SspI polymorphism in sequence encoding 3' untranslated region of the APC gene.
Explore the source record for details and available documents.
Adenomatous Polyposis Coli↗
Biomedical subjects
A H Wyllie
Publications and source records attributed to A H Wyllie.
Linked DNA markers for presymptomatic diagnosis of familial adenomatous polyposis.
41 symptom-free individuals aged 0-39 years who were at risk of familial adenomatous polyposis (FAP) were genotyped with six linked DNA probes. 28 individuals were informative for probes flanking the gene and 14 people assigned a probe-derived risk of over 0.93 were subsequently shown to be affected by clinical screening. 4 individuals who had been discharged from follow-up were designated high risk by this method. In those screened negative, risk was calculated from genotypic, colonic, and CHRPE findings and 89% of subjects had a risk below 0.003. An integrated risk analysis may have an important place in screening programmes for FAP.
Adenomatous Polyposis Coli↗
HPV in full thickness cervical biopsies: high prevalence in CIN 2 and CIN 3 detected by a sensitive PCR method.
A new type-specific, sensitive, non-radioactive assay is described for the detection of human papillomavirus (HPV) DNA in tissues. Sequences within the E6 gene were amplified by the polymerase chain reaction (PCR), using primer pairs which clearly distinguish HPV types, including those with close sequence homology such as 6b and 11. The amplified DNA products were identified by non-radioactive oligonucleotide hybridization and restriction endonuclease mapping, and the method was sufficiently sensitive to detect between 3 and 5 SiHa cells (each containing 1-2 copies of HPV 16 DNA) amongst 10,000 non-HPV-containing cells. Frozen and archival paraffin sections were equally acceptable substrates for the reaction. The assay was applied to frozen sections of full thickness cervical epithelium from 60 cases of cervical intraepithelial neoplasia (CIN) and 24 normal cervical controls. HPV DNA was detected in 60 per cent of cases of CIN 3 and CIN 2, in 25 per cent of cases of CIN 1, and in none of the normal controls. Prevalence of HPV 16 was similar (approximately 50 per cent) in both CIN 2 and CIN 3, and in the whole series HPV 16 was almost five-fold more common than HPV 18. Low-risk HPV types were present in 5 per cent of CIN 1, but 0 per cent of CIN 2 and CIN 3 biopsies. The data emphasize the biological similarity of CIN 2 and CIN 3 lesions, and their divergence from CIN 1.
Adolescent↗
Apoptosis: mechanisms and roles in pathology.
Explore the source record for details and available documents.
Animals↗
Stability of critical genetic lesions in human colorectal carcinoma xenografts.
Explore the source record for details and available documents.
Adenocarcinoma↗
MCC, a candidate familial polyposis gene in 5q.21, shows frequent allele loss in colorectal and lung cancer.
MCC is a gene located within human chromosome band 5q.21 that shows somatically acquired mutations in colorectal cancer, and may be identical to the gene responsible for inheritance of familial adenomatous polyposis. Here we demonstrate that alleles contiguous with or within MCC are deleted in a high proportion of sporadic colorectal carcinomas. Of 106 carcinomas that were informative concurrently at close-flanking sites both centromeric and telomeric to MCC, 41.5% showed acquired allele loss contiguous with MCC. Evidence is presented to show that the true frequency of loss of MCC alleles is higher still. In contrast, allele losses in chromosome 5 that were incompatible with involvement of MCC were very rare (2% of a total series of 201 informative tumours). Interstitial deletion was the commonest mechanism of allele loss, and L5.71-3, a probe known to include coding sequences of MCC, marks the most consistently deleted site. Moreover mapping of chromosome breakpoints with six probes within 5q.21 sited the common critical deletion in a 2.5 Mb region which included L5.71-3. However use of L5.71-3 itself suggested that critical deleted regions may lie on either side of the probed sequence. The simplest explanation for this unexpected finding is that MCC itself is the essential deleted gene, the lost exons lying sometimes centromeric to, sometimes telomeric to and occasionally within the region detected by L5.71-3. Tumours in which MCC-related alleles were lost by interstitial deletion were in general larger than those with other mechanisms of acquired homozygosity (e.g. mitotic recombination), but there were no other obvious associations with clinicopathological features. Between 20% and 25% of lung cancers also showed acquired allele losses contiguous with MCC. The significance of this observation is still to be determined, as lung tumours show allele losses at many other sites, but the specificity of the probes used in this study does establish that the 5q.21 losses in these tumours are compatible with involvement of MCC.
Adenomatous Polyposis Coli↗
p53 expression in colorectal tumors.
The expression of the nuclear phosphoprotein p53 was studied immunohistochemically in a series of 150 benign and malignant colorectal tumors. Using monoclonal antibody PAb1801, tumors divided unequivocally into two groups on the basis of immunohistochemistry. Forty of the carcinomas (46.5%) showed positive staining but only 4 of the adenomas (8.7%) were positive (P less than 0.001). The few positive adenomas always showed moderate or severe dysplasia. Metaplastic polyps (n = 9) and small familial adenomatous polyposis-related adenomas (n = 9) were uniformly negative. Carcinomas with p53 expression did not differ from those without in terms of site, differentiation or the prognostic indicators of Dukes' stage, DNA ploidy, or tumor histology. The improved morphologic resolution available in periodate lysine paraformaldehyde dichromate (PLPD)-fixed, paraffin-embedded tissue permitted several conclusions to be made: p53 is confined to neoplastic nuclei; staining in positive tumors is heterogeneous and often more marked at the infiltrative margins; and staining intensity is dramatically reduced in mitotic cells. It is concluded that expression of immunohistochemically detectable p53 (probably representing mutated forms of the protein) occurs in some adenomas around the time of transition to carcinoma. Therefore there is an association with the appearance of infiltrative behavior but not with degree of tumor progression (including metastasis) at the time of resection.
Adenomatous Polyposis Coli↗
Immunohistochemical demonstration of altered intracellular localization of the C-Myc oncogene product in human colorectal neoplasms.
The distribution of the c-myc oncogene product p62 was examined by immunohistochemistry using the monoclonal antibody Mycl-9E10 in a series of 50 colorectal resections for carcinoma. The specimens were specially handled to ensure rapid fixation in formalin, and a significant improvement was shown in the quality and localization of staining compared with routinely handled specimens. Non-neoplastic mucosa showed the presence of nuclear staining of epithelial cells in 93 per cent of the samples, whilst all carcinomas showed cytoplasmic staining and infrequent nuclear staining. Adenomas showed an intermediate pattern, with significantly more frequent cytoplasmic distribution than non-neoplastic mucosa, but less than carcinomas. The results show that whilst fixation conditions are important in the immunolocalization of the c-myc protein product, there may be a consistent difference between non-neoplastic mucosa and carcinoma in the manner of association of p62 with the nucleus.
Carcinoma↗
Papillomaviruses and human cancer.
Explore the source record for details and available documents.
Cell Transformation, Neoplastic↗
Genetic linkage map of six polymorphic DNA markers around the gene for familial adenomatous polyposis on chromosome 5.
A genetic linkage map of six polymorphic DNA markers close to the gene (APC) for familial adenomatous polyposis (FAP) on chromosome 5q is reported. One hundred fifty-five typed members of nine FAP kindred provided more than 90 meioses for linkage analysis. A number of crucial recombination events have been identified which are informative at three or more loci, allowing confident ordering of parts of the map. There was no evidence of genetic heterogeneity, with all families showing linkage of at least one chromosome 5 marker to the gene. Recombination data and two-point linkage analysis support a locus order of centromere-pi 227-C11P11-ECB27-L5.62-APC-EF5.44-YN5.48-telomer e, although EF5.44 could lie in the interval L5.62-APC or ECB27-L5.62. No recombinants were identified between APC and either EF5.44 or YN5.48, but published deletion mapping in colorectal carcinomas and linkage analysis in FAP suggest that YN5.48 is 1-3 cM from APC. The present study suggests that YN5.48 and L5.62 delineate a small region of chromosome 5 within which the EF5.44 locus lies very close to the APC gene. These data not only allow use of flanking markers for presymptomatic diagnosis of FAP but also provide a high-density map of the region for isolation of the APC gene itself and for further assessment of the role of chromosome 5 deletions in the biology of sporadic colorectal cancer.
Adenomatous Polyposis Coli↗
Apoptosis. The role of the endonuclease.
Cell death by apoptosis mediates several important physiologic and pathologic processes and appears to be intrinsically programmed. Its characteristic features are distinctive morphologic changes of nucleus and cytoplasm, along with cleavage of chromatin at regularly spaced sites. Here we study DNA organization and nuclear structure in apoptotic thymocytes to define the cleavage event and, by implication, the role of the responsible endonuclease. We show that in apoptosis, double-stranded cleavage of DNA generates two classes of chromatin fragments: 70% of DNA exists as long, H1-rich oligonucleosomes bound to the nucleus, and 30% comprises short oligonucleosomes and mononucleosomes, which are depleted in H1, enriched in HMG1 and 2, and not attached to the nucleus. This minority class probably derives from chromatin in a transcriptionally active configuration, which would allow better access to enzymes in the nucleoplasm, producing more complete digestion. The characteristic nucleolar morphology in apoptosis can also be explained in terms of cleavage of the transcriptionally active ribosomal genes, with conservation of the nucleolin-rich fibrillar center. The chromatin cleavage, nucleolar morphologic changes, and chromatin condensation were closely mimicked by micrococcal nuclease digestion of normal thymocyte nuclei in the presence of protease inhibitors. Thus, in apoptosis, selective activation of an endogenous endonuclease appears to be responsible not only for widespread chromatin cleavage but also for the major nuclear morphologic changes.
Animals↗
Glucocorticoids activate a suicide process in thymocytes through an elevation of cytosolic Ca2+ concentration.
Glucocorticoid hormones kill immature thymocytes through the induction of a suicide process commonly referred to as "apoptosis." A characteristic marker for this process is the stimulation of endogenous endonuclease activity which results in the extensive cleavage of cell chromatin. In an attempt to characterize the biochemical events involved in this process, we studied the role of Ca2+ in glucocorticoid-induced DNA fragmentation and cell killing in thymocytes. Treatment of thymocytes from immature rats with the synthetic glucocorticoid methylprednisolone resulted in extensive DNA fragmentation which was preceded by an early, sustained increase in cytosolic Ca2+ concentration. This increase in Ca2+ level was blocked by cycloheximide and actinomycin D, inhibitors of de novo protein and mRNA synthesis, respectively. Prevention of the Ca2+ increase by buffering cytosolic Ca2+ with quin-2, or through incubation of the thymocytes in a "Ca2+-free" medium, prevented endonuclease activation and cell killing. Inhibitors of calmodulin also prevented DNA fragmentation without inhibiting the glucocorticoid-stimulated elevation of cytosolic Ca2+ concentration. The Ca2+ increase appeared to be due to the action of a heat-labile cytosolic factor, synthesized in response to glucocorticoids, which facilitated the influx of extracellular Ca2+. Our findings suggest that glucocorticoids induce thymocyte suicide through an elevation of cytosolic Ca2+ concentration resulting in endonuclease activation, DNA fragmentation, and cell death.
Animals↗
Linkage analysis in familial adenomatous polyposis: order of C11P11 (D5S71) and pi 227 (D5S37) loci at the apc gene.
Linkage analysis with DNA probes C11P11 and pi 227 is reported in six Scottish families with familial adenomatous polyposis. Two families were informative for C11P11 and all six were at least partly informative for pi 227. Two C11P11-apc and two pi 227-apc recombinants were identified and one of these was recombinant for both C11P11-apc and pi 227-apc. A further possible combined C11P11-apc and pi 227-apc recombination event was also identified. Peak lod score for linkage of C11P11 to apc was 5.80 at a recombination fraction (theta) of 0.069 (95% probability limits 0.012-0.191) and for linkage of pi 227 to apc was 3.19 at theta = 0.110 (95% probability limits 0.023-0.286). Peak lod score for linkage of C11P11 to pi 227 was 1.79 at theta = 0.00. The data support a gene order of pi 227-C11P11-apc.
Chromosomes, Human, Pair 5↗
Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages.
Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of neutrophils and their potentially histotoxic contents is one prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the senescent neutrophil that are associated with their recognition by macrophages are the subject of this investigation. Over 24 h in culture an increasing proportion of human neutrophils from peripheral blood or acutely inflamed joints underwent morphological changes characteristic of programmed cell death or apoptosis. Time-related chromatin cleavage in an internucleosomal pattern indicative of the endogenous endonuclease activation associated with programmed cell death was also demonstrated. A close correlation was observed between the increasing properties of apoptosis in neutrophils and the degree of macrophage recognition of the aging neutrophil population, and a direct relationship between these parameters was confirmed within aged neutrophil populations separated by counterflow centrifugation into fractions with varying proportions of apoptosis. Macrophages from acutely inflamed joints preferentially ingested apoptotic neutrophils and histological evidence was presented for occurrence of the process in situ. Programmed cell death is a phenomenon of widespread biological importance and has not previously been described in a cell of the myeloid line. Because it leads to recognition of intact senescent neutrophils that have not necessarily disgorged their granule contents, these processes may represent a mechanism for the removal of neutrophils during inflammation that also serves to limit the degree of tissue injury.
Arthritis↗
Single strand DNA breaks in mitogen stimulated T lymphocytes are religated by a mechanism independent of accessory cells.
The phenomenon of religation of single-strand DNA breaks (nicks) in mitogenically stimulated human T lymphocytes is an event occurring within 8 h of mitogen stimulation. Many later events in lymphocyte activation are known to be dependent on accessory cells, whereas earlier events are often accessory-cell independent. To establish whether nick religation is dependent or independent of accessory-cell function, lymphocytes were stimulated with PHA in the presence of inhibitors thought to act, in part at least, on accessory cells (methylprednisolone and cyclosporine A), or under conditions in which accessory-cell function is limited (low-density culture, adherent-cell depleted populations). In each case DNA synthesis was inhibited but the religation process was retained, indicating that it is independent of accessory-cell function. Inhibition of DNA synthesis in these cells was shown to be readily reversible on addition of conditioned medium containing accessory-cell products, but there was no further change in ligation.
Antigen-Presenting Cells↗
High frequency of APC loss in sporadic colorectal carcinoma due to breaks clustered in 5q21-22.
Familial adenomatous polyposis is transmitted by a gene (APC) located within 5q21-22. Hemizygous loss of at least a part of 5q has been reported in 19-36% of sporadic colorectal carcinomas. This suggests that an anti-oncogene is located on that chromosome arm, but the probes used previously gave little information on the status of APC in the tumours. Using DNA probes homologous to polymorphic sequences flanking and close to the APC locus we show that more than half of a large series of carcinomas had lost at least one flanking allele. Mapping of allele losses provides data that imply clustering of breakpoints in a 10-15 megabase region around APC. The commonest chromosome defect responsible for APC loss was interstitial deletion. Mitotic recombination or partial arm loss were less frequent mechanisms. Whole chromosome loss was rare. This pattern contrasts with that reported in acquired homozygosity at other anti-oncogene loci in sporadic tumours and implies that APC loss is an early event in colorectal carcinogenesis. This view is also supported by the observations that 5q21-22 loss occurs with similar frequency in DNA diploid and DNA aneuploid tumours, and also in tumours at all clinical stages of progression.
Adenoma↗
Optimal preservation of p21 ras immunoreactivity and morphology in paraffin-embedded tissue.
Specific immunostaining of p21 ras protein by the well-characterized pan-ras antibody Y13-259 is achieved in paraffin sections of human and animal tissues fixed in periodate-lysine-paraformaldehyde-dichromate (PLPD). Intensity of staining is as good as in cryostat sections, with superior histological detail. Localization to plasma membrane is demonstrated in rodent cells genetically manipulated to express abundant p21 ras (the FHO5T1 cell line), both in preparations suspended in agar after culture in vitro and in those growing as tumour in vivo. Strong positive staining is observed in neoplasms of human breast and colon, tissues in which there is independent evidence of elevated ras gene expression. The superior morphology afforded by this technique allows clear characterization of p21 ras expression in small premalignant lesions for which other methods of detection of oncogene expression are not appropriate.
Animals↗
Stimulation of endogenous endonuclease activity in hepatocytes exposed to oxidative stress.
Incubation of freshly isolated rat hepatocytes with moderately toxic concentrations of menadione [2-methyl-1,4-naphthoquinone) resulted in chromatin condensation and progressive DNA fragmentation, suggestive of the stimulation of an endogenous endonuclease activity previously found to be involved in programmed cell death (apoptosis). Endonuclease activation followed upon a sustained increase in cytosolic Ca2+ concentration and preceded cell killing by 1-2 h. It is concluded that generation of oxidative stress in hepatocytes can activate processes similar to those observed during programmed cell death.
Animals↗