Prinzhorn Wiltrud, Stehle Michael, Kleiner Helga, Ruppenthal Sabrina, Müller Martin C, Hofmann Wolf-Karsten, Fabarius Alice, Seifarth Wolfgang
III. Medizinische Klinik (Hämatologie und Onkologie), Wissenschaftliches Labor, Medizinische Fakultät Mannheim der Universität Heidelberg, Pettenkofer Str. 22, 68169 Mannheim, Germany.
Biomark Res. 2016 Mar 2;4:5. doi: 10.1186/s40364-016-0059-2. eCollection 2016.
Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels.
In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter.
Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability.
基因组不稳定性和克隆进化是慢性髓性白血病（CML）进展的标志。最近，我们发现克隆进化和急变期与Separase的表达和活性改变相关，Separase是一种半胱氨酸内肽酶，是染色体分离和中心粒复制过程中的有丝分裂关键因子。人类造血细胞中Separase的过度激活与一种反馈机制有关，该机制在伊马替尼治疗导致Separase蛋白水平降低后，通过翻译后刺激Separase的蛋白水解活性。
为了寻找潜在的治疗反应性转录机制，我们研究了转录因子c-MYB在CML细胞系（LAMA-84、K562、BV-173）和临床样本中对Separase表达的作用。定量逆转录聚合酶链反应（RT-PCR）和蛋白质免疫印迹实验表明，c-MYB的表达水平以伊马替尼依赖的方式降低，并且与细胞系和临床CML样本中Separase的表达水平呈正相关。在CML细胞系中对c-MYB表达进行RNA沉默导致Separase蛋白水平降低。凝胶迁移和染色质免疫沉淀分析证实，c-MYB与ESPL1启动子内的一个假定的c-MYB结合序列结合。
我们的数据表明ESPL1/Separase是c-MYB的一个调控靶点。因此，已知在造血祖细胞的BCR-ABL依赖性转化和白血病发生中必需的c-MYB，也可能控制有丝分裂染色体分离和中心粒复制的Separase依赖性保真度，这对于维持基因组稳定性至关重要。
