Isolation and characterization of an L1210 cell line retaining the sodium-dependent carrier cif as its sole nucleoside transport activity.
Nucleoside permeation across mammalian cell membranes is complex with at least four distinct transporters known. Two of these (es and ei) are equilibrative (facilitated diffusion) carriers that have been studied is considerable detail. The other two (cif and cit) are concentrative, Na(+)-dependent carriers. A major obstacle to the characterization of the latter two mechanisms has been the lack of suitable model systems expressing only a single nucleoside transport activity. The present study describes the isolation of a cell line that has cif as its sole nucleoside transporter. L1210/MC5-1 cells, which have es and cif transport activity, were mutagenized and plated in soft agar containing two cytotoxic nucleosides (tubercidin (7-deazaadenosine) and cytosine arabinoside) that are substrates for es but not cif. A clonal line (L1210/MA-27.1) was isolated which retained the capacity for Na(+)-dependent [3H]formycin B transport but was unable to transport [3H]thymidine, a substrate for es but not cif. Failure of the mutant to transport thymidine was also demonstrated by the inability of thymidine (with adenine as a purine source) to rescue these cells from methotrexate toxicity. Furthermore, the mutant lacked nitrobenzylthioinosine (NBMPR) binding activity (an integral part of the es transporter) as demonstrated by reversible NBMPR binding and photoaffinity labeling with [3H]NBMPR. Loss of es transport activity was also demonstrated by the failure of NBMPR to affect the toxicity of 2-chlorodeoxyadenosine (IC50 approximately 30 nM) in L1210/MA27.1 cells. In contrast, NBMPR decreased the IC50 for 2-chlorodeoxyadenosine from 100 to 30 nM in the parental L1210/MC5-1 cell line. These results are consistent with the mechanism of NBMPR potentiation of 2-chlorodeoxyadenosine toxicity in L1210 cells being a blockade of efflux via es while the nucleoside is pumped into the cells by the concentrative cif carrier.
Crawford
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Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101.
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D015762:2-Chloroadenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000345:Affinity Labels / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D017338:Cladribine; D003839:Deoxyadenosines / Q000494:pharmacology; D007700:Kinetics; D007939:Leukemia L1210 / Q000378:metabolism*; D026901:Membrane Transport Proteins; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009154:Mutation; D009705:Nucleosides / Q000378:metabolism*; D012964:Sodium / Q000494:pharmacology*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000494:pharmacology; D013936:Thymidine / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
D000345:Affinity Labels; D002352:Carrier Proteins; D003839:Deoxyadenosines; D026901:Membrane Transport Proteins; D009705:Nucleosides; C065171:cif nucleoside transporter; D015762:2-Chloroadenosine; D013868:Thioinosine; D017338:Cladribine; D012964:Sodium; C001789:4-nitrobenzylthioinosine; D013936:Thymidine; D008727:Methotrexate