Ovechkina Sbs Ge Talk 2008

850 views

Published on

High Content GenoTox Screening using IN Cell Analyzer 1000

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total views
850
On SlideShare
0
From Embeds
0
Number of Embeds
9
Actions
Shares
0
Downloads
11
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide

Ovechkina Sbs Ge Talk 2008

  1. 1. High Content GenoTox Screening usingIN Cell Analyzer 1000 Yulia Ovechkina, Ph.D.SBS 12th Annual Conference & Exhibition September 18, 2006
  2. 2. Why test for genetic toxicity?Genetic toxicology measures a drug’s ability to induce genetic damage Genotoxicity Somatic cells Reproductive cells aging Infertility Spontaneous oncogenesis abortion atherosclerosis Genetic diseasesSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 2
  3. 3. Regulatory genotoxicity tests offered by MDS according to FDAtesting guidelines1. In vitro bacterial test – In vitro bacterial reverse mutation Ames test2. In vitro mammalian cell culture – In vitro mammalian cell gene mutation (mouse lymphoma) test – In vitro chromosomal aberration test in human lymphocytes – In vitro micronucleus mammalian test (OECD no. 487, draft)3. In vivo – In vivo bone marrow micronucleus test September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 3
  4. 4. High content in vitro micronucleus assay for early genotoxicity andcytotoxicity profiling in drug development•Quantitation of micronuclei induction, apoptosis and cellproliferation 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 usingautomated cell image acquisition and analysis•High correlation with in vivo micronucleus assaySeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 4
  5. 5. Micronuclei induction is a highly quantitative measurement ofchromosomal damageMicronuclei are small nuclei produced during cell division by a laggingchromosome fragment or entire chromosome + Aneugens cause spindle disruptions and loss of the entire chromosome from the daughter nuclei + Clastogens cause double stranded chromosomal breaks and loss of chromosomal fragments from the daughter nucleiSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 5
  6. 6. Panel of tested compounds Compound Classification Concentration range, mMMitomycin C Direct acting clastogen 0.003-2.5Cyclophosphamide Metabolism dependent clastogen 5-500Diethylstilbestrol Aneugen 0.014-15Colchicine Aneugen 0.003-2.5Etoposide Aneugen 0.003-2.5Erythromycin Non-genotoxin (Negative Control) 0.5-50DMSO Vehicle (Negative control) 0.004-4 %September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 6
  7. 7. IN Cell Analyzer 3000 micronucleus module is multifunctional anduser-friendly•Three channel detection for nuclei (andmicronuclei), cytoplasm and viabilityanalysis•Flexible nuclei and micronucleisegmentation parameters•Data heat map, image overlay and user-friendly image analysis settings•Compatible with/without cytokinesisblocked micronucleus assay images•Fast analysisSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 7
  8. 8. IN Cell Analyzer 3000 micronucleus module provides objective,consistent and fast micronuclei scoring 1 21. Mask nuclei2. Classify nuclei (Binucleated/Mononucleated)3. Segment cytoplasm 3 44. Search cytoplasm for micronuclei (arrow) of binucleated cells September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 8
  9. 9. Criteria for micronuclei identification•Diameter less than one-third ofnucleus•Separated from (or marginallyoverlaps) nucleus•Similar staining intensity tonucleusSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 9
  10. 10. Automated vs. manual micronuclei scoring 30 automated manual % of cells with MNs 20 10 0 -9 -8 -7 -6 -5 log [Etoposide], MSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 10
  11. 11. Two in vitro micronucleus assay formats: MMNA and CBMNA(OECD no. 487, draft) Mononucleated Micronucleus Assay (MMNA) +/- S9 plate add fix, stain plate add wash out fix, stain cells drug and analyze cells drug+S9 drug and analyze 24 hrs 40 hrs 21 hrs 3 hrs 40 hrs Cytokinesis-Blocked Binucleated Micronucleus Assay (CBMNA) +/- S9 plate add add fix, stain plate add wash out add fix, stain cells drug CytB and analyze cells drug+S9 drug CytB and analyze 24 hrs 24 hrs 16 hrs 21 hrs 3 hrs 24 hrs 16 hrs September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 11
  12. 12. Levels of micronuclei induction are consistent between twoformats of micronucleus assay, CBMNA and MMNA 40 CBMNA % cells with MNs 30 MMNA 20 10 0 1 10 100 1000 10000 [Etoposide] nMSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 12
  13. 13. Assessing cytotoxicity in MMNA and CBMNA micronucleusassayBoth growth index measured in MMNA assay and proliferation indexmeasured in CBMNA assay output similar valuesGrowth Index (GI) in the MMNA Growth Index, GIassay: 1.5 Fraction of control Proliferation Index, Rbm  GI = (Nx-Nto)/(Ncontrol-Nto) 1.0Proliferation index (Rbm) in the 0.5CBMNA assay: 0.0  Rbm= ratio of binucleated to -9 -8 -7 -6 -5 mononucleated cells log [Etoposide], MSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 13
  14. 14. Multiplexing micronucleus assay with cell proliferation assayminimizes counting of micronuclei in dying or dead cells 30 2 % of cells with MNs Growth Index, GI 20 1 10 0 0 -9 -8 -7 -6 -5 log [Etoposide], M Growth Index, GI % cells with MNsSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 14
  15. 15. Measurement of micronuclei induction at GI50 is a reproducible and statistically relevant way to quantify micronuclei 30 30 2% of cells with MNs 2 30 Growth Index, GI 2 20 20 20 1 1 1 10 +S9 10 10 0 0 0 0 0 0 -9 -8 -7 -6 -5 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 log [Etoposide], M log [Cyclophosphamide], M log [Erythromycin], M GI or Rbm % cells with MNs GI50 or Rbm EC50, mM Fold increase over MN background at the GI50 Etoposide 0.2 27.6 Cyclophosphamide + S9 304.8 12.9 Erythromycin >50 0.9 September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 15
  16. 16. High Content Apoptosis AssayAntibodies against active Caspase 3 detect both early and lateapoptotic stages % of 7-AAD+ cells % of cells with active Cas3 100 GI % cells with 30 % of 7-AAD+, Cas3+ activated Cas3 % of 7-AAD+;Cas3- Growth Index, GI % of cells 75 20 1 50 10 25 0 0 0 -8 -7 -6 -5 -4 -8 -7 -6 -5 -4 log [Etoposide], M log [Etoposide], MHela cells were treated with Etoposide for 48 hours, stained with 7-AAD, a marker forlate apoptotic and necrotic cells, fixed and stained with antibodies against activeCaspase 3.September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 16
  17. 17. Multiplexing micronucleus assay with apoptosis assay eliminatesfalse-positives by excluding apoptotic cells from micronuclei scoring 30 Active Caspase 3 positive % of cells with MNs apoptotic cell with a micronucleus 20 % of live cells with MNs % of total cells with MNs 10 0 -3 -2 -1 0 1 Active DNA log [DMSO], % Caspase 3September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 17
  18. 18. Background levels of micronuclei are higher in CBMNA assay thanthat in MMNA assay 4 % of cells with MNs 3 2 1 0 CBMNA MMNA 3.1% +/- 0.62 0.43% +/- 0.15 September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 18
  19. 19. Lower background results in higher sensitivity in MMNA assaythan that in CBMNA assayFold increase of micronuclei induction over background at the GI50 orRbm EC50 30 Fold increase MMNA CBMNA 20 10 0 Etoposide MitC Erythrom Cyclophos Colchicine Diethylst DMSOSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 19
  20. 20. CBMNA shortcomings are due to incorporating cytochalasin Btreatment•Cytochalasin B induces break down of actin cytoskeleton whichin turn leads to higher cytotoxicity•Cytochalasin B increases micronuclei background in the vehicle-treated cells which results in decreased sensitivity of CBMNAassays•Cytochalasin B can potentially interact synergistically with a testcompound which may lead to incorrect conclusion about thecompound MN induction activitySeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 20
  21. 21. Incorporation of cytochalasin B treatment may lead to falsenegative results in the CBMNA assay, e.g. colchicineColchicine•is a spindle poison (microtubule destabilizingagent)•blocks cell division prior to chromosomesegregation and cytokinesisCo-treatment of colchicine treated cells withcytochalasin B would not generate binucleatedcells since most cells are already blocked atthe mitosis stage as teraploid mononuclei. September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 21
  22. 22. Colchicine micronuclei induction activity can be missed in CBMNA assay MMNA CBMNA % of cells with MNs 30 30 Proliferation Index, Rbm 2 % of cells with MNs Growth Index, GI 10 20 20 1 5 10 10 0 0 0 0 -9 -8 -7 -6 -5 -9 -8 -7 -6 -5 log [Colchicine], M log [Colchicine], M GI or Rbm % cells with MNs Colchicine GI50 or Rbm EC50, Fold increase over MN background at the GI50 mM or Rbm EC50 MMNA 0.3 7.9 CBMNA 0.9 1.1 September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 22
  23. 23. ConclusionsThe automated in vitro micronucleus mammalian assay providesaccelerated 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 scoringSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 23
  24. 24. State-of-the-art cellular imaging and automation technologyAutomated cell imaging system, GE Healthcare IN Cell Analyzer 1000,integrated with robotic plate handlingSeptember 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 24
  25. 25. BioCel® Velocity 11 is non-contact dispensing automation systemfor 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) Labcyte® Echo™ 550September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 25
  26. 26. Automated compound addition using non-contact acoustic based system, Labcyte® Echo™ 550Inverted 384-wellcell plateCompound DMSOplatePiezoelectrictransducer No pre-dilution in cell media; Minimum compound consumption September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 26
  27. 27. MDS Pharma Services advantages•Quantitation of micronuclei induction, apoptosis and cell proliferationin one assay well over 10 concentrations•Evaluation of test compounds in the absence and presence of in vitrometabolic 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 September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 27
  28. 28. Cell image-based High Content Analysis assays: an integral partof the drug development scheme Enzyme Cells Tissues Systems Clinic assaysMDS Pharma Services offers a variety of High Content Screening assays•GenoTox screening •Immunohistology slide analysis•CytoTox screening •Biomarker Analysis•Cell cycle analysis•Angiogenesis•Quantitation of p65NF-kB andphospho-p38/MAPK activation andtranslocation September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 28
  29. 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.September 18, 2006 Presentation title “High Content GenoTox Screening using IN Cell Analyzer 1000” 29
  30. 30. Booth: #2701Two poster presentations:  P7083 ‘Advanced Technology for Drug Discovery’ session on Tuesday, 12:30pm - 2:30pmDevelopment and validation of automated in vitro micronucleus/genotoxicityand cytotoxicity screening assays  P13024 ‘High Content Analysis’ session on Wednesday 12:30pm - 2:30pmFluorescence microscopy assays for localization of phosphorylated p38/MAPKand cytoplasmic to nuclear translocation of p65 NF-kB September 18, 2006 Presentation title 30

×