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Three established Burkitt's lymphoma (BL) cell lines (Daudi, Raji and DG-75) and three B-non-Hodgkin's lymphoma (B-NHL) of other types (Pfeiffer, Farage and Toledo) were analyzed with respect to the presence of somatic point mutations in their rearranged immunoglobulin Vkappa genes. Two of the Vkappa sequences of BL and two of those of the B-NHL were heavily mutated (up to 11%), when compared with their closest germline variable region counterparts ("clonal mutations"). Only one of the six cell lines contained an unmutated germline Vkappa sequence. The clonal mutations have features characteristic of the mutation machinery operating in the course of the T-dependent immune response, such as a preference of mutations in purine bases, more transitions than transversions and targeting to CDR and to known "hotspot" motifs. Sequence variations among different Vkappa PCR clones isolated from each of the cell lines ("intraclonal mutations") showed that the Vkappa of Toledo exhibited about 5-fold higher mutation frequency (MF) than the background level of Taq polymerase error (approximately 0.12% mut/bp). Similarly, the MF of Vkappa of two of the BL cell lines was 3-4-fold higher than the Taq polymerase misincorporation rate. In contrast, the mutation frequencies of the Vkappa of DG-75, Farage and Pfeiffer did not significantly exceed the level of Taq polymerase error. Our combined results show that 5 out of the 6 B-cell lines studied originated from B-cells that have already somatically mutated in vivo their rearranged Vkappa genes. Moreover, two of the Burkitt's and one of the B-NHL cell lines exhibit intraclonal variation indicating that the process of somatic hypermutation continued following the neoplastic event, either in vivo or in culture. These results are in accord with the presumed origin of the majority of the BL and some types of the B-NHL, from centrocytes or centroblasts of the germinal centers in which the process of somatic hypermutation is taking place.

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Analysis of the alternatively spliced isoforms of the human and mouse oct-1 genes, combined with their exon-intron structure, show a high level of evolutionary conservation between these two species. The differential expression of several oct-1 isoforms was examined by reverse transcription-polymerase chain reaction performed on the 3' region of the murine oct-1 cDNA. Variations in the relative levels and patterns of expression of the isoforms were found among different tissues. Three novel isoforms originating from the 3'-distal region of oct-1, were isolated and sequenced: Two were derived from testis, and one from myeloma cells. Splicing out of different exons as revealed in the structure of these isoforms results in reading frameshifts that presumably lead to the expression of shortened Oct-1 proteins, with distinct C-terminal tails. Altogether, six out of the eight known murine oct-1 isoforms may have distinct C-termini, implying that these multiple tails have different functional roles in cellular differentiation and physiology.

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Non-Hodgkin's B-lymphomas (B-NHL) are a very heterogeneous group of B-cell neoplasias originating from the germinal centers of lymphatic follicles. Thus, they represent a suitable experimental model to study the molecular basis of certain key events which take place in the lymphatic follicles, including somatic hypermutation and heavy chain isotypic switch. An unusual B-NHL cell line ("Farage") not producing Ig polypeptide chains was previously shown to rearrange its IgH and Igkappa genes and transcribe seemingly normal size mu and kappa mRNAs. In an attempt to characterize the phenotype of Farage cells better and to elucidate the molecular basis of the failure of Farage cells to synthesize Ig chains, we sequenced its VH and Vkappa rearranged gene segments by PCR and RT-PCR. It was found that both V genes are somatically, heavily mutated compared to their germline counterparts. In addition, this rearranged VDJ gene of the heavy chain is not transcribed. Instead, the Farage cells express a low level of a new family of germline transcripts starting with a VH like sequence, continuing with a small segment of the 3'VH germline flanking region, and ending within the Cmu region. These transcripts lack D and J segments and do not contain the open reading frame of the full-length Cmu protein. Thus, Farage cells fail to produce mu heavy chains due to silencing of the expression of the conventional VDJCmu transcript and expression of unusual Cmu-germline transcripts. In contrast to the IgH genes, the rearranged VJ gene of Farage is transcribed and gives rise to a full-size kappa-mRNA. This transcript, however, is not translated to a full-length kappa-chain, as it contains a stop codon in its coding region. All the above show that Farage cells are unable to produce Ig polypeptide chains, due to somatic mutations altering the kappa-chain gene, and mutations and/or regulatory events that shutoff the transcription of the IgH gene. The heavily mutated Vkappa and Vkappa genes found, support the conclusion that the Farage cell line originated either from germinal center cells or from the mantle zone of the lymphoid follicle.

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"Farage" is a cell line derived from a patient who had a diffuse and mixed type malignant lymphoma. In a previous study it was shown that Farage cells expressed B-cell markers, but not membrane IgM. Karyotypic analysis showed that in contrast to most follicular cell lymphomas, Farage did not have the 14;18 chromosomal translocation. In the present work Farage was further characterized by Southern and Northern blot analyses. Two rearranged heavy chain alleles and one rearranged kappa chain gene were detected. The cells expressed both mu and kappa mRNA, even though at a 3-7 fold lower level than that found in the control Daudi and DG-75 Burkitt lymphomas. Farage cells did not express the terminal deoxynucleotydyl transferase gene (TdT), nor the recombination activating genes RAG-1 and RAG-2, known as markers of the pre-B cell stage. These results show that Farage represents a mature B-cell rather than a pre-B cell. Despite the presence of C kappa and C mu RNAs, no Ig polypeptide chains were produced by Farage as judged by immunoblotting and biosynthesis labeling assays. Ig mRNAs were detected on the polysomal fraction, but at a lower level relative to Daudi cells. Our combined results suggest that in Farage cells translation of Ig mRNA is not fully blocked at the stage of translation initiation. Farage cells may express "germline" or mutated variants of Ig mRNAs. The unusual phenotype of Farage may reflect a normal as yet unknown stage of B-cell differentiation, or it may be due to an aberrant expression developed after malignant transformation.

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In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of the differentiation-specific traits of the non-fibroblast parental cell. To explore the genetic basis of this phenomenon, we have studied a series of somatic cell hybrids between mouse myeloma and fibroblasts. All the hybrids were adherent having a fibroblast-like phenotype. Molecular analysis revealed that plasma cell specific genes like the productively rearranged Ig genes, the J chain gene and genes for the cell surface markers CD20 and PC1, were extinguished in the hybrids. In contrast, fibroblast specific genes like fibronectin, alpha 2(I) and III collagens, as well as the receptor for fibroblast growth factor (flg), were expressed. Extinction was not due to chromosomal loss or lack of the relevant genes. To learn about the mechanism(s) of this phenomenon we have looked for the presence of positive and negative transcription factors in our hybrids. Expression of the PU.1 transcription factor, a member of the Ets transcription factor family normally expressed in B cells and macrophages, was lost in the cell hybrids. Interestingly, we found that the B-cell-specific Oct-2 transcription factor was still expressed at somewhat variable levels in several of the hybrid cell lines. In contrast, expression of the recently identified octamer coactivator BOB.1/OBF.1 was extinguished in all cell hybrids. This supports a critical role of this transcriptional coactivator for B-cell-specific gene expression. In addition, the Id and HLH462 genes coding for proteins known to repress bHLH transcription factors by formation of heterodimers, were found to be expressed at increased levels in fibroblasts and in the hybrids, indicating that their increased levels might also contribute to the suppression of myeloma-specific genes. Our results show that in myeloma x fibroblast hybrids, the phenotype of the fibroblast is dominant. It is suggested that fibroblasts contain regulatory "master" genes that are responsible for activation of the fibroblast differentiation pathway and suppress differentiation programs of other cell types.

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Expression of tissue-specific genes can be altered upon fusion of mammalian cells of different types. To resolve the genetic basis of this phenomenon and to identify components of the regulatory circuits that are involved, we have established a series of somatic cell hybrids between mouse T cells and L cells. These hybrids have an unusual and interesting phenotype. Unlike many hybrid cells studied, in which the expression of an entire set of tissue-specific genes was coordinately extinguished, in our T x L-cell hybrids only two out of seven T-cell-restricted genes were completely extinguished, whereas the other genes were repressed to various degrees. These hybrids extinguish the production of TCR beta and Thy-1 mRNA, repress the expression of TCR alpha, GATA-3, TCF-1, and LEF-1 genes to different extents, exhibit small changes in the level of CD3-epsilon mRNA, and continue to express the fibroblast-specific fibronectin gene, and the ets-1 gene. In this study we have evaluated for the first time the molecular mechanisms that underlie the repression of TCR alpha and TCR beta chain genes in T x L-cell hybrids. We have shown that multiple repression mechanisms, both direct and indirect, contribute to TCR alpha and TCR beta suppression. Repression of the expression of these genes correlated not only with the downregulation of GATA-3, TCF-1, and LEF-1 transcription factor expression, and with a change in the chromatin structure, but more importantly, with the activation of the silencer activity. Our study provides evidence for the existence of at least two negatively regulating elements, located at the TCR alpha enhancer-containing fragment and at the silencer region, which are active in our hybrid cells. We have shown that there was no correlation between the levels of GATA-3, TCF-1, and LEF-1 expression versus the level of TCR alpha mRNA in the independent hybrids. In contrast, both the silencer activity and the ability of the TCR alpha enhancer to downregulate thymidine kinase (TK) promoter activity were found to be in an inverse correlation with the ability of the different hybrid cells to express TCR alpha mRNA.

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Oct-1 is a ubiquitously expressed regulatory gene of the POU domain family. The Oct-1 protein binds to the octamer motif present in the control regions of a variety of genes such as the immunoglobulins, histone H2B and snRNAs. To learn about Oct-1 and its possible role in B-cell maturation, we have used oct-2 cDNA to screen a murine pre-B cell, cDNA library. Two cDNA clones were identical in their POU-homeo box DNA binding domain, but differed in their 3'-region. Whereas one clone (oct-1a) was very similar to its human oct-1 homologue, the other (oct-1b), contained an additional 72 bp sequence (designated E1) at the serine threonine rich coding region (position 1485 of the human oct-1), and a deletion of another 72 bp sequence (designated E2) downstream (position 1920). These changes preserve the protein reading frame. DNA blot analysis indicates that murine oct-1 is a single copy gene and that the two oct-1 isoforms oct-1 is expressed as a large approximately 10 kb transcript in all the cell are generated by alternative RNA splicing. RNA blots showed that oct-1 is expressed as a large approximately 10 kb transcript in all the cell lines tested. PCR analysis of the E1 and E2 72 bp regions, indicated the presence of a third isoform containing both E1 and E2 (Oct-1c). Oct-1a and Oct-1b were present in all cell types examined, but the level of expression was lower in liver and spleen as compared to testis, thymus and kidney. The ratio of Oct-1b to Oct-1a ranged between 0.2 to 0.5, for all tissues examined except for testis which expressed higher amounts of oct-1b and/or oct-1c. Our findings thus show that the pattern of expression of the oct-1 gene is more complex than hitherto thought.

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The extracellular organic matrix of developing human enamel is composed of two major classes of proteins, the hydrophobic amelogenins and the acidic enamelins. In order to identify, purify, and characterize the amelogenins from this complex mixture of proteins, and to study their ultrastructural localization and their pathways of synthesis, secretion, and degradation, specific and sensitive probes are needed. In the present paper the production of a monoclonal antibody against human amelogenin employing an intrasplenic primary immunization protocol is described. The monoclonal antibody produced is IgM and recognizes major human amelogenin protein bands in Western immunoblot assays. It also recognizes amelogenin protein bands from other species, specifically bovine and porcine. Indirect immunohistochemical studies showed the monoclonal antibody to react specifically with the extracellular matrix of human developing enamel. It did not react with the underlying dentin layer.

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Using a subtractive cDNA approach we have identified a gene, PC326, expressed in 13 of 14 murine plasmacytoma cell lines, but not in any B- or pre-B-lymphoma cell lines. It expresses 4.6-kb and 5.2-kb mRNAs that encode a 747 amino acid protein containing two highly acidic domains flanking a novel, moderately acidic 20 amino acid sequence that is repeated 7.5 times. Sequence comparison identifies an additional 43 amino acid domain that is homologous to a repeated sequence found in the members of the beta-transducin gene family. The PC326 mRNA is detectable in testis but in no other murine tissues, including plasma cells induced by lipopolysaccharide stimulation of splenocytes. Somatic cell hybrids derived from plasmacytomas and fibroblast or T-cell lines have a fibroblastic or T-cell phenotype respectively. Unlike B-cell-specific genes (e.g. immunoglobulin), the expression of which is extinguished in these hybrids, PC326 mRNA appears to be irreversibly turned on in these hybrids. Since PC326 is not expressed in normal plasma cells, it appears that its expression is a cause or consequence of the tumorigenic process that generates murine plasmacytomas.

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The production of heparanase, an endoglycosidase capable of degrading heparan sulfate from the subendothelial extracellular matrix (ECM), was investigated in various murine B-lymphoid tumors representing distinct maturation stages of the B-cell lineage. We found that heparanase is produced and released by 3 out of 4 pre-B lymphomas and by 4 B lymphomas examined. In contrast, 5 plasmacytomas and resting normal B lymphocytes, expressed little, if any, heparanase activity. Treatment with LPS resulted in high expression of the enzyme by normal B-lymphocytes, but there was no effect on the constitutive production of heparanase by myeloma or B-lymphoma cells. Our results indicate that heparanase is produced by B cells during discrete stages of their maturation. We suggest that heparanase may play a role in B-cell migration by enabling pre-B and B lymphocytes to leave the bone-marrow compartment and recirculate among peripheral lymphoid organs.

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Nine monoclonal antibodies directed against class A beta-lactamases were detected and selected by a novel screening procedure based on assaying the modifications in the catalytic and stability properties of beta-lactamase in solution. Unlike conventional screening, e.g., ELISA or immunoprecipitation, the present method does not depend on firm binding and thus favors detection of low affinity antibodies. Individual antibodies were found to affect the enzymatic activity in various ways including stimulation, neutralization, protection and stabilization. Class A beta-lactamases show only 20% among members of this class. In contrast, two of our monoclonal antibodies cross-reacted with different beta-lactamases and thus demonstrate the presence of shared structural epitopes in this class of enzymes. One of the cross-reacting antibodies was elicited by sequential immunization with two different beta-lactamases. Taken together, our findings stress the importance of the screening method in antibody selection and illustrate the use of 'functional' monoclonal antibodies in the study of the structure-function relationship in an enzyme.

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Media from murine pre-B and B lymphoma cell cultures, but not from myeloma cell cultures, was cytotoxic to WEHI 164 cells, causing these TNF-sensitive targets to release 51Cr. The cytotoxic activity in the culture medium reached maximum levels approximately 4 days after the cell culture was initiated. The constitutive production of the factors was not influenced by depletion of serum from the medium or by the addition of either phorbol ester or bacterial endotoxin. The factor has a Mr greater than 10 kDa, and its cytotoxicity was abolished by anti-serum against murine TNF. Northern blot analysis with the use of cDNA probes to murine tumor necrosis factor (TNF-alpha) and lymphotoxin (LT, TNF-beta) showed high levels of TNF-mRNA in the pre-B cell lines, lower levels in the mature B cell lines and no TNF-mRNA in the myeloma cell lines. LT mRNA was present in pre-B cell lines, at a much lower concentration in only one of the B cell lines, and was not present in three other B lymphomas or in the myelomas tested. The results show a positive correlation between the presence of TNF and/or LT mRNA and the 51Cr-releasing activity present in the cell culture medium. Our data indicate that TNF and LT can be produced by murine B cells and that the synthesis of these cytokines may be restricted to certain differentiation stages of the B cell lineage.

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In most instances, fusion of differentiated cell types with fibroblasts has resulted in the extinction of differentiation-specific traits of the nonfibroblast parental cell. To explore the genetic basis of this phenomenon, we have used a series of somatic cell hybrids between myeloma cells and fibroblasts. Previous findings show that in these hybrids expression of the immunoglobulin (Ig) genes was extinguished at the transcriptional level. Our present results show that NF-kappa B transcription factor, known to be critical for kappa-chain enhancer activity, is present although in a lower amount, in the nucleus and in the cytosolic fraction of most of these hybrids (probably attached to the previously postulated I-kappa B inhibitor). In contrast, the expression of the NF-A2/OTF-2 transcription factor encoded by the oct-2 gene, which binds to the octameric motif located in the Ig promoters and heavy chain gene enhancer, is extinguished at the transcriptional level. Our data thus suggest that extinction of Ig genes expression occurs via an indirect mechanism in which a fibroblast factor suppresses transcription factor(s) which are critical for Ig transcription.

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The role of Na + transport systems in the mitogenic signal induced by growth factors was studied, and it was shown that two Na + transport systems contribute to the early increase in cytoplasmic Na + in response to serum growth factors, namely the amiloride-sensitive Na+/H+ antiport and the bumetanide-sensitive Na+/K+/Cl- cotransport. Bumetanide or amiloride, when added separately, inhibited part of the increase in cytoplasmic Na +, as a response to the addition of serum to quiescent BALB/c mouse 3T3 fibroblasts. Each drug also suppressed part of the stimulation of the ouabain-sensitive Rb + influx, which was controlled by intracellular Na +. However, when both drugs were added together with serum growth factors, a complete inhibition of the early increase in [Na +], and subsequently a complete blockage of Na+/K+ pump stimulation was obtained. Amiloride or bumetanide, when added separately, only partially inhibited DNA synthesis induced by serum, 24% and 8% respectively. However, when both drugs were added together, at the time of serum addition to the quiescent cells, cell entry into S-phase was completely inhibited. To investigate the mode of cell-cycle inhibition, analysis was done of the possible role of early Na + fluxes in the mitogenic signal transduced from cell membrane receptors to the nucleus. The effects of the two drugs amiloride and bumetanide on induction of three genes--c-fos, c-myc, and ornithin decarboxylase (ODC)--was measured during cell transition through the G1-phase. Amiloride and bumetanide, when added separately or in combination, did not inhibit the induction of c-fos, c-myc, and ODC mRNAs. These results suggest that stimulation of Na + fluxes by serum growth factors is essential for cell transition into the S-phase of cell cycle, but it plays no apparent role in the growth factor signal transduced from the cell surface to the interior of the cell, as manifested by c-fos, c-myc, and ODC genes induction.

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The immune system is capable of establishing an enormous repertoire of antibodies before its first contact with antigen. Most antibodies that express germ-line sequences are of relatively low affinity. Once antigen enters the system, it stimulates a somatic mutational mechanism that generates antibodies of higher affinity and selects for the expression of those antibodies to produce a more effective immune response. The details of the mechanism and regulation of somatic hypermutation remain to be elucidated.

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Most murine plasma-cell tumors show a t(12;15) reciprocal chromosomal translocation which truncates the first exon of one of the myc gene alleles and fuses it to one of the switch regions of the immunoglobulin (Ig) heavy-chain locus. This results in constitutive activation of the translocated myc gene and the production of smaller-sized mRNA molecules, which are initiated at new sites in the first myc intron. The normal myc allele is not expressed in these myeloma cells. We have studied the expression of the translocated myc gene in somatic cell hybrids between mouse myeloma and L-cells. Our previous findings show that Ig gene expression is extinguished in such hybrids. In the present work we found that the hybrids contain the normal and translocated myc genes. In contrast to the myeloma parental cells which express the translocated myc gene, the hybrids are similar to the L-cells in expressing only the normal myc allele. Our results suggest that the L-cell, fibroblast-like phenotype, is dominant in these hybrids, and show that the translocated myc gene is expressed in a tissue-specific manner in the context of the myeloma cell, and is not expressed when subjected to a fibroblast-like cellular environment.

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Adherent hybrids between immunoglobulin-producing mouse myeloma cells and fibroblasts do not produce immunoglobulin polypeptide chains. These hybrids retained the actively rearranged immunoglobulin genes of the myeloma parental cells but lacked immunoglobulin heavy- and light-chain RNA transcripts. We conclude that the shutoff of immunoglobulin production in these hybrids occurs at the transcription or early processing level.

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The present study describes the development of a new IgM monoclonal autoantibody reactive with the Thy-1 antigen. The C16-31 monoclonal antibody (mAb) was considered as autoreactive because it reacted with thymus cells of both the C3H and BALB/c strains, which were involved in the development of the antibody. The antibody was reactive with thymus cells in both immunofluorescent and cytotoxic tests. It also showed a weak immunofluorescent reactivity with peripheral T-lymphocytes. The identification of the specificity detected by the C16-31 mAb as the Thy-1 antigen was based on the following criteria: C16-31 mAB displayed a preferential reactivity with Thy-1.2 bearing thymus cells, rather than with Thy-1.1 bearing thymus cells. The tissue distribution of the antigen detected by the C16-31 antibody by direct tests and by direct tests and by adsorption experiments was in accordance with that characteristic for Thy-1. It was high on brain tissue and on thymus cells, and considerably lower on peripheral T-lymphocytes. Coating of thymocytes with C16-31 antibody blocked their reactivity with other monoclonal Thy-1 antibodies. Conversely, coating of thymus cells with rabbit anti-brain serum (RABR) inhibited the binding of C16-31. The C16-31 mAb differed from the Thy-1 autoantibodies described previously in its relatively strong reactivity with brain tissue and its considerably weaker reactivity with peripheral T-lymphocytes. Moreover, C16-31 mAb showed a preferential allospecificity for Thy-1.2, only in its reactivity with thymocytes. In contrast, it reacted equally well with brain tissue from either Thy-1.2 or Thy-1.1 mice.

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This report describes an immunoradiometric assay for Plasmodium falciparum in infected blood, based on a cross-reacting monoclonal antibody (mAb) raised against P. berghei. In this assay, binding of the mAb to intact P. berghei parasites coated on microtiter plates is inhibited by solubilized P. falciparum infected red blood cells. The use of P. berghei parasites in conjunction with monoclonal antibodies should facilitate the development of an inexpensive and reproducible test for the immunodiagnosis of malaria.

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