Human Burkitt Lymphoma Cell Line Research Articles

Preparative high-resolution two-dimensional electrophoresis enables the identification of RNA polymerase B transcription factor 3 as an apoptosis-associated protein in the human BL60-2 Burkitt lymphoma cell line.

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified RNA polymerase B transcription factor 3 (BTF3), which is associated with anti-IgM antibody-mediated apoptosis, using a subclone of the human Burkitt lymphoma cell line BL60. To identify the transcription factor BTF3, which is expressed only in minor amounts, we used preparative high-resolution two-dimensional gel electrophoresis (2DE) employing carrier ampholytes for isoelectric focusing. Comparison of the 2DE protein patterns from apoptotic and nonapoptotic cells showed BTF3 as a predominantly altered protein spot. The characterization of the differentially expressed transcription factor and 13 marker proteins described in this study were performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The proteome analysis was significantly improved by performing the newly developed preparative high-resolution two-dimensional gels employing high protein concentrations.

Identification of Apoptosis-associated Proteins in a Human Burkitt Lymphoma Cell Line: CLEAVAGE OF HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN A1 BY CASPASE 3

Apoptosis or programmed cell death is essential in the process of controlling lymphocyte growth and selection. We identified proteins that are involved in anti-IgM antibody-mediated apoptosis using a subclone of the human Burkitt lymphoma cell line BL60. Apoptosis-associated proteins were detected by high resolution two-dimensional gel electrophoresis on a micropreparative scale. Comparison of the high resolution two-dimensional gel electrophoresis protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots including protein modifications. Analysis of the predominantly altered proteins was performed by internal Edman microsequencing and/or by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. Analysis was significantly improved by using new micropreparative high resolution two-dimensional gels employing high protein concentrations. The following 12 apoptosis-associated proteins were identified: heterogeneous nuclear ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, FUSE-binding protein, dUTPase, lymphocyte-specific protein LSP1, UV excision repair protein RAD23 homologue B (HHR23B), 60 S acidic ribosomal protein P0 (L10E), heterochromatin protein 1 homologue alpha (HP1alpha), nucleolin, lamin, neutral calponin, and actin. Fragmentation of actin, hnRNP A1, hnRNP C1/C2, 60 S acidic ribosomal protein P0, lamin, and nucleolin could be inhibited by benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone, a selective irreversible inhibitor of CPP32 (caspase 3).

Inhibition of CPP32 blocks surface IgM-mediated apoptosis and D4-GDI cleavage in human BL60 Burkitt lymphoma cells

Apoptosis (programmed cell death) is instrumental in the process of controlling lymphocyte growth and selection. Negative selection, mediated by surface IgM (sIgM) signaling after encountering self antigen, eliminates autoreactive B cells. To identify proteins which are potentially involved in anti-IgM-mediated apoptosis, we used an anti-IgM-sensitive subclone of the human Burkitt lymphoma cell line BL60. After anti-IgM treatment and separation of apoptosis-committed cells, we performed high resolution two-dimensional gel electrophoresis (2-DE). Comparison of the 2-DE protein patterns from apoptotic and non-apoptotic cells showed differences in approximately 80 spots. Subsequent analysis of these proteins was performed by mass spectrometry and Edman microsequencing. We report that one of these spots which disappears after sIgM cross-linking turned out to be D4-GDI. D4-GDI is an abundant hematopoietic cell GDP dissociation inhibitor for the Ras-related Rho family GTPase. D4-GDI was rapidly truncated to a 23-kDa fragment in BL60 cells. By using a Rho-GDI-specific antiserum, which cross-reacts with D4-GDI, we observed the onset of cleavage after 8 h of stimulation with anti-IgM. Cleavage and apoptosis could be completely inhibited by z-DEVD-fmk, a selective irreversible inhibitor of CPP32 (caspase-3), whereas ac-YVAD-cmk, an inhibitor for interleukin-1β-converting enzyme-like proteases, did not block cleavage of D4-GDI or apoptosis. Our results revealed the functional importance of caspases and a new target protein in the process of anti-IgM-mediated apoptosis.

Establishment of a monoclonal antibody directed against Gb3Cer/CD77: a useful immunochemical reagent for a differentiation marker in Burkitt's lymphoma and germinal centre B cells.

A new monoclonal antibody (TU-1) directed against the Gal alpha 1-4Gal beta 1-4Glc residue of the Gb3Cer/CD77 antigen was prepared by the hybridoma technique following immunization of mice with an emulsion composed of monophosphoryl lipid A, trehalose dimycolate, and Gb3Cer isolated from porcine erythrocytes. TU-1 showed reactivity towards Gb3Cer and lyso-Gb3Cer(Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Sph), although the reactivity towards lyso-Gb3Cer was about 10-fold lower than that to Gb3Cer. But it did not react with other structurally-related glycolipids, such as LacCer (Gal beta 1-4Glc beta 1-1'Cer), Gg3Cer, Gg4Cer, Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1-1'Cer), galactosylparagloboside (Gal alpha 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer), sulfatide (HSO3-3Gal beta 1-1'Cer), other gangliosides (GM3, GM2, GM1a, GD1a and GT1b), or P1 antigen (Gal alpha 1-4Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc beta 1-1'Cer) among neutral glycolipids prepared from P1 phenotype red blood cells. Furthermore, TU-1 reacted with viable lymphoma cells, such as human Burkitt lymphoma cell line, Daudi, and Epstein-Barr virus (EBV)-transformed B cells by the immunofluorescence method, and also with germinal centre B cells in human tonsil and vessel endothelial cells in human thymus histochemically. These results indicate that TU-1 is a monoclonal antibody directed against Gb3Cer/CD77 antigen and can be utilized as a diagnostic reagent for Burkitt's lymphoma and also for detection of the blood group Pk antigen in glycolipid extracts of erythrocytes.

A role for the cyclin-dependent kinase inhibitor p21 in the G1 cell cycle arrest mediated by the type I interferons.

Type I interferons (IFN), such as IFN-alpha, are potent antiproliferative and antitumor agents. IFN-tau, originally identified as a pregnancy recognition hormone, is a type I IFN that is related to IFN-alpha. We examine here the mechanism of the antiproliferative effects of IFN-alpha and IFN-tau in terms of their effects on intracellular events that regulate the cell cycle. Both IFN inhibited proliferation of the human Burkitt lymphoma cell line, Daudi, causing accumulation of cells in the G1 phase of the cell cycle. IFN-alpha was more effective than IFN-tau in this regard. Both IFN were found to inhibit the kinase activity of the cyclin-dependent kinase cdk2 in a manner that correlated with their relative abilities to cause cells to accumulate in the G1 phase of the cell cycle. Further, IFN treatment did not affect the expression of cdk2 protein, suggesting that the IFN modulated cdk2 activity through a cdk inhibitor. Consistent with this conclusion, both IFN induced the expression of the cyclin-dependent kinase inhibitor protein p21. The levels of p21 induced also correlated with the relative abilities of the IFN to inhibit cdk2 activity and to arrest cell growth in the G1 phase of the cell cycle. Moreover, following IFN treatment, increased levels of p21 were found complexed with cdk2, consistent with its role in the inhibition of cdk2 activity. These data suggest that p21-mediated inhibition of cdk2 activity plays an important role in the antiproliferative activity of type I IFN. The findings highlight interesting similarities between these cytokines and the products of tumor suppressor genes, such as p53, and may indicate a mechanism for the antitumor effects of the type I IFN.

Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo.

Human Burkitt lymphoma cell lines give rise to progressively growing subcutaneous tumors in athymic mice. These tumors are induced to regress by inoculation of Epstein-Barr virus-immortalized normal human lymphocytes. In the present study, analysis of profiles of murine cytokine/chemokine gene expression in Burkitt tumor tissues excised from the nude mice showed that expression of the murine alpha-chemokine interferon-inducible protein-10 (IP-10) was higher in the regressing than in the progressive Burkitt tumors. We tested the effects of IP-10 on Burkitt tumor growth in nude mice. Inoculation of established Burkitt tumors either with crude preparations of murine IP-10 or with purified human IP-10 caused visible tumor necrosis in a proportion of the animals, although no complete tumor regressions were observed. Constitutive expression of murine IP-10 in Burkitt cells reduced their ability to grow as subcutaneous tumors, and caused visible tumor necrosis in a proportion of the animals. Histologically, IP-10-treated and IP-10-expressing Burkitt tumors had widespread evidence of tumor tissue necrosis and of capillary damage, including intimal thickening and vascular thrombosis. Thus, IP-10 is an antitumor agent that promotes damage in established tumor vasculature and causes tissue necrosis in human Burkitt lymphomas established subcutaneously in athymic mice.

Oligonucleotides complementary to c-myb messenger RNA inhibit growth and induce apoptosis in human Burkitt lymphoma cells

A 24-mer (antisense) phosphorothioate oligonucleotide (ODN) corresponding to the codons 2-9 of the c-myb gene was evaluated for its effects on the growth of a human Burkitt lymphoma cell line (Raji) in vitro. Raji cells incubated with different concentrations of c-myb antisense ODN (5-15 mu g/ml) for 24-72 h showed a significant dose-dependent decrease in growth. The same concentrations of control (sense) or scrambled c-myb phosphorothioate ODNs did not inhibit Raji cell growth. The c-myb antisense ODN, but not the control ODNs, significantly decreased c-myb mRNA levels in treated cells as determined by RT-PCR. Additionally, the c-myb antisense ODN induced apoptosis of Raji cells as demonstrated by i) flow cytometry to enumerate the A(o) (apoptotic cell population) population of propidium iodide stained cells; ii) electron microscopy to evaluate the cell morphology; and iii) DNA fragmentation pattern. Thus, an antisense c-myb ODN causes significant growth inhibition of Burkitt lymphoma cells, and one mechanism of growth inhibition is the induction of apoptosis of the lymphoma cells. In addition, antisense c-myb ODN did not reduce CFU-GM or BFU-e colony-forming ability of normal hematopoietic stem/progenitor cells. Because the inhibition is sequence-specific and Burkitt lymphoma cell selective, evaluation of the therapeutic effects of c-myb antisense ODN against Burkitt lymphoma is warranted.

A dual role for both CD40-ligand and TNF-alpha in controlling human B cell death.

Members of the TNF-R family are instrumental in controlling lymphoid cell death and survival. A major role in the regulation of murine and human B cell survival and differentiation has been attributed to CD40/CD40-ligand (CD40-L) interactions, but recent in vitro and in vivo data implicate that other receptor-ligand pairs might also be involved. We have used the human Burkitt lymphoma cell line Ramos as a model system to identify additional TNF-R/TNF family members that are implicated in the regulation of B cell apoptosis. Ligation of B cell receptor (BCR) with anti-IgM mAb for 48 h induced apoptosis in approximately 68% of the Ramos B cells. Interestingly, not only CD40 mAb but also rTNF-alpha could efficiently inhibit BCR-induced B cell death. In addition, activated T cells also prevented BCR-triggered apoptosis, and this effect was inhibited completely by a combination of blocking Abs against CD40-L and TNF-alpha. In contrast to the strong effect of BCR ligation, APO-1 mAb induced apoptosis in only +/- 18% of the Ramos cells after 48 h. Noticeably, addition of CD40 mAb or rTNF-alpha increased the percentage of cells (+/- 46%) undergoing apoptosis, which correlated with an increase of Fas/APO-1 membrane expression induced by CD40 or TNF-R ligation. Taken together, we show that CD40/CD40-L and TNF-R/TNF-alpha interactions not only postpone or prevent B cell death, but are also involved in sensitizing B cells for Fas-ligand (Fas-L)-dependent death.

Lyn tyrosine kinase signals cell cycle arrest but not apoptosis in B-lineage lymphoma cells.

Signal transduction initiated by binding of antibodies to cell surface molecules can have an important impact on the growth of tumor cells. The malignant behavior of the murine lymphoma BCL1 can be suppressed and the neoplastic cells can be induced to enter a dormant state by in vivo ligation of membrane immunoglobulin. Anti-CD19 antibodies can prolong the survival of SCID mice challenged with the human Burkitt lymphoma cell line, Daudi. Here, we show that cross-linking of membrane immunoglobulin on both murine BCL1 and human Daudi cells initiates a cascade of signals leading to the induction of both apoptosis and cell cycle arrest in vitro. Using antisense oligonucleotides, we demonstrate that the immunoglobulin-associated Lyn tyrosine kinase is required for anti-immunoglobulin-mediated cell cycle arrest but is not required for the signal leading to apoptosis. These results define a branch point in the cytosolic signaling pathways mediating cell cycle arrest and apoptosis. In Daudi cells, Lyn is also critical for cell cycle arrest induced by anti-CD19 signaling. Thus, the Lyn tyrosine kinase may be an important mediator of cell cycle arrest in neoplastic B lymphocytes and, perhaps, other cell types.

Transcription of the tumor necrosis factor alpha gene is rapidly induced by anti-immunoglobulin and blocked by cyclosporin A and FK506 in human B cells.

The human tumor necrosis factor alpha (TNF-alpha) gene encodes a cytokine whose activities have been implicated in many immunopathological processes, including the activation and differentiation of lymphocytes. Originally identified as a monocyte factor, our studies and those of others have demonstrated that B and T lymphocytes produce TNF-alpha when stimulated by a variety of inducers. We report here that TNF-alpha gene transcription is rapidly and highly induced in three independently derived human Burkitt lymphoma cell lines, as well as in freshly isolated human splenic B cells, activated by antibodies to surface immunoglobulin. This burst in TNF-alpha gene transcription is associated with an induction of TNF-alpha bioactivity in the culture supernatants from stimulated splenic B cells. Moreover, induction of TNF-alpha gene transcription by anti-immunoglobulin was blocked by the immunosuppressants cyclosporin A and FK506. These studies demonstrate that TNF-alpha production is an early event in B-cell activation and they establish the efficacy of using immunosuppressants as probes in dissecting transcriptional activation pathways in human B cells.

Rapid Purification and Structural Study of DNA Topoisomerase I from Human Burkitt Lymphoma Raji Cells1

We have developed a rapid purification method for DNA topoisomerase I from Raji cells, a human Burkitt lymphoma cell line, using ammonium sulfate fractionation followed by chromatography on a Mono S column (FPLC, Pharmacia). By this method, the enzyme could be purified to near homogeneity within one day. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel revealed that the final preparation is mainly composed of a 100-kDa protein. The major enzyme activity sedimented through a glycerol density gradient at 5.7S, accompanied with a minor peak at 8.7S. The former may correspond to the monomer of the 100-kDa polypeptide, and the latter, to its dimeric form. The gel filtration study of the crude extract revealed an active molecular species of 200 kDa, in addition to 100 kDa, and lower molecular weight forms. These results suggest that DNA topoisomerase I is largely in monomeric form, but also has a minor population of the dimeric form.

Characterization of the human type I interferon receptor by ligand blotting.

The human type I interferon (IFN) receptor was characterized by ligand blotting. In this method, plasmalemma proteins or detergent-lysed whole-cell extracts from human Burkitt lymphoma cell lines were separated on polyacrylamide gels and subsequently transferred onto nitrocellulose sheets. Probing the blots with 3 x 10(-10) M 125I-labeled recombinant IFN-alpha A (125I-rIFN-alpha A) revealed an IFN-alpha-binding protein with an apparent molecular mass of 95 kDa (p95). Performing the electrophoretic run under reducing conditions completely abrogated the signal on the blot, indicating that the type I IFN receptor contains a disulfide bond essential for IFN binding. Optimal binding of 125I-rIFN-alpha A occurred at pH 9. The specificity of the binding reaction was established by simultaneously adding an excess of unlabeled IFN during incubation of the blots with 125I-rIFN-alpha A. The addition of either unlabeled IFN-alpha or IFN-beta, but not IFN-gamma, abolished the binding of 125I-rIFN-alpha A to p95. 125I-IFN-gamma at 1.25 x 10(-11) M bound to two proteins distinct from p95 with apparent molecular mass of 92 and 87 kDa, respectively. Saturability of 125I-rIFN-alpha A binding was demonstrated by probing a constant amount of membrane proteins with increasing amounts of 125I-rIFN-alpha A. Scatchard analysis of the binding data yielded an apparent Kd of 5.4 x 10(-10) M for the immobilized type I IFN receptor. The expression of p95 on IFN-alpha-resistant and -sensitive cells was indistinguishable. We conclude that p95 is the IFN-alpha/beta receptor and that two proteins (p92 and p87) can specifically bind IFN-gamma. These results indicate that ligand blotting is a versatile method for characterization of unmodified IFN receptors and IFN-receptor interaction and could also provide a new investigational approach for other cytokine receptor systems.

FK506

WARTY, VIJAY; DIVEN, WARREN; CADOFF, EVAN; TPDO, SATORU; STARZL, THOMAS; SANGHVI, AJIT Author Information

Inhibition of homotypic aggregation of a human Burkitt‐lymphoma cell line

The molecules involved in homotypic aggregation of the human Burkitt-lymphoma cell line Raji were investigated by inhibition of reaggregation with carbohydrates and glycoconjugates, by inhibition of glycosylation, and enzyme treatment of the cell surface. Complete inhibition of reaggregation was achieved with bovine submaxillary mucin. Asialomucin, on the other hand, was not effective in this assay. Another potent inhibitor of reaggregation was the ganglioside GMI. The common carbohydrate structure of these molecules is NeuNAc-(gal)-galNAc. Lactosamine, fucosyllactosamine, sialyllactosamine, complex mannose type, or Thomsen-Friedenreich antigen sequences are not involved in aggregation. Neuraminidase and chloroquine also abolished agglutination of cells. The finding that mucin, but not asialomucin, inhibits the reaction, demonstrates the importance of sialic acid in this process. Homotypic aggregation was shown to be resistant to trypsin. Using the glycosylation inhibitor tunicamycin we show that N-glycosidically linked carbohydrate chains are involved in aggregation. Swainsonine or castanospermine, which inhibit processing of terminal sialyllactosamines to the mannose core, did not interfere with the reaction supporting the results of the inhibition assay. The data presented suggest the involvement of 2 molecules in homotypic aggregation of human Burkitt-lymphoma cells. One component is a lectin-like molecule containing N-linked carbohydrate chains. The other component carries the neuraminidase-sensitive and trypsin-resistant determinant NeuNAc-(gal)-galNAc and, therefore, appears to be a glycolipid. This proposed lectin-carbohydrate interaction in homotypic aggregation is further supported by the frequently observed dependence of lectins on divalent cations as indicated by inhibition of aggregation with EDTA and EGTA.

Roles of globotriosyl- and galabiosylceramide in verotoxin binding and high affinity interferon receptor.

The cellular specificity of the Escherichia coli-derived verotoxin is of particular interest because of its extreme toxicity and high selectivity toward certain primate cells. The human Burkitt lymphoma cell line (Daudi) is highly susceptible to the cytotoxicity of verotoxin and contains large amounts of the verotoxin-binding glycolipids on its surface. A mutant selected from Daudi cells for verotoxin resistance was found to be deficient in the verotoxin-binding glycolipids, globotriosylceramide and galabiosylceramide, and failed to bind verotoxin to its surface; interestingly, these mutant cells were found to be cross-resistant to inhibition of growth by alpha-interferon. Mutant cells also lack the high affinity component of alpha-interferon binding. These observations suggest that, in addition to providing the functional cell-surface receptor for verotoxin, these glycolipids may also play a role in the modulation of the affinity of alpha-interferon for its membrane protein receptors.

Elevated DNA topoisomerase II activity in nitrogen mustard-resistant human cells.

A human Burkitt lymphoma cell line, Raji-HN2, made 10-fold more resistant to nitrogen mustard (HN2) than the parental Raji cell line, exhibited the following characteristics when compared to the parental Raji cells: (i) decreased HN2-induced DNA interstrand crosslinking; (ii) increased (3-fold) DNA topoisomerase II [DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3] activity; (iii) increased (4- to 11-fold) sensitivity to topoisomerase II inhibitors; (iv) increased (2-fold) glutathione content; and (v) increased (2-fold) cell doubling time. The resistant phenotype was unstable and was maintained by weekly treatment of the cells with HN2. Growing the resistant cells in the absence of HN2 resulted in a time-dependent decrease in both resistance to HN2 and topoisomerase II activity and an increase in DNA interstrand crosslinking induced by HN2. We hypothesize that HN2 resistance is due to enhanced monoadduct repair with resultant decreased DNA crosslinking and that this process is mediated by topoisomerase II.

Kinetics of uptake and intracellular binding of cyclosporine a in RAJI cells, in vitro

Uptake characteristics of Cyclosporine A (CsA), an immunosuppressive agent widely used in organ transplantation, have been evaluated in RAJI cells, a human Burkitt lymphoma cell line which (i) does not bear T-cell markers, (ii) is insensitive to CsA after a 1 hr exposure to concentrations up to 50 μg/ml, and (iii) does not metabolize CsA. CsA is rapid;y accumulated inside the cells until a near steady-state is achieved (within 1–3 min). This uptake is characterized by two components: one linear process saturable at low drug concentrations (lower than 1 μg/ml) and another not saturable component even at hogh drug concentrations (up to 50 μg/ml). Uptake of CsA is temperature-dependent and unaffected by the presence of CsD, a structural CsA analog (50 μg/ml CsD) or sodium azide (10 mM) in the extracellular compartment. Intracellular accumulation of CsA is associated with the rapid appearance of a cytosolic drug-protein complex, which is responsible at least in part, for the large amount of total drug accumulated inside the cells. Chromotographic analysis of this ( 3H)CsA-macromolecule complex on a Bio-Gel P-60 exclusion column demonstrates that the molecular weight of this protein(s), likely cyclophilin, is around 15,000–20,000 daltons. Using Lineweaver-Burk analysis of binding equilibrium data, the dissociation constant of CsA for this binding site was approximately 2.2 μM. These studies, which demonstrate that CsA (i) is rapidly accumulated inside the cells as free drug but is also specifically bound to an intracellular macromolecule, and (ii) is selectively retained in the intracellular compartment after the extracellular drug is removed, could explain the intense distribution of CsA in the organs and the slow disappearance of CsA from plasma after CsA therapy in humans.

Effects of the [formula omitted][formula omitted] siderophore vibriobactin on the growth characteristics of L1210 cells

The microbial iron chelator vibriobactin, N-[3-(2,3-dihydroxybenzamido)propyl]-1,3-bis[2-(2,3-dihydroxyphenyl)- trans -5-methyl-2-oxazoline-4-carboxamido]-propane, is shown to inhibit the growth of L1210 cells in culture, with an IC 50 value of 2 μM. Its biological activity is assigned to the ligand's ability to chelate iron as indicated by the disappearance of its antimitotic properties on iron chelate preformation or on O-methylation. The ligand is shown to have pronounced effects on cell cycle kinetics introducing a G 1 S phase block. When treated cells are washed free of the ligand with fresh media they cascade into a high S phase in 5 hrs. Furthermore, after exposure of L1210 cells to vibriobactin (10 μM) for 5 hrs followed by removal of the drug, cells display different boubling times relative to untreated controls, with lower bromodeoxyuridine (BrdUrd) incorporation and no apparent cell death as shown by a 51Cr release assay. The effects on cell kinetics are consistent with inhibition of ribonucleotide reductase. Finally, vibriobactin is also shown to be active with a human Burkitt lymphoma cell line (Daudi).

Internalization and processing of antibodies to surface antigens on human B cells. Monoclonal anti-IgM antibodies are processed differently than monoclonal antibodies towards non-Ig surface receptors.

The internalization and intracellular processing of monoclonal antibody to immunoglobulin mu heavy chain (Mamu) have been investigated in two human Burkitt lymphoma cell lines (Ramos and Raji), in a human B cell lymphoma and in normal human peripheral blood B cells. In addition to the degradation of 125I-labeled Mamu to trichloroacetic acid (TCA) soluble material, a distinct pattern of larger 125I-Mamu fragments was detected in all sources of B cells tested. The particular fragmentation pattern, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, involved the cleavage of both peptide bonds and disulfide bridges. This type of antibody fragmentation appeared to be a selective mechanism associated with sIgM, as no other degradation than that leading to TCA-soluble material could be detected after the internalization and degradation of radiolabeled monoclonal antibodies towards a variety of non-Ig B cell surface receptors. Three fragments of 125I-Mamu degradation were also detected in the supernatant of Ramos cells, implying that the recycling and exocytosis of certain 125I-Mamu fragments also took place.

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