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Biomedical subjects

Peter A Jones

Publications and source records attributed to Peter A Jones.

At least 19 recordsLinked to original sources
PubMed2003

Establishment of conditional vectors for hairpin siRNA knockdowns.

Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown.

Base Sequence↗
PubMed2003

Inhibition of DNA methylation and reactivation of silenced genes by zebularine.

BACKGROUND: Gene silencing by abnormal methylation of promoter regions of regulatory genes is commonly associated with cancer. Silenced tumor suppressor genes are obvious targets for reactivation by methylation inhibitors such as 5-azacytidine (5-Aza-CR) and 5-aza-2'-deoxycytidine (5-Aza-CdR). However, both compounds are chemically unstable and toxic and neither can be given orally. We characterized a new demethylating agent, zebularine [1-(beta-D-ribofuranosyl)-1,2-dihydropyrimidin-2-one], which is a chemically stable cytidine analog. METHODS: We tested the ability of zebularine to reactivate a silenced Neurospora crassa gene using a hygromycin gene reactivation assay. We then analyzed the ability of zebularine to inhibit DNA methylation in C3H 10T1/2 Cl8 (10T1/2) mouse embryo cells as assayed by induction of a myogenic phenotype and in T24 human bladder carcinoma cells, using the methylation-sensitive single nucleotide primer extension (Ms-SNuPE) assay. We also evaluated the effects of zebularine (administered orally or intraperitoneally) on growth of EJ6 human bladder carcinoma cells grown in BALB/c nu/nu mice (five mice per group) and the in vivo reactivation of a methylated p16 gene in these cells. All statistical tests were two-sided. RESULTS: In N. crassa, zebularine inhibited DNA methylation and reactivated a gene previously silenced by methylation. Zebularine induced the myogenic phenotype in 10T1/2 cells, which is a phenomenon unique to DNA methylation inhibitors. Zebularine reactivated a silenced p16 gene and demethylated its promoter region in T24 bladder carcinoma cells in vitro and in tumors grown in mice. Zebularine was only slightly cytotoxic to T24 cells in vitro (1 mM zebularine for 48 hours decreased plating efficiency by 17% [95% confidence interval (CI) = 12.8% to 21.2%]) and to tumor-bearing mice (average maximal weight change in mice treated with 1000 mg/kg zebularine = 11% [95% CI = 4% to 19%]). Compared with those in control mice, tumor volumes were statistically significantly reduced in mice treated with high-dose zebularine administered by intraperitoneal injection (P<.001) or by oral gavage (P<.001). CONCLUSIONS: Zebularine is a stable DNA demethylating agent and the first drug in its class able to reactivate an epigenetically silenced gene by oral administration.

Administration, Oral↗
PubMed2003

Epigenetics in carcinogenesis and cancer prevention.

Carcinogenesis is a stepwise process of accumulation of genetic and epigenetic abnormalities that can lead to cellular dysfunction. It has become clear that epigenetic changes are equally important for this multistep process to produce its results. This article describes the different roles that epigenetic modulation may play during carcinogenesis and how an early detection and chemopreventive intervention strategy that takes both sides of the equation into account would be advantageous.

Cell Transformation, Neoplastic↗
PubMed2003

The CpG island searcher: a new WWW resource.

Clusters of CpG dinucleotides in GC rich regions of the genome called "CpG islands" frequently occur in the 5' ends of genes. Methylation of CpG islands plays a role in transcriptional silencing in higher organisms in certain situations. We have established a CpG-island-extraction algorithm, which we previously developed [Takai and Jones, 2002], on a web site which has a simple user interface to identify CpG islands from submitted sequences of up to 50kb. The web site determines the locations of CpG islands using parameters (lower limit of %GC, ObsCpG/ExpCpG, length) set by the user, to display the value of parameters on each CpG island, and provides a graphical map of CpG dinucleotide distribution and borders of CpG islands. A command-line version of the CpG islands searcher has also been developed for larger sequences. The CpG Island Searcher was applied to the latest sequence and mapping information of human chromosomes 20, 21 and 22, and a total of 2345 CpG islands were extracted and 534 (23%) of them contained first coding exons and 650 (28%) contained other exons. The CpG Island Searcher is available on the World Wide Web at http://www.cpgislands.com or http://www.uscnorris.com/cpgislands/cpg.cgi.

Algorithms↗
PubMed2002

Histone H3-lysine 9 methylation is associated with aberrant gene silencing in cancer cells and is rapidly reversed by 5-aza-2'-deoxycytidine.

Epigenetic modifications of cytosine residues in DNA and the amino termini of histone proteins have emerged as key mechanisms in chromatin remodeling, impacting both the transcriptional regulation and the establishment of chromosomal domains. Using the chromatin immunoprecipitation (ChIP) assay, we demonstrate that aberrantly silenced genes in cancer cells exhibit a heterochromatic structure that is characterized by histone H3 lysine 9 (H3-K9) hypermethylation and histone H3 lysine 4 (H3-K4) hypomethylation. This aberrant heterochromatin is incompatible with transcriptional initiation but does not inhibit elongation by RNA polymerase II. H3-K9 methylation may, therefore, play a role in the silencing of tumor-suppressor genes in cancer. Treatment with 5-aza-2'-deoxycytidine (5-Aza-CdR), previously known for its ability to inhibit cytosine methylation, induced a rapid and substantial remodeling of the heterochromatic domains of the p14ARF/p16INK4a locus in T24 bladder cancer cells, reducing levels of dimethylated H3-K9 and increasing levels of dimethylated H3-K4 at this locus. In addition, 5-Aza-CdR increased acetylation and H3-K4 methylation at the unmethylated p14 promoter, suggesting it can induce chromatin remodeling independently of its effects on cytosine methylation.

Acetylation↗
PubMed2002

Demethylation of a hypermethylated P15/INK4B gene in patients with myelodysplastic syndrome by 5-Aza-2'-deoxycytidine (decitabine) treatment.

p16 and p15, 2 inhibitors of cyclin-dependent kinases, are frequently hypermethylated in hematologic neoplasias. Decitabine, or 5-Aza-2'-deoxycytidine, reverts hypermethylation of these genes in vitro, and low-dose decitabine treatment improves cytopenias and blast excess in ~50% of patients with high-risk myelodysplastic syndrome (MDS). We examined p15 and p16 methylation status in bone marrow mononuclear cells from patients with high-risk MDS during treatment with decitabine, using a methylation-sensitive primer extension assay (Ms-SNuPE) to quantitate methylation, and denaturing gradient gel electrophoresis (DGGE) and bisulfite-DNA sequencing to distinguish individually methylated alleles. p15 expression was serially examined in bone marrow biopsies by immunohistochemistry. Hypermethylation in the 5' p15 gene region was detected in 15 of 23 patients (65%), whereas the 5' p16 region was unmethylated in all patients. Among 12 patients with hypermethylation sequentially analyzed after at least one course of decitabine treatment, a decrease in p15 methylation occurred in 9 and was associated with clinical response. DGGE and sequence analyses were indicative of hypomethylation induction at individual alleles. Immunohistochemical staining for p15 protein in bone marrow biopsies from 8 patients with p15 hypermethylation revealed low or absent expression in 4 patients, which was induced to normal levels during decitabine treatment. In conclusion, frequent, selective p15 hypermethylation was reversed in responding MDS patients following treatment with a methylation inhibitor. The emergence of partially demethylated epigenotypes and re-establishment of normal p15 protein expression following the initial decitabine courses implicate pharmacologic demethylation as a possible mechanism resulting in hematologic response in MDS.

Adult↗
PubMed2002

Identification and characterization of alternatively spliced variants of DNA methyltransferase 3a in mammalian cells.

CpG methylation is mediated by the functions of at least three active DNA methyltransferases (DNMTs). While DNMT1 is thought to perform maintenance methylation, the more recently discovered DNMT3a and DNMT3b enzymes are thought to facilitate de novo methylation. Murine Dnmt3a and 3b are developmentally regulated and a new Dnmt3a isoform, Dnmt3a2, has been recently shown to be expressed preferentially in mouse embryonic stem (ES) cells. Here we have characterized four alternatively spliced variants of human and mouse DNMT3a. These transcripts included a novel exon 1 (1beta) that was spliced into the same exon 2 acceptor splice site used by the original exon 1 (1alpha). Cloning and sequencing of the 5' region of the human DNMT3a gene revealed that exon 1beta was situated upstream of exon 1alpha and that the entire region was contained within a CpG island. We also identified other alternatively spliced species containing intron 4 inclusions that were associated with either exon 1alpha or 1beta. These were expressed at low levels in mouse and human cells. All transcripts were highly conserved between human and mouse. The levels of Dnmt3a mRNA containing exon 1beta were 3-25-fold greater in mouse ES cells than in various somatic cells as determined by semiquantitative reverse transcription-polymerase chain reaction analysis, while the levels of exon 1alpha-containing transcripts were slightly higher in human and mouse somatic cells. The preferential expression of the beta transcript in ES cells suggests that this transcript, in addition to Dnmt3a2, may also be important for de novo methylation during development.

3T3 Cells↗
PubMed2002

DNA methylation and cancer.

There is tremendous ferment in the field of epigenetics as the relationships between chromatin structure and DNA methylation patterns become clearer. Central to this activity is the realization that the 'histone code', which involves the post-translational modification of histones and which has important ramifications for chromatin structure, may be linked to the DNA cytosine methylation pattern. New discoveries have suggested that histone lysine 9 methylation is implicated in the spread of heterochromatin in Drosophila and other organisms. Very recently it has been found that histone lysine 9 methylation is also necessary for some DNA methylation in Neurospora and plants. There is therefore the possibility that these two processes are closely linked, suggesting ways in which DNA methylation patterns may be established during normal development. Understanding these processes is fundamental to understanding what goes awry during the process of aging and carcinogenesis where DNA methylation patterns become substantially altered and contribute to the malignant phenotype.

Animals↗
PubMed2002

DNA methylation and breast carcinogenesis.

Knowledge about breast carcinogenesis has accumulated during the last decades but has barely been translated into strategies for early detection or prevention of this common disease. Changes in DNA methylation have been recognized as one of the most common molecular alterations in human neoplasia and hypermethylation of gene-promoter regions is being revealed as one of the most frequent mechanisms of loss of gene function. The heritability of methylation states and the secondary nature of the decision to attract or exclude methylation support the idea that DNA methylation is adapted for a specific cellular memory. According to Hanahan and Weinberg, there are six novel capabilities a cell has to acquire to become a cancer cell: limitless replicative potential, self-sufficiency in growth signals, insensitivity to growth-inhibitory signals, evasion of programmed cell death, sustained angiogenesis and tissue invasion and metastasis. This review highlights how DNA-methylation contributes to these features and offers suggestions about how these changes could be prevented, reverted or used as a 'tag' for early detection of breast cancer or, preferably, for detection of premalignant changes.

Breast Neoplasms↗
PubMed2002

Cell division is required for de novo methylation of CpG islands in bladder cancer cells.

Cell division is essential for tumor development and progression. Methylation-mediated silencing caused by aberrant de novo methylation of CpG islands located in the promoter regions of growth regulatory genes occurs frequently in human cancers. We investigated the relationship between cell division and de novo methylation to determine whether de novo methylation can occur in the absence of cell division in cancer cells. We treated T24 bladder carcinoma cells with 5-Aza-2'-deoxycytidine to induce a transient demethylation and then compared the timing and kinetics of remethylation of the p16 gene locus under conditions of either G(0)-G(1) growth arrest induced by serum starvation and confluence or continuous cell proliferation in complete medium. Variable levels of remethylation were detected in CpG poor regions of DNA, as well as repetitive DNA elements in the absence of cell division, yet no remethylation occurred at CpG islands under these conditions. This correlated with continuous expression of p16 protein in these cells. DNA methyltransferase (DNMT)1 and DNMT3b3 proteins were undetectable in 5-Aza-2'-deoxycytidine-treated and untreated nondividing cells, and their mRNA transcripts were down-regulated in these cells. Although DNMT3a mRNA levels were also reduced, they recovered to original levels in nondividing cells after drug treatment. Our results suggest that cell division is required for de novo methylation of CpG islands and that DNMT3a may play a role in methylating CpG poor regions or repetitive DNA elements outside of the S phase of the cell cycle.

Antimetabolites, Antineoplastic↗
PubMed2002

Comprehensive analysis of CpG islands in human chromosomes 21 and 22.

CpG islands are useful markers for genes in organisms containing 5-methylcytosine in their genomes. In addition, CpG islands located in the promoter regions of genes can play important roles in gene silencing during processes such as X-chromosome inactivation, imprinting, and silencing of intragenomic parasites. The generally accepted definition of what constitutes a CpG island was proposed in 1987 by Gardiner-Garden and Frommer [Gardiner-Garden, M. & Frommer, M. (1987) J. Mol. Biol. 196, 261-282] as being a 200-bp stretch of DNA with a C+G content of 50% and an observed CpG/expected CpG in excess of 0.6. Any definition of a CpG island is somewhat arbitrary, and this one, which was derived before the sequencing of mammalian genomes, will include many sequences that are not necessarily associated with controlling regions of genes but rather are associated with intragenomic parasites. We have therefore used the complete genomic sequences of human chromosomes 21 and 22 to examine the properties of CpG islands in different sequence classes by using a search algorithm that we have developed. Regions of DNA of greater than 500 bp with a G+C equal to or greater than 55% and observed CpG/expected CpG of 0.65 were more likely to be associated with the 5' regions of genes and this definition excluded most Alu-repetitive elements. We also used genome sequences to show strong CpG suppression in the human genome and slight suppression in Drosophila melanogaster and Saccharomyces cerevisiae. This finding is compatible with the recent detection of 5-methylcytosine in Drosophila, and might suggest that S. cerevisiae has, or once had, CpG methylation.

5' Flanking Region↗
PubMed2002

Analysis of gene induction in human fibroblasts and bladder cancer cells exposed to the methylation inhibitor 5-aza-2'-deoxycytidine.

Hypermethylation of the promoters of cancer-related genes is often associated with their inactivation during tumorigenesis. Several preclinical and clinical trials have been developed to use DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (5-Aza-CdR) in attempts to reactivate silenced genes in human cancers. We used high-density oligonucleotide gene expression microarrays to examine the effects of 5-Aza-CdR treatment on human fibroblast cells (LD419) and a human bladder tumor cell line (T24). Data obtained 8 days after recovery from 5-Aza-CdR treatment showed that more genes were induced in tumorigenic cells (61 genes induced; >or=4-fold) than nontumorigenic cells (34 genes induced; >or= 4-fold). Approximately 60% of induced genes did not have CpG islands within their 5' regions, suggesting that some genes activated by 5-Aza-CdR may not result from the direct inhibition of promoter methylation. Interestingly, a high percentage of genes activated in both cell types belonged to the IFN signaling pathway, confirming data from other tumor cell types.

Antimetabolites, Antineoplastic↗
PubMed2002

Quantitative methylation analysis using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE).

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that allows for rapid and simultaneous quantitation of the degree of methylation at several CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. A strand-specific polymerase chain reaction product is then generated to provide a suitable DNA template for quantitative methylation analysis using Ms-SNuPE. Single-nucleotide primer extension is performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being analyzed. The Ms-SNuPE technique can be adapted for high-throughput methylation analysis and therefore represents a novel approach for rapid quantitation of cytosine methylation suitable for a wide range of biological investigations.

CpG Islands↗
PubMed2002

Identification of DNA methylation differences during tumorigenesis by methylation-sensitive arbitrarily primed polymerase chain reaction.

The ability to detect methylation changes associated with oncogenic transformation is of critical importance in understanding how DNA methylation may contribute to tumorigenesis. We have developed a simple and reproducible fingerprinting method called methylation-sensitive arbitrarily primed polymerase chain reaction (AP-PCR) to screen for DNA methylation changes. This technique relies on digesting genomic DNA with methylation-sensitive and -insensitive restriction enzymes (e.g., HpaII and MspI) prior to AP-PCR amplification. Matched normal and tumor DNAs were compared to identify differential methylation. After the PCR products were resolved on high-resolution polyacrylamide gels, regions of genomic DNA that showed hypo- and hypermethylation associated with tumors were detected. These fragments were then isolated, cloned, and sequenced. Novel CpG islands were found to be frequently hypermethylated in bladder and colon tumors. We have demonstrated that this technique is a rapid and efficient method that can be used to screen for altered methylation patterns in genomic DNA and to isolate specific sequences associated with these changes.

Blotting, Southern↗
PubMed2002

The fundamental role of epigenetic events in cancer.

Patterns of DNA methylation and chromatin structure are profoundly altered in neoplasia and include genome-wide losses of, and regional gains in, DNA methylation. The recent explosion in our knowledge of how chromatin organization modulates gene transcription has further highlighted the importance of epigenetic mechanisms in the initiation and progression of human cancer. These epigenetic changes -- in particular, aberrant promoter hypermethylation that is associated with inappropriate gene silencing -- affect virtually every step in tumour progression. In this review, we discuss these epigenetic events and the molecular alterations that might cause them and/or underlie altered gene expression in cancer.

Chromosome Mapping↗
PubMed2002

Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements.

We used mouse embryonic stem (ES) cells with systematic gene knockouts for DNA methyltransferases to delineate the roles of DNA methyltransferase 1 (Dnmt1) and Dnmt3a and -3b in maintaining methylation patterns in the mouse genome. Dnmt1 alone was able to maintain methylation of most CpG-poor regions analyzed. In contrast, both Dnmt1 and Dnmt3a and/or Dnmt3b were required for methylation of a select class of sequences which included abundant murine LINE-1 promoters. We used a novel hemimethylation assay to show that even in wild-type cells these sequences contain high levels of hemimethylated DNA, suggestive of poor maintenance methylation. We showed that Dnmt3a and/or -3b could restore methylation of these sequences to pretreatment levels following transient exposure of cells to 5-aza-CdR, whereas Dnmt1 by itself could not. We conclude that ongoing de novo methylation by Dnmt3a and/or Dnmt3b compensates for inefficient maintenance methylation by Dnmt1 of these endogenous repetitive sequences. Our results reveal a previously unrecognized degree of cooperativity among mammalian DNA methyltransferases in ES cells.

Animals↗