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• W1995287795 abstract "The polycystic kidney (PCK) rat represents a liver and kidney cyst pathology corresponding to Caroli's disease with congenital hepatic fibrosis and autosomal recessive polycystic kidney disease. We previously reported that an epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of biliary epithelial cells of PCK rats in vitro. This study investigated the effects of gefitinib on cyst pathogenesis of the PCK rat both in vitro and in vivo. A three-dimensional culture model of biliary epithelial cells in the collagen gel matrix was used for in vitro analysis. For in vivo experiments, PCK and control rats were treated with gefitinib between 3 and 10 weeks of age. In vitro, gefitinib had strong inhibitory effects on biliary cyst formation of PCK rats. In vivo, treatment with gefitinib significantly inhibited the cystic dilatation of the intrahepatic bile ducts of PCK rats, which was accompanied by improvement of liver fibrosis. By contrast, no beneficial effects were observed on renal cyst development because of the treatment. These results suggest that signaling pathways mediated by epidermal growth factor receptor are involved in biliary dysgenesis of the PCK rat, with the mechanisms of cyst progression being different between the liver and kidney. The polycystic kidney (PCK) rat represents a liver and kidney cyst pathology corresponding to Caroli's disease with congenital hepatic fibrosis and autosomal recessive polycystic kidney disease. We previously reported that an epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of biliary epithelial cells of PCK rats in vitro. This study investigated the effects of gefitinib on cyst pathogenesis of the PCK rat both in vitro and in vivo. A three-dimensional culture model of biliary epithelial cells in the collagen gel matrix was used for in vitro analysis. For in vivo experiments, PCK and control rats were treated with gefitinib between 3 and 10 weeks of age. In vitro, gefitinib had strong inhibitory effects on biliary cyst formation of PCK rats. In vivo, treatment with gefitinib significantly inhibited the cystic dilatation of the intrahepatic bile ducts of PCK rats, which was accompanied by improvement of liver fibrosis. By contrast, no beneficial effects were observed on renal cyst development because of the treatment. These results suggest that signaling pathways mediated by epidermal growth factor receptor are involved in biliary dysgenesis of the PCK rat, with the mechanisms of cyst progression being different between the liver and kidney. The polycystic kidney (PCK) rat, an animal model of human autosomal recessive polycystic kidney disease (ARPKD), represents a liver fibrocystic pathology corresponding to Caroli's disease with congenital hepatic fibrosis.1Nakanuma Y Terada T Ohta G Kurachi M Matsubara F Caroli's disease in congenital hepatic fibrosis and infantile polycystic disease.Liver. 1982; 2: 346-354Crossref PubMed Scopus (81) Google Scholar, 2Sanzen T Harada K Yasoshima M Kawamura Y Ishibashi M Nakanuma Y Polycystic kidney rat is a novel animal model of Caroli's disease associated with congenital hepatic fibrosis.Am J Pathol. 2001; 158: 1605-1612Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar ARPKD is a form of inherited childhood nephropathy, with an incidence of 1 in 20,000 live births.3Wilson PD Polycystic kidney disease.N Engl J Med. 2004; 350: 151-164Crossref PubMed Scopus (622) Google Scholar, 4Harris PC Rossetti S Molecular genetics of autosomal recessive polycystic kidney disease.Mol Genet Metab. 2004; 81: 75-85Crossref PubMed Scopus (63) Google Scholar The disease is characterized by the fusiform dilatation of collecting tubules and by biliary dysgenesis and hepatic fibrosis. Fetal or neonatal death is often caused, owing to tremendous bilateral enlargement of the kidneys, impaired lung formation, and pulmonary hypoplasia. Progression to end-stage renal disease occurs in 20 to 45% of cases within 15 years. A proportion of the patients maintain renal function into adulthood, when complications of liver disease predominate.4Harris PC Rossetti S Molecular genetics of autosomal recessive polycystic kidney disease.Mol Genet Metab. 2004; 81: 75-85Crossref PubMed Scopus (63) Google Scholar The liver and kidney lesions in ARPKD patients and in PCK rats are caused by mutations to orthologous genes, PKHD1/Pkhd1.5Ward CJ Hogan MC Rossetti S Walker D Sneddon T Wang X Kubly V Cunningham JM Bacallao R Ishibashi M Milliner DS Torres VE Harris PC The gene mutated in autosomal recessive polycystic kidney disease encodes a large, receptor-like protein.Nat Genet. 2002; 30: 259-269Crossref PubMed Scopus (594) Google Scholar, 6Onuchic LF Furu L Nagasawa Y Hou X Eggermann T Ren Z Bergmann C Senderek J Esquivel E Zeltner R Rudnik-Schoneborn S Mrug M Sweeney W Avner ED Zerres K Guay-Woodford LM Somlo S Germino GG PKHD1, the polycystic kidney and hepatic disease 1 gene, encodes a novel large protein containing multiple immunoglobulin-like plexin-transcription-factor domains and parallel beta-helix 1 repeats.Am J Hum Genet. 2002; 70: 1305-1317Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar PKHD1 is a large gene from which multiple transcripts may be generated by alternative splicing.4Harris PC Rossetti S Molecular genetics of autosomal recessive polycystic kidney disease.Mol Genet Metab. 2004; 81: 75-85Crossref PubMed Scopus (63) Google Scholar, 7Bergmann C Senderek J Kupper F Schneider F Dornia C Windelen E Eggermann T Rudnik-Schoneborn S Kirfel J Furu L Onuchic LF Rossetti S Harris PC Somlo S Guay-Woodford L Germino GG Moser M Buttner R Zerres K PKHD1 mutations in autosomal recessive polycystic kidney disease (ARPKD).Hum Mutat. 2004; 23: 453-463Crossref PubMed Scopus (124) Google Scholar In common with other PKD-related proteins, the ARPKD protein fibrocystin is localized to the primary cilia of renal epithelial cells and is often absent in ARPKD tissue.8Zhang MZ Mai W Li C Cho SY Hao C Moeckel G Zhao R Kim I Wang J Xiong H Wang H Sato Y Wu Y Nakanuma Y Lilova M Pei Y Harris RC Li S Coffey RJ Sun L Wu D Chen XZ Breyer MD Zhao ZJ McKanna JA Wu G PKHD1 protein encoded by the gene for autosomal recessive polycystic kidney disease associates with basal bodies and primary cilia in renal epithelial cells.Proc Natl Acad Sci USA. 2004; 101: 2311-2316Crossref PubMed Scopus (141) Google Scholar, 9Wang S Luo Y Wilson PD Witman GB Zhou J The autosomal recessive polycystic kidney disease protein is localized to primary cilia, with concentration in the basal body area.J Am Soc Nephrol. 2004; 15: 592-602Crossref PubMed Scopus (127) Google Scholar, 10Ward CJ Yuan D Masyuk TV Wang X Punyashthiti R Whelan S Bacallao R Torra R LaRusso NF Torres VE Harris PC Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.Hum Mol Genet. 2003; 12: 2703-2710Crossref PubMed Scopus (263) Google Scholar In intrahepatic bile ducts of normal rats, each cholangiocyte has a single cilium that expresses fibrocystin, whereas the cilia of PCK rats show an abnormal morphology devoid of fibrocystin.11Masyuk TV Huang BQ Masyuk AI Ritman EL Torres VE Wang X Harris PC Larusso NF Biliary dysgenesis in the PCK rat, an orthologous model of autosomal recessive polycystic kidney disease.Am J Pathol. 2004; 165: 1719-1730Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 12Masyuk TV Huang BQ Ward CJ Masyuk AI Yuan D Splinter PL Punyashthiti R Ritman EL Torres VE Harris PC LaRusso NF Defects in cholangiocyte fibrocystin expression and ciliary structure in the PCK rat.Gastroenterology. 2003; 125: 1303-1310Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar The link between cyst development and ciliary dysfunction attributable to the lack of fibrocystin has been suggested, although the precise role of fibrocystin in cyst development remains unclear.13Moser M Matthiesen S Kirfel J Schorle H Bergmann C Senderek J Rudnik-Schoneborn S Zerres K Buettner R A mouse model for cystic biliary dysgenesis in autosomal recessive polycystic kidney disease (ARPKD).Hepatology. 2005; 41: 1113-1121Crossref PubMed Scopus (74) Google Scholar It has been well established that the epidermal growth factor (EGF)/transforming growth factor (TGF)-α/EGF receptor (EGFR) pathway and the adenosine 3′,5′-cyclic monophosphate (cAMP) pathway play important roles in promoting the renal tubular epithelial cell proliferation and cyst formation in ARPKD as well as in the autosomal dominant form of PKD (ADPKD).14MacRae Dell K Nemo R Sweeney Jr, WE Avner ED EGF-related growth factors in the pathogenesis of murine ARPKD.Kidney Int. 2004; 65: 2018-2029Crossref PubMed Scopus (50) Google Scholar, 15Richards WG Sweeney WE Yoder BK Wilkinson JE Woychik RP Avner ED Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease.J Clin Invest. 1998; 101: 935-939Crossref PubMed Scopus (174) Google Scholar, 16Sweeney Jr, WE Avner ED Functional activity of epidermal growth factor receptors in autosomal recessive polycystic kidney disease.Am J Physiol. 1998; 275: F387-F394PubMed Google Scholar, 17Marfella-Scivittaro C Quinones A Orellana SA cAMP-dependent protein kinase and proliferation differ in normal and polycystic kidney epithelia.Am J Physiol. 2002; 282: C693-C707Crossref PubMed Scopus (20) Google Scholar, 18Yamaguchi T Pelling JC Ramaswamy NT Eppler JW Wallace DP Nagao S Rome LA Sullivan LP Grantham JJ cAMP stimulates the in vitro proliferation of renal cyst epithelial cells by activating the extracellular signal-regulated kinase pathway.Kidney Int. 2000; 57: 1460-1471Crossref PubMed Scopus (286) Google Scholar, 19Hanaoka K Guggino WB cAMP regulates cell proliferation and cyst formation in autosomal polycystic kidney disease cells.J Am Soc Nephrol. 2000; 11: 1179-1187PubMed Google Scholar Consequently, a number of studies have examined the effects of blocking these pathways in animal models of PKD. For example, inhibition of the EGFR tyrosine kinase activity inhibited renal cyst development in bpk and orpk mice (models of ARPKD) and in Han:SPRD rat (a model of ADPKD).15Richards WG Sweeney WE Yoder BK Wilkinson JE Woychik RP Avner ED Epidermal growth factor receptor activity mediates renal cyst formation in polycystic kidney disease.J Clin Invest. 1998; 101: 935-939Crossref PubMed Scopus (174) Google Scholar, 20Sweeney WE Chen Y Nakanishi K Frost P Avner ED Treatment of polycystic kidney disease with a novel tyrosine kinase inhibitor.Kidney Int. 2000; 57: 339-340Crossref PubMed Scopus (190) Google Scholar, 21Sweeney Jr, WE Hamahira K Sweeney J Garcia-Gatrell M Frost P Avner ED Combination treatment of PKD utilizing dual inhibition of EGF-receptor activity and ligand bioavailability.Kidney Int. 2003; 64: 1310-1319Crossref PubMed Scopus (63) Google Scholar, 22Torres VE Sweeney Jr, WE Wang X Qian Q Harris PC Frost P Avner ED EGF receptor tyrosine kinase inhibition attenuates the development of PKD in Han:SPRD rats.Kidney Int. 2003; 64: 1573-1579Crossref PubMed Scopus (114) Google Scholar The administration of a vasopressin V2 receptor (VPV2R) antagonist lowered the renal cAMP level, and inhibited renal cystogenesis in pcy mouse (a model of nephronophthisis) and in Pkd2−/tm1Som mouse (a model of ADPKD).23Gattone II, VH Wang X Harris PC Torres VE Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist.Nat Med. 2003; 9: 1323-1326Crossref PubMed Scopus (537) Google Scholar, 24Torres VE Wang X Qian Q Somlo S Harris PC Gattone II, VH Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease.Nat Med. 2004; 10: 363-364Crossref PubMed Scopus (404) Google Scholar Recently, it has been shown that that the VPV2R antagonist improved the renal disease development and progression of the PCK rat.25Wang X Gattone II, V Harris PC Torres VE Effectiveness of vasopressin V2 receptor antagonists OPC-31260 and OPC-41061 on polycystic kidney disease development in the PCK rat.J Am Soc Nephrol. 2005; 16: 846-851Crossref PubMed Scopus (265) Google Scholar In these studies, however, the VPV2R antagonist did not improve the fibrocystic liver disease of the PCK rat. Inhibition of PKD of the PCK rat by the use of EGFR tyrosine kinase inhibitors (EKI-785 and EKB-569) resulted in no effect or a worsened PKD as well as no significant effect on the fibrocystic liver disease.26Torres VE Sweeney Jr, WE Wang X Qian Q Harris PC Frost P Avner ED Epidermal growth factor receptor tyrosine kinase inhibition is not protective in PCK rats.Kidney Int. 2004; 66: 1766-1773Crossref PubMed Scopus (67) Google Scholar EGFR tyrosine kinase activation triggers numerous downstream signaling pathways, such as the extracellular-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK), and the phosphoinositide-3-kinase (PI3K)/Akt pathways.27Wells A EGF receptor.Int J Biochem Cell Biol. 1999; 31: 637-643Crossref PubMed Scopus (899) Google Scholar Recently, we demonstrated that biliary epithelial cells (BECs) isolated from the PCK rat were hyperreactive to EGF, which was accompanied by the activation of the MAPK pathway consisting of MAPK/ERK kinase 5 (MEK5)/ERK5 in vitro.28Sato Y Harada K Kizawa K Sanzen T Furubo S Yasoshima M Ozaki S Ishibashi M Nakanuma Y Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease.Am J Pathol. 2005; 166: 49-60Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar We also showed that an EGF tyrosine kinase inhibitor, gefitinib (Iressa), significantly inhibited the abnormal growth of cultured BECs of the PCK rat. The PCK rat is regarded as a slowly progressive model of ARPKD. Therapies for liver lesions of ARPKD are particularly important because liver disease becomes a major cause of morbidity and mortality in elderly patients of ARPKD.4Harris PC Rossetti S Molecular genetics of autosomal recessive polycystic kidney disease.Mol Genet Metab. 2004; 81: 75-85Crossref PubMed Scopus (63) Google Scholar To date, there has been no study that has successfully inhibited the fibrocystic liver disease of the PCK rat. This study aimed to inhibit the cystic dilation of the intrahepatic bile ducts and hepatic fibrosis as well as the PKD of the PCK rat by the use of a novel tyrosine kinase inhibitor gefitinib. PCK rats were maintained at the Laboratory Animal Institute of Kanazawa University Graduate School of Medicine. Control (Sprague-Dawley) rats were purchased from Charles River Japan (Sagamihara, Japan). The rats were maintained on a standard laboratory rat diet and water ad libitum. All studies were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Takara-machi Campus of Kanazawa University, Kanazawa, Japan. Intrahepatic BECs were isolated, purified, and cultured from 8-week-old rats as described previously.28Sato Y Harada K Kizawa K Sanzen T Furubo S Yasoshima M Ozaki S Ishibashi M Nakanuma Y Activation of the MEK5/ERK5 cascade is responsible for biliary dysgenesis in a rat model of Caroli's disease.Am J Pathol. 2005; 166: 49-60Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar Because the dilatation of the intrahepatic large bile ducts has been regarded as an essential feature of Caroli's disease, BECs were isolated from intrahepatic large bile ducts. The fourth subcultured BECs were used for the study. A three-dimensional cell culture in a collagen gel matrix was performed according to the previously described method with some modifications.29Kawamura Y Yoshida K Nakanuma Y Primary culture of rabbit gallbladder epithelial cells in collagen gel matrix.Lab Invest. 1989; 61: 350-356PubMed Google Scholar In brief, BECs were dispersed and directly embedded in a fluid collagen gel matrix (Cellmatrix Type 1-A; Nitta Gelatin, Osaka, Japan). The collagen gel matrix was composed of 0.3% Cellmatrix Type 1-A, 10× concentrated Dulbecco's modified Eagle's medium and nutrient mixture F-12 (1:1; Life Technologies, Inc., Rockville, MD) containing an 8:1:1 ratio of 0.05 N sodium hydroxide, 260 mmol/L sodium bicarbonate, and 200 mmol/L HEPES. The cellular density at the beginning of the culture was 1 × 105 cells/ml. The fluid collagen gel was planted on six-well plates. This collagen fluid soon became gelatinous. The cultures were then covered with a culture medium composed of Dulbecco's modified Eagle's medium and nutrient mixture F-12 (Life Technologies, Inc.), 10% Nu-Serum (Becton Dickinson, Bedford, MA), 1% ITS+ (Becton Dickinson), 5 μmol/L forskolin (Wako Pure Chemical, Osaka, Japan), 12.5 mg/ml bovine pituitary extract (Kurabo Industries, Osaka, Japan), 1 μmol/L dexamethasone (Sigma, St. Louis, MO), 5 μmol/L triiodo-thyronine (Sigma), 5 mg/ml glucose (Sigma), 25 mmol/L sodium bicarbonate (Sigma), 1% antibiotics-antimycotic (Life Technologies, Inc.), and 20 ng/ml EGF (Upstate Biotechnology, Lake Placid, NY) at 37°C in an atmosphere of 5% CO2. The basal culture medium was changed every 2 days. The effects of gefitinib on biliary cyst formation were examined using the three-dimensional cell culture system. On day 7 after the beginning of cell culture, the culture medium was changed to that containing 20 ng/ml EGF and 0.1, 0.5, or 1 μmol/L gefitinib (Iressa, ZD1839; provided by AstraZeneca, Macclesfield, UK). Incubation was continued for a further 12 days. The culture medium was changed every 2 days. The cultured cells were observed daily under phase-contrast microscopy (Olympus, Tokyo, Japan), and their morphological changes were recorded using a digital camera (model DXC-S500; Sony, Tokyo, Japan). The recorded images were reproduced on a computer, and cyst size was determined using image analysis software (Win ROOF version 3.6; Mitani Corp., Tokyo, Japan). Measurement was performed for 10 well-developed cysts for each time after gefitinib treatment, and the mean was calculated. The number of cysts was counted under phase-contrast microscopy at ×40 magnification, and the total cyst number of five fields was determined at each time. To determine whether gefitinib can prevent biliary cyst formation when it is administrated before BECs start to undergo cyst formation, 0.1 or 1 μmol/L gefitinib was administrated at the beginning of the three-dimensional cell culture in the presence of 20 ng/ml EGF. The culture medium was changed every 2 days. For 12 days after starting the treatment, the number and size of the cysts were determined as above. The effect of gefitinib on apoptosis of cultured BECs was determined using the single-stranded DNA (ssDNA) apoptosis enzyme-linked immunosorbent assay kit (Chemicon Int., Temecula, CA) according to the manufacturer's instructions. This method is based on the selective denaturation of DNA in apoptotic cells by formamide, which is a gentle agent that denatures DNA in apoptotic cells, but not in necrotic cells or in cells with DNA breaks in the absence of apoptosis.30Frankfurt OS Krishan A Identification of apoptotic cells by formamide-induced DNA denaturation in condensed chromatin.J Histochem Cytochem. 2001; 49: 369-378Crossref PubMed Scopus (155) Google Scholar In brief, a total of 1250 cells per well were seeded in a 96-well collagen-coated plate. After a 48-hour preincubation with the basal medium, the medium was exchanged for that containing appropriate concentrations of gefitinib and incubated at 37°C in an atmosphere of 5% CO2 for a further 24 or 72 hours. The cells were fixed with 80% methanol in phosphate-buffered saline (PBS) on the wells and incubated with formamide at 75°C for 20 minutes. For negative control wells, S1 nuclease (10 U/well) (Takara Bio, Otsu, Japan) was added and incubated at 37°C for 30 minutes for the removal of single-stranded regions in DNA-DNA hybrids. After washing with PBS, the wells were incubated with 2.5% bovine serum albumin at 37°C for 1 hour to block nonspecific binding sites and then incubated with antibody mixture (primary monoclonal to ssDNA and horseradish peroxidase-labeled anti-mouse IgM; provided in the kit) at room temperature for 30 minutes. After washing, color development was performed with 2,2′-azino-bis(3-ethylbenziazoline-6-sulfoic acid) solution, and its absorbance at 405 nm was measured using a microplate reader. As an additional experiment, DNA fragmentation attributable to apoptosis was detected using a terminal dUTP nick-end labeling (TUNEL) method. Three-dimensional cell culture in collagen gel matrix was performed as above. On day 7 after the beginning of cell culture, the culture medium was changed for those containing appropriate concentrations of gefitinib. The culture medium was changed every 2 days. On days 0, 4, 8, and 12 after gefitinib treatment, formalin-fixed, paraffin-embedded sections (4 μm thick) of the collagen gel matrix were prepared. After proteinase K digestion and endogenous peroxidase blocking, the sections were stained by using a commercial kit (TdT-FragEL DNA fragmentation detection kit; Calbiochem, San Diego, CA). After color development with 3,3′-diaminobenzidine tetrahydrochloride, sections were counterstained with methyl green. A total of 30 male rats were used. At 3 weeks of age, 15 normal and 15 PCK rats were divided into one control and two experimental groups. The experimental groups (five rats per group) were intraperitoneally administered 2 or 10 mg/kg gefitinib daily between 3 and 10 weeks of age. The dosage was determined based on our preliminary experimental data, as well as the recommendation of the AstraZeneca group of companies. The control group received vehicle (2% Tween 80 and 0.5% methylcellulose in water) alone. At 10 weeks of age, rats were weighed and anesthetized with diethylether. Blood was obtained by cardiac puncture for the determination of serum levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, albumin, blood urea nitrogen, and creatinine. The liver and kidney were weighed and immersed in 10% formalin neutral buffer solution (pH 7.4), and the tissues were embedded in paraffin for histological analysis. Parts of the tissues were immediately frozen in liquid nitrogen to use for Western blot analysis, the reverse transcriptase-polymerase chain reaction (RT-PCR), and collagen content measurement. The three-dimensional observation of the intrahepatic biliary tree was performed using a silicon rubber cast study as previously described.2Sanzen T Harada K Yasoshima M Kawamura Y Ishibashi M Nakanuma Y Polycystic kidney rat is a novel animal model of Caroli's disease associated with congenital hepatic fibrosis.Am J Pathol. 2001; 158: 1605-1612Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar In brief, a cannula was inserted into the extrahepatic bile duct, and Microfil compound (MV-112; Flow Tech. Inc., Carver, MA) was injected using a syringe. The Microfil compound-injected liver specimens were placed in a refrigerator at 4°C overnight to allow polymerization. Then they were immersed in 25% ethanol for 24 hours. At 24-hour intervals, the ethanol concentration was raised to 50, 75, 95, and 100%. Finally, the specimens were immersed in methyl salicylate for the cleaning of the tissue. Total RNA (1 μg) was extracted from the liver using an RNA extraction kit (RNeasy Mini; Qiagen, Tokyo, Japan) and was used to synthesize cDNA with reverse transcriptase (ReverTra Ace; Toyobo Co., Osaka, Japan). PCR amplification was performed in a total volume of 25 μl containing 1 μl of cDNA, 0.2 mmol/L dNTPs, 1 μmol/L each of 5′- and 3′-primers, and 2.5 U of TaqDNA polymerase (Takara EX Taq; Takara Bio) with an annealing temperature of 60°C. The sequences of the rat primers were as follows: connective tissue growth factor (CTGF), 5′-GAAAGACAGGTACTAGCTGA-3′ (forward) and 5′-GAACAATAGGCACAAACGTC-3′; TGF-β1, 5′-CAATTCCTGGCGTTACCTTG-3′ (forward) and 5′-GAAGCAGTAGTTGGTATCCA-3′ (reverse); and β-actin, 5′-ACCTTCAACACCCCAGCCATGTACG-3′ (forward) and 5′-CTGATCCACATCTGCTGGAAGGTGG-3′ (reverse). The number of PCR cycles for CTGF, TGF-β1, and β-actin were 30, 35, and 25, respectively. For each reaction, an initial denaturation cycle of 94°C for 3 minutes and a final cycle of 72°C for 10 minutes were incorporated. The PCR products were subjected to 2% agarose gel electrophoresis and stained with ethidium bromide. Semiquantitative analysis of the gel image was performed using the public domain NIH Image software in the exponential range of each PCR amplification. The fold difference compared with β-actin expression was calculated. Formalin-fixed, paraffin-embedded sections (4 μm thick) were deparaffinized. Antigen retrieval was performed for the antibodies against phosphorylated (p-)ERK1/2, p-ERK5, cytokeratin, and Ki-67 protein by microwaving in 10 mmol/L citrate buffer (pH 6.0). After blocking of the endogenous peroxidase, the sections were incubated overnight at 4°C with individual primary antibodies: anti-p-ERK1/2 (1:200) (44-680, rabbit polyclonal; Biosource Int., Camarillo, CA), anti-p-ERK5 (1:100) (KAS-MA002, rabbit polyclonal; Stressgen, San Diego, CA), anti-cytokeratin (1:600) (A0575, rabbit polyclonal; DAKO, Glostrup, Denmark), anti-ssDNA (1:400) (A4506, rabbit polyclonal; DAKO), and anti-Ki-67 protein (1:50) (MIB-5, mouse monoclonal; Immunotech, Marseille, France). Then the sections were incubated with secondary antibody conjugated to the peroxidase-labeled polymer, EnVison+ system (DAKO). Color development was performed using 3,3′-diaminobenzidine tetrahydrochloride, and the sections were counterstained with hematoxylin. Control sections were evaluated by substitution of the primary antibodies with nonimmunized serum, resulting in no signal detection. Formalin-fixed, paraffin-embedded sections were prepared for the liver and kidney, and whole tissue sections were used to measure cyst volumes and liver fibrosis. Cyst volumes of the liver and kidney were assessed using immunostained sections with anti-cytokeratin antibody and hematoxylin and eosin (H&E) staining of sections, respectively. Liver fibrosis was assessed using picrosirius red staining. Stained sections were visualized under an Olympus light microscope, and the digital images were acquired and reproduced on a computer using the image processing software Viewfinder Lite (version 1.0; Pixera Corp., Los Gatos, CA). Image analysis was performed in software using Win ROOF (Mitani Corporation). A color threshold was applied at a level that separated cysts from noncystic tissue or the picrosirius red-stained material from the background to calculate the volume of the cysts or fibrosis. The areas of interest were expressed as a percentage of the total tissue. Evaluation of apoptosis was performed using sections stained using the TUNEL method. Liver sections and sections of three-dimensional cell culture of BECs were used for the analysis. More than 500 BECs and hepatocytes were surveyed in liver sections, and more than 200 BECs for each section of collagen gel matrix were surveyed. The percentage of BECs positive for TUNEL was expressed as the TUNEL-labeling index (LI). To evaluate the cell proliferative activity, Ki-67 protein-positive signals were similarly counted for BECs of the Ki-67-immunostained liver sections and sections of collagen gel matrix. The percentage of BECs positive for Ki-67 protein was expressed as the Ki-67-LI. Proteins were extracted from the liver specimens and cultured BECs using T-PER tissue protein extraction reagent (Pierce Chemical Co., Rockford, IL), and the total protein was measured spectrometrically. First, 100 μg of the protein was subjected to 10% SDS-polyacrylamide electrophoresis and then electrophoretically transferred onto a nitrocellulose membrane. The membrane was incubated with primary antibodies against EGFR (1:100) (sc-03, rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA), p-EGFR (1:100) (sc-12351, rabbit polyclonal; Santa Cruz Biotechnology), p-ERK1/2 (1:100) (Biosource International), p-ERK5 (1:400) (Stressgen), and actin (1:100) (sc-10731, rabbit polyclonal; Santa Cruz Biotechnology). The protein expression was detected using an EnVison+ system (DAKO). 3,3′-Diaminobenzidine tetrahydrochloride was used as the chromogen. Liver specimens (40 to 60 mg, wet weight) were homogenized with T-PER tissue protein extraction reagent (Pierce Chemical Co.). Homogenates were centrifuged and filtered through a 0.22-μm sterile filter. The collagen content of the specimens was measured using the Sircol collagen assay kit (Biocolor Ltd., Belfast, UK) according to the manufacturer's instructions. In brief, Sirius red reagent (50 μl) was added to each liver homogenate (50 μl) and mixed for 30 minutes. The collagen-dye complex was precipitated by centrifugation at 15,000 × g for 5 minutes, washed with ethanol, and dissolved in 0.5 mol/L sodium hydroxide. Finally, the samples were introduced into a microplate reader, and the absorbance was determined at 540 nm. The mean ± SD was calculated for all parameters. Statistical differences were determined using the Mann-Whitney U-test or analysis of variance. A P value <0.05 was accepted as the level of statistical significance. The three-dimensional cell culture system was used for the determination of the effects of gefitinib on biliary cyst formation. When BECs were cultured in collagen gel matrix with the medium that did not contain gefitinib, the cells became spherical or elliptical small cystic masses 2 to 3 days later as observed under phase-contrast microscopy (Figure 1, A and D). The microcysts gradually enlarged and transformed to multicellular cysts forming well-developed cysts (Figure 1, B and E). The size and number of the biliary cysts increased throughout a period of 2 weeks. Gefitinib was" @default.
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• W1995287795 title "Inhibition of Intrahepatic Bile Duct Dilation of the Polycystic Kidney Rat with a Novel Tyrosine Kinase Inhibitor Gefitinib" @default.
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