Yuji Matsuzawa

Peroxisome Proliferator-activated Receptor γ Induces Growth Arrest and Differentiation Markers of Human Colon Cancer Cells

Peroxisome proliferator‐activated receptor γ (PPARγ), one of the nuclear receptors expressed in adipose tissue, plays an important role in adipocyte differentiation. In this study, we investigated the expression of PPARγ and its role in cellular growth and differentiation in six colon cancer cell lines: HT‐29, CaCo‐2, SW‐480, DLD‐1, LoVo, and T‐84. All six expressed PPARγ mRNA and protein, shown respectively on northern and western blot analyses. Luciferase assay in HT‐29 cells, which strongly express PPARγ showed that troglitazone, a selective ligand for PPAR?, transacti‐vated the transcription of a peroxisome proliferator response element (PPRE)‐driven promoter. Furthermore, troglitazone caused a marked decrease in [3H] thymidine incorporation and G1 cell‐cycle arrest determined by flow cytometry. Finally, troglitazone induced expression of mRNAs for villin and intestinal alkaline phosphatase, markers for enterocyte differentiation. In conclusion, human colon cancer cells express PPARγ, the ligands of which inhibit cell growth and induce differentiation markers.

STAT3 mediates the survival signal in oncogenic ras-transfected intestinal epithelial cells.

The oncogenic ras mutation is a common and critical step in gastrointestinal carcinogenesis. In a previous study, we demonstrated that oncogenic ras activated the EGF‐related peptide autocrine loop and that the apoptosis resistance observed in the oncogenic ras‐stimulated cell (IEC‐ras cell) was dependent on this activated EGF‐related peptide autocrine loop. STATs (signal transducers and activators of transcription), first identified as intracellular signal transducers stimulated by cytokines, are known to also be activated by EGF. However, the role of STATs in the survival signal of IEC‐ras cells is not clear. In the present study, we demonstrate that STAT3 is constitutively activated in ras‐stimulated cells and that STAT3 activation is considerably suppressed by the EGF‐specific receptor kinase inhibitor AG1478. We also show that disruption of the STAT3 pathway by introduction of a dominant‐negative STAT3 mutant abolishes the apoptosis resistance against UVC and MMC treatment observed in IEC‐ras cells without affecting proliferation. Moreover, the expression of Bcl‐2 and Bcl‐xL, apoptosis‐suppressive proteins, is reduced in dominant‐negative STAT3‐transfected cells. Thus, STAT3 appears to be an important mediator of the anti‐apoptotic signal in IEC‐ras cells. Int. J. Cancer 78:326–330, 1998.© 1998 Wiley‐Liss, Inc.

Gastrin Induces Heparin-Binding Epidermal Growth Factor-like Growth Factor in Rat Gastric Epithelial Cells Transfected With Gastrin Receptor

BACKGROUND & AIMSnParietal cells express heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF). However, it is unknown whether HB-EGF mediates the trophic action of gastrin. The purpose of this study was to determine whether gastrin modulates the expression of HB-EGF, which mediates the proliferative effects of gastrin on gastric epithelial cells.nnnMETHODSnRGM1 cells, a rat gastric epithelial cell line, were transfected with a human gastrin receptor complementary DNA. Gastrin induction of messenger RNAs (mRNAs) for EGF-related polypeptides was assayed by Northern blotting. Processing of cell surface-associated proHB-EGF and secretion of HB-EGF were determined by flow cytometry and Western blotting, respectively. Tyrosine phosphorylation of the EGF receptor was assayed by immunoprecipitation and Western blotting with an antiphosphotyrosine antibody. Cell growth was evaluated by [3H]thymidine incorporation.nnnRESULTSnGastrin induced expression of HB-EGF mRNA, processing of proHB-EGF, release of HB-EGF into the medium, and tyrosine phosphorylation of the EGF receptor. The growth-stimulatory effects of gastrin were partly inhibited by anti-rat HB-EGF serum and completely blocked by AG1478, an EGF receptor-specific tyrphostin.nnnCONCLUSIONSnThe findings suggest that HB-EGF at least partially mediates the proliferative effects of gastrin on gastric epithelial cells.

High Fas ligand expression on lymphocytes in lesions of ulcerative colitis

Background—The pathogenesis of ulcerative colitis is unclear, but cytotoxic T lymphocytes infiltrating the mucosa have been implicated in mucosal damage. The Fas ligand (FasL), expressed on cytotoxic T lymphocytes, induces apoptosis in cells expressing Fas. Aim—To analyse FasL expression in affected colonic mucosa to ascertain Fas-FasL interaction in ulcerative colitis. Methods—FasL mRNA was quantified in colonic mucosal specimens from healthy subjects and patients with ulcerative colitis or Crohn’s disease, using the competitive reverse transcription polymerase chain reaction. FasL mRNA localisation was determined by in situ hybridisation. Expression of Fas in colonic mucosa was analysed immunohistochemically. Phenotypes of lamina propria lymphocytes that expressed FasL were analysed by flow cytometry. Results—FasL mRNA was strongly expressed in active ulcerative colitis lesions, but not in those associated with active Crohn’s disease or active proctitis-type ulcerative colitis. In situ hybridisation showed that FasL mRNA expression occurred in mononuclear cells infiltrating lesions. Fas was expressed in epithelial cells in ulcerative colitis and Crohn’s disease, and in normal subjects. Cytometry showed that FasL was expressed in CD3 lymphocytes infiltrating the lamina propria in active lesions. Conclusions—FasL is expressed in CD3 lymphocytes infiltrating into ulcerative colitis but not Crohn’s disease lesions, suggesting that Fas-FasL induced apoptosis participates in the mucosal damage of ulcerative colitis.

Modulation of apoptosis by endogenous Bcl-xL expression in MKN-45 human gastric cancer cells

This study was designed to clarify the role of endogenous Bcl-xL expression in modulating apoptosis of malignant cells. Administration of bcl-x-antisense oligonucleotides decreased Bcl-xL protein levels in the MKN-45 human gastric cancer cell line. The decrease in Bcl-xL protein content resulted in increased cell death induced by serum deprivation or Fas-antibody administration. Flow cytometric analysis revealed that the increased apoptotic cell death was more prominent in bcl-x-antisense-treated cells as compared to control cells, bcl-x-sense-treated cells, or bcl-x-nonsense-treated cells. To inhibit the effect of intrinsic Bcl-xL protein, we overexpressed Bak, which binds Bcl-xL and inhibits the anti-apoptotic effect of Bcl-xL, by transfection into MKN-45 cells. Bak-overexpressing cells showed increased apoptotic cell death induced by Fas-antibody when compared to parent cells and MKN-neo-transfected cells. Bak-overexpressing cells also showed greater sensitization to 5-fluorouracil and cisplatin than parent cells and MKN-neo-transfected cells. u2003In conclusion, we demonstrated that administration of bcl-x-antisense oligonucleotides or overexpression of Bak protein induces sensitization to apoptosis in MKN-45 gastric cancer cells, suggesting that endogenous Bcl-xL expression in cancer cells is an important modulator of apoptosis.

Morphological and functional restoration of parietal cells in Helicobacter pylori associated enlarged fold gastritis after eradication

BACKGROUND/AIM Helicobacter pylori infections are associated with hypochlorhydria in patients with pangastritis. It has previously been shown that eradication of H pylori leads to an increase in acid secretion in H pylori associated enlarged fold gastritis, suggesting that H pylori infection affects parietal cell function in the gastric body. The aim of this study was to evaluate the effects ofH pylori infection on parietal cell morphology and function in hypochlorhydric patients. PATIENTS/METHODS The presence of H pylori infection, mucosal length, and inflammatory infiltration were investigated in six patients with enlarged fold gastritis and 12 patients without enlarged folds. Parietal cell morphology was examined by immunohistochemistry using an antibody against the α subunit of H+,K+-ATPase and electron microscopy. In addition, gastric acid secretion and fasting serum gastrin concentration were determined before and after the eradication ofH pylori. RESULTS In theH pylori positive patients with enlarged fold gastritis, fold width, foveolar length, and inflammatory infiltration were increased. In addition, the immunostaining pattern of H+, K+-ATPase was less uniform, and the percentage of altered parietal cells showing dilated canaliculi with vacuole-like structures and few short microvilli was greatly increased compared with that in H pylori positive patients without enlarged folds. After eradication, fold width, foveolar length, and inflammatory infiltrates decreased and nearly all parietal cells were restored to normal morphology. On the other hand, altered parietal cells were negligible in H pylori negative patients. In addition, the basal acid output and tetragastrin stimulated maximal acid output increased significantly from 0.5 (0.5) to 4.1 (1.5) mmol/h and from 2.5 (1.2) to 13.8 (0.7) mmol/h (p<0.01), and fasting serum gastrin concentrations decreased significantly from 213.5 (31.6) to 70.2 (7.5) pg/ml (p<0.01) after eradication in patients with enlarged fold gastritis. CONCLUSION The morphological changes in parietal cells associated withH pylori infection may be functionally associated with the inhibition of acid secretion seen in patients with enlarged fold gastritis.

Mucosal interleukin-1β production and acid secretion in enlarged fold gastritis

Background: We have previously shown that eradication of Helicobacter pylori increases acid secretion in H. pylori‐associated enlarged fold gastritis.

Calphostin C induces expression of amphiregulin mRNA via reactive oxygen species in IEC-6 cells.

Calphostin C, a secondary metabolite of the fungus Cladosporium cladosporioides, is generally used as a specific inhibitor of protein kinase C. It is known that 12-O-tetradecanoyl-13-phorbol acetate (TPA), a protein kinase C activator, induces expression of mRNA for amphiregulin (AR), a member of EGF-related polypeptides, in mammalian epithelial cells. In this work, we determined the effect of calphostin C on AR mRNA expression in IEC-6 cells, a rat intestinal epithelial cell line, and unexpectedly found that this compound enhanced the TPA-induced expression of AR mRNA. Moreover, calphostin C alone induced expression of AR mRNA in a light-dependent manner, and this effect was abrogated by pretreatment with N-acetylcysteine. These results suggest that calphostin C can upregulate expression of AR mRNA via reactive oxygen species.

Gastrin induces heparin-binding EGF-like growth factor in rat gastric epithelial cells transfected with gastrin receptor

• G3672 GASTRIN INDUCES HEPARIN-BINDING EGF-LIKE GROWTH FACTOR IN RAT GASTRIC EPITHELIAL CELLS TRANSFECTED WITH GASTRIN RECEPTOR. Yoshiii Mivazaki. Yasuhisa Shinomura, Shusaku Tsutsui, Shinichiro Zushi, Yoshifumi Higashimoto, Shuji Kanayama, Shigeki Higashiyama*, Naoyuki Taniguchi*, and Yuji Matsuzawa. Second Department of Internal Medicine and Department of Biochemistry*, Osaka University Medical School, 2-2 Yamadaoka, Suita Osaka 565, Japan.