The Histone Variant H2A.W Defines Heterochromatin and Promotes Chromatin Condensation in Arabidopsis

SummaryHistone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation

AZD2014 and rapamycin inhibited cell proliferation, colony formation and mTOR signaling in lung cancer cells.

<p>Inhibitory effects of AZD2014 and rapamycin on cell proliferation in A549, PC-9 and SK-MES-1 cells (a, b). (c) Influence of AZD2014 and rapamycin on colony-forming ability of A549 and PC-9 cells, evaluated by clonogenic assay. (d) AZD2014 and rapamycin inhibited mTOR signaling in A549 and SK-MES-1 cells as shown by the decreased phosphorylation of mTORand S6 after treatment for 24h. Cell viability was assessed by MTT, and Colony-forming assay was done as described in Materials and Methods. Columns, mean of three determinations; bars, SD. Results shown are representative of three independent experiments. **, <i>P</i>< 0.01; ***, <i>P</i> < 0.001, control versus AZD2014 or rapamycin-treated cells.</p

Synergistic Effects between mTOR Complex 1/2 and Glycolysis Inhibitors in Non-Small-Cell Lung Carcinoma Cells

<div><p>Cancer metabolism has greatly interested researchers. Mammalian target of rapamycin (mTOR) is dysregulated in a variety of cancers and considered to be an appealing therapeutic target. It has been proven that growth factor signal, mediated by mTOR complex 1 (mTORC1), drives cancer metabolism by regulating key enzymes in metabolic pathways. However, the role of mTORC2 in cancer metabolism has not been thoroughly investigated. In this study, by employing automated spectrophotometry, we found the level of glucose uptake was decreased in non-small-cell lung carcinoma (NSCLC) A549, PC-9 and SK-MES-1 cells treated with rapamycin or siRNA against Raptor, indicating that the inhibition of mTORC1 attenuated glycolytic metabolism in NSCLC cells. Moreover, the inhibition of AKT reduced glucose uptake in the cells as well, suggesting the involvement of AKT pathway in mTORC1 mediated glycolytic metabolism. Furthermore, our results showed a significant decrease in glucose uptake in rictor down-regulated NSCLC cells, implying a critical role of mTORC2 in NSCLC cell glycolysis. In addition, the experiments for MTT, ATP, and clonogenic assays demonstrated a reduction in cell proliferation, cell viability, and colony forming ability in mTOR inhibiting NSCLC cells. Interestingly, the combined application of mTORC1/2 inhibitors and glycolysis inhibitor not only suppressed the cell proliferation and colony formation, but also induced cell apoptosis, and such an effect of the combined application was stronger than that caused by mTORC1/2 inhibitors alone. In conclusion, this study reports a novel effect of mTORC2 on NSCLC cell metabolism, and reveals the synergistic effects between mTOR complex 1/2 and glycolysis inhibitors, suggesting that the combined application of mTORC1/2 and glycolysis inhibitors may be a new promising approach to treat NSCLC.</p></div

mTOR inhibitors combined with glycolysis inhibitor decreased the colony formation and cell viability in lung cancer cells.

<p>(a) Inhibitory effects of rapamycinorAZD2014 combined with 2-DGon cell proliferation in A549 cells. (b) Impact of rapamycinor AZD2014combined with 2-DG on colony forming ability of A549 cells, as evaluated by clonogenic assay. Data represent mean ± SD (n = 3). **, P< 0.01; ***, P < 0.001; control versus AZD2014- or rapamycin-treated cells.</p

Inhibition of mTOR pathway decreased the level of glycolytic metabolism in lung cancer cells.

<p>(a) A549 cells were treated with AZD2014 and rapamycin. (b) PC-9 cells were treated with AZD2014 and rapamycin. (c) SK-MES-1 cells were treated with AZD2014 and rapamycin. After 24 h, the cells were counted and the glucose in the culture media was immediately tested. Then the results were normalized to the number of cells, and performed as pmol/cell. Data represent mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; control versus AZD2014- or rapamycin-treated cells. (d) and (e) A549 cells were treated with AZD2014 and rapamycin. After 24 h, relative ATP content was determined by using a bioluminescence assay. Then the results were normalized to the number of cells. Data represent mean ± SD (n = 3). *<i>P</i><0.05; **<i>P</i><0.01; control versus AZD2014- or rapamycin-treated cells (see Fig 2d). Western blot analysis showed that AZD2014 and rapamycin decreased the level of PKM2 protein in a dose dependent manner. Three independent experiments were performed with similar results, and representative data are shown (see Fig 2e).</p

The combination index of mTOR inhibitors and glycolysis inhibitor on cell viability reduction in NSCLC cells.

<p>(a) and (b) The dose–response curve of each drug was determined and the combination index (CI) values for rapamycin/2-DG concentration ratios (2:1) and AZD2014/2-DG concentration ratios(1:1) were calculated according to Chou–Talalay’s method at 48 h time point. CI < 1, CI = 1, and CI > 1 indicate synergistic, additive and antagonistic effects, respectively. The effect ranges from 0 (no inhibition) to 1 (complete inhibition). The data are representative of three independent experiments.</p

AZD2014 and rapamycin inhibited cell proliferation, colony formation and mTOR signaling in lung cancer cells.

<p>Inhibitory effects of AZD2014 and rapamycin on cell proliferation in A549, PC-9 and SK-MES-1 cells (a, b). (c) Influence of AZD2014 and rapamycin on colony-forming ability of A549 and PC-9 cells, evaluated by clonogenic assay. (d) AZD2014 and rapamycin inhibited mTOR signaling in A549 and SK-MES-1 cells as shown by the decreased phosphorylation of mTORand S6 after treatment for 24h. Cell viability was assessed by MTT, and Colony-forming assay was done as described in Materials and Methods. Columns, mean of three determinations; bars, SD. Results shown are representative of three independent experiments. **, <i>P</i>< 0.01; ***, <i>P</i> < 0.001, control versus AZD2014 or rapamycin-treated cells.</p

Knockdown of mTOR, Raptor and Rictor decreased the level of glycolytic metabolism in lung cancer cells.

<p>(a) and (b) A549 and PC-9 cells were treated with negative control (NC) siRNA or one of those siRNAs targeting Raptor, Rictor, and mTOR. After 24 h and 48 h transfection, the cells were counted and the glucose in the culture media was immediately tested. The results were then normalized to the cells number, and performed as pmol/cell. Data represent mean ± SD (n = 3). *<i>P</i><0.05, **<i>P</i><0.01. (c) and (d) Western blot analysis showed that siRNAs against Raptor, Rictor, and mTOR decreased the protein expression of Raptor, Rictor, mTOR, AKT, p-AKT, S6, and p-S6, respectively. Three independent experiments were performed with similar results, and representative data are shown.</p