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Mays A.N.,Kings College London | Osheroff N.,Vanderbilt University | Xiao Y.,University of California at San Francisco | Wiemels J.L.,University of California at San Francisco | And 14 more authors.
Blood | Year: 2010

Therapy-related acute promyelocytic leukemia (t-APL) with t(15;17)(q22;q21) involving the PML and RARA genes is associated with exposure to agents targeting topoisomerase II (topoII), particularly mitoxantrone and epirubicin. We previously have shown that mitoxantrone preferentially induces topoII-mediated DNA damage in a "hotspot region" within PML intron 6. To investigate mechanisms underlying epirubicin-associated t-APL, t(15; 17) genomic breakpoints were characterized in 6 cases with prior breast cancer. Significant breakpoint clustering was observed in PML and RARA loci (P = .009 and P = .017, respectively), with PML breakpoints lying outside the mitoxantrone-associated hotspot region. Recurrent breakpoints identified in the PML and RARA loci in epirubicin-related t-APL were shown to be preferential sites of topoII-induced DNA damage, enhanced by epirubicin. Although site preferences for DNA damage differed between mitoxantrone and epirubicin, the observation that particular regions of the PML and RARA loci are susceptible to these agents may underlie their respective propensities to induce t-APL. © 2010 by The American Society of Hematology.

Subauste M.C.,rmott Center for Human Growth and Development | Sansom O.J.,Beatson Institute | Porecha N.,rmott Center for Human Growth and Development | Raich N.,U 654 Hopital A. Trousseau | And 2 more authors.
Molecular Carcinogenesis | Year: 2010

In the treatment of colon cancer, the development of resistance to apoptosis is a major factor in resistance to therapy. New molecular approaches to overcome apoptosis resistance, such as selectively upregulating proapoptotic proteins, are needed in colon cancer therapy. In a mouse model with inactivation of the adenomatous polyposis coli (Apc) tumor suppressor gene, reflecting the pathogenesis of most human colon cancers, the gene encoding feminization-1 homolog b (Fem1b) is upregulated in intestinal epithelium following Apc inactivation. Fem1b is a proapoptotic protein that interacts with apoptosis-inducing proteins Fas, tumor necrosis factor receptor-1 (TNFR1), and apoptotic protease activating factor-1 (Apaf-1). Increasing Fem1b expression induces apoptosis of cancer cells, but effects on colon cancer cells have not been reported. Fem1b is a homolog of feminization-1 (FEM-1), a protein in Caenorhabditis elegans that is regulated by proteasomal degradation, but whether Fem1b is likewise regulated by proteasomal degradation is unknown. Herein, we found that Fem1b protein is expressed in primary human colon cancer specimens, and in malignant SW620, HCT-116, and DLD-1 colon cancer cells. Increasing Fem1b expression, by transfection of a Fem1b expression construct, induced apoptosis of these cells. We found that proteasome inhibitor treatment of SW620, HCT-116, and DLD-1 cells caused upregulation of Fem1b protein levels, associated with induction of apoptosis. Blockade of Fem1b upregulation with morpholino antisense oligonucleotide suppressed the proteasome inhibitor-induced apoptosis of these cells. In conclusion, the proapoptotic protein Fem1b is downregulated by the proteasome in malignant colon cancer cells and mediates proteasome inhibitor-induced apoptosis of these cells. Therefore, Fem1b could represent a novel molecular target to overcome apoptosis resistance in therapy of colon cancer. © 2009 Wiley-Liss, Inc.

No statistical methods were used to predetermine sample size. YapΔ/Δ and TazΔ/Δ mice were generated by crossing Yap or Taz floxed mice30 with the villin-cre line (Jackson Laboratory), the villin-creERT2 line (S. Robine, Institut Curie-CNRS) or the Lgr5-creERT line (Jackson Laboratory). The Rosa26-lox-STOP-lox-rtta-IRES-EGFP and Rosa26 lacZ mouse lines were obtained from Jackson Laboratory. The YapTg transgenic line described in this study was generated by introducing a HA-tagged wild-type Yap cDNA downstream of 7 Tet-repressor elements in the pTRE2 vector (J. Whitsett, Cincinnati Children’s Hospital Medical Center). The transgenic Yap construct was linearized and microinjected in ICR embryos. As shown in Extended Data Fig. 3a, activation of Cre deletes a neo cassette and allows for expression of the rtTA gene. In the presence of doxycycline the rtTA activates transcription of HA-Yap. Apc floxed mice were obtained from O. Sansom (Beatson Institute). Lats1 and Lats2 floxed alleles were obtained from R. Johnson (MD Anderson Cancer Center) and crossed with villin-creERT2 mice to obtain Lats1Δ/Δ;Lats2Δ/Δ mice. To measure polyp formation, YapΔ/Δ mice were backcrossed to a Bl/6 background for 4 generations before crossing to ApcMin/+ mice. Polyps from Yap+/+ (Yap+/+;villin-cre;ApcMin/+), Yap+/Δ (Yapfl/+;villin-cre;ApcMin/+) and YapΔ/Δ (Yapfl/fl;villin-cre;ApcMin/+) mice were counted 16 weeks after birth or when animals appeared moribund. Survival of ApcMin mice was measured by the number of days before mice were euthanized due to poor health. In vivo assays comparing control and Yap mutant animals were performed between age- and sex-matched pairs. No method of randomization was followed and no animals were excluded in this study. The investigators were not blinded to allocation during experiments and outcome assessment. Inducible Cre-mediated deletion of genes was performed by intraperitoneal injections of >5-week-old mice with 200 μl tamoxifen in corn oil at 10 mg ml−1. To create mosaic expression of Yap, Yapfl/fl;villin-creERT2 mice were induced with a single injection of 200 μl of tamoxifen at a suboptimal dose typically between 0.5 and 2.0 mg ml−1. For in vivo regeneration assays, mice were given a single dose of 10 or 12 Gy using a GammaCell 40 irradiator. Animals were maintained and handled under procedures approved by the Canadian Council on Animal Care. The immunohistochemistry stainings and standard colorimetric in situ hybridization were carried out according to methods described elsewhere31. Staining experiments were repeated on independent tissue sections prepared from separate mice as indicated by n values in figure legends. The following primary antibodies were used for immunostaining: rat anti-Ki67 (Dako, Cat. no. M7249, 1:1,000), rabbit anti-Yap/Taz (Cell Signaling, Cat. no. 8418, 1:100), rabbit anti-Yap (Cell Signaling, Cat. no. 14074, 1:300), mouse anti-Yap (Santa Cruz, Cat. no. sc-101199, 1:100), rabbit anti-Lef (Cell Signaling, Cat. no. 2230, 1:300), phosphor-Egfr (Tyr1092) (Abcam, Cat. no. ab40815, 1:300), anti-cleaved caspase-3 (Cell Signaling, Cat. no. 9664, 1:300) and anti-lysozyme (Dako, Cat. no. A0099, 1:1,000). Detection of primary antibodies was achieved using the Dako Envision plus system. Multi-colour fluorescence in situ hybridization with tyramide signal amplification (TSA) was done essentially as described elsewhere32, 33, 34. In brief, RNA probes from hybridized sections were detected using appropriate hapten-specific HRP-conjugated antibodies (anti-digoxigenin-HRP (Roche, Cat. no. 11207733910, 1:500), anti-dinitrophenyl-HRP (PerkinElmer, Cat. no. NEL747A001KT, 1:300), and anti-fluorescein-HRP (Life Technologies, A21253, 1:500)). After overnight incubation with antibodies at 4°C (or 2 h at room temperature for anti-fluorescein-HRP detection of cryptin1) sections were washed in PBS, and rinsed twice in 100 mM borate pH 8.5 plus 0.1% BSA. TSA reaction was performed by applying 300 μl per slide of the following mixture: 100 mM borate pH 8.5, 2% dextran sulfate, 0.1% Tween-20 and 0.003% H O , 450 μg ml−1 4-iodophenol: 1:250 Tyramide product (that is, DyLight633-tyramide, Dylight488-tyramide, Dylight 555-tyramide). The TSA reaction was allowed to proceed for 20 min and then terminated by washing slides in 100 mM glycine pH 2.0 for 15 min. Sections were washed further in PBS for the next round of detection. To synthesize tyramide products, the following succinimidyl esters were used for conjugation with tyramine: DyLight 633 NHS-Ester (Thermo Scientific Cat#46414), DyLight 550 NHS-Ester (Thermo Scientific Cat#62262), DyLight 488 NHS-Ester (Thermo Scientific Cat. no. 46402). The synthesis reaction was carried out as described previously32. The following in situ hybridization probes were obtained from the collection of MGC clones at the Lunenfeld Tanenbaum Research Institute: TweakR (BC025860), Ly6c1 (BC092082), Edn1 (BC029547), Areg (BC009138), Ereg (BC027838), Il1rn (BC042532), Il33 (BC003847), Msln (BC023753) and Cyr61 (BC066019). The Olfm4 and cryptdin1 probes were a gift from H. Clevers (Hubrecht Institute). Before fixing organoids, 10 μM Edu was added to the culture media for 1 h. Then organoids were fixed in 10% buffered formalin for 30 min, permeabilized in 0.5% Triton for 20 min and blocked in 2% BSA. Incorporated Edu was detected using the ClickIt EDU Imaging kit (Invitrogen) according to the manufacturer’s instructions. The primary antibodies used for immunostaining were mouse anti-Yap (Santa Cruz, Cat # sc-101199, 1:100), mouse anti-HA (Sigma-Aldrich, Cat. no. H9658, 1:1,000), and chicken anti-β-gal (Abcam, Cat. no. ab9361, 1:300). The secondary antibodies used in immunostaining were: CF555-donkey anti-mouse (Biotium, Cat. no. 20037, 1:400) and CF647 donkey anti-rabbit (Biotium, Cat. no. 20047, 1:400). Organoids were counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Sigma-Aldrich) before mounting onto slides for visualization. Images were acquired using a 20×/NA oil immersion objective lens (HCX PL APO, Leica), an EM-CCD camera (ImagEM, Hamamatsu) on an inverted microscope (DMIRE2, Leica) with a spinning disk confocal scanner (CSU10, Yokogawa) and Volocity. De novo crypts were scored as any protrusions, typically containing Paneth cells, budding from the initial sphere formed after seeding isolated crypts. Crypts were counted from bright-field images using Image J. At least four independent cultures derived from four different mice per genotype were used for quantification. Survival of crypts in Fig. 1 was determined by Ki67 staining of cross-sections of proximal portions of the small intestine at 3 days post-irradiation (10 Gy or 12 Gy). Values in Fig. 1b represent average number of fully labelled Ki67+ crypts per intestinal circumference based on counts from at least two sections per mouse and assays were repeated in 6 independent mice per genotype for both 10 Gy and 12 Gy treatments. The percentage of surviving Yap-positive versus negative Lgr5+ ISCs in Fig. 1d was performed by counting 587 β-gal+ crypts from a total of 7 untreated YapΔLgr5-cre mice and 394 β-gal+ crypts from a total of 9 irradiated YapΔLgr5-cre mice. In Yap;ApcΔLgr5-cre mice tumour initiating cells were visualized by staining for the Wnt target gene, Lef. As shown in Extended Data Fig. 10b, Lef is undetected in wild-type crypts and highly upregulated in Apc-null cells and thus serves as a robust marker of Apc deletion31. The percentage of Paneth cells in Lef+ foci (Fig. 4a) was assessed by preparing consecutive sections stained for Lyz, Lef and Yap, respectively. Lysozyme-positive Paneth cells from a total of 207 Yap wild-type and 201 Yap mutant Lef+ foci were counted from 5 Yap;ApcΔLgr5-cre mice (10–16 days after tamoxifen injection) using Image J and the percentage of total cells within the boundaries of a given Lef+ lesion was calculated. Relative activation of Egfr was quantified in consecutive sections from 5 Yap;ApcΔLgr5-cre mice stained for Lef, Yap and phospho-Egfr. For assessing Phospho-Egfr, staining intensity in Lef+ foci was assessed in a blinded fashion. For this, consecutive sections stained for Yap were masked from the observer scoring phospho-Egfr staining intensity. Lef+ foci were scored as ‘+’ if phospho-Egfr expression was elevated compared to wild-type adjacent crypts at comparable levels within the crypt–villus axis (see Extended Data Fig. 10f, panels xi and xii). Lef+ foci were scored as ‘++’ if staining intensity was very strong even relative to the stem cell compartment in normal crypts and/or displayed prominent apical staining (see Yap-positive foci in Fig. 4c, panel iv, and Extended Data Fig. 10f, panels v and vi). Lef+ foci were scored as ‘–’ if staining intensity was undetected or unchanged relative to adjacent wild-type crypts (see Yap mutant foci in Fig. 4c and Extended Data Fig. 10f). In Extended Data Fig. 1, caspase 3 and BrdU positive crypt cells were counted from at least six sections per mouse in 4 independent mice per genotype and expressed as a percentage of total crypt cells. All data are presented as average values with s.e.m. Mann–Whitney (two-tailed) U-test was used to determine statistical significance. Calculations were performed using GraphPad Prism 5 software. RNA was isolated from organoids cultured for 24 h after seeding in Matrigel. RNA samples were pooled from at least three organoid cultures derived from at least three independent mice per genotype (Yapfl/+;villin-cre, Yapfl/fl;villin-cre and YapTg). Quality of RNA was verified by running samples on a Bioanalyzer. High-throughput sequencing was performed using the Illumina HiSeq 2000 at the Lunenfeld Tanenbaum Research Institute (LTRI) sequencing facility. Raw sequencing reads in Fastq formats were mapped onto mouse genome (mm9) using Tophat 1.4.1 and the RPKMs (reads per kilobase of exon model per million mapped reads) were calculated using a customized script. RNA-seq data are presented in Supplementary Table 1. Combined fold change presented in Extended Data Fig. 3d and Supplementary Table 1 was calculated using the following formula: combination fold change = log [(YapΔ/Δ/Yap+/Δ)/(Dox+/Dox−)]. R, Cluster 3.0 and Java TreeView were used for data visualization. Gut organoids were cultured according to a previously described protocol established by Sato and Clevers7. Briefly, crypts were harvested by incubating opened small intestines in PBS containing 2 mM EDTA. The epithelium was released by vigorous shaking and crypts separated using a 70 μm cell strainer. Crypts were seeded in growth factor reduced Matrigel (BD Biosciences) and grown in Advanced DMEM/F12 (Invitrogen) supplemented with 2 mM GlutaMax (Invitrogen), 100 U ml−1 Penicillin/100 μg ml−1 Streptomycin (Invitrogen), N2 Supplement (Invitrogen), B-27 Supplement (Invitrogen Cat), mouse recombinant Egf (R&D Systems), 100 ng ml−1 mouse recombinant Noggin (Peprotech), 150 ng ml−1 human Rsp1 (R&D Systems). Apc-deficient organoids were harvested from Yapfl/+;Apcfl/fl;villin-creERT, Yapfl/fl;Apcfl/fl;villin-creERT or YapTg;Apcfl/fl;villin-creERT mice injected with tamoxifen and seeded 48 h later in basal growth medium without Egf, Rsp1 or Noggin. To induce Yap expression in YapTg organoids, 1.5 μg ml−1 doxycycline was added to the culture medium on day 0. Egf (R&D Systems, Cat. no. AF2028), Areg (R&D Systems, Cat. no. AF989) and Ereg (R&D Systems, Cat. no. 1068-EP-050) were added to the culture medium at a final concentration of 0.5 μg ml−1. The following inhibitors were used: PD153053 (0.5 μM, Tocris Bioscience), U0126 (10 μM, Merck Millipore). To examine pErk1/2 levels, organoids were harvested at day 2 in cold PBS containing 5 mM EDTA, 1 mM NaVO , 1.5 mM NaF and protease inhibitors. Organoids were incubated at 4°C for 30 min to dissolve Matrigel and then lysed in TNTE buffer (50 mM Tris/HCl pH 7.6, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA) containing standard protease and phosphatase inhibitors. Protein concentrations were measured and samples were subjected to SDS–PAGE. Total RNA was extracted by removing culture medium and directly lysing organoids in wells using RTL buffer of the Rneasy Mini Kit (Qiagen). RNA was purified using columns and genomic DNA was removed by treatment with RNase-Free DNase (Qiagen).

Salji M.,NHS Greater Glasgow and Clyde | Salji M.,University of Glasgow | Jones R.,University of Glasgow | Jones R.,Cancer Research Glasgow Center | And 13 more authors.
British Journal of Cancer | Year: 2014

Background: Salvage therapeutic options for biochemical failure after primary radiation-based therapy include radical prostatectomy, cryoablation, high-intensity focused ultrasound (HIFU), brachytherapy (for post-EBRT patients) and androgen deprivation therapy (ADT). ADT and salvage prostate cryoablation (SPC) are two commonly considered treatment options for RRPC. However, there is an urgent need for high-quality clinical studies to support evidence-based decisions on treatment choice. Our study aims to determine the feasibility of randomising men with RRPC for treatment with ADT and SPC.Methods:The randomised controlled trial (CROP) was developed, which incorporated protocols to assess parameters relating to cryotherapy procedures and provide training workshops for optimising patient recruitment. Analysis of data from the recruitment phase and patient questionnaires was performed.Results:Over a period of 18 months, 39 patients were screened for eligibility. Overall 28 patients were offered entry into the trial, but only 7 agreed to randomisation. The majority reason for declining entry into the trial was an unwillingness to be randomised into the study. 'Having the chance of getting cryotherapy' was the major reason for accepting the trial. Despite difficulty in retrieving cryotherapy temperature parameters from prior cases, 9 of 11 cryotherapy centres progressed through the Cryotherapists Qualification Process (CQP) and were approved for recruiting into the CROP study.Conclusions:Conveying equipoise between the two study arms for a salvage therapy was challenging. The use of delayed androgen therapy may have been seen as an inferior option. Future cohort studies into available salvage options (including prostate cryotherapy) for RRPC may be more acceptable to patients than randomisation within an RCT. © 2014 Cancer Research UK. All rights reserved.

Zimmermann G.,Max Planck Institute of Molecular Physiology | Zimmermann G.,TU Dortmund | Schultz-Fademrecht C.,Lead Discovery Center | Kuchler P.,Max Planck Institute of Molecular Physiology | And 10 more authors.
Journal of Medicinal Chemistry | Year: 2014

K-Ras is one of the most frequently mutated signal transducing human oncogenes. Ras signaling activity requires correct cellular localization of the GTPase. The spatial organization of K-Ras is controlled by the prenyl binding protein PDEδ, which enhances Ras diffusion in the cytosol. Inhibition of the Ras-PDEδ interaction by small molecules impairs Ras localization and signaling. Here we describe in detail the identification and structure guided development of Ras-PDEδ inhibitors targeting the farnesyl binding pocket of PDEδ with nanomolar affinity. We report kinetic data that characterize the binding of the most potent small molecule ligands to PDEδ and prove their binding to endogenous PDEδ in cell lysates. The PDEδ inhibitors provide promising starting points for the establishment of new drug discovery programs aimed at cancers harboring oncogenic K-Ras. © 2014 American Chemical Society.

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