Upstream events that trigger initiation of cell division, at a point called START in yeast, determine the overall rates of cell proliferation. The identity and complete sequence of those events remain unknown. Previous studies relied mainly on cell size changes to identify systematically genes required for the timely completion of START. Here, we evaluated panels of non-essential single gene deletion strains for altered DNA content by flow cytometry. This analysis revealed that most gene deletions that altered cell cycle progression did not change cell size. Our results highlight a strong requirement for ribosomal biogenesis and protein synthesis for initiation of cell division. We also identified numerous factors that have not been previously implicated in cell cycle control mechanisms. We found that CBS, which catalyzes the synthesis of cystathionine from serine and homocysteine, advances START in two ways: by promoting cell growth, which requires CBS's catalytic activity, and by a separate function, which does not require CBS's catalytic activity. CBS defects cause disease in humans, and in animals CBS has vital, non-catalytic, unknown roles. Hence, our results may be relevant for human biology. Taken together, these findings significantly expand the range of factors required for the timely initiation of cell division. The systematic identification of non-essential regulators of cell division we describe will be a valuable resource for analysis of cell cycle progression in yeast and other organisms.
Nanocomposite electrodes made of sol-gel-derived ceramic-carbon nanotube are fabricated by doping mutliwalled carbon nanotubes (MWNTs) into a silicate gel matrix. The electrochemical behavior and potential electrochemical applications of the ceramic-carbon nanotube nanocomposite electrodes (CCNNEs) are also studied. The as-prepared CCNNEs exhibit a tunable dimension ranging from conventional electrode to nanoelectrode ensemble (NEE), depending on the amount of the MWNT dispersed in the silica sol and finally doped within the gel matrix. A high content of the MWNT (i.e., higher than 1.5 mg/mL in the sol) leads to the formation of the CCNNE characteristic of an electrode of conventional dimension, while a low content (typically lower than 0.10 mg/mL) essentially yields the CCNNE like a nanoelectrode ensemble. The NEE is demonstrated to possess good electrocatalytic activity toward the oxidation of ascorbic acid (AA), and the CCNNE of conventional dimension is found to possess remarkable electrocatalytic activity toward the oxidation of glutathione (both reduced and oxidized forms, GSH and GSSG). These properties of the CCNNEs essentially offer a new electrochemical approach for the detection of AA, GSH, and GSSG. The possible essence of the tailor-made dimensions of the CCNNEs is also presented and discussed.
The functional significance of Wnt antagonist DKK1 has not been investigated in renal cell carcinoma (RCC). Therefore, we hypothesized that DKK1 may be a tumor suppressor gene and is epigenetically silenced, thus decreased DKK1 may cause progression of RCC. To assess the function of DKK1, we established stable DKK1 transfected cells and monitored them regarding cell viability, colony formation, apoptosis, cell cycle, and invasive capability. RCC cell lines had decreased levels of DKK1, which were increased after treatment with 5-Aza-2 0 -deoxycytidine and trichostatin A. In chromatin immunoprecipitation assay, the level of dimethyl H3K9 and trimethyl H3K27 was decreased after 5-Aza-2 0 -deoxycytidine/trichostatin A treatment in RCC cell lines. Increased methylation was also associated with higher pathological stages in primary RCC tissues. T-cell factor/lymphoid enhancer factor activity and nuclear beta-catenin expression were not changed in DKK1 transfectants. Also the expression of cyclinD1 and c-Myc was not changed in DKK1 transfectants. These results suggest that DKK1 may not be involved in the beta-catenin dependent pathway. We also evaluated the expression of various related genes. Cleaved caspase3, p53, p21 and puma expression were significantly upregulated in the DKK1 transfected cells. The population of apoptotic cells was increased in stable DKK1 cells and tumor growth suppression was also observed in nude mice with DKK1 transfected cells. In conclusion, this is the first report to show that DKK1 expression is epigenetically silenced in kidney cancer and its reexpression induces apoptosis and cell cycle arrest in RCC.Renal cell carcinoma (RCC) is the third leading cause of death among urologic tumors, accounting for 2% of adult malignancies. 1 Although the rate of detection of incidental RCC has increased with improved diagnostic techniques, metastatic lesions are still found at diagnosis in $30% of patients with RCC.2 Wnt/beta-catenin signaling is involved in RCC. Canonical Wnt ligands bind to frizzled (FZD) family receptors and the LRP5/LRP6 coreceptor, which stabilize beta-catenin. Subsequently, beta-catenin interacts with members of the lymphoid enhancer factor 1/T-cell factor (LEF1/ TCF) family, resulting in a functional transcription factor complex and the expression of downstream target genes. 3,4Noncanonical Wnt ligands also bind to FZD family receptors and ROR2 and RYK coreceptors. [4][5][6][7] This signaling is mainly involved in cytoskeletal reorganization during cancer cell invasion and metastasis. 6,7 Among the five Wnt antagonist families [secreted FZD-related protein (sFRP), Wnt inhibitory factor 1 (Wif1), Xenopus cerberus, Wise and Dickkopf (DKK) families], the DKK family consists of four main members (DKK1-4), which contain two distinct cysteine-rich domains. 3,8 Recently, our laboratory reported that Wnt antagonists including Wif1, sFRP1, sFRP2, sFRP4, sFRP5 and DKK3 could be used as epigenetic biomarkers for RCC detection.9 Similar to these Wnt antagonist genes, DKK1 is also silenced...
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