- Email: [email protected]
Gap junction protein connexin 32 and c-H-ras expression in rat gastric mucosa in carcinogenesis
A512
AGA ABSTRACTS
P53 IN COLORECTAL CANCER: TISSUE AND SERUM ANALYSIS. M. Mueller1, E.-M Afini-Awani I, Th. Lehnert 2, W. Stremmel 1 and P.R-Ga--'~. 1Department of Internal Medicine IV, 2Department of Surgery, University of Heidelberg, Heidelberg, Germany Reported molecular genetic abnormalities in colorectal carcinoma involve tumor-suppressor genes that undergo inactivation (e.g. apc, mcc, dcc, p53) and dominant-acting 0ncogenes (e.g. ras, src, and myc). We have investigated the p53 antigen-expression in colorectal cancers and its correlation with the occurrence of antip53 serum antibodies. Eighty-nine patients who underwent surgical resection of colorectal cancer at the Department of Surgery of the University of Heidelberg during 1990-1994 were studied. P53 Jn tumor tissue was detected by western blotting using the rabbit anti-p53 antiserum HZp53R (Hepatology 1993, 18:559). In 17 of the 89 patients serum was available for serological analysis for anti-p53 antibodies. Sera were tested by a western blot assay with recombinant p53 expressed in E. coli as antigen. A serological follow-up was performed for up to two years. Of a total of 89 tumors examined, p53 was detected in 34 (38.2 %). Anti-p53 antibodies could be detected in 7 out of 17 patients (41.2 %). In 6 of 17 patients both p53 antigen expression in the tumor tissue and presence of anti-p53 antibodies could be demonstrated. In 6/10 tumors positive for p53 antigen an anti-p53 antibody response developed; in 4/10 no anti-p53 antibodies were detectable. In 1 of 7 patients with a p53-negative tumor anti-p53 antibodies were present. The anti-p53 antibody status remained stable over an observation period of 2 years, a change of the antibody status from positive to negative or vice Versa was not seen. Increased steady state levels of p53 indicative for p53 gene mutations can be identified by immunoblotting in about 40 % of colorectal carcinomas. Only 60 % of these p53 alterations induce an anti-p53 serum antibody response. However the absence of p53 antibodies in 40 % of p53-positive tumors does not exclude involvement of p53 in tumorigenesis of colorectal carcinoma,
CARCINOGENESIS OF BARRETT'S ESOPHAGUS: FREQUENT IMPLICATION OF THE P53 TUMOUR SUPPRESSOR GENE, NO IMPLICATION OF THE K-RAS ONCOGENE. F. Muzeaul, J.-F. Fl~joul,3, C. Lagorcel, R. Hamelin2, D. Henin3, F. Potetl. 11NSERM U410, Facult~ Xavier Bichat, 21nstitut Curie, Paris. 3H6pital Beaujon; Clichy. France. The carcinogenesis of Barrett's esophagus follows a sequence metaplasia- dysplasia - cancer. Data available in the literature suggest that the p53 tumor suppressor gene is implicated during this process. Few data is available on K-ras oncogene mutation during Barrett's carcinogenesis. The aim of this study was to establish the frequency and timing of K-ras and p53.gene mutations during the neoplastic transformation of Barrett's esophagus. Frozen specimens of 28 surgically resected Barrett's adenocarcinomas were included. The exon 1 (codons 12 and 13) of K-ras was studied by PCR-RFLP, p53 was studied by PCR-DGGE (exons 5 to 8) and immunohistochemistry. Both malignant tissue and surrounding Barrett's mucosa were studied. No K-ras mutation was found, neither in the cancer nor in the surrounding Barrett's mucosa. Twenty seven cancers (96%) showed an abnormal pattern on p53 DGGE (26 cases) and/or an abnormal expression of p53 protein (22 cases), two features highly suggestive of p53 gene mutation. These changes were often present in dysplastic areas around the tumor, but were absent in non dysplastic Barrett's mucosa. In conclusion, the K-ras oncogene is not involved in the carcinogenesis of Barrett's esophagus, in contrast to the p53 tumor suppressor gene that shows frequent and early alteration. Therefore, the carcinogenesis of Barrett's esophagus appears as having common features with that observed in ulcerative colitis, an other preneoplastic chronic inflammatory disease of the digestive tract, distinct from the process observed in colonic adenomas.
GASTROENTEROLOGY, VoI. IO8, No. 4
: • ENDOSCOPIC PROCEDURES REDUCE THE RISK OF COLORECTAL CANCER. Astrid D. Miiller, Amnon Sonnenberg. VA Medical Center and The Medical College of Wisconsin, Milwaukee, WI. Background: Although several clinical and epidemiol0gic studies suggest that timely diagnostic procedures of the large bowelmay reduce the development colorectal cancer, the evidence for this relationship is primarily circumstantial, Methods: A case-control study was conducted among US military veterans to investigate whether endoscopic procedures of the large bowel, such as flexible sigmoidoscopy, colonoscopy, and polypectomy, were performed in the period preceding cancer diagnosis less frequently in cases than controls. A total of 8,722 patients with colon cancer and 7,629 patients with rectal cancer were extracted from the records of the Department of Veterans Affairs (VA) between 1988 and 1993. Control subjects were selected randomly from the main file of the identical fiscal year when the case was first diagnosed with colorectal cancer, each control subject being matched by age, sex, and race to his/her corresponding case subject. The numbers of endoscopic procedures and the number of patients with at least one procedure prior to cancer diagnoses were compared between the case and the control population by conditional logistic regression calculating an odds ratio and its 95 percent confidence interval for each diagnostic code. Results: Endoscopic procedures of the large bowel reduced both, the risk of colon cancer (OR=0.51, 95% confidence interval: 0.44-0.58) and the risk of rectal cancer (0.55, 0.47-0.64). Their protective effect lasted for 6 years. The protective influence of inpatient and outpatient procedures was similar. No statistically significant difference could be established between the variOUSr.procedural codes. After adjustment for the joint contribution of various procedures, colonoscopy appeared to be associated with strongest protective influence against colon cancer (0.47, 0.37-0.58), followed by flexible sigmoidoscopy (0.56, 0.46-0.67) and polypectomy (0.59, 0.45-0.78). In rectal cancer, polypectomy was associated with most risk reduction (0.48, 0.35-0.66), while colonoscopy (0.61, 0.48-0.77) and flexible sigmoidoscopy (0,61, 0.490.75) appeared equally effective. Conclusion: Flexible sigmoidoscopy, colonoscopy, and polypectomy reduce the future risk of developing colon and rectal cancer by 50%, their protective influence lasting 6 years.
GAP JUNCTION PROTEIN CONNEXIN 32 and c-H-ras EXPRESSION IN RAT GASTRIC MuCOSA IN CARCINOGENESIS. A.Nagahara, S.Watanabe, K.Endo, MHirose, N. Sato D~stroenterology, Juntendo Univ. School of Med. Tokyo, Japan Gap junction-mediated intercellular communication plays a pivotal role in cellproliferation differentiation and carcinogenesis. Although, in cancer cells, the gap junction is generally lost, the'role of gap junction in the process of carcinogenesis is still unclear. Previous studies showed that continuous administration of N-Methyl-N'-Nitro-Nitrosoguanidine (MNNG) induced atrophic gastritis was followed by carcinoma We reported that the length of connexin 32 (major gap junction protein,Cx32) positive mucosa was reduced and the intercellular communication between mucosal ceils would be expected to be decreased in the atrophic gastritis. This study was performed to investigate the relationship among expression of Cx 32, c-H-ras oncoprotein and cell proliferation during the process of gastric carcinogenesis using MNNG treated rat model. METHODS. Five control rats were given tap water during the experiment. Five rats were given 100~g/ml of MNNG in water for the initial 8 weeks and six rats were given MNNG for 30 weeks then were given water for 2 weeks. BrdU (100mg/kg) was injected 1 h before sacrifice. Subsequently, the stomach was removed and longitudinal sections were prepared and stained for Cx 32, c-H-ras, bematoxylin-eosm and BrdU. Morphometric analysis was performed using an image analyzer. RESULTS. 1) MNNG treatment decreased the number of pyloric glands in the mucosal layer in 8 weeks(20~4 glands/mm p<0.05, 27 +-2 in control) and 2 of 6 rats in 30 week-treatment occurred adenocarcinoma. 2) Proliferative zones identified by BrdU-posltive staining were located in the deep part of mucosa in controls(32-----4 ~z m) and were elongated in the MNNG- treated group(58 +- 14 tz m)(p<0.05). BrdU positive cells were detected diffusely' throughout the carcinoma portion. 3) Cx 32 was expressed strongly in the surface mucosa and the length of Cx32 positive mucosa was shorter than controls(176+- 16 # m) in the MNNG-treated gastritis group(154--+ 7 ~ m)(p<0.05). Cx 32 was not expressed in carcinoma portion. 4) c-H-ras was not detected in normal controls. In MNNG-treated gastritis group, c-H-ras was detected only in deep part of mucosa, where correspondent to proliferative zone. It was observed more abundantly in both carcinoma and non-carcinoma lesion of 30-weeks treated group. DISCUSSION. The expression of Cx 32 was reduced in atrophic gastritis and disappeared completely in gastric carcinoma in this model. These results suspected that intercellular communication between mucosal ceils was decreased even in atrophic gastritis. With the evidence that c-H-ras was already expressed in atrophic gastritis, atrophic gastritis induced by MNNG at 8 week is a key step of gastric carcinogenesis and the loss of cell-cell communication plays an important role in this mechanism.
AGA ABSTRACTS
P53 IN COLORECTAL CANCER: TISSUE AND SERUM ANALYSIS. M. Mueller1, E.-M Afini-Awani I, Th. Lehnert 2, W. Stremmel 1 and P.R-Ga--'~. 1Department of Internal Medicine IV, 2Department of Surgery, University of Heidelberg, Heidelberg, Germany Reported molecular genetic abnormalities in colorectal carcinoma involve tumor-suppressor genes that undergo inactivation (e.g. apc, mcc, dcc, p53) and dominant-acting 0ncogenes (e.g. ras, src, and myc). We have investigated the p53 antigen-expression in colorectal cancers and its correlation with the occurrence of antip53 serum antibodies. Eighty-nine patients who underwent surgical resection of colorectal cancer at the Department of Surgery of the University of Heidelberg during 1990-1994 were studied. P53 Jn tumor tissue was detected by western blotting using the rabbit anti-p53 antiserum HZp53R (Hepatology 1993, 18:559). In 17 of the 89 patients serum was available for serological analysis for anti-p53 antibodies. Sera were tested by a western blot assay with recombinant p53 expressed in E. coli as antigen. A serological follow-up was performed for up to two years. Of a total of 89 tumors examined, p53 was detected in 34 (38.2 %). Anti-p53 antibodies could be detected in 7 out of 17 patients (41.2 %). In 6 of 17 patients both p53 antigen expression in the tumor tissue and presence of anti-p53 antibodies could be demonstrated. In 6/10 tumors positive for p53 antigen an anti-p53 antibody response developed; in 4/10 no anti-p53 antibodies were detectable. In 1 of 7 patients with a p53-negative tumor anti-p53 antibodies were present. The anti-p53 antibody status remained stable over an observation period of 2 years, a change of the antibody status from positive to negative or vice Versa was not seen. Increased steady state levels of p53 indicative for p53 gene mutations can be identified by immunoblotting in about 40 % of colorectal carcinomas. Only 60 % of these p53 alterations induce an anti-p53 serum antibody response. However the absence of p53 antibodies in 40 % of p53-positive tumors does not exclude involvement of p53 in tumorigenesis of colorectal carcinoma,
CARCINOGENESIS OF BARRETT'S ESOPHAGUS: FREQUENT IMPLICATION OF THE P53 TUMOUR SUPPRESSOR GENE, NO IMPLICATION OF THE K-RAS ONCOGENE. F. Muzeaul, J.-F. Fl~joul,3, C. Lagorcel, R. Hamelin2, D. Henin3, F. Potetl. 11NSERM U410, Facult~ Xavier Bichat, 21nstitut Curie, Paris. 3H6pital Beaujon; Clichy. France. The carcinogenesis of Barrett's esophagus follows a sequence metaplasia- dysplasia - cancer. Data available in the literature suggest that the p53 tumor suppressor gene is implicated during this process. Few data is available on K-ras oncogene mutation during Barrett's carcinogenesis. The aim of this study was to establish the frequency and timing of K-ras and p53.gene mutations during the neoplastic transformation of Barrett's esophagus. Frozen specimens of 28 surgically resected Barrett's adenocarcinomas were included. The exon 1 (codons 12 and 13) of K-ras was studied by PCR-RFLP, p53 was studied by PCR-DGGE (exons 5 to 8) and immunohistochemistry. Both malignant tissue and surrounding Barrett's mucosa were studied. No K-ras mutation was found, neither in the cancer nor in the surrounding Barrett's mucosa. Twenty seven cancers (96%) showed an abnormal pattern on p53 DGGE (26 cases) and/or an abnormal expression of p53 protein (22 cases), two features highly suggestive of p53 gene mutation. These changes were often present in dysplastic areas around the tumor, but were absent in non dysplastic Barrett's mucosa. In conclusion, the K-ras oncogene is not involved in the carcinogenesis of Barrett's esophagus, in contrast to the p53 tumor suppressor gene that shows frequent and early alteration. Therefore, the carcinogenesis of Barrett's esophagus appears as having common features with that observed in ulcerative colitis, an other preneoplastic chronic inflammatory disease of the digestive tract, distinct from the process observed in colonic adenomas.
GASTROENTEROLOGY, VoI. IO8, No. 4
: • ENDOSCOPIC PROCEDURES REDUCE THE RISK OF COLORECTAL CANCER. Astrid D. Miiller, Amnon Sonnenberg. VA Medical Center and The Medical College of Wisconsin, Milwaukee, WI. Background: Although several clinical and epidemiol0gic studies suggest that timely diagnostic procedures of the large bowelmay reduce the development colorectal cancer, the evidence for this relationship is primarily circumstantial, Methods: A case-control study was conducted among US military veterans to investigate whether endoscopic procedures of the large bowel, such as flexible sigmoidoscopy, colonoscopy, and polypectomy, were performed in the period preceding cancer diagnosis less frequently in cases than controls. A total of 8,722 patients with colon cancer and 7,629 patients with rectal cancer were extracted from the records of the Department of Veterans Affairs (VA) between 1988 and 1993. Control subjects were selected randomly from the main file of the identical fiscal year when the case was first diagnosed with colorectal cancer, each control subject being matched by age, sex, and race to his/her corresponding case subject. The numbers of endoscopic procedures and the number of patients with at least one procedure prior to cancer diagnoses were compared between the case and the control population by conditional logistic regression calculating an odds ratio and its 95 percent confidence interval for each diagnostic code. Results: Endoscopic procedures of the large bowel reduced both, the risk of colon cancer (OR=0.51, 95% confidence interval: 0.44-0.58) and the risk of rectal cancer (0.55, 0.47-0.64). Their protective effect lasted for 6 years. The protective influence of inpatient and outpatient procedures was similar. No statistically significant difference could be established between the variOUSr.procedural codes. After adjustment for the joint contribution of various procedures, colonoscopy appeared to be associated with strongest protective influence against colon cancer (0.47, 0.37-0.58), followed by flexible sigmoidoscopy (0.56, 0.46-0.67) and polypectomy (0.59, 0.45-0.78). In rectal cancer, polypectomy was associated with most risk reduction (0.48, 0.35-0.66), while colonoscopy (0.61, 0.48-0.77) and flexible sigmoidoscopy (0,61, 0.490.75) appeared equally effective. Conclusion: Flexible sigmoidoscopy, colonoscopy, and polypectomy reduce the future risk of developing colon and rectal cancer by 50%, their protective influence lasting 6 years.
GAP JUNCTION PROTEIN CONNEXIN 32 and c-H-ras EXPRESSION IN RAT GASTRIC MuCOSA IN CARCINOGENESIS. A.Nagahara, S.Watanabe, K.Endo, MHirose, N. Sato D~stroenterology, Juntendo Univ. School of Med. Tokyo, Japan Gap junction-mediated intercellular communication plays a pivotal role in cellproliferation differentiation and carcinogenesis. Although, in cancer cells, the gap junction is generally lost, the'role of gap junction in the process of carcinogenesis is still unclear. Previous studies showed that continuous administration of N-Methyl-N'-Nitro-Nitrosoguanidine (MNNG) induced atrophic gastritis was followed by carcinoma We reported that the length of connexin 32 (major gap junction protein,Cx32) positive mucosa was reduced and the intercellular communication between mucosal ceils would be expected to be decreased in the atrophic gastritis. This study was performed to investigate the relationship among expression of Cx 32, c-H-ras oncoprotein and cell proliferation during the process of gastric carcinogenesis using MNNG treated rat model. METHODS. Five control rats were given tap water during the experiment. Five rats were given 100~g/ml of MNNG in water for the initial 8 weeks and six rats were given MNNG for 30 weeks then were given water for 2 weeks. BrdU (100mg/kg) was injected 1 h before sacrifice. Subsequently, the stomach was removed and longitudinal sections were prepared and stained for Cx 32, c-H-ras, bematoxylin-eosm and BrdU. Morphometric analysis was performed using an image analyzer. RESULTS. 1) MNNG treatment decreased the number of pyloric glands in the mucosal layer in 8 weeks(20~4 glands/mm p<0.05, 27 +-2 in control) and 2 of 6 rats in 30 week-treatment occurred adenocarcinoma. 2) Proliferative zones identified by BrdU-posltive staining were located in the deep part of mucosa in controls(32-----4 ~z m) and were elongated in the MNNG- treated group(58 +- 14 tz m)(p<0.05). BrdU positive cells were detected diffusely' throughout the carcinoma portion. 3) Cx 32 was expressed strongly in the surface mucosa and the length of Cx32 positive mucosa was shorter than controls(176+- 16 # m) in the MNNG-treated gastritis group(154--+ 7 ~ m)(p<0.05). Cx 32 was not expressed in carcinoma portion. 4) c-H-ras was not detected in normal controls. In MNNG-treated gastritis group, c-H-ras was detected only in deep part of mucosa, where correspondent to proliferative zone. It was observed more abundantly in both carcinoma and non-carcinoma lesion of 30-weeks treated group. DISCUSSION. The expression of Cx 32 was reduced in atrophic gastritis and disappeared completely in gastric carcinoma in this model. These results suspected that intercellular communication between mucosal ceils was decreased even in atrophic gastritis. With the evidence that c-H-ras was already expressed in atrophic gastritis, atrophic gastritis induced by MNNG at 8 week is a key step of gastric carcinogenesis and the loss of cell-cell communication plays an important role in this mechanism.