PLK1 inhibition down-regulates polycomb group protein BMI1 via modulation of the miR-200c/141 cluster.

The polycomb group protein BMI1 is an important regulator of cancer stem cell (CSC) phenotype and is often overexpressed in cancer cells. Its overexpression leads to increase in CSC fraction and therapy resistance in tumors. BMI1 functions via polycomb repressive complex 1 (PRC1)-mediated gene silencing and also via PRC1-independent transcriptional activities. At present, very little is known about the therapy reagents that can efficiently inhibit BMI1 expression, and the CSC phenotype. Here, we report that the polo-like kinase 1 (PLK1) regulates BMI1 expression, and that its inhibition can efficiently down-regulate BMI1 expression and PRC1 activity, and induce premature senescence in breast cancer cells. We also show that the exogenous BMI1 overexpression mitigates anti-oncogenic effects of PLK1 inhibition and overcomes senescence induction by PLK1 inhibitors. We further show that PLK1 inhibition down-regulates BMI1 by upregulating the miRNA-200c/141 cluster, which encodes miR-200c and miR-141, both of which are known to post-transcriptionally downregulate BMI1 expression. Thus, our data suggest that PLK1 inhibitors can be successfully used to inhibit growth of tumors in which PcG protein BMI1 is overexpressed or the PRC1 activity is deregulated.

MicroRNA-31 is a transcriptional target of histone deacetylase inhibitors and a regulator of cellular senescence.

MicroRNAs (miRNAs) have emerged as important regulators of tumorigenesis. Several miRNAs, which can function either as oncomiRs or tumor suppressive miRs are deregulated in cancer cells. The microRNA-31 (miR-31) has been shown to be overexpressed in metastatic breast cancer. It promotes multiple oncogenic phenotypes, including proliferation, motility, and invasion of cancer cells. Using a breast cancer-related miRNA array analysis, we identified miR-31 as a novel target of histone deacetylase inhibitors (HDACi) in breast cancer cells. Specifically, we show that sodium butyrate (NaB) and panobinostat (LBH589), two broad-spectrum HDAC inhibitors up-regulate hsa-miR-31 (miR-31). The up-regulation of miR-31 was accompanied by repression of the polycomb group (PcG) protein BMI1 and induction of cellular senescence. We further show that inhibition of miR-31 overcomes the senescence-inducing effect of HDACi, and restores expression of the PcG protein BMI1. Interestingly, BMI1 also acts as a repressor of miR-31 transcription, suggesting a cross-negative feedback loop between the expression of miR-31 and BMI1. Our data suggest that miR-31 is an important physiological target of HDACi, and that it is an important regulator of senescence relevant to cancer. These studies further suggest that manipulation of miR-31 expression can be used to modulate senescence-related pathological conditions such as cancer, and the aging process.

A positive feedback loop regulates the expression of polycomb group protein BMI1 via WNT signaling pathway.

Polycomb group protein BMI1 plays an important role in cellular homeostasis by maintaining a balance between proliferation and senescence. It is often overexpressed in cancer cells and is required for self-renewal of stem cells. At present, very little is known about the signaling pathways that regulate the expression of BMI1. Here, we report that BMI1 autoactivates its own promoter via an E-box present in its promoter. We show that BMI1 acts as an activator of the WNT pathway by repressing Dickkopf (DKK) family of WNT inhibitors. BMI1 mediated repression of DKK proteins; in particular, DKK1 led to up-regulation of WNT target c-Myc, which in turn further led to transcriptional autoactivation of BMI1. Thus, a positive feedback loop connected by the WNT signaling pathway regulates BMI1 expression. This positive feedback loop regulating BMI1 expression may be relevant to the role of BMI1 in promoting cancer and maintaining stem cell phenotype.

Regulation of Golgi structure and secretion by receptor-induced G protein ßγ complex translocation.

We show that receptor induced G protein betagamma subunit translocation from the plasma membrane to the Golgi allows a receptor to initiate fragmentation and regulate secretion. A lung epithelial cell line, A549, was shown to contain an endogenous translocating G protein gamma subunit and exhibit receptor-induced Golgi fragmentation. Receptor-induced Golgi fragmentation was inhibited by a shRNA specific to the endogenous translocating gamma subunit. A kinase defective protein kinase D and a phospholipase C beta inhibitor blocked receptor-induced Golgi fragmentation, suggesting a role for this process in secretion. Consistent with betagamma translocation dependence, fragmentation induced by receptor activation was inhibited by a dominant negative nontranslocating gamma3. Insulin secretion was shown to be induced by muscarinic receptor activation in a pancreatic beta cell line, NIT-1. Induction of insulin secretion was also inhibited by the dominant negative gamma3 subunit consistent with the Golgi fragmentation induced by betagamma complex translocation playing a role in secretion.

Bacterial infection causes stress-induced memory dysfunction in mice.

BACKGROUND: The brain-gut axis is a key regulator of normal intestinal physiology; for example, psychological stress is linked to altered gut barrier function, development of food allergies and changes in behaviour. Whether intestinal events, such as enteric bacterial infections and bacterial colonisation, exert a reciprocal effect on stress-associated behaviour is not well established. OBJECTIVE: To determine the effects of either acute enteric infection or absence of gut microbiota on behaviour, including anxiety and non-spatial memory formation. METHODS: Behaviour was assessed following infection with the non-invasive enteric pathogen, Citrobacter rodentium in both C57BL/6 mice and germ-free Swiss-Webster mice, in the presence or absence of acute water avoidance stress. Whether daily treatment with probiotics normalised behaviour was assessed, and potential mechanisms of action evaluated. RESULTS: No behavioural abnormalities were observed, either at the height of infection (10 days) or following bacterial clearance (30 days), in C rodentium-infected C57BL/6 mice. When infected mice were exposed to acute stress, however, memory dysfunction was apparent after infection (10 days and 30 days). Memory dysfunction was prevented by daily treatment of infected mice with probiotics. Memory was impaired in germ-free mice, with or without exposure to stress, in contrast to conventionally reared, control Swiss-Webster mice with an intact intestinal microbiota. CONCLUSIONS: The intestinal microbiota influences the ability to form memory. Memory dysfunction occurs in infected mice exposed to acute stress, while in the germ-free setting memory is altered at baseline.

Oocyte-expressed interleukin 7 suppresses granulosa cell apoptosis and promotes oocyte maturation in rats.

Development of ovarian follicles is regulated by pituitary-derived gonadotropins together with local ovarian paracrine factors. Based on DNA microarray data, we performed RT-PCR and immunostaining to demonstrate the expression of interleukin 7 transcripts in oocytes of preantral, antral, and preovulatory follicles in rats. We also found the expression of interleukin 7 receptor and the coreceptor interleukin 2 receptor gamma in granulosa cells, cumulus cells, and preovulatory oocytes. In cultured rat granulosa cells obtained from early antral and preovulatory follicles, treatment with interleukin 7 stimulated the phosphorylation of AKT, glycogen synthase kinase (GSK3B), and STAT5 proteins in a time- and dose-dependent manner. Furthermore, measurement of mitochondrial reductase activity indicated that treatment with interleukin 7, similar to gonadotropins, increased the number of viable granulosa cells during a 24-h culture period. Furthermore, monitoring of the activities of apoptotic enzymes (caspase 3/7) indicated that treatment with interleukin 7 suppressed apoptosis of cultured granulosa cells from both antral and preovulatory follicles following serum withdrawal. The apoptosis-suppressing actions of interleukin 7 were blocked by an inhibitor of the phosphoinositol-3-kinase (PIK3)/AKT pathway. Furthermore, treatment of cultured preovulatory follicles with interleukin 7, like treatment with human chorionic gonadotropin, induced germinal vesicle breakdown of oocytes. The stimulatory effect of interleukin 7 was also blocked by inhibitors of the PIK3/AKT pathway. The present findings suggest that oocyte-derived interleukin 7 could act on neighboring granulosa cells as a survival factor and promote the nuclear maturation of preovulatory oocytes through activation of the PIK3/AKT pathway.

Nod1 and Nod2 regulation of inflammation in the Salmonella colitis model.

The pattern recognition molecules Nod1 and Nod2 play important roles in intestinal homeostasis; however, how these proteins impact on the development of inflammation during bacterial colitis has not been examined. In the streptomycin-treated mouse model of Salmonella colitis, we found that mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources contributed to the pathology, and all phenotypes were recapitulated in mice deficient for the signaling adaptor protein Rip2. However, the influence of Rip2 was strictly dependent on infection conditions that favored expression of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as Rip2 was dispensable for inflammation when mice were infected with bacteria grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1 and Nod2 can modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis, but their role is dependent on the expression of the SPI-2 TTSS.

Delayed Hemorrhage of the Hepatic Artery Caused by Biliary Stenting after Concurrent Chemoradiotherapy.

Neoadjuvant concurrent chemoradiotherapy has been increasingly used to obtain secondary resectability for locally advanced pancreatic cancers. Although most patients require biliary decompression, only a few studies have investigated the safety of biliary stenting with chemoradiotherapy. Herein, we report a rare case of delayed hemorrhage of the hepatic artery caused by biliary stenting after chemoradiotherapy. The serial follow-up CT demonstrated that the biliary stent was approaching the right hepatic artery and eventually caused acute angulation and indentation. Diagnostic catheter angiography revealed contrast extravasation at the right hepatic artery, and endovascular embolization was performed. This report highlights the relevance of anatomical deformation after chemoradiotherapy, which can result in fatal complications. Indentation of the hepatic artery caused by biliary stents should be recognized as a warning sign of vascular injury.

Peritoneal Carcinomatosis and Its Mimics: Review of CT Findings for Differential Diagnosis.

Peritoneal carcinomatosis (PC) indicates the metastasis of a malignant neoplasm to the peritoneal surface. PC can be incidentally detected before discovery of the primary malignancy during an imaging study. There are other conditions that can mimic PC, such as pseudomyxoma peritonei, peritoneal lymphomatosis, peritoneal malignant mesothelioma, leiomyomatosis peritonealis disseminata, and tuberculous peritonitis. These diseases may appear similar on computed tomography (CT), but there are some clues for the differential diagnosis. This article will describe the CT findings of PC and its mimics for the differential diagnosis.

Right Paraduodenal Hernia with Midgut Malrotation.

Main teaching point: Right paraduodenal hernia typically occurs in the fossa of Waldeyer, with the herniated bowel sac located posterior to the right colic artery and vein.

Fibroblast growth factor 7 stimulates in vitro growth of oocytes originating from bovine early antral follicles.

Essential factors required for growing oocytes derived from bovine early antral follicles and their mechanisms of action are poorly understood. Fibroblast growth factor 7 (FGF7) is a member of the heparin-binding FGF family with a distinctive pattern of target-cell specificity. The effect of FGF7 on the stimulation of oocyte growth in a culture of cumulus-oocyte complexes with granulosa cells (COCGs, oocyte diameter; 90-100 microm) was investigated. The oocyte diameter of COCGs was increased significantly in the FGF7-containing medium (10 ng/ml; 117.2 +/- 3.2 microm, 50 ng/ml; 116.5 +/- 3.5 microm) compared to the control (0 ng/ml; 110.5 +/- 2.8 microm) after 16 days. However, there was no stimulatory effect of FGF7 on the proliferation of cumulus-granulosa cells. The FGF7 receptor, fibroblast growth factor receptor 2IIIb (FGFR2IIIb), was detected in cumulus-granulosa cells from COCGs. Messenger RNA expression of FGFR2IIIb was induced to cumulus-granulosa cells by FGF7. The mRNA expression levels of KIT ligand (KITLG), KIT (KIT), growth differentiation factor 9 (GDF9), and bone morphogenetic protein 15 (BMP15) in the cultured COCGs were determined in FGF7-treated (10 ng/ml) cultures using real time RT-PCR analysis. The levels of KITLG and KIT, but not GDF9 and BMP15 mRNA expression were stimulated by FGF7. Furthermore, neutralizing antibody for KIT attenuated the stimulatory action of FGF7 on the oocyte growth. These results strongly suggest that FGF7 may be an important regulator for oocyte growth and its action is mediated via the KIT/KITLG signaling pathway.

[Effects of octreotide on small bowel obstructions in rats].

BACKGROUNDS/AIMS: Gastrointestinal decompression by nasogastric or intestinal tubes developed in 1930s has been the only treatment modality for inoperable intestinal obstruction. We hypothesized that the octreotide, a potent inhibitor of intestinal secretion, has a therapeutic potential in intestinal obstruction. METHODS: Forty Sprague-Dawley rats were randomly assigned to four groups. The rats were subjected to complete or partial ileal obstruction. The treated rats received octreotide (100 microgram/kg) while the controls received the same quantity of saline every 12 hours for 24 or 48 hours. After 24 or 48 hours, the volumes of the small bowel contents were measured. The volumes of supernatant and the concentrations of electrolytes in the small bowel contents after centrifugation were also analyzed. The ileal segments proximal to obstruction were harvested, fixed, and stained, and the pathological changes were evaluated with mucosal damage scores. RESULTS: There were no statistical differences in the volume and the electrolyte composition of intestinal fluid among the 4 groups. In the 48 hour complete obstruction group, the octreotide-treated rats showed statistically lower mucosal damage scores than the control rats (p<0.05). CONCLUSIONS: Octreotide exerts mucosal protecting effect on the complete intestinal obstruction rat model.

microRNA-141 regulates BMI1 expression and induces senescence in human diploid fibroblasts.

Polycomb group protein BMI1 is an important regulator of senescence, aging, and cancer. On one hand, it is overexpressed in cancer cells and is required for self-renewal of stem cells. On the other hand, it is downregulated during senescence and aging. MicroRNAs have emerged as major regulators of almost every gene associated with cancer, aging, and related pathologies. At present, very little is known about the miRNAs that regulate the expression of BMI1. Here, we report that miR-141 posttranscriptionally downregulates BMI1 expression in human diploid fibroblasts (HDFs) via a miR-141 targeting sequence in the 3' untranslated region of BMI1 mRNA. We also show that overexpression of miR-141 induces premature senescence in HDFs via targeting of BMI1 in normal but not in exogenous BMI1-overexpressing HDFs. Induction of premature senescence in HDFs was accompanied by upregulation of p16INK4a, an important downstream target of BMI1 and a major regulator of senescence. Our results suggest that miR-141-based therapies could be developed to treat pathologies where BMI1 is deregulated.

Nod1 and Nod2 signaling does not alter the composition of intestinal bacterial communities at homeostasis.

Patients with inflammatory bowel diseases (IBD) harbour intestinal bacterial communities with altered composition compared with healthy counterparts; however, it is unknown whether changes in the microbiota are associated with genetic susceptibility of individuals for developing disease or instead reflect other changes in the intestinal environment related to the disease itself. Since deficiencies in the innate immune receptors Nod1 and Nod2 are linked to IBD, we tested the hypothesis that Nod-signaling alters intestinal immune profiles and subsequently alters bacterial community structure. We used qPCR to analyze expression patterns of selected immune mediators in the ileum and cecum of Nod-deficient mice compared with their Nod-sufficient littermates and assessed the relative abundance of major bacterial groups sampled from the ileum, cecum and colon. The Nod1-deficient ileum exhibited significantly lower expression of Nod2, Muc2, α- and ß-defensins and keratinocyte-derived chemokine (KC), suggesting a weakened epithelial barrier compared with WT littermates; however, there were no significant differences in the relative abundance of targeted bacterial groups, indicating that Nod1-associated immune differences alone do not promote dysbiosis. Furthermore, Nod2-deficient mice did not display any changes in the expression of immune markers or bacterial communities. Shifts in bacterial communities that were observed in this study correlated with housing conditions and were independent of genotype. These findings emphasize the importance of using F2 littermate controls to minimize environmental sources of variation in microbial analyses, to establish baseline conditions for host-microbe homeostasis in Nod-deficient mice and to strengthen models for testing factors contributing to microbial dysbiosis associated with IBD.

Alteration of Golgi structure in senescent cells and its regulation by a G protein γ subunit.

Cellular senescence is a process wherein proliferating cells undergo permanent cell cycle arrest while remaining viable. Senescence results in enhanced secretion of proteins that promote cancer and inflammation. We report here that the structure of the Golgi complex which regulates secretion is altered in senescent cells. In cells where senescence is achieved by replicative exhaustion or in cells wherein senescence has been induced with BrdU treatment dependent stress, the Golgi complex is dispersed. The expression of a G protein γ subunit, γ11, capable of translocation from the plasma membrane to the Golgi complex on receptor activation increases with senescence. Knockdown of γ11 or overexpression of a dominant negative γ3 subunit inhibits Golgi dispersal induced by senescence. Overall these results suggest that in cellular senescence an upregulated G protein gamma subunit mediates alterations in the structure of the Golgi.

Identification of an innate T helper type 17 response to intestinal bacterial pathogens.

Interleukin 17 (IL-17) is a central cytokine implicated in inflammation and antimicrobial defense. After infection, both innate and adaptive IL-17 responses have been reported, but the type of cells involved in innate IL-17 induction, as well as their contribution to in vivo responses, are poorly understood. Here we found that Citrobacter and Salmonella infection triggered early IL-17 production, which was crucial for host defense and was mediated by CD4(+) T helper cells. Enteric innate T helper type 17 (iT(H)17) responses occurred principally in the cecum, were dependent on the Nod-like receptors Nod1 and Nod2, required IL-6 induction and were associated with a decrease in mucosal CD103(+) dendritic cells. Moreover, imprinting by the intestinal microbiota was fully required for the generation of iT(H)17 responses. Together, these results identify the Nod-iT(H)17 axis as a central element in controlling enteric pathogens, which may implicate Nod-driven iT(H)17 responses in the development of inflammatory bowel diseases.

G protein subunit dissociation and translocation regulate cellular response to receptor stimulation.

We examined the role of G proteins in modulating the response of living cells to receptor activation. The response of an effector, phospholipase C-beta to M3 muscarinic receptor activation was measured using sensors that detect the generation of inositol triphosphate or diacylglycerol. The recently discovered translocation of G betagamma from plasma membrane to endomembranes on receptor activation attenuated this response. A FRET based G protein sensor suggested that in contrast to translocating G betagamma, non-translocating G betagamma subunits do not dissociate from the alpha q subunit on receptor activation leading to prolonged retention of the heterotrimer state and an accentuated response. M3 receptors with tethered alpha q induced differential responses to receptor activation in cells with or without an endogenous translocation capable gamma subunit. G protein heterotrimer dissociation and betagamma translocation are thus unanticipated modulators of the intensity of a cell's response to an extracellular signal.