Cell Survival Curves

Cell death

A cell that is able to proli f erate indefin itel y and form a large colony from a single cell is said to be clonogenic .

Tu m o r cells can be grown indefinitely in cell culture.

Normal cells must be transformed to grow indefinitely in culture.

For cells growing in culture, the loss of th e ability to continue growth is term ed

reproduct i ve death .

Following irradiation, cells may still be intact and able to produce proteins, synthesize new DNA and even go through one or two cell divisions. But if it has lost the capability to reproduce indefinitely , it is considered dead.

Very high radiation doses (10,000 rads or 100 Gy) can cause the breakdown of all cellular functions.

In contrast, the mean lethal dose for loss of reproductive capability is usually less than 2 Gy.

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Clonogenic Survival Assay :

 Cells from an actively growing stock ar e harvested by gentle scraping or by the use of trypsin.

 The num b er of cells per unit vo lume is determ ined manually (hem ocytometer) or electronically (Coulter Counter).

 Known num bers of cells can then be plat ed into fresh dishes. If allowed t o incubate for 1-2 weeks, clonogenic ce lls will form macroscopically visible colonies that can be fixe d, stained and counted.

 Not every cell seeded will form a colony, even in the absence of irradiation, due to factors such as errors in coun ting, stress of manipulation, subopt i m al growth m e dium , etc. The plating efficiency (PE) is defined as the num ber of colonie s observed/ the num ber of cells plated.

 PE =

colonies number of

observed cells plated

 Parallel dishes are seeded with cells that have been exposed to increasing doses of radiation. The num ber of ce lls plated is increased so t h at a countable num ber of colonies results. Surviving fraction (SF ) is the colonies counted divided by the num b er of colonie s plated with a correction for the plating efficiency.

 SF =

colonies

counted

cells seeded

x ( PE / 100 )

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Cell survival curves

Cell survival data ar e gene rally plotted as logarithm of the surviving fraction versus dose.

For com p aring curve s , it is convenient to represent them math ematically, based on hypothetical m odels for the me chanism s behind lethality.

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The interpretation of the sh ape of the cell survival curve is still debated, as is the b e st way to fit these types of data mathematically.

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Radiation sensitivity and the cell cycle

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Example of cell cycle times

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Target T h eory

Target theory origin ated from work with exponenti al dose response curves .

It was assumed that each “hit” results in an inactivation, i.e., a “single-hit, single- target m odel”.

 Each cell has a single target.

 Inactivation of the target kills the cell.

Linear Su rvival Curves

Irradiation of cells with high-LET r a diati on produces linear s u rv ival curves. The relationshi p between the survivi ng fraction S and the dose D is t h en:

S  e   D

where:

S is the num b e r of surviving cells, – α is the slope,and

D is the radiation dose delivered.

This relationshi p is m o re comm only represented as by defining D 0 as 1/ α .

S  e  D / D 0

When D = D 0 ,

S  e  1  0 . 37

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Poisson Distribution

All calculations of hit probability are governed by Poisson statistics, where the probabil i t y of n events is give n by

( e  x )( x n )

P ( n ) 

n !

where x = the averag e nu mber of events and n = the specific num ber of events

If each “hit” is assumed to res u lt in ce ll inactivation, then the probability of survival is the probability of not being hit, P(0).

From the Poisson relationshi p, where x = 1, and n = 0,

P ( 0 ) 

( e  1  1 0 ) 0 !

 e  1

 37 %

For this re ason, D 0 is often called the mean lethal dose , or the dose that delivers, on average, one letha l event per target .

Exponenti a l dose response relationships are found in certain situations

 Certain types of sensitive cells (e.g., haem opoietic stem cells)

 Synchroni zed populations in M and G 2

 Irradiation with high-LET radiation

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Cell Survival Curves with Shoulders

Survi v al curves for m o st mammalian cells exposed to low - LET radiation show some cu rvature .

The initial low dose region in which ther e is less cell inactivation per unit dose than at high doses is called the shoulder .

Often the higher-dose region tends towards a straight line.

The par a meter D 0 can then be used to characterize the radios ensitivity in the linear (high dose) region of the curve.

Extrapolation of the term inal straight line porti on of the curve b ack to the abscissa defines a value, n , the extrapolation number .

In the shoulder region of the curve th e proporti on of the cells killed by a given dose increases with the dose alr eady give n. Two interpretations are possible:

 Cell death results from the accumulation of events that are indivi duall y incapable of killing the cell, but which beco m e lethal when ad ded together (target m odels).

 Lesions are individually repairable but beco me irrepairable and kill the cell if the efficiency of the en z y m a tic repair mech anisms di min i shes with nu m ber of lesions and therefor e the dose (repair m odels).

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Linear-Quadratic Model (two component model):

The linear quadratic model ha s evolved from t w o simil a r formulations, each with roots in target theory.

Theory of Dual Radiation Act i on

 Lesions responsible for cell inactiva tion result from the interaction of sublesions.

 At least two sublesions are req u ired for cell inactivation.

 Sublesions can be produced by the passa ge of one or two radiation tracks.

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Molecular Theory of Cell Inactivati o n

(Chadwick and Leenhouts, 1981)

 Cell inactivation results from unr epaired DNA double-strand breaks.

 At low-LET, a dsb can result from e ither a single event (linear com ponent) or two separate events (quadrati c com ponent).

 Alternatively, cell inactivation re sults from chrom o some aberrations.

 Some ab errations are produced by a single event.

 Som e aberrations are produced by two separate breaks.

Observations of chrom o som e dam a ge led to the assum p tion th at since DNA has 2 strands, it m u st tak e two events to break a strand.

Linear-quadratic model

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The linear quadratic m odel assumes that a cell can be killed in two ways.

 Single lethal event

 Accum u lation of sublethal events

If these modes of cel l death ar e assumed to be independent,

S = S 1 S 2

Where S 1 is the single event

killing or

e   D

And S 2 is the tw o ev ent k illing which can be represented as

e   D 2

The m o st comm on expression i s

S  e  (  D   D 2 )

S is the fraction of cells survivi ng a dose D and  and  are constants.

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Linear-quadratic model

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Useful parameters from linear qua dratic cell survival curves:

 D 1 , the initial slope , due to single event killing, the dose to reduce survival to 37%

 D 0 , the final slope , interpreted as m u ltiple-event killing, the dose to reduce survi v al by 67% from any point on the linear portion of the curve.

 some quantity to describe the w i dth of the shoulder . The extrapolation of the final slope D 0 , b a ck to the y axis yields n, the extrapolation number . The larger the value of n, the larg er the shoulder on t h e surviva l curve.

Other Target Models

Single-hit, multi-tar g et model:

Assum p tions:

 Each cell contains n distinct and identical targets.

 Each target can be inactivated b y the passage of a charged particle (a hit).

 Inactivation of a target is a sublethal event.

 All n targets m u st be inactivated to kill the cell.

 For a dose D 0 there i s on average one hit per target .

Therefore, for a dose D 0 , the probability that a target is undamaged is

e  D / D 0

If the probability that a target survives is the target is hit is:

P ( h )  1  e  D / D 0

S  e  D / D 0

, then the probability that

and the probability that all n targets are h it is

P ( h )  ( 1  e  D / D 0 ) n

Therefore the probability that all targets w ill not be hit, i.e., the probability of survival, is:

S  1  ( 1  e  D / D 0 ) n

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[Alpen, 1990]

As D   , S  ne  D / D 0 , the linear portion of this cu rve thus yields a slope of –

1/D 0 , and a y intercept of n (extrapolation num ber).

Thus, in this target theory m odel, the parameters D 0 and n define the radiosensitivity of the sublethal ta rgets and their num b er (n) per cell.

Quadratic model (tw o hits, single target):

Single target

Two independent “hits” are required for inactivation.

The mean number of lethal ev ents is then proportional to the square of the dose.

  D 2

, wher e β is the parameter relating dose to the probability of a lethal

event.

These two m odels (single hit, m u lti ta rget; two hits, single target) are good representations at high doses.

But they predict al most zero slope at lo w doses (underestimat e the effect of low doses).

Models based on Repair

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Linear survival curves are easy to understand

The curvature in the shoulder c ontinues to be “interpreted”

Repair is definitely involved…

Classic split-dose experime nts, Elkind and Sutton, 1959

Tw o doses of low - LET radiation

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Lo w - LET follo w e d by high-LET

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High-LET follow e d by low - LET

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Other Dose Response Assays

Mitotic arrest: cell division delay

 Block of progression of ce lls through the cell cycle

 Cells are usually bloc ked in the G2 phase of the cell cycle ( G 1 also possible)

 The block is usually tem porary, lengt h of block depends on radiation dose.

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Apoptosis: programmed cell death

A distinct m ode of cell death, different from necrosis in term s of m o rphology, biochem i stry and inci dence. Generally considered to be an active process of gene- directed self-destruction.

Normal process for rem oval of cells.

Occurs spontaneously in both norm al tissues and tum o rs.

Endpoints used to measure apoptosis: m i croscopy, TUNEL, flow cytometry, specific staining (caspase, annexin)

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Multiple pathways are involved in apoptosis.

Different pathways may be involved in the same cell type depending on the stim ulus as well as in the response of diff erent cell types to the same stim ulus.

Interruption of norm a l apoptosis path way is involved in many cancers.

Comparision of Features of Apoptosis and Necrosis

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Growth rate as says t o m e asure cell survi val

Exam ples: direct cell counts

Incorporation of radiolabeled biochem i cal substrates (e.g., 3 H-Thd) Tetrazolium (MTT) colorim e tric assay

Quantification of cell growth based on a ssum p tion that only viable cells reduce MTT to a blue form azan product. Am ou nt of product (color) is directly proportional to num ber of viable cells.

Procedure:

Cells plated in m u ltiwell plates, treated w ith radiation (or drugs), allowed to grow for a period of time. Cells are t h en treat ed with MTT, solub ilized and ab sorbance is measured.

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Some Adv a ntages:

 Relatively sim p le and rapid

 Requires small num b er of cells

 May be sem i autom a ted

Som e Disadvantages:

 Direct correlation to clonogenic assa ys requires gre a t attention to experimental details.

 Technical difficulties doing experim e nts in hypoxia

 Only accurate over a 1-1.5 log of response

 Cannot distinguish norm al from tum o r cells in a biopsy.

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Current area of active research: low dose hypersensitivity/adaptive response

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Effects of Radiation on Chromosomes

Chrom o some damage can be:

 m o rphologically visible, e.g., changes in num ber or structure

 or not visible but with func tional consequences: m u tations.

Methods of chrom o some analysis:

 Standard staining after adding an agen t that blocks m itosis in metaphase.

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 Banding techniques: various techniques to stain the chrom o somes produce visible bands.

 Incorporation of [ 3 H]Thd followed by autoradiography.

Techniques to look at chromosome aberrations

Premature ch romosome cond ensation :

 Irradiate cells

 Fuse irradiated cells with m itotic cells

 Factors in the m itotic cells force the chromoso mes of the irradiated interphase cells to condense.

 This visualizes all damage including some that may have been repaired.

 Visualizes severe damage that may ha ve been detected before m itosis and prevented the cell from entering m itosis.

Micronucleus formation

 Chem ically block cytokinesis to yield binucleat ed cells.

 Stain DNA.

 Fragments of chromosom e s with no cen tromere not associated with nuclei are m i cronuclei.

 Score % o f binucleated cells wi th mi cronuclei.

Fluorescence in situ hybridiz ation (FISH):

Uses fluorescently tagged chromoso me p r obes (compli m entary DNA) t o specific chrom o somes or regions of chrom o som e s.

Types of chromosome dama ge observ ed

 Term inal deletions

 Intrachromoso me exchange

 Interchrom osome exchange

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Chromosome aberrations and cell d e ath

 Chromosome aberrat i ons can be detect ed after doses as low as 0.1 Gy.

 Chrom o some aberrations reflect both the initial damage and the repair. (m isrepair), because the chromosom e s are not visible until the cells enter m itosis.

 Doses in the 0.5 – 2 Gy range, prod uce on the average one chr o moso me aberration per cell.

 This dose range is, on the average, the mean lethal d o se for cel ls.

 The frequency of chrom o som e aberrations is a linear quadratic function of radiation dose.

 There are considerable data showing a relationship betw een cell killing and the induction of chro m o some aberrations.

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Historically, these led to the theory of dual radiation action.

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