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1.
Oncogene ; 32(10): 1305-15, 2013 Mar 07.
Article in English | MEDLINE | ID: mdl-22543585

ABSTRACT

Disruption of glandular architecture associates with poor clinical outcome in high-grade colorectal cancer (CRC). Phosphatase and tensin homolog deleted on chromosome ten (PTEN) regulates morphogenic growth of benign MDCK (Madin Darby Canine Kidney) cells through effects on the Rho-like GTPase cdc42 (cell division cycle 42). This study investigates PTEN-dependent morphogenesis in a CRC model. Stable short hairpin RNA knockdown of PTEN in Caco-2 cells influenced expression or localization of cdc42 guanine nucleotide exchange factors and inhibited cdc42 activation. Parental Caco-2 cells formed regular hollow gland-like structures (glands) with a single central lumen, in three-dimensional (3D) cultures. Conversely, PTEN-deficient Caco-2 ShPTEN cells formed irregular glands with multiple abnormal lumens as well as intra- and/or intercellular vacuoles evocative of the high-grade CRC phenotype. Effects of targeted treatment were investigated. Phosphatidinylinositol 3-kinase (PI3K) modulating treatment did not affect gland morphogenesis but did influence gland number, gland size and/or cell size within glands. As PTEN may be regulated by the nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ), cultures were treated with the PPARγ ligand rosiglitazone. This treatment enhanced PTEN expression, cdc42 activation and rescued dysmorphogenesis by restoring single lumen formation in Caco-2 ShPTEN glands. Rosiglitazone effects on cdc42 activation and Caco-2 ShPTEN gland development were attenuated by cotreatment with GW9662, a PPARγ antagonist. Taken together, these studies show PTEN-cdc42 regulation of lumen formation in a 3D model of human CRC glandular morphogenesis. Treatment by the PPARγ ligand rosiglitazone, but not PI3K modulators, rescued colorectal glandular dysmorphogenesis of PTEN deficiency.


Subject(s)
Anilides/pharmacology , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , PPAR gamma/antagonists & inhibitors , PTEN Phosphohydrolase/deficiency , Thiazolidinediones/pharmacology , Apoptosis/drug effects , Apoptosis/physiology , Caco-2 Cells , Cell Growth Processes/drug effects , Cell Growth Processes/physiology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , HCT116 Cells , Humans , Ligands , Madin Darby Canine Kidney Cells , Molecular Targeted Therapy , PPAR gamma/genetics , PPAR gamma/metabolism , PTEN Phosphohydrolase/genetics , PTEN Phosphohydrolase/metabolism , Rosiglitazone , Signal Transduction , Transfection , cdc42 GTP-Binding Protein/metabolism
2.
Br J Cancer ; 105(9): 1313-21, 2011 Oct 25.
Article in English | MEDLINE | ID: mdl-21952626

ABSTRACT

BACKGROUND: Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) regulation of the Rho-like GTPase Cdc42 has a central role in epithelial polarised growth, but effects of this molecular network on apoptosis remain unclear. METHODS: To investigate the role of Cdc42 in PTEN-dependent cell death, we used flow cytometry, in vitro pull-down assays, poly(ADP ribose) polymerase (PARP) cleavage and other immunoblots in isogenic PTEN-expressing and -deficient colorectal cells (HCT116PTEN(+/+), HCT116PTEN(-/-), Caco2 and Caco2 ShPTEN cells) after transfection or treatment strategies. RESULTS: The PTEN knockout or suppression by short hairpin RNA or small interfering RNA (siRNA) inhibited Cdc42 activity, PARP cleavage and/or apoptosis in flow cytometry assays. Transfection of cells with wild-type or constitutively active Cdc42 enhanced PARP cleavage, whereas siRNA silencing of Cdc42 inhibited PARP cleavage and/or apoptosis. Pharmacological upregulation of PTEN by sodium butyrate (NaBt) treatment enhanced Cdc42 activity, PARP cleavage and apoptosis, whereas Cdc42 siRNA suppressed NaBt-induced PARP cleavage. Cdc42-dependent signals can suppress glycogen synthase kinase-ß (GSK3ß) activity. Pharmacological inhibition of GSK3ß by lithium chloride treatment mimicked effects of Cdc42 in promotion of PARP cleavage and/or apoptosis. CONCLUSION: Phosphatase and tensin homologue deleted on chromosome 10 may influence apoptosis in colorectal epithelium through Cdc42 signalling, thus providing a regulatory framework for both polarised growth and programmed cell death.


Subject(s)
Cell Cycle Proteins/metabolism , PTEN Phosphohydrolase/metabolism , RNA-Binding Proteins/metabolism , Apoptosis , Caco-2 Cells , Gene Knockout Techniques , HCT116 Cells , Humans , Poly(ADP-ribose) Polymerases/metabolism , RNA Splicing Factors , RNA, Small Interfering/pharmacology , Signal Transduction
3.
Clin Exp Immunol ; 164(2): 202-10, 2011 May.
Article in English | MEDLINE | ID: mdl-21361912

ABSTRACT

Identification of immune modifiers of inherited cancer syndromes may provide a rationale for preventive therapy. Cowden disease (CD) is a genetically heterogeneous inherited cancer syndrome that arises predominantly from germline phosphatase and tensin homologue deleted on chromosome 10 (PTEN) mutation and increased phosphoinositide 3-kinase/mammalian target of rapamycin (PI3K/mTOR) signalling. However, many patients with classic CD diagnostic features are mutation-negative for PTEN (PTEN M-Neg). Interferon (IFN)-γ can modulate the PI3K/mTOR pathway, but its association with PTEN M-Neg CD remains unclear. This study assessed IFN-γ secretion by multi-colour flow cytometry in a CD kindred that was mutation-negative for PTEN and other known susceptibility genes. Because IFN-γ responses may be regulated by killer cell immunoglobulin-like receptors (KIR) and respective human leucocyte antigen (HLA) ligands, KIR/HLA genotypes were also assessed. Activating treatments induced greater IFN-γ secretion in PTEN M-Neg CD peripheral blood lymphocytes versus healthy controls. Increased frequency of activating KIR genes, potentially activating KIR/HLA compound genotypes and reduced frequency of inhibitory genotypes, were found in the PTEN M-Neg CD kindred. Differences of IFN-γ secretion were observed among PTEN M-Neg CD patients with distinct KIR/HLA compound genotypes. Taken together, these findings show enhanced lymphocyte secretion of IFN-γ that may influence the PI3K/mTOR CD causal molecular pathway in a PTEN mutation-negative CD kindred.


Subject(s)
Hamartoma Syndrome, Multiple/metabolism , Interferon-gamma/metabolism , Female , Flow Cytometry , Genotype , HLA Antigens/biosynthesis , Hamartoma Syndrome, Multiple/genetics , Haplotypes/genetics , Humans , Ionomycin/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Male , PTEN Phosphohydrolase/analysis , Pedigree , Phenotype , Phosphatidylinositol 3-Kinases/metabolism , Polymerase Chain Reaction , Receptors, KIR/physiology , Signal Transduction , TOR Serine-Threonine Kinases/metabolism
4.
Biochem Pharmacol ; 79(1): 1-9, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19737544

ABSTRACT

Substantive evidence implicates vitamin D receptor (VDR) or its natural ligand 1alpha,25-(OH)2 D3 in modulation of tumor growth. However, both human and animal studies indicate tissue-specificity of effect. Epidemiological studies show both inverse and direct relationships between serum 25(OH)D levels and common solid cancers. VDR ablation affects carcinogen-induced tumorigenesis in a tissue-specific manner in model systems. Better understanding of the tissue-specificity of vitamin D-dependent molecular networks may provide insight into selective growth control by the seco-steroid, 1alpha,25-(OH)2 D3. This commentary considers complex factors that may influence the cell- or tissue-specificity of 1alpha,25-(OH)2 D3/VDR growth effects, including local synthesis, metabolism and transport of vitamin D and its metabolites, vitamin D receptor (VDR) expression and ligand-interactions, 1alpha,25-(OH)2 D3 genomic and non-genomic actions, Ca2+ flux, kinase activation, VDR interactions with activating and inhibitory vitamin D responsive elements (VDREs) within target gene promoters, VDR coregulator recruitment and differential effects on key downstream growth regulatory genes. We highlight some differences of VDR growth control relevant to colonic, esophageal, prostate, pancreatic and other cancers and assess the potential for development of selective prevention or treatment strategies.


Subject(s)
Neoplasms/metabolism , Neoplasms/pathology , Receptors, Calcitriol/physiology , Animals , Cholecalciferol/metabolism , Cholecalciferol/physiology , Dimerization , Disease Progression , Gene Targeting , Humans , Ligands , Neoplasms/drug therapy , Neoplasms/genetics , Organ Specificity/genetics , Organ Specificity/physiology , Protein Isoforms/physiology , Receptors, Calcitriol/metabolism , Response Elements/genetics , Retinoid X Receptors/physiology , Signal Transduction/genetics , Transcription, Genetic
5.
Biochim Biophys Acta ; 1763(4): 381-92, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16713447

ABSTRACT

Both male and female rat growth plate cartilage cells possess estrogen receptors (ERs), but 17beta-estradiol (E(2)) activates protein kinase C (PKC) and PKC-dependent biological responses to E(2) only in cells from female animals. PKC signaling can elicit genomic responses via mitogen activated protein kinase (MAPK) and E(2) has been shown to activate ERK MAPK in many cells, suggesting that MAPK may play a role in growth plate chondrocytes as well. We tested if E(2) increases MAPK activity and if so, whether the response is limited to female cells, if it is PKC-dependent, and if the mechanism involves traditional ER pathways. We also determined the contribution of MAPK to the biological response of growth plate chondrocytes and assessed the relative contributions of ERK, p38 and JNK MAPKs. Female rat costochondral cartilage cells were treated with E(2) and MAPK-specific activity determined in cell layer lysates. The mechanism of MAPK activation was determined by treating the cells with E(2) conjugated to bovine serum albumin (E(2)-BSA) to assess if membrane receptors were involved; stereospecificity was determined using 17alpha-estradiol; PKC and phospholipase C (PLC) dependence was determined using specific inhibitors; and the ER agonist diethylstilbestrol, the ER antagonist ICI 182780, and tamoxifen were used to assess the role of traditional ER pathways. E(2) regulation of ERK1/2 MAPK was assessed and the relative roles of ERK1/2, p38 and JNK MAPKs determined using specific inhibitors. E(2) caused a rapid dose-dependent activation of MAPK that was greatest in cells treated for 9 min with 10(-9) M hormone; activity remained elevated for 3 h. E(2)'s effect on MAPK was stereospecific and comparable to that of E(2)-BSA. It was insensitive to DES and ICI 182780, dependent on PKC and PLC, blocked by tamoxifen and it did not require gene transcription or translation. E(2) had no effect on ERK1 or ERK2 mRNA or protein but it caused a rapid phosphorylation of ERK1/2 at 9 min. Inhibition of ERK1/2 and p38 MAPK reduced the stimulatory effects of E(2) on alkaline phosphatase activity and [(35)S]-sulfate incorporation. These results suggest that E(2) regulates MAPK through a sex-specific membrane-mediated mechanism that does not involve cytosolic ERs in a traditional sense and that ERK1/2 and p38 mediate the downstream biological effects of the hormone.


Subject(s)
Chondrocytes/enzymology , Estradiol/physiology , Growth Plate/enzymology , MAP Kinase Signaling System/physiology , Sex Characteristics , Animals , Cells, Cultured , Enzyme Activation/physiology , Female , Growth Plate/cytology , Male , Rats , Rats, Sprague-Dawley
6.
Endocrinology ; 143(7): 2775-86, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12072413

ABSTRACT

Membrane-mediated increases in protein kinase C (PKC) activity and PKC-dependent physiological responses of growth plate chondrocytes to vitamin D metabolites depend on the state of endochondral maturation; 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] regulates growth zone (GC) cells, whereas 24R,25-(OH)(2)D(3) regulates resting zone (RC) cells. Different mechanisms, including protein kinase A signaling, mediate the effects of 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) on PKC, suggesting that different mechanisms may also regulate any MAPK involvement in the physiological responses. This study used confluent cultures of rat costochondral chondrocytes as a model. 1alpha,25-(OH)(2)D(3) stimulated MAPK specific activity in GC in a time- and dose-dependent manner, evident within 9 min. 24R,25-(OH)(2)D(3) stimulated MAPK in RC; increases were dose dependent, occurred after 9 min, and were greatest at 90 min. In both cells the effect was due to ERK1/2 activation (p42 > p44 in GC; p42 = p44 in RC). MAPK activation was dependent on PKC, but not protein kinase A. The effect of 1alpha,25-(OH)(2)D(3) required phospholipase C, and the effect of 24R,25-(OH)(2)D(3) required phospholipase D. Inhibition of cyclooxygenase activity reduced the effect of 1alpha,25-(OH)(2)D(3) on MAPK in GC and enhanced the effect of 24R,25-(OH)(2)D(3) in RC. Based on MAPK inhibition with PD98059, ERK1/2 MAPK mediated the effect of 24R,25-(OH)(2)D(3) on [(3)H]thymidine incorporation and [(35)S]sulfate incorporation by RC, but only partially mediated the effect of 1alpha,25-(OH)(2)D(3) on GC. ERK1/2 was not involved in the regulation of alkaline phosphatase specific activity by either metabolite. This paper supports the hypothesis that 1alpha,25-(OH)(2)D(3) regulates the physiology of GC via rapid membrane-mediated signaling pathways, and some, but not all, of the response to 1alpha,25-(OH)(2)D(3) is via the ERK family of MAPKs. In contrast, 24R,25-(OH)(2)D(3) exerts its effects on RC via PKC-dependent MAPK. Whereas 1alpha,25-(OH)(2)D(3) increases MAPK activity via phospholipase C and increased prostaglandin production, 24R,25-(OH)(2)D(3) increases MAPK via phospholipase D and decreased prostaglandin production. The cell specificity, metabolite stereospecificity, and the dependence on PKC argue for the participation of membrane receptors for 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in the regulation of ERK1/2 in the growth plate.


Subject(s)
24,25-Dihydroxyvitamin D 3/pharmacology , Calcitriol/pharmacology , Chondrocytes/physiology , Growth Plate/cytology , Growth Plate/physiology , Mitogen-Activated Protein Kinases/physiology , Protein Kinase C/metabolism , Signal Transduction/drug effects , Alkaline Phosphatase/metabolism , Animals , Blotting, Northern , Blotting, Western , Cells, Cultured , Chondrocytes/drug effects , Enzyme Activation/drug effects , Gene Expression Regulation, Enzymologic/drug effects , Growth Plate/drug effects , Indicators and Reagents , Male , Mitogen-Activated Protein Kinases/biosynthesis , Mitogen-Activated Protein Kinases/genetics , Phospholipases/metabolism , Phosphorylation , Prostaglandin-Endoperoxide Synthases/metabolism , Proteoglycans/metabolism , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley
7.
Vet Rec ; 148(8): 233-7, 2001 Feb 24.
Article in English | MEDLINE | ID: mdl-11289550

ABSTRACT

Hypomagnesaemic tetany was induced in non-lactating and lactating ewes by feeding them semi-synthetic low magnesium diets containing additional potassium chloride and citric acid. Aqueous and vitreous humour were sampled from one eye at the time of death (fresh) and from the second eye after the head had been stored at ambient temperature for 24 hours (24-hour). There were significant relationships between the concentrations of magnesium in cerebrospinal fluid and plasma and its concentrations in fresh aqueous humour and fresh vitreous humour. Magnesium concentrations of < 0.33 mmol/litre in fresh aqueous humour and < 0.50 mmol/litre in 24-hour aqueous humour were associated with severe hypomagnesaemia and tetany. However, the concentration of magnesium in aqueous humour is relatively unstable and, unless the time of death was known accurately, its interpretation would be difficult. Magnesium concentrations of < 0.60 mmol/litre in fresh vitreous humour and < 0.65 mmol/litre in 24-hour vitreous humour were associated with severe hypomagnesaemia and tetany in adult sheep. The concentration of magnesium in vitreous humour was relatively stable for up to 48 hours postmortem.


Subject(s)
Aqueous Humor/chemistry , Magnesium Deficiency/veterinary , Magnesium/analysis , Sheep Diseases/diagnosis , Tetany/veterinary , Vitreous Body/chemistry , Animals , Biomarkers/analysis , Biomarkers/blood , Biomarkers/cerebrospinal fluid , Diet , Female , Lactation/metabolism , Magnesium/blood , Magnesium/cerebrospinal fluid , Magnesium Deficiency/complications , Magnesium Deficiency/metabolism , Sheep , Sheep Diseases/etiology , Tetany/diagnosis , Tetany/etiology , Time Factors
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