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Wet Lab Radiation-Induced Chromosome Damage and Rejoining

Wet Lab Radiation-Induced Chromosome Damage and Rejoining. Background Equipment Supplies Procedures Troubleshooting and tips Lab Demonstrations. Background. Types of chromosome aberrations Radiation dose response curves. G1. G2M. Rx. Rx. Background. THE CELL CYCLE. M.

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Wet Lab Radiation-Induced Chromosome Damage and Rejoining

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

  2. Background • Types of chromosome aberrations • Radiation dose response curves

  3. G1 G2M Rx Rx Background

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

  6. Background

  7. Background

  8. Background

  9. Background

  10. Background

  11. 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 G1 irradiation Centric ring + fragment Acentric ring or interstitial deletion Background

  14. Acentric ring or interstitial deletion Ring chromosome Background

  15. Chromatid deletions in CHO chromosomes after irradiation in S or G2 Iso-chromatid deletion Chromatid deletion Background

  16. Chromatid exchanges in CHO chromosomes after irradiation in S or G2 complex exchange asymmetrical quadra-radial Background

  17. Background

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

  19. Multi-color FISH in human lymphocyte chromosomes Non-irradiated Irradiated From: Dr. J.D. Tucker 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 Symmetrical (balanced) gene rearrangement Asymmetrical (not balanced) fragment usually lost Polycentric 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 + aD k = background a = proportionality Fate: Deletions lost at mitosis 200 Chromatid breaks 100 Aberrations per 100 cells 1 2 Dose (Gy) Background

  23. 2. Exchange-type rearrangements > 2 hits required; dependent upon: Space = proximity Time = interaction P (2 hits) = D x D = D2 Y(yield) = k + bD2 3. Total yield Y = k + aD + bD2 Background

  24. Low dose dominated by linear component assume k (background) constant Y = k + aD + bD2 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 2.0 As dose rate aberrations Why? Repair Breaks rejoined, thus unavailable for further interaction 4Gy/hr 1.0 Dicentrics/Cell 0.1 Gy/hr 0 2 4 Dose (Gy) 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 + aD • 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 + aD + bD2

  27. Equipment • Sterile tissue culture hood • Incubator (CO2, 37oC, humidified) • Centrifuge • Microscopes( upright, inverted) • Slide warmer (hot plate) • Radiation source

  28. Supplies • Tissue culture flasks (dishes) • Tissue culture medium • Glass slides • Colchicine • Methanol (ethanol), acetic acid • KCl, sodium citrate

  29. Procedures • Cell culture • Cell growth and division leading to a good mitotic index • Harvesting • 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 spreading • Drops of cell suspension are placed on a slide, and allowed to dry in a controlled fashion, leading to chromosome spreading • Aging • dry heat and/or ethanol to denature the proteins, remove water/fixative , and enhance the adherence of the chromosomes to the glass slide

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

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

  33. Procedure Dropping/drying process • Cells touch the glass surface and become immobile. • Fixative starts drying. • 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.

  34. Procedure Dropping/drying process • 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.

  35. Troubleshooting and tips • Fixatives • 1:1, 3:1, 6:1, methanol:acetic acid • 3:1 ethanol:acetic acid • Hypotonic buffers • Drying temperatures • Humidity conditions

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

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

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

  39. Tips Pre-dropping conditions • Dry slides at room temperature. • Cold slides (0oC) when humidity is low. • Slightly warm slides (37oC) when humidity is 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).

  40. Tips Post-dropping: Drying • Room temperature • Heat plate: rainy days or high humidity • Hot water vapor (75-80oC): dry atmospheric conditions

  41. Lab Demonstrationsroom B-6624, B-6625 • Chromosome spreading • Counting aberrations-microscope • Counting aberrations-video image

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