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Helicobacter pylori induces activation of extracellular regulated kinases and enhanced AP-1 binding activity in gastric epithelial cells in vitro
A762 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
4096
4098
HEUCOBACTER PYLORI INDUCES ACTIVATION OF EXTRA-
HEUCOBACTER INFECION INCREASES DEVELOPMENT OF
CELLULAR REGULATED KINASES AND ENHANCED AP·I BINDING ACTIVITY IN GASTRIC EPITHELIAL CELLS IN
DYSPLASIA AND INFLAMMATION OF THE STOMACH IN MICE TREATED WITH N-ETHYL-N'-NITRO-N-NITROSOGUANIDINE. Tomoyuki Ohno, Masakazu Kita, Yoshi Yamaoka, Naoki Sawai, Toshihito Tanahashi, Kyoko Sakai, Shigeyoshi Imamura, Shoji Mitsufuji, Tadashi Kodama, Jiro Imanishi, Kei Kashima, Kyoto Prefectural Univ of Medicine, Kyoto, Japan; Kyoto Prefectural Univ of Medicine, Kyot, Japan.
VITRO. Barbara Obst, Siegfried Wagner, Winfried Beil, Medicine Hochschule Hannover, Hannover, Germany. H. pylori infection is recognized as a risk factor for gastric adenocarcinoma. However, the underlying pathogenic mechanisms are unknown. Extracellular regulated kinases (ERKs) playa pivotal role in the regulation of proliferation in response to extracellular mitogens. An enhanced cell proliferation has been shown in patients infected with H. pylori. This change in cellular proliferation of the gastric epithelium may playa critical role in carcinogenesis. Therefore we investigated the effect of H. pylori on ERK acitivation in gastric epithelial cells. Methods: Prior to incubation with H. pylori gastric epithelial cells (AGS) were starved for 20 hours in RPMI medium containing 0,1 % fetal calf serum (FCS). Incubation with H. pylori (107 to 5 X 108 bacteria/ml) was performed up to 60 minutes. PKC inhibitor Cal- phostin C (100 nM) and MEK inhibitor PD 98059 (10 ILM) were added to the culture medium one hour prior to addition of H. pylori. ERK activation was detected by western blotting using phospho-specific antibodies. DNA-binding of the transcription factor AP-l was determined by gel mobility shift analysis. Results: Incubation with H. pylori resulted in a time dependent increase in ERK activity with a maximum at 30 minutes and a decline near to control levels after 60 minutes. ERK activation was dose dependent with a 4-fold increase at 5 x 108 bacteria/ml. No difference between cag+ and cag- strains was observed. ERK activation was followed by a marked increase in AP-I binding activity which occured after I hour incubation with H. pylori and reached their peak at 3 - 4 hours. The increased AP-I binding activity was significantly inhibited by PD 98059 but not by Calphostin C. Conclusions: Incubation of gastric cells with H. pylori results in increased ERK activation with a concomittant enhanced DNA binding activity of AP-I, a transcription factor, which is involved in the expression of numerous genes responsible for cell proliferation. These may be mechanisms by which cell proliferation is upregulated in H. pylori infected patients and which may contribute to the development of gastric cancer.
4097 THE PROTEIN KINASE C ACTIVATORS CIS-9,10-METHYLENEOCTADECANOIC ACID AND TPA INHIBIT HEUCOBACTER PYWRI INDUCED APOPTOSIS IN GASTRIC EPITHELIAL CELLS IN VITRO. Barbara Obst, Siegfried Wagner, Winfried Beil, Medicine Hochschule Hannover, Hannover, Germany. H. pylori infection has been linked with gastric carcinoma. However, the underlying mechanisms have not yet been elucidated. Apoptosis plays an important role in the control of gastric epithelial cell turnover, e.g in the removement of lethally damaged cells and a dysregulated apoptosis is thought to contribute to the development of cancer. Protein kinase C (PKC) has been implicated in inhibition of cellular apoptosis. We have previously shown that H. pylori induces apoptosis in gastric epithelial cells and that the H pylori fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) activates PKC. Therefore the possible interplay between H. pylori induced apoptosis and PKC activation by MOA was investigated. Methods:. H. pylori cytosol was prepared by sonification and following centrifugation at 14.000 g. For assessment of apoptosis gastric epithelial cells (AGS) were incubated with viable H pylori (107 bacteria/ml) or H. pylori cytosol (40 ILg/ml) in the absence and presence of I - 10 ILM MOA or I - 5 nM TPA. Apoptosis was determined by Hoechst staining and with a histone enzymelinked immunosorbent assay. Cell viability was assessed by release of LDH. PKC activity in the cytosol and particulate cell fraction of AGS cells was determined by the incorporation of ['Y32p]ATP in GS-peptide. Results: MOA (50 ILM) induced a transient 20 % reduction in cytosolic PKC activity of AGS cells with no decrease in PKC activity after 90 minutes. In contrast, TPA (10 nM) induced a longlasting PKC activation with a 37 % reduction in PCK activity after 15 minutes and 22 % decrease after 90 minutes. Incubation of gastric cells with H. pylori and H. pylori cytosol caused a twofold increase in apoptosis. Preincubation of AGS cells with both PKC activators resulted in a dose dependent decrease of both H. pylori and H. pylori cytosol induced apoptosis with a complete inhibition at 10 ILM MOA and 5 nM TPA, respectively. No significant increase in LDH release was observed. Conclusion: In this study we show that the PKC activators TPA and H pylori fatty acid MOA inhibit H. pylori induced apoptosis. During H. pylori infection several substances occur, which have the potency to cause DNA damage. Lethally damaged cells are normally removed by apoptosis. Inhibition of gastric epithelial apoptosis during H. pylori infection by MOA may prevent mutated cells from being eliminated and hence may contribute to gastric carcinogenesis.
Introduction: Epidemiological studies indicate that Helicobacter pylori (Hpylori) infection is closely associated to development of gastric cancer in humans, however, it is still unknown whether Helicobacter infection really affects gastric carcinogenesis. To clarify the relationship between Helicobacter species and gastric carcinogenesis, the effects of bacteria on experimental carcinogenesis were investigated using N-ethyl-N' -nitro-Nnitrosoguanidine (ENNG), which is thought to be a chemical carcinogen. Materials and Methods: Six-week-old male specific-pathogen-free C57BLl6 mice were divided into 4 groups. Grou£ I (n= 15) mice were given Helicobacter felis (Hjelis:ATCC49179) (10 CPU) 3 times on alternate days and after I week were given lOOppm of ENNG in their drinking water for 16 weeks. Group 2 (n=15) mice were given only Hfelis. Group 3 (n= 15) mice were given only ENNG for 16 weeks and Group 4 (n= 10) mice were given saline solution and served as controls. The animals were sacrificed under anesthesia 50 weeks after inoculation for histologic examination and determination of serum levels of anti-Hjelis IgG antibody. Results: All inoculated animals (Group I and 2) were infected with Hjelis. Control animals (Group 4) showed no abnormal findings. Inflammation was more severe in Group I than in any of the other 3 groups (p
GASTROENTEROLOGY Vol. 118, No.4
4096
4098
HEUCOBACTER PYLORI INDUCES ACTIVATION OF EXTRA-
HEUCOBACTER INFECION INCREASES DEVELOPMENT OF
CELLULAR REGULATED KINASES AND ENHANCED AP·I BINDING ACTIVITY IN GASTRIC EPITHELIAL CELLS IN
DYSPLASIA AND INFLAMMATION OF THE STOMACH IN MICE TREATED WITH N-ETHYL-N'-NITRO-N-NITROSOGUANIDINE. Tomoyuki Ohno, Masakazu Kita, Yoshi Yamaoka, Naoki Sawai, Toshihito Tanahashi, Kyoko Sakai, Shigeyoshi Imamura, Shoji Mitsufuji, Tadashi Kodama, Jiro Imanishi, Kei Kashima, Kyoto Prefectural Univ of Medicine, Kyoto, Japan; Kyoto Prefectural Univ of Medicine, Kyot, Japan.
VITRO. Barbara Obst, Siegfried Wagner, Winfried Beil, Medicine Hochschule Hannover, Hannover, Germany. H. pylori infection is recognized as a risk factor for gastric adenocarcinoma. However, the underlying pathogenic mechanisms are unknown. Extracellular regulated kinases (ERKs) playa pivotal role in the regulation of proliferation in response to extracellular mitogens. An enhanced cell proliferation has been shown in patients infected with H. pylori. This change in cellular proliferation of the gastric epithelium may playa critical role in carcinogenesis. Therefore we investigated the effect of H. pylori on ERK acitivation in gastric epithelial cells. Methods: Prior to incubation with H. pylori gastric epithelial cells (AGS) were starved for 20 hours in RPMI medium containing 0,1 % fetal calf serum (FCS). Incubation with H. pylori (107 to 5 X 108 bacteria/ml) was performed up to 60 minutes. PKC inhibitor Cal- phostin C (100 nM) and MEK inhibitor PD 98059 (10 ILM) were added to the culture medium one hour prior to addition of H. pylori. ERK activation was detected by western blotting using phospho-specific antibodies. DNA-binding of the transcription factor AP-l was determined by gel mobility shift analysis. Results: Incubation with H. pylori resulted in a time dependent increase in ERK activity with a maximum at 30 minutes and a decline near to control levels after 60 minutes. ERK activation was dose dependent with a 4-fold increase at 5 x 108 bacteria/ml. No difference between cag+ and cag- strains was observed. ERK activation was followed by a marked increase in AP-I binding activity which occured after I hour incubation with H. pylori and reached their peak at 3 - 4 hours. The increased AP-I binding activity was significantly inhibited by PD 98059 but not by Calphostin C. Conclusions: Incubation of gastric cells with H. pylori results in increased ERK activation with a concomittant enhanced DNA binding activity of AP-I, a transcription factor, which is involved in the expression of numerous genes responsible for cell proliferation. These may be mechanisms by which cell proliferation is upregulated in H. pylori infected patients and which may contribute to the development of gastric cancer.
4097 THE PROTEIN KINASE C ACTIVATORS CIS-9,10-METHYLENEOCTADECANOIC ACID AND TPA INHIBIT HEUCOBACTER PYWRI INDUCED APOPTOSIS IN GASTRIC EPITHELIAL CELLS IN VITRO. Barbara Obst, Siegfried Wagner, Winfried Beil, Medicine Hochschule Hannover, Hannover, Germany. H. pylori infection has been linked with gastric carcinoma. However, the underlying mechanisms have not yet been elucidated. Apoptosis plays an important role in the control of gastric epithelial cell turnover, e.g in the removement of lethally damaged cells and a dysregulated apoptosis is thought to contribute to the development of cancer. Protein kinase C (PKC) has been implicated in inhibition of cellular apoptosis. We have previously shown that H. pylori induces apoptosis in gastric epithelial cells and that the H pylori fatty acid cis-9,10-methyleneoctadecanoic acid (MOA) activates PKC. Therefore the possible interplay between H. pylori induced apoptosis and PKC activation by MOA was investigated. Methods:. H. pylori cytosol was prepared by sonification and following centrifugation at 14.000 g. For assessment of apoptosis gastric epithelial cells (AGS) were incubated with viable H pylori (107 bacteria/ml) or H. pylori cytosol (40 ILg/ml) in the absence and presence of I - 10 ILM MOA or I - 5 nM TPA. Apoptosis was determined by Hoechst staining and with a histone enzymelinked immunosorbent assay. Cell viability was assessed by release of LDH. PKC activity in the cytosol and particulate cell fraction of AGS cells was determined by the incorporation of ['Y32p]ATP in GS-peptide. Results: MOA (50 ILM) induced a transient 20 % reduction in cytosolic PKC activity of AGS cells with no decrease in PKC activity after 90 minutes. In contrast, TPA (10 nM) induced a longlasting PKC activation with a 37 % reduction in PCK activity after 15 minutes and 22 % decrease after 90 minutes. Incubation of gastric cells with H. pylori and H. pylori cytosol caused a twofold increase in apoptosis. Preincubation of AGS cells with both PKC activators resulted in a dose dependent decrease of both H. pylori and H. pylori cytosol induced apoptosis with a complete inhibition at 10 ILM MOA and 5 nM TPA, respectively. No significant increase in LDH release was observed. Conclusion: In this study we show that the PKC activators TPA and H pylori fatty acid MOA inhibit H. pylori induced apoptosis. During H. pylori infection several substances occur, which have the potency to cause DNA damage. Lethally damaged cells are normally removed by apoptosis. Inhibition of gastric epithelial apoptosis during H. pylori infection by MOA may prevent mutated cells from being eliminated and hence may contribute to gastric carcinogenesis.
Introduction: Epidemiological studies indicate that Helicobacter pylori (Hpylori) infection is closely associated to development of gastric cancer in humans, however, it is still unknown whether Helicobacter infection really affects gastric carcinogenesis. To clarify the relationship between Helicobacter species and gastric carcinogenesis, the effects of bacteria on experimental carcinogenesis were investigated using N-ethyl-N' -nitro-Nnitrosoguanidine (ENNG), which is thought to be a chemical carcinogen. Materials and Methods: Six-week-old male specific-pathogen-free C57BLl6 mice were divided into 4 groups. Grou£ I (n= 15) mice were given Helicobacter felis (Hjelis:ATCC49179) (10 CPU) 3 times on alternate days and after I week were given lOOppm of ENNG in their drinking water for 16 weeks. Group 2 (n=15) mice were given only Hfelis. Group 3 (n= 15) mice were given only ENNG for 16 weeks and Group 4 (n= 10) mice were given saline solution and served as controls. The animals were sacrificed under anesthesia 50 weeks after inoculation for histologic examination and determination of serum levels of anti-Hjelis IgG antibody. Results: All inoculated animals (Group I and 2) were infected with Hjelis. Control animals (Group 4) showed no abnormal findings. Inflammation was more severe in Group I than in any of the other 3 groups (p