Proc. NatL Acad. Sci. USA Vol. 79, pp. 7842-7846, December 1982 Genetics

Two human c-one are located on the long arm of 8 (oncogenes/ mapping/cancer/chromosomal translocations/cytogenetics) BENJAMIN G. NEEL*, SURESH C. JHANWARt, R. S. K. CHAGANTIt, AND WILLIAM S. HAYWARDtt *Comell University Medical College, 1300 York Avenue, New York, New York 10021; and tMemorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, New York 10021 Communicated by Paul A. Marks, October 7, 1982

ABSTRACT We have used in situ chromosome hybridization associated chromosomal abnormalities (24). However, whether techniques to map the human cellular counterparts (c-onc genes) these chromosomal rearrangements are causally related to these of the transforming genes of two RNA tumor viruses on human tumors is not known. meiotic pachytene and somatic metaphase . We find We have shown that placing a strong viral transcriptional sig- that the human c-mos geneis located on at a position nal adjacent to a particular avian c-onc gene, c-myc, can cause corresponding to band 8q22 on the somatic map. The human c-myc increased transcription of that gene, resulting in neoplasia (5). gene is found on chromosome 8 at position 8q24. These regions on One possible explanation for the association of specific chro- the long arm of chromosome 8 have been previously reported to mosomal be involved in specific translocations found in the M-2 subset of abnormalities with specific human cancers is that re- acute nonlymphoblastic leukemias, Burkitt lymphoma, and other arrangements might result in the transfer of a c-one gene from forms of non-Hodgkin lymphoma, and a familial abnormality that a region that is transcriptionally silent to a region that is actively predisposes to renal cell carcinoma. These results suggest that transcribed (5, 16, 17, 25). This could result from placement of translocations of the human c-mos or c-myc genes may be causally a strong transcriptional signal adjacent to the c-onc gene. To test related to neoplastic transformation. this hypothesis, it would be important to determine the posi- tions of c-onc genes in the normal human chromosome com- The contains a set of genes (c-one genes) that plement. Recently, techniques have been developed for the are homologous to the transforming genes (v-onc genes) ofavian localization of single-copy genes by in situ molecular hybrid- and mammalian rapidly transforming RNA tumor viruses (1). ization (26-34). We have used a variation of these techniques These genes have been highly conserved throughout vertebrate to determine the chromosomal locations ofthe human c-mos and evolution. Although their precise roles in normal cell metabo- c-myc genes in both meiotic prophase and somatic metaphase lism remain to be elucidated, c-onc genes presumably encode chromosomes. We find that both of these c-onc genes are lo- functions important in the control of cell growth, differentia- cated on the long arm ofchromosome 8 near presumed break- tion, or development. It is clear from studies of both rapidly points for known neoplasia-associated chromosome abnormal- transforming (2) and slowly transforming (3-7) RNA tumor vi- ities. ruses, as well as DNA transfection experiments (8-11), that MATERIALS AND METHODS abnormally high expression of c-onc genes can result in neo- Molecular Probes. The plasmid pHumos was generously plastic transformation ofhost cells.- In addition, several groups supplied by M. Oskarsson and G. Vande Woude (National In- (12-14) have recently shown that several so-called human stitutes ofHealth). This plasmid contains a 2.75-kilobase insert "transforming genes" (genes isolated from human tumor cell consisting of human c-mos and adjacent cellular DNA cloned lines that are capable of inducing morphologic transformation into the EcoRI site ofpBR322. The sequence ofthe c-mos seg- oftissue culture cell lines upon transfection) are actually human ment has been completely determined (35), and it corresponds c-onc genes. It is not yet clear how these genes are involved in closely to that of the murine c-mos gene. human neoplasia, although recent evidence (R. A. Weinberg, The plasmid pv-myc was generously supplied by T. Papas personal communication) indicates that, for at least one ofthese (National Institutes ofHealth). This plasmid contains the entire genes, structural gene alteration, rather than enhanced expres- avian v-myc gene and its properties have been fully described sion, may be involved in conferring the neoplastic phenotype. elsewhere (36-38). For use as probe for in situ hybridization Nevertheless,' alltofthese studies point to an important role for experiments, plasmid DNA was labeled to high specific activity c-onc genes in the pathogenesis of human cancer. by nick-translation (39) with [3H]dTTP (99 Ci/mmol) [3H]dATP In the past 20 years, -much cytogenetic evidence has been (51 Ci/mmol), and 13H]dCTP (44 Ci/mmol) (New England accumulated linking specific chromosome abnormalities, es- Nuclear; 1 Ci = 3.7 X 10'° becquerels). Each 3H-labeled deoxy- pecially translocations, to specific forms of human cancer (15- nucleoside triphosphate was present at a concentration of 20 17). The classic example ofthese abnormalities is the 9,22 trans- pM in a 25-pul reaction mixture containing 60 j.M dGTP, 50 mM location that generates the Philadelphiachromosome associated Tris-HCl (pH 8), 5 mM MgCl2, 10 mM 2-mercaptoethanol, and with chronic myelogenous leukemia (18). Specific chromosome bovine serum albumin at 50 jig/ml, plus 250 ng of plasmid abnormalities have also been associated with Burkitt lymphoma DNA, 50 pg of DNase I, and 5-10 units of DNA polymerase (19-21), acute nonlymphoblastic leukemia (15), salivary tumors I. Incubation was carried out at 14WC for 2 hr, and unpolymer- (22), and small cell carcinoma ofthe lung (23). Recent evidence ized nucleotides were removed by gel filtration over Sephadex suggests that all human hematopoietic malignancies may have G-75. Specific activities of 2-6 X 107 dpm/,4g were routinely obtained. The publication costs ofthis article were defrayed in partby page charge payment. This article must therefore be hereby marked "advertise- Abbreviation: NaCl/Cit, 150 mM NaCl/15 mM sodium citrate. ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. t To whom reprint requests should be addressed. 7842 Downloaded by guest on September 29, 2021 Genetics: Neel et al. Proc. Natl. Acad. Sci. USA 79 (1982) 7843 Chromosome Preparations. Meiotic chromosome prepara- tions were made directly from biopsy samples ofnormal human 4. testes removed from men undergoing orchiectomy as part of -ot,. their treatment for carcinoma ofthe prostate (40). Somatic chro- mosome preparations were made from phytohemagglutinin- stimulated normal human peripheral blood lymphocytes (40). ._:0*e In Situ Hybridization. In situ hybridization of molecular 4_ probes to somatic and meiotic chromosomes was performed by a modification of the technique of Kirsch et aL (33). The chro- t x ~~16% @ mosome preparations were treated with RNase A (Sigma) at 100 8. jug/ml in 2x NaCl/Cit (1x NaCl/Cit = 150 mM NaCl/15 mM sodium citrate) at 370C for 1 hr, rinsed in 2x NaCl/Cit, and dehydrated with ethanol. Chromosomal DNA was denatured by treating slides with 70% (vol/vol) formamide/2x NaCl/Cit, A pH 7.0, at 70'C for 3-5 min, followed by dehydration in qp ethanol. Hybridization reaction mixtures contained 3H-labeled .I p . probe at 50-100 ng/ml in 50% formamide/2x NaCI/Cit/ f .T 0.2% polyvinylpyrrolidone/0.2% Ficoll/0.2% bovine serum S. =) .- albumin/250-300 pug ofyeast carrier RNA per ml/5-10% dex- *1 . tran sulfate. A 30- to 50-1.l aliquot ofhybridization mix was ap- plied to each chromosome preparation and hybridization was carried out at 370C for 12-18 hr in a closed Petri dish saturated II with 50% formamide/2x NaCl/Cit. After hybridization, slides were washed three times for 5 min each in 50% formamide/ ft 2X NaCl/Cit at 39°C, followed by 3 washes (5 min each) in 2X NaCl/Cit at 39°C, and then 15-20 washes in 2X NaCl/Cit at C D room temperature over 3-4 hr. Washed slides were dehydrated in ethanol and air dried. FIG. 1. Autoradiographs of pachytene cells hybridized in situ with Autoradiography. The hybridized and washed slides were pHumos (A andB) and pv-myc (C andD). All chromosomes with grains coated with NTB2 nuclear track emulsion (Kodak) and exposed are identified and grains at sites of specific label are indicated by ar- for 1-4 weeks at 4°C in a light-proof box. Exposed slides were rows. developed in Kodak Dektol developer for 3-5 min, fixed, and rinsed in tap water. chromomere site in these arms was six (four for 9q). This is in Microscopy and Chromosome Identification. Slides were marked contrast to 8q, where most (59/66) were localized in a stained by using the method ofChandler and Yunis (41). All cells discrete region, at the chromomere position corresponding to exhibiting label were photographed, and grain analysis was per- q22 ofthe somatic metaphase banding pattern (Fig. 3A). On the formed by the method ofJhanwar et al. (42). Individual pachy- somatic chromosomes, significantly more than the expected tene bivalents were identified on the basis ofchromomere pat- number of grains were found only on 8q (P ' 0.001), and most terns described by Jhanwar et al (40). These patterns are ofthese (29/33) were in one region, namely, q21-q23. Because comparable to high-resolution metaphase bands (40, 43). So- of the greater contraction of chromosomes and the suboptimal matic chromosomes were identified according to the Interna- quality ofbanding obtained after hybridization, the localization tional System for Human Cytogenetics Nomenclature (44). could not be made more precise on the somatic chromosomes. We conclude that specific hybridization ofpHumos takes place at 8q22. RESULTS Localization of c-myc. Chromosomal localization of the Localization of C-_M8. 3H-Labeled.pHumos was hybridized c-myc gene was determined in a manner identical to that of c- to pachytene and somatic metaphase chromosomes. Silver mos described above, using pv-myc as probe. Twenty-one cells grains were seen on autoradiographs of many cells within 1-6 at pachytene and 25 cells at somatic metaphase were used for weeks. Most of the grains were situated on the chromosomes, the analysis. Representative hybridizations are illustrated in although occasional grains were found scattered elsewhere on Fig. 1 C and D. In total, 100 grains were scored on the pachy- the slides. Representative hybridizations of pachytene chro- tene cells and 250 grains on somatic cells. Their distribution mosomes are illustrated in Fig, 1 A and B. Analysis of grains is shown in Fig. 2B. Grains significantly in excess of the ex- was performed on autoradiographs of 33 well-spread meiotic pected numbers were seen on chromosome arms lp (P 0.01) cells and 15 somatic cells. The criterion for selection of these and 8q (P ' 0.001) at pachytene and lp (P ' 0.001), 1q (P ' cells was that all chromosomes with grains should be identifi- 0.01), and 8q (P < 0.001) at metaphase. On both pachytene and able: this criterion was satisfied for most ofthe cells present in somatic chromosomes, except chromosome 8, the grains were the preparations. In total, 203 grains were scored on the pachy- distributed randomly among the various regions (Fig. 2B), with tene cells and 130grains on the somatic cells. Their distribution a maximum offour grains at pachytene and seven grains at meta-- in, all autosomal regions is shown in Fig. 2A. In pachytene chro- phase at any given chromomere/band site. In the case ofchro- mosomes, among all chromosome arms, 8q and 9q showed a mosome 8, however, essentially all grains (23/25 on the pachy- significantly larger number of grains than would be expected tene bivalents and 59/61 on somatic chromosomes) were at one on the basis ofa random distributiot proportional to the lengths site, namely, the chromomere region on the meiotic chromo- of chromosome arms (P ' 00Onl, 'The grains on 9q, as with some corresponding to band q24, and the region q23-q24 on grains on all chromosome arms with the exception of 8q, were the somatic chromosomes (Fig. 3B). We conclude that specific situated randomly; the maximal number of grains seen on any hybridization of pv-myc takes place at band 8q24. Downloaded by guest on September 29, 2021 P7107844 Genetics: Neel et al. Proc. Natl. Acad. Sci. USA 79 (1982)

70 r 60 F 50 F 40 F 30 V . CS 20 F Pachytene 0 5) ~0 '10h E O z 0 11 .n..fI.fl11|..1X.IW~~~HnnH~.1.i ~~~~f I~~~' n4q.Ip' n5~~~~~~ n p~~qnII- --llll-nnnnLP'JI- ,p-qJ14nHnjp'~~~~jqH n nnn 11n 1ki~l,nn I.|ZAl*en ~:~pJ1ull,nnnnn nnnnnr,pq~qIqFinn IPjqIPI 2 tsq I t 3ll-qI | 4q* &IP| |5q| sI | |6 |qIk S { s~~~~~~~~~~~~~~~~~~~'7 iI salp 8q 9Pg IP10qIPI 11qP 12q >13 P14 15 I16 17 18192w 2 AI ouu1 L JI u-u u U U U u u[]u U~u UtuuU lt 1i10 °FJ2'U U' U U''U U U' '' U U' U' U UUUU'U' LJUJL 'U'UjJU' UUU UU' U'U' UUULUu''U''U'U' 2 sot u ~~~~~~~~~~~~~~~Metaphase 310 0I1rlll11II1AII llI lI&II 11 IIV 1 II&& 1rl AHfiI1, itlmtla1lHU Unn I1 I1 11 1lu l f l1 30 B

I[0 Pachytene

0iinon1nn..ninn * lnn*alnn nnn[1 *-in n.....n* 1n,,fn,,..n.n.n . ,nnln, n nnnnalln-n CDa 2 q I p q p' 4q lpT 5 P6q I 71q1 8 Pq p1q Ip 1P12 13 14 15 16 178

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60 L FIG. 2. Histograms showing autoradiographic grain distribution with pHumos (A) and pv-myc (B) hybridizations. An idiogram (45, 46) of au- tosomes is shown in the middle of each diagram; the bars represent total grains seen in each of the chromosomal regions. Data are shown for both pachytene chromosomes (bars above the idiogram) and somatic chromosomes (below the idiogram). DISCUSSION pseudogenes contain too little myc information to be detected We have used the in situ hybridization technique to determine in our assay. The possibility that the slightly increased-incidence the chromosomal location of the human cellular counterparts ofgrains at lp reflects the presence ofa pseudogene(s) ofc-myc of the transforming genes of two RNA tumor viruses, Moloney cannot be ruled out. Resolution of this question awaits in situ hybridization experiments using clones of the human c-myc murine sarcoma virus (c-mos) and myelocytomatosis virus 29 pseudogenes. (c-myc). We find that both of these c-onc genes are located on Meiotic chromosomes at the pachytene stage- offer special the long arm ofchromosome 8, c-mosat 8q22 and c-myc at 8q24. opportunities for gene mapping. In direct preparations ofsper- Similar results were obtained with either meiotic (pachytene) matocyte or oocyte chromomere spreads, the chromosomes are or somatic chromosomes., Only a single copy of c-mos infor- extended in nature and exhibit easily recognizable chromomere mation is present per haploid human genome (35). Thus we patterns without the need for banding techniques. Maps of hu- would expect only one chromosomal site to be detected by in man autosomal bivalents have shown an excellent correspon- situ hybridization. However, analyses ofthe human c-myc gene dence between chromomere patterns and high resolution so- (47) have shown that in addition to a gene that contains most matic banding patterns (40). Pachytene chromosomes offer of the information present in the v-myc gene, the human ge- several.distinct advantages over somatic chromosomes in map- nome contains several pseudogenes containing smaller amounts ping single-copy genes by in situ hybridization: (i) a possible ofmyc information. We cannot say from our data whether these enhanced chromosomal signal for hybridization due to homol- myc-related genes are all contained at 8q24 or whether the ogous pairing that results in close apposition of allelic gene se- Downloaded by guest on September 29, 2021 Genetics: Neel et d Proc. NatL Acad. Sci. USA 79 (1982) 7845 A immunoglobulin (Ig) genes (33, 34, 55-57). The immunoglob- 8 21 ulin heavy chain genes (14q32) (33) and K light chain genes (2pl2) (34) have been precisely located by in situ hybridization; their locations correspond to the breakpoints in chromosomes C 2 and 14, respectively, involved in the neoplasia-associated translocations with 8q24. One intriguing possibility is that these translocations place the c-myc gene at 8q24 under the control Hq22 ofthe same transcriptional element as the Ig genes. We suspect that the A light chain genes, which are known to be located on $*90_q222_ rh~ chromosome 22 (55-57), will, eventually, be found in the 22q11 8 t(8q;2 lq) region (i.e., the region involved in the 8,22 translocation as- sociated with some forms of Burkitt lymphoma). Because Ig genes are efficiently expressed in B lymphoid cells, such a re- B to c- 14 arrangement might lead constitutively high expression of 8 myc and thus induce neoplasia. As mentioned earlier, a similar situation is found in avian lymphoid leukosis. In these tumors, C which, like Burkitt lymphoma, are B-cell lymphomas, the avian c-myc gene is placed under the control ofa strong transcriptional _61 signal, in this case the long terminal repeat ofan avian leukosis virus provirus (5). The high expression of c-myc presumably *1''t2!t9 # lq32 results in neoplasia. ~~~q24 I __I The recent finding of Lenoir et aL (58) of a correlation be- 8 t (8q;14q) tween the type of Ig light chain expression and the particular translocation [t(2;8) or t(8;22)] in Burkitt lymphoma cell lines FIG. 3. Comparisons of positions of c-mos and c-myc on chromo- supports this interpretation. We have studied the level of some 8 with breakpoints in t(8;21) and t(8;14) are shown in A and B, expression ofthe c-myc gene in a lymphoma cell line containing respectively. In the left of each panel, from left to right are two biva- lents with specific hybridization, a bivalent from an unhybridized the t(8, 14) translocation. Our preliminary results indicate that preparation for comparison, a chromomere idiogram, and a high-res- c-myc expression is higher in these cells than in several other olution somatic band idiogram (40), respectively. C, centromere. Ac- human lymphoid cell lines. However, we have no evidence that tual grain distribution is also noted along the idiograms. In the right this expression is a consequence of the 8,14 translocation. We of each panel, the respective translocations are representedbybanding could detect no expression of c-mos in any of these cell lines. patterns seen at conventional resolution [400-band stage (44)], which isthe level of resolution usually met inpreparationsof neoplastic cells. The c-myc gene may also be involved in otherforms ofhuman In the translocation diagrams, normal chromosomes are shown on the neoplasia. Collins and Groudine (59) and Dalla-Favera et aL (47) outside and the rearranged chromosomes on the inside. Correspon- have shown that the c-myc gene is amplified in the human acute dence between gene positions and breakpoints is indicated by arrows; promyelocytic leukemia-derived line HL-60. The latter group breakpoints on rearranged chromosomes are indicated by arrowheads. has also shown that this amplification is present in the original cells from which HL-60 was derived. The level of c-myc RNA quences in the cell; (ii) the higher resolution of many chro- in HL-60 cells is higher than that found in a number ofhuman mosome regions compared with banded somatic chromosomes; lymphoid and nonlymphoid cell lines (47, 59, 60). and (iii) the elimination of a potential source of confusion in While this manuscript was in preparation, a number of re- mapping due to possible intragenomic movement ofsingle-copy ports of localization of human c-onc genes by the somatic cell genes in somatic cells. hybridization method have appeared. The c-sis gene (simian Our reason for determining the chromosomal location of c- sarcoma virus) was localized to chromosome 22 (61), the c-fes onc genes was to compare their positions with known break- (Snyder-Theilen strain of feline sarcoma virus) to chromosome points for specific chromosomal rearrangements known to be 15 (60), and c-myb (avian myeloblastosis virus) to chromosome associated with neoplasia. Chromosome 8 has long been rec- 6 (62). In addition, in agreement with our results, Prakash et ognized as one of the chromosomes preferentially involved in ad have reported that c-mos is on chromosome 8 (63). It is in- aberrations in neoplastic cells. Trisomy ofthis chromosome has triguing that each of these chromosomes has been implicated been found associated with the blastic phase ofchronic myelog- in neoplasia-associated chromosome abnormalities. enous leukemia, acute nonlymphoblastic leukemia (22), and Somatic cell hybridization methods can, in general, only es- adenocarcinoma of the large intestine (48). Translocations in- tablish synteny, not precise chromosomal localization. The pre- volving the long arm of chromosome 8, the site of both c-mos cise localization of these genes by in situ hybridization, as de- and c-myc, characterize several forms of neoplasia. The region scribed here, should aid in determining the general significance to which we have mapped c-mos (8q22) is involved in a specific of chromosome abnormalities in the genesis of neoplasia. translocation found in the M-2 subset ofacute nonlymphoblastic We thank Anne Manwell and Ahamindra Jain for excellent technical leukemias [t(8,21)(q22;q22)] (49) (Fig. 3A). Translocations in- assistance, Seeta Chaganti for performing the statistical analyses, and volving the 8q24 region, the site ofc-myc, have been associated Lauren O'Connor for help in preparation ofthe manuscript. This work with non-Hodgkin lymphomas, especially of the Burkitt type was supported by Grant CA34502 from the National Institutes ofHealth (19-21, 23) (Fig. 3B) and a familial translocation that predisposes and by the Flora E. Griffin Memorial Fund. B.G.N. is a biomedical to renal cell carcinoma [t(3:8)(p21;q24)] (50). fellow in the Medical Scientist Training Program of the National Insti- In Burkitt lymphoma and its leukemic manifestation L-3 tutes of Health. acute three different translocations lymphoblastic leukemia, 1. Klein, G., ed. (1982) Advances in Viral Oncology: Oncogene involving 8q24 have been described: [t(8,14)(q24;q32), Studies (Raven, New York), Vol. 1. t(8,22)(q24;qll), and t(2,8)(p12;q24)] (23, 51-54). Recent map- 2. Hanafusa, H. (1981) Harvey Lect. 75, 255-275. ping studies have shown that the chromosomes involved in 3. Neel, B. G., Hayward, W. S., Robinson, H. L., Fang, J. & As- translocations with 8q24 (chromosomes 2, 14, and 22) contain trin, S. M. (1981) Cell 23, 323-334. Downloaded by guest on September 29, 2021 7846 Genetics: Neel et al. Proc. Natl. Acad. Sci. USA 79 (1982)

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