Sampling strategies to capture single-cell heterogeneity.

Advances in single-cell technologies have highlighted the prevalence and biological significance of cellular heterogeneity. A critical question researchers face is how to design experiments that faithfully capture the true range of heterogeneity from samples of cellular populations. Here we develop a data-driven approach, illustrated in the context of image data, that estimates the sampling depth required for prospective investigations of single-cell heterogeneity from an existing collection of samples.

Suppression of Ser/Thr phosphatase 4 (PP4C/PPP4C) mimics a novel post-mitotic action of fostriecin, producing mitotic slippage followed by tetraploid cell death.

UNLABELLED: Fostriecin is a natural product purified from Sterptomyces extracts with antitumor activity sufficient to warrant human clinical trials. Unfortunately, difficulties associated with supply and stable drug formulation stalled further development. At a molecular level, fostriecin is known to act as a catalytic inhibitor of four PPP-family phosphatases, and reports describing the design of molecules in this class suggest derivatives targeting enzymes within the fostriecin-sensitive subfamily can be successful. However, it is not clear if the tumor-selective cytotoxicity of fostriecin results from the inhibition of a specific phosphatase, multiple phosphatases, or a limited subset of fostriecin sensitive phosphatases. How the inhibition of sensitive phosphatases contributes to tumor-selective cytotoxicity is also not clear. Here, high-content time-lapse imaging of live cells revealed novel insight into the cellular actions of fostriecin, showing that fostriecin-induced apoptosis is not simply induced following a sustained mitotic arrest. Rather, apoptosis occurred in an apparent second interphase produced when tetraploid cells undergo mitotic slippage. Comparison of the actions of fostriecin and antisense-oligonucleotides specifically targeting human fostriecin-sensitive phosphatases revealed that the suppression PP4C alone is sufficient to mimic many actions of fostriecin. Importantly, targeted suppression of PP4C induced apoptosis, with death occurring in tetraploid cells following mitotic slippage. This effect was not observed following the suppression of PP1C, PP2AC, or PP5C. These data clarify PP4C as a fostriecin-sensitive phosphatase and demonstrate that the suppression of PP4C triggers mitotic slippage/apoptosis. IMPLICATIONS: Future development of fostriecin class inhibitors should consider PP4C as a potentially important target. Mol Cancer Res; 11(8); 845-55. ©2013 AACR.

Cantharidin-induced mitotic arrest is associated with the formation of aberrant mitotic spindles and lagging chromosomes resulting, in part, from the suppression of PP2Aalpha.

Cantharidin, a natural vesicant, inhibits the activity of several PPP family phosphatases, displays antitumor activity, and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that before apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G(1)-S or S-G(2) phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G(2) or early M phase. Immunostaining indicates that cell cycle arrest occurs before the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live cell imaging with time-lapse microscopy shows that cantharidin disrupts the metaphase alignment of chromosomes and produces a prolonged mitotic arrest, with the onset of apoptosis occurring before the onset of anaphase. To explore the contribution of individual phosphatases, antisense oligonucleotides and small interfering RNA were developed to suppress the expression of cantharidin-sensitive phosphatases. The suppression of PP2Aalpha, but not PP2Abeta, is sufficient to induce metaphase arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10- to 15-hour delay, the cells enter anaphase, suggesting that an additional cantharidin-sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis.

Fundamental role of the fostriecin unsaturated lactone and implications for selective protein phosphatase inhibition.

Key derivatives and analogues of fostriecin were prepared and examined that revealed a fundamental role for the unsaturated lactone and confirmed the essential nature of the phosphate monoester. Thus, an identical 200-fold reduction in protein phosphatase 2A (PP2A) inhibition is observed with either the saturated lactone (7) or with an analogue that lacks the entire lactone (15). This 200-fold increase in PP2A inhibition attributable to the unsaturated lactone potentially may be due to reversible C269 alkylation within the PP beta12-beta13 active site loop accounting for PP2A/4 potency and selectivity.