Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Mitotic spindle checkpoint genes are broadly overexpressed in human urothelial carcinoma. A, Human samples of normal urothelium (N=10) and urothelial carcinoma of the bladder (N=8) were subjected to RNA microarray. A subset of 13 gene transcripts related to the mitotic spindle checkpoint, including Aurora A and B, were upregulated at least 5-fold in the urothelial carcinoma compared to the normal urothelium. B, Upregulation of these genes was validated by two-step quantitative real-time PCR on a separate set of human samples of urothelial carcinoma (N=3) and normal urothelium (N=3). 10 of 13 genes (asterisked) demonstrated statistical significance (T-test P-value < 0.05) for differential expression in urothelial carcinoma compared to normal urothelium.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Microarray, Real-time Polymerase Chain Reaction, Quantitative Proteomics

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: MLN8237 induces cell cycle arrest and aneuploidy of bladder cancer cell lines. A, MLN8237 inhibited expression of phospho-Aurora A-T288 at mitotic spindles. B, MLN8237 demonstrated specificity for inhibiting Aurora A, as expression of histone-H3 and phospho-histone-H3, markers of Aurora B function, was maintained. C, Propidium iodide staining with flow cytometry analysis was performed to assess cell cycle changes. T24, UM-UC-3, and RT4 cells were treated with 10 nM to 1 μM MLN8237 for 48 h. All three cell lines demonstrated dramatic cell cycle arrest and increase in the 4N cell population in a dose-dependent manner. T24 and UM-UC-3 cells also demonstrated a considerable increase in aneuploidy, whereas RT4 cells did not.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Expressing, Staining, Flow Cytometry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Cellular phenotypes of T24 and RT4 cells differ after MLN8237 treatment. A, MLN8237 induces a dramatic increase in cell size in T24 cells but not RT4 cells. B, Immunocytochemistry and fluorescence microscopy of T24 and RT4 cells revealed the formation of aberrant spindle figures upon MLN8237 treatment, with multipolar spindle apparatuses and failure of localization of chromatids to a single metaphase plate. C, T24 cells demonstrate a phenotype of increased cell size and ploidy, whereas RT4 cells do not. D, T24 cells also exhibit a subpopulation of cells exhibiting marked cytoplasmic aurora A expression, whereas RT4 cells lacked cytoplasmic aurora A expression. E, Real-time imaging of T24 and RT4 cells treated with MLN8237 was performed over 48 h. T24 cells exhibited dramatic increases in cell size as a result of repeated cell cycle progressions without separation of daughter cells. RT4 cells appeared to become arrested after one failed mitotic attempt, preventing repeated cell cycle progressions that could otherwise result in increased ploidy.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: Immunocytochemistry, Fluorescence, Microscopy, Expressing, Imaging

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: MLN8237 induces cytotoxicity and differential apoptotic processes. A, MTS assay was used to calculate IC50 values for each cell line following treatment over a range of MLN8237 concentrations for 48 h. MLN8237 exhibited highest potency in T24 and UM-UC-3 cells (IC50 of 31 nM and 45 nM, respectively) and lowest potency in RT4 cells (IC50 of 120 nM). B, Western blot analysis of T24 and RT4 cells for apoptotic markers revealed induction of p53 expression in RT4 cells, and induction of p73, but not p53, expression in T24 cells. Both cell lines showed increased expression of the apoptotic marker cleaved PARP starting 24 h after initiation of treatment. C, Annexin V staining with flow cytometry analysis of T24 and RT4 cells revealed an increased apoptotic cell fraction at 48 and 72 h after initiation of MLN8237 treatment. D, Clonogenic assays of T24 and RT4 cells showed 90% inhibition of long-term clone forming capability at 100 nM MLN8237.

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: MTS Assay, Western Blot, Expressing, Marker, Staining, Flow Cytometry, Inhibition

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: The investigational Aurora kinase A inhibitor MLN8237 induces defects in cell viability and cell cycle progression in malignant bladder cancer cells in vitro and in vivo

doi: 10.1158/1078-0432.CCR-12-2383

Figure Lengend Snippet: Interactions of MLN8237 with paclitaxel and gemcitabine in vitro are schedule-dependent. MLN8237 (MLN) was combined with either paclitaxel (PTX) or gemcitabine (Gem) in T24 cells. Drugs were administered either simultaneously for 48 h (left column), or sequentially, with one drug for 48 h, followed by washout and the other drug for 48 h (middle and right columns). MTS assay was utilized to quantify the effect on cell viability of these combination treatments. MLN8237 demonstrated synergistic effects with paclitaxel and gemcitabine when dosed sequentially (middle and right columns), and antagonistic effects when dosed simultaneously (left column).

Article Snippet: Human urothelial carcinoma cell lines T24, UM-UC-3, and RT4 were purchased from the American Type Culture Collection (ATCC) and cultured at 37 °C and 5% CO2 in RPMI 1640 media (Gibco) supplemented with 10% FBS (Gibco).

Techniques: In Vitro, MTS Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

doi: 10.1007/s00018-010-0433-4

Figure Lengend Snippet: Role of ADAM10 and ADAM17 for CX3CL1-induced transmigration. Wt- and CX3CL1-ECV304 cells were stably transduced with lentivirus carrying shRNA for knockdown of ADAM10 and ADAM17 (A10shRNA or A17shRNA) or scrambled shRNA control, respectively. a Efficient and sustained downregulation of ADAM10 and ADAM17 surface expression was controlled by flow cytometry and is shown as representative histograms. b Stably transduced CX3CL1-ECV304 cells were incubated for 2 h, and subsequently released CX3CL1 in the supernatants and cell-associated CX3CL1 in the lysates were quantified by ELISA. The amount of residual cell-expressed CX3CL1 was calculated in relation to the total content of CX3CL1 in both the lysates and supernatants. c Stably transduced CX3CL1-ECV304 cells were investigated for transmigration by wt- and CX3CR1-L1.2 cells. d HUVEC were transduced with A10shRNA, A17shRNA or scramble shRNA control. Cells were stimulated with IFN-γ and TNF-α (both 10 ng/ml) or left unstimulated for 16 h and subsequently investigated for transmigration by wt- and CX3CR1-L1.2 cells. Data are shown as mean plus SD obtained from three independent experiments. Statistically significant differences versus the controls receiving scrambled shRNA are indicated by asterisks (p < 0.05)

Article Snippet: The adherent human bladder carcinoma cell line ECV304 (derivative of epithelial bladder carcinoma cell line T-24, DSMZ, Braunschweig, Germany) [ 23 ] was stably transfected with CX3CL1 (CX3CL1–ECV304) as previously described and cultured in M199 medium supplemented with 10% FCS [ 9 ].

Techniques: Transmigration Assay, Stable Transfection, Transduction, shRNA, Knockdown, Control, Expressing, Flow Cytometry, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

doi: 10.1007/s00018-010-0433-4

Figure Lengend Snippet: Transmembrane CX3CL1 promotes transmigration. a Wt- or CX3CL1-ECV304 cells were grown in transwell inserts and washed to remove free CX3CL1. As a control, recombinant soluble CX3CL1 was again added to the lower transwell compartment. Subsequently, cells were assayed for transmigration of freshly prepared PBMC that were added to the upper transwell compartment. PBMC that transmigrated into the lower compartment were counted and results were expressed as transmigration index representing the fold increase over random migration. b Wt- or CX3CL1-ECV304 cells were pretreated with recombinant soluble CX3CL1 (10 nM) that was added to the upper transwell compartment. After 1 h, cells were washed and assayed for transmigration of PBMC in the absence or presence of recombinant soluble CX3CL1 in the lower compartment. c HUVEC were left unstimulated or stimulated with IFN-γ and TNF-α (both 10 ng/ml) for 16 h and subsequently treated with neutralizing antibodies against CX3CL1 and ICAM-1 or isotype control (10 μg/ml each) for 1 h. After washing to remove free antibody, HUVEC were transferred into fresh medium and assayed for PBMC transmigration. Data are shown as mean plus SD of three independent experiments. The asterisks indicate statistically significant differences versus the controls (untransfected, unstimulated or isotype, respectively, p < 0.05)

Article Snippet: The adherent human bladder carcinoma cell line ECV304 (derivative of epithelial bladder carcinoma cell line T-24, DSMZ, Braunschweig, Germany) [ 23 ] was stably transfected with CX3CL1 (CX3CL1–ECV304) as previously described and cultured in M199 medium supplemented with 10% FCS [ 9 ].

Techniques: Transmigration Assay, Control, Recombinant, Migration

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

doi: 10.1007/s00018-010-0433-4

Figure Lengend Snippet: CX3CR1 mediates transmigration. a Wt- and CX3CR1-L1.2 were investigated for transmigration across wt- or CX3CL1-ECV304 cells cultured in transwell inserts. b Neutralizing monoclonal antibody to CX3CL1 or isotype control (10 μg/ml each) was added to the lower or upper compartment of transwells containing wt- or CX3CL1-ECV304 cells. After 15 min, the antibody was removed and the cells were investigated for transmigration of CX3CR1-L1.2 cells. c HUVEC were stimulated with IFN-γ and TNF-α (both 10 ng/ml) for 16 h or left untreated. Subsequently, wt- and CX3CR1-L1.2 cells were assayed for transmigration across the HUVEC layer. d Wt- and CX3CR1-L1.2 cells were pretreated for 30 min with soluble CX3CL1 (10 nM) or 2 h with PTX (100 ng/ml) and subsequently assayed for transmigration across wt- or CX3CL1-ECV304 cells. Data are shown as mean plus SD of three independent experiments. The asterisks indicate statistically significant differences versus the controls (untransfected, unstimulated or isotype, respectively, p < 0.05)

Article Snippet: The adherent human bladder carcinoma cell line ECV304 (derivative of epithelial bladder carcinoma cell line T-24, DSMZ, Braunschweig, Germany) [ 23 ] was stably transfected with CX3CL1 (CX3CL1–ECV304) as previously described and cultured in M199 medium supplemented with 10% FCS [ 9 ].

Techniques: Transmigration Assay, Cell Culture, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

doi: 10.1007/s00018-010-0433-4

Figure Lengend Snippet: Role of the DRY motif and the C-terminus for CX3CR1-mediated adhesion and transmigration. The ability of the receptor variants to mediate adhesion to CX3CL1 was tested under static (a) and flow (b) conditions. a For static adhesion assays, calcein-labeled HEK293 cells expressing the indicated receptor variants were seeded onto wt- or CX3CL1-ECV304 cells. After washing, the fluorescence signal of the adhering cells was measured. The adhesion index was determined by calculating the ratio of the fluorescence signal from adherent HEK293 cells bound to CX3CL1-ECV304 and from that bound to wt-ECV304 cells. b For flow adhesion experiments, labeled HEK293 cells expressing the indicated receptor variants were perfused over a layer of cytokine-stimulated HUVEC for 1 min. c L1.2 cells expressing the indicated receptor variants were tested for their chemotactic response towards increasing concentrations of soluble CX3CL1 (0.1–100 nM) in a modified Boyden chamber assay. d L1.2 cells expressing the indicated receptor variants were studied for transmigration through CX3CL1-ECV304 cells. e HUVEC were stimulated with IFN-γ and TNF-α (both 10 ng/ml) for 16 h and subsequently probed for transmigration of L1.2 cells in response to CX3CR1 or its R127N variant. Three independent experiments were performed and data are shown as mean plus SD. The asterisks indicate statistically significant differences versus the untransfected control (p < 0.05)

Article Snippet: The adherent human bladder carcinoma cell line ECV304 (derivative of epithelial bladder carcinoma cell line T-24, DSMZ, Braunschweig, Germany) [ 23 ] was stably transfected with CX3CL1 (CX3CL1–ECV304) as previously described and cultured in M199 medium supplemented with 10% FCS [ 9 ].

Techniques: Transmigration Assay, Labeling, Expressing, Fluorescence, Modification, Boyden Chamber Assay, Variant Assay, Control

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Requirements for leukocyte transmigration via the transmembrane chemokine CX3CL1

doi: 10.1007/s00018-010-0433-4

Figure Lengend Snippet: Effect of the metalloproteinase inhibitor GW280264X on CX3CL1-mediated transmigration. a Wt- or CX3CL1-ECV304 cells were grown on transwell inserts and pretreated with GW280264X (10 μM) or DMSO control for 1 h. After removal of the inhibitor, the lower compartments of the transwell systems received soluble CX3CL1 (10 nM) or were left without stimulus. Subsequently, cells were assayed for transmigration of wt- and CX3CR1-L1.2 cells. b CX3CL1-ECV304 cells were pretreated with GW280264X (10 μM) or DMSO for 1 h, washed and co-incubated with freshly prepared PBMC for 3 h. Cells were harvested and analyzed for CX3CL1 surface expression by flow cytometry. Surface expression was expressed in relation to that of CX3CL1-ECV304 cells receiving no inhibitor and no PBMC. c Wt- or CX3CL1-ECV304 cells were incubated for 3 h in the presence or absence of L1.2 cells expressing the indicated CX3CR1 variants and subsequently conditioned media were analyzed for the presence of shed soluble CX3CL1 by ELISA. Results are expressed in relation to the control receiving no L1.2 cells. d Wt- or CX3CL1-ECV304 cells were pretreated with DMSO control or GW280264X (10 μM) for 1 h, washed and subsequently analyzed for adhesion of PBMC under flow. e Wt- or CX3CL1-ECV304 cells were grown on transwell inserts, pretreated with DMSO control or GW280264X (10 μM) for 1 h, and subsequently probed for transmigration of PBMC. f HUVEC were stimulated with IFN-γ and TNF-α (both 10 ng/ml) or left unstimulated for 16 h. Subsequently, cells were pretreated with DMSO control or GW280264X (10 μM) for 1 h and then analyzed for flow-resistant adhesion of PBMC. g Unstimulated and IFN-γ/TNF-α-stimulated HUVEC were pretreated with DMSO control or GW280264X and subsequently probed for transmigration of PBMC. Data are shown as mean plus SD obtained from three independent experiments. Statistically significant differences between inhibitor-treated and DMSO-treated cells are indicated by asterisks (p < 0.05)

Article Snippet: The adherent human bladder carcinoma cell line ECV304 (derivative of epithelial bladder carcinoma cell line T-24, DSMZ, Braunschweig, Germany) [ 23 ] was stably transfected with CX3CL1 (CX3CL1–ECV304) as previously described and cultured in M199 medium supplemented with 10% FCS [ 9 ].

Techniques: Transmigration Assay, Control, Incubation, Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay