Normal Japanese individuals harbor polymorphisms in the p14 ARF /INK4 locus promoters and/or other gene introns. — Variation in nucleotide sequences in each individual

In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced promoters in the p14 ^ARF /INK4 (p16 ^INK4a and p15 ^INK4b ) locus genes and introns in murine double minute 2, vitamin D receptor, and presenilin 1 genes. We found nucleotide alterations in the promoters, including substitutions at positions ?2610 and ?1536 and deletions at positions ?4489, ?4488 to ?4483, ?2241 to ?2240, and ?2221 to ?2218 in p14 ^ARF and substitutions at positions ?1643 and ?1270 in p15 ^INK4b , and we found that each individual harbored polymorphisms in the locus promoters and/or introns. Some polymorphic nucleotides were included in the same set of associatively altered nucleotides. Reporter gene analysis by using luciferase revealed that altered nucleotides, including those containing the set, changed luciferase gene activity in some cell lines. The results of this study show that multi-nucleotide sequences are altered in each normal Japanese individual.     

Polymorphisms in the 5′-UTR of <Emphasis Type="Italic">PTEN</Emphasis> and other gene polymorphisms in normal Japanese individuals

Polymorphisms are distributed differently in populations, including those of regions, ethnic groups, and diseased patients. In order to investigate variation in nucleotide sequences in normal individuals, we isolated genomic DNA from the blood of healthy Japanese individuals and sequenced the 5′-untranslated region (5′-UTR) of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene and the gene promoter, intron, and exon nucleotides of p53, p14 ^ARF , murine double minute 2 (MDM2), and the β₂- and β₃-adrenoceptor (?AR). We found polymorphisms in these regions, including a deletion at positions ?465 to ?463 and a substitution at position ?404 in PTEN and a substitution at position ?4924 in p14 ^ARF , in normal individuals. Individuals with or without the PTEN polymorphism harbored a different distribution of polymorphisms, including simultaneous alterations in nucleotides of p53, MDM2, and β₃-AR, and also harbored some polymorphic nucleotides located in the same set of associatively altered nucleotides. Our results show that multiple nucleotides, including the PTEN nucleotides, are altered in normal Japanese individuals and provide useful information for genotyping studies in individuals and populations.     

Methylation profile of INK4B-ARF-INK4A locus in atherosclerosis

Single-nucleotide polymorphisms (SNPs) in the 9p21.3 locus have recently been demonstrated to be strongly associated with atherosclerosis. However, the pathophysiology of this locus is insufficiently studied. Here, the methylation profile of the nearest mapped genes for cyclin-dependent kinase inhibitors CDKN2A (p16^INK4a, p14^ARF) and CDKN2B (p15^INK4b) in the tissues of the carotid artery in patients with atherosclerosis was evaluated for the first time. Aberrant DNA methylation of the analyzed loci was not established in either the atherosclerotic plaques or in the tissues from the macroscopically intact vascular wall in the same patients.     

FOXM1 induces a global methylation signature that mimics the cancer epigenome in head and neck squamous cell carcinoma

The oncogene FOXM1 has been implicated in all major types of human cancer. We recently showed that aberrant FOXM1 expression causes stem cell compartment expansion resulting in the initiation of hyperplasia. We have previously shown that FOXM1 regulates HELLS, a SNF2/helicase involved in DNA methylation, implicating FOXM1 in epigenetic regulation. Here, we have demonstrated using primary normal human oral keratinocytes (NOK) that upregulation of FOXM1 suppressed the tumour suppressor gene p16^INK4A (CDKN2A) through promoter hypermethylation. Knockdown of HELLS using siRNA re-activated the mRNA expression of p16^INK4A and concomitant downregulation of two DNA methyltransferases DNMT1 and DNMT3B. The dose-dependent upregulation of endogenous FOXM1 (isoform B) expression during tumour progression across a panel of normal primary NOK strains (n = 8), dysplasias (n = 5) and head and neck squamous cell carcinoma (HNSCC) cell lines (n = 11) correlated positively with endogenous expressions of HELLS, BMI1, DNMT1 and DNMT3B and negatively with p16^INK4A and involucrin. Bisulfite modification and methylation-specific promoter analysis using absolute quantitative PCR (MS-qPCR) showed that upregulation of FOXM1 significantly induced p16^INK4A promoter hypermethylation (10-fold, P<0.05) in primary NOK cells. Using a non-bias genome-wide promoter methylation microarray profiling method, we revealed that aberrant FOXM1 expression in primary NOK induced a global hypomethylation pattern similar to that found in an HNSCC (SCC15) cell line. Following validation experiments using absolute qPCR, we have identified a set of differentially methylated genes, found to be inversely correlated with in vivo mRNA expression levels of clinical HNSCC tumour biopsy samples. This study provided the first evidence, using primary normal human cells and tumour tissues, that aberrant upregulation of FOXM1 orchestrated a DNA methylation signature that mimics the cancer methylome landscape, from which we have identified a unique FOXM1-induced epigenetic signature which may have clinical translational potentials as biomarkers for early cancer screening, diagnostic and/or therapeutic interventions.     

Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-β signaling

We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-β signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-β signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-β1 levels, phosphorylation of Smad2/3, and increased expression of p15^INK4B and p57^KIP2. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-β1 induced dephosphorylation of Smad2/3, and diminished the level of p15^INK4B and p57^KIP2. Moreover, Bmi-1 expression led to the inhibition of TGF-β-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15^INK4B, and p57^KIP2. In addition, an exposure of senescent NHOK to TGF-β receptor I kinase inhibitor or anti-TGF-β antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-β signaling pathway in NHOK.     

Epigenetic regulation of the INK4b-ARF-INK4a locus: In sickness and in health

The INK4b-ARF-INK4a locus encodes for two cyclin-dependent kinase inhibitors, p15^INK4b and p16^INK4a, and a regulator of the p53 pathway, ARF. In addition ANRIL , a non-coding RNA, is also transcribed from the locus. ARF, p15^INK4b and p16^INK4a are well-established tumor suppressors which function is frequently disabled in human cancers. Recent studies showed that single nucleotide polymorphisms mapping in the vicinity of ANRIL are linked to a wide spectrum of conditions, including cardiovascular disease, ischemic stroke, type 2 diabetes, frailty and Alzheimer disease. The INK4b-ARF-INK4a locus is regulated by Polycomb repressive complexes (PRCs) and its expression can be invoked by activating signals. Other epigenetic modifiers such as the histone demethylases JMJD3 and JHDM1B, the SWI/SNF chromatin remodeling complex and DNA methyltransferases regulate the locus interplaying with PRCs. In view of the intimate involvement of the INK4b-ARF-INK4a locus on disease, to understand its regulation is the first step for manipulate it to therapeutic benefit.Key words: senescence, p16^INK4a, ARF, p15^INK4b, ANRIL, polycomb, histone demethylases, DNA methylation     

Association between P16INK4a Promoter Methylation and HNSCC: A Meta-Analysis of 21 Published Studies

Background

The p16^INK4a is an important tumor suppressor gene (TSG) and aberrant methylation of promoter is known to be a major inactivation mechanism of the tumor suppressor and tumor-related genes. Aberrant TSG methylation was considered an important epigenetic silencing mechanism in the progression of head and neck squamous cell carcinoma (HNSCC). However, some studies have reported differences in the methylation frequencies of P^16INK4a promoter between cancer and the corresponding control group. Therefore, we conducted a meta-analysis to better identify the association.

Methods

PubMed, Ovid, ISI Web of Science, and EMBASE were searched to identify eligible studies to evaluate the association of p16^INK4a promoter methylation and HNSCC. Odds ratio (ORs) and 95% confidence intervals (95%CI) were calculated to evaluate the strength of association between p16^INK4a promoter methylation and HNSCC.

Results

A total of twenty-one studies with 1155 cases and 1017 controls were included in the meta-analysis. The frequencies of p16^INK4a promoter methylation in the cancer group were significantly higher than those in the control group (cancer group: median: 46.67%, range = 7.84%-95.12%; control group: median: 18.37%, range = 0–83.33%; respectively). The pooled odds ratio was 3.37 (95%CI = 2.32–4.90) in the cancer group versus the corresponding control group under the random-effects model.

Conclusion

This meta-analysis of 21 published studies identified that aberrant methylation of p16^INK4a promoter was found to be significantly associated with HNSCC.     

Methylation of p15INK4b and Expression of ANRIL on Chromosome 9p21 Are Associated with Coronary Artery Disease

Background

Genome-wide association studies have identified that multiple single nucleiotide polymorphisms on chromosome 9p21 are tightly associated with coronary artery disease (CAD). However, the mechanism linking this risk locus to CAD remains unclear.

Methodology/Principal Findings

The methylation status of six candidate genes (BAX, BCL-2, TIMP3, p14^ARF, p15^INK4b and p16^INK4a) in 205 patients and controls who underwent coronary angiography were analyzed by quantitative MethyLight assay. Rs10757274 was genotyped and expression of INK4/ARF and antisense non-coding RNA in the INK4 locus (ANRIL) was determined by real-time RT-PCR. Compared with controls, DNA methylation levels at p15^INK4b significantly increased in CAD patients (p = 0.006). To validate and dissect the methylation percentage of each target CpG site at p15^INK4b, pyrosequencing was performed, finding CpG +314 and +332 remarkably hypermethylated in CAD patients. Further investigation determined that p15^INK4b hypermethylation prevalently emerged in lymphocytes of CAD patients (p = 0.013). The rs10757274 genotype was significantly associated with CAD (p = 0.003) and GG genotype carriers had a higher level of ANRIL exon 1–5 expression compared among three genotypes (p = 0.009). There was a stepwise increase in p15^INK4b and p16^INK4a methylation as ANRIL exon 1–5 expression elevated (r = 0.23, p = 0.001 and r = 0.24, p = 0.001, respectively), although neither of two loci methylation was directly linked to rs10757274 genotype.

Conclusions/Significance

p15^INK4b methylation is associated with CAD and ANRIL expression. The epigenetic changes in p15^INK4b methylation and ANRIL expression may involve in the mechanisms of chromosome 9p21 on CAD development.