ra
ns
-sm
a,* a a a
We report here the development of a polyclonal antibody for human equilibrative nucleoside transporter 1 (hENT1)
and myelosuppression [1]. Gemcitabine is a cell- mination, and is probably the main mechanism
underlying the cytotoxicity of gemcitabine [2].
Mammalian cells express two subtypes of nucleo-
side transporters, human equilibrative nucleoside
transporter (hENT) and human concentrative nucle-
ights reserved.
* Corresponding author. Tel.: +81 52 853 8216; fax: +81 52 852
0849.
E-mail address: t-oguri@med.nagoya-cu.ac.jp (T. Oguri).
Cancer Letters 256 (20070304-3835/$ - see front matter  2007 Elsevier Ireland Ltd. All rand assess the expression of hENT1 in non-small cell lung cancer (NSCLC) patients who were treated with gemcita-
bine-containing chemotherapy. hENT1 expression was analyzed by immunohistochemical staining in 24 NSCLC biopsy
samples of formalin-fixed, paraffin-embedded tissues. The hENT1-positive staining in NSCLC samples was significantly
associated with response to gemcitabine-containing chemotherapy. Responses to gemcitabine-containing chemotherapy
were evident in none of the seven patients with no hENT1 expression. These results indicate that the absence of hENT1
expression may be useful to predict NSCLC patients who will not respond to gemcitabine-containing chemotherapy.
 2007 Elsevier Ireland Ltd. All rights reserved.
Keywords: hENT1; Immunohistochemistry; Gemcitabine; NSCLC
1. Introduction
Gemcitabine is one of the most commonly used
chemotherapeutic agents in non-small cell lung can-
cer (NSCLC), particularly as a first-line therapy. Its
toxicities include fatigue, myalgia, fluid retention,
cycle-dependent (S-phase-specific) deoxycytidine
analogue of the antimetabolite class and must first
be transported into the cell, where it is phosphory-
lated to its active, triphosphate form. Incorporation
of gemcitabine triphosphate into DNA causes the
addition of a single deoxynucleotide and chain ter-Tetsuya Oguri , Hiroyuki Achiwa , Hideki Muramatsu , Hiroaki Ozasa ,
Shigeki Sato a, Shigeki Shimizu a,b, Hideko Yamazaki b,
Tadaaki Eimoto b, Ryuzo Ueda a
a Department of Medical Oncology and Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi,
Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
b Department of Pathology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-cho, Mizuho-ku,
Nagoya 467-8601, Japan
Received 6 March 2007; received in revised form 24 May 2007; accepted 5 June 2007
AbstractThe absence of human equilib
expression predicts nonrespo
chemotherapy in nondoi:10.1016/j.canlet.2007.06.012tive nucleoside transporter 1
e to gemcitabine-containing
all cell lung cancer
) 112–119
www.elsevier.com/locate/canlet

physiological activity of nucleosides and in the trans-
carcinoma cell lines H23/GEM-R were established as
Thirty micrograms of whole-cell lysates was subjected to
T. Oguri et al. / Cancer Letters 256 (2007) 112–119 113described previously [10]. Further gemcitabine-resistant
ACC-LC-94/GEM-R cells selected from ACC-LC-94
human lung adenocarcinoma cells by gemcitabine was
established by continuous exposure to stepwise-increasing
concentrations of the drug. The human SCLC cell line
PC-6 and cells from their 7-ethyl-10-hydroxycamptothe-
cin (SN-38)-resistant subline PC-6/SN2-5 and paclitaxel
(TAX)-resistant subline PC-6/TAX1-1 were kindly pro-
vided by Daiichi Pharmaceutical (Tokyo, Japan; ref.
[11]). The human lung adenocarcinoma cell line PC-9
and cisplatin (CDDP)-resistant human lung adenocarci-
noma cell line PC-9/CDDP were used as described previ-
ously [12]. Cells were cultured in RPMI 1640
supplemented with 10% heat-inactivated FBS and 1%
(v/w) penicillin/streptomycin. Gemcitabine was provided
from Eli Lilly Pharmaceuticals (Indianapolis, IN).
2.2. Total RNA extraction and RT-PCR
Total RNA extraction and RT-PCR were performed
as described previously [10]. The sequences of the hENT1port of many therapeutic nucleoside drugs used as
cancer treatments [3–5]. Gemcitabine uptake is med-
iated by hENT1, hENT2, hCNT1, and hCNT3, with
hENT1 – which is ubiquitously expressed – being the
major route for transporting gemcitabine [6–9]. We
previously showed that hENT1 is a determinant of
gemcitabine sensitivity in NSCLC in vitro [10], but
whether hENT1 expression can be used as a bio-
marker for clinical gemcitabine treatment in NSCLC
is still under investigation. Because gemcitabine is
widely used forNSCLC chemotherapy, we employed
immunohistochemical analysis to examine the useful-
ness of hENT1 as a sensitivitymarker of clinical gem-
citabine treatment in NSCLC.
2. Materials and methods
2.1. Cell lines and chemicals
The following human NSCLC cell lines were used in
this study: nine adenocarcinomas (NCI-H23, RERF-LC-
AI, RERF-LC-MT, RERF-LC-OK, RERF-LC-MS,
ACC-LC-94, ACC-LC-176, ACC-LC-314, and SK-LC-
10), three squamous-cell carcinomas (PC10, Calu1, and
QG56), and two large-cell carcinomas (NCI-H460 and
SK-LC-6). The gemcitabine-resistant human lung adeno-oside transporter (hCNT),which canbedelineatedby
their sensitivity to inhibition by nitrobenzylmercap-
topurine ribonucleoside [3,4]. The nucleoside trans-
porters play an important role in modulating theprimers were 5 0-tgtttccagccgtgact-3 0 (forward) and 5 0-cag-SDS–PAGE and transferred to a nitrocellulose membrane
(Amersham Biosciences, Buckinghamshire, UK). After
protein transfer, the blots were incubated with the block-
ing solution and probed with the anti-hENT1 antibody.
Immunoreactive bands were visualized using the appro-
priate secondary horseradish-peroxidase-conjugated
antibodies and enhanced chemiluminescence (Amersham
Biosciences), with the density of each band measuredgccacatgaatacag-3 0 (reverse). Amplified products were
separated by 2% agarose gel electrophoresis, and bands
were visualized by staining with ethidium bromide. We
also performed real-time PCR with the LightCycler Fast-
Start DNA SYBR Green kit (Roche Diagnostics, India-
napolis, IN) as described previously [12]. The data from
each sample were normalized to those for the housekeep-
ing gene GAPDH.
2.3. Concentration of gemcitabine for 50% cell survival
Cells were cultured at 5000 cells/well in 96-well tissue
culture plates. To assess cell viability, stepwise ten-fold
dilutions of the anticancer drug were added 2 h after plat-
ing, and the cultures were incubated at 37 C for 96 h. At
the end of the culture period, 20 ll of MTS [3-(4,5-
dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt] solution (CellTit-
er 96 AQueous One Solution Cell Proliferation Assay,
Promega, Madison, WI) was added, and cells were incu-
bated for a further 4 h with the absorbance then measured
at 490 nm using an ELISA plate reader. Mean values were
calculated from three independent experiments performed
in quadruplicate. Chemosensitivity is expressed here as
the 50% cell survival (IC50) as determined from the
concentration–effect relationship using Graph Pad
Prism (version 4, GraphPad Software, San Diego, CA,
USA).
2.4. Production of a polyclonal antibody against hENT1
A polyclonal antibody specific to the hENT1 protein
was produced by immunizing rabbits with a synthetic pep-
tide-keyhole limpet hemocyanin conjugate. The synthetic
peptide corresponded to amino acids 254–271 of the pre-
dicted intracellular loop between transmembrane domains
6 and 7 of hENT1 as reported previously [13]. Sample sera
were routinely checked for immunoreactivity against the
antigen by ELISA. The immunoreactive antisera were
purified before being used in subsequent assays.
2.5. Western blot analysis
All cancer cells were lysed with CelLyticTM-M Mamma-
lian Cell Lysis/Extraction Reagent (Sigma–Aldrich, St.
Louis, MO) according to the manufacturer’s instructions.using a densitometer.