Efficient detection of hereditary nonpolyposis colorectal cancer gene carriers by screening for tumor microsatellite instability before germline genetic testing.

BACKGROUND & AIMS: The optimal strategy for the detection of hereditary nonpolyposis colorectal cancer (HNPCC) gene carriers remains uncertain. We evaluated whether microsatellite instability (MSI) analysis or MSH2 and MLH1 protein immunostaining of tumors will screen individuals efficiently for germline MSH2 and MLH1 testing. METHODS: We performed a case-series study of 114 eligible families enrolled in our high-risk colorectal cancer (CRC) registry. Medical history data were collected on probands and relatives. MSI analysis was performed on proband tumors, and MSH2 and MLH1 protein immunostaining was assessed. Denaturing gradient gel electrophoresis was used to identify germline MSH2 or MLH1 mutations in probands found to have tumors with high-frequency MSI. RESULTS: Tumor tissue and adequate clinical data were available in 109 of the 114 families. Amsterdam criteria and Bethesda guidelines were met by 23% and 70% of the families, respectively. High-frequency MSI was identified in the proband tumors in 47 of the 109 families (43%). Germline MSH2 and MLH1 gene testing was carried out in the probands of 32 of 47 families with MSI-H tumors. Mutations were detected in 16 families (9 in MSH2 and 7 in MLH1) and sequence variants of uncertain significance in 5 families (1 in MSH2 and 4 in MLH1). Germline mutations or sequence variants of uncertain significance were detected in 15 of 19 (79%) of our Amsterdam families and in 6 of 13 (46%) of our non-Amsterdam families with MSI-H tumors. MSH2 and MLH1 protein immunostaining was assessed in 38 of the 47 MSI-H tumors. Unequivocal loss of hMLH1 expression was found in 20 tumors and loss of MSH2 expression in 9 tumors. Corresponding loss of protein expression was seen in 17 of 18 (94%) of tumors from probands with germline mutations or variants. CONCLUSIONS: The detection of high-frequency MSI or the loss of MSH2 or MLH1 immunostaining in CRCs are both useful criteria for selecting high-risk patients who should be tested for germline mutations in MSH2 or MLH1.

Phorbol 12-myristate 13-acetate induces alteration in mucin gene expression and biological properties of colon cancer cells.

Phorbol esters such as phorbol 12-myristate 13-acetate (PMA) have been reported to modulate diverse cellular responses through signal transduction pathways including the protein kinase C (PKC) pathway. In the present study, we sought to determine the effect of PMA on mucin gene expression and on the biological properties of a human colon cancer cell line, HM3. The cells were treated for 8 and 24 h with various concentrations of PMA and total RNA was extracted and Northern and slot blot analyses were carried out using MUC2, MUC3 and MUC5AC mucin cDNA probes to assess the steady state levels of mRNA. Spent media were collected and the level of cancer associated carbohydrate antigens (T, Tn, sialyl Tn, sialyl Lex, and sialyl Lea) and matrix-degrading metalloproteinase (MMPs) activity were examined. Trypsinized cells were used for assessing in vitro invasion, motility and adhesion to matrigel. Our results showed that PMA caused upregulation of steady state mRNA levels of MUC2, MUC3 and MUC5AC which was inhibited after treatment with protein synthesis inhibitors. Calphostin C, a highly specific inhibitor of protein kinase C significantly inhibited the PMA induced induction of mRNA levels of MUC2, MUC3, and MUC5AC. The levels of all cancer-associated mucin carbohydrate antigens examined in the media were increased by PMA treatment. PMA also caused an increase in MMPs activity and in in vitro invasion and motility properties, but did not affect adhesion of HM3 cells to matrigel. Thus, PMA caused a significant increase in the expression of all three mucin genes through signaling pathways involving protein kinase C and increased secretion of mucin associated carbohydrate antigens. These changes were associated with increases in MMP activity as well as by increases in the invasive and motility properties of HM3 colon cancer cells. These data suggest that protein kinase C signaling pathways may be involved in mucin gene regulation and in modulating the invasive and metastatic properties of colon cancer cells.

Biological properties and expression of mucins in 5-fluorouracil resistant HT29 human colon cancer cells.

We have previously reported that HT29 human colon cancer cells selected by adaptation to 5-fluorouracil (5FU) (HT29-5FU cells) express increased levels of a major intestinal mucin MUC2 mRNA compared with parental HT29 cells. In this study, we examined in detail the changes in synthesis and secretion of mucin that occur in these cells and accompanying changes in the expression of cancer associated mucin related carbohydrate antigens and cell lineage associated biochemical markers. We further investigated their relationship to biological properties of cells. Northern blot analysis revealed a markedly increased level of MUC2 mRNA but no significant change in the mRNA levels of other mucins in HT29-5FU cells compared with parental HT29 cells. Labeling with radiolabeled precursors demonstrated increased synthesis and secretion of mucin glycoproteins by HT29-5FU cells. Immunoblot analysis showed a higher expression of mucin associated carbohydrate antigens such as T, Tn, sialyl Tn, sialyl Lea, sialyl Lex and non-O-acetylated sialic acid concomitant with significant increases in the expression of goblet cell lineage marker, MUC2 apomucin and a panepithelial cell marker, carcinoembryonic antigen. HT29-5FU cells showed significantly higher adhesion to E-selectin and to matrigel and in vitro invasive properties and significantly increased liver colonization capacity in nude mice following splenic vein injection. Nude mouse xenograft tumors produced by HT29-5FU cells showed a greater degree of differentiation, consisting of mucin secreting glands than those produced by parental HT29 cells. These results indicate that predominantly colonic type mucin, MUC2, has been selectively induced in HT29-5FU cells and that altered regulation of mucin genes associated with altered synthesis and secretion of mucin glycoproteins and the degree of differentiation in cancer cells may be responsible for the altered biological properties of these cells.

Chemoprevention of colon cancer carcinogenesis by balsalazide: inhibition of azoxymethane-induced aberrant crypt formation in the rat colon and intestinal tumor formation in the B6-Min/+ mouse.

Non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin have been shown to suppress colon carcinogenesis and in some cases reduce the size of colorectal polyps. Balsalazide disodium (BSZ) is a colon-specific prodrug of the salicylate, 5-aminosalicylic acid. The aim of the present study was to test the chemopreventive activity of BSZ in two established animal models of colon tumorigenesis, azoxymethane-induced aberrant crypt formation in the rat and intestinal tumor formation in the B6-Min/+ mouse. Aberrant crypt foci (ACF) were induced in Fischer 344 rats via 2 subcutaneous injections of azoxymethane (20 mg/kg). BSZ was supplied in the drinking water for 8 weeks and ACF quantitated. B6-Min/+ mice were treated from 55 days of age for 90 days and intestinal tumors scored for number, size and location. BSZ treatment of AOM-injected rats reduced ACF formation in a dose-dependent manner by 60% with the greatest effect observed on ACF with 4 or more crypts. In B6-Min/+ mice a dose-dependent reduction of intestinal tumor number was observed which reached 80% in the distal small intestine and colon. A preliminary mechanistic study in cultured human colon cancer cells showed that both BSZ and 5-ASA inhibited colon cancer cell proliferation in vitro. However, 5-ASA but not BSZ produced changes consistent with the induction of apoptosis. BSZ produces a dose-dependent chemopreventive effect on colon carcinogenesis. A possible mechanism is consistent with the inhibition of cellular proliferation and the induction of apoptosis.

Fifty landmark discoveries in gastroenterology during the past 50 years. A brief history of modern gastroenterology at the millennium: Part II. Gastrointestinal motility, nutrition, and diseases of the lower gastrointestinal tract, liver, and pancreas.

During the last half century, many outstanding discoveries have revolutionized the clinical practice and science of gastroenterology. Although the scientific results are widely disseminated, the discoverers have received inadequate recognition and the history of their discoveries is largely unstudied and unknown. At the millennium, a committee selected 50 landmark discoveries in gastroenterology during the past 50 years. A brief history of each landmark discovery is presented. Part I was presented in the previous issue of Gastroenterology Clinics of North America. Part II presents landmark discoveries in gastrointerintal (GI) motility, clinical trials, nutrition, and diseases of the lower GI tract, liver, biliary tree, and pancreas.

Fifty landmark discoveries in gastroenterology during the past 50 years. A brief history of modern gastroenterology at the millennium: Part I. Gastrointestinal procedures and upper gastrointestinal disorders.

During the last half century, many outstanding discoverers have revolutionized the clinical practice and science of gastroenterology. Whereas the scientific results are widely disseminated, the discoverers have received inadequate recognition, and the history of their discoveries is poorly known. At the millennium, a committee selected the 50 landmark discoveries in gastroenterology during the past 50 years. A brief history of each landmark discovery is presented. Part I presents the landmark discoveries in gastrointestinal (GI) procedures and in upper GI disorders. Part II of this presentation, which covers landmark discoveries in other areas of gastroenterology, will publish in Part II of the volume on High Risk Gastrointestinal Bleeding.

Genetic testing in hereditary colorectal cancer: indications and procedures.

Approximately 25% of colorectal cancers occur in younger individuals or those with a personal or family history of the disease, suggesting a heritable susceptibility. The minority of these cases are accounted for by one of the well-described hereditary colorectal cancer syndromes, familial adenomatous polyposis (FAP) and hereditary nonpolyposis colorectal cancer (HNPCC). The recent identification and cloning of the genes responsible for FAP and HNPCC, along with other colon cancer susceptibility genes, has led to the wide-spread availability of genetic testing for hereditary colorectal cancer. Genetic testing raises clinical, ethical, legal, and psychosocial questions that must urgently be discussed. This review highlights areas of knowledge and uncertainty about genetic predisposition testing for colorectal cancer and provides clinicians with practical recommendations regarding the proper indications and procedures for this testing.

Mucins secreted by cell lines derived from colorectal mucinous carcinoma and adenocarcinoma.

Mucinous (colloid) carcinoma and well- to moderately-differentiated adenocarcinoma of the colon differ in the pattern and the amount of mucin secretion and perhaps in their behaviour and clinical outcome. To ascertain why these differences exist and to elucidate the mechanisms of tumour progression, we examined two model human cell lines derived from colorectal mucinous carcinoma (C1a) and moderately differentiated adenocarcinoma (HM3) which show typical pathological and mucin staining patterns of the respective type of carcinomas to nude mouse tumour xenografts. Specifically, we sought to determine if there were quantitative and qualitative differences in mucin synthesis, in mucin gene expression and in biological properties between the two model cell lines. Northern blot analysis showed that MUC2 mRNA levels were significantly higher in C1a cells compared with HM3 cells, while those of MUC3, -5 and -6 mRNA were lower. C1a cells secreted approximately five times more radiolabelled apomucin and 1.5 times more glycosylated apomucin than HM3 cells. When the carbohydrate side-chain length of secreted mucins by these cell lines were examined by beta-elimination followed by P4 column chromatography, C1a mucins had mostly short carbohydrate side-chains, while HM3 cells had predominantly longer side-chains. Western blot analysis of the cell homogenate showed higher expression of MUC2 apomucin and mucin-associated carbohydrate antigens, such as T, Tn and sialyl Tn, with decreased sialyl Le(x) expression in C1a cells compared with HM3. Immunohistochemical analysis of 35 colorectal adenocarcinoma and 25 mucinous colorectal carcinoma tissues also demonstrated increased MUC2 apomucin, T, Tn and sialyl Tn antigens in the mucinous cancer specimens. Examination of the biological properties of these cell lines showed that C1a cells had significantly higher in vitro invasive activity in assays of invasion and collagenase activity and significantly lower E-selectin binding and liver colonisation activities in nude mice. These results indicate that colorectal mucinous carcinoma cells differ considerably from colorectal adenocarcinoma cells, both qualitatively and quantitatively, in the pattern of mucin gene expression and in the synthesis and secretion of mucin. In addition, biological studies showed that mucinous carcinoma cells have a greater degree of invasiveness, but less liver colonising activity. These results suggest that the biological and mucin characteristics of mucinous carcinoma cells contribute to extensive local invasion through tissue stroma as the predominant mechanism of tumour progression, while the biological and mucin characteristics of well- to moderately-differentiated colorectal adenocarcinoma contribute to progression via distant metastasis formation.

Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis.

BACKGROUNDS & AIMS: Patients with mucinous colon cancers often have a poor prognosis. The aim of this study was to determine whether metastatic potential depends on specific alterations in mucin-associated carbohydrate structures. METHODS: A quantitative scoring system was used to examine the expression of mucin-associated carbohydrates in paired human primary colon cancers and metastases and in cecal tumors and liver metastases from an animal model of metastasis. Adhesion of metastatic cells to basement membrane and endothelial ligands was examined. RESULTS: Metastases expressed a decrease in mucin core structures Tn and T, a reciprocal increase in sialyl T and sialyl Tn, and an increase in peripheral sialyl Le(x) compared with the primary tumors from which they arose. Altered expression of sialylated mucin structures resulted from selective metastasis of cells that produce sialomucins. Antibodies to sialylated epitopes or desialylation inhibited adhesion of metastatic cells to basement membranes. Neutralizing antibody to endothelial-associated E-selectin (a ligand for sialyl Le(x)) inhibited adhesion of metastatic cells to cytokine-activated hepatic endothelial cells, and inhibition of sialomucin with antisense to the MUC2 gene inhibited adhesion to E-selectin. CONCLUSIONS: Increased sialylation of mucin-associated carbohydrates is characteristic of colon cancer cells that are most likely to metastasize. Sialylated carbohydrate structures on mucin play a role in adhesive interactions involving both basement membrane and endothelial-associated ligands.

Screening for colorectal cancer.

In the past few years, knowledge of the clinical, biologic, and molecular genetic characteristics of colorectal cancer has greatly increased. Although the most cost-effective approach remains to be identified, screening for colorectal cancer can decrease mortality due to this disease by detecting cancers at earlier stages and allowing the removal of adenomas, thus preventing the subsequent development of cancer. Molecular studies that have helped define the genetic basis for this disease hold great promise for the development of better and more powerful methods to identify populations at risk. Individually, these technological, clinical, and basic-science advances are exciting; together, they promise to move us closer to the goal of substantially reducing mortality due to colorectal cancer.

Effects of sodium butyrate on human colonic adenocarcinoma cells. Induction of placental-like alkaline phosphatase.

We have examined the effects of the "differentiating agent," sodium butyrate, on the induction of alkaline phosphatase in human colonic tumor cell line LS174T. Culture of these cells in the presence of 2 mM butyrate caused this activity to increase from less than 0.0001 unit/mg of protein to greater than 0.7 unit/mg of protein over an 8-day period. This induction proceeded in a nonlinear fashion with a lag time of 2-3 days occurring before enzymatic activity began to rise. These increases in activity were accompanied by elevations in the content of a placental-like isozyme of alkaline phosphatase as demonstrated by "Western" immunoblots. Dome formation, indicative of differentiation in cultured cells, also required 3 days treatment with butyrate before becoming evident. The rate of biosynthesis of the enzyme, examined using metabolic labeling with L-[35S]methionine and immunoprecipitation, was found to increase continuously between days 2 and 6 of butyrate treatment. "Northern" blot analysis indicated that treatment of these cells with butyrate caused greater than 20-fold induction of a 2700-base mRNA that hybridized to a cDNA probe for placental alkaline phosphatase. The mRNA for alkaline phosphatase produced by these cells upon butyrate treatment was approximately 300-400 bases smaller than the mRNA for alkaline phosphatase found in placenta. Human small intestine also contained two mRNAs that hybridized relatively weakly with the placental alkaline phosphatase probe. These results indicate that a placental alkaline phosphatase-like protein and mRNA are induced by butyrate in LS174T cells with a time course consistent with cellular differentiation preceding induction.

Comparison of brush border membrane glycoproteins and glycoenzymes in the proximal and distal rat small intestine.

Brush border membranes isolated from the proximal and distal portions of the rat small intestine were examined to see whether qualitative differences exist in their glycoprotein constituents. After SDS-polyacrylamide gel electrophoresis distinct differences were observed, indicating that the protein and glycoprotein profiles of the distal intestine are less complex. A competitive radioassay of lectin receptors revealed that there are significantly more wheat germ agglutinin and succinylated wheat germ agglutinin receptors present on brush border membranes from proximal intestine as compared to distal intestine. However, binding of Ricinus communis agglutinin I to brush border membranes of distal intestine was 2-times higher than that of proximal intestine. These segmental differences were also reflected in the binding patterns of individual brush border membrane hydrolases to wheat germ agglutinin and R. communis agglutinin I. Carbohydrate analysis demonstrated that the overall sugar content of brush border membranes is higher in distal intestine, with more galactose and sialic acid residues. No difference was found in the content of N-acetylglucosamine between the two segments. When brush border membranes from both segments were used as acceptors for galactosyltransferase, those from proximal intestine were better acceptors. Neuraminidase treatment significantly enhanced galactose oxidase/sodium borotritide labeling of brush border membranes from distal intestine and altered the electrophoretic mobility of dipeptidyl aminopeptidase IV and aminopeptidase N. No significant changes in labeling or enzyme electrophoretic mobility were noted in brush border membranes from proximal intestine after neuraminidase treatment. These studies indicate that the glycoproteins from brush border membranes of proximal and distal intestine are qualitatively different and that the glycoproteins from distal intestine may have more completed oligosaccharide side chains.

Effect of lectins on the activity of brush border membrane-bound enzymes of rat small intestine.

Highly purified microvillus membrane vesicles isolated from rat small intestine were enriched in sucrase, maltase, and aminopeptidase activities. Approximately 90-95% of each enzyme was released from the membrane fraction by treatment with detergent (Triton X-100) and sonication. Using untreated and solubilized preparations, the effect of lectin binding on the activity of each of the three enzymes was measured. It was observed that wheat germ agglutinin (WGA) and phytohemagglutinin (PHA) dramatically enhanced the activity of membrane-bound maltase but had much less effect on the detergent solubilized enzyme. Under the same conditions aminopeptidase activity was inhibited by WGA and PHA while sucrase activity was not affected. These alterations in enzyme activity occurred at lectin concentrations that also precipitated each solubilized enzyme from solution. Inhibitory sugars prevented the alterations in enzyme activity suggesting that the effect is due to the binding of lectin to specific carbohydrate structures. Enhancement of membrane-bound maltase activity by WGA and PHA was shown to be temperature dependent indicating that the lipid environment of the microvillus membrane may play a role in mediating the lectin effect. A kinetic analysis of the changes in maltase activity induced by these two lectins was due solely to an increase in Vmax. Two other lectins used in this study (concanavalin A and Ricinus communis agglutinin) did not readily precipitate the enzymes in question or alter their activity. These results show that binding of lectins to brush border membranes can induce variable changes in the activity of several membrane associated hydrolases, and suggest that similar changes may occur in vivo in the presence of dietary lectin.

Effect of growth and sodium butyrate on brush border membrane-associated hydrolases in human colorectal cancer cell lines.

The activities of brush border membrane-associated hydrolases such as alkaline phosphatase (Alkpase), aminopeptidase, dipeptidyl aminopeptidase IV (DAP-IV), sucrase, lactase, and trehalase were studied in 14 different human colorectal cancer cell lines. The effect of sodium butyrate, a known differentiating agent, and cell growth on the activities of these enzymes was also examined. All 14 cell lines exhibited brush border membrane enzyme activities, and in general, the activity of Alkpase, aminopeptidase, and DAP-IV was much higher than the disaccharidases. However, the specific enzyme activities varied among different cell lines. The induction of Alkpase activity by sodium butyrate occurred in most of the 14 cell lines (2- to 123-fold), while induction of the other enzyme activities was observed in several (1.5- to 3.5-fold). In some instances, butyrate caused a decrease in enzyme activity. There was no statistically significant correlation between the induction of Alkpase activity and that of other enzyme activities by sodium butyrate. Levels of aminopeptidase and DAP-IV activity were found to be dependent on cell density and increased 3- to 4-fold by the tenth day in most of the cell lines. Sodium butyrate altered the subcellular distribution pattern of the disaccharidases, causing a significant increase in activity associated with the soluble (cytoplasmic) fraction. Other enzymes such as Alkpase and DAP-IV continued to be predominantly associated with the membrane fraction in butyrate-treated cells. These data suggest that brush border membrane hydrolase activity and the effect of sodium butyrate may provide useful information regarding the differentiation of human colorectal cancer cells.

Protein digestion and absorption after massive small bowel resection.

The digestion, absorption, and intestinal propulsion of a 14C-labeled protein meal were examined in rats with a 70% proximal small bowel resection at one and at six weeks after surgery. In addition, rats were studied one week after 70% small bowel resection plus ileocecal valve bypass. Comparison with sham-operated controls showed no significant impairment of digestion or absorption of the protein meal in both groups of resected rats. Intestinal transit was measured by a nonabsorbable marker 51CrCl3, and there was no evidence of early appearance of the meal in the colon even in resected animals with ileocecal valve bypass. We conclude that, as early as one week after massive proximal small bowel resection, significant compensatory adaptative and motility changes have occurred, allowing protein digestive and absorptive function to be maintained.

Intestinal assimilation of a proline-containing tetrapeptide. Role of a brush border membrane postproline dipeptidyl aminopeptidase IV.

The mechanism of hydrolysis and absorption of a proline-containing tetrapeptide, Leu-Pro-Gly-Gly (10 mM) by rat intestine was examined in vivo by using jejunal perfusion methods. The peptide substrate and hydrolysis products were analyzed by use of an automated amino acid analyzer. Leucine, proline, and glycine were absorbed by the intestine at a significantly higher rate from the tetrapeptide than from an equivalent amino acid mixture. The analysis of the hydrolytic products in the lumen during in vivo perfusion of the tetrapeptide showed that two dipeptides, Leu-Pro and Gly-Gly, were the major products. These two dipeptides were also the major hydrolytic products when a purified rat intestinal brush border membrane preparation was incubated with Leu-Pro-Gly-Gly. The rate of hydrolysis of the tetrapeptide was much higher than that for several other proline-containing peptides (Leu-Pro, Pro-Leu, and Pro-Gly-Gly) that were tested. Studies using Gly-Pro-beta-naphthylamide, a specific substrate for postproline dipeptidyl aminopeptidase IV, showed that this enzyme is mainly localized to the brush border membrane and is responsible for the hydrolysis of the tetrapeptide into the two dipeptides Leu-Pro and Gly-Gly. Thus, brush border membrane dipeptidyl aminopeptidase IV very likely plays an important role at the intestinal mucosal cell surface in the final stages of digestion of proline-containing peptides.