Inactivation of p16 in Human Mammary Epithelial Cells by CpG Island Methylation

Proliferation of human mammary epithelial cells (HMEC) is limited to a few passages in culture due to an arrest in G₁ termed selection or mortality stage 0, M0. A small number of cells spontaneously escape M0, continue to proliferate in culture, and then enter a second mortality stage, M1, at which they senesce. Evidence that M0 involves the Rb pathway comes from the observation that expression of human papillomavirus type 16 E7 alleviates the M0 proliferation block, and we further show that the Rb-binding region of E7 is required to allow cells to bypass M0. In contrast, E6 does not prevent HMEC from entering M0 but, rather, is involved in M1 bypass. Here we show that inactivation of the D-type cyclin-dependent kinase inhibitor p16^INK4A is associated with escape from the M0 proliferation block. Early-passage HMEC express readily detectable amounts of p16 protein, whereas normal or E6-expressing HMEC that escaped M0 expressed markedly reduced amounts of p16 mRNA and protein. This initial reduction of p16 expression was associated with limited methylation of the p16 promoter region CpG island. At later passages, a further reduction in p16 expression occurred, accompanied by increased CpG island methylation. In contrast, reduction of p16 expression did not occur in E7-expressing HMEC that bypassed M0, due to inactivation of Rb. These observations in the E6-expressing HMEC correlate well with the finding that CpG island methylation is a mechanism of p16 inactivation in the development of human tumors, including breast cancer.     

p16基因甲基化状态与散发性大肠癌的相关性研究

为探讨p1 6基因甲基化状态与散发性大肠癌发生发展的关系 ,用甲基化特异性的聚合酶链反应 (methylati omspecificPCR ,MSP)结合测序检测散发性大肠癌及相应癌旁组织p1 6基因甲基化状态。研究发现p1 6基因在散发性大肠癌中甲基化率为 2 8 9% (1 3 4 5 ) ,有 8例癌及癌旁组织都发生了甲基化 ;有淋巴结及远处转移的甲基化率为5 0 % (8 1 6 ) ,高于无转移的甲基化率 2 0 8(5 2 4 ) (P <0 0 5 )。p1 6基因高甲基化是散发性大肠癌中常见的分子改变之一 ,大肠癌中p1 6基因高甲基化可能发生在癌变早期并与大肠癌的恶性进展有相关性     

Curcumin Modulates DNA Methylation in Colorectal Cancer Cells

Aim

Recent evidence suggests that several dietary polyphenols may exert their chemopreventive effect through epigenetic modifications. Curcumin is one of the most widely studied dietary chemopreventive agents for colon cancer prevention, however, its effects on epigenetic alterations, particularly DNA methylation, remain unclear. Using systematic genome-wide approaches, we aimed to elucidate the effect of curcumin on DNA methylation alterations in colorectal cancer cells.

Materials and Methods

To evaluate the effect of curcumin on DNA methylation, three CRC cell lines, HCT116, HT29 and RKO, were treated with curcumin. 5-aza-2′-deoxycytidine (5-aza-CdR) and trichostatin A treated cells were used as positive and negative controls for DNA methylation changes, respectively. Methylation status of LINE-1 repeat elements, DNA promoter methylation microarrays and gene expression arrays were used to assess global methylation and gene expression changes. Validation was performed using independent microarrays, quantitative bisulfite pyrosequencing, and qPCR.

Results

As expected, genome-wide methylation microarrays revealed significant DNA hypomethylation in 5-aza-CdR-treated cells (mean β-values of 0.12), however, non-significant changes in mean β-values were observed in curcumin-treated cells. In comparison to mock-treated cells, curcumin-induced DNA methylation alterations occurred in a time-dependent manner. In contrast to the generalized, non-specific global hypomethylation observed with 5-aza-CdR, curcumin treatment resulted in methylation changes at selected, partially-methylated loci, instead of fully-methylated CpG sites. DNA methylation alterations were supported by corresponding changes in gene expression at both up- and down-regulated genes in various CRC cell lines.

Conclusions

Our data provide previously unrecognized evidence for curcumin-mediated DNA methylation alterations as a potential mechanism of colon cancer chemoprevention. In contrast to non-specific global hypomethylation induced by 5-aza-CdR, curcumin-induced methylation changes occurred only in a subset of partially-methylated genes, which provides additional mechanistic insights into the potent chemopreventive effect of this dietary nutraceutical.     

FHIT基因在宫颈癌细胞中的表达及其甲基化调控

目的：分析FHIT基因在宫颈癌细胞中表达情况以及甲基化的调控情况。方法：对RJC-1、SiHa、CS1213以及C4-1细胞进行培养,提取这些细胞的DNA并经过亚硫酸氢盐修饰,进行PCR反应和产物的检测。分析FHIT基因在宫颈癌细胞中表达情况以及甲基化的调控情况。结果：RJC-1、CS1213细胞仅有甲基化引物扩增出了目的条带,为完全甲基化状态。其他细胞则是甲基特异性引物与非甲基特异性引物共同扩增出73bp的目的条带,其状态为甲基化杂合性。通过5-aza-CdR处理细胞后,通过实时定量PCR检测FHIT mRNA的表达,显示处理后各种细胞中的FHIT mRNA的表达升高。结论：FHIT基因的甲基化是其表达下调的重要机制之一,是临床研究宫颈癌细胞的重要方向之一。     

脱氢表雄酮调节HepG2/GFP-HBx细胞p16基因甲基化及其与细胞周期和凋亡的相关性

目的探讨HBx与p16基因甲基化的关系,研究脱氢表雄酮（DHEA）对p16基因甲基化以及细胞周期和细胞凋亡的调节作用。方法以HepG2、HepG2/GFP和HepG2/GFP-HBx三种细胞为材料,采用MTT法检测细胞生长;流式细胞术检测细胞周期和凋亡率;MSP-PCR检测p16基因甲基化水平。比较分析HBx与p16基因启动子甲基化、DHEA与各检测指标的关系。结果HepG2/GFP-HBx细胞与HepG2细胞和HepG2/GFP细胞相比,细胞的增殖速度提高（P〈0.05）,G0/G1期细胞减少,S期细胞增多（P〈0.05）,凋亡率降低（P〈0.05）;HepG2/GFP-HBx细胞的p16基因启动子呈现高水平甲基化,HepG2和HepG2/GFP细胞呈现高水平非甲基化。100μmol/L的DHEA使三种细胞的增殖速度降低（P〈0.05）,G0/G1期细胞增加,S期细胞减少（P〈0.05）,凋亡率提高（P〈0.05）。DHEA可下调HepG2/GFP-HBx细胞的p16基因启动子甲基化水平,但对HepG2和HepG2/GFP细胞的p16基因启动子甲基化水平不产生影响。结论HBx引起肝癌细胞p16基因甲基化,并缩短细胞周期抑制细胞凋亡;DHEA可明显下调HBx引起的p16基因甲基化水平,延长细胞周期促进细胞凋亡,在无HBx基因整合的情况下,DHEA对肝癌细胞生长、细胞周期和凋亡的影响不通过p16基因甲基化的途径实现。     

Regulation and Methylation of Tumor Suppressor MiR-124 by Androgen Receptor in Prostate Cancer Cells

Prostate cancer (PCa) is the most frequently diagnosed cancer for men in the developed world. Androgen receptor signaling pathway plays an important role in prostate cancer progression. Recent studies show that microRNA miR-124 exerts a tumor suppressive function in prostate cancer. However, the relationship between AR and miR-124 is unclear. In the present study, we found a negative feedback loop between AR and miR-124 expression. On one hand, miR-124 was a positively regulated target gene of the AR, on the other hand, overexpression of miR-124 inhibited the expression of AR. In addition, we found that miR-124-2 and miR-124-3 promoters were hypermethylated in AR-negative PCa cells. Furthermore, overexpression of miR-124 inhibited proliferation rates and invasiveness capacity of PCa cells in vitro, and suppressed xenograft tumor growth in vivo. Taken together, our results support a negative feedback loop between AR and miR-124 expression. Methylation of miR-124-2 and miR-124-3 may serve as a biomarker for AR-negative PCa cells, and overexpression of miR-124 might be of potential therapeutic value for the treatment of PCa.     

Effects of Methylation Status of CpG Sites within the HPV16 Long Control Region on HPV16-Positive Head and Neck Cancer Cells

ObjectiveTo map comprehensively the methylation status of the CpG sites within the HPV16 long control region (LCR) in HPV-positive cancer cells, and to explore further the effects of methylation status of HPV16 LCR on cell bioactivity and E6 and E7 expression. In addition, to analyze the methylation status of the LCR in HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) patients.ResultsHypermethylation status of the LCR in UM-SCC47 (79.8%) and CaSki cells (90.0%) and unmethylation status of the LCR in SiHa cells (0%) were observed. Upon demethylation, the cells with different methylation levels responded differently during growth, apoptosis, and cell cycle arrest, as well as in terms of their E6 and E7 expression. In HPV16-positive OPSCC patients, the methylation rates were 9.5% in the entire LCR region, 13.9% in the 5′-LCR, 6.0% in the E6 enhancer, and 9.5% in the p97 promoter, and hypermethylation of p97 promoter was found in a subset of cases (20.0%, 2/10).ConclusionsOur study revealed two different methylation levels of the LCR in HPV16-positive cancer cells and OPSCC patients, which may represent different carcinogenesis mechanisms of HPV-positive cancers cells. Demethylating the meCpGs in HPV16 LCR might be a potential target for a subgroup of HPV16-positive patients with head and neck squamous cell carcinoma.     

p16基因甲基化与表达在子宫内膜癌中的意义

肿瘤抑制基因p16定位于9号染色体短臂2区1带,编码细胞周期调节蛋白p16,p16基因失活将导致细胞增殖失控。研究证实肿瘤抑制基因启动子区域5CpG岛甲基化是导致转录水平上基因失活的重要机制。为了研究p16基因甲基化状态及其表达异常与子宫内膜癌发生的关系,采用甲基化特异性PCR(MSP)、免疫组化及PCR方法检测62例子宫内膜癌及相应癌旁组织、10例相应年龄正常子宫内膜中p16基因5′cpG岛甲基化状态、p16蛋白表达及p16基因外显子E,和E：表达缺失情况。结果表明癌旁及正常子宫内膜p16基因无甲基化,且无p16蛋白、外显子1和2的表达异常。62例子宫内膜癌中,15例甲基化,占24、2％;33例p16蛋白表达下降或无表达,占54．8％;p16基因外显子1缺失率16．1％(10／62),外显子2缺失率为30．6％(19／62),两者均缺失9．68％(6／62),至少其中1种缺失46、6％(29／62)。提示P16基因失活在子宫内膜癌中多见且与病理分级、临床分期密切相关。D16基因甲基化在子宫内膜癌的发生中起着重要作用。MSP法测定基因甲基化状态准确且简便可行。     

Identification of Global DNA Methylation Signatures in Glioblastoma-Derived Cancer Stem Cells

Glioblastoma(GBM)is the most common and most aggressive primary brain tumor in adults.The existence of a small population of stem-like tumor cells that efficiently propagate tumors and resist cytotoxic therapy is one proposed mechanism leading to the resilient behavior of tumor cells and poor prognosis.In this study,we performed an in-depth analysis of the DNA methylation landscape in GBMderived cancer stem cells(GSCs).Parallel comparisons of primary tumors and GSC lines derived from these tumors with normal controls(a neural stem cell(NSC)line and normal brain tissue)identified groups of hyper- and hypomethylated genes that display a trend of either increasing or decreasing methylation levels in the order of controls,primary GBMs,and their counterpart GSC lines,respectively.Interestingly,concurrent promoter hypermethylation and gene body hypomethylation were observed in a subset of genes including MGMT,AJAP1 and PTPRN2.These unique DNA methylation signatures were also found in primary GBM-derived xenograft tumors indicating that they are not tissue culture-related epigenetic changes.Integration of GSC-specific epigenetic signatures with gene expression analysis further identified candidate tumor suppressor genes that are frequently down-regulated in GBMs such as SPINT2,NEFM and PENK.Forced re-expression of SPINT2 reduced glioma cell proliferative capacity,anchorage independent growth,cell motility,and tumor sphere formation in vitro.The results from this study demonstrate that GSCs possess unique epigenetic signatures that may play important roles in the pathogenesis of GBM.     

Comparative Anatomy of Chromosomal Domains with Imprinted and Non-Imprinted Allele-Specific DNA Methylation

Allele-specific DNA methylation (ASM) is well studied in imprinted domains, but this type of epigenetic asymmetry is actually found more commonly at non-imprinted loci, where the ASM is dictated not by parent-of-origin but instead by the local haplotype. We identified loci with strong ASM in human tissues from methylation-sensitive SNP array data. Two index regions (bisulfite PCR amplicons), one between the C3orf27 and RPN1 genes in chromosome band 3q21 and the other near the VTRNA2-1 vault RNA in band 5q31, proved to be new examples of imprinted DMRs (maternal alleles methylated) while a third, between STEAP3 and C2orf76 in chromosome band 2q14, showed non-imprinted haplotype-dependent ASM. Using long-read bisulfite sequencing (bis-seq) in 8 human tissues we found that in all 3 domains the ASM is restricted to single differentially methylated regions (DMRs), each less than 2kb. The ASM in the C3orf27-RPN1 intergenic region was placenta-specific and associated with allele-specific expression of a long non-coding RNA. Strikingly, the discrete DMRs in all 3 regions overlap with binding sites for the insulator protein CTCF, which we found selectively bound to the unmethylated allele of the STEAP3-C2orf76 DMR. Methylation mapping in two additional genes with non-imprinted haplotype-dependent ASM, ELK3 and CYP2A7, showed that the CYP2A7 DMR also overlaps a CTCF site. Thus, two features of imprinted domains, highly localized DMRs and allele-specific insulator occupancy by CTCF, can also be found in chromosomal domains with non-imprinted ASM. Arguing for biological importance, our analysis of published whole genome bis-seq data from hES cells revealed multiple genome-wide association study (GWAS) peaks near CTCF binding sites with ASM.     

NDRG2基因启动子甲基化与肺腺癌细胞中mRNA表达相关性的实验研究

目的:探讨肺腺癌细胞中NDRG2基因启动子甲基化状态及其与基因表达的关系。方法:甲基化焦磷酸测序技术检测启动子区域甲基化状态,荧光定量PCR技术检测不同药物浓度下培养细胞中NDRG2基因mRNA的表达水平,分析启动子区域甲基化与基因表达之间的关系。结果:在体外培养细胞中检测到NDRG2基因启动子区域呈现不同程度的甲基化,甲基化频率分别为肺癌A549细胞71.8%、GLC-82细胞86.1%、人脐静脉内皮ECV-304细胞36.8%、胃上皮GES-1细胞42.9%。NDRG2基因mRNA表达与其启动子甲基化程度成反比,甲基转移酶抑制剂5-杂氮-2-脱氧胞苷(5-Aza-CdR)作用于细胞后,A549和GLC-82细胞中NDRG2基因的mRNA转录明显上调,至72 h差异显著(P0.05)。结论:肺腺癌细胞中NDRG2基因启动子CpG岛存在高甲基化,甲基化程度与该基因的表达具有负相关性,5-Aza-CdR能在一定程度上提高NDRG2的转录水平。     

Allele-Specific Transcriptome and Methylome Analysis Reveals Stable Inheritance and Cis-Regulation of DNA Methylation in Nasonia

Gene expression divergence between closely related species could be attributed to both cis- and trans- DNA sequence changes during evolution, but it is unclear how the evolutionary dynamics of epigenetic marks are regulated. In eutherian mammals, biparental DNA methylation marks are erased and reset during gametogenesis, resulting in paternal or maternal imprints, which lead to genomic imprinting. Whether DNA methylation reprogramming exists in insects is not known. Wasps of the genus Nasonia are non-social parasitoids that are emerging as a model for studies of epigenetic processes in insects. In this study, we quantified allele-specific expression and methylation genome-wide in Nasonia vitripennis and Nasonia giraulti and their reciprocal F1 hybrids. No parent-of-origin effect in allelic expression was found for >8,000 covered genes, suggesting a lack of genomic imprinting in adult Nasonia. As we expected, both significant cis- and trans- effects are responsible for the expression divergence between N. vitripennis and N. giraulti. Surprisingly, all 178 differentially methylated genes are also differentially methylated between the two alleles in F1 hybrid offspring, recapitulating the parental methylation status with nearly 100% fidelity, indicating the presence of strong cis-elements driving the target of gene body methylation. In addition, we discovered that total and allele-specific expression are positively correlated with allele-specific methylation in a subset of the differentially methylated genes. The 100% cis-regulation in F1 hybrids suggests the methylation machinery is conserved and DNA methylation is targeted by cis features in Nasonia. The lack of genomic imprinting and parent-of-origin differentially methylated regions in Nasonia, together with the stable inheritance of methylation status between generations, suggests either a cis-regulatory motif for methylation at the DNA level or highly stable inheritance of an epigenetic signal in Nasonia.     

Novel Pharmacologic Targeting of Tight Junctions and Focal Adhesions in Prostate Cancer Cells

Cancer cell resistance to anoikis driven by aberrant signaling sustained by the tumor microenvironment confers high invasive potential and therapeutic resistance. We recently generated a novel lead quinazoline-based Doxazosin® derivative, DZ-50, which impairs tumor growth and metastasis via anoikis. Genome-wide analysis in the human prostate cancer cell line DU-145 identified primary downregulated targets of DZ-50, including genes involved in focal adhesion integrity (fibronectin, integrin-α6 and talin), tight junction formation (claudin-11) as well as insulin growth factor binding protein 3 (IGFBP-3) and the angiogenesis modulator thrombospondin 1 (TSP-1). Confocal microscopy demonstrated structural disruption of both focal adhesions and tight junctions by the downregulation of these gene targets, resulting in decreased cell survival, migration and adhesion to extracellular matrix (ECM) components in two androgen-independent human prostate cancer cell lines, PC-3 and DU-145. Stabilization of cell-ECM interactions by overexpression of talin-1 and/or exposing cells to a fibronectin-rich environment mitigated the effect of DZ-50. Loss of expression of the intracellular focal adhesion signaling effectors talin-1 and integrin linked kinase (ILK) sensitized human prostate cancer to anoikis. Our findings suggest that DZ-50 exerts its antitumor effect by targeting the key functional intercellular interactions, focal adhesions and tight junctions, supporting the therapeutic significance of this agent for the treatment of advanced prostate cancer.     

Replacement of Oct4 by Tet1 during iPSC Induction Reveals an Important Role of DNA Methylation and Hydroxymethylation in Reprogramming

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Highlights? Tet1 facilitates iPSC induction by promoting Oct4 demethylation and reactivation ? Tet1 can replace Oct4 to generate fully pluripotent TSKM iPSCs ? Both 5mC and 5hmC increase on the genome during TSKM 2° reprogramming ? 5hmC promotes the demethylation of pluripotency genes during reprogramming     

Comparison of Aberrant Methylation of CpG Islands in the p16/CDKN2A and p14/ARF Promoters in Non-Small Cell Lung Cancer and Acute Lymphoblastic Leukemia

Multiplex methylation-sensitive (MSe-PCR) and methylation-specific (MSp-PCR) PCRs were used to detect aberrant methylation of CpG islands in the promoter regions and first exons of p16/CDKN2A and p14/ARF in non-small cell lung cancer (NSCLC, 54 specimens) and B-cell acute lymphoblastic leukemia (B-ALL, 61 specimens). A difference in CpG methylation was observed for individual specimens and for the two malignancies. A high methylation frequency of the first exon of p16/CDKN2A was detected both in NSCLC (68%) and in B-ALL (55%). The CpG island of the p14/ARF first exon proved to be nonmethylated in both malignancies. Particular CpG-rich fragments were examined in the p16/CDKN2A and p14/ARF promoters. It was shown that methylation frequency can differ between the 5 regions of one promoter. The sensitivity was compared for MSe-PCR and MSp-PCR, which are commonly employed in methylation analysis.     

The Induction of microRNA-16 in Colon Cancer Cells by Protein Arginine Deiminase Inhibition Causes a p53-Dependent Cell Cycle Arrest

Protein Arginine Deiminases (PADs) catalyze the post-translational conversion of peptidyl-Arginine to peptidyl-Citrulline in a calcium-dependent, irreversible reaction. Evidence is emerging that PADs play a role in carcinogenesis. To determine the cancer-associated functional implications of PADs, we designed a small molecule PAD inhibitor (called Chor-amidine or Cl-amidine), and tested the impact of this drug on the cell cycle. Data derived from experiments in colon cancer cells indicate that Cl-amidine causes a G1 arrest, and that this was p53-dependent. In a separate set of experiments, we found that Cl-amidine caused a significant increase in microRNA-16 (miRNA-16), and that this increase was also p53-dependent. Because miRNA-16 is a putative tumor suppressor miRNA, and others have found that miRNA-16 suppresses proliferation, we hypothesized that the p53-dependent G1 arrest associated with PAD inhibition was, in turn, dependent on miRNA-16 expression. Results are consistent with this hypothesis. As well, we found the G1 arrest is at least in part due to the ability of Cl-amidine-mediated expression of miRNA-16 to suppress its'' G1-associated targets: cyclins D1, D2, D3, E1, and cdk6. Our study sheds light into the mechanisms by which PAD inhibition can protect against or treat colon cancer.     

p53 Suppresses Metabolic Stress-Induced Ferroptosis in Cancer Cells

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