Characterizing the prevalence of chromosome instability in interval colorectal cancer.

A substantial proportion of colorectal cancers (CRCs) are interval CRCs (I-CRCs; i.e., CRCs diagnosed soon after a colonoscopy). Chromosomal instability (CIN) is defined as an increase in the rate of which whole chromosomes/large chromosomal fragments are gained or lost and is observed in 85% of non-hereditary CRCs. The contribution of CIN to the etiology of I-CRCs remains unknown. We established a fluorescence in situ hybridization (FISH) approach to characterize CIN by enumerating specific chromosomes and determined the prevalence of numerical CIN in a population-based cohort of I-CRCs and control (sporadic) CRCs. Using the population-based Manitoba Health administrative databases and Manitoba Cancer Registry, we identified an age, sex, and colonic site of CRC matched cohort of I-CRCs and controls and retrieved their archived paraffin-embedded tumor samples. FISH chromosome enumeration probes specifically recognizing the pericentric regions of chromosomes 8, 11, and 17 were first used on cell lines and then CRC tissue microarrays to detect aneusomy, which was then used to calculate a CIN score (CS). The 15th percentile CS for control CRC was used to define CIN phenotype. Mean CSs were similar in the control CRCs and I-CRCs; 82% of I-CRCs exhibited a CIN phenotype, which was similar to that in the control CRCs. This study suggests that CIN is the most prevalent contributor to genomic instability in I-CRCs. Further studies should evaluate CIN and microsatellite instability (MSI) in the same cohort of I-CRCs to corroborate our findings and to further assess concomitant contribution of CIN and MSI to I-CRCs.

Novel cytopathological structures induced by mixed infection of unrelated plant viruses.

ABSTRACT When two unrelated plant viruses infect a plant simultaneously, synergistic viral interactions often occur resulting in devastating diseases. This study was initiated to examine ultrastructural virus-virus interactions of mixed viral infections. Mixed infections were induced using potyviruses and viruses from other plant virus families. Novel ultrastructural paracrystalline arrays composed of co-infecting viruses, referred to as mixed virus particle aggregates (MVPAs), were noted in the majority of the mixed infections studied. When the flexuous rod-shaped potyvirus particles involved in MVPAs were sectioned transversely, specific geometrical patterns were noted within some doubly infected cells. Although similar geometrical patterns were associated with MVPAs of various virus combinations, unique characteristics within patterns were consistent in each mixed infection virus pair. Centrally located virus particles within some MVPAs appeared swollen (Southern bean mosaic virus mixed with Blackeye cowpea mosaic virus, Cucumber mosaic virus mixed with Blackeye cowpea mosaic virus, and Sunn hemp mosaic virus mixed with Soybean mosaic virus). This ultrastructural study complements molecular studies of mixed infections of plant viruses by adding the additional dimension of visualizing the interactions between the coinfecting viruses.

Viral and bacterial agents associated with experimental transmission of infectious proventriculitis of broiler chickens.

Proventriculitis of broilers can be reproduced by oral inoculation of day-old chicks with a proventricular homogenate from affected 3-wk-old broilers. The objective of the following studies was to isolate from this homogenate viral and bacterial isolates that could produce proventriculitis. A monoclonal antibody to infectious bursal disease virus (IBDV) was used to precipitate virus from the homogenate. A primary chicken digestive tract cell culture system was also used to isolate virus from a 0.2-microm filtrate of the homogenate, and a bacterium was also isolated from the homogenate. In trial 1, day-old birds were orally inoculated with either proventriculus homogenate or monoclonal antibody immunoprecipitated IBDV (MAB-IBDV). At 4, 7, 14, and 21 days postinfection (PI), 12 birds from each treatment group were subjected to necropsy. In trial 2, day-old birds were orally inoculated with either infectious proventriculus homogenate, suspect virus isolated in cell culture and propagated in embryo livers and spleens, or a bacterial isolate. Twelve birds from each treatment were subjected to necropsy at days 7, 14, 21, and 28 PI. In trial 3, treatments were maintained in negative pressure isolation chambers, and an additional treatment included virus plus bacterial isolate. Twenty-four birds from each treatment were subjected to necropsy at day 21 PI. In trial 1, infectious homogenate decreased body weight and relative gizzard weights at 4, 7, 14, and 21 days PI. Proventriculus relative weight was increased at days 7, 14, and 21 PI, and proventriculus lesion scores were increased at days 14 and 21 PI. Bursa/spleen weight ratios were decreased at day 14, and feed conversion was increased at days 4 and 21. The MAB-IBDV treatment decreased proventriculus and gizzard relative weights at day 4 PI, increased proventriculus lesion scores and bursa/spleen weight ratios at day 14, and decreased heterophil/lymphocyte ratios at day 21. In trial 2, all infected birds had significantly higher mean relative proventriculus weights at 21 days PI and had higher 4-wk mean proventriculus scores as compared with both control groups. In trial 3, birds treated with homogenate and birds treated with both suspect virus and the bacterial isolate had significantly higher proventriculus lesion scores; higher relative weights of proventriculus, gizzard, liver, and heart; lower body weights; and lower relative bursa weights compared with the saline control group. These studies suggest that infectious proventriculitis has a complex etiology involving both viral and bacterial infection.

Dynamic analysis of proviral induction and De Novo methylation: implications for a histone deacetylase-independent, methylation density-dependent mechanism of transcriptional repression.

Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

The chicken beta-globin 5'HS4 boundary element blocks enhancer-mediated suppression of silencing.

A constitutive DNase I-hypersensitive site 5' of the chicken beta-globin locus, termed 5'HS4 or cHS4, has been shown to insulate a promoter from the effect of an upstream enhancer and to reduce position effects on mini-white expression in Drosophila cells; on the basis of these findings, it has been designated a chromatin insulator. We have examined the effect of the cHS4 insulator in a system that assays both the level of gene expression and the rate of transcriptional silencing. Because transgenes flanked by insulator elements are shielded from position effects in Drosophila cells, we tested the ability of cHS4 to protect transgenes from position effects in mammalian cells. Flanking of an expression vector with the cHS4 insulator in a colony assay did not increase the number of G418-resistant colonies. Using lox/cre-based recombinase-mediated cassette exchange to control integration position, we studied the effect of cHS4 on the silencing of an integrated beta-geo reporter at three genomic sites in K562 erythroleukemia cells. In this assay, enhancers act to suppress silencing but do not increase expression levels. While cHS4 blocked enhancement at each integration site, the strength of the effect varied from site to site. Furthermore, at some sites, cHS4 inhibited the enhancer effect either when placed between the enhancer and the promoter or when placed upstream of the enhancer. These results suggest that the activity of cHS4 is not dominant in all contexts and is unlikely to prevent silencing at all genomic integration sites.