P16 Methylation as an Early Predictor for Cancer Development From Oral Epithelial Dysplasia: A Double-blind Multicentre Prospective Study

AbstractBackgroundSilencing of P16 through methylation and locus deletion is the most frequent early events in carcinogenesis. The aim of this study is to prospectively determine if early P16 methylation is a predictor for oral cancer development.MethodsPatients (n=181) with mild or moderate oral epithelial dysplasia (OED) were recruited into the double blind multicentre cohort. P16 methylation was analyzed using the MethyLight assay. Progression of OEDs was monitored for a minimum 3year follow-up period.FindingsP16 methylation-informative cases (n=152) were enrolled in the prospective multicenter cohorts with an ultimate compliance of 96.7%. OED-derived squamous cell carcinomas were observed in 21 patients (14.3%) during the follow-up (median, 41.0months). The cancer progression rate from the P16 methylation-positive patients was significantly increased when compared to P16 methylation-negative patients [27.1% vs 8.1%; adjusted odds ratio=4.6; P=0.006]. When the P16 methylation-positive criteria were used as a biomarker for early prediction of cancer development from OEDs, sensitivity and specificity of 62% and 76% were obtained, respectively.InterpretationP16 methylation is unequivocally a marker for determining the malignant potential of OED and there is no need for further research regarding this aspect.FundingNational Basic Research Programs of China (2011CB504201 and 2015CB553902), Beijing Science and Technology Commission (Z090507017709016), and Beijing Municipal Administration of Hospital (XM201303) to Dajun Deng. The funding agencies have no role in the actual experimental design, patient recruitment, data collection, analysis, interpretation, or writing of this manuscript

P14AS upregulates gene expression in the CDKN2A/2B locus through competitive binding to PcG protein CBX7

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 < 0.05), but not in samples (n = 37) with low level of CBX7 expression, nor in paired surgical margin tissues. In addition, the results of RNA immunoprecipitation (RIP)- and chromatin immunoprecipitation (ChIP)-PCR analyses revealed that lncRNA P14AS could competitively bind to CBX7 protein which prevented the bindings of CBX7 to both lncRNA ANRIL and the promoters of P16INK4A, 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

Prevalence of A2143G mutation of H. pylori-23S rRNA in Chinese subjects with and without clarithromycin use history

<p>Abstract</p> <p>Background</p> <p>A2143G mutation of <it>23S rRNA </it>gene of <it>H. pylori </it>results in clarithromycin (CLR) resistance. To investigate the prevalence of the CLR resistance-related A2143G mutation of the <it>H. pylori</it>-specific <it>23S rRNA </it>gene in Chinese subjects with and without CLR use history, 307 subjects received the treatment with amoxicillin and omeprazole (OA) and 310 subjects received a placebo in 1995, and 153 subjects received a triple therapy with OA and CLR (OAC) in 2000. DNA was extracted from fasting gastric juice at the end of the intervention trial in 2003. <it>H. pylori </it>infection was determined by <it>H. pylori</it>-specific <it>23S rRNA </it>PCR, ELISA, and¹³C-urea breath test assays. Mutations of the <it>23S rRNA </it>gene were detected by RFLP assays.</p> <p>Results</p> <p>The presence of <it>23S rRNA </it>due to <it>H. pylori </it>infection in the OA group remained lower than that in the placebo group 7.3 yrs after OA-therapy [51.1% (157/307) vs. 83.9% (260/310), p = 0.0000]. In the OAC group, the <it>23S rRNA </it>detection rate was 26.8% (41/153) three yrs after OAC-treatment. The A2143G mutation rate among the <it>23S rRNA</it>-positive subjects in the OAC group [31.7% (13/41)] was significantly higher than that in the OA group [10.2% (16/157)] and the placebo group [13.8% (36/260)]. The frequency of the AAGGG → CTTCA (2222–2226) and AACC → GAAG (2081–2084) sequence alterations in the OAC group was also significantly higher than those in the OA group and the placebo group.</p> <p>Conclusion</p> <p>Primary prevalence of the A2143G mutation was 10~14% among Chinese population without history of CLR therapy. Administration of CLR to eliminate <it>H. pylori </it>infection increased the prevalence of the A2143G mutation in Chinese subjects (32%) significantly.</p

Polycomb CBX7 Directly Controls Trimethylation of Histone H3 at Lysine 9 at the p16 Locus

BACKGROUND: H3K9 trimethylation (H3K9me3) and binding of PcG repressor complex-1 (PRC1) may play crucial roles in the epigenetic silencing of the p16 gene. However, the mechanism of the initiation of this trimethylation is unknown. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we found that upregulating the expression of PRC1 component Cbx7 in gastric cancer cell lines MGC803 and BGC823 led to significantly suppress the expression of genes within the p16-Arf-p15 locus. H3K9me3 formation was observed at the p16 promoter and Regulatory Domain (RD). CBX7 and SUV39H2 binding to these regions were also detectable in the CBX7-stably upregulated cells. CBX7-SUV39H2 complexes were observed within nucleus in bimolecular fluorescence complementation assay (BiFC). Mutations of the chromodomain or deletion of Pc-box abolished the CBX7-binding and H3K9me3 formation, and thus partially repressed the function of CBX7. SiRNA-knockdown of Suv39h2 blocked the repressive effect of CBX7 on p16 transcription. Moreover, we found that expression of CBX7 in gastric carcinoma tissues with p16 methylation was significantly lower than that in their corresponding normal tissues, which showed a negative correlation with transcription of p16 in gastric mucosa. CONCLUSION/SIGNIFICANCE: These results demonstrated for the first time, to our knowledge, that CBX7 could initiate H3K9me3 formation at the p16 promoter

H∞ Filter Design for Networked Control Systems: A Markovian Jump System Approach

This paper puts forward a method to design the H∞ filter for networked control systems (NCSs) with time delay and data packet loss. Based on the properties of Markovian jump system, the packet loss is treated as a constant probability independent and identically distributed Bernoulli random process. Thus, the stochastic stability condition can be acquired for the filtering error system, which meets an H∞ performance index level γ. It is shown that, by introducing a special structure of the relaxation matrix, a linear representation of the filter meeting an H∞ performance index level for NCSs with time delay and packet loss can be obtained, which uses linear matrix inequalities (LMIs). Finally, numerical simulation examples demonstrate the effectiveness of the proposed method

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

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

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