Surget Sylvanie, Descamps Géraldine, Brosseau Carole, Normant Vincent, Maïga Sophie, Gomez-Bougie Patricia, Gouy-Colin Nadège, Godon Catherine, Béné Marie C, Moreau Philippe, Le Gouill Steven, Amiot Martine, Pellat-Deceunynck Catherine
CRCNA, INSERM, UMR 892, Nantes F-44000, France.
BMC Cancer. 2014 Jun 14;14:437. doi: 10.1186/1471-2407-14-437.
The aim of this study was to evaluate the efficacy of the p53-reactivating drugs RITA and nutlin3a in killing myeloma cells.
A large cohort of myeloma cell lines (n = 32) and primary cells (n = 21) was used for this study. This cohort contained cell lines with various TP53 statuses and primary cells with various incidences of deletion of chromosome 17. Apoptosis was evaluated using flow cytometry with Apo2.7 staining of the cell lines or via the loss of the myeloma-specific marker CD138 in primary cells. Apoptosis was further confirmed by the appearance of a subG1 peak and the activation of caspases 3 and 9. Activation of the p53 pathway was monitored using immunoblotting via the expression of the p53 target genes p21, Noxa, Bax and DR5. The involvement of p53 was further studied in 4 different p53-silenced cell lines.
Both drugs induced the apoptosis of myeloma cells. The apoptosis that was induced by RITA was not related to the TP53 status of the cell lines or the del17p status of the primary samples (p = 0.52 and p = 0.80, respectively), and RITA did not commonly increase the expression level of p53 or p53 targets (Noxa, p21, Bax or DR5) in sensitive cells. Moreover, silencing of p53 in two TP53(mutated) cell lines failed to inhibit apoptosis that was induced by RITA, which confirmed that RITA-induced apoptosis in myeloma cells was p53 independent. In contrast, apoptosis induced by nutlin3a was directly linked to the TP53 status of the cell lines and primary samples (p < 0.001 and p = 0.034, respectively) and nutlin3a increased the level of p53 and p53 targets in a p53-dependent manner. Finally, we showed that a nutlin3a-induced DR5 increase (≥ 1.2-fold increase) was a specific and sensitive marker (p < 0.001) for a weak incidence of 17p deletion within the samples (≤ 19%).
These data show that RITA, in contrast to nutlin3a, effectively induced apoptosis in a subset of MM cells independently of p53. The findings and could be of interest for patients with a 17p deletion, who are resistant to current therapies.
本研究旨在评估p53激活药物RITA和nutlin3a对骨髓瘤细胞的杀伤效果。
本研究使用了大量骨髓瘤细胞系（n = 32）和原代细胞（n = 21）。该队列包含具有不同TP53状态的细胞系以及具有不同17号染色体缺失发生率的原代细胞。使用Apo2.7染色的流式细胞术评估细胞系的凋亡情况，或通过原代细胞中骨髓瘤特异性标志物CD138的缺失来评估凋亡。通过出现亚G1峰以及半胱天冬酶3和9的激活进一步确认凋亡。通过免疫印迹法监测p53靶基因p21、Noxa、Bax和DR5的表达来监测p53途径的激活。在4种不同的p53沉默细胞系中进一步研究p53的作用。
两种药物均诱导骨髓瘤细胞凋亡。RITA诱导的凋亡与细胞系的TP53状态或原代样本的del17p状态无关（分别为p = 0.52和p = 0.80），并且RITA在敏感细胞中通常不会增加p53或p53靶标（Noxa、p21、Bax或DR5）的表达水平。此外，在两种TP53（突变）细胞系中沉默p53未能抑制RITA诱导的凋亡，这证实了RITA诱导的骨髓瘤细胞凋亡不依赖于p53。相比之下，nutlin3a诱导的凋亡与细胞系和原代样本的TP53状态直接相关（分别为p < 0.001和p = 0.034），并且nutlin3a以p53依赖的方式增加p53和p53靶标的水平。最后，我们表明nutlin3a诱导的DR5增加（≥ 1.2倍增加）是样本中17p缺失发生率较低（≤ 19%）的特异性和敏感标志物（p < 0.001）。
这些数据表明，与nutlin3a不同，RITA能在一部分骨髓瘤细胞中独立于p53有效诱导凋亡。这些发现可能对目前治疗耐药的17p缺失患者具有意义。
