1. Volume 120, Issue 7, Pages 1689-1699 (June 2001)
LST-2, A human liver-specific organic anion transporter, determines methotrexate sensitivity in gastrointestinal cancers 
Takaaki Abe, Michiaki Unno, Tohru Onogawa, Taro Tokui, Tohru Noriko Kondo, Rie Nakagomi, Hisanobu Adachi, Koh Fujiwara, Mitsunori Okabe, Takehiro Suzuki, Kazuo Nunoki, Eiichi Sato, Masayuki Kakyo, Toshiyuki Nishio, Junichi Sugita, Naoki Asano, Masayuki Tanemoto, Makoto Seki, Fumiko Date, Katsuhiko Ono, Yoshiaki Kondo, Kenichi Shiiba, Masanori Suzuki, Haruo Ohtani, Tooru Shimosegawa, Kazuie Iinuma, Hiroshi Nagura, Sadayoshi Ito, Seiki Matsuno 
Gastroenterology 
Volume 120, Issue 7, Pages (June 2001)
DOI: /gast
Copyright © 2001 American Gastroenterological Association Terms and Conditions

2. Fig. 1
Sequence alignment and analysis of LST-2. (A) Amino acid alignment of LST-1 and LST-2 (GenBank accession no. AF187815). The sequences are aligned with single-letter notation by inserting gaps (–) to achieve the maximum homology. Exact matches and conservative substitutions are shown by bars and colons, respectively. The 12 putative transmembrane segments (I to XII) were assigned on the basis of hydrophobicity analysis. Potential N-glycosylation sites (triangles) and possible phosphorylation sites (asterisks) are indicated. (B) Phylogenetic relationship between LST-1, LST-2, the oatp family, and the PG transporter. Branch lengths are drawn to scale.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

3. Fig. 2
Northern blot analysis of the human LST-2 mRNA. Human multiple tissue Northern blots were hybridized with the 3'-noncoding region of human LST-2.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

4. Fig. 3
Localization of LST-2 in the liver. (A) Western immunoblot of LST-2 in the human liver. Western blot analysis of human liver membrane was performed with affinity-purified antibody against LST-2. Immunohistochemical analysis of LST-1 and LST-2 in normal human liver. (B) The H&E staining. (C) The LST-2 immunoreactivity shown around the central vein. (D) LST-1 immunoreactivity was seen in all hepatocytes. (E) By immunofluorescent microscopy, LST-2 immunoreactivities were observed in the basolateral membranes of the hepatocytes (green). Canalicular membrane marker protein CD13 was simultaneously stained (red). (F) High magnification of the same slide. Note that no combined signal was detected (yellow). (G) Immunoelectron microscopy for LST-2 in hepatocytes. The immunoreactivity for LST-2 (black) was localized along the plasma membrane of hepatocytes (arrowhead). CV, central vein; PV, portal vein. Scale bar, (B–E) 200 μm and (F and G) 10 μm.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

5. Fig. 4
Genomic organization of human LST-2. Exons in the human LST-2 are represented by closed rectangles. HRE, hypoxia responsive element.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

6. Fig. 5
(A) Northern blot analysis of the LST-2 mRNA in the gastric cancer cell lines (20 μg of total RNAs; lane 1, NUGC-4; lane 2, KatoIII; lane 3, AZ521; lane 4, MKN7) and normal stomach poly (A)+ RNA (2 μg; lane 5). LST-1– and RFC-1–specific probes were also hybridized. For human RFC-1 analysis, full coding region was used as a probe. (B) Western immunoblot of LST-2 in KatoIII (lane 1) and the human liver (lane 2). Western blot analysis of the membrane was performed with affinity-purified antibody against LST-2. Ten micrograms of membrane was separated on 10% SDS–polyacrylamide gel under denaturing condition and subjected to immunoblotting with affinity-purified antibody. (C) Immunofluorescent study of KatoIII cells. Permeabilized (left) and nonpermeabilized (right) cells were stained with LST-2–specific antibody. Scale bar, 50 μm. (D) Putative membrane topology of LST-2. The 12 transmembrane domains were indicated. Note that the specific antibody recognizes the C-terminus of LST-2 (arrow). Signal was detected only when the cell membrane was permeabilized, indicating that the C-terminus of LST-2 is located intracellularly.
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

7. Fig. 6
Northern blot analysis of the LST-2 mRNA in human cancer cell lines. Human cancer cell lines were cultured and total RNA was isolated. Lane 1, DLD-1; lane 2, MIP-101; lane 3, COLO320-DM; lane 4, Clone A; lane 5, CX-1; lane 6, Caco-2; lane 7, MIA-Paca2; lane 8, PK-1; lane 9, BXPC-1; lane 10, ASPC-1; lane 11, PK-8; lane 12, PK-9; lane 13, PK-45H; lane 14, PK-45P; lane 15, PK-59; lane 16, PANC-1; lane 17, HepG2; lane 18, Hep3B; lane 19, HT-17; lane 20, Li-7; lane 21, HuH7; lane 22, HUH-28; lane 23, HuCCT1; lane 24, OcuchLM1; lane 25, TFK-1; lane 26, A549; lane 27, 1-87; lane 28, 11-8; lane 29, LK-2; lane 30, EBC-1; lane 31, Sq-19; lane 32, LU65; lane 33, LK79; lane 34, S1; lane 35, Lu99; lane 36, MCF-7; lane 37, CRL1500; lane 38, TE-2; lane 39, 8505C; lane 40, A172; lane 41, Caki-1; lane 42, OVK18#102; lane 43, T24; lane 44, ME180; lane 45, HL-60; lane 46, K-562; lane 47, MOLT-4; lane 48, Burkitt's (Raji). Additional information of cell lines can be seen on the website of Cancer Cell Repository Institute of Development, Aging and Cancer, Tohoku University (
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions

8. Fig. 7
(A) Northern blot analysis of the LST-2 mRNA in the specimens from human gastric (8 cases), colon (5 cases), and pancreatic (1 case) cancers. Immunohistochemical analysis of LST-2 in the Northern positive specimens. Positive immunostaining of LST-2 was detected in (B) gastric cancer, (C) pancreatic cancer, and (D) colon cancer. (E) The metastatic lymph node of the same colon cancer was also LST-2 positive. Scale bar, 500 μm.
Gastroenterology  , DOI: ( /gast )
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9. Fig. 8
Uptake of MTX by LST-2. (A) Transport of compounds in LST-1– and LST-2–expressing oocytes (60 minutes). (B) Transport of MTX in LST-2–expressing oocytes was measured (60 minutes). A representative of 3 experiments is shown. The LST-2–mediated MTX uptake is followed by Michaelis–Menten kinetics. (C) Inhibition by BSP (100 μmol/L) of LST-2–mediated MTX uptake (1 μmol/L) in oocytes. The uptake experiments were performed at the concentration indicated for 60 minutes. All data are means ± SEM of values from 8 to 11 oocytes; *P < 0.05 vs. water-injected control. (D) Transport of MTX in LST-2/MDCK (○) and mock-transfected (●) cells were measured (60 minutes). The net LST-2–mediated uptake is also indicated (▴). (E) Sensitivity to MTX in vitro. LST-2– or mock-transfected cells were plated, and 100 μL of medium containing various concentrations of MTX were added and incubated for 72 hours. Mock-transfected (●), LST-2/MDCK (○), BSP 10 μmol/L (▴), BSP 50 μmol/L (▵), BSP 100 μmol/L (■). All data are means ± SEM of values from 6 to 8 wells. *P < 0.05, LST-2/MDCK vs. mock control; †P < 0.05, LST-2/MDCK vs. LST-2/MDCK with BSP (100 μmol/L).
Gastroenterology  , DOI: ( /gast )
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10. Fig. 9
MTX sensitivity of KatoIII and other gastrointestinal cancers. (A) The uptake of MTX (1 μmol/L) in KatoIII with or without BSP (100 μmol/L). (B) Transport of methotrexate in KatoIII cells with or without BSP (100 μmol/L) were measured (60 minutes). BSP(−) (○), BSP 100 μmol/L (●), net LST-2–mediated uptake (▴). Antiproliferative activity of MTX in (C) KatoIII, colon cancer, (D) Clone A, and (E) pancreatic cancer, PK-8 cell lines. Cells were treated with various concentration of MTX with (○) or without (●) 100 μmol/L BSP. The statistical analysis was evaluated by one-way analysis of variance followed by the Student t test (*P < 0.05).
Gastroenterology  , DOI: ( /gast )
Copyright © 2001 American Gastroenterological Association Terms and Conditions