Perp expression stabilizes active p53 via modulation of p53-mdm2 interaction in uveal melanoma cells

ABSTRACT The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53

apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the

aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we

identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP,

p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription

(including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53

and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53

levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity. SIMILAR CONTENT BEING VIEWED BY OTHERS LOSS OF POLYCOMB REPRESSIVE

COMPLEX 1 ACTIVITY AND CHROMOSOMAL INSTABILITY DRIVE UVEAL MELANOMA PROGRESSION Article Open access 13 September 2021 EXPRESSION AND FUNCTIONAL SIGNIFICANCE OF PHOSPHOENOLPYRUVATE

CARBOXYKINASE 1 IN UVEAL MELANOMA Article Open access 26 April 2024 PRAME INDUCES GENOMIC INSTABILITY IN UVEAL MELANOMA Article Open access 29 November 2023 MAIN The regulation of p53 and of

its impressive array of interacting and target genes holds a central place among the molecular mechanism(s) influencing the choice between cell cycle arrest and apoptosis.1 The ability to

engage in apoptosis is critical to the tumour-suppressor role of p53 that is strongly supported by the presence of p53 mutations in over half of human cancers2, 3 and the compromised p53

activity by other mechanisms in the majority of other cancers.3 The latter scenario points towards the deregulation of downstream p53 targets as a mechanism employed by tumour cells to evade

apoptosis. PERP (p53 apoptosis effector related to PMP-22) was identified as a p53 transcriptional target that is distinctively induced during apoptosis, and not cell cycle arrest,4 and

consequently emerged as a prime candidate effector in the p53-dependent apoptotic pathway. Subsequent studies confirmed the pro-apoptotic role for PERP in a variety of cell types and

tissues,5, 6, 7, 8 with reported engagement of caspase-dependent pathways.5, 8 However, the precise function of PERP – a tetraspan protein primarily localized at the plasma membrane – in

eliciting an apoptotic response remains unknown. Cellular p53 levels are principally determined by the rate of degradation – mostly but not entirely linked to the E3 ligase activity of MDM2

– rather than synthesis.3, 9 Increased p53 stability in response to stress is achieved primarily through decreased MDM2 protein levels and reduced p53-MDM2 interaction, while the ensuing

elevated levels of transcriptionally active p53 result in increased MDM2 transcription.10, 11 This p53-MDM2 regulatory feedback loop is essential in maintaining tight regulation of p53

levels both in unstressed and stressed cells, with additional control through posttranslational modifications, most notably phosphorylation of key p53 serine (Ser) residues,12 some of which

influence the p53-MDM2 interaction13 and the nature of p53-target genes transcribed.14 (Co)localization and nuclear-cytoplasmic shuttling of p53 and MDM2 also contribute to regulation of p53

activity.15, 16 We have shown that PERP is an important molecular determinant of apoptosis in primary uveal melanoma (UM) tumours that is significantly downregulated in the aggressive

monosomy-3 type, compared with less aggressive disomy-3 type of UM.5, 17 Downregulation of PERP (THW18) was also reported in tumours of the ovary, uterus and breast, and in cutaneous

melanoma, pancreas and mammary carcinoma cell lines, compared with the respective normal tissues and non-metastasizing cell lines.18 The aim of this study was to investigate the effect of

increased PERP expression on its own upstream transcriptional regulator, p53. For this purpose, we used the UM cell line MEL20219 that provides a biological background of low endogenous PERP

protein with intact cell death machinery downstream of PERP.5 Our results show that PERP expression causes nuclear localization of p53 and increases the level of transcriptionally active

p53 protein, which also presents posttranslational modifications known to influence the p53–MDM2 interaction and to enhance the pro-apoptotic gene transcription. Together, these results

propose a novel role for PERP in enhancing functional p53 levels and reveal a potential new target for exploitation in the development of new therapeutic agents aimed at increasing the

endogenous p53 protein pool in neoplastic cells. RESULTS PERP EXPRESSION AUGMENTS P53 PROTEIN LEVEL To investigate the effect of increased levels of PERP protein on its transcriptional

regulator p53, lysates of MEL202 cells expressing green fluorescent protein (GFP)–PERP were analyzed by western blotting alongside lysates from control non-transfected (NT) and

GFP-only-transfected cells. Significantly increased levels of p53 protein were observed in MEL202 cells expressing GFP–PERP compared with control cells, in which p53 was barely detectable

(Figure 1a). Furthermore, the increased p53 protein levels in response to PERP expression were accompanied by an increase in the p53-negative regulator MDM2. As PERP is a transcriptional

target of p53, the increase in p53 protein in response to PERP expression suggested a role for PERP in the positive feedback regulation of p53. To determine if the observed increase in p53

and MDM2 proteins was transcription-driven, p53 and MDM2 transcriptional levels were determined by real-time quantitative PCR (Q-PCR) in control and GFP–PERP-expressing cells. There was no

significant increase in p53 mRNA in cells expressing PERP compared with GFP-only-expressing cells (_T_-test, _P_≥0.19). However, MDM2 mRNA was significantly higher in cells expressing PERP

at the three time points analyzed (_T-_test, _P_≤0.004; Figure 1b). Taken together, the results suggested that the increased p53 protein levels detected in response to PERP expression may be

a consequence of increased p53 protein stability rather than transcription, and that the ensuing p53 pool is likely to be transcriptionally active resulting in increased MDM2 at both the

transcriptional and protein level. There was also evidence that endogenous PERP protein (21 kDa) was slightly elevated in MEL202 cells at 72 h following transfection with GFP–PERP (Figure

1c). Owing to the low basal level of endogenous PERP in MEL202 cells and similar intensities of nonspecific proteins, the level of endogenous PERP in response to GFP–PERP expression was

further investigated in the wild-type p53-expressing cell line U2OS. An increase in endogenous PERP was apparent in U2OS cells at 48 and 72 h following transfection with GFP–PERP (Figure

1c). It was conceivable that the observed increase in endogenous PERP was due to the GFP–PERP-induced increased activity of its transcriptional regulator, p53. Consequently, it was important

to characterize the functional status of the raised p53 protein following GFP–PERP expression. PERP EXPRESSION INFLUENCES THE NUCLEAR TRANSLOCATION AND THE P53-DRIVEN EXPRESSION OF MDM2 The

effect of increased PERP expression described above was found in transiently transfected cell populations where, on average, the transfection efficiency was 13%. To refine our findings, we

assessed the effect of PERP expression on the subcellular localization and expression of the p53-transcriptional target MDM2 in individual cells by co-transfecting GFP–PERP with an

expression plasmid in which MDM2 was fused to yellow fluorescent protein (YFP) under the control of the human MDM2 native promoter that was previously shown to mirror the kinetic behaviour

of endogenous MDM2.20 The first striking observation was the almost exclusive nuclear localization of MDM2–YFP when co-expressed with GFP–PERP (in 96.8% of co-transfected cells; _T-_test,

_P_=0.02 and 0.04 _versus_ MDM2–YFP or MDM2–YFP and GFP-only; Figures 2a and b). In contrast, MDM2–YFP expression alone or in combination with GFP-only expression showed an additional

diffuse cytoplasmic localization of MDM2 in many cells (28 and 31%, respectively; Figures 2a and b). Control cells transfected with YFP-only presented a diffuse YFP expression throughout the

cytoplasm and nucleus, which was maintained following co-expression of GFP–PERP (98% cells; Figures 2a and b). To determine the effect of PERP on the expression of MDM2, YFP fluorescence

was measured in cells co-expressing GFP–PERP and MDM2–YFP and compared with that in control cells (MDM2–YFP-transfected and GFP-only plus MDM2–YFP co-transfected cells). The level of YFP

fluorescence – and therefore MDM2 expression – was significantly higher in cells co-expressing GFP–PERP compared with cells co-expressing GFP-only and MDM2–YFP (_T_-test, _P_<0.0001), or

expressing MDM2–YFP alone (_T_-test, _P_<0.0001; Figures 2c and d). To directly assess the role of p53-driven transcription of MDM2 in response to PERP expression, cells co-transfected

with GFP–PERP and MDM2–YFP were treated with pifithrin-_α_ (PFT_α_), a reversible inhibitor of p53-dependent gene transcription.21 The level of MDM2–YFP fluorescence in cells co-expressing

GFP–PERP was significantly reduced in cells treated with PFT_α_ (_T_-test, _P_<0.0001; Figures 2e and f), indicating that the induction of MDM2 expression in cells expressing GFP–PERP was

likely through p53-dependent transcriptional activation. No significant difference was seen in control cells co-expressing GFP-only and MDM2–YFP with or without PFT_α_ treatment (_T_-test,

_P_=0.95). PERP EXPRESSION LOCALIZES P53 IN THE NUCLEUS WITH EVIDENCE OF OSCILLATORY ACTIVITY To monitor the effect of PERP expression on the cellular localization of p53, MEL202 cells were

co-transfected with GFP–PERP and an expression plasmid in which p53 was fused to red fluorescent protein (RFP) placed under the control of a zinc-inducible promoter. Cells transfected with

p53–RFP-only served as controls. In the absence of GFP–PERP expression, p53–RFP was localized primarily in the cytoplasm with low expression in the nucleus (65% of transfected cells; Figures

3a and b), compared with 35% of cells showing nuclear-only p53–RFP expression. However, following GFP–PERP expression, p53–RFP was localized predominantly in the nucleus in a significantly

higher proportion of co-transfected cells (58%; _T_-test, _P_=0.04; Figures 3a and b). Nuclear expression of p53–RFP when co-expressed with GFP–PERP was also detected in the absence of ZnCl2

induction, albeit at much lower expression levels, and ZnCl2 had no effect on the expression or localization of GFP–PERP (Figure 3c). The PERP-induced nuclear localization of p53 suggests

that p53 is likely to wield its transcriptional activity – a scenario that is also supported by the p53-dependent upregulation of MDM2 transcription in the presence of elevated PERP (Figure

2). Furthermore, the highest nuclear MDM2–YFP fluorescence in cells co-transfected with MDM2–YFP and p53–RFP occurred when p53–RFP was also localized primarily in the nucleus, while

cytoplasmic p53 was associated (fluorescence colocalized) with cytoplasmic MDM2–YFP (Figure 3d), illustrating the nuclear-cytoplasmic shuttling of both p53 and MDM2 that is central to their

activities.15 As we had shown that the increase in MDM2 expression in the presence of elevated PERP was p53-dependent (Figure 2e), we next addressed the behaviour of p53 in the presence of

elevated PERP over time in cells co-expressing GFP–PERP and MDM2–YFP. These cells were monitored by time-lapse fluorescence confocal microscopy and the MDM2–YFP fluorescence was assessed as

a measure of the kinetic behaviour of p53.20 Full acquisition of YFP fluorescence measurements in a single cell over the entire time-lapse period (usually at least 18 h) proved difficult

because of the high motility of MEL202 cells, which resulted in many cells leaving the field of view. However, we were able to detect dampened oscillations in MDM2–YFP fluorescence at

regular 5–6 h intervals (Figure 3e), which corresponded to previous findings albeit in response to DNA damage.20 We also observed cell death in approximately 31% of cells expressing GFP–PERP

and MDM2–YFP over the time frame of imaging, which corresponded to approximately 30–48-h post-transfection (PT). These levels of cell death were comparable with our previous findings5 and

confirmed that the observations of MDM2–YFP expression in this study reflect the endogenous response to GFP–PERP expression. ENDOGENOUS MDM2 AND P53 ARE PREDOMINANTLY NUCLEAR IN THE PRESENCE

OF ELEVATED PERP EXPRESSION To verify that the prevalent nuclear localization of MDM2–YFP and p53–RFP seen in MEL202 cells following GFP–PERP expression reflected the subcellular

localization of endogenous MDM2 and p53 in the presence of elevated PERP, MEL202 cells were transfected with GFP–PERP and fixed 24-h PT. Endogenous MDM2 and p53 were detected by

immunofluorescence. Every cell-expressing GFP–PERP that was detected (52 cells) exhibited nuclear localization of MDM2 and p53, whereas a mixture of nuclear, nuclear/cytoplasmic or

cytoplasmic expression of MDM2 and p53 was seen in NT cells (Figure 4). The fixation and immunofluorescence procedure caused a significant detachment of GFP–PERP-transfected cells. This is

likely due to the pro-apoptotic effect of PERP on MEL202 cells5 that causes more readily detachment during fixation and subsequent immunodetection. P53 ELEVATED BY PERP EXPRESSION IS

MODIFIED ON KEY PHOSPHORYLATION SITES To determine if expression of PERP results in specific p53 posttranslational modification, western blotting was used to assess the phosphorylation

status of key p53 Ser residues in the presence of enhanced PERP expression. Phosphorylation at Ser15 was detected in control cells (NT and GFP-only-transfected), but was significantly

reduced in GFP–PERP-expressing cells (_T_-test, _P_≤0.003 _versus_ GFP-only-transfected cells; Figure 5a). No significant change in phosphorylation at Ser37 was detected. In response to DNA

damage, phosphorylation by ataxia telangiectasia mutated and ataxia telangiectasia and Rad3 related at Ser15 and Ser37 can impair the interaction between p53 and MDM2, promoting the

accumulation and activation of p53.12, 22 Consequently, reduction of phosphorylation at Ser15, and insignificant detection of Ser37P suggest that impairment of the p53–MDM2 interaction by

phosphorylation at these two Ser residues does not contribute to the increased p53 protein seen in response to PERP expression. However, a significant increase in the level of Ser20

phosphorylation was observed in cells expressing GFP–PERP (_T_-test, _P_≤0.004 _versus_ GFP-only-transfected cells; Figure 5a). As p53Ser20P is known to interfere with p53 binding to

MDM2,23, 24 it is possible that this modification may contribute to the PERP-related increased p53 accumulation. Total p53 protein level, detected using anti-p53 antibody (clone 7F5)

confirmed the upregulation of p53 protein in cells transfected with GFP–PERP detected previously with a different anti-p53 antibody (clone DO-1; Figure 1a), albeit with a slightly higher p53

level in NT cells (Figure 5a).The detection of p53Ser15P in cells in which total p53 was low/undetectable (NT and GFP-only-transfected) is likely due to differences in antibody specificity.

Phosphorylation of p53 at Ser46 was characterized as a specific phosphorylation event that irreversibly commits cells to apoptosis.14, 25 We detected the presence of p53Ser46P in control

MEL202 cells with significantly higher levels in GFP–PERP-expressing cells (_T_-test, _P_≤0.03 _versus_ GFP-only-transfected cells; Figure 5b), indicating that the p53 protein elevated in

response to PERP expression is likely to be active in apoptosis regulation. The effect of PERP expression on homeodomain-interacting protein kinase 2 (HIPK2) and p38 mitogen-activated

protein kinase (MAPK) (p38), both previously implicated in the induction of p53 Ser46 phosphorylation,26, 27, 28, 29 was also assessed by western blotting. No significant changes were

detected in HIPK2 expression or in the level of phospho-p38 (Thr180/Tyr182), suggesting the possibility that different pathway(s) may be involved. Q-PCR analysis of p53 target genes revealed

a statistically significant upregulation of death receptor 4 (DR4) and leucine-rich repeats and death domain containing (LRDD), both pro-apoptotic genes30 in MEL202 cells expressing

GFP–PERP compared with GFP-only-expressing cells (Figure 5c). No significant changes occurred in the level of cyclin-dependent kinase inhibitor 1A (p21) gene, involved in cell cycle

arrest.30 DISCUSSION The initial findings that elevated PERP expression resulted in increased p53 protein levels in MEL202 cells suggested a role for PERP in the positive feedback regulation

of its own transcriptional regulator, with potential implications for the regulation of p53 and the vast array of p53-targets involved in the tight control of cell cycle arrest and

apoptosis. Consequently, the study aimed to investigate, in real-time and live cells, the nature of the observed p53 upregulation and the functionality of the elevated p53. The active status

of the p53 elevated in response to PERP expression was initially indicated by the simultaneous increase in the p53-negative regulator protein, MDM2. Q-PCR analysis confirmed increased MDM2

transcription, which most likely occurs through the well-characterized autoregulatory p53–MDM2 feedback loop.10, 11 Single cell analysis corroborated that elevated levels of PERP protein

positively influenced the transcription level of MDM2, which was p53 dependent. Thus, the p53 that is augmented in response to PERP expression is transcriptionally active and able to

function in its transcriptional regulatory role. In the absence of exogenous PERP, p53 was localized primarily in the cytoplasm of MEL202 cells. P53 localization outside the nucleus occurs

in many tumour cells3 and was proposed as a mechanism employed by neoplastic cells to inactivate p53 transactivation function.2 Cytoplasmic retention of p53 renders tumour cells less

responsive to genotoxic stress15 and correlates with resistance to chemo- and radiotherapy in many tumour types,15, 31, 32 although this does not preclude the transcriptional-independent,

mitochondrial-related p53 function leading to apoptosis.33 Notably, GFP–PERP expression resulted in predominantly nuclear localization of p53 and MDM2, as well as higher MDM2 transcription.

Colocalization of p53 and MDM2, in the cytoplasm or the nucleus, can promote the MDM2-mediated proteasomal degradation of p53 without nuclear to cytoplasmic transport.34 However, cytoplasmic

localization of MDM2 in control cells lacking GFP–PERP expression suggested that nuclear-cytoplasmic shuttling of MDM2 is also occurring.16 Furthermore, time-lapse analysis of individual

cells co-expressing GFP–PERP and MDM2–YFP provided evidence of the oscillatory nature of p53 regulation, manifested in regular pulses in MDM2 at 5–6-h intervals, analogous with previously

reported oscillatory behaviour of p53 in DNA-damaged cells.20 Cell death following co-expression of GFP–PERP with MDM2–YFP was consistent with the previously observed level of apoptosis in

GFP–PERP-expressing MEL202 cells,5 substantiating the finding that elevated PERP is sufficient to induce apoptosis in MEL202 cells via the caspase-dependent pathway,5 downstream of the

p53/MDM2 feedback loop junction. Consistent apoptotic levels also confirm that the transcriptional changes in MDM2–YFP following GFP–PERP expression are representative of endogenous

processes in MEL202 cells. Taken together, current findings indicate that the pro-apoptotic effect of PERP involves and may be amplified by the effect PERP expression has on its own

transcriptional regulator p53. In line with our previous observations, PERP was targeted to the plasma membrane of MEL202 cells.5 A cytoplasmic and nuclear distribution of PERP has been

recently described in renal tubular epithelial cells8 and the potential for interaction of PERP with p53 and/or MDM2 in these subcellular compartments cannot be ruled out at present.

Evidence that differential phosphorylation of key Ser residues of p53 occurs following PERP expression indicates that the elevated p53 is subject to posttranslational modifications that

associate with p53 activation. Phosphorylation at p53Ser15, Ser20 and Ser37 can lower the affinity between p53 and MDM2, resulting in their reduced interaction and the subsequent

accumulation of p53.13, 22, 23 Our results showed that p53Ser15 phosphorylation is reduced in cells expressing PERP, however, a slight but significant increase in the level of p53Ser20

phosphorylation was detected in the presence of elevated PERP. Phosphorylation at Ser20 enhances p53 tetramerization, nuclear accumulation, stability and transcriptional activity,23, 24 and

therefore it is likely that this modification contributes to the stability and increased levels of nuclear p53 seen in response to PERP expression. Of particular interest was the increased

level of p53Ser46 phosphorylation; this modification is specifically involved in regulating the ability of p53 to induce apoptosis25 by preferentially activating the transcription of

pro-apoptotic p53-target genes.14, 33 The p53 affinity switch from promoters of cell cycle arrest-related genes to apoptotic-related ones usually requires severe, non-repairable DNA

damage.14, 25, 35 The increase in p53Ser46P induced by PERP expression is likely insufficient to drive the switch of gene–promoter targets to the level seen in response to genotoxic stress.

However, we observed an upregulation of DR4 and LRDD, both p53-induced pro-apoptotic genes,30 with no change in the cell cycle arrest-related gene, p21. Detection of p53Ser46P in control

cells (NT and GFP-only-transfected) suggests that other cell signals are contributing to p53 phosphorylation induction. PERP expression may enhance these signals or may render p53 more

susceptible to Ser46 phosphorylation. Initial experiments suggest that p53Ser46 phosphorylation in response to PERP may occur by processes other than HIPK2 or p38 activation. Further studies

will be required to elucidate the precise mechanism of p53 phosphorylation in response to PERP expression. Our findings that PERP can act by increasing p53 protein, which is both

transcriptionally active and subject to apoptosis-linked phosphorylation events, suggest the existence of an amplifying positive feedback loop at the functional level of p53 that may tip the

balance in favour of p53-dependent apoptosis rather than cell cycle arrest and/or lead to increased PERP levels that in turn promote apoptosis. The finding that PERP can only partially

reconstitute the level of apoptosis in p53-null cells4 also supports the existence of a PERP–p53 positive regulatory loop. Consequently, cells lacking functional PERP may also be impaired in

their ability to regulate levels of p53. Downregulation of PERP correlates with the aggressive monosomy-3 type of UM,17 a disease in which the upstream signalling to p53 is reportedly

intact, but functional defects downstream of p53 were inferred.36 Many tumours express p53 mutants with a reduced ability to activate p53-target gene expression that normally respond to

wild-type p53 activation.3, 37 This study demonstrates the transcriptional activity of p53 in MEL202 cells and reflects the reported low frequency of p53 mutations in primary UMs.36, 38, 39

Our previous findings that UM cells, including MEL202 cells, readily commit to apoptosis in response to upregulation of PERP expression5 imply that the downstream cell death pathway required

for PERP-induced apoptosis is intact and functional in UM cells. The scenario of a downstream target of p53, such as PERP, influencing levels of its own transcriptional regulator is

conceivable because loss of PERP function in these cells would not only have a detrimental effect on downstream signalling involving caspase-mediated apoptosis,5 but may also adjust p53

levels that influence the fine balance between cell death and survival. Deregulation of PERP appears as a mechanism engaged by UM cells to escape death by apoptosis.5, 17 In summary, this

study identifies a novel role for PERP in stabilizing active p53 levels and places PERP at a signalling junction that can influence both upstream and downstream pathways of p53. Although

interpretation of experiments involving manipulation of expression levels of p53-linked proteins should always be made with caution because of potentially abnormal or unregulated

interactions with other proteins,3 our data show that increased PERP expression affects several aspects of p53 regulation, including increased protein stability, apoptosis-promoting

posttranslational modifications, enhanced nuclear accumulation, and oscillatory regulation. Our findings highlight a potentially important target for development of cancer therapies aimed at

enhancing p53 apoptotic activity and reconstituting the p53-dependent apoptotic pathway. MATERIALS AND METHODS CELL CULTURE Tissue culture media and supplements were from Invitrogen

(Paisley, UK), unless otherwise stated. The UM cell line MEL20219 was grown in RPMI-1640 medium with 2 mM L-glutamine and 25 mM HEPES supplemented with 10% v/v heat-inactivated fetal calf

serum (FCS; Biosera, East Sussex, UK), 1 mM sodium pyruvate and 1% v/v non-essential amino acids. U2OS cells (ATCC No: HTB-96; LGC Standards, Teddington, Middlesex, UK) were grown in

Dulbecco's modified Eagle's medium with L-glutamine and 10% FCS. Cells were grown at 37 °C in controlled humidity and 5% CO2 atmosphere. PLASMID CONSTRUCTS AND TRANSIENT

TRANSFECTION OF CELL LINES The pcDNA-DEST53 plasmid containing GFP fused to the N-terminus of the full-length open-reading frame (ORF) of human PERP (GFP–PERP) under the CMV promoter was

constructed as described previously.5 The pEGFP-N1 vector (Clontech, Palo Alto, CA, USA) encoding GFP-only and pG-A-EYFP vector encoding YFP-only (kindly supplied by Louise Ashall,

University of Liverpool, UK) served as controls. The pU293 plasmid encoding human MDM2 fused to enhanced YFP under the control of the human MDM2 native promoter (MDM2–YFP20) and pU265

containing p53 ORF fused to DsRed-Express RFP under a zinc-inducible pMT promoter (p53-RFP) were kind gifts from Galit Lahav (Weizmann Institute of Science, Israel) and Rachel Nelson

(University of Liverpool, UK), respectively. To induce p53–RFP expression, zinc chloride (Sigma-Aldrich, Poole, UK) was added to cell media in a final concentration of 100 _μ_M, 6-h PT. The

pp53-EGFP Vector (Clontech) encoded p53 fused to enhance GFP (p53–GFP) under the CMV promoter. Transient transfections were performed using GeneJuice Transfection Reagent (Novagen, Merck

Biosciences Ltd., Darmstadt, Germany). Cells were transfected with 1 _μ_g plasmid DNA per 35 mm dish. For co-transfections, the total amount of plasmid DNA used was always 1 _μ_g. LASER

SCANNING CONFOCAL FLUORESCENCE MICROSCOPY OF LIVE TRANSFECTED CELLS Live cell imaging of transfected cells was performed in an incubator XL (PeCon GnbH) on the microscope stage that was

humidity controlled at 37 °C with 5% CO2. Cells were imaged using a LSM 5 EXCITER Laser Scanning Microscope (Carl Zeiss, Welwyn Garden City, Hertfordshire, UK) using a Fluar 40X/1.3 NA oil

immersion objective. Images were taken from 20-h PT. Excitation of EGFP was achieved with a 488 nm laser, and the emitted light was collected through a 505–530 nm bandpass filter from a 545 

nm dichroic mirror (515 nm dichroic mirror when in combination with YFP); YFP was excited at 514 nm (or at 488 nm when in combination with RFP) and its emission detected through a 515 nm

dichroic mirror and a 505–550 nm BP filter; RFP was excited at 543 nm and its emission detected through a 545 nm dichroic mirror and a 560 nm longpass filter. Data acquisition and analysis

were carried out with LSM510 AIM software, version 4.2 (Carl Zeiss) and ImageJ 1.43h.40 Cell death was assessed as described previously.5 PFT_Α_ TREATMENT Cells were treated with 30 _μ_M

PFT_α_ (Sigma-Aldrich) at 6-h PT and images were taken from 20-h PT. WESTERN BLOT ANALYSIS AND ANTIBODIES Lysates were prepared from cells at the indicated times as described previously5 and

resolved by SDS-PAGE before transfer to nitrocellulose membrane. The membranes were incubated overnight at 4 °C with the appropriate primary antibody diluted with 5% w/v non-fat dry milk or

5% bovine serum albumin (BSA) in tris-buffered saline-Tween-20 buffer (TBS-T) buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.6 with 0.1% v/v Tween-20) as recommended by the manufacturer. The

primary antibody dilutions used were as follows: anti-PERP polyclonal (1 : 1000 dilution) and anti-GAPDH monoclonal (clone 6C5, 1 : 10 000) (Abcam, Cambridge, UK); anti-p53 monoclonal (clone

DO-1, 1 : 1000) and anti-MDM2 monoclonal (clone 2A10, 1 : 1000) (Calbiochem, Merck Biosciences Ltd., Darmstadt, Germany); anti-HIPK2 monoclonal (clone F-189, 1 : 200) (Santa Cruz

Biotechnology, Inc., Santa Cruz, CA, USA); anti-phospho-p38 MAPK monoclonal (Thr180/Tyr182) (clone 3D7, 1 : 1000) and Phospho-p53 Antibody Sampler Kit including anti-phospho-p53 (Ser15

monoclonal; Ser20, Ser37, Ser46, polyclonal; 1 : 1000) and anti-p53 monoclonal (clone 7F5, 1 : 1000) (Cell Signaling Technology, Beverly, MA, USA). Immunocomplexes were detected with

horseradish peroxidase-conjugated secondary antibody (Sigma-Aldrich) by a chemiluminescent method (Amersham ECL Western Blotting Detection Reagents, GE Healthcare Life Sciences, Little

Chalfont, Buckinghamshire, UK), with 2-min exposure to autoradiography films unless otherwise stated. Sequential probing of blots with different antibodies was achieved by incubation in

stripping solution (62.5 mM Tris-HCl, pH 6.8, 100 mM _β_-mercaptoethanol, 2% w/v SDS) at 65oC for 30 min, followed by 2 × 10 min washes in TBS-T at room temperature, before blocking in 5%

milk/TBS-T and reprobing with antibody. Detection of phospho-p53 products was carried out using separate blots of identical samples rather than sequential probing of the same blot to prevent

carry-over of previously detected p53 products. Blots were probed with anti-GAPDH to confirm equal loading of samples. Relative intensities of the protein bands were obtained by

densitometry using ImageJ (Rasband, WS, ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2007) and normalized to endogenous GAPDH. Western blot

images presented are representative of three independent experiments. IMMUNOFLUORESCENCE MEL202 cells were seeded in eight-well Lab-Tek Chamber Slide (Nunc, Rochester, NY, USA) 24 h before

transfection. Media was removed 24 h following transfection and cells were washed in phosphate-buffered saline (PBS). Cells were fixed with freshly prepared 4% paraformaldehyde in PBS for 10

 min followed by 3 × 5 min washes. Following permeabilisation with 0.3% Triton X-100 in PBS for 5 min and a 5 min PBS wash, cells were incubated in blocking buffer (5% normal goat serum in

PBS) for 60 min before incubating with primary antibody diluted in antibody dilution buffer (1% BSA in PBS) for 60 min. Cells were washed for 3 × 5 min in PBS before addition, where

necessary, of diluted fluorochrome-conjugated secondary antibody for 45 min, followed by 3 × 5 min washes in PBS. All incubations were carried out at room temperature. Cells were

coverslipped with Fluorescence Mounting Medium (Dako, Cambridgeshire, UK) and examined using a LSM 5 EXCITER Laser Scanning Microscope (Carl Zeiss) with excitation at 633 nm. The primary

antibody dilutions used were: anti-MDM2 monoclonal (clone 2A10, 1 : 100) (Calbiochem, Merck Biosciences); anti-p53 Alexa Fluor 647 Conjugate monoclonal (clone 1C12, 1 : 50) (Cell Signaling

Technology). The secondary antibody used for detection of MDM2 was Alexa Fluor 633 goat anti-mouse IgG (H+L; 1 : 500) (Invitrogen). REAL-TIME QUANTITATIVE PCR RNA was isolated from cells

using RNeasy Kit (Qiagen Ltd. West Sussex, UK) with on-column DNase I treatment for removal of genomic DNA. First-strand cDNA was synthesized from 1 _μ_g RNA using Superscript II Reverse

Transcriptase (Invitrogen). Real-time Q-PCR was performed using qPCR Core Kit for SYBR Green I (Eurogentec, Southampton, UK) as described previously.5 The gene-specific primer set used for

PERP was 5′-GGCTTCATCATCCTGGTGAT-3′ and 5′-ACAGCAGCCAAGGCAAGGAG-3′; for p53, 5′-ATCTACAAGCAGTCACAGCAC-3′ and 5′-TTCCTTCCACTCGGATAAGATGC-3′; and for MDM2, 5′-CCTGGCTCTGTGTGTAATAAG-3′ and 5′-

ATCCAACCAATCACCTGAATG-3′. Primer sets for the p53-target genes30 PUMA, DR4, DR5, LRDD and p21 were purchased from SABiosciences (Qiagen). Q-PCR data were normalized to GAPDH (primer set:

5′-AACAGCCTCAAGATCATCAG-3′ and 5′-TGAGTCCTTCCACGATACC-3′). Amplifications were performed in triplicate using a Stratagene MX3000P QPCR System (Stratagene, La Jolla, CA, USA). Q-PCR data are

expressed relative to a calibrator sample assigned an arbitrary quantity of 1. ABBREVIATIONS * PERP: p53 apoptosis effector related to PMP-22 * Ser: serine * NT: non-transfected * GFP: green

fluorescent protein * Q-PCR: real-time quantitative PCR * YFP: yellow fluorescent protein * PFT_α_: pifithrin-_α_ * RFP: red fluorescent protein * HIPK2: homeodomain-interacting protein

kinase 2 * p38: p38 mitogen-activated protein kinase (MAPK) * DR4: death receptor 4 * LRDD: leucine-rich repeats and death domain containing * p21: cyclin-dependent kinase inhibitor 1A * UM:

uveal melanoma * ORF: open-reading frame * PT: post-transfection * BSA: bovine serum albumin * TBS-T buffer: tris-buffered saline-Tween-20 buffer * PBS: phosphate-buffered saline REFERENCES

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ACKNOWLEDGEMENTS We are grateful to Bruce Ksander and Martine Jager for the gift of the MEL202 cell line, Galit Lahav and Rachel Nelson for the generous gifts of MDM2-YFP and p53-RFP plasmid

constructs, respectively, and Louise Ashall for the YFP-only plasmid. The technical support given by Daniel Brotchie is acknowledged. This work was generously supported by The Humane

Research Trust, UK. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Eye and Vision Sciences, Institute of Ageing and Chronic Disease, University of Liverpool, Liverpool, UK L

Davies, I Grierson & L Paraoan * Centre for Cell Imaging, Institute of Integrative Biology, University of Liverpool, Liverpool, UK D Spiller & M R H White Authors * L Davies View

author publications You can also search for this author inPubMed Google Scholar * D Spiller View author publications You can also search for this author inPubMed Google Scholar * M R H White

View author publications You can also search for this author inPubMed Google Scholar * I Grierson View author publications You can also search for this author inPubMed Google Scholar * L

Paraoan View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to L Paraoan. ETHICS DECLARATIONS COMPETING INTERESTS The

authors declare no conflict of interest. ADDITIONAL INFORMATION Edited by A Stephanou RIGHTS AND PERMISSIONS This work is licensed under the Creative Commons Attribution-NonCommercial-No

Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/ Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE

Davies, L., Spiller, D., White, M. _et al._ PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells. _Cell Death Dis_ 2, e136 (2011).

https://doi.org/10.1038/cddis.2011.19 Download citation * Received: 30 August 2010 * Revised: 28 January 2011 * Accepted: 17 February 2011 * Published: 31 March 2011 * Issue Date: March 2011

* DOI: https://doi.org/10.1038/cddis.2011.19 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * PERP * apoptosis * uveal melanoma * p53 * fluorescent

protein