From the Laboratory for Cancer Medicine and
University Department of Medicine, Western Australian Institute for
Medical Research and Centre for Medical Research, the University of
Western Australia and ** Department of Endocrinology and
Diabetes, Royal Perth Hospital, Perth, Western Australia 6001, Australia and
Vascular Biology Program, Department of
Physiology, University of Connecticut Health Center, Farmington,
Connecticut 06030
Received for publication, August 19, 2002, and in revised form, November 11, 2002
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ABSTRACT |
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Despite promoting growth in
many cell types, epidermal growth factor (EGF) induces growth
inhibition in a variety of cancer cells that overexpress its
receptor. The cyclin-dependent kinase inhibitor
p21WAF1 is a central component of this pathway. We
found in human MDA-468 breast cancer cells that EGF up-regulates
p21WAF1 mRNA and protein, through a combination of
increased mRNA stability and transcription. The decay rate of a
hybrid luciferase reporter full-length p21WAF1
3'-untranslated region (UTR) mRNA was significantly faster than that of a control mRNA. Transfections with a variety of
p21WAF1 3'-UTR constructs identified multiple
cis-acting elements capable of reducing basal reporter
activity. Short wavelength ultraviolet light induced reporter
activity in constructs containing the 5' region of the
p21WAF1 3'-UTR, whereas EGF induced reporter activity in
constructs containing sequences 3' of the UVC-responsive region. These
cis-elements bound multiple proteins from MDA-468 cells,
including HuR and poly(C)-binding protein 1 (CP1). Immunoprecipitation
studies confirmed that HuR and CP1 associate with p21WAF1
mRNA in MDA-468 cells. Over- and underexpression of HuR in MDA-468 cells did not affect EGF-induced p21WAF1 protein expression
or growth inhibition. However, binding of HuR to its target 3'-UTR
cis-element was regulated by UVC but not by EGF, suggesting
that these stimuli modulate the stability of p21WAF1
mRNA via different mechanisms. We conclude that EGF-induced
p21WAF1 protein expression is mediated largely by
stabilization of p21WAF1 mRNA elicited via multiple
3'-UTR cis-elements. Although HuR binds at least one of
these elements, it does not appear to be a major modulator of
p21WAF1 expression or growth inhibition in this system.
CP1 is a novel p21WAF1 mRNA-binding protein that
may function cooperatively with other mRNA-binding proteins to
regulate p21WAF1 mRNA stability.
Inhibition of human tumor cell growth is mediated by a variety of
cell cycle-related proteins and tumor suppressors. p53, a well
characterized tumor suppressor, activates transcription of a number of
target genes, including p21WAF1 (wild-type p53
activated fragment-1) (1, 2), which
encodes a protein of Mr 21,000 (p21), also known
as cyclin-dependent kinase-interacting protein 1. p21WAF1 inhibits cyclin-cyclin-dependent kinase
activity, preventing phosphorylation of critical
cyclin-dependent kinase substrates, blocking transition
from G1 to S phase of the cell cycle (3), as well as
inducing apoptosis (4). Recent evidence suggests that factors other
than p53, such as EGF1
(16), can induce p21WAF1 expression in various cell
types (p53-independent pathways). Because most human tumors lack p53
function (5), investigation of the mechanisms that regulate
p21WAF1 expression through alternative growth
factor-induced pathways has become an important focus in cancer
research. In particular, a major goal is to devise approaches that
would increase expression of p21WAF1 in tumors to reduce
proliferation and tumor growth.
Although EGF is typically growth-proliferative in breast cancer cells
(6), some cancer cells are growth-inhibited by EGF (e.g.
MDA-468 breast (7, 8), A431 epidermoid (9, 10)). EGF-induced growth
inhibition of these cells is associated with EGF receptor (EGFR)
overexpression (11) and appears to be mediated by induction of
p21WAF1 mRNA and protein (8). Multiple reports show
conclusively that the regulation of p21WAF1 expression by
growth factors and other ligands occurs predominantly at the level of
mRNA stability (12-17). However, there is little understanding of
the specific RNA-protein interactions involved in this process,
particularly in breast cancer cells. Thus, the EGF-induced
up-regulation of p21WAF1 mRNA provides an ideal system
to investigate the mechanisms governing p21WAF1 mRNA decay.
The regulation of mRNA decay is a critical mechanism in the control
of gene expression (reviewed in Hollams et al. (18)) that
involves interactions between cis-acting sequences that
confer instability to mRNA and the trans-acting protein
factors that bind them. Many cis-acting sequences consist of
AU-rich elements (AREs), most often located in the 3'-untranslated
region (3'-UTR) of labile mRNAs. However, cis-acting
elements are also found within the coding regions and 5'-UTRs of
various mRNAs (e.g. c-fos, c-myc) (19). AREs often contain single or multiple repeats of pentamer (AUUUA)
sequences, and inclusion of the AUUUA pentamer motif often targets the
mRNA for rapid cytoplasmic degradation (20). One well characterized
cis-acting element is the AU-rich sequence found within the
3'-UTR of granulocyte monocyte colony-stimulating factor mRNA,
which is able to reduce the half-life of Multiple proteins have been identified that can bind to AU- and U-rich
regions (reviewed in Hollams et al. (18)). These include
AUBF (24), AUF1 (hnRNP D) (25), Hel-N1 (26), hnRNP C (27), hnRNP A1
(27), AUH (28), HuR (29), HuD (30), tristetraprolin (31), and
poly(A)-binding protein (32). Of the AU- and U-rich-binding proteins,
only a few have been shown to definitively regulate mRNA stability:
AUF1, HuR, and other Hu/ELAV proteins and tristetraprolin and its
family members.
HuR, a ubiquitously expressed member of the Hu/ELAV family, is
involved in the shuttling of transcripts from the nucleus into the
cytoplasm (33-35), as well as in the regulation of mRNA stability (17, 35-38). In RKO colorectal carcinoma cells, HuR mediates UVC-induced stabilization of p21WAF1 mRNA (17), and of
interest, HuD, a neuron-specific member of the Hu/ELAV family (30), has
been shown to bind to a 42-nt sequence within the 3'-UTR of
p21WAF1 mRNA (39). It seemed possible, therefore, that
HuR would play an important role in the regulation of
p21WAF1 mRNA stability in breast cancer cells.
Here, we show that the 3'-UTR of p21WAF1 mRNA contains
multiple cis-acting regions that reduce basal reporter
activity and confer EGF- and UVC-induced changes to reporter constructs
in a region-specific manner. These 3'-UTR elements are the target for a
number of RNA-binding proteins, including HuR and CP1, from MDA-468
breast cancer cells. Despite its role in the mediation of
p21WAF1 mRNA stabilization and p21WAF1
expression by UVC in other cell systems, HuR does not appear to have a
major role in EGF-induced p21WAF1 expression in MDA-468
breast cancer cells (EGFR overexpressed, mutant p53).
Cell Culture--
The MDA-468 (HTB 132) human breast cancer cell
line was obtained from ATCC (Manassas, VA). Cells were routinely
cultured in Dulbecco's modified Eagle's medium/F-12 medium
supplemented with 10% fetal calf serum (Invitrogen). BING cells
(40) were cultured in Dulbecco's modified Eagle's medium supplemented
with 10% fetal calf serum. All cell lines were cultured in the
presence of penicillin (50 units/ml) and streptomycin (50 µg/ml).
Cells were utilized within 12 passages of the original stock received
from ATCC for all experiments.
Cell Cycle Analysis--
For cell cycle analysis, EGF-treated
(25 ng/ml, 4 nM) and control MDA-468 cells were harvested
by trypsinization and then permeabilized and stained for DNA in
phosphate-buffered saline (PBS) containing 0.1% Nonidet P-40, 5 mM EDTA, 5 mM EGTA, 5 µg/ml propidium iodide,
and 100 µg/ml RNase A. Flow cytometry was performed on a Coulter
EPICS XL-MCL (Coulter Corp., Hialeah, FL), and cell cycle analysis was
performed with MultiPlus AV MultiParameter data analysis software
(Phoenix Flow Systems, San Diego, CA).
Plasmid Clones, cDNA Probes, Riboprobes, Fusion Proteins, and
Expression Clones--
The p21WAF1 plasmid cDNA
(pCEP-WAF1) (from Dr. B. Vogelstein) contained the 5'-UTR, coding
region, and 3'-UTR of p21WAF1 (see Fig.
1A; nucleotide sequence is in
the GenBankTM data base under accession number
U03106) (1) and was digested with EcoRI and NotI
to liberate a 1-kb cDNA fragment, which for Northern analysis, was
random prime-labeled using [32P]dCTP (~3000 Ci/mmol;
Amersham Biosciences). A 1.1-kb 18 S rRNA cDNA probe was used as a
loading control. Plasmids WAF1-1/7, WAF1-2/7, WAF1-6/7, WAF1-879,
WAF1-1512, and WAF1-1/6 (Fig. 1A) were constructed by
cloning PCR-amplified sequences from the 3'-UTR of the
p21WAF1 cDNA into either the XbaI site of
pGL3-control luciferase reporter vector (Promega) for transfection
experiments or into the BamHI/HindIII-digested pBluescript II KS+ vector (Stratagene) for the generation of labeled riboprobes. The plasmid containing the HuD binding site (WAF1-HuD) were
constructed by subcloning annealed 42-mer sense (nt 657-698, 5'-UCU
UAA UUA UUA UUU GTG UUU UAA UUU AAA CAC CUC CUC AUG-3') (39) and
antisense oligonucleotides corresponding to this region of
p21WAF1 3'-UTR (see Fig. 1, A and B)
into the BamHI/HindIII sites of the pBluescript
vector. The c-fos AU-rich element that generates an unstable
mRNA was also cloned into the XbaI site of pGL3-control and used in transfection assays (22). pRL-SV40 (Promega) was utilized
as a control for transfection efficiency in reporter assays. Some
plasmids contained three AU-rich sequences (shown in Fig. 1,
A and B) and are denoted A (nt
742-758, 5'-AAU UAU UUA AAC AAA AA-3'), B (nt 797-809,
5'-AUU UUU AUU UUA U-3'), and C (nt 811-824, 5'-AAA UAC UAU
UUA AA-3'). For some RNA gel shifts (RNA electrophoretic mobility shift
assays) the plasmid c-fos-HuD (5'-AUA UUU AUA UUU UUA UUU
UAU UUU UUU-3') (29) was also used (Fig. 1B). All
pBluescript plasmid clones were linearized with HindIII for
transcription with T7 RNA polymerase (Invitrogen) in reactions
containing [32P]UTP (3000 Ci/mmol; Amersham Biosciences),
as described (41), to produce riboprobes with a specific activity of
~2 × 109 cpm/µg RNA that included 66 nt of
pBluescript, in addition to the corresponding portion of the
p21WAF1 3'-UTR. Unlabeled RNA transcripts were
synthesized as above except with 2.5 mM rNTPs, quantified
by spectrophotometry and verified by PAGE. pGEX-2T-HuR (from Dr.
H. Furneaux) generated a fusion protein (GST-HuR) that contained amino
acids 2-326 of human HuR (29). pGEX-6P- RNA Isolation and Northern Analysis--
MDA-468 cells were
solubilized in 4 M guanidinium isothiocyanate, and total
RNA was isolated using the method of Chomczynski and Sacchi (43). RNA
(10-15 µg per sample) was size-fractionated on a 1%
agarose-formaldehyde gel and transferred to Hybond-N+ membrane
(Amersham Biosciences). RNA was UV cross-linked to the membrane, which
was prehybridized for 4 h at 42 °C in a buffer containing 50%
formamide, 0.75 M NaCl, 0.075 M sodium citrate, pH 7.0, 5× Denhardt's solution, 1% SDS, and 200 µg/ml salmon sperm DNA and then hybridized in the same buffer overnight at 42 °C with
32P-labeled p21WAF1 cDNA probe at
106 cpm/ml. The membrane was washed sequentially in 2×
SSC/0.1% SDS for 20 min at 22 °C, 0.2× SSC/0.1% SDS for 20 min at
22 °C, and finally in 0.2× SSC/0.1% SDS at 65 °C for 5 min.
Membranes were imaged with a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA) and quantified using ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). In all experiments an 18 S rRNA cDNA
probe was used for normalization.
mRNA Turnover Studies--
MDA-468 cells (70-80%
confluent) were treated with EGF (25 ng/ml) (Promega) or cycloheximide
(10 µg/ml) for 2 h followed by the addition of the transcription
inhibitor actinomycin D (ActD) at 7.5 µg/ml (Sigma). Total RNA was
isolated from the cells at 0-, 2-, 4-, and 8-h time intervals after
addition of ActD and subjected to Northern analysis as described
earlier. p21WAF1 mRNA half-life was determined using
linear regression analysis.
Nuclear Run-on Transcription Assay--
MDA-468 cells (70-80%
confluent) were treated with EGF (25 ng/ml) for 2 h. Nuclei were
isolated as described previously (44), rapidly frozen, and stored at
Immunoblot Assay for p21WAF1, HuR, and Actin
Protein--
Control and EGF (25 ng/ml)-treated MDA-468 cells were
harvested and lysed in ice-cold radioimmune precipitation assay lysis buffer (1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 150 mM NaCl, 50 mM NaF, 1 mM DTT, 50 mM Tris, pH 8.0), containing freshly added protease
inhibitors (1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 2 µg/ml aprotinin (Roche Molecular Biochemicals)). After 10 min on ice, the lysate was centrifuged at 750 × g for 10 min at 4 °C, after which the supernatant was
recovered and stored at Real-time PCR Assay for Luciferase mRNA Decay--
MDA-468
cells (50% confluent) were transiently transfected with 8 µg of
pGL3-control, pGL3-WAF1-1/7, or pGL3-c-fos ARE using FuGENE
(Roche Molecular Biochemicals), according to the manufacturer's instructions. The cells were passaged, and 38 h after transfection they were treated with ActD (7.5 µg/ml) (Sigma) for 0-4 h. Total MDA-468 RNA was harvested using TRIzol (Invitrogen). To generate cDNA, 2 µl of RNA (denatured at 70 °C for 10 min) was
reverse-transcribed in a 20-µl reaction containing 5 mM
MgCl2, 1× avian myeloblastosis virus reverse transcriptase
buffer, 1 mM dNTPs, 20 units of RNasin, 10 units of avian
myeloblastosis virus reverse transcriptase, and 250 ng of
oligo(dT)15 primer at 42 °C for 30 min. PCR was then
performed for both luciferase and Transfection and Luciferase Assays--
MDA-468 cells (50%
confluent) were transiently transfected with 8 µg of pGL3 ± various p21WAF1 3'-UTR regions (see Fig. 1A) and
100 ng of pRL-SV40 as a control, using FuGENE 6 as above. Some cells
were cultured following treatment with EGF (25 ng/ml) or UVC (254 nM, 20 J/m2), for 8 or 6 h respectively,
prior to lysate extraction. Cells were washed in PBS, harvested by
trypsinization, and lysed, and supernatant luciferase activity was
measured using the dual luciferase reporter assay kit (Promega) and a
Wallac Victor 1420 multilabel counter (Wallac Oy; Turku, Finland),
according to the manufacturer's instructions. Firefly luciferase
(pGL3) activity was normalized against Renilla luciferase (pRL-SV40)
activity to yield the relative luciferase activity.
Preparation of Cytoplasmic Extracts for RNA Gel Shift
Assays--
MDA-468 cells were grown to 70-80% confluence in 10-cm
culture dishes. Cytoplasmic extracts were prepared as described
previously (41). Briefly, cells were scraped from the culture dishes in chilled PBS, centrifuged at 450 × g for 4 min at
4 °C, washed again with PBS, and then incubated for 20 min with cold
cytoplasmic extract buffer (CEB; 10 mM HEPES, 3 µM MgCl2, 40 mM KCl, 5%
glycerol, 0.2% Nonidet P-40, 1 mM DTT), containing freshly
added protease inhibitors (0.5 mM PMSF, 10 µg/ml
leupeptin, 2 µg/ml aprotinin). Lysates were cleared by centrifugation
at 4 °C for 10 min at 12,100 × g, and the
supernatant was snap-frozen in liquid nitrogen and stored at
Preparation of Whole Cell Extracts for RNA Gel Shift
Assays--
MDA-468 cells were grown to 70-80% confluence in 10-cm
culture dishes. Medium was removed, the cell monolayer was washed twice in ice-cold PBS, and the cells were lysed in 0.5 ml of chilled lysis
buffer (containing 50 mM Tris, pH 7.5, 5 mM
EDTA, pH 8.5, 150 mM NaCl, 1% Triton X-100, 10 µg/ml
aprotinin, 10 µg/ml leupeptin, 1 mM PMSF, 2 mM NaVO4, 50 mM NaF, and 10 mM Na2MoO4·2H2O) on
ice for 10 min. Cells were scraped and transferred to Eppendorf tubes, and lysates were then cleared by centrifugation at 4 °C for 10 min
at 12,100 × g and stored at RNA Electrophoretic Mobility Shift Assay (REMSA)--
Binding
reactions were performed as described previously (41) with 5 µg of
cytoplasmic extract or 200 ng of recombinant protein and
105 cpm of 32P-labeled RNA (~2-5 pg).
Briefly, binding reactions were incubated at 22 °C for 30 min, after
which 0.3 units of RNase T1 (Roche Molecular Biochemicals) was added
for 10 min, followed by the addition of heparin (final concentration 5 mg/ml) (Sigma) for 10 min. Complexes were separated by 4% PAGE,
visualized by PhosphorImager, and analyzed by ImageQuant software
(Molecular Dynamics, Sunnyvale, CA). In competition assays, extracts
were incubated with an unlabeled competitor RNA (~100-fold excess)
for 30 min prior to addition of the 32P riboprobe. For
supershift assays with the HuR antibody, the method used was as
described previously (46).
UV Cross-linking (UVXL) of RNA-Protein
Complexes--
RNA-protein binding reactions were carried out as
described above, using 20 µg of whole cell extract or 100-500 ng of
recombinant protein, 1.5 × 105 cpm (10-15 pg) of
32P riboprobe (41), and 2-5 µg of tRNA. Following the
addition of heparin, samples were placed on ice in a microtiter tray
and UV-irradiated 1 cm below the Stratalinker UV light source (240 nm
UV-bulb; Stratagene) for 10 min. After UVXL, samples were incubated with RNase A (final concentration 100 µg/ml) (Roche Molecular Biochemicals) at 37 °C for 15 min. The samples were boiled for 3 min
in SDS sample buffer (50% glycerol, 0.25 M Tris, pH 6.8, 10% SDS, 4% Preparation of GST Fusion Proteins--
GST fusion proteins were
prepared essentially as described (47). Briefly, 1-liter cultures of
DH5 IP-RT-PCR Assay--
MDA-468 cells were grown to 50% confluence
in 10-cm dishes, and cytoplasmic extracts were harvested as described
above. Cytoplasmic extract (200 µg) was added to 10 µg of HuR, CP1,
or EGFR antibody. After incubation on ice for 45 min, 5 µg each of
protein A (Amersham Biosciences) and protein G (Sigma) beads was added
to all tubes, which were mixed for a further 45 min at 4 °C. The
tubes were centrifuged at 2,000 × g for 2 min, and the
supernatants were removed for RNA extraction. The pelleted beads were
washed with cold CEB (10 × 1 ml), and RNA was extracted using
TRIzol reagent. Reverse transcription was performed using random
hexamers (Promega) and standard procedures. PCR was performed for 33 cycles, comprising five cycles of denaturation at 94 °C/30 s,
annealing at 66 °C/30 s, and extension at 72 °C/1 min, followed
by 28 cycles of denaturation at 94 °C/30 s, annealing at 55 °C/30
s, and extension at 72 °C/1 min, with the primers 481F (5'-GAC TCT
CAG GGT CGA AAA CG-3') and 585R (5'-CTT CCT GTG GGC GGA TTA G-3') from
within the coding sequence of p21WAF1. These primers
produce an amplicon that spans an intron, allowing the discrimination
between cDNA- and genomic DNA-related products in these PCR
experiments. PCR products were resolved on an ethidium bromide-stained
3% agarose gel.
Retroviral Expression of HuR--
Full-length HuR cDNA was
cloned into the pBabe puro vector (42) in the sense and antisense
orientation and then transiently transfected into the retroviral
packaging cell line BING (40) using FuGENE according to the
manufacturer's protocol. Retroviral-containing conditioned medium was
collected from the BING cells at ~48 h after transfection. Following
filtration (0.45 µm) and the addition of 4 µg/ml polybrene
(hexadimethrine bromide; Sigma), the retroviral-containing medium was
incubated overnight with the target cells (MDA-468). Cells were
selected in 1 µg/ml puromycin (Sigma) starting 48 h after
infection. Pools of puromycin-resistant cells were analyzed by Western
blotting to confirm transgene expression. All subsequent experiments
were performed using pools of infected cells.
Colony Formation Assays--
Colony formation assays were
performed as described previously (58). Briefly, MDA-468 sublines were
plated in triplicate at a density of 5000 cells per 10-cm plate and
then incubated overnight. A single dose of EGF (25 ng/ml) or PBS
control was added at 24 h, and the cells were allowed to grow for
a further 10 days. After fixation in methanol:acetic acid (3:1),
colonies were stained with Giemsa (Fluka) and counted using a
Quantimet 520 image analyzer (Leica).
Statistical Analysis--
Transfection and luciferase mRNA
decay data are shown as mean ± S.D. Statistical analysis was
performed using Student's t test, with a p value
of <0.05 regarded as significant.
EGF Up-regulates p21WAF1 Expression in MDA-468
Cells--
The MDA-468 breast cancer cell line, which contains mutant
p53 and overexpresses the EGFR (7), provides an excellent model system
to investigate the mechanisms underlying p21WAF1 gene
expression and its regulation by EGF. To establish the validity of this
cell line as a model of EGF-induced cell cycle arrest and growth
inhibition, the cells were treated with EGF (25 ng/ml) for 8 h,
and the proportion of cells in S-phase of the cell cycle was determined
using flow cytometry (see "Materials and Methods"). EGF treatment
reduced the proportion of cells in S phase from 30 to 12.5% (data not
shown). Furthermore, in a colony formation assay (see "Materials and
Methods"), EGF treatment led to >98% reduction in the number of
detectable colonies (data not shown).
We next examined the effect of EGF on endogenous p21WAF1
mRNA and protein levels in MDA-468 cells. EGF rapidly up-regulated
p21WAF1 protein (Fig.
2A) and p21WAF1
mRNA (Fig. 2B) within 2 h. To determine whether
this up-regulation of p21WAF1 mRNA occurred at the
post-transcriptional level, we treated MDA-468 cells with EGF for
2 h and performed ActD chase studies. In the absence of EGF, the
basal half-life of p21WAF1 mRNA was relatively short
(2.7 h) (Fig. 2C). However, in the presence of EGF the
half-life was increased to 11.5 h (Fig. 2C). Treatment
of the cells with cycloheximide, a translational inhibitor, also
stabilized p21WAF1 mRNA (Fig. 2C),
suggesting that (i) ongoing translation is required for maintenance of
the short basal half-life, and/or (ii) existing cellular proteins
mediate the stabilization of p21WAF1 mRNA.
Nuclear run-on assays were employed to evaluate the effect of EGF on
p21WAF1 transcriptional activity. After treatment of
MDA-468 cells with EGF, p21WAF1 transcription increased by
~2-fold (Fig. 2D). Taken together, these data suggest that
the EGF-induced growth arrest in MDA-468 cells is associated with a
rapid increase in p21WAF1 mRNA and protein, which
results from a combination of increased mRNA stability and
increased transcription.
Identification of cis-Acting Elements in the 3'-UTR of
p21WAF1 mRNA--
Based on the observation that the
stabilization of p21WAF1 mRNA is a major contributor to
the overall up-regulation of p21WAF1 protein expression in
EGF-treated MDA-468 cells, we next sought to elucidate some of the
mechanisms underlying this effect. In breast cancer cells, little is
known of the cis-acting elements or trans-acting
factors involved in the regulation of p21WAF1 mRNA
stability. Transfection studies with ActD chase and real-time PCR
demonstrated that WAF1-1/7 (entire p21WAF1 3'-UTR; see
Fig. 1A) destabilized luciferase mRNA significantly, in
a manner equivalent to that of the highly unstable c-fos ARE (Fig. 3). These data provided strong
evidence that the p21WAF1 3'-UTR contains one or more
cis-elements that confer basal mRNA instability and
potentially contribute to the regulation of p21WAF1
mRNA stability.
The 3'-UTR of p21WAF1 contains an AU-rich region at the 5'
end that spans ~250 nt, termed WAF1-1/6 (see Fig. 1, A
and B), which contains at least one known HuR binding site
(17). Within WAF1-1/6 is a 42-nt sequence, termed WAF1-HuD, which
contains an imperfect consensus nonamer and is the target for HuD
binding (39). The WAF1-1/6 region also contains several smaller
stretches of AU-rich sequence, denoted A, B, and
C (see Fig. 1, A and B, and see
"Materials and Methods") and was therefore a candidate
cis-acting sequence.
To further dissect the cis-activity of the
p21WAF1 3'-UTR, we generated several reporter constructs
containing portions of the 3'-UTR of p21WAF1 (Fig.
1A). In transfection assays using MDA-468 cells, the
full-length 3'-UTR, WAF1-1/7, reduced basal reporter activity by
~85% (Fig. 4, A and
B), supporting our ActD-luciferase mRNA data (Fig. 3). Subsequent analysis of the three major components of the 3'-UTR (WAF1-1/6, WAF1-879, WAF1-1512) showed that each reduced reporter activity but that the major effect was 3' of the previously identified AU-rich region contained within WAF1-1/6 (Fig. 4B). In
support of this observation, clones WAF1-2/7 and WAF1-6/7, which
harbored deletions of the AU-rich regions, reduced reporter activity
similarly to WAF1-1/7. Taken together, these results suggest that the
WAF1-1/6 region is not the sole determinant of basal
p21WAF1 mRNA stability in MDA-468 cells and that the
four AU-rich regions (HuD; see Fig. 1, A-C) are
not major contributors to basal turnover of p21WAF1
mRNA in MDA-468 cells. Furthermore, although WAF1-879 and
WAF1-1512 each decrease reporter activity, neither is as effective as
the combined sequence (WAF1-6/7) (Fig. 4B). This suggests
the presence of multiple cis-elements within the 3'-UTR of
p21WAF1 mRNA.
We next examined the effect of EGF on the reporter activity of each of
these constructs in MDA-468 cells. EGF increased reporter activity by
~60% in the case of WAF1-1/7, with most of this effect being
contained within the WAF1-6/7 region (Fig. 4C). Both the WAF1-879 and WAF1-1512 constructs conferred EGF-induced up-regulation of luciferase reporter activity, whereas the WAF1-1/6 construct appeared to be relatively EGF-unresponsive (Fig. 4C). Taken
together, these results suggest that the predominant
cis-element(s) within the 3'-UTR of p21WAF1 that
are responsible for both basal mRNA instability and
EGF-inducibility in MDA-468 cells reside downstream of the WAF1-1/6 sequence.
To compare these results with another regulator of p21WAF1
mRNA stability, we tested the effect of UVC treatment on reporter
activity using the same constructs in transfection experiments with
MDA-468 cells. The full-length 3'-UTR (WAF1-1/7) increased reporter
activity by ~70% after UVC treatment (Fig. 4D). Further
analysis revealed that the UVC-mediated up-regulation of reporter
activity occurred predominantly through sequences contained within the
WAF1-1/6 construct, with a lesser contribution from sequences
downstream of WAF1-1/6 (Fig. 4D). This result suggests that
the WAF1-1/6 region is the major 3'-UTR determinant of UVC-induced
stabilization of p21WAF1 mRNA in MDA-468 cells. These
data illustrate that even within the one cell type, different stimuli
may lead to the preferential regulation of different and specific
cis-elements within the p21WAF1 3'-UTR,
presumably via different sets of RNA-protein interactions.
MDA-468 Cells Contain Proteins That Bind Specifically to
p21WAF1 mRNA--
To investigate whether the
cis-acting p21WAF1 mRNA 3'-UTR elements were
a target for cytoplasmic RNA-binding proteins from MDA-468 breast
cancer cells, we tested each region (WAF1-1/6, WAF1-HuD, WAF1-879,
WAF1-1512) with REMSA. Cytoplasmic proteins from MDA-468 cells bound
specifically to these probes (Fig.
5A, lanes 2,
6, 8, and 10), whereas no RNA-protein
complexes were observed with 32P-labeled vector control
(pBluescript; see Fig. 5A, lane 12). Furthermore,
addition of ~100-fold molar excess unlabeled pBluescript competitor
RNA did not diminish the formation of the RNA-protein complexes
significantly (WAF1-HuD; see Fig. 5B, lane 4)
(data not shown for the other riboprobes). However, addition of excess unlabeled self RNA (WAF1-HuD; see Fig. 5B, lane
3) (data not shown for the other riboprobes) virtually abolished
RNA-protein complexes in all cases, demonstrating the specificity of
the interaction.
We next utilized UVXL assays to characterize the individual
p21WAF1 RNA-binding proteins. We found that multiple
proteins targeted the WAF1-1/6, WAF1-HuD, WAF1-879, and WAF1-1512
probes (Fig. 5C, lanes 1 and 3; Fig.
5D, lanes 2 and 3, respectively). A
similar set of RNA-protein complexes (RPCs) was identified with the
WAF1-1/6 and WAF1-HuD probes, but the relative intensity of most of
the RPCs was significantly different between the two probes (Fig. 5C, lanes 1 and 3). This suggested
that the majority of these proteins bind to the WAF1-HuD region, which
is contained within WAF1-1/6. Interestingly, a smaller range of RPCs
was detected with the WAF1-879 and WAF1-1512 probes (Fig.
5D, lanes 2 and 3), and again the
relative intensities of the RPCs differed between the two probes.
HuR and CP1 Bind to p21WAF1 mRNA--
Previous
studies in other cell types have demonstrated that the WAF1-HuD region
within p21WAF1 mRNA is a target for members of the ELAV
RNA-binding protein family (e.g. HuD) (39). Wang et
al. (17) showed that increased binding of HuR to the WAF1-1/6
element mediated the UVC-induced stabilization of p21WAF1
mRNA (17). This suggested that the ~36-kDa band observed in UVXL
studies with the WAF1-HuD and WAF1-1/6 probes and using MDA-468 breast
cancer extracts (Fig. 5C, lanes 1 and
3) contained HuR. In addition, a preponderance of potential
CP1 binding sites within the p21WAF1 3'-UTR (see Fig.
1A), together with the observed RPCs at ~42 kDa using the
p21WAF1 3'-UTR riboprobes (Fig. 5C, lanes
1 and 3; Fig. 5D, lanes 2 and 3), suggested that CP1 protein might target
p21WAF1 mRNA.
To investigate the association among HuR, CP1, and p21WAF1
mRNA in MDA-468 cells, we utilized an immunoprecipitation-RT-PCR
assay with primers that target the p21WAF1 coding region
(see "Materials and Methods"). Using HuR and CP1 antibodies, we
were able to co-immunoprecipitate p21WAF1 mRNA from
MDA-468 cells (Fig. 6A,
lanes 2 and 3). However, no p21WAF1-specific PCR product was identified when using an
unrelated antibody (EGFR) (Fig. 6A, lane 4) or
with no antibody (beads alone) (Fig. 6A, lane 1).
Controls were used routinely in these assays: positive (assay of
supernatant following immunoprecipitation; see Fig. 6A,
lane 5 and plasmid p21WAF1 DNA; see Fig.
6A, lane 6) and negative (H2O; see
Fig. 6A, lane 7). These data provide definitive
evidence that HuR and CP1 interact closely with p21WAF1
mRNA in MDA-468 cells.
To definitively map the binding site of HuR to WAF1-HuD we performed a
UVXL assay using MDA-468 extracts and immunoprecipitated the resultant
RPCs with HuR antibody. We identified a single major RPC with a
molecular mass of ~36 kDa that was not present when the RPCs
were precipitated with GST antibody or with beads alone (Fig.
6B, lanes 3-5). Similar results were
obtained with the WAF1-1/6 probe (Fig. 6B, lanes
8-10). These findings provided strong evidence that
the 36-kDa RPC detected in UVXL in Fig. 5C represents HuR bound to the WAF1-HuD and WAF1-1/6 probes. When tested,
thrombin-cleaved recombinant GST-HuR bound to the WAF1-HuD and
c-fos HuD probes (Fig. 6C, lanes 1 and
3), and in each case, the RPC could be supershifted with HuR
antibody (Fig. 6C, lanes 2 and 4).
To investigate the binding of CP1 to the WAF1-1/6 element, we
performed a UVXL assay with thrombin-cleaved recombinant GST-CP1. CP1
protein bound to the WAF1-1/6 riboprobe (Fig. 6C,
lane 8); binding could be displaced using poly(C)
ribohomopolymer (Fig. 6C, lanes 9 and
11) but not using poly(A) ribohomopolymer (Fig. 6C, lanes 10 and 12), confirming the
specificity of this protein species for C-rich sequences. These data
suggest that CP1 may target one or more motifs within the
UVC-responsive WAF1-1/6 element.
We next used MDA-468 whole cell extracts treated with EGF or UVC in
UVXL experiments with WAF1-HuD and WAF1-1/6 probes. In each case, UVC
up-regulated binding of HuR (~36 kDa) to the probe, whereas EGF did
not (WAF1-HuD; see Fig. 6D, lanes 1-5 and
WAF1-1/6; see Fig. 6D, lanes 6-10). A similar
UVC-induced increase in binding of HuR to p21WAF1 mRNA
has been observed in RKO colorectal carcinoma cells (17). However, no
significant change was seen in the pattern of binding for any of the
other p21WAF1 RNA-binding proteins. These data suggest an
important role for HuR in the UVC-induced up-regulation of
p21WAF1 mRNA stability and implicate a lesser role for
HuR in the EGF-mediated changes in p21WAF1 mRNA
turnover. These results support our transfection data (Fig. 4,
C and D) in which EGF up-regulated luciferase
reporter activity through sequences downstream of WAF1-HuD and WAF-1/6,
whereas UVC regulated luciferase activity primarily through sequences within WAF1-1/6, presumably those which bind HuR.
To determine the functional role of HuR in the regulation of
p21WAF1 expression and control of cell cycle in MDA-468
cells, we used retroviral vectors to generate stable pools of MDA-468
cells expressing antisense or sense HuR. HuR protein levels varied
significantly between the antisense and sense MDA-468 sublines. Despite
this, no significant difference was observed in either
p21WAF1 or actin protein levels following EGF treatment
(Fig. 7A). We also used flow
cytometry to examine the effect of HuR levels on the cell cycle
profile. However, no difference was seen between the sublines (Fig.
7B), where the proportion of cells in S phase was unaffected
by increasing (sense) or decreasing (antisense) HuR levels. The
EGF-induced reduction in S phase content was also unaffected by HuR
levels (Fig. 7B). Similarly, in a colony formation assay
(not shown), EGF induced >98% reduction in the number of colonies
with each subline. Taken together, these data suggest that although HuR
binds to the WAF1-HuD sequence of p21WAF1 mRNA (within
the context of the larger WAF1-1/6 region) in MDA-468 cells,
modification of cellular HuR levels has little or no effect on the
regulation of p21WAF1 protein levels or progression of
cells through the cell cycle.
p21WAF1 plays a central role in various models of
growth inhibition, although the molecular mechanisms that regulate
p21WAF1 expression in breast cancer cells are not well
understood. Here we have shown that EGF-induced growth inhibition in
MDA-468 breast cancer cells (mutant p53) is associated with a rapid
increase in p21WAF1 protein and mRNA expression, which
results from the combination of increased transcription and
stabilization of p21WAF1 mRNA. Significantly, we
established that the 3'-UTR of p21WAF1 contains
cis-acting elements that modulate the stability of a heterologous reporter mRNA. Through transfection studies, distinct regions have been defined within the p21WAF1 3'-UTR that
basally regulate, and confer UVC- and EGF-inducible changes to,
heterologous reporter activity. Furthermore, these cis-elements are the target for several RNA-binding
proteins, including HuR and CP1. Despite its documented role in
mediating the stabilization of a number of transcripts, including
p21WAF1 mRNA in UVC-treated RKO colorectal carcinoma
cells (17), and in promoting cyclooxygenase-2 expression in colon
carcinoma cells (59), our binding assays, together with sense and
antisense expression studies, suggest that HuR does not play a major
role in the EGF-induced expression of p21WAF1 in MDA-468
breast cancer cells.
The data presented herein support the notion that post-transcriptional
pathways are a major regulator of p21WAF1 gene expression.
Several compounds have been shown to increase the stability of
p21WAF1 mRNA in a variety of cells. These include
phorbol ester (phorbol 12-myristate 13-acetate; ~8-fold increase in
p21WAF1 mRNA stability in human ovarian carcinoma
SKOV-3 cells) (12), tumor necrosis factor Our data provides definitive mRNA decay evidence implicating the
p21WAF1 3'-UTR in cis, supporting the findings
of Liu et al. (48). Previous studies into the effect of
shorter elements, such as WAF1-HuD or WAF1-1/6, demonstrated that the
WAF1-HuD region did not destabilize a CAT reporter RNA in transfected
HepG2 cells significantly (48). Similarly, Li et al. (48)
found that the ARE motifs present within the p21WAF1 3'-UTR
(HuD; A, B, and C in Fig. 1,
A and B) did not contribute substantially toward
message instability in breast cancer cells (14). Instead, they found
that the predominant basal instability sequences were contained
downstream of the WAF1-1/6 region thought responsible for UVC-induced
stabilization of p21WAF1 mRNA. Together with our data,
these findings suggest that the p21WAF1 3'-UTR is a
composite cis-acting sequence, with contributions to basal
turnover from each of the WAF1-1/6, WAF1-879, and WAF1-1512 regions,
but that the majority of the effect is because of sequences downstream
of WAF1-1/6 (cf. with Li et al. (14)).
Interestingly, EGF and UVC augmented reporter activity preferentially
via different components of the 3'-UTR. In particular, EGF-induced
up-regulation of reporter activity occurred predominantly via a
combination of WAF1-879 and WAF1-1512, with little effect via
WAF1-1/6. In contrast, UVC augmented reporter activity predominantly
through WAF1-1/6, consistent with the findings of Wang et
al. (17), together with smaller, yet significant, up-regulation
via WAF1-879 and WAF1-1512 sequences. Further analysis of each of
these three components of the 3'-UTR will be required in different cell
types and with different stimuli to develop a definitive understanding of the mechanisms underlying p21WAF1 mRNA turnover.
We have produced several lines of evidence in support of the
association of HuR and CP1 with p21WAF1 mRNA in MDA-468
breast cancer cells. These include the immunoprecipitation of
endogenous HuR bound to the WAF1-HuD and WAF1-1/6 riboprobes and the
immunoco-purification of p21WAF1 mRNA from MDA-468 cell
extracts using HuR and CP1 antibodies. These assays (UVXL-IP and
IP-RT-PCR) provide the first definitive evidence for a close
association of HuR and CP1 with p21WAF1 mRNA in MDA-468 cells.
Based on these observations and the findings of others, we presumed
there would be a significant role for HuR in the EGF-induced regulation
of p21WAF1 mRNA stability in breast cancer cells. For
example, HuR and other members of the ELAV protein family have been
shown to stabilize AU-rich mRNAs in several other cell systems
(35). These include the stabilization of VEGF mRNA (36), GLUT-1
mRNA (49), and p21WAF1 mRNA in UVC-treated
colorectal carcinoma cells (17). In the latter report, the shortest
riboprobe that the authors used was equivalent to our WAF1-1/6 probe
(see Fig. 1A). HuR was one of only two proteins detected by
UVXL, and HuR antibody produced a partial supershift in cell extracts.
Furthermore, antisense HuR-expressing clones (with a 4-6-fold
reduction in HuR levels) demonstrated a decrease in both basal and
UV-induced p21WAF1 expression (mRNA stability and
protein levels). However, we observed that the WAF1-1/6 region did not
have a major role in modulating EGF-induced reporter activity in
MDA-468 cells. Moreover, we found that treatment of MDA-468 cells with
UVC, but not EGF, regulated the binding of HuR to the WAF1-HuD and
WAF1-1/6 riboprobes. Thus, we were not surprised to find that
modification of HuR expression in MDA-468 cells had no detectable
effect on EGF-induced p21WAF1 protein levels, cell cycle,
or growth. Taken together, these observations suggest that in these
cells, HuR plays a relatively minor role in the regulation of basal and
EGF-induced expression of p21WAF1.
In this context, Liu et al. (48) found that non-HuR
RNA-binding proteins (24 and 52 kDa) mediated the induction of
p21WAF1 mRNA stability by the The poly(C)-binding proteins, CP1 and CP2, are members of the hnRNP
K-homology domain family of RNA-binding proteins (50) and
regulate the stability of a variety of transcripts, including We have identified a U- and C-rich cis-element in the 3'-UTR
of the human androgen receptor mRNA that is the target for
simultaneous, co-operative binding of HuR and CP1/CP2 (47). The UVXL
assays presented herein with recombinant CP1 suggest that CP1 may bind to the UVC-responsive WAF1-1/6 element. The close proximity of HuR and
CP1 binding sites within WAF1-1/6 might allow both proteins to
participate in coordinated mRNA decay. It also emphasizes the need
to examine the functional role of CP1 in p21WAF1 mRNA
turnover in MDA-468 cells.
In summary, EGF increases p21WAF1 mRNA expression in
p53 mutant breast cancer cells through a combination of mRNA
stabilization and transcriptional up-regulation. We have identified
cis-elements within the p21WAF1 3'-UTR that are
distinctly EGF- or UVC-inducible in MDA-468 cells. This implies that
different stimuli can regulate p21WAF1 mRNA stability
via independent cis-elements. HuR binding modulates p21WAF1 expression in UVC-treated RKO cells but not in
EGF-treated MDA-468 cells. This indicates that there is an
HuR-independent, cell type-specific mechanism through which EGF induces
p21WAF1 expression via stabilization of p21WAF1
mRNA. CP1 and other RNA-binding proteins associate with
p21WAF1 mRNA in MDA-468 cells and may direct its
turnover. The cloning and characterization of these proteins are the
subject of further investigation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-globin mRNA from many
hours to less than 30 min (20). Other studies have shown the
UUAUUUA(A/)U(A/U) nonamer sequence to be more predictive of rapid
mRNA decay than the AUUUA pentamer motif (21, 22). Several other
RNA binding motifs have been identified, including the C-rich motif
that is the target for the poly(C)-binding proteins (CPs) (23). To
date, the cis-activity of the 3'-UTR of p21WAF1
has not been characterized extensively.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
CP1 (from Dr. M. Kiledjian)
generated a fusion protein (GST-CP1) that contained amino acids 13-347
of human CP1 (60). For HuR over-/underexpression studies, the
retroviral vector pBabe puro (42) was used. The sequence of all plasmid constructs was confirmed by dideoxy sequencing.
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Fig. 1.
Schematic of p21WAF1 3'-UTR and
clones used in vitro. A, schematic
representation of the p21WAF1 mRNA 3'-UTR sequence and
clones used for transfection (pGL3) and REMSA (pBluescript). The 42-nt
HuD binding sequence (39) and three AU-rich regions (A,
B, and C) are delineated, together with a number
of C-rich motifs (shown beneath the 3'-UTR sequence, denoted
C). The numbers refer to nucleotide positions
within the p21WAF1 mRNA sequence (GenBankTM
accession number U03106). B, sequences of c-fos
HuD (nt 3399-3425 of GenBankTM accession number V01512),
WAF1-HuD (nt 657-698 of GenBankTM accession number
U03106), and WAF1-1/6 (nt 571-829 of GenBankTM accession
number U03106). Within WAF1-1/6, WAF1-HuD is underlined and
italicized, a CCUCC consensus motif for CP1 is shown in
bold italics, and the three AU-rich sequences, A,
B, and C, are shaded. C,
schematic representation of the GST fusion proteins used for in
vitro assays. HuR contains three RNA recognition motif domains
(RRMI, RRMII, RRMIII). CP1 contains
three K-homology domains (KHI, KHII,
KHIII). RNA-binding domains are shown to scale, and the
amino acid boundaries of each are defined.
85 °C. The transcription assay was performed as described
previously (45). Briefly, the nuclei were thawed on ice, resuspended in
100 µl of reaction buffer (10 mM Tris-HCl, pH 8.0, 5 mM MgCl2, 300 mM KCl, 5 mM dithiothreitol (DTT), 0.5 mM each of ATP,
CTP, and GTP, and 100 µCi of [32P]UTP (3000 Ci/mmol;
Amersham Biosciences)), and incubated at 30 °C for 30 min. Labeled
RNA was isolated and hybridized to nitrocellulose filters onto which 5 µg of p21WAF1 and 18 S rRNA cDNAs had been blotted.
Filters were washed and then analyzed by PhosphorImager and ImageQuant software.
85 °C. Total protein concentrations of
lysates were determined using the Bio-Rad protein assay, and 10 µg of
proteins were separated on 10% BisTris acrylamide gels (Invitrogen) in 1× MES SDS running buffer, pH 7.3 (Invitrogen), and transferred to
polyvinylidene difluoride membranes (Osmonics) in 1× NuPAGE transfer buffer, pH 7.2, according to the manufacturer's instructions. Membranes were blocked with 10% skim milk in TBS-T (20 mM
Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20) at 22 °C
for 1 h, prior to addition of either anti-p21WAF1
monoclonal antibody (1:1000) (15091A; BD Biosciences), anti-HuR monoclonal antibody 19F12 (1:2000) (from Henry Furneaux), or anti-actin monoclonal antibody (1:2000) (I-19, sc-1616; Santa Cruz Biotechnology, Santa Cruz, CA), diluted in 10% skim milk/TBS-T, for 1 h at
22 °C. Membranes were then washed in 10% skim milk/TBS-T, incubated for 1 h in appropriate peroxidase-conjugated secondary antibody (1:10000; Amersham Biosciences), and washed again with TBS-T, prior to
detection with ECL Plus detection reagents (Amersham Biosciences) on
ECL-Hyperfilm (Amersham Biosciences). Protein bands were quantified
using an Eastman Kodak Co. digital DCS-420C camera and ImageQuant software.
-actin cDNA (luciferase sense,
5'-TAC TGG GAC GAA GAC GAA CAC-3'; luciferase antisense, 5'-GTT CAC CGG
CGT CAT CGT CG-3';
-actin sense, 5'-GCC AAC ACA GTG CTG TCT GG-3';
-actin antisense, 5'-TAC TCC TGC TTG CTG ATC CA-3') using a Bio-Rad
iCycler iQ real-time PCR detection system (Bio-Rad). Data were
normalized using results obtained for
-actin and the ratio of
luciferase mRNA remaining for pGL3-WAF1-1/7 and pGL3-c-fos-ARE expressed relative to pGL3-control as a
function of time after ActD treatment.
80 °C. Protein concentrations were determined using the Bio-Rad
protein assay kit.
80 °C. Protein
concentrations were determined by Bio-Rad protein assay kit.
-mercaptoethanol) and subjected to SDS-PAGE (gels ranging from 8.5% to 12%), and RNA-protein complexes were detected by
PhosphorImager. In some competition experiments, recombinant proteins
were incubated with ribohomopolymers (poly(C), poly(A)) for 20 min,
prior to addition of riboprobe. For UVXL immunoprecipitation assays,
UVXL was performed as described above, and the reactions were then
incubated for 1 h with HuR or GST antibody at 4 °C, followed by
incubation with protein A and G beads (Sigma) for 45 min at 4 °C.
After washing, RNA-protein complexes were resolved by SDS-PAGE (gels
ranging from 8.5 to 12%) and detected by PhosphorImager. In all UVXL
experiments, 14C Rainbow molecular mass markers (Amersham
Biosciences) were used.
Escherichia coli expressing GST, GST-HuR, or
GST-CP1 fusion constructs (described above) were induced at
A600 of 0.6 with
isopropyl-
-D-thiogalactopyranoside (0.5 mM)
at 30 °C for 2 h. GST fusion protein was purified from bacterial pellets, lysed in 10 mM Tris, pH 7.8, 0.5 mM EDTA, 100 mM glucose, 0.4 mg/ml lysozyme,
0.13% Triton X-100, 0.5 mM PMSF, 1 µg/ml aprotinin, and
1 µg/ml leupeptin, using glutathione beads (Sigma). GST protein was
eluted in buffer containing 20 mM HEPES, pH 7.6, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 µg/ml aprotinin, 2 µg/ml leupeptin, and 25 mM glutathione. To cleave HuR
from the GST-HuR fusion protein, 0.1 units/µl of thrombin (Amersham
Biosciences) was incubated with the GST-HuR on glutathione beads
overnight at 4 °C, the sample was centrifuged at 12,100 × g at 4 °C for 2 min, and the supernatant was collected. CP1 was cleaved from GST-CP1 with PreScission protease (Amersham Biosciences), according to the manufacturer's instructions.
Purified proteins were quantified by Bio-Rad protein assay, and purity was ascertained by SDS-PAGE.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 2.
EGF increases p21WAF1 mRNA
and protein levels and p21WAF1 mRNA stability and
transcription in MDA-468 cells. A, Western blot
analysis showing p21WAF1 protein levels in MDA-468 cells
following treatment with EGF (25 ng/ml). Cell lysates (10 µg) were
subjected to SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and probed with anti-p21WAF1 and actin
antibodies. ECL-generated images were quantified using ImageQuant and
p21WAF1 protein levels graphed over time. B,
Northern blot analysis of total RNA extracted from MDA-468 cells after
treatment with EGF (25 ng/ml) for the times indicated. Following
hybridization with a 32P-labeled p21WAF1
cDNA probe, each blot was normalized using a
32P-labeled 18 S ribosomal RNA cDNA probe.
Quantification was performed using a PhosphorImager and ImageQuant
software, and p21WAF1 mRNA levels were graphed over
time. C, ActD chase studies in MDA-468 cells. Cells were
grown to 50% confluence and treated with 25 ng/ml EGF or 10 µg/ml
cycloheximide (CHX) for 2 h and then 7.5 µg/ml ActD.
Total RNA was extracted from the cells at 0, 2, 4, or 8 h after
ActD treatment and analyzed by Northern blot as in B.
p21WAF1 mRNA was normalized against 18 S rRNA (image
not shown). Half-life of p21WAF1 mRNA was 11.5 h
(EGF-treated cells) and 2.7 h (control cells). **,
p < 0.01. D, transcription run-on analysis
of MDA-468 cells after treatment with EGF (25 ng/ml) for 2 h.
p21WAF1 transcription rates were measured in isolated
nuclei by run-on transcription assays, and the results were analyzed by
PhosphorImager and ImageQuant and shown in this figure relative to 18 S
rRNA transcription levels. CON, control; EGF, 2-h
EGF treatment.
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Fig. 3.
The 3'-UTR of p21WAF1 mRNA is
a cis-acting sequence. MDA-468 cells were
transfected with 8 µg of pGL3-control, pGL3-WAF1-1/7 (see Fig. 1),
or pGL3-c-fos ARE, and 38 h after transfection they
were treated with ActD (7.5 µg/ml) for 0, 2, and 4 h. Total
MDA-468 RNA was harvested, and cDNA was generated by RT and used in
real-time PCR with luciferase- and -actin-specific primers. Data
were normalized using results obtained for
-actin, and the ratio of
luciferase RNA remaining for pGL3-WAF1-1/7 and
pGL3-c-fos-ARE was expressed relative to pGL3-control. The
data are representative of three separate experiments performed in
triplicate. **, p < 0.001.
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Fig. 4.
The 3'-UTR of p21WAF1 mRNA
contains elements that modulate basal luciferase reporter activity and
confer UVC and EGF inducibility to reporter constructs.
A, MDA-468 cells were transfected with 8 µg of pGL3 ± p21WAF1 3'-UTR elements and 100 ng of pRL-SV40 as a
control. Cells were cultured in the presence or absence of EGF (25 ng/ml) or UVC (20 J/m2) treatment. Firefly and Renilla
luciferase activity was measured as described under "Materials and
Methods." B, luciferase reporter activity for MDA-468
cells transfected with pGL3 ± p21WAF1 3'-UTR elements
expressed relative to pGL3-control. *, p < 0.002. C, luciferase reporter activity for EGF-treated MDA-468
cells transfected with pGL3 ± p21WAF1 3'-UTR
elements. Values were expressed relative to untreated cells transfected
with the same cis-element (untreated self). *,
p < 0.002; **, p < 0.03. D, luciferase reporter activity for UVC-treated MDA-468
cells transfected with pGL3 ± p21WAF1 3'-UTR
elements. Values were expressed relative to untreated cells transfected
with the same cis-element (untreated self). *,
p < 0.001; **, p < 0.02. The
graphs are representative of at least three separate
experiments, each performed in triplicate. All values were normalized
using Renilla luciferase activity.
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Fig. 5.
Proteins from MDA-468 cells bind specifically
to cis-acting elements from the 3'-UTR of
p21WAF1 mRNA. MDA-468 extracts were incubated with
a panel of 32P-labeled p21WAF1 or vector
control (pBluescript) riboprobes, and REMSA (4% PAGE) or UVXL (10 min
of UVXL exposure, SDS-PAGE 8.5 to 12%) was performed as described
under "Materials and Methods." A, REMSA utilizing
various p21WAF1 (lanes 1, 2, and
5-10), vector control (pBluescript) (lanes
11 and 12), or c-fos HuD (lanes 3 and 4) riboprobes in the presence or absence of MDA-468
cytoplasmic extract. B, REMSA with WAF1-HuD riboprobe and
MDA-468 extracts in the absence of competitor RNA (lane 2)
and in the presence of unlabeled self RNA (lane 3) or
unlabeled vector control RNA (lane 4). C, UVXL
with MDA-468 cell extracts and WAF1-1/6 (lane 1) or
WAF1-HuD (lane 3) riboprobes, and compared to
14C molecular mass markers (lane 2).
D, UVXL with MDA-468 cell extracts and WAF1-879
(lane 2) and WAF1-1512 (lane 3)
riboprobes; 14C molecular mass markers (lane
1).
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Fig. 6.
HuR and CP1 associate with
p21WAF1 mRNA in MDA-468 cells. A,
MDA-468 cytoplasmic extract (200 µg) was incubated with 10 µg of
HuR (lane 2), CP1 (lane 3), or EGFR (lane
4) antibody, and the resultant immune complexes were precipitated
using protein A and G beads. Co-purifying RNA was isolated following a
series of washes and analyzed by RT-PCR using
p21WAF1-specific primers (see "Materials and Methods").
MDA-468 RNA from an immunoprecipitation supernatant (lane 5)
and p21WAF1 plasmid DNA (lane 6) were used as
positive controls. Immunoprecipitation with beads alone (no antibody)
(lane 1) and water (lane 7) were included as
negative controls. p21WAF1-specific product (105 bp) is
indicated with an arrow relative to DNA markers
(lane 8). B, UVXL as in Fig. 5 with MDA-468 cell
extract and WAF1-HuD (lanes 2-5) or WAF1-1/6 (lanes
7-10) riboprobes, followed by immunoprecipitation with anti-HuR
mAb (lanes 3 and 8), anti-GST mAb (lanes
4 and 9), and with beads alone (lanes 5 and
10) prior to 8.5% SDS-PAGE and PhosphorImager analysis
compared with 14C molecular mass markers (lanes
1 and 6). C, REMSA-supershift assay
with cleaved recombinant HuR protein and WAF1-HuD (lanes 1 and 2) or c-fos HuD (lanes 3 and
4) riboprobes. HuR antibody was used to shift RPCs
(lanes 2 and 4). UVXL assay (lanes
6-12) using WAF1-1/6 riboprobe and recombinant GST (lane
7) or cleaved CP1 protein (lanes 8-12), in the
presence of either poly(C) (lanes 9 and 11) or
poly(A) (lanes 10 and 12) is shown compared with
14C molecular mass markers (lane 5).
D, UVXL assay with either WAF1-HuD or WAF1-1/6 riboprobes
and control (lanes 2 and 8), EGF (lanes
4 and 9)-, or UVC (lanes 5 and
10)-treated MDA-468 cell extracts compared with
14C molecular mass markers (lanes 3 and
6).
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Fig. 7.
HuR does not regulate EGF-induced
p21WAF1 expression or cell cycle profile in MDA-468
cells. A, Western blot analysis of HuR,
p21WAF1, and actin protein levels in pools of retrovirally
infected MDA-468 cells that stably express sense (lane 2) or
antisense (lane 3) HuR and were treated for 4 h with
EGF (25 ng/ml). Analysis of parental MDA-468 cells is included for
comparison (lane 1). AS, antisense. B,
MDA-468 sublines indicated in A, as well as vector
control-infected cells (Puro), were incubated ± EGF
(25 ng/ml) for 16 h and then subjected to flow cytometry analysis
(as described under "Materials and Methods") to determine % of
cells in S phase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(~5-fold increase in
human myeloid leukemic KG-1 cells) (13), a novel retinoid CD437
(~3-fold increase in MDA-468 and MCF-7 breast cancer cells) (14), UV
light (~4-fold increase in mouse embryonal fibroblasts) (15),
phenylephrine (~3-fold increase in transfected HepG2 cells, mediated
by p42/44 MAP kinase) (48), and of direct relevance to this work, EGF (~2-fold in A431 human epidermoid carcinoma cells) (16). This response to EGF may only be apparent in cells that overexpress EGFRs,
such as MDA-468 cells and A431 cells, as EGF did not modulate p21WAF1 mRNA stability in MCF-7 cells, which express
lower levels of EGFR (16). In each of these reports, where measured,
the transcriptional increase in p21WAF1 contributed less
than the increase in mRNA stability to the total mRNA level.
1
adrenergic agonist, phenylephrine (48). They did not observe binding of
cellular HuR to their riboprobe, which was identical to our WAF1-HuD
riboprobe. We therefore presume that the role of HuR in regulating
p21WAF1 expression varies according to the mode of stimulus
and is dependent upon cell type. It also emphasizes that RNA-binding
proteins other than HuR can regulate p21WAF1 mRNA turnover.
-globin, tyrosine hydroxylase, and erythropoeitin (51-55), as well
as regulating translation of 15-lipoxygenase and human papillomavirus (56, 57). Co-immunopurification of p21WAF1 mRNA from
MDA-468 cells using CP1 antibody suggests that CP1 protein binds to one
or more of the motifs distributed throughout the p21WAF1
3'-UTR (see Fig. 1A). UVXL analysis of WAF1-1/6, WAF1-879,
and WAF1-1512 demonstrates the presence of RPCs at ~42 kDa, which may contain CP1 and/or CP2. CP1 may therefore play a role in the regulation of p21WAF1 mRNA stability in MDA-468 cells
through interactions with sequences within and/or downstream of
WAF1-1/6.
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ACKNOWLEDGEMENTS |
---|
We thank Bert Vogelstein for the p21WAF1 cDNA plasmid, Maria Czyzyk-Krzeska for the CP1 antibody, Mike Kiledjian for the GST-CP1 construct, and Robert Medcalf for advice on the HuR supershift assay. We are also grateful to Romano Krueger for assistance with the cell cycle analysis and Britt Granath for advice with the statistical analysis.
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FOOTNOTES |
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* This work was funded in part by the Raine Medical Research Foundation, the Kathleen Cuningham Foundation for Breast Cancer Research, the Medical Research Foundation of Royal Perth Hospital, and the Cancer Foundation of Western Australia.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.
§ Supported by a Faculty of Dentistry and Medicine scholarship from the University of Western Australia.
¶ Supported by Raine Medical Research Foundation fellowship.
To whom correspondence should be addressed: University
Department of Medicine, Royal Perth Hospital, GPO Box X2213, Perth, Western Australia 6001, Australia. Tel.: 61-8-9224-0323; Fax: 61-8-9224-0246; E-mail: peterl@cyllene.uwa.edu.au.
Published, JBC Papers in Press, November 12, 2002, DOI 10.1074/jbc.M208439200
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ABBREVIATIONS |
---|
The abbreviations used are: EGF, epidermal growth factor; EGFR, EGF receptor; UVC, short wavelength ultraviolet light; ActD, actinomycin D; ARE, AU-rich element; CP, poly(C)-binding protein; DTT, dithiothreitol; ELAV, embryonic lethal abnormal vision; GST, glutathione S-transferase; REMSA, RNA electrophoretic mobility shift assay; RPC, RNA-protein complex; UTR, untranslated region; UVXL, UV cross-linking; nt, nucleotide; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1, 3-diol; MES, 4-morpholineethanesulfonic acid; RT, reverse transcriptase; IP, immunoprecipitation.
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