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.

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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 serumand glucocorticoid-inducible kinase 1 (SGK1) in insulinsecreting cells, an effect reversed by mifepristone (RU486), an antagonist of the nuclear glucocorticoid receptor. G lucocorticoids are known to induce diabetes (1-3). In addition to peripheral insulin resistance and increased hepatic glucose production by stimulating gluconeogenesis (4), glucocorticoids interfere with insulin secretion of pancreatic ␤-cells (5-7). Despite extensive (8 -12) studies, the molecular mechanism is still a matter of debate. Increased expression of ␣ 2 -adrenoceptors has been proposed to account for dexamethasone-induced inhibition of insulin secretion (9). Thus, transgenic mice overexpressing glucocorticoid receptors in ␤-cells show 30% more UK14304 binding, a selective adrenoceptor agonist, than wild-type islets (2). These mice are glucose intolerant and have reduced plasma insulin levels. Since pertussis toxin and cAMP overcome dexamethasone inhibition of glucose-induced insulin release, decreased cAMP levels during dexamethasone treatment may be responsible for inhibition of secretion (6,13). Furthermore, dexamethasone was reported to decrease Glut2 protein abundance at the plasma membrane, a change that may contribute to impaired glucose-induced insulin secretion (8). Dexamethasone also downregulates glucokinase mRNA in an insulin-secreting cell line (14). Mifepristone (RU486), a nuclear glucocorticoid receptor antagonist, completely abolished dexamethasone-induced inhibition of insulin secretion (5,6), pointing to the involvement of glucocorticoid-dependent gene expression. Glucocorticoid-sensitive genes include the serum-and glucocorticoid-inducible kinase 1 (SGK1) (rev. in 15). The kinase is expressed in virtually all human tissues tested. Unlike its isoforms SGK2 and SGK3 and the related kinase protein kinase B, SGK1 is under strong transcriptional control of glucocorticoids (15) and mineralocorticoids (16). SGK1 has been shown to regulate a variety of ion channels including K ϩ channels such as voltage-gated K v channels (17).Ion channel activity is in turn decisive for insulin secretion from pancreatic ␤-cells. 4-AP, 4-aminopyridine; GAPDH, glyceraldehyde-3-phsophate dehydrogenase; SGK1, serum-and glucocorticoid-inducible kinase 1; TEA, tetraethylammonium.

BACKGROUND AND PURPOSEFenamates 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 APPROACHFirstly, 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 insulinsecreting INS-1E cells, which endogenously express TRPM3, we validated the effect of mefenamic acid on cytosolic Ca 2+ and insulin secretion. KEY RESULTSWe 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 IMPLICATIONSThis study reveals that mefenamic acid selectively inhibits TRPM3-mediated calcium entry. This selectivity was further confirmed using insulin-secreting cells. KATP 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 b-cell function. Our results strongly suggest mefenamic acid is the most selective fenamate to interfere with TRPM3 function.

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.

OBJECTIVEIn vitro models suggest that free fatty acid–induced apoptotic β-cell death is mediated through protein kinase C (PKC)δ. To examine the role of PKCδ signaling in vivo, transgenic mice overexpressing a kinase-negative PKCδ (PKCδKN) selectively in β-cells were generated and analyzed for glucose homeostasis and β-cell survival.RESEARCH DESIGN AND METHODSMice 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 PKCδ 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.RESULTSPKCδKN 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 β-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 PKCδKN mice. PKCδKN 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 PKCδKN was accompanied by an increased phosphorylation of FOXO1 at Ser256 and a significant reduction of FOXO1 protein.CONCLUSIONSOverexpression of PKCδKN in β-cells protects from HFD-induced β-cell failure in vivo by a mechanism that involves inhibition of fatty acid–mediated apoptosis, inhibition of mitochondrial dysfunction, and inhibition of FOXO1 activation.

Appropriate insulin secretion depends on β-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 µmol/l), an inhibitor of PI3 kinase. In contrast, MAP kinase inhibitor PD98059 (10 µmol/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 effects of hyperglycemia on insulin secretion override the more subtle effects of genetic variation in the G6PC2 locus on insulin secretion.