The human cancer and stem cell marker podocalyxin interacts with the glucose-3-transporter in malignant pluripotent stem cells

Podocalyxin, an integral plasma membrane cell-adhesion glycoprotein, is a marker of human pluripotent and multipotent stem cells. Podocalyxin is also a marker of many types of cancers and its expression correlates with an aggressive and poor-prognosis tumor phenotype. The function of podocalyxin in stem cells and malignant cells is unknown. Protein sequence data obtained from purified podocalyxin protein isolated from embryonal carcinoma cancer stem cells reveals peptide sequence data for the glucose-3-transporter. Protein-precipitation experiments of embryonal carcinoma protein extracts identify a podocalyxin/glucose-3-transporter protein complex. Cell imaging studies demonstrate co-localization of podocalyxin and glucose-3-transporter and confirm the interaction in vivo. Finally, siRNA podocalyxin-knockdown experiments show decreased expression levels of the glucose-3-transporter. These findings suggest a novel interaction of the glucose-3-transporter and the cell-adhesion protein podocalyxin. In pluripotent stem cells and in human cancer disease, podocalyxin may function in part to regulate and maintain the cell surface expression of the glucose-3-transporter.     

Purification of a tumor-specific PNA-binding glycoprotein, gp200, from a human embryonal carcinoma cell line.

A 200-kDa peanut agglutinin (PNA)-binding glycoprotein, gp200, has been purified and partially characterized from the human embryonal carcinoma cell line, HT-E (833k). Tissue distribution analysis of this molecule by lectin blotting with PNA of detergent-extracted proteins from human cell lines and tissues demonstrated expression limited to nonseminomatous germ cell tumors. The 200-kDa protein was purified with lectin affinity and gel filtration chromatography. Purification to apparent homogeneity was demonstrated by one- and two-dimensional gel electrophoresis. Characterization of gp200 revealed it to be a surface integral membrane glycoprotein; however, gp200 could also be purified from the culture media of EC cells, suggesting gp200 has an extracellular role. The carbohydrate groups of gp200 are N-linked and partially sialylated and contain terminal galactose residues. These initial studies suggest that the PNA-defined glycoprotein, gp200, is a candidate for a nonseminomatous germ cell tumor marker.     

Characterization of a new human cell line derived from a xenografted embryonal carcinoma

Summary A human cell line has been established from a transplantable xenografted human testicular tumor, which, both in the original tumor and in the xenograft, exhibited the histological characteristics of an undifferentiated malignant teratoma (embryonal cell carcinoma). The cells in culture were undifferentiated by biochemical, morphological, and ultrastructural criteria, growing as small islands of cells that tended to form aggregates at high density. The cells showed some variation in chromosome number with 30 to 40% of the cells having a normal human karyotype. The cells expressed high levels of alkaline phosphatase, which by heat inactivation and inhibition studies was 40 to 50% placental type alkaline phosphatase. None of the cultures produced human chorionic gonadotrophin, alphafetoprotein, carcinoembryonic antigen, or fibronectin, although at high cell densities plasminogen activator could be detected at low levels. Cell surface studies showed that the cells shared antigens with the murine embryonal carcinoma cell line F9, expressedβ ₂-microglobulin at very low and variable levels, and bound the lectin peanut agglutinin. These studies suggest that this cell line has some of the characteristics described for murine embryonal carcinoma cell lines.     

Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen.

The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 was previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. Cross-linking of the APO-1 antigen by anti-APO-1 induced programmed cell death, apoptosis, of APO-1 positive cells. To characterize the APO-1 cell surface molecule and to better understand its role in induction of apoptosis, the APO-1 protein was purified to homogeneity from membranes of SKW6.4 B lymphoblastoid cells by solubilization with sodium deoxycholate, affinity chromatography with anti-APO-1 antibody, and reversed phase high performance liquid chromatography. Each purification step was followed by an APO-1-specific solid phase enzyme-linked immunosorbent assay using the monoclonal antibody anti-APO-1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the APO-1 antigen was found to be a membrane glycoprotein of 48-kDa. Endoproteinase-cleaved peptides of the APO-1 protein were subjected to amino acid sequencing, and corresponding oligonucleotides were used to identify a full-length APO-1 cDNA clone from an SKW6.4 cDNA library. The deduced amino acid sequence of APO-1 showed sequence identity with the Fas antigen, a cysteine-rich transmembrane protein of 335 amino acids with significant similarity to the members of the tumor necrosis factor/nerve growth factor receptor superfamily. The APO-1 antigen was expressed upon transfection of APO-1 cDNA into BL60-P7 Burkitt's lymphoma cells and conferred sensitivity towards anti-APO-1-induced apoptosis to the transfectants.     

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.     

Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells

Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.     

Partial characterization of a collagen-binding, differentiation-related glycoprotein from skeletal myoblasts

A 46-kDa glycoprotein, gp46, which binds collagen has been purified to homogeneity from L6 rat skeletal myoblasts. The procedure involves extraction of crude myoblast membranes with 1% sodium dodecyl sulfate followed by concanavalin A affinity chromatography and preparative gel electrophoresis. The sequence of 15 N-terminal amino acids had some resemblance to a sequence in myosin light chains. The oligosaccharide chains of the glycoprotein can be released by treatment with endoglycosidase H, suggesting that gp46 has high-mannose type of glycans. Galactose and sialic acid are not detected in the purified protein. gp46 is widely distributed and conserved in different cell lines as determined by immunoblotting using a monoclonal anti-gp46 antibody. High levels of gp46 were found in several fibroblastic and myogenic cell lines, but not in a hematopoietic cell line. Undifferentiated F9 embryonal carcinoma cells lacked gp46 but the glycoprotein was induced when the cells were made to differentiate in the presence of retinoic acid. Broad survey of gp46 in different cell lines also suggests that it is present mainly in those cell lines which attach to the substratum and produce collagens. Although the function of gp46 is not yet known, the evidence suggests that it is developmentally regulated and is probably involved in the synthesis or assembly of collagen in the endoplasmic reticulum.     

t-Complex-Associated Embryonic Surface Antigen Homologous to mLAMP-1

Previously we described an embryonic cell surface glycoprotein, ESGp, associated with the t-embryonic lethal alleles of the mouse t complex. This antigen is expressed on the cell surface of both early mouse embryos and embryonal carcinoma (EC) cell lines. The antigen is localized to areas of cell–cell contact in EC lines and redistributes to the outer edges of the blastomeres during compaction, thereby indicating a potential role in embryonic cell–cell interaction. We now report that this t-complex-associated ESGp is homologous to the mouse lysosomal-associated membrane protein-1 (LAMP-1). Limited protein sequence analyses of the amino terminal and an internal peptide indicate considerable homology with the LAMP-1 protein. Biochemical parameters such as protein core size, sulfation and phosphorylation status, and resistance to proteolysis also demonstrate homology. While we detect only a single message with a mouse LAMP-1 cDNA probe via Northern blotting, Southern analyses indicate the existence of at least two homologous LAMP-1 genes. Additionally, we present evidence suggesting that ESGp/LAMP-1 serves as a substrate which may be differentially glycosylated by the activities of the gene products of the different t-lethal alleles.     

Detection and characterization of mouse mammary tumor virus cell surface antigens: estimation of antigen abundance by protein A assay.

Antisera against the following mouse mammary tumor virus (MMTV) structural proteins were used to detect MMTV cell surface antigens: (i) the 27,000-dalton nucleoid protein, p27; (ii) the 36,000-dalton envelope glycoprotein, gp36; and (iii) the 52,000-dalton exterior envelope glycoprotein, gp52. We report here the development of an adherent-cell isotopic staphylococcal protein A (SPA) test (ISPAT) for MMTV structural proteins which allows for the detection of an MMTV membrane-associated antigen as well as an estimate of its relative abundance on the cell surface. This test demonstrated that the gp52 was the predominant MMTV cell surface antigen detected on both C3H and GR mouse mammary tumor cells. In a comparative study with anti-gp52 and anti-gp36 sera, SPA-specific binding with anti-gp36 serum was found to be only 5 to 6% of that obtained for the external virion glycoprotein, gp52. Both direct and indirect ISPAT indicated the presence of a low but detectable number of gp36 determinants on GR-MMTV cells; however, these gp36 determinants, unlike gp52 determinants, appeared to be exposed by the fixation procedure used. Only 0.9 to 1.1% of the gp52-specific binding was detected when anti-gp36 serum was allowed to react with viable cells. The binding of [125I]SPA achieved with anti-p27 serum was even less than that detected with gp36-directed reagents, indicating that p27 is not a cell surface antigen. The use of fluoresceinated SPA further demonstrated that p27 and gp36 reactivity was only associated with a small number of cells in each of the mammary cultures tested. When N-[4-(5-nitro-2-furyl)-2-thiazoly]-formamide-induced C3H bladder tumor cells were subjected to a gp52-directed ISPAT, the failure to detect gp52-specific binding demonstrated the specificity of this assay for MMTV gp52-expressing cells. In addition to detecting and characterizing MMTV cell surface antigens, the newly developed adherent cell assay could measure changes in the abundance of cell surface gp52. When dexamethasone-treated and untreated GR cells were compared, measurements of gp52-specific SPA binding indicated that dexamethasone stimulation leads to a 12.2-fold increase in the amount of cell surface gp52 detected.     

A collagen-binding protein in the endoplasmic reticulum of myoblasts exhibits relationship with serine protease inhibitors.

Several cDNA clones encoding a 46-kDa collagen-binding glycoprotein (gp46) from rat skeletal myoblasts were isolated and sequenced. The cDNA encoded a 17-amino acid signal peptide and a 400-amino acid mature protein, containing three potential N-linked oligosaccharide attachment sites. The cDNA sequence of gp46 shows 93% identity in the coding region with J6, a retinoic acid-inducible gene coding for a protein of unknown function described from embryonal carcinoma F9 cells. The first 41 NH2-terminal amino acids of the predicted J6 sequence are, however, different from the gp46 sequence as a result of a 7-base pair insertion in the gp46 cDNA. In addition, the NH2-terminal amino acid sequence of hsp47, a collagen-binding protein found in chick embryo fibroblasts, shows 64% identity to gp46 over 36 residues. Interestingly, this alignment begins 10 residues inward from the first amino acid in the mature form of gp46. A significant sequence similarity was observed between gp46 and members of the serine protease inhibitor (serpin) family. Unlike other serpins, however, gp46 is both a heat shock and a collagen-binding protein and is localized to the lumen of the endoplasmic reticulum, as suggested by the presence of the RDEL sequence at the COOH terminus. This sequence is similar to other proposed endoplasmic reticulum retention signals.     

Molecular cloning of gp49, a cell-surface antigen that is preferentially expressed by mouse mast cell progenitors and is a new member of the immunoglobulin superfamily.

gp49 is a Mr 49,000 glycoprotein expressed on the surface of mouse bone marrow-derived mast cells, which are progenitors for the major in vivo mast cell subclasses, typified by intestinal mucosal mast cells and serosal mast cells. The amino-terminal amino acid sequence of gp49 was determined after isolation of the solubilized membrane protein by affinity chromatography with the B23.1 anti-gp49 monoclonal antibody. Redundant oligonucleotides were used to isolate a full-length 1.3-kilobase cDNA from a mouse mast cell library. The predicted amino acid sequence contains a signal peptide of 23 residues, an extracellular domain of 215 residues with three potential sites of N-linked glycosylation, a transmembrane domain of 23 residues, and a cytoplasmic tail of 42 residues. Hybridization of the gp49 cDNA was limited to mRNA extracted from those cell types that also bound the B23.1 monoclonal antibody as assessed by cytofluorographic analyses. The predicted extracellular domain of gp49 contains two regions of 48 and 51 amino acids, each flanked by cysteine residues. Both regions meet criteria for being C2-type domains of the immunoglobulin superfamily based upon the alignment of consensus amino acids and their predicted secondary structure organization. Thus, gp49, a membrane glycoprotein preferentially expressed by the progenitor mast cell population, is a new member of the immunoglobulin superfamily.     

Expression of membrane glycoproteins in normal keratinocytes and squamous carcinoma cell lines

Con A acceptor glycoproteins were analyzed by 2D-PAGE and 125I-Con A overlay in three squamous carcinoma cell lines and compared with those in the simian virus (SV40)-transformed keratinocyte cell line SVK-14 and in normal keratinocytes. The majority of the glycoproteins identified by this technique were expressed at similar levels in all of the cells examined, independent of the culture conditions used. A cell surface glycoprotein gp34 (MW 34 kDa, pI 5.1) was increased in the tumor cells compared with normal keratinocytes and expression varied with the culture density. Another glycoprotein, gp21 (MW 21 kDa, pI 6.3), was found to be increased in expression in normal keratinocytes and stratified hyperconfluent cultures of squamous carcinoma cell lines. This paper describes the potential of this technique to identify membrane glycoproteins which may be expressed as a function of proliferation or differentiation.     

Molecular cloning of two CD7 (T-cell leukemia antigen) cDNAs by a COS cell expression system.

The human CD7 antigen (gp40) is a cell surface glycoprotein found on thymocytes and mature T-cells. It is one of the earliest antigens to appear on cells of the T-lymphocyte lineage, and the most reliable clinical marker of T-cell acute lymphocytic leukemia. This report describes the isolation and nucleotide sequence of a full length CD7 cDNA, and of a cDNA for an unusual intron-bearing precursor. The DNA sequence of the clone predicts a highly glycosylated membrane protein with homology to members of the immunoglobulin superfamily, and no relationship to known oncogenes. Over-expression of CD7 RNA was observed in only one T-cell tumor line, and genomic DNA rearrangement was not observed in any lines. Prompted by a recent suggestion that CD7 plays a role in IgM binding, COS cells expressing CD7 were tested and found not to bind IgM or IgM immune complexes.     

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.     

CD200: a putative therapeutic target in cancer

CD200 was recently described as a new prognosis factor in multiple myeloma and acute myeloid leukemia. CD200 is a membrane glycoprotein that imparts an immunoregulatory signal through CD200R, leading to the suppression of T-cell-mediated immune responses. We investigated the expression of CD200 in cancer using publicly available gene expression data. CD200 gene expression in normal or malignant human tissues or cell lines was obtained from the Oncomine Cancer Microarray database, Amazonia database and the ITTACA database. We found significant overexpression of CD200 in renal carcinoma, head and neck carcinoma, testicular cancer, malignant mesothelioma, colon carcinoma, MGUS/smoldering myeloma, and in chronic lymphocytic leukemia compared to their normal cells or their tissue counterparts. Moreover, we show that CD200 expression is associated with tumor progression in various cancers. Taken together, these data suggest that CD200 is a potential therapeutic target and prognostic factor for a large array of malignancies.     

Immunoelectron microscopic localization of mammary tumor virus (MTV) antigens in normal and cancerous mammary glands of mice

Peroxidase-labeled Fab' fragments of rabbit antisera against gp52 (major envelope protein) and A-particles of mammary tumor virus (MTV) were prepared and used for investigation by immunoelectron microscopy of the replication cycle of MTV-specific envelope and core antigens in normal and malignant mammary gland cells of female mice. The specificity of the antisera was proven by absorption tests and lack of reactivity to MTV-free mammary tissues. Periodate-lysine-paraformaldehyde (PLP) fixation sufficiently preserved the antigenicity of gp52, while Zamboni's fixative was useful to preserve the core antigen. Saponin pretreatment was necessary to reveal the intracellular antigen of A particles but had no influence on gp52. In addition to its presence at the envelope of D particles, gp52 was clearly associated with the biomembrane system, including the nuclear membrane, endoplasmic reticulum, Golgi apparatus and plasma membrane independent of the presence of virus particles. In mammary tumors, a significant level of gp52 antigen was often expressed on the entire cell surface membrane. In contrast, it was localized only to the apical plasma membrane in normal mammary gland cells. A particle antigens were confined to the intracytoplasmic A particles, usually visible as clusters, and to the inner part of B particles. These ultrastructural findings support the available biochemical data on the morphogenesis of MTV particles.     

EMMPRIN/CD147-encriched membrane vesicles released from malignant human testicular germ cells increase MMP production through tumor–stroma interaction

Background

Elevated levels of EMMPRIN/CD147 in cancer tissues have been correlated with tumor progression but the regulation of its expression is not yet understood. Here, the regulation of EMMPRIN expression was investigated in testicular germ cell tumor (TGCTs) cell lines.

Methods

EMMPRIN expression in seminoma JKT-1 and embryonal carcinoma NT2/D1 cell lines was determined by Western blot, immunofluorescence and qRT-PCR. Membrane vesicles (MVs) secreted from these cells, treated or not with EMMPRIN siRNA, were isolated by differential centrifugations of their conditioned medium. MMP-2 was analyzed by zymography and qRT-PCR.

Results

The more aggressive embryonic carcinoma NT2/D1 cells expressed more EMMPRIN mRNA than the seminoma JKT-1 cells, but surprisingly contained less EMMPRIN protein, as determined by immunoblotting and immunostaining. The protein/mRNA discrepancy was not due to accelerated protein degradation in NT2/D1 cells, but by the secretion of EMMPRIN within MVs, as the vesicles released from NT2/D1 contained considerably more EMMPRIN than those released from JKT-1. EMMPRIN-containing MVs obtained from NT2/D1, but not from EMMPRIN-siRNA treated NT2/D1, increased MMP-2 production in fibroblasts to a greater extent than those from JKT-1 cells.

Conclusion and general significance

The data presented show that the more aggressive embryonic carcinoma cells synthesize more EMMPRIN than seminoma cells, but which they preferentially target to secreted MVs, unlike seminoma cells which retain EMMPRIN within the cell membrane. This cellular event points to a mechanism by which EMMPRIN expressed by malignant testicular cells can exert its MMP inducing effect on distant cells within the tumor microenvironment to promote tumor invasion. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties.     

Molecular cloning and characterization of the rat liver IL-6 signal transducing molecule, gp130.

Interleukin-6 (IL-6) is a multifunctional cytokine that exerts its effects on different target cells by interacting with a specific receptor. This interaction leads to the association and activation of a second membrane glycoprotein, gp130, which is the IL-6 signal transducing molecule. The nucleotide sequence of gp130 from a human B-cell line has been reported. We report here the cloning and sequence analysis of the gp130 molecule derived from rat liver. Comparison of gp130 molecules from the different species and cell types reveals 78% overall amino acid homology and 94% identity in the growth factor signaling domain. Two gp130 mRNA species, a moderately abundant species of 7.5 kb and a lesser one of 9.0 kb, were present in rat hepatocytes. Ribonuclease protection analyses demonstrated the presence of gp130 mRNA in four different nontransformed cell types: hepatocytes, astrocytes, fibroblasts, and endothelial cells. The sequences between both gp130s in the different cell types are quite similar, supporting the prediction that the different responses initiated by IL-6 on different target cells are modulated by cell-specific proteins distal to the activated gp130 molecule.