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.
OBJECTIVE
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 [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was studied in Y-79 and ARPE-19 cells. Further, saturation kinetics of [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] 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 [(3)H] Folic acid, [(3)H] Biotin, and [(14)C] Arginine was found to be significantly higher in Y-79 cells relative to ARPE-19 cells. [(3)H] 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. [(3)H] 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. [(14)C] 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.
Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri 64108, USA.
Jwala
J
Jwala
Ramya Krishna
RK
Vadlapatla
Aswani Dutt
AD
Vadlapudi
Sai Hanuman Sagar
SH
Boddu
Dhananjay
D
Pal
Ashim K
AK
Mitra
D026905:Amino Acid Transport Systems / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism; D058975:Folate Receptor 1 / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D005492:Folic Acid / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D019572:Retinal Neoplasms / Q000235:genetics / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism*; D012175:Retinoblastoma / Q000235:genetics / Q000378:metabolism*; D027981:Symporters / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism
D026905:Amino Acid Transport Systems; D058975:Folate Receptor 1; D012333:RNA, Messenger; D027981:Symporters; C119981:biotin transporter; D001710:Biotin; D005492:Folic Acid; D001120:Arginine