radiation cell killing, mamdouh alenazi

8/14/2019 Radiation Cell Killing, Mamdouh Alenazi

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Radiation Cell KillingRadiation Cell Killing


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For cells proliferatingFor cells proliferating in vitroin vitro, define cell death as, define cell death as

loss ofloss ofreproductive abilityreproductive ability

Refers to cell losing its ability to exhibit unlimitedRefers to cell losing its ability to exhibit unlimited

cell divisioncell division

Clonogenic cell: cell that has reproductive ability, canClonogenic cell: cell that has reproductive ability, can

divide indefinitely to produce a large colony or clonedivide indefinitely to produce a large colony or clone


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In vivoIn vivo, predominant form of cell death following irradiation, predominant form of cell death following irradiationoccurs 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 highdividing 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 programmedApoptosis or programmed cell death: involves programmedsequence of events controlled by specific genes. Can occur atsequence of events controlled by specific genes. Can occur atlow doses of radiationlow doses of radiation


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Construction of anConstruction of an in vitroin vitro cellcell

survival curvesurvival curve

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 formEach single cell will divide several times to formcolonies that are fixed, stained and countedcolonies that are fixed, stained and counted

Count colonies containingCount colonies containing 50 cells (550 cells (5--66

generations of proliferation) to exclude cells thatgenerations of proliferation) to exclude cells thathave limited growth as a result of starting tohave limited growth as a result of starting todifferentiate or being subdifferentiate or being sub--lethally damagedlethally damaged


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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 duringerrors in cell counting; damage to cells during

trypsinizationtrypsinization

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


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Effective Cell Survival CurvesEffective Cell Survival Curves If radiation dose is delivered in aIf radiation dose is delivered in a

series of equal fractions (F),series of equal fractions (F),

separated by a time interval thatseparated by a time interval thatallows complete SLD repair, theallows complete SLD repair, theeffective dose survival curveeffective dose survival curvebecomes an exponential function ofbecomes an exponential function ofdosedose

Shoulder of the survival curve isShoulder of the survival curve isrepeated many times; the effectiverepeated many times; the effectivesurvival curve is a straight line fromsurvival curve is a straight line fromthe origin through point on thethe origin through point on thesinglesingle--dose survival curvedose 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), hasa value close to 3 Gy for humana value close to 3 Gy for humancells.cells.


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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


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Tumor contains 10Tumor contains 1099 cells. Effective dosecells. Effective dose--response curve has noresponse curve has no

shoulder, Dshoulder, D00 = 3Gy= 3GyWhat 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 killl

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 killTherefore, total dose for 10 decades of cell kill

= 10 x 6.9 = 69 Gy= 10 x 6.9 = 69 Gy


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Lethal DamageLethal Damage

Irreversible and irreparableIrreversible and irreparable

Leads to cell deathLeads to cell death


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Potentially Lethal DamagePotentially Lethal Damage

Component of radiation damage that can beComponent of radiation damage that can be

modified by postirradiation environmentalmodified by postirradiation environmentalconditionsconditions


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Varying environmental conditions after exposing cellsVarying environmental conditions after exposing cells

to Xto X--rays can influence proportion of cells that surviverays can influence proportion of cells that survivea given dose due to the expression or repair of PLDa given dose due to the expression or repair of PLD

Damage considered to be potentially lethal since underDamage considered to be potentially lethal since underordinary circumstances leads to cell deathordinary circumstances leads to cell death

However, if survival is increased followingHowever, if survival is increased followingmanipulation of the postirradiation environment, PLDmanipulation of the postirradiation environment, PLDis considered to have been repairedis considered to have been repaired


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MechanismMechanism

Cells maintained in subCells maintained in sub--optimal conditions do notoptimal conditions do not

have to attempt mitosis while chromosomes arehave to attempt mitosis while chromosomes are

expressing radiationexpressing radiation--induced injuryinduced injury

Delay leads to repair of the DNA damage andDelay leads to repair of the DNA damage and

increased survival. Relevance to clinical RT remainsincreased survival. Relevance to clinical RT remains

questionablequestionable


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Sublethal Damage RepairSublethal Damage RepairRepair of SLD in 2Repair of SLD in 2 in vivoin vivomammalian cell systems.mammalian cell systems.

A: SplitA: Split--dose experiments with P388dose experiments with P388lymphocytic leukemia cell in thelymphocytic leukemia cell in themouse. Onemouse. One--day tumors containday tumors containmainly oxic cells; 6mainly oxic cells; 6--day oldday oldtumors contain hypoxic cellstumors contain hypoxic cells(Belli(Belli et alet al JNCI 38:673JNCI 38:673--82,82,1967).1967).

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


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Sublethal Damage RepairSublethal Damage Repair

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 surviveore cells survivethan for the same total dose given in a single fraction, becausethan for the same total dose given in a single fraction, because the shoulder ofthe shoulder of

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


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Sublethal Damage RepairSublethal Damage Repair As time interval between 2 FAs time interval between 2 F

increases see rapid increase inincreases see rapid increase in

SF, usually complete within 2SF, usually complete within 2h in culture but longerh in culture but longerin vivoin vivo,,particularly for some lateparticularly for some late--responding tissuesresponding tissues

As time interval increases mayAs time interval increases maysee dip in SF due tosee dip in SF due tomovement of surviving cellsmovement of surviving cellsthrough the cell cycle; onlythrough the cell cycle; onlyobserved in cycling cellsobserved in cycling cells

If time interval exceeds theIf time interval exceeds thecell cycle, see increase in SFcell cycle, see increase in SFdue to proliferation.due to proliferation.


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Repair and Radiation QualityRepair and Radiation Quality

Since the presence of a shoulder on a cellSince the presence of a shoulder on a cell

survival curve is dependent on the quality ofsurvival curve is dependent on the quality of

radiation used, the amount of SLD repair isradiation 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, isHigh LET radiation, e.g., neutrons, isassociated with little repair of SLDassociated with little repair of SLD


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DoseDose--Rate EffectRate Effect


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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 associateddwith an increase in cell killwith an increase in cell kill

Mechanism:Mechanism:

At dose ratesAt dose rates 0.3 Gy/h cells tend to progress through the cell0.3 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 thecell cyclecell cycle

At higher dose rates the cells stay in the region of the cellAt 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 lowowdose rates they continue to cycle into Gdose rates they continue to cycle into G22 and thus becomeand thus becomemore radiosensitive.more radiosensitive.


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Summary of DoseSummary of Dose--Rate EffectRate Effect


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

Track average: obtained by dividing the trackTrack average: obtained by dividing the track

into equal lengths, calculating the energyinto 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 trackEnergy average: obtained by dividing the track

into equal energy increments and averaging theinto equal energy increments and averaging the

lengths of track over which these energylengths of track over which these energy

increments are depositedincrements are deposited


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Relative Biological EffectivenessRelative Biological Effectiveness

To compare the biological effect of differentTo compare the biological effect of different

types of radiation use xtypes of radiation use x--rays as the standardrays 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 effect

dose of test radiation to produce a given effectdose of test radiation to produce a given effect


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RBE as a function of LETRBE as a function of LET

As LET increases radiationAs LET increases radiationproduces more cell kill perproduces more cell kill per

Gy.Gy.

As LET increases survivalAs LET increases survival

curves become steeper andcurves become steeper and

the shoulder becomesthe shoulder becomes

progressively smaller.progressively smaller.


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Optimal LETOptimal LET LET radiation > 100LET radiation > 100

keV/keV/m results in wastedm results in wasted

energy or overkillenergy or overkill

Very high LET radiation isVery high LET radiation isinefficient since it depositsinefficient since it deposits

more energy than needed inmore energy than needed incritical sitescritical sites

These cells are overkilledThese cells are overkilled

and /Gy there is lessand /Gy there is lesslikelihood that other cellslikelihood that other cellswill be killed, leading to awill be killed, leading to areduced biological effectreduced biological effect


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Oxygen Effect and LETOxygen Effect and LET


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Oxygen Effect and LETOxygen Effect and LET

At low LET, corresponding to xAt low LET, corresponding to x--rays orrays or 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/m.m.OER then falls rapidly, reaching unity when LET around 200 keV/OER then falls rapidly, reaching unity when LET around 200 keV/m.m.

radiation cell killing, mamdouh alenazi

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