1. IAEA International Atomic Energy Agency Premature Chromosome Condensation (PCC) Analysis Lecture Module 6

2. IAEA 2 Introduction Biological dosimetry is generally performed by analyzing dicentrics and/or translocations at first mitosis following in vitro PHA blastic transformation Conventional dicentric assay may not provide accurate dose estimate in case of accidental exposure above 6 Gy due to lymphopaenia, mitotic delay and cell death during culture

3. IAEA Following exposure to high doses At high doses, number of lymphocytes in blood is reduced and cell cycle is delayed. Therefore sometimes it is not possible to make chromosome preparations containing sufficient number of metaphase cells necessary for dose estimation by routine dicentric assay in cytogenetic dosimetry LymphopaeniaMitotic delay Cell death 3

4. IAEA Premature Chromosome Condensation (1) PCC is research tool to probe immediate post- irradiation processes and kinetics of chromosomal break restitution and/or misrepair to form aberrations Dicentrics, complete and incomplete translocations and acentric fragments observed at metaphase are formed in G 0 at differing times, dependent on dose 4

5. IAEA Premature Chromosome Condensation (2) In X- irradiated human lymphocytes: at low doses (1-2 Gy), dicentrics and translocations are formed rapidly at higher doses (4-6 Gy), frequencies of chromosome exchanges increase proportionally to restitution of chromosome breaks (repair) 5

6. IAEA Premature Chromosome Condensation: Techniques (1) Techniques that cause chromatin condensation before first mitosis are termed premature chromosome condensation (PCC) assays 6

7. IAEA Premature Chromosome Condensation: Techniques (2) I. Fusing interphase cells to mitotic Chinese hamster ovary (CHO) or HeLa cells using Sendai virus or polyethylene glycol (PEG) as the fusing agent II. Chemical methods, using inhibitors of DNA phosphorylation such as okadaic acid or calyculin A. Most of these methods require cells to be cycling in culture Premature condensation can be induced by: 7

8. IAEA Premature Chromosome Condensation: Techniques – Mitotic Fusion Fusing lymphocytes with CHO mitotic cells in presence of PEG, enables chromosomal aberrations to be seen immediately following irradiation without need for mitogen stimulation or culturing This PCC method, in combination with C banding or FISH with chromosome specific DNA libraries with or without pan-centromeric probe, permits detection of breaks, dicentrics and rings as well as translocations 8

9. IAEA Techniques of Mitotic Fusion Applications in biodosimetry useful to determine exposure to low doses and life threatening high acute doses of low and high LET radiation discriminate between total and partial body exposures efficient for detecting even small spared fraction (as low as 5%) able to quantify small localized burns from partial body exposures 9

10. IAEA Mitotic fusion: Cell culture and cell fusion conditions CHO mitotic cells from stock lines Cells can be prepared in large quantities in advance and kept frozen before use Isolated lymphocytes using Ficoll Hypaque, some can be frozen for future use Use PEG as fusing agent, at 40%-50% w/v Fuse lymphocytes with mitotic CHO cells (ratio 5:1) in presence of PEG Fusion process takes 4 min, followed by 1h incubation in complete medium with Colcemid at 37ºC Fixation and slide preparations by standard techniques 10

11. IAEA Mitotic fusion: Choose the staining technique according to the end point PCC fragments, Giemsa (1) dicentrics, C banding (2) translocations, FISH (3) 3 12 11

12. IAEA Mitotic Fusion - Analysis PCC spreads can be located manually or by automated metaphase finder systems Analysis involves counting number of chromosome elements (single chromatids) Number in excess of 46 indicates fragments Dicentrics can be highlighted With FISH assay, cot-1 hamster DNA can be used to mask all signals in CHO chromosomes so that only appropriate human PCC are highlighted 12

13. IAEA Mitotic fusion - Scoring criteria Appearance of PCC defines cell cycle position of lymphocyte at time of its treatment G 0 /G 1 appear as single chromatid; S, as pulverized chromosomes and G 2 has two chromatids For biological dosimetry only spreads comprising single chromatids G 0 /G 1 are scored 13

14. IAEA Techniques: Chemical induction Rapid Interphase Chromosome Assay (RICA) Isolated lymphocytes are placed in culture medium containing phosphatase inhibitor (okadaic acid or calyculin A), adenosine triphosphate and p34 cdc2 /cyclin B kinase and incubated at 37ºC for 3 h Fixation and spread preparation follows normal procedures for metaphases Radiation induced damage is analyzed after in situ hybridization and chromosome painting by fluorescence microscopy 14

15. IAEA RICA images 15

16. IAEA Techniques - Chemical induction PCC ring assay (1) Simple and useful procedure: involves scoring of rings in Giemsa-stained chromosomes Requires lymphocytes to be cultured Particularly applicable to high overdoses in range where dose response for conventional dicentric assay shows signs of saturation 16

17. IAEA Techniques - Chemical induction PCC ring assay (2) Chemicals: okadaic acid (OA) or calyculin A is dissolved in dimethylsulphoxide (DMSO), diluted with medium and stored at -20°C as stock solution (5–10  M) Calyculin A is about 20 times more effective than OA Culture: PCC induction in lymphocytes requires cells to be cycling Procedure is to PHA stimulate and culture cells for 48 h PCC can be induced in whole blood cultures or using isolating lymphocytes 17

18. IAEA Technique – Chemical induction PCC ring assay: Protocol Okadaic acid (500 nM) or calyculin A (20–50 nM) is added to cultures during final 1-2 h, producing mixture of PCC cells in all stages of first cell cycle Concentration and period of optimal treatment should be determined based on yield of PCC cells and morphology quality of chromosomes Fixation, slide preparation and Giemsa staining procedures are similar to methods used for metaphases 18

19. IAEA Techniques – Chemical induction PCC ring assay: Scoring criteria (1) Select for analysis PCC cells with 46 or more chromosomal objects In 48 h cultures of highly irradiated lymphocytes most PCC cells are between late G 2 and metaphase With low-dose exposures there may be contamination with cells at anaphase Frequencies of PCC rings are not significantly different between late G 2 and anaphase cells therefore these data can be pooled 19

20. IAEA Techniques – Chemical induction PCC ring assay: Scoring criteria (2) 2. M/A-PCC cell with 1 ring 1. G2-PCC cell with 2 rings 20

21. IAEA Techniques – Chemical induction PCC ring assay: Scoring criteria (3) Ring with visible hole with or without centromere and pair of rings either separate or joined by centromere in PCC spreads is scored as single ring Analyze minimum of 100 PCC-r or 500 PCC-cells Similar to dicentric assay, overdispersed intercellular distribution of PCC rings compared with Poisson can indicate heterogeneous exposure to low LET radiation 21

22. IAEA 22 Discussion Drug induced PCC assay takes 48 h culturing + 2h for processing and analysis In the dose range is 0-6 Gy conventional dicentric assay may be better dose indicator than PCC assay, but is less suitable for higher dose exposures requiring rapid dose estimate Cell fusion induced PCC assay was not used in radiation accident biodosimetry due to highly demanding technical skill and poor yield

23. IAEA 23 Conclusions Frequency of radiation induced rings and fragments in Giemsa stained PCC preparations provides useful biodosimetry tool to evaluate acute whole/partial body exposures in high dose radiation emergencies compared to conventional dicentric method PCC assay is: easy to implement amenable to automation only requires conventional light microscope cost-effective 23