To identify transcriptionally regulated mediators associated with the cell cycle, we adopted the differential mRNA display technique for cell cultures of Tetrahymena pyriformis synchronized by cyclic heat treatment. One cDNA fragment that was expressed differently during synchronous cell division had a greatly decreased expression at 30 min after the end of heat treatment (EHT). Using this fragment as a probe, we isolated the fulllength cDNA for T. pyriformis acetyl-CoA synthetase (TpAcs) which encodes a 651 amino acid polypeptide with a predicted molecular mass of 72. 8 kDa. The deduced amino acid sequence of T. pyriformis ACS shows 42% sequence identity compared with that of Lysobacter sp. acetyl-CoA synthetase (ACS), an enzyme which catalyses the formation of acetyl-CoA from acetate via an acetyl-adenylate intermediate. The deduced sequence is also 41% and 40% identical compared with those of Pseudomonas putida and Coprinus cinereus ACS, respectively. Thededuced sequence of T. pyriformis ACS also shares similar characteristics of the conserved motifs I and II in the ACS family. To further investigate the actions of the gene encoding this enzyme, mRNA expression was determined during the course of synchronized cell division in T. pyriformis. Northern blot results show that themRNAlevel was dramatically decreased at 30min after EHT prior to entering synchronous cell division (which occurs 75 min after EHT), suggesting that mRNA expression of the TpAcs was associated with the cell cycle and that the down-regulated expression of TpAcs at 30min after EHT would be required for the initiation of the oncoming synchronous cell division in T. pyriformis.

To identify genes responsive to cold stress, we employed the differential display mRNA analysis technique to isolate a novel gene from Tetrahymena thermophila which encodes a protein kinase of 430 amino acids. A homolog of this kinase with 90% amino acid sequence identity was also found in T. pyriformis. Both kinases contain 11 subdomains typical of protein kinases. Sequence analysis revealed that the predicted amino acid sequences resemble those of mitogen-activated protein kinase (MAPK), especially p38 and stressactivated protein kinase which are known to be involvedin various stress responses. However, it should be noted that the tyrosine residue in the normally conserved MAPK phosphorylation site (Thr-X-Tyr) is replaced by histidine (Thr226-Gly-His228) in this MAPK-related kinase (MRK). The recombinant MRK expressed in Escherichia coli phosphorylated myelin basic protein (MBP) and became autophosphorylated. However, the mutated recombinant protein in which Thr226 was replaced by Ala lost the ability to phosphorylate MBP, suggesting that Thr226 residue is essential for kinase activity. The MRK mRNA transcript in T. thermophila increased markedly upon temperature downshift from 35 to 15℃ (0.8℃/min). Interestingly, osmotic shock either by sorbitol (100-200mm) or NaCl (25-100 mM) also induced mRNA expression of the MRK in T. pyriformis. In addition, the activity of the kinase as determined by an immune complex kinase assay using MBP as a substrate was also induced by osmotic stress. This is the first demonstration of a MAPK-related kinase in the unicellular eukaryotic protozoan Tetrahymena that is induced by physical stresses such as cold temperature and osmolarity. The present results suggest that this MRK may function in the stress-signaling pathway in Tetrahymena cells.

With the intention of investigating the signal-transduction pathway that mediates the cold-stress response in Tetrahymena, we isolated a gene that encodes a novel protein kinase of 561 amino acids, termed Tetrahymena pyriformis NIMA (never-inmitosis in Aspergillus nidulans)-related protein kinase (TpNrk), by differential display from Tetrahymena cells exposed to temperature shift-down. TpNrk possesses an N-terminal protein kinase domain that is highly homologous with other NIMArelated protein kinases (Neks) involved in the control of the cell cycle. The TpNrk protein is 42%identical in its catalytic domain with human Nek2, 41% identical with mouse Nek1 and 37% with A. nidulans NIMA. In addition, TpNrk and these NIMArelated kinases have long, basic C-terminal extensions and are therefore similar in overall structure. In order to further explorethe function of the TpNrk gene and the association of the cold stress with the cell cycle of Tetrahymena, changes of TpNrk mRNA were determined during the course of the synchronous cell division induced by the intermittent heat treatment. The level of TpNrk transcription increased immediately after the end of the heat treatment, with a peak at 30min, and declined thereafter reaching the minimum level when nearly 80% of the cells synchronously entered cell division (75min after the end of heat treatment). The accumulation of TpNrk mRNA starting from 0min to 30min after the end of the heat treatment was assumed to be a prerequisite for the start of synchronous cell division. These results suggest that TpNrk may have a role in the cell cycle of Tetrahymena, and that mRNA expression, at least, is under tight cell-cycle control.

TpMRK was identified as a stress-responsive mitogen activated protein kinase (MAPK)-related kinase and has been shown to play a critical role in the stress signalling in Tetrahymena cells. Here, we found that the mRNA expression of TpMRK was correlated with cell division of Tetrahymena with decreased expression occurring in cells prior to entering synchronous cell division induced by heat treatment. Notably, cell division was delayed with a lower division index of 40% after exposure to hydrogen peroxide while 85% of cells underwent cell division synchronously at 75 min after heat treatment without the oxidative exposure. Furthermore, inactivation of TpMRK signalling by p38 MAPK inhibitor SB203580 or MEK inhibitor PD 98059 partially derepressed cell division induced by hydrogen peroxide. Our data suggest that oxidative stimuli might cause aberration of synchronous cell division of Tetrahymena through activating the TpMRK cascade. Copyright # 2009 John Wiley & Sons, Ltd.

Tumor necrosis factor–related apoptosis–inducing ligand (TRAIL) has attracted interest as an anticancer treatment, when used in conjunction with standard chemotherapy. We investigated the mechanistic basis for combining low-dose TRAIL with microtubule-targeting agents that invoke the mitotic checkpoint. Treatment of T98G and HCT116 cells with nocodazole alone resulted in a robust mitotic block with initially little cell death; low levels of cell death were also seen with TRAIL alone at 10ng/mL final concentration. In contrast, the addition of low-dose TRAIL to nocodazole was associated with maximally increased caspase-3, caspase-8, and caspase-9 activation, which efficiently abrogated the mitotic delay and markedly increased cell death. In contrast, the abrogation of mitotic checkpoint and increased cell death were blocked by inhibitors of caspase-8 and caspase-9 or pan-caspase inhibitor. The addition of TRAIL to either nocodazole or paclitaxel (Taxol) reduced levels of the mitotic checkpoint proteins BubR1 and Bub1. BubR1 mutated for the caspase cleavage sites, but not wild-type BubR1, was resistant to cleavage induced by TRAIL added to nocodazole, and partially blocked the checkpoint abrogation. These resultssuggest that adding a relatively low concentration of TRAIL to antimicrotubule agents markedly increases complete caspase activation. This in turn accentuates degradation of spindle checkpoint proteins such as BubR1 and Bub1, contributes to abrogation of the mitotic checkpoint, and induces cancer cell death. These results suggest that TRAIL may increase the anticancer efficacy of microtubule-targeting drugs. [Cancer Res 2008;68 (9):3440–9]