Effect of inhibitors of protein synthesis and DNA replication on the induction of proteolytic activities, caspase-like activities and cell death in the unicellular chlorophyte Dunaliella tertiolecta

When the chlorophyte alga Dunaliella tertiolecta is placed in darkness, a form of programmed cell death with many similarities to apoptosis (including the induction of caspase-like proteases) is induced. Many uncertainties about this process remain, two of which are whether it requires protein synthesis and whether there is potential viral involvement. In order to examine the relationship between the induction and/or activation of proteolytic activities and the cell death event, we used inhibitors of cytoplasmic protein synthesis (cycloheximide, CHX), organellar protein synthesis (chloramphenicol, CAP) and DNA synthesis (mitomycin C, MMC). Use of MMC also allowed us to examine whether temperate viruses were present in the D. tertiolecta isolate, since MMC treatment has been shown to induce lytic cycles. Addition of protein synthesis inhibitors (100 mM CHX or 1500 mM CAP applied singly or both inhibitors added together) did not prevent cell death from occurring when cultures were placed in the dark. There were no differences in caseinolytic activities visualized using zymograms, or in caspase 1, 3, 8 and 9 -like activities. Surprisingly, 100 mM MMC prevented the cell death event. Caseinolytic activities that appeared in darkness in controls did not appear in MMC-treated cultures, and caspase-like activities remained the same as in controls maintained in the light. The lack of effect of CHX and CAP suggests that the cell death programme we observe does not depend on protein synthesis, but rather on post-translational modification of preexisting proteins. Results for MMC discount the involvement of temperate virus, but are difficult to interpret. MMC affects DNA synthesis and presumably transcription, but since inhibitors of translation did not prevent cell death, it is not clear why inhibiting transcription would. MMC affects cell cycle progression and cell division cycles, thus these processes may play as yet unexplained roles in mediating the cell death process observed.