Boutonnat J, Bonnefoix T, Mousseau M, Seigneurin D, Ronot X
Equipe de Cytologie Quantitative, Institut Albert Bonniot, La Tronche, France.
Anticancer Res. 1998 Jul-Aug;18(4C):2993-9.
Cross resistance to multiple natural cytotoxic products represents a major obstacle in myeloblastic acute leukaemia (AML). Multidrug resistance (MDR) often involves overexpression of plasma membrane drug transporter P-glycoprotein (PGP) or the resistance associated protein (MRP). Recently, a protein overexpressed in a non-PGP MDR lung cancer cell line and termed lung resistance related protein (LRP) was identified. These proteins are known to be associated with a bad prognosis in AML. We have developed a triple indirect labelling analysed by flow cytometry to detect the coexpression of these proteins. Since no cell line expressing all three antigens is known, we mixed K562 cells (resistant to Adriblastine, PGP+, MRP-, LRP-) with GLC4 cells (resistant to Adriblastine, PGP-, MRP+, LRP+) to create a model system to test the method. The antibodies used were UIC2 for PGP, MRPm6 for MRP and LRP56 for LRP. They were revealed by Fab'2 coupled with Fluoresceine-isothiocyanate, Phycoerythrin or Tricolor with isotype specificity. Cells were fixed and permeabilized after PGP labelling because MRPm6 and LRP56 recognize intracellular epitopes. PGP and LRP were easily detected. MRP is expressed at relatively low levels and was more difficult to detect because in the triple labelling the non specific staining was higher than in a single labelling. Despite the increased background in the triple labelling we were able to detect coexpression of PGP, MRP, LRP by flow cytometry. This method appears to be very useful to detect coexpression of markers in AML. Such coexpression could modify the therapeutic approach with revertants.
对多种天然细胞毒性产物的交叉耐药是髓细胞性急性白血病（AML）的一个主要障碍。多药耐药（MDR）通常涉及质膜药物转运蛋白P-糖蛋白（PGP）或耐药相关蛋白（MRP）的过表达。最近，在一种非PGP MDR肺癌细胞系中过表达的一种蛋白被鉴定出来，称为肺耐药相关蛋白（LRP）。已知这些蛋白与AML的不良预后相关。我们开发了一种通过流式细胞术分析的三重间接标记法来检测这些蛋白的共表达。由于目前尚无表达所有三种抗原的细胞系，我们将K562细胞（对阿霉素耐药，PGP+，MRP-，LRP-）与GLC4细胞（对阿霉素耐药，PGP-，MRP+，LRP+）混合，以创建一个模型系统来测试该方法。所用抗体分别为针对PGP的UIC2、针对MRP的MRPm6和针对LRP的LRP56。它们通过与异硫氰酸荧光素、藻红蛋白或具有同型特异性的三色荧光素偶联的Fab'2来显示。PGP标记后对细胞进行固定和通透处理，因为MRPm6和LRP56识别细胞内表位。PGP和LRP很容易检测到。MRP表达水平相对较低，更难检测，因为在三重标记中非特异性染色高于单一标记。尽管三重标记中背景有所增加，但我们仍能够通过流式细胞术检测到PGP、MRP、LRP的共表达。该方法对于检测AML中标志物的共表达似乎非常有用。这种共表达可能会改变对逆转剂的治疗方法。
