Plasmacytoma Cell Line Research Articles

Restrictin-P/stromal activin A, kills its target cells via an apoptotic mechanism.

We have recently found that the inhibitor of plasmacytoma cell growth, restrictin-P, is a stroma derived activin A and that it is an antagonist of interleukin-6 and interleukin-11. The present study was aimed at determining the mode by which this cytokine kills its target cells. On addition of the cytokine there was little or no net increase in cell number, depending on the specific target cells. All plasmacytoma cell lines tested exhibited a similar time dependent inhibition of DNA synthesis and a G0/G1 shift in the cell cycle. Electron microscope examination revealed classical apoptotic features i.e. chromatin condensation and membrane blebbing. DNA fragmentation, measured qualitatively and quantitatively, occurred in all cytokine treated plasmacytoma cell lines. Bovine activin A had an identical capacity to reduce cell viability, to induce G0/G1 shift and to cause DNA fragmentation. X-ray microanalysis of intracellular ions revealed an increase in calcium ions, following exposure of plasmacytoma cells to restrictin-P, accompanied by a decrease in phosphor ions. The cytotoxicity of the inhibitor was augmented in an additive manner by cycloheximide (CHX) indicating that the process did not require de novo protein synthesis. This study thus shows that restrictin-P/stromal activin A kills its target cells by inducing apoptosis. This effect was mediated by subnanogram concentrations and therefore may represent one physiological function of this pleiotropic cytokine.

Interleukin-11 (IL-11) and its receptor: biology and potential clinical applications in thrombocytopenic states.

Hematopoietic microenvironment consists of many different cell types including fibroblasts, macrophages, adipocytes, osteoblasts, and endothelial cells. It is believed that the steady-state equilibrium of hematopoiesis results from the interplay between these stromal cells and stem/progenitor cells through cell-cell contact and local production of cytokines. We have analyzed the growth-factor production of a primate stromal fibroblast cell line, PU-34 [1], and isolated by functional expression cloning a novel cDNA clone (named IL-11) conferring the mitogenic activity on an IL-6-dependent mouse plasmacytoma cell line, T1165 [2]. Subsequent molecular and biological characterization of this cytokine has indicated that IL-11 represents a multifunctional growth factor possessing biological activities overlapping with many growth factors in the gpl30 cytokine family [3–5]. The expression of IL-11 in PU-34 cells can be induced by IL-1, a macrophage-derived cytokine. IL-11 secreted by PU-34 cells following IL-1 stimulation can in turn exhibit inhibitory effect on adipocytes [6,7]. Since these cell types are major components in the hematopoietic microenvironment, the investigation of the control mechanisms of gene expression and signal transduction of IL-11 will advance our understanding of the cell/growth-factor interactions in hematopoiesis.

Cell death in bioreactors: A role for apoptosis

The incidence of apoptotic and necrotic cell death was compared in CHO, SF9 insect cells and murine plasmacytoma (J558L) and hybridoma (TB/C3) cells during in vitro cultivation in batch cultures. Acridine orange staining and fluorescence microscopy enabled the visualization of a classic morphological feature of apoptotic cell, the presence of condensed and/or fragmented chromatin. DNA gel electrophoresis was employed to show an additional characteristic of the process, the endonuclease-mediated fragmentation of DNA into multiples of 180 base pairs. The levels of apoptosis at the end of batch cultures of plasmacytoma and hybridoma cell lines were found to be 60% and 90% of total dead cells, respectively. However, employing the above-mentioned techniques, the biochemical and morphological features of apoptosis were not found in CHO and SF9 insect cells. Some factors affecting the induction of apoptosis during the batch culture of the hybridoma and plasmacytoma cell lines were identified. The most effective inducer was found to be glutamine limitation, followed by (in order of importance) serum limitation, glucose limitation, and ammonia toxicity. Blockage of the cell cycle of the plasmacytoma and hybridoma cells using thymidine resulted in the induction of apoptosis. This has important implications for the development of cell culture processes that minimize cell division and thereby increase specific productivity.

Evidence that the pathway of transferrin receptor mRNA degradation involves an endonucleolytic cleavage within the 3' UTR and does not involve poly(A) tail shortening.

The stability of transferrin receptor (TfR) mRNA is regulated by iron availability. When a human plasma-cytoma cell line (ARH-77) is treated with an iron source (hemin), the TfR mRNA is destabilized and a shorter TfR RNA appears. A similar phenomenon is also observed in mouse fibroblasts expressing a previously characterized iron-regulated human TfR mRNA (TRS-1). In contrast, mouse cells expressing a constitutively unstable human TfR mRNA (TRS-4) display the shorter RNA irrespective of iron treatment. These shorter RNAs found in both the hemin-treated ARH-77 cells and in the mouse fibroblasts are shown to be the result of a truncation within the 3' untranslated regions of the mRNAs. The truncated RNA is generated by an endonuclease, as most clearly evidenced by the detection of the matching 3' endonuclease product. The cleavage site of the human TfR mRNA in the mouse fibroblasts has been mapped to single nucleotide resolution to a single-stranded region near one of the iron-responsive elements contained in the 3' UTR. Site-directed mutagenesis demonstrates that the sequence surrounding the mapped endonuclease cleavage site is required for both iron-regulated mRNA turnover and generation of the truncated degradation intermediate. The TfR mRNA does not undergo poly(A) tail shortening prior to rapid degradation since the length of the poly(A) tail does not decrease during iron-induced destabilization. Moreover, the 3' endonuclease cleavage product is apparently polyadenylated to the same extent as the full-length mRNA.

Expression of immunoglobulin heavy chain-ricin a chain fusions in mammalian cells

Mammalian cell lines were transfected with antibody heavy (H) chain-ricin A chain gene fusions in attempts to assemble a recombinant immunotoxin. We found that a light chain-secreting mouse plasmacytoma cell line can be transfected stably with such a chimaeric gene, but only if the ricin A chain portion is disarmed by genetic means prior to transfection; if not, stable transfection appears to select for genetic inactivation of the transfected gene. Co-expression of an antibody heavy chain-ricin A chain fusion with light chain in non-lymphoid cells results in cell death. We conclude that the ricin A chain moiety retains biological activity precluding the expression of biologically active antibody-ricin A chain fusion proteins in mammalian cells.

Cloning and functional characterization of early B-cell factor, a regulator of lymphocyte-specific gene expression.

Early B-cell factor (EBF) was identified previously as a tissue-specific and differentiation stage-specific DNA-binding protein that participates in the regulation of the pre-B and B lymphocyte-specific mb-1 gene. Partial amino acid sequences obtained from purified EBF were used to isolate cDNA clones, which by multiple criteria encode EBF. The recombinant polypeptide formed sequence-specific complexes with the EBF-binding site in the mb-1 promoter. The cDNA hybridized to multiple transcripts in pre-B and B-cell lines, but transcripts were not detected at significant levels in plasmacytoma, T-cell, and nonlymphoid cell lines. Expression of recombinant EBF in transfected nonlymphoid cells strongly activated transcription from reporter plasmids containing functional EBF-binding sites. Analysis of DNA binding by deletion mutants of EBF identified an amino-terminal cysteine-rich DNA-binding domain lacking obvious sequence similarity to known transcription factors. DNA-binding assays with cotranslated wild-type and truncated forms of EBF indicated that the protein interacts with its site as a homodimer. Deletions delineated a carboxy-terminal dimerization region containing two repeats of 15 amino acids that show similarity with the dimerization domains of basic-helix-loop-helix proteins. Together, these data suggest that EBF represents a novel regulator of B lymphocyte-specific gene expression.

Abnormal κB-binding protein in the cytoplasm of a plasmacytoma cell line that lacks nuclear expression of NF-κB

The transcription factor NF-κB appears to play an important role in immunoglobulin gene expression and lymphokine production, and may play a role in primary B cell activation. Constitutive nuclear expression of NF-κB has been found in all mature B cell lines with the notable exception of the murine plasmacytoma, S107. We report herein that S107 cells express cytoplasmic κB-binding material detected by electrophoretic mobility shift assay that by several criteria represents authentic NF-κB. Despite the presence of cytoplasmic NF-κB, several stimuli known to induce nuclear translocation of NF-κB failed to do so in S107 cells, including: the PKC agonist, PMA; the protein synthesis inhibitor, cycloheximide; and LPS. Transfection of S107 cells with a κB-CAT reporter gene construct confirmed the absence of functional activity. Importantly, a global failure of nuclear transcription factor expression was ruled out by the ability of PMA to induce nuclear expression of another trans -acting factor, AP-1. Thus, rather than lacking NF-κB altogether, S107 cells manifest disordered regulation of NF-κB in which cytoplasmic material is incapable of translocation to the nucleus. While Northern analysis failed to reveal a gross defect in the mRNA coding for the DNA binding subunit of NF-κB, UV-photo-cross-linking followed by denaturing gel electrophoresis demonstrated the presence of a cytoplasmic κB-binding protein of abnormally elevated molecular size. This finding suggests that the abnormal regulation of NF-κ B in S107 cells is associated with the appearance of an unusual κB-binding molecule.

Macrophage colony‐stimulating factor gene transfer into tumor cells induces macrophage infiltration but not tumor suppression

In order to analyze the effect of a high local concentration of macrophage colony-stimulating factor (M-CSF; CSF-1) on tumor growth, the plasmacytoma cell line J558L was transfected with the human M-CSF gene and injected into syngeneic BALB/c mice. In contrast to the parental tumors, M-CSF transfectants were heavily infiltrated by macrophages as evidenced by immunohistochemistry with antibodies to Mac-1 and Mac-3 and by isolation of the macrophages from the tumor. Nevertheless, tumor growth was only slightly affected by M-CSF and M-CSF-producing cells grew as tumor in all cases. The growth retardation of M-CSF-producing cells varied depending on the experiment and seemed to be due to an indirect effect because the growth rate of the cells in vitro had not changed upon gene transfer. Attempts to activate the tumor-infiltrating macrophages for tumor suppression by systemic application of interferon-gamma and/or lipopolysaccharide were not successful. Altogether, our results suggest that M-CSF is a potent chemoattractant for macrophages in vivo but alone is not sufficient to activate these macrophages for tumoricidal activity.

Regulation of IL-6 receptor and GP130 expression on human cell lines of lymphoid and myeloid origin

The expression of 80 kDa interleukin-6 receptor (IL-6R) and the associated molecule gp130 has been studied on human cell lines by FACS- and Northern blot analysis. The effects of dexamethasone, dibutyric-(DB)-cAMP and phorbol-12-myristate-13-acetate (TPA) have been studied on plasmacytoma cell line U266, B cell line BMNH and monocytoid cell line U937. Our data show a definite downregulation of IL-6R and gp130 expression by TPA in U266 and BMNH at both mRNA and cell surface protein levels. In U937 TPA inhibits only the IL-6R expression, without affecting that of gp130. DB-cAMP decreases the expression of both proteins in U937, slightly inhibits the IL-6R expression in U266, but is uneffective in BMNH. Dexamethasone induces considerable upregulation of gp130 only in U266. Our findings suggest separate regulation of IL-6R and gp130 on U266, BMNH and U937 cell lines.

Use of IL-2 gene transfer in local immunotherapy of cancer

Insertion of functional interleukin-2 (IL-2) gene into a plasmacytoma cell line X63-Ag8.653 substantially reduced tumorigenicity of the resulting cloned cells, designated as X63-m-IL-2. Peritumoral administration of the X63-m-IL-2 cells, producing constitutively large quantities of IL-2, resulted in regressions of established X63-Ag8.653 plasmacytomas growing in the peritoneal cavity of syngeneic mice. In vitro activation of BALB/c spleen cells by co-culture with X63-m-IL-2 cells or their supernatants gave rise to cytotoxic lymphocytes with lymphokine-activated killer (LAK) activity against syngeneic X63-Ag8.653 plasmacytoma and other tumor targets. In contrast, peritumoral administration of X63-Ag8.653 cells carrying an inserted interleukin-4 (IL-4) gene (designated X63-m-IL-4 cells) and producing constitutively large quantities of IL-4 did not result in a therapeutic effect. Moreover, the admixture of the X63-m-IL-4 and X63-m-IL-2 cells substantially diminished the X63-m-IL-2 cell-mediated therapeutic effect. Similarly, IL-4-containing supernatants generated from X63-m-IL-4 cell cultures substantially diminished LAK activation by X63-m-IL-2 cell produced supernatants.

Lack of a role of interleukin 11 in the growth of multiple myeloma

Interleukin (IL)11 is a recently described lymphokine which, like IL-6, stimulates normal hematopoietic murine and human hematopoietic progenitor cells and therefore has potential value for either enhancing hematopoiesis in disease states or augmenting hematopoietic recovery after myeloablative theorapies. Since IL-6 is known to promote the growth of human myeloma, either in an autocrine or paracrine fashion, we examined the effect of IL-11 on the growth of a murine plasmacytoma cell line, human myeloma-derived cell lines, and freshly isolated human myeloma cells. Interleukin 11 does increase DNA synthesis by the murine plasmacytoma line T10 in the presence of neutralizing antibody to IL-6. However, neither human myeloma cells nor derived cell lines express IL-11 mRNA; secrete IL-11; express IL-11 cell surface receptors; or augment either DNA synthesis or Ig secretion in response to exogenous IL-11. These findings strongly suggest that IL-11 does support the growth of a murine plasmacytoma cell line but does not play a role in the growth of either freshly isolated human myeloma cells or derived cell lines.

Interleukin-11: A novel stroma-derived cytokine

Interleukin-11 (IL-11) is a novel stroma-derived cytokine that acts on both hematopoietic progenitors and stromal cells. IL-11 was originally identified in a medium conditioned by the macaque bone marrow-derived stromal cell line PU-34 and cloned as a growth factor for the IL-6-dependent plasmacytoma cell line T1165. IL-11 stimulates T-cell dependent development of antibody-producing B cells and is synergistic with IL-3 to stimulate megakaryocyte colony formation. Adipogenesis inhibitory factor (AGIF) was cloned from the human bone marrow-derived stromal cell line KM-102. The AGIF cDNA sequence was revealed to be identical to that of the IL-11 cDNA. AGIF inhibits the process of adipogenesis of the bone marrow-derived preadipocyte cell line H-1 A . Other biological activities such as stimulation of stem-cell proliferation, erythropoiesis, lymphohematopoiesis and hepatic acute-phase response are also summarized. The human IL-11 gene consists of five exons and four introns, and was mapped on chromosome 19 at band 19q13.3-q13.4. A single class of high-affinity IL-11 receptor (IL-11R) of 151 kDa is present on 3T3-L1 preadipocytes. A protein-tyrosine kinase pathway may be involved in the initiation of the IL-11R-mediated signal transduction.

The cloning and biological characterization of recombinant human interleukin 11.

Interleukin 11 (IL-11) is a pleiotropic hematopoietic growth factor with stimulatory effects on multiple hematopoietic progenitor cells. An immortalized primate bone marrow stromal cell line was established to facilitate the analysis of interactions between hematopoietic progenitors and the microenvironment. This line was found to produce a novel growth factor that directed the proliferation of a murine plasmacytoma cell line. A cDNA encoding this activity was isolated through functional expression cloning in mammalian cells. Preliminary characterization of the cytokine revealed that IL-11 stimulated T cell-dependent B cell immunoglobulin secretion and synergized with IL-3 in murine megakaryocyte formation. Subsequent analysis of the clone has demonstrated that it also synergizes with IL-3 and steel factor in the stimulation of early progenitors and affects early megakaryocyte formation and maturation. In addition, IL-11 induces secretion of acute phase proteins in the liver and may function in the hematopoietic microenvironment as an adipogenic antagonist in a paracrine manner. Further in vitro and in vivo analysis of IL-11 will be necessary to determine the biologic function and potential therapeutic use of the cytokine.

Interleukin 7 induces CD4+ T cell-dependent tumor rejection.

The potential of interleukin 7 (IL-7) to induce an antitumor response in vivo was analyzed. Therefore, the IL-7 gene was expressed in the plasmacytoma cell line J558L. Although the growth of IL-7-producing cells was not retarded in vitro, the IL-7-producing cells were completely rejected upon injection into mice. Tumor rejection was observed only in syngeneic but not in nude mice. The tumor-suppressive effect could be abolished by the parallel injection of an anti-IL-7 monoclonal antibody. Immunohistochemical analysis revealed IL-7-dependent infiltration of the tumor tissue by CD4+ and CD8+ T lymphocytes, and also type 3 complement receptor-positive (CR3+) cells, predominantly macrophages. Depletion of T cell subsets in tumor-bearing mice showed the absolute dependence of the antitumor response on CD4+ cells, whereas tumor rejection was unaffected by depletion of CD8+ cells. In addition to CD4+ cells, CR3+ cells were also needed for tumor rejection. The antitumor effect of IL-7 was confirmed by expression of the IL-7 gene in a second tumor cell line of different cellular origin. Together, our results demonstrate that a high local IL-7 concentration at the tumor site obtained by tumor cell-targeted gene transfer leads to tumor rejection involving a cellular mechanism that seems to be different from the ones observed in analogous experiments with other cytokines.

Effects of sodium butyrate on the rearranged c- myc expression in mouse plasmacytoma cells

The expression of c- myc mRNA was examined after 4 h of sodium butyrate treatment in a mouse plasmacytoma (MPC) cell line (S194). Steady-state levels of rearranged c- myc mRNA were suppressed by the agent in S194 cells. Run-on assay demonstrated that the suppression of the rearranged c- myc mRNA in the MPC was correlated with the transcriptional downregulation of the gene. The suppression was also accompanied by the reduced DNase I sensitivity of the gene. These findings suggest that the rapid downregulation of c- myc mRNA by sodium butyrate is subject to regulation at the transcriptional level following the alteration of the DNase I sensitive chromatin structure in mouse plasmacytoma cells.

Efficient gene transfer and expression in primary B lymphocytes

In this study we have used a panel of vectors expressing the chloramphenicol acetyltransferase (CAT) reporter gene under the control of different regulatory elements to optimize gene transfer and expression in primary B lymphocytes. The Moloney murine leukemia virus long terminal repeat (MoMLV LTR) and the SV40 early region promotors, while functional in transfected plasmacytoma cell lines, did not give rise to detectable CAT activity following transfection into primary activated mouse or human B lymphocytes. In contrast, the human cytomegalovirus immediate-early (HCMV-IE) enhancer/ promoter functioned in both established and primary B cells. The highest expression levels in the primary cells were obtained with vectors containing the Adenovirus 2 major late promoter or the HCMV-IE enhancer/promoter in combination with the Adenovirus 2 tripartite leader and VA genes. These latter expression cassettes were placed in a retrovival vector with the aim of combining their capacity for high-level gene expression with the efficient stable gene transfer afforded by retroviral infection. Several retroviral constructs were made, some of which were able to generate high virus titers. However all of these underwent deletions during the process of retroviral iifection, as judged by Southern analysis of infected cells, indicating that they were not optimal gene transfer vectors. The HCMV enhancer/promoter, which was the most active of the other expression cassesttes tested in the primary B cells, was inserted into a retroviral vector which also expressed the hph gene under the transcription control of the retroviral LTR. This vector did not undergo rearrangement during the process of retroviral infection, as judged by Southern analysis. The CAT gene was inserted downstream of the HCMV promoter in this vector, and a high-titer retroviral stock was generated. Primary B lymphocytes infected with this vector gave high levels of CAT activity, under conditions in which parallel experiments with the hph drug resistance marker showed that one in 20 of the cells were infected. These experiments demonstrate efficient gene transfer and expression in primary B lymphocytes in vitro.

N-linked oligosaccharides of the murine transferrin receptor from a plasmacytoma cell line. Comparison with total cellular N-glycans

The N-linked oligosaccharides synthesised by the murine plasmacytoma cell line NS-1 have been analysed by lectin affinity chromatography on columns of immobilised concanavalin A (Con A), Lens culinaris (lentil), Ricinus communis agglutinin (RCA) and leuko-phytohemagglutinin (L-PHA). The majority of complex N-glycans in this transformed cell line were branced structures with only a low level of biantennary complex chains detected. The analysis showed the major complex N-glycan fraction consisted of a minimum sialylated triantennary structure. [ 3H]Mannose-labelled transferrin receptor was isolated from NS-1 cells by immunoprecipitation followed by electroelution from SDS polyacrylamide gels. The isolated receptor was digested with Pronase and the 3H-labelled glycopeptides analysed by lectin affinity chromatography. Analysis by Con A-Sepharose indicated that approx. 50% of the labelled glycopeptides were branced complex N-glycans (unbound fraction) while the remainder were oligomannose structures (strongly bound). The presence of tri and/or tetraantennary structures in the Con A unbound fraction was further suggested by the interaction of 61% of the fraction with L-PHA. The lectin profiles obtained for the complex N-glycans of the transferrin receptor glycopeptides were similar to those for the total cellular glycopeptides of NS-1 cells. Reverse-phase HPLC analysis of tryptic glycopeptides of the isolated [ 3H]mannose-labelled transferrin receptor gave three 3H-labelled peaks, indicating that all three potential N-glycosylation sites on the receptor are utilised. The Con A-Sepharose profiles of the three fractions indicated the presence of branched complex N-glycans and high mannose chains at each site. The profiles of two of the tryptic glycopeptide fractions were very similar, while the third had a higher content of oligomannose oligosaccharides.

Molecular cloning of a cDNA encoding interleukin 11, a stromal cell-derived lymphopoietic and hematopoietic cytokine.

Hematopoiesis occurs in close association with a complex network of cells loosely termed the hematopoietic microenvironment. Analysis of the mechanisms of microenvironmental regulation of hematopoiesis has been hindered by the complexity of the microenvironment as well as the heterogeneity of hematopoietic stem cells and early progenitor cells. We have established immortalized primate bone marrow-derived stromal cell lines to facilitate analysis of the interactions of hematopoietic cells with the microenvironment in a large animal species. One such line, PU-34, was found to produce a variety of growth factors, including an activity that stimulates the proliferation of an interleukin 6-dependent murine plasmacytoma cell line. A cDNA encoding the plasmacytoma stimulatory activity was isolated through functional expression cloning in mammalian cells. The nucleotide sequence contained a single long reading frame of 597 nucleotides encoding a predicted 199-amino acid polypeptide. The amino acid sequence of this cytokine, designated interleukin 11 (IL-11), did not display significant similarity with any other sequence in the GenBank data base. Preliminary biological characterization indicates that in addition to stimulating plasmacytoma proliferation, IL-11 stimulates the T-cell-dependent development of immunoglobulin-producing B cells and synergizes with IL-3 in supporting murine megakaryocyte colony formation. These properties implicate IL-11 as an additional multifunctional regulator in the hematopoietic microenvironment.

Mouse plasmacytoma growth in vivo: enhancement by interleukin 6 (IL-6) and inhibition by antibodies directed against IL-6 or its receptor.

Murine plasmacytomas show a striking dependence on interleukin 6 (IL-6) for their growth in vitro. Here, we present evidence suggesting that IL-6 also plays an essential role in the in vivo development of these tumors. This conclusion is based on the finding that the tumorigenicity of an IL-6-dependent plasmacytoma cell line was increased approximately 100-fold on transfection with an IL-6 expression vector, whereas it was inhibited in animals treated with monoclonal antibodies capable of blocking the binding of IL-6 to its receptor. Injection of these antibodies 1 d before tumor challenge protected greater than 50% of the mice and retarded tumor growth in all animals. Tumors arising in antibody-treated mice retained their IL-6 dependence in vitro, suggesting that the level of protection could be improved if stronger IL-6 antagonists were available.

Studies on monoclonal anti-isotypic and anti-idiotypic antibodies against leukemia and myeloma: I. study on monoclonal anti-idiotypic antibodies against λ chain protein of myeloma

We used lambda chain extracted from urine of patient with myeloma (IgD lambda) as antigen for immunizing BALB/C mice, and 86 hybridoma cell clones secreting monoclonal antibodies (McAb) were obtained after fusing twice and cloning 3-4 times. 15 of these clones secreted monoclonal anti-idiotypic antibodies (anti-Id McAb). The results showed that 12 of 15 anti-Id antibodies reacted only with homologous lambda chain and IgD, not with lambda chain, kappa chain, IgG, IgA, IgM, IgD, IgE, albumin and paraglobulin from normal subjects. Indirect immunofluorescent assay demonstrated that positive reaction rate between anti-Id McAbs and peripheral blood lymphocytes or bone marrow cells of the patient with myeloma was up to 23%. No reaction was observed between anti-Id McAb and peripheral blood lymphocytes or bone marrow cells from normal subjects. Some of these McAbs presented positive reaction with plasmacytoma cell lines cultured in our laboratory.