The MRN complex is transcriptionally regulated by MYCN during neural cell proliferation to control replication stress

The MRE11/RAD50/NBS1 (MRN) complex is a major sensor of DNA double strand breaks, whose role in controlling faithful DNA replication and preventing replication stress is also emerging. Inactivation of the MRN complex invariably leads to developmental and/or degenerative neuronal defects, the pathogenesis of which still remains poorly understood. In particular, NBS1 gene mutations are associated with microcephaly and strongly impaired cerebellar development, both in humans and in the mouse model. These phenotypes strikingly overlap those induced by inactivation of MYCN, an essential promoter of the expansion of neuronal stem and progenitor cells, suggesting that MYCN and the MRN complex might be connected on a unique pathway essential for the safe expansion of neuronal cells. Here, we show that MYCN transcriptionally controls the expression of each component of the MRN complex. By genetic and pharmacological inhibition of the MRN complex in a MYCN overexpression model and in the more physiological context of the Hedgehog-dependent expansion of primary cerebellar granule progenitor cells, we also show that the MRN complex is required for MYCN-dependent proliferation. Indeed, its inhibition resulted in DNA damage, activation of a DNA damage response, and cell death in a MYCN- and replication-dependent manner. Our data indicate the MRN complex is essential to restrain MYCN-induced replication stress during neural cell proliferation and support the hypothesis that replication-born DNA damage is responsible for the neuronal defects associated with MRN dysfunctions.Cell Death and Differentiation advance online publication, 12 June 2015; doi:10.1038/cdd.2015.81

HDAC inhibitor confers radiosensitivity to prostate stem-like cells

Background: Radiotherapy can be an effective treatment for prostate cancer, but radiorecurrent tumours do develop. Considering prostate cancer heterogeneity, we hypothesised that primitive stem-like cells may constitute the radiation-resistant fraction. Methods: Primary cultures were derived from patients undergoing resection for prostate cancer or benign prostatic hyperplasia. After short-term culture, three populations of cells were sorted, reflecting the prostate epithelial hierarchy, namely stem-like cells (SCs, α2β1integrinhi/CD133+), transit-amplifying (TA, α2β1integrinhi/CD133−) and committed basal (CB, α2β1integrinlo) cells. Radiosensitivity was measured by colony-forming efficiency (CFE) and DNA damage by comet assay and DNA damage foci quantification. Immunofluorescence and flow cytometry were used to measure heterochromatin. The HDAC (histone deacetylase) inhibitor Trichostatin A was used as a radiosensitiser. Results: Stem-like cells had increased CFE post irradiation compared with the more differentiated cells (TA and CB). The SC population sustained fewer lethal double-strand breaks than either TA or CB cells, which correlated with SCs being less proliferative and having increased levels of heterochromatin. Finally, treatment with an HDAC inhibitor sensitised the SCs to radiation. Interpretation: Prostate SCs are more radioresistant than more differentiated cell populations. We suggest that the primitive cells survive radiation therapy and that pre-treatment with HDAC inhibitors may sensitise this resistant fraction

ATRX dysfunction Induces replication defects in primary mouse cells

The chromatin remodeling protein ATRX, which targets tandem repetitive DNA, has been shown to be required for expression of the alpha globin genes, for proliferation of a variety of cellular progenitors, for chromosome congression and for the maintenance of telomeres. Mutations in ATRX have recently been identified in tumours which maintain their telomeres by a telomerase independent pathway involving homologous recombination thought to be triggered by DNA damage. It is as yet unknown whether there is a central underlying mechanism associated with ATRX dysfunction which can explain the numerous cellular phenomena observed. There is, however, growing evidence for its role in the replication of various repetitive DNA templates which are thought to have a propensity to form secondary structures. Using a mouse knockout model we demonstrate that ATRX plays a direct role in facilitating DNA replication. Ablation of ATRX alone, although leading to a DNA damage response at telomeres, is not sufficient to trigger the alternative lengthening of telomere pathway in mouse embryonic stem cells

DnaC Inactivation in Escherichia coli K-12 Induces the SOS Response and Expression of Nucleotide Biosynthesis Genes

Background: Initiation of chromosome replication in E. coli requires the DnaA and DnaC proteins and conditionally-lethal dnaA and dnaC mutants are often used to synchronize cell populations. Methodology/Principal Findings: DNA microarrays were used to measure mRNA steady-state levels in initiation-deficient dnaA46 and dnaC2 bacteria at permissive and non-permissive temperatures and their expression profiles were compared to MG1655 wildtype cells. For both mutants there was altered expression of genes involved in nucleotide biosynthesis at the non-permissive temperature. Transcription of the dnaA and dnaC genes was increased at the non-permissive temperature in the respective mutant strains indicating auto-regulation of both genes. Induction of the SOS regulon was observed in dnaC2 cells at 38uC and 42uC. Flow cytometric analysis revealed that dnaC2 mutant cells at non-permissive temperature had completed the early stages of chromosome replication initiation. Conclusion/Significance: We suggest that in dnaC2 cells the SOS response is triggered by persistent open-complex formation at oriC and/or by arrested forks that require DnaC for replication restart

Retrograde traffic in the biosynthetic-secretory route

In the biosynthetic-secretory route from the rough endoplasmic reticulum, across the pre-Golgi intermediate compartments, the Golgi apparatus stacks, trans Golgi network, and post-Golgi organelles, anterograde transport is accompanied and counterbalanced by retrograde traffic of both membranes and contents. In the physiologic dynamics of cells, retrograde flow is necessary for retrieval of molecules that escaped from their compartments of function, for keeping the compartments’ balances, and maintenance of the functional integrities of organelles and compartments along the secretory route, for repeated use of molecules, and molecule repair. Internalized molecules may be transported in retrograde direction along certain sections of the secretory route, and compartments and machineries of the secretory pathway may be misused by toxins. An important example is the toxin of Shigella dysenteriae, which has been shown to travel from the cell surface across endosomes, and the Golgi apparatus en route to the endoplasmic reticulum, and the cytosol, where it exerts its deleterious effects. Most importantly in medical research, knowledge about the retrograde cellular pathways is increasingly being utilized for the development of strategies for targeted delivery of drugs to the interior of cells. Multiple details about the molecular transport machineries involved in retrograde traffic are known; a high number of the molecular constituents have been characterized, and the complicated fine structural architectures of the compartments involved become more and more visible. However, multiple contradictions exist, and already established traffic models again are in question by contradictory results obtained with diverse cell systems, and/or different techniques. Additional problems arise by the fact that the conditions used in the experimental protocols frequently do not reflect the physiologic situations of the cells. Regular and pathologic situations often are intermingled, and experimental treatments by themselves change cell organizations. This review addresses physiologic and pathologic situations, tries to correlate results obtained by different cell biologic techniques, and asks questions, which may be the basis and starting point for further investigations

Extracorporeal carbon dioxide removal: the future of lung support lies in the history.

Extracorporeal organ support in patients with dysfunction of vital organs like the kidney, heart, and liver has proven helpful in bridging the patients to recovery or more definitive therapy. Mechanical ventilation in patients with respiratory failure, although indispensable, has been associated with worsening injury to the lungs, termed ventilator-induced lung injury. Application of lung-protective ventilation strategies are limited by inevitable hypercapnia and hypercapnic acidosis. Various alternative extracorporeal strategies, proposed more than 30 years ago, to combat hypercapnia are now more readily available. In particular, the venovenous approach to effective carbon dioxide removal, which involves minimal invasiveness comparable to renal replacement therapy, appears to be very promising. The clinical applications of these extracorporeal carbon dioxide removal therapies may extend beyond just lung protection in ventilated patients. This article summarizes the rationale, technology and clinical application of various extracorporeal lung assist techniques available for clinical use, and some of the future perspectives in the field