[Mechanisms of development of resistance to methotrexate in choriocarcinoma cells]

Methotrexate (MTX) is extensively used, both as single agent and in combination regimens, for the treatment of human choriocarcinoma. However, development of resistance to MTX occurs frequently following continued administration, resulting in the failure of chemotherapy. Thus, the present study was undertaken to explore the mechanisms acquiring MTX resistance in choriocarcinoma cells. Seven MTX-resistant sublines were selected stepwise from two human choriocarcinoma cell lines (HCCM and CCl). The development of resistance to MTX was associated with an impaired transport of MTX into the cell and a ten-fold increase of dihydrofolate reductase (DHFR) activity. The former mechanism was responsible for cells resistant to the low MTX concentration. An increase in DHFR activity has been observed in cells resistant to 10(-6) M MTX concentration. Southern blot analysis of DNA from parent and resistant lines demonstrated the 8.7-fold amplification of the DHFR gene in the line resistant to 10(-7) M MTX concentration. Thus, the gene amplification preceded an increase of DHFR activity. Those results suggested that amplification of DHFR gene led the increase of DHFR m-RNA and of DHFR protein, providing the MTX resistance of human choriocarcinoma cells. The incidence of double-minute chromosomes (DMS) in metaphasic cells paralleled with the resistant MTX concentrations. Since DMS were also present in cells not showing DHFR gene amplification, however, mechanisms other than DHFR gene amplification was responsible for the de novo synthesis of DMS.