2. 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.
3. Cell Death
Cell death can have different meanings:
loss of a specific function - differentiated cells
muscle, secretory cells)
(nerve,
loss of the ability to divide - proliferating cells such as stem
cells in hematopoietic system or intestinal epithelium
loss of reproductive integrity - “reproductive death”
3
4. Proof
single
naked
of reproductive integrity - the capability of a
cell to grow into a large colony, visible to the
eye
A surviving cell that has retained its reproductive
integrity and is able to proliferate indefinitely is said to
be clonogenic
6
5. Estimating Survival
In order to determine the surviving fraction, we
must know the plating efficiency
PE is the percentage of cells
grow into colonies
P.E.= no of cells counted
no. of cells seeded
(in control batch) that
in other words,
Process
those cells that survive the plating
7
6. Relevant Dose
100 Gy
destroys cell function in non-proliferating
example: nerve, muscle cells)
Gy
systems (for
2
mean lethal dose for loss of proliferative capacity for
proliferating cells
4
7. Derivation of Survival Curves
Always will have a control
batch to determine PE.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
8
8. Surviving Fraction
Equal to the fraction of cells that plate
successfully and survive irradiation (without
losing their reproductive integrity) to grow
into colonies
Colonies counted
Surviving fraction
cells seeded PE/100
9
10. Quantization of cell killing
A dose of radiation that
introduces an average of one
lethal event per cell leaves
37%
dose.
still viable is called D0
Cell killing follows exponential
relationship. A dose which
50%reduces cell survival to
will, if repeated,
to 25%, and
reduce
similarlysurvival
to 12.5% from a third
exposure.
This means Surviving
never becomes zero.
fraction
11. A straight line results when cell
equal dose
logarithmic
survival (from a series of
fractions) is plotted on a
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
12. Survival Curve Features
Simple to describe qualitatively
Difficulty lies in explaining underlying
events
biophysical
Many models have been proposed
Steepness of curve represent the radio-
sensitiveness.
14
13. Survival Curve Shape
•Dose plotted on a linear scale
and surviving fraction on a
logarithmic scale.
• At High LETs, such as α-particles
or low-energy neutrons, the
curve is a straight line.
• For sparsely ionizing (low LET)
radiations, such as x-rays starts
out straight with a finite initial
slope ,so surviving fraction is an
exponential funtion of dose.
•At higher doses,curve bends.
•and at very high doses it tends
to straighten again thus surviving
fraction returns to being an
exponential function
14.
15. Mammalian Cell Survival Curve
Shoulder Region
Shows accumulation of SUB-
LETHAL DAMAGE.
The larger the
more
shoulder
dose willregion, the
initally be needed to kill the
same proportion of cells.
Beyond the shoulder region
The D0 dose, or the inverse of
the slope of the
curve,indicates radiosensitivity.
The smaller the D0 dose, the
greater the radiosensitivity.
16. Survival Curve Models
Linear-quadratic model
“dual radiation action”
first component - cell killing is proportional to dose
second component - cell killing is proportional
dose squared
Multi-target model
based on probability of hitting the “target”
widely used for many years; still has merit
to
17
21. Linear Quadratic Model
2
e-( D + D ) S =
where:
S represents the fraction of cells surviving
D represents dose
and are constants that characterize the slopes of the
two l portions of the semi-log survival curve
biological endpoint is cell death
22
22. Linear Quadratic Model
Linear and quadratic contributions to cell
killing are equal when the dose is equal to the
ratio of to
D = / or
D2D =
component is representative of damage caused
break,by a single event (hit, double-strand
“initiation / promotion” etc.)
component is representative of damage caused
by multiple events (hit/hit, 2 strand breaks, initiation
then promotion, etc.)
23
25. Multi-target Model
Quantified in terms of:
measure of
D1
measure of
D0
width of the
initial slope due to single-event killing,
final slope due to multiple-event killing,
shoulder, Dq or n
28
26. D1 and D0 are
1. reciprocals of
final slopes
the initial and
2. the doses required to reduce
the fraction of surviving cells
by 37%
3. the dose required to
onedeliver, on average,
inactivating event per cell
4. D1,reduces survivivig fraction
to 0.37
D0, from 0.1 to 0.037, or from
0.01 to 0.0037 ,and so on.
5.
27. 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
100% survival
the extrapolation number (n)
axis at
This value is obtained by extrapolating
the exponential portion of the
the vertical line.
curve to
“broad shoulder” results in larger value
of n
“narrow shoulder” results in small value
of n
30
28. Multi-Target Model
n or Dq represents the size
Or width of shoulder
Dq
100
10-1
due to single-event
killing10-2
10-3
10-4
0 3 6 9 12
Dose, Gy
31
Survival
n
Initial
slope,D1
three parameters,
n, D0, and Dq, are
related by the
expression
Log en = Dq /D0
Final slope,D0 due to
multiple event killing
29. •For oxygenated mammalian cells, D0 is
about 150 rads (1.5 Gy).
•The D0 of the X-ray survival curves for most
cells cultured in vitro is 1-2 Gy.
Exceptions are cells of cancer prone
syndromes, eg. A.T., with D0 of 0.5 GY.
• Dq :defined as the dose at which the
straight portion of the survival curve,
extrapolated backward, cuts the dose axis
drawn through a survival fraction of unity.
30. Linear –quadratic model Multi-target model
Neither the L-Q not the M-T model
biological basis.
has any established
At high doses the LQ model predicts a survival curve
that bends continuosly,
becomes linear
whereas the M-T model
At low doses the LQ model describes a curve that bends
more than a M-T curve.
31. • Defined as “the induction of biologic effects
in cells that are not directly traversed by
a charged particle, but are in close proximity
to cells that are.”
• ~30% of bystander cells can be killed in this
situation.
• Presumably due to cytotoxic molecules
released into the medium.
BYSTANDER EFFECT
33. LET
Low-LET radiations:
low dose region
shoulder region appears
high dose region
survival curve becomes linear and surviving
fraction
surviving
to an exponential function of dose
fraction is a dual exponential function
e-( D+ D2)S =
34
35. 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
36. Fractionation
If the dose is delivered as
equal fractions with sufficient
time ,repair of sub-lethal
damage ocurs
n = exp[D / D ]q 0
104
103
Elkind‟ s Recovery takes place 102
between radiation exposure ,
cell now acts as a fresh
target.
q
101
100
-1
10
Elkind & Sutton showed that
when two exposure were given
few hours apart ,the shoulder
-2
10 5 10 2515 20
Dose (Gy)
reappeared.
Dq
n = e
D
D0
-
37. 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.
38. •For calculation
purposes, it is often
useful to
use the D10, the dose
required to kill 90%
of the population.
For example:
D10 = 2.3 × D0
in which 2.3 is the
natural logarithm of
10.
Showing ~28 Gy in 14
fractions.
39. 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
40. Intrinsic radiosensitivity
Mammalian cells are significantly more radio-sensitive
than microorganisms:
Due to the differences in DNA content
represents bigger target for radiation damage
Sterilizing radiation dose for bacteria is 20,000 Gy
43. 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
followed be a sensitive period in late G1
early G1
Cells are usually sensitive to radiation in G2 (almost as
sensitive as in M)
44. Radiation & Micro-organisms
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
radiodurans.
if radiation is used as a
method of
sterilization, 20,000 Gy
necessary.
45. Greek word meaning “falling off,” as in petals from
flowers or leaves from trees.
• First, apoptosis after radiation seems
commonly to be a p53-dependent proces.Here,the DNA
Electrophoresis pattern is like a ladder since here, the
DSBs occur in the liner region between nucleosomes to
produce DNA fragments that are multiples of 185 base
pairs.
• Mitotic death (commonest form of death): Cells die
attempting
to divide because of damaged chromosomes
The primary target for radiation induced lethality is,
specifically the DNA.
APOTOSIS AND MITOTIC DEATH
46. 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.
47. Summary
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
