Double immunoglobulin production in cloned somatic cell hybrids between two human lymphoid cell lines.

Several clones of independently established somatic cell hybrids between two human lymphoid cell lines, Raji and Namalwa, were examined for surface immunoglobulin expression. Double-antibody radioimmunoassays were established for kappa and lambda light chains. Immunoglobulins were detergent-extrated by Triton X-100 and quantified by radioimmunoassay. The Raji parent expressed small amounts of kappa chains on its surface, and the Namalwa parent a 10 fold greater amount of lambda chains. We show that the majority of the hybrid clones co-express both parental phenotypes.     

Evidence for platelet-activating factor receptors in several B lymphoblastoid cell lines.

Previous studies have shown that Raji, an Epstein-Barr virus (EBV)-immortalized Burkitt lymphoma B cell line, contains functional platelet-activating factor (PAF) receptors. Twelve other lymphoid cell lines, including Burkitt and non-Burkitt B cell lines, T cell lines, and a non B, non T cell line were tested for the presence of PAF receptors. Radioligand binding studies conducted at 4 degrees C revealed that six lymphoid cell lines of B cell origin (Raji, P3HR-1, BJAB, BJA/HR-1, Dakiki and PB-1) could specifically bind [3H]PAF. Treatment of four (Raji, P3HR-1, Dakiki and PB-1) of the above lymphoid cell lines with PAF resulted in an increase in free intracellular calcium, indicating that these specific PAF binding sites were functional PAF receptors. Other B cell lines (Daudi, B95-8, sfBT, CB-1), T cell lines (MOLT-4, CCRF-CEM) and a non B, non T cell line (NALM-6) had no PAF binding sites and showed no PAF-induced increase in intracellular calcium levels. These studies demonstrate evidence for the presence of PAF receptors on several B lymphocyte cell lines.     

DNA repair, DNA synthesis and cell cycle delay in human lymphoblastoid cells differentially sensitive to the cytotoxic effects of nitrogen mustard

Two cloned human lymphoblastoid cell lines, Raji and TK6, differ in their sensitivity to the cytotoxic effects of nitrogen mustard (HN2). Raji cells exhibit a biphasic response with an initial D value of 0.06 microgram/ml and a final slope of 0.25 microgram/ml. TK6 cells were considerably more sensitive, D0 value 0.02 microgram/ml. Dose-response relationships for delay in cell cycle progression were measured using flow cytometry. Delay in S-phase traverse was concentration-dependent in both cell lines, and at a given concentration was 2-fold greater in TK6 than in Raji. Numbers of crosslinks (determined by alkaline elution) increased linearly with increasing HN2 concentration and were approximately 2-fold higher in TK6 than in Raji. At equal levels of DNA crosslinks, rates of removal were similar in both cell lines. Inhibition of [3H]TdR uptake was concentration-dependent and the extent of inhibition was similar in both cell lines. Recovery from HN2-induced inhibition of cell cycle progression markedly preceded recovery from inhibition of [3H]TdR incorporation suggesting that nucleotide pools are markedly perturbed in HN2-treated cells. The difference in sensitivity of these two cell lines cannot be adequately explained by differences in amounts of initial DNA damage, rates of repair, differential S-phase delay or rate of loss of DNA crosslinks.     

Karyologic study of continuous cell lines. IV. Study of human cell lines using the C-method of staining chromosomes

With the aid of the C-method of chromosome staining marker chromosomes three classes of human continuous cell lines were studied: 1) HeLa and HeLa-like cell lines (HEp-2, U, KB); 2) non-HeLa cell lines, with type B mobility of glucose-6-phosphate-dehydrogenase (HOS, A-549, A-204); 3) lymphoblastoid cell lines (Raji, Namalva, L-101). Two C-marker chromosomes were observed in two investigated cell lines A-204 and KB, one C-marker chromosome was observed in HEp-2, HeLa, U, A-549, Namalva cell lines; C-markers were absent in HOS and L-101 cell lines. Y-chromosome was found in Raji, A-549 and L-101 cell lines. The C-method of chromosome staining is a simple method, promoting an intraspecific identification of human cell lines.     

Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions

Identification of cytosolic proteins differentially expressed between types of leukemia and lymphoma may provide a molecular basis for classification and understanding their cellular properties. Two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry have been used to identify proteins that are differentially expressed in cytosolic extracts from four human leukemia and lymphoma cell lines: HL-60 (acute promyelocytic leukemia), MEC1 (B-cell chronic lymphocytic leukemia), CCRF-CEM (T-cell acute lymphoblastic leukemia) and Raji (B-cell Burkitt's lymphoma). A total of 247 differentially expressed proteins were identified between the four cell lines. Analysis of the data by principal component analysis identified 22 protein spots (17 different protein species) differentially expressed at more than a 95% variance level between these cell lines. Several of these proteins were differentially expressed in only one cell line: HL-60 (myeloperoxidase, phosphoprotein 32 family member A, ras related protein Rab-11B, protein disulfide-isomerase, ran-specific GTPase-activating protein, nucleophosmin and S-100 calcium binding protein A4), and Raji (ezrin). Several of these proteins were differentially expressed in two cell lines: Raji and MEC1 (C-1-tetrahydrofolate synthase, elongation factor 2, alpha- and beta-tubulin, transgelin-2 and stathmin). MEC1 and CCRF-CEM (gamma-enolase), HL-60 and CCRF-CEM (ubiquitin-conjugating enzyme E2 N). The differentially expressed proteins identified in these four cell lines correlate with cellular properties and provide insights into the molecular basis of these malignancies.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H2O2 but not O2*- produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H2O2 as ROS stress thereafter. The H2O2-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2-3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. alpha-Cyano-4-hydroxycinnamate, a specific inhibitor of the H+-monocarbohydrate transporter, increased the H2O2-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

ATP independent proteasomal degradation of NQO1 in BL cell lines

Human NAD(P)H: quinone oxidoreductase 1 (NQO1) catalyzes the obligatory two-electron reduction of quinones. For this peculiar catalytic mechanism, the enzyme is considered an important cytoprotector. The NQO1 gene is expressed in all human tissues, unless a polymorphism due to C609T point mutation is present. This polymorphism produces a null phenotype in the homozygous condition and reduced enzyme activity in the heterozygous one. We previously demonstrated that two cell lines of haematopoietic origin, HL60 and Raji cells, possess the same heterozygous genotype, but different phenotypes; as expected for a heterozygous condition the HL60 cell line showed a low level of enzyme activity, while the Raji cell line appeared as null phenotype. The level of NQO1 mRNA was similar in the two cell lines and the different phenotype was not due to additional mutations or to expression of alternative splicing products. Here we show that in Raji BL cell line with heterozygous genotype the null NQO1 phenotype is due to 20S proteasome degradation of wild type and mutant protein isoforms and is not directly linked to C609T polymorphism. This finding may have important implications in B-cell differentiation, in leukaemia risk evaluation and in chemotherapy based on proteasome inhibitors.     

Role of the production of natural killer cell stimulatory factor (NKSF/IL-12) in the ability of B cell lines to stimulate T and NK cell proliferation.

We have previously used human Epstein Barr virus (EBV)-transformed B lymphoblastoid cell lines for the identification and purification of a novel cytokine, natural killer cell stimulatory factor (NKSF/IL-12), that has pleiotropic effects on human lymphocytes. B cell lines are also routinely employed as feeder cells for the culture of T and natural killer (NK) cells. In this report we describe the ability of two NKSF/IL-12 producing B cell lines (RPMI-8866 and Cess) and two nonproducing lines (Raji and Daudi) to stimulate the proliferation of T and NK cells in 8-day PBL cultures. We demonstrate, using an anti-NKSF/IL-12 neutralizing monoclonal antibody, that the endogenous production of NKSF/IL-12 in these cultures can significantly enhance the proliferation and cytotoxic activity of T and NK cells. We also report that the addition of exogenous rNKSF/IL-12 can greatly increase the number of T and NK cells obtained from the cultures following stimulation by the B cell lines. Aside from the possible practical applications, the enhanced proliferation of T and NK cells consistently observed in the presence of endogenously produced NKSF/IL-12 or exogenously added rNKSF/IL-12 in this system may further our understanding of the role of this cytokine during an in vivo immune response.     

A surface-modified baculovirus vector with improved gene delivery to B-lymphocytic cells

A short peptide motif from gp350/220 of Epstein-Barr virus, EDPGFFNVEI, which was known to bind to CD21, a surface protein on B-lymphocyte, was inserted into the baculovirus surface protein gp64. The recombinant virus carrying the hybrid gp64/gp350 gene, vAc-gp350EGFP, was obtained, and the expression of gp64/gp350 protein was confirmed with immunoblot using anti-gp350 antibody. When compared with a control virus with wild type gp64, vAc-gp350EGFP showed increased transduction efficiency in B cell lines Raji, HR1, B95-8, BJAB, and DG75, regardless of their being EBV-positive or EBV-negative. No such increase was seen in non-B cell lines HEK293 and HeLa. When Raji cells were transduced with increased amount of vAc-gp350EGFP, transduction became saturated when the multiplicity of infection was higher than 20pfu/cell. The transduction of Raji cells by vAc-gp350EGFP was dose-dependently inhibited by pre-treatment of cells with anti-CD21 antibody. These results showed that vAc-gp350EGFP entered B cells by interacting with CD21.     

Functional characterization of cell hybrids generated by induced fusion of primary porcine mesenchymal stem cells with an immortal murine cell line

Bone marrow mesenchymal stem cells (MSC) integrate into various organs and contribute to the regeneration of diverse tissues. However, the mechanistic basis of the plasticity of MSC is not fully understood. The change of cell fate has been suggested to occur through cell fusion. We have generated hybrid cell lines by polyethylene-glycol-mediated cell fusion of primary porcine MSC with the immortal murine fibroblast cell line F7, a derivative of the GM05267 cell line. The hybrid cell lines display fibroblastic morphology and proliferate like immortal cells. They contain tetraploid to hexaploid porcine chromosomes accompanied by hypo-diploid murine chromosomes. Interestingly, many hybrid cell lines also express high levels of tissue-nonspecific alkaline phosphatase, which is considered to be a marker of undifferentiated embryonic stem cells. All tested hybrid cell lines retain osteogenic differentiation, a few of them also retain adipogenic potential, but none retain chondrogenic differentiation. Conditioned media from hybrid cells enhance the proliferation of both early-passage and late-passage porcine MSC, indicating that the hybrid cells secrete diffusible growth stimulatory factors. Murine F7 cells thus have the unique property of generating immortal cell hybrids containing unusually high numbers of chromosomes derived from normal cells. These hybrid cells can be employed in various studies to improve our understanding of regenerative biology. This is the first report, to our knowledge, describing the generation of experimentally induced cell hybrids by using normal primary MSC.     

Sister chromatid exchange in cell lines from malignant lymphomas (lymphoma lines)

Summary The frequency of sister chromatid exchanges (SCEs) was studied in cells from three freshly established lymphoma lines, derived from two patients with Hodgkin's disease and one patient with non-Hodgkin lymphoma. These values were compared to SCE rates found in cells from two long-established lymphoma lines (Raji and BJAB) and to those recorded in control cell lines of normal human donors. The highest SCE levels were demonstrated in the freshly established lymphoma lines, the lowest SCE values separated the lymphoblastoid cell lines from healthy controls, and the older lymphoma lines Raji and BJAB presented rates in between. The influences of BUDR concentration and of the duration of BUDR treatment on the frequency of SCEs were tested. Furthermore, the dependence of the SCE rate on the time interval between establishment of the cell line and its SCE investigation was considered. The connection between elevated SCE rates and the neoplastic nature of lymphoma lines is discussed.     

Somatic cell genetic analysis of the interferon system.

Current advances in the use of somatic cell hybrid systems have enhanced the value of these systems for studying eukaryotic cell functions. We have reviewed the use of somatic cells to investigate the human interferon system. It has been shown that interspecific heterokaryons and hybrid cells can produce interferon(s) of both parental types and may be protected from viral challenge by interferon(s) from either parent. Using mouse-human hybrid cells we have assigned a human gene(s) responsible for regulating interferon to chromosome 21 and genes involved in the production of human interferon to chromosomes 2 and 5. Our data also suggest possible assignment of a locus involved in control of interferon production to chromosome 16. Suggested further uses of the somatic cell system for interferon studies include study of the subunit structure of interferons and the development of hybrid lines that produce human interferon at high levels (interferon/somatic cell hybrids/human gene assignment.     

Glycosidases from the interferon producing lymphoid cell line Namalva and from three other lymphoid cell lines

1. The occurrence of lysozyme, neuraminidase and fourteen other glycosidases was investigated in the three lymphoma cell lines Namalva, Raji and Daudi derived from a Burkitt's lymphoma and the lymphoblastoid cell line Robinson from Epstein-Barr virus transformed normal peripheral blood lymphocytes. High activity of beta-N-acetyl-D-glucosaminidase was found in three of the cell lines, which also showed fairly high activities of beta-N-acetyl-D-galactosaminidase, alpha-D-mannosidase and beta-D-mannosidase. In Daudi the highest glycosidase activity was found for beta-D-mannosidase. 2. Neuraminidase and lysozyme were not detected in any of the four cell lines. 3. These cell lines showed characteristic enzyme patterns and enzyme ratios which may be used for the identification of the cell lines. 4. When calculated on a protein basis no statistically significant change in glycosidase activities of the cells could be recorded during interferon production.     

Properties of a hybrid between lines sensitive and insensitive to contact inhibition of cell division

Hybrid cell lines have been prepared between 3T3, a line highly sensitive to contact inhibition of division, and cl 1-D, an L cell derivative which is not sensitive. A number of hybrid clones isolated were found to be quite sensitive, indicating that in this respect the 3T3 behavior is the more fully expressed in the hybrid. On serial subculture, the hybrid lines gave rise to variants less sensitive to contact inhibition.     

Microbubble‐mediated sonoporation for highly efficient transfection of recalcitrant human B‐ cell lines

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult‐to‐transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B‐cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP‐positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP‐positive cells, respectively. Here, we present sonoporation as a feasible non‐viral strategy for stable and highly efficient heterologous transfection of recalcitrant B‐cell lines. This is the first demonstration of a non‐viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B‐cell lines.     

Pyruvate secreted by human lymphoid cell lines protects cells from hydrogen peroxide mediated cell death

Reactive oxygen species (ROS) released from polymorphonuclear leukocytes and macrophages could cause DNA damage, but also induce cell death. Therefore inhibition of cell death must be an important issue for accumulation of genetic changes in lymphoid cells in inflammatory foci. Scavengers in the post culture medium of four lymphoid cell lines, lymphoblastoid cell lines (LCL), Raji, BJAB and Jurkat cells, were examined. Over 80% of cultured cells showed cell death 24 h after xanthine (X)/xanthine oxidase (XOD) treatment, which was suppressed by addition of post culture medium from four cell lines in a dose-dependent manner. H₂O₂ but not O^·-₂ produced by the X/XOD reaction was responsible for the cytotoxity, thus we used H₂O₂ as ROS stress thereafter. The H₂O₂-scavenging activity of post culture media from four cell lines increased rapidly at the first day and continued to increase in the following 2–3 days for LCL, Raji and BJAB cells. The scavenging substance was shown to be pyruvate, with various concentrations in the cultured medium among cell lines. Over 99% of total pyruvate was present in the extracellular media and less than 1% in cells. α-Cyano-4-hydroxycinnamate, a specific inhibitor of the H⁺-monocarbohydrate transporter, increased the H₂O₂-scavenging activity in the media from all four cell lines via inhibition of pyruvate re-uptake by cultured cells from the media. These findings suggest that lymphoid cells in inflammatory foci could survive even under ROS by producing pyruvate, so that accumulation of lymphoid cells with DNA damage is possible.     

Complementation between two temperature-sensitive mammalian cell mutants, each defective in the G1 phase of the cell cycle

Two mammalian temperature-sensitive (ts) G1 cell cycle mutants of different species origin (Syrian hamster and mouse) have been tested for complementation using somatic cell hybrid analysis. All hamster-mouse hybrid clones tested were found to exhibit normal growth properties at the restrictive temperature, while neither mutant alone was capable of normal growth at this temperature. The two mutant lines therefore complement for growth in a somatic cell hybrid and most likely represent ts lesions in different cellular functions specific to the G1 phase of the cell cycle.     

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.     

Effect of UV-C mediated oxidative stress in leukemia cell lines and its relation to ubiquinone content

UV-C radiation is able to impair cellular functions by directly damaging DNA, and by inducing an increased formation of reactive oxygen species that leads to a condition of oxidative stress. In this study we evaluated different responses to UV insult of two leukemia cell lines, HL-60 and Raji, and the relationship with their CoQ10 content. DNA damage was monitored by means of the alkaline single cell gel electrophoresis (Comet assay); intracellular levels of ROS, mitochondrial depolarization and cell viability was measured by flow cytometry. Raji cells appeared more resistant to the UV insult; moreover, they did not show any increase in ROS content and the extent of mitochondrial depolarisation was much lower than in HL 60 cell line. Raji cells also contained significantly higher levels of CoQ10 and their ability to incorporate and to reduce exogenous CoQ10 added to the culture medium was remarkably elevated compared with HL 60.