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M A Knowles

Publications and source records attributed to M A Knowles.

At least 37 records · Page 2Linked to original sources
PubMed1999

Partial allelotype of schistosomiasis-associated bladder cancer.

In Egypt and other regions of the Middle East where the trematode Schistosoma haematobium is endemic, bladder cancer is the most common adult cancer. Unlike bladder cancers in Western countries, which are predominantly transitional-cell carcinoma (TCC), these schistosomiasis-associated bladder cancers are predominantly squamous-cell carcinoma (SCC). Our aim was to assess a large series of schistosomiasis-associated bladder tumours for genetic alterations commonly found in TCC in the United Kingdom and the United States. We have carried out a partial allelotype of 70 tumours from patients with schistosomiasis. LOH was found on all chromosome arms studied (3p, 4p, 4q, 8p, 9p, 9q, 11p, 11q, 13q, 14q, 17p, 18q). The most frequent regions of LOH were 9p (65%), 17p (58%), 3p (40%), 9q (39%) and 8p (37%). LOH on 17p, where the TP53 gene is located, was more common in Egyptian TCC than in SCC. Similarly, 8p LOH was more common in TCC than SCC. The most striking difference between this group of tumours and TCCs from the United Kingdom and the United States was the high frequency of 9p LOH in the region of the CDKN2 gene (65%) and the relatively low frequency of 9q LOH (39%); 15 of 43 tumours with LOH of at least one marker on chromosome 9 showed LOH of 9p only. This suggests that a 9p gene, possibly CDKN2, may contribute to the development of the majority of schistosomiasis-associated bladder tumours but that genes on 9q play a much less important role.

Adenocarcinoma↗
PubMed1999

Two novel regions of interstitial deletion on chromosome 8p in colorectal cancer.

We have investigated interstitial deletions of chromosome 8 in 70 colorectal carcinomas and 11 colonic adenomas using 11 microsatellite markers, including eight spanning the centromeric region of chromosome 8p (p11.2-p12). Allelic loss or imbalance was observed in 38 (54%) cancers and four (36%) adenomas. Twenty-eight (40%) of the cancers had deletions of 8p11.2-p12. Two distinct and independent regions of interstitial loss were found within this region. Fluorescent in situ hybridization, using an alpha satellite repeat probe to the centromere of 8p and two probes to the P1 region, was performed in four tumours that demonstrated allelic imbalance. Localized heterozygous deletions were confirmed in all four tumours. Eleven (16%) cancers had localized deletion in the region ANK-1 to D8S255 (P1) and a further eleven (16%) cancers had a less well localized deletion in the region defined by the markers D8S87 to D8S259 (P2). Loss of both centromeric loci was identified in a further six (9%) tumours. A functional significance for these two deletion regions was sought by correlation with primary and secondary tumour characteristics. Isolated P2 deletion was associated with 'early' T1 cancers (2p=0.0002), and were also identified in 3/11 adenomas. Conversely, interstitial deletions of the P1 locus were more frequently seen in 'locally invasive' T3/4 cancers (2p=0.015), and isolated P1 deletions were also associated with the presence of liver metastases (2p=0.016). Our data provide evidence of at least two genes within the 8p11.2-p12 region, mutations in which may confer different and independent roles in the pathogenesis of colorectal cancer.

Adenocarcinoma↗
PubMed1999

Identification of novel bladder tumour suppressor genes.

Many genetic alterations have recently been identified in transitional cell carcinoma (TCC) of the bladder. These include alterations to known proto-oncogenes and tumour suppressor genes and the identification of multiple sites of nonrandom chromosomal deletion which are predicted to define the location of as yet unidentified tumour suppressor genes. This review summarises recent efforts to define the location of novel bladder tumour suppressor genes using loss of heterozygositiy (LOH) and homozygous deletion analyses and to isolate the genes targeted by these deletions. For three of the four regions of deletion on chromosome 9, the most frequently deleted chromosome in TCC, candidate genes have been identified. It is anticipated that the identification of the genes and/or genetic regions which are frequently altered in TCC will provide useful tools for diagnosis, prediction of prognosis, patient monitoring and novel therapies.

Alleles↗
PubMed1999

Urological malignancies and the proteomic-genomic interface.

The urological malignancies, renal, bladder and prostate cancer, account for approximately 16% of all cancer cases. Unfortunately 5-year survival rates are relatively poor, largely a result of many cases not being diagnosed before the tumour has metastasised. There is a clear need for the identification of markers which will allow earlier detection of disease, and predict prognosis and response to therapy. In addition, they may be of use as therapeutic targets. Current advances in molecular biology are allowing the identification of a number of tumour-associated changes which could be of clinical use in the future. However, with the rapid technological advances being made in the field of proteomics, this approach could be integrated with genomics providing a complementary alternative, overcoming disparities between mRNA levels and protein production, and additionally allowing the identification of tumour-associated post-translational modifications. These approaches have already been used to identify novel genes and other cancer-related changes involved in the pathogenesis of urological malignancies. This review describes current progress in the genomic and proteomic study of urological malignancies, and highlights the potential of using proteomic technologies in the study of this group of diseases.

Genome↗
PubMed1999

Somatic mutation of PTEN in bladder carcinoma.

The tumour suppressor gene PTEN/MMAC1, which is mutated or homozygously deleted in glioma, breast and prostate cancer, is mapped to a region of 10q which shows loss of heterozygosity (LOH) in bladder cancer. We screened 123 bladder tumours for LOH in the region of PTEN. In 53 informative muscle invasive tumours (> or = pT2), allele loss was detected in 13 (24.5%) and allelic imbalance in four tumours (overall frequency 32%). LOH was found in four of 60 (6.6%) informative, non-invasive tumours (pTa/pT1). We screened 63 muscle invasive tumours for PTEN mutations by single-strand conformation polymorphism (SSCP) analysis and for homozygous deletion by duplex quantitative polymerase chain reaction (PCR). Two homozygous deletions were identified but no mutations. Of 15 bladder tumour cell lines analysed, three showed homozygous deletion of all or part of the PTEN gene, but none had mutations detectable by SSCP analysis. Our results indicate that PTEN is involved in the development of some bladder tumours. The low frequency of mutation of the retained allele in tumours with 10q23 LOH suggests that there may be another predominant mechanism of inactivation of the second allele, for example small intragenic deletions, that hemizygosity may be sufficient for phenotypic effect, or that there is another target gene at 10q23.

Carcinoma, Transitional Cell↗
PubMed1999

Homozygous deletion at the 9q32-33 candidate tumor suppressor locus in primary human bladder cancer.

Loss of heterozygosity (LOH) on chromosome arm 9q is the most frequent genetic alteration found in superficial and invasive transitional cell carcinoma (TCC) in a previous microsatellite-based deletion mapping study of the bladder and upper urinary tract, indicating the presence of one or more important tumor suppressor genes (TSGs). One of the putative tumor suppressor loci on 9q (DBC1) was mapped to 9q32-33 and the candidate region was localized within a single YAC. We report here a case of superficial papillary TCC, which showed a homozygous deletion encompassing this candidate tumor suppressor region. The region of homozygous deletion spanned the interval between D9S275 and AFMA239XA9 at 9q32-33, and was estimated to be </=6 cM. Although homozygous deletion mapping did not narrow down the candidate tumor suppressor region, this case provides further support for the presence of a TSG for TCC in this chromosomal region. To our knowledge, this is the first report to show homozygous deletion at 9q32-33 in TCC or any other type of human tumor. Genes Chromosomes Cancer 26:171-175, 1999.

Carcinoma, Transitional Cell↗
PubMed1998

Structure and methylation-based silencing of a gene (DBCCR1) within a candidate bladder cancer tumor suppressor region at 9q32-q33.

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, indicating the presence of one or more relevant tumor suppressor genes. We previously mapped one of these putative tumor suppressor loci to 9q32-q33 and localized the candidate region within a single YAC 840 kb in size. This locus has been designated DBC1 (for deleted in bladder cancer gene 1). We have identified a novel gene, DBCCR1, in this candidate region by searching for expressed sequence tags (ESTs) that map to YACs spanning the region. Database searching using the entire DBCCR1 cDNA sequence identified several human ESTs and a few homologous mouse. ESTs. However, the predicted 761-amino-acid sequence had no significant homology to known protein sequences. Mutation analysis of the coding region and Southern blot analysis detected neither somatic mutations nor gross genetic alterations in primary TCCs. Although DBCCR1 was expressed in multiple normal human tissues including urothelium, mRNA expression was absent in 5 of 10 (50%) bladder cancer cell lines. Methylation analysis of the CpG island at the 5' region of the gene and the induction of de novo expression by a demethylating agent indicated that this island might be a frequent target for hypermethylation and that hypermethylation-based silencing of the gene occurs in TCC. These findings make DBCCR1 a good candidate for DBC1.

Amino Acid Sequence↗
PubMed1997

Identification and characterization of the human homologue of SH3BP2, an SH3 binding domain protein within a common region of deletion at 4p16.3 involved in bladder cancer.

In a search for candidate tumor suppressor genes within a 30-kb common region of deletion previously identified in bladder cancer cell lines, we isolated a 2.4-kb cDNA clone comprising 13 exons that spanned approximately 16 kb of genomic DNA. Mutation analysis was carried out by single-strand conformation polymorphism analysis on DNA from 12 bladder carcinoma cell lines and 26 bladder tumors with LOH on chromosome 4p. Direct sequencing of the transcript in 4 bladder carcinoma cell lines with deletions in this region was also carried out. Two polymorphisms in exons 2 and 5 were identified, but no tumor-specific mutations were found. Sequence analysis identified a high degree of homology with the mouse sh3bp2 gene, which is abl-binding, suggesting that this gene is the human homologue. The predicted amino acid sequence of the putative gene product contains a Src homology 2 domain, a Src homology 3 binding domain, and a pleckstrin homology domain, suggesting a possible role in signal transduction. No evidence was found to indicate that SH3BP2 is the tumor suppressor gene at 4p16.3 involved in bladder cancer. However, this study has identified an interesting human gene that is a potential negative regulator of the abl oncogene.

Adaptor Proteins, Signal Transducing↗
PubMed1997

DAP-kinase loss of expression in various carcinoma and B-cell lymphoma cell lines: possible implications for role as tumor suppressor gene.

DAP-kinase is a novel calmodulin dependent serine/threonine kinase that carries ankyrin repeats and the death domain. It was recently isolated, by a functional selection approach of gene cloning, as a positive mediator of programmed cell death. In this study the expression of DAP-kinase was examined in the cell lines derived from various human neoplasms. DAP-kinase mRNA and protein expression were below the limit of detection in eight out of ten neoplastic derived B-cell lines. In six out of 14 examined bladder carcinoma, in three out of five renal cell carcinoma, and in four out of ten tested breast carcinoma cell lines, the DAP-kinase protein levels were below detection limits or lower than 1% compared to the positive cell lines. Interestingly, DAP-kinase expression could be restored in some of the negative bladder carcinoma and B-cell lines by treatment of cells with 5'-azadeoxycytidine that causes DNA demethylation. The high frequency of loss of DAP-kinase expression in human tumor cell lines, and the occasional involvement of methylation in this process raise the possibility that this novel mediator of cell death may function as a tumor suppressor gene.

Apoptosis Regulatory Proteins↗
PubMed1997

Mutation analysis of 8p genes POLB and PPP2CB in bladder cancer.

The DNA polymerase beta gene (POLB), which encodes a DNA polymerase believed to be involved in short gap-filling DNA synthesis, has been mapped to the proximal region of 8p (8p12-p11), a region commonly deleted in bladder carcinoma and a wide variety of other neoplasms. Also mapped to this region (8p12-p11.2) is the gene encoding the beta isoform of the catalytic subunit of protein phosphatase 2A (PPP2CB), a major serine/threonine phosphatase thought to play a regulatory role in many cellular pathways. The known functions of these proteins make them good candidates for 8p tumor suppressor genes. To test this hypothesis, we assessed a series of bladder tumors and bladder tumor cell lines for sequence variation in POLB and PPP2CB. Single strand conformation polymorphism (SSCP) analysis and direct sequencing of POLB cDNA derived from cell lines and tumors, many with known deletions of proximal 8p, revealed one sequence variant that was shown to represent a normal sequence polymorphism. No tumor-specific sequence variants were identified. The promotor sequence in genomic DNA from tumors with 8p LOH was also screened by SSCP. Four polymorphisms were identified but no tumor-specific mutations were found. PPP2CB was analyzed by SSCP analysis of all 7 coding exons in genomic DNA of bladder tumors and cell lines. Polymorphisms were detected in exons 4 and 5 but no tumor-specific mutations were found. We conclude that these genes are unlikely to be the suppressor genes for bladder cancer targeted by deletions of chromosome arm 8p.

Carcinoma, Transitional Cell↗
PubMed1997

A novel candidate tumour suppressor locus at 9q32-33 in bladder cancer: localization of the candidate region within a single 840 kb YAC.

Loss of heterozygosity (LOH) on chromosome 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder, implicating the presence of a tumour suppressor gene or genes on 9q. To define the location of a tumour suppressor locus on 9q in TCC, we screened 156 TCCs of the bladder and upper urinary tract by detailed deletion mapping using 31 microsatellite markers on 9q. Partial deletions of 9q were found in 10 TCCs (6%), and LOH at all informative loci on 9q was found in 77 TCCs (49%). In five low grade superficial bladder tumours, the partial deletion was localized to D9S195 located at 9q32-33, with retention of heterozygosity at all other informative loci including D9S103, D9S258, D9S275 and GSN. We constructed a yeast artificial chromosome (YAC) contig covering the deleted region in these five tumours and placed another four unmapped microsatellite markers on this contig map. Using these markers, we further defined the common deleted region to the interval between D9S1848 and AFMA239XA9. The region is covered by a single YAC (852e11), whose size is estimated to be 840 kb. Our data should expedite further fine mapping and identification of the candidate tumour suppressor gene at 9q32-33.

Animals↗
PubMed1996

Deletion mapping implicates two tumor suppressor genes on chromosome 8p in the development of bladder cancer.

Chromosome 8 loss of heterozygosity (LOH) in cancer of the urinary bladder is associated with high tumour grade and stage. We have screened 193 cases of transitional cell carcinoma (TCC) of the bladder using 30 microsatellite polymorphisms on chromosome 8. Forty three cases (22%) showed LOH on 8p, the majority of which (72%) had lost the entire short arm. Using 12 tumours with partial deletions of 8p, we have defined a minimum telomeric region of deletion between D8S264 and D8S133 (8p2l.1-pter). We also found LOH in the region 8pl1.2-12, where we have defined at least one centromeric target for deletion within a 4 cM interval. Two tumours were identified with loss of the telomeric region only, whereas all cases with loss in the centromeric region also had telomeric deletion. Chromosome 8p deletions have also been described in prostate, colorectal, hepatocellular, lung and endometrial carcinoma and collecting duct carcinoma of the kidney. It has been postulated that loss or inactivation of at least 2 tumour suppressor genes on 8p may play an important role in progression of both prostate and colorectal carcinomas. The regions of deletion we have identified in bladder tumours are compatible with these, suggesting that at least two genes on 8p may play a role in the development of many common solid tumours. Our findings significantly refine the localisation of the more centromeric of these loci.

Carcinoma, Transitional Cell↗
PubMed1996

Fluorescence in situ hybridization deletion mapping at 4p16.3 in bladder cancer cell lines refines the localisation of the critical interval to 30 kb.

An allelotype analysis of transitional cell carcinoma of the bladder identified loss of heterozygosity (LOH) on chromosome arm 4p in 22% of tumours. In a more detailed LOH study of 178 bladder carcinomas, a 750 kb common region of deletion was identified between the markers D4S43 and D4S127 just telomeric to the Huntington disease locus. To refine this region of deletion at 4p16.3, we have carried out detailed fluorescence in situ hybridisation (FISH) analysis of 12 bladder cancer cell lines by using a chromosome 4 centromeric probe combined with a series of cosmid probes from contigs spanning the 750 kb region of deletion. A common 30 kb region of deletion was identified at 4p16.3 in over one-third of the bladder cancer cell lines analysed. The present study has refined the localisation of the critical region of deletion from 750 kb to approximately 30 kb, providing a precise starting point for positional cloning of the gene(s) involved in bladder cancer from within a very gene-rich region on chromosome band 4p16.3. This study demonstrates that FISH can be used for fine deletion mapping of potential tumour suppressor gene regions. The utilisation of FISH analysis to map chromosomal deletions should facilitate positional cloning of other genes as bacterial artificial chromosome (BAC) and yeast artificial chromosome (YAC) contigs of the human genome are established.

Chromosome Deletion↗
PubMed1996

High frequency of chromosome 9 deletion in ovarian cancer: evidence for three tumour-suppressor loci.

We have screened 33 ovarian tumours of various grades and stages for the loss of heterozygosity (LOH) of markers on chromosome 9. LOH was detected in 26 cases (79%). Eleven tumours (33%) showed LOH of all informative markers. The remaining 15 cases had partial deletions. Of these, six (18%) had losses on 9p only, three (9%) had LOH confined to 9q and six (18%) had losses on both chromosome arms, four of which had a retention of hetereozygosity in between. There was no association between tumour grade stage or histopathology and any losses. High-density deletion mapping was carried out in 12 selected cases that had partial deletions of 9p and/or 9q. The deleted region on 9p included the cyclin-dependent kinase inhibitor 2 (CDKN2) locus and one tumour was found to have a homozygous deletion of CDKN2. LOH on 9q extended over a larger region. We found evidence for two regions of deletion on 9q, one at 9q34 and the other encompassing the nevoid basal cell carcinoma (Gorlin) syndrome locus on proximal 9q.

Adenocarcinoma↗
PubMed1995

Detailed deletion mapping of chromosome 9q in bladder cancer: evidence for two tumour suppressor loci.

Loss of heterozygosity (LOH) at loci on chromosome 9p and/or 9q is the most frequent genetic alteration in transitional cell carcinoma (TCC) of the bladder. However, localisation of the tumour suppressor locus or loci on 9q has been hampered by the relative infrequency of tumours with subchromosomal deletions. We have used 24 microsatellite markers to examine LOH in 70 new cases of TCC of the bladder and upper urinary tract. Forty tumours (57%) showed LOH at one or more loci on 9q and partial deletions were detected in five tumours (7%). Combined data from the five cases with partial deletions place one tumour suppressor locus at 9q34 between D9S61 and D9S66 (an estimated distance of 13-14 cM). This region is frequently deleted in other sporadic tumours and encompasses one of the loci for tuberous sclerosis (TSC1). One tumour contained a distinct deletion between D9S153 and D9S109 (9q13-q31), which encompasses the locus for the familial nevoid basal cell carcinoma syndrome (Gorlin syndrome). This may indicate the presence of another tumour suppressor locus on 9q for TCC. Our findings significantly reduce the regions of 9q within which suppressor genes for TCC may reside. The possible involvement of two deletion targets on 9q in addition to the locus at 9p21 implicated in TCC may explain why LOH at all loci on chromosome 9 is frequent in TCC.

Chromosome Mapping↗
PubMed1995

Deletion mapping of chromosome 11 in carcinoma of the bladder.

Deletions of the short arm of chromosome 11 have been identified by both cytogenetic and molecular criteria in bladder and other types of solid tumor, indicating the presence of one or more suppressor loci in this region. To localize the 11p deletion target(s) more precisely and to screen for loss of heterozygosity (LOH) on the long arm of the chromosome, 100 bladder tumors were analyzed for LOH on chromosome 11 using restriction fragment length polymorphisms (RFLPs) and microsatellite markers mapped to both 11p and 11q. Thirty-four tumors were found to have LOH at 1 or more loci. Of these, 17 had LOH restricted to 11p, 13 had LOH of both 11p and 11q, and 4 had LOH of 11q only. Eight tumors showed LOH at all informative loci indicating probable loss of an entire copy of chromosome 11. A common region of deletion was defined on 11p between D11S922 (11p15.5) and D11S569 (11p15.1-15.2). This region does not include the HRAS or WT1 loci (at 11p15.5 and 11p13, respectively). Seventeen tumors had LOH on 11q, 4 of which had LOH on 11q only. The common region of deletion on 11q was between FGF3 and D11S490 (11q13-q23.2). Two tumors showed LOH on both 11p and 11q with a clear region of retention of heterozygosity between, indicating the existence of two deletion targets on chromosome 11.

Alleles↗