By immunocytochemistry of PDE2A, it was suggested that PDE2A localized in cytoplasm. cAMP analogue, did not. Invasion, but not growth, was stimulated by A-kinase anchor protein (AKAP) St-Ht31 inhibitory peptide. Based on these results, PDE2 appears to play an important role in growth and invasion of the human malignant melanoma PMP cell collection. Selectively suppressing PDE2 might possibly inhibit growth and invasion of other malignant tumor cell lines. value of less than 0.05. 3. Results 3.1. Effects of 8-bromo-cAMP and 8-bromo-cGMP on cell growth and invasion 8-bromo-cAMP (8-Br-cAMP) suppressed cell growth and cell invasion in a dose-dependent manner (Fig. 1A and B). However, 8-bromo-cGMP (8-Br-cGMP) experienced no significant effect on cell growth or cell invasion (Fig. 1C and D). Open in a separate window Fig. 1 Effects of 8-Br-cAMP or 8-Br-cGMP on cell growth and invasion. Cell growth was measured using the MTS assay. Cells were cultured in the absence or presence of 8-Br-cAMP (0.1 to 1 1 mM) or 8-Br-cGMP (0.1 to 1 1 mM) for 5 days. Cell invasion was examined by Matrigel invasion assays. Cells were transferred to 8 m pore Matrigel pre-coated inserts, and 8-Br-cAMP (0.1 to 1 1 mM) or 8-Br-cGMP (0.1 t 1 mM) was added. After a 16 h incubation, invaded cells were stained with May-Grnwald-Giemsa stain and TC-E 5002 counted. Data in graphs are means of three impartial experiments, each performed in duplicate. (A) Effect of 8-Br-cAMP on cell growth. (B) Effect of 8-Br-cAMP on cell invasion. (C) Effect of 8-Br-cGMP on cell growth. (D) Effect of 8-Br-cGMP on cell invasion. The error bars represent means SD, = 3. The treatments that differ significantly from control are noted (*, < 0.01). 3.2. Identification of PDEs in PMP cells Total cAMP PDE activity in PMP cell homogenates was inhibited about 20% by EHNA, but was stimulated about three-fold by cGMP, indicating the presence of PDE2. This increase was suppressed by EHNA, a PDE2 inhibitor. PDE activity was minimally affected by cilostamide (PDE3 inhibitor), but was inhibited by about 55% by rolipram (PDE4 inhibitor) (Fig. 2A). Therefore, PMP cells exhibited PDE2 and PDE4 activities, but PDE3 activity was very low. Stimulated PDE activity was suppressed about 40% by 0.1 mM 8-Br-cAMP, 80% by 0.5 mM 8-Br-cAMP and 90% by 1 mM 8-Br-cAMP (Fig. 2B). Total cAMP PDE activity was suppressed about 45% by 0.1 mM and TC-E 5002 0.5 mM 8-Br-cAMP, and 60% by 1 mM 8-Br-cAMP. 8-Br-cAMP did not add to the inhibitory effect of EDC3 rolipram on PDE activity (Fig. 2C). Furthermore, RT-PCR was performed to ascertain the expression of PDE2, PDE3, and PDE4 mRNAs (Fig. 2D). Bands were seen for PDE2A, 4A, 4B, and 4C mRNAs. However, bands for PDE3A, 3B, and TC-E 5002 4D were not seen. Open in a separate window Fig. 2 Expression of PDEs and effects of 8-Br-cAMP on PDE activity in PMP cells. Data in graphs are means of three impartial experiments, each performed in triplicate. (A) PDE activities were analyzed by cAMP PDE activity assay with or without each specific PDE inhibitor. The error bars represent means SD (= 3). The concentrations of each reagents were: EHNA, 20 M; cGMP, 10 M; cilostamide, 0.5 M; rolipram, 10 M. (B) Effect of 8-Br-cAMP on cGMP-stimulated PDE activity in PMP cells. cGMP (10 M) and 8-Br-cAMP (0.1 to 1 1 mM) were used. The error bars represent.
