A Reversible Gene-Targeting Strategy Identifies Synthetic Lethal Interactions between MK2 and p53 in the DNA Damage Response In Vivo

A fundamental limitation in devising new therapeutic strategies for killing cancer cells with DNA damaging agents is the need to identify synthetic lethal interactions between tumor-specific mutations and components of the DNA damage response (DDR) in vivo. The stress-activated p38 mitogen-activated protein kinase (MAPK)/MAPKAP kinase-2 (MK2) pathway is a critical component of the DDR network in p53-deficient tumor cells in vitro. To explore the relevance of this pathway for cancer therapy in vivo, we developed a specific gene targeting strategy in which Cre-mediated recombination simultaneously creates isogenic MK2-proficient and MK2-deficient tumors within a single animal. This allows direct identification of MK2 synthetic lethality with mutations that promote tumor development or control response to genotoxic treatment. In an autochthonous model of non-small-cell lung cancer (NSCLC), we demonstrate that MK2 is responsible for resistance of p53-deficient tumors to cisplatin, indicating synthetic lethality between p53 and MK2 can successfully be exploited for enhanced sensitization of tumors to DNA-damaging chemotherapeutics in vivo.National Institutes of Health (U.S.) (Grant ES015339)National Institutes of Health (U.S.) (Grant GM60594)National Institutes of Health (U.S.) (Grant GM59281)National Institutes of Health (U.S.) (Grant CA112967)Janssen Pharmaceutical Ltd.Massachusetts Institute of Technology. Center for Environmental Health Sciences (Core Grant P30-CA14051)Massachusetts Institute of Technology. Center for Environmental Health Sciences (Core Grant ES-002109

Polo-like kinase 3 regulates CtIP during DNA double-strand break repair in G1

DNA double-strand breaks (DSBs) are repaired by nonhomologous end joining (NHEJ) or homologous recombination (HR). The C terminal binding protein–interacting protein (CtIP) is phosphorylated in G2 by cyclin-dependent kinases to initiate resection and promote HR. CtIP also exerts functions during NHEJ, although the mechanism phosphorylating CtIP in G1 is unknown. In this paper, we identify Plk3 (Polo-like kinase 3) as a novel DSB response factor that phosphorylates CtIP in G1 in a damage-inducible manner and impacts on various cellular processes in G1. First, Plk3 and CtIP enhance the formation of ionizing radiation-induced translocations; second, they promote large-scale genomic deletions from restriction enzyme-induced DSBs; third, they are required for resection and repair of complex DSBs; and finally, they regulate alternative NHEJ processes in Ku−/− mutants. We show that mutating CtIP at S327 or T847 to nonphosphorylatable alanine phenocopies Plk3 or CtIP loss. Plk3 binds to CtIP phosphorylated at S327 via its Polo box domains, which is necessary for robust damage-induced CtIP phosphorylation at S327 and subsequent CtIP phosphorylation at T847

Phenotypic plasticity and water flux rates of Citrus root orders under salinity

Knowledge about the root system structure and the uptake efficiency of root orders is critical to understand the adaptive plasticity of plants towards salt stress. Thus, this study describes the phenological and physiological plasticity of Citrus volkameriana rootstocks under severe NaCl stress on the level of root orders. Phenotypic root traits known to influence uptake processes, for example frequency of root orders, specific root area, cortical thickness, and xylem traits, did not change homogeneously throughout the root system, but changes after 6 months under 90 mM NaCl stress were root order specific. Chloride accumulation significantly increased with decreasing root order, and the Cl− concentration in lower root orders exceeded those in leaves. Water flux densities of first-order roots decreased to <20% under salinity and did not recover after stress release. The water flux densities of higher root orders changed marginally under salinity and increased 2- to 6-fold in second and third root orders after short-term stress release. Changes in root order frequency, morphology, and anatomy indicate rapid and major modification of C. volkameriana root systems under salt stress. Reduced water uptake under salinity was related to changes of water flux densities among root orders and to reduced root surface areas. The importance of root orders for water uptake changed under salinity from root tips towards higher root orders. The root order-specific changes reflect differences in vulnerability (indicated by the salt accumulation) and ontogenetic status, and point to functional differences among root orders under high salinity

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

A Reversible Gene-Targeting Strategy Identifies Synthetic Lethal Interactions between MK2 and p53 in the DNA Damage Response In Vivo

A fundamental limitation in devising new therapeutic strategies for killing cancer cells with DNA damaging agents is the need to identify synthetic lethal interactions between tumor-specific mutations and components of the DNA damage response (DDR) in vivo. The stress-activated p38 mitogen-activated protein kinase (MAPK)/MAPKAP kinase-2 (MK2) pathway is a critical component of the DDR network in p53-deficient tumor cells in vitro. To explore the relevance of this pathway for cancer therapy in vivo, we developed a specific gene targeting strategy in which Cre-mediated recombination simultaneously creates isogenic MK2-proficient and MK2-deficient tumors within a single animal. This allows direct identification of MK2 synthetic lethality with mutations that promote tumor development or control response to genotoxic treatment. In an autochthonous model of non-small-cell lung cancer (NSCLC), we demonstrate that MK2 is responsible for resistance of p53-deficient tumors to cisplatin, indicating synthetic lethality between p53 and MK2 can successfully be exploited for enhanced sensitization of tumors to DNA-damaging chemotherapeutics in vivo

PMCA1 mRNA expression in rat aortic myocytes: A real-time RT-PCR study

The plasmalemmal Ca2+ adenosine triphosphatase (PMCA) is a key regulator of Ca2+ efflux in vascular smooth muscle. In these studies are developed a realtime reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay for assessing PMCA1 mRNA levels in rat primary cultured aortic myocytes. This assay detected fetal bovine serum-induced increases in PMCA1 mRNA (relative to 18S rRNA) 4, 8, and 24 h after stimulation. Early fetal bovine serum-induced increases in PMCA1 mRNA were insensitive to the Ca2+ channel blockers nifedipine, flunarizine, and SKF-96365. These studies demonstrate the feasibility of real-time RT-PCR to assess mRNA levels of PMCA1 and illustrate dynamic regulation of this Ca2+ pump isoform in rat primary cultured aortic myocytes, (C) 2000 Academic Press

Analysis of aSYN levels.

<p>(A) qRT-PCR data showing aSYN mRNA expression levels in healthy controls and iPD lines. No significant differences are detected between the groups. (B) Representative western blot of aSYN and GAPDH loading control in DANs derived from 4 iPD patients and 4 healthy controls. Labels C/C (risk allele) and T/T (non-risk allele) denote the genotype of iPD patients at LRRK2-RS1491923. mDANs were treated with a DMSO vehicle control or 2 μM CZC (CZC) for 24h. (C) Western blots from three independent experiments (n = 3) were quantified by densitometry and aSYN normalized to a loading control. Graphs show all individual measurements. Stratification for T/T and C/C genotype is also included. (D) Ratio of aSYN (normalized to loading control) following CZC treatment/untreated. Significance was calculated using Mann-Whitney test.</p

Neurite outgrowth analysis.

<p>(A) Example of neurite outgrowth experimental setup. 5 positions per line were selected and imaged every 9 minutes over a period of 3 hours. Neurite length was assessed using Fiji’s plugin mTrackJ. (B) Neurite outgrowth in isogenic LRRK2 G2019S and gene-corrected WT iPSC-derived DANs. (C) Neurite outgrowth in neurons with <i>LRRK2</i> G2019S in the presence or absence of the LRRK2 inhibitor CZC-25146. (D) Neurite outgrowth in LRRK2 G2019S lines with and without the LRRK2 inhibitor HG-10-102-01. (E) Neurite outgrowth in iPD lines with and without the LRRK2 inhibitor CZC-25146. (F) Neurite outgrowth in healthy control lines with and without the LRRK2 inhibitor CZC-25146. (G) Neurite outgrowth in iPD lines with and without the LRRK2 inhibitor HG-10-102-01. (H) Neurite outgrowth in healthy control lines with and without the LRRK2 inhibitor HG-10-102-01. Graphs show mean with standard error of mean (S.E.M). Significance was calculated using Mann Whitney test. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001. n = 20 neurites analyzed from neurons of 3 independent cultures.</p

Investigation of autophagy.

<p>(A) Representative western blot of LC3B-I, LC3B-II and GAPDH loading control in DANs derived from 4 iPD patients and 4 healthy controls. Labels C/C (risk allele) and T/T (non-risk allele) denote the genotype of iPD patients at LRRK2-RS1491923. mDANs were treated with a DMSO vehicle control or 100 nM bafilomycin (baf) for 24 h. (B) Western blots from three independent experiments (n = 3) were quantified by densitometry and LC3B-II normalized to a loading control. All measurements are shown. Data is grouped as healthy vs iPD as well as iPD stratified for T/T and C/C genotype. (C) Ratio of LC3B-II (normalized to loading control) following bafilomycin treatment/untreated. (D) Representative western blot of LAMP1 and α-tubulin loading control in mDANs derived from iPD patients and healthy controls. (E) Western blots from three independent experiments (n = 3) were quantified by densitometry and LAMP1 normalized to a loading control. All data is shown, including data stratified for T/T and C/C genotype. (F) Ratio of LAMP1 (normalized to loading control) following bafilomycin treatment/untreated.</p