In a previous study, we demonstrated that the
protein phosphatase inhibitors,
okadaic acid and
calyculin A, induced apoptosis in human
osteosarcoma cell lines, Saos-2 and MG63 cells. In the present study, to determine if new gene transcription and
protein synthesis are required for
okadaic acid-induced apoptosis in Saos-2 and MG63 cells, the cells were treated for 48h with varying concentrations of the inhibitors of
protein or
RNA synthesis, i.e.,
cycloheximide,
actinomycin D, and
puromycin, in the presence of a fixed dose of
okadaic acid. All these
reagents in different concentrations prevented the
okadaic acid-induced apoptosis in MG63 cells in a dose-dependent fashion. The same concentrations of
cycloheximide,
actinomycin D, or
puromycin alone did not induce any apoptotic features in MG63 cells. However, not all the aforementioned
reagents affected
okadaic acid-induced apoptosis in Saos-2 cells.
Okadaic acid-induced and
cycloheximide-prevented apoptosis was shown by phase-contrast microscopy, WST-1 assay, direct visualization of nuclear condensation and fragmentation of
chromatin, and the characteristic
DNA ladder formation on
agarose gel electrophoresis. The present results indicate that the induction of new cell death genes and ongoing
protein synthesis may have a role in
okadaic acid-induced apoptosis in MG63 cells and that such
proteins are not required in Saos-2 cells.