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Murine plasmacytomas can be adapted to continuous in vitro culture by alternate passage between culture and animal. We have found that the kinetics of adaptation reflect a selection for the growth of variant plasmacytoma cells. The inclusion of an altered immunoglobulin phenotype in such variant cells could explain the Ig-producing variants that we observed in two of six transplantable lines of plasmacytomas that were adapted to culture. The first variant, an IgM-producing cell line (104-76), was adapted from a transplanted line of MOPC 104E that had stopped producing IgM with binding specificity for alpha1-3 Dextran. Unlike MOPC 104E, the IgM of 104-76 contains kappa- instead of lambda-light chains and probably contains an altered or different mu- heavy chain. A second variant (352-57) was found in an IgG2b-producing tumor (MOPC 352) which was induced in a BALB/c mouse strain (CB-6) that carried Ig genes of the C57BL/Ka allotype. This cell line apparently switched from producing IgG2b molecules of the C57BL allotype (H9) and of a known idiotype to IgG1 molecules of the BALB/c allotype (F19) without the idiotype marker. The propagation of a biclonal plasmacytoma from the time of original tumor induction does not appear as a likely explanation for these results. Rather, we seem to be dealing with plasmacytoma variants or with the possible induction of secondary tumors of host origin.

In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.

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Upon activation, B cells divide, form a germinal center, and express the activation induced deaminase (AID), an enzyme that triggers somatic hypermutation of the variable regions of immunoglobulin (Ig) loci. Recent evidence indicates that at least 25% of expressed genes in germinal center B cells are mutated or deaminated by AID. One of the most deaminated genes, c-Myc, frequently appears as a translocation partner with the Ig heavy chain gene (Igh) in mouse plasmacytomas and human Burkitt's lymphomas. This indicates that the two genes or their double-strand break ends come into close proximity at a biologically relevant frequency. However, the proximity of c-Myc and Igh has never been measured in germinal center B cells, where many such translocations are thought to occur. We hypothesized that in germinal center B cells, not only is c-Myc near Igh, but other mutating non-Ig genes are deaminated by AID because they are near Ig genes, the primary targets of AID. We tested this “collateral damage” model using 3D-fluorescence in situ hybridization (3D-FISH) to measure the distance from non-Ig genes to Ig genes in germinal center B cells. We also made mice transgenic for human MYC and measured expression and mutation of the transgenes. We found that there is no correlation between proximity to Ig genes and levels of AID targeting or gene mutation, and that c-Myc was not closer to Igh than were other non-Ig genes. In addition, the human MYC transgenes did not accumulate mutations and were not deaminated by AID. We conclude that proximity to Ig loci is unlikely to be a major determinant of AID targeting or mutation of non-Ig genes, and that the MYC transgenes are either missing important regulatory elements that allow mutation or are unable to mutate because their new nuclear position is not conducive to AID deamination.

Plasmacytomas expressing immunoglobulin A are rare and not well characterized. In this study, nine cases of IgA-positive plasmacytomas presenting in lymph node and three in extranodal sites were analyzed by morphology, immunohistochemistry, and PCR examination of immunoglobulin heavy and kappa light chain genes. Laboratory features were correlated with clinical findings. There were seven males and five females; age range was 10 to 66 years (median, 32 years). Six of the patients were younger than 30-years-old, five of whom had nodal disease. 67% (6/9) of the patients with nodal disease had evidence of immune system dysfunction, including human immunodeficiency virus (HIV) infection, T-cell deficiency, autoantibodies, arthritis, Sjögren’s syndrome, and decreased B-cells. An IgA M-spike was detected in 6/11 cases, and the M-protein was nearly always less than 30 g/L. All patients had an indolent clinical course without progression to plasma cell myeloma. Histologically, IgA plasmacytomas showed an interfollicular or diffuse pattern of plasma cell infiltration. The plasma cells were generally of mature Marschalko type with little or mild pleomorphism and exclusive expression of monotypic IgA. There was an equal expression of kappa and lambda light chains (ratio 6:6). Clonality was demonstrated in 9 of 12 cases: by PCR in 7 cases, by cytogenetic analysis in 1 case, and by immunofixation in 1 case. Clonality did not correlate with pattern of lymph node infiltration. Our results suggest that IgA plasmacytomas may represent a distinct form of extramedullary plasmacytoma characterized by younger age at presentation, frequent lymph node involvement and low risk of progression to plasma cell myeloma.

Plasma cells secrete immunoglobulins other than immunoglobulin M (IgM) after a deletion and recombination in which a portion of the immunoglobulin heavy-chain locus (IgH), from the 5'-flanking region of the mu constant-region gene (C mu) to the 5'-flanking region of the secreted heavy-chain constant-region gene (CH), is deleted. The recombination step is believed to be targeted via switch regions, stretches of repetitive DNA which lie in the 5' flank of all CH genes except delta. Although serum levels of IgD are very low, particularly in the mouse, IgD-secreting plasmacytomas of BALB/c and C57BL/6 mice are known. In an earlier study of two BALB/c IgD-secreting hybridomas, we reported that both had deleted the C mu gene, and we concluded that this deletion was common in the normal generation of IgD-secreting cells. To learn how such switch recombinations occur in the absence of a switch region upstream of the C delta 1 exon, we isolated seven more BALB/c and two C57BL/6 IgD-secreting hybridomas. We determined the DNA sequences of the switch recombination junctions in eight of these hybridomas as well as that of the C57BL/6 hybridoma B1-8. delta 1 and of the BALB/c, IgD-secreting plasmacytoma TEPC 1033. All of the lines had deleted the C mu gene, and three had deleted the C delta 1 exon in the switch recombination event. The delta switch recombination junction sequences were similar to those of published productive switch recombinations occurring 5' to other heavy-chain genes, suggesting that nonhomologous, illegitimate recombination is utilized whenever the heavy-chain switch region is involved in recombination.

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We describe the unique features of an aberrantly rearranged mu immunoglobulin heavy chain gene isolated from MPC-11 cells (a gamma 2b producing Balb/c plasmacytoma). A novel rearrangement has occurred 1.5 Kb 5' of the MPC-11 mu gene (denoted 18b mu) resulting in the deletion of the majority of the repetitive switch region (S mu) and 5' flanking DNA including the Joining (JH) sequences. The remainder (275 bp) of the S mu repeat has undergone a complete sequence inversion. DNA sequences 5' of the inverted S mu sequence do not resemble Variable (VH), Diversity (D), JH or their conserved flanking sequences. A DNA sequence localized 5' of the inverted S mu sequence, (p18b mu-1.4) detects a small family of homologous sequences in Balb/c DNA. The 18b mu-1.4 like sequences lack homology to S mu, exhibit flanking sequence polymorphisms in 5 out of 6 inbred mouse strains and undergo partial or complete deletion in 5 out of 10 plasmacytomas tested. Two 18b mu-1.4 homologous sequences display a higher copy number in C57Bl/6, AL/N and CAL9 mouse strains.

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The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.

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Immunoglobulin heavy-chain switching is effected by recombination events between sites associated with tandemly repeated switch sequences located 5' to immunoglobulin heavy-chain genes. Using the band mobility shift assay, we have identified two distinct sites 5' to the alpha heavy-chain switch sequence with affinity for a single B-cell-specific DNA-binding protein, S alpha-BP. S alpha-BP was present in nuclear extracts from pre-B and B cells but was not detected in extracts from plasmacytomas, B-cell hybridomas, T-cell lymphomas, or a macrophage cell line. It was also not detectable in other nonlymphoid cells tested. Evidence suggests there are S alpha-BP-binding sites near other immunoglobulin switch sequences. As with the S alpha sites, these sites appear to be distinct from the consensus tandem repeats characteristic of immunoglobulin switch sequences. The possible functions of S alpha-BP on contacting its binding sites are discussed in the context of immunoglobulin heavy-chain switch recombination.

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Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments. We conclude that PAb is an effective agent for in vitro and in vivo induction of apoptosis in multiple myeloma and that exploratory clinical trials may be warranted.

In chromosome translocations characteristic of Burkitt lymphomas (BL) and murine plasmacytomas, c-myc genes become juxtaposed to immunoglobulin heavy-chain (IgH) sequences, resulting in aberrant c-myc transcription. Translocated c-myc alleles that retain the first exon exhibit increased transcription from the normally minor c-myc promoter, P1, and increased transcriptional elongation through inherent pause sites proximal to the major c-myc promoter, P2. We recently demonstrated that a cassette derived from four DNase I-hypersensitive sites (HS1234) in the 3′Cα region of the IgH locus functions as an enhancer-locus control region (LCR) and directs a similar pattern of deregulated expression of linked c-myc genes in BL and plasmacytoma cell lines. Here, we report that the HS1234 enhancer-LCR mediates a widespread increase in histone acetylation along linked c-myc genes in Raji BL cells. Significantly, the increase in acetylation was not restricted to nucleosomes within the promoter region but also was apparent upstream and downstream of the transcription start sites as well as along vector sequences. Histone hyperacetylation of control c-myc genes, which was induced by the deacetylase inhibitor trichostatin A, mimics the effect of the HS1234 enhancer on expression from the c-myc P2 promoter, but not that from the P1 promoter. These results suggest that the HS1234 enhancer stimulates transcription of c-myc by a combination of mechanisms. Whereas HS1234 activates expression from the P2 promoter through a mechanism that includes increased histone acetylation, a general increase in histone acetylation is not sufficient to explain the HS1234-mediated activation of transcription from P1.

A mouse DNA clone containing the constant part of the immunoglobulin gamma 2b heavy chain was isolated from a mouse gene library. The library was constructed in Charon 4A from a partial EcoRI digest of mouse embryo DNA and was screened with a plasmid (p gamma (11)7) containing a cDNA insert of the heavy chain constant region of the plasmacytoma MPC-11 (1). The Charon 4A clone contains a 14 kb insert which is cleaved by EcoRI into a 6.8 kb and 7.2 kb fragments, of which only the 6.8 kb contains the sequence for gamma 2b heavy chain. Restriction analysis and partial sequence of the insert in p gamma (11) 7 enabled us to obtain three fragments corresponding to the 5' (amino acid 161-302) middle (amino acid 302-443) and 3' (mostly non coding 107 bp) regions of the constant region. Restriction analysis of the Charon 4A clone and hybridisation to these nick translated fragments revealed that the gamma 2b constant region gene contains about 1.5 kb and has three intervening sequences.

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Prototypical class switching in mouse and human immunoglobulin heavy chains occurs through recombination of tandem blocks of short repeats located 5' to each heavy chain constant region (CH) except C delta. Deletion of C mu in immunoglobulin D (IgD)-secreting murine plasmacytomas occurs illegitimately. We demonstrate here that in human IgD-secreting myeloma cells freshly isolated from patient bone marrow and in normal peripheral blood B lymphocytes, an IgD switch can occur through homologous recombination of a direct repeat consisting of a 442-bp sequence 1.5 kbp 3' of the JH complex and a 443-bp sequence that is duplicated almost perfectly (96% similarity) 1.7 kbp 5' of the C delta gene (442/443-base-pair [bp] repeat). This homologous recombination mechanism is not exclusive for IgD switching, since C mu deletion endpoints in two established IgD-secreting myeloma cell lines fall outside the 442/443-bp repeat. The 442/443-bp mediated recombination shows cell type specificity, and we propose that it represents a unique mode for increased levels of IgD secretion in humans.

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Extramedullary plasmacytoma of the liver is a rare tumour, only two cases of which have been reported so far. A third case arising in a 22 year old woman, who presented with abdominal pain and enlargement of the liver, is described. Ultrasound and a computed tomography scan showed a solitary hepatic mass, 12 cm diameter, involving both lobes of the liver. Serum immunoelectrophoresis revealed an IgG kappa monoclonal gammopathy. Histologically, the tumour was composed of mature plasma cells with mild atypia. The plasma cells infiltrated the liver parenchyma and showed kappa light chain restriction. The monoclonal nature of the tumour was also demonstrated by PCR amplification of the immunoglobulin heavy chain genes. There was no evidence of bone involvement and repeated bone marrow aspirates and biopsy specimens were normal. The patient was treated with eight courses of chemotherapy. One year after diagnosis, the patient is well, the size of the tumour has decreased and the paraproteinaemia has disappeared.

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The rearranged lambda 2 gene of the mouse plasmacytoma cell line MOPC315 has been cloned and sequenced. A comparison of its sequence with the sequence of the unrearranged (germ-line) V, J and C gene segments shows that the sequences of the V gene segments differ at six positions. The sequence of the J and C gene segments remained unchanged. These results add support to the hypothesis that somatic mutations occur in immunoglobulin in genes and that these mutations do not involve the C gene segment. The degree of homology of the elements of the lambda 2 gene with those of the lambda 1 gene and C lambda 3 and C lambda 4 gene fragments suggest a pathway of evolution by gene duplication of the immunoglobulin lambda light chain locus. According to this scheme the original structure V0-J0C0 gave rise to a structure V0-J1C1-J11C11 by duplication of the J0C0 region. A second duplication encompassing the whole region resulted in the present structure: V1-J3C3-J1C1/V2-J2C2-J4C4.

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Extramedullary plasmacytoma of the liver is a very rare tumor. Although a few cases of extramedullary plasmacytoma of the liver have been reported, we could not find any report on truly localized extramedullary plasmacytoma of the liver in the literature. The patient was a 63-yr-old man who exhibited a solitary liver mass on dynamic computed tomography and magnetic resonance imaging. Histologically, the tumor was composed of mature plasma cells with mild atypia. Immunohistochemistry demonstrated monoclonal IgG and Kappa light chain expression. Bone marrow examination revealed no abnormalities. There was no evidence of a monoclonal protein in the serum and urine, lytic bone lesions, anemia, renal insufficiency, and hypercalcemia. The patient was treated with 5,000 cGy of radiotherapy, and the tumor disappeared 6 months after treatment.

Immune responsiveness to phosphorylcholine (PC) in BALB/c mice has been characterized by combining (a) usuage of highly sensitive radioimmunoassays for quantitation of antibody, heavy-chain class, and idiotype on a weight basis; (b) isolation of PC-specific B cells in fragment cultures; and (c) stimulation in a carrier-primed environment with the PC hapten coupled to carrier through a tripeptide spacer in order to maximize carrier recognition. The specificity and accuracy of the radioimmunoassays have veen verified by specific inhibition, lack of nonspecific binding, and excellent concordance of values for monoclonal antibody concentration obtained independently for Fab and idiotype content. The latter evidence also serves as strong confirmation of the monoclonality of in vitro monofocal responses as well as the preservation of the idiotype on antibodies of differing immunoglobulin classes. The results indicate that while B cells expressing the TEPC 15 idiotype predominate, other idiotypes may be represented by 2-50% of PC-specific precursors, and monoclonal antibodies even of the TEPC 15 idiotype are produced in both the IgM and IgG1 immunoglobulin classes. These findings are confirmed by the analysis of serum antibodies produced in carrier-primed mice immunized with hapten coupled through a tripeptide spacer, thus re-emphasizint the enhancement of primary responsiveness, particularly IgG1 production, by maximizing carrier recognition. The finding of idiotype diversity in the PC response, as well as diversity of expression in terms of quantity and immunoglobulin class of antibody synthesized by the clonal progeny of B cells within the TEPC 15 clonotype, emphasize the heterogeneity of the B-cell population both in terms of specificity repertoire and the physiological state of cells even within a single clonotype.

Chromosomal translocation requires formation of paired double strand DNA breaks (DSBs) on heterologous chromosomes. One of the most well characterized oncogenic translocations juxtaposes c-myc and the immunoglobulin heavy chain locus (IgH) and is found in Burkitt’s lymphomas in humans and plasmacytomas in mice. DNA breaks in IgH leading to c-myc/IgH translocations are created by activation induced cytidine deaminase (AID) during antibody class switch recombination or somatic hypermutation. However, the source of DNA breaks at c-myc is not known. Here we provide evidence for the c-myc promoter region being required in targeting AID-mediated DNA damage to produce DSBs in c-myc that lead to c-myc/IgH translocations in primary B lymphocytes. Thus, in addition to producing somatic mutations and DNA breaks in antibody genes, AID is also responsible for the DNA lesions in oncogenes that are required for their translocation.

To study the potential involvement of IL-6 in the development of plasmacytomas, a number of plasmacytoma lines were analyzed for alterations in the IL-6 locus. A DNA rearrangement due to the insertion of an intracisternal A particle retrotransposon 18 bp 5' of the transcriptional start site was detected in the cell line MPC11. The IL- 6 gene is constitutively expressed in MPC11, suggesting the involvement of IL-6 in the development of certain myeloma/plasmacytomas according to the "autocrine growth hypothesis".

AL (amyloid light chain) amyloidosis is a rare hematologic disorder characterized by the accumulation of a misfolded monoclonal immunoglobulin light chain (LC) as fibrillar protein deposits. Current treatments, including cytotoxic chemotherapy and immunomodulatory therapy, are directed at killing the plasma cells that produce the LCs, but have significant toxicity for other cell types. We have designed small interfering RNAs (siRNAs) targeting the amyloidogenic LC mRNA in order to reduce expression of the amyloid precursor protein. Using nanomolar concentrations of siRNAs, we have inhibited synthesis of LC in transfected cells in vitro in a dose-dependent fashion. Furthermore, in an in vivo plasmacytoma mouse model of AL amyloidosis, we have demonstrated that these siRNAs can significantly reduce local production and circulating levels of LC. This model system highlights the therapeutic potential of siRNA for AL amyloidosis.

A monoclonal anti-idiotypic antibody (Ab2) whose antibody combining site contained a surrogate image of the meningococcal group C capsular polysaccharide was developed. To accomplish this, a monoclonal antibody against the group C capsular polysaccharide was developed by the fusion of splenocytes from mice immunized with Neisseria meningitidis group C strain MP13 with Sp2/0-Ag14 plasmacytoma cells. Monoclonal antibody 1E4, an immunoglobulin M isotype, demonstrated binding to the serogroup C polysaccharide in enzyme-linked immunosorbent assay (ELISA). Monoclonal antibody 1E4 reacted with 30 of 30 group C strains and 1 of 36 group B strains in immunodot assay, slide agglutination, inhibition ELISA, and bactericidal assay. This monoclonal antibody was selected as idiotype (Ab1) for the development of hybridomas producing an anti-idiotype antibody. One of the hybridomas developed, designated 6F9, was capable of over 70% inhibition of 1E4 in binding in the meningococcal C polysaccharide-specific ELISA. Studies with convalescent human serum demonstrated 100% inhibition of a serogroup C-specific ELISA with 200 micrograms of 6F9 per ml and 50% inhibition of this ELISA was achieved with 50 micrograms of 6F9 per ml. Monoclonal anti-idiotype antibodies (Ab3) with specificities similar to Ab1, 1E4 were generated from BALB/c mice immunized with the Ab2 (6F9). Immunization of rabbits with 6F9 resulted in an immunoglobulin G response which was significantly greater than that of control to a titer of 1:160. These studies indicate that monoclonal 6F9 contained a surrogate image on the combining antibody site which mimicked meningococcal C polysaccharide. This surrogate image is capable of evoking antibodies to the meningococcal C polysaccharide in syngenic and xenogenic species.

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Previous studies have indicated that immunoglobulin enhancers are essential for establishing transcriptional competence but not for maintaining the activity of constitutively transcribed genes. To understand the basis for this developmental shift away from dependence on enhancer function, we have investigated the relationship between transcriptional activity and methylation status of the immunoglobulin kappa light-chain genes (kappa genes) in mouse cell lines representing different stages of B-cell maturation. Using pre-B-cell lines in which the level of a critical kappa enhancer-binding factor, NF-kappa B, was controlled by the administration or withdrawal of lipopolysaccharide and plasmacytoma lines that either contain or lack this factor, we studied the properties of endogenous kappa genes and of transfected kappa genes which were stably integrated into the genomes of these cells. In the pre-B cells, the exogenous (originally unmethylated) kappa genes, as well as endogenous kappa genes, were fully methylated and persistently dependent on enhancer function, even after more than 30 generations in a transcriptionally active state. In plasmacytoma cells, the endogenous kappa genes were invariably hypomethylated, whereas exogenous kappa genes were hypomethylated only in cells that contain NF-kappa B and are thus permissive for kappa enhancer function. These results indicate that the linkage of hypomethylation to enhancer-dependent activation of kappa transcription occurs after the pre-B-cell stage of development. The change in methylation status, together with associated changes in chromatin structure, may suffice to eliminate or lessen the importance of the enhancer for the maintenance of the transcriptionally active state.

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The human lympholine osteoclast activating factor (OAF) is thought to be involved in several bone-destroying diseases. The current studies were designed to produce monoclonal antibodies against OAF for use in the subsequent design of immunoassays for OAF in clinical samples. Spleen cells from mice immunized with purified human OAF were hybridized with mouse plasmacytoma cells in vitro to yield hybridomas. Several clones of these hybridomas secreted into the culture medium antibodies, which neutralized the biological activity of OAF at dilutions as high as 1:100,000 relative to the initial culture medium. These antibodies did not interfere with the activities of parathyroid hormone in the same systems. These results represent the first report of monoclonal antibodies against a human lympholine, and validate the concept that hybridoma production is a useful technique for developing antibodies against weak or scarce antigens.

We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.

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Many immunoglobulin (Ig)-producing cells retain the DNA that separates Ig variable (V) and constant (C) region genes in the germline. This "remnant" DNA must be moved during the recombination process that joins V and C genes via a joining (J) segment. We have analyzed remnant DNAs in several Ig-producing cell lines. The nucleotide sequences of kappa (kappa) light chain remnant DNAs indicate close relationships to V-J joining. We find fused V kappa and J kappa recognition sequences in five remnant DNAs, suggesting reciprocal relationships to the fused V kappa and J kappa segments produced by V-J joining. However, of sixteen plasmacytoma remnant DNAs analyzed, all involve only recombination with J kappa l. Thus, in most cell lines, remnant DNAs are not directly reciprocal to recombined kappa-genes. On the other hand, our analyses of some myelomas do indicate indirect relationships between remnant DNAs and kappa-genes. Our results suggest that multiple steps of DNA recombination occur during Ig-gene rearrangement. Because remnant DNA joining sites do not exhibit the flexibility that has been observed in Ig-gene V-J joining, our findings also suggest that the joining mechanism may involve endonuclease, exonuclease and ligase activities.

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Background

Gene-targeted iMycEμ mice that carry a His6-tagged mouse Myc(c-myc)cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Eμ, are prone to B cell and plasma cell neoplasms, such as lymphoblastic B-cell lymphoma (LBL) and plasmacytoma (PCT). Cell lines derived from Myc-induced neoplasms of this sort may provide a good model system for the design and testing of new approaches to prevent and treat MYC-driven B cell and plasma cell neoplasms in human beings. To test this hypothesis, we used the LBL-derived cell line, iMycEμ-1, and the newly established PCT-derived cell line, iMycEμ-2, to evaluate the growth inhibitory and death inducing potency of the cancer drug candidate, CDDO-imidazolide (CDDO-Im).

Methods

Morphological features and surface marker expression of iMycEμ-2 cells were evaluated using cytological methods and FACS, respectively. mRNA expression levels of the inserted MycHis and normal Myc genes were determined by allele-specific RT-PCR and qPCR. Myc protein was detected by immunoblotting. Cell cycle progression and apoptosis were analyzed by FACS. The expression of 384 "pathway" genes was assessed with the help of Superarray© cDNA macroarrays and verified, in part, by RT-PCR.

Results

Sub-micromolar concentrations of CDDO-Im caused growth arrest and apoptosis in iMycEμ-1 and iMycEμ-2 cells. CDDO-Im-dependent growth inhibition and apoptosis were associated in both cell lines with the up-regulation of 30 genes involved in apoptosis, cell cycling, NFκB signaling, and stress and toxicity responses. Strongly induced (≥10 fold) were genes encoding caspase 14, heme oxygenase 1 (Hmox1), flavin-containing monooxygenase 4 (Fmo4), and three members of the cytochrome P450 subfamily 2 of mixed-function oxygenases (Cyp2a4, Cyp2b9, Cyp2c29). CDDO-Im-dependent gene induction coincided with a decrease in Myc protein.

Conclusion

Growth arrest and killing of neoplastic mouse B cells and plasma cells by CDDO-Im, a closely related derivative of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid, appears to be caused, in part, by drug-induced stress responses and reduction of Myc.