Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Sato, Jun; Cao, Hui-ting; Kayada, Yoshiaki; Cabot, Myles C.; Sato, Gordon; Okamoto, Tomoyoshi; Welsh, Clement J.

doi:10.1007/bf02624194

Cited by 25 publications

(17 citation statements)

References 19 publications

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“…Decreased cholesterol levels in multiple myeloma patients have been previously described, probably representing increased LDL clearance and utilization of lipids and cholesterol by myeloma cells (24). In fact, it has been shown that NS0 myeloma cells are naturally cholesterol dependent; probably due to a biochemical deficiency in the demethylation of lanosterol to C-29 sterols (25,26). Previous studies (27,28) have also shown that lysine and glutamine are absolutely required for myeloma cells growth.…”

Section: Discussionmentioning

confidence: 91%

Multiple Myeloma Patients Have a Specific Serum Metabolomic Profile That Changes after Achieving Complete Remission

Puchades‐Carrasco

Lecumberri

Martínez‐López

et al. 2013

Clinical Cancer Research

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Purpose: Multiple myeloma remains an incurable disease. New approaches to develop better tools for improving patient prognostication and monitoring treatment efficacy are very much needed. In this study, we aimed to evaluate the potential of metabolomics by 1 H-NMR to provide information on metabolic profiles that could be useful in the management of multiple myeloma. Experimental Design: Serum samples were collected from multiple myeloma patients at the time of diagnosis and after achieving complete remission. A matched control set of samples was also included in the study. The 1 H-NMR measurements used to obtain the metabolic profile for each patient were followed by the application of univariate and multivariate statistical analyses to determine significant differences.Results: Metabolic profiles of multiple myeloma patients at diagnosis exhibited higher levels of isoleucine, arginine, acetate, phenylalanine, and tyrosine, and decreased levels of 3-hydroxybutyrate, lysine, glutamine, and some lipids compared with the control set. A similar analysis conducted in multiple myeloma patients after achieving complete remission indicated that some of the metabolic changes (i.e., glutamine, cholesterol, lysine) observed at diagnosis displayed a variation in the opposite direction upon responding to treatment, thus contributing to multiple myeloma patients having a closer metabolic profile to those of healthy individuals after the disappearance of major disease manifestations.Conclusions: The results highlight the potential of metabolic profiles obtained by 1 H-NMR in identifying multiple myeloma biomarkers that may be useful to objectively discriminate individuals with and without multiple myeloma, and monitor response to treatment.

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Section: Discussionmentioning

confidence: 91%

Multiple Myeloma Patients Have a Specific Serum Metabolomic Profile That Changes after Achieving Complete Remission

Puchades‐Carrasco

Lecumberri

Martínez‐López

et al. 2013

Clinical Cancer Research

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“…Replacement of bovine lipids with non-animal lipid mix and elimination of rHA NS0 cells are auxotrophic for cholesterol (Sato et al 1988;Keen and Steward 1995). While the medium described above were free from animal-derived insulin, transferrin and albumin, they still contained Excyte, a bovine lipoprotein product, as the lipid source for NS0 cultures.…”

Section: Resultsmentioning

confidence: 99%

“…NS0 cells were derived from NS-1 cells that were deficient in 3-ketosteroid reductase activity thus unable to convert lathosterol to cholesterol (Sato et al 1988). Keen and Steward (1995) found that a large portion of the NS0 population were able to adapt to cholesterol-independence, suggesting that this adaptation was likely due to the reactivation of the relevant gene rather than due to a revertant mutation.…”

Section: Applicability To Cryopreservationmentioning

confidence: 99%

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture

Zhang

Robinson

2005

Cytotechnology

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There has been a recent boom of monoclonal antibodies on the market, and a significant portion of them were produced by NS0 cell lines. As regulations become more stringent in ensuring production processes are free of potential contamination by adventitious agents, it is highly desirable to further develop serumfree media into ones that do not contain any components of animal origin, or 'animal-free media'. Using a shake-flask batch culture system, recombinant proteins (human albumin and human insulin) and synthetic compounds (tropolone and ferric ammonium citrate) were identified to be capable of replacing the animalsourced proteins commonly found in serum-free media for NS0 cell culture, namely bovine albumin, insulin and transferrin. The cholesterol requirement of NS0 cells was satisfied by the use of a commercially available non-proteinaceous, non-animal sourced cholesterol/fatty acid mix in place of bovine lipoproteins, which in effect also eliminated the need for recombinant albumin. In the animal-free medium thus formulated, NS0 cell lines, either the host or recombinant constructs, were all able to grow in batch culture to 1$ 3 · 10 6 viable cells/ml for multiple passages, with no requirement for gradual adaptation even when seeded from 10% serum-containing cultures. It was surprising to observe that the recombinant insulin was essentially ineffective as sodium salt compared to its zinc salt. Studies showed that the zinc deficiency in the former resulted in a rapid decline of cell viabilities. Supplementation of zinc ions greatly improved growth, and even led to the total replacement of recombinant insulin and hence the formulation of a protein-free medium. When the cell lines were adapted to cholesterol-independent growth which eliminated the need for any lipid source, a completely chemically-defined animal-free medium was formulated. In all cases, antibody production by various GS-NS0 constructs in animal-free media was stable for multiple passages and at least similar to the original serum-free medium containing the animal-sourced proteins. The medium also served well for cryopreservation of NS0 cells in the absence of serum.

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“…However, despite the success of Birch et al, to the best of our knowledge this is the first industrial application to demonstrate the successful use of cholesterol-free, protein-free NS0 cells for recombinant antibody production. The underlying mechanism of cholesterol auxotrophy in NS-1 cells (the precursor to NS0) has been identified as a deficiency in 3-ketosteroid reductase (Sato et al, 1988). Recently, it has been reported that cholesterol auxotrophy in NS0 cells is due to reduced expression of the Hsd17b7 gene, encoding a 3-ketosteroid reductase (Seth et al, 2006a).…”

Section: Discussionmentioning

confidence: 98%

“…85110503), require serum for growth. NS0 cells, like the other P3K-derived subclones (P3-X63/Ag8 and NS-1), require exogenous cholesterol, which must be supplemented to the growth medium either on its own or via a natural or synthetic carrier such as serum, low density lipoprotein (LDL), bovine serum albumin (BSA), or b-cyclodextrin (Gramer and Maas, 2003;Sato et al, 1988;Walowitz et al, 2003). An alternative strategy using plant sterols complexed to b-cyclodextrin has also been described (Gorfien et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

Derivation and characterization of cholesterol‐independent non‐GS NS0 cell lines for production of recombinant antibodies

Hartman

Sar

Genereux

et al. 2006

Biotech & Bioengineering

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Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.

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Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Cited by 25 publications

References 19 publications

Multiple Myeloma Patients Have a Specific Serum Metabolomic Profile That Changes after Achieving Complete Remission

Multiple Myeloma Patients Have a Specific Serum Metabolomic Profile That Changes after Achieving Complete Remission

Development of Animal-free, Protein-Free and Chemically-Defined Media for NS0 Cell Culture

Derivation and characterization of cholesterol‐independent non‐GS NS0 cell lines for production of recombinant antibodies

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