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Sequential Measurements of Catalytic Activities of Multi-Drug-Resistance Transporters and Cytochrome P450 Enzymes by Cytometry of Reaction Rate Constant
bioRxiv - Cancer Biology Pub Date : 2020-10-15 , DOI: 10.1101/2020.10.15.340976
Vasilij Koshkin , Mariana Bleker de Oliveira , Sven Kochmann , Chun Peng , Sergey N. Krylov

Cytometry of reaction rate constant (CRRC) is an accurate and robust approach to characterize cell-population heterogeneity using rate constants of cellular processes for which kinetic mechanisms are known. We work on a CRRC-based method to develop predictors of tumor chemoresistance driven by two processes: drug extrusion by multi-drug-resistance (MDR) transporters and drug inactivation by cytochrome-P450 enzymes (CYP). Each of the two possess is studied with its specific substrate and the process activity is characterized by a corresponding unimolecular rate constant. Due to the incompatibility of MDR and CYP assays, MDR and CYP activities may be difficult to measure simultaneously suggesting that they may need to be measured sequentially. The sequential measurements may also impose a problem: the results of the second assay may be affected by artifacts exerted by the first assay. The goal of this work was to understand whether the cells have a memory of the first assay that significantly affects the results of the second assay. To achieve this goal, we compared CRRC results for two orders of sequential measurements: the MDR→CYP order in which MDR activity is measured before CYP activity and the CYP→MDR order in which CYP activity is measured before MDR activity. It was found that the results of the CYP assay were similar in both orders; on the contrary, the results of the MDR assay were significantly different. Our findings suggest that MDR and CYP activity can be studied sequentially provided that MDR activity is measured first and CYP activity second.

中文翻译:

反应速率常数的细胞计数法连续测量多种药物转运蛋白和细胞色素P450酶的催化活性

反应速率常数(CRRC)的细胞计数法是一种准确而可靠的方法,可利用已知动力学机制的细胞过程速率常数来表征细胞种群的异质性。我们研究基于CRRC的方法,以开发由两个过程驱动的肿瘤化学抗性的预测因子:通过多药抗性(MDR)转运蛋白进行的药物挤出和通过细胞色素P450酶(CYP)进行的药物灭活。用其特定的底物研究了两者各自的特征,并且通过相应的单分子速率常数来表征过程活性。由于MDR和CYP检测方法不兼容,因此MDR和CYP活性可能难以同时测量,表明它们可能需要顺序测量。顺序测量也可能会带来问题:第二次测定的结果可能会受到第一次测定所施加的伪影的影响。这项工作的目的是了解细胞是否具有对第一次测定的记忆力,从而显着影响第二次测定的结果。为了实现此目标,我们比较了两个顺序测量的CRRC结果:MDR→CYP顺序,其中MDR活性先于CYP活性进行测量; CYP→MDR顺序,其中CYP活性先于MDR活性进行测量。发现CYP测定的结果在两个顺序上是相似的。相反,MDR分析的结果却有显着差异。我们的发现表明,只要先测量MDR活性,然后再测量CYP活性,就可以顺序研究MDR和CYP活性。这项工作的目的是了解细胞是否具有对第一次测定的记忆力,从而显着影响第二次测定的结果。为了实现此目标,我们比较了两个顺序测量的CRRC结果:MDR→CYP顺序,其中MDR活性先于CYP活性进行测量; CYP→MDR顺序,其中CYP活性先于MDR活性进行测量。发现CYP测定的结果在两个顺序上是相似的。相反,MDR分析的结果却有显着差异。我们的发现表明,只要先测量MDR活性,然后再测量CYP活性,就可以顺序研究MDR和CYP活性。这项工作的目的是了解细胞是否具有对第一次测定的记忆力,从而显着影响第二次测定的结果。为了实现此目标,我们比较了两个顺序测量的CRRC结果:MDR→CYP顺序,其中MDR活性先于CYP活性进行测量; CYP→MDR顺序,其中CYP活性先于MDR活性进行测量。发现CYP测定的结果在两个顺序上是相似的。相反,MDR分析的结果却有显着差异。我们的发现表明,只要先测量MDR活性,然后再测量CYP活性,就可以顺序研究MDR和CYP活性。我们比较了两个顺序测量的CRRC结果:MDR→CYP顺序,其中MDR活性先于CYP活性进行测量; CYP→MDR顺序,其中CYP活性先于MDR活性进行测量。发现CYP测定的结果在两个顺序上是相似的。相反,MDR分析的结果却有显着差异。我们的发现表明,只要先测量MDR活性,然后再测量CYP活性,就可以顺序研究MDR和CYP活性。我们比较了两个顺序测量的CRRC结果:MDR→CYP顺序,其中MDR活性先于CYP活性进行测量; CYP→MDR顺序,其中CYP活性先于MDR活性进行测量。发现CYP测定的结果在两个顺序上是相似的。相反,MDR分析的结果却有显着差异。我们的发现表明,只要先测量MDR活性,然后再测量CYP活性,就可以顺序研究MDR和CYP活性。
更新日期:2020-10-17
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