Impact of the interplay between stemness features, p53 and pol iota on replication pathway choices.

Using human embryonic, adult and cancer stem cells/stem cell-like cells (SCs), we demonstrate that DNA replication speed differs in SCs and their differentiated counterparts. While SCs decelerate DNA replication, differentiated cells synthesize DNA faster and accumulate DNA damage. Notably, both replication phenotypes depend on p53 and polymerase iota (POLι). By exploring protein interactions and newly synthesized DNA, we show that SCs promote complex formation of p53 and POLι at replication sites. Intriguingly, in SCs the translocase ZRANB3 is recruited to POLι and required for slow-down of DNA replication. The known role of ZRANB3 in fork reversal suggests that the p53-POLι complex mediates slow but safe bypass of replication barriers in SCs. In differentiated cells, POLι localizes more transiently to sites of DNA synthesis and no longer interacts with p53 facilitating fast POLι-dependent DNA replication. In this alternative scenario, POLι associates with the p53 target p21, which antagonizes PCNA poly-ubiquitination and, thereby potentially disfavors the recruitment of translocases. Altogether, we provide evidence for diametrically opposed DNA replication phenotypes in SCs and their differentiated counterparts putting DNA replication-based strategies in the spotlight for the creation of therapeutic opportunities targeting SCs.

The transient production of anti-TNF-α antibody Adalimumab and a comparison of its characterization to the biosimilar Cinorra.

Recombinant antibodies have emerged over the last few decades as the fastest growing class of therapeutic proteins for autoimmune diseases. Post-translation modifications of antibodies produced by human cell lines are highly consistent with those existing in natural human proteins and this is a major advantage of utilizing these cell lines. Cinorra is a biosimilar form of the antibody Adalimumab, which is an antagonist of TNF-α used for the treatment of autoimmune diseases. Adalimumab and Cinorra were produced by stable expression from CHO cells. The aim of this study was to select HEK cells as a host for producing Adalimumab to reveal whether the antibody produced by this human-derived cell line has similar characterization to Cinorra. Adalimumab was transiently produced in HEK-293T cells, characterized and analyzed for its properties. Circular dichroism spectroscopy confirmed a strong structural similarity of the expressed antibody with Cinorra. Likewise its binding activity and kinetic affinity to TNF-α (EC50 = 416.5 ng/ml, KD = 3.89 E-10 M,) were highly similar to that of Cinorra (EC50 = 421.2 ng/ml and KD = 3.34 E-10 M,). Additionally there was near identical neutralization of TNF-α-mediated cellular cytotoxicity (IC50 of the expressed = 4.93 nM; IC50 of Cinorra = 4.5 nM). Results indicate that Adalimumab produced by HEK-293T cells possesses a similarly efficient function and biological activity to Cinorra. Consequently, human-derived host cells with human post-translational modifications might potentially provide a basis for the development of Adalimumab with pharmaceutical properties for research and therapeutic use.

Amniotic Fluid Cells, Stem Cells, and p53: Can We Stereotype p53 Functions?

In recent years, great interest has been devoted to finding alternative sources for human stem cells which can be easily isolated, ideally without raising ethical objections. These stem cells should furthermore have a high proliferation rate and the ability to differentiate into all three germ layers. Amniotic fluid, ordinarily discarded as medical waste, is potentially such a novel source of stem cells, and these amniotic fluid derived stem cells are currently gaining a lot of attention. However, further information will be required about the properties of these cells before they can be used for therapeutic purposes. For example, the risk of tumor formation after cell transplantation needs to be explored. The tumor suppressor protein p53, well known for its activity in controlling Cell Prolif.eration and cell death in differentiated cells, has more recently been found to be also active in amniotic fluid stem cells. In this review, we summarize the major findings about human amniotic fluid stem cells since their discovery, followed by a brief overview of the important role played by p53 in embryonic and adult stem cells. In addition, we explore what is known about p53 in amniotic fluid stem cells to date, and emphasize the need to investigate its role, particularly in the context of cell tumorigenicity.

Nucleotide excision repair driven by the dissociation of CAK from TFIIH.

The transcription/DNA repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during nucleotide excision repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/excision of the damaged oligonucleotide and thereby the repair of the DNA. Following repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.

Hierarchical Micro-Nano Surface Topography Promotes Long-Term Maintenance of Undifferentiated Mouse Embryonic Stem Cells.

Understanding of stem cell-surface interactions and, in particular, long-term maintenance of stem cell pluripotency on well-defined synthetic surfaces is crucial for fundamental research and biomedical applications of stem cells. Here, we show that synthetic surfaces possessing hierarchical micro-nano roughness (MN-surfaces) promote long-term self-renewal (>3 weeks) of mouse embryonic stem cells (mESCs) as monitored by the expression levels of the pluripotency markers octamer-binding transcription factor 4 (Oct4), Nanog, and alkaline phosphatase. On the contrary, culturing of mESCs on either smooth (S-) or nanorough polymer surfaces (N-surfaces) leads to their fast differentiation. Moreover, we show that regular passaging of mESCs on the hierarchical MN-polymer surface leads to an increased homogeneity and percentage of Oct4-positive stem cell colonies as compared to mESCs grown on fibroblast feeder cells. Immunostaining revealed the absence of focal adhesion markers on all polymer substrates studied. However, only the MN-surfaces elicited the formation of actin-positive cell protrusions, indicating an alternative anchorage mechanism involved in the maintenance of mESC stemness.

Expression screening using a Medaka cDNA library identifies evolutionarily conserved regulators of the p53/Mdm2 pathway.

BACKGROUND: The p53 tumor suppressor protein is mainly regulated by alterations in the half-life of the protein, resulting in significant differences in p53 protein levels in cells. The major regulator of this process is Mdm2, which ubiquitinates p53 and targets it for proteasomal degradation. This process can be enhanced or reduced by proteins that associate with p53 or Mdm2 and several proteins have been identified with such an activity. Furthermore, additional ubiquitin ligases for p53 have been identified in recent years. Nevertheless, our understanding of how p53 abundance and Mdm2 activity are regulated remains incomplete. Here we describe a cell culture based overexpression screen to identify evolutionarily conserved regulators of the p53/Mdm2 circuit. The results from this large-scale screening method will contribute to a better understanding of the regulation of these important proteins. METHODS: Expression screening was based on co-transfection of H1299 cells with pools of cDNA's from a Medaka library together with p53, Mdm2 and, as internal control, Ror2. After cell lysis, SDS-PAGE/WB analysis was used to detect alterations in these proteins. RESULTS: More than one hundred hits that altered the abundance of either p53, Mdm2, or both were identified in the primary screen. Subscreening of the library pools that were identified in the primary screen identified several potential novel regulators of p53 and/or Mdm2. We also tested whether the human orthologues of the Medaka genes regulate p53 and/or Mdm2 abundance. All human orthologues regulated p53 and/or Mdm2 abundance in the same manner as the proteins from Medaka, which underscores the suitability of this screening methodology for the identification of new modifiers of p53 and Mdm2. CONCLUSIONS: Despite enormous efforts in the last two decades, many unknown regulators for p53 and Mdm2 abundance are predicted to exist. This cross-species approach to identify evolutionarily conserved regulators demonstrates that our Medaka unigene cDNA library represents a powerful tool to screen for these novel regulators of the p53/Mdm2 pathway.

Mdm2 facilitates the association of p53 with the proteasome.

The ubiquitin ligase Mdm2 targets the p53 tumor suppressor protein for proteasomal degradation. Mutating phosphorylation sites in the central domain of Mdm2 prevents p53 degradation, although it is still ubiquitylated, indicating that Mdm2 has a post-ubiquitylation function for p53 degradation. We show that Mdm2 associates with several subunits of the 19S proteasome regulatory particle in a ubiquitylation-independent manner. Mdm2 furthermore promotes the formation of a ternary complex of itself, p53, and the proteasome. Replacing phosphorylation sites within the central domain with alanines reduced the formation of the ternary complex. The C-terminus of Mdm2 was sufficient for interaction with the proteasome despite an additional proteasome binding site in the Mdm2 N-terminus. In addition to binding to the proteasome, the C-terminus of Mdm2 bound to the central domain, possibly competing with, and therefore blocking, Mdm2/proteasome interaction. We propose that Mdm2 facilitates, or at least enhances, the association of p53 with the proteasome and that phosphorylation of the central domain of Mdm2 regulates this process.

TRIM25 targets p300 for degradation.

p300 is an important transcriptional co-factor. By stimulating the transfer of acetyl residues onto histones and several key transcription factors, p300 enhances transcriptional initiation and impacts cellular processes including cell proliferation and cell division. Despite its importance for cellular homeostasis, its regulation is poorly understood. We show that TRIM25, a member of the TRIM protein family, targets p300 for proteasomal degradation. However, despite TRIM25's RING domain and E3 activity, degradation of p300 by TRIM25 is independent of TRIM25-mediated p300 ubiquitination. Instead, TRIM25 promotes the interaction of p300 with dynein, which ensures a microtubule-dependent transport of p300 to cellular proteasomes. Through mediating p300 degradation, TRIM25 affects p300-dependent gene expression.

Identification of an Imidazopyridine-based Compound as an Oral Selective Estrogen Receptor Degrader for Breast Cancer Therapy.

The pro-oncogenic activities of estrogen receptor alpha (ERα) drive breast cancer pathogenesis. Endocrine therapies that impair the production of estrogen or the action of the ERα are therefore used to prevent primary disease metastasis. Although recent successes with ERα degraders have been reported, there is still the need to develop further ERα antagonists with additional properties for breast cancer therapy. We have previously described a benzothiazole compound A4B17 that inhibits the proliferation of androgen receptor-positive prostate cancer cells by disrupting the interaction of the cochaperone BAG1 with the AR. A4B17 was also found to inhibit the proliferation of estrogen receptor-positive (ER+) breast cancer cells. Using a scaffold hopping approach, we report here a group of small molecules with imidazopyridine scaffolds that are more potent and efficacious than A4B17. The prototype molecule X15695 efficiently degraded ERα and attenuated estrogen-mediated target gene expression as well as transactivation by the AR. X15695 also disrupted key cellular protein-protein interactions such as BAG1-mortalin (GRP75) interaction as well as wild-type p53-mortalin or mutant p53-BAG2 interactions. These activities together reactivated p53 and resulted in cell-cycle block and the induction of apoptosis. When administered orally to in vivo tumor xenograft models, X15695 potently inhibited the growth of breast tumor cells but less efficiently the growth of prostate tumor cells. We therefore identify X15695 as an oral selective ER degrader and propose further development of this compound for therapy of ER+ breast cancers. Significance: An imidazopyridine that selectively degrades ERα and is orally bioavailable has been identified for the development of ER+ breast cancer therapeutics. This compound also activates wild-type p53 and disrupts the gain-of-function tumorigenic activity of mutant p53, resulting in cell-cycle arrest and the induction of apoptosis.

Meeting report: Signal transduction meets systems biology.

In the 21st century, systems-wide analyses of biological processes are getting more and more realistic. Especially for the in depth analysis of signal transduction pathways and networks, various approaches of systems biology are now successfully used. The EU FP7 large integrated project SYBILLA (Systems Biology of T-cell Activation in Health and Disease) coordinates such an endeavor. By using a combination of experimental data sets and computational modelling, the consortium strives for gaining a detailed and mechanistic understanding of signal transduction processes that govern T-cell activation. In order to foster the interaction between systems biologists and experimentally working groups, SYBILLA co-organized the 15th meeting "Signal Transduction: Receptors, Mediators and Genes" together with the Signal Transduction Society (STS). Thus, the annual STS conference, held from November 7 to 9, 2011 in Weimar, Germany, provided an interdisciplinary forum for research on signal transduction with a major focus on systems biology addressing signalling events in T-cells. Here we report on a selection of ongoing projects of SYBILLA and how they were discussed at this interdisciplinary conference.