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Sugars in thymidine phosphorylase overexpressing cells 37°C. TPI was provided by Taiho Pharmaceutical, Co., Ltd. (Tokushima, Japan). TdR, thymine, dR, dR‐1‐P, dR, dR‐5‐P and G3P were obtained from Sigma Aldrich Chemicals (Zwijndrecht, the Netherlands). These chemicals were dissolved in PBS in stock solutions of 1‐20 mM and stored at ‐20 °C. 13 C6‐glucose‐6‐phosphate (prepared as previously described [21]) and 13 C2‐xylulose were obtained from Omicron biochemicals (South Bend, IN, USA). Ethoxyamine was obtained from Acros (Geel, Belgium) and pyridine from Merck (Darmstadt, Germany). 5'‐ 3 H‐TdR (specific activity of 18.8 mCi/mmol, conc. 12.9 µg/ml) was purchased from Moravek Biochemicals Inc. (Brea, CA, USA). The exact moiety where the sugar of the TdR is labelled is indicated in Fig. 1. The thymidine phosphorylase antibody was purchased from R&D systems Inc. (Minneapolis, MN USA) and the thymidine kinase antibody was purchased from QED Bioscience (San Diego, CA, USA). The secondary antibody, goat‐α‐mouse‐IRDye (800CW;#926‐32210 and 680;#926‐32220) was obtained from Westburg (Leusden, the Netherlands). Treatment of the cells For measurement of TdR and its sugars, Colo320TP1 and RT112/TP cells were seeded at 2x10 6 cells/flask. After 48 h, cells were exposed to 100 µM TdR for 0 min, 15 min, 30 min and 60 min. TPI (10 µM) was added 24 h prior to TdR exposure, after which samples were incubated for 60 min. After treatment, cells were trypsinized, washed in PBS and centrifuged for 5 min at 323 g at room temperature (RT). The medium and the cell pellets were stored separately at ‐80°C until sample preparation. Measurement of TP enzymatic activity and of TdR and thymine HPLC measurement of TP enzymatic activity was performed as described previously [22] and is based on the detection of both TdR and thymine [23]. After incubation with thymidine, 25 µl 80% trichloroacetic acid (TCA) was added to the samples, which were left on ice for 20 min. Subsequently, samples were centrifuged at 14000 g at 4°C for 10 min. The supernatant was transferred to a new vial, and the pH was neutralized. Samples were mixed and centrifuged for 1 min at 14000 g. The upper aqueous layer was used for analysis of TdR and thymine by HPLC analysis for nucleosides with UV detection as described previously [23]. Measurement of sugar‐phosphates For sample preparation, the cell pellet was dissolved in 500 µl MQ and subsequently samples were sonificated 3 x for 3 seconds on ice. 5 µM 13 C6‐glucose‐6‐phosphate was used as an internal standard. The samples were centrifuged through a filter with 10 kDa cut off (Ultracel YM‐10; #42407; Amicon Micron, Millipore, Billerica, MA, USA). Subsequently, the samples were injected into the LC‐MS/MS as described previously [24]. The amount of sugars was expressed based on the total protein content of each sample ‐ 121 ‐
Chapter 7 that was measured before centrifugation through the 10 kDa cut off filter. Protein concentration was determined using the BioRad Protein assay (#500‐0006; Bio‐Rad Laboratories, Veenendaal, The Netherlands) according to manufacture’s instruction. Measurement of dR For preparation of the samples for the dR measurement, the cell pellet was dissolved in 150 µL distilled water. 13 C2‐xylulose was used as internal standard. 17 µl of 20 µM 13 C2‐ xylulose was added to 50 µl of the sample. To deproteinize the sample, 200 µl methanol was added. After 15 min, the sample was centrifuged for 10 min at 10 000 g at 4 °C. Following this, the supernatant was transferred to a new vial and evaporated to dryness under a slight stream of nitrogen. Ethoxime derivatives of dR were formed by treating the residue with 2 mg ethoxyamine in 100 µl pyridine at 60 °C for 30 min. After cooling down to RT, the hydroxygroups were acetylated by adding 100 µl acetic anhydride at 80°C for 60 min. This solution was evaporated to dryness and the residue was redissolved in ethylacetate. 1‐2 µl was injected into the GC‐MS operating under positive chemical ionization in the single ion monitoring mode. Separation of cell compartments Colo320 TP1 and RT112/TP cells (1.5x10 6 cells) were exposed to 200 µM TdR (hot:cold (1:21) of which the batch of [5'‐ 3 H]‐TdR was mixed with 1 mM unlabeled TdR). After incubation for 1, 6 or 24 h or 1 h plus 24 h [5'‐ 3 H]‐TdR‐free medium at 37 °C, cell fractions were separated using a ProteoExtract® Subcellular Proteome Extraction Kit according to manufacture’s instructions (Calbiochem, San Diego, CA). Of every cell fraction, including from the medium above the cells after the designated incubation times, 5 µl was counted. To determine to which fraction (protein or non‐protein fraction) secreted [5'‐ 3 H]‐TdR‐ derived metabolites accumulated, 100 µl of the medium above the cells after the retention was precipitated with 60 µl 35% TCA for 20 min on ice. Subsequently, samples were centrifuged for 5 min at 300 g at 4°C and 5 µl of the supernatant (non‐protein fraction) was counted. In addition, the pellet (protein fraction) was recovered in 200 µl MQ, of which 5 µl was counted. Western blotting Colo320, Colo320 TP1, RT112 and RT112/TP cells were washed twice with ice‐cold PBS and lysed in lysis buffer (Cell Signalling Technology Inc., Denver, USA). Cell lysates were scraped, transferred into a vial and centrifuged at 11 000 g at 4 °C for 10 min. Supernatants were transferred to a new vial and protein amounts were determined by the Bio‐Rad assay, according to the manufacturer’s instruction (Bio‐Rad Laboratories, Veenendaal, the Netherlands). From each condition 30 µg of protein was separated on a 10% SDS‐PAGE and electroblotted onto polyvinylidenedifluoride (PVDF) membranes ‐ 122 ‐
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TRAIL-induced kinases activation an
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TRAIL‐induced kinases activation
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“In order to be irreplaceable one
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‐ 8 ‐
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Chapter 1 GENERAL INTRODUCTION Canc
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Chapter 1 OUTLINE OF THE THESIS As
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Chapter 1 Reference List 1. Jemal A
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Chapter 2 ABSTRACT Tumor necrosis f
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Chapter 2 INTRODUCTION The death li
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Chapter 2 In preclinical studies, a
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Chapter 2 Table 1| Summary of the k
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Chapter 2 proliferation could be pr
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Chapter 2 Figure 2. Canonical and n
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Chapter 2 associated with TRAIL res
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Chapter 2 adhesion molecules [109].
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Chapter 2 Reference List 1. Ashkena
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Chapter 2 37. Tolcher AW, Mita M, M
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Chapter 2 melanoma cells from TRAIL
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Chapter 2 and ERK pathways. Circula
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Chapter 3 inhibitors and TRAIL rece
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Chapter 3 of amplification of the t
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Chapter 3 P38 and JNK have opposing
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Chapter 3 Figure 2 (continued). (e)
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Chapter 3 previously established H4
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Chapter 3 effect on TRAIL‐induced
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Chapter 3 induced apoptosis in H460
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Chapter 3 Reference List 1. Jemal A
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Chapter 3 40. Domina AM, Vrana JA,
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Chapter 4 role in the activation of
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Chapter 4 the tip with a diameter o
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Chapter 4 RESULTS TRAIL induces a s
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Chapter 4 vehicle control or with 5
Page 71 and 72: Chapter 4 Non‐canonical TRAIL res
Page 73 and 74: Chapter 4 A549‐shRIP1 cells clear
Page 75 and 76: Chapter 4 Figure 7. Inhibition of S
Page 77 and 78: Chapter 4 DISCUSSION The TRAIL rece
Page 79 and 80: Chapter 4 Src‐independent mechani
Page 81 and 82: Chapter 4 variants. Cell Death Dis
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Page 85 and 86: Chapter 5 ABSTRACT TRAIL is an inte
Page 87 and 88: Chapter 5 targets and subsequent pr
Page 89 and 90: Chapter 5 RESULTS Synergistic activ
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Page 93 and 94: Chapter 5 by sub‐G1 levels in the
Page 95 and 96: Chapter 5 DISCUSSION In the present
Page 97 and 98: Chapter 5 Reference List 1. Jemal A
Page 101 and 102: Chapter 6 ABSTRACT TRAIL is a tumor
Page 103 and 104: Chapter 6 various stress stimuli [1
Page 105 and 106: Chapter 6 Subsequently, the membran
Page 107 and 108: Chapter 6 B H460 A549 Figure 1. Rep
Page 109 and 110: Chapter 6 TRAIL‐dependent cell de
Page 111 and 112: Chapter 6 B H460 A549 C Figure 4 (c
Page 113 and 114: Chapter 6 Figure 5 (continued). Mec
Page 115 and 116: Chapter 6 regulation and checkpoint
Page 117 and 118: Chapter 6 mechanism of immune evasi
Page 119 and 120: Chapter 7 ABSTRACT Thymidine phosph
Page 121: Chapter 7 Figure 1. Schematic overv
Page 125 and 126: Chapter 7 RESULTS TdR conversion to
Page 127 and 128: Chapter 7 dR is secreted from the c
Page 129 and 130: Chapter 7 Figure 4. Accumulation of
Page 131 and 132: Chapter 7 angiogenic properties. Ho
Page 133 and 134: Chapter 7 activity of enzymes. Natu
Page 137 and 138: Chapter 8 SUMMARIZING DISCUSSION AN
Page 139 and 140: Chapter 8 Recently, various differe
Page 141 and 142: Chapter 8 in phase II clinical tria
Page 143 and 144: Chapter 8 Reference List 1. Herbst
Page 147 and 148: Chapter 9 NEDERLANDSE SAMENVATTING
Page 149 and 150: Chapter 9 waargenomen. Het gebruik
Page 151 and 152: Chapter 9 CONCLUSIE Het activeren v
Page 153 and 154: zelfs na werktijden (lees 23:45) ko
Page 155 and 156: Verder wil ik ook dr. Eric Ronken b
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