Multiple Transport Pathways for L1210 Cells: Uptake of PTT.119, a Bifunctional Alkylator with Carrier Amino Acids

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Abstract
Delivery of the bifunctional alkylating agent, PTT.119 [p-F-L-Phe-m-bis-(2-chloroethyl)amino-L-Phe-Met-ethoxy-HCl], into tumor cells is significantly greater compared to L-phenylalanine mustard (L-PAM) as demonstrated by the 2-fold reduction in PTT.119 dosage required to reduce the viable L1210 cell fraction by 50% (TCD50). This increased uptake and consequent cytolytic efficacy observed in Dul-becco’s phosphate buffer was more apparent in culture medium; under this physiologic condition the TCD50 concentration of PTT.119 was 5 times lower than L-PAM. PTT.119 entry into leukemia cells was examined using competition transport assays assessing the ability of the tripeptide to compete with various amino acids and nonmetabolizable substrates for carrier receptors of the L, A and ASC transport systems. A 1-min exposure to a 1- to 50-fold excess of PTT.119 prior to addition of radiolabeled substrates significantly reduced within 60 s both sodium-dependent and sodium-independent uptake of leucine, methionine, threonine and α-[1-14C]-aminoisobutyric acid (AIB), but not MeAIB. In complimentary studies, L1210 cells were protected from PTT.119 cytolysis by an 8,000-fold excess of AIB, whereas β-2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) only abrogated tripeptide cytotoxicity by 95–97% even at BCH:PTT.119 ratios of 200,000. Leucine and methionine protection were significantly less effective; the TCD50 of leucine and methionine were 3.23 and 2.4 μM, respectively, compared to 11.41 μM for AIB and 7.96 μM for BCH. In addition, MeAIB and phenylalanine were totally unable to protect LI210 cells from PTT.l 19-induced cytolysis. The data indicate that L1210 cells actively transport PTT.119 primarily by the BCH-sensitive, AIB-sensitive, MeAIB-insensitive L carrier system. A second, BCH-insensitive, AIB-sensitive and MeAIB-insensitive carrier which is also involved in tripeptide uptake is probably the ASC system.

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