Ross D D, Wooten P J, Sridhara R, Ordóñez J V, Lee E J, Schiffer C A
University of Maryland Cancer Center, Baltimore, MD 21201.
Blood. 1993 Aug 15;82(4):1288-99.
This work was designed to discern the frequency of expression of classical multidrug resistance (MDR) in acute myeloid leukemia (AML) at the time of diagnosis, using Western blotting for P-glycoprotein (Pgp) and functional assays for an MDR phenotype (enhancement of daunorubicin [DNR] accumulation/retention and cytotoxicity by the known MDR modulators verapamil, cyclosporin A, and progesterone). Blast cells were studied from 49 newly diagnosed AML patients who were subsequently treated with the "3 and 7" combination of cytosine arabinoside (ara-C) and DNR. DNR accumulation (1 microgram/mL, 3 hours) and retention (16 hours) were determined by flow cytometry. Cyclosporin A (CsA, 5 mumol/L) or verapamil (6.6 mumol/L) each caused significant enhancement of DNR accumulation and retention in these blast cell samples (P < .001, Wilcoxon's test). Verapamil or CsA caused greater than 20% enhancement of DNR accumulation or retention in over 25% or 50% of these patients, respectively; however, there was no correlation with the presence or degree of enhancement and response to treatment. Progesterone (10 mumol/L) caused no significant enhancement of DNR accumulation or retention. The effects of the MDR modulators on the cytotoxicity of DNR was also determined in blast cells from 40 of the patients, using a flow cytometric assay. CsA alone was cytotoxic (caused an approximate 20% decrease in cell survival compared with control, P < .001); CsA or verapamil caused enhancement of 1 mumol/L DNR cytotoxicity (P < .001). Greater than 40% enhancement of cell kill by CsA or verapamil was observed in over 75% of patients studied. There was no difference in the degree of enhancement of cytotoxicity between patients clinically sensitive or resistant to treatment. Progesterone caused no enhancement in DNR cytotoxicity. In contrast to the functional assays, highly sensitive immunoblots using the C219 antibody to Pgp showed evidence of low level expression of Pgp in blast cells from only 3 of these patients: 1 was chemotherapy resistant, 2 were sensitive. Thus, although the functional assays suggest a high frequency of expression of a classic MDR phenotype in AML patients at the time of diagnosis, with enhancement by CsA obtained at a clinically relevant concentration (5 mumol/L), the frequency of Pgp expression detectable by C219 Western blots was low in these patients. This could be interpreted either that the method used was not sufficiently sensitive to detect Pgp in all of the blast cell specimens that actually overexpressed mdr1, or that the accumulation/efflux-based MDR phenotype observed is not always mediated by Pgp in these previously untreated patients.
本研究旨在通过蛋白质免疫印迹法检测P-糖蛋白（Pgp）以及对多药耐药（MDR）表型进行功能测定（使用已知的MDR调节剂维拉帕米、环孢素A和孕酮增强柔红霉素[DNR]的积累/滞留及细胞毒性），来确定急性髓系白血病（AML）诊断时经典MDR的表达频率。对49例新诊断的AML患者的原始细胞进行了研究，这些患者随后接受了阿糖胞苷（ara-C）和DNR的“3+7”联合治疗。通过流式细胞术测定DNR的积累（1μg/mL，3小时）和滞留（16小时）情况。环孢素A（CsA，5μmol/L）或维拉帕米（6.6μmol/L）均能显著增强这些原始细胞样本中DNR的积累和滞留（P<.001，Wilcoxon检验）。维拉帕米或CsA分别使超过25%或50%的患者DNR积累或滞留增强超过20%；然而，增强的存在或程度与治疗反应之间并无相关性。孕酮（10μmol/L）未显著增强DNR的积累或滞留。还使用流式细胞术检测法在40例患者的原始细胞中测定了MDR调节剂对DNR细胞毒性的影响。单独使用CsA具有细胞毒性（与对照组相比导致细胞存活率下降约20%，P<.001）；CsA或维拉帕米可增强1μmol/L DNR的细胞毒性（P<.001）。在超过75%的研究患者中观察到CsA或维拉帕米使细胞杀伤增强超过40%。对治疗临床敏感或耐药的患者，细胞毒性增强程度无差异。孕酮未增强DNR的细胞毒性。与功能测定相反，使用针对Pgp的C219抗体进行的高灵敏度免疫印迹显示，这些患者中只有3例患者的原始细胞有低水平Pgp表达的证据：1例对化疗耐药，2例敏感。因此，尽管功能测定表明AML患者诊断时经典MDR表型的表达频率较高，在临床相关浓度（5μmol/L）下CsA可增强其表达，但在这些患者中通过C219蛋白质免疫印迹法可检测到的Pgp表达频率较低。这可以解释为所使用的方法对在所有实际过度表达mdr1的原始细胞样本中检测Pgp不够敏感，或者在这些未经治疗的患者中观察到的基于积累/外排的MDR表型并不总是由Pgp介导。
