Radiation Cell Killing Cell... · 2017-10-02 · Radiation Cell Killing In vivo, predominant form...

Radiation Cell KillingRadiation Cell Killing


For cells proliferating For cells proliferating in vitroin vitro, define cell death as , define cell death as loss of loss of reproductive abilityreproductive ability

Refers to cell losing its ability to exhibit unlimited Refers to cell losing its ability to exhibit unlimited cell divisioncell division

Clonogenic cell: cell that has reproductive ability, can Clonogenic cell: cell that has reproductive ability, can divide indefinitely to produce a large colony or clonedivide indefinitely to produce a large colony or clone


In vivoIn vivo, predominant form of cell death following irradiation , predominant form of cell death following irradiation occurs at mitosis, requires a dose of ~ 2 Gyoccurs at mitosis, requires a dose of ~ 2 Gy

Irradiating nonIrradiating non--dividing or rarely dividing cells with very high dividing or rarely dividing cells with very high doses, ~ 100 Gy can cause loss of cell function and death, i.e.,doses, ~ 100 Gy can cause loss of cell function and death, i.e.,Interphase DeathInterphase Death

Apoptosis or programmed cell death: involves programmed Apoptosis or programmed cell death: involves programmed sequence of events controlled by specific genes. Can occur at sequence of events controlled by specific genes. Can occur at low doses of radiationlow doses of radiation

Construction of an Construction of an in vitroin vitro cell cell survival curvesurvival curve

General TechniqueGeneral Technique

Add trypsin (enzyme) to a flask containing Add trypsin (enzyme) to a flask containing the cells. Cells will round up and detach from the cells. Cells will round up and detach from the surface of the flaskthe surface of the flaskCount the number of cells/mL (manually can Count the number of cells/mL (manually can use a hemocytometer; electronically can use use a hemocytometer; electronically can use a Coulter Counter)a Coulter Counter)


Add a known number of cells to a new flask; Add a known number of cells to a new flask; incubate for 1incubate for 1--2 weeks2 weeks

Each single cell will divide several times to form Each single cell will divide several times to form colonies that are fixed, stained and countedcolonies that are fixed, stained and counted

Count colonies containing Count colonies containing ≥≥ 50 cells (550 cells (5--6 6 generations of proliferation) to exclude cells that generations of proliferation) to exclude cells that have limited growth as a result of starting to have limited growth as a result of starting to differentiate or being subdifferentiate or being sub--lethally damagedlethally damaged

Plating EfficiencyPlating Efficiency

Not all cells plated out will form coloniesNot all cells plated out will form colonies

Inability reflects suboptimal growth medium; Inability reflects suboptimal growth medium; errors in cell counting; damage to cells during errors in cell counting; damage to cells during trypsinizationtrypsinization

Need to determine the Plating Efficiency (PE)Need to determine the Plating Efficiency (PE)

Plating EfficiencyPlating Efficiency

PE = PE = Mean number of colonies/dish Mean number of colonies/dish Number of cells plated/dishNumber of cells plated/dish


Hall, Radiobiology for the Radiologist

Cell Survival CurveCell Survival Curve

To construct a cell survival curve use a range of doses and To construct a cell survival curve use a range of doses and determine the surviving fraction, SF, after each dosedetermine the surviving fraction, SF, after each dose

SF after dose D = SF after dose D = mean number of colonies after dose D/dishmean number of colonies after dose D/dishmean number of cells plated/dish x PEmean number of cells plated/dish x PE

Number of cells seeded per dish needs to be adjusted so that a Number of cells seeded per dish needs to be adjusted so that a countable number of colonies is obtainedcountable number of colonies is obtained

Mammalian Cell Survival CurvesMammalian Cell Survival Curves

Effective Cell Survival CurvesEffective Cell Survival CurvesIf radiation dose is delivered in a If radiation dose is delivered in a series of equal fractions (F), series of equal fractions (F), separated by a time interval that separated by a time interval that allows complete SLD repair, the allows complete SLD repair, the effective dose survival curve effective dose survival curve becomes an exponential function of becomes an exponential function of dosedose

Shoulder of the survival curve is Shoulder of the survival curve is repeated many times; the effective repeated many times; the effective survival curve is a straight line from survival curve is a straight line from the origin through point on the the origin through point on the singlesingle--dose survival curve dose survival curve corresponding to the daily dose Fcorresponding to the daily dose F

DD00 (the reciprocal of the slope), has (the reciprocal of the slope), has a value close to 3 Gy for human a value close to 3 Gy for human cells. cells.


For calculations, useful to use the DFor calculations, useful to use the D1010

Dose required to kill 90% of populationDose required to kill 90% of population

DD1010 = 2.3 x D= 2.3 x D00

where 2.3 is the natural log of 10where 2.3 is the natural log of 10


Tumor contains 10Tumor contains 1099 cells. Effective dosecells. Effective dose--response curve has no response curve has no shoulder, Dshoulder, D00 = 3Gy= 3Gy

What total dose is required to give 90% chance of tumor cure?What total dose is required to give 90% chance of tumor cure?

90% probability of tumor control requires 10 decades of cell kil90% probability of tumor control requires 10 decades of cell kill l

Dose resulting in one decade of cell kill, DDose resulting in one decade of cell kill, D1010,,= 2.3 x D= 2.3 x D00 = 2.3 x 3 = 6.9 Gy= 2.3 x 3 = 6.9 Gy

Therefore, total dose for 10 decades of cell kill Therefore, total dose for 10 decades of cell kill = 10 x 6.9 = 69 Gy= 10 x 6.9 = 69 Gy

Repair of Radiation DamageRepair of Radiation Damage

In mammalian cells consider 3 types of radiation In mammalian cells consider 3 types of radiation damage:damage:

Lethal damageLethal damageSublethal damageSublethal damagePotentially lethal damagePotentially lethal damage

Lethal DamageLethal Damage

Irreversible and irreparableIrreversible and irreparable

Leads to cell deathLeads to cell death

Potentially Lethal DamagePotentially Lethal Damage

Component of radiation damage that can be Component of radiation damage that can be modified by postirradiation environmental modified by postirradiation environmental conditionsconditions


Varying environmental conditions after exposing cells Varying environmental conditions after exposing cells to Xto X--rays can influence proportion of cells that survive rays can influence proportion of cells that survive a given dose due to the expression or repair of PLD a given dose due to the expression or repair of PLD

Damage considered to be potentially lethal since under Damage considered to be potentially lethal since under ordinary circumstances leads to cell death ordinary circumstances leads to cell death

However, if survival is increased following However, if survival is increased following manipulation of the postirradiation environment, PLD manipulation of the postirradiation environment, PLD is considered to have been repairedis considered to have been repaired


XX--ray survival curves for densityray survival curves for density--inhibited stationaryinhibited stationary--phase cells, phase cells, subcultured either immediately or 6subcultured either immediately or 6--12 h 12 h after irradiation (Little after irradiation (Little et alet al RadiolRadiol106:689106:689--94, 1973)94, 1973)


Mechanism Mechanism Cells maintained in subCells maintained in sub--optimal conditions do not optimal conditions do not have to attempt mitosis while chromosomes are have to attempt mitosis while chromosomes are expressing radiationexpressing radiation--induced injury induced injury

Delay leads to repair of the DNA damage and Delay leads to repair of the DNA damage and increased survival. Relevance to clinical RT remains increased survival. Relevance to clinical RT remains questionablequestionable

Sublethal DamageSublethal Damage

Under normal circumstances can be repaired in hours, Under normal circumstances can be repaired in hours, usually considered to be complete within 24 h usually considered to be complete within 24 h

If additional sublethal damage added within this time If additional sublethal damage added within this time then can interact to form lethal damagethen can interact to form lethal damage

Sublethal damage repair observed as an increase in Sublethal damage repair observed as an increase in survival if a dose of radiation is split into 2 equal survival if a dose of radiation is split into 2 equal fractions separated by a time intervalfractions separated by a time interval

Sublethal Damage RepairSublethal Damage Repair

Term used to describe the Term used to describe the increase in cell survival seen if increase in cell survival seen if a given radiation dose is split a given radiation dose is split into 2 equal fractions into 2 equal fractions separated by a time interval.separated by a time interval.


Repair of SLD in 2 Repair of SLD in 2 in vivo in vivo mammalian cell systems.mammalian cell systems.

A: SplitA: Split--dose experiments with P388 dose experiments with P388 lymphocytic leukemia cell in the lymphocytic leukemia cell in the mouse. Onemouse. One--day tumors contain day tumors contain mainly oxic cells; 6mainly oxic cells; 6--day old day old tumors contain hypoxic cells tumors contain hypoxic cells (Belli (Belli et alet al JNCI 38:673JNCI 38:673--82, 82, 1967). 1967).

B: SplitB: Split--dose experiments with skin dose experiments with skin epithelial cells in the mouse epithelial cells in the mouse (Emery(Emery et alet al Radiat Res 41:450, Radiat Res 41:450, 1970).1970).


If dose is split into 2 fractions separated by a time interval mIf dose is split into 2 fractions separated by a time interval more cells survive ore cells survive than for the same total dose given in a single fraction, becausethan for the same total dose given in a single fraction, because the shoulder of the shoulder of

the curve must be repeated each time. the curve must be repeated each time.

Sublethal Damage RepairSublethal Damage RepairAs time interval between 2 F As time interval between 2 F increases see rapid increase in increases see rapid increase in SF, usually complete within 2 SF, usually complete within 2 h in culture but longer h in culture but longer in vivoin vivo, , particularly for some lateparticularly for some late--responding tissuesresponding tissues

As time interval increases may As time interval increases may see dip in SF due to see dip in SF due to movement of surviving cells movement of surviving cells through the cell cycle; only through the cell cycle; only observed in cycling cellsobserved in cycling cells

If time interval exceeds the If time interval exceeds the cell cycle, see increase in SF cell cycle, see increase in SF due to proliferation.due to proliferation.

Repair and Radiation QualityRepair and Radiation Quality

Since the presence of a shoulder on a cell Since the presence of a shoulder on a cell survival curve is dependent on the quality of survival curve is dependent on the quality of radiation used, the amount of SLD repair is radiation used, the amount of SLD repair is similarly dependent on the quality of radiationsimilarly dependent on the quality of radiation

High LET radiation, e.g., neutrons, is High LET radiation, e.g., neutrons, is associated with little repair of SLDassociated with little repair of SLD

DoseDose--Rate EffectRate Effect

For XFor X-- or or γγ rays, dose rate is one of the most rays, dose rate is one of the most important factors that determine the biologic effect of important factors that determine the biologic effect of a given dosea given dose

As dose rate is lowered and exposure time increased, As dose rate is lowered and exposure time increased, biologic effect is in general reducedbiologic effect is in general reduced

Seen over a dose range of 1 Gy/min to 0.3 Gy/h; with Seen over a dose range of 1 Gy/min to 0.3 Gy/h; with decreasing dosedecreasing dose--rate see loss of shoulderrate see loss of shoulder



Inverse DoseInverse Dose--Rate EffectRate Effect

Seen in some situations when lowering the dose rate is associateSeen in some situations when lowering the dose rate is associated d with an increase in cell kill with an increase in cell kill

Mechanism: Mechanism: At dose rates At dose rates ≤≤ 0.3 Gy/h cells tend to progress through the cell 0.3 Gy/h cells tend to progress through the cell cycle and become arrested in Gcycle and become arrested in G22, a radiosensitive part of the , a radiosensitive part of the cell cyclecell cycle

At higher dose rates the cells stay in the region of the cell At higher dose rates the cells stay in the region of the cell cycle they were in at the time of irradiation. In contrast, at lcycle they were in at the time of irradiation. In contrast, at low ow dose rates they continue to cycle into Gdose rates they continue to cycle into G22 and thus become and thus become more radiosensitive.more radiosensitive.

Summary of DoseSummary of Dose--Rate EffectRate Effect

Linear Energy Transfer (LET)Linear Energy Transfer (LET)

LET is the energy transferred per unit length of LET is the energy transferred per unit length of tracktrack

Unit is the kiloelectron volt per micrometer Unit is the kiloelectron volt per micrometer (keV/(keV/μμm) of unit density materialm) of unit density material

LET is an average value that can be calculated LET is an average value that can be calculated in different waysin different ways


Track average: obtained by dividing the track Track average: obtained by dividing the track into equal lengths, calculating the energy into equal lengths, calculating the energy deposited in each length, and finding the meandeposited in each length, and finding the mean

Energy average: obtained by dividing the track Energy average: obtained by dividing the track into equal energy increments and averaging the into equal energy increments and averaging the lengths of track over which these energy lengths of track over which these energy increments are depositedincrements are deposited


For X rays or monoenergetic charged particles For X rays or monoenergetic charged particles the two methods give similar resultsthe two methods give similar results

However, very different for 14However, very different for 14--MeV neutrons; MeV neutrons; track average is ~12 keV/track average is ~12 keV/μμm, energy average m, energy average LET is ~ 75 keV/LET is ~ 75 keV/μμmm

Biological properties of neutrons tend to Biological properties of neutrons tend to correlate best with the energy average.correlate best with the energy average.

Relative Biological EffectivenessRelative Biological Effectiveness

The amount of radiation dose is expressed in terms of The amount of radiation dose is expressed in terms of absorbed energy; dose in Gy is a measure of energy absorbed energy; dose in Gy is a measure of energy absorbed/unit mass of tissueabsorbed/unit mass of tissue

However, equal doses of different types of radiation However, equal doses of different types of radiation DO NOTDO NOT produce equal biological effectsproduce equal biological effects

Key to the difference lies in the pattern of energy Key to the difference lies in the pattern of energy deposition at the microscopic leveldeposition at the microscopic level


To compare the biological effect of different To compare the biological effect of different types of radiation use xtypes of radiation use x--rays as the standard rays as the standard

RBE is formally defined as follows:RBE is formally defined as follows:

RBE = RBE = dose of xdose of x--rays to produce a given effectrays to produce a given effectdose of test radiation to produce a given effectdose of test radiation to produce a given effect


RBE is not a single valueRBE is not a single value

Depends on the level of biological damage Depends on the level of biological damage (and thus the dose) chosen(and thus the dose) chosen

In general, RBE In general, RBE as dose as dose until limiting until limiting value reachedvalue reached



RBE as a function of LETRBE as a function of LET

As LET increases radiation As LET increases radiation produces more cell kill per produces more cell kill per Gy. Gy.

As LET increases survival As LET increases survival curves become steeper and curves become steeper and the shoulder becomes the shoulder becomes progressively smaller. progressively smaller.

RBE as a function of LETRBE as a function of LET

If plot RBE as a function of LET, RBE increases slowly at first,If plot RBE as a function of LET, RBE increases slowly at first, then more then more rapidly as LET > 10 keV/rapidly as LET > 10 keV/μμm. m. RBE then increases rapidly to a peak value of ~ 100 keV/RBE then increases rapidly to a peak value of ~ 100 keV/μμm, after which RBE m, after which RBE decreases rapidly. decreases rapidly. The LET at which RBE peaks is essentially the same for a wide vaThe LET at which RBE peaks is essentially the same for a wide variety of riety of mammalian cells.mammalian cells.

Optimal LETOptimal LET

Why is radiation with an LET of 100 keV/Why is radiation with an LET of 100 keV/μμm optimal?m optimal?

At this density of ionization, average separation At this density of ionization, average separation density between ionizing events roughly coincides density between ionizing events roughly coincides with diameter of DNA double helix, i.e., 2nm (20with diameter of DNA double helix, i.e., 2nm (20ΑΑ))

Radiation of this density has the greatest probability Radiation of this density has the greatest probability of causing a doubleof causing a double--strand break by the passage of a strand break by the passage of a single charged particle; doublesingle charged particle; double--strand breaks are the strand breaks are the basis for most biologic effects.basis for most biologic effects.

Optimal LETOptimal LET

LET radiation > 100 LET radiation > 100 keV/keV/μμm results in wasted m results in wasted energy or overkillenergy or overkill

Very high LET radiation is Very high LET radiation is inefficient since it deposits inefficient since it deposits more energy than needed in more energy than needed in critical sites critical sites

These cells are overkilled These cells are overkilled and /Gy there is less and /Gy there is less likelihood that other cells likelihood that other cells will be killed, leading to a will be killed, leading to a reduced biological effectreduced biological effect

Factors that determine RBEFactors that determine RBE

Radiation quality (LET)Radiation quality (LET)Radiation doseRadiation doseNumber of dose fractionsNumber of dose fractionsDose rateDose rateBiologic system or endpoint.Biologic system or endpoint.

Oxygen Effect and LETOxygen Effect and LET

Oxygen Effect and LETOxygen Effect and LET

At low LET, corresponding to xAt low LET, corresponding to x--rays or rays or γγ rays, OER is 2.5rays, OER is 2.5--3. 3. As LET increases, OER decreases slowly until the LET > ~60 keV/As LET increases, OER decreases slowly until the LET > ~60 keV/μμm. m. OER then falls rapidly, reaching unity when LET around 200 keV/OER then falls rapidly, reaching unity when LET around 200 keV/μμm.m.

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Radiation Cell Killing Cell... · 2017-10-02 · Radiation Cell Killing In vivo, predominant form...

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