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HIF-2alpha downregulation in the absence of functional VHL is not sufficient for renal cell differentiation.

Hughes MD, Kapllani E, Alexander AE, Burk RD, Schoenfeld AR - Cancer Cell Int. (2007)

Bottom Line: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation.These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation.We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Adelphi University, Garden City, NY 11530-0701, USA. hughes@adelphi.edu

ABSTRACT

Background: Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary as well as sporadic clear cell renal carcinomas. The product of the VHL gene, pVHL, acts to target hypoxia-inducible factor alpha (HIF-alpha) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2alpha in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2alpha toward cellular pVHL activities.

Results: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation. However, cellular morphological changes and downregulation of integrins alpha5 and beta1, which were seen upon pVHL replacement, were not faithfully phenocopied by HIF-2alpha reduction. Moreover, fibronectin deposition and expression of renal cell differentiation markers were observed in cells containing replaced pVHL, but not in HIF-2alpha knockdown cells, indicating that these pVHL functions may occur independently of HIF-2alpha downregulation.

Conclusion: These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation. We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

No MeSH data available.


Related in: MedlinePlus

Knockdown of HIF-2α mediates cell cycle arrest on collagen I. (A) Parental 786-O cells (786-O), 786-O cells stably transfected with VHL (VHL +), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were grown for 2 weeks after attaining confluence on gels of polymerized collagen I. Flow cytometry was performed with a FACScan instrument. Percentages of cells in S phase are shown for each cell line. (B) FACS analysis was performed as in (A) using A498 cell lines. (C) Graphical representation of the FACS analysis in (B) with arrows highlighting cells with aneuploid DNA content. (D) Complete cell cycle profiles of 786-O and A498 cell lines (diploid cells only) from FACS analyses presented in (A), (B), and (C).
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Figure 2: Knockdown of HIF-2α mediates cell cycle arrest on collagen I. (A) Parental 786-O cells (786-O), 786-O cells stably transfected with VHL (VHL +), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were grown for 2 weeks after attaining confluence on gels of polymerized collagen I. Flow cytometry was performed with a FACScan instrument. Percentages of cells in S phase are shown for each cell line. (B) FACS analysis was performed as in (A) using A498 cell lines. (C) Graphical representation of the FACS analysis in (B) with arrows highlighting cells with aneuploid DNA content. (D) Complete cell cycle profiles of 786-O and A498 cell lines (diploid cells only) from FACS analyses presented in (A), (B), and (C).

Mentions: Downregulation of HIF-2α in 786-O and A498 cells has been shown to block tumor formation in nude mice [24,30]. One mechanism for this tumor suppression could involve cell cycle arrest. It has been previously shown that 786-O cells with replaced VHL arrest in the G0-G1 phase of the cell cycle when cultured on collagen I, whereas VHL negative cells do not [26]. Cell cycle arrest is not seen when these cells are cultured by standard methods (i.e., on plastic). To investigate whether HIF-2α plays a role in cell cycle arrest, 786-O and A498 cell lines were plated on collagen I and grown to high cell density (2 weeks post-confluency) and analyzed by FACS to assess cell cycle distributions. Note that under these conditions, the cell lines utilized formed multilayers of cells. Nonetheless, VHL replacement in 786-O cells led to a decreased percentage of cells in S-phase (Figures 2a and 2d) and an increased G1 population, as previously reported. Strikingly, knockdown of HIF-2α levels led to an even further decrease in the fraction of S-phase cells. Similar results were obtained using the A498 cell lines (Figures 2b and 2d). Of note, both parental and control-infected A498 cells contained a significant percentage of aneuploid cells, however A498 cells with VHL replaced and those with shRNA-lowered HIF-2α had no aneuploid cells (Figure 2c). On the whole, removal of HIF-2α appears to be sufficient to mediate cell cycle arrest and suppression of anueploidy in this experimental setting, suggesting that the in vivo tumor suppression observed may occur at least in part through this mechanism. However, while these findings indicate that HIF-2α removal is sufficient to block growth of VHL-associated renal tumors, it is not clear whether HIF-2α dysregulation alone can lead to the initiation of renal carcinomas.


HIF-2alpha downregulation in the absence of functional VHL is not sufficient for renal cell differentiation.

Hughes MD, Kapllani E, Alexander AE, Burk RD, Schoenfeld AR - Cancer Cell Int. (2007)

Knockdown of HIF-2α mediates cell cycle arrest on collagen I. (A) Parental 786-O cells (786-O), 786-O cells stably transfected with VHL (VHL +), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were grown for 2 weeks after attaining confluence on gels of polymerized collagen I. Flow cytometry was performed with a FACScan instrument. Percentages of cells in S phase are shown for each cell line. (B) FACS analysis was performed as in (A) using A498 cell lines. (C) Graphical representation of the FACS analysis in (B) with arrows highlighting cells with aneuploid DNA content. (D) Complete cell cycle profiles of 786-O and A498 cell lines (diploid cells only) from FACS analyses presented in (A), (B), and (C).
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Related In: Results  -  Collection

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Figure 2: Knockdown of HIF-2α mediates cell cycle arrest on collagen I. (A) Parental 786-O cells (786-O), 786-O cells stably transfected with VHL (VHL +), and 786-O cells infected with an empty retrovirus (pSuperRetro) or doubly infected with both HIF-2α shRNAs (HIF2α shRNA 1+2) were grown for 2 weeks after attaining confluence on gels of polymerized collagen I. Flow cytometry was performed with a FACScan instrument. Percentages of cells in S phase are shown for each cell line. (B) FACS analysis was performed as in (A) using A498 cell lines. (C) Graphical representation of the FACS analysis in (B) with arrows highlighting cells with aneuploid DNA content. (D) Complete cell cycle profiles of 786-O and A498 cell lines (diploid cells only) from FACS analyses presented in (A), (B), and (C).
Mentions: Downregulation of HIF-2α in 786-O and A498 cells has been shown to block tumor formation in nude mice [24,30]. One mechanism for this tumor suppression could involve cell cycle arrest. It has been previously shown that 786-O cells with replaced VHL arrest in the G0-G1 phase of the cell cycle when cultured on collagen I, whereas VHL negative cells do not [26]. Cell cycle arrest is not seen when these cells are cultured by standard methods (i.e., on plastic). To investigate whether HIF-2α plays a role in cell cycle arrest, 786-O and A498 cell lines were plated on collagen I and grown to high cell density (2 weeks post-confluency) and analyzed by FACS to assess cell cycle distributions. Note that under these conditions, the cell lines utilized formed multilayers of cells. Nonetheless, VHL replacement in 786-O cells led to a decreased percentage of cells in S-phase (Figures 2a and 2d) and an increased G1 population, as previously reported. Strikingly, knockdown of HIF-2α levels led to an even further decrease in the fraction of S-phase cells. Similar results were obtained using the A498 cell lines (Figures 2b and 2d). Of note, both parental and control-infected A498 cells contained a significant percentage of aneuploid cells, however A498 cells with VHL replaced and those with shRNA-lowered HIF-2α had no aneuploid cells (Figure 2c). On the whole, removal of HIF-2α appears to be sufficient to mediate cell cycle arrest and suppression of anueploidy in this experimental setting, suggesting that the in vivo tumor suppression observed may occur at least in part through this mechanism. However, while these findings indicate that HIF-2α removal is sufficient to block growth of VHL-associated renal tumors, it is not clear whether HIF-2α dysregulation alone can lead to the initiation of renal carcinomas.

Bottom Line: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation.These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation.We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, Adelphi University, Garden City, NY 11530-0701, USA. hughes@adelphi.edu

ABSTRACT

Background: Mutational inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene has been linked to hereditary as well as sporadic clear cell renal carcinomas. The product of the VHL gene, pVHL, acts to target hypoxia-inducible factor alpha (HIF-alpha) subunits for ubiquitination and subsequent degradation. Using an RNA interference approach to lower levels of HIF-2alpha in two different renal cell lines that lack functional pVHL, we have tested the contribution of HIF-2alpha toward cellular pVHL activities.

Results: Knockdown of HIF-2alpha resulted in cell cycle arrest of renal cells that were grown on collagen I, indicating that this pVHL function is dependent on HIF-2alpha regulation. However, cellular morphological changes and downregulation of integrins alpha5 and beta1, which were seen upon pVHL replacement, were not faithfully phenocopied by HIF-2alpha reduction. Moreover, fibronectin deposition and expression of renal cell differentiation markers were observed in cells containing replaced pVHL, but not in HIF-2alpha knockdown cells, indicating that these pVHL functions may occur independently of HIF-2alpha downregulation.

Conclusion: These results indicate that HIF-2alpha regulation is not sufficient for pVHL-induced renal cell differentiation. We hypothesize that in addition to HIF-2alpha dysregulation, abrogation of additional pVHL functions is required for the initiation of renal carcinogenesis.

No MeSH data available.


Related in: MedlinePlus