Direct activation of PDE5 by cGMP: long-term effects within NO/cGMP signaling.

In platelets, the nitric oxide (NO)-induced cGMP response is indicative of a highly regulated interplay of cGMP formation and cGMP degradation. Recently, we showed that within the NO-induced cGMP response in human platelets, activation and phosphorylation of phosphodiesterase type 5 (PDE5) occurred. Here, we identify cyclic GMP-dependent protein kinase I as the kinase responsible for the NO-induced PDE5 phosphorylation. However, we demonstrate that cGMP can directly activate PDE5 without phosphorylation in platelet cytosol, most likely via binding to the regulatory GAF domains. The reversal of activation was slow, and was not completed after 60 min. Phosphorylation enhanced the cGMP-induced activation, allowing it to occur at lower cGMP concentrations. Also, in intact platelets, a sustained NO-induced activation of PDE5 for as long as 60 min was detected. Finally, the long-term desensitization of the cGMP response induced by a low NO concentration reveals the physiological relevance of the PDE5 activation within NO/cGMP signaling. In sum, we suggest NO-induced activation and phosphorylation of PDE5 as the mechanism for a long-lasting negative feedback loop shaping the cGMP response in human platelets in order to adapt to the amount of NO available.

Celecoxib-induced growth inhibition in SW480 colon cancer cells is associated with activation of protein kinase G.

Although it is often assumed that the antitumor effects of nonsteroidal anti-inflammatory drugs (NSAIDs) are due to inhibition of cyclooxgenase (COX) activity, specifically COX-2, there is accumulating evidence that COX-2 independent mechanisms can also play an important role. Studies with sulindac sulfone (Aptosyn) and related derivatives have revealed a novel pathway of tumor growth inhibition and apoptosis mediated by activation of the guanosine 3',5' monophosphate (cGMP)-dependent enzyme protein kinase G (PKG). The present study indicates that concentrations of the NSAIDs celecoxib, indomethacin, and meclofenamic acid that inhibit growth of SW480 human colon cancer cells inhibit subcellular cGMP-phosphodiesterase (PDE) enzymatic activity and in intact cells induce a two- to threefold increase in intracellular levels of cGMP. This is associated with phosphorylation of the protein VASP, a marker of PKG activation, activation of JNK1 and a decrease in cellular levels of cyclin D1; effects seen with other agents that cause activation of PKG in these cells. On the other hand even a high concentration of the COX-2 specific inhibitor rofecoxib (500 microM) did not inhibit growth of SW480 cells. Nor did rofecoxib inhibit cGMP-PDE activity or cause other changes related to PKG activation in these cells. Since activation of the PKG pathways by celecoxib, indomethacin, and meclofenamic acid in this cell culture system required high concentrations of these compounds, it remains to be determined whether activation of this pathway contributes to the in vivo antitumor effects of specific NSAIDs.

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 Ialpha: PKG IalphaS65D, a constitutively activated point mutant; PKG IalphaDelta, a constitutively activated N-terminal truncated mutant; and PKG IalphaK390R, a dominant-negative point mutant. A similar series of mutants of PKG Ibeta 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 Ibeta, and to a lesser extent PKG Ialpha, 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 Ibeta displayed growth inhibition, whereas derivatives that stably expressed the dominant-negative mutant (KR) of PKG Ibeta grew more rapidly and were more resistant to Aptosyn-induced growth inhibition than vector control cells. Stable overexpression of PKG Ibeta was associated with decreased cellular levels of beta-catenin and cyclin D1 and increased levels of p21(CIP1). Reporter assays indicated that activation of PKG Ibeta inhibits the transcriptional activity of the cyclin D1 promoter. We also found that transient expression of the constitutively activated mutants of PKG Ibeta inhibited cell migration. Taken together, these results indicate that activation of PKG Ibeta 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 p21(CIP1).

Phase I and II study of exisulind in combination with capecitabine in patients with metastatic breast cancer.

PURPOSE: We studied the safety and clinical activity of exisulind in combination with capecitabine in 35 patients with metastatic breast cancer (MBC). PATIENTS AND METHODS: All patients had received previous anthracycline and taxane chemotherapies. Two dose levels of exisulind were explored, 125 and 250 mg orally bid as continuous daily therapy, concomitant with capecitabine 2,000 mg/m2 for 14 days in 21-day cycles. In the phase I study, the dose-limiting toxicities were hand-foot syndrome and diarrhea. The 125-mg bid dose was selected for phase II testing. RESULTS: The most common nonhematologic grade 2 to 3 adverse events were hand-foot syndrome (57%) and fatigue (48%). The most frequent grade 2 to 3 laboratory abnormality was granulocytopenia. No death, unexpected adverse events, or cumulative toxicity were encountered. One complete and four partial responses were achieved (objective response rate, 16%) in the 31 patients assessable for response. The median duration of response was 31 weeks; three patients experienced stable disease longer than 26 weeks. Overall clinical benefit (complete response, partial response, or stable disease > 26 weeks) was 23%. Fourteen specimens were available for immunohistochemical assessment of phosphodiesterase-5 isoenzyme (PDE-5) and PDE-2 expression, which are the targets of exisulind. Eighty percent of tumors showed some expression of PDE-5 in the invasive cancer cells including 35% that showed moderate or strong staining. PDE-2 showed moderate or strong staining in 78% of tumors. There was no apparent association between tumor response and staining intensity. CONCLUSION: Exisulind (125 mg orally bid) in combination with capecitabine is well tolerated and the combination has anticancer activity similar to that of capecitabine alone in heavily pretreated patients with MBC.

Specific cGMP binding by the cGMP binding domains of cGMP-binding cGMP specific phosphodiesterase.

The structure of cyclic GMP (cGMP)-binding (cGB), cGMP specific phosphodiesterase (PDE5) comprises several domains. We have used RT-PCR methods to clone the noncatalytic cGB domains of PDE5 from human colon cancer cell RNA and constructed glutathione-S-transferase (GST) fusion proteins to express and study the domains. One fragment showed 94% identity to bovine PDE5 and coded for the high affinity cGB domain of PDE5 (Val(156)-Asp(394), cGB-I). Another cloned fragment showed 92% identity to bovine PDE5 and coded for the phosphorylation site plus both high and low affinity cGB domains of PDE5 (Val(36)-Glu(529), cGB-II). Both fragments expressed as GST-cGB fusion proteins bound cGMP specifically, as determined by competitive [3H]-cGMP ligand binding. We found that cGB-I showed high affinity cGMP binding with K(d)=0.33 microM. cGB-II showed two cGMP binding sites with similar affinities and specificity to the native enzyme. cGB-II was phosphorylated by cGMP-dependent protein kinase (PKG) as reported for bovine PDE5. These data show that recombinant regulatory regions of PDE5 form cGB sites similar to native enzyme sites and confirm proposed domain functions. These results establish that recombinant fusion proteins of PDE5 domains may be used to further characterize the structure of PDE5.

Exisulind and related compounds inhibit expression and function of the androgen receptor in human prostate cancer cells.

In recent studies, we found that sulindac sulfide (SS), exisulind, CP248, and CP461 induce growth inhibition and apoptosis in a series of human prostate cancer cell lines, irrespective of cyclooxygenase expression, p53 mutations, or bcl-2 overexpression. Exisulind also inhibited the growth of the androgen-dependent LNCaP human prostate cancer cell line when grown as a xenograft in nude mice. This study demonstrates that doses of these compounds that induce growth inhibition and apoptosis in LNCaP cells also cause decreased prostate-specific antigen (PSA) secretion and decreased cellular levels of PSA. These effects appear to be a result, at least in part, of inhibition of the androgen receptor (AR) signaling pathway because the treated cells also display decreases in the level of the AR protein and mRNA and inhibition of transcription of an AR promoter luciferase reporter in transient transfection assays. SS and exisulind were more effective in inhibiting the expression of PSA and the AR than CP248 or CP461, apparently because of differential effects of these compounds on specific transcription factors. These findings suggest that the growth inhibition by these compounds in human prostate cancer cells may be mediated, in part, by inhibition of AR signaling. Thus, these compounds may provide a novel approach to the prevention and treatment of human prostate cancer.

Exisulind in combination with docetaxel inhibits growth and metastasis of human lung cancer and prolongs survival in athymic nude rats with orthotopic lung tumors.

Docetaxel, a semisynthetic taxane, improves the survival of stage IIIB and IV non-small cell lung cancer patients. However, the 5-year survival remains poor, and few patients experience a complete remission. In this report, we evaluated the effects of exisulind, a novel proapoptotic agent that is a sulfone metabolite of sulindac, in combination with docetaxel on the growth of the human non-small cell lung cancer cell line A549 in vitro and in vivo. Exisulind is a novel sulindac metabolite in that it does not inhibit cyclooxygenase enzymes and has been shown to induce apoptosis in a variety of human cancers by inhibiting cyclic GMP-dependent phosphodiesterase. Exisulind alone increased the fraction of cells in the G(1) phase of the cell cycle from 46% to 65%, whereas it decreased the fraction of cells in the S phase from 38% to 14%. Docetaxel increased the fraction of cells in the S phase from 17% to 19%, and 10 nM docetaxel increased the G2-M phase by 23%. Docetaxel alone induced apoptosis from 11% to 64% at 12-24 h after incubation. The combination of exisulind with concentrations of docetaxel (in concentrations that alone did not alter cell cycle distribution) reduced the G(1) accumulation induced by exisulind, increased the fraction of cells in G(2)-M (9-17%), and increased apoptosis (5-62%). The IC(50) for in vitro growth inhibition by exisulind alone was approximately 200 microM and 2.5 nM for docetaxel. The in vitro combination of exisulind and docetaxel produced an additive to synergistic growth inhibition. In athymic nude rats with A549 orthotopic lung cancers, both exisulind and docetaxel alone moderately prolonged survival, inhibited tumor growth and metastases, and increased apoptosis compared with control animals treated with a carrier. However, the combination of exisulind with docetaxel significantly prolonged survival (P = < 0.0004), inhibited tumor growth and metastases (P = < 0.0001), and increased apoptosis (P = < 0.001) when compared with control animals. These results provide rationale for conducting clinical trials using the combination of exisulind and docetaxel in patients with advanced lung cancer.

Vasodilator-stimulated phosphoprotein (VASP) phosphorylation provides a biomarker for the action of exisulind and related agents that activate protein kinase G.

Recent studies provide evidence that exisulind and two potent derivatives, CP461 and CP248, induce apoptosis in colon cancer cells by inhibiting cyclic GMP (cGMP)-specific phosphodiesterases (phosphodiesterases 2 and 5). This causes an increase in intracellular levels of cGMP, thus activating the cGMP-dependent protein kinase G (PKG), which then activates pathways that lead to apoptosis. To further examine this mechanism and to provide a potential in vivo biomarker for activation of this pathway, we examined phosphorylation of the vasodilator-stimulated phosphoprotein (VASP), a ubiquitously expressed endogenous substrate for PKG. We found that VASP was phosphorylated after treating SW480 colon cancer cells with exisulind, CP461, or CP248. CP248-induced VASP phosphorylation was inhibited by a specific PKG inhibitor but not by a protein kinase A inhibitor. The drug 3-(5'-hydroxymethyl-2'-furyl)-benzylindazole and nitric oxide donors that activate cellular guanylyl cyclase and thus increase cellular levels of cGMP also caused VASP phosphorylation. With all of these agents, the phosphorylation of VASP was associated with increased intracellular levels of cGMP and the induction of apoptosis. We also demonstrated direct in vivo phosphorylation of VASP with constitutively activated mutants of PKG. These results suggest that VASP phosphorylation can provide a useful endogenous cellular biomarker for anticancer agents that cause cGMP-mediated apoptosis.

CP248, a derivative of exisulind, causes growth inhibition, mitotic arrest, and abnormalities in microtubule polymerization in glioma cells.

Exisulind (sulindac sulfone) and two potent derivatives, CP248 and CP461, have been shown previously to cause growth inhibition and apoptosis in several types of human carcinoma cell lines. These and related compounds have not been previously studied with respect to glioma cell lines. In the present study, we found that these three compounds caused marked growth inhibition in four rat glioma and eight human glioma cell lines, with IC50 values of 150, 1, and 0.075 microm, respectively. When studied at these concentrations exisulind and CP461 had no significant effect on the cell cycle profile of glioma cells, but CP248 caused marked arrest in mitosis. Detailed studies of CP248 in the 9L rat gliosarcoma cell line indicated that treatment with 0.075 microM CP248 caused abnormalities in the spindle apparatus and activation of the spindle assembly check point. In interphase glioma cells, CP248 stabilized microtubules (MTs) at low concentrations (0.075 microM) and depolymerized MTs at higher concentrations (0.2-0.4 microM). In NIH 3T3 fibroblasts, 0.1 microM CP248 caused extensive MT depolymerization. CP248 also caused MT depolymerization when added to assembled MTs in vitro, which indicated that it can directly affect MTs, perhaps because it shares certain structural similarities with Colcemid. In glioma cells, the effects of CP248 on MTs were independent of the previously reported effects of this compound on activation of protein kinase G. Therefore, CP248 is a novel MT-active agent that may be useful in the treatment of glioblastoma, and possibly other types of cancer, because of its dual effects on protein kinase G and MTs.

Exisulind-induced apoptosis in a non-small cell lung cancer orthotopic lung tumor model augments docetaxel treatment and contributes to increased survival.

We reported previously a significant increase in survival of nude rats harboring orthotopic A549 human non-small cell lung cancer tumors after treatment with a combination of exisulind (Sulindac Sulfone) and docetaxel (D. C. Chan, Clin. Cancer Res., 8: 904-912, 2002). The purpose of the current study was to determine the biochemical mechanisms responsible for the increased survival by an analysis of the effects of both drugs on A549 orthotopic lung tumors and A549 cells in culture. Orthotopic A549 rat lung tissue sections from drug-treated rats and A549 cell culture responses to exisulind and docetaxel were compared using multiple apoptosis and proliferation analyses [i.e., terminal deoxynucleotidyl transferase-mediated nick end labeling, active caspase 3, the caspase cleavage products cytokeratin 18 and p85 poly(ADP-ribose) polymerase, and Ki-67]. Immunohistochemistry was used to determine cyclic GMP (cGMP) phosphodiesterase (PDE) expression in tumors. The cGMP PDE composition of cultured A549 cells was resolved by DEAE-Trisacryl M chromatography and the pharmacological sensitivity to exisulind, and additional known PDE inhibitors were determined by enzyme activity assays. Exisulind inhibited A549 cell cGMP hydrolysis and induced apoptosis of A549 cells grown in culture. PDE5 and 1 cGMP PDE gene family isoforms identified in cultured cells were highly expressed in orthotopic tumors. The in vivo apoptosis rates within the orthotopic tumors increased 7-8-fold in animals treated with the combination of exisulind and docetaxel. Exisulind increased the in vivo apoptosis rates as a single agent. Docetaxel, but not exisulind, decreased proliferative rates within the tumors. The data indicate that exisulind-induced apoptosis contributed significantly to the increased survival in rats treated with exisulind/docetaxel. The mechanism of exisulind-induced apoptosis involves inhibition of cGMP PDEs, and these results are consistent with a cGMP-regulated apoptosis pathway.

beta-Catenin signaling: therapeutic strategies in oncology.

Activated Wnt signaling pathways have been found in various human cancers, including those of the colon, liver, endometrium, ovary, prostate, and stomach. As a result, beta-catenin is accumulated and becomes transcriptionally active for proliferative genes and oncogenes. Wnt pathway mutations result in biochemical mechanisms yielding inefficient phosphorylation of beta-catenin by GSK3beta due to APC, beta-catenin and/or axin mutations. Therefore, the needs and the opportunity to develop new cancer therapies exist through reversing oncogenic APC/beta-catenin/Lef/Tcf signals. Exisulind and analogues are inhibitors of cyclic GMP phosphodiesterases (PDE) that have been shown to activate and induce protein kinase G. The data show PKG regulation of beta-catenin in wnt signaling, accounting, at least in part, for apoptosis induction in treated colon cancer cells carrying either APC or beta-catenin mutations. Exisulind and analogs reduce beta-catenin via a novel, GSK3beta independent processing mechanism. Activated PKG directly phosphorylate beta-catenin at its C-terminal domain and causes proteasome dependent degradation of the protein. Since this pathway is independent of APC and GSK3beta, exisulind and analogs provide a superior approach to circumvent the molecular defects of wnt signaling pathway and to treat cancers with such defects.

Activation and induction of cyclic AMP phosphodiesterase (PDE4) in rat pulmonary microvascular endothelial cells.

Regulation of the rolipram-sensitive cAMP-specific phosphodiesterase 4 (PDE4) gene family was studied in rat pulmonary microvascular endothelial cells (RPMVECs). Total PDE4 hydrolysis was increased within 10 min after addition of forskolin (10 microM), reached a maximum at 20-40 min, and then gradually declined in the cells. A similar activation of PDE4 activity was observed using a protein kinase A (PKA) activator, N(6)-monobutyryl cAMP. Both the forskolin and the N(6)-monobutyryl cAMP activated PDE4 activities were blocked by the PKA-specific inhibitor, H89. This forskolin-stimulated and PKA-mediated short-term activation of PDE4 activity was further confirmed by in vitro phosphorylation of 87kDa PDE4A6 and 83kDa PDE4B3 polypeptides using exogenous PKA Calpha. Increased immunoreactivity of phosphorylated PDE4A6 in situ was detected in Western blots by a PDE4A-phospho antibody specific to the putative PKA phosphorylation sites. Following long-term treatment of RPMVECs with rolipram and forskolin medium (RFM) for more than 60 days, PDE4 activity reached ten-fold higher values than control RPMVECS with twenty-fold increases detected in intracellular cAMP content. The RFM cells showed increased immunoreactivities of the constitutive 4A6 and 4B3 isoforms plus two novel splice variants at 101kDa (4B1) and 71kDa (4B2). Treatment with H89 did not inhibit the PDE4 elevation in RFM cells. In addition to the increased levels of PDE4 in RFM cells, immunofluorescence showed a translocation of PDE4A and 4B to a nuclear region, which was normally not observed in RPMVECs. The PDE4 activity in RFM cells decayed rapidly with an even faster decline of intracellular cAMP content when forskolin/rolipram were removed from the medium. These results suggest that both the activation (short-term) and induction (long-term) of PDE4A/4B isoforms in RPMVECs are closely modulated by the intracellular cAMP content via both post-translational and synthetic mechanisms.

Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4).

We report here studies on the regulation of the metabolism of adenosine 3',5'-monophosphate (cAMP) in established and primary cultures of rat pulmonary microvascular endothelial cells (RPMVEC). Inhibition by rolipram, a selective inhibitor of cAMP phosphodiesterase (PDE) of the PDE4 gene family, was required to achieve maximal cAMP accumulation induced by direct or receptor-mediated adenylate cyclase activation when measured by [3H]-adenine prelabeling. Rolipram increased cAMP accumulation more effectively than did forskolin, isoproterenol, or adenosine derivatives alone, although extensive synergy was seen with combined agents. High-affinity PDE4 inhibitors, but not low-affinity or non-selective inhibitors, were effective inducers of cAMP accumulation in intact cells. The maximum effects (i.e. intrinsic activities) of these agents in the intact cell did not correlate with their in vitro PDE4 inhibitory affinities. RPMVEC were shown to express almost exclusively the PDE4 gene family isoforms A6 and B3. Guanosine 3',5'-monophosphate hydrolysis, observed in other types of endothelial cells was not found in early or late passage RPMVEC. Reverse transcription-polymerase chain reaction identification of mRNAse supported these conclusions with the exception that PDE2 and PDE4D mRNA isoform transcripts were present. These studies also support the conclusion that the mechanism of rolipram reversal of rat lung ischemia-reperfusion-induced permeability involves PDE4 inhibition in the microvascular endothelial cells of the lung.

Pro-apoptotic actions of exisulind and CP461 in SW480 colon tumor cells involve beta-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.

Characterization of 3',5' cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma cells: absence of functional PDE6.

PURPOSE: Previous studies identified rod photoreceptor cyclic GMP phosphodiesterase (PDE6) transcripts in the human Y79 retinoblastoma cell line. To assess the potential to utilize this cell line for structure/function studies of PDE6, we analyzed 3',5' cyclic nucleotide phosphodiesterase activity focusing on expression of PDE6. METHODS: DEAE-chromatography was used to fractionate PDE activity from Y79 cell homogenates. PCR was performed on cDNA generated from Y79 cells and retina with PDE isoform specific primers. Western blots were performed with antibodies to PDE1, PDE4, or rod PDE6. DNA sequencing and protein truncation tests were performed with plasmids containing the entire coding region of Y79 rod PDE6 transcripts. Proteasome mediated degradation of PDE6 subunits was analyzed with a pathway specific inhibitor. Polysome isolation was performed by fractionation on sucrose gradients followed by RT-PCR for the PDE6 transcripts. RESULTS: Of three peaks of PDE activity, peaks 1 and 2 were activated by Ca2+/calmodulin, inhibited by dipyridamole and zaprinast, and were reactive with a PDE1 antibody. Peak 3 hydrolyzed only cAMP and was rolipram sensitive, indicative of PDE4. Transcripts for rod and cone PDE6 isoforms were detected in Y79 total RNA, however PDE6 antibodies recognized only a single 99 kDa polypeptide from immunoprecipitated 35S labeled Y79 extracts. DNA sequencing of PDE6 alpha, beta, gamma, and PDE6 associated delta-subunit cDNA revealed some polymorphism, but no apparent mutations. Each of the PDE6 transcripts could be translated into protein of the correct length. The concentration of cGMP in the cells was greatly reduced in comparison to that reported in the photoreceptor cell. Addition of cyclic nucleotide analogues, zinc, or butyrate did not enhance the expression of PDE6. Transduction into Y79 cells of adenovirus expressing PDE6 subunits failed to produce functional enzyme CONCLUSIONS: PDE1 and PDE4 enzyme activities predominate in Y79 cells. Despite the presence of PDE6 transcripts and the ability to translate each into protein in vitro, a functional PDE6 enzyme could not be detected. Attempts to enhance expression with cell culture or with introduction of virus expressing PDE6 were not successful. The results indicate that expression of a fully active stable PDE6 enzyme requires other post-transcriptional events that do not occur or are inhibited in Y79 cells.

Exisulind and CP248 induce growth inhibition and apoptosis in human esophageal adenocarcinoma and squamous carcinoma cells.

We examined the effects of exisulind (sulindac sulfone) and a potent derivative CP248 on the Barrett's esophagus (BE)-related adenocarcinoma cell lines Seg-1 and Bic-1, and on HCE7 esophageal squamous carcinoma cells. Marked growth inhibition and apoptosis occurred in all cell lines with IC50 values of 100-300 microM for exisulind and 100 nM for CP248. Bic-1 and HCE7 cells were more sensitive to the growth inhibitory properties of exisulind. Treatment of all cell lines with CP248 for 24 h increased the proportion of cells in mitosis. Exisulind had no effect on cell-cycle progression. Treatment with either compound induced rapid activation of the c-Jun NH2-terminal kinase 1 (JNK1), suggesting that JNK1 activation plays a role in the induction of apoptosis by these compounds. Only Seg-1 cells expressed a detectable basal level of cyclooxygenase-2 (cox-2), providing further evidence that cox-2 is not the critical target for the growth inhibitory and apoptotic effects of these compounds. Cellular levels of reduced glutathione (GSH) increased approximately five-fold in all cell lines after 24 h of treatment with either compound. These studies provide support for the use of exisulind in BE chemoprevention trials, and of exisulind or CP248 in the therapy of patients with esophageal carcinoma.

Adenylyl and guanylyl cyclase assays.

This unit presents two basic protocols to determine adenylyl cyclase and guanylyl cyclase activity in tissue and cell homogenates, permeabilized cells, or subcellular fractions. Each method is divided into two parts: the enzyme reaction that causes the formation of the labeled cyclic nucleotide, and the separation of cyclic nucleotide products from unreacted nucleotide triphosphates and metabolites using Dowex 50 resin and aluminum oxide chromatographies. In the case of guanylyl cyclase, alternative separation protocols are also provided. Additionally, protocols are provided that describe preparation of both the columns used in the assays and the tissue or cells to be assayed.

Suppression of cyclic GMP-specific phosphodiesterase 5 promotes apoptosis and inhibits growth in HT29 cells.

Phosphodiesterase 5 (PDE5) is a major isoform of cGMP phosphodiesterase in a variety of human tumor cell lines and plays a key role in regulating intracellular cGMP concentrations ([cGMP]i). Here, we demonstrate that suppression of PDE5 gene expression by antisense pZeoSV2/ASP5 plasmid transfection results in a sustained increase in [cGMP]i, growth inhibition, and apoptosis in human colon tumor HT29 cells. With stable transfection, antisense transcripts exhibited a specific suppression in PDE5 activity, mRNA levels, and a 93 kDa hPDE5A1 protein. In cloned antisense cells, prolongation of the cell growth doubling times correlate positively with suppressed PDE5 activity and increased [cGMP]i. The growth inhibition in PDE5 antisense clones is due to an increased apoptotic rate and delayed cell-cycle progression. These results corroborate previous findings with the PDE5 inhibitor exisulind and its derivatives showing that sustained [cGMP]i induces apoptosis and growth inhibition in tumor cells. Furthermore, an inducible mitotic inhibitor p21WAF1/CIP1 has been found to account for the delay of cell-cycle progression in PDE5 antisense clones at G2/M phase. A proteolytic cleavage of p21WAF1/CIP1 in the antisense clones is also increased at the later stage of serum stimulation. The protein kinase G (PKG) inhibitor, KT5823, can prevent the cleavage of p21(WAF1/CIP). These data substantiate a pivotal role for PDE5 as a modulator of apoptosis and cell-cycle progression for human carcinoma via a mechanism involving the activation of [cGMP]i/PKG signaling pathways.

Phosphodiesterase 3 activity is reduced in dog lung following pacing-induced heart failure.

We hypothesized that decreases in expression and/or activity of cAMP-specific phosphodiesterases (PDE) contribute to protective adaptations observed in lung after heart failure. In this study, we compared PDE activity in lung parenchyma isolated from control dogs and those paced to heart failure by assaying cyclic nucleotide hydrolysis in fractions of homogenate supernatant eluted from DEAE-Trisacryl columns. Cyclic nucleotide hydrolysis due to PDE3, PDE4, and PDE5 isoforms was predominant in both control and paced groups. The ratio of PDE3 activity to total cAMP PDE activity was decreased in the paced group compared with control (P < 0.05), whereas PDE4 or PDE5 activity ratios were not different between the two groups. With the use of RT-PCR, message expression for PDE3A or PDE3B did not differ between the two groups. Cilostamide, a selective PDE3 inhibitor, and forskolin, a nonspecific agonist for adenylyl cyclase, both inhibited thapsigargin-induced increases in endothelial permeability in control lung. We conclude that PDE3 activity, but not mRNA expression, is reduced in lung from dogs paced to heart failure, a change that could contribute to heart failure-induced attenuation of the lung endothelial permeability response to injury.

Phosphorylation of isolated human phosphodiesterase-5 regulatory domain induces an apparent conformational change and increases cGMP binding affinity.

Substrate binding to the phosphodiesterase-5 (PDE5) catalytic site increases cGMP binding to the regulatory domain (R domain). The latter promotes PDE5 phosphorylation by cyclic nucleotide-dependent protein kinases, which activates catalysis, enhances allosteric cGMP binding, and causes PDE5A1 to apparently elongate. A human PDE5A1 R domain fragment (Val(46)-Glu(539)) containing the phosphorylation site (Ser(102)) and allosteric cGMP-binding sites was studied. The rate, cGMP dependence, and stoichiometry of phosphorylation of the PDE5 R domain by the catalytic subunit of cAMP-dependent protein kinase are comparable with that of the holoenzyme. Migration in native polyacrylamide gels suggests that either cGMP binding or phosphorylation produces distinct conformers of the R domain. Phosphorylation of the R domain increases affinity for cGMP approximately 10-fold (K(D) values 97.8 +/- 17 and 10.0 +/- 0.5 nm for unphospho- and phospho-R domains, respectively). [(3)H]cGMP dissociates from the phospho-R domain with a single rate (t(12) = 339 +/- 30 min) compared with the biphasic pattern of the unphospho-R domain (t(12) = 39.0 +/- 4.8 and 265 +/- 28 min, for the fast and slow components, respectively). Thus, cGMP-directed regulation of PDE5 phosphorylation and the resulting increase in cGMP binding affinity occur largely within the R domain. Conformational change(s) elicited by phosphorylation of the R domain within the PDE5 holoenzyme may also cause or participate in stimulating catalysis.