Figure 1.
Schematic representation of the irradiation set-up.
Schematic representation of the microcollimator irradiation and DNA damage foci on 3D skin cultures (A). The cultures were either half shielded or exposed to microcollimated low LET irradiation from the apical side. For DNA damage foci cultures were 4% paraformaldehyde fixed and paraffin embedded, then sectioned as on (A) for immunofluorescence. For western blot experiments, cultures were cut in halves and lysed immediately as described in Materials and methods. On the bottom panels stained for 53 BP1 sections (green, 53BP1, blue – DAPI nuclear staining). Dose profile of the lines of irradiation as detected on gafchromic film after 30 kVp, 45 mA, 1 µm microcollimator exposure (B). Scale bar 20 µm.
Figure 2.
53BP1 foci induction in 2 D N/TERT-1 keratinocytes cultures.
Directly irradiated (A) and under the shielding ≈2.2 mm away from irradiation (B) *p<0.05, **p<0.01, ***p<0.001 indicate significant statistical difference to background; Two-way ANOVA analysis, Bonferoni post test.
Table 1.
Two phase exponential repair of 53BP1 foci in 2D N/TERT-1 keratinocytes.
Figure 3.
53BP1 foci 30/TERT-1 3D organotypic skin cultures.
A – typical examples of foci, B – dose-dependance in foci induction; statistical analysis (n = 3) t-test: *p<0.05; **p<0.01 difference to background level. Panel C, D show 53BP1 repair kinetics of 2D cultures Vs 3D model for both direct (C) and bystander effect (D). Green staining- 53BP1, blue – DAPI nuclear staining.
Figure 4.
53BP1 foci induction and repair in 2D N/TERT-1 keratinocytes and N/TERT-1 derived 3D skin cultures.
Foci in directly irradiated (A) and bystander (B) cells after 30 kVp X-rays micro-irradiation (1 µm slits, 8 min exposure, dose ∼0.095 Gy, (n = 2)). **-p<0.1; ***-p<0.001; t-test.
Figure 5.
p21 expression after 2 Gy irradiation.
Expression in N/TERT-1 keratinocytes in 2D (A) and 3D conditions (B). Densitometry results from two independent experiments in replicates (C and D).
Figure 6.
Morphological analysis of half shielded 3D organotypic skin cultures 7 days post irradiation.
H&E staining (A). Quantification of the cornified layer thickness of the H&E stained samples (B). * - p<0.05; **- p<0.01; ***- p<0.001, One-way ANOVA analysis, Tukey post-test.
Figure 7.
Cytokeratin 1 and filaggrin expression in half-shielded 3D cultures 7 days post irradiation.
Cytokeratin 1 expression (A–D) and filaggrin expression (E–H). Results (D and H) are mean from 2 independent experiments, 2 replicate slides per experiment and 5 visual fields with 145 µm length per each slide;*** - p<0.001, One way ANOVA analysis, Tukey post-test. Scale bar 20 µm.
Figure 8.
NF-κB phospho-p65 nuclear translocation.
Pannels show localization of the p-p65 unit 1 and 4 h after half-shielded 2 Gy irradiation of 3D skin model. Green – p-p65.
Figure 9.
COX-2 expression in 2D N/TERT-1 cells and 3D organotypic cultures.
COX-2 induction was observed in both irradiated and shielded areas of the 2D and 3D cultures and confirmed by quantification of the relative COX-2 expression (A, C). Dose-dependent induction of COX-2 in 3D skin cultures (B) Graphs represent densitometry analysis of of A, B and C respectively and are the mean of two independent experiments in replicates.
Figure 10.
p-p38 expression in half-shielded 2D (A) and 3D (B) N/TERT-1 keratinocyte cultures after 2 Gy irradiation.
(B) Quantification of the p-p38 relative levels, normalized to GAPDH in 2D (B) and 3D N/TERT-1 cell cultures (D). Results mean from two independent experiments in replicates. SEM-standard error bars. *-p<0.05, One-way ANOVA, Tukey post test.
Figure 11.
Scheme of the proposed radiation-induced signalling in directly irradiated and bystander cells in 3D organotypic skin cultures.
NF-κB is activated in the directly targeted cells from the DNA damage. It leads to secretion of TNF-α which binds to receptors in non-targeted cells and activates the intracellular NF-κB and p38 MAPK. Cx43 GJIC is also feasibly involved in the bystander signal transmission. Active NF-κB and p-p38 activate COX-2 expression and the product of COX-2 PGE2 is responsible for amplification of the pro-inflammatory signaling throughout the tissue. The red symbols show possible target points for inhibition in order to control the signaling.