Abstract
We found that when the hybridoma cell line HB4C5 was stimulated with wheat germ agglutinin (WGA), loss of production of the original λ light chain occurred, followed by production of new light chain, which mirrored the reaction when stimulated with concanavalin A (ConA). We previously reported that the RAG genes are expressed not only in HB4C5 and its ConA-treated variant subclones, but also in the in the parental Namalwa cells, which are known to be in the plasma state. However, the new λ light chains were expressed only in the HB4C5 cells and not in the parental Namalwa cells. Here we found that the RAG genes are expressed in HB4C5 cells after continuous stimulation with WGA. To further investigate the mechanism of this loss of original λ light chain production by stimulation with lectins in HB4C5 cells, which leads to a sIg-negative subpopulation, we analyzed the differences between HB4C5 and Namalwa cells. In this present study, we found that a 70 kDa phosphorylated protein in HB4C5 cells became undetectable after stimulation with lectins (WGA and ConA), and was not detected in Namalwa cells before or after lectin stimulation. It has been believed that the RAG genes and loss of original λ light chain production are required to induce expression of a new λ light chain in the HB4C5 cells. We suggested that the phosphorylated 70 kDa protein in HB4C5 cells play important roles in regulating the production of new λ light chains which is induced by lectins.
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References
Abe T, Tsuge I, Kamachi Y, Torii S, Utsumi K, Akahori Y, Ichihara Y, Kurosawa Y and Matsuoka H (1994) Evidence for defects in V(D)J rearrangement in patients with severe combined immunodeficiency. J Immunol 152: 5504–5513.
Bothwell A, Paskind M, Reth M, Imanish-Kari T, Rajewsky K and Baltimore D (1981) Heavy chain variable region contribution to the Npb family of antitodies: Somatic mutation evident in a γ 2a variable region. Cell 24: 625–637.
Cambier J, Justement L, Newell K, Chen Z, Harris L, Sandoval V, Klemsz M and Ransam J (1987) Transmembrane signals and intracellular ‘second messengers’ in the regulation of quiescent B lymphocyte activation. Immunol Rev 95: 37–42.
Chen Z, Coggeshall K and Cambier J (1986) Translocation of protein kinase C during membrane immunoglobulin-mediated transmembrane signaling in B lymphocytes. J Immunol 136: 2300–2304.
Feddersen R, Martin D and Van Ness D (1990) The frequency of multiple recombination events occurring at the human IgκL chain locus. J Immunol 144: 1088–1093.
Goldstein I, Hollerman C and Smith E (1965) Protein-carbohydrate interaction II, inhibition studies on the interaction of concanavalin A with polysaccharides. Biochemistry 4: 876–883.
Hesketh R, Moore T, Pennington S, Smith G and Metcalfe J (1990) Analysis of the primary signals required for activation of the mitogenic pathway in murine thymocytes from protein phosphorylation patteins. J Immunol 145: 2571–2580.
Kato T, Kokuho T, Tamura T and Nariuchi H (1994) Mechanisms of T cell contact-dependent B cell activation. J Immunol 152: 2130–2138.
Kitamura D and Rajewsky K (1992) Targeted disruption of M chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356: 154–156.
Krungkasem C, Tachibana H and Shirahata S (1997) Indentification of an antibody-secreting human B cell line undergoing ‘light chain shifting’. Animal cell technology: Basic and applied aspects Vol. 8, K Funatsu et al. (eds.) pp. 639–643, Kluwer Academic Press.
Larrick J, Danielsson L, Brenner C, Wallace E, Abrahamson M, Fry K and Borrebaeck C (1989) Polymerase chain reaction using mixed primers: cloning of human monoclonal antibody variable region genes from single hybridoma cells. Biotechnology 7: 934–938.
Lewis S, Rosenberg N, Alt F and Baltimore D (1982) Continuing kappa-gene reanangement in a cell line transformed by Abelson murine leukemia virus. Cell 30: 807–816.
Ma A, Fisher, Dildrop R, Oltz E, Rathbun G, Achacoso P, Stall A and Alt F (1992) Surface IgM mediated regulation of RAG gene expression in Eμ-N-myc B cell lines. EMBO J 11: 2727–2734.
Manz J, Denis K, Witte O, Brinster R and Storb U (1988) Feedback inhibition of immunoglobulin gene rearrangement membrane μ, but not secreted μ heavy chain. J Exp Med 168: 1363–1381.
Maxam A and Gilbert W (1980) Sequence end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65: 499–560.
Mitchell P and Tjian R (1989) Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245: 371–378.
Murakami H, Masui H, Sato G, Sueoka N, Chow T and Kano-Sueoka T (1982) Growth of hybridoma cells in serum-free medium: Ethanolamine is an essential component. Proc Natl Acad Sci USA 79: 1158–1162.
Murakami H, Hashizume S, Ohashi H, Shinohara K, Yasumoto K, Nomoto K and Omura H (1985) Human-human hybridomas secreting antibodies specific to human lung carcinoma. In Vitro. Cell Develop Biol 21: 593–596.
Nussenzweiz M, Shaw A, Sinn E, Danrer D, Holmes K, Morse H and Leder P (1987) Allelic exclusion in transgenic mice that express the membrane form of immunoglobulin. Science 236: 816–819.
Oettinger M, Schatz D, Gorka C and Baltimore D (1990) RAG-1 and RAG-2 adjacent genes that synergistically activate V(D)J recombination. Science 248: 1517–1523.
Osawa T and Tsuji T (1987) Fractionation and structural assessment of oligosaccharides and glycopeptides by use of immobilized lectins. Annu Rev Biochem 56: 21–36.
Papavasiliou F, Misulovin Z, Suh H and Nussenzweig M (1995) The role of Igβ inprecursor B cell transition and allele exclution. Science 268: 408–411.
Schatz D, Oettinger M and Baltimore D (1989) The V(D)J recombination activating gene, RAG-1. Cell 59: 1035–1048.
Schatz D, Oettinger M and Schlissel M (1992) V(D)J recombination: Molecular and regulation. Annu Rev Immunol 10: 359–383.
Stiernholm N and Berinstein N (1993) Up-regulated recombination-activating gene expression in sIg-variants of a human mature B cell line undergoing secondary Igλ rearrangements in cell culture. Eur J Immunol 23: 1501–1504.
Storb U (1987) Transgenic mice with immunologlobulin genes. Annu Rev Immunol 5: 151–174.
Tachibana H, Seki K and Murakami H (1993) Identification of hybritype carbohydrate chains on the light chain of human monoclonal antibody specific to lung adenocarcinoma. Biochem Biophys Acta 1182: 257–263.
Tachibana H, Kido I and Murakami H (1994) Heterogeneous expression of human antibody λ chains by concanavalin A-resistant hybridomas lead to changed antigen binding. J Biol Chem 269: 29061–29066.
Tachibana H, Ushio Y, Krungkasem C and Shirahata S (1996) Inducing the loss of immunoglobulin λ light chain production and the rearrangement of a previously excluded allele in human plasma B cell lines with Concanavalin A. J Biol Chem 271: 17404–17410.
Van Gent D, McBlane J, Ramsgen D, Sadofsky M, Hesse J and Gellert M (1995) Initiation of V(D)J recombination in a cell-free system. Cell 81: 925–934.
Wamer G, Gaur A and Scott D (1991) A polyclonal model for B-cell tolerance II. Linkage between signaling of B-cell egress from Go, class II upregulation and unresponsiveness. Cell Immunol 138: 404–412.
Yancopoulos G, Nolan G, Pollock R, Prockop LS, Henzenberg L and Alt F (1990) A novel fluorescence-based system for assaying and separating live cells according to V(D)J recombinase activity. Mol Cell Biol 10: 1697–1704.
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Krungkasem, C., Ueda, K., Tachibana, H. et al. Further investigation of the light chain shifting phenomenon: Light chain replacement through secondary rearrangement induced by lectin stimulation in the hybridoma cell line HB4C5. Cytotechnology 25, 145–154 (1997). https://doi.org/10.1023/A:1007943228587
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DOI: https://doi.org/10.1023/A:1007943228587