2. DEFINITION
• Cell survival curve describes the relationship between the radiation dose
and the proportion of cells that survive.
• This graph is obtained by plotting the surviving fraction, SF, along the
logarithmic y-axis and the dose along the linear x-axis.
• 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
3. Cell Death
• Cell death can have different meanings:
• Differentiated cells (nerve, muscle, secretory cells): loss of
a specific function.
• Lethal dose: 100 Gy
• Proliferating cells such as stem cells in hematopoietic
system or intestinal epithelium: loss of the ability to divide
- loss of reproductive integrity - “reproductive death”.
• Lethal dose: upto 2Gy
4. Survival
4
Conversely - “Survival” means retention of reproductive
integrity.
the capacity for sustained proliferation in cells that proliferate.
Clonogenic : Survivor able to proliferate indefinitely to produce
a large clone or colony.
Proof of reproductive integrity - the capability of a single cell to
grow into a large colony, visible to the naked eye.
• Cell Survival can be estimated by knowing
1. Plating efficiency
2. Survival fraction
5. Estimating Survival
5
• In order to determine the surviving fraction, we must
know the PLATING EFFICIENCY.
• PE is the percentage of cells that grow into colonies
• in other words, those cells that survive the plating process
• may be close to 100% in some established cell lines but 1% or
less for fresh explants of human cell
6. Derivation of Survival Curves
6
• Cells are 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 are counted
for survival data.
Always will have a control
batch to determine PE.
7. Surviving Fraction
• Equal to the fraction of cells that plate successfully and
survive irradiation (without losing their reproductive
integrity) to grow into colonies
• A cell survival curve is graph plotted between surviving
fraction on Y axis & absorbed dose on X axis
PE/100
seeded
cells
counted
Colonies
fraction
Surviving
7
9. 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 IRRADIATION
IRRADIATED
10. Modes of Radiation Injury
• Primarily by ionization (direct) and free radicals(indirect)
mechanism
• Low LET (X- and gamma-rays) damage by free radicals
• High LET (protons and a particles) damage by ionization
11. • The Poisson model was proposed in 1961 (Munro &
Gilbert)
• It states “the object of treating a tumour by radiotherapy is
to damage every single potentially malignant cell to such
an extent that it cannot continue to proliferate.”
• When enough radiation is delivered to the tumour mass
such that 1 lethal hit would be expected per cell,… the
likely percentage of cells receiving one lethal hits is 63%.
Therefore, 37% of cells would receive no lethal hits and
therefore the tumour would most likely not be cured.
• The probability of zero surviving tumor cell is given by
the tumour control probability (TCP)
Poisson Model of probability of cell death
12. • As no of lethal hits increases the probability of survival decreases
geometrically with dose.
• For example, if 100 clonagens were present within the tumour, an average of 37
clonagens would survive. Therefore the TCP would be e-37
• As the lethal hits per clonagen increases, the TCP also increases.
• The increase occurs rapidly once the lethal hits per clonagen exceeds 3 – 4 hits.
(likelihood of surviving clonagens begins to approach zero at this point).
13. 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 curve.
In exponential curve, the number of cells doubles with
each generation, and so exponential growth is faster than
arithmetic growth
14. 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
Reminder: All survival curves are plotted on semi-
log plots.
15. 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.
16. Log-linear scale: straight line in invitro
cell culture
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%,
D0 usually lies between 1 and 2 Gy
D50, the dose to reduce survival to
50%,
D10, the dose to reduce survival to
10%. D10= 2.3 x D0
17. Mammalian cell Survival Curve Shape
• Two Component: shoulder and
exponential curve
• Initial portion has a shoulder and
terminal portion become straight
line.
• Shows accumulation of SUB-
LETHAL DAMAGE.
• The larger the shoulder
region, the more dose will
initially be needed to kill the
same proportion of cells, so
less radiosensitive
Shoulder
Exponential
curve
18. Mammalian Cell Survival Curve
• Beyond shoulder region
• Terminal portion follow
exponential relationship means
same dose increment result
into equal reduction in
surviving fraction.
• The D0 dose, or the inverse of
the slop of the curve, indicates
the relative radio sensitivity.
The smaller the D0 dose, the
greater the radio sensitivity.
19. Two General Survival Models
19
• Linear-quadratic Model
• “Dual radiation action”
• First component - cell killing is proportional to dose
• Second component - cell killing is proportional to dose
squared
• Target Model:
• Single-target Model
• Multi-target Model
• based on probability of hitting the “target”
• widely used for many years; still has merit
20. 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.
21. Single Target Single Hit Inactivation Model
• single hit on a single target within the
cell leads to cell death.
• This generates an exponential cell
survival curve which appears as a
straight line on a semi-logarithmic
scale.
• This model is useful for highly
sensitive human tissues, if high LET
radiation is used.
• Mammalian cells usually have a
shoulder on their cell survival curves,
which is not seen in this model.
If, at dose D0 there is an average of one
lethal event/cell, then
S = e-D/D0 where D0 is called the mean
lethal dose
Simple Target Theory: ln S = -D/D0
22. • Each cell contain more than one target (may be assumed n number
of target and n may be any number more than one)
• In order to bring cell death by radiation, all the target should be deactivated.
• If n-1 targets are hit then cell survives.
• There are two type of cell killing taking place
Low Dose Region
Simultaneously to inactivate n target resulting into cell death.
• Cell kill by single hit event (SHE)
• Cell kill by multiple hit event (MHE)- High Dose Region
Multi Target Model
23. SHOULDERED SURVIVAL CURVE WITH NON ZERO 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.
24. SHOULDERED SURVIVAL CURVE WITH NON ZERO INITIAL SLOPE
• 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.
27. Multi-target Model
27
• 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
Dose, Gy
100
10-1
10-4
0 12
3 6 9
Survival
10-3
10-2
Initial slope measure, D1,
due to single-event killing
Final slope measure, D0,
due to multiple-event killing
Dq
n
n or Dq represents the size
or width of the shoulder
28. 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 surviving fraction to
0.37
5. D0, from 0.1 to 0.037, or from
0.01 to 0.0037 ,and so on.
29. D0 = dose that decreases the surviving fraction to 37%.
This is the dose required to induce an average damage
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.
30. Multi-target Model
30
• 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]
31. N=extrapolation number =represent number of
target in a cellwhichare required to be hit to bring
celldeath
Dq
Dq – Quasithreshold dose->which is
defined as dose of radiation which do
not produceanycell killing.
Butaswe know that nodoseof
radiation issafeand it alwaysproduces
somedamagessothe name isQuasi
SF
10
8
6
3
0
QuasithresholdDose
Extrapolation Number
Dose
Parametersusedto describe thesecurves
32. Single-target, Single Hit/Multi-target,
Single Hit Model
• Major problem with this model is that
there are too many parameters D1;D0;Dq
• Need a mathematically simpler model
with fewer “unknown” parameters
• The linear-quadratic (L-Q) model
meets these needs
33. Linear-quadratic model
•The linear quadratic model uses a polynomial
equation (αD+βD2).
•The probability of survival is equal to the exponential of this
–
ie:S = e−(αD+βD)2.
•The generated curve is perhaps the best approximation of
the actual cell kill seen after radiation exposure.
•It has the added benefit of two constants (α and β) which
can be determined for specific tissues and cancers to predict
dose response
37. Linear Quadratic Model
37
• S = e-(aD + bD
2
)
• where:
• S represents the fraction of cells surviving
• D represents dose
• a and b are constants that characterize the slopes of the
two portions of the semi-log survival curve
• biological endpoint is cell death
38. Linear Quadratic Model
38
• 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.)
100
10-1
10-2
0 12
3 6 9
a/b
bD2
aD
Dose, Gy
Survival
39. Killingby
SHE=Killingbytwo
hit event
Linear=Quadratic
aD =bD2
a/b D2
/D
a/b D
Soa/b canbe defined asthe
doseat which contribution by
single hit killbecomesequal to
double hit kill.
Dose
LinearHit (a cellkill)
S
F
It representthe
dosebeyondwhich
the doublehit kill
becomesmainmode
of cellkill andbefore
that the cellkill is
mainlybysinglehit.
a Kill
b Kill
Quadratic Hit(b cellkill)
40. Clinical significance of a/b
Early responding tissues:
Are rapidly proliferating tissues.
Early reactions are reduced by:
Lengthening OTT.
Keeping intervals between fractions to more than 5-6 hours to allow
for normal repair.
41. Clinical significance of a/b
Late responding tissues:
Are slowly proliferating tissues.
Changes in OTT does not have much effect.
Late reactions are reduced by:
• Reducing dose per fraction
42. Clinical significance of a/b
Tumor tissues:
Behave like early responding tissues.
Maybe spared if dose is too low.
Maybe spared if OTT is too long.
When changing one dose schedule to other, one has to be careful.
When we change fraction size (dose/fraction) we need to take into account the
a/b ratio of normal tissues for calculating the equivalent dose.
Usually a/b ratio of late responding tissues is used for calculating the equivalent
doses.
If calculated by a/b value of Early reacting tissues, then it leads to more damage
to late reacting tissues.
43.
44.
45. Biological Effective Dose(BED)
• If dose per fraction remain same then RE will depend inversely to a/b value.
• (BED) =
• Which means, that same dose per fraction will affect late reacting tissue more
than
• early reacting tissue as average a/b value for late reacting tissue is 3 as compare
to
• 10 for early reacting tissue.
• So increasing the dose per fraction will have more effect on late reacting tissues
• Similarly the BED for a fixed fractionation schedule will be more for late reacting
• tissue as compare to early reacting tissue.
46.
47. a) 70 Gy in 35 fractions, a/b- 3
b) 45 Gy in 25 fractions ,a/b- 10
c) 30 Gy in 10 fractions ,a/b-3
d) 10 Gy in 5 fractions ,a/b-10
49. Comparison of the L-Q and target
theory models
• Neither the L-Q not the M-T model has any established
biological basis.
• L-Q curves retain curvature even at very high doses
• Target Theory curves become linear at high doses
• With high-LET radiations, both theories result in straight lines
( irreparable damage dominates)
50. Linear –quadratic model Multi-target model
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.
51. Factors affecting cell survival curve
1. Mechanism of cell death
2. LET
3. Fractionation
4. Dose rate effect
5. Intrinsic radio sesitivity
6. Cell cycle stage
7. Oxygen presence
52. 1-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:
53.
54. 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.
• Using single particle microbeams, a bystander effect has been demonstrated for chromosomal
aberrations, cell killing, mutation, oncogenic transformation, and alteration of gene expression.
• The effect is most pronounced when the bystander cells are in gap-junction communication
with the irradiated cells.
• The effect being due, presumably, to cytotoxic molecules released into the medium.
55. • 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”.
• Irradiated cells secrete a molecule into the medium that is capable of killing
cells when that medium is transferred onto unirradiated cells.
56. Mitotic death results (principally) from exchange-type
chromosomal aberration.
survival has both linear quadratic function of dose
Survival curve is log-linear plot with broad shoulder
Characterized by dose-rate effect
Apoptotic death result from programmed death.
straight line on log-linear plot.
Characterized by exponential function of dose.
Survival curve is straight & shoulderless
little or no dose-rate effect.
SURVIVAL-CURVE SHAPE AND
MECHANISM OF CELL DEATH
57. <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.
• 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.
58. Mechanisms of Cell Killing
1. To cease communicating with its neighbors
2. Rounds up and detaches from its neighbors.
3. Condensation of the chromatin at the nuclear membrane and
fragmentation of the nucleus.
4. The cell shrinks because of cytoplasmic condensation(crosslinking of
proteins and loss of water).
5. The cell separates into several membrane-bound fragments of differing
sizes : apoptotic bodies
6. 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.
7. Necrosis causes a diffuse “smear” of DNA in gels.
59. 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)
• One-to-one correlation.
The experiment was carried out in a cell line where
apoptosis is not observed.
60. • 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.
61. 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.
62. 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.
63. 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.
64. 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.
66. Survival Curve Shape and Mechanisms of Cell Death
Radioresistant
Large dose-rate
effect
Radiosensitive
No dose-rate effect
Laddering
(after 10 Gy)
67. 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.
68. 2-LINEAR ENERGY TRANSFER
68
• Rate at which energy is transferred to cell during irradiation
• Low-LET radiations:
• At low dose region
• shoulder region appears
• At high dose region
• survival curve becomes linear and surviving fraction is an exponential function
of dose
• surviving fraction is a dual exponential
S = e-(aD+bD2)
69. 69
• High-LET radiations:
• survival curve is linear
• surviving fraction is a pure exponential function of dose
S = e-(aD)
70. Survival Curves and LET
70
• 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
71. 3- Fractionation
• If the dose is delivered in equal
fractions with sufficient time
interval, repair of sub-lethal
damage occurs
• Recovery takes place between
radiation exposure , remaining
cell act as fresh target.
• Elkind & Sutton showed that
when two exposure were given
few hours apart ,the shoulder
reappeared.
Dq
Dq
Dose (Gy)
5 10 15 20 25
104
103
102
101
100
10-1
10-2
D0
n = exp[Dq / D0]
72. The Effective Survival Curve:
Fractionation
72
• If the dose is delivered as
equal fractions with
sufficient time interval for
repair of 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.
Showing ~28 Gy
in 14 fractions.
73. 4-Dose-rate effect
Dose rate determines biological impact
At low dose rates, DNA repair processes are able to repair sub lethal
damage during the radiation treatment.
At very low dose rates other radiobiological effects (re-oxygenation,
redistribution and repopulation) may also begin to play a role. This
lessens the effect of a particular dose of radiation.
Low dose rates tend to negate the β component of the linear quadratic
equation, and the line becomes straighter on a logarithmic scale.
Reduction in dose rate generally reduces survival-curve slope
(D0 increases).
75. Composite survival curves for 40 Human Cell Lines
Survival Curves show
greater variation,
Greater range of repair
Less variation
in cell survival
curve due to
less repair
76. 5-Intrinsic radiosensitivity
Due to the differences in
DNA content
Represents bigger target
for radiation damage
Sterilizing radiation dose
for bacteria is 20,000 Gy
whereas for mammalian
cell is 1-2 Gy
Mammalian cells are significantly more radio-sensitive than
microorganisms:
77. GENETIC CONTROL OF RADIOSENSITIVITY
Inherited Human Syndromes associated with sensitivity to X-
rays
• Ataxia-telangiectasia(AT)
• Basal cell nevoid syndrome
• Cockayne syndrome
• Down syndrome
• Fanconi’s anaemia
• Usher syndrome
• Nijmegen breakage syndrome
78. 6-Survival Curve: Effect Of Cell Cycle
Stage
Late S:
least senstive.
M>G2>G1>early
S>late S for sensitivity
Difference caused by
cell cycle are similar to
difference caused by
Oxygen effect
The range of
senstivity between the
most senstive (M) &
most resistant (S) phase
is of the same order as
oxygen effect
The broken line is cell survival curve for
mitotic cell plotted under hypoxia
Slope is 2.5 times shallower than
aerated cell
79. Oxygen modifies biological effects of ionizing radiation
OER – oxygen enhancement ratio: ratio of hypoxic doses : aerated
doses needed to achieve the same biological effect
OER is absent for high LET radiations like alpha-particles and is
intermediate for fast neutron.
Low LET radiation (eg. photons, electrons) are highly dependent
on the presence of oxygen to ‘fix’ damage caused by free radicles.
The oxygen effect shows more cell killing in oxic conditions. This is
seen in cell survival curves as a shift in the steepness of the curve.
For low LET X-Rays/γ-Rays at high doses OER is 2.5-3.5
at lower doses OER is ~2.5
7-The Oxygen Effect
80. OER – oxygen enhancement ratio: ratio of hypoxic doses : aerated doses
needed to achieve the same biological effect
84. Cell survival curve depends
Factors that make cells less radiosensitive are:
Cell survival curve is used to calculate
1. on nature of radiation (LET);
2. Type of cell death
3. dose;
4. dose rate
5. cell type;
6. cell cycle stage
7. oxygen presence
1. removal of oxygen to create a hypoxic state,
2. the addition of chemical radical scavengers,
3. the use of low dose rates or multif ractionated irradiation,
4. cells synchronized in the late S phase of the cell cycle.
1. no.of tumor cell killed/ survived
2. tumor control probability
3. calculation of time dose and fractions
4. calculating Biologically effective dose