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Tumor Suppressor Protein p53 Regulates the Stress Activated BilirubinOxidase Cytochrome P450 2A6

Hao Hu, Ting Yu, Satu Arpiainen, Matti A. Lang, Jukka Hakkola, A’edahAbu-Bakar

PII: S0041-008X(15)30073-9DOI: doi: 10.1016/j.taap.2015.08.021Reference: YTAAP 13457

To appear in: Toxicology and Applied Pharmacology

Received date: 11 August 2015Revised date: 31 August 2015Accepted date: 31 August 2015

Please cite this article as: Hu, Hao, Yu, Ting, Arpiainen, Satu, Lang, Matti A., Hakkola,Jukka, Abu-Bakar, A’edah, Tumor Suppressor Protein p53 Regulates the Stress Ac-tivated Bilirubin Oxidase Cytochrome P450 2A6, Toxicology and Applied Pharmacology(2015), doi: 10.1016/j.taap.2015.08.021

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

http://dx.doi.org/10.1016/j.taap.2015.08.021http://dx.doi.org/10.1016/j.taap.2015.08.021

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Tumor Suppressor Protein p53 Regulates the Stress Activated Bilirubin Oxidase

Cytochrome P450 2A6

Hao Hua, Ting Yu

a, Satu Arpiainen

b, Matti A. Lang

a, Jukka Hakkola

b,

and A’edah Abu-Bakara*

aThe University of Queensland, National Research Centre for Environmental Toxicology

(Entox), 4072 Brisbane, Queensland, Australia. hao.hu1@uqconnect.edu.au;

t.yu2@uq.edu.au; m.lang@uq.edu.au; a.abubakar@uq.edu.au.

bInstitute of Biomedicine, Department of Pharmacology and Toxicology and Medical

Research Center Oulu, Oulu University Hospital and University of Oulu, Oulu, Finland.

Jukka.hakkola@oulu.fi; Satu.Juhila@orion.fi.

aAddress correspondence to: A’edah Abu-Bakar, Entox, The University of Queensland, 39

Kessels Road, Coopers Plains, 4108 Queensland, Australia. Phone: +617 0419 301 740; Fax:

+617 3274 9003; E-mail: a.abubakar@uq.edu.au

Abbreviations used: BaP, Benzo[α]pyrene; BR, bilirubin; CYP, cytochrome P450; CYP2A5,

cytochrome P450 2A5; CYP2A6, cytochrome P450 2A6; HMOX1, heme oxygenase-1; Nrf2,

nuclear factor erythroid 2-like 2; TF, transcription factor.

Running title: p53 regulates CYP2A6 gene

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Abstract

Human cytochrome P450 (CYP) 2A6 enzyme has been proposed to play a role in cellular

defence against chemical-induced oxidative stress. The encoding gene is regulated by various

stress activated transcription factors. This paper demonstrates that p53 is a novel

transcriptional regulator of the gene. Sequence analysis of the CYP2A6 promoter revealed six

putative p53 binding sites in a 3 kb proximate promoter region. The site closest to

transcription start site (TSS) is highly homologous with the p53 consensus sequence.

Transfection with various stepwise deletions of CYP2A6-5’-Luc constructs—down to -160 bp

from the TSS—showed p53 responsiveness in p53 overexpressed C3A cells. However, a

further deletion from -160 to -74 bp, including the putative p53 binding site, totally abolished

the p53 responsiveness. Electrophoretic mobility shift assay with a probe containing the

putative binding site showed specific binding of p53. A point mutation at the binding site

abolished both the binding and responsiveness of the recombinant gene to p53. Up-regulation

of the endogenous p53 with benzo[α]pyrene—a well-known p53 activator—increased the

expression of the p53 responsive positive control and the CYP2A6-5’-Luc construct

containing the intact p53 binding site but not the mutated CYP2A6-5’-Luc construct. Finally,

inducibility of the native CYP2A6 gene by benzo[α]pyrene was demonstrated by dose-

dependent increases in CYP2A6 mRNA and protein levels along with increased p53 levels in

the nucleus. Collectively, the results indicate that p53 protein is a regulator of the CYP2A6

gene in C3A cells and further support the putative cytoprotective role of CYP2A6.

Keywords: CYP2A6, p53, oxidative stress, bilirubin oxidase, bilirubin

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Introduction

Human cytochrome P450 (CYP) 2A6 is a key enzyme responsible for the metabolism

of nicotine and coumarin (review in Abu-Bakar et al., 2013). It can also metabolically

activate tobacco-specific nitrosamines (Yamazaki et al., 1992). The bile pigment bilirubin

(BR) has been suggested to be the endogenous substrate for CYP2A6 (Abu- Bakar et al.,

2012). The enzyme is predominantly expressed in the liver and commonly found in sex

steroid-responsive tissues such as breast, ovary, uterus, testis, and adrenal gland (Nakajima et

al., 2006), lung, nasal mucosa, oesophagus, trachea and skin (Janmohamed et al., 2001;

Koskela et al., 1999; Su et al., 1996).

The CYP2A6 expression is induced by structurally unrelated chemicals that are toxic to

the liver, such as pyrazole, aminotriazole, griseofulvin and cobalt chloride (Donato et al.,

2000). It is also induced in fatty, inflamed and cirrhotic liver associated with microorganisms

infections and harmful alcohol consumption (Fisher et al., 2009; Kirby et al., 1996; Niemela

et al., 2000; Raunio et al., 1998; Satarug et al., 1996). Given the unrelated nature of the

inducers, it is probable that CYP2A6 induction is not directly related to the nature of the

inducing agents, but instead a consequence of specific cellular events associated with

exposure to the inducers. This is indeed reflected in the complex regulation of CYP2A6,

where it is controlled transcriptionally and/or post-transcriptionally, depending on the nature

of cellular perturbations (Christian et al., 2004; Raunio & Rahnasto-Rilla, 2012).

The post-transcriptional regulation of CYP2A6 is mediated by the interaction of

heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) with the 3’-UTR of the CYP2A6

mRNA and the transcript is stabilized when transcription is impaired by Actinomycin D

(Christian et al., 2004). The up-regulation of CYP2A6 can also be achieved by protein

stabilization (Abu-Bakar et al., 2012). This is demonstrated in a study with HepG2 cells

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where treatment with BR—an endogenous substrate for CYP2A6—increased the half-life of

CYP2A6 protein but did not alter the CYP2A6 mRNA level (Abu-Bakar et al., 2012).

The transactivation of CYP2A6 is regulated by transcription factors (TFs) involved in

cellular homeostasis and cytoprotection. For example, TFs implicated in bile acid/steroid

homeostasis and energy metabolism, such as pregnane-X-receptor (PXR), peroxisome

proliferator-activated receptor ɤ coactivator 1α (PGC-1α) and estrogen receptor α (ERα)

(Goodwin et al., 2003; Staudinger et al., 2001; Xie et al., 2001) mediate upregulation of

CYP2A6 expression by interacting with the relevant response elements in the distal promoter

of CYP2A6 (> -2400 bp) (Higashi et al., 2007; Itoh et al., 2006).

Similarly, stress activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) mediates

transcriptional activation of CYP2A6 by direct interaction with the antioxidant response

element (ARE) at position -1212 of the 5’-flanking region of the gene (Yokota et al., 2011).

The study indicates a potential role for CYP2A6 in adaptive response to cellular stress as

Nrf2 is a key regulator of stress responsive genes. This is in agreement with the suggested

role of CYP2A6 in the regulation of intracellular antioxidant capacity during oxidative stress

(Abu-Bakar et al., 2013; Muhsain et al., 2015).

Another key transcription factor in cellular defence regulation is the tumor suppressor

protein, p53, which is activated by many cellular perturbations to regulate the transcription of

various genes that encode products with pro-oxidant or antioxidant functions (review in Saha

et al., 2015). Upon activation, p53 induces apoptosis to remove severely damaged cells

(Zuckerman et al., 2009). If the damage is limited, p53 invokes cell-cycle arrest to activate

DNA-repair pathways to correct the defects (Helton and Chen, 2007). A recent study

demonstrated that p53 not only functions to remove or fix damaged cells but also regulates

antioxidant mechanisms to prevent cell death (Nam and Sabapathy, 2011). It does this by

directly upregulating transactivation of a stress responsive gene heme oxygenase 1 (HMOX1).

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Nam and Sabapathy (2011) demonstrated that p53-dependent HMOX1 expression was

specifically induced by oxidative stress, nitrosative stress, hypoxia, and endoplasmic

reticulum stress but not by common chemotherapeutic DNA-damaging agents. Based on

these findings and the fact that some of the p53 activators are also inducers of CYP2A6, we

hypothesized that p53 could be involved in the regulation of CYP2A6 gene.

In the present study we demonstrate that CYP2A6 is a direct target gene of p53 and

describe the molecular mechanisms involved. The findings describe a novel mechanism by

which unspecific cellular stress, such as DNA damage and transcriptional arrest—triggers of

p53 activation—upregulated the CYP2A6.

Materials and Methods

Chemicals and antibodies. Benzo[α]pyrene (BaP), DMSO, PBS, dithiothreitol (DTT),

phenylmethylsulfonyl fluoride (PMSF), MgCl2, KCl, KOH, NaCl, Hepes, EDTA, IGEPAL,

cyclohexanediamine tetraacetic acid, Triton X-100, p-coumaric acid and luminol were

purchased from Sigma-Aldrich (Sydney, NSW, Australia). Mouse monoclonal anti-p53

antibody (sc-126), rabbit plyclonal anti-HNF4 (sc-8987) and A-431 whole cell lysate (sc-

2201) were obtained from Santa Cruz Biotechnology Inc. (Dallas, Texas, USA). Mouse

monoclonal anti-CYP2A6 (ab56069) and anti-HMOX1 (ab13248), and rabbit polyclonal anti

-β-actin (ab8227) were sourced from Abcam (Cambridge, UK). Goat anti-mouse and goat

anti-rabbit antibodies conjugated with horseradish-peroxidase were from Thermo Fisher

Scientific Inc. (Melbourne, Victoria, Australia). All cell culture consumables were sourced

from Invitrogen (Scoresby, Victoria, Australia).

Culturing and treatment of C3A cells. The C3A human hepatocellular carcinoma cells

(ATCC, Manassas, VA, USA) were propagated in T75 flasks in William’s Medium E

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containing 10% FBS, 1% Glutamax and 1% Penicillin-Streptomycin. Human breast cancer

cell line, MCF-7, were grown in Dulbecco's Modified Eagle Medium supplemented with

10% FBS, 1% Glutamax and 1% Penicillin-Streptomycin. The cells were maintained in a

humidifier incubator at 37°C in 5% CO2 and were subcultured at least two times per week.

Cells with passage number between 5 and 15 were used in all studies.

Plasmids and transient transfection assays. The CYP2A6 5’-flanking region -2901/+9 was

amplified from a human DNA sample using high-fidelity PCR polymerase and cloned in

front of the luciferase cDNA in pGL3-basic vector (Promega, Madison, WI). The 5’-

truncated fragments of the CYP2A6 promoter were produced by PCR amplification using the

cloned longer construct as template. The primers used for the amplification were listed in

Table 1. Promoter constructs were co-transfected with either the human p53 expression

plasmid (pcDNA3-hp53) (Christian et al., 2008) or the empty expression plasmid pcDNA3.

The pGL4.38[luc2P/p53 RE/Hygro] vector (Promega, Madison, WI) was used as the positive

control for the luciferase activity assay.

Polyethylenimine (PEI) (Polysciences Inc., PA, USA) was used to transiently transfect

cultured cells with the various promoter constructs. PEI stock solution (1 μg/μl) was prepared

by dissolving 100 mg of PEI in 100 ml of sterilised water at 50°C and pH adjusted to 7 with

HCl. The solution was passed through a 0.22 μm filter (Millipore, USA) and stored at -80°C.

The PEI solution was warmed up to room temperature before being added to DNA plasmid

solution at DNA:PEI ratio of 1:2. The DNA-PEI mixture was then incubated at room

temperature for 15 min before being added to each well of the 24-well plates.

Cells were seeded in 24-well plates (1.2 × 105 cells/well) for 24 h. Thereafter, the cells

were washed once in 500 µl PBS, followed by addition of 500 µl of fresh FBS-free medium

to each well. The cells were then co-transfected with 0.1 µg of pMAX-GFP (transfection

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control), 0.1 µg of pcDNA3 or pcDNA3-hp53, and 0.3 µg of CYP2A6-5’-Luc constructs.

The transfected cells were incubated for 24 h in a humidifier incubator at 37°C in 5% CO2.

Thereafter, the luciferase activity was measured (BriteliteTM

plus Reporter Gene Assay

System, PerkinElmer, USA). Transfection under each condition was performed in quadruplet.

The fluorescent and luminescence signals were captured on a FLUOstar Omega multimode

microplate reader with automatic gain settings.

BaP treatment of the transfected MCF-7 cells. Human breast cancer MCF-7 cells were

seeded in 24-well plates at 1.2 × 105 cells/well and 100 mm culture dish (Corning, USA) at 3

× 106 cells/well for 24 h. Cells in the 24-well plates were co-transfected with pMAX-GFP

and pGL4.38[luc2P/p53 RE/Hygro] or CYP2A6-5’-Luc constructs at a fixed DNA:PEI ratio

of 1:8. The amount of each plasmid used was 100 ng per well and 2.5 µg per dish. The cells

were transfected for 4 h before being treated with various concentrations of BaP dissolved in

DMEM containing 10% charcoal stripped FBS, 1% Glutamax, 1% Penicillin-Streptomycin,

and 0.1% DMSO. Twenty four hours after treatment luciferase activity was measured and

nuclei protein extracted.

Isolation of nuclear and cytoplasmic proteins. Nuclear and cytoplasmic extracts from

cultured cells were prepared as described previously (Abu-Bakar et al., 2007). Briefly, 5 ×

106 cells in a 100 mm culture dish were washed and resuspended in 5 ml cold PBS. The cell

suspension was centrifuged at 1600 x g for 10 min at 4oC. The resulting pellet was

resuspended in 200 µl of buffer A (10 mM Hepes-KOH, pH 7.9, 1.5 mM MgCl2, 10 mM

KCl, 0.5 mM DTT, 0.2 mM PMSF, 10µg/ml leupeptine, 0.4% IGEPAL) and kept on ice for 1

h. Thereafter, the cell suspension was vortexed, homogenized and centrifuged at 12,000 x g

for 10 min at 4oC. The supernatant containing cytoplasmic proteins was aliquoted and stored

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at -80oC. The pellet containing nuclei was resuspended in 50 µl of cold buffer B (20 mM

Hepes-KOH, pH 7.9, 25% glycerol, 1.5 mM MgCl2, 420 mM NaCl, 0.2 mM EDTA, 0.5 mM

DTT, 0.2 mM PMSF, 0.4% IGEPAL) and gently agitated with magnetic stirrer for 30 min at

4oC. The suspension was homogenized and centrifuged at 15,000 x g for 15 min, at 4

oC. The

supernatant containing nuclear proteins was diluted 1:1 with the dilution buffer (20 mM

Hepes-KOH, pH 7.9, 15% glycerol, 0.2 mM EDTA, 0.5 mM DTT, 0.2mM PMSF), aliquoted

and stored at -80oC.

Protein analysis. Protein concentrations of the subcellular fractions were measured by the DC

protein assay kit (Bio-Rad Laboratories, USA). The p53 protein and CYP2A6 apoprotein

levels in the subcellular fractions were measured by Western immunoblotting. In brief, 30 µg

of cytosolic or nuclei proteins were separated by 4%-15% SDS-polyacrylamide gel (Bio-Rad

Laboratories, USA) electrophoresis. The proteins were electrophoretically transferred on to a

Hybond ECL nitrocellulose membrane (Amersham Biosciences UK, Ltd) and blocked

overnight in Tris-buffered saline containing 0.1% Tween 20 (TBS-T) and 5% non-fat milk.

The membrane was then incubated with the relevant antibody (1:500 dilution) for 2 h,

washed with TBS-T, and incubated for 1 h with horseradish peroxidase-conjugated goat anti-

mouse IgG (1:3000 dilution). After further washing with TBS-T, the membrane was

incubated with luminol solution (1 M Tris pH 8.5, 90 mM p-coumaric acid, 250 mM luminol,

and 3% H2O2) for 5 min and immunoreactive bands were visualized and captured by the

ChemiDocTM

MP imaging system (Bio-Rad Laboratories, USA).

RNA extraction and mRNA analysis. Total cellular RNA was extracted from C3A cells with

TRIzol reagent (Gibco, Australia) in accordance with the manufacturer’s protocol. Total

RNA was treated with RNA-free DNAse prior to mRNA measurement by quantitative real

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time RT-PCR. One microgram of total RNA from the control and treated C3A cells was used

to synthesise first-strand cDNA with the Invitrogen SuperScript®

III First-Strand Synthesis kit

(Life Technologies, Vic, Australia). Two microliters of the cDNA solution was used as

template in 20 µl PCR mixture containing 10 µl of the 2 X iQ SYBR Green Supermix (Bio-

Rad Laboratories, USA) and 2 µl of each reverse and forward primers (Table 1) (0.2 µM final

concentration). The PCR mixtures were incubated at 95oC for 3 min, followed by 40 cycles

of 95oC for 20 s and 63.2

oC (for CYP2A6 primers), 65.7

oC (for HMOX1 primers), or 60.4

oC

(for GAPDH primers) for 1 min in Corbett RotorGene 3000 (QIAGEN, Germany). The

specificity of the PCR products was confirmed by melt curve analysis and amplicon size

(2.5% agarose gel electrophoresis).

Electrophoretic mobility shift assay. Double-stranded oligonucleotide corresponding to the -

149 to -55 (Probe 1) region of the 5’-flanking CYP2A6 promoter were generated by

polymerase chain reaction (PCR). The probe was amplified by the primer 1 and primer 2

(Table 1). The primers, double-stranded oligonucleotides of putative and consensus p53RE

and of the -127 to -98 region of the 5’-flanking CYP2A6 promoter (Probe 2) were obtained

from Sigma-Genosys (Sydney, Australia). The PCR product was purified by using the QIA

quick PCR purification kit (QIAGEN, Germany) in accordance with the manufacturer’s

protocol. The probes were labelled with digoxigenin on the 3’ end of the oligonucleotide

using DIG gel shift kit (Roche, Germany). Thereafter, 32 fmol/µl of digoxigenin-labelled

probe was incubated with 16 µl of binding buffer and 4 µg of nuclei protein—extracted from

C3A cells transfected with p53 expression plasmid—for 15 min at room temperature. In

antibody shift assay, 1 µl of anti-p53, Nrf2 or HNF4 antibody was added to the reaction

mixture and was incubated for 15 min. The samples were loaded to a pre-electrophoresed,

non-denaturing 4-15% polyacrylamide gel in 0.5 × TBE buffer (44 mM Tris-HCl, pH 8, 44

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mM boric acid, 1 mM EDTA). Samples were separated electrophoretically at 80V for 1 h and

transferred to a positive-charged nylon membrane Hybond-N (Amersham Biosciences, UK,

Ltd) by an electro-blotting device (Bio-Rad Laboratories, USA) set at 1 h at 30V, 300mA.

Thereafter, the membrane was baked at 140oC for 15 min and crosslinked in a UV

StratalinkerTM

1800 (Integrated Sciences, Australia) at 120 mJ. The membrane was briefly

rinsed for 3 min in washing buffer and was blocked in blocking solution for 30 min before

immunological detection. The digoxigenin-labelled probes on the membrane were detected

by incubating the membrane with an anti-digoxigenin-AP antibody (1:10,000). The

chemiluminescence signals of the labelled probes were captured by ChemiDoc™ MP

imaging system after applying the alkaline phosphatase substrate CSPD on the membrane.

Competition binding assay was performed to validate the specificity of protein–DNA

interactions. In this assay, 125-fold molar excess of unlabelled oligonucleotides was added to

the incubation mixture. The double-stranded consensus p53 oligonucleotide

5’AGACATGCCTAGACATGCCT 3’ (underlined bases are core sequence) was used as

positive control for p53 specific binding.

Site directed Mutagenesis. The potential p53 sites on the proximal CYP2A6 promoter was

analysed by site-directed mutagenesis. The core sequences of the putative p53RE 5’-

ATTCATGGTGGGGCATGTAG-3’ (core sequences, bold) was mutated to 5’-

ATTACCAGTGGGGATCCTAG-3’ (mutated sequences, underlined) by the two steps PCR.

The CYP2A6 construct from -250 to the p53RE mutated sites was amplified from the

template plasmid CYP2A6 5’ -437/+9 by priming with the forward primer CYP2A6 5’-250

FW and reverse primer CYP2A6-p53mut (Table 1). The CYP2A6 construct from p53RE

mutated site to +9 was amplified from the template plasmid CYP2A6 5’ -437/+9 by priming

with the forward primer CYP2A6-p53mut and the reverse primer CYP2A6 5’+9 NheI RV

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(Table 1). Equal amount of PCR products from the two reactions were mixed together, which

acted as a template to amplify the mutated p53RE. The CYP2A6-160/+9 construct containing

the mutated p53RE was generated by priming with the forward CYP2A6 5’ -160 KpnI FW

and the reverse primer CYP2A6 5’+9 NheI RV (Table 1). The p53RE mutated CYP2A6

construct was ligated to the pGL3-basic plasmid. The validity of the mutated p53RE on the

CYP2A6 5’ -160/+9 construct was confirmed by gene sequencing.

Statistical analysis. Two group comparisons were analysed by Student’s t test. Multiple

group comparisons were done with one-way analysis of variance (ANOVA) followed by the

least significant difference post hoc test. Differences were considered significant when p <

0.05.

Results

CYP2A6 putative p53REs are responsive to p53

Putative TF binding site search with ―MatInspector‖ (http://www.genomatix.de/)

revealed regions of the CYP2A6 promoter with high sequence similarity with the 10 bp

consensus p53RE [5’-RRRC(A/T)(T/A)GYYY-3’], where ―R‖ represents purine and ―Y‖

represents pyrimidine (el-Deiry et al., 1992). Five putative distal sites were identified at

positions -2582, -2461, -2208, -1617, -1056, and one putative proximal site at position -122

(Fig. 1A). We then tested p53 involvement in transcriptional regulation of the CYP2A6 gene

by transiently transfecting C3A cells with luciferase reporter plasmid containing either the

proximal (CYP2A6-5’-437/+9-Luc) or distal (CYP2A6-5’-2901/+9-Luc) CYP2A6 promoter

region. The cells were co-transfected with p53 expression plasmid (pcDNA3-hp53) or empty

control plasmid (pcDNA3). C3A cells were preferred over other cell lines because p53

overexpression was the strongest in C3A compared with HepG2 and MCF-7, and because it

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was challenging to produce consistent induction of the CYP2A6-Luc constructs in the HepG2

cells (Supplementary Figure1).

Figure 1B shows that the p53 protein levels in cells co-transfected with the pcDNA3-

hp53 plasmid were significantly higher than in the control cells (pcDNA3). The p53 co-

transfection induced the distal CYP2A6-5’-2901/+9-Luc construct by ~4-fold relative to the

control, which is similar to the fold induction observed in the positive control cells

(transfection with consensus p53RE-Luc construct) (Fig. 1C). The induction was weaker with

the proximal CYP2A6-5’-437/+9-Luc construct, by about 2.4-fold relative to the control (due

partly to the elevated control levels) (Fig. 1C). No significant luciferase activity was observed

in cells transfected with the pGL3-basic plasmid (negative control) (Fig. 1C). These

observations suggest that both the proximal and distal regions of the CYP2A6 promoter are

responsive to p53.

We note that in normal cells—where p53 was not overexpressed (opened bar)—the

luciferase activity of the distal promoter was significantly weaker than that of the proximal

promoter (Fig 1C). For this reason and the fact that luciferase activity of the proximal

promoter was almost equal to that of the positive control in p53 overexpressed cells, we

focused on describing the functionality of this site and its significance in the regulation of the

CYP2A6 gene.

It is also noted that the activity of the distal promoter (-2901-LUC construct) is

substantially lower than the vector control (p53RE-LUC) in both normal and p53-

overexpressed cells. This suggests presence of suppressor region(s) upstream the CYP2A6

promoter, which is confirmed by previous studies (Higashi et al., 2007; Itoh et al., 2006;

Pitarque et al., 2005).

Proximal p53RE mediates p53 response of the CYP2A6 gene.

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A series of 5’-truncated proximal CYP2A6 promoter-luciferase reporter plasmids were

constructed and transfected into the C3A cells. The cells were then co-transfected with p53

expression plasmid (pcDNA-hp53) or empty control plasmid (pcDNA3). P53 co-transfection

up-regulated the CYP2A6-5’-437/+9-Luc construct 2.5-fold relative to control (Fig. 2A). A

similar response was detected with the series of constructs equal to or longer than -160 bp but

not with the shorter construct (-74 bp) (Fig. 2A). This suggests that the p53RE is located in

the region from -160 to -74, which is in agreement with the MatInspector TF binding site

search that revealed one putative p53RE at position -122 (Fig. 1A). This putative p53RE

contains the two copies of p53 binding core sequence of the consensus p53RE, ―CATG‖ (Fig.

2B, bold letters), however, the satellite sequence of the putative p53RE is different from that

of the consensus p53RE (Fig. 2B).

To establish the functional significance of the putative binding site, we assessed the

luciferase expression after mutating the site, followed by assessment of p53 binding activity

by way of electrophoretic mobility shift assay (EMSA).

The core sequence, CATG, critical for p53 binding was mutated in the -122 bp site of

the CYP2A6-5’-160/+9-Luc and CYP2A6-5’-2901/+9-Luc constructs to ACCA. The mutant

constructs were then co-transfected with the p53 expression plasmid into the C3A cells. As

expected, p53 co-transfection induced luciferase expression driven by the two constructs by

about 2.6- to 3.9-fold relative to control (Fig. 2C). Point mutation at the core binding

sequence dramatically reduced the p53 response of both the proximal and distal promoter

activity (Fig. 2C), which suggests that the core binding sequence within the putative p53RE

element at the -122 bp site is crucial in mediating the response.

To confirm that the observed response was due to direct interaction of p53 with the

putative site, EMSA was performed with nuclei fractions from C3A cells transfected with

p53 expression plasmid using three oligonucleotide probes. Probe 1 is a longer

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oligonucleotide containing the putative binding site from -149 to -55 bp (Fig. 3A). Probe 2 is

a shorter oligonucleotide spanning the putative site from -127 to -98 bp. The third probe

(Probe1Mut) is the longer oligonucleotide with the core sequence, CATG, critical for p53

binding, mutated to ACCA.

As shown in Fig. 3B, the transfection produced a substantial amount of p53 in the

nucleus. Two distinct DNA-protein complexes were formed when Probe 1 was incubated

with the C3A nuclear extract (Fig. 3C, lane 2). The lower complex (closed arrow) was super-

shifted with an anti-p53 antibody (Fig. 3C, lane 3), indicating that the DNA-protein complex

contains p53. In the competitive assays, both complexes were competed out by a 125-fold

excess of the unlabelled Probe 1 (S) (Fig. 3C, lane 4, closed and opened arrows). However,

only the lower complex was competed out by increasing concentrations of the unlabelled

consensus p53RE oligonucleotide (C) (Fig. 3C, lanes 5-7, closed arrow). The results suggest

presence of p53 in the lower complex and indicate that a protein other than p53 is forming the

higher complex (opened arrow) with Probe 1.

Indeed, detailed analysis of the probe sequence revealed a putative binding site for

hepatocyte nuclear factor 4α (HNF4α) (Fig. 3A, opened line square). Incubation with anti-

HNF4α antibody shifted the migration of the higher complex (opened arrow) (Fig. 3C, lane

8), confirming the presence of HNF4α.

We included two other controls in the analysis to confirm specific binding of p53 and

HNF4α. Migrations of the two complexes were not shifted by an anti-Nrf2 antibody (Fig. 3C,

lane 9, closed and opened arrows) and the smaller complex was not formed with the mutated

Probe 1 (Fig. 3C, lane 10). To further confirm p53 specific binding, nuclei proteins were

incubated with the shorter Probe 2, which contains only the putative p53 binding site. As

expected, only one DNA-protein complex was formed with Probe 2 (closed arrow) (Fig. 3C,

lane 12) and the migration of the complex (closed arrow) was shifted by anti-p53 antibody

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but not with anti-HNF4α antibody (Fig. 3C, lanes 13 & 14). These observations strongly

indicate specific interaction of p53 with the putative site at the proximal CYP2A6 promoter.

Collectively, the EMSA analysis and the luciferase reporter gene assays demonstrate

that the proximal CYP2A6 gene promoter contains a functional p53RE that potentially

mediates transcriptional regulation of the gene. The functional p53RE appears to reside close

to the previously described HNF4α binding site (Pitarque et al., 2005).

Endogenous p53 activates expression of CYP2A6

To demonstrate that endogenous p53 can mediate expression of the CYP2A6-5’-

160/+9-Luc construct, as well as the transcriptional regulation of native CYP2A6 gene, we

treated transfected MCF-7 and C3A cells with different concentrations of BaP, a known

inducer of p53 (Biswal et al., 2003) for 24 h. BaP increased p53 protein levels in the nucleus

of MCF-7 by 4-fold relative to control (Fig. 4A&B). Accordingly, BaP increased luciferase

activity dose-dependently in cells transfected with either CYP2A6-5’-160/+9-Luc construct or

reporter construct containing the consensus p53RE (pGL4.38-p53RE) (Fig. 4C). The

response was totally abolished when the two copies of the CYP2A6 p53RE core sequence

were mutated (pCYP2A6-5’-160/+9_p53mut-Luc) (Fig. 4C). These observations align with

the results shown in Fig. 2C and Fig. 3, and indicate that the proximal CYP2A6 p53RE is

responsive to induced endogenous p53.

It is unlikely that the induced luciferase activity was mediated by HNF4α as its level

remained constant after BaP treatment (Fig. 4A&B). This is in agreement with previous

observation that HNF4α regulates basal expression of CYP2A6 (Pitarque et al., 2005), and

corresponds with our observation of constant luciferase activity in cells transfected with

pCYP2A6-5’-160/+9_p53mut-Luc (Fig. 4C, closed triangle), where the HNF4α binding site is

intact.

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Finally, BaP induced p53 dose-dependently in the nucleus of hepatocellular carcinoma

cells (C3A) by 4-fold relative to control (Fig. 5A&C). This confirmed previous observations

of BaP-dependent induction of p53 at mRNA and protein levels in human lung

adenocarcinoma (A549), mouse hepatoma (Hepa-1c1c7) and fibroblast (NIG3T3) cell lines,

as well as in mouse skin (Huang et al., 2012; Pei et al., 1999; Serpi & Vähäkangas, 2003).

Importantly, recent investigation reported that BaP-induced CYP1A1 expression was

regulated through p53 binding to a p53 response element in the CYP1A1 promoter region

(Wohak et al., 2014).

It is noted that p53 induction by BaP aligned with dose-dependent increased of the

CYP2A6 mRNA and apoprotein ranging from 2 to 4-fold relative to control (Fig. 5). Similar

pattern of HMOX1 induction at mRNA and protein levels (another p53 target gene) was also

observed (Fig. 5). This confirmed previous observations of BaP-dependent induction of

HMOX1 expression in various human cell lines (Lee et al., 2009; Spink et al., 2002).

These results together with our transfection data suggest that p53 mediates

transcriptional activation of CYP2A6. The fact that p53 responsiveness was observed in three

different cell lines (Suppl. Fig. 1) suggests that p53-dependent regulation of CYP2A6 is not

cell specific.

Discussion

In the present study, we have for the first time demonstrated that the tumor suppressor

protein, p53, is a regulator of the CYP2A6 gene. The finding provides novel insights into the

complex regulation of CYP2A6 in response to cellular perturbations. It is not entirely

surprising that p53 is a regulator of the CYP2A6 because the gene is known to be upregulated

by conditions that commonly activate p53 (Abu-Bakar et al., 2013).

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The presence of several putative p53REs on the CYP2A6 promoter suggests that p53 is

an important regulator for the gene. However, the sequence similarity of the five putative

p53REs at the distal CYP2A6 promoter with the consensus p53RE is not as high as that of the

proximal CYP2A6 p53RE. The functional significance of the distal sites in regulating

CYP2A6 expression is still obscured but our present observations supported by published data

suggest a role for negative regulation. For instance, the basal expression of the distal CYP2A6

promoter was significantly weaker than that of the proximal promoter (Fig 1C), which is in

accordance with previous findings of potential suppressor region(s) upstream the CYP2A6

promoter (Higashi et al., 2007; Itoh et al., 2006; Pitarque et al., 2005). This region (up to -

2901) also contains functional Nrf2 binding sites (Yokota et al., 2011). Importantly, previous

investigations have demonstrated direct interaction of p53 with Nrf2 response elements in

suppressive regulation of stress responding genes including the Phase II metabolizing genes,

NQO1 and GST-1 (Faraonio et al., 2006; Wakabayashi et al., 2010).

It is thus plausible that interplay between p53 and Nrf2 at the distal promoter mediates

suppression of CYP2A6 activation. Indeed, our preliminary observations in C3A cells

indicated that overexpression of Nrf2 substantially supressed the p53-mediated response of

the distal CYP2A6 promoter (manuscript in preparation), which contains a functional Nrf2

binding site in close proximity with one of the putative p53REs. Inevitably, further work is

warranted to confirm and understand in detail the possible Nrf2/p53 interaction on the

CYP2A6 promoter and to investigate the functional significance of such interaction in

CYP2A6 regulation, especially in the context of cellular perturbations.

Our present data clearly shows that the putative p53RE at position -122 is crucial for

the p53-dependent activation of the CYP2A6 because mutation of the core sequence of this

site negated the activities of both proximal and distal promoters (Fig. 2C). Additionally,

DNA-protein complex was not formed in gel shift assay with mutated p53RE probe (Fig. 3C,

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lane 10). Furthermore, BaP, a transcriptional activator of p53 (Huang et al., 2012; Pei et al.,

1999), increased the proximal promoter (CYP2A6-5’-160/+9-Luc) activity dose-dependently,

which is associated with dose-dependent increased of p53 protein (Fig. 4). The promoter

activity induction was abolished when the p53RE at position -122 was mutated, i.e., the

activity was maintained at basal level (Fig. 4C, closed triangle). It has been reported that the

basal expression of CYP2A6 is governed by interplay between the transcription factors

HNF4α, C/EBP α, C/EBPβ, and Oct-1 (Pitarque et al., 2005). The functional response

elements for these transcription factors reside within the proximal promoter, spanning from -

112 to -61 (Fig. 1A). Thus, the plausibility of p53 interacting with some or all of these

transcription factors in mediating transactivation of CYP2A6 cannot be ruled out by our

present study and worthy of further investigation.

We note that p53 also regulates other hepatic CYP genes involved in various metabolic

pathways (Goldstein et al., 2013; Wohak et al., 2014). Eleven members from five different

CYP subfamilies were induced by p53 (Goldstein et al., 2013). These include CYP4Fs and

CYP19A1, enzymes that metabolize lipids and sex steroids, respectively (Goldstein et al.,

2013); and CYP21A2, a catalyst of glucocorticoid synthesis (Goldstein et al., 2013). These

findings aligned with recent advancement in p53 research that acknowledges a critical role

for p53 in regulating metabolism and maintaining cellular homeostasis (reviewed in Meek,

2015). Additionally, p53 role in regulating drug metabolism was recognised in a recent work

demonstrating direct p53 interaction on the p53REs of the CYP3A4 promoter, leading to

transactivation of the gene (Goldstein et al., 2013). The p53-dependent activation of CYP3A4

activity, the main enzyme responsible for clearing chemotherapeutics, was heightened

following a chemotherapeutic stimulus (Goldstein et al., 2013). This observation indicates a

systemic role for p53 in response to chemotherapy.

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As CYP2A6 is not a major drug metabolizing enzyme, what is the physiological

significance of p53-mediated transactivation of CYP2A6? At present, we have no conclusive

explanation. However, as p53 plays an important role in maintaining cellular homeostasis

(Meek, 2015), our findings—along with our previous observation that CYP2A6 efficiently

oxidizes bilirubin (BR) to a less toxic biliverdin (Abu-Bakar et al., 2012)—indicate a

potential role for the CYP2A6/p53 pathway in regulating bilirubin homeostasis maintenance.

Furthermore, the observation that BaP—a carcinogen and transactivator of p53 (Pei et

al., 1999)—concurrently induced CYP2A6 and HMOX1 expression (Fig. 5) suggests a

potential role for CYP2A6 in cellular stress management. HMOX1 catalyses heme

breakdown to biliverdin (BV), which is reduced to BR by biliverdin reductase. Bilirubin is an

efficient scavenger of reactive oxygen and nitrogen species (Jansen et al., 2010; Liu et al.,

2003), but apoptotic at highly elevated concentrations (Rodrigues et al., 2002). Hence,

activation of HMOX1 by p53 may lead to drastic elevation of BR concentration, which in

turn, may overwhelm BR oxidant scavenging activity. Consequently, there is a need for a

protective mechanism where BR is enzymatically oxidized to BV to curb its cytotoxicity, and

to be reduced back to BR by biliverdin reductase when needed. CYP2A6 is potentially a good

BR oxidase candidate under this condition as the Km for BR is within the subtoxic range

(Abu-Bakar et al., 2012).

Importantly, the proposition of CYP2A6 role in regulating BR homeostasis during

chemical onslaught is further supported by a recent finding that BR elimination through

conjugation was not elevated during the initial stage of cellular stress (Muhsain et al., 2015).

Thus, transcriptional activation of CYP2A6 gene through p53 may function as a part of the

cellular protection to re-establish BR homeostasis.

We conclude that the novel findings presented in this paper implied important role for

p53-mediated CYP2A6 and HMOX1 activation in regulating hepatic BR homeostasis. Given

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the dual role of p53 in cytoprotection and apoptosis (Bensaad and Vousden, 2007), and the

antioxidant and pro-apoptotic effects of BR (Jansen et al., 2010; Liu et al., 2003; Ollinger et

al., 2007), it is of fundamental interest to understand how these seemingly contradictory

events are related to the regulation of CYP2A6 and HMOX1. In this regard the possible

interaction of Nrf2 and p53 in the regulation of CYP2A6 and HMOX1 might prove to be

critical as has been shown for some other stress responsive genes (Faraonio et al., 2006;

Wakabayashi et al., 2010). Understanding the molecular mechanisms of this interaction is the

main focus of our current research.

Conflict of interest statement

The authors declare that they have no conflict of interest in this work.

Acknowledgements

The authors would like to thank Professor Greg Monteith, Pharmacy Australia Centre of

Excellence, University of Queensland, for the provision of the MCF-7 cells. This work was

supported by the Cooperative Research Centre for Contamination and Remediation of the

Environment (CRC-CARE), Australia [2.1.04.11/12] and the University of Queensland Early

Career Researcher Grants Scheme [UQECR2013002328]. HH is a recipient of the CRC-

CARE PhD scholarship [2.1.04.11/12]. The National Research Centre for Environmental

Toxicology is a partnership between Queensland Health and the University of Queensland.

References

Abu-Bakar, A., Arthur, D.M., Wikman, A.S., Rahnasto, M., Juvonen, R.O., Vepsalainen, J.,

Raunio, H., Ng, J.C., Lang, M.A., 2012. Metabolism of bilirubin by human cytochrome

P450 2A6. Toxicol. Appl. Pharmacol. 261, 50-58.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

21

Abu-Bakar, A., Hakkola, J., Juvonen, R., Rahnasto-Rilla, M., Raunio, H., Lang, M.A., 2013.

Function and regulation of the Cyp2a5/CYP2A6 genes in response to toxic insults in the

liver. Curr. Drug Metab. 14, 137-150.

Abu-Bakar, A., Lämsä, V., Arpiainen, A., Moore, M.R., Lang, M., Hakkola, J., 2007.

Regulation of Cyp2a5 gene by the transcription factor nuclear factor (erythroid-derived

2)-like 2. Drug Metab. Dispos. 35, 787-794.

Bensaad, K., Vousden, K.H., 2007. p53: new roles in metabolism. TRENDS Cell Biol. 17,

286-291.

Biswal, S., Maxwell, T., Rangasamy, T., Kehrer, J.P., 2003. Modulation of benzo[α]pyrene-

induced p53 DNA activity by acrolein. Carcinogenesis. 24, 1401-1406.

Christian, K., Lang, M., Maurel, P., Raffalli-Mathieu, F., 2004. Interaction of heterogeneous

nuclear ribonucleoprotein A1 with cytochrome P450 2A6 mRNA: implications for post-

transcriptional regulation of the CYP2A6 gene. Mol. Pharmacol. 65, 1405-1414.

Christian, K.J., Lang, M.A., Raffalli-Mathieu, F., 2008. Interaction of heterogenous nuclear

ribonucleoprotein C1/C2 with a novel cis-regulatory element wihtin p53 mRNA as a

response to cytostatic drug treatment. Mol Pharmacol. 73, 1558-1567.

Donato, M.T., Vititala, P., Rodriguez-Anotona, C., Lindfors, A., Castell, J.V., Raunio, H.,

Gomez-Lechon, M.J., Pelkonen, O., 2000. Cyp2a5/CYP2A6 expression in mouse and

human hepatocytes treated with various in vivo inducers. Drug Metab. Dispos. 28, 1321-

1326.

el-Deiry, W.S., Kern, S.E., Pietenpol, J.A., Kinzler, K.W., Vogelstein, B., 1992. Definition of

a consensus binding site for p53. Nat. Genet. 1, 45-49.

Faraonio, R., Vergara, P., Di Marzo, D., Pierantoni, G.M., Napolitano, M., Russo, T.,

Cimino, F., 2006. P53 supresses the Nrf2-dependent transcription of antioxidant response

genes. J. Biol. Chem. 52, 39776-39784.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

22

Fisher, C.D., Lickteig, A.J., Augustine, L.M., Ranger-Moore, J., Jackson, J.P., Ferguson,

S.S., Cherrington, N.J., 2009. Hepatic cytochrome P450 enzyme alterations in humans

with progressive stages of nonalcoholic fatty liver disease. Drug Metab. Dispos. 37,

2087-2094.

Goldstein, I., Rivlin, N., Shoshana, O.Y., Ezra, O., Madar, S., Goldfinger, N., Rotter, V.,

2013. Chemotherapeutic agents induce the expression and activity of their clearing

enzyme CYP3A4 by activating p53. Carcinogenesis 34, 190-198.

Goodwin, B., Gauthier, K.C., Umetani, M., Watson, M.A., Lochansky, M.I., Collins, J.L.,

Leitersdorf, E., Mangelsdorf, D.J., Kliewer, S.A., Repa, J.J., 2003. Identification of bile

acid precursors as endogenous ligands for the nuclear xenobiotic pregnane X receptor.

PNAS. 100, 223-228.

Helton, E.S., Chen, X. (2007). p53 modulation of the DNA damage response. J. Cell.

Biochem. 100, 883-896.

Higashi, E., Fukami T., Itoh, M., Kyo, S., Inoue, M., Yokoi, T., Nakajima, M., 2007. Human

CYP2A6 is induced by estrogen vis estrogen receptor. Drug Metab. Dispos. 35, 1935-

1941.

Huang, M.C., Chen, F.Y., Chou, M.T., Su, J.G.J., 2012. Fluoranthene enhances p53

expression and decreases mutagenesisi induced by benzo[α]pyrene. Toxicol. Letts. 208,

214-224.

Itoh, M., Nakajima, M., Higashi, E., Yoshida, R., Nagata, K., Yamazoe, Y., Yokoi, T., 2006.

Induction of human CYP2A6 is mediated by the pregnane X receptor with peroxisome

proliferator-activated receptor-γ coactivator 1α. J. Pharmacol. Exp. Ther. 319, 693-702.

Janmohamed, A., Dolphin, C.T., Phillips, I.R., Shephard, E.A., 2001. Quantification and

cellular localization of expression in human skin of genes encoding flavin-containing

monooxygenases and cytochromes P450. Biochem. Pharmacol. 62, 777-786.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

23

Jansen, T., Hortmann, M., Oelze, M., Opitz, B., Steven, S., Schell, R., Knorr, M., Karbach,

S., Schuhmacher, S., Wenzel, P., Munzel, T., Daiber, A., 2010. Conversion of biliverdin

to bilirubin by biliverdin reductase contributes to endothelial cell protection by heme

oxygenase-1-evidence for direct and indirect antioxidant actions of bilirubin. J. Mol.

Cell. Cardiol. 49, 186-195.

Kansanen, E., Kuosmanen, S.M., Leinonen, H., Levonen, A.L., 2013. The Keap1-Nrf2

pathway: Mechanisms of activation and dysregulation in cancer. Redox Biol. 1, 45-49.

Kirby, G.M., Batist, G., Alpert, L., Lamoureux, E., Cameron, R.G., Alaoui-Jamali, M.A.,

1996. Overexpression of cytochrome P-450 isoforms involved in aflatoxin B1 activation

in human liver with cirrhosis and hepatitis. Toxicol. Pathol. 24, 458-467.

Koskela, S., Hakkola, J., Hukkanen, J., Pelkonen, O., Sorri, M., Saranen, A., Anttila, S.,

Fernandez-Salguero, P., Gonzalez, F., Raunio, H., 1999. Expression of CYP2A genes in

human liver and extrahepatic tissue. Biochem. Pharmacol. 57, 1407-1413.

Lee, S.H., Lee, S.E., Ahn, H.J., Park, C.S., Cho, J.J., Park, Y.S., 2009. Identification of

atherosclerosis related gene expression profiles by treatment of benzo(a)pyrene in human

umbilical vein endothelial cells. Mol. Cell. Toxicol. 5, 113-119.

Liu, Y., Zhu, B., Wang, X., Luo, L., Li, P., Paty, D.W., Cynader, M.S., 2003. Bilirubin as a

potent antioxidant suppresses expereimental autoimmune encephalomyelitis:

implications for the role of oxidative stress in the development of multiple sclerosis. J.

Neuroimmunol. 139, 27-35.

Meek, D.W., 2015. Regulation of the p53 and its relationship to cancer. Biochem. J. 469,

325-346.

Muhsain, S.N.F., Lang, M.A., Abu-Bakar, A., 2015. Mitochondrial targeting of bilirubin

regulatory enzymes: An adaptive response to oxidative stress. Toxicol. Appl. Pharmacol.

282, 77-89.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

24

Nakajima, M., Itoh, M., Sakai, H., Fukami, T., Katoh, M., Yamazaki, H., Kadlubar, F.F.,

Imaoka, S., Funae, Y., Yokoi, T., 2006. CYP2A13 expressed in human bladder

metabolically activates 4-aminobiphenyl. Int. J. Cancer. 119, 2520–2526.

Nam, S.Y., Sabapathy, K., 2011. p53 promotes cellular survival in a context-dependent

manner by directly inducing the expression of heme oxygenase 1. Oncogene 30, 4476-

4486.

Niemela, O., Parkkila, S., Juvenon, R.O., Viitala, K., Gelbion, H.V., Pasanen, M., 2000.

Cytochromes P450 2A6, 2E1, and 3A and production of protein-aldehyde adducts in the

liver of patients with alcoholic and non-alcoholic liver diseases. J. Hepatol. 33, 893-901.

Ollinger, R., Kogler, P., Troppmair, J., Hermann, M., Wurm, M., Drasche, A., Konigsrainer,

I., Amberger, A., Weiss, H., Ofner, D., Bach, F.H., Margreiter, R., 2007. Bilirubin

inhibits tumor cell growth via activation of ERK. Cell Cycle. 6, 3078-3085.

Pei, X.H., Nakanishi, Y., Takayama, K., Bai, F., Hara, N., 1999. Benzo[a]pyrene activates

the human p53 gene through induction of nuclear factor β activity. J. Biol. Chem. 274,

35240-35246.

Pitarque, M., Rodríguez-Antona, C., Oscarson, M., Ingelman-Sundberg, M., 2005.

Transcriptional regulation of the human CYP2A6 gene. J. Pharmacol. Exp. Ther. 313,

814-822.

Raunio, H., Juvenon, R., Pasanen, M., Pelkonen, O., Paakko, P., Soini, Y., 1998. Cytochrome

P4502A6 (CYP2A6) expression in human hepatocellular carcinoma. Hepatology. 27,

427-432.

Raunio, H, and Rahnasto-Rilla, M., 2012. CYP2A6: genetics, structure, regulation, and

function. Drug Metab. Drug Interact. 27, 73-88.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

25

Rodrigues, C.M., Sola, S., Brito, M.A., Brites, D., Moura, J.J., 2002. Bilirubin directly

disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat

mitochondria. J. Hepatol. 36, 335-341.

Saha, T., Kar, R.K., Sa, G., 2015. Structural and sequential context of p53: A review of

experimental and theoretical evidence. Prog. Biophys. Mol. Biol. Article in press.

doi:10.1016/j.pbiomolbio.2014.12.002, 1-14.

Satarug, S., Lang, M.A., Yongvanit, P., Sithithaworn, P., Mairiang, E., Mairiang, P.,

Pelkonen, P., Bartsch, H., Haswell-Elkins, M.R., 1996. Induction of cytochrome P450

2A6 expression in humans by the carcinogenic parasite infection, Opisthorchiasis

Viverrini. Cancer Epi. Biomark. Prev. 5, 795-800.

Serpi, R., and Vähäkangas, K., 2003. Benzo(a)pyrene-induced changes in p53 and related

proteins in mouse skin. Pharmacol. Toxicol. 92, 242-245.

Spink, D.C., Katz, B.H., Hussain, M.M., Spink, B.C., Wu, S.J., Liu, N., Pause, R., Kaminsky,

L.S., 2002. Induction of CYP1A1 and CYP1B1 in T-47D human breasy cancer cells by

benzo(a)pyrene is diminished by arsenite. Drug Metab. Dispos. 30, 262-269.

Staudinger, J.L., Goodwin, B., Jones, S.A., Hawkins-Brown, D., MacKenzie, K.I., LaTour,

A., Liu, Y., Klaassen, C.D., Brown, K.K., Reinhard, J., Willson, T.M., Koller, B.H.,

Kliewer, S.A., 2001. The nuclear receptor PXR is a lithocholic acid sensor that protects

against liver toxicity. PNAS. 98, 3369-3374.

Su, T., Sheng, J.J., Lipinskas, T.W., Ding, X., 1996. Expression of CYP2A genes in rodent

and human nasal mucosa. Drug Metab. Dispos. 24, 884-890.

Wohak, L.E., Krais, A.M., Kucab, J.E., Stertmann, J., Øvrebø, S., Seidel, A., Phillips, D.H.,

Arlt, V.M., 2014. Carcinogenic polycyclic aromatic hydrocarbons induce CYP1A1 in

human cells via a p53-dependent mechanism. Arch. Toxicol. [Epub ahead of print] DOI

10.1007/s00204-014-1409-1.

ACC

EPTE

D M

ANU

SCR

IPT

ACCEPTED MANUSCRIPT

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Xie, W., Radominska-Pandya, A., Shi, Y., Simon, C.M., Nelson, M.C., Ong, E.S., Waxman,

D.J., Evans, R.M., 2001. An essential role for nuclear receptor SXR/PXR in detoxication

of cholestatic bile acids. PNAS. 98, 3375-3380.

Yamazaki, H., Inui, Y., Yun, C.H., Guengerich, F.P., Shimada, T., 1992. Cytochrome P450

2E1 and 2A6 enzymes as major catalysts for metabolic activation of N-

nitrosodialkylamines and tobacco-related nitrosamine in human liver microsomes.

Carcinogenesis. 13, 1789–1794.

Yokota, S., Higashi, E., Fukami, T., Yokoi, T., Nakajima, M., 2011. Human CYP2A6 is

regulated by nuclear factor-erythroid 2 related factor 2. Biochem. Pharmacol. 81, 289-

294.

Zuckerman, V., Wolyniec, K., Sionov, R.V., Haupt, S., Haupt, Y., 2009. Tumour suppression

by p53: the importance of apoptosis and cellular senescence. J. Pathol. 219, 3-15.

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Figure legends

Figure 1: Putative p53REs at the CYP2A6 promoter and effect of p53 co-transfection on

CYP2A6-5’-Luc construct activities in C3A cells. (A) A schematic representation of the 2.9

kb CYP2A6 promoter region indicating the putative p53RE sites. The region spanning -112 to

-61 contains overlapping response elements for DR-4, C/EBP, C/EBPβ, Oct-1, and HNF4

(Pitarque et al., 2005). (B) The six putative p53 binding sites identified by MatInspector

(http://www.genomatix.de/) and the p53 consensus sequence. Putative p53RE core sequences

are shown in bold. R = purine; Y = pyrimidine. (C) Western blot and densitometry of p53

and β-actin (loading control) in C3A cells transfected with p53 expression plasmid (pcDNA-

hp53) and empty expression plasmid (pcDNA3). Each transfection was done in triplicate.

Positive controls (+) = A-431 whole cell lysate for p53 and β-actin peptide. Relative p53

expression (bar graph) = the intensity of p53 band relative to the intensity of β-actin band. (D)

Effect of p53 co-transfection (pcDNA-hp53) on CYP2A6 distal and proximal driven

luciferase activities. The firefly luciferase activities were measured 24 h after transfection.

The measured activites were normalized against pMAX-GFP activity (transfection control

plasmid). The values (n = 3) represent means ± S.D. p53 response of each reporter construct

is indicated by -fold of activity to control co-transfection with pcDNA3. Mean difference is

significant from control group at ***, p < 0.0005; **, p < 0.005; *, p < 0.05 (Student’s t test).

Figure 2: Functionality test of the p53RE site on the proximal CYP2A6 promoter. (A) C3A

cells were co-transfeceted with a series of 5’-truncated proximal CYP2A6 promoter-luciferase

reporter plasmids and p53 expression plasmid (pcDNA3-hp53) or empty expression plasmid

(pcDNA3). (B) The difference between putative and the consensus p53RE sequence. The

boxed region contains the p53 binding core sequence. (C) Effect of p53 co-transfection on the

mutated and wildtype CYP2A6 proximal and distal promoters activities. The potential

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proximal p53 binding sites were mutated as described: TTCATG → TTACCA and

GGCATG → GGATCC in CYP2A6-5’-160/p53mut-Luc, with the mutated bases in bold and

the underlined bases representing the core sequence. The mutated and wildtype CYP2A6-5’-

Luc constructs were transfected to C3A cells and luciferase activities were measured 24 h

after transfection. The measured luciferase activities were normalized against co-transfected

pMAX-GFP control plasmid activities. The values (n = 4) represent the means ± S.D. p53

response of each reporter construct is indicated by -fold of activity to control co-transfection

with pcDNA3. Mean difference is significant from control group at ****, p < 0.0001; **, p <

0.005 (Student’s t test).

Figure 3: EMSA analysis of p53 protein/CYP2A6 promoter interactions. (A) A schematic

representation of the -160 to +1 region in the proximal CYP2A6 promoter. The closed arrows

indicate orientation of the EMSA probe. The closed line square highlights sequence of the

potential p53RE that was revealed by the MatInspector transcription factor binding site

search (http://www.genomatix.de/). The opened line square highlights sequence of the

HNF4α response element. (B) Western blot of p53 in the nuclei fractions of C3A cells

transfected with p53 expression plasmid (pcDNA3-hp53). Positive control (+) = A-431

whole cell lysate. (C) EMSA blot. Nuclei proteins extracted from C3A cells transfected with

pcDNA3-hp53 expression plasmid were incubated with digoxigenin-labelled probe the

orientation of which was shown in (A). Probe 1 = 94 bp oligonucleotide containing the

putative binding site from -149 to -55 bp. Probe 2 = 29 bp oligonucleotide spanning the

putative binding site from -127 to -98 bp. Probe1Mut = Probe 1 with the core sequence,

CATG, critical for p53 binding, mutated to ACCA. Lanes 1 & 11 represent incubation

without nuclei proteins. Lanes 3 & 13 represent incubation with nuclei proteins and anti-p53

antibody. Lane 9 represents incubation with nuclei proteins and anti-Nrf2 antibody. Lanes 8

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& 14 represent incubation with nuclei proteins and anti- HNF4α antibody The reactions with

nuclear extracts were competed with 125-fold excess of unlabelled probe (S) (lane 4) and

descending concentrations of unlabelled consensus p53RE oligonucleotides (C) (lanes 5-7).

Lane 10 represents incubation with nuclei proteins digoxigenin-labelled mutated probe. The

closed arrow indicates smaller DNA-protein complex, and the opened arrow indicates bigger

complex. The experiment was repeated three times, and similar results were obtained.

Figure 4: Effect of benzo[a]pyrene (BaP) treatment on endogenous p53 activation and the

proximal CYP2A6-luciferase construct activities in MCF-7 cells. The cells were transfected

with pGL4.38-p53-Luc, CYP2A6-5’-160/+9-Luc, or CYP2A6-5’-160/p53mut-Luc for 4 h

before being treated with various concentrations of BaP. Twenty-four hours after treatment

nuclei proteins were extracted and luciferase activities were measured. (A) Western blot of

p53, HNF4 and β-actin (loading control) in MCF-7 cells treated with various concentrations

of BaP. Positive controls (+) = A-431 whole cell lysate for p53, HepG2 whole cell lysate for

HNF4 and β-actin peptide. Each blot represents one of three blots (each blot showed the

same pattern of induction). (B) Densitometry analysis of the Western blots. Fold induction =

Relative p53 or HNF4 expression of treated samples / Relative p53 or HNF4 expression of

control (0 µM BaP). Relative p53 or HNF4 expression = the intensity of p53 or HNF4

band relative to the intensity of β-actin band. The values (n = 3) represent the means ± S.D.

*Mean difference is significant from control group at p < 0.05 (Student’s t test). (C) Effect of

BaP treatment on the luciferase activities of the putative CYP2A6 p53RE constructs. The

firefly luciferase activities were measured 24 h after BaP treatment. The measured activites

were normalized against pMAX-GFP activity (transfection control plasmid). The values (n =

4) represent means ± S.D. *Mean difference is significant from control group at p < 0.05

(Student’s t test).

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Figure 5: Effect of BaP on endogenous p53, native CYP2A6 and HMOX1 expression in

C3A cells. The cells were treated with various concentrations of BaP for 24 h. Thereafter

total RNA, nuclei and cytosolic proteins were extracted. Quantitative real time RT-PCR was

used to detect CYP2A6 and HMOX1 mRNA expression. (A) Western blot and densitometry

of nuclei p53 and cytosolic CYP2A6 and HMOX1. β-actin was used as loading control.

Each blot represents one of three blots (each blot showed the same pattern of induction).

Positive controls (+) = A-431 whole cell lysate for p53; peptides for β-actin and HMXO1;

and recombinant CYP2A6 yeast microsomes. Fold induction = Relative protein expression of

treated samples / Relative protein expression of control (0 µM BaP). Relative protein

expression = the intensity of protein band relative to the intensity of β-actin band. (B)

Migration of RT-PCR products in 2.5% agarose gel. Lane 1 = 50 bp DNA ladder. Lanes 2, 5

and 8 are products from control cells; lanes 3, 6 and 9 are products from treated cells; and

lanes 6, 7 and 10 are non-template control (NTC). GAPDH = housekeeping gene. The sizes

of the PCR products align with the amplified regions by the primers. (C) Fold induction of

p53 proteins, CYP2A6 and HMOX1 mRNA and protein in control and treated cells.

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Figure 1

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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Table 1: Oligonucleotides used for amplifications in Real-time PCR, EMSA and site-directed

mutagenesis experiments.

Primers Sequences

PCR Amplification:

CYP2A6 5’ -368 KpnI FW

CYP2A6 5’ -308 KpnI FW

CYP2A6 5’ -250 KpnI FW

CYP2A6 5’ -160 KpnI FW

CYP2A6 5’ -74 KpnI FW

CYP2A6 5’ +9 NheI RV

CYP2A6 5’ -2901KpnI

CYP2A6 5’ -437KpnI

5’-GACGGTACCCCCCCAGATCCACAACTTTG-3’

5’-GACGGTACCGTGCTCCCCTATGCAAATATTC-3’

5’-GACGGTACCTCCTAAATCCACAGCCCTGC-3’

5’-GACGGTACCTTATCCTCCCTTGCTGGCTG-3’

5’-GACGGTACCATCAGCCAAAGTCCATCCCTC-3’

5’-GACGCTAGCGGTGGTAGTGGGATGATAGATG-3’

5’-GACGGTACCCATCCATCCGCTTCATCCTACAG-3’

5’-GACGGTACCCCTAAATGCACAGCCACACTTTG-3’

mRNA analysis:

CYP2A6 NM_000762

GAPDH NM_002046

HMOX1 NM_002133

F: 5’-AAGATCAGTGAGCGC TATGG-3’

R: 5’-TGAATACCACGCATAGCCT-3’

Amplicon size: 178 bp

F: 5’- CATGAGAAGTATGACAACAGCCT-3’

R: 5’-AGTCCTTCCACGATACCAAAGT-3’

Amplicon size: 113 bp

F: 5’- CAGTGCCACCAAGTTCAAGC-3'

R: 5’-GTTGAGCAGGAACGCAGTCTT-3'

Amplicon size: 112 bp

EMSA:

Primer 1

Primer 2

5’-TGCTGGCTGTGTCCCAAGCTAG-3’

5’-GGGATGGACTTTGGCTGATTAC-3’

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Site-directed mutagenesis:

CYP2A6-p53mut RV

CYP2A6-p53mut FW

5’-TAGGATCCCCACTGGTAATCCTGCCTAGCTTGG-3’

5’-ACCAGTGGGGATCCTAGTTGGGAGGTGAAATG-3’

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Highlights

CYP2A6 is an immediate target gene of p53

Six putative p53REs located on 3 kb proximate CYP2A6 promoter region

The region -160 bp from TSS is highly homologous with the p53 consensus sequence

P53 specifically bind to the p53RE on the -160 bp region

HNF4 may interact with p53 in regulating CYP2A6 expression