Apolipoprotein B100 (apoB), the structural protein component of triglyceride-rich very low density lipoprotein (VLDL) and atherogenic low density lipoprotein, is considered an important risk indicator of atherosclerosis. In insulin resistant states, hepatic overproduction of apoB leads to metabolic dyslipidemia, characterized by high circulating VLDL and hypertriglyceridemia. Since the mitogen-activated protein kinases (MAPKs) and the inhibitor of NFkappaB kinase (IKK)-NFkappaB cascades are perturbed in insulin resistance, we hypothesized that the MAPKs (ERK, p38 and JNK) and the IKK-NFkappaB pathways regulate hepatic apoB output. We modulated these pathways in HepG2, a human hepatoma cell line, and primary hamster hepatocytes using chemical inhibitors and protein overexpression. ApoB synthesis and secretion were examined by metabolic pulse labeling. HepG2 is typically defective in secreting apoB as large VLDL particles and secretes smaller triglyceride-poor apoB-particles. Under continuous pulse labeling, ERK inhibition not only increased apoB secretion, it enabled HepG2 to secrete VLDL-sized particles in the presence of exogenous fatty acid (oleate). Concomitant with the increased apoB-particle size, ERK inhibition raised intracellular triglyceride level and diacylglycerol acyltransferase (DGAT) 1 and DGAT2 mRNA levels. Conversely, ERK activation decreased VLDL-apoB secretion from primary hepatocytes. In contrast to ERK, p38 or JNK inhibition decreased apoB secretion without affecting apoB-particle size from oleate-treated HepG2 cells. JNK inhibition also modulated apoB levels in primary hamster hepatocytes. Interestingly, the development of diet-induced hepatic insulin resistance was associated with decreased ERK, and enhanced p38 and NFkappaB activities. Thus we investigated the role of the NFkappaB pathway in regulating hepatic apoB production. IKK inhibition decreased and IKK overexpression increased apoB levels by modulating apoB mRNA translation and protein stability. IKK inhibition also suppressed hepatic apoB overproduction in an insulin resistance model, the fructose-fed hamster. Altogether, our results suggest that among the MAPK cascades, the MEK-ERK pathway is crucial in regulating apoB-lipoprotein assembly, possibly by modulating lipid availability to newly-synthesized apoB. The inflammatory IKK-NFkappaB cascade is also involved in regulating apoB synthesis and secretion. We postulate that dysregulation in the MAPK or NFkappaB cascades in insulin resistant and inflammatory states may contribute to hepatic apoB overproduction, and the common phenotype of hypertriglyceridemia and dyslipidemia.

In this thesis, I describe studies identifying and characterizing two putative stem cell populations of the neural and pancreatic lineages. The mechanisms governing the emergence of the earliest mammalian neural cells during development and the ontogeny of neural stem cells remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition, however an instructive process has also been proposed. I utilized mouse ES cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals, and what cellular fate will result. Individual ES cells were found to rapidly transition directly into neural cells by a default mechanism, a process shown to be independent of suggested instructive factors. Further, I provide evidence that the default neural identity is that of a primitive neural stem cell, the earliest identified stem cell of the neural lineage. The exiguous conditions used to reveal the default state were found to present primitive neural stem cells with a survival challenge, which could be mitigated by survival factors or genetic interference with apoptosis. I also report the clonal identification of multipotent precursor cells, PMPs, from the adult mouse and human pancreas. These cells proliferate in vitro to form clonal colonies and display both pancreatic and neural cell multipotentiality. Importantly, the newly generated beta cells demonstrate glucose-dependent calcium responsiveness and regulated insulin release. PMP colonies do not express markers of embryonic stem cells, nor genes suggestive of mesodermal or neural crest origins. Moreover, genetic lineage-labeling experiments excluded the neural crest, and established the embryonic pancreatic lineage, as the developmental source of PMPs. The PMP cell was further found to express insulin in vivo, and insulin+ stem cells were shown to contribute to multiple pancreatic and neural cell populations in vivo. These findings demonstrate that the adult mammalian pancreas contains a population of insulin+ multipotent stem cells, capable of contributing to the pancreatic and neural lineages. In the final section of this thesis, I consider the relationships between neural and pancreatic tissues, as well as discussing the relevance of these two novel stem cell populations.

Neural crest stem cells (NCSCs) are embryonic multipotent cells that give rise to a wide range of cell types that include those forming the peripheral neural cells and the mesodermal cells of the face including the facial bones. In neonatal and adult skin, skin-derived precursor cells (SKPs) are multipotent dermal precursors that share similarities with NCSCs and can differentiate into peripheral neural and mesodermal cells, such as adipocytes. Based on the similarities between SKPs and NCSCs, I asked, in this thesis, whether rodent or human SKPs can differentiate into skeletal mesodermal cell types by determining their ability to differentiate into osteoblasts and chondrocytes. In culture, rodent and human SKPs differentiated into alkaline phosphatase-, osteopontin- and type-I collagen-positive osteoblasts that produced mineral deposits and into type-II collagen expressing chondrocytes. Clonal analysis showed that SKPs are multipotent for the osteogenic and chondrogenic lineages. To ask whether SKPs can generate these cells in vivo, genetically-tagged naïve rat SKPs were transplanted into a tibia bone fracture model. Six weeks post-transplantation, SKP-derived osteoblasts and osteocytes were present in the newly formed bone, showing their osteogenic differentiation in vivo. At three weeks post-transplantation, some of the injected cells differentiated into hypertrophic chondrocytes in the callus and others into perivascular cells in areas just outside the callus. To test whether it is the local environment that dictates the phenotype of transplanted SKPs, GFP-tagged undifferentiated rat SKPs were injected into the hypodermis of the skin, an adipogenic environment. Four weeks post-transplantation, SKPs differentiated into adipocytes, but not in inappropriate cell types. These results further the known differentiation potential of SKPs, show that local environment of a bone fracture or the hypodermis of the skin is sufficient to induce the differentiation of undifferentiated SKPs into appropriate cell types and suggest the use of SKPs as source of mesodermal precursor cells for cell therapy.

Bone Morphogenetic Proteins (BMPs) regulate numerous biological processes including neuronal development. The growth and morphological differentiation of dendrites are critical events in the establishment of proper neuronal connectivity and neural function. One extrinsic factor, BMP7, has been shown to specifically affect dendritic morphogenesis. Here, I describe the elucidation of novel signalling mechanisms involving the BMP type II receptor, BMPRII, during BMP7-dependent dendrite formation. The carboxy-terminal tail of BMPRII binds regulators of the cytoskeleton, and acts as a scaffold to localize and coordinate cytoskeletal remodelling. BMP7-induced localized remodelling of the actin and microtubule cytoskeleton mediated by BMPRII-bound LIMK1 and JNK, contributes to BMP7-dependent dendrite formation. In addition, in efforts to understand how BMP-induced activation of LIMK1 and JNK is regulated, upstream regulators, Rho GTPase, Cdc42, a Rho GTPase effector, PAK1, and a guanine exchange factor (GEF), αPIX, were identified as novel interacting partners of the BMP type I receptor, ALK2. Thus, elucidation of the molecular mechanisms governing BMP7-dependent dendrite formation, defines the extracellular cue, BMP7, as a critical regulator of cytoskeletal dynamics in neurons.

The acquisition and maintenance of cell division potential are important characteristics of tumorigenesis. Human telomerase reverse transcriptase (TERT) and telomerase RNA (TR) can immortalize cells through telomere maintenance, and telomerase activity is one factor that contributes to the in vitro transformation of normal cells. In vitro and in vivo evidence suggest that telomerase maintains telomeres as a functional multimer. In addition, hTERT may possess telomere maintenance-independent functions. To examine the effects of hTERT loss upon an in vitro generated tumorigenic cell line we created a tumorigenic cell line from human embryonic kidney cells through expression of the SV40 early region, H-RasG12V and a Cre-mediated excisable hTERT. These immortalized cells exhibited robust anchorage-independent colony growth and tumor formation in immuno-deficient mice. Cre recombinase expression resulted in the excision of hTERT from the tumorigenic cell lines, restoring cell mortality. A return to immortality was conferred by the re-introduction of wild-type hTERT, but not with hTERT point mutations in the N-terminus (hTEN) and reverse transcriptase (RT) domains that impair in vitro telomere elongation activity. The onset of cell mortality was not immediate, and the hTERT-excised tumorigenic cells exhibited clonal variation in the anchorage-independent colony growth assay and upon tumor formation in immuno-deficient mice. We hypothesized that tumorigenic potential was not related strictly to hTERT presence, but rather telomere length and/or integrity. To investigate this possibility we maintained the tumorigenic cell lines in the continuous presence of hTERT to permit telomere elongation prior to hTERT excision; subsequently, after hTERT excision tumor formation persisted, thus demonstrating a dependence on telomere length and not hTERT presence per se. To investigate the functional multimerization of hTERT in vivo we tested if two defective hTERT polypeptides with mutations in the hTEN and RT domains could restore telomere elongation activity in vivo. Unfortunately, during the course of these experiments an unanticipated recombination event occurred that restored a catalytically active hTERT transgene. Further in vivo analysis of hTERT multimerization should be carefully designed, accounting for the selective survival advantage bestowed by wild-type hTERT. This study provides compelling evidence that hTERT does not possess telomere maintenance-independent functions.

DNA damage represents a major challenge to the faithful replication and transmission of genetic information from one generation to the next. Cells utilize a highly integrated network of pathways to detect and accurately repair DNA damage. Mutations arise when DNA damage persists undetected, unrepaired, or repaired improperly. Mutations are a driving force of carcinogenesis and therefore many of the DNA damage surveillance and repair mechanisms guard against the transformation of normal cells into cancer cells. Central to the detection and repair of DNA damage is the relocalization of DNA damage surveillance proteins to DNA damage where they assemble into subnuclear foci and are capable to producing a signal that the cell interprets to induce cellular modifications such as cycle arrest and DNA repair which are important DNA damage tolerance. In this work, I describe my quest to understand the mechanisms underlying the assembly, maintenance, and disassembly of these DNA damage-induced foci and how they affect DNA damage signaling in Saccharomyces cerevisiae. First, I describe phenotypic characterization of a novel mutation that impairs assembly of the 9-1-1 checkpoint clamp complex into foci. Second, I describe my work to further understand the roles of the histone phosphatase Pph3 and phosphorylated histone H2A in modulating DNA damage signaling. Third, I include my work to uncover the possible mechanism by which the helicase Srs2 works to enable termination of DNA damage signaling. In summary, this thesis documents my efforts to understand the cellular and molecular nature of DNA damage signaling and how signaling is turned off in coordination with DNA damage repair.

DNA double-strand breaks (DSBs) are a threat to cell survival and genome integrity. In addition to canonical DNA repair systems, DSBs can be converted to telomeres by telomerase. This process, herein termed telomere healing, endangers genome stability since it usually results in chromosome arm loss. Therefore, cells possess mechanisms that prevent the untimely action of telomerase on DSBs. In this work, I reported the completion of a transposon mutagenesis screen in budding yeast and the identification of five novel genes (RRD1, CIK1, CTF18, RTS1, and IRC6) critical for telomere healing. The characterization of Rrd1 led to the surprising finding that Rrd1 facilitates telomere healing at DSBs with little or no TG-rich sequences but not at DSBs with long tracts of telomeric sequences. Pph3, a PP4 phosphatase, acts in conjunction with Rrd1 to promote telomere healing. Conversely, Mec1, the ATR ortholog, phosphorylates Cdc13 on its S306 residue to suppress its accumulation at DSBs. Rrd1 and Pph3 oppose Cdc13 S306 phosphorylation and are necessary for the efficient accumulation of Cdc13 at DSBs. Next, I found that Cik1 and its kinesin partner Kar3 are both important for telomere healing. Importantly, Kar3 contributes to telomere healing through its motor function. In contrast to Rrd1, Kar3 contributes to telomere healing regardless of telomeric sequence lengths adjacent to the break. Finally, Cik1 and Kar3 have a general role in DNA repair and physically associate with DSBs, which is dependent on the process of anchoring DSBs to nuclear periphery. In conclusion, I identified a mechanism by which the ATR family of kinases enforces genome integrity, a phosphoregulatory loop that underscores the contribution of Cdc13 to the fate of DNA ends, and a kinesin complex critical for the spatial organization of DNA repair.

It has been hypothesized that tumours are caricatures of normal tissue organization, where a minority cell population, the ‘stem cell’ of cancer, holds the exclusive ability for tumour propagation. These cancer stem cells (CSCs), or tumour-initiating cells, possess extensive self-renewal ability, through which they ensure maintenance of the tumourigenic clone. Such cells have been identified in various cancers, including colorectal, and have been proposed to be the source of tumour re-initiation following therapy. An important and currently unanswered question in solid tumours is whether all CSCs are equal or whether there is a gradient of potency within the CSC compartment. Using primary human colon tumour cells and sensitive in vivo functional assays, we have determined that colon CSCs are not uniform; rather, they vary with respect to their proliferative capacity, which is also linked to their response to chemotherapy. These findings hold therapeutic implications for colorectal cancer treatment since all types of CSCs must be eradicated to remove the risk of tumour relapse. While the CSC model may provide attractive answers to some challenging questions, it remains controversial. Ascertaining the importance of CSCs will come from targeted CSC therapies. Here we demonstrate that human colorectal CSC function is dependent on the self-renewal regulator BMI-1. Down-regulation of BMI-1 inhibits the ability of colorectal tumour-initiating cells to self-renew resulting in the abrogation of their tumourigenic potential. Treatment of primary colorectal cancer xenografts with small molecule BMI-1 inhibitors resulted in colorectal tumour-initiating cell loss with long-term and irreversible impairment of tumour growth. Targeting the BMI-1 related self-renewal machinery provides the basis for a new therapeutic approach in the treatment of colorectal cancer. Collectively, we have advanced the CSC field in two areas of importance. We show for the first time that the CSC pool encompasses a gradient of proliferative potential linked to chemotherapeutic response. Second, we provide critical proof for the clinical relevance of CSCs by inhibiting tumour growth through targeting of the self-renewal machinery. This body of work significantly advances our understanding of colorectal tumour-initiating cells.

Dictyostelium is a well established model for the study of differentiation and morphogenesis. It has previously been shown that Ca2+ and its primary sensor calmodulin (CaM) have roles in cell differentiation and morphogenesis in Dictyostelium and higher eukaryotes. Here I further elucidated the role of Ca2+ and CaM in cell differentiation in Dictyostelium. No previous work existed on the regulation of CaM-binding proteins (CaMBPs) or their binding partners by developmental morphogens. First, I gained insight into the developmental role of nucleomorphin (NumA1), a novel CaMBP, as well as its binding partners Ca2+-binding protein 4a (CBP4a) and puromycin-sensitive aminopeptidase A (PsaA). I showed that NumA1 and CBP4a expression is co-regulated by differentiation-inducing factor-1 (DIF-1), a stalk cell morphogen. Both proteins likely have a role in prestalk-O cell differentiation. On the other hand, I showed that PsaA expression is regulated by cAMP and PsaA regulates spore cell differentiation. Thus, NumA1 likely differentially regulates stalk and spore cell differentiation by interacting with CBP4a and PsaA, respectively. I also used Dictyostelium as a model to gain insight into the mechanism of action of colchicine, a microtubule disrupting agent that has been shown to affect differentiation and morphogenesis in many organisms. I identified that colchicine affects cell motility, disrupts morphogenesis, inhibits spore cell differentiation and induces stalk cell differentiation through a Ca2+ and CaM-dependent signal transduction pathway. It specifically induced differentiation of ecmB expressing stalk cells, independent of DIF-1 production. Lastly, I analyzed for the first time the role of Ca2+ and CaM in ecmB expression in vivo. I showed that Ca2+ and CaM regulate ecmB expression in intact and regenerating slugs and that Ca2+ and CaM also regulate cell differentiation, motility and slug shape. In conclusion, Ca2+ and CaM play integral roles in cell motility, cell differentiation and morphogenesis in Dictyostelium.

The AC133 epitope of the pentaspan transmembrane glycoprotein CD133 has been used as a cell-surface marker for normal and cancer stem cells from a broad range of tissue types. Despite the utility of CD133 as a marker, little is known regarding its regulation and biological function. To study these poorly understood aspects of CD133, I took two main experimental approaches: RNA interference (RNAi) screening and affinity purification coupled with mass spectrometry (AP-MS) to identify CD133 regulatory genes and CD133 protein-protein interactions (PPIs), respectively. Both of these experimental approaches relied on a human embryonic kidney (HEK) 293 cell line that exogenously expresses affinity tagged CD133 (HEK293/AC133). This cell line allowed me to perform a large-scale RNAi screen to interrogate 11,248 genes for their involvement in cell-surface AC133 recognition. This resulted in the identification of the N-glycosylation pathway as a direct contributor to CD133 plasma membrane localization and cell-surface AC133 detection. I used the same RNAi screening approach on the colon adenocarcinoma cell line Caco-2, which express CD133 from its native promoter, to identify factors that regulate endogenous CD133 transcription. I was able to demonstrate that AF4 promotes CD133 transcription in a number of cancer cell lines. Furthermore, I showed that CD133 expression in an acute lymphoblastic leukemia (ALL) cell line SEM, which is dependent on the mixed-lineage leukemia (MLL)-AF4 gene fusion, is critical for the viability of these cells. To gain further insight into the function of CD133, I performed AP-MS using HEK293/AC133 cells to identify CD133 PPIs. I identified histone deacetylase 6 (HDAC6) as a CD133 protein interaction partner. I found that HDAC6 negatively regulates CD133 trafficking into the endosomal-lysosomal degradation pathway. CD133 binds HDAC6 to prevent inhibition of HDAC6 deacetylase activity by phosphorylation. Protection of HDAC6 from phosphorylation promotes HDAC6 deacetylation of β-catenin, which results in β-catenin dependent signalling and the suppression of cancer cell differentiation. My thesis provide functional roles for CD133 as a pro-proliferative protein and as a key signalling protein in certain cancer cell lines.