von Haefen Clarissa, Gillissen Bernhard, Hemmati Philipp G, Wendt Jana, Güner Dilek, Mrozek Alicja, Belka Claus, Dörken Bernd, Daniel Peter T
Department of Hematology, Oncology and Tumor Immunology, University Medical Center Charité, Humboldt University, Berlin.
Oncogene. 2004 Oct 28;23(50):8320-32. doi: 10.1038/sj.onc.1207971.
The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.
死亡配体TRAIL可与化疗药物或电离辐射等DNA损伤疗法产生协同作用。在此，我们表明TRAIL与5-氟尿嘧啶（5-FU）的协同作用以及TRAIL与5-FU在诱导细胞凋亡方面的交叉致敏作用，完全取决于人DU145和HCT116癌细胞中Bax的功能正常。由于突变而失去Bax蛋白表达的DU145前列腺癌细胞在暴露于TRAIL和5-FU时，无法释放细胞色素c，也无法激活caspase-3和-9。相反，在重新构建Bax表达后，TRAIL对5-FU诱导的细胞凋亡产生致敏作用，反之亦然。在增加TRAIL浓度时对5-FU的ED50剂量进行等效线图分析显示，在表达Bax的细胞中TRAIL和5-FU具有明显的协同作用。相比之下，在缺乏Bax的DU145细胞中，这种作用仅仅是相加的。值得注意的是，DU145和HCT116 Bax缺陷细胞仍然表达Bak。这表明Bak不足以介导5-FU和TRAIL之间的交叉致敏作用和协同作用。在DU145中稳定过表达Bak可使其对表柔比星诱导的细胞凋亡产生致敏作用，但未能赋予TRAIL和5-FU之间的协同作用。此外，我们通过使用EGFP标记的Bax和Bak表明，TRAIL和5-FU协同触发Bax的寡聚化和聚集，但不触发Bak的寡聚化和聚集。这些数据清楚地确立了Bax和Bak在将TRAIL死亡受体途径与线粒体凋亡信号级联联系起来方面的不同作用，并描绘了多结构域Bcl-2同源物在细胞死亡信号传导中的更高特异性程度。
