1. Wet Lab Radiation-Induced Chromosome Damage and Rejoining Background Background Equipment Equipment Supplies Supplies Procedures Procedures Troubleshooting and tips Troubleshooting and tips Lab Demonstrations Lab Demonstrations

2. Background Types of chromosome aberrationsTypes of chromosome aberrations Radiation dose response curvesRadiation dose response curves

3. G1G2M Rx Background

4. THE CELL CYCLE Single chromatid Mitosis G1 S phase DNA replication G2 P MA T Background

5. Type of cytogenetic damage observed dependents upon where in the cell cycle irradiation occurs Chromosome Aberrations G1 irradiation Both sister chromatids involved Chromatid Aberrations S or G2 irradiation Usually only 1 chromatid involved Background

12. Multiple mis-rejoining events occurring in CHO chromosomes after G1 irradiation tricentric dicentrics Background

13. Mis-rejoining of 2 breaks on one chromatid after G 1 irradiation Centric ring + fragment Acentric ring or interstitial deletion Background

14. Ring chromosome Acentric ring or interstitial deletion Background

15. Chromatid deletions in CHO chromosomes after irradiation in S or G 2 Chromatid deletion Iso-chromatid deletion Background

16. Chromatid exchanges in CHO chromosomes after irradiation in S or G 2 asymmetrical quadra-radial complex exchange Background

18. Chromosomal rearrangement is the best biomarker for radiation exposure Sensitive Reliable Large data base Application of new techniques Fluorescence In Situ Hybridization (FISH) Background

19. From: Dr. J.D. Tucker Multi-color FISH in human lymphocyte chromosomes Non-irradiatedIrradiated Background

20. Aneupolid cell following FISH with probes for human chromosome 2 and human centromeres From: Dr. J.L. Schwartz Background

21. Fate of rearranged chromosomes 1. Deletions Lost at mitosis - micronuclei 2. Exchange-type rearrangements S ymmetrical (balanced) gene rearrangement A symmetrical (not balanced) fragment usually lost P olycentric chromosomes bridge-breakage-fusion fail mitosis cell death aneuploidy Background

22. Yield of radiation-induced chromosome damage Deletion: Terminal deletion = 1 hit Chromatid deletion = 1 hit Interstitial deletion = 2 hit Yield (Y) ~ linear Y = k +  D k = background  = proportionality Fate: Deletions lost at mitosis  Dose (Gy) Aberrations per 100 cells 100 200 Chromatid breaks Background

23. 2. Exchange-type rearrangements > 2 hits required; dependent upon: Space = proximity Time = interaction P (2 hits) = D x D = D 2 Y(yield) = k +  D 2 3. Total yield Y = k +  D +  D 2 Background

24. Low dose dominated by linear component assume k (background) constant Y = k +  D +  D 2 Y(0.1 Gy) = 0.1 + (0.1) 2 = 0.11 Y(0.2 Gy) = 0.2 + (0.2) 2 = 0.24 Y(1.0 Gy) = 1.0 + (1.0) 2 = 2.0 Y(2.0 Gy) = 2.0 + (2.0) 2 = 6.0 Y(3.0 Gy) = 3.0 + (3.0) 2 = 12.0 Y(6.0 Gy) = 6.0 + (6.0) 2 = 42.0 Background

25. Dose Rate Effects One hit aberrations - no dose rate effect Two hit aberrations - dose and fractionation effect As dose rate aberrations Why? Repair Breaks rejoined, thus unavailable for further interaction  Dose (Gy) Dicentrics/Cell 1.0 2.0 0 4Gy/hr 0.1 Gy/hr Background

26. SUMMARY Chromosomal rearrangements observed at first metaphase after irradiation, or after PCC Chromosome aberrations- G1 damage Chromatid aberrations - S or G2 damage Deletion type aberrations - lost at mitosis Y = k +  D Exchange-type aberrations Dose rate and LET dependent Symmetric - gene rearrangement complexity revealed by FISH role in carcinogenesis? Asymmetric - generally lethal mitotic failure, aneuploidy bridge - breakage - fusion cycles Y = k +  D +  D 2

27. Equipment Sterile tissue culture hoodSterile tissue culture hood Incubator (CO 2, 37 o C, humidified)Incubator (CO 2, 37 o C, humidified) CentrifugeCentrifuge Microscopes( upright, inverted)Microscopes( upright, inverted) Slide warmer (hot plate)Slide warmer (hot plate) Radiation sourceRadiation source

28. Supplies Tissue culture flasks (dishes)Tissue culture flasks (dishes) Tissue culture mediumTissue culture medium Glass slidesGlass slides ColchicineColchicine Methanol (ethanol), acetic acidMethanol (ethanol), acetic acid KCl, sodium citrateKCl, sodium citrate

29. Procedures Cell cultureCell culture –Cell growth and division leading to a good mitotic index HarvestingHarvesting –Metaphase by colchicine –Hypotonic treatments to swell and weaken the cell membrane –Fixation: (3:1 methanol:acetic acid) to make cell membrane very fragile.

30. Procedures Chromosome spreadingChromosome spreading –Drops of cell suspension are placed on a slide, and allowed to dry in a controlled fashion, leading to chromosome spreading AgingAging –dry heat and/or ethanol to denature the proteins, remove water/fixative, and enhance the adherence of the chromosomes to the glass slide

31. Tissue culture and harvesting Peripheral blood (whole blood)Peripheral blood (whole blood) 1640 RPMI with 10% FCS, PHA stimulation for 72 hrs1640 RPMI with 10% FCS, PHA stimulation for 72 hrs Colchicine for 40 minColchicine for 40 min Re-suspending in 0.075 M KCl hypotonic buffer for 15 min at 37 o CRe-suspending in 0.075 M KCl hypotonic buffer for 15 min at 37 o C Add fixatives to the cell suspension and wash 3x with fixativesAdd fixatives to the cell suspension and wash 3x with fixatives Cell pellets were stored at -20 o C in fixativeCell pellets were stored at -20 o C in fixative Procedure

32. Hypotonic treatment Lymphocytes: 0.075 M KCl, 37 o C, 12-20 minLymphocytes: 0.075 M KCl, 37 o C, 12-20 min Fibroblasts: 1:1 0.4% KCl:0.8% sodium citrate, 37 o C, 12-20 minFibroblasts: 1:1 0.4% KCl:0.8% sodium citrate, 37 o C, 12-20 min Longer time or more hypotonicLonger time or more hypotonic –Longer, thicker, sticker, less refractive chromosome Shorter time or less hypotonicShorter time or less hypotonic –Cell membranes and cell/nuclear debris around the chromosomes Procedure

33. Dropping/drying process Cells touch the glass surface and become immobile.Cells touch the glass surface and become immobile. Fixative starts drying.Fixative starts drying. Cells with metaphase chromosomes start flattening and spread their content (chromosome spreading).Cells with metaphase chromosomes start flattening and spread their content (chromosome spreading). As the fixative continues to dry, the nucleated cells continue to flatten slowly.As the fixative continues to dry, the nucleated cells continue to flatten slowly. Procedure

34. Dropping/drying process Dropping cells from different height helps distributing the cells more evenly on the slide, but does not influence chromosome spreading.Dropping cells from different height helps distributing the cells more evenly on the slide, but does not influence chromosome spreading. Most of chromosome spreading takes place at the time when the fixative evaporates from the spherical surfaces of cells.Most of chromosome spreading takes place at the time when the fixative evaporates from the spherical surfaces of cells. Procedure

35. Troubleshooting and tips FixativesFixatives –1:1, 3:1, 6:1, methanol:acetic acid –3:1 ethanol:acetic acid Hypotonic buffersHypotonic buffers Drying temperaturesDrying temperatures Humidity conditionsHumidity conditions

36. Metaphases spreading Breaking too easily:Breaking too easily: –Fragile cell pellets –Lowering the drying temperature Do not spread well:Do not spread well: –Extension of the drying period –Increasing the humidity Tips

37. High speed centrifugation Higher recoveryHigher recovery Better cell pellet for droppingBetter cell pellet for dropping 6,000-7,000 rpm in 2 ml microfuge vials with round bottom for 2-5 min6,000-7,000 rpm in 2 ml microfuge vials with round bottom for 2-5 min After addition of fixativeAfter addition of fixative Tips

38. Glass preparation Commercially available pre-cleaned glassCommercially available pre-cleaned glass Successive washed in aceton, HCl/ethanol and triple distilled waterSuccessive washed in aceton, HCl/ethanol and triple distilled water Dust or cardboard/paper residueDust or cardboard/paper residue Tips

39. Pre-dropping conditions Dry slides at room temperature.Dry slides at room temperature. Cold slides (0 o C) when humidity is low.Cold slides (0 o C) when humidity is low. Slightly warm slides (37 o C) when humidity is high.Slightly warm slides (37 o C) when humidity is high. Pre-soaking the slides with fixative when humidity is very high.Pre-soaking the slides with fixative when humidity is very high. Adding few drops of acetic acid on the slides to improve chromosome spreading in high humidity conditions (increase membrane fragility).Adding few drops of acetic acid on the slides to improve chromosome spreading in high humidity conditions (increase membrane fragility). Tips

40. Post-dropping: Drying Room temperatureRoom temperature Heat plate: rainy days or high humidityHeat plate: rainy days or high humidity Hot water vapor (75-80 o C): dry atmospheric conditionsHot water vapor (75-80 o C): dry atmospheric conditions Tips

41. Lab Demonstrations room B-6624, B-6625 Chromosome spreadingChromosome spreading Counting aberrations- microscopeCounting aberrations- microscope Counting aberrations- video imageCounting aberrations- video image