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Cyclic 3′,5′-Nucleotide Phosphodiesterases: Potential Targets for Anticancer Therapy Doris Marko, Gudrun Pahlke, Karl-Heinz Merz, and Gerhard Eisenbrand* Department of Chemistry, Division of Food Chemistry and Environmental Toxicology, University of Kaiserslautern, Erwin-Schroedinger-Strasse 52, D-67663 Kaiserslautern, Germany Received April 17, 2000
3′,5′-Cyclic Nucleotide Phosphodiesterases. The second messenger cAMP is involved in a multitude of cellular processes, including growth and differentiation. Intracellular cAMP is regulated by adenylate cyclases and cAMP phosphodiesterases (PDEs),1 the latter being responsible for cAMP hydrolysis and thus termination of cAMP signaling (Figure 1). Many tumor cells exhibit significantly decreased cAMP levels as a consequence of overexpression of cyclic nucleotide phosphodiesterases. These enzymes represent a superfamily comprising 10 enzyme families characterized by differences in sequence homology, substrate specificity, Km value, and sensitivity toward enzyme selective modulators (Table 1) (1, 2). We have analyzed the NCI panel of 60 human tumor cell lines for PDE enzyme expression by typing the cytosolic fraction with the appropriate inhibitors or activators of cAMP hydrolysis. Until now, only a few primary cell models have been assayed, namely, human and murine primary keratinocytes (3), human chorion villi cells, and Syrian hamster embryo cells. These exhibited cAMP PDE activities of 50 pmol min-1 (mg of protein)-1. The highest activities with mean values of 120-330 pmol min-1 (mg of protein)-1 were detected in cell lines originating from the central nervous system (CNS), lung, breast, and melanomas. In 41 of the 60 lines, cAMP-specific PDE4 represented the highest cAMP-hydrolyzing activity in the cytosol. PDE1 in Tumor Cells. In five of the six CNS lines, PDE1 was the predominant enzyme family, whereas PDE4 activity was minor. These cell lines were comprised of highly malignant dedifferentiated glioblastomas. Our RT-PCR results indicate that PDE1C represents the predominant PDE enzyme in these lines. In contrast, the levels of signals for PDE4 transcripts were low, which is in good agreement with our PDE activity data. PDE1C has a high affinity for cAMP, similar to PDE4. We conclude, therefore, that in malignant glioblastoma cells PDE1C plays the key role for cAMP homeostasis whereas in epithelial tumor cells PDE4 is the main player. This can be highly relevant for antitumor therapy, provided that PDE1 is demonstrated to have promise as a therapeutic target similar to that shown below for PDE4. PDE4 in Tumor Cells. Because lung tumor cells were found to represent models with high PDE activity, we selected this tumor cell type for our further studies. We 1
Abbreviations: PDE, phosphodiesterase; PKA, protein kinase A; CRE, cAMP-responsive element; MAP, mitogen-activated protein; CNS, central nervous system; DC-TA-46, PDE4 inhibitor 7-(benzylamino)6-chloro-2-piperazino-4-pyrrolidinopteridine; SRB, sulforhodamine B.
first examined whether there are differences in PDE expression as a result of tumor cells being held in longterm culture as compared with solid human tumor xenografts exclusively passaged in animals (4). As one example, we found that in the solid large cell lung carcinoma LXFL529, both cytosolic and particulate PDE activities were much higher than in the respective cell lines. Furthermore, in both the tumor tissue and cell lines, rolipram-sensitive PDE4 was found to represent the highest activity (Table 2). Human PDE4 is encoded by four different genes, PDE4A-D. As a result of alternative splicing and the use of different transcription starting points, more than 14 different PDE4 proteins have been identified (1). Therefore, we investigated whether expression patterns of PDE4 enzymes differ in the cell line compared to the respective human solid xenograft tumor. Via RT-PCR using subtype- and isoform-specific primers, no differences were found. Semiquantitative RT-PCR (Real Time TaqMan PCR, ABI) showed PDE4D to be the subtype predominantly expressed in both the cell line and solid tumor (Figure 2). In contrast to the marked differences in PDE activities between the tumor tissue and cell line, no differences in the level of transcripts for the different PDE4 subtypes were detected. Obviously, posttranslational processes such as phosphorylation (5) play a pivotal role at the PDE4 protein level. Using murine keratinocytes, we examined whether changes in PDE activity are associated with an increasing level of the malignant phenotype. We determined cAMP levels and PDE expression levels in normal primary keratinocytes and cells corresponding to different stages of epidermal tumor development in mouse skin. No significant differences in soluble PDE activity and intracellular cAMP were found in two benign papilloma cell lines as compared with primary keratinocytes. In contrast, a highly malignant spindle cell carcinoma line (CarB cells) displayed significantly increased PDE activity, concomitant with a very low cAMP level. Stable transfection of immortal human keratinocytes (HaCaT) with the c-Ha-ras oncogene (6) was found to be associated with increased PDE activity and a significantly reduced intracellular cAMP level. The transfected clones exhibited marked changes in the expression pattern of PDE enzymes. The expression of enzymes of the PDE1 family was shut off, whereas distinct PDE4 subtypes were upregulated.2 Cellular Effects of PDE4 Inhibition. In a broad panel of tumor cell lines of human and animal origin, 2
Manuscript in preparation.
10.1021/tx000090l CCC: $19.00 © 2000 American Chemical Society Published on Web 09/29/2000
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Figure 1. cAMP pathway. Rec, receptor; G, G-protein; AC, adenylate cyclase; R, regulatory subunit of PKA; C, catalytic subunit of PKA. Table 1. PDE Isoenzyme Families enzyme family
properties
inhibitory compounds
PDE1 PDE2 PDE3 PDE4 PDE5 PDE6 PDE7 PDE8 PDE9 PDE10
Ca2+/calmodulin-stimulated, hydrolyzes cAMP and cGMP cGMP-stimulated cGMP-inhibited cAMP-specific, rolipram-sensitive cGMP-specific, zaprinast-sensitive PDE of the photoreceptor, cGMP-specific cAMP-specific, rolipram-insensitive cAMP-specific, IBMX-insensitive cGMP-specific, IBMX-insensitive cAMP-inhibited, hydrolyzes cGMP and cAMP, IBMX-sensitive
vinpocetine, 8-MeoM-IBMX EHNA milrinone, cilostamide rolipram, Ro-20-1724 zaprinast, dipyridamole zaprinast, dipyridamole none known dipyridamole SCH 51866, zaprinast zaprinast, dipyridamole
Table 2. Phosphodiesterase Activity of Solid Tumor Tissue of the Human Large Cell Lung Tumor Xenograft LXFL529 Compared with the Respective Cell Line LXFL529La tumor tissueb
cytosol particulate nonsolublee
cell linec
PDE activity [pmol min-1 (mg of protein)-1]
rolipram-sensitive PDE activity (%)d
PDE activity [pmol min-1 (mg of protein)-1]
rolipram-sensitive PDE activity (%)
1088.0 ( 135.1 361.7 ( 114.0 6.5 ( 1.3
77.5 ( 6.1 67.7 ( 6.6 83.7 ( 2.7
426.2 ( 71.0 172.5 ( 35.2 ndf
88.3 ( 6.4 79.7 ( 4.6 ndf
a PDE activity was determined as described previously (3, 8) using 1 µM cAMP as the substrate. The assays were performed in triplicate. The tumor tissue of three animals was analyzed independently. Values are given as means ( the SD. c For each experiment, three Petri dishes were treated in parallel. The whole experiment was repeated three times. Values are given as means ( the SD. d PDE activity was determined in the presence of 10 µM rolipram. e Nonsoluble in buffer containing 0.5% Triton X-100. f Not detectable.
b
treatment with the potent PDE4 inhibitor 7-(benzylamino)-6-chloro-2-piperazino-4-pyrrolidinopteridine (DCTA-46) results in dose-dependent growth inhibition in the low micromolar range (3, 7, 8). Treatment of the highly malignant murine spindle cell carcinoma cell line CarB for 24 h induces an arrest in the G1/G0 phase of the cell cycle. At concentrations of g4 µM, induction of apoptosis is observed. Similar results have been obtained from
treatment of a set of human tumor cell lines. In all human tumor cell lines tested so far [large cell lung carcinoma (LXFL529L and LXFL1072L), small cell lung carcinoma (LCLS DMS237, LCLS DMS114, and LXFS650L), lung adenocarcinoma (LXFA526L), and mammary carcinoma (MCF-7)], treatment with DC-TA-46 induced G1/G0 arrest as shown representatively for the large cell lung carcinoma cell line LXFL529L (Figure 3).
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Figure 2. Semiquantification of PDE4 transcripts in LXFL529 tumor tissue and the corresponding cell line. Polyadenylated RNA was reverse transcribed following standard protocols. Real Time TaqMan PCR (ABI PRISM Sequence Detection System, Perkin-Elmer) analysis was carried out using PDE4 subtypespecific primers and specific fluorescence-labeled probes. For normalization, β-actin was used. The data generated from samples of solid tumor tissue represent the means of two different tumor passages. From each tumor sample, two independent experiments were performed, each carried out in triplicate. Values are presented as means ( the SD. No transcripts for PDE4C were detected.
Figure 3. LXFL529L cells were treated for 24 h with DC-TA46. After DAPI staining, the cell cycle distribution was determined by flow cytometry as described previously (3). The data represent means ( the SD of three independent experiments, each carried out in duplicate.
After a 24 h treatment with DC-TA-46 at concentrations of g6 µM, “sub-G1” cells were detected by flow cytometry
Marko et al.
Figure 4. Two-dimensional chart of the flow cytometry of LXFL529L cells after double staining with DAPI and sulforhodamine 101 as described previously (3). Prior to staining, LXFL529L cells were treated for 24 h with DC-TA-46. The charts represent the results from a typical experiment of three independent experiments.
(Figures 3 and 4). In morphological studies, these cells exhibited the typical features of apoptotic cells with reduced cell volume and highly condensed or fragmented chromatin but a cellular membrane that was still intact (data not shown). Effects on Downstream Signaling Cascades. In the murine spindle cell carcinoma cell line CarB, treatment with DC-TA-46 at growth-inhibitory concentrations inhibits the intracellular PDE activity, resulting in a rapid and long-lasting increase in the cAMP level (3). In LXFL529L cells, we found a time- and dose-dependent increase in the cellular cAMP level as a result of DCTA-46 treatment, accompanied by activation of protein kinase A (PKA). After activation, the catalytic subunits of PKA can translocate into the nucleus, phosphorylating and thus activating the respective transcription factors (Figure 1). In addition, cytosolic targets of PKA might be relevant, such as Raf-1, a serine-threonine kinase of the mitogen-activated protein kinase (MAP) cascade. We therefore searched for potential targets of PKA that might be involved in the observed cellular effects such as cell cycle arrest and apoptosis. Nuclear extracts of LXFL529L cells treated with DCTA-46 were incubated with an oligonucleotide containing the cAMP-responsive element (CRE) consensus sequence TGACGTCA. We found a dose- and time-dependent increased level of binding of nuclear proteins to the CRE sequence, which indicates that treatment with DC-TA46 triggers gene expression via the PKA pathway.2 Furthermore, we investigated the effect of DC-TA-46 on the MAP kinase cascade, a signaling pathway playing a central role in cellular proliferation. One element of this signaling cascade, Raf-1, has been reported to be inactivated by PKA phosphorylation (9, 10). One of the nuclear substrates of MAP kinase is the transcription factor Elk-1. We assayed the activity of the MAP kinase cascade by assessing the phosphorylation of Elk-1 in a reporter gene assay and found that at growth-inhibitory
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Figure 6. Structural elements of DC-TA-46.
Figure 5. Reporter gene assay for Elk-1 phosphorylation (PathDetect, Stratagene, La Jolla, CA). A431 cells (human vulva carcinoma cell line) were cotransfected with a plasmid encoding a fusion protein of Elk-1 and the DNA-binding domain of GAL-4 as well as a plasmid containing the upstream activating sequence of GAL-4 and luciferase as a reporter gene. The transfected cells were treated for 5 h with the respective compounds. Tyrphostin AG1478, a known inhibitor of the EGF receptor, was used as a positive control. The data represent means ( the SD of three independent experiments, each carried out in duplicate.
concentrations DC-TA-46 inhibited phosphorylation of Elk-1 (Figure 5).2 These results indicate that PKA-mediated phosphorylation not only directly triggers the activity of transcription factors as described above but also targets cytosolic signaling elements such as Raf-1. Notably, we found that 2-(aminoethylamino)-7-(benzylamino)-6-chloro-4-pyrrolidinopteridine (7k), a derivative with a growth-inhibitory potential equal to that of DC-TA-46 but lacking PDEinhibitory properties (8), induced no changes in nuclear factor binding to the CRE consensus sequence. Moreover, at growth-inhibitory concentrations, Elk-1 phosphorylation was very poorly inhibited. Thus, these DC-TA-46induced cellular effects correlate well with the PDE4inhibitory properties. Substituted Pteridines as PDE4 Inhibitors. To unravel the structural elements of DC-TA-46 that are relevant to interaction with the target enzyme, we systematically modified the molecule. We tested the impact of these modifications on enzyme activity by evaluating the ability of derivatives to inhibit cAMP hydrolysis by PDE4 purified from solid LXFL529 tumors. In tumor cells, PDE inhibition was assayed in cytosolic and particulate fractions. Growth inhibition of tumor cells was assessed by the sulforhodamine B (SRB) assay. We focused on potential pharmacophores (Figure 6) and examined the relevance of (1) the size of the substituents in positions 4 and 7, (2) the presence of a basic center in the substituent at position 2, as exemplified by N4′ of piperazine in DC-TA-46, (3) the pteridine ring nitrogens, and (4) the chloro substituent in position 6. For structure-activity studies, we developed a synthetic approach enabling directed successive nucleophilic aromatic substitution of the chlorines in 2,4,6,7-tetra-
chloropteridine (8). The main results of these structureactivity studies of substituted pteridines are given below. Replacement of pyrimidine nitrogen atoms in the pteridine system with C atoms yielded chinoxalines that turned out to be rather weak inhibitors of PDE (IC50 > 10 µM) or growth (IC50 > 20 µM). Similarly, poor activities were displayed by some substituted chromones (data not shown). Within the substituted pteridine series, DC-TA-46 was found to be an extremely effective inhibitor of isolated PDE4 (IC50 ) 16 nM), whereas its potency to inhibit PDE4 in tumor cells was about 2 orders of magnitude lower. The corresponding intracellular IC50 was about the same as that observed for cell growth inhibition. This discrepancy appears to reflect the property of the compound to accumulate to a large extent in the membranes of the endoplasmatic reticulum. As a consequence, only minute proportions are present in the cytosolic compartment, where about 80% of the target enzyme, PDE4, is localized (11). Permutation of the substituents at positions 4 and 7 of the pteridine system revealed that both substituents need a certain minimal size. For instance, replacement with a dimethylamino group of the substituent in either position resulted in a marked reduction of PDE4 and tumor cell growth-inhibitory properties. Replacement of chlorine in position 6 of DC-TA-46 with a methyl group had no strong influence, but replacement by hydrogen reduced the level of PDE4 inhibition 20-fold. However, because the level of growth inhibition was reduced by a factor of only 3, it appears that removal of the chlorine in position 6 leads to a new compound that addresses other cellular targets. Substituent modifications at position 2 of the pteridine system had the strongest impact. In particular, removal of the basic center in position 4′ of the piperazine ring resulted in a marked reduction in both PDE4- and growth-inhibitory properties. Compound 7k carries an aminoethylamino group at position 2 of the pteridine system. This modification has a very strong impact on the inhibition of isolated PDE4, decreasing the activity by more than 100-fold. Moreover, in tumor cells, no PDE inhibition was observed after treatment. This is in agreement with results shown above, which demonstrated that compound 7k does not affect downstream targets of PKA. However, because the compound exhibits about the same level of cell growth inhibition as DC-TA46, it can be concluded that 7k represents a new entity that addresses other cellular targets. Perspectives. In conclusion, the results summarized here support our view that cyclic nucleotide phosphodiesterases, especially, PDE1 and PDE4, represent cellular targets of promise for antitumor treatment.
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Acknowledgment. These studies were supported by Grants Ei 172/5-2 and Ei 172/7-1 from the Deutsche Forschungsgemeinschaft (DFG).
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Marko et al. (6) Boukamp, P., Stanbridge, E. J., Yin Foo, D., Cerutti, P. A., and Fusenig, N. E. (1990) c-Ha-ras oncogene expression in immortalized human keratinocytes (HaCaT) alters growth potential in vivo but lacks correlation with malignancy. Cancer Res. 50, 28402847. (7) Drees, M., Zimmermann, R., and Eisenbrand, G. (1993) 3′,5′-Cyclic nucleotide phosphodiesterase in tumor cells as potential target for tumor growth inhibition. Cancer Res. 53, 3058-3061. (8) Merz, K.-H., Marko, D., Regiert, T., Reiss, G., Frank, W., and Eisenbrand, G. (1998) Synthesis of 7-benzylamino-6-chloro-2piperazino-4-pyrrolidinopteridine and novel derivatives free of positional isomers. Potent inhibitors of cAMP-specific phosphodiesterase and of malignant tumor cell growth. J. Med. Chem. 41, 4733-4743. (9) Cook, S. J., and McCormick, F. (1993) Inhibition by cAMP of rasdependent activation of Raf. Science 262, 1069-1072. (10) Wu, J., Dent, P., Jelinek, T., Wolfman, A., Weber, M., and Sturgill, T. W. (1993) Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3′,5′-monophosphate. Science 262, 1065-1069. (11) Marko, D., Merz, K.-H., Tarasova, N., and Eisenbrand, G. (1997) Subcellular localization of a pteridine-based inhibitor of cyclic adenosine monophosphate specific phosphodiesterase. J. Cancer Res. Clin. Oncol. 123 (Suppl. 1), 11.
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