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Discrete probability distribution Given number of events In a fixed interval of time and/or space Known average rate Independently of the time since the last event k is the number of occurrences of an event — the probability of which is given by the function λ is a positive real number, equal to the expected number of occurrences during the given interval.Simeon Denis Poisson(1781-1840)
Overview• Reproductive Integrity• The In Vitro Survival Curve• The Shape of the Survival Curve• Mechanisms of Cell Killing – DNA as the Target – The Bystander Effect – Apoptotic and Mitotic Death – Autophagic Cell Death – Senescence• Survival Curves for Various Mammalian Cells in Culture• Survival Curve Shape and Mechanisms of Cell Death• Oncogenes and Radioresistance• Genetic Control of Radiosensitivity• Intrinsic Radiosensitivity and Cancer Stem Cells• Effective Survival Curve for a Multifraction Regimen• Calculations of Tumor Cell Kill• The Radiosensitivity of Mammalian Cells Compared with Microorganisms
Reproductive Integrity• Cell survival curve describes the relationship between the radiation dose and the proportion of cells that survive. – Cell ―Death‖ : loss of reproductive integrity• Clonogenic : survivor able to proliferate indefinitely to produce a large clone or colony
Reproductive Integrity• Mitotic death : death while attempting to divide(dominant following irradiation)• Apoptosis : programmed cell death• In general, a dose of 100 Gy is necessary to destroy cell function in nonproliferating systems.• By contrast, the mean lethal dose for loss of proliferative capacity is usually less than 2 Gy.
The Shape of the Survival Curve• At ―low doses‖ for sparsely ionizing (low-linear energy transfer[LET]) radiations, such as x-rays, the survival curve starts out straight on the log-linear plot with a finite initial slope. – The surviving fraction is an exponential function of dose.• At higher doses, the curve bends.• At very high doses, the survival curve often tends to straighten again. (usually not occur in RT)
The Shape of the Survival Curve A:The linear quadratic model. B:The multitarget model. A. Good fit to experimental data for the first few decades of survival.
The Shape of the Survival Curve• For densely ionizing (high-LET) radiations, such as α- particles or low-energy neutrons, the cell survival curve is a straight line from the origin.• Many attempts to deduce a curve shape that is consistent with experimental data, but it is never possible to choose among different models or theories based on goodness of fit to experimental data.
The Shape of the Survival Curve• 1. The multitarget model – D1 : initial slope(from single-event killing) – D0 : final slope(from multiple-event killing) – n or Dq : the width of the shoulder – D1, D0 are the dose required to reduce the fraction of surviving cells to 37% of its previous value. • Ex) D1 : 1 0.37 , D0 : 0.1 0.037 or 0.01 to 0.0037 – The surviving fraction is on a logarithmic scale and the survival curve becomes straight at higher doses, the dose required to reduce the cell population by a given factor (to 0.37) is the same at all survival levels. (On average, the dose required to deliver one inactivating event per cell.)
The Shape of the Survival Curve• 2. The linear-quadratic model – Direct development of the relation used to describe exchange-type chromosome aberrations that are clearly the result of an interaction between two separate breaks. – Assumes that there are two components to cell killing by radiation. • One that is proportional to dose • One that is proportional to the square of the dose
The Shape of the Survival Curve• The components of cell killing that are proportional to dose and to the square of the dose are equal if αD = βD2 or D = α/β• The linear and quadratic contributions to cell killing are equal at a dose that is equal to the ratio of α to β.• A characteristic of the LQ formulation is that the resultant cell survival curve is continuously bending; there is no final straight portion.
Mechanisms of Cell Killing <DNA as the Target>• The principal sensitive sites for radiation-induced cell lethality are located in the nucleus as opposed to the cytoplasm.• The evidence implicating the chromosomes, specifically the DNA, as the primary target for radiation-induced lethality may be summarized as follows:
Mechanisms of Cell Killing• 1. Cells are killed by radioactive tritiated thymidine incorporated into the DNA. The radiation dose results from short-range α-particles and is therefore very localized.
Mechanisms of Cell Killing• 2. Certain structural analogues of thymidine, particularly the halogenated pyrimidines, are incorporated selectively into DNA in place of thymidine if substituted in cell culture growth medium. This substitution dramatically increases the radiosensitivity of the mammalian cells to a degree that increases as a function of the amount of the incorporation. Substituted deoxyuridines, which are not incorporated into DNA, have no such effect on cellular radiosensitivity.
Mechanisms of Cell Killing• 3. Factors that modify cell lethality, such as variation in the type of radiation, oxygen concentration, and dose rate, also affect the production of chromosome damage in a fashion qualitatively and quantitatively similar. This is at least prima facie evidence to indicate that damage to the chromosomes is implicated in cell lethality.
Mechanisms of Cell Killing• 4. Early work showed a relationship between virus size and radiosensitivity; later work showed a better correlation with nucleic acid volume. The radiosensitivity of a wide range of plants has been correlated with the mean interphase chromosome volume, which is defined as the ratio of nuclear volume to chromosome number. The larger the mean chromosome volume, the greater the radiosensitivity.
Mechanisms of Cell Killing <The Bystander Effect>• Defined as the induction of biologic effects in cells that are not directly traversed by a charged particle, but are in proximity to cells that are.• Nagasawa and Little, 1992 – Low dose of α-particles, a larger proportion than estimated of cells showed an biologic change.
Mechanisms of Cell Killing• The use of sophisticated single-particle microbeams, which make it possible to deliver a known number of particles through the nucleus of specific cells.• The bystander effect has also been shown for protons and soft x-rays.• The effect is most pronounced when the bystander cells are in gap-junction communication with the irradiated cells.• For example, up to 30% of bystander cells can be killed in this situation.
Mechanisms of Cell Killing• The effect being due, presumably, to cytotoxic molecules released into the medium.• The existence of the bystander effect indicates that the target for radiation damage is larger than the nucleus and, indeed, larger than the cell itself.• Its importance is primarily at low doses, where not all cells are ―hit‖.
Mechanisms of Cell Killing• In addition to the experiments described previously involving sophisticated single-particle microbeams, there is a body of data involving the transfer of medium from irradiated cells that results in a biologic effect(cell killing) when added to unirradiated cells. – Suggest that irradiated cells secrete a molecule into the medium that is capable of killing cells when that medium is transferred onto unirradiated cells.
Mechanisms of Cell Killing <Apoptotic and Mitotic Death>• Apoptosis in Greek word : ―falling off‖• Programmed cell death• Occurs in normal tissues, also can be induced in some normal tissues and in some tumors by radiation.
Mechanisms of Cell Killing• For example, tadpoles lose their tails. – To cease communicating with its neighbors – Rounds up and detaches from its neighbors. – Condensation of the chromatin at the nuclear membrane and fragmentation of the nucleus. – The cell shrinks because of cytoplasmic condensation(crosslinking of proteins and loss of water). – The cell separates into several membrane-bound fragments of differing sizes : apoptotic bodies – Double-strand breaks(DSBs) occur in the linker regions between nucleosomes, producing DNA fragments that are multiples of approximately 185 base pairs. Laddering in gels. – Cf) necrosis causes a diffuse ―smear‖ of DNA in gels.
Mechanisms of Cell Killing• Apoptosis is highly cell-type dependent.• Hemopoietic and lymphoid cells are particularly prone to rapid radiation-induced cell death by the apoptotic pathway.• Apoptosis after radiation seems commonly to be a p53- dependent process; Bcl-2 is a suppressor or apoptosis.
Mechanisms of Cell Killing• The most common form of cell death from radiation is mitotic death. – Cells die attempting to divide because of damaged chromosomes. – The log of the surviving fraction – The average number of putative ―lethal‖ aberrations per cell(asymmetric exchange-type aberrations such as rings and dicentrics) The experiment was carried out in a cell – One-to-one correlation. line where apoptosis is not observed.
Mechanisms of Cell Killing• Data such as these provide strong circumstantial evidence to support the notion that asymmetric exchange-type aberrations represent the principle mechanism for radiation-induced mitotic death in mammalian cells.
Mechanisms of Cell Killing• At low doses, the two breaks may result from the passage of a single electron set in motion by the absorption of a photon of x- or γ- rays. Linear curve• At higher doses, may result from two separate electrons. bending curve.
Mechanisms of Cell Killing <Autophagic Cell Death>• Autophagy : self-digestive process that uses lysosomal degradation of long- lived proteins and organelles to restore or maintain cellular homeostasis.• Stress-inducing condition can also promote autophagic, or what has been termed programmed type II, cell death.• The combination of endoplasmic stress- inducing agents and ionizing radiation could enhance cell killing by inducing autophagic cell death.
Mechanisms of Cell Killing <Senescence>• The change in the biology of an organism as it ages after its maturity.• Has been classified as a tumor suppressor mechanism that prevents excessive cellular divisions in response to inappropriate growth signals or division of cells that have accumulated DNA damage.
Survival Curves for Various Mammalian Cells in Culture• The first in vitro survival curve for mammlian cells irradiated with x- rays.• All mammalian cells studied to date, normal or malignant, regardless of their species of origin, exhibit x-ray survival curves similar to those in figure. Initial shoulder
Survival Curves for Various Mammalian Cells in Culture• The D0 of the x-ray survival curves for most cells cultured in vitro falls in the range of 1 to 2 Gy.• The exceptions are cells from patients with cancer-prone syndromes such as ataxia-telangiectasia(AT); these cells are much more sensitive to ionizing radiations, with a D0 for x-rays of about 0.5 Gy.
Survival Curves for Various Mammalian Cells in Culture• Some researchers were skeptical that these in vitro techniques, which involved growing cells in petri dishes in very artificial conditions, would ever benefit clinical radiotherapy. – A metaphor of ―Robinson Crusoe‖• The in vitro culture technique measured the reproductive integrity of cells and that there was no reason to suppose that Robinson Crusoe’s reproductive integrity was any different on his desert island from what it would have been had he remained in York.
Survival Curves for Various Mammalian Cells in Culture• In more recent years, extensive studies have been made of the radiosensitivity of cells of human origin, both normal and malignant, grown and irradiated in culture. – In general, cells from a given normal tissue show a narrow range of radiosensitivities if many hundreds of people are studied. – By contrast, cells from human tumors show a very broad range of D0 values.
Survival Curves for VariousMammalian Cells in Culture
Survival Curve Shape and Mechanisms of Cell Death• Mammalian cells cultured in vitro vary considerably in their sensitivity to killing by radiation.
Survival Curve Shape and Mechanisms of Cell Death Radioresistant Large dose- rate effectRadiosensitiveNo dose-rate effect Laddering (after 10 Gy)
Survival Curve Shape and Mechanisms of Cell Death• Although asynchronous cells show this wide range of sensitivities to radiation, mitotic cells from all of these cell lines have essentially the same radiosensitivity. – In interphase, the radiosensitivity differs because of different conformations of the DNA.
Survival Curve Shape and Mechanisms of Cell Death• Characteristic laddering is indicative of programmed cell death or apoptosis during which the DNA breaks up into discrete lengths as previously described.• Comparing Fig.A and B, it is evident that there is a close and impressive correlation between radiosensitivity and the importance of apoptosis.• Increased ―laddering‖ = Increased radiosensitivity
Survival Curve Shape and Mechanisms of Cell Death• Mitotic death results (principally) from exchange-type chromosomal aberrations; the associated cell survival curve, therefore, is curved in a log-linear plot, with a broad initial shoulder.
Oncogenes and Radioresistance• Numerous reports have appeared in the literature that transfection of activated oncogenes into cells cultured in vitro increases their radioresistance, as defined by clonogenic survival.• It is by no means clear that oncogene expression is directly involved in the induction of radioresistance, and it is far less clear that oncogenes play any major role in radioresistance in human tumors.
Genetic Control of Radiosensitivity• A radiosensitive mutant can result from a mutation in a single gene that functions as a repair or checkpoint gene.• In many but not all cases, their sensitivity to cell killing by radiation has been related to their greatly reduced ability to repair DNA DSBs.
Genetic Control of Radiosensitivity <Inherited Human Syndromesassociated with sensitivity to X-rays>• Ataxia-telangiectasia(AT)• Seckel syndrome• Ataxia-telangiectasia-like disorder• Nijmegen breakage syndrome• Fanconi’s anemia• Homologues of RecQ-Bloom syndrome, Wernder syndrome, and Rothmund-Thompson syndrome
Intrinsic Radiosensitivity and Cancer Stem Cells• It has been well accepted that the radiosensitivity of cells changes as they undergo differentiation. – The more differentiated tumor cells, the more survival? – No. – In fact, cancer stem cells may be more resistant to radiation than their more differentiated counterparts. – Due to high intensity of free radical scavenger. – Needs further evaluations and tests.
Effective Survival Curve for a Multifraction Regimen
Calculations of Tumor Cell Kill• Problem 1• A tumor consists of 108 clonogenic cells. The effective dose-response curve given in daily dose fractions of 2 Gy has no shoulder and a D0 of 3 Gy. What total dose is required to give a 90% chance of tumor cure?
• Problem 2• Suppose that, in the previous example, the clonogenic cells underwent three cell doublings during treatment. About what total dose would be required to achieve the same probability of tumor control?
• Problem 3• During the course of radiotherapy, a tumor containing 109 cells receives 40 Gy. If the D0 is 2.2 Gy, how many tumor cells will be left?
• Problem 4• If 107 cells were irradiated according to single-hit kinetics so that the average number or hits per cell is one, how many cells would survive?
The Radiosensitivity of Mammalian Cells Compared with Microorganisms• It is evident that mammalian cells are exquisitely radiosensitive compared with microorganisms.• The most resistant is Micrococcus radiodurans, which shows no significant cell killing even after a dose of 1,000 Gy. A,Mammalian cells;B,E.coli;C,E.coli B/r;D,yeast;E,phage staph E;F. Bacillus megatherium;G,potato virus;H,M.radiodurans.
The Radiosensitivity of Mammalian Cells Compared with Microorganisms• 1. The dominant factor that accounts for this huge range of radiosensitivities is the DNA content. Mammalian cells are sensitive because they have a large DNA content, which represent a large target for radiation damage.
The Radiosensitivity of Mammalian Cells Compared with Microorganisms• 2. DNA content is not the whole story, however. E. coli and E. coli B/r have the same DNA content but differ in radiosensitivity because B/r has a mutant and more efficient DNA repair system. In higher organisms, mode of cell death -that is, apoptotic versus mitotic-also affects radiosensitivity.
The Radiosensitivity of Mammalian Cells Compared with Microorganisms• 3. The figure explains why, if radiation is used as a method of sterilization, doses of the order of 20,000 Gy are necessary. Even is objects are socially clean, such huge doses are necessary to reduce the population of contaminating microorganisms because of their extreme radioresistance.