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The human intestinal mucin gene MUC2 encodes multiple mRNA transcripts
April 2000
AGAA283
1594
1596
RED MEAT AND COLON CANCER: DIETARY HEME, BUT NOT FAT, HAS CYTOTOXIC AND HYPERPROLIFERATIVE EFFECTS ON THE COLONIC EPITHELIUM. Aloys L. Sesink, Denise S. Termont, Jan H. Kleibeuker, Roelof Van der Meer, Wageningen Ctr for Food Sci, Ede, Netherlands; Univ Hosp, Groningen, Netherlands.
TRANSIT INDUCED CHANGES IN SERUM DEOXYCHOLIC ACID STIMULATE INCREASED MITOSES WITHIN COLONIC CRYPTS: CONTROLS VS PATIENTS WITH ADENOMATOUS POLYPS. Mario Stellini, Jeremy D. Sanderson, Anthony Mallet, Hermon R. Dowling, Guys Hosp Campus GKT Sch of Medicine, London, United Kingdom; Univ of Greenwich, London, United Kingdom.
High intake of red meat is associated with increased colon cancer risk. It is suggested that fat from red meat is involved because it can increase fecal bile acid concentration. Experimental studies did not unequivocally support such a role for fat. Recently, we showed that dietary heme iron increased colonic cytotoxicity and epithelial proliferation (Cane Res 59, 5704). In this study, we wanted to clarify whether dietary fat affects colon cancer risk by modulating the detrimental heme effects. Methods: Rats were fed control or heme-supplemented diets with 10, 25 or 40 energy% derived from fat for 14 days. Feces were collected for biochemical analyses. Colonic cytotoxicity was determined from the degree of lysis of erythrocytes b~ fecal water. Colonic epithelial proliferation was measured in vivo using H-thymidine incorporation. Results (mean ± SE)are given in the table. Dietary heme increased fecal cation content and cytotoxicity of fecal water, suggesting that the colonic mucosa of heme-fed rats was exposed to high amounts of luminal irritants. This effect was smaller on the low fat diet. In addition, heme increased proliferation at all fat levels, though not significantly on the low fat diet. The heme-effects were not mediated by bile acids. Dietary fat itself did not affect colonic mucosa, though it increased total fecal bile acid excretion. However, bile acid concentration in fecal water was not increased by fat. Conclusions: Because in Western society 30-40% of the ingested energy is supplied by fat, our results suggest that the association between red meat consumption and colon canceris mainly due to the heme iron, and is largely independent of the fat content of the diet.
Diet
10%, control 10%,heme 25%,control 25%, heme 40%, control 40n%, heme
fecal cations (J.lmoUg)
cytotoxicity (%)
proliferation (dpmlJ.lg DNA)
totalbile acids ().lmol/day)
bileacids fecal water (mM)
195±19 428 ± 34' 244±27 858 ± 25' 227 ± 24 1199±27'
O±O 63± 15' 1± 1 100±0' 2±2 99± l'
4O±4 52±4 4O±4 66±4' 45± 7 64±3'
10.7 ± 0.6 15.1 ± 1.3' 13.1 ± 1.0 15.3± 1.0 17.0± 1.3 16.5± 1.3
2.0± 0.2 1.7±0.1 2.2 ± 0.2 1.2±0.l' 2.2±0.1 1.0±0.l'
, significant different from fat-matched control
1595 COMPLIANCE WITH DAILY ASPIRIN IN A CHEMOPREVENTION STUDY IN PATIENTS WITH PRIOR ADENOMAS: MEASUREMENT OF SALICYLATE LEVELS, PILL COUNTS AND SELF-REPORTS. Frank A. Sinicrope, Nancy Logan, Dory Sample, Michael Wargovich, UT MD Anderson Cancer Ctr, Houston, TX. BACKGROUND. Epidemiologic studies indicate that regular intake of aspirin reduces colorectal cancer incidence. Aspirin inhibits prostaglandin (PG) synthesis and measurement of mucosal PGs is utilized as an intermediate biomarker endpoint. Poor compliance with daily aspirin in a chemoprevention study was previously reported. PURPOSE. We conducted a phase II trial to determine the lowest dose of aspirin that markedly suppressed mucosal PGE2 levels. Compliance was assessed by correlating aspirin dose with serum salicylate levels, pill counts, and patient self reports. METHODS. Sixty men (n=34) and women (n=26) with a history of colorectal adenoma(s) within the preceding 5 years were recruited and randomly assigned to one of 4 aspirin doses (placebo, 81 mg, 325 mg, 650 mg) for a 28 day period of continuous drug therapy. Flexible sigmoidoscopy with rectal biopsies was performed at study entry and on the last day of drug administration. PGE2 levels were assayed in mucosal biopsies by competitive enzyme immunoassay. Serum salicylic acid levels were measured by high pressure liquid chromatography. Subjects were asked to record drug intake in a daily calendar and pill counts were performed. All subjects were paid for participation. RESULTS. Fifty-five patients were evaluable (14-placebo, 13-8Img, 14-325mg, 14-650mg). No significant toxic effects occurred. Mean and median PGE2 levels were significantly suppressed by aspirin relative to placebo (mean suppression was 77%8lmg, 70%-325mg, 79%-650mg), but did not differ between treatment groups (p> 0040). Serum salicylate levels correlated with aspirin dose. Salicylate levels indicated that 50 of 55 (91%) subjects were compliant. Pill counts revealed that >98% of the aspirin doses were taken by all subjects and self-reports were concordant with pill counts. CONCLUSION: The 81 mg aspirin dose was equally effective at suppressing PGE2 levels as was higher doses, and supports the use of this dose for chemoprevention studies. Compliance with daily aspirin exceeded 90% using salicylate levels and 98% by pill counts and self-reports. These data support the feasibility of daily aspirin for chemoprevention trials.
Background: The secondary bile acid, deoxycholic acid (DCA), is implicated in the pathogenesis of colonic adenomata and carcinomata. It is formed in the colon by bacterial enzymes, and its synthesis rate, pool size and proportions (% oftotal bile acids) in serum and bile, are linearly related to large bowel transit time (LBTT). Moreover, physiological concentrations (20-50f.l,M) of DCA stimulate proliferation of human colon cancer cells in vitro, while we and others have shown that fasting serum DCA is significantly increased in patients with colonic adenomata. Hypothesis: We postulated therefore that in patients with colonic adenomata, prolongation of LBTT increases colonic luminal (and serum) DCA which, in turn, stimulates colonocyte proliferation and promotes a hyperplasia-adenomacarcinoma sequence. Methods: To test this, we measured LBTT using radio-opaque makers, fasting serum DCA by GC-MS and mitotic counts in microdissected crypts (colonoscopic biopsies) from caecum, transverse and sigmoid colon of: (i) 24 controls (normal endoscopy) and (ii) 15 patients with colonic adenomata (past or present). Results: Although there was no significant difference in LBTT between the control (20.5±SEMI.7Ih) and the adenoma (24.1 ±2A) groups, the mean serum unconjugated (newlyformed) DCA concentration in the polyp patients (0.76± 0.08 f.l,M) was 85% higher than that in the controls (OAI±0.5 p
1597 THE HUMAN INTESTINAL MUCIN GENE MUC2 ENCODES MULTIPLE MRNA TRANSCRIPTS. Lawrence R. Sternberg, Christopher C. Yunker, Robert S. Bresalier, Henry Ford Health Sci Ctr, Detroit, MI. Background. Human colon cancers and cancer-derived cell lines can differ significantly in the amount of human intestinal mucin (MUC2) synthesized. Quantitative and qualitative differences in colon cancer mucin may also affect the biological behavior of colon cancer cells. A clear demonstration of the molecular mechanisms for these observations is lacking because it is unclear whether single or multiple MUC2 mRNA species are translated into protein. Complicating factors include the large size and repetitive nature of the MUC2 gene and its mRNA and protein products, allelic variation between MUC2 loci and difficulties in the stable propagation of MUC2 cDNAs containing MUC2 repetitive sequences (TRI and TR2). A complete set of MUC2 cDNAs has yet to be assembled, but a hypothetical eDNA sequence has been proposed that spans 15,721 base pairs including a 5,200+ amino acid open reading frame. Methods. Western analyses using antibodies directed against different portion of MUC2 apoprotein, immunoprecipitation, PCR based methods (RT-PCR, genomic PCR and 3 RACE), eDNA sequencing, multi-probe Northern analyses and antisense inhibition techniques were used to examine whether separable forms of MUC2 are produced in clonal derivatives of human colon cancer cells as well as the mechanism(s) involved. Results. Western analyses and subtractive immunoprecipitation indicate that two distinct classes of MUC2 proteins are produced, one containing TR2 and another lacking this domain. RT-PCR and Northern blot analyses demonstrate the expression of corresponding mRNA species. cDNA sequencing indicates that one of these species is produced by alternate splicing and suggests that another arises by alternative polyadenylation. Genomic PCR data indicate that these results cannot be explained by simple allelic polymorphism. Subsequent Northern hybridization experiments confirm expression of multiple MUC2 encoded transcripts in normal colon. Antisense inhibition studies indicate the potential for selective inhibition of individual MUC2 encoded mRNA species. Conclusion . The human MUC2 gene encodes multiple mRNA transcripts and apoprotein products. These data have important implications for the study of normal and neoplastic processes in the gastrointestinal tract. Supported by NIH grant ROlCA69480 and the Research Service of Henry Ford Health Sciences Center.
AGAA283
1594
1596
RED MEAT AND COLON CANCER: DIETARY HEME, BUT NOT FAT, HAS CYTOTOXIC AND HYPERPROLIFERATIVE EFFECTS ON THE COLONIC EPITHELIUM. Aloys L. Sesink, Denise S. Termont, Jan H. Kleibeuker, Roelof Van der Meer, Wageningen Ctr for Food Sci, Ede, Netherlands; Univ Hosp, Groningen, Netherlands.
TRANSIT INDUCED CHANGES IN SERUM DEOXYCHOLIC ACID STIMULATE INCREASED MITOSES WITHIN COLONIC CRYPTS: CONTROLS VS PATIENTS WITH ADENOMATOUS POLYPS. Mario Stellini, Jeremy D. Sanderson, Anthony Mallet, Hermon R. Dowling, Guys Hosp Campus GKT Sch of Medicine, London, United Kingdom; Univ of Greenwich, London, United Kingdom.
High intake of red meat is associated with increased colon cancer risk. It is suggested that fat from red meat is involved because it can increase fecal bile acid concentration. Experimental studies did not unequivocally support such a role for fat. Recently, we showed that dietary heme iron increased colonic cytotoxicity and epithelial proliferation (Cane Res 59, 5704). In this study, we wanted to clarify whether dietary fat affects colon cancer risk by modulating the detrimental heme effects. Methods: Rats were fed control or heme-supplemented diets with 10, 25 or 40 energy% derived from fat for 14 days. Feces were collected for biochemical analyses. Colonic cytotoxicity was determined from the degree of lysis of erythrocytes b~ fecal water. Colonic epithelial proliferation was measured in vivo using H-thymidine incorporation. Results (mean ± SE)are given in the table. Dietary heme increased fecal cation content and cytotoxicity of fecal water, suggesting that the colonic mucosa of heme-fed rats was exposed to high amounts of luminal irritants. This effect was smaller on the low fat diet. In addition, heme increased proliferation at all fat levels, though not significantly on the low fat diet. The heme-effects were not mediated by bile acids. Dietary fat itself did not affect colonic mucosa, though it increased total fecal bile acid excretion. However, bile acid concentration in fecal water was not increased by fat. Conclusions: Because in Western society 30-40% of the ingested energy is supplied by fat, our results suggest that the association between red meat consumption and colon canceris mainly due to the heme iron, and is largely independent of the fat content of the diet.
Diet
10%, control 10%,heme 25%,control 25%, heme 40%, control 40n%, heme
fecal cations (J.lmoUg)
cytotoxicity (%)
proliferation (dpmlJ.lg DNA)
totalbile acids ().lmol/day)
bileacids fecal water (mM)
195±19 428 ± 34' 244±27 858 ± 25' 227 ± 24 1199±27'
O±O 63± 15' 1± 1 100±0' 2±2 99± l'
4O±4 52±4 4O±4 66±4' 45± 7 64±3'
10.7 ± 0.6 15.1 ± 1.3' 13.1 ± 1.0 15.3± 1.0 17.0± 1.3 16.5± 1.3
2.0± 0.2 1.7±0.1 2.2 ± 0.2 1.2±0.l' 2.2±0.1 1.0±0.l'
, significant different from fat-matched control
1595 COMPLIANCE WITH DAILY ASPIRIN IN A CHEMOPREVENTION STUDY IN PATIENTS WITH PRIOR ADENOMAS: MEASUREMENT OF SALICYLATE LEVELS, PILL COUNTS AND SELF-REPORTS. Frank A. Sinicrope, Nancy Logan, Dory Sample, Michael Wargovich, UT MD Anderson Cancer Ctr, Houston, TX. BACKGROUND. Epidemiologic studies indicate that regular intake of aspirin reduces colorectal cancer incidence. Aspirin inhibits prostaglandin (PG) synthesis and measurement of mucosal PGs is utilized as an intermediate biomarker endpoint. Poor compliance with daily aspirin in a chemoprevention study was previously reported. PURPOSE. We conducted a phase II trial to determine the lowest dose of aspirin that markedly suppressed mucosal PGE2 levels. Compliance was assessed by correlating aspirin dose with serum salicylate levels, pill counts, and patient self reports. METHODS. Sixty men (n=34) and women (n=26) with a history of colorectal adenoma(s) within the preceding 5 years were recruited and randomly assigned to one of 4 aspirin doses (placebo, 81 mg, 325 mg, 650 mg) for a 28 day period of continuous drug therapy. Flexible sigmoidoscopy with rectal biopsies was performed at study entry and on the last day of drug administration. PGE2 levels were assayed in mucosal biopsies by competitive enzyme immunoassay. Serum salicylic acid levels were measured by high pressure liquid chromatography. Subjects were asked to record drug intake in a daily calendar and pill counts were performed. All subjects were paid for participation. RESULTS. Fifty-five patients were evaluable (14-placebo, 13-8Img, 14-325mg, 14-650mg). No significant toxic effects occurred. Mean and median PGE2 levels were significantly suppressed by aspirin relative to placebo (mean suppression was 77%8lmg, 70%-325mg, 79%-650mg), but did not differ between treatment groups (p> 0040). Serum salicylate levels correlated with aspirin dose. Salicylate levels indicated that 50 of 55 (91%) subjects were compliant. Pill counts revealed that >98% of the aspirin doses were taken by all subjects and self-reports were concordant with pill counts. CONCLUSION: The 81 mg aspirin dose was equally effective at suppressing PGE2 levels as was higher doses, and supports the use of this dose for chemoprevention studies. Compliance with daily aspirin exceeded 90% using salicylate levels and 98% by pill counts and self-reports. These data support the feasibility of daily aspirin for chemoprevention trials.
Background: The secondary bile acid, deoxycholic acid (DCA), is implicated in the pathogenesis of colonic adenomata and carcinomata. It is formed in the colon by bacterial enzymes, and its synthesis rate, pool size and proportions (% oftotal bile acids) in serum and bile, are linearly related to large bowel transit time (LBTT). Moreover, physiological concentrations (20-50f.l,M) of DCA stimulate proliferation of human colon cancer cells in vitro, while we and others have shown that fasting serum DCA is significantly increased in patients with colonic adenomata. Hypothesis: We postulated therefore that in patients with colonic adenomata, prolongation of LBTT increases colonic luminal (and serum) DCA which, in turn, stimulates colonocyte proliferation and promotes a hyperplasia-adenomacarcinoma sequence. Methods: To test this, we measured LBTT using radio-opaque makers, fasting serum DCA by GC-MS and mitotic counts in microdissected crypts (colonoscopic biopsies) from caecum, transverse and sigmoid colon of: (i) 24 controls (normal endoscopy) and (ii) 15 patients with colonic adenomata (past or present). Results: Although there was no significant difference in LBTT between the control (20.5±SEMI.7Ih) and the adenoma (24.1 ±2A) groups, the mean serum unconjugated (newlyformed) DCA concentration in the polyp patients (0.76± 0.08 f.l,M) was 85% higher than that in the controls (OAI±0.5 p
1597 THE HUMAN INTESTINAL MUCIN GENE MUC2 ENCODES MULTIPLE MRNA TRANSCRIPTS. Lawrence R. Sternberg, Christopher C. Yunker, Robert S. Bresalier, Henry Ford Health Sci Ctr, Detroit, MI. Background. Human colon cancers and cancer-derived cell lines can differ significantly in the amount of human intestinal mucin (MUC2) synthesized. Quantitative and qualitative differences in colon cancer mucin may also affect the biological behavior of colon cancer cells. A clear demonstration of the molecular mechanisms for these observations is lacking because it is unclear whether single or multiple MUC2 mRNA species are translated into protein. Complicating factors include the large size and repetitive nature of the MUC2 gene and its mRNA and protein products, allelic variation between MUC2 loci and difficulties in the stable propagation of MUC2 cDNAs containing MUC2 repetitive sequences (TRI and TR2). A complete set of MUC2 cDNAs has yet to be assembled, but a hypothetical eDNA sequence has been proposed that spans 15,721 base pairs including a 5,200+ amino acid open reading frame. Methods. Western analyses using antibodies directed against different portion of MUC2 apoprotein, immunoprecipitation, PCR based methods (RT-PCR, genomic PCR and 3 RACE), eDNA sequencing, multi-probe Northern analyses and antisense inhibition techniques were used to examine whether separable forms of MUC2 are produced in clonal derivatives of human colon cancer cells as well as the mechanism(s) involved. Results. Western analyses and subtractive immunoprecipitation indicate that two distinct classes of MUC2 proteins are produced, one containing TR2 and another lacking this domain. RT-PCR and Northern blot analyses demonstrate the expression of corresponding mRNA species. cDNA sequencing indicates that one of these species is produced by alternate splicing and suggests that another arises by alternative polyadenylation. Genomic PCR data indicate that these results cannot be explained by simple allelic polymorphism. Subsequent Northern hybridization experiments confirm expression of multiple MUC2 encoded transcripts in normal colon. Antisense inhibition studies indicate the potential for selective inhibition of individual MUC2 encoded mRNA species. Conclusion . The human MUC2 gene encodes multiple mRNA transcripts and apoprotein products. These data have important implications for the study of normal and neoplastic processes in the gastrointestinal tract. Supported by NIH grant ROlCA69480 and the Research Service of Henry Ford Health Sciences Center.