Characterization of a Novel Role for the Tousled- like Kinase in Kinetochore Assembly and Function in Caenorhabditis elegans

Chromosome segregation is a critical step during cell division to avoid aneuploidy and promote proper organismal development. Correct sister chromatid positioning and separation during mitosis helps to achieve faithful transmission of genetic material to daughter cells. This prevents improper chromosome partitioning that can potentially result in extrachromosomal fragments, increasing the tumorigenic potential of the cells. The kinetochore is a protenaicious structure responsible for the initiation and orchestration of chromosome movement during mitosis. This highly conserved structure among eukaryotes is required for chromosome attachment to the mitotic spindle and failure to assemble the kinetochore results in aberrant chromosome segregation. Thus elucidating the mechanism of kinetochore assembly is important to have a better understanding of the regulation that controls chromosome segregation. Our previous work identified the C. elegans Tousled-like kinase (TLK-1) as a mitotic kinase and depletion of TLK-1 results in embryonic lethality, characterized by nuclei displaying poor mitotic chromosome alignment, lagging chromosome, and chromosome bridges during anaphase. Additionally, previous studies from our group revealed that TLK-1 is phosphorylated independently by Aurora B at serine 634, and by CHK-1 at threonine T610. The research presented herein reveals that both phosphorylated forms of TLK-1 associate with the kinetochore during mitosis. Moreover, by systematic depletion of kinetochore proteins, I uncovered that pTLK-1 is bona fide kinetochore component that is located at the outer kinetochore layer, influencing the microtubule-binding interface. I also demonstrated that TLK-1 is necessary for the kinetochore localization of the microtubule interacting proteins CLS-2 and LIS-1 and I show that embryos depleted of TLK-1 presented an aberrant twisted kinetochore pattern. Furthermore, I established that the inner kinetochore protein KNL-2 is an in vitro substrate of TLK-1 indicating a possible role of TLK-1 in regulating centromeric assembly. Collectively, these results suggest a novel role for the Tousled-like kinase in regulation of kinetochore assembly and microtubule dynamics and demonstrate the necessity of TLK-1 for proper chromosome segregation in C. elegans.

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

Nuclear proteins interacting with an AP-1/CRE-like promoter sequence in the human TNF$\alpha$ gene

Monocyte developmental heterogeneity is reflected at the cellular level by differential activation competence, at the molecular level by differential regulation of gene expression. LPS activates monocytes to produce tumor necrosis factor-$\alpha$ (TNF). Events occurring at the molecular level necessary for TNF regulation have not been elucidated, but depend both on activation signals and the maturation state of the cell: Peripheral blood monocytes produce TNF upon LPS stimulation, but only within the first 72 hours of culture. Expression of c-fos is associated with monocytic differentiation and activation; the fos-associated protein, c-jun, is also expressed during monocyte activation. Increased cAMP levels are associated with down regulation of macrophage function, including LPS-induced TNF transcription. Due to these associations, we studied a region of the TNF promoter which resembles the binding sites for both AP-1(fos/jun) and CRE-binding protein (or ATF) in order to identify potential molecular markers defining activation competent populations of monocytic cells.^ Nuclear protein binding studies using extracts from THP-1 monocytic cells stimulated with LPS, which stimulates, or dexamethasone (Dex) or pentoxyfilline (PTX), which inhibit TNF production, respectively, suggest that a low mobility doublet complex may be involved in regulation through this promoter region. PTX or Dex increase binding of these complexes equivalently over untreated cells; approximately two hours after LPS induction, the upper complex is undetectable. The upper complex is composed of ATF2 (CRE-BP1); the lower is a heterodimer of jun/ATF2. LPS induces c-jun and thus may enhance formation of jun-ATF2 complexes. The simultaneous presence of both complexes may reduce the amount of TNF transcription through competitive binding, while a loss of the upper (ATF2) and/or gain of the lower (jun-ATF2) allow increased transcription. AP-1 elements generally transduce signals involving PKC; the CRE mediates a cAMP response, involving PKA. Thus, this element has the potential of receiving signals through divergent signalling pathways. Our findings also suggest that cAMP-induced inhibition of macrophage functions may occur via down regulation of activation-associated genes through competitive binding of particular cAMP-responsive nuclear protein complexes. ^

Involvement of heparin-like glycosaminoglycans and expression of a novel heparan sulfate binding protein (p24) during human placentation and in a model for human implantation

Previous studies in our laboratory have indicated that heparan sulfate proteoglycans (HSPGs) play an important role in murine embryo implantation. To investigate the potential function of HSPGs in human implantation, two human cell lines (RL95 and JAR) were selected to model uterine epithelium and embryonal trophectoderm, respectively. A heterologous cell-cell adhesion assay showed that initial binding between JAR and RL95 cells is mediated by cell surface glycosaminoglycans (GAG) with heparin-like properties, i.e., heparan sulfate and dermatan sulfate. Furthermore, a single class of highly specific, protease-sensitive heparin/heparan sulfate binding sites exist on the surface of RL95 cells. Three heparin binding, tryptic peptide fragments were isolated from RL95 cell surfaces and their amino termini partially sequenced. Reverse transcription-polymerase chain reaction (RT-PCR) generated 1 to 4 PCR products per tryptic peptide. Northern blot analysis of RNA from RL95 cells using one of these RT-PCR products identified a 1.2 Kb mRNA species (p24). The amino acid sequence predicted from the cDNA sequence contains a putative heparin-binding domain. A synthetic peptide representing this putative heparin binding domain was used to generate a rabbit polyclonal antibody (anti-p24). Indirect immunofluorescence studies on RL95 and JAR cells as well as binding studies of anti-p24 to intact RL95 cells demonstrate that p24 is distributed on the cell surface. Western blots of RL95 membrane preparations identify a 24 kDa protein (p24) highly enriched in the 100,000 g pellet plasma membrane-enriched fraction. p24 eluted from membranes with 0.8 M NaCl, but not 0.6 M NaCl, suggesting that it is a peripheral membrane component. Solubilized p24 binds heparin by heparin affinity chromatography and $\sp{125}$I-heparin binding assays. Furthermore, indirect immunofluorescence studies indicate that cytotrophoblast of floating and attached villi of the human fetal-maternal interface are recognized by anti-p24. The study also indicates that the HSPG, perlecan, accumulates where chorionic villi are attached to uterine stroma and where p24-expressing cytotrophoblast penetrate the stroma. Collectively, these data indicate that p24 is a cell surface membrane-associated heparin/heparan sulfate binding protein found in cytotrophoblast, but not many other cell types of the fetal-maternal interface. Furthermore, p24 colocalizes with HSPGs in regions of cytotrophoblast invasion. These observations are consistent with a role for HSPGs and HSPG binding proteins in human trophoblast-uterine cell interactions. ^

Integrins in human T cell function: Transdominant regulation, conformational constraints, and intracellular effectors

Integrin adhesion molecules have both positive and negative potential in the regulation of peripheral blood T cell (PB T cell) activation, yet their mechanism of action in the mediation of human T lymphocyte function remains largely undefined. The goals of this study then were to elucidate integrin signaling mechanisms in PB T cells.^ By ligating $\beta$1 integrins with mAb 18D3, it was demonstrated that costimulation of PB T cell proliferation induced by coimmobilizing antibodies specific for $\beta$1, $\beta$2, and $\beta$7 integrin subfamilies in conjunction with the anti-CD3 mAb OKT3 was inhibited. Costimulation of T cell proliferation induced by non-integrins CD4, CD26, CD28, CD44, CD45RA, or CD45RO was unaffected. Inhibition of costimulation correlated with diminished IL-2 production. In his manner, $\beta$1 integrins could regulate heterologous integrins of the $\beta$2 and $\beta$7 subfamilies in a transdominant fashion. It was also demonstrated that integrin costimulation of T cell activation was acutely sensitive to the structural conformation of $\beta$1 integrins. Using the cyclic hexapeptide CWLDVC (TBC772, which is based on the $\alpha4\beta1$ integrin binding site in fibronectin) in soluble form, it was shown that integrins locked into a conformation displaying a neo-epitope called the ligand induced binding site (LIBS) recognized by mAb 15/7 were inhibited from sending mitogenic signals to T cells. When BSA-conjugated TBC772 was coimmobilized with anti-CD3 mAb OKT3, costimulation of proliferation occurred. This suggested that temporally uncoupling integrin receptor occupancy from receptor crosslinking inhibited $\beta$1 integrin signaling mechanisms. When subsets of PB T cells were examined to determine those initially activated by integrins within 6 hours of activation, costimulation induced intracellular accumulation of IL-2 predominantly in the CD4$\sp+$ and CD45RO$\sp+$ T cell subsets. This was similar to a number of PB T cell costimulatory molecules including CD26, CD43, CD44. Only CD28 costimulated IL-2 production from both CD45RA$\sp+$ and CD45RO$\sp+$ subpopulations.^ The GTPase Rho has been implicated in regulating integrin mediated stress fiber formation and anchorage dependent growth in fibroblasts, so studies were initiated to determine if Rho played a role in integrin dependent T cell function. In order to perform this, a technique based on scrape-loading was developed to incorporate macromolecules into PB T cells that maintained their functional activity. With this technique, C3 exoenzyme from Clostridium botulinum was incorporated into PB T cells. C3 ADP-ribosylates Rho proteins on Asn$\sp{41},$ which is in close proximity to the Rho effector domain, rendering it inactive. It was demonstrated that functional Rho is not required for basal or upregulated PB T cell adhesion to $\beta$1 integrin substrates, however PB T cell homotypic aggregation induced by PMA, which is an event mediated predominantly by the integrin $\rm\alpha L\beta2,$ was delayed. PB T cells lacking Rho function displayed altered cell morphology on $\beta$1 integrin ligands, producing stellate, dendritic-like pseudopodia. Rho activity was also found to be required for integrin dependent costimulation of proliferation. When intracellular accumulation of IL-2 was measured, inactivation of Rho prevented both integrin and CD28 costimulatory activity. Rho was identified to lie upstream of signals mediating PKC activation and Ca$\sp{++}$ fluxes, as PMA and ionomycin activation of PB T cells was unaffected by the inactivation of Rho. ^

REGULATION OF BRAIN NORADRENERGIC-RECEPTOR FUNCTION BY ADRENOCORTICOTROPHIN AND ANTIDEPRESSANT DRUGS (ADRENERGIC RECEPTOR, CYCLIC-AMP, ADENYLATE CYCLASE, IMIPRAMINE, STRESS)

Human behavior appears to be regulated in part by noradrenergic transmission since antidepressant drugs modify the number and function of (beta)-adrenergic receptors in the central nervous system. Affective illness is also known to be associated with the endocrine system, particularly the hypothalamic-pituitary-adrenal axis. The aim of the present study was to determine whether hormones, in particular adrencorticotrophin (ACTH) and corticosterone, may influence behavior by regulating brain noradrenergic receptor function.^ Chronic treatment with ACTH accelerated the increase or decrease in rat brain (beta)-adrenergic receptor number induced by a lesion of the dorsal noradrenergic bundle or treatment with the antidepressant imipramine. Chronic administration of ACTH alone had no effect on (beta)-receptor number although it reduced norepinephrine stimulated cyclic AMP accumulation in brain slices. Treatment with imipramine also reduced the cyclic AMP response to norepinephrine but was accompanied by a decrease in (beta)-adrenergic receptor number. Both the imipramine and ACTH treatments reduced the affinity of (beta)-adrenergic receptors for norepinephrine, but only the antidepressant modified the potency of the neurotransmitter to stimulate second messenger production. Neither ACTH nor imipramine treatment altered Gpp(NH)p- or fluoride-stimulated adenylate cyclase, cyclic AMP, cyclic GMP, or cyclic GMP-stimulated cyclic AMP phosphodiesterase, or the activity of the guanine nucleotide binding protein (Gs). These findings suggested that post-receptor components of the cyclic nucleotide generating system are not influenced by the hormone or antidepressant. This conclusion was verified by the finding that neither treatment altered adenosine-stimulated cyclic AMP accumulation in brain tissue.^ A detailed examination of the (alpha)- and (beta)-adrenergic receptor components of norepinephrine-stimulated cyclic AMP production revealed that ACTH, but not imipramine, administration reduced the contribution of the (alpha)-receptor mediated response. Like ACTH treatment, corticosterone diminished the (alpha)-adrenergic component indicating that adrenal steroids probably mediate the neurochemical responses to ACTH administration. The data indicate that adrenal steroids and antidepressants decrease noradrenergic receptor function by selectively modifying the (alpha)- and (beta)-receptor components. The functional similarity in the action of the steroid and antidepressants suggests that adrenal hormones normally contribute to the maintenance of receptor systems which regulate affective behavior in man. ^

Functional studies of insulin-like growth factor binding protein 2 pathway in glioma progression

Comparison of gene expressing profiles between gliomas with different grades revealed frequent overexpression of insulin-like growth factor binding protein 2 (IGFBP2) in glioblastomas (GBM), in which uncontrolled cell proliferation, angiogenesis, invasion and anti-apoptosis are hallmarks. Using the glia-specific gene transfer transgenic mouse and the stable LN229(BP2) GBM cell lines, we found that IGFBP2 by itself cannot transform cells in vitro and in vivo. IGFBP2 had growth inhibitory effects on mouse primary neural progenitors, but overexpression of IGFBP2 had no effect on GBM cells. ^ Although IGFBP2 does not initiate gliomagenesis, using tissue array technology, we observed strong correlation between IGFBP2 overexpression and VEGF up-regulation in human diffuse gliomas. Furthermore, overexpression of IGFBP2 in GBM cells not only enhanced VEGF expression but also increased the malignant potential of U87 MG cells in our angiogenesis xenograft animal model. ^ In parallel to these studies, using established stable SNB19 GBM cells that overexpress IGFBP2, we found that IGFBP2 significantly increased invasion by induction of matrix metalloproteinase-2 (MMP-2) as well as other invasion related genes, providing evidence that IGFBP2 contributes to glioma progression in part by enhancing MMP-2 gene transcription and in turn tumor cell invasion. ^ Finally, we found that primary filial cells infected with an anti-sense IGFBP2 construct have markedly increased sensitivity to γ irradiation and reduced Akt activation. On the other hand, SNB19(BP2) stable lines have consistently increased levels of Akt and NFkB activation, suggesting that one possible mechanism for anti-apoptosic function of IGFBP2 is through the activation of Akt and NFkB. Beside this, what is especially interesting is the finding that Akt protein was cleaved and inactivated during apoptosis by caspases, and IGFBP2 can prevent Akt cleavage, revealing another possible mechanism through it IGFBP2 exhibit strong antiapoptotic effects. Our data showed that IGFBP2 is a specific substrate for caspase-3, raising the possibility that IGFBP2 may inhibit apoptosis by a suicide mechanism. ^ In summary, using cellular, genomics, and molecular approaches, this thesis documented the potential roles of IGFBP2 in glioma progression. Our findings shed light on an important biological aspect of glioma progression and may provide new insights useful for the design of novel mechanism-based therapies for GBM. ^

A blood-borne PDGF/VEGF-like ligand initiates wound-induced epidermal cell migration in Drosophila larvae

Wound healing is a conserved survival response whose function is to restore the integrity of the tissue after physical trauma. Despite numerous studies in the wound healing field, the signals and pathways that orchestrate and control the wound healing program are still not entirely known. To identify additional signals and pathways that regulate epidermal wound repair in Drosophila larvae, we performed a pilot in vivo RNAi screen using a live reporter for epidermal morphology and a wounding assay. From our pilot screen we identified Pvr, the Drosophila homolog of the vertebrate PDGF/VEGF receptors, and six other genes as epidermal wound healing genes. Morphological analysis of wound-edge cells lacking Pvr or the Drosophila Jun N-terminal Kinase (JNK), previously implicated in larval wound closure, suggest that Pvr signaling leads to cell process extension into the wound site while JNK mediates transient dedifferentiation of wound-edge epidermal cells. Furthermore, we found that one of the three known Pvr ligands, Pvf1, is also required for epidermal wound closure. Through tissue-specific knock down and rescue experiments, we propose a model in which epidermally-produced Pvf1 may be sequestered into the hemolymph (blood) and that tissue damage locally exposes blood-borne Pvf1 to Pvr receptors on epidermal cells at the wound edge, thus initiating epidermal cell process extension and migration into the wound gap. Together, our data suggest that the Pvr and JNK signaling pathways act in parallel to control different aspects of wound closure and that PDGF/VEGF ligands and receptors may have a conserved autocrine role in epidermal wound closure. ^

Polycomb repressive complex 2 and induced basal-like breast cancer phenotype mediated by cyclin E/Cdk2

Despite of much success of breast cancer treatment, basal-like breast cancer subtype still presented as a clinical challenge to mammary oncologist for its lack of available targeted therapy owing to their negative expression of targeted molecules, such as PgR, ERα and Her2. These molecules are all critical regulators in mammary gland development. EZH2, a histone methyltransferase, by forming Polycomb Repressive Complex 2(PRC2) can directly suppress a large array of developmental regulators. Overexpression of cyclin E has also been correlated with basal-like (triple-negative) breast cancer and poor prognosis. We found an important functional link between these two molecules. Cyclin E/Cdk2 can enhance PRC2 function by phosphorylating a specific residue of EZH2, threonine 416 and increasing EZH2's ability to complex with SUZ12. This regulation would further recruit whole PRC2 complex to core promoter regions of these developmental regulators. The local enrichment of PRC2 complex would then trimethylate H3K27 around the core promoter regions and suppress the expression of targeted genes, which included PgR, ERα, erbB2 and BRCA1. This widespread gene suppressive effect imposed by highly active PRC2 complex would then transform the lumina) type cell to adopt a basal-like phenotype. This finding suggested deregulated Cdk2 activity owing to cyclin E overexpression may contribute to basal phenotype through enhancing epigenetic silencing effects by regulating PRC2 function. Inhibition of Cdk2 activity in basal-like cancer cells may help release the suppression, reexpress the silenced genes and become responsive to existing anti-hormone or anti-Her2 therapy. From this study, the mechanisms described here provided a rationale to target basal-like breast cancer by new combinational therapy of Cdk2 inhibitors together with Lapatinib, or Aromatin. ^

Structure-function analysis of human Integrator subunit-4

Structure-function analysis of human Integrator subunit 4 Anupama Sataluri Advisor: Eric. J. Wagner, Ph.D. Uridine-rich small nuclear RNAs (U snRNA) are RNA Polymerase-II (RNAPII) transcripts that are ubiquitously expressed and are known to be essential for gene expression. snRNAs play a key role in mRNA splicing and in histone mRNA expression. Inaccurate snRNA biosynthesis can lead to diseases related to defective splicing and histone mRNA expression. Although the 3′ end formation mechanism and processing machinery of other RNAPII transcripts such as mRNA has been well studied, the mechanism of snRNA 3′ end processing has remained a mystery until the recent discovery of the machinery that mediates this process. In 2005, a complex of 14 subunits (the Integrator complex) associated with RNA Polymerase-II was discovered. The 14subunits were annotated Integrator 1-14 based on their size. The subunits of this complex together were found to facilitate 3′ end processing of snRNA. Identification of the Integrator complex propelled research in the direction of understanding the events of snRNA 3’end processing. Recent studies from our lab confirmed that Integrator subunit (IntS) 9 and 11 together perform the endonucleolytic cleavage of the nascent snRNA 3′ end to generate mature snRNA. However, the role of other members of the Integrator complex remains elusive. Current research in our lab is focused on deciphering the role of each subunit within the Integrator complex This work specifically focuses on elucidating the role of human Integrator subunit 4 (IntS4) and understanding how it facilitates the overall function of the complex. IntS4 has structural similarity with a protein called “Symplekin”, which is part of the mRNA 3’end processing machinery. Symplekin has been thoroughly researched in recent years and structure-function correlation studies in the context of mRNA 3’end processing have reported a scaffold function for Symplekin due to the presence of HEAT repeat motifs in its N-terminus. Based upon the structural similarity between IntS4 and Symplekin, we hypothesized that Integrator subunit 4 may be behaving as a Symplekin-like scaffold molecule that facilitates the interaction between other members of the Integrator Complex. To answer this question, the two important goals of this study were to: 1) identify the region of IntS4, which is important for snRNA 3′ end processing and 2) determine binding partners of IntS4 which promote its function as a scaffold. IntS4 structurally consists of a highly conserved N-terminus with 8 HEAT repeats, followed by a nonconserved C- terminus. A series of siRNA resistant N and C-terminus deletion constructs as well as specific point mutants within its N-terminal HEAT repeats were generated for human IntS4 and, utilizing a snRNA transcriptional readthrough GFP-reporter assay, we tested their ability to rescue misprocessing. This assay revealed a possible scaffold like property of IntS4. To probe IntS4 for interaction partners, we performed co-immunoprecipitation on nuclear extracts of IntS4 expressing stable cell lines and identified IntS3 and IntS5 among other Integrator subunits to be binding partners which facilitate the scaffold like function of hIntS4. These findings have established a critical role for IntS4 in snRNA 3′ end processing, identified that both its N and C termini are essential for its function, and mapped putative interaction domains with other Integrator subunits.

Modulation of replicative senescence of skeletal muscle satellite cells by insulin -like growth factor-I (IGF-I)

Growth and regeneration of postnatal skeletal muscle requires a population of mononuclear myogenic cells, called satellite cells to add/replace myonuclei, which are postmitotic. Wedged between the sarcolemma and the basal lamina of the skeletal muscle fiber, these cells function as the stem cells of mature muscle fibers. Like other normal diploid cells, satellite cells undergo cellular senescence. Investigations of aging in both rodents and humans have shown that satellite cell self-renewal capacity decreases with advanced age. As a consequence, this could be a potential reason for the characteristically observed age-associated loss in skeletal muscle mass (sarcopenia). This provided the rationale that any intervention that can further increase the proliferative capacity of these cells should potentially be able to either delay, or even prevent sarcopenia. ^ Using clonogenicity assays to determine a cell's proliferation potential, these studies have shown that IGF-I enhances the doubling potential of satellite cells from aged rodents. Using a transgenic model, where the mice express the IGF-I transgene specifically in their striated muscles, some of the underlying biochemical mechanisms for the observed increase in replicative life span were delineated. These studies have revealed that IGF-I activates the PI3/Akt pathway to mediate downregulation of p27KIP1, which consequently is associated with an increase in cyclin E-cdk2 kinase activity, phosphorylation of pRb, and upregulation of cyclin A protein. However, the beneficial effects of IGF-I on satellite cell proliferative potential appears to be limited as chronic overexpression of IGF-I in skeletal muscles did not protect against sarcopenia in 18-mo old mice, and was associated with an exhaustion of satellite cell replicative reserves. ^ These results have shown that replicative senescence can be modulated by environmental factors using skeletal muscle satellite cells as a model system. A better understanding of the molecular basis for enhancement of proliferative capacity by IGF-I will provide a rational basis for developing more effective counter-measures against physical frailty. However, the implications of these studies are that these beneficial effects of enhanced proliferative potential by IGF-I may only be over a short-term period, and other alternative approaches may need to be considered. ^

Function of GCIP (CCNDBP1), a helix -loop -helix leucine -zip protein, in cell differentiation and tumorigenesis

Cell growth and differentiation are complex and well-organized processes in which cells respond to stimuli from the environment by carrying out genetic programs. Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation and tumorigenesis. This study has examined the roles of GCIP (CCNDBP1) in cell differentiation and tumorigenesis. GCIP is a recently identified HLH-leucine zipper protein without a basic region like the Id family of proteins. However, GCIP shares little sequence homology with the Id proteins and has domains with high acidic amino acids and leucine-rich regions following the HLH domain like c-Myc. Here we firstly demonstrate that GCIP is a transcription regulator related to muscle differentiation program. Overexpression of GCIP in C2C12 cells not only promotes myotube formation but also upregulates myogenic differentiation biomarkers, including MHC and myogenein. On the other hand, our finding also suggests that GCIP is a potential tumor suppressor related to cell cycle control. Expression of GCIP was significantly down-regulated in colon tumors as compared to normal colon tissues. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation on soft agar assays while silencing of GCIP expression by siRNA can promote cell proliferation and colony formation. In addition, results from transgenic mice specifically expressing GCIP in liver also support the idea that GCIP is involved in the early stage of hepatocarcinogenesis and decreased susceptibility to chemical hepatocarcinogenesis. ^

Heregulin -beta 1 directly associates with RhoA via the immunoglobulin -like domain thereby modulating breast cancer metastasis-related properties

Heregulins constitute a family of growth factors belonging to the epidermal growth factor (EGF) family. Breast cancers that overexpress specific members of the EGF receptor family (EGFR, ErbB2, ErbB3, ErbB4) have increased metastatic potential, and Heregulin-β1 (HRGβ1), a ligand for ErbB3 and ErbB4, has also been shown to induce metastasis-related properties in breast cancer cells in vitro. The secreted form of the HRGβ1 is composed of five distinct structural domains, including the N-terminal domain, an immunoglobulin-like domain (IgG-like), a glycosylation domain, an EGF-like domain, and a β1-specific domain. Of these, the EGF-like domain is well characterized for its function in metastasis-related properties as well as its structure. However, the contributions of the other HRGβ1 domains in breast cancer metastasis remains unclear. ^ To investigate this, HRGβ1 proteins with targeted domain deletions were purified and subjected to assays for metastasis-related properties, including aggregation, invasion, activation of EGFR family members, and motility of breast cancer cells. These assays showed that retaining the EGF-like domain of HRGβ1 is important for activation of EGFRs. Interestingly, the HRGβ1 protein lacking the IgG-like domain (NGEB) led to a decrease in breast cancer cell motility, indicating the IgG-like domain modulates cell motility, an important step in cancer metastasis. ^ To understand the underlying mechanisms, I performed protein sequence and structural analysis of HRGβ1 and identified that the IgG-like domain of HRGβ1 shares sequence homology and three-dimensional structural similarity with the IgG-like domain of TRIO. TRIO is a cytoplasmic protein that directly associates with RhoA, a GTPase involved in cell reorganization and cell motility. Therefore, I hypothesized that HRGβ1 may translocate inside the breast cancer cells through receptor mediated endocytosis and bind to RhoA via its IgG-like domain. I show wild type HRGβ1 but not NGEB binds RhoA in vitro and in vivo, leading to RhoA activation. Inhibition of HRG-β1 internalization via endocytosis disrupted HRGβ1 binding to RhoA. Additionally, breast cancer cell motility induced by HRG-β1 is reduced after treatment with inhibitors to both endocytosis and RhoA function, similar to levels seen with NGEB treatment. ^ Thus, in addition to the well-known role of HRGβ1 as an extracellular stimulator of the EGFR family members, HRGβ1 also functions within the cell as a binding partner and activator of RhoA to modulate cancer cell motility. ^