DNA polymerase delta Exo domain stabilizes mononucleotide microsatellites in human cells.

In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.

Fat-specific protein 27 is a novel target gene of liver X receptor α.

Fat-specific protein 27 (FSP27) is highly expressed in the fatty liver of genetically obese ob/ob mice and promotes hepatic triglyceride (TG) accumulation. The nuclear hormone receptor liver X receptor α (LXRα) also plays a critical role in the control of TG levels in the liver. The present study demonstrated transcriptional regulation of Fsp27a and Fsp27b genes by LXRα. Treatment with the LXR ligand T0901317 markedly increased Fsp27a and Fsp27b mRNAs in wild-type C57BL/6J and ob/ob mouse livers. A reporter assay indicated that two LXR-responsive elements (LXREs) are necessary for LXRα-dependent induction of Fsp27a and Fsp27b promoter activities. Furthermore, the LXRα/retinoid X receptor α complex is capable of directly binding to the two LXREs both in vitro and in vivo. These results suggest that LXRα positively regulates Fsp27a and Fsp27b expression through two functional LXREs. Fsp27a/b are novel LXR target genes in the ob/ob fatty liver.

The Role of Cholecystokinin in Peripheral Taste Signaling in Mice.

Cholecystokinin (CCK) is a gut hormone released from enteroendocrine cells. CCK functions as an anorexigenic factor by acting on CCK receptors expressed on the vagal afferent nerve and hypothalamus with a synergistic interaction between leptin. In the gut, tastants such as amino acids and bitter compounds stimulate CCK release from enteroendocrine cells via activation of taste transduction pathways. CCK is also expressed in taste buds, suggesting potential roles of CCK in taste signaling in the peripheral taste organ. In the present study, we focused on the function of CCK in the initial responses to taste stimulation. CCK was coexpressed with type II taste cell markers such as Gα-gustducin, phospholipase Cß2, and transient receptor potential channel M5. Furthermore, a small subset (~30%) of CCK-expressing taste cells expressed a sweet/umami taste receptor component, taste receptor type 1 member 3, in taste buds. Because type II taste cells are sweet, umami or bitter taste cells, the majority of CCK-expressing taste cells may be bitter taste cells. CCK-A and -B receptors were expressed in both taste cells and gustatory neurons. CCK receptor knockout mice showed reduced neural responses to bitter compounds compared with wild-type mice. Consistently, intravenous injection of CCK-Ar antagonist lorglumide selectively suppressed gustatory nerve responses to bitter compounds. Intravenous injection of CCK-8 transiently increased gustatory nerve activities in a dose-dependent manner whereas administration of CCK-8 did not affect activities of bitter-sensitive taste cells. Collectively, CCK may be a functionally important neurotransmitter or neuromodulator to activate bitter nerve fibers in peripheral taste tissues.

Insulin Represses Fasting-Induced Expression of Hepatic Fat-Specific Protein 27.

The fat-specific protein 27 (Fsp27) gene belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue as well as the fatty liver of ob/ob mice. Fsp27 is directly regulated by the peroxisome proliferator-activated receptor γ (PPARγ) in livers of genetically obese leptin deficient ob/ob mice. In the present study, Fsp27 was markedly induced by 24 h fasting in genetically normal mouse livers and repressed by refeeding a high sucrose diet. In contrast with the liver, Fsp27 expression was decreased in adipose tissue by fasting and increased by refeeding. Interestingly, fasting-induced Fsp27 liver expression was independent of PPARγ. Moreover, Fsp27 expression was induced in the insulin-depleted livers of streptozotocin-treated mice. Finally, Fsp27 expression was repressed by direct injection of glucose or insulin in fasting mice. These results suggest that insulin represses Fsp27 expression in the fasting liver.

Crizotinib, a MET inhibitor, prevents peritoneal dissemination in pancreatic cancer.

Peritoneal dissemination is a frequent occurrence in pancreatic cancer, which is associated with a poor prognosis. MET is associated with the progression of pancreatic cancer; therefore, we evaluated the effect of a MET inhibitor, crizotinib, on peritoneal dissemination of pancreatic cancer. Crizotinib inhibited the growth of 8 pancreatic cancer cell lines with the IC50 ranging from 1.4 to 4.3 µM. Invasion of the pancreatic cancer cell line Suit-2, was suppressed in vitro at a concentration of 1.0 µM, which is sufficient for the inhibition of MET phosphorylation. This effect on cell invasion was also recapitulated by the reduction of MET expression in Suit-2 with siRNA. Crizotinib also inhibited RhoA activation in addition to MET phosphorylation. We further evaluated the effect of crizotinib on peritoneal dissemination of pancreatic cancer in vivo. Crizotinib reduced tumor burden and ascites accumulation due to development of peritoneal dissemination after inoculation of Suit-2. Taken together, crizotinib may be a potent drug for treating peritoneal dissemination of pancreatic cancer by inhibiting cancer cell proliferation and invasion, at least in part through the suppression of HGF/MET signaling and RhoA activation.

Ephedrae herba stimulates hepatocyte growth factor-induced MET endocytosis and downregulation via early/late endocytic pathways in gefitinib-resistant human lung cancer cells.

The MET tyrosine kinase receptor and its ligand, hepatocyte growth factor (HGF), are known to be overexpressed in a variety of malignant tumor cells, and are implicated in the development of gefitinib-resistance in human non-small cell lung cancer (NSCLC) cells. Ephedrae herba was previously reported to prevent HGF-induced cancer cell motility by directly suppressing HGF/MET signaling through the inhibition of MET tyrosine kinase, and treatment with its extract also considerably reduced MET protein levels. To further investigate the mechanism underlying the Ephedrae herba-induced inhibition of MET phosphorylation as well as its degradation and subsequent disappearance, we examined the effect of Ephedrae herba on HGF-stimulated MET endocytosis and downregulation via early/late endocytic pathways in an NSCLC cell line. Using immunofluorescence microscopy, we found that pretreatment of cells with Ephedrae herba extract dramatically changed the intracellular distribution of plasma membrane-associated MET, and that the resultant MET staining was distributed throughout the cytoplasm. Pretreatment of the cells with Ephedrae herba extract also led to the rapid loss of MET and phosphorylated (p)-MET in HGF-stimulated cells. In contrast, inefficient endocytic delivery of MET and p-MET from early to late endosomes was observed in the absence of Ephedrae herba extract, since considerable amounts of the internalized MET accumulated in the early endosomes and were not delivered to lysosomes up to 1 h after HGF-stimulation. Furthermore, large amounts of MET and p-MET that had accumulated in late endosomes of Ephedrae herba-pretreated cells after HGF stimulation were observed along with bafilomycin A1. Therefore, we inferred that degradation of MET occurred in the late endosome/lysosome pathway. Moreover, western blot analysis revealed the accelerated degradation of MET and p-MET proceeds in cells pretreated with Ephedrae herba extract. Collectively, our results suggest that some components of Ephedrae herba have a novel role in promoting HGF-stimulated MET and p-MET endocytosis followed by its downregulation, likely mediated by the early/late endocytic pathways.

EGF­stimulated AKT activation is mediated by EGFR recycling via an early endocytic pathway in a gefitinib­resistant human lung cancer cell line.

The receptor tyrosine kinase epidermal growth factor receptor (EGFR) and its ligand epidermal growth factor (EGF) are known to play important roles in malignant tumor cells, and the EGFR signaling pathway is one of the most important targets in various tumors, including non-small cell lung cancer (NSCLC). We reported recently that an aberration in certain steps of EGF-stimulated phosphorylated epidermal growth factor receptor (pEGFR) endocytic trafficking from the early endosomes to the late endosomes occurs in the gefitinib-resistant NSCLC cells, in which large amounts of sorting nexin 1 (SNX1) are colocalized with EGFR in the aggregated early endosomes where the internalized pEGFR is also accumulated of these cells. To further investigate the role of SNX1 in EGF­stimulated pEGFR endocytosis, followed by downstream signaling leading to the activation of phosphatidylinositol 3-kinase (PI3K)--the serine/threonine kinase AKT pathway, we examined the effect of depletion of SNX1 knock-down expression by siRNA and an inhibition of targeting membrane recycling using monensin. Using immunofluorescence, we observed an efficient endocytic transport of pEGFR from early endosomes to late endosomes/lysosomes after EGF-stimulation in the cells transfected with siRNA­SNX1, whereas the delayed endocytic delivery of pEGFR was evident in the siRNA-control-transfected cells. Furthermore, a large amount of endocytosed pEGFR was accumulated in the presence of monensin in the early endosomes of the SNX1 knock-down cells. In western blot analysis, EGF stimulation of both control and cells transfected with siRNA-SNX1 resulted in rapid phosphorylation of EGFR and enhanced AKT phosphorylation. Monensin-dependent inhibition of AKT phosphorylation was stronger in SNX1 knock-down cells than in controls. In contrast, however, monensin had no effect on AKT phosphorylation triggered by activation of the MET receptor tyrosine kinase. Collectively, we suggest that EGF-stimulated recycling of EGFR to the plasma membrane induces downstream signaling leading to AKT phosphorylation. Suppression of EGFR membrane recycling by SNX1 appears to be critical for the activation of EGFR/PI3K/AKT signaling pathway in human lung cancer cells.

Hepatic PPARγ and LXRα independently regulate lipid accumulation in the livers of genetically obese mice.

The nuclear hormone receptors liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ (PPARγ) play key roles in the development of fatty liver. To determine the link between hepatic PPARγ and LXRα signaling and the development of fatty liver, a LXRα-specific ligand, T0901317, was administered to normal OB/OB and genetically obese (ob/ob) mice lacking hepatic PPARγ (Pparγ(ΔH)). In ob/ob-Pparγ(ΔH) and OB/OB-Pparγ(ΔH) mice, as well as ob/ob-Pparγ(WT) and OB/OB-Pparγ(WT) mice, the liver weights and hepatic triglyceride levels were markedly increased in response to T0901317 treatment. These results suggest that hepatic PPARγ and LXRα signals independently contribute to the development of fatty liver.

Involvement of CXCL14 in osteolytic bone metastasis from lung cancer.

To investigate the molecular mechanisms of lung cancer-induced bone metastasis, we established a bone-seeking subclone (HARA-B4) from a human squamous lung cancer cell line (HARA) using an in vivo selection method. We compared comprehensive gene expression profiles between HARA and HARA-B4, and identified the critical factors for the formation of bone metastasis using in vitro and in vivo assays. The number of bone metastatic colonies in the hind legs was significantly higher in HARA-B4-inoculated mice than in HARA-inoculated mice at 4 weeks after inoculation. In addition, visceral (adrenal) metastases were not found in HARA-B4-inoculated mice at autopsy, suggesting an increase in cancer cell tropism to bone in HARA-B4. Based on a comprehensive gene expression analysis, the expression level of CXC chemokine ligand 14 (CXCL14) was 5-fold greater in HARA-B4 than in HARA. Results of a soft agar colony formation assay showed that anchorage-independent growth ability was 4.5-fold higher with HARA-B4 than with HARA. The murine pre-osteoblast cell line MC3T3-E1 and the pre-osteoclast/macrophage cell line RAW264.7 migrated faster toward cultured HARA-B4 cells than toward HARA cells in a transwell cell migration assay. Interestingly, CXCL14 was shown to be involved in all events (enhancement of cancer cell tropism to the bone, anchorage-independent growth and/or recruitment of bone marrow cells) based on siRNA experiments in HARA-B4 cells. Furthermore, in clinical specimens of lung cancer-induced bone metastasis, expression of CXCL14 was observed in the tumor cells infiltrated in bone marrow in all specimens examined. CXCL14 was able to promote bone metastasis through enhancement of cancer cell tropism to the bone and/or recruitment of bone marrow cells around metastatic cancer cells.

Evidence that depletion of the sorting nexin 1 by siRNA promotes HGF-induced MET endocytosis and MET phosphorylation in a gefitinib-resistant human lung cancer cell line.

The receptor tyrosine kinase MET and its ligand HGF are known to be overexpressed in malignant tumor cells, and they have been implicated in gefitinib resistance in lung cancer cells. We recently found that sorting nexin 1 (SNX1), a protein that interacts with EGFR, exhibited negative regulation of EGFR trafficking out of early to late endosomes in gefitinib-resistant NSCLC cell lines. To investigate the role of SNX1 on HGF-stimulated MET endocytosis and its downregulation via the early/late endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in NSCLC cells. Using immunofluorescence, we found that the silencing of SNX1 by siRNA caused a dramatic change in the intracellular distribution of plasma membrane-associated MET and that the resultant MET staining was spread throughout the cytoplasm, and it co-localized well with the endocytosed Texas red-labeled transferrin in the siRNA-SNX1-transfected cells. We also found efficient MET phosphorylation and rapid endocytic delivery of phosphorylated MET from early endosomes to late endosomes in the siRNA-SNX1-transfected cells. By contrast, the siRNA-control transfected cells showed inefficient endocytic delivery of phosphorylated MET from early endosomes to late endosomes. Furthermore, large amounts of phosphorylated MET that had accumulated in late endosomes were seen even after 60 min of HGF-stimulation in the presence of bafilomycin A1, indicating that degradation of phosphorylated MET proceeds in a late endosome/lysosome pathway. Western blot analysis revealed that depletion of SNX1 by siRNA induced a maximal and dramatic increase in phosphorylated MET at 60 min, followed by an accelerated degradation of phosphorylated MET after HGF stimulation in the cells. Taken together, we suggest that SNX1 plays a suppressive role in the regulation of HGF-stimulated MET/phosphorylated MET endocytosis and downregulation via the early/late endocytic pathway in the gefitinib-resistant NSCLC cells.

Expression of hepatic fat-specific protein 27 depends on the specific etiology of fatty liver.

Fat-specific protein 27 gene (FSP27), isolated by screening for genes specifically expressed in fully differentiated mouse adipocytes, belongs to the cell death-inducing DNA fragmentation factor, alpha subunit-like effector family. FSP27 is induced in not only adipose tissue but also the liver of ob/ob mice, and it promotes the development of fatty liver. The FSP27 gene is expressed in a fatty liver-specific manner and is not detected in the normal mouse liver. FSP27 expression is directly regulated by the induction of the hepatic peroxisome proliferator-activated receptor γ (PPARγ) in ob/ob fatty liver. In the present study, expression of hepatic FSP27 mRNA was determined in non-genetic fatty liver models. The FSP27 gene was markedly induced in the high-fat- or methionine- and choline-deficient (MCD) diet-induced fatty liver, but it was not elevated in alcohol-induced fatty liver. Interestingly, the induction of FSP27 mRNA due to the MCD diet was independent of PPARγ levels and completely absent in the liver from PPARγ-null mice. These results suggest that FSP27 mRNA expression in the liver depends on the etiology of fatty liver.

Infant satiety depends on transient expression of cholecystokinin-1 receptors on ependymal cells lining the third ventricle in mice.

Cholecystokinin (CCK) is a hypothetical controller for suckling and infancy body weight, although the underlying mechanisms remain unclear. Therefore, the present study analysed the mechanisms using mice lacking the CCK-1 receptor (CCK1R-/-). Although CCK1R-/- mice displayed normal weights at birth and adulthood, CCK1R-/- pups had enlarged adipocytes and were overweight from the first to second week after birth, regardless of maternal genotype. The lacZ reporter gene assay and/or calcium imaging analysis demonstrated that CCK-1 receptors were abundant in satiety-controlling regions such as the hypothalamus, brainstem, nodose ganglion and pylorus in adults, whereas these signals were few to lacking at pre-weanling stages. At postnatal day (PD) 6, the increase in cFos expression in the medullary nucleus tractus solitarius was similarly triggered by gastrointestinal milk- or saline filling in both genotypes, further indicating immature CCK-1 receptor function in an ascending satiety-controlling system during infancy. Conversely, third ventricle ependymal tanycyte-like cells expressed CCK-1 receptors with expression peaking at PD6. At PD6, wild-type but not CCK1R-/- mice had increased cFos immunoreactivity in ependymal cells following gastrointestinal milk filling whereas the response became negligible at PD12. In addition, ependymal cFos was not increased by saline filling, indicating that these responses are dependent on CCK-1 receptors, developmental stage and nutrients. Furthermore, body weights of wild-type pups were transiently increased by blocking ependymal CCK receptor function with microinjection of a CCK-1 antagonist, but not a CCK-2 antagonist. Hence, we demonstrate de novo functions of ependymal CCK-1 receptors and reveal a new aspect of infant satiety-controlling mechanisms.

Functional compensation between cholecystokinin-1 and -2 receptors in murine paraventricular nucleus neurons.

Cholecystokinin (CCK) and its receptor subtypes CCK-1 and -2 have diverse homeostatic functions. CCK-1 and -2 receptors share a common phosphatidylinositol signaling pathway, yet little is known regarding their possible functional coupling. We focused on CCK-mediated Ca(2+) signaling in parvocellular paraventricular nucleus (PVN) cells, which control satiety and other autonomic functions. Analysis of mouse hypothalamic slices demonstrated that the general CCK receptor agonist CCK-8s (10 nM) triggered Ca(2+) transients most significantly in the posterior subregion of the PVN (PaPo). This 10 nM CCK-8s-induced response was absent in CCK-1 receptor knock-out (CCK1R(-/-)) slices, showing that the response is mediated by CCK-1 receptors. CCK-8s concentrations higher than 30 nM triggered a Ca(2+) rise similarly in wild-type and CCK1R(-/-) slices. The large CCK-8s (100 nM)-induced Ca(2+) responses in CCK1R(-/-) slices were blocked by a CCK-2 receptor antagonist (CI-988), whereas those in wild-type slices required a mixture of CI-988 and lorglumide (a CCK-1 receptor antagonist) for complete antagonism. Therefore, CCK-1 and -2 receptors may function synergistically in single PaPo neurons and deletion of CCK-1 receptors may facilitate CCK-2 receptor signaling. This hypothesis was supported by results of real-time RT-PCR, immunofluorescence double labeling and Western blotting assays, which indicated CCK-2 receptor overexpression in PaPo neurons of CCK1R(-/-) mice. Furthermore, behavioral studies showed that intraperitoneal injections of lorglumide up-regulated food accesses in wild-type but not in CCK1R(-/-) mice, whereas CI-988 injections up-regulated food accesses in CCK1R(-/-) but not in wild-type mice. Compensatory CCK signaling via CCK-2 receptors in CCK1R(-/-) mice shed light on currently controversial satiety-controlling mechanisms.

Silencing of SNX1 by siRNA stimulates the ligand-induced endocytosis of EGFR and increases EGFR phosphorylation in gefitinib-resistant human lung cancer cell lines.

Gefitinib is known to suppress the activation of EGFR signaling, which is required for cell survival and proliferation in non-small cell lung cancer (NSCLC) cell lines. We previously demonstrated that the gefitinib-sensitive NSCLC cell line PC9 shows efficient ligand-induced endocytosis of phosphorylated EGFR (pEGFR). In contrast, the gefitinib-resistant NSCLC cell lines QG56 and A549 showed internalized pEGFR accumulation in the aggregated early endosomes, and this was associated with SNX1, a protein that interacts with and enhances the degradation of EGFR upon EGF stimulation. In the present study, to investigate the role of SNX1 on EGF-stimulated EGFR/pEGFR endocytosis via the endocytic pathway, we examined the effect of depletion of SNX1 expression by siRNA in human NSCLC cell lines. Using immunofluorescence, we demonstrated that transfection of SNX1 siRNA into gefitinib-resistant NSCLC cells resulted in the disappearance of a large amounts of SNX1 staining. In addition, upon 15 min of EGF stimulation, we observed an efficient EGFR phosphorylation and a rapid endocytic delivery of pEGFR from early endosomes to late endosomes. Further, western blot analysis revealed that silencing of SNX1 expression by siRNA in the gefitinib-resistant cells leads to an accelerated degradation of EGFR along with a dramatic increase in the amounts of pEGFR after EGF stimulation. Based on these findings, we suggest that SNX1 is involved in the negative regulation of ligand-induced EGFR phosphorylation and mediates EGFR/pEGFR trafficking out of early endosomes for targeting to late endosomes/lysosomes via the early/late endocytic pathway in human lung cancer cells.

The bisphosphonate incadronate inhibits intraperitoneal dissemination in an in vivo pancreatic cancer model.

Pancreatic cancer is characterized by intraperitoneal dissemination and often by large volumes of ascites. Aminobisphosphonates exhibit potent antitumor effects and are currently being tested against human solid tumors. Several aminobisphosphonates inhibit cancer cell migration by preventing the activation of Rho through inhibition of the mevalonate pathway. We evaluated the ability of an aminobisphosphonate, incadronate, to inhibit the growth of disseminated pancreatic cancer in vivo. We established an in vivo pancreatic cancer model with i.p. carcinomatosis in nude mice. Incadronate administration started from the day of tumor inoculation, and reduced tumor burden and ascites accumulation. Further, we evaluated the effect of incadronate on the inhibition of pancreatic cancer cell proliferation, migration and invasion in vitro. Incadronate induced growth inhibition and apoptotic death of pancreatic cancer cells. It also inhibited migration presumably by preventing the activation of Rho by lysophosphatidic acid. Thus, the in vivo antitumor effect may result from the inhibition of cancer cell proliferation and migration. The potent effects of incadronate in reducing tumor burden and ascites suggest that it will be of value in regimens for the treatment of pancreatic cancer.

A humanized anti-IGF-1R monoclonal antibody (R1507) and/or metformin enhance gemcitabine-induced apoptosis in pancreatic cancer cells.

Pancreatic cancer is a disease with a dismal prognosis and treatment options are limited. This study investigated the interaction of gemcitabine with R1507 and/or metformin and the induction of an inhibitor of apoptosis protein by this com-bination. Pancreatic cancer cells were treated with gemcitabine, R1507 and metformin alone or in combination. The effects of treatments were evaluated for cell proliferation, apoptosis, and the expression of genes related to inhibition of apoptosis and chemotherapy resistance. Combination of gemcitabine with R1507 and/or metformin additively interacted with the inhibition of cell proliferation in human pancreatic ductal adenocarcinoma cell lines, SUIT-2 and MIAPaCa-2 with differential gemcitabine resistance, and assessment of apoptosis demonstrated that drug associations increased the apoptotic index in both cell lines. Treatment with gemcitabine induced the expression of survivin and XIAP in both cell lines, indicating the induction of chemoresistance. In conclusion, these data demonstrate that the combination of gemcitabine with R1507 and/or metformin has an additive effect in pancreatic cancer cell lines with differential sensitivity to gemcitabine; however, gemcitabine may induce chemotherapy resistance.

Administration of ursodeoxycholate failed to prevent sludge and/or gallstone formation in cholecystokinin-1(A) receptor-deficient mice.

Gallstone disease is one of the most prevalent digestive diseases. The frequency of gallstone disease is about 10% in middle-age persons and 20% in aged persons. Gallbladder dysmotility and stasis of bile flow promote sludge and/or gallstone formation. Gallbladder contraction depends on cholecystokinin (CCK) via CCK-1 receptors (R)s. Previously, we raised CCK-1R deficient (-/-) mice and observed sludge and/or gallstone formation in more than 30% at 12-24 months of age. As ursodeoxycholate (UDCA) is commonly used for patients with gallstone disease, we expected that continuous administration of UDCA could prevent sludge and/or gallstone formation in CCK- 1R(-/-) mice. A diet containing 0.1% UDCA was administered in age-matched CCK-1R(-/-) and wild-type male and female mice for 8 months. Administration of UDCA decreased the frequency of sludge and/or gallstone formation compared with the control (CRF-1) diet (39%→26% in male, 35%→25% in female mice); however, these effects did not attain a level of statistical significance. Although the body weight was significantly higher in UDCA-fed than CRF-1-fed male mice regardless of genotypes, the plasma lipid concentrations did not differ between the two diets. In conclusion, administration of UDCA was less effective than expected at preventing sludge and/or gallstone formation in CCK-1R(-/-) mice.

Interaction between lung cancer cells and astrocytes via specific inflammatory cytokines in the microenvironment of brain metastasis.

The incidence of brain metastasis is increasing, however, little is known about molecular mechanism responsible for lung cancer-derived brain metastasis and their development in the brain. In the present study, brain pathology was examined in an experimental model system of brain metastasis as well as in human brain with lung cancer metastasis. In an experimental model, after 3-6 weeks of intracardiac inoculation of human lung cancer-derived (HARA-B) cells in nude mice, wide range of brain metastases were observed. The brain sections showed significant increase in glial fibrillary acidic protein (GFAP)-positive astrocytes around metastatic lesions. To elucidate the role of astrocytes in lung cancer proliferation, the interaction between primary cultured mouse astrocytes and HARA-B cells was analyzed in vitro. Co-cultures and insert-cultures demonstrated that astrocytes were activated by tumor cell-oriented factors; macrophage migration inhibitory factor (MIF), interleukin-8 (IL-8) and plasminogen activator inhibitor-1 (PAI-1). Activated astrocytes produced interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interleukin-1 ß (IL-1ß), which in turn promoted tumor cell proliferation. Semi-quantitative immunocytochemistry showed that increased expression of receptors for IL-6 and its subunits gp130 on HARA-B cells. Receptors for TNF-α and IL-1ß were also detected on HARA-B cells but down-regulated after co-culture with astrocytes. Insert-culture with astrocytes also stimulated the proliferation of other lung cancer-derived cell lines (PC-9, QG56, and EBC-1). These results suggest that tumor cells and astrocytes stimulate each other and these mutual relationships may be important to understand how lung cancer cells metastasize and develop in the brain.

Pancreatic hypertrophy in Ki-ras-induced actin-interacting protein gene knockout mice.

OBJECTIVES: Pancreatic functions were determined in a Ki-ras-induced actin-interacting protein (KRAP)-deficient (-/-) mouse mutant. METHODS: Pancreatic enzyme, protein, and DNA contents were measured, and histological examinations were conducted. The mixture of bile-pancreatic juice was collected, and amylase and bile acid outputs were determined. Oral glucose tolerance test was determined. Moreover, the gene expression of KRAP was determined in cholecystokinin (CCK)-A(1) receptor (-/-) mice. RESULTS: The body weight was smaller, and the ratio of pancreatic wet weight/body weight was higher in KRAP(-/-) mice compared with wild-type mice. The enzyme contents, but not DNA content, in the pancreas of KRAP(-/-) mice were higher than those of wild-type mice. Histological examination revealed the increase in the number of zymogen granules in the pancreatic acinar cells of KRAP(-/-) mice. Amylase secretions in response to CCK-octapeptide sulfate were significantly higher in KRAP(-/-) than wild-type mice, whereas the basal secretion did not differ between the 2 genotypes. A normal glucose tolerance was observed in KRAP(-/-) mice. The gene expression of KRAP in CCK-A(1) receptor (-/-) mice was significantly lower than in wild-type mice. CONCLUSIONS: The lack and/or decrease in KRAP level in the pancreas may promote the pancreatic growth and hypertrophy.

Gastric acid secretion in cholecystokinin-1 receptor, -2 receptor, and -1, -2 receptor gene knockout mice.

Gastrin is important for stimulating acid secretion as well as differentiating gastric mucosal cells via cholecystokinin-2 receptors (CCK-2Rs). In turn, CCK acts preferably via CCK-1R to release somatostatin, and somatostatin has been postulated to exhibit a tonic inhibition of gastrin bioactivity. The present study was designed to examine the hypothesis that CCK-1R and 2R may act in opposite directions in gastric acid secretion. Having generated CCK-1R(-/-), 2R(-/-), and 1R(-/-)2R(-/-) mice, we examined the regulation of gastric acid secretion in four genotypes including wild-type mice. Parietal cells possess histamine receptors, muscarinic receptors, and CCK-2Rs. Since histamine increases cAMP and carbachol increases calcium, the responses of gastric acid secretion to graded doses of histamine, carbachol, and a combination of histamine + carbachol were determined. The sensitivity to histamine did not differ among the four genotypes, while the maximal acid secretion was lower in CCK-2R(-/-) mice than in wild-type mice. In addition, sensitivity to carbachol was impaired in mice without CCK-2R. The interaction of histamine and carbachol was conserved in all genotypes. In conclusion, CCK-2R is necessary to respond to carbachol as well as to produce the maximal acid secretion, while the role of CCK-1R in acid secretion is less important.