Journal: bioRxiv

Article Title: MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

doi: 10.1101/573931

Figure Lengend Snippet: a) Quantification of the time in mitosis from the onset of pro-phase until the end of cytokinesis in U2OS-H2B-GFP cells treated with 10 μM TH588 and 0.5 μM Reversine for 24 h. Individual cells shown from a representative experiment, horizontal line indicates the mean. b) Relative tail moment in the modified comet assay of U2OS cells treated with 10 μM TH588 and 0.5 μM Reversine for 24 h. Data from two independent experiments shown with 400 cells analysed in total, horizontal line indicates the median, statistical testing was performed using the Mann-Whitney test (****, p<0.001). c) Viability assay of U2OS cells treated with TH588 with or without 0.5 μM Reversine for 72 h. Mean of 3 independent experiments shown, error bars indicate SD.

Article Snippet: The H2B-GFP vector was constructed by amplifying H2B from the H2B-RFP pENTR1A vector (Addgene # 22525) by PCR and subcloning the product into the pENTR1A-GFP-N2 vector (Addgene # 9364) at the HindIII and BamH1 restriction sites.

Techniques: Modification, Single Cell Gel Electrophoresis, MANN-WHITNEY, Viability Assay

Journal: bioRxiv

Article Title: MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

doi: 10.1101/573931

Figure Lengend Snippet: a) Quantification of the time in mitosis from onset of pro-phase until end of cytokinesis. Data from two experiments, 50 cells analysed in total, horizontal line indicates the mean. Statistical testing performed with unpaired t-test (****p>0.001). b) Pol δ replacement-H2B-GFP cells (WT, wild type; EP, error-prone) were treated with 10 ng/ml doxycyclin for 4 days before treatment with 2.5 μM TH588 and subsequent initiation of the time-lapse microscopy after 30 min. Images were acquired every 10 min for 24 h. Individual cells were followed manually and scored for defects in mitosis including mitotic slippage/polynucleation (MS/PN), micronuclei formation (G1/MN), mitotic slippage (MS) or cell death during mitosis (DiM). Representative of two independent experiments shown.

Article Snippet: The H2B-GFP vector was constructed by amplifying H2B from the H2B-RFP pENTR1A vector (Addgene # 22525) by PCR and subcloning the product into the pENTR1A-GFP-N2 vector (Addgene # 9364) at the HindIII and BamH1 restriction sites.

Techniques: Time-lapse Microscopy

Journal: bioRxiv

Article Title: MTH1 inhibitor TH588 induces mitosis-dependent accumulation of genomic 8-oxodG and disturbs mitotic progression

doi: 10.1101/573931

Figure Lengend Snippet: a) Quantification of the time in mitosis from the onset of pro-phase until the end of cytokinesis followed treatment with 2 nM Paclitaxel (PTX). Individual cells shown from a representative experiment, horizontal line indicates the mean. Statistical testing performed using unpaired t-test (ns, not significant). b) Time-lapse imaging of Pol δ replacement-H2B-GFP cells induced with 10 ng/ml doxycycline for 4 days prior to treatment with 2 nM Paclitaxel (PTX). Images were acquired every 10 min for 24 h. Individual cells were followed manually and scored for defects in mitosis including mitotic slippage/polynucleation (MS/PN), micronuclei formation (G1/MN), mitotic slippage (MS) or cell death during mitosis (DiM). Representative experiment shown. c) Quantification of the time in mitosis of Pol δ replacement-H2B-GFP cells induced with 10 ng/ml doxycycline for 4 days prior to treatment with 1 nM Vincristine or 20 nM CENP-Ei. Individual cells shown from a representative experiment, horizontal line indicates the mean. Statistical testing performed using unpaired t-test (ns, not significant). d) Mechanism of action of TH588. We propose MTH1 inhibition during S phase does not cause replication stress is related to overall low ROS levels also in cancer cells. Prolonged mitotic arrest causes mitophagy and ROS , likely above physiological levels (dotted line). We propose TH588 is causing ROS because of mitotic arrest mediated by directly interfering with tubulin polymerization or breaking MTH1-tubulin interactions. Through MTH1 inhibition, TH588 prevents 8-oxodGTP sanitization resulting in 8-oxodG incorporation into DNA during mitotic replication. We speculate cancer-specific toxicity of TH588 is related to replication stress associated in cancer, likely triggering mitotic replication, i.e., repair synthesis of under replicated regions.

Article Snippet: The H2B-GFP vector was constructed by amplifying H2B from the H2B-RFP pENTR1A vector (Addgene # 22525) by PCR and subcloning the product into the pENTR1A-GFP-N2 vector (Addgene # 9364) at the HindIII and BamH1 restriction sites.

Techniques: Imaging, Inhibition