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 phosphory lation or dephosphorylation status and apoptosis in salivary gland cells, w e examined the effects of okadaic acid and calyculin A, the protein phospha tase inhibitors, on cultured human submandibular gland ductal cell line, HS G cells. METHODS: HSG cells at subconfluent stages were exposed to varying concentra tions of okadaic acid or calyculin A. Apoptoses were analysed in HSG cells by phase-contrast microscopy, WST-1 cytotoxicity assay, Hoechst 33342 stain ing, and DNA ladder formation. RESULT: Both okadaic acid and calyculin A induced cell death in HSG cells i n a dose-dependent fashion. Marked nuclear condensation and fragmentation o f chromatin was observed in HSG cells. DNA ladder formation was also detect ed in HSG cells by treatment with okadaic acid or calyculin A. The induced DNA ladder formation was dose-dependent with maximal effect at concentratio ns 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 presenc e of 20 nM okadaic acid. Neither inhibitor protected the cells against okad aic acid-induced apoptosis. CONCLUSION: Based on the known selectivity of okadaic acid and calyculin A, our results indicate that the pathway of the apoptosis in the cultured sal ivary gland cells is regulated by protein phosphatase type 1 or type 2A. Ou r results also suggest that new protein synthesis and/or mRNA expression ar e not involved in okadaic acid-induced apoptosis in HSG cells.