Patterns of Cross-Resistance to the Antifolate Drugs Trimetrexate, Metoprine, HomofoÃ-ate,and CB3717 in Human Lymphoma and Osteosarcoma Cells Resistant to Methotrexate1

Methotrexate (MTX)-resistant sublines of malignant human cells were selected in vitro by stepwise increase in drug concen tration in the medium. By this procedure a subline of Burkitt's lymphoma cells (RAJI) was made 290-fold resistant (RAJI/MTXR), T-cell leukemia cells (CCRF-CEM) were obtained 210-fold resistant (CEM/MTX-R), and 3 MTX-resistant human osteosarcoma lines were selected: TE-85/MTX-R (19-fold resistant; rel ative to wild-type); MG-63/MTX-R (8-fold resistant); and SAOS2/MTX-R (200-fold resistant). We also studied a B-cell lymphoblastoid line, Wl-L2/m4, that was 13,000-fold resistant. Assay of cellular dihydrofolate reducÃ-ase(DHFR) showed the following pattern of activity in resistant cell lines, relative to parental cell activity: RAJI/MTX-R, 550-fold increased; OEM/ MTX-R, unchanged; TE-85/MTX-R, 4-fold increased; MG-63/ MTX-R, 6-fold increased; SAOS-2/MTX-R, unchanged; and WlL2/m4, 110-fold increased. Measurement of MTX membrane transport showed decreased uptake in CEM/MTX-R and SAOS2/MTX-R, relative to parental cell lines. The other DHFR-overproducmg cells all gave normal initial MTX uptake rates but increased total uptake. The DHFRoverproducing lines all had significant cross-resist ance to both metoprine and trimetrexate; the two lines with defective MTX transport were not cross-resistant, and the CEM/ MTX-R cells showed collateral sensitivity to these agents. Only minor cross-resistance to hornofolic acid was found in all MTXresistant lines. The highly MTX-resistant RAJI/MTX-R and WlL2/m4 cells showed minor cross-resistance to the dual inhibitor of thymidylate synthetase and DHFR, CB3717 (5and 15-fold, respectively). These studies demonstrated that, depending upon the mechanism of resistance, MTX-resistant human tumor cells may be effectively killed by antifolates with different routes of uptake into cells, or with a different enzyme target. Thus, there are at least three functionally distinct classes of folate antagonist with antitumor activity.