Limits...
Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth.

Kondo K, Kim WY, Lechpammer M, Kaelin WG - PLoS Biol. (2003)

Bottom Line: Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities.Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells.These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2alpha variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

Show MeSH

Related in: MedlinePlus

HIF2α Overrides Tumor Suppression by pVHL(A) 786-O subclones that were transfected to produce wild-type pVHL (WT8) or with an empty plasmid (PRC3) cells, as well as WT8 cells infected with an empty retrovirus (Empty) or retroviruses encoding the indicated HIF2α variants [ (P→A)2 = P405A;P531A and * = bHLH mutation] were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of WT8 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of WT8 cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC300692&req=5

pbio.0000083-g001: HIF2α Overrides Tumor Suppression by pVHL(A) 786-O subclones that were transfected to produce wild-type pVHL (WT8) or with an empty plasmid (PRC3) cells, as well as WT8 cells infected with an empty retrovirus (Empty) or retroviruses encoding the indicated HIF2α variants [ (P→A)2 = P405A;P531A and * = bHLH mutation] were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of WT8 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of WT8 cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.

Mentions: 786-O renal carcinoma cells lack wild-type pVHL and overproduce HIF2α (Iliopoulos et al. 1995; Maxwell et al. 1999). HIF1α is not detectable in these cells (Maxwell et al. 1999). Reintroduction of wild-type pVHL into 786-O cells by stable transfection does not affect cell growth in vitro under standard serum-rich growth conditions, but leads to downregulation of HIF2α protein levels, suppression of hypoxia-inducible gene expression, and impaired tumorigenesis in vivo (Iliopoulos et al. 1995, 1996; Gnarra et al. 1996; Schoenfeld et al. 1998; Maxwell et al. 1999; Davidowitz et al. 2001). A 786-O subclone stably transfected to produce wild-type pVHL (WT8) (Iliopoulos et al. 1995) was infected with a retrovirus encoding HIF2α P405A;P531A or HIF2α P405A;P531A;bHLH* and grown under hypoxic (1% oxygen) or normoxic (21% oxygen) conditions. As expected, HIF2α was only detectable under hypoxic conditions in WT8 cells and in WT8 cells infected with an empty retrovirus (Figure 1A). In contrast, HIF2α was readily detectable under both hypoxic and normoxic conditions in WT8 cells infected to produce either of the two HIF2α P405A;P531A variants. Indeed, the levels of HIF2α present in these cells approximated those seen in a 786-O subclone (PRC3) (Iliopoulos et al. 1995) that, unlike WT8 cells, was stably transfected with an empty expression plasmid and hence still lacks wild-type pVHL.


Inhibition of HIF2alpha is sufficient to suppress pVHL-defective tumor growth.

Kondo K, Kim WY, Lechpammer M, Kaelin WG - PLoS Biol. (2003)

HIF2α Overrides Tumor Suppression by pVHL(A) 786-O subclones that were transfected to produce wild-type pVHL (WT8) or with an empty plasmid (PRC3) cells, as well as WT8 cells infected with an empty retrovirus (Empty) or retroviruses encoding the indicated HIF2α variants [ (P→A)2 = P405A;P531A and * = bHLH mutation] were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of WT8 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of WT8 cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC300692&req=5

pbio.0000083-g001: HIF2α Overrides Tumor Suppression by pVHL(A) 786-O subclones that were transfected to produce wild-type pVHL (WT8) or with an empty plasmid (PRC3) cells, as well as WT8 cells infected with an empty retrovirus (Empty) or retroviruses encoding the indicated HIF2α variants [ (P→A)2 = P405A;P531A and * = bHLH mutation] were grown in the presence of 21% or 1% oxygen and immunoblotted (IB) with the indicated antibodies.(B) In vitro proliferation of WT8 cells infected with the indicated retroviruses.(C) Tumor weights approximately 9 wk after subcutaneous implantation of WT8 cells infected with the indicated retroviruses in nude mice. Number of tumors analyzed is shown in parentheses. Error bars = one standard error.
Mentions: 786-O renal carcinoma cells lack wild-type pVHL and overproduce HIF2α (Iliopoulos et al. 1995; Maxwell et al. 1999). HIF1α is not detectable in these cells (Maxwell et al. 1999). Reintroduction of wild-type pVHL into 786-O cells by stable transfection does not affect cell growth in vitro under standard serum-rich growth conditions, but leads to downregulation of HIF2α protein levels, suppression of hypoxia-inducible gene expression, and impaired tumorigenesis in vivo (Iliopoulos et al. 1995, 1996; Gnarra et al. 1996; Schoenfeld et al. 1998; Maxwell et al. 1999; Davidowitz et al. 2001). A 786-O subclone stably transfected to produce wild-type pVHL (WT8) (Iliopoulos et al. 1995) was infected with a retrovirus encoding HIF2α P405A;P531A or HIF2α P405A;P531A;bHLH* and grown under hypoxic (1% oxygen) or normoxic (21% oxygen) conditions. As expected, HIF2α was only detectable under hypoxic conditions in WT8 cells and in WT8 cells infected with an empty retrovirus (Figure 1A). In contrast, HIF2α was readily detectable under both hypoxic and normoxic conditions in WT8 cells infected to produce either of the two HIF2α P405A;P531A variants. Indeed, the levels of HIF2α present in these cells approximated those seen in a 786-O subclone (PRC3) (Iliopoulos et al. 1995) that, unlike WT8 cells, was stably transfected with an empty expression plasmid and hence still lacks wild-type pVHL.

Bottom Line: Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities.Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells.These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

View Article: PubMed Central - PubMed

Affiliation: Department of Adult Oncology, Dana-Farber Cancer Institute and Brigham and Womens Hospital, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Biallelic inactivation of the von Hippel-Lindau tumor suppressor gene (VHL) is linked to the development of hereditary (VHL-associated) and sporadic clear-cell renal carcinomas as well as other abnormalities. The VHL gene product, pVHL, is part of an E3 ubiquitin ligase complex that targets the alpha subunits of the heterodimeric transcription factor HIF (hypoxia-inducible factor) for degradation in the presence of oxygen. Here we report that a HIF2alpha variant lacking both of its two prolyl hydroxylation/pVHL-binding sites prevents tumor inhibition by pVHL in a DNA-binding dependent manner. Conversely, downregulation of HIF2alpha with short hairpin RNAs is sufficient to suppress tumor formation by pVHL-defective renal carcinoma cells. These results establish that tumor suppression by pVHL is linked to regulation of HIF target genes.

Show MeSH
Related in: MedlinePlus