The protein phosphatase inhibitors, okadaic acid and calyculin A, induce apoptosis in human submandibular gland ductal cell line HSG cells.
OBJECTIVE
To investigate a possible relationship between protein phosphorylation or dephosphorylation status and apoptosis in salivary gland cells, we examined the effects of okadaic acid and calyculin A, the protein phosphatase inhibitors, on cultured human submandibular gland ductal cell line, HSG cells.


METHODS
HSG cells at subconfluent stages were exposed to varying concentrations of okadaic acid or calyculin A. Apoptoses were analysed in HSG cells by phase-contrast microscopy, WST-1 cytotoxicity assay, Hoechst 33342 staining, and DNA ladder formation.


RESULTS
Both okadaic acid and calyculin A induced cell death in HSG cells in a dose-dependent fashion. Marked nuclear condensation and fragmentation of chromatin was observed in HSG cells. DNA ladder formation was also detected in HSG cells by treatment with okadaic acid or calyculin A. The induced DNA ladder formation was dose-dependent with maximal effect at concentrations of 50 nM okadaic acid and 2 nM calyculin A, respectively, and were time-dependent from 14 h to 48 h. To further determine if new gene transcription and protein synthesis regulate okadaic acid-induced apoptosis in HSG cells, the cells were treated with cycloheximide or actinomycin D in the presence of 20 nM okadaic acid. Neither inhibitor protected the cells against okadaic acid-induced apoptosis.


CONCLUSIONS
Based on the known selectivity of okadaic acid and calyculin A, our results indicate that the pathway of the apoptosis in the cultured salivary gland cells is regulated by protein phosphatase type 1 or type 2A. Our results also suggest that new protein synthesis and/or mRNA expression are not involved in okadaic acid-induced apoptosis in HSG cells.
Morimoto
Y
Y
First Department of Oral Anatomy, Kyushu Dental College, Kitakyushu, Japan.
Morimoto
H
H
Kobayashi
S
S
Ohba
T
T
Haneji
T
T
D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D053938:DNA Fragmentation; D003609:Dactinomycin / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000633:toxicity*; D006801:Humans; D008387:Marine Toxins; D019319:Okadaic Acid / Q000633:toxicity*; D010080:Oxazoles / Q000633:toxicity*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D018987:Salivary Ducts / Q000166:cytology; D013363:Submandibular Gland / Q000166:cytology / Q000187:drug effects*
D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; D011500:Protein Synthesis Inhibitors; D003609:Dactinomycin; D019319:Okadaic Acid; C059041:calyculin A; D003513:Cycloheximide; D010749:Phosphoprotein Phosphatases