Pharmacologic Inhibition of the Anaphase-Promoting Complex Induces A Spindle Checkpoint-Dependent Mitotic Arrest in the Absence of Spindle Damage

SummaryMicrotubule inhibitors are important cancer drugs that induce mitotic arrest by activating the spindle assembly checkpoint (SAC), which, in turn, inhibits the ubiquitin ligase activity of the anaphase-promoting complex (APC). Here, we report a small molecule, tosyl-L-arginine methyl ester (TAME), which binds to the APC and prevents its activation by Cdc20 and Cdh1. A prodrug of TAME arrests cells in metaphase without perturbing the spindle, but nonetheless the arrest is dependent on the SAC. Metaphase arrest induced by a proteasome inhibitor is also SAC dependent, suggesting that APC-dependent proteolysis is required to inactivate the SAC. We propose that mutual antagonism between the APC and the SAC yields a positive feedback loop that amplifies the ability of TAME to induce mitotic arrest

Arginine residues in and near the D-box are required for cyclin B1 localization to mitotic chromosomes.

<p><b>A</b>. Representative images of mitotic BS-C-1 cells expressing cyclin B1 containing mutations in and near the D-box. White arrows indicate the position of the metaphase plate as seen in the DIC image. Scale bar = 10 µm. <b>B</b>. CER box-and-whisker plots for the constructs shown in part A. The red dotted line indicates the threshold of 1.6. N  = 10–14, * indicates p<0.05, ** indicates p<0.01 and *** indicated p<0.001 compared to WT^1–433. Further details including mean CER, standard deviations, and Wilcoxon exact test p-values can be found in Table S2. <b>C</b>. Representative images of mitotic BS-C-1 cells expressing cyclin B1 fragments bearing R42A or Δ3–8/R42A double mutations. White arrows indicate the position of the metaphase plate as seen in the DIC image. Scale bar = 10 µm. <b>D</b>. CER box-and-whisker plots for the constructs shown in part C. The red dotted line indicates the threshold of 1.6. N = 9–13, * indicates p<0.05, ** indicated p<0.01 and ***indicates p<0.001 compared to WT counterpart. Further details including mean CER, standard deviations, and Wilcoxon exact test p-values can be found in Table S2.</p

Model summarizing key findings.

<p>A schematic representation of the Cyclin B1 protein. The numbers along the top indicate amino acid positions. D = D-box sequence, CRS = cytoplasmic retention sequence, * = MRAIL motif. This paper identifies amino acids 3–8 (LRVTRN) and arginine residues near the D-box as sequence motifs that promote cyclin B1 localization to chromosomes, whereas downstream regions, including the MRAIL motif, the CRS and the cyclin box may antagonize chromosome association.</p

The first 20 amino acids of cyclin B1 can promote association with mitotic chromosomes.

<p><b>A</b>. Representative images of mitotic BS-C-1 cells expressing GFP fusions of WT^1–433 cyclin B1, GFP only, or fragments of cyclin B1 that lack the C-terminus. The GFP signal is detected by the FITC channel (top) and the position of the metaphase plate (indicated with white arrows) can be identified with DIC optics (bottom). Scale bar  =  10 µm. <b>B</b>. Box-and-whisker plots of Chromosome Enrichment Ratios (CER) for the constructs shown in part A. The CER is calculated as the ratio of the mean fluorescence intensity of the chromosomal region to the mean fluorescence intensity of the entire cell. The diamond indicates the mean CER. The box represents the 2^nd and 3^rd quartiles of the data, with the horizontal line representing the median and the whiskers representing the range. The red dotted line indicates the threshold of 1.6 that correlates with chromosome association in the qualitative assay. N = 8–14, ** indicates p < 0.01 and *** indicates p<0.001 compared to WT^1–433. Further details including mean CER, standard deviations, and Wilcoxon exact test p-values can be found in Table S2. <b>C</b>. Representative mitotic BS-C-1 cells expressing GFP fusions of cyclin B1 fragments that lack different regions of the N- and C-termini. The metaphase plate is indicated by white arrows. Scale bar = 10 µm. <b>D</b> CER plots for the constructs shown in part C. The red dotted line indicates the threshold of 1.6. N = 10–11, *** indicates p<0.001 compared to the WT^1–433, WT^1–166, WT^1–110, respectively. Further details including mean CER, standard deviations, and Wilcoxon exact test p-values can be found in Table S2.</p

The Δ3–8 mutation causes defects in chromosome localization of N-terminal cyclin B1 fragments.

<p><b>A</b>. The Δ3–8 mutation disrupts the chromosome association of cyclin B1 truncations in a context-dependent fashion. Representative images of mitotic BS-C-1 cells expressing mutated cyclin B1. White arrows indicate position of metaphase plate as seen in the DIC image. Scale bar = 10 µm. <b>B</b>. CER box-and-whisker plots for the constructs shown in part A. The red dotted line indicates the threshold of 1.6. N  = 8–14, ** indicates p<0.01 and *** indicates p<0.001 compared to WT counterpart. Further details including mean CER, standard deviations, and Wilcoxon exact test p-values can be found in Table S2. <b>C</b>. Biochemical fractionation of HeLa cells stably expressing WT^1–41-GFP or Δ3–8^1–41-GFP. (1–41)-GFP was detected with a cyclin B1 antibody. (Abbreviations as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059169#pone-0059169-g001" target="_blank">Figure 1</a>).</p

Growth differentiation factor 11 (GDF11) has pronounced effects on skin biology.

Growth differentiation factor 11 (GDF11) belongs to the TGF-β superfamily of proteins and is closely related to myostatin. Recent findings show that GDF11 has rejuvenating properties with pronounced effects on the cardiovascular system, brain, skeletal muscle, and skeleton in mice. Several human studies were also conducted, some implicating decreasing levels of circulating GDF11 with age. To date, however, there have not been any reports on its role in human skin. This study examined the impact of GDF11 on human skin, specifically related to skin aging. The effect of recombinant GDF11 on the function of various skin cells was examined in human epidermal keratinocytes, dermal fibroblasts, melanocytes, dermal microvascular endothelial cells and 3D skin equivalents, as well as in ex vivo human skin explants. GDF11 had significant effects on the production of dermal matrix components in multiple skin models in vitro and ex vivo. In addition, it had a pronounced effect on expression of multiple skin related genes in full thickness 3D skin equivalents. This work, for the first time, demonstrates an important role for GDF11 in skin biology and a potential impact on skin health and aging

Naphthoquinones Oxidize H2S to Polysulfides and Thiosulfate, Implications for Therapeutic Applications

1,4-Napththoquinones (NQs) are clinically relevant therapeutics that affect cell function through production of reactive oxygen species (ROS) and formation of adducts with regulatory protein thiols. Reactive sulfur species (RSS) are chemically and biologically similar to ROS and here we examine RSS production by NQ oxidation of hydrogen sulfide (H2S) using RSS-specific fluorophores, liquid chromatography-mass spectrometry, UV-Vis absorption spectrometry, oxygen-sensitive optodes, thiosulfate-specific nanoparticles, HPLC-monobromobimane derivatization, and ion chromatographic assays. We show that NQs, catalytically oxidize H2S to per- and polysulfides (H2Sn, n = 2&ndash;6), thiosulfate, sulfite and sulfate in reactions that consume oxygen and are accelerated by superoxide dismutase (SOD) and inhibited by catalase. The approximate efficacy of NQs (in decreasing order) is, 1,4-NQ &asymp; juglone &asymp; plumbagin &gt; 2-methoxy-1,4-NQ &asymp; menadione &gt;&gt; phylloquinone &asymp; anthraquinone &asymp; menaquinone &asymp; lawsone. We propose that the most probable reactions are an initial two-electron oxidation of H2S to S0 and reduction of NQ to NQH2. S0 may react with H2S or elongate H2Sn in variety of reactions. Reoxidation of NQH2 likely involves a semiquinone radical (NQ&middot;&minus;) intermediate via several mechanisms involving oxygen and comproportionation to produce NQ and superoxide. Dismutation of the latter forms hydrogen peroxide which then further oxidizes RSS to sulfoxides. These findings provide the chemical background for novel sulfur-based approaches to naphthoquinone-directed therapies