10/28/2015 survival curves; modifiers p. 1 of 96 illinois institute of technology physics 561...

*Survival Curves; Modifiersp. * of 96Illinois Institute of TechnologyPHYSICS 561Radiation BiophysicsLecture 5:Survival Curves and Modifiers of ResponseAndrew Howard17 June 2014

Survival Curves; Modifiers

*Survival Curves; Modifiersp. * of 96Looking forwardBe alert for changes in the posted assignments: I may add a few thingsMidterm will cover chapters 1-7 and the bit of extra material on free radicals that we discussed last week; therefore, it will not include the material from todays lecture


*Survival Curves; Modifiersp. * of 96Survival CurvesWe discussed models for cell survival last timeWe looked at various ln(S/S0) vs. dose models and the logic behind themToday well focus on the graphical implications and how we can look at the numbersThen well talk about cell cycles and other good solid cell-biology topics.(Warning: Im more of a biochemist than a cell biologist, so dont expect high expertise in this later section!)


*Survival Curves; Modifiersp. * of 96Errata in Chapter 8Page 169, Paragraph 2, 1st sentence: Until the later 1950s it was not possible to use Two sentences later: Bacteroides BacillusFig. 8.1, p. 173: The label that says Dq is pointing at the wrong thing: it should be pointing at the place where the dashed line crosses the (Surviving faction = 1.0) value.


What Fig. 8.1 should have said*Survival Curves; Modifiersp. * of 96Dose, GySurviving FractionABSlope = k = 1/D0Dq = D0lnn = quasi-thresholdn


*Survival Curves; Modifiersp. * of 96Shoulder of the Survival CurveWe recognize that with MTSH dose-response we have a region where the slope is close to zero. We describe that region as a shoulder:


*Survival Curves; Modifiersp. * of 96Slopes in the MTSH modelRemember that the MTSH model says ln(S/S0) = ln(1-(1-exp(-D/D0))n)Because S/S0 = 1-(1-exp(-D/D0))nSo what is the slope of the S/S0 vs. D curve? and what is the slope of the ln(S/S0) vs. D curve?In particular, what is the slopes behavior at low dose?Answer: calculate dS/S0/ dD and d(ln(S/S0))/dD and investigate their behavior at or near D = 0.Note: were looking here at the n>1 case.


*Survival Curves; Modifiersp. * of 96Slope investigation, part IFor S/S0 itself, d(S/S0)/dD = d/dD(1-(1-exp(-D/D0))n)The 1 out front doesnt affect the derivative: d(S/S0)/dD = -d/dD(1-exp(-D/D0))n)Well do the rest of this calculation now based on the general formulasdun/dx = nun-1du/dxdeu/dx = eudu/dx


*Survival Curves; Modifiersp. * of 96Arithmetic & Calculusof Survival ModelsMTSH says S/S0 = 1 - (1-e -D/D0)nWhat I want to investigate is the slope at low dose, I.e. for D > D0.But are we interested in the slope of S/S0 vs. D or ln(S/S0) vs. D?Both!Slope = derivative with respect to D. SoSlope = d/dD(1 - (1-e -D/D0)n) = -d/dD(1-e -D/D0)n


*Survival Curves; Modifiersp. * of 96MTSH slope, continuedRecalling that dun/dx = nun-1du/dx, for n>1,Slope = -n(1-e -D/D0)n-1 d/dD (1-e -D/D0) = -n(1-e -D/D0)n-1 [-d/dD(e -D/D0 )]But we know deu/dx = eu du/dx, so d/dD(e -D/D0 ) = e -D/D0 (-1/D0) = -1/D0 e -D/D0Therefore Slope = -n(1-e -D/D0)n-1 (-)(-1/D0) e -D/D0i.e. Slope = (-n/D0)(1-e -D/D0)n-1e -D/D0


*Survival Curves; Modifiersp. * of 96Slope at D D0For small D, i.e. for D 1 this is = (-n/D0)(1-1)n-11 = 0. Shazam.


*Survival Curves; Modifiersp. * of 96Slope of ln(S/S0) vs DThe behavior of the slope of the ln(S/S0) vs D curve is not much harder to determine.Recall d lnu /dx = (1/u)du/dx. We apply this here:d ln(S/S0) / dD = (1/(S/S0)) d(S/S0)/dD.For very small D, S/S0 = 1, so d ln(S/S0) / dD = (1/1) * d(S/S0)/dD = d(S/S0)/dD .But weve just shown that that derivative is zero, so d ln(S/S0) / dD = 0.


*Survival Curves; Modifiersp. * of 96High-dose caseWeve covered the low-dose case.What happens at high dose, i.e. D >> D0?What wed like to show is that the slope of lnS/S0 vs. D is -1/D0. Lets see if we can do that.Slope = d ln(S/S0) / dD = (1/(S/S0) d/dD(S/S0)Thus slope = (1 - (1-e -D/D0)n-1)d/dD(1 - (1-e -D/D0)n)For D >> D0, D/D0 is large and -D/D0 is a large negative number; therefore e-D/D0 is close to zero.


*Survival Curves; Modifiersp. * of 96High-dose case, continuedSlope = limD {(1 - (1-e -D/D0)n)-1(-n/D0)(1-0)n-1e-D/D0)}Thats messy because the denominator and the numerator both go to zero. There are ways to do that using LHpitals rule, but there are simpler ways that dont involve limits.The trick is to recognize that we can do a binomial expansionIve done that in the HTML notesThe result will be slope = -1/D0 and ln n is the Y intercept of the extrapolated curve


*Survival Curves; Modifiersp. * of 96What constitutes a high dose here?The only scaling of the dose that occurs in the formula is the value of D0, so we would expect that we are in that high-dose regime provided that D >> D0.In practice the approximation that the slope is -1/D0 is valid if D > 5 D0.


*Survival Curves; Modifiersp. * of 96Linear-Quadratic ModelThis is simpler. ln(S/S0) = aD + bD2Therefore slope = d/dD (ln(S/S0)) = d/dD(aD + bD2)Thus slope = a + 2bD. Thats a pretty simple form.At low dose, |a| >> 2|b|D, so slope = a.At high dose (what does that mean?) |a| |a / (2b|Thus if dose >> |a / 2b|, then slope = 2bD.


*Survival Curves; Modifiersp. * of 96Implications of this modelAt low dose slope = a is independent of dose but is nonzero; thus ln(S/S0) is roughly linear with dose.At high dose slope = 2bD, i.e. its roughly quadratic.How can we represent this easily? We discussed this last time: ln(S/S0) / D = a + bD,so by plotting ln(S/S0) / D versus D, we can get a simple linear relationship.


*Survival Curves; Modifiersp. * of 96Y-intercept = aSlope = bln(S/S0)/DDLQ: Plot of ln(S/S0) / D versus DBy observation, both a and b are negative. a < 0 tells us that radiation harms cells; b < 0 is an observed fact. Thus the intercept is below the axis and the slope is negative.


*Survival Curves; Modifiersp. * of 96LQ graphical analysis: one step furtherWe can read -a/b directly off an extrapolation of the plot: at D = Dz, ln(S/S0)/D = 0, a + bDz = 0, a = -bDz,Dz = -a / b. Note a < 0, b < 0, so -a/b < 0.Y-intercept = aSlope = bln(S/S0)/DDDz


*Survival Curves; Modifiersp. * of 96Where do linear and quadratic responses become equal?At what dose does the linear response equal the quadratic response?At that dose, aD = bD2, D = a/bSo the value we read off the X-intercept of the previous curve is simply the opposite of the dose value at which the two influences are equal.We mentioned this last time, but were reminding you now


*Survival Curves; Modifiersp. * of 96How plausible is all this?Model studies suggest reasons to think that ln(S/S0) = aD + bD2 is a good approach.Much experimental data are consistent with the modelSome of these LQ approaches allow for time-dependence to be built in.


*Survival Curves; Modifiersp. * of 96LQ vs MTSH and thresholdsHow does the question of comparing the LQ model to the MTSH model relate to the question of threshold doses?A typical real-world question is a dose-response relationship for which the only reliable experimental results are obtained at high doses; at lower doses the confounding variables render the experiments uninformative.DoseResponse


*Survival Curves; Modifiersp. * of 96Dose-Response in Epidemiology#2 is the linear non-threshold (LNT) modelIs #3 more realistic?This has regulatory consequences!Human health effectBaselineDoseExperimental data#3#2#1ThresholdTolerable dose #3Tolerable dose #2Tolerable dose #1Limit of reliable measurementExtrapolations


*Survival Curves; Modifiersp. * of 96Studying repairWeve been suggesting that LQ models and even some MTSH models are dependent on the idea that some DNA damage can be repaired accurately.Lets look for approaches to studying DNA damage that might provide a fuller understanding of the effects of repair.


*Survival Curves; Modifiersp. * of 96The Elkind-Sutton ExperimentProvides a way of probing repair functions in cellsProcedure:Irradiate and establish survival curve (conditioning dose)Take cells surviving at S=0.1 and subject them to further irradiation at varying time intervals after reaching S=0.1If repair is taking place, then the appearance of a curve similar to the original shoulder is indicative of full recovery


*Survival Curves; Modifiersp. * of 96Results


*Survival Curves; Modifiersp. * of 96InterpretationIf slope and implied n value are equivalent to the original curve, then repair is completeSmaller n values indicate insufficient time has elapsedn=1 implies repair has not begun


*Survival Curves; Modifiersp. * of 96Elkind-Sutton and LETWe might expect more complicated results if we vary the LET for the two dosing regimensLow-LET first, high-LET second gives two lines of different slope, independent of the time intervalHigh-LET first, low-LET second gives line followed by usual Elkind-Sutton distribution


*Survival Curves; Modifiersp. * of 96Low-LET followed by High-LET


*Survival Curves; Modifiersp. * of 96High-LET, then Low-LET


*Survival Curves; Modifiersp. * of 96The Cell CycleCells have a definite cycle over which specific activities occur.Particular activities are limited to specific parts of the cycleHoward and Pelc (1953) characterized four specific phases:M (mitosis, i.e cell division)G1 (growth prior to DNA replication)S (synthesis, i.e DNA replication)G2 (preparation for mitosis)Mitosis (M)Presynthetic (G1)Synthesis (S)Post-synthetic (G2)


*Survival Curves; Modifiersp. * of 96What happens in S phase?DNA is replicated; thus, we have twice as much DNA at the end of S as at the beginning.During mitosis the two duplexes of DNA can separateOne goes to one daughter cell, the other to the other


*Survival Curves; Modifiersp. * of 96How much time do these segments take?Depends on the overall mitotic rate and the type of cell:Cell Cycle Times in hours:SegmentCHOHeLaM11G1111S68G234------------------------------------------TOTAL1124


*Survival Curves; Modifiersp. * of 96Pie charts of cycle percentagesThe point is that different kinds of cells spend differing amounts of time in the various phases


*Survival Curves; Modifiersp. * of 96Phase SensitivityMany cells are much more sensitive to radiation in some parts of the cell cycle than they are in others.Why?Repair is more vigorous in some stagesUnrepaired damage has more opportunity to manifest itself as clonal alteration close to mitosisAccess of repair enzymes to damaged DNA is sometimes influenced by how organized the DNA is.


*Survival Curves; Modifiersp. * of 96What phases are sensitive?In general, cells are radioresistant when they are synthesizing DNA.Cells that are synthesizing DNA are taking up label; the time that theyre doing that is correlated with survival:% of labeled cellsSurviving Fraction% of labeled cellsSurviving Fraction0.490Time in Hours024


*Survival Curves; Modifiersp. * of 96Survival Curves in Various PhasesSee fig. 8.8:Late S is least radiosensitiveEarly S next leastG-1 somewhat sensitiveG-2 and M most radiosensitiveM and G2 curves are essentially straight lines (log-linear dose-response), suggesting that repair is unavailable or of little influence


*Survival Curves; Modifiersp. * of 96Figure 8.8, reimagined with LQ model


fig8.8

0000

-0.3257208614-0.1824036824-0.1224710439-0.0173717793

-0.6514417229-0.3821791441-0.264051045-0.0555896937

-0.9771625843-0.599326385-0.4247400033-0.1146537432

-1.3028834457-0.8338454053-0.6045379188-0.1945639279

-1.6286043071-1.0857362048-0.8034447915-0.2953202477

-1.9543251686-1.3549987835-1.0214606214-0.4169227026

-2.28004603-1.6416331416-1.2585854086-0.5593712927

-2.6057668914-1.9456392789-1.5148191529-0.7226660179

-2.9314877528-2.2670171955-1.7901618544-0.9068068782

-3.2572086143-2.6057668914-2.0846135131-1.1117938737

-3.5829294757-2.9618883666-2.3981741291-1.3376270043

-3.9086503371-3.335381621-2.7308437022-1.58430627

M and G2:a=0.75, b=0

G-1:a=0.4, b=0.02

Early S:a=0.26, b=0.022

Late S:a=0.16, b=0.024

M and G2

G1

Early S

Late S

Dose, Gy

log10 of survival fraction

CHO V79 survival curves (fig. 8.8)

Sheet1

DoseM and G2G1Early SLate S

00000

1-0.3257208614-0.1824036824-0.1224710439-0.0173717793

2-0.6514417229-0.3821791441-0.264051045-0.0555896937

3-0.9771625843-0.599326385-0.4247400033-0.1146537432

4-1.3028834457-0.8338454053-0.6045379188-0.1945639279

5-1.6286043071-1.0857362048-0.8034447915-0.2953202477

6-1.9543251686-1.3549987835-1.0214606214-0.4169227026

7-2.28004603-1.6416331416-1.2585854086-0.5593712927

8-2.6057668914-1.9456392789-1.5148191529-0.7226660179

9-2.9314877528-2.2670171955-1.7901618544-0.9068068782

10-3.2572086143-2.6057668914-2.0846135131-1.1117938737

11-3.5829294757-2.9618883666-2.3981741291-1.3376270043

12-3.9086503371-3.335381621-2.7308437022-1.58430627

alpha0.750.40.260.016

beta00.020.0220.024

Sheet2

Sheet3

*Survival Curves; Modifiersp. * of 96Radiation-InducedCell Progression DelayNote that various biochemical signals regulate progression from one phase of the cycle to another.To study this, you need synchronized cells . . .Sample study (Leeper, 1973):CHO cells exposed to 1.5 Gy in mid-G1 experienced a delay of 0.5 h in cell division1.5 Gy in late S or early G2 caused a delay of 2-3 hDose-dependent: (4h for 3Gy, 6-7h for 6 Gy)


*Survival Curves; Modifiersp. * of 96Shape of the culture matters!How the cells grow influences how much the cells progression is altered by radiationMonolayers progression is altered less than cells in a multicellular spheroid geometry0.22 hours per Gray0.53 hours per Gray


*Survival Curves; Modifiersp. * of 96Is that such a big deal?Probably not:The cells in the spheroidal mass divide half as fast even in the absence of radiation, possibly due to contact inhibition.Therefore it may simply be that the whole mitotic clock has been slowed down, including the clock as its been influenced by radiation.


*Survival Curves; Modifiersp. * of 96Causes for these effectsWhy are cells more radiosensitive in M and G2?Reduced availability of repair enzymesRepackaged DNA is hard to repairHow is cell progression influenced by radiation?Damage to protein kinases and cyclins involved in cellular checkpointsPremature degradation of p21, maybeSample 1994 study: Edgar et al, Genes Dev. 440: 52 (1994)


*Survival Curves; Modifiersp. * of 96Effectors of Radiation SensitivityBiologicalCells go through life cycles & are much more sensitive to radiation damage at some stages than at othersChemicalPhysical


*Survival Curves; Modifiersp. * of 96Assignment related to amino acids1. There are exactly twenty amino acids that serve as the building blocks for proteins in almost all organisms. The general formula for 19 of these 20 amino acids is +NH3-CHR-COO-, where R is any of 19 different side-chain groups. The simplest of these R groups is H, for which the amino acid is called glycine; the most complicated is a moiety known as indole, for which the amino acid is called tryptophan.


*Survival Curves; Modifiersp. * of 96Upcoming Problem 1, contdMuch of the chemistry that these R groups participate in in proteins is ionic in nature, involving charges or partial charges; but usually these ionic interactions involve pairs of electrons rather than unpaired electrons. An exception is the amino acid tyrosine, which can participate in free-radical (unpaired-electron) interactions. Draw a structure of the tyrosyl free radical and explain why it might have a reasonably long lifetime, as compared to a hydroxyl radical or some other short-lived free radical.


*Survival Curves; Modifiersp. * of 96Second upcoming problem2. Most biochemical oxidation-reduction reactions involve transfers of pairs of electrons and therefore do not involve free radical mechanisms. A sizeable minority, however, do involve free radicals. Which of the following biochemical oxidizing or reducing agents are capable of participating in single-electron (free-radical) reactions, and which are not? Explain briefly. You may need to look up the structures of some of these compounds. (a) ferric iron, Fe3+ (b) nicotinamide adenine dinucleotide, oxidized form (NAD) (c) flavonamide mononucleotide (FMN) (also spelled flavin amide mononucleotide; its an instance of what are generally known as flavin prosthetic groups)


*Survival Curves; Modifiersp. * of 96ErrataPage 205, in the EXAMPLE: 4.0 M l-1 should be 4.0 M, or 4.0 mol l-1Page 206, last sentence: If the lesions produced by high LET radiation are predominantly of type II (irrepairable), then m-1 will be disappearingly small and no oxygen sensitization will be detectable.Page 213, last paragraph: cysteine, not crysteine


*Survival Curves; Modifiersp. * of 96Cellular Life Cycles (review)PhasesMitoticM(short)SensitivePresyntheticG1(variable)Radiation causes 1/2 h delay hereSyntheticS(4 - 8 h)DNA synthesisLeast sensitivePostsyntheticG2(usually short 1 - 2h)Radiation causes 3 - 4 h delay hereEnd of G2 sensitive__________Overall Process14 h


*Survival Curves; Modifiersp. * of 96What happens in G1?Routine cellular metabolismBoth buildup of new cellular structures and gathering energy to do so, i.e. both catabolism and anabolism:Metabolism as a whole consists of:CatabolismAnabolismEnergy-producingEnergy-requiringBreakdown of complexBuild-up of complex molecules into simplermolecules from simpler ones, producing ATPprecursors, using ATP


*Survival Curves; Modifiersp. * of 96A mitotic cellNucleus (DNA location; cellular organization)Mitochondrion (energy metabolism)Ribosomes (protein synthesis)Mitotic SpindleOther organelles


*Survival Curves; Modifiersp. * of 96Timescales of damage (fig. 4.1)Physics (~10-16 s)Primary interaction event with biomolecule or H2OExcitations & ionizationsFast Chemistry (10-15 - 10-7 s)Water radicals and other quasi-stable species formReactive species diffuse and cause damageSlower chemistry (10-7 to 10-3 s)Further damage via diffusionChemical restitution & repairBiochemistry (msec-min): enzymatic repair of damageBiology (hrs-days): biological repopulation from surviving cells


*Survival Curves; Modifiersp. * of 96Physical & Chemical EffectorsWaterfree radicalshn + H2OOH, HO2-, etcionized speciesPure water


*Survival Curves; Modifiersp. * of 96How does water matter?In a dry setting, the damage must be directIn a wet environment, water-derived free radicals and ions are the source of much of the chemical damageExperiments that can eliminate secondary (radical-mediated) damage show much reduced radiosensitivity


*Survival Curves; Modifiersp. * of 96An example from my fieldProtein crystals must be irradiated wet because they fall apart when theyre dry.Protein crystals irradiated at 300K are destroyed:In days on a conventional X-ray sourceIn minutes on a 2nd-generation synchrotronIn seconds on a 3rd-generation synchrotronTheyre essentially immortal at 110K except on a 3rd-generation synchrotron source, where they live for 5-100 minutes


*Survival Curves; Modifiersp. * of 96Radiosensitivity of EnzymesMost enzymes are more radiosensitive in the presence of substantial hydrations than when driedData cant readily be measured below ~4% hydrationBut those arent very physical anyway (except maybe with integral membrane proteins)


*Survival Curves; Modifiersp. * of 96Why does this work this way?Normal responseArylesterase: less water means less damageDry conditions mean that radiosensitivity depends entirely on direct, not indirect damageAbnormal response: Cholinesteraseharder to explainAugustinsson suggests: at low [H2O], free radicals are detoxified by nonfunctional sulfhydryls in the proteinAt higher water concentrations the sulfhydryls cant get to the damage in time


*Survival Curves; Modifiersp. * of 96Do we care?Not much:We cant really alter the hydration of most cell systems without destroying themWe definitely cant influence the hydration states of whole organisms without doing damage that is probably much more significant than that of the radiationBut these studies may help us understand mechanisms of damage, and that could be relevant


*Survival Curves; Modifiersp. * of 96Do we care, vol. IIDNA and RNA are heavily hydrated because theyre charged and because they tend to associate directly with solvent or with soluble proteins like histonesProteins vary a lot in their hydration:Soluble proteins are heavily hydratedIntegral membrane proteins have very little water around themIs there a difference in radiosensitivity?Not much research that I could findOne instance: Takts et al. (1993) J.Rad.Res. 34:141


*Survival Curves; Modifiersp. * of 96Temperature-sensitivityDirect damage will be essentially temperature-independentIndirect damage should be temperature dependent because it relies on diffusion of radicals and ions from the site of their production to the macromoleculeIonizations and excitations may display different temperature dependencies because once the molecule is excited, its chemistry may depend on thermal interactions


*Survival Curves; Modifiersp. * of 96Arrhenius plotsWe can examine temperature dependence via the Arrhenius plot, wherein we expect k = Qexp(-G/RT)Thus ln k = ln[Qexp(-G/RT)] = lnQ - G/RT, so if we plot ln(k) as a function of 1/T then the relationship should be linear, and the slope will be -G/R, i.e it will be proportional to the activation energy G.If multiple processes are involved we may get a non-log-linear response.Over temperature ranges typical of the internals of homeothermic organisms, were not going to see much effect!


*Survival Curves; Modifiersp. * of 96Physical meaning of GChemical reactions are characterized by an activation energy barrier, i.e., an energy that must be put into the system in order to get from reactant to product or vice versa.G is the height of that activation barrier.EnergyReaction coordinateReactantProductG


*Survival Curves; Modifiersp. * of 96Radiation & TemperatureKinetics

37oC = 310K27oC = 300Kp199 T < 100K 100 < T < 170 K 170 < T < 420 K


*Survival Curves; Modifiersp. * of 96Radiation and Temperature Effects in various temperature ranges:TminTmaxEffects0K100Ktemperature-insensitive; no charge migration, target must be hit.100K170Kexcitation localized at site; exciton migration is crucial170K420Kdisruption of disulfides, ionization Does this matter much with biological systems (esp. in homeotherms), where T ~ 310K almost all the time? Probably not, other than for understanding mechanisms.


*Survival Curves; Modifiersp. * of 96Oxygen and RadiationFACT: O2 is a radiation-sensitive molecule e.g. O2 + e- O2- (superoxide) interactions with macromoleculesIts tempting to think that all biology occurs in the presence of 19-21% O2, but it doesnt!H2O free-radical chemistry in the presence of O2 is different from H2O free radical chemistry in the absence of O2. (recall Fricke dosimetry story)P(O2) in tissue varies widelyHemoglobin transports O2Myoglobin stores O2


*Survival Curves; Modifiersp. * of 96Damage Fixation by OxygenR + O2 RO2semi stable10-7 - 10-3 sec?________________________________________Mitigators of O2 fixationradical presenceR + RSH R-H + R-SR-S + R-S R-S-S-R(dimerization)


*Survival Curves; Modifiersp. * of 96Minor syntactic pointBeware of the word fixationIt doesnt mean correction: it refers to stabilizationStabilizing a radical doesnt make it less dangerous; it makes it more dangerous!So if we say that oxygen is involved in fixation of damage, we mean that it makes the damage worse, not better!


*Survival Curves; Modifiersp. * of 96 Experiments on Oxygen SensitivityMore cells are killed in air than in N2:Survival in the absence of oxygenSurvival in the presence of oxygen


*Survival Curves; Modifiersp. * of 96LQ: oxic vs. anoxic


*Survival Curves; Modifiersp. * of 96Radiosensitivity as function of P(O2)Shigella experiments show threshold in P(O2) below which few cells are killed Alpen, fig. 9.3


*Survival Curves; Modifiersp. * of 96Oxygen Enhancement RatioOER [dose in N2 for surviving fraction S/So] /_____________________________dose in O2 for surviving fraction S/So

if S = So/10, then OER = DN / DO This definition is somewhat arbitrary, but it works!


*Survival Curves; Modifiersp. * of 96Quantitative Oxygen SensitivityPaul Howard-Flanders and Tikvah Alper: Define S/SN to be the ratio of the 10% survival dose under experimental conditions to the 10% survival dose in N2, i.e. in the absence of oxygenS/SN = (m[O2] + K) / ([O2] + K)for any concentration of oxygen.m and K are separately determined for each systemm is dimensionless, K is a concentrationm represents maximum relative sensitivity so m 1.


*Response Modifiers; Tumorsp. * of 96Extrema of S/SNAt [O2] = 0, S/SN = (m*0 + K) / (0 + K) = 1For [O2] >> K, S/SN = (m[O2] + K) / ([O2] + K) = m[O2] / [O2] = mThus justifying description of m as maximum relative sensitivity

Response Modifiers; Tumors

*Response Modifiers; Tumorsp. * of 96How to compute m and Km is available from asymptotic behaviorIts equal to S/SN for [O2] >> K.In practice most systems are equally radiosensitive from about 1% [O2] on upTo compute K, note that if [O2] = K, then SK/SN = (mK + K) / (K+K) = (m+1)/2Therefore if we know m from asymptotic behavior, we can examine a curve like 9.3 to find the point where S/SN = (m+1)/2 and read off K from the abscissa


*Response Modifiers; Tumorsp. * of 96Howard-Flanders CoefficientsCoefficients m and K for various organisms:Organismm K, MShigella flexneri Y6R2.94.0 Escherichia coli B/r3.14.7 Saccharomyces cerevisiae2.45.8 Can be read off curves like fig. 9.3


*Response Modifiers; Tumorsp. * of 96Curve-fits for Organismal DataThese are idealizations, of course!Note that 11.3 M = 1%


*Response Modifiers; Tumorsp. * of 96Why does this happen?Alpers model: 2 types of damage from primary radiation eventType I: lesion requires oxygen for lethalityType II: always lethal, independent of oxygenThus: type I can be chemically restitutedRestitution competes with oxygen fixationPosit: n1 type I lesions, n2 type II lesions, krep = rate of repair, kfix = rate of O2 fixation, then:m-1 = n1/n2 and K = krep/kfix


*Response Modifiers; Tumorsp. * of 96What happens with high-LET?Essentially all damage is type 2Nothing depends on restitutionTherefore n1/n2

*Response Modifiers; Tumorsp. * of 96Time-DependenceStudy the kinetics via short bursts of radiationWe look to see whether providing O2 at a certain time-point before or after irradiation sensitizes cellsOxygen sensitizes the cell if present before irradiationIf available till ~3 msec after irradiation, it still mattersIf oxygen is made available later than a few msec post-irradiation, it doesnt sensitize the cellThis suggests that the oxygen-dependent free radicals have lifetimes shorter than 3 msec.


*Response Modifiers; Tumorsp. * of 96Michaels results with SerratiaTime constant ~ 0.5 msec for sensitizationDose: 280 Gy of electrons


*Response Modifiers; Tumorsp. * of 96What radicals are involved?The short time-span shown in this experiment suggests that free radicals are involved: but which ones?Michael suggests that superoxide (O2-), hydroperoxyl (H-O=O), and eaq- have shorter lifetimes than is consistent with 0.5 msec timingOther researchers continue to plug for superoxide and hydroperoxyl as candidatesOH doesnt interact with O2 that much except via CO2-: OH + HCO2- -> CO2- + H2O CO2- + O2 -> CO2 + O2-


*Response Modifiers; Tumorsp. * of 96SuperoxideWere still not sure if superoxide is a major actor in oxygen-mediated damageBut this is how superoxide is detoxified by superoxide dismutase (SOD):2 O2- + 2H+ H2O2 + O2 (catalyzed by SOD)H2O2 H2O + (1/2)O2 (catalyzed by catalase)Exogenous SOD doesnt help alleviate damage (but maybe thats because it isnt tied to catalase, so peroxide can build up to toxic levels?)Manipulating constitutive levels of SOD by genetic means gives muddy resultsHuman Cu-Zn SOD


*Response Modifiers; Tumorsp. * of 96Thiol MitigatorsWe expect that oxygen competes with thiols, such that the more thiol mitigation is involved, the less oxygen-dependent fixation of damage can occur:1. [macro]-R + RSH [macro]-R-H + RS2RS R-S-S-R

2. [small] + RSH [small]-H + RS2RS R-S-S-R


*Response Modifiers; Tumorsp. * of 96Glutathione as mediatorGlutathione can readily dimerize under oxidizing conditions: R-SH + HS-R R-S-S-HReasonably prevalent in cells (millimolar: [glut] >> [cys])In principle cysteine could also operate this way, but its cellular concentration is too lowCysteineglutathione


*Response Modifiers; Tumorsp. * of 96Are thiols really important?Some argument about thatReveszs results support the competition modelMaybe only exogenous thiols really influence radiosensitivityIntroducing cysteine or synthetic thiols does definitely mitigate damage: RSH + OH RS + H2O RS- + OH RS + OH-. . . And then these RS radicals recombine as disulfides


*Response Modifiers; Tumorsp. * of 96Dose reduction factorsWe envision the effects of these exogenous thiols as involving dose reductionThe quantitation is equivalent to reducing the available dose to influence the biological systemExample, WR2721 or amifostine:Works well in vitroLimited utility in vivoFairly high toxicity


*Response Modifiers; Tumorsp. * of 96Sensitization by NitroaromaticsHow do we make the cells in the rapidly growing tumor as rad-sensitive as they would be if PO2 were higher?Nitroaromatics react with radicals to fix (stabilize) the damage metronidazolemisonidazole


*Response Modifiers; Tumorsp. * of 96Nitroaromatic actionLike oxygen, nitroaromatics react with short-lived radicals to produce longer-lived and therefore more reactive radicalsConcentrations required to enhance radiosensitivity are much higher than with O2These compounds have other applications, but they can be used therapeutically as potentiators of radiation damage


*Response Modifiers; Tumorsp. * of 96Oxygen vs. Mizonidazole (fig. 9.5)oxygenmisonidazole


*Response Modifiers; Tumorsp. * of 965-Halogen-substituted pyrimidinesThese are molecules that resemble thymineThe halogen at the 5- position looks like the methyl group in thymine and can be incorporated in place of thymine in DNAMost common: 5-bromodeoxyuridineSensitization produced by ready reaction with the aqueous electron


*Response Modifiers; Tumorsp. * of 96The 5-BrdU radicalSemi-stable radical is actually resident in the DNA and can influence chemistry in neighboring basesOther mechanisms are probably acting too.NHNROBr+ eaq- NHNROBr-NHNRO+ Br-


*Response Modifiers; Tumorsp. * of 96Cell DeathClonogenic cell death: inability to produce several generations worth of progenyAcute pathological cell death: necrosisCells typically swell, then lyseAccompanied by inflammationApoptosisProgrammed cell deathShrinkage, fragmentation, phagocytosisp53 is activator of genes that regulate it


*Response Modifiers; Tumorsp. * of 96Gilbert & Lajthas cell typesABCDEFSee next slide for explanation!


*Response Modifiers; Tumorsp. * of 96Cell populations in TissueA. Simple transit populationCells in, cells outSpermatozoa, blood cellsB. Decaying population (e.g. oocytes)C. Closed, static population (neurons?)

D. Dividing, transit populationSome cell division, so more leave than enterDifferentiating blood cellsE. Stem cell population (many kinds)F. Closed, dividing populationNo cells in or out just a lot of divisionTumors, eye-lens epithelial cells


*Response Modifiers; Tumorsp. * of 96Cell population kineticsCell types that divide are the most sensitive.Cells are most sensitive during G2 and M, so cells that spend a lot of time in G2 and M are more sensitiveIf a cell population is exposed to radiation, the outcome depends on there being an adequate number of (clonogenically) surviving cells.


*Response Modifiers; Tumorsp. * of 96Growth FractionLajtha (1963): described G0 phase in cell cycle: cell is not engaged in proliferation but could later re-enter proliferative stageGrowth fraction is defined as fraction of total cellular population that is clonogenically competent and actually in the process of DNA replication and cell division.Measurement: uses 3H-thymidine uptakeSignificance: cells in G0 have time to repair DNA damageWorks even if [repair enzymes] is low during G0This is suspected but not proven


*Response Modifiers; Tumorsp. * of 96The expanded cell cycle (Lajtha)G0 is seen as an alternative to normal cyclingCells may re-enter the cycle after a change in environmental conditions or upon receiving a signalMG1SG2G0


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10/28/2015 survival curves; modifiers p. 1 of 96 illinois institute of technology physics 561...

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