HRI/188/03 Life and Death of the Cell: Determinants of Cell Fate: Control of cell fate in the mammary gland; cytokine: growth factor interactionsHRI/190/03 Life and Death of the Cell: Determinants of Cell Fate: The role of proteases involved in extracellular matrix modelling in the determination of cell death or tumorigenesis
HRI/104/03 Lifestyle, metabolism and health: Fatty acid metabolism in the liver
HRI/105/03 Lifestyle, metabolism and health: Molecular characterisation of genes and gene-products involved in fatty acid metabolism
HRI/129/03 Life and death of the cell: Determinants of cell fate: Seeking a mechanistic explanation for the protective effect of lactation against mammary cancer
HRI/130/03 Tissue Growth and Development: The role of amino acid transport in mammary epithelial cell survival: importance of system L in normal and cancerous human mammary epithelial cells.
HRI/131/03 Animal physiology and product quality: Mammary Gland: Potassium and chloride transport in normal and tumorigenic mammary epithelial cells
HRI/158/03 Regulation of control and development: Mechanism and consequences of ectopic lipid accretion: contribution of de novo synthesis and oxidative rates
HRI/187/02 Life and Death of the Cell: Structure and function of some rheomorphic phosphoproteins
HRI/189/03 Life and death of the cell: Determinants of cell fate: Adipose tissue: mammary gland interactions; implications of maternal obesity for mammary gland function, cancer risk and neonatal development
HRI/188/03 Life and Death of the Cell: Determinants of Cell Fate: Control of cell fate in the mammary gland; cytokine: growth factor interactions
Final Costs: £700,360
This programme seeks to identify the key role of prolactin, growth hormone and the insulin-like growth factor (IGF) axis in the control of mammary cell death, in order to examine its potential utility for controlling the life span and determining the fate of mammary epithelial cells. In particular we will focus on the role of insulin-like growth factor binding proteins (IGFBPs) which can bind to and inhibit IGFs and thus exhibit the potential to induce cell death via apoptosis. We have demonstrated that IGFBP-5 is upregulated during involution of the mammary gland and proposed that this serves to inhibit IGF-mediated cell survival. We therefore produced a transgenic mouse expressing IGFBP-5 in the mammary gland and showed that it induces premature apoptosis. However, during differentiation of the mammary cell, IGFBP-5 synthesis is also increased although at lower levels. We hypothesise that IGF-I acts as a mitogen for mammary epithelial cells in the absence of IGFBP-5, that low levels of IGFBP production can switch IGF signalling to a differentiation pathway and that,with high concentrations of IGFBP (such as occur in involution), IGF signalling is completely blocked and cell death via apoptosis ensues These studies will thus provide a mechanistic insight into the fundamental processes which control the life and death of the cell and thereby offer potential therapeutic approaches for the control of tumourigenesis.
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HRI/190/03 Life and Death of the Cell: Determinants of Cell Fate: The role of proteases involved in extracellular matrix modelling in the determination of cell death or tumorigenesis
Final Costs: £635,932
Natural involutionary processes which occur in the mammary gland at the end of lactation involve programmed cell death and remodelling of the extracellular matrix (ECM). These processes are clearly coordinated and are controlled principally by prolactin, growth hormone (GH) and the IGF system. The process of ECM remodelling involves increased expression and activation of plasmin and the matrix metalloproteinases (MMPs). The MMPs are in fact activated by plasmin and we now have evidence that an IGF binding protein, IGFBP-5, can stimulate this system. IGFBP-5 can thus inhibit IGF-mediated cell survival and activate tissue remodelling. As such we propose that IGFBP-5 has a central coordinating role in tissue death. Using mutant forms of this protein we will explore its potential to influence cell-matrix interactions, cell migration (metastatic) potential and to compare these with its apoptotic actions. Such mutant IGFBPs will provide important information about IGF actions in tissue remodelling but will also offer the possibility of developing anti-metastatic as well as anti-tumorigenic therapies. In addition, by exploring the control of protease gene expression during remodelling of the mammary gland we aim to identify novel therapeutic control points to regulate the activity of these important proteases, since their expression or activation is often deranged during tumourigenesis and metastasis.
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HRI/104/03 Lifestyle, metabolism and health: Fatty acid metabolism in the liver
Final Costs: £444,653
One of the major causes of morbidity in Western societies, and the West of Scotland in particular, is the oversupply of fatty acids and triglycerides to tissues. Excessive secretion of triglycerides by the liver is a cause of hyperlipidaemia. Steatosis in the liver (non-alcohol induced fatty liver) is emerging as an important cause of diet- and other lifestyle-related morbidity. The occurrence of hyperlipidaemia and non-alcoholic fatty liver is highly related with the incidence of insulin resistance in peripheral tissues. The objective of this project is to study the role of fatty acid metabolism, and its regulation in the control of the pathways that determine the rates of secretion and storage of triglyceride in the liver, and the rate of fatty acid oxidation to products released by the liver. Conditions in which there is imbalance between the processes of fat oxidation and the synthesis of complex lipids result in metabolic derangements in all tissues, but are particularly important in the liver, as this tissue orchestrates whole-body lipid metabolism, and has the ability to precipitate the occurrence of coronary heart disease and type-2 diabetes through excessive secretion of triglycerides within very-low-density lipoproteins.
The regulation of the key proteins involved in the control of fatty acid oxidation, and the partitioning of triglycerides between storage and secretion will be studied in order to determine the factors that affect the excessive storage and/or secretion of lipids by the liver. These studies are already sponsored by the pharmaceutical industry, and are expected to lead to further attainments in developing dietary and pharmacological strategies for the alleviation of metabolic diseases, such as diabetes and obesity.
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HRI/105/03 Lifestyle, metabolism and health: Molecular characterisation of genes and gene-products involved in fatty acid metabolism
Final Costs: £341,232
We have shown that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now proved the existence of such N-C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N-C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N-C interactions. This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N-C interactions. Finally, we have shown that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser24-->Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40-47) as being involved in the chemical N-C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.
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HRI/129/03 Life and death of the cell: Determinants of cell fate: Seeking a mechanistic explanation for the protective effect of lactation against mammary cancer
Final Costs: £607,597
The project investigated the hypothesis that lactation might protect against breast cancer by effecting a permanent alteration in cell kinetics in the mammary gland. Mouse models were used and cell kinetics were measured using immunohistochemical techniques. The hypothesis was supported; measurements of cell proliferation and cell death made at several stages of the estrous cycle indicated reduced and increased levels, respectively, in mice that had previously lactated. Both factors potentially reduce the risk of mammary tumours arising.
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HRI/130/03 Tissue Growth and Development: The role of amino acid transport in mammary epithelial cell survival: importance of system L in normal and cancerous human mammary epithelial cells.
Final Costs: £309,022
The main objective of this project was to examine the transport of amino acids and their metabolism by human breast cancer cells. Breast cancer cells require an adequate supply of amino acids to survive. Therefore, a detailed knowledge of the systems responsible for transporting amino acids into cancer cells could help develop strategies aimed at limiting tumour growth. The project concentrated on the functional and molecular characteristics of a transport mechanism known as system L. This transporter is responsible for the uptake of several essential amino acids and is therefore a potentially important therapeutic target. During the course of the project it was established that human breast cancer cells express system L at relatively high levels and confirmed that this mechanism is at the forefront of supplying the cells with a number of essential amino acids. However, the expression of the transporter differed between estrogen receptor-positive and -negative cells. Indeed the differential expression of system L could potentially be used as an important diagnostic tool. It was also established that system L supplied breast cancer cells with tryptophan., an amino acid which is the main substrate of an enzyme called indoleamine 2,3-dioxygenase. This enzyme may be involved in helping cancer cells escape the attention of the immune system. Therefore, inhibition of tryptophan transport and/or indoleamine 2,3-dioxygenase activity in breast cancer cells may be a way of helping the immune system mount an attack on breast tumours.
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HRI/131/03 Animal physiology and product quality: Mammary Gland: Potassium and chloride transport in normal and tumorigenic mammary epithelial cells
Final Costs: £377,179
Potassium (K+) and chloride (Cl-) efflux from human breast cancer cells may be important in determining cell death, and hence may be linked to tumour proliferation. Thus a knowledge of the physiology of these processes in breast cells may give a better understanding of their role (if any) in breast disease. We used human breast cancer cell lines MDA MB 231 (estrogen insensitive) & MCF-7 (estrogen sensitive) to study factors affecting potassium K+ and Cl- efflux using the radioactive isotopes of rubidium (Rb+) and iodine (I-) as markers. When cells were swollen by reducing external sodium, K+ efflux occurred in both MDA MB 231 & MCF 7 cells, though to a lesser extent in the latter. Adenosine triphosphate (ATP) stimulated K+ efflux from MDA MB 231 cells, but not MCF 7 cells, despite causing a rise in calcium in both; the effect of ATP was modulated in part by calcium influx. Uridine triphosphate (UTP) also stimulated K+ efflux from MDA MB 231 cells, and the effect of ATP was inhibited by suramin, suggesting the effect was mediated by ATP P2Y2 receptor subtype in these cells. A combination of cell swelling and ATP treatment caused great K+ efflux in MDA MB 231 cells than the sum of the individual treatments.
Blocking Cl- channels with diiodosalicylate reduced the efflux of taurine which was stimulated by cell swelling in both cell lines. Another blocker of Cl- and related channels 4,4'-Diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) had a greater effect on MCF 7 cells than MDA MB 231; the profiles of taurine, Cl- and I- efflux were different, suggesting taurine does not use the same efflux pathway as the anions. ATP stimulated taurine efflux from MDA MB 231 cells (but not MCF 7) by a suramin-sensitive path, as did UTP. Suramin also inhibited swelling-induced taurine efflux in the absence of added ATP in both cell types, suggesting cell-released ATP may be acting on membrane receptors in an autocrine manner. Raising cell calcium with ionomycin enhanced volume-stimulated taurine efflux in MDA MB 231 but not MCF 7 cell. These results indicate that ATP could regulate ion and taurine efflux and thus volume regulation in an estrogen insensitive human breast cancer cell line, but to a lesser extent in estrogen sensitive human mammary cancer cells.
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HRI/158/03 Regulation of control and development: Mechanism and consequences of ectopic lipid accretion: contribution of de novo synthesis and oxidative rates
Final Costs: £668,343
Accumulation of fat in a variety of tissues other than adipose tissue is associated with a number of pathological conditions, notably insulin resistance, type 2 diabetes, cardiovascular and renal disease, and appears to contribute to impaired tissue integrity coincident with these disease states. Consequently, strategies aimed at alleviating this so-called ectopic lipid accumulation are postulated to restore tissue integrity and contribute to the regaining of normal metabolic function. Lipid accumulation is postulated to occur as a result of enhanced uptake of lipid from the blood or enhanced de novo synthesis of fatty acids within the affected tissue coincident with an impaired ability to metabolise the lipid through mitochondrial ß-oxidation. The synthesis of fatty acids and their oxidation in the mitochondrial pathway is controlled by the metabolite, malonyl-CoA, the product of the enzyme Acetyl-CoA carboxylase (ACC). Consequently, strategies to regulate malonyl-CoA levels by modulating the activity of ACC in the affected tissues may contribute to restoration of metabolic function. ACC activity results from the transcription of two related genes. ACC-alpha is localised mainly in the cytosol where it appears to be fundamental in the synthesis of fatty acids; ACC-beta is the principal regulator of ß-oxidation and is postulated to exist in a unique relationship with mitochondria. We have addressed the roles of these enzyme systems in regulating malonyl-CoA levels in key subcellular compartments of the cell as means to understand the metabolic dysfunctions resulting in ectopic lipid accumulation resulting from impaired adipocyte function.
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HRI/187/02 Life and Death of the Cell: Structure and function of some rheomorphic phosphoproteins
Final Costs: £1,023,600
The project resulted in the development of an equilibrium thermodynamic model of the interaction of calcium, phosphate and casein in milk in which the micellar calcium phosphate is assumed to be in the form of calcium phosphate nanoclusters. A generalized empirical formula for the nanocluster is used to define the molar ratios of small ions (Ca, Mg, P(i) and citrate) to a casein phosphorylated sequence (phosphate centre, PC). From this model, a method of calculating the partition of milk salts into diffusible and non-diffusible fractions is obtained. No arbitrary assumptions are made, no fitting of adjustable parameters is done and the PCs in the caseins are defined by inspection of their primary structures. In addition to the salt partition, the mole fractions of the individual caseins not complexed to the calcium phosphate through one or more of their PCs are computed and a generic stability rule for milks is derived. The use of the model is illustrated by calculations of the partition of salts in a standard milk and by comparison with experimental data on the partition of salts in the milk of individual cows. The generic stability rule is applied to the individual milks to determine whether the micellar calcium phosphate is thermodynamically stable. According to the calculations, compositions that might lead to pathological calcification in the lumen of the mammary gland were seldom found in primiparous healthy cows in early or mid lactation but occurred more often in multiparous animals, in late lactation and during mastitic infection.
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HRI/189/03 Life and death of the cell: Determinants of cell fate: Adipose tissue: mammary gland interactions; implications of maternal obesity for mammary gland function, cancer risk and neonatal development
Final Costs: £523,025
Recent epidemiological evidence identifies obesity as a major risk factor for breast cancer. In this project we studied mice that are obese either as a result of a genetic defect (which causes leptin deficiency and hence over-eating) or through feeding a high-fat, high-carbohydrate diet. In both cases obesity lead to an aberrant development of the mammary gland, with a reduced amount of secretory tissue and hence an impaired lactation potential.
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