Characterization of a candidate bcl-1 gene.

The t(11;14)(q13;q32) translocation has been associated with human B-lymphocytic malignancy. Several examples of this translocation have been cloned, documenting that this abnormality joins the immunoglobulin heavy-chain gene to the bcl-1 locus on chromosome 11. However, the identification of the bcl-1 gene, a putative dominant oncogene, has been elusive. In this work, we have isolated genomic clones covering 120 kb of the bcl-1 locus. Probes from the region of an HpaII-tiny-fragment island identified a candidate bcl-1 gene. cDNAs representing the bcl-1 mRNA were cloned from three cell lines, two with the translocation. The deduced amino acid sequence from these clones showed bcl-1 to be a member of the cyclin gene family. In addition, our analysis of expression of bcl-1 in an extensive panel of human cell lines showed it to be widely expressed except in lymphoid or myeloid lineages. This observation may provide a molecular basis for distinct modes of cell cycle control in different mammalian tissues. Activation of the bcl-1 gene may be oncogenic by directly altering progression through the cell cycle.     

The gene that encodes the human CD20 (B1) differentiation antigen is located on chromosome 11 near the t(11;14)(q13;q32) translocation site

The human CD20 gene (B1) encodes a B lymphocyte-specific, cell-surface molecule that is involved in B cell activation and differentiation. We report that the CD20 gene is located on human chromosome 11 at position q12-q13. The location of CD20 was determined by in situ hybridization and was further confirmed by Southern blot analysis of DNA from rodent/human hybrids that contained only portions of human chromosome 11. This localization places the CD20 gene near the site of the t(11;14)(q13;q32) translocation that is found in a subgroup of B cell-lineage malignancies. The site of this translocation has been previously identified by DNA cloning and termed bcl-1. The CD20 gene was found to lie on the centromeric side of bcl-1 on chromosome 11 and to be separated from bcl-1 by at least 50 kb of DNA. These results raise the possibility that alterations in the expression of the CD20 gene may result after the t(11;14) chromosomal alteration.     

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.     

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)     

Fc receptor gene translocation in a t(1;19) pre-B ALL cell line

We have recently mapped the human FCGR2 gene to chromosome 1 bands q23-q24. In situ hybridization of FCGR2 cDNA with a cell line containing a t(1;19)(g23;p13) derived from a patient with pre-B ALL has allowed a more accurate localization of this gene to chromosome 1 band q23. Furthermore, this study indicated a splitting of the FCGR2 gene or gene cluster by the t(1;19). However, Southern analysis showed no genetic rearrangement when compared with a karyotypically normal Epstein-Barr virus (EBV)-transformed cell line from the same patient. This suggests that the translocation breakpoint does not occur within the coding region of this gene.     

Definition of subchromosomal intervals around the myotonic dystrophy gene region at 19q

The localization to 19q of the gene causing myotonic dystrophy (DM) has been defined more precisely by refinement of the physical location of several linked markers. A somatic cell hybrid mapping panel from cells with t(1;19), t(12;19), and t(X;19) translocation products was constructed to define five different intervals across 19q. In addition, we have derived a series of cell hybrids by irradiation of a der(19)-only hybrid to further subdivide the cen-q13.1 region. Using an array of 36 cloned genes, anonymous DNAs, and enzyme markers, we have tested the location of the panel breakpoints and refined the regional assignment of several of these markers. All markers tightly linked to DM are localized mainly within 19q13.2, thus suggesting that the DM gene is also close to this region.     

Site-specific recombination of the tal-1 gene is a common occurrence in human T cell leukemia.

The tal-1 gene is altered as a consequence of the t(1;14) (p32;q11) chromosome translocation observed in 3% of patients with T cell acute lymphoblastic leukemia (T-ALL). tal-1 encodes a helix-loop-helix (HLH) domain, a DNA binding and dimerization motif found in a number of proteins involved in cell growth and differentiation. We now report that an additional 25% of T-ALL patients bear tal-1 gene rearrangements that are not detected by karyotype analysis. These rearrangements result from a precise 90 kb deletion (designated tald) that arises independently in different patients by site-specific DNA recombination. Since the deletion junctions resemble the coding joints of assembled immunoglobulin genes, tald rearrangements are likely to be mediated by aberrant activity of the immunoglobulin recombinase. Moreover, t(1;14)(p32;q11) translocations and tald rearrangements disrupt the coding potential of tal-1 in an equivalent manner, and thereby generate a common genetic lesion shared by a significant proportion of T-ALL patients.     

Gene mapping by enzymatic amplification from flow-sorted chromosomes

A new approach to gene mapping which combines enzymatic amplification with high-resolution flow sorting of human chromosomes has been devised. Reliable amplification from as few as 200 chromosomes has been demonstrated. This method, with particular application to mapping the position of chromosomal translocations, has been used to show that the breakpoint for the constitutional translocation t(11;22)(q23;q11) lies proximal to the genes c-ets-1, Thy-1, and T3 delta and distal to the int-2 gene. The mapping was confirmed by Southern analysis to much larger numbers of chromosomes sorted from the same cell line. Control reactions for the bcl-2 gene on chromosome 18 and the C alpha gene of the IGH locus on chromosome 14 demonstrated the discrimination which can be achieved.     

NOTCH2 Is Neither Rearranged nor Mutated in t(1;19) Positive Oligodendrogliomas

The combined deletion of 1p and 19q chromosomal arms is frequent in oligodendrogliomas (OD) and has recently been shown to be mediated by an unbalanced t(1;19) translocation. Recent studies of 1p/19q co-deleted OD suggest that the NOTCH2 gene is implicated in oligodendrocyte differentiation and may be involved in this rearrangement. The objective of the present study was to analyze the NOTCH2 locus either as a chromosomal translocation locus that may be altered by the 1p/19q recurrent rearrangement or as a gene that may be inactivated by a two hit process. We performed an array-CGH analysis of 15 ODs presenting 1p/19q co-deletion using a high-density oligonucleotide microarray spanning 1p and 19q pericentromeric regions with 377 bp average probe spacing. We showed that the 1p deletion extends to the centromere of chromosome 1 and includes the entire NOTCH2 gene. No internal rearrangement of this gene was observed. This strongly suggests that the t(1;19) translocation does not lead to an abnormal NOTCH2 structure. The analysis of the entire NOTCH2 coding sequence was performed in four cases and did not reveal any mutation therefore indicating that NOTCH2 does not harbor genetic characteristics of a tumor suppressor gene. Finally, the detailed analysis of chromosome 19 pericentromeric region led to the identification of two breakpoint clusters at 19p12 and 19q11–12. Interestingly, these two regions share a large stretch of homology. Together with previous observations of similarities between chromosome 1 and 19 alphoid sequences, this suggests that the t(1;19) translocation arises from complex intra and interchromosomal rearrangements.This is the first comprehensive deletion mapping by high density oligo-array of the 1p/19q co-deletion in oligodendroglioma tumors using a methodological approach superior to others previously applied. As such this paper provides clear evidence that the NOTCH2 gene is not physically rearranged by t(1;19) translocation of oligodendroglioma tumors.     

Paxillin's LD4 motif interacts with bcl-2

Bcl-2 is the founding member of a family of proteins which influences cell survival in response to a variety of stimuli including those from growth factor receptors and integrins. However, how these activities are coordinated through bcl-2 requires further investigation. bcl-2 interacts with paxillin, potentially linking cell survival and cell adhesive pathways. Paxillin is an adapter protein implicated in growth factor and integrin-mediated signal transduction pathways. Previous work in this laboratory demonstrated that loss of bcl-2 affects cell adhesion and migration characteristics of renal epithelial cells, perhaps through disruption of its interaction with paxillin. Here studies were performed to determine the bcl-2 binding motif in paxillin. The amino-terminal portion of paxillin, specifically its LD4 motif, was found to associate with bcl-2. However, the amino-terminal portion of paxillin with the LD4 domain deleted did not associate with bcl-2. The corresponding LD motif in other paxillin family members, Hic-5 and leupaxin, did not associate with bcl-2. Mutations in paxillin's LD4 motif made to mimic Hic-5 and leupaxin LD4-like motifs (E(268) --> R or S(272) --> H) abolished its association with bcl-2. Incubation of embryonic kidneys with paxillin's LD4 motif disrupted ureteric bud branching and morphogenesis, while incubation with the comparable Hic-5 LD motif did not significantly affect morphogenesis. These data suggest that paxillin's association with bcl-2 plays a unique role during kidney development that other paxillin family members may not be able to fulfill.