The Radiobiology of Radiation Therapy

Type of Injuries Nuclear DNA is major target Nuclear DNA is major target Cellular membrane damage – minor Cellular membrane damage – minor Nuclear membrane damage – minor Nuclear membrane damage – minor Cellular organelle injury – minor Cellular organelle injury – minor Mitochondrial DNA ??Mitochondrial DNA ??

Mechanism Two mechanisms of injury Two 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 micro-environment of the DNA DNA damaged by free radicals formed in the micro-environment of the DNA Water is most important source Water is most important source Oxygen is important in fixating injury Oxygen is important in fixating injury Sulfhydryl compounds promote repair Sulfhydryl compounds promote repair

Types of DNA Injury Base pair injury Base pair injury Base pair deletion Base pair deletion Base pair cross linkage Base pair cross linkage Single strand break in backbone Single strand break in backbone Double strand break in backbone Double strand break in backbone Gene suppression or activation Gene suppression or activation

Base Pair Injury Damage to one of the pairs of nitrogenous bases in the DNA sequence. Damage to one of the pairs of nitrogenous bases in the DNA sequence. Easily repaired by cellular repair mechanisms. Easily repaired by cellular repair mechanisms. Repair is error free Repair is error free

Base Pair Deletion Complete destruction of a pair of the nitrogenous bases in the sequence Complete destruction of a pair of the nitrogenous bases in the sequence Rapidly repaired by cellular repair mechanisms Rapidly repaired by cellular repair mechanisms Not necessarily error free repair. Not necessarily error free repair.

Base Pair Crosslinkage Injury Abnormal pairing of the nitrogenous bases. Abnormal pairing of the nitrogenous bases. May effect conformation of DNA May effect conformation of DNA Repaired efficiently Repaired efficiently

Single Strand Break Result of ionization of the sugar- phospate rail of the DNA molecule Result of ionization of the sugar- phospate rail of the DNA molecule Most is easily repaired unless base pairs are also lost Most is easily repaired unless base pairs are also lost Repair is rapid and accurate but some is not repairable. Repair is rapid and accurate but some is not repairable.

Double Strand Break Breakage of both strands of the DNA backbone in close proximity to each other. Breakage of both strands of the DNA backbone in close proximity to each 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 Activation Radiation injury may result in upregulation of some genes. Radiation injury may result in upregulation of some genes. Tumor Promoter genesTumor Promoter genes Tumor Suppressor genesTumor Suppressor genes Radiation injury may result in down regulation of the same genes Radiation injury may result in down regulation of the same genes Down regulation of genes controlling intracellular repair. Down regulation of genes controlling intracellular repair.

Cell Survival Curves Cell survival curve expressed on a log/linear plot. Cell survival curve expressed on a log/linear plot. Developed through many years of experimentation Developed through many years of experimentation Different curves are derived for different types of radiation. Different curves are derived for different types of radiation.

Cell survival, neutrons vrs. xrays

Single Hit Killing Lethal damage to DNA by single photon. Lethal damage to DNA by single photon. Mostly due to double strand breaks Mostly due to double strand breaks May be due to pro apoptotic gene activation May be due to pro apoptotic gene activation Represented by the initial straight portion of the photon survival curve Represented by the initial straight portion of the photon survival curve

Multi-hit Killing Lethal injury to the DNA following multiple hits of the DNA by photon radiation Lethal injury to the DNA following multiple hits of the DNA by photon radiation Coincident single strand breaks result in a double strand break Coincident single strand breaks result in a double strand break Activation of pro apoptotic genes Activation of pro apoptotic genes Increases with dose Increases with dose Represented by steep part of curve Represented by steep part of curve

Survival Curve Shoulder Represents the transition zone between single and multiple hit killing Represents the transition zone between single and multiple hit killing The shoulder is representative of the repair capability of the cell population The shoulder is representative of the repair capability of the cell population Wider in slowly dividing cells Wider in slowly dividing cells Narrower in rapidly dividing cells Narrower in rapidly dividing cells

Alpha/Beta Ratio Really is determined by a dose point Really is determined by a dose point Point on survival curve where single and multi-hit killing are equal Point on survival curve where single and multi-hit killing are equal Larger in cell lines with a wider repair shoulder. Larger in cell lines with a wider repair shoulder.

Alpha/Beta Ratio

LET and Effect on Survival LET = Linear Energy Transfer LET = Linear Energy Transfer Measured in keV/micronMeasured in keV/micron Characteristic of particulate radiationCharacteristic of particulate radiation High LET radiation increase killing per unit energy deposited. High LET radiation increase killing per unit energy deposited. Results in severe repair deficienciesResults in severe repair deficiencies Effectively removes the repair shoulder Effectively removes the repair shoulder

LET and Effect on Survival High LET radiation is densely ionizing High LET radiation is densely ionizing Averages >1 ionization event within the span of a DNA molecule. Averages >1 ionization event within the span of a DNA molecule. High ionization density increases probability of double strand breaks. High ionization density increases probability of double strand breaks. Reaches a maximum effect at about 100 keV/micron. Reaches a maximum effect at about 100 keV/micron.

LET and Effect on Survival Photons have an average LET of about 1. Photons have an average LET of about 1. <1 ionization event within the diameter of a DNA Molecule. <1 ionization event within the diameter of a DNA Molecule. Single strand breaks predominate Single strand breaks predominate Repair is permitted Repair is permitted

LET and Effect on Survival

Cell Cycle and Radiation Injury M phase – mitosis very sensitive to radiation injury M phase – mitosis very sensitive to radiation injury G1 phase – resting phase, moderately resistant G1 phase – resting phase, moderately resistant S phase – DNA synthesis, moderately resistant to radiation S phase – DNA synthesis, moderately resistant to radiation G2 resting phase – sensitive G2 resting phase – sensitive G0 non cycling cells – moderate resistance G0 non cycling cells – moderate resistance

Cell Cycle and Radiation Injury Mitosis Mitosis Chromosomes are condensedChromosomes are condensed DNA is closely packed – bigger target DNA 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 DNACell may loose large segments of DNA Fragments excluded from nucleus Fragments excluded from nucleus

Cell Cycle and Radiation Injury S phase S 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 damage Increases repair of radiation damage Lasts about 5 hours.Lasts about 5 hours.

Cell Cycle and Radiation Injury G1 G1 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 interface Goes down as cell nears the G1-S interface Point in cell cycle where apoptosis occurs Point in cell cycle where apoptosis occurs Cell death at this point is referred to as interphase deathCell death at this point is referred to as interphase death Longest part of cycle.Longest part of cycle. Lasts hours to years Lasts hours to years

Cell Cycle and Radiation Injury G2 G2 Short rest phase before MShort rest phase before M Quite radiation sensitiveQuite radiation sensitive Short time allows little for injury repairShort time allows little for injury repair Radiation injury incurred in S-phase may be repairedRadiation injury incurred in S-phase may be repaired May result in a mitotic delay in G2 May result in a mitotic delay in G2 Apoptosis-like death may also occurApoptosis-like death may also occur

The Four R’s Repair Repair Reassortment Reassortment Reoxygenation Reoxygenation Repopulation Repopulation

Repair Rapid repair of injury Rapid repair of injury Initiated within seconds of injury Initiated within seconds of injury Complete by 6 hours after injury Complete by 6 hours after injury Can be modified by environmental conditions Can be modified by environmental conditions Presence or absence of oxygen or free radical scavengers.Presence or absence of oxygen or free radical scavengers. Responsible for shoulder of survival curve Responsible for shoulder of survival curve

Reassortment When cells killed in sensitive phases it leave a gap in the cell population for those phases. When cells killed in sensitive phases it leave a gap in the cell population for those phases. Within two cycles cells from less sensitive parts of cycle replace them Within two cycles cells from less sensitive parts of cycle replace them Some non-cycling cells may be recruited into the cycling pool. Some non-cycling cells may be recruited into the cycling pool.

Reoxygenation Most tumors larger than 1 cm have some hypoxic cells in them Most tumors larger than 1 cm have some 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 oxygenated cells (O 2 fixation of injury) Radiation preferentially kills oxygenated cells (O 2 fixation of injury) Major contributor to tumor radiation resistance. Major contributor to tumor radiation resistance.

Reoxygenation

Reoxygenation

Repopulation Following killing of cells in a population by any means there is either replacement or repopulation of the cells killed Following killing of cells in a population by any means there is either replacement or repopulation of the cells killed Usually there is days to weeks delay before this begins Usually there is days to weeks delay before this begins Tissues with large clonogenic populations are able to do this better Tissues with large clonogenic populations are able to do this better

Repopulation Tends to be a low dose phenomenon Tends to be a low dose phenomenon Usually is most important in rapidly cycling cell population. Usually is most important in rapidly cycling cell population. This includes tumorsThis includes tumors Rapid repopulation may reduce level of repair Rapid repopulation may reduce level of repair

Tissue Level Radiation Effects All mammalian cells equally sensitive in cycling populations in cell culture All mammalian cells equally sensitive in cycling populations in cell culture However, in tissue the rate of cell replacement is variable However, in tissue the rate of cell replacement is variable Some cell populations turn over every 3-5 days and some never do. Some cell populations turn over every 3-5 days and some never do. Cell growth fractions and cell death fractions should be in balance.Cell growth fractions and cell death fractions should be in balance.

Tissue Effects Radiation response at tissue level is tied to cell death Radiation response at tissue level is tied to cell death Cell death is mostly tied to cell reproductionCell death is mostly tied to cell reproduction Apoptosis Apoptosis Radiation induction of apoptosis pathwaysRadiation induction of apoptosis pathways Mitotic linked death Mitotic 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 Effects Long cell cycle times promote repair and slow repopulation Long cell cycle times promote repair and slow repopulation Short cell cycle times promote repopulation and blunt repair Short cell cycle times promote repopulation and blunt repair Large non-cycling populations blunt radiation response Large non-cycling populations blunt radiation response Dose required to inhibit function is much higher than that for reproductive inhibition or failure. Dose required to inhibit function is much higher than that for reproductive inhibition or failure.

Tissue Effects At the tissue level the ultimate survival of the tissue depends on: At the tissue level the ultimate survival of the tissue depends on: The number of cycling cellsThe number of cycling cells The ability of the tissue to repair the injury.The ability of the tissue to repair the injury. The ability of the tissue to repopulate the tissue with the original cell type.The ability of the tissue to repopulate the tissue with the original cell type.

Tissue effects Repopulation is most important at low doses; Repopulation is most important at low doses; Early responding tissues tend to have more repopulation Early responding tissues tend to have more repopulation Late responding tissues tend to have limited repopulation capability Late responding tissues tend to have limited repopulation capability Therefore sensitive to larger doses of radiation.Therefore sensitive to larger doses of radiation.

Tissue Effects

Radiation Delivery Treatment with a number smaller doses improves normal tissue response and increases total dose that can be given to a tumor Treatment with a number smaller doses improves normal tissue response and increases total dose that can be given to a tumor Reduces hypoxiaReduces hypoxia Promotes repopulation in late responding tisuesPromotes repopulation in late responding tisues Promote reassortmentPromote reassortment Promotes repair of DNA injuryPromotes repair of DNA injury

Fractionation

Fractionation Optimal dose is that which is just about midway through the repair shoulder. Optimal dose is that which is just about midway through the repair shoulder. Usually approximately equal to the Do dose Usually approximately equal to the Do dose Must wait at least 6 hours for repair to be complete. Must wait at least 6 hours for repair to be complete.