From the Huntsman Cancer Institute and Departments of
§ Oncological Science and ¶ Medicinal Chemistry,
University of Utah, Salt Lake City, Utah 84112-5550
Received for publication, October 30, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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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.
Cyclopentenone prostaglandins
(PG),1 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
Here 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 Materials--
We used Dulbecco's modified Eagle's medium and
McCoy's 5A medium and supplements (Invitrogen); 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 Biosciences); 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 Cell Culture--
We maintained HCT 116 p53+/+,
p53+/ Isolation of Proteins Labeled by PGA1-ABP--
For
Neutravidin sequestration, we treated RKO and HCT 116 cells with
60 µM PGA1-APB for 4 h, or as described
in 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× CompleteTM 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 for 5 min to
isolate the neutravidin-biotin complexes. We washed the beads 5 times
with 1 ml of PBS/0.4% Tween 20. We fractionated samples by SDS-PAGE
and detected the biotinylated proteins with streptavidin-horseradish
peroxidase. We also reprobed the membrane with antibodies directed
against Trx, TrxR, p53, p50/p105 of NF 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 twice with 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 pCMV Thioredoxin Reductase Activity--
We lysed RKO cells in 50 mM Tris, pH 7.4/0.1 M NaCl/2 mM EDTA/1%
SDS/1% deoxycholate/1 mM NaF/1 mM sodium
orthovanadate/1× CompleteTM 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 Escherichia coli thioredoxin, 120 nM NADPH and 5.5 mM EDTA. We monitored the
oxidation of NADPH at 340 nm for 0-5 min at 25 °C or longer to
ensure we measured the linear portion of the progress curve.
Caspase-3 Activity--
We treated 106 HCT116 cells
for 48 h 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.
To determine whether electrophilic PGs react with TrxR or other
proteins we used PGA1 amidopentyl biotin
(PGA1-APB). PGA1-APB retains the
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-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
PGA2 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 NF
B 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.
-carbons (15-keto-PG, 4-hydroxy-2-nonenal, and
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
-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).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase
expression vector (pCMV
; Clontech).
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, UK) in Dulbecco's modified Eagle's medium 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% (v/v) fetal
bovine serum. In certain experiments, cells were metabolically
labeled with 100 µCi of 75Se, obtained from the
University of Missouri Research Reactor, for 48 h to ensure that
selenoproteins were labeled to steady state.
B, and IKK
to determine
whether they adhered to the Neutravidin beads.
in 3 µl of FuGENE 6. After 48 h, we incubated cells for 6 h with vehicle (Me2SO) and 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
-galactosidase activity in the
supernatant fractions.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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,
-unsaturated ketone substituent and the electrophilic
-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 (Ref. 6) (Fig. 1).
View larger version (10K):
[in a new window]
Fig. 1.
Structure of PGA1 and
PGA1-ABP. The electrophilic -carbon is
indicated with a star.
To calibrate the utility of PGA1-ABP, we examined its
interaction with IKK, 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 h;
sequestered any proteins with biotin epitopes on Neutravidin beads;
fractionated these same biotinylated proteins by SDS-PAGE; and
identified them immunochemically. Cells contained ~15 proteins
labeled de novo with PGA1-APB under these conditions; two of these proteins were IKK
(Fig.
2) and the p50/p105 subunit of NF
B
(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.
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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 (Fig. 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 (Fig. 2A, lanes
3). Incubation of cells with PGA1 alone, or
aminopentyl biotin alone, did not generate proteins with biotin
epitopes inserted de novo (Fig. 2A, lanes
2 and 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 labeled 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 (Fig. 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 in which we
immunoprecipated p53 with anti-p53 antibodies; fractionated the
precipitate by SDS-PAGE; and probed with neutravidin-horseradish
peroxidase, 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 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-horseradish peroxidase, 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 (Fig.
3). 4-Hydroxy-2-nonenal (4-HNE), a
decomposition product of linoleic or arachidonic acid hydroperoxides
was the most potent inhibitor followed by J- and A-series
cyclopentenone PG; ethacryinic acid, and 15-keto PGF2, the main pulmonary metabolite of PGF2
. Under our
experimental conditions malodialdehyde and PGB1 were
inactive, as expected, because exogenously added malodialdehyde does
not penetrate cell membranes readily and PGB1 has an inert
-carbon.
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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 h to
initiate genomic damage and time-dependent accumulation of
wild-type p53. We added PGA1 simultaneously with etoposide
at t = 0 h or at 1, 2 and 4 h after the addition of
etoposide. PGA1 impaired p53 transcription maximally when
present throughout the entire 6-h duration of the experiment. When
added at intervals after etoposide, PGA1 impaired p53
transactivation proportionately to exposure time (Fig.
4A). For example, when present
only for the final two h, t = 4 - 6 h, PGA1 did
not impair p53 transactivation or conformation significantly (95%
versus 100%).
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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 1 µM 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) (Fig. 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 (Fig. 4C), analogous to the electrophilic lipids depicted in Fig. 3.
Bunz and colleagues recently established that p53 haplosufficiency is
proportional to p53-mediated apoptosis in vitro using a
panel of isogenic HCT 116 cells in which 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
/
) (Fig. 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
(Fig. 5, bars to left). Apoptosis in HCT 116 p53+/+ cells treated with 6-20 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 |
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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 PGA2
impair the conformation, transactivation, and function of p53 (1); (ii) PGA1-ABP reacts covalently with TrxR and Trx (Fig. 2); and
(iii) TrxR-Trx coupling maintains p53 and several other redox-sensitive proteins and transcription factors in an active state (8-10). Collectively, these data suggest a model with several testable predictions. First, our model predicts that many other chemical agents
with an ,
-unsaturated carbonyl and accessible, electrophilic
-carbons should impair p53 conformation and function. Second, these
chemical agents should impair p53 transcription and 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 with which 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 convert p53 from an active to an
inactive conformation. Kinetic experiments affirmed this prediction.
Last, the molecular mechanism of action we propose can resolve a
paradox. Namely, A-series PG antagonize 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, such as 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 that 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 ,
-unsaturated carbonyl compounds with electrophilic
carbons are known risk factors for cancer, e.g. acrolein,
malodialdehyde, and 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
,
-unsaturated
ketones and
,
-unsaturated aldehydes are considered safer because
they have low rates of reaction with DNA and reduced glutathione (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 reassessing 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 , and dehydration products, e.g.
PGA2 or
12-PGJ2) and agents derived from the
lipoxygenase pathways (e.g. 4-HNE generated by decomposition of hydroperoxyoctadecadienoic 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-defense function, inflammation inevitably exposes proximal
epithelial and stromal cells to substances with mutagenic potential
in vitro (18). Individual eicosanoids with
,
-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
,
-unsaturated aldehydes derived from
eicosanoid biosynthesis or lipid peroxidation (4-HNE);
,
-unsaturated ketones derived from eicosanoid metabolism
(15-keto-PGF2
, 15-keto-PGE2, 5-, 12-, and
15-oxo-ETE); and
,
-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 TrxR. The level of endogenous 4-HNE in tissues ranges
from 0.1 to 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 the lipid mediator class switching hypothesis recently proposed by Serhan and colleagues (37). It has been suggested that J-series cyclopentenone PG 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 NFB (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
B (39) in situ.
Investigators have inferred that repression of NF
B in cells derives
from this mechanism. However, they have demonstrated adduct formation
only with isolated NF
B and I
B 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 signaling 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 medium should maintain TrxR activity and thereby spare p53 from inactivation by electrophilic lipids. We affirmed this prediction. Clark and 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 NFB pathways and TrxR-Trx in the case of p53 and HIF.
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ACKNOWLEDGEMENTS |
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Dr. B. Vogelstein generously provided HCT116 cell lines and p53 expression construct, pC53-SN3. Dr. G. L. Semanski generously provided the HIF-1 luciferase reporter construct.
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FOOTNOTES |
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* This work was supported by the Huntsman Cancer Foundation and United States Public Health Services Grant R01 AI26730.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: Guilford Pharmaceuticals, Baltimore, Maryland 21224.
** Dee Glenn and Ida W. Smith Chair for Cancer Research. To whom correspondence should be addressed: Huntsman Cancer Institute, 2000 Circle of Hope, University of Utah, Salt Lake City, UT 84112-5550; Tel.: 801-581-6204; Fax: 801-585-0101; E-mail: frank.fitzpatrick@hci.utah.edu.
Published, JBC Papers in Press, November 6, 2002, DOI 10.1074/jbc.M211134200
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ABBREVIATIONS |
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The abbreviations used are:
PG, prostaglandins;
4-HNE, 4-hydroxy-2-nonenal;
Trx, thioredoxin;
TrxR, thioredoxin
reductase;
IKK, I-kappa- kinase;
APB, amidopentyl biotin;
PBS, phosphate-buffered saline.
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