Post-cellular biotic evolution in a nutshell

BackgroundDifferentiation and fusion of skeletal muscle myoblasts into multi-nucleated myotubes is required for neonatal development and regeneration in adult skeletal muscle. Herein, we report novel findings that protein kinase C theta (PKCθ) regulates myoblast differentiation via phosphorylation of insulin receptor substrate-1 and ERK1/2.ResultsIn this study, PKCθ knockdown (PKCθshRNA) myotubes had reduced inhibitory insulin receptor substrate-1 ser1095 phosphorylation, enhanced myoblast differentiation and cell fusion, and increased rates of protein synthesis as determined by [3H] phenylalanine incorporation. Phosphorylation of insulin receptor substrate-1 ser632/635 and extracellular signal-regulated kinase1/2 (ERK1/2) was increased in PKCθshRNA cells, with no change in ERK5 phosphorylation, highlighting a PKCθ-regulated myogenic pathway. Inhibition of PI3-kinase prevented cell differentiation and fusion in control cells, which was attenuated in PKCθshRNA cells. Thus, with reduced PKCθ, differentiation and fusion occur in the absence of PI3-kinase activity. Inhibition of the ERK kinase, MEK1/2, impaired differentiation and cell fusion in control cells. Differentiation was preserved in PKCθshRNA cells treated with a MEK1/2 inhibitor, although cell fusion was blunted, indicating PKCθ regulates differentiation via IRS1 and ERK1/2, and this occurs independently of MEK1/2 activation.ConclusionCellular signaling regulating the myogenic program and protein synthesis are complex and intertwined. These studies suggest that PKCθ regulates myogenic and protein synthetic signaling via the modulation of IRS1and ERK1/2 phosphorylation. Myotubes lacking PKCθ had increased rates of protein synthesis and enhanced myotube development despite reduced activation of the canonical anabolic-signaling pathway. Further investigation of PKCθ regulated signaling may reveal important interactions regulating skeletal muscle health in an insulin resistant state.

Differential expression of the GLUT1 and GLUT4 glucose transporters during differentiation of L6 muscle cells

The Xenobiotic Transporter ABCG2 Plays a Novel Role in Differentiation of Trophoblast-like BeWo Cells

The syncytiotrophoblast is the major component of the human placenta, involved in feto-maternal exchanges and secretion of pregnancy-specific hormones. Multinucleated syncytiotrophoblast arises from fusion of mononuclear cytotrophoblast cells. In trisomy 21-affected placentas, we recently have shown that there is a defect in syncytiotrophoblast formation and a decrease in the production of pregnancy-specific hormones. Due to the role of oxygen free radicals in trophoblast cell differentiation, we investigated the role of the key antioxidant enzyme, copper/zinc superoxide dismutase, encoded by chromosome 21 in in vitro trophoblast differentiation. We first observed that overexpression of superoxide dismutase in normal cytotrophoblasts impaired syncytiotrophoblast formation. This was associated with a significant decrease in mRNA transcript levels and secretion of hCG and other hormonal markers of syncytiotrophoblast. We confirmed abnormal cell fusion by overexpression of green fluorescence protein-tagged superoxide dismutase in cytotrophoblasts. In addition, a significant decrease in syncytin transcript levels was observed in superoxide dismutase-transfected cells. We then examined superoxide dismutase expression and activity in isolated trophoblast cells from trisomy 21-affected placentas. Superoxide dismutase mRNA expression (P < 0.05), protein levels (P < 0.01), and activity (P < 0.05) were significantly higher in trophoblast cells isolated from trisomy 21-affected placentas than in those from normal placentas. These results suggest that superoxide dismutase overexpression may directly impair trophoblast cell differentiation and fusion, and superoxide dismutase overexpression in Down's syndrome may be responsible at least in part for the failure of syncytiotrophoblast formation observed in trisomy 21-affected placentas.

PASTICCINO1 (PAS1) is a high molecular weight FK506-binding protein (FKBP) involved in the control of cell proliferation and differentiation during plant development. Mutations in the C-terminal region of PAS1 result in severe developmental defects. We show here that the C-terminal domain of PAS1 controls the subcellular distribution of this protein. We also demonstrated in vitro and in vivo, by Forster resonance energy transfer, that this C-terminal region is required for interaction with FAN (FKBP-associated NAC), a new member of the plant-specific family of NAC transcription factors. PAS1 and FAN are translocated into the nucleus upon auxin treatment in plant seedlings. The nuclear translocation of PAS1 is dependent on the presence of the C terminus of the protein. Finally, we showed that FAN is involved in PAS1-regulated processes because FAN overproduction partly complemented the pas1 phenotype. We suggest that PAS1 regulates the function of this NAC-like transcription factor by controlling its targeting to the nucleus upon plant cell division.

BackgroundEukaryotic chromatin consists of nucleosome core particles connected by linker DNA of variable length. Histone H1 associates with the linker DNA to stabilize the higher-order chromatin structure and to modulate the ability of regulatory factors to access their nucleosomal targets. In Saccharomyces cerevisiae, the protein with greatest sequence similarity to H1 is Hho1p. However, during vegetative growth, hho1∆ cells do not show any discernible cell growth defects or the changes in bulk chromatin structure that are characteristic of chromatin from multicellular eukaryotes in which H1 is depleted. In contrast, the yeast high mobility group (HMGB) protein HMO1 has been reported to compact chromatin, as evidenced by increased nuclease sensitivity in hmo1∆ cells. HMO1 has an unusual domain architecture compared to vertebrate HMGB proteins in that the HMG domains are followed by a lysine-rich extension instead of an acidic domain. We address here the hypothesis that HMO1 serves the role of H1 in terms of chromatin compaction and that this function requires the lysine-rich extension.ResultsWe show here that HMO1 fulfills this function of a linker histone. For histone H1, chromatin compaction requires its basic C-terminal domain, and we find that the same pertains to HMO1, as deletion of its C-terminal lysine-rich extension renders chromatin nuclease sensitive. On rDNA, deletion of both HMO1 and Hho1p is required for significantly increased nuclease sensitivity. Expression of human histone H1 completely reverses the nuclease sensitivity characteristic of chromatin isolated from hmo1∆ cells. While chromatin remodeling events associated with repair of DNA double-strand breaks occur faster in the more dynamic chromatin environment created by the hmo1 deletion, expression of human histone H1 results in chromatin remodeling and double-strand break repair similar to that observed in wild-type cells.ConclusionOur data suggest that S. cerevisiae HMO1 protects linker DNA from nuclease digestion, a property also characteristic of mammalian linker histone H1. Notably, association with HMO1 creates a less dynamic chromatin environment that depends on its lysine-rich domain. That HMO1 has linker histone function has implications for investigations of chromatin structure and function as well as for evolution of proteins with roles in chromatin compaction.

The Mesoproterozoic era (1,600–1,000 million years ago (Ma)) has long been considered a period of relative environmental stasis, with persistently low levels of atmospheric oxygen. There remains much uncertainty, however, over the evolution of ocean chemistry during this period, which may have been of profound significance for the early evolution of eukaryotic life. Here we present rare earth element, iron-speciation and inorganic carbon isotope data to investigate the redox evolution of the 1,600–1,550 Ma Yanliao Basin, North China Craton. These data confirm that the ocean at the start of the Mesoproterozoic was dominantly anoxic and ferruginous. Significantly, however, we find evidence for a progressive oxygenation event starting at ~1,570 Ma, immediately prior to the occurrence of complex multicellular eukaryotes in shelf areas of the Yanliao Basin. Our study thus demonstrates that oxygenation of the Mesoproterozoic environment was far more dynamic and intense than previously envisaged, and establishes an important link between rising oxygen and the emerging record of diverse, multicellular eukaryotic life in the early Mesoproterozoic. The oxygenation of deeper continental shelf waters during the Mesoproterozoic coincided with the appearance of multicellular eukaryotes, according to geochemical and sedimentological analyses of the Yanliao Basin, China.

&lt;div&gt;Abstract&lt;p&gt;MYC regulates a myriad of genes controlling cell proliferation, metabolism, differentiation, and apoptosis. MYC also controls the expression of DNA double-strand break (DSB) repair genes and therefore may be a potential target for anticancer therapy to sensitize cancer cells to DNA damage or prevent genetic instability. In this report, we studied whether MYC binds to DSB repair gene promoters and modulates cell survival in response to DNA-damaging agents. Chromatin immunoprecipitation studies showed that MYC associates with several DSB repair gene promoters including &lt;i&gt;Rad51, Rad51B, Rad51C, XRCC2, Rad50, BRCA1, BRCA2, DNA-PKcs, XRCC4, Ku70&lt;/i&gt;, and &lt;i&gt;DNA ligase IV&lt;/i&gt;. Endogenous MYC protein expression was associated with increased RAD51 and KU70 protein expression of a panel of cancer cell lines of varying histopathology. Induction of MYC in G&lt;sub&gt;0&lt;/sub&gt;-G&lt;sub&gt;1&lt;/sub&gt; and S-G&lt;sub&gt;2&lt;/sub&gt;-M cells resulted in upregulation of &lt;i&gt;Rad51&lt;/i&gt; gene expression. MYC knockdown using small interfering RNA (siRNA) led to decreased RAD51 expression but minimal effects on homologous recombination based on a flow cytometry direct repeat green fluorescent protein assay. siRNA to MYC resulted in tumor cell kill in DU145 and H1299 cell lines in a manner independent of apoptosis. However, MYC-dependent changes in DSB repair protein expression were not sufficient to sensitize cells to mitomycin C or ionizing radiation, two agents selectively toxic to DSB repair–deficient cells. Our results suggest that anti-MYC agents may target cells to prevent genetic instability but would not lead to differential radiosensitization or chemosensitization. Cancer Res; 70(21); 8748–59. ©2010 AACR.&lt;/p&gt;&lt;/div&gt;

Our study was to elucidate the mechanisms whereby BMS-345541 (BMS, a specific IκB kinase β inhibitor) inhibits the repair of DNA double-strand breaks (DSBs) and evaluate whether BMS can sensitize MCF-7 breast cancer cells (MCF-7 cells) to ionizing radiation (IR) in an apoptosis-independent manner. In this study, MCF-7 cells were exposed to IR in vitro and in vivo with or without pretreatment of BMS. The effects of BMS on the repair of IR-induced DSBs by homologous recombination (HR) and non-homologous end-joining (NHEJ) were analyzed by the DR-GFP and EJ5-GFP reporter assays and IR-induced γ-H2AX, 53BP1, Brca1 and Rad51 foci assays. The mechanisms by which BMS inhibits HR were examined by microarray analysis and quantitative reverse transcription PCR. The effects of BMS on the sensitivity of MCF-7 cells to IR were determined by MTT and clonogenic assays in vitro and tumor growth inhibition in vivo in a xenograft mouse model. The results showed that BMS selectively inhibited HR repair of DSBs in MCF-7 cells, most likely by down-regulation of several genes that participate in HR. This resulted in a significant increase in the DNA damage response that sensitizes MCF-7 cells to IR-induced cell death in an apoptosis-independent manner. Furthermore, BMS treatment sensitized MCF-7 xenograft tumors to radiation therapy in vivo in an association with a significant delay in the repair of IR-induced DSBs. These data suggest that BMS is a novel HR inhibitor that has the potential to be used as a radiosensitizer to increase the responsiveness of cancer to radiotherapy.

Purpose: To test the hypothesis that differences in DNA double-strand breaks (DSB) repair fidelity underlies differences in radiosensitivity.Materials and methods: A primary fibroblast culture (C42) derived from a pediatric cancer patient treated with reduced radiation doses consequent to a family history of radiosensitivity reminiscent of chromosomal fragility syndrome, was compared to a normal control (C29). DNA DSB rejoining and repair fidelity were studied by Southern blotting and hybridization to specific fragments: Alu repetitive sequence representing the overall DSB rejoining capacity in the genome and a 3.2 Mbp NotI restriction fragment on chromosome 21 for DSB repair fidelity.Results: Although both assays showed statistically significant difference (p ≤ 0.05) between the two cell strains in residual misrepaired (un-or mis-rejoined) DSB (24 h after 30 or 80 Gy), the residual damage was lower in the Alu enriched genome assay compared to NotI assay (0.01–0.07 and 0.10–0.37, respectively).Conclusions: These results suggest that, in comparison to classic DSB repair experiment, an assay of measuring DNA DSB repair fidelity can provide better resolution and a more accurate estimate of misrepair of radiation-induced DNA damage, which underlies genomic instability and increased radiosensitivity.

Bone Marrow–Derived Hepatic Oval Cells Differentiate Into Hepatocytes in 2-Acetylaminofluorene/Partial Hepatectomy–Induced Liver Regeneration

Ataxia telangiectasia (A-T) is a human genetic disorder characterized by progressive cerebellar degeneration, hypersensitivity to ionizing radiation (IR), immunodeficiency, and high cancer risk. At the cellular level, IR sensitivity and increased frequency of spontaneous and IR-induced chromosomal breakage and rearrangements are the hallmarks of A-T. The ATM gene, mutated in this syndrome, has been cloned and codes for a protein sharing homology with DNA-PKcs, a protein kinase involved in DNA double-strand break (DSB) repair and DNA damage responses. The characteristics of the A-T cellular phenotypes and ATM gene suggest that ATM may play a role similar to that of DNA-PKcs in DSB repair and that there is a primary DNA repair defect in A-T cells. In the current study, the function of ATM in DNA DSB repair was evaluated in an in vitro system using two plasmids, carrying either an EcoRI-induced DSB within the lacZalpha gene or various endonuclease-induced DSB in the SupF suppressor tRNA gene. We found that the DSB repair efficiency in A-T nuclear extracts was comparable to, if not higher than, that in normal nuclear extracts. However, the repair fidelity in A-T nuclear extracts was decreased when repairing DSB with short 5' and 3' overhangs (<4 base pairs (bp)) or blunt ends, but not 5' 4-bp overhangs. Sequencing of the mutant plasmids revealed that deletions involving 1-6 nucleotide microhomologies were the major class of mutations in both A-T and normal extracts. However, the size of the deletions in plasmids from A-T nuclear extracts was larger than that from normal nuclear extracts. Expression of the ATM protein in A-T cells corrected the defect in DSB repair in A-T nuclear extracts. These results suggest that ATM plays a role in maintaining genomic stability by preventing the repair of DSB from an error-prone pathway.

Visualizing the First Steps of Human Double-Strand Break Repair on a Crowded DNA Track

Diamond Blackfan anemia (DBA) is caused by germline heterozygous loss-of-function pathogenic variants (PVs) in ribosomal protein (RP) genes, most commonly RPS19 and RPL5 . In addition to red cell aplasia, individuals with DBA are at increased risk of various cancers. Importantly, the mechanism(s) underlying cancer predisposition are poorly understood. We found that DBA patient-derived lymphoblastoid cells had persistent γ-H2AX foci following ionizing radiation (IR) treatment, suggesting DNA double-strand break (DSB) repair defects. RPS19- and RPL5-knocked down (KD) CD34+ cells had delayed repair of IR-induced DSBs, further implicating these RPs in DSB repair. Assessing the impact of RPS19- and RPL5-KD on specific DSB repair pathways, we found RPS19-KD decreased the efficiency of pathways requiring extensive end-resection, whereas RPL5-KD increased end-joining pathways. Additionally, RAD51 was reduced in RPS19- and RPL5-KD and RPS19- and RPL5-mutated DBA cells, whereas RPS19-deficient cells also had a reduction in PARP1 and BRCA2 proteins. RPS19-KD cells had an increase in nuclear RPA2 and a decrease in nuclear RAD51 foci post-IR, reflective of alterations in early, critical steps of homologous recombination. Notably, RPS19 and RPL5 interacted with poly(ADP)-ribose chains noncovalently, were recruited to DSBs in a poly(ADP)-ribose polymerase activity-dependent manner, and interacted with Ku70 and histone H2A. RPL5's recruitment, but not RPS19's, also required p53, suggesting that RPS19 and RPL5 directly participate in DSB repair via different pathways. We propose that defective DSB repair arising from haploinsufficiency of these RPs may underline the cancer predisposition in DBA.

Purpose: Ionizing radiations of different qualities (e.g. high-LET and low-LET) might differently interact with structurally and functionally distinct higher order chromatin domains (discussed in [1] and citations therein); this might be reflected by DNA double strand break (DSB) repair efficiency and the mechanism of how cancerogenous chromosomal translocations (CHT) form. Therefore, we compared the DSB repair kinetics and formation of γH2AX/p53BP1 repair clusters upon the action of γ-rays [2, 3], protons (15 and 30 MeV) [4], and 20Ne ions (preliminary data). Consequently, we discuss biological impacts of these clusters.Material and methods: Immunostaining methods in combination with high-resolution confocal microscopy, performed on 3D-fixed normal human skin fibroblasts [2–4], were used to study initial distributions of γH2AX and p53BP1 repair foci and their changes during the post-irradiation (PI) time, with a special concern on foci clustering. Irradiations with γ-rays, protons of different energies (15 and 30 MeV), and high-LET 20Ne ions was performed in IBP ASCR Brno (CR), NPI AVCR Řež (CR) and JINR Dubna (Russia), respectively.Results: Upon irradiating cells with 20Ne ions, tracks of multiple clustered γH2AX and p53BP1 repair foci appeared immediately after the irradiation; these clusters, called here as the ‘primary clusters’, were rare in cells irradiated with γ-rays or protons (submitted). Though γH2AX/p53BP1 foci were positionally quite stable [2], ‘secondary clusters’ occasionally appeared after all kinds of irradiation during about 30 min PI. The formation of secondary clusters usually appeared due to the heterochromatin decondensation at the sites of heterochromatic DNA double-strand breaks (hcDSBs), followed by their protrusion into a limited space of nuclear subdomains of low density-chromatin (discussed in [1, 2, 5]).Conclusions: Primary clusters appear in cell nuclei immediately PI as the consequence of highly localized energy deposition, while secondary clusters develop during (and because of) DSB repair. Primary DSB clusters probably represent the main cause of chromosomal translocations induced with high-LET radiations while secondary clusters seem to be more important for low-LET γ-rays and protons. Secondary clusters of primary clusters (higher-order clusters) observed for 20Ne ions might explain frequent formation of complex translocations upon the action of high-LET radiations. Finally, we suggest [1, 2, 4] a model that describes the relationship between the higher order chromatin structure, DSB formation, repair and misrepair.