|Home | About | Journals | Submit | Contact Us | Français|
PPAR δ was identified as a target of APC through the analysis of global gene expression profiles in human colorectal cancer (CRC) cells. PPARδ expression was elevated in CRCs and repressed by APC in CRC cells. This repression was mediated by β-catenin/Tcf-4-responsive elements in the PPARδ promotor. The ability of PPARs to bind eicosanoids suggested that PPARδ might be a target of chemopreventive non-steroidal anti-inflammatory drugs (NSAIDs). Reporters containing PPARδ-responsive elements were repressed by the NSAID sulindac. Furthermore, sulindac was able to disrupt the ability of PPARδ to bind its recognition sequences. These findings suggest that NSAIDs inhibit tumorigenesis through inhibition of PPARδ, the gene for which is normally regulated by APC.
Colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States. Recent advances in the understanding of CRC have raised expectations that this growing knowledge might lead to improved cancer prevention. In this regard, the identification of genetic alterations that underlie the initiation of colorectal tumors and of drugs which can prevent colorectal tumors show particular promise. Here, we suggest a molecular basis for the convergence of these two heretofore separate lines of investigation.
A growing body of evidence has shown that nonsteroidal anti-inflammatory drugs (NSAIDs) can suppress colorectal tumorigenesis in both humans and rodents (reviewed in Smalley and DuBois, 1997; Thun, 1997). In the general population, epidemiological studies have documented a decreased risk of CRC deaths associated with use of the NSAID aspirin. In individuals with familial adenomatous polyposis (FAP), an inherited predisposition to multiple colorectal polyps, the NSAID sulindac can reduce both the size and number of colorectal tumors. Likewise, sulindac and other NSAIDs have proven to be effective in prevention of intestinal tumorigenesis in mouse models of FAP. The molecular basis for these striking chemopreventive effects has been attributed to inhibition of cyclooxygenases (COX) and the resulting decrease in prostaglandin production (reviewed in Prescott and White, 1996). Consistent with this, expression of COX-2 is elevated in human colorectal tumors (Eberhart et al., 1994; Sano et al., 1995), and inactivation of the COX-2 gene in mice is associated with decreased intestinal tumorigenesis (Oshima et al., 1996).
However, other observations are difficult to reconcile with COX being the sole target of NSAIDs in the colon. For example, NSAID derivatives that lack the ability to inhibit COX have been shown to inhibit colon tumor growth in vivo and in vitro (Piazza et al., 1995, 1997; Mahmoud et al., 1998; Reddy et al., 1999). Conversely, colon cancer cells totally devoid of COX activity are growth inhibited as effectively as cells producing COX (Hanif et al., 1996; Elder et al., 1997). Likewise, COX-1 and COX-2 null mouse embryo fibroblast cells remain sensitive to the antiproliferative and antineoplastic effects of NSAIDs (Zhang et al., 1999). In those colon cancer cells producing COX, COX-produced prostaglandins cannot rescue cells from NSAID-associated growth arrest in vivo or in vitro (Narisawa et al., 1984; Hanif et al., 1996; Chan et al., 1998). The concentration of NSAIDs that inhibit growth is 10 to 100 times higher than that required to inhibit COX activity, suggesting the existence of additional cellular targets (Hanif et al., 1996; Ahnen, 1998; Charalambous et al., 1998; Simmons et al., 1999). Finally, many studies have demonstrated that the COX-2 protein is elevated in the neoplastic epithelial cells of human tumors (Eberhart et al., 1994; Sano et al., 1995), while COX-2 expression in mouse intestinal tumors is confined to nonneoplastic stromal cells (Oshima et al., 1996; Shattuck-Brandt et al., 1999). Thus, the mouse and human COX-2 proteins are located within disparate cellular populations of the colon, yet tumorigenesis in both species is prevented by the same NSAIDs. This suggests either that it is coincidental that the same agents can exert chemopreventive effects in mouse and human, or that other targets of NSAIDs, common to the neoplastic cells of both species, might exist and provide a link to the molecular pathogenesis described below.
Molecular genetic studies have identified a series of genetic alterations associated with the development of benign tumors (adenomas) and their progression to malignant disease (carcinomas) (reviewed in Kinzler and Vogelstein, 1996). In terms of prevention, the alterations that occur early in this process are of most interest. Inactivating mutations of the APC tumor suppressor pathway occur early and are found in most colorectal adenomas and carcinomas. Moreover, inherited mutations of APC cause FAP, characterized by the development of hundreds to thousands of colorectal adenomas. Several studies have suggested that APC’s association with β-catenin (Rubinfeld et al., 1993; Su et al., 1993) might be critical to its tumor-suppressive effects. In the colon, β-catenin binds to the Tcf-4 transcription factor, providing a domain that activates genes containing Tcf-4-binding sites in their regulatory regions (Behrens et al., 1996; Molenaar et al., 1996). Wild-type APC can promote the degradation of β-catenin (Munemitsu et al., 1995) and inhibit β-catenin/Tcf-4 regulated transcription (CRT), while disease-associated APC mutants are deficient in this ability (Korinek et al., 1997; Morin et al., 1997). In tumors lacking APC mutations, oncogenic mutations of β-catenin can lead to increased CRT (Morin et al., 1997; Rubinfeld et al., 1997).
The targets of this increased CRT are therefore likely to provide insights into APC’s suppressive effects. We have recently used SAGE technology to analyze changes in gene expression following inhibition of CRT by APC in human CRC cells and identified the c-myc oncogene as a direct target of CRT (He et al., 1998a). However, like many other critical regulators of cell growth, APC is likely to exert its effects through several effectors. Here, we report the identification of another target of the APC pathway, peroxisome proliferator-activated receptor δ (PPARδ; a.k.a. PPARβ, NUC1, and FAAR) (Schmidt et al., 1992; Amri et al., 1995; Jow and Mukherjee, 1995), which provides a link between NSAID-mediated chemoprevention and the genetic alterations identified in colorectal tumors.
PPARδ belongs to the nuclear receptor superfamily, which includes the steroid hormone, thyroid hormone, retinoid, and PPAR subfamilies as well as a growing number of orphan receptors (Kastner et al., 1995; Mangelsdorf et al., 1995; Lemberger et al., 1996). The PPAR subfamily comprises at least three distinct subtypes found in vertebrate species: PPARα, PPARδ, and PPARγ. The nuclear receptor family members function as ligand-dependent sequence-specific activators of transcription (Mangelsdorf et al., 1995; Lemberger et al., 1996). We found that the NSAIDs sulindac and indomethacin could mimic the effects of APC by downregulating the transcriptional activity of PPARδ. This inhibition appeared to be due to disruption of the DNA binding ability of PPARδ/RXR heterodimers. These observations demonstrate that APC and NSAIDs inhibit a mutual target, PPARδ, thereby providing a link between the genetic alterations underlying tumor development and cancer chemoprevention.
The effects of APC on gene expression were explored using SAGE as previously reported (He et al., 1998a). In brief, gene expression was examined in a human CRC cell line with inducible APC (HT29-APC) and a control cell line with an inducible lacZ gene (HT29-β-gal) 9 hr after induction. SAGE analysis of 55,233 and 59,752 tags from APC-expressing and control cells, respectively, led to the identification of 14,346 different transcripts, the majority of which were not differentially expressed. Because biochemical studies have indicated that APC directly represses CRT, we focused on the repressed transcripts. One of the most highly repressed tags corresponded to PPARδ (24 tags in HT29-β-gal versus 5 tags in HT29-APC).
To confirm the SAGE data, we performed Northern blot analysis of RNA from HT29-APC and HT29β-gal cells using PPAR probes (Figure 1A). Repression of PPARδ was evident as early as 3 hr after APC induction, whereas no change was detectable in HT29 β-gal cells even 9 hr after induction. In contrast, expression of PPARγ was not affected by expression of APC, and the other known PPAR subfamily member, PPARα, was not expressed at detectable levels (Figure 1A and data not shown). The ability of APC to repress PPARδ expression suggested that expression of PPARδ should be elevated in primary CRCs, where CRT is often increased by mutations in APC or β-catenin. Northern blot analysis of PPAR expression in CRCs and normal colorectal mucosa revealed a marked increase in PPARδ expression in each of four cancers studied (Figure 1B). In contrast, there was no increase in PPARγ expression in the cancers of these patients (Figure 1B).
To explore the basis for the repression of PPARδ, we isolated and sequenced a 3.1 kb genomic region upstream of the PPARδ transcription start site (GenBank accession #AF187850) and used it to analyze APC responsiveness (Figure 2). A luciferase reporter construct containing this fragment (BE) upstream of a minimal promotor was markedly repressed by APC expression (Figures 2A and 2B). Analysis of nested deletions and pro-motor fragments identified two APC-responsive fragments (fragment NH and HD, Figures 2A and 2B). The sequence of these fragments revealed two putative Tcf-4-binding sites, one (TRE1) located 1543 bp upstream of the PPARδ transcription start site in fragment NH and the other (TRE2) located 759 bp upstream in fragment HD. A fragment spanning these two sites conferred marked APC repression, which was completely abrogated by disruption of the putative Tcf-4-binding sites (NP versus mNP, Figure 2C). Moreover, either of the putative Tcf-4-binding sites in isolation could confer APC responsiveness in a sequence-specific manner (TRE1 versus mTRE1, and TRE2 versus mTRE2 in Figure 2C).
As noted above, an obvious basis for the APC responsiveness of PPARδ would be inhibition of CRT. Consistent with this, there was a perfect concordance between the ability of APC to repress PPARδ promotor fragments (Figures 2B and 2C) and the ability of oncogenic β-catenin to induce transcriptional activity (Figure 2D). Likewise, there was a perfect concordance between APC responsiveness and the ability of a dominant-negative Tcf-4 (dnTcf) expression vector to inhibit transcriptional activity (Figures 2B and 2C). As with APC responsiveness, the β-catenin transactivation and the dnTcf repression were abrogated by mutation of the putative Tcf-4-binding sites (Figures 2C and 2D). The ability of Tcf-4 to directly bind to the PPARδ TRE sites was demonstrated by gel electrophoresis mobility shift assays (GEMSA). Both putative binding sites demonstrated significant Tcf-4 binding, which was inhibited by their cognate wild-type binding sequences but not by their mutant counterparts (Figure 2E).
The above results suggested that APC repressed PPARδ expression by interfering with CRT and that alterations in this pathway could lead to increased expression of PPARδ in CRCs. To further evaluate the generality of this pathway, we examined the ability of dnTcf to interfere with PPARδ expression in other human CRC cell lines. Like the HT29 cells in which PPARδ expression was first identified (Figure 1A), SW480 and DLD1 cells contain inactivating mutations of APC. HCT116 cells have an activating mutation of β-catenin. As expected from the study of primary tumors (Figure 1B), PPARδ expression was detected in all the lines (Figure 1C). Moreover, PPARδ expression was inhibited in each line by infection with an adenovirus containing a dnTcf expression cassette but not by a control adenovirus containing a GFP expression cassette (Figure 1C). In contrast, PPARγ expression was barely detectable in SW480 cells, and dnTcf had no effect on PPARγ expression in any of the lines tested.
To explore the functional significance of PPARδ repression, we developed reporters for PPARδ function. Although downstream targets of PPARδ are unknown, studies of other PPAR family members have defined a prototypic response element. Maximum DNA binding and activation are achieved through heterodimerization between a PPAR protein and RXR (Gearing et al., 1993). Accordingly, the prototypic PPAR response element ACO from the acyl-CoA oxidase gene promotor contains two copies of the core binding sequence AGGTCA separated by one base pair (Tugwood et al., 1992; Mangelsdorf et al., 1995; Lemberger et al., 1996; Juge-Aubry et al., 1997). PPARα and PPARγ bind this consensus efficiently, whereas PPARδ does not (see below). To define a PPARδ-responsive element, we performed in vitro binding site selection for both PPARδ and RXRα. Analysis of 28 binding sites selected with a RXRα GST fusion protein identified (A/G)GGTCA as the core consensus for RXR (Figure 3A). Analysis of 20 sites selected with a PPARδ GST fusion protein revealed a novel binding consensus (CGCTCAC), which was distinct from the previously defined PPARα/γ consensus (Figure 3B).
The combination of the PPARδ and RXRα consensus sequences should form a PPARδ-binding element in vitro and a PPARδ-responsive element in vivo. We generated a putative PPARδ-responsive element (DRE, 5′-CGCTCACAGGTCA-3′) by joining the PPARδ- and RXRα-binding sites. GEMSA analysis of DRE revealed binding to PPARδ GST fusion protein but not to PPARα or PPARγ GST fusion proteins (Figure 3C). In contrast, the prototypic PPAR-responsive element ACO (5′-AGG ACAAAGGTCA-3′) bound PPARα and PPARγ but not PPARδ GST fusion proteins (Figure 3C). RXRα GST fusion protein demonstrated weaker binding to both responsive elements. To further test the specificity of these elements, we performed GEMSA analysis with in vitro translated PPARδ/RARα and PPARγ/RARα heterodimers in the presence or absence of ligand stimulation. Under the conditions used, binding of PPARδ/RXRα and PPARγ/RXRα heterodimers to their cognate elements could not be detected in the absence of ligand. However, PPARδ/RXRα binding to DRE was markedly induced by the PPARδ ligand cPGI, and PPARγ/RXRα binding to ACO was induced by the PPARγ ligand BRL49653 (Figure 3D). In contrast, PPARγ/RXRα heterodimers did not bind DRE in the presence of BRL49653 nor did PPARδ/RXRα bind ACO in the presence of cPGI. To test the specificity of these response elements in cells, we constructed luciferase reporters containing either the DRE or ACO elements. Transfection of 293 cells with PPARδ resulted in activation of the DRE reporter that was enhanced by cPGI (Figure 3E) but did not activate the ACO reporter even in the presence of cPGI (Figure 3F). In contrast, expression of PPARγ did not activate the DRE reporter (Figure 3E) but did activate the ACO reporter that was enhanced by BRL 49653 (Figure 3F).
The above findings suggested that PPARδ activity was regulated by APC/β-catenin/Tcf-4 pathway at the transcriptional level. To address the functional consequences of this transcriptional regulation in CRC cells, we used the PPARδ-specific reporters described above. Transfection of APC into a human CRC cell line resulted in downregulation of the PPARδ reporter DRE but had no effect on the PPARα/γ-responsive reporter ACO (Figure 4A). The lack of any effect on PPARα/γ demonstrated the specificity of this inhibition and made it unlikely that the effects were due to nonspecific toxicity of a tumor suppressor. Transfection of a dnTcf-4 expression vector also specifically repressed the PPARδ reporter but not the PPARα/γ reporter. To further eliminate the possibility of nonspecific toxic effects, we tested β-catenin’s ability to positively regulate PPARδ activity. Expression of oncogenic β-catenin mutants activated the PPARδ reporter but did not activate the PPARα/γ reporter (Figure 4B).
Suppression of colorectal tumorigenesis by NSAIDs suggested that these compounds may be linked to the genetic alterations that drive tumorigenesis. The identification of PPARδ as a target of the APC tumor suppressor pathway suggested a specific link. Both precursors and products involved in eicosanoid metabolism have been shown to be ligands for PPARs (Keller et al., 1993; Yu et al., 1995; Forman et al., 1997; Kliewer et al., 1997). The ability of NSAIDs to perturb eicosanoid metabolism suggested that PPARs may be an ultimate target of NSAIDs in suppressing tumorigenesis (Prescott and White, 1996), and the above findings suggest that PPARδ could be a specific target. To explore this possibility, we tested the effects of the NSAID sulindac on PPARδ function. As noted in the Introduction, sulindac has been shown to suppress intestinal tumorigenesis in humans and mice. In culture, sulindac sulfide, the active metabolite of sulindac, has been shown to induce apoptosis in CRC cells (Piazza et al., 1995; Shiff et al., 1995; Hanif et al., 1996; Chan et al., 1998). Sulindac sulfide treatment resulted in a dose-dependent repression of PPARδ activity in CRC cells, as assessed with the DRE reporter (Figure 4C). A greater than 2-fold repression was observed at low concentrations of sulindac sulfide, and a greater than 10-fold reduction was noted at levels of sulindac sulfide that induced substantial degrees of apoptosis in these cells (Figures 4C and and5D).5D). In contrast, sulindac sulfide had only a modest effect (less than 25% repression at the highest concentration tested) on PPARα/γ activity, assessed with the ACO reporter (Figure 4C). A similar dose-dependent suppression of PPARδ was observed with indomethacin, another NSAID (Figure 4C).
If suppression of PPARδ activity were contributing to sulindac-induced apoptosis, overexpression of PPARδ might be expected to protect against sulindac sulfide–induced apoptosis. We constructed an adenovirus (Ad-PPARδ) expressing PPARδ and a green fluorescent protein (GFP) marker using AdEasy technology (He et al., 1998b). The ability of AdPPARδ to suppress sulindac sulfide–induced apoptosis was compared to that of AdGFP, which contained only the GFP marker gene. AdPPARδ produced nearly a 5-fold decrease in apoptosis in HCT116 cells treated with 100 or 125 μM sulindac sulfide (Figures 5A–5D). Similar results were obtained with the SW480 cell line (Figure 5D). However, the suppression of apoptosis could be overridden at higher concentrations of sulindac sulfide (150 μM, Figure 5D). The results were further confirmed and extended by the ability of AdPPARδ to rescue inhibition of clonal cell growth by sulindac sulfide. Treatment of cells with sulindac sulfide resulted in a greater than 4-fold decrease in the number of colonies (Figure 5E). At 100 or 125 μM sulindac sulfide but not higher doses, this decrease could be completely rescued by infection with Ad-PPARδ, which actually resulted in a slightly increased number (~15%) of colonies. In contrast, the APC target and prototypic oncogene c-myc could not rescue the sulindac-mediated inhibition of clonal growth.
To determine whether sulindac could inhibit the transcription of PPARδ like APC, we examined expression of PPARδ following sulindac sulfide treatment. Concentrations of sulindac sulfide that resulted in suppression of PPARδ activity and apoptosis had no effect on the level of PPARδ transcripts, excluding this possibility (Figure 6A). Because PPARs can bind eicosanoids (Keller et al., 1993; Yu et al., 1995; Forman et al., 1997; Kliewer et al., 1997), the effects of NSAIDs on PPARδ could be due to their ability to perturb eicosanoid metabolism. However, several studies (see Discussion) have suggested that the chemopreventive effects of NSAIDs are not simply related to their ability to suppress prostaglandin synthesis. We therefore considered the possibility that NSAIDs directly inhibit PPARδ activity. This notion was supported by the ability of some NSAIDs to activate PPARα/γ (Lehmann et al., 1997) and of indomethacin to alter the ligand responsiveness of TetR/PPARδ fusion proteins (Yu et al., 1995). We tested the ability of sulindac to inhibit cPGI-stimulated PPARδ/RXRα heterodimer DNA binding activity in vitro. Sulindac sulfide was able to inhibit binding of PPARδ/RXRα heterodimers to the DRE element (Figure 6B). Binding to DRE was also inhibited by the NSAID indomethacin and, at higher concentrations, the sulindac sulfide–related compound sulindac sulfone (Figure 6B). The relative concentrations of sulindac sulfide, indomethacin, and sulindac sulfone required to inhibit binding to DRE were roughly concordant with the concentrations required to induce apoptosis in CRC cells, with sulindac sulfide being the most potent and sulindac sulfone the least (Figures 5D and and6B;6B; data not shown). None of these drugs had any effect on BRL49653-stimulated binding of PPARγ/RXRα heterodimers in an analogous assay performed with the ACO element (Figure 6C).
The above results demonstrate that PPARδ is a target of both APC and NSAIDs and suggest a model of how APC and NSAIDs operate to suppress intestinal tumorigenesis (Figure 7). In most CRCs, inactivating mutations of APC lead to elevated levels of CRT (Korinek et al., 1997; Morin et al., 1997). In rare CRCs without APC mutations, β-catenin mutations that render it resistant to APC-mediated degradation result in elevated β-catenin/Tcf-4-mediated transcription (Morin et al., 1997). In either case, this increased β-catenin/Tcf-4 activity leads to increased transcription of growth-promoting genes. Accordingly, restoration of APC function to CRC cells with defective APC function results in growth suppression and apoptosis (Morin et al., 1996). The genes that have been postulated to mediate the growth-promoting effects of β-catenin/Tcf-4 activity include those encoded by the c-myc oncogene (He et al., 1998a), the cyclin D1 gene (Tetsu and McCormick, 1999), and others (WISP, c-jun, and fra-1) (Pennica et al., 1998; Mann et al., 1999). The present findings suggest that PPARδ represents a β-catenin/Tcf-4 target with particular importance for chemoprevention. Whereas APC or β-catenin mutations can result in increased PPARδ activity, NSAIDs can compensate for this defect by suppressing PPARδ activity and promoting apoptosis. This suppression of PPARδ is mediated in part by the ability of some NSAIDs to directly inhibit the DNA binding activity of PPARδ. In addition, because fatty acids and eicosanoids can act as ligands and modifiers of PPAR activity (Keller et al., 1993; Yu et al., 1995; Prescott and White, 1996; Forman et al., 1997; Kliewer et al., 1997), PPARδ activity might be repressed by the NSAID-mediated changes in eicosanoid metabolism.
The above model can help explain several features of NSAID-mediated chemoprevention. First, the effectiveness of some NSAIDs in the prevention of colorectal adenomas can now be linked to genetic defects that underlie the initiation of these tumors and to the ability of NSAIDs to counterbalance the consequences of these genetic defects. Second, although NSAID functions have been linked to their inhibition of COX activity and the resulting inhibition of prostaglandin synthesis, several studies have suggested that the chemopreventive and apoptosis-inducing activities of NSAIDs are not entirely related to the inhibition of COX or to the decreased levels of prostaglandins (see Introduction). These results may be explained by the ability of NSAIDs to directly inhibit PPARδ. Indeed, the sulindac derivative sulindac sulfone, which is devoid of COX inhibitory activity, has apoptotic activity in vitro and chemopreventive activity in vivo and has been proposed as a chemopreventive agent that lacks the toxicity associated with traditional NSAIDs (Piazza et al., 1995, 1997; Mahmoud et al., 1998). Sulindac sulfone inhibited PPARδ activity, albeit at higher concentrations than that required for sulindac sulfide, consistent with its reduced chemopreventive and apoptosis-promoting activity. Third, recent studies have demonstrated that PPARγ agonists promote intestinal tumorigenesis in the Min mouse (Lefebvre et al., 1998; Saez et al., 1998), while the same agonists inhibit the growth of human CRC cells (Brockman et al., 1998; Sarraf et al., 1998). Although the conclusions of these studies were contradictory, they clearly demonstrated the ability of PPAR ligands to modify intestinal tumor growth. A role for PPARs in intestinal tumorigenesis is further suggested by the recent identification of loss-of-function mutations in one allele of PPARγ in 4 of 55 sporadic CRCs (Sarraf et al., 1999). However, unlike PPARδ, neither PPARγ nor PPARα is a target of the APC/β-catenin pathway. Whereas PPARδ is increased in expression in cancers, downregulated by APC, and upregulated by β-catenin, PPARγ and PPARα do not display these properties. Fourth, the ability of COX2 expression to modulate apoptosis (Tsujii and Dubois, 1995) and intestinal tumorigenesis (Oshima et al., 1996) may be partially related to its ability to alter the spectrum of ligands for PPARδ and other PPARs. In this regard, it is interesting to note that the PPARδ ligand cPGI can partially rescue infertility resulting from COX-2 deficiency (Lim et al., 1999). Finally, the ability of dietary fatty acids and secreted phospholipases to modify the spectrum of PPARδ ligands and thus alter PPARδ activity could account for their ability to affect CRC risk (Willett et al., 1990; Dietrich et al., 1993; MacPhee et al., 1995; Vanden Heuvel, 1999).
In addition to explaining several features of NSAID-mediated chemoprevention, our observations may have important ramifications for the development of chemopreventive agents. In particular, it is conceivable that the development of drugs that specifically target PPARδ might lead to more efficacious and less toxic means for CRC chemoprevention.
Human CRC cells (HT29, HCT116, SW480, and DLD1) and embryonic kidney cells (293) were obtained from ATCC (Manassas, VA). BRL49653 and cPGI were purchased from American Radiolabeled Chemicals and Cayman Chemical Company, respectively. Sulindac derivatives and indomethacin were purchased from BIOMOL. Unless otherwise indicated, all chemicals were purchased from Sigma (St. Louis, MO).
GST fusion proteins containing the N-terminal DNA-binding domains of human PPARδ and RXRα were constructed by PCR amplifying codons 1–249 of PPARδ and 1–224 of RXRα and cloning them into pGEX-2TK vector. As controls, GST fusion proteins containing the DNA-binding domains of human PPARα (aa 1–249) and PPARγ (aa 1–248) were also constructed. To identify the potential consensus DNA sequence motifs recognized by PPARδ and RXRα, a previously described in vitro site selection procedure was utilized (Zawel et al., 1998). Selected PCR products were cloned in to pZero2.1, tested for binding, and sequenced as described.
DNA-binding assays supplemented with Poly dIdC (6 μg/ml) were performed essentially as described (Zawel et al., 1998). For binding to PCR products derived from in vitro site selections, 1.0–1.5 μg of protein and 50 ng of DNA were used. For competitions, a 100-fold excess of unlabeled probe was used. For GEMSA with GST fusion proteins, 0.3–0.5 μg of fusion protein and 0.5 ng of 32P kinase labeled (~106 dpm) DNA were used. The probes for Tcf-4 binding were as previously reported (Korinek et al., 1997). For GEMSA with in vitro translated proteins, 0.1 to 0.2 μl of programmed lysate and 32P-labeled probe (~106 dpm) was used. The DRE probe was formed by annealing 5′-GCGTGAGCGCTCACAGGTCAATTCG-3′ and 5′-CCGAATT GACCTGTGAGCGCTCACG-3′. The ACO probe was formed by annealing 5′-GCGGACCAGGACAAAGGTCACGTTC-3′ and 5′-CGA ACGTGACCTTTGTCCTGGTCCG-3′.
The following oligonucleotides containing PPARδ and RXR recognition motifs that were identified from in vitro site selection approach were synthesized: 5′-CTAGCGTGAGCGCTCACAGGTCAATTCGGTGAGCGCTCACAGGTCAATTCG-3′ and 5′-CTAGCGAATTGACCTGTGAGCGCTCACCGAATTGACCTGTGAGCGCTCACG-3′. As a control, the following oligonucleotides containing a PPARα and PPARγ-responsive element from the acyl-CoA oxidase promotor were also synthesized: 5′-CTAGCGGACCAGGACAAAGGTCACGTTCGGACCAGGACAAAGGTCACGTTCG-3′ and 5′-CTAGCGAACGTGACCTTTGTCCTGGTCCGAACGTGACCTTTGTCCTGGTCCG-3′. The oligonucleotide cassettes were dimerized and cloned into pBV-Luc, a luciferase reporter plasmid with very low basal activity (He et al., 1998a). All constructs were verified by DNA sequencing.
Three independent BAC clones containing the PPARδ promotor sequence were obtained by screening a BAC library (Research Genetics). For the construction of PPARδ promotor reporters, corresponding restriction fragments (illustrated in Figure 2A) were sub-cloned into pBV-Luc. The mNP reporter was constructed by cloning a PCR product into pBV-Luc, whereas p4XTRE1-Luc, p4XmTRE1-Luc, p4XTRE2-Luc, and p4XmTRE2-Luc were constructed from oligonucleotides. Details of construction and oligonucleotide sequences are available upon request or at www.coloncancer.org/ppar.htm.
Reporter plasmid, effector plasmid, and β-gal control plasmid were transfected into cells using LipofectAmine (Life Technologies). Twenty-four hours after transfection, cells were lysed and collected for assays of luciferase activity using Promega’s Luciferase Assay System.
The full-length proteins of PPARδ, PPARγ, and RXRα were generated by in vitro transcription-coupled translation of PCR products using the TNT T7 Quick-Coupled Transcription/Translation System (Promega). PCR primer sequences are available upon request or at www.coloncancer.org/ppar.htm.
A PCR product of human PPARδ was cloned into pCMV-HAHA and verified by sequencing. This expression cassette was used to generate recombinant adenovirus using the AdEasy system as previously described (He et al., 1998b). Additional details of the construction are available upon request or at www.coloncancer.org/ppar.htm. AdMyc was generated in a similar fashion and was a gift of H. Hermeking.
We thank Carlo Rago, Christopher Torrance, Victor Velculescu, Leigh Zawel, Lin Zhang, and Wei Zhou for their help and advice and Heiko Hermeking for providing the AdMyc. This work is supported by National Institutes of Health grants CA57345 and CA62924. B. V. is an investigator of the Howard Hughes Medical Institute. K. W. K. received research funding from Genzyme Molecular Oncology (Genzyme). Under a licensing agreement between the Johns Hopkins University and Genzyme, the SAGE technology was licensed to Genzyme, and K. W. K. and B. V. are entitled to a share of royalties received by the University from sales of the licensed technology. The SAGE technology is freely available to academia for research purposes. K. W. K. and B. V. are consultants to Genzyme. The University and researchers (K. W. K. and B. V.) own Genzyme stock, which is subject to certain restrictions under University policy. The terms of this arrangement are being managed by the University in accordance with its conflict of interest policies.