towards improved death receptor targeted therapy for ... - TI Pharma

More documents

Recommendations

Info

Sugars in thymidine phosphorylase overexpressing cells detectable in this fraction. In the nuclear fraction, the radioactivity was increased after 24 h, which was about 55% of the total intracellular radioactivity. This is possibly in part due to metabolic activation of TdR by thymidine kinase, resulting in incorporations into the DNA and nuclear trapping. Interestingly, TdR‐derived radioactivity was present in the cytoskeletal protein fraction, which increased in time up to about 45% after 24 h. In RT112/TP cells, the radioactivity in the cytosolic fraction decreased in time, with about 45% of [5’‐ 3 H]‐products still detectable after 24 h (Fig. 4). This is in agreement with the lower metabolism of the sugars in these cells, as measured by LC‐MS/MS (Table 1). Comparable to Colo320 TP1 cells, the radioactive products in the nuclear fraction increased in RT112/TP cells, although at lower levels; up to 25% after 24 h. Both the membrane and cytoskeleton fraction increased in time, with a maximum of about 25% and 10% after 24 h incubation, respectively. When the [5’‐ 3 H]‐TdR was washed away following 1 h incubation, the radioactivity was found to a high extent in the membrane (9.6 and 13.6%) and cytoskeletal (73.5 and 39.4%) compartments of both Colo320 TP1 and RT112/TP cells, respectively (data not shown). Hardly any of the radioactive label retained in the nuclear fraction, which may be related to a lack of activation of TdR by TK within 1 h exposure. In summary, the TdR‐ sugars are used in the cytoskeletal and membrane, which may possibly be related to protein‐glycation. Secreted products are in the non‐protein fraction To determine whether products were secreted after conversion of TdR, Colo320 TP1 and RT112/TP cells were exposed to [5’‐ 3 H]‐TdR for 1 h, after which the medium was refreshed and cells were washed to remove the excess of [5’‐ 3 H]‐TdR. The amounts of secreted products were determined by analyzing the amount of radioactivity in the medium fraction. Both Colo320 TP1 and RT112/TP cells secreted radioactive products, with higher levels for RT112/TP (24%) than for Colo320 TP1(15%) cells. To analyze whether the secreted products were in the protein or in the non‐protein fraction, proteins were precipitated by adding TCA (Fig. 4B). In both Colo320 TP1 and RT112/TP cells, the largest part of the secreted radioactive products was found in the non‐ protein fraction. This indicates that the sugars may not be used to a high extent for protein‐glycation of secreted proteins, but may be more important for processes intracellularly. ‐ 127 ‐
Chapter 7 Figure 4. Accumulation of TdR‐related sugars. (A) Accumulation in subcellular compartments of the cytosol, membrane, nucleus and cytoskeleton. Values represent means of three independent experiments ± SEM. nd = not detectable. (B) Percentage of secreted TdR‐related sugars in the medium above the cells, relative to the total radioactivity intra‐ and extracellular. Cells were exposed for 1 h to TdR after which the medium was refreshed and incubated for another 24 h. Subsequently the medium was analyzed for TdR‐related sugars total, in the protein and in the non‐ protein fraction. Values are mean % of total formed thymine of 3 independent experiments ± SEM. ‐ 128 ‐
Page 2 and 3:
TRAIL-induced kinases activation an
Page 4 and 5:
TRAIL‐induced kinases activation
Page 6:
“In order to be irreplaceable one
Page 9 and 10:
‐ 8 ‐
Page 11 and 12:
Chapter 1 GENERAL INTRODUCTION Canc
Page 13 and 14:
Chapter 1 OUTLINE OF THE THESIS As
Page 15 and 16:
Chapter 1 Reference List 1. Jemal A
Page 17 and 18:
Chapter 2 ABSTRACT Tumor necrosis f
Page 19 and 20:
Chapter 2 INTRODUCTION The death li
Page 21 and 22:
Chapter 2 In preclinical studies, a
Page 23 and 24:
Chapter 2 Table 1| Summary of the k
Page 25 and 26:
Chapter 2 proliferation could be pr
Page 27 and 28:
Chapter 2 Figure 2. Canonical and n
Page 29 and 30:
Chapter 2 associated with TRAIL res
Page 31 and 32:
Chapter 2 adhesion molecules [109].
Page 33 and 34:
Chapter 2 Reference List 1. Ashkena
Page 35 and 36:
Chapter 2 37. Tolcher AW, Mita M, M
Page 37 and 38:
Chapter 2 melanoma cells from TRAIL
Page 39 and 40:
Chapter 2 and ERK pathways. Circula
Page 41 and 42:
Page 43 and 44:
Chapter 3 inhibitors and TRAIL rece
Page 45 and 46:
Chapter 3 of amplification of the t
Page 47 and 48:
Chapter 3 P38 and JNK have opposing
Page 49 and 50:
Chapter 3 Figure 2 (continued). (e)
Page 51 and 52:
Chapter 3 previously established H4
Page 53 and 54:
Chapter 3 effect on TRAIL‐induced
Page 55 and 56:
Chapter 3 induced apoptosis in H460
Page 57 and 58:
Chapter 3 Reference List 1. Jemal A
Page 59 and 60:
Chapter 3 40. Domina AM, Vrana JA,
Page 61 and 62:
Page 63 and 64:
Chapter 4 role in the activation of
Page 65 and 66:
Chapter 4 the tip with a diameter o
Page 67 and 68:
Chapter 4 RESULTS TRAIL induces a s
Page 69 and 70:
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
Page 83 and 84: Chapter 4 ‐ 82 ‐
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
Page 91 and 92: Chapter 5 ng/ml TRAIL (Fig. 3B). Ap
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 and 122: Chapter 7 Figure 1. Schematic overv
Page 123 and 124: Chapter 7 that was measured before
Page 125 and 126: Chapter 7 RESULTS TdR conversion to
Page 127: Chapter 7 dR is secreted from the c
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
show all

towards improved death receptor targeted therapy for ... - TI Pharma

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?