Increase in synthesis of human monoclonal antibodies by transfected Sp2/0 myeloma mouse cell line under conditions of microgravity

Microgravity can influence cell growth and function. A transfected Sp2/0 myeloma cell line P3A2 producing a human IgG1 anti-TNF monoclonal antibody was cultivated in static culture, spinner flasks and simulated microgravity using a rotating wall vessel bioreactor. Microgravity significantly decreased cell growth (from 1.7×10⁶ to 7.9×10⁵ cells/ml), but facilitated the synthesis of antibodies, (1.8, 1.3 and 0.5 g of anti-TNF hmAb per 10⁶ viable cells for cells cultivated under microgravity, in spinner flasks and static cultures, respectively). The results suggest that microgravity could be applied to improve the specific productivity of cell lines producing potentially important therapeutic proteins.     

Karyotypic characteristics of the murine myeloma line sp2/0-Ag14

Using methods of G- and C-banding, a study was made of the karyotype of mouse myeloma cell line sp2/0-Ag14. The number of chromosomes varies from 58 to 65, the modal class being 61-62. 50 per cent of chromosomes are rearranged. Normal chromosomes 6, 12 and X were not detected in either examined cell of this line. Among the marker chromosomes there are an isochromosome (19/19), three dicentric markers and one marker with two interstitial C-bands. There is a specific marker t (12; 15) of mouse plasmacytomas in the karyotype sp2/0-Ag14. A possible association of specific translocations and segregations of the normal chromosomes with the phenotype of line sp2/0-Ag14 is discussed. The results obtained may be useful for cytogenetic analysis of hybridomas.     

Thiol antioxidants increase the intracellular level of reactive oxygen species and prolifetaion of SP2/0 mouse myeloma cells in serum-free medium

The connections between the presence of low molecular weight RSH-antioxidants (N-acetylcysteine, glutathione) in serum-free medium, generation of reactive oxygen species (ROS), and proliferation of SP2/0-SF mouse myeloma cells have been demonstrated. It is shown that the presence of RSH compounds in the medium within the studied range of concentrations changed the contents of ROS in cells and had a dose-dependent effect on cell proliferation. Stimulation of the proliferative activity did not depend on the nature of an RSH compound. The optimal concentration for the both antioxidants was 0.2 mM. A further increase of the concentration led to inhibition of cell proliferation to different degrees for N-acetylcysteine and glutathione.     

Inhibition of caspase activity delays apoptosis in a transfected NS/0 myeloma cell line

The productivity of NS/0 myeloma batch cultures is often compromised by the premature induction of apoptosis, now established to be the predominant method of cell death during culture decline. Caspase proteases have recently been shown to play a major role in the transmission of signals for apoptotic cell death. Using a specific inhibitor that targets a range of caspases (Z-VAD-fmk) we assessed whether inhibition of caspase activity could prolong the viability of NS&vbar;h=0 cells under conditions that cause apoptotic cell death in batch cultures. Z-VAD-fmk was found to significantly reduce apoptotic cell death (by approximately 50%) induced by cytotoxins and to preserve membrane integrity to a similar extent. In conditions of low serum, Z-VAD-fmk reduced certain features of apoptosis (e.g., DNA fragmentation), but only marginally improved viability. In medium-depleted batch cultures, Z-VAD-fmk afforded a delay of between 24 and 48 h in both the induction of apoptosis and loss of viability. Despite an apparent increase in viability in Z-VAD-fmk-treated NS&vbar;h=0 cultures, no improvement in productivity could be demonstrated, suggesting that at least some normal pathways for protein production are shut down upstream of caspase activation. An examination of mitochondrial membrane potential (Deltapsim) in Z-VAD-fmk-treated and untreated NS&vbar;h=0 cells revealed only a small initial difference (5%) in the levels of Deltapsim depolarization. Similar levels of mitochondrial dysfunction, despite caspase inactivity, may therefore be responsible for the comparable productivity in untreated and Z-VAD-fmk-treated cultures. Thus, this study suggests that, while a delay in cell death due to caspase inhibition may reduce problems associated with cellular disintegration, it does not permit productivity improvements in this type of culture.     

天然纳米材料凹凸棒土促进Sp2/0细胞增殖的研究

目的:研究天然纳米材料凹凸棒土(ATP)对Sp2/0细胞增值的作用,并探讨这种作用与ATP优化细胞培养环境相关。方法:通过CCK8法和细胞计数方法,比较正常培养方式和添加天然纳米材料凹凸棒土培养方式下Sp2/0细胞的生长曲线、细胞活性,除外,Sp2/0细胞培养过程中谷氨酰胺的消耗以及细胞代谢产物NH4+浓度随时间的变化。结果:一定浓度范围的天然纳米材料凹凸棒土可有效的提高了Sp2/0细胞的细胞活性和细胞密度,促进了Sp2/0细胞的增殖。结论:天然纳米材料凹凸棒土通过降低细胞代谢产物中铵离子浓度,优化了细胞培养环境,促进了Sp2/0细胞增值。     

Aberrant imprinting in mouse trophoblast stem cells established from somatic cell nuclear transfer-derived embryos

Although phenotypic abnormalities frequently appear in the placenta following somatic cell nuclear transfer (SCNT), mouse trophoblast stem cells (TSCs) established from SCNT embryos reportedly show no distinct abnormalities compared with those derived from normal fertilization. In this study, we reexamined SCNT–TSCs to identify their imprinting statuses. Placenta-specific maternally imprinted genes (Gab1, Slc38a4, and Sfmbt2) consistently showed biallelic expression in SCNT–TSCs, suggesting their loss of imprinting (LOI). The LOI of Gab1 was associated with decreased DNA methylation, and that of Sfmbt2 was associated with decreased DNA methylation and histone H3K27 trimethylation. The maternal allele of the intergenic differentially methylated region (IG–DMR) was aberrantly hypermethylated following SCNT, even though this region was prone to demethylation in TSCs when established in a serum-free chemically defined medium. These findings indicate that the development of cloned embryos is associated with imprinting abnormalities specifically in the trophoblast lineage from its initial stage, which may affect subsequent placental development.     

Maintenance of Epstein-Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2.

Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein-Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.     

Fluctuation of the SP/non-SP phenotype in the C6 glioma cell line

Using the C6 glioma cell as a paradigm, we found that (i) the clonogenicity of C6 cells is several orders of magnitude higher than the percentage of SP cells; (ii) non-SP cells are able to generate SP cells, and conversely SP cells generate non-SP cells; (iii) non-SP sorted cells behave as tumorigenic cells. Hence, in C6 cells cultured in serum-containing medium, SP cells can be generated from non-SP cells. This dynamic equilibrium explains in C6 cells the maintenance of the SP phenotype with cell passaging and demonstrates the existence of tumorigenic non-SP cells.     

Expression and purification of a recombinant human glycoprotein in mouse hybridoma SP2/0-AG14 cells

The cDNA for human erythropoietin (hEPO) inserted into the mammalian expression vector BMCGSNeo was introduced into SP2/0-Ag14 cells and a transformant producing large amounts of hEPO was established. The recombinant hEPO in conditioned medium was purified by immunoaffinity and gel filtration column chromatographies. The purified hEPO had full in vitro biological activity, but low in vivo biological activity.     

Metabolic characterization of a hyper-productive state in an antibody producing NS0 myeloma cell line

Metabolic profiling or metabolomics is the analysis of a larger number of small metabolic compounds within cells. While this technique has been utilized to study microbial and yeast strains under different physiochemical conditions, very little has been reported regarding its application in mammalian cell culture. Here, the physiological and metabolic changes observed during the proliferation arrest of an antibody producing GS-NS0 mouse myeloma cell line were studied using conventional biochemical analysis and one-dimensional nuclear magnetic resonance (NMR)-based metabolic profiling. Proliferation-arrested cells had increased antibody productivity, enhanced normalized mitochondrial membrane potential, and showed changes in the consumption of several amino acids. Further investigation into these physiological changes was carried out by ¹H NMR profiling followed by principle component analysis (PCA). The resulting data showed a clear separation of the arrested and control spectra that related to the altered metabolic state of the arrested culture. Metabolites associated with phosphatidylcholine homeostasis, lipid and fatty acid metabolism, and ascorbate formation were found to be present in significant amount in these cultures. Taken together, the results suggested that there was a link between the metabolic alterations and the hyper-productive state, possibly relating to vesicle recycling and secretory functions, and mechanism to counteract against the generation of reactive oxygen species. While the use of metabolic profiling is still in its infancy, its potential to enhance the understanding of physiological processes in mammalian cell lines used for antibody production is certain.     

Selection of a highly aggressive myeloma cell line by an altered bone microenvironment in the C57BL/KaLwRij mouse

In multiple myeloma (MM), bone marrow microenvironment has an important role for the survival and growth of plasma cells. We previously showed that a high bone turnover, induced by ovariectomy, increased MM cells growth in the 5T2MM model. The present study characterized a new plasma cell line (5THL) which was isolated from 5T2MM mice previously ovariectomized. Cells were propagated unchanged in normal C57BL/KaLwRij mice during six generations. 5THL was compared to the original 5T2MM phenotype. Paraproteinemia was detected 6 weeks post injection in 5THL mice and after 8 weeks in 5T2MM mice. All 5THL mice developed a hind-limb paralysis after 10 weeks. 5T2MM mice were euthanized at 16 weeks, due to a more progressive development of the disease. In 5THL mice, osteolytic lesions were observed after 8 weeks and severe bone destruction was evidenced at 10 weeks. In 5T2MM mice, minimal lesions were observed only after 10 weeks. Like in 5T2MM mice, no extra osseous lesions were observed in 5THL mice. The 5THL MM model closely mimics human myeloma with higher and faster bone aggressiveness. This new aggressive cell line, with a preserved phenotype, was selected by an altered microenvironment due to an increased bone turnover.     

Metabolic and proteomic study of NS0 myeloma cell line following the adaptation to protein-free medium

Proteomics and metabolomics technologies are potentially useful tool for the study of the very complex process of cell adaptation to protein-free medium. In this work, we used the iTRAQ technology to analyze different protein levels in adapted and non-adapted NS0 myeloma cell line. Several proteins with differential expression profile were characterized and quantified. Carbohydrate metabolism, protein synthesis and membrane transport were the principal pathways that change after the adaptation. Changes in lactate production rate with respect to glucose consumption rate were observed according to the changes observed by proteomic.     

Sialyltransferase activity in AF10 myeloma cell line

A STA was observed in a human-derived myeloma cell line, AF10, that produces IgE paraprotein. The STA in the culture medium of the AF10 myeloma cell line was associated in the 24 to 48 hr period of incubation with IgE biosynthesis, and increased thereafter up to 72 hr, while IgE remained stable. These data support our previous observations that human myeloma cells are associated with a relatively high production of sialyltransferase, which is released to the medium, presumably by a mechanism of shedding.     

Nucleotide sequence of an unequal sister chromatid exchange site in a mouse myeloma cell line.

The mouse myeloma cell line MPC 11 carries two C gamma 2a immunoglobulin heavy-chain genes on the expressed chromosome, a duplication shown to have occurred through unequal sister chromatid exchange (USCE). In the present report, we present the nucleotide sequence of the USCE joint and show that both breaks occurred within tracts of repeated TC dinucleotides. Additional TC dinucleotide tracts and two oligonucleotide segments (N sequences) were inserted at the USCE site.     

Novel nucleolar and nuclear morphology in a vincristine-dependent human leukemia cell line (L100).

Acquired resistance to chemotherapeutic drugs by tumor cells is an important obstacle to effective therapy of human malignancy. We now describe a vincristine (VCR)-induced multidrug-resistant (MDR) human acute lymphatic leukemia cell line, the sustained in vitro growth of which is dependent on vincristine. The doubling time for parental drug-sensitive cells (L0) is 40.2 +/- 13.2 h and for the MDR subline (L100) 62.5 +/- 11.3 h. L100 cells have similar G2 and mitotic phase to parental cells, express the MDR phenotype, and are characterized by novel morphologic features with multilobulated nuclei and multiple small nucleoli. Compared with L0 cells which have 2-3 nucleoli per cell, L100 cells have 7-8 nucleoli per cell. Average nucleolar area is 11.3 +/- 7.3 microns 2 for L0 and 2.5 +/- 2.4 microns 2 for L100 cells determined by the laser scanning method. The striking morphologic abnormalities of L100 cells suggest a drug-induced cytoskeletal abnormality. The relationship of these abnormalities to the VCR growth dependence of L100 cells is discussed.     

Correlation between nuclear morphology and rate of deoxyribonucleic acid synthesis in a normal cell line.

Chinese hamster fibroblasts were investigated for the existence of correlations between proliferative activity and nuclear morphology. As a proliferative parameter, the rate of DNA synthesis of individual cells was determined by quantitative 14C-autoradiography. In a second step the images of the Feulgen-stained nuclei were digitized for extraction of features of morphology and texture. These features were correlated with the corresponding DNA synthesis rate values. The following relationships were found: Round nuclei have higher rates of DNA synthesis than flat ones. The more chromatin is packed at the nuclear rim, possibly representing heterochromatin, the lower the rate of DNA synthesis. The DNA synthesis rate also correlates with the graininess of chromatin. Larger areas of condensed chromatin are associated with lower rate values. A fine and irregular network of chromatin, as is typical of immature cell types, is associated with a high rate of DNA synthesis. Although these results are presently confined to the cell line investigated, parallels seem to exist to other cell types, such as erythropoietic cells, which await further investigation.     

Chromosome mapping in cattle using mouse myeloma/calf lymph node cell hybridomas

A study correlating the presence of bovine isozymes in mouse myeloma/calf hybridomas with specific banded chromosomes of their bovine complement has enabled tentative assignments to be made of the bovine isozyme locus for peptidase C (PEP C) to chromosome 5 and the syntenic group lactate dehydrogenase B/peptidase B (LDH B/PEP B) to chromosome 19. There was some evidence for the association of LDH A with one of the last seven small pairs (23–29) of the complement and of superoxide dismutase 1 (SOD 1) with chromosome 13.