P16 Gene Promoter Methylation Research Articles

Hepatitis B virus X protein induces p16 gene promoter methylation through upregulation of DNA methylation transferases DNMT1 and DNMT3A.

As a tumor suppressor, p16 can competitively block the cyclin D1-CDK4/6 complex to arrest the cell cycle in the G1 phase. Lack of p16 gene expression can lead to infinite cell proliferation and malignant transformation. To determine whether the hepatitis B virus X protein (HBx) regulates the methylation and expression of the p16 gene. We constructed a eukaryotic expression vector carrying the HBx gene and green fluorescent protein (GFP), and transfected it into HepG2 cells to build cell lines stably expressing GFP and GFP-HBx. The p16 protein level and p16 gene methylation were measured in these cell lines. We further detected the mRNA expression of DNA methyltransferases (DNMTs) 1, 3A and 3B. The DNMT1, DNMT3A and DNMT3B gene promoter sequences were inserted into a reporter vector (pGL3-Basic) to build recombinant vectors, which were then transiently transfected into different cell lines. After 48 h, the luciferase activity was measured. The level of p16 protein in GFP-HBx/HepG2 cells was significantly lower than in HepG2 and GFP/HepG2 cells. The CpG methylation was present in the p16 gene promoter region of GFP-HBx/HepG2 cells. The DNMT1 and DNMT3A mRNA levels in GFP-HBx/HepG2 cells were significantly elevated compared to that in the HepG2 cells (p = 0.0495). The luciferase activity in GFP-HBx/HepG2 cells transfected with the pGL3-DNMT1/3A pro plasmid was significantly higher than in HepG2 and GFP/HepG2 cells (both p < 0.05). The HBx can induce p16 gene promoter methylation and inhibit the expression of p16 in HepG2 cells. This occurs due to HBx activation of DNMT1 and DNMT3A promoters and the induction of p16 promoter methylation, which downregulates the expression of p16 protein.

Methylation of p16 gene and reduced expression of p16 protein in insulinoma associated with clinicopathological features

Objective: To study the alterations of p16 gene and its expression in insulinoma and to correlate the findings with clinicopathological characteristics. Methods: Expression of p16 protein was detected in 72 insulinomas and 49 para-tumoral or normal pancreatic tissues by immunohistochemical staining. Genomic DNA was isolated from 32 tumor tissue and 17 paired pancreatic tissues and bisulfite-modified. Promoter methylation status of p16 gene was detected in 32 tumor tissue and 17 paired pancreatic tissues by methylation specific PCR. The findings were correlated with the clinicopathological features. Results: There were 30 males and 42 females in all 72 patients, aged (46.5±14.0) years. Loss or reduced expression of p16 protein was found in 42 of 72 insulinomas (58.3%) while loss or reduced expression of p16 was seen in only 34.7% (17/49) of para-tumoral or normal pancreatic tissues (χ²=6.52, P=0.011). Promoter methylation of p16 gene was found in 13 of 32 insulinomas (40.6%) and only 2 of 17 (11.8%) para-tumoral tissues (χ²=4.35, P=0.037). The expression of p16 protein in insulinoma was not associated with clinicopathological features such as gender, age, tumor size and tumor grade. Conclusions: Loss or reduced expression of p16 protein was found in insulinomas, and associated with p16 gene promoter methylation.

Effects of the p16/cyclin D1/CDK4/Rb/E2F1 pathway on aberrant lung fibroblast proliferation in neonatal rats exposed to hyperoxia.

p16INK4a (p16) inhibits the vital G1 to S phase transition during cell cycle progression through the p16/cyclin D1/CDK4/retinoblastoma(Rb)/E2F1 pathway. Hyperoxia can suppress the G1/S checkpoint and induce more lung fibroblasts (LFs) to transition from the G1 phase to the S phase and undergo cell proliferation. The present study investigated the rate of p16 gene promoter methylation and the protein expression levels of p16, cyclin D1, CDK4, Rb and E2F1 in LFs from the lungs of rats exposed to hyperoxia and normoxia on postnatal days 3, 7 and 14. In the hyperoxia-exposed group, the methylation rate was 50 and 80% on days 7 and 14, respectively. Cyclin D1 and CDK4 overexpression was associated with p16 loss and Rb inactivation by phosphorylation. Rb phosphorylation induced E2F1 release in the G1 phase, which promoted cell proliferation. No methylation was observed in the normoxia-exposed group. These observations suggested that p16 loss may stimulate aberrant LF proliferation via the p16/cyclin D1/CDK4/Rb/E2F1 pathway.

The association between PON1 and GSTM1 genetic variation with methylation of p16 gene promoter among Javanese farmers exposed to pesticides at Magelang Regency, Central Java, Indonesia

Occupational exposure to pesticides leads to the development of cancer. Aberrant DNA methylation plays a crucial role in cancer. The manifestation of the carcinogenic effect of pesticides could be determined by the variation of genes encoding enzyme, including PON1 Q192R and GSTM1. The goal of this study was to find out polymorphism of PON1 Q192R and methylation of p16 gene promoter, and their correlation on Javanese farmers in the agricultural area of Ngablak Subdistrict, Magelang Regency, Central Java. Seventy-eight pesticide-exposed farmers enrolled in the study. Polymorphism of PON Q192R was determined using PCR-RFLP and variation of GSTM1 was examined using conventional PCR. The methylation of the p16 gene promoter was determined using methylation-specific PCR. The result revealed 94.9% polymorphism of PON1 Q192R, which was higher in the R/R (Arg/Arg) genotypes than Q/R (Gln/Arg) and lowest in Q/Q (Gln/Gln) genotypes. We also found 82.1% GSTM1 null genotype among the farmers enrolled in the study. As many as 26.9% methylations of p16 gene promoter were found among farmers. Genetic variation of PON1 Q192R and GSTM1 were not found to be correlated to the methylation status of p16 gene promoter in the Javanese population.

Study on the Expression and Significance of P16 Gene Methylation Based on Information Technology Statistics in Female Cervical Cancer in Hunan Povince

To explore the significance of p16 gene methylation and abnormal expression in cervical cancer, Methylation specific PCR (MSP) was used to detect 5 ′ CpG island methylation of p16 gene promoter in 60 cervical cancer tissues of different pathological types and clinical stages, who was chosen in Human province. Detection of homozygous deletion of exon 1 (E1) and exon 2 (E2) of p16 gene by PCR. Immunohistochemical analysis of p16 protein expression. The results showed that there was no methylation of p16 gene in normal control tissues and paracancerous tissues and no E1 and E2 deletions and abnormal p16 protein expression were found. The methylation rate of 60 cervical cancer samples was 21.67% (13 / 60), the deletion rate of p16 gene was 15.00% (9 / 60), and the expression rate of p16 protein was 51.67% (31 / 60). It can be seen that the positive expression rate of p16 gene protein decreased with the increase of clinical stage. The results suggest that p16 gene inactivation is common in cervical cancer and closely related to the pathological grade. p16 gene methylation plays a role in the development of cervical cancer.

P16 promoter methylation, expression, and its association with estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 subtype of breast carcinoma.

The purpose of the study is to investigate p16 protein expression and promoter methylation of p16 gene and their association with molecular subtypes based on parameter such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). A total of 114 breast cancer tissue biopsies were collected for methylation-specific polymerase chain reaction (MSP) and immunohistochemical (IHC) analysis. Seven tissue microarrays were constructed. p16 protein expression was studied in 114 cases, of which 35/114 (30.7%) cases showed strong expression and the majority of them had ER-positive tumor (57.6%), and it was statistically significant (P < 0.0074). Similarly, p16 expression was reduced in the majority of PR-negative tumors (83.9%) and the association was statistically significant (P = 0.0026). p16 methylation was studied in 114 cases and was positive in 71.0% cases. High p16 protein expression was associated with ER-positive, PR-negative, and HER2-negative tumors which is associated with poor prognosis. p16 protein expression may be used as a prognostic indicator to predict treatment response to hormonal therapy.

Promoter DNA Methylation and mRNA Expression Level of p16 Gene in Oral Squamous Cell Carcinoma: Correlation with Clinicopathological Characteristics.

The aim of this study was to investigate the relationship between p16 methylation and its expression in oral squamous cell carcinoma (OSCC). Also the contribution of clinicopathological factors, HPV infection and smoking in p16 expression and promoter methylation has been investigated. In this study 67 consecutive OSCC patients and 59 normal individuals were enrolled. All patients were candidates for surgery of oral cavity and fresh tumor biopsies were collected and processed for DNA and RNA extraction. Normal gingival tissues were collected from individuals referred to dentistry clinic and considered as controls. All the cases and controls were checked for HPV infection and then promoter methylation and expression of p16 gene were determined using Methylation-specific PCR (MSP) and real-time PCR (QPCR), respectively. Methylation of p16 in tumors and normal tissues were 59.7 and 38.9%, respectively. Most of hypermethylated samples (>82%) were in high grades. P16 methylation was comparable in HPV+ and HPV- patients or smokers. P16 was overexpressed (~3 fold; p = 0.044) in HPV+ tumors, but it was significantly down-regulated in smoker patients (40% of all tumors). Comparison of P16 expression in OSCC tumors with different degrees of promoter methylation further suggest the relationship of methylation rate and down-regulation of P16 expression. The p16 methylation and expression was differentially affected in patients with HPV infection and the smoker cases. Regardless of the influence of environmental factors, it appears that P16 status is useful for classifying patients with OSCC and for influencing treatment strategies in accordance with this classification. Moreover, targeting the upregulation of p16 could be a promising therapeutic option.

Association of GSTP1 and P16 promoter methylation with the risk of HBV-related hepatocellular carcinoma: a meta-analysis.

BackgroundStudy on the relationship between glutathione-S-transferase Pi 1 (GSTP1) and P16 promoter region methylation and the risk of hepatitis B virus-related hepatocellular carcinoma (HBV-related HCC) has produced inconsistent results.ObjectivesTo assess the correlation between GSTP1 and P16 promoter methylation frequency and HBV-related HCC susceptibility.MethodsAll relevant studies were identified by searching PubMed, Embase, Web of Science, and China National Knowledge Infrastructure literature databases before December, 2017. The OR and the corresponding 95% CI were calculated to investigate the risk of GSTP1 and P16 promoter methylation rate and HBV-related HCC. Sensitivity analysis was performed and publication bias was estimated using the Begg’s and Egger’s test.ResultsOur meta-analysis identified the relationships of GSTP1 (six studies including 213 HBV-related HCC tumor tissues) and P16 (nine studies with 287 HBV-related HCC tumor tissue) promoter methylation with HCC risk. Compared with normal liver tissue and cirrhosis, the pooled ORs of GSTP1 promoter region methylation in HBV-related HCC cancer tissues were 6.05 (95% CI =1.20–30.52) and 5.21 (95% CI =2.19–12.41), respectively. Compared with paracancerous tissue, normal liver tissue, cirrhosis, and chronic hepatitis B as controls, the pooled ORs of P16 promoter region methylation in HBV-related HCC cancer tissues were 7.18 (95% CI =2.31–22.33), 24.89 (95% CI =3.38–183.03), 5.92 (95% CI =1.78–19.68), and 12.12 (95% CI =0.75–196.50).ConclusionIn summary, our meta-analysis found strong associations between GSTP1 and P16 gene promoter methylation and an increased HBV-related HCC susceptibility. Moreover, GSTP1 and P16 methylation in promoter region could obviously increase the risk of HBV-related HCC in patients with cirrhosis, indicating that these would be promising biomarkers for early clinical diagnosis of HBV-related HCC.

Liquid-based cytology: do ancillary techniques enhance detection of epithelial abnormalities?

Cervical cancer is the fourth most common cancer in women worldwide with very high incidence in India. Liquid-based cytology (LBC) provides the use of ancillary techniques in addition to a good morphology and detection of cytologic abnormalities. The current study was designed to assess the diagnostics of P16INK4a immunoexpression, p16 promoter hypermethylation, human papilloma virus (HPV), and DNA ploidy in LBC samples with cervical precancer and cancer. A series of LBC samples categorised by Bethesda system including 22 atypical squamous cells of undetermined significance (ASC-US), 21 low-grade squamous intraepithelial lesion (LSIL), 41 high-grade squamous intraepithelial lesion (HSIL), 54 squamous cell carcinoma (SCC), and 26 controls with normal cytology were included. Ancillary techniques evaluated included P16INK4a immunoexpression, p16 promoter methylation DNA ploidy by flow cytometry, and HPV was detected using PGMY09/PGMY11 primers. The test positivity rate of p16 expression in women with ASC-US, LSIL, HSIL, and SCC was 21.1, 39.0, 67.7, and 85.4%. For the p16 methylation the corresponding test positivity rate was 36.4, 76.2, 92.7, and 92.6%. The test positive rate of HPV in women with ASC-US, LSIL, HSIL, and SCC was 45.5, 76.2, 87.8, and 92.6%. Diploid G1 and diploid S values significantly (p < 0.05 or p < 0.01) discriminate LSIL versus HSIL and LSIL versus. SCC. P16 gene promoter methylation and HPV seem more sensitive in detection of ASC-US and LSIL cytology with higher specificity. Diploid G1 and diploid S phase study provides progressive change in parameters with progression from LSIL to HSIL and SCC.

Detection of p16 Promoter Hypermethylation by Methylation-Specific PCR.

DNA methylation plays a decisive role in the regulation and control of gene expression. DNA methylation is a covalent modification, in which a methyl group is attached to the 5th carbon of the cytosine ring of a CpG dinucleotide that is located upstream from the promoter region of a gene. Promoter hypermethylation (gain of DNA methylation) of the p16 gene may cause silencing of gene expression and plays an important role in cancer. Therefore, detection of the methylation status of p16 gene is an important tool in epigenetic studies of various human cancers. The methylation-specific PCR (MSP) is the most commonly used technique for studying DNA methylation. This technique is based on bisulfite modification of DNA, which converts unmethylated cytosine (C) into uracil (U) and leaving methylated cytosine (Cm) unchanged. Here we describe the bisulfite modification of DNA samples and detection of promoter methylation of p16 gene from bisulfite-treated DNA using MSP. In MSP, modified DNA samples are subjected to PCR amplification using methylated and unmethylated specific primers for the p16 gene separately. The PCR amplified products are then analyzed in a 2.5-3% agarose gel containing ethidium bromide. The PCR amplified band generated by specific sets of primers is used to determine the methylation status of the p16 gene.

The synergic effect of HPV infection and epigenetic anomaly of the p16 gene in the development of cervical cancer.

Cervical cancer is the most common cancer in Indian women. Infection with a high-risk human papillomavirus (HR-HPV) is the greatest risk factor for developing cervical cancer. The genetic and epigenetic changes in the tumor suppressor p16 gene is play an important role in the development of cervical cancer. To evaluate the expression and promoter methylation of p16 gene in HR-HPV infected squamous cell carcinoma of the uterine cervix. To find out p16INK4a expression and methylation status 105 squamous cell carcinoma of the uterine cervix were investigated by using immunohistochemistry and Methylation Specific PCR techniques. HPV16/18 was amplified in 83.8% cases of the cervix. 80% of them were positive for HPV type 16, while only 3.8% were positive for HPV type 18. Promoter CpG island hypermethylation of p16 gene was detected in 20.9% tissue samples of cervical carcinoma. Of these hypermethylated samples 90.9% cases showed nil/very low p16INK4a expression (P= 0.001). Overexpression of p16INK4a was observed in 73.3% cases of HR-HPV infected squamous cell carcinoma of the cervix. An association between p16 methylation, expression, and HR-HPV infection suggested the compliance of HPV infection and aberration of p16 gene have a synergic effect on initiation and progression of cervical carcinoma.

Application of artificial neural network model combined with four biomarkers in auxiliary diagnosis of lung cancer.

The purpose of the study was to explore the application of artificial neural network model in the auxiliary diagnosis of lung cancer and compare the effects of back-propagation (BP) neural network with Fisher discrimination model for lung cancer screening by the combined detections of four biomarkers of p16, RASSF1A and FHIT gene promoter methylation levels and the relative telomere length. Real-time quantitative methylation-specific PCR was used to detect the levels of three-gene promoter methylation, and real-time PCR method was applied to determine the relative telomere length. BP neural network and Fisher discrimination analysis were used to establish the discrimination diagnosis model. The levels of three-gene promoter methylation in patients with lung cancer were significantly higher than those of the normal controls. The values of Z(P) in two groups were 2.641 (0.008), 2.075 (0.038) and 3.044 (0.002), respectively. The relative telomere lengths of patients with lung cancer (0.93±0.32) were significantly lower than those of the normal controls (1.16±0.57), t=4.072, P<0.001. The areas under the ROC curve (AUC) and 95% CI of prediction set from Fisher discrimination analysis and BP neural network were 0.670 (0.569-0.761) and 0.760 (0.664-0.840). The AUC of BP neural network was higher than that of Fisher discrimination analysis, and Z(P) was 0.76. Four biomarkers are associated with lung cancer. BP neural network model for the prediction of lung cancer is better than Fisher discrimination analysis, and it can provide an excellent and intelligent diagnosis tool for lung cancer.

Promoter Methylation and Relative mRNA Expression of the p16 Gene in Cervical Cancer in North Indians.

Cervical carcinoma is one of the main causes of mortality in women worldwide as well as in India. It occurs as a result of various molecular events that develop from the combined influences of an individual's genetic predisposition and external agents such as smoking and menstrual hygiene, for example. However, infection with human papillomavirus (HPV) is the established major risk factor. The aim of the current study was to investigate p16 CpG island methylation and establish any correlation with mRNA expression in a north Indian population. We analyzed 196 woman volunteers out of which 98 were cases and 98 healthy controls. For the analysis of methylation pattern, DNA extracted from blood samples was modified with a bisulfate kit and used as template for methylation specific PCR (MSP). Quantitative real-time PCR (QRT-PCR) was performed to check mRNA expression. Correlation between methylation status of p16 gene and poor menstrual hygiene was significant (p=0.006), high parity cases showed methylation of p16 gene (p=0.031) with increased risk up to 1.86 times for cervical cancer and smoking was a strong risk factor associated with cervical cancer. We analyzed methylation pattern and found 60.3% methylation in cases with low mRNA expression level (0.014) as compared to controls (1.24). It was also observed that promoter methylation of p16 gene was significantly greater in FIGO stage III. We conclude that p16 methylation plays an important role in cervical cancer in the north Indian population and its methylation decreases mRNA expression. It can be used as an important and consistent blood biomarker in cervical cancer patients.

Lack of Correlation between Aberrant p16, RAR-β2, TIMP3, ERCC1, and BRCA1 Protein Expression and Promoter Methylation in Squamous Cell Carcinoma Accompanying Candida albicans-Induced Inflammation

Hyperplastic candidiasis is characterized by thickening of the mucosal epithelia with Candida albicans infection with occasional progression to squamous cell carcinoma (SCC). C. albicans is a critical factor in tumor development; however, the oncogenic mechanism is unclear. We have previously produced an animal model for hyperplastic candidiasis in the rat forestomach. In the present study, we investigate whether impaired DNA methylation and associated protein expression of tumor suppressor and DNA repair genes are involved in the SCC carcinogenesis process using this hyperplastic candidiasis model. Promoter methylation and protein expression were analyzed by methylation specific PCR and immunohistochemical staining, respectively, of 5 areas in the forestomachs of alloxan-induced diabetic rats with hyperplastic candidiasis: normal squamous epithelia, squamous hyperplasia, squamous hyperplasia adjacent to SCC, squamous hyperplasia transitioning to SCC, and SCC. We observed nuclear p16 overexpression despite increases in p16 gene promoter methylation during the carcinogenic process. TIMP3 and RAR-β2 promoter methylation progressed until the precancerous stage but disappeared upon malignant transformation. In comparison, TIMP3 protein expression was suppressed during carcinogenesis and RAR-β2 expression was attenuated in the cytoplasm but enhanced in nuclei. ERCC1 and BRCA1 promoters were not methylated at any stage; however, their protein expression disappeared beginning at hyperplasia and nuclear protein re-expression in SCC was observed only for ERCC1. These results suggest that aberrant p16, RAR-β2, TIMP3, ERCC1, and BRCA1 expression might occur that is inconsistent with the respective gene promoter methylation status, and that this overexpression might serve to promote the inflammatory carcinogenesis caused by C. albicans infection.

Retraction of Relationships Between p16 Gene Promoter Methylation and Clinicopathologic Features of Colorectal Cancer: A Meta-Analysis of 27 Cohort Studies (DOI: 10.1089/dna.2013.2253).

DNA and Cell BiologyVol. 34, No. 12 RetractionsFree AccessRetraction of Relationships Between p16 Gene Promoter Methylation and Clinicopathologic Features of Colorectal Cancer: A Meta-Analysis of 27 Cohort Studies (DOI: 10.1089/dna.2013.2253)This article retracts the following:Relationships Between p16 Gene Promoter Methylation and Clinicopathologic Features of Colorectal Cancer: A Meta-Analysis of 27 Cohort StudiesPublished Online:15 Dec 2015https://doi.org/10.1089/dna.2013.2253.retAboutSectionsPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail All of the authors of two articles published in DNA and Cell Biology have requested the retraction of this meta-analysis article. The reason they state is “severe conflicts of author sequences.”Chen, Y.-Z. et al. (2014). Relationships Between p16 Gene Promoter Methylation and Clinicopathologic Features of Colorectal Cancer: A Meta-Analysis of 27 Cohort Studies. DNA Cell Biol 33(10):729–738. DOI: 10.1089/dna.2013.2253. Epub 2014 Jun 30.On PubMed.gov, the abstract is accompanied by a comment from Guillaume Filion indicating that the paper bears striking similarity to other publications.These authors have also recently retracted three papers, one from Molecular Biology Reports and two from Tumor Biology.DNA and Cell Biology is dedicated to upholding the strictest standards of scientific publishing, and will not tolerate any improprieties.FiguresReferencesRelatedDetailsCited byRetraction Notices: Who Authored Them?3 January 2018 | Publications, Vol. 6, No. 1Related articlesRelationships Between p16 Gene Promoter Methylation and Clinicopathologic Features of Colorectal Cancer: A Meta-Analysis of 27 Cohort Studies29 Sep 2014DNA and Cell Biology Volume 34Issue 12Dec 2015 InformationCopyright 2015, Mary Ann Liebert, Inc.To cite this article:Retraction of Relationships Between p16 Gene Promoter Methylation and Clinicopathologic Features of Colorectal Cancer: A Meta-Analysis of 27 Cohort Studies (DOI: 10.1089/dna.2013.2253).DNA and Cell Biology.Dec 2015.743-743.http://doi.org/10.1089/dna.2013.2253.retPublished in Volume: 34 Issue 12: December 15, 2015PDF download

Role of Glutathione-S-Transferase Polymorphism on Arsenic Induced DNA Hypermethylation

People chronically exposed to environmental arsenic through their drinking water experience various arsenic induced clinical manifestations and there is modulation of methylation level in their genome. However the response varies widely among persons. The present manuscript deals with the evaluation of role of individual's GST status in altering the degree of DNA methylation after chronic arsenic exposure in human. To study whether Glutathione-S-Transferase (GST) gene polymorphism plays any role in this variation, a total of 92 study subjects were recruited from the villages of southern region of West Bengal, India. Concentration of arsenic in their urine and water, extent of clinical manifestation, GST status and p53 and p16 gene promoter methylation status were determined. Results showed that genetic polymorphism of GSTM1 and T1 were significantly associated (p<0.05) with degree of methylation in p53 and p16 gene promoter region and urinary arsenic in higher exposure group. Persons having null genotype have significantly decreased urinary arsenic and increased DNA methylation level relative to persons with GSTM1 or GSTT1 nonnull genotype of same arsenic exposure group. In the present work we are therefore trying to show that individual's GST polymorphic status may alter the degree of DNA methylation after arsenic exposure.

Retraction Note to: Role of p16 gene promoter methylation in gastric carcinogenesis: a meta-analysis

This meta-analysis was performed to evaluate the relationships between promoter DNA methylation in tumor suppressor gene p16 and gastric carcinogenesis. The PubMed, CISCOM, CINAHL, Web of Science, Google Scholar, EBSCO, Cochrane Library and CBM databases were searched for relevant articles published before November 1st, 2013 without any language restrictions. Meta-analysis was conducted using the STATA 12.0 software. Crude odds ratios (ORs) with 95 % confidence intervals (95 % CI) were calculated. Forty-seven clinical cohort studies that met all inclusion criteria were included in this meta-analysis. A total of 2,813 gastric cancer (GC) patients were assessed. Our meta-analysis results revealed that the frequencies of p16 promoter methylation in the GC tissues were higher than those of normal and adjacent tissues (Normal: OR = 23.04, 95 % CI = 13.55–39.15, P < 0.001; Adjacent: OR = 4.42, 95 % CI = 1.66–11.76, P = 0.003; respectively). Furthermore, we observed significant associations of p16 promoter methylation with TNM stage, histologic grade, invasive grade, lymph node metastasis of GC (TNM stage: OR = 3.60, 95 % CI: 2.17–5.98, P < 0.001; Histologic grade: OR = 2.63, 95 % CI: 1.55–4.45, P < 0.001; Invasive grade: OR = 3.44, 95 % CI: 1.68–7.06, P = 0.001; Lymph node metastasis: OR = 2.68, 95 % CI: 1.66–4.32, P < 0.001; respectively). However, there were no correlations of p16 promoter methylation with the TNM stage and Helicobacter pylori (HP) infection of GC (Tumor size: OR = 0.76, 95 % CI: 0.14–4.07, P = 0.746; HP infection: OR = 1.31, 95 % CI: 0.75–2.27, P = 0.342; respectively). Our findings provide empirical evidence that p16 promoter methylation may play an important role in gastric carcinogenesis. Thus, p16 promoter methylation may be a promising potential biomarker for the early diagnosis of GC.

Oxidative stress levels are correlated with P15 and P16 gene promoter methylation in myelodysplastic syndrome patients.

Oxidative stress and abnormal DNA methylation have been implicated in some types of cancer, namely in myelodysplastic syndromes (MDS). Since both mechanisms are observed in MDS patients, we analyzed the correlation of intracellular levels of peroxides, superoxide anion, and glutathione (GSH), as well as ratios of peroxides/GSH and superoxide/GSH, with the methylation status of P15 and P16 gene promoters in bone marrow leukocytes from MDS patients. Compared to controls, these patients had lower GSH content, higher peroxide levels, peroxides/GSH and superoxide/GSH ratios, as well as higher methylation frequency of P15 and P16 gene promoters. Moreover, patients with methylated P15 gene had higher oxidative stress levels than patients without methylation (peroxides: 460±42 MIF vs 229±25 MIF, p=0.001; superoxide: 383±48 MIF vs 243±17 MIF, p=0.022; peroxides/GSH: 2.50±0.08 vs 1.04±0.34, p<0.001; superoxide/GSH: 1.76±0.21 vs 1.31±0.10, p=0.007). Patients with methylated P16 and at least one methylated gene had higher peroxide levels as well as peroxides/GSH ratio than patients without methylation. Interestingly, oxidative stress levels allow the discrimination of patients without methylation from ones with methylated P15, methylated P16, or at least one methylated (P15 or P16) promoter. Taken together, these findings support the hypothesis that oxidative stress is correlated with P15 and P16 hypermethylation.

A meta-analysis of serum p16 gene promoter methylation for diagnosis of nonsmall cell lung cancer.

To evaluate the diagnostic value of serum p16 gene promoter methylation for diagnosis of nonsmall cell lung cancer (NSCLC). By searching the databases of PubMed and CNKI, we included all the published articles related serum p16 gene promoter methylation and nonsmall lung cancer. The true positive, false positive, false negative, and true negative data for each included publication were extracted by the reviewers. The diagnostic sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, and area under the receiver operating characteristic (ROC) were pooled by MetaDiSc1.4 software. Finally, 13 manuscripts with 1440 subjects were involving in this diagnostic meta-analysis. The pooled sensitivity and specificity were 0.25 (95% confidence interval [CI]: 0.18-0.32) and 0.95 (95% CI: 0.93-0.97), respectively, with randomized effect model. The pooled positive likelihood ratio and negative likelihood ratio were 5.08 (95% CI: 3.00-8.62) and 0.69 (95% CI: 0.62-0.77) with fixed effect model and randomized effect model, respectively. The diagnostic ROC curve for the included 13 publications was pooled by statistical software MetaDiSc14.0 according to the Bayes theorem. The pooled area under the ROC was 0.72 with its standard error of 0.10. According to the published articles, high specificity and low sensitivity were found in this meta-analysis for the p16 gene promoter methylation in the diagnosis of NSCLC.

Relationships between p16 gene promoter methylation and clinicopathologic features of colorectal cancer: a meta-analysis of 27 cohort studies.

Many existing studies have demonstrated that p16 promoter methylation might be correlated with the clinicopathologic features of colorectal cancer (CRC), but individually published results are inconclusive. This meta-analysis aimed to derive a more precise estimation of the relationships between p16 promoter methylation and the clinicopathologic features of CRC. We searched the CISCOM, CINAHL, Web of Science, PubMed, Google Scholar, EBSCO, Cochrane Library, and CBM databases from inception through August 1, 2013. Meta-analysis was performed using the STATA 12.0 software. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated under fixed- or random-effects models. Twenty-seven clinical cohort studies were included with a total of 3311 CRC patients. Our meta-analysis results revealed that p16 promoter methylation was associated with pathological characteristics of CRC (tumor, nodes, metastasis stage: OR=1.55, 95% CI: 1.14-2.13, p=0.006; lymph node metastasis: OR=2.40, 95% CI: 1.37-4.19, p=0.002; histologic grade: OR=2.72, 95% CI: 1.63-4.54, p<0.001; Dukes stage: OR=2.06, 95% CI: 1.57-2.71, p=0.002; tumor size: OR=1.99, 95% CI: 1.03-3.85, p=0.041; location: OR=2.49, 95% CI: 1.95-3.18, p<0.001, respectively). Subgroup analysis by ethnicity suggested that there were also significant correlations between p16 gene promoter methylation and pathological characteristics of CRC among both Caucasian and Asian populations (all p<0.05). Our meta-analysis suggests that promoter methylation of the p16 gene may be strongly correlated with the clinicopathologic features of CRC. Thus, p16 gene promoter methylation may be a potential biomarker for CRC.