Biomedical subjects

V J Bubb

Publications and source records attributed to V J Bubb.

17 recordsLinked to original sources

PubMed2003

The serotonin transporter intronic VNTR enhancer correlated with a predisposition to affective disorders has distinct regulatory elements within the domain based on the primary DNA sequence of the repeat unit.

We have demonstrated that a variable number tandem repeat domain (VNTR) within intron 2 of the serotonin transporter gene is a transcriptional regulatory domain which is potentially correlated with a predisposition to affective disorders and other behavioural conditions. This correlation based on copy number of the VNTR alone (nine, 10 or 12 copies of 16/17 base-pair element) has been controversial and not reproduced in all studies. We demonstrate that individual repeat elements within the VNTR domain differ in their enhancer activity in an embryonic stem cell model. This has implications for both the mechanism by which these VNTRs are correlated with the progression of the disease and suggests that clinical analysis should now be extended to correlate sequence variation within the VNTR with the disorder. The latter may resolve some of the conflicting data published to date.

Animals↗

PubMed2002

Neuron restrictive silencer factor as a modulator of neuropeptide gene expression.

We hypothesize that the transcription factor neuron restrictive silencer factor (NRSF) is an important determinant of the expression of the preprotachykinin (PPTA) gene (encoding substance P and Neurokinin A) and arginine vasopressin (AVP) both in neuronal and nonneuronal cells. NRSF, a zinc finger repressor protein, binds the NRSE motif found in many neuronal specific genes at a variety of promoter locations. However, it is found in a similar location at the major transcriptional start site, within both PPTA and AVP peptide promoters. We have correlated modulation of NRSF activity with expression of AVP and PPTA in a variety of cell types, indicating the general mechanism by which this protein may regulate expression. Specifically, they are as follows:(1). Expression of NRSF dramatically represses PPTA promoter activity in reporter gene constructs in primary cultures of DRG neurons.(2). The PPTA promoter activity is regulated differentially in osteoarthritic compared to normal chondrocytes. This regulation correlates with the region containing the NRSE site.(3). We have correlated a splice variant of NRSF with the establishment and progression of small cell lung carcinoma (SCLC) and demonstrated that NRSF variants can directly affect the activity of the AVP promoter in reporter gene constructs. If the deregulated expression of peptides in these diseases point to the mechanism determining the pathology, then perhaps targeting protocols that correct this deregulation may also reverse the specific disease phenotypes. Our data would indicate that modulation of NRSF activity would be a target for such intervention.

Animals↗

PubMed2001

Kirsten ras mutations in patients with colorectal cancer: the 'RASCAL II' study.

Researchers worldwide with information about the Kirsten ras (Ki-ras) tumour genotype and outcome of patients with colorectal cancer were invited to provide that data in a schematized format for inclusion in a collaborative database called RASCAL (The Kirsten ras in-colorectal-cancer collaborative group). Our results from 2721 such patients have been presented previously and for the first time in any common cancer, showed conclusively that different gene mutations have different impacts on outcome, even when the mutations occur at the same site on the genome. To explore the effect of Ki-ras mutations at different stages of colorectal cancer, more patients were recruited to the database, which was reanalysed when information on 4268 patients from 42 centres in 21 countries had been entered. After predetermined exclusion criteria were applied, data on 3439 patients were entered into a multivariate analysis. This found that of the 12 possible mutations on codons 12 and 13 of Kirsten ras, only one mutation on codon 12, glycine to valine, found in 8.6% of all patients, had a statistically significant impact on failure-free survival (P = 0.004, HR 1.3) and overall survival (P = 0.008, HR 1.29). This mutation appeared to have a greater impact on outcome in Dukes' C cancers (failure-free survival, P = 0.008, HR 1.5; overall survival P = 0.02, HR 1.45) than in Dukes' B tumours (failure-free survival, P = 0.46, HR 1.12; overall survival P = 0.36, HR 1.15). Ki-ras mutations may occur early in the development of pre-cancerous adenomas in the colon and rectum. However, this collaborative study suggests that not only is the presence of a codon 12 glycine to valine mutation important for cancer progression but also that it may predispose to more aggressive biological behaviour in patients with advanced colorectal cancer.

Adolescent↗

PubMed2001

Adenomatous polyposis coli (APC), beta-catenin, and cadherin are expressed in human bone and cartilage.

AIMS: Members of the cadherin and catenin families are involved in chondrogenesis and catenin gene mutations have been detected in malignant tumours of bone. This study was undertaken to assess in detail expression of cadherin, beta-catenin and the associated tumour suppressor gene product APC in bone and cartilage at different stages of human skeletal maturity and in non-neoplastic and neoplastic osteoarticular disease. METHODS AND RESULTS: Immunohistochemical staining of formalin-fixed paraffin-embedded normal and osteoarthritic adult articular cartilage, fetal growth plate and a series of tumours of bone and cartilage was undertaken with a panel of antibodies against APC, beta-catenin, and pan-cadherin. This study demonstrated expression of APC, beta-catenin and cadherin in normal and diseased bone and cartilage. APC was present both in osteoblasts and osteoclasts but not in osteocytes. Although only weak APC staining of occasional growth plate hypertrophic chondrocytes and normal articular chondrocytes was seen, APC staining was increased in osteoarthritic articular cartilage. beta-catenin and pan-cadherin staining was strongly positive in osteoclasts and osteoblasts, with expression being lost when bone cells differentiated into osteocytes. Expression of APC, beta-catenin and pan-cadherin in bone tumours was similar to that of non-neoplastic adult tissues. CONCLUSIONS: These findings suggest previously unrecognized roles for APC in regulation of function of chondrocytes, osteoblasts and osteoclasts and support the view that catenin-cadherin interactions are important in regulation of bone cell activity. Abnormalities of expression or function of these molecules may be important in formation of bone tumours and their clinical behaviour.

Adenomatous Polyposis Coli Protein↗

PubMed2000

Familial adenomatous polyposis coli in South Africa--molecular basis and diagnosis.

OBJECTIVE: To determine the molecular basis and establish a routine molecular diagnostic service for familial adenomatous polyposis coli (FAP) families in South Africa. DESIGN: The coding region of the adenomatous polyposis coli (APC) gene in affected FAP kindreds was screened using heteroduplex analysis, single-strand conformation polymorphism analysis and the protein truncation test. SETTING: Department of Human Genetics, University of Stellenbosch, and the Cancer Research Campaign Laboratories, Department of Pathology, University of Edinburgh and Molecular Medicine Centre, Western General Hospital, Edinburgh, Scotland (academic visit of 6 months). SUBJECTS: FAP-affected individuals and at-risk family members in 28 apparently unrelated South African families. RESULTS: A total of nine different APC mutations was identified, allowing DNA-based diagnosis in 20 families. Three of these mutations have not been described previously in other populations. CONCLUSION: Pre-symptomatic diagnosis using direct mutation detection is cost-effective and surgical intervention has the potential to prevent cancer in at-risk individuals from FAP families.

Adenomatous Polyposis Coli↗

PubMed1999

Dysregulated expression of beta-catenin marks early neoplastic change in Apc mutant mice, but not all lesions arising in Msh2 deficient mice.

We have analysed the pattern of beta-catenin expression by immunohistochemistry in mice singly or multiply mutant for Apc, p53 and Msh2. We observed increased expression of beta-catenin in all intestinal lesions arising on an ApcMin+/- background. In all categories of lesion studied mosaic patterns of beta-catenin expression were observed, with the proportion of cells showing enhanced expression decreasing with increasing lesion size. p53 status did not alter these patterns. We also show that beta-catenin dysregulation marks pancreatic abnormalities occurring in ApcMin+/- and (ApcMin+/-, p53-/-) mice. In these mice both adenomas and adenocarcinomas of the pancreas arose and were characterized by increased expression of beta-catenin. We have extended these analyses to intestinal lesions arising in mice mutant for the mismatch repair gene Msh2. In these mice, increased expression of beta-catenin was again observed. However, in contrast with ApcMin+/- mice, a subset of lesions retained normal expression. Taken together, these findings show that increased expression of beta-catenin is an efficient marker of early neoplastic change in both murine intestine and pancreas in Apc mutant mice. However, we also show that dysregulation of beta-catenin is not an obligate step in the development of intestinal lesions, and therefore that genetic events other than the loss of Apc function may initiate the transition from normal to neoplastic epithelium.

Adenomatous Polyposis Coli Protein↗

PubMed1999

Caspase-mediated cleavage of APC results in an amino-terminal fragment with an intact armadillo repeat domain.

During the effector phase of apoptosis, caspase activation appears to be responsible for the distinctive structural changes of apoptosis and perhaps for some of the changes in function of the doomed cells. There is therefore interest in identifying caspase substrates and the details of the cleavage events. Here we define precisely the event responsible for generation of a stable 90 kDa fragment from the oncosuppressor protein adenomatous polyposis coli (APC). Using synthetic radiolabeled APC peptides as substrate, we demonstrate cleavage by cytosolic extracts from preapoptotic cells. This cleavage was reproduced by recombinant caspase-3 and blocked by a tetrapeptide inhibitor Ac-DEVD-CHO, which is specific for caspase-3 family members. Inhibitors specific for caspase-1 and -8 however, were less effective in blocking APC cleavage. Mutation of a candidate DNID caspase-3 target site completely abolished cleavage. This cleavage may be of biological importance since the 90 kDa fragment consists of a sequence that is highly conserved in the human, rat, mouse, Xenopus, and Drosophila APC, although wide sequence divergence is observed in Drosophila immediately carboxy-terminal to the DNID site. Furthermore, cleavage at this site separates two significant functional domains: an amino-terminal armadillo repeat and an adjacent series of beta-catenin binding sites. Further circumstantial evidence for the significance of APC-related pathways in apoptosis is provided by the observation that apoptosis also induces cleavage of beta-catenin itself, a protein known to accumulate in cells depleted in functional APC and that appears to link cell-cell signaling to changes in transcription and cell movement.

Adenomatous Polyposis Coli↗

PubMed1999

Apoptosis and carcinogenesis.

Apoptosis has long been known to be effected through a common sequence of structural changes, despite the wide variety of initiating stimuli. These common structural events appear to depend upon activation of a set of enzymes (caspases) which direct a strongly conserved, terminal effector pathway. The regulation of this pathway, and in particular its coupling to DNA damage, appears to be critical in maintaining at low levels the number of mutated cells within tissues. The frequency with which tumours (experimental and human) bear deficiency in p53 or MSH-2 repair function may indicate the importance of these proteins in coupling DNA damage to apoptosis.

Animals↗

PubMed1997

The pattern of K-ras mutation in pulmonary adenocarcinoma defines a new pathway of tumour development in the human lung.

Codon 12 of the K-ras oncogene was screened for mutations in 65 surgically-resected primary pulmonary adenocarcinomas and in 32 tissue foci of alveolar atypical hyperplasia (AAH) by a polymerase chain reaction (PCR)-based method. Mutations in either position 1 or position 2 of codon 12 were detected in 16 tumours (25 per cent). When analysed by site of origin, mutations were seen in 9/26 (35 per cent) parenchymal and in 0/12 bronchial adenocarcinomas (P < 0-02), K-ras mutations were seen in five AAH lesions from four patients. DNA sequencing showed that the great majority of mutations in both adenocarcinomas and AAH were G-T transversions. These findings provide support for the classification of pulmonary adenocarcinomas into bronchial and parenchymal subtypes and also provide molecular evidence to support the importance of AAH in the development of parenchymal cancers.

Adenocarcinoma↗

PubMed1997

APC expression in normal human tissues.

The tumour suppressor gene APC codes for a 2843-amino acid protein whose precise functions are still poorly understood. This paper describes the development of two new antisera to APC (to amino- and carboxy-terminal epitopes) which permit localization of the protein by immunohistochemistry in archival paraffin sections. The protein is expressed in a wide variety of normal epithelial tissues. Its distribution frequently coincides with the location of post-replicative cells within tissues. Staining patterns demonstrate that the APC protein, although often diffusely cytoplasmic in distribution, may also accumulate in the apical and immediately subapical regions, or along the lateral margins of certain cells. These results indicate that APC is significant in many tissues in addition to the colorectal epithelium. They are compatible with a function related to signalling at the adherens junction and possibly with other more complex roles in cells committed to terminal differentiation.

Adenomatous Polyposis Coli Protein↗

PubMed1996

Microsatellite instability and the role of hMSH2 in sporadic colorectalcancer.

Microsatellite instability (MSI) occurs in most tumours from patients with hereditary non-polyposis colorectal cancer (HNPCC) and in around 17% of sporadic colorectal cancers. Germline defects in mismatch repair (MMR) genes are responsible for the majority of large HNPCC families, with hMSH2 accounting for at least 50%. MMR gene defects also occur in a small proportion of sporadic colorectal tumours with MSI. Here we report a systematic analysis of mismatch repair deficiency in 215 Scottish patients with sporadic colorectal tumours. We found that 16.4% of tumours exhibited MSI; survival analysis by Cox proportional hazards method showed a substantial survival advantage for patients with tumours showing MSI, independent of other prognostic factors. Tumours with MSI were screened for hMSH2 mutations and although 61% were found to have alterations, of these only 1/24 was exonic. The majority of these changes were reductions in length at intronic mononucleotide tracts and we postulate that these alterations are the result of a genetic defect elsewhere, although they may compromise hMSH2 function as a second step in tumourigenesis. Our findings indicate that instability confers an improved prognosis in colorectal cancer and, despite the fact that these two groups of tumours share similar biological characteristics, the genetic basis of HNPCC and sporadic colorectal cancer with MSI is different.

Base Sequence↗

PubMed1996

Polymorphism in serotonin transporter gene associated with susceptibility to major depression.

UNLABELLED: BACKGROUND; The serotonin transporter of the brain provides the primary target for the action of selective antidepressant drugs. We set out to identify polymorphisms of the serotonin transporter gene and to find out whether there was a relation between any such polymorphisms and the occurrence of affective disorder. METHODS: A comparison of a polymorphic region of the human serotonin transporter gene was carried out between two groups. The study group comprised 83 patients (39 unipolar depressive disorder, 44 bipolar disorder) with major affective disorder. The control group comprised 122 anonymous blood donors, and 71 volunteers who had been screened for psychiatric disorders. FINDINGS: We detected three novel alleles of the variable-number-tandem-repeat (VNTR) region (STin2.9, STin2.10) and Stin2.12) containing nine, ten and 12 copies of the VNTR element, respectively. The frequencies of the different forms of the allele in the control group were compared with those in the affective disorder group. There was a significant difference between the control and affective disorder groups, largely explained by the excess of the STin2.9 allele in the unipolar group (chi2=10.05, p<0.004 [Bonferroni corrected]). The presence of the allele with nine copies of the repeat was significantly associated with risk of unipolar disorder (odds ratio=6.95 [95% CI 1.8-27.2]). INTERPRETATION: This association, for an obvious candidate gene, may provide a critical starting point for an understanding of the likely polygenic contributions towards susceptibility to affective disorder.

Alleles↗

PubMed1996

Loss of heterozygosity at 5q21 in non-small cell lung cancer: a frequent event but without evidence of apc mutation.

Four genetic polymorphisms in the APC and MCC genes at chromosome 5q21 were analysed for loss of heterozygosity (LOH) in 97 primary squamous carcinomas and adenocarcinomas of the lung. LOH was identified in at least two polymorphic loci in 41 percent of informative cases. There was no significant difference in the frequency of LOH between squamous carcinomas and adenocarcinomas. Within the adenocarcinoma group, however, LOH appeared to be more common in tumours having a bronchial origin (5/9; 56 per cent) than in parenchymal adenocarcinoma (6/21; 29 per cent). All 32 tumours showing LOH at one or more polymorphic sites were examined for mutations in the mutation cluster region (MCR) of APC by single-strand conformational polymorphism (SSCP) analysis. Mutations were not detected in any of these cases. We therefore propose that it is likely that a tumour suppressor gene on 5q other than APC is involved in the pathogenesis of lung cancer.

Adenocarcinoma↗

PubMed1996

Germline APC mutation (Gln1317) in a cancer-prone family that does not result in familial adenomatous polyposis.

Germline mutations of the adenomatous polyposis coli gene are associated with the dominantly inherited syndrome of familial adenomatous polyposis. Somatic mutations in this gene are an early event in sporadic colorectal tumorigenesis. Here we report a family with genetic characteristics that do not conform exactly to either of these situations. The index case and three siblings presented with colorectal cancer, and another sibling had lung cancer. There was no evidence of colorectal cancer susceptibility in previous generations, although one case of gastric cancer was observed. Using restriction fragment length polymorphism, single-strand conformational polymorphism, and sequencing analysis, we screened each living family member for alterations in the mutation cluster region of exon 15 of the APC gene. A constitutional single base pair substitution at codon 1317 was observed in two of the siblings with colorectal cancer, but neither exhibited any colonic features typical of FAP nor an early onset of cancer. This constitutional change is a missense mutation and therefore does not result in the truncation of the APC protein, the most commonly observed result of mutation in this gene. We present evidence that this change is not a polymorphism and may be capable of conferring a growth advantage. This particular germline APC mutation does not completely cosegregate with cancer in this family; therefore, we conclude that another gene locus may be responsible for the increased cancer risk observed.

Adenocarcinoma↗

PubMed1996

Extracolonic features of familial adenomatous polyposis in patients with sporadic colorectal cancer.

We have investigated the occurrence of attenuated extracolonic manifestations (AEMs) of familial adenomatous polyposis (FAP) in patients with non-polyposis colorectal cancer. In a prospective case-control study, we observed that significantly more colorectal cancer patients exhibited AEM than did age and sex-matched controls (19.5% vs 7.5%, P < 0.004). However patients with AEMs do not have occult FAP, as we found no heterozygous adenomatous polyposis coli (APC) gene mutations despite extensive analysis of constitutional DNA. Genome-wide DNA replication errors (RERs) occur in a proportion of colorectal cancers, particularly right-sided lesions and in almost all tumours from hereditary non-polyposis colorectal cancer (HNPCC) patients. As AEMs have been reported in familial colon cancer cases, we investigated the relationship of AEMs to tumour RER phenotype. There was indeed an excess of AEMs in patients with right-sided tumours (30.2% of 53 patients vs 14.7% of 116 patients, P < 0.03) and in those with RER tumours (3 out of 12 patients with RER tumours vs none out of 21 patients with non-RER tumours, P < 0.05). Two patients with AEM were from HNPCC families compared with none of those without AEM (P < 0.05). The association of AEMs with colorectal cancer is intriguing, and we speculate that it may be a manifestation of mutational mosaicism of the APC gene, perhaps associated with a constitutional defect in DNA mismatch pair.

Adenomatous Polyposis Coli↗

PubMed1994

Loss of heterozygosity of MCC is not associated with mutation of the retained allele in sporadic colorectal cancer.

The adenomatous polyposis coli (APC) gene, which transmits familial adenomatous polyposis, is frequently mutated in sporadic colorectal tumours. Acquired somatic mutations have also been reported in a second gene, mutated in colorectal cancer (MCC), which lies within 500 kb of APC on chromosome 5q21 and has thus been implicated in tumour development. Further evidence for an oncosuppressor gene other than APC on chromosome 5q comes from recent studies of lung, renal and hepatic cancers in which there is loss of heterozygosity of 5q21 but no somatic APC mutations. To investigate the relative importance of APC and MCC in sporadic colorectal cancer, we have assessed the extent of 5q21 allelic loss in 80 carcinomas. All informative tumours exhibiting allelic loss had deletions which included both APC and MCC. In 21 tumours with loss of heterozygosity in MCC we have screened the entire coding region of the gene for mutation of the retained allele and found no evidence for mutation. The data indicate that independent loss of MCC is a rare event, and that in cases where allele loss occurs mutation of the retained allele is uncommon. This suggests that MCC does not function as an independent tumour suppressor in the majority of colorectal cancers.

Alleles↗

PubMed1993

The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein.

The human papillomavirus (HPV) E5 proteins are predicted from DNA sequence analysis to be small hydrophobic molecules, and the HPV type 6 (HPV-6) and HPV-11 E5 proteins share several structural similarities with the bovine papillomavirus type 1 (BPV-1) E5 protein. Also similar to the BPV-1 E5 protein, the HPV-6 and HPV-16 E5 proteins exhibit transforming activity when assayed on NIH 3T3 and C127 cells. In this study, we expressed epitope-tagged E5 proteins from both the "low-risk" HPV-6 and the "high-risk" HPV-16 in order to permit their immunologic identification and biochemical characterization. While the HPV-6 and HPV-16 E5 proteins fail to form disulfide-linked dimers and oligomers, they did resemble the BPV-1 E5 protein in their intracellular localization to the Golgi apparatus, endoplasmic reticulum, and nuclear membranes. In addition, the HPV E5 proteins also bound to the 16-kDa pore-forming protein component of the vacuolar ATPase, a known characteristic of the BPV-1 E5 protein. These studies reveal a common intramembrane localization and potential cellular protein target for both the BPV and HPV E5 proteins.

Amino Acid Sequence↗