4F2 monoclonal antibody recognizes a surface antigen on spread human fibroblasts of embryonic but not of adult origin

The 4F2 monoclonal antibody (mAb) has been shown to recognize a 120- kilodalton glycoprotein expressed on the cell surface of human peripheral blood monocytes, activated (but not resting) T or B cells, and T and B lymphoblastoid cell lines. In this report we show that 4F2 mAb specifically binds to the surface of adherent human embryonic fibroblasts but fails to bind to normal adult fibroblasts. Moreover, 4F2 antigen was expressed on sarcoma-derived or SV40-transformed adult fibroblastic cells. Finally, addition of 4F2 mAb inhibited the growth of cultured HT-1080 fibrosarcoma cell line, but had no inhibitory effect on various embryonic and adult normal or transformed fibroblasts.     

Membrane glycoproteins involved in neurite fasciculation

Lectin affinity chromatography combined with mAb production was used to identify chick neural cell surface molecules related to L1 antigen, a mouse neural glycoprotein implicated in cell-cell adhesion (Rathjen, F. G., and M. Schachner, 1984, EMBO (Eur. Mol. Biol. Organ.) J., 3:1-10). A glycoprotein, G4 antigen, isolated by mAb G4 from adult chick brain is described which comprises a major 135-kD component, a minor doublet at 190 kD, and diffusely migrating bands at 80 and 65 kD in SDS PAGE. This molecule is structurally related to mouse L1 antigen according to NH2-terminal amino acid sequence (50% identity) as well as the behavior of its components in two-dimensional IEF/SDS PAGE gels. A second chicken glycoprotein, F11 antigen, was isolated from adult chick brain using mAb F11. This protein has also a major 135-kD component and minor components at 170 kD and 120 kD. Both immunotransfer analysis with polyclonal antibodies to mAb G4 and to mAb F11 isolate and the behavior on IEF/SDS PAGE gels indicates that the major 135-kD component of F11 antigen is distinct from G4 antigen components. However, the 135-kD component of F11 antigen shares with G4 antigen and the neural cell adhesion molecule (NCAM) the HNK-1/L2 carbohydrate epitope. In immunofluorescence studies, G4 and F11 antigenic sites were found to be associated mainly with the surface of process-bearing cells, particularly in fiber-rich regions of embryonic brain. Although Fab fragments of polyclonal antibodies to mAbs G4 or F11 immunoaffinity isolate only weakly inhibit the Ca2+-independent aggregation of neural cells, they strongly inhibit fasciculation of retinal axons. Together these studies extend the evidence that bundling of axons reflects the combined effects of a group of distinct cell surface glycoproteins.     

Preparation of fibroblast-free cytotrophoblast cultures utilizing differential expression of the CD9 antigen

Summary The leukemia-associated antigen CD9 is present on a variety of normal cells, with apparent variable expression on normal human fibroblasts. In this study, we demonstrate by immunoperoxidase staining and direct binding studies that the CD9 antigen is uniformly expressed on normal human fibroblasts grown from first trimester and term placenta, embryonic fetal fibroblasts, and from human adult and fetal skin fibroblasts. Higher CD9 expression was present on fetal cells. CD9 antigen was not present on trophoblast. Over 99% of fibroblasts could be absorbed onto antibody to the CD9 antigen conjugated to magnetic beads. By applying this selective immunoadsorption of fibroblasts to term placental cytotrophoblast preparations, we demonstrated that fibroblast contamination could be nearly completely eliminated. This is a novel technique for purifying primary trophoblast cultures and may have wider applicability in cell culture of other cell types.     

Fibroblast surface antigen (SF): Molecular properties,distribution in vitro and in vivo,and altered expression in transformed cells

We have recently described a cell type-specific surface (SF) antigen that is deleted in chick fibroblasts transformed by Rous sarcoma virus. SF antigen is a major surface component and makes up about 0.5% of the total protein on normal cultured fibroblasts. The antigen is shed from normal cells and is present in circulation (serum, plasma), and in vivo, also, in tissue boundary membranes. The molecular equivalents of both cellular and serum SF antigen are distinct, large polypeptides, one of which (SF210, MW 210,000) is glycosylated and, on the cell surface, highly susceptible to proteases and accessible to surface iodination. Immunofluorescence and scanning electron microscopy have indicated that the antigen is located in fibrillar structures of the cell surface, membrane ridges, and processes. Human SF antigen is present in human fibroblasts and in human serum. We have recently shown that human SF antigen is identical to what has been known as the “cold-insoluble globulin” and that it shows affinity toward fibrin and fibrinogen. Our results also indicate that loss of the transformation-sensitive surface proteins is due not to loss of synthesis but to lack of insertion of the protein in the neoplastic cell surface. Both normal and transformed cells produce the SF antigen, but the latter do not retain it in the cell surface. The loss of SF antigen, a major cell surface component, from malignant cells creates an impressive difference between the surface properties of normal and malignant cells. The possible significance of SF antigen to the integrity of the normal membrane and its interaction to surrounding structures is discussed.     

Expression of differentiation and age-related antigens on chicken erythroleukemia cells transformed by avian erythroblastosis virus (AEV)

Immature circulating chicken red cells express on their surface two antigenic molecules referred to as Im 48 kD and Im 140 kD antigens. The Im 140 kD antigen is not present beyond the erythroblast stage while the expression of Im 48 kD antigenic molecule remains detectable on circulating erythrocytes of embryos and young chickens, but not on erythrocytes of adult animals. In addition to Im 48 kD and Im 140 kD antigens, the avian erythroblastosis virus (AEV)-transformed erythroid cells express two novel high molecular weight (MW) immature antigens referred to as Im 150 kD and Im 160 kD. Since the transformed erythroid cells are apparently blocked at a stage close to the colony-forming units erythrocytic (CFU-E), these molecules might be expressed on these progenitor cells. The age-related antigenic molecules referred to as E1 48 kD and A 40 kD/A 85 kD antigens are detected on erythrocytes of embryos (and young chickens) and adult animals respectively. The E1 48 kD antigen as well as an antigen related to the A 40 kD were also detected on AEV-transformed erythroid cells deriving from both young chicken bone marrow and yolk sac. The presence of an adult antigen on the embryonic cells might well be related to the transformation by AEV, since the yolk sac CFU-E progenitor cells do not bear the adult antigenicity.     

Localisation on human chromosome 19 of three genes for cell surface antigens defined by monoclonal antibodies

Expression of three distinct human cell surface antigens defined by monoclonal antibodies (mAbs) was examined in a series of rodent-human somatic cell hybrids retaining different subsets of human chromosomes. Cell surface reactivity with mAbs F8 and G253, detecting a 95 kilodalton (kD) glycoprotein (gp95); with mAbs F10 and A103, detecting a 50 kD glycoprotein (gp50); and with mAb S7 was found to cosegregate with human chromosome 19. However, differential antigen expression was observed with hybrids containing fragments of the 19 and hybrids constructed with different human cell types. Comparison of results from the serological typing with the presence of a number of chromosome 19 DNA markers in hybrid cells and cytogenetic analysis suggests that MSK20, the gene coding for the F10/A103 antigen gp50, is located in chromosome region 19pter----19p13.2. The genes coding for the F8/G253 antigen, gp95 (gene symbol MSK19) and the S7 antigen (MSK37) are located in region 19p13.2----19q13.2. Thus, the cell surface antigens described in this study may be used as selectable markers for specific portions of human chromosome 19.     

Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

Cytoskeleton organization of normal,scar, and embryonic human fibroblasts spread on extracellular matrix proteins

We assayed the cytoskeleton organization of normal, scar, and embryonic human fibroblasts spread on major proteins of the extracellular matrix (ECM), type-I and-IV collagens, laminin 2/4, and fibronectin. Confocal fluorescent microscopy showed that fibroblasts of different origins were distinguished by their organization of actin structures and focal contacts visualized with antibodies to vinculin. It was found that different fibroblasts spread on identical ECM proteins had a common spatial organization of their cytoskeletons and some modifications of their actin structures and focal contacts. Variations in the organization of actin microfilaments indicate differences in cell interactions with various ECM proteins. The difference may be dependent on the integrin combination exposed on the cell membrane. It is suggested that fibroblasts of different origins differ in their morphogenetic functions.     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Distribution and modulation of a human leukemia-associated antigen (CALLA)

CALLA is a 100,000-dalton surface glycoprotein expressed by malignant cells of patients with clinically important subtypes of acute leukemia. Incubation of human leukemic cells expressing CALLA with specific monoclonal antibody (J5) at 37 degrees C causes rapid and selective internalization and degradation of this antigen (antigenic modulation). In these studies we show that CALLA-specific monoclonal antibodies also identify a cell surface glycoprotein having a m. w. of approximately 100,000 on 2 to 6% of nonmyeloid nucleated cells of normal adult bone marrow, on normal fibroblasts in tissue culture, and on cells of several nonhematopoietic human tumor cell lines. J5 antibody similarly modulates the surface expression of CALLA on nonleukemic cell populations, although the extent of modulation at a given concentration of antibody varied considerably. Modulation was almost complete for CALLA on cells of normal bone marrow, but was highly variable for cells of nonhematopoietic cell lines, possibly reflecting variability in antibody access to surface antigen. Using fluoresceinated or iodinated J5 antibody to modulate expression of CALLA on cells of leukemic cell lines, we show that antibody-antigen complexes undergo a temperature-dependent redistribution on the cell surface during modulation to form microaggregates. Antibody as well as antigen is then internalized. Studies of [35S]methionine-labeled cells indicate that synthesis of CALLA continues despite modulation of its surface expression by specific antibody, implying that the presence of CALLA on the cell surface reflects a dynamic equilibrium between the processes of surface expression of newly synthesized glycoprotein and its spontaneous and antibody-mediated clearance. The implications of these observations for immunotherapy are discussed.     

Genes that cooperate with tumor promoters in transformation

Tumor-promoting phorbol esters, like growth factors, elicit pleiotropic responses involving biochemical pathways that lead to different biological responses. Genetic variant cell lines that are resistant to mitogenic, differentiation, or transformation responses to tumor promoters have been valuable tools for understanding the molecular bases of these responses. Studies using the mouse epidermal JB6 cell lines that are sensitive or resistant to tumor promoter-induced transformation have yielded new understanding of genetic and signal transduction events involved in neoplastic transformation. The isolation and characterization of cloned mouse promotion sensitivity genes pro-1 and pro-2 is reviewed. A new activity of pro-1 has been identified: when transfected into human cancer prone basal cell nevus syndrome fibroblasts but not normal fibroblasts mouse pro-1 confers lifespan extension on these cells. Recently, we have found that a pro-1 homolog from a library of nasopharyngeal carcinoma, but not the homolog from a normal human library, is activated for transferring promotion sensitivity. The many genetic variants for responses to tumor promoters have also proved valuable for signal transduction studies. JB6 P- cells fail to show the 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-induced synthesis of two proteins of 15 and 16 kD seen in P+ cells. P-, P+, and TPA transformed cells show a progressive decrease in both basal and TPA-inducible levels of a protein kinase C substrate of 80 kD. P- cells are relatively resistant both to anchorage-independent transformation and to a protein band shift induced by the calcium analog lanthanum. It appears that one or more calcium-binding proteins and one or more pro genes may be critical determinants of tumor promoter-induced neoplastic transformation.     

Recombinant adenoviral vectors can induce expression of p73 via the E4-orf6/7 protein

Despite the utility of recombinant adenoviral vectors in basic research, their therapeutic promise remains unfulfilled. Most engineered adenoviral vectors use a heterologous promoter to transcribe a foreign gene. We show that adenoviruses containing the cytomegalovirus immediate-early promoter induce the expression of the proapoptotic cellular protein TAp73 via the cyclin-dependent kinase-retinoblastoma protein-E2F pathway in murine embryonic fibroblasts. Cells transduced with these vectors also expressed high levels of the adenoviral E4-orf6/7 and E2A proteins. By contrast, adenoviruses containing the ubiquitin C promoter failed to elicit these effects. E4-orf6/7 is necessary and sufficient for increased TAp73 expression, as shown by using retrovirus-mediated E4-orf6/7 expression and adenovirus with the E4-orf6/7 gene deleted. Activation of TAp73 likely occurs via E4-orf6/7-induced dimerization of E2F and subsequent binding to the inverted E2F-responsive elements within the TAp73 promoter. In addition, adenoviral vectors containing the cytomegalovirus immediate-early promoter, but not the ubiquitin C promoter, cooperated with chemotherapeutic agents to decrease cellularity in vitro. In contrast to murine embryonic fibroblasts, adenoviruses containing the ubiquitin C promoter, but not the cytomegalovirus immediate-early promoter, induced both E4-orf6/7 and TAp73 in human foreskin fibroblasts, emphasizing the importance of cellular context for promoter-dependent effects. Because TAp73 is important for the efficacy of chemotherapy, adenoviruses that increase TAp73 expression may enhance cancer therapies by promoting apoptosis. However, such adenoviruses may impair the long-term survival of transduced cells during gene replacement therapies. Our findings reveal previously unknown effects of foreign promoters in recombinant adenoviral vectors and suggest means to improve the utility of engineered adenoviruses by better controlling their impact on viral and cellular gene expression.     

An integral glycoprotein associated with the membrane attachment sites of actin microfilaments

An integral membrane protein associated with sites of microfilament-membrane attachment has been identified by a newly developed IgG1 monoclonal antibody. This antibody, MAb 30B6, was derived from hybridoma fusion experiments using intact mitotic cells of chick embryo fibroblasts as the immunization vehicle as well as the screening probe for cell surface antigens. In immunofluorescent experiments with fixed cells, MAb 30B6 surface labeling is uniquely correlated with microfilament distributions in the cleavage furrow region of dividing chick embryo fibroblasts and cardiac myocytes in culture. The MAb 30B6 antigen in addition is associated with microfilament-membrane attachment sites in interphase fibroblasts at the dorsal surface, the adhesion plaque region at the ventral surface, and at junction-like regions of cell-cell contact. It is also found co-localized with the membrane-dense plaques of smooth muscle. The MAb 30B6 antigen is expressed in a wide number of chicken cell types (particularly smooth muscle cells, platelets, and endothelial cells), but not in erythrocytes. Some of the molecular characteristics of the MAb 30B6 antigen have been determined from immunoblotting, immunoaffinity chromatography, immunoprecipitation, cell extraction, and charge shift electrophoresis experiments. It is an integral sialoglycoprotein with an apparent molecular mass of 130 kD (reduced form)/107 kD (nonreduced form) in SDS PAGE. Another prominent glycoprotein species with an apparent molecular mass of 175 kD (reduced form)/165 kD (nonreduced form) in SDS PAGE is co-isolated on MAb 30B6 affinity columns, but appears to be antigenically distinct since it is not recognized by MAb 30B6 in immunoblotting or immunoprecipitation experiments. By virtue of its surface distributions relative to actin microfilaments and its integral protein character, we propose that the MAb 30B6 antigen is an excellent candidate for the function of directly or indirectly anchoring microfilaments to the membrane.     

Functional analysis of posttranslational cleavage products of the neuron-glia cell adhesion molecule, Ng-CAM

Neuron-glia cell adhesion molecule (Ng-CAM) mediates cell adhesion between neurons homophilically and between neurons and glia heterophilically; it also promotes neurite outgrowth. In the chick brain, Ng-CAM is detected as glycoproteins of 190 and 210 kD (Ng- CAM200) with posttranslational cleavage products of 135 kD (F135, which contains most of the extracellular region) and 80 kD (F80, which includes the transmembrane and the cytoplasmic domains). To examine the functions of each of these components, we have expressed Ng-CAM200, F135, and F80 in murine L cells, and F135 and F80 as GST fusion proteins in the pGEX vector in bacteria. Appropriately transfected L cells expressed each of these proteins on their surfaces; F135 was also found in the media of cells transfected with Ng-CAM200 and F135. In addition to binding homophilically, cells transfected with Ng-CAM200 and F135 bound heterophilically to untransfected L cells, suggesting that there is a ligand for Ng-CAM on fibroblasts that may be related to the glial ligand. Detailed studies using the transfected cells and the fusion proteins indicated that both the homophilic and the heterophilic binding activities of Ng-CAM are localized in the F135 fragment of the molecule. The results also indicated that proteolytic cleavage of Ng- CAM200 is not required either for its expression on the cell surface or for cell adhesion and that there is an "anchor" for F135 on L cells (and presumably on neurons). In contrast to the cell binding results, the F80 but not the F135 fusion protein enhanced the outgrowth of neurites from dorsal root ganglion cells; this activity was associated with the FnIII repeats of F80. The observations that a protein corresponding to F135 contains the cell aggregation sites whereas one corresponding to the F80 has the ability to promote neurite outgrowth suggest that proteolytic cleavage may be an important event in regulating these Ng-CAM activities during embryonic development and neural regeneration.     

Molecular cloning of the cell surface antigen identified by the osteoprogenitor-specific monoclonal antibody,HOP-26

Bone is a highly organized structure comprising a calcified connective tissue matrix formed by mature osteoblasts, which develop from the proliferation and differentiation of osteoprogenitor cells. The osteogenic cell lineage is thought to arise from a population of uncommitted multipotential stromal precursor cells (SPC) which reside close to all bone surfaces, in the bone marrow spaces and the surrounding connective tissue. These SPC also give rise to related cell lineages which form cartilage, smooth muscle, fat, and fibrous tissue. Due to the lack of well defined cell surface markers, little is known of the precise developmentally regulated changes in phenotype which occur during the differentiation and maturation of human osteoprogenitor cells into functional osteoblasts and ultimately, terminally differentiated osteocytes. In order to identify antibody reagents with greater specificity for osteoprogenitors we generated a series of antibodies following immunization with freshly isolated human bone marrow stromal fibroblasts. One such antibody, HOP-26, reacts with a cell surface antigen expressed by SPC and developing bone cells. We now demonstrate that this mAb identifies a member of the tetraspan family of cell surface glycoproteins, namely CD63. Western blot analysis of human bone marrow stromal cells (HBMSC) has revealed that like a well defined CD63 mAb 12F12, HOP-26 interacts with a heavily glycosylated cell surface protein with an apparent molecular weight of 50-60 kD.     

Complementation of multiple sulfatase deficiency in somatic cell hybrids

Multiple sulfatase deficiency (MSD) is an inherited disorder characterized by deficient activity of seven different sulfatases. Genetic complementation for steroid sulfatase (STS), arylsulfatase A, and N-acetylgalactosamine 6-SO4 sulfatase was demonstrated in somatic cell hybrids between MSD fibroblasts and mouse cells ( LA9 ) or Chinese hamster cells ( CHW ). In an electrophoretic system that separates human and rodent STS isozymes, enzyme from hybrids migrated as human enzyme. We concluded that the rodent cell complemented the MSD deficiency and allowed normal expression of the STS structural gene. Some MSD- LA9 hybrids showed significant levels of human arylsulfatase A activity, as shown by the immunoprecipitation of active enzyme by human-specific antiserum. Complementation was also suggested for N-acetylgalactosamine 6- sulfatate sulfatase (GalNAc-6S sulfatase) in several MSD- LA9 hybrids by the demonstration of a significant increase in activity (10-fold) over that of the GalNAc-6S sulfatase-deficient parental mouse and MSD cells. Thus, it was possible to demonstrate complementation for more than one sulfatase in a single MSD-rodent hybrid. Normal levels of sulfatase activity in hybrids indicate that the sulfatase structural genes are intact in MSD cells.