Fusao Makishima

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG1 which is reported stable, and IgG1 production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM λ1 chain and cultured at nonpermissive temperature to enhance production of λ1. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.

Partial purification and characterization of phospholipid N-methyltransferases from murine thymocyte microsomes☆

Significant amounts of phospholipid N-methyltransferase activity in murine thymocytes were found to be distributed on the plasma membrane. The enzyme activity had an optimum pH of 9. The presence of divalent cations, Mg2+ (10 mM) or Ca2+ (1 mM), and EGTA separately in the assay had only a small effect on the enzyme activity. However, addition of both 10 mM Mg2+ and 1 mM Ca2+ increased the enzyme activity. The presence of two enzymes for each conversion of phosphatidylethanolamine (PE) to phosphatidylmonomethylethanolamine (PME) and PME to phosphatidylcholine (PC) was suggested by the result of the determination of the incorporated radioactivity into PME, phosphatidyldimethylethanolamine (PDE) and PC; the apparent Km values for S-adenosyl-L-methionine were 20 and 400-500 microM for the conversion of PE to PME and for the conversion of PME to PC they were 5 microM and 40 microM. S-Adenosyl-L-homocysteine (AdoHcy), a known inhibitor of enzymatic methylation, competitively inhibited [14C]methyl incorporation into total lipid. The apparent Ki value for AdoHcy was 44.7 microM. Two phospholipid N-methyltransferases were partially purified by extraction with sodium deoxycholate, gel filtration on Sephadex G-75, and affinity column chromatography on AdoHcy-Sepharose. One enzyme, mainly catalyzing the formation of PME, was purified approximately 1548-fold and the other catalyzing the formation of PDE and PC, was purified approximately 629- to 703-fold. However, the former still contained a little activity for PDE and PC formation and the latter contained a little activity for PME formation. In these partially purified phospholipid N-methyltransferase preparations, little contaminating protein O-carboxylmethyltransferase activity was observed; however, significant PC-phospholipase A2 activity was detected. This result may suggest that phospholipid N-methyltransferases associate with phospholipase A2 in the thymocyte plasma membrane.

Enhancingin vitro antibody production rate per cell by applying mouse peritoneal factors

SummaryMonoclonal antibody production rateper hybridoma cell was measured as much larger in mouse peritoneal cavity thanin vitro culture. To identify factors enhancing antibody productivity per cell, a hybridoma was culturedin vitro with ascites and peritoneal exudate cells(PEC). Both the ascites and the PEC improved the productivity per cellin vitro. Each chromatograph-fraction of ascites was assayed for the enhancing activity.

Enhancing effect of mouse peritoneal exudate cells and their products on antibody productivity of hybridoma cells: Application of in vivo factors to in vitro culture

Mouse peritoneal exudate cells induced by casein enhanced in vitro antibody production rate per cell of a hybridoma in co-culture. Culture supernatant of the exudate cells also enhanced three-fold the antibody productivity when added to cultures of a hybridoma at 10% (v/v). Hence the enhancement of antibody productivity by the exudate cells seemed to be caused by soluble enhancing factors secreted by the exudate cells. The exudate cells maximally secreted the enhancing factors when harvested from mice on day 4 of the induction period following the injection of casein. A semi-continuous culture of the hybridoma demonstrated the applicability of the culture supernatant to enhance antibody production by producing a two-fold increase over the control for seven days when supplemented with the supernatant at 5%. Significant amounts of interleukin-6 were detected in culture supernatant of the exudate cells. Interleukin-6 obtained from other sources enhanced the antibody productivity two-fold when added to the hybridoma culture at the concentration of 5 unit/ml. Interleukin-6, therefore, is expected to be one of the principal antibody enhancing factors secreted by the exudate cells. Other interleukins examined, that is, interleukin-1 to-5 did not enhance the antibody productivity.

Induction of increased calcium uptake in liposomes having membrane proteins of chicken erythrocytes by S-adenosylmethionine

Liposomes having membrane proteins of chicken erythrocytes were prepared and the effect of S-adenosylmethionine on 45Ca2+ uptake into the liposomes was investigated. S-Adenosylmethionine, a donor of methyl groups in enzymatic methylation, induced an increase of 45Ca2+ uptake into the proteoliposomes with membrane proteins but not into the liposomes without membrane proteins. Increased release of 45Ca2+ from the inside of the proteoliposomes was also induced by S-adenosylmethionine. These increases of uptake and release of 45Ca2+ were inhibited by S-adenosylhomocystein, an inhibitor of enzymatic methylation. Furthermore, membrane proteins from chicken erythrocytes showed protein and phospholipid methyltransferase activities. The uptake of other materials, 3-0-[methyl-3H]glucose, alpha-[1-14C]aminoisobutyric acid, 42K+ and 54Mn2+, into the proteoliposomes was not increased by S-adenosylmethionine. These results suggest that enzymatic methylation of membrane components may have an important role in the regulation of calcium transport in the chicken erythrocyte membrane and this regulation is rather specific for calcium.

Enhancing Effect of Interleukin-6 on Antibody Productivity of a Hybridoma Cell Line

For the commercial scale production of monoclonal antibody (MoAb) by hybridoma cells, enhancement of production rates in unit volume and in unit number of cells is desirable and important. Interleukin-6(IL-6) is a cytokine, which induces the final maturation of B cells into Ig-secreting cells, the production of acute phase proteins by hepatocytes, etc. Though IL-6 has been shown to stimulate growth of hybridoma cells, we found that purified recombinant human IL-6 (rhIL-6) suppressed growth rate of a hybridoma cell line (2E3-O) cultured in serum-free medium. The cells could be in a viable state for a remarkably long period in the growth suppressed batch culture. And the IL-6 also enhanced MoAb production rate per cell. At the optimum condition the cells cultured with rhIL-6 produced about six times as much MoAb as the control. In a perfusion culture, the cell cultured with rhIL-6 produced about twice as much MoAb as the control.

Statistical Mechanical Approach for Predicting the Transition to Non-B DNA Structures in Supercoiled DNA

Supercoiling causes global twist of DNA structure and the supercoiled state has wide influence on conformational transition. A statistical mechanical approach was made for prediction of the transition probability to non-B DNA structures under torsional stress. A conditional partition function was defined as the sum over all possible states of the DNA sequence with basepair 1 and basepair n being in B-form helix and a recurrence formula was developed which expressed the partition function for basepair n with those for less number of pairs. This new definition permits a quick enumeration of every configuration of secondary structures. Energetic parameters of all conformations concerned, involving B-form, interior loop, cruciform and Z-form, were included in the equation. The probability of transition to each non-B conformation could be derived from these conditional partition functions. For treatment of effects of superhelicity, supercoiling energy was considered, and a twist of each conformation was determined to minimize the supercoiling energy. As the twist itself affects the transition probability, the whole scheme of equations was solved by renormalization technique. The present method permits a simultaneous treatment of several types of conformations under a common torsional stress. A set of energetic parameters of DNA secondary structures has been chosen for calculation. Some DNA sequences were submitted to the calculation, and all the sequences that we submitted gave stable convergence. Some of them have been investigated the critical supercoil density for the transition to non-B DNA structures. Even though the reliability of the set of parameters was not enough, the prediction of secondary structure transition showed good agreement with reported observation. Hence, the present algorithm can estimate the probability of local conformational change of DNA under a given supercoil density, and also be employed to predict some specific sequences in which conformational change is sensitive to superhelicity.

Efficient selection of μ m − mutants from μm-expressing myeloma cells by treatment with ricin A-conjugated anti-μ antibody

We have developed an efficient system for obtaining myeloma mutants defective intrans-acting factors required for immunoglobulin (Ig) gene expression. The system consists of a myeloma cell line designed for this purpose and an efficient method for selecting mutants from it. The cell line is X63.653 transfected with the μ gene, whose tailpiece sequence was replaced with the transmembrane sequence of human EGF receptor to hold μ on the cell surface and whose CH1 sequence was removed to prevent μ from being retained in the endoplasmic reticulum. It efficiently and stably expressed μ chains of IgM on the cell surface (μm+) without light chains. To obtain mutants lacking μm (μm−) from the μm+ cell line by selectively killing μm+ cells, a method with ricin A-conjugated anti-μ antibody was more reliable than complement lysis mediated by anti-μ antibody. Applying the system, we obtained a variety of μm− mutants.

EFFECT OF CYTOKINES ON GROWTH OF A MOUSE HYBRIDOMA CELL LINE AND ITS ANTIBODY PRODUCTION

Three cytokines, interleukin 6 (IL-6), leukemia inhibitory factor(LIF), and oncostatin M (OSM), that bind to composite receptors including a common signal transducer gpl30 suppressed growth of a mouse B-cell hybridoma cell line 2E3-O cultured in serum-free medium, while they enhanced antibody production of the cells. The specific growth rate of the cells reduced from 1.0 day-1 for control to 0.6 day-1 for the cultures supplemented with 1 ng/ml of IL-6, 4ng/ml of LIF, or 20ng/ml of OSM, respectively. The antibody productivity increased five-fold when the cells were cultured with l ng/ml of IL-6, 25ng/ml of LIF, or 20ng/ml of OSM, respectively. Transforming growth factor β 1 (TGF- β 1) similarly suppressed growth of the cells at the concentration of 5 ng/ml, while it did not enhance the antibody production. As a whole, this work suggests that gp-130, which is commonly involved in each receptor for IL-6, LIF, and OSM, transduce signals for suppressing growth and possibly for enhancing antibody production in the hybridoma cells.

Selection of Regulatory Mutants of μ Gene Expression from Myeloma Cells Transfected with the Modified μ Gene

Cells of the murine immunoglobulin non-producing myeloma cell line could stably express the μ chain on the surface (µm) without a light chain by transfection of the modified µ gene that had the transmembrane sequence of the human epidermal growth factor receptor and that lacked the CH1 region. We aim to find regulatory mutants of the μ gene expression in the μm - mutants that are selected from μm + cells harboring the modified μ gene on the Bovine papillomavirus vector as extrachromosomal DNA. We established the method of selectively killing μm + cells with ricin A-conjugated anti-μ antibody for efficient selection of the μm - mutants.