cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines

Accepted Manuscript Title: cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines Author: Sabine Wolter Christina Kloth Marina Golombek Fanni Dittmar Lisa F¨orsterling Roland Seifert PII: DOI: Reference:

S0006-2952(15)00541-9 http://dx.doi.org/doi:10.1016/j.bcp.2015.08.096 BCP 12352

To appear in:

BCP

Received date: Accepted date:

21-5-2015 17-8-2015

Please cite this article as: Wolter Sabine, Kloth Christina, Golombek Marina, Dittmar Fanni, F¨orsterling Lisa, Seifert Roland.cCMP causes caspasedependent apoptosis in mouse lymphoma cell lines.Biochemical Pharmacology http://dx.doi.org/10.1016/j.bcp.2015.08.096 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

cCMP causes caspase-dependent apoptosis in mouse lymphoma cell lines

Sabine Wolter1,#, Christina Kloth1, Marina Golombek1, Fanni Dittmar1, Lisa Försterling1, and Roland Seifert1

1

Institute of Pharmacology, Hannover Medical School, Carl-Neuberg-Str. 1, D-30625 Hannover, Germany

Email addresses: Sabine Wolter, [email protected]; Christina Kloth, [email protected]; Marina Golombek, [email protected]; Fanni Dittmar, [email protected]; Lisa Försterling, [email protected]; Roland Seifert, [email protected]

#

Corresponding author: Institute of Pharmacology, Hannover Medical School, Carl-

Neuberg-Str. 1, D-30625 Hannover, Germany, Phone: +49-511-532-2887, Fax: +49-511-5324081, Email: [email protected]

Graphical abstract

Abstract

cCMP is a cyclic pyrimidine nucleotide which binds to and activates cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG). In S49 lymphoma cells, cAMP induces apoptosis via PKA. In our present study, we examined the effect of cCMP on apoptosis in S49 mouse lymphoma cells and in PKA-deficient S49 kin- cells. These two cell lines also lack PKG, hyperpolarization-activated cyclic nucleotide-gated channel 2 and 4 (HCN2 and HCN4) as assessed by real-time PCR. The cell-permeable analog cCMP-AM induced PKA- and PKG-independent apoptosis in S49 cells. In contrast, exchange protein

activated by cAMP (Epac) activation did not induce apoptosis. cCMP induced caspasedependent apoptosis via the intrinsic pathway, led to cytochrome c release from mitochondria and also activated the ER stress pathway. On the contrary, the extrinsic apoptotic pathway was not involved. Autophagy was not detectable after treatment with cCMP-AM in both cell lines. cAMP-AM, cGMP-AM, cUMP-AM as well as the cyclic nucleotides lacking the acetoxymethylester (AM)-group had no effect. cCMP-AM altered gene expression of the apoptotic-relevant gene Gadd45 and the immediate early response genes cFos and Nr4A1 in S49 wild-type (wt) cells. In conclusion, cCMP induces apoptosis of S49 lymphoma cells, independently of hitherto known cCMP target proteins. Abbreviations AM, acetoxymethylester ; Bcl-2, B-cell lymphoma 2 ; BFA, brefeldin A ; Bim, Bcl-2 interacting protein ; cAMP, adenosine 3´,5´-cyclic monophosphate; cCMP, cytidine 3´,5´cyclic monophosphate ; cGMP, guanosine 3´,5´-cyclic monophosphate; cNMP, nucleoside 3´,5´-cyclic monophosphate; cUMP, uridine 3´,5´-cyclic monophosphate; CHX, cycloheximide ; db, dibutyryl ; Epac, exchange protein activated by cAMP ; ER, endoplasmatic reticulum ; FITC, fluorescein isothiocyanate ; Gadd45, growth arrest and DNA damage-inducible protein 45  ; HCN, hyperpolarization-activated cyclic nucleotidegated channel ; HPLC-MS/MS, high performance liquid chromatography quadrupole tandem mass spectrometry ; IBMX, 3-Isobutyl-1-methylxanthine; MRP, multidrug resistance protein ; Nr4A1, nuclear receptor subfamiliy 4, group A, member1 ; PARP, poly(ADP-ribose) polymerase ; PDE, phosphodiesterase ; PI, propidium iodide ; PKA, cAMP-dependent protein kinase ; PKG, cGMP-dependent protein kinase ; sAC, soluble adenylyl cyclase; SNP, sodium nitroprusside ; wt, wild-type

Keywords: cyclic CMP; apoptosis; S49 mouse lymphoma cells 1. Introduction The cyclic purine nucleotides cAMP and cGMP are well characterized and established second messengers playing important roles in intracellular signal transduction of various external stimuli. Target molecules of cAMP and cGMP are PKA, PKG, HCN channels, and Epac for cAMP [1]. PKA consists of two regulatory and two catalytic subunits and different isoforms of each subunit exist [2]. In mammalian cells, two PKG genes encode PKG I and PKG II, PKG I existing in two splice variants [3]. Besides cAMP and cGMP, the cyclic pyrimidine nucleotide cCMP can also function as second messenger [4]. The occurrence of cCMP in intact mammalian cells, in vivo in organs of mice, and after infection with Pseudomonas aeruginosa containing the nucleotidyl cyclase toxin ExoY, were verified by highly specific mass spectrometry methods [5,6]. In HEK293 and B103 cells the soluble adenylyl cyclase is responsible for high basal levels of cCMP [7]. So far, only the phosphodiesterase 7A1 (PDE7A1) has been identified as a cCMP-degrading enzyme [8]. Another mechanism for cCMP elimination in cells is the export by multidrug resistance protein 5 (MRP5) [9]. However, little is known about the cellular functions of cCMP. A useful tool is cCMP-AM. Inside the cell, the AM-group is cleaved and cCMP is released [10,11]. To analyze the signaling pathways, holistic dynamic mass distribution assays were used [11]. In HEK293 and B103 cells cCMP is effective in these assays, and certain cCMP effects are independent of PKA and PKG [11]. Desch et al. showed the induction of smooth muscle relaxation by the membrane-permeable cCMP-analog dibutyryl-cCMP (db-cCMP) as well as activation of PKGI in aortic tissue lysates and inhibition of platelet aggregation [12]. Very recently, MAPK was identified as another cCMP-interacting protein and activation of the MAPK pathway by db-cCMP in mouse lysates was reported [13]. cCMP binds to the regulatory subunits of PKA of different species [14] and affects the function of HCN2 and 4 channels [15]. Low-potency

activation of PKGI, the regulatory subunits of PKA, RI and RII by cCMP and different cCMP-analogs was observed in vitro [16,17]. Up to now, nothing is known about the potential of cCMP to induce apoptosis and if so, whether activation of PKA is necessary or involved. For these investigations, S49 cells are a useful model. S49 cells are of thymic origin; the cells were established by van Daalen Wetters and Coffino; derived from lymphoma of tumors of oil-treated BALB/c mice [18]. Under basal conditions cAMP and cCMP occur, but neither cGMP nor cUMP are detectable in S49 cells [5]. After incubation with DB-cAMP or other cAMP-increasing compounds, S49 wt cells arrested in the G1 phase of the cell cycle, followed by delayed cytolysis which finally led to cell death [19]. S49 cells are a useful tool to study the role of cAMP in apoptosis, because S49 kin- cells are resistant to cAMP-induced apoptosis [19]. The mRNA for the catalytic subunits of PKA is expressed at normal level, but the catalytic subunit protein is degraded rapidly. Therefore, the catalytic subunit is not detectable in S49 kin- cells at the protein level and, subsequently, the cells have no PKA activity [20]. After incubation with cAMP-analogs or cAMP-increasing stimuli, S49 kin- cells show neither G1-phase arrest, nor mitochondriadependent apoptosis, nor induction of PDE or other apoptosis-relevant proteins [19]. Apoptosis is a cellular mechanism for programmed cell death characterized by membrane blebbing, condensation of the cytoplasm and nucleus, DNA fragmentation and cell shrinkage [21]. Apoptosis is important for normal and embryonic development, tissue homeostasis and immune response. Caspases are the central enzymes in apoptosis. Generally there are three ways of inducing apoptosis. The extrinsic pathway is activated by death ligands like TNF or FasL, which bind to specific receptors and lead to activation of the initiator caspase 8. Activated caspase 8 initiates apoptosis by cleaving other downstream executioner caspases, such as caspase 3 and caspase 7 [22]. The intrinsic mitochondrial pathway is initiated within the cell by irreparable genetic damage, severe oxidative stress, hypoxia or deprivation of survival factors increasing the mitochondrial permeability [23]. This

leads to the release of pro-apoptotic factors, such as cytochrome c, out of the mitochondria into the cytoplasm, the loss of integrity of the outer membrane caused by pro-apoptotic factors of the Bcl-2 family, and results in activation of caspase 3 [23]. Another pathway is the intrinsic endoplasmic reticulum (ER) pathway leading to activation of caspase 12. This pathway is triggered by ER stress caused by misfolded proteins. Stimulation with brefeldin A (BFA), an inhibitor of the intracellular protein transport, induces ER stress [24,25]. Disordered apoptosis results in pathophysiological diseases like cancer. Understanding the cellular mechanisms can give the opportunity to treat diseases or develop drugs targeting apoptotic genes and pathways by cCMP. Autophagy is a catabolic degradative process and often associated with anti-proliferative mechanism and physiolological processes like differentiation, development and cancer [26]. In this process, cytoplasmatic components are delivered to lysosomes and disposed. Important enzymes in autophagy are autophagy-related genes (ATG) and LC3 (light chain 3) A/B proteins, which function as autophagy marker proteins. Using the S49 cell model, we characterized the mechanism of cCMP action in apoptosis and autophagy. 2. Materials and Methods 2.1.

Materials cAMP-AM, cCMP-AM, cGMP-AM, cUMP-AM, cAMP, cCMP, cGMP, cUMP, PO4-

AM3, db-cAMP, db-cCMP, Sp-cAMPS-AM, Sp-8-Br-cAMPS-AM and 8-pCPT-2'-O-MecAMP (Epac agonist) were obtained from Biolog LSI (Bremen, Germany), Z-VAD-fmk was obtained from R&D Systems (Minneapolis, MN, USA). Staurosporine was purchased from Enzo (Lörrach, Germany), TNF was from eBioscience (San Diego, CA, USA), forskolin was from LC Laboratories (Woburn, MA, USA), brefeldin A, cycloheximide, 3-Isobutyl-1methylxanthine (IBMX), sodium nitroprusside dihydrate (SNP), isoproterenol, trypan blue,

propidium iodide (PI), saponin and all other chemicals were purchased from Sigma-Aldrich (Seelze, Germany) and of analytical grade if not otherwise mentioned.

2.2.

Cell culture S49 wt and S49 kin- cells were obtained from the Cell Culture Facility of the University

of California (San Francisco, CA, USA). Cells were grown in suspension culture with Dulbecco’s modified Eagle’s medium (high glucose) supplemented with 10 % (v/v) heatinactivated horse serum (PAN Biotech, Aidenberg, Germany), 1 mM sodium pyruvate, 200 µg/ml L-glutamine, 100 U/ml penicillin, and 0.1 mg/ml streptomycin in a humidified atmosphere containing 8 % (v/v) CO2 at 37°C. S49 cells were described in detail [18-20].

2.3.

Determination of the cell number S49 wt and S49 kin- cells were seeded at 1 x 105 cells/ml in a 12-well plate with 2 ml of

media and treated with the reagents. After 24, 48 and 72 h aliquots were taken. Cell number was estimated using a hemacytometer. Viable cells were distinguished from dead cells using the trypan blue dye exclusion method.

2.4.

Flow cytometry S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of

media and treated with the reagents. After the incubation times the samples were transferred to 1.5 ml tubes and centrifuged for 10 min at 300 x g. The supernatants were discarded and the cells were resuspended in 100 µl binding buffer (HEPES 10 mM, NaCl 140 mM, CaCl 2 2.5 mM, pH 7.4) containing 5 % (v/v) annexinV-APC (MabTag, Friesoythe, Germany) and transferred into FACS tubes. After light protected incubation for 20 min at room temperature 200 µl binding buffer were added. Immediately before the measurement 1.7 µg/ml of PI was

added to each sample. The samples were analyzed with a MAQS Quant Analyzer (Miltenyi Biotech, Bergisch Gladbach, Germany).

2.5.

Cytochrome c release S49 wt and S49 kin- cells were seeded at 2.5 x 105 cells/ml in a 12-well plate with 2 ml

of media and treated with reagents. After incubation time, cells were sedimented by centrifugation for 10 min at 300 x g and 4°C. The pellets were resuspended in 50 µl PFA (PBS + 4 % (v/v) para-formaldehyde) and incubated for 10 min on ice. The cells were washed two times with 300 µl PAB (PBS with 0.5 % (w/v) BSA and 0.01 % (w/v) NaN3). The pellets were resuspended in 100 µl PAB containing 0.5 % (w/v) saponin, and 1.5 µl of anticytochrome c-FITC antibody (eBioscience, San Diego, CA, USA) was added. After lightprotected incubation for 30 min at room temperature the cells were washed twice with 300 µl PAB containing 0.1 % (w/v) saponin. The cells were resuspended in 200 µl PAB and analyzed with the MAQS Quant Analyzer.

2.6.

Western blotting S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 6-well plate with 5 ml of

media and treated with cCMP-AM (150 µM) and as a control with PO4-AM3 (50 µM) for 20 h. Thereafter the cells were sedimented by centrifugation at 300 x g for 10 min at 4°C. The cells were resuspended in PBS containing protease and phosphatase inhibitors (Roche, Penzberg, Germany) and lysed by sonification. Ten microliters of protein solution were taken for quantification of protein concentration with Bradford protein assay. Equal protein quantities were separated by SDS-PAGE using a gel containing 10 % (m/v) acrylamide for PKA Ireg, PKA IIreg, PKA C pan, PARP, caspase 12 and tubulin; 12.5 % (m/v) acrylamide for caspase 3, caspase 7, caspase 9, CREB and p44/p42 MAPK and 15 % (m/v) acrylamide for LC3 A/B detection. Following transfer to nitrocellulose membranes, the

proteins were analyzed with specific antibodies: anti-PKA Ireg, sc-136231; anti-PKA IIreg, sc-136262 (Santa Cruz, Heidelberg, Germany), anti-PKA C pan (MAB4175, R&D systems, Minneapolis, Minnesota, USA), anti-caspase-3, #9662; anti-caspase-7, #9492; anticaspase-9, #9504, anti-cleaved caspase-9, #9509; anti-caspase-12, #2202; anti-PARP, #9542; anti-cleaved PARP, #9544; anti-phospho p44/42 MAPK Erk1/2, #4370; anti-phospho CREB, #9198; anti-CREB, # 4820 (all from cell signaling, New England Biolabs, Frankfurt/Main, Germany); anti-PKG, ADI-KAP-PK005 from Enzo (Lörrach, Germany) and anti-tubulin, sc8035 was from Santa Cruz, Heidelberg, Germany. For analyzing activation of CREB and MAPK pathways equal cell numbers were used, stimulated for 0.5 h or 1 h, sedimented by centrifugation and resuspended in SDS loading puffer. After sonification, the whole cell extract was separated by SDS-PAGE. The second antibodies were obtained from cell signaling. Proteins were detected by using the SuperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Schwerte, Germany). The images were analyzed with ImageJ software. The protein bands were normalized to the expression of tubulin and S49 wt control was calculated to 1.

2.7.

Caspase 8 assay S49 wt and S49 kin- cells were seeded at 2.5 x 105 cells/ml in a 6-well plate with 4 ml

of media and incubated with cCMP-AM (150 µM), PO4-AM3 (50 µM) and Brefeldin A (4 µM) for 20 h. As positive control the cells were treated with cycloheximide (100 ng/ml) and TNF (1 ng/ml) (eBioscience, San Diego, CA, USA) for 4 h. The activity of caspase 8 was analyzed with the Caspase-8/FLICE Fluorometric Assay Kit (ALX-850-222-KI01, Enzo, Lörrach, Germany) according to the manufacturer’s instructions.

2.8.

Gene expression analysis

S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of media and incubated with cCMP-AM (150 µM) and PO4-AM3 (50 µM) for 1 h, 2 h, 3 h and 4 h or were left untreated. The cells were sedimented by centrifugation and RNA was prepared using NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany) according to the manufacturer’s instructions. RNA was reverse-described into cDNA and analyzed with TaqMan probes for the expression of Actb (Mm00607939_s1), Bcl2 (Mm00477631_m1), Bim (Mm00437796_m1), Gadd45 (Mm00432802_m1), Hcn2 (Mm00468538_m1), Hcn4 (Mm01176086_m1), (Mm00435938_m1)

cFos and

(Mm00487425_m1), Nr4a1

Prkg1

(Mm01300401_m1)

all

(Mm00440954_m1), from

Life

Prkg2

Technologies

(Frankfurt/Main, Germany) using the qPCRBIO Probe Mix (Nippon Genetics, Düren, Germany) according to the manufacturer’s instructions. The data were analyzed using the Ct method.

2.9.

Stimulation of S49 wt and S49 kin- cells and measurement of cAMP, cGMP and

cCMP concentration by HPLC-MS/MS To analyze if specific drugs can enhance cCMP concentration in S49 cells, S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of media, preincubated with IBMX (100 µM) for 10 min and stimulated with forskolin (10 µM), isoproterenol (1 µM) for 1 h and SNP (100 µM) for 1 h and 3 h or were left untreated. The concentrations of cAMP, cCMP and cGMP were measured by HPLC-MS/MS [27].

2.10. Statistical analysis Data are presented as means ± SD and are based on at least 3 independent experiments. GraphPad Prism 5.01 software (San Diego, CA, USA) was used for calculation of p-values by means of one-way ANOVA followed by Dunnett’s test; Bonferroni’s multiple comparison

test or the unpaired t test was used to compare selected pairs of columns: *, p < 0.05; **, p < 0.01 and ***, p < 0.001; n.s., not significant. 3.

Results

3.1. S49 kin- cells lack PKA-activity and S49 wt as well as S49 kin- cells do not express mRNA for PKG, HCN2 and HCN4

S49 cells were analyzed regarding the expression of PKA subunits by western blot and by real-time PCR. Equally, S49 cells were investigated for the mRNA expression of already identified cCMP targets (PKGI, PKGII, HCN2 and HCN4) by real-time PCR. The regulatory subunits of PKA, RI and RII were expressed both in S49 wt and S49 kin- cells, whereby the PKA RI subunit showed lower expression in S49 wt cells. The catalytic subunits of PKA, analyzed with an antibody which recognized ,  and  subunits, were detectable only in S49 wt cells but not in S49 kin- cells (Fig. 1A). The expression of PKG was measured by western blotting. No specific protein expression of PKG was detectable in S49 cells (data not shown). The mRNAs of PKA RI, PKA RI, PKA RII, PKA RII, PKA CPKA C and PKA C were expressed in S49 wt and S49 kin- cells, but mRNA for PKGI, PKGII, HCN2 and HCN4 was not detectable either in S49 wt or in S49 kin- cells (Fig. 1B).

3.2. Reduced cell number in S49 cells by cCM P-AM S49 wt and S49 kin- cells were incubated with db-cAMP, db-cCMP, sodium butyrate, cCMP-AM and PO4-AM 3 72 h. Cell number was determined after trypan blue staining. Reduced cell number was observed in S49 wt cells with the membrane-permeable cAMP anlog like db-cAMP. db-cCMP altered the cell number neither in S49 wt nor in S49 kin- cells (Fig. 2). In contrast, a significant reduction in cell number occurred in S49 wt and also in S49

kin- cells after incubation with the cell-permeable cCMP-AM. The control reagents, sodium butyrate and PO4-AM 3, did not change the number of cells either in S49 cells or in S49 kincells.

3.3. Concentr ation-dependent induction of apoptosis by cCM P-AM S49 wt and S49 kin- cells were incubated with different concentrations of cCMP-AM and the control substance PO4-AM 3 for 18 h. Apoptosis was analyzed after annexinV-APC/PI staining by flow cytometry analysis. AnnexinV binds to phosphatidylserine that is translocated by early apoptotic cells from the inner to the outer membrane. Early-apoptotic cells are positive for annexinV staining and negative for PI staining. cCMP-AM induced a concentration-dependent apoptosis in both cell types, whereas PO4-AM 3 had no effect (Fig. 3). The curves of cCMP-AM for S49 wt and S49 kin- cells were similar and steep. The induction of apoptosis started at around 100 µM cCMP-AM. At 200 µM cCMP-AM 82 % of S49 wt and 88 % of S49 kin- cells became apoptotic whereas only at higher concentrations of PO4-AM 3 around 30 % were affected.

3.4. cCM P-AM -induced apoptosis is caspase-dependent: Caspase inhibitor Z-VAD-fmk abr ogates cCM P-induced apoptosis Incubation of the cells with the control reagent PO4-AM 3 showed about 10 % of apoptotic cells in S49 wt and S49 kin- cells (Fig. 4). After addition of 100 µM cCMP-AM to the cells, the percentage of apoptotic cells increased to 56.9 % and to 36.0 %, respectively. The activation of caspases occurred at an early stage of apoptosis, and was inhibited by the pan-caspase inhibitor Z-VAD-fmk to 19.7 % in S49 wt and to 24.2 % in S49 kin- cells. Representative dot blots from S49 wt and S49 kin- cells treated with the control reagent PO4-

AM 3, cCMP-AM as well as pre-incubation with Z-VAD-fmk and subsequent treatment with cCMP-AM were shown in Fig 4A. The amount of early apoptotic cells from five independent experiments were analyzed by one-way ANOVA followed by Dunnett’s test. Bonferroni’s multiple comparison tests were used to compare control and also Z-VAD-fmk-treated cells with cCMP-AM treated cells (Fig. 4B). Pre-incubation with Z-VAD-fmk showed significant reduction of apoptotic cells in S49 wt and S49 kin- cells.

3.5. Autophagy is not induced by cCM P-AM S49 wt and S49 kin- cells were incubated with cCMP-AM and PO4-AM 3 for 24 h and analyzed with autophagy-specific antibodies against LC3 A/B and normalized to tubulin expression. If autophagy had been induced, the protein expression of LC3 A/B would have been enhanced. However, we detected no alteration in protein expression of LC3 A/B after cCMP-AM treatment (Fig. 5A and 5B).

3.6. cNM Ps fail to induce apoptosis in S49 wt and S49 kin- cells; induction of apoptosis is specific for cCM P-AM S49 wt and S49 kin- cells were incubated with cCMP-AM, PO4-AM 3, cAMP-AM, cUMPAM and cGMP-AM for 24 h and 48 h and analyzed by flow cytometry. Except for cCMPAM, all other substances failed to induce apoptosis in the cells treated for 24 h (Fig. 6A) and 48 h (Fig. 6B). Incubation of S49 wt and S49 kin- cells with cAMP, cCMP, cGMP and cUMP did not induce apoptosis either after 24 h (Fig.7A) or after 48 h (Fig. 7B) of treatment.

3.7. PDE-r esistant cAM P-AM analogs activate the PK A signaling pathway and induce apoptosis in S49 wt cells S49 wt and S49 kin- cells were stimulated with cAMP-AM, PO4-AM 3 and membranepermeable and PDE-resistant cAMP-AM analogs. Induction of apoptosis was analyzed by flow cytometry. The PDE-resistant cAMP-AM analog, Sp-8-Br-cAMPS-AM, and to a lower extent, also Sp-cAMPS-AM, induced apoptosis in S49 wt cells (Fig. 8A).The activation of the PKA pathway led to phosphorylation of the transcription factor CREB. This was analyzed by western blot. The phospho-CREB expression was normalized to total CREB expression (Fig. 8C). Activation of PKA signaling was detectable only in S49 wt cells and not in S49 kin- cells after treatment with Sp-8-Br-cAMPS-AM.

3.8. I dentification of the apoptotic pathways by wester n blotting: cCM P-AM activates the intr insic pathway and also induces ER str ess-mediated apoptosis Caspases are important enzymes involved in the apoptotic pathways [22]. By western blotting the activation of the executioner caspases 3 and 7 after treatment with cCMP-AM in S49 wt and S49 kin- cells was detectable. The full length proteins were reduced and specific cleaved products of caspase 3 as well as the cleavage of nuclear poly (ADP-ribose) polymerase (PARP), one of the main targets of caspase 3, occurred (Fig. 9A). Furthermore, activation of caspase 9 and caspase 12, the reduction of the full length form and the specific cleaved protein of caspase 9 was observed as well. Significant blots were shown in Fig. 7A. Protein expression from three independent experiments was quantified with ImageJ software. After cCMP-AM treatment the cleaved caspase 3 proteins increased and the caspase 12 protein decreased (Fig. 9B); no significant alteration was detected for the full length caspase 3 protein.

3.9. cCM P-AM induces r elease of cytochr ome c fr om mitochondr ia During mitochondrial apoptosis, cytochrome c is redistributed from mitochondria to the cytosol in intact cells. S49 wt and S49 kin- cells were treated with cCMP-AM, PO4-AM3 and as a positive control with staurosporine, fixed and stained with a cytochrome c-FITC antibody and analyzed by flow cytometry with a MAQS Quant Analyzer. Both S49 cells treated with cCMP-AM and staurosporine showed two distinct cell populations with a decrease in the FITC signal. No effect was detectable after treatment with the control substance PO4-AM3 in S49 wt and S49 kin- cells. Representative dot-blots were shown in Fig. 10A. Data from four independent experiments were analyzed by one-way ANOVA followed by Dunnett’s test. Significant increase in the lower FITC signal as a criterion for the release of cytochrome c out of mitochondria was detected in S49 wt and S49 kin- cells after treatment with cCMP-AM and staurosporine compared to untreated cells (Fig. 10B). The release of cytochrome c from the mitochondria into the cytoplasm implies the involvement of the intrinsic apoptotic pathway in cCMP-AM-treated S49 wt and S49 kin- cells.

3.10. Caspase 8 is not involved in cCM P-AM -mediated apoptosis Besides the intrinsic and the ER-mediated apoptotic pathways, also the receptormediated extrinsic pathway could be responsible for cCMP-induced apoptosis. S49 wt and S49 kin- cells were treated with cCMP-AM, PO4-AM 3, and as a positive control with a combination of cycloheximide and TNFand as a control regarding the specificity of this assay, also with BFA. With a fluorometric assay, activation of caspase 8 was measured in the cycloheximide and TNF–treated cells only (Fig. 11). BFA activated the ER stress apoptotic pathway and led to activation of caspase 12. Activation of caspase 8 occurred neither in the BFA-stimulated cells nor in the cCMP-AM- or PO4-AM 3-treated cells.

3.11. cCM P-AM induces differ ent apoptosis-r elated genes After exposure of S49 wt and S49 kin- cells to cCMP-AM and PO4-AM 3 for the indicated time points the expression of Bim and other important apoptotic genes was analyzed by quantitative real-time PCR. The pro-apoptotic protein Bim belongs to the Bcl-2 protein family and is induced after cAMP stimulation in S49 wt cells [28]. No significant induction of Bim or Bcl-2 was detectable in S49 wt or in S49 kin- cells (Fig. 12). Other apoptotic relevant genes were induced by cCMP-AM in S49 wt cells; mRNA of Gadd45increased after 2 h about 3-fold, the expression of the immediate-early genes cFos and Nr4A1 showed a rapid and strong induction about 15-fold for cFos and about 12-fold for Nr4A1. No increase of mRNA expression of analyzed genes was detected in S49 kin- cells. Treatment with PO4-AM 3 did not alter gene expression in both cell lines.

3.12. Epac is not involved in cCM P-induced apoptosis Another target of cAMP is Epac. To elucidate whether Epac activation is involved in cCMP-induced apoptosis, we quantified the number of cells using the trypan blue dye exclusion method. S49 wt and S49 kin- cells were incubated with 100 µM 8-pCPT-2'-O-MecAMP (Epac agonist) for 72 h. Cell number was determined after trypan blue staining after 24 h, 48 h and 72 h. No significant reduction in cell number occurred either in S49 wt or in S49 kin- cells incubated with the Epac agonist (Fig. 13).

3.13. cCM P-AM fails to activate the M APK -p44/p42 pathway

Recently, activation of p44/p42 MAPK pathway by db-cCMP in jejunum and lung tissue lysates from mice was reported [13]. To analyze if activation of this pathway also occurs in S49 cells, S49 wt and S49 kin- cells were treated with cCMP-AM, PO4-AM 3, db-cCMP and sodium butyrate for 0.5 h. Activation of the MAPK pathway was analyzed by western blotting (Fig. 14A). Phosphorylation of p44/p42 was normalized to total protein amount of p44/p42 (Fig. 14B). No significant activation occurred in S49 wt or S49 kin- cells.

3.14. Var ious dr ugs fail to enhance cCM P concentration in S49 cells For future studies on cCMP function in S49 cells, enhancement of cCMP concentration after stimulation by specific drugs could be helpful. Therefore, we treated S9 wt and S49 kincells with forskolin, an adenylyl cyclase activator, the -adrenoreceptor agonist isoproterenol and the NO-activator sodium nitroprusside and measured the cAMP, cGMP and cCMP concentration of the S49 cells by HPLC-MS/MS. As expected, forskolin significantly increased the cAMP concentration in S49 wt and S49 kin- cells [29] (table 1). Isoproterenol stimulation led to a slight increase of the cAMP concentration in S49 wt and S49 kin- cells. Forskolin and also isoproterenol did not enhance cCMP concentration. Sodium nitroprusside failed to increase cAMP, cGMP or cCMP concentration. 4.

Discussion Occurrence of cCMP has been verified by high specific mass spectrometry in several

cell lines, primary cells and in vivo [5,6]. Extensive studies were performed resulting in detection of generators, target molecules, degrading enzymes and export mechanisms for the newly emerging second messenger cCMP and also cUMP [10]. However, the function of cCMP is still elusive. Here we show for the first time that cCMP induces apoptosis in S49 wt and kin- mouse lymphoma cell lines. cCMP induced apoptosis via the intrinsic apoptotic pathway and also via the ER stress pathway, whereas the extrinsic apoptotic pathway is not

involved and some other proteins, PKG, Epac, HCN2, HCN4, and in the S49 kin- cells, also PKA were excluded as cCMP targets (Fig. 15). The process of autophagy is not activated by cCMP-AM in S49 cells. S49 cells were used before to analyze the function of cAMP in promoting apoptosis [19]. Moreover, S49 cells revealed to be an excellent system to investigate the contribution of PKA in apoptosis since a PKA-deficient cell line is available which is resistant to cAMP-promoted apoptosis [19]. Interestingly, treatment with cCMP-AM induced apoptosis in S49 wt and S49 kin- cells. Induction of apoptosis was specific for cCMP, because all other cNMP-AMs used, i.e. cAMP-AM, cGMP-AM, and also cUMP-AM, failed to activate apoptotic pathways in both S49 cell types. The target molecule of cCMP is not PKA, because the catalytic subunit is absent in S49 kin- cells [20], although we have found normal level of mRNA for all PKA subunits. Several other known target molecules for cAMP can be already excluded as cCMP target molecules. Activation of Epac did not reduce S49 cell number and aborted induction of apoptosis; moreover cCMP failed to activate Epac [11]. cCMP activates PKGI in vitro [16]. But for induction of apoptosis this activation is not relevant due to the lack of PKGI and PKGII mRNA in S49 wt and S49 kin- cells. Furthermore, the regulation of the HCN2 and HCN4 channels by cCMP was shown [15]. However, this function can also be excluded for apoptosis induction by cCMP since we neither found mRNA for HCN2 nor for HCN4 in S49 cells. We did not find any extracellular effects from cCMP or other cNMPs, because all tested cNMPs failed to induce apoptosis in S49 cells as well. These results indicate that binding of cCMP to specific membrane receptors is not involved in apoptosis. Surprisingly, we found different effects using db-cAMP and db-cCMP in the determination of the cell number. As expected, db-cAMP reduced the cell number of S49 wt cells, but treatment with db-cCMP had no effect although some function of db-cCMP in muscle relaxation has been lately shown

by Desch et al. [12]. Recently, activation of p44/p42 MAPK pathway has been reported [13].These studies were performed with mouse tissue lysates. In contrast, in S49 cells we did not detect any activation of this pathway by cCMP-AM or db-cCMP. db-cCMP is cleaved by esterases and the main metabolite 4-MB-cCMP arises, which is different from cCMP [17]. Probably the bulky monobutyryl substituent of N4-monobutyryl-cCMP prevents activation of cCMP effector proteins. Treatment with the cell-permeable cCMP-analog cCMP-AM, where the negative charge is masked by an acetoxymethylester (AM)-group and whose cleavage results in cCMP [11], resulted in a reduced cell number and apoptosis in S49 wt and also in S49 kin- cells. These cells were often used in previous investigations to elucidate the function of cAMP. cAMP functions both as pro- and anti-apoptotic factor, depending on the circumstances of the cells [30]. In S49 cells, cAMP acts as pro-apoptotic stimulus and operates via PKA to induce G1 phase cell cycle arrest and apoptosis [19]. Surprisingly, we did not find apoptosis in S49 wt cells after treatment with cAMP-AM. This might be due to fast degradation of cAMP by phosphodiesterases, because co-incubation with inhibitors for phosphodiesterases, like IBMX or rolipram, increased apoptosis in S49 cells. Moreover, activation of PKA pathway and induction of apoptosis after treatment with PDE-resistant and cell-permeable cAMP-AM analogs occurred in S49 wt cells. cCMP-induced apoptosis is caspase-dependent because the apoptotic mechanism is partly abrogated by using a pan-caspase inhibitor. Caspases are the relevant enzymes in apoptosis and the apoptotic mechanisms can be partly distinguished by the activated and cleaved caspases [21]. The cCMP-activated caspases were identified by western blot analysis. Intracellular cCMP activated caspases 9 and 12. Caspase 9 is an initiator caspase of the intrinsic apoptotic pathway [22]. The importance of this pathway for cAMP-induced apoptosis in S49 wt cells and the release of cytochrome c were shown by Zhang et al. [31]. cCMP also induced cytochrome c release, as was shown by flow cytometry analysis with a specific antibody. Surprisingly, we found cytochrome c release in S49 wt and to a lesser

amount also in S49 kin- cells. Responsible for the stronger release of cytochrome c in S49 wt cells could be PKA activation by cCMP that is absent in S49 kin- cells. But for cytochrome c release, PKA activity is not absolutely required. Intracellular cCMP also activated and cleaved caspase 12. Caspase 12 is involved in the ER stress apoptotic pathway [32]. In preliminary studies we identified calnexin as a cCMPbinding protein [33]. Calnexin is a molecular chaperone that plays an essential role in folding membrane proteins correctly; misfolded or incompletely built proteins bind longer to calnexin [34]. Binding between calnexin and intracellular cCMP may decrease the calnexin concentration inside the cell and, therefore, increase the amount of unfolded or incorrectly built proteins which induces ER stress and apoptosis. Calnexin is cleaved in apoptotic cells and the cleavage products inhibit apoptosis [34]. This feedback mechanism could also be disrupted by cCMP binding to calnexin. Further investigations have to be done to analyze the mechanism between cCMP and calnexin binding and the induction of apoptosis. The extrinsic apoptotic pathway is not involved in cCMP-promoted apoptosis, since activation of caspase 8 was not detected after cCMP-AM treatment (Fig. 15). Another approach to analyze the cCMP-activated mechanism is the identification of altered gene expression in S49 cells after cCMP-AM-treatment. The pro-apoptotic factor Bim is induced in S49 wt cells after exposure to cAMP-analogs or cAMP-generating agents like forskolin [28]. After treatment with cCMP-AM, induction of Bim or Bcl-2 was neither detectable in S49 wt nor in S49 kin- cells. Possibly, other Bcl-2-related proteins are involved in cCMP-induced apoptosis. The Bcl-2 protein family consists of about 15 members with either pro- or anti-apoptotic functions [35] Additional investigations are in progress to analyse Bcl-2 proteins in detail. Until now, no genes induced or repressed by cAMP have been identified in S49 kin- cells [36,37]. However, differences in gene expression due to differences in cAMP signalling were shown under basal condition in S49 wt and S49 kin-

cells [38]. In our study, we identified cCMP-AM-dependent gene alterations in S49 wt cells only, induction of the apoptotic-relevant gene Gadd45 and of the immediate early response genes cFos and Nr4A1. Induction of apoptosis by cCMP-AM is partly different from cAMP action, whereas Bim expression was not increased, but cCMP-AM as well cAMP-analogs induced Gadd45]. Identification of additional cCMP-altered genes in S49 wt and also in S49 kin- cells could be helpful to understand the mechanism. In the future, additional studies will have to be performed to investigate further functional activities and the mechanism of cCMP. First results about cCMP-induction of apoptosis in a human cell line were obtained from HEL cells [39]. Some cCMP-resistant cell lines were detected, as well. Additional studies about human cells will be performed in the future. Furthermore, we will apply cCMP-agarose or cCMP-biotin as matrices to identify additional cCMP target molecules. S49 kin- cells are an excellent model for these investigations, because they lack all known cCMP targets but after cCMP-AM treatment apoptosis occurred. It is conceivable that cCMP could play an important role in future tumor therapy. To address this question, some more tumour cell lines have to be treated with cCMP-AM and to be analyzed for apoptosis and other cell functions. Many investigations on cAMP and the antiproliferative ability in tumours were performed [40]. In chronic lymphocytic leukemia, cAMP is pro-apoptotic and alters the gene expression of proteins belonging to the Bcl-2 family [36]. Comparable studies for cCMP should be performed to identify cCMP target molecules or cCMP-altered genes. Probably, cCMP is involved in the beginning of the apoptotic process. It is not known yet how cytochrome c release occurs in detail, but Bcl-2 proteins are involved [21]. One could envisage an alteration of genes belonging to the Bcl-2 family or a direct interaction between cCMP and Bcl-2 proteins. However, only one cCMP-degrading PDE, PDE7A1, has been described so far [8], nothing is known about the stability of cCMP inside a cell. Therefore, further experiments with PDE-resistant analogs should be performed in S49

wt, S49 kin- cells and also in other tumor and human cell lines. For further investigations about cCMP function the availability of cCMP-enhancing drugs would be helpful. In conclusion, we described for the first time that cCMP is effective in inducing apoptosis and we identified the apoptotic pathways activated by cCMP.

Acknowledgements: We thank Mrs. Tina Hagedorn for excellent technical support. We thank the MHH Research Core Unit Metabolomics for measurement of cNMP concentrations and the reviewers for their constructive critique. References [1]

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[17] Wolter S, Dove S, Golombek M, Schwede F, Seifert R. N4-monobutyryl-cCMP activates PKA RI and PKA RII more potently and with higher efficacy than PKG I in vitro but not in vivo. Naunyn Schmiedebergs Arch Pharmacol 2014; 387:1163-75. [18] van Daalen Wetters T, Coffino P. Cultered S49 mouse T lymphoma cells. Methods Enzymol 1987; 151:9-19. [19] Yan L, Herrmann V, Hofer JK, Insel PA. -Adrenergic receptor/cAMP-mediated signaling and apoptosis of S49 lymphoma cells. Am J Physiol Cell Physiol 2000; 279: C1665-C1674. [20] Orellana SA, McKnight GS. The S49 Kin- Cell Line Transcribes and Translates a Functional mRNA Coding for the Catalytic Subunit of cAMP-dependent Protein Kinase. J Biol Chem 1990; 265:3048-53. [21] Hengartner MO. The biochemistry of apoptosis. Nature 2000; 407:770-6. [22] Elmore S. Apoptosis: A review of programmed cell death. Toxicol Pathol 2007; 35:495516. [23] Martinou J-C, Youle RJ. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev Cell 2011; 21:92-101. [24] Kaufman RJ. Stress signalling from the lumen of the endoplasmic reticulum: coordination of gene transcriptional and translational controls. Genes Dev 1999; 13:1211-33. [25] Szegezdi E, Fitzgerald U, Samali A. Caspase-12 and ER-stress-mediated apoptosis: the story so far. Ann N Y Acad Sci 2003; 1010:186-94. [26] Reggiori F, Klionsky DJ. Autophagy in the Eukaryotic Cell. Eukaryot Cell 2002; 1:1121. [27] Hartwig C, Bähre H, Wolter S, Beckert U, Kaever V, Seifert R. cAMP, cGMP, cCMP and cUMP concentrations across the tree of life: High cCMP and cUMP levels in astrocytes. Neurosci Lett 2014; 579:183-7.

[28] Zhang L, Insel PA. The pro-apoptotic protein Bim is a convergence point for cAMP/protein kinase A- and glucocorticoid-promoted apoptosis of lymphoid cells. J Biol Chem 2004; 279:20858-65. [29] Zhang L, Insel PA. Bcl-2 protects lymphoma cells from apoptosis but not growth arrest promoted by cAMP and dexamethasone. Am J Physiol 2001; 281:C1642-C1674. [30] Insel PA, Zhang L, Murray F, Yokouchi H, Zambon AC. Cyclic AMP is both a proapoptotic and anti-apoptotic second messenger. Acta Physiol 2012; 204:277-87. [31] Zhang L, Zambon AC, Vranizan K, Pothula K, Conklin BR, Insel PA. Gene expression signatures of cAMP/Protein Kinase A (PKA)-promoted, Mitochondrial-dependent Apoptosis. J Biol Chem 2008; 283:4304-13. [32] Nakagawa T, Zhu H, Morishima N, Li E, Xu J, Yankner BA, et al. Caspase 12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid- Nature 2000; 403:98-103. [33] Neumann M, Schwede F, Pich A, Wolter S. Seifert R. Identifying cUMP-binding proteins. Naunyn Schmiedebergs Arch Pharmacol 2014; 387 (Suppl 1):S71. [34] Takizawa T, Tatematsu C, Watanabe K, Kato K, Nakanishi Y. Cleavage of calnexin by apoptotic stimuli: Implication for the regulation of apoptosis. J Biochem 2004; 136:399405. [35] Siddiqui WA, Ahad A, Ahsan H. The mystery of BCL2 family: Bcl-2 proteins and apoptosis: an update. Arch Toxicol 2015; 89:289-317. [36] Insel PA, Wilderman A, Zhang L, Keshwani MM, Zambon AC. Cyclic AMP/PKApromoted apoptosis: Insights from studies of S49 lymphoma cells. Horm Metab Res 2014; 46:854-62. [37] Zambon AC, Zhang L, Minovitsky S, Kanter JR, Prabhakar S, Salomonis N, et al. Gene expression patterns define key transcriptional events in cell-cycle regulation by cAMP and protein kinase A. Proc Natl Acad Sci USA 2005; 102:8561-6.

[38] Guo Y, Wilderman A, L. Zhang, Taylor SS, Insel PA. Quantitative proteomics analysis of the cAMP/protein kinase A signaling pathway. Biochemistry 2012; 51:9323-31. [39] Dittmar F, Wolter S, Hartwig C, Schwede F, Seifert R. The cyclic nucleotide cCMP affects proliferation and apoptosis of the human erythroleukemia cell line HEL 92.1.7. Naunym-Schmiedeberg’s Arch Pharmacol (2014) 387 (Suppl 1): 137 [40] Murray F, Insel PA. Targeting cAMP in chronic lymphocytic leukemia: a pathwaydependent approach for the treatment of leukemia and lymphoma. Expert Opin Ther Targets 2013; 17:937-49.

Figure legends Fig. 1: The catalytic subunits of PKA are not detectable in S49 kin- cells, and the mRNAs for PKGI, PKGII, HCN2 and HCN4 are not expressed in S49 wt and S49 kincells. S49 wt and S49 kin- cells were analyzed by western blot and real-time PCR for the expression of the PKA subunits and for mRNA expression of PKGI, PKGII, HCN2 and HCN4 by real-time PCR using TaqMan probes. A, a representative western blot is shown , *: indicates the regulatory subunits RI and RII from PKA; a pan antibody recognize the ,  and  subunits of PKA and also an unspecific protein; equal protein loading is shown with the tubulin expression. B, data show mRNA expression, the expression of Actb was used as housekeeping gene. To determine the mRNA expression of the genes Ct values were calculated according to the following equation: Ct= Ct (gene) – Ct (Actb). Data shown are means ± SD of four experiments.

Fig. 2: Reduced cell number in S49 wt and S49 kin- cells after cCMP-AM treatment. S49 wt and S49 kin- cells were seeded at 105 cells/ml in 200 µl and treated with 100 µM dbcAMP, 1 mM db-cCMP, 1mM sodium butyrate, 100 µM cCMP-AM and 33 µM PO4-AM3 for

72 h. Cell number was determined using the trypan blue dye exclusion method. Shown are data as means ± SD from 4 independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test (***, p < 0.001).

Fig. 3: Concentration-dependent induction of apoptosis by cCMP-AM. S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of media and treated in a volume of 1 ml with 50 µM, 75 µM, 100 µM, 150 µM and 200 µM cCMP-AM and as control with 16.7 µM, 25 µM, 33.3 µM, 50 µM and 66.6 µM PO4-AM3 for 18 h. The cells were sedimented by centrifugation and stained with annexinV-APC/PI and analyzed by flow cytometry. The early apoptotic cells were positive for annexinV-APC and negative for PI staining. Shown are the percentages of early apoptotic cells from 4 independent experiments as means ± SD. Data were analyzed by one-way ANOVA followed by the unpaired t test to compare cCMP-AM- and corresponding PO4-AM3-treated cells (*, p < 0.05; **, p < 0.01 and ***, p < 0.001).

Fig. 4: Apoptosis induced by cCMP-AM is caspase dependent. S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of media and were treated with 100 µM cCMP-AM and 33.3 µM PO4-AM3 for 18 h. Cells were preincubated with 20 µM ZVAD-fmk for 1h. Cells were sedimented by centrifugation and stained with annexinVAPC/PI for analyzing via flow cytometry. A, representative dot-blots from S49 wt and S49 kin- cells treated with PO4-AM3, cCMP-AM and cCMP-AM preincubated with the caspase inhibitor Z-VAD-fmk are shown. The percentage of early apoptotic cells is indicated. B, data are shown as means ± SD from five independent experiments, data were analyzed by one-way ANOVA followed by Dunnett’s test; Bonferroni’s multiple comparison test were used to compare selected columns (*, p < 0.05, **, p < 0.01 and **, p < 0.01).

Fig. 5: Autophagy is not induced by cCMP-AM in S49 wt and S49 kin- cells. S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 3 ml of media and were treated with 150 µM cCMP-AM and 50 µM PO4-AM3 for 24 h. The cells were sedimented by centrifugation. Protein expression was assessed as described in "Material and Methods”. A, representative immunoblots of LC3 A/B and tubulin protein expression are shown. B, protein expression was quantified using ImageJ software. The protein bands were normalized to the expression of tubulin and S49 wt control was calculated to 1. Shown are data as means ± SD from three independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test.

Fig. 6: Other cNMP-AMs than cCMP-AM failed to induce apoptosis in S49 wt and S49 kin- cells. 2 x 105 S49 wt and S49 kin- cells/ml were treated with 150 µM cCMP-AM and 50 µM PO4-AM3 in a volume of 5 ml for 20 h. Protein expression was assessed as described in "Material and Methods”. A, representative immunoblots of the LC3 A/B and tubulin protein expression are shown. B, shown are data as means ± SD from four independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test.

Fig. 7: Cell membrane-impermeable cNMPs have no apoptosis-inducing potential. S49 wt and S49 kin- cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of media and incubated with 150 µM cCMP, cAMP, cGMP and cUMP for 24 h (A) and 48 h (B). The cells were sedimented by centrifugation, stained with annexinV-APC/PI and analyzed by flow cytometry. Shown are data as means ± SD from four (A) and three (B) independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test.

Fig. 8: Activation of PKA pathway by PDE-resistant cAMPS-AM analogs in S49 wt cells. A, S49 wt cells were seeded at 2 x 105 cells/ml in a 12-well plate with 2 ml of media

and incubated with 150 µM cAMP-AM, 50 µM PO4-AM3, 150 µM Sp-8-Br-cAMPS-AM or Sp-cAMPS-AM for 24 h. The cells were sedimented by centrifugation, stained with annexinV-APC/PI and analyzed by flow cytometry. Shown are data as means ± SD from four experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test (***, p < 0.001). B and C, for western blotting 2 x 105 S49 wt and S49 kin- cells/ml were treated with the same reagents as in A in a volume of 2 ml for 0.5 h. Cells were sedimented, resuspended in SDS-loading buffer and lysed by sonification and separated by SDS-PAGE. B, representative immunoblots are shown. C, protein expression was quantified using ImageJ software. The protein bands were normalized to the expression of CREB and S49 wt control was calculated to 1. Data shown are as means ± SD from four independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test (*, p < 0.05).

Fig. 9: Activation of caspase 3, 7, 9 and 12 and cleavage of PARP by cCMP-AM in S49 wt and S49 kin- cells. 2 x 105 S49 wt and S49 kin- cells/ml were treated with 150 µM cCMPAM and 50 µM PO4-AM3 in a volume of 5 ml for 20 h. Protein expression was assessed as described in "Material and Methods”. A, representative immunoblots are shown. Indicated by arrows are the full length protein of caspase 3 and the cleaved form of caspase 3, both forms are detected by the caspase 3 antibody. B, protein expression was quantified using ImageJ software. The protein bands were normalized to the expression of tubulin and S49 wt control was calculated to 1. Shown are data as means ± SD from three independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test (*, p < 0.05 and ***, p < 0.001).

Fig. 10: Cytochrome c release after cCMP-AM treatment. 2.5 x 105 S49 wt and S49 kincells/ml were treated in 2 ml with 150 µM cCMP-AM, 50 µM PO4-AM3 and 0.05 µM staurosporine for 24 h. After fixation, the cells were stained with an anti-cytochrome c-FITC

antibody and analyzed by flow cytometry. A, SSC-A (side scatter-area) and the FITC signal of representative dot blots are shown. B, data as means ± SD from four independent experiments are shown. Data were analyzed by one-way ANOVA followed by Dunnett’s test (*, p < 0.05, p < 0.01 and ***, p < 0.001).

Fig. 11: No activation of caspase 8-dependent extrinsic apoptotic pathway by cCMPAM. 2.5 x 105 S49 wt and S49 kin- cells/ml were treated in 4 ml with 150 µM cCMP-AM, 50 µM PO4-AM3 and 4 µM BFA for 20 h. As positive control S49 wt and S49 kin- cells were incubated with 100 ng/ml cycloheximide and 1 ng/ml TNF. Caspase 8 activity was measured in a fluorometer by 400 nm excitation and 505 nm emission. Shown are data as means ± SD from five independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test (***, p < 0.001).

Fig. 12: Increased expression of apoptosis-related genes after incubation with cCMPAM in S49 wt cells. 5 x 105 S49 wt and S49 kin- cells were incubated in 2 ml medium with cCMP-AM (150 µM) and PO4-AM3 (50 µM) for 1 h, 2 h, 3 h and 4 h or were left untreated (c: control). The gene expressions were analyzed with real-time PCR using TaqMan probes. Expression of Actb was used as a housekeeping gene. The data were analyzed using the Ct method. Shown are data as means ± SD from three independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test (*, p < 0.05; **, p < 0.01 and ***, p < 0.001).

Fig. 13: Activation of Epac caused no reduced cell number. S49 wt and S49 kin- cells were seeded at 105 cells/ml in 200 µl and treated with 100 µM 8-pCPT-2'-O-Me-cAMP for 72 h. Cell number was determined using the trypan blue dye exclusion method. Shown are data as

means ± SD from three independent experiments. Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison tests of control cells and Epac-treated cells.

Fig. 14: No activation of p44/p42 MAPK by cCMP-AM or db-cCMP in S49 wt and S49 kin- cells. 2 x 105 S49 wt and S49 kin- cells/ml were treated with 150 µM cCMP-AM, dbcCMP, sodium butyrate and 50 µM PO4-AM3 in a volume of 2 ml for 1 h. The cells were sedimented, resuspended in SDS-loading buffer and lysed by sonification and separated by SDS-PAGE. A, representative immunoblots are shown. B, protein expression was quantified using ImageJ software. The protein bands were normalized to the expression of p44/p42 MAPK and S49 wt control was calculated to 1. Data shown are as means ± SD from four independent experiments. Data were analyzed by one-way ANOVA followed by Dunnett’s test.

Fig. 15: Apoptotic pathways activated by cCMP-AM in S49 wt and S49 kin- cells. Schematic representation of the cCMP-activated pathways: cCMP-AM is cleaved inside the cells by esterases. The resulting cCMP activates the intrinsic and the ER stress apoptotic pathways. Cytochrome c is released out of the mitochondrium, specific caspases are activated and apoptosis occurred. Not involved in cCMP-induced apoptosis were PKG, HCN2 and HCN4, Epac, and in S49 kin- cells, also PKA.

Table 1: cAMP, cGMP and cCMP concentration in S49 wt and S49 kin- cells

cNMP (pmol/mg protein) S49 wt cAMP

control

forskolin

cGMP cCMP

5.30 ± 597.4 ± 580.3* 11.88 0.27 ± 0.38 0.14 ± 0.16 0 2.93 ± 2.61

S49 kincAMP cGMP cCMP

4.02 ± 5.46 0.29 ± 0.19 0.27 ± 0.48

isoproterenol

SNP 1h

72.52 ± 76.48

6.07 ± 2.44 3.91 ± 1.92

0.13 ± 0.14 0.16 ± 0.28

0.18 ± 0.22 0.14 ±0.16 0.07 ± 0.13 0.08 ± 0.14

765.7 ± 757.6*** 113.7 ± 137.6 0.28 ± 0.25 0.26 ± 0.27 4.64 ± 6.47 0.80 ± 0.848

SNP 3h

15.0 ± 7.53 15.88 ± 10.02 0.12 ± 0.12 0.16 ±0.20 0.28 ± 0.48 0.16 ± 0.28

S49 wt and S49 kin- cells were preincubated with of IBMX (100 µM) for 10 min and stimulated with forskolin (10 µM), isoproterenol (1 µM) for 1h and SNP (100 µM) for 1h and 3h. The concentrations of cAMP, cGMP and cCMP were analyzed by HPLCMS/MS and normalized to protein concentrations [27]. Shown are means ± SD of 3 assays performed in triplicates as pmol/mg protein. Data were analyzed by one-way ANOVA followed by Dunnett’s test (*, p < 0.05 and ***, p < 0.001). .

Figure 1

number of cells [%]

150

100

S49 wt S49 kin-

50 *** ***

***

3

M

PO

4 -A

AM cC MP -

m

bu ty

rat e

MP so diu

db -cC

MP db -cA

co ntr ol

0

Figure 2

S49 kin - cells

S49 wt cells

***

100

*** apoptotic cells [%]

apoptotic cells [%]

100 80 60

*

40 20 0

***

***

cCMP-AM PO4-AM3

80

*

60 40 20 0

0

50

100

150

compound (µM)

Figure 3

200

250

0

50

100

150

compound (µM)

200

250

A S49 wt

S49 kin

p r o p i d p r o p i d

9.7 %

56.9 %

19.7 %

9.9 %

36.0 %

24.2 %

annexin

-

annexin

S49 kin-

S49 wt ***

**

40

20

0

Figure 4

***

60

apoptotic cells [%]

apoptotic cells [%]

60

B

annexin

*

40

20

0

c

ol tr on cC

P M

M -A PO

M -A 4

3

Z-

D VA

PM cC

M A

+

Z-

D VA

nt co

l ro

AM P-

M cC

M3 -A 4 O P

VA Z-

D

A P-

cC

M

M

+

VA Z-

D

Figure 5

S49 kin -

S49 wt 100

***

80

apoptotic cells [%]

apoptotic cells [%]

100

60 40 20 0

***

80 60 40 20 0

c

ol tr n o

M cC

M A P-

M M M M A A A PP-A P4 M M M PO cU cG cA 3

c

ol tr n o

M cC

M A P-

PO

S49 wt

M M M A A A PPPM M M cA cU cG

M3

S49 kin -

***

***

100

apoptotic cells [%]

apoptotic cells [%]

100

-A

4

80 60 40 20 0

80 60 40 20 0

l ro

co

Figure 6

nt

M cC

P

M -A PO

M -A

4

3

M cA

A

P-

M -A

M M cU

P

cG

M -A

M

P

nt co

l ro cC

M

AM P-

PO

-A

4

M3 cA

M

A P-

M M cU

A P-

M

PM cG

A

M

A

S49 kin -

S49 wt 10

apoptotic cells [%]

apoptotic cells [%]

10 8 6 4 2 0

8 6 4 2 0

l ro nt co

P M cC

P M cU

P M cA

l ro nt co

P M cG

P M cC

P M cU

P M cA

P M cG

P M cA

P M cG

B S49 kin-

S49 wt 15

apoptotic cells [%]

apoptotic cells [%]

15

10

5

0

Figure 8

5

0 l ro nt o c

Figure 7

10

P M cC

P M cU

P M cA

P M cG

l ro nt o c

P M cC

P M cU

Figure 9

Figure 10

100000

*** ***

E(405/505)

80000 60000 40000 20000 0

l 3  ro AM AM F FA t n P- 4TN B o c M PO + X cC H C

l 3 ro AM AM F BFA t n P- 4N o c M PO + T cC HX C

S49 kin-

S49 wt Figure 11

Bcl2 4

3

3

qRT-PCR

qRT-PCR

Bim 4

2 1

1

0

0

c 1h 2h 3h 4h

cCMP-AM

1h 2h 3h 4h

c 1h 2h 3h 4h

PO 4-AM3

cCMP-AM

S49 wt

1h 2h 3h 4h

S49 kin

cCMP-AM

PO 4 -AM3

S49 wt

-

* *** ***

*** *** ***

c 1h 2h 3h 4h 1h 2h 3h 4h c 1h 2h 3h 4h 1h 2h 3h 4h

PO 4 -AM3

Gadd45a 4

2

cCMP-AM

PO 4 -AM3

S49 kin

-

cFos

Nr4a1

20

20

* *** **

2

10

1

5

0

0 c 1h 2h 3h 4h

cCMP-AM S49 wt

Figure 12

1h 2h 3h 4h

PO 4 -AM3

c 1h 2h 3h 4h

cCMP-AM

1h 2h 3h 4h

PO 4 -AM3

S49 kin -

15

qRT-PCR

15

qRT-PCR

qRT-PCR

3

10 5 0

c 1h 2h 3h 4h

cCMP-AM

S49 wt

1h 2h 3h 4h

PO 4 -AM3

c 1h 2h 3h 4h

cCMP-AM

S49 kin

1h 2h 3h 4h

PO 4 -AM3 -

c 1h 2h 3h 4h

1h 2h 3h 4h

c 1h 2h 3h 4h

cCMP-AM

PO 4 -AM3

cCMP-AM

S49 wt

1h 2h 3h 4h

S49 kin

PO 4-AM3 -

S49 wt control S49 wt 8-pCPT-2'-O-M e-cAM P

cell number x 105

25

S49 kin - control S49 kin - 8-pCPT-2'-O-M e-cAM P

20 15 10 5

time [h] Figure 13

72

48

24

0

Figure 14

Figure 15