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Serum pepsinogens and antibody to helicobacter pylor in gastric diseases
A492
AGA ABSTRACTS
SERUM PEPSINOGENS AND ANTIBODY TO HELICOBACTER PYLORI IN GASTRIC DISEASES. J,¥, Koo, S.J. Park, Depts. of Internal Medicine, Kosin Medical College, Pusan, Korea. To investigate the role of serum pepsinogens and Helicobacter pylori(H, pylori) in the diagnosis and management of various gastric diseases, we have detemained the levels of serum Impsinogen I(PG-I), serum pepsinogen II(PG-II) and antibody to H. pylori in 151 cases of chronic gastritis(CG), 44 cases of peptic ulcer(gastric 26, duodenal 18), and 98 cases of gastric carcinoma (early 14, advanced 84) using radioimmunoassay for PG-I, PG-rr and enzyme immunoassas~ for H. pyloN, The results were correlated with gastric disea~s. RESULTS : 1) The serum levels of PG-I and PG-,K in peptic ulcer(PU) group(78.18+35.52, 22.95-+12.88, respectively) were significantly higher than those of CG(53.35 •+28.64, 15.55-+9.72, respectively) and CK;(50.14-+37A5, 18.12-+ 12.26, res0ectively)(P<0.05), but there was no difference between those of CG and GC. 2) serum PG I/lI ratio in CCgroup and PU group was 3.83-+1.52 and 3.77-+1.52, reslx~ctively, which were significantly higher than that of GC(2.94-+l.52)(P<0.05). 3) PG I/iI ratio of early gastric cancer group(3.80-+l.74) was higher than that of advanced gastric carcinoma group(2.80-+l.45), but was similiar to that of CG group(3.83-+1.52). 4) The serum levels of PG-I, PG-II and serum PG-I/PG-II ratio in scirrhous gastric carcinoma were 53.41-+20.39, 15.35-+8.33 and 2.39-+1.42, respectively, which were much lower than those of CG group(P<0.05), and the serum levels of PG-I, PG-II and serum PG-I/PG-II ratio of gastric ulcer group were higher than those of Borrmann's type 2 carcinoma group, but not statistically significant. 5) The serum levels of PG-I, PG-II and serum PG-I/PG-II ratio showed no significant differences accordig to the location, the gross type and the histdiogic differentiation in GC group. 6) Positivity of antibody to H. pylori Of CG group, PU group o o and GC group were 90.1%(136/151), 97.6%(40/41), 86.6%(84/97), resixctively, showing no significant.difference between each groups. CONCLUSION : Above data suggest that serum PG-I and PG-II may be a useful differential diagnistic marker in various gastric diseases, and especially PG-I/PG-II ratio may become a good ancillary marker in the differential diagnosis between scinhous cancer and chronic gastritis. Antibody to H. pylori was not useful in the differential diagnosis of several gastric diseases in Korea.
• ASPIRIN AND OTHER NSAIDs INHIBIT THE PROLIFERATION OF COLON ADENOCARCINOMA CELLS: EFFECTS ON CELL CYCLE AND APOPTOSIS Marko$ I Koutsos ', Steven J Shift "=, Liang Qiao t, Basil Rigas "= ' Department of Medicine, Cornell University Medical College, and ' The Rockefeller University New York, N.Y. 10021 INTRODUCTION: Aspirin and other NSAIDs reduce the incidence of and mortality from colon cancer by about 50%, but their mechanism of action remains unknown. We evaluated the effect Of aspirin and representative NSAIDs on cell proliferation, cell cycle, and apoptosis in a human colon cancer cell line. METHODS: HT-29 colon adenocarcinoma cells were treated for up to 96 hr with aspirin (400-1500 IzM), indomethacin (100-600 #M), piroxicam (100-600/zM) and naproxen (200-1000 #M). Cells were counted and their viability was assessed by the trypan blue exclusion method. The cell cycle distribution of these cells was determined by flow cytometry following staining with propidium iodide. The levels of cyclindependent kinases were determined by Western blotting. Apoptosis was evaluated by (a) determining the amount of sub-diploid DNA content by flow cytometry, (b) by staining cells with acridine orange and (c) by determining fragmentation of genomic DNA on 1.8% agarose gels. RESULTS: 1) All NSAIDs had a significant antiproliferative effect that was time- and concentration-dependent. The relative potencies of each compound for this response was I > P> N > ASA. 2) They all altered the cell cycle distribution of the HT29 cells: ASA and I increased G0/Gt phase by 35-45%, decreased the S phase by - 5 0 % , and decreased the GJM phase by - 3 0 % . Naproxen and piroxicam had a similar effect but for the decrease in G2/M phase. These changes were accompanied by morphological changes of the cells. 3) NSAIDs decreased substantially the levels of the cyclin-dependent kinases p34cdc2 and p33 car2. 4) All NSAIDs tested, except for aspirin, induced apoptosis, as determined by all three methods. CONCLUSIONS: In the HT29 colon cancer cells, aspirin and related NSAIDs 1) have an antiproliferative effect; 2) cause a Go/G~ cell cycle block, accompanied by a marked decrease in the levels of two kinases which control cell cycle transitions; and 3) induce apoptosis. These effects, if operative in vivo, may account in part for the protective effect of these compounds on colon cancer.
GASTROENTEROLOGY, Vol. 108, No. 4
REGULATION OF THE ADHESION MOLECULE EGP40 PROMOTER BY GAMMA INTERFERON IN COLON C A R C I N O M A CELLS. G. KOrtner, R. Arnold and B. Simon. Department of Internal Medicine, Philipps University, Marburg, Germany. Gamma Interferon (IFNT) is considered a regulator of tumorassociated and MHC class II antigen expression. We previously reported the isolation of the tumor-associated antigen EGP40 cDNA from a human colon carcinoma eeUine. Recent data suggested that EGP40 is a key calcium-independent adhesion molecule. In this study we investigated whether EGP40 expression is modulated by interferons in colon tumor cells. Methods: The colon carcinoma ceUine HT29 was cultured in the absence or presence of 50, 250 and 1000 U/mi IFN'y or IFNo~ for 12, 48 and 72 hours (h). EGP40 antigen expression was revealed by fluorescentactivated cell-sorting analysis using monoclonal antibody HEAl25. EGP40 mP,NA was determined by northern blot analysis, control hybridization was performed with 18S rRNA. To analyse the effect of IFNs on EGP40 transcription, 1.1 kb of the EGP40 5"-flanking DNA including the promoter region were isolated. The transcription start site was mapped by RNase protection assay. EGP40 5"-flanking DNA was cloned into the pCATbasic reporter gone (EGP1100-CAT). CAT activity was determined after 72 hours in HT29 cells after transient transfection with EGPll00-CAT and interferon treatment. Results: Maximum repression of EGP40 mRNA was observed by treatment with 1000 U/ml IFNy for 72h, while EGP40 surface antigen expression was reduced to 66.5+4.2% of controls. Interestingly, treatment induced a 10% subpopulation of cells to reduce EGP40 antigen expression to less than 2% of controls. In contrast, IFN(x had no effect on EGP40 mRNA nor antigen expression. IFN~/ revealed complete repression of EGP40 promoter activity in HT29 cells at a dose of 50 U/ml. IFNc~ had no significant effect on EGP40 promoter activity. Conclusion: The EGP40 promoter is differentially regulated by IFNs. This is the first description of an adhesion molecule transcriptionally repressed by IFNy. Further studies will allow to analyse the negative signalling pathway in response to IFN~/ and the identification of a potential interferon negative response element in the EGP40 promoter.
THE EFFECT OF URSODEOXYCHOLIC AND LITHOCHOLIC ACID ON CELL CYCLE AND APOPTOSIS IN HUMAN COLON ADENOCARCINOMA CELLS. M~rkg~ I Koutsos~, Steven J Shiffl'~, Basil Rigas ~,7. i Department of Medicine, Cornell University Medical College, and 2 The Rockefeller University New York, N.Y. 10021 INTRODUCTION: Bile acids have been implicated in the development of colorectal neoplasms by epidemiological, clinical and experimental studies. In rats, ursodeoxycholie acid (URSO) has a protective effect against colon cancer, while lithocholic acid (LCA) has tumorigenic properties. To evaluate the possible mechanisms by which URSO and LCA affect colon carcinogenesis, we assessed their effect on the the cell cycle and programmed cell death (apoptosis) in colon adenocarcinoma cells. METHODS: The HT29 colon cancer cell line was treated with both bile acids for 24, 48, and 72 hrs; their concentrations ranged from 50 ftM to 1 mM. URSO & LCA were dissolved in ethanol; LCA tended to fall out of solution at the higher concentrations. The cell cycle distribution was determined by flow cytometry after propidium iodide staining. Apoptosis was evaluated by a) the amount of sub-diploid DNA content by flow cytometry, b) acridine orange staining and c) a modified Wright staining. RESULTS: 1) URSO decreased the proportion of cells in the S phase and increased those in GJM. This effect, which was concentration- and time-dependent, suggests a block of transition into and through the M phase. 2) LCA increased the proportion of cells in Go/G~ and decreased those in the S and G J M phases of the cell cycle. This finding suggests either quiescence of the cells or a block into entering the S phase. 3) URSO, at concentrations ranging from 100 #M to 0.1 raM, induced apoptosis, as determined by all three methods. Apoptosis became apparent at 48 hr and maximal at 72 hrs. LCA did not exhibit any of these effects at all time points and concentrations. CONCLUSIONS: URSO and LCA have a differential effect on the cell cycle, while only URSO induced apoptosis. These differences may contribute to their different effects on colon carcinogenesis. It is conceivable that the apoptosis induced by URSO may play a role in its chemopreventive effect in colon cancer.
AGA ABSTRACTS
SERUM PEPSINOGENS AND ANTIBODY TO HELICOBACTER PYLORI IN GASTRIC DISEASES. J,¥, Koo, S.J. Park, Depts. of Internal Medicine, Kosin Medical College, Pusan, Korea. To investigate the role of serum pepsinogens and Helicobacter pylori(H, pylori) in the diagnosis and management of various gastric diseases, we have detemained the levels of serum Impsinogen I(PG-I), serum pepsinogen II(PG-II) and antibody to H. pylori in 151 cases of chronic gastritis(CG), 44 cases of peptic ulcer(gastric 26, duodenal 18), and 98 cases of gastric carcinoma (early 14, advanced 84) using radioimmunoassay for PG-I, PG-rr and enzyme immunoassas~ for H. pyloN, The results were correlated with gastric disea~s. RESULTS : 1) The serum levels of PG-I and PG-,K in peptic ulcer(PU) group(78.18+35.52, 22.95-+12.88, respectively) were significantly higher than those of CG(53.35 •+28.64, 15.55-+9.72, respectively) and CK;(50.14-+37A5, 18.12-+ 12.26, res0ectively)(P<0.05), but there was no difference between those of CG and GC. 2) serum PG I/lI ratio in CCgroup and PU group was 3.83-+1.52 and 3.77-+1.52, reslx~ctively, which were significantly higher than that of GC(2.94-+l.52)(P<0.05). 3) PG I/iI ratio of early gastric cancer group(3.80-+l.74) was higher than that of advanced gastric carcinoma group(2.80-+l.45), but was similiar to that of CG group(3.83-+1.52). 4) The serum levels of PG-I, PG-II and serum PG-I/PG-II ratio in scirrhous gastric carcinoma were 53.41-+20.39, 15.35-+8.33 and 2.39-+1.42, respectively, which were much lower than those of CG group(P<0.05), and the serum levels of PG-I, PG-II and serum PG-I/PG-II ratio of gastric ulcer group were higher than those of Borrmann's type 2 carcinoma group, but not statistically significant. 5) The serum levels of PG-I, PG-II and serum PG-I/PG-II ratio showed no significant differences accordig to the location, the gross type and the histdiogic differentiation in GC group. 6) Positivity of antibody to H. pylori Of CG group, PU group o o and GC group were 90.1%(136/151), 97.6%(40/41), 86.6%(84/97), resixctively, showing no significant.difference between each groups. CONCLUSION : Above data suggest that serum PG-I and PG-II may be a useful differential diagnistic marker in various gastric diseases, and especially PG-I/PG-II ratio may become a good ancillary marker in the differential diagnosis between scinhous cancer and chronic gastritis. Antibody to H. pylori was not useful in the differential diagnosis of several gastric diseases in Korea.
• ASPIRIN AND OTHER NSAIDs INHIBIT THE PROLIFERATION OF COLON ADENOCARCINOMA CELLS: EFFECTS ON CELL CYCLE AND APOPTOSIS Marko$ I Koutsos ', Steven J Shift "=, Liang Qiao t, Basil Rigas "= ' Department of Medicine, Cornell University Medical College, and ' The Rockefeller University New York, N.Y. 10021 INTRODUCTION: Aspirin and other NSAIDs reduce the incidence of and mortality from colon cancer by about 50%, but their mechanism of action remains unknown. We evaluated the effect Of aspirin and representative NSAIDs on cell proliferation, cell cycle, and apoptosis in a human colon cancer cell line. METHODS: HT-29 colon adenocarcinoma cells were treated for up to 96 hr with aspirin (400-1500 IzM), indomethacin (100-600 #M), piroxicam (100-600/zM) and naproxen (200-1000 #M). Cells were counted and their viability was assessed by the trypan blue exclusion method. The cell cycle distribution of these cells was determined by flow cytometry following staining with propidium iodide. The levels of cyclindependent kinases were determined by Western blotting. Apoptosis was evaluated by (a) determining the amount of sub-diploid DNA content by flow cytometry, (b) by staining cells with acridine orange and (c) by determining fragmentation of genomic DNA on 1.8% agarose gels. RESULTS: 1) All NSAIDs had a significant antiproliferative effect that was time- and concentration-dependent. The relative potencies of each compound for this response was I > P> N > ASA. 2) They all altered the cell cycle distribution of the HT29 cells: ASA and I increased G0/Gt phase by 35-45%, decreased the S phase by - 5 0 % , and decreased the GJM phase by - 3 0 % . Naproxen and piroxicam had a similar effect but for the decrease in G2/M phase. These changes were accompanied by morphological changes of the cells. 3) NSAIDs decreased substantially the levels of the cyclin-dependent kinases p34cdc2 and p33 car2. 4) All NSAIDs tested, except for aspirin, induced apoptosis, as determined by all three methods. CONCLUSIONS: In the HT29 colon cancer cells, aspirin and related NSAIDs 1) have an antiproliferative effect; 2) cause a Go/G~ cell cycle block, accompanied by a marked decrease in the levels of two kinases which control cell cycle transitions; and 3) induce apoptosis. These effects, if operative in vivo, may account in part for the protective effect of these compounds on colon cancer.
GASTROENTEROLOGY, Vol. 108, No. 4
REGULATION OF THE ADHESION MOLECULE EGP40 PROMOTER BY GAMMA INTERFERON IN COLON C A R C I N O M A CELLS. G. KOrtner, R. Arnold and B. Simon. Department of Internal Medicine, Philipps University, Marburg, Germany. Gamma Interferon (IFNT) is considered a regulator of tumorassociated and MHC class II antigen expression. We previously reported the isolation of the tumor-associated antigen EGP40 cDNA from a human colon carcinoma eeUine. Recent data suggested that EGP40 is a key calcium-independent adhesion molecule. In this study we investigated whether EGP40 expression is modulated by interferons in colon tumor cells. Methods: The colon carcinoma ceUine HT29 was cultured in the absence or presence of 50, 250 and 1000 U/mi IFN'y or IFNo~ for 12, 48 and 72 hours (h). EGP40 antigen expression was revealed by fluorescentactivated cell-sorting analysis using monoclonal antibody HEAl25. EGP40 mP,NA was determined by northern blot analysis, control hybridization was performed with 18S rRNA. To analyse the effect of IFNs on EGP40 transcription, 1.1 kb of the EGP40 5"-flanking DNA including the promoter region were isolated. The transcription start site was mapped by RNase protection assay. EGP40 5"-flanking DNA was cloned into the pCATbasic reporter gone (EGP1100-CAT). CAT activity was determined after 72 hours in HT29 cells after transient transfection with EGPll00-CAT and interferon treatment. Results: Maximum repression of EGP40 mRNA was observed by treatment with 1000 U/ml IFNy for 72h, while EGP40 surface antigen expression was reduced to 66.5+4.2% of controls. Interestingly, treatment induced a 10% subpopulation of cells to reduce EGP40 antigen expression to less than 2% of controls. In contrast, IFN(x had no effect on EGP40 mRNA nor antigen expression. IFN~/ revealed complete repression of EGP40 promoter activity in HT29 cells at a dose of 50 U/ml. IFNc~ had no significant effect on EGP40 promoter activity. Conclusion: The EGP40 promoter is differentially regulated by IFNs. This is the first description of an adhesion molecule transcriptionally repressed by IFNy. Further studies will allow to analyse the negative signalling pathway in response to IFN~/ and the identification of a potential interferon negative response element in the EGP40 promoter.
THE EFFECT OF URSODEOXYCHOLIC AND LITHOCHOLIC ACID ON CELL CYCLE AND APOPTOSIS IN HUMAN COLON ADENOCARCINOMA CELLS. M~rkg~ I Koutsos~, Steven J Shiffl'~, Basil Rigas ~,7. i Department of Medicine, Cornell University Medical College, and 2 The Rockefeller University New York, N.Y. 10021 INTRODUCTION: Bile acids have been implicated in the development of colorectal neoplasms by epidemiological, clinical and experimental studies. In rats, ursodeoxycholie acid (URSO) has a protective effect against colon cancer, while lithocholic acid (LCA) has tumorigenic properties. To evaluate the possible mechanisms by which URSO and LCA affect colon carcinogenesis, we assessed their effect on the the cell cycle and programmed cell death (apoptosis) in colon adenocarcinoma cells. METHODS: The HT29 colon cancer cell line was treated with both bile acids for 24, 48, and 72 hrs; their concentrations ranged from 50 ftM to 1 mM. URSO & LCA were dissolved in ethanol; LCA tended to fall out of solution at the higher concentrations. The cell cycle distribution was determined by flow cytometry after propidium iodide staining. Apoptosis was evaluated by a) the amount of sub-diploid DNA content by flow cytometry, b) acridine orange staining and c) a modified Wright staining. RESULTS: 1) URSO decreased the proportion of cells in the S phase and increased those in GJM. This effect, which was concentration- and time-dependent, suggests a block of transition into and through the M phase. 2) LCA increased the proportion of cells in Go/G~ and decreased those in the S and G J M phases of the cell cycle. This finding suggests either quiescence of the cells or a block into entering the S phase. 3) URSO, at concentrations ranging from 100 #M to 0.1 raM, induced apoptosis, as determined by all three methods. Apoptosis became apparent at 48 hr and maximal at 72 hrs. LCA did not exhibit any of these effects at all time points and concentrations. CONCLUSIONS: URSO and LCA have a differential effect on the cell cycle, while only URSO induced apoptosis. These differences may contribute to their different effects on colon carcinogenesis. It is conceivable that the apoptosis induced by URSO may play a role in its chemopreventive effect in colon cancer.