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M A Knowles

Publications and source records attributed to M A Knowles.

At least 55 records · Page 3Linked to original sources
PubMed1995

p16 (CDKN2) is a major deletion target at 9p21 in bladder cancer.

The p16 gene has been identified as a candidate tumour suppressor gene at 9p21, a region commonly deleted in bladder cancer. We screened 140 bladder tumours and 16 cell lines for deletions and sequence variants of p16. Eight cell lines showed homozygous deletion of p16 and two had small sequence variations. All 13 tumours with small defined deletions of 9p21, 18/31 (58%) of tumours with monosomy 9 and 9/91 (10%) of tumours with no chromosome 9 loss of heterozygosity had homozygous deletion of p16. No tumour-specific sequence variants were identified. Deletion mapping revealed a nested set of deletions focused on p16. Six deletions involved p16 but not the related and adjacent gene p15 and one tumour had an intragenic deletion of p16. All other deletions involved both p16 and p15. We conclude that p16 represents the major target for deletion at 9p21 in bladder cancer.

Base Sequence↗
PubMed1994

Allelotype of human bladder cancer.

To identify common regions of deletion in human bladder tumors, we have screened 83 cases of transitional cell carcinoma for loss of heterozygosity (LOH) on all autosomal chromosome arms. Seventy-two restriction fragment length polymorphism, variable number of tandem repeats, and minisatellite markers and 18 microsatellite markers were used to obtain a minimum of 50% informative results for each chromosome arm. A mean of 29.6 informative results per patient was obtained from 39 chromosome arms studied, representing information for 76% of chromosome arms. The most frequent losses were apparent monosomies of chromosome 9 (9p, 51%; 9q, 57%). Other frequent losses were on chromosomes 11p (32%), 17p (32%), 8p (23%), 4p (22%), and 13q (15%). LOH of 4p has not been reported previously in bladder carcinoma. The frequency of LOH on all other chromosome arms was < 12%. LOH on chromosome 8p showed a significant association with both high tumor grade and stage, and LOH on 13q showed a significant association with high tumor grade. Fractional allelic loss was calculated for all tumors and had a mean of 0.125 and a median of 0.110. A significant association was found between increased fractional allelic loss and higher tumor grade. An association was also found between LOH of chromosomes 8p and 9q and values for fractional allelic loss > or = the median value. No associations were found between LOH on different pairs of chromosome arms.

Alleles↗
PubMed1994

The spectrum of TP53 mutations in bladder carcinoma.

The mutational spectrum for the TP53 gene was investigated in a large series of bladder tumors and bladder tumor cell lines. Tumors and cell lines were screened for the presence of TP53 point mutations by single-strand conformational polymorphism analysis followed by direct sequencing. Mutations were detected in 16 of 88 (18%) tumors and 4 of 14 cell lines (28%). In total, twelve missense mutations, one nonsense mutation, three deletions, and two insertions were identified by direct sequencing. Of the thirteen point mutations sequenced, only one was a transition at a CpG site, whereas five G:C-->T:A transversions were found, suggesting a major role for exogenous mutagens in bladder tumorigenesis. Tumors were also examined for loss of heterozygosity (LOH) on chromosome arm 17p. LOH of one or more markers on 17p was detected in 31% of tumors. All eight tumors with a TP53 mutation from patients informative at TP53 had LOH, whereas nine tumors with LOH at TP53 did not have an identified mutation. Three tumors had LOH on 17p at sites distal to the TP53 locus but retained both TP53 alleles, suggesting the involvement of another tumor suppressor gene on 17p in bladder tumorigenesis in some tumors.

Alleles↗
PubMed1994

Kainic acid and 4-aminopyridine seizure models in mice: evaluation of efficacy of anti-epileptic agents and calcium antagonists.

Seizures may be induced in mice in response to stimulation of subtypes of glutamate receptors by kainic acid or inhibition of certain voltage-dependent potassium channels by 4-aminopyridine (4-AP). The anti-seizure efficacy of intraperitoneally administered anticonvulsants and Ca++ antagonists to CF-1 mice was tested using these models. The order of potency for prevention of kainate convulsions and the subsequent lethality was: dihydropyridine Ca++ antagonists (nicardipine, nisoldipine > nitrendipine > nifedipine > nimodipine) followed by verapamil > prenylamine > diltiazem > flunarizine > remacemide HCl > ethosuximide > valproate. In the 4-AP model the order of potency to prevent hind limb tonic extension was: MK801(+/-) > lamotrigine > phenytoin, phenobarbital > carbamazepine > FPL 12495AA (the desglycine metabolite of remacemide HCl), remacemide HCl > flunarizine > prenylamine >>> valproate. Therefore, compounds that limit activation of kainate receptors and voltage-operated linked calcium channels are active in the kainate model. Agents effective against maximal electroshock appear to be effective in the 4-AP model.

4-Aminopyridine↗
PubMed1994

Pre-septal cellulitis and facial erysipelas due to Moraxella species.

We describe a patient with an acute ulcerated pre-septal cellulitis with subsequent spread across the face, in whom conjunctival swabs demonstrated the presence of Moraxella species. Unusually, this organism was resistant to penicillin. Clinical improvement was only achieved when treatment with co-amoxiclav was commenced on the basis of the antibiotic sensitivities demonstrated by culture of this organism. Moraxella species are rare skin pathogens but may be underestimated as they usually respond to a wide range of antibiotics; they should be considered as a cause of facial cellulitis or erysipelas which responds poorly to conventional therapy.

Adult↗
PubMed1994

Deletion of two regions on chromosome 4 in bladder carcinoma: definition of a critical 750kB region at 4p16.3.

Loss of heterozygosity (LOH) of chromosome 4 has been identified in several human tumours including carcinomas of colorectum, ovary, liver and head and neck. LOH at loci on chromosome 4p has previously been identified in 22% of primary bladder tumours. We have assessed LOH in 178 bladder tumours using a panel of six microsatellite markers. Thirty-four tumours (19%) showed LOH at one or more loci. Twenty-three deletions involved restricted regions of 4p and could be used to define two common regions of deletion. A very small common region between D4S43 and D4S127 (estimated to be approximately 750 kB) was involved in 14/23 (61%) of 4p deletions. A second common region centromeric to D4S174 was deleted in 7/23 (30%) of tumours with deletions. Two tumours (9%) had deletions involving both regions independently. Previous functional studies have demonstrated both a senescence function and a suppressor of tumorigenicity on human chromosome 4. Localisation of the common regions of deletion in bladder tumours provides a starting point for positional cloning of the gene(s) concerned and for more precise comparative functional studies.

Chromosome Deletion↗
PubMed1994

Homozygous deletion mapping at 9p21 in bladder carcinoma defines a critical region within 2cM of IFNA.

We have screened 165 cases of primary transitional cell carcinoma of the bladder for loss of heterozygosity of markers on chromosome 9. High density deletion mapping was then carried out on a panel of 16 tumours with partial deletions of 9p using 15 microsatellite markers. Seven tumours had hemizygous deletions which included small interstitial losses and large terminal deletions. Homozygous deletions of one or more markers were identified in the remaining 9 tumours. By comparing the limits of hemi- and homozygous deletions in these tumours we have defined a single common region of 2cM flanked by IFNA and D9S171.

Chromosome Deletion↗
PubMed1994

Definition of two regions of deletion on chromosome 9 in carcinoma of the bladder.

Loss of heterozygosity (LOH) at loci on chromosome 9 can be detected in more than 50% of bladder tumours, suggesting the presence on this chromosome of one or more suppressor genes for bladder carcinogenesis. Localisation of the target gene(s) by deletion mapping has previously proved difficult due to the uneven distribution of polymorphic loci and the finding of LOH at all these loci in the majority of tumours. We have used a panel of 22 highly informative microsatellite markers, evenly distributed along chromosome 9 to analyse LOH in 95 cases of primary transitional cell carcinoma of the bladder. Forty nine tumours (53%) showed LOH at one or more loci. Of these, 30 had LOH at all informative loci, indicating probable monosomy 9. Nineteen tumours (22%) had subchromosomal deletions, 5 of 9p only, 9 of 9q only and 5 of both 9p and 9q with a clear region of retention of heterozygosity between. The patterns of LOH in these tumours indicated a common region of deletion on 9p between D9S126 (9p21) and IFNA (9p21). A single tumour showed a second site of deletion on 9p telomeric to IFNA indicating the possible existence of 2 target genes on 9p. All deletions of 9q were large, with a common region of deletion between D9S15 (9q13-q21.1) and D9S60 (9q33-q34.1). These results indicate a much higher frequency of subchromosomal deletion than has previously been detected and provide evidence for the simultaneous involvement of distinct suppressor loci on 9p and 9q in bladder carcinoma.

Carcinoma, Transitional Cell↗
PubMed1993

Preliminary mapping of the deleted region of chromosome 9 in bladder cancer.

Inactivation of a suppressor gene by deletion of chromosome 9 is a candidate initiating event in bladder carcinogenesis. We have used 13 polymorphic markers spanning the length of chromosome 9 in order to map the region of deletion in human bladder carcinomas. In the majority of tumors loss of heterozygosity was found at all informative sites along the chromosome, indicating deletion of the entire chromosome. Nine tumors had selective deletions of chromosome 9. Mapping of the deleted region in these tumors suggests that the target gene is located between D9S22 at 9q22 and D9S18 at 9p12-13.

Chromosome Deletion↗
PubMed1993

Loss of RB protein expression in primary bladder cancer correlates with loss of heterozygosity at the RB locus and tumor progression.

RB-protein status as determined by immunohistochemical analysis was compared with loss of heterozygosity (LOH) at the RB locus in 68 primary transitional-cell carcinomas of the bladder. Absence of RB-protein expression was found in 15 of 17 tumors in which LOH at the RB locus was identified, whereas 31 of 36 tumors from informative patients which showed no LOH had a normal RB-protein pattern (p < 0.001). Altered RB-protein expression was also more frequently seen in muscle-invasive and high-grade tumors (p < 0.003 and < 0.005, respectively). Our results indicate that LOH at the RB locus is highly correlated with loss of RB-protein expression in primary bladder carcinomas and further strengthens the notion that loss of RB function may be associated with more aggressive bladder tumors.

Carcinoma, Transitional Cell↗
PubMed1993

Mutation of H-ras is infrequent in bladder cancer: confirmation by single-strand conformation polymorphism analysis, designed restriction fragment length polymorphisms, and direct sequencing.

A series of 152 human bladder tumors, 14 bladder tumor cell lines, and 1 immortal urothelial cell line were examined by single-strand conformation polymorphism (SSCP) and designed restriction fragment.length polymorphism analyses for mutations in exons 1 and 2 of the H-ras gene. Nine tumors (6%) contained mutations. There was complete concordance between SSCP and restriction fragment length polymorphism analyses. Six mutations in exon 1 and three in exon 2 were identified by SSCP analysis. Subsequent restriction fragment length polymorphism analysis showed that of the exon 1 mutations, four were in codon 12 and two in codon 13, and all three exon 2 mutations were in codon 61. Eight mutations were confirmed by direct sequencing. One codon 13 mutation could not be identified by direct sequencing. Distinct strand mobility shifts detected by SSCP analysis identified specific point mutations, and in all cases, strands containing different mutations migrated differently. The base substitutions identified in these bladder tumors were diverse and included four transversions (three G-->T and one A-->T) and four transitions (two G-->A and two A-->G). This pattern of base substitutions is compatible with interactions of the urothelium with more than one class of environmental agent during bladder tumor development. No correlation was found between tumor grade and/or stage and the presence of H-ras mutation. We conclude that H-ras mutation does not play a role in the development of the majority of transitional cell tumors of the bladder.

Base Sequence↗
PubMed1993

Reduced expression of TGF beta is associated with advanced disease in transitional cell carcinoma.

The gene structure and expression of the related peptide regulatory factors TGF beta 1 and TGF beta 2 were studied in a panel of seven urothelial carcinoma cell lines and 40 transitional cell carcinomas. The latter comprised 15 grade 1, 18 grade 2 and 5 grade 3 tumours and two cases of carcinoma in situ. Control tissues included ten matched 'field' biopsies and 17 other biopsies including 11 biopsies of macroscopically normal urothelium, two of which were from patients with no history of bladder cancer. No amplification of rearrangements of either TGF beta 1 or TGF beta 2 were detected in any sample. A complex pattern of expression or the two genes was found in the urothelial cell lines. High, but variable levels of the 2.5 kb TGF beta 1 transcript were detected and lower and more variable levels of the three (4.1 kb, 5.1 kb and 6.5 kb) transcripts of TGF beta 2 were detected. Although those cell lines expressing most TGF beta 1 tended to express less TGF beta 2 transcript there was no clear-cut relationship. In comparison, no TGF beta 2 transcript was identified in any primary transitional cell carcinoma or control tissue. Markedly reduced or undetectable levels of TGF beta 1 transcript were detected in 4/15 (26%) grade 1, 5/18 (28%) grade 2 and 3/5 (60%) grade 3 tumours. There was no clear relationship to tumour stage, lymphocytic infiltration or stromal content of the tumours. Clinical review one year after the 2 year period of tumour collection showed that 6/9 (66%) of patients with tumours with reduced levels of transcript had died or had disease which was not controllable by local resection and 3/9 (33%) had developed tumour re-occurrences. In comparison, in the group with normal levels of expression of TGF beta 1, 3/18 (17%) had disease which was not controllable by local means, 9/18 (50%) had tumour re-occurrence and 6/18 (33%) had no evidence of disease. The association of reduced expression of TGF beta 1 and advanced disease was statistically significant P < 0.02 (Fisher's test). Although the sample size is small, we suggest that the loss of expression of TGF beta 1 may be a potential marker of progressive disease or prognosis in transitional cell carcinoma and warrants further study.

Blotting, Northern↗
PubMed1993

Initiation of bladder cancer may involve deletion of a tumour-suppressor gene on chromosome 9.

Although many genetic lesions including suppressor gene inactivation have been identified in sporadic tumours, the identity of the common initiating or early genetic events in most cases remains obscure. We have analysed tumour and leucocyte DNA from a series of 252 primary human bladder tumours of all grades and stages for deletions of chromosome 9. Probes from four polymorphic loci were used to detect loss of heterozygosity (LOH) in tumour specimens. Fifty-nine per cent of tumours from patients informative at one or more of these loci showed LOH. A comparison of LOH for markers on 9p and 9q indicated that proximal 9p or 9q is the likely site for a candidate bladder tumour-suppressor gene. Most strikingly, LOH was observed not only in tumours of high grade or stage but also in > 50% of grade 1 and 2 superficial (pTa) tumours. This is the first change to be identified at significant frequency in such tumours. Inactivation of a tumour-suppressor gene on chromosome 9 may therefore be an early genetic event in the development of bladder cancer.

Chromosome Deletion↗
PubMed1993

Deletion mapping of chromosome 8 in cancers of the urinary bladder using restriction fragment length polymorphisms and microsatellite polymorphisms.

We have used a combination of restriction fragment length polymorphism (RFLP) markers and highly informative microsatellite polymorphisms to map a common region of deletion on chromosome 8p in cancers of the urinary bladder. Analysis of loss of heterozygosity (LOH) using microsatellite polymorphisms was shown to be at least as sensitive as detection of RFLPs by Southern blotting. A total of 110 tumours was analysed for loss of heterozygosity (LOH) on 8p and 8q; 109 patients were informative for at least one marker on each chromosome arm and 29 tumours (26%) showed LOH of chromosome 8 markers, 26 of which (25%) showed LOH on 8p. Sixteen tumours (14%) showed LOH on 8q. Thirteen of these also had LOH on 8p. Of the 29 tumours with LOH, five had LOH at all informative loci, indicating loss of an entire copy of chromosome 8. An association was found between high tumour grade and stage and chromosome 8 LOH. Fifty-three per cent of grade 3 muscle-invasive tumours showed LOH compared with 11% of grade 1 non-invasive tumours (0.01 < P < 0.025 and 0.025 < P < 0.05 for grade and stage respectively). Deletion mapping of tumours with chromosome 8 LOH suggests the presence of a suppressor gene(s) for urothelial cancer within a region defined by the loci NEFL and PLAT (8p21-q11.2). If there is a common target for deletions in bladder and those in hepatocellular and colorectal tumours reported previously, this defines a common region of deletion at 8p21.3.

Base Sequence↗
PubMed1992

Human papillomavirus sequences are not detectable by Southern blotting or general primer-mediated polymerase chain reaction in transitional cell tumours of the bladder.

A large series of transitional cell tumours has been screened for the presence of human papillomavirus (HPV) sequences using Southern blotting and general primer-mediated polymerase chain reaction (GP-PCR). The latter technique allows the detection of a broad spectrum of both sequenced and unsequenced HPV types using two primer pairs located in the highly conserved L1 and E1 regions of the HPV genome. No evidence for HPV infection was found in 100 transitional cell tumours, 6 cases of carcinoma in situ, 2 adenocarcinomas and a squamous carcinoma of the bladder and 3 cases of cystitis. Similarly, 12 bladder tumour cell lines were HPV-negative in these assays. Cervical carcinoma cell lines containing from 1-3 to 600 copies of the HPV genome were used as positive controls and were scored positive in all assays by both Southern blotting and GP-PCR. It is concluded that despite the close proximity of the urothelium to the genital mucosa and the resemblance of some bladder tumours to known HPV-induced lesions in other tissues, HPV infection is absent or very uncommon in bladder tumours.

Blotting, Southern↗