Human Namalwa Research Articles

Determination of in vitro immunotoxic potencies of a series of perfluoralkylsubstances (PFASs) in human Namalwa B lymphocyte and human Jurkat T lymphocyte cells.

Exposure to PFASs is associated to several adverse health effects, such as immunotoxicity. Immunotoxic effects of PFOA and PFOS, including a reduced antibody response in both experimental animals and humans, have been reported. However, there is limited understanding of the underlying mechanisms involved. Moreover, there is only a restricted amount of immunotoxicity data available for a limited number of PFASs. In the current study the effects of 15 PFASs, including short- and long-chain perfluorinated carboxylic and sulfonic acids, fluorotelomer alcohols, and perfluoralkyl ether carboxylic acids were studied on the expression of recombinant activating gene 1 (RAG1) and RAG2 in the Namalwa human B lymphoma cell line, and on the human IL-2 promotor activity in Jurkat T-cells. Concentration-response data were subsequently used to derive in vitro relative potencies through benchmark dose analysis. In vitro relative potency factors (RPFs) were obtained for 6 and 9 PFASs based on their effect on RAG1 and RAG2 gene expression in Namalwa B-cells, respectively, and for 10 PFASs based on their inhibitory effect on IL-2 promotor activity in Jurkat T-cells. The most potent substances were HFPO-TA for the reduction of RAG1 and RAG2 gene expression in Namalwa cells (RPFs of 2.1 and 2.3 respectively), and PFDA on IL-2 promoter activity (RPF of 9.1). RAG1 and RAG2 play a crucial role in V (D)J gene recombination, a process for acquiring a varied array of antibodies crucial for antigen recognition. Hence, the effects observed in Namalwa cells might indicate a PFAS-induced impairment of generating a diverse range of B-cells essential for antigen recognition. The observed outcomes in the Jurkat T-cells suggest a possible PFAS-induced reduction of T-cell activation, which may contribute to a decline in the T-cell dependent antibody response. Altogether, the present study provides potential mechanistic insights into the reported PFAS-induced decreased antibody response. Additionally, the presented in vitro models may represent useful tools for assessing the immunotoxic potential of PFASs and prioritization for further risk assessment.

Perfluoroalkyl substances (PFASs) decrease the expression of recombination-activating genes (RAG1 and RAG2) in human B lymphoma Namalwa cells

Per- and polyfluoroalkyl substances (PFASs) are omnipresent and have been shown to induce a wide range of adverse effects, including hepatotoxicity, developmental toxicity and immunotoxicity. So far, little information is available about the mechanisms underlying the toxicity of PFASs, including those related to their immunotoxicity. Reported immunotoxic effects of PFASs include decreased antibody responses in experimental animals and humans, indicating that PFASs may, among others, affect B cell function. In the present study, we first assessed the effects of PFOA on the transcriptome of the human Namalwa B cell line using RNA seq analysis. Gene expression changes, analyzed using Ingenuity Pathway Analysis, pointed to various cellular processes affected by PFOA, including ‘B cell development’ and ‘Primary immunodeficiency signaling’. Interestingly, PFOA decreased the expression of RAG1 and RAG2, genes involved in immunoglobulin and T cell receptor V(D)J recombination. As a next step, time- and concentration-dependent changes in the expression of RAG1 and RAG2 upon exposure to PFOA, PFNA, PFHxS and PFOS were studied through RT-qPCR analysis. Analysis with the concentration–response modeling software PROAST resulted in the following potency ranking: PFNA > PFOA > PFOS > PFHxS. Altogether, the present in vitro study provides insights into the effects of selected PFASs on B cells, identifying RAG1 and RAG2 expression as possible relevant targets that may play a role in the immunotoxicity of PFASs.

Stabilization of human interferon-α1 mRNA by its antisense RNA

Antisense transcription is a widespread phenomenon in the mammalian genome and is believed to play a role in regulating gene expression. However, the exact functional significance of antisense transcription is largely unknown. Here, we show that natural antisense (AS) RNA is an important modulator of interferon-α1 (IFN-α1) mRNA levels. A ~4-kb, spliced IFN-α1 AS RNA targets a single-stranded region within a conserved secondary structure element of the IFN-α1 mRNA, an element which was previously reported to function as the nuclear export element. Following infection of human Namalwa lymphocytes with Sendai virus or infection of guinea pig 104C1 fetal fibroblasts with influenza virus A/PR/8/34, expression of IFN-α1 AS RNA becomes elevated. This elevated expression results in increased IFN-α1 mRNA stability because of the cytoplasmic (but not nuclear) interaction of the AS RNA with the mRNA at the single-stranded region. This results in increased IFN-α protein production. The silencing of IFN-α1 AS RNA by sense oligonucleotides or over-expression of antisense oligoribonucleotides, which were both designed from the target region, confirmed the critical role of the AS RNA in the post-transcriptional regulation of IFN-α1 mRNA levels. This AS RNA stabilization effect is caused by the prevention of the microRNA (miRNA)-induced destabilization of IFN-α1 mRNA due to masking of the miR-1270 binding site. This discovery not only reveals a regulatory pathway for controlling IFN-α1 gene expression during the host innate immune response against virus infection but also suggests a reason for the large number of overlapping complementary transcripts with previously unknown function.Electronic supplementary materialThe online version of this article (doi:10.1007/s00018-012-1216-x) contains supplementary material, which is available to authorized users.

Simultaneously targeting mitochondria and endoplasmic reticulum by photodynamic therapy induces apoptosis in human lymphoma cells

Photodynamic therapy (PDT) and photodetection with protoporphyrin IX (PpIX) precursors have widely been used in the diseases with abnormally proliferative cells, but the mechanism of the modality is not fully understood yet. In this study 70-95% of apoptotic cells after PDT with PpIX precursor, hexaminolevulinate (HAL) in two human lymphoma cell lines, Namalwa and Bjab, were confirmed by fluorescence microscopy, electron microscopy and flow cytometry. HAL-derived PpIX was mainly distributed in the mitochondria and endoplasmic reticulum (ER), both of which were initial targets after light exposure causing two major pathways simultaneously involved in the apoptotic induction. One was the mitochondrial pathway including the release of cytochrome c, cleavage of caspases-9/-3, poly(ADP-ribose) polymerase and DNA fragmentation factor. The other was the ER stress-mediated pathway triggering a transient increase in the cytosolic Ca(2+) level after photodamage to the ER calcium pump protein SERCA2. The released Ca(2+) further initiated the caspase-8 cleavage. The use of both extracellular Ca(2+) chelator EGTA and intracellular Ca(2+) chelator BAPTA-AM confirmed that such cytosolic Ca(2+) originated from the ER rather than extracellular Ca(2+)-containing medium. About 30% of the apoptosis was blocked with BAPTA-AM alone; while a complete inhibition of such apoptosis was achieved with a combination of the caspase-9 inhibitor Z-LEHD-FMK and caspase-8 inhibitor Z-IETD-FMK, thus quantifying each role of the mitochondrial and ER pathways.

HMG-CoA reductase inhibitor simvastatin overcomes bortezomib-induced apoptosis resistance by disrupting a geranylgeranyl pyrophosphate-dependent survival pathway

Simvastatin is a competitive inhibitor of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate pathway required for the biosynthesis of cholesterol and higher isoprenoids such as geranylgeranyl pyrophosphate (GGPP). Apart from its capacity to lower cholesterol plasma levels and to protect against cardiovascular disease, simvastatin induces apoptosis in various cancer cells. We have generated human Namalwa Burkitt lymphoma cells that display general apoptosis resistance and hyperproliferation due to increased expression and proteolytic activity of 26S proteasomes in response to continuous treatment of the cells with the proteasome inhibitor bortezomib. In these cells, simvastatin does not inhibit proteasome activity, but induces apoptosis, G2/M cell cycle arrest and accumulation of p21 Waf1/Cip1, and effectively inhibits hyperproliferation. These effects are reversed by the addition of GGPP. GGPP-dependent plasma membrane localization of the small GTPase RhoA that is required for RhoA-mediated oncogenic signaling is completely inhibited by simvastatin. Finally, bortezomib but not simvastatin induces accumulation and stabilization of the anti-apoptotic protein Mcl-1, which is known to confer resistance to apoptosis in cancer cells. Thus, simvastatin overcomes bortezomib-induced apoptosis resistance by inhibiting synthesis of GGPP and disrupting a GGPP-dependent survival pathway.

Increased expression and altered subunit composition of proteasomes induced by continuous proteasome inhibition establish apoptosis resistance and hyperproliferation of Burkitt lymphoma cells

The proteasome is the main protease for extralysosomal protein degradation in eukaryotic cells, and constitutes a sophisticated high molecular mass proteinase complex underlying a tightly coordinated expression and assembly of multiple subunits and subcomplexes. Here we show that continuous inhibition of proteasomal chymotrypsin-like peptidase activity by the proteasome inhibitor bortezomib induces in human Namalwa Burkitt lymphoma cells increased de novo biogenesis of proteasomes accompanied by increased expression of the proteasome maturation protein POMP, increased expression of 19S-20S-19S proteasomes, and abrogation of expression of beta 1i, beta 2i and beta 5i immunosubunits and PA28 in favor of increased expression of constitutive proteolytic beta1, beta2 and beta 5 subunits and 19S regulatory complexes. These alterations of proteasome expression and subunit composition are accompanied by an increase in proteasomal caspase-like, trypsin-like and chymotrypsin-like peptidase activities, not inhibitable by high doses of bortezomib. Cells harboring these proteasomal alterations display rapid proliferation and cell cycle progression, and acquire resistance to apoptosis induced by proteasome inhibitors, gamma-irradiation and staurosporine. This acquired apoptosis resistance is accompanied by de novo expression of anti-apoptotic Hsp27 protein and the loss of ability to accumulate and stabilize pro-apoptotic p53 protein. Thus, increased expression, altered subunit composition and increased activity of proteasomes constitute a hitherto unknown adaptive and autoregulatory feedback mechanism to allow cells to survive the lethal challenge of proteasome inhibition and to establish a hyperproliferative and apoptosis-resistant phenotype.

The Overexpression of ANKHD1 in Acute Leukemia Cells and Its Association with SIVA-1, a Proapoptotic Protein.

Ankyrin-repeat-containing proteins regulate multiple cellular functions including transcriptional and cell-cycle regulation, ion channel, cell survival and cell signaling and participate in protein-protein interactions via their repeat motifs. Ankyrin Repeat and KH Domain Containing 1-ANKHD1, was first identified in LNCaP, a human prostate cancer cell line. However, protein expression patterns or protein interactions of ANKHD1 have not yet been described. The aim of this studied was to characterize the mRNA and protein expression patterns of ANKHD1 in normal hematopoietic cells and acute leukemia cells, and also to identify new proteins that interact with ANKHD1 in hematopoietic cells. The National Ethical Committee Board approved the study and informed-written consent was obtained from all subjects. Real Time RT-PCR was performed and increased levels of ANKHD1 mRNA were detected in acute leukemia cell lines; KG-1, HEL, K562, NB4, HL-60 Jurkat, MOLT4, Raji, Daudi and Namalwa (up to 10-fold increase), compared to normal bone marrow cells. Additionally, ANKHD1 mRNA expression was significantly higher in samples from patients with a diagnosis of acute myeloid leukemia (n = 30) (medians: 1.97 versus 1.05, P = 0.004) when compared to normal hematopoietic cells (n = 9), and in samples from patients with a diagnosis of acute lymphoblastic leukemia (n = 7) (medians: 2.53 versus 1.05, P = 0.005) when compared to normal hematopoietic cells (n = 9). Regarding protein expression, immunoblot analysis detected a very low expression of ANKHD1 in Peripheral Blood Mononuclear Cells (PBMC) of normal donors. Conversely, a high expression of the protein was detected in the human acute leukemia cell lines, KG-1, HEL, K562, NB4, HL-60 Jurkat, MOLT4, Raji, Daudi and Namalwa. Laser confocal analysis of K562 and Jurkat cell lines and PBMC showed that ANKHD1 is located in the cytoplasm of leukemia and normal hematopoietic cells. This cytoplasmatic localization of ANKHD1 was further confirmed by immunoblotting performed with anti-ANKHD1 antibody and tissue fractions of the Jurkat cell line. In an attempt to identify the proteins associated with ANKHD1 in hematopoietic cells, yeast two-hybrid screening was used and AH109 was transformed with PGBKT7-ANKHD1 (amino acids 1130–1243) and used to screen a Matchmaker pACT2-cDNA library prepared from normal human bone marrow (Clontech). The screening identified a new protein interaction between ANKHD1 and SIVA-1. This interaction was further confirmed by immunoprecipitation and Western Blotting analysis in the Jurkat cell line. The finding that ANKHD1 interacts with SIVA-1, which has been recently identified as a proapoptotic protein up regulated in leukemia cells, as well as the high expression of ANKHD1 in acute leukemia, described in our study, gives rise to the hypothesis that ANKHD1 protein may be involved in leukemogenesis. The identification of new disease-specific targets for acute myeloid leukemia immunotherapy expands treatment options and increases our chances of successfully treating this heterogeneous disease and lowering the unacceptably high mortality rate.

CXCR4 is involved in multiple functions of cancer cells and is a compelling target for monoclonal antibody-based therapeutics

20013 Background: CXCR4 is over-expressed on > 75% of cancers and absent on most non-neoplastic cells. It is a G protein coupled receptor spanning the cell membrane with CXCL-12 as its ligand. It is uniquely involved in 3 vital aspects of cancer: 1) proliferation; 2) motility; and 3) metastasis. Our objective was to assess specific Abs as candidates for clinical development in preclinical models. Methods: Several human cancer cell lines were used to study whether either mAbs specific to the CXCR4 receptor or to CXCL12 could inhibit: 1) proliferation; 2) chemotaxis; and 3) invasion through a reconstituted basement membrane. The ability of anti-CXCR4 Ab to inhibit tumor growth in vivo was also tested. Female Nod/Scid mice were injected IP with 2 × 105 human Namalwa tumor cells with 10 mice per group. Mice were injected with: Group 1) 100μg irrelevant IgG2b isotype control Ab; Group 2) 100μg CX02 anti-CXCR4 Ab; and Group 3) 100 μg CX05 anti-CXCR4 Ab. All mice in each group received an injection of Ab at day 2, 8, 14 and 20. Mice were observed for tumor growth over the following 4–5 months. Results: We originally found that CXCR4 was over expressed in fresh surgical human Glioblastoma, then showing that it was also over expressed in multiple cancer types. Specific anti-CXCR4 monoclonal Abs, CX02 and CX05 as well as anti-CXCL12 Abs demonstrated the ability to block cell proliferation of a variety of human Glioblastoma tumor cells including GB1690, HTB16, and 5GB, but not the proliferation of cells that do not express CXCR4. CX02 and CX05 Abs bind to live tumor cells with high affinity and have an EC50 of 3.2 and 2.7 μg/ml respectively (effective concentration required to provide 50% maximal median fluorescence shift). Both Abs (10 μg/ml) also inhibited by over 90% the motility of HS Sultan cells as demonstrated by the standard chemotaxis assays. In addition, both Abs (10–40 μg/ml) also inhibited the ability of HS Sultan cells to penetrate Matrigel, a reconstituted basement membrane assay used to assess malignant potential of cancer cells. Finally, both Abs were successful in extensively prolonging survival of Nod/Scid mice injected with human Namalwa cells. Conclusions: CXCR4 represents a compelling target for mAb therapy; Phase I clinical trails are pending. [Table: see text]

Immune evasion by CD200: New approaches to targeted therapies for chronic lymphocytic leukemia (CLL)

2519 Background: Although the human immune system is capable of raising an immune response against many cancer types, that response is insufficient to eradicate the cancer in most patients, possibly due to immune evasion through negative regulation of the immune system by the tumor. We identified the immune-suppressive molecule CD200 to be upregulated 1.5–5.4-fold on CLL cells in all 80 patients examined. Interaction of CD200 with its receptor alters cytokine profiles from Th1 to Th2 in mixed lymphocyte reactions, and results in the induction of regulatory T cells, which are thought to hamper tumor-specific effector T cell immunity. We addressed whether CD200 expression on tumor cells plays a role in immune evasion, thereby preventing elimination of tumor cells by the immune system in a xenograft hu/SCID mouse model, and whether treatment with an antagonistic anti-CD200 antibody affects tumor growth. Methods: The human non-Hodgkins lymphoma cell lines RAJI and Namalwa were transduced with human CD200 and injected subcutaneously together with human peripheral blood lymphocytes (PBL) into NOD/SCID mice. Tumor growth over time was compared among mice that either received CD200-expressing tumor cells or received tumor cells lacking CD200 expression. In subsequent experiments, mice were treated with chimeric or humanized anti-CD200 antibodies (doses ranged from 1 to 20 mg/kg) by intravenous injection. Treatment was either started immediately or 7 days after tumor cell injection. Results: As expected, PBLs reduced CD200-negative RAJI or Namalwa tumor growth by up to 75%. In contrast, growth of RAJI or Namalwa tumors expressing CD200 at levels comparable to that of CLL was not reduced by PBLs. Administration of anti-CD200 antibodies at 5 mg/kg resulted in nearly complete tumor growth inhibition (1/10 mice developed a small tumor) over the course of the study even when treatment was started 7 days after tumor cell injection. Conclusions: CD200 expression on tumor cells inhibits the ability of human lymphocytes to eradicate tumor cells. Treatment of CD200-expressing tumors with antagonistic anti-CD200 antibodies inhibits tumor growth, indicating the potential for anti-CD200 therapy as a promising approach for CLL. No significant financial relationships to disclose.

VDAC1 Is a Transplasma Membrane NADH-Ferricyanide Reductase

Porin isoform 1 or VDAC (voltage-dependent anion-selective channel) 1 is the predominant protein in the outer mitochondrial membrane. We demonstrated previously that a plasma membrane NADH-ferricyanide reductase activity becomes up-regulated upon mitochondrial perturbation, and therefore suggested that it functions as a cellular redox sensor. VDAC1 is known to be expressed in the plasma membrane; however, its function there remained a mystery. Here we show that VDAC1, when expressed in the plasma membrane, functions as a NADH-ferricyanide reductase. VDAC1 preparations purified from both plasma membrane and mitochondria fractions exhibit NADH-ferricyanide reductase activity, which can be immunoprecipitated with poly- and monoclonal antibodies directed against VDAC(1). Transfecting cells with pl-VDAC1-GFP, which carries an N-terminal signal peptide, directs VDAC1 to the plasma membrane, as shown by confocal microscopy and FACS analysis, and significantly increases the plasma membrane NADH-ferricyanide reductase activity of the transfected cells. This novel enzymatic activity of the well known VDAC1 molecule may provide an explanation for its role in the plasma membrane. Our data suggest that a major function of VDAC1 in the plasma membrane is that of a NADH(-ferricyanide) reductase that may be involved in the maintenance of cellular redox homeostasis.

Mosquito cells bind and replicate hepatitis C virus.

Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells.

Mosquito cells bind and replicate hepatitis C virus

Several studies have demonstrated some hepatitis C virus (HCV) replication in lymphocyte and hepatocyte cell lines such as in African green monkey Vero cells. The aim of the present study was to select other cell lines able to bind and replicate HCV. Human hepatoma PLC/PRF/5 cells, human lymphoma Namalwa cells, Vero and mosquito AP61 cells were inoculated with HCV-positive plasma, washed six times and examined for the presence of the viral genome at different times post infection, using an RT-PCR method. Binding of HCV to cells was estimated by HCV RNA detection in cells 2 hr after inoculation and in the last wash of these cells. Successive virus passages in cells were carried out. All the cells studied were able to bind HCV but only AP61 and Vero cells provided evidence of replication and production of infectious virus: virus RNA was detected during 28 days post-infection in four successive virus passages. CD81 molecules, a putative HCV receptor, were detected by cytofluorometric analysis. Vero cells express CD81 molecules whereas these molecules were not detected on AP61 cells. It is suggested that other receptors are involved in HCV binding to Vero and AP61 cells. J. Med. Virol. 64:6–12, 2001. © 2001 Wiley-Liss, Inc.

Ectocellular CD38-catalyzed synthesis and intracellular Ca(2+)-mobilizing activity of cyclic ADP-ribose.

CD38 is a type-II transmembrane glycoprotein occurring in several hematopoietic and mature blood cells as well as in other cell types, including neurons. Although classified as an orphan receptor, CD38 is also a bifunctional ectoenzyme that catalyzes both the conversion of NAD+ to nicotinamide and cyclic ADP-ribose (cADPR), via an ADP-ribosyl cyclase reaction, and also the hydrolysis of cADPR to ADP-ribose (hydrolase). Major unresolved questions concern the correlation between receptor and catalytic properties of CD38, and also the apparent contradiction between ectocellular generation and intracellular Ca(2+)-mobilizing activity of cADPR. Results are presented that provide some explanations to this topological paradox in two different cell types. In cultured rat cerebellar granule neurons, extracellular cADPR (either generated by CD38 or directly added) elicited an enhanced intracellular Ca(2+)-response to KCl-induced depolarization, a process that can be qualified as a Ca(2+)-induced Ca2+ release (CICR) mechanism. On the other hand, in the CD38+ human Namalwa B lymphoid cells, NAD+ (and thiol compounds as well) induced a two-step process of self-aggregation followed by endocytosis of CD38, which resulted in a shift of cADPR metabolism from the cell surface to the cytosol. Both distinctive types of cellular responses to extracellular NAD+ seem to be suitable to elicit changes in the intracellular Ca2+ homeostasis.

Effect of hepatitis B virus on tumour necrosis factor (TNFα) gene expression in human THP-1 monocytic and Namalwa B-cell lines

In response to viruses, monocytes and B cells produce TNF alpha. Therefore, we investigated TNF alpha gene expression and protein secretion in a human monocytic cell line, THP-1, and a Burkitt's lymphoma B-cell line, Namalwa, in response to hepatitis B virus (HBV). Stimulation by phorbol myristate acetate (PMA) (100 ng/ml for 48 h) induced TNF alpha secretion in THP-1 and Namalwa cells (100 to 300 pg/ml). In THP cells, the optimum response (> 2000 pg/ml) was obtained in the presence of a second mitogenic signal such as lipopolysaccharide (LPS) (10 microg/ml for 24 h). In our activation conditions, Northern blot analysis revealed a marked accumulation of TNF alpha mRNA species at 1.7 kb in both cell lines. When PMA- or PMA+LPS-stimulated THP-1 cells were exposed to HBV, TNF alpha protein and mRNA significantly decreased (> 50%). In contrast, HBV exposure of PMA-activated Namalwa cells resulted in strongly increased TNF alpha protein secretion (1 ng/ml). In this case, HBV induced TNF alpha mRNA accumulation that consisted of two types: a regular 1.7 kb and two novel high molecular weight (HMW) species at 3.7 and 4.3 kb. Exposure of stimulated THP-1 and Namalwa cells to HBV resulted in HBs and pre-S1 antigen production in the supernatants. In addition, HMW HBV DNA forms were detected in both cell lines, but with distinct HindIII restriction patterns. These findings indicate that TNF alpha gene expression may be differently regulated by HBV in activated human macrophages and B cells, and thus TNF alpha may be involved in the pathogenesis of HBV.

NAD +-dependent internalization of the transmembrane glycoprotein CD38 in human Namalwa B cells

CD38 is a transmembrane glycoprotein involved as an orphan receptor in many physiological processes of lymphocytes. It is also a bifunctional enzyme that catalyzes at its ectocellular domain the synthesis from NAD + (cyclase) and the hydrolysis (hydrolase) of the calcium-mobilizing metabolite cyclic ADP-ribose (cADPR). A still unexplained paradox concerns the relationship between ectocellular localization of CD38 and intracellular calcium-releasing activity of its intermediate product cADPR. Incubation of CD38 + human Namalwa B cells with external NAD + elicited extensive membrane down-regulation of CD38 and its internalization in non-clathrin-coated vesicles. Since the internalized CD38 was demonstrated to be enzymatically active, this NAD +-dependent process is a hitherto unrecognized means for shifting cADPR metabolism from the cell surface to the intracellular environment.

Cloning and Mapping of a HumanRBP56Gene Encoding a Putative RNA Binding Protein Similar to FUS/TLS and EWS Proteins

TheEWSgene was found at the chromosome breakpoints in Ewing sarcoma, and theFUS/TLSgene was found at the breakpoints of myxoid liposarcoma and acute myeloid leukemia. These genes encode proteins that carry a highly homologous RNA binding domain. Fusion proteins made of the N-terminal half of EWS or FUS/TLS and transcriptional regulatory proteins, also derived from genes located at breakpoints, have been suggested to be involved in the pathogenesis of tumors. By PCR amplification of human Namalwa cell cDNA using degenerate primers made from the conserved amino acid sequences in the RNA binding domain of EWS and FUS/TLS, we obtained a cDNA fragment (RBP56cDNA), the predicted amino acid sequences of which were similar but not identical to those of EWS and FUS/TLS. Using this fragment as a probe, we obtained two isoforms of cDNAs consisting of 2144 and 2153 bp, respectively, which encode proteins consisting of 589 and 592 amino acid residues, respectively. The predicted amino acid sequences of RBP56 protein have a serine-, tyrosine-, glutamine-, and glycine-rich region in the N-terminal region, an RNA binding domain and a C2C2finger motif in the central region, and degenerate repeats of DR(S)GG(G)- YGG sequences in the C-terminal region. The expression ofRBP56mRNA was observed in all of the human fetal and adult tissues examined, as was the expression ofEWSandFUS/TLSmRNAs. TheRBP56gene was mapped to chromosome 17q11.2 to q12.

Expression and Purification of Recombinant, Glycosylated Human Interferon Alpha 2b in Murine Myeloma NSo Cells

We have expressed recombinant human interferon-α2b in mammalian cells and isolated cell lines constitutively secreting very high levels of biologically active protein. The expression system takes advantage of the strong human cytomegalovirus immediate early promoter in mouse myeloma NSo cells and glutamine synthetase as a selectable marker; spontaneous mutants with amplified gene copy numbers were selected by growth of primary transfectants in the presence of methionine sulfoximine. Using this procedure, we have isolated a recombinant NSo cell line which secretes human interferon at the rate of 20 μg/106cells/24 h and accumulates up to 120 μg/ml (∼2.4 × 107U/ml) following prolonged undiluted culture. The interferon (IFN) could be efficiently purified on a polyclonal bovine anti-human IFNα specific antibody column and the glycosylation pattern was found to be similar to that of nonrecombinant IFNα2b purified from virus-induced human Namalwa cells. The biological activity of the recombinant material was indistinguishable from that of natural IFN from Namalwa cells, and the specific antiviral activity, as assayed on human HeLa cells challenged with encephalomyocarditis virus, was 2 × 108IU/mg, similar to that of nonrecombinant IFN preparations. This represents the highest reported level of glycosylated, recombinant IFN expression in a stable mammalian system and is a significant advance in the large-scale production of these clinically important cytokines.

Cytotoxicity and Suppression of Immunoglobulin Production against Human Namalwa Cells Caused by Oxidized Cholesterol

The effects of oxidized cholesterols on proliferation and IgM production of human lymphoblastoid Namalwa cells were examined. An oxidized cholesterol mixture, in contrast to cholesterol, was a potent cytotoxin to Namalwa cells. Among oxidized cholesterols examined, 25-hydroxycholesterol was the most cytotoxic. However, no oxidized cholesterol examined suppressed IgM production, although cholestanetriol and 7-ketocholesterol did suppress it. Thus, oxidized cholesterols are cytotoxic to lymphocytes, while the influence on the immunoglobulin production may be marginal.

Transcription of interferon-α2 alleles from virus-induced human leucocytes and lymphoblastoid cells of African origin

It has been previously shown that the genomic DNA of both the lymphoblastoid cell line (Namalwa) and certain human donors (of African origin) contain sequences corresponding to two allelic variants, b and c, of the interferon-α2 gene (IFNA2). Little is known however about the relative expression of these two alleles in heterozygous cells. We have therefore examined the transcription of allelic variants of the human IFNA2 locus by both normal human leucocytes (from a heterozygous donor) and Namalwa cells. Analysis of cDNA clones identified sequences of both allelic variants, IFNA2b and c, indicating active transcription by both cell types. Analysis of tryptic and peptic peptides derived from purified IFN-α2 also demonstrated both IFN-α2b and IFN-α2c proteins. Populations of virus induced heterozygous cells can therefore effectively transcribe and secrete both forms of IFN-α2 simultaneously, with no apparent restrictions on expression of either allele.

Human cell mutants with very low mitochondrial DNA copy number ( d)

Mutants of human Namalwa cells are described, denoted rho d (mtDNA-depleted), which contain a very low mtDNA copy number. Three isolates, denoted FV02, FV03 and FV05, each selected on the basis of their pyruvate-dependent phenotype, were shown to have severely reduced mitochondrial respiratory functions consistent with results of histochemical and cytochrome analysis. Analyses of mtDNA by the polymerase chain reaction (PCR) and Southern blotting indicated that FV02, FV03 and FV05 each contained a full complement of mtDNA sequences, 16.6 kb in length, in circular form but at a greatly reduced level compared with that of parent rho + cells. Quantitative PCR tests showed first, that the parent Namalwa rho + cells contain about 1800 molecules of mtDNA and second, that cells of each of FV02, FV03 and FV05 contain between 100- and 1000-fold less mtDNA than rho + cells. Subclones of each of FV02, FV03 and FV05 all contained detectable mtDNA, thus eliminating the possibility that these isolates are mixtures of rho o (devoid of mtDNA) and rho + cells. Each of the rho d cell lines FV02, FV03 and FV05 had a more rapid growth rate than the corresponding rho o cells but less than that of the parent Namalwa rho + cells. The experimentally induced rho d cells described here may represent a useful model for human diseases in which severe depletion of cellular mtDNA levels in tissues is encountered.