8 Translocation in Burkitt Lymphoma Interrupts the VK Locus (Gene Localization/Immunoglobulin Genes/Genetics of B-Cell Neoplasia/In Situ Hybridization) BEVERLY S

Proc. Natl. Acad. Sci. USA Vol. 81, pp. 2444-2446, April 1984 Genetics

The 2p breakpoint of a 2;8 translocation in Burkitt lymphoma interrupts the VK locus (gene localization/immunoglobulin genes/genetics of B-cell neoplasia/in situ hybridization) BEVERLY S. EMANUEL*, JULES R. SELDENt, R. S. K. CHAGANTIt, SURESH JHANWARt, PETER C. NOWELL, AND CARLO M. CROCEt§ *Departments of Pediatrics and of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, and tThe Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, PA 19104; and MLaboratory of Cancer Genetics and Cytogenetics, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 Contributed by Peter C. Nowell, January 3, 1984

ABSTRACT The majority of chromosomal rearrange- have demonstrated translocation from 8q24 to 14q32 and ments observed in Burkitt lymphomas involve a translocation deregulation of transcription of the c-myc oncogene (18, 20). between 8q and 14q, while the remaining minority carry vari- Close proximity between immunoglobulin heavy chain and ant translocations between chromosome 8 and either 2 or 22. c-myc DNA sequences on the 14q+ chromosome are a result We have studied the JI Burkitt lymphoma cell line carrying of the translocation (15-18, 20). In Burkitt lines with the 8;22 the variant 2;8 chromosome translocation using a combination rearrangement, there is evidence for translocation of X se- of high-resolution and molecular cytogenetic techniques. We quences to the 8q+ chromosome (9, 10), resulting in tran- have determined that the chromosome 2 breakpoint of the 2;8 scriptional activation of the c-myc that remains on the in- translocation in these cells is in the distal portion of 2pll.2. In volved chromosome 8 (9). Thus, there is substantial cytoge- situ hybridization of a DNA probe for K light chain variable netic and molecular evidence to support the involvement of (V,.) region demonstrated that this 2pil.2 breakpoint is within immunoglobulin chain genes in the nonrandom chromosomal the V. region. There was significant hybridization of the probe changes and activation of transcription of the c-myc onco- to both the 2p- and 8q+ chromosomes, with 23% of all grains gene (9, 10, 12, 20). considered to be specific for V. located over the middle one- Our recent study with mouse-human hybrids of a Burkitt third of the long arm of the 8q+ chromosome. Thus, there is lymphoma line carrying a 2;8 translocation indicated that the translocation of the entire c constant (C) region and a portion c-myc oncogene remains on the involved chromosome 8, of the region carrying V,, genes from 2p to a region 3' of the c- that the CK gene moves from the short arm of chromosome 2 myc oncogene on the involved chromosome 8, resulting in tran- to a region 3' of the c-myc oncogene, and that the break on scriptional activation of the c-myc that is quite distant from the 2p occurs within the VK gene complex (13). High levels of 5' end of the C, gene. These results provide direct evidence for human c-myc transcription in hybrids were associated with translocation-related rearrangement of the K immunoglobulin the presence of the 8q+ chromosome, while the c-myc on the gene cluster in this Burkitt lymphoma and for the assignment normal 8 was silent (13). of the V,, locus to 2pll.2. Using the in situ hybridization technique, we have further investigated the Burkitt lymphoma cell line JI, which carries Cytogenetic studies of human lymphomas and leukemias the 2;8 translocation. The results of these studies confirm have demonstrated chromosomal translocations that occur the interruption and separation of V1K sequences as a result of with nonrandom frequency. Studies of Burkitt lymphomas the 2;8 translocation and provide direct evidence for the as- have shown that these cells carry one of several site-specific signment of the immunoglobulin K light chain variable locus chromosomal translocations. All of the rearrangements ex- to the chromosomal segment 2p11.2. amined have in common chromosome 8, with a breakpoint at 8q24. The other chromosome involved is most frequently 14 MATERIALS AND METHODS (1, 2) and less frequently a 2 or 22 (3-5). The breakpoints on Metaphase chromosome preparations for trypsin G-banding chromosome 14 at q32, on 2 at p1l -+ p12, and on 22 at qil and in situ hybridization were prepared from the JI Burkitt are in regions where the immunoglobulin heavy (6, 7), X (8- lymphoma cell line with a 2;8 translocation (13), using stan- 10), and K (11-13) light chain genes, respectively, have been dard methods and air-dried slides. Slides for G-band analysis mapped. These findings have led to suggestions of immuno- were incubated at 95°C for 15 min, cooled, pretreated for 1-5 globulin chain gene involvement in the nonrandom rear- sec in 0.025% trypsin (Difco 1;250) in isotonic saline, rinsed rangements of these B-cell malignancies (8-14). in saline, and stained for 6 min in a mixture of one part of In addition, analysis of somatic cell hybrids for the pres- 0.3% Wright's stain (Fisher) prepared in methanol to four ence of known retroviral oncogenes and in situ hybridization parts of Gurr's pH 6.8 phosphate buffer (Biomedical Special- of pv-myc to somatic and pachytene metaphase chromo- ties, Santa Monica, CA). Twenty metaphases were exam- somes led to the localization of the c-myc oncogene on the ined to confirm the karyotype of the cells (13) prior to their terminal portion of the long arm of chromosome 8, at band use for the in situ hybridization studies. 8q24 (15-19). This finding suggested that c-myc might have a The DNA probe for the in situ hybridization was a 598- role in the development of the Burkitt lymphomas, since this base-pair genomic DNA clone (HK 101/80) in pBR322 (12, is the segment of chromosome 8 that translocates to 14 in the 13). The probe was labeled with 3H by nick-translation (21) 8;14 rearrangements and to either 2 or 22 in the 2;8 and 8;22 to a specific activity of 2-4 x 107 cpm/,ug of DNA. rearrangements of the variant translocations. Metaphase chromosome preparations on glass slides were Studies of the 8;14 translocation Burkitt lymphoma cells hybridized with the 3H-labeled probe DNA at a final concen- tration of 0.035 ,ug/ml. After denaturation of chromosomal and probe DNA, hybridization was carried out in 50% (vol/ 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. §To whom reprint requests should be addressed. 2444 Downloaded by guest on September 24, 2021 Genetics: Emanuel et aL Proc. Natl. Acad. Sci. USA 81 (1984) 2445 vol) formamide in 0.3 M sodium chloride/0.03 M sodium ci- RESULTS trate, pH 7, in the presence of 10% dextran sulfate for 16- Cytogenetic Studies. The JI Burkitt lymphoma cells carry 18 hr as described (10, 19, 22). After hybridization and wash- the t(2;8) variant chromosome translocation in every cell ex- ing, slides were dipped in nuclear track emulsion (Kodak- amined. We have determined that the translocation break- NTB-2), stored in the dark, and developed after 6-14 days. points are at 2pll.2 and 8q24.1. Fig. 1 illustrates the normal Slides were stained with 0.25% Wright's stain and analyzed and abnormal no. 2 and 8 chromosomes from the JI cell line, microscopically for grain localization, using only intact, prepared by high-resolution techniques at the 550-band stage well-spread somatic cell metaphases where the chromo- of condensation and idiograms of these chromosomes dem- somal sites with grains were non-overlapped and identifiable onstrating the breakpoints. by G-band pattern or chromosomal morphology. In a previous report we have described the other consis- tent chromosomal changes in the karyotype (13). These in- clude, in varying proportions of the cells, trisomy 15 and 21, a marker chromosome derived from centric fusion of two 21s, an unidentified small acrocentric marker, and a deriva- tive chromosome 6 resulting in partial trisomy for lq and monosomy for 6q. In Situ Hybridization of V. Probe to JI Cells. In situ hybrid- _ _ ization studies were performed in two independent labora- ! ! tories (B.S.E. and R.S.K.C.) by using a single source of the HK 101/80 probe and of the JI cell line, with similar in situ 41 I.- -..tpll.2 hybridization techniques (10, 19, 22). The data from both laboratory 1 _ m laboratories are summarized in Table 1. In * U (B.S.E.), 50 metaphases were analyzed and a total of 124 grains were seen overlying chromosomes. Of these, 23 grains (19%) were on the pericentromeric region of the nor- _ U mal 2 (2p11 -+ 2q11), 25 grains (20%) were on the pericentromeric region of the 2p- chromosome (Fig. 2A), 2 were on the _ _ long arm of the normal 8, and a total of 8 grains (7%) were on *-_q24.1 the middle one-third of the long arm of the 8q+ chromosome (8q22 -* 8q24.1::2pll.2 -+ 2p15) near the translocationjunc- tion (Fig. 2B). The observed 7% of all grains over the midre- gion of the 8q+ is significantly higher (P < 0.01) than the proportion of grains observed at any other chromosomal region of comparable size if the distribution of other autosomal E grains is normalized to account for chromosome copy number. The next highest number of grains over a normal chromosomal segment was a total of 6 over both short arms of the no. 3 chromosome. In laboratory 2 (R.S.K.C.), among 23 grains found over all chromosomes in 15 metaphases, 2 were on the pericentro- 4. meric region of the normal 2, 4 were on the same region of the 2p- chromosome, and 8 were over the middle one-third SI. of the long arm of the 8q+ chromosome (Table 1). There were a total of 3 grains over both short arms of chromosome no. 1 in this study, and this was the only other site with >1 ,4- grain. Taken together, the data from both laboratories support the view that the translocation breakpoint interrupts the VK locus with a portion translocated to the involved 8q. DISCUSSION Previous studies have demonstrated that, in Burkitt lymphoma cells carrying the 8;14 rearrangement, translocation of the c-myc encoding exons to the immunoglobulin heavy chain locus results in high levels of constitutive c-myc 4 m. " expression, while the c-myc gene on the normal 8 is not tran- . studies of Burkitt lympho- I^ A- scribed (9, 13, 20, 24). Our recent '4 Table 1. Hybridization of VK probe to chromosomes of the JI cell line 8 8q Chromosomal Number of Number distribution of grains 2 2p- 8 8q+ Laboratory metaphases of grains 8q+ 8q 2p 2p- 1 (B.S.E.) 50 124 8 2 23 25 FIG. 1. Trypsin G-banded partial karyotypes demonstrating nor- 2 (R.S.K.C.) 15 23 8 0 2 4 mal and abnormal no. 2 and 8 chromosomes from the JI cell line at the 550-band stage of condensation and the ISCN (1981) idiogram 8q+ = grains over middle one-third of long arm. 8q = grains over for these chromosomes (23). The breakpoints are indicated on the long arm of normal chromosome 8. 2p = grains over pericentromeric chromosomal diagrams as well as on the translocation chromo- region of normal chromosome 2. 2p- = grains over pericentromeric somes. region of deleted chromosome 2. Downloaded by guest on September 24, 2021 2446 Genetics: Emanuel et aL Proc. NatL. Acad. Sci. USA 81 (1984) volved in the rearrangement in two independent laboratories A~~~~~~~~~~~~~~A, support assignment of the VK gene cluster to 2pll.2. Gene titration studies have indicated that there are between 15 and 20 VK gene loci (25). These individual VK genes are separated by a minimum of 7-10 kilobases of intervening sequence DNA. We have demonstrated that only a portion of the involved V1K complex translocates to the 8q+ chromosome, suggesting that a minimum of 50 and perhaps >100 kilobases of DNA separate the c-myc on distal 8q from the translocated CK gene complex. The c-myc gene on the involved chromosome 8 must thus be at a relatively long dis- tance from the immunoglobulin constant region gene, suggesting that a presumed "activating" influence of this gene can be effective over a considerable length of chromosome. 2p~~~~~~~~~~ This study was supported in part by National Cancer Institute Grants CA-10815, CA-16685, CA-35150, and CA-34775 and by Na- tional Institutes of Health Grant GM-20138 to the University of Pennsylvania Genetics Center. B 1. Manolov, G. & Manolova, Y. (1972) Nature (London) 237, 33- 36. 2. Zech, L., Haglund, V., Nilsson, N. & Klein, G. (1976) let. J. Cancer 17, 47-56. 3. Van den Berghe, H., Parloir, C., Gosseye, S., Eglebienne, V., Cornu, G. & Sokal, G. (1979) Cancer Genet. Cytogenet. 1, 9- 14. 4. Miyoshi, I., Hiraki, S., Kimura, I., Miyamoto, K. & Sato, J. OW "''I: (1979) Experientia 35, 742-743. .f 8q 5. Bernheim, A., Berger, R. & Lenoir, G. (1981) Cancer Genet. Cytogenet. 3, 307-316. 6. Croce, C. M., Shander, M., Martinis, J., Cicurel, L., D'An- cona, G. G., Dolby, T. W. & Koprowski, H. (1979) Proc. Natl. Acad. Sci. USA 76, 3416-3419. 7. Erikson, J., Finan, J., Nowell, P. C. & Croce, C. M. (1982) Proc. Natl. Acad. Sci. USA 79, 5611-5616. 8. Erikson, J., Martinis, J. & Croce, C. M. (1981) Nature (Lon- don) 294, 173-175. 9. Croce, C. M., Thierfelder, W., Erikson, J., Nishikura, K., Finan, J., Lenoir, G. M. & Nowell, P. C. (1983) Proc. Natl. FIG. 2. Representative autoradiographs from in situ hybridiza- Acad. Sci. USA 80, 6922-6926. tion of VK probe to metaphase chromosome preparations of the JI 10. Emanuel, B. S., Selden, J. R., Wang, E., Nowell, P. C. & cell line. (A) A portion of a metaphase with a grain over the 2p- Croce, C. M. (1984) Cytogenet. Cell Genet., in press. chromosome (arrow); (B) a portion of a metaphase with a grain over 11. McBride, D. W., Heiter, P. A., Hollis, G. F., Swan, D., Otey, the 8q+ chromosome (arrow). M. C. & Leder, P. (1982) J. Exp. Med. 155, 1680-1690. 12. Malcolm, S., Barton, P., Murphy, C., Ferguson-Smith, M. A., ma cells with the 8;22 translocation using both somatic cell Bentley, D. L. & Rabbitts, T. H. (1982) Proc. Natl. Acad. Sci. in situ hybridization techniques indicate that in USA 79, 4957-4961. genetic and 13. Erikson, J., Nishikura, K., ar-Rushdi, A., Finan, J., Emanuel, this translocation the c-myc oncogene remains on the 8q + B., Lenoir, G., Nowell, P. C. & Croce, C. M. (1983) Proc. chromosome and the C, locus translocates to a region 3' of Natl. Acad. Sci. USA 80, 7581-7585. the c-myc oncogene (9, 10). The c-myc oncogene located 14. Klein, G. (1981) Nature (London) 294, 313-318. proximal or 5' to the translocated CA locus is transcribed at 15. Dalla Favera, R., Bregni, M., Erikson, J., Patterson, D., high levels, while the myc on the normal 8 is transcriptional- Gallo, R. C. & Croce, C. M. (1982) Proc. Natl. Acad. Sci. ly silent (9). USA 79, 7824-7827. The present study confirms and extends our previous ob- 16. Taub, R., Kirsch, I., Morton, C., Lenoir, G., Swan, D., Tron- servations on the 2;8 translocation of JI Burkitt lymphoma ick, S., Aaronson, S. & Leder, P. (1982) Proc. Natl. Acad. Sci. cells. We have shown that in the 2;8, as in the 8;22 rearrange- USA 79, 7837-7841. 17. Marcu, K. B., Harris, L. J., Stanton, L. W., Erikson, J., ment, the Ig gene translocates 3' of the myc gene on 8q, and Watt, R. & Croce, C. M. (1983) Proc. Natl. Acad. Sci. USA we confirm previous suggestions that the translocation 80, 519-523. breakpoint is within the VK locus (13). 18. Dalla Favera, R., Martinotti, S., Gallo, R. C., Erikson, J. & Chromosomal breaks and DNA and chromosomal rear- Croce, C. M. (1983) Science 219, 963-967. rangements can occasionally occur in somatic cell hybrids. 19. Neel, B. G., Jhanwar, S. C., Chaganti, R. S. K. & Hayward, Our previous description of the presence of V1K genes in hy- W. S. (1982) Proc. Natl. Acad. Sci. USA 79, 7842-7846. brids that retained either the 2p- or the 8q+ chromosome in 20. Erikson, J., ar-Rushdi, A., Drwinga, fI. L., Nowell, P. C. & the absence of the normal chromosome 2 could be explained Croce, C. M. (1983) Proc. Natl. Acad. Sci. USA 80, 820-824. Efstradiatis, A. & Kafatos, F. C. on or chromosomal rearrangement unrelat- 21. Maniatis, T., Kee, S. G., the basis ofDNA (1976) Cell 27, 583-591. ed to the original translocation. The present study provides 22. Harper, M. E., Ullrich, A. & Saunders, G. F. (1981) Proc. direct cytologic evidence for the 2p- and 8q+ location of VK Natl. Acad. Sci. USA 78, 4458-4462. sequences in the JI parental cell line, lending additional sup- 23. ISCN (1981) Cytogenet. Cell Genet. 32, 1-23. port to the somatic cell hybrid data. The precise breakpoint 24. ar-Rushdi, A., Nishikura, K., Erikson, J., Watt, R., Rovera, identification by high-resolution banding coupled with hy- G. & Croce, C. M. (1983) Science 222, 390-393. bridization of the VK probe to both of the chromosomes in- 25. Bentley, D. L. & Rabbitts, T. H. (1981) Cell 24, 613-623. Downloaded by guest on September 24, 2021