| Objective:To evaluate the impact of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) genes on methotrexate(MTX) resistance in osteosarcoma cells(U-20S).Methods:Four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) were obtained by exposing parental cell line to in vitro high dose and stepwise increased MTX concentrations.The sensitivity of the MTX resistance was measured by MTT assay;the level of reduced folate carrier(RFC) and dihydrofolate reductase(DHFR) mRNA expression was assayed with RT-PCR.Results:The authors developed four MTX-resistant variants(U-2 OS/R1～U-2 OS/R4) that were resistant to MTX,the resistance factor(RF) in U-2 OS/R1～U-2 OS/R4 is 4,23,71,142 competed with the U-2 OS cell.RFC gene mRNA expression level of resistant cells was decreased gradually along with the concentration increasing of MTX in U-2 OS/R1～U-2 OS/R4 cell lines(72%,66%,64%,58%)(p＜0.05);with DHFR gene mRNA expression level increased 3.5,6.2, 18.0,31.2 folds(p＜0.05).Conclusions:The MTX resistance was associated with increased levels of DHFR and decrease of RFC gene expression.The DHFR and RFC gene expression level result in MTX resistance in human osteosarcoma cell U-2 OS which can be present at clinical onset in osteosarcoma patients.Simultaneous evaluation of RFC, DHFR gene status at the time of diagnosis may help for the identification of potentially MTX-unresponsive osteosarcoma patients;moreover these MTX resistent osteosarcoma cell sublines are useful models for further research of MTX resistence in osteosarcoma and for the development of treatment protocols. |
