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Biomedical subjects

D Lane

Publications and source records attributed to D Lane.

At least 19 recordsLinked to original sources
PubMed1992

The immune response to p53 in breast cancer patients is directed against immunodominant epitopes unrelated to the mutational hot spot.

Alteration of the p53 gene is the most frequent genetic feature of human cancer and leads to overexpression of the altered protein in the tumor cell nucleus. Two diagnostic procedures are currently available to assess p53 mutations: (a) molecular analysis of the gene sequence; and (b) immunohistochemical analysis of p53 protein accumulation. We now report a third approach, serological analysis. Fifteen % of primary breast cancer patients were found to have circulating antibodies to p53 protein by immunoprecipitation or immunoblotting. We have found a close correlation between the presence of such antibodies and bad prognosis such as high histological grade and the absence of hormone receptors. Furthermore, we found that the B-cell response to p53 protein is induced by two immunodominant regions located at the carboxy and amino termini of the protein, outside the central mutational hot spot region. These findings suggest that serological analysis, combined with molecular and histochemical methods, may be suitable for assessing the state of the p53 gene in cancer patients.

Antibodies, Neoplasm↗
PubMed1992

Over-expression of p53 nuclear oncoprotein in colorectal adenomas.

p53 is a nuclear phosphoprotein which controls normal cell growth. Normal p53 protein is undetectable by standard immunohistochemical staining and the over-expression found in neoplastic cells correlates with the presence of point mutations of evolutionary conserved regions of the p53 gene. We examined the expression of p53 protein in a series of 36 colorectal adenomas (13 tubular, 17 tubulovillous, 6 villous) showing different degrees of dysplasia (11 mild, 19 moderate, 6 severe), 11 moderately differentiated adenocarcinomas (6 Duke's A, 4 Duke's B, 1 Duke's C) and 5 metaplastic polyps using the polyclonal antibody CM1 which recognises p53 protein in conventionally fixed and processed histological material. We found that 15 out of 36 colorectal adenomas showed p53 immunoreactivity, although in 4 positive cases (26%) the staining was very focal (less than 0.1% positive cells). More than 80% of severely dysplastic adenomas showed strong p53 immunoreactivity and this over-expression was correlated with increased cell proliferative rate as detected by the proliferating-cell-nuclear-antigen (PCNA) staining. p53 nuclear staining was also seen in 8 out of 11 (65%) colorectal adenocarcinomas as previously shown. Our data suggest that the p53 gene mutation, with the subsequent over-expression of the protein, occurs in colorectal adenomas and may therefore be a fundamental genetic event underlying the dysplasia and loss of proliferative control that are characteristic of adenomas with malignant potential.

Adenoma↗
PubMed1992

Angiostatic activity and metabolism of cortisol in the chorioallantoic membrane (CAM) of the chick embryo.

There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37 degrees C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20 beta-dihydrocortisol (4-pregnen-11 beta,17 alpha,20 beta,21-tetrol-3-one). The cortisol metabolite, 20 beta-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent Km determined for cortisol metabolism was 12 microM and the observed Vmax was 1.4 mumol cortisol/mg protein/min. The majority of the 20 beta-reductase activity was found in the soluble (242,000 g) fraction of CAM homogenate. 20 beta-Reductase activity in chick embryo CAM has not been previously reported.

Allantois↗
PubMed1992

The relationship of p53 immunostaining to survival in carcinoma of the lung.

In this study 125 primary lung tumours have been immunostained with a panel of 5 anti-p53 antibodies (PAb240, PAb421, PAb1801, CM-1 and C19). These antibodies recognise different epitopes over the full extent of the p53 gene. It is generally believed that immunolabelling identifies only mutant p53 proteins due to the short half life of the wild type protein. The aims of this study were to confirm earlier studies of p53 positivity in human lung tumours and to establish whether or not this bore any relationship to survival. Immunostaining was demonstrated within the nuclei of affected cells in 54% of the 125 lung tumours (59% of 78 squamous cell carcinomas, 52% of 42 adenocarcinomas and 20% of five small cell carcinomas). This confirms previous smaller studies of p53 protein expression in human lung tumours. Survival curves have been drawn for all of the cases considered together and for squamous and adenocarcinomas separately. No differences in survival between p53 positive and negative cases were seen for any group of tumours. This indicates that although p53 may be of considerable importance in the initiation of malignancy it is probably of little significance once a tumour has developed.

Adenocarcinoma↗
PubMed1992

Overexpression of the p53 protein and allele loss at 17p13 in ovarian carcinoma.

Mouse monoclonal antibodies PAb 240 and PAb 1801 which specifically immunoprecipitate p53 protein, were used to examine 27 fresh ovarian tumours (16 serous adenocarcinomas, six endometrioid carcinomas, one mucinous adenocarcinoma, one mucinous borderline tumour and three benign adenomas). Eleven out of 16 (69%) serous adenocarcinomas and one endometrioid tumour showed positive staining with one or both antibodies and none of the mucinous or benign tumours stained with either antibody. DNA from tumour and peripheral blood leukocytes was used to identify allelic deletions on chromosome 17p in tumours. 11/12 positively staining tumours showed less of heterozygosity (LOH) on 17p at the nearest informative locus to the p53 gene. In this series of ovarian tumours, LOH on 17p correlates closely with the aberrant expression of the p53 protein in a high proportion of advanced stage serous adenocarcinomas. This observation suggests that the p53 tumour suppressor gene is involved in the evolution of epithelial ovarian cancer (EOC) and may have prognostic significance.

Adenocarcinoma↗
PubMed1992

Towards a phylogeny of the genus Vibrio based on 16S rRNA sequences.

The inter- and intrageneric relationships of the genus Vibrio were investigated by performing a comparative analysis of the 16S rRNAs of 10 species, including four pathogenic representatives. The results of immunological and 5S rRNA studies were confirmed in that the genus is a neighboring taxon of the family Enterobacteriaceae. With regard to the intrageneric structure, Vibrio alginolyticus, Vibrio campbellii, Vibrio natriegens, Vibrio harveyi, Vibrio proteolyticus, Vibrio parahaemolyticus, and Vibrio vulnificus form the core of the genus, while Vibrio (Listonella) anguillarum, Vibrio diazotrophicus, and Vibrio hollisae are placed on the outskirts of the genus. Variable regions around positions 80, 180, and 450 could be used as target sites for genus- and species-specific oligonucleotide probes and polymerase chain reaction primers to be used in molecular identification.

Base Sequence↗
PubMed1992

Use of gel retardation to analyze protein-nucleic acid interactions.

Protein-nucleic acid interactions are crucial in the regulation of many fundamental cellular processes. The nature of these interactions is susceptible to analysis by a variety of methods, but the combination of high analytical power and technical simplicity offered by the gel retardation (band shift) technique has made this perhaps the most widely used such method over the last decade. This procedure is based on the observation that the formation of protein-nucleic complexes generally reduces the electrophoretic mobility of the nucleic acid component in the gel matrix. This review attempts to give a simplified account of the physical basis of the behavior of protein-nucleic acid complexes in gels and an overview of many of the applications in which the technique has proved especially useful. The factors which contribute most to the resolution of the complex from the naked nucleic acid are the gel pore size, the relative mass of protein compared with nucleic acid, and changes in nucleic acid conformation (bending) induced by binding. The consequences of induced bending on the mobility of double-strand DNA fragments are similar to those arising from sequence-directed bends, and the latter can be used to help characterize the angle and direction of protein-induced bends. Whether a complex formed in solution is actually detected as a retarded band on a gel depends not only on resolution but also on complex stability within the gel. This is strongly influenced by the composition and, particularly, the ionic strength of the gel buffer. We discuss the applications of the technique to analyzing complex formation and stability, including characterizing cooperative binding, defining binding sites on nucleic acids, analyzing DNA conformation in complexes, assessing binding to supercoiled DNA, defining protein complexes by using cell extracts, and analyzing biological processes such as transcription and splicing.

Base Sequence↗
PubMed1992

Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli.

A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter. The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence. Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53. These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays. Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax. Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer. We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours.

Antibodies↗
PubMed1991

Expression of RB and p53 proteins in HPV-positive and HPV-negative cervical carcinoma cell lines.

Mutations within the tumor suppressor genes Rb-1 and p53 are commonly found in many human malignancies, and loss of wild-type function of both p53 and RB appear to be important events in the development of these malignancies. Interference with normal RB and p53 function in the cell has apparently also been exploited by the oncogenic genital human papillomaviruses (HPVs), which encode transforming proteins capable of binding cellular RB and p53 proteins. We have investigated the expression of RB and p53 in a series of eight cervical carcinoma cell lines, six of which contain HPV sequences and two of which have arisen apparently independently of HPV infection. In the six HPV-positive lines, no evidence of abnormal RB or p53 protein could be detected. However, there was evidence for abnormal RB and p53 in the two HPV-negative lines. These data are consistent with the hypothesis that loss of wild-type RB and p53 function is necessary for tumor development and that such loss can occur either by mutation within the cellular gene or by expression of viral proteins capable of complexing wild-type cellular proteins.

Carcinoma↗
PubMed1991

Asthma: a diagnostic and therapeutic challenge.

Asthma is a disease needing urgent action. Morbidity and mortality are rising, despite advances in our understanding of asthma and in its treatment. The lifetime prevalence of asthma in children varies from 5 to 31%, depending on the defining criteria, and even when recent prevalence (occurring in the past two years) is examined, rates from 5 to 18% have been found. There is growing evidence which shows that signs of reversible airways disease occur in the elderly with even greater frequency and it is likely that at least 10% of the population will need asthma treatment at some stage of their lives; this means that well over five million people in the UK or 250 patients in each 'average' general practice list of 2500 merit the diagnosis of asthma at some stage of their lives. The fact that an average practice is likely to have diagnosed less than half that number clearly implies that asthma is first and foremost an under-diagnosed disease.

Asthma↗
PubMed1991

Mutant p53, EGF receptor and c-erbB-2 expression in human breast cancer.

p53 expression was studied in 111 primary breast cancers with a monoclonal antibody (PAb240) that reacts with an epitope in mutant p53. Epidermal growth factor receptors (EGFr) and oestrogen receptors (ER) measured by ligand binding, c-erbB-2 expression assessed by immunochemistry and lymph node status were compared with p53 staining. Fifty-nine tumours (53%) were positive for p53, and this correlated with EGFr expression (P less than 0.02), which is a known poor prognostic factor. Eighteen out of 59 p53+ tumours expressed c-erbB-2 versus 4 out of 52 p53- tumours assessed on paraffin sections. However, assessing c-erbB-2 by Southern blotting and immunochemistry on frozen sections showed that 20 out of 59 p53+ tumours overexpressed or had amplified c-erbB-2 compared with 15 out of 52 p53- tumours (not significant). Mean EGFr concentration in p53+ tumours was 31 fmol per mg of membrane protein versus 14 fmol mg-1 in p53- tumours. Cytoplasmic staining was the most frequent pattern found for p53, but some cases showed cytoplasmic plus nuclear staining, or focal cells with predominantly nuclear staining. Thus, abnormal p53 is the commonest oncogene abnormality described in breast cancer. The association with EGFr expression suggests that these oncogenes interact in the pathogenesis of breast cancer.

Biomarkers, Tumor↗
PubMed1990

Increased expression of mutant forms of p53 oncogene in primary lung cancer.

Primary lung cancer samples of the major histological types were examined for expression of the tumor suppressor gene p53 by immunohistochemistry. Abnormalities in p53 expression were found in 28 of 40 carcinomas, 14 of 17 squamous tumours showing abnormal p53 expression, whereas no expression of p53 was detectable in 7 carcinoid tumours or in 10 normal lung samples. Direct evidence for homozygous expression of mutant p53 mRNA in representative carcinomas was obtained by means of an asymmetric polymerase chain reaction mRNA sequencing strategy, which allowed sequencing without any cloning step. All the mutations were G to T transversions resulting in mis-sense mutations in aminoacids highly conserved in evolution. Mutation of the p53 gene is the most frequently identified genetic change in human lung cancer; these findings suggest that simple immunohistological methods can provide strong evidence of such mutation.

Adenocarcinoma↗