W. Joseph Thompson

Activation of Protein Kinase G Is Sufficient to Induce Apoptosis and Inhibit Cell Migration in Colon Cancer Cells

The activation of protein kinase G (PKG) by cGMP has become of considerable interest as a novel molecular mechanism for the induction of apoptosis in cancer cells, because sulindac sulfone (exisulind, Aptosyn) and certain derivatives that inhibit cGMP-phosphodiesterases and thereby increase cellular levels of cGMP appear to induce apoptosis via this mechanism. However, other effects of these compounds have not been excluded, and the precise mechanism by which PKG activation induces apoptosis has not been elucidated in detail. To directly examine the effects of PKG on cell growth and apoptosis, we generated a series of mutants of PKG Iα: PKG IαS65D, a constitutively activated point mutant; PKG IαΔ, a constitutively activated N-terminal truncated mutant; and PKG IαK390R, a dominant-negative point mutant. A similar series of mutants of PKG Iβ were also constructed (Deguchi et al., Mol. Cancer Ther., 1: 803–809, 2002). The present study demonstrates that when transiently expressed in SW480 colon cancer, the constitutively activated mutants of PKG Iβ, and to a lesser extent PKG Iα, inhibit colony formation and induce apoptosis. We were not able to obtain derivatives of SW480 cells that stably expressed these constitutively activated mutants, presumably because of toxicity. However, derivatives that stably overexpressed wild-type PKG Iβ displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Iβ grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Iβ was associated with decreased cellular levels of β-catenin and cyclin D1 and increased levels of p21CIP1. Reporter assays indicated that activation of PKG Iβ inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Iβ inhibited cell migration. Taken together, these results indicate that activation of PKG Iβ is sufficient to inhibit growth and cell migration and induce apoptosis in human colon cancer cells and that these effects are associated with inhibition of the transcription of cyclin D1 and an increase in the expression of p21CIP1.

Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells involve β-catenin and cyclin D1 down-regulation

Exisulind and its analogues are inhibitors of cyclic GMP phosphodiesterases (PDEs) that have been shown to activate and induce protein kinase G, resulting in the induction of apoptosis in colon cancer cells. These drugs also reduce beta-catenin protein levels and decrease cyclin D1 mRNA levels in SW480 cells. Herein we report on studies pertaining to exisulind regulation of beta-catenin levels and activity in colon tumor cells. Exisulind and its higher-affinity PDE analogues, (Z)-5-fluoro-2-methyl-(4-pyridylidene)-3-(N-benzyl)-indenylacetamide hydrochloride (CP461) and (Z)-1H-indene-3-acetamide, 5-fluoro-2-methyl-N-(phenylmethyl)-1-[(3,4,5-trimethoxyphenyl)methylene] (CP248), reduced beta-catenin, including the nuclear beta-catenin in SW480 cells (EC(50) approximately 200 microM, 1 microM, and <1 microM, respectively). The 50% reduction of beta-catenin was seen in 8-14 hr. There was no change in beta-catenin mRNA. Exisulind-induced beta-catenin reduction was blocked by the proteasomal inhibitor MG132 (Z-leu-Leu-Leu-CHO), indicating that the effect of exisulind involved ubiquitin-proteasomal degradation. A consequence of reduced beta-catenin in SW480 cells was that exisulind, CP461, and CP248 caused a concentration- and time-dependent decrease in cyclin D1 levels (EC(50) approximately 300 microM, 1 microM, and <1 microM, respectively) in 4 hr. The effect was via decreased cyclin D1 mRNA levels. Exisulind-induced degradation of beta-catenin was not blocked by the inhibition of caspase-3 activity and/or apoptosis, and some SW480 cells showed a reduction in beta-catenin levels before the appearance of early apoptosis indicators. Expression of the N-terminal 170 amino acid fragment of beta-catenin reduced the effects of beta-catenin degradation, cyclin D1 reduction, and the apoptosis response to exisulind. These results indicate that exisulind-induced beta-catenin degradation precedes the induction of apoptosis and that the down-regulation of inappropriate beta-catenin-activated genes accounts in part for the pro-apoptotic effects of exisulind and CP461 in colon tumor cells.

Catalytic and kinetic properties of purified high-affinity cyclic AMP phosphodiesterase from dog kidney☆

Abstract High-affinity cyclic AMP phosphodiesterase purified to homogeneity from dog kidney was studied with respect to its stability, its catalytic and kinetic properties, and its sensitivity to pharmacological agents. The enzyme was shown to rapidly lose activity upon dilution to low protein concentrations in aqueous media, but this activity loss was largely prevented by the presence of bovine serum albumin or ethylene glycol. Similarly, maximum activity required bovine serum albumin to be present during incubation for activity analysis. Enzyme activity required a divalent cation; Mg 2+ , Mn 2+ , and Co 2+ each supported activity, but highest activity was obtained with Mg 2 . The temperature optimum ranged from 30 to 45 °C and depended on substrate concentration; the E a = 10,600 cal/mol. The pH optimum of the enzyme was broad, with a maximum from pH 8.0 to 9.5. The enzyme exhibits linear Michaelis-Menton kinetics for hydrolysis of cyclic AMP at all substrate concentrations tested and for hydrolysis of cyclic GMP at > 20 μ m . The K m for cyclic AMP hydrolysis was 2 μ m , and that for cyclic GMP hydrolysis was 312 μ m . The K i values for the competitive inhibition of hydrolysis of each substrate by the other were similar to their K m values suggesting a single active site. Cyclic AMP hydrolysis was weakly inhibited by cyclic GMP, cyclic IMP, adenine, and adenosine, but was not inhibited by the mono-, di, or trinucleotides of adenosine, guanosine, or inosine. Activity was competitively inhibited with K i values in the micromolar range by drugs representative of methylxanthines, isoquinolines, pyrazolopyridines, imidazolidinones, triazolopyrimidines, pyridylethylenediamines, phenothiazines, and calcium antagonists. The results are discussed with reference to the similarities and differences between high- and low-affinity phosphodiesterase forms.

Isolated rat gastric parietal cells: Cholinergic response and pharmacology

Abstract Isolated, partially purified or enriched rat gastric muscosal parietal cells were shown to respond to carbamycholine (EC 50 = 2 μ M) and other muscarinic cholinergic agonists as measured by an increased accumulation of 14 C-aminopyrine, an indirect measure of acid secretion. The secretory response to carbamylcholine was shown to be inhibited stereoselectively and reversibly by nanomolar concentrations of muscarinic cholinergic antagonists. Non-muscarinic antagonists, including cimetidine, were either ineffective or very weak inhibitors. The affinity constants calculated for cholinergic antagonist inhibition of 14 C-aminopyrine accumulation induced by carbamylcholine were similar to those previously calculated from direct binding studies on purified parietal cell particulate fractions using 3 H-QNB (1). These studies support the existence of specific parietal cell muscarinic cholinergic receptors with which the natural secretagogue acetylcholine interacts to regulate gastric acid secretion.

Characterization of particulate cyclic nucleotide phosphodiesterases of rat kidney.

Abstract Particulate cyclic nucleotide phosphodiesterases of rat kidney display some distinct kinetic and regulatory properties. Only a small portion (5–10%) of the total homogenate low K m cyclic AMP phosphodiesterase activity (measured with concentrations of cyclic AMP less than l μ m ) is tightly associated with kidney membranes. Cyclic GMP phosphodiesterase activity (measured with 0.25–200 μ m cyclic GMP) is readily detectable in these fractionated and washed membranes. Low concentrations of cyclic GMP stimulated the hydrolysis of cyclic AMP ( K a ∼- 0.5 μM), an effect not noted in most other membrane systems. High concentrations of cyclic GMP ( K i ∼- 450 μM) and cyclic AMP ( K i ∼- 150 μM ) inhibited the hydrolysis of each other noncompetitively. Solubilization of membrane bound activities by sonication or Sarkosyl L markedly alters enzyme kinetic properties and the responses to cyclic nucleotides and sulfhydryl reagents. Incubation of membrane fractions with dithiothreitol (5 m m ) or storage of the membranes at 4 °C results in a change in extrapolated kinetic constants for cyclic AMP hydrolysis and an increase in the rate of denaturation at 45 °C. Our findings raise the possibility that regulation of membrane-bound cyclic nucleotide phosphodiesterase activity involves interactions with cyclic nucleotides themselves, as well as oxidation and reduction of disulfide bonds and membrane-enzyme interactions.

Hormonal Regulation of Cyclic Nucleotide Phosphodiesterases

Publisher Summary This chapter explains the hormonal regulation of cyclic nucleotide phosphodiesterases. Cyclic nucleotide phosphodiesterase of mammalian tissues portray a complex picture of multiple enzyme forms, substrate specificities, and irregular kinetic behavior. Numerous types of regulation of enzyme forms and activities have been proposed, but perhaps, to the biomedical scientist, the most essential of these is that hormones have been established as chief regulators of phosphodiesterase activity in mammalian tissues. In most mammalian tissues, the phosphodiesterase activity consists of multiple enzyme forms, each having different substrate specificities, sizes, and charges; some with complex kinetic behavior, and all indicating a great deal of resistance to refinement. These studies support the hypothesis that hormones and mitogenic agents act together with membrane surfaces, receptors, or acceptors in such a manner to release or support the action of factor(s) that can kindle high-affinity cyclic AMP phosphodiesterases.

Comparison of the effects of secretory stimulants and inhibitors on gastric mucosal adenylyl cyclases of various species.

Summary Adenylyl cyclase activities were measured in mucosal 600g paniculate preparations from the fundic stomach of rats, dogs, rabbits and guinea pigs. In vitro incubation with prostaglandin E1 and NaF activated the enzyme in all species. Penta-gastrin and carbachol were ineffective activators in any of the species tested. Hista-mine activated mucosal adenylyl cyclase activity in the guinea pig but not in any of the other species tested. Histamine antagonists did not conclusively demonstrate the specificity of histamine activated adenylyl cyclase. Our results do not support the universal hypothesis that activation of adenylyl cyclase is the first step in the mechanisms of action of gastric acid secretagogues. Our findings do indicate that species variability and mucosal tissue heterogeneity constitute difficult problems that must be solved prior to the elucidation of the mechanisms of regulation of acid secretion. The authors are indebted to Mrs. Lily Chang for expert technical assistance. This research was supported by USPHS Grant Nos. HL16552 and AM 15997 and by a gift from The Upjohn Company.

Analysis of cyclic nucleotide phosphodiesterase(s) by radioimmunoassay

Abstract A high-affinity form of cyclic AMP phosphodiesterase, purified to apparent homogeneity from dog kidney, was labeled with 125 I using a solid-state lactoperoxidaseglucose oxidase system and its purity confirmed by acrylamide gel electrophoresis and isoelectric focusing. Sheep anti-cyclic AMP phosphodiesterase immunoglobulin fraction was analyzed for 125 I-enzyme binding and covalently bound to agarose A 1.5m for isotopically labeled antigen displacement. Anti-phosphodiesterase antiserum was purified by Sepharose 4B-cAPDE affinity chromatography and used for a radioimmunoassay employing second-antibody precipitation. The specificity of the anti-cyclic AMP phosphodiesterase antibody was established by its use as a covalently bound affinity ligand for cyclic AMP phosphodiesterase purification and analysis of sodium dodecyl sulfate-gel extracts of partially purified and purified dog kidney supernatants. Radioimmunoassay using a monospecific antibody preparation demonstrated the similarity of high-affinity cyclic AMP phosphodiesterase forms of different tissues and species that had been separated by DEAE-cellulose chromatography. Various purified preparations of calmodulin, as well as brain calcineurin, did not cross-react in the high-affinity cyclic AMP phosphodiesterase radioimmunoassay. However, higher molecular weight cyclic GMP/lower affinity cyclic AMP phosphodiesterase enzyme forms, partially purified by anion-exchange chromatography, gel filtration, and Cibacron blue adsorption, were shown to cross-react in the high-affinity cAMP phosphodiesterase radioimmunoassay. These studies suggest immunological similarities between the major forms of this enzyme system and the possibility of higher molecular weight complexes containing both cyclic GMP and cyclic AMP hydrolytic sites.

Select Cyclic Nucleotide Phosphodiesterase Inhibitors in Colon Tumor Chemoprevention and Chemotherapy

Exisulind, and later generation drugs including CP461, are members of a group of new anticancer compounds known as Selective Apoptotic Antineoplastic Drugs (SAANDs). These drugs act by selectively inducing apoptosis in precancerous and cancerous tissues. Exisulind is a metabolite of sulindac, a nonsteroidal antiinflammatory drug (NSAID). Although initially developed to prevent colon cancer, exisulind and CP461 are currently being tested in clinical trials as chemotherapeutics against many types of cancer, including breast, colon, lung, prostate, chronic lymphocytic leukemia, and renal cell carcinoma. SAANDs were screened as cyclic 5′ guanosine monophosphate (cGMP) phosphodiesterase (PDE) inhibitors with a preference for the PDE5 gene family. Published data have shown that in a colon cancer model, these drugs increase cGMP levels, activate cGMP-dependent protein kinase G (PKG) to stimulate the Jun kinase (JNK) regulatory cascade, and cause a decrease in accumulated nuclear and cytosolic β-catenin followed by reduced cyclin D1 transcription.

Regulation of gastric acid secretion

Despite the fact that increased gastric acid secretion is not diagnostic for peptic ulcer disease, it is generally believed that where there is no acid there is no ulcer. Thus, there remains a strong medical interest in the understanding of the cellular regulation of gastric acid secretion by the major secretagogues, histamine, gastrin and acetylcholine and therapy designed to interfere with this regulation. Released as a result of various physiological stimuli, including food, these paracrine, endocrine, and neurohumoral agents act by both direct and indirect pathways to effect the production and secretion of hydrogen ions from parietal cells located in the lower portion of the oxyntic glands in the fundic stomach. Recently, the study of gastric acid secretion has been advanced by the advent of methods to isolate gastric oxyntic glands and gastric parietal cells. These preparations have allowed the formulation of new concepts to explain in vivo and in vitro physiological and pharmacological data derived from studies on the interactions of hormones that regulate the acid secretory process14,17,20.