Co-expression of CD45 in part of stromal cells originated from bone marrow-derived cells.

<p>The representative images demonstrated that some GFP(+) BMDCs in the tumor stroma co-expressed CD45 (indicated by arrows), a hematopoietic cell marker, in a carcinogen-induced small intestinal tumor. Other GFP(+) cells without CD45 expression are indicated by filled arrows. The intestinal glands and stromal cells are also labeled.</p

Bone Marrow-Derived Cells May Not Be the Original Cells for Carcinogen-Induced Mouse Gastrointestinal Carcinomas

<div><p>Aim</p><p>It has been reported that bone marrow-derived cells (BMDC) can be original cells of mouse gastric cancers induced by <i>Helicobacter felis</i> (<i>H. felis</i>) infection. However, it is unknown whether BMDCs are also the original cells of mouse gastrointestinal cancers induced by gastric carcinogens <i>N</i>-nitroso-<i>N</i>-methylurea (NMU) and <i>H. felis</i> infection.</p><p>Methods</p><p>C57BL/6 recipient mice were initially irradiated with 10Gy X-ray, reconstituted with bone marrow cells from the C57BL/6-Tg (CAG-EGFP) donor mice to label BMDCs with green fluorescence protein (GFP). After 4 weeks of recovery, the bone marrow-transplanted mice were given NMU in drinking water (240 ppm) and subsequently infected with <i>H. felis</i> by gavage. Eighty weeks later, all mice were euthanized for pathological examination. The BMDCs expressing GFP were detected in tissues using direct GFP fluorescence confocal microscopy analysis and immunohistochemistry staining (IHC) assays.</p><p>Results</p><p>Neoplastic lesions were induced by NMU treatment and/or <i>H. felis</i> infection at the antrum of the glandular stomach and small intestine. In the direct GFP fluorescence confocal assay, GFP(+) epithelial cell cluster or glands were not observed in these gastrointestinal tumors, however, most GFP(+) BMDCs sporadically located in the tumor stromal tissues. Some of these GFP(+) stromal BMDCs co-expressed the hematopoietic marker CD45 or myofibroblasts markers αSMA and SRF. In the indirect GFP IHC assay, similar results were observed among 11 gastric intraepithelial neoplasia lesions and 2 small intestine tumors.</p><p>Conclusion</p><p>These results demonstrated that BMDCs might not be the source of gastrointestinal tumor cells induced by NMU and/or <i>H. felis</i> infection.</p></div

Bone marrow-derived infiltrating cells in the stromal tissue of gastrointestinal tumors displayed with GFP immunohistochemistry staining.

<p>(<b>A</b>) Normal tissues of the glandular stomach of a regular GFP(−) control mouse; (<b>B</b>) Normal tissues of the glandular stomach of a GFP(+) transgenic control mouse; (<b>C</b>) An induced gastric intraepithelial neoplasia (GIN) in a bone marrow transplanted mouse; (<b>D</b>) An induced small intestinal tumor in a bone marrow transplanted mouse. Hematoxylin was used to visualize nuclei.</p

Bone marrow-derived infiltrating cells in the stromal tissue of gastric intraepithelial tumor traced by GFP direct fluorescence.

<p>(<b>A</b>) Normal tissues of the glandular stomach of a regular GFP(−) control mouse; (<b>B</b>) Normal tissues of the glandular stomach of a GFP(+) transgenic control mouse; (<b>C, E, D, F</b>) An induced gastric intraepithelial neoplasia (GIN) in a bone marrow transplanted mouse. GFP(+) BMDCs tracked with direct fluorescence localized in the GIN stromal tissue are shown in <b>C</b> and <b>E.</b> The same GIN lesion slide stained by H&E after the fluorescence observation are shown in <b>D</b> and <b>F</b>. DAPI (<b>A</b>–<b>C</b> and <b>E</b>) and hematoxylin (<b>D</b> and <b>F</b>) are used to visualize nuclei, respectively. Locations of the images <b>C</b> and <b>D</b> in the images <b>E</b> and <b>F</b>, and the image <b>E</b> in the image <b>F</b> are marked in the corresponding color. The gastric glands and stromal cells are also labeled.</p

Stromal cells originated from bone marrow-derived cells in two small intestinal tumors.

<p>GFP(+) BMDCs indicated by white arrows were observed in the stromal tissues of two small intestinal tumors in the GFP direct-fluorescence assay (left panel). The histological images were further displayed with the H&E staining (right panel). H&E-staining images of paraffin-embedded tissue from the intestinal tumor used in this direct-GFP confocal analysis are displayed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079615#pone.0079615.s003" target="_blank">Figure S3</a>.</p

Average body weight of mice with different treatments.

<p>BMT, bone-marrow transplantation; Mock, normal mice without any treatment; <i>H.felis</i>, gavaged with <i>H. felis</i> suspension on three alternate days (one time/day); NMU, drinking water containing 240 ppm NMU in five alternate weeks.</p

Co-expression of αSMA and SRF in part of stromal cells originated from bone marrow-derived cells.

<p>(<b>A</b>) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed αSMA (indicated by arrows) in a carcinogen-induced small intestinal tumor. (<b>B</b>) Images demonstrating that some GFP(+) BMDCs in the tumor stroma co-expressed SRF (indicated by arrows) in the small intestinal tumor. A SRF(+) cell without GFP expression is indicated by a filled arrow.</p

DataSheet1_P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7.pdf

Background: It is well known that P16INK4A, P14ARF, P15INK4B mRNAs, and ANRIL lncRNA are transcribed from the CDKN2A/2B locus. LncRNA P14AS is a lncRNA transcribed from antisense strand of P14ARF promoter to intron-1. Our previous study showed that P14AS could upregulate the expression level of ANRIL and P16INK4A and promote the proliferation of cancer cells. Because polycomb group protein CBX7 could repress P16INK4A expression and bind ANRIL, we wonder whether the P14AS-upregulated ANRIL and P16INK4A expression is mediated with CBX7.Results: In this study, we found that the upregulation of P16INK4A, P14ARF, P15INK4B and ANRIL expression was induced by P14AS overexpression only in HEK293T and HCT116 cells with active endogenous CBX7 expression, but not in MGC803 and HepG2 cells with weak CBX7 expression. Further studies showed that the stable shRNA-knockdown of CBX7 expression abolished the P14AS-induced upregulation of these P14AS target genes in HEK293T and HCT116 cells whereas enforced CBX7 overexpression enabled P14AS to upregulate expression of these target genes in MGC803 and HepG2 cells. Moreover, a significant association between the expression levels of P14AS and its target genes were observed only in human colon cancer tissue samples with high level of CBX7 expression (n = 38, p INK4A, P14ARF and P15INK4B genes. The amounts of repressive histone modification H3K9m3 was also significantly decreased at the promoters of these genes by P14AS in CBX7 actively expressing cells.Conclusions: CBX7 expression is essential for P14AS to upregulate the expression of P16INK4A, P14ARF, P15INK4B and ANRIL genes in the CDKN2A/2Blocus. P14AS may upregulate these genes’ expression through competitively blocking CBX7-binding to ANRIL lncRNA and target gene promoters.</p

Boletín de Segovia: Número 84 - 1875 julio 7

Copia digital. Madrid : Ministerio de Cultura. Subdirección General de Coordinación Bibliotecaria, 200

Homeostatic Maintenance of Allele-Specific <i>p16</i> Methylation in Cancer Cells Accompanied by Dynamic Focal Methylation and Hydroxymethylation

<div><p>Aim</p><p><i>p16</i> Methylation frequently occurs in carcinogenesis. While it has been hypothesized that the <i>p16</i> methylation states are dynamically maintained in cancer cells, direct evidence supporting this hypothesis has not been available until now.</p><p>Methods</p><p>A fusion cell model was established which reprogrammed the native DNA methylation pattern of the cells. The methylation status of the <i>p16</i> alleles was then repeatedly quantitatively analyzed in the fusion monoclonal, parental cancer cell lines (<i>p16</i>-completely methylated-AGS and unmethylated-MGC803), and HCT116 non-fusion cell using DHPLC and bisulfite sequencing. Histone methylation was analyzed using chromatin immuno-precipitation (ChIP)-PCR. P16 expression status was determined using immuno-staining and RT-PCR.</p><p>Results</p><p>The methylation status for the majority of the <i>p16</i> alleles was stably maintained in the fusion monoclonal cells after up to 60 passages. Most importantly, focal de novo methylation, demethylation, and hydroxymethylation were consistently observed within about 27% of the <i>p16</i> alleles in the fusion monoclones, but not the homozygously methylated or unmethylated parental cells. Furthermore, subclones of the monoclones consistently maintained the same <i>p16</i> methylation pattern. A similar phenomenon was also observed using the <i>p16</i> hemi-methylated HCT116 non-fusion cancer cell line. Interestingly, transcription was not observed in <i>p16</i> alleles that were hydroxymethylated with an antisense-strand-specific pattern. Also, the levels of H3K9 and H3K4 trimethylation in the fusion cells were found to be slightly lower than the parental AGS and MGC803 cells, respectively.</p><p>Conclusion</p><p>The present study provides the first direct evidence confirming that the methylation states of <i>p16</i> CpG islands is not only homeostatically maintained, but also accompanied by a dynamic process of transient focal methylation, demethylation, and hydroxymethylation in cancer cells.</p></div