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Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase 1

Electrophilic Prostaglandins and Lipid Aldehydes Repress Redox Sensitive

Transcription Factors p53 and HIF by Impairing the Selenoprotein

Thioredoxin Reductase

Philip J. Moos1, Kornelia Edes, Pamela Cassidy2, Edmund Massuda, F.A.

Fitzpatrick1,2

Huntsman Cancer Institute, Department of Oncological Science1 & Medicinal

Chemistry2

2000 Circle of Hope

University of Utah

Salt Lake City, UT 84112-5550

Correspond with:

F.A. Fitzpatrick, Ph.D.

Dee Glenn and Ida W. Smith Chair of Cancer Research

Huntsman Cancer Institute

2000 Circle of Hope

University of Utah

Salt Lake City, Utah 84112-5550

Telephone: 801-581-6204

Fax: 801-585-0011

E.mail [email protected]

Running title: Cyclopentenone PG and 4-HNE inactivate thioredoxin reductase

1Abreviations used: PG, prostaglandins; 4-HNE, 4-hydroxy-2-nonenal; TrxR,

thioredoxin reductase; IKK, I-kappa-β kinase; APB, amidopentyl biotin; MDA,

malodialdehyde.

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SUMMARY

Tumor suppressor p53 exhibits an enigmatic phenotype in cells exposed to

electrophilic, cyclopentenone prostaglandins of the A- and J-series. Namely, cells

harboring a wild type p53 gene accumulate p53 protein that is conformationally

and functionally impaired. This occurs via an unknown molecular mechanism. We

report that electrophilic cyclopentenone prostaglandins covalently modify and

inhibit thioredoxin reductase, a selenoprotein that governs p53 and other redox

sensitive transcription factors. This mechanism accounts fully for the unusual p53

phenotype in cells exposed to electrophilic prostaglandins. Based on this

mechanism we derived, tested, and affirmed several predictions regarding the

kinetics of p53 inactivation; the protective effects of selenium; the structure-activity

relationships for inhibition of thioredoxin reductase and impairment of p53 by

electrophilic lipids; the susceptibility of hypoxia-inducible factor to inactivation by

electrophilic lipids; and the equivalence of chemical inactivation of p53 to deletion

of a p53 allele. Chemical precepts dictate that other electrophilic agents should

also inhibit thioredoxin reductase and impair its governance of redox sensitive

proteins. Our results provide a novel framework to understand how endogenous

and exogenous electrophiles might participate in carcinogenesis; how

selenoproteins and selenium might confer protection against cancer; how certain

tumors might acquire their paradoxical p53 phenotype, and how chronic

inflammation might heighten the risk for cancer.


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INTRODUCTION

Cyclopentenone prostaglandins (PG1), of the A- and J- series impair the

conformation and function of tumor suppressor p53 by a novel, but unknown,

mechanism of action (1,2). Cyclopentenone PG penetrate cells and accumulate in

the cytosol and nucleus (3) where they can react covalently with other molecules

via their electrophilic β carbon (4). Few of the proximal molecular targets of these

PG are established (5-7). There are two formal hypotheses to explain how

electrophilic PG might inactivate p53. First, they might act directly, via covalent

reaction with p53 itself. Second, they might act indirectly, via covalent reaction

with regulatory proteins that govern p53 conformation and function. A direct

mechanism of action is incompatible with our observation that PGA1 and A2

antagonize only the apoptosis mediated by p53 (1), but not the cell-cycle arrest.

These PG should antagonize all functions mediated by p53 if their molecular

mechanism of action involved its modification directly. Accordingly, we sought

candidate proteins consistent with an indirect molecular mechanism of action.

Thioredoxin reductase (TrxR) is notable from biological and chemical perspectives.

Biologically, TrxR-Trx cycling modulates sulfhydryl-disulfide isomerization

reactions that govern the conformation and function of p53, as well as several other

redox sensitive transcription factors, like NFκB and hypoxia-inducible factor (HIF)

(8-10). Trr1, the yeast ortholog of TrxR, is essential for transcription by p53

expressed ectopically in yeast (11,12). Chemically, TrxR is a selenoprotein.

Selenocysteine residues are typically more nucleophilic than cysteine under

comparable conditions. Chemical precepts dictate that electrophilic agents, like

cyclopentenone PG, should react readily with selenoproteins under conditions

encountered in cells.


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Herein we report that: i) a prototypical PGA analog forms a covalent adduct

with TrxR; ii) analogous to the cyclopentenone PG, several representative, naturally

occurring or synthetic aldehydes and ketones with electrophilic β carbons (15-keto-

PG, 4-hydroxy-2-nonenal, ethacrynic acid) impair p53 conformation and function,

indirectly, via inhibition of TrxR; iii) other redox-sensitive transcription factors

governed by TrxR-Trx cycling, e.g. HIF, are also susceptible to inactivation by lipid

aldehydes and ketones with electrophilic β carbons; iv) supplementation of cell

culture medium with inorganic Se spares p53 from inactivation by lipid

electrophiles; v) impairment of p53 by lipid electrophiles is comparable in severity

to loss of one allele of the p53 gene. Our results provide a novel framework to

understand how these agents and numerous chemically related, endogenous and

exogenous electrophiles might participate in carcinogenesis; how selenoproteins

and dietary selenium may confer protection against cancer (13); how cells might

acquire an unusual and unexplained p53 phenotype observed in some tumors (14-

17); and how chronic inflammation might heighten the risk for cancer (18).


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EXPERIMENTAL PROCEDURES

Materials. We used DMEM and McCoy’s 5A medium and supplements

(GIBCO/BRL); prostaglandins (Cayman Chemicals); Auranofin (ICN Biomedicals);

cobalt chloride and etoposide (Sigma); malondialdehyde (Fluka); 4-hydroxy-2-

nonenal (Oxis International, Inc.); protease inhibitor mixture and FuGene-6

transfection reagent (Roche Molecular Biochemicals); enhanced chemiluminescence

reagents (Amersham Pharmacia); luciferase reporter lysis buffer and reporter

detection reagents (Promega); monoclonal antibodies directed against p53 (Pab240

– Santa Cruz; Pab1620 (Ab5) – Oncogene Sciences); polyclonal antibodies against

TrxR (Upstate Biotechnology) and p53 (FL-393-G, Santa Cruz); horseradish

peroxidase-conjugated secondary antibodies; protein A/G PLUS-Agarose (Santa

Cruz Biotechnology); Neutravidin-conjugated beads (Pierce); Ac-DEVD-MCA

(Peptides International); luciferase reporter constructs for p53 (p53-Luc,

Stratagene) and HIF-1 (p2.1, ref 19; gift of G.L. Semenza, Johns Hopkins

University, Baltimore); and a β-galactosidase expression vector (pCMVβ;

CLONTECH).

Cell Culture. We maintained HCT 116 p53+/+, p53+/- and p53-/- (ref. 20 gift of

B. Vogelstein) cells in McCoy’s 5A medium and RKO cells (gift of M. Meuth,

Institute for Cancer Studies, University of Sheffield, Sheffield, U.K.) in DMEM at

37°C in a humidified incubator with 5% CO2. We supplemented media with 2 mM

L-glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin and streptomycin, and

10% (vol/vol) FBS. In certain experiments, cells were metabolically labeled with

100 µCi [75Se], obtained from the University of Missouri Research Reactor, for 48

hrs to ensure that selenoproteins were labeled to steady state.

Isolation of Proteins Labeled by PGA1-ABP: Neutravidin Sequestration. We

treated RKO and HCT 116 cells with 60 µM PGA1-APB for 4 hrs, or as described in


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the text. We lysed cells in 250 mM sucrose 50 mM Tris, pH 7.4/25 mM KCl/5 mM

MgCl2/1 mM EDTA/1× complete protease inhibitor/2 mM NaF/2 mM sodium

orthovanadate. We sonicated the lysate twice for 5 s at 4°C. After centrifugation at

13,000 × g, we incubated samples containing 200 µg of total cell lysate for 16 h at

4°C with 100 µl of Neutravidin beads in 1 ml of PBS with 0.4% Tween 20. We

centrifuged the samples at 500×g, 5 min to isolate the Neutravidin–biotin

complexes. We washed the beads 5 × 1 ml of PBS/0.4% Tween 20. We fractionated

samples by SDS/PAGE and detected the biotinylated proteins with Streptavidin-

HRP. We also reprobed the membrane with antibodies directed against Trx, TrxR,

p53, p50/p105 of NFκB, and IKKα to determine if they adhered to the Neutravidin

beads.

Immunoprecipitation of p53. We lysed cells and incubated 200 µg of total cell

lysate for 16 h at 4°C with 1 µg of either Pab240 or Pab1620, antibodies that

specifically recognize p53 in its mutant or wild-type conformation, respectively,

(21). We added 20 µl of protein A/G PLUS-Agarose in 1 ml of PBS with 0.4% Tween

20. We centrifuged the samples at 500× g, for 5 min to isolate the antigen–antibody

immune complexes. We washed the immunoprecipitate 2 × 1 ml of PBS/0.4%

Tween 20. We fractionated samples by SDS/PAGE and measured the amount of

conformationally mutant or wild type p53 in the immunoprecipitate by

hybridization with a separate anti-p53 polyclonal antibody (FL-393).

p53 and HIF-1 Transcriptional Activity. We transfected 105 RKO cells per well

with 1 µg of p53-Luc or p2.1 and 50 ng of pCMVβ in 3 µl of FuGene-6. After 48 h,

we incubated cells for 6 h with vehicle (DMSO), 0–60 µM of various electrophiles,

plus 50 µM etoposide or 100 µM CoCl2, respectively. We aspirated media, washed

cells with PBS (pH 7.4) at 4°C, and then lysed cells at 4°C in 100 µl of Reporter

Lysis Buffer. We centrifuged the lysate at 20,000×g for 15 min at 4°C and

quantified luciferase and β-galactosidase activity in the supernatant fractions.


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Thioredoxin reductase activity. We lysed RKO cells in 50 mM Tris, pH

7.4/0.1M NaCl/2 mM EDTA/1% SDS/1% deoxycholate/1 mM NaF/1 mM sodium

orthovanadate/1× complete protease inhibitors. We added 20 µl of protein lysate

(3-4 µg/µl) to 100 µl of 20 µM Tris, pH 7.4, containing 70 µM insulin, 66.6 µM E.

coli thioredoxin, and 120 nM NADPH and 5.5 mM EDTA. We monitored the

oxidation of NADPH at 340 nm for 0-5 min at 25oC, or longer to ensure we

measured the linear portion of the progress curve.

Caspase-3 Activity. We treated 106 HCT116 cells for 48 hrs with 20 µM

amethopterin plus 0, 6, or 20 µM PGA2. We measured caspase-3 activity as an

index of apoptosis (22). We verified that PGA2 did not interfere with caspase-3.


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RESULTS

To determine whether electrophilic PGs react with TrxR or other proteins we

used PGA1 amidopentyl biotin (PGA1-APB). PGA1-APB retains the α, β unsaturated

ketone substituent and the electrophilic β carbon of PGA1. Its C1 biotin amide,

instead of a C1 carboxyl group, facilitates the detection of any covalent adducts it

might form with proteins (6). [Figure 1]

To calibrate the utility of PGA1-ABP we examined its interaction with IKKα, a

protein putatively modified by cyclopentenone PG (7). We incubated intact RKO

and HCT 116 colon cancer cells with PGA1-ABP (60 µM) for 4 hr; sequestered any

proteins with biotin epitopes on neutravidin beads; fractionated these same

biotinylated proteins by SDS-PAGE; and identified them immunochemically. Cells

contained approximately 15 proteins labelled de novo with PGA1-APB under these

conditions - two of these proteins were IKKα [Figure 2] and the p50/p105 subunit

of NFκB [data not shown]. Our data are the first direct evidence that

cyclopentenone PG do covalently modify cellular IKK, as hypothesized (7), and they

suggest that PGA1-ABP is useful for isolating and identifying proteins that react

with cyclopentenone PG.

Accordingly, we determined if PGA1-APB reacted with TrxR, the strongest

candidate molecule for an indirect mechanism of action that leads to impairment of

p53. RKO cells and two HCT 116 cell lines contained a protein modified covalently

by PGA1-ABP and identified as TrxR immunochemically with anti-TrxR antibody

[Figure 2A, lanes 3]. The fact that HCT 116 p53+/+ and HCT 116 p53-/- cells each

contained this ∼ 56-kDA protein excludes the possibility that it is a p53-PGA1-ABP

adduct that migrates more slowly than p53 during fractionation by SDS-PAGE

[Figure 2A, lanes 3]. Incubation of cells with PGA1 alone, or aminopentyl biotin

alone, did not generate proteins with biotin epitopes inserted de novo [Figure 2A,


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lanes 2 & 4]. Thus, the biotin epitope on the 56-kDa TrxR protein and other

cellular proteins originates from their covalent reaction with PGA1-ABP, not its

hydrolysis products in cells. We exploited the fact that TrxR is a selenoprotein to

strengthen our conclusion that it forms an adduct with PGA1-ABP. We

metabolically labelled RKO and HCT 116 cells with 75Se to incorporate it into their

selenoproteins; repeated the previous experiment; and found a 75Se-labeled, 56 kDa

protein recognized by anti-TrxR antibody among the proteins sequestered on

neutravidin beads [Figure 2B].

RKO cells and HCT 116 p53+/+ cells that are haplosufficient in p53 contained

barely detectible amounts of p53 covalently modified by PGA1-APB when we

conducted experiments analogous to those described above. In corresponding

experiments where we immunoprecipated p53 with anti-p53 antibodies;

fractionated the precipitate by SDS-PAGE; and probed with neutravidin-HRP we

found no detectable biotinylated p53 adduct [data not shown]. Thus, PGA1-ABP

does not react to an appreciable extent with p53, itself, in intact cells. As expected,

the HCT 116 p53-/- cells that lack p53 did not contain any modified protein

corresponding to p53.

In addition to TrxR, we examined thioredoxin (Trx), the substrate of TrxR.

RKO cells contained a 12-kDa species labeled by PGA1-ABP and identified as Trx

with anti-Trx antibody [data not shown]. The 12-kDa species also

immunoprecipitated with anti-Trx antibody and reacted positively with

streptavidin-HRP, consistent with its annotation as a Trx:PGA1-ABP adduct.

Several representative electrophilic lipids also inhibited p53 transactivation,

p53 conformation, TrxR activity and HIF transactivation with a similar rank-order

of potency [Figure 3]. 4-Hydroxy-2-nonenal (4-HNE), a decomposition product of

linoleic or arachidonic acid hydroperoxides was the most potent inhibitor followed


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by J- and A-series cyclopentenone PG; ethacryinic acid, and 15-keto PGF2α, the

main pulmonary metabolite of PGF2α. Under our experimental conditions MDA and

PGB1 were inactive, as expected, because exogenously added MDA does not

penetrate cell membranes readily and PGB1 has an inert β-carbon.

We investigated the kinetic features of p53 inactivation by PGA1 to assess

their compatibility with inhibition of TrxR. We exposed RKO cells to 50 µM

etoposide for 6 hr to initiate genomic damage and time-dependent accumulation of

wild type p53. We added PGA1 simultaneously with etoposide at t = 0 hr or at 1, 2

and 4 hr after the addition of etoposide. PGA1 impaired p53 transcription

maximally when present throughout the entire 6 hr duration of the experiment.

When added at intervals after etoposide, PGA1 impaired p53 transactivation

proportionately to exposure time [Figure 4A]. For example, when present only for

the final two hr, t = 4 - 6 hr, PGA1 did not impair p53 transactivation or

conformation significantly (95% versus 100%).

TrxR is a selenoprotein whose cellular steady-state level depends on selenium

availability (23,24). Most tissue culture media is partially deficient in selenium

[∼ 0.1 µM]. Tumor suppressor p53 was less vulnerable to impairment by PGA2 when

cells were grown in media supplemented with 1µM inorganic Se. Half-maximal

impairment of p53 required exposure to ∼ 3-fold more PGA2 [IC50 = 60 µM PGA2] in

cells grown with supplemental selenium versus cells with no supplementation

[IC50= 20 µM PGA2] [Figure 4B].

Auranofin is chemically unrelated to cyclopentenone PG or other electrophilic

lipids, but it is an inhibitor of TrxR activity (23,25). Auranofin impaired the

conformation of p53 and transcription by p53 and HIF [Figure 4C], analogously to

the electrophilic lipids depicted in Figure 3.


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Bunz and colleagues recently established that p53 haplosufficiency is

proportional to p53-mediated apoptosis in vitro using a panel of isogenic HCT 116

cells whose p53 alleles were disrupted experimentally (20). We used these same

cell lines to compare the functional consequences of p53 inactivation by

electrophilic agents versus the functional consequences of genetic deletion of p53

alleles. Consistent with previous results (20) amethopterin induced apoptosis

proportional to p53 haplosufficiency (caspase-3 activity in HCT 116 p53+/+ > HCT

116 p53+/- > HCT 116 p53-/-) [Figure 5 bars to right]. When we incubated the HCT

116 p53+/+ cells with amethopterin plus PGA2, the PGA2 antagonized apoptosis in a

concentration-dependent manner [Figure 5 bars to left]. Apoptosis in HCT 116

p53+/+ cells treated with 6 to 20 µM PGA1 plus amethopterin corresponded

approximately to apoptosis in the HCT 116 p53+/- cells treated with amethopterin

alone. In other words, chemical impairment of p53 approximates the loss of at least

one allele of p53.


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DISCUSSION

We recently discovered that electrophilic cyclopentenone PG impair the p53

tumor suppressor by a novel mechanism that is distinct from mutation of the p53

gene or functional inactivation of p53 by oncoproteins like mdm-2. Since our

initial report (1) we have sought the precise molecular mechanism responsible for

this effect. Here we report that cyclopentenone PG act by covalently modifying and

inhibiting TrxR. This indirect mechanism can account fully for the p53 phenotype

in cells exposed to cyclopentenone PG, as well as other representative electrophilic

lipids.

To develop our mechanism of action hypothesis we integrated three

independent observations: i) PGA1 and A2 impair the conformation, transactivation

and function of p53 (1); ii) PGA1-ABP reacts covalently with TrxR and Trx [Figure 2];

and iii) TrxR-Trx coupling maintains p53 and several other several redox-sensitive

proteins and transcription factors in an active state (8-10). Collectively, these data

suggested a model with several, testable predictions. First, our model predicts that

many other chemical agents with an α,β unsaturated carbonyl and accessible,

electrophilic β carbons should impair p53 conformation and function. Second,

these chemical agents should impair p53 transcription, p53 conformation and

inhibit TrxR activity with the same rank-order of potency, if they act indirectly via

TrxR. Third, these agents should impair transactivation by other redox-sensitive

transcription factors, e.g. hypoxia-inducible factor (10,26) with the same rank-

order of potency that they impair TrxR and p53. Our results affirm each of these

predictions. Our model also predicts that PGA1 will impair p53 only as it

accumulates during the initial, early stage of the cellular response to DNA damage;

but not later, after it has assumed a transcriptionally active, wild type

conformation. By inhibiting the disulfide reductase activity of TrxR-Trx, PGA1

should prevent assembly of p53 into a mature conformation, but it should not


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convert p53 from an active to an inactive conformation. Kinetic experiments

affirmed this prediction. Lastly, the molecular mechanism of action we propose can

resolve a paradox. Namely, A-series PG antagonise p53-dependent apoptosis, but

not cell cycle arrest. These effects are fully compatible with inhibition of TrxR.

Inhibition of TrxR-Trx cycling deranges the assembly of p53 into a transcriptionally

competent form; this manifests as antagonism of p53 mediated processes, like

apoptosis. Inhibition of TrxR-Trx cycling can derange the redox status and

catalytic competence of ribonucleotide reductase; this manifests as cell cycle arrest

in G1 because ribonucleotide reductase is the rate-limiting enzyme in DNA

synthesis.

Our mechanistic framework is supported, but not necessarily proven, by our

data. Notably, our framework aligns well with yeast genetic experiments indicating

that TrxR is essential for transcription by p53 (11,12). In addition to its effects on

TrxR, we observed that PGA1-APB binds covalently to thioredoxin, the substrate of

TrxR. TrxR and Trx are vital components of a regulatory cycle and they act

coordinately to maintain p53 conformation and function. Biologically, the

reductase activity of TrxR maintains Trx in a reduced state so it is competent to

function as a sulfhydryl-disulfide isomerase (8). It is possible that covalent binding

to, and direct inactivation of Trx is also important for p53 inactivation. In other

words, electrophilic lipids inhibit Trx directly, via irreversible, covalent binding and

indirectly via their effects on TrxR.

Certain α,β unsaturated carbonyl compounds with electrophilic carbons are

known risk factors for cancer, e.g. acrolein, MDA, 4-hydroxy-2-nonenal. Their

carcinogenic mechanism is best understood in terms of their direct interaction with

DNA (27-29). Our data and model provide a new molecular basis for appreciating

their carcinogenic effects. Aside from these well-established carcinogens many

chemically complex α,β unsaturated ketones and α,β unsaturated aldehydes are


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considered safer because they have low rates of reaction with DNA and reduced

glutathione (GSH) (30). Dipple and colleagues have termed these agents "stealth

carcinogens" (31) because the unusual elements of this phenotype occur in tumors.

Our data and model provide a new molecular basis for appreciating, and re-

assessing the safety of such compounds. The cyclopentenone PG provoke an

unusual and distinctive phenotype typified by accumulation of p53 protein, in an

abnormal conformation that cannot support DNA binding and transcription.

We have tested a set of representative electrophilic chemicals that include:

agents derived from the cyclooxygenase pathway (malondialdehyde, metabolites of

prostaglandins, e.g. 15-keto-PGF2 α, and dehydration products, e.g. PGA2 or ∆12-

PGJ2) and; agents derived from the lipoxygenase (LOX) pathways (e.g. 4-HNE

generated by decomposition of hydroperoxy-octadecadienoic acids. Based on

chemical precepts we anticipate that electrophilic carbons on numerous other

compounds would confer a similar ability to inhibit TrxR. Cells can encounter

electrophilic chemicals via environmental exposure, dietary exposure, or normal

metabolic processes. We draw special attention to inflammation as one of these

processes. As part of their normal host-defence function inflammation inevitably

exposes proximal epithelial and stromal cells to substances with mutagenic

potential in vitro (18). Individual eicosanoids with α,β unsaturated ketone

substituents like PGA1 or PGA2 may not occur in µM concentrations at a site of

inflammation. However, inflammatory exudate contains a blend of electrophiles

typified by α,β unsaturated aldehydes derived from eicosanoid biosynthesis or lipid

peroxidation (4-HNE); α,β unsaturated ketones derived from eicosanoid metabolism

(15-keto-PGF2α, 15-keto-PGE2, 5-, 12-, and 15-oxo-ETE); and α,β unsaturated

ketones derived from albumin dehydrating PGE2 to PGA2 and, PGD2 to ∆12- PGJ2

and 15-deoxy-∆12-PGJ2 (32-35). Thus, inflammation likely exposes cells to a

mixture of electrophiles in quantities sufficient to impair TrxR. Note that 4-HNE, a

common product from lipid peroxidation potently inactivates the selenoprotein


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TrxR. The level of endogenous 4-HNE in tissues ranges from 0.1-3.0 µM and

increases to ∼ 10 µM in conditions of oxidative stress (36).

Although purely speculative at this point, our mechanistic framework aligns

well with lipid mediator class switching hypothesis recently proposed by Serhan

and colleagues (37). It has been suggested that J-series cyclopentenone PGs are

present during resolution phases of inflammation and attenuate the inflammatory

response (33). Electrophilic lipoxins would also be present and perhaps act

through a common mechanism involving TrxR inhibition.

The same electrophilic cyclopentenone PG and lipid aldehydes that impair

conformation and repress transcription by p53 can also repress transcription and

antagonize the effects of NF-κB (7,38-40). This may also occur via an irreversible,

covalent modification of proteins. For instance, cyclopentenone PG of the A- and J-

series, or 4-HNE can form adducts with IKK (7,40) or NF-κB (39), in situ.

Investigators have inferred that repression of NF-κB in cells derives from this

mechanism. However, they have demonstrated adduct formation only with isolated

NFκB and IκB proteins in situ, not in cells (7,38-40). With the PGA1-APB analog,

we have now demonstrated direct alkylation of IKK in cells. Therefore, electrophilic

lipids may resolve inflammation through direct and indirect mechanisms to

attenuate cellular signalling pathways. The fact that several of these types of

chemicals can impair the p53 tumor suppressor and that the functional

consequences of its impairment rival the loss of a p53 allele may have broad

implications for cancer progression.

The fact that electrophilic lipids act indirectly, via a selenoprotein may have

implications for cancer prevention. For instance, our model predicts that selenium

supplementation of culture media should maintain TrxR activity, and thereby spare

p53 from inactivation by electrophilic lipids. We affirmed this prediction. Clark and


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colleagues have reported that supplementation of dietary selenium lowers the risk

of prostate, colon and certain other cancers (41). The molecular basis for this

phenomenon, especially the role of selenoproteins, is uncertain. Our observation

that selenium spares an important tumor suppressor, p53, provides an explicit

mechanistic framework to understand how dietary selenium confers protection

against cancer. Approximately 50-60% of cancer patients have tumors harboring

mutations or deletions of p53. These patients typically have a poorer prognosis

than patients with tumors harboring wild type p53 (20,42). Likewise, not all

mutations are equally pernicious - certain classes may be worse than others. In

particular, mutations associated with an altered conformation of p53 protein

correlate with significantly shorter survival and poorer prognosis in patients with

colorectal cancer (43,44). We draw attention to reports about colon, breast and

neuroblastoma tumors with a wild type p53 gene that paradoxically express a

dysfunctional p53 protein with a mutant conformation (14,15,17,45-48). The

latency model of p53 function (49), in its current form, does not account for the

peculiar p53 phenotype described in these reports. We can recapitulate this

unusual and unexplained p53 phenotype in cells by impairing their TrxR-Trx

activity with electrophilic lipids, suggesting a potential role for TrxR-Trx, or related

disulfide reductases, in its emergence.

In summary, various electrophilic lipids have the capacity to repress

transactivation by several redox-responsive transcription factors by covalently

modifying regulatory proteins in the pathways - IKK in the case of the NFκB

pathways and TrxR-Trx in the case of p53 and HIF.


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ACKOWLEDGEMENTS:

Dr. B. Vogelstein genereously provided HCT116 cell lines and p53 expression

construct, pC53-SN3. Dr. G. L. Semanski generously provided the HIF-1 luciferase

reporter construct. We thank the Huntsman Cancer Foundation and R01 AI26730

for support of this work. F.A.F. holds the Dee Glenn and Ida W. Smith Chair for

Cancer Research.


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Figure 1. Structure of PGA1 and PGA1-ABP. The electrophilic β-carbon is indicated

with a star.

Figure 2. A) Neutravidin sequestration of PGA1-ABP-labeled proteins in RKO and

HCT 116 cells. The upper panel shows the proteins identified with anti-IKKα, anti-

TrxR and anti-p53 antibodies formed de novo in cells incubated with PGA1-ABP.

The lower panel shows the immunochemical identification of TrxR and p53 in the

whole cell lysates. IKKα and TrxR occurs as a PGA1-ABP conjugate in all three cell

lines and a minimal amount of p53 occurs as a PGA1-ABP conjugate in the HCT116

p53+/+ cells. Each cell line was incubated with lane 1) vehicle control (DMSO), lane

2) 60 µM PGA1, lane 3) 60 µM PGA1-ABP, and lane 4) 180 µM biotin pentylamine

linker. B) The identification of selenoprotein TrxR was further supported by

metabolically labelling RKO and HCT 116 cells with 75Se, and repeating the

experiment using PGA1-ABP and anti-TrxR antibodies: lane 5) RKO cells incubated

with PGA1, lane 6) RKO cells incubated with PGA1-ABP, lane 7) HCT 116 p53-/-

cells incubated with PGA1-ABP, and lane 8) HCT 116 p53+/+ cells incubated with

PGA1-ABP.

Figure 3. Electrophilic lipids attenuate A) p53 activity as measured by luciferase

reporter constructs in RKO cells in a dose-dependent manner. Electrophiles used

include: 4-HNE (filled square), ∆12-PGJ2 (filled up-triangle), 15-dideoxy-∆12, ∆14-

PGJ2 (filled down-triangle), PGA2 (filled diamond), 15-keto-PGF2α (filled circle), 15-

keto-PGE2 (open down-triangle), PGB1 (open up-triangle), and malondiadehyde (open

circle). B) These lipids also result in a change in conformation of p53 to the ‘mutant’

conformation. C) The lipids attenuate HIF-1 transcriptional with a similar rank

order of potency to p53. D) Electrophilic lipids attenuate TrxR activity as measured

by Trx-dependent NADPH oxidase activity in RKO cell lysates.

Figure 4. A) Selenium supplementation of RKO cells spares p53 from impairment

by PGA2. Cells were either incubated in media supplemented with 1 µM sodium


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selenite (filled squares) for 48 hr or maintained in standard media (filled circles) and

then stimulated with etoposide in the presence of PGA2. B) PGA2 attenuates

transcriptionally active p53 in a time-dependent manner. Luciferase activity was

monitored (right panel) from a p53_luc reporter construct in RKO cells that were

stimulated with 50 µM etoposide (dark bars, left panel) and 60 µM PGA2 was added

after 0, 1, 2 or 4 hr (light gray bars, left panel). PGA2 must be present during the

initial 1-2 hrs of the p53 response to attenuate p53 transcription. Delayed addition

of PGA2 , 4 hrs after the initiation of the p53 has no effect on p53 transcription. C)

Incubation of RKO cells with 10 µM auranofin for 6 hr deranges the conformation

(left panel) of p53 as determined by immunoprecipitations with conformation

sensitive antibodies. The content of p53 recognized by Pab240 (‘mutant’

conformation) increased; and the content of p53 recognized by Pab1620 (wild type

conformation) decreased. Auranofin also attenuated p53 and HIF-1 transcription

in RKO cells (right panel) as measured by luciferase reporter assays.

Figure 5. Electrophilic antagonism of apoptosis corresponds to the loss of a p53

allele. Caspase acitivity was measured as a determinant of apoptosis in HCT 116

p53+/+, p53+/-, and p53-/- cells incubated with 20 µM amethopterin for 48 hr (right

panel). PGA2 functionally impaired apoptosis in p53+/+ cells at a level comparable

to the p53+/-, haploinsufficient, cells (left panel).


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Figure 1:

SCOO

OH

NH

NH

CO

NH NH

O

O

OH

COOH

PGA1

PGA1-amidopentylbiotin

SCOO

OH

NH

NH

CO

NH NH

O

O

OH

COOH

PGA1

PGA1-amidopentylbiotin


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Figure 2:

NeutrAvidin Pull-down

1 2 4321432143

∝ -TrxR1

∝ -p53

TrxR

p53

RKO HCT 116p53+/+

HCT 116p53-/-

Whole Cell Lysate

∝ -IKKα

A

5 6 87

Whole Cell Lysate


5 6 87

TrxR

TrxR

∝ -TrxR1

[75Se] metabolic

label

B


1 2 4321432143

∝ -TrxR1

∝ -p53

TrxR

p53p53

RKO HCT 116p53+/+

HCT 116p53-/-

Whole Cell LysateWhole Cell Lysate

∝ -IKKα

A

5 6 87

Whole Cell Lysate


5 6 87

TrxR

TrxR

∝ -TrxR1

[75Se] metabolic

label

B


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Figure 3:

C

D

0 10 1000

20

40

60

80

100

120

Nor

mal

ized

HIF

-1 R

epor

ter A

ctiv

ity(6

hr C

obal

t Chl

orid

e, 1

00 µ

M)

Concentration (µM)

0 1000

20

40

60

80

100

Concentration (µM)

Nor

mal

ized

NAD

PHO

xida

se A

ctiv

ity

C

D

0 10 1000

20

40

60

80

100

120

Nor

mal

ized

HIF

-1 R

epor

ter A

ctiv

ity(6

hr C

obal

t Chl

orid

e, 1

00 µ

M)

Concentration (µM)

0 1000

20

40

60

80

100

Concentration (µM)

Nor

mal

ized

NAD

PHO

xida

se A

ctiv

ity

∆12

-PG

J 2

PGA 2

PGA 1

DMSO

blan

k

15-d

ideo

xy∆∆

PGJ 2

15-k

eto-

PGF 2

α

4-hy

drox

ynon

enal

Mal

ondi

alde

hyde

A

B

IP: 240

0 10 1000

20

40

60

80

100

Concentration

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(6 h

r Eto

posi

de, 5

0 µM

)

∆12

-PG

J 2

PGA 2

PGA 1

DMSO

blan

k

15-d

ideo

xy∆∆

PGJ 2

15-k

eto-

PGF 2

α

4-hy

drox

ynon

enal

Mal

ondi

alde

hyde

A

B

IP: 240

∆12

-PG

J 2

PGA 2

PGA 1

DMSO

blan

k

15-d

ideo

xy∆∆

PGJ 2

15-k

eto-

PGF 2

α

4-hy

drox

ynon

enal

Mal

ondi

alde

hyde

A

B

IP: 240

0 10 1000

20

40

60

80

100

Concentration

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(6 h

r Eto

posi

de, 5

0 µM

)


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Figure 4:

Aura

nofin

EtO

H

IP: 240

IP: 1620

Lysate

0

20

40

60

80

100

120

CoCl2+Auranofin

EtOH CoCl2Etop Etop+Auranofin

EtOH

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(% M

axim

um)

0

20

40

60

80

100

120

Nor

mal

ized

HIF

-1 R

epor

ter A

citiv

ity(%

Max

imum

)

B

C

20 40 60 80 100

Normalized p53 Reporter Activity(% Maximum)

PG

A 2 E

top

Trea

tmen

t

Treatment Time (hr)0 1 2 3 4 5 6

A

0 10 1000

20

40

60

80

100

PGA2 Concentration (µM)

B

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(6 h

r Eto

posi

de, 5

0 µM

)Au

rano

fin

EtO

H

IP: 240

IP: 1620

Lysate

0

20

40

60

80

100

120

CoCl2+Auranofin


EtOH

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(% M

axim

um)

0

20

40

60

80

100

120

Nor

mal

ized

HIF

-1 R

epor

ter A

citiv

ity(%

Max

imum

)

Aura

nofin

EtO

H

IP: 240

IP: 1620

Lysate

Aura

nofin

EtO

H

IP: 240

IP: 1620

Lysate

0

20

40

60

80

100

120

CoCl2+Auranofin


EtOH

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(% M

axim

um)

0

20

40

60

80

100

120

Nor

mal

ized

HIF

-1 R

epor

ter A

citiv

ity(%

Max

imum

)

B

C

20 40 60 80 100

Normalized p53 Reporter Activity(% Maximum)

PG

A 2 E

top

Trea

tmen

t

Treatment Time (hr)0 1 2 3 4 5 6

A

0 10 1000

20

40

60

80

100


B

Nor

mal

ized

p53

Rep

orte

r Act

ivity

(6 h

r Eto

posi

de, 5

0 µM

)


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Figure 5:

0 6 200

50

100

150

200

250

300

350

400

450HCT116 p53+/+

Amethopterin DMSO

Cap

ase

Activ

ity (p

mol

e/m

in/m

g pr

otei

n)(D

EVD

sub

stra

te)


0

50

100

150

200

250

300

350

400

450

+/++/--/-

HCT116 p53 status


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FitzpatrickPhilip J. Moos, Kornelia Edes, Pamela Cassidy, Edmund Massuda and Frank A.

reductasetranscription factors p53 and HIF by impairing the selenoprotein thioredoxin

Electrophilic prostaglandins and lipid aldehydes repress redox sensitive

published online November 6, 2002J. Biol. Chem.

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