Wet LabRadiation-Induced Chromosome

Damage and Rejoining

• BackgroundBackground• EquipmentEquipment• SuppliesSupplies• ProceduresProcedures• Troubleshooting and tipsTroubleshooting and tips• Lab DemonstrationsLab Demonstrations

BackgroundBackground

• Types of chromosome Types of chromosome aberrationsaberrations

• Radiation dose response Radiation dose response curvescurves

G1 G2M

Rx

Rx

Background

THE CELL CYCLE

Singlechromatid

Mitosis

G1

S phase

DNA replication

G2

P M A T

Background

Type of cytogenetic damage observed dependents upon where in the cell cycleirradiation occurs

Chromosome AberrationsG1 irradiationBoth sister chromatids involved

Chromatid AberrationsS or G2 irradiationUsually only 1 chromatid involved

Background

Background

Background

Background

Background

Background

Background

Multiple mis-rejoining events occurring in CHO chromosomes after G1 irradiation

tricentric

dicentrics

Background

Mis-rejoining of 2 breaks on one chromatid after G1 irradiation

Centric ring+ fragment

Acentric ringor interstitialdeletion

Background

Ring chromosomeAcentric ring or interstitial deletion

Background

Chromatid deletions in CHO chromosomes after irradiation in S or G2

Chromatid deletion

Iso-chromatid deletion

Background

Chromatid exchanges in CHO chromosomes after irradiation in S or G2

asymmetrical quadra-radial

complex exchange

Background

Background

Chromosomal rearrangement is thebest biomarker for radiation exposure

Sensitive Reliable Large data base

Application of new techniques

Fluorescence In Situ Hybridization (FISH)

Background

From: Dr. J.D. Tucker

Multi-color FISH in human lymphocyte chromosomes

Non-irradiated Irradiated

Background

Aneupolid cell following FISH with probes for human chromosome 2 and human centromeres

From: Dr. J.L. SchwartzBackground

Fate of rearranged chromosomes

1. Deletions Lost at mitosis - micronuclei

2. Exchange-type rearrangements Symmetrical (balanced) gene rearrangement

Asymmetrical (not balanced) fragment usually lost

Polycentric chromosomesbridge-breakage-fusionfail mitosis

cell deathaneuploidy

Background

Yield of radiation-induced chromosome damage

Deletion:

Terminal deletion = 1 hitChromatid deletion = 1 hitInterstitial deletion = 2 hit

Yield (Y) ~ linearY = k +D

k = background = proportionality

Fate: Deletions lost at mitosis

Dose (Gy)

Ab

erra

t io

ns

pe r

10 0

cel

l s

100

200

Chromatidbreaks

Background

2. Exchange-type rearrangements

> 2 hits required; dependent upon:

Space = proximity Time = interaction

P (2 hits) = D x D = D2

Y(yield) = k + D2

3. Total yield

Y = k + D + D2

Background

Low dose dominated by linear componentassume k (background) constant

Y = k + D + D2

Y(0.1 Gy) = 0.1 + (0.1)2 = 0.11Y(0.2 Gy) = 0.2 + (0.2)2 = 0.24

Y(1.0 Gy) = 1.0 + (1.0)2 = 2.0Y(2.0 Gy) = 2.0 + (2.0)2 = 6.0Y(3.0 Gy) = 3.0 + (3.0)2 = 12.0

Y(6.0 Gy) = 6.0 + (6.0)2 = 42.0

Background

Dose Rate EffectsOne hit aberrations - no dose rate effectTwo hit aberrations - dose and fractionation effect

As dose rate aberrations Why?

RepairBreaks rejoined, thusunavailable for furtherinteraction

Dose (Gy)

Dic

e ntr

ics /

Cel

l

1.0

2.0

0

4Gy/hr

0.1 Gy/hr

Background

SUMMARYChromosomal rearrangements observed at first metaphase after irradiation, or after PCCChromosome aberrations- G1 damageChromatid aberrations - S or G2 damageDeletion type aberrations - lost at mitosis

Y = k + DExchange-type aberrations

Dose rate and LET dependentSymmetric - gene rearrangement

complexity revealed by FISHrole in carcinogenesis?

Asymmetric - generally lethalmitotic failure, aneuploidybridge - breakage - fusion cycles

Y = k + D + D2

EquipmentEquipment

• Sterile tissue culture hoodSterile tissue culture hood

• Incubator (COIncubator (CO22, 37, 37ooC, humidified)C, humidified)

• CentrifugeCentrifuge

• Microscopes( upright, inverted)Microscopes( upright, inverted)

• Slide warmer (hot plate)Slide warmer (hot plate)

• Radiation sourceRadiation source

SuppliesSupplies

• Tissue culture flasks (dishes)Tissue culture flasks (dishes)

• Tissue culture mediumTissue culture medium

• Glass slidesGlass slides

• ColchicineColchicine

• Methanol (ethanol), acetic Methanol (ethanol), acetic acidacid

• KCl, sodium citrateKCl, sodium citrate

ProceduresProcedures

• Cell cultureCell culture– Cell growth and division leading Cell growth and division leading

to a good mitotic indexto a good mitotic index• HarvestingHarvesting

– Metaphase by colchicineMetaphase by colchicine– Hypotonic treatments to swell Hypotonic treatments to swell

and weaken the cell membraneand weaken the cell membrane– Fixation: (3:1 methanol:acetic Fixation: (3:1 methanol:acetic

acid) to make cell membrane very acid) to make cell membrane very fragile.fragile.

ProceduresProcedures

• Chromosome spreadingChromosome spreading– Drops of cell suspension are placed Drops of cell suspension are placed

on a slide, and allowed to dry in on a slide, and allowed to dry in a a controlled fashioncontrolled fashion, leading to , leading to chromosome spreadingchromosome spreading

• AgingAging– dry heat and/or ethanol to denature dry heat and/or ethanol to denature

the proteins, remove water/fixative , the proteins, remove water/fixative , and enhance the adherence of the and enhance the adherence of the chromosomes to the glass slidechromosomes to the glass slide

Tissue culture and Tissue culture and harvestingharvesting

• Peripheral blood (whole blood)Peripheral blood (whole blood)• 1640 RPMI with 10% FCS, PHA stimulation 1640 RPMI with 10% FCS, PHA stimulation

for 72 hrsfor 72 hrs• Colchicine for 40 minColchicine for 40 min• Re-suspending in 0.075 M KCl hypotonic Re-suspending in 0.075 M KCl hypotonic

buffer for 15 min at 37buffer for 15 min at 37oo C C• Add fixatives to the cell suspension and Add fixatives to the cell suspension and

wash 3x with fixativeswash 3x with fixatives• Cell pellets were stored at -20Cell pellets were stored at -20ooC in fixativeC in fixative

Procedure

Hypotonic treatmentHypotonic treatment

• Lymphocytes: 0.075 M KCl, 37Lymphocytes: 0.075 M KCl, 37ooC, 12-20 minC, 12-20 min• Fibroblasts: 1:1 0.4% KCl:0.8% sodium Fibroblasts: 1:1 0.4% KCl:0.8% sodium

citrate, 37citrate, 37ooC, 12-20 minC, 12-20 min• Longer time or more hypotonicLonger time or more hypotonic

– Longer, thicker, sticker, less refractive Longer, thicker, sticker, less refractive chromosomechromosome

• Shorter time or less hypotonicShorter time or less hypotonic– Cell membranes and cell/nuclear debris around Cell membranes and cell/nuclear debris around

the chromosomesthe chromosomes

Procedure

Dropping/drying processDropping/drying process

• Cells touch the glass surface and Cells touch the glass surface and become immobile.become immobile.

• Fixative starts drying.Fixative starts drying.• Cells with metaphase chromosomes Cells with metaphase chromosomes

start flattening and spread their start flattening and spread their content (chromosome spreading).content (chromosome spreading).

• As the fixative continues to dry, the As the fixative continues to dry, the nucleated cells continue to flatten nucleated cells continue to flatten slowly.slowly.

Procedure

Dropping/drying processDropping/drying process

• Dropping cells from different height Dropping cells from different height helps distributing the cells more helps distributing the cells more evenly on the slide, but does not evenly on the slide, but does not influence chromosome spreading.influence chromosome spreading.

• Most of chromosome spreading Most of chromosome spreading takes place at the time when the takes place at the time when the fixative evaporates from the fixative evaporates from the spherical surfaces of cells.spherical surfaces of cells.

Procedure

Troubleshooting and tipsTroubleshooting and tips

• FixativesFixatives– 1:1, 3:1, 6:1, methanol:acetic acid1:1, 3:1, 6:1, methanol:acetic acid– 3:1 ethanol:acetic acid3:1 ethanol:acetic acid

• Hypotonic buffersHypotonic buffers

• Drying temperaturesDrying temperatures

• Humidity conditionsHumidity conditions

Metaphases spreadingMetaphases spreading

• Breaking too easily: Breaking too easily: – Fragile cell pelletsFragile cell pellets– Lowering the drying temperatureLowering the drying temperature

• Do not spread well:Do not spread well:– Extension of the drying periodExtension of the drying period– Increasing the humidityIncreasing the humidity

Tips

High speed centrifugationHigh speed centrifugation

• Higher recoveryHigher recovery

• Better cell pellet for droppingBetter cell pellet for dropping

• 6,000-7,000 rpm in 2 ml microfuge 6,000-7,000 rpm in 2 ml microfuge vials with round bottom for 2-5 minvials with round bottom for 2-5 min

• After addition of fixativeAfter addition of fixative

Tips

Glass preparationGlass preparation

• Commercially available pre-cleaned Commercially available pre-cleaned glassglass

• Successive washed in aceton, Successive washed in aceton, HCl/ethanol and triple distilled waterHCl/ethanol and triple distilled water

• Dust or cardboard/paper residue Dust or cardboard/paper residue

Tips

Pre-dropping conditionsPre-dropping conditions

• Dry slides at room temperature.Dry slides at room temperature.• Cold slides (0Cold slides (0ooC) when humidity is low.C) when humidity is low.• Slightly warm slides (37Slightly warm slides (37ooC) when humidity C) when humidity

is high.is high.• Pre-soaking the slides with fixative when Pre-soaking the slides with fixative when

humidity is very high.humidity is very high.• Adding few drops of acetic acid on the Adding few drops of acetic acid on the

slides to improve chromosome spreading slides to improve chromosome spreading in high humidity conditions (increase in high humidity conditions (increase membrane fragility).membrane fragility).

Tips

Post-dropping: DryingPost-dropping: Drying

• Room temperatureRoom temperature

• Heat plate: rainy days or high Heat plate: rainy days or high humidityhumidity

• Hot water vapor (75-80Hot water vapor (75-80ooC): dry C): dry atmospheric conditionsatmospheric conditions

Tips

Lab DemonstrationsLab Demonstrationsroom B-6624, B-6625room B-6624, B-6625

• Chromosome spreadingChromosome spreading

• Counting aberrations-Counting aberrations-microscopemicroscope

• Counting aberrations-Counting aberrations-video imagevideo image