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

This laboratory previously described an L1210 leukemia cell line (MTX( r)A) selected for resistance to methotrexate by virtue of impaired tra nsport. In this line, the reduced folate carrier had unchanged affinit y for methotrexate, was present at the cell surface in usual quantity, but did not deliver drug into the cell, indicative of a functional de fect in the translocation process. In this study, we further character ize this cell line along with a subline (F2-MTX(r)A) selected for grow th in low levels of folic acid. This subline demonstrates continued hi gh resistance to methotrexate and very low influx of [H-3]methotrexate and 5-[H-3]formyltetrahydrofolate, indicating the persistence of the defect in the reduced folate carrier. Both MTX(r)A and F2-MTX(r)A are shown to overexpresses FBP2, the murine homolog of a folate-binding pr otein initially isolated from human placenta. Compared with parent L12 10 cells, Northern analysis revealed FBP2 expression to be elevated 40 fold in the MTX(r)A line and 500-fold in F2-MTX(r)A. The large increa se in FBP2 expression in the F2-MTX(r)A line correlates with a 10-fold increase in [H-3]folic acid membrane surface binding and a 1000-fold decrease in the folic acid growth requirement compared with parental L 1210 cells. Also, there are 20- and 500-fold decreases in the 5-formyl tetrahydrofolate growth requirement compared with parent L1210 and MTX (r)A cells, respectively. Finally, the genomic organization of the FBP 2 locus is presented. The results of Northern analyses using probes sp ecific to FBP2 5'-untranslated sequences or to a splice junction withi n this region suggest that the up-regulated FBP2-specific message in F 2- MTX(r)A utilizes 5'-noncoding se quences distinct from those used i n the message encoded in L1210 cell lines with low level FBP2 expressi on. The MTX(r)A cells provide an example of a line selected for primar y 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 identifi ed in placenta) provides cells with the ability to meet cellular folat e needs in a folate deprived environment.