Common Chemical Inductors of Replication Stress: Focus on Cell-Based Studies.

DNA replication is a highly demanding process regarding the energy and material supply and must be precisely regulated, involving multiple cellular feedbacks. The slowing down or stalling of DNA synthesis and/or replication forks is referred to as replication stress (RS). Owing to the complexity and requirements of replication, a plethora of factors may interfere and challenge the genome stability, cell survival or affect the whole organism. This review outlines chemical compounds that are known inducers of RS and commonly used in laboratory research. These compounds act on replication by direct interaction with DNA causing DNA crosslinks and bulky lesions (cisplatin), chemical interference with the metabolism of deoxyribonucleotide triphosphates (hydroxyurea), direct inhibition of the activity of replicative DNA polymerases (aphidicolin) and interference with enzymes dealing with topological DNA stress (camptothecin, etoposide). As a variety of mechanisms can induce RS, the responses of mammalian cells also vary. Here, we review the activity and mechanism of action of these compounds based on recent knowledge, accompanied by examples of induced phenotypes, cellular readouts and commonly used doses

Carfilzomib and dexamethasone versus bortezomib and dexamethasone for patients with relapsed or refractory multiple myeloma (ENDEAVOR): And randomised, phase 3, open-label, multicentre study

Background: Bortezomib with dexamethasone is a standard treatment option for relapsed or refractory multiple myeloma. Carfilzomib with dexamethasone has shown promising activity in patients in this disease setting. The aim of this study was to compare the combination of carfilzomib and dexamethasone with bortezomib and dexamethasone in patients with relapsed or refractory multiple myeloma. Methods: In this randomised, phase 3, open-label, multicentre study, patients with relapsed or refractory multiple myeloma who had one to three previous treatments were randomly assigned (1:1) using a blocked randomisation scheme (block size of four) to receive carfilzomib with dexamethasone (carfilzomib group) or bortezomib with dexamethasone (bortezomib group). Randomisation was stratified by previous proteasome inhibitor therapy, previous lines of treatment, International Staging System stage, and planned route of bortezomib administration if randomly assigned to bortezomib with dexamethasone. Patients received treatment until progression with carfilzomib (20 mg/m2 on days 1 and 2 of cycle 1; 56 mg/m2 thereafter; 30 min intravenous infusion) and dexamethasone (20 mg oral or intravenous infusion) or bortezomib (1·3 mg/m2; intravenous bolus or subcutaneous injection) and dexamethasone (20 mg oral or intravenous infusion). The primary endpoint was progression-free survival in the intention-to-treat population. All participants who received at least one dose of study drug were included in the safety analyses. The study is ongoing but not enrolling participants; results for the interim analysis of the primary endpoint are presented. The trial is registered at ClinicalTrials.gov, number NCT01568866. Findings: Between June 20, 2012, and June 30, 2014, 929 patients were randomly assigned (464 to the carfilzomib group; 465 to the bortezomib group). Median follow-up was 11·9 months (IQR 9·3-16·1) in the carfilzomib group and 11·1 months (8·2-14·3) in the bortezomib group. Median progression-free survival was 18·7 months (95% CI 15·6-not estimable) in the carfilzomib group versus 9·4 months (8·4-10·4) in the bortezomib group at a preplanned interim analysis (hazard ratio [HR] 0·53 [95% CI 0·44-0·65]; p<0·0001). On-study death due to adverse events occurred in 18 (4%) of 464 patients in the carfilzomib group and in 16 (3%) of 465 patients in the bortezomib group. Serious adverse events were reported in 224 (48%) of 463 patients in the carfilzomib group and in 162 (36%) of 456 patients in the bortezomib group. The most frequent grade 3 or higher adverse events were anaemia (67 [14%] of 463 patients in the carfilzomib group vs 45 [10%] of 456 patients in the bortezomib group), hypertension (41 [9%] vs 12 [3%]), thrombocytopenia (39 [8%] vs 43 [9%]), and pneumonia (32 [7%] vs 36 [8%]). Interpretation: For patients with relapsed or refractory multiple myeloma, carfilzomib with dexamethasone could be considered in cases in which bortezomib with dexamethasone is a potential treatment option. Funding: Onyx Pharmaceuticals, Inc., an Amgen subsidiary

ATP-Evoked Intracellular Ca Signaling of Different Supporting Cells in the Hearing Mouse Hemicochlea

Hearing and its protection is regulated by ATP-evoked Ca2+ signaling in the supporting cells of the organ of Corti, however, the unique anatomy of the cochlea hampers observing these mechanisms. For the first time, we have performed functional ratiometric Ca2+ imaging (fura-2) in three different supporting cell types in the hemicochlea preparation of hearing mice to measure purinergic receptor-mediated Ca2+ signaling in pillar, Deiters' and Hensen's cells. Their resting [Ca2+]i was determined and compared in the same type of preparation. ATP evoked reversible, repeatable and dose-dependent Ca2+ transients in all three cell types, showing desensitization. Inhibiting the Ca2+ signaling of the ionotropic P2X (omission of extracellular Ca2+) and metabotropic P2Y purinergic receptors (depletion of intracellular Ca2+ stores) revealed the involvement of both receptor types. Detection of P2X2,3,4,6,7 and P2Y1,2,6,12,14 receptor mRNAs by RT-PCR supported this finding and antagonism by PPADS suggested different functional purinergic receptor population in pillar versus Deiters' and Hensen's cells. The sum of the extra- and intracellular Ca2+-dependent components of the response was about equal with the control ATP response (linear additivity) in pillar cells, and showed supralinearity in Deiters' and Hensen's cells. Calcium-induced calcium release might explain this synergistic interaction. The more pronounced Ca2+ leak from the endoplasmic reticulum in Deiters' and Hensen's cells, unmasked by cyclopiazonic acid, may also suggests the higher activity of the internal stores in Ca2+ signaling in these cells. Differences in Ca2+ homeostasis and ATP-induced Ca2+ signaling might reflect the distinct roles these cells play in cochlear function and pathophysiology

ATR-Chk1-APC/C-dependent stabilization of Cdc7-ASK (Dbf4) kinase is required for DNA lesion bypass under replication stress

Cdc7 kinase regulates DNA replication. However, its role in DNA repair and recombination is poorly understood. Here we describe a pathway that stabilizes the human Cdc7–ASK (activator of S-phase kinase; also called Dbf4), its regulation, and its function in cellular responses to compromised DNA replication. Stalled DNA replication evoked stabilization of the Cdc7–ASK (Dbf4) complex in a manner dependent on ATR–Chk1-mediated checkpoint signaling and its interplay with the anaphase-promoting complex/cyclosome(Cdh1) (APC/C(Cdh1)) ubiquitin ligase. Mechanistically, Chk1 kinase inactivates APC/C(Cdh1) through degradation of Cdh1 upon replication block, thereby stabilizing APC/C(Cdh1) substrates, including Cdc7–ASK (Dbf4). Furthermore, motif C of ASK (Dbf4) interacts with the N-terminal region of RAD18 ubiquitin ligase, and this interaction is required for chromatin binding of RAD18. Impaired interaction of ASK (Dbf4) with RAD18 disables foci formation by RAD18 and hinders chromatin loading of translesion DNA polymerase η. These findings define a novel mechanism that orchestrates replication checkpoint signaling and ubiquitin–proteasome machinery with the DNA damage bypass pathway to guard against replication collapse under conditions of replication stress

Magneto-optical and optical spectroscopic ellipsometries of La2∕3Sr1∕3MnO3 thin films

International audienc

Magneto-optical and optical spectroscopic ellipsometries of La2∕3Sr1∕3MnO3 thin films

International audienc

Vascularization, High-Volume Solution Flow, and Localized Roles for Enzymes of Sucrose Metabolism during Tumorigenesis by Agrobacterium tumefaciens

Vascular differentiation and epidermal disruption are associated with establishment of tumors induced by Agrobacterium tumefaciens. Here, we address the relationship of these processes to the redirection of nutrient-bearing water flow and carbohydrate delivery for tumor growth within the castor bean (Ricinus communis) host. Treatment with aminoethoxyvinyl-glycine showed that vascular differentiation and epidermal disruption were central to ethylene-dependent tumor establishment. CO(2) release paralleled tumor growth, but water flow increased dramatically during the first 3 weeks. However, tumor water loss contributed little to water flow to host shoots. Tumor water loss was followed by accumulation of the osmoprotectants, sucrose (Suc) and proline, in the tumor periphery, shifting hexose-to-Suc balance in favor of sugar signals for maturation and desiccation tolerance. Concurrent activities and sites of action for enzymes of Suc metabolism changed: Vacuolar invertase predominated during initial import of Suc into the symplastic continuum, corresponding to hexose concentrations in expanding tumors. Later, Suc synthase (SuSy) and cell wall invertase rose in the tumor periphery to modulate both Suc accumulation and descending turgor for import by metabolization. Sites of abscisic acid immunolocalization correlated with both central vacuolar invertase and peripheral cell wall invertase. Vascular roles were indicated by SuSy immunolocalization in xylem parenchyma for inorganic nutrient uptake and in phloem, where resolution allowed SuSy identification in sieve elements and companion cells, which has widespread implications for SuSy function in transport. Together, data indicate key roles for ethylene-dependent vascularization and cuticular disruption in the redirection of water flow and carbohydrate transport for successful tumor establishment