DNA甲基化修饰与干细胞分化

干细胞自我更新及分化潜能一方面是内源性转录因子相互协调控制的结果,另一方面表观遗传修饰也起着重要的作用。该文综述了DNA甲基化修饰的机理、哺乳动物DNA甲基化的特点以及干细胞分化的DNA甲基化修饰。     

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.     

肿瘤干细胞及肝癌肿瘤干细胞

肿瘤干细胞理论认为只有存在于肿瘤中的少量干细胞性质的细胞群体对肿瘤发生和发展起着决定作用,肿瘤是由干细胞突变积累而形成的无限增殖的异常组织,这一理论的提出使人们对肿瘤发生机制的认识上升到了一个新的高度,也引起了研究者的广泛关注;肝癌是我国常见的恶性肿瘤之一,我国肝癌死亡率居世界之首,目前对肝癌的研究是我国恶性肿瘤防治的重点工作,现对当前肿瘤干细胞与肝癌肿瘤干细胞相关方面的最新研究进展作一概述。     

Epithelial to Mesenchymal Transition Is Mechanistically Linked with Stem Cell Signatures in Prostate Cancer Cells

Background

Current management of patients diagnosed with prostate cancer (PCa) is very effective; however, tumor recurrence with Castrate Resistant Prostate Cancer (CRPC) and subsequent metastasis lead to poor survival outcome, suggesting that there is a dire need for novel mechanistic understanding of tumor recurrence, which would be critical for designing novel therapies. The recurrence and the metastasis of PCa are tightly linked with the biology of prostate cancer stem cells or cancer-initiating cells that is reminiscent of the acquisition of Epithelial to Mesenchymal Transition (EMT) phenotype. Increasing evidence suggests that EMT-type cells share many biological characteristics with cancer stem-like cells.

Methodology/Principal Findings

In this study, we found that PCa cells with EMT phenotype displayed stem-like cell features characterized by increased expression of Sox2, Nanog, Oct4, Lin28B and/or Notch1, consistent with enhanced clonogenic and sphere (prostasphere)-forming ability and tumorigenecity in mice, which was associated with decreased expression of miR-200 and/or let-7 family. Reversal of EMT by re-expression of miR-200 inhibited prostasphere-forming ability of EMT-type cells and reduced the expression of Notch1 and Lin28B. Down-regulation of Lin28B increased let-7 expression, which was consistent with repressed self-renewal capability.

Conclusions/Significance

These results suggest that miR-200 played a pivotal role in linking the characteristics of cancer stem-like cells with EMT-like cell signatures in PCa. Selective elimination of cancer stem-like cells by reversing the EMT phenotype to Mesenchymal-Epithelial Transition (MET) phenotype using novel agents would be useful for the prevention of tumor recurrence especially by eliminating those cells that are the “Root Cause” of tumor development and recurrence.     

DNA甲基化与癌症

DNA甲基化是重要的表观遗传修饰,主要发生在DNA的CpG岛. DNA的甲基化通过DNA甲基转移酶（DNA methyltransferases, DNMTs）完成. DNA甲基化参与了细胞分化、基因组稳定性、X染色体失活、基因印记等多种细胞生物学过程.单基因水平及基因组范围内的DNA甲基化改变在肿瘤发生发展中亦发挥重要作用. 抑癌基因的异常甲基化引起的表达抑制,可导致肿瘤细胞的增殖失控和侵袭转移,并参与肿瘤组织的血管生成过程.在许多肿瘤的研究中都发现了基因组整体DNA低甲基化所导致的染色体不稳定性. 本文从DNA的异常高甲基化和低甲基化两方面论述了DNA甲基化在细胞恶变发生发展过程中的改变及其影响,并阐述了DNA甲基化改变在肿瘤诊断和治疗中的作用.     

Estimation of the Fraction of Cancer Cells in a Tumor DNA Sample Using DNA Methylation

Contamination of normal cells is almost always present in tumor samples and affects their molecular analyses. DNA methylation, a stable epigenetic modification, is cell type-dependent, and different between cancer and normal cells. Here, we aimed to demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample, using esophageal squamous cell carcinoma (ESCC) as an example. First, by an Infinium HumanMethylation450 BeadChip array, we isolated three genomic regions (TFAP2B, ARHGEF4, and RAPGEFL1) i) highly methylated in four ESCC cell lines, ii) hardly methylated in a pooled sample of non-cancerous mucosae, a pooled sample of normal esophageal mucosae, and peripheral leukocytes, and iii) frequently methylated in 28 ESCCs (TFAP2B, 24/28; ARHGEF4, 20/28; and RAPGEFL1, 19/28). Second, using eight pairs of cancer and non-cancer cell samples prepared by laser capture microdissection, we confirmed that at least one of the three regions was almost completely methylated in ESCC cells, and all the three regions were almost completely unmethylated in non-cancer cells. We also confirmed that DNA copy number alterations of the three regions in 15 ESCC samples were rare, and did not affect the estimation of the fraction of cancer cells. Then, the fraction of cancer cells in a tumor DNA sample was defined as the highest methylation level of the three regions, and we confirmed a high correlation between the fraction assessed by the DNA methylation fraction marker and the fraction assessed by a pathologist (r=0.85; p<0.001). Finally, we observed that, by correction of the cancer cell content, CpG islands in promoter regions of tumor-suppressor genes were almost completely methylated. These results demonstrate that DNA methylation can be used to estimate the fraction of cancer cells in a tumor DNA sample.     

肿瘤干细胞与胃癌

最近的一项研究报导,采用流式细胞仪分选技术从人胃癌细胞株中分离出CD44^＋胃癌干细胞. 20～30×10³个CD44＋细胞入NOD/SCID 鼠腹部皮下和胃浆膜下能形成胃癌移植瘤, 100×10³个CD44^－的细胞入NOD/SCID 鼠体内不形成肿瘤.采用无血清、无粘附间质的干细胞体外培养方法,发现CD44^＋的细胞能形成肿瘤微球体,具有自我更新能力,而CD44^－的细胞则不形成球形克隆.上述的实验结果说明,在人胃癌细胞株中存在胃癌肿瘤干细胞.据此可以相信,胃癌干细胞是胃癌细胞中具有自我更新及分化潜能的一小群细胞,不能被目前的化疗、放疗等抗癌治疗措施所杀灭,是胃癌术后复发、肿瘤进展扩散转移的根源.胃癌干细胞可能来源于骨髓干细胞.随着对胃癌肿瘤干细胞生物学研究的深入,必将为胃癌的临床诊断和治疗提供新的策略.     

Comparison of Methods for Quantification of Global DNA Methylation in Human Cells and Tissues

DNA methylation is a key epigenetic modification which, in mammals, occurs mainly at CpG dinucleotides. Most of the CpG methylation in the genome is found in repetitive regions, rich in dormant transposons and endogenous retroviruses. Global DNA hypomethylation, which is a common feature of several conditions such as ageing and cancer, can cause the undesirable activation of dormant repeat elements and lead to altered expression of associated genes. DNA hypomethylation can cause genomic instability and may contribute to mutations and chromosomal recombinations. Various approaches for quantification of global DNA methylation are widely used. Several of these approaches measure a surrogate for total genomic methyl cytosine and there is uncertainty about the comparability of these methods. Here we have applied 3 different approaches (luminometric methylation assay, pyrosequencing of the methylation status of the Alu repeat element and of the LINE1 repeat element) for estimating global DNA methylation in the same human cell and tissue samples and have compared these estimates with the “gold standard” of methyl cytosine quantification by HPLC. Next to HPLC, the LINE1 approach shows the smallest variation between samples, followed by Alu. Pearson correlations and Bland-Altman analyses confirmed that global DNA methylation estimates obtained via the LINE1 approach corresponded best with HPLC-based measurements. Although, we did not find compelling evidence that the gold standard measurement by HPLC could be substituted with confidence by any of the surrogate assays for detecting global DNA methylation investigated here, the LINE1 assay seems likely to be an acceptable surrogate in many cases.     

Aberrant DNA Methylation in ES Cells

Both mouse and human embryonic stem cells can be differentiated in vitro to produce a variety of somatic cell types. Using a new developmental tracing approach, we show that these cells are subject to massive aberrant CpG island de novo methylation that is exacerbated by differentiation in vitro. Bioinformatics analysis indicates that there are two distinct forms of abnormal de novo methylation, global as opposed to targeted, and in each case the resulting pattern is determined by molecular rules correlated with local pre-existing histone modification profiles. Since much of the abnormal methylation generated in vitro appears to be stably maintained, this modification may inhibit normal differentiation and could predispose to cancer if cells are used for replacement therapy. Excess CpG island methylation is also observed in normal placenta, suggesting that this process may be governed by an inherent program.     

DNA Methylation Markers in Lung Cancer

Lung cancer is the most common cancer and the leading cause of cancer-related morbidity and mortality worldwide. As early symptoms of lung cancer are minimal and non-specific, many patients are diagnosed at an advanced stage. Despite a concerted effort to diagnose lung cancer early, no biomarkers that can be used for lung cancer screening and prognosis prediction have been established so far. As global DNA demethylation and gene-specific promoter DNA methylation are present in lung cancer, DNA methylation biomarkers have become a major area of research as potential alternative diagnostic methods to detect lung cancer at an early stage. This review summarizes the emerging DNA methylation changes in lung cancer tumorigenesis, focusing on biomarkers for early detection and their potential clinical applications in lung cancer.     

Identification and Characterization of CXCR4-Positive Gastric Cancer Stem Cells

Diffuse-type solid tumors are often composed of a high proportion of rarely proliferating (i.e., dormant) cancer cells, strongly indicating the involvement of cancer stem cells (CSCs) Although diffuse-type gastric cancer (GC) patients have a poor prognosis due to high-frequent development of peritoneal dissemination (PD), it is limited knowledge that the PD-associated CSCs and efficacy of CSC-targeting therapy in diffuse-type GC. In this study, we established highly metastatic GC cell lines by in vivo selection designed for the enrichment of PD-associated GC cells. By microarray analysis, we found C-X-C chemokine receptor type 4 (CXCR4) can be a novel marker for highly metastatic CSCs, since CXCR4-positive cells can grow anchorage-independently, initiate tumors in mice, be resistant to cytotoxic drug, and produce differentiated daughter cells. In clinical samples, these CXCR4-positive cells were found from not only late metastasis stage (accumulated ascites) but also earlier stage (peritoneal washings). Moreover, treatment with transforming growth factor-β enhanced the anti-cancer effect of docetaxel via induction of cell differentiation/asymmetric cell division of the CXCR4-positive gastric CSCs even in a dormant state. Therefore, differentiation inducers hold promise for obtaining the maximum therapeutic outcome from currently available anti-cancer drugs through re-cycling of CSCs.     

Analysis of Age-Related Global DNA Methylation in Chicken

DNA methylation is an epigenetic modification that plays an important role in the normal development and function of organisms. The level of DNA methylation is species-, tissue-, and organelle-specific, and the methylation pattern is determined during embryogenesis. DNA methylation has also been correlated with age. The aim of this study was to determine the global DNA methylation levels and their correlation with age in the chicken, using a Polish autosexing chicken breed, Polbar. A quantitative technique based on an immunoenzymatic assay was used for global DNA methylation analysis. The results show increased global DNA methylation levels with older Polbar embryos. Global DNA methylation levels decrease with the age of hens in the postembryonic stage. This study expands the current knowledge of the Polbar epigenome and the general knowledge of the function of epigenetic mechanisms in birds.     

Global DNA Methylation of Ischemic Stroke Subtypes

Ischemic stroke (IS), a heterogeneous multifactorial disorder, is among the leading causes of mortality and long-term disability in the western world. Epidemiological data provides evidence for a genetic component to the disease, but its epigenetic involvement is still largely unknown. Epigenetic mechanisms, such as DNA methylation, change over time and may be associated with aging processes and with modulation of the risk of various pathologies, such as cardiovascular disease and stroke. We analyzed 2 independent cohorts of IS patients. Global DNA methylation was measured by luminometric methylation assay (LUMA) of DNA blood samples. Univariate and multivariate regression analyses were used to assess the methylation differences between the 3 most common IS subtypes, large-artery atherosclerosis (LAA), small-artery disease (SAD), and cardio-aortic embolism (CE). A total of 485 IS patients from 2 independent hospital cohorts (n = 281 and n = 204) were included, distributed across 3 IS subtypes: LAA (78/281, 59/204), SAD (97/281, 53/204), and CE (106/281, 89/204). In univariate analyses, no statistical differences in LUMA levels were observed between the 3 etiologies in either cohort. Multivariate analysis, adjusted by age, sex, hyperlipidemia, and smoking habit, confirmed the lack of differences in methylation levels between the analyzed IS subtypes in both cohorts. Despite differences in pathogenesis, our results showed no global methylation differences between LAA, SAD, and CE subtypes of IS. Further work is required to establish whether the epigenetic mechanism of methylation might play a role in this complex disease.