Cytofluorimetric analysis of a renal tubular cell line and its resistant counterpart

P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overex pression is often responsible of the development of multidrug resistance in cancer therapy. These proteins ale also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is deri ved from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been estab lished in our laboratory by chronic exposure to increasing doses of doxorub icin. Cytofluorimetric analysis of P-gp and MRP expression performed by C21 9 and MRPm6 immunofluorescence detection showed that these cells overexpres s P-gp, but nor MRP. The uptake of doxorubicin and rhodamine 123 has been q uantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp o r MRP overexpression. P388 sensitive cells and its resistant counterpart P3 88/ADR cells, which overexpress P-gp and PANC-1 cells, which express high l evels of MRP were used. A lower fluorescence intensity was evident with bot h doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells , that overexpresses P-gp, in comparison with the parental fines. The uptak e was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANG-I cells, confirming a selective effect of this inhibito r on P-gp. Propidium iodide staining, performed after doxorubicin treatment , confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.