Pattern of endogenous lectins in a human epithelial tumor

Salt and detergent extracts of a malignant epithelial tumor, obtained by extraction of acetone powder, were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan, fucose, and heparin. Successive elution by chelating reagent and specific sugar resulted in isolation of different Ca2+-dependent and Ca2+-independent endogenous carbohydrate-binding proteins, as analyzed by gel electrophoresis. It appears from the analysis that certain bands represent newly identified proteins capable of binding to lactose (at Mr 64,000), melibiose (at Mr 28,000), and fucose (at Mr 62,000 and 70,000). Other carbohydrate-binding proteins isolated from this human tumor have been identified in normal, especially embryonic, tissues of different nonhuman vertebrates. The carbohydrate-binding proteins are assayable as agglutinin with rabbit erythrocytes and show no detectable enzymatic activity. They can thus be defined as lectins. The presence of a complex pattern of endogenous lectins and their biochemical characteristics may contribute to an understanding of intercellular interaction during the complex process of metastatic spread and may furthermore allow a new tool for diagnosis and a lectin-based therapy.     

Localization of endogenous beta-galactoside-binding lectin in human cells and tissues

A rabbit antiserum raised against the 14.5-kilodalton (kDa) subunit of human splenic galaptin was used to probe protein blots of several tissue extracts. For all tissues examined, the only immunoreactive species detected was a 14.5-kDa polypeptide. This antiserum and a rabbit antiserum raised against native lung galaptin were used in immunohistochemical assays to determine the localization of galaptin in selected tissues and cells. In normal colon, galaptin was found prominently in the basement membrane and in the stroma. The cytoplasm of epithelial cells stained lightly for galaptin whereas mucous granules and secreted mucin were uniformly negative for galaptin. Hemagglutination inhibition assays also failed to demonstrate an interaction between galaptin and mucin. Macrophages stained conspicuously for galaptin in colonic and cutaneous tissue as did some capillary walls. In cutaneous tissue, the extracellular matrix and hair follicle cells contained abundant galaptin. Galaptin was absent in basal cell carcinoma and associated stroma. Galaptin was found throughout the cytoplasm of carcinoma cells of gynecologic origin present in effusions. Protein blot analysis of extracts of extracellular matrix synthesized in vitro by endothelial cells confirmed the presence of galaptin in matrix. The results show that: (1) galaptin is variably expressed by different cells and tissues; (2) its cellular location is not restricted to the cell surface; (3) galaptin is not associated with normal mucin; (4) the extracellular matrix is a major site of galaptin deposition, and (5) some malignant tissue may be characterized by a deficiency of galaptin.     

Differential expression of endogenous lectins on the surface of nontumorigenic, tumorigenic, and metastatic cells

A monoclonal antibody that was found to recognize endogenous galactoside-specific lectins of various tumor cells by immunoblot analysis was used for quantitative analyses of cell surface lectin on nontumorigenic, tumorigenic, and metastatic cells of diverse histological types and origin. Indirect immunofluorescent staining of viable cells followed by analysis with a fluorescence-activated cell sorter revealed marked differences in the amount of surface lectins between untransformed and malignant cells. While lectin was either absent or present in a very low density on the surface of normal cells, neoplastic cells were invariably stained by the antilectin antibodies. Furthermore, among related tumor cell variants of the K-1735 melanoma and UV-2237 fibrosarcoma tumor systems, cells exhibiting a higher lung-colonizing potential also expressed higher levels of cell surface lectin. These results suggest that the presence of a lectin on the cell surface may be related to neoplastic transformation and progression toward metastasis.     

Immunogenic cell variants of a mouse teratocarcinoma confer a protection against the original non-immunogenic transplantable tumor

We reported previously that, by mutagenesis of teratocarcinoma cell line PCC4. aza1, it is possible to obtain immunogenic cell variants [tum⁻ (incapable of forming progressive tumors)], that are rejected by syngeneic mice. These tum⁻ variants confer an immune protection against PCC 4.aza 1, even though this line does not elicit any rejection response in syngeneic mice.We show here that these variants also confer an immune protection against other malignant cell lines derived from the same transplantable teratocarcinoma as PCC 4.aza 1 and also against this transplantable tumor itself. These results rule out the possibility that the protection against PCC 4.aza 1 is caused by an artefactual antigen acquired in vitro. They suggest that it may be possible to use tum⁻ variants to elicit a rejection response against non-immunogenic tumors in the primary host.     

Comparison of endogenous lectins in human embryonic carcinoma and yolk sac carcinoma

Salt and detergent extracts of xenografted human embryonic carcinoma and a human yolk sac carcinoma were fractionated on different sets of Sepharose columns covalently derivatized with lactose, asialofetuin, melibiose, mannan and fucose. Successive elution by chelating reagent and specific sugar resulted in isolation of endogenous Ca2+-dependent and Ca2+-independent carbohydrate-binding proteins, as analyzed by gel electrophoresis. The salt extracts of the embryonic carcinoma contained a Ca2+-dependent carbohydrate-binding protein at Mr66,000, that was undetectable in yolk sac tumor extracts. Also, a Ca2+-independent fucose-binding protein at Mr62,000 could be purified. In general, the pattern of carbohydrate-binding proteins showed quantitative and qualitative differences as compared to normal tissue. The carbohydrate-binding proteins were assayable as agglutinin and showed no enzymatic activity. They can thus be defined as lectins. Their presence within certain stages of differentiation and developmental regulation may contribute to improvement of the classification, diagnosis and therapy of this tumor class. These new lectins may also be functionally related to the stage-specific embryonic antigens like SSEA-1.     

Endogenous galactoside-binding lectins: a new class of functional tumor cell surface molecules related to metastasis

The formation of secondary tumors by circulating cancer cells (blood-borne metastasis) correlates with an increased tendency of the cells to form emboli by aggregation with other tumor cells or with host cells. Although it is evident that cell-cell recognition and adhesion are mediated by cell surface components, the identity of these molecules is only now being unraveled. Over the last decade an increasing number of studies have demonstrated the presence of endogenous carbohydrate-binding proteins on the surface of various normal cells, and it has been proposed that such lectin-like molecules might be involved in intercellular adhesion.We have shown that various tumor cell lines contain endogenous galactose-specific lectins. Lectin activity was detected at the cell surface by the binding of asialofetuin. This glycoprotein also enhanced the aggregation of the tumor cells. After purification by affinity chromatography on immobilized asialofetuin the lectin activity was associated with two proteins of M_r 14.500 and 34,000. By using polyclonal and monoclonal antilectin antibodies in conjunction with various immunologic techniques we have demonstrated that the endogenous lectins are present on the surface of different tumor cells. Quantitation of cell surface lectins by flow cytometric analyses of antilectin antibody binding revealed that among related tumor cells those exhibiting a higher metastatic potential expressed more lectin on their surface. The binding of monoclonal antilectin antibodies to metastatic cells decreased asialofetuin-induced homotypic aggregation in vitro and suppressed the ability of the cells to form lung metastases after intravenous injection in the tail vein of syngeneic mice. These results strongly implicate the tumor cell surface lectins in cell adhesion and metastasis. We propose that such lectins can increase the ability of tumor cells that enter the blood stream to form aggregates with other tumor cells, or to adhere to host cells or the extracellular matrix and thereby increase their metastatic potential. Other contributing components to tumor cell-host cell interactions are cell surface carbohydrate-binding proteins that have been detected on lymphocytes, platelets, macrophages, hepatocytes, and endothelial cells. These lectin-like molecules might recognize and bind carbohydrates expressed on the surface of tumor cells and enhance emboli formation and organ colonization.     

Binding patterns of lectins to human retinoblastoma cells: preliminary findings

Lectin binding patterns of cultivated retinoblastoma cell lines Y-79 and Mac-7 and freshly isolated tumor tissue revealed membrane-associated sugar residues NAcD-Gal, D-Gal, mannose, and D-GlcNAc. Compared to normal retinal pigment epithelium, LCA positively bound to retinoblastoma cells. Similarities in the glycoconjugate patterns with LV-B-1 cells are shown.     

Implications of endogenous tumor cell surface lectins as mediators of cellular interactions and lung colonization

A monoclonal antibody (mAb) designated 5D7 that is directed against endogenous, galactoside-specific lectin and binds to the surface of various tumor cells was used to examine the involvement of cell surface lectin molecules in cellular interactions in vitro and in vivo. The mAb 5D7 was found to inhibit asialofetuin-induced homotypic aggregation of B16 melanoma and UV-2237 fibrosarcoma cell variants by up to 80%. The rate at which these cells, as well as the virally transformed fibroblasts (SVPy-3T3), adhere to tissue culture dishes was reduced in the presence of mAb 5D7 to less than 50% of the control. The anti-lectin mAb had no effect on the adhesion rate of untransformed 3T3 fibroblasts. Treatment of B16 and UV-2237 cells with mAb 5D7 in vitro before their injection into the tail vein of syngeneic mice resulted in a decrease of up to 90% in the appearance of tumor lung colonies. These findings imply that tumor cell surface lectins might play a role in mediation of cell-to-cell and cell-to-substratum adhesion in vitro as well as in similar interactions in vivo that are relevant for metastasis.     

Cell surface alterations in murine leukaemia P388 adriamycin-resistant cells: studies on lectin-induced agglutination and rearrangement of lectin receptors

Cell surface alteration was studied in a subline of murine lymphocytic leukaemia resistant to the broad-spectrum anticancer agent adriamycin (P388/ADR) employing concanavalin A(Con A)-induced agglutination and rearrangement of lectin receptors. Con A induced more agglutination of P388/ADR when compared to the drug-sensitive parental cell line (P388/S). Studies on the redistribution of Con A and Ricinus communis agglutinin-I revealed a high percentage of P388/ADR showing internalized fluorescence, while a majority of P388/S displayed a uniform distribution of fluorescence on the cell surface.     

Probing human malignant T cells with lectins: A comparison with their surface antigen patterns defined by monoclonal antibodies

Tumor cells from 40 children and 13 adults with T cell malignancies were assessed for staining with fluorescinated peanut agglutinin (PNA) and soybean agglutinin (SBA). These cell populations had also been characterized for surface antigens using a series of monoclonal antibodies (Mo. Ab.) that permit an assignment of malignant cells to discrete stages of normal T cell differentiation. We had previously shown a clear correspondence between lectin- and Mo. Ab.-defined cell compartments within thymuses from normal children and T cells in peripheral blood. We report here that the pattern of reactivity of malignant T cells populations with lectins correlates closely the degree of maturation, as assessed by Mo. Ab. Thus, utilization of lectins together with Mo. Ab., can be clinically useful to characterize T cell malignancies. This observation shows that, in spite of a high degree of heterogeneity of malignant T cell populations from one patient to the other, in their pattern of surface antigens, these populations seem essentially to conform to the scheme of normal T cell differentiation.     

Heterogeneity of the surface material in isolated cells of transplantable hamster melanomas

The heterogeneity of the surface material released by trypsin in isolated cells of melanotic and amelanotic melanomas was studied by the method of separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoretic patterns of the surface material derived from two kinds of melanoma showed some differences. The differences in the surface proteins and glycoproteins seem to be related to the biological properties of both melanomas.     

Cell surface glycoprotein analysis: a diagnostic tool in human leukemias.

We have radiolabelled surface glycoproteins of different types of leukemic cell. The labelled proteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Surface glycoprotein patterns discriminatory for acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) were found. We conclude that the analysis of the surface glycoprotein profile provides a useful method for the classification of leukemic cells according to cell type and stage of differentiation.     

Cell surface asparagine-linked sugar chains of human early myeloblastic leukemic cells (KG-1a)

The asparagine-linked sugar chains obtained from total cell surface membrane glycoproteins of human early myeloblastic leukemic cells (KG-1a cells) were studied. The sugar chains liberated by hydrazinolysis were purified by paper electrophoresis, paper chromatography, and Bio-Gel P-4 chromatography followed by analysis of exoglycosidase digestion and methylation study. Neutral oligosaccharides were all composed of high mannose type sugar chains. Acidic oligosaccharides were chiefly composed of typical bi-, tri-, and tetraantennary complex type sugar chains with Gal beta 1----4GlcNAc beta 1----groups and Neu-Ac alpha 2----3 or 6Gal beta 1----4GlcNAc beta 1----groups (in which Gal is galactosyl, GlcNac is N-acetylglucosamine, and NeuAc is N-acetylneuraminic acid) as side chains. Moreover the following two structures were identified in (in which Fuc is fucosyl): monosialyl bi- and triantennary sugar chains with a Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----group (X determinant) as one of the side chains; and monosialyl tetraantennary sugar chains with a Gal beta 1----4GlcNAc beta 1----3Gal beta 1----4GlcNAc group (repeating N-acetyllactosamine unit) as one of the side chains. These data together with our previous studies on sugar chains of K562 cells [early erythroblast], adult erythrocytes [H. Yoshima, N. Shiraishi, A. Matsumoto, S. Maeda, T. Sugiyama, and A. Kobata, J. Biochem. (Tokyo), 91: 233-246, 1982], and HL-60 cells [promyelocyte] [A. Mizoguchi, S. Takasaki, S. Maeda, and A. Kobata, J. Biol. Chem., 259: 11943-11957, 1984] strongly suggest that the cell surface asparagine-linked sugar chains alter in an orderly fashion, systematically in association with lineage and maturation stages during hematopoietic cell differentiation.     

PGM-1: a transplantable murine leukemia of granulocyte-macrophage progenitor cells

PGM-1 is a transplantable leukemia of C3H/HeJ mice growing as a population of undifferentiated blast cells with a predisposition to form subcutaneous tumors and to grow in lymphoid organs. Cell survival and proliferation in vitro are absolutely dependent on stimulation by hemopoietic growth factors, and up to 100% of tumor cells can form colonies of mature granulocytes and/or macrophages in semisolid cultures, the colonies containing no clonogenic cells. Most clonogenic cells in the leukemic population respond to stimulation by multi-colony-stimulating factor (IL-3) or GM-CSF, but some respond also to M-CSF, G-CSF, IL-4, IL-5, or IL-6. In their surface phenotype and proliferative characteristics in vitro, PGM-1 leukemic cells resemble normal granulocyte-macrophage progenitor cells, and the leukemia may be a useful model for human chronic myeloid leukemia.     

Chimeric potency of a mutator strain of mouse teratocarcinoma cells

The developmental potential of a mutator strain Ara Cr (1.5)4 of mouse teratocarcinoma cells was examined by injecting these cells into host blastocysts. Analysis of developing embryos at 9.5 days of gestation showed the mutator strain gave chimeras at a rate comparable with that of the parent strain. However, most of these chimeric embryos with the mutator strain were abnormal, and the extent of abnormality seems to be related to the proportion of the mutator-derived cells in the embryos. When injected embryos were allowed to develop to 14.5 days, the number of developing embryos decreased, and only a few were chimeric in limited tissues. The results suggest that the mutator strain is lethal to early gestational development, and only those chimeras with limited colonization of the strain can develop normally beyond this stage.     

Peanut lectin: a mitogen for normal human colonic epithelium and human HT29 colorectal cancer cells.

BACKGROUND: The protein peanut agglutinin (PNA) is a galactose-binding lectin whose receptor, the Thomsen-Friedenreich (TF) blood-group antigen, shows increased expression in hyperplastic and neoplastic colonic epithelium. PURPOSE: Our hypothesis was that, under these conditions, increased lectin receptors could interact with dietary lectins, which would act as tumor promoters by stimulating cell proliferation. This study was designed to confirm whether active PNA is recoverable from feces after ingestion of peanuts and to assess the mitogenic effect of PNA on proliferation of epithelial cells in the colon. METHODS: Peanut lectin was extracted from feces by lactose-agarose affinity chromatography and was assayed for hemagglutinating activity. Cultured explants of histologically normal biopsy specimens of colonic mucosa from 31 patients were examined. Crypt cell production rate and incorporation of [3H]N-acetylglucosamine into mucin were assessed as indicators of proliferative and metabolic responses to PNA. In addition, we evaluated the separate and combined effects of PNA and epidermal growth factor (EGF) on cell proliferation in human HT29 colorectal cancer cells, by using tritiated thymidine incorporation and cell counts. RESULTS: Peanut lectin extracted from feces showed hemagglutinating activity toward desialylated red blood cells similar to that of a lectin preparation extracted from raw peanuts. Evaluation of biopsy specimens of normal colonic mucosa demonstrated that PNA at a concentration of 25 micrograms/mL caused statistically significant increases in crypt cell production (31% [mean] +/- 5% [SD]; P = .00005) and mucus synthesis (77% +/- 12%; P less than .000001). At 7.5-100 micrograms/mL, PNA was mitogenic for the HT29 colorectal cancer cell line. At 25 micrograms/mL, PNA alone produced a statistically significant increase in thymidine incorporation (44% [mean] +/- 3.7% [SD]; P = .002). For PNA in combination with EGF at 100 pg/mL, the increase was significantly greater (222% +/- 11.2%) than that for EGF alone (57% +/- 5%; P = .003). CONCLUSIONS: These results suggest that expression of the PNA receptor, TF antigen, by hyperplastic or neoplastic colonic epithelium may affect cell proliferation. IMPLICATIONS: It is possible that dietary lectins such as PNA, which bind to the TF antigen, promote cell proliferation and thus cancerous growth, while galactose-containing vegetable fiber would inhibit this effect by competing for binding by these lectins.     

Expression of endogenous murine leukemia virus in transplantable tumor cells and DNA methylation of the viral sequences

A cell line derived from early passage of a transplantable tumor in athymic mice that initially had been injected with human breast adenocarcinoma cells produced a substantial amount of RNA sequences related to the Kirsten murine leukemia virus. The cell line derived from later passage of transplantation of these tumors diminished viral RNA production. Expression of the viral RNA sequences in these cell lines is inversely correlated with the level of viral DNA methylation in the mouse genome.