Article Figures & Data
Figures
- FIG. 1.
Expression of p53 in RCC cells with different VHL statuses. (A) Representative Western blot analysis to monitor p53 and pVHL expression in 786-O, UOK121 and UMRC6 renal cell carcinoma (RCC) cells, in which pVHL expression was restored by transfection (described for 786-O cells in reference 2 and in Materials and Methods for UOK121 and UMRC6). β-Actin signals on the same filters illustrate even loading of samples. VHL-, parental populations (VHL-deficient); VHL+, VHL-restored populations (expressing wild-type VHL through stable transfection). (B) Representative Northern blot analysis of p53 mRNA abundance in RCC cells with different VHL statuses. 18S rRNA signals served to monitor loading differences among samples. Relative abundances of p53 mRNA and protein are the mean values obtained from three independent experiments.
- FIG. 2.
Analysis of p53 stability and translation in cells with different VHL statuses. (A) We prepared 20 μg of whole-cell protein extracts from 786-O cells that either lacked (VHL-) or expressed (VHL+) VHL after treatment with 10 μM lactacystin for the times indicated. Western blot analysis of expression of p53 (top) and p21 (bottom), as well as β-actin (loading control) after resolving samples by electrophoresis in SDS-containing gels with either 12% (top) or 15% (bottom) polyacrylamide. (B) Cells were incubated for 20 min in the presence of l-[35S]methionine and l-[35S]cysteine, whereupon nascent p53 was visualized by immunoprecipitation as described in the Materials and Methods section. Samples were resolved by electrophoresis in SDS-containing 12% polyacrylamide gels. Radiolabeled p53 signal is indicated.
- FIG. 3.
Association of HuR with the p53 3′UTR. (A) Schematic of p53 mRNA, depicting U- and AU-rich regions in the 3′UTR (shaded regions), as well as transcripts prepared for RNA-binding analysis. CR, coding region. (B) Radiolabeled transcripts encompassing either the p53 3′UTR or the p53 coding region were incubated with cytoplasmic lysates from 786-O cells (10 μg each) and subjected to REMSA supershift assays in the presence of either a control antibody recognizing mitogen-activated protein kinase p38 [a protein that lacks RNA-binding activity (+αp38 lanes)] or an antibody recognizing HuR (+αHuR lanes).
- FIG. 4.
Association of endogenous HuR with synthetic p53 transcripts and with endogenous p53 mRNA in RCC cells. (A) Radiolabeled p53 3′UTR transcript was incubated with cytoplasmic lysates from 786-O cells and then immunoprecipitated in either the presence or absence of anti-HuR antibody (αHuR). MWM, size markers (in kilodaltons). (B) Biotinylated p53 transcripts (1 μg each) encompassing either the coding region or the 3′UTR were tested for their ability to pull down HuR from cytoplasmic lysates of 786-O cells with different VHL statuses, as described in the Materials and Methods section. The presence of β-actin in pull-down material was monitored in order to assess the specificity of the assay. (C) Immunoprecipitation with either anti-HuR antibody or IgG1 under conditions that preserve the association of RNA-binding proteins with target mRNAs was followed by reverse transcription-PCR analysis to detect endogenous p53 mRNA in the three RCC cell lines shown. PCR products were resolved by electrophoresis in 1.5% agarose gels and visualized by staining with ethidium bromide.
- FIG. 5.
Subcellular localization of HuR and AMPK activity in 786-O cells with different VHL statuses. (A) Whole-cell (20 μg), nuclear (10 μg), and cytoplasmic (40 μg) lysates were subjected to Western blot analysis to monitor the expression of HuR. Sequential hybridizations with antibodies against β-tubulin (a cytoplasmic protein) and HDAC1 (a nuclear protein) were carried out to assess the quality of the fractionation process and the uniformity in loading and transfer of samples. (B) Detection of HuR by immunofluorescence in RCC cells (786-O, UMRC6, and UOK121) with different VHL statuses. Top, HuR immunofluorescence; middle, Hoechst staining to visualize nuclei; bottom, phase contrast images. Representative photographs from two independent experiments are shown.
- FIG. 6.
AMPK activity in 786-O cells with different VHL statuses and influence of AMPK activation on the cytoplasmic levels of HuR and p53. (A) Whole-cell lysates prepared from VHL+ and VHL− cells were used to measure AMPK activity. AMPK was immunoprecipitated with a polyclonal antibody that recognizes the AMPK α1 and α2 subunits; the in vitro kinase assay was performed with the synthetic peptide SAMS (described in the Materials and Methods section). (B) Western blot analysis of HuR and p53 expression in lysates (40 μg) prepared from 786-O cells treated for 6 h in the presence of AMPK activator AICAR (2 mM).
- FIG. 7.
Cytoplasmic HuR and p53 mRNAs are associated with high-molecular-weight, actively translating polysomes in VHL+ cells. (A) Cytoplasmic lysates from 786-O cells were fractionated through sucrose gradients in order to separate unbound mRNA from mRNA bound to polysomes of increasing size. Top, representative profile. Bottom, representative Western blots depicting HuR signals in the unbound fraction 2, low-molecular-weight (LMW) polysome fraction 6, and high-molecular-weight (HMW) polysome fractions 8 and 10. Also shown are pVHL and p53 signals. (B) p53 mRNA is associated with high-molecular-weight, actively translating polysomes in VHL+ cells. Following fractionation of cytoplasmic lysates from 786-O cells, RNA was prepared from each of the 11 fractions and subjected to Northern blot analysis. Representative p53 mRNA signals are shown; β-actin mRNA signals were included to monitor differences in loading and transfer of samples.
- FIG. 8.
Small interfering RNA-mediated reduction of HuR expression causes a reduction in p53 levels in RCC cells. (A) 786-O cells were transfected with either control (Ctrl) or HuR-specific (HuR4) siRNA, and expression levels of HuR and p53 were assessed 2 days later in preparations of total protein or cytoplasmic protein (10 and 40 μg, respectively). *, second transfection with the same siRNA 24 h later; proteins were collected 2 days after the second transfection. (B) VHL+ RCC lines UOK121 and UMRC6 were transfected with either control (Ctrl) or HuR-specific (HuR4) siRNA, and expression levels of HuR and p53 were assessed 2 days later in whole-cell lysates (20 μg). Representative signals are shown. (C) Two days after transfection with either control siRNA or HuR-directed siRNA (HuR4), 786-O cells with different VHL statuses were incubated for 20 min in the presence of l-[35S]methionine and l-[35S]cysteine, whereupon nascent p53 was visualized by immunoprecipitation as described in the Materials and Methods section. Samples were resolved by electrophoresis in SDS-containing 12% polyacrylamide gels. Radiolabeled p53 signal is shown.
Tables
- TABLE 1.
mRNAs preferentially associated with polysomes in VHL-expressing cellsa
Unigene no. Gene Relative abundance Hs.1030 Ras inhibitor ++ Hs.75356 Transcription factor 4 + Hs.75162 MAPK-activated protein kinase 3 ++ Hs.118825 MAPK kinase 6 ++ Hs.1846 Tumor suppressor protein p53 +++ Hs.79362 Retinoblastoma-like protein 2 ++ ↵ a For each gene on the cDNA arrays (described at http://www.grc.nia.nih.gov/branches/rrb/dna/dnapubs.htm ), the relative presence of the encoded mRNA in polysome-bound and unbound fractions was compared between VHL-expressing (VHL+) and VHL− deficient (VHL−) 786-O cell lines. Cell fractionations and cDNA array analysis were performed in three independent experiments (16). The list includes genes potentially subject to translational upregulation by pVHL, since they are encoded by mRNAs that were significantly more abundant in polysome-bound fractions from VHL− cells than in those from VHL+ cells, as assessed through comparison of Z averages (described in reference 16). Briefly, for each gene on the array, mRNA abundance in each fraction (polysomal and nonpolysomal) was compared between VHL− and VHL+ cells. Data analysis was carried out as explained in the Materials and Methods section and in reference 16. Differences in Z averages (Z diff) served to assess the degree to which a given transcript was preferentially associated with polysomes in VHL+ cells and was represented as follows: +, Z diff between 0 and 0.25; ++, Z diff between 0.25 and 0.50; +++, Z diff between 0.50 and 0.75. Genes encoding mRNAs that exhibited no significant difference in their relative distribution (polysomal compared with nonpolysomal) in cells with different VHL statuses were described previously (16).
