Activation of p53 by Nutlin-3a Induces Apoptosis and Cellular Senescence in Human Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is the most common and aggressive primary brain tumor in adults. Despite concerted efforts to improve current therapies and develop novel clinical approaches, patient survival remains poor. As such, increasing attention has focused on developing new therapeutic strategies that specifically target the apoptotic pathway in order to improve treatment responses. Recently, nutlins, small-molecule antagonists of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Glioma cell lines and primary cultured glioblastoma cells were treated with nutlin-3a. Nutlin-3a induced p53-dependent G1- and G2-M cell cycle arrest and apoptosis in glioma cell lines with normal TP53 status. In addition, nutlin-arrested glioma cells show morphological features of senescence and persistent induction of p21 protein. Furthermore, senescence induced by nutlin-3a might be depending on mTOR pathway activity. In wild-type TP53 primary cultured cells, exposure to nutlin-3a resulted in variable degrees of apoptosis as well as cellular features of senescence. Nutlin-3a-induced apoptosis and senescence were firmly dependent on the presence of functional p53, as revealed by the fact that glioblastoma cells with knockdown p53 with specific siRNA, or cells with mutated or functionally impaired p53 pathway, were completely insensitive to the drug. Finally, we also found that nutlin-3a increased response of glioma cells to radiation therapy. The results provide a basis for the rational use of MDM2 antagonists as a novel treatment option for glioblastoma patients

Anàlisi “ex vivo” de mecanismes d’inducció d’apoptosi i resistència al tractament en gliomes malignes

El glioblastoma multiforme és una de les neoplàsies més agressives de l’adult, malgrat els esforços realitzats en millorar el seu tractament, la supervivència dels pacients continua sent dolenta. L’objectiu global d’aquesta tesi ha estat l’estudi dels mecanismes moleculars implicats en la inducció de mort, parada de cicle pels inhibidors de MDM2 i els inhibidors de survivina en el glioblastoma multiforme humà, a més ha permès l’establiment de cultius primaris de glioblastoma humà. L’efecte dels compostos s’ha estudiat ex vivo en els cultius primaris i línies cel•lulars de glioblastoma multiforme humà. Les Nutlines són antagonistes de MDM2 que actuen, amb una gran especificitat, desplaçant la unió de la proteïna p53 del seu inhibidor MDM2 a un rang de dosi nanomolar, el qual comporta l’estabilització de la molècula de p53 i la posterior activació de la seva via de senyalització. Els resultats del tractament de cultius primaris i línies cel•lulars de glioblastoma demostren que la Nultina-3a és capaç de induir parada de cicle cel•lular (p21), apoptosi (Noxa i Puma), a més de induir senescència (marcatge amb SA-βGal) en aquelles cèl•lules amb un p53 funcional. Per el contrari, les línies cel•lulars i cultius primaris amb un p53 no funcional, són resistents al tractament degut a l’incapacitat de aquestes cèl•lules per activar la via de p53. El pretractament amb Nutlina-3a sensibilitza la línia cel•lular U87-MG enfront la radioteràpia. Això suggereix que la utilització de inhibidors de MDM2 podria ser una nova estratègia terapèutica, ja sigui de forma individual o combinada amb radioteràpia en el tractament de pacients amb glioblastoma multiforme amb una proteïna p53 funcional. YM155 (1-(2-metoxietil)-2-metil-4,9-dioxo-3-(piracina-2 ymetil)-4,9-dihidro-1H-nafto[2,3-d ]bromuro de imidazolium), és una petita molècula inhibidora de la proteïna antiapoptòtica Survivina. YM155 va ser seleccionat mitjançant un cribratge d’alt rendiment com supressor específic de l'expressió de survivina per inhibició del seu promotor. La survivina es troba sobreexpressada en la majoria dels tumors. La survivina es troba altament expressada en gliomes a més està associada amb una menor supervivència i amb un augment de la recurrència tumoral. En aquest apartat de la tesi es va estudiar l’efecte de YM155 en línies cel•lulars i un cultiu primari de glioblastoma humà. Els resultats obtinguts mostren que el tractament amb YM155 provoca una disminució de la viabilitat cel•lular, així com inducció d’apoptosi, que s’acompanya d’una disminució dels nivells proteics de survivina en totes les línies cel•lulars de glioma avaluades. La incubació de les línies cel•lulars i del cultius primari mostra canvis en el patró de cicle cel•lular, provocant aturada tant en fase G0/G1 o en fase S depenent de la línia cel•lular avaluada. El pretractament amb YM155 abans de la radioteràpia indueix un augment en el nombre de focus de γ-H2AX. Els resultats de aquest apartat de la tesis suggereixen que la inhibició de la Survivina podria ser una nova eina terapèutica que permetria augmentar la sensibilitat a la radioteràpia en pacients amb glioblastoma multiforme.Glioblastoma multiforme is the most common and aggressive primary brain tumor in adults, despite efforts to improve their treatment, patients survival remains poor. The purpose of this thesis is the study of molecular mechanism involved in the induction of apoptosis, cells cycle arrest as a consequence of the treatment with MDM2 and Survivin inhibitors in human glioblatoma multiforme. This effect has been studied ex vivo in glioma cell lines and primary cultured glioblastoma. Nutlins small-molecule antagonist of MDM2, have been developed to inhibit p53-MDM2 interaction and activate p53 signaling in cancer cells. Nutlin-3a can induce cell cycle arrest (p21), apoptosi (PUMA and Noxa) in those cells with a functional p53 protein. In addiction Nutlin can induce cellular senescence (SA-βGal). In primary cultures and cells lines with a mutated p53 protein, Nutlin-3a fails to induce apoptosis and cell cycle arrest. Moreover Nutlin-3a pre-treatment in U87 cells sensitizes to radiotherapy. YM155 is a small molecule inhibitor of antiapoptotic protein survivin. Survivin is overexpressed in glioma and is associated with decreased survival and increases tumor recurrence. YM155 treatment induces a decrease in cell viability, as well as an apoptosis induction. This is accompanied by a decreased in survivin protein levels in all glioma cell lines tested. Glioma cell line and primary culture incubation show changes in the cell cycle pattern, causing arrest either in G0/G1 or S depending of the cell line tested

A Ran-binding protein facilitates nuclear import of human papillomavirus type 16.

Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex

Cell Uptake and Localization Studies of Squaramide Based Fluorescent Probes

Cell internalization is a major issue in drug design. Although squaramide-based compounds are receiving much attention because of their interesting bioactivity, cell uptake and trafficking within cells of this type of compounds are still unknown. In order to monitor the cell internalization process of cyclosquaramide compounds we have prepared two fluorescent probes by covalently linking a fluorescent dye (BODIPY derivative or fluorescein) to a noncytotoxic cyclosquaramide framework. These two probes (C2-BDP and C2-FITC) rapidly internalize across live cell membranes through endocytic receptor-mediated mechanisms. Due to its higher fluorescence and photochemical stability, C2-BDP is a superior dye than C2-FITC. C2-BDP remains sequestered in late endosomes allowing their fast and selective imaging in various live cell types. Cyclosquaramide–cell membrane interactions facilitate cell uptake and have been investigated by binding studies in solution as well as in live cells. Cyclosquaramide <b>1</b> (C2-BDP) can be used as a highly fluorescent probe for the rapid and selective imaging of late endosomes in live cells

N-(2-methyl-indol-1H-5-yl)-1-naphthalenesulfonamide: a novel reversible antimitotic agent inhibiting cancer cell motility

A series of compounds containing the sulfonamide scaffold were synthesized andscreened for their in vitro anticancer activity against a representative panel of human cancer cell lines, leading to the identification ofN-(2-methyl-1H-indol-5-yl)-1-naphthalenesulfonamide (8e) as a compound showing a remarkable activity across the panel, with IC50 values in the nanomolar-to-low micromolar range. Cell cycle distribution analysis revealed that 8e promoted a severe G2/M arrest, which was followed by cellular senescence as indicated by the detection of senescence-associated -galactosidase (SA-gal) in 8e-treated cells. Prolonged 8e treatment also led to the onset of apoptosis, in correlation with the detection of increased Caspase 3/7 activities. Despite increasing-H2A.X levels, a well-established readout for DNA double-strand breaks, in vitro DNA binding studies with 8e did not support interaction with DNA. In agreement with this, 8e failed to activate the cellular DNA damage checkpoint. Importantly, tubulin staining showed that 8e promoted a severe disorganization of microtubules and mitotic spindle formation was not detected in 8e-treated cells. Accordingly, 8e inhibited tubulin polymerization in vitro in a dose-dependent manner andwas also able to robustly inhibit cancer cell motility. Docking analysis revealed a compatible interaction with the colchicine-binding site of tubulin. Remarkably, thesecellular effects were reversible since disruption of treatment resulted in the reorganization of microtubules, cell cycle re-entry and loss of senescent markers. Collectively, our data suggest that this compound may be a promising new anticancer agent capable of both reducing cancer cell growth and motility

A central region in the minor capsid protein of papillomaviruses facilitates viral genome tethering and membrane penetration for mitotic nuclear entry

<div><p>Incoming papillomaviruses (PVs) depend on mitotic nuclear envelope breakdown to gain initial access to the nucleus for viral transcription and replication. In our previous work, we hypothesized that the minor capsid protein L2 of PVs tethers the incoming vDNA to mitotic chromosomes to direct them into the nascent nuclei. To re-evaluate how dynamic L2 recruitment to cellular chromosomes occurs specifically during prometaphase, we developed a quantitative, microscopy-based assay for measuring the degree of chromosome recruitment of L2-EGFP. Analyzing various HPV16 L2 truncation-mutants revealed a central chromosome-binding region (CBR) of 147 amino acids that confers binding to mitotic chromosomes. Specific mutations of conserved motifs (IVAL286AAAA, RR302/5AA, and RTR313EEE) within the CBR interfered with chromosomal binding. Moreover, assembly-competent HPV16 containing the chromosome-binding deficient L2(RTR313EEE) or L2(IVAL286AAAA) were inhibited for infection despite their ability to be transported to intracellular compartments. Since vDNA and L2 were not associated with mitotic chromosomes either, the infectivity was likely impaired by a defect in tethering of the vDNA to mitotic chromosomes. However, L2 mutations that abrogated chromatin association also compromised translocation of L2 across membranes of intracellular organelles. Thus, chromatin recruitment of L2 may in itself be a requirement for successful penetration of the limiting membrane thereby linking both processes mechanistically. Furthermore, we demonstrate that the association of L2 with mitotic chromosomes is conserved among the alpha, beta, gamma, and iota genera of <i>Papillomaviridae</i>. However, different binding patterns point to a certain variance amongst the different genera. Overall, our data suggest a common strategy among various PVs, in which a central region of L2 mediates tethering of vDNA to mitotic chromosomes during cell division thereby coordinating membrane translocation and delivery to daughter nuclei.</p></div

Chromosomal association of point mutants of HPV16 L2.

<p>(A) Full-length L2 is depicted with N-terminal, middle and C-terminal functional domains. The minimal chromosomal binding region (CBR) is highlighted in orange. Within the CBR (insert) the nuclear retention signal and the SUMO interaction motif are indicated at their relative location with the CBR. Conserved regions within the CBR are depicted in red, whereas regions with lower conservation are white. Single conserved residues are highlighted. The sites of the point mutations in the CBR are color-coded according to their impact on chromosomal association (black = interfering; grey = silent). (B) The alignment of the amino acid (aa) sequences of the NRS and upstream residues of HPV16, 18, 5, BPV1 and MnPV L2 are written in the single-letter code. The aa residue numbers, and mutated residues (bold font) are denoted for HPV16 L2. The conservation between the indicated L2 aa sequences was scored by PRALINE [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006308#ppat.1006308.ref078" target="_blank">78</a>], and obtained scores were grouped into five categories: no (white), low (blue), intermediate-low (green), intermediate-high (orange) and high (red) conservation. (C), (D) The aa substitutions IVAL286AAAA, RR297EE, RR302/5AA and RTR313EEE in HPV16 L2-EGFP were analyzed for chromosomal association during mitosis as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006308#ppat.1006308.g002" target="_blank">Fig 2C and 2D</a>. (C) Images of wild-type and mutant HPV16 L2-EGFP (upper row, green), H2B-mCherry (center row, red), and merges (lower row) are shown for representative cells. (D) The chromosomal association indices are depicted in a dot plot as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006308#ppat.1006308.g002" target="_blank">Fig 2D</a>. Statistical significances (two-tailed Student’s t-test) relative to wild-type HPV16 L2-EGFP were assessed (**P < 0.01, ***P < 0.001).</p

Mutant PsVs are defective in mitotic chromosome association of vDNA and membrane penetration of L2 C-terminus.

<p>(A), (B) Subcellular localization of incoming vDNA upon infection with EdU-HPV16, EdU-HPV16-L2(RTR313EEE) and EdU-HPV16-L2(IVAL286AAAA) in mitotic HeLa H2B-mCherry and HaCaT cells. Cellular chromatin in HaCaT cells was visualized by Hoechst staining. (A) Depicted are maximum intensity projections of confocal slices of host chromatin (left panel, red) and vDNA (center panel, green), and merge images (right panel). (B) Quantification of intensity-based colocalization (in percent ± SD) of vDNA from EdU-HPV16 and EdU-HPV16-L2(RTR313EEE) with the host chromatin. (C) Infection and translocation in HaCaT GFP-BAP cells infected with HPV16 L2, L2(RTR313EEE), L2(RR302/05AA), or L2(IVAL286AAAA)-BirA PsV at equal multiplicities of infection. GFP-biotin, total GFP, and intracellular L2-BirA were visualized by Western blot and immunostaining with neutravidin, anti-GFP, and anti-L2 (K4) respectively, as described in [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006308#ppat.1006308.ref047" target="_blank">47</a>]. (D) Infection values were determined by luciferase measurements and represent mean relative infection values compared to wt L2-BirA infected cells and normalized to GAPDH levels.</p

Association of L2 with mitotic chromosomes is conserved among PVs.

<p>(A) The phylogram illustrates the evolutionary relationships between the mucosal high-risk alpha-PVs HPV16 and HPV18, the cutaneous beta-PV HPV5, and the animal delta- and iota-PV BPV1 and MnPV, respectively, based on their L2 amino acid sequences. The branch length represents the amount of genetic changes (scale bar: 0.6 substitutions/site). (B) Images of L2-EGFP (left, green), H2B-mCherry (center, red), and merges (right) are shown for representative cells. (C) The chromosomal association indices for N-terminal deletion mutants are depicted as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006308#ppat.1006308.g002" target="_blank">Fig 2D</a>. As an exception, only 33 cells were analyzed for BPV1 L2.</p

Mutant HPV16 PsVs are impaired in nuclear delivery of vDNA.

<p>(A) HeLa cells and HaCaT cells were infected with low and high amounts of HPV16, HPV16-L2(RTR313EEE) and HPV16-L2(IVAL286AAAA) PsVs carrying a GFP reporter plasmid, fixed at 48 h p.i., and analyzed by flow cytometry. Depicted is the fraction of GFP-positive (i.e. infected) cells in percent ± SD. (B) Subcellular localization of incoming vDNA upon infection with HPV16, HPV16-L2(RTR313EEE) and HPV16-L2(IVAL286AAAA) PsVs carrying EdU-labeled vDNA in the absence or presence of 15 μM aphidicolin was analyzed in HeLa cells at 20 h p.i. by CLSM. Depicted are maximum intensity projections of three medial confocal slices of vDNA detected by EdU-click chemistry (green), host nucleus stained with Hoechst (blue), and the trans-Golgi network immunofluorescently stained for p230 (TGN, red). Shown are merge images of vDNA and nucleus (upper row), and vDNA and TGN in the absence and presence of aphidicolin (middle an lower row, respectively). (C) Quantification of intensity-based colocalization (in percent ± SD) of vDNA from EdU-HPV16, EdU-HPV16-L2(RTR313EEE) and EdU-HPV16-L2(IVAL286AAAA) with the host nucleus. (D) Quantification of intensity-based colocalization (in percent ± SD) of vDNA from EdU-HPV16, and EdU-HPV16-L2(RTR313EEE) with the TGN upon infection in the absence or presence of aphidicolin. (E) Subcellular localization of incoming vDNA upon infection with HPV16, HPV16-L2(RTR313EEE) and HPV16-L2(IVAL286AAAA) PsVs carrying EdU-labeled vDNA was analyzed in HaCaT cells at 20 h p.i. by CLSM. Depicted are maximum intensity projections of three medial confocal slices of vDNA detected by EdU-click chemistry (green) and host nucleus stained with Hoechst (blue). Shown are merge images of vDNA and nucleus (top panel), and vDNA with the nuclear outline (lower panel). (F) Quantification of intensity-based colocalization was performed as in (C).</p