A-Myb up-regulates Bcl-2 through a Cdx binding site in t(14;18) lymphoma cells

In follicular lymphoma, bcl-2 is translocated to the immunoglobulin heavy chain locus leading to deregulation of bcl-2 expression. We examined the role of Myb proteins in the regulation of bcl-2 expression in lymphoma cells. We showed that A-Myb up-regulates bcl-2 promoter activity. Northern and Western analyses demonstrated that A-Myb was expressed in the DHL-4 t(14;18) cell line. In t(14;18) cells and mature B cells, A-Myb up-regulated bcl-2 expression, whereas B- and c-Myb had little effect on bcl-2 gene expression. Deletion analysis of the bcl-2 5'-region identified a region responsive to A-Myb in t(14;18) cells. A potential binding site for the Cdx homeodomain proteins was located in this sequence. Analysis of the A-Myb-responsive region by UV cross-linking experiments revealed that a 32-kDa protein formed a complex with this region, but direct binding by Myb proteins could not be demonstrated. A-Myb could be recovered along with Cdx2 when nuclear extracts were passed over the Cdx site. Mutagenesis of the Cdx binding site abolished binding by the 32-kDa protein and significantly reduced the ability of A-Myb to induce bcl-2 expression. A strong induction of bcl-2 P2 promoter activity was observed in cotransfection studies of DHL-4 cells with the A-Myb and Cdx2 expression vectors, and increased endogenous Bcl-2 protein expression was observed in B cells transfected with A-Myb and/or Cdx2 expression constructs.     

bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation

Human follicular B cell lymphomas possess a t(14;18) interchromosomal translocation that juxtaposes the putative proto-oncogene bcl-2 with the immunoglobulin (Ig) heavy chain locus. We generated minigene constructs representing the bcl-2-Ig fusion gene found at this chromosomal breakpoint. These constructs were placed into the germ line of mice to assess the effects of the t(14;18) during development. The transgene demonstrates a lymphoid pattern of expression and uniformly results in an expanded follicular center cell population. Hyperplastic splenic follicles coalesce to form massive regions of splenic white pulp. Mice over 15 weeks of age demonstrate regional lymphadenopathy with abnormal cellular infiltrates. The expanded lymphoid compartment is composed predominantly of polyclonal B220-positive, IgM/IgD-positive B cells. Provocatively, the bcl-2-Ig transgene confers a survival advantage to a population of mature B cells assessed in vitro. bcl-2-Ig transgenic mice document a prospective role for the t(14;18) in B cell growth and the pathogenesis of follicular lymphoma.     

Amplifiedbcl-2/JH rearrangements in reactive lymphadenopathy

Using the polymerase chain reaction (PCR) to examine the occurrence ofbcl-2/JH joining produced by t(14;18) chromosomal translocation, amplified DNA was detected in 2 of 18 lymph nodes showing reactive lymphadenopathy. The PCR was repeated in these two lymphs nodes using the same DNA samples, but no amplification was detected at the second attempt. Thus the amplified DNA was considered to be derived from one copy of joinedbcl-2/JH in one cell, or from a few copies in a few clonal cells with the same joinedbcl-2/JH. These results suggest that false joining ofbcl-2/JH at the t(14;l8) junction may occur in reactive lymph nodes.     

CA repeats in the 3'-untranslated region of bcl-2 mRNA mediate constitutive decay of bcl-2 mRNA

An AU-rich element (ARE) in the 3'-untranslated region (UTR) of bcl-2 mRNA has previously been shown to be responsible for destabilizing bcl-2 mRNA during apoptosis through increasing AUF1 binding. In the present study, we investigated the effect of the region upstream of the ARE on bcl-2 mRNA stability using serial deletion constructs of the 3'-UTR of bcl-2. Deletion of 30 nucleotides mostly consisting of the CA repeats, located upstream of the ARE, resulted in the stabilization of bcl-2 mRNA abundance, in the absence or presence of the ARE. The specificity of the CA repeats in terms of destabilizing bcl-2 mRNA was proven by the substituting the CA repeats with other alternative repeats of purine/pyrimidine, but this had no effect on the stability of bcl-2 mRNA. CA repeats alone, however, failed to confer instability to bcl-2 or gfp reporter mRNAs, indicating a requirement for additional sequences in the upstream region of the 3'-UTR. Serial deletion and replacement of a part of the region upstream of the CA repeats revealed that the entire 131-nucleotide upstream region is an essential prerequisite for the CA repeat-dependent destabilization of bcl-2 mRNA. Unlike the ARE, CA repeat-mediated degradation of bcl-2 mRNA was not accelerated upon apoptotic stimulus. Moreover, the upstream sequences and CA repeats are conserved among mammals. Collectively, CA repeats contribute to the constitutive decay of bcl-2 mRNA in the steady states, thereby maintaining appropriate bcl-2 levels in mammalian cells.     

Both V(D)J coding ends but neither signal end can recombine at the bcl-2 major breakpoint region, and the rejoining is ligase IV dependent

The t(14;18) chromosomal translocation is the most common translocation in human cancer, and it occurs in all follicular lymphomas. The 150-bp bcl-2 major breakpoint region (Mbr) on chromosome 18 is a fragile site, because it adopts a non-B DNA conformation that can be cleaved by the RAG complex. The non-B DNA structure and the chromosomal translocation can be recapitulated on intracellular human minichromosomes where immunoglobulin 12- and 23-signals are positioned downstream of the bcl-2 Mbr. Here we show that either of the two coding ends in these V(D)J recombination reactions can recombine with either of the two broken ends of the bcl-2 Mbr but that neither signal end can recombine with the Mbr. Moreover, we show that the rejoining is fully dependent on DNA ligase IV, indicating that the rejoining phase relies on the nonhomologous DNA end-joining pathway. These results permit us to formulate a complete model for the order and types of cleavage and rejoining events in the t(14;18) translocation.     

Bcl-2 antisense oligodeoxynucleotide increases the sensitivity of leukemic cells to arsenic trioxide

Cell culture, tissue chemistry and flow cytometry were used to determine whether antisense bcl-2 oligodeoxynucleotides enhanced the sensitivity of leukemia cells to arsenic trioxide. A combination of arsenic trioxide with antisense bcl-2 oligodeoxynucleotides inhibited cell growth, induced apoptosis and induced bcl-2 protein expression in K562 and NB4 leukemic cells more significantly than either arsenic trioxide or the oligodeoxynucleotides on their own (P<0.01). Thus, bcl-2 antisense oligodeoxynucleotides increase the sensitivity of leukemic cells to arsenic trioxide. Combined use of the two agents could be a novel and attractive strategy in leukemia treatment.     

Analysis of ribosyl-modified,mixed backbone analogs of a bcl-2/bcl-xL antisense oligonucleotide

Progress in oligonucleotide chemistry has provided second-generation antisense oligonucleotides with increased efficacy and reduced non-antisense-related toxicity. The ability of the 2'-O-(2-methoxyethylribose) (2'-MOE)-modified phosphorothioate gapmer oligonucleotide 4625, which matches the bcl-2 mRNA and has three base-mismatches to bcl-xL, to inhibit bcl-2 and bcl-xL expression and induce tumor cell apoptosis has been described. Here we investigated the consequences of adding of 2'-MOE or 2'-Me modifications to ribonucleotides at either the two ends of the sequence, or the center region together with different combinations of phosphodiester/phosphorothioate backbones on the activity of oligonucleotide 4625. The ability of the various 4625 analogs, including the parental first-generation oligonucleotide 3005, to inhibit bcl-2 and bcl-xL expression, and diminish cell growth or induce tumor cell death was assessed in SW2 lung cancer cells using real-time PCR, Western blotting and cell viability assays. Only oligonucleotide 4625 exhibited a potent bispecific antisense activity against bcl-2 and bcl-xL, which effectively reduced tumor cell viability. The other antisense oligonucleotides were either uniquely active against bcl-2 or completely inactive. Our data suggest that the 2'-MOE modification in combination with the phophorothioate gapmer chemistry is the optimal format of the 4625 sequence in terms of antisense activity and biological efficacy.     

Flow cytometric detection of the mitochondrial BCL-2 protein in normal and neoplastic human lymphoid cells.

The bcl-2 proto-oncogene, rearranged and deregulated in B-cell lymphomas bearing the t(14;18) translocation, encodes an inner mitochondrial membrane protein that blocks apoptotic cell death. We have developed a sensitive immunofluorescence assay for the single- and multicolor flow cytometric analysis of bcl-2 protein in relation to other markers and cell cycle, based on a fixation-permeation step of cells with paraformaldehyde and Triton X100 and the use of a bcl-2 specific monoclonal antibody (MoAb). As an application of this method, we have examined the expression of bcl-2 in normal and neoplastic lymphoid cells. We have found that greater than 80% of normal T-and B-cells are bcl-2 positive; following in vitro mitogen activation, the bcl-2 reactivity decreased slightly in the former but markedly in latter cells. In both cases the bcl-2 expression was not restricted to a specific phase of the cell cycle, as evidenced by two-color analysis. On lymphoblastoid cell lines, the bcl-2 staining intensity was variable and not necessarily correlated to molecular rearrangements of the bcl-2 gene. Among fresh B-cell non-Hodgkin's lymphomas (B-NHL), most sporadic Burkitt's cases were bcl-2 negative. Of four centroblastic-centrocytic cases with rearrangements of the bcl-2 gene, only two presented elevated amounts of bcl-2 protein, indicating that the levels of bcl-2 are not diagnostic of the translocation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cellular localization of the bcl-2 protein and response to glucocorticoid stress

We performed immunoelectronmicroscopy, immunofluorescence and subcellular fractionation studies of insect cells (Spodopetra frugiperda or SF9) infected with recombinant baculovirus containing bcl-2 cDNA to determine the cellular localization of the bcl-2 product. Similar studies were also undertaken in pre-B cells carrying a bcl-2 gene activated by t(14;18) chromosomal translocation. By immunogold electron microscopy, bcl-2 was localized at several intracellular sites including the nuclear membrane, endoplasmic reticulum, mitochondria and plasma membrane. Immunofluorescence studies revealed the presence of the bcl-2 product throughout the cytoplasm, whereas biochemical fractionation studies indicated a similar pattern to that observed on electron microscopy. Our investigation clearly indicates that the bcl-2 product is expressed at several intracellular sites. Studies were also undertaken to determine any changes in the subcellular distribution of bcl-2 protein following glucocorticoid exposure of immature B lymphocytes. Although no major changes in the distribution of bcl-2 protein were observed, more aggregated patches of gold labelled bcl-2 particles were found under glucocorticoid stress. Aggregation of bcl-2 molecules might represent dimerization necessary to prevent apoptosis.     

Identification of nucleolin as an AU-rich element binding protein involved in bcl-2 mRNA stabilization

bcl-2 mRNA contains an AU-rich element (ARE) that functions in regulating bcl-2 stability. Our earlier studies indicated that taxol- or okadaic acid-induced bcl-2 mRNA destabilization in HL-60 cells is associated with decreased binding of trans-acting factors to the ARE. To identify factors that play a role in the regulation of bcl-2 mRNA stability, bcl-2 ARE-binding proteins were purified from HL-60 cells. Three polypeptides of 100, 70, and 32 kDa were isolated from a bcl-2 ARE affinity matrix. Matrix-assisted laser desorption ionization mass spectroscopy analysis identified these proteins as full-length nucleolin and proteolytic fragments of nucleolin. RNA gel shifts assays indicated that recombinant nucleolin (residues 284-707) binds specifically to bcl-2 ARE RNA. In addition, recombinant nucleolin decreases the rate of decay of mRNA in HL-60 cell extracts in an ARE-dependent manner. Taxol or okadaic acid treatment of HL-60 cells results in proteolysis of nucleolin in a similar time frame as drug-induced bcl-2 mRNA down-regulation. These findings suggest that nucleolin functions as a bcl-2-stabilizing factor and that taxol and okadaic acid treatment induces apoptosis in HL-60 cells through a process that involves down-regulation of nucleolin and destabilization of bcl-2 mRNA.