RBRT Test 1 Review PDF

Test 1 Review Introduction to Cancer & Radiation Physics Radiobiology: study of radiation on living things 0.0 Terminology DNA: the molecule that carries genetic information for the development and functioning o Tumours: groups of abnormal cells that form lumps or growths (benign or maligna...

Test 1 Review Introduction to Cancer & Radiation Physics Radiobiology: study of radiation on living things 0.0 Terminology DNA: the molecule that carries genetic information for the development and functioning o Tumours: groups of abnormal cells that form lumps or growths (benign or malignant) Cancer: any one of a number of diseases characterized by the development of abnormal ce and have the ability to infiltrate and destroy normal body tissue 1.0 The Nature of Cancer Carcinogenesis: the process that leads to the formation of cancer; can take years ○ Normal cell (10-20 microns) --> genomic alteration leading to uncontrolled replicati ^9 cells) --> tumour formation (radiologically detectable at 2mm^3) Tumours ○ Benign: stay in one place [fixed]; don't usually come back after removed; regular sm [capsule: keeps cell contained, easier to resect]; moved easily in the tissue ○ Malignant: mobile [metastasis]; angiogenesis; uncontrolled proliferative signalling, suppressors § Carcinoma: 80-90% □ Adenocarcinoma: glands/organs □ Squamous Cell Carcinoma: squamous epithelium § Sarcoma: connective tissue § Myeloma: plasma cells of bone marrow § Leukemia: "liquid/blood cancer" of bone marrow § Lymphoma: lymph cells □ Hodgkin's □ Non-Hodgkin's § Mixed types: combinations 1.1 Epidemiology The study of the distribution and determinants of disease in human populations ○ Incidence: number of new events in a defined population in a specific time period ○ Prevalence: number of previously + newly diagnosed events in a defined population ○ Mortality: death rate, usually disease-related or event-specific 1.2 Etiology The study of causation (what factors cause cancer?) ○ of an organism ells that divide uncontrollably, ion (clinically detectable at 1g/10 mooth shape and covering , resist cell death & growth n in a specific time  ○ Incidence: number of new events in a defined population in a specific time period ○ Prevalence: number of previously + newly diagnosed events in a defined population ○ Mortality: death rate, usually disease-related or event-specific 1.2 Etiology The study of causation (what factors cause cancer?) ○ Risk factors: variables associated with a likelihood of a specific endpoint (i.e. not a your risk); often disease-site specific § Intrinsic or Non-intrinsic 1.2.1 Diving Into Specific Risk Factors 1. Age: risk increases as age increases 2. Carcinogens: pharmaceuticals; biological agents [virus/bacteria]; metals, arsenic, dusts, fib alcohol, coal smoke, salted fish; chemical agents and related occupations 2.0 Screening A mechanism of identifying the presence of an undiagnosed disease that has yet to show a ○ Allows us to catch the disease early, which decreases mortality rate and increases su A good screening test possesses: ○ Sensitivity: the effectiveness of the test to detect cancer in those who have the diseas ○ Specificity: the effectiveness of the test to give a negative result in those who do not ○ Acceptability: the extent to which the target population agrees to be tested by this m complicated) Strategies include running screening programs (e.g. free in Canada for certain demographi ○ Breast screening: 40-74 years old; non-invasive mammogram; every 2 years ○ Colorectal: 50-74 years old; invasive (colonoscopy) or non-invasive (blood test); ev ○ Lung screening: 55-74 years old (smokers); non-invasive CT scan ○ Prostate: 50-74 years old; non-invasive PSA (blood) test 3.0 Therapeutic Ratio Tumour response for a fixed level of normal-tissue damage (benefits / costs) ○ Any intervention will have its disadvantages or side effects ○ The goal is to maximize the benefits of a given intervention 4.0 Radiation Physics 4.0.1 Terminology  n in a specific time direct cause, but can increase bers; radiation [UV]; tobacco, any signs or symptoms urvival rate se [true +] t have the disease [true -] method (not too invasive or ics) very 5 years  ○ Any intervention will have its disadvantages or side effects ○ The goal is to maximize the benefits of a given intervention 4.0 Radiation Physics 4.0.1 Terminology Radiation: energy that comes from a source and travels through space at the speed of light electric field and magnetic field associated with it, and wave-like properties ○ Electromagnetic radiation: radiation consisting of interacting electrical and magnet of light (e.g. light, radio waves, Xrays) ○ Particle radiation: fast-moving particles that have both energy and mass 4.1 Ionizing Radiation Can be both electromagnetic and particulate (particle) radiation; lose energy when interact ○ Cosmic, gamma, Xrays, and UV (high frequency, short wavelength) ○ Different energies are associated with different types/wavelengths of radiation (eV) § 1 eV = 1.6e-19 J = 1.6e-12 ergs § SI unit for absorbed dose = Gray (Gy) □ Where 1 Gy = 1 J/kg = 100 rads = 10 000 ergs = 1 Sv = 1000 mSv □ And absorbed dose (D) = dE/dm 4.1.1 Photon Interactions Photoelectric Effect: photon interaction with inner-shell electron [diagnostic, annihilati machines, >20MeV] ○ All photon interactions will set electrons in motion! 4.1.2 Stopping Power Bethe-Bloch Equation: gives the mean rate of energy loss [stopping power (W)] of a heav ○ Where 𝐵 = max and min energy loss + a relativistic correction (differs for electrons ** this is energy gained by the tissue per unit length 4.1.3 Measuring Deposition of Energy Linear Energy Transfer (LET): energy transferred per unit path length (** this is energy l ○ High LET = more energy in smaller area (e.g. nucleus); low LET = energy scattering ○ Different forms of energy have different LET values (increases as photon energy dec increases) t; energy has an associated tic waves that travel at the speed ting with matter 20MeV] ion (2x0.511MeV) [major vy charged particle and protons) lost by the beam per unit length) g (e.g. Xray) creases, and as net particle charge  ○ High LET = more energy in smaller area (e.g. nucleus); low LET = energy scattering ○ Different forms of energy have different LET values (increases as photon energy dec increases) § Therefore, we can consider the use of different particles in cancer treatment fo ○ Calculated in one of two ways: § Track Average: divide the track into equal lengths, calculating energy deposit § Energy Average: divide the track into equal energy increments & average the Relative Biological Effectiveness (RBE): the amount of radiation is expressed in terms of physical quantity (Gy [energy absorbed per unit mass of tissue]) ○ Equal doses of different types of radiation do not produce equal biologic effects (e.g § Due to the microscopic differences in patterns of energy deposition § Thus, RBE is used to be able to compare different types of radiation with resp ○ Factors that determine RBE: § Radiation quality (RBE): type of energy and its energy, charged or uncharged § Radiation dose: # of fractions or dose per fraction § Dose rate g (e.g. Xray) creases, and as net particle charge or more/less damage ted in each length & find mean lengths of track the absorbed dose (D) as a g. 1 Gy neutrons > 1 Gy Xrays) pect to their inherent differences! d  ○ Factors that determine RBE: § Radiation quality (RBE): type of energy and its energy, charged or uncharged § Radiation dose: # of fractions or dose per fraction § Dose rate § Biologic system itself: high vs low repair 4.1.4 RBE as a Function of LET As dose [LET] increases, the survival curve [RBE] (1) becomes steeper, (2) shoulder beco ○ High LET radiation (like alpha rays) deposits energy densely over a short distance -- more cells [graph 1] ○ As LET increases, RBE also increases… to a certain point, where RBE may plateau already killed off) [graph 2] Optimal LET is around 100keV/micrometer, when separation between ionizing events coi DNA double helix (~2nm) ○ Highest chance of DSB, the basis of most biologic effects! Radiation Chemistry 1.0 Stages of Radiolysis & Effect 1. Physical (incident Xray --> ionizing e-) 2. Physical-Chemical (--> free ion radicals) 3. Chemical (--> bond breakage in DNA) 4. Biological (effects apparent at cellular/organ level) 1.1 The Stages (+Jablonski Diagrams) Diagrams are used to show changes in the energy state of a molecule (energy diagram) a. Physical Stage: the initial reaction that occurs; primary products [free radicals] form i. Ionization (e- ejected), then excitation (increase energy state) of target atom b. Physical-Chemical Stage: formation of free radicals and other molecules; 10^-15 to d omes smaller (i.e. increases too) - thus, less dose is needed to kill u/decrease (saturation, most cells incides with the diameter of the med in < 10^-15 seconds o 10^-12 seconds; 3 possible 1.1 The Stages (+Jablonski Diagrams) Diagrams are used to show changes in the energy state of a molecule (energy diagram) a. Physical Stage: the initial reaction that occurs; primary products [free radicals] form i. Ionization (e- ejected), then excitation (increase energy state) of target atom b. Physical-Chemical Stage: formation of free radicals and other molecules; 10^-15 to processes can occur… i. Internal Conversion: molecule absorbs energy from the ionizing radiation & lower state it was before [singlet state: opposite e- spins] 1) No radiation emitted, energy lost as heat ii. Crossing Over: e- drops even lower & undergoes change of spin [triplet state: 1) Molecules shift between electronic states (singlet --> triplet) 2) Once shifted, molecule can release energy through phosphorescence/etc with other molecules iii. Disassociation: extra energy released if more than bond energy; molecule bre (radicals) 1) Radicals can then go on to participate in further chemical reactions c. Chemical Stage: where chemical reactions between free radicals and other molecule seconds; 3 possible processes can occur… i. Luminescence 1) Fluorescence: photon emitted has E = difference of lowest singlet & gr 2) Phosphorescence: photon emitted has E = difference of triplet & ground 3) Non-radiative thermal ii. Energy transfer 1) Intermolecular: from excited molecule A* to ground state G (A* + G -- 2) Intramolecular: from one part of molecule A* to another part of the sam iii. Charge transfer 1) Intermolecular: from molecule A to molecule B (A* + B --> B* + A) d. Biological Stage: takes place over minutes, hours, days, weeks, years; can lead to on i. Radiation never works immediately… various reactions over time = many mis 1.2 Radiation Chemistry of Water Humans are mostly water, therefore most reactions are in water ○ Indirect ionizing effects transfer through water/cytoplasm… thus, 80% radiation is i 1.2.1 Products of Water 1. e-aq [aqueous electron: formed by ionization of water (into H2O+ + e-) --> free e-] 2. H [hydrogen radical: formed by free e- interacting with H2O (into H + OH-)] 3. OH [hydroxyl radical: formed by ionized water + water (into H3O+ + OH )] 1.2.2 Spatial Loss of Energy Clusters of reactive water radicals form discrete deposits of energy ○ Spurs: deposit of 100eV (usually single ionizing event that affects a few molecules; ○ Blobs: deposit of 300-700eV (many molecules affected; bigger area) Secondary reactions can then occur in these clusters due to high local concentrations of wa ○ OH + OH --> H2O2 [hydrogen peroxide] ○ H + H --> H2 [hydrogen gas] med in < 10^-15 seconds o 10^-12 seconds; 3 possible is excited; then, e- relaxes back to : same e- spins] c., or go onto further reactions eaks apart into small fragments es take place; 10^-12 to 10^-6 round state d state -> A + G*) me molecule (A*-a --> A-a*) ncogenesis or cell death stakes = cancer growth indirect, 20% is direct DNA break ; small area) ater radicals  Clusters of reactive water radicals form discrete deposits of energy ○ Spurs: deposit of 100eV (usually single ionizing event that affects a few molecules; ○ Blobs: deposit of 300-700eV (many molecules affected; bigger area) Secondary reactions can then occur in these clusters due to high local concentrations of wa ○ OH + OH --> H2O2 [hydrogen peroxide] ○ H + H --> H2 [hydrogen gas] ○ e-aq + H3O+ --> H + H2O [more water + more hydrogen radical] § Do not want these in your cells! Plus, they can continue to interact and interac 1.2.3 Subsequent Radical Reactions of Water G-value: the number of molecules of product (radicals) formed per 100eV of energy absor radiation-induced chemical reactions; represents the number of surviving reactive species ○ Higher G = lower LET (sparse energy deposit/LET = less secondary reactions = mor area = increase G-value) Radical "R" = solute; water "H2O" = solvent ○ Can have dimerization of R, non-identical final products, or hydrogen transfer ○ Radicals are not normal in the body's aqueous environment, therefore we do not natu people take antioxidants!) § Note: this is why patients should not take antioxidants during treatment -- we 2.0 Central Dogma of Molecular Biology DNA --> (transcribed) --> RNA --> (translated) --> Protein ○ Reactions of radicals with: § DNA backbone = breaks (ss/ds/cross-linking [binding of 'sticky ends']) § Nucleic acids = base damage (= kinks in DNA) □ *Each base has its own sensitivity to radicals! (most - T > C > A > G - ○ Then goes on to multiply these damaged sites in DNA (transcription/translation) = ir 2.1 How do we know radiation causes DNA damage? Pulsed-field gel electrophoresis (PFGE): determine how much DNA damage has been do ○ Heavier/unbroken DNA will travel shorter distance (heavier) than fragmented/broke Single-cell gel electrophoresis (Comet Assay): also used to determine DNA breakage ○ Irradiate cell culture --> break nucleus [DNA hub] --> see how far DNA travels with ○ Graph -- comet tail shows DNA migrating out of the cell (greater length = more brea ; small area) ater radicals ct…. rbed; essentially the efficiency of (less radical recombination) re individual reactions over larger urally "clear them up" (this is why utilize their free radicals! least) rregular proteins one (breakage) en DNA (lighter) h electrical current akage)  ○ Graph -- comet tail shows DNA migrating out of the cell (greater length = more brea DNA damage-induced nuclear foci (Radiation-Induced Foci Assay): indirect way to dete proteins involved in DNA repair ○ If stain is present in imaging, repair proteins are present (thus, show up in imaging o Production of non-functional proteins: ionizing radiation can cause fragmentation and agg Cell and Molecular Effects 1.0 Types of Cell Death 1. Apoptosis: programmed cell death; mechanism in which a cell knows it's at its end; p53 tr a. Morphology: cells shrink; loss of organelle organization; DNA condensation; nuclea 2. Necrosis: death caused by inability to survive/perform regular functioning; "demise of cell a. E.g. cutting off blood supply to cells will kill them b. Morphology: cells swell; membrane ruptures; loss of organelles (chaotic & unorgani 2.0 Mechanisms of DNA Repair 1. Base Excision Repair (BER): single-strand repair akage) ermine DNA damage; stain for of irradiated cells!) gregation of protein molecules riggers downstream effects ar rupture (requires energy) ls otherwise not destined to die" ized)  2. Necrosis: death caused by inability to survive/perform regular functioning; "demise of cell a. E.g. cutting off blood supply to cells will kill them b. Morphology: cells swell; membrane ruptures; loss of organelles (chaotic & unorgani 2.0 Mechanisms of DNA Repair 1. Base Excision Repair (BER): single-strand repair a. Free radical attaches to base, making it appear different (like 'wrong base') b. BER removes wrong base + sugar backbone c. Then replaces with correct base based on present complementary strand 2. Nucleotide Excision Repair (NER): single-strand repair a. Removal of several nucleotides/bases (removal of bulky adducts) b. Synthesis of new base sequence to fill the gap c. Ligation (sticking it together) 3. Homologous Recombination Repair: double-strand repair a. Requires an undamaged DNA strand to serve as a template (error-free repair, just co i. Usually during late S/G2 phase (undamaged sister chromatid is available to ac 4. Non-Homologous End-Joining (NHEJ): double-strand repair a. Simply joins the broken DNA back together, disregarding if it is actually right b. Error-prone, but also active in late S/G2 (thus, may induce mutant progeny as well) i. Not sure why one mechanism (homologous vs non-homologous) is used over 5. Cross Link Repair: interstrand or intrastrand repair a. Repair proteins make incision at crosslink/fork b. Excision repair and homologous recombination allow for re-replication at damaged 6. Mismatch Repair (MMR): used to repair wrong base matches (when A-T and C-G not pai a. Sensors identify the mismatch & recruit MMR factors/proteins b. Nucleotides excised & correct base replaced 3.0 Cell Cycle 2 distinct phases: 1. Interphase: majority of cycle i. G1: pre-replicative; decision window to commit to DNA replication [checkpo ii. S: synthesis; replication of DNA (8h) iii. G2: pre-replicative; decision window to commit to mitosis [checkpoint] (3h) 2. Mitotic phase (M): segregation of DNA into 2 daughter cells (1h) i. Prophase ii. Metaphase iii. Anaphase iv. Telophase v. Cytokinesis ** total cycle t 3.1 Chromosomes DNA is organized in chromosomes in eukaryotes; 2 sister chromatids joined by a centrom  ls otherwise not destined to die" ized) opies!) ct as template) the other area ired) oint] (4h in mammalian cells) time in mammalian cells = 16h ** mere  iv. Telophase v. Cytokinesis ** total cycle t 3.1 Chromosomes DNA is organized in chromosomes in eukaryotes; 2 sister chromatids joined by a centrom ○ Human somatic cell = 46 chromosomes (23 pairs -- 22 autosomal, 1 sex) ** Note: "chromatins" --> (M) --> "chromosomes" Chromosomal damage [chromatid aberrations] by radiation can be studied at the first mit ○ Broken ends are 'sticky' (can interact with other chromosomes or molecules) Restitution: breaks rejoin to their original configuration Deletion: breaks fail to rejoin; no centromere [acentric] = loss of info fragmen Exchange: breaks rejoin to other broken ends ○ Lethal chromosomal aberrations: i. Dicentric: due to exchange (between two separate chromatids) 1) Break in each chromosome in early interphase & join together 2) Illegitimate Unions: one chromosome has two centromeres, the other ha 3) After replication, the dicentric chromosome goes on, and acentric is lost ii. Ring: break in both arms of the same chromatid in early interphase 1) Incorrect Unions: sticky ends join to form a ring, while produced fragm 2) Rings replicate & have some genetic impact down the line iii. Anaphase Bridge: chromosomal breaks after replication (in G2) 1) Sister Unions: breaks may occur in sister chromatids, such that sticky jo 2) The chromosome cannot be pulled apart at Anaphase, and so is stretched separated into two daughter cells) 3) Extra fragments lost [acentric] ○ Non-lethal chromosomal aberrations (but still problematic): § Symmetric Translocation: break in G1 chromatids, where broken fragments a □ E.g. Philadelphia Chromosome (9 & 22) § Small Interstitial Deletions: two breaks in the same arm of the same chromati the breaks is lost [deletion] 4.0 Laboratory Techniques for Studying Cells In-vitro assay: use of a growth medium that mimics the environment within the body ○ Growth medium usually supplemented with essential amino acids & some serum fro § Thus, mammalian cells can be grown in tissue culture outside the body! □ Cell strains: not immortal; senescent after 40-60 doublings [Hayflick Li □ Cell lines: immortal; able to divide indefinitely (like cancer cells) ○ Cells are trypsinized [detached from dish by breaking aggregates, using trypsin] to b ○ Cells grow exponentially, such that: § dN ~ Ndt (change in cell number [dN] is proportional to cell number [N] at tim § dN = kNdt (where k is the constant of proportionality; 'slope' of logN vs t grap □ k = 0.693/t2 (where t2 is doubling time) In-vivo assay: the use of living organisms ○ Usually inbred mice, since homozygous recessive = immunosuppression/deficiency grow human tumours § 98% homozygous after 20th cycle of inbreeding (F20)  time in mammalian cells = 16h ** mere tosis after exposure nts as none [acentric] t (not pulled at Anaphase) ments are lost [acentric] oins end in the same chromosome d across the cell (cannot be are exchanged between the two id, where the fragment between om an animal imit] be counted me interval dt); and so… ph = straight line) ["Nude" or "SCID"], used to  § dN ~ Ndt (change in cell number [dN] is proportional to cell number [N] at tim § dN = kNdt (where k is the constant of proportionality; 'slope' of logN vs t grap □ k = 0.693/t2 (where t2 is doubling time) In-vivo assay: the use of living organisms ○ Usually inbred mice, since homozygous recessive = immunosuppression/deficiency grow human tumours § 98% homozygous after 20th cycle of inbreeding (F20) § Certain characteristics can be selected for during inbreeding 4.1 Generation Time (Tg) Time for one cell to go around the cell cycle (G1 > S > G2 > M) ○ Can be less than doubling time for a cell population, since not all progeny are viable ○ Tg for mammalian cells = 16h 4.1.1 Determining Where Cells are in the Cell Cycle 1. Use of titrated thymidine (unstable; 1 p+ and 2 n0 --> decays to helium, giving off 5.5keV a. Labelling cells with tritium (H3) allows the isotope (labelled thymidine) to be selecti synthesis b. Decay & energy release can be imaged/tracked through the cell cycle! 2. BrdU Proliferation Assay a. Introducing radiolabelled nucleotides [5-bromo-20-deoxyuridine = thymidine analog incorporated in new DNA (as T base) b. BrdU can be fluorescently labelled via antibody & seen through fluorescence micros i. See more fluorescence as time goes on (more incorporation into DNA as it rep 4.2 Measuring Cell Viability and Survival In-Vitro 1. Calculate plating efficiency (human error) 2. Do multiple assays/plates to compare different conditions/irradiative doses me interval dt); and so… ph = straight line) ["Nude" or "SCID"], used to e V) ively taken by DNA during g] in S phase, such that they are scopy plicates)  2. Do multiple assays/plates to compare different conditions/irradiative doses 4.2.1 Survival Curves Looks at dose given vs number of surviving colonies ○ N: back extrapolation number of the straight portion of the survival curve, to the y-a § i.e. read the y-axis number [surviving fraction] at x = 0 ○ D37 or D1: dose of radiation to reduce survival by 37% of initial survival (i.e. 63% ○ Dq: quasi-threshold dose; back extrapolation of the survival curve to intercept the li 100%) § i.e. read the x-axis number [dose] at y = 1.0 ○ D0: dose of radiation to reduce survival to 37% (range of x from y = 1.0 to y = 0.37) 4.3 Measuring Cell Death In-Vivo 1. Mouse tumour models: immunodeficiency = no repair mechanisms to fight tumours a. Subcutaneous (Heterotopic Implantation): monitor tumour size externally i. Limited to localized cancers (no metastasis) b. Genetically engineered (GEMM): genetically modify embryo so cancer 'naturally' d i. Breeding required (embryo implanted in mouse, then mates with WT mouse; s axis (zero dose) remaining) ine from surviving fraction 1.0 (or ) develops wherever it wants select for homozygous offspring) 4.3 Measuring Cell Death In-Vivo 1. Mouse tumour models: immunodeficiency = no repair mechanisms to fight tumours a. Subcutaneous (Heterotopic Implantation): monitor tumour size externally i. Limited to localized cancers (no metastasis) b. Genetically engineered (GEMM): genetically modify embryo so cancer 'naturally' d i. Breeding required (embryo implanted in mouse, then mates with WT mouse; s 2. Tumour Re-growth Assay: all mice have tumour, then get some dose of radiation; measu of different dose groups a. Continue Tx after success observed (apoptosis), to see when cells begin to regrow (t i. Growth delay: time after irradiation that it takes a tumour to regrow to start siz ii. Index of radiation damage: time taken for tumour to grow from one point to a diameter of 7mm to 15mm) 1) Note: TCON = unirradiated control cells; Tx-ray = irradiated experimental 3. Tumour Cure (TCD50) Assay: mice with same size tumours separated into irradiation gr each Tx/fraction/dose a. Found that "single dose" (less time between radiation Tx) = more tumour control! (w b. Found that "10 doses" (more time between radiation Tx) = less tumour control (and i. Thus, there is some level of DNA repair in cancer cells; time between treatme increased regrowth) develops wherever it wants select for homozygous offspring) ure average tumour size post-Tx tumour re-formation) ze an experimental endpoint (e.g. cells roups, based on time between with less dose total!) more total dose administered) ents matters (increased time =  4. Dilution Assay: single-cell suspension prepared by diluting (counted, known #) cancer cel then injected into mice; determine how many irradiated cells are required to produce a tum a. TD50: number of cells required to transmit cancer/tumour to 50% of recipient anima b. E.g. mice experiments with lung & spleen 5. Other tumour model assays a. Cell Pellet Culture Method: use spheroids (cell sphere) for testing i. No mice needed! b. Organoids: cultured from stem cells; expanded, then differentiated until fully devel i. Compared to spheroids, contains stromal components and other growth factor ii. No mice needed! c. PDX: biopsy obtained, tumour cells isolated and cultured; injected into mice & obse d. Chick embryo (CAM): human cancer cells injected into CAM membrane of fertiliz human tumour with high vasculature, multiple cell types, and ECM is formed (suppl i. More ethical -- animal nerve fibers not fully developed ii. Naturally immunodeficient iii. But sensitive to environmental factors & cannot observe normal (live) animal/ Models of Cell Survival 1.0 Survival Curves (cont.) Shoulder of the curve = where sublethal repair takes place (repair mechanisms are triggere ○ Normal tissue has a wider shoulder, since it has proper repair mechanisms ○ Tumour tissue has a narrower shoulder, since it has less/nonfunctional repair mecha HeLa Cell Line: cell line from Henrietta Lacks (unknowingly extracted her cancer cells fo After radiation, there are two pathways for cell death: ○ Apoptosis ○ Mitotic death [caused by chromosomal damage and lethal aberrations; loss of geneti § In general, more lethal damage ~ less survival § BUT cells are generally more radioresistant during S phase and more radiosen □ Radioresistant cell lines show no evidence of apoptosis (wide shoulder, □ Radiosensitive cell lines show more apoptosis (narrow shoulder, less rep 1.1 Radiation-Induced Cell Death 1.1.1 Bystander Effect Not all cells are exposed to radiation, but there may be higher occurrence of cell death in m signals to unirradiated cells - "we're all dying!")  lls (grown & irradiated in-situ), mour al population loped structure/organoid rs (more realistic) erved zed chicken egg; in a few days, a lemental to tumour survival) /biological response ed) anisms or research) (24h cell cycle) ic info in progeny] nsitive during M more repair) pair) more cells (irradiated cells send 1.1 Radiation-Induced Cell Death 1.1.1 Bystander Effect Not all cells are exposed to radiation, but there may be higher occurrence of cell death in m signals to unirradiated cells - "we're all dying!") Defined as the induction of biological effects in cells that are not directly traversed by radi cells that are 1.1.2 Random Distribution Ionization events are random, with random distributions (everything is by chance) Characterized by the Poisson Distribution (events are random & can be independent of ea ○ Gives the probability 0-1 of an event happening a certain number of times (k) within ○ A function of lambda [the mean number of events] ○ Constraints: § The probability of one event does not affect the probability of another event § Lambda is assumed to be constant (although only calculated for one time perio § Only appropriate for count data [observations + non-negative integers] ○ Thus, we assume mean number of cells killed is proportional to the dose of radiation probability) § In cell survival curves, each hit is assumed to cause a killing (probability for a P(0) where x=1 = 37% = D0 = 1-2Gy) § Killing has exponential behaviour, where (1) a dose that reduces survival by 5 then 12.5%, respectively; and (2) steepness of the curve represents radiation e □ Curve is affected by… ® LET ® Fractionation ® Dose-rate effect ® Intrinsic radiosensitivity ® Cell age ® Oxygenation 1.2 Target Model Theory Hypothesis: critical target (typically DNA) is inactivated for a cell to be killed (function d 1.2.1 Single Target Single Hit If one hit per target destroys it, the number of survivors (N) is the dose in 'hits' = D37 = D0 more cells (irradiated cells send iation, but are in proximity to ach other) n a given time interval od) n administered (count on the a single cell survival is zero hit, 50% will then reduce it to 25%, effectiveness disrupted, break…) 0  ® Oxygenation 1.2 Target Model Theory Hypothesis: critical target (typically DNA) is inactivated for a cell to be killed (function d 1.2.1 Single Target Single Hit If one hit per target destroys it, the number of survivors (N) is the dose in 'hits' = D37 = D0 Survival curve has no shoulder (linear) Exponential decrease of N as dose increases 1.2.2 Multitarget-Single Hit Model Cell consists of multiple targets and each should be hit once to achieve cell death ○ Assumes DNA is just one of multiple targets ○ Does not consider sublethal repair or time-dependent qualities 1.2.3 Linear Quadratic Model Assumes killing has two components, (1) proportional to dose (linear part), (2) proportiona part) ○ Alpha/Beta ratio: linear damage is alpha, quadratic damage is beta; the ratio represe types of damage contribute equally to cell death § High ratio = more sensitive to radiation, less able to repair; more responsive to □ Should use high dose per fraction □ E.g. tumour tissues § Low ratio = more resistant to radiation, better repair; more responsive to quad □ Should use smaller doses over more fractions (hit at right time in cell cy □ E.g. normal tissues ○ We use BED [biological effective dose] to know biologically-equivalent effects of c § E.g. 78 Gy over 39 Fx = 40 Gy over 5 Fx [for one beam type, not comparing t Factors That Influence Cell Survival 1.0 Cyclin Dependent Kinase (CDKs) Cell cycle is regulated by periodic activation of various members of the CDK family (prot Phosphorylate specific proteins that drive key events (like DNA synthesis & mitosis) ○ But cells growing in-vitro grow asynchronously… how do we get many cells at the § Mitotic Harvest: mitotic cells are rounded & have less adherence to solution a § Hydroxyurea: blocks cells from entering S phase; accumulation of cells at G1 continue growing at same pace § Hypoxia: lack of O2 can be used as a sensitizer; cells need it to repair, so lack radiation effects 1.1 Cell Progression After Radiation Radiation induces two blocks to cell cycle to = more radioresistant (by signalling CDKs to ○ Late G1: delay allows for repair of DNA damage before S-phase (p53) ○ Late G2: delay allows for repair of DNA before chromosome condensation (p34/CD disrupted, break…) 0 al to square of the dose (quadratic ents the dose at which these two o linear damage [numerator!] dratic damage [denominator!] ycle, when more sensitive) changing dose amount & fractions two types!] tein complexes) same stage? around them 1/S border, collect, then let them k would result in more damaging o stop or continue) DK1)  § Hypoxia: lack of O2 can be used as a sensitizer; cells need it to repair, so lack radiation effects 1.1 Cell Progression After Radiation Radiation induces two blocks to cell cycle to = more radioresistant (by signalling CDKs to ○ Late G1: delay allows for repair of DNA damage before S-phase (p53) ○ Late G2: delay allows for repair of DNA before chromosome condensation (p34/CD 1.2 Classifications of Radiation Damage Potentially Lethal Damage (PLD): can be modified by post-irradiation environmental con Sublethal Damage: can normally be repaired in hours (needs some time) unless more SLD ○ If we treat with a second fractionation 30 mins after the first, cells will be in M (and § Note: shoulder is repeated every time dose is readministered Lethal damage: irreversible and irreparable; leads to cell death 1.2.1 Dose Rate Effect Biological effect of a dose decreases as time increases (we want to hit cells at their most ra much time for their repair) ○ More time between LDR fractions = increased cell survival :( 1.2.2 Inverse Dose Rate Effect Under continuous low-dose-rate irradiation, cells are stopped in their radiosensitive G2 ph ○ An asynchronous population of cells becomes a synchronous radiosensitive populati 1.2.3 The Oxygen Effect Cells are more sensitive to radiation when in oxygen ○ Also, ROS can be created in presence of O2 = lots of damage! ○ OER [oxygen enhancement ratio]: ratio of doses administered under hypoxic to aer achieve the same biologic effect That being said, hypoxia is a major radiation resistance factor ○ Tumours undergo angiogenesis for increased blood flow = increased O2 supply = ca ○ But smoking = hypoxia (blood binds to things other than O2, not good for Tx) We get patients to breathe O2 during some Tx, or simply give more dose to more hypoxic Note: as LET increases, OER decreases… but RBE increases… ○ Must find a common ground! Although if radiation is significantly working for a tum worry about 1.2.4 Reoxygenation Tumour surface has more vasculature, tumour core has less vasculature ○ But hypoxic core cells become oxygenated after a dose of radiation § Right after = hypoxic cell dominated § Given time = reoxygenation restores proportions to pre-rad level! (as core cell more radiosensitive than before □ Note: this can cause inflammation and swelling 1.3 Four Rs of Radiobiology 1. Repair (DNA repair; curve shoulder) k would result in more damaging o stop or continue) DK1) nditions D is added d more sensitive) = more damage! adiosensitive time, not giving hase ion rated conditions needed to an target areas of tumour mour, OER is not as important to ls move closer to surface) & are  § Given time = reoxygenation restores proportions to pre-rad level! (as core cell more radiosensitive than before □ Note: this can cause inflammation and swelling 1.3 Four Rs of Radiobiology 1. Repair (DNA repair; curve shoulder) 2. Redistribution (at different points in cell cycle, cells can be more or less radiosensitive; S 3. Repopulation (given time, cells will replicate into new progeny; M) 4. Reoxygenation (O2 is a radiosensitizer) Radiation Sensitivity and Risk Radiation is a normal component of our environment, in small doses ○ E.g. cosmic rays, building materials, earth's crust, ingested (in tiny doses) 1.0 Nuclear Accidents 1. Chernobyl: 1986, Ukraine; human error, safety system turned off for a reactor during che radius, minimal life; spread to other countries a. Population studies show… i. Thyroid disease: 25000 individuals followed in Belarus and Ukraine 1) 45 thyroid cancers detected in Ukraine 2) Excess relative risk of ~5 per 1Gy 3) Older age = decreased risk 4) Fraction of cancers attributed to radiation was 75% 2.0 Why Measure Radiation Risk? What is the likelihood of exposed individuals developing cancer? (epidemiological unders Quantify risks associated with different exposure scenarios (how much until symptoms de Establish public health and occupational safety policy (e.g. dosimeters/TLD) Research on teratogenesis & generational risks 2.1 Stochastic and Deterministic Effects Stochastic: severity of effect is independent of dose, but probability of effect occurring inc ○ i.e. more exposure = more likely to develop something ○ Probability always increasing with dose, but never definite (value) Deterministic: severity of effect increases with dose; practical thresholds [where probabili ○ E.g. 1Gy to eyes = cataracts; 1+ Gy to eyes = more severe cataracts ○ Probability is definite at some point (value) 2.2 Radiation Weighting Factors Radiation effects are related to dose and quality (type/LET) of radiation ○ Quality = weighting factor (WR), where a value is assigned to different types of radi § Photons, electrons, muons = 1 § Protons, charged pions = 2 ls move closer to surface) & are vs M) ecks; "exclusion zone" = 30km standing of carcinogenesis) evelop?) creases with dose; no threshold ity increases rapidly] iation 2.2 Radiation Weighting Factors Radiation effects are related to dose and quality (type/LET) of radiation ○ Quality = weighting factor (WR), where a value is assigned to different types of radi § Photons, electrons, muons = 1 § Protons, charged pions = 2 § A-particles, heavy ions = 20 § Neutrons = continuous curve as a function of neutron energy ○ Equivalent dose (Sv): product of the absorbed dose (averaged over the tissue/organ) Tissues vary in their sensitivity to radiation; tissue weighting factor (WT) accounts for this ○ The sum of all weighting factors equal to 1.0 [whole body] ○ Effective dose: the sum of all weighted doses in the irradiated tissue/organ 3.0 Risks from Radiation Exposure Skin cancer and leukemia reported in x-ray workers before safety standards Lung cancer is frequent in miners who dug out ore containing radium In Britain, radiotherapy to the spine to relieve pain increased leukemia incidences 3.1 Radiation Risk Modelling Absolute Risk Model: assumes the effect of radiation is to increase the natural incidence a Relative Risk Model: predicts a large number of radiation-induced cancers in old age 3.1.1 Quantitative Risk Estimates for Radiation-Induced Cancer 1. BIER [Biological Effects of Ionizing Radiation] 2. UNSCEAR [Scientific Committee on the Effects of Atomic Radiation] a. Both report increased mortality in females, decreased mortality in older age (for Ato 3. Mega-Mouse Project a. Compares radiation-induced mutations with those that occur spontaneously b. Russel & Russel (7 million mice used) c. Determined specific locus mutation rates under various irradiation conditions d. Four major conclusions: i. Radiosensitivity of different mutations varies, typically by gene size ii. There is a dose-rate effect, and spreading Tx over a period results in fewer mu iii. Female oocytes are killed instantly by radiation, so only males were used iv. Heritable consequences of a given dose can be reduced if a time interval is all conception (repair) 1) At least 2 months in males, more in females = significant reduction 2) In humans, this = 6 months (adjusted for the fact humans are exposed to iation ) and the WR s at all ages omic Bomb Hiroshima) utations than acute exposure lowed between irradiation and o LDR, and mice acute HDR)  iii. Female oocytes are killed instantly by radiation, so only males were used iv. Heritable consequences of a given dose can be reduced if a time interval is all conception (repair) 1) At least 2 months in males, more in females = significant reduction 2) In humans, this = 6 months (adjusted for the fact humans are exposed to 3.1.2 Recommended Dose Limits (stochastic) Occupational = 20mSv/year over 5 years Embryo = 1mSv total to abdomen Public exposure = 1mSv/year lowed between irradiation and o LDR, and mice acute HDR)