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  1. 1. © 2002 Oxford University Press Human Molecular Genetics, 2002, Vol. 11, No. 6 641–649 Disruption of a novel MFS transporter gene, DIRC2, by a familial renal cell carcinoma-associated t(2;3)(q35;q21) Daniëlle Bodmer*, Marc Eleveld, Ellen Kater-Baats, Irene Janssen, Bert Janssen, Marian Weterman, Eric Schoenmakers, Michael Nickerson1, Marston Linehan2, Berton Zbar1 and Ad Geurts van Kessel Department of Human Genetics, University Medical Center Nijmegen, Nijmegen, The Netherlands, 1Laboratory of Immunobiology, National Cancer Institute, Frederick Cancer and Development Center, Frederick, MD 21702 USA and 2Urologic Oncology Section Surgery Branch, National Institute of Health, Bethesda, MD 20892, USA Received October 29, 2001; Revised and Accepted January 16, 2002 DDBJ/EMBL/GenBank accession no. AK027690 Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 Previously, we described a family with a significantly increased predisposition for renal cell cancer co-segregating with a t(2;3)(q35;q21) chromosomal translocation. Several primary tumors of the clear cell type from different family members were analyzed at a molecular level. Loss of the derivative chromosome 3 was consistently found. In addition, different somatic Von Hippel Lindau (VHL) gene mutations were observed in most of the tumors analyzed, even within the same patient. Based on these results a multistep tumorigenesis model was proposed in which (non-disjunctional) loss of the derivative chromosome 3 represents an early event and somatic mutation of the VHL gene represents a late event related to tumor progression. More recently, how- ever, we noted that these two anomalies were absent in at least one early-stage tumor sample that we tested. Similar results were obtained in another family with renal cell cancer and t(3;6)(q12;q15), thus suggesting that another genetic event may precede these two oncogenetic steps. We speculate that deregulation of a gene(s) located at or near the translocation breakpoint may act as such. In order to identify such genes, a detailed physical map encompassing the 3q21 breakpoint region was constructed. Through a subsequent positional cloning effort we found that this breakpoint targets a hitherto unidentified gene, designated DIRC2 (disrupted in renal cancer 2). Computer predictions of the putative DIRC2 protein showed significant homology to different members of the major facilitator superfamily (MFS) of transporters. Based on additional DIRC2 expression and mutation analyses, we propose that the observed gene disruption may result in haplo-insufficiency and, through this mechanism, in the onset of tumor growth. INTRODUCTION the other chromosome 3 translocation-positive RCC families Hereditary renal cell carcinomas (RCCs) of the clear cell type (7,10–12). Based on these results a tumorigenesis model for mostly occur as a consequence of the Von Hippel Lindau the development of familial clear cell RCCs was proposed in (VHL) cancer syndrome in which patients carry a germline which (non-disjunctional) loss of the short arm of chromosome mutation in one allele of the VHL tumor suppressor gene (1). In 3 and somatic mutation of the remaining VHL allele on 3p addition, seven families have thus far been described with represent critical steps (8,11). In one early-stage tumor biopsy constitutional chromosome 3 translocations and hereditary in this family, however, neither der(3) loss nor VHL gene clear cell RCC (2–7). In one of these families, in which a mutations were observed (9). The same was observed in t(2;3)(q35;q21) translocation segregates, five RCC cases have several tumor biopsies in another RCC family with a thus far been identified in three generations (4,8,9). In all t(3;6)(q12;q15) translocation (12). These findings led us to instances the patients appeared to be translocation carriers. assume that the two oncogenetic steps mentioned above may Subsequent molecular studies on eight primary tumors in this be preceded by another genetic event related to tumor initiation family showed loss of the derivative chromosome 3 [der(3)], (9,12). Deregulation of a gene(s) located at or near the trans- containing the short arm of chromosome 3 (3p) in seven of location breakpoint(s) may act as such. them and different VHL mutations in the remaining 3p allele in Previous positional cloning efforts have led to the identification five of them (8,9). Similar observations were made in some of of breakpoint-spanning genes in the familial RCC-associated *To whom correspondence should be addressed. Tel: +31 24 3618847; Fax: +31 24 3540488; Email: d.bodmer@antrg.azn.nl Present address: Marian Weterman, Department of Pathology, Academic Medical Center, Amsterdam, The Netherlands
  2. 2. 642 Human Molecular Genetics, 2002, Vol. 11, No. 6 t(3;8)(p14;q24) (13,14). At the 3p14 breakpoint the FHIT der(2) and der(3)]. In order to confirm the breakpoint-spanning (fragile histidine triad) gene, which coincides with the nature of this YAC, end-fragments were generated by polymerase common fragile site FRA3B, was identified (13). The gene chain reaction (PCR) between a partially degenerate oligo- encodes a hydrolase with, preferably, Ap3A as substrate (15). nucleotide primer (DOP) and the vector primer, and used as Aberrant FHIT transcripts and FHIT genomic lesions were probes on Southern blots containing EcoRI digested DNA frequently observed in a variety of primary tumors and tumor- extracted from translocation carrier-derived somatic cell derived cell lines (13,16–18). Since similar FHIT alterations hybrids (A3KE-11C, A3KE-34B, A3KE-1A and A3KE-1D) were also observed in non-malignant cells (16,19–21), it was (8), independent monochromosomal hybrids containing suggested that fragility of the locus itself might account for the human chromosomes 2 (m2) and 3 (m3), Chinese hamster A3 observed anomalies (20,22). FHIT transfection studies, cells and translocation carrier-derived lymphocytes (7127). however, showed that replacement of FHIT in Fhit-deficient One of the end-fragments (944UL) hybridized to a 5.3 kb cancer cells in nude mice suppresses tumorigenicity, indicating EcoRI fragment in lymphocytes, both cell hybrids containing that FHIT may function as a tumor suppressor (23). As mutant chromosome 3 (Fig. 2B, lanes 7127, m3 and A3KE-1A), and FHIT, lacking enzyme activity, can also suppress tumori- the der(2) containing hybrid (Fig. 2B, lane A3KE-11C, arrow). genicity, it was proposed that another function of this protein, The other end-fragment (944UR) hybridized to a 2.2 kb EcoRI such as the binding to Ap3A or other proteins, might be related fragment in lymphocytes, both cell hybrids containing chromo- to tumor development (23). More recent studies reporting a some 3 (Fig. 2C, lanes 7127, m3 and A3KE-1A), and the reduced or even complete absence of FHIT protein expression der(3) containing hybrid (Fig. 2C, lane A3KE-1D, arrow). Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 in clear cell RCCs (24,25) are in agreement with these latter These results thus independently confirmed the breakpoint- notions. In addition, the increased incidence of carcinogen- spanning nature of this YAC. The breakpoint could be posi- induced tumor formation in Fhit-deficient mice similar to that tioned between the markers WI-5963 and D3S1760 (Fig. 1). observed in the human Muir–Torre tumor syndrome (MTS) These two markers, and two end-fragments of YACs 944h8 (26) supports such a role for FHIT. The chromosome 8 break- and 912f12, were subsequently used as probes to screen a point of the 3;8 translocation also disrupts a gene, TRC8, which human P1-derived artificial chromosome (PAC) library and a shows significant homology to the Drosophila patched gene human chromosome 3-specific cosmid library. Five PACs (14). Interestingly, inactivation of the human homolog of this (832H17, 860G10, 1173P12, 828J14 and 1040F10) and one patched gene (PTCH) leads to excessive growth, indicating cosmid (M1344) were selected. All shared the marker that also this gene most likely functions as a tumor suppressor D3S1760 (Fig. 1). FISH and Southern blot analyses of the (27). These observations underline the notion that deregulation t(2;3)-derived somatic cell hybrid panel revealed that four of of a gene(s) located at or near familial RCC-associated trans- the selected PACs (860G10, 1173P12, 828J14 and 1040F10) location breakpoints may be related to tumor development. and cosmid M1344 mapped distal to the 3q21 breakpoint and More recently, a new gene, DIRC1 (disrupted in renal cancer that PAC 832H17 was breakpoint-spanning (Fig. 1). The end- 1), was identified at the chromosome 2q33 breakpoint of a fragments of this latter PAC were again used as probes to constitutional RCC-related 2;3 translocation (28). The function screen the chromosome 3-specific cosmid library. Two of this gene is unknown. At the chromosome 3 breakpoint of cosmids (L1951 and O156) were isolated, of which L1951 this translocation no genes have yet been identified. mapped proximal and O156 mapped distal to the breakpoint. Here we report the physical mapping and positional cloning Thus, the breakpoint was narrowed down to the region of the t(2;3)(q35;q21) chromosomal breakpoint at 3q21 and the between cosmid L1951 and PAC 860G10. Long range PCR subsequent identification of a breakpoint-spanning gene, between sequences of the respective PAC and cosmid inserts DIRC2, which encodes a novel member of the major facilitator produced a fragment of 15 kb (Fig. 1, LR-15). Southern analysis superfamily (MFS) of transporters. The putative consequences confirmed that LR-15 was indeed breakpoint-spanning and, addi- of this gene disruption for RCC development are discussed. tionally, that the breakpoint coincides with a deletion of ∼4 kb (data not shown). Through database searches two sequenced RESULTS bacterial artificial chromosome (BAC) clones, RP11-90F21 and RP11-67L2 (GenBank accession nos AC011116 and Construction of a 3q21 breakpoint-spanning contig AC078794, respectively), were identified (Fig. 1). Primer sets were selected and, using the t(2;3)-derived somatic cell hybrid Previously, the t(2;3)-associated breakpoints were cytogenetically panel, the breakpoint region could be narrowed down to ∼500 bp assigned to bands 2q35 and 3q21, respectively (4). CEPH yeast on both sites of the deletion (Fig. 1, BP1 and BP2). artificial chromosome (YAC) clones from the 3q21 region were selected and positioned by fluorescence in situ hybridization A novel gene, DIRC2, is disrupted by the 3q21 (FISH) relative to the breakpoint using lymphoblastoid cell translocation breakpoint line-derived metaphase spreads. By doing so, four YACs (957g11, 766d8, 912f12 and 944d9) were mapped proximal to To identify genes at/near the 3q21 breakpoint, two kidney the 3q21 breakpoint and two YACs (790d8 and 827d3) were related cDNA libraries were screened with breakpoint-spanning mapped distal to the 3q21 breakpoint (Fig. 1). In addition, PAC 832H17 and breakpoint-flanking cosmid L1951. Two three overlapping YACs (944h8, 959e4 and 822a12) were cDNAs were isolated, of which the 3′ ends matched with found to span the breakpoint. In Figure 2A a typical FISH experi- several expressed sequence tags (ESTs) from UniGene cluster ment, with YAC 944h8 as a probe, is shown. Besides a signal on Hs.11360. Alignment of the isolated cDNAs with the different the normal chromosome 3, as expected, a split signal is seen at the EST sequences from the database revealed a consensus transcript breakpoint regions on both derivative chromosomes [Fig. 2A, of 1628 bp. Rapid amplification of cDNA ends (RACE) and a
  3. 3. Human Molecular Genetics, 2002, Vol. 11, No. 6 643 Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 Figure 1. Schematic representation of the 3q21 breakpoint region. Markers are shown in the upper part of the figure followed by the respective YAC, PAC and cosmid positions. Breakpoint-spanning YACs, PACs and cosmids are indicated by black bars, whereas the breakpoint-flanking YACs, PACs and cosmids are indicated by gray bars. LR-15 is the breakpoint-spanning genomic fragment that was isolated by long range PCR. The translocation breakpoints that coincide with a 4 kb deletion are indicated by BP1 and BP2 (arrows) on LR-15. The exons 6 and 7 of the DIRC2 gene are also positioned within LR-15. The genes in the breakpoint region, their respective transcriptional directions and relative sizes are shown in the lower part of the figure. modified reverse transcriptase (RT)–PCR, including Betaine revealed the presence of nine exons. The putative exon–intron and dimethyl sulfoxide (DMSO) (29), extended this cDNA boundaries and the sizes of the exons and introns are listed in sequence at the 5′ end with 420 bp (the latter with the primer the Supplementary Material (Table 1). An open reading frame sets 2–6 and 2–7; see Supplementary Material, Fig. 1). Simul- (ORF) of 1434 bp was predicted, of which the putative translation taneously, database searches revealed a human cDNA for the initiation codon ATG is embedded in a strong Kozak hypothetical protein FLJ14784 (GenBank accession no. consensus sequence (30) within the first exon (Supplementary AK027690) which showed extensive homology (97–100% Material, Fig. 2). The GC-rich first exon and 5′-untranslated identity) to our transcript and extended the composite region (5′-UTR) are indicative of a CpG island. The 3′-UTR is consensus sequence with another 58 bp. Alignment of the total 535 bp long and a highly conserved polyadenylation signal 2106 bp cDNA sequence with that of the two sequenced break- sequence (AATAAA) was observed 15 nucleotides upstream point-spanning BACs (RP11-90F21 and RP11-67L2) and other of the poly(A) addition site (Supplementary Material, Fig. 2). overlapping BAC clones in the database, i.e. RP11-396B8, LR-15 contains both the exons 6 and 7 of this gene and the RP11-457E6 and RP11-181G12 (Fig. 1; GenBank accession translocation breakpoint (BP1) was found to map 2 kb down- nos AC010858, AC048348 and AC069581, respectively), stream from exon 7, thereby indicating that this gene is
  4. 4. 644 Human Molecular Genetics, 2002, Vol. 11, No. 6 Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 Figure 2. FISH and Southern blot analyses of breakpoint-spanning YAC 944h8. (A) FISH analysis using YAC 944h8 as a probe (green) on t(2;3)-positive metaphase spreads showing a signal on the normal chromosome 3 and a split signal on both der(2) and der(3) (arrows). The centromere of chromosome 3 is detected in red. (B and C) Southern blot analyses using YAC end-fragments 944UL (B) and 944 UR (C) as probes. 7127 is a t(2;3)-positive lymphocytic cell line, m2 and m3 are independent monochromosomal human/rodent somatic cell hybrids containing a normal chromosome 2 and 3, respectively. A3KE-11C, A3KE-1D, A3KE-34B, and A3KE-1A are t(2;3)(q35;q21)-derived human/hamster somatic cell hybrids containing der(2), der(3), normal chromosome 2 and normal chromo- some 3, respectively. A3 is the hamster control. DNAs were digested with EcoRI. Fragment sizes are indicated in kb. disrupted by the translocation (Fig. 1). These results were 2 and 3. DIRC2 exhibits all these characteristics (Supplementary confirmed by Southern blot analyses using cDNAs corresponding Material, Fig. 2). Moreover, DIRC2 shows homology to an to this gene as probes on somatic cell hybrid and control DNAs unknown transporter in Leishmania major (44% similarity in (data not shown). This gene turned out to be anonymous and, amino acids 278–433; GenPept accession nos AAF34284) and therefore, we have designated it DIRC2. a sodium-dependent inorganic phosphate cotransporter in Arabidopsis thaliana (50% similarity in amino acids 156–215; DIRC2 is a member of the MFS superfamily of GenPept accession no. AAD32766). transporters DIRC2 is expressed in proximal tubular cells of the kidney The putative full-length DIRC2 cDNA contains an ORF of 1434 bp encoding a predicted protein of 478 amino acids To determine whether DIRC2 is expressed in a tissue-specific (Supplementary Material, Fig. 2). Different web-based protein manner, a cDNA probe was hybridized to a northern blot prediction programs defined 12 membrane-spanning domains prepared from eight different normal adult human tissues. and cytoplasmic N- and C-termini within the predicted protein Although DIRC2 expression levels appeared to be low in (Supplementary Material, Fig. 2). Furthermore, one putative general, a 2.2 kb DIRC2 transcript could be identified in most N-glycosylation site, two putative protein kinase C phos- of these tissues, including kidney (Fig. 3A). RT–PCR on RNA phorylation sites, and one putative casein kinase II phosphoryl- extracted from proximal epithelial tubule cell lines of different ation site could be predicted in different hydrophilic domains species (dog, pig and mouse) was performed since clear cell (Supplementary Material, Fig. 2). A proline-rich region was RCCs are thought to develop from this part of the nephron. observed in the large hydrophilic region between the Since different mouse and pig ESTs showed 85–95% membrane-spanning domains 6 and 7. Database searches with homology to the human DIRC2 sequence, respectively, the the predicted amino acid sequence of DIRC2 revealed 100 and primers used were designed from the human sequence. A PCR 98% identity to the human hypothetical protein FLJ14784 product was readily obtained from the mouse cell line using the (GenPept accession no. BAB55300) and a predicated protein first primer set (Fig. 3B, lane 3), whereas the second primer set from Macaca fascilaris (GenPept accession no. BAB17282), yielded PCR products from the pig and dog cell lines (Fig. 3B, respectively. A significant homology in the amino acid region lanes 1 and 2). Inadequate homology of the primer sequences 69–427 (43% similarity) was also found with the human feline to the respective RNAs may explain the observed absence of leukemia virus type C receptor (FLVCR; GenPept accession signals in some of the cell lines. In addition, placenta RNA was no. AAD45243). This receptor has been classified as a member included as a positive control for human DIRC2 expression of the ancient MFS of transporters, which contains carriers that (Fig. 3A, lane 6 and Fig. 3B, lane p). transport small solutes across membranes in response to chemical- osmotic gradients (31,32). The MFS family of proteins is DIRC2 mutation analysis in patients and tumors characterized by the presence of 12 membrane-spanning domains with a large hydrophilic region between the In order to further elucidate the role of DIRC2 in RCC develop- membrane-spanning domains 6 and 7 and a well conserved ment, we performed mutation analyses on tumors isolated from MFS-specific motif between the membrane-spanning domains two affected t(2;3) family members (II:7 and III:13) (4,8,9).
  5. 5. Human Molecular Genetics, 2002, Vol. 11, No. 6 645 Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 Figure 3. Expression of the DIRC2 gene in normal tissues and tumors. (A) Northern blot containing mRNAs extracted from adult human tissues and placenta (Clontech) hybridized with a 1.4 kb DIRC2 cDNA encompassing exons 2–9. Lanes: 1, pancreas; 2, kidney; 3, skeletal muscle; 4, liver; 5, lung; 6, placenta; 7, brain; 8, heart. (B) RT–PCR analyses of mRNAs extracted from proximal renal tubular cell lines from dog (lane 1), pig (lane 2) and mouse (lane 3). Lane p contains human placenta RNA (positive control) and lane mQ contains water (negative control). (C) Northern blot containing RNAs extracted from a t(2;3)-positive EBV cell line (lane 1), a t(2;3)-positive tumor (lane 2) and a sporadic renal clear cell tumor (lane 3), hybridized with the same 1.4 kb DIRC2 cDNA as in (A). Fragment sizes are indicated in kb. PCR products spanning exons 2–8 and the coding sequence of observed in an early stage tumor in this t(2;3)-positive RCC exon 9 were analyzed by direct sequencing. Reliable sequence family (9) and several tumor samples in another t(3;6)-positive data could not be obtained from the first exon due to its GC- RCC family (12), an additional translocation-associated onco- richness. One G→A sequence variant in intron 7 genetic event was suggested. Therefore, we set out to analyze (31 nucleotides upstream from the intron–exon boundary of the t(2;3)-related 3q21 breakpoint in more detail. Through exon 8) was identified in patient III:13, but not in patient II:7 positional cloning of this breakpoint we found that an anonymous (Supplementary Material, Fig. 1). Since patient II:7 is the gene, designated DIRC2, was disrupted. This gene contains father of patient III:13 (4), the G→A sequence variant must be nine exons of which the first exon is GC-rich and, thus, indicative derived from the unaffected mother of III:13 and thus, is of a CpG island. The 3q21 breakpoint maps within intron 7 of unlikely to be linked to RCC development. In addition, this gene and coincides with an ∼4 kb genomic deletion. northern blot analysis of a renal tumor sample obtained from a DIRC2 encodes a predicted protein of 478 amino acids with an t(2;3)-carrier (II:7) (4,8,9) revealed a normal DIRC2 transcript, overall significant degree of homology to the FLVCR and which was also detected in an unrelated sporadic renal tumor various putative transporters which are all members of the sample, but not in the t(2;3)-positive lymphoblastoid cell line MFS superfamily of transporters (31,32). The DIRC2 protein tested (Fig. 3C). No additional aberrant bands were observed has, in accordance to the MFS transporters, 12 membrane- in the translocation-positive tumor. spanning domains and a conserved MFS-specific signature The same G→A sequence variant was also encountered in between the membrane-spanning domains 2 and 3. DIRC2 also two of 10 unrelated sporadic clear cell RCC cell lines and in shows homology to an Arabidopsis thaliana sodium-dependent three of the 42 blood samples derived from RCC patients of 31 inorganic phosphate cotransporter. This latter type of transporter unrelated families. No other sequence variants were found in has been categorized to the anion–cation symporter (ACS) these samples. In addition, RT–PCR and northern blot analyses subfamily of the MFS superfamily (31). The ACS subfamily revealed normal DIRC2 transcripts in all sporadic RCCs that encompasses proteins that are widely distributed in both prokary- we tested (data not shown). otic and eukaryotic organisms. As a consequence of their ancient divergences, most of the ACS subfamily members are distantly related. Only four amino acid residues were found to DISCUSSION be conserved in all ACS family members and, as such, serve as Previous molecular studies in translocation chromosome an ACS signature (31). These residues were also found to be 3-positive RCC families showed that loss of the derivative present in DIRC2 (Supplementary Material, Fig. 2) and thus chromosome containing the 3p arm and somatic mutation of suggest that this protein may act as such a symporter. The high the VHL gene are important steps in the development of these degree of homology (>85%) of the DIRC2 transcript to ESTs and clear cell renal tumors (7–12). As none of these anomalies were transcripts of different eukaryotic species including monkey, pig,
  6. 6. 646 Human Molecular Genetics, 2002, Vol. 11, No. 6 dog and mouse, and the significant degree of homology the database, several matching ESTs were found for this gene (30–45%) of the predicted DIRC2 protein to different and the corresponding cDNA predicts a putative ORF predicted proteins in Leishmania major and Arabidopsis thaliana, encoding a protein of 484 amino acids. Subsequent database indicate that the gene has been strongly conserved throughout searches revealed 73% homology to PASS1 (Fig. 1), a rat evolution. The hydrophilic regions of the predicted DIRC2 protein that was previously found to be associated with the protein contain one glycosylation site and several phosphoryla- small stress protein hsp27 (GenPept accession no. AAD48846) tion sites which may be involved in the interaction of this (36). Small stress or heat shock proteins are thought to play protein with the extracellular and intracellular environments, critical roles in cellular stress responses and to render cells respectively. A proline-rich region was also observed in the stress-resistant. PASS1 is most abundantly expressed in kidney intracellular hydrophilic region between membrane-spanning and testis. In addition, two different PASS1 transcripts were domains 6 and 7. Previous studies have shown that Src encountered in kidney cells and alternative splicing has been homology 3 (SH3) domains from several proteins may recog- suggested as a causative mechanism (36). Another interesting nize these proline-rich (P-x-x-P) motifs as binding sites (33), observation is that PASS1 expression affects the ability of thereby mediating critical protein–protein interactions hsp27 to protect cells against shock. This, and the finding that involved in responses to extracellular signals. The DIRC2 hsp27 levels are elevated in renal tumors as compared to protein may, via this putative protein binding site, also be normal kidney tissues (37,38), turns PASS1 into another inter- involved in such cellular signaling pathways. Transport esting RCC candidate gene. Another gene of interest is systems for organic anions and cations are primarily involved SEMA5B, which is located ∼20 kb distal to DIRC2 exon 9 Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 in the secretion of drugs in renal tubules and, thus, play a critical (GenBank accession no. AK001234) (Fig. 1). Interestingly, role in protecting the organism against their potential toxic two other human semaphorin genes, SEMA3F and SEMA3B, effects (34). Disruption of such a transporter, as is the case in map in one of the so called tumor suppressor regions on the the t(2;3) family, may impose stress conditions onto renal short arm of chromosome 3 (39–42). As such, they have been epithelial cells resulting in dysplasia and/or genomic instability. considered as candidate genes involved in tumor development. Teleologically, such a condition may result in loss of the der(3) SEMA5B contains, besides the Sema domain (pfam01403) a chromosome and, thus, trigger the oncogenetic process. plexin repeat domain (pfam01437) and four thrombospondin According to the previously proposed multi-step model (8), type 1 domains (pfam00090). These latter domains are known subsequent VHL gene mutations may lead to full-blown tumor to bind the protein thrombospondin-1 (TSP-1) to transforming development. growth factor (TGF)β1 (43,44). A role for TGFβ1 in kidney To substantiate a role for DIRC2 in proximal renal tubular cancer is suggested since elevated levels of TGFβ1 have been epithelial cells, various expression studies and mutation analyses observed in patients with renal cell cancer (45,46) and an were performed. Multiple tissue northern blot analysis showed altered TGFβ1 signaling pathway has been observed in kidney that the DIRC2 gene is expressed in several tissues, including tumors (47). The possible involvement of SEMA5B and PASS1 the kidney. Subsequent RT–PCR analyses on RNAs extracted in RCC development through breakpoint related position from epithelial cells derived from the proximal kidney tubule effects remains to be investigated. demonstrated that this gene is also expressed in this part of the Although the translocation breakpoints of the seven RCC kidney. Northern blot analysis of t(2;3)-positive tumor cells families with constitutional chromosome 3 translocations revealed normal DIRC2 transcripts, indicating that the affect different genes, these may affect the same genetic remaining intact chromosome 3 allele is normally transcribed. pathway to RCC development. From these results, however, Since no additional abnormal transcripts were detected, we no obvious overlap in function or genetic pathway could be conclude that truncated and/or fusion transcripts are absent or made between the DIRC2 gene and/or one of its neighboring expressed at very low levels. Subsequent mutation analysis on genes and the other breakpoint genes, such as FHIT, TRC8 or the remaining DIRC2 allele (>80% of the gene) revealed one DIRC1. Finally, it also remains to be investigated whether G→A sequence variant in intron 7. As we could deduce that additional candidate genes are located in the 2q35 breakpoint this variant must be derived from an unaffected family region and whether deregulation of one or more of these genes member, we conclude that it is not disease-linked. The DIRC2 contributes to RCC development. gene was also normally expressed in several sporadic RCCs. Additional mutation analyses of these RCCs and blood samples of a series of unrelated RCC patients revealed no MATERIALS AND METHODS further mutations besides the G→A variant. As yet, mutations in the first exon cannot be excluded and, therefore, this option Patient material and cell lines remains to be tested. The apparent hemizygous state of DIRC2 From different family members, both carriers and non-carriers in t(2;3)-positive tumor cells leads us to suggest that, if disruption of the t(2;3)(q35;q21) translocation, Epstein–Barr virus of the DIRC2 gene contributes to tumor development, it must (EBV)-transformed lymphoblastoid cell lines were established be through a haplo-insufficiency scenario. We are currently (8). From one of these cell lines (7127) somatic cell hybrids investigating this option in further detail. were generated after fusion with thymidine kinase-deficient At this stage it can not be excluded that RCC initiation is Chinese hamster A3 cells. Four hybrids were selected, caused by a position effect, whereby the translocation break- containing either the normal chromosomes 2 or 3 (A3KE-34B point deregulates another gene located upstream or down- and A3KE-1A), the derivative chromosome 2 (A3KE-11C) or the stream from DIRC2 (35). Directly proximal to the first exon of derivative chromosome 3 (A3KE-1D), as described previously DIRC2, and thus ∼80 kb proximal to the translocation break- (8). In addition, we used independent monochromosomal point BP1, the first exon of a predicted gene was identified. In human–rodent somatic cell hybrids containing the normal
  7. 7. Human Molecular Genetics, 2002, Vol. 11, No. 6 647 chromosomes 2 or 3 as the only human constituents (Coriell 94°C, 10 cycles of 15 s at 94°C, 15 s at 64°C (decreasing 1°C Cell Repositories, Camden, NJ). Cell lines derived from the per cycle to 54°C), and 15 min at 68°C, 25 cycles of 15 s at renal proximal tubules included MDCK2 (dog), LLC-PK1 94°C, 15 s at 64°C and 15 min at 68°C, followed by a final (pig) and a conditionally immortalized cell line derived from a elongation step at 72°C for 10 min. transgenic H-2Kb-ts a58 mouse (48). Mutation analyses were performed on genomic DNAs from the renal tumors from the cDNA cloning and sequencing family-members II:7 and III:13, which were both t(2;3)(q35;q21) carriers (4,8,9). Additionally, mutation analyses were For the search of genes in the breakpoint region, two cDNA performed on DNAs isolated from 10 sporadic RCC-derived libraries derived from normal human kidney tissue and human cell lines of the clear cell subtype and blood samples collected kidney tumor tissue that were previously constructed in our from 42 patients in 31 unrelated families carrying renal tumors laboratory (52) were used. These cDNA libraries were with various histologies, including renal oncocytomas, unusual screened with genomic YAC and PAC clones. Subsequent papillary RCCs without germline MET mutations, and clear sequence analyses were performed using a Ready Reaction cell RCCs without germline VHL mutations. All human Dye Terminator Cycle sequencing kit (Perkin-Elmer) and run samples used for this study were obtained with informed on a ABI 3700 automated sequencer (Applied Biosystems). consent. BlastN searches against nr, dbEST, month and HTGS databases were performed at the NCBI server (http://www.ncbi.nlm.nih.gov/) Construction of physical YAC/PAC/BAC/cosmid map (53). Sequences of the breakpoint-spanning gene were assem- Downloaded from http://hmg.oxfordjournals.org by on July 26, 2010 bled with those obtained from the EST database using the YAC DNA isolations were performed using Zymolyase Staden Genome Assembly Package (54) accessed through the (Seikagaku). All YAC inserts were characterized using pulsed Nijmegen Center of Molecular and Biomolecular Informatics field gel electrophoresis (CHEF-DR III, Biorad). FISH analysis (www.cmbi.nl). was performed on metaphase spreads from t(2;3)-positive To extend the 5′ end of the DIRC2 cDNA, 5′ RACE was EBV cell lines essentially as described previously (12). For the performed with the Marathon cDNA amplification kit (Clontech) isolation of YAC end-fragments, a DOP-vector PCR protocol using cDNA prepared from human kidney. The cDNAs were was used (49) employing the partially degenerate primer 6-MW, amplified following the supplier’s manual using gene-specific the left YAC vector primer UL and the right YAC vector primers (GSPs) 782-GSP3 and 754-GSP3N (also see Supplementary primer UR. For nested PCR we used the left internal vector Material, Table 2 and Fig. 1). For this PCR reaction the AdvanTage primer LS2 and the right internal vector primer RA2. Primer cDNA PCR kit (Clontech) was used. sequences are given in the Supplementary Material (Table 2). The DOP-vector PCR protocol was also used to isolate end- To extend the GC-rich first exon, RT–PCR was performed fragments from PACs. The pCYPAC2 specific primers were with extra Betaine and DMSO again on the Marathon Ready used as described by Wu et al. (49). A gridded total human human kidney cDNA (Clontech) with different primers corre- PAC library (RPCI-5; BAC_PAC Resources, Roswell Park sponding to the 5′ genomic region (primers 1–4) and cDNA Cancer Institute, Buffalo, NY) (50) and a gridded chromosome specific primers corresponding to exons 2 and 3 (primers 5–7). 3-specific cosmid library (RessourcenZentrum/PrimärDatenbank, The sequences of these primers and their positions within the RZPD, Berlin) (51) were used for the selection of PACs and DIRC2 gene are shown in the Supplementary Material (Table 2 cosmids. DNA isolations from the isolated PACs and cosmids and Fig. 1, respectively). PCR reactions were carried out in 25 µl were performed using a plasmid purification kit (Qiagen). For reaction mixtures with 0.5 ng cDNA, 1× buffer (Gibco BRL), Southern blot analysis, genomic DNA was isolated from the 1.5 mM MgCl2 (Gibco BRL), 0.2 mM of each dNTP, 0.2 pmol different cell lines via standard SDS/proteinase K lysis, of each primer, 0.5 U Taq polymerase (Gibco BRL), 1 M Betaine phenol-chloroform extractions and ethanol precipitations. (Fluka) and 1.3% DMSO (Sigma) under standard conditions at After size-selection on agarose gels this DNA was blotted onto an annealing temperature of 58°C (29). Genescreen Plus membranes (Dupont). Probes were labeled by random priming with [α-32P]dCTP and, after pre-annealing Protein analysis with hybridime DNA (HT Biotechnology) for 5 h, hybridized EST predictions and BlastP searches against the nr, swissprot to the blots overnight at 65°C in 0.5 M sodium phosphate and month databases were performed at the NCBI server (54). buffer, 1 mM EDTA, 7% SDS. Subsequently, the blots were Transmembrane segments were identified by five different washed stepwise starting with 100 mM sodium phosphate, prediction programs: DAS at Stockholm University (http:// 0.1% SDS, which was decreased to 40 mM, and 0.1% SDS. www.sbc.su.se/~miklos/DAS/), TMHMM at the Technical Finally, the blots were exposed to X-ray films at –80°C using University of Danmark (http://www.cbs.dtu.dk/services/ intensifying screens. TMHMM-1.0/), PHD-htm at European Molecular Biology Laboratory (EMBL) (http://www.embl-heidelberg.de/predict- Long range PCR protein/), Tmpred at Swiss Institute for Experimental Long range PCR between cosmid L1951 and PAC 860G10 Cancer Research (ISREC) (http://ulrec3.unil.ch/software/ was performed using the primers LT3A-FOR and 860Sp6-REV TMPRED_form.html) and SOSUI at Tokyo University (http:// (Supplementary Material, Table 2). PAC 832H17 was used as sosui.proteome.bio.tuat.ac.jp/sosuiframe0.html). Protein motifs in a template. PCR was carried out in a mixture containing 100 ng the predicted amino acid sequence were identified by DNA, 1× ExTaq buffer (Takara), 0.2 mM of each dNTP, 0.2 pmol PROSITE at the Expert Protein Analysis System (ExPASy) of each primer, and 2.5 U ExTaq (Takara) in a final volume of server of the Swiss Institute of Bioinformatics (SIB) (http:// 50 µl. PCR cycles included an initial denaturation for 3 min at ca.expasy.org/tools/scnpsit1.html).
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Gemmill,R.M., West,J.D., Boldog,F., Tanake,N., Robinson,L.J., Bokhoven, H.Brunner, O.Madsen (UMC Nijmegen, The Smith,D.I., Li,F. and Drabkin,H.A. (1998) The hereditary renal cell carcinoma 3;8 translocation fuses FHIT to a patched-related gene, TRC8. Netherlands), M.Saier (UCSD, La Jolla, CA), L.Schmidt Proc. Natl Acad. Sci. USA, 95, 9572–9577. (NCI, Frederick, MD), G.Vriend and J.Leunissen (CMBI, 15. Barnes,L.D., Garrison,P.N., Siprashvili,Z., Guranowski,A., Nijmegen, The Netherlands) for advice and support. A.van der Robinson,A.K., Ingram,S.W., Croce,C.M. and Huebner,K. (1996) Meijden (Bosch Medicentrum, Den Bosch, The Netherlands), Fhit, a putative tumor suppressor in humans, is a dinucleoside 5′,5′′-P1, E.Oosterwijk and J.Schalken (UMC Nijmegen, The Netherlands) Pn-triphosphate hydrolase. Biochemistry, 35, 11529–11535. 16. Druck,T., Hadaczek,P., Fu,T.-B., Ohta,M., Siprashvili,Z., Baffa,R., are acknowledged for providing tumor material, R.Gemmill Negrini,M., Kastury,K., Veronese,M.L., Rosen,D. et al. (1997) Structure and H.Drabkin (UCHSC, Denver, CO) for 3q21 specific and expression of the human FHIT gene in normal and tumor cells. clones, I.Konings and C.van Os (UMC Nijmegen, The Nether- Cancer Res., 57, 504–512. lands) for the MDCK2 cell line, J.Petrijs (UMC Nijmegen, The 17. Baffa,R., Veronese,M.L., Santoro,R., Mandes,B., Palazzo,J.P., Rugge,M., Netherlands) for the LLC-PK1 cell line, and J.van Groningen Santoro,E., Croce,C.M. and Huebner,K. (1998) Loss of FHIT expression in gastric carcinoma. Cancer Res., 58, 4708–4714. (UMC Nijmegen, The Netherlands) for the mouse proximal 18. Sorio,C., Baron,A., Orlandini,S., Zamboni,G., Pederzoli,P., Huebner,K. kidney tubule cell line. This work was supported by the Dutch and Scarpa,A. 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