Murine pre-B Cell Line Research Articles

Class-switching from mu to gamma 3 or gamma 2b production at pre-B cell stage.

Abstract An Abelson virus-transformed murine pre-B cell line class-switched from mu to gamma 3 or gamma 2b at the pre-B stage during culture. A class-switch was mediated by the deletion mechanism of the intervening CH genes on the active chromosome. This study showed for the first time that class-switching at the pre-B cell stage was not always confined to gamma 2b production.

A labile inhibitor blocks immunoglobulin kappa-light-chain-gene transcription in a pre-B leukemic cell line.

The murine pre-B leukemic cell line 70Z/3 contains both an unrearranged immunoglobulin kappa-light-chain gene and a functionally rearranged but silent kappa-light-chain gene. Mitogenic stimulation of growing 70Z/3 cells with bacterial lipopolysaccharide (LPS) activates kappa-light-gene transcription and results in a 10- to 20-fold increase in cytoplasmic kappa-light-chain mRNA. The induction of kappa gene expression by LPS was probed by using an inhibitor of protein synthesis. Concomitant treatment of 70Z/3 cells with LPS and cycloheximide failed to block kappa-light-chain mRNA accumulation, indicating that new protein synthesis is not required for the activation of kappa-gene expression. Treatment of 70Z/3 cells with cycloheximide alone resulted in kappa-mRNA induction equivalent to those produced by LPS alone. The kappa mRNA synthesized in the presence of cycloheximide was intact and able to direct the synthesis of kappa light chains. Nuclear transcription assays revealed that cycloheximide, like LPS, activated kappa-gene transcription. These findings indicate that the trans-acting factors necessary for kappa-light-chain-gene transcription are present in pre-B cells, but their activity is blocked by short-lived inhibitory proteins.

Lipopolysaccharide and phorbol esters induce differentiation but have opposite effects on phosphatidylinositol turnover and Ca2+ mobilization in 70Z/3 pre-B lymphocytes.

We have recently shown that both lipopolysaccharide (LPS) and the phorbol ester, 12-O-tetradecanoyl phorbol 13-acetate (TPA) induce differentiation in the transformed murine pre-B lymphocyte cell line 70Z/3 by enhancing Na+-H+ exchange across the plasma membrane through an amiloride-sensitive transport system (Rosoff, P.M., Stein, L.F., and Cantley, L.C. (1984) J. Biol. Chem. 259, 7056-7060). These data suggested that the activation of protein kinase C indirectly by LPS and directly by TPA was the critical step in the initiation of differentiation in these cells. We extend these observations to show that LPS rapidly stimulates an increase in phosphatidylinositol turnover, leading to a rise in the levels of diacylglycerol and inositol 1,4,5-trisphosphate and a concomitant decrease in the amount of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate. There is also a rapid elevation of intracellular free [Ca2+] which is independent of the presence of extracellular Ca2+ or Na+. These results suggest that the increase in cytosolic [Ca2+] is due to release of cation from internal stores. TPA, which also causes differentiation in these cells, and the synthetic diacylglycerol, 1-oleoyl-2-acetylglycerol, have opposite effects from LPS on both phosphatidylinositol turnover and cellular Ca+ mobilization. These data suggest that protein kinase C inhibits the activity of phospholipase C. Thus protein kinase C plays a pivotal role in the regulation of mitogen-induced differentiation in these cells by both transducing a positive stimulus to the Na+-H+ exchange system as well as feedback regulating its own stimulatory pathway.

Confirmation of the assignment of the gene coding for the BA-2 antigen to human chromosome 12.

Roden t -human hybrids, segregating human chromosomes, have been shown to be useful tools for gene localization studies (McKusick 1980). Consti tutive ("house-hold") phenotypes of both fusion partners tend to be expressed coordinately, irrespective of the differentiat ion lineage of the fused cells (Davis and Ade lberg 1973). This is in contrast with the expres1 10 0 13 9 sion of differentiat ion-linked or specialized gene products 2 6 0 17 9 which depends on the differentiation lineage of the parental 3 10 1 13 8 rodent and human cells (Foug6re and Weiss 1978; Deisseroth 4 9 1 14 8 and Hendr ick 1978; Geurts van Kessel et al. 1983). In our at5 22 5 1 4 tempts to map the genes involved in the expression of B cell 6 22 7 1 2 associated antigens, we have chosen murine (precursor) B 7 10 2 13 7 cell lines as rodent partners for fusion with human (precursor) 8 19 5 4 4 B cells. One panel of 20 hybrid clones resulted from a fusion 9 10 1 13 8 of the murine pre-B cell line 18.81 (Siden et al. 1979) with 10 21 5 2 4 bone marrow cells f rom a patient with common acute lympho11 23 6 0 3 blastic leukemia (cALL), which is regarded as a precursor 12 23 i b 0 8 B cell leukemia (Korsmeyer et al. 1983). These 20 hybrid 13 9 2 14 7 clones covered only half of the human chromosomes. Ano the r 14 11 1 12 8 panel of 12 hybrid clones was obtained by fusing the murine 15 9 1 14 8 myeloma cell line P3 (K6hler and Milstein 1975) with the 16 10 1 13 8 human Burkitt lymphoma cell line ROS-1, isolated in our 17 10 1 13 8 laboratory. These P3 × ROS-1 clones covered all human 18 9 3 14 6 chromosomes. 19 10 0 13 9 The 32 hybrid clones were tested for human chromosome 20 12 3 11 6 content and reactivity with the monoclonal antibodies VIL-A1 21 20 4 3 5 (ant i -cALL antigen), B1, B2, BA-1, and BA-2 (Kersey et al. 22 10 1 13 8 1981; McKenzie and Zola 1983). The BA-2 antigen was reguX 22 9 1 0 lary expressed in both panels of hybrid clones, which allowed y 0 0 23 9 us to confirm the assignment of the BA-2 antigen to human 8q -c 7 1 3 1 chromosome 12 (Katz et al. 1983). The concordancy rate be14q+ ° 10 0 0 2 tween the absence and presence of the BA-2 antigen and chromosome 12 was more than 96% (Table 1). Regarding the other antigens tested, all P3 x ROS-1 clones were negative for the cALL, B1, B2, and BA-1 antigens, al though this panel of hybrids covered all human chromosomes. Katz et al. (1983) found that their P3 x c A L L clones were also negative for the c A L L antigen. These data

Site-specific recombination between immunoglobulin D and J H segments that were introduced into the genome of a murine pre-B cell line

A recombinant plasmid containing the herpes simplex virus thymidine kinase ( tk) gene, flanked on one side by two murine immunoglobulin heavy chain diversity (D) elements and on the other by two murine immunoglobulin heavy chain joining (J H) elements, was introduced into a tk − variant of a pre-B cell line transformed by Abelson murine leukemia virus. The four possible site-specific joining events between the D and J H segments within the integrated construct occurred frequently during passage of the cloned line under nonselective conditions, and deletion of the internal tk gene as a result of these joining events was, by far, the predominant mechanism of resistance to BUdR within this line. These studies demonstrate that a precise chromosomal location is not essential for the assembly of D and J H elements and provide a model system for mechanistic and genetic studies of this recombination process.

Immunoglobulin gene expression and DNA methylation in murine pre-B cell lines.

DNA modification accompanying immunoglobulin gene expression was examined in various Abelson murine leukemia virus (A-MuLV)-transformed cell lines, which were able to differentiate from the mu- to mu+ stage or to undergo an isotype switch during in vitro culture. The C mu genes were relatively demethylated in the A-MuLV-transformed cell lines examined irrespective of whether or not the C mu genes were expressed. Normal IgM-bearing B cells, as well as a T cell line, also showed a similar DNA methylation pattern and the C mu genes were relatively demethylated. In one of the mu+ clones, however, the expressed C mu gene was heavily methylated. The DNA methylation pattern did not change and remained hypermethylated before and after gamma 2b expression in the two cell lines which underwent class switch to gamma 2b during in vitro culture, suggesting that expression of the gamma 2b gene was not accompanied by demethylation of the C gamma 2b gene. Taken together, these results indicate that DNA demethylation within and around the CH gene may not be necessary for its expression.

Isotype switching in murine pre-B cell lines

Isotype switching at the pre-B cell stage was studied by employing A-MuLV-transformed cell lines. Two γ 2b- producing cell lines that did not have other cytoplasmic heavy chains or light chains were established from A-MuLV-transformed cell lines. One clone (SL2-1-52) arose spontaneously from a non-Ig-producing cell line (SL2-1) during in vitro culture. Another clone (AT11-2-24-6-99) underwent isotype switching from a μ-producing cell line (AT11-2-24-6), which had been derived from another non-lg-producing line (AT11-2). Southern blot analysis of two γ 2b- producing clones was performed in comparison to that of their respective parent clones. The results showed that isotype switching can operate at the stage of pre-B cells by a C H gene deletion mechanism without utilizing the switch region. In addition, the possibility is presented that deletion of the intervening C H genes could occur prior to the formation of the functional V region-coding DNA segment. This indicates that the prior expression of μ chains is not obligatory for the expression of other isotypes and that isotype commitment could occur in pre-B cells.

Induction of 19S IgM secretion in a murine pre-B cell line, 70Z/3, by cell hybridization with non-secreting myeloma cells

Somatic cell hybrids were prepared between the TK-deficient variant of murine pre-B cell line, 70Z/3, cells and the HGPRT-deficient variant of non-secreting myeloma cells. Several hybrid clones which secreted IgM but did not express surface IgM were isolated. LPS stimulation did not induce the expression of surface IgM. SDS-PAGE analysis indicated that the IgM secreted by one of the hybrid clones was a 19S pentamer and that the size of its μ-chains was the same as that of μ-chains from MOPC 104E myeloma IgM. Two-dimensional gel electrophoresis of biosynthetically labeled immunoglobulin showed that the same pattern was obtained with κ-chains from two hybrid clones and from the LPS-induced 70Z/3 cells. The result showed that cell hybridization could induce L-chain synthesis in the pre-B cell line. Anti-idiotypic antibody against the secreted IgM was prepared and it was shown that the surface IgM expressed on all LPS-stimulated 70Z/3 cells bore the same idiotype. Those results indicated that the specificity commitment has already occurred in 70Z/3 cells.

Relationship between the rearrangement of immunoglobulin genes, the appearance of a B lymphocyte antigen, and immunoglobulin synthesis in murine pre-B cell lines.

Eighteen Abelson virus-transformed immature B cell lines were established and immunoglobulin biosynthesis, expression of a B lymphocyte antigen detected by a monoclonal antibody, and rearrangement of immunoglobulin genes in these cell lines were studied. Only one cell line (A1) synthesized micro-chains but no light chains, and the other cell lines synthesized no detectable immunoglobulins. None of the cell lines established had detectable membrane-associated IgM. Fifteen cell lines expressed a B lymphocyte antigen on their cell surfaces. In three cell lines, however, the majority (greater than 99%) of cells did not express this antigen. Heavy chain genes were rearranged on both chromosomes in all the cell lines, although one heavy chain gene was deleted in three cell lines. In 12 of 18 cell lines, one or both kappa-chain genes were rearranged. In six cell lines, however, both kappa-chain genes remained in embryonic form; lambda-chain genes were in embryonic form in all the cell lines. These results suggested the hierarchy of Ig gene rearrangements, beginning with mu and proceeding to kappa and then to lambda. JH rearrangement was also shown to precede the appearance of a B lymphocyte antigen. In three cell lines (A1-A3), which were considered subclones derived from a single common precursor, it was suggested that one rearranged JH gene was functional, and the other was nonfunctional, indicating that allelic exclusion already operated in pre-B cells.

Control of IgM synthesis in the murine pre-B cell line, 70Z/3'.

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.

Insulin receptors on cultured murine lymphoid tumor cell lines.

Eight murine lymphoid tumor cell lines have been examined for the presence of high-affinity insulin receptors. The eight cell lines included two Abelson murine leukemia virus-transformed pre-B cell lines, three plasmacytoma cell lines, and three spontaneous T-cell lymphomas from AKR mice. All of the cell lines in the B-cell series had high-affinity insulin binding sites. The apparent equilibrium association constant (Ka) for the high-affinity binding sites on these cells was 1.3-3.3 X 10(9) M-1. Two of the T-cell lymphomas had high-affinity receptor levels so low as to be undetectable in the whole cell binding assay under the conditions used for assaying the other cell lines, although in binding assays performed at very high cell densities, these two cell lines did appear to have a small number of high-affinity insulin binding sites. These results indicate that the growth stimulus provided by the tumor virus in neoplastic transformation of the AKR thymic lymphocytes differs from that provided by lectins in blast transformation of lymphocytes in that the neoplastic transforming event does not always result in the emergence of large numbers of high-affinity insulin receptors. In addition, the existence of cell lines such as the T-cell lymphomas that have nearly exclusively low-affinity binding sites suggests that the low-affinity sites may represent a distinct receptor that is not freely interconvertible with the high-affinity receptor.

Induction and regulation of immunoglobulin expression in a murine pre-B cell line, 70Z/3. I. Cell cycle-associated induction of sIgM expression and kappa-chain synthesis in 70Z/3 cells by LPS stimulation.

Stimulation of a murine pre-B cell line, 70Z/3, with LPS induced sIgM expression and an increase of L kappa-chain mRNA within 12 hr. In synchronized 70Z/3 cells, the cell division cycle was 10 to 11 hr and LSP-stimulation did not affect the cell division cycle. LPS-signals given at G1/S boundary induced sIgM expression in the G2 to M-phase. On the other hand, LPS-signals given after M-phase did not induce sIgM expression. Inhibition of cell division with demecorcin did not affect sIgM expression in the M-phase. Induction of intracellular kappa-chain synthesis was also observed in the G2 to M-phase when the LPS-stimulation was provided at G1/S boundary, but LPS-signals given after the M-phase did not induce de novo synthesis of kappa-chain. These results showed that LPS-induced sIgM expression and synthesis of kappa-chain were cell cycle-related events and sIgM expression was associated wih de novo synthesis of kappa-chain.