Kazuko Nishikura , Jan Erikson , Abbas Ar - Rushdi , Kay Huebner , and Carlo

We hybridized Raji Burkitt lymphoma cells, which carry a t(8;14) chromosome translocation, with human lymphoblastoid cells to study the expression of the translocated cellular myc oncogene (c-myc) in the hybrid cells. In Raji cells the c-myc oncogene is translocated to a switch region of the y heavy chain locus (Sy). Because of sequence alterations in the 5' exon of the translocated c-myc oncogene in this cell line, it is possible to distinguish the transcripts of the translocated c-myc gene and of the normal c-myc gene. S1 nuclease protection experiments with a c-myc first exon probe indicate that Raji cells express predominantly the translocated c-myc gene, while the level of expression of the normal c-myc gene is less than 2% of that of the translocated c-myc gene. Somatic cell hybrids between Raji and human lymphoblastoid cells retain the lymphoblastoid phenotype and express only the normal c-myc oncogene. This result indicates that the activation of a c-mtyc oncogene translocated to a S region depends on the stage of B-cell differentiation of the cells harboring the translocated c-myc gene and not on alterations in the structure of the translocated c-myc oncogene. In Burkitt lymphomas with t(8;14) translocations, the cellular myc oncogene, c-myc, which is normally on band q24 of chromosome 8 (1-4), translocates to the heavy (H) chain locus (1, 3, 4) on chromosome 14 (5). In Burkitt lymphomas with variant t(8;22) and t(2;8) chromosome translocations, the c-myc gene remains in its germ-line configuration on the involved chromosome 8 (8q+), while either the A or the K light chain locus translocates to a region distal (3') to the involved c-myc oncogene (6, 7). The consequence of these different types ofrearrangements is deregulation oftranscription ofthe involved c-myc oncogene, which is expressed constitutively at elevated levels (6-8) in the lymphomas. Since c-myc deregulation or, in other terms, constitutive c-myc expression is a possible initial cause of these lymphomas, there is great interest in arriving at an understanding of the mechanism(s) of constitutive c-myc expression. The first obvious common denominator in the lymphoma is the translocation itself, which juxtaposes a c-myc gene and an immunoglobulin locus; the second common denominator is that the involved c-myc locus in cases thus far analyzed is always 5' ofthe involved immunoglobulin locus; and the third common denominator, aside from the constitutive expression of the involved c-myc gene, is the silence or near silence of the untranslocated c-myc allele in the lymphomas (8, 9). These common denominators may serve as clues in unraveling the mechanism(s) responsible for constitutive expression of the involved c-myc allele, but the heterogeneity of the breakpoints on chromosome 8 and within the immunoglobulin loci have led to differing proposals to explain constitutive c-myc expression. Because in many cases of Burkitt lymphoma with the t(8;14) translocation the c-myc oncogene translocates to a H-chain switch (S) region (predominantly S,) on the 14q+ chromosome, with concomitant translocation of the immunoglobtilin H-chain gene (IGH) enhancer [located between the H-chain joining region (JH) and the So region] to the 8qchromosome, it has been speculated that c-myc activation in Burkitt lymphoma is not the result of transcriptional enhancement (10, 11). Leder et al. (10) have proposed that the first exon of the c-myc oncogene and regions 5' to the first exon mediate negative regulation of c-myc transcription by binding to a putative repressor protein. According to this model, truncation of the c-myc oncogene at its 5' end or changes in its 5' exon would affect the binding of the putative repressor to the c-myc gene, resulting in its deregulation (10, 11); thus, the translocated c-myc gene would be insensitive to negative regulation (10, 11). Since we observed that a 5'-truncated and translocated c-myc oncogene is not expressed (i.e., not deregulated) when introduced into fibroblasts (12), it seemed unlikely that loss of the c-myc first exon is responsible per se for c-myc deregulation. These studies and others to be discussed below demonstrated that the constitutive expression of the translocated c-myc allele was observed only in a B-cell background; i.e., activation of the c-myc gene involved in the different chromosomal translocations was B-cell specific (8, 12-14). If constitutive c-myc expression from the translocated (or involved) c-myc allele were due solely to loss of or change in certain regulatory signals normally contained within or 5' of the c-myc gene so that the involved allele could no longer respond to negative regulation, one would expect the involved c-myc allele to remain activated even when placed within a phenotypically different cell type. Thus, we have proposed that the constitutive expression of the involved c-myc allele in Burkitt lymphomas must be due to B-cell stage-specific, cis-acting, positive-regulatory elements within the immunoglobulin loci (13, 14). In previous studies (15, 16), two Burkitt lymphoma cell lines, ST486 and CA46, in which the coding portion of the 5'-truncated c-myc gene (lacking its 5' exon) is translocated respectively to the S., or the Sa region, were hybridized with human lymphoblastoid cells; the hybrids retained the lymphoblastoid phenotype and expressed the normal but not the translocated c-myc gene (13). From these results we concluded that the translocated c-myc gene is under the control of thus far unidentified genetic elements within the H-chain locus capable of enhancing c-myc transcription in Burkitt lymphoma cells and plasma cells, but incapable of activating gene transcription in lymphoblastoid cells (13). These results also illustrate the point that loss ofthe 5' exon and oftheDNA region 5' to the c-myc gene does not necessarily result in deregulation of translocated c-myc genes. Recently we hybridized lymphoblastoid cells with Daudi cells in which the translocated c-myc gene on the 14q+ chromosome is located Abbreviations: c, cellular; S, switch; H, heavy; J, joining; C, constant; kb, kilobase(s). 2900 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 82 (1985) 2901 5' of the JH segment of the H-chain locus and, therefore, 5' of the immunoglobulin H-chain gene enhancer located between JH and SM (14). Interestingly, we observed that the hybrid cells expressed both the normal and the translocated c-myc genes. These results indicate that genetic elements 5' of S, (possibly the immunoglobulin H-chain gene enhancer between S. and JH) are capable of activating c-myc transcription not only in Burkitt lymphoma cells and plasma cells but also in lymphoblastoid cells (14). Thus, it seems likely that Burkitt-like translocations lead to malignant transformation by deregulation of c-myc transcription due to proximity to different genetic elements capable of enhancing gene transcription, some of which are more "promiscuous" and can activate gene transcription in B cells at different stages of B-cell differentiation from pre-B-cells (17) to plasma cells (13, 14), while others are able to activate gene transcription only in the more differentiated B cells (13). In this study we intended to assess the role of changes in the first c-myc exon in the deregulation of the c-myc gene. Therefore, we hybridized human lymphoblastoid cells with Raji cells which carry a translocated c-myc gene that is mutated in its 5' exon (11). If changes in the c-myc 5' exon or its flanking sequence were responsible for c-myc activation in Raji cells, we should observe expression of the translocated c-myc gene in the hybrids. On the contrary, if the genetic elements that are responsible for translocated c-myc activation in ST486 and CA46 cells are involved in the enhancement of expression of the translocated Raji c-myc gene, we should not observe translocated c-myc transcription in lymphoblastoid hybrid cells, since the putative enhancer elements that are active in CA46 and ST486 Burkitt lymphoma cells are not active in human lymphoblastoid cells (13). kb 23.0 _20.0 12.012 3 4 5 6