Gamma-actin is involved in regulating centrosome function and mitotic progression in cancer cells

<p>Reorganization of the actin cytoskeleton during mitosis is crucial for regulating cell division. A functional role for γ-actin in mitotic arrest induced by the microtubule-targeted agent, paclitaxel, has recently been demonstrated. We hypothesized that γ-actin plays a role in mitosis. Herein, we investigated the effect of γ-actin in mitosis and demonstrated that γ-actin is important in the distribution of β-actin and formation of actin-rich retraction fibers during mitosis. The reduced ability of paclitaxel to induce mitotic arrest as a result of γ-actin depletion was replicated with a range of mitotic inhibitors, suggesting that γ-actin loss reduces the ability of broad classes of anti-mitotic agents to induce mitotic arrest. In addition, partial depletion of γ-actin enhanced centrosome amplification in cancer cells and caused a significant delay in prometaphase/metaphase. This prolonged prometaphase/metaphase arrest was due to mitotic defects such as uncongressed and missegregated chromosomes, and correlated with an increased presence of mitotic spindle abnormalities in the γ-actin depleted cells. Collectively, these results demonstrate a previously unknown role for γ-actin in regulating centrosome function, chromosome alignment and maintenance of mitotic spindle integrity.</p

Assessment of Cholesterol-Derived <i>Ionic</i> Copolymers as Potential Vectors for Gene Delivery

A library of cholesterol-derived <i>ionic</i> copolymers were previously synthesized via reversible addition–fragmentation chain transfer (RAFT) polymerization as ‘smart’ gene delivery vehicles that hold diverse surface charges. Polyplex systems formed with anionic poly­(methacrylic acid-co-cholesteryl methacrylate) (P­(MAA-<i>co</i>-CMA)) and cationic poly­(dimethylamino ethyl methacrylate-co-cholesteryl methacrylate) (Q-P­(DMAEMA-<i>co</i>-CMA)) copolymer series were evaluated for their therapeutic efficiency. Cell viability assays, conducted on SHEP, HepG2, H460, and MRC5 cell lines, revealed that alterations in the copolymer composition (CMA mol %) affected the cytotoxicity profile. Increasing the number of cholesterol moieties in Q-P­(DMAEMA-<i>co</i>-CMA) copolymers reduced the overall toxicity (in H460 and HepG2 cells) while P­(MAA-<i>co</i>-CMA) series displayed no significant toxicity regardless of the CMA content. Agarose gel electrophoresis was employed to investigate the formation of stable polyplexes and determine their complete conjugation ratios. P­(MAA-<i>co</i>-CMA) copolymer series were conjugated to DNA through a cationic linker, oligolysine, while Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA complexes were readily formed via electrostatic interactions at conjugation ratios beginning from 6:1:1 (oligolysine-P­(MAA-<i>co</i>-CMA)-DNA) and 20:1 (Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA), respectively. The hydrodynamic diameter, ζ potential and complex stability of the polyplexes were evaluated in accordance to complexation ratios and copolymer composition by dynamic light scattering (DLS). The therapeutic efficiency of the conjugates was assessed in SHEP cells via transfection and imaging assays using RT-qPCR, Western blotting, flow cytometry, and confocal microscopy. DNA transfection studies revealed P­(MAA-<i>co</i>-CMA)-oligolysine-DNA ternary complexes to be ineffective transfection vehicles that mostly adhere to the cell surface as opposed to internalizing and partaking in endosomal disrupting activity. The transfection efficiency of Q-P­(DMAEMA-<i>co</i>-CMA)-GFP siRNA complexes were found to be polymer composition and N/P ratio dependent, with Q-2% CMA-GFP siRNA polyplexes at N/P ratio 20:1 showing the highest gene suppression in GFP expressing SHEP cells. Cellular internalization studies suggested that Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA conjugates efficiently escaped the endolysosomal pathway and released siRNA into the cytoplasm. The gene delivery profile, reported herein, illuminates the positive and negative attributes of each therapeutic design and strongly suggests Q-P­(DMAEMA-<i>co</i>-CMA)-siRNA particles are extremely promising candidates for <i>in vivo</i> applications of siRNA therapy

Macromolecular Hydrogen Sulfide Donors Trigger Spatiotemporally Confined Changes in Cell Signaling

Hydrogen sulfide (H₂S) is involved in a myriad of cell signaling processes that trigger physiological events ranging from vasodilation to cell proliferation. Moreover, disturbances to H₂S signaling have been associated with numerous pathologies. As such, the ability to release H₂S in a cellular environment and stimulate signaling events is of considerable interest. Herein we report the synthesis of macromolecular H₂S donors capable of stimulating cell signaling pathways in both the cytosol and at the cell membrane. Specifically, copolymers having pendent oligo­(ethylene glycol) and benzonitrile groups were synthesized, and the benzonitrile groups were subsequently transformed into primary aryl thioamide groups via thionation using sodium hydrosulfide. These thioamide moieties could be incorporated into a hydrophilic copolymer or a block copolymer (i.e., into either the hydrophilic or hydrophobic domain). An electrochemical sensor was used to demonstrate release of H₂S under simulated physiological conditions. Subsequent treatment of HEK293 cells with a macromolecular H₂S donor elicited a slow and sustained increase in cytosolic ERK signaling, as monitored using a FRET-based biosensor. The macromolecular donor was also shown to induce a small, fast and sustained increase in plasma membrane-localized PKC activity immediately following addition to cells. Studies using an H₂S-selective fluorescent probe in live cells confirmed release of H₂S from the macromolecular donor over physiologically relevant time scales consistent with the signaling observations. Taken together, these results demonstrate that by using macromolecular H₂S donors it is possible to trigger spatiotemporally confined cell signaling events. Moreover, the localized nature of the observed signaling suggests that macromolecular donor design may provide an approach for selectively stimulating certain cellular biochemical pathways

Induction of apoptosis in CEM and CEM/AKB cells.

<p>A) Levels of cleaved PARP in cells treated with indicated concentrations of ZM447439 for 24 h as determined by western blot. B) Proportion of apoptotic cells in both untreated and CEM and CEM/AKB cells treated with 16 µM ZM447439 for 24 h.</p

Levels of phosphorylated Histone H3 in CEM and CEM/AKB cells in the presence or absence of 16 µM ZM447439.

<p>Levels of phosphorylated Histone H3 in CEM and CEM/AKB cells in the presence or absence of 16 µM ZM447439.</p

Proliferation timecourse of CEM and CEM/AKB cells in the presence and absence of ZM447439.

<p>Cells were grown either in vehicle alone or in 4 µM ZM447439 and proliferation was determined at indicated timepoints as the corrected absorbance using the Alamar blue assay measured spectrophotometrically. Error bars represent the SEM of three independent experiments.</p

Relative resistance of CEM/AKB4 cells to cytotoxic agents compared to parental CCRF-CEM cells.

a<p>Determined by dividing the IC₅₀ for the resistant (CEM/AKB4) cell line by the IC₅₀ of the parent (CEM) cell line.</p

Resistance levels of CEM/AKB4.

<p>(A) Plot of cell viability against concentration of ZM447439 for both CEM/AKB4 and parental CEM cells as determined by cytotoxicity assay. (B) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030734#s3" target="_blank">Results</a> of the same experiment performed in the presence of the P-glycoprotein inhibitor verapamil. Points are the means, and bars are the SEM of at least three independent experiments.</p

Mitotic index of CEM and CEM/AKB4 cells in the presence and absence of 4 µM ZM447439.

<p>*Percentages of mitotic cells were calculated after counting at least 1000 cells in three independent experiments.</p

Expression and functional relevance of MDR1 (P-glycoprotein) in CEM/AKB cells.

<p>A) CEM/AKBMDR1 mRNA expression in ZM447439 resistant CEM cells as determined by real-time PCR. Expression is displayed as relative ΔΔCt values of CEM, CEM/AKB4, CEM/AKB4 and CEM/AKB4 cells compared to that for CEM/VCRR cells with Ct values normalised to the cyclophilin-A gene (PPIA). Error bars represent the SEM of three independent experiments. B) Cytotoxicity assays of doxorubicin against CEM and CEM/AKB4, 8, and 16 cells.</p