Bailly J D, Muller C, Jaffrézou J P, Demur C, Gassar G, Bordier C, Laurent G
Laboratoire de Pharmacologie et de Toxicologie Fondamentales, CNRS, Toulouse, France.
Leukemia. 1995 May;9(5):799-807.
In a panel of acute myeloblastic leukemia (AML) cell lines, representative of distinct differentiation stages, we investigated the possible correlation between drug-resistance and both expression and function of the multidrug resistance (MDR)-related P-glycoprotein (P-gp). The AML cell lines were KG1a, KG1, TF1, HEL, ML1, and two non drug-selected P-gp positive subclones originating from HL-60 (HL-60JD) and U937 (U937AQ). All these cells overexpressed the mdr1 gene (analyzed by RT-PCR) and displayed variable levels of P-gp expression. Flow cytometric semi-quantitative evaluation of P-gp with two P-gp specific monoclonal antibodies (MRK16 and UIC2) showed the following P-gp expression hierarchy: TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ; the latter expressing 13 times more P-gp than TF1. When P-gp function was assessed by Rhodamine 123 (Rh123) efflux kinetics, we found that only KG1a and KG1 cells, which have an early (immature) CD34+ CD33- CD38- phenotype, and to a lesser extent TF1, with an intermediate (CD34+ CD33+ CD38+) phenotype, displayed significant P-gp activity which could be inhibited by both verapamil and SDZ PSC 833. In contrast, the other more mature CD33+ CD34- AML cell lines presented no Rh123 efflux capacity although they expressed higher P-gp levels. Daunorubicin (DNR) accumulation studies showed that inhibitors of P-gp increased DNR accumulation only in the immature AML cells whereas they had no impact on the mature AML cell lines. MTT drug cytotoxicity assay confirmed that the immature AML cells were 10-15-fold more resistant to DNR than the mature AML cells. Although P-gp inhibitors were able to increase the cytotoxicity of DNR in AML cells which displayed functional P-gp, they could not increase DNR cytotoxicity to levels comparable to that of the CD34- CD33+ cells, suggesting that DNR resistance of immature AML cells may not solely be related to P-gp. With drug-selection, AML subclones displayed higher levels of P-gp expression and higher extruding capacities, and therefore chemoresistance, and this independently of their initial differentiation phenotype. Finally, this study provides evidence for a lack of correlation between expression and function of P-gp in AML cells; this relationship being dependent upon leukemic cell differentiation in unselected myeloid leukemic cells.
在一组代表不同分化阶段的急性髓性白血病（AML）细胞系中，我们研究了耐药性与多药耐药（MDR）相关P-糖蛋白（P-gp）的表达及功能之间的可能相关性。这些AML细胞系包括KG1a、KG1、TF1、HEL、ML1，以及源自HL-60（HL-60JD）和U937（U937AQ）的两个未经药物筛选的P-gp阳性亚克隆。所有这些细胞均过度表达mdr1基因（通过逆转录聚合酶链反应分析），并呈现出不同水平的P-gp表达。使用两种P-gp特异性单克隆抗体（MRK16和UIC2）通过流式细胞术对P-gp进行半定量评估，结果显示以下P-gp表达层次：TF1 < KG1a < HEL < KG1 < HL-60JD < ML1 < U937AQ；后者的P-gp表达量比TF1高13倍。当通过罗丹明123（Rh123）外排动力学评估P-gp功能时，我们发现只有具有早期（不成熟）CD34+ CD33- CD38-表型的KG1a和KG1细胞，以及在较小程度上具有中间（CD34+ CD33+ CD38+）表型的TF1细胞，表现出显著的P-gp活性，这种活性可被维拉帕米和SDZ PSC 833抑制。相比之下，其他更成熟的CD33+ CD34- AML细胞系尽管表达较高水平的P-gp，但没有Rh123外排能力。柔红霉素（DNR）蓄积研究表明，P-gp抑制剂仅增加未成熟AML细胞中的DNR蓄积，而对成熟AML细胞系没有影响。MTT药物细胞毒性测定证实，未成熟AML细胞对DNR的耐药性比成熟AML细胞高10 - 15倍。尽管P-gp抑制剂能够增加具有功能性P-gp的AML细胞中DNR的细胞毒性，但它们无法将DNR细胞毒性提高到与CD34- CD33+细胞相当的水平，这表明未成熟AML细胞对DNR的耐药性可能不仅仅与P-gp有关。通过药物筛选，AML亚克隆表现出更高水平的P-gp表达和更高的外排能力，因此具有化疗耐药性，且这与它们最初的分化表型无关。最后，本研究提供证据表明AML细胞中P-gp的表达与功能之间缺乏相关性；这种关系取决于未选择的髓性白血病细胞中的白血病细胞分化。
