12Solid tumors require an efficient decision-making mechanism to progress 13 through a gradient of hypoxia. Here, we show that an oxygen-sensory p53 14 tetramer-octamer switch makes cell decision for survival or death in variable 15 hypoxia. Trapping homo-oligomers from biosynthesis cycle, we found a 16 metastable p53 tetramer in cells. Under the operation of switch, tetramer 17 segregates the p53 character of a tumor suppressor and promoter. The p53 18 switch generates a pattern of its on-off state in time that is specific to the 19 strength of hypoxia. A bidirectional tetramer-octamer conversion in on state 20 decides the restoration of basal state by forward and programs apoptosis 21 upon the reverse shift via p53-MDM2 loop. However, reversible dimer-22 tetramer transitions in off state trigger chaperoning of HIF-1 complex by 23 tetramer in forward and oncogenic gain-of-function by prion-like dimers in 24 reverse direction. Temporal on-off patterns calibrate stabilized p53 pool by 25 defining the abundance of dimer, tetramer and octamer that ultimately 26 decides diverse cellular outcomes in hypoxia. Through multi-chromophore 27 FRET, we further show that chaperoning of HIF-1 may modulate angiogenesis 28 through a possible flip-flop of the p53T-HIF-1 complex upon DNA. Our results 29 demonstrate how p53 can sense oxygen and act upon its homo-30 oligomerization states to control cell fate in hypoxic tumors. 31 33 part of specific gene expression in DNA damage repair, cell cycle arrest, apoptosis and 34senescence, has led to propose multiple mechanisms (1). p53 is inactive with loss of its 35 apoptotic potential in the hypoxic region of solid tumors (2) and it remains unknown 36 whether p53 transforms this state in order to control tumorigenesis through a gradient of 37 oxygen. Variable tissue oxygenation in solid tumor triggers p53 conformational 38 transitions from wild-type (WT) to mutant-like (ML) states, or vice versa, in order to 39 control cancer progression (3) and it further modulates post-translational modifications 40 2 in myocardial infarction dictating survival or death of the cell (4). Besides, change in ROS 41 levels differentially controls p53 post-translational modifications in controlling p53-42 mediated apoptosis and necrosis (5). However, the mechanism of p53's varied dynamics 43 under oxygen stress is unknown. The cellular events operate through chemical 44 switchboards, gene-protein loops and protein circuits in response to external stimuli. 45Molecular switches such as bacterial iron-cluster protein that senses molecular oxygen, 46 iron, nitric oxide in countering to changing circumstances (6), GTP-binding between the 47 GDP-bound off and the GTP-bound on state (7,8) and ion-mediated conformational 48 switches and switchable lipids reversibly shifting between two or more stable states in 49 response to stimuli and in microenvironment have been reported (9,10). Protein Tox, a 50 decisive molecular switch for exhaustion mode of immune cells, activates a genetic 51 program that alters immun...
17Low oxygen induces wild type p53 inactivation and selects for mutant-like p53 18 phenotypes for aggressive tumor growth. Recently, we have shown wild type p53 as 19 a cellular oxygen-sensor that operates in switch-like fashion to transform its 20 characters of a tumor suppressor or promoter in a gradient of hypoxia. However, it is 21 unclear how hypoxic tumors select for wild type p53 phenotypes for oxygen-22 sensitive responses. Here, we show that oxygen-generated spatial distribution of the 23 cell population induces p53 phenotype-specific survival or death. We have found 24 that a dynamic state of spatial scatters or clustering patterns of cell populations 25 favor the survival of wild type more than the mutant phenotypes in a wide range of 26 oxygen fluctuation by affecting p53 subcellular localization. Our results demonstrate 27 how spatial distribution could function to establish wild type p53-mediated oxygen 28 sensing and cell fate decisions in a cell population with heterogeneous p53 allele 29 status. We anticipate that such behavior of cells in a gradient of oxygen can be 30 utilized by the hypoxic tumors to maintain distinct p53 alleles and determine the 31 release and metastasis of single or clustered circulating tumor cells (CTCs).32 33 129 Pune), and DU145 p53 WT/MT (NCCS, Pune) were cultured in Dulbecco's modified Eagle's medium 130 (Merck, D5648) supplemented with 10% (v/v) FCS (Thermofisher, 10438018), penicillin (100 131 U/ml), streptomycin (100 U/ ml) and L-glutamine (4 mM) (Merck, G3126), pH 7.4. For the 132 proliferating cell cultures, 5% CO 2 was maintained in a humidified incubator at 37 °C. Hypoxia 133 exposure to the cell culture was performed under a humidified condition in a hypoxia chamber 134 (Plas Labs inc., USA) equipped with O 2 and CO 2 sensors. O 2 and CO 2 levels were quickly restored 135 upon the detection of fluctuations (by ±0.2 units) from the set levels through the automated 136 purging of gases by inbuilt sensors. The normal atmospheric pressure was maintained through 137 the N 2 gas. Similarly, the integrated thermostat maintained the temperature at 37°C. Just before 138 the exposure, DMEM was replenished in the cultures. Re-oxygenation was performed by 139 transferring the cultures from the hypoxia chamber to the CO 2 (5%) incubator for a 24h period.140 Flow cytometry 141 Following the treatments, cells were rinsed with PBS and harvested by mild trypsinization 142 for 10-15 seconds at 37°C. The trypsinized cells were gently detached with chilled PBS 143 supplemented with 2% FBS. The cells were gently washed and diluted (approx. 30,000 cells per 144 173 were washed and blocked in 4% BSA in PBS (with 0.1% TX-100) overnight at 4°C. 174 Endogenous p53 was detected by anti-p53DO1 primary antibody (1:1000 dilutions) at 22°C 175 for 4h. Indirect immune labeling was performed by FITC-tagged secondary antibody (1:5000 176 dilutions) for 2h in dark at room temperature. From now onwards all procedures were 177 performed in dark. Secondary antibodies were washed and SlowFade antif...
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