Production and characterization of highly tumor‐specific rat monoclonal antibodies recognizing the extracellular domain of human l‐type amino‐acid transporter 1

Abstract

C luster of differentiation 98/4F2 is a heterodimeric protein with a relative molecular mass of 125 000 (GP125), comprising a 90-kDa hc and 35-kDa lc.(1-3) CD98 was originally identified as a cell-surface antigen associated with the activation of lymphocytes (2) and is expressed on the basal layer of the squamous epithelium and a wide variety of tumors,suggesting its functional involvement in lymphocyte activation, cell proliferation, and malignant transformation. In fact, mAb against rat and human CD98 hc inhibits the activation of lymphocytes and proliferation of tumor cells. (5,6) In addition, NIH3T3 and Balb3T3 cells transfected with cDNA of human and rat CD98 hc have shown various malignant phenotypes.(7-9) CD98 lc have been revealed to be amino acid transporters, (3,10) and multiple functions of CD98 hc, such as cell fusion, (11) regulation of β 1 integrin activation, (12) and induction of apoptosis, (13) have been demonstrated. Transporters corresponding to the amino acid transport system L, y + L, , and Asc have been shown to be CD98 lc, which require CD98 hc for their membrane-based expression. (3,9,14) Six amino acid transporters (LAT1, LAT2, y + LAT1, y + LAT2, Asc-1, and xCT) that belong to the SLC7 family, have been identified to be CD98 lc, and all CD98 lc are believed to be sorted to the plasma membrane via association with CD98 hc. (15 -21) l-type amino-acid transporter 1 is a 12-membrane pass non-glycosylated protein that was first identified as CD98 lc associated with CD98 hc glycoprotein, and mediates Na + -independent large amino acid transport (system L). (3,22) It is reported that mRNA of LAT1 is expressed widely on tumor cells in addition to in the testis, ovary, and brain. (3,(23)(24)(25) However, because specific mAb recognizing the extracellular domain of native human LAT1 protein have not been obtained until now, the precise expression profile of LAT1 protein in normal and cancer cells remains unsolved. In the present paper, we report the successful production of specific mAb against human LAT1 protein, and discuss the specificity and usefulness of anti-LAT1 mAb in cancer therapy. Materials and MethodsCell culture. Human leukemia cells (Molt-4, Jurkat, Daudi, Raji, CCRF-SB, K562, and U937), mouse myeloma cells (P3 × 63Ag8.653), and peripheral blood leukocytes from healthy volunteers were cultured in RPMI-1640 medium (Sigma-Aldrich, St Louis, MO, USA). Human tumor cell lines from the tongue (HEp2), larynx (HSC-3), lung (A549), esophagus (TE-3), breast , liver (HepG2, Hep3B, and HLF), pancreas (PK-1 and PaCa-1), stomach , colon (SW1116, HT29, DU145, and LS-174T), cervix (HeLa and ME180), prostate (PC-3), kidney (ACHN and TOS-1), and bladder (T24, J82, KU-1, KK47, and MGH-U1), glioblastoma cells (KNS-42), melanoma cells (SK-MEL-37), neuroblastoma cells (Tagawa), HEK293F human embryonic kidney cells (Invitrogen, Carlsbad, CA, USA), Int407 embryonic intestine cells, RH7777 rat hepatoma cells (kindly donated by Mitsubishi Tanabe Pharma, Yokohama, Japan), and RenCa mouse renal carcinoma ce...

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