High-dose methotrexate therapy with leucovorin rescue: in vitro investigations on human osteosarcoma cell lines

High-dose methotrexate (MTX) therapy with subsequent leucovorin (LV) rescue (HDMTX-LV) in the treatment of osteosarcoma is based on the assumption that this tumor has a deficient uptake system for MTX and reduced folates. To simulate features of HDMTX-LV therapy protocols in vitro, sensitive and MTX-resistant human osteosarcoma cell lines and a lymphoblastoid cell line were exposed to MTX and/or LV at various dosages and time schedules and the effects on DNA metabolism and on cell growth were evaluated. The data show that in osteosarcoma cells and in lymphoblasts the cytotoxic effects of 10(-6) M to 10(-7) M MTX can be substantially reversed by LV if the antidote is applied within the first 12 h of MTX exposure. The results are not consistent with the assumption mentioned above and should be taken into consideration when designing new therapeutic regimens. An alternative hypothesis for the efficacy of HDMTX-LV is discussed. It is concluded that HDMTX-LV therapy may be effective in the treatment of osteosarcoma, even when subpopulations of the tumor cells exhibit different mechanisms of resistance to MTX, such as elevated levels of dihydrofolate reductase or a deficient transport system for MTX, if high doses of MTX are applied long enough to ensure lethal intracellular MTX levels and low-dose LV schedules instituted after a long delay are used.