Demur C, Muller C, Cassar G, Bousquet C, Laroche M, Laurent G
Laboratoire d'Hématologie, CHU Purpan, Toulouse, France.
Leukemia. 1998 Feb;12(2):192-9. doi: 10.1038/sj.leu.2400925.
This study was designed to correlate the clonogenic capacity of acute myeloid leukemia (AML) cells with P-glycoprotein (P-gp) expression level and P-gp-mediated efflux capacity. Fifty AML cell samples were tested for P-gp expression using MRK16 monoclonal antibody and flow cytometry. Among them, 12 samples were selected for sorting experiments according to the following two criteria: their clonogenic capacity in methylcellulose in the presence of 5637 conditioned medium, and the heterogeneity of P-gp distribution in leukemic cells. For each of these 12 samples, leukemic cells which displayed the highest P-gp expression level (P-gp++) and P-gp- leukemic cells were sorted after MRK16 staining and seeded into methylcellulose for primary clonogenic assay. In each case, the number of CFU-L in the P-gp fraction was significantly higher than that of the P-gp++ fraction (P < 0.01); the median number of CFU-L for 10(5) seeded cells being 147 (range 3-1855) and 495 (range 60-4100) for P-gp++ and P-gp- populations, respectively. Furthermore, in order to correlate clonogenic capacity and P-gp function, AML cells were stained with rhodamine 123 (Rh 123), washed and then sorted after 4 h incubation at 37 degrees C in Rh 123-free media on the basis of their residual fluorescence intensity before plating. For each of six samples, we found that the number of CFU-L in the AML cell fraction which displayed the most efficient Rh 123 efflux capacity (Rh 123dull) was significantly higher compared to that of the AML cell fraction which displayed high residual fluorescence signal (Rh 123bright) (P = 0.05); the median number of CFU-L for 10(5) seeded cells being 1025 (range 250-2240) and 296 (range 11-838) for Rh 123dull and Rh 123bright populations, respectively. Altogether this study suggests that, for an individual AML cell population, the clonogenic fraction is preferentially recruited in AML cells which display low P-gp expression and high P-gp-mediated efflux capacity.
本研究旨在将急性髓系白血病（AML）细胞的克隆形成能力与P-糖蛋白（P-gp）表达水平及P-gp介导的外排能力相关联。使用MRK16单克隆抗体和流式细胞术检测了50个AML细胞样本的P-gp表达。其中，根据以下两个标准选择12个样本进行分选实验：它们在5637条件培养基存在下于甲基纤维素中的克隆形成能力，以及白血病细胞中P-gp分布的异质性。对于这12个样本中的每一个，在MRK16染色后分选显示最高P-gp表达水平的白血病细胞（P-gp++）和P-gp-白血病细胞，并接种到甲基纤维素中进行初次克隆形成测定。在每种情况下，P-gp组分中的集落形成单位-白血病（CFU-L）数量显著高于P-gp++组分（P < 0.01）；接种10(5)个细胞时，P-gp++和P-gp-群体的CFU-L中位数分别为147（范围3 - 1855）和495（范围60 - 4100）。此外，为了关联克隆形成能力和P-gp功能，用罗丹明123（Rh 123）对AML细胞进行染色，洗涤后，在37℃于无Rh 123的培养基中孵育4小时，然后根据接种前的残余荧光强度进行分选。对于六个样本中的每一个，我们发现显示最有效Rh 123外排能力的AML细胞组分（Rh 123dull）中的CFU-L数量显著高于显示高残余荧光信号的AML细胞组分（Rh 123bright）（P = 0.05）；接种10(5)个细胞时，Rh 123dull和Rh 123bright群体的CFU-L中位数分别为1025（范围250 - 2240）和296（范围11 - 838）。总之，本研究表明，对于单个AML细胞群体，克隆形成组分优先存在于显示低P-gp表达和高P-gp介导外排能力的AML细胞中。
