Shirley Lehnert

Biomolecular Action of Ionizing Radiation

INTRODUCTION BASIC RADIATION PHYSICS AND CHEMISTRY Ionization and Excitation Types of Ionizing Radiation Electromagnetic Radiation Particulate Radiations Processes of Energy Absorption Direct and Indirect Action of Radiation Radiolysis of Water Haber-Weiss Reaction Reactions of the Primary Radiolytic Products of Water with Target Molecules Solute Radicals Form Stable Products Linear Energy Transfer Relative Biological Effectiveness BASIC CELL BIOLOGY AND MOLECULAR GENETICS Basic Cell Biology Cell Membrane Cytoplasm Nucleus Mitochondria Endoplasmic Reticulum and Ribosomes Golgi Complex Cytoskeleton Lysosomes Extracellular Materials Molecular Genetics DNA Structure DNA Structure Is the Basis for Heredity Mechanism of DNA Replication Transcribing DNA to RNA From RNA to Protein Proteins METHODS OF CELL AND MOLECULAR RADIOBIOLOGY Methods of Classical Radiobiology Cell Survival In Vitro: The Clonogenic Assay Non-Clonogenic Assays Methods of Cell Synchronization Determination of Duration of Phases of the Cell Cycle Measuring Cell Survival In Vivo Methods for Detecting Damage to DNA Strand Break Assays Measurement of DNA Damage and Repair in Individual Mammalian Cells Tools and Techniques of Molecular Biology Hybridization of Nucleic Acids Restriction Enzymes Gel Electrophoresis and Blotting Techniques Polymerase Chain Reaction Putting New Genes into Cells: DNA-Mediated Gene Transfer Generation of a Cloned Probe or DNA Library Sequencing of DNA Single Nucleotide Polymorphisms Functional Inactivation of Genes Genomic Methods of Tumor Analysis Analysis of Proteins Production of Monoclonal Antibodies Proteomics: Analysis of Protein Structure and Function Analysis of Tissue Sections and Single Cells Laser Capture Microdissection IONIZING RADIATION EFFECTS TO THE CYTOPLASM Oxidative Stress Metabolic Oxidative Stress Ionizing Radiation-Induced ROS/RNS Demonstration of Radiation-Induced Intracellular ROS/RNS Mechanisms of Generation and Amplification of ROS/RNS Following Irradiation of the Cytoplasm Consequences of Radiation-Induced Generation of ROS/RNS Effects of Ionizing Radiation on the Cell Membrane Structure of the Cell Membrane Lipid Peroxidation in Plasma Membranes Consequences of Damage to Plasma Membrane Lipids Plasma Membrane Is a Target for Ionizing Radiation-Induced Apoptosis DAMAGE TO DNA BY IONIZING RADIATION Mechanisms of DNA Damage: Physicochemical Relationships Mechanisms of Damage Induction: Chemical End Points Mechanisms of Damage Induction: Cellular End Points Types of DNA Damage Simple Damages to DNA: Base Damage and Single-Strand Breaks Apurinic or Apyrimidinic Sites Modifiers of Radiation Effect DNA Strand Breaks Double-Strand Breaks and Other Multiply Damaged Sites Distribution of MDS Clustered Damage in DNA of Mammalian Cells DNA-Protein Cross-Links REPAIR OF RADIATION DAMAGE TO DNA Overview of DNA Repair Mechanisms Repair of Radiation-Induced DNA Damage Repair of Base Damage and Single-Strand DNA Breaks: Base Excision Repair Role of PARP Processing of Multiply Damaged Sites by BER Repair of DNA Double-Strand Breaks Homologous Recombination Nonhomologous End Joining Genes and Proteins Involved in NHEJ Telomere-Bound Proteins and DNA Repair Human Syndromes Involving DNA Repair Deficiency Relationship between DNA Repair and Cell Survival CELLULAR RESPONSE TO DNA DAMAGE Passing on the Message that DNA Has Been Damaged Signal Transduction Signal Transduction Cascade Initiated by Radiation-Induced DNA Damage ATM Protein Functions of ATM How Does ATM Respond to Radiation-Induced DNA Damage? Role of ATM in DNA Repair ATM and the MRN Complex Tumor Suppressor Gene p53 Turnover of p53: Mdm2 Modulation of p53 Stability and Activity Radiation-Induced Growth Arrest Cell Cycle: Cyclins and Cyclin Dependent Kinases. Radiation-Induced Cell-Cycle Arrest Oncogenes and Cell-Cycle Checkpoints Variation in Radiosensitivity through the Cell Cycle P53-Mediated Apoptosis CHROMATIN STRUCTURE AND RADIATION SENSITIVITY Cell Nucleus Hierarchical Structure of Chromatin Structure and Function: Chromatin and the Nuclear Matrix Protection of DNA from Radiation Damage by Nuclear Proteins DNA-Protein Cross-Link Formation DSB Yields and RBE Role of Polyamines Radiation Sensitivity and the Stability of the DNA-Nuclear Matrix Radiosensitivity of Condensed Chromatin Role of Chromatin in DNA DSB Recognition and Repair Histone 2AX ATM Signaling from Chromatin Modulation of Chromatin Structure and Function by Acetylation Radiosensitization by Histone Deacetylase Inhibitors RADIATION-INDUCED CHROMOSOME DAMAGE DNA, Chromosomes, and the Cell Cycle Organization of DNA into Chromatin and Chromosomes Cell Cycle Mitosis Radiation-Induced Chromosome Aberrations Nature of the Initial Lesion Partial Catalog of Chromosome and Chromatid Aberrations Visualization of Chromosome Breaks during Interphase: Premature Chromosome Condensation FISH, mFISH, SKY, mBAND FISH, and Chromosome Painting Results of Whole Chromosome Painting Mechanisms of Aberration Formation Chromosome Localization and Proximity Effects Implications of Chromosome Damage Genetics Carcinogenesis Cell Survival, Dose Rate, and Fractionation Response Genomic Instability Biodosimetry and Risk Estimation MODULATION OF RADIATION RESPONSE VIA SIGNAL TRANSDUCTION PATHWAYS Intracellular Signaling Transmembrane Receptors ErbB Family of Receptor Kinases Cytoplasmic Signaling Ras Proto-Oncogene Family Signal Transduction Cascades Modulation of Radiation Response by Interaction of Signal Transduction Pathways Activation of ErbB Receptors by Ionizing Radiation Mechanism of Receptor Activation by Ionizing Radiation Role of Other Growth Factors Effects of Activation of GF Receptors on Cell Survival Autocrine Signaling Radiosensitization by Modulation of Signal Transduction Intermediates: Molecular Radiosensitizers ErbB Family Signal Inhibitors Clinical Applications of EGFR Signal Inhibitors Inhibition of the Ras-Mediated Signaling Pathway Clinical Application of Farnesyl Transferase Inhibitors Clinical Implications of Radiation-Induced Cell Signaling: Accelerated Cell Proliferation RADIATION-INDUCED APOPTOSIS Apoptosis Mechanisms of Apoptosis Caspases Apoptotic Signaling Pathways Intrinsic Apoptotic Signaling: The Mitochondrial Pathway Extrinsic Apoptotic Signaling Extrinsic Apoptotic Signaling Initiated at the Plasma Membrane: The Ceramide Pathway Why Do Some Cells Die as the Result of Apoptosis and Not Others? Apoptotic Processes and the In Vivo Radiation Response Normal Tissue Tumor Response EARLY AND LATE RESPONDING GENES INDUCED BY IONIZING RADIATION Gene Expression Is Induced by Ionizing Radiation Transcription Factors Important Transcription Factors Activated by Radiation Radiation-Gene Therapy Early and Late Response Genes Induction of Late Response Genes by Ionizing Radiation Cytokine-Mediated Responses in Irradiated Tissues Late Effects: Radiation-Mediated Fibrosis Gene Expression Associated with Radiation-Mediated Vascular Damage Cytokines as Therapeutic Agents: Radioprotection and Radiosensitization Radiosensitization Radioprotection Cytokines as Biomarkers of Radiation Exposure CELL DEATH, CELL SURVIVAL, AND ADAPTATION Cell Death Modes of Cell Death in Nonirradiated Cells Radiation-Induced Cell Death Role of p53 Quantitating Cell Kill: Analysis of Cell Survival Curves Target Theory Linear Quadratic Model Lethal, Potentially Lethal Damage Model Repair Saturation Models Cell Survival at Low Radiation Doses Low Dose Hypersensitivity Adaptive Response Interactions of Adaptive Response and Bystander Effects Implications of Low Dose Effects for Risk Assessment Exposure to Background Radiation Adaptive Response and Neoplastic Transformation Clinical Implications of Low Dose Effects BYSTANDER EFFECTS AND GENOMIC INSTABILITY Dogma of Radiation Biology Bystander Effects Bystander Effects In Vitro Bystander Effects Seen after Transfer of Medium from Irradiated Cells Bystander Effects In Vivo Mechanisms Underlying Radiation-Induced Bystander Effects Implications in Risk Assessment Genomic Instability Genomic Instability In Vitro: Delayed Responses to Radiation Exposure Demonstration of Genomic Instability In Vivo Genomic Instability and Cancer Mechanisms Underlying Radiation-Induced Genomic Instability Relationship between Radiation-Induced Bystander Effects and Genomic Instability TUMOR RADIOBIOLOGY Tumor Radiobiology Unique Tumor Microenvironment Interstitial Fluid Pressure Tumor Hypoxia Tumor Acidosis Tumor Metabolism: Aerobic and Anaerobic Glycolysis Tumor Microenvironment Creates Barriers to Conventional Therapies Chemotherapy Radiotherapy Measurement of Tumor Hypoxia Radio-Sensitization by Modifying Tumor Oxygenation Effect of Hypoxia on Tumor Development and Progression Targeting the Ubiquitin/Proteasome System RADIATION BIOLOGY OF NONMAMMALIAN SPECIES: THREE EUKARYOTES AND A BACTERIUM Introduction: Lower Eukaryotes in Radiation Research Yeast, a Single-Celled Eukaryote Radiation Biology of Yeast Radiosensitive Mutants for the Study of DNA Repair DNA Damage Checkpoints Genome Wide-Screening for Radiation Response-Associated in Yeast Caenorhabditis elegans Apoptosis in C. elegans Cell Cycle Checkpoints in C. elegans DNA Repair in Celegans DNA Damage Responses in C. elegans Radiation-Induced Mutation Worms in Space Zebrafish Zebrafish for the Evaluation of Genotoxic Stress Effects of Ionizing Radiation on Brain and Eye Development Modulation of Radiation Response Gene Function during Embryonic Development Hematological Studies with Zebra Fish Deinococcus radiodurans Origins of Extremophiles Genetics of D. radiodurans Characteristics of D. radiodurans Predisposing to Radiation Resistance Regulation of Cellular Responses to Extensive Radiation Damage Double-Strand Break Tolerance An Economic Niche for D. radiodurans References Glossary Index * Each Chapter contains a Summary section and References

Implication of PBP74/mortalin/GRP75 in the radio-adaptive response

Purpose : To investigate the relationship between expression of the human peptide-binding protein PBP74 and the occurrence of an adaptive response to ionizing radiation. Materials and methods : Human tumour cell lines HT29 and MCF-7 were transfected with a PBP74 or PBP74 antisense construct. For demonstration of an adaptive response, cells lines were irradiated with a conditioning dose of 0.25 Gy cobalt-60 γ-rays followed by a second dose of 4.0 Gy after an interval of 4.5 h. Response was measured in terms of clonogenic survival. Results : Transfection of a PBP74 plasmid caused transient overexpression of PBP74 mRNA in both cell lines. The optimal dose for the induction of PBP74 in the cell lines investigated was 0.1-0.25Gy and PBP74 induction occurred within 30 min of irradiation. For both cell lines, the adaptive response was repressed when cells were transfected with the anti-PBP plasmid. However, the converse, an enhancement of the adaptive response in cell lines transfected with the PBP74 construct, was seen only for HT29 cells under certain experimental conditions. Conclusions : The results support the view that while PBP74 is necessary to the adaptive response, it may not by itself be sufficient for the adaptive response to occur.

Interstitial pneumonitis following total body irradiation for bone marrow transplantation using two different dose rates

A total of 22 patients with leukemia (10 ALL, 11 AML, 1 CML) have undergone allogeneic bone marrow transplantation (BMT) by the Quebec Co-operative Group for Marrow Transplantation from 1980 to 1982. All patients received 900 cGy total body irradiation (TBI), in a single fraction, on the day preceding BMT. The first 11 patients were treated on a cobalt unit at a constant dose rate of 4.7 to 6.3 cGy/min. Six of these patients developed interstitial pneumonitis (IP). The clinical course of three patients, two with idiopathic and one with drug-induced pneumonitis, was mild and recovery was complete in all. The other three patients developed severe infectious IP and two died. The next 11 patients were treated with a sweeping beam technique on a 4 MV linear accelerator delivering a total tumor dose of 900 cGy at an average dose rate of 6.0 to 6.5 cGy/min but an instantaneous dose rate of 21.0 to 23.5 cGy/min. Eight patients developed severe IP. Five of these were idiopathic and four died. Three were infectious and all died. The fatality of interstitial pneumonitis appeared to be greater in the group treated with the sweeping beam technique.

Radiation Response of Haematopoietic Cell Lines of Human Origin

Six human haematopoietic cell lines, five of leukaemic origin, including cells with myeloid, lymphoid and undifferentiated phenotype have been studied with respect to radiation response. The intrinsic radiosensitivity of the cells varied widely, the D0s ranging from 0.53 to 1.39 Gy. Five of the cell lines showed some capacity to accumulate sublethal damage; in three of these, enhanced survival was demonstrated in split-dose experiments. One cell line (HL-60) was anomalous in that although little accumulation of sublethal damage was demonstrable, survival was enhanced by fractionation of the dose. Five of the six cell lines studied were of leukaemic origin. The results support the belief that, in contrast to the almost constant radiosensitivity of normal haematopoietic cell progenitors, leukaemic cell progenitors may show a wide range of radiosensitivites.

Dose rate dependence of response of mouse lung to irradiation

The dose-rate dependence of lung damage in mice has been studied using LD50/50-180 as an index of the incidence of radiation pneumonitis. Mean lethal doses for 60Co gamma radiation to the thorax delivered at 100, 25 and 6 cGy/min were 1403, 1923 and 2488 cGy respectively. There were statistically significant differences between values obtained at 6 and 25 cGy/min and between those obtained at 25 and 100 cGy/min. An isoeffect plot of this data on a log-log graph shows the sparing effect of dose rate reduction to be greater for the lung than for more rapidly responding systems (colony forming units of small intestine and Chinese hamster cells in culture).

CHANGES IN GROWTH KINETICS OF JEJUNAL EPITHELIUM IN MICE MAINTAINED ON AN ELEMENTAL DIET

Changes in the kinetics of the intestinal epithelium were observed in mice maintained on an elemental diet containing hydrolysed protein and medium chain triglycerides. An increase in the length of the villi seen shortly after commencement of the diet was followed by a reduction in the rate of proliferation in the crypt. After 7 days on the diet, an equilibrium state was reached with the cellularity of the villi being 120% that of control while the number of proliferative cells/crypt was reduced by 35%. The proliferative response of the crypt following irradiation occurrred 16 hr later in diet‐fed mice than in controls. It was postulated that, because of the increased cellularity of the villus compartment in diet‐fed mice, additional time was required to reduce the number of villus cells to a critical level at which a proliferative response is induced in the crypt.

Mechanisms of radiation-induced sensorineural hearing loss and radioprotection

Patients that receive radiotherapy are at risk of late sensorineural hearing loss when the inner ear is included within the radiation field. Preclinical and human temporal bone studies have shown that there is differential damage to cochlear structures depending on the amount of dose delivered to the inner ear. In vitro studies have suggested that reactive oxygen species (ROS) are the main initial actors in radiation-induced damage. The interaction of ROS with different cellular components can result in different apoptotic pathways. Therefore, approaches to radioprotection are mainly aimed to reduce ROS production through antioxidants. This review summarizes recent research in the field that can improve the understanding and boost preventive efforts of this adverse effect.

Amplification of the graft-versus-host reaction by partial body irradiation

An experimental model has been developed for the study of combined effects of partial body irradiation (PBI) and graft-versus-host disease (GVHD) in which irradiation is delivered to the thorax 24 hr prior to induction of GVHD in hybrid mice by the injection of parental lymphoid cells. In mice irradiated to 1000 cGy or exposed to low doses of allogeneic lymphoid cells (20 X 10(6)), survival was 100% at 250 days. In contrast, combination of the two treatments, GVHD and PBI, resulted in a mortality of 83% and a mean survival time of 29 days, indicating synergy between GVHD and PBI. From histological studies of the lung it appeared that about 40% of the deaths occurring after combined GVHD/PBR treatment might be attributable to pneumonia. The cause of death in the remaining mice receiving combined treatment is not known. Mice receiving combined PBI/lymphoid cell treatment develop a characteristic skin lesion that is not seen in nonirradiated mice and is confined to the irradiated area. The effect of preinduction PBR on the timing and severity of GVHD is similar to that which would be produced by an increase in the number of effector cells.

In vivo toxicity of phenothiazines to cells of a transplantable tumor

SummaryThe toxicities of three phenothiazines, promazine, chlorpromazine, and trifluoperazine, towards cells of a mouse fibrosarcoma were quantified by means of an in vitro assay of clonogenic cell survival.For all three drugs cell kill was proportional to the amount of drug injected. Following injection of equimolar (0.2 mM/kg) amounts, cell survival was progressively reduced for a period of at least 48 h. On the basis of cell survival at 48 h after administration the ranking of the drugs for cytotoxicity, in ascending order, was trifluoperazine, chlorpromazine, promazine.A period of acute hypoxia prior to processing of the tumor did not enhance the toxicity of any of the drugs, and no change in the size of the hypoxic fraction of the tumor was seen 24 h after the injection of chlorpromazine. On this basis it was concluded that there was no evidence of enhanced toxicity of drugs for either chronically or acutely hypoxic tumor cells.A reduction in the number of clonogenic tumor cells per gram of tumor was largely the result of a fall in the number of viable cells recovered from the tumor. The plating efficiency of surviving cells remained constant or was only slightly depressed.

Radioprotection of Normal and Malignant Tissue in the Mouse by Diethylaminoreserpine

The protective effect of pre-irradiation injections of diethylaminoreserpine (DL-152) for normal and malignant tissues in the mouse has been investigated. Dose modifying factors (DMFs) obtained for normal tissue ranged from 1.0 for bone marrow CFUs to more than 1.8 for skin. The DMFs for two transplantable tumours investigated were 1.0 for the EMT6 adenocarcinoma and 1.70 for the KHT fibrosarcoma (at a surviving fraction of 0.1. Acutely hypoxic KHT tumours were protected to a slightly lesser extent than were aerated tumours. For the KHT tumour, the number of clonogenic cells recovered from non-irradiated tumours one hour after DL-152 injection was reduced to 60 per cent of the number covered fro saline-injected controls, while, if DL-152 injected mice were acutely hypoxic at the time of sacrifice, the number of clonogenic cells was further reduced. The survival of non-irradiated EMT6 tumour cells was unaffected by DL-152 injection prior to sacrifice.