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VHL Type 2B gene mutation moderates HIF dosage in vitro and in vivo.

Lee CM, Hickey MM, Sanford CA, McGuire CG, Cowey CL, Simon MC, Rathmell WK - Oncogene (2009)

Bottom Line: Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma.Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation.Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.

ABSTRACT
Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma. Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation. Murine embryonic stem (ES) cells in which the endogenous wild-type Vhl gene was replaced with the representative Type 2B VHL hotspot mutation R167Q (Vhl(2B/2B)) displayed preserved physiological regulation of both HIF factors with slightly greater normoxic dysregulation of HIF-2alpha. Differentiated Vhl(2B/2B)-derived teratomas overexpressed joint HIF targets Vegf and EglN3 but not the HIF-1alpha-specific target Pfk1. Vhl(2B/2B) teratomas additionally displayed a growth advantage over Vhl(-/-)-derived teratomas, suggestive of a tight connection between perturbations in the degree and ratio of HIF-1alpha and HIF-2alpha stabilization and cell growth. Vhl(2B/2B) mice displayed mid-gestational embryonic lethality, whereas adult Vhl(2B/+) mice exhibited susceptibility to carcinogen-promoted renal neoplasia compared with wild-type littermates at 12 months. Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

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Type 2B Vhl mutation knock-in strategy and genotyping. A. Human:Mouse pVHL protein alignment. The R167Q pVHL substitution representing human Type 2B VHL disease is embedded in a highly homologous region. B. Type 2B mutant murine Vhl containing the G518A (R167Q) representative missense mutation (star) was prepared with site-directed mutagenesis and then cloned into the targeting vector as shown. Following electroporation of the targeting vector into J1 ES cells, neomycin and gancyclovir resistance was used to select clones with homologous recombination at the endogenous murine locus (Neo-in). Cre recombinase exposure in vitro or in vivo resulted in excision of the floxed NeoR cassette (Neo-out). C. Southern blot genotyping distinguished between wild-type and targeted Vhl alleles based on the introduction of new HindIII restriction sites. HindIII-digested murine Vhl was detected with a probe recognizing the Vhl 3′ untranslated region (UTR, bar). D. Restriction PCR was used as an alternative genotyping strategy. Primers (arrows) flanking the targeted mutation generated a 300-bp PCR product. The novel restriction site introduced by the G518A mutation rendered the targeted Vhl-origin PCR product susceptible to HpyIV digestion.
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Figure 1: Type 2B Vhl mutation knock-in strategy and genotyping. A. Human:Mouse pVHL protein alignment. The R167Q pVHL substitution representing human Type 2B VHL disease is embedded in a highly homologous region. B. Type 2B mutant murine Vhl containing the G518A (R167Q) representative missense mutation (star) was prepared with site-directed mutagenesis and then cloned into the targeting vector as shown. Following electroporation of the targeting vector into J1 ES cells, neomycin and gancyclovir resistance was used to select clones with homologous recombination at the endogenous murine locus (Neo-in). Cre recombinase exposure in vitro or in vivo resulted in excision of the floxed NeoR cassette (Neo-out). C. Southern blot genotyping distinguished between wild-type and targeted Vhl alleles based on the introduction of new HindIII restriction sites. HindIII-digested murine Vhl was detected with a probe recognizing the Vhl 3′ untranslated region (UTR, bar). D. Restriction PCR was used as an alternative genotyping strategy. Primers (arrows) flanking the targeted mutation generated a 300-bp PCR product. The novel restriction site introduced by the G518A mutation rendered the targeted Vhl-origin PCR product susceptible to HpyIV digestion.

Mentions: The Arginine 167 → Glutamine (R167Q) missense mutation is a hotspot in the human VHL gene with a tight genotype-phenotype correlation to Type 2B disease (Maher et al., 1991; Zbar et al., 1996). Localized to the pVHL α helical domain (Figure 1A), R167 is predicted to stabilize the α/β domain interface and interaction with Elongin C (Ohh et al., 1999; Stebbins et al., 1999). In the murine Vhl allele, this mutation corresponds to a guanine to alanine transition at position 518 (G518A).


VHL Type 2B gene mutation moderates HIF dosage in vitro and in vivo.

Lee CM, Hickey MM, Sanford CA, McGuire CG, Cowey CL, Simon MC, Rathmell WK - Oncogene (2009)

Type 2B Vhl mutation knock-in strategy and genotyping. A. Human:Mouse pVHL protein alignment. The R167Q pVHL substitution representing human Type 2B VHL disease is embedded in a highly homologous region. B. Type 2B mutant murine Vhl containing the G518A (R167Q) representative missense mutation (star) was prepared with site-directed mutagenesis and then cloned into the targeting vector as shown. Following electroporation of the targeting vector into J1 ES cells, neomycin and gancyclovir resistance was used to select clones with homologous recombination at the endogenous murine locus (Neo-in). Cre recombinase exposure in vitro or in vivo resulted in excision of the floxed NeoR cassette (Neo-out). C. Southern blot genotyping distinguished between wild-type and targeted Vhl alleles based on the introduction of new HindIII restriction sites. HindIII-digested murine Vhl was detected with a probe recognizing the Vhl 3′ untranslated region (UTR, bar). D. Restriction PCR was used as an alternative genotyping strategy. Primers (arrows) flanking the targeted mutation generated a 300-bp PCR product. The novel restriction site introduced by the G518A mutation rendered the targeted Vhl-origin PCR product susceptible to HpyIV digestion.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667565&req=5

Figure 1: Type 2B Vhl mutation knock-in strategy and genotyping. A. Human:Mouse pVHL protein alignment. The R167Q pVHL substitution representing human Type 2B VHL disease is embedded in a highly homologous region. B. Type 2B mutant murine Vhl containing the G518A (R167Q) representative missense mutation (star) was prepared with site-directed mutagenesis and then cloned into the targeting vector as shown. Following electroporation of the targeting vector into J1 ES cells, neomycin and gancyclovir resistance was used to select clones with homologous recombination at the endogenous murine locus (Neo-in). Cre recombinase exposure in vitro or in vivo resulted in excision of the floxed NeoR cassette (Neo-out). C. Southern blot genotyping distinguished between wild-type and targeted Vhl alleles based on the introduction of new HindIII restriction sites. HindIII-digested murine Vhl was detected with a probe recognizing the Vhl 3′ untranslated region (UTR, bar). D. Restriction PCR was used as an alternative genotyping strategy. Primers (arrows) flanking the targeted mutation generated a 300-bp PCR product. The novel restriction site introduced by the G518A mutation rendered the targeted Vhl-origin PCR product susceptible to HpyIV digestion.
Mentions: The Arginine 167 → Glutamine (R167Q) missense mutation is a hotspot in the human VHL gene with a tight genotype-phenotype correlation to Type 2B disease (Maher et al., 1991; Zbar et al., 1996). Localized to the pVHL α helical domain (Figure 1A), R167 is predicted to stabilize the α/β domain interface and interaction with Elongin C (Ohh et al., 1999; Stebbins et al., 1999). In the murine Vhl allele, this mutation corresponds to a guanine to alanine transition at position 518 (G518A).

Bottom Line: Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma.Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation.Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC 27599-7295, USA.

ABSTRACT
Von Hippel-Lindau (VHL) disease is caused by germline mutations in the VHL tumor suppressor gene, with Type 2B missense VHL mutations predisposing to renal cell carcinoma, hemangioblastoma and pheochromocytoma. Type 2B mutant pVHL is predicted to be defective in hypoxia inducible factor (HIF)-alpha regulation. Murine embryonic stem (ES) cells in which the endogenous wild-type Vhl gene was replaced with the representative Type 2B VHL hotspot mutation R167Q (Vhl(2B/2B)) displayed preserved physiological regulation of both HIF factors with slightly greater normoxic dysregulation of HIF-2alpha. Differentiated Vhl(2B/2B)-derived teratomas overexpressed joint HIF targets Vegf and EglN3 but not the HIF-1alpha-specific target Pfk1. Vhl(2B/2B) teratomas additionally displayed a growth advantage over Vhl(-/-)-derived teratomas, suggestive of a tight connection between perturbations in the degree and ratio of HIF-1alpha and HIF-2alpha stabilization and cell growth. Vhl(2B/2B) mice displayed mid-gestational embryonic lethality, whereas adult Vhl(2B/+) mice exhibited susceptibility to carcinogen-promoted renal neoplasia compared with wild-type littermates at 12 months. Our experiments support a model in which the representative Type 2B R167Q mutant pVhl produces a unique profile of HIF dysregulation, thereby promoting tissue-specific effects on cell growth, development and tumor predisposition.

Show MeSH
Related in: MedlinePlus