Karyotypic characteristics of a number of clonal lines of an interspecific mouse-mink hybridoma

Using G-banding method, a study was made of the karyotypes of mouse myeloma cell line P3-X63-Ag8.653 and some cell hybrid lines originated from a fusion of mink immunized spleen cells and this myeloma. Normal chromosomes 6, 12 and X were not detected in either examined cell of the parental myeloma. The mink chromosomes are preferentially lost from cells of the hybrid lines. These lines varied significantly from cell to cell and from each other in the retention of some mink chromosomes. The karyological study of the hybrid lines revealed some cytogenetical specificities of mouse chromosome composition that were general for cells of hybrid lines, but atypical for myeloma parental cells. This data suggest the cells of myeloma parental line to take part in the processes of somatic hybridization and/or selection for mink immunoglobulin production nonrandomly.     

Maintenance of Fc receptors in hybrid cell lines obtained by fusing mouse splenic macrophages with mouse myeloma cells.

Mouse splenic macrophages were fused with cells of the mouse myeloma line P3-X63-Ag8 in the presence of inactivated Sendai virus. Two continuously growing hybrid cell lines were established from fusion mixtures. These hybrid cell lines exhibited macrophage-like morphology and continued to express macrophage derived Fc receptor activity even after prolonged culture in vitro.     

Morphocytochemical and electron microscopic research on murine hybridoma cells producing and not producing monoclonal antibodies

Cytochemical and ultrastructural features of mouse hybridomas and also of the parental cells--myeloma P3-X63-Ag8.653 and spleen cells of the Balb/c mice immunized with cell line RPMI-1788 have been studied. Differences in cytomorphological signs and activity of acid phosphatase, acid nonspecific esterase, nonspecific-alpha-naphthyl acetate esterase were shown in hybrid cell lines secreting and not secreting monoclonal antibodies.     

Enhanced formation of mouse hybridomas without hat treatment in a serum-free medium

Summary A newly developed, serum-free medium (NYSF-404) selects for antibody-producing hybridomas after fusion of antigen-sensitized mouse spleen cells with myeloma cell lines P3-X63-Ag8-U1 (P3-U1), P3-X63-Ag8-6.5.3 (Ag8.653), or P3-NSI/1-Ag4-1 (NS-1). Without the need for hypoxanthine-aminopterinthymidine (HAT) selection of hybrid cells, frequency of hybridoma formation in medium NYSF-404 is higher (twice) than that in serum- and HAT-containing medium. Colonies developed upon limiting dilution in the presence of the mortal parent myeloma cells in medium NYSF-404 and pure culture of antibody-secreting cells could be subsequently established. The results suggest that fusions can be done in serum-free medium and that the clonal growth of hybridomas is dependent on factors produced by parent myeloma cells under serum-free culture conditions. Such factors seem deficient in serum- and HAT-containing medium or are masked by serum.     

Prolongation of murine hybridoma cell survival in stationary batch culture by Bcl-xL expression

While the ectopic expression of the anti-apoptoticprotein Bcl-2 has been shown to significantly increaseboth cell viability and antibody production in batchculture, some cell lines are refractory to thesemanipulations. For example, the NS/O and theP3x63Ag8.653 murine myelomas, which express highendogenous levels of the Bcl-2 homologue Bcl-xL, areboth resistant to the anti-apoptotic effect of Bcl-2.This indicates that, in these cells, Bcl-2 and Bcl-xLmay be functionally redundant. In order to define therole which Bcl-xL plays in hybridoma cultures, we usedthe Sp2/0-Ag14 cell line. This murine hybridomaexpresses low levels of Bcl-xL and is highly sensitiveto apoptosis induction by cycloheximide (CHX) and byamino acid depletion. Bcl-xL-transfected Sp2/0-Ag14cells were more resistant than the wild type and theplasmid-containing cells to apoptosis induced by CHXand by glutamine depletion. Moreover, when compared tothe vector-transfected control, Bcl-xL-Sp2/0 cellsexhibited a substantial increase in viability instationary batch culture. Interestingly, Sp2/0-Ag14cells overexpressing Bcl-xL showed a growth behaviourthat was similar to the parent myeloma cell lineP3x63Ag8.653. Our results suggest that Bcl-xLexpression levels are sufficient to account for therelative robustness of some hybridoma cell lines instationary batch cultures.     

Growth and protein production kinetics of a murine myeloma cell line transfected with the human growth hormone gene

A model mammalian cell system for the production of recombinant proteins was investigated. Murine myeloma cells which had lost the ability to produce both heavy and light chain immunoglobulin molecules were transfected with a vector containing the immunoglobulin heavy chain promoter and enhancer elements linked to the human growth hormone gene. The growth kinetics of G32, a clonal isolate, were found to be similar to both the parent myeloma and hybridomas. However, production of hGH by G32 was growth associated, rather than as a secondary metabolite as is the case for hybridomas. In addition, G32 produced hGH at molar levels greater than most hybridomas.Abbreviations ELISA Enzyme Linked Immunosorbent Assay - Ig Immunoglobulin - MAb Monoclonal Antibody - X63 Murine Myeloma Cell Line P3X63-Ag8.653     

Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures

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.     

Demonstration of non-histone nuclear proteins in mouse myeloma. Trial comparative study

The non-histone chromosomal proteins from mouse myeloma cell line X 63-Ag 8.653 have been studied using two dimensional polyacrylamide gel electrophoresis. Our results were compared to previous analysis of other myeloma cell lines.     

Lack of lamins A and C in mammalian hemopoietic cell lines devoid of intermediate filament proteins

Using immunofluorescence microscopy and immunoblot analysis, we have examined the composition of the nuclear lamina in several murine and human cell lines. Whereas it was shown that intermediate filament-positive Ehrlich ascites tumor and HeLa-S3 cells contain the three major mammalian lamin subspecies, only lamin B could be detected in several myeloid- and lymphoid-derived cell lines representative of distinct stages in hemopoietic differentiation but all devoid of cytoplasmic intermediate filament proteins. These included the murine plasmacytoma cell types MPC-11 and MOPC-31C, murine myeloma cells X63-Ag8.6.5.3 and human promyelocytic leukemia cells HL-60. Our results provide the first evidence that mammalian somatic cells capable of normal proliferation may lack both cytoplasmic intermediate filament proteins and a normal complement of nuclear lamins.     

Differential expression of the c-myb proto-oncogene marks the pre-B cell/B cell junction in murine B lymphoid tumors

A series of murine B lymphoid tumor cell lines which are representative of the pre-B cell, immature and mature B cell, and plasma cell stages of B cell development have been examined for expression of c-myb proto-oncogene mRNA. The pre-B cell lymphoma cell lines express equivalent high steady state levels of c-myb mRNA. In contrast, the B cell lymphoma and plasmacytoma cell lines express steady state c-myb mRNA at levels which are 0.005 to 0.1 times that of the pre-B cell lymphoma lines. These results correlate high levels of c-myb mRNA expression with the pre-B cell stage of development. Subclones of the 1881 pre-B cell lymphoma which express K light chain and are surface IgM-positive as well as two types of hybrid B lymphoid cell lines have been used to demonstrate that surface immunoglobulin expression is not sufficient to result in the down-regulation of c-myb mRNA levels or changes in the expression N-myc mRNA, lambda 5 mRNA, or the BP-1 surface antigen which are markers of the pre-B cell stage of development. Thus, changes in the expression of genes which are independent of immunoglobulin expression are associated with transition from the pre-B cell to the immature B cell stage of development.     

Double immunoglobulin production in cloned somatic cell hybrids between two human lymphoid cell lines.

Several clones of independently established somatic cell hybrids between two human lymphoid cell lines, Raji and Namalwa, were examined for surface immunoglobulin expression. Double-antibody radioimmunoassays were established for kappa and lambda light chains. Immunoglobulins were detergent-extrated by Triton X-100 and quantified by radioimmunoassay. The Raji parent expressed small amounts of kappa chains on its surface, and the Namalwa parent a 10 fold greater amount of lambda chains. We show that the majority of the hybrid clones co-express both parental phenotypes.     

Rat monoclonal antitubulin antibodies derived by using a new nonsecreting rat cell line

Hybrid myeloma cell lines secreting monoclonal antibodies to tubulin have been prepared using rat myelomas and spleen cells from rats immunized with yeast tubulin. A comparison between the results obtained with the rat myeloma Y3-Ag 1.2.3., which secretes a light chain, and a new line, YB2/O, which does not, shows that they are both excellent parental lines and that the second produces hybrids with no myeloma chain components. The antitubulin antibodies in the serum of rats bearing two of the hybrid myeloma tumors gave titers of up to 1:10(6) from which large amounts of monoclonal antibodies could be easily purified. They recognized tubulin from yeast as well as from birds and mammals. The two antibodies gave clear immunofluorescent staining of yeast mitotic spindles as well as the interphase microtubule network of tissue culture cells. Some difference in the pattern of immunofluorescence staining of yeast cells and nuclei was observed between the two antibodies. The purified antibodies could be conjugated to colloidal gold particles and used for direct labeling of yeast microtubules for electron microscopy.     

An inhibitor of tumor cell growth from normal horse serum

Summary During our studies of cytostatic cytokines in the mixed leukocyte culture, we found that horse serum in the medium control contained a tumor cell growth-inhibitory factor. The fraction isolated by molecular sieving and ion exchange chromatography inhibited the growth and DNA synthesis of the primary culture and passaged cell line of the canine transmissible venereal sarcoma, murine T (L5178Y) and B (P3-X63-Ag8.653) lymphoid tumor cells, murine mammary tumor cells (RIII), bovine lymphoid tumor cells (BL3), and the nontransformed cell line of baby hamster kidney cells. Nontransformed cell lines such as African green monkey kidney (Vero) and Madin-Darby bovine kidney cells and normal mammary cells of the dog and goat were inhibited only at high concentrations. The active component is a protein with an R_f value of 0.09 upon electrophoresis in native 7.5% polyacrylamide gels. The eluate from the native gel could be further separated into three polypeptides with molecular weights of approximately 67, 65, and 55 kDa under reduced conditions in sodium dodecyl sulfate-polyacrylamide gels.     

A novel homodimeric molecule involved in human T cell activation

A mAb, 10D1, was obtained by fusing spleen cells from BALB/c mice immunized with a CD3/TCR- human T cell line, P12/ichikawa, to mouse myeloma cells, P3X63-Ag8-653. 10D1 mAb is specific for T cells in that it reacted with all the T cell lines tested, but not with B or myeloid cell lines. A small fraction of normal peripheral blood T cells, preferentially CD4+, was also reactive with 10D1 mAb. Biochemical studies revealed that 10D1 mAb recognizes a disulfide-linked homodimeric molecule composed of 90-kDa polypeptide. 10D1 mAb induced a substantial proliferation of peripheral blood T cells when cross-linked with goat anti-mouse Ig antibody. The elimination of CD4+ cells totally abrogated the proliferative response induced by 10D1 mAb, whereas the elimination of CD8+ cells rather enhanced it. The proliferative response of peripheral blood T cells induced by 10D1 mAb was almost completely inhibited after modulation of the CD3/TCR complex with anti-CD3 mAb. In addition, a prompt increase in intracellular [Ca2+] was observed in a CD3+ T cell line, Jurkat but not in its surface CD3- mutant when 10D1 mAb was added. These results indicate that the 10D1 molecule is involved in a novel pathway of human CD4+ T cell activation, which is associated with the CD3/TCR-mediated pathway.     

Immunologic characterization of hairy cell leukemias in continuous culture.

The immunobiologic characteristics of three continuous cell lines established from hairy cell leukemia cells were investigated. All three cell lines continued to produce tartrate-resistant acid phosphatase, the enzymatic marker of hairy cells. Two of these cell lines were B lymphoid in nature. They carried Fc and C receptors, had surface and internal immunoglobulin, and did not form spontaneous sheep red blood cell rosettes. Experiments employing biosynthetic radiolabeling of immunoglobulin demonstrated distinctive immunoglobulin kinetics for each of these two hairy cell lines. One cell line remained quite similar to the original hairy cells from which it was derived whereas the other B lymphoid hairy cell line had undergone a switch in the immunoglobulin isotype produced. The third hairy cell leukemia line was shown to be of thymic derivation. These cells formed spontaneous sheep red blood cell rosettes and did not carry Fc or C receptors. The spontaneous sheep red blood cell rosette-forming cells contained tartrate-resistant acid phosphatase. They did not possess surface on internal immunoglobulin and did not synthesize immunoglobulin in vitro. Hairy cell leukemia cells maintained in permanent cell culture retain their immunobiologic properties and offer the opportunity for indepth study of these unusual cells.     

Serum-free growth medium for the cultivation of a wide spectrum of mammalian cells in stirred bioreactors

A serum free medium was developed, that could be used for the large scale propagation of various cell lines in bioreactors. The medium is based on a 1:1 mixture of Iscove's Modified Dulbecco's Medium and Ham's Medium F12, supplemented with transferrin, insulin and a BSA/oleic acid complex. Several myelomas, hybridomas derived from different myelomas and spleen cells, and other lymphoid and non-lymphoid cell lines were cultivated at growth rates comparable to those observed using serum-supplemented media. There was furthermore no reduction in the formation of products such as monoclonal antibodies or recombinant human interleukin-2.Abbreviations Ag8 Mouse myeloma cell line P3-X63-Ag8.653 - BME Basal Medium Eagle - BSA Bovine Serum Albumin - DMEM Dulbecco's Modified Eagle's Medium - EDTA Ethylenediaminete-traacetic Acid - e-PC Phosphatidyl choline from egg yolk - FCS Fetal Calf Serum - FGF Fibroblast Growth Factor - GHL Glycyl-histidyl-lysine - HDL High Density Lipoprotein - HPL Human Plasma Lipid - IF 1:1 mixture of IMDM and Ham's F12 - IMDM Iscove's Modified Dulbecco's medium - LDL Low Density Lipoprotein - NS1 Mouse myeloma cell line NSI-1-Ag4-1 - PBS Phosphate Buffered Saline - s-PC Phosphatidylcholine from soy beans - s-PE Phosphatidylethanolamine from soy beans - s-lecithin lecithin from soy beans     

Properties and applications of monoclonal antibodies directed against determinants of they Thy-1 locus.

Fusion of cells of the mouse myeloma line, P3/X63-Ag8 with spleen cells from AKR/J mice immunized against C3H thymocytes or from (BALB/c x BALB.K)F1 mice immunized against AKR/J thymocytes gave rise to hybrid cell lines that continuously secrete antibodies specific for the Thy-1.2 and Thy-1.1 antigens, respectively. Monoclonal antibodies from four such cell lines were analyzed in detail. All were 19S IgM, and, in the presence of complement (C), had high lytic titers on T cells of the appropriate antigenicity. Their specificity was shown by lysis of thymocytes from Thy-1 congenic mouse strains, A/J(Thy-1.2) and A. Thy 1.1. Furthermore, they lyse only 60 to 70% of lymph node cells, suggesting cytotoxicity for mature T cells and not B cells. Treatment of peripheral lymphocyte populations with monoclonal antibody plus C eliminated effector cytotoxic T lymphocytes, their precursors, and the mitogenic response to Con A, but did not affect the response to LPS. Purified, fluorescein-labeled monoclonal anti-Thy-1 antibody could be used to distinguish T and B cells. Purified antibody coupled to Sepharose 6MB was used to separate viable T and B cells. Two independently isolated anti-Thy-1.2 hybridomas are indistinguishable and bind the same determinant whereas a third is unique and may bind a separate site.     

A new mammalian host cell with enhanced survival enables completely serum-free development of high-level protein production cell lines

With over 25 monoclonal antibodies (mAbs) currently approved and many more in development, there is considerable interest in gaining improved productivity by increasing cell density and enhancing cell survival of production cell lines. In addition, high costs and growing safety concerns with use of animal products have made the availability of serum-free cell lines more appealing. We elected to transfect the myeloma cell line Sp2/0-Ag14 with Bcl2-EEE, the constitutively active phosphomimetic mutant of Bcl2, for extended cell survival. After adaptation of the initial transfectants to serum-independent growth, a clone with superior growth properties, referred to as SpESF, was isolated and further subjected to iterative rounds of stressful growth over a period of 4 months. The effort resulted in the selection of a promising clone, designated SpESFX-10, which was shown to exhibit robust growth and resist apoptosis induced by sodium butyrate or glutamine deprivation. The advantage of SpESFX-10 as a host for generating mAb-production cell lines was demonstrated by its increased transfection efficiency, culture longevity, and mAb productivity, as well as by the feasibility of accomplishing the entire cell line development process, including transfection, subcloning, and cryopreservation, in the complete absence of serum.