32 research outputs found
Patients characteristics.
<p> =  sample identifier, sex  =  patient sex, KPS (score)  =  Karnofsky Performance Status score; Sympt. (mo)  =  symptom duration in month; Surgery (type)  =  origin of tumor tissue from the patient brain (temporal/parietal/occipital/frontal); ki67-%  = % of cells expressing ki-67; p53 =  p53 positivity (less than 5% of nuclei); MGMT  =  MGMT promoter metylation; EGFR  =  EGFR positivity (moderate-to-strong signal on >% of cells); PFS  =  progression-free survival; OS  =  overall survival; PTEN activ  =  PTEN activation group according to clustering of phospoproteomics profiles. Characteristics of patients from which samples were collected. Legend: Sample </p
Transcriptional and post-translational modulation of CDC25A, DUB3 and Wee1 during cell cycle in PTEN active cell lines.
<p>CDC25A mRNA is constantly produced throughout all phases while DUB3 expression increases in the G1/S phase and wee1 expression decreases during the phase M. CDC25A modulation occurs at post translational level with DUB3 inhibiting its ubiquitination and Wee1 controlling negatively the activity of Cyclin-B/Cdk1.</p
Transcripts fold change in differentiation.
<p>Green, red and black indicate transcripts that decrease, increase or maintain their concentration during induction respectively.</p
Immunophenotype of undifferentiated and differentiated cells.
<p>Immunofluorescence of undifferentiated GSCs neurosphere (left panels) and of differentiated cells (right panels) labelled with anti-human Sox2 (top panels), anti-human GFAP and anti-human TUBB3 (bottom panels).</p
<i>In vitro</i> inhibition of miR-221 and miR-222 reduces tumor growth of PC3 derived tumors in SCID mice.
<p>A Northern blot analysis of total RNA extracted from PC3 cells transfected <i>in vitro</i> with anti-miR-221+anti-miR-222 LNA oligonucleotides (anti-221/222). The hybridisation to snRNA U6 was used as a loading control. B Tumor growth curves measured after the injection of PC3 cells transfected with either anti-miR-221 and anti-miR-222 LNA oligonucleotides (anti-221/222) or a control LNA oligo (ctrl). The tumor volumes were calculated as v = L×l<sup>2</sup>×0.5, where L is the longer diameter, and l the shorter one. C Average volume fold increase of tumors derived from PC3 cells transfected with anti-miR-221+anti-miR-222 LNA oligonucleotides (anti-221/222) or with a negative control LNA oligonucleotide (ctrl). Values represent the ratio between the volumes at the day of sacrifice and the volumes measured 54 days before, when all tumors were clearly detectable and measurable. N = 4 for ctrl tumors and n = 5 for anti-miR treated tumors. Data in B and C are presented as the means±SEM. *, P<0.05.</p
Undifferentiated LMS cells display a Mesenchymal Stem Cell (MSC)-phenotype.
<p>Flow cytometric detection of the indicated antigens in undifferentiated LMS cells isolated as tumors spheres (A), as adherent cells in MSC-culture conditions (B), as differentiated cells obtained in standard culture conditions (C), in non tumoral mesenchymal stem cells (D) or in fresh tumor cells (E). Representative FACS dot plots (SSC vs FSC), 7-AAD staining and images of the corresponding cells are reported for each condition.</p
IRESSA treatment reduces LMS stem-like cell growth rate and sensitizes them to chemotherapy.
<p>A) <i>Effect of IRESSA on LMS cell chemo-sensitivity</i>. (Left) Stem-like cells were exposed to IRESSA and the indicated compounds and cell viability was evaluated after 72 hours by Cell Titer Glo assay (Promega). (Right) Cell viability of undifferentiated (stem) or differentiated (diff) LMS cells exposed to IRESSA/vincristine combination for 72 hours. Mean ± SD of 3 independent experiments is shown. ** p<0,01. B) Flow cytometric analysis for Annexin V of LMS stem-like cells untreated (control) or treated as indicated. C) Immunoblot for the indicated proteins or phosphoproteins of LMS stem-like cells left untreated or treated as indicated. D) <i>Effect of IRESSA on SP and drug efflux ability.</i> (Left) SP analysis of control or IRESSA-treated LMS stem-like cells. (Right) Cytofluorimetric profiles of control or IRESSA treated LMS stem-like cells after exposure to doxorubicin (Uptake), or after drug treatment followed by removal and overnight incubation in drug-free medium (Efflux).</p
Undifferentiated LMS cells are highly tumorigenic and reproduce the human tumor in immunocompromised mice.
<p>A) Tumor volumes of xenografts generated by injection of spheres or adherent undifferentiated LMS cells (6 weeks post-injection). Mean ± SD of 3 independent experiments is shown. ** p<0,01. B) Tumor growth rate of undifferentiated (adherent) or differentiated (diff) LMS cells injected subcutaneously in NOD-SCID mice at the indicated cell doses. Mean ± SD of 3 independent experiments is shown. ** p<0,01, <i>***p<</i>0,001. (C) Tumor growth rate of secondary tumors injected subcutaneously in NOD-SCID mice at the indicated cell doses. Mean ± SD of 3 independent experiments is shown. <i>***p<</i>0,001. D) Hematoxylin and eosin (H&E) or immunohistochemistry for the indicated antigens performed on patient tumor (D), tumor generated by subcutaneous injection of LMS spheres (E) or adherent undifferentiated cells (F). The original magnification for each image is indicated.</p
MiR-221 ectopic overexpression enhances the growth of LNCaP-derived tumors.
<p>A Northern blot analysis of LNCaP cells permanently transfected with p-221 or empty vector pCDNA3.1. The expression of miR-221 in the highly aggressive PC3 prostate carcinoma cell line is also shown, as a positive control. Hybridization to snRNA U6 is included as a loading control. Under each lane, a number indicates the relative miR-221 expression as compared to LNCaP cells transfected with the empty vector pCDNA3.1, where miR-221 endogenous expression is set as  = 1. B <i>In vivo</i> tumor growth in SCID mice. Average tumor volumes are represented (n = 6 for both experimental groups) starting from the first time point when tumor volumes were clearly measurable (t0) until the last measurement before sacrifice, performed 6 weeks later. C Average volume fold increase of the same tumors as in B at the moment of sacrifice (i.e. 6 weeks after the first measurement) as compared to values measured at time 0. Data are presented as the mean±SEM *, P<0.05, and are representative of 2 independent experiments. D–E Proliferation markers: mitotic index and Ki-67 expression. D Graph of the mitotic index and Ki-67 expression as percent of positive cells (10 fields, 2 sections for each tumor. *, P<0.01; **, P≪0.001). Grey bars: pCDNA3.1 transfected LNCaP cells; black bars: p-221 transfected LNCaP cells. E, left upper panel: LNCaP cells transfected with empty vector pCDNA3.1, haematoxylin eosin stained section (magnification 600×). Cells have epithelioid phenotype with low mitotic index. Right upper panel: tumor tissue from LNCaP cells transfected with p-221, haematoxylin-eosin stained section (magnification 600×). Cells have epithelioid phenotype with high mitotic index; arrows indicate mitotic pictures. Left lower panel: immunohistochemistry of the proliferation marker Ki-67 in tumor tissue from LNCaP ctrl cells; scattered cells with brown, granular nuclear staining considered to be positive for Ki-67 (magnification 200×). Right lower panel: immunohistochemistry of the proliferation marker Ki-67 in tumor tissue from LNCaP cells transfected with p-221: numerous cells with brown, granular nuclear staining positive for Ki-67 (magnification 200×). F Northern and Western blot analysis of RNA and proteins extracted from p-221 and control vector transduced tumors from two mice sacrificed at 6 weeks from the first measurement (as in B). The upper part of the panel (Northern blot) shows the persistent expression of miR-221 in p-221 transduced tumors, and the lower part (Western blot) shows the downregulation of p27 in miR-221 expressing tumors. U6 and β-actin are shown as loading controls for Northern and Western blot, respectively. The numerical values under each lane indicate the relative expression of miR-221 and of p27, where each p-221 transfected tumor is compared to its controlateral control (pCDNA3.1) tumor, whose miR-221 and p27 expression levels are set as  = 1. G p27 mRNA 3′UTR sites targeted by miR-221 and miR-222. The core annealing regions are located at nucleotides 201–208 and 274–281 of p27 3′UTR. Dotted vertical lines indicate G-U bonds.</p
Clinical aspects of prostate cancer cases used in this study.
a<p>Case: patients treated with radical prostatectomy.</p>b<p>PSA: Prostate Specific Antigen.</p>c<p>Gleason grade: Gleason's score.</p>d<p>TNM: Tumor, Nodes, Metastasis.</p><p>pT: pT category, N: lymphnodes, N0: not involved; M: metastasis, Mx: not reliable.</p