c-myc and immunoglobulin kappa light chain constant genes are on the 8q+ chromosome of three Burkitt lymphoma lines with t(2;8) translocations.

We have determined the localization of c-myc and the immunoglobulin kappa light chain genes on the 8q+/2p- chromosomes of the three Burkitt lymphoma lines BL21, LY66 and LY91 with t(2;8) translocation by in situ hybridization. BL21 is characterized by a complex translocation in which a piece of chromosome 9 appears to be located between the fragments of chromosome 8 and 2 on the 8q+ chromosome. Our data indicate that in all three cell lines the c-myc gene is located on the 8q+ chromosome proximal to the breakpoint in band 8q24. In all cell lines examined the cluster of kappa variable genes has remained on the 2p- chromosome. In LY91 cells the major part of the joining region remained on 2p-, while the joining region has moved to 8q+ in the cell lines BL21 and LY66. In all three cell lines the constant kappa light chain gene was found on the 8q+ chromosome. The fact that an essentially identical pattern was found in the cell line BL21, with the complex translocation, suggests that the insertion of the piece of chromosome 9 into the 8q+ chromosome might be a secondary event. Our present data fit into the concept that in all Burkitt lymphoma lines investigated so far, including cases with t(8;14) and the variant translocations t(2;8) and t(8;22), the c-myc gene becomes situated at the 5' side of an immunoglobulin constant gene. This may have implications for the generation of somatic mutations in the coding and non-coding part of the c-myc gene.     

N segment insertion and region-directed somatic hypermutation in a kappa gene of a t(2;8) chromosomal translocation.

A detailed molecular analysis of both reciprocal recombination products of the variant t(2;8) chromosomal translocation of the Burkitt lymphoma derived cell line JI and their germline counterparts was carried out. The breakpoint on chromosome 8 is localized 28 kb to the 3' side of the c-myc protooncogene, the breakpoint on chromosome 2 was found to be within an aberrantly rearranged VK gene (abbreviations ref. 1). Novel features of the immunoglobulin moiety involved in this process include insertion of extra nucleotides in the V-J junction which have the characteristics of a N segment as it has been found up to now only in heavy chain and T cell receptor genes; the occurrence of somatic mutations in 8q+ and not in 2p-. These data allow a reconstruction of the course of events in the cell line JI; remarkable sequence regularities at the chromosomal breakpoints consisting of symmetrically placed dinucleotides and elements related to the hepta- and nonanucleotide recombinase recognition sequences are discussed in the context of the translocation mechanism.     

Three breakpoints of variant t(2;8) translocations in Burkitt''s lymphoma cells fall within a region 140 kilobases distal from c-myc.

The variant translocations t(2;8) in Burkitt's lymphoma cells join band q24 of chromosome 8, distal from c-myc, to the Igkappa locus, with considerable variation in the location of the breakpoints on chromosome 8. We report the cloning and molecular characterization of a chromosome 8 region, distal from the c-myc locus, which encompasses the breakpoints of the Burkitt's lymphoma cell lines BL64, BL21, and LY91 within 11 kilobase pairs, termed provisionally bvr-1 (Burkitt's variants' rearranging region 1). Using probes from the c-myc, the bvr-1, and the human pvt-1 loci obtained by chromosome walking coupled with pulsed-field gel electrophoresis, we have constructed a physical map of the region 3' of c-myc. We map bvr-1 and pvt-1 about 140 and 260 kilobase pairs, respectively, distal from c-myc.     

Involvement of immunoglobulin heavy- and light-chain (kappa) gene clusters in a human B-cell translocation, t(2;14)

The breakpoints of a translocation, t(2;14)(p11;q32), detected in an Epstein-Barr virus-transformed lymphoid B-cell line were mapped by Southern analysis, field-inversion gel electrophoresis, and in situ hybridisation. The translocation involved the immunoglobulin light-chain (kappa) locus on chromosome 2 and the heavy-chain locus on chromosome 14. The breakpoint on chromosome 2 was between VK and CK, most likely within JK. The chromosome 14 break was located within the VH cluster, no more than 220 kb 5' of the productively rearranged JH locus. The translocation probably resulted from an aberrant rearrangement of the kappa light-chain genes.     

Burkitt lymphoma cell line carrying a variant translocation creates new DNA at the breakpoint and violates the hierarchy of immunoglobulin gene rearrangement.

The Burkitt lymphoma cell line KK124, which contains a reciprocal t(8;22) translocation, was shown to have rearranged in a region 3' to the c-myc proto-oncogene on chromosome 8 and 5' to the lambda constant region on chromosome 22. The breakpoint was cloned and sequenced, revealing that c-myc and a portion of its 3' region abutted a complete lambda variable gene that had undergone V-J recombination. Since this cell line expresses kappa light chain, this lambda rearrangement violates the previously proposed hierarchy of immunoglobulin gene rearrangement. A novel duplication of normal chromosome 8 sequences was also found at the breakpoint. The first exon of c-myc and its flanking sequence from the translocated allele was sequenced and compared with a normal counterpart. Extensive mutation was found within the first exon in contrast to its 3' and 5' flanking regions. S1 nuclease analysis revealed that it was the translocated c-myc being expressed and that there was a promoter shift from P2 to P1. The detailed structural analysis of this cell line provides clues concerning mechanisms of chromosomal translocation and c-myc deregulation in Burkitt lymphomas.     

Breakage on chromosome 2 brings the Ck gene to a region 3' of c-myc in a Burkitt's lymphoma line carrying a (2;8) translocation

We have shown using in situ hybridization that the constant region of the kappa light chain immunoglobulin gene (Ck) is translocated from chromosome 2 to chromosome 8 in Burkitt's lymphoma cells with a (2;8) translocation. The Ck gene then ends up adjacent to and on the 3' side of c-myc. The breakpoint probably falls between the gene for the variable region of the kappa light chain (Vk) and the Ck gene.     

Clustered somatic mutations in and around first exon of non-rearranged c-myc in Burkitt lymphoma with t(8;22) translocation.

We have examined the restriction map of the c-myc gene in 15 BL cell lines carrying the variant t(8;22) translocation in which c-myc is known to remain on chromosome 8. Using 3 restriction enzymes cutting outside the c-myc domain (EcoRI, BamHI, HindIII), we found no evidence for a c-myc/Ig lambda rearrangement in 14 BL cell lines. In the last one, BL 37, the 3' flanking region was rearranged corresponding to the already identified breakpoint located 400 pb downstream from the c-myc gene (9). Using 4 restriction enzymes cutting inside the c-myc gene (PvuII, PstI, SacI, HincII) we looked for discrete abnormalities within the gene limits, and we found in 9 BL cell lines several abolished and created sites, compatible with multiple independent somatic mutations. They are significantly clustered in the 5' non coding region, with a striking prevalence at the end of exon 1. The role of mutations in the non-coding first exon region for the deregulation of c-myc expression is discussed.     

The t(8;14) breakpoint of the EW 36 undifferentiated lymphoma cell line lies 5' of MYC in a region prone to involvement in endemic Burkitt's lymphomas

One of the best analyzed tumor-specific cytogenetic abnormalities is the t(8;14) chromosomal translocation observed in cases of Burkitt's and undifferentiated lymphomas (ULs), and acute lymphoblastic leukemias (ALLs). Here we analyze the cloned (8;14) chromosomal breakpoint of the UL cell line EW 36. We show that the region of chromosome 8 involved in the translocation is situated near a site previously demonstrated to harbor a cluster of endemic Burkitt's lymphoma breakpoints, approximately 50 kb 5' of MYC. In those cases, we demonstrated that malfunction of the V-D-J recombinase generated the translocations. However, in this case the isotype switch mechanism of translocation is implicated: at the breakpoint, S mu/S gamma and C gamma sequences are found on chromosome 14. Thus, the features of the EW 36 t(8;14) breakpoint are consonant with our model for B-cell lymphomagenesis which relates the precursor cell that gives rise to malignancy, the mechanism of translocation, and the phenotype of the tumor.     

Chromosome 8 breakpoint far 3'' of the c-myc oncogene in a Burkitt''s lymphoma 2;8 variant translocation is equivalent to the murine pvt-1 locus.

The 2;8 variant translocation of human Burkitt's lymphomas is closely related cytogenetically to the t(6;15) of murine plasmacytomas; both involve a reciprocal exchange between the Ig kappa locus and a band region indistinguishable from that bearing the c-myc oncogene. To define their molecular relationship, we have compared cloned chromosome 8 DNA from the t(2;8) breakpoint in the human Burkitt's lymphoma JBL2 with cloned DNA from the murine pvt-1 locus, the major chromosome 15 breakpoint region in murine t(6;15). DNA sequencing and Southern blot analysis shows that these two regions are homologous. Thus the t(2;8) in JBL2 is the molecular equivalent of many murine t(6;15). The murine pvt-1 locus lies an unknown distance 3' of c-myc; analysis of DNA from several tumours with c-myc amplification reveals that pvt-1 is co-amplified in at least one case, placing pvt-1 approximately 100-500 kb 3' of c-myc. The significance of these results with respect to the role of pvt-1 in tumorigenesis is discussed.     

Molecular involvement of the pvt-1 locus in a gamma/delta T-cell leukemia bearing a variant t(8;14)(q24;q11) translocation.

A highly malignant human T-cell receptor (TCR) gamma/delta+ T-cell leukemia was shown to have a productive rearrangement of the TCR delta locus on one chromosome 14 and a novel t(8;14)(q24;q11) rearrangement involving the J delta 1 gene segment on the other chromosome 14. Chromosome walking coupled with pulsed-field gel electrophoretic (PFGE) analysis determined that the TCR J delta 1 gene fragment of the involved chromosome was relocated approximately 280 kb downstream of the c-myc proto-oncogene locus found on chromosome band 8q24. This rearrangement was reminiscent of the Burkitt's lymphoma variants that translocate to a region identified as the pvt-1 locus. Sequence comparison of the breakpoint junctions of interchromosomal rearrangements in T-cell leukemias involving the TCR delta-chain locus revealed novel signal-like sequence motifs, GCAGA(A/T)C and CCCA(C/G)GAC. These sequences were found on chromosome 8 at the 5' flanking site of the breakpoint junction of chromosome 8 in the TCR gamma/delta leukemic cells reported here and also on chromosome 1 in T-cell acute lymphocytic leukemia patients carrying the t(1;14)(p32;q11) rearrangement. These results suggest that (i) during early stages of gamma delta T-cell ontogeny, the region 280 kb 3' of the c-myc proto-oncogene on chromosome 8 is fragile and accessible to the lymphoid recombination machinery and (ii) rearrangements to both 8q24 and 1p32 may be governed by novel sequence motifs and be subject to common enzymatic mechanisms.     

Variant (6;15) translocations in murine plasmacytomas involve a chromosome 15 locus at least 72 kb from the c-myc oncogene.

The variant (6;15) translocations in murine plasmacytomas join the myc oncogene-bearing band of chromosome 15 and the immunoglobulin kappa band of chromosome 6. We recently cloned a region from chromosome 15 linked to C kappa and have now used probes from that region to define the major locus of plasmacytoma variant translocations, which we denote pvt-1. In five of nine plasmacytomas we analysed, the 6;15 translocation resulted from reciprocal recombination between the C kappa locus and a 4.5-kb region of pvt-1. Moreover, nearby we located the region shown by others to have undergone a complex (15;12;6) translocation in plasmacytoma PC7183. All the chromosome 6 breakpoints fell between 1 and 3 kb 5' to C kappa but only two were near J kappa genes. Thus the J kappa -C kappa region appears to be a recombination 'hot spot' in lymphocytes, but the breaks are unlikely to be mediated via V/J recombination enzymes. Comparison of a cloned 108-kb region across pvt-1 and another of 52 kb across c-myc established that the pvt-1 breakpoints lie at least 72 kb from the c-myc promoters. Since c-myc is expressed at a substantial level, the 6;15 translocation apparently activates c-myc. Activation may occur directly, at a remarkable distance along the chromosome, or indirectly, via a putative pvt-1 gene product.     

In situ hybridization and translocation breakpoint mapping. II. Two unusual t(21;22) translocations

Using a combination of banding techniques, we examined two atypical 21;22 translocations, 46,XX or XY,t(21;22)(p11;q11). In situ chromosomal hybridization of a probe for the constant region of the lambda light chain locus demonstrated that the 22q11 breakpoints of both rearrangements were proximal to the C lambda gene cluster. These studies permitted us to distinguish the 22q11 breakpoints of these translocations from the breakpoint of the 22q--chromosome of chronic myelogenous leukemia.     

Localization of the rhabdomyosarcoma t(2;13) breakpoint on a physical map of chromosome 13.

Previous investigations of the pediatric soft tissue tumor alveolar rhabdomyosarcoma have identified a characteristic translocation t(2;13)(q35;q14). We have employed a physical mapping strategy to localize the site of this translocation breakpoint on chromosome 13. Using a panel of somatic cell hybrid and lymphoblast cell lines with deletions and unbalanced translocations involving chromosome 13, we have mapped numerous probes from the 13q12-q14 region and demonstrate that this region is divisible into five physical intervals. These probes were then mapped with respect to the t(2;13) rhabdomyosarcoma breakpoint by quantitative Southern blot analysis of an alveolar rhabdomyosarcoma cell line with two copies of the derivative chromosome 13 and one copy of the derivative chromosome 2. Our findings demonstrate that the t(2;13) breakpoint is localized within a map interval delimited by the proximal deletion breakpoints in lymphoblast lines GM01484 and GM07312. Furthermore, the breakpoint is most closely flanked by loci D13S29 and TUBBP2 within this map interval. These findings will facilitate chromosomal walking strategies for cloning the regions disrupted by the alveolar rhabdomyosarcoma translocation. In addition, this physical map will permit rapid determination of the proximity of new cloned sequences to the translocation breakpoint.     

Chromosome translocations in breast cancer with breakpoints at 8p12

Unbalanced chromosome translocations with breakpoints around 8p12, resulting in loss of distal 8p, are common in carcinomas. We have mapped the 8p12 breakpoints in three breast cancer cell lines, T-47D, MDA-MB-361, and ZR-75-1, using YACs and PACs between D8S540 and D8S255 by fluorescence in situ hybridization. All three lines had a breakpoint close to D8S505, proximal to HGL. Each breakpoint was distinct, but all were within 0.5 to 1.5 Mb of each other. The T-47D cell line had a straightforward translocation, but in MDA-MB-361 and ZR-75-1 the translocations were accompanied by local rearrangements of surprising complexity. Small regions of 8p from close to the breakpoint were duplicated or amplified as inserts in the attached chromosome fragment. ZR-75-1 also had retained a separate fragment of about 1 Mb, from the region 1 to 3 Mb telomeric to the common breakpoint, that included HGL. This line also had an interstitial deletion several megabases more centromeric. The data suggest that breakpoints on 8p12 are clustered in a small region and show that translocations breaking there may be accompanied by additional rearrangements.