Stacks and D-Brane Bundles

In this paper we describe explicitly how the twisted ``bundles'' on a D-brane worldvolume in the presence of a nontrivial B field, can be understood in terms of sheaves on stacks. We also take this opportunity to provide the physics community with a readable introduction to stacks and generalized spaces.Comment: 24 pages, LaTeX; v2: references adde

Role of Id-2 in the maintenance of a differentiated and noninvasive phenotype in breast cancer cells. Cancer Res

ABSTRACT Id proteins are inhibitors of basic helix-loop-helix transcription factors and generally stimulate cell proliferation and inhibit differentiation. We have shown that ectopic expression of Id-1 in murine mammary epithelial cells resulted in loss of differentiation and gain of invasive and proliferative abilities. Moreover, Id-1 was highly expressed in aggressive breast cancer cells in culture and in biopsies from infiltrating carcinomas. In contrast to Id-1, we found that, in vitro and in vivo, Id-2 mRNA and protein were up-regulated as mammary epithelial cells lost proliferative capacity and initiated differentiation. We further determined that this up-regulation of Id-2 was a necessary step toward a fully differentiated phenotype in breast cells. Here we show that one of the components of the extracellular matrix network, laminin, is responsible for the increase in Id-2 expression during differentiation. We also show that Id-2 expression is inversely correlated with the rate of proliferation in murine mammary epithelial cells and that Id-2 is expressed at a higher level in differentiated human breast cancer cells in comparison with very aggressive and metastatic cells. When reintroduced in aggressive breast cancer cells, Id-2 is able to reduce their proliferative and invasive phenotypes and decrease their level of matrix metalloproteinase 9 secretion as well as increase syndecan-1 expression. Moreover, little Id-2 protein expression is detectable in human biopsies from aggressive and invasive carcinomas in comparison with in situ carcinomas. In conclusion, Id-2 expression not only follows a pattern opposite to that of Id-1 during mammary gland development and breast cancer progression but also appears to act as an important protein for the maintenance of a differentiated and noninvasive phenotype in normal and transformed breast cells

Aberrant regulation of the BST2 (Tetherin) promoter enhances cell proliferation and apoptosis evasion in high grade breast cancer cells.

Normal cellular phenotypes that serve an oncogenic function during tumorigenesis are potential candidates for cancer targeting drugs. Within a subset of invasive primary breast carcinoma, we observed relatively abundant expression of Tetherin, a cell surface protein encoded by the Bone Marrow Stromal Cell Antigen (BST2) known to play an inhibitory role in viral release from infected immune cells of the host. Using breast cancer cell lines derived from low and intermediate histopathologic grade invasive primary tumors that maintain growth-suppressive TGFβ signaling, we demonstrate that BST2 is negatively regulated by the TGFβ axis in epithelial cells. Binding of the transcription factor AP2 to the BST2 promoter was attenuated by inhibition of the TGFβ pathway thereby increasing BST2 expression in tumor cells. In contrast, inherent TGFβ resistance characteristic of high grade breast tumors is a key factor underlying compromised BST2 regulation, and consequently its constitutive overexpression relative to non-malignant breast epithelium, and to most low and intermediate grade cancer cells. In both 2-dimensional and 3-dimensional growth conditions, BST2-silenced tumor cells displayed an enhancement in tamoxifen or staurosporine-induced apoptotic cell death together with a reduction in the S-phase fraction compared to BST2 overexpressing counterparts. In a subset of breast cancer patients treated with pro apoptotic hormonal therapy, BST2 expression correlated with a trend for poor clinical outcome, further supporting its role in conferring an anti apoptotic phenotype. Similar to the effects of gene manipulation, declining levels of endogenous BST2 induced by the phytoalexin - resveratrol, restored apoptotic function, and curbed cell proliferation. We provide evidence for a direct approach that diminishes aberrant BST2 expression in cancer cells as an early targeting strategy to assist in surmounting resistance to pro apoptotic therapies

BST2 immunolocalization in primary invasive breast cancer.

<p>The association between BST2 positive status and histological grade is significant (p<0.001).</p

Differential AP2 binding to the <i>BST2</i> promoter in primary breast cancer cell lines of varying histologic grade.

<p><b><i>A.</i></b> Schematic of the <i>BST2</i> promoter region spanning 239 bp upstream of the transcriptional start site, including 111 bp of exon 1. Numbering is relative to the translation start site, highlighted in green (+1). Potential <i>cis</i>-regulatory elements shown are either on the plus (+) or minus (–) DNA strands. Regulatory binding sequences: AP2 (blue), STAT1 (red) and STAT3 (purple). <b><i>B.</i></b><b><i>Top panel</i></b> - Chromatin immunoprecipitation (ChIP) performed with anti AP2, or control IgG in 8 breast cancer cell lines. Significant AP2 recruitment to the <i>BST2</i> promoter observed only in grade 1 (CCdl22, CCdl68, CCdl67) & grade 2 (CCdl66, CCdl61) cell lines. <b><i>Bottom panel</i></b><b> -</b> DNA from ChIP samples in top panel analyzed by QPCR. Primers encompassing putative AP2 binding sites from −221 to +6 were used. Melt curves were analyzed to ensure amplification of a single product. Each reaction was performed in triplicate. The plot represents relative binding efficiency determined by 2^-ΔΔC_T, where ΔC_T is the difference between input C_T and immunoprecipitated C_T; ΔΔC_T is the difference between AP2-immunoprecipitated ΔC_T and IgG-immunoprecipitated ΔC_T. Asterisks represent statistical significance (p<0.01). <b><i>C</i></b><i>.</i> Inhibition of AP2 binding to the <i>BST2</i> promoter in TGFβ-responsive primary breast cancer cell lines. Prior to processing for ChIP, cells were treated with vehicle, 4 ng/ml TGFβ, or 20 uM TGFβ inhibitor - SB-431542<b>.</b> Note striking reduction in AP2 binding in the presence of SB-431542 in 3 independent test cell lines. <b><i>D.</i></b> Hypothetical representation of <i>BST2</i> transcriptional regulation by the TGFβ axis. Intact TGFβ regulation mediated by AP2 binding to the <i>BST2</i> promoter enables maintenance of low baseline expression in grade 1 & 2 breast cancer cells.</p

Differential BST2 expression in primary breast cancer of varying histological grade is maintained in tumor-derived cell lines.

<p><b><b><i>A</i></b></b><i>.</i> QPCR based <i>BST2</i> transcript levels in 17 breast cancer cell lines normalized to expression in non-malignant breast epithelium. <b><i>B.</i></b> Western blot analysis of BST2 protein (25–35 kd) in breast cancer cells. Tubulin used as a loading control. <b><i>C.</i></b> Microscopic images of BST2 immunostaining (green) in fixed, permeabilized breast cancer cells. Nuclei counterstained with propidium iodide (red). Bar –50 µm. <b><i>D.</i></b> BST2 immunolocalized at the cell membrane in live, unfixed breast cancer cells. Bar –25 µm.</p

Functional consequences of endogenous BST2 overexpression in high grade primary breast cancer cells.

<p><b><i>A. Top panel</i></b> – Reduction of <i>BST2</i> expression after transient knockdown by <i>BST2</i> siRNA transfection of grade 3 tumor cells (CCdl54). Values normalized to <i>ACTB</i> expression. <b><i>Bottom panel</i></b> – BST2 immunolocalization (green) on day-6 post knockdown, showing sustained reduction in <i>BST2</i> siRNA transfected cells plated in Matrigel<b>;</b> nuclei counterstained with PI (red). Bar - 50 µm. <b><i>B</i></b><i>. BST2</i> knockdown enhances drug-mediated apoptotic cell death in 6-day old tumor cultures plated in Matrigel, and treated with 5 µM tamoxifen, or 5 µM staurosporine for 24 hrs. <b><i>Top panel</i></b> – immunolocalization of anti cleaved caspase 3 (green); nuclei counterstained with PI (red). Bar - 50 µm. <b><i>Bottom panel</i></b> – Data plotted as percent apoptotic cells in 2 independent BST2 overexpressing cell lines (CCdl54, CCdl672). Total number of PI-stained nuclei (>100), and proportion of cleaved caspase 3 positive tumor cells was averaged for 3 optical fields scanned with a 20× objective. Differences in apoptotic cell yield between NS siRNA vs. <i>BST2</i> siRNA-transfected cultures after treatment with each drug were significant (p<0.01). <b><i>C</i></b><i>.</i> Growth reduction induced by <i>BST2</i> knockdown measured by BrdU incorporation in primary tumor cells plated in Matrigel. <b><i>Top panel</i></b> - BrdU immunolocalization (yellow) in tumor nuclei (CCdl54) counterstained with PI (red) 7-day post transfection with <i>BST2</i> siRNA. Bar - 25 µm. <b><i>Middle panel</i></b> - Data plots represent the fraction of BrdU positive proliferating cells in control (>100) and <i>BST2</i> knockdown cultures averaged from 5 optical fields scanned with a 20× objective. Asterisk denotes a significant (p<0.01) difference. <b><i>Bottom panel</i></b> – Data plots demonstrate an increase in the number of small (4-cell) and medium (8- to 12-cell) colonies accompanied by a decrease in the number of large-sized colonies (>25-cell) in <i>BST2</i> siRNA-treated Matrigel cultures. <b><i>D.</i></b> Stratification of hormone-treated ER+ breast cancer (n = 66) based on <i>BST2</i> transcript levels. The Kaplan Meier plot suggests a trend whereby a relatively poor clinical outcome is conferred upon cases with moderate (red) or high (green) <i>BST2</i> expression compared to those with low gene expression (blue).</p

Regulation of <i>BST2</i> expression by TGFβ in breast cancer cells.

<p><b><i>A.</i></b> Reduction in baseline <i>BST2</i> expression detected in 5 grade 1 & 2 cell lines after 24 hr TGFβ (4 ng/ml) treatment. Each QPCR reaction was carried out in triplicate, and values normalized to <i>ACTB</i> expression and to vehicle-only controls. Asterisks represent statistically significant differences (p<0.01) between untreated and TGFβ-treated samples. <b><i>B.</i></b> TGFβ-induced shift in <i>AP2</i> and <i>STAT3</i> transcript levels under the same treatment conditions employed in panel A. <i>BST2</i> reduction in grade 1 (CCdl22) & 2 (CCdl66) cell lines is accompanied by a decline in <i>STAT3</i> levels, while <i>AP2</i> transcripts increase significantly. No significant changes were observed in grade 3 (CCdl54) cells. Each assay was done in triplicate. Asterisks indicate a significant difference (p<0.01) between untreated and TGFβ-treated samples. <b><i>C.</i></b> Inhibition of the TGFβ pathway with 24 hr SB-431542 treatment increased <i>BST2</i> expression in grade 1 cell lines (CCdl22, CCdl68). Data acquired in triplicate shows significant differences (p<0.01) between test and vehicle-only cell samples, indicated by asterisks.</p

BST2 overexpression is associated with high histological grade of primary breast cancer.

<p><b><b><i>A.</i></b></b> Low grade invasive primary breast tumor showing weak or no BST2 expressing cancer cells. Blue – hematoxylin counterstain. <b><i>B.</i></b> High grade invasive primary breast tumor displaying strong BST2 localization in cancer cells (brown). <b><i>C.</i></b> Homogeneous BST2 immunostaining in cells of coexisting ductal carcinoma <i>in situ</i> (DCIS), and invasive tumor. Bar –50 µm.</p