Project description:The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation, Polysome isolation was performed by separation of ribosome-bound mRNAs in sucrose gradients. Beckman Ultra-Clear centrifuge tubes were loaded with 5.5 ml of 50% sucrose in low salt buffer (LSB: 200 mM Tris pH7.4 in DEPC H2O, 100 mM NaCl, 30 mM MgCl2) with 1:1000 RNasin (Fermentas) and 100 μg/mL cycloheximide (CHX) in ethanol and incubated at 4°C horizontally overnight. Media was removed from cell cultures, replaced with media containing 100 μg/mL CHX, cells incubated at 37°C for 10 min to halt protein synthesis, and trypsinized in trypsin/EDTA containing 100 μg/mL CHX. Cells were washed twice in PBS containing CHX, RNasin and Roche complete EDTA-free protease inhibitor tablet, lysed in LSB with CHX and RNasin. Lysates were added to a Dounce homogenizer and incubated on ice for 3 min before addition of Triton detergent buffer (1.2% Triton N-100, 0.2M sucrose in LSB) and homogenization. Samples were transferred to cold sterile Eppendorf centrifuge tubes and centrifuged at 13,000 x RPM for 10 min at 4°C. Post-nuclear supernatants were transferred to centrifuge tubes containing 100 μL Heparin solution (10 mg/mL heparin, 1.5M NaCl in LSB) with RNasin and CHX, applied to a sucrose gradient. Gradients were centrifuged at 36K RPM at 4°C for 2 h, and supernatants recovered with an ISCO-UV fractionator. Samples were recovered into Eppendorf centrifuge tubes containing 40 μL RNase-free 0.5M EDTA and kept on ice. One volume acidic phenol-chloroform was added to each sample, which were then mixed and centrifuged at 10,000 x g for 15 min. The acidic phenol-chloroform extraction was repeated with the aqueous phase, which was then extracted twice via chloroform extraction. RNA was precipitated at -20°C overnight by addition of isopropanol with 0.1 vol NaOAc, pH5.2. The following day, RNA pellets were recovered by centrifugation, washed with 70% ethanol, air-dried and resuspended in sterile H2O. RNA from fractions was then pooled based on their relative rate of translation, with fractions containing 4 or more ribosomes considered heavily translated and used for gene chip analysis. The RNA quality was examined via the Agilent Technologies kit.

Project description:Genomic instability is the hallmark of various cancers with the increasing accumulation of DNA damage. The application of radiotherapy and chemotherapy in cancer treatment is typically based on this property of cancers. However, the adverse effects including normal tissues injury are also accompanied by the radiotherapy and chemotherapy. Targeted cancer therapy has the potential to suppress cancer cells' DNA damage response through tailoring therapy to cancer patients lacking specific DNA damage response functions. Obviously, understanding the broader role of DNA damage repair in cancers has became a basic and attractive strategy for targeted cancer therapy, in particular, raising novel hypothesis or theory in this field on the basis of previous scientists' findings would be important for future promising druggable emerging targets. In this review, we first illustrate the timeline steps for the understanding the roles of DNA damage repair in the promotion of cancer and cancer therapy developed, then we summarize the mechanisms regarding DNA damage repair associated with targeted cancer therapy, highlighting the specific proteins behind targeting DNA damage repair that initiate functioning abnormally duo to extrinsic harm by environmental DNA damage factors, also, the DNA damage baseline drift leads to the harmful intrinsic targeted cancer therapy. In addition, clinical therapeutic drugs for DNA damage and repair including therapeutic effects, as well as the strategy and scheme of relative clinical trials were intensive discussed. Based on this background, we suggest two hypotheses, namely "environmental gear selection" to describe DNA damage repair pathway evolution, and "DNA damage baseline drift", which may play a magnified role in mediating repair during cancer treatment. This two new hypothesis would shed new light on targeted cancer therapy, provide a much better or more comprehensive holistic view and also promote the development of new research direction and new overcoming strategies for patients.

Project description:The cellular response to DNA damage is mediated through multiple pathways that regulate and coordinate DNA repair, cell cycle arrest and cell death. We show that the DNA damage response (DDR) induced by ionizing radiation (IR) is coordinated in breast cancer cells by selective mRNA translation mediated by high levels of translation initiation factor eIF4G1. Increased expression of eIF4G1, common in breast cancers, was found to selectively increase translation of mRNAs involved in cell survival and the DDR, preventing autophagy and apoptosis (Survivin, HIF1α, XIAP), promoting cell cycle arrest (GADD45a, p53, ATRIP, Chk1) and DNA repair (53BP1, BRCA1/2, PARP, Rfc2-5, ATM, MRE-11, others). Reduced expression of eIF4G1, but not its homolog eIF4G2, greatly sensitizes cells to DNA damage by IR, induces cell death by both apoptosis and autophagy, and significantly delays resolution of DNA damage foci with little reduction of overall protein synthesis. While some mRNAs selectively translated by higher levels of eIF4G1 were found to use internal ribosome entry site (IRES)-mediated alternate translation, most do not. The latter group shows significantly reduced dependence on eIF4E for translation, facilitated by an enhanced requirement for eIF4G1. Increased expression of eIF4G1 therefore promotes specialized translation of survival, growth arrest and DDR mRNAs that are important in cell survival and DNA repair following genotoxic DNA damage. The purpose of this study was to identify mRNAs that are selectively increased in translation by high levels of the initiation factor eIF4G1, which occurs in many advanced human cancers, in response to DNA damage caused by ionizing radiation, Polysome isolation was performed by separation of ribosome-bound mRNAs in sucrose gradients. Beckman Ultra-Clear centrifuge tubes were loaded with 5.5 ml of 50% sucrose in low salt buffer (LSB: 200 mM Tris pH7.4 in DEPC H2O, 100 mM NaCl, 30 mM MgCl2) with 1:1000 RNasin (Fermentas) and 100 μg/mL cycloheximide (CHX) in ethanol and incubated at 4°C horizontally overnight. Media was removed from cell cultures, replaced with media containing 100 μg/mL CHX, cells incubated at 37°C for 10 min to halt protein synthesis, and trypsinized in trypsin/EDTA containing 100 μg/mL CHX. Cells were washed twice in PBS containing CHX, RNasin and Roche complete EDTA-free protease inhibitor tablet, lysed in LSB with CHX and RNasin. Lysates were added to a Dounce homogenizer and incubated on ice for 3 min before addition of Triton detergent buffer (1.2% Triton N-100, 0.2M sucrose in LSB) and homogenization. Samples were transferred to cold sterile Eppendorf centrifuge tubes and centrifuged at 13,000 x RPM for 10 min at 4°C. Post-nuclear supernatants were transferred to centrifuge tubes containing 100 μL Heparin solution (10 mg/mL heparin, 1.5M NaCl in LSB) with RNasin and CHX, applied to a sucrose gradient. Gradients were centrifuged at 36K RPM at 4°C for 2 h, and supernatants recovered with an ISCO-UV fractionator. Samples were recovered into Eppendorf centrifuge tubes containing 40 μL RNase-free 0.5M EDTA and kept on ice. One volume acidic phenol-chloroform was added to each sample, which were then mixed and centrifuged at 10,000 x g for 15 min. The acidic phenol-chloroform extraction was repeated with the aqueous phase, which was then extracted twice via chloroform extraction. RNA was precipitated at -20°C overnight by addition of isopropanol with 0.1 vol NaOAc, pH5.2. The following day, RNA pellets were recovered by centrifugation, washed with 70% ethanol, air-dried and resuspended in sterile H2O. RNA from fractions was then pooled based on their relative rate of translation, with fractions containing 4 or more ribosomes considered heavily translated and used for gene chip analysis. The RNA quality was examined via the Agilent Technologies kit.

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DNA Repair: Translation to the Clinic.

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DNA Repair: Translation to the Clinic.

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