Background e Fucci (Fluorescent Ubiquitination-Based Cell Cycle Indicator) cell is a recently created line of HeLa cells (cervix carcinoma) [1]. is system uses a couple of oscillating cell cycle proteins, cdt1 and geminin, respectively labeled with monomeric Kusabira Orange 2 *Corresponding author: Baptiste Bedessem, UJF-Grenoble 1, CNRS, Laboratory TIMC-IMAG/DyCTiM, UMR 5525, 38041 Grenoble, France, Tel: +33 0456520045; E-mail: baptiste.bedessem@imag.fr Citation: Bedessem B, Marie-Paule M, Hamel M, Giroud F, Stéphanou A (2015) Effects of the Hypoxia-Mimetic Agents DFO and CoCl 2 on HeLa-Fucci Cells. J Cell Biol Cell Metab 2: 004. Received: February 13, 2015; Accepted: March 12, 2015; Published: March 26, 2015 and monomeric Azami Green. As a consequence, the G1, S, and G2/M phases are labeled with different colors. is cell line is useful to quantify the duration of each phase of the cycle, and to determine the percentage of cycling and non-cycling cells in a non destructive way. Notably, it can be used to visualize in vivo the progression of the cycle [2,3]. In vitro, it is a good tool to observe the effects on cell dynamics of various environmental strains, such as hypoxia. e study of the cellular and molecular effects of hypoxia is one of the important challenge of modern biology. Indeed, it is well established that tumor cells undergo chronic hypoxia, due to the limit of oxygen diffusion in the cancer tissue [4,5]. is chronic hypoxia correlates with an increased resistance to chemotherapy [6,7]. Notably, hypoxia activates anti-apoptotic pathways [8] and can induce proliferation of cancer cells. Indeed, even though hypoxia is known to induce cell cycle arrest in G1 phase [9], it was shown that chronic hypoxia can also stimulate proliferation in various cell lines [10-13]. In a general way, it seems that a balance exists between death and survival/proliferating pathways, depending on the duration and severity of the hypoxic event [14]. e complexity of the cellular response to hypoxia is controlled by the HiF-1α factor, which accumulates when oxygen is lacking [15]. is factor activates signaling pathways which regulate proliferation and death. It was oſten observed that HiF-1α accumulates in cancer cells [16,17]. is accumulation can be due to the physiological chronic hypoxia of cancer cells or genetically determined, for instance by the mutations affecting the Von Hippel-Lindau (VHL) gene [18]. However, the study of the effects of hypoxia using HeLa-Fucci cell line is complex since the lack of oxygen was shown to modify the fluorescence of Kusabira Orange and Azami Green, as shown by Kaida et al., [19,20]. is artefact makes difficult the use of HeLa-Fucci cells in real hypoxic conditions. As a consequence, the study of the effect of the hypoxia pathway, notably the stabilization of HiF-1α, on cell cycle dynamics using this cell line could be done through the use of hypoxia-mimetic chemicals. Indeed, various molecules can be used to mimic hypoxia by inducing HiF-1α accumulation, such as Cobalt Chloride (CoCl 2), Dimethyloxalylglycine (DMOG), Desferrioxamine (DFO). ese molecules are also studied for their anti-cancer effect, as iron-chelators [21,22]. In a general way, they induce HiF-1α accumulation, but they also have numerous HiF-1α-independent effects [23]. Notably, they are known to induce apoptosis [8] and cell cycle arrest through complex interactions with regulating proteins [22]. CoCl 2 and DFO are certainly the most commonly used hypoxia-mimetic molecules [24]. As a consequence, their biological effects were relatively well tested over the last years. ey both induce apoptosis when their concentration is high enough [8,25,26]. However, CoCl 2 seems to promote cell survival during the first 6-8 hours of treatment [27,28]. Cell proliferation in presence of these agents was assessed using different cell lines. Dai et al., (2012) [25] have shown that in presence of CoCl 2, PC-2 cells growth was stimulated during 72h. en, a dose-dependent inhibition of growth was observed, with an increase of the cell death rate. In a general way, CoCl 2 and DFO are known to inhibit cell proliferation [24]. However, their precise influence on cell cycle is variable. In presence of CoCl2, some studies report an arrest in the G1 phase [24,29], or in the G2 phase [30]. In the case of DFO, it is not clear if DFO blocks the cell Bedessem B, et al., J Cell Biol Cell Metab 2015, 2: 004 HSOA Journal of Cell Biology and Cell Metabolism Research Article Abstract The Fucci system is a promising model to study the cell cycle in vivo and in vitro. Because it enables to follow the progression of the cycle using fluorescent microscopy, it can be used to precisely visualize the effects of external strains on proliferation. However, it was reported that the lack of oxygen strongly affects the fluorophores used in the HeLa-Fucci cell line. As a consequence, it limits its use in hypoxic conditions. To study the effects of hypoxia on cell proliferation by using this cell line, one could use HiF-1α inducers, such as CoCl2 and DFO, which are often used as hypoxia-mimetic agents. As these iron-chelators also have anti-cancer properties, their biological effects were studied in various experimental conditions. A great variability of actions were reported. In this study, we investigate for the first time their effects on HeLa-Fucci cells proliferation dynamics. We find that CoCl2 has a biphasic effect on the cell cycle, by promoting or inhibiting the progression into the G1-phase. In presence of DFO, we identified an arrest in the G2-phase, which follows a switch-like behavior. These observations bring original results since iron-chelators are mainly known to induce an arrest at the G1/S transition. They also provide specific information about the possibility to use Fucci cells to study the influence of hypoxia on the cell cycle. Finally, by considering the known genetic characteristics of HeLa cells we propose a hypothesis to explain the particular response of Fucci cells to DFO and CoCl2. The discussion proposes a plausible action of DFO and CoCl2 on the MAPK pathway. Keywords: CoCl2; DFO; HeLa Fucci-cells; Hypoxia; MAPK Baptiste Bedessem*, Marie-Paule Montmasson, Malika Hamel, Françoise Giroud and Angélique Stéphanou UJF-Grenoble 1, CNRS, Laboratory TIMC-IMAG/DyCTIM, UMR 5525, Grenoble, France Effects of the Hypoxia-Mimet- ic Agents DFO and CoCl 2 on HeLa-Fucci Cells