Induction Of Drug Resistance In Green Fluorescent Protein-labeled Human Leukemia K562 Cells And Observation Of The Biological Characteristics

Objective: In this study, the human leukemia K562 cells were to be labeled with green fluorescent protein (GFP) gene by transgene technology to establish a stable fluorescence leukemia cell model, and the fluorescent cells were induced for the drug-resistant phenotype, by long-term and continuous stepwise increments of adriamycin (ADM) concentration, to set up a GFP-labeled multidrug-resistant leukemia cell model. The research aim is to provide a drug studying and screening platform with the advantages of convenient, practical, economical and dynamic observation and to explore its application in the screening of anti-leukemia agents and drug-resistance modifiers.Methods:1. The GFP expression recombinant plasmid (pCI-neo-GFP) was constructed and transformed into K562 cells, and then the stable GFP-expressing K562 cells, named K562-GFP cells, were screened out. K562-GFP cells were selected for the ADM-resistant phenotype by long-term, intermittent, repeated and continuous stepwise increments of ADM concentration, from initial 0.01 mg/L, until the cells were steadily able to tolerate 8 mg/L ADM to set up a stable multidrug-resistant leukemia subline, named K562-GFP/ADM cells.2. MTT colorimetric assay was used to dynamically observe the proliferation activity of K562-GFP and K562-GFP/ADM cells, the sensitivity of the cells to vincristine, As2O3, doxorubicin, daunorubicin,5-fluorouracil, paclitaxel, pirarubicin and etoposide. The morphological and ultra-structural changes were observed with optical microscopy and electron microscopy, and the colony-forming ability was tested with methylcellulose semisolid colony formation. RT-PCR and Western blot were employed to detect the expression of multidrug-resistance-related genes/proteins, mdrl/P-gp、mrp1/MRP1 and bcrp/BCRP. The cell-cycle distribution, positive expression rate of P-gp and leukemia stem cells (CSC) proportion in cell population were assed by flow cytometry (FCM).3. K562-GFP and K562-GFP/ADM cells were used as cell models to study the anti-multidrug-resistant leukemia effects of resveratrol combined with AS2O3. MTT assay was used to examine cell proliferating inhibition, and cell apoptosis was detected with Annexin V/PI double staining. RT-PCR, FCM and Western blot were empolyed to assess the expression of apoptosis and drug-resistance-related genes/proteins NF-κB, Bcl-2, Bax, P53, Caspase-3, mdrl/P-gp, BCRP and MRP1.Results:1. K562 cells transfected with GFP gene, named K562-GFP cells, were able to express stably GFP protein with high fluorescence intensity, and the GFP-expressing cells were over 95%. There was no obvious difference in the characteristics of morphology and proliferation between K562-GFP cells and the parent K562 cells.2. The K562-GFP cells were induced with gradually and intermittently increased ADM concentration for 14 months, a multidrug resistant cell line, named K562-GFP/ADM, tolerated 8.0 mg/L ADM was successfully established. The resistance of K562-GFP/ADM cells to ADM was 115.81-times higher than that of the parental K562-GFP cells, and the cells were also cross-resistant to the other chemotherapeutics vincristine, daunorubicin, topiramate, doxoirubicin, 5-fluorouracil, etoposide and paclitaxel but not AS2O3. After the long-term induction with ADM, K562-GFP/ADM cells became smaller and increased nucleus/cytoplasm ratio, and the cell-cycle distribution displayed the increased G0/G1 phase and decreased S phase. After removal of ADM for 60 days, the cell morphology and cell-cycle distribution of K562-GFP/ADM cells recovered similar to that of K562-GFP cells. Compared with the parental K562-GFP cells, K562-GFP/ADM cells had stronger colony-formation ability and a 210-fold higher proportion of LSCs in cell population. During the ADM induction, the expression of drug-resistance-related genes mdrl, mrpl and bcrp in K562-GFP cells enhanced gradually and obviously. The expression of mdrl, mrpl and bcrp genes in 8.0 mg/L ADM-tolerated K562-GFP cells were 132.5,2.85 and 8.87 times over that of un-induced K562-GFP cells, respectively. The positive exprssion rate of P-gp in K562-GFP/ADM cells was almost 100% and remained stable in ADM-free culture.3. K562-GFP/ADM cells and K562-GFP cells were both sensitive to As2O3, and reseratrol could improve the sensitivity of the cells to AS2O3. After treatment of 20 μmol/L and 40 μmol/L resveratrol combined with 2 μmol/L As2O3 respectively for 24 to 72 hours, the result showed that resveratrol significantly enhanced the As2O3-induced proliferation inhibition and apoptosis in K562-GFP cells, especially in K562-GFP/ADM cells. The apoptotic rates induced by 2 μmol/L As2O3 in K562-GFP cells and K562-GFP/ADM cells were 51.88% and 54.88%, respectively, but if co-induced by combination of 2 μmol/L AS2O3 and 40 μmol/L resveratrol, the rates increased to 77.64% and 99.36%, respectively. The combined treatment of 2 μmol/L As2O3 and 40 umol/L resveratrol significantly reduced the expression of drug-resistance-related genes/proteins mdrl/P-gp, mrpl/MRP1 and bcrp/BCRP and apoptosis-inhibiting genes bcl-2, NF-κB and P53, and enhanced the expression of apoptotic gene bax and activation of Caspase-3.Conclusions:1. A fluorescence leukemia cell line stably expressed green fluorescent protein (GFP), K562-GFP cells, is successfully established, and the fluorescent cells are induced with ADM to acquire the characteristics of classical multidrug resistance to set up a fluorescent drug-resistant cell line, K562-GFP/ADM cells. The two fluorescent cell lines can be used for study and screening of anti-leukemia agents and drug-resistance modifiers.2. Resveratrol significantly enhances the sensitivity of drug-resistant K562-GFP/ADM cells to As2O3-induced proliferating-inhibition and apoptosis, and the mechanisms have a connection with down-regulation of drug-resistant and apoptosis-inhibiting genes and up-regulation of apoptosis genes in the drug-resistant leukemia cells.