Inhibition Of Mitotic Entry Research Articles

The live cell DNA stain SiR-Hoechst induces DNA damage responses and impairs cell cycle progression

SiR-Hoechst (SiR-DNA) is a far-red fluorescent DNA probe being used widely for time-lapse imaging of living cells that is reported to be minimally toxic at concentrations as high as 10–25 µM. However, measuring nuclear import of Cyclin B1, inhibition of mitotic entry, and the induction of γH2AX foci in cultured human cells reveals that SiR-Hoechst induces DNA damage responses and G2 arrest at concentrations well below 1 µM. SiR-Hoechst is useful for live cell imaging, but it should be used with caution and at the lowest practicable concentration.

The Auto-ubiquitylation of E3 Ubiquitin-protein Ligase Chfr at G2 Phase Is Required for Accumulation of Polo-like Kinase 1 and Mitotic Entry in Mammalian Cells

The E3 ubiquitin-protein ligase Chfr is a mitotic stress checkpoint protein that delays mitotic entry in response to microtubule damage; however, the molecular mechanism by which Chfr accomplishes this remains elusive. Here, we show that Chfr levels are elevated in response to microtubule-damaging stress. Moreover, G(2)/M transition is associated with cell cycle-dependent turnover of Chfr accompanied by high autoubiquitylation activity, suggesting that regulation of Chfr levels and auto-ubiquitylation activity are functionally significant. To test this, we generated Chfr mutants Chfr-K2A and Chfr-K5A in which putative lysine target sites of auto-ubiquitylation were replaced with alanine. Chfr-K2A did not undergo cell cycle-dependent degradation, and its levels remained high during G(2)/M phase. The elevated levels of Chfr-K2A caused a significant reduction in phosphohistone H3 levels and cyclinB1/Cdk1 kinase activities, leading to mitotic entry delay. Notably, polo-like kinase 1 levels at G(2) phase, but not at S phase, were ∼2-3-fold lower in cells expressing Chfr-K2A than in wild-type Chfr-expressing cells. Consistent with this, ubiquitylation of Plk1 at G(2) phase was accelerated in Chfr-K2A-expressing cells. In contrast, Aurora A levels remained constant, indicating that Plk1 is a major target of Chfr in controlling the timing of mitotic entry. Indeed, overexpression of Plk1 in Chfr-K2A-expressing cells restored cyclin B1/Cdk1 kinase activity and promoted mitotic entry. Collectively, these data indicate that Chfr auto-ubiquitylation is required to allow Plk1 to accumulate to levels necessary for activation of cyclin B1/Cdk1 kinase and mitotic entry. Our results provide the first evidence that Chfr auto-ubiquitylation and degradation are important for the G(2)/M transition.

CAMP signaling regulates histone H3 phosphorylation and mitotic entry through a disruption of G2 progression

cAMP signaling is known to have significant effects on cell growth, either inhibitory or stimulatory depending on the cell type. Study of cAMP-induced growth inhibition in mammalian somatic cells has focused mainly on the combined role of protein kinase A (PKA) and mitogen-activated protein (MAP) kinases in regulation of progression through the G1 phase of the cell cycle. Here we show that cAMP signaling regulates histone H3 phosphorylation in a cell cycle-dependent fashion, increasing it in quiescent cells but dramatically reducing it in cycling cells. The latter is due to a rapid and dramatic loss of mitotic histone H3 phosphorylation caused by a disruption in G2 progression, as evidenced by the inhibition of mitotic entry and decreased activity of the CyclinB/Cdk1 kinase. The inhibition of G2 progression induced through cAMP signaling is dependent on expression of the catalytic subunit of PKA and is highly sensitive to intracellular cAMP concentration. The mechanism by which G2 progression is inhibited is independent of both DNA damage and MAP kinase signaling. Our results suggest that cAMP signaling activates a G2 checkpoint by a unique mechanism and provide new insight into normal cellular regulation of G2 progression.

The Cyclin-Dependent Kinase Inhibitor Seliciclib (R-roscovitine; CYC202) Decreases the Expression of Mitotic Control Genes and Prevents Entry into Mitosis

The cyclin-dependent kinase (CDK) inhibitor seliciclib (R-roscovitine, CYC202) shows promising antitumor activity in preclinical models and is currently undergoing phase II clinical trials. Inhibition of the CDKs by seliciclib could contribute to cell cycle arrest and apoptosis seen with the drug. However, it is common for drugs to exert multiple effects on gene expression and biochemical pathways. To further our understanding of the molecular pharmacology of seliciclib, we employed cDNA microarrays to determine changes in gene expression profiles induced by the drug in HT29 human colon cancer cells. Concentrations of seliciclib were used that inhibited RB phosphorylation and cell proliferation. An increase in the mRNA expression for CJUN and EGR1 was confirmed by Western blotting, consistent with activation of the ERK1/2 MAPK pathway by seliciclib. Transcripts of key genes required for the progression through mitosis showed markedly reduced expression, including Aurora-A/B (AURK-A/B), Polo-like kinase (PLK), cyclin B2 (CCNB2), WEE1 and CDC25C. Reduced expression of these mitotic genes was also seen at the protein level. siRNA-mediated depletion of Aurora-A protein led to an arrest of cells in the G2/M phase, consistent with the effects of seliciclib treatment. Inhibition of mitotic entry following seliciclib treatment was indicated by a reduction of histone H3 phosphorylation, which is catalyzed by Aurora-B, and by decreased expression of mitotic markers, including phospho-protein phosphatase 1α. The results indicate a potential mechanism through which seliciclib prevents entry into mitosis. Gene expression profiling has generated hypotheses that led to an increase in our knowledge of the cellular effects of seliciclib and could provide potential pharmacodynamic or response biomarkers for use in animal models and clinical trials.

Cyclic AMP‐induced loss of histone H3 phosphorylation and disruption of cell cycle progression mediated through a novel cAMP binding protein

Cyclic AMP signaling is known to have a multitude of effects on cellular metabolism. It can be either inhibitory or stimulatory to cell growth depending on cell type. We have found that cAMP causes a rapid, significant loss of mitotic H3 phosphorylation at serines 10 and 28, and threonine 3. This loss is associated with cAMP-induced effects on cell cycle progression, including a delay in G2 and inhibition of mitotic entry. Interestingly, we have excluded the known cAMP binding proteins (protein kinase A, Epac, and cyclic nucleotide-gated ion channels) as mediators of this pathway through the use of specific inhibitors, antagonists, and agonists. We also excluded the possibility that a cAMP metabolite was the signaling molecule. We suggest that this may be a novel extracellular cAMP receptor because it responds efficiently to cAMP analogs with low lipophilicity and is activated by very low doses of membrane permeable cAMP derivatives. In fact, marked differences in analog lipophilicity do not correlate with significant changes in cellular response. Consistent with the idea that this may be an extracellular receptor, epinephrine induces rapid cAMP efflux from our cells. We conclude that cAMP signaling regulates cell cycle progression through a novel cyclic nucleotide binding protein. We are continuing with experiments to characterize and identify the receptor. Funding was provided by the National Cancer Institute in part through an Intramural Research Award to Dr. Catharine L. Smith.

Cell cycle inhibition of potato root tips treated with Imazethapyr

SUMMARYImazethapyr inhibits the progression of the cell cycle in potato (Solanum tuberosum L.) root tips. A variety of existing methods were developed and adapted for the determination of mitotic index in potatoes. Significantly lower numbers of mitotically dividing cells were recorded after just 30 min of incubation with imazethapyr (0.35 μM) and the effect was characterised as an inhibition of mitotic entry. Imazethapyr inhibits a key enzyme in the biosynthesis of the branched‐chain amino acids, valine, leucine and isoleucine. The inhibition of mitotic entry can be considered to be a secondary manifestation of a primary metabolic change induced by imazethapyr.