5′-Iodotubercidin represses insulinoma-associated-1 expression, decreases cAMP levels, and suppresses human neuroblastoma cell growth

Insulinoma-associated-1 (INSM1) is a key protein functioning as a transcriptional repressor in neuroendocrine differentiation and is activated by N-Myc in human neuroblastoma (NB). INSM1 modulates the phosphoinositide 3-kinase (PI3K)-AKT Ser/Thr kinase (AKT)-glycogen synthase kinase 3β (GSK3β) signaling pathway through a positive-feedback loop, resulting in N-Myc stabilization. Accordingly, INSM1 has emerged as a critical player closely associated with N-Myc in facilitating NB cell growth. Here, an INSM1 promoter-driven luciferase-based screen revealed that the compound 5′-iodotubercidin suppresses adenosine kinase (ADK), an energy pathway enzyme, and also INSM1 expression and NB tumor growth. Next, we sought to dissect how the ADK pathway contributes to NB tumor cell growth in the context of INSM1 expression. We also found that 5′-iodotubercidin inhibits INSM1 expression and induces an intra- and extracellular adenosine imbalance. The adenosine imbalance, which triggers adenosine receptor-3 signaling that decreases cAMP levels and AKT phosphorylation and enhances GSK3β activity. We further observed that GSK3β then phosphorylates β-catenin and promotes the cytoplasmic proteasomal degradation pathway. 5′-Iodotubercidin treatment and INSM1 inhibition suppressed extracellular signal-regulated kinase 1/2 (ERK1/2) activity and the AKT signaling pathways required for NB cell proliferation. The 5′-iodotubercidin treatment also suppressed β-catenin, lymphoid enhancer–binding factor 1 (LEF-1), cyclin D1, N-Myc, and INSM1 levels, ultimately leading to apoptosis via caspase-3 and p53 activation. The identification of the signaling pathways that control the proliferation of aggressive NB reported here suggests new options for combination treatments of NB patients.

cro ARTICLE an inhibitor (5Ј-iodotubercidin, 5Ј-IT) of adenosine kinase (ADKi). Treatment of NB with 5Ј-IT down-regulates INSM1 and inhibits NB cell growth. ADKi inhibits adenosine kinase (ADK) signaling and modulates intra-and extracellular adenosine metabolism, which is critical for NB tumor cell growth.
The primary function of ADK is to regulate intracellular/ extracellular adenine nucleotide pools mediated through the adenosine receptor (AR), inosine formation, and/or energysensing AMP-activated protein kinase (AMPK) signaling pathways. The mechanistic connection between ADKi and neuroblastoma (NB) tumor cell growth remains unclear. In this study, for the first time we demonstrate that 5Ј-iodotubercidin (5Ј-IT) blockage of the adenosine kinase (ADK) pathway inhibits INSM1 expression and promotes the AR signaling pathway contributing to the suppression of NB tumor cell viability. 5Ј-IT increases the intra-and extracellular adenosine levels that triggers the ARA 3 signaling pathway resulting in the inhibition of intracellular cAMP affecting NB tumor cell growth. 5Ј-IT modulates cAMP via ARA 3 leading to the activation of GSK3␤ and ␤-catenin phosphorylation. Additionally, 5Ј-IT and INSM1 suppression inhibit ERK1/2 phosphorylation. Inhibition of these two signaling pathways resulted in LEF-1, INSM1, N-Myc, and cyclin D1 down-regulation while activating caspase-3 and p53 to promote apoptosis.

Drug screen of NB using INSM1 promoter-driven luciferase assay
In a previous study, multiple signaling pathways were identified that regulate NB tumor cell growth in the context of two oncogenic proteins, N-Myc and INSM1 (12). Thus, stable cell lines in BE2-M17 or IMR-32 containing an INSM1 promoterdriven luciferase reporter gene was developed. Using this luciferase-based screening platform, we screened nine compounds previously selected for INSM1 functional study in our laboratory with a medium range of concentrations versus DMSO control (Fig. 1A). LY294002 is a strong nonselective inhibitor of phosphoinositide 3-kinase (PI3K) (13). TSA selectively inhibits the class I and II mammalian histone deacetylase (HDAC) family of enzymes and cell cycle progression (14). 5Ј-IT is an adenosine kinase (ADK) inhibitor that displays potent suppressive effects on INSM1 promoter activity. A 5Ј-IT dose-response study on  (Fig. 1E). This result strongly suggests that INSM1 and/or N-Myc expression is critically important for NB cell survival, which responds to 5Ј-IT treatment. Both IMR-32 and BE2-M17 showed 50% cell viability at 0.5 M, but little or no effect was observed in glioblastoma (U87MG), or myelogenous leukemia (K562) cells ( Fig. 2A). However, 5Ј-IT inhibits ADK and perturbs adenosine balance in cell metabolism that poses varying degrees of tumor cell cytotoxicity including NB. We performed the reporter assay in the presence or absence of cycloheximide in a shorter time point (3 or 6 h) with 5Ј-IT (1 M) treatment (Fig. 2B). Cycloheximide (10 g/ml) alone suppresses INSM1 promoter activity greatly, whereas 5Ј-IT suppresses INSM1 promoter activity significantly in 3 or 6 h. Suppression of INSM1 promoter activity by cycloheximide suggests that INSM1 promoter activity is depended upon de novo protein synthesis. Comparison of the cycloheximide alone and cycloheximide ϩ 5Ј-IT showed significant enhanced inhibition in BE2-M17 but not IMR-32 cells in 3 h. However, after 6 h treatment, both cell lines showed significant inhibitory enhancement suggesting that 5Ј-IT acts on INSM1 promoter activity more than de novo protein synthesis. The effect of 5Ј-IT on the INSM1 expression (3 h) precedes the growth inhibition (Ͼ24 h).

5-IT blockage of the ADK pathway increases intracellular adenosine and the consumption of extracellular adenosine through adenosine receptor-3 signaling pathway
How the ADK inhibitor (ADKi, 5Ј-IT) affects NB tumor cell growth was further investigated. The first step of this study is to dissect the functional effects of adenosine imbalance using specific inhibitors to block alternative signaling pathways controlling intra-and extracellular adenosine levels to establish how they contribute to the down-regulation of INSM1 leading to reduced NB tumor cell growth. Other studies have revealed that activation of the AMPK inhibits the proliferation of human endothelial cells (15). In the NB system, 5Ј-IT exhibits an inhibitory effect in contrast to facilitating NB tumor cell growth. Therefore, the 5Ј-IT blockage of the ADK pathway (AMPK) does not contribute to the negative effect on NB tumor cell growth. Other mechanisms that control NB cell viability via

ADKi suppresses INSM1 in neuroblastoma
5Ј-IT treatment could include increases of intracellular adenosine levels direct toward the formation of intracellular inosine through adenosine deaminase (ADA) or by directing a re-balance of the intra-and extracellular adenosine levels through the nucleoside transporter (NT). BE2-M17 or IMR-32 cells were treated with the ADA inhibitor (pentostatin) alone (Fig. 3A). ADA inhibitor alone has little cytotoxic effects to the cells. However, a combination of the ADA inhibitor with 5Ј-IT shows an opposite effect on BE2-M17 versus IMR-32 cells. Pentostatin slightly restores BE2-M17 cell viability in contrast to the slight decreases in IMR-32 cell viability suggesting that perturbation of intracellular adenosine levels through the ADA pathway could either reduce or enhance 5Ј-IT cytotoxicity (Fig. 3B). The NT inhibitor (dipyridamole) alone has no significant effects on cell viability. When an NT inhibitor (dipyridamole) and 5Ј-IT were used, the result shows a moderate lessening of 5Ј-IT cytotoxicity and increase in INSM1 promoter activity in both cell lines (Fig. 3, C and D). Addition of NT inhibitor in 5Ј-ITtreated cells also restores the down-regulated INSM1 protein expression partially in BE2-M17 cells (Fig. 3E). These results suggest that blockage of NT could reduce adenosine export and 5Ј-IT cytotoxicity. Using reverse phase HPLC analysis, intracellular adenosine concentration (nmol/mg protein) increased upon 5Ј-IT treatment for 24 h (Fig. 3F). Additionally, 5Ј-IT treatment blocks the extracellular ADK signaling pathway, which facilitates extracellular adenosine interaction with the adenosine receptor-3 (ARA 3 ) pathway. The control medium adenosine concentration at the 5-min time point reached ϳ4.5 M, which is higher than the ARA 3 activated concentration (EC 50 : 0.29 M) (16). Clearly, 5Ј-IT treatment facilitates the usage of extracellular adenosine to activate ARA 3 faster than the control group in a time period between 5 and 30 min (Fig. 3G).

Altered adenosine balance triggers adenosine receptor-3 (ARA 3 ) signaling pathway
An interesting observation reveals that human NB tumor cells (BE2-M17 and IMR-32) do not express ARA 2a , but express a moderate level of ARA 1 and ARA 3 , and high level of ARA 2b adenosine receptors. ARA 1 is coupled to pertussis toxin-sensitive G i proteins that lead to inhibition of adenylyl cyclase activity (17). ARA 2b is known to be a low-affinity AR playing a major role in inflammation (18), whereas ARA 3 is the only adenosine subtype to be overexpressed in inflammatory and cancer cells (19). Because ARA 1 , ARA 3 , or ARA 2b exhibit opposite effects of either inhibiting or activating adenylate cyclase, they are destined to modulate the cAMP signaling pathway. An agonist for ARA 1 (N 6 -cyclopentyladenosine (CPA)), ARA 3 (2-Cl-IB-MECA), or ARA 2b (BAY606583) was tested with or without  (Fig. 4B). In contrast, CPA or BAY606583 lessens 5Ј-IT cytotoxicity (Fig. 4, A and C). This result suggests that 5Ј-IT induces an intra-and extracellular adenosine imbalance that facilitates further interaction with ARA 3 . INSM1 promoter activity is inhibited by 5Ј-IT alone or showed and enhanced inhibitory effect in the presence of ARA 3 agonist (2-Cl-IB-MECA). Alternatively, the ARA 1 agonist (CPA), or ARA 2b agonist (BAY606583) lessens the inhibitory effect with 5Ј-IT on the NB cells suggesting that ARA 3 signaling mediated the post-5Ј-IT effects (Fig. 4D). Similarly, INSM1 protein inhibition is consistent with 5Ј-IT or 5Ј-IT ϩ 2-Cl-IB-MECA treatment (Fig. 4E). In contrast to 5Ј-IT treatment, exogenously added 8-bromo-cAMP does not change INSM1 protein expression (Fig. 4F).

5-IT triggers ARA 3 signaling pathway for the suppression of cAMP
Stimulation of the ARA 3 signaling pathway mediates suppression of adenylyl cyclase and cAMP levels. BE2-M17 or IMR-32 cells were treated with forskolin (as a stimulator of adenylyl cyclase) for 30 min, followed by treatment with 5Ј-IT (1.0 M) in the presence of 100 M Ro-20-1724 (a phosphodiesterase inhibitor) for an additional 30 min. Following treatment, cAMP content of the acid cell extracts was measured using a cAMP ELISA kit normalized with total cellular protein concentration. Because endogenous cAMP before or after 5Ј-IT treatment was too low to be reliably detected, forskolin was used to increase the baseline levels of cAMP in the cells. Therefore, cells were pretreated with forskolin before the addition of 5Ј-IT. Forskolin stimulation of cAMP was completely suppressed by 5Ј-IT treatment (Fig. 4G). 5Ј-IT down-regulates cAMP and INSM1 promoter activity. We measured INSM1 promoter activity using BE2-M17 and IMR-32 cells by adding forskolin, 8-bromo-cAMP (exogenous cAMP), 5Ј-IT, 5Ј-IT ϩ forskolin, or 5Ј-IT ϩ 8-bromo-cAMP. Forskolin or 8-bromo-cAMP slightly increases INSM1 promoter activity, whereas 5Ј-IT alone, 5Ј-IT and forskolin, or 5Ј-IT and 8-bromo-cAMP overrides INSM1 promoter activity. Addition of forskolin or 8-bromo-cAMP does not inhibit the INSM1 expression (Fig. 4, F and H).

ADKi suppresses INSM1 in neuroblastoma 5-IT modulates cAMP via ARA 3 signaling leading to cell growth inhibition
5Ј-IT-treated BE2-M17 cells trigger ARA 3 signaling leading to suppression of cAMP. Suppression of cAMP decreased AKT and GSK3␤ phosphorylation thus resulting in increased GSK3␤ activity triggering ␤-catenin phosphorylation and degradation (Fig. 5, A and C). Inversely, exogenously added forskolin activated adenylyl cyclase that stimulates cAMP, AKT, and GSK3␤ phosphorylation that peaked at 10 min (Fig. 5B). ␤-Catenin phosphorylation and LEF-1 expression were down-regulated upon 5Ј-IT treatment (Fig. 5C). Additionally, 5Ј-IT also downregulated ERK1/2 phosphorylation that is critical for cell proliferation and cross-talk with the AKT pathway (Fig. 5D). Blockage of the ERK1/2 pathway (U0126) inhibits INSM1 protein expression similar to the effect of 5Ј-IT supporting crosstalk between the AKT and ERK1/2 signaling pathways (Fig. 5E). 5Ј-IT induces apoptosis via down-regulation of INSM1 and cyclin D1 in addition to the observed activation of both p53 and caspase-3 (Fig. 5, F and G). These results suggest that the inhibition of ADK and ERK1/2 pathways resulted in INSM1 suppression, which plays a critical role in the inhibition of NB cell proliferation and induction of programmed cell death.

Discussion
Due to the importance of the transcriptional regulator INSM1 on the proliferation of NB cells, an INSM1 promoterdriven luciferase screening platform was used to target associated signaling pathways that lead to the down-regulation of INSM1 expression in NB. A novel ADKi, 5Ј-IT, was identified that exhibited potent inhibition of INSM1 expression and NB tumor cell growth. The molecular mechanisms underlying how 5Ј-IT suppressed the ADK pathway that contributes to the inhibition of NB cell growth was further investigated. Three different pathways associated with adenosine perturbation via ADK inhibition were tested. These pathways include the balance of adenosine and AMPK toward the energy-sensing pathway, increase of intracellular adenosine that triggers inosine formation via ADA, or a re-balance of the intra-and extracellular adenosine levels through the NT (Fig. 6). Adenosine was shown to induce apoptosis in human gastric cancer cells via activation of the AMP-activated protein kinase (20). Similarly, activation of the AMP-activated protein kinase inhibits the proliferation of human endothelial cells (15). In contrast, blockage of the AMP-activated protein kinase pathway by 5Ј-IT promotes beta-cell proliferation and cell growth (21), which is opposite from our observation that 5Ј-IT suppresses NB tumor cell growth. Therefore, it is unlikely that blockage of the AMPK signaling pathway contributes to NB cell death. Using the ADA inhibitor (pentostatin) that targets the inosine synthesis pathway reveals that 5Ј-IT alters adenosine concentration and could only slightly reduce or enhance the 5Ј-IT cytotoxicity in the NB cells. Whereas the NT inhibitor (dipyridamole) blocks adenosine export and consistently lessened the 5Ј-IT cytotoxicity to the NB cells. An interesting observation in our work reveals that 5Ј-IT re-balances intracellular adenosine and directs extracellular adenosine to trigger the ARA 3 signaling pathway. The

ADKi suppresses INSM1 in neuroblastoma
observation was confirmed by HPLC analyses of intra-and extracellular adenosine concentration as well as using 5Ј-IT and ARA 3 agonist for combined treatment of NB cells. Our data suggests that agonist activation of the ARA 3 signaling pathway potentiates the observed ADKi effect and INSM1 inhibition leading to reduced cell viability of NB cells.
Adenosine is considered a major regulator of local tissue function. There are four types of adenosine receptors including ARA 1 , ARA 2a , ARA 2b , and ARA 3 . Our study showed that inhibition of the ADK signaling pathway perturbs the balance of intra-and extracellular adenosine levels via the ARA 3 in NB cells. This hypothesis is further substantiated by use of the ARA 3 agonist (2-Cl-IB-MECA) that causes the down-regulation of cAMP via the ARA 3 signaling pathway. Suppression of cAMP decreases AKT (Ser-473) phosphorylation and activates GSK3␤ activity. GSK3␤ induces ␤-catenin phosphorylation, which thereby targets phosphorylated ␤-catenin for proteasomal degradation and subsequent NB cell apoptosis. Furthermore, we present evidence that 5Ј-IT inhibits the ERK1/2 signaling pathway, which is consistent with ARA 3 activation that inhibits cell proliferation via the PI3K/AKT-dependent inhibition of the ERK1/2 phosphorylation in A375 human melanoma cells (22). In parallel, INSM1 expression is positively correlated with AKT and ERK1/2 phosphorylation (12,23). Both downregulation of AKT and ERK1/2 phosphorylation by 5Ј-IT could be due to the inhibition of INSM1 expression as well as triggering the ARA 3 signaling pathway. Therefore, targeting ADK and the ARA 3 pathway could lead to the inhibition of NB cell growth and the promotion of apoptosis. These findings are significant because they could develop a novel pre-clinical treatment for aggressive forms of human NB (Fig. 6).
The current study established an INSM1 promoter-driven luciferase reporter assay to identify positive hits on regulation of INSM1 promoter activity, which in parallel correlates with NB tumor cell growth. Our most significant hit supports that the ADK signaling pathway inhibitor 5Ј-IT modulates INSM1 and N-Myc expression and targets NB cell growth through intra-and extracellular adenosine re-balance, which triggers the ARA 3 signaling pathway. Specifically, the ARA 3 agonist demonstrates anti-inflammatory and anti-cancer effects via a molecular mechanism that entails modulation of the Wnt and the NF-B signal transduction pathways (24,25). ARA 3 agonists were developed for the treatment of anti-tumor, inflammatory, ophthalmic, and liver diseases and demonstrate excellent safety and efficacy in phase 2 clinical studies (26 -28). The outcome of this study enhances our understanding of the underlying mechanisms that leads to NB tumor cell growth and provides us with new insights for the novel combinational treatment options against this aggressive tumor.
In summary, INSM1 is a specific NE tumor marker critical for aggressive NB tumor cell growth and invasion. Knockdown of INSM1 suppresses NB tumor growth in vivo (12). We used an INSM1 promoter-driven luciferase reporter assay to identify drugs that specifically inhibit INSM1 promoter activity. 5Ј-IT was identified to suppress INSM1 promoter and NB tumor cell growth. However, 5Ј-IT was originally considered as a general kinase inhibitor, especially ADK due to its affinity for the ATPbinding sites of these enzymes and has been shown to affect cell proliferation and survival. 5Ј-IT could cause DNA damage and activate the Atm-p53 pathway as a genotoxic drug with anticolon cancer potential (29). 5Ј-IT suppresses INSM1 promoter activity led us to investigate additional pathways associated

ADKi suppresses INSM1 in neuroblastoma
with INSM1 expression in aggressive NB. The outcomes of this study revealed that 5Ј-IT not only suppresses INSM1 expression, but also triggers ARA 3 signaling and AKT/ERK1/2 phosphorylation pathways, which could contribute to the inhibition of NB cell proliferation and the promotion of apoptosis.

Cell culture and reagents
Human NB cell lines, IMR-32 and BE2-M17, glioblastoma cell line, U87MG, and chronic myelogenous leukemia cell line, K562, were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (Atlanta Biological Inc., Flowery Branch, GA), 1ϫ penicillin/streptomycin in 5% CO 2 incubator at 37°C. All the cell lines were authenticated with Short Tandem  , and cyclin D1 were purchased from Cell Signaling Technology. Antibody to GAPDH was purchased from Life Technologies and actin antibody was obtained from Sigma. Horseradish peroxidase-conjugated secondary antibody was obtained from Bio-Rad Laboratories. ADK siRNA (sc-38902) was purchased from Santa Cruz Biotechnology and transfected with Lipofectamine RNAiMAX transfection protocol (Life Technologies).

Real-time RT-PCR
RNA were extracted with TRIzol reagent and treated with 2 units of DNase I (Promega, Madison, WI) to digest genomic DNA. cDNA synthesis using the High Capacity RNA-to-cDNA TM Kit (Life Tech.) followed the manufacturer's protocol. RNA was reverse-transcribed and analyzed by real-time PCR for the expression of INSM1, N-myc, and GAPDH. The relative RNA concentration of the target gene was normalized with GAPDH. Primers for INSM1: forward, 5Ј-ACGGAATTCTGCCACCTGTGCCC-AGTGTGCGGAGAG-3Ј, reverse, 5Ј-CACCTCGAGCTAGCA-GGCCGGGCGCACGGGCACCTGCAG-3Ј; and primers for N-Myc: forward, 5Ј-CCCTGAGCGATTCAGATGA-3Ј, reverse, 5Ј-GACGCACAGTGATGGTGAAT-3Ј, were used. The INSM1, N-Myc primers and probes for real-time PCR were purchased from Life Technologies.

MTS assay
MTS proliferation assay was carried out according to the manufacturer's protocol. In brief, each group of cells were treated with the indicated concentrations of compounds for 72 h. Treated cells were collected and incubated in medium containing 20 l of MTS assay reagent (Abcam, Cambridge, MA) at 37°C for 4 h. The assay was read at absorbance (490 nm) using a 96-well plate spectrophotometer to calculate cell viability.

INSM1 promoter-driven luciferase reporter assay
An INSM1 promoter (Ϫ426/ϩ40 bp) was inserted into pGL4.18-[luc2P/Neo] basic vector (Promega Co.) in front of a luciferase 2 (Luc2) gene. INSM1 reporter plasmid was linearized and transfected into BE2-M17 or IMR-32 NB cells with Lipofectamine 2000 (Invitrogen) for 48 h. Transfected cells were selected with a pre-determined G418 concentration in culture medium for 2 weeks. The G418-selected stable cells were further assayed for the INSM1 promoter-driven Luc2 activity. The strong luciferase activity reflects strong INSM1 promoter activity in each cell line. For the INSM1 promoter screening assay, cells were seeded in a 96-well plate overnight, then treated with each indicated test compound for 24 to 72 h. After treatment, the luciferase activity was analyzed with Firefly Luciferase Assay Kit 2.0 (Biotium Inc., Fremont, CA).

Western blot analysis
Cell lysates were extracted with lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM DTT, 0.2 mM PMSF, 1 g/ml of aproptinin, 1 g/ml of leupeptin, 1 mM Na 3 VO 4 , and 5 mM NaF) and separated by SDS-PAGE. The electrophoresed proteins were electrotransferred onto a nitrocellulose membrane (Bio-Rad) for Western blotting analyses. The membrane was blocked with 5% nonfat dry milk in TBST (20 mM Tris-HCl, pH 7.6, 137 mM NaCl, and 0.1% Tween 20), probed with the indicated primary antibody overnight at 4°C, and bound with horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. The blot was developed with chemiluminescence substrate (Bio-Rad) and exposed on X-ray film (Fuji Photo Film Co., Japan). The same blot was striped several times for subsequent antibody blotting. Western blot analysis was repeated a minimum of three separate experiments to ensure reproducibility.

Analysis of adenosine by RP-HPLC
BE2-M17 cells (1 ϫ 10 7 ) were treated with 5Ј-IT (1 M) for 10 -30-min intervals or the indicated time point. Culture medium or cell pellet was isolated and extracted with 0.4 M perchloric acid on ice for 5 min. The solution was centrifuged at 13,000 rpm for 2 min at 4°C and neutralized with ice-cold 2 M K 2 CO 3 to pH 7.5. Adenosine unknowns were run on a Thermo Scientific Dionex U3000 Ultimate system equipped with a quaternary pump, an autosampler, and a diode array detector, all controlled by Thermo Fisher Dionex Chromeleon 6.8 software. Run conditions were previously established with the following changes (30). The run consisted of an isocratic separation in 7% acetonitrile at 0.2 ml/min on a Dionex Acclaim 120 Bonded Silica C18 (5 m, 120 Å ϫ 2.1 ϫ 150 mm) column with a corresponding 5-mm guard column. Adenosine was monitored at 260 nm, bandwidth 20 nm, using a 20-min run time. A total of 16 adenosine standards were freshly prepared in 7% acetonitrile

ADKi suppresses INSM1 in neuroblastoma
by serial dilution between 1000 and 0.01 M. Standard curves were generated by linear regression analysis and accepted with a R 2 Ͼ 0.999. Unknowns were run 4 times each and standard deviation was calculated for each. Drift was monitored by repeating standards as unknowns throughout the run.

cAMP assay
BE2-M17 or IMR-32 cells were seeded in 24-well plate overnight. Cells were washed in 500 l of Hank's balanced salt solution for 10 min at room temperature. Cells were pre-treated with phosphodiesterase inhibitor Ro-20-1724 (100 M) for 30 min to inhibit endogenous phosphodiesterase activity. Forskolin (10 M) was added for 15 min before addition of 5Ј-IT (1 M) and incubated for an additional 30 min at 37°C. Cellular cAMP was extracted and measured using a cAMP ELISA kit (Cell Biolabs, Inc., San Diego, CA) according to the manufacturer's instructions.

Statistical analysis
The presented values were calculated and expressed relative to an untreated control group. All experiments were repeated at least three times. Results are presented as mean Ϯ S.D. Statistical analysis was performed using either the Student's t test when only two groups were in the experiment or by one-way analysis of variance comparison of multiple groups using the Tukey-Kramer test with differences at p values of less than 0.05 being considered significant.