Biomedical subjects

J K Cowell

Publications and source records attributed to J K Cowell.

At least 19 recordsLinked to original sources

PubMed2006

The EVI5 TBC domain provides the GTPase-activating protein motif for RAB11.

The human EVI5 gene was originally isolated through its involvement with a constitutional chromosome translocation in a patient with stage 4S neuroblastoma. Recently, it has been shown that EVI5 is a centrosomal protein in interphase cells, which relocalizes to the midbody during late phases of mitosis. Disruption of its function leads to incomplete cell division and the formation of multinucleate cells. The EVI5 protein contains a TBC (TRE2/BUB/CDC16 homology) motif located in the N-terminal region. Proteins containing a TBC domain have been shown in some cases to act as GTPase-activating proteins (GAPs) and function through the interaction with Rab-like small G proteins. Despite the identification of over 50 TBC-containing proteins, and over 70 Rab-like proteins, only three combinations have been shown to have Rab/GAP activity to date. In this study, using linear ion trap mass spectroscopy, we have demonstrated that EVI5 exists in a protein complex with Rab11. Further, using a specific Rab-binding assay, we have shown that EVI5 preferentially interacts with the guanosine triphosphate-bound form of Rab11, and in a GAP activity assay, we have confirmed that EVI5 functions as a GAP for the Rab11 GTPase.

Amino Acid Sequence↗

PubMed2006

Identifying candidate colon cancer tumor suppressor genes using inhibition of nonsense-mediated mRNA decay in colon cancer cells.

Inhibition of the nonsense-mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene identification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations. Genes containing frameshift mutations in colon cancer cell line have been identified using a modified version of GINI. To increase the efficiency of identifying mutant genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhibiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analysed colon cancer cell lines showing microsatellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC31L1, NCOR1, BAT3, PHF14, ZNF294, C19ORF5 genes as well as genes coding for proteins with yet unknown functions.

Caffeine↗

PubMed2005

Analysis of the RB1 gene in children with retinoblastoma having residential connections to West Cumbria, England.

Six of eight cases of retinoblastoma previously identified as having a residential association with West Cumbria, England, in which the Sellafield nuclear installation is situated, were examined for the presence of a constitutional RB1 mutation. No mutations were detected, thus providing strong evidence against an environmental or occupational genotoxic effect causing germline mutations in the parents of these children.

Child, Preschool↗

PubMed2004

Identification and characterisation of constitutional chromosome abnormalities using arrays of bacterial artificial chromosomes.

Constitutional chromosome deletions and duplications frequently predispose to the development of a wide variety of cancers. We have developed a microarray of 6000 bacterial artificial chromosomes for array-based comparative genomic hybridisation, which provides an average resolution of 750 kb across the human genome. Using these arrays, subtle gains and losses of chromosome regions can be detected in constitutional cells, following a single overnight hybridisation. In this report, we demonstrate the efficiency of this procedure in identifying constitutional deletions and duplications associated with predisposition to retinoblastoma, Wilms tumour and Beckwith-Wiedemann syndrome.

Bacteria↗

PubMed2003

Rapid detection of allelic losses in brain tumours using microsatellite repeat markers and high-performance liquid chromatography.

High-performance liquid chromatography (HPLC) is a recently introduced high-capacity automated method for detecting unknown mutations (denaturing HPLC) or for sizing DNA fragments under nondenaturing conditions. We have adapted the HPLC method for detection of loss of heterozygosity (LOH) and used glial tumours as a model to evaluate its sensitivity and specificity in comparison to conventional denaturing polyacrylamide gel electrophoresis. A total of 20 oligodendrogliomas (grades II and III), and five astrocytic tumours (grades III and IV) were analysed using 14 polymorphic microsatellite markers mapping to regions on chromosomes 1p, 19q, and 10q using both DNA-HPLC and denaturing gel electrophoresis. This study demonstrated complete concordance between both methods. However, unlike gel electrophoresis, HPLC is automated, does not require post-PCR processing, and does not require hazardous radioactive or expensive fluorescent labelling. Our data suggest that HPLC is a reliable and sensitive method for detection of allelic losses in tumour samples and it is a favourable alternative for high-sensitivity LOH detection in both research and diagnostic environments.

Brain Neoplasms↗

PubMed2001

A novel member of the WD-repeat gene family, WDR11, maps to the 10q26 region and is disrupted by a chromosome translocation in human glioblastoma cells.

Allelic deletions of 10q25-26 and 19q13.3-13.4 are the most common genetic alterations in glial tumors. We have identified a balanced t(10;19) reciprocal translocation in the A172 glioblastoma cell line which involves both critical regions on chromosomes 10 and 19. In addition, loss of an entire copy of chromosome 10 has occurred in this cell line suggesting that the translocation event may provide a highly specific critical inactivating event in a gene responsible for tumorigenesis. Positional cloning of this translocation breakpoint resulted in the identification of a novel chromosome 10 gene, WDR11, which is a member of the WD-repeat gene family. The WDR11 gene is ubiquitously expressed, including normal brain and glial tumors. WDR11 is composed of 29 exons distributed over 58 kilobases and oriented towards the telomere. The translocation resulted in deletion of exon 5 and consequently fusion of intron 4 of WDR11 to the 3' untranslated region of a novel member, ZNF320, of the Krüppel-like zinc finger gene family. Since ZNF320 is oriented toward the centromere of chromosome 19, both genes appeared on the same derivative chromosome der(10). The chimeric transcript encodes the WDR11 polypeptide, which is truncated after the second of six WD-repeats. ZNF320 is also expressed in A172 cells, although it is not clear if the translocation affects the expression of the altered gene because of the presence of another unrearranged gene on chromosome 19. We suggest that, because of its localization in a region frequently showing LOH and the observation of inactivation of this gene in glioblastoma cells, WDR11 is a candidate gene for the frequently proposed tumor suppressor gene in 10q25-26 which is involved in tumorigenesis of glial and other tumors showing frequent alterations in the distal 10q region.

Alleles↗

PubMed2001

Molecular characterization of the breakpoint region associated with a constitutional t(2;15)(q34;q26) in a patient with multiple myeloma.

The molecular cloning of the translocation breakpoints from constitutional chromosome rearrangements in patients with a variety of human diseases has consistently led to the isolation of genes important in the development of the phenotype. We used fluorescence in situ hybridization (FISH) to analyze the breakpoint region of a constitutional chromosome translocation involving regions 2q34 and 15q26 observed in a patient with multiple myeloma (MM), a malignant disorder of plasma cells secreting monoclonal immunoglobulin. FISH analysis of this rearrangement showed that the chromosome 2-specific yeast artificial chromosome (YAC) 914E7 and the chromosome 15-specific YAC 757H6 span the translocation breakpoints, respectively. In order to characterize the location of the breakpoints further, somatic cell hybrids were constructed between mouse NIH3T3 cells and t(2;15)-bearing lymphoblastoid cells. Using these somatic cell hybrids, we have shown that the breakpoint on chromosome 2 lies between D2S3007 and D2S3004 and the chromosome 15 breakpoint lies between D15S107 and WI5967 (D15S836). YAC fragmentation has been used to define a 350 kb region containing the 15q26 breakpoint.

3T3 Cells↗

PubMed2001

Molecular alterations in the neurofibromatosis type 2 gene and its protein rarely occurring in meningothelial meningiomas.

OBJECT: The neurofibromatosis Type 2 (NF2) gene is the only tumor suppressor gene that has been clearly implicated in the development of benign meningiomas. Interestingly, previous data obtained by the authors indicate that reduced NF2 protein expression seldom occurs in meningothelial meningiomas, the most common histological type of meningioma. The goal of the current study was to explore further the hypothesis of NF2 gene-independent tumorigenesis of meningothelial meningiomas. METHODS: The authors performed a mutational analysis of all 17 exons of the NF2 gene by using single-stranded conformational polymorphism (SSCP). In addition, expression levels of the NF2 protein and mu-calpain, a protease suggested to inactivate the NF2 protein, were determined by immunoblotting analysis of 27 meningiomas (20 meningothelial and seven nonmeningothelial). Mutations of the NF2 gene were found in only one (5%) of 20 meningothelial meningiomas and three (43%) of seven nonmeningothelial tumors (Fisher's exact test, p = 0.042). The levels of NF2 protein were severely reduced in six (28.5%) of 21 meningothelial meningiomas, in contrast to six (86%) of seven nonmeningothelial meningiomas (Fisher's exact test, p = 0.023). Activation of IL-calpain did not correlate with the status of NF2 protein expression in the meningiomas analyzed, demonstrating that mu-calpain activation does not account for the loss of NF2 protein in meningiomas with apparently normal NF2 genes. CONCLUSIONS: These results clearly demonstrate that NF2 gene mutations and decreased NF2 protein expression rarely occur in meningothelial meningiomas compared with other histological types of meningiomas. The clinical behavior of meningothelial meningiomas, however, is similar to that of other benign meningiomas. It is likely, therefore, that the tumorigenesis of meningothelial meningiomas is the result of deleterious alterations of genes that have final phenotypical effects similar to inactivation of the NF2 gene.

Adult↗

PubMed2001

Fine mapping of the PTGFR gene to 1p31 region and mutation analysis in human breast cancer.

The 1p31 chromosomal region shows loss of heterozygosity (LOH) in up to 50% of human breast cancer, indicating the presence of a tumor suppressor gene at this location. Many efforts have been made to identify candidate genes responsible for breast cancer on the short arm of chromosome 1. It was shown that prostaglandins have been implicated in the tumorigenesis pathway, perhaps via interactions with their cell surface receptors. The prostaglandin F2 receptor gene (PTGFR) was tentatively mapped to 1p31 adjacent to the region undergoing LOH in human breast cancer. We undertook a mutation study in 34 sporadic human breast tumors using a variant of SSCP, incorporation PCR SSCP (IPS). Several nucleotide variants were detected in different tumors. Here we report the nature of these nucleotide changes and the possible involvement of the PTGFR gene in the etiology of human cancer.

Base Sequence↗

PubMed2000

TTC4, a novel candidate tumor suppressor gene at 1p31 is often mutated in malignant melanoma of the skin.

A novel candidate tumor suppressor gene, TTC4, on chromosome 1p31 has been described recently. Since aberrations in this region have been detected in malignant melanoma, we investigated DNA of paraffin-embedded sections from 16 typical naevi, 19 atypical naevi, 32 primary melanomas (15 superficial spreading melanomas, 17 nodular melanomas) and 25 metastases and DNA from four melanoma cell lines by PCR and direct sequencing analysis for mutations in all exons of TTC4. Tumors comprised a wide range of thickness (Breslow index) and Clark levels. No mutations could be detected in typical or atypical naevi, but we found seven different point mutations in the tumor samples, six of them causing an amino acid change. Ten melanoma samples belonging to nine patients showed one or more of these mutations. In detail, in six of 25 metastases, in two of 17 nodular melanomas and in two of 15 superficial spreading melanomas point mutations could be detected. In two cell lines, a loss of a whole exon could be demonstrated and in one cell line we found a point mutation. In addition, three polymorphisms were found. Our findings indicate that TTC4 may participate in the pathogenesis of malignant melanomas of the skin.

Amino Acid Sequence↗

PubMed2000

A transcription map of the minimally deleted region from 13q14 in B-cell chronic lymphocytic leukemia as defined by large scale sequencing of the 650 kb critical region.

Extensive analysis of tumors has demonstrated homozygous and heterozygous deletions in chromosome region 13q14.3 in B-cell chronic lymphocytic leukemia (B-CLL), suggesting the site of a tumor suppressor gene. Since previous searches for this gene have not yielded any viable candidates, we now present the sequence of the BACs which span the minimally deleted approximately 650 kb region between markers D13S319 and D13S25. This sequence has allowed us to create the definitive transcription map for the region which reveals 93 ESTs and 12 Unigene clusters in this region. Using gene prediction programs, a further 19 potential genes are also identified. The genes show an asymmetrical distribution throughout the region with most of them clustering at the extreme ends. This sequencing effort provides for the definitive structure of the B-CLL deletion region and the identification of the vast majority of the potential candidate genes. Of all the genes identified, only three have homologies to known genes: two L1 repeat genes and rabbit epididymal protein 52. This 13q14.3 sequence provides the final substrate from which to characterize the B-CLL tumor suppressor gene.

Chromosomes, Artificial, Bacterial↗

PubMed2000

2-5A antisense directed against telomerase RNA produces apoptosis in ovarian cancer cells.

OBJECTIVE: RNase L is converted to an active form upon binding short 2',5'-oligoadenylates (2-5A). To direct RNase L to an RNA target, 2-5A is attached to an antisense oligonucleotide (2-5A antisense). This chimera can be directed against telomerase-an RNA-protein complex that elongates telomeric DNA and is involved in cellular immortalization. Our objective is to investigate the effect of 2-5A antisense by targeting telomerase RNA (hTR) in the ovarian cancer cell line, HEY-1B. METHODS: Baseline RNase L levels and telomerase activities were measured in both HEY-1B and normal ovarian epithelial cells (NOE). Cells were treated daily with chimeric oligonuclotides (ODN) directed against four different hTR sites, or control ODNs including nonchimeric antisense, 2-5A fused to a mismatched sequence, or inactive 2-5A fused to antisense. At 48 h, apoptosis was evaluated using the TUNEL assay. After six daily ODN administrations, telomerase activity was redetermined, and at 7 days viability counts were obtained. RESULTS: Both cell lines expressed similar levels of RNase L. Hey-1B displayed telomerase activity while NOE did not. After 7 days of transfection, 2-5A antisense ODNs caused profound cell death in the HEY-1B cells, but not in the NOE cells. This effect was seen regardless of hTR target site, and ODN controls showed no significant decrease in cell viability in either cell line. HEY1B cells treated with 2-5A antisense against hTR showed a decrease in telomerase activity and a profound induction of programmed cell death. CONCLUSIONS: The results suggest that 2-5A antisense directed against telomerase RNA results in apoptotic cell death in ovarian cancer cells, but not normal ovarian epithelial cells. The 2-5A antisense strategy may hold a considerable advantage over the conventional antisense approach in targeting cancer-causing genes.

Adenine Nucleotides↗

PubMed2000

Identification of the promoter, genomic structure, and mouse ortholog of LGI1.

The human LGI1 gene is a leucine-rich, repeat-containing gene that was cloned from the t(10:19) breakpoint of the T98G glioblastoma cell line. The LGI1 gene maps to 10q24, a region of peak LOH in malignant gliomas, and is inactivated during the transition from low to high-grade brain tumors. Here we report detailed studies of the genomic structure of the LGI1 gene and its promoter. We have also cloned and characterized the mouse lgil gene, which is 97% homologous to the human gene at the amino acid level and 91% homologous at the nucleotide level. LGI1 contains 8 exons, where each of the four leucine-rich repeat units is contained in an individual 72-bp exon. The cysteine-rich regions flanking the LRR and the single trans-membrane domain do not occupy individual exons. Approximately 5-kb of the genomic region 5' to LGI1 was sequenced, but conventional CAAT and TATA motifs were not present within this sequence. A 597-bp fragment of this 5' sequence was cloned upstream of a promoterless luciferase gene and was shown to be sufficient to drive transcription. SSCP analysis of the coding region of LGI1 in 20 glioblastomas and five cell lines did not reveal any mutations. Because LGI1 expression is considerably downregulated in gliomas, we also investigated whether this was owing to changes in the methylation status of the promoter. Southern blot analysis and 5-azacytidine treatment did not show any appreciable difference in methylation status between normal brain and glioblastomas.

3T3 Cells↗

PubMed2000

Cloning and characterization of the TATA-less promoter from the human GFI1 proto-oncogene.

The growth factor independent 1 (GFI1) gene encodes a zinc finger protein which acts as a transcriptional repressor and confers growth factor independence on tumor cells, as suggested by the study of its mouse ortholog, Gfi1. We previously isolated the human GFI1 gene but no information about the structure and location of the promotor of this gene has been reported. In this study we have cloned and characterized the human GFI1 promoter. The nucleotide sequence of the promoter region is GC-rich and does not contain a typical TATA or CAAT box. Several Sp1 sites are present and computer predictions indicate that either of the two Sp1 sites might serve as the sites for transcription initiation. Analysis of various lengths of the promoter region using the luciferase reporter assay identifies a functional promoter that is active in NIH3T3 cells. The strongest activity lies within a region 312-602 basepairs upstream from the translation start site.

3T3 Cells↗

PubMed2000

Radiation-induced meningioma: a distinct molecular genetic pattern?

Radiation-induced meningiomas arise after low-dose irradiation treatment of certain medical conditions and are recognized as clinically separate from sporadic meningioma. These tumors are often aggressive or malignant, they are likely to be multiple, and they have a high recurrence rate following treatment compared with sporadic meningiomas. To understand the molecular mechanism by which radiation-induced meningioma (RIM) arise, we compared genetic changes in 7 RIM and 8 sporadic meningioma (SM) samples. The presence of mutations in the 17 exons of the neurofibromatosis type 2 (NF2) gene, which has been shown to be inactivated in sporadic meningiomas, was analyzed in RIM and SM using single-strand conformation polymorphism (SSCP) and DNA sequencing. In contrast to SM, which showed NF2 mutations in 50% of specimens, no mutations were found in RIM. In addition, Western blot analysis of schwannomin/merlin protein, the NF2 gene product, demonstrated protein levels comparable to normal brain in 4/4 RIM tumor samples analyzed. Loss of heterozygosity (LOH) of genomic regions, which were reported for SM, was also analyzed in all cases of RIM using 22 polymorphic DNA markers. Allele losses were found on chromosomes 1p (4/7), 9p (2/7), 19q (2/7), 22q (2/7), and 18q (1/7). From these observations we conclude that unlike sporadic meningiomas, NF2 gene inactivation and chromosome 22q deletions are far less frequent in RIM, and their role in meningioma development following low dose irradiation is less significant. Other chromosomal lesions, especially loss of 1p, possibly induced by irradiation, may be more important in the development of these tumors.

Adult↗

PubMed2000

A new region of synteny between human chromosome 1p22 and mouse chromosome 5.

By constructing a physical map of the p22 region of human chromosome 1 we have been able to show the relative orientation of four genes; GFI1, NB4S/EVI5, RPL5 and DR1. Analysis of the mouse physical map shows that the murine orthologs of these genes are located on mouse chromosome 5. Through this analysis we have established a new region of synteny between mouse chromosome 5 and human chromosome region 1p22.

Animals↗

PubMed2000

Genomic structure of the human tetratricopeptide repeat-containing gene, TTC4, from chromosome region 1p31 and mutation analysis in breast cancers.

Loss of heterozygosity (LOH) in 1p31 is a frequent genetic alteration in breast tumors indicating the site of a tumor suppressor gene. We recently isolated a new member of the human tetratricopeptide repeat-containing family of genes, TTC4, which maps to this region. Other members of this gene family have been implicated in tumorigenesis suggesting that TTC4 may represent a breast cancer tumor suppressor gene. We now report the exon/intron structure of TTC4 and single strand conformation polymorphism (SSCP) analysis of DNA from 20 sporadic breast tumors. Although polymorphic variations were identified no mutations affecting the open reading frame of TTC4 were detected. Since the overall region of chromosome 1p31 which undergoes LOH can be relatively large, excluding involvement of newly isolated genes from this region in breast cancer tumorigenesis is an important process for the successful identification of the critical gene. Understanding the structure of TTC4 now makes mutation analysis possible for other cancers and diseases that map to this region.

Breast Neoplasms↗

PubMed1999

Identification of a novel gene (ADPRTL1) encoding a potential Poly(ADP-ribosyl)transferase protein.

Poly(ADP-ribosyl)ation of nuclear proteins plays a significant role in the maintenance of genomic DNA stability. To date, four poly(ADP-ribosyl)ating proteins have been identified in humans. We now report the full-length sequence, expression profile, and chromosomal localization of a novel gene, ADPRTL1, encoding an ADP-ribosyltransferase-like protein. The predicted open reading frame encodes a protein of 1724 amino acids with a molecular mass of 192.8 kDa. The protein contains a region showing homology to the catalytic domains of the nuclear-localized ADP-ribosyltransferase proteins (Adprt), two recently identified Adprt-like proteins (Adprtl2 and Adprtl3), and the telomere-associated protein tankyrase. Key amino acids known to be important for the activity of these enzymes are conserved within this region of the Adprtl1 protein, indicating that Adprtl1 is a functional poly(ADP-ribosyl)transferase. As has been noted for tankyrase, sequence analysis of the Adprtl1 protein suggests that it is not capable of binding DNA directly. Thus, the transferase activity of Adprtl1 may be activated by other factors such as protein-protein interaction mediated by the extensive carboxyl terminus. We have subsequently refined the location of the ADPRTL1 genomic locus to 13q11, close to the recently cloned ZNF198 gene.

Amino Acid Sequence↗