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A cell cycle role for the epigenetic factor CTCF-L/BORIS.
CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.
Document Type:

Reference

Product Catalog Number:

07-729
Product Catalog Name:

Anti-CTCF Antibody
Cell cycle- and cancer-associated gene networks activated by Dsg2: evidence of cystatin a deregulation and a potential role in cell-cell adhesion.
Cell-cell adhesion is paramount in providing and maintaining multicellular structure and signal transmission between cells. In the skin, disruption to desmosomal regulated intercellular connectivity may lead to disorders of keratinization and hyperproliferative disease including cancer. Recently we showed transgenic mice overexpressing desmoglein 2 (Dsg2) in the epidermis develop hyperplasia. Following microarray and gene network analysis, we demonstrate that Dsg2 caused a profound change in the transcriptome of keratinocytes in vivo and altered a number of genes important in epithelial dysplasia including: calcium-binding proteins (S100A8 and S100A9), members of the cyclin protein family, and the cysteine protease inhibitor cystatin A (CSTA). CSTA is deregulated in several skin cancers, including squamous cell carcinomas (SCC) and loss of function mutations lead to recessive skin fragility disorders. The microarray results were confirmed by qPCR, immunoblotting, and immunohistochemistry. CSTA was detected at high level throughout the newborn mouse epidermis but dramatically decreased with development and was detected predominantly in the differentiated layers. In human keratinocytes, knockdown of Dsg2 by siRNA or shRNA reduced CSTA expression. Furthermore, siRNA knockdown of CSTA resulted in cytoplasmic localization of Dsg2, perturbed cytokeratin 14 staining and reduced levels of desmoplakin in response to mechanical stretching. Both knockdown of either Dsg2 or CSTA induced loss of cell adhesion in a dispase-based assay and the effect was synergistic. Our findings here offer a novel pathway of CSTA regulation involving Dsg2 and a potential crosstalk between Dsg2 and CSTA that modulates cell adhesion. These results further support the recent human genetic findings that loss of function mutations in the CSTA gene result in skin fragility due to impaired cell-cell adhesion: autosomal-recessive exfoliative ichthyosis or acral peeling skin syndrome.
Document Type:

Reference

Product Catalog Number:

AB4065
Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage.
The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.
Document Type:

Reference

Product Catalog Number:

05-636
Product Catalog Name:

Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301
Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis.
8-Oxoguanine DNA glycosylase (OGG1) is a major DNA repair enzyme in mammalian cells. OGG1 participates in the repair of 8-oxoG, the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. In this study, antibodies directed against purified recombinant human OGG1 (hOGG1) or murine (mOGG1) protein were chemically conjugated to either the photosensitizer Rose Bengal or the fluorescent dye Texas red. These dye-protein conjugates, in combination with binding assays, were used to identify associations between mOGG1 and the cytoskeleton of NIH3T3 fibroblasts. Results from these binding studies showed that mOGG1 associates with the cytoskeleton by specifically binding to the centriole and microtubules radiating from the centrosome at interphase and the spindle assembly at mitosis. Similar results were obtained with hOGG1. Together results reported in this study suggest that OGG1 is a microtubule-associated protein itself or that OGG1 utilizes yet to be identified motor proteins to ride on microtubules as tracks facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage.
Document Type:

Reference

Product Catalog Number:

MAB1636
Cell lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration.
Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.
Document Type:

Reference

Product Catalog Number:

AB5977
Product Catalog Name:

Anti-Musashi-1 Antibody
Cell cycle regulation of DNA replication initiator factor Dbf4p.
The precise duplication of eukaryotic genetic material takes place once and only once per cell cycle and is dependent on the completion of the previous mitosis. Two evolutionarily conserved kinases, the cyclin B (Clb)/cyclin-dependent kinase (Cdk/Cdc28p) and Cdc7p along with its interacting factor Dbf4p, are required late in G1 to initiate DNA replication. We have determined that the levels of Dbf4p are cell cycle regulated. Dbf4p levels increase as cells begin S phase and remain high through late mitosis, after which they decline dramatically as cells begin the next cell cycle. We report that Dbf4p levels are sensitive to mutations in key components of the anaphase-promoting complex (APC). In addition, Dbf4p is modified in response to DNA damage, and this modification is dependent upon the DNA damage response pathway. We had previously shown that Dbf4p interacts with the M phase polo-like kinase Cdc5p, a key regulator of the APC late in mitosis. These results further link the actions of the initiator protein, Dbf4p, to the completion of mitosis and suggest possible roles for Dbf4p during progression through mitosis.
Document Type:

Reference

Product Catalog Number:

AP182F
Cell cycle regulated phosphorylation of the telomere-associated protein TIN2.
The protein TIN2 is a member of telomere-binding protein complex that serves to cap and protect mammalian chromosome ends. As a number of proteins in this complex are phosphorylated in a cell cycle-dependent manner, we investigated whether TIN2 is modified by phosphorylation as well. We performed phospho-proteomic analysis of human TIN2, and identified two phosphorylated residues, serines 295 and 330. We demonstrated that both these sites were phosphorylated during mitosis in human cells, as detected by Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2. Specifically, phosphorylation of TIN2 at both these residues was increased upon expression of RSK2 and reduced by an inhibitor of the RSK family of kinases. Moreover, RSK2 phosphorylated TIN2 in vitro. The identification of these specifically timed post-translational events during the cell cycle suggests a potential mitotic regulation of TIN2 by phosphorylation.
Document Type:

Reference

Product Catalog Number:

ABE1314
Product Catalog Name:

Anti-phospho TIN2 (Ser295) Antibody
Cell cycle arrest and proapoptotic effects of the anticancer cyclodepsipeptide serratamolide (AT514) are independent of p53 status in breast cancer cells.
In a search for new anticancer agents, we have identified serratamolide (AT514), a cyclodepsipeptide from Serratia marcescens 2170 that induces cell cycle arrest and apoptosis in various cancer cell lines. A cell viability assay showed that the concentrations that cause 50% inhibition (IC50) in human cancer cell lines range from 5.6 to 11.5 microM depending on the cell line. Flow cytometry analysis revealed that AT514 caused cell cycle arrest in G0/G1 or cell death, depending on the cell type and the length of time for which the cells were exposed to the drug. Subsequent studies revealed that AT514-induced cell death is caused by apoptosis, as indicated by caspases activation (8, 9, 2 and 3) and cleavage of poly (ADP-ribose) polymerase (PARP), release of cytochrome c and apoptosis inducing factor (AIF) from mitochondria, and the appearance of apoptotic bodies and DNA laddering. Alterations in protein levels of Bcl-2 family members might be involved in the mitochondrial disruption observed. AT514 induced p53 accumulation in wild-type p53 cells but cell death was observed in both deficient and wild-type p53 cells. Our results indicate that AT514 induces cell cycle arrest and apoptosis in breast cancer cells irrespectively of p53 status, suggesting that it might represent a potential new chemotherapeutic agent.
Document Type:

Reference

Product Catalog Number:

05-572
Cell cycle, oncogenic and tumor suppressor pathways regulate numerous long and macro non-protein-coding RNAs.
The genome is pervasively transcribed but most transcripts do not code for proteins, constituting non-protein-coding RNAs. Despite increasing numbers of functional reports of individual long non-coding RNAs (lncRNAs), assessing the extent of functionality among the non-coding transcriptional output of mammalian cells remains intricate. In the protein-coding world, transcripts differentially expressed in the context of processes essential for the survival of multicellular organisms have been instrumental in the discovery of functionally relevant proteins and their deregulation is frequently associated with diseases. We therefore systematically identified lncRNAs expressed differentially in response to oncologically relevant processes and cell-cycle, p53 and STAT3 pathways, using tiling arrays.We found that up to 80% of the pathway-triggered transcriptional responses are non-coding. Among these we identified very large macroRNAs with pathway-specific expression patterns and demonstrated that these are likely continuous transcripts. MacroRNAs contain elements conserved in mammals and sauropsids, which in part exhibit conserved RNA secondary structure. Comparing evolutionary rates of a macroRNA to adjacent protein-coding genes suggests a local action of the transcript. Finally, in different grades of astrocytoma, a tumor disease unrelated to the initially used cell lines, macroRNAs are differentially expressed.It has been shown previously that the majority of expressed non-ribosomal transcripts are non-coding. We now conclude that differential expression triggered by signaling pathways gives rise to a similar abundance of non-coding content. It is thus unlikely that the prevalence of non-coding transcripts in the cell is a trivial consequence of leaky or random transcription events.
Document Type:

Reference

Product Catalog Number:

12-370
Product Catalog Name:

Normal Rabbit IgG
Cell cycle-dependent activation of Ras.
BACKGROUND: Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated. RESULTS: To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) sub-group of mitogen-activated protein kinases were not temporally correlated during G1-phase progression. CONCLUSIONS: Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple