Characterization of p53 expression in rainbow trout

Comparative Biochemistry and Physiology, Part C 154 (2011) 326–332

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Comparative Biochemistry and Physiology, Part C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c b p c

Characterization of p53 expression in rainbow trout Michelle Liu a, Catherine Tee a, Fanxing Zeng a, James P. Sherry b, Brian Dixon a, Niels C. Bols a, Bernard P. Duncker a,⁎ a b

Department of Biology, University of Waterloo, Waterloo, Ontario, N2L 3G1, Canada Aquatic Ecosystem Protection Research Division, Environment Canada, Burlington, Ontario, L7R 4A6, Canada

a r t i c l e

i n f o

Article history: Received 21 May 2011 Received in revised form 29 June 2011 Accepted 30 June 2011 Available online 13 July 2011 Keywords: p53 Checkpoint DNA damage Rainbow Trout Biomarker

a b s t r a c t The tumour suppressor protein p53 is a critical component of cell cycle checkpoint responses. It upregulates the expression of cyclin-dependent kinase inhibitors in response to DNA damage and other cellular perturbations, and promotes apoptosis when DNA repair pathways are overwhelmed. Given the high incidence of p53 mutations in human cancers, it has been extensively studied, though only a small fraction of these investigations have been in non-mammalian systems. For the present study, an anti-rainbow trout p53 polyclonal antibody was generated. A variety of rainbow trout (Oncorhynchus mykiss) tissues and cell lines were examined through western blot analysis of cellular protein extracts, which revealed relatively high p53 levels in brain and gills. To evaluate the checkpoint response of rainbow trout p53, RTbrain-W1 and RTgill-W1 cell lines were exposed to varying concentrations of the DNA damaging agent bleomycin and ribonucleotide reductase inhibitor hydroxyurea. In contrast to mammals, these checkpoint-inducing agents provoked no apparent increase in rainbow trout p53 levels. These results infer the presence of alternate DNA damage checkpoint mechanisms in rainbow trout cells. © 2011 Elsevier Inc. All rights reserved.

1. Introduction To ensure proper DNA replication, surveillance mechanisms known as checkpoints are in place at various stages of the cell cycle. Specific checkpoints can be activated in the presence of genotoxic stress, including double strand breaks (DSBs), single strand breaks (SSBs), and stalled replication forks. Such perturbations typically activate several groups of proteins through a kinase cascade. The initial DNA damage or replication stress is detected by sensor proteins that proceed to stimulate the activity of transducer proteins, which in turn lead to the activation of downstream effector proteins, resulting in cell cycle arrest, DNA repair, or apoptosis (reviewed in Warmerdam and Kanaar, 2010). The tumor suppressor protein p53 is a key factor in the mammalian DNA damage checkpoint pathway. It is a transcription factor which stimulates the expression of target genes encoding proteins that can help lead the cells to a resolution of the checkpoint (reviewed in Brady and Attardi, 2010). As one of the main effector proteins in the checkpoint pathway, mutations in its functional domains can lead to tumorigenesis and cancer. Indeed, p53 mutations have been found in over 50% of human tumors (reviewed in Olivier et al., 2010). Due to its importance in cancer research, p53 has been extensively characterized

⁎ Corresponding author. Tel.: + 1 519 888 4567x33957; fax: + 1 519 746 0614. E-mail address: [email protected] (B.P. Duncker). 1532-0456/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpc.2011.06.018

in mammalian models; however, its regulation and mechanisms of action remain poorly understood for lower vertebrate species. Functional conservation relative to mammalian p53 has been shown in studies with zebrafish (reviewed in Storer and Zon, 2010). For example, it has been reported that p53 is necessary for apoptosis of cells in response to DNA damaging agents in developing zebrafish (Langheinrich et al., 2002; Berghmans et al., 2005). As in mammals, zebrafish p53 is negatively regulated by MDM2, and embryos that are depleted of MDM2 display developmental defects and an increased incidence of apoptosis (Langheinrich et al., 2002). Furthermore, zebrafish p53 has been found to regulate the transcription of several of the same genes as in mammals (Langheinrich et al., 2002; Lee et al., 2008), and homozygous p53 mutants develop tumors (Berghmans et al., 2005). Checkpoint proteins are attractive candidates as biomarkers for detecting the presence of genotoxic compounds in polluted waters, as they are often upregulated and/or post-translationally modified as part of the response to DNA damage. For example, we have previously shown that levels of rainbow trout CHK2 increase dramatically in a rainbow trout brain cell line, following exposure to the radiomimetic drug bleomycin (Steinmoeller et al., 2009). Given the conserved checkpoint role of fish p53, and the fact that mammalian p53 is known to be stabilized in response to genotoxic stress (reviewed in Kruse and Gu, 2009), it is important to assess the potential of p53 as a biomarker for genomic damage in aquatic ecosystems. To date, studies of p53 in fish have resulted in widely differing findings, with some reporting induction following exposure of cells to stressors (Lee et al., 2008;

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Brzuzan et al., 2009; Mai et al., 2010) and others observing no changes in response to the agents that normally upregulate mammalian p53 (Chen et al., 2001; Rau Embry et al., 2006). In reconciling differing outcomes for p53 induction trials, an important consideration is that p53 distribution and activity can vary greatly between different tissue types. Immunoblot analysis of total protein extracts from zebrafish embryos showed no change in global p53 levels following treatment with either R-roscovotine (a negative regulator of MDM2, found to stabilize p53 in human cells) and/or γ-irradiation. In contrast, the same study reported that immunohistochemical staining of p53 in embryos revealed an accumulation in gut epithelium, liver, pancreas and brain tissues following such treatments (Lee et al., 2008). Variability in p53 expression levels between different tissue types has also been observed during normal mouse and human development (Schmid et al., 1991; Lichnovsky et al., 1998). In the present study, we report the levels of p53 in rainbow trout tissues and cell lines, and assess its potential utility as a biomarker for genotoxic stress. 2. Materials and methods 2.1. Rainbow trout polyclonal p53 antibody production Rainbow trout (Oncorhynchus mykiss, Salmonidae) p53 coding sequence was cloned using RTgill-W1 cDNA as template, along with forward (5’-GACTTCTCGAGCTGGCGGAGAACGTGTCTCTTC-3’) and reverse (5’-GGACTTAAGCTTCACTCCGAAGTCCCGTTTGGC-3’) primers, including XhoI and HindIII restriction enzyme sites, respectively. These primers amplify a 1125 bp sequence, encoding residues 4–378 of the 396 aa rainbow trout p53 protein (Caron de Fromentel et al., 1992; Danilova et al., 2008). Following PCR amplification, the fragment was gel-purified and inserted into a pRSET A expression vector (Invitrogen) through digestion with XhoI and HindIII restriction enzymes. Ligation was then performed using T4 DNA ligase (Promega), resulting in a construct, pRSETA-RTp53, expressing rainbow trout p53 as a fusion protein with a polyhistindine tag and an Xpress epitope at the N-terminus. The construct was sequenced (Robarts Research Institute DNA Sequencing Facility), and found to contain a single point mutation, resulting in an amino acid substitution of cysteine to arginine in a non-conserved region of the protein (position 365). pRSETA-RTp53 was then transformed into competent (DE3)pLysS E. coli cells (Promega) for inducible expression of recombinant rainbow trout p53 according to the manufacturer's suggested protocol. Transformed cells were grown in SOB medium supplemented with 100 μg/mL ampicillin, shaken at 37 °C, 250 rpm to an optical density of 0.4–0.6, before a 4 h induction of recombinant protein expression with 1 mM IPTG. Cells were harvested by centrifugation (5000 g, 10 min) and lysed overnight at room temperature with 8 M urea denaturing buffer (100 mM NaH2PO4, 10 mM Tris–HCl, 8 M urea, pH 8). The lysate was then centrifuged (10,000 g , 6 min) at 4 °C before saving the supernatant and discarding the pellet. To purify recombinant p53, affinity chromatography was performed in an econo-column (Bio-Rad) with Ni–NTA resin (Qiagen). Binding of resin to the polyhistidine tag at the N-terminus of the recombinant protein was carried out through incubation with the lysate on a rotator at 4 °C for 2 h. After incubation, the flowthrough was discarded. The protein was then refolded on the column using a decreasing gradient of urea (6 M–0 M) before elution in its native form with 250 mM imidazole in 8 fractions of 1 mL each. 10 μL of each fraction was then run on a 12% SDS polyacrylamide gel and analyzed by Coomassie blue staining to determine recombinant protein concentration. Elution fractions were dialyzed overnight at 4 °C in 200 mL of 1× PBS. Following dialysis, recombinant protein samples were again analyzed by SDS-PAGE and Coomassie blue staining, and a Bradford assay was then performed to determine protein yield.

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The dialyzed recombinant protein sample fractions were then stored at −20 °C. Polyclonal antiserum production was performed essentially as described previously (Kales et al., 2006). Subcutaneous rabbit immunization was carried out with 200 μL recombinant p53 (0.7 mg/ mL) mixed with 200 μL Freund's complete adjuvant (Sigma), at four different injection sites. This was followed with booster shots of a similar emulsion, but with Freund's incomplete adjuvant, at three week intervals. Blood samples were obtained before each boost through the marginal ear vein. To separate the serum from the blood, samples were left at room temperature for 2 h and then overnight at 4 °C to allow the blood to clot. Samples were then centrifuged at 5000 g for 10 min at 4 °C to pellet the blood cells, and the serum was collected and assessed for antibody titre through ELISA analysis with purified recombinant p53. At the end of the twelfth week, exsanguination by carotid cannulation was carried out for final collection of total blood. Antibodies specific to recombinant rainbow trout p53 were purified from crude serum with a SulfoLink Immobilization Kit for Proteins (Pierce). The SulfoLink column was generated by binding recombinant rainbow trout p53 to the resin according to the manufacturer's suggested protocol with minor changes. To prepare the protein for coupling, 1 mL of recombinant p53 (0.7 mg/mL) was reduced with 2-mercaptoethylamine (2-MEA) and applied to the provided desalting column to remove remaining 2-MEA. For affinity purification, 2 mL of pure serum was used for each run. Each wash step was performed the maximum number of suggested times. Bound antibodies were eluted in 4 aliquots of 1 mL each, and concentration was determined through a Bradford assay. 2.2. Whole fish Rainbow trout (Oncorhynchus mykiss) were obtained from the Alma Aquaculture Research Station (University of Guelph) and acclimated for at least two weeks in 200 L aerated tanks with constant water flow at 13 °C and a 12 h light:12 h dark photoperiod, prior to experimentation. Organs were harvested and frozen on dry ice, then stored at − 80 °C. Lysates were prepared through homogenization and sonication of frozen tissues in 50 mM Tris–HCl buffer (pH 7.5) supplemented with protease inhibitor cocktail (Roche). Lysates were centrifuged at 16,000 g for 10 min to remove cell debris and insoluble proteins. The supernatant was retained and protein concentrations were determined through Bradford protein assay (Bio-Rad) according to the manufacturer's suggested protocol. 2.3. Fish cell lines Rainbow trout cell lines from the brain (RTbrain-W1), gastrointestinal tract (RTgut-GC), gill (RTgill-W1), gonad (RTG-2), liver (RTLW1) and spleen (RTS11) were used. Comprehensive descriptions for the development and characterization of RTgutGC, RTL-W1, and RTS11 have been presented respectively by Kawano et al. (2011), Lee et al. (1993), and Ganassin and Bols (1998). RTbrain-W1 was developed by the general methods outlined by Ganassin and Bols (1997) and was used by Steinmoeller et al. (2009). Development of RTgill-W1 is described in Bols et al (1994), and this cell line has been submitted to the American Type Culture Collection (ATCC) (Manassas, VA) from where it can be obtained as CRL-2532 . RTG-2 was obtained from ATCC as CCL-55 and its origins are described by Wolf and Quimby (1962). The routine growth of the cell lines was done at room temperature as described in detail previously (Bols and Lee, 1994). 2.4. Cell culture treatment regimes Cells were seeded in 25 cm 2 flasks at either 1.5 × 10 6 (RTbrain-W1) or 3 × 10 6 (RTgill-W1) cells per flask, 24 h prior to treatment.

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2.5. Protein extraction from cell lines Cells were harvested using a cell scraper and centrifuged at 1000 g for 5 min before lysing the pellet with 50 μL of RIPA buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate), supplemented with protease and phosphatase inhibitor cocktails (Roche). The lysate was then centrifuged at 16,000 g for 10 min to remove cell debris before determining protein concentration with a Bradford assay.

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2.6. Western blot analysis Each protein extract (20 μg, unless otherwise specified) was analyzed by SDS-PAGE on a 12% separating gel and transferred to a 0.2 μM nitrocellulose membrane (Bio-Rad). Membranes were blocked in 5% dry milk in 1X TBST (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 0.1% Tween 20) for 1 h at room temperature. Incubation of membranes with primary antibodies were carried out at the following dilutions: anti-Xpress (Invitrogen) 1:5000, affinity purified anti-rainbow trout p53 1:200, anti-human p53 1:200 (Santa Cruz Biotech), antiphospho-histone H2A.X (Ser139) (Santa Cruz Biotech) 1:200, antiphospho-(Ser/Thr) ATM/ATR substrate (Cell Signaling Technology) 1:1000, anti-actin-Cy3 (Sigma) 1:1000. For all primary antibodies except anti-actin-Cy3, membranes were subsequently incubated with either Alexa Fluor 488 goat anti-rabbit IgG secondary antibody (Invitrogen, 1:3000 dilution) or anti-rabbit-HRP secondary antibody (Bio-Rad, 1:3000 dilution) before detection with ECL Plus (Amersham) as per the manufacturer's suggested protocol, or anti-rabbit IgG-AP secondary antibody (Bio-Rad, 1:3000) followed by standard colorimetric detection. For the phospho-specific antibodies, incubations were carried out in 5% BSA, 1X TBST rocking overnight at 4 °C. All other antibodies were incubated for an hour at room temperature in 5% milk, 1X TBST. Because ubiquitously expressed reference gene products can demonstrate variability between tissue and cell types (Overgard et al., 2010), we used Ponceau S membrane staining as a measure of relative protein loading in addition to, or in place of, actin detection. Membrane imaging was performed using a Typhoon 9400 Variable Image Scanner (GE Healthcare). 2.7. Cell proliferation assay RTbrain-W1 and RTgill-W1 cells were added to 24 well plates at a density of 6 × 10 4 cells per well or 1.2 × 10 5 cells per well, respectively, 24 h prior to treatment using previously described culture conditions (Steinmoeller et al., 2009). Cells were dosed for the specified reagent concentrations and incubation times. At each time point being analyzed, cells were trypsinized and counted using a Coulter Counter Z2 (Beckman Coulter), set for counting cells between 8 μm and 20 μm. 3. Results

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Fig. 1. Western blot analysis of p53 antibodies. (A) 20 μg samples of RTbrain-W1 and RTgill-W1 protein extracts were subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane. Pre-immune serum, final bleed serum, and affinity purified anti-rainbow trout p53 antibody were used at a 1:200 dilution, while goat anti-rabbit IgG HRP conjugated secondary antibody (Bio-Rad) was used at a 1:3000 dilution. The molecular weights of the marker bands (M) are indicated in kDa to the left of the image (Fermentas). (B) 20 μg samples of RTgill-W1 protein extracts were subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane. Affinity purified antirainbow trout p53 antibody, as well as two lots of commercially available anti-human p53 antibody (Santa Cruz sc-6243, lots H3109 and B2410) were used at a 1:200 dilution, while goat anti-rabbit IgG-AP secondary antibody (Bio-Rad) was used at a 1:3000 dilution. The molecular weights of the marker bands are indicated in kDa to the left of the images (Fermentas).

migrating close to the 55 kDa size marker. To improve the specificity of detection, the final bleed serum was applied to a SulfoLink column (Pierce) coupled to recombinant rainbow trout p53. The resultant affinity purified rainbow trout p53 antibodies enabled a much clearer detection of the 55 kDa band, without the appearance of the lower bands that had been seen using unprocessed serum. To compare the ability of our antibody to detect rainbow trout p53 to that of a commercially available antibody raised to the human form of the protein, we carried out parallel detections for western blots of RTgillW1 extracts. In contrast to two different lot numbers of the antihuman p53 antibody, which both resulted in a multitude of bands, the anti-rainbow trout antibody produced a single prominent band of expected size (Fig. 1B).

3.1. Generation and use of polyclonal anti-rainbow trout p53 antibodies 3.2. Characterization of p53 levels in rainbow trout tissues and cell lines Recombinant rainbow trout p53 was produced and used to generate rabbit polyclonal antibodies as described in Materials and methods. To assess the ability of the antibodies to detect rainbow trout p53, final bleed serum was incubated with a western blot of protein extracts from rainbow trout brain (RTbrain-W1) and gill (RTgill-W1) cell lines (Fig. 1A). In contrast to a complete absence of signal with pre-immune serum, use of the final bleed serum resulted in the detection of multiple bands, including one of expected size

In order to determine the relative p53 levels in different rainbow trout tissues, protein extracts were prepared from a variety of tissue samples. Visual inspection of western blot analysis using anti-rainbow trout p53 antibodies revealed prominent p53 bands for brain and gill (Fig. 2). Although a strong p53 signal was also evident for heart, Ponceau S staining of the membrane revealed that this sample was more heavily loaded than the other samples. In contrast, p53 could

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Fig. 2. Western blot analysis of p53 levels in rainbow trout tissue samples. 20 μg of each protein extract from the indicated rainbow trout tissues was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. Detection was carried out with affinity purified rabbit anti-rainbow trout p53 primary and goat anti-rabbit IgG HRP conjugated secondary antibodies. The molecular weights of the marker bands (M) are indicated in kDa to the left of the image (Fermentas). Ponceau S staining was performed prior to detection to assess relative protein loading. These results are representative of samples from three fish.

only be faintly detected in the liver sample, while no expression was seen for spleen. To examine whether rainbow trout cell lines show a comparable cell type specificity for p53 levels, western blot analysis of protein extracts was conducted for gill (RTgill-W1), brain (RTbrain-W1), gonad (RTG-2), gut (RTgut-GC), liver (RTL-W1) and spleen (RTS-11) cells (Fig. 3). In good agreement with the whole tissue results, p53 levels were much higher in gill and brain cell lines compared to those corresponding to other tissues. It should be noted that the migration of p53 compared to the molecular weight standards was slightly slower for tissue relative to cell extracts (compare Figs. 2 and 3), which may reflect differences in posttranslational modifications, or simply a difference in the way the two types of samples run on SDS gels. 3.3. Exposure to checkpoint-inducing agents does not alter rainbow trout p53 levels

the radiomimetic drug bleomycin, which causes DNA strand breaks, and the ribonucleotide reductase inhibitor hydroxyurea, which results in the depletion of intracellular dNTP pools. Even at the highest dose of bleomycin (200 μg/mL), p53 levels in both cell types remained the same as for untreated controls (Fig. 4). To verify that the bleomycin treatment was actually causing DNA damage, we also checked the levels of phosphorylated histone H2AX (γH2AX), since phosphorylation of histone H2AX is associated with DNA double strand breaks in both mammalian and fish cells (Rogakou et al., 1998; Krumschnabel et al., 2010). Despite no change in p53, γH2AX levels clearly rose in response to bleomycin treatment for both RTbrain-W1 and RTgill-W1 cells, consistent with the generation of DNA damage. As with exposure to bleomycin, both cell types dosed with hydroxyurea showed no changes in the level of p53 (Fig. 5A). To verify the effectiveness of the hydroxyurea treatment, RTbrain-W1 and RTgill-W1 proliferation was monitored, comparing cells exposed to 200 mM hydroxyurea to untreated controls (Fig. 5B). For both cell types, hydroxyurea addition completely halted cell division, consistent with depletion of dNTPs. Given that neither DNA damage nor dNTP depletion provoked increased levels of p53 in rainbow trout cell lines, we wanted to further verify that treatment with these reagents was activating other checkpoint proteins. Two central checkpoint kinases that act upstream of p53 are ATM and ATR. ATM is activated in response to double strand DNA breaks, while ATR signaling is triggered by regions of single stranded DNA which can be caused by either DNA damage or replication fork stalling (reviewed in Warmerdam and Kanaar, 2010). Moreover, p53 has been identified as a downstream target of both (reviewed in Abraham, 2001). To investigate whether bleomycin and hydroxyurea treatment influences ATM/ATR activity in RTbrain-W1 and RTgill-W1 cells, we used an antibody which specifically detects proteins with a phosphorylated ATM/ATR substrate motif (Cell Signalling Technology; Schwartz et al., 2002). For both cell types, exposure to either reagent resulted in the appearance of prominent protein bands as judged by western blot detection with this antibody (Fig. 6), consistent with ATM/ATR activation. 4. Discussion For the first time, an antibody has been raised to rainbow trout p53 and used to assess the levels of this protein in extracts from both whole fish and fish cell lines representing a variety of tissue types. The expression of p53 has been shown to vary considerably between different tissue types during normal mammalian development (Schmid et al., 1991; Lichnovsky et al., 1998). Of the tissues examined,

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Studies in mammalian systems have shown that higher p53 levels can be triggered through cellular exposure to agents that damage DNA or deplete dNTP pools (reviewed in Pluquet and Hainaut, 2001). In order to examine whether such responses are conserved in rainbow trout, RTgill-W1 and RTbrain-W1 cells were treated with

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RTbrain-W1 Fig. 3. Western blot analysis of p53 levels in rainbow trout cell lines. 20 μg of protein extract from rainbow trout brain (RTbrain-W1), gill (RTgill-W1), gonad (RTG-2), gut (RTgut-GC), liver (RTL-W1) and spleen (RTS-11) cell lines was subjected to SDS-PAGE and then transferred to a nitrocellulose membrane. Detection was carried out with affinity purified rabbit anti-rainbow trout p53 primary and goat anti-rabbit IgG HRP conjugated secondary antibodies. The molecular masses of the marker bands (M) are indicated in kDa to the left of the image (Fermentas). Ponceau S staining was performed prior to detection to assess relative protein loading.

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Fig. 4. Rainbow trout p53 levels remain constant following treatment with bleomycin. RTbrain-W1 and RTgill-W1 cells were treated with the indicated concentrations of bleomycin for 24 h. Protein extracts were prepared and subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane. Detection was carried out with affinity purified rabbit anti-rainbow trout p53, anti-γH2AX and anti-actin antibodies as described in Materials and methods. Ponceau S staining was performed prior to detection to assess relative protein loading.

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rainbow trout p53 levels were highest in gill and brain. Detection of high p53 levels in the gill may reflect the need for elevated checkpoint readiness in a tissue that would be expected to be particularly accessible to genotoxic compounds in the aquatic environment. Exposure of mudskipper to ammonia was previously reported to result in a transient increase in p53 levels in gills (Ching et al., 2009), and we have similarly reported high CHK2 levels in rainbow trout gill (Steinmoeller et al., 2009). p53 has previously been shown to mediate cell-cycle arrest, DNA repair and apoptosis in neuronal cells during the course of normal neuronal development and in adult organisms in response to DNA damage (reviewed in Danilova et al., 2008; Tedeschi and Di Giovanni, 2009). In addition to protecting the genome, p53 might have important developmental and physiological functions in the brain. Preferential expression of p53 in the brain has previously been reported for both mouse and human embryos with levels declining during later stages of development (Gottlieb et al., 1997; Lichnovsky et al., 1998; Komarova et al., 2000). Similarly, at the pharyngula stage (24–48 h) of zebrafish development, the highest levels of p53 mRNA expression are seen in the tectum and retina (Thisse et al., 2000). Although the corresponding p53 protein was not detected in 48 h untreated zebrafish embryos, exposure to R-roscovitine, a reagent known to stabilize p53, resulted in a strong signal for p53 specifically in the retina, as well as in gut epithelium, and liver (Lee et al., 2008). The importance of p53 in neuronal development has been further highlighted by studies of p53-null mice, where many of those surviving to term have been found to exhibit exencephaly and/or abnormalities of the neural retina and lens (Armstrong et al., 1995; Sah et al., 1995). Recently, p53 has been shown in mammals to have a role in neurodegeneration and possibly in Alzheimer's disease (Proctor and Gray, 2010). Clearly, it will now be of interest to examine the extent to which p53 governs fish brain development and function. In contrast to mammalian p53, fish p53 expression can be recalcitrant to easy experimental modulation. Previously, it was reported that p53 transcript levels from both medaka fry and cell cultures were not altered following exposure to ultraviolet radiation (Chen et al., 2001). In our study, neither DNA damage through treatment with bleomycin nor depletion of dNTP pools with hydroxyurea provoked an increase in p53 levels. While we can't rule

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days Fig. 5. Rainbow trout p53 levels remain constant following treatment with hydroxyurea. (A) RTbrain-W1 and RTgill-W1 cells were treated with the indicated concentrations of hydroxyurea for 24 h. Protein extracts were prepared and 20 μg aliquots were subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane. Detection was carried out with affinity purified rabbit anti-rainbow trout p53 and anti-actin antibodies as described in Materials and methods. Ponceau S staining was performed prior to detection to assess relative protein loading. (B) Counts for RTbrain-W1 and RTgill-W1 cells treated with 200 mM hydroxyurea compared to untreated control cultures. Cells were counted at the indicated time points using a Coulter counter (Beckman Coulter). Error bars represent standard deviation for 3 replicate counts.

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Fig. 6. The profile of potential phosphorylated ATM/ATR substrates is altered in rainbow trout cell lines following treatment with bleomycin or hydroxyurea. RTbrain-W1 and RTgillW1 cells were treated with the indicated concentrations of bleomycin or hydroxyurea for 24 h. Protein extracts were prepared and 20 μg aliquots were subjected to SDS-PAGE followed by transfer to a nitrocellulose membrane. Detection was carried out with anti-phospho-(Ser/Thr) ATM/ATR substrate and anti-actin antibodies as described in Materials and methods. Ponceau S staining was performed prior to detection to assess relative protein loading.

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out that exposure to these reagents resulted in post-translational modifications, such as phosphorylation, no changes in p53 mobility were observed through western blot analysis with our polyclonal antiserum to rainbow trout p53. Rau Embry et al. (2006) obtained similar results with a commercial polyclonal antiserum to human p53 (sc-6243, Santa Cruz Biotechnology) which we found to vary considerably in its effectiveness for rainbow trout p53 depending on the lot number (Fig. 1B and data not shown). They saw no change in p53 levels for rainbow trout liver cell lines and primary hepatocytes, as well as desert topminnow hepatocellular carcinoma cells, exposed to various chemotherapeutic agents known to upregulate p53 in mammalian cells. In contrast to the findings with the genotoxic agents described above, exposure to oxidative stress, which can lead to DNA damage, has been reported to stimulate an increase in p53 expression in both whitefish liver and tilapia blood cells (Brzuzan et al., 2009; Mai et al., 2010). Given the lack of p53 induction in fish cells following exposure to many reagents known to cause DNA damage and DNA replication defects, one might question whether p53 holds the central checkpoint role it has become known for in other organisms. While p53 mutations are found at a very high rate in human cancers (reviewed in Olivier et al., 2010), it is not yet known whether this is the case for fish. Screening of medaka tumors induced by exposure to the DNA alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), did not uncover any mutations in the regions of the p53 gene equivalent to mutational hotspots in mammalian orthologs (Krause et al., 1997), however mutations in the p53 gene have been identified in European flounder living in polluted areas of the north-eastern Atlantic that had developed liver tumors (Cachot et al., 2000). Persuasive evidence of conserved checkpoint function in at least some cell types has been provided through the generation of p53 mutant zebrafish lines that were found to develop peripheral nerve sheath tumors (Berghmans et al., 2005), while medaka p53 knockouts exhibited a wide range of tumour types (Taniguchi et al., 2006). In both systems, mutant fish had impaired p53 target gene induction and a suppression of apoptosis following γ-irradiation. It is also important to keep in mind that stabilization of p53 and the resultant increase in its cellular levels is only one way that its activity can be regulated. For example, p53 phosphorylation was increased at serine 15, but total p53 levels remained unchanged, in axolotl cells exposed to either MNNG or UV radiation (Villiard et al., 2007). Treatment with R-roscovitine and/or γ-irradiation did not alter p53 levels in total protein extracts from zebrafish embryos, however immunohistochemical staining of p53 in embryo sections following exposure to both reagents did reveal localized increases within regions of certain tissue types, indicating a change in distribution (Lee et al., 2008). Thus, our findings add to a growing body of evidence that an increase in total p53 is not a consistent feature of checkpoint responses in lower vertebrates. A practical application of fish checkpoint proteins would be to use them as biomarkers for monitoring of genotoxic substances in polluted waters. Previously, we identified rainbow trout CHK2 as a possible indicator of DNA double strand breaks, as its levels increased following exposure of RTbrain-W1 cells to bleomycin (Steinmoeller et al., 2009). Based on our observations in the present study with a couple of rainbow trout cell lines, p53 accumulation seems to be a poor biomarker candidate for double strand breaks or replication fork stalling as a consequence of nucleotide depletion. In contrast, levels of γH2AX rose sharply following treatment of both RTbrain-W1 and RTgill-W1 cells with bleomycin, suggesting that it could be useful as a biomarker. Following treatment with bleomycin or hydroxyurea, western blot analysis of protein extracts using an antibody specific for ATM/ATR phosphosubstrates revealed a striking increase in relative intensity of numerous protein bands, the pattern of which was cell line specific. Future research aimed at determining the identity of these upregulated proteins would be a promising approach to reveal yet more biomarker candidates.

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