Marko D, Romanakis K, Zankl H, Fürstenberger G, Steinbauer B, Eisenbrand G
Department of Chemistry, University of Kaiserslautern, Germany.
Cell Biochem Biophys. 1998;28(2-3):75-101. doi: 10.1007/BF02737806.
In order to study potential changes in phosphodiesterase (PDE) activity associated with malignant transformation, normal primary keratinocytes and cells corresponding to different stages of epidermal tumor development in mouse skin were analyzed with respect to their 3',5'-cyclic adenosine monophosphate (cAMP) hydrolyzing activity. Expression of cAMP-specific PDE-4, intracellular cAMP content, and the sensitivity to the growth inhibitory effect of the PDE-4-specific inhibitor 7-benzylamino-6-chloro-2 piperazino-4-pyrrolidino-pteridine (DC-TA-46) were studied in the two papilloma cell lines, MSCP6 and 308, and in the highly malignant carcinoma cell line CarB. No significant difference in soluble PDE activity and in intracellular cAMP was found in the two papilloma cell lines when compared to primary keratinocytes. In contrast, the spindle-cell carcinoma cell line CarB exhibited significantly higher PDE activity, concomitant with the lowest cAMP level. In all cell lines and also in the primary keratinocytes, rolipram-sensitive PDE-4 activity accounted for the major cAMP-hydrolyzing activity. In primary keratinocytes and in MSCP6 cells, the PDE-4 inhibitor DC-TA-46 induced at best marginal growth inhibition, whereas cell growth of 308 cells was markedly affected at concentrations > 2 microM. The carcinoma cell line CarB showed the highest sensitivity to DC-TA-46 (IC50 = 0.8 +/- 0.3 microM). Treatment of CarB cells with DC-TA-46 strongly inhibits intracellular PDE activity, resulting in a marked and long-lasting rise of cAMP. After 24 h of treatment, arrest in the G0/G1 phase of the cell cycle is induced. Treatment with concentrations > 2 microM of this highly effective PDE inhibitor results in induction of apoptotic cell death, as detected by fluorescence microscopy, flow cytometry, and ELISA-based determination of fragmented DNA in intact cells.
为了研究与恶性转化相关的磷酸二酯酶（PDE）活性的潜在变化，对正常原代角质形成细胞以及小鼠皮肤中对应于表皮肿瘤发展不同阶段的细胞进行了3',5'-环磷酸腺苷（cAMP）水解活性分析。在两种乳头瘤细胞系MSCP6和308以及高恶性癌细胞系CarB中，研究了cAMP特异性PDE-4的表达、细胞内cAMP含量以及对PDE-4特异性抑制剂7-苄基氨基-6-氯-2-哌嗪基-4-吡咯烷基蝶啶（DC-TA-46）生长抑制作用的敏感性。与原代角质形成细胞相比，两种乳头瘤细胞系中的可溶性PDE活性和细胞内cAMP均未发现显著差异。相反，梭形细胞癌细胞系CarB表现出显著更高的PDE活性，同时cAMP水平最低。在所有细胞系以及原代角质形成细胞中，咯利普兰敏感的PDE-4活性占主要的cAMP水解活性。在原代角质形成细胞和MSCP6细胞中，PDE-4抑制剂DC-TA-46充其量只能诱导轻微的生长抑制，而当浓度>2 microM时，308细胞的生长受到明显影响。癌细胞系CarB对DC-TA-46表现出最高的敏感性（IC50 = 0.8 +/- 0.3 microM）。用DC-TA-46处理CarB细胞可强烈抑制细胞内PDE活性，导致cAMP显著且持久地升高。处理24小时后，诱导细胞周期停滞在G0/G1期。用浓度>2 microM的这种高效PDE抑制剂处理会导致凋亡细胞死亡，这可通过荧光显微镜、流式细胞术以及基于ELISA的完整细胞中DNA片段化测定来检测。
