Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy

Abstract

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4 kb. We established five homozygous LAT1-disrupted ( LAT1 −/−) cell clones, derived from a heterozygous LAT1 +/− clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1 −/− DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1 −/− cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1 −/− DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1 −/− DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1 +/− DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.