Burkitt Lymphoma Cell Line Daudi Research Articles

Non-viral TRAC-knocked-in CD19KICAR-T and gp350KICAR-T cells tested against Burkitt lymphomas with type 1 or 2 EBV infection: In vivo cellular dynamics and potency.

The ubiquitous Epstein-Barr virus (EBV) is an oncogenic herpes virus associated with several human malignancies. EBV is an immune-evasive pathogen that promotes CD8+ T cell exhaustion and dysregulates CD4+ T cell functions. Burkitt lymphoma (BL) is frequently associated with EBV infections. Since BL relapses after conventional therapies are difficult to treat, we evaluated prospective off-the-shelf edited CAR-T cell therapies targeting CD19 or the EBV gp350 cell surface antigen. We used CRISPR/Cas9 gene editing methods to knock in (KI) the CD19CAR.CD28z or gp350CAR.CD28z into the T cell receptor (TCR) alpha chain (TRAC) locus. Applying upscaled methods with the ExPERT ATx® MaxCyte system, KI efficacy was ~20% of the total ~2 × 108 TCR-knocked-out (KO) generated cells. KOTCRKICAR-T cells were co-cultured in vitro with the gp350+CD19+ BL cell lines Daudi (infected with type 1 EBV) or with Jiyoye (harboring a lytic type 2 EBV). Both types of CAR-T cells showed cytotoxic effects against the BL lines in vitro. CD8+ KICAR-T cells showed higher persistency than CD4+ KICAR-T cells after in vitro co-culture with BL and upregulation of the activation/exhaustion markers PD-1, LAG-3, and TIM-3. Two preclinical in vivo xenograft models were set up with Nod.Rag.Gamma mice injected intravenously (i.v.) with 2 × 105 Daudi/fLuc-GFP or with Jiyoye/fLuc-GFP cells. Compared with the non-treated controls, mice challenged with BL and treated with CD19KICAR-T cells showed delayed lymphoma dissemination with lower EBV DNA load. Notably, for the Jiyoye/fLuc-GFP model, almost exclusively CD4+ CD19KICAR-T cells were detectable at the endpoint analyses in the bone marrow, with increased frequencies of regulatory T cells (Tregs) and TIM-3+CD4+ T cells. Administration of gp350KICAR-T cells to mice after Jiyoye/GFP-fLuc challenge did not inhibit BL growth in vivo but reduced the EBV DNA load in the bone marrow and promoted gp350 antigen escape. CD8+PD-1+LAG-3+ gp350KICAR-T cells were predominant in the bone marrow. The two types of KOTCRKICAR-T cells showed different therapeutic effects and in vivo dynamics. These findings reflect the complexities of the immune escape mechanisms of EBV, which may interfere with the CAR-T cell property and potency and should be taken into account for future clinical translation.

Myeloid-Derived Suppressor Cells Specifically Suppress IFN-γ Production and Antitumor Cytotoxic Activity of Vδ2 T Cells.

γδ T cells represent less than 5% of circulating T cells; they exert a potent cytotoxic function against tumor or infected cells and secrete cytokines like conventional αβ T cells. As αβ T cells γδ T cells reside in the typical T cell compartments (the lymph nodes and spleen), but are more widely distributed in tissues throughout the body. For these reasons, some investigators are exploring the possibility of immunotherapies aimed to expand and activate Vδ2 T cells, or using them as Chimeric Antigen Receptor carriers. However, the role of immunosuppressive microenvironment on Vδ2 T cells during infections and cancers has not been completely elucidated. In particular, the effects of myeloid-derived suppressor cells (MDSC), largely expanded in such pathologies, were not explored. In the present work, we demonstrated that MDSC may inhibit IFN-γ production and degranulation of phosphoantigen-activated Vδ2 T cells. Moreover, the Vδ2 T cells cytotoxic activity against the Burkitt lymphoma cell line Daudi and Jurkat cell line were impaired by MDSC. The Arginase I seems to be involved in the impairment of Vδ2 T cell function induced by both tumor cells and MDSC. These data open a key issue in the context of Vδ2-targeted immunoteraphy, suggesting the need of combined strategies aimed to boost Vδ2 T cells circumventing tumor- and MDSC-induced Vδ2 T cells suppression.

Synergistic action of 5Z-7-oxozeaenol and bortezomib in inducing apoptosis of Burkitt lymphoma cell line Daudi

Treatment failure in cancer chemotherapy is largely due to the toxic effects of chemotherapeutic agents on normal cells/tissues. The proteasome inhibitor bortezomib has been successfully applied to treat multiple myeloma (MM), but there are some common adverse reactions in the clinic including peripheral neuropathy (PN). The TAK1 selective inhibitor 5Z-7-oxozeaenol has been widely studied in cancer therapy. Here, we investigated the potential synergy of bortezomib and 5Z-7-oxozeaenol in Burkitt's lymphoma (BL) cell lines. Cell viability assay showed that co-treatment of bortezomib at 8nM, representing a one-eighth concentration for growth arrest, and 5Z-7-oxozeaenol at 2μM, a dose that exhibited insignificant cytotoxic effects, synergistically induced apoptosis in the cell line Daudi. In parallel with the increasing dose of the bortezomib, and 5Z-7-oxozeaenol at 0.5μM, lower colony formation efficiencies were seen in the cell line Daudi. Western blotting analysis verified that TAK1 inhibition by 5Z-7-oxozeaenol completely blocked JNK, p38, Erk, IKK, and IκB phosphorylation, which was almost instantly activated by TAK1 both directly or indirectly. Both agents synergistically prevented nuclear translocation of NF-κB, a characteristic of NF-κB inactivation. Moreover, a synergistic effect of bortezomib and 5Z-7-oxozeaenol on Western blotting analysis and flow cytometry was disclosed. Collectively, our results indicated that the proteasome inhibitor bortezomib and the TAK1 inhibitor 5Z-7-oxozeaenol displayed synergy on inhibiting BL cell apoptosis by inhibiting NF-κB activity.

Re‐expression of microRNA‐150 induces EBV‐positive Burkitt lymphoma differentiation by modulating c‐Myb in vitro

Burkitt lymphoma (BL) is a highly aggressive B-cell lymphoma that includes two forms of BL differing in Epstein-Barr virus (EBV) infection status, EBV-positive and EBV-negative. Although many efforts, such as high-intensity, short-duration combination chemotherapy, have been devoted to improving therapy for this rapidly proliferating neoplasm, there are still significant treatment-associated toxicities. Therefore, there remains a need for novel effective therapeutic strategies. MicroRNAs play a role in "fine tuning" the physiological and pathological differentiation process, by which cells can rapidly regulate dynamic events such as cell-lineage decisions and morphogenesis. This unique miRNA feature shifts the traditional one drug target paradigm to a novel one drug multiple targets paradigm. Here, we found that BL cell lines showed an extremely low expression of microRNA-150 (miR-150), and then restored miR-150 expression at physiologic levels in BL cell lines Daudi, Raji, BJAB, and Ramos. The results showed that re-expression of miR-150 reduced proliferation of Daudi and Raji cells. Furthermore, Daudi and Raji, both of which are of EBV-positive germinal center B-cell origin, transduced with miR-150 can be rescued to differentiate toward B-cell terminal stage. However, no significant changes were observed in BJAB or Ramos cells, which are of EBV-negative germinal center B-cell origin. Of note, re-expression of miR-150 also resulted in decreasing c-Myb protein levels. Additionally, c-Myb knockdown in Daudi and Raji cell lines recapitulated the partial characteristics similar to that caused by re-expression of miR-150. Taken together, our findings show that miR-150 can induce EBV-positive BL differentiation by targeting c-Myb.

Base-pair resolution DNA methylome of the EBV-positive Endemic Burkitt lymphoma cell line DAUDI determined by SOLiD bisulfite-sequencing

Base-pair resolution DNA methylome of the EBV-positive Endemic Burkitt lymphoma cell line DAUDI determined by SOLiD bisulfite-sequencing

Abstract 4581: IMGN529 is an anti-CD37-maytansinoid conjugate with potent in vitro and in vivo activity against NHL and CLL cell lines

Abstract IMGN529 is an antibody-drug conjugate consisting of the functional anti-CD37 antibody K7153A conjugated by a non-cleavable SMCC linker to the highly cytotoxic maytansinoid, DM1. Expression of CD37 in normal tissues is mainly limited to B-cells in blood and lymphoid tissues such as spleen, tonsil and lymph nodes. To evaluate the potential for targeting CD37 in B-cell malignancies by IMGN529, CD37 expression was compared to that of CD20, a well-validated target for antibody-directed therapy in lymphoma. CD37 exhibited a similar prevalence as CD20 in subtypes of NHL such as follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL) and mantle cell lymphoma (MCL) as assessed by immunohistochemistry using tumor microarrays. In addition, CD37 and CD20 antigen levels were measured by quantitative flow cytometry in NHL and B-CLL cell lines. All of the CD20+ B-cell lines examined also expressed CD37 at antigen levels ranging from 17,000 to 360,000 ABC (antibodies bound per cell). The activity of IMGN529 and its antibody component (K7153A) were evaluated against NHL and CLL cell lines in vitro. K7153A was active against an array of NHL-derived lymphoma cell lines including the DLBCL cell line SU-DHL-4, the FL cell line DOHH-2, the MCL cell line Granta-519, as well as B-CLL cell lines such as JVM-2 and JVM-3. The antibody showed only minimal activity against the BL cell lines Daudi and BJAB. Importantly, IMGN529 demonstrated enhanced and specific cytotoxicity against these cell lines when compared to either unconjugated K7153A antibody or a non-targeted control conjugate (EC50 10 – 400 pM). In contrast, the anti-CD37 compound, TRU-016, had no direct effect on any of these cell lines. The anti-CD20 antibody rituximab showed less direct activity than K7153A in most of these cell lines despite the often higher CD20 expression levels. In vivo, while the K7153A antibody showed anti-tumor efficacy in established tumor xenograft models of DLBCL (SU-DHL-4), FL (DOHH-2) and B-CLL (JVM-3), IMGN529 was significantly more active in these models. In addition, IMGN529 was active against established BL-derived BJAB tumors at doses (5 or 10 mg/kg single dose) where the unconjugated antibody was not active. In the FL-derived DOHH-2 tumor model, a single treatment of IMGN529 at 10 mg/kg resulted in tumor growth delay that was similar to rituximab given at 5 mg/kg twice weekly for 3 weeks and was better than treatment with CVP (cyclophosphamide/vincristine/prednisone). A single 5 mg/kg dose of IMGN529 demonstrated better activity than the anti-CD20 antibody ofatumumab (5 mg/kg twice weekly × 3) or bendamustine (50 mg/kg x1) in the B-CLL-derived JVM-3 xenograft model. In conclusion, IMGN529 is highly active in vitro and in vivo against representative B-cell lines from NHL and CLL suggesting the utility of this functional antibody-maytansinoid conjugate as a therapeutic in these disease indications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4581. doi:10.1158/1538-7445.AM2011-4581

Interleukin-2 Gene Transduction in Human Natural Killer Cells Augments Their Survival and Anti-Leukemic Capacity

Novel therapies are required to circumvent the resistance of leukemic cells to chemotherapy and improve cure rates. Infusions of natural killer (NK) cells might enhance the anti-leukemic effects of chemotherapy and hematopoietic stem cell transplantation. Indeed, haploidentical NK cells were shown to induce remission in patients with high-risk acute myeloid leukemia (AML; Miller at al. Blood 105: 3051, 2005). Current NK-cell therapy protocols include administration of interleukin (IL)-2 to sustain the survival and activation of the infused NK cells in vivo. In this study, we tested whether enforcing the expression of the IL-2 gene in NK cells would abrogate their requirement for exogenous IL-2, prolong their survival and augment their anti-leukemic capacity., We previously reported a method that induces NK cells to proliferate and reliably allows their genetic modification (Imai et al, Blood 106: 376, 2005). We used this method to expand primary NK cell from healthy individuals for 7 days. After depletion of residual T cells, NK cells were transduced with a retroviral vector containing human IL-2 cDNA and GFP. Fourteen days after transduction, more than 99% of cells were CD3− CD56+ NK cells; median transduction efficiency, assessed by GFP positivity, was 70.0% (40.4%–91.4%, n=9). NK cells expressing IL-2 cDNA (NK-gIL2) showed intracellular expression of IL-2 and secreted IL-2 (1.3 ± 0.5 U/106 cells/24 hours). Karyotyping of NK-gIL2 cells propagated for more than 6 weeks revealed no chromosomal aberration. The cytotoxicity of NK-gIL2 cells against the AML cell line K562 was similar to that of NK cells transduced with a vector containing GFP only when the cytotoxicity assay was performed with 4-hour coculture. However, NK-gIL2 exerted a much more potent cytotoxicity if the cultures were prolonged to 7 days. For example, residual leukemia was 58.4% ± 1.3% versus 97.3% ± 2.1% in control culture at a 0.5:1 E:T ratio. The superiority of NK-gIL2 was particularly evident in experiments where exogenous IL-2 was withdrawn for 72 hours before testing. In 4-hour cytotoxicity assays performed this way, cytotoxicity against K562 cells, as well as the T-ALL cell line MOLT4 and the Burkitt lymphoma cell line Daudi was significantly higher than that exerted by control NK cells (e.g., K562 killing: 82.9% ± 1.1% versus 30.9% ± 4.1% at a 1:1 ratio). NK-gIL2 showed significantly lower apoptotic rates than control NK cells after 72 hours of culture without exogenous IL-2 by Annexin V/7AAD staining. Survival after 7 days of culture in the absence of exogenous cytokines was also much higher in NK-gIL2 than NK cells transduced with GFP only. Cell death of NK cells may occur after killing of K562. We found that this was significantly lower in NK-gIL2 than in control NK cells, thus explaining the more potent anti-leukemic activity of NK-gIL2 in long-term culture. The addition of more than 100 U/ml IL-2 was needed to suppress cell death in control NK cells to a degree similar to that measured for NK-gIL2 cells in cultures without exogenous IL-2, suggesting that secretion of low amounts of IL-2 in an autocrine/paracrine fashion could sustain NK cell survival better than exogenous IL-2. We also found that the expression of the natural cytotoxicity receptor p30 and p44 was higher in NK-gIL2 cells than in control NK cells after 72-hour of IL-2 depletion, in line with their higher cytotoxic activities after IL-2 withdrawal. Our results indicate that endogenous IL-2 production can replace the requirement for exogenous IL-2 in NK cells and significantly improve their survival and cytotoxicity. An in vivo infusion of 1010 NK-gIL2 cells would result in the secretion of 1 × 104 U IL-2, an amount much lower than that administered in most immunotherapy protocols (1–2 × 106 U/sqm/day). These data suggest that infusion of NK-gIL2 could be more effective than that of unmodified NK cells while minimizing adverse effects of IL-2 administration.

Regulation of CD20 expression by radiation-induced changes in intracellular redox status

Increasing the levels of CD20 expression in cells that harbor low CD20 levels may enhance their responsiveness to CD20-specific antibody therapies. Here, we examined the regulation of CD20 expression after treatment with 0.5–2.0 Gy X-irradiation and hydrogen peroxide (H 2O 2), in the presence or absence of known antioxidants, in the Burkitt lymphoma cell lines Daudi and Raji. Irradiation of cells enhanced cell-surface CD20 expression; the kinetics and extent of this change were cell-type specific and time-dependent. The kinetics of reactive oxygen species generation and changes in mitochondrial membrane potential after irradiation were also correlated with changes in CD20 expression. Raji and Daudi cells treated with H 2O 2 showed a 2-to 2.5-fold increase in CD20 expression at 12 and 20 h, respectively. Buthionine sulfoximine, which depletes glutathione, also increased surface CD20, whereas antioxidants, such as PEG-catalase, PEG-SOD, vitamin C, and amifostine, decreased CD20 expression induced by radiation or H 2O 2. The antioxidant-mediated decrease in CD20 expression induced by radiation or H 2O 2 suggests a mechanism involving redox regulation. These results demonstrate the critical role of radiation-induced oxidative stress in CD20 expression and may have implications for defining and improving the efficacy of CD20-targeted antibody therapy and radioimmunotherapy.

Proteolytic Disassembly Is a Critical Determinant for Reovirus Oncolysis

See page 1406Mammalian ortheoreoviruses are currently being investigated as novel cancer therapeutics, but the cellular mechanisms that regulate susceptibility to reovirus oncolysis remain poorly understood. In this study, we present evidence that virion disassembly is a key determinant of reovirus oncolysis. To penetrate cell membranes and initiate infection, the outermost capsid proteins of reovirus must be proteolyzed to generate a disassembled particle called an infectious subviral particle (ISVP). In fibroblasts, this process is mediated by the endo/lysosomal proteases cathepsins B and L. We have analyzed the early events of infection in reovirus-susceptible and -resistant cells. We find that, in contrast to susceptible glioma cells and Ras-transformed NIH3T3 cells, reovirus-resistant cancer cells and untransformed NIH3T3 cells restrict virion uncoating and subsequent gene expression. Disassembly-restrictive cells support reovirus infection, as in vitro-generated ISVPs establish productive infection, and pretreatment with poly(I:C) does not prevent infection in cancer cells. We find that the level of active cathepsin B and L is increased in tumors and that disassembly-restrictive glioma cells support reovirus oncolysis when grown as a tumor in vivo. Together, these results provide a model in which proteolytic disassembly of reovirus is a critical determinant of susceptibility to reovirus oncolysis.

The Stability and Anti-apoptotic Function of A1 Are Controlled by Its C Terminus

Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. We found that the C terminus of the anti-apoptotic family member A1 did not function as a membrane anchor. Instead, this stretch of the protein rendered A1 highly unstable by mediating its polyubiquitination and rapid proteasomal degradation. Moreover, the domain did not only function independently of its position within the A1 protein but when transferred could even destabilize unrelated proteins like enhanced green fluorescent protein and caspase-3. A1 was, however, much more stable in the presence of the Bcl-2 homology-only protein BimEL, suggesting that direct interaction of A1 with pro-apoptotic members of the Bcl-2 family strongly reduces its rate of turnover. We further show that the C-terminal end of A1 also contributes to the anti-apoptotic capacity of the protein. In conclusion, our data demonstrate that the C terminus serves a dual function by controlling the stability of A1 and by amplifying the capacity of the protein to protect cells against apoptosis.

Survivin—An attractive target for RNAi in non-Hodgkin's lymphoma, Daudi cell line as a model

RNA interference (RNAi) has been widely used in tumor gene therapy, antivirus and gene drug selection. Survivin gene is highly expressed in non-Hodgkin's lymphoma (NHL) tissues and high malignancy Burkitt's lymphoma cell line-Daudi and it is regarded as a potential target of gene therapy for NHL. This study used a vector-based short hairpin RNA (shRNA) technique to explore the effect of RNAi-mediated survivin gene silencing on apoptosis and proliferation of Daudi cells. Recombinant plasmid survivin-shRNA was transfected into Daudi cells transiently and stably. The expression of survivin was detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. The apoptosis of Daudi cells after transfection were evaluated by flow cytometry. After transfection of survivin-shRNA, the levels of survivin mRNA were significantly reduced by 64.20% (transient transfection) and 62.32% (stable transfection), respectively; The levels of survivin protein were significantly reduced by 63.50% (transient transfection) and 61.88% (stable transfection); compared with control-shRNA and PBS treated groups. Apoptosis of Daudi cells were significantly higher in the transfection group than in the control group, respectively 21.30 ± 2.96% (transient transfection) and 19.10 ± 2.15% (stable transfection). In conclusion, it was suggested that survivin could be an attractive target for new anti-cancer intervention of NHL and vector-based survivin-shRNA could effectively reduce the expression of survivin and induce cell apoptosis and growth inhibition of NHL cells.

The Initial Enzyme for Glycosylphosphatidylinositol Biosynthesis Requires PIG-Y, a Seventh Component

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by an unusually complex GPI-N-acetylglucosaminyltransferase (GPI-GnT) consisting of at least six proteins. Here, we report that human GPI-GnT requires another component, termed PIG-Y, a 71 amino acid protein with two transmembrane domains. The Burkitt lymphoma cell line Daudi, severely defective in the surface expression of GPI-anchored proteins, was a null mutant of PIG-Y. A complex of six components was formed without PIG-Y. PIG-Y appeared to be directly associated with PIG-A, implying that PIG-Y is the key molecule that regulates GPI-GnT activity by binding directly to the catalytic subunit PIG-A. PIG-Y is probably homologous to yeast Eri1p, a component of GPI-GnT. We did not obtain evidence for a functional linkage between GPI-GnT and ras GTPases in mammalian cells as has been reported for yeast cells. A single transcript encoded PIG-Y and, to its 5' side, another protein PreY that has homologues in a wide range of organisms and is characterized by a conserved domain termed DUF343. These two proteins are translated from one mRNA by leaky scanning of the PreY initiation site.

Functional and phenotypic differences between CD4+ and CD4- T cell receptor-gamma delta clones from peripheral blood.

CD4+ TCR-gamma delta+ T cells comprise a very small subset of TCR-gamma delta+ T cells. CD4+ TCR gamma delta+ T cell clones were established to study the phenotypical and functional characteristics of these cells. Thirty-four CD4+ TCR-gamma delta+ T cell clones were established after sorting CD4+ T cells from a pre-expanded TCR-gamma delta+ T cell population. These clones as well as the CD4- TCR-gamma delta+ T cells from the same donor used V gamma 2 and V delta 2. In a second cloning experiment CD4+ TCR-gamma delta+ T cells were cloned directly from freshly isolated TCR-gamma delta+ T cells using a cloning device coupled to a FACS sorter. Forty-three clones were obtained, which all expressed CD4 and TCR-gamma delta. Eleven of these clones used V delta 1 and three of them coexpressed V gamma 2. The other CD4+ TCR-gamma delta+ T cell clones used both V delta 2 and V gamma 2. CD4+ TCR-gamma delta+ T cell clones expressed CD28 irrespective of the V gamma or V delta usage, and were CD11b negative. Three CD4-CD8+ TCR-gamma delta+ clones expressed CD8 alpha but not CD8 beta and were CD11b positive. CD28 expression among CD4-CD8+ and CD4-CD8- was variable but lower than on CD4+ T cell clones. CD4- TCR-gamma delta+ T cell clones using V gamma 2 and V delta 2 specifically lyse the Burkitt lymphoma cell line Daudi and secrete low levels of IFN-gamma and granulocyte-macrophage-CSF upon stimulation with Daudi. In contrast, most CD4+ T cell clones that use V gamma 2 and V delta 2 had a very low lytic activity against Daudi cells and secrete high levels of IFN-gamma and granulocyte-macrophage-CSF after stimulation with Daudi cells. The NK-sensitive cell line K562 was killed efficiently by the CD4- TCR-gamma delta+ T cell clones, but not by CD4+ TCR-gamma delta+ T cell clones, and could not induce cytokine secretion in CD4+ or CD4- T cell clones. CD4+ TCR-gamma delta+ T cell clones, but not the CD4- clones, could provide bystander cognate T cell help for production of IgG, IgM, and IgA in the presence of IL-2 and IgE in the presence of IL-4. Thus, CD4+ TCR-gamma delta+ T cells are similar to CD4+ TCR-alpha beta+ T cells in their abilities to secrete high levels of cytokines and to provide T cell help in antibody production.(ABSTRACT TRUNCATED AT 400 WORDS)

The t(8;14) chromosome translocation of the Burkitt lymphoma cell line Daudi occurred during immunoglobulin gene rearrangement and involved the heavy chain diversity region.

Recent molecular analyses of Burkitt lymphomas carrying the t(8;14) chromosome translocation have indicated that a dichotomy exists regarding the molecular mechanisms by which the translocations occur. Most sporadic Burkitt tumors carry translocations that apparently arise due to mistakes in the immunoglobulin isotype-switching process. In contrast, there is evidence that the translocations of most endemic Burkitt lymphomas occur as a consequence of aberrant V-D-J recombination of variable, diversity, and joining gene segments, catalyzed by the recombinase enzymes. This phenomenon was first noted in follicular lymphomas and chronic lymphocytic leukemias of the B-cell lineage and has been described in T-cell malignancies as well. In each of these cases, analysis of the nucleotide sequence at chromosome breakpoints demonstrated the involvement of immunoglobulin heavy chain JH or T-cell-receptor alpha-chain J alpha gene segments in the translocation. We now have cloned and sequenced both the 8q- and 14q+ translocation breakpoints deriving from the t(8;14) translocation of the endemic Burkitt lymphoma line Daudi. Our data show that the translocation resulted from a reciprocal exchange between the DH region on chromosome 14 and sequences far 5' of the MYC protooncogene on chromosome 8. Features of the nucleotide sequences surrounding the breakpoint further implicate the V-D-J joining machinery in the genesis of chromosome translocations in endemic Burkitt lymphomas and, more generally, in other lymphoid malignancies as well.

The synthesis and expression of HLA-A and -B antigens in Xenopus laevis oocytes

Purified mRNA's encoding the HLA-A and -B antigen heavy chains or β 2-microglobulin were prepared from human B lymphoid cells by positive hybridization selection procedures. The role of chain association in the biosynthesis and intracellular transport of HLA-A and -B antigents was investigated by injecting these mRNA species into Xenopus laevis oocytes and following the fates of the translated products by immunoprecipitation. When mRNA encoding β 2-microglobulin from the B lymphoblastoid cell line MST was coinjected with mRNA encoding the HLA-A and -B antigen heavy chains from the Burkitt lymphoma cell line Daudi, fully assembled class I antigens were detected using the monoclonal antibody W6/32. This result suggested that there may be no defect in the mRNA encoding Daudi HLA-A and -B antigen heavy chains. When the state of maturity of the N-linked glycan units on these class I antigen heavy chains was assessed, they were found to have undergone some processing. In contrast, when mRNA encoding immunoglobulin M (IgM) was injected into oocytes, the glycan units of the IgM heavy chains were found to be in the unprocessed (high mannose) form. This result shows that Xenopus oocytes can process some eukaryotic glycoproteins of exogenous origin.

Expression of Burkitt lymphoma-associated antigen (defined by the monoclonal antibody 38.13) on both normal and malignant germinal-centre B cells.

Monoclonal antibody 38.13 was raised against the Burkitt lymphoma cell line Daudi and has been previously shown to recognize a globotriaosylceramide (Pk) determinant expressed selectively on Burkitt and a few other B-cell lymphomas. We report here that 38.13 binds to a subset of normal B lymphocytes in germinal centres of secondary lymphoid follicles as well as to blood vessels. In malignant populations equivalent to various developmental compartments of the B-cell lineage, only those with a follicular phenotype express the Pk determinant at detectable levels. The expression of this tumour-associated antigen, in common with many others, is therefore regulated in concert with cell lineage and maturation status.

The beta2-microglobulin mRNA in human Daudi cells has a mutated initiation codon but is still inducible by interferon.

The human Burkitt lymphoma cell line Daudi does not synthesize beta2-microglobulin (beta2m) and lacks the cell surface histocompatibility antigens. The cells, however, contain RNA hybridizing to a cloned human beta2m cDNA probe. cDNA from this Daudi beta2m RNA, was cloned and sequenced. By comparison with cDNA prepared from Ramos cells, which synthesized microglobulin, we determined the sequence of the 20 amino acid long leader peptide of pre-beta2m and show that in Daudi cells the initiator ATG has been mutated to ATC. Although Daudi beta2m RNA cannot be translated, interferon induces the beta2m RNA in Daudi cells as well as in normal human cells.