| Abstract: |
Studies are described examining further the decline in folate analogue influx mediated by the one-carbon reduced-folate transport system in HL-60 cells following induction of maturation by cytodifferentiation agents. To facilitate the investigation of the underlying basis of this phenomenon, we derived a variant (HL-60/LCV) with 4-5-fold elevated influx capacity (V(max)) for folate analogues. A commensurate increase in the putative transporter for this system was documented by affinity labeling of these cells with N- hydroxysuccinimide-[3H]aminopterin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity labeled plasma membrane in HL-60/LCV cells delineated a protein peak at M(r) = 75,000-80,000. This was substantially greater than the analogous transporter (M(r) = 45,000-47,000) we had delineated (Yang, C.-H., Sirotnak, F. M., and Mines, L. S. (1988) J. Biol. Chem. 263, 9703-9709) with the same methodology in the L1210 cell plasma membrane. In addition, the rate of translocation of the M(r) = 75,000- 80,000 transporter in HL-60 and HL-60/LCV cells was 2-fold lower than the rate of translocation determined for the M(r) = 45,000-47,000 transporter in L1210 cells. During induced maturation of HL-60/LCV cells toward the granulocyte pathway, [3H]methotrexate (MTX) influx capacity and the amount of the affinity labeled transporter decreased rapidly in a parallel fashion. The decrease in [3H]MTX influx and in affinity labeling and in the amount of the M(r) = 75,000-80,000 transporter was 5-fold following exposure to 210 mM dimethyl sulfoxide (Me2SO) for 5 days during growth in culture. Moreover, during cycloheximide treatment, the decay in [3H]MTX influx at 37 °C and in amount of affinity labeled transporter was the same (t( 1/2 ) = 144-155 min) for both control and Me2SO-treated HL-60/LCV cells. These results, which reveal no difference in metabolic turnover for control and Me2SO-treated cells, suggest that the decline in folate analogue influx in HL-60/LCV influx cells is a very early event in the program of differentiation and probably occurs by down-regulation of synthesis of the transporter for the one-carbon reduced-folate transport system. |
| Keywords: |
controlled study; carrier protein; human cell; methotrexate; cytology; cell maturation; down-regulation; membrane proteins; tumor cells, cultured; dimethyl sulfoxide; kinetics; cell membrane; down regulation; folic acid; cell transport; biological transport; polyacrylamide gel electrophoresis; electrophoresis, polyacrylamide gel; affinity labeling; protein synthesis regulation; turnover time; cycloheximide; cell strain hl 60; affinity labels; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.
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