Myc oncogene activation in B and T lymphoid tumours

The chromosome translocations characteristic of certain B lymphoid tumours associate the myc oncogene and immunoglobulin loci. The typical t(12;15) in murine plasmacytomas and analogous t(14;8) in Burkitt lymphomas couple the myc coding region to one of the switch recombination regions within the immunoglobulin heavy (H) chain locus; hence the switch machinery may promote some translocations. Significantly, translocation induces constitutive myc expression, the untranslocated myc allele remaining silent. The predilection for breakpoints near the 5' end of the c-myc gene may reflect selection for altered myc regulation. In most tumours, the stimulatory effect of the H locus context is not understood, but an H locus enhancer participates in some tumours, including one displaying a novel transposition. The variant (6;15) translocations found in about 15% of plasmacytomas involve the myc band and the region of chromosome 6 where the kappa locus lies. The t(6;15) is shown here to represent an exchange between C kappa and a chromosome 15 locus (designated pvt-1) which lies unexpectedly far from c-myc. The association of myc expression with pvt-1 alterations suggest that myc can be activated at a distance. Myc has also been implicated in some T lymphomas by detection of proviral inserts near myc and also, surprisingly, within the pvt-1 locus. Inserts near myc appear to activate its expression via the retroviral enhancer.     

Proviral integration site Mis-1 in rat thymomas corresponds to the pvt-1 translocation breakpoint in murine plasmacytomas.

Two loci independently implicated in T-and B-lymphocyte neoplasia are shown to be equivalent. The Mis-1 locus is a common proviral integration site in retrovirally induced rat T lymphomas, while the pvt-1 locus on murine chromosome 15 frequently translocates to the kappa locus in plasmacytomas bearing 6;15 translocations. By comparing cloned sequences, we show that pvt-1 is the murine homolog of Mis-1.     

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.     

c-myc Gene rearrangements involving gamma immunoglobulin heavy chain gene switch regions in murine plasmacytomas

In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.     

The chromosomal location of T-cell receptor genes and a T cell rearranging gene: possible correlation with specific translocations in human T cell leukaemia.

We have examined the chromosomal location of human T cell-specific genes which are involved in antigen recognition and of a gene which specifically rearranges in T cells. The genes encoding both the variable and constant region segments of the T cell receptor alpha chain are found on chromosome 14 while the delta chain gene of the T cell receptor-associated T3 complex is localised to chromosome 11. Further, the two tandemly arranged T cell-specific rearranging genes, designated gamma, were mapped to chromosome 7, but apparently not closely linked to the previously mapped T cell receptor beta-chain gene. The locations of the three different genes, which undergo rearrangement in T cells, may correlate with the chromosomal breakpoints known to be involved in translocations within abnormal human T cells.     

AID is required for c-myc/IgH chromosome translocations in vivo

Chromosome translocations between c-myc and immunoglobulin (Ig) are associated with Burkitt's lymphoma in humans and with pristane- and IL6-induced plasmacytomas in mice. These translocations frequently involve Ig switch regions, suggesting that they might be the result of aberrant Ig class switch recombination (CSR). However, a direct link between CSR and chromosome translocations has not been established. We have examined c-myc/IgH translocations in IL6 transgenic mice that are mutant for activation induced cytidine deaminase (AID), the enzyme that initiates CSR. Here we report that AID is essential for the c-myc/IgH chromosome translocations induced by IL6.     

Mapping of the c-myc, pvt-1 and immunoglobulin kappa genes in relation to the mouse plasmacytoma-associated variant (6;15) translocation breakpoint.

A variant mouse plasmacytoma (MPC)-associated translocation chromosome has arisen by pericentric inversion and exchange of the distal segments of a Robertsonian 6;15 fusion chromosome in the CAK TEPC 1198 mouse plasmacytoma, as described earlier. In situ hybridization was performed on the normal and the inverted Rb chromosomes, using myc and kappa probes. On the normal Rb chromosome, myc was in the 15 D2/3 region, whereas kappa hybridized in the 6 C2 area, as expected. On the inverted Rb chromosome, myc remains on the centrometric side of the translocation breakpoint on the chromosome 15-derived portion, whereas kappa has moved to the chromosome 6-derived segment that joined the same breakpoint on the telomeric side. Taken together with our recent demonstration that the murine c-myc locus is oriented 'head up' on chromosome 15, and with the results of Cory and co-workers concerning the relationship between the kappa gene and the associated pvt-1 region in the CAK TEPC 1198 tumor, the following conclusions can be drawn: (i) in the variant translocation of the CAK TEPC 1198 MPC, the breakage occurs 3' of the c-myc gene, as in the human Burkitt lymphoma-associated variant translocations; (ii) the pvt-1 gene on chromosome 15 is distal to the myc gene; (iii) the kappa light chain locus is oriented 'head up' on mouse chromosome 6 and faces pvt-1 and, beyond it, c-myc, in a head-to-tail configuration.     

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.     

Cytogenetic characteristics of a murine in vitro model for the human anaplastic large cell lymphoma (ALCL)

Anaplastic large cell lymphoma (ALCL) is an entity of non-Hodgkin lymphomas (NHL) that often occurs in young children and adolescents. In the majority of cases, ALCL are of T-cell origin and contain the t(2;5)(p23;q35) leading to an NPM-ALK fusion or variant ALK translocations. In addition, there is an ALK-negative subtype of ALCL. The anaplastic lymphoid cell line TS1G6 established by interleukin (IL)-9 transfection of T-helper cells represents a murine model of this subtype. Here, we describe the cytogenetic features of this cell line using spectral karyotyping (SKY) and single-color fluorescence in situ hybridization (FISH). We show that TS1G6 cells exhibit a hypotetraploid karyotype with complex structural alterations. Several unbalanced translocations involved the chromosomal region 14E5, and different translocation partners, i.e. X?A6, 3A3 and 8A1. FISH analysis using a BAC clone containing c-myc confirmed the presence of six copies, but also demonstrated that two loci were irregularly located, indicating that additional intrachromosomal rearrangements had occurred. Moreover, a duplication of the region XF2 approximately 3 was identified. Furthermore, six chromosomes 15 were found, representing a trisomy 15 in a tetraploid chromosome complement, indicating an altered gene dosage of the oncogene c-myc located in region 15D3.     

Development of a set of compensating Triticum aestivum-Dasypyrum villosum Robertsonian translocation lines

Dasypyrum villosum (L.) Candargy, a wild relative of bread wheat ( Triticum aestivum L.), is the source of many agronomically important genes for wheat improvement. Production of compensating Robertsonian translocations (cRobTs), consisting of D. villosum chromosome arms translocated to homoeologous wheat chromosome arms, is one of the initial steps in exploiting this variation. The cRobTs for D. villosum chromosomes 1V, 4V, and 6V have been reported previously. Here we report attempted cRobTs for wheat - D. villosum chromosome combinations 2D/2V, 3D/3V, 5D/5V, and 7D/7V. The cRobTs for all D. villosum chromosomes were recovered except for the 2VS and 5VL arms. As was the case with the 6D/6V combination, no cRobTs involving 2D/2V chromosomes were recovered; instead, cRobT T2BS·2VL involving a nontargeted chromosome was recovered. All cRobTs are fertile, although the level of spike fertility and hundred kernel weight (HKW) varied among the lines. The set of cRobTs involving 12 of the 14 D. villosum chromosomes will be useful in wheat improvement programs. In fact, among the already reported cRobTs, T6AL·6VS carrying the Pm21 gene is deployed in agriculture and many useful genes have been reported on other cRobTs including resistance to stem rust race UG99 on T6AS·6VL.     

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.     

Assignment of the human pancreatic colipase gene to chromosome 6p21.1 to pter

Pancreatic colipase is a 12-kDa polypeptide cofactor for pancreatic lipase (EC 3.1.1.3), an enzyme essential for the absorption of dietary long-chain triglyceride fatty acids. Colipase is thought to anchor lipase noncovalently to the surface of lipid micelles, counteracting the destabilizing influence of intestinal bile salts. Using primers derived from the known amino acid sequence, we have used the polymerase chain reaction to produce a cDNA clone corresponding to the complete coding region of the human procolipase mRNA. Southern blot analysis of genomic DNA from a panel of mouse-human somatic cell hybrids indicated that the colipase gene (CLPS) resides on human chromosome 6. Further analysis of somatic cell hybrids carrying chromosome 6 translocations permitted regional localization of CLPS to the 6p21.1-pter region.     

Relationship of the human protooncogene CBL2 on 11q23 to the t(4;11), t(11;22), and t(11;14) breakpoints

A probe identifying CBL2, the human cellular homolog of the murine oncogene v-cbl and murine cellular protooncogene Cbl-2, and panels of rodent X human somatic cell hybrids were used to study the relationship of this protooncogene to translocations associated with acute leukemia, lymphoma, and Ewing sarcoma. CBL2 was mapped to 11q23 and found to translocate from chromosome 11 to 4 in an acute leukemia cell line possessing a t(4;11)(q21;q23) and from chromosome 11 to 14 in a B-cell lymphoma with a t(11;14)(q23;q32). In an Ewing sarcoma cell line with a t(11;22)(q23;q12), however, CBL2 remained on chromosome 11. Additional studies of other genes in the region of 11q23 allowed the following ordering of these genes and breakpoints: 11cen--q23--NCAM--CD3(E-D-G)--[t(11;14), t(4;11)]--(THY1, CBL2, ETS1)--t(11;22)--11qter. The gross structure of the CBL2 sequences examined was not altered by either of the flanking breakpoints. Given that the 5' and 3' ends of the CBL2 gene are not known and are probably not evaluated by the v-cbl probe, these results do not rule out the possibility of CBL2 involvement in the pathogenesis of a subset of acute leukemias possessing a t(4;11), B-cell lymphomas possessing a t(11;14), or Ewing sarcomas possessing a t(11;22).     

Karyotypic characteristics of the murine myeloma line sp2/0-Ag14

Using methods of G- and C-banding, a study was made of the karyotype of mouse myeloma cell line sp2/0-Ag14. The number of chromosomes varies from 58 to 65, the modal class being 61-62. 50 per cent of chromosomes are rearranged. Normal chromosomes 6, 12 and X were not detected in either examined cell of this line. Among the marker chromosomes there are an isochromosome (19/19), three dicentric markers and one marker with two interstitial C-bands. There is a specific marker t (12; 15) of mouse plasmacytomas in the karyotype sp2/0-Ag14. A possible association of specific translocations and segregations of the normal chromosomes with the phenotype of line sp2/0-Ag14 is discussed. The results obtained may be useful for cytogenetic analysis of hybridomas.     

T15 D region germ line amino acid sequences distinguished by monoclonal anti-idiotope antibody

Monoclonal antibody NL16, prepared with phosphorylcholine (PC)-binding myeloma protein C.BBPC3 (C3), identified an idiotope (C3-16 Id) that was present on T15 IdX+ myeloma proteins (MP) C3, T15, and H8, but not the T15 IdX- MP M167 and M603. The binding of C3 to NL16 is PC inhibitable, indicating that C3-16 Id is site associated. Inhibition studies with PC-specific hybridoma proteins (HP) demonstrated that the T15-type L chain VK22 and elements of the H chain were required for C3-16 Id expression. Studies of amino acid sequences of these PC-binding HP and MP showed that VK22+, T15 IdX+ HP, and MP that use the T15 D region (YYGSS) sequences were always C3-16 Id+. However, the reverse was not true, because all but one VK22+, T15 IdX+ HP with D region sequence changes were C3-16 Id-. This suggested that NL16 defined a specificity mainly determined by the D region of the H chain. A direct test of this hypothesis with heterologous heavy/light chain recombinant molecules obtained from C3-16 Id+ and C3-16 Id- HP of known sequence, showed that the D region was critical to idiotope expression. Additionally, an examination of the amino acid sequences of VK22+, T15 IdX- HP, HPCG14, and HPCM6 suggest that profound changes in the D region may also alter the expression of T15 IdX (an Id defined by a multispecific antiserum from A/He mice). The C3-16 Id+ was found in anti-PC serum of most Ig haplotype-inbred strains except for CBA/J, C3H, and PL, which are all of the Igh-Cj haplotype. Amino acid sequences of PC-binding CBA and PL HP showed marked changes in the D region from the T15 type, and this may account for the C3-16 Id- character of Igh-Cj strains.