Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts
Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts
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DOI：
10.1007/bf02737806
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发表时间：
1998-01-01
影响因子：
2.6
通讯作者：
Eisenbrand, Gerhard
中科院分区：
文献类型：
--
作者：
Marko, Doris;Romanakis, Konstantinos;Eisenbrand, Gerhard
为了研究与恶性转化相关的磷酸二酯酶(PDE)活性的变化，对小鼠皮肤正常原代角质形成细胞和对应于表皮肿瘤不同发展阶段的细胞进行了3‘，5’-环磷酸腺苷(CAMP)的水解性分析。本文研究了cAMP-4特异性的PDE-4在MSCP6和308两种乳头状瘤细胞系和高度恶性癌细胞系CARB中的表达、细胞内cAMP含量以及对PDE-4特异性抑制剂7-苄氨基-6-氯-2-哌氨基-4-吡咯烷蝶啶(DC-TA-46)抑制生长作用的敏感性。与原代角质形成细胞相比，两种乳头状瘤细胞系的可溶性PDE活性和细胞内cAMP活性均无显著差异。相反，梭形细胞癌细胞系CARB表现出明显更高的PDE活性，伴随着最低的cAMP水平。在所有细胞系和原代角质形成细胞中，对罗利普兰敏感的PDE-4活性是cAMP水解酶的主要活性。在原代角质形成细胞和MSCP6细胞中，PDE-4抑制剂DC-TA-46的边缘生长抑制作用最强，而308细胞的生长在浓度为2µm时受到明显影响。癌细胞株CARB对DC-TA-46的敏感性最高(IC50=0.8±-0.3um)。用DC-TA-46处理CARB细胞后，细胞内PDE活性受到强烈抑制，导致cAMP显著而持久的升高。作用24小时后，细胞周期停滞于G0/G1期。用这种高效PDE抑制剂的浓度>；2um处理后，通过荧光显微镜、流式细胞仪和基于酶联免疫吸附试验的完整细胞中DNA片段的测定，可以检测到诱导细胞的凋亡。
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 muM. The carcinoma cell line CarB showed the highest sensitivity to DC-TA-46 (IC50 = 0.8 +- 0.3 muM). 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 muM 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.