ovechkina sbs ge talk 2008
DESCRIPTION
High Content GenoTox Screening using IN Cell Analyzer 1000TRANSCRIPT
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27 High Content GenoTox
Screening using
IN Cell Analyzer 1000
SBS 12th Annual Conference & Exhibition
September 18, 2006
Yulia Ovechkina, Ph.D.
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September 18, 2006 Presentation title 2
Why test for genetic toxicity?
Genetic toxicology measures a drug’s ability to induce genetic damage
Genotoxicity
Somatic cells Reproductive cells
aging oncogenesis
Infertility Spontaneous
abortion
Genetic diseases atherosclerosis
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September 18, 2006 Presentation title 3
Regulatory genotoxicity tests offered by MDS according to FDA
testing guidelines
1. In vitro bacterial test
– In vitro bacterial reverse mutation Ames test
2. In vitro mammalian cell culture
– In vitro mammalian cell gene mutation (mouse lymphoma) test
– In vitro chromosomal aberration test in human lymphocytes
3. In vivo
– In vivo bone marrow micronucleus test
– In vitro micronucleus mammalian test (OECD no. 487, draft)
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September 18, 2006 Presentation title 4
High content in vitro micronucleus assay for early genotoxicity and
cytotoxicity profiling in drug development
•Quantitation of micronuclei induction, apoptosis and cell
proliferation in one assay well
•Automated, robust and cost-effective
•Accelerated throughput screening (200 compounds per week)
•Minimum compound consumption for 384-well plate format
•Objective, consistent and fast scoring of micronuclei using
automated cell image acquisition and analysis
•High correlation with in vivo micronucleus assay
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September 18, 2006 Presentation title 5
Micronuclei induction is a highly quantitative measurement of
chromosomal damage
Clastogens cause double stranded chromosomal
breaks and loss of chromosomal fragments from
the daughter nuclei
+
+
Aneugens cause spindle disruptions and loss of
the entire chromosome from the daughter nuclei
Micronuclei are small nuclei produced during cell division by a lagging
chromosome fragment or entire chromosome
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September 18, 2006 Presentation title 6
Panel of tested compounds
0.004-4 % Vehicle (Negative control) DMSO
0.5-50 Non-genotoxin (Negative Control) Erythromycin
0.003-2.5 Aneugen Etoposide
0.003-2.5 Aneugen Colchicine
0.014-15 Aneugen Diethylstilbestrol
5-500 Metabolism dependent clastogen Cyclophosphamide
0.003-2.5 Direct acting clastogen Mitomycin C
Concentration range, mM Classification Compound
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September 18, 2006 Presentation title 7
IN Cell Analyzer 3000 micronucleus module is multifunctional and
user-friendly
•Three channel detection for nuclei (and
micronuclei), cytoplasm and viability
analysis
•Flexible nuclei and micronuclei
segmentation parameters
•Data heat map, image overlay and user-
friendly image analysis settings
•Compatible with/without cytokinesis
blocked micronucleus assay images
•Fast analysis
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September 18, 2006 Presentation title 8
IN Cell Analyzer 3000 micronucleus module provides objective,
consistent and fast micronuclei scoring
1. Mask nuclei
2. Classify nuclei
(Binucleated/Mononucleated)
3. Segment cytoplasm
4. Search cytoplasm for
micronuclei (arrow) of
binucleated cells
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September 18, 2006 Presentation title 9
Criteria for micronuclei identification
•Diameter less than one-third of
nucleus
•Separated from (or marginally
overlaps) nucleus
•Similar staining intensity to
nucleus
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September 18, 2006 Presentation title 10
-9 -8 -7 -6 -50
10
20
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Automated vs. manual micronuclei scoring
log [Etoposide], M
% o
f ce
lls w
ith
MN
s
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manual
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September 18, 2006 Presentation title 11
Two in vitro micronucleus assay formats: MMNA and CBMNA
(OECD no. 487, draft)
16 hrs
Cytokinesis-Blocked Binucleated Micronucleus Assay (CBMNA) +/- S9
add
drug
fix, stain
and analyze
24 hrs 24 hrs
plate
cells
16 hrs
add
CytB
plate
cells
add
drug+S9
fix, stain
and analyze
21 hrs 3 hrs
wash out
drug
24 hrs
add
CytB
“High Content GenoTox Screening using IN Cell Analyzer 1000”
Mononucleated Micronucleus Assay (MMNA) +/- S9 plate
cells
add
drug+S9
fix, stain
and analyze
21 hrs 40 hrs 3 hrs
wash out
drug add
drug
fix, stain
and analyze
24 hrs 40 hrs
plate
cells
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September 18, 2006 Presentation title 12
Levels of micronuclei induction are consistent between two
formats of micronucleus assay, CBMNA and MMNA
1 10 100 1000 10000
0
10
20
30
40
CBMNA
MMNA
[Etoposide] nM
% c
ells w
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MN
s
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September 18, 2006 Presentation title 13
Assessing cytotoxicity in MMNA and CBMNA micronucleus
assay
Growth Index (GI) in the MMNA
assay:
GI = (Nx-Nto)/(Ncontrol-Nto)
Proliferation index (Rbm) in the
CBMNA assay:
Rbm= ratio of binucleated to
mononucleated cells
-9 -8 -7 -6 -50.0
0.5
1.0
1.5Growth Index, GI
Proliferation Index, Rbm
log [Etoposide], M
Fra
cti
on
of
co
ntr
ol
Both growth index measured in MMNA assay and proliferation index
measured in CBMNA assay output similar values
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September 18, 2006 Presentation title 14
Multiplexing micronucleus assay with cell proliferation assay
minimizes counting of micronuclei in dying or dead cells
-9 -8 -7 -6 -50
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2
log [Etoposide], M
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ith
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s G
row
th In
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I
% cells with MNs Growth Index, GI
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September 18, 2006 Presentation title 15
Measurement of micronuclei induction at GI50 is a reproducible and
statistically relevant way to quantify micronuclei
-7 -6 -5 -4 -30
10
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2
log [Cyclophosphamide], M
Gro
wth
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, GI
log [Etoposide], M
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s
log [Erythromycin], M
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2
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10
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2
0.9 >50 Erythromycin
12.9 304.8 Cyclophosphamide + S9
27.6 0.2 Etoposide
Fold increase over MN
background at the GI50
GI50 or Rbm EC50, mM
GI or Rbm % cells with MNs
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September 18, 2006 Presentation title 16
High Content Apoptosis Assay
Antibodies against active Caspase 3 detect both early and late
apoptotic stages
Hela cells were treated with Etoposide for 48 hours, stained with 7-AAD, a marker for
late apoptotic and necrotic cells, fixed and stained with antibodies against active
Caspase 3.
-8 -7 -6 -5 -4 0
25
50
75
100 % cells with
activated Cas3 GI
0
1
log [Etoposide], M
% o
f c
ell
s w
ith
ac
tive
Cas
3
Gro
wth
Ind
ex
, GI
-8 -7 -6 -5 -4 0
10
20
30
% of 7-AAD+
cells
% of 7-AAD+
, Cas3+
% of 7-AAD+
;Cas3-
log [Etoposide], M
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September 18, 2006 Presentation title 17
Multiplexing micronucleus assay with apoptosis assay eliminates
false-positives by excluding apoptotic cells from micronuclei scoring
Active Caspase 3 positive
apoptotic cell with a micronucleus
% of live cells with MNs
% of total cells with MNs
-3 -2 -1 0 1 0
10
20
30
log [DMSO], %
% o
f c
ell
s w
ith
MN
s
Active
Caspase 3
DNA
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September 18, 2006 Presentation title 18
Background levels of micronuclei are higher in CBMNA assay than
that in MMNA assay
0.43% +/- 0.15 3.1% +/- 0.62
CBMNA MMNA 0
1
2
3
4
% o
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September 18, 2006 Presentation title 19
Lower background results in higher sensitivity in MMNA assay
than that in CBMNA assay
Fold increase of micronuclei induction over background at the GI50 or
Rbm EC50
0
10
20
30
Etoposide MitC Erythrom Cyclophos Colchicine Diethylst DMSO
MMNA CBMNA
Fo
ld in
cre
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September 18, 2006 Presentation title 20
CBMNA shortcomings are due to incorporating cytochalasin B
treatment
•Cytochalasin B induces break down of actin cytoskeleton which
in turn leads to higher cytotoxicity
•Cytochalasin B increases micronuclei background in the vehicle-
treated cells which results in decreased sensitivity of CBMNA
assays
•Cytochalasin B can potentially interact synergistically with a test
compound which may lead to incorrect conclusion about the
compound MN induction activity
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 21
Incorporation of cytochalasin B treatment may lead to false
negative results in the CBMNA assay, e.g. colchicine
Colchicine
•is a spindle poison (microtubule destabilizing
agent)
•blocks cell division prior to chromosome
segregation and cytokinesis
Co-treatment of colchicine treated cells with
cytochalasin B would not generate binucleated
cells since most cells are already blocked at
the mitosis stage as teraploid mononuclei.
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 22
Colchicine micronuclei induction activity can be missed in CBMNA assay
Colchicine GI50 or Rbm EC50,
mM
Fold increase over MN background at the GI50
or Rbm EC50
MMNA 0.3 7.9
CBMNA 0.9 1.1
MMNA
-9 -8 -7 -6 -5 0
10
20
30
0
1
2
log [Colchicine], M
% o
f c
ell
s w
ith
MN
s
Gro
wth
Ind
ex
, GI
CBMNA
-9 -8 -7 -6 -5 0
10
20
30
0
5
10
log [Colchicine], M
% o
f c
ell
s w
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MN
s
Pro
lifera
tion
Ind
ex, R
bm
GI or Rbm % cells with MNs
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September 18, 2006 Presentation title 23
Conclusions
The automated in vitro micronucleus mammalian assay provides
accelerated throughput of genotoxicity and cytotoxicity screening:
reproducible and accurate method to quantify micronuclei induction, apoptosis and cell
proliferation
quantitation of percentage of cells with micronuclei and fold increase over background
at the GI50 or EC50 of Rbm is an accurate way to determine micronuclei induction
activities
both MMNA and CBMNA formats are reproducible tools for genotoxicity analysis but
MMNA is superior to CBMNA format in continuously growing cell lines
multiplexing the micronucleus assay with the apoptosis assay eliminates false-positives
by excluding apoptotic cells from micronuclei scoring
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 24
State-of-the-art cellular imaging and automation technology
Automated cell imaging system, GE Healthcare IN Cell Analyzer 1000,
integrated with robotic plate handling
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September 18, 2006 Presentation title 25
BioCel® Velocity 11 is non-contact dispensing automation system
for compound addition, cell fixing and immunostaining
Titertek Multidrop
V-Spin Centrifuge
LiCONiC CO2
incubator
De-lidder
Carousel w/ 12 hotels, 16
slots each (not shown)
“High Content GenoTox Screening using IN Cell Analyzer 1000”
Labcyte® Echo™ 550
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September 18, 2006 Presentation title 26
Automated compound addition using non-contact acoustic
based system, Labcyte® Echo™ 550
Inverted 384-well
cell plate
Compound DMSO
plate
Piezoelectric
transducer
No pre-dilution in cell media; Minimum compound consumption
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 27
MDS Pharma Services advantages
•Quantitation of micronuclei induction, apoptosis and cell proliferation
in one assay well over 10 concentrations
•Evaluation of test compounds in the absence and presence of in vitro
metabolic activation system in pre-validated mammalian cell line
•Available in two formats: cytokinesis-blocked, binucleated (CBMNA)
and mononucleated (MMNA) micronucleus assays
•Accelerated throughput screening (200 compounds per week)
•Minimum compound consumption for 384-well plate format
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 28
Cell image-based High Content Analysis assays: an integral part
of the drug development scheme
Enzyme
assays Cells Tissues Systems Clinic
•GenoTox screening
•CytoTox screening
•Cell cycle analysis
•Angiogenesis
•Quantitation of p65NF-kB and
phospho-p38/MAPK activation and
translocation
•Immunohistology slide analysis
•Biomarker Analysis
MDS Pharma Services offers a variety of High Content Screening assays
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 29
Acknowledgements
MDS Pharma Services
David Kirk, Ph.D.
Christine O’Day, Ph.D.
Robert Keyser
Phuong TB Nguyen
Richard Rodriguez
Jaiver Alfonso
GE Healthcare Biosciences
Nick Thomas, Ph.D.
“High Content GenoTox Screening using IN Cell Analyzer 1000”
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September 18, 2006 Presentation title 30
Two poster presentations:
P7083 ‘Advanced Technology for Drug Discovery’ session on Tuesday,
12:30pm - 2:30pm
Development and validation of automated in vitro micronucleus/genotoxicity
and cytotoxicity screening assays
P13024 ‘High Content Analysis’ session on Wednesday 12:30pm - 2:30pm
Fluorescence microscopy assays for localization of phosphorylated p38/MAPK
and cytoplasmic to nuclear translocation of p65 NF-kB
Booth: #2701