Module 6 - International Atomic Energy Agency

Report
Premature Chromosome
Condensation (PCC) Analysis
Lecture
Module 6
IAEA
International Atomic Energy Agency
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
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Following exposure to high doses
Lymphopaenia
Mitotic delay
Cell death
• 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
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Premature Chromosome Condensation (1)
PCC is research tool to probe immediate postirradiation 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 G0 at differing times,
dependent on dose
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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)
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Premature Chromosome Condensation:
Techniques (1)
• Techniques that cause chromatin condensation
before first mitosis are termed premature
chromosome condensation (PCC) assays
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Premature Chromosome Condensation:
Techniques (2)
Premature condensation can be induced by:
I.
II.
Fusing interphase cells to mitotic Chinese
hamster ovary (CHO) or HeLa cells using
Sendai virus or polyethylene glycol (PEG) as
the fusing agent
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
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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
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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
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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
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Mitotic fusion: Choose the staining
technique according to the end point
• PCC fragments, Giemsa (1)
• dicentrics, C banding (2)
• translocations, FISH (3)
1
2
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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
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Mitotic fusion - Scoring criteria
• Appearance of PCC defines cell cycle
position of lymphocyte at time of its
treatment
• G0/G1 appear as single chromatid; S, as
pulverized chromosomes and G2 has two
chromatids
• For biological dosimetry only spreads
comprising single chromatids G0/G1are
scored
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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 p34cdc2/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
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RICA images
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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
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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
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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
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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 G2 and metaphase
• With low-dose exposures there may be
contamination with cells at anaphase
• Frequencies of PCC rings are not significantly
different between late G2 and anaphase cells
therefore these data can be pooled
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Techniques – Chemical induction
PCC ring assay: Scoring criteria (2)
1. G2-PCC cell
with 2 rings
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2. M/A-PCC cell
with 1 ring
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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
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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
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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
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