From the Departments of Molecular Biology and
§ Virology, Lerner Research Institute, Cleveland Clinic
Foundation, Cleveland, Ohio 44195 and ¶ Quark Biotech Inc.,
Cleveland, Ohio 44195
Received for publication, January 13, 2003, and in revised form, February 19, 2003
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ABSTRACT |
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Pifithrin Based on the analysis of p53-dependent effects caused
by ionizing radiation and chemotherapeutic drugs in mice, p53-mediated apoptosis was defined as a determinant of organism sensitivity to
systemic genotoxic stress associated with cancer treatment (1).
Temporary reversible pharmacological suppression of p53 was suggested
as an approach to reduce cancer treatment side effects. This hypothesis
was supported by isolation of a small molecule inhibitor of p53,
pifithrin PFT Reagents--
1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)- benzothiazolyl)ethanone
hydrobromide, molecular weight 367 (known as PFT Cell Lines and Animals--
Mouse fibroblast cell line ConA
carries the wild type p53 gene and the bacterial lacZ
reporter gene under the control of a p53-responsive promoter (2). Two
isogenic human colon cancer cell lines HCT116 p53 (wt-p53) and its
p53-deficient derivative, developed from the parental cell line by
targeted homologous recombination (8), were provided by I. Roninson
(University of Illinois at Chicago). HeLa cells and human
prostate cancer cell line PC3 (p53-deficient) were purchased from ATCC.
Short term cultures of primary thymocytes were prepared from the thymus
of 4-week-old C57BL/6 mice (wild type and p53-deficient), which were
purchased from Jackson Laboratory (Bar Harbor, ME).
Cell Viability Assay--
At the end of cell treatments, the
number of attached cells was estimated by staining with 0.25% crystal
violet in 50% methanol, followed by elution of the dye with 1% SDS.
Optical density (530 nm) reflecting the number of stained cells was
determined with a Bio-Tek EL311 microplate reader. Cell viability in
suspension of short term culture of primary thymocytes was determined
by their staining with 0.1% of methyl blue and microscopic counting of
blue (dead) cells.
Gel Shift Assay--
Gel shift assay was performed as described
earlier (9). Nuclear and total cellular extracts were prepared from
untreated or HS-treated (30 min, 42 °C) ConA cells with and without
PFT CAT Assay--
CAT assay was done as previously described (11).
ConA and HeLa cells were transfected with plasmids, containing CAT gene under the control of a minimal thymidine kinase promoter alone (Promega) or combined with HSF1-binding or GRE-binding sequences from
HSP70 (12) and LTR MMTV (13) promoters, respectively. Cells were
treated with HS (42 °C, 30 min) or Dex (0.1 µM, 1 h) with and without PFT Western Blot Analysis--
Western blot analysis was done as
described previously (12). Wild type and p53-deficient HCT116 cells
were incubated 15 min at 43 °C in the presence or in the
absence of PFT PFT
Activation of HSF1-containing transcription complex was determined by
gel-shift assay using 32P-labeled heat shock
element-derived oligonucleotides and total or nuclear extracts
of ConA cells growing under normal conditions or subjected to HS
(42 °C, 20 min) in the presence and in the absence of 15 µM of PFT
The results of gel-shift experiments were confirmed in a functional
transcription assay using HSF1-responsive construct with CAT reporter.
PFT
To determine whether the effect of PFT PFT
To analyze whether PFT
Consistently, presence of PFT
To test whether biochemical indications of inhibition of GR
activity by PFT
PFT PFT
In our study of PFT
Inhibition of transactivation ability of p53, HSF1, and GR by PFT
The dramatic differences between the effects of PFT (PFT
) is a chemical compound
isolated for its ability to suppress p53-mediated transactivation. It
can protect cells from p53-mediated apoptosis induced by various
stimuli and reduce sensitivity of mice to gamma radiation.
Identification of molecular targets of PFT
is likely to provide new
insights into mechanisms of regulation of p53 pathway and is important for predicting potential risks associated with administration of
PFT
-like p53 inhibitors in vivo. We found that PFT
,
in addition to p53, can suppress heat shock and glucocorticoid
receptor signaling but has no effect on nuclear factor-
B
signaling. PFT
reduces activation of heat shock transcription factor
(HSF1) and increases cell sensitivity to heat. Moreover, it reduces
activation of glucocorticoid receptor and rescues mouse thymocytes
in vitro and in vivo from apoptotic death after
dexamethasone treatment. PFT
affected both signaling pathways in
a p53-independent manner. These observations suggest that PFT
targets some unknown factor that is common for three major signal
transduction pathways.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(PFT
)1 that
was capable of rescuing mice from lethal genotoxic stress caused by
gamma radiation (2). Furthermore, inhibition of p53 was suggested as a
therapeutic approach to treatment of other pathological conditions
associated with p53 activation (3), some of which have already been
experimentally confirmed. Thus, PFT
was shown to protect neurons
from death induced by DNA-damaging agents, hypoxia and dopamine (4, 5):
it had therapeutic effects in animal models of Parkinson disease (6)
and acute renal failure (7). In all these works, biological effects of PFT
were attributed to its anti-p53 function, although not in all of
them has this conclusion been confirmed by genetic approaches. Accurate
interpretation of biological effects of PFT
requires identification
of its molecular target(s) and determination of molecular mechanisms of
its activity.
was isolated by screening of chemical library in a cell-based
readout system for its ability to reduce p53-dependent transactivation (2). This biological effect could be reached by
affecting p53 pathway at numerous points and therefore PFT
could act
by targeting one of numerous factors cooperating with p53 function.
Biological effects of PFT
on p53 pathway suggested that it acted by
interfering with nuclear accumulation of p53 (2). Many transcription
factors involved in other signal transduction pathways have the same
principles of regulation as p53: after activation in cytoplasm they are
translocated to the nucleus, followed by modulation of transcription of
the target genes. We were, therefore, interested to test whether PFT
would have an effect on other signal transduction pathways besides p53.
We found that, in fact, PFT
can also interfere with heat shock (HS)
and glucocorticoid receptor (GR) signaling but shows no effect on the
activity of NF-
B. This finding indicates that PFT
is not solely
specific to p53 and presumably targets some unknown cellular component
that is common for three major signal transduction pathways.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
), was
provided by Chembridge Corporation (San Diego, CA) and stored as 10 mM Me2SO frozen solution at
70 °C.
TNF, cycloheximide (CHI) and dexamethasone (Dex) were purchased from
Sigma and used at a concentration of 1.5 ng/ml, 1 µg/ml, and 0.5-2
µM, respectively.
(10 µM), or HeLa cells, untreated and treated with
TNF (1.5 ng/ml) in the presence or in the absence of 15 µM of PFT
or Dex (0.05-0.1 µM) for
4 h. Labeled double-stranded oligonucleotides, corresponding to
the sequences of the HSF1-binding site in HSP70 promoter (5'-TCG AGC
GGC TGG AAT ATT CCC GAC CTG GCA GCC-3'), NF-
B-binding region of the
mouse B cell light chain enhancer (9) (5'-AGT TGA GGG GAC TTT CCC AGG
C-3') and glucocorticoid-responsive element (GRE) in LTR MMTV (5'-GATCC
ACCTT ATTTA CATAA GCA-3') (10) were used as the probes.
(15 µM).
(15 µM) and total cell lysates were
prepared 3 and 6 h later. HSP70 was detected using goat polyclonal
antibodies K-20 (Santa Cruz Biotechnology).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and Heat Shock Response--
HS induces expression of a
large family of heat shock proteins (HSP70, HSP90, HSP43, HSP27, etc.),
many of which function as either molecular chaperones or proteases that
assist the cell in recovery either by repairing damaged proteins
(protein refolding) or by degrading them (14). Hsp gene
promoters contain heat shock elements responsible for binding with HSFs
mediating HS-inducible transcription, among which HSF1 seems to play a
major role in Hsp gene regulation (15). Under normal
conditions, HSF1 exists as an inactive monomer bound to multichaperone
complexes (HSP90, HSP70, and others) (16) but is readily activated
after HS by forming active trimers that are translocated into the
nucleus where they bind heat HS-responsive elements in cellular DNA and stimulate HS genes transcription (14). Thus, there are obvious similarities in regulation of HSF1 and p53 response pathways that justified testing effects of PFT
on HS signaling.
. The intensity of HSF1-specific band in the
lysates from HS-treated cells was substantially decreased if the cells
were incubated with PFT
during treatment (Fig.
1a).
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Fig. 1.
PFT inhibits HS
response. a, PFT
inhibits DNA binding activity of
HSF1. Results of gel-shift assay using nuclear and total cellular
extracts from either untreated or HS-treated (42 °C, 30 min) ConA
cells with and without PFT
(10 µM) are shown. Labeled
double-stranded oligonucleotides, corresponding to the sequences of the
HSF1-binding site in HSP70 promoter, were used. Untreated cells are
marked as "u/t." b, PFT
suppresses HSF1-mediated transactivation of reporter construct. ConA
cells were transfected with the plasmids, containing CAT gene under
minimal thymidine kinase promoter alone (marked "min
pr") or combined with the HSF1-binding sequence from HSP70
promoter (marked "HS pr"). 24 h after transfection
cells were incubated 30 min at 42 °C, with or without PFT
(15 µM) followed by preparation of lysates and estimation of
CAT activity. c, PFT
increases cell sensitivity to HS in
a p53-independent manner. Wild type p53 and p53-deficient HCT116
cells preincubated with the indicated concentrations of PFT
were
subjected to HS (45 °C, 25 min). Cell numbers were estimated 48 h after treatment using methylene blue assay. d, PFT
(15 µM) reduces induction of HSP70 by HS in a p53-independent
manner. Wild type p53 and p53-deficient HCT116 cells were incubated 15 min at 43 °C, and the amounts of HSP70 in cell lysates prepared
3 h after HS were estimated by western immunoblotting. A similar
effect was observed in mouse NMuMG cells shown in the lower
panel (lysed 6 h after HS). Western blots with HCT116
proteins were reprobed with anti-actin antibody (loading control); a
nonspecific band is shown as a loading control (marked
"LC") for NMuMG membrane.
(15 µM) caused a 2-fold reduction in CAT activity in ConA cells under conditions of HS (Fig. 1b), suggesting
that it might have similar effect on the expression of endogenous
Hsp genes. In fact, application of PFT
was accompanied by
an increased susceptibility of ConA cells to heat shock determined by
colony assay (data not shown) and reduction in accumulation of HSP70 (Fig. 1d).
on cell sensitivity to HS is
p53-dependent or p53-independent, we compared the effect of
the compound on HS sensitivity of two isogenic variants of human colon
cancer cell line HCT116 differing in their p53 status (8). Presence of
PFT
during HS treatment (45 °C, 30 min) significantly increased
HS-induced cytotoxicity in both p53-wt and p53-deficient cell lines in
a dose-dependent manner; similar doses of PFT
had no
toxic effect on either cell line under normal growth conditions (Fig.
1c). Consistently, PFT
caused a reduction in HS-induced accumulation of HSP70 protein in both p53 wild type and p53-deficient HCT116 cells (Fig. 1d). In addition to ConA and HCT116,
similar results were obtained with p53-deficient human prostate cancer cell line PC3 (data not shown). These observations indicate that PFT
has a p53-independent mechanism of activity directed against HSF1-mediated HS response.
and GR Signaling--
Glucocorticoid hormones are involved
in regulation of many important functions in the organism, including
development and function of the immune system. Signaling is mediated by
interaction of glucocorticoids with their receptor (GR),
ligand-dependent transcription factor, that is, as p53 and
HSF, regulated at the level of nuclear transport (17, 18). In the
absence of ligands, GR resides in the cytoplasm in a monomeric form
bound to cytoplasmic chaperones, such as HSP70 and HSP90. Binding of
the ligand typically results in a conformational change in GR,
dimerization and translocation to the nucleus, where GR homodimer binds
to a DNA motif termed a GRE and transactivates
glucocorticoid-responsive genes. In thymocytes, this results in
activation of proapoptotic genes and subsequent death that is
consistent with anti-inflammatory role of glucocorticoids (19).
has an effect on GR signaling we used the
same strategy as described above for HS signaling. GR activation was
tested in HeLa cells treated for 4 h with a range of
concentrations of synthetic glucocorticoid Dex using gel-shift assay
with oligonucleotide specific for GRE. As shown in Fig.
2a, the Dex-induced DNA
binding activity of GR was significantly inhibited by 15 µM PFT
.
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Fig. 2.
PFT inhibits
glucocorticoid-mediated signaling. a, PFT
inhibits
DNA binding activity of GR. Results of gel-shift assay using nuclear
cellular extracts from untreated cells or cells treated 4 h with
the indicated concentrations of Dex in the absence and in the presence
of PFT
(15 µM) are shown. Untreated cells are marked
as "u/t." b, PFT
suppresses
transactivation of GR-responsive reporter construct. HeLa cells were
transfected with the plasmids, containing CAT gene under the control of
minimal promoter alone ("min pr") or combined with
GRE-binding sequence from U3 region of LTR of mouse mammary tumor virus
("GR pr"). 24 h post-transfection cells were
treated 1 h by Dex (0.1 µM) with or without 15 µM of PFT
followed by estimation of CAT activity in
cell lysates. c, PFT
suppresses Dex-induced apoptosis in
primary thymocytes from wild type p53 and p53-deficient mice.
Cells were incubated 20 h with or without Dex (concentrations
indicated) and/or PFT
(10 µM) and then stained by
0.4% of trypan blue to estimate the proportion of live cells.
d, PFT
protects thymus from Dex in vivo. Dex
(4.5 mg/kg) was injected subcutaneously in C57BL6 mice, followed by
three intraperitoneal injections of PFT
at 0, 2, and 6 h (3.6 mg/kg each) after Dex. Mice were sacrificed 24 h after treatment,
and their thymuses were weighed. Numbers indicate relative
thymus weight calculated as mg/gram of body weight.
reduced CAT activity in the lysates of
ConA and HeLa cell transfected with the glucocorticoid-responsive construct with CAT reporter and treated with Dex (Fig.
2b).
reflect alterations of physiological function of GR,
we analyzed cell response to glucocorticoid in the presence and in the
absence of PFT
in vitro and in vivo. Primary
thymocytes are known to respond to glucocorticoid treatment by rapid
p53-independent apoptosis (20). We tested the effect of PFT
on
apoptosis induced in short term cultures of thymocytes by Dex
treatment. To distinguish between p53-dependent and
-independent effects, we compared thymocytes from p53 wild type and
p53-deficient mice. As it is shown in Fig. 2c, PFT
protected both p53 wild type and (to a lesser extent) p53-deficient
thymocytes from Dex-induced death, indicating that the protective
effect of PFT
against glucocorticoid is p53-independent (Fig.
2c).
also had a prominent protective effect in vivo,
inhibiting Dex-induced degeneration of the thymus. Subcutaneous
injection of Dex (4.5 mg/kg) resulted in almost 2-fold decrease in the
size of mouse thymus as early as 24 h after hormone
administration. This effect was almost completely reverted by PFT
that was intraperitoneally injected three times, 0, 2 and 6 h
(each dose was 3.6 µg/kg) after Dex (Fig. 2d).
Has No Effect on the Activity of NF-
B--
Transcription
factor NF-
B is a key component of a major anti-apoptotic signal
transduction pathway induced by a variety of physiological stimuli and
stresses. It plays an important role in regulating inflammation by
determining cell response to TNF (21) and mediating activation of
numerous inflammatory cytokines. It also determines anti-apoptotic
activity of AKT signaling, a major survival pathway that connects cell
viability with physiological conditions (i.e. availability
of growth factors) (22). It is also regulated at the level of nuclear
transport; however, the exact mechanism of this regulation is different
from p53, HSF1, or GR. Under normal conditions, it resides in the
cytoplasm as an inactive complex bound to inhibitory protein factor
I-
B, while p53, GR, and HSF1 are coupled with HS chaperone
complexes. Activation of NF-
B is followed by phosphorylation and
degradation of I-
B that leads to a release of NF-
B and its
translocation to the nucleus where it binds to specific binding sites
causing transcriptional activation of a set of NF-
B-responsive genes
that determine physiological cell response (23).
effect on NF-
B activity we followed the same
steps as with other signal transduction pathways, starting from gel
shift assays. Nuclear extracts were isolated from ConA and HeLa cells,
untreated and treated with TNF (1.5 ng/ml) in the presence or in the
absence of 15 µM of PFT
. Results of gel-shift analysis
using lysates of both cell lines and labeled oligonucleotide, corresponding to NF-
B-binding region of the mouse B cell light chain
enhancer, showed no affect PFT
on the induction of NF-
B (Fig.
3a, shown for ConA cells).
Similarly, PFT
had no effect of NF-
B transactivation as judged by
reporter transfection assays (data not shown).
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Fig. 3.
PFT does not
interfere with NF-
B activation by TNF.
a, PFT
does not affect TNF-induced nuclear accumulation
and DNA binding activity of NF-
B. Nuclear extracts were prepared
from HeLa cells, untreated and treated with TNF (1.5 ng/ml) in the
presence and in the absence of indicated concentrations of PFT
.
Labeled oligonucleotide, corresponding to the NF-
B-binding region of
the mouse B cell light chain enhancer, was used as a probe.
b, PFT
does not sensitize cells to TNF-induced apoptosis.
ConA cells were treated 24 h with the indicated combinations of
TNF (1.5 ng/ml), PFT
(15 µM), and CHI (1 µg/ml), and
cell viability was estimated. Untreated cells are marked as
"u/t."
was accompanied by suppression of their biological functions resulting
in suppression of apoptosis caused by genotoxic stress (p53),
sensitization to HS (HSF1), and resistance to Dex-mediate cell killing
(GR). We tested whether PFT
would affect the ability of activated
NF-
B to protect cells from TNF-induced apoptosis (23). CHI, an
inhibitor of translation, suppresses induction of NF
B and makes
cells highly sensitive to TNF-mediated apoptosis. If PFT
would
suppress NF-
B activation (as CHI does), its application should
sensitize cells to TNF. Analysis of three cell systems (ConA, NIH 3T3
cells, and short term primary culture of thymocytes), all known to be
TNF-resistant due to activation of NF-
B, did not show any effect of
PFT
on their sensitivity to TNF, while treatment with CHI had strong
sensitizing effect presumably by blocking NF-
B activation (23) (Fig.
3b). These observations are well in line with lack of PFT
effect on activity of the activation of NF-
B transcription factor
found in biochemical assays.
on p53, HSF1,
and GR on one hand and NF-
B, on the other, suggest the existence of
a common regulatory component(s) in those pathways that are affected by
the compound, which is not part of NF-
B signaling. Moreover, this
putative PFT
target is likely to act by affecting nuclear
accumulation of sensitive transcription factors as it was previously
shown for p53 (2). Although at this stage it is impossible to precisely
define the molecular target of PFT
, we can base our speculations on
the known properties of the studied pathways focusing on what differs
PFT
-sensitive pathways from NF-
B signaling. Cellular factors
belonging to this category are HSP complexes that participate in
holding inactive HSF1, GR, and p53 proteins in the cytoplasm but are
not likely to be involved in regulation of NF-
B that couples instead
with its "own" specific inhibitor I-
B. Many properties of HSPs
and HSP inhibitors (such as quercetin, a flavanoid that shares
structural similarity with PFT
(24)) are consistent with the
potential involvement of HSPs in PFT
activity. HSPs are common
participants of different apoptotic pathways, and they are induced by a
variety of stress agents, including UV and gamma radiation, HS,
glucocorticoids, cytotoxic drugs, etc. (17, 25). Moreover, cells could
be protected from gamma or UV radiation induced apoptosis by exposure
to HS or overexpression of HSP70 (17, 26), while quercetin, an HSP inhibitor, is known to enhance apoptosis in a variety of systems (24,
27). Thus, HSPs are obvious candidate targets of PFT
, and
this hypothesis remains to be experimentally tested.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant CA17579 and by a grant from Quark Biotech, Inc. (both to A. V. G.).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.
To whom correspondence should be addressed: Dept. of Molecular
Biology, Lerner Research Inst., Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-1205; Fax: 216-444-0512; E-mail: gudkov@ccf.org.
Published, JBC Papers in Press, March 12, 2003, DOI 10.1074/jbc.C300011200
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ABBREVIATIONS |
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The abbreviations used are:
PFT, pifithrin-
;
TNF, tumor necrosis factor;
CHI, cycloheximide;
HS, heat shock;
HSF, heat shock transcription factor;
HSP, heat shock
protein;
Dex, dexamethasone;
ConA, concanavalin A;
GR, glucocorticoid
receptor;
GRE, glucocorticoid responsive element;
NF, nuclear factor;
LTR, long terminal repeat;
MMTV, murine mammary tumor virus;
CAT, chloramphenicol acetyltransferase.
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