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A phenotypic similarity of small intestinal neoplasia with colonic epithelium as detected by a novel monoclonal antibody (mAb)
April 1998 In the other pts no malignant recurrency was observed (median follow-up 35 mos). One pt was lost to follow-up, one died of metastatic renal CA. Discussion: Endoscopic resection of large colorectal polyps (_>3 cm) is a safe procedure. Ivasive CA was found in only 10% and could be managed endoscopically in at least half of the cases irrespective of growth and histologic type. • G2319 FOLIC ACID: REGULATION OF GROWTH OF COLON AND GASTRIC CANCER CELL LINES. S. B. Bonnana. F.H. Sarkar, L. Liu, Y-W. Li, A. Khan, O. Kucuk, K. Mahakala, R. Jaszewski, and A.P.N. Majumdar. John D. Dingell VA Med. Ctr., and Depts. Med. and Pathol, Wayne State Univ. Sch. Med., Detroit, MI; J.S. Bertram, Cancer Res. Ctr. of Hawaii, Honolulu. HI Results from several laboratories, including our own, suggest that folic acid may be chemopreventive against colon cancer. However, little is known about the regulation of this process. We hypothesize that folic acid exerts its chemopreventive effect on the colon by inhibiting proliferation and enhancing, differentiation and/or apoptosis. To test this hypothesis, we examined the changes in proliferation and protein levels of bax (whose expression is positively related to apoptosis) and connexcin (C-43; involved in cell-cell communication; an indicator of differentiation)in two colon cancer cell lines (HCT-116 and CaCo-2). These parameters were also measured in a gastric cancer line (Kato-III) to determine whether folic acid may also be chemopreventive against gastric cancer. In each of these cell lines, folic acid inhibited proliferation in a dose-dependent manner. However, the 15o dose of folic acid was lower in colon cancer cell lines compared to the gastric cancer cells (125 ng/ml vs 626 ng/ml). In these cell lines, bax and Cx-34 levels, as assessed by Western-immunoblot, was increased significantly by 30-50% after 48 h of exposure to folic acid (625 ng/ml). We further hypothesize that EGF-receptor (EGFR) may play a role in regulating folic acid-induced changes in proliferation, differentiation and apoptotic processes. Indeed, we found folic acid (625 ng/ml) to significantly inhibit (40-60%) EGFR tyrosine kinase activity and tyrosine phosphorylation of EGFR as well as the relative concentration of the 14 kDa precursor form of TGF-a (one of the ligands of EGFR) in membranes of Kato-III as well as HCT-116 and CaCo-2 cells. Our data suggest a chemopreventive role for folic acid in colon and gastric cancers, as evidenced by the inhibition of proliferation and stimulation of differentiation and apoptotic processes in colon and gastric cancer cell lines. We also suggest that diminished activation of EGFR tyrosine kinase resulting from decreased membrane accumulation of TGF-a may partly be involved in regulating these processes. Supported by grants from the NIH/NIA G2320
ORALLY ADMINISTERED DEHYDROEPIANDROSTERONE~ULFATE INCREASES G6PD AND TRANSKETOLASE ACTIVITY IN TUMOR TISSUE. L.G. Boros, *B. Comin, *J. Puigjaner, LL. Brandes, P. Muscarella, F.I. Yusuf, R.D. Williams, +W.P. Lee, *M. Cascante, W.J. Schirmer and W.S. Melvin. The Ohio State University College of Medicine, Department of Surgery, Columbus, OH; *Department of Biochemistry, University of Barcelona, Barcelona, Spain; +Research and Education Institute, HarborUCLA Medical Center, Torrance, CA, USA Background: Glucose-6P dehydrogenase (G6PD) and transketolase (TK) are two key enzymes of the pentose cycle (PC). They are directly involved in the generation of NADPH, ribose and glyceraldehyde necessary for macromolecule synthesis and cell proliferation. We have previously shown that dehydroepiandrosterone-sulfate (DHEA-S), a reversible non-competitive inhibitor of G6PD, inhibits in vitro and in vivo growth of Mia pancreatic adenocarcinoma cells. Aims: In order to further characterize the antiproliferative effect of DHEA-S, we measured the specific activity of G6PD and TK in Mia tumor cells hosted in DHEA-S treated nude mice. Methods: Forty male, nude, athymic mice were fed either Teklad 22/5 rodent diet, or diet supplemented with 0.6% DHEA-S ad libitum. Four weeks after the institution of the experimental diets, the animals tight flanks were injected with lxl06 MiaPaCa-2 cells. After 5 weeks, tumors were excised, snap frozen in liquid nitrogen then homogenized. Tumor supematants were used in specific G6PD or TK enzyme activity assays which measure the nmols of G6PD or TK substrates metabolized by each mg of protein in tumor supernatants per minutes (units/mg protein). Results: Orally administered DHEA-S significantly reduced final tumor weight but increased the specific activity of G6PD by 55.3% (control: 56.3 +/- 3.27 n=3; DHEA: 87.4 +/- 13.05 n=3, p=0.009). TK specific activity was also increased by 27% (control: 20.3 +/- 4.2 n=5; DHEA-S: 25.7 +/- 5.9 n=5, p=0.064). Discussion: Since DHEA-S reversibly inhibits G6PD, it is not possible to predict the net effect of DHEA-S treatment on ribose synthesis through G6PD. However, our observations by mass isotopomer carbon flow studies through the PC suggest a reduction in total ribose synthesis through G6PD after DHEA-S treatment in cultured Mia cells [Cancer Research 57:4242-4248,1997]. Conclusions: Oral administration of DHEA-S increases the specific activity of G6PD in hosted tumors. Increased expression and production of this enzyme in tumors in response to the reversible inhibitory effect of DHEA-S could be a limiting
Gastrointestinal Oncology A567
factor for its use in tumor therapy. Histochemical techniques and studies with labeled substrates and mass isotopomer analysis may be necessary for gaining additional insights into metabolic changes and growth reduction in tumor ceils after treatments with PC enzyme inhibitors or other antineoplastic drugs. G2321 A PHENOTYPIC SIMILARITY OF SMALL INTESTINAL NEOPLASIA WITH COLONIC EPITHELIUM AS DETECTED BY A NOVEL MONOCLONAL ANTIBODY (mAb). S. B0rra, E.K. 0numa, V. Mohan, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ and Lyons VA Medical Center, Lyons, NJ. We earlier reported a colon epithelial specific mAb (mAb DAS-1) that specifically reacts with normal colon epithelium but not with any other parts of the gastrointestinal tract including the small intestinal enterocytes (SIE) from the jejunum and ileum (J Immunol 1987;139:77). Interestingly, mAb DAS-1 sensitively and specifically reacts with Barrett's epithelium (BE) and adenocarcinoma arising from BE, but does not react with normal gastroesophageal junctional mucosa (Ann Int Med 1994;120:753). AIMS & METHODS: In contrast to colon, small intestinal neoplasia, including adenomas and adenocarcinoma, are rare. To examine if there is a phenotypic change of SIE towards colonocytes in the small intestinal neoplastic tissue, we have utilized the mAb DAS-1. Twelve formalin fixed paraffin blocks of tissue consisting of adenomas (n=2), and hyperplastic polyps (n=2) of upper small intestine, non-specific chronic inflammatory mucosa of the small intestine (n=5), adenocarcinoma of the jejunum (n=3, one primary, 2 metastatic from outside the gastrointestinal tract) were examined by a sensitive immunoperoxidase assay using the mAb DAS-1. An isotype control mAb (MOPC-IgM) was used in parallel. Normal jejunal and colonic biopsy specimens (n=4) were also included as additional controls. RESULTS: mAb DAS-1 reacted strongly with the neoplastic epithelial cells in 2/2 small intestinal adenomas as well as the primary adenocarcinoma of the jejunum. However, the normal small intestinal mucosa adjacent to the adenomas and the carcinoma was totally negative. Each of the specimens with inflammatory mucosa, hyperplastic polyps, and the two metastatic carcinoma did not react with the mAb DAS-1. As expected, mAb DAS-1 reacted with colonic epithelium used as positive control and did not react with normal jejunal mucosa. Isotype control mAb did not react with any of the tissues. CONCLUSION: These data demonstrate a phenotypic change towards colonic epithelium in the small intestinal neoplasms, adenomas and adenocarcinoma. G2322 DELETIONS OF pl6/MTS-1 GENE IS A FREQUENT EVENT IN PANCREATIC JUICE FROM PATIENTS WITH PANCREATIC CANCER AND CHRONIC PANCREATITIS. M. Bouisson, P. Pages, L. Buscail, O. Croizet, C. Bureau, P. Berth61emy, J. Fourcade, N. Vaysse, J. Escourrou, L. Pradavrol. INSERM U151 and Department of Gastroenterology, CHU Rangueil, 31403 Toulouse, France. Background Mutations of K-ras found in most of pancreatic adenocarcinomas represent a critical event in oncogenesis of this aggressive cancer. We previously report the existence of K-ras oncogene activation before tumor visualization by ERCP. More precise definition of patients at risk of developing a pancreatic cancer (P.C.) and differential diagnosis between pseudo-tumorous chronic pancreatitis (C.P.) and P.C. might come from simultaneous determination of K-ras mutation and tumor suppressor gene(s) inactivation in pancreatic juice such as p 16/MTS 1 gene. Aim To determine p16 / MTS1 inactivation by exon 1 and/or exon 2 deletion or mutations and K-ras codon 12 mutations in pure pancreatic juice from patients with confirmed C.P. and P.C. Methods K-ras codon 12 and p16/MTS1 mutations were looked for by direct sequencing of PCR products from pancreatic juice DNA. Multiplex PCR was used for deletion studies. Results Forty nine pancreatic juice samples collected from 49 patients distributed into three groups: Group 1 - Non tumoral pancreato-biliary diseases (n=15), Group 2 - C.P. (n=19) and Group 3 - P.C. (n=15) were analyzed for both gene alterations. Ten out of 15 patient samples (67% CI 42 to 92) from P.C. group were found positive for K-ras mutation and p16 exon 2 deletion. These values were significantly different from those observed in Groupl (7% CI 0 to 20 K-ras, 20% CI 0 to 40 for p16 exon 2). Striking differences in these alterations were observed in patients from Group 2. Out of 19 patient samples from this group, one (5% CI 0 to 15) showed K-ras codon 12 mutation whilst nine (47% C130 to 76) were found deleted in p16 / MTS1 exon 2. Overall exon 1 deletions are few, they ranged from 0, Group 1 to 5, Group 2 and were only 2 out of 15 in P.C. Group. Conclusions Exon 2 deletions of p16 /MTS1 gene in DNA accumulated in pancreatic juice of C.P. patients is a frequent event, the incidence of this abnormality is not different from the one observed in pancreatic juice collected from patients with P.C. The frequent inactivation of p16 / MTS1 by exon 2 deletion observed in DNA from pure pancreatic juice of C.P. patients constitute a strong limitation for the use of this marker in early diagnosis of P.C. Its use in combination with K-ras codon 12 mutations research does not improve the sensitivity, specificity characteristics of diagnosis based on K-ras alone.
ORALLY ADMINISTERED DEHYDROEPIANDROSTERONE~ULFATE INCREASES G6PD AND TRANSKETOLASE ACTIVITY IN TUMOR TISSUE. L.G. Boros, *B. Comin, *J. Puigjaner, LL. Brandes, P. Muscarella, F.I. Yusuf, R.D. Williams, +W.P. Lee, *M. Cascante, W.J. Schirmer and W.S. Melvin. The Ohio State University College of Medicine, Department of Surgery, Columbus, OH; *Department of Biochemistry, University of Barcelona, Barcelona, Spain; +Research and Education Institute, HarborUCLA Medical Center, Torrance, CA, USA Background: Glucose-6P dehydrogenase (G6PD) and transketolase (TK) are two key enzymes of the pentose cycle (PC). They are directly involved in the generation of NADPH, ribose and glyceraldehyde necessary for macromolecule synthesis and cell proliferation. We have previously shown that dehydroepiandrosterone-sulfate (DHEA-S), a reversible non-competitive inhibitor of G6PD, inhibits in vitro and in vivo growth of Mia pancreatic adenocarcinoma cells. Aims: In order to further characterize the antiproliferative effect of DHEA-S, we measured the specific activity of G6PD and TK in Mia tumor cells hosted in DHEA-S treated nude mice. Methods: Forty male, nude, athymic mice were fed either Teklad 22/5 rodent diet, or diet supplemented with 0.6% DHEA-S ad libitum. Four weeks after the institution of the experimental diets, the animals tight flanks were injected with lxl06 MiaPaCa-2 cells. After 5 weeks, tumors were excised, snap frozen in liquid nitrogen then homogenized. Tumor supematants were used in specific G6PD or TK enzyme activity assays which measure the nmols of G6PD or TK substrates metabolized by each mg of protein in tumor supernatants per minutes (units/mg protein). Results: Orally administered DHEA-S significantly reduced final tumor weight but increased the specific activity of G6PD by 55.3% (control: 56.3 +/- 3.27 n=3; DHEA: 87.4 +/- 13.05 n=3, p=0.009). TK specific activity was also increased by 27% (control: 20.3 +/- 4.2 n=5; DHEA-S: 25.7 +/- 5.9 n=5, p=0.064). Discussion: Since DHEA-S reversibly inhibits G6PD, it is not possible to predict the net effect of DHEA-S treatment on ribose synthesis through G6PD. However, our observations by mass isotopomer carbon flow studies through the PC suggest a reduction in total ribose synthesis through G6PD after DHEA-S treatment in cultured Mia cells [Cancer Research 57:4242-4248,1997]. Conclusions: Oral administration of DHEA-S increases the specific activity of G6PD in hosted tumors. Increased expression and production of this enzyme in tumors in response to the reversible inhibitory effect of DHEA-S could be a limiting
Gastrointestinal Oncology A567
factor for its use in tumor therapy. Histochemical techniques and studies with labeled substrates and mass isotopomer analysis may be necessary for gaining additional insights into metabolic changes and growth reduction in tumor ceils after treatments with PC enzyme inhibitors or other antineoplastic drugs. G2321 A PHENOTYPIC SIMILARITY OF SMALL INTESTINAL NEOPLASIA WITH COLONIC EPITHELIUM AS DETECTED BY A NOVEL MONOCLONAL ANTIBODY (mAb). S. B0rra, E.K. 0numa, V. Mohan, K.M. Das. UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ and Lyons VA Medical Center, Lyons, NJ. We earlier reported a colon epithelial specific mAb (mAb DAS-1) that specifically reacts with normal colon epithelium but not with any other parts of the gastrointestinal tract including the small intestinal enterocytes (SIE) from the jejunum and ileum (J Immunol 1987;139:77). Interestingly, mAb DAS-1 sensitively and specifically reacts with Barrett's epithelium (BE) and adenocarcinoma arising from BE, but does not react with normal gastroesophageal junctional mucosa (Ann Int Med 1994;120:753). AIMS & METHODS: In contrast to colon, small intestinal neoplasia, including adenomas and adenocarcinoma, are rare. To examine if there is a phenotypic change of SIE towards colonocytes in the small intestinal neoplastic tissue, we have utilized the mAb DAS-1. Twelve formalin fixed paraffin blocks of tissue consisting of adenomas (n=2), and hyperplastic polyps (n=2) of upper small intestine, non-specific chronic inflammatory mucosa of the small intestine (n=5), adenocarcinoma of the jejunum (n=3, one primary, 2 metastatic from outside the gastrointestinal tract) were examined by a sensitive immunoperoxidase assay using the mAb DAS-1. An isotype control mAb (MOPC-IgM) was used in parallel. Normal jejunal and colonic biopsy specimens (n=4) were also included as additional controls. RESULTS: mAb DAS-1 reacted strongly with the neoplastic epithelial cells in 2/2 small intestinal adenomas as well as the primary adenocarcinoma of the jejunum. However, the normal small intestinal mucosa adjacent to the adenomas and the carcinoma was totally negative. Each of the specimens with inflammatory mucosa, hyperplastic polyps, and the two metastatic carcinoma did not react with the mAb DAS-1. As expected, mAb DAS-1 reacted with colonic epithelium used as positive control and did not react with normal jejunal mucosa. Isotype control mAb did not react with any of the tissues. CONCLUSION: These data demonstrate a phenotypic change towards colonic epithelium in the small intestinal neoplasms, adenomas and adenocarcinoma. G2322 DELETIONS OF pl6/MTS-1 GENE IS A FREQUENT EVENT IN PANCREATIC JUICE FROM PATIENTS WITH PANCREATIC CANCER AND CHRONIC PANCREATITIS. M. Bouisson, P. Pages, L. Buscail, O. Croizet, C. Bureau, P. Berth61emy, J. Fourcade, N. Vaysse, J. Escourrou, L. Pradavrol. INSERM U151 and Department of Gastroenterology, CHU Rangueil, 31403 Toulouse, France. Background Mutations of K-ras found in most of pancreatic adenocarcinomas represent a critical event in oncogenesis of this aggressive cancer. We previously report the existence of K-ras oncogene activation before tumor visualization by ERCP. More precise definition of patients at risk of developing a pancreatic cancer (P.C.) and differential diagnosis between pseudo-tumorous chronic pancreatitis (C.P.) and P.C. might come from simultaneous determination of K-ras mutation and tumor suppressor gene(s) inactivation in pancreatic juice such as p 16/MTS 1 gene. Aim To determine p16 / MTS1 inactivation by exon 1 and/or exon 2 deletion or mutations and K-ras codon 12 mutations in pure pancreatic juice from patients with confirmed C.P. and P.C. Methods K-ras codon 12 and p16/MTS1 mutations were looked for by direct sequencing of PCR products from pancreatic juice DNA. Multiplex PCR was used for deletion studies. Results Forty nine pancreatic juice samples collected from 49 patients distributed into three groups: Group 1 - Non tumoral pancreato-biliary diseases (n=15), Group 2 - C.P. (n=19) and Group 3 - P.C. (n=15) were analyzed for both gene alterations. Ten out of 15 patient samples (67% CI 42 to 92) from P.C. group were found positive for K-ras mutation and p16 exon 2 deletion. These values were significantly different from those observed in Groupl (7% CI 0 to 20 K-ras, 20% CI 0 to 40 for p16 exon 2). Striking differences in these alterations were observed in patients from Group 2. Out of 19 patient samples from this group, one (5% CI 0 to 15) showed K-ras codon 12 mutation whilst nine (47% C130 to 76) were found deleted in p16 / MTS1 exon 2. Overall exon 1 deletions are few, they ranged from 0, Group 1 to 5, Group 2 and were only 2 out of 15 in P.C. Group. Conclusions Exon 2 deletions of p16 /MTS1 gene in DNA accumulated in pancreatic juice of C.P. patients is a frequent event, the incidence of this abnormality is not different from the one observed in pancreatic juice collected from patients with P.C. The frequent inactivation of p16 / MTS1 by exon 2 deletion observed in DNA from pure pancreatic juice of C.P. patients constitute a strong limitation for the use of this marker in early diagnosis of P.C. Its use in combination with K-ras codon 12 mutations research does not improve the sensitivity, specificity characteristics of diagnosis based on K-ras alone.