Induction of apoptosis by ionizing radiation following p53 stabilization.

<p>H295R, SW-13 and SK-OV-3 cell lines were transfected with empty vector (mock) or p53-vector (WT) and irradiated at a dose of 6 Gy. (A) TUNEL assay was performed at 48 and 72 after transfection. In all cell lines, TUNEL-positive cells are detectable only in irradiated samples expressing the wild type form of <i>TP53</i>. Cells were also stained with Hoechst 33342 and then visualized with a fluorescence microscope at a magnification of 40×. Figure shown is representative of three experiments performed in duplicate. (B) Percentage of apoptosis in H295R, SW-13 and SK-OV-3 was calculated by comparing TUNEL-positive cells with total cells, stained with Hoechst 33342. Apoptosis increases in cells transfected with p53 vector compared with mock-irradiated samples, starting from 48 h since transfection and the effect strengthens after 72 h. (*, p<0.05; #, p<0.01). (C) <i>BCL2</i> expression was evaluated in H295R cell line by RT-PCR. p53 restoration induces a significant reduction in <i>BCL2</i> expression at 48 and 72 h after irradiation. (D) Inverted images relative to semi-quantitative RT-PCR analysis of <i>BCL2</i> gene expression performed on SW-13 cells, showing a significant reduction in <i>BCL2</i> signal in cells transfected with p53-vector following ionizing radiations treatment. (E) The effect of p53 restoration on <i>BCL2</i> mRNA levels in response to irradiation was evaluated in SK-OV-3 cell lines. p53 inhibits <i>BCL2</i> expression and the effect is stronger at 72 h after transfection. Images are representative of at least three independent experiments. <i>GAPDH</i> was used for normalization.</p

Ultrastructure of β cells.

<p>Trasmission electron micrographs of STZ and STZ+T3 treated pancreatic islets compared to control (Uranyl acetate/lead citrate; space bar 1 µm). Nu, nucleus; MB, myelinic bodies. The ultrastructure of the β cells was affected by STZ and maintained unaltered by the addition of T3. Data are from 1 or 2 experiments with similar results (n = 5 animals/group).</p

Phisiological parameters.

<p><b>A, B</b>: Analysis of blood glucose and Insulin levels after intra-peritoneal glucose tolerance test (upper panels). Glycemia was measured by glucometer, while Insulin concentration was assessed by ELISA assay, as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Oral administration of T3 significantly reduces severity and progression of STZ-induced diabetes in Balb/c mice and assured normal Insulin responsiveness. <b>C</b>: Insulin tolerance was performed (lower panel) after intra peritoneal glucose injection. Insulin was injected intraperitoneally after glucose to the different experimental groups of animals. Glycemia was measured by glucometer. Results represent the mean ± SE of three separate experiments. Grey: control black:STZ white:STZ+T3. <b>D</b> RT-PCR: Total RNA was extracted from liver from mice of the different experimental groups and RTPCR was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. A single product was obtained for each gene, as showed by agarose electrophoresis. All PCR products were of the expected size and sequence. The presence of T3 did not induce any change in the DIO1 expression, as normalized to 18s.</p

Effect of p53 over-expression and irradiation on cell proliferation in H295R, SW-13 and SK-OV-3 cells.

<p>Cell growth was evaluated in H295R, SW-13 and SK-OV-3 cell lines transfected with empty vector (mock) or p53-vector (WT), either irradiated or not. (A) In H295R cell lines (left), ionizing radiation (IR) treatment induces a significant inhibition of cell growth in cells expressing wild type p53, starting from 48 h after transfection (−18% compared to mock irradiated cells; p<0.05) and reaching −25% after 72 h (p<0.01). Irradiation does not induce a stable effect in SW-13 cells (right) transfected with empty vector, whereas cells expressing wild type p53 are significantly inhibited at 48 h after transfection (−28%; p<0.05) until the end of the experiment (−31%; p<0.01). In SK-OV-3 cells (bottom), p53 restoration induces an strong effect on cell growth, which is evident since 48 from transfection (−20% of growth inhibition in WT+IR sample compared with mock +IR; p<0.01) and endures up to 72 h (−28%; p<0.01). Data are mean ± S.D. of at least three independent experiments carried out by duplicates. (B) Western blotting analysis of cyclin E and cyclin D1 expression in H295R cells at 72 h after transfection revealed a down-regulation of these proteins in cells expressing ^wtp53 treated with IR at a dose of 6 Gy. (C) Analysis of cyclin E and cyclin D1 expression in SW-13 cell line by Western blotting shows a decrease in protein levels in samples transfected with p53-vector after irradiation. Bands' intensities were quantified with ImageJ software, using vinculin for normalization. (*, p<0.05).</p

Sequencing analysis of <i>TP53</i> coding sequence in H295R and SW-13 cell lines.

<p>(A) Representation of <i>TP53</i> mutations identified in H295R and SW-13 cell lines, showing a large deletion affecting 211 base pairs in H295R cells (top) and a homozygous point mutation in exon 6 (<i>r.577c>u</i>) in SW-13 cell line (bottom). SW-13 cells also exhibit a polymorphic site located in exon 4 (<i>r.215c>g</i>). (B) Inverted image relative to RT-PCR analysis of exons 7 to 10 of <i>TP53</i> gene. Electrophoresis on a 2% agarose gel revealed a major band of ∼575 bp, corresponding to SW-13 transcript, and a minor band of ∼364 bp, corresponding to H295R transcript (DNA ladder, 1 Kb plus). (C) Predicted amino-acid sequence of p53 in H295R and SW-13 cell lines. Deletion of exons 8 and 9 in H295R determines a frameshift starting at codon 261, creating a subsequent stop codon at position 274 (top). In SW-13 cells, a homozygous point mutation at codon 193 determines an amino-acid substitution (histidine to tyrosine) in the DBD of the protein (bottom). (D) Western blotting analysis of p53 in H295R and SW-13 cells, showing the presence of a shorter protein in H295R cell line, which molecular weight is ∼44 kDa.</p

Thyroid hormone receptors expression.

<p>Tissue sections from the different experimental groups of animals have been obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Indirect immunofluorescence for Thyroid Receptor (Red) and Insulin (Greeen) revealed the presence of TR α/β within the islets and the pancreatic tissue surrounding. Nuclei were counterstained with Hoechst (blue). Data are from 1 or 2 experiments with similar results (n = 5 animals/group). At least ten fields <i>per</i> chamber and three independent cultures were examined. Space bar: 100 µm. Western Blot analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> and a specific band corresponding to the Thyroid Receptor α/β was detected. The expression of B-actin was analyzed as a control for gel loading. At least three different experiments were performed, and a representative one is shown here. Densitometric absorbance values from three separate experiments were averaged (± SD), after they had been normalized to B-actin for equal loading. Data are presented on the right of the Western Blot panel in the histogram as Relative Densitometric Units (y axis). The different experimental groups are indicated on the x axis. A comparison of the individual treatment was conducted by using Student's <i>t</i> test. p = 0.003.</p

Effect of p53 stabilization on Akt activation after ionizing radiation treatment.

<p>(A) Western blotting analysis of total Akt and phosphorylated Akt levels, performed on irradiated H295R cells transfected with empty vector (mock) and p53-vector (WT) at 48 and 72 h after transfection. Densitometric analysis of Akt normalized to vinculin and p-Akt <i>versus</i> Akt shows that Akt expression is not influenced by p53 status, while the activated form of the protein is significantly reduced in ^wtp53-expressing cells. (B) Total cell lysates obtained from SW-13 cell line were subjected to Western blotting analysis using antibodies targeting Akt and Akt-pSer473. Phosphorylation levels of Akt are significantly reduced by p53 stabilization in response to ionizing radiation treatment. Results shown are representative of at least three experiments. Bands' intensities were quantified using ImageJ and bands' densitometry is shown in the right panels. (*, p<0.05; #, p<0.01).</p

RT-PCR analysis of ^wtp53 effect on <i>IGF2</i> expression.

<p>(A) Inverted images relative to semi-quantitative RT-PCR analysis of <i>IGF2</i> gene expression performed on H295R cells, showing a massive loss of <i>IGF2</i> signal in irradiated cells at 72 h after transfection of p53-vector (WT). (B) RT-PCR analysis of <i>IGF2</i> mRNA levels performed on SW-13 cells. A significant reduction in <i>IGF2</i> expression is detectable in cells transfected with p53-vector (WT) following ionizing radiations treatment. Images are representative of three independent experiments. <i>GAPDH</i> expression was used for normalization and bands' intensities were quantified using ImageJ. Band densitometry is shown in the right panels. (*, p<0.05; #, p<0.01).</p

Effect of ionizing radiations on p53 stabilization.

<p>Expression levels of <i>TP53</i> and p53 protein evaluated by Real Time RT-PCR and Western blotting in H295R, SW-13 and SK-OV-3 cells 72 h after transfection with empty vector (mock) or p53-vector (WT). Samples were treated with IR at a dose of 6 Gy where indicated. (A) RT-PCR (top) and Real Time RT-PCR (bottom) analysis of <i>TP53</i> expression in H295R cells. Samples transfected with pBABE-neo-p53 vector show a 5-fold increase in <i>TP53</i> mRNA compared with mock. (B) Western blotting analysis of p53 in H295R cells (top) revealed a major band of ∼53 kDa (^wtp53) and a minor band of ∼44 kDa, corresponding to the truncated form of p53 (p53^Δ8–9). Densitometry shows that ^wtp53 is stabilized by IR treatment (bottom). (C) RT-PCR analysis of <i>TP53</i> mRNA in SW-13 cells shows over-expression of the gene in samples transfected with pBABE-neo-p53 vector (top). Real Time RT-PCR analysis revealed a 3-fold increase in <i>TP53</i> transcript in samples transfected with p53 vector. The expression of <i>TP53</i> is not modulated by irradiation (bottom). (D) Levels of p53 protein in SW-13 cells significantly increase after IR treatment in cells transfected with pBABE-neo-p53 vector, while no change is detectable in samples transfected with empty vector. (E) In SK-OV-3 cells, no <i>TP53</i> transcript was detectable by RT-PCR in samples transfected with empty vector, while in cells transfected with pBABE-neo-p53 vector a strong signal, corresponding to <i>TP53</i> mRNA, is observable (top). Real Time RT-PCR analysis shows no modulation of <i>TP53</i> expression after irradiation (bottom). (F) Western blotting analysis of p53 in SK-OV-3 cells revealed a detectable band only in samples transfected with pBABE-neo-p53 vector (top). Densitometry shows that p53 levels increase after irradiation, indicating the stabilization of the protein (bottom). Results are representative of at least three independent experiments. <i>GAPDH</i> and vinculin were used for normalization. (*, p<0.05; #, p<0.01).</p

Glut-2 status.

<p>Immunofluorescence: Tissue sections from the different experimental groups of animals have been obtained as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> section. Indirect Immunofluorescence for Glut-2 (green) revealed the presence of the Glucose transporter within the islets, clearly detectable. Nuclei were counterstained with Hoechst (blue). As shown with T3 treatment, no differences either in the expression levels or in the localization of the Glucose transporter Glut-2 was observed. On the other hand, the presence of the STZ was sufficient to cause a strong disarray in both expression and localization of Glut-2 in the islets. Data are from 1 or 2 experiments with similar results (n = 5 animals/group). At least ten fields <i>per</i> chamber and three independent cultures were examined. Space bar: 100 µm. Western Blot: Western Blot analyses were performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019839#s2" target="_blank">Materials and Methods</a> on protein extracts from the CTR and the T3 treated animals and a specific band corresponding to Glut-2 was detected. The presence of T3 did not provoke any change in the Glut-2 expression, as compared to B-actin expression. The expression of B-actin was analyzed as a control for gel loading. At least three different experiments were performed, and a representative one is shown here. Densitometric absorbance values from three separate experiments were averaged (± SD), after they had been normalized to B-actin for equal loading. Data are presented on the right of the Western Blot panel in the histogram as Relative Densitometric Units (y axis). The different experimental groups are indicated on the x axis. A comparison of the individual treatment was conducted by using Student's <i>t</i> test. P<0.003.</p