Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands.

Precise mapping of DNA methylation patterns in CpG islands has become essential for understanding diverse biological processes such as the regulation of imprinted genes, X chromosome inactivation, and tumor suppressor gene silencing in human cancer. We describe a new method, MSP (methylation-specific PCR), which can rapidly assess the methylation status of virtually any group of CpG sites within a CpG island, independent of the use of methylation-sensitive restriction enzymes. This assay entails initial modification of DNA by sodium bisulfite, converting all unmethylated, but not methylated, cytosines to uracil, and subsequent amplification with primers specific for methylated versus unmethylated DNA. MSP requires only small quantities of DNA, is sensitive to 0.1% methylated alleles of a given CpG island locus, and can be performed on DNA extracted from paraffin-embedded samples. MSP eliminates the false positive results inherent to previous PCR-based approaches which relied on differential restriction enzyme cleavage to distinguish methylated from unmethylated DNA. In this study, we demonstrate the use of MSP to identify promoter region hypermethylation changes associated with transcriptional inactivation in four important tumor suppressor genes (p16, p15, E-cadherin, and von Hippel-Lindau) in human cancer.

Preferential isolation of DNA fragments associated with CpG islands.

We describe a procedure for preferential isolation of DNA fragments with G+C-rich portions. Such fragments occur in known genes within or adjacent to CpG islands. Since about 56% of human genes are associated with CpG islands, isolation of these fragments permits detection and probing of many genes within much larger segments of DNA, such as cosmids or yeast artificial chromosomes, which have not been sequenced. Cloned DNA fragments digested with four restriction endonucleases were subjected to denaturing gradient gel electrophoresis. Long G+C-rich sections in fragments inhibit strand dissociation after the fragments reach retardation level in the gradient; such fragments are retained in the gel after most others disappear. Nucleotide sequences of the retained fragments show that about half of these fragments appear to be derived from CpG islands. Northern analysis indicated the presence of RNA complementary to most of the retained fragments. A heuristic approach to the relation between base sequence and the kinetics of strand dissociation of partly melted molecules appears to account for retention and nonretention. The expectation that CpG island fragments will be enriched among fragments retained in a denaturing gradient is supported by rate estimates based on melting theory applied to known sequences. This method, designated SPM for segregation of partly melted molecules, is expected to provide a means for convenient and efficient isolation of genes from unsequenced DNA.

The properties of CpG islands in the putative promoter regions of human immunoglobulin (Ig) genes

CpG island is a GC-rich motif occurred in gene promoter region, which can play important roles in gene silencing and imprinting. Here, we present a set of discriminant functions that can recognize the structural and compositional features of CpG islands in the putative promoter regions (PPRs) of human and mouse immunoglobulin (Ig) genes. We showed that the PPRs of both human and mouse Ig genes irrespective of gene chromosomal localization are apparently CpG island poor, with a low percentage of the CpG islands overlapped with the transcription start site (TSS). The human Ig genes that have CpG islands in the PPRs show a very narrow range of CpG densities. 47% of the Ig genes fall in the range of 3.5-4 CpGs/100 bp. In contrast, the non-Ig genes examined have a wide range of the density of CpG island, with 10.5% having the density of 8.1-15 CpGs/100 bp. Meantime, five patterns of the CpG distributions within the CpG islands have been classified: Pat A, B, C, D, and E. 21.6% and 10.8% of the Ig genes fall into the Pat B and Pat D groups, respectively, which were significantly higher than the non-Ig genes examined (8.2% and 3.8%). Moreover, the length of CpG islands is shorter in human Ig genes than in non-Ig genes but is much longer than in mouse orthologues. These findings provide a clear picture of non-neutral and nonrandom occurrence of the CpG islands in the PPRs of human and mouse Ig genes, which facilitate rational recommendations regarding their nomenclature. (C) 2005 Elsevier B.V. All rights reserved.

Transduction of CpG DNA-stimulated primary human B cells with bicistronic lentivectors.

Recently, using HIV-1-derived lentivectors, we obtained efficient transduction of primary human B lymphocytes cocultured with murine EL-4 B5 thymoma cells, but not of isolated B cells activated by CD40 ligation. Coculture with a cell line is problematic for gene therapy applications or study of gene functions. We have now found that transduction of B cells in a system using CpG DNA was comparable to that in the EL-4 B5 system. A monocistronic vector with a CMV promoter gave 32 +/- 4.7% green fluorescent protein (GFP)+ cells. A bicistronic vector, encoding IL-4 and GFP in the first and second cistrons, respectively, gave 14.2 +/- 2.1% GFP+ cells and IL-4 secretion of 1.3 +/- 0.2 ng/10(5) B cells/24 h. This was similar to results obtained in CD34+ cells using the elongation factor-1alpha promoter. Activated memory and naive B cells were transducible. After transduction with a bicistronic vector encoding a viral FLIP molecule, vFLIP was detectable by FACS or Western blot in GFP+, but not in GFP-, B cells, and 57% of sorted GFP+ B cells were protected against Fas ligand-induced cell death. This system should be useful for gene function research in primary B cells and development of gene therapies.

Methylation of CpG island promoters in uveal melanoma.

BACKGROUND: Inactivation of tumour-related genes by promoter hypermethylation is a common epigenetic event in the development of a variety of tumours. AIM: To investigate in primary uveal melanoma the status of promoter methylation of genes thought to be involved in tumour development: p16, TIMP3, RASSF1, RARB, FHIT, hTERT and APC. METHODS: Gene promoter methylation was studied by methylation-sensitive single-strand conformation analysis and dot-blot assay in a series of 23 primary uveal melanomas. All DNA samples were obtained from paraffin-embedded formalin-fixed tissue blocks. RESULTS: hTERT promoter methylation was found with a relatively high frequency (52%). Promoter methylation of p16, TIMP3, RASSF1, RARB, FHIT and APC was a rare event. For none of these genes did promoter methylation exceed 15% of tumour samples, and, for some genes (FHIT and APC), no methylation was found at all. Furthermore, promoter methylation was absent in 39% (9/23) of cases. In only 22% (5/23) of cases was hypermethylation of at least two promoters observed. CONCLUSIONS: Promoter methylation of hTERT is a regular event in uveal melanoma. Hypermethylation of the other genes studied does not seem to be an essential element in the development of this tumour. As promoter methylation of APC, RASSF1 and RARB is often observed in cutaneous melanoma, these results suggest that different epigenetic events occur in the development of cutaneous and uveal melanoma.

IDENTIFICATION OF FACTORS INVOLVED IN DNA METHYLATION OF CPG-ISLAND-PROMOTERS

Repression of many tumor suppressor genes (TSGs) in cancer is mediated by aberrantly increased DNA methylation levels at promoter CpG islands (CGI). About one-fourth of empirically defined human promoters are surrounded by or contain clustered repetitive elements. It was previously observed that a sharp transition of methylation occurs between highly methylated repetitive elements (SINE or LINE) and unmethylated CGI-promoters (e.g. P16, VHL, CDH and RIL) in normal tissues. The functions that lead to increased CGI methylation in cancer remain poorly understood. We propose that CGI-promoters contain cis-elements for triggering de novo DNA methylation. In the first part of our project, we established a site-specific integration system with enforced local transcriptional repression in colorectal cancer cells and monitored the occurrence of de novo DNA methylation in exogenous fragments containing a CGI-promoter and repetitive elements. Initial de novo methylation was seeded at specific CG sites in a repetitive element, and accelerated by persistent binding of a KRAB-containing transcriptional repressor. Furthermore, additional repetitive elements (LINE and SINE) located adjacent to the promoter could confer DNA methylation spreading into the CGI particularly in the setting of KRAB-factor binding. However, a repressive chromatin alone was not sufficient to initiate DNA methylation, which required specific DNA sequences and was integration-site (and/or cell-line) specific. In addition, all the methylation observed showed slow and gradual accumulation over several months of culture. Overall, these results demonstrate a requirement for specific DNA sequences to trigger de novo DNA methylation, and repetitive elements as cis-regulatory factors to cooperate with strengthened transcriptional repression in promoting methylation spreading. In the second part, we re-introduced disrupted DNMT3B or DNMT1 into HCT116 DKO cells and mapped the remethylation pattern through a profiling method (DREAM). Moderate remethylation occurred when DNMT3B was re-expressed with a preference toward non-CGI and non-promoter regions. Hence, there exists a set of genomic regions with priority to be targets for DNMT3B in somatic cells.

Selective association of the methyl-CpG binding protein MBD2 with the silent p14/p16 locus in human neoplasia

DNA methylation of tumor suppressor genes is a common feature of human cancer. The cyclin-dependent kinase inhibitor gene p16/Ink4A is hypermethylated in a wide range of malignant tissues and the p14/ARF gene located 20 kb upstream on chromosome 9p21 is also methylated in carcinomas. p14/ARF (ARF, alternative reading frame) does not inhibit the activities of cyclins or cyclin-dependent kinase complexes; however, the importance of the two gene products in the etiology of cancer resides in their involvement in two major cell cycle regulatory pathways: p53 and the retinoblastoma protein, Rb, respectively. Distinct first exons driven from separate promoters are spliced onto the common exons 2 and 3 and the resulting proteins are translated in different reading frames. Both genes are expressed in normal cells but can be alternatively or coordinately silenced when their CpG islands are hypermethylated. Herein, we examined the presence of methyl-CpG binding proteins associated with aberrantly methylated promoters, the distribution of acetylated histones H3 and H4 by chromatin immunoprecipitation assays, and the effect of chemical treatment with 5-aza-2′-deoxycytidine (5aza-dC) and trichostatin A on gene induction in colon cell lines by quantitative reverse transcriptase–PCR. We observed that the methyl-CpG binding protein MBD2 is targeted to methylated regulatory regions and excludes the acetylated histones H3 and H4, resulting in a localized inactive chromatin configuration. When methylated, the genes can be induced by 5aza-dC but the combined action of 5aza-dC and trichostatin A results in robust gene expression. Thus, methyl-CpG binding proteins and histone deacetylases appear to cooperate in vivo, with a dominant effect of DNA methylation toward histone acetylation, and repress expression of tumor suppressor genes hypermethylated in cancers.

Epigenetic Silencing of CRABP2 and MX1 in Head and Neck Tumors

Head and neck squamous cell carcinoma (HNSCC) is a heterogeneous disease affecting the epithelium of the oral cavity, pharynx and larynx. Conditions of most patients are diagnosed at late stages of the disease, and no sensitive and specific predictors of aggressive behavior have been identified yet. Therefore, early detection and prognostic biomarkers are highly desirable for a more rational management of the disease. Hypermethylation of CpG islands is one of the most important epigenetic mechanisms that leads to gene silencing in tumors and has been extensively used for the identification of biomarkers. In this study, we combined rapid subtractive hybridization and microarray analysis in a hierarchical manner to select genes that are putatively reactivated by the demethylating agent 5-aza-2'-deoxycytidine (5Aza-dC) in HNSCC cell lines (FaDu, UM-SCC-14A, UM-SCC-17A, UM-SCC-38A). This combined analysis identified 78 genes, 35 of which were reactivated in at least 2 cell lines and harbored a CpG island at their 5' region. Reactivation of 3 of these 35 genes (CRABP2, MX1, and SLC15A3) was confirmed by quantitative real-time polymerase chain reaction (PCR; fold change, >= 3). Bisulfite sequencing of their CpG islands revealed that they are indeed differentially methylated in the HNSCC cell lines. Using methylation-specific PCR, we detected a higher frequency of CRABP2 (58.1% for region 1) and MX1 (46.3%) hypermethylation in primary HNSCC when compared with lymphocytes from healthy individuals. Finally, absence of the CRABP2 protein was associated with decreased disease-free survival rates, supporting a potential use of CRABP2 expression as a prognostic biomarker for HNSCC patients.

ADAM23 Negatively Modulates alpha(v)beta(3) Integrin Activation during Metastasis

The ADAM23 gene is frequently silenced in different types of tumors, and, in breast tumors, silencing is correlated with tumor progression, suggesting that it might be associated with the acquisition of a metastatic phenotype. ADAM23 exerts its function mainly through the disintegrin domain, because its metalloprotease domain is inactive. Analysis of ADAM23 binding to integrins has revealed a specific interaction with alpha(v)beta(3) integrin mediated by the disintegrin domain. Altered expression of alpha(v)beta(3) integrin has been observed in different types of tumors, and expression of this integrin in the activated form has been shown to promote metastasis formation. Here, we investigated the possibility that interaction between ADAM23 and alpha(v)beta(3) integrin might negatively modulate alpha(v)beta(3) activation during metastatic progression. ADAM23 expression was knocked down using short hairpin RNA in the MDA-MB-435 cell line, which has been extensively used as a model for alpha(v)beta(3) integrin activation. Ablation of ADAM23 enhanced alpha(v)beta(3) integrin activation by at least 2- to 4-fold and ADAM23 knockdown cells showed enhanced migration and adhesion to classic alpha(v)beta(3) integrin ligands. Ablation of ADAM23 expression also enhanced pulmonary tumor cell arrest in immunodeficient mice. To complement our findings with clinical evidence, we showed that silencing of ADAM23 gene by DNA promoter hypermethylation in a collection of 94 primary breast tumors was significantly associated with lower distant metastases-free and disease-specific survivals and was an independent prognostic factor for poor disease outcome. Our results strongly support a functional role of ADAM23 during metastatic progression by negatively modulating alpha(v)beta(3) integrin activation. [Cancer Res 2009;69(13):5546-52]

Integration pattern of HIV-1 based lentiviral vector carrying recombinant coagulation factor VIII in Sk-Hep and 293T cells

293T and Sk-Hep-1 cells were transduced with a replication-defective self-inactivating HIV-1 derived vector carrying FVIII cDNA. The genomic DNA was sequenced to reveal LTR/human genome junctions and integration sites. One hundred and thirty-two sequences matched human sequences, with an identity of at least 98%. The integration sites in 293T-FVIIIDB and in Sk-Hep-FVIIIDB cells were preferentially located in gene regions. The integrations in both cell lines were distant from the CpG islands and from the transcription start sites. A comparison between the two cell lines showed that the lentiviral-transduced DNA had the same preferred regions in the two different cell lines.

HPP1: A transmembrane protein-encoding gene commonly methylated in colorectal polyps and cancers

Adenomas are the precursors of most colorectal cancers. Hyperplastic polyps have been linked to the subset of colorectal cancers showing DNA microsatellite instability, but little is known of their underlying genetic etiology. Using a strategy that isolates differentially methylated sequences from hyperplastic polyps and normal mucosa, we identified a 370-bp sequence containing the 5' untranslated region and the first exon of a gene that we have called HPP1. Rapid amplification of cDNA ends was used to isolate HPP1 from normal mucose. Using reverse transcription-PCR, HPP1 was expressed in 28 of 30 (93%) normal colonic samples but in only seven of 30 (23%) colorectal cancers (P < 0.001). The 5' region of HPP1 included a CpG island containing 49 CpG sites, of which 96% were found to be methylated by bisulfite sequencing of DNA from colonic tumor samples. By COBRA analysis, methylation was detected in six of nine (66%) adenomas, 17 of 27 (63%) hyperplastic polyps, and 46 of 55 (84%) colorectal cancers. There was an inverse relationship between methylation level and mRNA expression in cancers (r = -0.67; P < 0.001), and 5-aza-2-deoxycytidine treatment restored HPP1 expression in two colorectal cancer cell lines. In situ hybridization of HPP1 indicated that expression occurs in epithelial and stromal elements in normal mucosa but is silenced in both cell types in early colonic neoplasia. HPP1 is predicted to encode a transmembrane protein containing follistatin and epidermal growth factor-like domains. Silencing of HPP1 by methylation may increase the probability of neoplastic transformation.

Reduced expression of intercellular adhesion molecule-1 in ovarian adenocarcinomas

Ovarian adenocarcinomas develop as the result of multiple genetic, and epigenetic changes in the precursor ovarian surface epithelial (OSE) cells which result in a malignant phenotype. We investigated changes in gene expression in ovarian adenocarcinoma using a cDNA array containing 588 known human genes. We found that intercellular adhesion molecule-1 (ICAM-1) was expressed at lower levels in the ovarian tumour cell lines OAW42, PEO1 and JAM than in the immortalised human ovarian surface epithelial cell line HOSE 17.1. Further investigation revealed ICAM-1 was expressed in the surface epithelium of normal ovaries and both mRNA and protein expression levels were reduced in the majority of ovarian adenocarcinoma cell lines and primary tumours. ICAM-1 expression was increased in 8/8 cell lines treated with the de novo methyltransferase inhibitor 5-aza-2'-deoxycytidiine, indicating that methylation of CpG islands may play a role in the down-regulation of its expression in primary tumours. 'There was a significant association between patients whose tumours expressed ICAM-1 and survival (P = 0.03), suggesting that expression levels of ICAM-1 may have clinical relevance. (C) 2001 Cancer Research Campaign.

Reciprocal relationship between methylation status and loss of heterozygosity at the p14 (ARF) locus in Australian and South African hepatocellular carcinomas

Chromosome 9p21, a locus comprising the tumor suppressor genes (TSG) p16(INK4) (a) and p14(ARF) , is a common region of loss of heterozygosity (LOH) in hepatocellular carcinoma (HCC). p14(ARF) shares exon 2 with p16 in a different reading frame. p14 binds to MDM2 resulting in a stabilization of functional p53 . This study examined the roles of p14, p16 and p53 in hepatocarcinogenesis, in 37 Australian and 24 South African patients. LOH at 9p21 and 17p13.1, p14 and p16 mutation analysis, p14 and p16 promoter methylation and p14, p16 and p53 protein expression was examined. LOH at 9p21 was detected more frequently in South African HCC (P = 0.04). Comparable rates of p53 LOH were observed in Australian and South African HCC (10/22, 45%vs 13/22, 59%, respectively). Hypermethylation of the p14 promoter was more prevalent in Australian HCC than in South African HCC (17/37, 46%vs 7/24, 29%, respectively). In Australian HCC the prevalence of p14 methylation increased with age (P = 0.03). p16 promoter methylation was observed in 12/37 (32%) and 6/24 (25%) in Australian and South African HCC, respectively. Loss of p16 protein expression was detected in 14/36 Australian HCC whereas p53 protein expression was detected in 9/36. Significantly, a reciprocal relationship between 9p21 LOH and p14 promoter hypermethylation was observed (P less than or equal to0.05 ). No significant association between p14 and p53 was seen in this study. The reciprocal relationship identified indicates different pathways of tumorigenesis and likely reflects different etiologies of HCC in the two countries. (C) 2002 Blackwell Science Asia Pty Ltd.

Methylation profile of TP53 regulatory pathway and mtDNA alterations in breast cancer patients lacking TP53 mutations.

The present study investigated promoter hypermethylation of TP53 regulatory pathways providing a potential link between epigenetic changes and mitochondrial DNA (mtDNA) alterations in breast cancer patients lacking a TP53 mutation. The possibility of using the cancer-specific alterations in serum samples as a blood-based test was also explored. Triple-matched samples (cancerous tissues, matched adjacent normal tissues and serum samples) from breast cancer patients were screened for TP53 mutations, and the promoter methylation profile of P14(ARF), MDM2, TP53 and PTEN genes was analyzed as well as mtDNA alterations, including D-loop mutations and mtDNA content. In the studied cohort, no mutation was found in TP53 (DNA-binding domain). Comparison of P14(ARF) and PTEN methylation patterns showed significant hypermethylation levels in tumor tissues (P < 0.05 and <0.01, respectively) whereas the TP53 tumor suppressor gene was not hypermethylated (P < 0.511). The proportion of PTEN methylation was significantly higher in serum than in the normal tissues and it has a significant correlation to tumor tissues (P < 0.05). mtDNA analysis revealed 36.36% somatic and 90.91% germline mutations in the D-loop region and also significant mtDNA depletion in tumor tissues (P < 0.01). In addition, the mtDNA content in matched serum was significantly lower than in the normal tissues (P < 0.05). These data can provide an insight into the management of a therapeutic approach based on the reversal of epigenetic silencing of the crucial genes involved in regulatory pathways of the tumor suppressor TP53. Additionally, release of significant aberrant methylated PTEN in matched serum samples might represent a promising biomarker for breast cancer.

Nineteen additional unpredicted transcripts from human chromosome 21.

The identification of all human chromosome 21 (HC21) genes is a necessary step in understanding the molecular pathogenesis of trisomy 21 (Down syndrome). The first analysis of the sequence of 21q included 127 previously characterized genes and predicted an additional 98 novel anonymous genes. Recently we evaluated the quality of this annotation by characterizing a set of HC21 open reading frames (C21orfs) identified by mapping spliced expressed sequence tags (ESTs) and predicted genes (PREDs), identified only in silico. This study underscored the limitations of in silico-only gene prediction, as many PREDs were incorrectly predicted. To refine the HC21 annotation, we have developed a reliable algorithm to extract and stringently map sequences that contain bona fide 3' transcript ends to the genome. We then created a specific 21q graphical display allowing an integrated view of the data that incorporates new ESTs as well as features such as CpG islands, repeats, and gene predictions. Using these tools we identified 27 new putative genes. To validate these, we sequenced previously cloned cDNAs and carried out RT-PCR, 5'- and 3'-RACE procedures, and comparative mapping. These approaches substantiated 19 new transcripts, thus increasing the HC21 gene count by 9.5%. These transcripts were likely not previously identified because they are small and encode small proteins. We also identified four transcriptional units that are spliced but contain no obvious open reading frame. The HC21 data presented here further emphasize that current gene prediction algorithms miss a substantial number of transcripts that nevertheless can be identified using a combination of experimental approaches and multiple refined algorithms.