DNA copy number profiles of gastric cancer precursor lesions

<p>Abstract</p> <p>Background</p> <p>Chromosomal instability (CIN) is the most prevalent type of genomic instability in gastric tumours, but its role in malignant transformation of the gastric mucosa is still obscure. In the present study, we set out to study whether two morphologically distinct categories of gastric cancer precursor lesions, i.e. intestinal-type and pyloric gland adenomas, would carry different patterns of DNA copy number changes, possibly reflecting distinct genetic pathways of gastric carcinogenesis in these two adenoma types.</p> <p>Results</p> <p>Using a 5K BAC array CGH platform, we showed that the most common aberrations shared by the 11 intestinal-type and 10 pyloric gland adenomas were gains of chromosomes 9 (29%), 11q (29%) and 20 (33%), and losses of chromosomes 13q (48%), 6(48%), 5(43%) and 10 (33%). The most frequent aberrations in intestinal-type gastric adenoma were gains on 11q, 9q and 8, and losses on chromosomes 5q, 6, 10 and 13, whereas in pyloric gland gastric adenomas these were gains on chromosome 20 and losses on 5q and 6. However, no significant differences were observed between the two adenoma types.</p> <p>Conclusion</p> <p>The results suggest that gains on chromosomes 8, 9q, 11q and 20, and losses on chromosomes 5q, 6, 10 and 13, likely represent early events in gastric carcinogenesis. The phenotypical entities, intestinal-type and pyloric gland adenomas, however, do not differ significantly (P = 0.8) at the level of DNA copy number changes.</p

Persistent DNA Damage after High Dose In Vivo Gamma Exposure of Minipig Skin

Exposure to high doses of ionizing radiation (IR) can lead to localized radiation injury of the skin and exposed cells suffer dsDNA breaks that may elicit cell death or stochastic changes. Little is known about the DNA damage response after high-dose exposure of the skin. Here, we investigate the cellular and DNA damage response in acutely irradiated minipig skin.IR-induced DNA damage, repair and cellular survival were studied in 15 cm(2) of minipig skin exposed in vivo to ~50 Co-60 γ rays. Skin biopsies of control and 4 h up to 96 days post exposure were investigated for radiation-induced foci (RIF) formation using γ-H2AX, 53BP1, and active ATM-p immunofluorescence. High-dose IR induced massive γ-H2AX phosphorylation and high 53BP1 RIF numbers 4 h, 20 h after IR. As time progressed RIF numbers dropped to a low of <1% of keratinocytes at 28-70 days. The latter contained large RIFs that included ATM-p, indicating the accumulation of complex DNA damage. At 96 days most of the cells with RIFs had disappeared. The frequency of active-caspase-3-positive apoptotic cells was 17-fold increased 3 days after IR and remained >3-fold elevated at all subsequent time points. Replicating basal cells (Ki67+) were reduced 3 days post IR followed by increased proliferation and recovery of epidermal cellularity after 28 days.Acute high dose irradiation of minipig epidermis impaired stem cell replication and induced elevated apoptosis from 3 days onward. DNA repair cleared the high numbers of DBSs in skin cells, while RIFs that persisted in <1% cells marked complex and potentially lethal DNA damage up to several weeks after exposure. An elevated frequency of keratinocytes with persistent RIFs may thus serve as indicator of previous acute radiation exposure, which may be useful in the follow up of nuclear or radiological accident scenarios

X-ray irradiation induces subtle changes in the genome-wide distribution of DNA hydroxymethylation with opposing trends in genic and intergenic regions

DNA hydroxymethylation has gained attention as an intermediate in the process of DNA demethylation. More recently, 5-hydroxymethylcytosine has been recognized as an independent epigenetic mark that can persist over time and that exerts influence on gene regulation and other biological processes. Deregulation of this DNA modification has been linked to tumorigenesis and a variety of other diseases. The impact of irradiation on DNA hydroxymethylation is poorly understood. In this study we exposed lung fibroblasts (IMR90) to 0.5 Gy and 2 Gy of X-rays, respectively. We characterized radiation induced changes of DNA hydroxymethylation 1 h, 6 h, 24 h and 120 h after exposure employing immunoprecipitation and subsequent deep sequencing of the genomic fraction enriched for hydroxymethylated DNA. Transcriptomic response to irradiation was analyzed for time points 6 h and 24 h post exposure by means of RNA sequencing. Irradiated and sham-irradiated samples shared the same overall distribution of 5-hydroxymethylcytosines with respect to genomic features such as promoters and exons. The frequency of 5-hydroxymethylcytosine peaks differentially detected in irradiated samples increased in genic regions over time, while the opposing trend was observed for intergenic regions. Onset and extent of this effect was dose dependent. Moreover, we demonstrated a biased distribution of 5-hmC alterations at CpG islands and sites occupied by the DNA binding protein CTCF. In summary, our study provides new insights into the epigenetic response to irradiation. Our data highlight genomic features more prone to irradiation induced changes of DNA hydroxymethylation, which might impact early and late onset effects of irradiation

IR-induced changes of proliferation index (Ki67) and in epidermal cellularity as displayed by nuclear counterstaining (DAPI, blue).

<p>(A) Control minipig skin samples showing Ki67-positive cells (pink) at the basal layer of the densely populated epidermis. (B) Minipig skin sample 3 days post IR displaying a dearth of replicating cells, while at 28 (C), 49 (D) and 70 (E) days proliferating cells are abundant. (F) At 98 days epidermal hyperplasia was associated with a drop of replicating cell number. Magnification bar for all details: 20 µm. (G) Frequency of Ki67 positive (+) cells per unit area revealing a significant reduction (**p<0.001) of Ki-67-positive cells 3 days after IR relative to control and 4 h. A raise of replicating cells was noted ≥ 49 days post IR being significant at day 70 relative to control and the earlier time points. The fraction of replicating cells was significantly reduced at 96 days post IR, being associated with epithelia hyperplasia. (H) Epidermal cellularity during the time window investigated as determined by enumeration of the total number of DAPI-positive nuclei (blue, A-F) of keratinocytes per 169 µm unit contour length (average of 3 pigs). There is a significant decrease in the cellularity of the epidermis from 3 days up to 28 days post IR, as compared to control. A constant raise of the cellularity of the epidermis was observed from 28 days post irradiation onwards. The mean and SD of 3 pigs is given, except for 3 days (2 pigs); *, p<0.05; **, p<0.001. (G,H) Significant difference: ^a relative to control, ^b relative to 98 d, ^c relative to all other time points, ^d relative to 4 h, 3d, 48d and 72 days.</p

Average number of DSB-related foci/cell in pig epidermis.

<p>−, sample not available. Con, non-irradiated control.</p

Acute IR exposure of the skin leads to caspase-dependent apoptosis.

<p>(A) TUNEL staining renders the epidermis largely positive for fragmented DNA in control (con) and (B) 50Gy-exposed skin sections (4 h post IR). (C-F) Immunofluorescence for activated-caspase 3 (a.casp.3; red) and γ-H2AX (green). Nuclei are counterstained with DAPI (blue). (C) Unirradiated control is largely lacking activated caspase 3-positive cells. (D) Three days post IR numerous activated-caspase 3-positive cells are present in the basal layer of the epidermis. (E) At 28 days post IR only few cells are positive for activated caspase 3. (F) 98 days post IR activated-caspase 3-positive cells often occur in the spinous layer of the epidermis (nuclei, blue) relative to the earlier time points. Activated-caspase 3-positive apoptotic cells at late time points often are also strongly positive for γ-H2AX nuclear staining (green; yellow due to signal overlap). Magnification bar: 20 µm.</p

DNA damage response in epidermis sections of 140 µm unit length after in vivo exposure to 50 Gy γ-irradiation studied by radiation-induced focus analysis.

<p>The mean and SD of 3 pigs are presented, except for the 20 h and 3 day values (2 pigs). (A) 4 h and 20 h post IR all cells contained RIF. This percentage dropped after 3days post IR. (B) Average number of γ-H2AX and 53BP1 RIF per cell in skin biopsies at the consecutive time points investigated. At 4 h post IR the focus number was derived from cells with distinct γ-H2AX labeling (see text, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039521#pone-0039521-g001" target="_blank">Fig.1A</a>). “Overlap” addresses foci with γ-H2AX and 53BP1 signal colocalization, which is complete at late time points. (C) Average foci numbers in keratinocytes that carry ≥1 RIF are significantly increased relative to control, 4 h and 20 h time points (**p<0.001). (D) Percentage of cells carrying ≥1 53BP1 RIF at 70 days post IR. There is a significant difference (**, p<0.001) between keratinocytes of the hair sheath (70d dermis) and all other conditions. (E) At ≥49 days post IR the mean 53BP1 foci diameter (maximum contour radius ± SD) is significantly increased (**, p<0.002). Error bars reflect differences of 3 different animals. Significant differences were noted ^a relative to control, ^b relative to 4 h, ^c to 3 days, ^e to 28 days, ^f to 49 days and, ^g relative to 70 days.</p

Haematoxilin and eosin staining (original magnification ×400) of intestinal-type (A) and pyloric gland (B) gastric adenomas

<p><b>Copyright information:</b></p><p>Taken from "DNA copy number profiles of gastric cancer precursor lesions"</p><p>http://www.biomedcentral.com/1471-2164/8/345</p><p>BMC Genomics 2007;8():345-345.</p><p>Published online 1 Oct 2007</p><p>PMCID:PMC2147033.</p><p></p> A. Intestinal-type adenoma of the stomach composed of irregularly arranged glands composed of intestinal-type epithelium with eosinophilic cytoplasm and enlarged nuclei. B. Pyloric gland adenoma of the stomach composed of densely back to back packed glands consisting of cells with pale cytoplasm and small round hyperchromatic nuclei