Anne Roobol

Jo Roobol

Martin J Carden

Amandine Bastide

Anne E Willis

Warwick Dunn

Royston Goodacre

C Mark Smales
email: c.m.smales@kent.ac.uk

Mark Smales

Ataxia-telangiectasia mutated and Rad3-related kinase (ATR) is activated by mild hypothermia in mammalian cells and subsequently activates p53

Keywords: cold shock, hypothermia, ATR, p53, lipid composition, Chinese hamster ovary cells

In vitro cultured mammalian cells respond to mild hypothermia (27-33 o C) by attenuating cellular processes and slowing and arresting the cell cycle. The slowing of the cell cycle at the upper range (31-33 o C) and its complete arrest at the lower range (27-28 o C) of mild hypothermia is effected by the activation of p53 and subsequent expression of p21. However, the mechanism by which cold is perceived in mammalian cells with the subsequent activation of p53 has remained undetermined. Here we report that the exposure of CHOK1 cells to mildly hypothermic conditions activates the Ataxia-telangiectasia mutated and Rad3-related kinase (ATR)-p53-p21 signalling pathway and is thus a key pathway involved in p53 activation upon mild hypothermia. In addition, we show that although p38 MAPK is also involved in activation of p53 upon mild hypothermia, this is likely the result of activation of p38 MAPK by ATR. Further, we show that cold-induced changes in cell membrane lipid composition are correlated with the activation of the ATR-p53-p21 pathway. We therefore provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in mammalian cells leading to p53 and p21 activation which is known to lead to cell cycle arrest.

INTRODUCTION

Under mildly hypothermic conditions (31-33 o C) mammalian cells proliferate slowly [START_REF] Underhill | The cold-shock response in mammalian cells: Investigating the HeLa cell cold-shock proteome[END_REF], generally attenuate the processes of transcription and mRNA translation [START_REF] Underhill | On the effect of transient expression of mutated eIF2 alpha and eIF4E eukaryotic translation initiation factors on reporter gene expression in mammalian cells upon cold-shock[END_REF] (although protein folding may actually improve [START_REF] Masterton | Posttranslational events of a model reporter protein proceed with higher fidelity and accuracy upon mild hypothermic culturing of Chinese hamster ovary cells[END_REF]) and the cell cycle proceeds at a much reduced rate [START_REF] Al-Fageeh | Control and regulation of the cellular responses to cold shock: the responses in yeast and mammalian systems[END_REF]. However, below 30 o C cells become arrested, predominantly in G1 phase [START_REF] Kaufmann | Influence of low temperature on productivity, proteome and protein phosphorylation of CHO Cells[END_REF][START_REF] Marchant | Metabolic rates, growth phase, and mRNA levels influence cell-specific antibody production levels from in vitro-cultured Mammalian cells at sub-physiological temperatures[END_REF]; normally the stage in the cell cycle when protein synthesis rates are optimal. Indeed, because of this, other strategies for inducing cell cycle arrest in late proliferative stage cultures of mammalian cells in an industrial sense have been investigated, including generation of cell lines with inducible expression of the general cyclin inhibitor, p21 [START_REF] Bi | Uncoupling of cell growth and proliferation results in enhancement of productivity in p21CIP1-arrested CHO cells[END_REF] and the addition of solvents such as dimethyl sulphoxide to the growth medium which also induces p21 expression [START_REF] Fiore | Dimethyl sulfoxide restores contact inhibition-induced growth arrest and inhibits cell density-dependent apoptosis in hamster cells[END_REF]. However, exposure to mildly hypothermic conditions remains the most economic and most effective way of extending the productive life of cultured mammalian cells for large-scale recombinant protein production [START_REF] Al-Fageeh | The cold-shock response in cultured mammalian cells: Harnessing the response for the improvement of recombinant protein production[END_REF].

The slowing of the cell cycle at the upper range of mild hypothermia (31-33 o C) and its complete arrest at the lower range of mild hypothermia (27-28 o C) is regulated by the expression of p21 [START_REF] Ohnishi | p53-dependent induction of WAF1 by cold shock human glioblastoma cells[END_REF]. There are numerous examples (reviewed in [START_REF] Gartel | The role of the cyclin-dependent kinase inhibitor p21 in apoptosis[END_REF]) of p21-induced cell cycle arrest protecting damaged or stressed cells from apoptosis, thus providing a time-window within which the damage may be repaired or stress conditions removed. This is certainly the case for mildly cold-stressed cells since they recover rapidly and fully on returning to 37 o C [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF]. It is also well established that p21 induction in mildly hypothermic cells is subsequent to an increase in the stability and hence amounts of the tumour suppressor protein p53 [START_REF] Ohnishi | p53-dependent induction of WAF1 by cold shock human glioblastoma cells[END_REF][START_REF] Matijasevic | Hypothermia causes a reversible, p53mediated cell cycle arrest in cultured fibroblasts[END_REF] and to changes in the p53 isoform array [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF], though the post-translational modification(s) generating these observed changes in p53 isoform pattern remain to be identified. Indeed, p53-deficient mammalian cells do not show cell cycle arrest at mildly hypothermic temperatures confirming the key role of p53 in regulating this process upon cold shock [START_REF] Ohnishi | p53-dependent induction of WAF1 by cold shock human glioblastoma cells[END_REF][START_REF] Matijasevic | Hypothermia causes a reversible, p53mediated cell cycle arrest in cultured fibroblasts[END_REF]. However, the mechanism(s) by which p53 phosphorylation and amounts are unregulated upon mammalian cells being placed under hypothermic conditions, or how these conditions are sensed, are currently unknown.

In addition to cell cycle arrest and the general attenuation of transcription and translation, changes to the cell membrane composition are also observed when both prokaryotic [14] and eukaryotic [START_REF] Pandero | A downshift in temperature activates the high osmolarity glycerol (HOG) pathway, which determines freeze tolerance in S.cerevisiae[END_REF] cells are exposed to hypothermic conditions. Essentially, cells respond to reduced temperature by increasing the polyunsaturated fatty acid content of membrane phospholipids thereby maintaining the fluidity under hypothermic conditions, so-called homeoviscous adaptation [START_REF] Sinensky | Homeoviscous adaptation -a homeostatic process that regulates the viscosity of membrane lipids in Escherichia coli[END_REF]. At 37 o C an increase in polyunsaturated fatty acid content of membrane phosphatidylcholines, induced by exposure to the Ca 2+ -dependent phospholipase A 2 inhibitor bromoenol lactone, has been reported to arrest mammalian cells in G1 by A c c e p t e d M a n u s c r i p t activation of the p53-p21 pathway [START_REF] Zhang | The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR[END_REF]. This was subsequently shown to be mediated by phosphorylation of p53 at Ser15 by a member of the phosphoinositide-3-kinase-related-kinase (PIKK) family, ATR (ataxia-telangiectasia mutated and Rad3-related kinase) [START_REF] Zhang | The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR[END_REF], a signalling pathway more usually associated with cell cycle arrest in response to compromised DNA replication [START_REF] Abraham | Cell cycle checkpoint signaling through the ATM and ATR kinases[END_REF]. Since there was no evidence of DNA damage in these cells with altered membrane composition, it was concluded that an increase in the ratio of polyunsaturated to saturated fatty acids in phosphatidylcholines in cell membranes independently activates the ATR-p53-p21 pathway [START_REF] Zhang | The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR[END_REF]. Further, the expression of the cold-shock protein CIRP (also known as hnRNP A18) is induced at mildly hypothermic temperatures in mammalian cells [START_REF] Al-Fageeh | Cold-inducible RNA binding protein (CIRP) expression is modulated by alternative mRNAs[END_REF] and binds to the 3'-UTRs of certain transcripts, increasing their translation [START_REF] Yang | Functional significance for a heterogenous ribonucleoprotein A18 signature RNA motif in the 3'-untranslated region of ataxia telangiectasia mutated and Rad3-related (ATR) transcript[END_REF]. CIRP protein binds to the 3'-UTR of ATR mRNA and over-expression of CIRP results in increased ATR protein levels [START_REF] Yang | Functional significance for a heterogenous ribonucleoprotein A18 signature RNA motif in the 3'-untranslated region of ataxia telangiectasia mutated and Rad3-related (ATR) transcript[END_REF].

In view of these reports, and our own previous observations that changes in p53 isoform pattern are observed upon mildly hypothermic conditions in mammalian cells as are the detection of lipid droplets at lower temperature [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF] we set out to investigate (i) if p53 activation upon mild hypothermia was at least in part mediated via the ATR kinase signalling pathway and (ii) whether mild hypothermia resulted in changes in lipid composition consistent with those previously reported to activate ATR (an increase in the ratio of polyunsaturated to saturated fatty acids), thus linking hypothermia-induced changes in membrane composition to hypothermia-induced cell cycle arrest. We show that p53 phosphorylation and activation in the commercially relevant CHOK1 cell line is mediated by the ATR-p53-p21 pathway and ATR signalling is thus a key pathway involved in p53 activation upon mild hypothermia, and further, that cold-induced changes in cell membrane lipid composition are associated with this. We therefore provide the first mechanistic detail of cell sensing and signalling upon mild hypothermia in CHO cells leading to p53 and p21 activation which are known to subsequently result in cell cycle arrest.

EXPERIMENTAL

Cells and cell maintenance

CHOK1 cells (originally sourced from the European Collection of Cell Cultures) were maintained in DMEM:F12 (Invitrogen), supplemented with 10% (v/v) dialyzed, heat inactivated foetal bovine serum (PAA, A15-507), glutamine, glutamate, aspartate, nucleosides and non-essential amino acids (Invitrogen), at 37 o C in a 5% CO 2 atmosphere as described previously [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF]. HeLa (Ohio) cells (sourced from ATCC) were maintained in DMEM supplemented with 10% (v/v) (PAA, A15-151), 2 mM glutamine and nonessential amino acids. [ 35 S]-labelled amino acid incorporation into proteins was assessed as described in [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF]. Exposure to mildly sub-physiological temperatures was undertaken in routine culture medium in appropriately regulated (+/-0.1 o C) incubators. Exposure to 15 M bromoenol lactone (Sigma) was for 6 h at 37 o C in normal growth medium. Caffeine (Sigma) was used at a final concentration of 2.5 mM, wortmannin (Sigma) at a final concentration of 20 M, the p38 kinase inhibitor SP203580 (Calbiochem) at a final concentration of 10 M, the ATM inhibitor KU0055933 at a final concentration of 10 M and the DNA-PK inhibitor NU7441 at a final concentration of 1 M. Cells were exposed to these inhibitors for 30 min at 37 o C prior to transfer, without removal of inhibitor, to mildly hypothermic conditions. For RNAi knockdown, cells were transfected with validated siRNAs for Hs ATR; Hs_ATR_11 (Qiagen) and Hs_ATR_12 (Qiagen), using HiPerfect reagent (Qiagen) according to the manufacturer's instructions and using a final siRNA concentration of 5 nM. When combining RNAi knockdown of ATR with inhibitor experiments, CHOK1 cells were first exposed to ATR siRNA for 48 h at 37 o C, then 10 M SP203580 added for a further 30 min prior to transfer, in the continued presence of siRNA and SP203580, to 32 o C or 27 o C for 10 h. Total RNA was extracted from intact cells using the commercially available Qiagen RNeasy kit as per the manufacturer's instructions. Cell lysates for protein analysis were prepared by lysing PBS-washed cells into ice-cold extraction buffer, 20 mM HEPES-NaOH, pH 7.2, containing 100 mM NaCl, 1% (w/v) Triton-X100, protease inhibitors (10 g/ml leupeptin, 2 g/ml pepstatin, 0.2 mM phenylmethylsulphonyl fluoride) and protein phosphatase inhibitors (50 mM NaF and 1 mM activated Na 3 VO 4 ). For each lipid extraction 5x10 6 CHOK1 cells (60% confluent) were washed with 10 ml of 0.8% (w/v) NaCl at the appropriate temperature then scraped into 600 l of dry-ice-chilled methanol followed by extraction of lipids by vortexing for 15 s with 600 l of dry-ice-chilled chloroform and then freezing in liquid N 2 for 1 min before thawing on ice. This freeze-thaw procedure was repeated twice more. The chloroform extract was then washed twice by adding 900 l of ice-cold H 2 O, vortexing for 30 s followed by centrifugation at 16,000 g for 15 min and removal of the aqueous layer. Samples were stored at -80 o C prior to transportation (as solutions) on dry ice to Manchester for lipid analysis.

Extraction of RNA, protein and lipids from cells

qRTPCR analysis of mRNA levels

Relative quantitation of mRNA levels was undertaken by qRT-PCR using the BioRad iScript one step kit according to the manufacturer's instructions with the following primers: Human ATR, Quantitect primer assay Hs_ATR_1_SG (Qiagen); CHO ATR, forward, GTTAATCCATGGTCGAGC, reverse, TTGTCATAGTACTTGGCAAGG; Human actin, forward, CCGAGGACTTTGATTGCAC, reverse, AGTGGGGTGGCTTTTAGGAT and CHO actin, forward, AGCTGAGAGGGAAATTGTGCG, reverse, GCAACGGAACCGCTCATT.

Reactions were carried out using a Mastercycler ep Realplex Thermocycler (Eppendorf) programmed for a reverse transcription incubation at 50 o C for 10 min, followed by a 95 o C hold for 5 min and a subsequent 40 cycles of 10 sec at 95 o C, 20 sec at 55 o C.

SDS-PAGE and immunoblot analysis

For SDS-PAGE analysis 10% separation gels were prepared according to the procedure of Laemmli [START_REF] Laemmli | Cleavage of structural proteins during the assembly of bacteriophage T4[END_REF] loading 20 g of protein lysate per lane. SDS-PAGE-resolved polypeptides were transferred to nitrocellulose which was then blocked with 5% (w/v) non-fat milk in 0.2% (w/v) Tween20-TBS. Primary antibodies were sourced as follows: anti-ATR, Santa Cruz (N19); anti-p53, Dako (clone DO-7); anti-p53 phosphorylated at Ser15, Cell Signalling (#9284); anti-p21, Santa Cruz (C19); anti--actin, Sigma (clone AC15). Peroxidase-conjugated secondary antibodies were detected by enhanced chemiluminescence using Hyperfilm ECL (GE Healthcare). Linearity of antibody response over the concentration range of target protein was established previously [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF].

Immunofluorescence microscopy

PBS-washed CHOK1 cells grown on 13 mm glass coverslips were fixed in 3% (w/v) paraformaldehyde in PBS for 15 min at 37 or 27 o C then permeabilised with 0.5% (w/v) Triton X-100 in PBS for 10 min at room temperature. After blocking in 0.1% (w/v) Tween-20-PBS containing 3% (w/v) BSA coverslips were incubated with anti-ATR (1:50) overnight at 4 o C. Further processing and detection were as in [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF].

Mass spectrometry analysis of lipids

Profiling of the lipid fraction of cell extracts was performed using direct infusion mass spectrometry (DIMS [START_REF] Goodacre | Metabolic profiling using direct infusion electrospray ionisation mass spectrometry for the characterisation of olive oils[END_REF]). Chloroform extracts (400 µl) were diluted in 600 µl of methanol. Samples were infused into an electrospray ThermoFisher Scientific LTQ-Orbitrap XL mass spectrometer operating in negative ion mode at a flow rate of 5 µl min -1 for one minute. Accurate mass data were acquired in the Orbitrap mass analyser operating at a mass resolution of 100 000 (at m/z 400) and a scan time of 1.2 s. All mass spectra were averaged to provide a single mass spectrum for each sample which was passed forward for further data processing and analysis. All mass peaks were binned to the nearest nominal mass (defined as mass bins).

Data analysis of lipid profiles

Multivariate Principal Components-Discriminant Function Analysis (PC-DFA) using cross validation was performed to inspect the clustering of sample classes as detailed in [START_REF] Jarvis | Rapid discrimination of bacteria using surface enhanced Raman spectroscopy[END_REF]. All data were normalised to a sum of 1. A PCA model was then constructed [24] with three of six samples per class; the first 10 PCs were extracted and these accounted for 99.8% of the total explained variance. Next these PCs were used by the DFA algorithm [25] with the a priori knowledge of the 6 different treatments. In order to validate this PC-DFA model it was tested by cross-validation by the projection of the three remaining samples; a process that allows cluster integrity to be assessed. That is to say, if the projected samples co-cluster with samples used to construct the PC-DFA model then the groupings observed and their relationships are valid. All multivariate analyses were performed in Matlab (The MathWorks Inc., Natwick, MA, USA). To define statistically significant differences Kruskal-Wallis analyses were performed. For those mass bins of statistical significance, further analysis was performed to determine the lipid accurate mass contributing to this statistical difference. Putative identification of lipids (as the deprotonated ion, sodiated or potassiated negatively charged adducts) was performed by matching to the accurate mass of lipids in the Lipid Maps database (http://www.lipidmaps.org/) with a mass accuracy less than 2 ppm.

RESULTS

p53 is phosphorylated at Ser15 when CHOK1 cells are exposed to mildly hypothermic conditions

When CHOK1 cells were transferred from 37 o C to 32 o C the subsequent growth rate was greatly reduced while cells transferred to 27 o C ceased to proliferate (Fig. 1A). Under both of these mildly hypothermic conditions expression of p21 was induced and maintained throughout a 5 day period whereas phosphorylation of p53 at Ser15 was initially dramatically increased but then decreased towards the end of the 5 day period (Fig. 1B). Notably, the levels of both p53 phosphorylation and p21 expression were greater at 27 o C compared to 32 o C in line with the proliferation status of the cells at these two temperatures and at 32 o C cell proliferation was observed towards the end of the 5 day period when Ser15 phosphorylation levels were once again decreased (Fig. 1B). A more detailed examination of the early period following temperature shift to 27 o C (Fig. 1C) clearly showed that the phosphorylation of p53 at Ser15 preceded a modest increase in p53 levels that, in turn, preceded the induction of p21. This is consistent with the response to hypothermia being due to stabilization of p53 consequent to its phosphorylation at Ser15 and this increased level of p53 then inducing p21 expression.

The ATR protein kinase regulates phosphorylation of p53 at Ser15 upon exposure to mild hypothermia

Having established that p53 is phosphorylated at Ser15 in response to mild hypothermia, we set out to establish the kinase(s) responsible for this phosphorylation. Phosphorylation at Ser15 of p53 can be mediated by several protein kinases, including ATM (Ataxia-telangiectasia-mutated kinase), ATR, DNA-PK and the stress response signalling pathway protein kinase, p38 MAPK [26]. To determine whether any of these was effecting p53 phosphorylation during mild hypothermia we used a combination of general and specific protein kinase inhibitors and siRNA knockdown. Initially we used caffeine, a well-known, though not very specific, inhibitor of the PIKK family of protein kinases [START_REF] Sarkaria | Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine[END_REF]. In the concentration range usually employed (low millimolar) it inhibits both ATM and ATR but DNA-PK is relatively resistant. However another PIKK family member, mTOR (mammalian target of rapamycin), a protein kinase that positively regulates protein synthesis in response to nutrient availability and growth factor signalling, is A c c e p t e d M a n u s c r i p t also inhibited by low millimolar concentrations of caffeine [START_REF] Sarkaria | Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine[END_REF]. This must be taken into account when assessing the effect of caffeine on hypothermia-induced p21 expression. In the short term, 2.5 mM caffeine inhibited phosphorylation of p53 at Ser15 when cells were transferred to 32 o C, but had little effect when cells were transferred to 27 o C (Fig. 2A). During longer term exposure to caffeine, phosphorylation of p53 at Ser15 was less sustained than in the absence of caffeine and p21 expression was reduced, under both hypothermic conditions investigated (32 and 27 o C) (Fig. 2B). When compared with the inhibition of general protein synthesis by caffeine (due to mTOR inhibition), the inhibition by caffeine of p21 expression was greater (Fig. 2D), consistent with either ATM or ATR being involved in hypothermia-induced expression of p21. More specific inhibition of DNA-PK with NU7441 [START_REF] Chen | Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break[END_REF] had no effect on either hypothermia-induced phosphorylation of p53 at Ser15 or induction of p21 (Fig. 3A and3B). Thus, of the potential PIKK kinases that could phosphorylate p53 at Ser15 upon mild-hypothermia, these data suggested that either ATM or ATR are responsible.

The fungal metabolite wortmannin is a widely used, irreversible inhibitor of phosphoinositide-3kinases and treatment of cells with micromolar concentrations of this compound causes inhibition of ATM, DNA-PK and mTOR [29]. However, ATR is relatively resistant to wortmannin and cells require exposure to concentrations in excess of 100 M before ATR in inhibited [29]. In agreement with the results from the caffeine work that suggested ATR might phosphorylate p53 at Ser15, 20 M wortmannin had no effect on hypothermia-associated phosphorylation of p53 at Ser15 and marginally inhibited p21 induction (Fig. 2C). However, in contrast to inhibition by caffeine, inhibition of general protein synthesis by wortmannin was not significantly different from inhibition of hypothermia-induced p21 expression by wortmannin (Fig. 2E). We then used a specific inhibition of ATM, KU0055933 [START_REF] Chen | Ataxia telangiectasia mutated (ATM) is essential for DNA-PKcs phosphorylations at the Thr-2609 cluster upon DNA double strand break[END_REF], and this inhibited neither hypothermia-associated phosphorylation of p53 at Ser15 nor induction of p21 (Fig. 3A and3B). Therefore, using specific inhibitors to DNA-PK and ATM we were able to demonstrate that neither is the primary kinase involved in the hypothermia-induced p53-p21 pathway.

Although these inhibitor data are consistent with a signalling pathway in which ATR is a key kinase in the hypothermia-induced p53-p21 pathway, they are not specific ATR inhibitors and therefore to test this hypothesis further, siRNA knockdown of ATR mRNA was employed. This approach has been shown to effectively reduce ATR protein levels by approximately 70% 24 h after transfection [START_REF] Casper | ATR regulates fragile site stability[END_REF][START_REF] Roshel | Activation of the ATRmediated DNA damage response by the HIV-1 viral protein R[END_REF] and therefore although this does not obliterate protein levels a knockdown would be expected to result in decreased Ser15 phosphorylated p53 in response to mild hypothermia if this kinase is responsible. Two commercial, validated siRNAs to human ATR were tested for their ability to knockdown CHOK1 ATR mRNA due to the lack of availability of such reagents for CHO ATR. As expected, both siRNAs efficiently decreased HeLa cell ATR mRNA over a 48 h period by between 67 and 77% (Fig. 4A). When tested in CHOK1 cells, exposure to one of these siRNAs for 48 h decreased CHO ATR mRNA by 77-87%. However, knockdown by the second siRNA was less effective and more variable in CHO cells (Fig. 4A). Knockdown of ATR mRNA was maintained at 72 h and, to a lesser degree, at 96 h posttransfection (Fig. 4A). We confirmed that knockdown of ATR mRNA resulted in a knockdown in ATR protein levels in both HeLa and CHO cells by western blotting which showed ATR protein levels were reduced by 55-85% after 48h exposure to ATR siRNA (Fig. 4B). Following transfection with these siRNAs, cells were maintained at 37 o C for 48 h before transfer to either 32 o C or 27 o C for a further 10 h. The decreases in ATR mRNA and protein observed after 48 h exposure to ATR siRNA were clearly mirrored by the decrease in the extent of phosphorylation at Ser15 of p53 under these mildly hypothermic conditions (Fig. 4C). Inhibition of cold-induced phosphorylation of p53 at Ser15 was still evident at 72 h, but not at 96 h, post-transfection (Fig. 4D), but at this last time point the hypothermia-associated phosphorylation of p53 is already in decline (Fig. 1B).(Fig. 4D). These data are consistent with the inhibitor data indicating that hypothermia induces p53 phosphorylation and p21 activation via the ATR-p53-p21 signalling pathway. Furthermore, the relative longevity (several days) of p53 phosphorylation at Ser15 during hypothermia is also consistent with this phosphorylation being regulated by ATR [START_REF] Tibbetts | A role for ATR in the DNA damage-induced phosphorylation of p53[END_REF]. We note that although knockdown of ATR protein was clearly achieved, ATR protein was still present and that some phosphorylated p53 was also present in the knock down experiments (Fig 4C and4D). We were unable to ascertain from these data whether the phosphorylated p53 present upon cold shock was A c c e p t e d M a n u s c r i p t due to the residue ATR protein present or as a result of an additional pathway not investigated here. Despite this, when cells were shifted to 27 o C following knockdown of ATR for 48 h at 37 o C by siRNA, those wells in which knockdown had been undertaken initially showed an increased in cell numbers 1 and 2 days after being placed at 27 o C above that observed in the mock knockdown (Supplemenatry figure 1). This further suggests that p53 activation via ATR is involved in the inhibition of cell proliferation upon cold shock at 27 o C. This effect was lost after 2 days at 27 o C, probably because at this stage the knockdown cells at higher cell concentration are beginning to experience nutrient and growth stresses that leads to a decrease in cell number as seen in supplementary figure 1.

Involvement of the p38 MAPK stress kinase signalling pathway in cell cycle arrest during mild hypothermia

Although our data shows that ATR is involved in the regulation of p53 Ser15 phosphorylation upon mild hypothermia and ruled out ATM and DNA-PK, this phosphorylation could also be effected by the stress response protein kinase p38 MAPK (Hog1 in yeast) either directly [START_REF] She | ERKs and p38 kinase phosphorylate p53 protein at serine15 in response to UV radiation[END_REF], or via its phosphorylation at Ser33 and Ser46 of p53 that, in turn, enhances phosphorylation at Ser15 [START_REF] Bulavin | Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation[END_REF]. In yeast this protein kinase is activated by osmotic stress or exposure to cold [START_REF] O'rourke | Yeast go the whole HOG for the hyperosmotic response[END_REF] while in mammalian cells it has also been shown to be activated by osmotic stress [START_REF] Kishi | Osmotic shock induces G1 arrest through p53 phosphorylation at ser33 by activated p38MAPK without phosphorylation at ser15 and ser20[END_REF]. p38 MAPK is also activated by hypoxia and it has been reported that mildly hypothermic mammalian cells are hypoxic [START_REF] Conrad | Selective activation of p38alpha and p38 gamma by hypoxia: Role in regulation of cyclin D1 by hypopxia in PC12 cells[END_REF]. Furthermore, ATR can also phosphorylate, and thereby activate, p38 MAPK [START_REF] Im | ATR-dependent activation of p38 MAP kinase is responsible for apoptotic cell death in cells depleted of cdc7[END_REF]. It was therefore considered important to determine whether the p38 MAPK protein kinase was involved, either independently, or via activation by ATR, in the p53-p21 pathway induced by mild hypothermia.

SP203580 is an inhibitor frequently used for assessing involvement of p38 MAPK in signalling pathways [START_REF] Bain | The selectivity of protein kinase inhibitors: a further update[END_REF]. Although this inhibitor can also inhibit casein kinase 1 (5), this kinase will not phosphorylate p53 at Ser15 [START_REF] Lacroix | p53 and breast cancer, an update[END_REF] and therefore this inhibitor allowed us to investigate potential p38 MAPK involvement in hypothermia induced phosphorylation of p53 Ser15. Treatment of CHOK1 cells with 10 M SP203580 for 30 min prior to transfer to 27 o C or 32 o C reduced both phosphorylation at Ser15 of p53 and expression of p21 at these temperatures (Fig. 5A). Since SP203580 had no effect on general protein synthesis (Fig. 5B), its inhibition of p21 expression suggested involvement of p38 MAPK in the hypothermia-induced p53-p21 pathway.

To determine whether this p38 mechanism was a second pathway leading to phosphorylation at Ser15 of p53 independent of the ATR route, treatment with SP203580 was combined with siRNA knockdown of ATR. The resulting effects of combined ATR knockdown and SP203580 treatment on hypothermia-induced phosphorylation at Ser15 of p53 and p53 isoform pattern (Fig. 6) mirrored those effects observed for ATR knockdown alone (Fig. 4). This suggests that the involvement of p38 MAPK in hypothermia-induced cell cycle arrest lies within, rather than acts independently from, the ATR pathway; otherwise, the effects of ATR knockdown and SP203580 treatment should have been additive. We therefore suggest that the p38 MAPK protein kinase is involved in phosphorylation of p53 at Ser15 upon mild hypothermia via activation by ATR.

How is ATR activated upon exposure of CHOK1 cells to mild hypothermia?

Our data confirmed that ATR is activated upon CHOK1 cells being exposed to mild hypothermia which in turns phosphorylates Ser15 of p53 and p21 induction. However, how might ATR itself be activated upon mild hypothermia? We used immunofluorescence to determine if there was any change in the localisation of ATR following cold shock (Supplementary Figure 2). Using this approach it was found that 2-48 h post cold shock at 27 o C ATR appeared to be concentrated into the nucleolus (Supplementary Figure 2). We also noted an overall increase in ATR-associated fluorescence throughout the cell, particularly between 6 and 24 h exposure to 27 o C.

In addition to localization studies, we investigated changes to the lipid content of cold shocked cells. When prokaryotic and lower eukaryotic cells are exposed to hypothermic conditions, the A c c e p t e d M a n u s c r i p t unsaturated fatty acyl content of cell membrane lipids has been reported to increase [14]. In mammalian cells exposure to the Ca 2+ -independent phospholipase A 2 inhibitor bromoenol lactone (BEL) at 37 o C also increases the unsaturated fatty acyl content of phosphatidylcholines and activates ATR [START_REF] Zhang | The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR[END_REF]. We therefore compared the effect of BEL treatment with that of hypothermia on cellular lipid composition to determine if a similar effect was observed that might offer an explanation of ATR activation upon mild hypothermia. To achieve this, mass spectrometric analysis of total lipids extracted from cells maintained at normal temperature (37 o C), after treatment with BEL, and at mildly hypothermic temperatures was performed. Multivariate analysis (PC-DFA) was applied to the resulting data with cross-validation as described in the methods section and shown in Figure 7. The results show that BEL treated and 37 o C control cells were different from all cells cultured at reduced temperatures and the chemical treatment and control cells dominated the separation of the second canonical variate (Fig. 7A). When PC-DF1 was plotted against PC-DF3 (Fig. 7B) each class was biologically distinct from other classes highlighting that the detectable lipid profile of each of the six classes was different and perturbations (chemical or temperature-based) results in phenotypic changes.

Further univariate analyses using Kruskal-Wallis analysis of variance to define the lipids which were statistically significantly changing (Supplemental Table 1) revealed that the positive control (treatment with bromoenol lactone) showed a different relative change to the control in the PC-DFA model when compared to the temperature-treated cells. Ten lipids were statistically different (P<0.05) and all showed an increase in their relative concentration in the BEL treated cells compared to the control. Cells treated at 27 and 32 o C (mild hypothermia) for 6 h showed a similar trajectory from the control samples, with the 27 o C samples showing a greater biological difference in multivariate space than the samples perturbed at 32 o C. However, more statistically significant changes were observed between control and 32 o C samples in the univariate analysis (37 vs 4 changes for 32 o C and 27 o C, respectively). All the changes showed an increase in concentration of a range of lipids, predominantly phospholipids (diacylglyceroserines, diacylglyceroinositols, diacylglycerophosphocholines). In most cases, though not exclusively, an increase in the unsaturated double-bond content was present in the lipids of increased abundance. This highlights a definitive increase in the production of a specific class of lipids in response to temperature-based perturbations. The increase in temperature after hypothermia (recovery) perturbation provides a change in the lipid profile from that of reduced temperature but this lipid profile is distinct from all other samples. This shows that an increase in temperature changes the lipid profile but not to a normal profile at 2 h after the return to 37 o C. Decreases in the relative concentration of lipids were observed in the change from lower to higher temperature, of the same classes of lipids as observed increasing as the temperature was decreased. This highlights the specific role of these lipids in the response to temperature perturbation and how their relative concentration is temperature dependent. Although many of the lipids were chemically identified we were unable to show significant changes in the overall unsaturated fatty acyl content of cell membrane lipids. However, we have shown an increase in polyunsaturated lipids upon mild-hypothermia consistent with a previous report showing an increase in phosphatidylcholines containing polyunsaturated fatty acids activates ATR-p53 signalling at 37 o C.

DISCUSSION

Although we [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF] and others [START_REF] Ohnishi | p53-dependent induction of WAF1 by cold shock human glioblastoma cells[END_REF][START_REF] Matijasevic | Hypothermia causes a reversible, p53mediated cell cycle arrest in cultured fibroblasts[END_REF] have documented that p53 activation of p21 is a key mechanism by which mammalian cells initiate cell cycle arrest upon being subjected to mild hypothermic temperatures, the mechanism by which p53 is activated and the cellular mechanisms that allow the perception of cold and subsequent activation of p53 have remained undetermined. Here we show that the exposure of CHOK1 cells to mildly hypothermic conditions activates the ATR kinase which subsequently activates p53 by phosphorylation at Ser15 and hence the ATR-p53-p21 signalling pathway. We note that although our experiments clearly show ATR regulation of p53 phosphorylation upon cold shock, in our ATR knockdown and inhibitor experiments some ATR protein and phosphorylated p53 still remained and we were unable to ascertain from these data whether the phosphorylated p53 present upon cold shock in these We speculate that the primary stimulus for the activation of the ATR-p53-p21 signalling pathway upon mild hypothermia may be changes in membrane rigidity [14] as a direct result of changes in membrane lipid composition (homeoviscous adaptation). Our data show changes in the levels of polyunsaturated fatty acids upon cold shock which are known to influence the fluidity of cellular membranes, and further, these changes correlated with the activation of ATR. As described above, a previous report has demonstrated that changes to cell membrane fluidity and increased polyunsaturation activates ATR and the authors of this study suggest this occurs as a result of ATR 'sensing' the change in the ratio of polyunsaturated to saturated hydrocarbons [START_REF] Zhang | The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR[END_REF]. The question is how might this change in lipid composition activate ATR? Zhang et al [START_REF] Zhang | The increase of cell-membranous phosphatidylcholines containing polyunsaturated fatty acid residues induces phosphorylation of p53 through activation of ATR[END_REF] suggest this is the result of changes in the fluidity and function of the nuclear envelop where by the nuclear localised ATR sensors this change and is activated. We further speculate that this leads to an intranuclear relocalization of ATR upon activation (as shown in supplementary figure 2), p53 activation and cell cycle arrest. Such intranuclear relocalization of ATR to nuclear foci has been documented in response to both hypoxia [START_REF] Hammond | Hypoxia links ATR and p53 through replication arrest[END_REF] and DNA damage [START_REF] Barr | ATR Kinase Activity Regulates the Intranuclear Translocation of ATR and RPA following Ionizing Radiation[END_REF]. The overall increase in ATR-associated fluorescence throughout the cell during early exposure to 27 o C without an increase in immunoblot detection of ATR also suggests that there may also be a conformational change in ATR upon exposure of the cell to cold that renders the protein more accessible to the anti-ATR antibody used.

CHOK1 p53 carries a single point mutation at codon 211 in exon 6 in the DNA binding domain of the molecule, though this mutation is not within an evolutionarily conserved region [START_REF] Hu | Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing[END_REF]. Further, CHOK1 p53 is rather more abundant and stable than wild-type p53. At 37 o C its half-life is 5.2 h [START_REF] Roobol | Biochemical insights into the mechanisms central to the response of mammalian cells to cold-stress and subsequent rewarming[END_REF] compared with the more usual range of 20-60 min for p53 half-lives. Furthermore, CHOK1 p53 is not further stabilised, and thereby increased in amount, by ionising radiation i.e. by the ATM signalling pathway alone [START_REF] Hu | Characterization of p53 in Chinese hamster cell lines CHO-K1, CHO-WBL, and CHL: implications for genotoxicity testing[END_REF]. Thus, even though CHOK1 p53 is relatively abundant it is not sufficient, under normal conditions, to activate transcription of p21. Even under mildly hypothermic conditions when p21 transcription is activated, increases in CHOK1 p53 total protein are very modest (Figs. 1, 2, 4 and6). What does change markedly in response to hypothermia is the phosphorylation status of p53. For wildtype p53, phosphorylation at Ser15 enhances p53 transactivation of p21 transcription by increasing the binding of p53 to its transcriptional co-activator, p300/CBP [44]. Furthermore, although phosphorylation at Ser15 of p53 is not itself sufficient to disrupt the interaction between p53 and Mdm2 that targets p53 for degradation, phosphorylation at this site is a prerequisite for phosphorylation at Ser20 of p53. Ser20 phosphorylation inhibits the binding of p53 to Mdm2 [START_REF] Chehab | Phosphorylation at ser20 mediates stabilisation of human p53 in response to DNA damage[END_REF]. The overall effect of phosphorylation of Ser15 of wild-type p53 is therefore 2-fold, i.e. enhanced stability and enhanced transcriptional activation ability. In the context of CHOK1 cells, this must mean that phosphorylation at Ser15 is sufficient to enhance the transcription factor activity of an already abundant p53, even though this transactivation activity might be compromised to some extent by the point mutation in the DNA binding domain of CHOK1 p53.

A consistent finding that has emerged from the numerous studies of p53 post-translational modifications is that phosphorylation and acetylation sites are seldom modified alone and that posttranslational modification at one site is often a prerequisite for further post-translational modifications elsewhere on p53 [26]. This activation of p53 at more than one site has been termed 'intramolecular phosphorylation site interdependence' [START_REF] Saito | Phosphorylation site interdependence of human p53 post-translational modifications in response to stress[END_REF] and is nearly always required before downstream transcriptional activation takes place. These data suggest that p53 transcriptional activation is tightly regulated by multiple modifications thus minimizing inappropriate transcriptional activation by p53 and providing a point of integration of signals from multiple protein kinases [START_REF] Saito | Phosphorylation site interdependence of human p53 post-translational modifications in response to stress[END_REF]. This appears to be the case for activation of p53 by mild hypothermia since we have found that p38 MAPK is also involved in the ATR-p53-p21 pathway.

While it is well established that ATR directly phosphorylates p53 at Ser15, there are conflicting reports regarding the ability of p38 MAPK to directly phosphorylate p53 at Ser15 [START_REF] She | ERKs and p38 kinase phosphorylate p53 protein at serine15 in response to UV radiation[END_REF][START_REF] Bulavin | Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation[END_REF]. However, some of the transient transfection experiments used to delineate this may have been complicated by the transfection vehicle itself eliciting a stress response involving phosphorylation of p53 and activation of p21 transcription [START_REF] Bulavin | Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation[END_REF]. We too have noted (unpublished) this effect of some transfection reagents on p53 activation and suggest that this, and indeed, the induction of p21 expression by addition of DMSO to the culture medium mentioned earlier, may be consequent to changes in membrane fluidity or composition. Nevertheless, it has been established that p38 MAPK phosphorylates p53 at Ser33 and Ser46 and that when p53 is doubly phosphorylated at these two sites, phosphorylation at Ser15 by other protein kinases is enhanced [START_REF] Bulavin | Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation[END_REF]. It has also been shown that in mammalian cells activation of p38 MAPK by hypoxia is mediated by ATR [START_REF] Hammond | The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation[END_REF].

These findings, when combined with the data we have presented here, suggest that mild hypothermia activates the transcription of p21 via ATR activation and subsequent phosphorylation of p53 at Ser15. At the same time, we suggest that ATR activates p38 MAPK , resulting in the phosphorylation of p53 at Ser33 and Ser46 that subsequently enhances Ser15 phosphorylation. Activated p53 in this way subsequently activates transcription of the downstream target p21 and induction p21 is known to lead to cell cycle arrest upon mild hypothermia.

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303 A c c e p t e d M a n u s c r i p t

  Biochemical Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303 A c c e p t e d M a n u s c r i p t experiments was due to the residual ATR protein present or as a result of an additional signalling pathway not investigated here.

Biochemical

  Journal Immediate Publication. Published on 02 Feb 2011 as manuscript BJ20101303 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101303 A c c e p t e d M a n u s c r i p t

FIGURE 1 .FIGURE 2 .FIGURE 3 .

 123 FIGURE 1. Mild hypothermia reduces cell proliferation, induces phosphorylation of p53 at Ser15 and subsequent induction of p21 expression. (A) Growth curves of CHOK1 cells maintained at the indicated temperatures. (B) Immunoblot detection of p53 phosphorylated at Ser15, total p53 protein and p21 in lysates of CHOK1 cells maintained at the indicated temperatures for the indicated number of days. (C) Immunoblot detection of p53 phosphorylated at Ser15, total p53 protein and p21 in lysates of CHOK1 cells maintained at 27 o C for the indicated number of hours. In B and C immunoblot detection of -actin served as an indicator of lysate protein loading.

FIGURE 4 .FIGURE 5 .FIGURE 6 .

 456 FIGURE 4. siRNA knockdown of ATR mRNA inhibits hypothermia induced phosphorylation of p53 at Ser15. (A) Hela and CHOK1 cells were transfected with 5 nM siRNAs against human ATR mRNA and then maintained at 37 o C for 48-96 h before undertaking qRT-PCR analysis of ATR mRNA levels in total RNA. (B) Immunoblot detection of ATR in Hela and CHOK1 cell lysates prepared after 48-96 h exposure to ATR siRNAs at 37 o C (m, mock transfected; 1, ATR siRNA1; 2, ATR siRNA 2). (C) Immunoblot detection of total p53 protein and p53 phosphorylated at Ser15 in cell lysates of CHOK1 cells 48 h after siRNA knockdown of ATR mRNA at 37 o C followed by 10 h at 27 or 32 o C (ut, untreated; m, mock transfected; 1, ATR siRNA 1; 2, ATR siRNA 2). (D) Inhibition of phosphorylation of p53 at Ser15 is maintained over longer periods of siRNA knockdown of ATR than 48 h. CHOK1 cells were transfected with siRNA 1 or mock transfected, incubated at 37 o C for 72 or 96 h and then maintained at 37 o C or transferred to 27 o C for a further 10 h prior to extraction for immunoblot detection of the indicated proteins.

FIGURE 7 .

 7 FIGURE 7. Exposure of CHOK1 cells mild hypothermia is associated with changes in the cellular lipid profile. Principal components-discriminant function analysis of all samples. (A) PC-DF1 plotted against PC-DF2 and (B) PC-DF1 plotted against PC-DF3. The first 10 PCs were used by the DFA algorithm and this accounted for 99.8% of the total explained variance. The multivariate model was constructed using 3 of 6 samples in each class (shown in red) and cross-validated by projection of the remaining 3 samples (shown in blue and with an asterisk). The level of agreement of the samples projected with those used to construct the model highlight the model is validated. Class 1control maintained at 37°C for 6 h with no treatment. Class 2 -control maintained at 37°C for 6 h with bromoenol lactone treatment. Class 3maintained at 27°C for 6 h. Class 4maintained at 32°C for 6 h. Class 5recovery at 37°C for 2 h after temperature of 27°C for 6 h. Class 6 -recovery at 37°C for 2 h after temperature of 32°C for 6 h.

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

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ACKNOWLEDGEMENTS

This work was part funded by grants BB/F018908/1 (Kent) and BB/F018738/1 (Nottingham/Leicester) from the Biotechnology and Biological Sciences Research Council (BBSRC), UK. The contribution from the Manchester Centre for Integrative Systems Biology was funded in particular by BBSRC and EPSRC (BB/C008219/1).