The Radiobiology of Radiation The Radiobiology of Radiation TherapyTherapy

Type of InjuriesType of Injuries

Nuclear DNA is major targetNuclear DNA is major target Cellular membrane damage – minorCellular membrane damage – minor Nuclear membrane damage – minorNuclear membrane damage – minor Cellular organelle injury – minorCellular organelle injury – minor

• Mitochondrial DNA ??Mitochondrial DNA ??

MechanismMechanism

Two mechanisms of injuryTwo mechanisms of injury• Direct Ionization of the DNA, ≈ 15%Direct Ionization of the DNA, ≈ 15%• Indirect Ionization of the DNA, ≈ 85% Indirect Ionization of the DNA, ≈ 85%

DNA damaged by free radicals formed in the DNA damaged by free radicals formed in the micro-environment of the DNAmicro-environment of the DNA

Water is most important sourceWater is most important source Oxygen is important in fixating injuryOxygen is important in fixating injury Sulfhydryl compounds promote repairSulfhydryl compounds promote repair

Types of DNA InjuryTypes of DNA Injury

Base pair injuryBase pair injury Base pair deletionBase pair deletion Base pair cross linkageBase pair cross linkage Single strand break in backboneSingle strand break in backbone Double strand break in backboneDouble strand break in backbone Gene suppression or activationGene suppression or activation

Base Pair InjuryBase Pair Injury

Damage to one of the pairs of Damage to one of the pairs of nitrogenous bases in the DNA nitrogenous bases in the DNA sequence. sequence.

Easily repaired by cellular repair Easily repaired by cellular repair mechanisms. mechanisms.

Repair is error freeRepair is error free

Base Pair DeletionBase Pair Deletion

Complete destruction of a pair of the Complete destruction of a pair of the nitrogenous bases in the sequencenitrogenous bases in the sequence

Rapidly repaired by cellular repair Rapidly repaired by cellular repair mechanismsmechanisms

Not necessarily error free repair.Not necessarily error free repair.

Base Pair Crosslinkage InjuryBase Pair Crosslinkage Injury

Abnormal pairing of the nitrogenous Abnormal pairing of the nitrogenous bases. bases.

May effect conformation of DNA May effect conformation of DNA Repaired efficiently Repaired efficiently

Single Strand BreakSingle Strand Break

Result of ionization of the sugar-Result of ionization of the sugar-phospate rail of the DNA moleculephospate rail of the DNA molecule

Most is easily repaired unless base Most is easily repaired unless base pairs are also lostpairs are also lost

Repair is rapid and accurate but Repair is rapid and accurate but some is not repairable.some is not repairable.

Double Strand BreakDouble Strand Break

Breakage of both strands of the DNA Breakage of both strands of the DNA backbone in close proximity to each backbone in close proximity to each other.other.

Difficult to repair Difficult to repair Repair is quite prone to errors.Repair is quite prone to errors. High dose and High LET event. High dose and High LET event.

Gene Suppression or ActivationGene Suppression or Activation

Radiation injury may result in Radiation injury may result in upregulation of some genes.upregulation of some genes.• Tumor Promoter genesTumor Promoter genes• Tumor Suppressor genesTumor Suppressor genes

Radiation injury may result in down Radiation injury may result in down regulation of the same genesregulation of the same genes

Down regulation of genes controlling Down regulation of genes controlling intracellular repair. intracellular repair.

Cell Survival CurvesCell Survival Curves

Cell survival curve expressed on a Cell survival curve expressed on a log/linear plot. log/linear plot.

Developed through many years of Developed through many years of experimentationexperimentation

Different curves are derived for Different curves are derived for different types of radiation.different types of radiation.

Cell survival, neutrons vrs. Cell survival, neutrons vrs. xraysxrays

Single Hit KillingSingle Hit Killing

Lethal damage to DNA by single Lethal damage to DNA by single photon. photon.

Mostly due to double strand breaksMostly due to double strand breaks May be due to pro apoptotic gene May be due to pro apoptotic gene

activationactivation Represented by the initial straight Represented by the initial straight

portion of the photon survival curveportion of the photon survival curve

Multi-hit KillingMulti-hit Killing

Lethal injury to the DNA following Lethal injury to the DNA following multiple hits of the DNA by photon multiple hits of the DNA by photon radiationradiation

Coincident single strand breaks Coincident single strand breaks result in a double strand breakresult in a double strand break

Activation of pro apoptotic genesActivation of pro apoptotic genes Increases with doseIncreases with dose Represented by steep part of curveRepresented by steep part of curve

Survival Curve ShoulderSurvival Curve Shoulder

Represents the transition zone Represents the transition zone between single and multiple hit between single and multiple hit killingkilling

The shoulder is representative of the The shoulder is representative of the repair capability of the cell repair capability of the cell populationpopulation

Wider in slowly dividing cellsWider in slowly dividing cells Narrower in rapidly dividing cellsNarrower in rapidly dividing cells

Alpha/Beta RatioAlpha/Beta Ratio

Really is determined by a dose pointReally is determined by a dose point Point on survival curve where single Point on survival curve where single

and multi-hit killing are equaland multi-hit killing are equal Larger in cell lines with a wider repair Larger in cell lines with a wider repair

shoulder. shoulder.

Alpha/Beta RatioAlpha/Beta Ratio

LET and Effect on SurvivalLET and Effect on Survival

LET = Linear Energy TransferLET = Linear Energy Transfer• Measured in keV/micronMeasured in keV/micron• Characteristic of particulate radiation Characteristic of particulate radiation

High LET radiation increase killing High LET radiation increase killing per unit energy deposited. per unit energy deposited. • Results in severe repair deficiencies Results in severe repair deficiencies

Effectively removes the repair Effectively removes the repair shouldershoulder

LET and Effect on SurvivalLET and Effect on Survival

High LET radiation is densely ionizingHigh LET radiation is densely ionizing Averages >1 ionization event within Averages >1 ionization event within

the span of a DNA molecule.the span of a DNA molecule. High ionization density increases High ionization density increases

probability of double strand breaks. probability of double strand breaks. Reaches a maximum effect at about Reaches a maximum effect at about

100 keV/micron.100 keV/micron.

LET and Effect on SurvivalLET and Effect on Survival

Photons have an average LET of Photons have an average LET of about 1. about 1.

<1 ionization event within the <1 ionization event within the diameter of a DNA Molecule. diameter of a DNA Molecule.

Single strand breaks predominateSingle strand breaks predominate Repair is permittedRepair is permitted

LET and Effect on SurvivalLET and Effect on Survival

Cell Cycle and Radiation InjuryCell Cycle and Radiation Injury

M phase – mitosis very sensitive to M phase – mitosis very sensitive to radiation injuryradiation injury

G1 phase – resting phase, G1 phase – resting phase, moderately resistantmoderately resistant

S phase – DNA synthesis, S phase – DNA synthesis, moderately resistant to radiationmoderately resistant to radiation

G2 resting phase – sensitiveG2 resting phase – sensitive G0 non cycling cells – moderate G0 non cycling cells – moderate

resistance resistance

Cell Cycle and Radiation InjuryCell Cycle and Radiation Injury

MitosisMitosis• Chromosomes are condensedChromosomes are condensed

DNA is closely packed – bigger targetDNA is closely packed – bigger target

• Repair mechanisms are shut downRepair mechanisms are shut down• Very compressed time scale = 1 hr.Very compressed time scale = 1 hr.• Any DNA injury is fixed in placeAny DNA injury is fixed in place• Cell may loose large segments of DNA Cell may loose large segments of DNA

Fragments excluded from nucleusFragments excluded from nucleus

Cell Cycle and Radiation InjuryCell Cycle and Radiation Injury

S phaseS phase• Phase of DNA synthesisPhase of DNA synthesis• Most radiation resistant phaseMost radiation resistant phase• Cellular repair mechanisms are activeCellular repair mechanisms are active

Increases repair of radiation damageIncreases repair of radiation damage

• Lasts about 5 hours. Lasts about 5 hours.

Cell Cycle and Radiation InjuryCell Cycle and Radiation Injury

G1G1• Functional part of cell cycleFunctional part of cell cycle• Resistance varies with part of phaseResistance varies with part of phase

Goes down as cell nears the G1-S interfaceGoes down as cell nears the G1-S interface Point in cell cycle where apoptosis occursPoint in cell cycle where apoptosis occurs

• Cell death at this point is referred to as Cell death at this point is referred to as interphase deathinterphase death

• Longest part of cycle. Longest part of cycle. Lasts hours to yearsLasts hours to years

Cell Cycle and Radiation InjuryCell Cycle and Radiation Injury

G2G2• Short rest phase before MShort rest phase before M• Quite radiation sensitive Quite radiation sensitive • Short time allows little for injury repairShort time allows little for injury repair• Radiation injury incurred in S-phase may Radiation injury incurred in S-phase may

be repaired be repaired May result in a mitotic delay in G2May result in a mitotic delay in G2

• Apoptosis-like death may also occurApoptosis-like death may also occur

The Four R’sThe Four R’s

RepairRepair Reassortment Reassortment ReoxygenationReoxygenation RepopulationRepopulation

RepairRepair

Rapid repair of injuryRapid repair of injury Initiated within seconds of injuryInitiated within seconds of injury Complete by 6 hours after injuryComplete by 6 hours after injury Can be modified by environmental Can be modified by environmental

conditionsconditions• Presence or absence of oxygen or free Presence or absence of oxygen or free

radical scavengers. radical scavengers. Responsible for shoulder of survival Responsible for shoulder of survival

curvecurve

ReassortmentReassortment

When cells killed in sensitive phases When cells killed in sensitive phases it leave a gap in the cell population it leave a gap in the cell population for those phases.for those phases.

Within two cycles cells from less Within two cycles cells from less sensitive parts of cycle replace themsensitive parts of cycle replace them

Some non-cycling cells may be Some non-cycling cells may be recruited into the cycling pool.recruited into the cycling pool.

ReoxygenationReoxygenation

Most tumors larger than 1 cm have Most tumors larger than 1 cm have some hypoxic cells in themsome hypoxic cells in them• Some tumor types have larger %Some tumor types have larger %• May be transient or chronicMay be transient or chronic

Radiation preferentially kills Radiation preferentially kills oxygenated cells oxygenated cells (O(O22 fixation of injury) fixation of injury)

Major contributor to tumor radiation Major contributor to tumor radiation resistance.resistance.

ReoxygenationReoxygenation

ReoxygenationReoxygenation

RepopulationRepopulation

Following killing of cells in a Following killing of cells in a population by any means there is population by any means there is either replacement or repopulation of either replacement or repopulation of the cells killedthe cells killed

Usually there is days to weeks delay Usually there is days to weeks delay before this beginsbefore this begins

Tissues with large clonogenic Tissues with large clonogenic populations are able to do this betterpopulations are able to do this better

RepopulationRepopulation

Tends to be a low dose phenomenonTends to be a low dose phenomenon Usually is most important in rapidly Usually is most important in rapidly

cycling cell population.cycling cell population.• This includes tumorsThis includes tumors

Rapid repopulation may reduce level Rapid repopulation may reduce level of repairof repair

Tissue Level Radiation EffectsTissue Level Radiation Effects

All mammalian cells equally sensitive All mammalian cells equally sensitive in cycling populations in cell culturein cycling populations in cell culture

However, in tissue the rate of cell However, in tissue the rate of cell replacement is variablereplacement is variable

Some cell populations turn over Some cell populations turn over every 3-5 days and some never do. every 3-5 days and some never do. • Cell growth fractions and cell death Cell growth fractions and cell death

fractions should be in balance.fractions should be in balance.

Tissue EffectsTissue Effects

Radiation response at tissue level is Radiation response at tissue level is tied to cell deathtied to cell death• Cell death is mostly tied to cell Cell death is mostly tied to cell

reproductionreproduction ApoptosisApoptosis

• Radiation induction of apoptosis pathwaysRadiation induction of apoptosis pathways Mitotic linked deathMitotic linked death

• Reproductive failure due to missing DNAReproductive failure due to missing DNA

• Long cell cycle times blunt responseLong cell cycle times blunt response

Tissue EffectsTissue Effects

Long cell cycle times promote repair Long cell cycle times promote repair and slow repopulationand slow repopulation

Short cell cycle times promote Short cell cycle times promote repopulation and blunt repairrepopulation and blunt repair

Large non-cycling populations blunt Large non-cycling populations blunt radiation responseradiation response

Dose required to inhibit function is Dose required to inhibit function is much higher than that for much higher than that for reproductive inhibition or failure.reproductive inhibition or failure.

Tissue EffectsTissue Effects

At the tissue level the ultimate At the tissue level the ultimate survival of the tissue depends on:survival of the tissue depends on:• The number of cycling cellsThe number of cycling cells• The ability of the tissue to repair the The ability of the tissue to repair the

injury.injury.• The ability of the tissue to repopulate The ability of the tissue to repopulate

the tissue with the original cell type. the tissue with the original cell type.

Tissue effectsTissue effects

Repopulation is most important at Repopulation is most important at low doses; low doses;

Early responding tissues tend to have Early responding tissues tend to have more repopulationmore repopulation

Late responding tissues tend to have Late responding tissues tend to have limited repopulation capabilitylimited repopulation capability• Therefore sensitive to larger doses of Therefore sensitive to larger doses of

radiation.radiation.

Tissue EffectsTissue Effects

Radiation DeliveryRadiation Delivery

Treatment with a number smaller Treatment with a number smaller doses improves normal tissue doses improves normal tissue response and increases total dose response and increases total dose that can be given to a tumorthat can be given to a tumor• Reduces hypoxiaReduces hypoxia• Promotes repopulation in late Promotes repopulation in late

responding tisuesresponding tisues• Promote reassortmentPromote reassortment• Promotes repair of DNA injuryPromotes repair of DNA injury

FractionationFractionation

FractionationFractionation

Optimal dose is that which is just Optimal dose is that which is just about midway through the repair about midway through the repair shoulder. shoulder.

Usually approximately equal to the Usually approximately equal to the Do doseDo dose

Must wait at least 6 hours for repair Must wait at least 6 hours for repair to be complete.to be complete.