Romano M F, Lamberti A, Tassone P, Alfinito F, Costantini S, Chiurazzi F, Defrance T, Bonelli P, Tuccillo F, Turco M C, Venuta S
Dipartimento di Biochimica e Biotecnologie Mediche, Università Federico II, Napoli, Italia.
Blood. 1998 Aug 1;92(3):990-5.
We analyzed the effect of CD40 triggering on the fludarabine-induced apoptosis of B chronic lymphocytic leukemia (B-CLL) cells. Peripheral blood samples obtained from 15 patients were incubated with fludarabine in the absence or the presence of the anti-CD40 monoclonal antibody (MoAb) G28-5. In 12 patients a significant proportion of apoptotic cells, ranging from 22% to 38% (mean +/- SE: 28.5 +/- 1.6), were detected after 3 days of culture. In 9 of these samples, the addition of G28-5 reduced apoptosis by at least 30.1% and by 57.1% +/- 7.8% on average (P = .0077). Because the CD40 antigen activates NF-kappaB/Rel transcription factors in B cells, and NF-kappaB/Rel complexes can inhibit cell apoptosis, we investigated whether the antiapoptotic effect of G28-5, in our system, could be related to modulation of NF-kappaB/Rel activity. As expected, B-CLL cells displayed significant levels of nuclear NF-kappaB/Rel activity; p50, RelA, and c-Rel components of the NF-kappaB/Rel protein family were identified in these complexes. After exposure to fludarabine, NF-kappaB/Rel complexes were decreased in the nuclei. The addition of G28-5 upregulated the NF-kappaB/Rel levels. To determine the involvement of NF-kappaB/Rel activity in the G28-5-mediated inhibition of apoptosis, we blocked the transcription factor with a decoy oligonucleotide, corresponding to the NF-kappaB/Rel consensus sequence. Cells incubated with the anti-CD40 MoAb in the presence of the decoy oligonucleotide but not a control oligonucleotide displayed a complete impairment of the G28-5 antiapoptotic effect, indicating that NF-kappaB/Rel activity was required for the inhibition of apoptosis. These results suggest that CD40 triggering in vivo could counteract the apoptotic effect of fludarabine on B-CLL cells and that its neutralization, or the use of NF-kappaB/Rel inhibitors, could improve the therapeutic effect of fludarabine.
我们分析了CD40激活对氟达拉滨诱导的B慢性淋巴细胞白血病（B-CLL）细胞凋亡的影响。从15例患者获取的外周血样本在不存在或存在抗CD40单克隆抗体（MoAb）G28-5的情况下与氟达拉滨一起孵育。培养3天后，在12例患者中检测到显著比例的凋亡细胞，范围为22%至38%（平均值±标准误：28.5±1.6）。在其中9个样本中，添加G28-5使凋亡减少至少30.1%，平均减少57.1%±7.8%（P = 0.0077）。由于CD40抗原在B细胞中激活NF-κB/Rel转录因子，且NF-κB/Rel复合物可抑制细胞凋亡，我们研究了在我们的体系中G28-5的抗凋亡作用是否与NF-κB/Rel活性的调节有关。正如预期的那样，B-CLL细胞显示出显著水平的核NF-κB/Rel活性；在这些复合物中鉴定出了NF-κB/Rel蛋白家族的p50、RelA和c-Rel成分。暴露于氟达拉滨后，细胞核中的NF-κB/Rel复合物减少。添加G28-5上调了NF-κB/Rel水平。为了确定NF-κB/Rel活性在G28-5介导的凋亡抑制中的作用，我们用与NF-κB/Rel共有序列对应的诱饵寡核苷酸阻断转录因子。在存在诱饵寡核苷酸而非对照寡核苷酸的情况下与抗CD40 MoAb一起孵育的细胞显示G28-5抗凋亡作用完全受损，表明NF-κB/Rel活性是抑制凋亡所必需的。这些结果提示，体内CD40激活可能抵消氟达拉滨对B-CLL细胞的凋亡作用，而中和CD40或使用NF-κB/Rel抑制剂可能会提高氟达拉滨的治疗效果。
