Trudi Harris

Refractive Index Measurement in Viable Cells Using Quantitative Phase-Amplitude Microscopy and Confocal Microscopy

The refractive index (RI) of cellular material provides fundamental biophysical information about the composition and organizational structure of cells. Efforts to describe the refractive properties of cells have been significantly impeded by the experimental difficulties encountered in measuring viable cell RI. In this report we describe a procedure for the application of quantitative phase microscopy in conjunction with confocal microscopy to measure the RI of a cultured muscle cell specimen.

The importance of ERK activity in the regulation of cyclin D1 levels and DNA synthesis in human cultured airway smooth muscle

The relationship between persistent ERK (extracellular signal‐regulated kinase) activity, cyclin D1 protein and mRNA levels and cell cycle progression in human cultured airway smooth muscle was examined in response to stimulation by ET‐1 (endothelin‐1), thrombin and bFGF (basic fibroblast growth factor). Thrombin (0.3 and 3 u ml−1) and bFGF (0.3 and 3 nM) increased ERK activity for more than 2 h and increased cell number, whereas ET‐1 (100 nM) transiently stimulated ERK activity and was non‐mitogenic. The MEK1 (mitogen‐activated ERK kinase) inhibitor, PD 98059 (30 μM), inhibited both ERK phosphorylation and activity, and either prevented (thrombin 0.3 and 3 u ml−1, bFGF 300 pM) or attenuated (bFGF 3 nM) DNA synthesis. Thrombin and bFGF increased both cyclin D1 mRNA and protein levels. PD 98059 decreased cyclin D1 protein levels stimulated by the lower but not higher thrombin concentrations. Moreover, increases in cyclin D1 mRNA levels were unaffected by PD 98059 pretreatment, irrespective of the mitogen or its concentration, suggesting that inhibition of cyclin D1 protein levels occurred by a post‐transcriptional mechanism. These findings indicate that the control of cyclin D1 protein levels may occur independently of the MEK1/ERK signalling pathways. The inhibition of S phase entry by PD 98059 at higher thrombin concentrations appears to result from effects on pathways downstream or parallel to those regulating cyclin D1 protein levels. These findings suggest heterogeneity in the signalling of DNA synthesis in human cultured airway smooth muscle.

Mitogenic actions of endothelin-1 and epidermal growth factor in cultured airway smooth muscle.

1. Hyperplasia of airway smooth muscle contributes to the increase in bronchomotor responsiveness that characterizes asthma. We have investigated the mitogenic potential of endothelin‐1 (ET‐1) and epidermal growth factor (EGF) in guinea‐pig cultured airway smooth muscle and the relationship of these actions to tyrosine phosphorylation and increases in intracellular calcium (Ca2+i).

PPARγ ligands, 15‐deoxy‐Δ12,14‐prostaglandin J2 and rosiglitazone regulate human cultured airway smooth muscle proliferation through different mechanisms

The influence of two peroxisome proliferator‐activated receptor γ (PPARγ) ligands, a thiazolidinedione, rosiglitazone (RG) and the prostaglandin D2 metabolite 15‐deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2) on the proliferation of human cultured airway smooth muscle (HASM) was examined. The increases in HASM cell number in response to basic fibroblast growth factor (bFGF, 300 pM) or thrombin (0.3 U ml−1) were significantly inhibited by either RG (1–10 μM) or 15d‐PGJ2 (1–10 μM). The effects of RG, but not 15d‐PGJ2, were reversed by the selective PPARγ antagonist GW9662 (1 μM). Neither RG nor 15d‐PGJ2 (10 μM) decreased cell viability, or induced apoptosis, suggesting that the regulation of cell number was due to inhibition of proliferation, rather than increased cell death. Flow‐cytometric analysis of HASM cell cycle distribution 24 h after bFGF addition showed that RG prevented the progression of cells from G1 to S phase. In contrast, 15d‐PGJ2 caused an increase in the proportion of cells in S phase, and a decrease in G2/M, compared to bFGF alone. Neither RG nor 15d‐PGJ2 inhibited ERK phosphorylation measured 6 h post mitogen addition. The bFGF‐mediated increase in cyclin D1 protein levels after 8 h was reduced in the presence of 15d‐PGJ2, but not RG. Although both RG and 15d‐PGJ2 can inhibit proliferation of HASM irrespective of the mitogen used, only the antiproliferative effects of RG appear to be PPARγ‐dependent. The different antimitogenic mechanisms of 15d‐PGJ2 and synthetic ligands for PPARγ may be exploited to optimise the potential for these compounds to inhibit airway remodelling in asthma.

Collagen‐induced resistance to glucocorticoid anti‐mitogenic actions: a potential explanation of smooth muscle hyperplasia in the asthmatic remodelled airway

Glucocorticoids (GCS) inhibit mitogenesis of airway smooth muscle (ASM) cells grown on plastic. We have now evaluated the effects of GCS on proliferation of ASM grown on extracellular matrix proteins (ECM) abundant in noninflamed airways (laminin) and in fibrotic asthmatic airways (collagen type I). Dexamethasone inhibited basic fibroblast growth factor (bFGF)‐induced proliferation in cells maintained on laminin, but not collagen. Cells grown on collagen were resistant to the anti‐mitogenic actions of fluticasone propionate. In addition, dexamethasone did not inhibit thrombin‐induced proliferation. Thus, resistance induced by collagen is not dependent on the mitogen and appears to be a class effect on GCS. The inhibition of bFGF‐induced granulocyte–macrophage colony‐stimulating factor production was unaffected by the ECM type on which cells were grown. The impaired anti‐mitogenic activity of GCS in cells maintained on collagen may be due to a lack of efficacy against the collagen‐amplified mitogenesis, rather than any defect in responsiveness that is specific to glucocorticoid receptor mechanisms.

Annexin-1 signals mitogen-stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2

The role of the calcium‐ and phospho‐lipid‐binding protein annexin I (ANXA1) in cell cycle regulation has been investigated in estrogen receptor (ER)‐positive MCF‐7 and ER‐negative MDA‐MB‐231 breast tumor cell lines. In MCF‐7 cells, ANXA1‐targeting small interfering RNA (siRNA) reduced ANXA1 mRNA and protein levels and attenuated cell proliferation induced by FCS, estradiol, or epidermal growth factor. Well‐characterized agonists for the known ANXA1 receptor, FPR2, including the ANXA1 N‐terminal proteolytic product ANXA12_26, lipoxin A4 (LXA4), and the synthetic peptide, Trp‐Lys‐Tyr‐Met‐Val‐D‐Met (WKYMVm), stimulated proliferation of MCF‐7 and MDA‐MB‐231 cells that was attenuated by incubation with FPR2 antagonists WRW4 (1 µM) or Boc2 (100 nM) or by siRNA against FPR2. FCS‐induced mitogenic responses were attenuated by each of the FPR antagonists and by siRNAagainst FPR2 and, to a lesser extent, FPR1. LXA4 increased phosphorylation of Akt, p70S6K but not ERK1/2. Increases in cyclin D1 protein induced by FCS or LXA4 were blocked by the PI3 kinase inhibitor, LY294002, and attenuated by FPR2 antagonism using Boc2. In invasive breast cancer, immunohis‐tochemistry revealed the presence of ANXA1 and its receptor, FPR2, in both tumor epithelium and stromal cells. These observations suggest a novel signaling role for ANXA1 in mitogen‐activated proliferation of breast tumor epithelial cells that is mediated via activation of FPR1 and FPR2.—Khau, T., Langenbach, S. Y., Schu‐liga, M., Harris, T., Johnstone, C. N., Anderson, R. L., Stewart, A. G. Annexin‐1 signals mitogen‐stimulated breast tumor cell proliferation by activation of the formyl peptide receptors (FPRs) 1 and 2. FASEB J. 25, 483_496 (2011). www.fasebj.org

2-Methoxyestradiol Is an Estrogen Receptor Agonist That Supports Tumor Growth in Murine Xenograft Models of Breast Cancer

Purpose: 2-Methoxyestradiol (2MEO) is being developed as a novel antitumor agent based on its antiangiogenic activity, tumor cell cytotoxicity, and apparent lack of toxicity. However, pharmacologic concentrations of 2MEO bind to estrogen receptors (ER). We have therefore examined the ER activity of 2MEO. Experimental Design: Estrogenic actions of 2MEO were evaluated by changes in gene expression of the ER-positive (MCF7) breast tumor cell line and, in vivo, estrogenicity was assessed in breast tumor xenograft models and by measuring endocrine responses in uterus and liver. Results: In the ER-positive breast tumor cell line (MCF7), microarray experiments revealed that 269 of 279 changes in gene expression common to 2MEO and estradiol were prevented by the ER antagonist, ICI 182,780. Changes in the expression of selected genes and their sensitivity to inhibition by ICI 182,780 were confirmed by quantitative reverse transcription–PCR measurement. Activation of ER in MCF7 cells by 2MEO was further confirmed by stimulation of an estrogen response element–dependent reporter gene that was blocked by ICI 182,780 (1 μmol/L). Doses of 2MEO (15-150 mg/kg) that had no antitumor efficacy in either nu/nu BALB/c or severe combined immunodeficient mice bearing ER-negative MDA-MB-435 tumors had uterotropic and hepatic estrogen-like actions. In female nu/nu BALB/c mice inoculated with the estrogen-dependent MCF7 tumor cells, 2MEO (50 mg/kg/d) supported tumor growth. Conclusions: Tumor growth enhancement by 2MEO at doses generating serum levels (100-500 nmol/L) that have estrogenic activity suggests that a conservative approach to the further clinical evaluation of this agent should be adopted and that its evaluation in breast cancer is inappropriate.

Quantitative phase microscopy: a new tool for measurement of cell culture growth and confluency in situ

Quantitative phase microscopy (QPM) is a recently developed computational approach that provides quantitative phase measurements of specimen images obtained under bright-field conditions without phase- or interference-contrast optics. To perform QPM, an in-focus bright-field image is acquired, together with one positive and one negative de-focus image. An algorithm is then applied to produce a specimen phase map. In this investigation we demonstrate that manipulation of the phase map intensity histogram using novel, non-subjective thresholding and segmentation methods provides enhanced delineation of cells in culture. QPM was utilised to measure the growth behaviour of cultured airway smooth muscle cells over a 92-h period. There was a high degree of correlation between parallel QPM-derived confluency measurements and haemocytometry-derived counts of airway smooth muscle cells over this time period. Using QPM, translucent cells can be visualised with improved cell boundary definition allowing precise and reproducible measurements of cell culture confluency. Quantitative phase imaging provides a rapid, optically simple and non-destructive approach for measurement of cellular morphology. Further development of the QPM-based analysis methodology has the potential to provide even more refined measures of cellular growth.

Collagen impairs glucocorticoid actions in airway smooth muscle through integrin signalling

Airway wall remodelling in asthma is characterised by a number of structural changes, including an increase in the volume of airway smooth muscle (ASM), and the abundance of the extracellular matrix (ECM) protein, collagen, is increased. We have investigated the mechanism of collagen‐induced glucocorticoid resistance of proliferation, and migration of ASM.

Proliferation is not increased in airway myofibroblasts isolated from asthmatics

Airway mesenchymal cells, such as myofibroblasts and airway smooth muscle cells, contribute to inflammation, airway remodelling and hyperresponsiveness in asthma by excessive proliferation and inflammatory mediator production. Using endobronchial biopsies obtained from both nonasthmatic and asthmatic subjects, in situ proliferation was assessed by immunostaining for cyclin D1. The number of immunoreactive cells increased with asthma severity and was restricted to the epithelium and subepithelial connective tissue. Despite increases in smooth muscle area, cyclin D1 was not detected in cells in intact muscle bundles. Biopsy-derived cell cultures were characterised as predominantly myofibroblasts, and were assessed to determine whether proliferation and cytokine production varied with asthma status. Cell enumeration showed that basal proliferation was similar in cells from nonasthmatics and asthmatics, and mitogenic responses to fibroblast growth factor-2, thrombin or serum were either reduced or unchanged in cells from asthmatics. Interleukin (IL)-1-dependent granulocyte-macrophage colony-stimulating factor and IL-8 release was increased in cell supernatants from asthmatics. Thus, increased rates of cellular proliferation identified in situ in the asthmatic airway occurred outside the expanded smooth muscle compartment. Although reduced proliferative responses were observed in cultured myofibroblasts from asthmatics, the increased cytokine production by these cells suggests that this contributes to and may perpetuate ongoing inflammation in asthma.