Mouse Plasmacytoma Cells Research Articles

TPA induces apoptosis in MPC‐11 mouse plasmacytoma cells grown in serum‐free medium

Apoptotic‐like events could be rapidly induced by the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) in cells of the mouse plasmacytoma cell line MPC‐11 grown in serum‐free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so‐called ‘DNA ladder’. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin‐positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC‐11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.

Active suppression of the class II transactivator-encoding AIR-1 locus is responsible for the lack of major histocompatibility complex class II gene expression observed during differentiation from B cells to plasma cells.

In this study the genetic control of major histocompatibility complex (MHC) class II gene expression during the transition from B cell to plasma cell has been analyzed. Class II molecules are not expressed in plasma cells because of an active suppression resulting in the abrogation of class II gene transcription. We show here that the plasma cell-specific repressor function, designated SIR (suppressor of immune response genes), does not act directly on the transcription of class II genes, but instead on the transcription of the AIR-1 gene, whose product, the class II transactivator (CIITA), is fundamental for the regulation of the constitutive and inducible expression of MHC class II genes. This was unambiguously demonstrated by the fact that plasmacytoma x B cell hybrids carrying an AIR-1 locus derived from CIITA-expressing cells do not express CIITA-specific transcripts. Transfection of a cDNA containing the human CIITA coding sequence under the control of an heterologous promoter restores expression of human MHC class II genes in the hybrids and is responsible for de novo expression of mouse MHC class II genes in both the mouse plasmacytoma cell line and the hybrids. These results confirm and extend the notion of the functional conservation of the AIR-1 gene product across species barriers. Interestingly, in CIITA-transfected cell hybrids, cell surface expression of the human HLA-DQ heterodimer was not observed. This result was not attributable to lack of HLA-DQ alpha or -DQ beta transcription, because both transcripts were present in the CIITA-transfected hybrids, although at reduced levels. These findings further support our previous observations on the distinct regulation of expression of the human HLA-DQ class II subset, which may be thus controlled at the posttranscriptional level by a CIITA-independent mechanism.

Suppression of Lck protooncogene expression in murine somatic cell hybrids between T lymphoma cells and fibroblasts

Somatic cell hybrids were obtained by cell fusions between Lck-positive EL4 mouse T lymphoma cells and Lck-negative B82 mouse fibroblasts of S194 mouse plasmacytoma cells to examine negative control of lck gene expression in the resulting hybrids. Western blot analysis using a monoclonal antibody against the Lck protein showed a marked decrease in p56lck expression in B82 x EL4 (BEL) hybrids. In contrast to BEL hybrids, the level of p56lck was not changed significantly in S194 x EL4 (SEL) hybrids and was approximately one-half of that seen in EL4 cells. Diminished expression of the Lck protein in BEL hybrids paralleled downregulation of lck mRNA, which was exclusively transcribed from the distal promoter in EL4 cells. It is unlikely that the suppression was simply a consequence of chromosome segregation critical for lck gene expression, since BEL hybrids retained the EL4-derived lck gene and most of the chromosomes from both parental cells. The results from treatment of BEL hybrids with actinomycin D or cycloheximide suggested that suppression of lck gene expression in the hybrids might not be due to posttranscriptional control. DNA methylation status in the lck distal promoter and the coding regions did not appear to correlate with the expression of the gene. Our results suggest that negative control of lck gene expression differs between fibroblasts and B cells, in that lck gene expression in T cells can be shut down by transfer of a putative repressor factor or factors in fibroblasts but not in B cells.

Aberrant regulation of the IgH 3′ enhancer by c-myc in plasmacytoma cells

The identification of enhancers at the 3′ end of the IgH locus has prompted a re-evaluation of the regulation of Ig gene expression. Moreover, these elements may provide a possible explanation as to how the c-myc oncogene becomes dysregulated upon translocation into the IgH locus with the IgH intragenic enhancer on the reciprocal chromosome. These 3′ enhancers have also been shown to redirect promoter utilization on c-myc reporter gene constructs in transient transfection experiments. This region of the locus also contains the B cell specific IgH 3′ enhancer. This temporally regulated enhancer has been implicated in the mechanisms that control class switch recombination. Here we demonstrate that overexpression of the c-myc protein in mouse plasmacytoma cells (MPC-11) and HeLa cells can transcriptionally upregulate a reporter gene construct driven by a subregion (domain C) of the IgH 3′ enhancer. Domain C contains a functional dual symmetry E-box motif, CACGTG. The DNA binding experiments demonstrate that USF was the major factor interacting with this motif. Based on these observations, we speculate that the c-myc protein may upregulate expression of translocated c-myc in mouse plasmacytomas possibly via an USF-binding E-box motif in the IgH 3′ enhancer.

Scintigraphic imaging of oncogenes with antisense probes: does it make sense?

Based on the specificity of the Watson-Crick base pairing formation, antisense deoxyoligonucleotides have been used to inhibit the expression of oncogenes in various cancer cells. Activation of an oncogene by means of amplification leads to an increased, detectable amount of the mRNA transcript in the cytoplasm. The aim of this study was to demonstrate that cells which are expressing a particular mRNA transcript do preferentially and specifically retain the antisense probe targeting that mRNA. Using a mouse plasmacytoma cell line (MOPC315) which produces high levels of IgA heavy chain mRNA, a control mouse pre B cell line (7OZ/3B), a human mammary cell line (MCF7) which expresses the erbB2 or neu oncogene, MOPC315 cells as neu-negative controls, and antisense DNA oligonucleotides complementary to the 5' region of the mRNAs and the sense sequence, we have shown that there is a preferential, specific retention of the IgA and neu antisense sequence in MOPC315 and MCF7 cells, respectively. We have further demonstrated that this retention is time and concentration dependent with a maximum at 24 h. We conclude that cancer cells which express a particular oncogene are suitable targets for radiolabeled antisense deoxyoligonucleotides directed toward the oncogene transcript. This work and recent developments in the antisense field lead to the expectation of a new class of radiopharmaceuticals with unique specificity.

Okadaic acid co‐induces vimentin expression and cell cycle arrest in MPC‐11 mouse plasmacytoma cells

The effect of the tumor promoter okadaic acid on cell cycle progression and on vimentin expression in MPC-11 mouse plasmacytoma cells was compared with that of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Cell cycle progression of asynchronously grown MPC-11 cells was inhibited by both agents, but, in contrast to the G1 phase arrest caused by TPA, okadaic acid gave rise to G2/M phase and S phase arrest. This effect of okadaic acid was delayed significantly compared to the TPA-caused arrest. Furthermore, okadaic acid was able to induce vimentin expression to an extent comparable to the TPA response. However, vimentin expression was markedly delayed in okadaic acid-treated relative to TPA-treated cells. Another protein phosphatase inhibitor, calyculin A, also induced cell cycle changes and vimentin expression at concentrations at or above 1 x 10(-9) M. Based on these observations, we suggest an involvement of protein phosphatase 1 (possibly also phosphatase 2A and/or other phosphatases) in both the G2/M cell cycle block and the induction of vimentin expression in MPC-11 cells by okadaic acid.

Structure-Function Relationships in Human Interleukin-11: IDENTIFICATION OF REGIONS INVOLVED IN ACTIVITY BY CHEMICAL MODIFICATION AND SITE-DIRECTED MUTAGENESIS

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met58, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met58 is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys41 and Lys98, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met58, Lys41 and Lys98 are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modifications. By analogy with human growth hormone, we postulate that Met58 and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys41 and Lys98 may be a part of binding site II.

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.

High resolution analysis of cell cycle‐correlated vimentin expression in asynchronously grown, TPA‐treated MPC‐11 cells by the novel flow cytometric multiparameter BrdU‐hoechst/PI and immunolabeling technique

High resolution, multiparameter analysis using the flow cytometric BrdU/Hoechst quenching technique has been applied to study cell cycle kinetics and vimentin expression in individual cells of asynchronously grown MPC-11 mouse plasmacytoma cell cultures treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) to induce in vitro differentiation. BrdU treatment up to 16 h in the absence or presence of TPA did not affect either cell cycle progression or the kinetics or quantity of vimentin expression. TPA-treated cells became arrested in G1 phase of the second cell cycle; however, this G1 phase arrest was transient only. In addition, G1 phase cells located prior to a putative transition point at the beginning of TPA treatment were completely blocked in cell cycle progression. There is also evidence that cells located in G1 or G2/M phase at the beginning of TPA treatment finally expressed low levels of vimentin. On the contrary, cells located in S phase at TPA exposure showed high vimentin levels after treatment. The results presented here show that, with the flow cytometric BrdU/Hoechst quenching technique, one can correlate time-dependent protein expression at the single cell level in asynchronously grown cultures not only with the actual cell cycle state, but also with the history of cell replication.

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.

Identification and characterization of a soluble form of the plasma cell membrane glycoprotein PC-1 (5'-nucleotide phosphodiesterase).

PC-1 is a membrane glycoprotein, found on the surface of plasma cells and a few types of nonlymphoid cells, which has recently been found to have 5'-nucleotide phosphodiesterase activity. In this paper, we demonstrate the existence of enzymically active water-soluble forms of PC-1 in ascites from plasmacytoma-bearing mice, normal mouse serum, and in supernatants of cultured mouse plasmacytoma cells and mouse L cells transfected with a cDNA encoding the membrane form of PC-1. The water-soluble enzyme activity can be specifically immunoprecipitated by a monoclonal antibody to an allotypic determinant on the membrane form of PC-1, and resides on a slightly smaller polypeptide than membrane PC-1. Biosynthetic studies revealed a single, monomeric, endoglycosidase-H-sensitive membrane PC-1 precursor, which was gradually converted to a disulphide-bonded, endoglycosidase-H-resistant form over a period of about 2 h. Soluble PC-1 was first detectable in the supernatant after about 2 h. A distinct intracellular form of soluble PC-1 was not seen. The soluble form of PC-1 does not appear to arise by proteolytic cleavage from the cell surface, although cleavage inside the cell remains a possibility. When taken together with the structure of the relevant portions of PC-1 gene exons, the data suggest that the most likely site of cleavage is between Pro152 and Ala153.

Effect of arachidonic acid and hydrocortisone on the intracellular concentration of free Ca2+ ions in JW mouse plasmacytoma cells

Effect of arachidonic acid and hydrocortisone on the intracellular concentration of free Ca2+ ions in JW mouse plasmacytoma cells

Reactivation of a major histocompatibility complex class II gene in mouse plasmacytoma cells and mouse T cells.

Terminally differentiated plasma cells and mouse T cells do not express major histocompatibility complex (MHC) class II genes although class II gene expression is observed in pre-B and mature B cells as well as in activated human T cells. Transient heterokaryons were prepared and analyzed to investigate the mechanisms of inactivation of MHC class II gene in mouse plasmacytoma cells and mouse T cells. The endogenous MHC class II genes in both mouse plasmacytoma cells and mouse T cells can be reactivated by factors present in B cells. This reactivation of class II gene is also observed by fusion with a human T cell line which expresses MHC class II genes, but not with a class II negative human T cell line. It appears that the loss of MHC class II gene expression during the terminal differentiation of B cells or T cell lineage is due to absence of positive regulatory factor(s) necessary for class II transcription.

Immunoglobulin kappa gene enhancers synergistically activate gene expression but independently determine chromatin structure.

Previous studies have located transcriptional enhancer elements within both the intron and 3'-region of the mouse kappa immunoglobulin gene. Here we address the role of these two enhancers in specifying gene activity and specific chromatin structures. MOPC41 kappa gene constructs, either intact or containing deletions of one or both enhancers, were introduced into S194 mouse plasmacytoma cells for transient and stable expression studies. Transient expression assays revealed that the basal level expression exhibited by enhancerless constructs was activated 100-200-fold by the two enhancers together in a synergistic fashion. A similar trend was observed when both enhancers were present in stably integrated constructs, although the synergy was less pronounced. Analysis of DNase I hypersensitive sites in the chromatin revealed that stably integrated constructs established hypersensitive sites about the enhancer sequences. These sites demonstrated the same nuclease susceptibility as those associated with the endogenous gene(s), and their establishment was independent of the presence of the other enhancer. Thus, although both enhancers are required for maximal gene expression, the elements act independently in determining specific chromatin structures.

Preparation and characterization of a murine monoclonal antibody (MDR3M) reactive with mdr3 gene product.

A murine monoclonal antibody (MDR3M) (isotype: IgM) reactive with mdr3 gene product was generated by immunizing mice with mdr3‐specific peptide (H2N‐12WRPTSAEGDFELGISSKQKRKKTKTVKMI41G‐COOH) and hybridizing the primed mouse splenic B cells with X63‐Ag8,6.5.3 mouse plasmacytoma cells. MDR3M did not cross‐react with mdr1 gene product. This monoclonal antibody may be useful for analyzing the role of mdr3 gene product in cells and tissues.

Cell cycle-dependent vimentin expression in elutriator-synchronized, TPA-treated MPC-11 mouse plasmacytoma cells

We correlated cell cycle progression and vimentin expression at the single cell level by multiparameter flow cytometry in populations of MPC-11 cells enriched in different cell cycle phases by centrifugal elutriation and subsequently treated with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Synchronized, untreated cultures showed a uniform, synchronous progression through the cell cycle during further cultivation. A 6-h TPA treatment of G1-phase-enriched cultures induced both a partial G1-phase arrest in the same cycle and a moderate fraction of cells to become vimentin positive. However, nearly all cells of the cultures enriched in S- or in G2/M-phase cells could be arrested by TPA treatment at the earliest in the G1 phase of the second cell cycle and displayed higher fractions of positive cells as well as higher average levels of vimentin. After 20 h of treatment, the G1-phase arrest was almost complete. In terms of fractions of vimentin-positive cells as well as of average cellular vimentin content, the differences between the cultures resembled, albeit on a higher level, those between the respective cultures treated with TPA for 6 h. These observations might explain the striking bimodal distribution of individual cellular vimentin content detectable in G1-phase fractions of asynchronous, TPA-treated cultures. The pattern of vimentin mRNA accumulation in synchronized cultures after short-term TPA treatment strongly suggests that the cell cycle-dependent pattern of vimentin expression is caused, at least in part, by different levels of vimentin mRNA accumulated in the cells. Since proteinaceous mediator(s) are obviously involved in TPA-induced vimentin expression in MPC-11 cells, cell cycle-dependent vimentin expression in these cells may be dependent on cell cycle-dependent regulation of the activity and/or concentration of such mediator(s).

Monoclonal Antibody AP-282 Recognizes a Marker for Human Polymorphonuclear Granules

Hybridoma AP-282 was produced by fusing mouse plasmacytoma cells with splenocytes of mice immunized against purified human polymorphonuclear cells. The secreted monoclonal antibody (MAb), AP-282, a mouse IgG1, was found to react strongly with all neutrophilic granulocytes, their bone marrow precursors, weakly with blood monocytes and not with eosinophils. The antigen was resistant to formalin fixation but was destroyed by exposure to fixatives containing acetic acid. Using the APAAP technique, antibody AP-282 strongly labelled neutrophils on sections of frozen cut or paraffin embedded tissues. No staining was seen of non hematopoietic tissues. AP-282 recognized an internal antigen associated to cytoplasmic granules. Chemical investigations on dot blots of whole or of purified cellular extracts indicated that the antigen idenfied by MAb AP-282 was different from those recognized by usual antigranulocyte antibodies, i.e. myeloperoxidase, elastase, cathepsin G and lactoferrin. Thus, antibody AP-282 constitutes a new cytoplasmic marker of neutrophils.

Inability of insulin and insulinlike growth factor-1 to stimulate sugar or amino acid transport and thymidine incorporation in cultured myeloma cells

NS-1 mouse plasmacytoma cells were examined for their insulin and insulinlike growth factor-1 (IGF-1) binding characteristics and ability to produce peptide-dependent cellular effects. At concentrations of labelled insulin (i.e., 1.7 x 10(-10) M) or IGF-1 (i.e., 1.5 x 10(-10) M), NS-1 cells specifically bind 0.2 +/- 0.06 fmol insulin per 10(6) cells (n = 7), where little, if any, IGF-1 specific binding was observed (0.02 +/- 0.01 fmol/10(6) cells) (n = 3). Additionally, the data indicate that the total number of insulin binding sites per cell was 3200 +/- 390 (n = 3). Insulin was employed at various concentrations (6.7-667 nM) and failed to stimulate either sugar or amino acid transport. Insulin at low concentrations (i.e., 6.7 or 67 nM) did not stimulate DNA synthesis, yet a small but significant increase was observed at a concentration of 667 nM insulin. IGF-1 did not stimulate DNA synthesis at all concentrations employed (1.4-143 nM). In summary, there exists a small but significant number of insulin receptors, little insulin-stimulated DNA synthesis, and no apparent insulin stimulation of sugar or amino acid transport. Also, since there is no significant IGF-1 binding and no IGF-1 stimulation of DNA synthesis, these findings indicate that this cell line might be a good candidate for the study of insulin receptor function as a transfection recipient of insulin receptor genes.

Re-transformation of non-transformed hybrids between c-myc-activating mouse plasmacytoma cells and normal fibroblasts by transfection with activated c-Ha-ras but not c-myc

In a mouse plasmacytoma S194, c-myc oncogene is rearranged with Ig gene by chromosomal translocation and is consequently activated. We previously reported that transformation of phenotype and expression of rearranged c-myc were repressed in independently isolated hybrid clones, I-1 and IV-10, between S194 and normal fibroblasts. In order to investigate the relationship between transformation of phenotype and oncogene expression, transcriptionally enhanced c-myc or activated c-Ha-ras was transfected into I-1 or IV-10I, a subclone of IV-10. Transfectants expressing high levels of c-myc were found to retain the non-transformed phenotypes. On the other hand, transfectants expressing activated c-Ha-ras showed the transformed phenotypes. These results suggest that enhanced expression of c-myc is not sufficient for re-transformation of the non-transformed hybrid clones between c-myc-activating plasmacytoma cells and normal fibroblasts, but expression of activated c-Ha-ras could diminish or overcome the tumor-suppressive activity of normal fibroblasts.

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.