DNA Damage Strength Modulates a Bimodal Switch of P53 Dynamics For
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Chen et al. BMC Biology 2013, 11:73 http://www.biomedcentral.com/1741-7007/11/73 RESEARCH ARTICLE Open Access DNA damage strength modulates a bimodal switch of p53 dynamics for cell-fate control Xi Chen1, Jia Chen2, Siting Gan2, Huaji Guan1, Yuan Zhou1, Qi Ouyang2* and Jue Shi1* Abstract Background: The p53 pathway is differentially activated in response to distinct DNA damage, leading to alternative phenotypic outcomes in mammalian cells. Recent evidence suggests that p53 expression dynamics play an important role in the differential regulation of cell fate, but questions remain as to how p53 dynamics and the subsequent cellular response are modulated by variable DNA damage. Results: We identified a novel, bimodal switch of p53 dynamics modulated by DNA-damage strength that is crucial for cell-fate control. After low DNA damage, p53 underwent periodic pulsing and cells entered cell-cycle arrest. After high DNA damage, p53 underwent a strong monotonic increase and cells activated apoptosis. We found that the damage dose-dependent bimodal switch was due to differential Mdm2 upregulation, which controlled the alternative cell fates mainly by modulating the induction level and pro-apoptotic activities of p53. Conclusions: Our findings not only uncover a new mode of regulation for p53 dynamics and cell fate, but also suggest that p53 oscillation may function as a suppressor, maintaining a low level of p53 induction and pro-apoptotic activities so as to render cell-cycle arrest that allows damage repair. Background known to upregulate p53 by attenuating its interaction An efficient and precisely controlled DNA-damage re- with Mdm2, the E3 ubiquitin ligase that targets p53 for sponse is crucial for cell survival. In mammalian cells, proteasome-mediated degradation [5,6]. Less clear is the p53 regulatory pathway, consisting mainly of the how the severity of DNA damage modulates the degree tumor suppressor protein p53 and its downstream tran- of attenuation in p53-Mdm2 interactions, giving rise to scriptional targets, plays an essential role in mediating differential p53 expression that results in these alterna- this crucial stress response to chromosomal damage tive cell fates. Also not clear is how the dynamics of p53 [1-3]. Depending on the type and severity of DNA dam- differ with variation in damage level, and what role any age, the p53 pathway activates either cell-cycle arrest changes in the dynamics might play in the downstream that allows repair of the damage or alternatively, death function of p53. Most previous studies of p53 dynamics of the cell through apoptosis, but the mechanism by have focused only on the response to transient DNA which variation in DNA-damage strength differentially damage induced by gamma or UV irradiation, which has regulates p53 pathway dynamics, and the effect that this been shown to induce an intriguing oscillatory behavior of has on cell fate, are poorly understood. p53, which is largely independent of DNA damage level Mild DNA damage generally induces a moderate in- [7-11]. Moreover, although recent results, including our crease in p53 level, and results in transient cell-cycle ar- own [11,12], have shown that p53 dynamics can be altered rest that allows damage repair, whereas severe and by using nutlin to inhibit Mdm2, the observed change in possibly irreparable DNA damage leads to a large in- p53 dynamics was again independent of damage severity. crease in p53, followed by cell death [4]. DNA damage is To investigate whether and how DNA-damage strength modulates p53 dynamics and subsequently alters cell-fate outcome, we performed a dose–response study by meas- * Correspondence: [email protected]; [email protected] 2 uring the real-time p53 dynamics under variable DNA Center for Quantitative Biology and State Key Laboratory for Mesoscopic Physics, Peking University, 100871, Beijing, P. R. China damage generated by etoposide, a chemotherapeutic com- 1Center for Quantitative Systems Biology and Department of Physics, Hong Kong pound that induces sustained DNA damage. Using time- Baptist University, 224 Waterloo Road, Kowloon Tong, Kowloon, Hong Kong, China © 2013 Chen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Chen et al. BMC Biology 2013, 11:73 Page 2 of 11 http://www.biomedcentral.com/1741-7007/11/73 lapse microscopy, we quantified p53 dynamics at the the degree of DNA damage increased with increasing dos- single-cell level, and correlated the dynamics with cell age of etoposide from 1 μmol/l to 75 to 100 μmol/l. fates. Our results showed that p53 dynamics exhibit To verify that dynamics of the p53-Venus reporter ‘switch-like’ behavior, changing from oscillatory dynamics construct in our clonal reporter line mimicked the en- at low damage to monotonic increase at high damage. dogenous p53, we compared the dynamics of the en- Moreover, this damage dose-dependent, bimodal switch of dogenous and fluorescently tagged p53 by western p53 dynamics was found to regulate cell fate mainly by blotting. The dynamics of the p53-Venus construct that modulating the p53 induction level and its pro-apoptotic we used have been extensively studied previously in activities as a function of DNA-damage strength. Our re- MCF7 cells, which all showed that the reporter con- sults suggest that under certain conditions, p53 oscilla- struct behaved similarly to the endogenous p53 [9,10]. tions may act as a mechanism to suppress p53 induction, Our data from the U-2 OS clone also showed similar thereby restraining its pro-apoptotic activities and pro- induction dynamics between p53-Venus and its en- moting cell-cycle arrest that allows DNA-damage repair. dogenous counterpart (Figure 1b; note that the level of p53-Venus reporter is about half of the endogenous p53. Results For quantified results of western blots, see Additional Dynamics of nuclear p53 is damage dose-dependent file 1: Figure S1), when cells were treated with both low To obtain data on p53 dynamics and cell fate as a func- and high doses of etoposide. Therefore, the fluorescent tion of DNA-damage strength, we performed dose titra- p53 reporter is probably regulated similarly to the en- tion of a DNA-damaging drug, etoposide, on U-2 OS dogenous wild-type p53, and can be used to infer p53 cells expressing a fluorescent p53 reporter. The reporter dynamics in response to DNA damage. cell line was generated by infecting U-2 OS cells with From time-lapse microscopy movies, we quantified the lentivirus encoding an established wild-type p53-Venus real-time induction dynamics of p53 by measuring aver- reporter construct [9]. For the study, we selected an iso- age Venus fluorescence in the nucleus and cytoplasm, genic clone that exhibited behaviors most similar to the respectively. In general, the fluorescent signal of p53- parental line (refer to discussion below). We used Venus in the nucleus was significantly higher than that etoposide as the DNA-damage stimulus, as it is known to in the cytoplasm. Single-cell p53 fluorescence trajector- trigger DNA double-strand breaks (DSBs) by inhibiting ies showed two distinctive dynamic features: 1) nuclear topoisomerase II [13,14]. To select the appropriate range p53 exhibited periodic pulsing at low drug dose, but of etoposide dosage for activating variable amounts of monotonic increase at high dose; and 2) the amplitude of DNA damage, we first measured the level of DNA damage monotonically elevated p53 was substantially higher than under different etoposide doses. DNA damage, as indi- that of the pulsing mode (Figure 1c; see Additional file 1: cated by the DNA-damage marker, phospho-H2A.X, was Figure S2). These bimodal, damage dose-dependent p53 present after 12 hours of drug treatment (Figure 1a), and dynamics are in stark contrast to the dose-independent Figure 1 Differential p53 dynamics as a function of DNA-damage strength. (a) Phospho-H2A.X levels (DNA damage marker) in U-2 OS cells treated with increasing doses of etoposide for 12 hours. Actin served as a loading control. (b) Induction kinetics of p53-Venus and endogenous p53 in the clonal U-2 OS report line treated with 1 and 100 μmol/l etoposide at the indicated time points (in hour). The upper band is p53-Venus (around 80 kDa) and the lower band is endogenous p53 (approximately 53 kDa). (c) Single-cell trajectories of p53 dynamics at the nucleus quantified from p53-Venus fluorescence. U-2 OS cells were treated with either 1 μmol/l (black lines) or 100 μmol/l (red lines) of etoposide at time 0. Individual cells were tracked for 72 hours or until cell death occurred. The abrupt end of p53 trajectories before 72 hours corresponds to the time of death. Chen et al. BMC Biology 2013, 11:73 Page 3 of 11 http://www.biomedcentral.com/1741-7007/11/73 oscillatory response of p53 to gamma irradiation previ- into cell-cycle arrest, and more than 83% of those with ously identified in single mammalian cells [8]. The obser- monotonic p53 induction died. Our data thus suggest that vation of a switch from oscillatory dynamics to a the bimodal switch and its correlation with alternative cell monotonic increase in p53 as DNA damage increased also fate are common in at least some mammalian cells in re- suggests an intriguing new transitional regulation of p53. sponse to variable DNA damage. We noted that although the correlation between cell The distinct mode of p53 dynamics strongly correlates fate and p53 induction dynamics was evident across the with cell fate entire cell population, there was strong cell-to-cell vari- Besides p53 dynamics, cumulative survival curves (quan- ation in terms of the quantitative characteristics.