Identification of reciprocal translocation sites within the c-myc oncogene and immunoglobulin mu locus in a Burkitt lymphoma

The association between certain human tumours and characteristic chromosomal abnormalities has led to the hypothesis that specific cellular oncogenes may be involved and consequently 'activated' in these genetic recombinations. This hypothesis has found strong support in the recent findings that some cellular homologues of retroviral onc genes are located in chromosomal segments which are affected by specific tumour-related abnormalities (see ref. 4 for review). In the case of human undifferentiated B-cell lymphoma (UBL) and mouse plasmacytomas, cytogenetic and chromosomal mapping data have identified characteristic chromosomal recombinations directly involving different immunoglobulin genes and the c-myc oncogene (for review see refs 5, 6). In UBLs carrying the t(8:14) translocation it has been shown that the human c-myc gene is located on the region of chromosome 8 (8q24) which is translocated to the immunoglobulin heavy-chain locus (IHC) on chromosome 14. Although it is known that the chromosomal breakpoints can be variably located within or outside the c-myc locus and within the IHC mu (refs 9, 11) or IHC gamma locus, the recombination sites have not been exactly identified and mapped in relation to the functional domains of these loci. We report here the identification and characterization of two reciprocal recombination sites between c-myc and IHC mu in a Burkitt lymphoma. Nucleotide sequencing of the cross-over point joining chromosomes 8 and 14 on chromosome 14q--shows that the onc gene is interrupted within its first intron and joined to the heavy-chain mu switch region. This recombination predicts that the translocated onc gene would code for a rearranged mRNA but a normal c-myc polypeptide.     

T-cell control of Epstein-Barr virus-infected B cells is lost during P. falciparum malaria

Endemic Burkitt's lymphoma, a tumour of children in which B lymphocytes are infected with Epstein-Barr virus (EBV), is common in areas of Africa where malaria is holoendemic. The tumour is characterized by chromosome translocations; usually the terminal portion of chromosome 8 containing the c-myc gene is translocated to chromosome 14, near the enhancer of the immunoglobulin heavy-chain locus. Less frequent are translocations of chromosome 8 to the kappa light-chain locus of chromosome 2 or to the lambda light-chain locus of chromosome 22. In vitro, EBV induces B cells to proliferate and secrete immunoglobulin and antibody. However, in vivo the infected B lymphocytes are under immunological control, so that abnormal proliferation is found only in immunosuppressed patients. Such patients are subsequently liable to develop lymphomas. Burkitt believed that the tumour he had described resulted from interaction between a virus(es) and a "reticuloendothelial system altered by chronic and heavy infection by malarial or other parasites". We report here that during an attack of Plasmodium falciparum malaria, T-cell subpopulations are radically altered so that, in vitro, B lymphocytes infected with EBV proliferate abnormally to secrete large amounts of immunoglobulin and antibody. This phenomenon offers some explanation for the increased incidence of Burkitt's tumour and the high levels of immunoglobulin found in people living in areas where P. falciparum malaria is common.     

Bcl-2 gene promotes haemopoietic cell survival and cooperates with c-myc to immortalize pre-B cells

A common feature of follicular lymphoma, the most prevalent haematological malignancy in humans, is a chromosome translocation (t(14;18] that has coupled the immunoglobulin heavy chain locus to a chromosome 18 gene denoted bcl-2. By analogy with the translocated c-myc oncogene in other B-lymphoid tumours bcl-2 is a candidate oncogene, but no biological effects of bcl-2 have yet been reported. To test whether bcl-2 influences the growth of haematopoietic cells, either alone or together with a deregulated c-myc gene, we have introduced a human bcl-2 complementary DNA using a retroviral vector into bone marrow cells from either normal or E mu-myc transgenic mice, in which B-lineage cells constitutively express the c-myc gene. Bcl-2 cooperated with c-myc to promote proliferation of B-cell precursors, some of which became tumorigenic. To determine how bcl-2 expression impinges on growth factor requirements, the gene was introduced into a lymphoid and a myeloid cell line that require interleukin 3 (IL-3). In the absence of IL-3, bcl-2 promoted the survival of the infected cells but they persisted in a G0 state, rather than proliferating. These results argue that bcl-2 provided a distinct survival signal to the cell and may contribute to neoplasia by allowing a clone to persist until other oncogenes, such as c-myc, become activated.     

Clustering of breakpoints on chromosome 11 in human B-cell neoplasms with the t(11;14) chromosome translocation

The t(11;14) (q13;q32) chromosome translocation has been reported in diffuse small and large cell lymphomas and in chronic lymphocytic leukaemia (B-CLL) and multiple myeloma. Because chromosome band 14q32 is involved in this translocation, as well as in the t(8;14) (q24;q32) translocation of the Burkitt tumour, interruption of the immunoglobulin heavy-chain locus was postulated for this rearrangement. We have cloned the chromosomal joinings between chromosomes 11 and 14 and also between chromosomes 14 and 18, in B-cell tumours carrying translocations involving these chromosomes, and suggested the existence of two translocated loci, bcl-1 and bcl-2, normally located on chromosomes 11 (band q13) and 18 (band q21) respectively, involved in the pathogenesis of human B-cell neoplasms. The results indicate that in the leukaemic cells from two different cases of CLL, the breakpoints on chromosome 11 are within 8 nucleotides of each other and on chromosome 14 involve the J4-DNA segment. Because we detected a 7mer-9mer signal-like sequence with a 12-base-long spacer on the normal chromosome 11, close to the breakpoint, we speculate that the t(11;14) chromosome translocation in CLL may be sequence specific and may involve the recombination system for immunoglobulin gene segment (V-D-J) joining.     

Variant (6 ; 15) translocation in a murine plasmacytoma occurs near an immunoglobulin kappa gene but far from the myc oncogene

Chromosome translocations in B-lymphoid tumours are providing intriguing insights and puzzles regarding the role of immunoglobulin genes in the activation of the myc oncogene (reviewed in refs 1, 2). The 15 ; 12 translocations found in most murine plasmacytomas and the analogous 8 ; 14 translocation in human Burkitt's lymphomas involve scissions of murine chromosome 15 (human chromosome 8) near the 5' end of the c-myc gene and subsequent fusion near an immunoglobulin heavy-chain gene. The less well characterized 'variant' translocations found in about 15% of such tumours also involve the myc-bearing chromosome band, but exchange occurs with a chromosome bearing an immunoglobulin light-chain locus--in mice, the kappa-chain locus bearing chromosome 6 (refs 3-5) and, in man, chromosome 2 (or 22), at the same band at which the kappa (or lambda) locus lies (reviewed in ref. 1). The Burkitt variant translocations involve scissions 3' of c-myc; one 8 ; 22 translocation placed the C lambda locus just 3' of c-myc, but usually the chromosome 8 breakpoint is a greater, but unknown, distance away from c-myc, more than 20 kilobases (kb) in one 8 ; 2 translocation involving the C kappa gene. Little is known about the murine 6 ; 15 translocations, although a C kappa gene cloned from one plasmacytoma (PC7183) is linked, via chromosome 12 sequences, to an unidentified region of chromosome 15 (ref. 11). We describe here the chromosome fusion region from plasmacytoma ABPC4, which displays the typical reciprocal 6;15 translocations. We find that the chromosome 6 breakpoint is near C kappa but, unlike those in the heavy-chain locus, not at a position where immunoglobulin genes normally recombine. Moreover, the chromosome 15 sequences involved in the ABPC4 translocation are not derived from the vicinity of c-myc.     

Nucleotide sequence of cloned cDNA of human c-myc oncogene

Like other transforming genes of retroviruses, the v-myc gene of the avian virus, MC29, has a homologue in the genome of normal eukaryotic cells. The human cellular homologue, c-myc, located on human chromosome 8, region q24 leads to qter (refs 1, 2), is translocated into the immunoglobulin heavy-chain locus on human chromosome 14 (ref. 3) in Burkitt's lymphoma, suggesting that c-myc has a primary role in transformation of some human haematopoietic cells. In addition, c-myc is amplified in the human promyelocytic leukaemia cell line, HL60 (refs 6, 7) which also contains high levels of c-myc mRNA. Recently, Colby et al. reported the nucleotide sequence of the human c-myc DNA isolated from a genomic recombinant DNA library derived from human fetal liver. This 4,053-base pair (bp) sequence includes two exons and one intron of the myc gene, and the authors have suggested the existence of a human c-myc mRNA of 2,291 nucleotides that has a coding capacity for a protein of molecular weight (Mr) 48,812. We have approached the problem of accurately defining the characteristics of the human c-myc mRNA and c-myc protein by determining the sequence of the c-myc cDNA isolated from a cDNA library prepared from mRNA of a clone of the K562 human leukaemic cell line. K562 cells are known to contain c-myc mRNA which is similar in size to the c-myc mRNA of other human cell types. We report here the sequence of 2,121 nucleotides of a human c-myc mRNA and demonstrate that its 5' noncoding sequence does not correspond to the sequence of the reported genomic human sequence. However, our data confirm that the intact human c-myc mRNA can encode a 48,812-Mr protein with a sequence identical to that reported by Colby et al.     

Altered nucleotide sequences of a translocated c-myc gene in Burkitt lymphoma

The nucleotide sequence of a translocated c-myc gene in a Burkitt lymphoma reveals multiple base changes in the coding region. Twenty-five base changes, generating 16 codon alterations, were found in the first coding exon; no changes occur in the second coding exon. These changes are probably the result of somatic mutations that occurred during and after translocation, and may contribute to oncogenesis by allowing synthesis of an altered c-myc gene product.     

Elevated c-myc expression facilitates the replication of SV40 DNA in human lymphoma cells

The v-myc oncogene can induce tumours in haematopoietic, mesenchymal and epithelial tissues. The corresponding c-myc proto-oncogene can contribute to the genesis and/or the progression of an equally wide variety of tumours when activated by retroviral insertions, chromosomal translocations or gene amplification. The c-myc gene product is a DNA-binding, nuclear phosphoprotein that is involved in the control of cell proliferation and possibly in DNA synthesis. The replication of Simian virus 40 (SV40) is a useful model system to study eukaryotic DNA replication as the virus relies almost entirely on cellular DNA replication apparatus. The SV40-based vector, pSVEpR4, replicates poorly in the human BJAB lymphoma line and in most human cells, but replicates well in Burkitt lymphoma lines, which have fused immunoglobulin and c-myc genes, resulting in high c-myc expression. Cotransfection of the BJAB cells with a c-myc-expressing construct (pI4-P6) increased the replication of pSVEpR4 tenfold. Our findings indicate that overexpression of the c-myc gene product allows the replication of SV40 in human lymphoma cells, suggesting that c-myc is involved in the control of replication.     

Breakpoints in the human T-cell antigen receptor alpha-chain locus in two T-cell leukaemia patients with chromosomal translocations

Specific chromosomal translocations have been observed in several human and animal tumours and are believed to be important in tumorigenesis. In many of these translocations the breakpoints lie near cellular homologues of transforming genes, suggesting that tumour development is partly due to the activation of these genes. The best-characterized example of such a translocation occurs in mouse plasmacytoma and human B-cell lymphoma, where c-myc, the cellular homologue of the viral oncogene myc, is brought into close proximity with either the light- or heavy-chain genes of the immunoglobulin loci, resulting in a change in the regulation of the myc gene. T-cell malignancies also have characteristic chromosomal abnormalities, many of which seem to involve the 14q11-14q13 region. This region has recently been found to contain the alpha-chain genes of the human T-cell antigen receptor. Here we determine more precisely the chromosome breakpoints in two patients whose leukaemic T cells contain reciprocal translocations between 11p13 and 14q13. Segregation analysis of somatic cell hybrids demonstrates that in both patients the breakpoints occur between the variable (V) and constant (C) region genes of the T-cell receptor alpha-chain locus, resulting in the translocation of the C-region gene from chromosome 14 to chromosome 11. As the 11p13 locus has been implicated in the development of Wilms' tumour, it is possible that either the Wilms' tumour gene or a yet unidentified gene in this region is involved in tumorigenesis and is altered as a result of its translocation into the T-cell receptor alpha-chain locus.     

High-affinity binding site for a specific nuclear protein in the human IgM gene

Proteins binding to specific regions of DNA with high affinity frequently govern or regulate reactions at the gene level. We have identified a high-affinity binding site in the immunoglobulin mu gene that binds a specific nuclear protein, and have now characterized it fully using nuclear factor 1 (NF-1), a protein purified from the nuclei of HeLa cells and required for the in vitro replication of adenovirus (Ad) DNA. NF-1 protects a 25-base pair (bp) double-stranded segment of DNA which shares a consensus sequence, 5' TGGA/CNNNNNGCCAA 3', with similar binding sites in the Ad-5 terminal repeat and the human c-myc gene. Although this site differs from the enhancer region, a biological function is suggested by the fact that it is DNase I hypersensitive in immunoglobulin-producing lymphoblastoid cells. The binding site for the NF-1 protein in the mu gene, by analogy with the site in the Ad-5 terminal repeat, may represent one component of a cellular origin of replication; alternatively, it may be responsible for the activation of the chromatin in this region.     

The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice

Transgenic mice bearing the cellular myc oncogene coupled to the immunoglobulin mu or kappa enhancer frequently develop a fatal lymphoma within a few months of birth. Since the tumours represent represent both immature and mature B lymphocytes, constitutive c-myc expression appears to be highly leukaemogenic at several stages of B-cell maturation. These myc mice should aid study of lymphoma development, B-cell ontogeny and immunoglobulin regulation.     

Murine T lymphomas with retroviral inserts in the chromosomal 15 locus for plasmacytoma variant translocations

The frequent trisomy of murine chromosome 15 in T lymphomas suggests that it bears one or more genes conducive to T-cell neoplasia. One such gene seems to be c-myc, the oncogene frequently activated in B-lymphoid tumours either by retroviral insertion, as in the avian bursal lymphomas, or by a translocation to the immunoglobulin heavy-chain locus, as in the predominant t(12; 15) of murine plasmacytomas and the analogous t(14; 8) of human Burkitt lymphomas. The c-myc gene was strongly implicated in T-cell neoplasia when 15-25% of T lymphomas arising in AKR mice, a strain prone to leukaemia, were found to have retroviral inserts near c-myc. Proviruses near c-myc were also found in several T lymphomas induced by murine leukaemia viruses (MuLV) in both mice and rats, but many of the rat thymomas bear an insert instead at one of several other common sites, at least two of which have murine homologues on chromosome 15. We show here that some murine T lymphomas contain proviral inserts in the recently identified chromosome 15 locus for plasmacytoma variant (6; 15) translocations, which we have denoted pvt-1. Although 6; 15 breakpoints map cytogenetically to the same chromosome band as c-myc, the alterations of pvt-1 in tumours occur at least 72 kilobases (kb) from the c-myc promoters. The insertions in T lymphomas suggest that an altered pvt-1 locus is conducive to neoplasia in T cells as well as B cells, possibly via long-range effects on c-myc expression.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.