PIK3CA mutation spectrum in urothelial carcinoma reflects cell context-dependent signaling and phenotypic outputs.

Although activating mutations of PIK3CA are frequent in urothelial carcinoma (UC), no information is available on their specific effects in urothelial cells or the basis for the observed mutation spectrum, which has a large excess of helical domain mutations. We investigated the phenotypic and signaling consequences of hotspot and UC-specific rare PIK3CA mutations in immortalized normal human urothelial cells (NHUC) and mouse fibroblasts (NIH3T3). Our results indicate that in NHUC, rare mutant forms and all three hotspot mutant forms of PIK3CA can activate the PI3K/AKT pathway. The relative frequency at which helical domain and kinase domain mutations are found in UC is related to their potency in inducing signaling downstream of AKT and to the phenotypic effects induced in this cell type (E545K>E542K>H1047R). Helical domain mutations E542K and E545K conferred a significant proliferative advantage at confluence and under conditions of nutrient depletion, and increased cellular resistance to anoikis. Both helical and kinase domain mutants induced increased NHUC cell motility and migration towards a chemoattractant, though no significant differences were found between the mutant forms. In NIH3T3 cells, the kinase domain mutant H1047R induced high levels of AKT activation, but helical domain mutants were significantly less potent and this was reflected in their relative abilities to confer anchorage-independent growth. Our findings indicate that the effects of mutant PIK3CA are both cell type- and mutation-specific. Helical domain mutations in PIK3CA may confer a selective advantage in the urothelium in vivo by overcoming normal contact-mediated inhibitory signals and allowing proliferation in nutrient-limiting conditions. Mutant forms of PIK3CA may also stimulate intraepithelial cell movement, which could contribute to spread of cells within the urothelium.

AKT1 mutations in bladder cancer: identification of a novel oncogenic mutation that can co-operate with E17K.

The phosphatidylinositol-3-kinase (PI3 kinase)-AKT pathway is frequently activated in cancer. Recent reports have identified a transforming mutation of AKT1 in breast, colorectal, ovarian and lung cancers. We report here the occurrence of this mutation in bladder tumours. The AKT1 G49A (E17K) mutation was found in 2/44 (4.8%) bladder cancer cell lines and 5/184 (2.7%) bladder tumours. Cell lines expressing mutant AKT1 show constitutive AKT1 activation under conditions of growth factor withdrawal. We also detected a novel AKT1 mutation G145A (E49K). This mutation also enhances AKT activation and shows transforming activity in NIH3T3 cells, though activity is weaker than that of E17K. Enhanced activation of AKT1 when E17K and E49K mutations are in tandem suggests that they can co-operate.

Social and ethnic differences in attendance for antenatal care in England.

OBJECTIVES: Evidence about sociodemographic factors associated with late attendance for antenatal care in the UK is of poor quality. This study aimed to identify any social or ethnic differences in access to antenatal care, and to quantify the effect of any such differences using data collected in a survey of women's experiences of antenatal screening. STUDY DESIGN: Cross-sectional survey using a postal questionnaire. METHODS: A stratified clustered random sampling strategy was used. Hospitals in England were stratified according to ethnic mix. In order to ensure inclusion of an adequate number of women from Black and Minority Ethnic (BME) backgrounds, hospitals with >or= 15% of women of BME origin were oversampled. Pregnant women aged >or= 16 years, receiving care in 15 participating hospitals, were sent a postal questionnaire at 27-31 weeks of gestation. Logistic regression was used to estimate odds ratios (ORs) comparing social and ethnic groups for attendance for antenatal care, adjusting for sociodemographic and clinical factors. RESULTS: In total, 839 women (57%) returned completed questionnaires. Compared with all women giving birth in 2005 in England and Wales, the survey sample contained fewer women aged <20 years (5.8% vs 6.9%), more women aged >35 years (24.1% vs 19.6%) and fewer women who were born outside the UK (14.8% vs 20.8%). Five percent of responders were late attenders for their first antenatal appointment. The odds of late initiation of antenatal care were higher for women born outside the UK [OR 4.37, 95% confidence interval (CI) 2.25-8.52; P=0.0004] and for women living without a husband/partner (OR 2.74, 95% CI 1.81-4.16; P=0.0002). In total, 2.5% of women were late attenders for their booking appointment. The odds of late booking were higher for Black women (OR 5.92, 95% CI 2.97-11.83) and women living without a husband/partner (OR 1.95, 95% CI 0.97-3.93; P=0.06). CONCLUSIONS: A small proportion of women initiate and/or book late for antenatal care. This study provides recent, good-quality evidence that women born outside the UK and those living without a husband/partner may be at particular risk of late attendance for antenatal care.

MCAK associates with EB1.

The microtubule (MT)-associated protein EB1 localizes to and promotes growth at MT plus ends. The MT depolymerizing kinesin MCAK has also been reported to track growing MT plus ends. Here, we confirm that human MCAK colocalizes with EB1 at growing MT ends when expressed as a GFP fusion protein in transfected cells. We show that MCAK associates with the C-terminus of EB1 and EB3 but much less efficiently with RP1. EB1 associates with the N-terminal localization and regulatory domain in MCAK but not with the motor domain of the protein. The interaction is competitive with the binding of other EB1 ligands and does not require MTs. Knockdown of EB1 expression using siRNA impaired the ability of GFP-MCAK to localize to MT tips in transfected cells. We propose that MCAK is targeted to growing MT ends by EB1, that MCAK is held in an inactive conformation when associated with EB1 and that this could provide the basis for a mechanism that facilitates rapid switching between phases of MT growth and depolymerization.

Adenomatous polyposis coli localization is both cell type and cell context dependent.

The adenomatous polyposis coli (APC) tumor suppressor protein is mutated in most colorectal carcinomas. In addition to its role in WNT signaling it is proposed to be involved in both cell migration and mitosis. Although a variety of studies have shown an APC localization along lateral membranes of adjacent epithelial cells the existence of a cortical APC localization in mammalian cells remains controversial. To address this we have used matched rat epithelial (NRK-52E) and fibroblast (NRK-49F) cell lines to investigate the localization of APC. Subconfluent cultures of NRK-52E and -49F cells displayed microtubule-associated APC populations by immunostaining. However, confluent NRK-52E, but not -49F monolayers, exhibited a cortical APC distribution. Cortical APC localized in close proximity to a number of cell junction proteins in a microtubule-independent manner while calcium switch experiments suggested that APC was recruited to the cortex only when junction assembly was complete. Confluent NRK-49F and -52E cells also showed contrasting APC localizations in response to monolayer wounding. Our data suggests APC cortical localization is a feature of confluent epithelioid cells and that the subcellular distribution of APC is therefore dependent upon both cell type and context.

Evidence that an interaction between EB1 and p150(Glued) is required for the formation and maintenance of a radial microtubule array anchored at the centrosome.

EB1 is a microtubule tip-associated protein that interacts with the APC tumor suppressor protein and components of the dynein/dynactin complex. We have found that the C-terminal 50 and 84 amino acids (aa) of EB1 were sufficient to mediate the interactions with APC and dynactin, respectively. EB1 formed mutually exclusive complexes with APC and dynactin, and a direct interaction between EB1 and p150(Glued) was identified. EB1-GFP deletion mutants demonstrated a role for the N-terminus in mediating the EB1-microtubule interaction, whereas C-terminal regions contributed to both its microtubule tip localization and a centrosomal localization. Cells expressing the last 84 aa of EB1 fused to GFP (EB1-C84-GFP) displayed profound defects in microtubule organization and centrosomal anchoring. EB1-C84-GFP expression severely inhibited microtubule regrowth, focusing, and anchoring in transfected cells during recovery from nocodazole treatment. The recruitment of gamma-tubulin and p150(Glued) to centrosomes was also inhibited. None of these effects were seen in cells expressing the last 50 aa of EB1 fused to GFP. Furthermore, EB1-C84-GFP expression did not induce Golgi apparatus fragmentation. We propose that a functional interaction between EB1 and p150(Glued) is required for microtubule minus end anchoring at centrosomes during the assembly and maintenance of a radial microtubule array.

EB1 identifies sites of microtubule polymerisation during neurite development.

EB1 is a microtubule associated protein which interacts with the APC tumour suppressor protein and components of the cytoplasmic dynein/dynactin complex. EB1 is also a specific marker of growing microtubule tips. Here we demonstrate that EB1 protein levels are increased during axon but not dendrite formation in differentiated N2A neuroblastoma cells, and that EB1 localises to microtubule tips throughout extending neurites in these cells. In N2A axons, analysis of the ratio of EB1/beta-tubulin fluorescence demonstrated that the distal tip region contained the highest proportion of polymerising microtubules. Time-lapse confocal imaging of an EB1-GFP fusion protein in transfected N2A cells directly revealed the dynamics of microtubule extension in neurites, and demonstrated the existence of unusual, discrete knots of microtubule polymerisation at the periphery of non-process bearing cells which may represent an early event in neurite outgrowth. We conclude that EB1 localisation can be used to identify and analyse sites of microtubule polymerisation at a high resolution during neurite development, a process to which it may contribute.

EB 1 immunofluorescence reveals an increase in growing astral microtubule length and number during anaphase in NRK-52E cells.

Spindle positioning in animal cells is thought to rely upon the interaction of astral microtubules with the cell cortex. Information on the dynamics of astral microtubules during this process is scarce, in part because of the difficulty in visualising these microtubules by light microscopy. EB1 is a protein which specifically localises to growing microtubule distal tips. Immunostaining for EB1 therefore represents a powerful method for visualising the distribution of growing microtubule tips within cells. In this study we used EB1 immunostaining in mitotic NRK-52E cells to quantitatively analyse the length and number of growing astral microtubules during metaphase and anaphase. We observed a dramatic increase in growing astral microtubule length and number during anaphase. Furthermore, drug treatments which specifically destroyed astral microtubules resulted in an increase in misaligned anaphase but not metaphase spindles. We suggest that an anaphase-specific increase in growing astral microtubule length and number facilitates the maintenance of a correctly aligned spindle in mitotic NRK-52E cells.

Regulation and function of the interaction between the APC tumour suppressor protein and EB1.

The interaction between the adenomatous polyposis coli (APC) tumour suppressor and the microtubule-associated protein EB1 was examined. Immunoprecipitation suggested that APC and EB1 were not associated in cultures of HCT116 cells arrested in mitosis. The C-terminal 170 amino acids of APC, purified as a bacterial fusion protein, precipitated EB1 from cell extracts, significantly refining the location of the EB1 interaction domain in APC. In vitro phosphorylation of this fusion protein by either protein kinase A or p34cdc2 reduced its ability to bind to EB1. Expression of GFP fusions to C-terminal APC sequences lacking or including the APC basic domain but encompassing the EB1 binding region in SW480 cells revealed a microtubule tip association which co-localized with that of EB1. Expression of the basic domain alone revealed a non-specific microtubule localization. In vitro interaction studies confirmed that the APC basic domain did not contribute to EB1 binding. These findings strongly suggest that the interaction between APC and EB1 targets APC to microtubule tips, and that the interaction between the two proteins is down-regulated during mitosis by the previously described mitotic phosphorylation of APC.

Truncated adenomatous polyposis coli (APC) tumour suppressor protein can undergo tyrosine phosphorylation.

Numerous mutations in the adenomatous polyposis coli (APC) gene have been described in colorectal cancer. The vast majority introduce nonsense codons leading to the production of truncated N-terminal APC fragments. Mutations occurring before APC codon 158, have been associated with an attenuated form of familial adenomatous polyposis whereas those occurring at codon 168 or beyond lead to the characteristic form of the disease. These 10 amino acid residues of APC contain a YYAQ motif which appears to constitute a potential SH2 binding domain similar to a sequence present in tyrosine kinase receptors that activate STAT 3 when phosphorylated. We have expressed a recombinant, N-terminal APC fragment in bacterial cells, and shown that it can indeed undergo tyrosine phosphorylation in this domain. We used site-directed mutagenesis to confirm the specificity of the reaction. These observations raise the possibility that tyrosine phosphorylation may be another mechanism involved in controlling APC function.

Consensus development methods: a review of best practice in creating clinical guidelines.

BACKGROUND: Although there is debate about the appropriate place of guidelines in clinical practice, guidelines can be seen as one way of assisting clinicians in decision-making. Given the likely diversity of opinion that any group of people may display when considering a topic, methods are needed for organising subjective judgements. Three principal methods (Delphi, nominal group technique, consensus development conference) exist which share the common objective of synthesising judgements when a state of uncertainty exists. OBJECTIVES: To identify the factors that shape and influence the clinical guidelines that emerge from consensus development methods and to make recommendations about best practice in the use of such methods. METHODS: Five electronic databases were searched: Medline (1966-1996), PsychLIT (1974-1996), Social Science Citation Index (1990-1996), ABI Inform and Sociofile. From the searches and reference lists of articles a total of 177 empirical and review articles were selected for review. RESULTS: The output from consensus development methods may be affected by: the way the task is set (choice of cues, recognition of contextual cues, the focus of the task, the comprehensiveness of the scenarios); the selection of participants (choice of individuals, degree of homogeneity of the group, their background, their number); the selection and presentation of scientific information (format, extent to which its quality and content is assessed); the way any interaction is structured (number of rating rounds, ensuring equitable participation, physical environment for meetings); and the method of synthesising individual judgements (definition of agreement, rules governing outliers, method of mathematical aggregation). CONCLUSIONS: Although a considerable amount of research has been carried out, many aspects have not been investigated sufficiently. For the time being at least, advice on those aspects has, therefore, to be based on the user's own commonsense and the experience of those who have used or participated in these methods. Even in the long term, some aspects will not be amenable to scientific study. Meanwhile, adherence to best practice will enhance the validity, reliability and impact of the clinical guidelines produced.

EB1, a protein which interacts with the APC tumour suppressor, is associated with the microtubule cytoskeleton throughout the cell cycle.

The characteristics of the adenomatous polyposis coli (APC) associated protein EB1 were examined in mammalian cells. By immunocytochemistry EB1 was shown to be closely associated with the microtubule cytoskeleton throughout the cell cycle. In interphase cells EB1 was associated with microtubules along their full length but was often particularly concentrated at their tips. During early mitosis, EB1 was localized to separating centrosomes and associated microtubules, while at metaphase it was associated with the spindle poles and associated microtubules. During cytokinesis EB1 was strongly associated with the midbody microtubules. Treatment with nocodazole caused a diffuse redistribution of EB1 immunoreactivity, whereas treatment with cytochalasin D had no effect. Interestingly, treatment with taxol abolished the EB1 association with microtubules. In nocodazole washout experiments EB1 rapidly became associated with the centrosome and repolymerizing microtubules. In taxol wash-out experiments EB1 rapidly re-associated with the microtubule cytoskeleton, resembling untreated control cells within 10 min. Immunostaining of SW480 cells, which contain truncated APC incapable of interaction with EB1, showed that the association of EB1 with microtubules throughout the cell cycle was not dependent upon an interaction with APC. These results suggest a role for EB1 in the control of microtubule dynamics in mammalian cells.

Expression of beta-catenin and the adenomatous polyposis coli tumour suppressor protein in mouse neocortical cells in vitro.

Beta-catenin is known to associate with the tumour suppressor protein adenomatous polyposis coli (APC), which is highly expressed in developing brain. We have therefore investigated the distribution of beta-catenin and APC in primary cultures of mouse neocortex. Western blotting demonstrated the presence of a single beta-catenin species in our cultures. Immunocytochemistry showed that beta-catenin was plasma membrane associated and concentrated in growth cones in cultured neurons. The APC tumour suppressor protein was also concentrated in growth cones. In glial cells, beta-catenin was localised at cell-cell contacts in a manner similar to that previously described in other cell types. This data suggests a role for both APC and beta-catenin in neuronal growth cones, and for beta-catenin in the formation of cell to cell contacts between glia.

The cellular distribution of the adenomatous polyposis coli tumour suppressor protein in neuroblastoma cells is regulated by microtubule dynamics.

The adenomatous polyposis coli tumour suppressor protein is highly expressed in developing rodent brain, but its function is unclear. Recent studies have suggested a role for this protein in regulating microtubule dynamics. Neuro 2A mouse neuroblastoma cells were previously thought not to express this protein. Using immunochemical techniques, this report corrects this observation. Immunoreactive bands of a size consistent with that of the full-length protein were observed by western blotting. Using immunocytochemistry, punctate immunoreactivity localized to areas of the cell containing microtubules, particularly neurite growth cones, in a distribution suggesting a role in neuritogenesis and growth cone extension. The protein did not localize to actin-rich cellular structures, and perturbation of the actin cytoskeleton had no effect upon this distribution. Treatment of cells with taxol to stabilize microtubules caused the concentration of the immunoreactive puncta to the tips of microtubules and areas along the axis of potential microtubule assembly. Treatment of cells with the microtubule disrupting reagent nocodazole showed that over shorter times the punctate distribution was not dependent upon polymerized microtubules. However, at longer incubation times a decrease in punctate immunostaining was observed. These results indicate that the intracellular distribution of the adenomatous polyposis coli protein is dependent upon microtubule but not actin dynamics. A role for this protein in the regulation of directed microtubule assembly is suggested.

Ageing in black and ethnic minorities: a challenge to service provision.

Health services should not be colour-blind or ethnicity-blind. Service providers need to be aware of any differences in need between ethnic groups as well as between genders, age groups, and so on. There will be a greatly increased number of older people among minority ethnic groups in Britain in the next 10-20 years. This article reviews some of the reasons why this is a challenge to the health and care services.

Professionals' criteria for accepting people as patients.

KIE: Interviews with health professionals caring for stroke patients, and other data, were examined to determine some of the criteria used by health professionals in accepting, rejecting, or referring patients. Four categories of criteria used in the decision making process were identified: the nature of the medical condition, the personal characteristics of the patient, the patient's environment, and the type of health service setting. Nineteen more specific selection criteria were derived from these four. Askham concludes with a discussion of the implications of her findings on professional decision making, and with suggestions of areas for further research.^ieng