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Cell cycle abnormalities in small bowel tumors in Israel
A272 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
1546
1548
CELL CYCLE ABNORMALITIES IN SMALL BOWEL TUMORS IN ISRAEL. Leonor E. Trejo, Meital Shaked, Hanina Hibshoosh, Eyal Ozeri, Limor Baron, Hanna Strul, Marc Umanski, Fred M. Konikoff, Alfred I. Neugut, Nadir Arber, Tel-Aviv Med Ctr, Tel-Aviv, Israel; Columbia Univ, NewYork, NY.
REDUCED UNCOUPLING PROTEIN (UCP)-3 IN HUMAN CO· LONIC MALIGNANCY. Andrew 1. Bennett, Deborah A. Briggs, Lucina M. Jackson, Chris J. Hawkey, David A. White, Univ of Nottingham, Nottingham, United Kingdom; Univ Hosp Nottingham. Nottingham, United Kingdom. INTRODUCTION: Uncoupling proteins (UCPs) are mitochondrial transporters which dissipate proton gradients generated during electron transport, releasing energy as heat. UCP3 is expressed at high level in skeletal muscle where it is regulated by peroxisome proliferated-activated receptor (PPAR)a and may function in substrate selection. Given the emerging role of PP ARa and the importance of dietary fat in colonic neoplasia, we have investigated expression of UCP3 by Western Blotting and RT-PCT in normal and malignant human colonic mucosa. METHODS: We studied normal and malignant tissue obtained from 8 patients undergoing surgery for colorectal cancer. Samples were snap frozen and stored at -70°C. Human skeletal muscle was used as a positive control. UCP3 protein was separated using SDS-PAGE of tissue protein extract and visualised by Western Blotting with polyclonal anti-UCP3 antibodies, which showed a single band of approximately 35 kDa. Total RNA was extracted by the guanidine thiocyanate method and reverse transcribed. UCP3 mRNA was detected after PCR (28 cycles), using specific primers designed to identify expression of both the long and short forms of UCP3 mRNA. RESULTS: The long and short form of UCP3 mRNA and UCP protein were detected reliably in the positive control human skeletal muscle. UCP3 protein was detected at high levels in normal colonic mucosa, but substantially reduced or absent in 7 of the 8 paired tumour specimens. Only the short form of UCP3 mRNA was detectable in either normal or malignant colonic mucosa. CONCLUSIONS: UCP3 (short form) is expressed in normal human colon. It is substantially reduced in colorectal cancer tissue. Given its close relationship to PP ARa and potential role in fat metabolism, it may play an important role in the pathogenesis of malignant change.
Background and Purpose: When compared to the high incidence of adenomas (A) and adenocarcinomas (AC) in the human large bowel, the incidence of similar tumors in the small bowel is quite rare. Moreover, the reason that epithelial cells of the small bowel appear to be more resistant to the development of both benign and malignant tumors is not known. Recently, we reported on cell cycle abnormalities in small bowel tumors in the US (CEBP 12/99), however little is known regarding the molecular genetics of these tumors, outside of the US. Patients and Methods: A search of the records covering the past decade, from the Tel Aviv Medical Center computer files was carried out. Nine cases of A and 30 cases of AC were found. Epidemiology data was obtained and protein levels determined by immunohistochemistry. Results: 28 males and 11 females, mean age 69, were studied. 83% of the tumors were located in the duodenum. 4 were Duke stage A, 8 were B, 6 were C and 12 were D stage. The 3 year survival rate was 23%. Age, alcohol consumption and smoking were independent risk factors for SB tumors. Interestingly, 63% of the patients had a second primary mostly in the colon. 27% had received adjuvant therapy. Tumor incidence increased from 0.6/100,000 in 1990 to 1.2/100,000 in 1998.All of the normal mucosa samples expressed only p27 protein. About 30% of the tumors demonstrated a decreased expression of p27. Ki-67 was detected in 97% of the tumors and increased p53 expression was associated with tumor progression in 45% of the A and 68% of the AC (P<0.05). Increased expression of cyclin DI (40%), {3-catenine (30%) and Her-2/neu (33%) were closely associated and seen at approximately equal frequencies in A and AC. Advanced age and p53 expression were associated with poor survival, {3-catenine with poorly differentiated tumors, cyclin DI with advanced age, and decreased p27 expression was seen in alcohol consumers (p<0.05). Conclusions: 1. The epidemiology and molecular genetics of small bowel tumors in Israel are similar to that seen in the US. 2. This study confirms the increase in the incidence of SB tumors seen in the US over the last decade. 3. Small bowel tumors in Israel, like those in the US, frequently display abnormalities in the expression of cell cycle related proteins, as previously described for colorectal cancer. 4. This is the first report regarding increased expression of Her-2/neu and nuclear f3-catenine in 30% of small bowel tumors, which is similar to that seen in colorectal cancer. 1547 CHEMOPREVENTIVE ACTION OF RESVERATROL ON GASTRIC ADENOCARCINOMA CELLS VIA PKC-MEDIATED INDUCTION OF P53. Mary Jo Atten, Bashar M. Attar, Matthew Zopel, Oksana Holian, Cook County Hosp, Chicago, IL. Resveratrol, a phytoalexin present in grapes and wine, is a promising chemopreventive agent shown to block each stage of carcinogenesis. In gastric adenocarcinoma cells, resveratrol inhibits DNA synthesis and cellular proliferation, and inhibits cellular protein kinase C (PKC) activity (Gastro 116:A373,1999). We present evidence that resveratrol suppresses cellular proliferation through a p53-dependent pathway by inducing p2l cip, waf-\ expression. We identify a panel of PKC isozymes present in SNU-I cells and determine the relationship between changes in subcellular distribution of specific PKC isozymes and cellular expression of p53 and p21 elicited by resveratrol treatment. METHODS: Human gastric adenocarcinoma SNU-l cells (ATCC CRL-597l) were maintained in RPMI-I640 media supplemented with 10% FBS, and were treated with resveratrol in increasing concentrations and increasing exposure times. Cell cycle distribution was analyzed by flow cytometry. Expression of p21 and p53 products was performed by Western blot analysis following SDS-PAGE. Expression of PKC isozymes in subcellular fractions of SNU-I cells was also determined br Western blotting, while DNA synthesis was assessed by measurement of H-thymidine incorporation into live cells. RESULTS: Expression of the tumor suppressor gene product p53 increased with increasing concentrations or duration of exposure to resveratrol. Concomitant with the increase in p53, we observed increased expression of the cell cycle inhibitor p21 and arrest of SNU-I cells in GJG J phase. Maximal levels of both p53 and p21 were detected after 48 hours of cell treatment with resveratrol at which time the apoptotic index reached 50%. Expression of p53 and p21, cell cycle arrest, and apoptosis were preceded by a decrease in PKC activity and membrane-associated PKC-cS, and an increase in cytosolic PKC-a. CONCLUSIONS: Resveratrol-induced suppression of SNU-I cell proliferation follows a specific sequence of events: 1) inhibition of PKC activity, apparently resulting from the decrease in membraneassociated, phospholipid-activated PKC-cS and a simultaneous increase in cytosolic, inactive PKC-a; and 2) PKC signal-generated increase in expression of p53 and p21, culminating in SNU-I cell arrest in GJG\ phase. These findings suggest that p53-dependent transcriptional activity may be associated with resveratrol-triggered inhibition of tumor growth.
1549 DETERMINATION OF HER2 GENE PRODUCT (PI85) AND ADENYLOSUCCINATE LYASE (ASL) ACTIVITY IN HUMAN PRENEOPLASTIC AND NORMAL COLONIC MUCOSA. Antonio De Martino, Francesco Cetta, Giulia Montalto, Remo Vernillo, Filippo Carlucci, Antonella Tabucchi, Fausto Rosi, Enrico Marinello, Institute of Surg Clinics, Siena, Italy; Institute of Surg, Siena, Italy; Institute of Biochemistry and Enzymology, Siena, Italy. Background and Aims: The HER2 gene has been found amplified in various human carcinomas leading to overexpression of the encoded product, pI85 protein. Adenylosuccinate lyase (ASL, EC 4.3.2.2) is an enzyme involved in purine nucleotide biosynthesis an is known to increase in malignancies such as colorectal, breast, and prostate cancer. In order to evaluate the potential of ASL as a tumor marker, its activity was determined and compared with the expression of pI85 in pre-neoplastic and normal colonic mucosa. Materials and Methods: 18 patients (pts) (16 males, 2 females, mean age 55, range 22-75 years) were analyzed. 13 had tubular adenomas, 4 tubulovillous adenomas and 1 multiple polyps. p185 was determined by immunoenzymatic staining by the avidin-biotin peroxidase method, using murine 300G 9 antibody, that is specific for the extrcellular domain of human HER2 oncogene. ASL activity was analyzed in 23 specimens of normal mucosa and in 14 of 18 patients with adenomas. ASL activity was evaluated in dialyzed supernatants, following the disappearance of substrate (adenylosuccinate AMP-S) and the formation of product (adenosine 5 -monophophathe-AMP), separated by high performance liquid chromatography (HPLC). Results: Twelve patients showed HER2 overexpression, in particular in dysplastic glands; 4 patients showed diffuse, positive staining mainly basal; 2 patients had heterogeneous staining of weak intensity. High ASL activity (4974 + 900 nmol/hlg) was found in preneoplastic lesions vs normal mucosa (2074 + 416 nmol/hlg) (p<0.05). Conclusion: Overexpression of pl85 and elevated ASL activity were observed in tubular and tubulovillous adenomas. They may be associated with the early stages of colorectal cancer. 1550 EXPRESSION OF E-CADHERIN-CATENIN COMPLEX IN THE PRECANCEROUS LESIONS IN GASTRIC CANCER. Annie 00 Chan, Shiu-Kum Lam, Hui-Yao Lan, Wai-Kong Chan, YuiHung Yueng, Lawerence Hou, Wai-Mo Hui, Univ of Hong Kong, Hong Kong, Hong Kong. Introduction: E-cadherin is an adhesion molecule responsible for cell-cell adhesion. Alpha- and b-catenin bind to E-cadherin to form the cytoskeleton. Under-expression of the cadherin-catenin complex has been found in gastric cancer. Downregulation of b-catenin and E-cadherin has been postulated to be potential good prognostic marker in gastric cancer. Aims: To study the change in the expression of the cadherin-catenin complex in the precancerous stages of gastric cancer. Materials and Methods: Gastrectomy specimens were taken from 17 patients with gastric cancer. Serial sections from areas with chronic gastritis (n= 17), chronic atrophic gastritis (n=8), intestinal metaplasia (n=8), dysplasia (n=8), gastric carcinoma (n=I7) and lymph node metastasis (n=I5) were taken from paraffin blocks. Immunostaining using ABC method with E-cadherin, a-, and b-catenin monoclonal antibodies were performed. Each section was paired with a negative control and each batch with a positive control from the
GASTROENTEROLOGY Vol. 118, No.4
1546
1548
CELL CYCLE ABNORMALITIES IN SMALL BOWEL TUMORS IN ISRAEL. Leonor E. Trejo, Meital Shaked, Hanina Hibshoosh, Eyal Ozeri, Limor Baron, Hanna Strul, Marc Umanski, Fred M. Konikoff, Alfred I. Neugut, Nadir Arber, Tel-Aviv Med Ctr, Tel-Aviv, Israel; Columbia Univ, NewYork, NY.
REDUCED UNCOUPLING PROTEIN (UCP)-3 IN HUMAN CO· LONIC MALIGNANCY. Andrew 1. Bennett, Deborah A. Briggs, Lucina M. Jackson, Chris J. Hawkey, David A. White, Univ of Nottingham, Nottingham, United Kingdom; Univ Hosp Nottingham. Nottingham, United Kingdom. INTRODUCTION: Uncoupling proteins (UCPs) are mitochondrial transporters which dissipate proton gradients generated during electron transport, releasing energy as heat. UCP3 is expressed at high level in skeletal muscle where it is regulated by peroxisome proliferated-activated receptor (PPAR)a and may function in substrate selection. Given the emerging role of PP ARa and the importance of dietary fat in colonic neoplasia, we have investigated expression of UCP3 by Western Blotting and RT-PCT in normal and malignant human colonic mucosa. METHODS: We studied normal and malignant tissue obtained from 8 patients undergoing surgery for colorectal cancer. Samples were snap frozen and stored at -70°C. Human skeletal muscle was used as a positive control. UCP3 protein was separated using SDS-PAGE of tissue protein extract and visualised by Western Blotting with polyclonal anti-UCP3 antibodies, which showed a single band of approximately 35 kDa. Total RNA was extracted by the guanidine thiocyanate method and reverse transcribed. UCP3 mRNA was detected after PCR (28 cycles), using specific primers designed to identify expression of both the long and short forms of UCP3 mRNA. RESULTS: The long and short form of UCP3 mRNA and UCP protein were detected reliably in the positive control human skeletal muscle. UCP3 protein was detected at high levels in normal colonic mucosa, but substantially reduced or absent in 7 of the 8 paired tumour specimens. Only the short form of UCP3 mRNA was detectable in either normal or malignant colonic mucosa. CONCLUSIONS: UCP3 (short form) is expressed in normal human colon. It is substantially reduced in colorectal cancer tissue. Given its close relationship to PP ARa and potential role in fat metabolism, it may play an important role in the pathogenesis of malignant change.
Background and Purpose: When compared to the high incidence of adenomas (A) and adenocarcinomas (AC) in the human large bowel, the incidence of similar tumors in the small bowel is quite rare. Moreover, the reason that epithelial cells of the small bowel appear to be more resistant to the development of both benign and malignant tumors is not known. Recently, we reported on cell cycle abnormalities in small bowel tumors in the US (CEBP 12/99), however little is known regarding the molecular genetics of these tumors, outside of the US. Patients and Methods: A search of the records covering the past decade, from the Tel Aviv Medical Center computer files was carried out. Nine cases of A and 30 cases of AC were found. Epidemiology data was obtained and protein levels determined by immunohistochemistry. Results: 28 males and 11 females, mean age 69, were studied. 83% of the tumors were located in the duodenum. 4 were Duke stage A, 8 were B, 6 were C and 12 were D stage. The 3 year survival rate was 23%. Age, alcohol consumption and smoking were independent risk factors for SB tumors. Interestingly, 63% of the patients had a second primary mostly in the colon. 27% had received adjuvant therapy. Tumor incidence increased from 0.6/100,000 in 1990 to 1.2/100,000 in 1998.All of the normal mucosa samples expressed only p27 protein. About 30% of the tumors demonstrated a decreased expression of p27. Ki-67 was detected in 97% of the tumors and increased p53 expression was associated with tumor progression in 45% of the A and 68% of the AC (P<0.05). Increased expression of cyclin DI (40%), {3-catenine (30%) and Her-2/neu (33%) were closely associated and seen at approximately equal frequencies in A and AC. Advanced age and p53 expression were associated with poor survival, {3-catenine with poorly differentiated tumors, cyclin DI with advanced age, and decreased p27 expression was seen in alcohol consumers (p<0.05). Conclusions: 1. The epidemiology and molecular genetics of small bowel tumors in Israel are similar to that seen in the US. 2. This study confirms the increase in the incidence of SB tumors seen in the US over the last decade. 3. Small bowel tumors in Israel, like those in the US, frequently display abnormalities in the expression of cell cycle related proteins, as previously described for colorectal cancer. 4. This is the first report regarding increased expression of Her-2/neu and nuclear f3-catenine in 30% of small bowel tumors, which is similar to that seen in colorectal cancer. 1547 CHEMOPREVENTIVE ACTION OF RESVERATROL ON GASTRIC ADENOCARCINOMA CELLS VIA PKC-MEDIATED INDUCTION OF P53. Mary Jo Atten, Bashar M. Attar, Matthew Zopel, Oksana Holian, Cook County Hosp, Chicago, IL. Resveratrol, a phytoalexin present in grapes and wine, is a promising chemopreventive agent shown to block each stage of carcinogenesis. In gastric adenocarcinoma cells, resveratrol inhibits DNA synthesis and cellular proliferation, and inhibits cellular protein kinase C (PKC) activity (Gastro 116:A373,1999). We present evidence that resveratrol suppresses cellular proliferation through a p53-dependent pathway by inducing p2l cip, waf-\ expression. We identify a panel of PKC isozymes present in SNU-I cells and determine the relationship between changes in subcellular distribution of specific PKC isozymes and cellular expression of p53 and p21 elicited by resveratrol treatment. METHODS: Human gastric adenocarcinoma SNU-l cells (ATCC CRL-597l) were maintained in RPMI-I640 media supplemented with 10% FBS, and were treated with resveratrol in increasing concentrations and increasing exposure times. Cell cycle distribution was analyzed by flow cytometry. Expression of p21 and p53 products was performed by Western blot analysis following SDS-PAGE. Expression of PKC isozymes in subcellular fractions of SNU-I cells was also determined br Western blotting, while DNA synthesis was assessed by measurement of H-thymidine incorporation into live cells. RESULTS: Expression of the tumor suppressor gene product p53 increased with increasing concentrations or duration of exposure to resveratrol. Concomitant with the increase in p53, we observed increased expression of the cell cycle inhibitor p21 and arrest of SNU-I cells in GJG J phase. Maximal levels of both p53 and p21 were detected after 48 hours of cell treatment with resveratrol at which time the apoptotic index reached 50%. Expression of p53 and p21, cell cycle arrest, and apoptosis were preceded by a decrease in PKC activity and membrane-associated PKC-cS, and an increase in cytosolic PKC-a. CONCLUSIONS: Resveratrol-induced suppression of SNU-I cell proliferation follows a specific sequence of events: 1) inhibition of PKC activity, apparently resulting from the decrease in membraneassociated, phospholipid-activated PKC-cS and a simultaneous increase in cytosolic, inactive PKC-a; and 2) PKC signal-generated increase in expression of p53 and p21, culminating in SNU-I cell arrest in GJG\ phase. These findings suggest that p53-dependent transcriptional activity may be associated with resveratrol-triggered inhibition of tumor growth.
1549 DETERMINATION OF HER2 GENE PRODUCT (PI85) AND ADENYLOSUCCINATE LYASE (ASL) ACTIVITY IN HUMAN PRENEOPLASTIC AND NORMAL COLONIC MUCOSA. Antonio De Martino, Francesco Cetta, Giulia Montalto, Remo Vernillo, Filippo Carlucci, Antonella Tabucchi, Fausto Rosi, Enrico Marinello, Institute of Surg Clinics, Siena, Italy; Institute of Surg, Siena, Italy; Institute of Biochemistry and Enzymology, Siena, Italy. Background and Aims: The HER2 gene has been found amplified in various human carcinomas leading to overexpression of the encoded product, pI85 protein. Adenylosuccinate lyase (ASL, EC 4.3.2.2) is an enzyme involved in purine nucleotide biosynthesis an is known to increase in malignancies such as colorectal, breast, and prostate cancer. In order to evaluate the potential of ASL as a tumor marker, its activity was determined and compared with the expression of pI85 in pre-neoplastic and normal colonic mucosa. Materials and Methods: 18 patients (pts) (16 males, 2 females, mean age 55, range 22-75 years) were analyzed. 13 had tubular adenomas, 4 tubulovillous adenomas and 1 multiple polyps. p185 was determined by immunoenzymatic staining by the avidin-biotin peroxidase method, using murine 300G 9 antibody, that is specific for the extrcellular domain of human HER2 oncogene. ASL activity was analyzed in 23 specimens of normal mucosa and in 14 of 18 patients with adenomas. ASL activity was evaluated in dialyzed supernatants, following the disappearance of substrate (adenylosuccinate AMP-S) and the formation of product (adenosine 5 -monophophathe-AMP), separated by high performance liquid chromatography (HPLC). Results: Twelve patients showed HER2 overexpression, in particular in dysplastic glands; 4 patients showed diffuse, positive staining mainly basal; 2 patients had heterogeneous staining of weak intensity. High ASL activity (4974 + 900 nmol/hlg) was found in preneoplastic lesions vs normal mucosa (2074 + 416 nmol/hlg) (p<0.05). Conclusion: Overexpression of pl85 and elevated ASL activity were observed in tubular and tubulovillous adenomas. They may be associated with the early stages of colorectal cancer. 1550 EXPRESSION OF E-CADHERIN-CATENIN COMPLEX IN THE PRECANCEROUS LESIONS IN GASTRIC CANCER. Annie 00 Chan, Shiu-Kum Lam, Hui-Yao Lan, Wai-Kong Chan, YuiHung Yueng, Lawerence Hou, Wai-Mo Hui, Univ of Hong Kong, Hong Kong, Hong Kong. Introduction: E-cadherin is an adhesion molecule responsible for cell-cell adhesion. Alpha- and b-catenin bind to E-cadherin to form the cytoskeleton. Under-expression of the cadherin-catenin complex has been found in gastric cancer. Downregulation of b-catenin and E-cadherin has been postulated to be potential good prognostic marker in gastric cancer. Aims: To study the change in the expression of the cadherin-catenin complex in the precancerous stages of gastric cancer. Materials and Methods: Gastrectomy specimens were taken from 17 patients with gastric cancer. Serial sections from areas with chronic gastritis (n= 17), chronic atrophic gastritis (n=8), intestinal metaplasia (n=8), dysplasia (n=8), gastric carcinoma (n=I7) and lymph node metastasis (n=I5) were taken from paraffin blocks. Immunostaining using ABC method with E-cadherin, a-, and b-catenin monoclonal antibodies were performed. Each section was paired with a negative control and each batch with a positive control from the