Purification of the Epstein-Barr virus-determined nuclear antigen from Epstein-Barr virus-transformed human lymphoid cell lines.

The Epstein-Barr virus-determined nuclear antigen (EBNA) was purified from extracts of the human lymphoid cell lines Raji, Namalwa, and B95-8/MLD by two different methods. In the first approach, the apparently native antigen was purified 1,200-fold by a four-step procedure involving DNA-cellulose chromatography, blue dexptran-agarose chromatography, hydroxyapatite chromatography, and gel filtration, employing complement fixation as the assay procedure. Such EBNA preparations specifically inhibited the anticomplement immunofluorescence test for EBNA and bound to methanol/acetic acid-fixed metaphase chromosomes. The purified antigen, which has a molecular weight of 170,000 to 200,000, yielded a single protein band of molecular weight about 48,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. These data indicate that native EBNA has a tetrameric structure. In the second purification method, EBNA-containing cell extracts containing radioactively labeled proteins were incubated with anti-EBNA-positive sera, and antigen-antibody complexes were adsorbed to matrix-bound staphylococcal protein A. The bound proteins were then released with an SDS-containing buffer, and denatured EBNA was separated from antibody chains by SDS-polyacrylamide gel electrophoresis and visualized by fluorography. The denatured EBNA obtained in radiochemically pure form by this procedure has a molecular weight of about 48,000, so both methods yield an EBNA monomer of the same size.     

Biochemical and ultrastructural findings in Epstein-Barr virus-transformed lymphoid cell lines from type 1 Gaucher disease

Lymphoid cell lines established by Epstein-Barr Virus (EBV)-transformation of peripheral blood B-lymphocytes from patients affected with type 1 Gaucher disease showed a severe deficiency of glucosylceramide-beta-glucosidase activity (residual activity around 15%-30% of control activity). Ultrastructural investigations showed, in these lymphoid cell lines from type 1 Gaucher disease, the presence of numerous membrane-bound inclusion bodies characteristic of Gaucher cells.     

Biochemical and ultrastructural studies on an Epstein-Barr virus-transformed lymphoid cell line from a Niemann-Pick disease type C patient

Human lymphoid cell lines established from normal subjects and from a Niemann-Pick disease type C patient were investigated from a triple point of view of enzymology, metabolism and ultrastructure: Sphingomyelinase activities, isoenzyme electrofocusing profiles and properties of the major enzyme were quite similar in type C and normal lymphoid cell lines. Similarly, no significant difference was observed in non-specific phosphodiesterases hydrolysing bis(methylumbelliferyl)phosphate and bis(methylumbelliferyl)pyrophosphate. The study of the lipid composition of type C cells showed no obvious accumulation of sphingomyelin or other phospholipid, but only a higher amount of glycolipids (mainly GlcCer and GbOse3Cer), as visualized by bidimensional thin-layer chromatography. Ultrastructural studies demonstrated, in type C cells, the presence of an obvious lysosomal storage of amphiphilic lipids quite similar to that observed in tissues of type C patients. These studies, which demonstrate the validity of lymphoid cell lines as an experimental model system for type C disease, agree with the current opinion that an impairment of sphingomyelin catabolism is not the primary defect in type C disease.     

beta-Glucosidase isoenzymes in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and patients with type 1 Gaucher disease

Lymphoid cell lines (LCL) from 3 adult patients with non-neuropathic Gaucher disease were established by Epstein-Barr virus (EBV) transformation and were investigated from the view of enzymology. Glucosylceramide-beta-glucosidase (GlcCer-beta-glucosidase) was present in soluble and particulate fraction of LCL from normal subjects and was deficient in type 1 Gaucher LCL; the deficiency of all molecular forms, shown by electrofocusing, indicates that they are coded by the same gene. The existence of two non-specific beta-glucosidases, one soluble (minor), the other membrane-bound (major), was demonstrated in leucocytes and LCL from normals; in Gaucher LCL, these were also present in a normal range. Characteristic properties of the non-specific membrane-bound beta-glucosidase were defined: lability at acidic pH and strong inhibitory effect by detergents. These properties allowed to discriminate it from the lysosomal GlcCer-beta-glucosidase and to define optimal assay conditions for determination of residual GlcCer-beta-glucosidase activity in Gaucher disease, using artificial substrate, without interference of non-specific membrane-bound beta-glucosidase. These results demonstrate that EBV-transformed LCL represent an accurate model system for enzymatic studies of Gaucher disease.     

Metabolism of pyrenedecanoic acid in Epstein-Barr virus-transformed lymphoid cell lines from normal subjects and from a patient with multisystemic lipid storage myopathy

A lymphoid cell line has been established from a patient with multisystemic lipid storage myopathy and showed a major triacylglycerol storage, whereas the content of other neutral lipids and phospholipids was in the normal range. The metabolism of the triacylglycerols has been investigated in this lymphoid cell line from multisystemic lipid storage myopathy as well as in control cells through pulse-chase experiments using 10-(1-pyrene)decanoic acid (P10), a fluorescent fatty acid derivative, as precursor. After 1 h incubation, the uptake of P10 was not significantly different in multisystemic lipid storage myopathy and control lymphoid cells. The amount of fluorescent lipids synthesized by the lymphoid cells was proportional to the concentration of P10 in the culture medium. After 24 h incubation, at any extracellular concentration of P10, the content of P10-labelled triacylglycerols was much higher in multisystemic lipid storage myopathy cells than in controls. Chase experiments showed an impairment in the rate of degradation of biosynthesized triacylglycerols in multisystemic lipid storage myopathy lymphoblasts compared to controls with time of chase (the ratio P10-triacylglycerols/P10-phospholipids increased in mutant cells while it decreased in normal cells). Elsewhere, no enzyme deficiency of the neutral triacylglycerol lipase activity, has been found in multisystemic lipid storage myopathy lymphoid cells.     

Ultrastructural investigations on two lymphoid cell lines from Niemann-Pick disease type B

Lymphoid cell lines (LCL) were established by Epstein-Barr Virus (EBV) transformation of blood B-lymphocytes from two different patients affected with Niemann-Pick disease (NPD) type B. Those lines were severely deficient in sphingomyelinase activity (8% and 10% residual activity). Ultrastructural investigations showed in both these lines the presence of numerous osmiophilic, dense and pleiomorphic inclusions characteristic of lysosomal storage (due to the accumulation of amphiphilic lipids) similar to those observed in tissues from NPD.     

Lysosomal acid hydrolases in established lymphoblastoid cell lines,transformed by Epstein-Barr virus,from patients with genetic lysosomal storage diseases

Summary Lysosomal acid hydrolases were determined in established lymphoblastoid cell lines, transformed in vitro by Epstein-Barr virus (EBV) from lymphocyte-rich cell populations isolated from the peripheral blood of patients with genetic lysosomal storage diseases—Hurler syndrome, Scheie syndrome, G_M1-gangliosidosis type 1 and type 2, Tay-Sachs disease, and I-cell disease—and from obligate heterozygotes for these diseases.The respective enzyme activity was undectectable in lymphoblastoid cells from the patients, but not from controls. Obligate heterozygotes could not always be distinguished from controls in lymphoblastoid cells as well as in leukocytes. These results suggest that established lymphoblastoid cell lines are useful material for the enzymatic study of genetic lysosomal storage diseases.     

Acid lipase and carboxylesterases in EBV-transformed lymphoid cell line from Wolman's disease: influence of fatty acid structure of substrate

Lymphoid cell lines established by Epstein-Barr virus transformation of blood B lymphocytes from a patient with Wolman's disease exhibited the acid lipase deficiency characteristic for this disease. Comparison of hydrolysis by normal and Wolman's cells of 4-methylumbelliferyl-acyl esters with variable chain length demonstrates that: (1) the best substrates for acid lipase were characterized by an acyl chain length of 12-18 carbon atoms; (2) the acid residual activity in Wolman's cells showed a slightly different substrate specificity and this is probably due to an acid carboxylesterase different from the lysosomal acid lipase, and (3) the 'nonspecific' carboxylesterases (at pH 6.0 and 8.0) not inhibited by taurocholate showed a characteristic substrate specificity for short-chain fatty acids. In the used assay conditions (optimal for acid lipase), methylumbelliferyl-palmitate, -elaidate and -lignocerate are the most accurate synthetic substrates for the diagnostic of Wolman's disease.     

The effects of mitogens on the expression of Epstein-Barr virus antigens in human lymphoid cell lines.

Treatment of human lymphoblastoid cells with either phytohemagglutinin (PHA), concanavalin A, Staphylococcus protein A, or polyinosinic acid-polycytidylic acid, in combination with 5-iodo-2' deoxyuridine (IUdR) markedly increased the expression of Epstein-Barr virus (EBV) early antigen (EA) relative to IUdR alone. Such treatment did not, however, modify the production of virus capsid antigen in any of the lymphoid cell lines tested. The effect of PHA on EA induction in Raji cells was not accompanied by changes in the incorporation of labeled precursors into cellular DNA, or in the intracellular concentration of either adenosine 3'5' cyclic monophosphate or guanosine 3'5' cyclic monophosphate. However, those mitogens that stimulated EA expression in Raji cells also increased the fluorescence polarization of 1,6 diphenyl 1,3,5-hexatriene-labeled Raji cells. The possible role of cell surface changes in the mitogen activation of latent EBV in human lymphoblastoid cells is discussed.     

Detection of acyl esterase activity on electrophoresis membranes and separation from hyaluronidase.

A method is described for the detection of acyl esterase activity on cellulose acetate membranes following electrophoresis of the enzyme. It uses the indigogenic substrate, indoxyl acetate, which directly forms the colored product visualized in the test. This substrate also detects activity of acetyl cholinesterase and pseudocholinesterase. With this method, bovine testicular hyaluronidase is shown to contain acyl esterase activity. By electrophoresis of hyaluronidase preparations at pH 6.8, esterase and hyaluronidase activities are separated, further assuring the specificity of the method for hyaluronidase.     

Receptors for hyaluronate on the surface of parent and virus-transformed cell lines : Binding and aggregation studies

Two sets of parent and virus-transformed cell lines (3T3 vs SV-3T3; BHK vs PY-BHK) were compared with respect to the extent of divalentcation independent aggregation which previously has been shown to depend upon the interaction of endogenous hyaluronate with specific receptors on the cell surface. When measured under conditions of physiological ionic strength, a significant amount of hyaluronidase-inhibitable aggregation was found in the virus-transformed cell lines (SV-3T3 and PY-BHK) but not in their parent counterparts (3T3 and BHK). However, when the same experiment was performed in a high ionic strength solution (0.5 M NaCl), the hyaluronidase inhibitable aggregation was detected in all of the cell lines. The differences in the aggregation between the various cell lines was also reflected in the binding of [³H]hyaluronate. In physiological saline, the virus-transformed cells bound greater amounts of hyaluronate (higher B_max) with a greater affinity (lower k_d) than did their untransformed counterparts. Increasing the ionic strength to 0.5 M NaCl increased the binding of [³H]hyaluronate by each cell line; however, the relative differences between the cell lines remained. These results indicate that variations in the ability of the cells to bind hyaluronate can partially account for the differences between the parent and the virus-transformed cells with respect to their ability to aggregate.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Comparative studies on cell lines established from normal and radiation-exposed miniature swine.

Cloned cell lines were established from two swine with radiation-induced myeloproliferative disorders, including one cell culture from an animal with myelogenous leukemia and one from an animal with myeloid metaplasia. A third cloned cell line with similar morphology was established from pooled normal fetal swine cornea to compare the growth characteristics of cells from normal and irradiated swine. All three cell lines grew as foci of aggregated cells and were able to form macroscopic colonies in semisolid agar medium. The lack of normal mechanisms of contact inhibition and the observed aneuploidy indicated that these cells were morphologically transformed. Further, the cloned cells caused tumors in nude mice, clearly indicating that these cells were also malignantly transformed. A major difference between these cell lines was that type C viruses were observed only in the cells derived from swine with myeloproliferative disorders.     

The effect of cell synchronization upon the detection of T and B lymphoid cell receptors on two continuous lymphoid cell lines.

In the present study, the effect of the cell synchronization on the detection of T and B cell surface markers of two continuous lines of lymphoid cells (FL-74 and CT45-S) was examined. Suspension cultures were synchronized by deprivation of isoleucine and surface markers were quantitated by T rosette formation with guinea pig erythrocytes (E) and B rosette formation with an erythrocyte-antibody-complement (EAC) complex. After 24 hr, cells were resuspended in complete culture medium. Virtually 100% of FL-74 cells expressed the T cell marker at time 0, with a progressive decline to 80% at saturation density. A bell-shaped curve for expression of the EAC marker on CT45-S cells was seen with maximum expression in the logarithmic phase of the growth cycle. Spent culture medium was examined for the presence of free soluble receptor. Preincubation of E and EAC in appropriate old medium resulted in 42% inhibition of E rosettes and 42% inhibition of EAC rosettes with FL-74 and CT45-S cells, respectively. Thus quantitation of lymphocyte subpopulations as B, T or null cells with these cellular markers may be influenced by the age of the cell examined, phase of the cell cycle and the amount of free receptor present in the surrounding medium.     

Protein-DNA interaction and CpG methylation at rep*/vIL-10p of latent Epstein-Barr virus genomes in lymphoid cell lines.

The viral interleukin-10 promoter (vIL-10p), overlapping the rep* element in the Epstein-Barr virus (EBV) genome, is a promoter element active mostly in the late phase of the lytic cycle and immediately upon infection of B cells. rep* was, through transfection experiments with small plasmids, characterised as a cis element supporting oriP replicative function. In this study, in vivo protein binding and CpG methylation at rep*/vIL-10p were analysed in five cell lines that harbour strictly latent EBV genomes. Contrary to the invariably unmethylated dyad symmetry element (DS) of oriP, rep*/vIL-10p was highly methylated and showed only traces of protein binding in all examined cell lines. This result is in agreement with vIL-10p being an inactive promoter of EBV genomes, and makes it less likely that rep* functions as a replicative element of latent EBV genomes.