Influence of extracellular factors on p53-mediated DNA damage responses

Cells have evolved sophisticated mechanisms to maintain genomic stability after cellular stress. Activation of DNA damage response pathways, and most importantly p53, leads to adaptive responses that can be influenced by different extracellular factors. The aim of this project was to study how extracellular factors modulate p53 cell-fate decisions after DNA damage, with particular interest in oxygen tensions and vitamin A metabolites. First, we focused on how physiological oxygen tensions (5% 02) may influence cellular responses to genotoxic stress. We showed that normal and cancer cells cultured at 5% 02 had a reduction in p53-mediated apoptosis after exposure to different genotoxic stresses. This was not due to a decrease p53 protein levels or its transactivation activity, and the oxidative damage caused by DNA damaging agents was not affected by oxygen tensions. We also found a p53-independent activation of MAPK at 5% 02, which when inhibited restored levels of p53-induced apoptosis. HIF-1α, a transcription factor induced at lower O2 concentrations, was present and active at 5% 02. However, this did not affect MAPK activation and HIF-1α was not involved in the resistance to apoptosis under these conditions, although MAPK was necessary for HIF-1α expression and activation. We next explored the effect of the vitamin A (retinol) pathway on the cellular responses to DNA damage. We showed that Stra6, a retinol-inducible gene, is upregulated by p53 after DNA damage. While overexpression of Stra6 sensitized cells to p53-induced apoptosis independently of the retinoic acid signalling, its inhibition resulted in decreased apoptosis after DNA damage and less induction of oxidative stress. This shows that both oxygen tensions and vitamin A metabolites, through Stra6, are potent modulators of the p53 responses to DNA damage

Inhibition of HIF-1α expression by DNA damaging agents.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells treated with 1.25 µM U0126 and 0.4 µg/ml doxorubicin, and cultured at 20% or 5% O₂ for 2 days. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α in HCT116 and HCT116 p53^−/− in lysates collected 24 hours after treatment with 1 µg/ml Actinomicyn D (ActD) or 200 µM tert-Butyl Hydroperoxyde (tBH) for 2hours (<b>C</b>) Western blot showing the protein levels of HIF-1α, p53 and p21 in HCT116 and MCF-7 cells treated with 0.4 µg/ml doxorubicin or 10 Gy γ-radiation for 24 hours at 20% or <1% O₂ (hypoxia).</p

The role of the HIF-1α transcription factor in increased cell division at physiological oxygen tensions

HIF-1 is a transcription factor that mediates the cellular responses to low oxygen environments, mainly as a result of having an oxygen-labile subunit, HIF-1α. HIF-1α has been carefully studied in the context of severe hypoxic stresses (<1% O[subscript 2]), but it is also known to be present at oxygen tensions commonly found in normal tissues in vivo (∼1-13% O[subscript 2]), albeit at much lower levels. Its role under these physiological conditions is not fully understood. Here, we show that a transcriptionally active HIF-1α was up-regulated at 5% O[subscript 2], both in normal and cancer cells, but only some of its target genes were elevated as a result. HIF-1α induction was in part dependent on the activation of the ERK1/2 MAPK signalling pathway, which we have previously shown is active at 5% O[subscript 2]. We also found that HIF-1α does not contribute to the protection against DNA damage that can be observed in low oxygen environments, and that there are certain DNA damaging agents, such as doxorubicin and actinomycin D, that prevent HIF-1α induction independently of p53. Moreover, absence of HIF-1α significantly reduced the growth advantage of cells cultured at 5% O[subscript 2]. In view of these data, we conclude that HIF-1α can be induced and activated at physiological oxygen tensions in a MAPK-dependent manner and that, although this does not lead to pro-survival responses to stress, it determines the increased cell proliferation rates that are common under these conditions

HIF-1α has no effect on the activation of MAPK at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α, p53, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 cells transfected with 200 pmol of siRNA against HIF-1α and treated with doxorubicin (0.4 µg/ml). Control cells were transfected with a luciferase siRNA instead. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>B</b>) Western blot showing the protein levels of HIF-1α, phosphorylated ERK 1/2 (P-MAPK) and ERK 1/2 (MAPK) in HCT116 treated with 40 µM YC-1 and/or 0.4 µg/ml doxorubicin and cultured at 20% or 5% O₂ for 24 hours. Cells treated with 500 µM CoCl₂ for 16 hours were used as a positive control for the induction of HIF-1α (H). (<b>C</b>) Representative FACS plots of HCT116 cells stained with PI. Cells were transfected with 50 pmol of siRNA against HIF-1α and treated with 0.4 µg/ml doxorubicin. Cells were cultured at 20% or 5% O₂ for 24 hours. Percentages indicate number of subG₁ events (dead cells).</p

Chemical inhibition of MAPK reduces the activation of HIF-1α.

<p>Western blot showing the protein levels of HIF-1α and phosphorylated (active) ERK 1/2 MAPK in HCT116 cultured at 20% or 5% O₂for 12 to 48 hours, in the presence of 1.25 µM U0126. U0126 was added at the same time cells were transferred to 5%O₂. Total MAPK levels are provided as loading control.</p

HIF-1α contributes to increased proliferation of cells at physiological oxygen tensions.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116 HIF^+/+ and HIF^−/−cultured either at 20% O₂ or under hypoxic stress (<0.1% O₂) for 16 hours. (<b>B</b>) Proliferation curves of HIF^+/+ and HIF^−/− HCT116 cells cultured at 20% or 5% O₂ from 2 to 8 days. Values represent ratio of cell numbers normalized to the initial seeded cells (10⁶). (<b>C</b>) Representative colony formation assay for HCT116 HIF^+/+ and HIF^−/−cultured at 20% or 5% O₂. 200 cells were seeded in each plate and 14 days later they were stained with Giemsa. Media was not changed during the process. (<b>D</b>) Percentage of EdU positive HCT116 HIF^+/+ and HIF^−/− cells as assessed by immunofluorescence (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097938#pone.0097938.s002" target="_blank">Figure S2</a>). Cells were incubated with EdU for 30 minutes in the corresponding oxygen tensions. Results represent means of two independent experiments. Two microscope fields were scored in each experiment. Error bars represent standard error. P value (unpaired t-test): 0.0127 (*), (<b>E</b>) Proposed model of the roles of HIF-1 at different oxygen concentrations.</p

Physiological oxygen tensions induce HIF-1α expression and activity.

<p>(<b>A</b>) Western blot showing the protein levels of HIF-1α in HCT116. Cells were cultured at 20% or 5% O₂ for 72 hours. (<b>B</b>) Western blot showing the protein levels of HIF-1α in normal human keratinocytes cultured for 1 to 4 days at 5% O₂ or treated 500 µM of chemical hypoximimetic CoCl₂ for 16 hours (H). (<b>C</b>) Luciferase assay showing HIF-1α in HCT116 cells. HCT116 were transfected with a PGK-1 luciferase reporter plasmid and a β-galactosidase control plasmid and then cultured for 48 hours at 5% O₂. β-galactosidase activity was used to normalize luciferase activity. Luciferase activity is expressed as a ratio to 20% O₂ levels. Results show mean values of 3 independent experiments and error bars represent standard deviation (<b>D</b>) qRT-PCR showing mRNA levels of Glut-1, PGK-1 and VEGF in HCT116 cells cultured at 20% and 5% O₂ for 24 hours. Results show mean values of 3 independent experiments and error bars represent standard deviation. P values (unpaired t-tests): 0.03 (*), 0.001 (**), 0.7 (ns). (E) Western blot of lysates of HCT116 cultured at 20% or 5% O₂ for 72 hours, showing expression of Glut-1 and PHD2.</p

Reactive oxygen species and mitochondrial sensitivity to oxidative stress determine induction of cancer cell death by p21.

p21(Waf1/Cip1/Sdi1) is a cyclin-dependent kinase inhibitor that mediates cell cycle arrest. Prolonged p21 up-regulation induces a senescent phenotype in normal and cancer cells, accompanied by an increase in intracellular reactive oxygen species (ROS). However, it has been shown recently that p21 expression can also lead to cell death in certain models. The mechanisms involved in this process are not fully understood. Here, we describe an induction of apoptosis by p21 in sarcoma cell lines that is p53-independent and can be ameliorated with antioxidants. Similar levels of p21 and ROS caused senescence in the absence of significant death in other cancer cell lines, suggesting a cell-specific response. We also found that cells undergoing p21-dependent cell death had higher sensitivity to oxidants and a specific pattern of mitochondrial polarization changes. Consistent with this, apoptosis could be blocked with targeted expression of catalase in the mitochondria of these cells. We propose that the balance between cancer cell death and arrest after p21 up-regulation depends on the specific effects of p21-induced ROS on the mitochondria. This suggests that selective up-regulation of p21 in cancer cells could be a successful therapeutic intervention for sarcomas and tumors with lower resistance to mitochondrial oxidative damage, regardless of p53 status

Protection of cells in physiological oxygen tensions against DNA damage-induced apoptosis.

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy