Ratiometric two-photon fluorescent probe for quantitative detection of ß-galactosidase activity in senescent cells.

We reported a ratiometric two-photon fluorescent probe (SG1) for ß-galactosidase (ß-gal) and its application to quantitative detection of ß-gal activity during cellular senescence in live cells and in aged tissues. This probe is characterized by a significant two-photon excited fluorescence, a marked blue-to-yellow emission color change in response to ß-gal, easy loading, insensitivity to pH and reactive oxygen species (ROS), high photostability, and low cytotoxicity. In addition, we show that SG1 labeling is an effective tool for quantitative detection of senescence-associated ß-gal activity at the subcellular level in situ. This finding demonstrates that SG1 will find useful applications in biomedical research, including studies of cell aging.

The Cancer Clinical Library Database (CCLD) from the Korea-Clinical Data Utilization Network for Research Excellence (K-CURE) Project.

The common data model (CDM) has found widespread application in healthcare studies, but its utilization in cancer research has been limited. This article describes the development and implementation strategy for Cancer Clinical Library Databases (CCLDs), which are standardized cancer-specific databases established under the Korea-Clinical Data Utilization Network for Research Excellence (K-CURE) project by the Korean Ministry of Health and Welfare. Fifteen leading hospitals and fourteen academic associations in Korea are engaged in constructing CCLDs for 10 primary cancer types. For each cancer type-specific CCLD, cancer data experts determine key clinical data items essential for cancer research, standardize these items across cancer types, and create a standardized schema. Comprehensive clinical records covering diagnosis, treatment, and outcomes, with annual updates, are collected for each cancer patient in the target population, and quality control is based on six-sigma standards. To protect patient privacy, CCLDs follow stringent data security guidelines by pseudonymizing personal identification information and operating within a closed analysis environment. Researchers can apply for access to CCLD data through the K-CURE portal, which is subject to Institutional Review Board and Data Review Board approval. The CCLD is considered a pioneering standardized cancer-specific database, significantly representing Korea's cancer data. It is expected to overcome limitations of previous CDMs and provide a valuable resource for multicenter cancer research in Korea.

Analysis of gene expression levels in individual bacterial cells without image segmentation.

Studies of stochasticity in gene expression typically make use of fluorescent protein reporters, which permit the measurement of expression levels within individual cells by fluorescence microscopy. Analysis of such microscopy images is almost invariably based on a segmentation algorithm, where the image of a cell or cluster is analyzed mathematically to delineate individual cell boundaries. However segmentation can be ineffective for studying bacterial cells or clusters, especially at lower magnification, where outlines of individual cells are poorly resolved. Here we demonstrate an alternative method for analyzing such images without segmentation. The method employs a comparison between the pixel brightness in phase contrast vs fluorescence microscopy images. By fitting the correlation between phase contrast and fluorescence intensity to a physical model, we obtain well-defined estimates for the different levels of gene expression that are present in the cell or cluster. The method reveals the boundaries of the individual cells, even if the source images lack the resolution to show these boundaries clearly.

Temperature dependent infrared nano-imaging of La0.67Sr0.33MnO3 thin film.

We investigate the temperature dependence of infrared properties at nanometer length scales in La0.67Sr0.33MnO3 (LSMO) thin film with a thickness of 47 unit cells grown on SrTiO3 substrate. The infrared nano-imaging experiments were performed using a near-field optical microscope in conjunction with a variable temperature heating stage. The near-field infrared data is consistent with the bulk of the LSMO film undergoing the thermally-driven non-percolative second-order transition from a metallic, ferromagnetic phase to an insulating, paramagnetic phase. We find persistent infrared contrast on the nanoscale that is independent of temperature and which we attribute to two novel phases with different conductivities coexisting in the vicinity of the film-substrate interface. These two coexisting phases at the film-substrate interface do not undergo the metal-insulator transition (MIT) and hence are different from the metallic, ferromagnetic and insulating, paramagnetic phases in the bulk of the film. At temperatures approaching the nominal MIT temperature, repeated scans of the same microscopic area at constant temperature reveal bimodal fluctuation of the near-field infrared amplitude. We interpret this phenomenon as slow, critical fluctuations of the conductivity in the bulk of the LSMO film.

Survivin protects fused cancer cells from cell death.

Tetraploidy, a potential precursor of cancer-associated aneuploidy, is produced either by cell fusion or failure of cytokinesis. In this study, low p53-expressing HeLa cells were used to address the fate of cancer cells after fusion. We found that massive cell death or growth arrest occurred a few days after fusion. Interestingly, cells with larger nuclei preferentially died after fusion, suggesting that a larger deviation of DNA content is a strong inducer of apoptosis. Notably, a fraction of cells escaped cell death. Also, the stability of survivin increased, and its localization changed preferentially to the cytosol in fused cells. Knockdown of survivin decreased the survival of fused cells, more than observed in unfused cells, showing increased dependency of fused cells on survivin. Collectively, after cancer cell fusion, some fused cells avoid the apoptotic crisis partly owing to survivin, and continue to proliferate, a process that contributes to human cancer progression. [BMB Reports 2017; 50(7): 361-366].

Nuclear accumulation of globular actin as a cellular senescence marker.

We evaluated the nuclear actin accumulation as a new marker of cellular senescence, using human diploid fibroblast (HDF), chondrocyte primary cultures, Mv1Lu epithelial cells, and Huh7 cancer cells. Nuclear accumulation of globular actin (G-actin) and dephosphorylated cofilin was highly significant in the senescent HDF cells, accompanied with inhibition of LIM kinase (LIMK) -1 activity. When nuclear export of the actin was induced by 12-O-tetradecanoylphorbol-13-acetate, DNA synthesis of the senescent cells increased significantly, accompanied with changes of morphologic and biochemical profiles, such as increased RB protein phosphorylation and decreased expressions of p21(WAF1), cytoplasmic p-extracellular signal-regulated kinase 1/2, and caveolins 1 and 2. Significance of these findings was strengthened additionally by the fact that nuclear actin export of young HDF cells was inhibited by the treatment with leptomycin B and mutant cofilin transfection, whose LIMK-1 phosphorylation site was lost, and the old cell phenotypes were duplicated with nuclear actin accumulation, suggesting that nuclear actin accumulation was accompanied with G1 arrest during cellular senescence. The aforementioned changes were observed not only in the replicative senescence but also in the senescence induced by treatment of HDF cells, Mv1Lu, primary culture of human chondrocytes, or Huh7 cells with H-ras virus infection, hydroxyurea, deferoxamine, or H(2)O(2). Nuclear actin accumulation was much more sensitive and an earlier event than the well-known, senescence-associated beta-galactosidase activity.

Epigallocatechin-3-gallate inhibits paracrine and autocrine hepatocyte growth factor/scatter factor-induced tumor cell migration and invasion.

Aberrant activation of hepatocyte growth factor/scatter factor (HGF/SF) and its receptor, Met, is involved in the development and progression of many human cancers. In the cell-based screening assay, (-)epigallocatechin-3-gallate (EGCG) inhibited HGF/SF-Met signaling as indicated by its inhibitory activity on HGF/SF-induced cell scattering and uPA activation (IC50=15.8 microgram/ml). Further analysis revealed that EGCG at low doses specifically inhibited HGF/SF-induced tyrosine phosphorylation of Met but not epidermal growth factor (EGF)-induced phosphorylation of EGF receptor (EGFR). On the other hand, high-dose EGCG decreased both Met and EGFR proteins. We also found that EGCG did not act on the intracellular portion of Met receptor tyrosine kinase, i.e., it inhibited InlB-dependent activation of Met but not NGF-induced activation of Trk-Met hybrid receptor. This inhibition decreased HGF-induced migration and invasion by parental or HGF/SF-transfected B16F10 melanoma cells in vitro in either a paracrine or autocrine manner. Furthermore, EGCG inhibited the invasion/metastasis of HGF/SF-transfected B16F10 melanoma cells in mice. Our data suggest the possible use of EGCG in human cancers associated with dysregulated paracrine or autocrine HGF/SF-Met signaling.

Constitutive induction of p-Erk1/2 accompanied by reduced activities of protein phosphatases 1 and 2A and MKP3 due to reactive oxygen species during cellular senescence.

The mechanism of senescence-associated cytoplasmic induction of p-Erk1/2 (SA-p-Erk1/2) proteins in human diploid fibroblasts was investigated. p-Erk1/2 proteins were efficiently dephosphorylated in vitro by protein phosphatases 1 and 2A (PP1/2A) and MAPK phosphatase 3 (MKP3). Specific activity of PP1/2A and MKP3 activity significantly decreased during cellular senescence, whereas their protein expression levels did not. To investigate possible mechanism of phosphatase inactivation, we measured reactive oxygen species (ROS) generation by fluorescence-activated cell sorting analysis and found it was much higher in mid-old cells than the young cells. Treating the young cells once with 1 mm H2O2 remarkably induced p-Erk1/2 expression; however, it was transient unless repeatedly treated until 72 h. Multiple treatment of the cells with 0.2 mm H2O2 significantly duplicated inactivation of PP1/2A; however, thiol-specific reagents could reverse the PP1/2A activities, suggesting the oxidation of cysteine molecule in PP1/2A by the increased ROS. When the cells were pretreated with 10 mm N-acetyl-l-cysteine for 1 h, Erk1/2 activation was completely blocked. To elucidate which cysteine residue and/or metal ion in PP1/2A was modified by H2O2, electrospray ionization-tandem mass spectrometry analyses were performed with purified PP1C-alpha and found Cys62-SO3H and Cys105-SO3H, implicating the tertiary structure perturbation. H2O2 inhibited purified PP1C-alpha activity by both oxidation of Cys residues and metal ion(s), evidenced by dithiothreitol and ascorbate-restoration assay. In summary, SA-p-Erk1/2 was most likely due to the oxidation of PP1/2A, which resulted from the continuous exposure of the cells to vast amounts of ROS generated during cellular senescence by oxidation of Cys62 and Cys105 in PP1C-alpha and metal ion(s).