From the Department of Pharmacology and the
§ Experimental and Clinical Therapeutics Program, Barbara
Ann Karmanos Cancer Institute, Wayne State University School of
Medicine, Detroit, Michigan 48201 and ¶ the Department of
Pharmaceutical Science, St. Jude Children's Research Hospital,
Memphis, Tennessee 38101
Received for publication, June 16, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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The relationship between loss of functional p53
and human reduced folate carrier (hRFC) levels and function was
examined in REH lymphoblastic leukemia cells, which express wild type
p53, and in p53-null K562 cells (K562pTet-on/p53)
engineered to express wild type p53 under control of a
tetracycline-inducible promoter. Activation of p53 in REH cells by
treatment with daunorubicin was accompanied by decreased (~5-fold)
levels of hRFC transcripts and methotrexate transport. Treatment of
K562pTet-on/p53 cells with doxycycline resulted in a
dose-dependent expression of p53 protein and transcripts,
increased p21 protein, decreased dihydrofolate reductase, and
G1 arrest with decreased numbers of cells in S-phase. p53
induction was accompanied by up to 3-fold decreases in hRFC transcripts
transcribed from the upstream hRFC-B promoter and similar losses of
hRFC protein and methotrexate uptake capacity. Expression of p15 in an
analogous inducible system in K562 cells resulted in a nearly identical
decrease of S-phase cells and dihydrofolate reductase without effects
on hRFC levels or activity. When the hRFC-B promoter was expressed as
full-length and basal promoter-luciferase reporter constructs in
K562pTet-on/p53 cells, induction of p53 with doxycycline
resulted in a 3-fold loss of promoter activity, which was reversed by
cotransfection with a trans-dominant-negative p53. These studies show
that wild type p53 acts as a repressor of hRFC gene
expression, via a mechanism that is independent of its effects on cell
cycle progression.
p53 is a 53-kDa nuclear phosphoprotein involved in control of cell
growth and apoptosis. Inactivation of the p53 tumor
suppressor gene, either by mutation or deletion, has frequently been
found in a variety of human malignancies (1-3). Loss of p53 function leads to increased cell proliferation and genetic instability, and
reduced capacity for apoptosis (1-3). A major function of p53 is to
transcriptionally regulate downstream effector genes (1-3). p53 has
been reported to transactivate genes involved in cell proliferation or
apoptosis such as Gadd45 (4), MDM2 (5),
p21waf1 (6), Bcl2 (7), Bax
(8), IGF-BP3 (9), and cyclin G (10).
Transcriptional activation is mediated by p53 binding to a consensus
site consisting of two copies of a
10-bp1 binding sequence
(5'-PuPuPuC(A/T)(T/A)GPyPyPy), separated by up to 13 bp (11, 12). p53
has also been reported to repress transcription of a number of genes,
including genes encoding proteins involved in the response of
chemotherapeutic drugs such as topoisomerase II Although one of the major roles of p53 is to stimulate apoptosis,
the relationships between wild type p53 and sensitivity to
chemotherapeutic drugs are frequently complex and contradictory. A
number of studies suggest that loss of wild type p53 function leads to
resistance to chemotherapy and radiation therapy due to decreased
susceptibility to apoptosis (24), however, this has not always been
found to be the case. For example, loss of wild type p53 has been
reported to sensitize mammalian cells to taxol (25) and cisplatin (26).
Furthermore, the anti-purine inhibitor, AG2034, was selectively
cytotoxic toward cells with nonfunctional p53 that were lacking a
functional G1 checkpoint (27). In H35 rat hepatoma cells
expressing a trans-dominant-negative p53, levels of P-glycoprotein and
mdr1a transcripts were markedly elevated, resulting in resistance to
P-glycoprotein substrates (16). Mtx sensitivity was reported to be
increased in H35 cells characterized by loss of wild type p53 function
(16). However, the basis for this finding was not considered further.
Mtx and related antifolates (Tomudex,
5,10-dideaza-5,6,7,8-tetrahydrofolate) are transported into cells by
the RFC (28, 29). For Mtx, RFC-mediated transport is a key element in
antitumor activity, reflecting its role in generating intracellular
unbound Mtx for complete inhibition of DHFR and for Mtx polyglutamate synthesis (28, 29). Given our interest in the mechanisms of transcriptional control of RFC, we began a systematic investigation to
explore the possibility that wild type p53 may regulate RFC. Accordingly, we expressed wild type p53 in p53-null K562 cells under
control of a tetracycline-inducible promoter. As described herein, in
this system, wild type p53 acts as a potent repressor of
hRFC gene expression, via a mechanism that seems to be
independent of its well-established effects on cell cycle progression.
Rather, decreased hRFC levels and function upon p53 induction are
likely due to an inactivation of transcription from the upstream hRFC-B promoter (30-32). We also show that induction of endogenous wild type
p53 in REH leukemia cells is accompanied by a dramatic decrease in hRFC
expression and Mtx transport.
Chemicals and Reagents--
[
Wild type human p53 cDNA (34) was obtained from Dr. Bert Vogelstein
(The Johns Hopkins University School of Medicine, Baltimore, MD), and
the p15 cDNA (35) was obtained from Dr. David Beach (Cold Spring
Harbor Laboratories, Cold Spring Harbor, NY). The TDN-p53 construct was
described previously (16). The p53-responsive reporter construct,
p53CON, containing the p53 consensus binding sequence (36), was
obtained from Dr. Jerry Shay (The University of Texas Southwestern
Medical Center, Dallas, TX). Antiserum to purified recombinant DHFR
(provided by Dr. R. Blakley, Memphis, TN) was prepared in rabbits by
the Pocono Rabbit Farm and Laboratory (Canadensis, PA).
Cell Culture--
K562 human erythroleukemia cells and REH
B-lymphoblastic leukemia cells were purchased from the American Type
Culture Collection (Rockville, MD). K562 and REH cells were grown in
RPMI 1640 medium (Sigma Chemical Co.) containing 10% heat-inactivated
iron-supplemented calf serum (Hyclone), 2 mM
L-glutamine, 100 units/ml penicillin, and 100 µg/ml
streptomycin. Cells were cultured in a 37 °C humidified atmosphere
containing 5% CO2/95% air. For cytotoxicity
determinations, cells were cultured in complete RPMI 1640 medium
containing 10% dialyzed fetal bovine serum. Cell numbers were
determined after equivalent numbers of doublings by direct microscopic
counting with a hemacytometer.
Expression of an Inducible p53 Construct in K562 Cells--
A
commercial "Tet-on" (CLONTECH) kit was used to
express wild type p53 in p53-null K562 cells under control of a
tetracycline/Dox-inducible promoter (37). The K562pTet-on
cell line was developed from wild type K562 cells by transfection with
the regulator pTet-on plasmid (5 µg) using Lipofectin (Life Technologies, Inc.), as instructed by the manufacturer. Colonies were
isolated in soft agar in the presence of 1 mg/ml G418 (38). G418-resistant clones were expanded and screened by transient transfection with a pTRE-luc reporter plasmid. For reporter gene assays, cells were transfected with a 2.5 µg of pTRE-luc plasmid and
treated with and without Dox (2 µg/ml) for 24 h, followed by
additional incubation for 48 h. The cell lysates were prepared and
tested for luciferase activity, as described below.
To construct the pTRE/p53 plasmid, wild type p53 was cloned into a pTRE
response plasmid at the BamHI site. The plasmid was then
cotransfected into the K562pTet-on cell line with the
pcDNA3.1/Zeo plasmid (0.25 µg, Invitrogen). Colonies were
isolated in soft agar, expanded in the presence of 1 mg/ml G418 and 150 µg/ml zeocin, and tested for p53 expression in the presence and
absence of Dox (2 µg/ml) by Western blotting, as described below. A
"double-stable" resistant clone (designated K562pTet-on/p53), which exhibited no background and a high
level of Dox-induced p53 expression, was identified and used for
further analysis. K562 pTRE "vector control" cells were generated
by transfecting K562pTet-on cells with an empty pTRE
vector. An analogous p15 "Tet-on"-inducible cell line
(K562p15Tet-on-8) was prepared by transfecting p15- and
p16-null K562pTet-on cells with a pTRE/p15 plasmid
construct.2
Western Blot Analysis of p53, p21, DHFR, and hRFC--
Soluble
protein extracts were prepared by sonication in hypotonic buffer (10 mM Tris-Cl, pH 7.0) containing 1% SDS and proteolytic inhibitors and subjected to SDS-PAGE (39). p53, p21, and DHFR proteins
were electrophoretically transferred to PVDF membranes (DuPont (40))
and immunoblotted with anti-p53 monoclonal (DO-1) antibody (Oncogene
Research Products), p21waf1 antibody (Oncogene Research
Products), and antiserum to purified recombinant human DHFR,
respectively. Immunoreactive proteins were visualized by enhanced
chemiluminescence (Pierce, Rockford, IL).
For hRFC protein expression, sucrose-gradient-purified plasma membranes
were prepared as previously described (41-43). Membrane proteins were
solubilized in 10 mM Tris-Cl (pH 7.0) hypotonic buffer and
1% SDS in the presence of proteolytic inhibitors (41-43). Aliquots
(100 µg) were subjected to a 7.5% SDS-PAGE and transferred to PVDF
membranes followed by immunoblotting. hRFC proteins were detected by
chemiluminescence (Lumi Light, Roche Molecular Biochemicals) using
protein A-purified glutathione S-transferase-hRFC antibody (43). All antibody incubations were performed in Tween 20/Tris-buffered saline that was supplemented with 0.5% nonfat milk. Light emission was
recorded on x-ray film with various exposure times, and the signal was
quantitated on a Kodak Digital Science 440CF Image Station.
Northern Analysis--
Total RNAs were isolated from cells with
TRIzol reagent (Life Technologies, Inc.). Equal amounts (20 µg) of
total RNAs were loaded onto a 0.7% formaldehyde/agarose gel and
transferred to nylon membranes (GeneScreen, PerkinElmer Life Sciences),
followed by baking the membranes at 80 °C until dry. The
32P-labeled probes (i.e. hRFC, p53, and p15)
were prepared by random-priming (Roche Molecular Biochemicals) using
[ Membrane Transport Methodology--
Exponentially growing cells
were treated with Dox over a range of concentrations. After 48 h
of incubation, ~1 × 107 cells were harvested for
[3H]Mtx (0.5 µM) transport assays (41, 43,
44). Cells were washed twice with D-PBS and resuspended in 2 ml of
Hanks' balanced salts. Cells were incubated with [3H]Mtx
at 37 °C for 180 s. The reactions were quenched with ice-cold D-PBS, and the cells were washed three times with D-PBS. The cell pellets were dissolved in 1 ml of 0.5 N NaOH. The levels of
intracellular radioactivity were expressed in units of pmol/mg of
protein, calculated from direct measurements of radioactivity and
protein contents of the cell homogenates as determined by the method of
Lowry et al. (45).
Transient Transfections of hRFC Promoter-luciferase Reporter Gene
Constructs--
The full-length hRFC-B (previously, Pro43; positions
Flow Cytometry Analysis--
Approximately 1-2 × 106 of K562pTet-on/p53,
K562p15Tet-on-8, or pTRE vector cells were harvested after
treatment with and without Dox (2 µg/ml) for 48 h, washed twice
with D-PBS, and fixed in 70% ethanol. For DNA fluorescence staining,
cells were stained in propidium iodide by the method of Crissman and
Steinkamp (46) and analyzed by flow cytometry (Becton Dickinson
FACScan). Apoptosis was assayed by annexin V staining and flow
cytometry using a commercial kit (R&D Systems).
RT-PCR of the hRFC 5'-UTRs--
Total RNAs were isolated from
K562pTet-on/p53 cells, treated with and without Dox, and
used for the synthesis of cDNA (PerkinElmer Life Sciences RT-PCR
kits). cDNAs were amplified with Taq polymerase (Promega) in the presence of GC-Rich reagent (Roche Molecular Biochemicals) and specific 5'-UTR primers and compatible coding sequence primers. Primer pairs for hRFC 5'-UTR were based on the DNA
sequence for the 5'-untranslated exons (30, 44) and the hRFC cDNA
sequence (Ref. 44; GenBankTM accession number U19720) and are as
follows (listed as sense, antisense): KS6
(5'-cggggccctggggtgagt-3', 5'-gccatggtgacgctgtagaa-3'); KS32-1
(5'-tctggaggaaagcgtggat-3', 5'-tgaagccgtagaagcaaaggta-3'); KS32-2
(5'-gcaatcccgaggcgtctcag-3', 5'-ccagcagcggccaggtaggagta-3'); KS32-3
(5'-ctgtccatcggaaactcctgtc-3', 5'-gccatggtgacgctgtagaa-3'); and KS43
(5'-cgagtcgcaggcacagtgtcac-3', 5'-ccagcacggccaggtaggagta-3'). Three
separate KS32 primers (designated KS32-1, -2, and -3) were employed to
identify the different putative KS32 splice forms, as previously
described (30). A 194-bp fragment of the hRFC open reading frame
(positions Effects of Induction of Endogenous p53 on hRFC Levels in REH
Cells--
Loss of functional wild type p53 was originally reported to
lead to enhanced Mtx sensitivity in H35 rat hepatoma cells (16). Although this was independent of changes in DHFR in this model (16), it
could involve any combination of other determinants of drug response
(e.g. Mtx polyglutamylation, Mtx membrane transport by RFC,
or levels of thymidylate synthase (28)) and/or secondary effects on
cell cycle progression, which manifest as decreased sensitivity to
Mtx.
To further explore the relationship between induction of
endogenous p53 and hRFC, we used the REH B-lymphoblastic
leukemia subline, which expresses high levels of hRFC transcripts and
protein, and wild type p53 (47). REH cells were treated with 0.5 µM daunorubicin to induce p53. p53 levels increased over
time and were maximal by 6 h (Fig.
1A). Notably, hRFC transcripts
also decreased in a time-dependent fashion (~5-fold by
9 h; Fig. 1B) and were accompanied by a similar loss of
[3H]Mtx transport (not shown). However, there were no
changes in the levels of transcripts for the housekeeping gene,
GAPDH (Fig. 1C), suggesting that the loss of hRFC
in REH cells is comparatively specific. These data strongly suggest a
suppression of hRFC levels and function by wild type p53.
Generation and Characterization of Double-stable
K562pTet-on/p53 Cells--
To better study the
relationships between p53 and hRFC levels and function, we generated a
double-stable transfectant (designated K562pTet-on/p53) in p53-null K562 cells, using a commercial
Tet-on system (37) to express wild type p53 under control of a
tetracycline/doxycycline-inducible promoter. We initially measured the
time course induction of p53 expression following exposure to a maximum
dose of Dox (2 µg/ml). In the absence of Dox, p53 was undetectable in
K562pTet-on/p53 cells on Western (Fig.
2A) or Northern blots (not
shown). Following Dox treatment, we found that p53 transcripts and
protein were detectable as early as 6 h and that induction by Dox
was maximal after 48 h of treatment. The induction of p53 protein
was dose-dependent over a wide range of Dox concentrations
(0.015-2 µg/ml; Fig. 2A), and p21 expression paralleled
the induction of p53 on Western blots (Fig. 2B), indicating
that p53 was transcriptionally active.
At 2 µg/ml Dox, there was obvious G1 arrest with a
3-fold loss in the number of cells in S-phase (Table
I) and an ~30% decrease in growth rate
(data not shown), compared with cells without Dox treatment. By annexin
V staining and flow cytometry analysis, there was no detectable
increase in the numbers of apoptotic cells after 48 h of Dox
treatment (not shown), indicating that expression of high levels of p53
in K562pTet-on/p53 cells did not stimulate apoptosis.
Collectively, our results establish the tightly regulated nature of p53
expression and downstream pathways in K562pTet-on/p53 cells
treated with Dox.
In growth inhibition assays, preinduction of p53 (with 2 µg/ml
Dox) for 48 h was accompanied by insignificant differences in Mtx sensitivities over uninduced cells (IC50 values of
8.83 ± 1.64 and 7.70 ± 0.70 nM (S.E.;
n = 3) with and without Dox, respectively). However,
unlike H35 cells (16), DHFR levels were significantly decreased
(~3-fold) in K562pTet-on/p53 cells in the presence of p53
(Fig. 2F). This was, presumably, due to downstream effects
on DHFR transcription (48).
hRFC Expression and Function in K562pTet-on/p53 Cells
following p53 Induction--
The lack of a significant change in Mtx
sensitivity despite significantly reduced DHFR (Fig. 2F),
initially suggested that decreased levels of this enzyme target must
somehow be offset by alterations in drug uptake. To directly explore
the possibility that p53 may regulate hRFC, K562pTet-on/p53
cells were treated with a wide range of Dox concentrations (0.015-2 µg/ml). After 48 h of incubation, total RNAs were prepared and probed on Northern blots with 32P-labeled full-length hRFC
cDNA. hRFC transcripts decreased accompanying expression of p53 and
in a dose-dependent manner with increasing concentrations
of Dox (Fig. 2C). At a maximum dose of Dox (2 µg/ml), there was an ~70% loss in hRFC transcripts. Likewise, there was a
significant loss of immunoreactive hRFC protein, detected with hRFC-specific antibody on Western blots (Fig.
3). Furthermore, there was a nearly
identical dose-dependent decrease in [3H]Mtx
influx capacity (Fig. 4A) in
K562pTet-on/p53 cells treated with Dox. At 2 µg/ml Dox,
hRFC transport activity was only ~30% of the level for the untreated
control. By contrast, neither hRFC transcripts (Fig. 2E),
hRFC protein (not shown), nor Mtx transport activity (Fig.
4B) significantly differed in pTRE vector control cells in
the presence and absence of 2 µg/ml Dox.
Effects of p15 Induction in K562p15Tet-on-8 Cells on
S-phase Fraction and hRFC Levels--
hRFC levels and function have
been linked to cell proliferation (49). Thus, the changes in hRFC
levels and function upon p53 induction (Figs. 2C, 3, and
4A) could potentially be due to indirect effects of elevated
p21, resulting in the loss of S-phase fraction (Table I) and
S-phase-dependent hRFC expression. In such a case, an
identical effect should result from elevated levels of p15, another
inhibitor of cyclin-dependent kinases-4 and -6 (34).
However, for the analogous p15 Tet-on-inducible model (K562p15Tet-on-8), hRFC mRNAs were unchanged on
Northern blots accompanying p15 induction with 2 µg/ml Dox (Fig.
5), even though there was a nearly
identical ~3-fold loss of S-phase cells (Table I). Likewise, Mtx
transport by hRFC was completely unaffected upon p15 induction (1.23 versus 1.31 pmol/mg of protein with and without Dox
pretreatment, after 180-s incubation with 0.5 µM
Mtx). Thus, a simple cell cycle arrest does not appear to
explain the decreased hRFC levels in cells that up-regulate wild type
p53.
It was of interest that DHFR also decreased (~2.5 fold) upon p15
induction in K562p15Tet-on-8 cells (Fig. 5D),
yet this was not accompanied by a corresponding increased Mtx
sensitivity (6.85 ± 0.15 and 9.00 ± 1.00 nM, in the presence and absence of Dox, respectively). The basis of this paradoxical result is not entirely certain, however, it may relate to
additional downstream effects of p15 (and also p53) on other folate-dependent pathways that impact Mtx response. For
instance, elevated p15 (and p21) might result in decreased thymidylate
synthase (50), analogous to DHFR, however, this would increase Mtx
resistance (51). Clearly, changes in levels of p15 or p53 can
potentially cause a multiplicity of effects in different cells and
tissues, involving hRFC, DHFR, and, possibly, thymidylate synthase,
which are not easily reconciled in terms of their Mtx sensitivities.
Effects of p53 Induction in K562pTet-on/p53 Cells on
hRFC Transcript Stability--
p53 has been reported to exhibit RNA
binding activity (52), which could conceivably affect mRNA
stabilities. Accordingly, the loss of hRFC transcripts in
K562pTet-on/p53 cells treated with Dox could possibly
reflect downstream effects of p53 on hRFC transcript stability. To
directly assess this possibility, K562pTet-on/p53 cells
were pretreated with Dox (2 µg/ml) for 24 h and subsequently with 10 µg/ml actinomycin D. Changes in hRFC transcript levels were
followed over 12 h on Northern blots (data not shown). Under these
conditions, the rates of hRFC transcript turnover were identical and
independent of p53 induction (half-lives of 5.2 and 5.9 h in the
presence and absence of Dox, respectively).
Effects of p53 on hRFC Involve Transcriptional Repression of the
Upstream hRFC-B Promoter--
The hRFC gene encodes
transcripts characterized by heterogeneous 5'-UTRs, reflecting multiple
transcription starts and variable splicing of alternative exons,
transcribed from dual TATA-less and CAAT-less promoters (30-32, 44).
By 5'-RACE (rapid amplification of cDNA ends) analysis, hRFC
transcripts in K562 cells contain predominantly (~93%) the KS43
5'-UTR from exon 1, transcribed from the upstream hRFC-B ("Pro43")
promoter (53). When total RNAs from K562pTet-on/p53 cells
were reverse-transcribed and PCR-amplified to identify the unique
5'-UTRs (previously designated KS6 and KS43, and the KS32 splice forms
I, II, and III (32, 45)), transcripts with the KS43 5'-UTR again
predominated (>90%; Fig.
6A). When normalized to
To further examine the mechanism(s) involved with this effect,
full-length (positions
These results show that induction of p53 suppresses hRFC-B promoter
activity and that this can be reversed with a trans-dominant-negative form of p53. Furthermore, the inhibitory effects of p53 can be localized to the hRFC-B minimal promoter region. The finding that transcriptional repression by p53 can be abolished by mutating the
highly conserved GC-box suggests a direct or indirect effect of p53 on
Sp1/Sp3 transactivation of the hRFC-B promoter.
Mechanisms of p53 Transcriptional Repression of the Basal hRFC-B
Promoter--
The mechanism by which p53 typically regulates cellular
functions involves its role as a transcriptional regulatory
activator/repressor (1-3) and may involve binding to specific
consensus sequences (11, 12, 14) and/or direct binding with individual
transcription factors (18-21). Although the p53 effects on hRFC
transcription can be localized to the basal hRFC-B promoter, no p53
response element in this region can be identified.
An important role for the Sp1 family of transcription factors in
activating basal hRFC-B transcription (via the conserved GC-box) has
been established by gel shift analyses of nuclear extracts prepared
from HepG2 and HT1080 cells and by transient cotransfections in
Drosophila SL2 cells with a pPac-Sp1 expression vector and
hRFC-B promoter constructs in pGL3-Basic vector (32). p53 has been
reported to bind with Sp1 (20), and its repressive effects on
Sp1-regulated promoters (MRP1 (17) and thymidylate synthase (18)) have
been suggested to occur by direct association and functional antagonism
of promoter transactivation. Consistent with this notion, in
Drosophila cells cotransfected with Sp1, p53, and the hRFC-B
promoter, a potent suppressive effect on promoter activity by p53 was
observed (data not shown).
However, on gel shifts with a 46-bp oligonucleotide (positions
Of course, putative Sp1·p53 interactions may not necessarily be
detectable on gel shifts, due to a lack of sensitivity and/or use of
suboptimal binding conditions. Alternatively, the repressive effects of
p53 on hRFC transcription may include downstream effects on Sp1 or Sp3
levels or structure (e.g. phosphorylation or
O-glycosylation (54, 55)) not detectable on gel shifts or
effects of p53 on accessory transcription proteins. Indeed, any
repressive effect of p53 can best be interpreted within the overall
context of hRFC-B promoter structure and function. The nature of the
putative inhibitory complex that forms ultimately depends not only on
the levels of individual transcription factors but on the formation of
the transcription initiation complex, and assorted other elements such
as transcription factor accessibility and post-translational
modifications (see above), the presence of upstream inhibitory or
activating elements, and the overall promoter architecture.
Conclusions--
In summary, our results document a suppression of
hRFC levels and function by wild type p53, suggesting that hRFC is an
important cellular target for this tumor suppressor. Wild type p53
represses hRFC expression at the level of transcription, and it appears to involve direct or indirect effects on the transactivation of the
hRFC-B minimal promoter. The mechanism of p53-mediated transcriptional repression of hRFC and its similarity to the p53 repression of other
TATA-less promoters is not well established. However, for hRFC, it
seems to be independent of its effects on cell cycle progression.
Although our findings suggest that treatment with DNA-damaging agents
(e.g., chemotherapy, irradiation) could possibly attenuate sensitivities to antifolates due to decreased hRFC for cells that express wild type p53, the ramifications of this likely depend on the
intracellular enzyme target and may vary in different tumors and
tissues. Likewise, decreased uptake of reduced folates by this
mechanism may exacerbate the effects of DNA-damaging agents, due to
perturbations of nucleotide pools required for DNA repair. Clearly, a
better understanding of the regulation of hRFC expression by wild type
p53 should provide insights into the chemotherapeutic efficacy of
antifolates in combination with other anticancer drugs and into the
relationships between nutritional folate deficiency, genetic
instability, and malignancy.
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(13, 14),
P-glycoprotein (MDR1 (15, 16)), MRP (17), and thymidylate synthase (18). Transcriptional repression by p53
is likely due to protein·protein interactions with transcription factors such as TATA-binding protein (19), Sp1 (20), and CCAAT-binding factor (21). In other cases, p53 repression has been mapped to a
p53-negative response element (14). Most recently, histone deacetylases
were implicated in transcriptional silencing of the p53 target genes,
MAP4 (22) and stathmin (23), through the formation of "repression complexes" composed of the Sin3a
corepressor, histone deacetylases, and p53. However, the extent to
which these mechanisms of transcriptional repression by p53 can be
generalized is unclear.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
-32P]dCTP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences.
[3',5',7-3H]Mtx (20 Ci/mmol) was purchased from Moravek
Biochemicals (Brea, CA). Unlabeled Mtx was provided by the Drug
Development Branch, National Cancer Institute, Bethesda, MD. Both
labeled and unlabeled Mtx were purified by high pressure liquid
chromatography prior to use (33). Restriction enzymes were purchased
from Promega (Madison, WI). Synthetic oligonucleotides were obtained
from Genosys Biotechnologies, Inc. (The Woodlands, TX).
-32P]dCTP, and blots were autoradiographed after
hybridization. Densitometry of autoradiographs on x-ray films was
performed on the Kodak Digital Science 440CF Image Station. Equal
loading of the RNAs was established by ethidium bromide staining of 28 S and 18 S RNAs or by probing with 32P-labeled GAPDH cDNA.
2016 to
959) and the basal hRFC-B (positions
1088 to
960;
numbering based on nonintron sequence relative to ATG
translation start site) constructs were subcloned into pGL3-Basic
plasmid (Promega) as previously described (30, 32).
K562pTet-on/p53 cells (~2.5 × 106 cells
per 6-well dish) were transfected with 5 µg of hRFC-B luciferase fusion gene constructs in pGL3-Basic, p53CON
(5'-GGACATGCCCGGGCATGTCC-3' (36)), or empty pGL3-Basic, along with 25 ng of pRL-SV40, using Lipofectin. Lipofection treatments were for
24 h, followed by Dox induction and an additional incubation for
48 h in complete RPMI 1640 media containing 20% iron-supplemented
calf serum. Cell lysates were assayed with a Dual-Luciferase reporter
assay system (Promega) on a TD-2420 luminometer (Turner Designs),
according to the manufacturer's protocol. The relative luciferase
activity was obtained by normalizing to Renilla luciferase
activity expressed from the pRL-SV40 construct. For certain
experiments, cells were cotransfected with 2.5 µg of a TDN-p53
construct (16).
46 to 148) was amplified as a measure of relative
hRFC expression (RFC/P8, 5'-cagtgtcaccttcgtcccctccg-3'; and RFC/JW,
5'-gggtgatgaagctctcccctgg-3').
-Actin was amplified with commercial
primers (CLONTECH) as an additional control. For all primer sets, PCR conditions were: 1 cycle (95 °C for 4 min) and
32 cycles (95 °C for 30 s, 67 °C for 30 s, 72 °C for
45 s). In separate control experiments, all primer sets were found
to amplify from purified cDNA templates with similar efficiencies (not shown).
RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Fig. 1.
Effects of induction of endogenous wild type
p53 on hRFC levels in REH cells. REH cells were treated with 0.5 µM daunorubicin (DNR) to induce endogenous
p53. Soluble protein extracts were prepared, and total RNAs were
extracted at the indicated times for Western and Northern blots
analysis, respectively. A, expression of p53 protein in REH
cells was assayed on Western blots with anti-p53 monoclonal antibody
(DO-1). B, levels of hRFC transcripts on Northern blots were
probed with 32P-labeled hRFC cDNA. C, equal
loading of the RNAs in B was established by probing with
32P-labeled GAPDH cDNA.
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Fig. 2.
Induction of wild type p53 in
K562pTet-on/p53 cells decreases the levels of hRFC
transcripts and increases DHFR protein.
K562pTet-on/p53 cells were treated with various Dox
concentrations (0-2 µg/ml) for 48 h. A, the results
of a Western blot analysis of p53 protein with increasing doses of Dox
are shown. B, induction of p21 in response to increasing p53
was assayed on Western blots with anti-p21waf antibody.
C, total RNAs were subjected to Northern blot analysis and
probed with a 32P-labeled hRFC cDNA. The
arrow indicates the major 3.1-kb hRFC transcript.
D, ethidium bromide staining of 28 S and 18 S RNAs for the
blot in C were used to assess equal RNA loading.
E, total RNAs from both induced (+Dox) and
uninduced ( Dox) vector control cells were subjected to
Northern analysis to assess the levels of hRFC transcripts. 28 S and 18 S ribosomal RNAs were stained with ethidium bromide as controls for
equal loading. F, soluble proteins (20 µg) from induced
(+Dox) and uninduced (
Dox) cells were analyzed
on Western blots with anti-DHFR antiserum.
Cell cycle distribution of K562pTet-on/p53 and
K562p15pTet-on-8 cells
) Dox (2 µg/ml) for 48 h. Portions of
the cells were stained with propidium iodide and analyzed for DNA
contents as described under "Materials and Methods."
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Fig. 3.
Expression of wild type p53 in
K562pTet-on/p53 cells is associated with a loss of hRFC
protein. K562pTet-on/p53 cells were treated with 2 µg/ml of Dox for 48 h, and sucrose gradient-purified plasma
membranes of Dox-induced cells (+) were prepared in parallel with the
uninduced cells ( ). Aliquots of membrane proteins (100 µg) were
electrophoresed on a 7.5% SDS-PAGE gel, transferred to PVDF membrane,
and probed with a protein A-purified hRFC-specific antibody. Enhanced
chemiluminescence was used for visualization. The broadly migrating
~85-kDa hRFC protein band (38, 43, 44), detectable with hRFC
antiserum, is indicated.
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Fig. 4.
Influx of [3H]Mtx by hRFC in
K562pTet-on/p53 (A) and
vector control (B) cells. Following exposure to a
wide range of Dox concentrations (0.015-2 µg/ml), cells were
harvested and incubated with tritiated Mtx (0.5 µM) for
180 s at 37 °C. Transport activity was expressed in units of
pmol/mg of protein as a function of Dox concentration (in µg/ml) and
are presented as mean values (±S.E.) from three experiments in
duplicate.
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Fig. 5.
Effects of p15 induction in
K562p15Tet-on-8 cells on hRFC levels.
K562p15Tet-on-8 cells were induced with 2 µg/ml Dox for
48 h followed by total RNA extraction. When these cells were
subjected to flow cytometry analysis, a G1 arrest and an
~3-fold loss of S-phase fraction were detected (Table I).
A, total RNAs were fractionated on a 0.7%
formaldehyde/agarose gel and hybridized with 32P-labeled
hRFC cDNA to detect the major 3.1-kb hRFC transcript. B,
the blot was then stripped and reprobed with 32P-labeled
p15 cDNA to assess the levels of p15 transcripts in the presence
(+) and absence ( ) of Dox. C, 28 S and 18 S ribosomal RNAs
were stained with ethidium bromide as controls for equal loading.
D, soluble proteins (20 µg) from induced (+Dox)
and uninduced (
Dox) cells were analyzed on Western blots
with anti-DHFR antiserum.
-actin (Fig. 6C), both the KS43 5'-UTR and open reading
frame PCR products were significantly decreased (~70%) in cells
treated with Dox (+Dox; panel B) compared with
untreated cells (
Dox; panel A). Thus, the loss
of hRFC transcripts in K562pTet-on/p53 cells upon p53
induction is completely due to effects on KS43 transcripts transcribed
from the hRFC-B promoter.
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Fig. 6.
RT-PCR analysis of hRFC-B transcripts for
5'-UTR utilization. Total RNAs were extracted from
K562pTet-on/p53 cells treated for 48 h with
(B) and without (A) Dox and used directly for
synthesis of the cDNA. cDNAs were PCR amplified with primer
sets specific for the KS6 (lane 1), KS32 (I, II, and III;
lanes 2-4, respectively), and KS43 (lane 5)
5'-UTRs (30, 44), and the hRFC open reading frame (lane 6).
PCR products were fractionated on a 2% agarose gel and detected with
ethidium bromide staining. A 100-bp DNA ladder (M) and the
sizes of the major PCR products are indicated. C, a
commercial primer set was used to amplify -actin (838 bp) to
normalize the relative levels of hRFC PCR products in the presence and
absence of Dox.
2016 to
959) and basal (positions
1088 to
960) hRFC-B promoter constructs (in pGL3-Basic) (32), containing a
luciferase reporter gene, were transiently expressed in
K562pTet-on/p53 cells in the presence and absence of Dox (2 µg/ml). For both full-length and basal promoter constructs, induction
of p53 resulted in an ~3- to 4-fold decrease in relative luciferase
activity (Fig. 7, A and
B, respectively). p53 repression of the low residual levels
of basal activity was completely abolished in a promoter construct in
which a highly conserved GC-box element (cccgccc; positions
1081 to
1075 (32)) was mutated (to cccgaac; data not shown). Luciferase
activity was completely unaffected by Dox for the vector control cells
(not shown). When a TDN-p53 expression construct was cotransfected with
the full-length hRFC-B construct, the repressive effects of p53 were
largely abolished (Fig. 7A). A similar effect of TDN-p53 on
basal hRFC-B activity was also observed (not shown). Fig. 7C
shows that induction of p53 results in a potent stimulation of
luciferase activity in K562pTet-on/p53 cells transfected
with the p53-responsive p53CON reporter plasmid (36).
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Fig. 7.
Effects of p53 on hRFC levels in
K562pTet-on/p53 cells involve transcriptional repression of
both full-length and basal hRFC-B promoters. Full-length
(A) and basal (B) hRFC-B promoter constructs
linked to a luciferase reporter gene in pGL3-Basic, were transiently
expressed in K562pTet-on/p53 cells with (+) and without
( ) 2 µg/ml Dox. Five micrograms of each hRFC promoter construct was
cotransfected with 25 ng of pRL-SV40 plasmid for normalization. Also
shown in A are the effects of cotransfections with the
TDN-p53 construct (2.5 µg) on luciferase activity of the full-length
hRFC-B promoter construct (5 µg). C shows the effects of
p53 induction on promoter activity of the p53-responsive luciferase
reporter construct, p53CON, as a positive control. All data are
presented mean values (±S.E.) from three experiments in
duplicate.
1088
to
1043) encompassing the Sp1 binding site (
1081 to
1075) (32)
and nuclear extracts prepared from K562pTet-on/p53 cells,
treated with and without Dox (2 µg/ml), there were no notable
differences in the levels or migrations of the DNA·Sp1 or DNA·Sp3
complexes (not shown). The DNA·Sp1 and DNA·Sp3 complexes could be supershifted with antibodies specific for Sp1 and Sp3, respectively, however, p53 antibody did not effect a supershift (not shown).
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Bert Vogelstein, Jerry Shay, and David Beach for providing plasmid constructs for our studies and Dr. Ray Blakley for providing recombinant human DHFR protein for antiserum preparation. The assistance of Dr. Surreyya Savasan with the apoptosis assays is appreciated.
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FOOTNOTES |
---|
* This study was supported by Grants CA53535, CA77641, and ES05851 from the National Institutes of Health, and by the American Lebanese Syrian Associated Charities.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: Experimental and
Clinical Therapeutics Program, Karmanos Cancer Institute, 110 E. Warren
Ave., Detroit, MI 48201. Tel.: 313-833-0715 (ext. 2407); Fax:
313-832-7294; E-mail: matherly@kci.wayne.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M005248200
2 S. Shah, J. Taub, T. L. Witt, J. Pollock, B. C. Ding, M. Amylon, J. Pullen, Y. Ravindranath, and L. H. Matherly, manuscript submitted.
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ABBREVIATIONS |
---|
The abbreviations used are: bp, base pair(s); DHFR, dihydrofolate reductase; Dox, doxycycline; D-PBS, Dulbecco's phosphate-buffered solution; RFC, reduced folate carrier; hRFC, human RFC; Mtx, methotrexate; MRP, multidrug resistance protein; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene difluoride; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcription-polymerase chain reaction; TDN, trans-dominant-negative; UTR, untranslated region; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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