premature chromosome condensation (pcc) analysis (dose estimation …€¦ · pcc cells are...

Radiation Biology IREM/HU


Hirosaki University Chromosome Research Group

Premature Chromosome Condensation (PCC) analysis

(Dose estimation by PCC method)

Mitsuaki Yoshida

Department of Radiation Biology Institute of Radiation Emergency Medicine Hirosaki University (IREM/HU)

BIODOSIMETRY IN THE 21st CENTURY Training Meeting HICARE in collaboration with the International Atomic Energy Agency Hiroshima, Japan, June 10-14, 2013.




Radiological Accident ? Nuclear Plants Nuclear terrorism Nuclear Bomb Factory Medical field Scientific Research

6

Fukushima Dai- Ichi NPS

Atomic Bomb

CT scan laboratory

terrorism JCO in Tokai- Mura

http://gigazine.jp/img/2011/03/14/fukushima_daiichi_3rd_movie/movie06.jpg

http://ja.wikipedia.org/wiki/%E3%83%95%E3%82%A1%E3%82%A4%E3%83%AB:Nagasakibomb.jpg




1 . Medical systems for the radiation emergency 2. The system for Radiation Measurement Biological dose estimation Bioassay Physical dose estimation 3. Education of the professional responder

Preparedness for Radiation Emergency Medicine




Most important thing in radiological accident is “medicine” to save a life of exposed person. The second is “to estimate the radiation dose”.

Why? Clinical symptom such as acute radiation syndrome depends on radiation dose. The information of the estimated dose will be useful for both the medical treatment and the protection of damage in the irradiated persons by suspecting the degree of damage.




Biological dose estimation 1. Clinical symptom 2. Number of lymphocyte 3. Chromosome analysis -dicentric -translocation -PCC-ring -micronucleus

10

➸Chromosome is the most reliable biological marker (direct evidence) for the measurement of radiation damage （Gold standard)





Dose estimation (Dosimetry)

if possible

➸ quickly and accurately

8 4




Dicentric assay: acute exposure (external) Translocation assay : old or long term (chronic) exposure (external)

Atomic bomb survivor in Hiroshima and Nagasaki Residents in high- background radiation area in China Children in Namie- Town of Fukushima Prefecture




Dose estimation by PCC-ring method

suspected high dose exposure


5 10 15 20

A: <1 Sv B: 1-2 Sv C: 2-5 Sv D: >5 Sv Days after the exposure

Lymphocyte

Thrombocyte(blood platelet)

Granulocyte After the cells are irradiated with high dose, the cell cycle will be stopped in G2 stage (G2 arrest) and will not proceed at the metaphase. or such cells will be dead (apoptosis) Chromosomes will not be Identified and analyzed. Radiation dose cannot be estimated.


G1 S G2 M DNA replication

P M P M P M P M P M P M P M P M

metaphase

de-condensation

DNA

centromere

T-lymphocyte （G0 stage）

+PHA（The cell cycle starts by the stimulation of PHA)

The dynamics of DNA in the cell cycle.

P :Paternal M :Maternal




5

underestimate

: Y=αD2+βD+c

: real data of dic/cell

There is the difference between standard curve obtained from the dic analysis in the dose range from 0 – 5Gy data and the real data of dic/cell at the dose of over 5Gy.

If there is a possibility that the exposed dose may over 5Gy, the radiation dose will be underestimated.

Also, when lymphocytes are exposed By high dose radiation, apoptosis and G2 arrest are induced and chromosome Figure cannot be obtained. Therefore, in such case of possible high dose exposure, other technique should be applied in order to estimate radiation Dose.

PCC

M. S. Sasaki, et al.




PCC= Premature Chromosome Condensation

PCC is induced by fusing human lymphocytes with Chinese hamster ovary (CHO) mitotic cells in the presence of polyethylene glycol (PEG). PCC is also chemically induced by the treatment with a phosphatase inhibitor such as Okadaic acid or Calyculin A.


Fusion by virus or PEG

Chromosome in Metaphase

Prematurely condensed chromosome

MPF: Mitosis Promoting Factor

Interphase Metaphase

Chromosome condensation is induced by activated MPF in interphase cell fused with metaphase cell.

PCC by cell fusion


Functions of MPF

Triggers the formation of mitotic spindle through microtubule instability. Promotes mitosis i.e. chromatin condensation through phosphorylation of condensins. Causes nuclear envelope breakdown by phosphorylating the lamins that form an intermediate filament-type network (nuclear lamina) underlying the inner nuclear membrane. The three lamins present in the nuclear lamina, lamin A,B & C, are phosphorylated by MPF at serine amino residues. This leads to depolymerisation of the nuclear lamina & breakdown of nuclear envelope into small vesicles. Causes phosphorylation of GM130, which leads to the fragmentation of the Golgi and the ER


Development of PCC assay - fusion PCC assay -

Johnson RT. Nature, 1970 Pantelias GE. Somatic Cell Genet, 1983

Virus-mediated PEG-mediated

Fusion PCC method has several issues, such as complicated operation, low condensation index, and maintenance of cells for fusion.


Development of PCC assay - chemical induced PCC assay -

Gotoh E. Biomed Res, 1995 Dyban AP. Mol Reprod Dev, 1993

Mouse embryo Human PBL

PCC induced by okadaic acid PCC induced by calyculin A


cdc25 cdc25

P

Protein Phosphatase Type 2A (PP2A) PP2A

CA, OA

Inactive MPF (Cyclin B/cdc2 complex)

P Cyclin B cdc2

inactive active

Cyclin B cdc2

Active MPF (Cyclin B/cdc2 complex)

dephosphorylation

Phosphorylation

promotion

inhibition

Chemical induced PCC（Hypothesis)

condensation


Okadaic Acid : 500nM for 1hr

Calyculin A : 50nM for 1hr




Premature Chromosome Condensation induced by Protein Phosphatase inhibitors

Okadaic acid Calyculin A

treat for 5～60min

PCC induced in lymphocytes at various stages of the cell cycle. A: G2/Meta-PCC, B: Meta/Ana-PCC, C: Ana-PCC Kanda et al.




Typical images of G1-, S-, G2/M-, M/Ana, and Ana/Telo-PCC cells.

G1 S G2 M

PCC cells are classified into 5 categories as follows; G1-PCC cells showed univalent chromosomes, S-PCC cells had a "pulverized" appearance, G2/M-PCC cells displayed bivalent chromosomes, M/Ana-PCC cell showed separated sister chromatids, and Ana/ Telo-PCC cells had more distant separated sister chromatids than those in M/Ana-PCC cells.




Problems in ring analysis by PCC 1. Chromosomes are over-condensed and become fuzzy. solution : optimization of chemical treatment (concentration and time) 2. Identification of ring chromosome solution: determination of criteria to identify ring

decreasing of detection sensitivity of ring chromosome




Ana/Tel PCC G2/M PCC

It may be difficult to identify a ring chromosome




Optimization Of

chemical treatment


Conditions of calyculin A treatment used for chromosome aberration study by PCC assay

Cells Conc. Period Parameters References Whole blood 50 nM 30 min Translocation George K. Int J Radiat Biol, 2001

PBL 50 nM 30 min Chr #, PCC index Gotoh E. Int J Radiat Biol, 2005

PBL 50 nM 30 min Chromosome length ratio

Gotoh E. Int J Radiat Bio, 2005

Cell line 50 nM 5 min Chromatid break Bryant P. Mutagenesis, 2007

Whole blood 500 nM 60 min Chr #, chromosome length ratio

Wang ZZ. Radiat Environ Biophys, 2007

Cell line 80nM 60 min Fragment, translocation

Bergs JW, J Radiat Res (Tokyo), 2008

Cell line 50 nM 5 min Chromatid break Bryant P. Mutat Res, 2008

Whole blood 100 nM 60 min Fragment Wang ZZ. Mutat Res, 2009

Biopsy 80nM 60 min Chr #, translocation Darroudi F. Cancer Lett, 2010

Condition of calyculin A treatment in PCC assay is not fixed yet.



G2/M-PCC images in cell spread prepared using the optimized calyculin A treatment

0 Gy

7 Gy 20 Gy

3 Gy

Treatment with 50 nM Calyculin A 30 min

before harvesting

T. Miura and W. F. Blakely, Cytometry 2011




Optimized condition of chemical treatment

Okadaic Acid : 1000nM for 60 min Calyculin A : 25 or 50nM for 30 min




Dose response of PCC-ring in human lymphocytes

I. Hayata et al., J. RADIAT. RES., 42:SUPPL., S149-S155(2001)




The criticality accident in a uranium conversion test plant in Tokai-mura On September 30, 1999, at around 10:35 a. m., the criticality accident occurred in a uranium conversion test plant of the JCO Ltd., in Tokai-mura, Ibaraki Prefecture, Japan. As two workers (A and B) were pouring uranyl nitrate into a precipitation tank, the solution emitted a flash of blue light and the alarms went off to warn against gamma rays. Another worker (C) who was in the corridor next to the room immediately recognized that a criticality accident had taken place and ordered the two workers to evacuate. 60

61

Two workers (A and B) were pouring uranyl nitrate into a precipitation tank, the solution emitted a flash of blue light and the alarms went off to warn against gamma rays.


Hematological change in the 3 patients in Tokai-mura criticality accident

A B C

62


Biodosimetry by PCC assay in the JCO accident

Hayata I. J Radiat Res (Tokyo), 2001

Pt. A

Pt. B

Pt. C




Preparedness for

Cytogenetic biodosimetry in

Hirosaki University



Culture after stimulation by PHA

Taking Blood (heparinized)

Harvest

Staining

CO2 incubator （48 h）

Whole blood or

Isolated lymphocytes

Hypotonic treatment and

Cell fixation

Slide preparation Observation/analysis

Colcemid treatment to stop the cell cycle

Flow of chromosome analysis




Equipment for the cytogenetic biodosimetry laboratory

1. Culture

Centrifuge : for isolation of lymphocytes

Safety cabinet CO2 incubator

2. Harvest: hypotonic treatment and fixation

Centrifuge and water bath

Cell Harvester ：HANABI-PIII ADStec. Inc., Japan

Chromosome spreader : HANABI-PIV ADStec. Inc. Japan




Automated Chromosome Spreader （HANABI-PIV, ADStec. Inc. Japan)

Automated Cell Harvester (HANABI-PII, ADStec. Inc., Japan)

A total of 24 blood samples are processed by this machine at one time.

A total of 96 slides can be prepared by this machine (Max. 96 samples, 1slide/sample).




Slide preparation Scanning and analysis

Microscope and PC

Freezer Refrigerator

Phase contrast microscope

Manual HANABI 5 slides




High yield chromosome preparation technique (isolated lymphocytes culture) １．Isolation of lymphocytes from 3 ml PB using CPT tube ２．Culture for 48 hrs in 20%FCS/RPMI medium containing PHA and/or Colcemid (３．Add Calyculin A for 30 min ~1 hour before starting harvest) ４．Hypotonic treatment with 0. 075M KCl for 20 min ５．Fix the cell with methanol- acetic acid (3: 1 ) sol. ６．Slide preparation by chromosome spreader

15ml tube


Isolation of the lymphocytes from whole blood

BD CPT Tube Remove PHA in tube Add 20%FCS culture medium (1 ml) Add the whole blood (3ml)

Centrifugation 3000rpm for 1 5min

Remove the lymphocytes Culture (6ml) + PHA with or without Colcemid

Washing and Centrifugation 1 500rpm for 5min


Heparinized whole blood 3~5ml

Remove this solution

Add 1ml of 20%FCS/RPMI1640

1. taking whole blood using heparin

2. Preparation of CPT tube (Becton Dickinson)


3. Add 3ml of whole blood into CPT tube

Remove plasma and add 5~6ml of 2%FCS/ RPMI1640(4℃） and Pipetting to take lymphocytes

Centrifuge this CPT tube at 3000rpm for 15min at room temperature

Red blood cells

Lymphocytes You cannot see the layer of lymphocytes.

plasma

Transfer solution including lymphocyte Into 15ml tube




Education and Training experiences of Hirosaki University Chromosome Research Group for Cytogenetic biodosimetry

Laboratory of Dept. of Radiation Biology 1. Is a member of the REMPAN of WHO. 2. Is member of the exchange program of researcher between Japan and Asian countries supported by the MEXT in Japan. October, 2012-March 2013: Ms. Benchawan Rungsimaphorn from Thailand 2013, Exchange program by the MEXT 3. Is supporting the establishment of cytogenetic biodosimetry in Asian countries. Thailand, Korea, Taiwan 4. Is accepting researchers and technologist from Asian countries. 2010, October- December, Dr. Wol-Soon, Jo, Korea 2013, Researcher from Thailand, supported by the IAEA program




Preparedness of

Radiation Emergency Medicine in

Hirosaki University

National Institute of Radiological Sciences Hiroshima

Hirosaki City Hirosaki University

Tokyo

Location of Hirosaki City Aomori Prefecture

Nagasaki

Hirosaki University


Chromosome Research Group

Fukushima NPP

Nuclear power facilities in Aomori Prefecture

Hirosaki University



Hirosaki University Radiation Safety

Organization

Graduate School of Medicine

School of Medicine

Graduate School of Health Science

Advanced Emergency & Disaster Medical Center in University Hospital

Institute of Radiation Emergency Medicine (IREM/HU)

Administrative office Hirosaki University



Advanced Emergency and Disaster Medical Center

Hirosaki University



July 1st, 2010 Advanced Emergency and Disaster Medical Center

The victims in radiological accident are transferred by helicopter.

Decontamination Room

Patient treatment room

body surface contamination monitor Hand and foot cross monitor Refrigerator for specimens

Whole body counter

Total body Thyroid monitor

WBC-301B ALOKA

Radiation Measurement room (Bioassay)

Equipment for bioassay

βmeasuring instruments ( liquid scintillation counter)ALOKA LSC-LB5 α measuring instrument

Low backgroundα/β automated counter ALOKA LBC-4302

Gamma measuring instruments

Sterilized room and treatment room for the victims with a burn




Co-medical Education Program in Radiation Emergency Medicine

The grant of the Ministry of Education, Culture, Sports, Science and Technology, JAPAN （2008 - 2012）

The Projects in Hirosaki University

文部科学省科学技術振興調整費地域再生人材創出拠点の形成

被被ばばくく医医療療ププロロフフェェッッシショョナナルル育育成成計計画画 MEXT Special Coordination Funds for Promoting Science and Technology Re-establishment of Vital regions through Fostering Talents

Education Program for Professionals in Radiation Emergency Medicine

The grant of the Ministry of Education, Culture, Sports, Science and Technology, JAPAN （2010 - 2014）

Hirosaki University

Aomori Prefecture


Education Programs for Radiation Emergency Medicine

Seminar in “Education program for Professional in REM”

Dr. Makoto Akashi, NIRS, Japan Dr. Joan Francesc Barquinero, Barcelona University, Spain

Dr. Reiko Kanda, NIRS, Japan

Exercise and training on REM


Education Programs for Radiation Emergency Medicine

International Conference, March 1 9th, 201 0 Dr. Gordon Livingston, REAC/TS, USA Dr. William F. Blakely, AFRRI, USA Dr. Horst Romm, Bundesamt fuer Strahlenschutz, Germany Dr. Ruth Wilkins, Health Canada, Canada

4th Seminar for “Education Program for Professional in Radiation Emergency Medicine”, January 24th, 201 1 Dr. Zhanat Carr, WHO Dr. David Lloyd, HPA , England Dr. Firouz Darroudi, Leiden University the Netherlands

Thank you for your attention!! Mt. Iwaki in Hirosaki

Hirosaki University



premature chromosome condensation (pcc) analysis (dose estimation …€¦ · pcc cells are...

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