cell survival curve
DESCRIPTION
It describes relationship between radiation dose and the fraction of cells that “survive” that dose. This is mainly used to assess biological effectiveness of radiation. To understand it better, we need to know about a few basic things e.g. Cell Death Estimation of Survival / Plating Efficiency Nature of Cell killing etc. A cell survival curve is the relationship between the fraction of cells retaining their reproductive integrity and absorbed dose. Conventionally, surviving fraction on a logarithmic scale is plotted on the Y-axis, the dose is on the X-axis . The shape of the survival curve is important. The cell-survival curve for densely ionizing radiations (α-particles and low-energy neutrons) is a straight line on a log-linear plot, that is survival is an exponential function of dose. The cell-survival curve for sparsely ionizing radiations (X-rays, gamma-rays has an initial slope, followed by a shoulder after which it tends to straighten again at higher doses.TRANSCRIPT
Cell Survival Curve
Dr. Vandana, King George’s Medical University, Lucknow
Cell Survival Curve It describes relationship between radiation
dose and the fraction of cells that “survive” that dose.
This is mainly used to assess biological effectiveness of radiation.
To understand it better, we need to know about a few basic things e.g. Cell Death Estimation of Survival / Plating Efficiency Nature of Cell killing etc.
3
Cell Death
Cell death can have different meanings: loss of a specific function - differentiated cells
(nerve, muscle, secretory cells)
loss of the ability to divide - proliferating cells such as stem cells in hematopoietic system or intestinal epithelium loss of reproductive integrity - “reproductive death”
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Relevant Dose 100 Gy
destroys cell function in non-proliferating systems (for example: nerve, muscle cells)
2 Gy mean lethal dose for loss of proliferative capacity
for proliferating cells
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Survival
Conversely - “Survival” means retention of reproductive integrity the capacity for sustained proliferation in cells
that proliferate
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Proof of reproductive integrity - the capability of a single cell to grow into a large colony, visible to the naked eye
A surviving cell that has retained its reproductive integrity and is able to proliferate indefinitely is said to be clonogenic
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Estimating Survival
In order to determine the surviving fraction, we must know the plating efficiency
PE is the percentage of cells (in control batch) that grow into colonies in other words, those cells that survive the
plating process
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Derivation of Survival Curves
Cells have been taken from stock culture and placed in seed dishes
Then irradiated (0 Gy to 6 Gy)and allowed to grow into colonies for 1-2 weeks
Colonies have been counted for survival data
Always will have a controlbatch to determine PE.
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Surviving Fraction
Equal to the fraction of cells that plate successfully and survive irradiation (without losing their reproductive integrity) to grow into colonies
PE/100seededcellscountedColonies
fractionSurviving
10
Dose (Gy)
SurvivingFraction
2 64
0.01
0.1
1
0.001
SF(2) = = 0.272 colonies
400 seeded x 0.9 plated
As Ionizations produced within cells by irradiation are distributed randomly.
So consequently, cell death follows random probability statistics, the probability of survival decreasing geometrically with dose.
Quantization of cell killing A dose of radiation that
introduces an average of one lethal event per cell leaves 37% still viable is called D0 dose.
Cell killing follows exponential relationship. A dose which reduces cell survival to 50% will, if repeated, reduce survival to 25%, and similarly to 12.5% from a third exposure.
This means Surviving fraction never becomes zero.
A straight line results when cell survival (from a series of equal dose fractions) is plotted on a logarithmic scale as a function of dose on linear scale.
The slope of such a semi-logarithmic dose curve could be described by the D0, the dose to reduce survival to 37%, D50, the dose to reduce survival to 50%, the D10, the dose to reduce survival to 10%.
D0 usually lies between 1 and 2 Gy
D10= 2.3 x D0
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Survival Curve Features Simple to describe qualitatively
Difficulty lies in explaining underlying biophysical events
Many models have been proposed
Steepness of curve represent the radio-sensitiveness.
Survival Curve Shape general shapes of survival
curves for mammalian cells exposed to radiation
Initial portion has a shoulder and terminal portion become straight line.
In low dose region ,some dose of radiation goes waste.
Terminal portion follow exponential relationship means same dose increment result into equal reduction in surviving fraction.
Mammalian Cell Survival Curve Shoulder Region
Shows accumulation of SUB-LETHAL DAMAGE.
The larger the shoulder region, the more dose will initally be needed to kill the same proportion of cells.
Beyond the shoulder region The D0 dose, or the inverse
of the slop of the curve, indicates the relative radiosensitivity. The smaller the D0 dose, the greater the radiosensitivity.
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Two General Survival Models
Linear-quadratic model “dual radiation action” first component - cell killing is proportional to
dose second component - cell killing is
proportional to dose squared Multi-target model
based on probability of hitting the “target” widely used for many years; still has merit
L-Q Model
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Linear Quadratic Model
S = e-(aD + bD2)
where: S represents the fraction of cells surviving D represents dose a and b are constants that characterize the slopes
of the two l portions of the semi-log survival curve biological endpoint is cell death
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Linear Quadratic Model
Linear and quadratic contributions to cell killing are equal when the dose is equal to the ratio of a to b D = a/ b or aD = b D2
a component is representative of damage caused by a single event (hit, double-strand break, “initiation / promotion” etc.)
b component is representative of damage caused by multiple events (hit/hit, 2 strand breaks, initiation then promotion, etc.)
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a and b Determination
100
10-1
10-2
0 123 6 9
/a b
bD2
aD
Dose, Gy
Su
rviv
al
Multi-target Model
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Multi-target Model
Quantified in terms of:
measure of initial slope due to single-event killing, D1
measure of final slope due to multiple-event killing, D0
width of the shoulder, Dq or n
D1 and D0 are
1. reciprocals of the initial and final slopes
2. the doses required to reduce the fraction of surviving cells by 37%
3. the dose required to deliver, on average, one inactivating event per cell
4. D1,reduces survivivig fraction to 0.37
5. D0, from 0.1 to 0.037, or from 0.01 to 0.0037 ,and so on.
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Multi-target Model
Shoulder-width measures: the quasi-threshold dose (Dq)
the dose at which the extrapolated line from the straight portion of the survival curve (final slope) crosses the axis at 100% survival
the extrapolation number (n) This value is obtained by extrapolating
the exponential portion of the curve to the vertical line.
“broad shoulder” results in larger value of n
“narrow shoulder” results in small value of n
n = exp[Dq / D0]
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Multi-Target Model
Dose, Gy
100
10-1
10-4
0 123 6 9
Su
rviv
al
10-3
10-2
Initial slope measure, D1,due to single-event killing
Final slope measure, D0,due to multiple-event killing
Dq
nn or Dq represents the sizeor width of the shoulder
Linear –quadratic model Multi-target model
Neither the L-Q not the M-T model has any established biological basis.
At high doses the LQ model predicts a survival curve that bends continuosly, whereas the M-T model become linear.
At low doses the LQ model describes a curve that bends more than a M-T curve.
Factors affecting cell survival curve
1. LET
2. Fractionation
3. Dose rate effect
4. Intrinsic radiosesitivity
5. Cell age
6. Oxygen presence
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LET
Low-LET radiations: low dose region
shoulder region appears high dose region
survival curve becomes linear and surviving fraction to an exponential function of dose
surviving fraction is a dual exponential
S = e-(aD+bD2)
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High-LET radiations: survival curve is linear surviving fraction is a pure exponential function of
dose
S = e-(aD)
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Survival Curves and LET
Increasing LET: increases the
steepness of the survival curve
results in a more linear curve
shoulder disappears due to increase of killing by single-events
Fractionation
If the dose is delivered as equal fractions with sufficient time ,repair of sub-lethal damage ocurs
Elkind’ s Recovery takes place between radiation exposure , cell act as fresh target.
Elkind & Sutton showed that when two exposure were given few hours apart ,the shoulder reappeared.
Dq
Two D
oses
Single Dose
Dq
Dose (Gy)5 10 15 20 25
104
103
102
101
100
10-1
10-2
D0
n = exp[Dq / D0]
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The Effective Survival Curve: Fractionation
If the dose is delivered as equal fractions with sufficient time between for repair of the sub-lethal (non-killing) damage, the shoulder of the survival curve is repeated many times.
The effective survival curve becomes a composite of all the shoulder repetitions.
Dose required to produce the same reduction in surviving fraction increases.
D0 is 3 Gy
Showing ~28 Gyin 14 fractions.
Dose-rate effect
Dose rate determines biological impact
reduction in dose rate causes reduced cell killing, due to repair of SLD
reduction in dose rate generally reduces survival-curve slope (D0 increases)
inverse dose-rate effect occurs in some cell lines at ‘optimal’ dose rate due to accumulation of cells in G2
Dose-Rate Effect in CHO Cells
Dose rate effect is more dramatic in CHO than in HeLa Cells
Broad shoulder to survivalcurve
Composite survival curves for 40 Human Cell Lines
Low Dose Rate: SurvivalCurves show greater variation,Greater range in repair times
Less variation
Evidence for Dose Rate Effect in vivo: Crypt Cells
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4.5
0.92
0.54 rad/min
Crypt cells: rapid dividingDramatic dose rate effectExposure time is longer thanCell cycle (repopulation)
Intrinsic radiosensitivity
Due to the differences in DNA content
represents bigger target for radiation damage
Sterilizing radiation dose for bacteria is 20,000 Gy
Mammalian cells are significantly more radio-sensitive than microorganisms:
Age response:Cell Cycle
Late S—least sens.
M>G2>G1>early S>late S for sensitivityDifference caused by cell cycle are similar to difference caused by Oxygen effect
Cells are most sensitive to radiation at or close to M
Cells are most resistant to radiation in late S
For prolonged G1 a resistant period is evident early G1 followed be a sensitive period in late G1
Cells are usually sensitive to radiation in G2 (almost as sensitive as in M)
Oxygen modifies the biological effects of ionizing radiation
02 effect does not require that 02 be present during radiation – just added within 5 msec after generation of free radical
OER – oxygen enhancement ratio: ratio of hypoxic: aerated doses needed to achieve the same biological effect
X-Rays/γ-Rays at high doses is 2.5-3.5 OER is ~2.5 at lower doses
OER is absent for high LET radiations like alpha-particles and is intermediate for fast neutron.
OER is lower for types of radiation predisposed to killing cells by single-hit mechanisms
The Oxygen Effect
Cells are more sensitive toRadiation in the presence ofOxygen than in its absence High dose region of survival
curve
Low dose region of survivalcurve
Summary A cell survival curve is the relationship between the
fraction of cells retaining their reproductive integrity and absorbed dose.
Conventionally, surviving fraction on a logarithmic scale is plotted on the Y-axis, the dose is on the X-axis . The shape of the survival curve is important.
The cell-survival curve for densely ionizing radiations (α-particles and low-energy neutrons) is a straight line on a log-linear plot, that is survival is an exponential function of dose.
The cell-survival curve for sparsely ionizing radiations (X-rays, gamma-rays has an initial slope, followed by a shoulder after which it tends to straighten again at higher doses.
At low doses most cell killing results from “α-type” (single-hit, non-repairable) injury, but that as the dose increases, the“β –type” (multi-hit, repairable) injury becomes predominant, increasing as the square of the dose.
Survival data are fitted by many models. Some of them are: multitarget hypothesis, linear-quadratic hypothesis.
The survival curve for a multifraction regimen is also an exponential function of dose.
The D10, the dose resulting in one decade of cell killing, is related to the Do by the expression D10 = 2.3 x Do
Cell survival also depends on the dose, dose rate and the cell type
Summary
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