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Oncogene (1999) 18, 1529 ± 1535 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Alternate choice of initiation codon produces a biologically active product of the von Hippel Lindau gene with tumor suppressor activity Catherine Blankenship1,4, Joseph G Naglich2, Jean M Whaley3, Bernd Seizinger1 and Nikolai Kley*,1 1 Department of Functional Genomics, Genome Therapeutics Corporation, 100 Beaver Street, Waltham, Massachusetts 02154, USA; 2Department of Oncology, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08540, USA; 3 Department of Metabolic Diseases, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08540 USA The VHL tumor suppressor gene has previously been reported to encode a protein of 213 amino acid residues. Here we report the identi®cation of a second major VHL gene product with an apparent molecular weight of 18 kD, pVHL18, which appears to arise from alternate translation initiation at a second AUG codon (codon 54) within the VHL open reading frame. In vitro and in vivo studies indicate that the internal codon in the VHL mRNA is necessary and sucient for production of pVHL18. pVHL18 can bind to elongin B, elongin C, and Hs-CUL2. When reintroduced into renal carcinoma cells that lack a wild-type VHL allele, pVHL18 suppresses basal levels of VEGF expression, restores hypoxiainducibility of VEGF expression, and inhibits tumor formation in nude mice. These data strongly support the existence of two distinct VHL gene products in VHL tumor suppression. Keywords: pVHL; VEGF; elongins; Hs-CUL2 Introduction Inactivation or loss of the VHL gene predisposes aected individuals to various neoplasias associated with von Hippel Lindau disease (Latif et al., 1993), characteristically retinal angiomas, hemangioblastomas of the cerebellum and spinal cord, pheochromocytomas, and renal cell carcinomas (Zbar, 1995, Maher and Kaelin, 1997). In addition, somatic inactivation of the VHL gene has been detected in sporadic renal cell carcinomas and sporadic hemangioblastomas (Zbar, 1995; Gnarra et al., 1994; Shuin et al., 1994; Whaley et al., 1994; Kanno et al., 1994; Herman et al., 1994). Most notably, inactivation of the VHL gene by mutation or hypermethylation has been detected in approximately 80% of sporadic clear cell renal carcinomas. Restoration of wild-type VHL function in VHL mutant renal carcinoma cell lines inhibits their ability to form tumors in nude mice (Iliopoulos et al., 1995). These ®ndings attribute the VHL gene a major role in the development of the most common form of human kidney cancers. *Correspondence: N Kley 4 Current address: Department of Molecular Biology, Princeton University, Princeton, NJ 08540, USA Received 13 July 1998; revised 25 September 1998; accepted 6 October 1998 Inhibition of hypoxia-inducible mRNAs has been reported as an important mechanism associated with pVHL tumor suppression (Ilioupoulos et al., 1996; Gnarra et al., 1996; Siemeister et al., 1996; Mukhopadhay et al., 1997). Thus, pVHL inhibits expression of mRNAs encoding vascular endothelial growth factor (VEGF), mitogenic factors such as PDGF-B and TGFa (Knebelmann et al., 1998), and the GLUT1 glucose transporter. VHL associated neoplasms are highly vascular and have been shown to overproduce VEGF (Wizigmann-Voos et al., 1995). Thus, regulation of VEGF by pVHL is likely to represent a critical component of its tumor suppressor activity. Recently, a role for pVHL in assembly of an extracellular ®bronectin matrix has been proposed, a second mechanism whereby pVHL could in¯uence tumor neovascularization (Ohh et al., 1998). Previous studies have shown that the VHL gene encodes a protein of 213 residues with an apparent molecular weight of 30 kD, pVHL30 (Latif et al., 1993; Iliopoulos et al., 1995). pVHL30 localizes primarily to the cytoplasm but under certain experimental conditions may shuttle between the cytosol and the nucleus (Iliopoulos et al., 1995; Los et al., 1996; Lee et al., 1996; Corless et al., 1997). Although the deduced amino acid sequence of the predicted VHL protein has provided no clue to its function, functional studies have recently provided insights into biochemical properties of the VHL protein. pVHL has been shown to physically interact with elongin subunits B and C of the elongin SIII complex (Duan et al., 1995a; Kibel et al., 1995), a heterotrimeric protein complex that regulates transcription elongation in vitro. Whether elongin B and C regulate transcription elongation in vivo, however, remains to be determined. Certain, but not all, tumor-derived mutants of pVHL are defective in binding to the B/C complex (Duan et al., 1995b; Kishida et al., 1995; Kibel et al., 1995). In addition, wild-type pVHL appears to interact with the human Hs-CUL2 protein (Pause et al., 1997; Lonergan et al., 1998), a member of a conserved gene family believed to play a role in ubiquitin-mediated protein degradation and growth control in lower eukaryotes. Binding of pVHL to HS-CUL2 appears to be dependent on binding of pVHL to elongin B/C (Lonergan et al., 1998), suggesting that the pVHL/B/C/Hs-CUL2 complex may aect ubiquitination of target proteins regulating tumorigenic growth. Biochemical studies have shown that interaction of pVHL with elongin B, elongin C and Hs-CUL2 require an intact C-terminus of pVHL (Kibel et al., 1995; pVHL isoforms and tumor suppression C Blankenship et al 1530 Lonergan et al., 1998; Kishida et al., 1995). The role of the characteristic acidic N-terminus of pVHL, containing eight repeats of the Gly-X-Glu-Glu-X motif, in tumor suppression is so far unclear. Studies comparing the mouse and human VHL genes predicted a mouse pVHL protein that only retains one of eight acidic GlyX-Glu-Glu-X motifs present at the N-terminus of pVHL30 (Latif et al., 1993; Gao et al., 1995; Duan et al., 1995b). All known VHL mutations map downstream of this region and a conserved AUG codon (codon 54) in the VHL open reading frame (Zbar, 1995; Maher and Kaelin, 1997). Interestingly, this conserved AUG codon appears in the context of a more conserved Kozak consensus sequence. Taken together with previous observations that anti-VHL antibodies speci®cally detected multiple proteins in addition to pVHL30 (Iliopoulos et al., 1995; Kibel et al., 1995), these observations led us to investigate whether the VHL gene encodes protein products in addition to pVHL30, and whether protein products could arise from internal translation initiation at the conserved AUG codon within the VHL mRNA. Here we show that the VHL gene encodes a second major and biologically active protein, pVHL18, which arises from translation initiation at an internal AUG codon (codon 54) and can inhibit tumor formation in nude mice. Results Previous studies have shown that the VHL gene encodes a protein product of 213 amino acid residues, pVHL, with apparent molecular weight of 30 kD (pVHL30). A hypothetical VHL protein arising from internal initiation at codon 54 of the predicted VHL open reading frame, has a predicted molecular weight of approximately 17 kD and more hydrophobic characteristics. To test whether detergent-assisted extraction would reveal such a protein by immunoblot and immunoprecipitation analysis, tTA-786-0 VHL7/7 renal carcinoma cells were transfected with tetOPplasmids encoding pVHL (1 ± 213), pVHL (54 ± 213) or control plasmid, and selected for stable and tetracycline-inducible expression of VHL transgenes (TG cell lines), as described (Gossen and Bujard, 1992). Figure 1a shows a representative immunoblot analysis of VHL proteins expressed in the presence or absence of tetracycline, using the previously described anti-VHL polyclonal antibody pABVHLr1 (Gao et al., 1995). Analysis of TG1-AR, harboring a VHL transgene encoding pVHL 1 ± 213 (and thus the acidic repeat region, AR), revealed at least two major immunoreactive proteins of approximately 25 kD (corresponding to the previously reported 30 kD pVHL protein) and 18 kD. For TG2 and TG3, encoding pVHL (54 ± 213), a comigrating 18 kD protein was detected in each case (indicating that the faster migrating protein detected in TG1-AR lysates may have arisen by internal translation initiation). As previously reported (Gao et al., 1995), no immunoreactive proteins were detected with preimmune serum (data not shown). Importantly, no immunoreactive proteins were detected in the presence of tetracycline, which eectively inhibited transgene expression by interfering with the tTA transcription activator expressed in these cells, or parental 786-0 carcinoma cells. Thus, NP40 eectively facilitates extraction of both pVHL30 and pVHL18 proteins. When native VHL products from VHL+/+ 293 human embryonic kidney were analysed using the same protocol, speci®c comigrating proteins of 25 and 18 kD (predominant form) were detected (Figure 1a, lane 1 or 1c, lane 1). Similar observations were made upon analysis of native proteins from normal human diploid ®broblasts (FB), Hela cells, and monkey COS7 cells (COS) (Figure 1b). Detection of the 25 kD protein was somewhat variable. The 5' region of the native VHL mRNA has a high GC content (*80%), predicting an extensive secondary structure possibly interfering with eective use of the ®rst translation start codon. To test the possibility that the 18 kD protein detected in transgenic TG1-AR cells or 293 cells arose from internal translation initiation, stably transfected sublines of TG1-AR were established with plasmids in which the second ATG in the VHL open reading frame was mutated to ATT. As shown in Figure 1c, a 25 kD but no 18 kD pVHL protein was detected in three distinct cell lines carrying such mutated transgenes. Similar results were obtained by analysis of in vitro-translated proteins (not shown). Thus, pVHL18 is not a breakdown product of pVHL25. To further con®rm the identity of the native 18 kD pVHL protein detected in 293 cells and other human cell types, 293 cells or Hela cells were metabolically labeled with 35S-methionine and lysates immunoprecipitated with anti-VHL monoclonal antibody Figure 1 Identi®cation of cellular pVHL18 protein. (a) Immunoblot analysis of VHL proteins expressed in parental or clonal VHL7/7 RCC-786-0 cells carrying VHL transgenes encoding VHL(1 ± 213) (TG1AR); VHL(54 ± 213) (TG2 and TG3); or human VHL+/+293 cells. Transgene expression was regulated by addition of tetracycline (1 mg/ml) to the cell culture medium as indicated. (b) Immunoblot analysis of VHL proteins detected in various cells (COS: SV40 T antigen-expressing COS7 monkey kidney cells. FB: normal WI38 diploid human ®broblasts. Hela: human HPV-transformed cervical carcinoma cells. (c) Eect of mutation of the second AUG codon in the VHL open reading frame on expression of pVHL18 in clonal 786-0 cells (TG*4 ± 6AR) as compared to TG1AR cells. Immunoblot analysis was performed using polyclonal antibody pAbVHLr1. Mobility of the MW markers (in kilodaltons) is indicated in the right margin pVHL isoforms and tumor suppression C Blankenship et al IG32 (Kibel et al., 1995). Immune complexes were resolved by SDS ± polyacrylamide gel electrophoresis and detected by autoradiography. As shown in Figure 2a, IG32 immunoprecipitates an 18 kD protein from VHL+/+ 293 cells and Hela cells, as compared to VHL7/7 786-0 renal carcinoma cells. As expected, this protein migrated faster than the HA-tagged VHL(54 ± 213) protein expressed in TG8 clonal cells. Similar results were obtained by immunoprecipitation using anti-VHL polyclonal antibody. No immunoreactive 18 kD bands were detected with control antibody. IP-Western analysis with IG32 and polyclonal antibody con®rmed immunoreactivity of the 18 kD proteins (not shown). Identity of the 18 kD protein as pVHL18 was further analysed by comparing peptide maps of native 18 kD pVHL and in vitro-translated pVHL18 (54 ± 213). Proteins were metabolically labeled in vivo or in vitro, immunoprecipitated, comigrating 18 kD proteins excised from the gel and digested with Staph. aureus V8 protease. The partial proteolytic peptide maps of the two proteins were identical (Figure a 2b). Taken together, these results suggest that the endogenous 18 kD protein identi®ed in human cells (e.g. 293 cells) is produced by alternate translation initiation and encodes residues 54 ± 213 of the conceptual VHL open reading frame. To study the tumor suppressor function of pVHL18, we determined its eect on renal carcinoma cell growth. Extensive analysis of the in vitro growth properties of stably transfected 786-0 cells showed that expression of pVHL18 has no signi®cant eect on the growth of these cells in monolayers (not shown). However, pronounced inhibition of their tumorigenic growth was observed when assayed in nude mice. In total, 81 mice were injected with either control cell lines tranfected with control plasmid (cTG lines) or plasmids expressing pVHL25, pVHL18 or pVHL18 fused to the HA-tag (hemaglutinin tag). Expression of VHL proteins in all cell lines was con®rmed by Western blot analysis (not shown). As previously observed for pVHL30 (9) or pVHL25 (Table 1), pVHL18 eectively suppressed tumor formation as compared to control cells (Table 1). Thus, pVHL18 encodes the necessary domains for tumor suppression. pVHL30 has been shown previously to inhibit expression of proteins encoded by hypoxia-inducible mRNAs (10 ± 12), such as vascular endothelial growth factor (VEGF). Figure 3 shows that pVHL18 equally inhibits VEGF mRNA expression. Both untagged and HA-tagged forms of pVHL18 eectively suppressed basal VEGF expression in those cell lines previously tested in the nude mouse assay. Although pVHL18 reduces the overall levels of VEGF mRNA, it does not appear to inhibit induction of VEGF mRNA by the phorbol ester TPA (Figure 3a). These results are consistent with previous ®ndings that pVHL30 inhibits VEGF mRNA at the post-transcriptional level, whereas TPA induces VEGF mRNA at the transcriptional level. The eect of pVHL18 on VEGF b Table 1 pVHL18 inhibits tumorigenic growth in nude mice Clone Figure 2 Endogenous pVHL18 peptide map. (a) Immunoprecipitation of 35S-methionine-labeled endogenous pVHL18 from 293 and Hela cells, HA-tagged VHL(54 ± 213) from clonal 786-0 cells (TG8), and associated proteins. Proteins were immunoprecipitated using monoclonal antibody IG32 or IgG. Arrows in the left margin indicate the locations of pVHL18 and pVHL25. The mobility of the molecular weight markers (in kilodaltons) is indicated in right margins. B and C, elongins B and C. (b) Endoproteinase Glu-C (V8 protease) digestion of 293 cell endogenous 35S-labeled pVHL18 and in vitro translated (IVT) pVHL (54 ± 213). Excised proteins were digested with 0, 0.02 or 0.2 mg of protease and resolved by 15% SDS ± PAGE T/M Approximate median tumor weight (mg) Day 45 Day 60 Control cell lines c151-B 10/10 cTG-1 6/6 cTG-2 8/8 cTG-3 8/8 cTG-4 6/6 cTG-5 6/6 726 188 198 196 847 663 1614 538 725 1311 NA NA VHL-transgenic TG-1 (AR) TG-2 TG-3 TG-7 (HA) TG-8 (HA) TG-9 (HA) TG-10 (HA) 35 520 520 0 0 0 0 66 520 520 0 0 0 0 lines 2/5 2/5 4/5 0/6 0/6 0/6 0/6 Typically, 107 RCC-786-0 cells were implanted at a single site on each mouse and tumors were measured at the indicated times (see Materials and methods). Cells: 151-B: parental RCC-786-0 cell line expressing the tTA transgene: cTG1-5: clonal RCC-786-0/151-B cells transfected with control mutant VHL transgene (see Materials and methods); TG1(AR): clonal RCC-786-0/151-B cells expressing pVHL(1 ± 213). TG2, TG3: clonal RCC-786-0/151-B cells expressing pVHL(54 ± 213). TG7-10: clonal RCC-786-0/151-B cells expressing HA tagged VHL(54 ± 213). T/M: tumors/mice 1531 pVHL isoforms and tumor suppression C Blankenship et al 1532 Figure 3 Inhibition of VEGF mRNA expression by wild-type pVHL18 compared to tumor-derived mutant pVHL18 proteins. (a) pVHL18 inhibits basal but not phorbol ester TPA-mediated induction of VEGF mRNA. Total RNA (5 mg/lane) isolated from each of the indicated transgene-expressing cell lines was analysed by Northern blot using a human VEGF or GAPDH probes. Lanes 1 ± 6 contain total RNA from cell lines expressing untagged or tagged wt VHL proteins (lane 1:TG1AR, lane 2: TG2, lane 3:TG3, lanes 4 ± 6: TG 7 ± 9). Lanes 7 ± 10 contain total RNA from control cell lines transfected with mutant VHL transgenes (TG1-4, respectively; see Materials and methods and Table 1). Cell lines were maintained in the absence of tetracycline and phorbol ester TPA (upper panel a) or presence of TPA (100 ng/ ml, 8 h) (lower panel b) prior to harvesting RNA. (b) Tetracycline regulated expression of VEGF mRNA by tumor-derived mutant of pVHL18 (VHLR167Q). Total RNA (*5 mg/lane) isolated from each of the indicated transgene-expressing cell lines grown in the presence (+) or absence (7) of 1 mg/ml tetracycline was analysed by Northern blot using a human VEGF, GAPDH or VHL cDNA probes, as indicated. mTG11 and mTG12 are clonal RCC-786-0/151-B cells expressing a mutant form of HA-VHL (54 ± 213). Mutation is at codon 167 (R167Q) of the published VHL ORF. (c) pVHL18 restores hypoxia-inducibility of VEGF mRNA. Total RNA (*5 mg/lane) was isolated from VHL transgene-expressing cell lines grown in normoxic (N) or hypoxic expression appears to be very pronounced. In multiple experiments we failed to consistently observe tetracycline regulated VEGF expression in response to pVHL18 (or pVHL25) expression in the presence or absence of tetracycline, despite the apparent eective regulation of the expression of the VHL transgene (Figure 3b). Although these ®ndings initially raised concerns with regard to the physiological role of pVHL in VEGF mRNA regulation, subsequent analysis showed that very low level expression of pVHL could be detected even in the presence of tetracycline (not shown). Thus, leaky pVHL expression is presumably sucient to downregulate VEGF mRNA. In contrast, regulated VEGF mRNA expression was observed with the naturally occurring mutant pVHL18-R167Q (Figure 3b). Thus, this mutant pVHL18 protein is compromised in function as compared to wild-type, but retains partial activity that can be detected in overexpression assays. In this context it should be noted that certain pVHL mutants, including R167W, retain partial elongin binding activity (Kibel et al., 1995). Taken together, these ®ndings indicate that pVHL18 is likely to inhibit tumor formation, at least in part, by inhibiting VEGF expression. Furthermore, expression of pVHL18 (and pVHL-R167Q) restored induction of VEGF mRNA by hypoxia (Figure 3c), indicating that pVHL18 shares this function with pVHL25 (which corresponds to the previously reported isoform pVHL30). pVHL30 has been shown previously to bind to at least four cellular proteins, elongin B, elongin C, HsCUL2, and ®bronectin (Duan et al., 1995a; Kibel et al., 1995; Pause et al., 1997; Lonergan et al., 1998; Ohh et al., 1998). To test whether pVHL18 retains similar functions, 786-0 transgenic subclones transfected expressing pVHL25 or pVHL18 were metabolically labeled with 35S-methionine, and lysates immunoprecipitated with IG32 monoclonal antibody. Bound proteins were resolved by SDS ± polyacrylamide gel electrophoresis and visualized by autoradiography. As previously reported, pVHL25 speci®cally coimmunoprecipitates with elongin B, elongin C and an 80 kD protein, p80, presumably Hs-CUL2. Similarly, pVHL18 bound to all three of these proteins (Figure 4a). A p80 protein was also detected in immunoprecipitates from 293 cells and Hela cells (Figure 2a), indicating that p80, as previously shown, interacts with endogenous VHL proteins. To con®rm the identity of p80, parallel large scale experiments were performed with pVHL18 (HA) expressing 786-0 cells (TG8) and vector transfected control cells (cTG2). Lysates were immunoprecipitated with anti-HA monoclonal antibody, and bound proteins resolved by gradient gel electrophoresis. As observed with metabolically labeled proteins (Figure 4a,b), p80 was speci®cally immunoprecipitated from TG8 vs control cells, as visualized by copper staining. Proteolytic digestion of p80 and mass spectrometric analysis of peptides revealed four (H) conditions, as indicated (see Materials and methods). Northern blots were hybridized with VEGF and GAPDH cDNA probes pVHL isoforms and tumor suppression C Blankenship et al a labeled p80 (Figure 4c). These results demonstrate that pVHL18 indeed binds to cellular Hs-CUL2. Discussion Figure 4 Binding of pVHL18 to cellular proteins. (a) Clonal 7860/151-B cells expressing VHL (54 ± 213) (lane 1) or VHL(1 ± 213) (lane 2) proteins, or control cells lines (cTG2, cTG3), were metabolically labeled with 35S-methionine, lysed and immunoprecipitated with anti-VHL IG32 monoclonal antibody. Immune complexes were resolved by 4 ± 20% SDS ± PAGE. The mobility of the molecular weight markers (in kilodaltons) is indicated in left margin. Speci®c pVHL co-immunoprecipitating proteins are indicated: elongin B and C (B/C) and p80. (b) TG8 clonal cells expressing HA tagged VHL(54 ± 213) protein, or control cTG2 cells, were used for large scale immunoprecipitation experiments for puri®cation and peptide sequencing of p80. As shown, four peptides with sequences corresponding to the published Hs-CUL2 sequence were identi®ed. In vitro translated Hs-CUL2 is also shown to comigrate in SDS ± PAGE with 35S-labeled p80 protein immunoprecipitated as a HA-pVHL/ p80 complex with anti-HA monoclonal antibody 12CA5. (c) V8-protease map of p80. Digestion of 35S-labeled endogenous p80 from TG8 cells and in vitro translated 35S-labeled Hs-CUL2 with endoproteinase Glu-C (V8 protease) revealed identical peptide maps. Proteins were digested with 0, 0.02 or 0.2 mg of protease and resolved by 15% SDS ± PAGE peptides with sequences identical to regions of the predicted Hs-CUL2 protein. In vitro-translated 35SHsCUL2 comigrated with metabolically labeled p80 in SDS ± PAGE (Figure 4b). Furthermore, identical V8 protease peptide maps were obtained for in vitrotranslated Hs-CUL2 protein and in vivo metabolically Previous studies have shown that the VHL gene encodes a protein of 213 amino acid residues with an acidic N-terminus (residues 1 ± 54) characterized by a Gly-X-Glu-Glu-X motif repeated eight times (pVHL30 or, as reported here, pVHL25). In this study we identi®ed a second abundant VHL protein, pVHL18, which appears to encode residues 54 ± 213 of the previously characterized pVHL30 product and also functions as a tumor suppressor. Two VHL mRNAs have been detected in human cells (Gnarra et al., 1994; Shuin et al., 1994; Whaley et al., 1995). Isoform I contains exon 1 (which encodes both Met1 and Met54), 2 and 3. In isoform II, exon 1 is fused to exon 3. Certain renal carcinoma cell lines produce only transcript isoform II (Gnarra et al., 1994), suggesting that even if made (this transcript has not been shown to encode a protein), it lacks tumor suppressor activity. As shown in this study, (a) pVHL18 comigrates with pVHL(54 ± 213) which retains tumor suppressor activity, (b) the proteolytic peptide maps of these two proteins are identical, and (c) coexpression of pVHL18 with pVHL30 in cells (or in vitro) expressing an extended VHL transgene is inhibited when the Met54 codon is mutated to encode isoleucine. Thus, pVHL18 does not appear to arise from alternative mRNA splicing or proteolytic degradation of pVHL30 but most likely is produced by ribosomal scanning and internal translation initiation at Met54 of the VHL mRNA open reading frame. Similar observations were made by others working independently from us (Dr William G Kaelin, personal communication). As previously reported for pVHL30 (Iliopoulos et al., 1995), pVHL18 had no apparent eect on renal carcinoma cell monolayer growth in vitro, indicating that its ability to inhibit tumor formation in nude mice is not an unspeci®c growth inhibitory eect. Inhibition of tumor growth correlated with downregulation of VEGF mRNA, indicating that, at least in part, the tumor suppressor function of pVHL18 has an antiangiogenic component. It has previously been shown that inhibition of VEGF expression by pVHL30 correlates with its ability to bind to the cellular proteins elongin B, elongin C, and Hs-CUL2. Likewise, we show here that pVHL18 binds to these cellular proteins, indicating that it shares common mechanisms of action with pVHL30. As a result of lacking the N-terminal acidic region, which is part of pVHL30, pVHL18 is predicted to exhibit a more hydrophobic character, and may not only share properties with pVHL30 but possibly also encode distinct biological activities. The production of protein products as a result of internal translation initiation has been reported for an increasing number of viral and cellular mRNAs, and in various cases these encode proteins with distinct subcellular localization and biological functions. Examples of such kind include, for instance, the FGF-2, int-2, the MOD5 and drosophila antennapeadia genes (Acland et al., 1990; Gillman et al., 1991; Iizula et al., 1995; 1533 pVHL isoforms and tumor suppression C Blankenship et al 1534 Vagner et al., 1995). Thus, future studies related to pVHL18 may reveal novel functions associated with the VHL gene. We and others have shown previously that the region corresponding to residues 54 ± 213 of the VHL open reading frame is the region of highest homology between human, mouse and rat VHL genes (Gao et al., 1993). Furthermore, all known mutations in the VHL gene invariably map downstream of the internal AUG codon-54 (Zbar, 1995, Maher and Kaelin, 1997), indicating that no known mutations would selectively target pVHL30. Thus, germline and somatic mutations inactivate both pVHL30 and pVHL18, which, as shown here, have tumor suppressor activity. Materials and methods Cell lines and cDNA cloning COS-7 (CRL 1651), NIH3T3 (CRL1658), Hela (CCL-2), 293 (CRL 1573), WI-38 (CCL 75), NRK (CRL 6509) and 786-0 (CRL 1932) cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and maintained as recommended. VHL7/7 RCC-786-0 renal carcinoma cells were transfected with pUHD15-1Neo (tTA-encoding tetracycline activator). Colonies were isolated, expanded and analysed for tetracycline-mediated regulation of gene expression by transient transfection with tetOP-reporter plasmids. One clone was subsequently cotransfected with tTA-responsive expression plasmids, pUHD10-3 or pUHD10-3HA (containing 5' HA epitope tag), encoding various VHL proteins: VHL(1 ± 213) (TG1AR, cDNA spanning nucleotides (nt) 184 ± 1673 of the ®rst published VHL sequence (Latif et al., 1993); VHL(54 ± 213) (TG2,TG3: cDNA spanning nt 296 ± 955 of the ®rst reported human VHL cDNA); mutant VHL(1 ± 213) (TG*4 ± 6: cDNA spanning nt 184 ± 1673 and containing a mutation at codon 54 (ATG to ATT) changing Met54 to Ile54); HA-tagged VHL(54 ± 213) (TG7-10, HA-tag fused to Met54); HA-tagged mutant VHL(54 ± 213) containing tumor derived missense mutation R167Q. Control cell lines contain a mutant VHL transgene generated by insertion of a cassette containing stop codons in all three reading frames into the unique NotI restriction site at nt 384, adjacent to the second ORF ATG codon (codon 54). Individual colonies were ampli®ed and assayed for tetracycline (1 mg/ml)-regulated VHL protein expression by Western blot analysis. Plasmids pUHD151Neo (tTA) and pUHD10-3 (tTA-responsive expression vector) were obtained from Gossen and Bujard (1992). Marathon 5' RACE was used to clone the 5' end of HsCUL2 and full length Hs-CUL2, using polyA RNA from human kidney (Clontech) according to manufacturer's instructions (Clontech). Hs-CUL2 cDNA was cloned into pCR3.1 by TA cloning (Invitrogen, Carlsbad, CA, USA). Recombinant clones were con®rmed by sequencing of both strands. The cDNA sequence obtained corresponded to the recently published Hs-CUL2 cDNA sequence (Lonergan et al., 1998; unpublished data). Protein analysis Antibodies and cell-free protein translation assays Anitypuri®ed polyclonal rabbit antiserum to human VHL protein (pAbVHLr1) was described previously (Gao et al., 1995). Anti-VHL monoclonal antibody IG32 was purchased from PharMingen (San Diego, CA, USA), anti-tubulin antibody from Oncogene Science (Cambridge, MA, USA), and anti-HA monoclonal antibody (12CA5) from Boehringer Mannheim (Indianapolis, IN, USA). 35S-methionine-labeled proteins were produced by coupled in vitro transcription/translation using reticulocyte lysates, as recommended by the manufacturer (Promega, Madison, WI, USA). Proteins were resolved by SDS ± PAGE under denaturing conditions employing either 4 ± 20% or 15% acrylamide gels. Immunoprecipitation analyses Immunoprecipitation analyses were performed essentially as described by Kibel et al. (1995). Brie¯y, transfected RCC, HeLa or 293 cells were labeled metabolically using DMEM minus methionine and cysteine (Gibco/BRL, Gaithersburg, MD, USA) supplemented with L-[35S]-methionine and L-[35S]-cysteine [0.5 mCi of Promix (41000 Ci/mmol; Amersham, Arlington Heights, IL, USA) per ml] for 5 h at 378C, 5% CO2. Cells were lysed in ice-cold EBC buer [50 mM Tris-Cl (pH 8.0), 120 mM NaCl, 0.5% Nonidet P-40] containing protease inhibitors. The radiolabeled supernatants were mixed with an equal volume of ice-cold NETN [20 mM Tris-Cl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, protease inhibitors] containing the appropriate antibody and protein A sepharose. The immune complexes were then washed ®ve times at 48C and resolved by SDS ± PAGE. Immunoblot analysis Cells were washed once with PBS (pH 7.4) and collected by centrifugation. Cell pellets were resuspended in 26Laemmli sample buer and incubated at 958C (5 min). Cellular lysate material was passed ®ve times through a 25 gauge syringe needle and resolved directly by SDS ± PAGE. Alternatively, cells were lysed, as described for immunoprecipitation analyses and resolved by SDS ± PAGE. Immunoblot reactions were performed as previously described (Gao et al., 1995). Peptide microsequencing VHL immune complexes were resolved by 7% SDS ± PAGE. p80 was detected by copper staining (BioRad, according to manufacturer's instructions), and the corresponding band was excised, destained, digested with trypsin and microsequenced (William S Lane, Harvard Microchemistry Facility). V-8 protease maps Partial proteolytic digests of in vitro translated pVHL(54 ± 213) and Hs-CUL2 proteins, and endogenous p18 (VHL) and p80 (Hs-CUL2), were performed as described by Harlow and Lane (1988). Hypoxia treatment, TPA treatment and RNA analysis Cell lines expressing VHL transgenes were plated in complete medium, and 12 h later cells were either placed in GasPak pouches (BBL Microbiology Systems) and incubated for 18 h under hypoxic conditions at 378C, as indicated by methylene blue decolorization (less than 2% O2 is achieved under these conditions (BBL)), or incubated under standard growth conditions (5% CO2, 378C). For treatments with the phorbol ester TPA, cells were plated in complete medium, and 24 h later the medium was changed to complete medium+100 ng/ml TPA. At 24 h following TPA addition, cells were harvested for RNA analyses. Total RNA was isolated from cultured cells and analysed by Northern blot as previously described (Buckbinder et al., 1995). A human VEGF cDNA corresponding to the coding region of VEGF isoform 165 was kindly provided by Dr M Klagsbrun, and a human GAPDH probe was obtained from Clontech (Palo Alto, CA, USA). Probes were labeled using 32P-dCTP (New England Nuclear, Boston, MA, USA). pVHL isoforms and tumor suppression C Blankenship et al Nude mouse xenograft assay Renal carcinoma cells stably expressing VHL transgenes were treated with trypsin:EDTA and suspended in basal culture medium. Cells (107 cells/site) were implanted subcutaneously into the axillary region of nude mice (nu/ nu; Harland Sprague Dawley). 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