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ANTICANCER RESEARCH 29: 1119-1122 (2009)

Characterization of 1 in Human Malignant Melanoma Lines

KASUMI SHIMIZU, TAKU MURATA, YOSHIHIRO WATANABE, CHU SATO, HIROSHI MORITA and TOSHIRO TAGAWA

Department of Oral and Maxillofacial Surgery, Division of Reparative and Regenerative Medicine, Institute of Medical Science, Mie University Graduate School of Medicine, Tsu, Mie 514-8507, Japan

Abstract. Background: Differentiation-inducing factor 1 in either or the original organism until we revealed [DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl) that cyclic phosphodiesterase 1 (PDE1) is a hexan-1-one] from Dictyostelium discoideum exhibits pharmacological and specific target of DIF-1 (8). antiproliferative activity in mammalian cells. We have By catalyzing the hydrolysis of cyclic , PDEs previously shown that phosphodiesterase 1 (PDE1) is a regulate the intracellular concentrations and effects of these pharmacological target of DIF-1, but there are no reports of secondary messengers. Eleven PDE families (PDE1- PDE1 in human malignant melanoma cells. Therefore, we 11) have been identified, and some PDE isoforms are characterized PDE1 in human malignant melanoma MAA selectively expressed in various tissues and cell types (9). cells. Materials and Methods: PDE1 mRNA expression was PDE1s hydrolyze cAMP and cGMP and their activity is investigated in MAA cells. The full open reading frames for increased by binding to Ca2+ and , a feature human PDE1C1 and PDE1C3 were cloned. Cell growth was unique to the PDE1 family of PDEs (9, 10). Three PDE1 determined by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- (PDE1A, PDE1B, and PDE1C) have been identified, carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, and several isoforms are generated from these genes inner salt] assay. Results: PDE1C mRNA expression was by alternative splicing. The PDE1A and PDE1B isoforms detected in MAA cells. The nucleotide sequence of PDE1C1 have significantly higher affinities for cGMP than for cAMP, was identical to that of human PDE1C1, previously whereas PDE1C isoforms have similar affinities for both published. At nucleotide 2246 in PDE1C3, A was replaced substrates (9, 10). PDE1s have a broad distribution, but are by G, but this did not change the encoded . Cell particularly common in testis, heart and neural system tissues growth was inhibited by the PDE1 inhibitor vinpocetin. (9, 10). However, there are no data on the expression and Conclusion: PDE1C mRNA is expressed and may play an function of PDE1s in human malignant melanoma cells; important role in human malignant melanoma MAA cells. therefore, we have characterized PDE1 in human malignant melanoma MAA cells. Differentiation-inducing factor 1 [DIF-1; 1-(3,5-dichloro-2,6- dihydroxy-4-methoxyphenyl)hexan-1-one] is a signal Materials and Methods that induces stalk-cell differentiation in Dictyostelium discoideum (1). Although antiproliferative Cell culture. Human malignant melanoma MAA cells (11) were activities have been demonstrated for DIF-1, which can also maintained in RPMI-1640 containing 10% fetal bovine serum occasionally induce cell differentiation in mammalian cells (Invitrogen Corp., Carlsbad, CA, USA) at 37˚C in a humidified 5% CO atmosphere. (2-7), the target molecule(s) of DIF-1 had not been identified 2 Preparation of cell extracts. MAA cells were seeded at 1×106 cells/ 25 cm2 flask. After 3 days, the cells were washed twice with phosphate buffered saline (PBS). The Cells were homogenized in ice- Correspondence to: Kasumi Shimizu, Department of Oral and cold homogenization buffer (1 ml; 100 mM TES pH 7.4, 10 μg/ml Maxillofacial Surgery, Division of Reparative and Regenerative each of pepstatin, leupeptin and aprotinin, 1 mM benzamidine, Medicine, Institute of Medical Science, Mie University Graduate School 0.5 mM pefabloc, 1 mM EDTA, 0.1 mM EGTA, 5 mM MgSO4 of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507, Japan. Tel: +81 and 10% glycerol). 592321111, Fax: +81 592315207, e-mail: [email protected] u.ac.jp cAMP PDE assay. cAMP PDE activity was then assayed as described elsewhere (8). Briefly, samples were incubated at 30˚C Key Words: Phosphodiesterase, phosphodiesterase 1, malignant for 10 min in a total volume of 0.3 ml containing 50 mM Hepes pH 3 melanoma. 7.5, 0.1 mM EGTA, 8.3 mM MgCl2, and 0.5μM [ H] cAMP

0250-7005/2009 $2.00+.40 1119 ANTICANCER RESEARCH 29: 1119-1122 (2009)

Table Ⅰ. Primer sequence for PDE1s.

PDE1A 5’-CAC TGG CTC ACT GAA CTG GA-3’ 5’-GGA CAT GGT TTT GGC TCT GT-3’ PDE1B 5’-GCC GAT GTT ACC CAG ACA GT-3’ 5’-TTG GGC TTG TCA ATC CTC TC-3’ PDE1C 5’-GAC GGA GCT GGA GAT CTT TG-3’ 5’-TGC TGC AGA GCA GTC TTC AT-3’ PDE1C1 5’-CAA TAC CTG CAA CCG GAA CAG-3’ 5’-CAG TCT TGG ACC TCA GAA GG-3’ PDE1C3 5’-GAA CAC GGG CAA ACA AAA GT-3’ 5’-GCT TGA TGA CTG GCA ACG TAG-3’ PDE1C1 (full) 5’-GAA AAT GGA TCC GGT TCT GTG-3’ 5’-AAA CCA ACC CCC AAA GAT TC-3’ PDE1C3 (full) 5’-GTG CAG GCT GGG ATA TTT GT-3’ 5’-CAA GGG TCT TGG AGG TGT GTC-3’

(18,000 cpm) with or without 0.2 mM CaCl2, 10 mU calmodulin and PDE1 inhibitor (vinpocetin). Figure 1. PDE1 activity in human malignant melanoma MAA cells. Homogenates were prepared and assayed as described in the Materials and Methods section. Data are shown as mean±S.D. of three different RT-PCR. The cells were seeded at 1×106 cells/25-cm2 flask. After experiments. Ca, ; Calmo, Calmodulin; Vinpo, Vinpocetin. 3 days, total RNA was isolated using the RNeasy® Mini Kit (Qiagen, Hilden, Germany). Human heart total RNA was purchased from Ambion (Applied Biosystems, Foster City, CA, USA). First-strand cDNA was generated from total RNA using TaqMan® Reverse Transcription Reagents (Applied Biosystems). Results PCR was performed with specific primer pairs for PDE1A, PDE1B, PDE1C, PDE1C1, PDE1C3, PDE1C1 (full) and PDE1C3 PDE1 activity in MAA cells. PDE1 activity is known to be (full) (Table Ⅰ). PCR amplification was carried out using stimulated by Ca2+ and calmodulin and inhibited by the HotStarTaq® Master Mix Kit (Qiagen) and 0.5 μM sense and antisense primers. HotStarTaq™ DNA Polymerase was activated PDE1 inhibitor vinpocetin (10). As shown in Figure 1, PDE by incubation at 95˚C for 15 min followed by 35 cycles of activity in the MAA cells was stimulated approximately two- amplification (94˚C for 1 min, 62˚C for 1 min and 72˚C for 1 fold by Ca2+ with calmodulin. This increase was suppressed min). Products were subjected to electrophoresis on 2% agarose by the PDE1 inhibitor vinpocetin. gels and visualized by SYBR® Green I staining (Molecular Probes, Inc., Eugene, OR, USA). PDE1 expression in MAA cells. Using specific primers (Table Ⅰ) based on published cDNA sequences, Sequencing of PDE1Cs. First-strand cDNA from MAA cells was used. PCR amplification was carried out using Easy-A® High- PDE1C mRNA, but not PDE1A or PDE1B mRNA, was Fidelity PCR Cloning (Agilent Technologies, Santa Clara, detected by RT-PCR (Figure 2). No fragments were amplified CA, USA) and 0.2 μM sense and antisense primers [PDE1C1 (full) in the absence of reverse transcriptase or RNA. These findings and PDE1C3 (full)]. The enzyme was activated by incubation at are consistent with the PDE activity. 95˚C for 2 min followed by 35 cycles of amplification (95˚C for 1 min, 58˚C for 1 min and 72˚C for 1 min). Products were subjected Sequencing of PDE1Cs in MAA cells. As two human to electrophoresis on 1% agarose gels and visualized by SYBR® PDE1C variants, PDE1C1 and PDE1C3, have been Green I staining (Molecular Probes). The purified PCR product was introduced into the plasmid pCR II-TOPO vector (Invitrogen) and reported (12), we investigated the expression of PDE1C1 verified by DNA sequencing. and PDE1C3 in MAA cells using specific oligonucleotide primers (PDE1C1 and PDE1C3): both variants were Cell growth experiment. The cells were plated at 5×102 cells/well detected (Figure 3). We then examined the nucleotide in a 96-well plate and allowed to adhere for 24 h. The cells were sequences of PDE1C1 and PDE1C3 from MAA cells. The cultured in the presence or absence of different concentrations of nucleotide sequence of PDE1C1 was identical to that of vinpocetin for 5 days. MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- the previously published human PDE1C1. In PDE1C3, A carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assays were performed using the CellTiter 96® Aqueous One was replaced with G at nucleotide 2246 (12), but this did Solution Cell Proliferation Assay (Promega, Madison, WI, USA), not change the encoded amino acid, leucine (data not and the cell numbers were calculated. shown).

1120 Shimizu et al: PDE1 in Human Malignant Melanoma

Figure 2. Expression of PDE1 mRNA in human malignant melanoma MAA cells. Total RNA was extracted as described in the Materials and Methods section. cDNA was generated from 1 μg total RNA and amplified by PCR, using oligonucleotide primer sets based on sequences from PDE1A(1A), PDE1B(1B) and PDE1C(1C). The products were separated on agarose gels and photographed after staining by SYBR® Green I staining. H, human heart; M, MAA cells; Mr, molecular markers.

Figure 4. Effect of the PDE1 inhibitor vinpocetin on human malignant melanoma MAA cells. Cells were plated in 96-well plates and cultured with different concentrations of vinpocetin. The cells were counted as described in the Materials and Methods section. Data are shown as mean±S.D. of three different experiments.

activity was inhibited by the PDE1 inhibitor vinpocetin, indicating the presence of PDE1 . Splicing variants of PDE1C in human malignant melanoma Figure 3. PDE1 mRNAs are expressed in some malignant tumor MAA cells. Total RNA was extracted as described in the Materials and Methods section. cDNA was generated from 1 μg total RNA and cells: PDE1A and PDE1B in human retinoblastoma Y79 cells amplified by PCR, using oligonucleotide primer sets based on sequences (14); PDE1A and PDE1C in human osteosarcoma MG-63 from PDE1C1 and PDE1C3. The products were separated on agarose cells (15); PDE1B in lymphoblastoid B-cell RPMI-8392 gels and photographed after staining by SYBR® Green I staining. 1C1, cells (16); and PDE1C in human glioblastoma SNB75 and PDE1C1; 1C3, PDE1C3; M, molecular markers. SF295 cells (17). However, it was not clear whether PDE1 mRNA was expressed in human malignant melanoma cells. In this study, we found that PDE1C mRNA, but not (or at much lower concentration) PDE1A or PDE1B mRNA, was Cell growth experiment. As there have been no previous expressed in human malignant melanoma MAA cells (Figure reports of PDE1 function in malignant melanoma cells, we 2). These data are consistent with the observed PDE1 examined the effect of the PDE1 inhibitor vinpocetin on the activity. MAA cells and found it to inhibit cell growth in a dose- There are no reported mutants of PDE1C in malignant dependent manner (Figure 4). tumor cells. In MAA cells, we found that A was replaced by G at nucleotide 2246 in PDE1C3 (12). If siRNAs can be Discussion made for this point, they would specifically inhibit the function of PDE1C3 in MAA cells without influencing The PDE1 family was one of the first PDE families to be PDE1C3 in normal cells. discovered and is well characterized (10). Although PDE1 In mouse insulinoma βTC3 cells, PDE1C constitutes a activity was isolated from the supernatant fractions of B16 glucose-dependent feedback mechanism for the control of murine melanoma cells by Mono Q HR5/5 ion-exchange secretion (18). Furthermore, it has been reported that column (13), there was no report of PDE1 expression in the induction of PDE1C promoted the proliferation of arterial human malignant melanoma cells. As shown in Figure 1, cells (SMCs) and that PDE1C inhibitors PDE activity was stimulated by calcium and calmodulin in might target proliferating SMCs in lesions of extracts of MAA cells, and the calmodulin-stimulated or restenosis (19). However, the functions of PDE1C in

1121 ANTICANCER RESEARCH 29: 1119-1122 (2009) malignant melanoma cells are not known. As the PDE1 9 Francis SH, Turko IV and Corbin JD: inhibitor vinpocetin inhibited proliferation of the MAA cells, : relating structure and function. Prog Nucleic PDE1C might regulate the proliferation of MAA cells, but Acid Res Mol Biol 65: 1-52, 2001. 10 Bender AT: Calmodulin-stimulated cyclic nucleotide phospho- further investigations will be necessary to determine its diesterases. In: Cyclic Nucleotide Phosphodiesterases in Health functions. and Disease. Beavo JA, Francis SH and Houslay MD (eds.). In conclusion, these data suggest that PDE1C is Boca Raton, CRC Press, pp. 35-59, 2007. transcripted in human malignant melanoma MAA cells, and 11 Kamei T, Inui M, Nakamura S, Okumura K, Goto A and Tagawa might have an important role in the function of these cells. T: Interferon-γ and anti-Fas antibody-induced apoptosis in human melanoma cell lines and its relationship to bcl-2 cleavage Acknowledgements and bak expression. Melanoma Res 13: 153-159, 2003. 12 Loughney K, Martins TJ, Harris EA, Sadhu K, Hicks JB, Sonnenburg WK, Beavo JA and Ferguson K: Isolation and This work was supported by a Grant-in-Aid for Young Scientists characterization of cDNAs corresponding to two human calcium, (B) and a Grant-in-Aid for Scientific Research (B). calmodulin-regulated, 3’,5’-cyclic nucleotide phosphodiesterases. J Biol Chem 271: 796-806, 1996. References 13 Drees M, Zimmermann R and Eisenbrand G: 3’,5’-Cyclic nucleotide phosphodiesterase in tumor cells as potential target 1 Morris HR, Taylor GW, Masento MS, Jermyn KA and Kay RR: for tumor growth inhibition. Cancer Res 53: 3058-3061, 1993. Chemical structure of the morphogen differentiation inducing 14 White JB, Thompson WJ and Pittler SJ: Characterization of 3’,5’ factor from Dictyostelium discoideum. Nature 328: 811-814, cyclic nucleotide phosphodiesterase activity in Y79 retinoblastoma 1987. cells: absence of functional PDE6. Mol Vis 10: 738-749, 2004. 2 Kubohara Y, Saito Y and Tatemoto K: Differentiation-inducing 15 Ahlstrom M, Pekkinen M, Huttunen M, Lamberg-Allardt C: factor of D. discoideum raises intracellular calcium concentration Dexamethasone down-regulates cAMP-phosphodiesterase in and suppresses cell growth in pancreatic AR42J cells. FEBS human osteosarcoma cells. Biochem Pharmacol 69: 267-275, Lett 359: 119-122, 1995. 2005. 3 Kubohara Y: DIF-1, putative morphogen of D. discoideum, 16 Jiang X, Li J, Paskind M and Epstein PM: Inhibition of suppresses cell growth and promotes retinoic acid-induced cell calmodulin-dependent phosphodiesterase induces apoptosis in differentiation in HL-60. Biochem Biophys Res Commu 236: human leukemic cells. Proc Natl Acad Sci USA 93: 11236- 418-422, 1997. 11241, 1996. 4 Kubohara Y: Effects of differentiation-inducing factors of 17 Vetter S, Pahlke G, Deitmer JW and Eisenbrand G: Differential Dictyostelium discoideum on human leukemia K562 cells: DIF- phosphodiesterase expression and cytosolic Ca2+ in human CNS 3 is the most potent anti-leukemic agent. Eur J Pharmacol 381: tumour cells and in non-malignant and malignant cells of rat 57-62, 1999. origin. J Neurochem 93: 321-329, 2005. 5 Kubohara Y and Hosaka K: The putative morphogen, DIF-1, of 18 Han P, Werber J, Surana M, Fleischer N and Michaeli T: The Dictyostelium discoideum activates Akt/PKB in human leukemia calcium/calmodulin-dependent phosphodiesterase PDE1C down- K562 cells. Biochem Biophys Res Commun 263: 790-796, 1999. regulates glucose-induced insulin secretion. J Biol Chem 274: 6 Miwa Y, Sasaguri T, Kosaka C, Tabe Y, Ishida A, Abumiya T and 22337-22344, 1999. Kubohara Y: Differentiation-inducing factor-1, a morphogen of 19 Rybalkin SD, Rybalkina I, Beavo JA and Bornfeldt KE: Cyclic Dictyostelium, induces G1 arrest and differentiation of vascular nucleotide phosphodiesterase 1C promotes human arterial smooth muscle cells. Circ Res 86: 68-75, 2000. smooth muscle cell proliferation. Circ Res 90: 151-157, 2008. 7 Kanai M, Konda Y, Nakajima T, Izumi Y, Kanda N, Nanakin A, Kubohara Y and Chiba T: Differentiation-inducing factor-1 (DIF-1) inhibits STAT3 activity involved in gastric cancer cell proliferation via MEK-ERK dependent pathway. Oncogene 22: 548-554, 2003. 8 Shimizu K, Murata T, Tagawa T, Takahashi K, Ishikawa R, Abe Y, Hosaka K and Kubohara Y: Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) is a pharmacological Received August 24, 2008 target of differentiation-inducing factor-1, an antitumor agent Revised December 29, 2008 isolated from Dictyostelium. Cancer Res 64: 2568-2571, 2004. Accepted February 2, 2009

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