CELL SURVIVAL CURVE

PRESENTER :DR.VIJAY.P.RATURIMODERATOR :- MR.TEERTHRAJ

SIR

J.R 2 ,KGMU lucknow

DEFINITION

• Cell survival curve describes the relation-ship between the radiation dose and the proportion of cells that survive.– Cell “Death” : loss of reproductive in-

tegrity

• Clonogenic : Survivor able to proliferate indefinitely to produce a large clone or colony.

• 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 prolifer-ative capacity is usually less than 2 Gy.

The In Vitro Survival Curve

• Plating efficiency– PE = x 100

• Surviving fraction– SF =

• 100 cells are seeded into an unirradiated culture, and 10 colonies are formed, then the PE is 10/100.

• If there are 5 colonies after a 450 cGy dose of radiation, the SF is 5/[100 × 10/100] = 1/2. Thus, the SF of 450 cGy is 50%.

NO IRRIDATION

IRRADIATED

The In Vitro Survival Curve

The number of cells in cell lines within cell cultures can

increase in one of two way: Arithmetically or exponentially (geometrically).

The number of cells increases linearly (by a constant number) with each generation in an arithmetic.

In exponential , the number of cells doubles with each generation, and so exponential growth is faster than arithmetic growth

If the SF is calculated for various doses, then it can be presented as a cell–dose plot. Combining the points on the plot leads to a cell survival curve.

SIGMOID CURVE SEMILOGARITHMIC CURVE

EXPONENTIAL SURVIVAL CURVE

Survival curves resulting from the single target–single hit hypothesis of target theory .They show that cell death dueto irradiation occurs randomly.

At certain doses with one unit increase, both same number of cell deaths and same proportion of cell death occur.

D0 = dose that decreases the surviving fraction to 37%.

This is the dose required to induce an average dam-age

per cell.

A D0 dose always kills 63% of the cells in the region in

which it is applied, while 37% of the cells will survive.

1/D0 = the slope of the survival curve.

As the value of D0 decreases → 1/D0 increases → slope → radiosensitive cell.

As the value of D0 increases → 1/D0 decreases → slope → radioresistant cell.

SHOULDERED SURVIVAL CURVES WITH ZERO INITIAL SLOPE

These survival curves are based on the multiple target–single hit hypothesis of target theory

They are produced by the hypothesis of requiring multiple targets per cell, and only one of these targets needs to be hit to kill the cell.

Dq: half-threshold dose → the region of the survival curve where the shoulder Starts (indicates where the cells start to die exponentially) (= quasi-threshold dose).

n: extrapolation number (the number of D0 doses that

must be given before all of the cells have been killed).

Dq → the width of the shoulder region.

Dq = Do log n 2.7

If n increases → Dq increases → a wide shouldered curve is observed.

If n decreases → Dq decreases → a narrow shouldered

curve is observed.

If Dq is wide and D0 is narrow, the cell is radiores-istant.

The D0 and Dq values for the tumor should be smal-ler

than those of normal tissue to achieve clinical suc-cess.

SHOULDERED SURVIVAL CURVE WITH NON ZERO INITIAL SLOPE

COMPONENTS OF SHOULDERED SUR-VIVAL CURVES WITH NONZERO INITIAL SLOPE • Component corresponding to the single target–single hit model (blue in the figure)- This shows lethal damage.- This shows the cells killed by the direct effect of the radiation.- This shows the effect of high-LET radiation.

• Component corresponding to the multiple target–single hit model (red in the figure)- This shows the accumulation of SLD.- This shows the cells killed by the indirect effect of

the radiation.- This shows the effect of low-LET radiation.

SHAPE OF THE SURVIVAL CURVE

• At “low doses” for sparsely ionizing(low 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.

• 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.

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.

MECHANISMS OF CELL KILLING

• 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 radiation-induced lethality may be summarized as follows:

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.

Certain structural analogues of thymidine, particu-larly the halogenated pyrimidines, are incorpo-rated selectively into DNA in place of thymidine if substituted in cell culture growth medium. This substitution dramatically increases the ra-diosensitivity of the mammalian cells.

Factors that modify cell lethality, such as variation 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.

The radiosensitivity of a wide range of plants has been correlated with the mean interphase chromo-some volume, which is defined as the ratio of nu-clear volume to chromosome number. The larger the mean chromosome volume, the greater the radiosensitivity

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 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.

• 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”.

APOPTOTIC 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.

• Double-strand breaks(DSBs) occur in the linker re-gions between nucleosomes, producing DNA frag-ments that are multiples of approximately 185 base pairs. Laddering in gels.

• Apoptosis is highly cell-type dependent.

• Hemopoietic and lymphoid cells are particu-larly 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 suppres-sor or apoptosis.

MITOTIC DEATH

The most common form of cell death from radiation is mi-totic death.

– Cells die attempting to divide because of damaged chromo-somes.

– The log of the surviving fraction

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

– Data such as these provide strong circumstantial evidence to support the notion that asymmetric exchange-type aber-rations represent the principle mechanism for radi-ation-induced mitotic death in mammalian cells.

RELATION BETWEEN CHROMOSOMAL ABERRA-TION & SURVIVAL CURVE

SURVIVAL CURVES FOR VARIOUS MAMMALIAN CELLS IN CULTURE

• 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 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 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.

• In more recent years, extensive studies have been made of the radiosensitivity of cells of human ori-gin, both normal and malignant, grown and irradi-ated in culture.

– In general, cells from a given normal tissue show a narrow range of radiosensitivity if many hun-dreds of people are studied.

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

SURVIVAL CURVES FOR VARIOUS MAMMALIAN CELLS IN CULTURE

SURVIVAL CURVE SHAPE AND MECHANISMS OF CELL DEATH

Radioresis-tantLarge dose-rate effect

RadiosensitiveNo dose-rate ef-fect Laddering

(after 10 Gy)

• 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 radiosensitivity

• Mitotic death results (principally) from ex-change-type chromosomal aberrations; the associated cell survival curve, therefore, is curved in a log-linear plot, with a broad initial shoulder.

GENETIC CONTROL OF RA-DIOSENSITIVITY

Inherited Human Syndromes associated with sen-sitivity to X-rays

• Ataxia-telangiectasia(AT)• Basal cell nevoid syndrome• Cockayne syndrome• Down syndrome• Fanconi’s anaemia• Usher syndrome• Nijmegen breakage syndrome

EFFECTIVE SURVIVAL CURVE FOR A MULTIFRACTION REGIMEN

• The effective survival curve is an exponential function of dose whether the single-dose sur-vival 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 human origin).

• D10(dose required to kill 90% of the population)

– D10 = 2.3 D0

EFFECTIVE SURVIVAL CURVE FOR A MULTIFRAC-TION REGIMEN

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 ra-diodurans, 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, B. megatherium; G, potato virus; H, Micrococcus radi-odurans.

THE RADIOSENSITIVITY OF MAMMALIAN CELLS COMPARED WITH MICROORGANISMS

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.

E. coli and E. coli B/r have the same DNA con-tent but differ in radiosensitivity because B/r has a mutant and more efficient DNA repair sys-tem.

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