Early de novo gene expression is required for 15-deoxy-Delta 12,14-prostaglandin J2-induced apoptosis in breast cancer cells.

Cyclopentenone prostaglandin derivatives of arachidonic acid are potent inducers of apoptosis in a variety of cancer cell types. Several investigators have shown that the terminal derivative of prostaglandin J(2) (PGJ(2)) metabolism, 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)), induces apoptosis in breast cancer cells and is a potent activator of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma (PPARgamma), but 15dPGJ(2) effects can be mediated by PPARgamma-dependent and PPARgamma-independent mechanisms. Here we report that 15dPGJ(2) regulates early gene expression critical to apoptosis. Specifically, 15dPGJ(2) induces potent and irreversible S phase arrest that is correlated with expression of genes critical to cell cycle arrest and apoptosis, including the cyclin-dependent kinase inhibitor p21(Waf1/Cip1) (p21). Inhibition of RNA or protein synthesis abrogates apoptosis induced by 15dPGJ(2) in breast cancer cells but potentiates apoptosis induced by tumor necrosis factor-alpha or CD95/Fas ligand. Additionally, 15dPGJ(2) induces caspase activation that is blocked by peptide caspase inhibitors. These data show that de novo gene transcription is necessary for 15dPGJ(2)-induced apoptosis in breast cancer cells. Critical candidate genes are likely to be revealed through analysis of differential cDNA array expression.

perhaps via direct physical interaction between these transcription factors (21) or competition for limited transcriptional co-activators (22). Activation of PPAR␥ or inhibition of NFB blocks angiogenesis of endothelial cells and suppresses transcriptional activation of COX-2 (23,24). Together these observations suggest a variety of cellular pathways through which cyclopentenone prostaglandins may exert potent anti-inflammatory and antineoplastic properties in diverse cell types. Moreover, it was recently shown that cyclopentenone compounds are produced in vivo (25)(26)(27)(28). Thus, it is critical to determine which mechanism(s) predominate to further develop these compounds as anticancer agents.
These studies demonstrate that 15dPGJ 2 -induced apoptosis in breast cancer cells requires expression of specific gene products. Blocking de novo mRNA and protein expression inhibited 15dPGJ 2 -induced apoptosis. Among the genes up-regulated by 15dPGJ 2 , which may account, at least in part, for cell cycle arrest and apoptosis, are the cyclin-dependent kinase inhibitors p21 Waf1/Cip1 (p21) and p27 Kip1 (p27).
Flow Cytometry for Markers of Apoptosis-Cells were incubated with or with out actinomycin D (1 g/ml) or cycloheximide (2 g/ml) for 1 h and then exposed to 15dPGJ 2 (10 M) or vehicle (ethanol). After 36 h, cells were collected by trypsinization and pelleted, and the percentage of cells undergoing apoptosis was determined by flow cytometry using a TACS annexin V-fluorescein isothiocyanate kit (Trevegin, Gaithersburg, MD) according to the protocol of the manufacturer.
Histochemistry and Immunofluorescence-Cells were incubated with or with out actinomycin D (1 g/ml) or cycloheximide (2 g/ml) for 1 h and then exposed to 15dPGJ 2 (10 M) or vehicle (ethanol). After 36 h, cells were stained with annexin V-fluorescein isothiocyanate monoclonal antibody (Roche Molecular Biochemicals) followed by overnight incubation with 0.1 g/ml 4Ј,6-diamidino-2-phenylindole dihydrochloride in 10% formaldehyde. Dual stained cells were washed and mounted under a glass coverslip, and digital images were obtained using a Zeiss Axiplan-2 epifluorescence microscope equipped with UV excitation filters and a Spot digital camera.
Differential Display Analysis-Cells were incubated with or with out 15dPGJ 2 (10 M) for 2 h. Total RNA was isolated using Trizol Reagent (Life Technologies, Inc.), and 1.0 g of total RNA was used to create radiolabeled cDNA by RT-PCR and hybridized to a Human Broad Coverage cDNA Array 1.2 (CLONTECH, Palo Alto, CA) according to the protocol of the manufacturer. Relative gene expression level and the mean from three separate experiments were determined using AtlasIm-age 2.0 software (CLONTECH). Relative gene expression level is highly reproducible between experiments (31,32).
Western Blot Analysis-MDA-MB-231 cells were incubated with or with out actinomycin D (1 g/ml) or cycloheximide (2 g/ml) for 1 h and then exposed to 15dPGJ 2 (10 M) or vehicle (ethanol). After 9 h, total protein was isolated in lysis buffer, and 50 g of total protein was separated by gel electrophoresis in 10 -20% SDS-polyacrylamide precast gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Bio-Rad). Antibodies to p21 Waf1/Cip1 , p27 Kip1 , and procaspase-3 were from Santa Cruz Biotechnologies (Santa Cruz, CA). Bands were visualized using ECL-Plus (Amersham Pharmacia Biotech) and Kodak BioMax film (Eastman Kodak Co.).
Northern Blot Analysis-MDA-MB-231 cells were incubated with or without actinomycin D (1 g/ml) or cycloheximide (2 g/ml) for 1 h and then exposed to 15dPGJ 2 (10 M) or vehicle (ethanol). After 2 h, total RNA was isolated in Trizol reagent (Life Technologies, Inc.), and 10 g was separated by gel electrophoresis and transferred to ZetaProbe GT membranes (Bio-Rad). cDNA probes to human p21 and ␤-actin were amplified by RT-PCR using Gene Amp Gold RNA PCR kit (PerkinElmer Life Sciences), cloned into TOPO cloning vectors (Invitrogen, Carlsbad, CA), and labeled and hybridized using a Strip-EZ RNA kit (Ambion, Austin, TX). Autoradiograph images were obtained with BioMax film (Kodak) by overnight exposure at Ϫ70°C.
Caspase Activity Assays-Cells were incubated with 15dPGJ 2 (10 M) with or with out the indicated caspase inhibitors (10 M). After 36 h, phosphatidylserine translocation was determined by flow cytometry as described above, and immunoblotting for procaspase-3 (Santa Cruz Biotechnologies) expression was performed as described above.

Inhibition of RNA or Protein Synthesis Blocks 15dPGJ 2induced Apoptosis-Previous experiments in our laboratory
showed that MDA-MB-231 cells undergo apoptosis as early as 8 h after exposure to exogenous 15dPGJ 2 (2). Here we investigated the minimum amount of exposure time necessary to induce apoptosis. Cell growth was markedly inhibited after cells were incubated with 15dPGJ 2 for as little as 2 h (Fig. 1A). Incubation of cells with 15dPGJ 2 or the phosphatase inhibitor staurosporin for 36 h induced marked apoptosis that was blocked by actinomycin D and cycloheximide, respectively (Fig.  1B). However, TNF␣ and CD95/Fas ligand induced apoptosis only when RNA and protein synthesis was blocked. NFB mediates a mechanism of protection via expression of cytoprotective genes, but TNF␣ pretreatment of breast cancer cells does not rescue cells from 15dPGJ 2 -induced apoptosis (data not shown).
Further investigation showed that treatment of cells with 15dPGJ 2 for as little as 5 h irreversibly induced apoptosis, which was markedly inhibited by pretreatment with actinomycin D or cycloheximide (Fig. 1C). Additionally, phosphatidylserine translocation and nuclear condensation, hallmarks of apoptosis, were attenuated when RNA or protein synthesis was blocked (Fig. 1D) in 15dPGJ 2 -treated cells. Together these data show that early de novo transcription of genes is required for 15dPGJ 2 -induced apoptosis.
Genes Regulated by 15dPGJ 2 -Recently DuBois and colleagues (33) investigated the expression of PPAR␥ target genes in colon cancer at 24 h and 6 days. Our data suggested that critical genes are transcribed much earlier. We investigated the 15dPGJ 2 -mediated expression of genes critical to apoptosis by differential display analysis at 2 h (Table I). The expression of gene products encoding proteins involved in cell cycle arrest and apoptosis, including Bag-1, a promoter enhancer and Bcl 2binding protein; the cysteine protease caspases 3, 4, and 8; the transcription factors hEGR1, AP-1, and c-Jun; antioxidative genes heme oxygenase and SOD1; and the cyclin-dependent kinase inhibitor p21 was increased Ն2.0-fold. Additionally, the expression of genes that are negative prognostic indicators in breast, colon, and liver cancer, including BRCA2, hepatomaderived growth factor, and MCC, and genes involved in DNA maintenance and repair, including ERCC1, Rad52, Rad23A, DAXX, Dap3, DNA ligase 1, and GADD45 were decreased Ն1.5-fold. Thus, 15dPGJ 2 likely exerts potent antiproliferative and proapoptotic responses, at least in part, via activation of genes critical to cell cycle arrest and apoptosis.
Expression of p21 Waf1/Cip1 and p27 Kip1 Is Increased by 15dPGJ 2 -Of the genes identified by differential display analysis, expression of p21 and p27 has clinical relevance in breast cancer. The expression of p21 and p27 is associated with better prognosis and disease-free survival of breast cancer (34,35). Additionally, the p21 gene contains a potential conserved consensus PPAR␥ response element (PPRE) in the promoter region (36). Although Fitzpatrick and colleagues (37) showed that p21 mRNA expression is not increased after 6 h of treatment in RKO cells with the 15dPGJ 2 precursor ⌬ 12 -prostaglandin J 2 (⌬12PGJ 2 ), we have observed activation of p21 mRNA as early as 2 h using 15dPGJ 2 . Consistent with this finding, 15dPGJ 2 induces rapid and irreversible S phase arrest in synchronized breast cancer cells ( Fig. 2A), which correlated with the rapid expression of p21 and p27 protein (Fig. 2B). Furthermore, both actinomycin and cycloheximide blocked 15dPGJ 2 -induced p27 protein expression (Fig. 2C). However, 15dPGJ 2 -induced p21 protein expression was blocked by cycloheximide only, despite evidence by Northern blot analysis that actinomycin did block 15dPGJ 2 -induced p21 mRNA (Fig. 2D, see "Discussion").
Caspase Activation Is Mediated by 15dPGJ 2 -The activation of caspases by 15dPGJ 2 has been shown in a variety of cancer cell types (8,9,12,23). We show here that the pan-caspase inhibitor ZVAD-fmk blocked 15dPGJ 2 -induced apoptosis more effectively than specific caspase-3, caspase-8, or caspase-9 inhibitors DEVD-fmk, IETD-fmk, or LEHD-fmk, respectively (Fig. 3C). These data suggest that consequent to 15dPGJ 2 - induced gene transcription, caspase activation is induced that cannot be completely blocked by an individual caspase inhibitor.

DISCUSSION
The cyclopentenone prostaglandins possess potent antiproliferative and antitumor activities, but their mechanisms of action are complex and not well understood. Recently it was shown that 15dPGJ 2 induces intracellular oxidative stress in human neuroblastoma cells and hepatic myofibroblasts (38,39) and that 15dPGJ 2 induces expression of antineoplastic enzymes, such as glutathione S-transferases (40). Additionally, Fitzpatrick and colleagues (37) showed that ⌬12PGJ 2 , the immediate precursor to 15dPGJ 2 , inhibits isopeptidase activity of the proteosome pathway. We have shown that 15dPGJ 2 induces important and seemingly opposite biological responses, including proliferation, differentiation, and apoptosis in breast cancer cells and that these phenotypes correlate with increasing levels of PPRE-mediated transcription (1, 41). The studies presented here show that 15dPGJ 2 -induced apoptosis in breast cancer cells requires the rapid synthesis of new gene products (Ͻ5 h after exposure) that irreversibly leads to apoptosis. Differential cDNA array analysis suggests candidate genes including p21 and p27, which are potentially critical to this process given that 15dPGJ 2 induces an S phase arrest.
In addition to cell cycle arrest, p21 and p27 may play a critical role in apoptosis. Growth factor withdrawal leads to a proapoptotic feedback loop involving p21, p27, and caspase-3 in human endothelial cells (42,43). Furthermore, caspase-3-mediated cleavage of p21 is an early event after DNA damage (44). Moreover, dominant negative mutants of p21 abrogated apoptosis in these studies. Together these reports suggest that transcriptional activation of p21 and p27 followed by caspase-3-mediated cleavage represents a potential mechanism of action for 15dPGJ 2 . However, our data suggest the relationship is not so straightforward, at least for p21. Actinomycin clearly blocks 15dPGJ 2 -induced apoptosis but does not reduce early 15dPGJ 2 -induced p21 protein levels. One explanation for this may be that arachidonic acid and many of its metabolites TABLE I Summary of gene expression after exposure of MDA-MB-231 cells to 15dPGJ 2 for 2 h 15-Deoxy-⌬ 12,14 -PGJ 2 modulates the expression of critical cell cycle arrest and apoptosis genes. Total RNA was isolated from MDA-MB-231 cells treated with vehicle or 15dPGJ 2 (10 M) for 2 h. Radiolabeled cDNA was created by RT-PCR and hybridized to a Human Broad Coverage cDNA Array 1.2. Relative gene expression was determined using Atlas Image 2.0 and is reported as the mean from three separate experiments.
increase protein kinase C activity (45), which has been shown to stabilize p21 mRNA (46). Further research is needed to reconcile these contradictory data.
Recent investigations have shown that PPAR␥ agonists differentially regulate genes associated with cell growth and differentiation. 15dPGJ 2 negatively regulates myogenesis in part by inhibition of MyoD gene expression (47), and DuBois and colleagues (33) used microarray technology to show that inhibition of colon cancer cell growth by the PPAR␥-selective ligand BRL49653 is associated with inhibition of RegIA and Gob4, genes critical to growth and maturation of colonic epithelial cells. Our data suggest that early expression of p21 and p27 may be required for 15dPGJ 2 -induced apoptosis in breast cancer cells. These studies show, however, that expression of gene products is likely agonist-and cell type-specific.
Regulation of arachidonic acid (AA) metabolism is critical to the growth and survival of all cell types. 15dPGJ 2 inhibits transcriptional activation of COX-2 by a negative feedback loop mediated through PPAR␥ (26,48), which may lead to increased intracellular levels of free AA. Blocking arachidonate-phospholipid remodeling, by inhibition of CoA-independent transacylase (30,49,50) or inhibition of fatty acid-CoA ligase 4 (51), may produce nonenzymatic oxidized AA metabolites via increased levels of unesterified AA, which results in apoptosis of several cancer cell types. In addition, hexadecyl azelaoyl phosphatidylcholine, a novel phospholipase A 1 digestion product of alkyl phosphatidylcholines in low density lipoprotein (54), and (15S)-hydroxyeicosatetraenoic acid ((15S)-HETE), the major oxidized derivative of AA from 15-lipoxygenase-2, can be generated in monocytes and macrophages that activate PPAR␥ and mediate transcription of CD36, a PPAR␥-dependent gene (55). Moreover, 15-lipoxygenase-2 and the formation of (15S)-HETE from AA is decreased in prostate adenocarcinoma (56), and addition of exogenous (15S)-HETE induces apoptosis of prostate cancer cells (57). Inhibition of COX-2 by cyclopentenone prostaglandins with coordinate increases in intracellular oxidative stress could lead to the generation of oxidized AA metabolites as endogenous activators of PPARs or other transcription factors, creating a positive feedback loop of PPAR activation resulting in expression of gene products critical to apoptosis. This may account for the fact that 15dPGJ 2 is a far more potent activator of PPRE-mediated transcription than any other PPAR␥ agonist.
While many of the hypotheses presented here are being tested in our laboratory, deeper understanding of how AA metabolites induce cancer cell growth or death is essential. Common chemotherapeutic drugs including alkylating agents and nucleoside analogues work by blocking cellular replication at the level of DNA or interfere with ubiquitous cellular process FIG. 2. p21 Waf1/Cip1 and p27 Kip1 expression is increased by 15deoxy-⌬ 12,14 -prostaglandin J 2 and blunted by actinomycin D or cycloheximide. A, MDA-MB-231 cells were synchronized and treated with or without 15dPGJ 2 , and cell cycle progression was determined by flow cytometry. B, total protein was isolated from synchronized cells at the indicated times, and the expression of p21 and p27 was determined by immunoblotting. C, total protein from asynchronous cells treated with the indicated compounds was isolated at 9 h, and the expression of p21 and p27 was determined by immunoblotting. D, total RNA from asynchronous cells treated with the indicated compounds was isolated at 2 h, and the expression of p21 was determined by Northern blot analysis. The expression of ␤-actin was used as a control. The lack of new p21 mRNA expression during treatment with 15dPGJ 2 and actinomycin suggests that p21 protein detected under similar conditions (C) is from preformed p21 mRNA (see "Discussion"). Chx, cycloheximide; Act, actinomycin; 15d, 15dPGJ 2 .
FIG. 3. 15-Deoxy-⌬ 12,14 -prostaglandin J 2 -induced apoptosis requires caspase-3. A, MDA-MB-231 cells were exposed to 15dPGJ 2 . Total protein was isolated at the indicated times and analyzed for expression of procaspase-3 (Pro Cas-3) by immunoblotting. B, MDA-MB-231 cells were grown in the presence of 15dPGJ 2 (15d) with or without ZVAD-fmk. Total protein was isolated after 36 h and analyzed for expression of procaspase-3 by immunoblotting. C, MDA-MB-231 cells were grown in the presence of 15dPGJ 2 with or without the pan-caspase inhibitor ZVAD-fmk, the caspase-3 inhibitor DEVD-fmk, the caspase-8 inhibitor IETD-fmk, or the caspase-9 inhibitor LEHDfmk. After 36 h, the percentage of cells undergoing apoptosis was determined by flow cytometry. such as microtubule formation. This strategy reduces cell proliferation, but relapse and the development of resistance to chemotherapy suggest that cancer cells are not eradicated. The data presented here demonstrate that gene expression may be required for the induction of apoptosis and thus eradication of some cancer cell types. In addition, potent bioactive derivatives of AA, such as cyclopentenone prostaglandins, acting through multiple pathways may represent a promising class of therapeutic molecules for the treatment of cancer. However, the potential of these compounds to act as proliferators of tumorigenesis via PPAR␥ (58,59) suggests that it is critical to further clarify the mechanisms that regulate the antiproliferative and proapoptotic activities of these compounds. The data presented here strongly suggest that new gene synthesis is required for 15dPGJ 2 -induced apoptosis. Whether this is mediated via PPAR␥ or some other critical regulator of gene synthesis remains to be defined.