Antigen-induced lymphomagenesis: identification of a murine B cell lymphoma with known antigen specificity

CH12, a murine B cell lymphoma derived in B10 H-2aH-4bp/Wts mice after transfer of SRBC hyperimmunized spleen cells into an adult-thymectomized, sublethally irradiated, syngeneic recipient, is demonstrated to bear surface IgM specific for a determinant found on SRBC and ChRBC. The Ig specificity has been demonstrated by rosetting assays and complement-dependent hemolysis. The removal of CH12 surface IgM by capping with anti-mu or with anti-CH12 idiotype, but not with anti-gamma or with irrelevant anti-idiotype, eliminated the formation of rosettes between CH12 and SRBC or ChRBC. The absorption of CH12 Ig produced in vitro, with either SRBC or ChRBC but not with HRBC, removed all hemolysin activity against SRBC, demonstrating that only one CH12 product was responsible for the reactivity with both SRBC and ChRBC. CH12 has a surface phenotype of a relatively mature B cell expressing surface Ig (IgM-mu,kappa) and la antigens, but lacking Thy-1 or detectable Fc or C3 receptors. CH12 also expresses the antigen Lyt-1. Growth of CH12 in vivo or in vitro results in the generation of up to 3% direct PFC and serum hemolysin, which shows that CH12 is not irretrievably "frozen". The generation of PFC and serum hemolysin is associated with increased population density, and the rate of PFC and serum hemolysin accumulation cannot be explained by simple cell division. A continuously secreting hybridoma derived from CH12 was used to purify the CH12 IgM to facilitate studies of protein sequence and idiotype.     

A role for Lys-His-Gly-NH2 in avian and murine B cell development

Lys-His-Gly-NH2 has been claimed to selectively induce B cell precursors to differentiate into mature B lymphocytes. In the present study, the effects of this tripeptide and a control compound having the reverse sequence (Gly-His-Lys-NH2) on growth and differentiation of chicken and mouse B cell precursors were investigated. When chicken bone marrow (BM) cells from 15-day-old embryos were treated for 18 hr with either of the tripeptides, the frequency of Bu-1 antigen-bearing cells increased. Moreover, when embryonic bursa cells were stimulated in vitro with phorbol myristate acetate, which induces them to proliferate and undergo terminal differentiation into immunoglobulin (Ig)-secreting cells, these compounds caused a 10-fold increase in the number of Ig-secreting cells but did not increase cell proliferation. They had no effect on neonatal or adult bursa cells. Embryonic bursa cells were cultured in the presence of either of the tripeptides and metabolically labeled with [35S]methionine. When immunoprecipitated Ig was analyzed by two-dimensional gel electrophoresis, no differences in mu heavy or lambda light chain diversity patterns could be detected, indicating that neither of these compounds enhances Ig diversification. The effect of these tripeptides on murine B cell precursors was assayed in cultures of BM cells depleted of mature B cells by 5-fluorouracil. When precursor cells were incubated without adherent BM stromal cells, they did not respond to the tripeptides. However, after incubation of precursors with adherent stromal BM cells for 2 days, followed by treatment with either of the two tripeptides, differentiation into lipopolysaccharide-reactive mature B cells took place. Incubation of precursors with adherent stromal BM cells in the absence of tripeptides was not sufficient to allow the precursors to complete differentiation. In addition, both tripeptides acted synergistically with interleukin 1 or interleukin 3. In conclusion, these tripeptides seem to enhance precursor B cell differentiation in a lineage-nonspecific manner rather than to function as lineage-specific differentiation hormones.     

Transgenic expression of Helios in B lineage cells alters B cell properties and promotes lymphomagenesis

Helios, a member of the Ikaros family of DNA-binding proteins, is expressed in multipotential lymphoid progenitors and throughout the T lineage. However, in most B lineage cells, Helios is not expressed, suggesting that its absence may be critical for B cell development and function. To test this possibility, transgenic mice were generated that express Helios under the control of an Ig mu enhancer. Commitment to the B cell lineage was unaltered in Helios transgenic mice, and numbers of surface IgM(+) B cells were normal in the bone marrow and spleen. However, both bone marrow and splenic B cells exhibited prolonged survival and enhanced proliferation. B cells in Helios transgenic mice were also hyperresponsive to Ag stimulation. These alterations were observed even though the concentration of ectopic Helios in B lineage cells, like that of endogenous Helios in thymocytes, was well below the concentration of Ikaros. Further evidence that ectopic Helios expression contributes to B cell abnormalities was provided by the observation that Helios transgenic mice developed metastatic lymphoma as they aged. Taken together, these results demonstrate that silencing of Helios is critical for normal B cell function.     

Identification of macrophage external membrane proteins and their possible role in cell adhesion

Starch-activated mouse peritoneal macrophages (STpMAC) plated on plastic demonstrate the adhesive properties typical for activated pMAC: attaching as round cells and, within 15 min, spreading out with marginal membrane ruffles. These attached STpMAC were labeled by lactoperoxidase-catalysed 125I surface iodination, sodium dodecyl- sulfate-lysed, and the lysates electrophoresed on polyacrylamide gels which were examined by autoradiography. The STpMAC morphological phenotype correlates with the labeling of a particular protein (195,000, estimated mol wt). Normal pMAC (NpMAC), from unstimulated mice, do not spread and do not display the 195,000 band. Both pMAC band patterns, including the 195,000 band, are relatively resistant to trypsin digestion, as is pMAC adhesion itself trypsin-resistant. Neither class of pMAC exhibits fibronectin (Cell Adhesion Factor, LETS protein) which is a component in the adhesive matrix of cells forming trypsin-sensitive monolayers. When pMAC are tested against antifibronectin antibody, these cells do not give immunofluorescent staining. In summary, two functions in pMAC adhesion, enzyme resistance and the ability to spread, appear related to molecular properties distinctive for pMAC surface protein.     

Helicoidal pattern in secondary cell walls and possible role of xylans in their construction

The helicoidal organization of secondary cell walls is overviewed from several examples. Both the plywood texture and the occurrence of characteristic defects strongly suggest that the wall ordering is relevant of a cholesteric liquid-crystal assembly that is rapidly and strongly consolidated by lignification. A preferential localization of glucuronoxylans, major matrix components, and in vitro re-association experiments emphasize their preeminent role: (1) during the construction of the composite as directing the cellulose microfibrils in a helicoidal array; (2) during the lignification of the composite as a host structure for lignin precursors.     

Lipopolysaccharide-induced murine B cell proliferation: a role of protein kinase C

Lipopolysaccharide (LPS)-induced murine B cell proliferation was blocked by 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (H7), an inhibitor of protein kinase C (PKC), in a dose- and time-dependent manner. The maximum inhibition of B cell proliferation was observed when H7 was added at the initiation of cultures. H7-induced inhibition was prolonged and irreversible. Furthermore, pretreatment of B cells with phorbol myristate acetate ester, a process that degrades membrane-associated PKC, rendered them unresponsive to LPS. These data strongly suggest that the activation of PKC is one of the mechanisms of LPS-induced murine B cell proliferation.     

Clonal dominance and progression in Abelson murine leukemia virus lymphomagenesis.

We examined the clonality of tumors induced by an acutely transforming retrovirus which carries a single oncogene. Contrary to our expectation, tumors induced by the Abelson murine leukemia virus (A-MuLV) showed one to four major proviral integration events. To further investigate the process by which clonality was established, we analyzed the number of cells infected and transformed by A-MuLV at various times after in vivo infection. At the midpoint of tumor latency (14 days postinfection), we found that infection of total bone marrow cells by A-MuLV was efficient and polyclonal. However, only a minority of these infected cells were transformed as assayed in cell culture, and clonal dominance had already been established in this transformed cell population. Examination of the in vitro growth properties of transformed cells recovered from preleukemic and leukemic mice indicated that preleukemic cells had lower cloning efficiencies than primary tumor cells. Our results suggest that the rate-limiting step in this system of lymphomagenesis is the initial transformation of bone marrow target cells and that these cells undergo subsequent changes in cloning ability during the course of the disease that lead to an autonomous neoplastic state.     

Regulatory elements in the immunoglobulin heavy chain gene 3'-enhancers induce c-myc deregulation and lymphomagenesis in murine B cells

Burkitt's lymphoma is invariably associated with chromosomal translocations that juxtapose the c-myc proto-oncogene with regulatory elements of the immunoglobulin heavy (IgH) or light chain loci resulting in the deregulation of c-myc expression. However, the enhancer elements mediating c-myc deregulation in vivo remain largely unidentified. To investigate the role of the IgH 3'-enhancers in c-myc deregulation, we used gene targeting to generate knock-in mice in which four DNase I hypersensitive regions from the murine IgH 3'-region were integrated into the 5'-region of the c-myc locus. The IgH 3'-enhancers induced the up-regulation of c-myc expression specifically in B cells of IgH-3'-E-myc mice. After approximately 10 months, the mice developed a Burkitt-like B cell lymphoma with the phenotype of B220+, IgM+, and IgD(low). Analysis of immunoglobulin gene rearrangements indicated that the lymphoma cells were of clonal origin. The presence of a rapidly expanding population of B cells in the spleen and bone marrow of young knock-in mice at 2-4 months of age was observed. Premalignant splenic B cells of knock-in mice showed higher spontaneous and induced apoptosis; however, malignant B cells were more resistant to apoptosis. The p53-ARF-Mdm2 pathway was disabled in half of the lymphomas examined, in most cases through Mdm2 overexpression. Although c-myc expression was increased in premalignant B cells, the promoter shift from P2 to P1 was observed only in malignant B cells. Our studies demonstrate that the IgH 3'-enhancers play an important role in c-myc deregulation and B cell lymphomagenesis in vivo.     

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.