Introduction

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Cyclic GMP levels are determined by soluble and particulate guanylyl cyclases (GCs: GC-A, GC-B, and GC-C), cGMP PDE (gene families 1, 2, 3, 5, 6, 9, 10, and 11), and cGMP export pumps (Patel et al., 1995; Jedlitschky et al., 2000). In the intestine GC synthesis of cGMP from GTP can be activated by agonists such as guanylin, uroguanylin, bacterial toxins, nitric oxide, CO, and YC-1. Cyclic GMP PDE hydrolysis can control the intensity and duration of responses. In intestine and colon epithelium cells, GC-C is highly expressed and has been studied as a diagnostic marker for metastatic colorectal tumors in human extraintestinal tissues (Carrithers et al., 1996). PDE5 and PDE2 also show enhanced immunoreactivity in colon, lung, pancreatic, and bladder tumor tissues (Piazza et al., 2000, 2001a,b,c). Thus, cGMP metabolism in colon tumor cells may be a useful therapeutic target.

Exisulind (sulindac sulfone) is a proapoptotic drug that causes regression and prevents recurrence of polyps in patients with familial adenomatous polyposis (Stoner et al., 1999). Exisulind and its analogs CP461, CP78, and CP248 inhibit cell growth and induce apoptosis in SW480 colon tumor cells without cyclooxygenase (I or II) inhibition (Thompson et al., 2000b). The analogs of exisulind were developed as inhibitors of cGMP PDEs with a preference for PDE5, 2, and 1 gene families. It is apparently important to maintain cross-reactivity among these isoforms, because highly selective PDE5 inhibitors do not induce apoptosis in tumor cell lines (Thompson et al., 2000b). These drugs inhibit cGMP PDEs 5 and 2 expressed by SW480 cells and PDE5 expressed by HT29 colon tumor cells (Soh et al., 2000; Thompson et al., 2000b). Because these agents maintained similar rank orders of potency for apoptosis induction, growth inhibition, cGMP PDE5 and 2 inhibition, and also caused sustained intracellular cGMP increases in the colon tumor cells (Thompson et al., 2000b), we proposed a cGMP-mediated mechanism underlying the actions of exisulind and analogs on apoptosis in neoplastic cells. Soh et al. (2000) found that cGMP mediated apoptosis in SW480 and HT29 cells by mechanisms involving activation of c-Jun NH₂-terminal kinase 1 (JNK1). The studies reported here extend our initial finding that exisulind and analogs also activated PKG (Thompson et al., 2000b). Recent studies by Soh et al. (2001) have shown that PKG activates JNK1 via a novel PKG phosphorylation and activation of MEKK1.

The possible involvement of cGMP and PKG in apoptosis is supported by studies in rat myocytes (Wu et al., 1997), pancreatic -cells (Loweth et al., 1997), and endothelial cells (Suenobu et al., 1999), as well as data showing that transfection of PKG increases the sensitivity of vascular smooth muscle cells to apoptosis inducers (Chiche et al., 1998). Boerth et al. (1997) have shown that PKG also plays a major role in the phenotype and morphology of vascular smooth muscle cells with the involvement of extracellular signal receptor-activated kinase activation with increased proliferation (Komalavilas et al., 1999). To test our hypothesis and further explore cGMP-induced apoptosis, we have used a sensitive solid phase assay to determine PKG activity changes from cell supernatants treated with exisulind and a higher affinity analog, CP461, as well as the GC activators, YC-1 and guanylin. CP461, YC-1, and guanylin increased PKG and, like exisulind, induced cell growth inhibition and apoptosis in colon tumor cells.

Exisulind and CP461 decrease -catenin in colon tumor cells expressing either mutated or wild-type adenomatous polyposis coli (APC) genes (Thompson et al., 2000a,b). Because -catenin phosphorylation is required for its ubiquitination and subsequent proteolysis, phosphorylation of -catenin, an oncogenic protein, by purified PKG and exisulind-activated PKG in SW480 cells was studied as a downstream target and pathway responsive to cyclic GMP-controlled apoptosis. The results show exisulind and CP461 induce -catenin phosphorylation and processing, providing a mechanism to circumvent -catenin accumulation from colon genetic mutations.

	Experimental Procedures

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Materials. Cyclic GMP was obtained from ICN (Costa Mesa, CA). 8-Br-cGMP and Rp-8-Br-cGMPs were obtained from BIOMOL (Plymouth Meeting, PA). 8-Br-cGMP was further purified with Sephadex G-25 (Amersham Pharmacia Biotech, Piscataway, NJ) chromatography (Corbin et al., 1988). [3-(5'-Hydroxymethyl-2'-furyl)-benzylindazole] (YC-1), human guanylin, and forskolin (FSK) were purchased from Alexis Biochemicals (San Diego, CA). E4021 was from Eisai Co., Ltd. (Tokyo, Japan). Exisulind (sulindac sulfone) and CP461 were synthesized by Cell Pathways, Inc. (Horsham, PA). Isopropyl--D-thiogalactoside and GSH-Sepharose 4B were from Amersham Pharmacia Biotech. Purified PKG I, catalytic subunit of cAMP-dependent protein kinase (PKA, mouse recombinant), GSK-3 (recombinant), and PKA inhibitor PKI (5-24) were obtained from Calbiochem-Novabiochem (San Diego, CA).

Cell Culture. SW480, HCT116, HT29, and T84 colon tumor cells were obtained from American Type Culture Collection (Rockville, MD). SW480, HCT116, and HT29 cells were grown in RPMI 1640 media supplemented with 5% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 µg/ml amphotericin. T84 cells were grown in 47% Ham's F-12 media (American Type Culture Collection) and 47% Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 5% fetal bovine serum, 8.4 mM sodium bicarbonate, 100 units/ml penicillin, 100 units/ml streptomycin, and 0.25 µg/ml amphotericin, pH 7.25. Cells were harvested at 70 to 90% confluence with either trypsin/EDTA or pancreatin and used immediately. Exisulind and CP461 were solubilized in 100% DMSO and diluted with media to obtain a final DMSO concentration of 0.5% or less.

Cloning and Expression of GST-PDE5_36-529 for PKG Activity Assay. RT-PCR methods were used to obtain a domain of PDE5_36-529 (Val₃₆-Glu₅₂₉ relative to bovine PDE5) (McAllister-Lucas et al., 1993) from HT-29 cells as a PKG substrate. The forward primer (GTT-AGA-AAA-GCC-ACC-AGA-GAA-ATG) and the reverse primer (AGC-TCT-CTT-GTT-TCT-TCC-TCT-GCT) defined a 1484-base pair fragment containing phosphorylation and high- and low-affinity cGMP binding sites of PDE5 (PDE5_36-529). The PCR product was cloned into a pGEX-5X-3 GST fusion vector (Amersham Pharmacia Biotech) with EcoRI and XhoI cloning sites. The DNA construct was sequenced by Applied Biosystems (Foster City, CA) model 377 Prism DNA sequencers at the DNA Sequencing and Synthesis Facility at Iowa State University, Ames, IA.

Apparent K_m Determination of GST-PDE5_36-529 Phosphorylation. The protein phosphorylation assay was performed in 10 mM potassium phosphate buffer, pH 6.8, containing 190 µM [-³²P]ATP (3000 Ci/mmol; PerkinElmer Life Science Products, Boston, MA) and 4.5 mM MgCl₂. The phosphorylation reaction was initiated by adding PKG I to affinity-purified GST-PDE5_36-529 in phosphorylation buffer with added cGMP (20 µM) unless indicated otherwise. Incubation was at 30°C and the reaction was terminated by spotting 50 µl of reaction mixture onto Whatman P-81 phosphocellulose paper (Roskoski, 1983). After four washes with 75 mM phosphoric acid, the paper was air-dried and counted on a Beckman Coulter LS 6500 scintillation counter (Beckman Coulter, Inc., Fullerton, CA). The phosphate incorporation of GST-PDE5_36-529 was also analyzed by SDS-PAGE followed by phosphor imaging (Cyclone; Packard, Meriden, CT).

Cell PKG Activity Assay. SW480, HCT116, HT29, and T84 colon tumor cells were treated with compounds or DMSO (0.5%) by using culture conditions as described above. Cells (~1 × 10⁷) were washed with cold PBS and lysed with cold modified RIPA buffer (400 µl) containing 50 mM Tris-HCl, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 1 mM NaF, 500 µM 3-isobutyl-1-methylxanthine, and Complete protease inhibitor cocktail (Roche Molecular Biochemicals, Palo Alto, CA). Cell supernatants were obtained by centrifugation of the cell lysates at 14,000 rpm with an Eppendorf model 5417C for 15 min at 4°C. Protein was quantitated by Bio-Rad DC protein assay (Hercules, CA). PKG activities from cell supernatants were measured using, as a phosphate acceptor, the fusion protein fragment of PDE5 (GST-PDE5_36-529) bound to GSH-Sepharose affinity beads. Cell supernatant (100 µg), substrate (20 µg of bound protein), 0.5 µM PKI, 4.5 mM Mg²⁺, and [-³²P]ATP (10 µCi, 190 µM) were mixed with or without added cGMP and incubated at 30°C for 30 min. The phosphorylated PDE5 fragment on beads (~85 kDa) was resolved on 7.5% SDS-PAGE and ³²P incorporation quantitated (digital light units) by using a phosphor imaging system (Cyclone; Packard). Exposure times were optimized to maintain a linear range. In addition digitized images in the figures are normalized to no drug treatment (fold versus no drug).

Western Blot. Total protein per sample (50 µg) of cell supernatant prepared as described above was loaded onto 10% NuPAGE Bis-Tris gels (Novex, San Diego, CA) and transferred. Western blots were probed with affinity-purified rabbit polyclonal antibody (PK002; Stressgen Biotechnologies, Victoria, BC, Canada) specific for human PKG I and detecting an 80-kDa band. Another affinity-purified peptide antibody (PK005; Stressgen Biotechnologies) detecting both human PKG I (75-kDa) and I (80-kDa) isoforms has been studied. Bound antibody was identified with the corresponding horseradish peroxidase-conjugated anti-IgG secondary antibodies by using BM blue substrate (Roche Molecular Biochemicals). The results were analyzed using an AlphaImager 2000 and software (Alpha Innotech Corporation, San Leandro, CA).

Radioimmunoassay for cGMP and cAMP. Approximately 5 × 10⁶ cells were used for each assay. Cells were plated on 100-mm dishes and drugs were added after 2 days of growth at specified times and doses. This was followed by a rapid wash, 0.2 N HCl/50% methanol extraction (1 ml), and drying. The dried samples were reconstituted in water and acetylated before radioimmunoassay with anti-cGMP and anti-cAMP antibodies (Brooker et al., 1979).

Cell Growth Inhibition and DNA Fragmentation. Cell growth inhibition was determined by plating cells at 1000 cells/well in 96-well plates. Cells were dosed after 24 h and incubated for six more days. Cells were fixed with 50% trichloroacetic acid at 4°C for 1 h, rinsed five times with deionized H₂O, and incubated for 10 min with 0.4% sulforhodamine B in 1% acetic acid. Plates were rinsed four times with 1% acetic acid, dried 30 min, and solubilized in 10 mM Tris. Absorbance was determined at 540 nM by using a Molecular Devices Spectra Max 340 plate reader. For apoptosis assay, cells were seeded at 10,000 cells/well in 96-well plates. After 24 h, cells were dosed and grown for an additional 48 h. DNA fragmentation was measured using a double antibody ELISA kit (Roche Molecular Biochemicals) that detects histone protein and fragmented DNA.

-Catenin Phosphorylation in Vitro and in Intact Cells. SW480 cells were lysed using modified RIPA buffer with protease inhibitors and cell supernatants were obtained as described above. From 500 µg of supernatant, -catenin was immunoprecipitated using 4 µg of rabbit anti--catenin IgG for 2 h at 4°C followed by an additional overnight incubation with 100 µl of protein A-agarose beads. The immunoprecipitates were washed three times with cell lysis buffer and one time with kinase assay buffer. Human -catenin (2-698) (Hulsken et al., 1994) was expressed as a GST fusion protein in Escherichia coli (BL21) by using -catenin cDNA cloned by RT-PCR from SW480 cells and purified by GSH-Sepharose 4B affinity chromatography.

	Results

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Solid Phase PKG Activity Assay. The solid phase substrate used to determine PKG activity was GST-PDE5_36-529 bound as a GSH conjugate to Sepharose 4B beads (Fig. 1A). PKI, a specific inhibitor of PKA, was added to the assay mixture to block phosphorylation by PKA in cell lysates. As shown in Fig. 1B, 0.5 µM PKI completely blocked PKA phosphorylation of GST-PDE5_36-529 without influencing PKG phosphorylation. Phosphorylation of GST-PDE5_36-529 by SW480 cell supernatants was time- and cGMP-dependent (Fig. 1, C and D). The assay was linear for up to 60 min and 100 µM cGMP activated PKG maximally. Rp-8-Br-cGMP, a PKG-specific inhibitor, blocked the phosphorylation of GST-PDE5_36-529 by SW480 cell supernatants (Fig. 1E). The apparent K_m for PKG I phosphorylation of GST-PDE5_36-529 was 3 µM (Fig. 2A), indicating a higher affinity than BPDEtide at 68 µM (Colbran et al., 1992) and suggesting that GST-PDE5_36-529 is an improved substrate over BPDEtide for PKG. PKG phosphorylation showed 0.9 moles of phosphate incorporated per mole of GST-PDE5_36-529 protein after 1 h (Fig. 2B), data consistent with the phosphorylation of bovine PDE5 at Ser92 (Thomas et al., 1990; Colbran et al., 1992). Moreover, mutation of Ser92 to Ala (S92A) in GSTPDE5_36-529 substrate completely prevented PKG phosphorylation of the protein (Fig. 2B, inset). Because of the low K_m, quantitative and site-specific phosphorylation of GST-PDE5_36-529, this solid phase assay provides a specific and sensitive measure of PKG activity from cell supernatant without enzyme purification.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Solid phase PKG assay. A, solid phase PKG substrate: GST-PDE536-529 bound to GSH-Sepharose 4B beads. The protein bound to the beads was resolved in 10% SDS-PAGE and stained with Coomassie blue. B, catalytic subunit of PKA (20 ng) or PKG I (2 ng) was incubated in PKG assay buffer with solid phase substrate (20 µg) in the absence or presence of 0.5 µM PKI and analyzed by 7.5% SDS-PAGE and autoradiography. C, SW480 cell supernatant (100 µg) was incubated with PKG assay reaction mixture containing 20 µg of substrate in the absence or presence of cGMP (100 µM) as described under Experimental Procedures for the time indicated at 30°C. Phosphorylation was quantitated with phosphor imaging system. D, cyclic GMP (1-1000 µM) was added to SW480 cell supernatant (100 µg) in the PKG assay reaction mixture, incubated at 30°C for 30 min, and the phosphorylation was quantitated by determination of the 32P incorporation in protein bands with a scintillation counter. E, Rp-8-Br-cGMPs (1 mM), a PKG inhibitor, was added to the SW480 cell supernatant (100 µg) reaction mixture in the absence or presence of cGMP (100 µM). The reaction was incubated at 30°C for 30 min. The phosphorylated proteins were resolved by 7.5% SDS-PAGE, quantitated, and visualized by phosphor imaging.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Specific phosphorylation of GST-PDE536-529. A, GST-PDE536-529 (0.14-7 µM) was incubated with cGMP (20 µM) and 45 ng of purified bovine PKG I in the presence of [-32P]ATP in phosphorylation buffer at 30°C for 30 min. The reaction was stopped by blotting to P81 filter paper and phosphate incorporated was determined as described under Experimental Procedures. Apparent Km was calculated by double reciprocal plot. B, GST-PDE536-529 (10 µM) was incubated with 20 µM cGMP and 45 ng of PKG I in the phosphorylation reaction as described under Experimental Procedures at 30°C for 0 to 65 min. Inset, 2 µg of WT or Ser92 mutant (S92A) GST-PDE536-529 was incubated with 20 µM cGMP, 5 ng PKG I, and [-32P]ATP in phosphorylation buffer at 30°C for 15 min. The phosphorylated protein was analyzed by 7.5% SDS-PAGE followed by exposure to phosphor imager.

Exisulind-Induced PKG Activation in Intact Colon Tumor Cells. SW480 cells were treated with various concentrations of exisulind for 40 min and PKG activities determined using the solid phase assay. Treatment with exisulind showed a concentration-dependent increase in PKG activity both in the absence or presence of cGMP in the assay (Fig. 3A). E4021, a drug that did not sustain cGMP increases, inhibit cell growth, or induce apoptosis (Thompson et al., 2000b), had no effect on PKG activity in SW480 cells. The doses of exisulind that activate PKG (200-600 µM) are consistent with concentrations needed to induce apoptosis or inhibit cell growth (Thompson et al., 2000b).

View larger version (59K): [in this window] [in a new window]   Fig. 3.   Increased PKG activity in SW480 colon tumor cells induced by exisulind treatment. SW480 cells were treated with exisulind and E4021 at 37°C for 40 min (A), with exisulind (400 µM) for 0 to 24 h (B), and with exisulind and E4021 for 48 h (C). Bar graphs on the right of each figure show 32P incorporation measured in the phosphor imager (digital light units). PKG activities were measured using solid phase substrate as described under Experimental Procedures. Cell supernatant (100 µg), substrate (20 µg), PKI (0.5 µM), Mg2+ (4.5 mM), and [-32P]ATP (10 µCi, 200 µM) with or without added cGMP were mixed and incubated at 30°C for 30 min. The phosphorylated GST-PDE536-529 was resolved by 7.5% SDS-PAGE, quantitated, and visualized by phosphor imaging. Fold activation from basal activity was determined by quantitating the ratio of drug treated to the no-drug control in the cG panel. Fold activation for +cG is compared with cG above. The data represent three separate experiments.

Exisulind-Induced PKG Protein Expression. PKG activity increases were studied further in SW480 cells treated with exisulind by using antibody specific for the human I isoform (Fig. 4). Induction of PKG I protein was detectable at 8 h after exisulind (600 µM) treatment. Exisulind continued to increase PKG expression in the cells attached to the plates for up to 72 h (Fig. 4A). PKG I protein induction by exisulind was dose-dependent (Fig. 4B). Consistent with a lack of effect on cGMP accumulation, the kinase was not induced by treatment with E4021 treatment. A different affinity-purified peptide antibody detecting both human PKG I and I isoforms showed I 80-kDa immunoreactivity but no band at PKG I 75 kDa by Western blot (data not shown). RT-PCR with published primers (Selvaraj et al., 2000) detected PKG II mRNA in these colon tumor cells (data not shown). PKG II isoforms have not been studied further due to lack of commercially available antibody.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Western blot of PKG I induction from SW480 cells. Cell supernatants (50 µg) from SW480 cells treated with exisulind or E4021 were loaded on 10% NuPAGE gel. Anti-PKG I was used to quantitate PKG protein expression as described under Experimental Procedures.

Lack of Direct Effect of Exisulind or CP461 on PKG Activity. To determine whether exisulind or CP461 had a direct effect on PKG to activate the enzyme and therefore, would not require an intact cell for activation, we studied PKG activation in vitro. No direct activating effects were seen when exisulind or CP461 was added to the phosphorylation reaction mixtures (Fig. 5). Neither exisulind nor CP461 was effective with recombinant PKG I added to solid phase substrate (Fig. 5A). PKG in untreated SW480 cell supernatants was not directly activated by either drug after the cells were lysed (Fig. 5B). Furthermore, PKG in supernatants from cells treated with exisulind for 48 h was not activated by adding exisulind or CP461 directly to the assay (Fig. 5C). To ensure enzyme integrity cGMP (10 µM) and 8-Br-cGMP (10 µM) were added to activate PKG in the same phosphorylation reaction mixtures (Fig. 5, A and C).

View larger version (37K): [in this window] [in a new window]   Fig. 5.   No direct effects on PKG activity. Exisulind, CP461, cGMP, or 8-Br-cGMP were added to PKG I (5 ng, A) or cell supernatants (100 µg) from SW480 cells treated with DMSO (0.5%, B) or 500 µM exisulind for 48 h (C). The PKG activity was measured with 20 µg of GST-PDE536-529 bound to GSH-Sepharose 4B, 0.5 µM PKI, 4.5 mM Mg2+, and [-32P]ATP (10 µCi, 200 µM) at 30°C for 30 min. The phosphorylated proteins were resolved by 7.5% SDS-PAGE and visualized by autoradiography.

PKG Activation and Induction by Exisulind in Multiple Colon Tumor Cell Lines. Exisulind also increased PKG activity in HT29, HCT116, and T84 colon tumor cells (Fig. 6A). Although the basal activity varied from cell line to cell line, PKG activity was increased at 1-h drug treatment. After 48-h drug treatment, all four colon tumor cell lines showed PKG I induction by Western blot analysis (Fig. 6B).

View larger version (67K): [in this window] [in a new window]   Fig. 6.   Exisulind induced PKG activity and PKG I immunoreactivity from different colon tumor cells. A, SW480, HT-29, HCT116, and T84 cells were treated with exisulind (500 µM) for 1 h. Cells were washed and lysed with modified RIPA buffer. Supernatant protein (100 µg) of the lysate (C, DMSO control; Exi, exisulind) was used for PKG activity assay as described under Experimental Procedures. B, PKG I protein expression after 48-h exisulind (500 µM) treatment determined by Western blots as described under Experimental Procedures.

CP461 and YC-1 Activation of PKG in SW480. CP461 inhibits cGMP PDE isoforms with more selectivity than exisulind and shows higher affinity for PDE5 and PDE2 than exisulind (IC₅₀ = 3 and 14 µM for CP461, respectively, and 114 and 335 µM for exisulind, respectively). Because YC-1 and CP461 have been shown to increase cGMP, inhibit cell growth, and induce apoptosis in SW480 cells (Thompson et al., 2000b), we studied PKG activation by these agents. CP461 and YC-1 increased PKG activity in SW480 cells after 40-min and 24-h treatments (Fig. 7, A and B) and induced more PKG I protein after 24-h drug treatments (Fig. 7C). FSK (10 µM) increased cellular cAMP 32-fold in SW480 cells, but did not increase cellular cGMP, inhibit cell growth, induce apoptosis, or activate PKG after 24-h treatment (Fig. 7B). Exisulind-, CP461-, and YC-1-induced PKG activity remained sensitive to activation by 100 µM cGMP in the assay.

View larger version (36K): [in this window] [in a new window]   Fig. 7.   CP461 and YC-1 activation and induction of PKG in SW480. SW480 cells were treated with exisulind (Exi) or CP461 for 40 min (A) and with CP461, YC-1, and FSK for 24 h (B and C). Cells were washed with cold PBS and lysed with modified RIPA buffer. Supernatants (100 µg) of the lysate from 40-min drug treatment (A) and 24-h drug treatment (B) were used for PKG activity assay as described under Experimental Procedures. C, PKG I protein expression after 24-h drug treatment in SW480 cells was determined by Western blots. Supernatant protein (50 µg) of each cell lysate was loaded on 10% NuPAGE gel. Anti-PKG I was used to quantitate PKG protein expression as described under Experimental Procedures.

Guanylin Increased cGMP and Induced Apoptosis in T84 Cells. T84 colon tumor cells express membrane-bound GC-C (Singh et al., 1991). Guanylin is a 15-amino acid peptide homolog of bacterial heat-stable enterotoxin and an endogenous activator of GC-C. When T84 colon tumor cells were treated with 200 nM guanylin for 40 min, cellular cGMP was increased 3.6-fold (Fig. 8A). Exisulind (400 µM) increased cGMP in T84 cells at 40 min and an additive accumulation of cGMP was detected in T84 cells treated with guanylin in combination with exisulind (Fig. 8A). Consistent with cGMP increases, guanylin and exisulind also induced apoptosis in T84 cells and showed an additive effect on apoptosis in combination at 48-h treatment (Fig. 8B). Guanylin (200 nM) also increased PKG activity 2-fold in T84 cells after 1-h incubation (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 8.   Guanylin increased cGMP and apoptosis induction in T84 cells. T84 cells were treated with compounds (control, 0.5% DMSO; Gua, 200 nM human guanylin; Exi, 400 µM exisulind; Gua + Exi, 200 nM guanylin + 400 µM exisulind) for 40 min (A) and 48 h (B). Intracellular cGMP levels (A) were measured by radioimmunoassay and DNA fragmentation induction (B) was measured by ELISA as described under Experimental Procedures. Data shown are representative of three separate experiments. *P < 0.05 compared with control group, **P < 0.05 compared with either guanylin or exisulind treatment alone; two-tailed, unpaired Student's t test.

-Catenin Phosphorylation by PKG. Because we have postulated that -catenin is a downstream target of PKG activated by exisulind or CP461 that could mediate regulation of apoptosis pathways (Thompson et al., 2000b), -catenin phosphorylation was investigated further. PKG I phosphorylated -catenin immunoprecipitated from SW480 cells (Fig. 9A) or purified GST--catenin (Fig. 9B). GSK-3, known to require -catenin complexed to APC and axin proteins to phosphorylate -catenin, was not effective in the immunoprecipitates (Fig. 9A). PKG from SW480 cells activated by exisulind for 48 h increased GST--catenin phosphorylation (Fig. 9C), indicating the induced PKG also showed higher specific activity with -catenin substrate.

View larger version (61K): [in this window] [in a new window]   Fig. 9.   In vitro phosphorylation of -catenin. Autoradiography staining -catenin from SW480 (A) was prepared by immunoprecipitation with anti--catenin IgG. GST--catenin2-698 (B and C) was cloned and affinity purified as described under Experimental Procedures. -Catenins were phosphorylated with purified kinase (A and B) or exisulind (48 h)-treated cell supernatant (C) in [-32P]ATP (10 µCi, 200 µM) and MgCl2 (4.5 mM) buffer. Phosphorylated -catenins were resolved by 7.5% SDS-PAGE and quantitated by phosphor imaging. A, -catenin immunoprecipitates from SW480 (50 µl/assay) were phosphorylated with PBS buffer (C), purified PKG I (49 ng, 2 µM cGMP), or GSK-3 (800 ng) at 30°C for 30 min. B, GST--catenin2-698 was phosphorylated by PKG I (13 ng) at 30°C for 30 min. C, GST--catenin bound to Sepharose beads (20 µg) was phosphorylated at 30°C for 20 min by cell supernatant (100 µg) from SW480 cells treated with DMSO (0.5%, C) or exisulind (500 µM, Exi) for 48 h.

View larger version (32K): [in this window] [in a new window]   Fig. 10.   In vivo phosphorylation of -catenin in SW480 cells. SW480 cells were treated with DMSO (0.2%, C) or exisulind (500 µM) for 18 h in 32P-RPMI medium with 5% fetal bovine serum. Cells were washed and lysed with modified RIPA buffer. -Catenin was immunoprecipitated with anti--catenin IgG, and a Western blot with anti--catenin IgG was used to normalize the amount of -catenin loaded in each lane. [32P]-catenin was resolved on 7.5% SDS-PAGE and quantitated by phosphor imaging.

Summary Correlations of Exisulind and CP461 on Colon Cell cGMP and Apoptosis. Table 1 shows a summary of data published previously and the current study on colon tumor cell lines with exisulind and CP461 effects on cGMP-mediated apoptosis. Two of the cell lines show major cGMP PDE isoform expressions of PDE5 and PDE2 and two of the lines express only PDE5. Inhibition constants for fractionated isoforms correlate with growth inhibition and apoptosis determined by DNA fragmentation ELISA assays. PKG I is increased by both drugs in all the cell lines at similar concentrations and cGMP is increased and sustained from 1 to 72 h in three of the four cell lines. Not shown on this table are cAMP values. Cyclic AMP values for SW480, T84, and HT29 cells were in the 4000 to 5000 fmol/mg range or approximately 50 times the level of cGMP, which varied from 50 to 200 fmol/mg in the three cell lines. Cyclic AMP levels remain unchanged in T84 and SW480 cells and decreased in HT29 cells with drug treatment for 1 to 72 h.

	Discussion

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These studies support the proposed involvement of cGMP and PKG in exisulind- and CP461-mediated apoptosis in colon tumor cells (Thompson et al., 2000b; Soh et al., 2000, 2001). Although cGMP is a mediator of signal transduction with intracellular targets, including several cGMP PDEs, cGMP-gated cation channels, and PKG, Wu et al. (1997) have suggested a possible role for PKG and cGMP in smooth muscle cell apoptosis in addition to the more well studied platelet aggregation and smooth muscle relaxation events. Exisulind and CP461 caused PKG activation in SW480, HT-29, HCT116, and T84 colon tumor cell lines at concentrations that correlated with cGMP PDE inhibition constants, cGMP increases, and apoptosis induction and cell growth inhibition. Exisulind administration to patients with familial adenomatous polyposis induced apoptosis in dysplastic epithelial but not normal-appearing colonic mucosa, thus leading to the term selective, apoptotic, and antineoplastic drugs (SAANDs) for this class of compounds (Stoner et al., 1999).

The solid phase assay used to assess PKG activity in supernatants was validated with respect to linearity in crude cell fractions and appropriate cGMP and analog activation and inhibition criteria. The substrate (GST-PDE5_36-529) was specifically phosphorylated by PKG with one phosphorylation site. The phosphorylation site of Ser92 was further confirmed by site-directed mutagenesis. The higher affinity of GST-PDE5_36-529 fusion protein (3 µM) versus BPDEtide (68 µM) suggests that interaction between PKG and GST-PDE5_36-529 involves additional contacts aside from those in the immediate phosphorylation site. Therefore, the solid phase assay provided a higher affinity substrate with PKG specificity, as well as rapid and stable processing by being bound to beads.

Activation of PKG by exisulind and CP461 in SW480 cells was both rapid (within minutes) and sustained (continued through 72 h). Exisulind and CP461 have both been shown to increase cGMP and sustain the increased cGMP levels in SW480, HT29, and T84 cells for up to 72 h (Thompson et al., 2000b), suggesting that PKG activation may be mediated by cGMP. In addition, Deguchi et al. (2001) confirmed PKG activation by exisulind, CP461, and YC-1 in intact SW480 cells by using the in vivo substrate vasodilator-stimulated phosphoprotein phosphorylation. The drugs have no direct effect on supernatant PKG activity or on exisulind-activated PKG activity and therefore require an intact cell for activation of the enzyme.

This novel mechanism whereby exisulind and CP461 induces PKG activation and subsequently apoptosis is supported by the effects of YC-1, an activator of soluble GC, and guanylin, an activator of membrane-bound GC. Both agents increased cellular cGMP, activated PKG, and induced apoptosis in colon tumor cells. 8-Br-cGMP, a PKG activator, also showed apoptosis induction in SW480 cells by using a 7-day morphology assay (data not shown). Related studies are also supportive because transfection of constitutively active PKG I into SW480 inhibited cell growth and induced apoptosis through phosphorylation of MEKK1 and activation of JNK1 (Soh et al., 2000, 2001; Deguchi et al., 2001). Moreover, Shailubhai et al. (2000) found that another GC-C activator, uroguanylin, induced apoptosis in T84 cells via cGMP, and when administered to the Min/+ mouse model of colorectal cancer inhibited the formation of polyps. During the preparation of this manuscript, Pitari et al. (2001) showed that both E. coli heat-stable enterotoxin and uroguanylin treatment inhibited serum and/or L-glutamine-stimulated T84 colon tumor cell growth. However, their data showed a cGMP-mediated effect to delay cell cycle progression with no increase in apoptosis in the serum-starved and restimulated cell model. Because increased cGMP can induce apoptosis in unstimulated T84 cells (Shailubhai et al., 2000; present study), the experimental conditions used to study T84 cells appear critical for apoptosis induction.

Exisulind and CP461 appear to increase cellular cGMP, and not cAMP, by inhibiting cGMP PDEs for which they show a PDE5 preference, but not specificity, because in vitro both drugs inhibit PDE2 and 1 gene family isoforms in addition to PDE5. The colon tumor cell lines tested all prominently expressed PDE5 with two of the lines showing PDE2. Analogs of exisulind are proapoptotic drugs developed from screening for inhibitors of PDE5 and 2 while maintaining apoptosis and growth-inhibiting activity. It is not clear what isoforms of PDE are effected by exisulind and analogs when cGMP is increased in colon tumor cells; however, inhibitors lacking the chemical structure to inhibit both isoforms in vitro lose apoptosis-inducing properties. Thus, as shown previously, very selective PDE5 inhibitors in vitro, such as E4021, are not proapoptotic drugs and these agents do not sustain cGMP increases in intact cells or activate PKG in colon tumor cells. We have not found PDE 1, 9, 10, or 11 activities in these colon tumor cells (Li et al., 2000), although as reported earlier, mRNAs for all PDE gene families can be detected by RT-PCR in these cell lines. PDE5, and to a lesser extent PDE2, show enhanced immunoreactivity in biopsies of human colon adenomas and adenocarcinomas, suggesting a role for these enzymes in colon cell survival (Piazza et al., 2000). As discussed elsewhere (Thompson et al., 2000b), the less selective PDE5 inhibitors, such as SAANDs, may be more effective as antineoplastic agents because of the multiple inductive effects of PDE inhibitors on alternative isoforms that could circumvent selective inhibitor actions.

Although the mechanism of PKG activation in intact colon tumor cells is under investigation, in vitro activation models show that activity of PKG I and  isozymes is increased by cGMP binding and autophosphorylation (Hofmann et al., 1985; Francis et al., 1996; Smith et al., 1996; Chu et al., 1998). These studies showed that cGMP binding stimulated basal kinase activity and increased the sensitivity of the enzyme to cGMP. Autophosphorylation as a result of increased cGMP has been shown to prolong increased kinase activity after cellular cGMP declines. Because exisulind and CP461 cause sustained cGMP increases, cGMP and autophosphorylation could both provide mechanisms to sustain an increased activation of the enzyme by the drugs until protein induction at approximately 8 h of drug treatment. Our homogenization conditions apparently preserve the active state of PKG 1 induced by exisulind or CP461. No evidence for PKG I induction by exisulind treatment was seen in SW480 cells. SAANDs are the first class of drugs shown to induce the synthesis of PKG. Because most cultured cells appear to adapt to little or no PKG expression (Cornwell et al., 1994), regulation of PKG protein synthesis needs further study to determine the role of cGMP and PKG in the induction mechanism, if any. The concentrations of exisulind and CP461 required for PKG induction were consistent with cGMP PDE inhibition and PKG activating concentrations. E4021, did not induce PKG consistent with its lack of PKG activation, further suggesting that the sustained effects of exisulind and CP461 are important for apoptosis induction.

Exisulind and analogs decrease -catenin levels in SW480 cells, suggesting a mechanism to regulate apoptosis (Thompson et al., 2000b). Cytosolic and nuclear -catenin accumulations occur in a variety of tumors, including SW480, HCT116, HT29, and T84 colon tumor cells due to mutations in the protein and defective phosphorylation from APC mutations (Morin et al., 1997; Efstathiou et al., 1998). Based on in vitro phosphorylation data, we have proposed previously that -catenin might be a downstream target of PKG (Thompson et al., 2000b). Ubiquitination, and thus turnover of -catenin via proteosomal processing, requires phosphorylation of the protein. The results of the current studies show that purified PKG phosphorylated immunoprecipitated -catenin from SW480 cells, as well as recombinant -catenin expressed as a GST fusion protein. Furthermore, cell supernatants activated by exisulind treatment of SW480 cells for 48 h showed increased phosphorylation of -catenin compared with untreated cell supernatant phosphorylation. In intact cells prelabeled with ³²P, exisulind treatment resulted in a 3-fold increase in -catenin phosphorylation. These data indicate that PKG activation by exisulind results in -catenin phosphorylation to initiate degradation by the ubiquitin-proteosomal system. The PKG phosphorylation site(s) on -catenin, a protein that contains multiple potential sites, is under investigation. Reduced -catenin in SW480 cells would provide a mechanism leading to enhanced apoptosis by exisulind and other SAANDs. Activation of the proapoptotic Jun kinase by exisulind and analogs through PKG activation and phosphorylation of MEKK1 (Soh et al., 2000, 2001) would be expected to complement -catenin reduction to provide antineoplastic cell killing by SAANDs.