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Biomedical subjects

C L Clarke

Publications and source records attributed to C L Clarke.

At least 19 recordsLinked to original sources

Mapping loss of heterozygosity in normal human breast cells from BRCA1/2 carriers.

We have studied loss of heterozygosity at the BRCA1 and BRCA2 loci in 992 normal cell clones derived from topographically defined areas of normal tissue in four samples from BRCA1/BRCA2 mutation carriers. The frequency of loss of heterozygosity in the clones was low (1.01%), but it was found in all four samples, whether or not a tumour was present. Topographical mapping revealed that the genetic changes were clustered in some breast samples. Our study confirms the previous finding that a field of genetic instability can exist around a tumour, suggesting that sufficient tissue must be removed at surgery to avoid local recurrence. We also demonstrate that such a field of genetic change can exist in morphologically normal tissue before a tumour develops and, for the first time, we demonstrate that the field is of a size greater than one terminal duct-lobular unit. The genetic changes are not identical, however, which suggests that genetic instability in these regions may play an early role in tumour development. We also confirm and extend our original observation of loss of the wild-type BRCA1 allele in some clones, and loss of the mutant allele in others, demonstrating that loss of either allele is a stochastic event.

Breast↗

Tamoxifen treatment promotes phosphorylation of the adhesion molecules, p130Cas/BCAR1, FAK and Src, via an adhesion-dependent pathway.

Reports that the adhesion-associated molecule p130Cas/BCAR1 promotes resistance to tamoxifen suggested that adhesion-mediated signalling may be altered by tamoxifen treatment. We find that p130Cas/BCAR1 phosphorylation is enhanced in tamoxifen-treated estrogen receptor (ER)-positive MCF-7 breast cancer cells. The effects of estrogen and tamoxifen were assessed independently and in combination, and the results demonstrate that tamoxifen antagonizes estrogen regulation of p130Cas/BCAR1 phosphorylation. Phosphorylation correlates with tamoxifen ER antagonist effects, as phosphorylation effects are replicated by the pure antiestrogen ICI 182, 780. Correspondingly, phosphorylation is not changed in ER-negative cells exposed to tamoxifen. We show that deletion of the p130Cas/BCAR1 substrate domain substantially reduces tamoxifen-induced phosphorylation of p130Cas/BCAR1 and confers enhanced sensitivity to tamoxifen. P130Cas/BCAR1 forms a phosphorylation-dependent signalling complex with focal adhesion kinase (FAK) and Src kinase that promotes adhesion-mediated cell survival. Therefore, we examined the kinetics of p130Cas/BCAR1, Src and FAK phosphorylation over a 14-day time course and find sustained phosphorylation of these molecules after 7 days exposure to tamoxifen. Inhibition of Src kinase is shown to reduce tamoxifen-promoted p130Cas/BCAR1 phosphorylation and reduce cell viability. Stimulation of the Src/FAK/p130Cas/BCAR1 adhesion signalling pathway in tamoxifen-treated MCF-7 cells does not cause increased migration; however, there is Src-dependent phosphorylation of the cell survival molecule Akt. Correspondingly, Akt inhibition reduces cell viability in cells treated with tamoxifen. We propose that prolonged activation of adhesion-dependent signalling may confer a survival advantage in response to additional cellular insults or alternatively, may poise cells to develop a migratory phenotype in response to additional cellular cues.

Antineoplastic Agents, Hormonal↗

Histopathologic indicators of breast cancer biology: insights from population mammographic screening.

Histopathologic features of breast cancer such as tumour size, grade and axillary lymph node (LN) status variably reflect tumour biology and time. Recent evidence suggests that the biological character of breast cancer is established at an early stage and has a major impact on clinical course. The aim of this study was to distinguish the impact of biology on breast cancer histopathology by comparing features of breast cancers diagnosed following population mammographic screening with prevalent vs incident detection and screening interval. Central histopathology review data from 1147 cases of ductal in situ and/or invasive breast cancer were examined. Size, grade and LN status of invasive cancers were positively correlated (P < 0.001). Prevalent invasive cancers were larger (P < 0.001) and more likely to be LN positive (P = 0.02) than incident cases, but grade was not associated with screening episode (P = 0.7). Screening interval for incident cancers was positively associated with invasive cancer size (P = 0.05) and LN status (P = 0.002) but not grade (P = 0.1). Together, these data indicate that biology and time both impact on size and LN status of invasive breast cancer, but grade reflects biology alone. In view of the clinical importance of breast cancer biology, grade as its most direct indicator assumes particular significance.

Adult↗

Effect of progesterone receptor a predominance on breast cancer cell migration into bone marrow fibroblasts.

Women exposed to exogenous progesterone have increased breast cancer risk, but the mechanisms of progesterone involvement in breast cancer development are unknown. In human breast and endometrium, progesterone receptor (PR) isoform expression is disrupted in premalignant lesions and predominance of one isoform, usually PRA, in invasive cancers is associated with poorer prognosis. Disrupted PR isoform expression results in disrupted progestin regulation of cell morphology, including rounded morphology and decreased adherence of cells to tissue culture flasks. The purpose of this study was to test the hypothesis that predominance of PRA affects the interaction of breast cancer cells with a physiologically relevant stromal tissue, bone marrow stroma. T-47D breast cancer cells demonstrated the ability to migrate into bone marrow fibroblasts and this was inhibited by progestin treatment. The antiprogestin RU38486 abrogated the progestin effect on migration, demonstrating that it was PR-mediated. In cells expressing a predominance of PRA, after induction of a stably integrated inducible PRA construct, the ability of progestin to inhibit breast cancer cell migration was lost. A number of integrins were progestin regulated in T-47D cells, but there was no difference in the progestin effect in cells with PRA predominance, nor were the levels of focal adhesion proteins altered in these cells. This suggested that the lack of inhibition by progestin of breast cancer cell migration in cells with PRA predominance was not mediated by PRA effects on the membrane components of the adherens junctions. In summary, this study has shown that PRA predominance has a striking functional effect on breast cancer cell migration into stromal layers. PRA predominance may render breast cancer cells relatively resistant to the inhibitory effects of progestins and one consequence of this may be increased invasion of stroma. If borne out in vivo, these findings suggest that tumours with PRA predominance may be predisposed to cancer progression and this may signal a poorer prognosis in patients.

Bone Marrow Neoplasms↗

Cytoskeletal responsiveness to progestins is dependent on progesterone receptor A levels.

Changes in the cell cytoskeleton occur in cell transformation and recent data suggest the involvement of ovarian hormones, which are implicated in cancer development and progression. In human breast and endometrial tumors, there is disrupted expression of progesterone receptor (PR) isoforms and predominance of one isoform, usually PRA. PRA predominance is an early event in carcinogenesis, and in cancers is associated with poor clinical features. Overexpression of PRA in vitro causes altered progestin regulation of cell morphology, suggesting that PRA overexpression may provoke deleterious changes in cell functioning. This study aimed to identify pathways of cytoskeleton regulation responsive to progestins and to determine whether these are perturbed when PRA is overexpressed to the levels seen in cancers. Progestin treatment of PR-positive breast cancer cells caused increased cell surface area whereas after induction of a stably integrated PRA construct, cells became rounded and the cell surface was decreased. The effect of PRA induction on cell rounding was reversed by the anti-progestin RU38486. Altered tropomyosin (Tm) isoforms were implicated in these morphological differences, as there was a PRA-mediated alteration in Tm5 isoform levels, and transfection of Tm5a mimicked progestin-mediated cell rounding in PRA-overexpressing cells. Ezrin was redistributed from the membrane to cytoplasmic locations in the presence of progestin, and discrete focal localization was evident in cells with PRA predominance. Progestin effects on the cytoskeleton in PRA-overexpressing cells provide evidence for novel endocrine regulation of aspects of actin microfilament composition, suggesting that changes in the cytoskeleton known to be associated with cancer development and progression may be regulated in part by altered PRA expression which develops early in carcinogenesis.

Actin Cytoskeleton↗

Loss of co-ordinate expression of progesterone receptors A and B is an early event in breast carcinogenesis.

Progesterone receptor (PR) mediates the effects of progesterone in mammary tissues and plays a crucial role in normal breast development and in breast cancer. PR proteins are expressed as two isoforms, PRA and PRB, that have different capacities to activate target genes, yet it is unknown whether progesterone action in normal and malignant breast is mediated by PRA and/or PRB. This study determines the relative expression of PRA and PRB in normal breast and in benign, premalignant and malignant archival breast lesions by dual immunofluorescent histochemistry. In normal breast and in proliferative disease without atypia (PDWA) PRA and PRB were co-expressed within the same cells in comparable amounts, implicating both isoforms in progesterone action. In atypical lesions, however, there was a significant increase in predominant expression of PRA or PRB, with lesion progression from the normal state to malignancy. PR isoform predominance, especially PRA predominance, was evident in a high proportion of ductal carcinomas in situ (DCIS) and invasive breast lesions. In the normal breast and in PDWA, the relative expression of PRA and PRB in adjacent cells was homogenous. There was a significant increase in cell-to-cell heterogeneity of PR isoform expression in ADH and DCIS lesions and in the majority of breast cancers. Heterogeneous cell-to-cell expression of PR isoforms occurred prior to overall predominant expression of one isoform in premalignant breast lesions, demonstrating that loss of control of relative PRA:PRB expression is an early event in the development of breast cancer. PRA:PRB ratios within a breast lesion are likely to be important as both markers and effectors of tumor growth and development, and progressively aberrant PR isoform expression may play a role in the etiology of breast cancer.

Adult↗

Relative expression of progesterone receptors A and B in endometrioid cancers of the endometrium.

The nuclear receptor for the female hormone progesterone (PR) is widely expressed in uterine cancer. PR is expressed as two proteins (PRA and PRB) with different functions, and in vitro evidence reveals PRA to inhibit PRB function, so the cellular ratio of PRA:PRB is likely to be an important determinant of progesterone action. The relative expression of PRA and B and their involvement in the pathogenesis of endometrial cancer is not known. The aims of this study were to determine PRA and B expression by dual immunofluorescent histochemistry in endometrial adenocarcinomas compared with expression in normal and hyperplastic glands, and to correlate expression in tumors with clinical features including grade. Significantly lower PR levels were found in tumors compared with normal glands and areas of complex atypical hyperplasia within the same specimen. The normal glands expressed both of the isoforms at similar levels, whereas there was increased predominance of one isoform in hyperplastic areas and in tumors, which suggested that the loss of coordinated expression of PR isoforms was an early event in tumor progression. The majority of tumors [27 (58%) of 46] expressed only one PR isoform, and the proportion expressing either PRA or B was the same [14 (30%) of 46, and 13 (28%) of 46, respectively]. One-half of all tumors ([23 (50%) of 46] expressed either PRA only or a predominance of PRA, and a few tumors [10 (22%) of 46] expressed comparable levels of PRA and B. Similar levels of PRA and B were noted only in FIGO grade 1 tumors, whereas higher grades (2 and 3) were associated with a predominance of one isoform. In summary, expression of only one PR isoform was common in endometrial cancers, which indicates that the decreased PR levels observed in these cancers arise from the loss of one PR isoform. Expression of a single PR isoform was associated with higher clinical grade, which suggests a relationship between the loss of PR isoform expression and features of poorer prognosis. Disruption of relative PR isoform expression was observed in complex atypical hyperplasia, which suggests that early alterations in the ratio of PRA:PRB may precede and/or be implicated in the development of endometrial adenocarcinoma. Alterations in the ratio of PR isoform expression are likely to cause disordered regulation of target genes, resulting in altered progestin action in the uterus, and this may be involved in the pathogenesis of endometrial cancer.

Adult↗

Professional and organizational learning: analysing the relationship with the development of practice.

BACKGROUND: Organizational and professional learning are interrelated processes that underpin the contemporary drive for a quality evidence-based delivery of health care in the United Kingdom (UK). DESIGN: A soft systems methodology was used to explore the pervasiveness of practice developments. Three case study sites were identified using matrix sampling and data collected through 29 individual interviews and two focus group interviews, with the interviews augmented with a tool designed to maximize analysis of the processes of developing practice. FINDINGS: The resultant model of developing health care practice includes three processes: using and creating knowledge, understanding and practice of patient care, and effecting development. The whole model was underpinned by professional and organizational learning in which 'expert thinkers' engaged in double loop learning to reconceptualize care rather than just perpetuate existing patterns of care delivery.

Attitude of Health Personnel↗

Detection of progesterone receptor forms A and B by immunohistochemical analysis.

AIM: The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR. METHODS: PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions. RESULTS: Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR. CONCLUSIONS: Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens.

Animals↗

The hD52 (TPD52) gene is a candidate target gene for events resulting in increased 8q21 copy number in human breast carcinoma.

Chromosome band 8q21 is frequently overrepresented in human cancer, but to date no 8q21 target gene has been proposed. The hD52 (TPD52) gene is of potential significance in breast and other cancers due to its location and expression pattern. Fine mapping of hD52 placed this locus within the peak of the 8q21 amplicon delineated in the SK-BR-3 breast carcinoma cell line, and a positive association between hD52 gene dosage and transcript levels was subsequently demonstrated in four breast carcinoma cell lines, including SK-BR-3. Increased copy number (ICN) was measured using Southern blot analyses in 3/32 human breast carcinomas at hD52, and the related hD54 gene in 20q13.2-q13.3. Subsequent immunohistochemical analysis of hD52 expression in 19 breast carcinomas with varying hD52 gene dosages demonstrated a significant positive association between hD52 dosage and hD52 expression using a Spearman rank correlation coefficient (r(s) = 0.573, alpha = 0.01) and a Wilcoxon rank-sum test (alpha = 0.05). On the basis of its map location and expression pattern in breast carcinoma, we therefore propose hD52 as a candidate target gene at chromosome band 8q21.

Aneuploidy↗

Heterogeneity of progesterone receptors A and B expression in human endometrial glands and stroma.

The human progesterone receptor (PR) is expressed as two isoforms, PRA and PRB, which function as ligand-activated transcription factors. In-vitro studies suggest that the isoforms differ functionally and that their relative expression in a target cell may determine the nature and magnitude of response to progesterone. We have shown recently that PRA and PRB are co-expressed in target cells of the human endometrium. The purpose of this study was to investigate the homogeneity of expression of PRA and PRB in target cells of the human uterus throughout the menstrual cycle. In the functionalis, PRA and PRB were expressed in comparable levels in glandular epithelium during the proliferative phase of the cycle, whereas there was persistence of PRB but not PRA in the glands during mid-secretory phase. In the stroma, there was predominance of the PRA isoform throughout the cycle. There was remarkable homogeneity in the relative expression of PRA and PRB in adjacent cells within the same tissue compartment, suggesting that the mechanisms regulating relative PR isoform expression are similarly active in these cells. By contrast, heterogeneity between glands was observed under some circumstances in the functionalis of the endometrium, suggesting PR isoform down-regulation by progesterone to be asynchronous. Heterogeneity was also seen between the glands of the basalis and functionalis of the endometrium implying region-specific responses to hormonal stimuli. This study demonstrates adjacent cell homogeneity in the relative expression of PRA and PRB in normal human endometrial tissue and a differential response to ovarian steroid hormones between cell types and between different regions within the same tissue.

Endometrium↗

Hearing the voices of children with chronic illness.

This qualitative study has aimed to explore children's, parents' and health professionals' experience of childhood chronic illness. Seven families and their professional carers participated in semistrucured interviews. The children's interviews were augmented with a 'drawing' technique. A grounded theory approach facilitated data collection and analysis. This paper debates the perceived passivity of the voice of children in health care and research and illustrates data collection methods that seek to give children and other disadvantaged groups a voice. The children who participated in this study are described as competent interpreters of their world.

Adaptation, Psychological↗

Histone deacetylase inhibitors decrease proliferation and modulate cell cycle gene expression in normal mammary epithelial cells.

Full-term pregnancy early in reproductive life is protective against breast cancer in women. The protective effects of parity have variously been attributed to the differentiation that accompanies pregnancy and lactation, alterations in ovarian hormone receptor levels, and altered sensitivity to ovarian hormones. Butyrate, a short-chain fatty acid, induces differentiation in breast cancer cell lines and decreases hormone receptor expression. Butyrate also inhibits proliferation in breast cancer cell lines and modulates expression of key cell cycle-regulatory proteins including cyclin D1. Given these properties, butyrate could be considered a promising agent for breast cancer prevention. Therefore, this study aimed to determine the effects of butyrate on normal human breast epithelial cells and to compare the effects of two stable butyrate derivatives with more favorable pharmacological properties: phenylacetate and its p.o. active precursor phenylbutyrate. Treatment with each agent resulted in concentration-dependent growth inhibition in a normal breast epithelial cell line and two breast cancer cell lines (MCF-7 and MDA-MB-231). Phenylbutyrate and butyrate inhibited proliferation to a similar extent, but phenylacetate was less effective in all of the cell lines. All three of the agents induced differentiation (accumulation of lipid droplets) in normal as well as in breast cancer cells and caused a decrease in estrogen receptor (ER) mRNA in MCF-7 cells. The butyrates decreased expression of cyclin D1, increased expression of p21(Waf1/Cip1), and hypophosphorylated pRB in the normal mammary epithelial cells. The effects on cyclin D1 expression correlated with the effects on cell proliferation, which suggests that modulation of cyclin D1 expression may underpin the antiproliferative effects of butyrates. We have shown that butyrate and butyrate-like agents are able to decrease proliferation and induce differentiation in normal breast cells as well as in malignant breast cells (ER-positive and ER-negative) and, as such, may be considered as candidate chemopreventative agents for women at high risk of developing breast cancer.

Anticarcinogenic Agents↗

Localization of the delta-opioid receptor and dopamine transporter in the nucleus accumbens shell: implications for opiate and psychostimulant cross-sensitization.

Opiate- and psychostimulant-induced modulation of dopamine transmission in the nucleus accumbens shell (AcbSh) is thought to play a key role in their potent reinforcing and locomotor effects. To investigate the cellular basis for potential functional interactions involving opiates active at the delta-opioid receptor (DOR) and psychostimulants that bind selectively to the dopamine transporter (DAT), we examined the electron microscopic localization of their respective antisera in rat AcbSh. DOR immunoperoxidase labeling was seen primarily, and DAT immunogold particles exclusively, in axon terminals. In these terminals, DOR immunoreactivity was prominently associated with discrete segments of the plasma membrane and the membranes of nearby small synaptic and large dense core vesicles. DAT immunogold particles were almost exclusively distributed along nonsynaptic axonal plasma membranes. Thirty-nine percent DOR-labeled profiles (221/566) either apposed DAT-immunoreactive terminals or also contained DAT. Of these 221 DOR-labeled profiles, 13% were axon terminals containing DAT and 15% were dendritic spines apposed to DAT-immunoreactive terminals. In contrast, 70% were morphologically heterogeneous axon terminals and small axons apposed to DAT-immunoreactive terminals. Our results indicate that DOR agonists in the AcbSh can directly modulate the release of dopamine, as well as postsynaptic responses in spiny neurons that receive dopaminergic input, but act principally to control the presynaptic secretion of other neurotransmitters whose release may influence or be influenced by extracellular dopamine. Thus, while opiates and psychostimulants mainly have differential sites of action, cross-sensitization of their addictive properties may occur through common neuronal targets.

Animals↗

N-methyl-D-aspartate receptor 1 in the caudate-putamen nucleus: ultrastructural localization and co-expression with sorcin, a 22,000 mol. wt calcium binding protein.

Entry of calcium through N-methyl-D-aspartate-type glutamate receptors in the caudate-putamen nucleus is essential for normal motor activity, but can produce cytotoxicity with continued stimulation and subsequent release of intracellular calcium. To determine potential functional sites for N-methyl-D-aspartate receptor activation in this region, we examined the ultrastructural localization of the R1 subunit of the N-methyl-D-aspartate receptor (NMDAR1) in rat brain. In addition, we comparatively examined the localization of NMDAR1 and sorcin, a 22,000 mol. wt calcium binding protein present in certain striatal neurons and involved in calcium-induced calcium release. NMDAR1-like immunoreactivity was seen at synaptic and non-synaptic sites on neuronal plasma membranes. Of 1514 NMDAR1-labeled profiles, 62% were dendrites and dendritic spines and the remainder were mainly unmyelinated axons and axon terminals. Sorcin-like immunoreactivity was present in 39% of the profiles that contained NMDAR1 labeling, most (533/595) of which were dendrites and dendritic spines. Of 1807 sorcin-labeled profiles, 42% were identified, however, as small processes including spine necks and unmyelinated axons or axon terminals. These profiles also occasionally contained NMDAR1 or showed synaptic or appositional contacts with other NMDAR1-immunoreactive neurons. The results of this study suggest that in the caudate-putamen nucleus, activation of NMDA receptors permits calcium influx at plasmalemmal sites mainly on dendrites where sorcin may play a role in calcium-induced calcium release. The presence of sorcin in some, but not all NMDA-containing neurons in the caudate-putamen nucleus has potential implications for the known differential vulnerability of certain striatal neurons to excitotoxins.

Animals↗

Absence of progesterone receptor associated with secondary breast cancer in postmenopausal women.

The relationship between expression of receptors for oestrogen and progesterone (ER and PR) and disease progression in breast cancer was investigated by comparing immunocytochemical determinations of ER and PR in fine needle aspirates from primary and secondary breast tumours. Rates of receptor expression were significantly higher in primary than in secondary lesions: for ER 63.3% (n = 689) compared with 45.3% (n = 223), and for PR 53.7% (n = 443) compared with 33.1% (n = 121). The effect of menopausal status was examined by subdividing the patient cohort into those over or under the age of 50 years. In both instances, ER expression in secondary tumours was relatively low; however, only postmenopausal patients had significantly lower rates of PR expression in secondary tumours. Consistent with this, an increase in the ER+PR- profile in secondary tumours compared with primary cases from postmenopausal patients was seen, and in a multivariate analysis, a specific absence of PR expression in secondary tumours was revealed. Comparison of ER and PR expression in simultaneously sampled primary tumours and lymph node metastases from the same patient showed that receptor expression was stable with progression to a metastatic site as results were concordant for ER in 92% (n = 88) and PR in 93.8% of cases (n = 65). These results suggest that absence of PR expression in primary breast cancer is associated with disease progression and may be a marker of an aggressive tumour phenotype.

Adult↗