The Mycotoxin Fumonisin B1 Transcriptionally
Activates the p21 Promoter through a
cis-Acting Element Containing Two Sp1 Binding
Sites*
Yange
Zhang
§,
Martin B.
Dickman§, and
Clinton
Jones
¶
From the
Center for Biotechnology, Department of
Veterinary and Biomedical Sciences, and the § Department of
Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503
 |
ABSTRACT |
Fumonisin B1 (FB1)
is a food-borne mycotoxin produced by Fusarium moniliforme.
Structurally FB1 resembles sphingoid bases, and ingestion
of FB1 causes several animal diseases. FB1 will cause hepatic carcinoma in rats and is implicated as a cofactor in
esophageal or hepatic carcinoma. Previous studies concluded that
FB1 repressed cyclin-dependent kinase 2 (CDK2)
activity but induced CDK inhibitors
p21Waf1/Cip1, p27Kip1,
and p57Kip2 in monkey kidney cells (CV-1). In
contrast, CV-1 cells transformed by simian virus 40 are resistant to
the antiproliferative or apoptotic effects of FB1.
Consequently, FB1 treatment of CV-1 cells leads to cell
cycle arrest and apoptosis. In this study, we demonstrate that
FB1 transcriptionally activates the p21
promoter. Functional analysis of the p21 promoter by
reporter gene assays mapped the FB1-responsive region to
124 to
47. DNase I footprinting analysis revealed two protected
motifs that span the FB1-responsive region,
124 to
101
(footprint II) and
89 to
67 (footprint III). Further studies
demonstrated that DNA sequences from
124 to
101 were sufficient for
FB1 stimulation. DNA sequences from
124 to
101 contain
two Sp1 binding sites, and gel shift assays provided evidence that
nuclear factors specifically bind to this region. Disruption of the two
Sp1 binding sites abrogated the binding of nuclear proteins and
prevented activation by FB1. Taken together, these results
suggest that Sp1 or Sp1-related proteins mediate
FB1-induced activation of the p21 promoter.
 |
INTRODUCTION |
Fumonisin B1
(FB1)1 is a
mycotoxin, produced by Fusarium moniliforme, a fungal
pathogen that causes stalk and ear rot of maize (1). F. moniliforme is prevalent throughout the world and as such is an
important plant pathogen. FB1 ingestion is associated with
increased incidence of esophageal cancer in southern regions of Africa
and parts of China (2, 3). FB1 is hepatocarcinogenic and
causes primary hepatocellular carcinoma or cholangiocarcinoma in rats
(4). FB1 is also a potent tumor promoter in rat liver after
initiation with diethylnitrosoamine (5, 6). FB1 causes equine leukoencephalomalacia, nephrotoxicity (7), and porcine pulmonary
edema (8) and is hepatotoxic in rats (9). FB1 is also
nephrotoxic to several animal species, and kidney tissue is very
sensitive to its toxic effects (10, 11). An in vivo study
demonstrated that FB1 induced apoptosis in rat kidney or livers (12, 13). Thus, ingestion of FB1 by humans or food animals is a health concern.
Structurally, FB1 is similar to sphinganine or other
sphingoid bases (14, 15) and is the first known naturally occurring inhibitor of sphingolipid biosynthesis. Sphinganine is an intermediate in the biosynthesis of the sphingosine backbone of ceramide,
sphingomyelin, cerebrosides, gangliosides, and sulfatides.
FB1 inhibits ceramide synthase, which alters
sphinganine:sphingosine ratios (16, 17) and inhibits the synthesis of
ceramide. Sphingolipids are involved in the regulation of cell contact,
cell growth, and differentiation primarily since sphingomyelin is part
of a signal transduction pathway generating ceramide as second
messenger (18). Thus disruption of sphingolipid biosynthesis by
FB1 is likely to be important for the toxic effects of
FB1.
FB1 is cytotoxic to certain mammalian cells including baby
hamster kidney cells, rat primary hepatocytes, turkey lymphocytes, or
chicken macrophages (19-22). The most sensitive cell lines are rat
hepatoma H4TG and Madin-Darby canine kidney cells with IC50 values of 4 and 2.5 µg/ml, respectively (23, 24). FB1
treatment leads to cell cycle arrest and apoptosis in monkey kidney
cells (CV-1) (25), inhibits certain protein kinase C isoforms (26), inhibits cyclin-dependent kinase 2 (CDK2) activity, and
reduces cyclin E protein levels (27). In contrast, CDK inhibitors,
p21Waf1/Cip1, p27Kip1,
and p57Kip2 are induced after FB1
treatment. Consequently dephosphorylation of the retinoblastoma protein
(RB) occurs.
In recent years, numerous studies have established that cell cycle
progression is controlled by CDKs and cyclins (28, 29). CDK inhibitors,
such as p21, bind CDK-cyclin complexes and inhibit CDK activity
(30-32). Induction of p21 expression occurs in response to a variety
of antiproliferative stimuli thus resulting in cell cycle arrest or
apoptosis (33, 34). p21 expression is induced by the p53 protein upon
DNA damage (35, 36). However, p53-independent expression of p21 occurs
during cell growth and differentiation (37, 38), suggesting that the
function of p21 is not limited to cell cycle arrest
following DNA damage. At low concentrations, p21 promotes the assembly
of CDK4-cyclin D complexes, whereas at high concentrations it inhibits
kinase activity (39). Furthermore, p21 can interact with proliferating
cell nuclear antigen to inhibit DNA replication by preventing
proliferating cell nuclear antigen binding to DNA polymerases (40).
The p21 promoter contains several cis-acting
elements that are transcriptionally activated by different
transcription factors in response to various agents. For example, two
p53 binding sites are located at
2.2 and
1.3 kilobases
upstream of the transcription start site (41). These sites are required
for p53-dependent transactivation of the p21
promoter. MyoD is a skeletal muscle-specific transcription factor that
recognizes sequences between
1749 and
1717. During skeletal muscle
differentiation, MyoD induces p21 expression, resulting in
cell cycle arrest (42). Stat1 is a cytokine-induced signal transducer
and an activator of transcription which recognizes DNA sequences
between
698 and
689 in response to interferon-
-induced growth
suppression (43). The proximal promoter of p21 contains a
GC-rich region with several potential Sp1 binding sites that regulate
p21 promoter activity (44-46).
Although extensive studies have been conducted concerning the toxicity
and biological effects of FB1, the molecular mechanisms of
FB1 action are not well understood. To understand the means by which FB1 induces expression of p21, we analyzed the
region of the p21 promoter necessary for FB1
activation. These studies demonstrated that the two Sp1 binding sites
within
124 to
101 were necessary and sufficient for
FB1-induced p21 transcription in CV-1 cells.
Mutagenesis of the Sp1 binding sites abrogated the binding of nuclear
factors and inhibited FB1 activation. Thus FB1
induces cell cycle arrest and apoptosis by transcriptionally activating
p21 via Sp1 binding sites.
 |
MATERIALS AND METHODS |
Cells and Chemicals--
CV-1 cells (African green monkey kidney
cells) were grown in Earle's modified Eagle's medium supplemented
with 5% fetal bovine serum. FB1 was obtained from Sigma,
R. D. Plattner (>99% pure) (USDA, Peoria, IL), or R. Eppley
(>99.9% pure) (USDA). FB1 was dissolved at 5 mM in calcium- and magnesium-free phosphate-buffered saline
(80 mM Na2HPO4, 20 mM
NaH2PO4, 100 mM NaCl, pH 7.5) and kept in the dark at 4 °C.
Oligonucleotides--
Four p21 promoter fragments,
124 to
47,
124 to
101 ( (FP II),
89 to
67 (FP III), and
124 to
101 in which the two Sp1 binding sites were mutated (FP
II-mut), were synthesized with 5'-CTAG overhangs and purified from 15%
urea-polyacrylamide gel. The purified complementary strands of
oligonucleotides were annealed at 50 mM in 20 mM Tris-HCl, pH 8.3, 0.1 M NaCl by heating at
95 °C for 5 min followed by incubating at 5 °C below the melting point for 1 h, and subsequent storage at 4 °C.
Construction of p21 Promoter Reporter Plasmids--
The
full-length human p21 promoter construct pWWP and a series
of 5'-processive deletion constructs were obtained from Dr. Bert
Vogelstein. The 3'-end of all of the promoter segments extended to +16
from transcription start site (47). To insert p21 promoter fragments upstream of a heterologous promoter, the full-length p21 promoter from
2328 to +16 was released from plasmid
pWWP by HindIII digestion, and this 2.4-kilobase fragment
was gel purified and digested with PstI. The resulting two
fragments corresponding to
2328 to
210 and
210 to +16 were
recovered and inserted into the respective sites of vector pBLcat
(Promega, Madison, WI). To clone multimers of the p21
promoter fragment upstream of the thymidine kinase (TK) promoter, the
respective double-stranded oligonucleotides were phosphorylated by T4
polynucleotide kinase before ligation into the unique Xba I
site of pBLcat. The copy numbers of each insert were determined by both
2% agarose gel and 8% polyacrylamide gel electrophoresis. All
plasmids were prepared by alkaline lysis followed by two CsCl gradients.
Transient Transfection and CAT Assays--
p21
promoter reporter constructs were transfected into CV-1 cells by the
calcium phosphate precipitation method (48). Briefly, cells were plated
at a 1:5 ratio into 100-mm culture dishes the day before transfection.
3 h before transfection the old medium was removed and replaced
with fresh medium. For each transfection, 21 µg of reporter plasmid
was used to form DNA-calcium phosphate coprecipitates. This solution
was incubated with cells for 12 h and then replaced with fresh
medium. 24 h later, cells were split at a 1:2 or 1:4 ratio; half
of the cells were treated with 5 µM FB1 and
incubated for 24 h. Total cell lysate was prepared by three
freeze-thaw cycles in 0.25 M Tris, pH 8.0. CAT activity was
measured in the presence of 0.2 µCi of
[14C]chloramphenicol and 0.5 mM
acetylcoenzyme A for 3 h or longer. All forms of chloramphenicol
were separated by thin layer chromatography. The amount of acetylated
chloramphenicol was measured with a PhosphorImager (Molecular Dynamics,
Sunnyvale, CA).
Preparation of Nuclear Extract--
Nuclear extract was prepared
as described by Dignam et al. (49) with the following
modifications. The crude nuclei obtained from at least 5 × 107 cells were resuspended in 500 µl (equal to half the
nuclei pellet volume) of buffer B (20 mM Hepes, pH 7.9, 20 mM KCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 0.5 mM dithiothreitol,
0.5 mM phenylmethylsulfonyl fluoride, and 5 µg/ml
proteinase inhibitor mixture containing leupeptin, pepstatin, and
antipain, respectively). Another 500 µl of buffer C (the same as
buffer B except for 1.2 M KCl) was added drop by drop with
continuous agitation. The nuclei were incubated for 30 min and cleared
by centrifugation at 14,000 rpm for 30 min. The supernatant was
dialyzed for 1 h and cleared by centrifugation at 14,000 rpm for
20 min. The resulting supernatant was aliquoted and stored at
110 °C. The protein concentration was determined by the Bradford
assay (50).
DNase I Footprinting Analysis--
The p21 promoter
segment from
210 to +16 was cloned into pBLcat and subjected to DNase
I footprinting analysis. To label the coding strand, the construct was
digested by HindIII, and the overhang was filled in with
[
-32P]dATP and Klenow polymerase. The radiolabeled
fragment was released by digestion with XhoI and purified
from a 5% native acrylamide gel. The noncoding strand was labeled by
digestion with XhoI, incubation with
[
-32P]dCTP and Klenow to fill in the 5'-overhang, and
release of the fragment by HindIII digestion. The binding
reaction was performed in a total volume of 50 µl containing 12 mM Hepes, pH 7.9, 12% glycerol, 60 mM KCl, 2.5 mM MgCl2, 0.1 mM EDTA, 0.3 mM dithiothreitol, and 0.3 mM
phenylmethylsulfonyl fluoride. 75 µg of nuclear extract was incubated
with 2 µg of double-stranded poly(dI-dC) at room temperature for 10 min prior to the addition of 0.5-0.6 ng of radiolabeled probe. The
binding reactions were incubated at room temperature for 30 min.
CaCl2 was added to a final concentration of 2.5 mM, and freshly diluted DNase I was subsequently added to
digest the probe. For the naked probe, 0.11, 0.036, or 0.012 unit of
DNase I was used in separate reactions. For reactions in the presence
of nuclear extracts, 1, 0.33, or 0.11 unit of DNase I (Roche Molecular
Biochemicals) was used. The digestion reactions were allowed to proceed
for 1 min at room temperature and were stopped by adding equal volume
of stop buffer (100 mM Tris-HCl, pH 7.6, 100 mM
EDTA, 1% SDS).150 µg of proteinase K was then added and incubated at
56 °C for 45 min. The samples were extracted with
phenol/chloroform/isoamyl alcohol (50:49:1) followed by ethanol
precipitation. The digested probes were analyzed on 6%
urea-polyacrylamide gel. Chemical (G+A) sequencing analysis was
performed by the Maxam and Gilbert method (51).
Electrophoretic Mobility Shift Assay--
The double-stranded
oligonucleotides were end-labeled with [
-32P]dCTP and
Klenow polymerase and purified by passing through a Sephadex G-50
column (Amersham Pharmacia Biotech). DNA-protein binding reactions were
performed in a total volume of 20 µl containing 12 mM
Hepes, pH 7.9, 12% glycerol, 60 mM KCl, 0.12 mM EDTA, 0.3 mM dithiothreitol, 0.3 mM phenylmethylsulfonyl fluoride, and 1 µg of
double-stranded poly(dI-dC). 10 µg of crude nuclear extract was
incubated with 250 pg of end-labeled probe at room temperature for 20 min, and the reactions were subsequently resolved on a 5% native
acrylamide gel in 0.25 × TBE running buffer. For competition experiments, a 100-fold molar excess of unlabeled oligonucleotides was
added to the binding reactions and incubated at room temperature for 10 min prior to the addition of radiolabeled probes.
 |
RESULTS |
Localization of p21 Promoter Region Responsible for the
Transcription Activation by FB1--
A previous study
demonstrated that expression of the p21 protein was induced by
FB1 treatment (27). To investigate whether the
p21 promoter was transcriptionally activated by
FB1, a series of deletion constructs spanning the
p21 promoter (Fig.
1A) was transfected into CV-1
cells, and promoter activities were compared in cells treated with
FB1 or without. As shown in Fig. 1B,
p21 promoter activity (
2328/+16) was stimulated by
FB1 after 24 h of treatment. The deletion constructs
were also stimulated when cells were treated with FB1. When
p21 promoter sequences spanning
124 to
47 were inserted
at the 5'-terminus of a TK construct (3×
124/
47), FB1
reproducibly stimulated this construct up to 10-fold. In contrast,
sequences from
2328 to
210 were not stimulated when linked to the
TK promoter. Thus, p21 promoter sequences between
124 and
47 contained crucial elements that were necessary for activation by
FB1 treatment of CV-1 cells.

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Fig. 1.
Localization of the
FB1-responsive region in the p21
promoter. Panel A, schematic diagram of the
various p21 promoter constructs that were fused to the CAT
reporter gene. The two p53 consensus binding sites are located at the
5'-end of the full-length p21 promoter. Two additional
constructs were created by cloning three copies of the 124 to 47
fragment or 2328 to 210 region of the p21 promoter
upstream of the herpes simplex virus (HSV)-TK promoter in vector
pBLcat. Panel B, the respective constructs (21 µg) were
transfected into CV-1 cells. 24 h after transfection, cells were
split in a 1:4 or 1:2 ratio. Half of the cultures were then treated
with 5 µM FB1 for 24 h. Total cell
lysates were prepared and CAT activity measured using thin layer
chromatography. The fold activation induced by FB1 is shown
on the bottom. Each transfection experiment was repeated at
least three times, and these results are representative of all
experiments.
|
|
Identification and Characterization of Nuclear Proteins Binding to
p21 Promoter--
To determine if FB1 induces nuclear
proteins that interact with DNA sequences in the region between
124
and
47, in vitro DNase I footprinting analysis was
performed. To generate strand-specific probes for DNase I footprinting,
sequences from
210 to +16 were obtained from the pWWP construct and
inserted into pBLcat. Nuclear extracts were prepared from CV-1 cells
that were treated with 5 µM FB1 for 48 h. As a control, nuclear extracts were prepared from untreated CV-1
cells. Three distinct areas of the p21 promoter were
protected from DNase I digestion regardless of whether the nuclear
extracts were prepared from normal or FB1-treated cells. Analysis on both strands showed nearly identical protection regions (Fig. 2), and these were designated I,
II, or III, respectively. No dramatic or reproducible differences were
detected in the footprints from CV-1 cells relative to CV-1 cells
treated with FB1. Two canonical Sp1 binding sites were
contained within FP II, and one Sp1 binding site was contained within
FP III.

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Fig. 2.
Footprinting analysis of the p21 promoter
210 to +16 region. Panel A, p21 promoter
sequences spanning 210 to +16 were obtained from the full-length
fragment and recloned into pBLcat. Labeling was performed on coding or
noncoding strands by filling in the restriction sites with
[ -32P]dNTP and Klenow enzyme. Each probe was subjected
to chemical G+A sequence analysis. DNase I footprinting analysis was
performed in the absence of nuclear extract (naked) or in
the presence of nuclear extracts from CV-1 cells (CV-1) or
CV-1 cells treated with 5 µM FB1 for 48 h (CV-1+FB1). Arrows indicate
increasing amounts of DNase I (0.012, 0.036, or 0.11 unit for the naked
probe and 0.11, 0.33, or 1 unit for CV-1 or CV-1+FB1) used
to digest the probes. The boxed areas represented the three
footprints (I, II, or III) detected on both strands. Panel
B, the protected sequences are shown at the bottom, and
the underlined sequences indicate the consensus Sp1 binding
sites within FP II or III.
|
|
Because FP II and FP III were within the
124 to
47 region that was
necessary for FB1 stimulation, gel shift assays were performed using oligonucleotides spanning FP II or III (Fig.
3B). An oligonucleotide
containing mutated Sp1 binding sites from
124 to
101(FP II-mut) was
also utilized in the gel shift assay. Four specific shifted complexes
(A, B, C, and D) were detected using FP II. Complex A or B was
efficiently competed by FP II and a Sp1 oligonucleotide but not by FP
II-mut, suggesting that A or B was bound by proteins that bind
consensus Sp1 sites. This hypothesis is supported by the finding that
FP II-mut did not contain complex A or B but did contain complexes C
and D. It is also possible that D was bound by a protein that binds Sp1
sites because the Sp1 oligonucleotide diminished the intensity of D. Two specific DNA-protein complexes were detected using the FP III
oligonucleotide. Consistent with the DNase I footprinting analysis, no
difference was detected in DNA-binding proteins using nuclear extracts
prepared from FB1-treated or untreated CV-1 cells.

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Fig. 3.
Gel shift analysis of FP II and III.
Sequences corresponding to FP II, FP II-mut, or FP III were used to
perform gel shift assays. For each binding reaction, 10 µg of nuclear
extracts from CV-1 cells (0) or CV-1 cells treated with FB1
for 6, 24, 48 h was incubated with the radiolabeled probe for 20 min at room temperature. For the competition experiment, a 100-fold
molar excess of the unlabeled competitor was incubated with the nuclear
extract for 10 min before the addition of the probes. Binding reactions
were then run on a 4% acrylamide gel with 0.25 × TBE as running
buffer. Arrows indicated the four specific DNA-protein
complexes (A, B, C, and D). The unbound oligonucleotides were run off
the gel to resolve A and B.
|
|
Sp1 or Sp1-related Transcription Factors Were Involved in
FB1-induced p21 Transcription--
To determine whether FP
II, FP II-mut, or FP III contributed to FB1-induced
transcription of the p21 promoter, the three
oligonucleotides were multimerized, inserted into the 5'-terminus of
the TK promoter, and CAT expression measured. For each construct, two
clones containing different copies of the oligonucleotides were
selected to transfect CV-1 cells. Transfected cells were then treated
with 5 µM FB1 for 24 h, and promoter
activity was compared with that of untreated cells. Constructs
containing FP II were consistently activated up to 5-fold by
FB1 treatment. However, promoter activity of constructs containing FP II-mut or FP III were not activated by FB1
(Fig. 4). In fact, CAT activity of FP
II-mut or FP III was reduced compared with that of untreated controls.
Based on this result, we concluded that DNA sequences within FP II were
necessary for FB1-induced transcription of the
p21 promoter and that the two Sp1 consensus binding sites
within FP II were important.

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Fig. 4.
Sequences that bind Sp1 or Sp1-related
transcription factors were responsible for FB1
transactivation of p21 promoter. Multimers of FP
II, FP II-mut, or FP III were cloned upstream of the HSV-TK promoter to
drive expression of CAT. For each construct, two clones containing
different copies of the insert were selected to transfect CV-1 cells.
The exact copy number is indicated at the top of the figure.
21 µg of each construct was transfected into CV-1 cells. 24 h
after transfection, cells were split in a 1:2 ratio, and half of the
cultures were treated with 5 µM FB1 for
24 h. Cell extracts were prepared and CAT activities measured. The
fold activation number induced by FB1 is given at the
bottom.
|
|
 |
DISCUSSION |
p21 expression is induced by many agents including
transforming growth factor
,
12-O-tetradecanoylphorbol-13-acetate, growth factors,
interleukin 2, retinoids, and calcium or vitamin D (44-46, 52-54). In
this study, we demonstrated that FB1, a fungal mycotoxin, transcriptionally activated p21 promoter activity. To
investigate the mechanism involved in the stimulation of p21 by
FB1, deletion analysis of the p21 promoter
demonstrated that the FB1-responsive region was localized
between
124 and
101 (FP II). Induction of p21 promoter
activity by FB1 was p53-independent because the two
p53 binding sites were not required for FB1
stimulation. This result is consistent with our previous observations
that p53 protein levels do not increase dramatically after treatment
with FB1 (27). DNA sequences containing two Sp1 sites were
necessary for FB1-induced transcription. This conclusion
was based on the following results. 1) The FB1-responsive
region from
124 to
101 contains two consensus Sp1 binding sites. 2)
Nuclear proteins specifically recognized this region, and a Sp1
oligonucleotide competed for binding. 3) Mutations of the two Sp1
binding sites prevented protein binding and abolished stimulation by
FB1. However, the binding pattern and intensity of the Sp1
binding proteins were not changed dramatically after FB1
treatment. Although FB1 does not appear to stimulate the
synthesis of Sp1 or enhance the binding of Sp1 to its recognition site,
we propose that post-translational modification of Sp1-like factors or
protein-protein interactions occurs after FB1 treatment, and these changes are responsible for p21 promoter
activation. A previous study has demonstrated that neoplastic CV-1
cells (COS-7) are resistant to the apoptotic and antiproliferative
effects of FB1. Consistent with the observation is the fact
that p21 levels in COS-7 cells are low and not induced
following FB1 treatment (27). Thus, the ability of
FB1 to induce p21 levels correlates with growth
arrest and as such may play a role in the pathogenic potential of
FB1.
Sp1 or Sp1-related proteins transactivate numerous cellular or viral
genes (55-57), and Sp1-mediated transactivation is complex. The
activity of Sp1 is regulated by O-linked glycosylation and phosphorylation (58, 59). Glycosylation appears to regulate transcriptional activation but not DNA binding. Sp1 is phosphorylated by a DNA-dependent protein kinase, but phosphorylation does
not appear to affect DNA binding or transcription activation.
Protein-protein interactions also regulate Sp1 activity. For
example, Sp1 activation requires coactivator dTAF II110, which
associates with TATA-binding protein (60). Sp1 also interacts with the
cell cycle-regulated transcription factor, E2F, and this interaction
leads to activation of Sp1 or E2F consensus sites (61, 62). E2F also
binds to GC-rich regions of promoters and activates these sequences
(63), suggesting that E2F-Sp1 interactions can activate promoters by more than one mechanism. Finally, a negative regulator of Sp1 is
released by the tumor suppressor retinoblastoma protein
(64, 65). Previous studies have shown that FB1
induced dephosphorylation of retinoblastoma protein (27). It is not
known if E2F or retinoblastoma protein interaction with Sp1 is altered
in CV-1 cells after FB1 treatment.
The Sp1 binding sites in the p21 promoter are required for
induction by phorbol ester, okadaic acid, or transforming growth factor
(44, 45). Because FB1 represses certain protein kinase C isoforms (26), and phorbol esters induce protein kinase C activity,
it appears that activation of the p21 promoter by phorbol esters is not the same as FB1. Transforming growth factor
activation of the p21 promoter requires the Sp1 binding
site located between
84 and
74. These DNA sequences are contained
within FP III and were not required for FB1 activation.
FB1 can activate mitogen-activated protein kinase (66).
However, the induction of p21 by FB1 was not affected by
several mitogen-activated protein kinase inhibitors (data not shown),
suggesting that mitogen-activated protein kinase does not play a role
in p21 activation by FB1. Recent reports have demonstrated
that the tumor suppressor BRCA1 activates the p21 promoter
and that DNA sequences located between
117 and
93 (FP II) are
necessary for activation (67). Because BRCA1 is a transcription factor
that associates with RNA polymerase II (68), it is tempting to
speculate that FB1 regulates p21 promoter activity by a BRCA1-dependent mechanism. In summary, our
results show that FB1 induces transcription of
p21 via two Sp1 sites in the p21 promoter. The
activity is likely mediated by a post-translational modification of Sp1
factors which has not yet been identified. The induction of p21
activity by FB1 is consistent with G1 arrest and apoptosis, both of which occur following toxin treatment.
 |
ACKNOWLEDGEMENTS |
We are very grateful to Dr. Bert Vogelstein
for generous gifts of the p21 promoter constructs.
 |
FOOTNOTES |
*
This work was supported by a grant from the Institute of
Agriculture and Natural Resources, University of Nebraska-Lincoln Center for Biotechnology and by United States Department of Agriculture Grant NRICGP 9602186.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. Tel.:
402-472-1890; Fax: 402-472-9690; E-mail: cj{at}unlinfo.unl.edu.
 |
ABBREVIATIONS |
The abbreviations used are:
FB1, fumonisin B1;
CDK, cyclin-dependent kinase;
FP, footprint;
CAT, chloramphenicol acetyltransferase;
TK, thymidine
kinase.
 |
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