Mapping of Transforming Growth Factor a Gene on Human Chromosome 2 Close to the Breakpoint of the Burkitt's Lymphoma T(2;8) Variant Translocation1

(CANCER RESEARCH 45, 5593-5597, November 1985]

Mapping of Transforming Growth Factor a Gene on Human Chromosome 2 Close to the Breakpoint of the Burkitt's Lymphoma t(2;8) Variant Translocation1

Jane E. Brissenden, Rik Derynck, and Uta Francke

Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 [J. E. B., U. F.], and Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080 [R. D.]

ABSTRACT or EGF to induce transformation of normal rabbit kidney indicator cells (8, 9) and do not compete with EGF for binding to EGF Transforming growth factors (TGFs) are defined as biologically receptors although they do induce an increase of EGF receptor active polypeptides which reversibly confer the transformed phe- numbers on normal rabbit kidney cells in culture (11). notype onto untransformed cultured cells. TGF-a shows se TGFs-a have been suggested to play a physiological role in quence homology with epidermal growth factor and competes embryonic development (12), and many observations have linked with epidermal growth factor for binding to the epidermal growth their secretion to the malignant transformation of cells. For factor receptor, stimulating the phosphorylation of the receptor. example, cells transformed with a temperature sensitive Kirsten TGF-a is secreted by many transformed cells and may be murine sarcoma virus secrete TGF-a only when phenotypic involved in embryonic development. transformation occurs at the permissive temperature (13). Intro A cloned human TGF-a gene was used to map the locus for duction of the DNA segment coding for middle T-antigen of the TGF-a precursor to the short arm of human chromosome 2, polyoma virus into cells is sufficient to induce both a transformed region 2p11-Â»2p13, by Southern blotting techniques with DNA phenotype and TGF-a secretion (14). Limited clinical studies prepared from rodent x human somatic cell hybrids. These have also suggested the presence of a characteristic TGF-a hybrids contained different subsets of human chromosomes and species in the urine of cancer patients (15,16). These and other included a set with a translocation between human chromo data, including TGF-a production by many tumor cells in a somes 1 and 2 [t(1 ;2) (q32;q13)]. In situ hybridization of the TGF- transformation-dependent manner, have indicated an important role for TGFs-a in cellular transformation, perhaps by an auto a probe to normal human metaphase spreads confirmed these data and localized TGF-a more precisely to bands 2p11->2p13. crine (feedback) mechanism (2,17). Comparison of the amino acid sequences of the purified 50- Breakpoints in the variant Burkitt lymphoma translocation t(2;8) amino acid-long rodent TGFs-a and human TGF-a to human and occur within these bands. Such a t(2;8) translocation could place 7GF-Â«next to c-myc in band 8q24. The possibility is raised that murine EGF has demonstrated considerable sequence identity TGF-a might contribute to tumor progression in these cases of (18-20). These results could suggest a common evolutionary Burkitt's lymphoma. origin and the possibility that the M, 6,000 TGFs-a and EGF may have arisen by gene duplication from a common ancestral mol ecule (18). Brissenden ef a/. (21) have recently assigned the gene INTRODUCTION coding for human EGF to the distal long arm of human chromo some 4, bands 4q21-*qter, which overlaps the region to which The presence of growth factors produced by sarcoma virus or the human T-cell growth factor (interieukin 2) has been mapped chemically transformed cells, as well as by spontaneously oc (22). The possibility that EGF and TGF-a might have arisen from curring human tumors, was first suspected because such cells a common DNA sequence prompted us to determine if human were blocked in their ability to bind epidermal growth factor or TGF-Â«also maps to human chromosome 4 or perhaps to chro multiplication stimulating activity (insulin-like growth factors) (1, mosome 7, which carries the locus for the EGF receptor (23, 2). Many such cells have now been shown to produce TGFs2 (3, 24). 4). TGFs are defined as biologically active polypeptides which The availability of a cloned DNA probe coding for human TGF- reversibly confer the transformed phenotype onto untransformed a (20) has allowed us to map the human gene for TGF-a to the cultured cells, as assayed by induction of anchorage independent short arm or to the proximal portion of the long arm of chromo growth (5). They have been subdivided into two classes, TGFs- some 2 using somatic cell hybrids and Southern transfer tech a and -ÃŸ(5, 6). Type a TGFs compete with EGF for binding to niques. Since chromosome 2 contains almost 8% of the total the EGF receptor and stimulate tyrosine phosphorylation of this human chromosomal material and several interesting genes re receptor (7). TGFs-a do not seem to be secreted by normal cells lated to oncogenesis or growth control (25-29), we carried out in culture or tissues but have been found in the supernatant in situ hybridizations to normal human mitotic chromosomes to medium of a wide variety of transformed cell lines. Type ÃŸTGFs further refine the map location of the major hybridizing sequence have been isolated from nonneoplastic (5, 8, 9) and neoplastic and to detect minor sites of hybridization to sequences that are tissues (10). Type ÃŸTGFs require the presence of either TGF-a partially homologous to the probe.

1This work was supported by the National Institute of General Medical Sciences (NIH Research Grant GM26105). MATERIALS AND METHODS 2The abbreviations used are: TGF, transforming growth factor; EGF, epidermal growth factor; BL, Burkitt's-type leukemia-lymphoma; SSC, standard saline citrate. A 670-base pair Sacl-Sa/l genomic DNA fragment containing a 121- Received 5/7/85; revised 7/24/85; accepted 7/29/85. base pair TGF-a exon (20) was used for hybridization. This probe was

CANCER RESEARCH VOL. 45 NOVEMBER 1985 5593

labeled with [a-^PJdCTP and [Â«-"PJdATP as described (30). Somatic fragment was detected and was absent when the human band cell hybrids were derived from six different human-rodent hybrid series was absent (Table 1). While there was no discordance for which have been described previously (31 -36). The rodent parental cell chromosome 2 in all 19 clones analyzed, each of the other line was Chinese hamster lung fibroblast V79/380-6. The human cells, human chromosomes was discordant with the TGF-a band in at either fibroblasts or leukocytes, were all heterozygous for defined chro least 25% of the hybrids. mosomal rearrangements. The resulting somatic cell hybrid clones con To more precisely define the location of the TGF-a gene on tained different subsets of human chromosomes defined by karyotyping, enzyme marker, and DMA marker studies. chromosome 2, we used two different approaches. The TGF-a DNA was extracted from these hybrid clones as described previously probe was used for Southern blotting of DNA from somatic cell (37), digested to completion with BamHI (International Biotechnologies, hybrids containing defined regions of human chromosome 2 and, Inc.), and separated in 0.8% agarose gels followed by blotting onto secondly, was hybridized directly to chromosomes from normal nitrocellulose filters (38). The filters were hybridized overnight under individuals. DNAs from two hybrid clones derived from a human stringent conditions (5 x SSC and 67Â°C)in the presence of 7% dextran cell strain with a balanced reciprocal translocation between chro sulfate. After hybridization, they were rinsed twice in 2 x SSC plus 0.1% mosomes 1 and 2 [t(1;2) (q32;q13)] (31, 41) were analyzed by sodium dodecyl sulfate followed by three changes of 0.1 x SSC plus 0.1% sodium dodecyl sulfate at 56Â°Cover 2 h. Southern hybridization. In the absence of a normal chromosome 2, the human TGF-a band was present when the short arm and In situ hybridization was carried out with normal human metaphase spreads. The same TGF-Â«DNA fragment used for the filter hybridization, proximal long arm of chromosome 2 (region 2pterâ€”>q13)was subcloned in pBR322, was nick-translated with three tritium-labeled present, but this band was absent when only the reciprocal nucleotides ([3H]dATP, [3H]dCTP, and [3H]dTTP) to a specific activity of translocation product (region 2q13â€”Â»qter)wasretained (Fig. 2). 2 x 107 cpm/fzg as described (39). The procedures of hybridization and Thus the locus coding for TGF-a maps to the region 2pterâ€”Â»q13. staining with quinacrine and Wright's stain were as described (39, 40). In Situ Hybridization to Metaphase Chromosomes. Analysis One hundred metaphase spreads from one individual were completely of 100 normal male metaphase spreads hybridized with the analyzed, and the results were confirmed by analysis of 50 metaphase tritiated TGF-a probe revealed a total of 220 silver grains distrib spreads from a second individual. uted over all human chromosomes (Chart 1). Of these grains, 33 (15%) were situated over the short arm of chromosome 2, and RESULTS 25 of those (75.8%) occurred in the region 2p11-Â»2p13 (Chart 2; Fig. 3). The number of grains observed over the chromosome Southern Analysis of Somatic Cell Hybrids. Under the strin 2 short arm was compared with the number expected (based on gent conditions used, a single SamHI fragment of about 12 the relative length of the short arm) by x2 analysis (42). The x2 kilobases was detected on autoradiograms in lanes containing 106.7 with 1 d.f. gives a probability of occurrence by chance of human DNA (Fig. 1). No cross-reacting bands were detected in less than 0.0001. Analysis of 50 metaphase spreads from a lanes containing Chinese hamster DNA. Only human chromo normal female confirmed these findings. Thirteen of the 115 some 2 was consistently present when the human 12-kilobase silver grains found on chromosomes (12%) were over region 2p11-Â»2p13 [x2 = 26.6; P < 0.0001]. Twenty-eight % of cells 1 2 3 4 5 6 7 8 9 10 11 12 13 14 were labeled at 2p11â€”Â»2p13.Noother chromosomal site was labeled above background (Chart 1). Even though TGF-Â« is partially homologous to EGF (18-20), no specific label, at the stringency used here, was detected over distal 4q where EGF sequences have been mapped. Taken together, these results map 7GF-0 to the short arm of human chromosome 2, region 2p11-*2p13(Chart2). Fig. 1. Hybridization of M, 6000 human TGF-a probe to human, rodent and hybrid cell DNA. Human control DNA (Lanes 1 and 14) produced a single SamHI band (arrow). Chinese hamster DNA (Lane 73) showed no cross-hybridizationwith DISCUSSION the TGF-n probe. Chinese hamster human hybrids in Lanes 2,4,7,8, and 77 were positive for the TGF-a band and those in Lanes3,5,6,9,70 and 72were negative. Hybrid lanes were loaded with 10 ng of DNA with the exception of Lanes 7 and 70 Results from both Southern blotting analysis and in situ hy which contained 7 Â¡Â¿geach. bridization to normal human chromosomes gave no evidence for

1 M,Hybridization/ChromiCorrelation of human TGF-ahybrids547 sequences with human chromosomes in Chinese hamster x human somatic cell ofchromosomes8102 hybrids for human

some|V13

9 11 3 8 87 2 9 9 3 7 4 3 6 5 6 6 6 4 3 2 0 0 2 446470471 2 B 6 1 141344 0 4 1 5 2 452057232171 27X4 1 4 228 037 86000453Table 186 8No931. 2116 6127 6148 8154 5167 6173 5185 4194 62264 5Y44 4

Total discordant 4085848 10 6875 10 897 10 6957968

Total informative 16 19 18 18 19 15 17 18 18 19 16 19 18 19 19 19 19 17 19 17 18 19 13 16

% of discordant hybrids 25 0 44 28 42 27 47 56 33 42 44 26 56 42 47 37 53 35 47 29 39 47 46 50 " The numbers of hybrids showing concordant (+/+ and -/-) and discordant (+/- and -/+) segregation with TGF-Â«aregiven for each chromosome. Only intact human chromosomes present in at least 20% of cells were included. Data on rearrangedchromosomes or chromosomes present at low frequencies were excluded.

CANCER RESEARCH VOL. 45 NOVEMBER 1985 5594

uU, |P q l p q l p q I p q | p 1 2

Chart 1. Distribution of silver grains over all human chromosomes. The greatest number of grains occurred at the proximal short arm (p) of chromosome 2.

123456 B

TGFa Fig. 2. Regional mapping of TGF-a on human chromosome 2. Hybrids in Lanes 2 and 4 were derived from Chinese hamster cells and a human donor with a t(1 ;2) (q32;q13) translocation (31). The hybrid in Lane 4 had retained the derivative chromosome with the long arm of human chromosome 2 and does not have the TGF-n restriction fragment. The hybrid in Lane 2 had retained both derivative chromosomes and was positive for the TGF-a band (arrow). Lanes 1, 3, and 6 contain DMA from human controls, and Lane 5 contains DMA from the Chinese hamster parental cell line. Hybrid lanes were loaded with 10 >ig DMA. the presence of multiple genes closely related to the TGF-a sequence that was used as a probe. There was only a single hybridizing band on Southern blots of human DMA. Furthermore after in situ hybridization with the TGF-a probe, only one human chromosomal region was labeled at a level greater than that expected by chance. We found that the locus for human M, 6,000 TGF-a is not syntenic with either EGF (on chromosome 4), to which TGF-a appears to be related, or with the gene for the EGF receptor (on chromosome 7), to which TGF-a binds. Instead we have determined by two independent approaches that TGF-a maps to the short arm of human chromosome 2 (region 2p11-Â»2p13). Chromosome 2 contains other genes involved in growth reg Chart 2. Localization of the gene for M, 6000 TGF-a on human chromosome 2 ulation and/or tumorigenesis. A sequence related to the c-myc as determined by in situ hybridization to normal human mitotic chromosomes (A) and by Southern blot analysis of somatic cell hybrid DNA (B). Arrow, breakpoint of protooncogene (termed N-myc) was isolated from homogene the translocation present in the human hybrid cell donor. ously staining regions situated primarily on the short arm of chromosome 1 in human neuroblastoma lines. This sequence a close relationship between N-myc and TGF-a based on their which was amplified within the homogeneously staining regions location is unlikely since these loci are separated by about 12 (N-myc) was mapped by in situ hybridization to the short arm of chromosome bands at the 850-band stage [possibly 40,000 human chromosome 2 (25) in both normal and neuroblastoma kilobases, assuming 3300 kilobases per band (43)]. cells. A similar N-myc probe was assigned to chromosome 2 and Using species-specific monoclonal antibodies a gene, or localized to region 2p23-Â»2p24 by Schwab ef a/. (26). Although genes, responsible for the detection of human plasma fibronectin some neuroblastoma cells have recently been demonstrated to in tissue culture medium was assigned to chromosome 2 (44, produce TGFs, including EGF-potentiated TGFs, in culture (27), 45). Its intrachromosomal position has not yet been determined.

postulated negative control element that operates in frans (52) allowing overproduction of a normal c-myc message or a shift of transcription to an alternate promotor generating an altered c- myc mRNA. Damage to the normal control element has been postulated to occur either by separation of c-myc from its promotor by the translocation event or by mutation of the promotor by the same mechanism that introduces somatic mutations into variable positions of the immunoglobulin genes in normal B-cells (51, 54). However, despite the detailed knowledge of c-myc and immunoglobulin gene rearrangements in lymphoma cells, the functional role for c-myc has remained elusive, c-myc is known to be inactive in resting cells (51) and is stimulated by mitogens and some growth factors (55-57). N-ras, an active ras protoon cogene, has been found to coexist with an altered c-myc in a BL, and the two genes were shown to be capable of acting synergistically in vitro (58). These results suggested that the c- myc gene product may interact with some growth factors and oncogene products, but not with others, in producing its effects. The expected physical proximity of TGF-a and c-myc genes after a t(2;8) translocation in BL raises the possibility that these two genes may interact in producing their effects, one of which may be to stimulate the growth of B-lymphocytes. A possible role of TGF-a in tumor progression of some cases of BL might be worth further investigation.

ACKNOWLEDGMENTS

We thank Dr. Teresa Yang-Feng for her help with the in situ hybridization.

REFERENCES

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CANCER RESEARCH VOL. 45 NOVEMBER 1985 5597

Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1985 American Association for Cancer Research. Mapping of Transforming Growth Factor α Gene on Human Chromosome 2 Close to the Breakpoint of the Burkitt's Lymphoma t(2;8) Variant Translocation

Jane E. Brissenden, Rik Derynck and Uta Francke

Cancer Res 1985;45:5593-5597.

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