Oncogene (1997) 15, 79 ± 86  1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00

The FHIT is alternatively spliced in normal kidney and renal cell carcinoma

Xudong Luan1, Guangping Shi1, Mahnaz Zohouri1, William Paradee2, David I Smith2, H Jochen Decker3 and Linda A Cannizzaro1

1Department of Pathology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Bronx, New York 10461, USA; 2Department of Internal Medicine, Division of Hematology & Oncology, Wayne State University School of Medicine, 540 E. Can®eld, Detroit, Michigan 48201, USA; 3Department of Medicine, Division of Hematology/Oncology, Johannes Gutenberg University, Langenbeck-Str.1, D 55313 Mainz, Germany

FHIT (Fragile Histidine Triad), a putative tumor As most common cancers, RCC occurs in either a suppressor gene, was cloned from fetal brain and colon sporadic or inherited manner. Histologically, the cDNA libraries. Portions of this gene are deleted in majority (80 ± 85%) of these tumors are of the non- esophageal, colon, lung and breast tumors, but this gene papillary, clear cell variety (Kovacs and Ishikawa, 1993). has not been found altered in sporadic renal cell Clues regarding which may be responsible for the carcinomas. We report here an alternatively spliced initiation and progression of RCC have come from form of this gene cloned from a kidney cDNA library. cytogenetic and molecular anlayses performed on both This cDNA is 1189 bp in length, and contains an tumor types. Consistent loss of alleles has been detected additional 94 bp exon, designated exon 2a (E2a). This most frequently on the short arm of 3 (3p) novel sequence is located between exon 2 and exon 3 of (Kovacs and Hoene, 1988; Kovacs and Frisch, 1989; the FHIT gene's untranslated region and exon 2a is Anglard et al., 1991; Morita et al., 1991). In addition, present in all normal kidney tissues and cell lines. several families have been identi®ed in which members Analyses performed on sporadic renal cell carcinoma with constitutional chromosome rearrangements even- (RCC) tissues and cell lines, show consistent loss of exon tually acquire RCC (Cohen et al., 1979; Pathak et al., 8 of the FHIT cDNA in almost 60% of the cases. 1982; Kovacs and Hoene, 1988, 1989; Li et al., 1993). Interestingly, in a familial, as well as, in a metastatic Cohen et al. (1979) reported one family with ten RCC, derived from a patient with the sporadic form, members who had an apparently balanced exon 2a and exon 3 are also deleted. Northern analyses t(3;8)(p14.2;q24.1) chromosome translocation, and all with the exon 2a of the familial and the metastatic RCC of whom developed RCC in their third decade of life. demonstrates concurrent loss of expression of a 4.4 kb Follow up studies of several of these family members, transcript with the loss of the E2a sequence, suggesting revealed eventual loss of the derivative chromosome 8 that exon 2a of the FHIT gene may play an important containing genetic material from the 3p14 to terminal role in the oncogenesis of renal cell carcinoma. region, as their disease progressed (Li et al., 1993). Comparative hybridization analyses performed on Keywords: FHIT; renal cell carcinoma non-metastatic, non-papillary clear cell RCC's, have revealed that a signi®cant portion of 3p is deleted from the 3p14.2 breakpoint of the 3;8 RCC familial translocation, extending through the terminal region Introduction of 3p, including the Von ± Hippel ± Lindau (VHL) at 3p25 (Moch et al., 1996). A strong association exists Renal cell carcinoma (RCC) comprises about 85% of between the manifestation of VHL, an autosomal all tumors which occur in the kidney (Olson et al., dominant disorder, with the development of RCC, 1984). The incidence of RCC is still increasing since 57% of clear cell RCC's show mutations of the throughout the world at an annual rate of 2%. In VHL gene (Kovacs et al., 1991; Moch et al., 1996). the United States, this malignancy is diagnosed in more In a search for the candidate than 27 000 individuals each year, and is responsible for RCC, the 3p14.2 locus has been intensively studied for more than 11 000 deaths (Boring et al., 1994). The (Yamakawa et al., 1992; Boghosian-Sell and Canniz- etiology has not yet been discerned, however, increased zaro, 1992; LaForgia et al., 1991, 1993; Atchison et al., risk to develop RCC is associated with tobacco use, 1995; Gnarra et al., 1995, Shi and Cannizzaro, 1996). and exposure to environmental carcinogens, such as Even though multiple markers which span this asbestos. Restrospective analyses of patients under- breakpoint region have been isolated, a gene has yet going long term kidney dialysis have uncovered an to be identi®ed in RCC, with altered expression as a increased frequency of RCC among them. Recent risk result of the 3;8 rearrangement. One cDNA clone, assessments estimate these patients have approximately HCRA1, was identi®ed which mapped adjacent to the four times the risk of eventually acquiring RCC than 3;8 translocation, but is not rearranged or expressed in the general population (Fallon et al., 1989). RCC patients (Boldog et al., 1993; Wilke et al., 1994). A second gene, FHIT, has been mapped by hybridiza- tion to markers from within the t(3;8) RCC breakpoint Correspondence: LA Cannizzaro PhD to the 3p14.2 region. This gene is interrupted by the Received 26 December 1996; revised 19 March 1997; accepted 20 translocation breakpoint region, and portions of it are March 1997. deleted in esophageal, colon, lung and breast tumors FHIT alternatively spliced in RCC XLuanet al 80 (Ohta et al., 1996; Sozzi et al., 1996; Negrini et al., 1996). However, the prototypic FHIT gene, isolated from fetal brain and colon cDNA libraries, was not altered in two cell lines and two uncultured renal cell carcinomas established from patients with the sporadic form of RCC (Ohta et al., 1996). In addition, the mutations of FHIT observed so far in multiple tumor types other than kidney, have not revealed any speci®c pattern in association with the type or stage of malignant progression of these tumors. Our laboratory has cloned an alternatively spliced version of the FHIT gene from a kidney cDNA library. This gene contains an additional 94 bp exon not found in colon and fetal brain FHIT cDNA (Ohta et al., 1996). Sequencing analyses of the kidney FHIT reveals that the novel 94 bp sequence maps between exon 2 and exon 3, and is designated as Exon 2a (E2a). Figure 1 FISH analysis showing hybridization of cosmids, 1B7 RT ± PCR analyses performed on normal kidney and 1C12, to cell line 9944, containing the t(3;8)(p14.2;q24.1) RCC translocation. Signal is detected on the normal chromosome tissues and cell lines demonstrates the presence of exon 3, the derivative 3 and, the derivative 8 (arrows) 2a in all of them. RT ± PCR analyses show loss of the exon 2a in a familial RCC cell line which contains a t(3;8)(p14.2;q24.1) translocation, as well as, in a metastatic tumor derived from a patient with the the human kidney cDNA library were PCR ampli®ed sporadic form of RCC. In addition, almost 60% of the using the lambda gt10 forward and reverse primers, RCC sporadic tumors and cell lines, show loss of exon then screened with the biotin labeled cosmids on the 8 from the translated region of the kidney FHIT. streptavidin magnetic particles surface. After three Northern expression studies with an exon 2a probe rounds of selection, the PCR products were cloned detected the loss of a major 4.4 kb transcript in the into a plasmid vector and re-screened with the cosmids. familial RCC and in the metastatic RCC tumor, Forty-four positive clones were selected, and a partial suggesting that the exon 2a sequence may be a cDNA of 334 bp was sequenced. A polymerase chain potential RCC tumor suppressor site which is reaction strategy was employed to obtain the positioned on the 5' side of the familial RCC 3;8 remainder of the full length cDNA. Two primers, C- chromosome translocation breakpoint region. Thus, F and C-R, were derived from the 334 bp cDNA. PCR these studies provide strong evidence that the exon 2a ampli®cations were performed by using these two sequence and the FHIT gene may play an important primers, along with the lambda gt10 forward and role in the oncogenesis of renal cell carcinoma. reverse primers, to screen the human kidney cDNA library. The resulting PCR products were cloned, then, screened with the PCR products of C-F and C-R. A full length cDNA of 1189 bp in size was obtained from Results this screening (Figure 2a). Nucleic acid homology searches of the nonredun- Mapping a cosmid contig to the t(3;8) breakpoint by dant databases were performed using BLAST through ¯uorescence in situ hybridization the National Center for Biotechnology Information Fluorescence in situ hybridizations (FISH) were server. An additional 94 bp segment located between performed on metaphase chromosomes from the RCC the FHIT gene's exon 2 and exon 3 at the centromeric cell line, 9944, established from a family member with 5' end of the cDNA was detected which was not the t(3;8)(p14.2;q24.1) translocation, to determine the homologous to any other known sequence (Figure 2a position of each cosmid of a four cosmid contig: 1B7, and b). This exon was designated as exon 2a (E2a). 4H6, 6H8 and 1C12, in relation to the translocation The Grail and GeneWork 2.4 programs were used to breakpoint. Two of the cosmids, 1B7 and 1C12, which analyse the cDNA's open reading frame (ORF). mapped to the distal and proximal side of the contig, Although the cloned cDNA sequence is 94 bp longer were labeled with biotin by nick translation and used as than the FHIT gene, the predicted ORF is identical to probes. Signals were detected on the short arm of both that of the FHIT gene (Figure 2a) (Ohta et al., 1996). the normal and derivative , as well as, on the long arm of the derivative chromosome 8, which Analysis of the alternative splicing of the FHIT gene in contains the 3p14.2 to the 3p terminal region. No kidney by RT ± PCR signal was detected in the normal chromosome 8 (Figure 1). These results con®rm that 1B7 and 1C12 Total RNAs were prepared from normal kidney and span the 3;8 translocation breakpoint region and map RCC tumor tissues, normal kidney and RCC cell to the 3p14.2 side of the translocation. lines. Initial RT ± PCR analyses were performed with the primers C1-F and C1-R (Figure 3a). The PCR products were puri®ed and then used as a template for Cloning of an alternatively spliced FHIT cDNA from a nested PCR. Primers N-F and N-R were constructed kidney cDNA library to detect mutations in exons 1 through 6 of the This cosmid contig was then used to select cDNAs kidney FHIT gene (Figure 3a). Nested PCR with the from a kidney cDNA library. 16107 recombinants of N-F/N-R primers resulted in a 475 bp product, which FHIT alternatively spliced in RCC XLuanet al 81

Figure 2 Structure of kidney FHIT cDNA. (a) Nucleotide and predicted amino acid sequence of the cDNA. Amino acids are shown in single letter code. The 94 bp E2a sequence is underlined. The primers for PCR are shown above the sequence. (b)A schematic presentation of (2) the kidney FHIT cDNA with the additional 94 bp E2a sequence, and (1) the FHIT cDNA obtained from colon and fetal brain contained the 94 bp segment, and a 381 bp product oligo from the 94 bp sequence was designed as a detected in all normal human kidney tissues and cell probe for Southern analyses of the nested PCR lines (Figure 4a). The 475 bp product, however, was products to determine which portion of the kidney not detected in the metastatic renal cell carcinoma FHIT gene was missing in the 9944 cell line. The tissue, A95-50 (Figure 4a, lane 4) suggesting that the resulting Southern with the E2a probe detected signal 94 bp/exon 2a sequence was deleted in this tumor. in normal kidney tissues and cell lines, but a signal Instead, a smaller product at 327 bp was detected in was not detected in either the metastatic RCC tissue, the metastatic tissue (Figure 4a, lane 4). Cloning and A95-50, or the 9944 cell line (Figure 4c, lane 4 and sequencing of the 327 bp product revealed a deletion lane 6). These results con®rm the loss of the 94 bp/ of exon 2a and exon 3 in the metastatic tumor (Figure E2a segment in both the familial RCC, as well as, the 3b). Nested PCR analyses performed on the 9944 cell metastatic RCC. line with the N-F/N-R primers did not show any Primers N2-F and N2-R were constructed to detect product since it contained the t(3;8) chromosome alterations between exons 6 and 10 of the translated rearrangement (Figure 4a, lane 13). Thus, a 20 bp region of the kidney FHIT (Figure 3a). After nested FHIT alternatively spliced in RCC XLuanet al 82

Figure 3 Kidney FHIT alterations in RCC tissues and cell lines. (a) Locations of the RT ± PCR and nested PCR primers. N-F and N-R (spans exons 1 ± 6), and, primers N2-F and N2-R (spans exons 5 ± 10). (b) RCC tissues and cell lines showing spliced portions of exons located in the translated and untranslated regions of the kidney FHIT cDNA

PCR, a 445 bp PCR product was detected in all 1.5 kb transcript may indicate production of a normal kidney and RCC tumor tissues and cell lines. truncated transcript resulting from the splicing out of In addition, a 376 bp product was detected in the the E2a portion of the kidney FHIT. metastatic RCC, A95-50, the sporadic RCC tissues, Since nested PCR analyses revealed that the 94 bp/ 96150 and 937, as well as, the sporadic RCC cell lines, E2a sequence is deleted in the familial and metastatic ST94, 126, UMRC, RCC-2 and 786-0 (Figure 4b, lanes RCC, one can postulate that production of the 4.4 kb 4, 6, 10, 15, 16, 17 and 18). Cloning and sequencing of transcript is partially a result of the presence of the E2a the 376 bp product revealed a deletion of exon 8 in sequence in the untranslated region of the kidney each of these RCC tumor tissues and cell lines (Figure FHIT cDNA. The deletion of exon 8, on the other 3b). hand, does not appear to alter the expression of either the 4.4 kb or the 1.5 kb transcript. However, the splicing out of exon 8 in the translated region of the Northern blot analysis of the Exon 2a expression kidney FHIT cDNA could produce a frameshift In order to assess the a€ect, if any, these exon deletions mutation shortening the length of the protein sequence had on the expression of the 94 bp+FHIT transcript which could lead to diminished or altered protein in each of these tumors, a 20 bp oligo from the 94 bp activity. segment was used as a probe for Northern blot Additional hybridizations were performed with a analyses to normal kidney tissues and cell lines, as FHIT cDNA which did not contain the E2a sequence, well as, kidney tumor tissues and cell lines. Two to determine which portion of the cDNA was transcripts were detected by Northerns using the E2a responsible for the expression of the two transcripts. probe, a major transcript at 4.4 kb and a minor Results of these hybridizations are shown in Figure 5. transcript at 1.5 kb. The 4.4 kb transcript was Figure 5A.2 demonstrates selective expression of the expressed in normal kidney tissue and cell line, A94-8 1.5 kb transcript in both the normal and RCC kidney and 346, the sporadic RCC tissue, S95-89, and the tissues and cell lines using the kidney FHIT cDNA sporadic RCC cell line, JD1. However, this 4.4 kb without the E2a sequence. Figure 5B.1 is a series of transcript was not expressed in either the familial t(3;8) sporadic RCC tissues and cell lines which demonstrate containing RCC cell line, 9944, or the metastatic RCC selective expression of the 1.5 kb transcript, again tumor tissue, A95-50 (Figure 5A.1, lanes 4 and 6). It using the FHIT cDNA without the E2a sequence. also appears that the expression level of the 1.5 kb Figure 5B.2 shows expression of both transcripts in a transcript is less in the familial and metastatic RCC series of sporadic RCC tissues and cell lines using the than the level detected in the normal and abnormal kidney FHIT with the E2a sequence. This series of kidney tissues and cell lines which retain the E2a Northerns clearly demonstrates that the 4.4 kb sequence. This di€erence in expression level of the transcript contains the E2a sequence, and that the FHIT alternatively spliced in RCC XLuanet al 83 a M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

— 475 — 381 — 327

b M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

— 445 — 376

c 1 2 3 4 5 6

— 475

FHIT cDNA in di€erent kidney tissues and cell lines. (a) Nested PCR products of primers N-F and N-R from normal kidney tissue 1068 (lane 1), sporadic RCC tissue 1068 (lane 2), normal kidney tissue, A95-8 (lane 3), metastatic RCC tissue, A95-50 (lane 4), normal kidney tissue, A95-89 (lane 5), sporadic RCCs 96150, 1000, 1022, 1088 and 937 (lanes 6 ± 10), oncocytomas, 814 and 906 (lanes 11, 12), familial RCC cell line, 9944 with the t(3;8) (lane 13), sporadic RCC cell lines, JD1, ST94 126, UMRC, RCC-2, 786-0, and ST94-138 (lanes 14 ± 19). Loss of the 475 bp PCR product is detected in the metastatic RCC (lane 4). A second PCR product is detected at 327 bp. In the familial RCC cell line, 9944 loss of both PCR products is observed (lane 13). (b) Nested PCR products of primers N2-F and N2-R from the above kidney tissues and cell lines in the same order as (a). An abnormal band is detected in lanes 4, 6, 10, 15, 16, 17 and 18 which correspond to the metastatic RCC tissue, A95-50; sporadic RCC tissues 96150, 937, and sporadic RCC cell lines ST94 126, UMRC, RCC-2 and 786-0, respectively (c). Southern analyses with 20 bp oligo derived from the E2a sequence hybridized to the nested PCR products of primers N-F and N-R. Lane 1: normal kidney cell line, 346; lane 2: sporadic RCC cell line, JD1; lane 3: normal kidney tissue, A95-8; lane 4: metastatic RCC tissue, A95-50; lane 5: sporadic RCC tissue, A95- 89; lane 6: familial RCC cell line, 9944. Signal is not detected in either the metastatic RCC, A95-50, or the familial RCC cell line, 9944, indicating the loss of exon 2a sequence in each

1.5 kb is the major transcript expressed by the FHIT RT ± PCR analyses performed on a series of familial cDNA which does not contain the alternatively spliced and sporadic RCC tumors demonstrate selective E2a sequence. alterations within the kidney FHIT gene. A deletion of the E2a sequence was observed in an RCC cell line derived from a family member with a t(3;8) chromo- Discussion some rearrangement, as well as, in a metastatic RCC tissue taken from a patient with the sporadic form of An alternatively spliced variant of human FHIT cDNA RCC. has been cloned, and has been detected in normal A high rate of exon 8 deletion was also detected by kidney derived tissues, as well as, in sporadic and RT ± PCR analysis in sporadic RCC tumors and cell familial forms of renal cell carcinoma. This alternative lines, occurring in almost 60% of these tumors. form of the FHIT gene contains a novel 94 bp exon, Previous RT ± PCR analyses of lung and breast designated exon 2a (E2a), which is located between tumors have also shown loss of exon 8 in the FHIT exon 2 and exon 3. This is the ®rst such documentation cDNA. However, in these tumors, a deletion of exon 8 of an alternative form of the FHIT gene and this was usually observed in conjunction with deletions of alternatively spliced variant of FHIT has not been one or several other exons from the translated region detected in the prototypic FHIT cDNA isolated from of the prototypic FHIT gene (Ohta et al., 1996; Sozzi fetal brain and colon cDNA libraries (Ohta et al., et al., 1996). Since the FHIT gene spans the common 1996). fragile site region at 3p14.2, it is not surprising that FHIT alternatively spliced in RCC XLuanet al 84 1 2 3 4 5 6 1 2 3 4 5 6

— 4.4 kb

— 1.5 kb

A.1 A.2

1 2 3 4 5 6 1 2 3 4 5 6

— 4.4 kb

— 1.5 kb

B.1 B.2 Figure 5 Expression of the kidney FHIT cDNA by Northern analyses. (A.1) Using an oligo probe containing the E2a sequence, 1.5 kb and 4.4 kb sized transcripts are detected in lane 1, normal kidney cell line 346, lane 2, sporadic RCC cell line JD1, lane 3, normal kidney tissue, A95-8, lane 5, kidney papillary transitional RCC tissue, A95-89. The 4.4 kb transcript is not detected in either lane 4, the metastatic RCC tissue, A95-50, or in lane 6, the familial RCC cell line, 9944. (A.2). Using the FHIT cDNA without the E2a sequence as a probe, only the 1.5 kb transcript is detected in each of the above RCC tissues and cell lines. (B.1). Using the FHIT cDNA without the 94 bp/E2a sequence as probe, the 1.5 kb transcript is the only signal in sporadic RCC cell lines, UMRC, RCC-2, and 786-0 (lanes 1, 2, 3) and sporadic RCC tissues, 1000, 1022 and 1088 (lanes 4, 5 and 6). (B.2). Using the oligo derived from the E2a sequence as probe, strong signal is detected for the 4.4 kb sized transcript in sporadic RCC cell lines, UMRC, RCC-2 and 786-0 (lanes 1 ± 3), and the RCC tissues, 1000, 1022 and 1088 (lanes 4 ± 6). A weak signal is detected for the 1.5 kb transcript in all the RCC cell lines and tissues

deletions of di€erent portions of FHIT have been likely mechanism responsible for regulating the found in multiple tumor types. Due to the composition expression of this large 4.4 kb transcript. Since the of sequences in fragile sites, the DNA in these regions E2a sequence does contain an internal stop codon, loss is considered to be a `hot spot' for mutative events. of E2a may result in premature termination and The discovery of a gene like FHIT which spans a ultimate loss of expression of the 4.4 kb transcript. fragile site, may provide information regarding the Primers have been constructed to the 5' end of the molecular mechanism responsible for the development kidney FHIT and chromosome walking is in progress of oncogenicity and the progression of events which to isolate an upstream promoter sequence which may lead to development of a metastatic lesion. In addition, be responsible for regulating the E2a portion of the this discovery may uncover the signi®cance of fragile kidney FHIT gene. sites in the genome, a topic of controversy over the last We have shown that the kidney FHIT gene is decade. strongly homologous to the colon and fetal FHIT Northern analyses performed with the 94 bp/E2a cDNA, but di€ers signi®cantly due to the alternative sequence as a probe, show that a 4.4 kb transcript splicing represented by the presence of the 94 bp/E2a appears to be the major expression product of the sequence. Even though the predicted protein sequence kidney FHIT cDNA, and that the 94 bp/E2a exon is of the alternatively spliced FHIT is structurally similar contained within this transcript. Further evidence to the prototypic FHIT, it is possible that the protein regarding this is shown by RT ± PCR and Northern produced by the alternative form may di€er in activity. studies of familial and metastatic RCC's, where loss of Interestingly, the splicing out of exon 8 in a large the E2a/94 bp sequence is detected in conjunction with proportion of sporadic tumors does not alter the the loss of expression of the 4.4 kb transcript. A expression of either the 4.4 kb or the 1.5 kb transcript. promoter sequence 5' to the E2a sequence is the most Since exon 8 is within the open reading frame (ORF) FHIT alternatively spliced in RCC XLuanet al 85 of the FHIT gene, its deletion in RCC tumors would 5mMNH4Cl at room temperature for 5 min for four times, shorten the amino acid sequence as a result of a then, blocked with 50 mg denatured human Cot-I DNA frameshift mutation, thereby causing an alteration of (Gibco BRL), 7.5 mg HeaII digested cosmid vector DNA in 750 mM NaCl, 50 mM NaPO ,56Denhardt's reagent, the 94 bp+FHIT proteins' conformation. Conforma- 4 tional analyses must be undertaken to determine how 0.05% SDS, 5 mM EDTA, and 50% formamide. The particles were then washed with 200 ml washing bu€er for the FHIT protein is a€ected by the alternative splicing, 5 min at room temperature and, pelleted with a magnet as well as, any mutations occurring in either the (Promega). 16107 recombinants of a human kidney cDNA untranslated/translated portions of the kidney FHIT. library (Clontech) were ampli®ed by PCR using lambda gt Because of the position of the E2a sequence, it is 10 forward and reverse primers (Sambrook et al., 1989). possible that a gene upstream may function in The PCR products were puri®ed (PCR Puri®cation Kit, regulating the alternatively spliced variant of the Qiagen), and, denatured at 958C for 5 min after adding 1/ FHIT gene. This as-yet-to-be identi®ed gene along 20 volume of 1 M KCl, chilled on ice for 3 min, then with the alternative variant of FHIT may play a hybridized to the biotin labeled cosmid on the magnetic signi®cant role in the development of the transforma- particles at 658C for 14 h. The particles were washed in tion of kidney derived cells which leads to manifesta- 26SSC for 15 min at room temperature, 26SSC for 15 min at 688C, with a ®nal wash in 0.26SSC for 15 min tion of renal cell carcinoma. Functional analyses are at 688C ®ve times. Speci®c cDNAs were eluted from the now underway to determine the role the kidney FHIT particles at 708C for 15 min in 100 mlH2O and ampli®ed by gene plays in kidney development, as well as, to PCR. The PCR products were hybridized another two determine how the E2a sequence contributes to tumor times with the biotin labeled cosmids using the same suppressor activity in renal cell carinoma. conditions. The ®nal PCR ampli®ed speci®c cDNAs were cloned into pBluescript plasmid vector (Stratagene) and sequenced with Sequenase Version 2.0 (USB) according to the manufacturer's instructions. Materials and methods PCR assays

Cosmids, kidney tissues and cell lines Reactions were performed in 10 mM TrisCl, pH 8.3, 50 mM

A cosmid contig of four cosmids, designated 1B7, 4H6, KCl, 1.5 mM MgCl2, 200 mM dNTP mix, 200 nM of each 6H8 and 1C12, which covered the t(3;8) translocation primer, 100 ± 200 ng template DNA and 5 U Taq DNA breakpoint region at 3p14.2, were used to select cDNAs. polymerase in a 100 ml volume. For ampli®cation of human Tissues and cell lines used included: normal kidney tissues, kidney cDNA library and, selected cDNAs, the PCR A95-8 and 1068 (kindly provided by Dr Howard Ratech); conditions were: a 3 min denaturation at 938C followed sporadic renal cell carinoma tissues, A95-89, 937, 96150, by 10 cycles of denaturation at 938C for 45 s, annealing at 1000, 1022 and 1088 (kindly provided by Dr Howard 588C for 1 min, extension at 728C for 3 min, followed by Ratech); oncocytoma tissues, 814 and 906; normal kidney another 10 cycles of denaturation at 938Cfor45s, cell line, 346; sporadic RCC cell lines, ST94 126, UMRC, annealing at 588C for 1 min, extension at 728Cfor4min RCC-2 and 786-0 (kindly provided by Dr Robert Burk); a and, a ®nal 8 min extension at 728C. For ampli®cation of sporadic RCC cell line with a t(3;6) translocation, JD1; a the remainder of the full length cDNA and for nested familial RCC cell line, 9944 with a t(3;8) translocation PCR, the PCR conditions were the same as those used for (kindly provided by Drs Robert Gemmill and Frederick RT ± PCR ampli®cation described below. For ampli®cation Li); and a metastatic RCC tissue, A95-50, established from of the segment between primers C-F and C-R, the PCR a patient with a sporadic RCC. conditions were: a 5 min denaturation at 948C followed by 30 cycles of denaturation at 948C for 30 s, annealing at 568C for 45 s, extension at 728C for 1 min, followed by a FISH ®nal extension at 728C for 8 min. PCR reactions were Fluorescence in situ hybridization (FISH) was performed performed on a Programmable Thermal Control Cycler on metaphase chromosomes obtained from cell line 9944 (MJ Research, Inc). PCR products were analysed by gel with the t(3;8)(p14.2;q24.1) translocation to determine the electrophoresis using 1.5% agarose. PCR products for position of each cosmid in relation to the translocation cloning were puri®ed using PCR puri®cation columns breakpoint. Cosmids were labeled with biotin-14-dATP by (Qiagen). Cloning of PCR products was performed with nick translation (BioNick Labeling System, Gibco BRL) a PCR Cloning Kit (Promega). using chromosome 3 alpha-satellite (D3Z1)(ONCOR) and chromosome 8 alpha-satellite (D8Z2)(ONCOR) to distin- guish the normal and derivative chromosomes (Shi and PCR primers Cannizzaro, 1996). The hybridization signals were cyto- Lambda gt10 forward primer, 5'-AGCAAGTTCAGCCTG- chemically detected with FITC-labeled Avidin (ONCOR) GTTAAG-3'; lambda gt10 reverse primer, 5'-CTTATGAG- and the chromosomes were counterstained with Propidium TATTTCTTCCAGGGTA-3';C-F:5'-TACATCCAGAC- Iodide (PI)(ONCOR). GGTGGAAGG-3';C-R:5'-GGCTTGATGAGATGTT- GG C-3';C1-F:5'-GCTGTCAACATCCTGGAAGC-3'; C1-R: 5'-TCACTGGTTGAAGAATACAGG-3';N-F:5'- Direct cDNA selection, cloning and sequencing ATCCT GGAAGCTTTGAAGCTCA-3';N-R:5'-ATCAG- One mg of each cosmid DNA was labeled with biotin GACGCAGGTCATGG-3';N2-F:5'-AGGACATGTCCT- (BioNick Labeling System, Gibco BRL) and puri®ed with a TGTGT GCC-3';N2-R:5'-TGCTGATTCAGTTCCTCTT- G-50 sephadex column (Boehringer Mannheim). The four GG-3'. The sequence of the 20 bp oligo probe is 5'-CTGT- biotin labeled cosmids were pooled together, denatured at AGCC GCTGAAGAGAGG-3'. 958C for 5 min after adding KCl to 100 mM ®nal concentration, then annealed to the streptavidin magnetic particles (Promega) at room temperature for 1 h. The Southern blot analyses particles were washed with 200 ml washing bu€er contain- Human genomic DNA (Promega) and genomic DNA from ing 100 mM KCl, 1.5 mM MgCl2,10mM TrisHCl pH 8.6, cell lines/tissues were isolated with the Genomic DNA FHIT alternatively spliced in RCC XLuanet al 86 Isolation Kit (CLONTECH). DNAs were digested with RT ± PCR analyses restriction enzymes, separated on a 0.8% agarose gel, then First strand cDNA synthesis was performed at 428Cfor transferred to Hybond N+ nylon membrane (Amersham) 30 min under the following conditions: 50 mM Tris HCl, in 0.4 M NaOH, 1.5 M NaCl. Probes were labeled with pH 8.3, 75 mM KCl, 3 mM MgCl ,10mM DTT, 0.5 mM [32P]dCTP by random priming (Boehringer Mannheim) 2 dNTP mix, 1 mM reverse primer, 2 ± 5 mgtotalRNAand, and, puri®ed with G-50 Sephadex columns (Boehringer 200 units SuperScript II Reverse Transcriptase (Gibco Mannheim). Hybridizations were performed at 658Cin BRL) in a total volume of 20 ml. RNA, reverse primers 0.5 M NaHPO (pH 7.2), 1 mM EDTA, 1% BSA and 7% 4 and, H O were mixed, then heated to 708Cfor5min,and SDS for 16 h. Filters were washed in 26SSC, 0.1% SDS at 2 chilled on ice prior to the addition of the other ingredients. room temperature, then in 0.26SSC, 0.1% SDS at 658C Eight ml of the ®nal mixture was used in 45 mlvolumeof two times for 15 ± 20 min each. PCR. Hot-start PCR was carried out under the following conditions: 1 min denaturing at 948C, 1 min annealing at Isolation of RNA and Northern analyses 558C, 2 min extension at 728C for 35 cycles, and another 10 min extension at 728C. Total RNA was isolated from human kidney tissues and cell lines with TRIzol Reagent (Gibco BRL) (Gramza et al., 1995). Fifteen to 20 mg were denatured in formamide, then, electrophoresed on a 1% 6M formaldehyde agarose gel (MorthernMAX AMBION). After staining with ethidium bromide to visualize the ribosomal RNA bands, the gels were blotted onto nylon membrane in transfer Acknowledgements 32 bu€er. Oligo-probes were labeled with PbyT4 poly- This work was supported by grant VM-89 awarded by The nucleotide kinase (Gibco BRL) and puri®ed with G-50 American Cancer Society (LAC) and by grant De 356/3-2 sephadex columns. Hybridization of the Northern blots awarded by the German Research Council (DFG, were carried out according to the manufacturer's instruc- Deutsche Forschungsgemeinschaft to HJD). The authors tions. wish to thank Dr KH Ramesh for helpful discussions.

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