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Downregulation of protein kinase C inhibits indomethacin-induced apoptosis in gastric epithelial cells by differential regulation of apoptosis-related genes
April 1998
Gastrointestinal Oncology A709
identifiable in control untreated fundic mucosa (2 -+ 1/mm2 and 14 _+3/mm2, respectively. 2) In hyperplastic fundic mucosa, generated by 8 week Lox treatment, Cdk4 expression was significantly increased (86-+ 8/mm2), and immunoreactivity localized to the apical proliferating epithelium as well as the base of gastric glands, where the ECL cell hyperplasia was evident. In contrast, cyclin D1 expression was not significantly increased at this stage (8/mm2). 3) After 16 weeks Lox treatment, ECL cell neoplasia and carcinoid tumors were evident, and Cdk4 immunoreactivity w~s further increased (403/mm2) and defined in most of tumor ECL cells (>85%). In addition, cyclin D1 expression was now remarkably up-regulated (262/mm2) and exclusively localized to the transformed ECL cells. We thus conclude that increased Cdk4 expression is associated with proliferative activity of the fundic mucosa induced by hypergastrinemia. Whereas cyclin D1 expression appears to be particularly related to the phase progression of ECL cell transformation. • G2933 COMPARATIVE EFFECTS OF ANTIOXIDANT VITAMINS C, E AND 13-CAROTENE ON CELL PROLIFERATION AND APOPTOSIS IN A GASTRIC EPITHELIAL CELL LINE. ZW Zhang ], S Wilks2, D Davis3, S Carnaby 1, Paul Allen 2, JF Poschet t, SE Patchett 1, MJG Farthing I. Digestive Diseases Research Centre I and Department of Haematology2, St Bartholomew's & The Royal London School of Medicine & Dentistry and the FACS Laboratory, ICRF 3, London, UK. Previous studies suggest that antioxidant vitamins C, E and 13-carotene may be protective against gastric cancer. However, how these antioxidant vitamins interact with gastric mucosa remain unclear. In the present study, the effects of antioxidant vitamins C, E and 13-carotene on gastric epithelial cell proliferation and cell cycle were evaluated in vitro. The gastric epithelial cell line, AGS, was grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Cell viability and proliferation were determined by trypan blue dye exclusion analysis and by a BrdU incorporation assay (Boehringer Mannheim, UK). Cell cycle distribution was based on DNA content, measured after labelling with propidium iodide and quantified by flow cytometric analysis. Apoptosis was confirmed by the fluorescence microscopy with acridine orange and by the DNA ladder assay. Vitamin C (10-12001aM) and 13-carotene (1-40~aM) inhibited the proliferation of AGS cells in a concentration-dependent manner, while vitamin E (5-80~tM) had no effect (figure). The cell cycle distribution of viable ceils was not affected by these antioxidant vitamins, however, the fraction of subdiploid cells with oligonucleosomal DNA degradation characteristic of apoptosis was induced by both vitamin C and 13-carotene, but not vitamin E. P r elit'erlllnn of Saslr 1¢ f ~ ¢ b ¢ l i t | c d l l i n t (&GS)
9q, 10q, 19q, and 22q. Most of these areas contain candidate tumor suppressors or oncogenes, but have been missed by previous LOH studies. There were several instances in blocks of homozygosity where a robust marker did not amplify and many of these are probably areas of homozygous deletions. There are rare differences between two lines from the same patient (from primary and metastasis). LOH has been reported by other labs for several of the cell lines at various loci and in all cases this was confirmed in the present study. Cell line NCI-SNU-638 had microsatellite instability at 6% of the markers and the other lines had from 0 to 1.5% instability. In all, 4500 useful data points were obtained; 37% of the reactions showed homozygosity, and the failure rate of amplification was 4%. Conclusions: High density quantitative PCR has allowed construction of detailed maps of the genomes of 14 gastric adenocarcinoma cell lines. The maps show the likely positions of new tumor suppressor genes, the cell lines most likely to harbor alterations of known genes, and regions prone to deletion. • G2935
DOWNREGULATION OF PROTEIN KINASE C INHIBITS INDOMETHACIN-INDUCED APOPTOSIS IN GASTRIC EPITHELIAL CELLS BY DIFFERENTIAL REGULATION OF APOPTOSISRELATED GENES. GH Zhu, BCY Wong, CK Ching, KC Lai and SK Lam. Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong. Introduction: Nonsteroidal antiinflammatory drugs (NSAIDs) decrease the incidence of and mortality from gastrointestinal cancer. We previously identified that NSAIDs could inhibit cell proliferation and induce apnptosis in human gastric epithelial cells (Gastroenterology 1997;112:a683), which is considered to be relevant to their anti-tumor effects in vivo. Aim: In this study, we further explored the roles of protein kinase C pathway and apoptosisrelated genes in the mechanism mediated in indomethacin-induced apoptosis. Methods: A human gastric adenocarcinoma (AGS) cell line, bearing wild-type p53, was used. The cell was treated with 4001aM indomethacin alone or in combination with a PKC activator, 12-o-tetradecanoylphomol (TPA), for various time periods. Cell apoptosis was characterized by acridine orange staining, DNA fragmentation, flow cytometry analysis. The expression of p53, p21Wa~clPt, c-myc, bcl-2, and bax were detected by Northern and Western blotting. Results: Indomethacin could readily induce apoptosis in AGS cells. A progressive increase in c-myc mRNA and protein was noted as early as 2 hr and peak at 8 hr, while p53, p21wafl/ciP] kept constant during 24 hr indomethacin treatment. Treatment with indomethacin also increased bax level at 12 hr and peaked at 24 hr, while bcl-2 kept unchanged. Prolonged cotreatment (24 hr) of 100nM TPA could greatly reverse the apoptosis induction by indomethacin (>50%), along with increasing the expression of p21watVciPl, and decreasing the overexpression of c-my and bax by indomethacin. Conclusions: Indomethacin-induced apoptosis of gastric epithelial cells and its prevention by down-regulation of PKC is mediated by differential regulation of protooncogene and tumor suppressor genes. • G2936
These results suggest that vitamin C and 13-carotene, but not vitamin E inhibit gastric epithelial cell proliferation without affecting the cell cycle profile of the viable cells, and induce apoptosis in AGS cells, which may be implicated in the protective role of these antioxidant vitamins against gastric cancer. • G2934 DETAILED GENOME ANATOMY OF THE COMMONLY STUDIED GASTRIC ADENOCARCINOMA CELL LINES. Y.-L. Zheng, A. Herr, L.J. Ferrin. Genetics and Biochemistry Branch/NIDDK, National Institutes of Health, Bethesda, MD Backeround: Over the last several years, loss of heterozygosity (LOH) experiments have grown in popularity and have proven useful for identifying regions of DNA containing tumor suppressor genes. Use or extension of previous LOH studies of gastric adenocarcinoma, however, have proven difficult for several reasons. Probes or markers are often poorly characterized, and only a small number of them are usually used. The primary tumors and some of the cell lines used are not available. LOH has been reported for at least 11 different chromosomes, and the data is not available to evaluate the relative significance of the findings from different labs. This study was done to eliminate these shortcomings. Methods: All of the nine gastric adenocarcinoma cell lines from the American Type Culture Collection were used. These are the KATO-III, Hs 746T, RF-1, RF-48, AGS, NCI-N87, and NCI-SNU-1, -5, and -16 lines. Five additional lines (NCI-SNU-601, -620, -638, -668, and -719) were also used. DNA samples from the 14 lines were amplified by PCR using a set of 340 markers developed by the Cooperative Human Linkage Center. These markers are well characterized, highly polymorphic with an average heterozygosity of 0.76, evenly distributed throughout the human genome, amplified under a uniform set of conditions, and commercially available. Reactions were assembled using a robotic workstation and analyzed quantitatively on an automated sequencer. Results: All cell lines showed large blocks of LOH. While no marker was completely homozygous, 14 markers showed homozygosity in over twothirds of the cell lines. Four of the markers are in the p53 region. The 10 other markers define small discreet regions of LOH involving lq, 6p, 6q, 10q, 7q,
PHOTODYNAMIC THERAPY WITH 5-AMINOLAEVULINIC ACID FOR THE TREATMENT OF DYSPLASIA AND EARLY CARCINOMA IN THE UPPER GI-TRACT. T. Zoepf 1,3, G. Preclik 1, W. Boeck l, A. Rneck2, J.F. Riemann3, G. AdlerL 1) Dept. Internal Medicine I, University Hospital of Ulm, Germany. 2) Institute for Lasertechnologies at the University of Ulm, Germany. 3) Medical Dept. C, Klinikum Ludwigshafen, Germany Photodynamic therapy (PDT) is an experimental therapy for treatment of localized small malignancies. First results using hematoporphyrins as photosensitizers are encouraging, however there is a long lasting skin photosensitivity. 5-aminolaevulinic acid (5-ALA) induces protoporhyrin IX as an endogeneous photosensitizer, with skin photosensitivity of only 24-48h. Our initial results using 5-ALA-PDT in 8 patients are reported. Method: 3 male and 5 female patients (aged 43 to 80 years) gave their informed consent to participate in this clinical pilot study. All patients were locally staged by endosonography. 2 patients showed severe dysplasia in Barrett's esophagus, 4 patients showed Barrett's adenocarcinoma (lx CIS, lx T1N0, 2x TEN0), 1 patient showed CIS in squamouscell carcinoma of the esophagus and 1 patient showed early gastric cancer. 5 hours after oralsystemic application of 60mg /kg b.w. 5-ALA dissolved in fruit juice, the lesions were irradiated by 635nm laserlight from an argon-pumped dye laser through a 2,5cm cylindrical diffusor. The lightdose delivered per treatment session was 100-150J/cm2 with an output of 100 mW/cm2. Results: 7/8 patients showed reduction of tumor volume or of dysplasia. In all patients with Barrett's esophagus there was a marked reduction or eradication of Barrett's mucosa. 1 patient with a squamous cell carcinoma showed no respose to the treatment, but there appeared a mucosal edema immediately after starting the laser irradiation. All patients were treated twice in an interval of 5 weeks. There was no skin phototoxicity observed. The follow-up was 4-18 months. Conclusions: 5-ALA-PDT seems to be effective for the treatment of mucosal malignancies and dysplasia of the upper Gl-tract with little side effects. Studies comparing PDT with other ablative methods, like argon plasma coagulation, must confirm the theoretical advantages of PDT.
Gastrointestinal Oncology A709
identifiable in control untreated fundic mucosa (2 -+ 1/mm2 and 14 _+3/mm2, respectively. 2) In hyperplastic fundic mucosa, generated by 8 week Lox treatment, Cdk4 expression was significantly increased (86-+ 8/mm2), and immunoreactivity localized to the apical proliferating epithelium as well as the base of gastric glands, where the ECL cell hyperplasia was evident. In contrast, cyclin D1 expression was not significantly increased at this stage (8/mm2). 3) After 16 weeks Lox treatment, ECL cell neoplasia and carcinoid tumors were evident, and Cdk4 immunoreactivity w~s further increased (403/mm2) and defined in most of tumor ECL cells (>85%). In addition, cyclin D1 expression was now remarkably up-regulated (262/mm2) and exclusively localized to the transformed ECL cells. We thus conclude that increased Cdk4 expression is associated with proliferative activity of the fundic mucosa induced by hypergastrinemia. Whereas cyclin D1 expression appears to be particularly related to the phase progression of ECL cell transformation. • G2933 COMPARATIVE EFFECTS OF ANTIOXIDANT VITAMINS C, E AND 13-CAROTENE ON CELL PROLIFERATION AND APOPTOSIS IN A GASTRIC EPITHELIAL CELL LINE. ZW Zhang ], S Wilks2, D Davis3, S Carnaby 1, Paul Allen 2, JF Poschet t, SE Patchett 1, MJG Farthing I. Digestive Diseases Research Centre I and Department of Haematology2, St Bartholomew's & The Royal London School of Medicine & Dentistry and the FACS Laboratory, ICRF 3, London, UK. Previous studies suggest that antioxidant vitamins C, E and 13-carotene may be protective against gastric cancer. However, how these antioxidant vitamins interact with gastric mucosa remain unclear. In the present study, the effects of antioxidant vitamins C, E and 13-carotene on gastric epithelial cell proliferation and cell cycle were evaluated in vitro. The gastric epithelial cell line, AGS, was grown in RPMI 1640 medium supplemented with 10% fetal calf serum. Cell viability and proliferation were determined by trypan blue dye exclusion analysis and by a BrdU incorporation assay (Boehringer Mannheim, UK). Cell cycle distribution was based on DNA content, measured after labelling with propidium iodide and quantified by flow cytometric analysis. Apoptosis was confirmed by the fluorescence microscopy with acridine orange and by the DNA ladder assay. Vitamin C (10-12001aM) and 13-carotene (1-40~aM) inhibited the proliferation of AGS cells in a concentration-dependent manner, while vitamin E (5-80~tM) had no effect (figure). The cell cycle distribution of viable ceils was not affected by these antioxidant vitamins, however, the fraction of subdiploid cells with oligonucleosomal DNA degradation characteristic of apoptosis was induced by both vitamin C and 13-carotene, but not vitamin E. P r elit'erlllnn of Saslr 1¢ f ~ ¢ b ¢ l i t | c d l l i n t (&GS)
9q, 10q, 19q, and 22q. Most of these areas contain candidate tumor suppressors or oncogenes, but have been missed by previous LOH studies. There were several instances in blocks of homozygosity where a robust marker did not amplify and many of these are probably areas of homozygous deletions. There are rare differences between two lines from the same patient (from primary and metastasis). LOH has been reported by other labs for several of the cell lines at various loci and in all cases this was confirmed in the present study. Cell line NCI-SNU-638 had microsatellite instability at 6% of the markers and the other lines had from 0 to 1.5% instability. In all, 4500 useful data points were obtained; 37% of the reactions showed homozygosity, and the failure rate of amplification was 4%. Conclusions: High density quantitative PCR has allowed construction of detailed maps of the genomes of 14 gastric adenocarcinoma cell lines. The maps show the likely positions of new tumor suppressor genes, the cell lines most likely to harbor alterations of known genes, and regions prone to deletion. • G2935
DOWNREGULATION OF PROTEIN KINASE C INHIBITS INDOMETHACIN-INDUCED APOPTOSIS IN GASTRIC EPITHELIAL CELLS BY DIFFERENTIAL REGULATION OF APOPTOSISRELATED GENES. GH Zhu, BCY Wong, CK Ching, KC Lai and SK Lam. Department of Medicine, Queen Mary Hospital, the University of Hong Kong, Hong Kong. Introduction: Nonsteroidal antiinflammatory drugs (NSAIDs) decrease the incidence of and mortality from gastrointestinal cancer. We previously identified that NSAIDs could inhibit cell proliferation and induce apnptosis in human gastric epithelial cells (Gastroenterology 1997;112:a683), which is considered to be relevant to their anti-tumor effects in vivo. Aim: In this study, we further explored the roles of protein kinase C pathway and apoptosisrelated genes in the mechanism mediated in indomethacin-induced apoptosis. Methods: A human gastric adenocarcinoma (AGS) cell line, bearing wild-type p53, was used. The cell was treated with 4001aM indomethacin alone or in combination with a PKC activator, 12-o-tetradecanoylphomol (TPA), for various time periods. Cell apoptosis was characterized by acridine orange staining, DNA fragmentation, flow cytometry analysis. The expression of p53, p21Wa~clPt, c-myc, bcl-2, and bax were detected by Northern and Western blotting. Results: Indomethacin could readily induce apoptosis in AGS cells. A progressive increase in c-myc mRNA and protein was noted as early as 2 hr and peak at 8 hr, while p53, p21wafl/ciP] kept constant during 24 hr indomethacin treatment. Treatment with indomethacin also increased bax level at 12 hr and peaked at 24 hr, while bcl-2 kept unchanged. Prolonged cotreatment (24 hr) of 100nM TPA could greatly reverse the apoptosis induction by indomethacin (>50%), along with increasing the expression of p21watVciPl, and decreasing the overexpression of c-my and bax by indomethacin. Conclusions: Indomethacin-induced apoptosis of gastric epithelial cells and its prevention by down-regulation of PKC is mediated by differential regulation of protooncogene and tumor suppressor genes. • G2936
These results suggest that vitamin C and 13-carotene, but not vitamin E inhibit gastric epithelial cell proliferation without affecting the cell cycle profile of the viable cells, and induce apoptosis in AGS cells, which may be implicated in the protective role of these antioxidant vitamins against gastric cancer. • G2934 DETAILED GENOME ANATOMY OF THE COMMONLY STUDIED GASTRIC ADENOCARCINOMA CELL LINES. Y.-L. Zheng, A. Herr, L.J. Ferrin. Genetics and Biochemistry Branch/NIDDK, National Institutes of Health, Bethesda, MD Backeround: Over the last several years, loss of heterozygosity (LOH) experiments have grown in popularity and have proven useful for identifying regions of DNA containing tumor suppressor genes. Use or extension of previous LOH studies of gastric adenocarcinoma, however, have proven difficult for several reasons. Probes or markers are often poorly characterized, and only a small number of them are usually used. The primary tumors and some of the cell lines used are not available. LOH has been reported for at least 11 different chromosomes, and the data is not available to evaluate the relative significance of the findings from different labs. This study was done to eliminate these shortcomings. Methods: All of the nine gastric adenocarcinoma cell lines from the American Type Culture Collection were used. These are the KATO-III, Hs 746T, RF-1, RF-48, AGS, NCI-N87, and NCI-SNU-1, -5, and -16 lines. Five additional lines (NCI-SNU-601, -620, -638, -668, and -719) were also used. DNA samples from the 14 lines were amplified by PCR using a set of 340 markers developed by the Cooperative Human Linkage Center. These markers are well characterized, highly polymorphic with an average heterozygosity of 0.76, evenly distributed throughout the human genome, amplified under a uniform set of conditions, and commercially available. Reactions were assembled using a robotic workstation and analyzed quantitatively on an automated sequencer. Results: All cell lines showed large blocks of LOH. While no marker was completely homozygous, 14 markers showed homozygosity in over twothirds of the cell lines. Four of the markers are in the p53 region. The 10 other markers define small discreet regions of LOH involving lq, 6p, 6q, 10q, 7q,
PHOTODYNAMIC THERAPY WITH 5-AMINOLAEVULINIC ACID FOR THE TREATMENT OF DYSPLASIA AND EARLY CARCINOMA IN THE UPPER GI-TRACT. T. Zoepf 1,3, G. Preclik 1, W. Boeck l, A. Rneck2, J.F. Riemann3, G. AdlerL 1) Dept. Internal Medicine I, University Hospital of Ulm, Germany. 2) Institute for Lasertechnologies at the University of Ulm, Germany. 3) Medical Dept. C, Klinikum Ludwigshafen, Germany Photodynamic therapy (PDT) is an experimental therapy for treatment of localized small malignancies. First results using hematoporphyrins as photosensitizers are encouraging, however there is a long lasting skin photosensitivity. 5-aminolaevulinic acid (5-ALA) induces protoporhyrin IX as an endogeneous photosensitizer, with skin photosensitivity of only 24-48h. Our initial results using 5-ALA-PDT in 8 patients are reported. Method: 3 male and 5 female patients (aged 43 to 80 years) gave their informed consent to participate in this clinical pilot study. All patients were locally staged by endosonography. 2 patients showed severe dysplasia in Barrett's esophagus, 4 patients showed Barrett's adenocarcinoma (lx CIS, lx T1N0, 2x TEN0), 1 patient showed CIS in squamouscell carcinoma of the esophagus and 1 patient showed early gastric cancer. 5 hours after oralsystemic application of 60mg /kg b.w. 5-ALA dissolved in fruit juice, the lesions were irradiated by 635nm laserlight from an argon-pumped dye laser through a 2,5cm cylindrical diffusor. The lightdose delivered per treatment session was 100-150J/cm2 with an output of 100 mW/cm2. Results: 7/8 patients showed reduction of tumor volume or of dysplasia. In all patients with Barrett's esophagus there was a marked reduction or eradication of Barrett's mucosa. 1 patient with a squamous cell carcinoma showed no respose to the treatment, but there appeared a mucosal edema immediately after starting the laser irradiation. All patients were treated twice in an interval of 5 weeks. There was no skin phototoxicity observed. The follow-up was 4-18 months. Conclusions: 5-ALA-PDT seems to be effective for the treatment of mucosal malignancies and dysplasia of the upper Gl-tract with little side effects. Studies comparing PDT with other ablative methods, like argon plasma coagulation, must confirm the theoretical advantages of PDT.