18 research outputs found
Suspension Induces Phosphorylation of eIF2α and Translation Repression in Mammary and Kidney Epithelial Cells.
<p>(A) Whole cell lysates from MCF10A (upper left), HEK293T (upper right) and primary HMEC (lower panels) cells grown either in adhered (A) or suspended conditions (S) as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000615#s4" target="_blank">methods</a> section for the indicated time points, were immunoblotted for p-eIF2α and total eIF2α levels. Adhered MCF10A or HEK293T cells treated with 2 mM DTT or 5 µg/ml tunicamycin (Tn) respectively, were used as positive controls. (B) Quantification of the rate of DNA synthesis using a BrdU incorporation assay and flow cytometry to measure the percentage of BrdU-positive cells (filled diamonds) at different time points in suspension. The percentage of apoptotic cells was measured using propidium iodide staining and flow cytometry to identify the sub-G0 apoptotic fraction for adhered (dashed line) or suspended (dotted line) MCF10A cells for different time points. Data points show the mean±SD for BrdU–positive cells in each sample as a percentage of the total. (C) Autoradiogram of [<sup>35</sup>S] Met/Cys incorporation (right panel) into newly synthesized proteins in MCF10A cells adhered or suspended for 24 hrs (two independent samples). Coomassie Blue staining of an identical gel (left panel) shows equal protein loading. (D) Polysome profiles from 24 hr adhered (left) and suspended (right) MCF10A cells showing an increase and decrease in the monosome and polysome peaks, respectively in suspended cells. Absorbance at 254 nm (Y-axis, RNA concentration) was plotted against migration in the sucrose gradient (X-axis, bottom to top). Total RNA was isolated from individual fractions to visualize the 18S and 28S rRNAs by ethidium bromide staining.</p
Unscheduled Activation of PERK Restricts Acinar Growth and Promotes Apoptosis in 3D Matrigel.
<p>(A) Time-dependent increase in phosphorylation of eIF2α in MCF10A cells expressing an Fv2E-ΔNPERK construct, upon treatment with the dimerizing drug, AP20187 (2 nM). AP20187 has no effect on P-eIF2α levels in β-Gal cells. Total eIF2α was used as loading control. (B) Photomicrographs of Fv2E-ΔNPERK cells in 3D Matrigel treated with 2 nM AP20187 or equal volume of ethanol as control, added every 24 hrs from Day 4 up to Day 6 of morphogenesis; representative phase-contrast images depict the effect of forced PERK activation on acini development; (B-a and B-e) A×10 magnification image of several developing acini; (B-b) Normal acinus, (B-c) 2 cell cluster, (B-f) 4 cell cluster, (B-g) 4 cell cluster containing apoptotic cells. (B-d and h) Confocal images through the equatorial region of Fv2E-ΔNPERK cells in 3D Matrigel immunostained for active caspase-3 (red) or LN-5 (green) with (B-h) and without (B-d) treatment with 2nM AP20187 every 24 hrs, (B-h) cell cluster where the majority of cells have entered apoptosis. (C) Quantitation of phase contrast images of Fv2E-ΔNPERK cells on Day 6, treated every 24 hrs with or without 2nM AP20187. Over 400 acini were visually scored for the presence of apoptotic or growth arrested 2–4 celled acini and calculated as a percentage of the total number of acini; graph shows mean±SD. (D) Photomicrographs of β-Gal vector control cells treated with 2nM AP20187 or with equal volume EtOH as control, every 24 hrs from Day 4 up to Day 6 in Matrigel. Note that AP20187 treatment caused no noticeable changes in acini size or morphology, consistent with the absence of modulation of P-eIF2α levels in the same cells (<i>A</i>). (E) Confocal images showing Fv2E-ΔNPERK cells treated with (+AP) or without (-AP) AP20187 every 12 hrs stained for LN-5 (green) to delineate the acini and active caspase-3 (red). Note that a majority of cells even in large acini can be pushed into apoptosis by strong activation of PERK signaling. Scale bars = 10 µm.</p
PERK is Required to Maintain Normal Mammary Acini Growth and Morphogenesis through Regulation of Cell Proliferation.
<p>(A) Representative images through the equatorial cross sections of acini from myc-PERKΔC, myc-PERK-K618A or β-gal expressing MCF10A cells on Day 8 of culture in 3D Matrigel, using LSCM. Nuclei are stained green using CyQuant dye. Note the increase in acinar size, luminal filling and the multi-lobular nature of the PERKΔC structures as compared to the vector control acini. PERK-K618A expressing acini were also significantly larger and always showed luminal cells, but were not frequently multi-lobulated. Scale bars = 20 µm. (B) Acinar size was measured on different days in Matrigel for β-Gal and PERKΔC or PERK-K618A cells, using SPOT™ software to measure two perpendicular diameters per acinus and calculate acinar volume considering an ellipsoid structure; volume for 50–200 acini were quantitated per time point, shown as percentage of total for the given size range, Mean±SD. (C) Confocal images for β-Gal and PERKΔC cells immunostained for cleaved (*) caspase-3 (red, top panels) or Ki-67 (red, bottom panels) on Day 8 in Matrigel. Acini are outlined with white dotted lines to delineate the normal β-Gal vs. disorganized large PERKΔC acini, which shows Ki-67 positively stained cells in the lumen. Scale bars = 20 µm. (D) Graphs showing distribution of the percentage of cells per acinus that stained positively for Ki-67 or cleaved caspase-3 in β-Gal and PERKΔC cells on Day 8 in Matrigel; between 50–100 acini were scored and statistical significance was determined by the <i>t</i> test for independent samples with P<0.001 defined as statistically significant. #N.S-not significant.</p
GADD153 mRNA and Protein Levels Are Strongly Upregulated During Suspension Conditions.
<p>(A) RT-PCR analysis of GADD153 mRNA levels in MCF10A (left panel) and HEK293T (right panel) cells at different time points in either adhered (A) or suspended (S) conditions. Adhered MCF10A cells treated with 2mM DTT for 4h were used as positive control and GAPDH was used as a loading control. (B) MCF10A cells were transiently transfected with a GADD153 promoter-driven-EGFP reporter plasmid and EGFP fluorescence was analyzed 48 h post-transfection by FACS; total events captured: 2×10<sup>4</sup>. The graph shows the number of GFP-positive events in FLH2>10 (mean±SD). (C) Western blot for GADD153 protein in adhered (A) or suspended (S) MCF10A cells. (D and E) Immunofluorescence (D) and FACS (E) analysis of GADD153 (red) expression in MCF10A cells following growth in adhered or suspension conditions for the indicated times. Secondary antibody was used as negative control in E. (F) MCF10A (top) and HEK293T (lower) cells were transiently co-transfected with the GADD153-EGFP reporter plasmid and either a full-length Flag-tagged GADD34 plasmid or an empty vector as control for 24 hrs before being detached and left to reattach or put into suspension for an additional 48 hrs before FACS analysis. GFP fluorescence was analyzed 48 h post transfection by FACS where a total 2×10<sup>4</sup> events were captured. The graphs show the number of GFP positive events in FLH2>10 or the mean fluorescence intensity (MFI) in the PMT2-FITC channel (mean±SD). (G) RT-PCR for XBP-1 splicing (top panel) in MCF10A cells at different time points either adhered (A) or suspended (S). Adhered MCF10A cells treated with 2mM DTT for 4 hrs was used as positive control and GAPDH, shown in (A) was used as a loading control. Lower panels show RT-PCR for BiP, Hsp47 and Erp72/PDI chaperone mRNA levels in adhered (Adh) or suspended (Sus) MCF10A cells. GAPDH was used as a loading control.</p
PERK Inhibition in MCF10A Cells Favors Mammary Tumor Formation.
<p>(A) Western blot for ErbB1 (EGFR) and ErbB2 (Her2) levels in adhered β-Gal or PERKΔC cells show increased levels when PERK is inhibited. GAPDH was used as loading control. (B) β-Gal or PERKΔC cells were injected orthotopically into the contralateral abdominal mammary fat pads of 3 week-old female nude mice (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000615#s4" target="_blank">methods</a> for details). Post-implantation mice were monitored biweekly for tumor take and when detected tumor diameters were measured and the volume was calculated and plotted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000615#s4" target="_blank">methods</a>. Note that none of the mice implanted with β-Gal-MCF10A cells developed tumor nodules. (C) Histology of mammary glands and tumors in mice implanted with β-Gal and PERKΔC expressing MCF10A cells. (C-a) H&E staining of a cleared mammary fat pad inoculated with β-Gal cells. Only adipose tissue and occasional stromal cells that were negative for β-galactosidase activity was observed. (C-b) H&E staining of a PERKΔC tumor lesion. Note the disorganization of the fibrotic-epithelial tissue. Arrows depict the presence of epithelial cells forming acinar or duct-like structures within the PERKΔC tumor lesion. (C-c) Higher magnification of intratumoral accumulations of cells without a defined architecture but comprised of a mixture of epithelial (larger nuclei and pink cytoplasm) and inflammatory cells (smaller darkly stained nuclei). (C-d and e) PERKΔC cells form acini-like structures with an empty lumen or show hyperplastic growth and a repopulated lumen (C-e, top left and right insets). (C-f) Anti-Myc (αMyc) staining in control β-Gal injected mammary fat pads. Note that only a light background signal is observed in adipocytes. (C-g) Histological section of a PERKΔC acinus-like structure stained with a non-specific IgG (arrow denotes the lack of staining in these epithelial cells) or with anti-Myc 9E10 mAb (C-h and C-i); arrows denote the brown staining generated by Myc-tag detection. (D) β-Gal (left panel) or PERKΔC (middle and right panels) cells grown on 2D coverslips and fixed and stained with a non-specific IgG (IgG) or with an anti-Myc 9E10 mAb (α-Myc). The Myc staining was characteristic of intracellular cistern distribution.</p
Metastasis-free analysis for four clinical studies.
<p>(A, B) van de Vijver et al., (C, D) Loi et al., (E,F) Wang et al., (G, H) Pawitan et al. Kaplan Meier estimates of metastasis-free proportion among patients with high (upper third, green), medium (middle third, red), and low (bottom third, black) dormancy scores for patients with ER+ (A,C,E,F) and ER− (B,D,F,G) tumors.</p
PERK Inhibition Results in Increased Laminin-5 Production, Secretion and Deposition.
<p>(A) Confocal images showing the equatorial cross sections through β-Gal (A-a, A-b and A-f) and PERKΔC (A-c to A-e and A-g to A-i) acini stained for Laminin-5 (green), on Day 8 in 3D. Note the increased staining and disorganized Laminin-5 deposition in PERKΔC cells as compared to proper basal localization in the β-Gal control cells. Panels A-e and A-g through A-i (arrows) show details of intra-acinar deposition of Laminin-5 within and around cells in the filled lumen. Scale bars = 10 µm; A-h is a magnified image of an acinus shown in A-g. (B) Western blot analysis from β-Gal and PERKΔC cell lysates shows the precursor (P) and mature (M) forms of LN-5. BimEL was used as loading control. (C) Western blot for Laminin-5 secreted into the conditioned media by β-Gal and PERKΔC cells grown in 3D-Matrigel. For conditioned media, samples corresponding to days 4 and 8, the supernatant was collected after 96 hrs of 3D culture. For the conditioned media sample corresponding to day 10, the supernatant was collected after 48 hrs of 3D culture. ΔC = MCF10A-PERKΔC cells. Note that while in cell lysates (B) the precursor is predominantly detected, in the conditioned media there is stronger detection of the mature form of the Laminin-5-γ2 subunit.</p
GADD153 Upregulation During Acini Development in 3D Cultures is PERK-dependent.
<p>(Aa and Ab) Representative confocal images through the equatorial section of Day10 MCF10A acini depicts strong GADD153-positive staining (red) in cells in the luminal space (arrowheads) that also display condensed nuclei, co-stained with CyQuant (green) as well as in some cases, in cells that localize to the outer basal layer of cells (arrow) that do not have condensed nuclei. (Ac-f) Representative images of GADD153 staining (red) in β-Gal (Ac and Ae) and PERKΔC (Ad and Af), cells on Day 8 in Matrigel. Scale bars = 10 µm. (B) Graph showing the distribution and median of GADD153- positive acini/field; 21 fields adding up to a total of 215 acini were scored.* P<0.001 determined by <i>t</i> test for independent samples.</p
Clustering of dormancy signature scores.
<p>A) Dormancy score analysis in breast cancer cell lines. The cell lines are ordered by dormancy scores (low to high from left to right). The rows correspond to genes and the columns represent cell lines. Expression levels for positive dormancy genes (upregulated genes - top section) and negative dormancy genes (downregulated genes - bottom section) were clustered by a hierarchical clustering algorithm. The colors represent log<sub>2</sub> fold change compared to the average from −2.5 (blue, below average) to +2.5 (red, above average) with white as the average value. A, B, and L stand for Basal A, Basal B, and Luminal classifications, respectively. B) Correlation of cell line dormancy scores with proliferation indices from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035569#pone-0035569-t001" target="_blank">Table 1</a> of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035569#pone.0035569-Kenny1" target="_blank">[9]</a> with ER- lines plotted as squares and ER+ lines plotted as diamonds. Straight line fits of ER- (blue, Spearman correlation coefficient r = .027) and ER+ (red, r = −0.76) cell lines are plotted. The ER status of HCC1500 is unclear (ATCC indicates it as ER+ while it is ER- by gene expression and Western blot in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035569#pone.0035569-Neve1" target="_blank">[8]</a>) and it was not used in the analysis. MDA-MB-231 (large orange square), MCF7 (large green diamond) and T47D (large yellow diamond) are identified. C) Patient tumor analysis. The four clinical studies were clustered as in A. In the ER status bar, ER status is indicated by black (ER+), blue (ER−) or white (not determined) bars. The two genes for which probes were not present in the van de Vijver et al. data set are represented by gray bars. D) Comparison of clustering of cell lines and patient data. Top: Positive dormancy genes that are upregulated in high dormancy score cell lines or patients. Bottom: Negative dormancy genes that are up regulated in low dormancy score cell lines or patients.</p
Dormancy scores of ER positive and ER negative tumors.
*<p>The statistical significance of the difference in dormancy score between ER+ and ER− tumors was determined using the Mann-Whitney rank sum test.</p>#<p>Mann-Whitney rank sum test (van Elteren's test) stratified by studies using all samples from 4 studies.</p