Molecular Pharmacology, Vol 17, 382-387, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

The Interaction of Carrier-Bound Methotrexate with L1210 Cells

¹ Department of Biochemistry, Scripps Clinic and Research Foundation, La Jolla, California 92037

Methotrexate (MTX), covalently linked to two randomly chosen proteins (-chymotrypsinogen and bovine IgG), was as effective as free MTX and MTX bound to bovine serum albumin (MTX-BSA) in prolonging the life span of BDF₁ mice bearing the L1210 tumor, suggesting that MTX can act as an effective chemotherapeutic agent when covalently bound to a variety of proteins. MTX was more toxic than MTX-BSA to L1210 cells grown in vitro at 37°C, an observation which correlated with the relative internal drug concentrations recorded after incubation for 1 h with 4 µM free or derivatized MTX. Further in vitro experiments employing [³H]MTX-BSA as a prototype of a drug-protein carrier indicated that MTX-BSA was only partially dependent on the same transport mechanism for cellular uptake as MTX. For example, MTX transport into cells was almost zero at 0°C, whereas MTX-BSA entry was only inhibited 50%; additionally MTX-BSA uptake was much less inhibited by p-chloromercuribenzenesulfonate and 5-formyltetrahydrofolate than the parent compound. Experiments using [³H]MTX-BSA or MTX-¹²⁵I-BSA indicated that the MTX-BSA complex was not degraded on the external surface of the cell membrane, as 60-65% of both radioactive labels was found inside the cell lysate fraction with much of the iodinated material degraded to low-molecular-weight fragments. In contrast the ¹²⁵I marker remaining bound to the cell membrane was still present as a nondialyzable high-molecular-weight molecule. Experiments employing MTX bound to Dextran T70, an ineffective antitumor agent, indicated only limited cellular interaction and low drug uptake.