T-helper-1-cell cytokines drive cancer into senescence.

Cancer control by adaptive immunity involves a number of defined death and clearance mechanisms. However, efficient inhibition of exponential cancer growth by T cells and interferon-γ (IFN-γ) requires additional undefined mechanisms that arrest cancer cell proliferation. Here we show that the combined action of the T-helper-1-cell cytokines IFN-γ and tumour necrosis factor (TNF) directly induces permanent growth arrest in cancers. To safely separate senescence induced by tumour immunity from oncogene-induced senescence, we used a mouse model in which the Simian virus 40 large T antigen (Tag) expressed under the control of the rat insulin promoter creates tumours by attenuating p53- and Rb-mediated cell cycle control. When combined, IFN-γ and TNF drive Tag-expressing cancers into senescence by inducing permanent growth arrest in G1/G0, activation of p16INK4a (also known as CDKN2A), and downstream Rb hypophosphorylation at serine 795. This cytokine-induced senescence strictly requires STAT1 and TNFR1 (also known as TNFRSF1A) signalling in addition to p16INK4a. In vivo, Tag-specific T-helper 1 cells permanently arrest Tag-expressing cancers by inducing IFN-γ- and TNFR1-dependent senescence. Conversely, Tnfr1(-/-)Tag-expressing cancers resist cytokine-induced senescence and grow aggressively, even in TNFR1-expressing hosts. Finally, as IFN-γ and TNF induce senescence in numerous murine and human cancers, this may be a general mechanism for arresting cancer progression.

Fenamates as TRP channel blockers: mefenamic acid selectively blocks TRPM3.

BACKGROUND AND PURPOSE: Fenamates are N-phenyl-substituted anthranilic acid derivatives clinically used as non-steroid anti-inflammatory drugs in pain treatment. Reports describing fenamates as tools to interfere with cellular volume regulation attracted our attention based on our interest in the role of the volume-modulated transient receptor potential (TRP) channels TRPM3 and TRPV4. EXPERIMENTAL APPROACH: Firstly, we measured the blocking potencies and selectivities of fenamates on TRPM3 and TRPV4 as well as TRPC6 and TRPM2 by Ca(2+) imaging in the heterologous HEK293 cell system. Secondly, we further investigated the effects of mefenamic acid on cytosolic Ca(2+) and on the membrane voltage in single HEK293 cells that exogenously express TRPM3. Thirdly, in insulin-secreting INS-1E cells, which endogenously express TRPM3, we validated the effect of mefenamic acid on cytosolic Ca(2+) and insulin secretion. KEY RESULTS: We identified and characterized mefenamic acid as a selective and potent TRPM3 blocker, whereas other fenamate structures non-selectively blocked TRPM3, TRPV4, TRPC6 and TRPM2. CONCLUSIONS AND IMPLICATIONS: This study reveals that mefenamic acid selectively inhibits TRPM3-mediated calcium entry. This selectivity was further confirmed using insulin-secreting cells. K(ATP) channel-dependent increases in cytosolic Ca(2+) and insulin secretion were not blocked by mefenamic acid, but the selective stimulation of TRPM3-dependent Ca(2+) entry and insulin secretion induced by pregnenolone sulphate were inhibited. However, the physiological regulator of TRPM3 in insulin-secreting cells remains to be elucidated, as well as the conditions under which the inhibition of TRPM3 can impair pancreatic ß-cell function. Our results strongly suggest mefenamic acid is the most selective fenamate to interfere with TRPM3 function.

Protein kinase C delta (PKCδ) affects proliferation of insulin-secreting cells by promoting nuclear extrusion of the cell cycle inhibitor p21Cip1/WAF1.

BACKGROUND: High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in ß-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS: Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE: These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role.

The impact of genetic variation in the G6PC2 gene on insulin secretion depends on glycemia.

CONTEXT: Single-nucleotide polymorphisms (SNPs) within the G6PC2 locus are associated with fasting glucose and insulin secretion. These SNPs are not associated with type 2 diabetes risk. OBJECTIVE: Our objective was to investigate whether the impact of the SNP on variables of glucose-stimulated insulin secretion is influenced by glucose tolerance status. DESIGN, SETTING, PARTICIPANTS, AND INTERVENTION: In this cross-sectional study, we genotyped 1505 healthy Caucasian subjects [normal glucose tolerance (NGT), 1098; impaired glucose tolerance (IGT)/impaired fasting glucose (IFG), 407] for SNP rs560887 within the G6PC2 locus. A subgroup of 326 subjects underwent an iv glucose tolerance test, and 512 participants took part in a hyperinsulinemic-euglycemic clamp. For replication, SNP rs560887 was genotyped in 457 subjects (NGT, 265; IGT, 192) from four independent German and Dutch studies who underwent a hyperglycemic clamp. MAIN OUTCOME MEASURE: Insulin secretion was evaluated. RESULTS: Carriers of the major G-allele exhibited increased fasting glycemia (P<0.0001). Insulin sensitivity and secretion were not associated with the SNP (P≥0.06). Glucose tolerance status and genotype interacted on insulin secretion (P=0.036), such that in NGT subjects, the minor A-allele of rs560887 was associated with decreased insulinogenic index (P=0.044), which was not the case in subjects with IFG/IGT (P=1.0). During the iv glucose tolerance test, an association of A-allele carriers with decreased first-phase insulin secretion was also observed only in NGT subjects (P=0.0053). Likewise, in the hyperglycemic clamp group, the A-allele was associated with decreased first-phase insulin secretion only in the NGT group (P=0.022) but not in the IGT group. CONCLUSIONS: The effects of hyperglycemia on insulin secretion override the more subtle effects of genetic variation in the G6PC2 locus on insulin secretion.

Overexpression of kinase-negative protein kinase Cdelta in pancreatic beta-cells protects mice from diet-induced glucose intolerance and beta-cell dysfunction.

OBJECTIVE: In vitro models suggest that free fatty acid-induced apoptotic beta-cell death is mediated through protein kinase C (PKC)delta. To examine the role of PKCdelta signaling in vivo, transgenic mice overexpressing a kinase-negative PKCdelta (PKCdeltaKN) selectively in beta-cells were generated and analyzed for glucose homeostasis and beta-cell survival. RESEARCH DESIGN AND METHODS: Mice were fed a standard or high-fat diet (HFD). Blood glucose and insulin levels were determined after glucose loads. Islet size, cleaved caspase-3, and PKCdelta expression were estimated by immunohistochemistry. In isolated islet cells apoptosis was assessed with TUNEL/TO-PRO3 DNA staining and the mitochondrial potential by rhodamine-123 staining. Changes in phosphorylation and subcellular distribution of forkhead box class O1 (FOXO1) were analyzed by Western blotting and immunohistochemistry. RESULTS: PKCdeltaKN mice were protected from HFD-induced glucose intolerance. This was accompanied by increased insulin levels in vivo, by an increased islet size, and by a reduced staining of beta-cells for cleaved caspase-3 compared with wild-type littermates. In accordance, long-term treatment with palmitate increased apoptotic cell death of isolated islet cells from wild-type but not from PKCdeltaKN mice. PKCdeltaKN overexpression protected islet cells from palmitate-induced mitochondrial dysfunction and inhibited nuclear accumulation of FOXO1 in mouse islet and INS-1E cells. The inhibition of nuclear accumulation of FOXO1 by PKCdeltaKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein. CONCLUSIONS: Overexpression of PKCdeltaKN in beta-cells protects from HFD-induced beta-cell failure in vivo by a mechanism that involves inhibition of fatty acid-mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.

Common polymorphisms within the NR4A3 locus, encoding the orphan nuclear receptor Nor-1, are associated with enhanced beta-cell function in non-diabetic subjects.

BACKGROUND: Neuron-derived orphan receptor (Nor) 1, nuclear receptor (Nur) 77, and nuclear receptor-related protein (Nurr) 1 constitute the NR4A family of orphan nuclear receptors which were recently found to modulate hepatic glucose production, insulin signalling in adipocytes, and oxidative metabolism in skeletal muscle. In this study, we assessed whether common genetic variation within the NR4A3 locus, encoding Nor-1, contributes to the development of prediabetic phenotypes, such as glucose intolerance, insulin resistance, or beta-cell dysfunction. METHODS: We genotyped 1495 non-diabetic subjects from Southern Germany for the five tagging single nucleotide polymorphisms (SNPs) rs7047636, rs1526267, rs2416879, rs12686676, and rs10819699 (minor allele frequencies >or= 0.05) covering 100% of genetic variation within the NR4A3 locus (with D' = 1.0, r2 >or= 0.9) and assessed their association with metabolic data derived from the fasting state, an oral glucose tolerance test (OGTT), and a hyperinsulinemic-euglycemic clamp (subgroup, N = 506). SNPs that revealed consistent associations with prediabetic phenotypes were subsequently genotyped in a second cohort (METSIM Study; Finland; N = 5265) for replication. RESULTS: All five SNPs were in Hardy-Weinberg equilibrium (p >or= 0.7, all). The minor alleles of three SNPs, i.e., rs1526267, rs12686676, and rs10819699, consistently tended to associate with higher insulin release as derived from plasma insulin at 30 min(OGTT), AUCC C-peptide-to-AUC Gluc ratio and the AUC Ins30-to-AUC Gluc30 ratio with rs12686676 reaching the level of significance (p

Sphingomyelinase dependent apoptosis following treatment of pancreatic beta-cells with amyloid peptides Abeta(1-42) or IAPP.

Amyloid peptides interfere with survival of pancreatic beta-cells. In some cells apoptosis is paralleled by ceramide-dependent alterations of ion channel activity. The purpose of the present study was to elucidate the dependence of amyloid peptides Abeta(1-42) and islet amyloid polypeptide (IAPP)-induced cell death on ceramide formation and ion channel activity in murine pancreatic islet cells. As disclosed by TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining, apoptotic cell death was induced by Abeta(1-42), IAPP and exogenously added C2-ceramide in islet cells from wild type mice. In islet cells from acid sphingomyelinase-deficient mice (ASMKO) Abeta(1-42) and IAPP but not exogenously added N-acetyl-D-sphingosine (C2-ceramide, 20 microM) failed to stimulate apoptosis. Immunofluorescent staining revealed a stimulatory effect of Abeta(1-42) on ceramide formation. According to patch clamp experiments, administration of Abeta(1-42) and IAPP significantly decreased outwardly rectifying whole cell currents in wild type but not in ASMKO islet cells. C2-ceramide but not inactive di-ceramide (20 microM) mimicked the inhibitory effect on Kv channel current. In conclusion, amyloid peptides induce apoptosis of pancreatic islet cells at least in part through activation of acid sphingomyelinase resulting in production of ceramide and subsequent inhibition of ion channel activity.

Regulation of calcineurin activity in insulin-secreting cells: stimulation by Hsp90 during glucocorticoid-induced apoptosis.

Previously, we described that apoptotic cell death induced by the synthetic glucocorticoid dexamethasone (dex) is inhibited by calcineurin inhibitors, FK506 and deltamethrin, in insulin-secreting cells. The aim of the present study was to examine the mechanism of dex-dependent activation of calcineurin. In INS-1 cells cultured up to 4d with dex (100 nmol/l), the percentage of apoptosis, quantified by condensed nuclei and TUNEL positive cells, increased from 1% to 10.9%. FK506 inhibited dex-mediated cell death. Apoptosis was significantly higher at glucose concentrations that induce [Ca(2+)](i) oscillations than at low, non-stimulatory glucose. Dex had no acute effect on [Ca(2+)](i). Calcineurin activity, measured in control and dex-treated cell homogenates, revealed that maximal activity and the sensitivity to the substrate RII peptide was unaltered. However, dex treatment significantly increased enzyme activity at submaximal, physiological Ca(2+) concentrations. Dex did not stimulate the Ca(2+)-dependent protease calpain, known to activate calcineurin by cleavage, as no cleaved calcineurin was detectable. Furthermore, the calpain inhibitor ALLN did not counteract dex-dependent cell death. Western blotting revealed that in dex-treated cells heat shock protein 90 (Hsp90), a component of the glucocorticoid receptor (GR) known to stimulate calcineurin, was increased while calcineurin protein levels were unchanged. In immunoprecipitates with calcineurin antibodies, Hsp90 was only detected in dex-treated cell homogenates. These data suggest that dex-induced apoptosis involves release of Hsp90 from the stimulated GR complex, subsequent binding to and activation of calcineurin, that may contribute to dex-mediated cell death in the presence of high glucose.

IGF-1 protects against dexamethasone-induced cell death in insulin secreting INS-1 cells independent of AKT/PKB phosphorylation.

Appropriate insulin secretion depends on beta-cell mass that is determined by the balance between cell proliferation and death. IGF-1 stimulates proliferation and protects against apoptosis. In contrast, glucocorticoids promote cell death. In this study we examined molecular interactions of the glucocorticoid dexamethasone (dexa) with IGF-1 signalling pathways in insulin secreting INS-1 cells. IGF-1 (50 ng/ml) increased the growth rate and stimulated BrdU incorporation, while dexa (100 nmol/l) inhibited cell growth, BrdU incorporation and induced apoptosis. Dexa-induced cell death was partially antagonized by IGF-1. This protection was further increased by LY294002 (10 micromol/l), an inhibitor of PI3 kinase. In contrast, MAP kinase inhibitor PD98059 (10 micromol/l) significantly reduced the protective effect of IGF-1. The analysis of signalling pathways by Western blotting revealed that dexa increased IRS-2 protein abundance while the expression of PI3K, PKB and ERK remained unchanged. Despite increased IRS-2 protein,IRS-2 tyrosine phosphorylation stimulated by IGF-1 was inhibited by dexa. Dexa treatment reduced basal PKB phosphorylation. However, IGF-1-mediated stimulation of PKB phosphorylation was not affected by dexa, but ERK phosphorylation was reduced. LY294002 restored IGF-1-induced ERK phosphorylation. These data suggest that dexa induces apoptosis in INS-1 cells by inhibiting phosphorylation of IRS-2, PKB and ERK. IGF-1 counteracts dexa-mediated apoptosis in the presence of reduced PKB but increased ERK phosphorylation.

The KATP channel is critical for calcium sequestration into non-ER compartments in mouse pancreatic beta cells.

K(ATP) channel activity influences beta cell Ca(2+) homeostasis by regulating Ca(2+) influx through L-type Ca(2+) channels. The present paper demonstrates that loss of K(ATP) channel activity due to pharmacologic or genetic ablation affects Ca(2+) storage in intracellular organelles. ATP depletion, by the mitochondrial inhibitor FCCP, led to Ca(2+) release from the endoplasmic reticulum (ER) of wildtype beta cells. Blockade of ER Ca(2+) ATPases by cyclopiazonic acid abolished the FCCP-induced Ca(2+) transient. In beta cells treated with K(ATP) channel inhibitors FCCP elicited a significantly larger Ca(2+) transient. Cyclopiazonic acid did not abolish this Ca(2+) transient suggesting that non-ER compartments are recruited as additional Ca(2+) stores in beta cells lacking K(ATP) channel activity. Genetic ablation of K(ATP) channels in SUR1KO mice produced identical results. In INS-1 cells transfected with a mitochondrial-targeted Ca(2+)-sensitive fluorescence dye (ratiometric pericam) the increase in mitochondrial Ca(2+) evoked by tolbutamide was 5-fold larger compared to 15 mM glucose. These data show that genetic or pharmacologic ablation of K(ATP) channel activity conveys Ca(2+) release from a non-ER store. Based on the sensitivity to FCCP and the property of tolbutamide to increase mitochondrial Ca(2+) it is suggested that mitochondria are the recruited store. The change in Ca(2+) sequestration in beta cells treated with insulinotropic antidiabetics may have implications for beta cell survival and the therapeutic use of these drugs.

Dexamethasone increases Na+/K+ ATPase activity in insulin secreting cells through SGK1.

Glucocorticoids blunt insulin release, an effect partially due to activation of Kv channels. Similar to those channels Na+/K+ ATPase activity repolarizes the plasma membrane. The present study explored whether glucocorticoids increase the Na+/K+ ATPase activity in pancreatic beta-cells. The glucocorticoid dexamethasone (100 nmol/l for 1 day) significantly increased Na+/K+ ATPase alpha1/beta1-subunit transcript levels and ouabain-sensitive outward current reflecting Na+/K+ ATPase activity in INS-1 cells, effects blunted by glucocorticoid-receptor-blocker RU487 (1 micromol/l). Dexamethasone (100 nmol/l) increased K+ current in beta-cells from wild type mice but not from knockout mice lacking functional serum and glucocorticoid inducible kinase SGK1. Thus, glucocorticoids indeed up-regulate Na+/K+ ATPase activity, an effect requiring SGK1.

Dexamethasone induces cell death in insulin-secreting cells, an effect reversed by exendin-4.

Glucocorticoid excess induces hyperglycemia, which may result in diabetes. The present experiments explored whether glucocorticoids trigger apoptosis in insulin-secreting cells. Treatment of mouse beta-cells or INS-1 cells with the glucocorticoid dexamethasone (0.1 micromol/l) over 4 days in cell culture increased the number of fractionated nuclei from 2 to 7 and 14%, respectively, an effect that was reversed by the glucocorticoid receptor antagonist RU486 (1 micromol/l). In INS-1 cells, dexamethasone increased the number of transferase-mediated dUTP nick-end labeling-staining positive cells, caspase-3 activity, and poly-(ADP-) ribose polymerase protein cleavage; decreased Bcl-2 transcript and protein abundance; dephosphorylated the proapoptotic protein of the Bcl-2 family (BAD) at serine155; and depolarized mitochondria. Dexamethasone increased PP-2B (calcineurin) activity, an effect abrogated by FK506. FK506 (0.1 micromol/l) and another calcineurin inhibitor, deltamethrin (1 micromol/l), attenuated dexamethasone-induced cell death. The stable glucagon-like peptide 1 analog, exendin-4 (10 nmol/l), inhibited dexamethasone-induced apoptosis in mouse beta-cells and INS-1 cells. The protective effect of exendin-4 was mimicked by forskolin (10 micromol/l) but not mimicked by guanine nucleotide exchange factor with the specific agonist 8CPT-Me-cAMP (50 micromol/l). Exendin-4 did not protect against cell death in the presence of cAMP-dependent protein kinase (PKA) inhibition by H89 (10 micromol/l) or KT5720 (5 micromol/l). In conclusion, glucocorticoid-induced apoptosis in insulin-secreting cells is accompanied by a downregulation of Bcl-2, activation of calcineurin with subsequent dephosphorylation of BAD, and mitochondrial depolarization. Exendin-4 protects against glucocorticoid-induced apoptosis, an effect mimicked by forskolin and reversed by PKA inhibitors.

Effects of I(Ks) channel inhibitors in insulin-secreting INS-1 cells.

Potassium channels regulate insulin secretion. The closure of K(ATP) channels leads to membrane depolarisation, which triggers Ca(2+) influx and stimulates insulin secretion. The subsequent activation of K(+) channels terminates secretion. We examined whether KCNQ1 channels are expressed in pancreatic beta-cells and analysed their functional role. Using RT/PCR cellular mRNA of KCNQ1 but not of KCNE1 channels was detected in INS-1 cells. Effects of two sulfonamide analogues, 293B and HMR1556, inhibitors of KCNQ1 channels, were examined on voltage-activated outwardly rectifying K(+) currents using the patch-clamp method. It was found that 293B inhibited 60% of whole-cell outward currents induced by voltage pulses from -70 to +50 mV with a concentration for half-maximal inhibition (IC(50)) of 37 microM. The other sulfonamide analogue HMR1556 inhibited 48% of the outward current with an IC(50) of 7 microM. The chromanol 293B had no effect on tolbutamide-sensitive K(ATP) channels. Action potentials induced by current injections were broadened and after-repolarisation was attenuated by 293B. Insulin secretion in the presence but not in the absence of tolbutamide was significantly increased by 293B. These results suggest that 293B- and HMR1556-sensitive channels, probably in concert with other voltage-activated K(+) channels, influence action potential duration and frequency and thus insulin secretion.

Serum- and glucocorticoid-inducible kinase 1 (SGK1) mediates glucocorticoid-induced inhibition of insulin secretion.

Glucocorticoid excess predisposes to the development of diabetes, at least in part through impairment of insulin secretion. The underlying mechanism has remained elusive. We show here that dexamethasone upregulates transcription and expression of the serum- and glucocorticoid-inducible kinase 1 (SGK1) in insulin-secreting cells, an effect reversed by mifepristone (RU486), an antagonist of the nuclear glucocorticoid receptor. When coexpressed in Xenopus oocytes, SGK1 increases the activity of voltage-gated K(+) channel K(v)1.5. In INS-1 cells, dexamethasone stimulates the transcription of K(v)1.5, increases the repolarizing outward current, reduces peak values of [Ca(2+)](i) oscillations, and decreases glucose-induced insulin release. The latter effect is reversed by K(+) channel blockers 4-aminopyridine and tetraethylammonium and by a more selective K(v)1.5 channel inhibitor MSD-D. Dexamethasone also increases expression of K(v)1.5 in mouse islets and reduces glucose-induced insulin secretion, an effect reversed by MSD-D. In islets isolated from wild-type but not SGK1 knockout mice, dexamethasone significantly blunted glucose-, forskolin-, and phorbol myristic acid-induced insulin release. In conclusion, dexamethasone stimulates the transcription of SGK1, which in turn upregulates the activity of voltage-gated K(+) channels. Increased K(+) channel activity reduces Ca(2+) entry through voltage-gated Ca(2+) channels and insulin release.