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BCL-2 family proteins are expressed by pancreatic adenocarcinoma
GASTROFA~I'EROLOGYVol. 114, No. 4
A638 AGA ABSTRACTS G2626 WITHDRAWN.
• G2627 SHOULD THE SAME RULES OF COLONIC POLYP SURVEILLANCE APPLY TO PATIENTS WITH COLORECTAL CANCER AI:I'ER CURATIVE RESECTION? A. P. Manocha, R. E. Weesner. Veteran's Administration Medical Center, Cincinnati, OH. BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. However, recent literature seems to confirm the adenoma to carcinoma sequence, and supports that the time for this transformation to occur may be as long as 10 years. This new data has formed the cornerstone of the new CRC screening guidelines for average risk and high risk patients recently published in Gastroenterology. However, it is still unclear how to survey patients with a history of CRC after curative resection. It is also unclear whether these patients form colon polyps faster or whether these polyps progress to cancer at a different rate. The previous guidelines suggested follow-up colonoscopy after resection at 6 months to 1 year, followed by colonoscopy annually for two years and if negative then every 3 years. The current guidelines in February, 1997 Gastroenterology stated that if patients had a preoperative colonoscopy, then surveillance should be offered at 3 years and if negative every 5 years (similar to the adenoma follow-up). AIM: To determine if surveillance prior to 3 years in patients with a history of CRC after curative resection provides any significant advantage. METHOD: This was a retrospective review of all the patients with CRC who underwent a colonic resection with curative intent at the Veteran's Administration Medical Center in Cincinnati, Ohio (VAMC, Cin) from January 1987 to July 1996. RESULTS: In total, 76 patients were found, out of which 51 patients had a follow-up colonoscopy performed from 6 to 24 months after resection. 37 patients had both the index and follow-up colonoscopy and 2 patients had an index flexible sigmoidoscopy and followup colonoscopy performed at the VAMC, Cin; whereas, 12 patients had their initial exam elsewhere and follow up colonoscopy at VAMC, Cin. Of the 51 patients, there were 15 (29%) tubular or tubulovillous adenomas, 2 (4%) polyps with focal high grade dysplasia, 4 (8%) polyps with no pathology available, 2 (4%) recurrent malignancy, and 22 (43%) normal exams. In addition, there were 8 (16%) colon cancer recurrences at distant sites with an average time to recurrence of 19.5 months. Overall, within 2 years of the initial curative resection, 19 (37%) patients had significant findings on colonoscopy. Both of the recurrent colon cancers were rectosigmoid with the initial malignancy being rectosigmoid as well. In addition, 11 (65%) of the polyps were left sided. CONCLUSION: Based on this retrospective study, there was a high incidence of significant pathology within the 3 year surveillance period after curative resection for CRC that is currently recommended. There was a left sided predominance to these lesions with 13/19 (68%) of the lesions within the reach of a flexible sigmoidoscope. Perhaps, consideration should be given to retaining the initial 1 year colonoscopy or flexible sigmoidoscopy after resection, to rule out metachronous colonic lesions. G2628 BCL-2 FAMILY PROTEINS ARE EXPRESSED BY PANCREATIC
ADENOCARCINOMA. R.Manta, M.Piantelli**, G.Costamagna. M.Mutignani, F.M.Vecchio*, F.Crucitti. Department of Surgery and Pathology*, Catholic University, Rome. Department of Pathology**, University of Chieti, Italy. Back~,round: Many genes codify proteins for apoptosis regulation. The gene's expression of bcl2 proteins' family (anti-agonist: bcl2, mcll, bcl xl; agonist: bax, bcl xs) is an important group of these proteins. Some of these have been recently investigated as possible tumoral markers. The aim of this study was to investigate the presence of bcl2 proteins family and their possible correlation with apoptosis in pancreatic adenocarcinoma. Materials and methods: Eight patients with pancreatic adenocarcinoma who had undergone pancreatoduodenectomy were studied. Patients' characteristics are shown in the table below. Immunohistochemical colorations were performed on tissue slices from paraffin inclusions. The presence of p53 protein was also investigated. The evidence of bax, bclx s/1 and p53 proteins was obtained by the strepto-biotin-peroxidase method. Evidence of bcl2 and mcll proteins was obtained with the same procedure and previous microwave heating for 10 minutes. The expression of bcl 2, mcl 1, bax and bclx s/1 was measured subjectively according to the intensity of coloration. To determine the percentage of apoptosis, fragmentation of DNA was performed in situ by marking the break points of DNA by terminal deoxynucleotidyl-transferase mediated-biotin nick end labeling (TUNEL). P53 and apoptosis were expressed as percentage of positive cells. Results:
Sex/ neoplasm TNM GRAD. Bcl 2 mcl I bax bclx p 53 apoptosi~ age ~cm s/l % % F/63 2.5 T2N0 G3 +++ +++ +++ 78 1.0 F/68 3.0 T2N0 G3 ++ +++ +++ 42 1.5 F/70 3.5 T2N1 G3 + - +++ +++ +++ 0 2.5 M/61 3.5 T2N1 G2 +++ ++ ++ 13 1.5 M/53 3.0 T2N0 G3 ++ +++ +++ 1 4.0 M/75 3.5 T2N0 G3 +++ +++ +++ 2 2.0 M/56 4.5 T3N1 G3 + + ++ 68 12.0 F/64 2.0 T2N1 G3 +++ + ++ 89 1.5
Conclusions: This is the first report of the presence of some of the bcl2 family proteins in pancreatic adenocarcinoma. Absence of bcl2 protein expression was unexpected: bcl2 apoptogenic function in this setting could be performed by mcll, which was always present in this series. The percentage of apoptosis observed was the same as reported in the literature. These results need to be confirmed by analysis of large number of resected pancreatic adenocarcinoma specimen and by further evaluation with quantitative protein analysis techniques, like Western-blot and ELISA. • G2629
NEUROTENSIN AND NONPEPTIDE NEUROTENSIN RECEPTOR ANTAGONIST CONTROL HUMAN COLON CANCER CELL GROWTH IN CELL CULTURE AND IN CELLS XENOGRAFTED INTO NUDE MICE. J.J. Maoret, Y. Anini, C. Rouyer-Fessard, Paris, France; D. Gully, Toulouse, France; M. Laburthe, Pads, France. The intestine is a large endocrine organ but the dependence of colon cancer on hormones is still unknown. We demonstrate here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCTll6 and CI.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells that do not express NTR1. In SW480 cells neurotensin is active in the 10-1°-10-6 M concentration range (EDs0 = 0.47 riM) while the neurotensin (1-11) fragment is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692 which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54~tmoles/kg/24h) by osmotic pumps to nude mice that have been xenografted with SW480 ceils, results in a significant increase of tumor volume i.e. up to 255% of control at day 20 of treatment. Conversely, daily intraperitoneal injections of SR 48692 alone (17 lamoles/kg/24h) are able to reduce the development of tumors formed by xenografting SW480 cells in nude mice. A significant reduction of tumor volume of 35% is observed. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. These results support that colon cancer growth may be dependent on blood-borne neurotensin and suggest that nonpeptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies for colon cancer. • G2630 R~-~TANCE TO BUTYRATF~INDUCED APOPTOSIS AND DIFFERENTIATION DURING SPONTANEOUS CACO-2 CELL DIFFERENTIATION. JM Mariadason, KL Rickard, PR Gibson. Department of Medicine, University of Melbourne, The Royal Melbourne Hospital, Victoria, Australia. The short-chain fatty acid, butyrate, is a potent inducer of cell differentiation and apoptosis in colon cancer cell lines in vitro. Caco-2 cells undergo spontaneous cell differentiation in culture. Whether the response of colonic epithelial cells to butyrate varies according to their basal differentiation status is unknown. METHODS: Caco-2 cells of varying degrees of spontaneous differentiation (0-21 days post confluence) were treated with butyrate (2 raM) for 72 hours. Alkaline phosphatase (ALP), maltase, dipeptidypeptidase IV (DPPIV) and uPA activities were measured spectrophotometrically. CEA expression was measured by RIA, and apoptosis and IL-8 secretion by ELISA. Butyrate consumption by Caco-2 cells was measured by gas chromatography. RESULTS: Caco-2 cells underwent spontaneous differentiation in culture as shown by the increased activities of ALP, maltase, DPPIV and CEA. ALP activity increased spontaneously by 312±38%*, maltase activity by 446±43%*, DPPIV activity by 338 +69%* and CEA expression by 555 ± 163% after 21 days in culture (*P<0.05 compared to day 0, t test). Treatment of Caco-2 cells at confluence or 2-7 days thereafter with butyrate resulted in maximal stimulation of ALP (569 -+ 165%* of control, *P<0.05, t test) and uPA (218 -+ 116%*) activity, IL-8 secretion (1352 _+ 166%*), and apoptosis (734-+ 233%*). Butyrate, however, failed to significantly increase ALP (12-+7%, P=NS), uPA (-7±6%) IL-8 (202±31%) and apoptosis (95 ± 28%) when added to Caco-2 cells 14 days after confluence and thereafter. Gas chromatography studies revealed that this resistance to butyrate was not due to reduced butyrate consumption by Caco-2 cells. CONCLUSIONS: The response of colonic epithelial cells to butyrate varies according to their basal differentiation status. These observations have potential importance for understanding the intracellular mechanisms of butyrate's anti-tumour effects.
A638 AGA ABSTRACTS G2626 WITHDRAWN.
• G2627 SHOULD THE SAME RULES OF COLONIC POLYP SURVEILLANCE APPLY TO PATIENTS WITH COLORECTAL CANCER AI:I'ER CURATIVE RESECTION? A. P. Manocha, R. E. Weesner. Veteran's Administration Medical Center, Cincinnati, OH. BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer death in the United States. However, recent literature seems to confirm the adenoma to carcinoma sequence, and supports that the time for this transformation to occur may be as long as 10 years. This new data has formed the cornerstone of the new CRC screening guidelines for average risk and high risk patients recently published in Gastroenterology. However, it is still unclear how to survey patients with a history of CRC after curative resection. It is also unclear whether these patients form colon polyps faster or whether these polyps progress to cancer at a different rate. The previous guidelines suggested follow-up colonoscopy after resection at 6 months to 1 year, followed by colonoscopy annually for two years and if negative then every 3 years. The current guidelines in February, 1997 Gastroenterology stated that if patients had a preoperative colonoscopy, then surveillance should be offered at 3 years and if negative every 5 years (similar to the adenoma follow-up). AIM: To determine if surveillance prior to 3 years in patients with a history of CRC after curative resection provides any significant advantage. METHOD: This was a retrospective review of all the patients with CRC who underwent a colonic resection with curative intent at the Veteran's Administration Medical Center in Cincinnati, Ohio (VAMC, Cin) from January 1987 to July 1996. RESULTS: In total, 76 patients were found, out of which 51 patients had a follow-up colonoscopy performed from 6 to 24 months after resection. 37 patients had both the index and follow-up colonoscopy and 2 patients had an index flexible sigmoidoscopy and followup colonoscopy performed at the VAMC, Cin; whereas, 12 patients had their initial exam elsewhere and follow up colonoscopy at VAMC, Cin. Of the 51 patients, there were 15 (29%) tubular or tubulovillous adenomas, 2 (4%) polyps with focal high grade dysplasia, 4 (8%) polyps with no pathology available, 2 (4%) recurrent malignancy, and 22 (43%) normal exams. In addition, there were 8 (16%) colon cancer recurrences at distant sites with an average time to recurrence of 19.5 months. Overall, within 2 years of the initial curative resection, 19 (37%) patients had significant findings on colonoscopy. Both of the recurrent colon cancers were rectosigmoid with the initial malignancy being rectosigmoid as well. In addition, 11 (65%) of the polyps were left sided. CONCLUSION: Based on this retrospective study, there was a high incidence of significant pathology within the 3 year surveillance period after curative resection for CRC that is currently recommended. There was a left sided predominance to these lesions with 13/19 (68%) of the lesions within the reach of a flexible sigmoidoscope. Perhaps, consideration should be given to retaining the initial 1 year colonoscopy or flexible sigmoidoscopy after resection, to rule out metachronous colonic lesions. G2628 BCL-2 FAMILY PROTEINS ARE EXPRESSED BY PANCREATIC
ADENOCARCINOMA. R.Manta, M.Piantelli**, G.Costamagna. M.Mutignani, F.M.Vecchio*, F.Crucitti. Department of Surgery and Pathology*, Catholic University, Rome. Department of Pathology**, University of Chieti, Italy. Back~,round: Many genes codify proteins for apoptosis regulation. The gene's expression of bcl2 proteins' family (anti-agonist: bcl2, mcll, bcl xl; agonist: bax, bcl xs) is an important group of these proteins. Some of these have been recently investigated as possible tumoral markers. The aim of this study was to investigate the presence of bcl2 proteins family and their possible correlation with apoptosis in pancreatic adenocarcinoma. Materials and methods: Eight patients with pancreatic adenocarcinoma who had undergone pancreatoduodenectomy were studied. Patients' characteristics are shown in the table below. Immunohistochemical colorations were performed on tissue slices from paraffin inclusions. The presence of p53 protein was also investigated. The evidence of bax, bclx s/1 and p53 proteins was obtained by the strepto-biotin-peroxidase method. Evidence of bcl2 and mcll proteins was obtained with the same procedure and previous microwave heating for 10 minutes. The expression of bcl 2, mcl 1, bax and bclx s/1 was measured subjectively according to the intensity of coloration. To determine the percentage of apoptosis, fragmentation of DNA was performed in situ by marking the break points of DNA by terminal deoxynucleotidyl-transferase mediated-biotin nick end labeling (TUNEL). P53 and apoptosis were expressed as percentage of positive cells. Results:
Sex/ neoplasm TNM GRAD. Bcl 2 mcl I bax bclx p 53 apoptosi~ age ~cm s/l % % F/63 2.5 T2N0 G3 +++ +++ +++ 78 1.0 F/68 3.0 T2N0 G3 ++ +++ +++ 42 1.5 F/70 3.5 T2N1 G3 + - +++ +++ +++ 0 2.5 M/61 3.5 T2N1 G2 +++ ++ ++ 13 1.5 M/53 3.0 T2N0 G3 ++ +++ +++ 1 4.0 M/75 3.5 T2N0 G3 +++ +++ +++ 2 2.0 M/56 4.5 T3N1 G3 + + ++ 68 12.0 F/64 2.0 T2N1 G3 +++ + ++ 89 1.5
Conclusions: This is the first report of the presence of some of the bcl2 family proteins in pancreatic adenocarcinoma. Absence of bcl2 protein expression was unexpected: bcl2 apoptogenic function in this setting could be performed by mcll, which was always present in this series. The percentage of apoptosis observed was the same as reported in the literature. These results need to be confirmed by analysis of large number of resected pancreatic adenocarcinoma specimen and by further evaluation with quantitative protein analysis techniques, like Western-blot and ELISA. • G2629
NEUROTENSIN AND NONPEPTIDE NEUROTENSIN RECEPTOR ANTAGONIST CONTROL HUMAN COLON CANCER CELL GROWTH IN CELL CULTURE AND IN CELLS XENOGRAFTED INTO NUDE MICE. J.J. Maoret, Y. Anini, C. Rouyer-Fessard, Paris, France; D. Gully, Toulouse, France; M. Laburthe, Pads, France. The intestine is a large endocrine organ but the dependence of colon cancer on hormones is still unknown. We demonstrate here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCTll6 and CI.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells that do not express NTR1. In SW480 cells neurotensin is active in the 10-1°-10-6 M concentration range (EDs0 = 0.47 riM) while the neurotensin (1-11) fragment is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692 which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54~tmoles/kg/24h) by osmotic pumps to nude mice that have been xenografted with SW480 ceils, results in a significant increase of tumor volume i.e. up to 255% of control at day 20 of treatment. Conversely, daily intraperitoneal injections of SR 48692 alone (17 lamoles/kg/24h) are able to reduce the development of tumors formed by xenografting SW480 cells in nude mice. A significant reduction of tumor volume of 35% is observed. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. These results support that colon cancer growth may be dependent on blood-borne neurotensin and suggest that nonpeptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies for colon cancer. • G2630 R~-~TANCE TO BUTYRATF~INDUCED APOPTOSIS AND DIFFERENTIATION DURING SPONTANEOUS CACO-2 CELL DIFFERENTIATION. JM Mariadason, KL Rickard, PR Gibson. Department of Medicine, University of Melbourne, The Royal Melbourne Hospital, Victoria, Australia. The short-chain fatty acid, butyrate, is a potent inducer of cell differentiation and apoptosis in colon cancer cell lines in vitro. Caco-2 cells undergo spontaneous cell differentiation in culture. Whether the response of colonic epithelial cells to butyrate varies according to their basal differentiation status is unknown. METHODS: Caco-2 cells of varying degrees of spontaneous differentiation (0-21 days post confluence) were treated with butyrate (2 raM) for 72 hours. Alkaline phosphatase (ALP), maltase, dipeptidypeptidase IV (DPPIV) and uPA activities were measured spectrophotometrically. CEA expression was measured by RIA, and apoptosis and IL-8 secretion by ELISA. Butyrate consumption by Caco-2 cells was measured by gas chromatography. RESULTS: Caco-2 cells underwent spontaneous differentiation in culture as shown by the increased activities of ALP, maltase, DPPIV and CEA. ALP activity increased spontaneously by 312±38%*, maltase activity by 446±43%*, DPPIV activity by 338 +69%* and CEA expression by 555 ± 163% after 21 days in culture (*P<0.05 compared to day 0, t test). Treatment of Caco-2 cells at confluence or 2-7 days thereafter with butyrate resulted in maximal stimulation of ALP (569 -+ 165%* of control, *P<0.05, t test) and uPA (218 -+ 116%*) activity, IL-8 secretion (1352 _+ 166%*), and apoptosis (734-+ 233%*). Butyrate, however, failed to significantly increase ALP (12-+7%, P=NS), uPA (-7±6%) IL-8 (202±31%) and apoptosis (95 ± 28%) when added to Caco-2 cells 14 days after confluence and thereafter. Gas chromatography studies revealed that this resistance to butyrate was not due to reduced butyrate consumption by Caco-2 cells. CONCLUSIONS: The response of colonic epithelial cells to butyrate varies according to their basal differentiation status. These observations have potential importance for understanding the intracellular mechanisms of butyrate's anti-tumour effects.