Biomedical subjects

S H Doak

Publications and source records attributed to S H Doak.

10 recordsLinked to original sources

PubMed2006

Fluorescence in-situ hybridisation on biopsies from clam ileocystoplasties and on a clam cancer.

The incidence of carcinoma following an enterocystoplasty increases with time and is a major concern after such procedures. The aim of this study was to investigate genetic instability (in the form of numerical chromosomal aberrations) at the enterovesical anastomosis in patients who had undergone a clam ileocystoplasty using fluorescent in-situ hybridisation (FISH). Fluorescent in-situ hybridisation was performed on touch preparation samples prepared from fresh endoscopic biopsies obtained from the enterovesical anastomosis and native bladder remnant (control specimens) of 15 patients who had undergone a clam ileocystoplasty. Fluorescent in-situ hybridisation was also performed on one squamous cell cancer specimen. Significant aneusomic changes were found at the enterovesical anastomosis in all 15 patients. Alterations in chromosome 18 copy number were the most frequent abnormal finding (trisomy 18, n=8; monosomy 18, n=7). Nine patients were monosomic for chromosome 9. Isolated monosomy 8 and trisomy 8 were each found in one patient. The control specimens were all normal. An unusually high incidence of polysomic cells was found in the clam tumour specimen, reflecting the aggressive nature of this cancer. Chromosomal numerical abnormalities occur at the enterovesical anastomosis following a clam ileocystoplasty and chromosome 18 appears to be a particularly good marker of genetic instability. The results of this study indicate that morphologically normal tissue obtained from the enterovesical anastomosis displays evidence of chromosomal instability that may predispose to tumour formation. However, further prospective, blinded, longitudinal studies are required to establish whether predetermined FISH signal patterns in enterocystoplasty cells in urine or obtained by biopsy predict the presence or absence of tumour.

Adult↗

PubMed2006

Chromosome morphology after long-term storage investigated by scanning near-field optical microscopy.

Fluorescence in situ hybridization coupled with far-field fluorescence microscopy is a commonly used technique to visualize chromosomal aberrations in diseased cells. To obtain the best possible results, chromatin integrity must be preserved to ensure optimal hybridization of fluorescence in situ hybridization probes. However, biological samples are known to degrade and storage conditions can be critical. This study concentrates its investigation on chromatin stability as a function of time following fluorescence in situ hybridization type denaturing protocols. This issue is extremely important because chromatin integrity affects the fluorescence response of the chromosome. To investigate this, metaphase chromosome spreads of human lymphocytes were stored at both -20 and -80 degrees C, and were then imaged using scanning near-field optical microscopy over a nine month period. Using the scanning near-field optical microscope's topography mode, chromosome morphology was analysed before and after the application of fluorescence in situ hybridization type protocols, and then as a function of storage time. The findings revealed that human chromosome samples can be stored at -20 degrees C for short periods of time (approximately several weeks), but storage over 3 months compromises chromatin stability. Topography measurements clearly show the collapse of the stored chromatin, with variations as large as 60 nm across a chromosome. However, storage at -80 degrees C considerably preserved the integrity with variations in topography significantly reduced. We report studies of the fluorescent response of stored chromosomes using scanning near-field optical microscopy and their importance for gaining further understanding of chromosomal aberrations.

Cell Nucleus↗

PubMed2005

Do dose response thresholds exist for genotoxic alkylating agents?

The demonstration and acceptance of dose response thresholds for genotoxins may have substantial implications for the setting of safe exposure levels. Here we test the hypothesis that direct-acting DNA reactive agents may exhibit thresholded dose responses. We examine the potential mechanisms involved in such thresholded responses, particularly in relation to those of alkylating agents. As alkylating agents are representative model DNA reactive compounds with well characterized activities and DNA targets, they could help shed light on the general mechanisms involved in thresholded dose responses for genotoxins. Presently, thresholds have mainly been described for agents with non-DNA targets. We pay particular attention here to the contribution of DNA repair to genotoxic thresholds. A review of the literature shows that limited threshold data for alkylating agents are currently available, but the contribution of DNA repair in thresholded dose responses is suggested by several studies. The existence of genotoxic thresholds for alkylating agents methylmethanesulfonate is also supported here by data from our laboratory. Overall, it is clear that different endpoints induced by the same alkylator, can possess different dose response characteristics. This may have an impact on the setting of safe exposure levels for such agents. The limited information available concerning the dose response relationships of alkylators can nevertheless lead to the design of experiments to investigate the mechanisms that may be involved in threshold responses. Through using paired alkylators inducing different lesions, repaired by different pathways, insights into the processes involved in genotoxic thresholds may be elucidated. Furthermore, as alkyl-guanine-DNA transferase, base excision repair and mismatch repair appear to contribute to genotoxic thresholds for alkylators, cells deficient in these repair processes may possess altered dose responses compared with wild-type cells and this approach may help understand the contribution of these repair pathways to the production of thresholds for genotoxic effects in general. Finally, genotoxic thresholds are currently being described for acute exposures to single agents in vitro, however, dose response data for chronic exposures to complex mixtures are, as yet, a long way off.

Alkylating Agents↗

PubMed2005

Fluorescence in situ hybridisation analysis of chromosomal aberrations in gastric tissue: the potential involvement of Helicobacter pylori.

In this series of experiments, a novel protocol was developed whereby gastric cells were collected using endoscopic cytology brush techniques, and prepared, such that interphase fluorescence in situ hybridization (FISH) could be performed. In total, 80 distinct histological samples from 37 patients were studied using four chromosome probes (over 32,000 cells analysed). Studies have previously identified abnormalities of these four chromosomes in upper GI tumours. Using premalignant tissues, we aimed to determine how early in Correa's pathway to gastric cancer these chromosome abnormalities occurred. Aneuploidy of chromosomes 4, 8, 20 and 17(p53) was detected in histologically normal gastric mucosa, as well as in gastritis, intestinal metaplasia, dysplasia and cancer samples. The levels of aneuploidy increased as disease severity increased. Amplification of chromosome 4 and chromosome 20, and deletion of chromosome 17(p53) were the more common findings. Hence, a role for these abnormalities may exist in the initiation of, and the progression to, gastric cancer. Helicobacter pylori infection was determined in premalignant tissue using histological analysis and PCR technology. Detection rates were comparable. PCR was used to subtype H. pylori for CagA status. The amplification of chromosome 4 in gastric tissue was significantly more prevalent in H. pylori-positive patients (n=7) compared to H. pylori-negative patients (n=11), possibly reflecting a role for chromosome 4 amplification in H. pylori-induced gastric cancer. The more virulent CagA strain of H. pylori was associated with increased disease pathology and chromosomal abnormalities, although numbers were small (CagA+ n=3, CagA- n=4). Finally, in vitro work demonstrated that the aneuploidy induced in a human cell line after exposure to the reactive oxygen species (ROS) hydrogen peroxide was similar to that already shown in the gastric cancer pathway, and may further strengthen the hypothesis that H. pylori causes gastric cancer progression via an ROS-mediated mechanism.

Aged↗

PubMed2004

Generation of locus-specific probes for interphase fluorescence in situ hybridisation--application in Barrett's esophagus.

Despite the wide range of probes commercially available for interphase fluorescence in situ hybridisation (FISH), the supply of locus-specific probes is limited to genes or chromosomal regions commonly altered in genetic diseases or during carcinogenesis. Generation of these probes is therefore desirable to accommodate individual research requirements. Hence, we detail the methodology required to design and produce custom locus-specific interphase FISH probes for any human genomic region of interest and their application was illustrated in cytogenetic investigations of Barrett's tumourigenesis. Previously utilising FISH, we observed that Barrett's tissues demonstrated chromosome 4 hyperploidy [Gut 52 (2003) 623], but as centromeric probes were used in this analysis, it was not known if the whole chromosome was amplified. We consequently generated single-copy sequence probes for the 4p16.3 and 4q35.1 subtelomeric loci. Multicolour FISH was subsequently performed on interphase preparations originating from patients with Barrett's esophagus at varying histological grades, thus demonstrating the whole region of chromosome 4 was amplified within the tissues. Additionally, probes for the DNA methyltransferase genes were produced to determine if gene dosage alterations were responsible for increasing methylation activity during Barrett's neoplastic progression. No significant alterations at the DNMT1 and DNMT3a loci were detected. An increased copy number of these genes is therefore not the basis for the hypermethylation commonly observed in this premalignant lesion.

Adenocarcinoma↗

PubMed2003

The bile acid deoxycholic acid (DCA) at neutral pH activates NF-kappaB and induces IL-8 expression in oesophageal cells in vitro.

Barrett's oesophagus patients accumulate chromosomal defects during the histological progression to cancer, one of the most prominent of which is the amplification of the whole of chromosome 4. We aimed to study the role that the transcription factor NF-kappaB, a candidate cancer- promoting gene, present on chromosome 4, plays in Barrett's oesophagus, using OE33 cells as a model. Specifically, we wanted to determine if NF-kappaB was activated by exposure to bile acid (deoxycholic acid) in oesophageal cells. We employed pathway specific cDNA microarrays and real-time PCR, to first identify bile acid induced genes and specifically to investigate the role of NF-kappaB. An NF-kappaB reporter system was used, as well as an inhibitor of NF-kappaB (pyrrolidine dithiocarbamate) to confirm the activation of NF-kappaB by bile. We show that physiological levels of DCA (100-300 microM) were capable of activating NF-kappaB in OE33 cells and inducing NF-kappaB target gene expression (particularly IkappaB and IL-8). Other gene expression abnormalities were also shown to be induced by DCA. Importantly, preliminary experiments showed that NF-kappaB activation by bile occurred at neutral pH, but not at acid pH. Acidic bile did however cause over-expression of the c-myc oncogene, as reported previously. Hence, we present data showing that NF-kappaB may be a key mediator of carcinogenesis in bile exposed Barrett's tissues. In addition, neutral bile acids appear to play a significant part in reflux induced gene expression changes. We postulate that the activation of the survival factor NF-kappaB by bile may be linked to the previous cytogenetic data from our laboratory showing the amplification of NF-kappaB's chromosome (chromosome 4), during Barrett's cancer progression. Hence chromosome 4 amplification may provide a survival mechanism for bile exposed oesophageal tissues via NF-kappaB.

Adenocarcinoma↗

PubMed2003

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma.

p53 mutations and loss of heterozygosity have been commonly associated with oesophageal adenocarcinoma. In this investigation, the p53 status of a Welsh population of Barrett's-associated oesophageal adenocarcinomas were fully characterised at the gene sequence, chromosomal, mRNA and protein levels. In total, 31 tumours were examined for p53 gene sequence mutations using RFLP with sequencing, allelic loss of the gene was characterised by FISH, mRNA expression by p53 pathway signalling arrays and protein levels by p53 immunohistochemistry. In all, 9.6% of adenocarcinomas harboured p53 mutations, 24% displayed p53 allelic loss and 83% exhibited p53 protein accumulation. Point mutations and deletions of the gene did not coexist within the same samples. All samples containing p53 mutations also displayed positive immunostaining; however; in the majority of cases, p53 protein accumulation developed in the absence of mutations. The gene expression analysis demonstrated no differences in p53 and mdm-2 transcription levels between the p53 immunonegative and immunopositive samples, indicating other mechanisms underlie the proteins' overexpression. In conclusion, p53 mutations and deletions do not appear to be frequent events in oesophageal adenocarcinomas; however, abnormal accumulation of the protein is present in a vast majority of cases. P53 gene mutations are not the primary cause of protein overexpression--an alternative mechanism is responsible for the positive p53 immunohistochemistry detected.

Adenocarcinoma↗

PubMed2003

Early p53 mutations in nondysplastic Barrett's tissue detected by the restriction site mutation (RSM) methodology.

Barrett's oesophagus is a premalignant condition whose incidence is rising dramatically. Molecular markers are urgently needed to identify Barrett's patients at the highest risk of cancer progression. To this end, we have used a rapid molecular technique, restriction site mutation (RSM), to detect low-frequency mutations in the p53 tumour suppressor gene in premalignant Barrett's tissues of cancer-free patients. In total, 38 endoscopically diagnosed Barrett's patients with a range of histological stages of Barrett's progression, plus four control patients without Barrett's oesophagus, were analysed for early p53 mutations. Tissue samples taken from these patients (93 samples in total) were analysed for the presence of low-frequency p53 mutations at hotspot codons: 175, 213, 248, 249, 282. In total, 13 of the 38 Barrett's patients were shown to possess a p53 mutation in at least one sample (no mutations in the four control patients). Although no statistically significant associations were found, p53 mutations reflected histological progression in Barrett's patients with p53 mutations found in 30% of metaplasia patients (P=0.4) and low-grade dysplasia patients (P=0.33) and 45% of high-grade dysplasia patients (P=0.15). Detected p53 mutations were mainly GC to AT transitions at CpG sites.

Barrett Esophagus↗

PubMed2003

Chromosome 4 hyperploidy represents an early genetic aberration in premalignant Barrett's oesophagus.

BACKGROUND AND AIMS: Characterisation of the underlying molecular mechanisms that promote Barrett's progression may ultimately lead to identification of potential predictive genetic markers that classify patients' malignant risk. In an attempt to understand these causative pathways, fluorescence in situ hybridisation (FISH) was used in this study to determine when specific genetic alterations arise during Barrett's associated neoplastic progression. METHODS: Endoscopic cytology brushings were obtained from 28 patients with Barrett's metaplasia, 28 with dysplasia (20 low grade dysplasia (LGD) and eight with high grade dysplasia (HGD)), and seven with adenocarcinoma, together with paired control brushings from regions of normal proximal squamous cell epithelium. The exfoliated epithelial cells were washed and deposited onto slides. Probes specific for the centromeres of chromosomes 4, 8, 20, and Y, and locus specific probes for the tumour suppressor genes p16, p53, and Rb were subsequently hybridised. RESULTS: Aneuploidy was found early in progression, with metaplastic tissues displaying increased copy numbers of chromosomes 4 and 8. Chromosome 4 hyperploidy was found in 89%, 90%, 88%, and 100% of metaplasias, LGD, HGD, adenocarcinomas, respectively, while chromosome 8 hyperploidy occurred in 71%, 75%, 100%, and 100% of patients with the respective staging. Loss of the p16 tumour suppressor gene also presented in metaplastic epithelium (7%) but most other genetic aberrations were only seen in HGD. CONCLUSIONS: Genetic instability arises well before dysplasia in Barrett's oesophagus, with chromosome 4 and 8 hyperploidy representing the earliest and most common alterations identified. As these aberrations are widespread at all the premalignant stages, there may be genes on chromosomes 4 and 8 that are involved in both the initiation and progression of Barrett's oesophagus.

Adenocarcinoma↗

PubMed2002

Genetic pathways involved in the progression of Barrett's metaplasia to adenocarcinoma.

BACKGROUND: The prediction of which patients with Barrett's metaplasia will develop cancer is difficult. Better genetic characterization of the condition may aid clinicians in devising more effective management and follow-up strategies. METHODS: A review was undertaken of the accumulated genetic data relating to the progression of squamous epithelium to adenocarcinoma. The normal functions of a number of cancer-related genes are described and an explanation is given of how alterations in these genes interfere with normal cell processes and lead to cancer. RESULTS AND CONCLUSION: The main genetic alterations accompanying the progression through dysplasia to adenocarcinoma were collated from 135 papers. The principal genetic changes implicated are the loss of p16 gene expression (by deletion or hypermethylation), the loss of p53 expression (by mutation and deletion), the increase in cyclin D1 expression, the induction of aneuploidy and the losses of the Rb, DCC and APC chromosomal loci.

Adenocarcinoma↗