Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0026764 (multiple myeloma)
 36,148 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Growth of the murine B-lymphocyte cell line CC9C10 and the myeloma SP2/0 was enhanced significantly by the presence of the unsaturated fatty acids, oleic and linoleic acids in serum-free culture. The cellular content of linoleic and oleic acids gradually increased during continuous culture passage, with no evidence of regulatory control. Over 10 culture passages in the presence of these fatty acids, the unsaturated/saturated fatty acid ratio of all cellular lipid fractions increased substantially. Most of the fatty acid accumulated in the polar lipid fraction (more than 74%) and only a small proportion was oxidized to CO2 (0.5%). Linoleic acid caused a decrease to one-eighth in the rate of metabolism of glutamine and a 1.4-fold increase in the rate of metabolism of glucose. There was no change in the relative flux of glucose through the pathways of glycolysis, pentose phosphate or the tricarboxylic acid cycle. The changes in energy metabolism were reversed when the cells were removed from fatty acid-supplemented medium. The most plausible explanation for these effects is the observed decrease in the rate of uptake of glutamine into cells loaded with linoleic acid. Growth of the CC9C10 cells in linoleic acid caused the Km of glutamine uptake to increase from 2.7 to 23 mM, whereas glucose uptake was unaffected.
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PMID:Unsaturated fatty acids enhance cell yields and perturb the energy metabolism of an antibody-secreting hybridoma. 906 85

The physiology of cultured animal cells, in particular hybridoma, myeloma and insect cells, with respect to growth and proliferation, amino acid metabolism, energy metabolism and cellular responses to environmental stress is discussed in this paper. The rate of proliferation of hybridoma cells in serum-containing media is limited by growth factors at a surprisingly early stage of growth. To maintain exponential growth in a batch culture, it is necessary to stimulate cell proliferation with repeated additions of serum or pure growth factor. It is further suggested that proliferation of Spodoptera frugiperda (Sf9 insect cells), a normal cell line able to grow in a serum-free medium without any added growth factors, is regulated by autocrine growth factors and possibly by other regulatory mechanisms, as Sf9 cells secrete a growth factor (IGF-I) and the medium still appears nutritionally sufficient at the time of cessation of growth. The uptake and metabolism of amino acids is one of the determinants of growth and production. Wasteful overproduction of amino acids in myeloma and hybridoma cells is a result of excess glutamine, and can be avoided by glutamine limitation. Synthesis of amino acids may be conditional, as in Sf9 cells which synthesise glutamine provided that ammonium is supplied to the medium; and cysteine (from methionine) provided that a sufficiently young inoculum is used. Uptake of amino acids in Sf9 cells appears regulated in relation to the proliferative status as there is a distinct cessation of uptake even before growth ceases. The energy metabolism in myeloma, hybridoma and insect cells is a typically substrate-concentration-dependent overflow metabolism. Substrate limitation (glucose and glutamine) decreases by-product formation and increases metabolic efficiency in all these cell lines. However, glutamine limitation, as used in fed-batch cultures (or chemostat cultures) provokes cell death (in parallel to growth) in hybridoma cells in the concentration range below 0.05 mM.
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PMID:Physiology of cultured animal cells. 948 19

As part of the development of structured models for the metabolism of myeloma cells in suspension culture, a study was made of the subcellular localization of key enzymes of glucose and glutamine metabolism. Steady state chemostat cultures of the mouse myeloma SP2/0-Ag14 were used as a reproducible source of biomass. Homogenates of the cells, obtained via mechanical disruption, were separated into a mitochondrial and a cytosolic fraction via differential centrifugation. The following conclusions are drawn: (1) approximately one fifth of the hexokinase activity of cell-free homogenates is associated with the mitochondria; (2) a malate-aspartate shuttle may operate for oxidation of cytosolic NADH, as indicated by high levels of malate dehydrogenase and aspartate aminotransferase in both particulate and soluble fractions; (3) the pentose phosphate pathway and isocitrate dehydrogenase may contribute to the provision of cytosolic NADPH; (4) phosphoenolpyruvate carboxykinase and pyruvate kinase, which are present in high activities, are exclusively cytosolic and probably play a key role in glutamine metabolism; (5) oxidation of glutamine via these enzymes leads to the formation of pyruvate that enters the same pool as pyruvate generated by glycolysis. As a result, lactate and alanine formation can occur from both glucose and glutamine.
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PMID:Subcellular localization of enzyme activities in chemostat-grown murine myeloma cells. 965 Feb 85

Activities of enzymes in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, and glutaminolysis have been determined in the mouse myeloma SP2/0.Ag14. Cells were grown on IMDM medium with 5% serum in steady-state chemostat culture at a fixed dilution rate of 0.03 h-1. Three culture conditions, which differed in supply of glucose and oxygen, were chosen so as to change catabolic fluxes in the central metabolism, while keeping anabolic fluxes constant. In the three steady-state situations, the ratio between specific rates of glucose and glutamine consumption differed by more than twentyfold. The specific rates of glucose consumption and lactate production were highest at low oxygen supply, whereas the specific rate of glutamine consumption was highest in the culture fed with low amounts of glucose. Under low oxygen conditions, the specific production of ammonia increased and the consumption pattern of amino acids showed large changes compared with the other two cultures. For the three steady states, activities of key enzymes in glycolysis, the pentose phosphate pathway, glutaminolysis, and the TCA cycle were measured. The differences in the in vivo fluxes were only partially reflected in changes in enzyme levels. The largest differences were observed in the levels of glycolytic enzymes, which were elevated under conditions of low oxygen supply. High activities of phosphoenolpyruvate carboxykinase (E.C. 4.1.1.32) in all cultures suggest an important role for this enzyme as a link between glutaminolysis and glycolysis. For all enzymes, in vitro activities were found that could accommodate the estimated maximum in vivo fluxes. These results show that the regulation of fluxes in central metabolism of mammalian cells occurs mainly through modulation of enzyme activity and, to a much lesser extent, by enzyme synthesis.
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PMID:Fluxes and enzyme activities in central metabolism of myeloma cells grown in chemostat culture. 1009 11

The glutamine metabolism was studied in glucose-starved and glucose-sufficient hybridoma and Sp2/0-Ag14 myeloma cells. Glucose starvation was attained by cultivating the hybridoma cells with fructose instead of glucose, and the myeloma cells with a low initial glucose concentration which was rapidly exhausted. Glutamine used in the experiments was labeled with 15N, either in the amine or in the amide position. The fate of the label was monitored by 1H/15N NMR analysis of released 15NH+4 and 15N-alanine. Thus, NH+4 formed via glutaminase (GLNase) could be distinguished from NH+4 formed via glutamate dehydrogenase (GDH). In the glucose-sufficient cells a small but measurable amount of 15NH+4 released by GDH could be detected in both cell lines (0.75 and 0.31 micromole/10(6) cells for hybridoma and myeloma cells, respectively). The uptake of glutamine and the total production of NH+4 was significantly increased in both fructose-grown hybridoma and glucose-starved myeloma cells, as compared to the glucose-sufficient cells. The increased NH+4 production was due to an increased throughput via GLNase (1.6 -1.9-fold in the hybridoma, and 2.7-fold in the myeloma cell line) and an even further increased metabolism via GDH (4.8-7.9-fold in the hybridoma cells, and 3.1-fold in the myeloma cells). The data indicate that both GLNase and GDH are down-regulated when glucose is in excess, but up-regulated in glucose-starved cells. It was calculated that the maximum potential ATP production from glutamine could increase by 35-40 % in the fructose-grown hybridoma cells, mainly due to the increased metabolism via GDH.
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PMID:Elevated glutamate dehydrogenase flux in glucose-deprived hybridoma and myeloma cells: evidence from 1H/15N NMR. 1009 57

It is now well documented that apoptosis represents the prevalent mode of death in lymphoid cultures and occurs spontaneously in late-exponential phase of batch cultures following nutrient exhaustion. In an attempt to enhance the cell survival of these cell lines, we have initially engineered nonproducing NS/0 myeloma cells with a vector expressing the adenoviral E1B-19K protein. NS/0 cells transfected with E1B-19K were found to be more resistant to apoptosis occurring in the late phase of batch culture and under stressful conditions such as cultivation in glutamine-free medium or following heat shock. In this study, we have characterised a number of NS/0 subclones constitutively expressing different levels of E1B-19K, as well as several subclones in which the expression of E1B-19K was regulated by a tetracycline-controllable gene switch. We have found that a threshold E1B-19K level was required in order to achieve protection against apoptosis. The extent of resistance against cell death induced by nutrient deprivation in glutamine-free medium and in the late phase of batch cultures correlated with the level of E1B-19K expression up to an optimal level where further increases in E1B-19K levels did not result in significant additional protection. To assess the effects of E1B-19K on antibody productivity, an apoptosis-resistant NS/0 clone was then transfected with a chimeric antibody construct. Despite their improved viability, the antibody productivity of E1B-19K clones in batch culture was not significantly improved. Moreover, while the use of E1B-19K considerably delayed cell death, cells eventually died by apoptosis. Surprisingly, E1B-19K had no beneficial effect on the efficiency of fusion of NS/0 myelomas and splenocytes for the generation of hybridoma cells. Furthermore, the resulting hybridomas, although expressing E1B-19K at levels comparable to the myeloma parent, were no longer resistant to apoptosis. This indicates that the ability of E1B-19K to prevent apoptosis is not only dose-dependent but also seems to be cell-type dependent.
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PMID:Dose-dependent reduction of apoptosis in nutrient-limited cultures of NS/0 myeloma cells transfected with the E1B-19K adenoviral gene. 1039 8

Insulin-like growth factors (IGF) I and II are potent mitogens for a variety of cancer cells. The proliferative and anti-apoptotic actions of IGF are mediated by the IGF-I receptor (IGF-IR), to which both IGF-I and IGF-II bind with high affinity. To investigate the mitogenic and anti-apoptotic activities of IGF-IR and to achieve better inhibition of IGF-IR function, single-chain antibodies against human IGF-IR (alphaIGF-IR scFvs) were constructed and expressed. IgG cDNA encoding variable regions of light and heavy chains (VL and VH) from mouse IgG were cloned from a hybridoma producing the 1H7 alphaIGF-IR monoclonal antibody [Li et al., Biochem Biophys Res Commun 196: 92-98 (1993)]. The splice-overlap extension polymerase chain reaction was used to assemble a gene encoding the alphaIGF-IR scFv, including the N-terminal signal peptide, VL, linker peptide, VH, and C-terminal DYKD tag. Two types of soluble alphaIGF-IR scFvs, a prototype alphaIGF-IR scFv and its alternative type alphaIGF-IR scFv-Fc, were constructed and expressed in murine myeloma cells. alphaIGF-IR scFv-Fc, containing the human IgG1 Fc domain, was stably expressed in NS0 myeloma cells, using a glutamine synthase selection system, and purified from the conditioned medium of stable clones by protein-A--agarose chromatography. Levels of alphaIGF-IR scFv-Fc expression ranged from 40 mg/l to 100 mg/l conditioned medium. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis under reducing and nonreducing conditions indicated that alphaIGF-IR scFv-Fc is a dimeric antibody. alphaIGF-IR scFv-Fc retained general characteristics of the parental 1H7 monoclonal antibody except that its binding affinity for IGF-IR was estimated to be approximately 10(8) M(-1), which was one-order of magnitude lower than that of 1H7 monoclonal antibody. Injection of alphaIGF-IR scFv-Fc (500 microg/mouse, twice a week) significantly suppressed MCF-7 tumor growth in athymic mice. These results suggest that the alphaIGF-IR scFv-Fc is a first-generation recombinant alphaIGF-IR for the potential development of future alphaIGF-IR therapeutics.
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PMID:Single-chain antibodies against human insulin-like growth factor I receptor: expression, purification, and effect on tumor growth. 1094 7

F(ab')(2) fragments are desirable structural derivatives of monoclonal antibodies (MAbs) because of their pharmacokinetic properties and bivalent binding to antigen. Production of these fragments, however, has proven difficult because of the variable sensitivity of intact antibodies to proteolytic enzymes, which can result in very low yields and unstable product. To circumvent these problems, we attempted to apply genetic engineering methods to generate stable F(ab')(2) fragments in NSO murine myeloma cells using the glutamine synthase expression system. For these studies, the chimeric MAb, chTNT-3, directed against necrotic regions of solid tumors, was used to generate several F(ab')(2) variants, which contained between one and three cysteine residues at the end of the hinge region. In addition, two different affinity tags (his tag, streptactin tag) were used with each variant to determine the best tag for purification procedures. Stability was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and by antigen binding studies and the constructs were tested in vivo to measure their pharmacokinetic properties and biodistribution in normal organs and tumor. The results of these studies show that 3 cysteine residues are required to produce stable F(ab')(2) fragments and that either purification tag can be used with this variant to produce suitable reagents for in vivo studies. Those constructs containing one or two cysteines were found to be unstable and broke down to Fab fragments regardless of the purification tag used. These studies demonstrate that stable, clinically useful F(ab')(2) fragments of chTNT-3 can be produced in mammalian cells by genetic engineering methods.
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PMID:Stable, genetically engineered F(ab')(2) fragments of chimeric TNT-3 expressed in mammalian cells. 1199 12

While feeding protocols and ectopic expression of anti-apoptotic genes have been used to improve the viability of hybridoma cell lines, the effect of the expression levels of survival genes on the behavior of hybridomas following nutrient supplementation is unknown. In this study, we compared the behavior of the Sp2/0-Ag14 hybridoma (Bcl-xL(low)) and the P3x63-Ag8.653 myeloma (Bcl-xL(high)) following culture supplementation with the amino acid L-glutamine (L-Gln). Our data revealed that L-Gln addition substantially increased Sp2/0-Ag14 cell viability and total cell density, concomitant with a decrease in the rate of cell death. This effect was not seen when other amino acids or D-glucose (D-Glc) replaced L-Gln. The improvement in the culture behavior of Sp2/0-Ag14 cells was attributed to a reduction in the rate of accumulation of apoptotic cells. On the other hand, L-Gln supplementation had only a limited effect on the growth of the P3x63-Ag8.653 cells. Interestingly, Sp2/0-Ag14 cells over-expressing Bcl-xL showed a culture behavior upon L-Gln complementation that was similar to the P3x63-Ag8.653 myeloma. These results suggest that the anti-apoptotic gene expression profile of hybridoma cells can markedly impact on the beneficial effects afforded by nutrient supplementation.
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PMID:Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures. 1247 50

Although intact monoclonal antibodies (MAbs) are well suited as therapeutic reagents, their relatively slow clearance rates render them less useful for imaging applications. Over the last several years, our laboratory has developed a unique targeting approach to solid tumors that utilizes MAbs directed against DNA and its components to bind to degenerating cells and necrotic regions of tumors in a specific manner. Because these MAbs have considerable potential for the early diagnosis of cancer and for the monitoring of cytoreductive therapies, the availability of an effective imaging agent is highly desirable. To accomplish this goal, a series of genetically engineered derivatives of MAb chTNT-3 including the single-chain Fv, diabody, triabody, Fab, and F(ab')(2) were generated and expressed in NS0 myeloma cells using the Glutamine Synthetase Amplification System. Initial in vitro studies demonstrated that each of the antibody derivatives maintained its antigen binding in a stable manner. In vivo analyses after radiolabeling were then performed to evaluate their pharmacokinetic, biodistribution, and tumor-imaging properties in solid tumor-bearing mice. The results of these studies showed that compared with intact parental chTNT-3, which has a half-life of 134.2 h, the smaller derivatives were eliminated more rapidly (4.9-8.1 h). Importantly, the smaller derivatives were found to have significantly higher tumor-to-organ ratios, but lower overall uptake levels compared with parental (125)I-chTNT-3 in two different tumor models. A comparison of the five derivatives showed that the F(ab')(2) reagent consistently gave the best results in imaging and biodistribution studies. Based upon these results, further studies are warranted to demonstrate the potential of this reagent for the diagnosis and monitoring of solid tumors using noninvasive imaging techniques such as immunoscintigraphy and positron emission tomography (PET).
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PMID:Comparison of recombinant derivatives of chimeric TNT-3 antibody for the radioimaging of solid tumors. 1271 84


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