Uncontrolled accumulation of reactive oxygen species (ROS) causes oxidative stress and induces harmful effects. Both high ROS levels and p53 mutations are frequent in human cancer. Mutant p53 forms are known to actively promote malignant growth. However, no mechanistic details are known about the contribution of mutant p53 to excessive ROS accumulation in cancer cells. Herein, we examine the effect of p53R273H, a commonly occurring mutated p53 form, on the expression of phase 2 ROS-detoxifying enzymes and on the ability of cells to readopt a reducing environment after exposure to oxidative stress. Our data suggest that p53R273H mutant interferes with the normal response of human cells to oxidative stress. We show here that, upon oxidative stress, mutant p53R273H attenuates the activation and function of NF-E2-related factor 2 (NRF2), a transcription factor that induces the antioxidant response. This effect of mutant p53 is manifested by decreased expression of phase 2 detoxifying enzymes NQO1 and HO-1 and high ROS levels. These findings were observed in several human cancer cell lines, highlighting the general nature of this phenomenon. The failure of p53R273H mutant-expressing cells to restore a reducing oxidative environment was accompanied by increased survival, a known consequence of mutant p53 expression. These activities are attributable to mutant p53R273H gain of function and might underlie its well-documented oncogenic nature in human cancer.
Molecular oxygen (O2) and its homeostasis are essential for the survival of all aerobic organisms. Under normal physiological conditions, partially reduced oxygen metabolites, including hydrogen peroxide (H2O2), superoxide anion (O2−) and hydroxyl radical (OH−) are generated as metabolic by-products, termed the reactive oxygen species (ROS) (Cho et al., 2006). ROS can be either harmful or beneficial to living systems. On the one hand, low to moderate levels of ROS are involved in normal cellular processes. On the other hand, uncontrolled accumulation of ROS leads to “oxidative stress” which represents the harmful effects of ROS including damage to cell structure, nucleic acids, lipids and proteins (Cho et al., 2006; Valko et al., 2001; Valko et al., 2006). Accordingly, when ROS accumulate in a cell, a defense mechanism inactivates these ROS to prevent their possible harmful effect. This defense mechanism is associated with transcriptional activation of a variety of detoxifying enzymes and molecules such as superoxide dismutase (SOD) heme oxygenase-1 (HO-1), quinone oxidoreductase (NQO1) and glutathione (GSH) (Kobayashi and Yamamoto, 2005).
ROS-induced expression of detoxifying enzymes was shown to be mediated by the transcription factor erythroid 2 (NF-E2) related factor 2 (NRF2). Under normal conditions this transcription factor is maintained at low levels through constitutive proteosomal degradation mediated by the E3 ubiquitin ligase (Kobayashi et al., 2004; Kobayashi and Yamamoto, 2005). However, under oxidative stress, NRF2 is being stabilized, it translocates to the nucleus and induces the expression of antioxidant genes through interaction with specific antioxidant responsive elements (Kobayashi and Yamamoto, 2005) (Katoh et al., 2005; Kobayashi et al., 2004). NRF2 plays a central role in maintaining an intact balance of ROS activity in living cells preventing the damage to cellular structures and especially the DNA. Accordingly, NRF2 was suggested to play a role as a tumor suppressor protein. Indeed, it was demonstrated that NRF2−/− mice exhibit low detoxifying enzyme activity and are highly susceptible to tumor formation (Ramos-Gomez et al., 2001b).
More than 50% of human tumors acquire a mutation in p53 protein. Most of these mutations simultaneously lead to both loss of wild-type p53 tumor-suppressive activity and acquisition of new functions actively promoting malignant transformation. These oncogenic activities, known as mutant p53 “gain of function” may lead to enhanced cell proliferation, invasiveness, metastasis and resistance to a variety of anti-cancer drugs (Brosh and Rotter, 2009; Buganim et al., 2010; Kogan-Sakin et al., 2011; Muller et al., 2009; Stambolsky et al., 2010; Stambolsky et al., 2010). In the current study, we aimed to investigate how a hot-spot p53R273H mutation, known to possess oncogenic gain of function, affects cellular response to oxidative stress and expression of antioxidant defense enzymes. We report herein on a cross-talk between the NRF2 pathway and the p53R273H mutant. We observed a reduced expression of phase 2 enzymes following oxidative stress in mutant p53R273H-bearing cells. Furthermore, this reduction was associated with augmented ROS accumulation and resistance to cell death. The role of mutant p53R273H in increasing ROS levels through attenuating the NRF2 stress response pathway contributes to understanding mutant p53 gain of function in tumorigenic processes.
p53R273H mutant attenuates the induction of NQO1 and HO-1 following oxidative stress in HCT116 colon carcinoma cells
Numerous studies have indicated increased ROS activity in cancer cells when compared with normal cells (Valko et al., 2001; Valko et al., 2006; Vousden and Ryan, 2009). Increased ROS activity is usually associated with a repression of the antioxidant defense system including the ROS detoxifying enzymes and their regulators (Nguyen et al., 2009; Osburn et al., 2006). Furthermore, it is well established that more than 50% of human cancers lose wild-type p53 tumor suppressor while acquiring a mutated form of the protein (Brosh and Rotter, 2009). These two notions prompted us to explore whether mutant p53 is involved in the increased ROS activity observed in cancer cells. Therefore, we decided to evaluate the effect of mutant p53 on the expression of NQO1 and HO-1 detoxifying enzymes and their transcriptional regulator NRF2. To this end, we first used HCT116 colon carcinoma cells in which a hot-spot p53R273H mutant was overexpressed and green fluorescent protein (GFP) was used as a control (Fig. 1A). To induce oxidative stress, we applied diethylmaleate (DEM), a glutathione-depleting compound, which leads to the accumulation of ROS. Western blot analysis indicated that treatment of HCT116 cells with 100 µM DEM induced accumulation of the NRF2 protein in both control and p53R273H mutant expressing cells (Fig. 1A). Moreover, quantitative reverse transcriptase PCR (QRT-PCR) indicated that increased NRF2 protein levels were accompanied by an increase in mRNA encoding NQO1 and HO-1 (Fig. 1B,C). Importantly, both NQO1 and HO-1 were induced to a much lesser extent in HCT116 cells harboring the p53R273H mutant compared with HCT116 control cells following DEM treatment (Fig. 1B,C). This implies that p53R273H mutant diminishes the levels of phase 2 detoxifying enzymes following oxidative damage.
Since HCT116 cells bear wild-type p53, it was important to test whether the effect of mutant p53 on the expression of phase 2 enzymes was due to inactivation of wild-type p53 by the overexpressed mutant protein. Mutant p53 is known to promote its oncogenic effects by either inactivating the tumor suppressive functions of wild-type p53 in a dominant negative manner or by a gain of function mechanism whereby mutant p53 induces pro-cancerous effects by itself, independently of wild-type p53. To elucidate whether the observed inhibition of NQO1 and HO-1 by mutant p53R273H depends on wild-type p53 present in these cells, we took advantage of the HCT116 p53−/− cells that are devoid of wild-type p53. p53R273H mutant was overexpressed in HCT116 p53−/− cells (Fig. 1D) and this was followed by DEM treatment. As Fig. 1D shows, DEM administration induced an accumulation of NRF2 in both control and p53R273H mutant expressing cells (Fig. 1D) and increased the expression of NQO1 and HO-1 in HCT116 p53−/− (Fig. 1E,F). Interestingly, in mutant p53R273H cells the NRF2 protein induction seemed weaker than in the control cells (Fig. 1A,D). Furthermore, consistent with HCT116 containing wild-type p53, the p53R273H mutant attenuated the expression of both NQO1 and HO-1 in the HCT116 p53−/− (Fig. 1E,F). Hence, we concluded that the p53R273H mutant inhibits the induction of NRF2 target genes in response to oxidative stress due to a gain of function mechanism irrespective of wild-type p53.
To determine the role of wild-type p53, we compared the mRNA induction levels of NQO1 and HO-1 in HCT116 cells bearing wild-type p53 to those levels in HCT116 p53−/−. As supplementary material Fig. S1 shows, wild-type p53 was important for maximal induction of NQO1 (supplementary material Fig. S1A) and HO-1 (supplementary material Fig. S1B) upon DEM treatment.
p53R273H and p53R248Q mutant forms attenuate NRF2-dependent induction of phase 2 enzymes in H1299 lung cancer cells
Following the observation that p53R273H mutant attenuates the expression of NRF2 target genes in HCT116 cells via a gain of function mechanism, we aimed to examine whether mutant p53 leads to an impaired response to oxidative stress in additional cellular system. To this end, we used the H1299 lung cancer cells that are null for p53. We infected these cells with mutant p53R273H or GFP as a control for stable expression (Fig. 2A) and analyzed the mRNA levels of both NQO1 and HO-1 following 100 µM DEM treatment and in untreated cells. The DEM treatment strongly induced the expression of both NQO1 and HO-1 in the control H1299 GFP cells (Fig. 2B,C). In contrast, in cells expressing the p53R273H NQO1 levels remained similar to non-treated cells (Fig. 2B). HO-1 mRNA was induced in H1299-p53R273H to a much lesser extent than in the control cells (Fig. 2C). According to these data we propose a gain of function role for mutant p53R273H manifested by repressing the phase 2 detoxifying enzymes following DEM-induced oxidative stress.
Next, we wished to evaluate the effect of a direct activation of NRF2 protein in H1299 cells differentially expressing mutant p53. To attain this, we used sulforaphane (SF), an anti-oxidant compound which induces the expression of phase 2 enzymes similarly to DEM (Ahn et al., 2010). However, unlike DEM, SF directly stabilizes NRF2 and induces the expression of phase 2 enzymes even in an oxygen detoxified environment (Ahn et al., 2010). H1299 cells with and without mutant p53 were treated with 2 μM SF for 12 hours which induced the expression of NQO1 and HO-1 (Fig. 2D,E). Nevertheless, in mutant p53R273H expressing cells the level of induction of these enzymes following administration of SF was significantly reduced compared with the control cells (Fig. 2D,E). Next, we examined whether other hot-spot mutant, the p53R248Q would also reduce the antioxidant response. To this end, H1299 cells expressing the p53R248Q mutant (supplementary material Fig. S2A) were treated with DEM or SF and HO-1 expression levels were analyzed by QRT-PCR. This analysis indicated that mutant p53R248Q also lead to an attenuated HO-1 induction upon both DEM (supplementary material Fig. S2B) and SF (supplementary material Fig. S2C). Taken together these results suggest that in response to oxidative stress, mutant p53 forms interfere with normal antioxidant response via reduced induction of phase 2 detoxifying enzymes.
To confirm that the observed attenuation of phase 2 enzymes expression by mutant p53R273H is dependent upon NRF2, we knocked down NRF2 expression in p53 null cells, and examined whether the effect would be similar to that of mutant p53R273H overexpression. To this end, H1299 cells were transfected with NRF2-specific siRNA oligos. The downregulation of NRF2 was validated by QRT-PCR (Fig. 3A). DEM administration induced upregulation in NRF2 protein only in control cells but not in siNRF2-transfected cells (Fig. 3B). Accordingly, the induction of NQO1 and HO-1 mRNA following DEM treatment was much stronger in control cells than in siNRF2-transfected cells (Fig. 3C,D) indicating that DEM-stimulated expression of phase 2 enzymes is indeed dependent upon NRF2. This implies that the low induction levels of NQO1 and HO-1 in mutant p53 producing cells, that we observed, were due to attenuation of NRF2 response.
p53R273H mutant promotes survival of cells following oxidative damage
Our data hitherto suggested that p53R273H mutant inhibits the induction of phase 2 detoxifying enzymes in a gain of function mechanism. Mutant p53 forms were shown to exhibit gain of function in protecting cells from death induced by various genotoxic stimuli (Sigal and Rotter, 2000). Hence, our next aim was to evaluate whether mutant p53R273H also promotes the survival of DEM-treated cells, protecting them from cell death. To address this question, we tested the ability of H1299 cells expressing either mutant p53R273H or GFP to grow after DEM treatment. Both cultures were sparsely seeded and treated with 100 µM DEM for three days after which the DEM was removed and the cells were allowed to grow. After two weeks of incubation the resultant colonies were stained with Crystal Violet and counted. As Fig. 4A demonstrates, the control H1299-GFP cells barely formed colonies while H1299 cells expressing the mutant p53R273H were able of proliferating despite DEM treatment (Fig. 4A,B). These results suggest that DEM-induced oxidative stress blocks proliferation in H1299 and that mutant p53R273H diminishes this effect. To further examine whether mutant p53R273H protects cells from death induced by oxidative damage, we analyzed DEM-treated H1299 cells for cell death using the fluorescence-activated cell sorting (FACS) analyzer. To this end, GFP and p53R273H mutant-harboring H1299 cells were treated with 100 µM DEM for 48 hours and the percentage of cells at the sub G1 fraction representing the dead cells, was determined. As Fig. 4C illustrates, following DEM treatment H1299 cell population expressing GFP contained 22% of dead cells. In the H1299 p53R273H cells the fraction of dead cells was remarkably lower and consisted only of 10.5% (Fig. 4C). Thus, p53R273H mutant may protect cells from oxidative stress-induced cell death.
Impaired detoxification of ROS in p53R273H mutant-expressing cells
The above presented data suggest that, p53R273H mutant may promote the survival and growth of cells under the conditions of oxidative stress. In parallel, our data suggest that the p53R273H mutant reduced the expression of phase 2 enzymes following DEM-induced oxidative stress (Fig. 1B,C,E,F; Fig. 2B,C). These enzymes are known to play critical roles in defending cells from the accumulation of ROS (Nguyen et al., 2009). Accordingly, reducing the activation of these enzymes is usually associated with strong oxidative stress which further leads to DNA oxidation and mutations fostering malignant transformation (Johnson et al., 1996). Hence, we were now interested to examine whether mutant p53R273H-mediated reduction in the expression of phase 2 enzymes HO-1 and NQO1 is associated with increased ROS accumulation. To test this, we used dihydroethidium (DHE), a reagent which is commonly exploited to evaluate intracellular amounts of O2−. DHE is a cell permeable compound which reacts with O2− to form ethidium. Ethidium then translocates into the nucleus, intercalates with DNA and provides nuclear fluorescence that can be detected by FACS (Kitada et al., 2003). To evaluate whether mutant p53R273H affects the ROS accumulation, we exposed HCT116 and H1299 cells to DHE upon DEM. Interestingly, in the control (GFP) cells the levels of DHE fluorescence after DEM administration were comparable to those before DEM treatment (Fig. 5A,B) suggesting that O2− formed after DEM treatment was effectively detoxified in these cells. However, in the presence of p53R273H mutant in both the HCT116 and the H1299 cells greater DHE fluorescence was observed following DEM treatment as compared with non treated HCT116 p53R273H (Fig. 5A) and H1299 p53R273H (Fig. 5B) cells. These data suggest that the reduction of O2− after exposure to DEM is less efficient in mutant p53R273H harboring cells, proposing a gain of function mechanism for mutant p53 in preventing detoxification of ROS.
Finally, we validated the observed effect of mutant p53R273H on ROS accumulation also in WI-38 lung fibroblasts. Thus, the p53R273H mutant was introduced into the WI-38 cells in parallel with shp53 and shRNA against mouse Noxa as a control (shCon) (Fig. 6A). Since WI-38 cells express wild-type p53, it was important to knock down its expression as a control for a possible dominant negative effect exerted by the mutant protein over the endogenous wild-type p53. To test the effect of mutant p53 on the response of these cells to oxidative stress, WI-38 cells were treated with 300 µM DEM. As Fig. 6B shows, DEM treatment of these cells induced NRF2 protein stabilization. QRT-PCR analysis demonstrated induction of NQO1 and HO-1 mRNA following DEM treatment in both of the control cultures: the WI-38 shCon and WI-38 shp53 (Fig. 6C). However, in WI-38 p53R273H cells NQO1 mRNA levels were not induced following DEM treatment (Fig. 6C), and HO-1 induction was attenuated. Furthermore, we measured the expression levels of an additional NRF2 target gene MafG which showed a similar pattern of expression, i.e. attenuated MafG induction in WI-38 p53R273H as compared with the control cultures was evidenced (Fig. 6E). To measure ROS levels in DEM treated WI-38 cells, we used the 2′-7′-dichlorodihydrofluorescein diacetate (DCFDA). This reagent is highly sensitive to ROS. Upon oxidation DCFDA turns into highly fluorescent molecule DCF detectable by FACS (Eruslanov and Kusmartsev, 2010). Accordingly, the detected level of fluorescent DCF staining is proportional to the levels of ROS in the cells. To address the accumulation of ROS, WI-38 cells were treated with 300 µM DEM for 12 hours. The cells were then incubated with DCFDA and analyzed using the FACS analyzer. As Fig. 6F demonstrates both WI-38 shCon and WI-38 shp53 control cultures exhibited a slight change in the levels of DCF fluorescence following DEM treatment. However, the WI-38 p53R273H cells exhibited nearly twofold higher levels of DCF fluorescence following the exposure to DEM (Fig. 6F). Therefore, we could recapitulate the results obtained in HCT116 colon carcinoma cells and in H1299 lung cancer cells also in WI-38 lung fibroblasts. Taken together these data suggest that the detoxification of ROS generated by DEM-induced oxidative stress is attenuated by p53R273H mutant.
Next, we wished to investigate the mechanism through which mutant p53 attains the observed effects. To this end, we performed a time course DEM experiment and analyzed NRF2 protein levels by western blot analysis in WI-38 and HCT116 p53−/− cells. As Fig. 7 demonstrates, mutant p53R273H expression resulted in lower amounts of NRF2 protein levels following oxidative damage indicating impaired accumulation of NRF2 upon DEM-induced stress signal. Quantitative analysis of NRF2 protein in experiments described in previous sections indicated a similar effect (supplementary material Fig. S3). NRF2 mRNA did not show mutant p53-dependent downregulation (supplementary material Fig. S4). We therefore concluded that mutant p53R273H affected NRF2 protein stability rather than NRF2 mRNA, leading to reduced expression of NRF2 target phase 2 enzymes.
The results described above were obtained using ectopically expressed mutant p53. Thus, we wished to examine the response to oxidative damage in endogenous mutant p53 bearing cancer cells. To this end, we used the SW620 cells expressing p53R273H and DU145 cells expressing p53P223L and p53V274F stably knocked-down for p53 (Fig. 8A,D, respectively). In both cell lines downregulation of endogenous mutant p53 resulted in increased levels of NQO1 and HO-1 following DEM treatment compared to control cells (Fig. 8B,C,E,F). Hence, endogenous mutant p53 also attenuated the induction of phase 2 enzymes supporting the original observation obtained in cell lines ectopically expressing mutant p53.
Reducing environment within a living cell is of critical importance in order to avoid the destructive consequences of ROS accumulation potentially leading to tissue damage and initiation of carcinogenic process (Cho et al., 2006; Valko et al., 2001; Valko et al., 2006). This reducing environment is generally maintained by ROS detoxifying enzymes and molecules (Cho et al., 2006) (Jaiswal, 2004) induced mainly through the NRF2 pathway (Nguyen et al., 2009). In the present study we demonstrate that the oxidative stress-dependent NRF2 induction and the expression of NRF2 target genes were attenuated by mutant p53 in several cellular systems. Importantly, this effect was observed in tumor cell lines that either ectopically or endogenously express mutant p53. Furthermore, following exposure to oxidative stress cells with mutant p53R273H continued growing despite elevated ROS levels. The data presented here underscore a new role of p53 mutant in promoting malignant growth despite poor ROS detoxification.
Loss of NRF2 is strongly associated with a reduction in the expression of phase 2 detoxifying enzymes (Ramos-Gomez et al., 2001a) inducing the amplification of ROS which are well-established carcinogenic mediators (Ramos-Gomez et al., 2001a; Valko et al., 2006; Marnett, 2000). As 50% of human cancers harbor mutations in p53 protein (Brosh and Rotter, 2009; Buganim et al., 2006; Stambolsky et al., 2010), it is tempting to speculate that mutant p53 might be responsible for high levels of ROS observed in cancer cells. Furthermore, several studies suggest that stress induced by oxidative and nitrosative damage may cause p53 mutations at specific sites (Goodman et al., 2004; Hussain et al., 2007; Marrogi et al., 2001). Our findings suggest that mutant p53 enhances oxidative burden and counteracts the elimination of cells with elevated ROS resulting in greater amount of damaged cells. This is a novel mechanism of mutant p53 oncogenic function through which mutant p53 deteriorates the cellular integrity leading to tumor progression.
It was shown that mice expressing either mutant p53R270H (human p53R273H) or p53R172H (human p53R175H) developed broader spectrum of tumors that exhibited higher level of metastasis compared with either p53−/− or p53−/+ mice (Lang et al., 2004; Olive et al., 2004). Furthermore, a study conducted by Iida et al. (Iida et al., 2007) demonstrated that tumor susceptibility is synergistically exacerbated in NRF2−/−; p53+/− mice in comparison with either single mutant alone (NRF2−/− or p53−/+). It has been suggested that these differences in tumor susceptibility are due to a poor detoxification and accelerated proliferation. It is possible that, the increase in tumor spectra and invasiveness observed in mutant p53 animals is a consequence of increased ROS amounts because of a defect in NRF2 pathway activation similarly to NRF2−/−.
The major function of wild-type p53 tumor suppressor is to restrict the growth of cells that suffered from severe stress by triggering either permanent cell cycle arrest or cell death. Though, under low stress levels wild-type p53 is known to induce the expression of repair genes allowing the cell to overcome the stress and return to the original state. Such stress signal can be mediated by reactive electrophiles. Under mild oxidative stress conditions, wild-type p53 was shown to induce the expression of oxidative stress enzymes ensuring activation of repair mechanisms and cell survival (Cano et al., 2009; Sablina et al., 2005; Tan et al., 1999; Yoon et al., 2004). However, upon acute or sustained stress, p53 tumor suppressor undergoes stabilization and acts both to reduce the expression of detoxifying enzymes and to stimulate apoptosis (Faraonio et al., 2006; Johnson et al., 1996; Polyak et al., 1997; Sablina et al., 2005). Therefore, loss of wild-type p53 leads to higher ROS levels and less efficient apoptosis (Bensaad and Vousden, 2007; Sablina et al., 2005). Importantly, the data of the present study suggest that the acquisition of gain of function p53 mutant allele promotes ROS accumulation and cell survival beyond the phenotype observed on the background of p53−/− leading to the advancement of the malignant process.
Although we focused here on the role of mutant p53, examination of the HCT116 system allowed us to evaluate wild-type p53 responses to oxidative stress. We tested NQO1 and HO-1 induction upon oxidative stress in both HCT116 cells expressing wild-type p53 and HCT116 cells in which p53 was knocked out. Importantly, comparing NQO1 and HO-1 induction levels in HCT116 and HCT116 p53−/− demonstrated higher levels of these genes in wild-type p53 expressing cells (supplementary material Fig. S1). This implies that under oxidative stress the wild-type p53 increased the induction of NRF2 target genes. This may further establish the documented contribution of wild-type p53 to the expression of phase 2 enzymes under moderate oxidative stress conditions (Cano et al., 2009; Vousden and Ryan, 2009).
We propose here that mutant p53 interferes with proper NRF2 activation upon oxidative damage leading to high ROS levels and additionally, increases cell survival in oxidative environment. We assume that increased survival of tumor cells expressing mutant p53 under oxidative stress conditions occurs in an NRF2-independent manner. This statement can be supported by previous studies showing that NRF2 does not repress tumor survival, but rather suppresses tumorigenesis via activation of antioxidant response (Colburn and Kensler, 2008), while mutant p53 is known to protect cells from death induced by various signals (Sigal and Rotter, 2000).
In conclusion, our study provides data on mutant p53R273H crosstalk with NRF2 pathway which inhibits the normal function of NRF2 in ROS detoxification. Highly accumulated ROS levels which normally induce growth arrest and apoptosis fail to do so in mutant p53 expressing cells. Thus, mutant p53R273H not only leads to highly oxidative environment, but also to cell survival and proliferation of damaged cells. The novel finding of mutant p53-dependent ROS accumulation in cancer cells contributes to our understanding of a carcinogenic process and how oncogenic function of mutant p53 promotes this process.
Materials and Methods
Antibodies and reagents
Rabbit anti-NRF2 antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-p53 was produced in our laboratory. Mouse anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibody (MAB374) was purchased from Chemicon (Hampshire, UK). DMSO, DEM and anti-tubulin antibody were purchased from Sigma (St Louis, MO).
Ecotropic Phoenix retrovirus-producing cells were purchased from the American Type Culture Collection (ATCC) and maintained in DMEM supplemented with 10% fetal calf serum (FCS; Sigma). The immortalized primary human embryonic lung fibroblasts WI-38T/R (WI-38/hTERTfast/H-RasV12) were established and maintained as described (Milyavsky et al., 2003). Unless mentioned else WI-38T/R are designated as WI-38 cells. H1299 cells were obtained from the ATCC and maintained in RPMI supplemented with FCS and antibiotics. HCT116 and HCT116 p53−/− were kindly provided by Bert Vogelstein and were maintained in DMEM with 10% FCS and antibiotics. SW620 and DU145 were purchased from ATCC and maintained in DMEM supplemented with 10% fetal calf serum and antibiotics
Ecotropic Phoenix-packaging cells were transfected with 10 µg of pRetroSuper plasmid encoding for shRNA against either p53 or a control vector expressing shRNA against mouse Noxa protein that has no effect on human Noxa. The transfection was performed by a standard calcium phosphate procedure. Culture supernatants were collected 36–48 hours after transfection and filtered. WI-38 cells were infected with the filtered viral supernatants in the presence of 4 µg/ml polybrene (Sigma) for 12 hours, after which, the medium was changed. Fresh viral suspensions were added after 24 hours interval for an additional 12 hours. Infected cells were then selected with 10 µg/ml blasticidine (7 days). Similarly, WI-38, H1299, HCT116 and HCT116 p53−/− were infected with pWZL encoding mutant p53R273H. The cells were then selected with 5–10 ug/ml Blasticidine (7 days).
Knockdown of NRF2 was conducted by transfection with specific oligo-nucleotides (Dharmacon, Lafayette, CO) using DharmaFECT3 reagent (Dharmacon).
mRNA extraction, reverse transcription and quantitative real-time PCR
Total RNA was extracted using a Nucleospin RNA cell kit (Macherey Nagel, Easton, PA). Two micrograms of each RNA sample were reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI) and random hexamer primers. Real-time PCR was performed on an ABI 7300 real-time PCR system (Applied Biosystems, Lincoln Center, CA), using SYBR green PCR master mix (Invitrogene Carlsbad, CA). cDNA levels of each gene that was analyzed were normalized to GAPDH.
DHE and DCFDA staining
H1299 and HCT116 cells were seeded in six-well plates and treated with 100 uM DEM for 12 hours. The cells were then incubated with 10 uM DHE for 30 minutes at 37°C and analyzed for DHE fluorescent staining by FACS. WI-38 cells were seeded in six-well plates and treated with 300 uM DEM for 12 hours. Following the incubation with DCFDA for 30 minutes at 37°C the cells were analyzed for DCF fluorescent staining by FACS.
DEM-induced colony formation assay
H1299 cells (1×103) expressing either mutant p53R273H or GFP as control were seeded overnight in six-well plates and treated with 100 uM DEM for three days. Cells were then washed to remove DEM residuals and maintained in fresh medium for additional two weeks. Medium was refreshed every four days. Colonies were then stained with Crystal Violet and counted.
Cells were plated in six-well plates and treated with 100 uM DEM for 48 hours. Cells were subsequently trypsinized, washed and resuspended in PBS containing 50 µg/mL propidium iodide and 10 µg/mL RNase A. Samples were then subjected to FACS and analyzed for cell death.
Statistical significance was determined by Student's t-test. P-values are presented in figure legends. Asterisks indicate data for which statistical analysis was performed; P<0.05 was considered significant.
This research was supported by a Center of Excellence grant from Flight Attendant Medical Research Institute (FAMRI), EC FP6 [grant number LSHC-CT-2004-503576] and the Yad Abraham Center for Cancer Diagnosis and Therapy. This publication reflects the authors' views and not necessarily those of the European Community. The EC is not liable for any use that may be made of the information contained herein. V.R. is the incumbent of the Norman and Helen Asher Professorial Chair Cancer Research at the Weizmann Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.