Biomedical subjects

Dominic J Smiraglia

Publications and source records attributed to Dominic J Smiraglia.

10 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

Contour area filtering of two-dimensional electrophoresis images.

We describe an algorithm, Contour Area Filtering, for separating background from foreground in gray scale images. The algorithm is based on the area contained within gray scale contour lines. It can be viewed as a form of local thresholding, or as a seed growing algorithm, or as a type of watershed segmentation. The most important feature of the algorithm is that it uses object area to determine the segmentation. Thus, it is relatively impervious to brightness and contrast variations across an image or between different images. Contour Area Filtering was designed specifically for image analysis of 2D electrophoresis gels, although it can be applied to other gray scale images. A typical gel image is an electrophoretogram or a phosphor image of 1000-2500 spots representing protein or DNA restriction fragments. The images are quantitative with spot intensities reflective of the number of proteins or the DNA fragment copy number. The background intensity can vary widely across the image caused both by variation in spot density and by the physical laboratory process of creating a gel. Analyzing and comparing gel images entails extracting and segmenting spots, registering images and matching spots, and measuring differences between spots. We present experimental results which show that Contour Area Filtering is a quick, efficient method for separating electrophoresis gel background from foreground with extremely high accuracy.

Algorithms↗

PubMed2006

Epigenetic regulation of the tumor suppressor gene TCF21 on 6q23-q24 in lung and head and neck cancer.

The identification of tumor suppressor genes has classically depended on their localization within recurrent regions of loss of heterozygosity. According to Knudson's two-hit hypothesis, the remaining allele is lost, either genetically or, more recently identified, through epigenetic events. To date, retrospective analyses have determined promoter methylation as a common alternative alteration in cancer cells to silence cancer-related genes. Here we report an application of restriction landmark genomic scanning that allows for DNA methylation profiling along a region of recurrent loss of heterozygosity at chromosome 6q23-q24. This approach resulted in the identification of a tumor suppressor gene, TCF21, which is frequently lost in human malignancies. We demonstrate that TCF21 is expressed in normal lung airway epithelial cells and aberrantly methylated and silenced in the majority of head and neck squamous cell carcinomas and non-small-cell lung cancers analyzed. TCF21 is known to regulate mesenchymal cell transition into epithelial cells, a property that has been shown to be deficient in carcinomas. We further demonstrate that exogenous expression of TCF21 in cells that have silenced the endogenous TCF21 locus resulted in a reduction of tumor properties in vitro and in vivo.

Animals↗

PubMed2003

A comprehensive search for DNA amplification in lung cancer identifies inhibitors of apoptosis cIAP1 and cIAP2 as candidate oncogenes.

Amplification of oncogenes is an important mechanism that can cause gene overexpression and contributes to tumor development. The identification of amplified regions might have both prognostic and therapeutic significance. We used primary lung carcinomas and lung cancer cell lines for restriction landmark genomic scanning (RLGS) to identify novel amplified sequences. Enhanced RLGS fragments that indicate gene amplification were observed in primary tumors and lung cancer cell lines of both non-small cell lung cancer and small cell lung cancer. We identified one novel amplicon on chromosome 11q22, in addition to previously reported amplicons that include oncogenes MYCC, MYCL1 and previously identified amplification of chromosomal regions 6q21 and 3q26-27. Amplification of 11q22 has been reported in other types of cancer and was refined to an approximately 1.19 Mbp region for which the complete sequence is available. Based on a patient sample with a small region of low-level amplification we were able to further narrow this region to 0.92 Mbp. Genes localized in this region include two inhibitors of apoptosis (cIAP1 and cIAP2). Immunohistochemistry and western blot analysis identified cIAP1 and cIAP2 as potential oncogenes in this region as both are overexpressed in multiple lung cancers with or without higher copy numbers.

Blotting, Southern↗

PubMed2003

Aging results in hypermethylation of ribosomal DNA in sperm and liver of male rats.

There is a concern that increased paternal age may be associated with altered fertility and an increased incidence of birth defects in man. In previous studies of aged male rats, we have found abnormalities in the fertility and in the embryos sired by older males. Aging in mammals is associated with alterations in the content and patterns of DNA methylation in somatic cells; however, little is known in regard to germ cells. A systematic search for global and gene-specific alterations of DNA methylation in germ cells and liver of male rats was done. Restriction landmark genomic scanning, a method used to determine specific methylation patterns of CpG island sequences, has revealed a region of the ribosomal DNA locus that is preferentially hypermethylated with age in both spermatozoa and liver. In contrast, all single copy CpG island sequences in spermatozoa and in liver remain unaltered with age. We further demonstrate that a large proportion of rat ribosomal DNA is normally methylated and that regional and site-specific differences exist in the patterns of methylation between spermatozoa and liver. We conclude that patterns of ribosomal DNA methylation in spermatozoa are vulnerable to the same age-dependent alterations that we observe in normal aging liver. Failure to maintain normal DNA methylation patterns in male germ cells could be one of the mechanisms underlying age-related abnormalities in fertility and progeny outcome.

Aging↗

PubMed2003

The development of CpG island methylation biomarkers using restriction landmark genomic scanning.

CpG island hypermethylation is a common occurrence in cancer. Because this is a stable molecular alteration of the DNA, which can be detected easily from very small amounts, DNA methylation is an attractive candidate to use as a molecular biomarker. Recent studies have used DNA methylation of genes known to be targets of genetic disruption in cancer as biomarkers for early detection of cancer, classification of malignancies, response to drug treatment, and as markers predictive of outcome. Since many of the currently used targets of methylation are methylated at rather low frequencies in various cancer types even though the gene may be frequently disrupted by other mechanisms, it would be useful to develop additional markers that are methylated at high frequency in the cancer being studied. Restriction landmark genomic scanning has been used for the identification of frequent targets of methylation in multiple malignancies. These markers, which can be either cancer type-specific or nonspecific, may prove to be effective biomarkers for diagnostic or prognostic purposes, or for midpoint analysis of intervention strategies.

Biomarkers↗

PubMed2002

The study of aberrant methylation in cancer via restriction landmark genomic scanning.

Restriction landmark genomic scanning (RLGS) has been used to study DNA methylation in cancer for nearly a decade. The strong bias of RLGS for assessing the methylation state of CpG islands genome wide makes this an attractive technique to study both hypo- and hypermethylation of regions of the genome likely to harbor genes. RLGS has been used successfully to identify regions of hypomethylation, candidate tumor suppressor genes, correlations between hypermethylation events and clinical factors, and quantification of hypermethylation in a multitude of malignancies. This review will examine the major uses of RLGS in the study of aberrant methylation in cancer and discuss the significance of some of the findings.

Animals↗

PubMed2002

Distinct epigenetic phenotypes in seminomatous and nonseminomatous testicular germ cell tumors.

The genetic nature of testicular germ cell tumors and the molecular mechanisms underlying the morphological and clinical differences between the two subtypes, seminomas and nonseminomas, remains unclear. Genetic studies show that both subtypes exhibit many of the same regional genomic disruptions, although the frequencies vary and few clear differences are found. We demonstrate significant epigenetic differences between seminomas and nonseminomas by restriction landmark genomic scanning. Seminomas show almost no CpG island methylation, in contrast to nonseminomas that show CpG island methylation at a level similar to other solid tumors. We find an average of 1.11% of CpG islands methylation in nonseminomas, but only 0.08% methylated in seminomas. Furthermore, we demonstrate that seminomas are more highly hypomethylated than nonseminomas throughout their genome. Since both subtypes are thought to arise from primordial germ cells, the epigenetic differences seen between these subtypes may reflect the normal developmental switch in primordial germ cells from an undermethylated genome to a normally methylated genome. We discuss these findings in relation to different developmental models for seminomatous and nonseminomatous testicular germ cell tumors.

Blotting, Southern↗

PubMed2002

Restriction landmark genome scanning.

Restriction landmark genome scanning (RLGS) is a quantitative approach that is uniquely suited for simultaneously assessing the methylation status of thousands of CpG islands. RLGS separates radiolabeled NotI fragments (or other CpG-containing restriction enzyme fragments) in two dimensions and allows distinction of single-copy CpG islands from multicopy CpG-rich sequences. The methylation sensitivity of the endonuclease activity of NotI provides the basis for differential methylation analysis, and NotI sites occur primarily in CpG islands and genes. RLGS has been used to identify novel imprinted genes, novel targets of DNA amplification and methylation in human cancer, and to identify deletion, methylation, and gene amplification in a mouse model of tumorigenesis. Such massively parallel analyses are critical for pattern recognition within and between tumor types, and for estimating the overall influence of CpG island methylation on the cancer cell genome. RLGS is also a useful method for integrating methylation analyses with high-resolution gene copy number analyses.

Brain Neoplasms↗

PubMed2002

An AscI boundary library for the studies of genetic and epigenetic alterations in CpG islands.

Knudson's two-hit hypothesis postulates that genetic alterations in both alleles are required for the inactivation of tumor-suppressor genes. Genetic alterations include small or large deletions and mutations. Over the past years, it has become clear that epigenetic alterations such as DNA methylation are additional mechanisms for gene silencing. Restriction Landmark Genomic Scanning (RLGS) is a two-dimensional gel electrophoresis that assesses the methylation status of thousands of CpG islands. RLGS has been applied successfully to scan cancer genomes for aberrant DNA methylation patterns. So far, the majority of this work was done using NotI as the restriction landmark site. Here, we describe the development of RLGS using AscI as the restriction landmark site for genome-wide scans of cancer genomes. The availability of AscI as a restriction landmark for RLGS allows for scanning almost twice as many CpG islands in the human genome compared with using NotI only. We describe the development of an AscI-EcoRV boundary library that supports the cloning of novel methylated genes. Feasibility of this system is shown in three tumor types, medulloblastomas, lung cancers, and head and neck cancers. We report the cloning of 178 AscI RLGS fragments via two methods by use of this library.

Cloning, Molecular↗