Differential Expression of Folate Receptor-Alpha,Sodium-Dependent Multivitamin Transporter,and Amino Acid Transporter (B (0,+) ) in Human Retinoblastoma (Y-79) and Retinal Pigment Epithelial (ARPE-19) Cell Lines

Abstract

Abstract

Purpose:

The overall objective of this study was to investigate the differential expression of folate receptor-alpha (FR-α), sodium-dependent multivitamin transporter (SMVT), and amino acid transporter [B ^{(0, +)}] in retinoblastoma (Y-79) and retinal pigment epithelial (ARPE-19) cells.

Methods:

Polymerase chain reaction (PCR) analysis was performed to confirm the existence of FR-α, SMVT, and B ^{(0, +)} in Y-79 and ARPE-19 cell lines. Quantitative real-time PCR was also performed to determine the relative expression of FR-α, SMVT, and B ^{(0, +)} at mRNA level in these cell lines. Quantitative uptake of [³H] Folic acid, [³H] Biotin, and [¹⁴C] Arginine was studied in Y-79 and ARPE-19 cells. Further, saturation kinetics of [³H] Folic acid, [³H] Biotin, and [¹⁴C] Arginine was performed in the presence of various concentrations of respective cold substrates to determine the kinetic parameters (K_m and V_max) in Y-79 and ARPE-19 cells.

Results:

PCR analysis had confirmed the existence of FR-α, SMVT, and B ^{(0, +)} in Y-79 and ARPE-19 cells. Quantitative real-time PCR analysis had shown significantly higher expression of FR-α, SMVT, and B ^{(0, +)} mRNA levels in Y-79 cells compared with ARPE-19 cells. Quantitative uptake of [³H] Folic acid, [³H] Biotin, and [¹⁴C] Arginine was found to be significantly higher in Y-79 cells relative to ARPE-19 cells. [³H] Folic acid uptake process followed saturation kinetics with apparent K_m of 8.29 nM and V_max of 393.47 fmol/min/mg protein in Y-79 cells and K_m of 80.55 nM and V_max of 491.86 fmol/min/mg protein in ARPE-19 cells. [³H] Biotin uptake process also displayed saturation kinetics with K_m of 8.53 μM and V_max of 14.12 pmol/min/mg protein in Y-79 cells and K_m of 138.25 μM and V_max of 38.85 pmol/min/mg protein in ARPE-19 cells. [¹⁴C] Arginine uptake process followed saturation kinetics with K_m of 16.77 μM and V_max of 348.27 pmol/min/mg protein in Y-79 cells and K_m of 52.03 μM and V_max of 379.21 pmol/min/mg protein in ARPE-19 cells.

Conclusions:

This work demonstrated for the first time the higher expression and affinity of FR-α, SMVT, and B ^{(0, +)} mRNA levels in retinoblastoma (Y-79) cells compared with retinal pigment epithelial (ARPE-19) cells.