Dose-Response Modeling of Etoposide-Induced DNA Damage Response

Abstract

The 2007 National Research Council Report "Toxicity Testing in the 21st Century: A Vision and A Strategy" recommended an integrated, toxicity pathway-oriented approach for chemical testing. As an integral component of the recommendation, computational dose-response modeling of toxicity pathways promises to provide mechanistic interpretation and prediction of adverse cellular outcomes. Among the many toxicity pathways, the DNA damage response is better characterized and thus more suited for computational modeling. In the present study, we formulated a minimal mathematical model of this pathway to examine the dose response for etoposide (ETP), an anticancer drug that causes DNA double strand breaks (DSBs). In the model, DSB results from inhibition of topoisomerase by ETP and p53 is activated by a bistable switch composed of a positive feedback loop between ATM and γH2AX. Our stochastic model recapitulated the dose response for several molecular biomarkers measured with flow cytometry in HT1080 cells, including phosphorylated p53, ATM, γH2AX, and micronuclei. Model simulations were consistent with a bimodal pattern of p53 activation and a graded population-averaged response at high ETP concentrations. The graded response was a result of heterogeneous activation of individual cells due to molecular stochasticity. This work shows the value of combining data collection on single cell responses and mechanistic, stochastic modeling to develop and test hypothesis for the circuitry of important toxicity pathways. Future studies will determine how well this initial modeling effort agrees with a broader set of experimental studies on pathway responses by examining a more diverse group of DNA-damaging compounds.

miR-15a/miR-16 down-regulates BMI1, impacting Ub-H2A mediated DNA repair and breast cancer cell sensitivity to doxorubicin

“…Double strand break repair follows two major pathways; homologous recombination (HR) and non-homologous end-joining (NHEJ) 48 . BMI1contributesto DNA double-stranded break repair by homologous recombination 25 .…”

Chemical exposure and infant leukaemia: development of an adverse outcome pathway (AOP) for aetiology and risk assessment research

“…There should be some information about the concentration-response relationships of the selected tool chemical in the development of the AOP. Molecular dose-response modelling of etoposideinduced DNA damage response, based on comprehensive in vitro high content imaging in the HT1080 cell model, was developed by Li et al (2014). The model was based on the hypothesis that cells are capable of clearing low-level DNA damage with existing repair capacity; however, when the number of double strand breaks (DSBs) exceeds a threshold value, ataxia telangiectasia mutated (ATM) is recruited and becomes fully activated through a reversible mechanism, leading to elevated repair capacity as a result of phosphorylation (activation) of several target proteins, including p53 and other tumour suppressor proteins.…”

“…The model was able to quantitatively capture the doseresponse relationships of a number of markers observed with etoposide. Especially interesting are the dose-response relationships for activation of p53 and the formation of micronuclei in the target cell model, which indicate point-of-departure concentrations of etoposide in the range of 0.01 to 0.1 µM (Li et al, 2014). This range is in agreement with the finding that in human foetal liver CD34 + cells an increase in DSBs was observed at a concentration of 0.14 µM (Bueno et al, 2009) and MLL-r were detectable by FISH or flow cytometry at higher concentrations (Moneypenny et al, 2006).…”

“…(phosphorylated form of the histone H2AX) or the Comet assay (Li et al, 2014, Schroeter et al, 2015.…”

Assessment of mechanisms driving non-linear dose–response relationships in genotoxicity testing

“…As part of the ''Tox21'' 1647 program [70,148,209], that recommends testing based on adverse 1648 or toxicity pathways by using primarily in vitro experiments, dose-1649 response of some genotoxicants (MMS, H 2 O 2 , etoposide, quercetin 1650 and curcumin) have been studied. The aim is to quantitatively 1651 assess a genotoxic response in relation to potential biochemical 1652 toxicity pathways to mechanistically underline a non-linear dose 1653 effect relationship [148]. In an extensive in vitro experiment, the 1654 dose-response of etoposide-induced MN was studies.…”