The Use of Quantitative, Cell-based, High Content ...

The Use of Quantitative, Cell-based, High Content Screening Assays to Detect in vitro Genotoxicity and the Effect on Cell Cycle Checkpoints 50 μM Etoposide

Phospho H2AX nuclear Intensity

Control

Bhaskar S. Mandavilli, Suk J. Hong, Krishna M. Vattem, and Richik N. Ghosh

DNA Damage Sensing and Repair - Histone 2AX Phosphorylation and Ku 70/80 Activation A. 50 μM Etoposide 600

Etoposide EC50 = 68  1M

500

Camptothecin EC50 = 0.3 M

400 300 200 100 0

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-5

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-3

-2

-1

0

1

2

Ku 70/80 nuclear Intensity

400 200 0

Etoposide EC50 = 18  2 M Camptothecin EC50 = 1.5 M

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Multiplexed p53 & p21 Kits, Cat # 8400601, 8400602

Measures p53 & p21 activation.

Singleplex p21 detection kit, Cat # 8400901, 8400902

Measures p21 activation.

Singleplex p53 kit, Cat # 8400801, 8400802

Measures p53 activation

Multiplex p53/MDM2 kits, Cat# 8401801; 8401802,; 8401901; 8401902

Measures MDM2 and p53 activation

Ku70/80 activation kits, Cat# 8403101; 8403102

Measures Ku70/80 dimers upon DNA damage

Phospho-H2AX Activation kits, Cat# 8402901; 8402902

Measures activation of H2AX phosphorylation

Phospho Chk2 activation kits, Cat# 8402801; 8402802

Measures activation of Chk2 phosphorylation

Phospho-ATM activation kits, Cat# 8403001; 8403002

Measures activation of ATM kinase

Phospho ATM/p53 duplex activation kit, Cat# 8405701; 8405702

New! MeasuresATM and p53 activation in the same cell

Phospho Rb detection kit, Cat# 8404501; 8404502

New! Measures activation of Rb protein

Phospho Plk-1 detection kit, Cat # 8404801; 8404802

New! Measures activation of Plk-1

Cyclin B1 Activation kit, Cat # 8404401; 8404402

New! Measures activation of Cyclin b1

New Kits available in 3 sizes: • Sufficient material for 1 X 96 wells • Sufficient material for 5 X 96 wells • Custom larger size Please visit www.cellomics.com for more information

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200

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Blue: Nuclei; Red: phospho-Rb

B. Control

Nocodazole

100

20 0

-6

-5

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-2

-1

0

1

2

3

50 μM Etoposide

Control

100

50 μM Etoposide

Etoposide EC50 = 1.76  0.05 M

80

Camptothecin EC50 = 0.6 M

60 40 20

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Control

Nocodazole

Phospho ATM p53

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2 1

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0

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-5

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Nocodazole is an anti-neoplastic agent which induces G2/M cell cycle arrest by depolymerizing microtubules in the cell.

EC50 for ATM = 19  5 M EC50 for p53 = 36  6 M

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Phospho p53 intensity

2 1

-2

-1

0

1

2

EC50 for ATM = 19  5 M EC50 for p53 = 36  6 M

50

1.5 μM Camptothecin

Phospho p53

75 50 25

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-2 -1 0 Log M) Camptothecin

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-2 -1 0 Log M) Camptothecin

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-1

0

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Log (M), Camptothecin

EC50 for p53 = 0.58  0.05 M EC50 for MDM2 = 9.70  0.50 M

CyclinB1, EC50 = 46.3 nM -4.5

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-2.5

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1.5

MDM2 p53

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Conclusions

200 100

• Quantitative cell based HCS assays have been developed for a range of DNA damage -5

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EC50 for p53 = 0.58  0.05 M EC50 for MDM2 = 9.70  0.50 M

MDM2 μM suppressor Camptothecin Figure 4:Control (A) p53 phosphorylation: p53 is a1.5 tumor protein and can be activated in response to several stressors including DNA damage. In response to DNA damage, p53 is phosphorylated (ser15) by ATM kinase and leads to cell cycle p53 arrest. A549 cells were treated with vehicle (DMSO) or 1.0 mM camptothecin for 24 300hrs. After staining cells with Cellomics phospho-p53 detection kit, Images were acquired and analyzed. Z-factor for phosphor-p53 is 0.50 ± 0.03 and %COV is 8 ± 1. Camptothecin dose response curves show that EC50 for p53 phosphorylation is 0.35 mM. (B) MDM2 activation: MDM2, a p53 specific E3 ubiquitin ligase is an antagonist of p53 and limits its growth-suppressive function. Disruption of MDM2-p53 complex by DNA damage leads to p53 induction. A549 cells were treated with vehicle 200 (DMSO) or 1.5 mM camptothecin, for 24 hrs. After staining cells with Cellomics MDM2 activation kit, Images were acquired and analyzed for MDM2. Z-factor for MDM2 is 0.58 ± 0.03 and %COV is 6 ± 1. Camptothecin dose response curves show that EC50 for MDM2 activation is 9.7 mM. Camptothecin is a topoisomerase I inhibitor resulting in DNA strand breaks. 100

Phospho-PLK1, EC50 = 41.8 nM

1

Blue: Nuclei; Green: p53; Red: MDM2 400

50

and cell cycle check point control signaling pathways.

Log (M), Camptothecin

1

Blue: Nuclei; Red: phospho-p53

Phospho-Rb, EC50 = 37.2 nM

Log [M], Nocodazole

400

0 EC50 = 0.35  0.03 M

75

25

EC50 = 0.35  0.03 M

Blue: Nuclei; Red: phospho-p53 Control

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Blue: Nuclei; Red: Cyclin B1

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Etoposide (M )

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Figure 5: (A) Rb phosphorylation: Rb, a retinoblastoma susceptibility protein is regulated by phosphorylation. When hypophosphorylated, Rb inhibits cell cycle progression by repression of E2F transcription factor until the restriction (R) point in G1 phase. After R point, Rb becomes hyperphosphorylated leading to cell cycle progression. A549 cells were treated with vehicle (DMSO) or 0.5 mg/ml nocodazole for 16 hrs. Nuclear fluorescence intensity of phospho-Rb increases when cells are arrested in G2/M phase. Z-factor for phospho Rb is 0.53 ± 0.04. The normalized fluorescence intensity in drug treated cells was used for EC50. EC50 for phosphor-Rb is 37 nM. (B) PLK1 phosphorylation: Polo like kinase 1 (PLK1) is activated during cell cycle arrest at G2/M. A549 cells were treated with vehicle (DMSO) or 0.5 mg/ml of nocodazole for 16 hrs and then Plk1 phosphorylation was measured after staining with phospho-PLK1 detection kit. Z-factor for phospho-Plk1 is 0.37 ± 0.04. EC50 for phospho-PLK1 is 42 nM. (C) Cyclin B1 activation: Cyclin B1 in cooperation with Cdc2 (Cdk1) plays essential role in mitosis in eukaryotic cells. It is also target of multiple mitotic checkpoints with DNA damage. A549 cells were treated with vehicle (DMSO) or 0.5 mg/ml of nocodazole for 16 hrs and then Cyclin B1 activation was measured after staining with Cyclin B1 activation kit. Z-factor for cyclin B1 is 0.46 ± 0.06. EC50 for cyclin B1 is 46 nM.

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Blue: Nuclei; Green: p53; Red: phospho-ATM

1 μM Camptothecin

Blue: Nuclei; Green: p53; Red: MDM2

C.

B.

B. Control

Blue: Nuclei; Red: phospho-PLK-1

% Control

Control

40

Fold change (Nuclear Intensity)

A.

A.

Table 1: DNA damage and cell cycle check point signaling kits available from ThermoFisher Scientific.

Nocodazole

Etoposide EC50 = 18  2 M Camptothecin EC50 = 1.5 M

4

Blue: Nuclei; Red: Ku 70/80

Phospho p53 intensity

BrdU Cell Proliferation Kit – Singleplex Kit Containing Orange BrdU Probes

0

Blue: Nuclei; Red: phospho-Chk2

Nuc-Cytoplasm Intensity

Singleplex Cell Proliferation Kit, Cat # 8401201, 8401202

-1

400

Etoposide (M ) Transduction of DNA Damage Signal to the Cell Cycle Checkpoint – Activation of MDM2 and Phosphorylation of p53 (Ser15) 0

Catalog number BrdU and ki67 Cell Proliferation Kit – Multiplex Kit containing Green BrdU & Orange Ki67 Probes

-2

Figure 3: (A) Chk2 phosphorylation: Chk2 is phosphorylated by ATM kinase in response to DNAPhospho damage ATM and is important in DNA damage induced checkpoint signaling. A549 cells were treated with vehicle (DMSO) or 50 mM etoposide for 3 hrs. After Control 50 μM Etoposide staining cells with Cellomics Chk2 activation kit, images were acquired and analyzed. 5Z-factor for of etoposide1 or camptothecin treatment for Chk2 phosphorylation is 1.8 mM and 0.6 mM p53phospho-Chk2 is 0.49 ± 0.03 and %COV is 11.2 ± 0.4. EC 50 Control μM Camptothecin respectively. Camptothecin is a topoisomerase I inhibitor and etoposide is a topoisomerase II inhibitor resulting in DNA strand breaks. (B) Activation of ATM and p53: ATM kinase is auto-phosphorylated100 in response to DNA damage and is involved in DNA damage induced activation of p53 and cell cycle arrest. This is a multiplex kit,4enabling the user to simultaneously measure both phospho-ATM and p53. A549 cells were treated with vehicle (DMSO) or 50 mMPhospho etoposide for 24 hrs. After staining p53 cells with Cellomics phospho-ATM/p53 duplex activation kit, images were acquired and analyzed. Z-factor for phospho-ATM = 0.46 ± 0.11 and %COV = 11 ± 3. Z-factor for p53 = 0.44 ± 0.03 and %COV = 13 ± 1. EC50 of etoposide treatment for ATM 75 3 activation is 19 mM and EC50 for p53 activation is 36 mM. Etoposide is a topoisomerase II inhibitor resulting in DNA strand breaks.

DNA damage and cell cycle check point signaling kits available from ThermoFisher Scientific

Multiplex cell proliferation kits, Cat # 8401101, 8401102

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600

0

Blue: Nuclei; Red: phospho-Chk2

p21, Phospho-Rb, Cyclin B1, Phospho-PLK1

Monitors genotoxicity and monitors changes in nuclear morphology and cell permeability.

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800

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Transduction of DNA Damage Signal to the Cell Cycle Checkpoint - Activation of ATM, Chk2 and p53

Figure 1: DNA Damage and Cell Cycle Check Point Signaling.

Phospho-ATM, Phospho-Chk2, p53, Phospho-p53, MDM2

Micronucleus , Cat # K11-0001-1

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then treated with vehicle (DMSO) or 50 mM etoposide for 1 hr. After cells were stained with Cellomics phospho-H2AX kit, images were acquired and analyzed. Z-factor for phospho-H2AX is 0.63 ± 0.04 and %COV is 9.6 ± 0.4. EC50 =of1.76 etoposide or Etoposide EC50  0.05 M camptothecin treatment for H2AX activation is 68 mM and 0.3 mM respectively. (B)800 Ku 70/80 activation: Ku 70/80 is a heterodimer and plays an important role in DNA double strand break repair by non-homologous end joining (NHEJ). A549 cells were 80 Camptothecin EC50 = 0.6 M treated with vehicle (DMSO) or 75 mM etoposide for 24 hrs. After staining cells with Cellomics Ku 70/80 kit, images were acquired and analyzed. Z-factor for Ku 70/80 is 0.47 ± 0.03 and %COV is 11.7 ± 1.0. EC50 of etoposide or camptothecin treatment for Ku 70/80 activation is 18 mM and 1.5 mM respectively. Camptothecin is a topoisomerase I inhibitor and etoposide is a topoisomerase II inhibitor resulting in DNA strand breaks. 60 600

Blue: Nuclei; Green: p53; Red: phospho-ATM

Target

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Control 50 μM Etoposide Control 75 μM Etoposide Figure 2: (A) H2AX phosphorylation: Histone 2AX is rapidly phosphorylated (also known as gH2AX) in response to DNA damage and is a good sensor of DNA double strand breaks. A549 cells were plated in triplicate in 96 well plates overnight and

Phospho-H2AX, Ku 70/80

Cell Cycle Checkpoint Control and Cell Cycle Arrest

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Blue: Nuclei; Red: Ku 70/80

Fold change (Nuclear Intensity)

Checkpoint Signaling

Cell Cycle Checkpoint Control – Phospho Rb, Cyclin B1 and PLK1 Activation

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75 μM Etoposide

Nuc-Cytoplasm Intensity

DNA Damage Sensing and Repair

200

A. Control Control

% cells with p-Chk2 staining

Cellomics HCS Kits

300

Ku 70/80 nuclear Intensity

Control

DNA Damage and Cell Cycle Check Point Signaling Cell Function

Camptothecin EC50 = 0.3 M

B.

Introduction

We developed several high content screening (HCS) assays for measuring DNA damage induced cell cycle check point signaling events. These assays use Cellomics HCS kits that have been validated for robust, multiplexed HCS assay performance, and contain all the key reagents to deliver ease of use, turnkey assay implementation to the end user.

500

Thermo Fisher Scientific • 3747 N. Meridian Rd., PO Box 117, Rockford, Illinois U.S.A. 61105

Blue: Nuclei; Red: phospho-H2AX

Blue: Nuclei; Red: phospho-H2AX

Cells activate DNA damage induced checkpoint signaling in response to DNA double strand breaks. The cell cycle checkpoints trigger cell cycle arrest to repair damaged DNA before transferring DNA to daughter cells. Mutations resulting in a breakdown in the cell cycle checkpoint and arrest machinery cause DNA damage to be inherited by daughter cells, and have been implicated in many cancers as well as in certain aspects of aging in humans. Manipulating DNA damage is also used in cancer therapy. As shown in Figure 1, the DNA damage response involves sensing DNA damage and then transducing it to downstream effectors to cause cell cycle arrest at the appropriate check point. This signaling pathway involves the molecules that sense DNA damage and its repair (phospho H2AX, Ku 70/80); molecules that transduce the DNA damage information to the check point (p53, MDM2, p21, ATM and Chk2) and molecules involved in cell cycle checkpoint control and cell cycle arrest at G1 or G2 (phospho-Rb, Cyclin B1 or phospho-PLK1).

Etoposide EC50 = 68  1M

400

% cells with p-Chk2 staining

DNA damage-induced checkpoint signaling plays an important role in the regulation of genomic integrity and the cell cycle. Chromosomal DNA damaged by either genotoxic agents or DNA replication inhibitors triggers multiple checkpoint signaling events to arrest cell cycle progression at G2/M, which allows cells to repair the damaged DNA. Specifically, DNA damage response activates phosphorylation of ATM and Chk2, resulting in p53 activation and cell cycle arrest. Phosphorylated retinoblastoma (Rb) and polo like kinase 1 (Plk1) proteins also play important roles in cell cycle arrest in G1/S and G2/M, respectively. Automated, quantitative, cell-based imaging high-content screening (HCS) assays enable multiparametric measurements of DNA damage, activation of cell cycle checkpoints and cell cycle arrest in individual cells. Using a Thermo Scientific Cellomics® ArrayScan® HCS Reader and Thermo Scientific Cellomics HCS Reagent Kits, we measured the phosphorylation and activation of ATM, Chk2, p53, Rb and Plk1 following treatment of cells with different compounds. Dose- and time-dependent responses were measured, and compounds were ranked according to their ability to activate DNA damageinduced checkpoint signaling events. These multi-parametric assays comprise an efficient platform to evaluate cell cycle arrest and checkpoint signaling events triggered by genotoxic compounds.

Phospho H2AX nuclear Intensity

Abstract

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• These assays use Cellomics HCS reagent kits which are well characterized for optimum performance and work robustly with Z’ values of ≥ 0.3 and COV of less than 20%. • The kits work robustly with various drugs and cell lines. • All kits contain validated kit components to enable ease-of-use implementation of the assay for the end user.