mortality was most likely attributable to a chemical competitive mechanism, as control corals with algal mimics showed no mortality. Our results suggest that coral (Acropora) reefs may become increasingly susceptible to seaweed proliferation under ocean acidification, and processes regulating algal abundance (e.g. herbivory) will play an increasingly important role in maintaining coral abundance.

Infrastructure development and economic growth in China

China is the fastest growing country in the world for last few decades and one of the defining features of China's growth has been investment-led growth. China's sustained high economic growth and increased competitiveness in manufacturing has been underpinned by a massive development of physical infrastructure. In this context, we investigate the role of infrastructure in promoting economic growth in China for the period 1975 to 2007. Overall, the results reveal that infrastructure stock, labour force, public and private investments have played an important role in economic growth in China. More importantly, we find that Infrastructure development in China has significant positive contribution to growth than both private and public investment. Further, there is unidirectional causality from infrastructure development to output growth justifying China's high spending on infrastructure development since the early nineties. The experience from China suggests that it is necessary to design an economic policy that improves the physical infrastructure as well as human capital formation for sustainable economic growth in developing countries.

Promoting mixed stands through conversion treatments. Effect of holm oak coppices thinning on black pine regeneration

The effects of conversion treatments, depending on ecological factors and silvicultural parameters (thinning intensity, thinning type and rotation, among others) have been studied during the last fifteen years in an experimental trial in Central Spain. The general climate is continental Mediterranean; soils are low depth and limy; vegetation is an homogeneous dense coppices of Quercus ilex with isolated Pinus nigra trees. The experimental design (three locations) includes different thinning intensities (from 0 to 100% of extracted basal area). Inventories have been carried out in 1994 and 2010; thinning treatments were done in 1995 and 2011. Analysis of the effects of the conversion treatment show the increment of diameter and height growth rates, the canopy recovery and the stand resprouting, finding differences in these effects between thinning treatments. Besides the induced changes at holm oak stand, the application of conversion treatment clearly changed the woodland dynamics. Fifteen years after the thinnings, floristic composition varied and an abundant pine regeneration was installed in the woodland. In this work we describe the changes between inventories in tree species composition and diameter distribution, specially in the case of black pine. The conversion treatment caused changes in forest dynamics in the short term, increasing biodiversity and diversifying the forest structure. The fast installation of Pinus regeneration suggests the potential of the zone for the establishment of multipurpose mixed Quercus-Pinus stands in wide areas where Quercus species were favoured by human populations for firewood production. Conversion treatment of coppices, with the creation of mixed stands, constitutes a good management alternative for extensive areas and an interesting technique to adaptation to global change.

Distinct roles for E2F proteins in cell growth control and apoptosis

E2F transcription activity is composed of a family of heterodimers encoded by distinct genes. Through the overproduction of each of the five known E2F proteins in mammalian cells, we demonstrate that a large number of genes encoding proteins important for cell cycle regulation and DNA replication can be activated by the E2F proteins and that there are distinct specificities in the activation of these genes by individual E2F family members. Coexpression of each E2F protein with the DP1 heterodimeric partner does not significantly alter this specificity. We also find that only E2F1 overexpression induces cells to undergo apoptosis, despite the fact that at least two other E2F family members, E2F2 and E2F3, are equally capable of inducing S phase. The ability of E2F1 to induce apoptosis appears to result from the specific induction of an apoptosis-promoting activity rather than the lack of induction of a survival activity, because co-expression of E2F2 and E2F3 does not rescue cells from E2F1-mediated apoptosis. We conclude that E2F family members play distinct roles in cell cycle control and that E2F1 may function as a specific signal for the initiation of an apoptosis pathway that must normally be blocked for a productive proliferation event.

Cloning, expression, and characterization of a cDNA encoding a novel human growth factor for primitive hematopoietic progenitor cells

Multiple growth factors synergistically stimulate proliferation of primitive hematopoietic progenitor cells. A human myeloid cell line, KPB-M15, constitutively produces a novel hematopoietic cytokine, termed stem cell growth factor (SCGF), possessing species-specific proliferative activities. Here we report the molecular cloning, expression, and characterization of a cDNA encoding human SCGF using a newly developed λSHDM vector that is more efficient for differential and expression cloning. cDNA for SCGF encodes a 29-kDa polypeptide without N-linked glycosylation. SCGF transiently produced by COS-1 cells supports growth of hematopoietic progenitor cells through a short-term liquid culture of bone marrow cells and exhibits promoting activities on erythroid and granulocyte/macrophage progenitor cells in primary semisolid culture with erythropoietin and granulocyte/macrophage colony-stimulating factor, respectively. Expression of SCGF mRNA is restricted to myeloid cells and fibroblasts, suggesting that SCGF is a growth factor functioning within the hematopoietic microenvironment. SCGF could disclose some human-specific mechanisms as yet unidentified from studies on the murine hematopoietic system.

The cancer growth suppressor gene mda-7 selectively induces apoptosis in human breast cancer cells and inhibits tumor growth in nude mice

A differentiation induction subtraction hybridization strategy is being used to identify and clone genes involved in growth control and terminal differentiation in human cancer cells. This scheme identified melanoma differentiation associated gene-7 (mda-7), whose expression is up-regulated as a consequence of terminal differentiation in human melanoma cells. Forced expression of mda-7 is growth inhibitory toward diverse human tumor cells. The present studies elucidate the mechanism by which mda-7 selectively suppresses the growth of human breast cancer cells and the consequence of ectopic expression of mda-7 on human breast tumor formation in vivo in nude mice. Infection of wild-type, mutant, and null p53 human breast cancer cells with a recombinant type 5 adenovirus expressing mda-7, Ad.mda-7 S, inhibited growth and induced programmed cell death (apoptosis). Induction of apoptosis correlated with an increase in BAX protein, an established inducer of programmed cell death, and an increase in the ratio of BAX to BCL-2, an established inhibitor of apoptosis. Infection of breast carcinoma cells with Ad.mda-7 S before injection into nude mice inhibited tumor development. In contrast, ectopic expression of mda-7 did not significantly alter cell cycle kinetics, growth rate, or survival in normal human mammary epithelial cells. These data suggest that mda-7 induces its selective anticancer properties in human breast carcinoma cells by promoting apoptosis that occurs independent of p53 status. On the basis of its selective anticancer inhibitory activity and its direct antitumor effects, mda-7 may represent a new class of cancer suppressor genes that could prove useful for the targeted therapy of human cancer.

The Fibrinogen Globe of Tenascin-C Promotes Basic Fibroblast Growth Factor-induced Endothelial Cell Elongation

To investigate the potential role of tenascin-C (TN-C) on endothelial sprouting we used bovine aortic endothelial cells (BAECs) as an in vitro model of angiogenesis. We found that TN-C is specifically expressed by sprouting and cord-forming BAECs but not by nonsprouting BAECs. To test whether TN-C alone or in combination with basic fibroblast growth factor (bFGF) can enhance endothelial sprouting or cord formation, we used BAECs that normally do not sprout and, fittingly, do not express TN-C. In the presence of bFGF, exogenous TN-C but not fibronectin induced an elongated phenotype in nonsprouting BAECs. This phenotype was due to altered actin cytoskeleton organization. The fibrinogen globe of the TN-C molecule was the active domain promoting the elongated phenotype in response to bFGF. Furthermore, we found that the fibrinogen globe was responsible for reduced cell adhesion of BAECs on TN-C substrates. We conclude that bFGF-stimulated endothelial cells can be switched to a sprouting phenotype by the decreased adhesive strength of TN-C, mediated by the fibrinogen globe.

Angiostatin gene transfer: Inhibition of tumor growth in vivo by blockage of endothelial cell proliferation associated with a mitosis arrest

The antitumoral effects that follow the local delivery of the N-terminal fragment of human plasminogen (angiostatin K3) have been studied in two xenograft murine models. Angiostatin delivery was achieved by a defective adenovirus expressing a secretable angiostatin K3 molecule from the cytomegalovirus promoter (AdK3). In in vitro studies, AdK3 selectively inhibited endothelial cell proliferation and disrupted the G2/M transition induced by M-phase-promoting factors. AdK3-infected endothelial cells showed a marked mitosis arrest that correlated with the down-regulation of the M-phase phosphoproteins. A single intratumoral injection of AdK3 into preestablished rat C6 glioma or human MDA-MB-231 breast carcinoma grown in athymic mice was followed by a significant arrest of tumor growth, which was associated with a suppression of neovascularization within and at the vicinity of the tumors. AdK3 therapy also induced a 10-fold increase in apoptotic tumor cells as compared with a control adenovirus. Furthermore, we showed that systemic injection of AdK3 delayed C6 tumor establishment and growth, confirming that angiostatin can function in a paracrin manner. Our data support the concept that targeted antiangiogenesis, using adenovirus-mediated gene transfer, represents a promising alternative strategy for delivering antiangiogenic factors as their bolus injections present unsolved pharmacological problems.

Insulin-like growth factor 1 regulates developing brain glucose metabolism

The brain has enormous anabolic needs during early postnatal development. This study presents multiple lines of evidence showing that endogenous brain insulin-like growth factor 1 (Igf1) serves an essential, insulin-like role in promoting neuronal glucose utilization and growth during this period. Brain 2-deoxy-d- [1-14C]glucose uptake parallels Igf1 expression in wild-type mice and is profoundly reduced in Igf1−/− mice, particularly in those structures where Igf1 is normally most highly expressed. 2-Deoxy-d- [1-14C]glucose is significantly reduced in synaptosomes prepared from Igf1−/− brains, and the deficit is corrected by inclusion of Igf1 in the incubation medium. The serine/threonine kinase Akt/PKB is a major target of insulin-signaling in the regulation of glucose transport via the facilitative glucose transporter (GLUT4) and glycogen synthesis in peripheral tissues. Phosphorylation of Akt and GLUT4 expression are reduced in Igf1−/− neurons. Phosphorylation of glycogen synthase kinase 3β and glycogen accumulation also are reduced in Igf1−/− neurons. These data support the hypothesis that endogenous brain Igf1 serves an anabolic, insulin-like role in developing brain metabolism.

A serologically identified tumor antigen encoded by a homeobox gene promotes growth of ovarian epithelial cells

Ovarian carcinomas are thought to arise from cells of the ovarian surface epithelium by mechanisms that are poorly understood. Molecules associated with neoplasia are potentially immunogenic, but few ovarian tumor antigens have been identified. Because ovarian carcinomas can elicit humoral responses in patients, we searched for novel tumor antigens by immunoscreening a cDNA expression library with ovarian cancer patient serum. Seven clones corresponding to the homeobox gene HOXB7 were isolated. ELISAs using purified recombinant HOXB7 protein revealed significant serologic reactivity to HOXB7 in 13 of 39 ovarian cancer patients and in only one of 29 healthy women (P < 0.0001). Ovarian carcinomas were found to express HOXB7 at markedly higher levels than normal ovarian surface epithelium, suggesting that immunogenicity of HOXB7 in patients could be associated with its elevated expression in ovarian carcinomas. Overexpression of HOXB7 in immortalized normal ovarian surface epithelial cells dramatically enhanced cellular proliferation. Furthermore, HOXB7 overexpression increased intracellular accumulation and secretion of basic fibroblast growth factor, a potent angiogenic and mitogenic factor. These results reveal HOXB7 as a tumor antigen whose up-regulated expression could play a significant role in promoting growth and development of ovarian carcinomas.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.