Thesis of Dana Simiuc

Amphithéâtre Pierre Glorieux (CERLA) Thesis defense of Dana Simiuc - laboratory Phlam The sensitivity of cancer cells to oxidative stress: systemic approach to study the interplay between metabolic flux and oxidative stress Keywords: stress dose, dynamics, metabolism, adaptation; hydrogen peroxide, single cell Abstract : Living cells, when constantly exposed to stress, are able to respond in a complex manner involving various intracellular regulation networks. Their regulation control for instance the cellular fate outcome in response to an oxidative stress. When defensive mechanisms manage to cope against stress, a negative feedback is involved and cell survive, otherwise cell dies. One of a key defensive mechanism relies on the interplay between metabolic flux and oxidative stress exploiting the dualistic role of hydrogen peroxide, acting both as signalling and damaging molecule.
Our work aims to identify key molecules involved in cellular fate and to monitor their dynamics at the single cell level, using fluorescent microscopy. In a first step, we design an experimental system inspired by chemotaxis studies to constantly control the dose applied to breast cancer cell line (MCF7). The choice of the stimulation method plays an important role in our study. Indeed, in order to deliver a constant concentration of stimulus to mammalian cells, non-consuming H2O2 cell culture medium is chosen. Using a fluidic system, the intracellular H2O2 production rate is controlled by varying the external H2O2 concentration. Stimulus delivery and removal is thus performed fast enough (faster than cellular consumption) to study the dynamical cellular responses.
During constant stimulation, adaptation dynamics are notified, suggesting that negative feedbacks are involved in the cellular protection against stress. Cell-to-cell variability is observed and can be quantified using identified adaptation parameters. The fluorescent signal is processed and preliminary results of pH modulation dependence by the cellular metabolic state are discussed. The adaptation features are not depicted when the carbon sources are completely removed from external medium. This result underlines the role of glucose in the cellular defensive mechanism. Another important result is that the feedback dynamics is depending by the H2O2 dose applied to cells: stronger stimulation implies stronger response. It is a first limiting factor we identified while quantifying the cell death response to H2O2 stress. The results of cell death dose response are suggesting that the cell fate (survival or death) is also depending by both the control of the stimulus and the cellular metabolic state. In order to identify the metabolic pathways involved in the negative feedback induced by the oxidative stress, key molecules regulating the Phosphate Pentose Pathway (PPP) are modulated. We
conclude that the orchestration of molecular network is more complex and PPP is the main but not the only network involved in the cellular defence.
In this manuscript an experimental design is presented in order to study the adaptation responses to oxidative stress in real time. Our experiments are confirming the fast adaptation kinetics of NAD(P)H already observed in literature. We identify, for the first time, a second regulation mechanism where the glutathione system is restoring within 30 min during controlled H2O2 stimulation. The glucose metabolism is supporting the regeneration of this antioxidant system and PPP network is thus identified as the main negative feedback in the molecular adaptation here observed.