Heparan sulfate degradation: relation to tumor invasive and metastatic properties of mouse B16 melanoma sublines.

After transport in the blood and implantation in the microcirculation, metastatic tumor cells must invade the vascular endothelium and underlying basal lamina. Mouse B16 melanoma sublines were used to determine the relation between metastatic properties and the ability of the sublines to degrade enzymatically the sulfated glycosaminoglycans present in the extracellular matrix of cultured vascular endothelial cells. Highly invasive and metastatic B16 sublines degraded matrix glycosaminoglycans faster than did sublines of lower metastatic potential. The main products of this matrix degradation were heparan sulfate fragments. Intact B16 cells (or their cell-free homogenates) with a high potential for lung colonization degraded purified heparan sulfate from bovine lung at higher rates than did B16 cells with a poor potential for lung colonization. Analysis of the degradation fragments indicated that B16 cells have a heparan sulfate endoglycosidase. Thus the abilities of B16 melanoma cells to extravasate and successfully colonize the lung may be related to their capacities to degrade heparan sulfate in the walls of pulmonary blood vessels.

Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions.

We have examined the expression of chemokines and their receptors in the atopic dermatitis-like (AD-like) lesions of NC/Nga mice. Such lesions develop when the mice are kept in conventional conditions, but not when they are kept isolated from specific pathogens. The thymus- and activation-regulated chemokine TARC is unexpectedly highly expressed in the basal epidermis of 14-week-old mice with lesions, whereas it is not expressed in the skin without lesions. Production of TARC by keratinocytes was confirmed by culturing murine keratinocytic cell line cells (PAM212) with TNF-alpha, IFN-gamma, or IL-1beta. Expression of another Th2 chemokine, macrophage-derived chemokine (MDC), was observed in the skin from mice kept in both conventional and pathogen-free conditions, but expression of MDC was increased severalfold in the skin with lesions. The cellular origin of MDC was identified to be dermal dendritic cells. Infiltration of the skin by IL-4-producing T cells and mast cells, and the increase of CCR4 mRNA in the skin, coincided with the development of AD lesions. These observations indicate that TARC and MDC actively participate in the pathogenesis of AD-like lesions in NC/Nga mice and that these Th2 chemokines could be novel targets for intervention therapy of AD in humans.

Involvement of cell surface glycans in adhesion of human colon carcinoma cells to liver tissue in a frozen section assay: role of endo-beta-galactosidase-sensitive structures.

Adhesion of human colon carcinoma variant cell lines expressing different levels of the cell surface sialyl Lewis X (sLeX) antigen to frozen sections of mouse liver was examined. KM12-HX cells that bound the monoclonal antibody (mAb) FH6 (anti-sLeX) and thus expressed a high level of sLeX demonstrated a greater degree of adhesion to liver sections than their low-binding counterparts, KM12-LX cells. The adhesion of KM12-HX cells to liver sections was partially blocked by mAb FH6, but not by another anti-sLeX mAb, KM93. The adhesion was Ca2+ dependent but was not inhibited by anti-E-selectin. Endo-beta-galactosidase treatment significantly reduced adhesion and resulted in the loss of cell surface binding sites for mAb FH6. O-linked oligosaccharides from KM12-HX cells incubated in the presence of p-nitrophenyl-N-acetylgalactosaminide were fractionated by a combination of gel filtration, anion exchange chromatography, and normal phase high-performance liquid chromatography. The structure of a mAb FH6-reactive and endo-beta-galactosidase-sensitive glycan was estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in a post source decay mode and by glycosidase digestions to be NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc-NAc beta1-3Gal beta1-4(+/-Fuc alpha1-3)GlcNAc beta1-6(NeuAc alpha2-3Gal beta1-3)GalNAc-pNP. Mild detergent lysates of mouse liver surface-labeled with sulfo-NHS biotin were incubated with glutaraldehyde-fixed monolayers of KM12-HX cells, and bound components were isolated after EDTA treatment. A Mr 49,000 component that bound only to KM12-HX cells and not to KM12-LX cells was identified.

Carbohydrate chain analysis by lectin binding to electrophoretically separated glycoproteins from murine B16 melanoma sublines of various metastatic properties.

Cellular glycoprotein carbohydrate chains of B16 melanoma sublines of various metastatic colonization capacities were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and direct lectin staining, combined with chemical modification of carbohydrate chains in situ. For these studies, we utilized B16 sublines selected for low (B16-F1) or high lung (B16-F10), high brain (B16-B15b), or high ovary (B16-O13) colonization properties, or high tissue invasiveness in vitro (B16-BL6). The major B16 cell surface sialoglycoproteins were of Mr approximately 115,000, approximately 90,000, approximately 82,000, and 60,000 to 65,000, and were detectable by periodate NaB3H4 labeling and binding of 125I-wheat germ agglutinin (WGA). Terminal sialic acid residues in the carbohydrate chains were responsible for WGA binding, since chemical removal of sialic acid prevented WGA labeling of the glycoproteins. However, removal of sialic acid residues followed by Smith degradation resulted in reappearance of WGA-binding sites on these sialoglycoproteins, indicating that the carbohydrate chains possessed at least one branching point at an outer alpha-mannosyl residue. This structural feature was further indicated by the failure of 125I-Lens culinaris hemagglutinin to bind to these sialoglycoproteins. The fact that the carbohydrate residues of the Mr approximately 115,000, approximately 90,000, and approximately 82,000 sialoglycoproteins were of the complex type was confirmed by their reactivity with 125I-Ricinus communis agglutinin I, which preferentially binds to Gal leads to GlcNAc sequences after removal of sialic acid in situ. In contrast, 125I-peanut (Arachis hypogaea) agglutinin, specific for Gal leads to GalNAc sequences, failed to bind to the major WGA-reactive sialoglycoproteins, but strongly interacted after removal of sialic acid with Mr approximately 51,000 and approximately 56,000 glycoproteins from sublines B16-F1, -F10, and -BL6 and with a Mr approximately 63,000 glycoprotein from sublines B16-F10, -BL6, -O13, and -B15b. Thus, the small, mucin-type carbohydrate chains were expressed almost exclusively on these lower Mr sialoglycoproteins, and very little on the Mr approximately 82,000, approximately 90,000, and approximately 115,000 sialoglycoproteins. Differences in lectin binding to glycoproteins were observed with different sublines. These glycoproteins included: (a) a WGA-binding Mr 60,000 to 75,000 sialoglycoprotein prominent on B16-B15b cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Ulex europeus agglutinin I-reactive high molecular weight glycoproteins of adenocarcinoma of distal colon and rectum and their possible relationship with metastatic potential.

A Ulex europeus agglutinin I (UEAI)-reactive glycoprotein(s) with molecular weight higher than 300,000 was detected by direct binding of 125I-labeled UEAI to lysates of rectal or sigmoid colon cancer tissues separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. This was the major UEAI-reactive molecule in tumor tissues and different tumors possessed varying reactivities. Very little UEAI binding was detected to lower molecular weight components. Histochemical localization of UEAI confirmed that the UEAI-reactive molecules were mostly localized to the surface of carcinoma cells. A total of 135 fresh tissue samples, including those from adenocarcinoma, villous adenoma, and normal mucosa in surgical specimens (69 patients), were examined to determine the reactivities of UEAI to the high molecular weight component in the tissue extracts. The quantitative binding of 125I-UEAI was compared according to the stage of colorectal cancer at the time of surgery. The relative amount of UEAI-reactive high molecular weight substance was significantly higher in the carcinomas than in normal mucosa. UEAI binding to high molecular weight regions of the polyacrylamide gel was significantly lower in primary colorectal adenocarcinomas from stage C or D patients than in those from stage B1 patients. Therefore, increased expression of the UEAI-reactive molecule was related to transformation of colorectal epithelial cells and decreased expression appeared to be associated with progression and metastatic potential.

Effects of tunicamycin on B16 metastatic melanoma cell surface glycoproteins and blood-borne arrest and survival properties.

The role of cell surface glycoproteins in determining in vivo blood-borne arrest and survival characteristics of murine melanoma sublines of low (B16-F1) or high (B16-F10) potential to form experimental lung metastases after injection i.v. was assessed after inhibiting tumor cell protein glycosylation with tunicamycin. Incubation of B16-F1 or B16-F10 cells with 0.5 micrograms (or above) tunicamycin per ml for 12 to 36 hr inhibited significantly lung tumor colony formation. Examination of B16 cells in the presence of 0.5 micrograms drug per ml indicated that complex oligosaccharide synthesis was inhibited greater than 90%, while protein synthesis remained at about 50% of the control levels. Tunicamycin induced morphological changes in B16-F1 and B16-F10 cells such as cellular rounding. Cell growth was also inhibited by tunicamycin. These effects were reversible, and B16 cells recovered their normal morphologies and growth rates within 24 hr after removal of the drug. Exposed cell surface protein analyzed by lactoperoxidase-catalyzed 125I iodination-sodium dodecyl sulfate-polyacrylamide gel electrophoresis-autoradiography showed few changes after tunicamycin treatment; however, sialogalactoproteins (detected by the binding of 125I-labeled R. communis agglutinin I to polyacrylamide gels containing desialized B16 cell surface components) were reduced dramatically by the drug. The adhesive properties of untreated and tunicamycin-treated B16 cells were assessed by the binding of 51Cr-labeled B16 cells to endothelial cell monolayers. Tunicamycin-treated B16-F1 and B16-F10 cells adhered at lower rates to endothelial cells such that after 24 to 36 hr of drug (0.5 micrograms/ml) treatment adhesion was almost completely blocked, suggesting that tunicamycin-induced cell surface glycoprotein changes in B16 melanoma cells may interfere with tumor cell-host cell interactions that lead to arrest and survival of blood-borne malignant cells.

Modification of cell surface glycoproteins, macrophage cytostasis, and blood-borne metastatic properties of the murine RAW117 large cell lymphoma by virus superinfection.

Highly metastatic sublines of the murine RAW117-P large cell lymphoma parental line have been selected sequentially in vivo for enhanced blood-borne organ colonization. We found that the subline RAW117-H10, selected 10 times for liver colonization, formed more than 200 times as many liver tumor nodules than the RAW117-P line and showed loss of cell surface RNA tumor virus Mr 70,000 envelope glycoprotein and sensitivity to activated macrophage-mediated cytostasis. Superinfection of RAW117-H10 cells with endogenous RNA tumor virus isolated from RAW117-P cells caused increases in Mr 70,000 envelope glycoprotein expression and sensitivity to activated macrophage-mediated cytostasis concomitant with loss of liver metastatic properties. The results suggest that RAW117 cell surface Mr 70,000 envelope glycoprotein is involved in host macrophage-mediated surveillance mechanisms and metastatic properties.

Tumor cells with organ-specific metastatic ability show distinctive trafficking in vivo: analyses by positron emission tomography and bioimaging.

To elucidate the behavior of various metastatic tumor cells with different characteristics in the blood flow, we have developed a system to investigate real-time trafficking using positron emission tomography. In this study, positron-labeled cells, i.e., lung-metastatic B16BL6 melanoma and two sublines of liver-metastatic RAW117 large cell lymphoma, were injected i.v., and the trafficking of these cells was noninvasively determined. All sublines tested accumulated in the lungs immediately after injection, presumably because the lungs were the first organ passed through after i.v. injection. The elimination of RAW117 cells from the lungs, however, was fast compared with that of B16BL6 cells. The latter showed a release rate from the lungs of less than 1%/min, whereas that of RAW117 cells was greater than 2%/min. Reflecting the elimination from lungs, RAW117 cells accumulated in the liver in a time-dependent manner. Biodistribution of metastatic cells was also analyzed by whole-body autoradiography after injection of 5-[125I]iodo-2'-deoxyuridine-labeled cells, using a bioimaging analyzer system. The method is invasive; however, it enables a precise determination of the biodistribution of metastatic cells. Bioimaging analyzer system analysis also showed the organ-specific accumulation of these metastatic cells. Furthermore, colonized distribution of B16BL6 cells in the lungs and that of RAW117 cells in the liver were observed. The present data suggest that the trafficking of metastatic tumor cells greatly influences the organ specificity of cancer metastasis.

Positron emission tomography analysis of metastatic tumor cell trafficking.

Organ-specific colonization of metastatic tumor cells is regulated through complex interactions of specific adhesion molecules on the tumor cell surface with organ specific microvascular endothelium. The present paper shows the real time tumor cell trafficking under the actual blood flow, which became able to be determined using a new technology, positron emission tomography. This technology would be useful to evaluate the interaction of characteristic tumor cells with various organs in vivo. High and low metastatic rat mammary adenocarcinoma cell variants, MTLn3 and MTC, respectively, were labeled with [2-18F]2-fluoro-2-deoxy-D-glucose in vitro. The labeled cells preferentially accumulated in the lungs, in which the disposition was more intense for MTLn3 than for MTC cells especially for the first 2-10 min after injection, apparently reflecting the organ specific interaction of metastatic tumor cells which may lead to metastasis. Such a short time change of cell disposition is easily determined in a living animal by this new technique. Furthermore, sialidase treatment of MTLn3 cells suppressed the accumulation of these cells in lungs, suggesting that some sialyl glycoconjugates on the MTLn3 cell surface play an important role in cell adhesion to lung endothelium. Positron emission tomography scanning thus enables the noninvasive study of the interaction of characteristic tumor cells with specific endothelium in a living animal.

Human colonic sulfomucin identified by a specific monoclonal antibody.

Since the 1960s, the loss of sulfomucin from colonic epithelium has been considered to be an indicator of an early stage of carcinogenesis; yet, the biochemical basis for this phenomenon has never been elucidated. We recently prepared a monoclonal antibody (mAb) 91.9H that immunoprecipitates the normal colonic mucins metabolically incorporating [35S]-sulfate. This mouse IgG1 antibody did not cross-react with colon carcinoma mucins that lack sulfate groups. Using normal colonic epithelia unlabeled or radiolabeled with [35S]sulfate and [3H]glucosamine, we purified a high molecular weight glycoprotein that reacts with mAb 91.9H. This was achieved by a combination of DEAE-cellulose anion-exchange chromatography, consecutive treatments with chondroitinase ABC plus heparitinase and with sodium dodecyl sulfate plus 2-mercaptoethanol, and gel filtration on Sepharose CL-2B in the presence of 8 M urea. Antibody reactivity was found in acidic but not neutral high molecular weight glycoproteins. After Sepharose CL-2B fractionation, the mAb 91.9H-reactive fractions consisted of a component with an approximate molecular weight of 500,000-900,000. A purified sulfomucin contained protein, neutral sugar, amino sugar, sialic acid, and sulfate in an approximate ratio of 2.5:1.0:1.1:0.4:0.5. The polypeptide portion was rich in hydrophilic amino acids, particularly threonine. Binding of mAb 91.9H in solid-phase assays was inhibited to 50% by purified normal colon acidic mucin at doses of 5-50 micrograms/ml, depending on different preparations. Various glycosaminoglycans or sulfatides did not show inhibitory activity. Sulfomucin reactivity with mAb 91.9H, as determined by solid-phase-binding inhibition and by dot blot assays, was significantly reduced by chemical desulfation of sulfomucins with anhydrous hydrochloric acid, suggesting that sulfate groups served as a portion of the immunochemical determinant for this antibody. Sulfate residues were apparently linked to alkaline-sensitive carbohydrate chains, but alkaline-released carbohydrate chains did not react with mAb 91.9H. Immunohistochemical examinations showed that mAb 91.9H bound normal colonic epithelial cells, which also stained with high-iron diamine, more strongly than it bound colon carcinoma cells.

Differential production of high molecular weight sulfated glycoproteins in normal colonic mucosa, primary colon carcinoma, and metastases.

Sulfated macromolecules synthesized in tumor and mucosa tissues derived from colorectal cancer patients were labeled with [35S]sulfate and separated into two fractions on DEAE-Sephacel: the slightly acidic peak (peak I) was eluted with 0.2 M NaCl and the highly acidic peak (peak II) was eluted with 0.5 M NaCl. A total of 40 specimens, which included primary colon cancer, liver metastases, and normal mucosa obtained at surgery (16 patients), were examined regarding the amount of peak I and peak II. The amount of peak I significantly decreased in the order of normal mucosa greater than primary tumors greater than metastases, while the amount of peak II did not significantly change among the tissues. Peak I was mostly resistant to chondroitinase ABC and nitrous acid treatment under acidic conditions, whereas combined chondroitinase-sensitive materials and nitrous acid-sensitive materials were greater than 80% of the radioactivity in peak II. The major radioactive component of peak I migrated at a position corresponding to Mr greater than 300,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and became Mr less than 40,000 after alkaline borohydride treatment. The major component of peak I was likely to be a sulfated glycoprotein containing sulfate groups on alkaline labile carbohydrate chains. Peak II consisted of a mixture of heparan sulfate proteoglycans and chondroitin sulfate proteoglycans. Differential incorporation of [35S]sulfate into peak I among normal mucosa, primary colon carcinoma, and colon carcinoma metastasis was observed. Therefore, decreased peak I production may be a biochemical change associated with colorectal cancer progression and metastasis.

Differential expression of a sialoglycoprotein with an approximate molecular weight of 900,000 on metastatic human colon carcinoma cells growing in culture and in tumor tissues.

Wheat germ agglutinin (WGA)-binding cellular glycoproteins produced by HT-29 human colon carcinoma and its variant cells established from liver metastases in nude mice after intrasplenic injection were analyzed by polyacrylamide gel electrophoresis. On 5.5% polyacrylamide gels five major sialoglycoproteins (approximate Mr 115,000, 145,000, 190,000, 450,000, and 740,000) reactive with WGA were common to the parental and metastatic sublines. There was an additional component of Mr approximately 900,000 that was prominent in cells established from liver metastases. Specific removal of sialic acid from the glycoproteins eliminated WGA binding, indicating that all the WGA-binding glycoproteins including the Mr 900,000 component were sialoglycoproteins. Smith degradation following mild acid hydrolysis resulted in formation of WGA-binding carbohydrate chains on Mr 115,000, 145,000, 190,000, and 900,000 components, but not on Mr 450,000 and 740,000 components, which indicated that these two sialoglycoproteins bore different oligosaccharides from the other sialoglycoproteins. The Mr 900,000 component was more prominent with HT-29 cells growing in nude mice than those growing in vitro. WGA binding to the Mr 900,000 component of metastasis-derived HT-29 cells growing in a nude mouse was higher than that of parental cells growing in nude mice. The expression in liver metastases derived from parental as well as metastatic cells was higher than the primary tumor growing in the spleen of the same mouse, indicating that the levels of Mr 900,000 sialoglycoprotein (SGP = 900) were regulated by intrinsic and environmental factors. The influence of organ microenvironmental factors was confirmed by analyzing sialoglycoproteins of HT-29 cells growing in the liver of a nude mouse following intrahepatic injection. Analyses of human colorectal carcinoma tissues and liver metastases revealed a polydisperse WGA-reactive high-molecular-weight component similar to that seen in tumors growing in nude mice. The mean value of WGA binding to high-molecular-weight glycoproteins in the primary tumors of stage B1 patients was smaller than that of all other primary tumors. Comparison of primary tumors with liver metastases from the same patients indicated that the level of SGP-900-like high-molecular-weight glycoproteins in metastases was not always higher than those in primary tumors.

Increased expression of sialyl-dimeric LeX antigen in liver metastases of human colorectal carcinoma.

We collected a total of 78 tissue specimens, including primary colorectal carcinoma, normal colonic mucosa, and liver metastases of colon carcinoma, to examine whether the extracts of these tissues inhibited the binding of a monoclonal antibody FH6, specific for sialyl-dimeric LeX antigen. The results of inhibition assays demonstrated that: (a) contents of FH6-reactive molecules were greater in carcinoma tissues than in normal colonic mucosa; (b) metastatic foci in livers contained more FH6-reactive molecules than primary tumors; (c) primary tumors from Dukes' stage B1 patients contained less FH6-reactive molecules than primary tumors from Dukes' stage D patients. The inhibitory activity of these tumor tissue extracts against the binding of a monoclonal antibody FH6 to cultured colon carcinoma cells was eliminated by prior treatment of the extracts with sialidase, confirming that the FH6-reactive materials were sialyl-dimeric LeX antigen. Electrophoretic separation of tumor tissue extracts on 3% polyacrylamide gels followed by direct staining with monoclonal antibody FH6 revealed that very high molecular weight glycoproteins, presumably mucins, contained sialyl-dimeric LeX antigen.

Monoclonal antibody against human colonic sulfomucin: immunochemical detection of its binding sites in colonic mucosa, colorectal primary carcinoma, and metastases.

Previous studies using metabolic labeling of fresh colonic mucosa and colorectal carcinoma with [35S]sulfate followed by biochemical analysis demonstrated that the amount of a sulfated high-molecular-weight glycoprotein expressed in primary colorectal carcinoma was lower than that in normal mucosa, and that the amount further decreased in liver metastases. This suggested that this sulfated molecule represented a sulfomucin previously defined by histochemical reactivity with a cationic dye. We have extracted and partially purified this high-molecular-weight sulfated glycoprotein from normal human colonic mucosa. We immunized mice with the partially purified sulfomucin and generated hybridomas. One cloned hybridoma, designated as 91.9H, produced a monoclonal antibody strongly reactive with a component which migrated at an identical position as the metabolically [35S]sulfate-labeled high-molecular-weight glycoprotein after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reactive molecules appeared to have a polydisperse nature with a molecular weight ranging between 400,000 and 900,000. The [35S]sulfate-labeled high-molecular-weight glycoprotein was bound to Staphylococcus Protein A-agarose coated with this monoclonal antibody but did not bind to unconjugated Protein A-agarose. The immunoprecipitated substance also migrated at an apparent molecular weight range of 400,000 to 900,000. The reactivity of monoclonal antibody 91.9H with the extracts of normal mucosa, colorectal primary carcinoma, and metastasis was compared by dot blot assay on a nitrocellulose membrane. This antibody was more reactive with the extracts of mucosa adjacent to carcinoma tissues than with the carcinoma extracts. Primary tumors showed higher reactivity than metastases in most of the cases. These results strongly suggest that this antibody is specific to colonic sulfomucins or at least to mucins closely related to colonic mucins previously identified by metabolic labeling with [35S]sulfate.

Increased content of an endogenous lactose-binding lectin in human colorectal carcinoma progressed to metastatic stages.

The quantity and localization of two lactose-binding lectins with molecular weights of 31,000 and 14,500 in human colorectal carcinoma tissue specimens obtained by surgical resection have been studied using specific polyclonal antibodies. Electrophoretic separation and blotting of detergent extracts of tumor tissues (48 specimens), followed by the binding of an antibody that recognizes both of these lectins, demonstrated that the contents of Mr 31,000 and 14,500 lectins vary from one specimen to another. The Mr 31,000 lectin content was higher in tumor specimens classified as Dukes' stage D than in those from other stages. A significant correlation was found between Mr 31,000 lectin levels and the levels of carcinoembryonic antigen in the patients' sera at the time of surgery. Immunohistochemical staining with antibodies specific for each lectin was performed with 20 colon carcinoma tissues and 5 colonic adenoma tissues. The results showed that the Mr 31,000 lectin localizes in the cytoplasm of colorectal carcinoma cells and normal epithelial cells, whereas antibody binding to Mr 14,500 lectin is observed in a limited number of carcinoma specimens and is mainly associated with luminal surfaces and secretory products. Adenoma cells were reactive with Mr 14,500 anti-lectin antibody at their luminal surfaces or cytoplasms, but they did not stain with Mr 31,000 anti-lectin antibody. These results suggest that a correlation exists among the level of the Mr 31,000 lectin, the serum level of carcinoembryonic antigen, and the stage of progression of colorectal carcinomas.

Increased expression of sialyl Lewisx antigen correlates with poor survival in patients with colorectal carcinoma: clinicopathological and immunohistochemical study.

We have previously shown that sialyl Lewisx antigen (sLex) (NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAC-R) has an important functional role in defining the invasion and metastasis of human colorectal carcinoma. The results were derived from the clinical specimens obtained at surgery or experimental metastasis of human colon carcinoma variant expressing different levels of sLex in nude mice. In the present study, we immunohistochemically examined 132 human colorectal carcinomas for the expression of sLex to investigate whether this antigen expression could serve as a prognostic parameter. The tumors were divided into two groups: sLex positive and sLex negative. The incidence of sLex positive was correlated with the depth of tumor invasion, the presence of the lymph node metastasis, lymphatic invasion, and the disease stage. The difference was statistically significant (P = 0.0026; P = 0.0002; P = 0.003; P = 0.0013; respectively). Based on the data on 114 patients who underwent curative resections, incidence of the disease recurrence was assessed. The sLex-positive patients had higher incidence of recurrence in distant organs, especially in the liver, than that of the sLex-negative patients. The 5-year disease free survival rates of sLex-positive and -negative patients were 57.7 and 89.1%, respectively (P = 0.0002). The difference of 5-year overall survival rates between the two were also significant (sLex positive, 58.3%; sLex negative, 93.0%: P < 0.0001). By Cox multivariate analysis, sLex expression levels remained the best discriminant of disease-free survival (P = 0.035) and overall survival (P = 0.0081). These results suggest that increased expression of sLex is correlated with the extent of malignancy and high incidence of recurrence and consequently with survival of colorectal carcinoma patients. Thus sLex may prove to be a potent marker of recurrence in colorectal carcinoma patients.

Inhibition of cancer cell growth by polyinosinic-polycytidylic acid/cationic liposome complex: a new biological activity.

A complex of polyinosinic-polycytidylic acid [poly(I) x poly(C)] and cationic liposome (LIC) inhibited the growth of many tumor cell lines at low concentration in vitro, but poly(I) x poly(C) alone had no such antiproliferative effect. The IC50 values of LIC against the tumor cells ranged from 0.1 to 1000 ng/ml. LIC had strong cytotoxic effects on malignant cells of epithelial and fibroblastic origin from various tissues and was also effective against Adriamycin-resistant tumor cells. LIC did not significantly affect the growth of lymphoma cells, leukemia cells, normal diploid fibroblasts, or primary liver cells at concentrations up to 10 microg/ml. The mechanism of the antiproliferative effect of LIC against malignant cells was the induction of apoptosis. LIC induced the fragmentation of nuclear DNA and the degradation of rRNA in tumor cells. The DNA fragmentation occurred within 1-5 h after the addition of LIC, and both the fragmentation and the inhibition of cancer-cell growth were suppressed by a nuclease inhibitor. In contrast, caspase inhibitors did not affect the antiproliferative activity of LIC. These results suggest that LIC induced apoptosis in malignant cells through the direct activation of nucleases and not through the activation of caspases. LIC reduced the incidence and the size of metastatic liver-cancer tumors in two different mouse metastatic liver-cancer models using human colon carcinoma cells. Histochemical analysis revealed that the KM12-HX cells in the tumor nodules were undergoing apoptosis; therefore, LIC also induced the apoptosis of tumor cells in vivo. In these animal models, LIC caused no observed changes in normal hepatocytes.

CD44 stimulation induces integrin-mediated adhesion of colon cancer cell lines to endothelial cells by up-regulation of integrins and c-Met and activation of integrins.

For cancer metastasis, tumor cells present in the circulation must first adhere to the endothelium. Integrins lymphocyte function-associated antigen (LFA) 1 and very late antigen 4 play a central role in leukocyte adhesion to the endothelium and subsequent migration into tissues. The majority of tumor cells derived from solid cancers including colorectal cancer do not express suitable adhesion receptors, LFA-1 and very late antigen 4. We investigated the mechanisms of adhesion and transendothelial migration of cancer cells using colorectal carcinoma cell lines. Our results showed the following novel features of CD44 on the cells: (a) colon cancer cells express high levels of CD44; (b) stimulation of cancer cells by CD44 cross-linking or fragmented hyaluronan markedly induces the expression of LFA-1s, some of which reveal an activation epitope on the cells; (c) CD44 cross-linking induces F-actin polymerization in the cell cortex; (d) fragmented hyaluronan induces up-regulation of the activation epitope of LFA-1, which is mediated through protein kinase C; (e) stimulation of CD44 augments the LFA-1-mediated adhesion of cancer cells to endothelial cells and intercellular adhesion molecule 1-transfected cells and facilitates transendothelial migration; (f) stimulation of CD44 also induces expression of the hepatocyte growth factor (HGF) receptor c-Met on cancer cells; and (g) HGF further amplifies the LFA-1-mediated adhesion of cells prestimulated by CD44-derived signaling. Our results indicated that stimulation by CD44 induces "outside-in signaling," which consists of a direct pathway via CD44 and an alternate pathway through the induction of c-Met expression via HGF. Such stimuli augment the expression and trigger the function of integrins via "inside-out signaling" in colon cancer cells, which leads to amplification of integrin-mediated adhesion to the vessel wall and subsequent transendothelial migration.

Soluble factor in normal tissues that stimulates high-molecular-weight sialoglycoprotein production by human colon carcinoma cells.

The stimulation of high molecular weight sialoglycoprotein synthesis by a soluble factor derived from normal colon tissues was studied in vitro with human colon carcinoma cell lines, HT-29 P and a metastatic variant HT-29 LMM. The synthesis of all three high-molecular-weight sialoglycoproteins (approximate Mr 900,000, 740,000, and 450,000) by HT-29 P cells or HT-29 LMM cells growing in vitro was enhanced by supplementing the culture medium with a conditioned medium of fresh human colon organ culture. Changes were detected by polyacrylamide gel electrophoresis of lysates from [3H]glucosamine-labeled cells on 3% gels followed by fluorography, or by electrophoresis of lysates from unlabeled cells followed by incubation with 125I-labeled wheat germ agglutinin and autoradiography. No changes were detected in the major protein components or in glycoproteins at Mr less than 200,000 as revealed by polyacrylamide gel electrophoresis. The treated cells did not change their growth rate or morphology. The connective tissue portions of the colon tissues were apparently responsible for the production of this stimulatory substance. The stimulatory activity was preserved at 56 degrees C but was inactivated by heating at 100 degrees C. The substance was eluted from a Sephacryl S-200 column at a position between the elution positions of ovalbumin and trypsinogen. The colon carcinoma cells treated with the conditioned medium and producing increased amounts of high-molecular-weight sialoglycoproteins were less sensitive to the cytolytic effects of recombinant interleukin 2-activated human peripheral blood lymphocytes than untreated cells were. The treated colon carcinoma cells induced stronger platelet aggregation than their untreated counterparts did. Therefore, this substance may represent one of the normal host tissue factors that can influence and modulate malignant behavior of carcinoma cells growing in vivo.

Distribution of ferritin-conjugated lectins on sialidase-treated membranes of human erythrocytes.

The labeling of sialidase-treated, human erythrocyte membranes with ferritin-conjugates of four plant lectins, concanavalin A, Ricinus communis hemagglutinin, Bauhinia purpurea hemagglutinin and Arachis hypogoea hemagglutinin, is reported. Among these ferritin-conjugated lectins, ferritin-conjugated concanavalin A and ferritin-conjugated R. communis hemagglutinin were found in clusters on the sialidase-treated membranes, whereas ferritin-conjugated B. purpurea hemagglutinin and ferritin-conjugated A. hypogoea hemagglutinin were found in a random distribution on the membranes. Furthermore, when the membranes were labeled with a mixutre of concanavalin A and ferritin-conjugated B. purpurea hemagglutinin, ferritin particles were found in clusters, indicating that the membrane receptors for B. purpurea hemagglutinin were forced to more together with those for concanavalin A. A method for the quantitative estimation of the clustering of ferritin particles on the membranes was also devised and applied to the labeling of sialidase-treated, human erythrocyte membranes with the above four ferritin-conjugated lectins.