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P J Coates

Publications and source records attributed to P J Coates.

At least 19 recordsLinked to original sources
PubMed2006

Regulating p73 isoforms in human tumours.

Although mutations in the TP73 gene are extremely rare in human tumours, altered expression is common. In some tumours, most notably leukaemias and lymphomas, expression of TP73 is reduced, suggesting a tumour suppressor role. In contrast, TP73 is over-expressed in many other tumour types, implying that it has oncogenic functions in human tumourigenesis. These conflicting scenarios can be reconciled by the observations that the TP73 gene produces p53-like isoforms (TAp73) and anti-p53 isoforms (DeltaTAp73). Thus, loss of TAp73 or over-expression of DeltaTAp73 should each promote oncogenic transformation, and the balance of expression of the opposing isoforms is the crucial factor. The mechanisms that regulate expression of TP73 isoforms are therefore of great interest. Recent data provide evidence for interacting roles of ZEB1, p300, and a polymorphic 73 bp deletion in intron 1 of the human TP73 gene in this process. Importantly, alterations to the proposed regulatory pathway for controlling TP73 isoform expression in colorectal cancer are associated with adverse clinico-pathological characteristics. Because p73 is also associated with tumour chemosensitivity, these new findings should provide prognostic information and have the potential to guide future therapeutic decisions.

DNA-Binding Proteins↗
PubMed2005

Discriminating functional and non-functional p53 in human tumours by p53 and MDM2 immunohistochemistry.

Mutation and/or loss of the TP53 tumour suppressor gene is the single most common genetic abnormality in human cancer. The majority of TP53 mutations lead to stabilization of the protein, so that immunohistochemical staining for p53 can suggest mutation status in many cases. However, various false-positive and false-negative situations mean that simple immunostaining for p53 is not informative in a substantial number of tumours. In the present study, a series of 119 human cancers were immunostained using a highly sensitive technique that detects the low levels of wild-type protein expressed in normal cells, such that homozygous gene deletion or non-sense TP53 mutation can be identified by an absence of staining. TP53 gene status was also assessed using FASAY as a genetic/functional screen and in selected cases by direct sequencing. A quantitative scoring system was employed to assess p53 levels, and p53 post-translational modification was evaluated using antibodies that detect specific phosphorylation sites. Phosphorylated p53 correlated with total p53 levels and did not improve the prediction of TP53 mutation status. The transcriptional activity of TP53 was determined by staining for two downstream target genes, p21(WAF1) and MDM2, and statistical correlations between MDM2/p21(WAF1) and p53 were found in tumours with wild-type, but not mutant TP53. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost-effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data.

Adenocarcinoma↗
PubMed2003

The yeast two-hybrid system for identifying protein-protein interactions.

The yeast two-hybrid assay is a system for identifying and analysing protein-protein interactions. Since the original description in 1989, the technique has provided insight into many biological pathways. A variety of adaptations to the technique have been developed that allow analysis of protein-DNA, protein-RNA, or small molecule-protein interactions. Recent developments now allow the use of these technologies to perform global analyses of all such interactions that occur in cells. The information gained from these approaches is uncovering many aspects of the complex networks that underlie normal cellular processes and how they are perturbed in disease states.

Animals↗
PubMed2003

Differential expression of p73alpha in normal ectocervical epithelium, cervical intraepithelial neoplasia, and invasive squamous cell carcinoma.

The TP73 gene is a member of the p53 family and through differential promoter usage and alternative splicing can encode a number of different isoforms that have distinct properties. p73 proteins are widely expressed in neural, epithelial, and hemopoietic cells and are proposed to have roles in the development and differentiation of various cell types and in tumorigenesis. The authors have developed a novel monoclonal antibody that is specific for p73alpha to study the expression of this individual isoform in normal and neoplastic cervical epithelium. In normal epithelium, p73alpha is restricted to nonproliferating cells at the base of the epithelium, whereas other p73 isoforms are found in the proliferative zones higher up in the epithelium. In cervical cancers, p73alpha expression is commonly lost, although other p73 isoforms are present at high levels. In particular, the authors found that invasive islands lose p73alpha expression when compared with the overlying intraepithelial lesion. These results show a tight regulation of p73 isoform expression in cervical epithelium and imply that different isoforms of p73 enhance or suppress neoplastic cell growth. These data raise the possibility that reactivation of p73alpha might be beneficial in cervical carcinoma. In addition, the absence of p73alpha in cervical cancer represents a potentially useful tool for the diagnosis of this disease.

Antibodies, Monoclonal↗
PubMed2003

Radiation-induced genomic instability in immortalized haemopoietic stem cells.

PURPOSE: To investigate the expression of radiation-induced delayed reproductive death and chromosomal instability using an immortalized cell line (R-M26/2-1) with the characteristics of long-term repopulating haemopoietic stem cells established from the CBA mouse strain. MATERIALS AND METHODS: R-M26/2-1 cells were gamma-irradiated and maintained in culture for up to 41 population doublings. At intervals, measurements were made of cloning efficiency and cells were examined for cytogenetic aberrations at eight and 24 population doublings. The p53 status and p53 phosphorylation were investigated by Western analysis and immunocytochemistry and production of intracellular reactive oxygen species was investigated by use of the fluorescent probe DCFH-DA. RESULTS: The descendants of cells surviving 4Gy gamma-irradiation exhibited a reduced colony-forming efficiency and expressed chromosomal instability independent of p53 function and with no evidence of enhanced production of reactive oxygen species. The delayed reproductive death phenotype persisted at a constant rate of 12% clonogenic cell loss when colony formation was assessed in anchorage-dependent conditions on tissue culture substrates. However, R-M26/2-1 cells, like normal haemopoietic cells, can be cultured in anchorage-independent conditions and this type of assay demonstrated a 50% or greater persisting clonogenic cell loss. There was no significant delayed reproductive death or chromosomal instability in cultures established with 0.5 Gy gamma-irradiated R-M26/2-1 cells. CONCLUSIONS: A radiation-induced genomic instability phenotype, independent of p53 function and with no evidence of oxidative stress, was demonstrated in the descendants of 4Gy gamma-irradiated R-M26/2-1 cells and unstable aberrations characteristic of radiation-induced chromosomal instability may account for a component of the delayed reproductive death phenotype. Colony-forming efficiency and expression of the delayed death phenotype determined using an anchorage-independent assay was significantly greater than that determined using an anchorage-dependent assay indicating that some aspect of adherence influences these endpoints. The absence of significant instability in the descendants of 0.5 Gy gamma-irradiated cells implies a threshold for these endpoints in this haemopoietic stem cell line.

Animals↗
PubMed2002

The mitochondrial PHB complex: roles in mitochondrial respiratory complex assembly, ageing and degenerative disease.

Although originally identified as putative negative regulators of the cell cycle, recent studies have demonstrated that the PHB proteins act as a chaperone in the assembly of subunits of mitochondrial respiratory chain complexes. The two PHB proteins, Phblp and Phb2p, are located in the mitochondrial inner membrane where they form a large complex that represents a novel type of membrane-bound chaperone. On the basis of its native molecular weight, the PHB-complex should contain 12-14 copies of both Phblp and Phb2p. The PHB complex binds directly to newly synthesised mitochondrial translation products and stabilises them against degradation by membrane-bound metalloproteases belonging to the family of mitochondrial triple-A proteins. Sequence homology assigns Phb1p and Phb2p to a family of proteins which also contains stomatins, HflKC, flotillins and plant defence proteins. However, to date only the bacterial HflKC proteins have been shown to possess a direct functional homology with the PHB complex. Previously assigned actions of the PHB proteins, including roles in tumour suppression, cell cycle regulation, immunoglobulin M receptor binding and apoptosis seem unlikely in view of any hard evidence in their support. Nevertheless, because the proteins are probably indirectly involved in ageing and cancer, we assess their possible role in these processes. Finally, we suggest that the original name for these proteins, the prohibitins, should be amended to reflect their roles as proteins that hold badly formed subunits, thereby keeping the nomenclature already in use but altering its meaning to reflect their true function more accurately.

Aging↗
PubMed2001

Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Animals↗
PubMed2001

Induction of endogenous beta-galactosidase by ionizing radiation complicates the analysis of p53-LacZ transgenic mice.

Studies of the response of p53-lacZ transgenic mice have uncovered an unexpected induction of endogenous acid-beta-galactosidase activity following whole body irradiation. Strong induction of endogenous enzyme activity is seen in a variety of mouse strains commonly used in the production of transgenes. The induction of endogenous enzyme activity therefore complicates the analysis of p53-lacZ transgenes and may also influence the analysis of radiation responses in other lacZ-reporter mice.

Animals↗
PubMed2001

Differential post-translational modification of the tumour suppressor proteins Rb and p53 modulate the rates of radiation-induced apoptosis in vivo.

Ionizing radiation induces p53-dependent apoptosis in the spleen, providing a model system to study p53 regulated events in a normal cell type. We have developed an in vivo model that identifies genetic differences in the regulation of p53-mediated apoptosis and addresses whether altered post-translational events in the p53-p21/Rb axis modulate the sensitivity of cells to radiation-induced cell death in vivo. Splenocytes from mice with distinct genetic backgrounds (DBA/2 and C57BL/6) exhibit differences in the rate of apoptosis. Whilst no obvious strain differences in protein levels of Bcl-2 or the cyclin-CDKs were observed, early post-translational regulatory events in the p53-p21/Rb axis showed striking differences in the two mouse strains. Cells from C57BL/6 animals undergo more rapid apoptosis after irradiation resulting from elevated levels and rapid induction of p53, pronounced Rb-cleavage, and the absence of a sustained induction of p21. In contrast, cells from DBA/2 animals have a reduced rate of apoptosis following irradiation with elevated levels of hyperphosphorylated Rb and a sustained induction of the p21 protein that is coincident with the C-terminal phosphorylation of p53. These data suggest that quantitative differences in the level of p21 protein can affect the rate of apoptosis in vivo, consistent with the view that p21 is an anti-apoptotic effector of p53. However, striking differences in the Rb protein-caspase cleavage or hyperphosphorylation-in the same cell type, but in different genetic backgrounds, demonstrates that p53-dependent apoptosis can be modulated in vivo by genetic factors that impinge upon the pro- or anti-apoptotic potential of Rb. In addition, we show that Rb cleavage is p53-dependent and that its phosphorylation status can be uncoupled from p21 expression. This study highlights the possibility that genetic factors can be identified that affect differential sensitivity of cells to ionizing radiation in vivo.

Animals↗
PubMed2001

Mammalian prohibitin proteins respond to mitochondrial stress and decrease during cellular senescence.

The two prohibitin proteins, Phb1p and Phb2p(BAP37), have been ascribed various functions, including cell cycle regulation, apoptosis, assembly of mitochondrial respiratory chain enzymes, and aging. We show that the mammalian prohibitins are present in the inner mitochondrial membrane and are always bound to each other, with no free protein detectable. They are coexpressed during development and in adult mammalian tissues, and expression levels are indicative of a role in mitochondrial metabolism, but are not compatible with roles in the regulation of cellular proliferation or apoptosis. High level expression of the proteins is consistently seen in primary human tumors, while cellular senescence of human and chick fibroblasts is accompanied by heterogeneous decreases in both proteins. The two proteins are induced by metabolic stress caused by an imbalance in the synthesis of mitochondrial- and nuclear-encoded mitochondrial proteins, but do not respond to oxidative stress, heat shock, or other cellular stresses. The gene promoter sequences contain binding sites for the Myc oncoprotein and overexpression of Myc induces expression of the prohibitins. The data support conserved roles for the prohibitins in regulating mitochondrial respiratory activity and in aging.

Animals↗
PubMed2000

Characterization of the expression pattern of p63 alpha and delta Np63 alpha in benign and malignant oral epithelial lesions.

The p53 homologue p63 is essential for ectodermal differentiation, such that p63-/- mice lack all squamous epithelia and teeth. The p63 gene expresses at least 6 different transcripts, but information regarding the expression, regulation and function of the different isoforms has remained sparse, due to the lack of adequate reagents directed specifically against the individual proteins. Here we characterize the expression of p63 alpha/delta Np63 alpha in benign and malignant lesions of the oral epithelium, using a specific antibody raised against a peptide derived from the C-terminus of p63 alpha, which does not cross-react with p53 or the other p53 homologue, p73. By immunohistochemical analysis, we show that these p63 isoforms are expressed in the nucleus of many cells. In normal and benign lesions, p63 alpha/delta Np63 alpha-expressing cells are mainly found suprabasally, whereas p53-expressing cells are restricted to the basal-cell layer. By RT-PCR, we show that delta Np63 alpha is the predominant isoform in cell lines from squamous-cell carcinomas of the head and neck, confirming our immunochemical observations. Our data are consistent with studies suggesting a role for p63 in the transit-amplifying population of epidermal cells. Over-expression of p63 alpha, and in particular the delta N form, was frequently seen in carcinomas. Taken together with previous analyses of p63 expression, our data suggest distinct roles for different p63 isoforms in the regulation of growth and/or differentiation of epithelial cells. Moreover, our data are compatible with the notion that p63 can act to promote neoplastic growth in the oral epithelium.

Amino Acid Sequence↗
PubMed2000

Prohibitins act as a membrane-bound chaperone for the stabilization of mitochondrial proteins.

Prohibitins are ubiquitous, abundant and evolutionarily strongly conserved proteins that play a role in important cellular processes. Using blue native electrophoresis we have demonstrated that human prohibitin and Bap37 together form a large complex in the mitochondrial inner membrane. This complex is similar in size to the yeast complex formed by the homologues Phb1p and Phb2p. In yeast, levels of this complex are increased on co-overexpression of both Phb1p and Phb2p, suggesting that these two proteins are the only components of the complex. Pulse-chase experiments with mitochondria isolated from phb1/phb2-null and PHB1/2 overexpressing cells show that the Phb1/2 complex is able to stabilize newly synthesized mitochondrial translation products. This stabilization probably occurs through a direct interaction because association of mitochondrial translation products with the Phb1/2 complex could be demonstrated. The fact that Phb1/2 is a large multimeric complex, which provides protection of native peptides against proteolysis, suggests a functional homology with protein chaperones with respect to their ability to hold and prevent misfolding of newly synthesized proteins.

Amino Acid Sequence↗
PubMed1998

Fen1 expression: a novel marker for cell proliferation.

The identification of antigens whose expression is associated with the cell cycle is a particularly attractive method with which to define proliferative populations in histological and cytological preparations. A polyclonal antibody 3220 has been raised which recognizes the structure-specific endonuclease Fen1 and can be used for a wide range of applications including western blotting, immunoprecipitation and immunohistochemical analysis. This antibody has been used to examine Fen1 levels by immunoblotting and its subcellular localization in cultured cells and tissue samples by immunostaining. Although the role Fen1 plays in DNA replication has been well characterized, its function in DNA repair is not so clear. The possible roles of Fen1 in repair have been investigated by examining any changes in level or localization of Fen1 in response to DNA damaging agents. We find that Fen1 is a nuclear antigen, that it is expressed by cycling cells, and that it co-localizes with PCNA and polymerase alpha during S phase. Fen1 expression is topologically regulated in vivo and is associated with proliferative populations. No change has been found in either patterns or levels of Fen1 expression induced by DNA damaging agents, either in vivo or in vitro. This anti-Fen1 antiserum is well suited to the analysis of proliferation in histological material, since (1) the proportion of labelled cells equals the experimentally determined growth fraction in an experimental xenograft system and (2) unlike markers such as PCNA, Fen1 is not induced by DNA damage.

3T3 Cells↗
PubMed1997

The prohibitin family of mitochondrial proteins regulate replicative lifespan.

Cellular senescence is determined by multiple factors, including the genetic regulation of metabolism and responses to endogenous and exogenous stresses [1-4]. Recent studies implicate a limited number of gene products in elongating lifespan in yeast and Caenorhabditis elegans [2-4]; these include the C, elegans gene cik-1, a central regulator of metabolism [5], and yeast RAS2, which controls the response to ultraviolet irradiation and other stresses [3]. Another gene postulated to effect senescence is PHB1, the yeast homologue of prohibitin [3], a rodent gene initially identified as a potential regulator of growth arrest and tumour suppressor [6-8]. Highly conserved prohibitin homologues have been identified in mammals [9], Drosophila [10], C. elegans [9], plants [11] and yeast. A second mammalian gene, encoding BAP37, a protein with sequence similarity to prohibitin, is thought to be involved in lymphocyte function [9]. Here, we show that the nuclear-encoded mammalian prohibitin and BAP37 proteins are present in mitochondria, are co-expressed, and interact physically with each other. Deletion of the Saccharomyces cerevisiae homologues, PHB1 and PHB2, results in a decreased replicative lifespan and a defect in mitochondrial membrane potential. Our observations highlight the relationship between the metabolic efficiency of cells and the ageing process, and provide evidence for its evolutionary conservation.

Amino Acid Sequence↗
PubMed1997

Distinct nuclear assembly pathways for lamins A and C lead to their increase during quiescence in Swiss 3T3 cells.

The expression of A-type lamins coincides with cell differentiation and as A-type lamins specifically interact with chromatin, a role in the regulation of differential gene expression has been suggested for A-type lamins. Using the mouse Swiss 3T3 cell line as a model, the change in two A-type lamins, lamins A and C, during cellular quiescence has been investigated. This well established model system mimics the first stages of differentiation when cells exit the cell cycle. In fact, quiescence in Swiss 3T3 cells was accompanied by a significant increase (2.6-fold) in lamin A protein levels and a smaller but reproducible increase (1.4-fold) in lamin C. These effects were fully reversible upon restimulation of the cells with serum. No effect upon lamin B levels was observed. Conversely, levels of A-type lamin mRNA decreased markedly as a result of quiescence suggesting transcriptional mechanisms are involved in the change in levels of lamins A and C. No difference in the incorporation of microinjected human lamin A into nuclei of quiescent or proliferating cells was observed. These data suggest A-type lamin binding sites were not limiting and indicated little difference between A-type lamin assembly mechanisms in quiescent and proliferating cells. The data did demonstrate lamin A and lamin C incorporation into the nuclear lamina proceeded by different pathways when microinjected in Swiss 3T3 cells. The incorporation of recombinant lamin C into the nuclear lamina was delayed compared to lamin A and proceeded via intranuclear foci. Such foci were not seen with microinjected lamin A. Instead, recombinant lamin A was rapidly (<20 minutes) incorporated into the nuclear lamina. Comicroinjection of lamin A with lamin C did not prevent foci formation but assisted in the rapid clearing (t1/2=30 minutes) of these structures and the incorporation of both lamins A and C into the lamina. These data suggest that the incorporation of lamin C into the lamina is facilitated by lamin A. They demonstrate a distinct difference in the nuclear assembly pathways of lamins A and C and show for the first time a functional distinction for these two splice variants of the A-type lamin gene. From the differences in assembly pathways and changes in protein levels accompanying quiescence in 3T3 cells, we suggest distinct roles for lamin A and lamin C in proliferating and quiescent states of the cell cycle.

3T3 Cells↗
PubMed1996

Identification of the antigen recognized by the monoclonal antibody BU31 as lamins A and C.

The murine monoclonal antibody BU31 binds to the nuclear membrane of many cell types. The expression of the BU31 antigen has previously been shown to have an inverse correlation with the proliferative index in lung tumours, defined by Ki67 staining. The distribution of BU31-positive cells is now shown to parallel the distribution of non-dividing cells in a range of normal human and rat tissues, although neuroendocrine cells and germ cells in the testis show no reactivity. Cells grown in culture and induced to undergo growth arrest show a higher level of labelling with BU31 than their proliferating counterparts. Confocal laser scanning microscopy reveals that the BU31 antigen is distributed predominantly along the nuclear lamina, with occasional internal foci. This distribution is very similar to that of the nuclear membrane proteins lamin A and lamin C, suggesting that the BU31 antigen and lamins A and C could be one and the same. Immunoblotting using recombinant lamin proteins confirmed this proposal. Moreover, a monoclonal antibody to the non-proliferation-associated antigen, statin, also recognizes lamins A and C. These data indicate that the demonstration of lamins A and C can be used to provide information on the proliferative activity of normal and neoplastic tissues. These data also suggest a role for nuclear lamins A and C during cellular quiescence, possibly through the reorganization and maintenance of nuclear structure, or more directly through interactions with the retinoblastoma gene product or related proteins.

3T3 Cells↗
PubMed1996

The association between cell proliferation and apoptosis: studies using the cell cycle-associated proteins Ki67 and DNA polymerase alpha.

The process of apoptosis is associated with the inappropriate expression of cell cycle regulatory proteins, which has led to the proposal that the apoptotic pathway represents an abortive attempt to pass through the cell proliferation cycle. To investigate this hypothesis, we examined the expression of two proliferation-associated antigens in apoptotic cells. Apoptotic bodies seen in a range of normal and pathological tissues are often positive for the Ki67 antigen, indicating that these cells were in the cell cycle during the period that they died. In contrast, spontaneous apoptosis of human polymorphonuclear leukocytes maintained in culture was not associated with the expression of either Ki67 or DNA polymerase a. In addition, apoptotic bodies in the pre-menstrual endometrium did not express the Ki67 antigen. These results indicate that, contrary to previous suggestions, apoptosis does not always depend on cell cycle entry. The use of antibodies to Ki67 should be valuable in defining the association of apoptosis with proliferation in a wide range of cells and tissues.

Apoptosis↗