ABO blood type predicts the cytolocalization of anti-P-glycoprotein monoclonal antibody reactivity in human colon and ureter.

Classic multidrug resistance is mediated by a P-glycoprotein. Using monoclonal antibody C219 (MAb C219) in an immunohistochemical study, we found high levels of putative Golgi P-glycoprotein in normal columnar and transitional epithelium in subpopulations of patients with specific blood types. For example, Golgi staining was present in blood type A patients in 46% of normal colon samples (N = 21) and 88% of normal ureter samples (N = 17). In comparison, Golgi staining was present in blood group O patients in only 6% of normal colon samples (N = 34) and in 0% of normal ureter samples (N = 19). The association of MAb C219 Golgi staining with blood type A and lack of Golgi staining with blood type O was statistically significant in normal colon (P = .001) and normal ureter (P less than .0001). Inappropriate hyperexpression of P-glycoprotein was frequently found in colon carcinomas. Additional evidence that Golgi MAb C219 reactivity represents P-glycoprotein is presented. This includes (1) immunostaining of Golgi with two anti-P-glycoprotein MAbs, C219 and JSB-1, and (2) experiments in which Mab C219 Golgi reactivity was blocked by preincubation of MAb C219 with a specific P-glycoprotein epitope-containing peptide. The high degree of association of Golgi P-glycoprotein with blood type A may suggest a role for P-glycoprotein in processing or trafficking of specific blood group antigens.

Changes in glycosaminoglycan synthesis and in heparan sulfate deposition in human colorectal adenocarcinomas.

Biosynthesis of glycosaminoglycans (GAGs) was studied in morphologically normal colonic mucosa, in peritumoral and tumoral areas, and in colorectal polyps of tumor-bearing patients. After GAG purification, overall biosynthesis was determined: the general trend was a decrease in GAG production in neoplastic colon, lowest GAG synthesis being observed in Dukes' stage C tumors. Separation by ion-exchange chromatography of various GAG species and further characterization revealed the presence of hyaluronic acid (HA) and heparan sulfate (HS) molecules in all specimens studied. Chondroitin-4 sulfate (CS4) was occasionally found in tumor samples. The relative proportion of HA and HS was modified in tumor tissue: i.e. increased HA and decreased HS were observed. Differences in DEAE-chromatographic behavior were obvious in pathological samples as compared to controls, the hydrodynamic form of HA and the charge density of HS being decreased. The latter could be attributed to undersulfatation of HS molecules. Immunocytochemical detection of HS proteoglycan molecules revealed regular and bright labelling at epithelial-stromal interface in control samples. In pathological samples, staining was patchy and discontinuous, showing large areas of basement membrane interruption.

Binding of tissue plasminogen activator to human aortic endothelial cells.

The experiments described in this paper were designed to examine the specific binding of tissue plasminogen activator (tPA) to cultured human aortic endothelial (HAE) cells. When 125I-labelled tPA was incubated with the cells at 4 degrees C, binding was found to plateau within 90 min after incubations were begun. Binding was saturable and the bound enzyme dissociated from the sites with a half-time of approx. 48 min. Scatchard analyses were performed using tPA molecules isolated from human melanoma and colon cells as well as from C127 and Chinese hamster ovary cells that had been transfected with the human tPA gene. These enzymes showed very similar binding characteristics in spite of the fact that they differ substantially in the types of sugars which comprise their side chains. Neither the chainedness of the molecules (one-chain or two-chain) nor the sites at which they are glycosylated (type I or type II) appear to affect their ability to interact with binding sites. The tPA molecules were found to have an average equilibrium dissociation constant of (1.15 +/- 0.10) x 10(-9) M and HAE cells appeared to have a single, homogeneous population of independent binding sites present at a concentration of (1.57 +/- 0.13) x 10(6) sites per cell. Lowering the pH of the binding buffer from 7.4 to 6.5 resulted in a reversible increase in specific binding of between 2-fold and 7-fold depending upon the particular preparation of cells. Preincubation of tPA with plasminogen activator inhibitor 1 (PAI-1) was found to have little effect on binding, suggesting that tPA interacts at sites distinct from surface-bound PAI-1. No evidence for either internalization or degradation of tPA was observed in assays run at 37 degrees C. This suggests that, like urokinase, tPA remains on cell surfaces for an extended period of time.

Second generation monoclonal antibodies to the human integrin alpha 6 beta 4.

Second generation monoclonal antibodies to the alpha 6 beta 4 subunits of human integrins have been prepared. MAbs 450-9D, 10D, and 11A1 react at different sites on the beta 4 molecule and MAbs 450-30A1 and 33D react at the same site on the alpha 6 subunit. Double determinant (two-site) radioimmunoassays using combinations of these MAbs have been developed. Two assays for beta 4 distinguish between the whole beta 4 molecule and the beta 4 molecule truncated from the C-terminus (form c) while another assay measures the presence of alpha 6 subunits. Data from the two-site assays support the following conclusions: (1) Colon tumors and normal colon mucosa express large amounts of alpha 6 beta 4 although only form c of the beta 4 was detected; (2) There is no evidence for alpha 6 beta 1 expression in colon; however, some of this complex may be present in certain lung tumors. The extracellular domains of alpha 6 and beta 4 can associate with each other even if the cytoplasmic domain of the beta 4 subunit is not present. MAbs to specific domains of the beta 4 molecule may be useful in analyses of forms a and c in normal and malignant tissue. The fact that only the largest beta 4 molecule "a" retains the phosphorylation site may have functional significance.

Increased colonic mucosal angiotensin I and II concentrations in Crohn's colitis.

To define a potential role for the angiotensin system in Crohn's colitis, the colonic mucosal levels of angiotensin I and II were measured in endoscopic biopsy samples from patients with active Crohn's colitis (n = 20), ulcerative colitis (n = 13), other forms of colitis (n = 3), and normal controls (n = 17). Colonic mucosal levels of angiotensin I and II were greater in patients with Crohn's colitis than in normal subjects (p less than 0.001 and p less than 0.001, respectively). Mucosal levels of angiotensin I and II were also higher in Crohn's colitis than in ulcerative colitis (p less than 0.001 and p less than 0.001, respectively), and levels of angiotensin II were higher in Crohn's than in other forms of colitis (p = 0.014). Mucosal levels of angiotensin I and II correlated well with the degree of macroscopic inflammation in Crohn's colitis (r = 0.86, p less than 0.001 and r = 0.68, p less than 0.001, respectively). Mucosal levels of angiotensin I correlated fairly well with the Crohn's Disease Activity Index (r = 0.46, p less than 0.05) while angiotensin II levels correlated poorly. These studies suggest that angiotensin I and II may have a role in the inflammation associated with Crohn's colitis.

The carbohydrate composition of mucin in colonic cancer.

Mucins synthesized in colonic cancer are known to be different from those in the normal colon; however, the biochemical differences between these mucins have not been defined. We have purified mucins from samples of nonneoplastic (normal) human colon and colon cancer and found that the carbohydrate content of the cancer-associated mucins is 48% of that in the normal colon, including significant reductions in galactose, N-acetylglucosamine, N-acetylgalactosamine, and fucose. By subjecting the mucins to alkaline degradation, we determined that there are 19% fewer oligosaccharide chains per milligram of cancer-associated colonic mucin than there are in mucins from normal colons. We also found a reduction in mean oligosaccharide chain length in cancer-associated mucin (5.83 carbohydrate residues per chain) compared with those derived from normal colons (10.2 residues). Total and individual amino acid contents were greater in cancer-associated mucins, with the exception of three amino acids (threonine, serine, and proline), two of which represent the O-linked glycosylation sites for glycoproteins. Thus, mucins are aberrantly glycosylated in colon cancer, both in terms of the number and mean chain length of the oligosaccharide moiety. Because of their relative abundance in colonic tissue, mucins appear to be useful molecular species in the study of the derangements in protein glycosylation that occur during neoplasia.

Tissue carcinoembryonic antigen and DNA aneuploidy in precancerous and cancerous colorectal lesions.

Chronic inflammatory bowel disease (CIBD) and colorectal adenoma are considered as precancerous conditions and lesions of large bowel carcinoma, respectively. They, therefore, may be used to study the behavior of such different factors as tumor-associated antigens and nuclear DNA content abnormalities in colorectal carcinogenesis. Tissue concentrations of carcinoembryonic antigen (CEA) were significantly higher in those precancerous lesions (CIBD: 61 +/- 11.2 ng/mg, adenoma: 70 +/- 6 ng/mg; mean +/- standard error of the mean) than in normal colonic mucosa (36 +/- 4.7 ng/mg). Colorectal carcinoma had still higher tissue levels (437 +/- 108.2 ng/mg). No correlation between tissue CEA and tumor differentiation could be found, but there was a significant difference between aneuploid (747 +/- 354 ng/mg) and diploid (139 +/- 43 ng/mg) tumors. Using flow cytometry DNA aneuploidy was present in 31.6%, 10.5%, and 51.6% of CIBD, colorectal adenoma, and carcinoma, respectively. These data suggest that the occurrence of aneuploidy is not strongly dependent on a malignant transformation, but it may also be present in premalignant colorectal lesions without cellular dysplasia.

The effect of endotoxin on 1,2-dimethylhydrazine-induced colonic tumors in rats.

The effect of endotoxin on colon tumors was studied in male Sprague-Dawley rats. Colon tumors were induced in weanling rats by the administration of 20 weekly subcutaneous injections of 1,2-dimethylhydrazine (DMH). When colon tumors were detected by colonoscopy in 80% of the rats around week 24 after DMH injection, the animals were divided randomly into two groups. One group served as the control. The other group received six endotoxin (Escherichia coli) treatments every fifth day. The first dose was 50 micrograms/100 g (intraperitoneally); the remaining doses were 100 micrograms/100 g (subcutaneously). Rats were killed 2 weeks after the last endotoxin injection. Endotoxin treatments resulted in larger colon tumors. The median tumor size was 71 mm2 for endotoxin-treated and 31 mm2 for untreated rats (P less than 0.02). Endotoxin treatments also resulted in a significantly higher incidence (P less than 0.05) of ulcer development in the small intestine, that is 47% in the endotoxin-treated versus 23% in the untreated rats. After a single subcutaneous injection of endotoxin (100 micrograms/100 g), the colon mucosal reduced glutathione (GSH) level was raised by 21% at 16 hours, reached a peak on day 2, then decreased to baseline by day 4. The increased GSH level in the colon mucosa was maintained up to the third endotoxin injection. By the fifth injection, no increase in the GSH level was observed. These results suggest that the growth of colon tumors in rats induced by DMH could be enhanced by endotoxin treatments. The enhanced tumor growth may be due to an increase in the colon GSH level and/or other mediators released by macrophages as a result of endotoxin treatments.

Expression of two types of receptor for insulinlike growth factors in human colonic epithelium.

The presence of receptors for insulinlike growth factor I and II in human colonic epithelium is demonstrated. Scatchard analysis of binding data obtained with 125I-insulinlike growth factor I showed an insulinlike growth factor I receptor with a dissociation constant of 8.6 nM and a binding capacity of 0.8 pmol/mg membrane protein. Distinct insulinlike growth factor II receptors labeled with 125I-insulinlike growth factor II were also found with a dissociation constant of 6.9 nM and a binding capacity of 4.7 pmol/mg membrane protein. Two sets of observations make it possible to discriminate between the two types of insulinlike growth factor receptors. (a) Unlabeled insulinlike growth factor I was 3 times more potent than insulinlike growth factor II in inhibiting [125I] insulinlike growth factor I binding. Conversely, unlabeled insulinlike growth factor II was 10 times more potent than insulinlike growth factor I in competing with 125I-insulinlike growth factor II for binding to membranes. Insulin and proinsulin did not compete with either of the tracers. (b) Affinity labeling of membranes followed by sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions, revealed a radio-ligand-receptor complex of molecular weight 130,000 and 250,000 using 125I-insulinlike growth factor I and 125I-insulinlike growth factor II, respectively. These observations indicate that adult human colonic epithelium is abundantly equipped with two sets of receptors that recognize preferentially either insulinlike growth factor I or insulinlike growth factor II.

Intracellular free amino acid patterns in duodenal and colonic mucosa.

We report for the first time the concentrations of free amino acids in human intestinal biopsies obtained by routinely performed endoscopy. We studied 15 medical patients with no changes of the mucosa and six HIV-infected persons with duodenitis. The mean (and SD) sum of all amino acids, taurine excepted, was 61.9 (5.4) mmol/kg dry weight in duodenal biopsies of HIV-negative subjects (n = 11) and 82.9 (0.6) mmol/kg in colonic specimens: 50% (44%) of the total (minus taurine) consisted of aspartate and glutamate and 14% (12%), of the essential amino acids. The relative amino acid pattern in duodenum and colon differed completely from that for muscle: aspartate was fourfold higher; glutamate, phenylalanine, glycine, valine, leucine, and isoleucine were about twofold higher. In contrast, glutamine amounted only to 4% (duodenum) to 14% (colon) of muscle glutamine. In duodenal biopsies of the HIV-infected persons, we found significantly (P less than 0.01, except glutamine: P less than 0.025) increased concentrations of glutamate (24.1 vs 17 mmol/kg dry weight), ornithine (1.4 vs 0.4), valine (2.2 vs 1.7), and glutamine.

Deglycosylation of neutral and acidic human colonic mucin.

Human colonic mucin has been isolated from normal colonic mucosa by a phenol-water extraction procedure and purified by Sepharose 2B column chromatography. The mucin was further purified by cesium bromide density gradient centrifugation. Sodium dodecyl sulfate-polyacrylamide gel (5%) electrophoresis of this material showed high-molecular-weight mucin component(s) at the top of the gel. Chemical analyses of this preparation indicated a typical mucin profile of amino acids and carbohydrates. Ion-exchange chromatography resulted in the separation of two major fractions, one being more acidic than the other. Chemical deglycosylation of the purified preparation at 20 degrees C for 3 1/2 showed loss of sialic acid, fucose, galactose, and N-acetylglucosamine, whereas traces of N-acetylgalactosamine were still detected. High-pressure liquid chromatography of the deglycosylated material resulted in the purification of a major peptide, P1, with high levels of threonine, serine, and proline, resembling, in most respects, the profile of native mucin. The molecular weight of the peptide was determined to be approximately 97 kDa and serine was the single NH2 terminus.

Polyamines in colorectal cancer. Evaluation of polyamine concentrations in the colon tissue, serum, and urine of 50 patients with colorectal cancer.

Total, free, and acetylated polyamine concentrations were measured simultaneously in colon tissue, serum, and urine of 50 patients with histologically proven colorectal cancer, 40 patients with nonmalignant gastrointestinal diseases, and 30 healthy volunteers. Compared with histologically unaffected colon tissue, concentrations were significantly (P less than 0.001) higher for putrescine, elevated for cadaverine, and nearly identical for spermidine and spermine in colon carcinoma, whereas N1-acetylated and N8-acetylated spermidine were detectable in cancer tissue only. Serum and urine concentrations of all polyamines except total cadaverine and spermine in serum and free spermine in urine were significantly elevated compared with healthy controls and highest sensitivity for colon cancer was found for total spermidine (89.15%) in serum and acetylputrescine (84.5%), total putrescine (84.0%), N1-acetylspermidine (79.3%), and total spermidine (92.1%) in urine. However, nonmalignant gastrointestinal diseases partly showed similar elevations which resulted in a low specificity for polyamines in colorectal cancer. Therefore, polyamines are of little value only as diagnostic markers in colorectal carcinoma. Since polyamine concentrations in serum and urine normalized in patients after curative operation while they were further elevated in patients with proven tumor relapse or metastases, these substances might play a clinical role in predicting therapeutic success or indicating relapse of the tumor. Although a significant dependency of polyamine concentrations in serum or urine to Dukes' classification, tumor localization, CEA, CA 19-9, or CA 125 did not exist, a significant linear correlation was found for tumor size.

c-myc overexpression is a tumor-specific phenomenon in a subset of human colorectal carcinomas.

The transcriptional activity of the c-myc proto-oncogene was examined in 25 primary human colorectal carcinomas and their corresponding normal mucosae. The purpose was to determine whether the elevated levels of c-myc expression, frequently detected in this type of tumor, might be the consequence of alterations in the cell growth rate or the effect of a real transcriptional deregulation of the gene. In about 44% of the tumors the elevated c-myc expression was consequent to the enhanced growth rate of the neoplastic tissue, as estimated by the expression of the S-phase-specific histone H3 gene. In the other 56%, c-myc overexpression did not entirely depend on the proliferative activity of the neoplastic population. In this latter group, c-myc deregulation did not reside in structural modifications of the putative regulatory regions of the gene. Therefore, c-myc overexpression, at least in a subset of colorectal cancer, seems to be consequent to alterations in transregulative phenomena exerted on the c-myc gene by other genetic loci.

Transcription of human endogenous retroviral long terminal repeat (LTR) sequence in a lung cancer cell line.

The human genome carries several endogenous retroviral sequences. One of them that we named 'HERV-A', carries almost the complete sequence of the long terminal repeat (LTR), and is located in the 5' region of the amylase genes (M.Emi, A.Horii, N.Tomita, T.Nishide, M.Ogawa, T. Mori and K.Matsubara, Gene 62: 229-235, 1988). Using this sequence as a probe, we found a 1.4 kb LTR transcript(s) in a lung cancer cell line. No corresponding transcript was observed in control cells. Two partial, but different cDNA clones were obtained, and each one was found to be a transcript starting within human sequences at 5' upstream from the LTR and ending within the LTR sequence.

Structural characterization of canine PYY.

PYY was purified from canine colonic mucosa by sequential steps of reverse phase HPLC and ion-exchange FPLC. Microsequence, amino acid and mass spectral analyses of the purified peptide and its tryptic fragments were consistent with the structure: YPAKPEAPGEDASPEELSRYYASLRHYLNLVTRQRY-amide. Canine PYY(1-36) has the identical sequence as porcine and rat PYY but differs from human PYY at position 3, with Ala instead of Ile, and position 18, with Ser instead of Asn. A smaller form, PYY(3-36), was also purified and characterized. It may differ in its biological activity from the intact peptide and could act as a partial antagonist or agonist of PYY(1-36).

Distribution of hyaluronic acid and chondroitin sulfate proteoglycans in the presumptive aganglionic terminal bowel of ls/ls fetal mice: an ultrastructural analysis.

The terminal colon of the ls/ls mouse is aganglionic because an intrinsic defect prevents its colonization by cells migrating from the neural crest. Previous studies showed that laminin, type IV collagen, and glycosaminoglycans accumulate in the region of the presumptive aganglionic ls/ls bowel through which crest-derived cells would be expected to migrate. It was suggested that crest-derived cells might fail to enter the abnormal bowel because they receive inappropriate signals from a defective extracellular matrix. This hypothesis was evaluated by analyzing the ultrastructure of the extracellular matrix in mutant and control gut. Tissue was fixed in the presence of ruthenium red before or after selective enzymatic digestion. Heparan sulfate proteoglycan (diameter approximately equal to 15 nm) and chondroitin sulfate proteoglycan (diameter approximately equal to 20-50 nm) granules were found in both control and presumptive aganglionic gut. The heparan sulfate proteoglycan granules were primarily located within formed basal laminae, while chondroitin sulfate proteoglycan granules decorated plasma membranes and 5 nm hyaluronic acid microfibrils that formed a network in the extracellular matrix. At day E11.5, the mutant gut differed from the control in the following: 1) Hyaluronic acid microfibrils were longer and more numerous. 2) There were larger numbers of chondroitin sulfate proteoglycan granules associated with cell membranes and with hyaluronic acid microfibrils. By day E13 the spaces between mesenchymal cells of the outer wall of the control bowel contained a regular lattice of hyaluronic acid microfibrils studded with chondroitin sulfate proteoglycan granules. Instead of this lattice, tangles of excessively long hyaluronic acid microfibrils, coated more heavily than in the control with chondroitin sulfate proteoglycan granules, were found in the presumptive aganglionic gut. These results confirm that the extracellular matrix is abnormal in the presumptive aganglionic bowel of the ls/ls mouse; moreover, they also indicate that the defect involves not one, but several components of the extracellular matrix, as well as their distribution. The defective extracellular matrix is apparent at a time when crest-derived cells would be expected to be migrating in the terminal bowel and is located in their path. The observations thus support the idea that a localized abnormality of the extracellular matrix interferes with the colonization of the terminal bowel by crest-derived cells in the ls/ls mouse.

Calbindin D28k is essentially located in the colonic part of the toad intestine.

The distribution of calbindin D28k in the digestive system and the urinary bladder of the toad was investigated using immunohistochemistry and Western blotting. By analogy with mammals and birds, the protein was expected to be located preferentially in the duodenal part of the intestine. Interestingly, absorptive cells of the duodenum were totally devoid of calbindin D28k while the colon contained high amounts of the calcium-binding protein. This reversed polarity of calbindin D28k content in the toad intestine should obviously correspond to a different scheme of calcium absorption regulation between amphibians and higher vertebrates. Calbindin D28k containing neuroendocrine-like cells were found scattered in the proximal parts of the gut with a similar distribution to what has been described in rat and chick intestine. The oesophagus, the stomach, and the intrinsic nervous system of the intestine were negative. No significant amounts of the proteins were found in the urinary bladder, which is known to be a site of Ca2+ active transport.