Journal of historical geography

Follistatin is a potent regulator of the inflammatory response and binds to and inhibits activin A action. Activin A is a member of the TGFβ protein superfamily which has regulatory roles in the inflammatory response and in the fibrotic process. Fibrosis can occur following cell injury and cell death induced by agents such as ionizing radiation (IR). IR is used to treat cancer and marked fibrotic response is a normal tissue (non-tumour) consequence in a fraction of patients under the current dose regimes. The discovery and development of a therapeutic to abate fibrosis in these radiosensitive patients would be a major advance for cancer radiotherapy. Likewise, prediction of which patients are susceptible to fibrosis would enable individualization of treatment and provide an opportunity for pre-emptive fibrosis control and better tumour treatment outcomes. The levels of activin A and follistatin were measured in fibroblasts derived from patients who developed severe radiation-induced fibrosis following radiotherapy and compared to fibroblasts from patients who did not. Both follistatin and activin A gene expression levels were increased following IR and the follistatin gene expression level was lower in the fibroblasts from fibrosis patients compared to controls at both basal levels and after IR. The major follistatin transcript variants were found to have a similar response to IR and both were reduced in fibrosis patients. Levels of follistatin and activin A secreted in the fibroblast culture medium also increased in response to IR and the relative follistatin protein levels were significantly lower in the samples derived from fibrosis patients. The decrease in the follistatin levels can lead to an increased bioactivity of activin A and hence may provide a useful measurement to identify patients at risk of a severe fibrotic response to IR. Additionally, follistatin, by its ability to neutralise the actions of activin A may be of value as an anti-fibrotic for radiation induced fibrosis

Synchrotron X-Ray Radiation-Induced Bystander Effect: An Impact of the Scattered Radiation, Distance From the Irradiated Site and p53 Cell Status

Synchrotron radiation, especially microbeam radiotherapy (MRT), has a great potential to improve cancer radiotherapy, but non-targeted effects of synchrotron radiation have not yet been sufficiently explored. We have previously demonstrated that scattered synchrotron radiation induces measurable γ-H2AX foci, a biomarker of DNA double-strand breaks, at biologically relevant distances from the irradiated field that could contribute to the apparent accumulation of bystander DNA damage detected in cells and tissues outside of the irradiated area. Here, we quantified an impact of scattered radiation to DNA damage response in "naïve" cells sharing the medium with the cells that were exposed to synchrotron radiation. To understand the effect of genetic alterations in naïve cells, we utilised p53-null and p53-wild-type human colon cancer cells HCT116. The cells were grown in two-well chamber slides, with only one of nine zones (of equal area) of one well irradiated with broad beam or MRT. γ-H2AX foci per cell values induced by scattered radiation in selected zones of the unirradiated well were compared to the commensurate values from selected zones in the irradiated well, with matching distances from the irradiated zone. Scattered radiation highly impacted the DNA damage response in both wells and a pronounced distance-independent bystander DNA damage was generated by broad-beam irradiations, while MRT-generated bystander response was negligible. For p53-null cells, a trend for a reduced response to scattered irradiation was observed, but not to bystander signalling. These results will be taken into account for the assessment of genotoxic effects in surrounding non-targeted tissues in preclinical experiments designed to optimise conditions for clinical MRT and for cancer treatment in patients

Alternative transcripts in DNA repair genes are induced by IR.

<p>Alternative transcripts in the DNA repair genes, XPC (A) and RRM2B (B) in response to IR are shown. Graph axes are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g001" target="_blank">figure 1</a>; PCR products from 5′-RML-RACE were run on a 2% agarose gel. An arrow indicates the amplicon from the alternative initiated transcript that was sequenced (gel picture to the right of the panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone.0053358.s004" target="_blank">Figure S4</a>). Diagrams of the predominant transcripts (initiating by P1) and the alternatively initiated (P2) transcripts after IR are shown below. Primer locations for 5′ RLM-RACE are indicated below exon 2 (arrow pointing to the left).</p

Induction of DNA repair genes at the exon level four hours after treatment with 10 Gy IR in primary fibroblast cells.

<p>The DNA repair genes: <i>XPC</i> (A), <i>POLH</i> (B), <i>DDB2</i> (C), <i>PCNA</i> (D) and <i>RRM2B</i> (E) as identified using Partek Genomics Suite 6.6 statistical package. Relative fluorescence (y-axis; log₂) is plotted for each PSR (x-axis). Core PSRs are labelled numerically in a 5′ to 3′ direction (left to right). Samples were either sham irradiated (red) or irradiated (blue) with 10 Gy of radiation from a ¹³⁷Cs source. RNA was collected 4 hours following treatment. Arrow indicates the PSR region to which primers were designed for qRT-PCR used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g003" target="_blank">figure 3B</a>. Error bars = SEM (n = 12).</p

DNA repair genes transcriptionally modulated in LCLs at 4 hr post-IR (p&lt;0.05).

<p>DNA repair genes transcriptionally modulated in LCLs at 4 hr post-IR (p&lt;0.05).</p

Dose response and time course of selected DNA repair genes in human cell lines.

<p>PSR hybridization signals are shown for a two DNA repair genes that are induced following radiation. These are <i>POLH</i> (A, B) and <i>DDB2</i> (C–F) in LCLs (A–D) or fibroblasts (E, F). Relative fluorescence is plotted on the y-axis and PSRs are plotted evenly across the x-axis in a 5′ to 3′ direction (left to right). Samples from different individuals were either sham irradiated (red) or irradiated with 1 Gy (blue), 2 Gy (green), 5 Gy (purple), 10 Gy (orange) or 20 Gy (aqua) of radiation (A, C and E; n = 4). RNA was collect 4 hours following treatment. Time course of DNA repair genes were either sham irradiated (red) or irradiated with 10 Gy and RNA isolated 2 hrs (blue), 4 hrs (green), 8 hrs (purple), 24 hours (orange) or 48 hours (aqua) after irradiation (B, D and F; n = 4). Error bars = SEM.</p

Induction of DNA repair genes at the exon level four hours after treatment with 10 Gy IR in LCLs.

<p>The top five DNA repair genes: <i>XPC</i> (A), <i>POLH</i> (B), <i>DDB2</i> (C), <i>PCNA</i> (D) and <i>RRM2B</i> (E) as identified using Partek Genomics Suite 6.6 statistical package. Relative fluorescence (y-axis; log₂) is plotted for each PSR (x-axis). Core PSRs are labelled numerically in a 5′ to 3′ direction (left to right). Samples were either sham irradiated (red) or irradiated (blue) with 10 Gy from a ¹³⁷Cs source. Arrow indicates the PSR region to which primers were designed for qRT-PCR used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g003" target="_blank">figure 3A</a>. Error bars = SEM (n = 12).</p

DNA repair genes transcriptionally modulated in primary fibroblasts at 4 hr post-IR (p&lt;0.05).

<p>DNA repair genes transcriptionally modulated in primary fibroblasts at 4 hr post-IR (p&lt;0.05).</p

Expression levels of the top five DNA repair genes as determined by qRT-PCR in LCLs over different IR doses and times.

<p>Relative gene expression is shown for samples that were either sham irradiated or irradiated with 1 Gy, 2 Gy, 5 Gy, 10 Gy or 20 Gy of radiation at 4 hours post-IR, or sham irradiated or irradiated with 10 Gy at 2 h, 4 h, 8 h, 24 h or 48 h post-IR.</p

Expression levels of the top five DNA repair genes as determined by qRT-PCR in fibroblasts over different IR doses and times.

<p>Relative gene expression is shown for samples that were either sham irradiated or irradiated with 1 Gy, 2 Gy, 5 Gy, 10 Gy or 20 Gy of radiation at 4 hours post-IR, or sham irradiated or irradiated with 10 Gy at 2 h, 4 h, 8 h, 24 h or 48 h post-IR.</p