Quantitative assessment of promoter hypermethylation during breast cancer development

The aberrant methylation of cytosine residues in the promoter region of growth regulatory genes is now widely recognized as an additional mechanism for gene inactivation in cancer cells. In this study we analyzed the methylation status of four growth regulatory genes (p16, RASSF1A, cyclinD2, 14-3-3zeta) during breast cancer progression. For this purpose invasive and noninvasive tumor cell populations as well as hyperplastic cell proliferations were isolated from a series of archival breast tissue specimens (n = 57) using laser-assisted microdissection. A new real-time polymerase chain reaction-based assay was used for the sensitive and quantitative determination of the cell-specific methylation status. We found that aberrant promoter methylation was already prevalent in pure intraductal carcinoma with different frequencies and different methylation levels for the four genes analyzed. For RASSF1A and 14-3-3zeta promoter methylation was also demonstrated in epithelial hyperplasia and intraductal papillomas. By contrast, aberrant methylation of cyclinD2 and p16 was restricted to cancerous epithelium. Increased methylation of the cyclinD2 gene was significantly associated with a higher van Nuys grade. Furthermore, when intraductal and invasive tumor cells were compared, significant quantitative changes in the methylation level were detected primarily within the cyclinD2 gene. These results demonstrate that promoter methylation is an early and frequent event in breast cancer development, but displays great quantitative and gene-specific differences, and changes in a gene-specific manner during tumor progression.     

Loss of E-cadherin expression in gastric intestinal metaplasia and later stage p53 altered expression in gastric carcinogenesis

Gastric cancers are commonly subdivided into intestinal and diffuse subtypes on a morphologic basis, supported by corollary evidence of differences at the pathogenetic and molecular levels. Chronic atrophic gastritis with intestinal metaplasia is a common precursor lesion for the intestinal type of carcinoma. To identify early molecular changes, in this study we have examined 13 surgical specimens both for the expression of E-cadherin, p53 and beta-catenin by immunohistochemistry and for methylation of the CDH1 promoter (E-cadherin) by bisulfite genomic sequencing of laser capture microdissected samples. Each specimen examined contained areas of normal (nonmetaplastic) gastric mucosa, as well as areas of intestinal metaplasia and/or carcinoma. Reduced or absent E-cadherin and partial to complete methylation of one to multiple CpG sites examined in the CDH1 promoter were observed in all of the metaplasia samples. Thus, the methylation status of the CDH1 promoter and expression of E-cadherin together provide strong evidence that loss of E-cadherin is an early event in intestinal type gastric carcinogenesis. In contrast, expression of p53, assumed to be mutant p53, was generally not detected (except for isolated cells) until the carcinoma stage in tissues from these patients. These results suggest that mutation of p53 is a late event in intestinal type gastric cancer. The level of beta-catenin expression did not appear to change between normal, metaplastic and carcinoma cells of intestinal type, and no nuclear staining was visible in any of the tissues. These results suggest that the Wnt signaling pathway is not upregulated in this type of cancer.     

NDRG-1基因甲基化与乳腺癌发生的关系

目的 对NDRG-1基因启动子区甲基化状态进行研究,探讨NDRG-1基因甲基化在乳腺癌发生中的作用.方法 采用双色荧光杂交微阵列基因芯片技术对33例乳腺癌样本和癌旁组织样本进行NDRG-1基因启动子区甲基化状态研究.结果 33例肿瘤样本和癌旁组织样本中NDRG-1基因启动子区CpG位点均有不同程度的甲基化,肿瘤组织中NDRG-1基因启动子区CpG位点甲基化率显著高于相应的癌旁样本组织(t=14.12,P＜0.05).结论 DNA甲基化作为NDRG-1基因的重要调控机制,NDRG-1基因启动子区过甲基化可能在乳腺癌的发生过程中起重要作用.     

乳腺癌发生过程中NOEY2基因启动子区甲基化及mRNA表达

目的 探讨乳腺癌发生过程中抑癌基因NOEY2启动子区甲基化状态及其对mRNA表达的影响。方法 应用甲基化特异性PCR及双亚硫酸钠基因测序技术检测MCF10模型中乳腺增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCFIODCIS．com、浸润癌细胞系MCF10CA1a、MCF10CA1d、MCF10CA1h及正常乳腺组织中NOEY2基因启动子区CpG岛Ⅰ甲基化状态,然后用RT-PCR和实时PCR技术检测上述样品的mRNA表达水平。结果 MCF10模型的增生细胞系、癌前细胞系、导管内癌细胞系、浸润癌细胞系均发生该基因启动子区CpG岛Ⅰ高度甲基化;与正常乳腺组织相比,上述细胞系mRNA表达显著减少。结论 NOEY2基因启动子区高度甲基化及相应的mRNA表达减少是乳腺癌发生过程中的早期事件,与乳腺癌发生有关,可能成为早期诊断乳腺癌的潜在分子生物学标记。     

p53 mutations and expression in breast carcinoma in situ

The p53 tumor suppressor gene is altered in approximately half of human cancers. Although p53 mutations are common in invasive breast carcinoma, few have been identified in breast carcinoma in situ (intraductal breast carcinomas). Most studies of p53 in breast carcinoma in situ are immunohistochemical studies of p53 staining in paraffin-embedded tissue sections. Few studies have isolated the tumor cells and subjected them to DNA sequence analysis. The current study was undertaken to characterize p53 in a cohort of breast carcinoma in situ cases, both with and without invasive disease. Fifty-eight frozen breast biopsy samples were used for these investigations. Twenty-seven cases had only ductal carcinoma in situ (CIS) and 31 cases had evidence of both invasive and in situ carcinoma. DNA sequence alterations in exons 2 through 11 of p53 were screened by the single-strand conformational polymorphism technique. Exons with altered mobility were sequenced. Among breast CIS cases without invasive disease, 22% had p53 mutations and 7% had DNA sequence alterations of unknown significance. Analysis of breast CIS with concurrent invasive disease demonstrated p53 mutations in 19% of cases and one (3%) DNA alteration of unknown significance. Each carcinoma having a p53 mutation in the breast CIS component had the identical mutation in the invasive component of the same tumor indicating a clonal relationship between the two tumor components. p53 protein overexpression was identified in 22% of pure intraductal breast carcinomas and in 35% of breast CIS with invasive disease. Comparison of immunostaining and DNA sequence alterations showed a significant association between overexpression and mutations (P = 0. 0037) in cases of CIS without invasion, and similarly between overexpression and mutations in cases of CIS with invasion (P = 0. 007). p53 mutations and p53 overexpression were relatively common in intraductal breast carcinomas but were not observed in adjacent normal breast lobules or ducts in 9 cases available for DNA analysis. The frequency of p53 alterations when comparing breast CIS with and without an invasive component indicated that p53 mutations usually occur before invasion during the progression of breast cancer, as is observed for a number of other adult solid tumors.     

Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancers

It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high-frequency MSI (MSI-H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI-H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI-H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI-H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation.     

Relationship between p53 pathway and estrogen receptor status in endometrioid-type endometrial cancer

We analyzed the mechanism of estrogen receptor (ER) loss and status of the p53 pathway in 64 cases of endometrial cancer. 26.6% (17 of 64) of endometrial cancers lost ER. Methylation of the ER CpG island was significantly related to ER status (P = 0.0074). However, the methylation site of the ER CpG island differed between breast and endometrial cancers. The abnormal expression rate of p14ARF, MDM2, p53, and the p53 pathway were 7.8% (5 of 64), 32.8% (21 of 64), 25.0% (16 of 64) and 53.1% (34 of 64), respectively. There was no significant difference in the overexpression of MDM2 between p53-positive cases (43.8%: 7 of 16) and p53-negative cases (29.2%; 14 of 48) (P = 0.3595). Abnormal p53 was higher in grade 3 tumors (55.6%; 5 of 9) than in grade 1 and 2 tumors (20.0%; 11 of 55) (P = 0.0364). The abnormality of the p53 pathway was higher in grade 3 tumors (88.9%; 8 of 9) than in grade 1 and 2 tumors (47.3%; 26 of 55) (P = 0.0294). However, there was no significant difference in abnormal p53 pathway between ER-negative and ER-positive cases. In endometrial cancer, ER CpG island methylation was the important mechanism of ER loss. However, there was no significant relationship between the p53 pathway and ER status.     

Patterns of cellular and HPV 16 methylation as biomarkers for cervical neoplasia

Aberrant promoter methylation of biologically relevant genes in cervical cancer and uneven CpG distribution within the human papillomavirus 16 (HPV 16) enhancer region have been reported. Cervical samples and questionnaires from 151 women screened for cervical cancer in Appalachian Ohio were analyzed. Methylation was measured by bisulfite sequencing in candidate gene sites in ESR1, DCC, p16, and LINE1 elements. Among 89 HPV 16-positive women, CpG sites in the E6 promoter and enhancer regions and the L1 region of the HPV 16 genome were measured. Methylation levels were compared by cervical cytology and HPV 16 status. HPV methylation was low regardless of cytology status, however E6 methylation was significantly higher in women with normal cytology. ESR1 and DCC methylation were significantly higher in HPV 16-positive women. Increased methylation at sites in the E6 promoter region was associated with lower odds of abnormal cytology. Increased methylation in candidate genes was associated with higher odds of abnormal cytology, particularly DCC region 2.4, DCC region 2.6, ESR1 region 3.2, and LINE1 site 1.2. HPV 16 genome CpG methylation was low except for the L1 region. In general, lower HPV 16 methylation and higher candidate gene methylation levels were associated with higher odds of abnormal cytology.     

Tumor suppressor gene p16 (CDKN2A) mutation status and promoter inactivation in head and neck cancer.

The p16INK4A (CDKN2A/MTS1) putative tumor suppressor gene encodes a cyclin dependent kinase inhibitor which plays an important role in the regulation of the G1/S phase cell cycle checkpoint. A high frequency of various p16 gene alterations were consequently observed in many primary tumors. P16 can be inactivated by different mechanisms: i) homozygous deletion, ii) methylation of the promoter region or iii) point mutation. In order to investigate p16 alterations in head and neck cancer (HNC) we analyzed 70 primary tumors of the larynx, pharynx and oral cavity including their corresponding normal mucosa for mutation inactivation by direct sequencing exon 2. We detected only one so far undescribed transversion G to T at position 322 (Asp108Tyr) and a known polymorphism (Ala148Thr) in five cases. The methylation status of the p16 promoter region was analyzed by an improved highly sensitive methylation-specific PCR protocol. P16 methylation inactivation was found in 16 of 55 cases (29%). Our data indicate that p16 point mutations in HNC are less frequent, but inactivation by methylation of the promoter region could be involved in genesis and progression of HNC.     

大肠癌组织p14ARF与p53基因变异研究

目的：观察大肠癌组织p14ARF基因遗 传和表观遗传变化及p53基因突变状况，探讨两者改变的关系及p14ARF-p53通路功能 破坏在大肠癌发生中的作用。方法：应用PCR、直接测序、甲基化特异PC R和RT-PCR分别检测56例原发性大肠癌及相应癌旁正常组织p14ARF基因纯合性缺失、 突变、5′CpG岛甲基化、mRNA表达及p53基因突变状况。结果：①大肠 癌组织p14ARF总变异率为27% (15/56)，其中1例纯合性缺失，14例5′CpG岛甲基化,未见突变发生。②15例p14ARF基因变异的大肠癌组织其相应mRNA表达阴性（13例）或 低水平表达（2例），41例未发生基因变异的大肠癌组织和所有癌旁正常组织p14ARF mRNA均显示明显表达。③大肠癌组织p53突变率为48% (27/56)。④56例大肠癌组织中，12例 仅发生p14ARF变异，24例仅显示p53突变，3例同时有p14ARF 5′CpG岛甲基化 和p53突变，17例p14ARF和p53均无变异，p14ARF-p53通路总变异率为70%（39/ 56），p14ARF 5′CpG岛高甲基化与野生型p53状态有关（P＜0.05）。⑤低分化 腺癌组p14ARF变异率（44%，7/16）明显高于高中分化腺癌组（20%，8/40）（P＜ 0.05），但两组间p53突变率无显著差异（P＞0.05）。结论:①大肠癌p14ARF基因5′ CpG岛高甲基化是其表达失活的主要机制；②大肠癌p14 ARF基因高甲基化与p53基因突变可能是相互独立的事件，两者的失活可能各自出现于不同 的大肠癌亚组中；③p14ARF高甲基化或p53突变引起的p14ARF-p53通路功能 破坏在大肠癌的发生中具有重要作用。     

Differences in methylation patterns in the methylation boundary region of IDS gene in Hunter syndrome patients: implications for CpG hot spot mutations

Hunter syndrome, an X-linked disorder, results from deficiency of iduronate-2-sulfatase (IDS). Around 40% of independent point mutations at IDS were found at CpG sites as transitional events. The 15 CpG sites in the coding sequences of exons 1 and 2, which are normally hypomethylated, account for very few of transitional mutations. By contrast, the CpG sites in the coding sequences of exon 3, though also normally hypomethylated, account for much higher fraction of transitional mutations. To better understand relationship between methylation status and CpG transitional mutations in this region, the methylation patterns of 11 Hunter patients with transitional mutations at CpG sites were investigated using bisulfite genomic sequencing. The patient cohort mutation spectrum is composed of one mutation in exon 1 (one patient) and three different mutations in exon 3 (10 patients). We confirmed that in normal males, cytosines at the CpG sites from the promoter region to a portion of intron 3 were hypomethylated. However, specific CpG sites in this area were more highly methylated in patients. The patients with p.R8X (exon 1), p.P86L (exon 3), and p.R88H (exon 3) mutations had a hypermethylated condition in exon 2 to intron 3 but retained hypomethylation in exon 1. The same trend was found in four patients with p.A85T (exon 3), although the degree of hypermethylation was less. These findings suggest methylation patterns in the beginning of IDS genomic region are polymorphic in humans and that hypermethylation in this region in some individuals predisposes them to CpG mutations resulting in Hunter syndrome.     

银屑病患者皮损p16INK4a基因CpG甲基化位点和频度

目的研究皮损表皮p16INK4a甲基化阳性的银屑病患者基因启动子区CpG甲基化的位点和频度。方法采集50例银屑病患者皮损处皮肤,分离表皮,提取DNA。采用甲基化特异性PCR和测序法检测p16INK4a基因启动子区CpG甲基化的位点和频度。结果p16INK4a基因启动子甲基化阳性的患者,约50%CpG发生甲基化,均出现于特定位点。结论p16INK4a甲基化阳性银屑病患者p16INK4a基因启动子并非完全发生甲基化。     

乳腺癌发生模型MCF10各阶段细胞系中TIMP3基因启动子区甲基化分析

目的探讨抑癌基因TIMP3失活与乳腺癌发生和进展的关系。方法用甲基化特异性PCR技术和亚硫酸盐测序技术检测乳腺癌发生模型MCF10的增生细胞系MCF10A、癌前细胞系MCF10AT、导管内癌细胞系MCF10DCIS.com、浸润癌细胞系MCF10CA1a和转移癌细胞系MCF10CA1d、MCF10CA1h中TIMP3启动子区甲基化状态。结果甲基化特异性PCR分析显示,在上述各细胞系中,TIMP3启动子区均呈高度甲基化状态。亚硫酸盐测序显示,在上述各细胞系中,测序区内的68个CG位点几乎全部发生了甲基化,且甲基化累及了绝大部分等位基因。结论TIMP3基因启动子区甲基化在乳腺癌的发生和进展中起重要作用,可能成为早期诊断乳腺癌和判断乳腺癌预后的分子生物学标记。     

Methylation profile of the MLH1 promoter region and their relationship to colorectal carcinogenesis

Methylation of the MLH1 promoter region has been suggested to be a principal mechanism of gene inactivation in sporadic microsatellite instability (MSI)-positive colorectal carcinoma. Recently, we have shown a novel methylation profile of the MLH1 promoter region (i.e., full, partial, and no methylation), among which full methylation was strongly associated with MSI. In this study, to confirm whether methylation requires the involvement of both alleles, we studied the MLH1 promoter region concerning the methylation profile and allelic loss. Furthermore, we studied correlations of methylation profiles with genetic alternations such as loss of heterozygosity (LOH) of the TP53 locus and KRAS mutation. Eighty-eight tumors were classified as full (n = 14), partial (n = 26), and no methylation (n = 48). Full methylation was observed in 78% (14/18) of high-frequency MSI, in which all CpG sites in the promoter region were methylated. Full methylation differed significantly from partial methylation regarding absence of TP53 LOH (0/12) and KRAS mutation (0/14). In cases with full methylation, we could show biallelic methylation by use of a single-base nucleotide polymorphism in the promoter. However, this did not accompany LOH of the MLH1 locus. In contrast, there were no significant differences in molecular features between partial and no methylation, except for low frequencies of LOH of the MLH1 locus (P = 0.02). In conclusion, biallelic extensive methylation of the MLH1 promoter region plays a significant role in gene inactivation and is independent of KRAS mutation and TP53 LOH.     

BRCA1基因复杂甲基化模式

应用体外甲基化酶M-SssI处理和甲基化敏感单链构象分析法(Methylation-sensitive single-strand conformation     

GSTP1 promoter hypermethylation is an early event in breast carcinogenesis

Promoter hypermethylation in precursor lesions of the breast cancer may be biomarkers of cancer risk and targets for cancer chemoprevention. Pi-class glutathione-S-transferases (GSTP1) is inactivated by promoter hypermethylation in invasive breast cancers. However, little is known about epigenetic silencing of GSTP1 gene by promoter hypermethylation in precursor lesions. To determine the significance of GSTP1 promoter hypermethylation in breast carcinogenesis, methylation status of GSTP1 gene was studied by nested methylation-specific polymerase chain reaction, and GSTP1 expression was studied by immunohistochemistry in invasive ductal carcinoma (IDC), ductal carcinoma in situ (DCIS), usual ductal hyperplasia (UDH), and normal breast tissue. GSTP1 promoter hypermethylation was detected in 4/24 (16.7%) of UDH, 18/49 (36.7%) of DCIS, and 14/36 (38.9%) of IDC. No hypermethylation was detected in normal breast tissues. GSTP1 promoter hypermethylation was found to be progressively elevated during breast carcinogenesis (p < 0.01). GSTP1 promoter hypermethylation was associated with loss of GSTP1 expression (p < 0.01 for UDH, p < 0.001 for DCIS and IDC). Our results suggest that GSTP1 promoter hypermethylation is an early event in breast carcinogenesis and appears to functionally silence GSTP1 expression. GSTP1 promoter hypermethylation in the precursor lesions of breast cancer may be used as a target for cancer chemoprevention.