Biomedical subjects

James Flanagan

Publications and source records attributed to James Flanagan.

9 recordsLinked to original sources

PubMed2006

Microarray-based DNA methylation profiling: technology and applications.

This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions using microarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340 000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.

Chromosome Mapping↗

PubMed2005

Mutation analysis of FANCD2, BRIP1/BACH1, LMO4 and SFN in familial breast cancer.

INTRODUCTION: Mutations in known predisposition genes account for only about a third of all multiple-case breast cancer families. We hypothesized that germline mutations in FANCD2, BRIP1/BACH1, LMO4 and SFN may account for some of the unexplained multiple-case breast cancer families. METHODS: The families used in this study were ascertained through the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab). Denaturing high performance liquid chromatography (DHPLC) analysis of the coding regions of these four genes was conducted in the youngest affected cases of 30 to 267 non-BRCA1/2 breast cancer families. In addition, a further 399 index cases were also screened for mutations in two functionally significant regions of the FANCD2 gene and 253 index cases were screened for two previously reported mutations in BACH1 (p. P47A and p. M299I). RESULTS: DHPLC analysis of FANCD2 identified six silent exonic variants, and a large number of intronic variants, which tagged two common haplotypes. One protein truncating variant was found in BRIP1/BACH1, as well as four missense variants, a silent change and a variant in the 3' untranslated region. No missense or splice site mutations were found in LMO4 or SFN. Analysis of the missense, silent and frameshift variants of FANCD2 and BACH1 in relatives of the index cases, and in a panel of controls, found no evidence suggestive of pathogenicity. CONCLUSION: There is no evidence that highly penetrant exonic or splice site mutations in FANCD2, BRIP1/BACH1, LMO4 or SFN contribute to familial breast cancer. Large scale association studies will be necessary to determine whether any of the polymorphisms or haplotypes identified in these genes contributes to breast cancer risk.

14-3-3 Proteins↗

PubMed2005

Single nucleotide extension technology for quantitative site-specific evaluation of metC/C in GC-rich regions.

The development and use of high throughput technologies for detailed mapping of methylated cytosines (metC) is becoming of increasing importance for the expanding field of epigenetics. The single nucleotide primer extension reaction used for genotyping of single nucleotide polymorphisms has been recently adapted to interrogate the bisulfite modification induced 'quantitative' C/T polymorphism that corresponds to metC/C in the native DNA. In this study, we explored the opportunity to investigate C/T (and G/A) ratios using the Applied Biosystems (ABI) SNaPshot technology. The main effort of this study was dedicated to addressing the complexities in the analysis of DNA methylation in GC-rich regions where interrogation of the target cytosine can be confounded by variable degrees of methylation in other cytosines (resulting in variable C/T or G/A ratios after treatment with bisulfite) in the annealing site of the interrogating primer. In our studies, the mismatches of the SNaPshot primer with the target DNA sequence resulted in a biasing effect of up to 70% while these effects decreased as the location of the polymorphic site moved upstream of the target cytosine. We demonstrated that the biasing effect can be corrected with the SNaPshot primers containing degenerative C/T and G/A nucleotides. A series of experiments using various permutations of quantitative C/T and G/A polymorphisms at various positions of the target DNA sequence demonstrated that SNaPshot is able to accurately report cytosine methylation levels with <5% average SD from the true values. Given the relative simplicity of the method and the possibility to multiplex C/T and G/A interrogations, the SNaPshot approach may become a useful tool for large-scale mapping of metC.

Base Composition↗

PubMed2004

The EBNA-3 gene family proteins disrupt the G2/M checkpoint.

The Epstein-Barr nuclear antigens (EBNA), EBNA-3, -4 and -6, have previously been shown to act as transcriptional regulators, however, this study identifies another function for these proteins, disruption of the G2/M checkpoint. Lymphoblastoid cell lines (LCLs) treated with a G2/M initiating drug azelaic bishydroxamine (ABHA) did not show a G2/M checkpoint response, but rather they display an increase in cell death, a characteristic of sensitivity to the cytotoxic effects of the drug. Cell cycle analysis demonstrated that the individual expression of EBNA-3, -4 or -6 are capable of disrupting the G2/M checkpoint response induced by ABHA resulting in increased toxicity, whereas EBNA-2, and -5 were not. EBNA-3 gene family protein expression also disrupted the G2/M checkpoint initiated in response to the genotoxin etoposide and the S phase inhibitor hydroxyurea. The G2 arrest in response to these drugs were sensitive to caffeine, suggesting that ATM/ATR signalling in these checkpoint responses may be blocked by the EBNA-3 family proteins. The function of EBNA-3, -4 and -6 proteins appears to be more complex than anticipated and these data suggest a role for these proteins in disrupting the host cell cycle machinery.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2004

The Epstein-Barr virus nuclear antigen-6 protein co-localizes with EBNA-3 and survival of motor neurons protein.

The Epstein-Barr virus nuclear antigen (EBNA)-6 protein is essential for Epstein-Barr virus (EBV)-induced immortalization of primary human B-lymphocytes in vitro. In this study, fusion proteins of EBNA-6 with green fluorescent protein (GFP) have been used to characterize its nuclear localization and organization within the nucleus. EBNA-6 associates with nuclear structures and in immunofluorescence demonstrate a punctate staining pattern. Herein, we show that the association of EBNA-6 with these nuclear structures was maintained throughout the cell cycle and with the use of GFP-E6 deletion mutants, that the region amino acids 733-808 of EBNA-6 contains a domain that can influence the association of EBNA-6 with these nuclear structures. Co-immunofluorescence and confocal analyses demonstrated that EBNA-6 and EBNA-3 co-localize in the nucleus of cells. Expression of EBNA-6, but not EBNA-3, caused a redistribution of nuclear survival of motor neurons protein (SMN) to the EBNA-6 containing nuclear structures resulting in co-localization of SMN with EBNA-6.

Cell Line, Tumor↗

PubMed2004

Identification of the nuclear localization signals within the Epstein-Barr virus EBNA-6 protein.

Epstein-Barr virus nuclear antigen (EBNA)-6 is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. Previous studies have shown that EBNA-6 acts as a transcriptional regulator of viral and cellular genes; however at present, few functional domains of the 140 kDa EBNA-6 protein have been completely characterized. There are five computer-predicted nuclear localization signals (NLS), four monopartite and one bipartite, present in the EBNA-6 amino acid sequence. To identify which of these NLS are functional, fusion proteins between green fluorescent protein and deletion constructs of EBNA-6 were expressed in HeLa cells. Each of the constructs containing at least one of the NLS was targeted to the nucleus of cells whereas a construct lacking all of the NLS was cytoplasmic. Site-directed mutation of these NLS demonstrated that only three of the NLS were functional, one at the N-terminal end (aa 72-80), one in the middle (aa 412-418) and one at the C-terminal end (aa 939-945) of the EBNA-6 protein.

Amino Acid Sequence↗

PubMed2003

Analysis of the transcription regulator, CNOT7, as a candidate chromosome 8 tumor suppressor gene in colorectal cancer.

Loss of heterozygosity (LOH) on the short arm of chromosome 8 occurs at high frequencies in many tumor types, including colorectal carcinoma. We have previously used microcell-mediated chromosome transfer (MMCT) to map an approximately 7.7 Mb colorectal cancer suppressor region (CRCSR) at 8p22-23.1. We have now taken a candidate gene approach to identify the putative tumor suppressor gene located within the CRCSR. CNOT7 encodes a subunit of the CCR4-Not transcription complex and is located at 8p22. We showed that CNOT7 is expressed in normal colonic mucosa and in colonic crypt cells, as well as in colorectal cell lines and primary tumors. We assembled a panel of 88 primary colorectal tumors comprising 20 MSI-high (high microsatellite instability), 19 MSI-low and 49 MSS (microsatellite stable) tumors for mutation analysis of the CNOT7 gene. Denaturing high-performance liquid chromatography (DHPLC) analysis of the entire coding region of the CNOT7 gene revealed only one somatic missense mutation in an MSS tumor. The rarity of somatic mutations in CNOT7, and its expression in primary colorectal tumors and cell lines, suggests that CNOT7 is not the target tumor suppressor gene in the 8p22-23.1 CRCSR.

Carcinoma, Squamous Cell↗

PubMed2003

Transfer of chromosome 8 into two breast cancer cell lines: total exclusion of three regions indicates location of putative in vitro growth suppressor genes.

Loss of heterozygosity (LOH) of the short arm of chromosome 8 occurs frequently in breast tumors. Fine mapping of the smallest regions of overlap of the deletions indicates that multiple tumor suppressor genes may be located in this region. We have performed microcell-mediated chromosome transfer of chromosome 8 into two breast cancer cell lines, 21MT-1 and T-47D. Twenty-two of the resulting hybrids were characterized extensively with chromosome 8 microsatellite markers and a subset were assayed for growth in vitro and soft agar clonicity. There was no evidence in any of the hybrids for suppression of growth or clonicity that could be attributed to the presence of particular regions of chromosome 8; however, none of the 22 hybrids examined had taken up all of the donor chromosome 8, and in fact there were three regions that contained only one allele of the markers genotyped in all 22 hybrids. These results are consistent with the presence of suppressor genes on the short arm of chromosome 8 causing strong growth suppression that is incompatible with growth in vitro; that is, multiple suppressor genes may exist on the short arm of chromosome 8.

Breast Neoplasms↗

PubMed2003

Localization of the Epstein-Barr virus protein LMP 1 to exosomes.

The Epstein-Barr virus latent membrane protein (LMP 1) functions as a constitutively active signalling molecule and associates in lipid rafts clustered with other signalling molecules. Using immunofluorescent confocal microscopy, LMP 1 was shown to have an heterogeneous distribution among individual cells which was not related to the cell cycle stage. LMP 1 was shown to localize to intracellular compartments in cells other than the plasma membrane. Co-labelling of cells with both an LMP 1 antibody and an antibody to the Golgi protein GS15 revealed that the intracellular LMP 1 partly co-localized with the Golgi apparatus. Further confirmation of intracellular LMP 1 localization was obtained by immunoelectron microscopy with rabbit polyclonal LMP 1 antibodies and cryosectioning. As well as being present in intracellular foci, LMP 1 co-localized in part with MHC-II and was present on exosomes derived from a lymphoblastoid cell line. Preparations of LMP 1 containing exosomes were shown to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that LMP 1 could be involved in immune regulation. This may be of particular relevance in EBV-associated tumours such as nasopharyngeal carcinoma and Hodgkin's disease, as LMP 1-containing exosomes may be taken up by infiltrating T-lymphocytes, where LMP 1 could exert an anti-proliferative effect, allowing the tumour cells to evade the immune system.

B-Lymphocytes↗