Study of Resistance Mechanisms of All-trans Retinoic Acid in Acute Promyelocytic Leukemia

本文研究了急性早幼粒细胞白血病（APL）患者全反式维甲酸（ATRA）耐药与p糖蛋白（P-gp）相关多药耐药（MDR）之间的关系。本研究使用的细胞包括NB4 （MDR1、MDR相关蛋白和肺耐药蛋白阴性）、耐atra的NB4 （NB4/RA）、MDR1 cdna转导的NB4 （NB4/MDR）和MDR1 cdna转导的NB4/RA (NB4/RA/MDR)，以及来自10例APL患者的胚细胞。在多激光设备的流式细胞仪上，通过紫外线激发ATRA的特征发射曲线来测定细胞内ATRA的积累。通过CD11b、NBT还原活性、细胞周期分布和形态学观察ATRA诱导细胞凋亡和分化的作用。NB4/MDR和NB4/RA/MDR细胞中rhodamin -123 （Rh123）的积累量分别低于NB4和NB4/RA细胞，但细胞内ATRA浓度差异无统计学意义。PSC833是一种MDR修饰剂，可增加NB4/MDR和NB4/RA/MDR细胞内Rh123的积累，但不影响ATRA。NB4细胞与NB4/MDR细胞、NB4/RA细胞与NB4/RA/MDR细胞间CD11b的表达、NBT还原活性、凋亡细胞比例及形态学均无差异。在atra诱导的完全缓解后复发患者的APL细胞分析中也获得了类似的结果。虽然有少数报道称ATRA耐药的APL细胞比ATRA敏感的APL细胞表达更多的P-gp，但我们在这里直接证明了P-gp的表达不影响细胞内ATRA浓度，P-gp和ATRA耐药是独立存在的。

We present here the relationship between all-trans retinoic acid (ATRA)-resistance and P-glycoprotein (P-gp)-associated multidrug resistance (MDR) in acute promyelocytic leukemia (APL). Cells used in this study were NB4 (negative for MDR1, MDR related protein and lung resistant protein), ATRA-resistant NB4 (NB4/RA), MDR1 cDNA-transduced NB4 (NB4/MDR) and MDR1 cDNA-transduced NB4/RA (NB4/RA/MDR), and blast cells from 10 patients with APL. Intracellular ATRA accumulation was determined by the characteristic emission curve of ATRA, which was excited by ultra violet in multi-laser-equipped flow cytometer. The apoptosis- and differentiation-introducing effects of ATRA were determined by CD11b, NBT reduction activity, cell cycle distribution and morphology.While NB4/MDR and NB4/RA/MDR cells accumulated less Rhodamine-123 (Rh123) than NB4 and NB4/RA cells, respectively, there is no difference in the intracellular ATRA concentration between them. PSC833, a MDR modifier, increased the intracellular accumulation of Rh123 in NB4/MDR and NB4/RA/MDR cells, but not of ATRA. The expression of CD11b, the NBT reduction activity, the proportion of apoptotic cells and the morphology were not different between NB4 and NB4/MDR cells and between NB4/RA and NB4/RA/MDR cells. Similar results were obtained in the analysis of APL cells from patients relapsed after ATRA-induced complete remission.Although there are a few reports that ATRA-resistant APL cells expressed more P-gp than ATRA-sensitive ones, we here directly show that the expression of P-gp does not influence the intracellular concentration of ATRA and that P-gp and ATRA-resistance independently exist.