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J. Biol. Chem., Vol. 276, Issue 50, 47131-47135, December 14, 2001

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Early de Novo Gene Expression Is Required for 15-Deoxy-^12,14-prostaglandin J₂-induced Apoptosis in Breast Cancer Cells^*

Carl E. Clay^§¶, Gen-ichi Atsumi^§, Kevin P. High^**, and Floyd H. Chilton^§

From the  Department of Internal Medicine, ^§ Section of Pulmonary Critical Care,  Section of Infectious Diseases, and  Department of Physiology and Pharmacology, Wake Forest University Baptist Medical Center, Winston Salem, North Carolina 27157

Received for publication, June 19, 2001, and in revised form, October 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 J₂ (PGJ₂) metabolism, 15-deoxy-^12,14-PGJ₂ (15dPGJ₂), induces apoptosis in breast cancer cells and is a potent activator of the nuclear hormone receptor peroxisome proliferator-activated receptor  (PPAR), but 15dPGJ₂ effects can be mediated by PPAR-dependent and PPAR-independent mechanisms. Here we report that 15dPGJ₂ regulates early gene expression critical to apoptosis. Specifically, 15dPGJ₂ 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 15dPGJ₂ in breast cancer cells but potentiates apoptosis induced by tumor necrosis factor- or CD95/Fas ligand. Additionally, 15dPGJ₂ induces caspase activation that is blocked by peptide caspase inhibitors. These data show that de novo gene transcription is necessary for 15dPGJ₂-induced apoptosis in breast cancer cells. Critical candidate genes are likely to be revealed through analysis of differential cDNA array expression.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cyclopentenone prostaglandins potently induce cell cycle arrest and apoptosis in a number of cancer cell types (1-13). The terminal derivative of prostaglandin J₂ (PGJ₂)¹ metabolism, 15-deoxy-^12,14-PGJ₂ (15dPGJ₂), is a potent agonist for the peroxisome proliferator-activated receptor  (PPAR) (14, 15) and inhibits NFB, AP-1, nuclear factor of activated T-cells, and signal transducers and activators of transcription (16-20), 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 (25-28). Thus, it is critical to determine which mechanism(s) predominate to further develop these compounds as anticancer agents.

These studies demonstrate that 15dPGJ₂-induced apoptosis in breast cancer cells requires expression of specific gene products. Blocking de novo mRNA and protein expression inhibited 15dPGJ₂-induced apoptosis. Among the genes up-regulated by 15dPGJ₂, 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).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents and Cell Culture-- 15dPGJ₂ was purchased from Cayman Chemical (Ann Arbor, MI). 15dPGJ₂ is rapidly interconverted to a mixture of at least five active isomers (29). Actinomycin D, cycloheximide, 4',6-diamidino-2-phenylindole dihydrochloride, TNF, staurosporin, and CD95/Fas ligand were purchased from Immunotech (St. Louis, MO). Caspase inhibitors were purchased from Calbiochem. MDA-MB-231 breast cancer epithelial cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% penicillin, 1% streptomycin, and 1% L-glutamine (Life Technologies, Inc.). For cell synchronization experiments, cells were synchronized as described previously (30).

Flow Cytometry for Markers of Apoptosis-- Cells were incubated with or with out actinomycin D (1 µg/ml) or cycloheximide (2 µg/ml) for 1 h and then exposed to 15dPGJ₂ (10 µM) 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 µg/ml) or cycloheximide (2 µg/ml) for 1 h and then exposed to 15dPGJ₂ (10 µM) 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 µg/ml 4',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 15dPGJ₂ (10 µM) for 2 h. Total RNA was isolated using Trizol Reagent (Life Technologies, Inc.), and 1.0 µg 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 AtlasImage 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 µg/ml) or cycloheximide (2 µg/ml) for 1 h and then exposed to 15dPGJ₂ (10 µM) or vehicle (ethanol). After 9 h, total protein was isolated in lysis buffer, and 50 µg 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 µg/ml) or cycloheximide (2 µg/ml) for 1 h and then exposed to 15dPGJ₂ (10 µM) or vehicle (ethanol). After 2 h, total RNA was isolated in Trizol reagent (Life Technologies, Inc.), and 10 µg 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 70 °C.

Caspase Activity Assays-- Cells were incubated with 15dPGJ₂ (10 µM) with or with out the indicated caspase inhibitors (10 µM). 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Inhibition of RNA or Protein Synthesis Blocks 15dPGJ₂-induced Apoptosis-- Previous experiments in our laboratory showed that MDA-MB-231 cells undergo apoptosis as early as 8 h after exposure to exogenous 15dPGJ₂ (2). Here we investigated the minimum amount of exposure time necessary to induce apoptosis. Cell growth was markedly inhibited after cells were incubated with 15dPGJ₂ for as little as 2 h (Fig. 1A). Incubation of cells with 15dPGJ₂ 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 15dPGJ₂-induced apoptosis (data not shown).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   15-Deoxy-12,14-prostaglandin J2 rapidly induces apoptosis and requires RNA and protein synthesis. A, MDA-MB-231 cells were incubated with 15dPGJ2 for the indicated times, washed, and allowed to grow in fresh medium. After 96 h, total cell number was determined. B, MDA-MB-231 cells were incubated with the indicated compounds, and after 36 h the percentage of cells undergoing apoptosis was determined by flow cytometry. C and D, MDA-MB-231 cells were pretreated for 1 h with actinomycin D (Act) or cycloheximide (Chx) and then provided 15dPGJ2 or vehicle. After 5 h, cells were washed, and medium was replaced with fresh medium containing no drug. After 36 h, the percentage of cells undergoing apoptosis was determined by flow cytometry, and changes in cell surface and nuclear morphology were determined by histochemistry. DAPI, 4',6-diamidino-2-phenylindole dihydrochloride; FITC, fluorescein isothiocyanate.

Further investigation showed that treatment of cells with 15dPGJ₂ 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 15dPGJ₂-treated cells. Together these data show that early de novo transcription of genes is required for 15dPGJ₂-induced apoptosis.

Genes Regulated by 15dPGJ₂-- 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 15dPGJ₂-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 Bcl₂-binding 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 2.0-fold. Additionally, the expression of genes that are negative prognostic indicators in breast, colon, and liver cancer, including BRCA2, hepatoma-derived 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 1.5-fold. Thus, 15dPGJ₂ likely exerts potent antiproliferative and proapoptotic responses, at least in part, via activation of genes critical to cell cycle arrest and apoptosis.

View this table: [in this window] [in a new window]   Table I Summary of gene expression after exposure of MDA-MB-231 cells to 15dPGJ2 for 2 h 15-Deoxy-12,14-PGJ2 modulates the expression of critical cell cycle arrest and apoptosis genes. Total RNA was isolated from MDA-MB-231 cells treated with vehicle or 15dPGJ2 (10 µM) 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.

Expression of p21^Waf1/Cip1 and p27^Kip1 Is Increased by 15dPGJ₂-- 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 15dPGJ₂ precursor ¹²-prostaglandin J₂ (12PGJ₂), we have observed activation of p21 mRNA as early as 2 h using 15dPGJ₂. Consistent with this finding, 15dPGJ₂ 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 15dPGJ₂-induced p27 protein expression (Fig. 2C). However, 15dPGJ₂-induced p21 protein expression was blocked by cycloheximide only, despite evidence by Northern blot analysis that actinomycin did block 15dPGJ₂-induced p21 mRNA (Fig. 2D, see "Discussion").

View larger version (63K): [in this window] [in a new window]   Fig. 2.   p21Waf1/Cip1 and p27Kip1 expression is increased by 15-deoxy-12,14-prostaglandin J2 and blunted by actinomycin D or cycloheximide. A, MDA-MB-231 cells were synchronized and treated with or without 15dPGJ2, 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 15dPGJ2 and actinomycin suggests that p21 protein detected under similar conditions (C) is from preformed p21 mRNA (see "Discussion"). Chx, cycloheximide; Act, actinomycin; 15d, 15dPGJ2.

Caspase Activation Is Mediated by 15dPGJ₂-- The activation of caspases by 15dPGJ₂ 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 15dPGJ₂-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 15dPGJ₂-induced gene transcription, caspase activation is induced that cannot be completely blocked by an individual caspase inhibitor.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   15-Deoxy-12,14-prostaglandin J2-induced apoptosis requires caspase-3. A, MDA-MB-231 cells were exposed to 15dPGJ2. 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 15dPGJ2 (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 15dPGJ2 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 LEHD-fmk. After 36 h, the percentage of cells undergoing apoptosis was determined by flow cytometry.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 15dPGJ₂ induces intracellular oxidative stress in human neuroblastoma cells and hepatic myofibroblasts (38, 39) and that 15dPGJ₂ induces expression of antineoplastic enzymes, such as glutathione S-transferases (40). Additionally, Fitzpatrick and colleagues (37) showed that 12PGJ₂, the immediate precursor to 15dPGJ₂, inhibits isopeptidase activity of the proteosome pathway. We have shown that 15dPGJ₂ 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 15dPGJ₂-induced apoptosis in breast cancer cells requires the rapid synthesis of new gene products (<5 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 15dPGJ₂ 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 15dPGJ₂. However, our data suggest the relationship is not so straightforward, at least for p21. Actinomycin clearly blocks 15dPGJ₂-induced apoptosis but does not reduce early 15dPGJ₂-induced p21 protein levels. One explanation for this may be that arachidonic acid and many of its metabolites 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. 15dPGJ₂ 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 15dPGJ₂-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. 15dPGJ₂ 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 A₁ 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 15dPGJ₂ 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 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 15dPGJ₂-induced apoptosis. Whether this is mediated via PPAR or some other critical regulator of gene synthesis remains to be defined.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant RO1AI42022 and American Institute of Cancer Research Grant 97B108.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.

^¶ Received support through Grant DAMD17-00-1-0489 from the United States Army Medical Research Acquisition Activity (USAMRAA).

^** To whom correspondence should be addressed: Dept. of Internal Medicine, Section of Infectious Diseases, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157-1042. Tel.: 336-716-4584; Fax: 336-716-3825; E-mail: khigh@wfubmc.edu.

Published, JBC Papers in Press, November 1, 2001, DOI 10.1074/jbc.C100339200

	ABBREVIATIONS

The abbreviations used are: PGJ₂, prostaglandin J₂; 12PGJ₂, ¹²-prostaglandin J₂; 15dPGJ₂, 15-deoxy-^12,14-prostaglandin J₂; PPAR, peroxisome proliferator-activated receptor; p21, p21^Waf1/Cip1; p27, p27^Kip1; AA, arachidonic acid; COX-2, cyclooxygenase-2; (15S)-HETE, (15S)-hydroxyeicosatetraenoic acid; NFB, nuclear factor B; TNF, tumor necrosis factor; RT, reverse transcription; PPRE, PPAR response element; ZVAD-fmk, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone.

	REFERENCES

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