The Mycotoxin Fumonisin B1 Transcriptionally Activates the p21 Promoter through a cis-Acting Element Containing Two Sp1 Binding Sites*

Yange ZhangDagger §, Martin B. Dickman§, and Clinton JonesDagger

From the Dagger  Center for Biotechnology, Department of Veterinary and Biomedical Sciences, and the § Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68503

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-gamma -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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 [alpha -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 [alpha -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 [alpha -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.

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INTRODUCTION
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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 [alpha -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.


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p21 expression is induced by many agents including transforming growth factor beta , 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 beta  (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 beta  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.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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