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In a prospective study, the DNA content of Feulgen-stained nuclei obtained from fresh samples of 211 colorectal adenocarcinomas was evaluated by means of image analysis. The DNA histogram classification took into account aneuploidy and S-phase fraction for diploid cases. No significant relationship was found between ploidy and sex, age, preoperative carcinoembryonic antigen (CEA), size of the tumor, histologic differentiation, or Dukes' stage. Aneuploidy was more frequently encountered in distal tumors. Preoperative CEA, histologic differentiation, Dukes' stage, and ploidy were individually associated with overall survival. In Dukes' A, B, and C tumors, patients with normal and elevated CEA had no significant difference in overall survival. A relationship was apparent between disease-free survival and site, histologic differentiation, Dukes' stage, and ploidy. Multivariate overall survival analysis did not reveal independent prognostic significance of ploidy when all Dukes' stages were considered. In contrast, Dukes' stage, differentiation, and ploidy were good indicators of higher risk of colorectal cancer-related death in patients undergoing curative surgery. Dukes' stage and ploidy were also indicators for recurrence. Thus, routine histopathologic characteristics should be used in combination with quantitative cytologic features for the definition of a relevant prognostic index in colorectal cancer.

The metabolism of human low-density lipoproteins was studied in 2 subpopulations deriving from cells of HT29, a human colon carcinoma cell line. When grown on standard medium (25 mM glucose), about 95% of these cells are undifferentiated (G+ cells). From this heterogeneous population, a subpopulation with features of differentiated small-intestinal cells was selected by glucose deprivation (G- cells). The characteristics of the LDL receptor were first investigated. The results showed that the binding of 125I-LDL to G+ and G- cells performed at 4 degrees C was saturable and specific. The Kd values were not statistically different in the 2 cell subpopulations. The Bmax of G+ cells was 55 +/- 6 ng 125I-LDL/mg cell protein and showed no changes whatever the phase of culture. In G- cells, the Bmax was higher during the exponential phase of culture and decreased in the post-confluent phase (82 +/- 5 versus 15 +/- 6.8 ng 125I-LDL/mg cell protein). Cellular degradation of 125I-LDL was effective in both cell subpopulations but time-course studies showed that, in post-confluent G- cells, degradation was slowed as compared to G+ cells (4 hr vs. 2 hr to reach maximal degradation). The rate of LDL processing at 37 degrees C was enhanced by pre-incubation with FCS-supplemented medium, suggesting the existence of a serum component which stimulates the total degradation of 125I-LDL. Concerning regulation of the LDL receptor activity, we demonstrated that pre-incubation of G+ cells with LDL induced 80% down-regulation of receptor number in both phases of culture. This was also observed in G- cells during the exponential phase while only a 20% decrease of the receptor number was observed in post-confluent G- cells. The LDL degradation of G+ cells resulted in an inhibition of the cholesterogenic activity by 30% and 60% depending on the phase of culture. In G- cells, LDL pre-incubation inhibited cholesterol synthesis to the same extent (45%) in the exponential phase but did not affect the rate of cholesterol synthesis when cells were confluent. The defective regulatory role of LDL on receptor number and cholesterol synthesis suggests that, in the post-confluent differentiated cells, cholesterol derived from LDL does not reach the regulatory pool. Taken together, our findings indicate the existence of functional LDL receptors in the HT29 cell line, either in the differentiated or in the undifferentiated form.

Cell surface receptors for laminin may play an important role in tumor migration and metastasis. To evaluate laminin receptor/laminin-binding protein expression in human colon carcinoma, surgical specimens of primary colon cancers and liver metastases were examined by blot hybridization of total RNA with a complementary DNA clone which encodes a Mr 32,000 human laminin-binding protein. The mRNA level of the laminin-binding protein was higher in primary colon carcinoma than in adjacent normal colonic epithelium in 20 of 21 cases. In all 6 cases of colon cancer liver metastases, the laminin-binding protein mRNA level was more than 3-fold greater in tumor than in adjacent normal liver tissue. The tumor/normal ratio of this laminin-binding protein mRNA expression in primary colon cancer has significant correlation with Dukes' classification (P less than 0.001). Our results suggest that mRNA expression of the laminin-binding protein may be a marker of human colorectal cancer progression and biological aggressiveness.

In a series of 130 cases of adenocarcinoma of the large intestine, enterochromaffin (EC) cells were detected in 54 cases (41.5%) by immunocytochemistry with anti-chromogranin monoclonal antibody. Among the 54 cases, 30 were found positive for serotonin, 14 for somatostatin, 11 for glucagon, 5 for pancreatic polypeptide, and only one for gastrin. The cases with EC cells (++) or polypeptide positive cells exhibited higher grade of differentiation, earlier stage of tumour extension and higher survival rate than those without EC cells. A significant difference of the EC cell population pattern among different histological grades of the tumours and nonneoplastic mucosa was found. The proportion of hormone, especially polypeptide positive cells was the highest in the mucosa and lowest in the moderately poorly differentiated carcinomas. The incidence, methodology and clinicopathological significance of EC cells found in the tumours are discussed.

Recently, it was reported that there is a significant elevation of total plasma apolipoprotein(a) level in cancer patients. Because of their structural homology and immunological cross-reactivity, localization of the plasminogen and the apolipoprotein(a) was comparatively studied in cancerous tissues of breast (N = 4) and colon (N = 3) by immunofluorescence. The following results were obtained: 1) Tumor cells isolated or in nodules were strongly stained in all cancerous samples using antiserum against Pg, absorbed or not with Lp(a). 2) Tumor cells were lightly stained in two breast carcinomas, using anti-Lp(a) serum. All other carcinomas were negative. The staining was abolished when anti-Lp(a) serum was absorbed with Pg. 3) Blood vessels were strongly stained using antiserum against Lp(a) even when it was absorbed with Pg. Anti-Pg serum decorated only a few capillaries. These results show that the two proteins have different localizations: Lp(a) is seen exclusively in the vascular system. The component present at the surface of tumor cells is plasminogen (or plasmin) but not Lp(a).

Colonic epithelial tumors (101) including villoglandular adenomas, carcinomas in situ, adenocarcinomas, and neuroendocrine (NE) carcinomas were studied immunohistochemically with monoclonal antibodies (MoAb) RAP-5 and RAS-10 recognizing altered and unaltered ras oncogene products. In addition, 20 samples from multiple polyposis including adenomas with and without dysplasia, carcinomas in situ, and invasive carcinomas were studied. Using immunostaining techniques, normal mucosa was weakly stained, whereas the mucosa in the vicinity of tumors or inflammation showed enhanced staining. More tumors stained intensely with MoAb RAP-5 than with MoAb RAS-10. With MoAb RAP-5, most benign and malignant tumors showed enhanced staining. No significant differences in staining were noted in relation to superficial versus deeply invasive carcinomas or clinical staging. Immunostaining was also noted in some metastases. No significant differences in enhanced staining were found in carcinomas. Interestingly, the most extensive and enhanced immunostaining was noted in the villoglandular adenomas, dysplastic adenomas, and carcinomas in situ. The authors conclude that (1) ras protein expression is detectable in most benign, borderline, and malignant epithelial tumors of the colon as determined with MoAb RAP-5 and RAS-10, whereas enhanced expression is more often detected with RAP-5; (2) enhanced ras product expression in colon carcinomas does not seem to correlate with advanced tumor stages or with exocrine, NE, or phenotypically mixed tumors; and (3) the finding of the most intensely enhanced ras products expression in villoglandular polyps and carcinomas in situ suggests a possibly significant role for the oncogene in the early phases of transformation.

The oestrogen receptor content of colorectal adenocarcinoma was investigated using an established ligand binding biochemical assay and two more recently introduced techniques using specific monoclonal antibodies (Abbott ER-EIA and ER-ICA assay kits). Twenty nine tumours were investigated by the ligand binding assay. Only one (3.4%) tumour gave a weakly positive result (11 fmol/mg cytosol protein); the rest were all negative. Where sufficient tissue was available, the receptors were also determined by a quantitative immunoassay in 18 patients and an immunohistochemical method in 13 patients. The results were similarly all negative. It is concluded that most colorectal carcinomas, irrespective of sex, are oestrogen receptor negative, and it is thus unlikely that hormonal manipulation would have an influence on the course of the disease.

Metabolic pathways of glucose utilization have been investigated in a human colon adenocarcinoma cell line (HT29) using carbon-13 Nuclear Magnetic Resonance spectroscopy. HT29 cells were adapted to grow on a polystyrene beaded microcarrier and were perfused when attached to the beads in a specially designed NMR cell. Abnormalities in carbohydrate metabolism already observed in several cancer cells were studied in HT29 cells fed with (1-13C)-enriched glucose. The cells were first perfused with a glucose-free medium for 2 h in order to deplete the intracellular store of glycogen, and they were subsequently perfused with a medium containing enriched glucose at an initial concentration of 5.5 mM. Sequential 13C-NMR spectra, recorded at 100.5 MHz (5 min accumulation), show that HT29 cells were able to utilize glucose through the glycolytic pathway while storing glucose as glycogen (glucose was utilized at a rate of 3.9 mumol/mg protein/hr). The glycolytic activity determined by the amount of lactic acid produced was 4.6 microns/mg protein/hr, corresponding to the formation of 1.2 lactic acid per glucose molecule. Glycogen accumulation corresponded to 16 micrograms/mg of protein. Treatment of HT29 with 10 nM vasoactive intestinal peptide (VIP) induced a transient decrease in the level of labelled glycogen to 50% of the initial value. Control level was recovered 12 min after VIP loading.

Binding characteristics of peripheral benzodiazepine binding sites (PBS) in membrane homogenates of samples taken at abdominal surgery from colonic adenocarcinoma and from healthy colonic tissues in 17 patients were determined. [3H]PK 11195, an isoquinoline carboxamide derivative, which exhibits high affinity to PBS, was used as a radioligand for the binding assay. A robust increase (3.1-fold) was found in membranes from the tumor sites as compared to normal control colon, whereas the affinity of [3H]PK 11195 to PBS was similar in both tissues.

The invasion of malignant cells through the basement membrane is a critical step in local infiltration and metastasis. Adhesion and invasion of malignant cells may be modulated by their receptor mediated binding to the basement membrane glycoprotein laminin. We studied the specific adhesion of human colon adenocarcinoma derived HT 29 cells to laminin and its proteolytic fragments. The major cell adhesion domain of laminin was localised in the central part of the cross shaped molecule. Immunoblotting experiments on separated HT 29 cell membranes using specific antibodies or radiolabelled laminin fragments revealed two major laminin-binding cell surface components with Mr of 67,000 and 69,000 D similar to the putative laminin receptor described for other tissues. Using a nitrocellulose filter disk assay, the specific interaction between cell surface binding proteins and proteolytic fragments originating from the central core of the laminin molecule could be further corroborated. In contrast, interaction of HT 29 cell membranes with the pentapeptide YIGSR (tyr-ile-gly-ser-arg), a sequence domain of the B1-chain of the laminin molecule, thought to be responsible for cell adhesion, was significantly weaker.

Lectins were used to characterize mucin glycoproteins and other secretory glycoconjugates synthesized by a human colon adenocarcinoma-derived cell line which expresses a goblet cell phenotype. Despite being clonally derived, HT29-18N2 (N2) cells, like normal goblet cells in situ were heterogeneous in their glycosylation of mucin. Only wheat-germ agglutinin, which recognizes N-acetylglucosamine and sialic acid residues, and succinylated wheatgerm agglutinin, which binds N-acetylglucosamine, stained the contents of all secretory granules in all N2 goblet cells. The N-acetylgalactosamine binding lectins Dolichos biflorus and Glycine max stained 20% and 21% of N2 goblet cells respectively. Ricinus communis I, a galactose-binding lectin, stained 67% of N2 goblet cells although staining by another galactose-binding lectin, Bandeiraea simplicifolia I, was limited to 19%. Peanut agglutinin, a lectin whose Gal(beta 1-3)GalNAc binding site is not present on mucins produced in the normal colon but which is found on most mucins of cancerous colonic epithelia, stained 68% of the cells. Ulex europeus I, a fucose-binding lectin, did not stain any N2 goblet cells. Four lectins (Lens culinaris, Pisum sativum, Phaseolus vulgaris E, Phaseolus vulgaris L) which recognize sugars normally present only in N-linked oligosaccharides stained up to 38% of N2 goblet cells. The binding of these lectins indicates either both O-linked and N-linked oligosaccharide chains are present on the mucin protein backbone or the co-existence of non-mucin N-linked glycoproteins and O-linked mucins within the goblet cell secretory granule.

Procoagulant activity (PA) and fibrinolytic activity (FA) were investigated in 14 specimens of gastric cancer (GC) and in 26 specimens of colorectal cancer (CC) and simultaneously, in samples of the normal mucosa obtained from the same organs. Histochemical localization of plasminogen activator was performed at the tumor borderline. The PA of the tumor extracts was in most cases lower than in the corresponding normal mucosa from GC as well as CC patients. The FA of the extracts of CC was also lower than that of normal mucosa. On the other hand, 11 of 14 cases with GC showed a significantly higher FA in the tumor extracts than that in the corresponding normal mucosa. The histochemical localization of plasminogen activator revealed that its activity is connected with small and medium-sized blood vessels, but not with the cells of the two kinds of cancer investigated. Morphological and histochemical findings from both kinds of cancer demonstrate that the FA of their extracts depends on the vascularity of specimens of the respective tumors.

The mucosal scrapings of the normal human colons and the colonic adenocarcinoma tissues were digested with pronase, and the glycosaminoglycan fractions were prepared by fractionation with cetylpyridinium chloride after treatment with deoxyribonuclease. Chondroitin sulfate A/C, heparan sulfate, and hyaluronic acid were contained in the glycosaminoglycan fraction of the normal tissue, but dermatan sulfate was undetectable. In contrast, the glycosaminoglycan fractions derived from the tumors contained substantial amounts of dermatan sulfate together with the foregoing three glycosaminoglycans. The total content of glycosaminoglycans per wet tissue weight was elevated in the tumor tissues, which was consistent with histochemical findings.

The synthesis of carcinoembryonic antigen (CEA) in human tumour cell cultures has been investigated at the ultrastructural level in the colon carcinoma cell line LS 174 T. By application of a CEA-specific antibody fraction, gained from goat serum which was directly coupled with horse radish peroxidase (HRP), the expression of CEA could be followed in the cell culture both intra- and extracellularly with ultra-immunohistochemical methods, utilizing the pre-embedding technique. Morphometric and ultra-immunohistochemical investigations showed the possible involvement of the Golgi systems in CEA production. The changes in the volume fraction of the Golgi apparatus related to the total cell volume showed a direct correlation with the CEA content in the cell culture supernatants. Freeze-etch control experiments revealed negligible structural changes in the cells following ethyl-dimethyl-aminopropylcarbodiimide (EDC)-and saponin permeation techniques.

A new approach to the problem of monitoring patients with chronic ulcerative colitis is presented and discussed in connection with a case report. When annual colonoscopies are performed, biopsies are taken for histopathological examination and DNA measurements are made using flow-cytometric techniques (FCM). Using the latter approach, gross chromosomal aberrations indicating malignant transformation in a cell population may be detected. In a 46 year old man with a long history of ulcerative colitis, an area with slight mucosal dysplasia at light microscopy was accompanied by two aneuploid cells lines - that is, colonic mucosa cells with an abnormal amount of DNA in the nuclei. An operation one year later revealed a 5 X 2 mm large adenocarcinoma in the corresponding area of the colon. We suggest that flow-cytometric techniques can be used as a complement to already practised methods for monitoring the colorectal mucosa in colitic patients for the early detection of malignancy.

Tumor tissue samples from 25 patients with adenocarcinoma of the colon, twelve related samples of normal colons as well as five serum specimens from the same patients were analyzed for ferritin. The average ferritin content of the tumor tissue was 788 ng/mcp with a range of 47-1,745 ng/mcp. The average ferritin content of normal colon mucosa was 115 ng/mcp with a range of 32-230 ng/mcp. Two specimens of metastatic colon cancer taken from the retroperitoneal space and liver, respectively, contained 3,867 and 2,827 ng/mcp of ferritin. The ferritin content of the tumor tissue was higher than that of the normal colon in 8 of 9 patients who had specimens obtained from both sites. The amount of ferritin found in tumor tissue was independent of sex, age, and the site of the original tumor. This study shows that the ferritin content of colon neoplasms is elevated and indicates that the tumor tissue may be the direct source of elevated serum levels of ferritin previously observed in cancer patients.

Glycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against gangliotriaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetraosylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used. The glycolipid fractions of each cell line separated by Iatrobeads column chromatography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.

The structure of colonic mucin, which is thought to be important in several diseases, including ulcerative colitis and colon cancer, is poorly understood. Mucin was isolated from nude mouse xenografts of the LS174T colonic adenocarcinoma cell line by gel filtration and CsCl density gradient centrifugation. The isolated mucin had a high content of threonine, serine, and proline, with 28% of the total amino acids O-glycosylated. The carbohydrates present were fucose, sialic acid, galactose, N-acetyl-glucosamine, and N-acetyl-galactosamine in the ratio of 0.4:1.5:1.0:0.9:1.4. Rabbit antibodies were prepared that recognized primarily protein-dependent determinants. By DEAE-cellulose chromatography, the purified mucin was found to be heterogeneous, with three major components that had small differences in carbohydrate composition. LS174T was antigenically and chromatographically similar to mucins in colon cancer tissue specimens and in nonmalignant colonic mucosae.

The distribution of Coomassie blue-stained proteins from uninvolved regions of 4 human livers, from 1 hepatocellular carcinoma, and from 4 samples each of uninvolved colon, primary adenocarcinoma of the colon, and colon cancer metastatic to the liver was analyzed by two-dimensional protein electrophoresis. From a comparison of acidic proteins between pI 3.5 and 6.5, we conclude (1) that the majority (66 of 82) of denoted acidic proteins from 4 normal liver samples were represented in the hepatocellular carcinoma. Fifty-one of 58 proteins denoted in the 4 colon samples were detected in each of the 4 primary colon cancers; (2) that the "normograms" of proteins from normal colon and normal liver differed in many details, and their dissimilar patterns identified the source of the sample; (3) that hepatoma and primary adenocarcinoma of the colon were easily distinguished by their distribution of proteins; (4) that colon cancer metastatic to the liver contained a majority (50/58) of acidic proteins enumerated in primary colon cancer. These results indicate that uninvolved liver and colon and their primary or secondary cancers can be identified by their distribution of electrophoresed acidic proteins.

The association of N1-acetylspermidine with human colorectal adenocarcinomas has been evaluated in this study. Free polyamines and their monoacetylated forms in adenocarcinomas, adenomas, and apparently healthy mucosae were determined using high-performance ion-exchange chromatography. The N1-acetylspermidine levels in well- and moderately differentiated adenocarcinomas were 27.30 +/- 3.13 (S.E.) (n = 99) and 22.86 +/- 3.60 (n = 22) nmol/g, wet weight, respectively. These values were significantly higher than those of benign adenomas (5.38 +/- 0.85 nmol/g, n = 31) and of control mucosae. The N1-acetylspermidine levels in control mucosae on the oral and anal side of adenocarcinomas were 5.84 +/- 1.44 (n = 57) and 7.92 +/- 2.89 (n = 50) nmol/g, respectively; no significant difference was observed between control mucosae and adenomas. The mean levels of three polyamines, putrescine, spermidine, and spermine in both adenomas and adenocarcinomas were about twice as high as those of control mucosae. The molar ratios of spermidine to spermine were significantly greater in both adenomas and adenocarcinomas than in control tissues. There was no obvious correlation between the free polyamine concentrations and the degree of malignancy of the colorectal tumors. These results suggest that the metabolism of N1-acetylspermidine in colorectal adenocarcinomas is quite different from that in adenomas and in nonneoplastic mucosae and that N1-acetylspermidine can be a promising biochemical marker of cancer in the human large intestine.

The cell surface glycopeptides from an epithelial cell line (CCL 239) derived from normal human colon were compared with those from three cell lines (HCT-8R, HCT-15, and CaCo-2) derived independently from human colonic adenocarcinomas. Cells were incubated with D-[2-3H]mannose or L-[5,6-3H]fucose for 24 h and treated with trypsin to release cell surface components which were then digested exhaustively with Pronase and fractionated on Bio-Gel P-6 before and after treatment with endo-beta-N-acetylglucosaminidase H. The most noticeable difference between the labeled glycopeptides from the tumor and CCL 239 cells was the presence in the former of an endo-beta-N-acetylglucosaminidase H-resistant high molecular weight glycopeptide fraction which was eluted in the void volume of Bio-Gel P-6. This fraction was obtained with both labeled mannose and fucose as precursors. However, acid hydrolysis of this fraction obtained after incubation with [2-3H]mannose revealed that as much as 60-90% of the radioactivity was recovered as fucose. Analysis of the total glycopeptides (cell surface and cell pellet) obtained after incubation with [2-3H]mannose showed that from 40-45% of the radioactivity in the tumor cells and less than 10% of the radioactivity in the CCL 239 cells was recovered as fucose. After incubation of the HCT-8R cells with D-[1,6-3H]glucosamine and L-[1-14C]fucose, strong acid hydrolysis of the labeled glycopeptide fraction excluded from Bio-Gel P-6 produced 3H-labeled N-acetylglucosamine and N-acetylgalactosamine. This high molecular weight glycopeptide fraction was susceptible to mild alkaline borohydride reduction, yielding a mixture of labeled oligosaccharides which contained N-acetylgalactosaminitol. Thus, the HCT-8R cells are expressing fucosylated mucin-type glycoproteins on their surface.

Immunoperoxidase staining for carcinoembryonic antigen (CEA) was performed on 192 colorectal carcinomas to determine: whether tissue staining can be substituted for preoperative serum CEA levels, and whether patient survival can be predicted by these parameters. The overall incidence of positive tissue staining was 75 percent, which was similar to the elevated serum level percentage of 73 percent. Both the serum CEA level and the CEA tissue stain correlated with patient survival in Dukes' stage C patients. There was no correlation between tissue CEA stain and tumor differentiation. Positive tissue stain and elevated preoperative serum CEA identified patients with poor prognosis in Dukes' stage D only. This study shows that tissue staining with immunoperoxidase may be substituted for preoperative serum levels for CEA. The combination of these two parameters, however, does not identify patients at greater risk for recurrence than either procedure alone.

Normal, transitional, and carcinoma areas of five colons resected for carcinoma were examined morphologically, histochemically, and biochemically. The transitional area contained a larger amount of non-sulphated acid mucin than the normal mucosa as verified histochemically. Normal mucosa contained mainly sulphated mucin. The hexosamine-containing macromolecules present in different areas were isolated and characterized. They were divided into the following groups: 1) acid glycosaminoglycans, 2) high-molecular-weight glycopeptides, and 3) low-molecular weight glycopeptides. The concentration of the total hexosamine-containing material was in the carcinoma area twice as high as in normal areas. Acid glycosaminoglycans were identified as hyaluronate, heparan sulphate, dermatan sulphate, and chondroitin 4-(6)-sulphate. Their concentraitons were found to increase from normal to transitional and from transitional to carcinoma areas. The high-molecular-weight glycopeptide was composed of fucose, galactose, glucosamine, galactosamine, sialic acid, and variable amounts of sulphate. The sulphation degree of the glycopeptide was higher in normal mucosa than in transitional or carcinoma areas: The low-molecular-weight glycopeptides consisted of about a half of the total hexosamine-containing substances. The concentration of saline-insoluble fraction of the low-molecular-weight glycopeptides was in transitional areas about two times, and in carcinoma areas about four times, higher than in normal mucosa.

RNA-containing vesicles, recovered from the supernatant of high-density cell samples of human colon carcinoma, produce a high-resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters. Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor-bearing patients [(1985) Proc. Natl. Acad. Sci. USA 82, 3455-3459; (1986) FEBS Lett. 203, 164-168]. Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown [(1985) Dev. Biol. 3, 33-57]. Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles.

Because it is a common prerequisite for steroid responsiveness in target tissue, we investigated the presence of specific 1,25-DR in spontaneous human colorectal adenocarcinomas (ADC) and adjacent normal-appearing mucosa (NAM) from 23 operative specimens (12 male and 11 female patients). 1,25-DR was determined in cytosol by a DCC assay technique. 1,25-DR was present in 21 of 23 NAM and in only 4 of 23 HCRA. All positive ADC were well differentiated. Receptor content expressed in femtomoles/mg of protein (mean +/- SEM) was respectively 63.9 +/- 7.6 for right colon NAM and 51.3 +/- 12.9 for left colon or rectum NAM. When we compared all NAM specimens, receptor content was 56.7 +/- 8.0 femtomoles/mg of protein. No difference in 1,25-DR NAM level was observed between right colon and left colon or rectum. In adenocarcinoma the mean content was 66.5 +/- 14 fmoles/mg of protein. Scatchard analysis showed a single class of specific high-affinity saturable 1,25-DR with a dissociation constant (Kd) of 0.97 +/- 0.57 and 1.03 +/- 0.39 chi 10(-10) M in NAM and ADC respectively. These preliminary data represent the first demonstration of 1,25-DR throughout the entire human colon and indicate that the receptivity for this hormone is often lost during malignant transformation of the human colorectal mucosa. In addition, 1,25-DR could be a marker of differentiation in ADC. These preliminary results provide evidence supporting the addition of Vitamin D to the roster of developmental cancer chemopreventative agents.

Large amounts of particulate material, mostly lipid vesicles, are released by human colon adenocarcinoma HCT-8R cells when they are packed at high density in saline solution. RNA is also present in the released structures. Vesicle sheding is displayed only by healthy and viable cells. The process, in our experimental conditions, lasts up to 40 min, and can be restored by supplementing cells with nutrients and oxygen. RNA and lipids give rise to IH and 3IP NMR signals. The process is somehow related to a thermotropic transition observed by means of IH NMR spectroscopy for peculiar lipid domains in the plasma membrane. Analysis of 3IP NMR spectra of the phosphodiester groups, upon pH variation, indicates strong interaction between RNA and proteins in an assembled structure. A constant amount of polyA+ RNA can be recovered from the vesicles. The electrophoretic pattern and in vitro protein synthesis indicate that mRNA can be isolated as a functionally active molecule with a major 5 Kb fraction.

Fucose-containing glycoproteins and glycolipids were compared in three human colon cancer cell lines and five human fetal intestinal epithelial cell lines. Cells were labeled by culturing cells in the presence of L-[3H]-fucose. Fucose was incorporated into both the membrane and cytoplasmic fractions of all three colon cancer cell lines to a much lesser extent than into fetal cells. When the relative fucose labeling of glycolipids and glycoproteins were examined, a much greater proportion of fucose labeling in the membrane was associated with lipid in colon cancer cells (11.6-16.7%) compared to fetal intestinal cells (1.3-2.5%). Fluorographic analysis of SDS-polyacrylamide gel electrophoresis of fucose-labeled glycoproteins revealed a rather uniform labeling pattern of fetal intestinal cells which was distinct from those of colon cancer cells. Thin-layer chromatographic analysis of fucose labeled glycolipids of all three colon cancer cell lines indicated the presence of fucose-containing glycolipids with carbohydrate chain lengths greater than five sugars. Glycolipids of the SKCO-1 cells in particular appear to consist predominantly of complex fucose-containing glycolipids. These results indicate that significant qualitative differences in the fucose-containing glycoproteins and glycolipids exist between the membranes of human colon cancer cells and fetal intestinal cells and that complex fuco-glycolipids with long carbohydrate side chains are present in the three human colon cancer cell lines.

We have constructed cDNA libraries from the poly(A)+ RNA of normal colonic mucosa and a liver metastasis from a colonic adenocarcinoma. Differential screening of these libraries using 32P-labelled cDNAs transcribed from poly(A)+ RNAs isolated from specimens of four normal colonic mucosae, five adenocarcinomas, and three liver metastases by Grunstein-Hogness and dot-blot hybridization has identified a number of recombinant cDNA clones homologous to mRNAs that appear to differ significantly in abundance between normal and neoplastic colon and metastases. These cDNA clones, and others identified in the libraries, may be of considerable importance both as diagnostic tools and in defining the phenotypic changes associated with tumour progression and metastasis.

Among a series of fucolipids accumulating in human colonic and liver adenocarcinoma, two neutral fucolipids have been isolated and their carbohydrate structures have been characterized by methylation analysis, mass spectrometry, enzymatic degradation, and proton NMR spectrometry as shown in Structures 1 and 2 below. These glycolipids are either absent or present in very small quantity in normal colonic mucosa and normal liver tissue. (formular; see text) Difucosyl neolactonorhexaosylceramide (Structure 1) gives a doublet or a triplet on high performance thin layer chromatography and can be separated into four to five fractions on high pressure high performance liquid chromatography. These components have the same carbohydrate structure, but have different fatty acid composition. Fractions are characterized by a predominance of either alpha-hydroxy C16 fatty acid or alpha-hydroxy C24 fatty acid. Trifucosyl neolactonoroctaosylceramide (structure 2) gives two discrete bands, which are designated Z3 and Z4, on high performance thin layer chromatography. Methylation analysis and nuclear magnetic resonance spectra show that the Z3 and Z4 have identical carbohydrate structures. All the cases of cancer tissue that accumulate di- or trifucosyl structure (1 or 2 above) also accumulated lactofucopentaose(III)ceramide. A possible enhancement in a specific synthetic pathway including type 2 chain elongation coupled with alpha 1----3 fucosylation in human cancer is discussed.

The nuclear DNA content was determined with a cytophotometric technique in colonic mucosa of 15 patients with long-standing ulcerative colitis. The epithelial lesions were classified into inactive colitis, low grade and high grade dysplasia and adenocarcinoma. The histogram pattern varied between narrow unimodal in quiescent colitis, broad unimodal in low grade dysplasia with some hypertetraploid values in three cases (27%) and aneuploid in 62.5% of the lesions with high grade dysplasia. In well-differentiated adenocarcinoma the histograms were broad unimodal, whereas the curves of moderately and poorly differentiated carcinomas were wider and aneuploid. The technique can be used for a prognostic purpose: in dysplastic lesions, the detection of aneuploidy is important because it is frequently found in the presence of invasion although it does not allow its prediction. Carcinomas with polyploid DNA distribution have a better outcome than tumours with aneuploid distribution.

Sequential studies of 6 tumour markers in an adenomatous polyp which eventually metastesized as adenocarcinoma are described. The findings suggest that with the development of malignancy, clones of cells with an increasing number of markers are favoured for continued growth and that this probably represents progressive gene derepression.

The presence of human chorionic gonadotropin in large bowel cancers was studied immunohistochemically using an immunoperoxidase technique. HCG-positive tumour cells were present in 42 of 194 adenocarcinomas examined (22.0% of colon cancer and 21.2% of rectal cancers). On histological grading, the hCG-positive rate tended to rise as the degree of differentiation decreased. HCG was detected more frequently in cancers invading the total bowel wall (27%) than in those invading the partial wall (17.1%). Lymph node, liver or peritoneal metastases were present more frequently in hCG-positive tumours than in hCG-negative tumours. Furthermore, there was an intimate correlation between the presence of hCG-positive tumour cells and CEA doubling times in nine cases with untreated liver metastasis. The survival rate for patients with tissue hCG-positive cells was lower than for those with hCG-negative tumours. Thus, the presence of tissue hCG in colorectal cancers may be a biological marker of prognostic significance.

The DNA content of 37 colorectal carcinomas was studied prospectively by flow cytometry. The DNA content from the same tumors was also studied in disintegrated paraffin sections. The methods gave similar information on the DNA content and the correlation was 0.94. In eight patients tumor imprints were prepared and the DNA content was analyzed with static cytometry. A correlation coefficient of 0.83 between flow cytometry on fresh tumor tissue and static cytometry was found. Sixty-two percent of the tumors were aneuploid and had significantly higher S-phase values than diploid tumors. Histologic grade was not related to ploidy level, whereas Dukes' C tumors often were aneuploid. When the clinical course of patients was analyzed by log rank test (mean follow-up, 30.4 months), the patients with diploid tumors showed a clear advantage in terms of survival; only two patients in this group developed progressive disease. It is concluded that the DNA content is an independent prognostic predictor of colorectal carcinoma and that DNA can be analyzed by several methods. Of these, the method using disintegrated paraffin sections appears most attractive.

Tumor-associated glycosphingolipids expressed on human colorectal adenocarcinomas were surveyed by staining of glycolipid bands on thin layer chromatogram with several horse radish peroxidase-labeled lectins. Glycolipids reacting with Ricinus communis agglutinin-I were found in all of the tumors from 9 patients with various types of colorectal adenocarcinomas, but only very weakly in the normal mucosa. Susceptibility to glycosidases and chromatographic mobilities indicated that these glycolipids were lactosylceramide, paragloboside and probably higher homologues of neolactoseries glycolipids.

The purpose of this study was to compare the expression of O-acetylated sialic acids on normal colonic epithelial cells to that on primary and metastatic human adenocarcinoma of the colon and rectum. In 24 cases, the relative percentages of biosynthetically labeled non-, mono-, di-, and tri-O-acetylated sialic acids were measured after hydrolytic release, separation, and identification by paper chromatography. In one case, the presence of di- and tri-O-acetylated sialic acids was confirmed by fast atom bombardment-mass spectral analysis. Differences were observed in the expression of sialic acids between normal colonic epithelium, "uninvolved" colon mucosa remote to a colonic adenocarcinoma, and colonic adenocarcinoma. The levels of mono- and tri-O-acetylated sialic acids accounted for the difference in the ratios of sialic acids expressed between normal and "uninvolved" colonic mucosa, while the total amount of O-acetylation was unchanged. However, no difference was observed in the relative amounts of non- and O-acetylated sialic acids between either fresh and tissue culture-established colon carcinomas, or fresh and tissue culture-established liver metastasis derived from carcinoma of the colon. The relative expression of these O-acetylated sialic acids molecules appears to vary according to tissue type. This study suggests that individuals with adenocarcinoma of the colon express a field defect resulting in abnormal ratios of O-acetylated sialic acids.

Cells of the normal colonic mucosa express several types of cytokeratins, the characteristic intermediate filament proteins of epithelial cells. An immunohistochemical study was designed to examine the expression of two distinct groups of cytokeratins, recognized by monoclonal antibodies AE1 and AE3, in the colonic mucosa and to compare the findings with those obtained with a large number of polypoid lesions (adenomatous and hyperplastic) and carcinomas of the colon. AE1 and AE3 immunostaining was found in the surface epithelium and upper portions of the crypts of Lieberkühn (functional zone) of normal colonic mucosa, whereas the lower portions of the crypts (proliferative compartment) were unreactive with both AE1 and AE3. Polypoid lesions of the colonic mucosa can be placed into two categories based on their patterns of cytokeratin expression. Solitary tubular adenomas and hyperplastic polyps are composed of AE1 and AE3 nonexpressing cells with only few, patchy areas of AE1 and AE3 expressing cells present within glands and in the surface epithelium. In contrast, villous adenomas show strong AE1 and AE3 reactivity throughout the glands. Furthermore, tubular and villous adenomas, and even histologically normal mucosa in patients with familial polyposis, show AE1/AE3 expression throughout the glands and surface epithelium. Colonic carcinomas show a predominance of AE1/AE3 expressing cells. Thus, cytokeratins recognized by monoclonal antibodies AE1 and AE3 represent molecular markers of cellular maturation in the normal colonic mucosa, that are expressed in colonic carcinomas and, in addition, serve as markers that distinguish colonic mucosa and adenomas with a high risk for development of cancers from those with a lower risk.

Binding of FITC conjugated lectins to mucin in benign and malignant colon lesions was studied by fluorescence microscopy. In 145 cases of colonic adenocarcinoma, PNA (peanut agglutinin) was found to be bound to 75% of cases, while DBA (dolichos biflorus agglutinin) to 13% only. By contrast, DBA was bound to 94% of normal colon mucosa, while PNA to 15% only. Chronic colitis, simple adenoma and inflammatory polyps had the same binding pattern as normal mucosa, but villous adenoma, mixed polyps and multiple polyposis which are considered as premalignant lesions in colon had high positive rate of PNA binding and low binding percentage of DBA. These results indicate that an exposed carbohydrate structure Gal B1-2----GlNAc is expressed in the mucin produced by colonic adenocarcinoma. Meanwhile, some normal mucin component disappears from the malignant colonic epithelium. The mechanism of alteration of human colonic mucin present in malignant transformation is briefly discussed.

Human colonic adenocarcinoma tissue and derived cell lines have been characterized by an abundance of different type 1 and 2 lacto series glycolipid antigens which are either low or not found in normal colonic mucosa. The enzymatic basis for the expression of contrasting glycolipid compositions between adenocarcinomas and normal colonic mucosa, as well as between derived cell lines, has been studied. The following results were of particular interest. (i) Abundant activities of beta 1----4galactosyltransferase associated with synthesis of both lactosylceramide and lactoneotetraosylceramide, beta 1----3galactosyltransferase for synthesis of lactotetraosylceramide, and an alpha 1----3/4fucosyltransferase responsible for synthesis of Lex and Lea antigens were found in normal colonic mucosa or in a normal mucosal epithelial cell line HCMC, or in both. Variable levels of these activities were found in adenocarcinoma tissues and in various established adenocarcinoma cell lines. In striking contrast, significant activity of a beta 1----3N-acetylglucosaminyltransferase responsible for synthesis of lactotriaosylceramide (Lc3) was found in various cases of colonic adenocarcinoma and cell lines, but was undetectable in normal colonic epithelial cells. (ii) In situ transfer of galactose to Lc3 was performed on histologic sections by preincubation of the tissue with acceptor glycolipid followed by incubation with UDP-galactose. The biosynthesized glycolipid was revealed by indirect immunofluorescence with the monoclonal antibody 1B2 which defines lactoneotetraosylceramide antigen. In these studies, histologic sections prepared from frozen normal proximal colon tissue were shown to lack native type 2 chain structures. However, transfer of galactose from UDP-galactose could be demonstrated in the epithelial cells of normal proximal colon after incorporation of Lc3 into the membranes, indicating the ability of normal colonic epithelial cells to synthesize type 2 chain core structures if the precursor Lc3 is available. In contrast, adenocarcinoma tissues showed significant native immunofluorescence with the antibody. These data suggest that an accumulation of both type 1 and 2 chain lacto series glycolipids with alpha 1----3- or alpha 1----4fucosyl substitution in human adenocarcinoma is due to enhanced beta 1----3N-acetylglucosaminyltransferase rather than enhancement of other enzymes. This enzyme may play a key role in regulating the level of various types of lacto series tumor-associated antigens with the lacto type 1 or 2 chain.

A new fucoganglioside, 6B, which accumulates in human colonic adenocarcinoma but is absent in normal colonic mucosa, was isolated from a monosialoganglioside fraction of colonic adenocarcinomas. The structure of this ganglioside was identified as shown below by methylation analysis, direct probe mass spectrometry, and enzymatic degradation followed by examination of the degradation products with specific monoclonal antibodies. (formula; see text) The hybridoma (FH6) secreting a monoclonal IgM antibody directed to this glycolipid was selected by reactivity of the antibody with this ganglioside and lack of reactivity with other glycolipids having a closely related structure, such as sialosyllactoneotetraosylceramide (IV3NeuAcnLc4), sialosyllactofucopentaosy(III)ceramide (IV3NeuAcIII3FucnLc4), sialosyllactofucopentaosy(II)ceramide (sialosyl-Lea glycolipid; IV3NeuAcIII4FucLc4), and 6C fucoganglioside (sialosyl 2 leads to 6 fucoganglioside; VI6NeuAcIII3FucnLc6). The antibody was highly reactive with a large variety of human cancer cells, but was less reactive or did not react with a variety of normal cells.

The purpose of this study was to determine whether in man unusual types of concentrations or bile acids were present in colonic polyps, colon carcinomas, or the adjacent, apparently normal tissue. Methods for the determination of soluble and tissue-bound bile acids were validated. Of 14 polyps analyzed, eight contained detectable levels of bile acid, predominantly chenodeoxycholic acid; no lithocholic acid was observed in either the tissue-bound or soluble bile acid fractions. Bile acids were found in four of nine samples of colon carcinoma; in one tumor, tissue-bound lithocholic acid was present. Bile acids were similarly found in seven of 10 samples of normal bowel taken adjacent to the carcinoma. In the soluble bile acid fraction, cholic acid was more abundant than chenodeoxycholic acid. There was no correlation between tissue histology and bile acid composition or concentration. Under the conditions used, this study did not disclose a relationship between tissue bile acids and colorectal histology.

Clinical course of 77 surgical cases of cancer of the large bowel was evaluated versus levels of estrogen, androgen, progesterone and glucocorticoid hormone receptors. Within the follow-up period (range 6-41 months), 7 (9%) patients died of tumor progression, 16 (21%) developed recurrence or metastases, while the rest 54 (70%) cases continued in remission. Patients with steroid hormone receptor-positive tumors revealed more favorable clinical course as well as longer disease-free and overall survival than cases of receptor-negative cancer. Estrogen receptor status was found to be of the highest prognostic value in terms of clinical course and survival, as compared to all other hormone receptors studied.

The human colon adenocarcinoma cell-line HT 29 has been shown to possess functional alpha 2-adrenergic receptors. Here, [3H] clonidine was used as radioligand to study the evolution of alpha 2-adrenergic receptivity during the time course of HT 29 cell culture. Scatchard analysis of the saturation curves indicates that the number of [3H]clonidine binding sites increases throughout the 17 day culture period. The maximal number of alpha 2-adrenoceptors is found during the stationary phase of growth, when cell density is high and mitotic rate low. Moreover, the use of adrenaline and clonidine as alpha 2-adrenergic agonists reveals a relationship between the number of receptors and the intensity of the biological effect associated with their stimulation (inhibition of the VIP-induced cyclic AMP accumulation).

Modal DNA (ploidy) and sensitivity of DNA in situ to denaturation by acid have been analyzed by flow cytometry of 10 colorectal adenomas and 35 adenocarcinomas; 39 normal mucosa samples served as controls. A new method was developed to denature DNA in chromatin of the freshly isolated, intact, and unfixed individual cell nuclei from surgically resected material. The sensitivity of DNA denaturation (T alpha) was assayed by metachromatic staining with acridine orange and calculated as a ratio of the alpha t index of the tumor sample to the alpha t index of normal mucosa; the alpha t index is that fraction of DNA, following treatment at pH 1.4, that stains metachromatically with acridine orange at pH 2.6. All adenomas were diploid and in nine of 10 the T alpha value was close to 1.00, indicating no difference from control specimens in DNA sensitivity to denaturation. Forty-nine% of adenocarcinomas were aneuploid. Forty-six% of adenocarcinomas differed from normal in sensitivity of DNA to denaturation; the T alpha value was lower than 0.90 indicating that chromatin of the tumor cells was more resistant to denaturation than control cells. There was no correlation between sensitivity to denaturation of DNA and incidence of aneuploidy. However, there was a correlation between T alpha and the pathologically determined stage of disease. There was increased resistance to denaturation in 58% of tumors classified as Dukes' C/D stage, in 36% of tumors classified as Dukes' B, and in 20% classified as Dukes' A stage of the disease. Statistical analysis of these results revealed significant differences between distributions of T alpha in noninvasive (Adenomas and Dukes' A) versus invasive (Dukes' B and C/D) tumors with level of significance at P = 0.02. The data suggest that acid denaturation of DNA in situ may be a valuable adjunct in assessing the biology of colon cancer. The molecular basis for this phenomenon is discussed.

The distribution of fibronectin (FN) and collagen type III (IIIC) have been compared in a series of epithelial neoplasms from the gastrointestinal tract and salivary gland. The difficulty of distinguishing between FN of epithelial and fibroblastic origin is emphasised and evidence is presented for the validity of this distinction. In carcinomas FN was sometimes, but not invariably, lost from epithelial cell surfaces. Both FN and IIIC were increased in reactive connective tissue stroma. It is concluded that loss of cell surface FN is unlikely to be a useful diagnostic marker for malignancy, but that the occurrence of this phenomenon in vivo as in vitro indicates that it is biologically significant.