Analysis of L-type amino acid transporter in canine hepatocellular carcinoma

Abstract

Analysis of L-type amino acid transport expression of hepatocellular carcinoma cells (HCCs) of the dog was performed. The leucine transport activity of canine HCCs was 0.628 ± 0.018 nmol/mg protein/min. The inhibitor of LAT 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) reduced 90% of the activity at 1 mM. The deduced amino acid sequences of canine LAT2, LAT3 and LAT4 were well conserved in mammalians, exhibiting 89, 88 and 77% homology, respectively. RT-PCR revealed distinct LAT1 expression compared with normal hepatocytes. Western blotting analysis confirmed the potent LAT1 expression in canine HCCs but not hepatocytes, and real-time RT-PCR analysis indicated that canine HCCs possessed 28 times higher LAT1 expression than hepatocytes. These results indicated that the leucine transport activity of canine HCCs was due to LAT1.

Fig. 1.

Histologic section from the specimen from the liver of the dog. HE stain (A, B). Hepatocellular carcinoma (_*) and cholangiocellular carcinoma (arrow) (A). In part, hepatic cells with a normal pattern were observed (arrow). The tumor cells contained various-sized cord-like structures and conspicuous nucleoli (_*). Bar=20 µm. (B). Histologic section of the nude mouse with a transplanted tumor. HE stain (C, D). Bar=100 µm (C). The cells of 2 nuclei were observed in various-sized and inequality (arrow). Bar=20 µm (D).

Fig. 2.

Leucine transport activity of canine HCCs in the presence or absence of 1 mM BCH. The values represent the means and SD of 4 individual experiments.

Fig. 3.

Amino acid sequences of canine LAT4 were compared with those of the human and mouse. Multiple sequence alignments were performed using the GENETYX program (ver. 10). Asterisks and dots indicate identical residues and conservative substitutions, respectively. The cDNA sequences data of canine LAT2 were deposited in the DNA Data Bank of Japan (accession numbers AB923978).

Fig. 4.

Amino acid sequences of canine LAT3 were compared with those of the human and mouse. Multiple sequence alignments were performed using the GENETYX program (ver. 10). Asterisks and dots indicate identical residues and conservative substitutions, respectively. The cDNA sequences data of canine LAT3 were deposited in the DNA Data Bank of Japan (accession numbers AB924118).

Fig. 5.

Amino acid sequences of canine LAT4 were compared with those of the human and mouse. Multiple sequence alignments were performed using the GENETYX program (ver. 10). Asterisks and dots indicate identical residues and conservative substitutions, respectively. The cDNA sequences data of canine LAT4 were deposited in the DNA Data Bank of Japan (accession numbers AB924119).

Fig. 6.

Amino acid sequences of the four isotypes of canine LATs were compared. Multiple sequence alignments were performed using the GENETYX program (ver. 10). Asterisks and dots indicate identical residues and conservative substitutions, respectively.

Fig. 7.

RT-PCR analysis of the four isotypes of LATs in canine HCCs and normal hepatocytes. Integrity of RNA was examined by using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (A). Control experiments of LAT2-LAT4 were also examined.

Fig. 8.

Real-time RT-PCR analysis of mRNA of canine LAT1 in canine HCCs and normal hepatocytes (A) and Western blot analysis of LAT1 expression using antiserum against the C-terminus of canine LAT1 peptides (B).