sepharose and Cell-Transformation--Viral

Chondrocytes, the only cell type present in articular cartilage, regulate tissue homeostasis by a fine balance of metabolism that includes both anabolic and catabolic activities. Therefore, the biology of chondrocytes is critical for understanding cartilage metabolism. One major limitation when studying primary chondrocytes in culture is their loss of phenotype. To overcome this hurdle, limited attempts have been made to develop human chondrocyte cell lines that retain the phenotype for use as a good surrogate model. In this study, we report a novel approach to the establishment and characterization of human articular cartilage-derived chondrocyte cell lines. Adenoviral infection followed by culture of chondrocytes in 3-dimensional matrix within 48 h post-infection maintained the phenotype prior to clonal selection. Cells were then placed in culture either as monolayer, or in 3-dimensional matrix of alginate or agarose. The clones were characterized by their basal gene expression profile of chondrocyte markers. Based on type II collagen expression, 21 clones were analyzed for gene expression following treatment with IL-1 or BMP-7 and compared to similarly stimulated primary chondrocytes. This resulted in selection of two clones that retained the chondrocyte phenotype as evidenced by expression of type II collagen and other extra-cellular matrix molecules. In addition, one clone (AL-4-17) showed similar responses as primary chondrocytes when treated with IL-1 or BMP-7. In summary, this report provides a novel procedure to develop human articular cartilage-derived chondrocyte cell lines, which preserve important characteristics of articular chondrocytes and represent a useful model to study chondrocyte biology.

Topics: Adenoviridae; Aged; Alginates; Bone Morphogenetic Protein 7; Cartilage, Articular; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Proliferation; Cell Separation; Cell Shape; Cell Transformation, Viral; Chondrocytes; Clone Cells; Collagen Type II; Female; Gene Expression Profiling; Gene Expression Regulation; Glucuronic Acid; Hexuronic Acids; Humans; Interleukin-1; Male; Middle Aged; Phenotype; RNA, Messenger; Sepharose; Time Factors

Epstein-Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807-2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+ LMP2- recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2- virus to immortalize primary human B cells when compared to that of wild-type virus.

Topics: Animals; B-Lymphocytes; Callithrix; Cell Division; Cell Line; Cell Transformation, Viral; Cells, Cultured; Cysteine Endopeptidases; Gene Deletion; Green Fluorescent Proteins; Herpesvirus 4, Human; Humans; Luminescent Proteins; Proteins; Sepharose; Tumor Cells, Cultured

The CD23 molecule is a low-affinity receptor for IgE and has a marked homology in amino acid sequence with C-type animal lectins, including asialoglycoprotein receptor. We tested whether the CD23 antigen can indeed interact with the sugar chain of glycoproteins. Detergent extract of the membrane component from Epstein-Barr Virus (EBV)-transformed human B-cell line, L-KT9 cells, was incubated with asialofetuin (ASF)-coupled Sepharose, and bound proteins were effectively eluted by 0.3 M lactose or galactose which were among the competitive sugars tested. In this eluate, the CD23 molecule was detected by an immunoblotting technique. Because fetuin has both an N- and O-type sugar chain on the molecule, we tested which type of sugar chain can interact with CD23. The CD23 molecule interacted with asialocasein having a sugar chain with the Gal-GalNAc structure with asialobovine submaxillary mucin having the GalNAc structure, and also with ASF; however, it faintly interacted with ASF after removal of the O-type sugar chain by beta-elimination. These results showed that the CD23 molecule can, indeed, interact with the galactose residue, especially with the Gal-GalNAc rather than the Gal-GlcNAc structure of the terminal sugar chain of glycoproteins.

Topics: Acetylgalactosamine; alpha-Fetoproteins; Asialoglycoproteins; B-Lymphocytes; Cell Line; Cell Transformation, Viral; Electrophoresis, Polyacrylamide Gel; Fetuins; Galactose; Humans; Immunoblotting; Membrane Glycoproteins; Receptors, IgE; Sepharose

Using affinity chromatography on ATP-agarose, we have identified a major ATP-binding protein in Nonidet P-40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP-agarose beads with high-ionic-strength buffer (0.4 M NaCl), the 37-kD protein was shown to be one of the major ATP-binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP-agarose. This protein consisted of several spots on two-dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat-shock treatment of BALB/3T3 cells, the major heat-shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP-binding fraction. Besides hsp70, a 37-kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with alpha,alpha'-dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP-binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c-Ha-ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP-binding protein of Mr = 37,000 is a heat-inducible protein and that the synthesis of this protein is regulated by malignant transformation.

Topics: 2,2'-Dipyridyl; Adenosine Triphosphate; Animals; Arsenic; Arsenites; Calcimycin; Carrier Proteins; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Chick Embryo; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum Chaperone BiP; Fibroblasts; Heat-Shock Proteins; Methionine; Mice; Mice, Inbred BALB C; Precipitin Tests; Sepharose; Sulfur Radioisotopes; Tetradecanoylphorbol Acetate; Tunicamycin

The Epstein-Barr virus (EBV)-negative Burkitt lymphoma (BL) line BL-41, and 5 independently established EBV-converted sublines, derived by infection with a transforming (B95-8) or a nontransforming (P3HR1) strain of EBV, were compared for clonability in semi-solid agarose and for tumorigenicity in immuno-suppressed mice. One P3HR1 viral convertant and 3 out of 4 B95-8 virus-converted sublines had a high (greater than 40%) agarose clonability, like the BL 41 parent, and were slightly more tumorigenic than BL-41. In contrast, the fourth B95-8 converted subline, BL-41/95, was virtually non-tumorigenic and its agarose clonability was much lower (3-23%). It showed a more drastic shift towards an LCL-like phenotype than the other convertants as reflected by high HLA class-I and EBV-encoded latent membrane protein (LMP) expression. BL 41/95 still contains the 8;14 IgH/myc translocation, carried by the parental line, and maintains the same relatively high steady-state level of c-myc mRNA and protein as the highly tumorigenic convertants. We conclude that the tumorigenicity of BL41/95 has been suppressed by a gene that acts at a level beyond the expression of the activated oncogene, in the same way as the revertants isolated from ras and SV-40-transformed cultures (Klein, 1987b; Bassin and Noda, 1987).

Topics: Animals; Burkitt Lymphoma; Cell Transformation, Viral; Cloning, Molecular; Culture Media; Mice; Oncogene Protein p55(v-myc); Phenotype; Retroviridae Proteins; RNA, Messenger; RNA, Neoplasm; Sepharose; Translocation, Genetic; Tumor Cells, Cultured

Human monoclonal antibodies against DNA were specifically purified from the culture medium of an EBV transformant of SLE patients' lymphocytes using a DNA-coupled Sepharose 4B affinity column. The monoclonal antibodies were eluted from the column with 5% dimethylsulfoxide (pH 10.7) containing 0.5 M NaCl without loss of immunological activity and without contamination by other proteins.

Topics: Antibodies, Antinuclear; Antibodies, Monoclonal; Cell Transformation, Viral; Cells, Cultured; Chromatography, Affinity; Culture Media; Dimethyl Sulfoxide; DNA; Herpesvirus 4, Human; Humans; Lupus Erythematosus, Systemic; Lymphocytes; Sepharose

Simian virus 40 early region mutants which are partially or completely replication defective were tested for their ability to transform postcrisis mouse fibroblasts. All mutants tested were capable of generating anchorage-independent transformants. We have previously reported the presence of a variant tumor antigen of 100,000 Mr (100K protein) generated upon transformation by wild-type simian virus 40 virions which correlates with anchorage-independent growth (Chen et al., Mol. Cell. Biol. 1:994-1006, 1981). In this study, none of the mutants tested produced the 100K variant protein at early (before the fifth) passage. Long-term passage (greater than 20 weeks) permitted the expression of this 100K variant in half of the transformants. Thus the phenotype of these mutants is different from both wild-type simian virus 40 (frequently production of 100K by the third passage, and always by the tenth passage) and the origin-minus class of mutants (no production of 100K at any passage).

Topics: Animals; Antigens, Polyomavirus Transforming; Antigens, Viral, Tumor; Cell Line; Cell Transformation, Viral; DNA Replication; Fibroblasts; Genes, Viral; Mice; Molecular Weight; Mutation; Oncogene Proteins, Viral; Sepharose; Simian virus 40; Transfection; Virus Replication

We report the development of a method for cloning human EBV-transformed cells which has greater efficiency than techniques used presently. In this new method lymphoblastoid cells are cultured in semisolid agarose in close physical association with human fibroblasts. The results indicate a 10-fold increase in the cloning efficiencies. The average cloning efficiency, depending on the age of cell lines, was from 1 to 14%, and colonies appeared 7-9 days sooner than in the traditional soft agarose method. The new method has allowed us to develop several stable lymphoblastoid cell lines producing antibody cytotoxic to human B lymphocytes. This method may make it more practical to obtain monoclonal human antibodies from lymphoblastoid cell lines which had previously been unstable due to heterogeneity.

Topics: Antibody-Producing Cells; Cell Line; Cell Transformation, Viral; Clone Cells; Culture Media; Fibroblasts; Herpesvirus 4, Human; Humans; Lymphocytes; Sepharose

Recombinant human interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) were compared for their ability to influence the proliferative capacity of tumor-derived cell lines and of normal B lymphocytes infected in vitro by Epstein-Barr virus (EBV). EBV-induced B-cell proliferation was suppressed almost completely when 10(2) U/ml IFN-alpha were added to the culture medium while the same dose of IFN-gamma had significantly lower inhibitory activity. The pure IFNs differed in their ability to influence the growth of three Burkitt lymphoma-derived cell lines, Raji, Daudi, and Namalwa, depending on whether the cells were propagated in suspension or in semisolid cultures. IFN-alpha inhibited cell proliferation under both culture conditions with thresholds of sensitivity characteristics for each cell line. In contrast, IFN-gamma had no effect on the growth in suspension but it abolished the clonogenic potential of tumor cell lines in semisolid agarose. The results suggest that the two IFN types may exert their growth inhibitory activity through different mechanisms of action.

Topics: B-Lymphocytes; Cell Division; Cell Line; Cell Transformation, Viral; Clone Cells; Herpesvirus 4, Human; Humans; Interferon Type I; Interferon-gamma; Lymphocyte Activation; Sepharose

Both newly formed and long-term culture-generated substratum adhesion sites, generated by EGTA-mediated detachment of Balb/c SVT2 cells, were extracted with an eta-octyl-beta-D-glucopyranoside buffer containing salt and several protease inhibitors under conditions which result in maximal solubilization of the sulfate-radiolabeled proteoglycans. Because of the functional importance of heparan sulfate proteoglycans in the fibronectin-dependent cell-substratum adhesion processes of these cells, these proteoglycans were fractionated on affinity columns of octyl-Sepharose or of the heparan sulfate-binding proteins platelet factor 4 or plasma fibronectin. These affinity matrices resolved a number of both binding and nonbinding classes of heparan sulfate proteoglycan from both types of adhesion sites. In particular, the platelet factor 4 column could resolve several proteoglycans with differing binding affinities. Approximately twice as much heparan sulfate proteoglycan from newly formed sites bound to all three matrices as proteoglycan from longterm sites. The proteoglycan which bound to one matrix was then tested for binding to a second matrix; this approach resolved a number of biochemically distinct species. For example, one-half of the fibronectin-Sepharose-binding fraction from the long-term sites could also bind to platelet factor 4-Sepharose; however, over 90% of the fibronectin-binding fraction from newly formed sites could bind to platelet factor 4. A major portion of the octyl-Sepharose-binding fractions of the original extracts could bind to fibronectin-Sepharose. These studies indicate that some of these proteoglycans have overlapping affinities for fibronectin, platelet factor 4, and octyl-Sepharose and that a portion of the heparan sulfate proteoglycan from these adhesion sites cannot bind to any of these affinity matrices. These results are discussed with regard to the functional significance of these various heparan sulfate proteoglycans in mediating adhesion to extracellular matrices containing fibronectin or platelet factor 4.

Topics: Animals; Cell Adhesion; Cell Transformation, Viral; Chondroitin Sulfate Proteoglycans; Chromatography, Affinity; Fibroblasts; Fibronectins; Glycosaminoglycans; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Mice; Mice, Inbred BALB C; Platelet Factor 4; Proteoglycans; Sepharose; Simian virus 40; Surface Properties

Topics: Cell Line; Cell Separation; Cell Transformation, Viral; Clone Cells; Culture Media; Herpesvirus 4, Human; Humans; Lymphocytes; Sepharose

Topics: Animals; Cell Line; Cell Transformation, Viral; Cells, Cultured; Fibroblasts; Glycosaminoglycans; Heparitin Sulfate; Humans; Lung; Mice; Polyomavirus; Sepharose; Simian virus 40

We have compared the growth in agarose medium of two EBV-negative Burkitt lymphoma lines, BJAB and Ramos, and that of their EBV-converted derivatives. Optimal conditions for growth in agarose medium are described. The original EBV-negative lines can grow in agarose to a high efficiency, provided a critical level of cell concentration is attained. Below this level, there is no growth at all. EBV-converted sublines form colonies at a much lower cell concentration, and their maximal plating efficiency is higher. These differences may be related to a super-transformation state induced in these cells by EBV.

Topics: Burkitt Lymphoma; Cell Count; Cell Line; Cell Transformation, Viral; Clone Cells; Culture Media; Herpesvirus 4, Human; Humans; Polysaccharides; Sepharose

Normal C57 Black mouse embryo cells did not form colonies in agarose, but rare variant (ar+) cells able to grow in agarose were detected. Fluctuation analysis showed that ar+ variants arose by spontaneous mutation in the cultured cells. The frequency of ar+ variants was increased by treating cells with N-methyl-N'nitro-N-nitrosoguanidine or ethyl methane sulphonate, or by abortive infection by human adenovirus type 5. Induced ar+ cells were fibroblastic; most grew slowly and had slightly reduced saturation density and increased serum requirement, but formed colonies in agarose. Fourteen of twenty ar+ clones induced by Ad5 were T antigen negative and two of these were also negative when tested for viral DNA. Six clones contained a few cells that were T antigen positive when first tested, but were negative when retested later. The ar+ variants were tumorigenic in athymic and in normal syngeneic mice. The results suggest that the ar+ phenotype can arise by spontaneous or chemically-induced mutation, and can be induced by adenovirus by a process different from classical transformation.

Topics: Adenoviruses, Human; Animals; Cell Division; Cell Line; Cell Transformation, Neoplastic; Cell Transformation, Viral; Cells, Cultured; Culture Media; Ethyl Methanesulfonate; Fibroblasts; Genetic Variation; Methylnitronitrosoguanidine; Mice; Mutagens; Mutation; Phenotype; Polysaccharides; Sepharose