Differential Molecular Response In Mice And Human Thymocytes ...

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Abstract

In the quest for more effective radiation treatment options that can improve both cell killing and healthy tissue recovery, combined radiation therapies are lately in the spotlight. The molecular response to a combined radiation regime where exposure to an initial low dose (priming dose) of ionizing radiation is administered prior to a subsequent higher radiation dose (challenging dose) after a given latency period have not been thoroughly explored. In this study we report on the differential response to either a combined radiation regime or a single challenging dose both in mouse in vivo and in human ex vivo thymocytes. A differential cell cycle response including an increase in the subG1 fraction on cells exposed to the combined regime was found. Together with this, a differential protein expression profiling in several pathways including cell cycle control (ATM, TP53, p21CDKN1A), damage response (γH2AX) and cell death pathways such as apoptosis (Cleaved Caspase-3, PARP1, PKCδ and H3T45ph) and ferroptosis (xCT/GPX4) was demonstrated. This study also shows the epigenetic regulation following a combined regime that alters the expression of chromatin modifiers such as DNMTs (DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3L) and glycosylases (MBD4 and TDG). Furthermore, a study of the underlying cellular status six hours after the priming dose alone showed evidence of retained modifications on the molecular and epigenetic pathways suggesting that the priming dose infers a "radiation awareness phenotype" to the thymocytes, a sensitization key to the differential response seen after the second hit with the challenging dose. These data suggest that combined-dose radiation regimes could be more efficient at making cells respond to radiation and it would be interesting to further investigate how can these schemes be of use to potential new radiation therapies.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

In vivo proliferation study of…

Figure 1

In vivo proliferation study of mouse thymocytes after a single or combined radiation…

Figure 1 In vivo proliferation study of mouse thymocytes after a single or combined radiation regime. (A) Cell cycle analysis using flow cytometry. Propidium iodide (PI) staining of thymocytes for each radiation regime. Representative plot depicted for each time point (left panels) and the mean of 3 biological replicates (right panels). Control cells, single dose (1.75 Gy) and combined regime (0.075 Gy + latency + 1.75 Gy) at the different times assayed after the end of each regime, 30 min (upper panels), 4 h (center panels) and 6 h (lower panels). (B) Western blot analysis of several proteins of the ATM-TP53-phosphoserine-18-TP53 axis. Radiation dose-response and time-dependent protein expression of ataxia telangiectasia mutated protein (ATM) (350 kDa); tumor protein p53 (TP53) (53 kDa); phosphoserine-18-TP53 and p21CDKN1A (21 kDa). Blots show 3 biological replicates for each radiation regime. β-Actin was probed as a loading control for each membrane as shown in annexed Fig. 1. Original blots are presented in supplementary information.
Figure 2

Figure 2

Study of the DNA damage…

Figure 2

Study of the DNA damage response and cell death pathways in thymocytes in…

Figure 2 Study of the DNA damage response and cell death pathways in thymocytes in vivo after a single or combined radiation scheme. Time and dose dependent accumulation of DNA damage and apoptotic pathways markers as determined 30 min, 4 and 6 h after irradiation in control and irradiated thymocytes (with single and combine schemes) by immunoblotting. Damage response marker: (A) γH2AX-Ser139 (15 kDa). Apoptosis response markers: (B) Caspase 3 activated subunits (19 and 17 kDa). (C) Poly (ADP-ribose) polymerase-1 (PARP1) full length (116 kDa) and its cleavaged form (89 kDa fragment). (D) PKCδ full length (78 kDa) and its catalytic fragment (41 kDa). (E) Phosphorylation of H3T45 (H3T45ph) (15 kDa). Ferroptosis response markers: (F) xCT (55 kDa) and (G) GPX4 (17 kDa). β-Actin was probed as a loading control for each membrane in annexed Fig. 2. Original blots are presented in supplementary information.
Figure 3

Figure 3

Study of the response of…

Figure 3

Study of the response of chromatin remodelers to a combined radiation regime versus…

Figure 3 Study of the response of chromatin remodelers to a combined radiation regime versus control or single irradiated mouse thymocytes in vivo. Western blots of 3 biological replicates showing time-dependent changes (30 min, 4 and 6 h) in the expression of several chromatin remodelers such as DNA methylases: DNMT1 (200 kDa), DNMT2 (55 kDa), DNMT3A (130 kDa), DNMT3B (96 kDa) and DNMT3L (49 kDa) and glycosylases such as MBD4 (Methyl Binding Domain) (60–65 kDa) and TDG (Thymine DNA glycosylase) (55–65 kDa) in control thymocytes, thymocytes exposed to a single dose (1.75 Gy) or thymocytes exposed to a radiation combined regime (0.075 Gy + latency + 1.75 Gy). β-Actin was probed as a loading control for each membrane in annexed Fig. 3 Original blots are presented in supplementary information.
Figure 4

Figure 4

Study of the remnant radiation…

Figure 4

Study of the remnant radiation response 6 h after exposure to the priming…

Figure 4 Study of the remnant radiation response 6 h after exposure to the priming dose (0.0075 Gy) in mice in vivo. (A) Cell cycle analysis using flow cytometry. Propidium iodide (PI) staining of thymocytes for control cells and thymocytes exposed to the priming dose (0.0075 Gy) 6 h after exposure. Representative plot (left panel) and the mean of 3 biological replicates (right panel). (B) Western blot analysis of several proteins of the ATM-TP53-phosphoserine-18-TP53 axis. Expression changes of ataxia telangiectasia mutated protein (ATM); tumor protein p53 (TP53); phosphoserine-18-TP53 and p21CDKN1A. Blots show 3 biological replicates for each radiation regime. (C) Markers of the damage response and cell death pathways were determined by immunoblotting with antibodies specific against γH2AX-Ser139 (15 kDa), Caspase 3 activated subunits (19 and 17 kDa), Poly (ADP-ribose) polymerase-1 (PARP1) full length (116 kDa) and its cleavaged form (89 kDa fragment), PKCδ full length (78 kDa) and its catalytic fragment (41 kDa) and Phosphorylation of H3T45 (H3T45ph) (15 kDa). (D) Chromatin remodellers study 6 h after the priming dose. Comparative analysis of the endogenous expression of DNMTs DNMT1 (200 kDa), DNMT2 (55 kDa), DNMT3A (130 kDa), DNMT3B (96 kDa) and DNMT3L (49 kDa) and the glycosylases MBD4 (Methyl Binding Domain) (60–65 kDa) and TDG (Thymine DNA glycosylase) (55–65 kDa) in mouse thymocytes 6 h after the priming dose. β-Actin was probed as a loading control for each membrane in annexed Fig. 4. Original blots are presented in supplementary information.
Figure 5

Figure 5

Ex vivo response to a…

Figure 5

Ex vivo response to a combined regime in human thymocytes. ( A )…

Figure 5 Ex vivo response to a combined regime in human thymocytes. (A) Cell cycle analysis using flow cytometry of human thymocytes exposed to radiation ex-vivo. Propidium iodide (PI) staining of thymocytes for each radiation regime; control cells, single dose (1.75 Gy) and combined regime (0.075 Gy + latency + 1.75 Gy) 30 min after exposure. Representative plot depicted in the left panel. The mean of 3 biological replicates is shown in the right panel. (B). Western blot analysis of several proteins of the ATM-TP53-phosphoserine-18-TP53 axis, damage response and cell death pathways in human thymocytes. Expression changes of phosphoserine-15-TP53, p21CDKN1A, γH2A.X-Ser139 (15 kDa) and caspase 3 activated subunits (19 and 17 kDa). Blots show 1 representative biological replicate. (C) Chromatin remodellers study in human thymocytes. Protein profiling of DNMT1 (200 kDa), DNMT2 (55 kDa), DNMT3A (130 kDa) and TDG (55–65 kDa) after the radiation treatment with either a single dose (1.75 Gy) or the combined regime (0.075 Gy + latency + 1.75 Gy) 30 min after exposure. Representative blots are presented. (E) Priming dose study in human thymocytes. Protein status of phosphoserine-15-TP53, p21CDKN1A, γH2AX-Ser139 (15 kDa) and caspase 3 activated subunits (19 and 17 kDa) 6 h after the priming dose (0.0075 Gy). (F). Chromatin remodellers study 6 h after the priming dose in human thymocytes. Protein expression study of DNMT1, DNMT2, DNMT3A and TDG. Data shows 2 biological replicates for the control cells and 2 biological replicates for cells irradiated with the priming dose after a latency of 6 h. β-Actin was probed as a loading control for each membrane in annexed Fig. 5. Original blots are presented in supplementary information.
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