Establishment of human multidrug resistant hepatocarcinoma cell line transfected human multidrug resistance-associated protein gene and its biological features

Abstract

Objective Treatment of hepatocellular carcinoma(HCC)is hampered by resistance to chemotherapy,which might be mediated by muhidrug resistance-associated proteins(MRP).The recombinant mammalian expression vector(pCI-neo)carrying human full lengh MRP cDNA was construtted and transfected into human hepatocellular carcinoma cell line HeDG2.The transfergene multidrug resistant cell Iine(HepG2/mrp1)was established.Methods The human full-length MRP cDNA was obtained from cloning vector pGEM-mrp1,which was cloned into pCI-neo multiple cloning site and the recombinant expression vector(pCI-mrp1)was constructed,which transfected into HepG2 and established the multidrug resistant cell line HepG2/mrp1.The chemosensitivity of HepG2/mrp1 was determined bv MTT assay.The intracellular concentration of DNR and MRP was determined by flow cytometry.The MRP mRNA was assessed with reverse transcriptase polymerase chain reaction(RTPCR).Results The resistance index of ADM and DNR was 11.4 and 8 times in HepG2/mrpl cell line,higher than that of parent cell line HeDG2.The MRP and MRP mRNA positive expression increased and the introcellular concentration of DNR decreased compared with parent cell line HepG2 respectively.Conclusion Hepatocarcinoma cell line HepG2/mrpl transfected MRP cDNA with stably over-expressed MRP presents typical MDR phenotype.It provides a good model for the study of MRP mediated MDR. Key words: Carcinoma,hepatocellular; Multidrug resistance; Multidrug resistance-associated proteins; Plasmid; Transfer-gene