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Separase activity distribution can be a marker of major molecular response and proliferation of CD34+ cells in TKI-treated chronic myeloid leukemia patients.
Annals of Hematology ( IF 3.5 ) Pub Date : 2020-04-06 , DOI: 10.1007/s00277-020-04007-4
Birgit Spiess 1, 2 , Helga Kleiner 1 , Johanna Flach 1 , Alice Fabarius 1 , Susanne Saussele 1 , Wolf-Karsten Hofmann 1 , Wolfgang Seifarth 1
Affiliation  

Separase, a cysteine endopeptidase, is a key player in mitotic sister chromatid separation, replication fork dynamics, and DNA repair. Aberrant expression and/or altered separase proteolytic activity are associated with aneuploidy, tumorigenesis, and disease progression. Since genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML), we have comparatively examined separase proteolytic activity in TKI-treated chronic phase CML. Separase proteolytic activity was analyzed on single cell level in 88 clinical samples and in 14 healthy controls by a flow cytometric assay. In parallel, BCR-ABL1 gene expression and replication fork velocity were measured by qRT-PCR and DNA fiber assays, respectively. The separase activity distribution (SAD) value indicating the occurrence of MNCs with elevated separase proteolytic activity within samples was found to positively correlate with BCR-ABL1 gene expression levels and loss of MMR (relapse) throughout routine BCR-ABL1 monitoring. Analyses of CD34+ cells and MNCs fractionized by flow cytometric cell sorting according to their separase activity levels (H- and L-fractions) revealed that CD34+ cells with elevated separase activity levels (H-fractions) displayed enhanced proliferation/viability when compared with cells with regular (L-fraction) separase activity (mean 3.3-fold, p = 0.0011). BCR-ABL1 gene expression positivity prevailed in MNC H-fractions over L-fractions (42% vs. 8%, respectively). Moreover, expanding CD34+ cells of H-fractions showed decreased replication fork velocity compared with cells of L-fractions (p < 0.0001). Our data suggests an association between high separase activity, residual BCR-ABL1 gene expression, and enhanced proliferative capacity in hematopoietic cells within the leukemic niche of TKI-treated chronic phase CML.

中文翻译:

分离酶活性分布可能是TKI治疗的慢性粒细胞白血病患者主要分子应答和CD34 +细胞增殖的标志。

Separase是一种半胱氨酸内肽酶,是有丝分裂姐妹染色单体分离,复制叉动力学和DNA修复的关键参与者。异常表达和/或分离酶蛋白水解活性改变与非整倍性,致瘤性和疾病进展相关。由于基因组不稳定性和克隆进化是进行性慢性粒细胞白血病(CML)的标志,因此我们比较研究了TKI治疗的慢性期CML中的Separase蛋白水解活性。通过流式细胞术分析了88个临床样品和14个健康对照中单细胞水平的Separase蛋白水解活性。平行地,分别通过qRT-PCR和DNA纤维测定法测量BCR-ABL1基因表达和复制叉速度。在整个常规BCR-ABL1监测过程中,发现样品中的Separase活性分布(SAD)值表明MNC具有较高的Separase蛋白水解活性,与BCR-ABL1基因表达水平和MMR丢失(复发)呈正相关。通过流式细胞术分选的CD34 +细胞和MNC的分离酶活性水平(H和L分数)分析发现,与分离的CD34 +细胞和MNC相比,分离酶活性水平(H分数)升高的CD34 +细胞显示出增强的增殖/活力。有规律的(L分数)分离酶活性(平均3.3倍,p = 0.0011)。BNC-ABL1基因表达阳性在MNC H-馏分中优于L-馏分(分别为42%和8%)。此外,与L级分的细胞相比,H级分的扩增CD34 +细胞显示出降低的复制叉速度(p <0.0001)。我们的数据表明,高分离酶活性,残留的BCR-ABL1基因表达与TKI治疗的慢性期CML白血病患者体内造血细胞增殖能力增强之间存在关联。
更新日期:2020-04-20
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