A novel role for the condensin II complex in cellular senescence

<p>Although cellular senescence is accompanied by global alterations in genome architecture, how the genome is restructured during the senescent processes is not well understood. Here, we show that the hCAP-H2 subunit of the condensin II complex exists as either a full-length protein or an N-terminus truncated variant (ΔN). While the full-length hCAP-H2 associates with mitotic chromosomes, the ΔN variant exists as an insoluble nuclear structure. When overexpressed, both hCAP-H2 isoforms assemble this nuclear architecture and induce senescence-associated heterochromatic foci (SAHF). The hCAP-H2ΔN protein accumulates as cells approach senescence, and hCAP-H2 knockdown inhibits oncogene-induced senescence. This study identifies a novel mechanism whereby condensin drives senescence via nuclear/genomic reorganization.</p

Comprehensive analysis of the ubiquitinome during oncogene-induced senescence in human fibroblasts

<div><p>Oncogene-induced senescence (OIS) is an important tumor suppression mechanism preventing uncontrolled proliferation in response to aberrant oncogenic signaling. The profound functional and morphological remodelling of the senescent cell involves extensive changes. In particular, alterations in protein ubiquitination during senescence have not been systematically analyzed previously. Here, we report the first global ubiquitination profile of primary human cells undergoing senescence. We employed a well-characterized <i>in vitro</i> model of OIS, primary human fibroblasts expressing oncogenic RAS. To compare the ubiquitinome of RAS-induced OIS and controls, ubiquitinated peptides were enriched by immune affinity purification and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). We identified 4,472 ubiquitination sites, with 397 sites significantly changed (>3 standard deviations) in senescent cells. In addition, we performed mass spectrometry analysis of total proteins in OIS and control cells to account for parallel changes in both protein abundance and ubiquitin levels that did not affect the percentage of ubiquitination of a given protein. Pathway analysis revealed that the OIS-induced ubiquitinome alterations mainly affected 3 signaling networks: eIF2 signaling, eIF4/p70S6K signaling, and mTOR signaling. Interestingly, the majority of the changed ubiquitinated proteins in these pathways belong to the translation machinery. This includes several translation initiation factors (eIF2C2, eIF2B4, eIF3I, eIF3L, eIF4A1) and elongation factors (eEF1G, eEF1A) as well as 40S (RPS4X, RPS7, RPS11 and RPS20) and 60S ribosomal subunits (RPL10, RPL11, RPL18 and RPL35a). In addition, we observed enriched ubiquitination of aminoacyl-tRNA ligases (isoleucyl-, glutamine-, and tyrosine-tRNA ligase), which provide the amino acid-loaded tRNAs for protein synthesis. These results suggest that ubiquitination affects key components of the translation machinery to regulate protein synthesis during OIS. Our results thus point toward ubiquitination as a hitherto unappreciated regulatory mechanism during OIS.</p></div

Shizukaol D, a Dimeric Sesquiterpene Isolated from <i>Chloranthus serratus</i>, Represses the Growth of Human Liver Cancer Cells by Modulating Wnt Signalling Pathway

<div><p>Natural products have become sources of developing new drugs for the treatment of cancer. To seek candidate compounds that inhibit the growth of liver cancer, components of <i>Chloranthus serratus</i> were tested. Here, we report that shizukaol D, a dimeric sesquiterpene from <i>Chloranthus serratus</i>, exerted a growth inhibition effect on liver cancer cells in a dose- and time-dependent manner. We demonstrated that shizukaol D induced cells to undergo apoptosis. More importantly, shizukaol D attenuated Wnt signalling and reduced the expression of endogenous Wnt target genes, which resulted in decreased expression of β-catenin. Collectively, this study demonstrated that shizukaol D inhibited the growth of liver cancer cells by modulating Wnt pathway.</p></div

Shizukaol D inhibited the growth of liver cancer cells.

<p>(A-D). Cells were seed in a 96 well plate and treated with a series concentration of shizukaol D. Cell proliferation was measured by CCK-8 assay. (A) The viability of Focusand SMMC-7721cells were decreased when the cells were treated with increasing concentration of shizukaol D for 48h.(B) The viability decreased faster when treated with shizukaol D than treated with same concentration of 5-FU in SMMC-7721 cells for 48 h. The cell viability decreased in Focus (C) and SMMC-7721 (D) cells treated by shizukaol D in a dose- and time-dependent manner. (E) The transparent version of Focus cells treated with 0.00, 12.50, 25.00 and 50.00 μmol/L of shizukaol D for 48 hours. (F) Colony formation assay of SMMC-7721 cells. The cells were treated with 0.00, 3.13, 6.25, 12.50 and 25.00 μmol/L of shizukaol D for 48 hours and cells for each concentration were collected respectively, and plated in 6-well plates as 500/well. The cells were cultured with normal medium for 10 to 15 d then. (G) Statistics of cloning efficiency in Figure 2F. **: P<0.01. Values are presented as mean±S.D.</p

Modulation of Wnt pathway of liver cancer cells by shizukaol D.

<p>(A) Shizukaol D decreased TOPflash activity and attenuated TOPflash activity in HEK-293T cells cultured in RPMI-1640 medium containing 50mmol/L of LiCl. Renilla plasmid was included in all the transfections as control. (B-C) Western blot analysis of expression of β-catenin expression in Focus (B) and SMMC-7721 (C) cells treated with shizukaol D. (D) Immunofluorescence analysis of β-catenin in liver cancer cells with 10μmol/L shizukaol D treatment for 24 h. Red: staining for active β-catenin, blue: nuclear staining by DAPI. Upper: Focus cells; lower: SMMC-7721 cells. (E) Western blot analysis of the expression of endogenous Wnt target genes. SMMC-7721 cells were treated with 20μmol/L shizukaol D for 24 h before collectedand GAPDH was set as control. (F) mRNA expression level of endogenous Wnt target genes,c-myc, cyclin D, Tcf-1, LEF1, wnt3a and FGF18, by QPCR. Total RNA of SMMC-7721 cells was isolated after treated by 20μmol/L shizukaol D for 6 or 12 h, and then was transcripted reversely to cDNA. GAPDH was set as control. **: P<0.01. Values are presented as mean±S.D.</p

IC₅₀ values of shizukaol D on liver cancer cell lines.

<p>IC₅₀ values of shizukaol D on liver cancer cell lines.</p

Shizukaol D inducedliver cancer cells to apoptosis.

<p>(A) The statistics of the sub-G1 ratio of Focus by FCM. Cells were incubated with shizukaol D for 48 hours before being collected and diluted in PBS with 50μg/ml PI and 0.03% TritonX-100. **: P<0.01. Values are expressed as mean±S.D. (B) Western blot analysis of the expression of PARP. Focus cells were treated with shizukaol D for 48 hbefore collected and then the total protein was subjected to 10% SDS-PAGE and transferred onto the nitrocellulose membrane. β-actin antibody was used to confirm equivalent loading.</p