Cultured Myeloma Cells Research Articles

A model of multiple myeloma: culture of 5T33 murine myeloma cells and evaluation of tumorigenicity in the C57BL/KaLwRij mouse.

The 5T33 multiple myeloma is one of a series of transplantable murine myelomas arising spontaneously in C57BL/KaLwRij mice. This study describes the establishment and characterisation of the 5T33 murine myeloma in vitro as a cultured cell line in terms of its morphology, growth rate, expression of paraprotein (IgG2b) and tumorigenicity in syngeneic animals. The 5T33 cell line has been in continuous culture for over 10 months and has achieved more than passage 34. In culture, 5T33 myeloma grows as single cells or in small clusters of loosely adherent cells on an adherent stromal cell layer. Maximum doubling time is approximately 25 h, and over 90% of the cells express cytoplasmic IgG2b paraprotein. The cultured 5T33 myeloma cells are highly tumorigenic in C57BL/KaLwRij mice with as few as 500 cells inducing paralysis and death as early as day 36 post-tumour inoculation. Kinetics of tumour development and detection of IgG2b paraprotein are dose dependent. Two weeks following intravenous inoculation of 5 x 10(5) cultured 5T33 myeloma cells, tumour cells were readily identified in the bone marrow. By 3 weeks post-tumour inoculation, 5T33 myeloma cells were found in various tissues throughout the animal. Studies are now underway to determine the sensitivity of this cell line to various therapeutic modalities.

Resistance of ADP-ribosylated histones and HMG proteins to proteases.

Calf thymus histones (individually isolated or mixtures) and high mobility group proteins were ADP-ribosylated in vitro using [32P]NAD+ and immobilized purified poly(ADP-ribose) polymerase. The modified histones were then subjected to V8 protease or alpha-chymotrypsin digestion and the resulting peptides were separated by electrophoresis on acetic acid-urea-Triton gels. It was found that in vitro ADP-ribosylated histones were much more resistant to proteases than unmodified histones. A similar approach was applied to histones modified by the endogenous poly(ADP-ribose) polymerase in permeabilized NS-1 mouse myeloma cells in culture. In this case, the proteases could not discriminate between modified and unmodified histones and putative mono(ADP-ribosyl)ated peptides appeared in a digestion frame corresponding to that of bulk peptides. These differences are most probably due to the specificity or number of ADP-ribose groups added to the histones by the endogenous or exogenous poly(ADP-ribose) polymerase. Thus, depending on the size of poly(ADP-ribose) attached to nuclear proteins, these modified proteins might display different degrees of resistance to proteolysis.

Evaluation of Lymphoid-Specific Enhancer Addition or Substitution in a Basic Retrovirus Vector

Two novel retroviral vectors bearing lymphoid-specific enhancers were tested for improved expression of human adenosine deaminase (hADA) in tissue culture cells and in mouse bone marrow transplant recipients. These vectors carried either an added human T-cell receptor alpha-chain enhancer (delta N2TADA) or a substitution of the Moloney long terminal repeat (LTR) enhancer with the murine immunoglobulin mu heavy-chain first intron enhancer (delta N2 mu ADA). Each vector was produced at a titer of approximately 10(6) infectious units/ml and efficiently transduced hADA into murine fibroblast and myeloma cells in culture. No quantitative difference in expression was observed between the enhancer modified vectors and the basic retrovirus vector (delta N2ADA). In addition, each vector efficiently conferred hADA expression in lymphoid, myeloid, and erythroid cells of long-term transplanted mice. The majority of the transduced-marrow recipients demonstrated expression of the human enzyme for 4-8 months with each of the three vectors.

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.

Characterization of an interleukin-6-mediated autocrine growth loop in the human multiple myeloma cell line, U266

It has been reported recently that freshly isolated human myeloma cell cultures proliferate in response to added interleukin-6 (IL-6). Endogenous levels of IL-6 found in the same cultures suggested that an autocrine growth loop may contribute to cell growth. However, the lack of homogenous cell populations in primary myeloma cultures has made it difficult to distinguish between paracrine and autocrine growth mechanisms. To precisely address the autocrine growth issue we have evaluated the growth of the human myeloma cell line, U266. We have found that a neutralizing anti-IL-6 monoclonal antibody can inhibit U266 proliferation. Furthermore, the addition of IL-6 antisense oligonucleotides also inhibits U266 proliferation. These effects are reversed by adding IL-6, suggesting the presence of an autocrine loop. Using bioassays with two different IL-6-dependent cell lines, we were able to detect IL-6 in concentrated U266 supernatants. IL-6 mRNA was detected by polymerase chain reaction amplification of cDNA. Cell cycle parameter analysis shows that IL-6 acts to release a block in G1. Taken together these results present conclusive evidence for IL-6-mediated autocrine growth in the U266 human myeloma cell line.

Characterization of an Interleukin-6-Mediated Autocrine Growth Loop in the Human Multiple Myeloma Cell Line, U266

It has been reported recently that freshly isolated human myeloma cell cultures proliferate in response to added interleukin-6 (IL-6). Endogenous levels of IL-6 found in the same cultures suggested that an autocrine growth loop may contribute to cell growth. However, the lack of homogenous cell populations in primary myeloma cultures has made it difficult to distinguish between paracrine and autocrine growth mechanisms. To precisely address the autocrine growth issue we have evaluated the growth of the human myeloma cell line, U266. We have found that a neutralizing anti-IL-6 monoclonal antibody can inhibit U266 proliferation. Furthermore, the addition of IL-6 antisense oligonucleotides also inhibits U266 proliferation. These effects are reversed by adding IL-6, suggesting the presence of an autocrine loop. Using bioassays with two different IL-6-dependent cell lines, we were able to detect IL-6 in concentrated U266 supernatants. IL-6 mRNA was detected by polymerase chain reaction amplification of cDNA. Cell cycle parameter analysis shows that IL-6 acts to release a block in G1. Taken together these results present conclusive evidence for IL-6-mediated autocrine growth in the U266 human myeloma cell line.

Detection of Interleukin-1β and Interleukin-6 in Human Multiple Myeloma by Fluorescent in situ Hybridization.

Using fluorescent in situ hybridization together with cell surface marker staining, we studied the expression of mRNA of IL-6 and mRNA of IL-1β in bone marrow samples from human multiple myeloma patients. It is known that IL-6 can stimulate B cell growth and differentiation and recently it has been suggested that IL-6 is responsible for autocrine growth stimulation of myeloma cells and that IL-1 may play a role in bone resorption. These interleukins have previously been detected in the supernatants of cultured myeloma cells. Here we report the expression of IL-1β mRNA by plasma cells, T cells and macrophages according to morphology and immunologic marker analysis, suggesting that not only myeloma cells but other cell types can also contribute to the production of IL-1β and thus to bone-resorption. IL-6 mRNA could not be detected in plasma cells from bone marrow aspirates but were present in monocytes and T cells, suggesting that in vivo IL-6 stimulates the growth of myeloma cells in a paracrine instead of an autocrine way.

Production of tumor necrosis factor (TNF-alpha) and lymphotoxin (TNF-beta) by murine pre-B and B cell lymphomas.

Media from murine pre-B and B lymphoma cell cultures, but not from myeloma cell cultures, was cytotoxic to WEHI 164 cells, causing these TNF-sensitive targets to release 51Cr. The cytotoxic activity in the culture medium reached maximum levels approximately 4 days after the cell culture was initiated. The constitutive production of the factors was not influenced by depletion of serum from the medium or by the addition of either phorbol ester or bacterial endotoxin. The factor has a Mr greater than 10 kDa, and its cytotoxicity was abolished by anti-serum against murine TNF. Northern blot analysis with the use of cDNA probes to murine tumor necrosis factor (TNF-alpha) and lymphotoxin (LT, TNF-beta) showed high levels of TNF-mRNA in the pre-B cell lines, lower levels in the mature B cell lines and no TNF-mRNA in the myeloma cell lines. LT mRNA was present in pre-B cell lines, at a much lower concentration in only one of the B cell lines, and was not present in three other B lymphomas or in the myelomas tested. The results show a positive correlation between the presence of TNF and/or LT mRNA and the 51Cr-releasing activity present in the cell culture medium. Our data indicate that TNF and LT can be produced by murine B cells and that the synthesis of these cytokines may be restricted to certain differentiation stages of the B cell lineage.

Hyperammonemia in Multiple Myeloma

Two patients with multiple myeloma who appeared to be producing ammonia are reported. Both patients showed hyperammonemia and amino acid disturbances, such as a low Fischer ratio. One patient had Bence Jones protein (lambda) type myeloma and became comatose, but the hyperammonemia and disturbance of consciousness were improved by chemotherapy for the myeloma. The other patient had IgA kappa type myeloma and somnolence and died of malignant pleurisy despite intensive chemotherapy. Autopsy showed widespread multiple myeloma and an almost normal liver. Ammonia levels in the supernatant of cultured myeloma cells from the patient's pleural effusion increased almost linearly from the time of cell seeding. These observations showed that ammonia was produced at a high level by these human myeloma cells. We also found that one of the common myeloma cell lines, RPMI 8226, could produce ammonia as well.

Elimination of mycoplasmas from mouse myeloma cells by intraperitoneal passage in mice and by antibiotic treatment.

Intraperitoneal passage in mice and antibiotic treatment were evaluated alone and in combination for elimination of mycoplasma contamination of mouse myeloma cell cultures. Intentional infections were established by inoculating Mycoplasma arginini, M. fermentans, M. hyorhinis and M. orale into cell cultures. Successful elimination of mycoplasmas was achieved with all strains tested by intraperitoneal passage in mice, however, cells infected with M. hyorhinis did not survive the infection long enough to be tested. Clindamycin and lincomycin cured cells infected with M. arginini, M. hyorhinis, M. orale but not M. fermentans. M. fermentans were resistant to all antibiotics tested, but could be partially suppressed by clindamycin long enough to permit curing by in vivo passage. M. arginini was eliminated by all antibiotics tested. In vivo passage and treatment with antibiotics is an efficient combination of methods for mycoplasma elimination from cell cultures and has the advantage of being simple and inexpensive.

Effects of a triazine antifolate (NSC 127755) on serine hydroxymethyltransferase in myeloma cells in culture

The effect of the triazine antifolate NSC 127755 on serine hydroxymethyltransferase activity in mouse myeloma X63 cells in culture was determined. The enzyme was inhibited, apparently irreversibly, with IC 50 ≈ 5 × 10 −8 M. A similar concentration-dependency for inhibition of [6- 3H]deoxyuridine incorporation into DNA was observed in these cells in culture. Long-term culture of myeloma cells with NSC 127755 resulted in a progressive decrease in the number of viable cells. The study emphasises the significance of serine hydroxymethyltransferase as a target for anticancer chermotherapy.

Amylase-producing multiple myeloma

The first autopsy case of amylase-producing IgA-lambda-type multiple myeloma is described. Immunohistochemically, amylase and alpha and lambda chains of immunoglobulin were demonstrated in the cytoplasm of the myeloma cells. Secretion of amylase by cultured myeloma cells obtained from the patient's pleural effusion was clearly demonstrated by the starch film method. Immunoelectron microscopically, positive reaction products for amylase and the alpha chain of immunoglobulin were observed in the well developed endoplasmic reticulum. Since no secretory granules were observed, we postulated that the secretory process of amylase was not via the zymogen granules but via a mechanism similar to that for immunoglobulin.

Ectopic production of salivary-type amylase by a lga-λ-type multiple myeloma

A patient with IgA-lambda-type multiple myeloma who appeared to be secreting salivary-type amylase ectopically is reported. Both the patient's serum and urine contained high levels of amylase. Amylase activity in the supernatant of cultured myeloma cells, obtained from the patient's pleural effusion while he had malignant pleuritis, increased almost linearly from the time of cell seeding. The presence of IgA and S-type amylase in the myeloma cells was demonstrated cytochemically and by immunoelectron microscopy. These observations showed that amylase was produced by these human myeloma cells.

Phycocyanin: laser activation, cytotoxic effects, and uptake in human atherosclerotic plaque.

Phycocyanin is a phycobiliprotein with peak absorption at 620 nm. The laser activation, cytotoxic effects, and uptake into atherosclerotic plaque of phycocyanin was studied. Optimal activation was produced by argon dye laser at 0.5 W and a total energy dose of 300 J/cm2 at 620 nm and 650 nm, irradiated through blood with a hematocrit of 8%. Activation was evidenced by reduction of optical density by 0.3 units at 340 nm caused by oxidation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) in a buffered reaction solution containing 0.1 mg/ml of phycocyanin. Cytotoxicity was evaluated by measuring viability of mouse myeloma cells in culture after incubation with phycocyanin (0.25 mg/ml) and irradiated by 300 J/cm2 at 514 nm. After 72 hours post-treatment the cells showed 15% viability compared to 69% and 71% for control cells exposed to laser only or phycocyanin only, respectively. Atherosclerotic artery segments obtained within 5 hours postmortem were perfused with 0.1 mg/ml phycocyanin in oxygenated Krebs Ringer solution at 30 mm Hg for 5 minutes followed by washout with phycocyanin-free Krebs for 10 minutes. Artery sections examined histologically by light and fluorescence microscopy showed specific fluorescence localization within the plaque particularly at the elastic laminae and to a larger extent at the internal elastic lamina but not in the medial muscle layer. In conclusion, phycocyanin is a cytotoxic photosensitizer that exhibits specific binding to plaque and is activated at a wavelength minimally absorbed by blood. These properties suggest potential therapeutic use for plaque localization and regression.

At least 60 ADP-ribosylated variant histones are present in nuclei from dimethylsulfate-treated and untreated cells.

The level of histone adenosine diphospho (ADP) ribosylation was studied in isolated nuclei from mouse myeloma cells in culture. The cells were treated with dimethylsulfate (DMS), a DNA-methylating agent, and histones were analyzed using two-dimensional gel electrophoresis. Seventeen or more bands probably representing mono-to heptadeca (ADP-ribosylated) histones could be visualized for each major variant histone. DMS treatment, by increasing the number of chromatin sites undergoing repair, greatly enhanced histone ADP-ribosylation. When histones were labeled in a cell lysate rather than in isolated nuclei, mono- and oligo(ADP-ribosylated) histone forms prevailed. The presence of approximately 87 ADP-ribosylated variant histone forms in cell lysates and of approximately 170 in isolated nuclei is shown for the first time in this work. Previous studies show multiple ADP-ribosylated forms for only histone H1. The theoretical number of variegated nucleosomes is thus much higher than previously thought, provided that histone-histone contacts are not disrupted at up to a certain level of histone ADP-ribosylation.

Cell-mediated toxicity of interleukin-2-activated lymphocytes against autologous and allogeneic human myeloma cells.

We studied the sensitivity of human myeloma (plasma cell leukemia) toward autologous and allogeneic lymphokine-activated killer (LAK) cells. Fresh plasma cell leukemia (PCL)-derived peripheral blood mononuclear cells (PBMC) and PBMC from 3 normal donors were cultured in the presence of recombinant interleukin-2 (rIL2; 1,000 U/ml) for subsequent use as cytotoxic effectors against fresh and continuously cultured myeloma cells. Target cell lysis was measured in a 4-hour 51Cr radioisotope release assay. At an effector to target (E:T) ratio of 50:1, rIL2-induced PCL-PBMC lysed 48 +/- 19% (mean +/- 1 SD) of autologous myeloma targets, as compared to 89 +/- 5, 95 +/- 15, and 100 +/- 9% lysis of standard LAK-sensitive Daudi cells and allogeneic myeloma cell lines SKO-007, and RPMI-8226, respectively. Normal PBMC-derived rIL2-induced (LAK) cells exhibited a slightly lower cytotoxic reactivity against allogeneic targets (61 +/- 9, 60 +/- 6, and 81 +/- 8% cytolysis of SKO-007, RPMI-8226, and Daudi cells, respectively, at a 50:1 E:T ratio). Cytotoxicity against myeloma (PCL) of autologous PCL-derived killer cells could be significantly (at least 2-fold) enhanced when rIL-2-induced effector cells were preincubated for 18 h in the presence of recombinant Interferon-alpha rIFN-alpha; 1,000 U/ml). In summary, our results indicate the potential antitumor efficacy of rIL2- and rIL2 + rIFN-alpha-activated killer cells in human myeloma (PCL).

Antitrichomonal activity of alpha-difluoromethylornithine.

alpha-Difluoromethylornithine, an inhibitor of polyamine biosynthesis, affected the pathogenicity of Trichomonas vaginalis in two model systems. Firstly, it blocked the cytotoxic effect of the parasite towards mammalian cells in culture: at a concentration of 1 mM it prevented the death of mouse myeloma cells in mixed culture with trichomonads. Secondly, when administered orally to mice (multiple doses of 750 mg/kg body wt), the drug delayed the development of subcutaneous abscesses due to T. vaginalis infection. The same drug dose did not cure intravaginal infections by the parasite. The implications of these findings with respect to the value of the model systems used and the potential of DFMO as an antitrichomonal drug are discussed.

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium.

A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterin-thymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.

CDNA cloning of the murine transferrin receptor: sequence of trans-membrane and adjacent regions.

We previously purified the murine transferrin receptor from cultured myeloma cells and determined the amino acid sequence of six tryptic peptides. We now report the cloning of cDNA encoding the murine transferrin receptor. A tryptic peptide containing a region of six consecutive amino acids, all encoded by four or fewer codons, was used to design two overlapping oligonucleotide probes, 17 and 14 nucleotides in length. These probes were used to screen a lambda gt10 cDNA library from NS-1 myeloma cells. Of approximately 400,000 plaques screened, two hybridized strongly to both probes. A subfragment of one clone that hybridized with both oligonucleotide probes was found to encode the tryptic peptide from which the probes were derived, as well as another sequenced tryptic peptide. Comparison of the sequence with that of the human transferrin receptor shows a high degree of conservation of the sequences surrounding and penetrating the membrane, including cysteine residues that may be involved in interchain disulfide bonding and/or covalent attachment of lipid. The current data, when combined with the published sequence of the human receptor, allow assignment of all six tryptic peptides to a single chain, supporting the idea that the receptor is a homodimer. A 4.9-kb messenger RNA was found in several cultured murine and human tumor cell lines, but transferrin receptor messenger RNA was not detectable in murine spleen. An additional RNA species of 2.7 kb was present in approximately equal abundance in the murine myelomas NS-1 and C118 but was absent from T lymphomas TIKAUT, ST-1, and ST-4.

Application of monoclonal antibodies in basic research, diagnosis, and therapy.

Monoclonal antibodies are the natural products of hybrid cells called hybridomas, which are obtained from fusion of cultured myeloma cells with spleen cells from an immunized animal. Monoclonal antibodies can exhibit high specificity and affinity for distinct determinants (epitopes) in low-molecular-weight compounds and macromolecules. The availability of an inexhaustible and easily reproducible source of such antibodies, which can be harvested from cell culture supernatants of permanent hybrid cell lines and animal ascites fluids, promises to advance considerably our knowledge of biology at the cellular, subcellular, and molecular levels. In the future, hybridoma technology should allow preparation of many well-characterized diagnostic tools and development of novel therapeutic reagents. This presentation provides an overview of the rapidly growing areas in which monoclonal antibodies are being implemented. A few specific examples illustrate their value in advancing our knowledge of biomedical systems.