Increased expression and genomic organization of a folate-binding protein homologous to the human placental isoform in L1210 murine leukemia cell lines with a defective reduced folate carrier

Abstract

This laboratory previously described an L1210 leukemia cell line (MTXrA) selected for resistance to methotrexate by virtue of impaired transport. In this line, the reduced folate carrier had unchanged affinity for methotrexate, was present at the cell surface in usual quantity, but did not deliver drug into the cell, indicative of a functional defect in the translocation process. In this study, we further characterize this cell line along with a subline (F2-MTXrA) selected for growth in low levels of folic acid. This subline demonstrates continued high resistance to methotrexate and very low influx of [3H]methotrexate and 5-[3H]formyltetrahydrofolate, indicating the persistence of the defect in the reduced folate carrier. Both MTXrA and F2-MTXrA are shown to overexpress FBP2, the murine homolog of a folate-binding protein initially isolated from human placenta. Compared with parent L1210 cells, Northern analysis revealed FBP2 expression to be elevated 40-fold in the MTXrA line and 500-fold in F2-MTXrA. The large increase in FBP2 expression in the F2-MTXrA line correlates with a 10-fold increase in [3H]folic acid membrane surface binding and a 1000-fold decrease in the folic acid growth requirement compared with parental L1210 cells. Also, there are 20- and 500-fold decreases in the 5-formyltetrahydrofolate growth requirement compared with parent L1210 and MTXrA cells, respectively. Finally, the genomic organization of the FBP2 locus is presented. The results of Northern analyses using probes specific to FBP2 5'-untranslated sequences or to a splice junction within this region suggest that the up-regulated FBP2-specific message in F2-MTXrA utilizes 5'-noncoding sequences distinct from those used in the message encoded in L1210 cell lines with low level FBP2 expression. The MTXrA cells provide an example of a line selected for primary resistance to methotrexate that also exhibits concomitant increased expression of a folate-binding protein. Further overexpression of this folate-binding protein (which has homology to that initially identified in placenta) provides cells with the ability to meet cellular folate needs in a folate-deprived environment.