Chinese hamster V79-171B cells grown for about 24 hours in suspension culture display increased resistance to cell killing by ionizing radiation compared with cells grown as monolayers, an observation originally termed the "contact effect". More recently, development of that resistance was shown to be accompanied by changes in the conformation of the DNA which reduce its denaturation rate in high salt/weak alkali. These changes in DNA conformation, mediated by the cellular micro-environment, appear to be responsible for the contact effect. The conditions necessary for the development of the effect are not, however, completely understood. In particular, when cells grown as monolayers on petri plates are suspended in spinner culture flasks, their growth characteristics change in three distinct ways. First, cells in suspension no longer have a solid substrate, so they remain round. Second, after several hours, they begin to aggregate to form "spheroids", so that three-dimensional intercellular cell contact develops. Third, cells in the stirred suspension cultures are not subjected to high local concentrations of metabolic by-products or surrounded by a zone depleted of nutrients, as are cells in monolayer culture. The studies described here were designed to determine how each of these factors influence changes in DNA conformation, as assayed using the alkali unwinding technique. Our results indicated that a round shape may not be an essential requirement, since cells spread out on the surface of cytodex beads in suspension culture, and sparsely-seeded cells in monolayer culture demonstrated at least a partial contact effect. Three-dimensional intercellular contact does not always seem necessary for the development of the contact effect. Cells grown in a methyl cellulose matrix developed radioresistance, even though the cells formed only small clusters of less than five cells. Similarly, suspension culture cells which were prevented from aggregating by frequent exposure to trypsin, also developed the contact effect. There was no evidence that nutrient depletion plays a role in the failure of cells grown as monolayers to develop a contact effect. However, cells grown as spheroids in the presence of monolayer cells, or in monolayer cell-conditioned medium, did not display a full contact effect. This indicates a role for monolayer cell-produced factors (possibly extracellular matrix proteins) in preventing the development of the contact effect. We conclude that changes in DNA conformation and the increase in radiation resistance, seen in V79-171b cells grown as spheroids, are not the result of intercellular contact or round shape of the cells. This radioresistance appears to be the result of an absence of monolayer cell-produced factors which could control both cell shape and DNA conformation. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

Thesis (MSc)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: This study’s focus is on the determination and quantization of radiation damage on a cellular level due to the decay of the Auger electron-emitting 123I and the replication of this energy deposition using Geant4 Monte Carlo simulations. The relatively short half-life of 123I (13.2 hours) makes it ideal for studies of Auger electrons which induce biological damage similar to that of high linear energy transfer radiations, when permitted to deposit their energy in close proximity to DNA. Due to small cellular dimensions, direct dose measurements are impossible but estimates may be made from Monte Carlo simulations. In this investigation the thymidine analogue 5-[123I]-iodo-2-deoxyuridine (123IUdR) was used to incorporate the 123I into the cellular DNA of T-lymphocytes from two human donors. Radiation induced micronuclei were numerated in binucleated cells using fluorescence microscopy. The energy deposition per decay of 123I was calculated within a spherical geometry, having the same size and density as a human lymphocyte, using the open source Geant4 toolkit. The absorbed energy per disintegration was used to convert the incorporated 123I activity (Bq) into absorbed dose (Gy) values, in order to compare the biological damage caused by the radioactive iodine to 60Co γ-radiation. A linear relationship between micronuclei frequency and 123I activity could be established. The linear dose-response noted for Auger electrons in the study is indicative of the high-LET nature of these particles. Using the linear-quadratic dose-response curve for micronuclei frequencies following exposure to graded doses of 60Co γ-rays, the relative biological effectiveness (RBE) of the DNA incorporated 123I estimated in this work was found to range from 19 ± 10 to 32 ± 7 for lymphocyte donor 1 and 15 ± 6 to 42 ± 11 for donor 2. The dose limiting RBE (RBEM) for lymphocyte donor 1 and 2 are respectively 34 ± 8 and 50 ± 15 and follows the expected shift in terms of the inherent radiosensitivity of the donors. We also considered the inclusion of the S-phase fraction of the lymphocytes in the dosimetry calculations. The resultant RBEs of the dose points of lymphocyte donor 1 ranges from 4 ± 2 to 7 ± 2, and those of donor 2 ranges from 3 ± 1 to 9 ± 2. The RBEM for lymphocyte donor 1 and 2 are respectively 7 ± 2 and 11 ± 3. The inclusion of the S-phase fraction reduces the calculated RBEs significantly and these observed RBE values relate well to those obtained in studies with fibroblasts and 125IUdR. / AFRIKAANSE OPSOMMING: Hierdie studie fokus op die bepaling en kwantisering van stralingskade op 'n sellulêre vlak as gevolg van die verval van 123I wat Auger elektrone afgee, asook die simulering van hierdie energie afsetting met behulp van die Geant4 Monte Carlo program. Die relatiewe kort half-leeftyd van 123I (13.2 uur) maak dit ideaal vir studies van Auger elektrone wat biologiese skade soortgelyk aan dié van 'n hoë lineêre-energie-oordrag uitstraling veroorsaak, indien die energie van die elektrone naby sellulêre DNA geabsorbeer word. As gevolg van die klein sellulêre dimensies is direkte dosis metings egter onmoontlik, maar skattings kan gemaak word met behulp van Monte Carlo simulasies. Die timidien analoog 5-[123I]-jodo-2-deoxyuridien (123IUdR) was in hierdie ondersoek gebruik om die 123I in die DNA van menslike T-limfosiete in te bou. Mikrokerne in dubbel-kernige selle wat vorm as gevolg van die Auger elektrone was getel met behulp van fluoressensie mikroskopie. Die energie afsetting per 123I verval was bereken binne ‘n sferiese geometrie, met dieselfde grootte en digtheid as 'n menslike limfosiet, met behulp van die Geant4 sagteware. Die geabsorbeerde energie per verval was gebruik om die geïnkorporeerde 123I aktiwiteit (Bq) om te skakel na ‘n waarde van geabsorbeerde dosis (Gy), ten einde die biologiese skade wat veroorsaak word deur die radioaktiewe jodium-123 met kobalt-60 gamma straling te vergelyk. ‘n Lineêre verwantskap tussen die mikrokerne frekwensies en die 123I aktiwiteit is vasgestel. Hierdie verwantskap vir Auger elektrone is 'n aanduiding van die hoë lineêre-energie-oordrag van hierdie deeltjies. Die lineêr-kwadratiese dosis-effek krommes vir mikrokerne frekwensies na blootstelling aan 60Co γ-strale was gebruik om die relatiewe biologiese doeltreffendheid (RBE) van die DNA geïnkorporeerde 123I te beraam. RBE waardes wissel van 19 ± 10 tot 32 ± 7 vir limfosiete van skenker 1 en 15 ± 6 tot 42 ± 11 vir skenker 2. Die dosis beperkte RBE (RBEM) vir limfosiet skenker 1 en 2 is onderskeidelik 34 ± 8 en 50 ± 15 en volg die verwagte skuif in terme van die inherente radiogevoeligheid van die skenkers. Die fraksie van limfosiete wat in S-fase was tydens die blootstelling aan 125IUdR was ingesluit in verdere dosimetrie berekeninge. Die gevolglike RBEs van die dosispunte van limfosiete van skenker 1 wissel van 4 ± 2 tot 7 ± 2 en dié van skenker 2 wissel van 3 ± 1 tot 9 ± 2. Die RBEM vir limfosiet skenker 1 en 2 is onderskeidelik 7 ± 2 en 11 ± 3. Die insluiting van die S-fase fraksie verminder die berekende RBEs aansienlik en die RBE waardes waargeneem hou goed verband met die wat in studies met fibroblaste en 125IUdR verkry is.

For over 100 years, cancer radiation therapy has provided patients with increased survival rates. Despite this success, radiation exposure poses a threat to the progeny of exposed parents. It causes transgenerational genome instability that is linked to transgenerational carcinogenesis. The exact mechanisms in which this instability occurs have yet to be discovered. Current evidence points to their epigenetic nature, specifically changes in DNA methylation. Using mouse and rat models, this thesis investigated the transgenerational effects of radiation in the offspring from parents who received whole body or localized exposure to ionizing radiation (IR). Both types of exposure resulted in significant global DNA hypomethylation in the somatic tissues of the progeny. These changes were paralleled by the significantly decreased levels of methyltransferases and methyl-CpG-binding protein. In summary, our results suggest that both localized and whole body parental exposures to IR result in transgenerational epigenetic instability within the unexposed offspring. / vii, 106 leaves : ill. ; 29 cm.

Bystander effects are traditionally known as a phenomenon whereby unexposed cells exhibit the molecular symptoms of stress exposure when adjacent or nearby cells are traversed by ionizing radiation. However, the realm of bystander effects can be expanded to include any systemic changes to cellular homeostasis in response to a number of biotic or abiotic stresses, in any molecular system. This thesis encompasses three independent experiments looking at bystander and bystander-like responses in both plant and animal models. In plants, an investigation into the regulation of small RNAs has given us some insights into the regulation of the plant hormone auxin in both stress-treated and systemic (bystander) leaves. Another plant model shows that a bystander-like plant-plant signal can be induced upon ionizing radiation to increase the genome instability of neighbouring unexposed (bystander) plants. In animals, it is shown that the microRNAome is largely affected in the bystander cells in a three-dimensional human tissue model. In silico and bioinfomatic analysis of this data provide us with clues as to the nature of bystander signalling in this human ‘in vivo’ model. / xiv, 141 p. : ill. ; 29 cm.

Thesis (MTech (Biomedical Technology))--Cape Technikon, Cape Town, 2004 / The need for radiobiological research was bom from the discovery that high doses of radiation could cause cancer and other health effects. However, recent developments in molecular biology uncovered the effects of low doses of radiation on different biological systems and as a result new techniques have been developed to measure these effects. The aim of this study was thus to validate biomarkers of initial DNA strand breaks, micronucleus formation, and the different pt ;ases of apoptosis as biological indicators of low-dose radiation damage. Furthermore, the difference in response of blood cells to different qualities and doses of radiation was investigated by irradiating cells with low- and high-LET radiation simultaneously. Blood from one donor was irradiated with doses between 0 and 4 Gy gamma- and neutron radiation. The alkaline single-cell gel electrophoresis (comet) assay was performed on different cell preparations directly after irradiation for the detection of initial DNA strand breaks. Radiation-induced cytogenetic damage was investigated using the cytokinesis-blocked micronucleus assay while different features of apoptosis were investigated by measuring caspase activation, enzymatic DNA fragmentation, and cellular morphology. The comet assay was sensitive enough to detect DNA strand breaks above 0.25 Gy and showed that the Iymphocyte isolation process induced some endogenous damage in cells, detected by the formation of highly damaged cells and hedgehogs in isolated cell preparations only.

Approximately 45% of men will develop cancer during their lifetime; some of which will be of reproductive age (Canadian Cancer Society, 2008). Current advances in treatment regimens such as radiotherapy have significantly lowered cancer-related mortality rates; however, one major quality-of-life issue in cancer survivors is the ability to produce healthy offspring. Exposure to ionizing radiation (IR) leads to genomic instability in the germline, and further to transgeneration genome instability in unexposed offspring of preconceptionally exposed parents. The results presented in this thesis define, in part, the molecular consequences of direct and indirect irradiation for the male germline. Direct exposure results in a significant accumulation of DNA damage, altered levels of global DNA methylation and microRNAome dysregulation of testis tissue. Localized cranial irradiation results in a significant accumulation of unrepaired DNA lesions and loss of global DNA methylation in the rodent (rat) germline. Biological consequences of the changes observed are discussed. / xii, 121 leaves : ill. ; 29 cm.

Indiana University-Purdue University Indianapolis (IUPUI) / The hematopoietic system is highly proliferative, making hematopoietic stem and progenitor cells (HSPC) sensitive to radiation damage. Total body irradiation and chemotherapy, as well as the risk of radiation accident, create a need for countermeasures that promote recovery of hematopoiesis. Substantive damage to the bone marrow from radiation exposure results in the hematopoietic syndrome of the acute radiation syndrome (HS-ARS), which includes life-threatening neutropenia, lymphocytopenia, thrombocytopenia, and possible death due to infection and/or hemorrhage. Given adequate time to recover, expand, and appropriately differentiate, bone marrow HSPC may overcome HS-ARS and restore homeostasis of the hematopoietic system. Prostaglandin E2 (PGE2) is known to have pleiotropic effects on hematopoiesis, inhibiting apoptosis and promoting self-renewal of hematopoietic stem cells (HSC), while inhibiting hematopoietic progenitor cell (HPC) proliferation. We assessed the radiomitigation potential of modulating PGE2 signaling in a mouse model of HS-ARS. Treatment with the PGE2 analog 16,16 dimethyl PGE2 (dmPGE2) at 24 hours post-irradiation resulted in increased survival of irradiated mice compared to vehicle control, with greater recovery in HPC number and colony-forming potential measured at 30 days post-irradiation. In a sublethal mouse model of irradiation, dmPGE2-treatment at 24 hours post-irradiation is associated with enhanced recovery of HSPC populations compared to vehicle-treated mice. Furthermore, dmPGE2-treatment may also act to promote recovery of the HSC niche through enhancement of osteoblast-supporting megakaryocyte (MK) migration to the endosteal surface of bone. A 2-fold increase in MKs within 40 um of the endosteum of cortical bone was seen at 48 hours post-irradiation in mice treated with dmPGE2 compared to mice treated with vehicle control. Treatment with the non-steroidal anti-inflammatory drug (NSAID) meloxicam abrogated this effect, suggesting an important role for PGE2 signaling in MK migration. In vitro assays support this data, showing that treatment with dmPGE2 increases MK expression of the chemokine receptor CXCR4 and enhances migration to its ligand SDF-1, which is produced by osteoblasts. Our results demonstrate the ability of dmPGE2 to act as an effective radiomitigative agent, promoting recovery of HSPC number and enhancing migration of MKs to the endosteum where they play a valuable role in niche restoration.

PiRNAs and their Piwi family protein partners are part of a germline specific epigenetic regulatory mechanism essential for proper spermatogenesis, silencing of transposable elements, and maintaining germline genome integrity, yet their role in the response of the male germline to genotoxic stress is unknown. Ionizing radiation (IR) is known to cause transgenerational genome instability that is linked to carcinogenesis. Although the molecular etiology of IR-induced transgenerational genomic instability is not fully understood, it is believed to be an epigenetically mediated phenomenon. IR-induced alterations in the expression pattern of key regulatory proteins involved in the piRNA pathway essential for paternal germline genome stability may be directly involved in producing epigenetic alterations that can impact future generations. Here we show whole body and localized X-irradiation leads to significant altered expression of proteins that are necessary for, and intimately involved in, the proper functioning of the germline specific piRNA pathway in mice and rats. In addition we found that IR-induced alterations to piRNA pathway protein levels were time and dose dependent. / ix, 123 leaves : ill. (some col.) ; 29 cm