Induction of apoptosis by an inhibitor of cAMP-specific PDE in malignant murine carcinoma cells overexpressing PDE activity in comparison to their nonmalignant counterparts.
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.
Marko
D
D
Department of Chemistry, University of Kaiserslautern, Germany.
Romanakis
K
K
Zankl
H
H
Fürstenberger
G
G
Steinbauer
B
B
Eisenbrand
G
G
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002277:Carcinoma / Q000737:chemistry / Q000201:enzymology / Q000473:pathology; D002453:Cell Cycle; D002455:Cell Division / Q000187:drug effects; D048429:Cell Size / Q000187:drug effects; D002471:Cell Transformation, Neoplastic; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000032:analysis*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D053938:DNA Fragmentation / Q000187:drug effects*; D007527:Isoenzymes / Q000378:metabolism; D015603:Keratinocytes / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D051379:Mice; D010212:Papilloma / Q000737:chemistry / Q000201:enzymology / Q000473:pathology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D010879:Piperazines / Q000494:pharmacology; D011621:Pteridines / Q000494:pharmacology; D011760:Pyrrolidinones / Q000494:pharmacology; D020889:Rolipram; D012878:Skin Neoplasms / Q000737:chemistry / Q000201:enzymology* / Q000473:pathology; D014407:Tumor Cells, Cultured
D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D010879:Piperazines; D011621:Pteridines; D011760:Pyrrolidinones; C081896:7-benzylamino-6-chloro-2-piperazino-4-pyrrolidinopteridine; C010192:9-(2-hydroxy-3-nonyl)adenine; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D000225:Adenine; D020889:Rolipram