Biomedical subjects

Adam R Karpf

Publications and source records attributed to Adam R Karpf.

9 recordsLinked to original sources

PubMed2006

DNA methylation pathway alterations in an autochthonous murine model of prostate cancer.

We examined the DNA methylation pathway in an autochthonous murine prostate cancer model, transgenic adenocarcinoma of mouse prostate (TRAMP). We observed that, compared with strain-matched normal prostates, primary and metastatic TRAMP tumors display increased cytosine DNA methyltransferase (Dnmt) activity, Dnmt1 and Dnmt3b protein expression, and Dnmt1, Dnmt3a, and Dnmt3b mRNA expression. Increased expression of Dnmt genes correlates with increased expression of cyclin A and E2F target genes, implicating increased cell proliferation and Rb inactivation in Dnmt overexpression. We analyzed DNA methylation in TRAMP and found that global levels of 5-methyl-2'-deoxycytidine are unaltered, whereas specific tumors display centromeric repeat hypomethylation. To interrogate locus-specific methylation, we did restriction landmark genomic scanning (RLGS) on normal prostates and primary tumors. In primary tumors, 2.3% of approximately 1,200 analyzed loci display aberrant DNA hypermethylation, whereas a considerably smaller number of events show hypomethylation. The pattern of RLGS changes was nonrandom, indicating a coordinated methylation defect. Two specific genes identified by RLGS were studied in detail. Surprisingly, methylation of a downstream exon of p16(INK4a) (p16) was the highest frequency hypermethylation event identified in TRAMP, where it is associated with increased p16 mRNA and protein expression. In contrast, hypermethylation of the 5' CpG island region of the homeobox gene Irx3 in TRAMP is associated with reduced gene expression. In summary, our data reveal a systemic DNA methylation pathway defect in TRAMP reminiscent of human prostate cancer, supporting the use of this model to investigate the functional role of DNA methylation pathway alterations in prostate cancer development.

Adenocarcinoma↗

PubMed2006

Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication.

Chromatin methylation is necessary for stable repression of gene expression during mammalian development. During cell division, DNMT1 maintains the DNA methylation pattern of the newly synthesized daughter strand, while G9a methylates H3K9. Here, DNMT1 is shown to directly bind G9a both in vivo and in vitro and to colocalize in the nucleus during DNA replication. The complex of DNMT1 and G9a colocalizes with dimethylated H3K9 (H3K9me2) at replication foci. Similarly, another H3K9 histone methyltransferase, SUV39H1, colocalizes with DNMT1 on heterochromatic regions of the nucleoli exclusively before cell division. Both DNMT1 and G9a are loaded onto the chromatin simultaneously in a ternary complex with loading factor PCNA during chromatin replication. Small interfering RNA (siRNA) knockdown of DNMT1 impairs DNA methylation, G9a loading, and H3K9 methylation on chromatin and rDNA repeats, confirming DNMT1 as the primary loading factor. Additionally, the complex of DNMT1 and G9a led to enhanced DNA and histone methylation of in vitro assembled chromatin substrates. Thus, direct cooperation between DNMT1 and G9a provides a mechanism of coordinated DNA and H3K9 methylation during cell division.

Animals↗

PubMed2006

An improved synthesis of psammaplin A.

The marine natural product, psammaplin A, was first isolated from the Psammaplinaplysilla sponge in 1987. Since that time, psammaplin A has shown a wide spectrum of biological activities that include enzyme inhibitory activities resulting in antibacterial and antitumor effects. An improved synthesis of psammaplin A has been developed, making the compound more easily accessible for further biological evaluations. In this context, we find that psammaplin A is an effective DNA methyltransferase inhibitor in vitro but fails to alter genomic DNA methylation levels in treated human cancer cells.

Cell Line, Tumor↗

PubMed2006

Zebra fish Dnmt1 and Suv39h1 regulate organ-specific terminal differentiation during development.

DNA methylation and histone methylation are two key epigenetic modifications that help govern heterochromatin dynamics. The roles for these chromatin-modifying activities in directing tissue-specific development remain largely unknown. To address this issue, we examined the roles of DNA methyltransferase 1 (Dnmt1) and the H3K9 histone methyltransferase Suv39h1 in zebra fish development. Knockdown of Dnmt1 in zebra fish embryos caused defects in terminal differentiation of the intestine, exocrine pancreas, and retina. Interestingly, not all tissues required Dnmt1, as differentiation of the liver and endocrine pancreas appeared normal. Proper differentiation depended on Dnmt1 catalytic activity, as Dnmt1 morphants could be rescued by active zebra fish or human DNMT1 but not by catalytically inactive derivatives. Dnmt1 morphants exhibited dramatic reductions of both genomic cytosine methylation and genome-wide H3K9 trimethyl levels, leading us to investigate the overlap of in vivo functions of Dnmt1 and Suv39h1. Embryos lacking Suv39h1 had organ-specific terminal differentiation defects that produced largely phenocopies of Dnmt1 morphants but retained wild-type levels of DNA methylation. Remarkably, suv39h1 overexpression rescued markers of terminal differentiation in Dnmt1 morphants. Our results suggest that Dnmt1 activity helps direct histone methylation by Suv39h1 and that, together, Dnmt1 and Suv39h1 help guide the terminal differentiation of particular tissues.

Animals↗

PubMed2005

Genetic disruption of cytosine DNA methyltransferase enzymes induces chromosomal instability in human cancer cells.

DNA methyltransferase 1 (DNMT1)-deficient mice are tumor-prone, and this has been proposed to result from the induction of genomic instability. To address whether loss of DNMT1, or the related protein DNMT3b, results in genomic instability in human cancer cells, we used a near-diploid human colorectal cancer cell line, HCT116, in which one or both DNMT genes were disrupted by homologous recombination. Array-based comparative genomic hybridization analyses indicated that double, but not single, DNMT knock-out cells display two specific alterations in regional DNA copy number, suggesting that DNMT deficiency and genomic DNA hypomethylation are not associated with widespread genomic amplifications or deletions in human cancer cells. However, spectral karyotype analyses revealed that DNMT-deficient HCT116 cells are highly unstable with respect to large-scale chromosomal alterations; furthermore, this effect is characterized by a high degree of individual cell heterogeneity. The induction of chromosomal alterations in DNMT-deficient cells was evidenced both by aneuploidy and by large increases in the number of novel chromosomal translocations. Studies of double knock-out cells indicated that the generation of chromosomal alterations is spontaneous and persistent in vitro, meeting the formal definition of genomic instability. In summary, we show that DNMT deficiency in human cancer cells results in constitutive genomic instability manifested by chromosomal translocations.

Aneuploidy↗

PubMed2005

Evaluation of a 7-day continuous intravenous infusion of decitabine: inhibition of promoter-specific and global genomic DNA methylation.

PURPOSE: The nucleoside analog 5-aza-2'-deoxycytidine (5-aza-CdR, decitabine) is a potent inhibitor of DNA methylation in vitro. Cellular treatment with this agent induces the re-expression of methylation-silenced genes. It remains unclear to what extent this compound inhibits DNA methylation in vivo. A clinical study was designed to examine the molecular effects and toxicity of a continuous 1-week intravenous infusion of decitabine in solid tumor patients. METHODS: Ten patients with refractory solid tumors were included in this study. Decitabine was administered at 2 mg/m(2)/d [DOSAGE ERROR CORRECTED] via continuous infusion for 168 hours. Quantitative polymerase chain reaction and high performance liquid chromatography were utilized to measure promoter-specific and global DNA methylation in peripheral-blood cells before and after treatment. RESULTS: Transient grade III/IV neutropenia (two patients) and grade II thrombocytopenia (one patient) was observed at the lowest planned dose step (2 mg/m2/d for 7 days). Nonhematologic toxicities were not observed. Quantitative polymerase chain reaction demonstrated significant MAGE-1 promoter hypomethylation by 14 days after the start of treatment in all 13 treatment cycles examined. Significant genomic DNA hypomethylation was also seen by day 14 in 11 of 13 treatment cycles analyzed. Genomic DNA methylation reverted to baseline levels by 28 to 35 days after the start of treatment, demonstrating that inhibition of DNA methylation by decitabine is transient. CONCLUSION: A 168-hour continuous infusion of decitabine is well tolerated and results in the inhibition of promoter-specific and genomic DNA methylation in vivo. This treatment schedule is suitable for evaluation of decitabine in combination with agents whose activity may be enhanced by the reversal of DNA methylation-mediated gene silencing.

Aged↗

PubMed2005

Specific method for the determination of genomic DNA methylation by liquid chromatography-electrospray ionization tandem mass spectrometry.

Herein we report a novel method for determining genomic DNA methylation that utilizes liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to measure 5-methyl-2'-deoxycytidine levels following enzymatic hydrolysis of genomic DNA. LC separation of 5-methyl-2'-deoxycytidine from the four deoxyribonucleosides, the four ribonucleosides, and 5-methyl-2'-cytidine, a RNA methylation product, has been achieved within 15 min. In combination with ESI-MS/MS detection, the reported method is highly specific and extremely sensitive with a limit of detection (LOD) of 0.2 fmol and a quantification linearity range from 1 fmol to 20 pmol. Genomic DNA methylation was expressed as the ratio of 5-methyl-2'-deoxycytidine to 2'-deoxyguanosine and was determined directly using 2'-deoxyguanosine as the internal standard. Because deoxycytidine methylation typically ranges from 2 to 6% in mammalian genomes, and pharmacological or genetic manipulations have not achieved levels lower than 0.1%, we validated the assay for methylation levels ranging from 0.05 to 10%. Importantly, both RNA contamination and incomplete DNA hydrolysis had no appreciable effect on 5-methyl-2'-deoxycytidine quantification. LOD studies indicate that only 4 ng of DNA is required for this assay. This LOD should permit the use of this method for applications having limiting amounts of DNA that were not previously candidates for global genomic DNA methylation analysis, e.g., clinical trial samples, or cells collected by laser capture microdissection.

Cell Line, Tumor↗

PubMed2004

Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine.

It remains unclear to what extent drugs targeting transcriptional repressor complexes affect global gene expression in cells derived from target and nontarget human tissues. To address this question, we used genome-wide expression analysis using microarrays to analyze the response of three tumor and one normal epithelial cell line to treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR). Notably, we found that 5-aza-CdR treatment induced a limited number of genes (mean, 0.67%; range, 0.17-1.8% of 25,940 genes screened) in each cell line tested. The majority of the gene expression changes that followed 5-aza-CdR treatment were conserved in tumor and normal cells, including genes that function in cell proliferation, differentiation, immune presentation, and cytokine signaling. In contrast, 5-aza-CdR treatment induced the expression of cancer-testis class tumor antigens only in tumor cell lines. To explain this tissue-specific response, we analyzed the mechanism of transcriptional regulation of the prototype member of this tumor antigen gene family, MAGE-1. Taken from our analysis of MAGE-1 gene regulation, we propose that 5-aza-CdR-mediated gene activation has two distinct requirements: 1) the reversal of promoter hypermethylation, and 2) the presence of transcriptional activators competent for the activation of hypomethylated target promoters. This latter requirement for gene activation by 5-aza-CdR is probably mediated by sequence-specific transcription factors and may account for the limited number of human genes induced by 5-aza-CdR treatment. This revised model for gene activation by 5-aza-CdR has important implications for the use of DNA methyltransferase inhibitors in clinical settings.

Antigens, Neoplasm↗

PubMed2002

Reactivating the expression of methylation silenced genes in human cancer.

DNA methylation alterations are now widely recognized as a contributing factor in human tumorigenesis. A significant number of tumor suppressor genes are transcriptionally silenced by promoter hypermethylation, and recent research implicates alterations in chromatin structure as the mechanistic basis for this repression. The enzymes responsible for catalyzing DNA-cytosine methylation, as well as the proteins involved in interpreting the DNA methylation signal, have now been elucidated. Technological advances, including gene expression microarrays and genome scanning techniques, have allowed the comprehensive measurement of DNA methylation changes in human cancers. An important distinction between DNA methylation (epigenetic) and mutation or deletion (genetic) tumor suppressor gene inactivation is that epigenetic inactivation can be abrogated by small molecules, including DNA methyltransferase and histone deacetylase inhibitors. Further, strategies have been developed that combine treatments with drugs that reactivate silenced gene expression with secondary agents that target the re-expressed genes and/or reconstituted signal transduction pathways. In this review, we will discuss in detail the mechanisms of gene silencing by DNA methylation, the techniques used to decipher the complement of methylation-inactivated genes in human cancers, and current and future strategies for reactivating the expression of methylation-silenced genes.

Antineoplastic Agents↗