Biomedical subjects

J K Cowell

Publications and source records attributed to J K Cowell.

At least 55 records · Page 3Linked to original sources

PubMed1998

Antisense telomerase treatment: induction of two distinct pathways, apoptosis and differentiation.

Telomerase, the enzyme that elongates telomeric DNA (TTAGGG)n, may be involved in cellular immortality and oncogenesis. To investigate the effect of inhibition of telomerase on tumor cells, we transfected the antisense vector against the human telomerase RNA into human malignant glioma cells exhibiting telomerase activity. After 30 doublings, some subpopulations of transfectants expressed a high level of interleukin-1beta-converting enzyme (ICE) protein and underwent apoptosis. In contrast, other subpopulations also showed enhanced ICE protein but escaped from apoptotic crisis and continued to grow, although their DNA synthesis, invasive ability, and tumorigenicity in nude mice were significantly reduced. Surviving cells demonstrated increased expression of glial fibrillary acidic protein and decreased motility, consistent with a more differentiated state. These cells also contained enhanced expression of the cyclin-dependent kinase inhibitors (CDKIs) p21 and p27. Treatment of surviving nonapoptotic cells with antisense oligonucleotides against p27, but not p21, induced apoptotic cell death, suggesting that p27 may have protected differentiating glioma cells from apoptosis. These data show that treatment with antisense telomerase inhibits telomerase activity and subsequently induces either apoptosis or differentiation. Regulation of these two distinct pathways may be dependent on the expression of ICE or CDKIs.

Animals↗

PubMed1998

Comparative genomic hybridization and its application to Wilms' tumorigenesis.

Eighty sporadic Wilms' tumor samples were analyzed by comparative genomic hybridization (CGH) to identify chromosomal regions involved in the etiology of the disease. Twenty percent of the samples showed chromosomal gains or losses. The majority of chromosomal gains and losses were similar to those identified through molecular and cytogenetic studies. Gains were observed on chromosomes 1q, 7q, 8, and 12, whereas losses were found on chromosomes 1p, 4p, 4q, 7p, 16q, 18q, 21q, and 22q. Other genetic aberrations identified in this study included deletions of chromosomes 5p and 15q, as well as gains of discrete loci on chromosomes 3p and 3q. These latter regions have not been previously implicated in Wilms' tumorigenesis and may contain novel genes relevant to the development and/or progression of this disease.

Chromosome Aberrations↗

PubMed1998

Characterization of the breakpoints in unbalanced t(5;11)(p15;p15) constitutional chromosome translocations in two patients with beckwith-wiedemann syndrome using fluorescence in situ hybridisation.

Although the majority of patients with Beckwith-Wiedemann syndrome (BWS) have a normal karyotype, the study of those rare patients with a cytogenetic abnormality has given considerable insight into the genetics of this condition. The karyotypic abnormalities found include partial chromosome duplications of paternal origin and maternally derived translocations which usually involve the 11p15 region and provide one of the lines of evidence for the location of the BWS gene(s). Because the extent of the duplicated region in these patients is variable, the phenotypic expression of BWS is presumably due to the presence of a common duplicated region. Two unrelated patients with BWS were both noted to have a similar unbalanced t(5;11)(p15;p14) translocation. The parents in both families were unaffected but both fathers carried a balanced translocation involving the same chromosomes. Since the extent and nature of the duplication apparently determines the complex phenotypes seen in these patients, we undertook a detailed analysis of the extent of the triplicated region using fluorescent in situ hybrisation (FISH). Despite having markedly different phenotypes and presenting in disimilar ways the two patients had apparently identical duplication breakpoints.

Beckwith-Wiedemann Syndrome↗

PubMed1997

Molecular characterization of the t(8;13)(p11;q12) translocation associated with an atypical myeloproliferative disorder: evidence for three discrete loci involved in myeloid leukemias on 8p11.

A reciprocal chromosome translocation between 13q12 and 8p11 is the consistent cytogenetic abnormality seen in a nonspecific myeloproliferative disorder that is associated with T-cell leukemia/lymphoma and peripheral blood eosinophilia. Detailed molecular analyses of the translocation breakpoints associated with this rearrangement have not been reported to date. We have now generated somatic cell hybrids from a newly described patient with this specific structural rearrangement and analyzed the breakpoints on the derivative chromosomes. We have shown that the breakpoint on chromosome 13 lies within a 300- to 500-kb region defined by the KIAA177 gene and D13S1123 marker. In addition, we have identified a 1.2-Mb YAC, 959A4, that crosses the translocation breakpoint on the short arm of chromosome 8 in this patient. The location of this breakpoint in 8p11 is distinct from the t(8;16) and t(8;22) translocations associated with M4/M5 myeloid leukemias, and suggests that three distinct loci located within 8p11 are involved in the pathogenesis of myeloid neoplasias.

Adult↗

PubMed1997

Direct isolation of human transcribed sequences from yeast artificial chromosomes through the application of RNA fingerprinting.

The identification of cDNA clones from genomic regions known to contain human genes is usually the rate-limiting factor in positional cloning strategies. We demonstrate here that human genes present on yeast artificial chromosomes (YACs) are transcribed in yeast host cells. We have used the arbitrarily primed RNA (RAP) fingerprinting method to identify human-specific, transcribed sequences from YACs located in the 13q12 chromosome region. By comparing the RAP fingerprints generated using defined, arbitrary primers from various fragmented YACs, megaYACs, and host yeast, we were able to identify and map 20 products transcribed from the human YAC inserts. This method, therefore, permits the simultaneous isolation and mapping of novel expressed sequences directly from whole YACs.

Chromosomes, Artificial, Yeast↗

PubMed1997

Cloning of the human Gfi-1 gene and its mapping to chromosome region 1p22.

Recently the rat and mouse Growth Factor Independence (Gfi-1) genes have been cloned (Gilks et al., 1993; Zoring et al; 1996). This gene allows cells in culture to overcome the depletion of growth factors in the culture medium and maintain their proliferative potential. As part of a cloning strategy to isolated genes from human chromosome 1p22 which are associated with a constitutional chromosome translocation from a patient with stage 4S neuroblastoma, we have identified the human homologue of the Gfi gene and defined a 50 Kb map position within a well characterised YAC contig from the region. The full length cDNA sequence is 81% homologous with the rodent counterparts and, at the protein level, is even more highly conserved.

Amino Acid Sequence↗

PubMed1997

The use of SCID mice for the growth of retinoblastoma cell lines and for the establishment of xenografts from primary tumours.

We investigated the possibility of growing primary retinoblastoma tumour tissue in SCID mice. In preliminary experiments with the WERI retinoblastoma cell line injected subcutaneously in SCID mice, tumours arose at only 25% of the inoculation sites. After mixing these cells with a reconstituted basement membrane extracellular matrix (Matrigel) prior to inoculation, tumours arose at 100% of inoculated sites. When primary retinoblastoma cells were injected subcutaneously into SCID mice in the presence of Matrigel, tumours arose in 5/8 cases. On average, the latency period was 4 months before the tumours were palpable. Histopathological examination of the tumours showed that they resembled surgically resected human retinoblastomas and one of the tumours formed pseudo-rosettes which is a characteristic of these tumours. Unfortunately, when these xenografted tumours were introduced into tissue culture, it was not possible to establish cell lines directly and the cultures were soon overgrown by mouse cells which could clearly be shown to be infiltrating the tumour. The ability to grow retinoblastoma cell lines and primary tissue subcutaneously in SCID mice offers a convenient model system to study the genetics of tumorigenesis in this tumour type and possibly an opportunity to study the role of chemotherapy in the treatment and progression of the disease.

Animals↗

PubMed1997

Fine structure physical mapping of a 1.9 Mb region of chromosome 13q12.

Through linkage analysis and the identification of structural chromosome rearrangements, a number of disease genes have been mapped to the pericentromeric region of the long arm of chromosome 13. Structural rearrangements, or deletions, of the 13q12 region have been implicated in a range of myeloproliferative neoplasms, and other haematopoietic malignancies. In particular, seven cases of a t(8;13)(p11;q12.1) rearrangement have been noted in patients with an atypical myeloproliferative disorder associated with T-cell leukemia and eosinophilia. We have previously identified a CEPH mega YAC, 943E4, which crosses the translocation breakpoint in archival tumour samples from two patients with this t(8;13) translocation. As an initial step in the characterisation of this translocation breakpoint, we have generated a fine structure physical map of this 1.9 Mb YAC. We have used the method of YAC fragmentation to generate a series of deletion constructs of known size, which provide discreet physical landmarks convenient for mapping genetic markers along the 943E4 YAC. Analysis of these deletion constructs defined the order of ESTs and microsatellite markers in 943E4 as: cen-NIB1257-(ATP1AL1/D13S283)-D13S179E-(D13S5 04E/D13S505E)-D13S824E-D13S182E -D13S221-tel. These markers have also been assigned to physically defined regions relative to the fragmented YAC endpoints and a derived NotI restriction map.

Base Sequence↗

PubMed1996

Regional localization of 192 genic markers on human chromosome 1.

A panel of somatic cell hybrids has been used to localize 192 novel eSTS markers to seven individual subregions of human chromosome 1. The positions of the breakpoints in each of these hybrids have been determined relative to the genetic linkage map of chromosome 1, and so the approximate locations of the genes from which the eSTS markers have been derived can be determined. Although the distribution of the eSTS markers is relatively random, 23% were assigned to the 1p34-p36 region. The hybrid mapping panel does not subdivide the 1q24-q44 region, which contains 36% of the eSTS markers. This analysis, therefore, provides a series of genic markers in which to search for candidates for a variety of human genetic disorders and recessive oncogenes mapped to the same relative position on the chromosome.

Chromosome Mapping↗

PubMed1996

A novel mutation in the promotor region in a family with a mild form of retinoblastoma indicates the location of a new regulatory domain for the RB1 gene.

We describe a family segregating the retinoblastoma phenotype where the affected individuals have only unifocal tumours and where linkage analysis has identified unaffected mutant gene carriers. DNA from members of this 'low penetrance' pedigree was subjected to an exon-by-exon SSCP analysis of the RB1 gene. No mutations were found in the 27 exons of the coding region but an SSCP band shift was seen for PCR products covering the RB1 promoter region. Sequencing identified a G-->C change within a GGGCGG motif which is the core of the recognition sequence of the SP1 transcription factor. Electromobility shift assays demonstrated that SP1 does not bind to oligomers from this region of the RB1 promoter but bandshifts were seen for an, as yet, unidentified protein(s) which was not seen using an oligomer containing the G-->C mutation. Thus, identification of a naturally occurring mutation in a family with only 'mild' phenotypes has identified another regulatory sequence in the RB1 promoter which binds an endogenous cellular protein(s). Identification of this protein should allow a better understanding of the control of expression of the RB1 gene.

Base Sequence↗

PubMed1996

The expression pattern of Wilms' tumour gene (WT1) product in normal tissues and paediatric renal tumours.

WT1, the Wilms' tumour suppressor gene located at chromosome 11p13, plays an important role in the development of the kidney. It is mutated in 10 per cent of Wilms' tumours (WTs) and their putative precursors called nephrogenic rests (NRs). A sensitive immunohistochemical technique was established to localize the WT1 gene product in archival normal tissues and paediatric renal tumour samples. Strong nuclear signal was seen in the various sites such as the kidneys, gonads, and decidua. Nuclear immunoreactivity of variable intensity was also seen in the skeletal muscle, smooth muscle of urinary bladder, ureter, and arteries. All 28 WTs (including the anaplastic variant) showed the WT1 gene product in a varying proportion of the blastema, epithelium, and stroma. The anaplastic nuclei in six WTs showed an intensity of staining comparable to their adjacent favourable histology counterparts. All the intralobar and perilobar types of NR demonstrated the WT1 gene product. All three malignant rhabdoid tumours were positive, while three of four mesoblastic nephromas and a clear cell sarcoma were negative. These findings provide an insight into the interrelationships of these renal tumours.

Child↗

PubMed1996

Constitutional nonsense germline mutations in the RB1 gene detected in patients with early onset unilateral retinoblastoma.

The 'two-hit' hypothesis for the development of the childhood eye cancer, retinoblastoma (Rb), predicts that bilaterally affected individuals will carry germline mutations. The second suggestion is that patients with early presentation of unilateral tumours also carry predisposing mutations. We have used SSCP analysis to study the 27 individual exons of the RB1 gene in constitutional DNA from 3 patients whose tumours were treated under the age of 12 months. Bandshifts on SSCP gels were detected in 2 of these patients which, on sequencing, were shown to be a C-->T transition converting a CGAarg to a TGAstop codon in exon 17 and an 8 bp deletion in exon 20 resulting in a downstream stop codon. The mutations seen in these patients are reminiscent of those seen in patients with hereditary Rb and confirms that at least some early onset unilateral cases carry constitutional mutations, which has important implications for genetic screening and counselling of these individuals.

Base Sequence↗

PubMed1996

Characterisation of a human chromosome 1 somatic cell hybrid mapping panel and regional assignment of 6 novel STS.

A somatic cell hybrid mapping panel has been constructed which allows subdivision of human chromosome 1 into 8 distinct subregions. All of the hybrids carry copies of chromosome 1 with specific deletions and the position of the breakpoints has been determined relative to the location of microsatellite markers in the genetic linkage map produced by Genethon. The majority of the breakpoints can be positioned between adjacent loci on the map. The usefulness of this hybrid panel for physical mapping has been demonstrated by the regional assignment of 6 novel STS markers made from Alu-PCR clones generated from a hybrid which contains the short arm of chromosome 1.

Chromosome Breakage↗

PubMed1996

Regional assignment of EST sequences on human chromosome 13.

We have used a panel of somatic cell hybrids carrying structural rearrangements of human chromosome 13 to regionally localise 10 expressed sequence tags (ESTs) from this chromosome. Three of these genes were not present in the somatic cell hybrid PGME, which contains chromosome 13 as its major human component. Using a panel of somatic cell hybrids, two of these genes were localised to chromosome 8 and one to chromosome 16. Using the PCR primers as published, it was impossible to map one EST; consequently, a YAC containing this gene was isolated, and fluorescence in situ hybridisation was used to map the gene to 13q14. The distribution of the remaining six chromosome 13-specific ESTs was nonrandom; one mapped to 13q14 and the remaining five to 13q12, with three mapping within the proximal portion and two mapping within the distal portion of this region.

Chromosome Mapping↗

PubMed1995

A yeast artificial chromosome contig that spans the RB1-D13S31 interval on human chromosome 13 and encompasses the frequently deleted region in B-cell chronic lymphocytic leukemia.

Abnormalities involving chromosome 13 have been reported as the only cytogenetic change in B-cell chronic lymphocytic leukemia (BCLL). Deletions are the most common cytogenetic abnormality and always involve 13q14, but when translocations are seen, the consistent breakpoint is always in 13q14. It is now established that deletions, distal to the RB1 gene in 13q14, are invariably associated with these translocations. We have recently described the smallest such deletion from a series of rearrangements from these tumors isolated in somatic cell hybrids, which spans approximately 1 Mb. In this report, we present the results of a series of a chromosome walking experiments using YACs and have been able to span this small deletion, which must contain the gene that is frequently deleted in BCLL. Four probes from 13q14 (RB1-mgg15-D13S25-D13S31) were used to isolate corresponding YACs for each of the markers. The chromosomal location of these YACs was verified using FISH, which also demonstrated their nonchimeric nature. Vectorette end rescue was then used to demonstrate the overlap of the YACs and to isolate new clones to complete the contig. The extremes of the contig were shown to cross the chromosome 13 translocation breakpoints isolated in somatic cell hybrids that carry the derivatives of chromosome 13 involved in the smallest BCLL deletion. This YAC contig covers the entire deletion and will prove a valuable resource to begin isolating genes from this region. In addition, we have isolated YACs corresponding to the RB1 locus, which extends the contig over a 3.8-cM distance on the chromosome.

Base Sequence↗

PubMed1995

Independent constitutional germline mutations occurring in the RB1 gene in cousins with bilateral retinoblastoma.

The inheritance of a genetic susceptibility to the development of retinoblastoma generally follows an autosomal mode of inheritance with high penetrance. Rare families, however, show evidence of incomplete penetrance where individuals can transmit the mutant gene without being affected themselves. In these families formal proof of this dogma requires the identification of the predisposing mutation. In this study we have identified the mutations in cousins with bilateral (hereditary) disease. Using SSCP and DNA sequencing, different constitutional mutations were detected in the affected cousins in this pedigree. One cousin carries a C-->T mutation in exon 8 generating a stop codon directly which was also present in his affected mother whereas the other cousin carries an 8 base pair deletion in exon 20. Neither half of the family carried the same mutation as the other. The mother of the patient with the 8 bp deletion carried neither of the mutations. Thus, we have demonstrated that the retinoblastomas in this family have developed as a result of independent, sporadic genetic events which occurred coincidentally in the same extended family rather than being due to a common mutation which manifests as incompletely penetrant. These observations have important implications for genetic counselling in this type of family.

Base Sequence↗

PubMed1995

Integration of the physical and genetic linkage map for human chromosome 13.

We have used a panel of somatic cell hybrids containing different rearrangements of human chromosome 13 to integrate genetic and physical maps of this chromosome. The positions of 17 translocation/deletion breakpoints on human chromosome 13 have been determined relative to the microsatellite markers on the genetic linkage map compiled by Généthon. Because markers on maps from several other Consortium groups have also been analyzed using many of the same hybrids, it was possible to relate these with the Généthon map. The position of all of the chromosome breakpoints have been placed, wherever possible, between two adjacent markers on the genetic linkage maps using PCR analysis for the presence/absence of the markers in the somatic cell hybrids. The positions of the breakpoints have already been determined cytogenetically, and some of these breakpoints are located at landmark positions on the chromosome. The relative density of markers along the chromosome differs between independently derived maps, and, based on the known locations of certain breakpoints in the physical map, inconsistencies in the genetic maps have been identified.

Chromosome Banding↗

PubMed1995

Identification of YAC clones for human chromosome 1p32 and physical mapping of the infantile neuronal ceroid lipofuscinosis (INCL) locus.

Infantile neuronal ceroid lipofuscinosis (INCL, CLN1) is a neurodegenerative disorder in which the biochemical defect is unknown. We earlier assigned the disease locus to chromosome 1p32 in the immediate vicinity of the highly informative HY-TM1 marker by linkage and linkage disequilibrium analysis. Here we report the construction of PFGE maps on the CLN1 region covering a total of 4 Mb of this relatively poorly mapped chromosomal region. We established the order of loci at 1p32 as tel-D1S57-L-myc-HY-TM1-rlf-COL9A2-D1S193-D1S6 2-D1S211-cen by combining data obtained from analysis of a chromosome 1 somatic cell hybrid panel, PFGE, and interphase FISH. We isolated YACs and constructed two separate YAC contigs, the loci L-myc, HY-TM1, rlf, and COL9A2 being present on a 1000-kb contig and the markers D1S193, D1S62, and D1S211 on a YAC contig spanning a maximum of 860 kb. Within the 1000-kb contig we were able to identify five CpG islands in addition to those associated with the earlier cloned genes. The YAC contigs as well as the physical map provide us with tools for the identification of the INCL gene.

Base Sequence↗