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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The growth of myeloma cells in Leibovitz medium supplemented with 20% serum was limited by the depletion of glutamine. A simple modification of the Leibovitz medium by increasing the concentrations of glutamine, lysine, isoleucine, leucine, sodium pyruvate, galactose, and vitamins resulted in over 100% increase in cell growth yield. The total myeloma protein produced by the cells was increased by approximately 90% in modified Leibovitz media. Analysis of spent culture media for 19 amino acids showed that the concentrations of 8 amino acids were reduced; those of 5 amino acids were increased and the other 6 did not change significantly.
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PMID:Amino acid metabolism of myeloma cells in culture. 96 31

Hybridoma cells usually grow to fairly low cell densities in batch cultures (1-3 x 10(6) cells/ml). The reason for this is either that essential nutritional components of the medium are consumed, or that the cells produce some kind of inhibitory or toxic metabolite. This investigation presents evidence for the latter. Spent medium from cultures of hybridoma cells did not support growth of cells at lower cell densities (1-3 x 10(5) cells/ml). The ability to support cell growth could not be restored by adding additional serum, energy sources (glucose, pyruvate) or L-glutamine. Furthermore, the consumption of amino acids could not account for this growth inhibition. On the contrary, the spent medium contained a substance that inhibited cell growth. This substance or metabolite was found in a fraction eluted from a gel filtration column when spent medium was applied to the column. This substance was found in the spent medium from all hybridoma and myeloma cell lines that were tested. The molecular weight of the substance was about 5 kD. Spent medium from two hybridoma cell lines also contained a substance that was eluted in the same fraction as albumin (67 kD). It is likely that this (or these) substance(s) is responsible for the growth limitation in hybridoma cell cultures.
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PMID:Growth limitation in hybridoma cell cultures: the role of inhibitory or toxic metabolites. 136 98

The murine myeloma cell line Sp 2/0-Ag 14 was cultured in an ordinary batch culture and in a glutamine limited fed-batch culture. In batch culture, the overflow metabolism of glutamine ends in excess production of ammonium and the amino acids alanine, proline, ornithine, asparagine, glutamate, serine and glycine. This pattern was dramatically changed in the fed-batch culture. Glutamine limitation halved the cellular ammonium production and reduced the ratio of NH4+/glutamine. The excess production of alanine, proline and ornithine was reduced by a factor of 2-6 while asparagine was not produced at all. In contrary to the other amino acids glycine production was increased. These results are discussed in view of the different nature of glutamine metabolism in the mitochondrial compartment vs. the cytosolic. Furthermore, essential amino acids were used more efficiently in the fed-batch as judged by the increase in the cellular yield coefficients in the range of 1.3-2.6 times for seven of the 11 consumed ones. In all, this leads to a more efficient use of the energy sources glucose and glutamine as revealed by an increase in the cellular yield coefficient for glucose by 70% and for glutamine by 61%.
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PMID:Glutamine limited fed-batch culture reduces the overflow metabolism of amino acids in myeloma cells. 136 3

We report a method for introducing a glutamine synthetase (GS) selectable marker into myeloma cells in which transfectants are selected by growth in a glutamine-free medium. Vector amplification can subsequently be selected using the specific inhibitor of GS, methionine sulphoximine (MSX). Using this system, DNA sequences encoding a chimeric B72.3 IgG4 antibody were expressed from hCMV-MIE promoters in NSO myeloma cells. A cell line was isolated after a single round of selection for vector amplification which contains approximately 4 copies of the vector, secretes 10-15 pg/cell/day cB72.3 antibody during exponential growth and can accumulate 560 mg/l antibody in a fed-batch air-lift fermentation system. Productivity is stable in the absence of MSX selection.
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PMID:High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. 136 77

A cell line of plasma cells with high ammonia (NH3) production (KHM-4) was established from a patient with multiple myeloma complicated by hyperammonemia and abnormal serum concentrations of amino acids. Surface marker studies of KHM-4 cells showed that the cells were positive for cytoplasmic immunoglobulins (IgA kappa), HLA-DR, and T 10. Secretion of ammonia by the KHM-4 cells was detected by the addition of L-glutamine and L-arginine into the culture medium of amino acid-free RPMI 1640. In the presence of L-glutamine, KHM-4 cells secreted a greater amount of ammonia than the T cell line, CEM. However, production of ammonia by L-arginine was not observed in other cell lines. These observations provide evidence for the existence of a peculiar amino acid metabolism in the myeloma cells causing hyperammonemia and serum amino acid disturbance.
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PMID:Human myeloma cell line (KHM-4) established from a patient with multiple myeloma associated with hyperammonemia. 161 Nov 84

Alterations of ras, c-myc and bcl-1 have been described in hematologic malignancies of lymphoid origin. We investigated the structure of these genes and evaluated the frequency of point mutations involving H-, K- or N-ras in bone marrow samples from patients with multiple myeloma. No abnormalities were detected in the c-myc and bcl-1 genes, but two of 17 patients were found to have N-ras mutations by differential oligonucleotide hybridization and dideoxynucleotide sequencing following amplification by polymerase chain reaction. Bone marrow DNA from both patients had identical missense mutations of N-ras codon 61 changing CAA to AAA, resulting in a substitution of lysine for glutamine in the encoded protein. Multiple myeloma is the first mature B cell neoplasm found to harbor ras mutations.
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PMID:Oncogenes in multiple myeloma: point mutation of N-ras. 226 33

Monoclonal antibodies (MAbs), for human use require chemical and biological purity. The best approach seems in vitro cultivation in a serum-, protein-free medium. A basal defined culture medium has been developed to sustain optimal hybridoma cell growth and MAb secretion. It consists of Iscove's Dulbecco's modified, Eagle's, Ham's F12 and NCTC 135 media in a 5:5:1 mixture (v/v/v), to which glucose is added to reach a final concentration of 25 mM, glutamine to 4-6 mM, 2-mercaptoethanol to 50 microM, Pluronic F68 to 0.01-0.1% (w/v), Hepes to 25 mM and NaHCO3 to 3 g/l. Hybridoma cells, derived from Sp 2/0 myeloma and secreting a MAb to a human milk fat globule membrane-associated high molecular weight glycoprotein, were cloned in this medium containing 1% (v/v) fetal calf serum and then sequentially adapted in serum-free medium further supplemented with transferrin and insulin, both at 10 micrograms/ml. Clones producing immunoreactive MAbs secrete a mean of 50 micrograms IgG/ml, i.e., ca. 80% of the concentration reached in Dulbecco's modified Eagle's medium containing 10% serum. When cells were cultured in spinner flasks with a semi-continuous mode of cultivation (with a daily removal of 20% of the volume and its replacement by fresh culture medium), in serum-free medium further supplemented with 10 nM estradiol, a mixture of trace elements and albumin (at 30 micrograms/ml) complexed to linoleic acid, MAb secretion reached 100 micrograms/ml and became equal or higher to that obtained in serum-containing medium. MAb secretion was not decreased and was even significantly increased during the growth phase, when transferrin was replaced by another iron source, i.e., ferric citrate at 500 microM associated with 20 microM ascorbic acid. Finally, deletion of insulin and of albumin-linoleic acid did not affect significantly cell density nor MAb secretion. In conclusion, it appears from this study that semi-continuous cultivation in spinner flasks of hybridoma cells, after cloning and progressive adaptation, in a chemically defined, serum- and protein-free medium, permitted MAb secretion to be increased to a mean of 144 micrograms/ml, i.e., multiplied by a factor of ca. 1.5 compared to culture of these cells in serum-containing medium under the same conditions and by a factor of ca. 2.4 compared to cultivation in serum-containing medium in flasks.
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PMID:Optimisation of hybridoma cell growth and monoclonal antibody secretion in a chemically defined, serum- and protein-free culture medium. 264 56

Two new human myeloma cell lines have been established from a 36-year-old woman with refractory IgG kappa multiple myeloma in whom bilateral malignant pleural effusions developed. The malignant plasma cells from each effusion were set up in a liquid culture using an L-15 medium containing catalase, glutathione, selenous acid, ascorbic acid, insulin, transferrin, additional glutamine hydrocortisone, and 2-mercaptoethanol and designated as M-3 medium. Two IgG kappa cell lines, LB -831 and LB-832, were established and proved to be Epstein-Barr virus negative using the internal repeat sequence DNA probe. Characteristic plasma cell morphology was evident by light and electron microscopy. Immunotyping revealed an IgG kappa , B1+, B2-, Ia (HLA-DR)+, CALLA+ phenotype for each cell line as well as for the original pleural fluid and bone marrow myeloma cells. The supernatants also contained IgG kappa, beta 2 microglobulin, and large amounts of osteoclast-activating factor (indicating bone-resorbing activity). Cytogenetic analysis of the LB-831 cell line revealed a nearly triploid highly abnormal karyotype with numerous clonal chromosomal abnormalities involving chromosomes 1, 3, 5, 7, 13, and 15; several structurally abnormal marker chromosomes; and a putative homogeneously staining region on chromosome 7p at band p22. Analysis of the LB-832 cell line revealed several additional clonal abnormalities. These additional cytogenetic changes suggest that in vivo sequential clonal evolution occurred in this patient. Therefore, two new but related cell lines have been established, which should prove useful for further biological studies.
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PMID:Establishment of two new myeloma cell lines from bilateral pleural effusions: evidence for sequential in vivo clonal change. 392 97

Plasma-cell tumour 5563 forms a single molecular species of immunoglobulin IgG(2)a, i.e. one variant of heavy chain and one variant of light chain. The molecules formed are labile and undergo alterations in charge properties, which rapidly lead to heterogeneity of the myeloma protein after synthesis. The single immunoglobulin species originally formed is found only after the shortest time-intervals tested, i.e. 10min incubation. Two types of changes in charge properties take place: (1) The originally formed protein (component o) is converted via an intermediate o' into the most basic form of 5563 myeloma protein found in serum (component a). Charge differences between these components are suppressed at pH8.9, but can be studied by chromatography at pH6.5 or by analysis of isoelectric points by isoelectric focusing in polyacrylamide gel. The conversion of components o and o' into component a apparently commences soon after assembly of the molecules and proceeds to completion extracellularly. (2) The second type of charge difference that distinguishes components a, b, c and d is exhibited over the pH range 6.0-8.9, but not at acid pH, and has been studied by electrophoresis at pH8.9, by chromatography and by isoelectric focusing. Conversion of component a into components b, c, d and e is only partial so that all five components can be found at decreasing concentrations in serum. Both types of charge alteration can be effected in vitro in the presence of serum, with optimum pH8.0. None of the charge differences could be attributed to the secretion process, since a component with the same isoelectric point as component o was found in secreted myeloma protein (1h). We have found no evidence to support the idea that the first type of change from component o to component a is due to ring formation of N-terminal [(14)C]glutamine into pyrrolid-2-one-5-carboxylic acid; however, our findings do not exclude this process happening very rapidly to a precursor of component o, possibly the polypeptide chain during or immediately after synthesis. In studying this point we noted that not only the heavy chains but also the kappa-type light chain of mouse 5563 myeloma protein have a blocked N-terminus.
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PMID:One cell-one immunoglobulin. Origin of limited heterogeneity of myeloma proteins. 541 99

To learn about the V lambda gene segments that are expressed in lambda 3 light chains, the most recently identified lambda-chain subtype in inbred mice, we determined partial amino acid sequences of the V regions of two of these chains, L5-8 and Lc49 . The partial sequences were extended by establishing the complete V region sequence of cDNA for the lambda-chain mRNA from the hybridoma (RZ 5-8) and the myeloma ( CBPC -49) that produce these chains. The primer extension method used to sequence the cDNA is described in detail, because the same primer (a synthetic heptadecadeoxynucleotide ) can be used for sequencing cDNA for lambda-chains of all three subtypes of inbred mice and probably for lambda-chains from some other vertebrate species as well. The results confirm earlier preliminary findings that for both chains the V region is encoded by the V lambda 1 and J lambda 3 gene segments. The unmutated germ-line sequences of these gene segments are present in both chains, but the two chains, nevertheless, differ at codon 97, the V lambda-J lambda boundary. A T/G difference in the third position of this codon resulted in a codon for histidine (CAT) in one chain (L5-8) and for glutamine (CAG) in the other chain ( Lc49 ). This difference can be accounted for by variation in the site of V lambda 1-J lambda 3 recombination. Though the V region amino acid sequences of L5-8 and Lc49 differ only by the His/Gln substitution at position 97, the two chains have been shown (manuscript in preparation) to differ in their ability to form an effective combining site for the 2,4-dinitrophenyl group.
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PMID:Amino acid and nucleotide sequences of variable regions of mouse immunoglobulin light chains of the lambda 3-subtype. 620 90


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