Forecasting Cell Death Dose-Response from Early Signal Transduction Responses In Vitro

Abstract

The rapid pharmacodynamic response of cells to toxic xenobiotics is primarily coordinated by signal transduction networks, which follow a simple framework: the phosphorylation/dephosphorylation cycle mediated by kinases and phosphatases. However, the time course from initial pharmacodynamic response(s) to cell death following exposure can have a vast range. Viewing this time lag between early signaling events and the ultimate cellular response as an opportunity, we hypothesize that monitoring the phosphorylation of proteins related to cell death and survival pathways at key, early time points may be used to forecast a cell's eventual fate, provided that we can measure and accurately interpret the protein responses. In this paper, we focused on a three-phased approach to forecast cell death after exposure: (1) determine time points relevant to important signaling events (protein phosphorylation) by using estimations of adenosine triphosphate production to reflect the relationship between mitochondrial-driven energy metabolism and kinase response, (2) experimentally determine phosphorylation values for proteins related to cell death and/or survival pathways at these significant time points, and (3) use cluster analysis to predict the dose-response relationship between cellular exposure to a xenobiotic and plasma membrane degradation at 24 h post-exposure. To test this approach, we exposed HepG2 cells to two disparate treatments: a GSK-3β inhibitor and a MEK inhibitor. After using our three-phased approach, we were able to accurately forecast the 24 h HepG2 plasma membrane degradation dose-response from protein phosphorylation values as early as 20 min post-MEK inhibitor exposure and 40 min post-GSK-3β exposure.

Utilizing early cellular changes to explore biological responses to individual chemical and mixture exposures

“…We have also shown that 24 h in vitro cell death dose-response can be forecasted at early time points (as early as 20 min or 40 min, depending upon the exposure) by using twoway cluster analysis of phosphorylation responses at time points relevant to changes in cellular bioenergetics and, consequently, perturbations in signal transduction (Vrana et al, 2014).…”

“…Therefore, it is the delicate balance/imbalance of these pathways that decides the cell's ultimate fate (Bononi et al, 2011;Currie et al, 2014). The highly dynamic and interconnected nature of signaling networks has made it increasingly difficult to elucidate and predict network responses to xenobiotic stress.…”

“…One of the most useful and remarkable features of NADH is that it strongly absorbs at 340 nm; therefore, real time kinetic measurements of cellular NADH can be obtained without the assistance of any fluorescent tag or probe (McComb et al, 1976;Vrana et al, 2014 Finally, one of the most critical components of bioenergetics, ATP can be measured to monitor cellular perturbations after xenobiotic exposure. Previously, assays for intracellular ATP have employed fluorescent tags, which can potentially disrupt endogenous intracellular activity (e.g., FRET) (Berg et al, 2009), or involve cell lysis (e.g., luciferase assay), making real-time in vitro ATP measurements not possible (Imamura et al, 2009).…”

“…This may be particularly true for HepG2 cells, since many cancer cells have been shown to have a high reliance on glycolysis due to the Warburg effect . To account for ATP production when there is limited or no oxygen, an "if then else loop" was used to calculate theoretical ATP This method has proven successful for two disparate xenobiotics, demonstrating a strong correlation to relative ATP measurements collected via the luciferase assay (Vrana et al, 2014).…”

“…shown that 24 h in vitro cell death dose-response can be forecasted at significantly earlier time-points (as early as 20 min or 40 min, depending upon the exposure) by using twoway cluster analysis of phosphorylation responses (Vrana et al, 2014 (disparate kinase inhibitors). The phosphoprotein targets used in this study are key kinases involved in cellular survival and/or death pathways.…”