Positions of Chromosome 3Pl'l2 Fragile Sites (FRA3B) Within the FHIT Gene'

(CANCERRESEARCH57.1166-1 170, March15. 1997) Positions of Chromosome 3pl'L2 Fragile Sites (FRA3B) within the FHIT Gene'

Drazen B. Zimonjic, Teresa Druck, Masataka Ohta,2 Kumar Kastury,3 Carlo M. Croce, Nicholas C. Popescu, and Kay Huebner4 Laboratory of Experinwntal Carcinogenesis. Division of Basic Sciences, National Cancer Institute, Bethesda, Maryland 20892 (D. B. 1, N. C. P.!; Kimmel Cancer institute, JeffersonMedicalCollege.ThomasJeffersonUniversity.Philadelphia,Pennsylvania19107fT. D..M.0.. K.K., C.M.C.,K.H.J

ABSTRACT constitutive familial clear-cell renal carcinoma-associated transloca tion t(3;8)(pl4.2;q24) (1 1, 12) has been found to be indistinguishable The FHIT gene spans approximately 1 Mb of DNA at chromosome from the most frequent of the common fragile sites, FRA3B (13). band 3p14.2, which includes the famffial renal ceHcarcinoma chromosome Nevertheless, evidence implicating fragile sites in the process of translocation breakpoint (between FHIT exons 3 and 4), the most Ire neoplastic development has been circumstantial. It has indeed been quently expressed human constitutive chromosomal fragile site (FRA3B, telomeric to the t(3;8) translocation), and numerous homozygous deletions argued that it is very unlikely that breaks in a fragile site should be in various human cancers, frequently involving FHIT exon 5. The FRA3B associated with cancer (14). has previously been shown to represent more than one specific site, and The constitutive ape-inducible fragile site at chromosome band some specific representatives of FRA3B breaks have been shown to fall in 3pl4.2 (FRA3B) is of special interest, because it is the most com two regions, which we know to be in FHIT introns 4 and intron 5. Because monly expressed in humans (15), and the region 3pi4.2 is involved in breakage and integration of exogenous DNA in this chromosome region is deletions in a variety of histologically different cancers (16â€”18),the frequent in aphidicolin-treated somatic cell hybrids, cancer cells, and, t(3;8) translocation in familial RCC, and Kaposi's sarcomas (19). In presumably, aphidicolin-treated normal lymphocytes that exhibit gaps or addition, consistent with previous cytogenetic observations linking breaks, we determined by one- and two color fluorescence in situ hybrid viral integration sites and fragile sites (8, 20), the first molecular ization, using cosmids covering specific regions of the FHIT gene, that evidence for HPV integration in FRA3B was provided recently (21). most of the aphidicolin-induced gaps at FRA3B fall within the FHIT gene, with the higjtest frequency ofgaps falling in intron 5 ofthe FHIT gene, less The positions of some ape-induced gaps and breaks in FRA3B have than 30 kb telomeric to FHIT exon 5. Gaps also occur in intron 4, where been determined by isolation of YAC clones encompassing the breaks a human papillomavirus 16 integration site has been localized, and in (22â€”25)and mapping ape-induced hybrid breaks (21, 26) or plasmid intron 3, where the t(3;8) break point is located. These results suggest that integration sites (27, 28) to regions of the YAC map. the cancer-specific deletions, which frequently involve introns 4 and 5, We have recently isolated the FHJT gene, which encompasses originated through breaks in fragile sites. perhaps 1 Mb, straddling the t(3;8) break, the FRA3B, and cancer cell-specific homozygous deletions (29), and have suggested that the INTRODUCTION FHIT gene may be the target of FRA3B-induced deletions in cancer cells. The FlIT gene is ex@pressedin all tissues thus far examined Chromosomal fragile sites are regions susceptible to breakage (29), including normal lymphocytes (30) and ape-induced lympho under specific experimental conditions. Antifolates, fluorodeoxyuri cytes, in which the normal-sized FHJT transcript was observed (data dine plus methotrexate, and ape,5 a DNA polymerase inhibitor (1), not shown). induce the expression of fragile sites during metaphase as nonstaining To determine the relationship of the FHJT gene to cancer cell gaps or breaks, usually involving both chromatids (for review, see deletions and FRA3B breaks and gaps, we have determined the posi Ref. 2). The latest compendium comprises 103 fragile sites classified tions of FHlTexons relative to the translocation, viral integration site, as common and rare, constitutive and inherited, that have been local cancer cell deletions, and FRA3B breaks described previously, and ized in all human chromosomes except chromosomes 21 and Y (3). have then used FHIT cosmid probes to determine the positions of Although fragile sites are dispersed throughout the genome, they tend specific FHJT exons relative to the ape-induced gaps and breaks in to cluster at G-light chromosome bands (4) that are known to contain human lymphocyte metaphase chromosomes. GC-nch Alu repeats and are constitutionally more relaxed and un folded during transcription (5). The location of fragile sites has been correlated cytogenetically with locations of recurrent translocation MATERIALS AND METHODS and deletion breakpoints in cancer cells, as well as the integration sites of oncogenic viruses (6â€”9).Moreover, structural rearrangements in Localization of STSs by PCR. Primer pair sequences for amplification of duced by mutagens or carcinogens through fragile sites can be cyto STSs were obtained from the Genome Data Base or were designed from genetically indistinguishable from those in cancer cells (10), and the published sequences in GenBank; the numbers beginning with U are GenBank accession numbers of the sequences. U39799 and U39804 primer pairs are Received I 1126/96;accepted 1/13/97. listed in Druck et a!. (30). Other newly designed primer pairs are: U39793F The costs of publication of this article were defrayed in part by the payment of page (5'-TGGATTGAmCACACC-3') and U39793R (5'-GCCAGAGG charges. This article must therefore be hereby marked advertisement in accordance with GAGA ri-i-i- iC-3') at bp 104 and 265 of the U39793 sequence, respectively; 18 U.S.C. Section 1734 solely to indicate this fact. U061 l8F (5'-CGUGTAGTFCTGA'ITFGCA-3') and U06l l8R (5'- I This work was supported partially by United States Public Health Service Grants CA51083, CA39860, and CA21124, and a gift from R. R. M. Carpenter Ill and Mary K. GGATA1T1'GTCACTGCC1TF-3') at nucleotides 20 and 330, respectively; Carpenter. U60203F (5'-GTGCTCAGAGATFACATGC-3' and U60203R (5'-GTATGT 2 Present address: Banyu Tsukuba Research Institute, Okubo 3, Tsukuba 300-33 Japan. CAGCCAAACACC-3') at positions 786 and 1069, respectively; and cSB1AF Phone: 81-298-77-2000; Fax: 81-298-77-2024. (5'-CAAAGCAGAAATAAAACTGC-3') and c5B 1AR (5'-AlTFCCAA L1 Present address: Department of Endocrinology. Charles R. Drew University of Medical Science, Room 3069, 162 1 E. I20th Street, MPO2, Los Angeles, CA 90059. CAAGCFGCCA-3'), which amplify a â€”2l8-bpproduct containing a TA Phone:(213)563-5959. dinucleotide repeat (locus name D3S4490; GenBank accession no. U76285). 4 To whom requests for reprints should be addressed, at Kimmel Cancer Institute, Each primer pair was used in a PCR reaction with template DNA containing Room 1008, Jefferson Medical College, Thomas Jefferson University, 233 South 10th some portion of the FHIT genomic locus. PCR amplifications were carried out Street, Philadelphia, PA 19107. Phone: (215) 503-4656; Fax: (215) 923-4498. in 12.5-pJ reaction volumes with 10 ng of cloned genomic DNA or 100 ng of 5 The abbreviations used are: ape. aphidicolin; HPV, human papillomavirus; YAC, yeast artificial chromosome; STS, sequence-tagged site; BAC, bacterial artificial chro total genomic DNA, 20 ng of each primer, 10 mMTris-HCI (pH 8.3), 50 mr@i mosome; RCC, renal cell carcinoma; FISH, fluorescence in situ hybridization. KC1,0.1 mg/rn)gelatin, 1.5mMMgCl2,0.2 mMeachdeoxynucleotidetriphos 1166

Telomere Centromere 4- pSVZn.o HPV16 -@

Q)0)Q) 5'PTPRG @ F:::I 118k; @ I II@I@ C,) LI) @ + @+J!@ U â€˜I, I II I II

@8E8@ll14â€”@â€”14-c06#7c05#9c05#163 E4 El.-4j..cX3â€”O-'cX2E3E2 .Iâ€”c84 .â€”ll-cX4 â€”II-cXl c19-20 â€” @ â€”c76 â€”c36 BAC

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Fig. 1. Map of the Fllfflocus. The chromosome 3pl4.2 region (â€”1Mb) is illustrated with approximate placement of the cosmid and BAC clones relative to each other. Positions of relevant STSs were determined by PCR amplification with cosmid and BAC DNA templates. Localization of the telomeric side of the lung carcinoma deletion (U60203), the pSV2neo plasmid integrations, the c13break, the HPVI6 integration site, the t(3;8) break, as well as 5'-PTPRG are shown (arrows). D3S4260 and IA6G/H primers are derived from the cosmid 1A6, to which 10 fragile site breaks in hybrid cells have been mapped (37).

phate, and 0.5 units of Taq polymerase. Amplifications were for 30 cycles of quences. The positions of the U39793, U39804, and U39799 markers, 94Â°Cfor30 s, 57Â°Cfor30 s, and 72Â°Cfor30 s. Products were analyzed by which represent the HPV16 integration site (21), were deduced by electrophoresis in ethidium bromide-stained 1.5%agarose gels. Presence of an amplification of appropriate fragments from the BAC clone (shown in amplification product determined localization of a STS to a particular genomic Fig. 1) and from knowledge of their positions relative to the nearby clone. Miul site and other markers that we mapped within the exon 4 Isolation of Genomic Clones. New cosmid clones were isolated as de scribed previously (29), and BAC clones were identified by PCR amplification cosmid. The hybrid cl3 break, representing our 3pl4.2 hybrid de novo of specific STS fragments from pooled BAC library template DNAs (Research break (35, 36), had previously been placed telomeric to FHJT exon 5 Genetics, Huntsville, AL); individual BAC clones were then ordered from based on Southern blot data (29), but we now know the break falls in Research Genetics, single colonies for specific STSs were identified and intron 4, as shown here. The U60203 marker falls in cosmid cP4 in grown, and end sequences were determined, as for cosmids. intron 5, based on amplification of a fragment from this template. This FISH Analysis. Normal peripheral lymphocytes and lymphoblastoid cells marker represents the telomeric side of a homozygous deletion ob derived from a member of the RCC family with the t(3;8) translocation were served in a lung carcinoma.6 Human sequences U40597 and U40401, cultivated in RPM! 1640 supplemented with 15% fetal bovine serum, penicil representing portions of plasmid integration flanking sites (27, 28) lin-streptomycin (50 mg/mI), and 0.2 m@i glutamine. The expression of FRA3B were mapped within 4 kb telomenc of FHIT exon 5 and in cosmids was induced by ape (0.2 p.M;Sigma Chemical Co., St. Louis, MO) dissolved in DMSO (31), or by combined ape and ethanol (0.2%) during the last 26 h of c36 and c76, respectively, by sequence identity; sequence U061l8, culture (32). Metaphase chromosomes were obtained after a 1-h colcemid (50 another plasmid flanking sequence, was mapped to cosmid c36; see ILg/ml) treatment by standard KC1 hypotonic incubation and acetic acid Fig. 1 for positions of each of these sequences in intron 5. Addition methanol fixation. Six previously described cosmid probes (25, 29) [19-20 ally, numerous tumor-specific deletion end points fall within introns 4 (representing the PTPRG 5' end, as shown in Fig. 1), cX3 (including FHJT and 5 (30), and seven hybrid breakpoints identified by Paradee et a!. exon 3), cX4 (exon 4), c76 (exon 5), c36, and c63 (intron 5)] labeled with (37) are in intron 5. The relationship of these various chromosome biotin or digoxigenin, as well as chromosome 3 and 8 painting probes (Oncor, landmarks to each other and to the FH!T gene is summarized in Gaithersburg, MD) were used for in situ hybridization. The conditions for Fig.1. FISH, detection ofthe signal, and digital imaging were carried out as described Position of Exons Relative to FRA3B Gaps. Metaphase spreads previously (33, 34). prepared from ape-treated peripheral lymphocytes derived from two normal donors were hybridized with the six cosmid probes; 19-20, RESULTS cX3, cX4, c76, c36, and c63 probes that span the t(3;8) and FRA3B The FHIT/FRA3B Map. We have previously described a YAC sites (25, 29); see Fig. 1 for a map of the region. To obtain a sufficient and partial cosmid contig for the FlIT locus, which extends from number of metaphases with informative signals at FRA3B, two or more than 300 kb centromeric to the t(3;8) break to an estimated 700 three slides were hybridized with each cosmid probe. Only symmet kb telomeric to the t(3;8) (25, 29). A map of the FHITlocus is shown rical signals were included in statistical analysis, although, due to the in Fig. 1, on which we have placed a number of relevant markers and chromatid displacement at expressed fragile sites, single fluorescent the cosmid probes and the position of the FH!T exons; the relative spots were observed frequently. The location of the hybridization distances of the FHIT exons 1, 2, and 3 relative to each other and the signal was assigned as medial (crossing the gap or break), proximal 5' end of the PTPRG gene is not known, nor is the position of c5BlA relative to exon 1 known. The positions of markers IA6G/H and 6 T. 0. Ong, K. Fong, S. Bader, J. Minus, M. Le Beau, T. McKeithan, and F. Rassool. D3S4260shownbetweenexons4and5(Fig.1,E4andES,respec Precise localization of the common fragile site at 3pl4.2 to the putative tumor suppressor gene FHJT and characterization of homozygous deletions in tumor cell lines within tively) were deduced by PCR amplification from our YAC, BAC, and FRA3B affecting FHIT transcription, unpublished, obtained from GenBank (accession cosmid templates, with pairs of primers derived from published se number U6O203). 1167

the fragile region and were retained on chromosome 3 in metaphases derived from lymphocytes with the t(3;8) translocation. The remain ing probes were found translocated to chromosome 8 in the t(3;8) lymphocytes. Among these, the signals produced with the probes c36 and c63 (both in FHJT intron 5) were located distal to the gaps, whereas probes cX4 (exon 4) and c76 (exon 5) hybridized proximal to the gaps in some metaphases but distal to the gaps in other met aphases, with different frequencies, as summarized in Table 1; exam pies are shown in Fig. 2, a, b, d, and e. Experiments with two-color detection of digoxigenin and biotin label were also carried out to distinguish the placement of pairs of the probes relative to the FRA3B gaps. The combination of two probes hybridized to individual met aphase slides revealed distinct separation of the doublets to sites proximal and distal to the gaps with paired probes cl9-20 and c63 (see Fig. 2c for an example) or cX3 and c36. Probe cX4 and c76 hybrid a b ization signals were not separable in cohybridized metaphase or prometaphase spreads. Signal at the median site (overlapping the gap) was observed with all cosmids, but the incidence varied significantly among the probes, ranging from 7.14% for probe 19-20 to 33.34% for probe c76 (see Table 1 for summary). The data presented in Table 1 are represented graphically in Fig. 3. The difference between the percentages at proximal and distal sites was plotted as an absolute value for each probe (Fig. 3, line), together with the values representing percentages of signals at the median site (Fig. 3, columns). Thus, probes 19-20 and cX3, and probes c36 and c63 flank, proximally and distally, respectively, the ape-induced gaps or breaks at FRA3B. On the other hand, probe cX4 and particularly c76 contain sequences crossing the fragile sites, and the signal gen crated with these probes could fall on either side of the breakpoint at FRA3B. This analysis delineates the FRA3B to a region of a few hundred kb between FHIT exon 3 (cosmid cX3) and FHJT intron 5 (c36), with the majority of gaps occurring in introns 4 and 5, flanking the first FHIT protein-coding exon.

C DISCUSSION By FISH analysis of chromosomes derived from normal lympho cytes treated with ape, we localized the majority of chromosome gaps, collectively known as FRA3B, to a region telomeric to the breakpoint of the t(3;8) translocation in RCC, encompassing the region involved in deletions in tumor cell lines and bordered distally by a portion of intron 5 of the recently identified FH!T gene (29). In fact, the majority of gaps map just to either side of FHJT exon 5, the exon that is most frequently lost in cancer cells with homozygous deletions in the FHJT gene (30). Rare folate-sensitive fragile sites that have been cloned (FRAXA, FRAXF, FRAJ6A, and FR@411B)involve unstable expansion of CCG trinucleotide repeats (38â€”42),and their expression is mediated by an excessive copy number of CCG repeats. Regions of 3pl4.2 thus far tested do not contain CCG repeats (21, 28), suggesting that the distinction between constitutive and heritable fragile sites is not only d e in inducing agent and frequency of expression within the population, Fig. 2. FISH localization of cosmid probes at FRA3B on metaphase chromosomes but also in molecular structure and the mechanisms mediating their derived from ape-treated normal peripheral lymphocytes. Partial and complete metaphase expression. spreads with fluorescent hybridization signal located proximal (a; probe cX3), distal (d; probe c36), and on both sides (c; probes c19-20 red, rhodamine, and c63 green; FITC Several groups of investigators have made important contributions detection) on the FRA3B. 4',6'-diamidino-2-phenylindole-generated chromosome band to our current understanding of the fragile region at 3pl4.2. A group ing (b and e) of the labeled spreads permits an accurate localization of the fluorescent signal relative to the gaps and breaks at FRA3B. of investigators studying FRA3B-associated break points induced in hamster-human hybrids (21, 22, 37) has most recently shown that two clusters of aphidicolin induced hybrid break points, both telomeric to (centromeric), and distal (telomenc) to the gaps or constrictions the t(3;8) 3pl4.2 break, are separated by more than 200 kb and flank marking the fragile sites, in a minimum of 60 metaphases with an Miul site estimated to be â€”185kb from t(3;8). We have mapped 4',6'-diamidino-2-phenylindole enhanced G-like banding (Fig. 2; Ta this M!ul site within intron 4, perhaps â€”100kb centromeric to exon ble 1). Probes 19-20 and cX3 hybridized predominantly proximal to 5 (see Fig. 1); thus, the two hybrid break point clusters flank FH!T 1168

FRA3BProbeNaN Table 1Distribution (percentage) offluorescent signals generated by cosmid probes spanning the

D)19-208478 (%) P N (%) D N (%) M(P - (7.14)92.86cX36248 (92.86) 0(0.00) 6 (19.35)73.59cX48860 (77.42) 2 (3.83) 12 (31.82)68.18c766614(21.21) (68.18) 0 (0.00) 28 22(33.34)24.24c36760 30(45.45) (15.79)84.21c63 (0.00) 64 (84.21) 12 (0.00) 60 (83.33) 12 (16.66) a Number of metaphase72 spreads examined0; P. proximal; D, distal; M, medial; P â€”D, percentage proximal minus percentage distal in absolute numbers.83.33

exon 5. In fact, the cluster of fragile sites represented by 10 hybrid and Rassool et a!. (28) have also performed FISH analyses using breaks in the proximal cluster map very close to FHJT exon 4, as probes from regions near their identified fragile sites, and their results shown in Fig. 1 by the position of markers D3S4260 and IA6GIH. We are largely in agreement with ours. Interestingly, c76 and cX4 cos were also able to determine that the HPV16 integration site (21, 37) mids, containing exons 5 and 4, respectively, can be proximal or distal falls in FHJT intron 4 as shown in Fig. 1; integration of HPV16 in this to the gaps or can overlap the gaps, as summarized in Fig. 3. Most site resulted in a large deletion (21) in the cervical carcinoma FlIT gaps occur within cosmid c76, the exon 5-containing cosmid, mirror locus, which included exon 5. Interestingly, our own cl3 human ing the findings in tumor cells (30), but a number of gaps fall within hamster hybrid with a de novo break in 3pl4.2 (35, 36) also falls in the exon 4 cosmid and even in the exon 3 cosmid, two cosmids that intron 4, probably not far telomeric to the HPV16 integration site and flank the t(3;8) break, suggesting that the t(3;8) translocation, as well between the two hybrid breakpoint clusters defined by Paradee et a!. as the loss and rearrangement of FHIT exons 3 and 4 observed in (37). cancer cells (30), could have derived from breaks in sequences that are A second group of investigators has been isolating DNA sequences â€œfragile,â€•althoughthe actual fragile sequences, the bases for breaks from FRA3B by cloning plasmid integration sites (27) and sequencing and gaps in this region, have not yet been identified. and mapping the flanking regions (28). 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Drazen B. Zimonjic, Teresa Druck, Masataka Ohta, et al.

Cancer Res 1997;57:1166-1170.

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