Dex decreases p53 and GR levels and inhibits expression of their target genes in HepG2cells

Cytoplasmic p53 in neuroblastoma cells is released from its complex with GR and translocates to the nucleus following treatment with GR antagonists (Sengupta et al. 2000b). We investigated whether cytoplasmic p53 in other tumor cell types, such as HepG2 (hepatoma) and SA1 (osteosarcoma) (Wasylyk et al. 1999; Lu et al. 2000), behaved similarly. Under normal growth conditions, Dex increased the amount of p53 and GR in the cytoplasm of both cell lines, although some p53 always remained nuclear. In contrast, RU486 significantly increased the amount of nuclear p53 (data not shown). Interestingly, in HepG2 cells, Dex caused a drastic decrease in the protein levels of both p53 and GR (Fig. ​(Fig.2A,2A, lanes 1,2). This effect was blocked by the proteasome inhibitor LLnL (Fig. ​(Fig.2A,2A, lanes 3,4), suggesting that the decrease is due to enhanced degradation. We investigated whether Dex can inhibit the expression of genes downstream from both p53 (Bax, Hdm2, and p21^WAF1/CIP1) and GR (phospho enol pyruvate carboxy kinase, PEPCK; Imai et al. 1990). Dex decreased Bax and p21^WAF1/CIP1 protein levels (Fig. ​(Fig.2B)2B) and promoter activity (reporter assays; data not shown). Dex did not affect either the level of Hdm2 protein (Fig. ​(Fig.2B),2B), or the activity of the promoter (reporter assay, data not shown), showing that Hdm2 expression is less sensitive than Bax or p21^WAF1/CIP1 to changes in p53 levels. Dex decreased PEPCK protein levels (Fig. ​(Fig.2B).2B). These results show that Dex down-regulates both p53 and GR protein levels and the expression of their downstream targets in HepG2 cells.

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Figure 2

p53 and GR undergo ligand and proteasome-dependent degradation in HepG2 cells. (A) HepG2 cells were grown for 12 h with vehicle (ethanol), 10⁻⁷ M Dex, and 50 μM LLnL, as indicated. Proteins (50 μg) were analyzed by Western blotting with antibodies to: (a) GR (E-20 and P-20); (b) p53 (DO-1); (c) TBP (3G3). (Lane 1) +Dex; (lane 2) (−, vehicle); (lane 3) +Dex+LLnL; (lane 4) (−, vehicle)+LLnL. (B) Lysates from cells incubated in the absence of LLnL were Western blotted with antibodies to: (a) Bax (N-20); (b) p21^WAF1/CIP1 (1WA-IC581); (c) Hdm2 (2A10); (d) PEPCK; (e) TBP (3G3). (Lane 1) +Dex; (lane 2) (−, vehicle).

Dex activation of endogenous GR inhibits p53 induction by hypoxia in normal endothelial cells(HUVEC)

HepG2 is a tumor cell line with undefined genetic alterations. Hence, we investigated in normal human cells (HUVEC) the response to hypoxia, a natural inducer of p53. Normally growing HUVEC express very little or no p53 protein (Fig. ​(Fig.3A,3A, lane 1). p53 was strongly induced after 12 h of hypoxia, and increased further after 18 h (Fig. ​(Fig.3A,3A, lanes 3,5). Dex inhibited the induction of p53 up to 36 h (Fig. ​(Fig.3A,3A, lanes 2–5; data not shown). The proteasome inhibitor, LLnL, decreased Dex-dependent inhibition of p53 induction, without substantially affecting the level of p53 in the absence of ligand (Fig. ​(Fig.3A,3A, lanes 6–9). p53 RNA levels detected by Northern blotting were not affected by Dex (Fig. ​(Fig.3B),3B), showing that the inhibitory effect of Dex on p53 is not due to changes in transcription or RNA stability. In conclusion, Dex decreases p53 stability by enhanced proteasomal degradation.

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Figure 3

Dex inhibits induction by hypoxia of p53 protein levels, expression of p53 target genes, and apoptosis. (A) HUVEC cells were grown for different times with, as indicated, vehicle (ethanol), 10⁻⁷ M Dex, and 50 μM LLnL, under normoxic (−, lane 1) or hypoxic (lanes 2–9) conditions. Lysates (50 μg) were analyzed by SDS-PAGE and Western blotting with antibodies against (a) p53 (DO-1); (b) TBP (3G3). (Lanes 1,3,5) (−, vehicle); (lanes 2,4) +Dex; (lanes 6,8) +Dex+LLnL; (lanes 7,9) (−, vehicle)+LLnL. (B) Total RNA was extracted from HUVEC cells grown with vehicle (ethanol) or 10⁻⁷ M Dex and hypoxia for 18 h. The RNAs (40 μg) were run on formaldehyde–agarose gels and northerns were hybridized with probes against (a) p53, (b) β-actin. (Lane 1) +Dex; (lane 2) (−, vehicle). (C) HUVEC were grown under normoxia (lanes 1,8–11) or hypoxia (lanes 2–7) with vehicle (ethanol) or Dex. Lysates (50 μg, lanes 1–7; 200 μg, lanes 8–11) were Western blotted with antibodies against (a,d) p21^WAF1/CIP1 (1WA-IC581); (b,e) Bax (N-20); (c,f) TBP (3G3). (Lanes 1, 3,5,7,9,11) (−, vehicle); (lanes 2,4,6,8,10) +Dex. (D) HUVEC were grown under normoxia (lane 1) or hypoxia (lanes 2–5) with vehicle (ethanol) or Dex. Lysates (50 μg) were Western blotted with antibodies against HIF-1α (C-19). (Lanes 1,3,5) (−, vehicle); (lanes 2,4) +Dex. CR, cross-reacting band. (E) HUVEC were grown under normoxia (lane 1) or hypoxia (lanes 2–9) with vehicle (ethanol), Dex, and 50 μM LLnL, as indicated. Lysates (50 μg) were Western blotted with antibodies to (a) Hdm2 (2A10), (b) TBP (3G3). (Lanes 1,3,5,7,9) (−, vehicle); (lanes 2,4,6,8) +Dex. (F) HUVEC were grown under normoxia (lane 1) or hypoxia (lanes 2–8). Lysates (50 μg) were Western blotted with polyclonal antibodies against PARP. (Lanes 1–5) (−, vehicle); (lanes 6–8) +Dex. FL, full length. The cleavage products were scanned and the values are the amount of the cleaved product relative to lane 5.

The products of the p53 downstream genes Bax and p21^WAF1/CIP1 were induced by hypoxia (Fig. ​(Fig.3C,3C, lanes 1,3,5,7). p21^WAF1/CIP1 has also been shown to be induced by hypoxia in normal fibroblasts but not in several transformed cell lines (Ashcroft et al. 2000; Koumenis et al. 2001). Bax and p21^WAF1/CIP1 induction was inhibited by Dex (Fig. ​(Fig.3C,3C, lanes 2,4,6). Under normoxia, in which p53 levels are very low, Dex had no effect, indicating that Dex inhibition of p53 downstream genes is p53 dependent (Fig. ​(Fig.3C,3C, lanes 8–11). HIF-1α stabilizes p53 in response to hypoxia (An et al. 1998), which raised the possibility that it, too, is affected by Dex. However, HIF-1α induction by hypoxia in HUVEC was not significantly affected by Dex (Fig. ​(Fig.3D).3D). The apparently weak induction of HIF-1α is consistent with the low level of HIF-1α RNA in endothelial cells (Tian et al. 1997). Dex did, however, inhibit Hdm2 induction until 12 h under hypoxia (Fig. ​(Fig.3E,3E, lanes 2,3). In the presence of LLnL the difference in the level of induction was no longer observed, indicating that the difference is due to Dex-induced proteasomal degradation (Fig. ​(Fig.3E,3E, lanes 6,7). After 18 and up to 36 h the levels of Hdm2 were similar, irrespective of the presence of Dex and LLnL (Fig. ​(Fig.3E,3E, lanes 4,5,8,9; data not shown). Hypoxia induces apoptosis of HUVEC in a p53-dependent manner (Stempien-Otero et al. 1999). We, therefore, investigated whether Dex affected apoptosis by following PARP cleavage and DNA fragmentation. PARP cleavage, an early marker of apoptosis, was detected at 18 h and increased up to 36 h (Fig. ​(Fig.3F,3F, lanes 1–5). Dex inhibited PARP cleavage up to 36 h under hypoxia (Fig. ​(Fig.3F,3F, lanes 6–8). Similar results were obtained using DNA fragmentation (data not shown). These results show that the activation of endogenous GR with Dex in HUVEC, as well as HepG2, dramatically decreases p53 downstream functions.

Dex-dependent GR degradation by a proteasome pathway and inhibition of its target genes underhypoxia

Hypoxia has been shown to affect GR functions and levels of expression in several systems (Jenq et al. 1996; Bauer et al. 1999). Therefore, we investigated whether hypoxia affects GR in HUVEC. Hypoxia alone caused a reproducible increase in GR protein levels (Fig. ​(Fig.4A,4A, lanes 1,3,7), which was down-regulated by Dex (Fig. ​(Fig.4A,4A, lanes 2,3,6,7). This down-regulation was blocked by the proteasome inhibitor LLnL (Fig. ​(Fig.4A,4A, lanes 4,5,8,9). As Dex had no effect on GR at the RNA level under hypoxia (data not shown), we conclude that GR is decreased at the protein level by a proteasome-mediated process in response to Dex under hypoxic conditions, similar to p53 (see above).

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Figure 4

Dex enhances proteasome-mediated degradation of GR and prevents up-regulation of its target genes under hypoxia. (A) HUVEC were grown with vehicle (ethanol), 10⁻⁷ M Dex, and 50 μM LLnL, as indicated under normal (lane 1) or hypoxic (lanes 2–9) conditions. Lysates (50 μg) were Western blotted with antibodies to (a) GR (E-20 and P-20); (b) TBP (3G3). (Lanes 1,3,5) (−, vehicle); (lanes 2,4) +Dex; (lanes 6,8) +Dex+LLnL; (lanes 7,9) (−, vehicle)+LLnL. (B) Total RNA from HUVEC grown in normoxia (lanes 1,2) or hypoxia (lanes 3,4) for 18 h with vehicle (ethanol) or 10⁻⁷ M Dex. Northerns were hybridized with probes for (a) PEPCK; (b) G-6-Pase; (c) β-actin. (Lanes 1,3) +Dex; (lanes 2,4) (−, vehicle).

We next investigated whether hypoxia affects Dex induction of the GR downstream genes PEPCK (Imai et al. 1990) and Glucose-6-Phosphatase (G-6-Pase) (Argaud et al. 1996). Whereas Dex induced RNA expression from both of these genes under normoxia (Fig. ​(Fig.4B,4B, lanes 1,2), hypoxia blocked this induction (Fig. ​(Fig.4B,4B, lanes 3,4). Similarly, hypoxia blocked PEPCK protein induction (data not shown). These results concur with studies in rat hepatocytes, where in vivo hypoxic exposure inhibits gluconeogenesis at the PEPCK level due to decreased PEPCK gene transcription (Pison et al. 1995).

p53, GR, and Hdm2 colocalize in the cytoplasm in the presence ofligand

Our experiments show that p53 and GR are degraded by proteasomes in HepG2 under normal growth conditions and in HUVEC under hypoxia. Because subcellular localization affects p53 degradation, we studied the localization of p53 and GR in HUVEC cells (Fig. ​(Fig.5).5). As expected, under normal conditions p53 was not detectable (Fig. ​(Fig.5A),5A), because of its very low level of expression (see above). GR was located in the cytoplasm in the absence of ligand, but accumulated in the nucleus in the presence of Dex. Under hypoxic conditions, in the absence of Dex, p53 was detectable and was localized to both the nucleus and the cytoplasm in the majority of cells (65%), and exclusively in the nucleus in about 25%, whereas GR remained in the cytoplasm (Fig. ​(Fig.5B).5B). In the presence of Dex, p53 was completely cytoplasmic in around 60% of the cells, and was colocalized extensively with GR. The immunofluorecence of both GR and p53 was, however, considerably lower, as expected from their degradation (see above). To confirm these data, LLnL was used to stabilize the proteins and increase the signal. Typical cells are shown in Figure ​Figure5C–E,5C–E, and the quantification in Figure ​Figure5F.5F. The presence of LLnL did not change the results overall, but the effects were more pronounced. p53 was detected in the nucleus in almost 90% of the cells in the absence of Dex. However, in the presence of ligand, p53 and GR colocalized extensively in the cytoplasm (Fig. ​(Fig.5C).5C).

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Figure 5

Dexamethasone induces cytoplasmic colocalization of p53, GR, and Hdm2 under hypoxia. (A–E) HUVEC were grown with vehicle (no ligand) or 10⁻⁷ M Dex for 18 h in normoxia (A) or hypoxia (B–E), without (A,B) or with (C–E) 50 μM LLnL. Fixed cells were incubated with antibodies against (A–C) GR (E-20 and P-200) and p53 (DO-1); (D) GR (E-20 and P-20) and Hdm2 (2A10); (E) p53 (588) and Hdm2 (2A10), secondary antibodies, and analyzed by confocal microscopy. (F) Quantitation of panels (C–E). The cells were grown without (a,c,e) or with (b,d,f) 10⁻⁷ M Dex. At least 500 cells were counted for each graph. The percentage of cells with protein in the nucleus (N), cytoplasm (C), and both (N+C) are shown. (a,b) p53+GR; (c,d) Hdm2+GR; (e,f) Hdm2+p53.

Hdm2 is involved in p53 degradation and export from the nucleus, raising the possibility that it may also be implicated in GR turnover and localization. As expected, Hdm2 was found to be predominantly nuclear under hypoxia and in the absence of ligand. Interestingly, Hdm2 was cytoplasmic and extensively colocalized with GR in the presence of ligand (Fig. ​(Fig.5D).5D). Similarly, Hdm2 was found to colocalize with p53 in the nucleus in the absence of ligand and in the cytoplasm in the presence of Dex (Fig. ​(Fig.55E).

The ligand-dependent trimeric complex between p53, GR, and Hdm2 enhances proteasomaldegradation

The colocalization of p53, GR, and Hdm2 in the cytoplasm led us to investigate whether they form a trimeric complex. Coimmunoprecipitation assays were performed on extracts from HUVEC that had been exposed to hypoxia in the presence of LLnL and in the presence or absence of Dex. Equivalent amounts of p53 were immunoprecipitated with the anti-p53 antibody, irrespective of the presence of ligand, as expected from the inhibition of p53 degradation by LLnL (Fig. ​(Fig.6A,6A, c). The p53-antibody immunoprecipitate contained both GR and Hdm2 in significantly higher amounts in the presence of Dex than in its absence (Fig. ​(Fig.6A,6A, a,b, lanes 3,4). In the reciprocal experiment, the GR-antibody immunoprecipitates contained equivalent amounts of GR irrespective of Dex treatment (Fig. ​(Fig.6A,6A, f). They also contained both p53 and Hdm2 in significantly higher amounts in the presence of Dex (Fig. ​(Fig.6A,6A, d,e, lanes 7,8). Similar results were obtained in HepG2 cells, where p53, GR and Hdm2 formed a trimeric complex more efficiently following Dex treatment of the cells (Fig. ​(Fig.6B).6B).

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Figure 6

Dex stimulates the formation of a trimeric complex between p53, GR, and Hdm2 and ubiquitylation of GR and p53 by the E3 ligase activity of Hdm2. (A) HUVEC were grown in the presence of 50 μM LLnL and vehicle (−, ethanol) or 10⁻⁷ M Dex for 18 h under hypoxic conditions. Lysates (500 μg) were immunoprecipitated with antibodies against p53 (421) or GR (E-20 and P-20). Immunoprecipitates (IP) were Western blotted with antibodies against GR and Hdm2 (for p53 IP; a,b) or Hdm2 and p53 (for GR IP; d,e). The efficiency of IP was verified with self-antibodies (c,f). DO-1 was used to reveal p53 on the Western blots. (Lanes 1,3,5,7) +Dex; (lanes 2,4,6,8) (−, vehicle). Input, 10% lysate used for IP. (B) HepG2 cells were grown in the presence of 50 μM LLnL and vehicle (−, ethanol) or 10⁻⁷ M Dex for 12 h. Lysates (500 μg) were immunoprecipitated with antibodies against GR (E-20 and P-20). The IPs were Western blotted with antibodies against Hdm2 and p53 (a,b). The efficiency of IP was checked with self-antibodies (c). (Lanes 1,3) +Dex; (lanes 2,4) (−, vehicle). Input, 10% lysate used for IP. (C) HUVEC cells were transfected (lanes 1–4) with HA-Ub, pBC–IP3.2, or pBC, and 24 h posttransfection subjected to hypoxia for 18 h with LLnL (50 μM) and either vehicle (−, ethanol) or 10⁻⁷ M Dex. Lysates (500 μg) were immunoprecipitated with antibodies against HA (12CA5). The IPs were Western blotted with antibodies against with GR (E-20 and p-20) (a) and p53 (DO-1) (b). (D) HCT116 p53 (WT) (lanes 1,2,5,6) or HCT116 p53^−/− (lanes 3,4,7,8) cells were transfected with pHA–Ub, pBC–IP3 (where indicated), and pEGGFP-C1 (lanes 5–8, to measure transfection efficiency). Twelve hours posttransfection the cells were incubated with 10⁻⁷ M Dex under hypoxia for 12 h, with (lanes 1–4) or without (lanes 5–8) 50 μM LLnL. Lysates (500 μg) from cells grown in the presence of LLnL were immunoprecipitated with antibodies against HA (12CA5). The IPs were Western blotted with antibodies against GR (E-20 and p-20; a). Lysates (50 μg) from cells with LLnL were Western blotted with antibodies to (b) GR (E-20 and P-20); (c) p53 (DO-1); (d) GFP (monoclonal). (Lanes 1,3,5,7) −IP3; (lanes 2,4,6,8) +IP3. (E) HCT116 p53^−/− cells were transfected with pcDNA3–GR, pRC p53 (WT) pRC p53(22,23), pxMDM2, and pEGGFP-C1 (to measure transfection efficiency). Twelve hours posttransfection the cells were subjected to hypoxia for 12 h with 10⁻⁷ M Dex. Lysates (50 μg) were Western blotted with antibodies against (a) GR (E-20 and P-20); (b) p53 (1801); c) Hdm2 (2A10); (d) GFP (monoclonal). (Lane 1) Mock transfection; (lane 2) GR; (lane 3) p53+GR; (lane 4) p53+GR+Hdm2; (lane 5) p53(22,23)+GR+Hdm2. (F) HCT116 p53^−/− cells were transfected with pcDNA3–GR, pRC p53 (WT), pxMDM2, px ΔR, and pxC462A. Twelve hours posttransfection the cells were subjected to hypoxia for 12 h with 10⁻⁷ M Dex. Lysates (50 μg) were Western blotted with antibodies against (a) Hdm2 (2A10); (b) GR (E-20 and P-20); (c) p53 (DO-1); (d) TBP (3G3). (Lane 1) Mock transfection; (lane 2) p53+GR; (lane 3) p53+GR+ΔR; (lane 4) p53+GR+Hdm2; (lane 5) p53+GR+C462A.

GR has been postulated to be degraded by the ubiquitin–proteasome pathway (Whitesell and Cook 1996; Segnitz and Gehring 1997), but there was no direct evidence. Our results do indicate, however, that during cross talk between the GR and p53 pathways, GR is degraded concurrently with p53 by a pathway involving the E3 ubiquitin ligase activity of MDM2. To test this hypothesis HUVEC cells were transfected with HA-tagged ubiquitin (HA-Ub) in the presence or absence of IP3, a small peptide that specifically disrupts the p53–Hdm2 interaction (Wasylyk et al. 1999). Twenty-four hours post-transfection, the cells were placed under hypoxic conditions in the presence of the proteasome inhibitor, LLnL. Western blotting of HA-immunoprecipitates with GR antibodies showed that GR was poly-ubiquitinated, but only when the cells were incubated in the presence of Dex (Fig. ​(Fig.6C,6C, a, lanes 1,2). IP3 expression inhibited GR poly-ubiquitylation, showing that it requires the p53–Hdm2 interaction (Fig. ​(Fig.6C,6C, a, lanes 1,3). The poly-ubiquitinated forms are specific, because they were not detected when HA-Ub was not transfected (Fig. ​(Fig.6C,6C, a, lanes 5,6). Interestingly, polyubiquitylation of p53 is also enhanced in the presence of Dex (Fig. ​(Fig.6C,6C, b, lanes 1,2). Polyubiquitylation of p53 was inhibited by the expression of IP3, as expected from the disruption of the p53–Hdm2 interaction (Fig. ​(Fig.6C,6C, b, lanes 1,3). These results show that Dex stimulates GR and p53 polyubiquitylation through a pathway involving Hdm2 and its interaction with p53.

The E3 ligase activity of Hdm2 is responsible for ligand-dependent degradation ofGR

To study GR degradation in transfection assays, in which exogenous proteins are expressed in cells, we used HCT116 isogenic cell lines in which p53 is either wild type or disrupted by homologous recombination (Bunz et al. 1998). The isogenic cell lines were transfected with recombinants that express HA-Ub and IP3. Posttransfection, the cells were subjected to hypoxia in the presence of Dex and LLnL. HA-antibody immunoprecipitates contained lower mobility HA-tagged polyubiquitylated forms of GR (Fig. ​(Fig.6D,6D, a, lane 1). IP3 expression inhibited ubiquitylation of GR (Fig. ​(Fig.6D,6D, a, lane 2). Ubiquitylated GR was not detected in cells lacking p53, whether or not IP3 was expressed (Fig. ​(Fig.6D,6D, a, lanes 3,4). These results indicate that GR ubiquitylation requires p53 and its interaction with Hdm2. In cells that were not treated with LLnL, IP3 expression increased the levels of both GR and p53 (Fig. ​(Fig.6D,6D, a, lanes 5,6), as expected from decreased degradation of both proteins by a common mechanism involving Hdm2. Irrespective of the presence or absence of IP3, GR levels were higher in the isogenic p53^−/− cells (Fig. ​(Fig.6D,6D, a, lanes 7,8), as expected from the lack of p53 that is required to recruit Hdm2.

To confirm that p53–Hdm2 interactions are required for GR degradation, we used p53(22,23), which cannot interact with Hdm2 (Lin et al. 1994). Expression of wild-type p53, Hdm2, and exogenous GR in HCT116 p53^−/− cells in the presence of Dex resulted in a significant decrease in GR levels (Fig. ​(Fig.6E,6E, lanes 1–4). In contrast, the GR levels remained unchanged when the p53(22,23) mutant was expressed (Fig. ​(Fig.6E,6E, lane 5). Interestingly, similar to GR, the levels of p53 and Hdm2 were also higher with p53(22,23) compared to p53 wild type (Fig. ​(Fig.6E,6E, lanes 5,6). These results suggest that Dex-dependent ternary complex formation induces degradation of all three proteins.

To determine whether the E3 ligase activity of Hdm2 is responsible for GR ubiquitylation and degradation, HCT116 p53^−/− cells were transfected with GR, p53, and the Mdm2 mutants ΔR and C462A, which abolish the E3 ligase activity of Mdm2 (Argentini et al. 2000). Mdm2 promoted the degradation of both p53 and GR, whereas the mutants protected both proteins from degradation (Fig. ​(Fig.6F).6F). These results show that Mdm2 E3 ligase activity is required for GR degradation.

Cells

HSC-2 (Momose et al. 1989); HUVEC, ATCC (CRL-1730); HepG2, ECACC (Ref. 85011430); HCT116 p53(WT) and HCT116 p53(−/−) (Bunz et al. 1998).

Protein expression, purification, and GSTinteractions

GST proteins were induced with 1 mM IPTG in E. coli DH5α grown at 25°C, and extracted by sonication in STE buffer (10 mM Tris-HCl at pH 8.0; 150 mM NaCl; 1 mM EDTA supplemented with 5 mM DTT, 1mM PMSF, 2.5 μg/mL proteinase inhibitors and 1.5% Sarkosyl). The sonicate was adjusted to 2% Triton X-100, agitated for 30 min at 4°C, centrifuged, and the crude extract stored in aliquots at −70°C. Glutathione-S-Sepharose beads were equilibrated in GST buffer (50 mM Tris-HCl at pH 7.5, 100 mM KCl, 10 mM MgCl₂, 5% glycerol, 0.5% NP-40 supplemented with 0.3 mM DTT, 1 mM PMSF and 2.5 μg/mL proteinase inhibitors), incubated with the crude extracts at 4°C for 90 min, and washed. Bound proteins were analyzed by SDS-PAGE and Coomassie staining. GR was produced by in vitro translation with pcDNA3GR and the TNT Quick coupled transcription/translation system (Promega) for 90 min at 30°C. Equivalent amounts of bound protein were incubated with 5 μL of in vitro translated GR for 1 h at 4°C in the presence of vehicle (ethanol), Dex, Cortisol, or RU486 (Sigma). The bound proteins were washed and analyzed by SDS-PAGE and fluorography.

Cell culture andtransfection

Cells were maintained in DMEM plus 10% serum and antibiotics at 37°C with 5% CO₂. HUVEC medium was supplemented with ECGF (50 μg/mL), Heparin (50 μg/mL), and Glutamine (1 mM). The HCT116 cell lines were grown in McKoy's 5A medium plus 10% serum, antibiotics, and 2 mM glutamine. HSC-2 were transfected by calcium phosphate precipitation (Chen and Okayama 1987) with 4 μg of plasmid in 36-mm plates (six-well cluster, Costar 3516). After 16 h the cells were washed with medium and incubated in full medium with ligands or vehicle (ethanol). To express HA-Ub and IP3, low-passage HUVEC (<15) was transfected with Superfect (QIAGEN) according to the manufacturer's instructions. After 3 h with Superfect the cells were incubated in normal medium for 24 h. HCT116 cells were transfected with Lipfectamine 2000 (Life Technologies) in the presence of serum for 6 h, washed, and incubated in normal medium for 12 h before hypoxic treatment. Post-transfection is defined as after the removal of the facilitator. Hypoxia experiments were carried out in anaerobic chambers (Anaerocult, Merck, Cat. no. 1.13829) at 37°C according to the manufacturer's instructions; 10 μM mitomycin C (Sigma) was used.

Transcriptional activity

Cell lysates were analyzed for luciferase and β-galactosidase activities as described previously (Sengupta et al. 2000a).

Western blots

Equal quantities of protein, estimated by the Bio-Rad assay on RIPA buffer lysates, were fractionated by SDS-PAGE, transferred to nitrocellulose membranes (Schleicher and Schuell), and Western blotted. Primary and secondary antibodies: Bax, N20 (Santa Cruz Biotechnology); p21^WAF1/CIP1, 1WA-IC581 (Sengupta et al. 2000a); TBP, 3G3 (Brou et al. 1993); p53, DO-1 (Vojtesek et al. 1992); p53, PAb 421 (Wade-Evans and Jenkins 1985); p53, #588, raised against a GST–human p53(320–393) (C. Wasylyk and the IGBMC core facility); GR, E-20 and P-20 (Santa Cruz Biotechnology); PARP (Kaufmann et al. 1993); HA, 12CA5 (Boerhinger); PEPCK (Zimmer and Magnuson 1990); Hdm2, 2A10 (Chen et al. 1993); HIF-1α, C-19 (Santa Cruz Biotechnology); peroxidase-coupled anti-rabbit or anti-mouse antibodies (Jackson Laboratories); p53, 1801 (Ab-2) (Oncogene Research Products); GFP (Clontech). The blots were revealed by chemiluminescence (Pierce Super Signal).

Northern blots

Forty micrograms of total RNA, extracted with Trizol (GIBCO BRL), was electrophoresed on 6% formaldehyde–1% agarose gels, transferred to Hybond N+ membranes, hybridized with random-primed probes at 55°C (for PEPCK and G-6-Pase) or 65°C (for p53, GR, and actin), washed, and autoradiographed.

Immunofluorescence

Cells were fixed with acetone:methanol (1:1), blocked with 10% goat serum, incubated with primary antibodies for 2 h, secondary antibodies linked to either Cy3 or FITC (Moll et al. 1996) and Hoechst (to stain nuclei), then visualized by confocal microscopy (Leica TCS 4D).

We thank Julia Young for critical reading of the manuscript; D. Bohmann, E. Conseiller, M.R. el-Maghrabi, T. Kietzmann, and J. Shay for the gifts of recombinants; N. Tsuchida for HSC-2 cells; D. Granner, G. de Murcia, and L. Tora for the gift of antibodies; the IGBMC core facilities for help and support; and BioAvenir (Aventis, Rhone-Poulenc), the Centre National de la Recherche Scientifique, the Institut National de la Santé et de la Recherche Médicale, the Hôpital Universitaire de Strasbourg, the Association pour la Recherche sur le Cancer, the Fondation pour la Recherche Médicale, the Ligue Nationale Française contre le Cancer (Equipe labellisée), the Ligue Régionale (Haut-Rhin) contre le Cancer, and the Ligue Régionale (Bas-Rhin) contre le Cancer for financial assistance.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.