Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation - PubMed (original) (raw)

Regulation of p53 by hypoxia: dissociation of transcriptional repression and apoptosis from p53-dependent transactivation

C Koumenis et al. Mol Cell Biol. 2001 Feb.

Abstract

Hypoxic stress, like DNA damage, induces p53 protein accumulation and p53-dependent apoptosis in oncogenically transformed cells. Unlike DNA damage, hypoxia does not induce p53-dependent cell cycle arrest, suggesting that p53 activity is differentially regulated by these two stresses. Here we report that hypoxia induces p53 protein accumulation, but in contrast to DNA damage, hypoxia fails to induce endogenous downstream p53 effector mRNAs and proteins. Hypoxia does not inhibit the induction of p53 target genes by ionizing radiation, indicating that p53-dependent transactivation requires a DNA damage-inducible signal that is lacking under hypoxic treatment alone. At the molecular level, DNA damage induces the interaction of p53 with the transcriptional activator p300 as well as with the transcriptional corepressor mSin3A. In contrast, hypoxia primarily induces an interaction of p53 with mSin3A, but not with p300. Pretreatment of cells with an inhibitor of histone deacetylases that relieves transcriptional repression resulted in a significant reduction of p53-dependent transrepression and hypoxia-induced apoptosis. These results led us to propose a model in which different cellular pools of p53 can modulate transcriptional activity through interactions with transcriptional coactivators or corepressors. Genotoxic stress induces both kinds of interactions, whereas stresses that lack a DNA damage component as exemplified by hypoxia primarily induce interaction with corepressors. However, inhibition of either type of interaction can result in diminished apoptotic activity.

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Figures

FIG. 1

FIG. 1

Hypoxia induces p53 accumulation but uncouples it from p21 accumulation. (A) Western blot analysis of p53 and p21 levels from RKO exposed to different levels of hypoxia for 6 h or following 5 h of reoxygenation after hypoxia treatment. (B) Western blot analysis of p53 and p21 protein levels in GM2184B lymphoblasts with wt p53 following treatment with 6 Gy of IR or hypoxia for the indicated times. Also shown are western blot analyses of p21 levels in RKO cells (C) and in MCF-7 cells (D) after treatment with 6 Gy of IR or with hypoxia for the indicated times.

FIG. 2

FIG. 2

IR but not hypoxia induces Mdm-2 protein and mRNA accumulation. (A) Time course of the regulation of Mdm-2 levels in cell extracts prepared at the indicated times under hypoxia or 3 h following treatment with 6 Gy of IR. (B) Northern blot analysis of Mdm-2 mRNA levels following treatment with hypoxia or 6 Gy of IR. Hybridization to 18S rRNA was used as a loading control (Con).

FIG. 3

FIG. 3

Hypoxia-induced p53 fails to transactivate endogenous effector gene mRNAs. MCF-7 cells were treated with hypoxia or with 6 Gy of IR, and at the times indicated whole-cell lysates and total RNA was prepared. (A) Western blot analysis of p53 levels following treatment of MCF-7 cells. (B) Multi-RPAs of mRNA levels of various p53 effectors and other genes involved in cellular stress and apoptotic responses. RPAs were performed in MCF-7 cells using RNA probes synthesized from a set containing various stress-induced genes (hStress set). Probes that are complementary to two housekeeping genes (ribosomal protein L32 and GAPDH) serve as normalization controls (Con).

FIG. 4

FIG. 4

Irradiation of MCF7 and RKO cells under hypoxia induces p53-dependent transactivation. Cells were grown in normoxic conditions (lanes 1 to 3, MCF-7, and lanes 1, 2, and 5, RKO) or exposed to hypoxia (lanes 4 to 6, MCF-7, and lanes 3 and 4, RKO). Cells in lanes 2, 3, 5, and 6 (MCF-7) and lanes 2 and 4 (RKO) received 3 or 10 Gy of IR, as indicated, 3 h before cell lysis. Cells in lane 5 (RKO) were treated with 20 μM ALLN for 3 h before cell lysis. Following cell lysis, total RNA was isolated and RPAs were performed as described in Materials and Methods.

FIG. 5

FIG. 5

Hypoxia-induced p53 is mainly localized to the nucleus. RKO cells were grown under normoxia (a to d), or exposed to hypoxia (e to h). Nuclei were visualized with DAPI (4′,6′-diamidino-2-phenylindole) counterstaining (a, c, e, and g), while p53 was visualized using the DO-1 monoclonal antibody and a fluorescein-conjugated mouse secondary antibody (d, f, and h). Panel b depicts fluorescence due to binding of the secondary antibody alone. Images in panels g and h were taken using a higher magnification objective (×60) than the other panels (×20).

FIG. 6

FIG. 6

Hypoxia induces p53-dependent transrepression. (A) Doxycycline (200 ng/ml) induces p53 expression in H1299 cells transfected with a _tet_-inducible construct (clone 30) under normoxic and hypoxic conditions. The slight increase in p53 levels under hypoxia in the absence of doxycycline is probably due to stabilization of residual p53 expressed due to the “leakiness” of the construct. (B) Northern analysis of β-tubulin (β-tubul.) mRNA levels in an H1299 clone expressing a _tet_-inducible p53 construct and the parental clone transfected with empty vector. Cells were treated with hypoxia, doxycycline, or doxycycline for 2 h followed by treatment with hypoxia. In this cell line, hybridization with the β-tubulin probe results in two signals, probably because of alternative splicing of the β-tubulin. At the bottom, a methylene blue-stained nitrocellulose membrane, indicating levels of 18S and 28S RNA is shown. (C) Northern blot analysis of the p53-transrepressed gene coding for α-tubulin after treatment of p53+/+ and p53−/− MEFs with hypoxia or with 10 J of UV radiation/m2. Hybridization to 18S rRNA was used as a control. This is a light PhosphorImager scan that better represents the repression levels of α-tubulin. Values represent relative β-tubulin levels.

FIG. 7

FIG. 7

Differences in p53 modifications induced by IR and hypoxia. (A) IR but not hypoxia induces acetylation of Lys382. RKO cells were treated with hypoxia, 6 Gy of IR, or 20 μM ALLN for the times indicated. Immunoblotting was performed with a rabbit polyclonal antibody raised against Lys382. (B) Both IR and hypoxia induce Ser15 phosphorylation of p53. Treatment times are indicated. The top panel shows an immunoblot using a rabbit polyclonal that recognizes p53 phosphorylated at Ser15. The bottom panel shows the same immunoblot after being stripped and reprobed with the DO-1 monoclonal antibody.

FIG. 8

FIG. 8

Interactions of p53, p300, and mSin3A after hypoxia and ionizing radiation exposure. (A) Immunoprecipitation of mSin3a or p300 was performed on extracts from cells treated with 3 Gy of IR (top) or hypoxia-ALLN (bottom) using polyclonal antibodies against the corresponding proteins. One-tenth of the cell extract was loaded in a lane of each set to assay for p53 levels. The blot was probed with the DO-1 anti-p53 monoclonal antibody. (B) Immunoblot on cell lysate (top) and immunoprecipitation (IP) followed by immunoblotting (bottom) of p53 levels in extracts of cells treated with doxorubicin (100 ng/ml) for 12 h, hypoxia for 12 and 24 h, or both doxorubicin (12 h) and hypoxia (24 h). In the third case, doxorubicin was added during the last 12 h of hypoxia. The same anti-p300 polyclonal antibody (N-15) that was used in the top panel of B was used here. The table at the bottom of the figure indicates the tail moment values from cells treated as described above as determined by the Comet assay. Values are averages of 75 cells per treatment group and are reported along with SEMs for each group.

FIG. 9

FIG. 9

(A) TSA inhibits p53-dependent apoptosis under hypoxia. H1299 cells transfected with a p53 expression vector under the control of the tetracycline promoter were treated with IR or hypoxia. One group of cells did not receive any additional treatment (CON). TSA (25 nM) was added to a second group 1 h prior to treatment. A third group was treated with doxycycline 2 h prior to treatment, while both TSA and doxycycline were added to a fourth group prior to treatments. At the end of the treatment, cells with apoptotic morphology were visualized with Hoechst 3342 and phosphatidyl inositol staining. The total number of cells and the number of apoptotic cells in four different fields in 60-mm-diameter dishes were counted and expressed as the percentage of the total number of cells. Numbers represent the average of three independent experiments. Error bars represent SEM (B) TSA inhibits p53 transrepression under hypoxia. RKO cells were treated as described above, with the exception that cells were lysed 6 h after IR treatment and 12 h after hypoxia treatment. Northern blot analysis was performed on total RNA using a probe for human α-tubulin and 18S rRNA as described above.

FIG. 10

FIG. 10

Model for the regulation of p53 function by genotoxic stress (top) and hypoxia (bottom). See text for details.

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