chapter3 cell survival curve

Chapter 3. Cell Survival Curves

2012.04.10Dahoon Jung

Korea Cancer Center Hospital

Radiobiology for the Radiologist, Hall, 7th ed

100 balls

10 x 10 lat-tice

Simeon Denis Poisson(1781-1840)

Discrete probability distributionGiven number of eventsIn a fixed interval of time and/or spaceKnown average rateIndependently of the time since the last event

k is the number of occurrences of an event — the probability of which is given by the functionλ is a positive real number, equal to the expected number of occurrences during the given interval.

http://en.wikipedia.org/wiki/Real_number

http://en.wikipedia.org/wiki/Expected_value

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 relation-ship 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 de-stroy cell function in nonproliferating systems.

• By contrast, the mean lethal dose for loss of pro-liferative capacity is usually less than 2 Gy.

The In Vitro Survival Curve

• Plating efficiency– PE = x 100

• Surviving fraction– SF =

2,000 cells seeded.Exposed to 8 Gy.Plating efficiency is 70%.1,400 cells grew.32 colonies on the dish.Surviving fraction is

The In Vitro Survival Curve

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 func-

tion 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)


A:The linear quadratic model. B:The multitarget model.A. Good fit to experimental data for

the first few decades of survival.


• For densely ionizing (high-LET) radiations, such as α-particles or low-energy neutrons, the cell sur-vival 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.


• 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.)


• n(extrapolation number)– Large(10 or 12) broad shoulder– Small(1.5 – 2) narrow shoulder

• Dq (quasithresholddose)– Dose at which the straight portion of the sur-

vival curve, extrapolated backward, cuts the dose axis drawn through a survival fraction of unity.


• 2. The linear-quadratic model– Direct development of the relation used to de-

scribe exchange-type chromosome aberrations that are clearly the result of an interaction be-tween 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 components of cell killing that are propor-tional 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 ra-tio of α to β.

• A characteristic of the LQ formulation is that the resultant cell survival curve is continuously bend-ing; 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 op-posed to the cytoplasm.

• The evidence implicating the chromosomes, specifically the DNA, as the primary target for ra-diation-induced lethality may be summarized as follows:


• 1. Cells are killed by radioactive tritiated thymi-dine incorporated into the DNA. The radiation dose results from short-range α-particles and is therefore very localized.


• 2. Certain structural analogues of thymidine, particularly the halogenated pyrimidines, are incorporated selec-tively 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 cellu-lar radiosensitivity.


• 3. Factors that modify cell lethality, such as varia-tion in the type of radiation, oxygen concentra-tion, 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.


• 4. Early work showed a relationship between virus size and radiosensitivity; later work showed a bet-ter correlation with nucleic acid volume. The ra-diosensitivity 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 ra-diosensitivity.


<The Bystander Effect>• Defined as the induction of biologic effects in cells

that are not directly traversed by a charged parti-cle, 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.


• The use of sophisticated single-particle mi-crobeams, 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 pro-tons and soft x-rays.

• The effect is most pronounced when the by-stander cells are in gap-junction communication with the irradiated cells.

• For example, up to 30% of bystander cells can be killed in this situation.


• 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 it-self.

• Its importance is primarily at low doses, where not all cells are “hit”.


• In addition to the experiments described previ-ously involving sophisticated single-particle mi-crobeams, there is a body of data involving the transfer of medium from irradiated cells that re-sults 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.


<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 radi-ation.


• 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 condensa-

tion(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.


• 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.


• The most common form of cell death from radiation is mitotic death.– Cells die attempting to di-

vide because of damaged chromosomes.

– The log of the surviving fraction

– The average number of putative “lethal” aberra-tions per cell(asymmetric exchange-type aberra-tions such as rings and dicentrics)

– One-to-one correlation.

The experiment was carried out in a cell line where apoptosis is not ob-served.


• 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.


• 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.


<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.


<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 inap-propriate growth signals or division of cells that have accu-mulated DNA damage.

Survival Curves for Various Mammalian Cells in Culture

• The first in vitro survival curve for mammlian cells irradi-ated 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 shoul-der


• 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 can-cer-prone syndromes such as ataxia-telangiecta-sia(AT); these cells are much more sensitive to ionizing radiations, with a D0 for x-rays of about 0.5 Gy.


• Some researchers were skeptical that these in vitro techniques, which in-volved growing cells in petri dishes in very artificial conditions, would ever benefit clinical radiotherapy.– A metaphor of “Robinson Crusoe”

• The in vitro culture technique mea-sured the reproductive integrity of cells and that there was no reason to suppose that Robinson Crusoe’s re-productive integrity was any different on his desert island from what it would have been had he remained in York.


• In more recent years, extensive studies have been made of the radiosensitivity of cells of hu-man origin, both normal and malignant, grown and irradiated in culture.– In general, cells from a given normal tissue show a nar-

row range of radiosensitivities if many hundreds of peo-ple are studied.

– By contrast, cells from human tumors show a very broad range of D0 values.

Survival Curve Shape and Mechanisms of Cell Death

• Mammalian cells cultured in vitro vary considerably in their sensitivity to killing by radiation.


Radioresis-tantLarge dose-rate effect

RadiosensitiveNo dose-rate ef-fect Laddering

(after 10 Gy)


• Although asynchronous cells show this wide range of sensitivities to radiation, mitotic cells from all of these cell lines have essentially the same radiosensi-tivity.

– In interphase, the radiosensitivity dif-fers because of different conformations of the DNA.


• Characteristic laddering is indicative of pro-grammed cell death or apoptosis during which the DNA breaks up into discrete lengths as previ-ously described.

• Comparing Fig.A and B, it is evident that there is a close and impressive correlation between ra-diosensitivity and the importance of apoptosis.

• Increased “laddering” = Increased radiosensitiv-ity


• 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 Radioresis-tance

• 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 radioresis-tance, and it is far less clear that oncogenes play any major role in radioresistance in human tu-mors.

Genetic Control of Radiosensi-tivity

• A radiosensitive mutant can result from a muta-tion 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 Radiosensi-tivity

<Inherited Human Syndromes as-sociated with sensitivity to X-rays>• Ataxia-telangiectasia(AT)• Seckel syndrome• Ataxia-telangiectasia-like disor-

der• Nijmegen breakage syndrome• Fanconi’s anemia• Homologues of RecQ-Bloom

syndrome, Wernder syndrome, and Rothmund-Thompson syn-drome

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 radi-

ation 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

• The effective survival curve is an exponential function of dose whether the single-dose survival curve has a constant terminal slope or is continuously bending.

• The D0 of the effective survival curve: the dose required to reduce the fraction of cells surviving to 37%(close to 3 Gy for cells of hu-man origin).

• D10(dose required to kill 90% of the population)– D10 = 2.3 D0

– (2.3 = )

Calculations of Tumor Cell Kill

• Problem 1

• A tumor consists of 108 clonogenic cells. The ef-fective 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?

• Answer

• To give a 90% probability of tumor control in a tumor containing 108 cells requires a cellular de-population of 10-9. The dose resulting in one decade of cell killing(D10) is given by

• D10 = 2.3 D0 = 2.3 3 = 6.9 Gy

• The total dose for 9 decades of cell killing, there-fore, is 9 6.9 = 62.1 Gy.

• 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?

• Answer

• Three cell doublings would increase the cell num-ber by

• 2 2 2 = 8• Consequently, about one extra decade of cell

killing would be required, corresponding to an ad-ditional dose of 6.9 Gy. The total dose is 62.1 + 6.9 = 69 Gy.

• Problem 3

• During the course of radiotherapy, a tumor con-taining 109 cells receives 40 Gy. If the D0 is 2.2 Gy, how many tumor cells will be left?

• Answer

• If the D0 is 2.2 Gy, the D10 is given by

• D10 = 2.3 D0

= 2.3 2.2 = 5 Gy Because the total dose is 40 Gy, the number of decades of cell killing is 40/5 = 8. The number of cells remaining is 109 10-8 = 10.

• 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?

• Answer

• A dose that gives an average of one hit per cell is the D0, that is, the dose that on the exponential region of the survival curve reduces the number of survivors to 37%. The number of surviving cells is therefore

The Radiosensitivity of Mammalian Cells Compared with Microorganisms

• It is evident that mam-malian cells are exquisitely radiosensitive compared with micro-organisms.

• The most resistant is Mi-crococcus 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 Mam-malian Cells Compared with

Microorganisms• 1. The dominant factor that accounts for this

huge range of radiosensitivities is the DNA con-tent. Mammalian cells are sensitive because they have a large DNA content, which represent a large target for radiation damage.


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 radiosensitiv-ity.


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 so-cially clean, such huge doses are necessary to re-duce the population of contaminating microorgan-isms because of their extreme radioresistance.

• Thank you for listening.

chapter3 cell survival curve

Technology

cell survival curves

resultantcell survival

cell survival curveis

culture survival curve

survival curvea

survival levels

components of cell

curve bends