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)

The dihydrofolate reductase activity has been studied cytochemically in various haematological diseases. The variation between normal controls, Hodgkin's disease, myeloma, polycythaemia vera, chronic lymphocytic leukaemia and chronic myeloid leukamia was not significant, comparing the same type of cells. In acute myeloid leukaemia and acute lymphoblastic leukaemia the blast cells were weakly positive or negative. This finding is very interesting as the blast cells are capable of division. Probably the dihydrofolate reductase appears in the blast cells in some stage of mitosis. Lymphocytes stimulated by phytohaemagglutinin showed increased enzyme activity compared with normal non-stimulated lymphocytes. The "blast like" cells were more strongly positive than the blast cells of leukaemic patients. The patients with acute lymphoblastic leukaemia or acute myeloid leukaemia treated with methotrexate showed increased dihydrofolate reductase activity cytochemically.
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PMID:Cytochemical demonstration of dihydrofolate reductase in leukaemia and other haematological diseases. 26 23

Murine/human chimeric gamma 1 and K Ig genes were cloned adjacent to the gene coding for methotrexate-resistant dihydrofolate reductase. These constructs were introduced into myeloma cells, and lines containing stably integrated genes were selected. The integrated Ig genes were then amplified by selection of the cells in increasing concentrations of methotrexate. The extent of gene amplification, mRNA accumulation, and production of Ig was studied in transfectomas containing introduced light chain genes, heavy chain genes, or both. When the light chain gene was introduced alone, it was expressed at low levels, but after selection with methotrexate, light chain expression was increased as much as 63-fold. In contrast, the transfected heavy chain genes were highly expressed, but production of the corresponding protein was increased a maximum of only fourfold by methotrexate treatment. Cellular toxicity of unassembled heavy chain monomer was not observed, even at amounts equivalent to 2% of total cellular protein. Cointroduction of the heavy and light chain constructs with subsequent amplification resulted in as much as 25-fold increase in secretion of intact antibody relative to unamplified cells. The results demonstrate that amplification of Ig genes can induce transfectomas to secrete antibody at nearly the rate of hybridomas.
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PMID:The effect of dihydrofolate reductase-mediated gene amplification on the expression of transfected immunoglobulin genes. 312 34

We report a detailed comparison of two commonly used stable, amplifiable mammalian expression systems (Chinese Hamster Ovary cells/dihydrofolate reductase and Mouse NSO myeloma/glutamine synthetase) used to express a humanized IgG1 monoclonal antibody. We compare copy number and steady state mRNA levels of both the selectable marker and heavy chain of the antibody throughout the selection and amplification process. In both cell lines, copy number and steady state levels of heavy chain and selectable marker increased during selection and were further increased during amplification. As expected, an increase in steady state mRNA levels of heavy chain correlated with an increase in expression of antibody whilst an increase in the steady state levels of mRNA of the selectable marker correlated with increased resistance to the selective agent. In NSO and CHO cells producing equivalent amounts of antibody, the copy number of the antibody genes and selectable marker was significantly higher in the CHO cells than in the NSO cells. However, the steady state mRNA levels of the heavy chain of the antibody were virtually identical. Rates of protein secretion in the two cell lines were also compared and found to be very similar. When the antibody purified from both systems was compared in a number of functional assays they behaved identically.
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PMID:Comparison of expression of a humanized monoclonal antibody in mouse NSO myeloma cells and Chinese hamster ovary cells. 753 24

We previously reported the expression of a mouse/human chimeric anti-ganglioside GD3 antibody, KM871 (IgG1,kappa) in mouse myeloma SP2/0 cells under the control of the ecotropic Moloney virus long terminal repeat by the co-transfection of chimeric heavy (H) and light (L) chain vectors (Shitara et al. (1993) Cancer Immunol. Immunother.). To establish an efficient and high level expression system for the chimeric antibody, we did comparative study on vector systems and host cells. An improved expression vector, named 'a tandem vector, pChi641HLGM4' was constructed, in which both of the chimeric H and L chain gene transcription units and a dihydrofolate reductase (dhfr) gene transcription unit were inserted. When two kinds of mouse myeloma cell lines, SP2/0 and P3U1, were used as host cells, frequency of the incidence of antibody-producing transfectants was markedly increased by the use of the tandem vector compared with the use of the mixture of each chimeric H vector and L chain vector. To select out appropriate host cells, transfection frequency and antibody production level were compared among SP2/0, P3U1 and rat myeloma YB2/0 cells by transfection of the tandem vector. YB2/0 cell was shown to have the highest potential in both the transfection frequency and the antibody production. Introduction of the tandem vector into YB2/0 cells and the subsequent amplification with 50-200 nM methotrexate gave rise to several clones that stably secreted 70-100 micrograms/10(6) cells per 24 h of the chimeric antibody. This productivity of the antibody is one of the highest levels which have been achieved by other investigators using transfected myeloma cells. Using this system it took only 2-3 months to establish the transfectant clones which stably produced the chimeric antibody.
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PMID:A new vector for the high level expression of chimeric antibodies in myeloma cells. 830 83

Process development for biopharmaceuticals is dictated by product quality, drug safety and economy of the manufacturing process. Not surprisingly, these factors also play a key role in the evaluation of mammalian cell expression systems to be used in the production of pharmacologically active glycoproteins. To date, the most prominent candidates for efficient expression of glycoproteins are mammalian cell lines such as mouse fibroblast cells (C 127-BPV), Chinese hamster ovary cells (CHO-DHFR, CHO-NEOSPLA, CHO-GS), mouse myeloma cells (NSO-GS) as well as transgenic animals carrying c-DNA or genomic DNA which codes for the protein of interest. The expression titer in the case of glycoproteins is mainly determined by the promoter construct, the site of integration into the chromosome, the copy number and the type of protein in question. Based on expression titer, CHO-NEOSPLA and NSO-GS expression systems are most effective in the production of monoclonal antibodies and, to a lesser extent, of recombinant DNA derived proteins. However, based on overall product yield, expression of recombinant DNA derived proteins in transgenic animals is by far the most promising system. Therefore, for proteins required in large quantities, transgenic expression systems offer an attractive choice. However, cost of goods for products for which the dosage or the overall annual quantities are low, is dominated by downstream processing, filling, lyophilization and packaging and not by the fermentation process. Such proteins are preferentially produced by classical mammalian cell culture systems. Concerns which have to be addressed with respect to drug safety in the transgenic animal approach are the size of the herd, genetic stability from animal to animal, variation in productivity and in impurity profiles during lactation periods, microbial, viral, mycoplasma and prion contaminants, the dependence on health status and the life span of the animal. In a number of cases glycosylation of the protein is relevant for the prevention of immunogenicity of the protein, the pharmacological activity, the pharmacokinetic profile, solubility and stability against proteolysis. The glycosylation pattern, depending on protein structure, is influenced by the enzymatic system of the host cell as well as by fermentation conditions. Therefore, selection of host cells and culture conditions must take into account the requirement for a specific and stable glycosylation pattern. For the assessment of glycovariants, a number of protein analytical methods such as peptide mapping, isoelectric focusing, oligosaccharide mapping, MALDI-TOF (matrix assisted laser desorption mass spectrometry-time of flight), capillary electrophoresis and specific potency assays are available. In our experiments, glycosylation of proteins expressed in CHO cells was demonstrated to be very stable. Only extreme process times, cultivation methods and ammonium ion concentrations had an influence on the glycosylation profile. Among the three products investigated--tissue plasminogen activator (t-PA), interferon omega and soluble intercellular adhesion molecule (s-ICAM)--t-PA expressed the most stable glycosylation pattern. Only at extreme ammonium concentrations an increase of mannose-5 structures was observed, whereas biantennary complex structures were reduced. On the other hand, interferon omega and s-ICAM showed greater susceptibility to increased ammonium concentrations and to adherent cultivation. Such conditions induced quantitative changes to the glycosylation pattern favoring the appearance of higher branched structures. Short cultivation times resulted in more heterogenous oligosaccharide structures. Since the glycosylation of the three proteins is different in the same host cell, the amino acid sequence of the protein apparently influences the glycosylation pattern and its sensitivity to culture conditions. In NSO-mouse myeloma cells, production of s-ICAM is two times as high as in CHO cells
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PMID:Appropriate mammalian expression systems for biopharmaceuticals. 974 18

Cells synthesize nucleotides through de novo and salvage pathways that require the activities of dihydrofolate reductase (DHFR) and hypoxanthine-guanine phosphoribosyltransfease (HGPRT), respectively. Aminopterin, an inhibitor of dihydrofolate reductase, has been demonstrated to allow HGPRT(-) cells to be negatively selected. However, the pathway by which aminopterin leads to cell death remains to be clarified. In this study, we characterized features of cellular responses induced by aminopterin treatment in P3-X63-Ag8.653, a mouse HGPRT(-) myeloma cell line. Upon treatment with aminopterin, the cells readily underwent an apoptotic process, as assessed by DNA fragmentation assay and electron microscopic analysis. Aminopterin-induced apoptosis was drastically reduced by addition of actinomycin D and cycloheximide, indicating that active RNA and protein synthesis is required for the apoptotic effect of aminopterin. Interestingly, the induction of c-myc gene expression preceded the activity of DNA fragmentation in aminopterin-treated cells. Taken together, these results suggest that cells deficient in the salvage pathway of purine biosynthesis are susceptible to aminopterin-induced apoptosis that requires de novo synthesis of proapoptotic factors, including Myc oncoprotein.
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PMID:Requirement of de novo protein synthesis for aminopterin-induced apoptosis in a mouse myeloma cell line. 1141 Feb 44

The promoter activity of the human c-fos and human cytomegalovirus (CMV) immediate early promoter was compared in transient and stable transfection experiments with six cell lines of mouse, human, and hamster origin which are all of commercial importance. The c-fos promoter was 1.8-5.6-fold stronger than the CMV promoter in BHK-A, BHK-B, CHO-DHFR(-), and mouse NIH-3T3 in stable transfectants and less effective in mouse myeloma or human 293 cells, suggesting a new transcriptional control element for high-level expression and protein production in mammalian cells. The induction profiles determined in the presence and absence of serum are dependent on the cell line used. Induction levels of up to 8-fold could be achieved in preselected cell pools.
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PMID:Human c-fos promoter mediates high-level, inducible expression in various mammalian cell lines. 1255 18

Tumor-specific genes delivered to dendritic cells (DCs) have been used for the generation of cytotoxic T cells (CTLs), but their application has been limited on the one hand by low viral titers resulting in low transduction efficiency and poor protein production, and on the other hand by immunogenicity of the selectable marker and poor viability of the DCs. We addressed these limitations by creating a multipurpose master vector (pMV) and cloning the tumor gene NY-ESO-1, which is highly expressed in more than 50% of advanced myeloma patients. pMV was constructed from a Moloney murine leukemia virus (Mo-MuLV)-based retroviral backbone with the following features: (1) an extended packaging signal to achieve high viral titers, (2) a splice acceptor region to facilitate protein production, (3) a nonimmunogenic selectable marker, dihydrofolate reductase-L22Y (DHFR(L22Y)), to exclude the generation of CTLs against the selectable marker, (4) an internal ribosomal entry site between the tumor-specific gene (NY-ESO-1) and the selectable marker DHFR(L22Y) for coexpression of two heterologous gene products from a single bicistronic mRNA, minimizing the possibility of differential expression of these two genes, and (5) human granulocyte-macrophage colony-stimulating factor (hGM-CSF) cDNA driven by the human T-lymphotropic virus promoter to enhance DC function and viability. Recombinant virus of pMV-NY-ESO-1 was generated with vesicular stomatitis virus G envelope protein (VSV-G) in the GP2-293 cell line for efficient transduction. We present evidence that the DC phenotype is unaltered after transduction and that more than 85% of DCs express NY-ESO-1, which secrete approximately 40 ng of GM-CSF per 10(6) DCs.
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PMID:High-level expression of cancer/testis antigen NY-ESO-1 and human granulocyte-macrophage colony-stimulating factor in dendritic cells with a bicistronic retroviral vector. 1450 68

We have established a large-scale manufacturing system to produce recombinant human alpha-thrombin. In this system, a high yield of alpha-thrombin is prepared from prethrombin-2 activated by recombinant ecarin. We produced human prethrombin-2 using mouse myeloma cells and an expression plasmid carrying the chicken beta-actin promoter and mutant dihydrofolate reductase gene for gene amplification. To increase prethrombin-2 expression further, we performed fed-batch cultivation with the addition of vegetable peptone in 50 liters of suspension culture. After five feedings of vegetable peptone, the expression level of the recombinant prethrombin-2 reached 200 micro g/ml. Subsequently, the recombinant prethrombin-2 could be activated to alpha-thrombin by recombinant ecarin expressed in a similar manner. Finally, recombinant alpha-thrombin was purified to homogeneity by affinity chromatography using a benzamidine-Sepharose gel. The yield from prethrombin-2 in culture medium was approximately 70%. The activity of the purified recombinant alpha-thrombin, including hydrolysis of a chromogenic substrate, release of fibrinopeptide A, and activation of protein C, was indistinguishable from that of plasma-derived alpha-thrombin. Our system is suitable for the large-scale production of recombinant alpha-thrombin, which can be used in place of clinically available alpha-thrombin derived from human or bovine plasma.
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PMID:Preparation of recombinant alpha-thrombin: high-level expression of recombinant human prethrombin-2 and its activation by recombinant ecarin. 1517 95

Osteolytic bone disease (OBD) in multiple myeloma (MM) is caused by interactions between MM cells and the bone marrow microenvironment and is characterized by increased osteoclastic bone resorption and decreased osteoblastic bone formation. Recently, the role of osteoblast inhibition has come into focus, especially the possible role of overexpression of DKK1, an inhibitor of the Wnt signalling pathway. Further, CKS2, PSME2 and DHFR have also been reported as candidate genes for OBD. We studied the gene expression by quantitative reverse transcription polymerase chain reaction of TNFSF11 (RANKL), TNFSF11A (RANK), TNFRSF11B (OPG), CCL3 (MIP1A), CCL4 (MIP1B), PTHR1 (PTHrp), DKK1, CKS2, PSME2 and DHFR in purified, immunophenotypic FACS-sorted plasma cells from 171 newly diagnosed MM patients, 20 patients with monoclonal gammopathy of undetermined significance and 12 controls. The gene expressions of the analysed genes were correlated with radiographically assessed OBD. Only overexpression of DKK1 was correlated to the degree of OBD. Myeloma cells did not express TNFSF11A, TNFSF11, or TNFRSF11B, and very rarely expressed CCL3 and PTHR11. CCL4, CKS2, PSME2 and DHFR were variably expressed, but the expression of these genes showed no correlation with OBD. In contrast, loss of PSME2 expression in MM plasma cells was significantly correlated with OBD.
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PMID:Myeloma cell expression of 10 candidate genes for osteolytic bone disease. Only overexpression of DKK1 correlates with clinical bone involvement at diagnosis. 1800 68


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