Distinct types of glial cells populate the Drosophila antenna

Background: The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix- loop- helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, we have investigated whether similar types of cells exist in the Drosophila antenna. Results: We have used different P( Gal4) lines to drive Green Fluorescent Protein ( GFP) in distinct populations of cells within the Drosophila antenna. Mz317:: GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional similar to 30 glial cells detected by GH146:: GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. We denote these distinct populations of cells as Mz317- glia and GH146- glia respectively. In the adult, processes of GH146- glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317- glia form a peripheral layer. Ablation of GH146- glia does not result in any significant effects on the patterning of the olfactory receptor axons. Conclusion: We have demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146- glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca.

Cellular delivery of hammerhead ribozymes

Hammerhead ribozymes are potent RNA molecules which have the potential to specifically inhibit gene expression by catalysing the trans-cleavage of mRNAs. However, they are unstable in biological fluids and cellular delivery poses a problem. Site-specific chemical modification of hammerhead ribozymes was evaluated as a means of enhancing biological stability. Chimeric, 2'-O-methylated ribozymes, containing only five unmodified ribonucleotides, were catalytically active in vitro (kcat = 1.46 min-1) and were significantly more stable in serum and lysosomal enzymes than unmodified (all-RNA) counterparts. Furthermore, they remained undegraded in cell-containing media for up to 8 hours. Stability enhancement allowed cellular uptake properties of radiolabelled ribozymes to be assessed following exogenous delivery. Studies in vulval and glial cell lines indicated that chimeric ribozymes became cell-associated via an inefficient process, which was energy and concentration dependant. A considerable proportion of ribozymes remained bound to cell-surface components, however, a small proportion (<1%) were internalised via mechanisms of adsorptive and / or receptor mediated endocytosis. Fluorescent microscopy indicated that ribozymes were localised within endosomal / lysosomal vesicles following cell entry. This was confirmed by immuno-electron microscopy, which allowed the detection of biotin-labelled ribozymes within the cell ultrastructure. Despite the predominant localisation within endocytic vesicles, a small proportion of internalised ribozymes appeared able to exit these compartments and penetrate target sites within the nucleus and cytoplasm. The ribozymes designed in this report were directed against the epidermal growth factor receptor mRNA, which is over-expressed in a malignant brain disease called glioblastoma multiforme. In order to examine the fate of ribozymes in the brain, the distribution of FITC-labelled ribozymes was examined following intra-cerebro ventricular injection to mice. FITC-ribozymes demonstrated high punctate pattern of distribution within the striatum and cortex, which appeared to represent localisation within cell bodies and dendritic processes. This suggested that delivery to glial cells in vivo may be possible. Finally, strategies were investigated to enhance the cellular delivery of ribozymes. Conjugation of ribozymes to anti~transferrin receptor antibodies improved cellular uptake 3-fold as a result of a specific interaction with transferrin receptors. Complexation with cationic liposomes also significantly improved cell association, however, some toxiclty was observed and this could be a limitation to their use. Overall, it would appear that hammerhead ribozymes can be chemically stabilised to allow direct exogenous administration in vivo. However, additional delivery strategies are probably required to improve cellular uptake, and thus, allow ribozymes to achieve their full potential as pharmaceutical agents. KEYWORDS: Catalytic

Synthesis and biological properties of 2-phenylbenzothiazole derivatives

2-Phenylbenzothiazoles have structural similarities to the antioestrogenic 2-phenylindole, zindoxifene and to the oestrogenic isoflavone, genistein which also inhibits tyrosine kinases. Hydroxylated 2-phenylbenzothiazole derivatives were therefore produced and tested for oestrogenic and tyrosine kinase inhibitory activity. Synthesis of methoxy substituted 2-phenylbenzothiazoles was via the Jacobson method, demethylation being effected by boron tribromide at -70oC. Three amino substituted 2-phenylbenzothiazoles were also synthesised and tested for activity. Data is presented for oestrogen receptor binding activity, aromatase inhibitory activity, epidermal growth factor receptor tyrosine kinase (EGFRTK) inhibitory activity and cytotoxicity to ANN-1, 3T3, MCF-7 and WIDR cells. Oestrogen receptor binding affinity (RBA) was shown by five of the nine compounds tested. 2-(4-hydroxy)-6-hydroxybenzo-thiazole was the most active of the benzothiazoles tested (RBA 0.7). This is low but comparable to that of genistein. EGFRTK inhibitory activity was shown by four of the six benzothiazole derivatives tested; activity was comparable to that of genistein. Cytotoxicity assays have shown no selective toxicity of 2-phenylbenzothiazoles to any of the cell lines tested. Toxicity to MCF-7 cells was similar to that for other cell lines despite some compounds showing oestrogen receptor binding capacity. Amino-substituted 2-phenylbenzothiazoles showed selective toxicity towards transformed ANN-1 cells compared to normal 3T3 cells but the mechanism of this selectivity has not been established. Molecular modelling techniques, including CHEM-X, QUANTA and MOPAC were used to compare known ATP-competitive tyrosine kinase inhibitors with a model of ATP built from the crystal structure of the ATP-phosphoglycerate kinase complex. Structural features thought to be important to kinase inhibition were found and used to suggest further 2-phenylbenzothiazole analogues which may have improved activity.

Evaluating the evidence for targeting FOXO3a in breast cancer:a systematic review

Background: Tumour cells show greater dependency on glycolysis so providing a sufficient and rapid energy supply for fast growth. In many breast cancers, estrogen, progesterone and epidermal growth factor receptor-positive cells proliferate in response to growth factors and growth factor antagonists are a mainstay of treatment. However, triple negative breast cancer (TNBC) cells lack receptor expression, are frequently more aggressive and are resistant to growth factor inhibition. Downstream of growth factor receptors, signal transduction proceeds via phosphatidylinositol 3-kinase (PI3k), Akt and FOXO3a inhibition, the latter being partly responsible for coordinated increases in glycolysis and apoptosis resistance. FOXO3a may be an attractive therapeutic target for TNBC. Therefore we have undertaken a systematic review of FOXO3a as a target for breast cancer therapeutics. Methods: Articles from NCBI were retrieved systematically when reporting primary data about FOXO3a expression in breast cancer cells after cytotoxic drug treatment. Results: Increased FOXO3a expression is common following cytotoxic drug treatment and is associated with apoptosis and cell cycle arrest. There is some evidence that metabolic enzyme expression is also altered and that this effect is also elicited in TNBC cells. FOXO3a expression serves as a positive prognostic marker, especially in estrogen (ER) receptor positive cells. Discussion: FOXO3a is upregulated by a number of receptor-dependent and -independent anti-cancer drugs and associates with apoptosis. The identification of microRNA that regulate FOXO3a directly suggest that it offers a tangible therapeutic target that merits wider evaluation.

Studying Key Amino Acids in the Function of Ras Interference 1 (Rinl) SH2 and Vps9 Domains

Receptor mediated endocytosis effectively removes the "ears" with which a cell would "hear" a signal conveyed by extracellular signaling molecules, but does not necessarily block the signaling pathway in which the endocytosed receptor participates. In the process of signal attenuation, this newly formed vesicle is fused with a phagosome and the receptor molecules are degraded. Receptor mediated endocytosis as a way to attenuate epidermal growth factor (EGF) and insulin signaling will be the focus here. Ras Interference 1 (Rin 1) is a multifunctional protein involved in intracellular membrane trafficking and receptor mediated endocytosis through its Rab5 Guanine Exchange Factor and SH2 domains. The goal of this investigation is to determine the role of key amino acids involved in the interaction of Rinl with Epidermal Growth Factor Receptor and Rab5. To elucidate this role, a number of point mutations have been created and the effects of each mutation on Rin 1 function will be investigated. Key amino acids in the SH2 and Vps9 Domain were identified and effects of mutations on rate of endocytosis were observed.

Novel insights into the mechanisms of regulation of tyrosine kinase receptors by Ras Interference 1

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBP&agr;. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

Le glucagon-like peptide-I : un facteur de croissance et une hormone anti-apoptotique pour la cellule pancréatique[bêta] : étude de la transduction du signal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le glucagon-like peptide-I : un facteur de croissance et une hormone anti-apoptotique pour la cellule pancréatique[bêta] : étude de la transduction du signal

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Targeting the actin cytoskeleton in HER2+ metastatic cancer cells using a macrolide toxin

Breast and ovarian cancers are among the leading causes of cancer related deaths in women worldwide. In a subset of these cancers, dysregulation of the human epidermal growth factor receptor 2 (HER2) leads to overexpression of the receptor on the cell surface. Previous studies have found that these HER2+ cancers show high rates of progression to metastatic disease. Metastasis is driven by cytoskeletal rearrangements that produce filamentous actin (F-actin) based structures that penetrate and degrade extracellular matrix to facilitate tumour invasion. Advancements in targeted therapy have made F-actin an attractive target for the development of new cancer therapies. In this thesis, we tested the actin-depolymerizing macrolide toxin, Mycalolide B (MycB), as a potential warhead for a novel antibody drug conjugate (ADC) to target highly metastatic HER2+ breast and ovarian cancers. We found that MycB treatment of HER2+ breast (SKBR3, MDA-MB-453) and ovarian (SKOV3) cancer cells led to loss of viability (IC50 values ≤ 64 nM). Sub-lethal doses of MycB treatment caused potent suppression of leading edge protrusions, migration and invasion potential of HER2+ cancer cells (IC50 ≤ 32 nM). In contrast, other F-actin based processes such as receptor endocytosis were less sensitive to MycB treatment. MycB treatment skewed the size of endocytic vesicles, which may reflect defects in F-actin based vesicle motility or maturation. Given that HER2+ cancers have been effectively targeted by Trastuzumab and Trastuzumab-based ADCs, we tested the effects of a combination of Trastuzumab and MycB on cell migration and invasion. We found that MycB/ Trastuzumab combination treatments inhibited motility of SKOV3 cells to a greater degree than either treatment alone. Altogether, our results provide proof-of-principle that actin toxins such as MycB can be used as a novel class of warheads for ADCs to target and combat highly metastatic cancers.

Prognostic role of the LCS6 KRAS variant in locally advanced rectal cancer: results of the EXPERT-C trial.

Background: Lethal-7 (let-7) is a tumour suppressor miRNA which acts by down-regulating several oncogenes including KRAS. A single-nucleotide polymorphism (rs61764370, T > G base substitution) in the let-7 complementary site 6 (LCS-6) of KRAS mRNA has been shown to predict prognosis in early-stage colorectal cancer (CRC) and benefit from anti-epidermal growth factor receptor monoclonal antibodies in metastatic CRC. Patients and methods: We analysed rs61764370 in EXPERT-C, a randomised phase II trial of neoadjuvant CAPOX followed by chemoradiotherapy, surgery and adjuvant CAPOX plus or minus cetuximab in locally advanced rectal cancer. DNA was isolated from formalin-fixed paraffin-embedded tumour tissue and genotyped using a PCR-based commercially available assay. Kaplan–Meier method and Cox regression analysis were used to calculate survival estimates and compare treatment arms. Results: A total of 155/164 (94.5%) patients were successfully analysed, of whom 123 (79.4%) and 32 (20.6%) had the LCS-6 TT and LCS-6 TG genotype, respectively. Carriers of the G allele were found to have a statistically significantly higher rate of complete response (CR) after neoadjuvant therapy (28.1% versus 10.6%; P = 0.020) and a trend for better 5-year progression-free survival (PFS) [77.4% versus 64.5%: hazard ratio (HR) 0.56; P = 0.152] and overall survival (OS) rates (80.3% versus 71.9%: HR 0.59; P = 0.234). Both CR and survival outcomes were independent of the use of cetuximab. The negative prognostic effect associated with KRAS mutation appeared to be stronger in patients with the LCS-6 TT genotype (HR PFS 1.70, P = 0.078; HR OS 1.79, P = 0.082) compared with those with the LCS-6 TG genotype (HR PFS 1.33, P = 0.713; HR OS 1.01, P = 0.995). Conclusion: This analysis suggests that rs61764370 may be a biomarker of response to neoadjuvant treatment and an indicator of favourable outcome in locally advanced rectal cancer possibly by mitigating the poor prognosis of KRAS mutation. In this setting, however, this polymorphism does not appear to predict cetuximab benefit.

A comparison of three methods for detecting KRAS mutations in formalin-fixed colorectal cancer specimens.

BACKGROUND: KRAS mutation testing is required to select patients with metastatic colorectal cancer (CRC) to receive anti-epidermal growth factor receptor antibodies, but the optimal KRAS mutation test method is uncertain. METHODS: We conducted a two-site comparison of two commercial KRAS mutation kits - the cobas KRAS Mutation Test and the Qiagen therascreen KRAS Kit - and Sanger sequencing. A panel of 120 CRC specimens was tested with all three methods. The agreement between the cobas test and each of the other methods was assessed. Specimens with discordant results were subjected to quantitative massively parallel pyrosequencing (MPP). DNA blends were tested to determine detection rates at 5% mutant alleles. RESULTS: Reproducibility of the cobas test between sites was 98%. Six mutations were detected by cobas that were not detected by Sanger, and five were confirmed by MPP. The cobas test detected eight mutations which were not detected by the therascreen test, and seven were confirmed by MPP. Detection rates with 5% mutant DNA blends were 100% for the cobas and therascreen tests and 19% for Sanger. CONCLUSION: The cobas test was reproducible between sites, and detected several mutations that were not detected by the therascreen test or Sanger. Sanger sequencing had poor sensitivity for low levels of mutation.

Small molecules exploiting emerging therapeutic opportunities for breast cancer treatment

Among the different types of breast cancer (BC), the estrogen receptor positive (ER+) subtype, which requires estrogens for its growth and proliferation, is the most common, while triple negative BC, characterized by the absence of ER, progesterone receptor and human epidermal growth factor receptor 2, often leads to poor prognosis. First-line therapies for the treatment of ER+ BC act either by suppressing estrogen production, through the inhibition of aromatase (AR) enzyme, or by blocking estrogen prooncogenic activity, via the modulation/degradation of ERs. The serious side effects and the intrinsic or acquired resistance phenomena that arise with prolonged use of these drugs limit their therapeutic application, stimulating the search for new strategies to face this disease. In this context, the development of dual acting aromatase inhibitors, able to target both the orthosteric and the recently identified allosteric pockets of AR could be an opportunity to fight ER+ BC. Another promising strategy could be the development of multitarget compounds, targeting both AR and ERs. In this scenario, here we designed and synthesized two series of new xanthones or more flexible benzophenones as potential dual acting aromatase inhibitors. Moreover, inspired from tamoxifen metabolites and a literature compound endowed with activity on both AR and ER, different structurally related series of potential multitarget compounds were developed. The biological results showed that some of the new molecules were promising candidates for further development. It was recently observed that the lately discovered histamine H4 receptor is expressed in human breast tissue, displaying a key role in biological processes mediated by histamine such as cell proliferation, senescence, and apoptosis in malignant cells, representing a potential target in triple negative BC. Thus, a broad series of methyl quinazoline sulfonamides, carrying different functional groups on the sulfonamide moiety, were designed and synthesized as potential H4 receptor ligands.

Phenotypic and molecular investigation of circulating tumor cells (CTCs) isolated from breast cancer patients at single cell resolution

Despite the paramount advances in cancer research, breast cancer (BC) still ranks one of the leading causes of cancer-related death worldwide. Thanks to the screening campaign started in developed countries, BC is often diagnosed at early stages (non-metastatic BC, nmBC), but disease relapse occurrence even after decades and at distant sites is not an uncommon phenomenon. Conversely, metastatic BC (mBC) is considered an incurable disease. The major perpetrators of tumor spread to secondary organs are circulating tumor cells (CTCs), a rare population of cells detectable in the peripheral blood of oncologic patients. In this study, CTCs from patients diagnosed with luminal nmBC and mBC (hormone receptor positive, Human Epidermal Growth Factor Receptor 2 (HER2) negative) were characterized at both phenotypic and molecular levels. To better understand the molecular mechanisms underlying their biology and their metastatic potential, next-generation sequencing (NGS) analyses were performed at single-cell resolution to assess copy number aberrations (CNAs), single nucleotide variants (SNVs) and gene expression profiling. The findings of this study arise hints in CTC detection, and pave the way to new application in CTC research.

Genetic- or transforming growth factor-beta 1-induced changes in epidermal peroxisome proliferator-activated receptor beta/delta expression dictate wound repair kinetics.

Advances in wound care are of great importance in clinical injury management. In this respect, the nuclear receptor peroxisome proliferator-activated receptor (PPAR)beta/delta occupies a unique position at the intersection of diverse inflammatory or anti-inflammatory signals that influence wound repair. This study shows how changes in PPARbeta/delta expression have a profound effect on wound healing. Using two different in vivo models based on topical application of recombinant transforming growth factor (TGF)-beta1 and ablation of the Smad3 gene, we show that prolonged expression and activity of PPARbeta/delta accelerate wound closure. The results reveal a dual role of TGF-beta1 as a chemoattractant of inflammatory cells and repressor of inflammation-induced PPARbeta/delta expression. Also, they provide insight into the so far reported paradoxical effects of the application of exogenous TGF-beta1 at wound sites.