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Biomedical subjects

A Thorburn

Publications and source records attributed to A Thorburn.

At least 19 recordsLinked to original sources
PubMed2006

Fatty acid synthase: a novel target for antiglioma therapy.

High levels of fatty acid synthase (FAS) expression have been observed in several cancers, including breast, prostate, colon and lung carcinoma, compared with their respective normal tissue. We present data that show high levels of FAS protein in human and rat glioma cell lines and human glioma tissue samples, as compared to normal rat astrocytes and normal human brain. Incubating glioma cells with the FAS inhibitor cerulenin decreased endogenous fatty acid synthesis by approximately 50%. Cell cycle analysis demonstrated a time- and dose-dependent increase in S-phase cell arrest following cerulenin treatment for 24 h. Further, treatment with cerulenin resulted in time- and dose-dependent decreases in glioma cell viability, as well as reduced clonogenic survival. Increased apoptotic cell death and PARP cleavage were observed in U251 and SNB-19 cells treated with cerulenin, which was independent of the death receptor pathway. Overexpressing Bcl-2 inhibited cerulenin-mediated cell death. In contrast, primary rat astrocytes appeared unaffected. Finally, RNAi-mediated knockdown of FAS leading to reduced FAS enzymatic activity was associated with decreased glioma cell viability. These findings suggest that FAS might be a novel target for antiglioma therapy.

Animals↗
PubMed2005

Recruitment of TRADD, FADD, and caspase 8 to double-stranded RNA-triggered death inducing signaling complexes (dsRNA-DISCs).

Rapid elimination of virus-infected cells by apoptosis is an efficient anti-viral strategy. Double-stranded RNA (dsRNA), a viral product, is potently and rapidly apoptogenic in susceptible cells. Caspase 8 plays an important role in the dsRNA-induced apoptosis; however, the mechanisms of caspase 8 activation in response to dsRNA are unknown. We demonstrate here that, in HeLa cells, the dsRNA-triggered activation of caspase 8 is independent of ongoing proteins synthesis (and is, therefore, independent of changes in pro- and anti-apoptotic gene expression) and involves the formation of multiprotein dsRNA-triggered death inducing signaling complexes (dsRNA-DISCs). DsRNA-DISCs contain FADD, TRADD, and caspase 8; however, several experimental approaches suggest that death ligands and death receptors (such as Fas/Apo1 and DR4/Apo2) are not involved in the formation of dsRNA-DISCs.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The adaptor protein TRADD activates distinct mechanisms of apoptosis from the nucleus and the cytoplasm.

TNFR1 associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain along with nuclear import and export sequences that cause shuttling between the cytoplasm and nucleus. The death domain of TRADD contains the nuclear import sequence and expression of the core death domain (nuclear TRADD) results in exclusive nuclear localization and activation of a distinct apoptotic pathway. Cytoplasmic TRADD activates apoptosis through Fas-associated death domain protein (FADD) and caspase-8 activation that was blocked by caspase inhibitors or dominant-negative FADD. These inhibitors did not inhibit death induced by nuclear TRADD, which could only be inhibited by combining caspase inhibitors and a serine protease inhibitor. The pathway activated by nuclear TRADD requires caspase-9 catalytic activity. However, apoptosis activating factor deficiency confers only partial protection from death. This pathway represents an alternate means by which TRADD can regulate cell death independently of FADD and caspase-8 that occurs from the nucleus rather than the cytoplasm.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Induction of apoptosis by tumor cell-targeted toxins.

Targeted toxins are fusion proteins that combine a targeting molecule that selectively binds to and enters tumor cells with a protein toxin that kills the target cells. These molecules represent an exciting approach to develop effective cancer-specific therapeutics that have few side effects on normal tissues and numerous such toxins are in various stages of pre-clinical and clinical development to treat a wide variety of tumors. In this review, we discuss this strategy, describe ways that the toxins activate the apoptosis machinery and discuss future developments in this field.

Antineoplastic Agents↗
PubMed2001

Retinoic acid-induced expression of autotaxin in N-myc-amplified neuroblastoma cells.

Neuroblastoma, the most common extracranial solid tumor in children, arises from precursors of the sympathetic nervous system. Neuroblastoma cell lines are responsive to the differentiation agent retinoic acid, which induces its effects by altering transcription rates of specific target genes. We identified autotaxin (ATX), which encodes an autocrine tumor motility-stimulating factor, as a gene whose expression is significantly induced by retinoic acid in neuroblastoma cells. ATX induction was specific for neuroblastoma cell lines that contain N-myc amplification, a cytogenetic feature commonly associated with aggressive neuroblastomas. Although ATX expression was associated with amplification of the N-myc locus, N-myc itself was neither sufficient nor required for ATX expression, suggesting that a coamplified gene is responsible. ATX induction by retinoic acid was due to increased transcription and required new protein synthesis.

Antineoplastic Agents↗
PubMed2001

Measurement of caspase activity in individual cells reveals differences in the kinetics of caspase activation between cells.

Most forms of apoptosis involve activation of caspases and it is likely that differences between cells in their ability to activate caspases contributes to the responsiveness of any given cell within a population to apoptotic stimuli. To study the molecular mechanisms that underlie such differences, it is necessary to measure caspase activity in individual cells. Here, we describe a method that allows the continuous monitoring of caspase activity in individual, living mammalian cells. This approach allows studies of the kinetics of caspase activation to be performed in individual cells within a population. We demonstrate that in a group of cells where some cells die and some cells survive in response to the same stimulus, the cells that die can be differentiated from those that survive based on the amount of caspase activity in each cell several hours before death occurs.

Apoptosis↗
PubMed2001

An apoptosis signaling pathway induced by the death domain of FADD selectively kills normal but not cancerous prostate epithelial cells.

The adaptor protein FADD directly, or indirectly via another adaptor called TRADD, recruits caspase 8 to death receptors of the tumor necrosis factor receptor family. Consequentially, a dominant-negative mutant (FADD-DN, which consists only of the FADD death domain) that binds to receptors but cannot recruit caspase 8 has been widely used to inhibit apoptosis by various stimuli that work via death receptors. Here, we show that FADD-DN also has another cell type- and cancer-dependent activity because it induces apoptosis of normal human prostate epithelial cells but not normal prostate stromal cells or prostate cancer cells. This activity is independent of FADD-DN's ability to bind to three known interacting proteins, Fas, TRADD or RIP suggesting that it is distinct from FADD's functions at activated death receptors. FADD-DN induces caspase activation in normal epithelial cells as demonstrated using a Fluorescence Resonance Energy Transfer assay that measures caspase activity in individual living cells. However, caspase-independent pathways are also implicated in FADD-DN-induced apoptosis because caspase inhibitors were inefficient at preventing prostate cell death. Therefore, the death domain of FADD has a previously unrecognized role in cell survival that is epithelial-specific and defective in cancer cells. This FADD-dependent signaling pathway may be important in prostate carcinogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

The effects of human GH and its lipolytic fragment (AOD9604) on lipid metabolism following chronic treatment in obese mice and beta(3)-AR knock-out mice.

Both human GH (hGH) and a lipolytic fragment (AOD9604) synthesized from its C-terminus are capable of inducing weight loss and increasing lipolytic sensitivity following long-term treatment in mice. One mechanism by which this may occur is through an interaction with the beta-adrenergic pathway, particularly with the beta(3)-adrenergic receptors (beta(3)-AR). Here we describe how hGH and AOD9604 can reduce body weight and body fat in obese mice following 14 d of chronic ip administration. These results correlate with increases in the level of expression of beta(3)-AR RNA, the major lipolytic receptor found in fat cells. Importantly, both hGH and AOD9604 are capable of increasing the repressed levels of beta(3)-AR RNA in obese mice to levels comparable with those in lean mice. The importance of beta(3)-AR was verified when long-term treatment with hGH and AOD9604 in beta(3)-AR knock-out mice failed to produce the change in body weight and increase in lipolysis that was observed in wild-type control mice. However, in an acute experiment, AOD9604 was capable of increasing energy expenditure and fat oxidation in the beta(3)-AR knock-out mice. In conclusion, this study demonstrates that the lipolytic actions of both hGH and AOD9604 are not mediated directly through the beta(3)-AR although both compounds increase beta(3)-AR expression, which may subsequently contribute to enhanced lipolytic sensitivity.

Adipose Tissue↗
PubMed2000

A Raf-induced, MEK-independent signaling pathway regulates atrial natriuretic factor gene expression in cardiac muscle cells.

The atrial natriuretic factor (ANF) gene is activated in cardiac myocytes by Ras and its effector Raf. However, MEK, the best-characterized Raf substrate, cannot efficiently activate ANF suggesting that Raf uses a MEK-independent pathway to activate ANF. By manipulating MEK and Raf activities so that they are equally effective at activating ERK, we now demonstrate that Raf activates at least two signaling pathways in cardiac myocytes that regulate the ANF promoter; the MEK-->ERK pathway inhibits ANF gene expression while a Raf-induced, MEK-independent pathway activates expression. This mechanism may provide increased ability to regulate ANF expression in response to hypertrophic stimuli.

Animals↗
PubMed2000

Dexamethasone inhibits insulin-like growth factor signaling and potentiates myoblast apoptosis.

In the critically ill, glucocorticoids induce myopathy, combining profound protein catabolism and mild myotubular death. Insulin-like growth factors (IGFs) inhibit muscle catabolism through activation of phosphatidylinositol 3-kinase (PI3K). Using rat L6 myoblasts, we show that IGF-I also acts through PI3K to inhibit apoptosis induced by hyperosmolar metabolic stress with 300 mM mannitol. We find that the glucocorticoid dexamethasone inhibits this antiapoptotic effect of IGF-I by impairing PI3K signaling. Dexamethasone induces overexpression of the PI3K subunit p85alpha, which, in turn, competes with the complete PI3K heterodimer for binding at insulin receptor substrate-1, inhibiting PI3K activation. Dexamethasone blocks IGF-I-induced phosphorylation of Akt, a PI3K-dependent process. Increased cellular p85alpha abundance, induced by either 10 microM dexamethasone or transient transfection with a plasmid coding for p85alpha, significantly inhibits IGF-I rescue from apoptosis induced by mannitol, as indicated by both loss of cell viability and increased activity of caspase-3 by fluorogenic assay. Conversely, constitutively active PI3K inhibits death induced by mannitol, even in the presence of dexamethasone. These findings may have particular relevance in the pathogenesis of acute steroid myopathy in critical illness, in which catabolic glucocorticoid effects combine with acute metabolic stressors, including sepsis, fasting, and chemical denervation.

Animals↗
PubMed1999

Activation of Ras by phorbol esters in cardiac myocytes. Role of guanine nucleotide exchange factors.

The relationship between protein kinase C (PKC) activation and Ras function was investigated in cardiac cells. Ras function was required for ERK activation by phorbol esters in cardiac myocytes, but not in cardiac fibroblasts. Accordingly, treatment with phorbol esters resulted in GTP loading of Ras in cardiac myocytes, but not fibroblasts. Ras activation by phorbol esters was abolished by a PKC specific inhibitor, but was insensitive to tyrosine kinase inhibitors. Ras activation was mediated by stimulation of guanine nucleotide exchange. These results suggest the existence of a novel pathway for Ras activation, specific to cardiac myocytes, with implications for myocardial hypertrophy.

Alkaloids↗
PubMed1999

Apoptosis induced by overexpression of hMSH2 or hMLH1.

Mutations of the mismatch repair genes hMSH2 and hMLH1 have been found in a high proportion of individuals with hereditary nonpolyposis colon cancer (HNPCC), establishing the link between mismatch repair and cancer. Tumor cell lines that are deficient in mismatch repair develop a mutator phenotype that appears to drive the accumulation of mutations required for tumor development. However, mutations of other mismatch repair genes such as hPMS2 can lead to a mutator phenotype, although inherited mutations of these genes are rare in HNPCC families. Here, we show that overexpression of hMSH2 or hMLH1 but not of hMSH3, hMSH6, or hPMS2 induces apoptosis in either repair-proficient or -deficient cells. Furthermore, primary mouse embryo fibroblasts derived from Msh2-deficient mice lose their ability to undergo apoptosis after treatment with N-methyl-N'-nitro-N-nitrosoguanidine. These results suggest that the mismatch repair proteins hMSH2 and hMLH1 may be components of a pathway that influences apoptosis. We consider the possibility that loss of apoptosis as a result of hMSH2 or hMLH1 deficiency may be an additional factor in cancer predisposition in HNPCC.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Transcriptional activation of the glucose transporter GLUT1 in ventricular cardiac myocytes by hypertrophic agonists.

Myocardial hypertrophy is associated with increased basal glucose metabolism. Basal glucose transport into cardiac myocytes is mediated by the GLUT1 isoform of glucose transporters, whereas the GLUT4 isoform is responsible for regulatable glucose transport. Treatment of neonatal cardiac myocytes with the hypertrophic agonist 12-O-tetradecanoylphorbol-13-acetate or phenylephrine increased expression of Glut1 mRNA relative to Glut4 mRNA. To study the transcriptional regulation of GLUT1 expression, myocytes were transfected with luciferase reporter constructs under the control of the Glut1 promoter. Stimulation of the cells with 12-O-tetradecanoylphorbol-13-acetate or phenylephrine induced transcription from the Glut1 promoter, which was inhibited by cotransfection with the mitogen-activated protein kinase phosphatases CL100 and MKP-3. Cotransfection of the myocytes with constitutively active versions of Ras and MEK1 or an estrogen-inducible version of Raf1 also stimulated transcription from the Glut1 promoter. Hypertrophic induction of the Glut1 promoter was also partially sensitive to inhibition of the phosphatidylinositol 3-kinase pathway and was strongly inhibited by cotransfection with dominant-negative Ras. Thus, Ras activation and pathways downstream of Ras mediate induction of the Glut1 promoter during myocardial hypertrophy.

Animals↗
PubMed1999

A low-affinity serum response element allows other transcription factors to activate inducible gene expression in cardiac myocytes.

Hypertrophic growth of cardiac muscle cells is induced by a variety of physiological and pathological stimuli and is associated with a number of changes, including activation of genes such as atrial natriuretic factor. We found that two serum response element (SRE)-like DNA elements, one of which does not meet the consensus sequence and binds serum response factor (SRF) with low affinity, regulate the activity of this promoter. Surprisingly, the ability to induce the promoter by two different physiologic stimuli, as well as various activated transcription factors, including SRF-VP16, was primarily dependent upon the nonconsensus rather than the consensus SRE. This SRE controls the induction of gene expression via an unusual mechanism in that it is required to allow some, but not all, active transcription factors at unrelated sites on the promoter to stimulate gene expression. Thus, in addition to regulation of SRF activity by growth stimuli, regulation of a low-affinity SRE element controls inducible gene expression by modulating the ability of other transcription factors to stimulate the transcription machinery.

Atrial Natriuretic Factor↗
PubMed1999

Cell density and contraction regulate p38 MAP kinase-dependent responses in neonatal rat cardiac myocytes.

In vitro cardiac myocyte hypertrophy is characterized by increased cell size, sarcomere organization, and induction of several genes including atrial natriuretic factor (ANF). The hypertrophic growth program has been associated with activation of various mitogen-activated protein kinase (MAP) kinase family members, one of which is a stress kinase, p38. In this study, we found that the p38-specific inhibitor SB-203580 failed to inhibit phenylephrine-induced ANF-driven gene expression in low-density myocyte cultures but did inhibit gene expression in higher density cultures. Dense myocyte cultures also had a higher metabolic activity and contraction rate than cells plated at low density. We found that mimicking this effect by rapid electrical pacing activated ANF-driven gene expression and that this expression was inhibited by inactivation of p38. However, addition of SB-203580 at time points ranging between 1 and 72 h suggests that the effect of p38 on the ANF promoter may be both direct and indirect. Electrical pacing induced a small, but consistent, increase in p38 phosphorylation (phospho-p38) at time points ranging from 30 min to 4 h, but at later times phospho-p38 levels were reduced. When myocytes were treated with phenylephrine or electrically paced in the presence of the p38 inhibitor, there was little discernible change in morphology or rates of protein synthesis from DMSO-treated cells at 48 or 72 h. These data indicate that cell density and myocyte contraction may modulate p38-dependent pathways for ANF gene expression, but these pathways may not be direct and have limited effects on hypertrophic morphology.

Animals↗
PubMed1998

Membrane-targeting sequences on AKAP79 bind phosphatidylinositol-4, 5-bisphosphate.

Protein kinases and phosphatases are targeted through association with anchoring proteins that tether the enzymes to subcellular structures and organelles. Through in situ fluorescent techniques using a Green Fluorescent Protein tag, we have mapped membrane-targeting domains on AKAP79, a multivalent anchoring protein that binds the cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and protein phosphatase 2B, calcineurin (CaN). Three linear sequences termed region A (residues 31-52), region B (residues 76-101) and region C (residues 116-145) mediate targeting of AKAP79 in HEK-293 cells and cortical neurons. Analysis of these targeting sequences suggests that they contain putative phosphorylation sites for PKA and PKC and are rich in basic and hydrophobic amino acids similar to a class of membrane-targeting domains which bind acidic phospholipids and calmodulin. Accordingly, the AKAP79 basic regions mediate binding to membrane vesicles containing acidic phospholipids including phosphatidylinositol-4, 5-bisphosphate [PtdIns(4,5)P2] and this binding is regulated by phosphorylation and calcium-calmodulin. Finally, AKAP79 was shown to be phosphorylated in HEK-293 cells following stimulation of PKA and PKC, and activation of PKC or calmodulin was shown to release AKAP79 from membrane particulate fractions. These findings suggest that AKAP79 might function in cells not only as an anchoring protein but also as a substrate and effector for the anchored kinases and phosphatases.

A Kinase Anchor Proteins↗
PubMed1998

Ras and rho are required for galphaq-induced hypertrophic gene expression in neonatal rat cardiac myocytes.

The hypertrophic response is characterized by increased myofibril/sarcomere organization, induction of the cardiac specific atrial natriuretic factor (ANF) and myosin light chain-2 (MLC-2v) genes, and an increase in total cell volume. The alpha1-adrenergic agonist phenylephrine induces both the morphological and biochemical markers of hypertrophy in cultured neonatal rat ventricular cardiomyocytes. Previous studies have suggested a functional requirement for the heterotrimeric G-protein, Galphaq, for a subset of the hypertrophic phenotypes. The small GTPases Ras and Rho have also been implicated in phenylephrine-induced hypertrophy. To further delineate the role of Galphaq in hypertrophy, a constitutively active mutant of Galphaq was transiently transfected in primary rat ventricular cardiomyocytes. This molecule was sufficient to induce ANF-, AP1-, and MLC-2-driven gene expression. Co-transfection of Galphaq and dominant negative Ras or dominant negative Raf resulted in dose-dependent inhibition of ANF-driven expression. Both dominant negative Rho, and the Rho inhibitor C3-transferase, also attenuated Galphaq- and Ras-induced ANF-driven gene expression. Cells transfected with active Galphaq did not show a detectable increase in activation of the mitogen activated protein kinases ERK or SAPK. However, activity of the MAP-kinases appears to be important for Galphaq-induced gene expression since the MAP-kinase phosphatase Clone 100 and catalytically inactive SAPK strongly inhibited Galphaq-induced ANF expression. Thus, our studies indicate Galphaq-induced hypertrophic gene expression requires the small G-proteins Ras and Rho. The data also indicates that Galphaq mediated gene expression is dependent on functional MAP-kinases and that multiple signaling pathways contribute to Galphaq-mediated cardiac cell hypertrophy.

Animals↗