Abstract

Medical Technology

SCHEDULE
Morning Session : 11:00 ~ 12:00
Session chair: Prof. Tsutom u Kawabe
1. Cancer-directed delivery of doxorubicin-rapamycin and cancer imaging by anti-Her2
immunoliposomes

Min Woo Kim

2. Variation of IgE-binding epitopes in the allergen after repeated immunization
Takehiro Yamaguchi
3. Evaluation of human papillomavirus oncogenic mRNA testing for diagnosis of
cervical cancer by using real-time NASBA

Geehyuk Kim

Afternoon Session : 13:30 ~ 16:00
Session chair: Prof. Takaaki Kondo
4.

Quantitationofneonicotinoid

metabolites

in

human

chromatography-mass spectrometry

urine

using

gas

Hiroshi Nomura

5. Comparison of virulence factors, phylogenetic groups and ciprofloxacin susceptibility
between Escherichia coli isolated from healthy students and uropathogenic E. coli
isolated from patients with urinary tract infections in Korea

Min Park

6. The relationship between Helicobacter pylori infection and medical history of
selected diseases among an urban area population of Japan	  	  Akina Hayashi– 10 minutes rest –Session chair: Assistant Prof. Ki Jong Rhee
7. Anti-HCV agent ribavirin modulates transcription elongation in clotting factor VII
expression

Atsuo Suzuki

8. Cervical condyloma acuminatum shows more progressive histopathological changes
than vulvar condyloma acuminatum

Satomi Mizuno

9. Parkin induces apoptotic cell death in TNF-α-treated cervical cancer cells
Yoonjung Cho– 5minutes rest –Session chair: Prof. Takashi M urate
10. Parkin induces MMP-3 expression in human cervical cancer cells
Byung Chul Jung

11. Induction of the IL-2-independent growth by KRAS G12A mutation
Naoki Mizutani
12. Changes of matrix metalloproteinases (MMPs) expression in triglyceride treatedmacrophages.

Jaewon Lim

CANCER-DIRECTED DELIVERY OF DOXORUBICIN-RAPAMYCIN AND
CANCER IMAGING BY ANTI-HER2 IMMUNOLIPOSOMESMin Woo Kim, Yeon Kyung Lee, Hwa Yeon Jung, Seong Jae Kang, and Yong Serk Park
Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju 220-710, Republic of Korea

Introduction
About 30% of human breast cancers overexpresses
Her2/neu, implying clinically bad prognosis. Althoughdoxorubicin, intercalating agent in the DNA double strand,
has been known as an effective anticancer drug, Her2/neuoverexpressing cell lines such as SK-BR-3 have resistance tothe drug via PI3K/AKT/mTOR signaling, associated withcell survival pathway. Therefore, we have tried to develop an
effective treatment procedure for Her2-positive breast cancer
by blocking of the PI3K/AKT/mTOR pathway. Rapamycin,
acting as an immunosuppressant drug, is known to directlybind the mTOR Complex, so it appears to be an appropriate
inhibitor to the PI3K/AKT/mTOR pathway. [2]Combinational therapy with rapamycin and doxorubicinwould presumably exhibit elevated anticancer efficacy toHer2-positive cancers. [3] One strategy to deliver the bothdrugs specifically to the cancer cells is to develop tumordirected liposomal formulations. Doxorubicin was
encapsulated into anti-Her2 immunoliposomes andrapamycin was incorporated in the lipid bilayers. Quantum
dots, semiconductor nanocrystals having unique opticalproperties, were also incorporated in the system for cancer
imaging.
Materials and Methods
Materials

POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine),
DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)2000]),
DSPE-PEG2000-MAL (1,2-distearoyl-sn-glycero-3phosphoethanolam ine-N -[m aleim ide (polyethylene
glycol)2000]) were purchased from Avanti Polar Lipid, Inc.
(Alabaster, USA). PD-10 column and sepharose CL-4B waspurchased from Amersham Bioscience (Uppsala, Sweden).
Inorganic Quantum dots (dissolved in chloroform) werepurchased from Nanosquare (Seoul, Korea).

Table 1.Components of anti-Her2 immunoliposomes

lipid
POPC
DSPE-PEG2000
DSPE-PEG2000-Mal
Cholesterolmole%
48.00%
3.80%
0.20%
48.00%

Direct coupling of thiolated anti-Her2 antibody to liposomes

Herceptin were thiolated for 1 hr at room temperature byreacting with Traut’s reagent in degassed HEPES-EDTA buffer(20 mM HEPES, 140 nM NaCl, 2 mM EDTA, pH 8.0).
Unreacted Traut’s reagent was removed by passing through PD10 column with the degassed HEPES buffer. Thiolated
antibodies were added and then conjugated to the preparedliposomes by incubation for 16 hr at 4℃ with continuous
agitation.
Doxorubicin encapsulation

Doxorubicin was encapsulated by the pH gradient method. [4]Briefly, the hydrated liposomes in a citrate buffer (150 mM, pH
4.0) were passed through the sepharose CL-4B column
equilibrated with the HEPES buffer (20 mM HEPES, 140 nM
NaCl, pH 7.5) to replace the extra-liposomal solution.
Doxorubicin was added to the liposomal solution (1:3 weightratio) and then incubated at 60℃ for 10 min with gentle mixing.
The incubation process at 60℃ was necessary for rapid andcomplete entrapment of doxorubicin inside lipis vesicles.
Results
Preperation of anti-Her2 multifunctional immunoliposomes

A conceptual illustration of the anti-Her2 immunoliposomes isshown in Figure 1.

Cell lines and cell culture

Her2/neu-positive SK-BR-3 (human breast cancer cell line)and Her2/neu-negative MDA-MB-231 (human breast cancer
cell line) were maintained in DMEM (Gibco, Carlsbad,USA). The culture media were supplemented with 10% fetal
bovine serum and 100 units/ml streptomycin.
Preperation of liposomes

PEGylated liposomes conjugated to Herceptin (Her2targeted humanized antibody) were prepared according to theethanol injection method. POPC, cholesterol, PEG-PE, PEGmaleimide and rapamycin were dissolved in 100 ul of
ethanol at 60℃ (Table 1) and quantum dots wereadditionally added to the ethanol solvent. Then, the ethanol
system was injected into 900 ul of degassed citrate buffer.
(150 mM, pH 4.0) [1]Figure 1. A diagram of Her2-targeted multifunctional immunoliposomes

After direct coupling of anti-Her2 antibodies, the liposomal
solutions were passed through a sepharose CL-4B gel filtration
column (Figure 2). The fraction 5-6 shows peak and represents
liposome confirmed by the phosphate assay (650 nm) to detectlipid components, and by the Lowry protein assay (750 nm) to
detect conjugated antibodies. Additionally, the fluorescence of
Quantum dots was visualized by UV illumination.

[2] Gnant M. The Role of Mammalian Target of Rapamycin (mTOR)
Inhibition in the Treatment of Advanced Breast Cancer. Curr Oncol Rep.
2012 Sep 28.
[3] Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S, Kogan
S, Cordon-Cardo C, Pelletier J, Lowe SW. Survival signalling by Akt
and eIF4E in oncogenesis and cancer therapy. Nature. 2004 Mar
18;428(6980):332-7[4] Nakamura K, Yoshino K, Yamashita K, Kasukawa H. Designing anovel in vitro drug-release-testing method for liposomes prepared by
pH-gradient method. Int J Pharm. 2012 Jul 1;430(1-2):381-7. Epub
2012 Apr 12

Author address
E-Mail: nanodream@yonsei.ac.kr
Figure 2. Elution profiles of anti-Her2 immunoliposomes.
Specific cellular binding of anti-Her2 immunoliposomes

Specific cellular binding of anti-Her2 multifunctional
immunoliposomes was examined by FACS analysis andconfocal microscopy. The anti-Her2 liposomes were able tospecifically bind to Her2-positive SK-BR-3 cells, but not toHer2-negative MDA-MB-231 cells (Figure 3).

Figure 3. Binding of anti-Her2 multifunctional immunoliposomes to
SK-BR-3 and MDA-MB-231 cancer cells.
A : MDA-MB-231, untreated
B : MDA-MB-231, treated with anti-Her2 immunoliposomes
C : SK-BR-3, untreated
D : SK-BR-3, treated with anti-Her2 immunoliposomes

Additionally,theanti-Her2
multifunctional
immunoliposomes containing quantum dots were added to
SK-BR-3 cells. After 3 hr incubation, red flurescences
representing quantum dots were observed by confocal
microsopy (Figure 4).

Figure 4. Binding of anti-Her2 multifunctional immunoliposomes
containing qantum dots to SK-BR-3 cancer cells.
A : SK-BR-3, no treatment
B : SK-BR-3, anti-Her2 multifunctional immunoliposomes

Reference[1] Muthu MS, Kulkarni SA, Raju A, Feng SS. Theranostic
liposomes of TPGS coating for targeted co-delivery of docetaxel andquantum dots. Biomaterials. 2012 Apr;33(12):3494-501. Epub 2012
Feb 4

Variation of IgE-binding epitopes in the allergen after repeated immunization
Takehiro Yamaguchi, Miyoko Matsushima, Nanako Ogasawara, Haruka Nose, Tsutomu Kawabe
Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya,Japan

Introduction
The production of IgE antibodies to exposed allergen isneeded to develop allergic responses. In humoral responses
after primary exposure to allergen, naive B cells aredifferentiated into plasma cells but also differentiated intomemory B cells. Plasma cells constitutively secrete antibody,which bind to the antigen. Memory B cells differentiate
quickly into antibody secreting cells that are enhanced in
production rate, titre and affinity from an original antibody
when re-exposed to allergen [1, 2, 3]. However it is not clear
whether memory B cells could continue to make antibody
responses following repeated immunization of allergen.
Since the antibody produced by one B cell recognizes only
one of the epitopes of the antigen that usually has multiple
epitopes, several antibodies would be generated against theset of epitopes. We usually detect the quantity of allergenspecific IgE antibodies by the ELISA tests. The problems ofthe current ELISA test results in false positive and false
negative with high frequency to diagnose allergen-specificallergic diseases because the current test detected only theconditions sensitized by allergen. Because the purified
whole proteins were used as allergen to detect allergenspecific IgE antibody levels, we hypothesized that the
information on the variations of epitopes recognized by IgE
antibodies to cause allergic reactions might improve theaccuracy to diagnose the allergy.
Recently the peptide microarray was used as versatilecommon tools for high throughput screening assay inbiomedical and pharmaceutical research [4, 5]. Peptide
arrays can evaluate the ligand–receptor interactions andepitope mapping. Using the peptide array, we can get more
precise data on allergens detected by specific IgE antibodies.
Therefore, the analysis of epitope patterns would identify theallergen-specific antibody to cause allergic reactions.
Furthermore, this will lead us to understand the clinicaltolerance of the allergy or the severity of the allergy
symptom.
In this study, we examined the distribution pattern ofallergen-specific IgE recognizing epitopes by using peptide
array after repeated immunization of allergen.
Materials and Methods
Animals: 6 to 8 week-old female BALB/c mice weremaintained in a temperature (22-24°C), humidity (55±5%),and light (12 hour light-dark cycle; lights on at 7:00)
regulated room with food and water ad libitum. Allprocedures were performed with the approval of Medicine,
Nagoya University in accordance with the Guidelines for
Animal Experimentation of Nagoya University.
Immunization: Mice were sensitized i.p. in 0, 2, 4, 6, 10, 12,
16 and 20 weeks, with 100 µg of β -lactoglobulin together
with 1 mg of alum in a final volume of 200 µl saline.
Control mice were injected 200 µl saline containing 1 mg ofalum. Before each immunization, mouse serum was collected.
Measurementofallergen-specificantibodies:
βlactoglobulin specific IgE levels were measured using ELISA.

Microtitre plates were coated with β-lactoglobulin (25 µg/ wellin 100 µl) overnight at 4 °C, and then blocked with 1% bovine
serum albumin (BSA) for 1 h at room temperature (RT). The
plates were washed with phosphate-buffered saline (PBS) with
0.05% Tween 20 (PBS-T). Serum samples were diluted 1 : 5 in
1% BSA, and incubated for 2 h at RT. Plates were then washedand incubated with biotin-labelled anti-mouse IgE antibodies
for 2 h at RT. Followed by incubation with HRP-labelledstreptavidin for 2 h at RT. Plates were developed using ophenylenediamine substrate and optical density was measured at
490 nm using microplate reader.
Alexa 647-Labeled
Anti-IgE Antibodies
Antigen-SpecificIgE Antibodies
Peptides(16 amino acids)

Fig1. IgE-binding analysis using the peptide arrayIgE-binding analysis using the peptide array: A library of
peptide used in this study consists of 16 amino acids
overlapping by 13 (3-offset) and corresponds to the primary
sequences of a β-lactoglobulin (UniprotKB P02754). Linear
peptides were individually dissolved in a 20 mM phosphatebuffer (pH 8.5) containing 0.1% sodium dodecyl sulfate (SDS).
After the addition of 1-ethyl-3- (3-dimethylaminopropyl)
carbodiimide hydrochloride (EDC), peptide solutions werespotted in at least triplicate on a succinimidyl-functionalized
glass slide using the piezoelectric ceramic micropump(GENESHOT, NGKInsulators, Ltd., Aichi, Japan). Peptide
array was blocked with blocking buffer containing 50 mM
ethanolamine for 90 min. Then, the peptide array was washed 3times for 5 min with PBS–T. Blood sera were diluted 1:10 in
PBS-T containing 1% ovalbumin to a final volume of 200 µl.
The diluted sera were then transferred to the surface of theslides and covered by a micro cover glass. The slides wereincubated for 1 h at 37 °C, followed by overnight incubation at
4 °C. Then, the peptide array was washed 3 times for 5 minwith PBS-T and incubated with Alexa 647-labeled anti-mouse
IgE diluted 1:500 in PBS-T containing 1% ovalbumin for 3 h at
RT in dark. The peptide arrays were finally washed 3 times in
PBS-T and in distilled water for 5 min, and dried bycentrifugation at 800 rpm for 3 min. All these steps were
performed at room temperature. An Agilent microarray scanner(model G2505B, software G2565BA/DA; Agilent Technologies,
Palo Alto, CA, USA) was used to analyse the fluorescence
images of Alexa 647-labeled anti-mouse IgE at an emission
wavelength of 665 nm. Fluorescence intensities were quantified
with GenePix Pro (Axon, Union City, CA, USA).

Results
Detection of the β-lactoglobulin specific IgE
We first examined the allergen-specific IgE antibodies
after immunization.
Mice were immunized with βlactoglobulin for 8 times. β-lactoglobulin-specific IgE weredetected after primary immunization. High levels of βlactoglobulin-specific IgE amount were maintained after
subsequent immunization.
Variation of IgE-binding epitopes after repeatedimmunization
We next analysed the IgE-binding peptide patterns using
mouse sera collected after each immunization (2, 6, 14, 18,
and 22 weeks). β-lactoglobulin-specific IgE antibodies onlybound to the set of peptides which include the peptide No. 20
and flanking peptides in 2 weeks after primary immunization.
Furthermore, IgE bound to the similar pattern of peptides in 6
to 22 weeks after subsequent immunization. However,
fluorescence intensities of these peptides was decreased in 6,
14, and 18 weeks but increased again in 22 weeks. We also
detected the binding of IgE to the new set of peptides which
include peptide No. 42 and flanking peptide in 14 weeks as
6th immunization proceeded. Similar pattern of epitopes was
also observed in 18 and 22 weeks. In 18 weeks, the set of
peptides which include peptide No.42 were detected thehighest fluorescence intensities. Whereas, the set of peptides
which include peptide No.33 were detected the highest
fluorescence intensities in 22 weeks.
Discussion
In this study, we investigated the variations of IgE-binding
epitopes after repeated immunization by using a peptide
array.
First, we detected the epitopes bound by the mouse serum
IgE using peptide array. The epitope recognized by anantibody was normally consisted of 7 to 8 amino acids, andwe established a peptide array which was consisted of 16
amino acids overlapping by 13 (3-offset) and corresponds tothe primary sequences of β-lactoglobulin. Therefore, wehypothesized that several fluorescence intensities weredetected in flanking peptides which contains the epitope foreach epitope that recognized by an antigen-specific IgE
antibody. Thus, we thought fluorescence intensity detected
in a single peptide was non-specific reaction to peptide thatnot contains an IgE-binding epitope.
Amazingly, we detected only one epitope recognized by βlactoglobulin-specific IgE after primary immunization since
several antibodies which recognize different epitopes was
supposed to be generated against the β-lactoglobulin that has
multiple epitopes. We also detected the similar pattern of
epitope distribution by antigen-specific IgE antibodies inmouse serum, suggesting that memory B cells might be
activated to produce β-lactoglobulin-specific IgE binding tosame epitopes with repeated immunization. Furthermore,
fluorescence intensities were decreased after consequent
immunization, suggesting the activation of memory B cells
was gradually declined in subsequent immunization.
However, we could not exclude the possibility that thegeneration of these antibodies were from the activation ofnaïve B cells. We also detected the binding of IgE to thedifferent set of epitopes as immunization proceeded. These
results suggested that another newly activated B cells withthe different epitopes recognized by β-lactoglobulin-specificIgE would be generated after second or subsequent
immunization. Moreover, the degree of memory or naïve B
cell activation might be different in every immunization
since there was a shift of the peptide which has the highest

fluorescence intensities in each serum. However, we could notdetect the conformation-dependent epitopes recognized by βlactoglobulin-specific IgE in our experimental conditions sinceantigen-specific IgE could bind only linear protein epitopes by
peptide array.
Furthermore, the part of amino acid sequences of epitopes
that we detected in this study were corresponded to the aminoacid sequences that were important in cow`s milk allergy which
previously reported [6].
To elucidate the role of memory B cells in repeatedimmunization, further studies should be done. Memory B cells
are differentiated in germinal center through a process involving
the acquisition of affinity maturation that enhance antigen
binding [7]. Therefore, the measurement of affinity of antigenspecific IgE antibodies to each epitope would reveal whether
memory B cell produces antibodies after repeated immunization.
Conclusions
In this study, we suggest that memory B cells seemed to be
activated to produce antibodies in repeated immunization.
However, there was a possibility that activation of memory B
cells was not necessarily maintained in high levels after
subsequent immunization. In parallel to the activation ofmemory B cells, allergen-specific IgE antibodies which
recognize different epitopes would be newly generated.
References[1] Benson, M.J., R. Elgueta, W. Schpero, M. Molloy, W.
Zhang, E. Usherwood, and R.J. Noelle. Distinction of thememory B cell response to cognate antigen versus bystander
inflammatory signals. J. Exp. Med. 2009; 206: 2013–2025.
[2] Dogan, I., B. Bertocci, V. Vilmont, F. Delbos, J. Mégret, S.
Storck, C.A. Reynaud, and J.C. Weill. Multiple layers of B
cell memory with different effector functions. Nat. Immunol.
2009; 10: 1292–1299.
[3] Pape, K.A., J.J. Taylor, R.W. Maul, P.J. Gearhart, and M.K.
Jenkins. Different B cell populations mediate early and latememory during an endogenous immune response. Science.
2011; 331:1203–1207.
[4] Andresen H, Zarse K, Gro¨ tzinger C, Hollidt JM,
Ehrentreich-Fo¨ rster E, Bier FF, et al. Development of
peptide microarrays for epitope mapping of antibodies
against the human TSH receptor. J Immunol Methods. 2006;
315:11–8.
[5] Nahtman T, Jernberg A, Mahdavifar S, Zerweck J,
Schutkowski M, Maeurer Met al. Validation of peptide
epitope microarray experiments and extraction of quality
data. J Immunol Methods. 2007; 328:1–13.
[6] Kirsi-Marjut Järvinen, Pantipa Chatchatee, Ludmilla
Bardina, Kirsten Beyer, and Hugh A. Sampson. IgE and IgG
Binding Epitopes on α -Lactalbumin and β -Lactoglobulin in
Cow’s Milk Allergy. Int Arch Allergy Immunol. 2001;
126:111–118[7] McHeyzer-Williams, L.J., and M.G. McHeyzer-Williams.
Antigenspecific memory B cell development. Annu Rev
Immunol. 2005; 23: 487–513.
Author address
E-Mail:yamaguchi.takehiro@c.mbox.nagoya-u.ac.jp

Evaluation of human papillomavirus oncogenic mRNA testing for
diagnosis of cervical cancer by using real-time NASBA
Yeonim Choi1), Geehyuk Kim1), Sangjung Park1), Sunghyun Kim1), Yeon Kim1), Dongsup Lee2), Jijgee
Munkhdelger3), Kwang Hwa Park3), and Hyeyoung Lee1)†1)Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 220701 Korea
2)Department of Clinical Laboratory Science, Hyejeon College, Hongseoung, Chungcheong Nam-do, Republicof Korea3)Department of Pathology, College of Medicine, Yonsei University, Wonju 220-701, Korea
Introduction
Human cervical cancer is the third most common cancer
among women worldwide [1]. In the past, cytopathological test, such as the Papanicolaou smear, was the
only way of detecting invasive cervical carcinoma. Several
decades ago, Human papillomaviruses (HPV) were foundout to be a major factor of cervical cancer [2]. This finding
was based on the observations that showed there is asignificant association between oncogenic HPV infections
and cyto-histologically defined pre-invasive lesions andinvasive cervical carcinoma [3, 4]. Ever since then, themethods detecting HPV in clinical specimens became
widely used for diagnosing cervical cancer.
Among those, DNA-based HPV genotyping assay has been
the method of the choice, since it has shown high analyticalsensitivity. However, the poor clinical specificity of the
HPV genoytyping based on cervical cancer diagnosis has
drawn serious clinical concerns. The latest results show
oncogenic HPV DNA appeared not only in cancerous
tissues, but also in the normal tissues according tocytological diagnosis.
Recently, a cervical diagnostic test employing real-time
NASBA has been commercialized. The test detects E6 andE7 mRNA of oncogenic HPV, which are known to be
responsible for oncogenesis of cervical cancer by HPV,
instead of oncogenic HPV DNA. In this study, weevaluated the clinical sensitivity and specificity of the testfor diagnosis of cervical cancer.

Materials and Methods
Cervical decidual cell samples were recruited at Yonsei
University Wonju Christian Hospital in Gangwon provinceof Korea. A total of 187 cervical decidual cell samples
were classified by 2001 Bethesda system (cyto-phathologic
analysis) and were performed with HPV DNA genotyping
test (DNA chip, Cancer Minichip kit, Goodgen).
For real-time NASBA test for diagnosis of cervical cancer,
RNA was extracted using automation machine (MagNA
Pure LC 2.0, Roche) and RNA Isolation High Performance
Kit (Roche). The NASBA kit (NucliSENS EasyQ HPV,
Biomerieux) used in this study targets E6 and E7 mRNA of
HPV genotype 16, 18, 31, 33, 45. The NASABA was
performed by the manufacturer's recommendation using
the EASY Q NASBA ANALYSER (Biomerieux).

Results
A total of 138 samples were high-risk HPV positive by
HPV	  DNA genotyping test and 49 samples were HPV
DNA negative. Real-time NASBA test was performed withall of 187 samples. The positive rate of real-time NASBA
test with high-risk HPV positive samples was 73.3% in
SCC & ADC (squamous cell carcinoma & adenocarcinoma)group, 73.9% in HSIL (High-grade squamous
intraepithelial lesion) group, 60% in ASC-H (Atypicalsquamous cells) group, 37.5 % in LSIL, 29.2% in ASC-US(Atypical squamous cells of undetermined significance)group, and 16.4% in normal group. On the other hand, thepositive rate of real-time NASBA in normal group samples
whose cytological findings were normal and HPV
genotyping results were also negative samples was 0%.

Conclusions
The data from this study seems clearly show that thereal-time NASBA for diagnosis of cervical cancer has
higher clinical specificity than HPV DNA genotyping test,
since the positivity rate detected by the real-time NASBA
in normal samples was significantly lower than that
detected by the HPV DNA genotyping test. However, thepositive rate detected by the real-time NASBA incancerous samples was much lower than that detected by
the HPV DNA genotyping test. In this respect, thesensitivity of the real-time NASBA for cervical cancer
diagnosis seems to be lower than current
HPV genotyping test. This fact can be problematic inclinical settings. Therefore, the results from this study seemto suggest that for a real-time NASBA based cervicalcancer test to be employed in clinical settings, more careful
evaluations with large numbers of clinical specimens will
be recommended.

References[1] Jemal A, Bray F, Center MM, Ferlay J, et al. Global
cancer statistics: GLOBOCAN 2008. CA Cancer J Clin.
2011;61:69–90[2] F. Xavier Bosch, M. Michele Manos, Nubia Munoz,
Mark Sherman, Angela M. Jansen, Julian Peto, Mark H.
Schiffman, Victor Moreno, Robert Kurman, Keerti V. Shah.
Prevalence of Human Papillomavirus in Cervical Cancer: a
Worldwide Perspective. Journal of the National Cancer
Institute. 1995;Vol. 87, No. 11, June 7.
[3] Walboomers JMM, Jacobs MV, Manos MM, Bosch FX,
Kummer A, Shah KV, Snijders PJF. Human
papillomavirus is a necessary cause of invasive cervicalcancer worldwide. J Pathol. 1999;189:12–19.
[4] Bohmer G, van den Brule AJ, Brummer O, Meijer CL,
Petry KU. No confirmed case of human papillomavirus
DNA negative cervical intraepithelial neoplasia Grade 3 orinvasive primary cancer of the uterine cervix among 511
patients. Am J Obstet Gynecol. 2003;189:118–120.
Author address
E-Mail : hyugiyoebi@naver.com
Corresponding author : hyelee@yonsei.ac.kr

Quantitation of neonicotinid metabolites in human urine
using gas chromatography-mass spectrometry
H. Nomura1), J. Ueyama1), S. Wakusawa1), T. Kondo1)
1) Department of Pathophysiological Laboratory Sciences, Nagoya University
Graduate School of Medicine, Nagoya, Japan

Introduction
Neonicotinoid insecticides (NEOs), developing as
relatively new pesticides, act selectively on insect nicotinic
acetylcholine receptors, and are widely used in agriculture as
foliar and seed treatments, for indoor and outdoor insect
control, home gardening and pet products (Jeschke et al.,2011). NEOs have from five to ten times higher selectivity
factors for insects versus mammals than theorganophopsphates, methylcarbamates, and organochlorines(Tomizawa and Casida, 2005). For this reason, NEOs are
considered to be harmless to mammals and to be a promising
candidate that could replace more toxic organophosphorus
insecticides (OPs). NEO-induced toxicity in the nervous
systems of vertebrates has been studied once in a while. It
has been reported that such NEOs as thiamethoxam andclothianidin induce dopamine release in the rat striatum via
nicotinic acetylcholine receptors (nAChRs) and thatthiamethoxam alters behavioral and biochemical processes
related to the rat cholinergic systems (Rodrigues et al., 2010).
These findings suggest a possibility that NEOs affectmammalian nAChRs to a greater extent than previously
believed based on general toxicity data obtained registration
requirements tests for pesticide; this warrants further studiesof the effect of NEOs on human health.
NEO metabolism has been addressed by Casida andcolleagues. As shown in Fig. 1, NEOs such as imidacloprid,nitenpyram, thiacloprid and acetamiprid have a common
structure chloropyridinyl, and these compounds metabolize
into 6-chloronicotinic acid (6-CN), which is conjugated withglycine or glucuronic acid. Similarly, thiamethoxam andclothianidin have a common structure chlorothiazole, andboth metabolize into 2-chloro-1,3-thiazole-5-carboxylic acid
(2CTCA) also conjugated with glycine or glucuronic acid.
For the determination of urinary 6CN, high-performancegas chromatography tandem mass spectrometry (GCMS/MS) combined with solid phase extraction (SPE) has
been developed (Uroz et al., 2001). Their method showed ahigh-sensitivity performance (limit of detection 0.02 mg/L ofurine), but required complicated sample procedures and GCMS/MS is not affordable even now.

Fig. 1.
Neonicotinoids andtheir urinary
metabolites(shaded areas)measured forbiologicalmonitoring.

The main object of this study is to develop and validate a simple
method for quantitation of urinary NEO metabolites, 6CN and2CTCA, in human urine with GC-MS. The sensitivity andreliability of our method will be examined in this study.
Materials and Methods
Chemicals and reagents: 6CN (purity >98.0 %),hydrochloric acid (6 mol/L), formic acid, methanol wereobtained from Wako Pure Chemicals (Osaka, Japan), and2CTCA (purity > 98.0%) was from Santa Cruz Biotechnology,
Inc (CA, USA). Isotope labeled 3-phenoxybenzoic acid(phenoxy-13C6, purity > 98%), for use as an internal standard(I.S.) substance, was purchased from Cambridge Isotope
Laboratories,
Inc
(Ma,
USA).
N,Obis(trimethylsilyl)trifluoroacetamide
with1%
trimethylchlorosilane(BSTFA-TMCS,
99:1)
and
trimethylsilyldiazomethane were purchased from Tokyo Kasei
Kogyo (Tokyo, Japan), acetonitrile and sulfuric acid were from
Kanto Chemicals(Tokyo, Japan).
1,1,1, 3, 3, 3Hexafluoroisopropanol(HFIP)
andN,Ndiisopropylcarbodiimide (DIC) were from Sigma-Aldrich (MO,USA). Polymeric strong cation exchange SPE products, theBond Elute PCX (30 mg) (Agilent Technologies, Inc. CO,USA), was used for NEO metabolite extraction from urine.
Sample preparation procedure: Two milliliters of urine
was pipetted into a 10-mL screw-top glass test tube, and 50 mL
H2SO4 and I.S. solution (1 mg/L isotope labeled 3-PBA,
d3PBA) were added. After gentle shaking, the test tube wasincubated at 86 oC in a heat block for 2 hour for deconjugation.
After cooling on ice, the test tube was centrifuged at 2,000×g
for 10 min, and the supernatant was applied to SPE procedure.
A SPE cartridge packed into a 1-mL solvent reservoir was
pre-conditioned with 500 mL methanol and followed by 500 mL
water wash. Then, the conditioned SPE was loaded with 2 mLurine sample and placed under a vacuum pressure and followed
by 500 mL formic acid solution (2%) wash. The SPE cartridge
was dried under vacuum for 3 min and eluted with 500 mL
methanol. The eluate was dried up with a gentle nitrogenstream at room temperature and the residue was resolved with
500 mL acetonitrile. Thirty minutes after the addition of 50 mL
derivatization reagent, BSTFA-TMCS, 1 mL sample wasanalyzed by GC-MS within 24 hours after the derivatization.
Results and discussion
Derivatization condition: First of all, to perform highly
sensitive determination, the optimum derivatization reagentsand relevant conditions including reaction solution andtemperature were examined. Previously, Uroz and colleagues(2001) derivatized 6CN with HFIP and DIC in hexane solution
at room temperature for GC-based analysis. In our results asshown in Table 1, higher total ion chromatograph peak areas of
2CTCA and 6CN were detected when BSTFA-TMCS in either
solution of acetonitrile or toluene was used for derivatization
than with other conditions. Increase of reaction temperaturefrom room temperature to 60oC did not affect the peak areas
dramatically. Toluene is considered to be an inconvenientsolution for derivatization procedure due to its relatively highvolatility and flammability. Our result indicates that use of
BSTFA-TMCS in acetonitrile is the most efficient forderivatization at room temperature for 30 min to determine
urinary NEO metabolites.
We selected this optimum
derivatization conditions in the later fundamental
examination and applications. The full-scan mass spectora oftrimethylsilyl-2CTCA and trimethylsilyl-6CN were shown in
Fig. 2.

Fig. 2. Mass spectrum of derivatized 2CTCA (left) and 6CN (light).

Fig. 3 shows selected ion chromatograms of pooled urine
samples spiked with 2CTCA (upper, m/z 220) and 6CN(lower, m/z 214). Our method yielded highly resolved peaks,
allowing clear identification of NEO metabolites even at low
concentration levels. No apparent interference peaks wereobserved. The most abundant ion derived from 2CTCA wasthe fragmentation ion at m/z 176 but its peak was interferedby an unknown peak. Thus, we selected the second largest
fragment ion at m/z 220 as Q-ion.

Fig. 3. GC-MS selected ion monitoring chromatogram of
2CTCA (top) and 6CN (bottom) in standard spiked pooled urine
samples.

Accuracy, precision and linearity: The accuracy, precisionand linearity parameters are summarized in Table 3. The
regression equations were y=0.0054x+0.0002 (r2=0.999) for
2CTCA and y=0.013x+0.0015 (r2=0.999) for 6CN. The

LOD and LOQ were calculated to be 0.1 and 0.3 mg/L for both
metabolites, respectively. The LOD value of 6CN was about
six times higher than previously reported ones using GCMS/MS (Uroz et al., 2001). Taira and colleagues reported a
6CN determination method with the LOQ of 1 mg/L using LCMS. Although the LOD levels of our method are not as low as
those in the previous reports, some advantages of our method
was confirmed; i.e., simple sample preparation and use ofcommonly-used apparatus that provide stable assay results. We
believe that this method might be useful for high-throughput
analyses. Besides, no method of determining 2CTCA in urine
has been previously reported to our knowledge. Our results
indicate that the assay had remarkable reproducibility withacceptable accuracy and precision.
Application of the method to field study samples: Our
method was applied for human spot urine obtained from nine
apple farmers collected in August 2009. Based on theirquestionnaire response, four farmers who worked for NEO
handling operation within 10 days before urine collection wereselected as the occupational NEO exposure group. Mean
concentrations of 6CN (above LOD) were 1.7 mg/L for theoccupational exposure group and 1.3 mg/L for the control group.
A 2CTCA concentration of 0.1 mg/L was detected only in one
person in the exposure group. To our knowledge, this is thefirst report about the determination method of urinary 2CTCA
and detection of 2CTCA from human urine.
In conclusion, a simple, sensitive and reliable method of
GC-MS with SPE sample preparation for the determination ofurinary 2CTCA and 6CN was developed. Since urinary 2CTCA
and 6CN can be measured simultaneously, our method is
expected to practically serve for the biomonitoring of NEO
exposure at the individual level.
References
P. Jeschke, R. Nauen, M, Schindler, A. Elbert, J Agric. Food
Chem. 59 (2011) 2897.
M. Tomizawa and J.E. Casida. Annu. Rev. Pharmacol. Toxicol.
45 (2005) 247
K.J. Rodrigues, M.B. Santana, J.L. Do Nascimento, D.L.
Picanco-Diniz, L.A. Maues, S.N. Santos, V.M. Ferreira,
M. Alfonso, R. Durán, L.R. Faro. Ecotoxicol. Environ.
Saf. 73 (2010) 101.
F.J. Uroz, F.J. Arrebola, F.J. Egea-González, J.L. MartínezVidal. Analyst. 126 (2001) 1355.
Author address
E-Mail: nomura.hiroshi@h.mbox.nagoya-u.ac.jp

Comparison of virulence factors, phylogenetic groups and ciprofloxacin susceptibility
between Escherichia coli isolated from healthy students and uropathogenic E. coli isolated
from patients with urinary tract infections in Korea
M. Park1), S. Park1), S-H. Kim1), G. Lee1), H. Woo1), Y. Uh2), W-D. Seo3) and J. Kim1)
1) Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju, Korea
2) Department of Laboratory Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea
3) Department of Functional Crop, NICS, Miryang, Korea
Introduction
Uropathogenic Escherichia coli (UPEC) is one of themost common etiological agents in urinary tract infections.
UTIs consist of such as asymptomatic bacteriuria, acutecystitis and acute pyelonephritis, etc. [1][2]. Causative agentof urinary tract infections include E. coli, Proteus spp.,
Enterobacteraerogenes,
Pseudomonas
aeruginosa,
Enterococcus
faecalis,
Staphylococcus
aureus,
Staphylococcus saprophyticus and Klebsiella pneumonia[3][4]. UPEC cause urinary tract infections with the rate for70-90% of community-acquired UTIs and approximately
40% of all nosocomial UTIs in the Unite States [5].
UPEC strains generally possess several genes encoding
virulence factors, which are mostly adhesions, toxins,
bacteriocin and siderophores [6]. Pathogenic E. coli strains,
including UPEC strains, belong mainly to groups B2 and D[7]. Fluoroquinolones are commonly used for UTIs treatment
in Korea, and resistance to fluoroquinolones is increasing,
especially resistance to ciprofloxacin. In the current study,
comparison of virulence factors (P and S fimbriae, type 1
fimbriae, α -hemolysin, uropathogen-specific protein andcytotoxic necrotizing factor 1), phylogenetic groups (A, B1,
B2 and D) and ciprofloxacin susceptibility were examined
between E. coli isolated from healthy students anduropathogenic E. coli isolated from patients with urinary tract
infections in Korea..

- Each strains was screened for susceptibility (by the disk
diffusion method) to ciprofloxacin

Results
The identified virulence factors (VFs), phylogenetic groups
and cipofloxacin resistance in 13 E. coli strains isolated fromhealthy students were papC (15.4%), fimG/H (76.9%), sfaD/E(30.8%), hlyA (23.1%), cnf1 (23.1%), usp (7.7%), phylogenetic
group A (23%), B1 (8%), B2 (46%), D (23%) and ciprofloxacin
resistance (7.7%) , while those of in 175 E. coli strains isolated
from patients with UTI were papC (41.1%), fimG/H (92.5%),
sfaD/E (30.3%), hlyA (10.3%), cnf1 (30.3%), usp (27.4%),phylogenetic group A (9.1%), B1 (5.1%), B2 (60.6%), D(25.1%) and ciprofloxacin resistance (29.7%).

Materials and Methods
Specimen collection- A total of 188 strains of 175 UPEC clinical isolates wascollected from Korean healthcare facility in 2010 and 13
strains were collected from healthy students in 2011.
Bacterial DNA extraction- To prepare DNA templates, brain heart infusion (Difco,
Detroit, USA) broth was inoculated with one colony of
UPEC strain. After 18 hours incubation at 37°C, 1 ml of theculture were centrifuged at 12,000g for 10 minutes. The
pellets were resuspended in 1 ml of saline, harvested bycentrifugation and suspended in 200 µl of sterile water,incubated at 100°C for 10 min, and centrifuged. The
supernatant was used in all PCRs as described below.
PCR (Polymerase Chain Reaction) amplification- Determination of phylogenetic group was performed by atriplex PCR method was performed, which identifies type A,
B1, B2 and D [7].
- Determination of genetic virulence profiles by multiplex
PCR method, which detect fragments of genes encoding
selected E. coli VFs (hly1, fimG/H, usp, cnf1, papC, andsfaD/E) [6]
Ciprofloxacin susceptilibity test

Conclusions
In our study, E. coli strains (76.9%) from healthy students
were found to possess more than one virulence factor associatedwith adhesion. In addition, one E. coli strain isolated fromhealthy students who had never been infected with UPEC
showed ciprofloxacin resistance (7.7%). Furthermore, UPEC
strains isolated from patients showed high level of ciprofloxacin
resistance. Because resistance to ciprofloxacin has approached30%, ciprofloxacin should be considered as a first-line
empirical treatment for UTIs after the proper antimicrobial
susceptibility test.

References[1]Fhin SD. Clinical practice. Acute uncomplicated urinary
tract infection in Women. N Engl J Med. 2003.
17;349(3):259-266.
[2]Lee WY, Kim JB. Serological studies on the specificantibodies against p-pili of uropathogenic Escherichia coli.
Korean J Biomed Lab Sci. 1996. 2(1):31-40[3]DeBoy JM, Wachsmuth IK, Davis BR. Hemolytic activity
in enterotoxigenic and non-enterotoxigenic strains ofEscherichia coli. J Clin Microbiol. 1988. 12(2):193-198.
[4]Gupta K, Hooton TM, Stamm WE. Increasing
antimicrobial resistance and the management of
uncomplicated community acquired urinary tract
infections. Ann Intern Med. 2001. 135(1):41-50.
[5]Li D, Liu B, Guo D, Guo X, Liu F, Feng L, Wang L. A
multiplex PCR method to detect 14 Escherichia coli
serogroups assocated with urinary tract infections. J
Microbiol Methods. 2010. 82(1);71-77.
[6]Adamus-Bialek W, Wojtasik A, Majchrzak M, Sosnowski
M, Parniewski P. (CGG)4-Based PCR as a Novel Tool for
Discrimination of Uropathogenic Escherichia coli strains:
Comparison with Enterobacterial Repetitive Intergenic
Consensus-PCR. J Clin Microbiol. 2009. 47(12);39373944.
[7]Clermont O, Bonacorsi S, Bingen E, Rapid and simple
determination of the Escherichia coli phylogenetic group.
Appl Environ Microbiol. 2000. 66(10);4555-4558.

Author address
E-Mail: pm1984@naver.com

The Relationship Between Helicobacter Pylori Infection and Medical History of
Selected Diseases among an Urban Area Population of Japan
A. Hayashi1), T. Kondo1)
1) Department of Pathophysiological Laboratory Sciences
Nagoya University Graduate School of Medicine, Nagoya, Japan

Introduction
Since successfully isolated and cultivated by Robin
Warren and Barry Marshall in 1983, Helicobacter pylori (H.
pylori) has become a high-profile organism. H. pylori is ahelix-shaped, microaerophilic Gram-negative bacterium. In
the infected stomach where normal secretion of urea usuallyoccurs, large amounts of urease produced by H. pylori breaks
down urea into ammonium to neutralize gastric acid, whichhelps H. pylori survive the acidic pH of the gastric lumen.
There has been established evidence indicating astrong association of H. pylori infection with thedevelopment of chronic active gastritis and peptic disease of
the stomach and duodenum. Also, evidence showing longstanding infection to be an essential risk factor for gastriccancer is growing, while mechanisms by which H. pylori
increases the risk of cancer have been vigorously
investigated. In addition, involvement of H. pylori infectionin the pathogenesis of extragastric disorders such as chronic
urticaria, iron deficiency anemia, and idiopathic
thrombocytopenic purpura has also been suggested.
H. pylori is so ubiquitous that over half of theworld's population are considered to harbor the organism,with high prevalence in developing countries and decreasing
incidence in industrialized countries. Unlike Western nations,however, Japan has an exceptionally high H. pylori infection
rate of presumably as high as 50% of the population, though
the rate is on the steady decline. Though the exact route oftransmission is still unknown, the contagion is most likely tooccur via either the oral-to-oral or fecal-to-oral contact. This
contact includes mouth-to-mouth feeding from infectedmothers to children at weaning ages and intake of fecallypolluted water or food. In the unsanitary environment, supplyof drinking water from wells, streams, or ponds, and sanitary
insects or animal waste can also be the source of infection.
A large body of literature has linked H. pylori
infection to chronic gastritis, peptic ulcer, and gastric cancer;
these associations were mostly studied in clinical or casecontrol research design, however. In Japan, prevalence of H.
pylori infection in general populations has been previously
studied in rural areas, but no such studies were performed inurban areas, with the exception of a study that we conductedin the Metropolitan City of Nagoya. The purpose of thisstudy is to examine the association between H, pylori
infection with medical history of various gastrointestinal andliver diseases.
Materials and Methods
In 2005, a long-term cancer cohort study, the Japan
Multi-Institutional Collaborative Cohort Study (J-MICC
Study), was launched, which mainly focuses on geneenvironment interactions for cancer incidence. As one of 12affiliated institutions, the Nagoya University Graduate
School of Medicine team enrolled 5,167 voluntary residents(1,467 males and 3,700 females) aged 35 to 69 years old
between June of 2008 and May of 2010. The enrolment took
place at the Daiko Medical Center of Nagoya University (Daiko
Study), and all the participants gave written informed consent.
The participants provided urine samples for thedetection of anti-H. pylori antibody by using the commerciallyavailable antibody kit Rapiran (Otsuka Pharmaceutical Co. Ltd,.
Tokyo, Japan). Positive test results were defined as presence of
persistent H. pylori infection, and negative results as absence inthis study. The questionnaire in this study covered various
aspects of health-related lifestyles such as smoking and drinking
history, dietary habit, and physical activity. Also included in thequestionnaire was a history of H. pylori test and eradication
treatments with or without successful results. History of pastand present illness was also elicited by the questionnaire. For
statistical analyses, we selected seven self-reported diseases ofstomach ulcer, duodenal ulcer, chronic gastritis, diabetes, colon
polyp, hepatitis B, and hepatitis C, which were regarded as
outcome events. To model the association between these eventsand persistent H. pylori infection, multivariate-adjusted odds
ratios were estimated using the logistic regression method. We
conducted statistical analyses by running a free software R on
Windows 7. The study protocol was approved by the ethics
committee of the Nagoya University School of Medicine.
Results
After exclusion of those with successful previous
eradication and unknown treatment results, a total of 4,753(1,283 males and 3,470 females) subjects were available for thecurrent analyses. Profiles of the subjects were presented inTable 1.
Table 1. Characteristics of the study population
Male (n=1,283)

Female (n=3,470)

Age (years)
35-39
40-44
45-49
50-54
55-59
60-64
65-69

175
146
166
174
180
207
235

553
462
510
446
437
526
536

average±SD53.2±10.3

52.0±10.3

779
504

2,246
1,224

Results of test for H. pylori
negative
positive

The overall H. pylori infection rate of 36.4% (39.2%
for males and 35.3% for females) seems to be lower than
expected, considering the relatively high mean age of our study
population.
For both sexes, the logistic regression analysis
revealed a statistically significant association between a historyof peptic disease and H. pylori infection as determined by thepresence of anti-H. pylori antibodies; i.e., the risk forstomach/duodenal ulcer seems to be elevated in association
with H. pylori infection (Table 2). History of chronic gastritis
was also predicted by the persistent infection in both maleand female subjects. A significant negative association wasdemonstrated between H. pylori infection and diabetes
history in males, while no such significant association wasobserved in females. History of colon polyp, hepatitis B, andhepatitis C failed to show any significant relationships withpersistent H. pylori infection in both sexes.

[5]

Multi-Institutional Collaborative Cohort Study (J-MICC
Study). Nagoya J Med Sci 2011;73:187-95.
Tamura T, Morita E, Kondo T, Ueyama J, Tanaka T, Kida
Y, Hori Y, Inoue S, Tomita K, Okada R, Kawai S,
Hishida A, Naito M, Wakai K, Hamajima N. Prevalenceof Helicobacter Pylori infection measured with urinary
antibody in an urban area of Japan, 2008-2010. Nagoya J
Med Sci 2012;74:63-70.

Author address

E-Mail: hayashi.akina@f.mbox.nagoya-u.ac.jp
Table 2. Odds ratio (OR) and 95% confidence interval (95%
CI) for the association between H. pylori infection andmedical history of selected diseases
Male
Stomach ulcer
Duodenal ulcer
Peptic ulcer
Chronic gastritis
Diabetes
Colon polypHepatitis B
Hepatitis C

OR
1.43
1.75
1.67
1.36
0.56
0.93
1.26
2.86

95% CI
1.03-1.97
1.20-2.55
1.25-2.22
0.95-1.95
0.31-0.84
0.65-1.33
0.52-3.02
0.75-1.38

OR
1.29
1.54
1.30
1.36
1.29
0.84
0.60
0.97

Female95% CI
1.00-1.66
1.10-2.17
1.04-1.63
1.09-1.69
0.82-2.04
0.59-1.19
0.29-1.15
0.35-2.55

Discussion
In consistent with previous findings, our results
indicated a strong association between H. pylori infection
and peptic ulcers of the stomach and duodenum. However,
the observed inverse association between the infection anddiabetes among male subjects remains to be explored. We
hypothesized that among our male subjects, who seem to beprone to peptic disease as compared with females, capabilityof sugar absorption in their intestine might be affected,
resulting in a lowered risk for the development of diabetes.
Conclusions
The current study demonstrated that residents inurban areas of Japan are less likely to harbor H. pylori thanthose in other areas. While the association between H. pylori
infection and such diseases as peptic ulcer and chronic
gastritis were found consistent with that from previous
reports, it remains an open question whether H. pylori
infection provides protection against type 2 diabetes.

References
[1]

[2]

[3]

[4]

Marshall BJ, Warren JR. Unidentified curved bacilli on
gastric epithelium in active chronic gastritis. Lancet
1983; 1:1273-75.
Hamajima N, Goto Y, Nishio K, Tanaka D, Kawai S,
Sakakibara H, Kondo T. Helicobacter Pylori
eradication as a preventive tool against gastric cancer.
Asian Pac J Cancer Prev 2004;5:246-52.
Hamajima N, Naito M, Kondo T, Goto Y. Genetic
factors involved in the development of Helicobacter
pylori-related gastric cancer. Cancer Sci 2006;97:112938.
Morita E, Hamajima N, Hishida A, Aoyama K, Okada
R, Kawai S, Tomita K, Kuriki S, Tamura T, Naito M,
Kondo T, Ueyama J, Kimata A, Yamamoto K, Hori Y,
Hoshino J, Hamamoto R, Tsukamoto S, Onishi J,
Hagikura S, Naito H, Hibi S, Ito Y, Wakai K. Study
profile on baseline survey of Daiko Study in the Japan

Anti-HCV agent ribavirin modulates transcription elongationin clotting factor VII expression
Atsuo Suzuki1, 2), Eriko Okuyama1), Moe Murata1), Yumi Ando1), Yuki Takagi1), Io Kato1),
Akira Takagi1), Takashi Murate1), and Tetsuhito Kojima1)
1) Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine
2) Japan Society for the Promotion of Science Research Fellow

Introduction
Factor VII (FVII), a vitamin K-dependent plasma
glycoprotein, is synthesized in the liver and secreted into theblood. Once vascular injury occurrs, FVII forms a complex
with tissue factor, and works as a trigger for blood
coagulation.
In 2006, Yamamoto et al. reported that the anti-hepatitis C
virus (HCV) agent ribavirin elevated the activity of FVII,
leading to reduced doses of clotting factors used inhemostatic therapy for haemopillia patients with chronic
hepatitis C [1]. They also demonstrated increased mRNA
expression of the FVII gene (F7) by ribavirin treatment in
human hepatoma cell lines (HepG2) and in cultured humanhepatocytes; however, the detailed molecular mechanisms forthis phenomenon remains unknown.
Ribavirin is a purine nucleoside analog used in anti-HCV
therapy. The candidate mechanisms for the anti-viral actionsof ribavirin include inhibition of RNA-dependent RNA
polymerase activity in HCV or of the activity of inosinemonophosphate dehydrogenase (IMPDH) to decrease theintracellular GTP pool, and modulating the immune system[2]. Although the actions of ribavirin are partially understood,
the mechanisms are not completely clear.
The aim of our study was to further investigate themolecular mechanisms underlying increased FVII mRNA
expression by ribavirin treatment in HepG2 cells. It is
important to elucidate the molecular basis of ribavirin before
understanding its in vivo functions.
Materials and Methods
Cell culture and quantitative RT-PCR– The humanhepatoma cell line (HepG2) was purchased from theAmerican Type Culture Collection (ATCC, Manassas, VA,USA) and cultured as described previously [3]. Total RNA
was extracted from the cells using the RNeasy mini kit(QIAGEN, GmbH, Germany) and the first strand cDNA wasprepared from 1 µg of total RNA using the PrimeScript RT
reagent kit (TaKaRa, Kyoto, Japan). Quantitative RT-PCR
was performed with SYBR Premix ExTaq II (TaKaRa).
Relative mRNA expression levels were calculated as therespective mRNA/GusB mRNA.
Nuclear run-on assay and nascent RNA capturing– The
nuclear run-on assay was performed as reported previouslywith minor modifications [4]. Newly synthesized RNAtranscripts were determined by the Click-iT Nascent RNA
Capture Kit (Invitrogen) according to the manufacturer’s
protocol. HepG2 cells were cultured with or without ribavirin(100 µg/mL) for 16 h and subsequently pulsed with 0.2 mM
5-ethynyl uridine (EU) at 37 °C for 8 h. The cells were
washed and collected, and total RNA was extracted. Clickreaction was performed using 5 µg of EU-labeled RNA and
0.5 mM biotin azide; the mixture was incubated at room
temperature for 30 min. Following RNA precipitation, theRNA was dissolved in 50 µL of RNase-free water. Biotin-labeled EU-RNA-binding pull-down assay was performed using
50 µL of Dynabeads MyOne Streptavidin and the bound RNA
was washed and used as a template for reverse transcription.
The captured nascent RNA was analyzed using qRT-PCR as
described above.
RNA interference– HepG2 cells were transfected with ELL3specific siRNA (siGENOME SMARTpool siRNA, Human
ELL3; Dharmacon, Lafayette, CO, USA) or nonspecific siRNA(siGENOME Non-Targeting siRNA#3; Dharmacon) using
Lipofectamine RNAiMAX (Invitrogen) by the reverse
transfection protocol according to the manufacturer’s protocol.
Chromatin immunoprecipitation (ChIP)– ChIP assay was
performed as described previously with minor modifications [3].
The chromatin supernatants were immunoprecipitated withrespective antibodies specific to Pol II 4H8, Pol II CTD pSer2,
Pol II CTD pSer5 (Abcam), CDK9, AFF4, ELL3, andnonspecific normal rabbit IgG (Santa Cruz Biotechnology) at
4 °C for 3 h. The protein–antibody complexes were incubatedwith Dynabeads Protein G at 4 °C for 1 h. Bound DNA was
purified used as a template for subsequent quantitative PCR.
Results and Discussion
We observed that ribavirin treatment increased FVII mRNA
expression in HepG2 cells in a dose- and time-dependent
manner. Ribavirin is a purine nucleoside analog, which could beincorporated into hepatocytes via equilibrative nucleosidetransporter (ENT) [5]. We treated HepG2 cells with ENT
inhibitor NBMPR together with ribavirin, and observed
NBMPR completely abolished ribavirin-induced FVII
upregulation.
Incorporated ribavirin is metabolited to ribavirinmonophosphate (RMP) or ribavirin triphosphate (RTP). RMP is
known as a inhibitor of IMPDH, which is a rate-limiting
enzyme in a purine metabolism. It is known that inhibition of
IMPDH causes intracellular GTP depletion, and that the GTP
reduction could be reversed by supplementation with guanosine
in a salvage pathway [6]. Then, we tested other IMPDH
inhibitors, mycophenolic acid (MPA) and 6-mercaptopurine (6MP), and found that they also induced FVII upregulation in
HepG2 cells. The upregulation was canceled by addition of
guanosine. Although we could not clarified why intracellular
GTP reduction caused FVII gene upregulation, there may be aclose relationship between GTP depletion caused by sequential
inhibition of IMPDH and FVII upregulation.
Next, we investigated how FVII transcription was modulated
by ribavirn treatment in HepG2 cells. In general, control of gene
expression in eukaryotic cells involves regulatory events at
multiple transcriptional and post-transcriptional stages.
Transcription by RNA polymerase II (Pol II) is divided into fourstages: initiation, promoter clearance, elongation, andtermination. We analyzed which step of transcription was
critical for ribavirin-induced FVII mRNA upregulation. First,
we found that ribavirin treatment did not affect the FVII mRNAstability. Second, we performed luciferase-reporter analyses,which reflects promoter initiation and clearance, to determine
the F7 promoter activity, but could not observed enoughactivation by ribavirin. In contrast, ribavirin treatment
sufficiently increased nascent FVII mRNA synthesis in
HepG2 cells, as analyzed by the nuclear run-on and nascentmRNA capturing assay, which reflects activation oftranscription elongation. The F7 upregulation was abolishedby inhibiting CDK9 by DRB. CDK9 is a component of
positive-transcription elongation factor b (P-TEFb), and PTEFb is a major transcription factor which contributes thetranscription elongation event [7, 8]. These findings
demonstrated that ribavirin-induced F7 upregulation ismainly induced by acceleration of transcription elongation
associated with P-TEFb.
Further chromatin immunoprecipitation (ChIP) assays
revealed that recruitment of Pol II and CDK9, andphosphorylation of Ser2 (pSer2) of the Pol II C-terminal
domain (CTD) were reinforced by ribavirin treatment. The
levels of pSer2 of CTD could be a marker of enhanced
transcription elongation phase [7, 8]. To further investigate
the other molecules which were necessary for increased FVIImRNA expression, we searched “ribavirin-responsive genes”
by cDNA microarray analysis. We found that transcription
elongation factor ELL3 expression was significantly
upregulated in HepG2 cells treated with ribavirin, and that
the ELL3 upregulation was induced in response to
intracellular GTP depletion. In addition, we found ribavirin
induced an increase in ELL3 mRNA expression before an
increase in FVII mRNA expression in HepG2 cells, andELL3 was recruited to the F7 by ribavirin treatment.
Knockdown of ELL3 by RNAi resulted in diminished FVII
upregulation. These results suggested that transcription
elongation factor ELL3 contributed ribavirin-induced FVII
upregulation in HepG2 cells.
Taken together, in this study we found that ribavirin
increased FVII mRNA expression by acceleration of its
transcription elongation in HepG2 cells. The enhanced
transcription elongation was induced by CDK9 kinase
activity of P-TEFb and associated with ELL3. Althoughfurther study is required to determine why intracellular GTP
depletion causes accelerated F7 transcription elongation, ourfindings have contributed to elucidate the precise
mechanisms of ribavirin-induced F7 upregulation.
References[1]
Yamamoto, K., Honda, T., Matsushita, T., Kojima, T.
and Takamatsu, J. (2006) Anti-HCV agent, ribavirin,
elevates the activity of clotting factor VII in patientswith hemophilia: a possible mechanism of decreased
events of bleeding in patients with hemophilia byribavirin. J. Thromb. Haemost. 4, 469-470[2]Feld, J. J. and Hoofnagle, J. H. (2005) Mechanism ofaction of interferon and ribavirin in treatment of
hepatitis C. Nature 436, 967-972[3]Suzuki, A., Sanda, N., Miyawaki, Y., Fujimori, Y.,
Yamada, T., Takagi, A., Murate, T., Saito, H. andKojima, T. (2010) Down-regulation of PROS1 gene
expression by 17β-estradiol via estrogen receptor α(ERα)-Sp1 interaction recruiting receptor-interacting
protein 140 and the corepressor-HDAC3 complex. J.
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Yamada, M., Horiguchi, K., Umezawa, R., Hashimoto,K., Satoh, T., Ozawa, A., Shibusawa, N., Monden, T.,
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Troglitazone, a ligand of peroxisome proliferatoractivated receptor-γ, stabilizes NUCB2 (Nesfatin)mRNA by activating the ERK1/2 pathway: isolation

[5]
[6]

[7]
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and characterization of the human NUCB2 gene.
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Fukuchi, Y., Furihata, T., Hashizume, M., Iikura, M.,
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systems in human hepatocytes. J. Hepatol. 52, 486-492
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Fuda, N. J., Ardehali, M. B. and Lis, J. T. (2009)
Defining mechanisms that regulate RNA polymerase IItranscription in vivo. Nature 461, 186-192

Conflict of interest
Authors have no conflicts of interest to declare.
Author address
E-mail: suzuki.atsuo@d.mbox.nagoya-u.ac.jp

CERVICAL CONDYLOMA ACUMINATUM SHOWS MORE PROGRESSIVEHISTOPATHOLOGICAL CHANGES THAN VULVAR CONDYLOMAACUMINATUM
S. Mizuno1), K. Hashimoto1), K. Kawai1), T. Nagasaka1)
1) Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya,Japan

Introduction
Condyloma acuminatum (CA) is a common, sexuallytransmitted disease caused by Human papillomavirus (HPV).
So far, more than 100 HPV genotypes have been identified,
40 of which infect the genital tract. The genital-mucosal
HPV types are classified on the basis of oncogenicity as low(e.g. HPV6 and 11), intermediate (e.g. HPV31, 33, 35, 51, 52
and 58) and high risk (e.g. 16 and 18) [1]. High-risk HPV is
responsible for malignant tumors, especially for cervicalcarcinoma, whereas low-risk HPV leads to benign lesions.
Previous studies of HPV-associated lesions have shownthat benign and low-grade lesions mostly contain the viral
DNA as episomes. In contrast, the viral genome is integrated
into the host-cell DNA in almost all high-grade lesions andcervical carcinomas [2, 3]. These observations suggest that
integration of HPV into the human genome is one of the key
events in the progression of neoplasia. HPV DNA integration
is associated with decreased expression of HPV L1 capsid
gene and increased expression of the E6 and E7 oncogene.
The E6 and E7 oncoproteins of HPV cause inactivation of
the tumor-suppressor proteins p53 and Rb. Rb inactivation
results in the overexpression of the cyclin-dependent kinase
inhibitor p16. Immunohistochemical staining for HPV-L1
capsid protein and p16 has been used for the detection of
HPV infection. It has been reported in some studies that
L1/p16 expression pattern could be helpful for estimating thebiologic potentiality of low-grade cervical lesions [4, 5].
CA is genital warts characterized by papillomatous
proliferations of the squamous epithelium. Approximately
90% of CA is related to HPV types 6 and 11. In women, CA
occurs on the vulva, vagina, cervix and around the anus.
Vulvar CA is commonly found but cervical CA is relatively
uncommon. Although CA occurs anywhere in the genitalarea, few studies have examined female CA in light of its siteof origin. The purpose of this study was to analyze
histopathological features of cervical CA by comparison withvulvar CA. We investigated nuclear morphology, physicalstatus of HPV DNA and protein expression (L1 and p16) inCA.
Materials and methods
Sample preparation: We obtained forty-seven formalinfixed paraffin-embedded (FFPE) blocks composed of vulvar
CA case (V-CA, n=33) and cervical CA case (CX-CA, n=14).
This research was approved by the Ethics Committee of the
School of Health Sciences, Nagoya University.
PCR analysis and sample selection: Genomic DNA wasextracted from each sample using QIAamp® DNA Mini Kit(QIAGEN). Adequate DNA quality was established bysuccessful PCR amplification of human ß-globin gene. HPV
type-specific primers (HPV6 and HPV11) and pU-1M/pU2R primers to amplify high-risk HPV (HPV16, 18, 31, 33, 35,
52b, and 58) DNA were used separately. Samples in which
HPV6 or HPV11 were detected and high-risk HPV notdetected by PCR were selected for analysis in this study (V-CA
n=22, CX-CA n=12).
H＆E staining and image analysis: Each section of samples
selected by PCR results stained with hematoxylin and eosin(H&E). Ten digital images were taken from each H&E stained
slide under high power field (HPF, x40 objective) of the
microscope. The nuclear area and circularity were measured
using software ImageJ 1.45s. For each sample, the mean of a
nuclear area and circularity were calculated. In ImageJ 1.45s,
the formula for circularity is 4π (area/perimeter2). A value of 1.0
indicates a perfect circle, and as the value approaches 0.0, it
indicates an increasingly elongated shape.
DNA in situ hybridization for low-risk HPV: in situ
hybridization (ISH) was performed using GenPoint™ (DAKO)
and DNA probe cocktails for HPV6/11 (DAKO). The HPV
signal patterns in nuclei were classified as punctate, diffuse, or
mixed. A punctate pattern was considered to indicate integrated
HPV, and a diffuse pattern was considered to represent
episomal HPV. We evaluated the physical state of HPV byclassifying into five categories: I (Integrated), E (Episomal),
E<I, E=I, and I<E. These categories correspond to HPV signal
patterns by ISH: punctate, diffuse, the predominance of punctate,
the same degree, and the predominance of diffuse, respectively.
L1, p16 immunohistochemistry: Immunohistochemical
staining was performed using a L1 monoclonal antibody (clone
K1H8, DAKO) and a p16 monoclonal antibody (clone E6H4,
CINTec). L1 and p16 staining was scored 0, 1+, 2+, and 3+.
Statistics: Statistical analysis was performed using SPSS
ver.14.0-J
and
MEPHAS(http://www.gen-info.osakau.ac.jp/MEPHAS/). Mann-Whitney test and Wilcoxon's ranksum test were used to compare between the V-CA group and the
CX-CA group. Kruskal-Wallis test with post-hoc Steel-Dwass
test was applied to examine the relation between HPV DNA
physical status and L1/p16 expression pattern.
Results
Nuclear morphology: The median nuclear areas were 41.71
and 39.39 µm 2 (p=0.454, Mann-Whitney test) and nuclear
circularity values were 0.872 and 0.835 (p<0.01, Mann-Whitneytest), respectively, in the V-CA and CX-CA group.
HPV DNA physical status: LR-HPV DNA was detected in
13 (65%) of 20 specimens of the V-CA group and all specimens(n=12) of the CX-CA group. Estimated results of HPV DNA
physical status were summarized in Figure 1. Wilcoxon's ranksum test showed a significantly different distribution (p<0.01)
in the physical status were category between the two groups, VCA and CX-CA. The physical status category was divided into
2 groups which defined E and I<E as Epi (Episomal) group, E=I,
E<I and I as Int (Integrated) group. There was a significant
difference (p<0.01, Fisher’s exact test) between the two groups.
L1, p16 staining pattern: The expression of L1 was nuclear
in superficial and intermediate cells, often with features of
koilocytosis. Of the 20 specimens in the V-CA group, L1
staining score was 0 in 10 cases (50%), 1+ in 4 cases (20%), 2+
in 2 cases (10%), and 3+ in 4 cases (20%). Of the 12 specimens

in the CX-CA group, L1 staining score was 0 in 8 cases(66.67%), 1+ in 1 case (8.33%), 2+ in 1 case (8.33%), and 3+
in 2 cases (16.67%).
The expression of p16 was cytoplasmic or nuclear andcytoplasmic. Of the 20 specimens in the V-CA group, thep16 staining score was 0 in 16 cases (80%), 1+ in 4 cases(20%), 2+, and 3+ in no case. Of the 12 specimens in the
CX-CA group, p16 staining score was 0 in no case, 1+ in 2
cases (16.67%), 2+ in 5 cases (41.67%) and 3+ in 5 cases(41.67%).
As for analysis of L1/p16 expression patterns, L1
staining score was classified into two groups which defined
L1 staining score 0 as negative and 1+, 2+, 3+ as positive,
and the p16 staining score was classified in the same way.
The L1 and p16 expression, positive (+) or negative (-),
could be divided into four categories as follows: L1-/p16-,
L1+/p16-, L1+/p16+, L1-/p16+. L1/p16 expression in thatorder is shifted with increasing histological severity.
Wilcoxon's rank-sum test showed a significantly different
distribution (p<0.01) in this categories between the twogroups, V-CA and CX-CA. L1-/p16- or L1+/p16- wascommon in the V-CX, L1+/p16+ or L1-/p16+ in the CX-CA
group (Figure 2).
Relation between HPV-DNA physical status and L1/p16
expression pattern: Little information related to therelationship of between L1/p16 expression pattern and HPV
DNA physical status is available. We examined therelationship of between L1/p16 expression pattern and ISH
result (Figure 3). The physical status was classified into threegroups: ISH negative (Neg), Epi, and Int. L1/p16 expression
pattern was classified by the above-mentioned four
categories. Among Neg, Epi and Int, the distribution L1/p16
expression pattern was analyzed using Kruskal-Wallis testwith post-hoc Steel-Dwass test. There were significant
differences between the Neg and Epi, Neg and Int, Epi andIng group (p<0.01).
Discussion
Results revealed that CX-CA showed histopathologicallydifferent features from those of V-CA. Lower nuclear
circularity, integrated HPV DNA and higher expression of
p16 were seen in CX-CA compared with V-CA.
HPV DNA physical status evaluated by ISH was relevant
to L1/p16 expression pattern. L1+/p16- or L1+/p16+ pattern
was observed in the presence of episomal HPV DNA, andL1-/p16+ observed in the presence of integrated HPV DNA.
All specimens with no detectable HPV DNA by ISH showed
the L1-/p16- pattern. Non-detection of HPV DNA and loss of
L1/p16 expression were considered consistent with a lowlevel or latent HPV infection. Collectively, these findings
indicate that histological progression, L1/p16 expression
pattern and HPV DNA physical status relate to each other.
CA is a benign tumor caused by low-risk HPV infection
and considered a lower-grade lesion among HPV-associatedlesions. Our results show that V-CA findings are consistent
with features of a low-grade lesion, whereas CX-CA findings
are not. CX-CA tended to show more progressive features,such as the abnormalities in nuclear morphology and HPV
DNA integration. This suggests that the potential of
integration could depend on HPV infection site. The
differences between vulvar skin and cervical mucous
membrane are suspected to be related to the potential of HPV
DNA integration.
Conclusions
Cervical condyloma acuminatum shows moreprogressive histopathological changes than vulvar condyloma

acuminatum. Integration of HPV DNA could depend on the
HPV infection site.
References[1] Muñoz N, Bosch FX, de Sanjosé S, et al. Epidemiologic
Classification of Human Papillomavirus Types Associatedwith Cervical Cancer. N Engl J Med. 2003;348:518-527.
[2] Cooper K, Herrington CS, Stickland JE, et al. Episomal	 and integrated human papillomavirus in cervical neoplasia
shown by non-isotopic in situ hybridisation. J Clin Pathol.
1991:44(12):990–996.
[3] Lehn H, Villa LL, Marziona F, et al. Physical state andbiological activity of human papillomavirus genomes inprecancerous lesions of the female genital tract. J Gen Virol.
1988:69:187–196.
[4] Negri G, Bellisano G, Zannoni GF, Rivasi F, Kasal, A,
Vittadello F, Antoniazzi S, et al. p16 ink4a and HPV L1
immunohistochemistry is helpful for estimating the
behavior of low-grade dysplastic lesions of the cervix uteri.
Am J Surg Pathol. 2008;32(11):1715–1720.
[5] Hoshikawa S, Sano T, Yoshida T, Ito H, Oyama T, et al.
Immunohistological analysis of HPV L1 capsid protein andp16 protein in low-grade dysplastic lesions of the uterine
cervix. Pathol Res Pract. 2010;206(12):816–820.
Author address
E-Mail: mizuno.satomi@e.mbox.nagoya-u.ac.jp

Figure 1 Distribution of HPV DNA physical status

Figure 2 Distribution of L1/p16 expression patternFigure 3 Relation between HPV DNA physical status
and L1/p16 expression pattern

Parkin induces apoptotic cell death in TNF-α-treated cervical cancer cells
Yoonjung Cho1, Min Ho Lee1, Yeo Wool Kang1, Dongsup Lee1,2,
Ki-Jong Rhee1, Tae Ue Kim1, Yoon Suk Kim1
1) Department of Biomedical Laboratory Science, College of Health Science, Yonsei University, Wonju, Korea
2) Department of Clinical Laboratory Science, Hyejeon Colleage, Hongseong, Chungcheongnam-do 350-702,
Korea
Introduction
Parkin protein (parkin) was originally implicated in
Parkinson’s disease [1]. The parkin gene (PARK2) is locatedin chromosome 6q25.2-6q27, and loss of heterozygosity(LOH) in 6q26 was reported in ovarian, breast,
hepatocellular and squamous cell lung cancers [2-4]. In
addition, in a variety of cancer cells including brain, liver,colorectal, ovarian, cervical pancreas, kidney and breast
cancers, alternative transcripts were found due to the deletionand duplication in PARK2 gene [5-8]. Recent reports not
only shows suppressed Parkin expression have an essential
role in tumorigenesis but also suggest Parkin is a putative
tumor
suppresor.
OverexpressionofParkininhepatocarcinoma, glioma, lung cancer, breast cancer andcolon cancer cell lines repressed growth of the cells. In
glioblastoma and other cancers, Parkin overexpression resultsin ubiquitin mediated cyclinE degradation and subsequentcell cycle arrest. Mechanisms of Parkin for its role in tumor
suppression is partly come to light but still unclear.
We mainly used HeLa, a cervical cancer cell line, forexperiment, because HeLa is Parkin gene mutated andresistant to TNF-α. TNF-α is one of the cytokines secreted byimmune response to suppress cancer growth in the body.
However, as the cancer develops, the cancer becomes TNF-α
resistant.
Our hypothesis is that Parkin expression sensitizes naturally
resistant HeLa cells to TNF-α-induced tumor regression. This
study will suggest a role of Parkin as a tumor suppressor in
the reactions accompanied by immune reaction.

Materials and Methods
Materials

Cells (2x105/well) were seeded in 6-well culture plates,
cultured for 24 h and then infected with Parkin or Mock for an
additional 24 h. The cells were then treated with TNF-α (5ng/ml) for 24 h and the cells trypsinized, stained with trypan
blue dye solution and viable cells counted on a hemocytometer.

Western blot analysis
Cells were lysed with a PBS buffer containing 1% Triton X100 and protease inhibitor cocktail. The supernatant wascollected after centrifugation and the protein concentration
determined using the Lowry protein assay (Bio-Rad). Protein
samples were separated by SDS-PAGE, transferred to anitrocellulose membrane and the membrane incubated withprimary antibodies overnight and then with appropriate
secondary antibodies for 1 h. Bands were visualized using ECL(Thermo, Waltham, MA, USA). β-actin was used as an internal
control.

RNA extraction and reverse transcriptase PCR (RTPCR)
Total RNA was extracted from cells using Trizol. cDNA wassynthesized using 2 µg of total RNA, 0.25 µg of random
hexamers and 200 U of MMLV-RT. PCR was performed using
0.2 U of Taq polymerase (Cosmogenetech, Korea) and specificprimers. GAPDH was used as an internal control. PCR products
were electrophoresed on 1.5% (w/v) agarose gels containing
ethidium bromide and images taken using Gel Doc (Bio-Rad).

Results

Dulbecco’s modified Eagle’s medium (DMEM), fetal
bovine serum (FBS), penicillin-streptomycin, trypsin-EDTA,
and trypan blue stain solution were from Gibco BRL.
Recombinant human TNF-α was purchased from R&D
Systems. DMSO and protease inhibitor cocktail were from
Sigma-Aldrich. Propidium iodide (2.5 mg/ml) was purchasedfrom BD Biosciences. Trizol reagent, random hexamer, and
MMLV-RT were purchased from Invitrogen.

Cell culture and viral infection with parkinexpressing adenovirus
HeLa (human cervical adenocarcinoma cells, ATCC) cells
were infected with adenoviruses as described previously [9].
In brief, cells were seeded in 6-well plates at a density of 2 x
105/well. After 24 h, cells were infected with 150 multiplicity
of infection (M.O.I.) of parkin-expressing virus (Parkin) or
mock virus (Mock) for 24 h. For dose-dependent experiments,
cells were infected with 0, 19, 38, 75, and 150 M.O. of
Parkin. Various concentrations of Parkin were mixed with
Mock and normalized for a to tal of 150 M.O.I. per infection..

Trypan blue dye exclusion assay

Figure 1. Expression of parkin restores susceptibility to
TNF- α -induced cell death.

Figure 2. Caspase -8, -9, -3, -7 and Poly ADP ribose
polymerase (PARP), cleaved form of which induces
apoptosis, is cleaved by combination of Parkin expression
and TNF-alpha treated in HeLa cells.

Figure 3. Parkin reduces mRNA expression of survivinin TNF-alpha treated HeLa cells.

Conclusions
Although, parkin has been proposed to be a tumor
suppressor, the mechanisms by which parkin functions as atumor suppressor are unclear. In the current study, weinvestigated the role of parkin in TNF-α-induced apoptosis of
HeLa cells. HeLa cells are inherently resistant to TNF-αinduced cell death and also lack parkin expression. We foundthat i) parkin expression in HeLa cells restored TNF-αinduced cell death and ii) TNF-α-induced cell death was
mediated via activation of the apoptotic pathway involving
survivin, caspase-8, -9, -3,-7, and PARP.
In conclusion, we report that restoration of parkin
expression in the parkin-deficient HeLa cell line restored
susceptibility to TNF-α-induced cell death. Although further
studies are needed to determine the exact mechanisms bywhich parkin expression restores TNF-α-induced cell death,
we have identified signaling pathways that will provide aclue to understanding how parkin acts as a tumor suppressor.

References[1] Shimura, H., Schlossmacher, M. G., Hattori, N., Frosch,M. P., Trockenbacher, A., Schneider, R., Mizuno, Y.,
Kosik, K. S. and Selkoe, D. J. (2001) Ubiquitination of a
new form of alpha-synuclein by parkin from humanbrain: implications for Parkinson's disease. Science 293,263-269.
[2] Kong, F. M., Anscher, M. S., Washington, M. K., Killian,
J. K. and Jirtle, R. L. (2000) M6P/IGF2R is mutated insquamous cell carcinoma of the lung. Oncogene 19, 15721578.
[3] Oates, A. J., Schumaker, L. M., Jenkins, S. B., Pearce, A.
A., DaCosta, S. A., Arun, B. and Ellis, M. J. (1998) The
mannose 6-phosphate/insulin-like growth factor 2 receptor(M6P/IGF2R), a putative breast tumor suppressor gene.
Breast Cancer Res Treat 47, 269-281.
[4] Shridhar, V., Staub, J., Huntley, B., Cliby, W., Jenkins,
R., Pass, H. I., Hartmann, L. and Smith, D. I. (1999) A
novel region of deletion on chromosome 6q23.3 spanningless than 500 Kb in high grade invasive epithelial ovariancancer. Oncogene 18, 3913-3918.
[5] Denison, S. R., Callahan, G., Becker, N. A., Phillips, L. A.
and Smith, D. I. (2003) Characterization of FRA6E and its
potential role in autosomal recessive juvenile parkinsonism
and ovarian cancer. Genes Chromosomes Cancer 38, 40-52.
[6] Denison, S. R., Wang, F., Becker, N. A., Schule, B., Kock,
N., Phillips, L. A., Klein, C. and Smith, D. I. (2003)
Alterations in the common fragile site gene Parkin in
ovarian and other cancers. Oncogene 22, 8370-8378.
[7] Poulogiannis, G., McIntyre, R. E., Dimitriadi, M., Apps, J.
R., Wilson, C. H., Ichimura, K., Luo, F., Cantley, L. C.,
Wyllie, A. H., Adams, D. J. and Arends, M. J. (2010)
PARK2 deletions occur frequently in sporadic colorectal
cancer and accelerate adenoma development in Apc mutant
mice. Proc Natl Acad Sci U S A 107, 15145-15150.
[8] Tay, S. P., Yeo, C. W., Chai, C., Chua, P. J., Tan, H. M.,
Ang, A. X., Yip, D. L., Sung, J. X., Tan, P. H., Bay, B. H.,
Wong, S. H., Tang, C., Tan, J. M. and Lim, K. L. (2010)
Parkin enhances the expression of cyclin-dependent kinase 6
and negatively regulates the proliferation of breast cancer
cells. J Biol Chem 285, 29231-29238.
[9] Kim, Y. S., Patel, S. and Lee, S. J. (2006) Lack of direct roleof parkin in the steady-state level and aggregation of alphasynuclein and the clearance of pre-formed aggregates. Exp
Neurol 197, 538-541.

Author address
E-Mail: black5579@naver.com

Parkin induces MMP-3 expression in human cervical cancer cells
Byung Chul Jung1, Min Ho Lee1, Hyun-Kyung Kim2, Ju Yeon Lee1, In-Soo Lee3,
Ki-Jong Rhee1, and Yoon Suk Kim1,*
1

Department of Biomedical Laboratory Science, College of Health Sciences,
Yonsei University, Wonju, Gangwon-do 220-710, Republic of Korea
2
Department of Biomedical Laboratory Science, College of Natural Science, Gimcheon University, 214 Daehakro,
Gimcheon City, Gyungbuk 740-704, Republic of Korea
3
Department of Clinical Laboratory Science, Hyejeon College, Hongseong, Chungcheongnam-do, 350-702
Introduction
The parkin gene (PARK2) was first described in Parkinson’s
disease (Shimura et al., 2001). It is located on chromosome
6q25.2-6q27 which is implicated as a common fragile site
(Denison et al., 2003b). Loss of heterozygosity (LOH) on
chromosome 6q26 was reported in hepatocellular carcinoma,
ovarian, breast, lung, and squamous cell lung cancers (Konget al., 2000; Oates et al., 1998; Shridhar et al., 1999).
Furthermore, alternative transcripts of parkin gene were
found in a variety of cancers including ovarian, brain, liver,colorectal, cervical, pancreas, kidney, and breast cancer due
to deletion or duplication of the gene (Denison et al., 2003a;
Denison et al., 2003b; Poulogiannis et al., 2010; Tay et al.,2010; Veeriah et al., 2010; Wang et al., 2004). These reports
attracted interest in the role of parkin as a putative tumor
suppressor.
Matrix metalloproteases (MMP) are zinc-dependent
endopeptidases that can degrade various components of theextracellular matrix thereby influencing cell proliferation,
cell death, cell differentiation, cell migration, and cell–cellinteractions (Van Lint and Libert, 2007). MMPs are
zymogens which become enzymatically active upon after
proteolytic cleavage. Active MMP-3 degrades various
collagens (types II, III, IV, IX, X), proteoglycans, fibronectin,
laminin, and elastin (Murphy and Nagase, 2008). In addition,
MMP-3 can also activate other MMPs such as MMP-1,
MMP-7, and MMP-9 (Ye et al., 1996). MMP-3 is involved inwound repair, progression of atherosclerosis, and tumor
initiation (Murphy and Nagase, 2008). In general, MMPs are
secreted proteins and exert its function in the extracellularenvironment. However, recent data suggest that MMP-3 canalso act intracellularly. It has been reported that endoplasmic
reticulum stress results in activation of caspase-12 which inturn activates intracellular MMP-3. Activated MMP-3 in turn
directly activates caspase-3 which results in apoptosis (Choiet al., 2008; Kim et al., 2010).
In this study, we investigated the role of MMP-3 during
TNF-α-mediated apoptosis in parkin expressing HeLa cells.
We report that parkin induces expression of MMP-3 andinhibition of MMP-3 activity alleviates TNF-α-induced cell
death in parkin expressing cells.

Materials and Methods
1. Materials
Recombinant adenoviral vector including parkin gene
(Parkin virus) was produced as previously described by Kim
et al. (Kim et al., 2006). Recombinant human TNF-α waspurchased from R&D System (Minneapolis, USA). Ro 317549, PD 98059, Ly 294002, SB 203580, GW 5074, JNK
inhibitor II, and N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycylhydroxamic Acid (NNGH) were purchased from
Calbiochem (Darmstadt, Germany). BAY 11-7085 waspurchased from Enzo life science (New York, USA). U73122
was purchased from Cayman (Michigan, USA).

2. Cell lines and cell culture
HeLa (Human cervical adenocarcinoma cells, ATCC) was
grown in Dulbecco’s modified Eagle’s medium (DMEM)(Gibco BRL, Carlsbad, USA) supplemented with 10% fetal
bovine serum (FBS) (Gibco BRL, Carlsbad, USA) andstreptomycin-penicillin (Gibco BRL, Carlsbad, USA). Cells
were maintained at 37℃ in humidified atmosphere with 5%
CO2.
3. Parkin gene expression
HeLa cells (2 x 105) were seeded into each well of a 6-wellplate and 24 h later infected with different concentrations ofParkin virus and Mock virus in serum-free DMEM. After an
additional 90 min 10% FBS-DMEM was added to each well. In
Parkin virus dose-dependent experiments, cells were infectedwith Parkin virus at different multiplicity of infection (M.O.I.)(0,25, 50, 100, 200). To compensate for the effect of the viral
vector Mock virus was added with the Parkin virus to maintainequal concentration of 200 M.O.I. A non-infected group wasadded as a negative control.
4. Trypan blue dye exclusion assay
The cell culture medium was removed and treated withtrypsin-EDTA (Gibco BRL, Carlsbad, USA) for 2 min. Cells
were dislodged and an equal volume of cell suspension mixed
with 0.4% trypan blue stain solution (Gibco BRL, Carlsbad,USA). Viable cells were enumerated using a hematocytometer(Marienfeld, Lauda-Königshofen, Germany).
5. RT-PCR (Reverse transcriptase - polymerase chain
reaction)
Total RNA was extracted from cultured cells using Trizol
reagent (Invitrogen, New York, USA) according to the
manufacturer’s instructions. cDNA was synthesized by reverse
transcription with 2 µg total RNA, 0.25 µg of random hexamer(Invitrogen, New York, USA) and 200 U of Moloney Murine
Leukemia Virus-Reverse Transcriptase (MMLV-RT; Invitrogen,
New York, USA) for 50 min at 37℃ and 15 min at 70℃.
Subsequent PCR amplification using 0.2 U of Taq polymerase(Cosmogenetech, Seoul, Korea) was performed in athermocycler using specific primers. The sequences of the PCRprimers are as follows: 5’-ggc cag gga tta atg gag at-3’(forward) and 5’-gct gac agc atc aaa gga ca-3’ (reverse) for
MMP-3 and 5’-cgg gaa gct tgt cat caa tgg-3’ (forward) and 5’ggc agt gat ggc atg gac tg-3’ (reverse) for GAPDH. PCR
products were electrophoresed on 1.5% agarose gels, stained for
10 min with ethidium bromide and destained for 20 min.
Product size were determined by comparing to a 100 bp DNA
ladder marker (Bioprince, Georgia, USA). The intensity of eachband was quantitated using a Gel Doc (Bio-Rad, California,
USA).

Results

Figure 1. Parkin expression restores susceptibility to
TNF-α-induced death in HeLa cells.
HeLa cells were infected with either Parkin virus or Mockvirus (200 M.O.I.) for 24 h and then treated with TNF-α (5ng/ml) for 24 h. Viable cells were enumerated by trypan blue
dye exclusion assay. The number of viable cells in the noninfected group was set as 100 %. Data are from three
independent experiments.

Figure 2. Parkin induces prolonged expression of MMP-3
in TNF-α-treated HeLa cells.

2009). Caspases play a central role in the transduction of TNFα-induced apoptotic signals. The initiator caspases, caspase-8(mitochondria independent) and caspase-9 (mitochondria
dependent) and the effector caspases, caspase-3 and caspase-7
are crucial signaling molecules involved in this apoptotic
pathway (Fesik and Shi, 2001). A recent study reported that
MMP-3 activates caspase-3 leading to neuronal apoptosis (Choiet al., 2008). Therefore, in the current study we investigated theexpression of MMP-3 during TNF-α-induced cell death inparkin expressing HeLa cells. In this study, we showed that
expression of parkin in HeLa cells induces a slight increase in
MMP-3 expression which increases further when treated with
TNF−α (Fig. 2A). Our results imply parkin affects upregulation of MMP-3 in TNF-α−treated HeLa cells. In addition,
we found that a positive correlation between increase in MMP-3
activity and TNF-α-induced cell death in parkin-expressing
HeLa cells (Fig. 3B, C).
Interestingly, we found a dramatic but short-lived increase in
MMP-3 expression when HeLa cells were infected with the
Mock virus and then treated with TNF-α (Fig. 2B). One
possibility is that infection with the adenovirus itself issufficient to induce a transient increase in MMP-3 expression.
We are unaware of any reports to this effect. However, a recent
report showed that introduction of Poly (I:C), a synthetic analog
of viral double-stranded RNA, into corneal fibroblasts
upregulated MMP-3 (Kimura et al., 2010). The adenovirus
vector that we used in this study is a double-stranded DNA
virus (Philipson et al., 1975). It remains to be seen whether
dsDNA is sufficient to induce MMP-3 expression.
Moreover, the interaction between parkin and MMP-3 withother apoptotic proteins remains to be determined. Further
studies seem to be needed to elucidate mechanism(s) by which
MMP-3 plays a role in Parkin+TNF-α-induced cell death and it
would be interesting to determine if other MMPs are also
regulated by Parkin.

References

Figure 3. Parkin expression-induced reduction of cell
viability in TNF-α-stimulated HeLa cells was partially
recovered by MMP-3 specific inhibitor.

Discussion
Many studies suggest a tumor suppressive role of Parkin
but the mechanism by which it acts is still unclear. In thisstudy, we investigated the implication of MMP-3 in parkininduced TNF-α-treated HeLa cell death. We found that i)parkin induces expression of MMP-3, and ii) inhibition of
MMP-3 activity alleviates Parkin+TNF-α-treated HeLa cell
death.
TNF-α is a potent activator of apoptosis and a defect in theapoptotic pathway contributes to uncontrolled cellproliferation resulting in cancer development (Balkwill,

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Induction of the IL-2-independent growth by KRAS G12A mutation
N. Mizutani1), T.Murate1)
1) Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan
Introduction
MOTN-1 and PLT-2 are human T-cell large granularlymphocyte (T-LGL) leukemia cell lines derived from thesame patient at defferent disease stage.1),2) MOTN-1 wasestablished at the indolent and chronic stage, and is IL-2
dependent, whereas PLT-2 was established from theaggresssive and termnal stage and is IL-2 independent.
Karyotype analysis of the two cell lines showed that PLT-2
cells retain the basic abnormalities observed in MOTN-1
cells. In the present study, we analyzed the signaltransduction pathway of these two cell lines with or without
IL-2 treatment, and found that the constitutively active ERK
pathway is important for the survival of PLT-2. We detected
KRAS mutation in parallel with the increased Ras activity of
PLT-2 but not MOTN-1 cells.
Materials and Methods
Cells and reagents
MOTN-1 and PLT-2 cells were derived from Kochi
University School of Medicine. MOTN-1 cells were cultured
in 10% FCS and IL-2 (100 U/ml) in RPMI1640. IL-15 waspurchased from Peprotech (Rocky Hill, NJ, USA). Cellsignaling inhibitors: SB203580, was purchased from Wako
(Osaka, Japan), JAK Inhibitor I was from Merck chemicals(Darmstadt, Germany), LY294002, PD90859, and U0126
were from Cell Signaling Technology (Beverly, MA, USA)
respectively. NIH3T3 cells and its transfectants were
cultured in 5% FCS in DMEM.
Cell viability
Viable cell number was counted by the trypan blue dye
exclusion method.
Western blotting
Anti-Bcl-2 antibody, anti-Bcl-XL antibody (BD Transduction
Laboratories, Franklin Lakes, NJ, USA), anti-p15 antibody,anti-p27 antibody, anti-cyclin D3 antibody, anti-p-STAT3
antibody, anti-cyclin dependent kinase (CDK) 4 and 6
antibodies (Cell Signaling), anti-STAT3 antibody, antiSTAT5 antibody, anti-p-STAT5 antibody (Sigma), anti-Akt
antibody, anti-p-Akt antibody, anti-ERK 1/2 antibody, antip-ERK 1/2 antibody (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), and anti-β-actin antibody (Bio vision, Milpitas,
CA, USA).
Ras activity assay
Ras activity was measured using an Active Ras Pull-Down
and Detection kit (Thermo Scientific, Billerica, MA, USA)according to the manufacturer’s instructions.
MAPK/ERK pathway activation
PathDetect Elk1 trans-reporting system (Agilent
Technologies, La Jolla, CA, USA) was used to determine theactivated state of the MAPK/ERK pathway according to the
manufacturer’s manual.
RAS and PTPN11 mutation study
N, K and HRAS and PTPN11 mutations were analyzed by
RT-PCR followed by direct sequencing. Reported hot spotsof RAS (corresponding to the amino acid 12, 13 and 61) weresurveyed. The whole coding sequence of PTPN11 wasanalyzed.
Transfection and colony formation of NIH3T3
NIH3T3 cells were transfected with pcDNA 3.1 expression
vectors containing wild-type KRAS, KRAS G12A and KRAS
G12V, respectively. After G418 selection, suitable subclones
were selected by determining respective phosphorylated
ERK level. In order to directly demonstrate the growthadvantage of NIH3T3 cells transfected with mutated KRAS,
we performed the colony formation assay. Throughpreliminary experiments, the suitable initial cell density for
plating and the culture period were determined. Briefly,

stably transfected-NIH3T3 cells were placed in a tissue culture
plate (35 mm diameter) in triplicate. After 10 days, tissue
culture plates were stained with May-Giemsa staining dye.
Colony number was counted under the inverted microscope.
KRAS Eexpression vectors and transfection for MOTN-1
KRAS wild type, KRAS G12V, and KRAS G12A were inserted
into the suitable multi-cloning site of Original MSCV-GFP
vector. After obtaining culture supernatant containing
retroviruses, MOTN-1 cells were transfected with culture
supernatant containing MSCV-GFP-KRAS (wild, G12A, andG12V) or MSCV-GFP (as the mock vector). Cells were sortedby FACS according to their GFP fluorescent intensity. Strongly
positive cells were collected and used for further analysis.
Results
Difference of cytokine dependence between MOTN-1 and PLT-2
IL-2 and IL-15 receptors are composed of α, β and γ subunits,
and share β and γ chains. Consistently, MOTN-1 proliferated
with either IL-2 or IL-15 but stopped proliferation by depletingIL-2 or IL-15. However, PLT-2 did not require either of thesecytokines for proliferation.
Status of signaling pathway and Ras analysis
Examination of apoptosis- and cell cycle-related proteins
revealed that p15 and p27 were increased after depletion of IL-2
in MOTN-1, whereas cyclin D3 and CDK6 did not show
significant changes by IL-2 depletion. In MOTN-1, activated
STAT3 and STAT5 were observed in IL-2 (+) cells. STAT
activation was also observed in IL-2 (+) but not in IL-2 (-) PLT2, suggesting that JAK2/STAT pathway was not involved in its
IL-2 independent growth.
However, activated ERK pathway was observed in PLT-2 cells
as compared with that of MOTN-1. AKT signaling did not show
significant change between MOTN-1 and PLT-2 regardless ofcytokine treatment. Increased Ras activity of PLT-2 parallels
ERK activation compared with MOTN-1. Direct sequence of N,
K and HRAS revealed KRAS G12A mutation in PLT-2 but not in
MOTN-1. Our preliminary analysis using allele-specific PCR
showed that this mutation was only present in a population of
the original leukemia cell DNA in the aggressive phase.
In Noonan syndrome and JMML, mutations of PTPN11 that
encode SHP2, an SH-2 domain containing tyrosine phosphatase,have been reported, and its mutation leads to the enhanced
Ras/MEK signaling.3) However, PTPN11 mutation was notdetected in PLT-2. Moreover, MEK inhibitor, PD98059 andU0126, and farnesylation inhibitor FTI277, inhibited cell
growth of PLT-2 cells. PI3K inhibitor, LY294002, but not p38
inhibitor, SB203580 and JAK inhibitor I, showed someinhibition. These results demonstrate the importance ofRas/MAPK signaling pathway of PLT-2 cell proliferation.
Overexpression of KRAS mutattions
In order to examine the role of KRAS G12A found in PLT-2,
we transfected various KRAS expression vectors into NIH3T3
and MOTN-1 cells. The effect of KRAS G12V, a KRAS
common mutation observed in various cancer cells, was also
analyzed. High ERK activation was observed in KRAS G12A
and G12V transfectants of NIH3T3 as compared with mockand wild-type KRAS expressed cells, respectively. Enhanced
Ras activity of KRAS G12A and G12V leading to activation of
ERK pathway was also demonstrated using Elk-1 transreporting system. Colony formation of respective KRAS
transfected-NIH3T3, suggesting the significantly enhanced
colony formation of KRAS G12A and G12V but not of wildtype KRAS NIH3T3 .
ERK activation by mutated KRAS was also observed in KRAS
retrovirus-transfected MOTN-1. High Ras activity was observedin mutated KRAS-transfectants. KRAS G12V and KRAS G12A
exhibited higher proliferation in the absence of IL-2 andresistance against both serum and IL-2 depletion during ourobservation period.

Discussion
LGL leukemia features a clonal expansion of antigenprimed, competent, cytotoxic T lymphocytes. IL-15, IL-2 andPDGF are included in the recent network model of survival
signaling of this leukemia.4) IL-2 and IL-15 share their β andγ chains, and IL-2 and IL-15 α receptor subunits are coexpressed in T cells.5) Our results indicate that the
proliferation signals of MOTN-1 utilized these common
chains.
Comparison between MOTN-1 and PLT-2 cultured with orwithout IL-2 revealed increased levels of cell cycle
regulators, p27 and p15, in MOTN-1 by IL-2 depletion,
which is consistent with the growth arrest of MOTN-1.
However, we could not detect the change of cyclins, CDK,
and apoptosis-related proteins of MOTN-1 under our
experimental conditions. Apoptosis related proteins (Bcl-2,
Bcl-xL, Bax and Bad) also showed no significant changes
after IL-2 depletion. These data might be related with the factthat IL-2-depleted MOTN-1 cells were relatively resistant toapoptosis during the short-term observation period.
Considering a similar TCR rearrangement pattern between
MOTN-1 and PLT-2 and shared chromosome abnormalities,6,7)
PLT-2 was thought to be derived from MOTN-1 or at least
maintain the basic cellular characteristics. Our analysis
revealed that IL-2-independent growth of PLT-2 was at least
partially due to the constitutive activation of the MAPK/ERK
pathway but not the activated AKT and STAT pathway.
MEK inhibitor or farnesylation inhibitor inhibited cell
growth of PLT-2 cells. The coupling of ERK and activatedRas has been reported previously in lymphoproliferative
disease of granular lymphocyte.6)
Enhanced Ras activity of PLT-2 was thought to be due to
KRAS G12A mutation, because no HRAS, NRAS or PTPN11
mutation was observed. The possibility of autocrine
production of IL-2 or IL-15 was less likely, because culture
medium of PLT-2 did not sustain cell proliferation of
MOTN-1. Ras pathway is important for survival,
proliferation, senescence and differentiation. Ras activation
causes Raf recruitment and activation by phosphorylation,
followed by activation of the MEK and ERK sequentially.
Mutation of Ras family (HRas, NRas and KRas) induced
abnormal cellular signaling, deregulation of gene expression
and oncogenensis.7) Oncogenic KRAS mutation initiates
leukemia in hematopoietic stem cells.8)
Among three KRAS mutation hot spots, KRAS codon 12
mutation induces higher resistance to apoptosis andpredisposition to anchorage-independent growth than codon
13 mutation.9) KRAS G12A is relatively rare in clinicalcancer samples and cell lines,10) and the transformation
activity of KRAS G12A remains to be disclosed.11) Our
results showed increased Ras activity and enhanced ERK
signaling by KRAS G12A was clearly shown usingtransfected NIH3T3 cells and MOTN-1 cells. The analysis ofrelapsed childhood acute lymphoblastic leukemia revealed
that KRAS mutation was observed only in relapsed patients.12)
Such patients with KRAS mutation profit from MEK
inhibitors.13) RAS mutation analysis of CMML patientssuggests that RAS mutation is a secondary event that
contributes to the expansion of s malignant clone withproliferative advantage.14) PLT-2 is assumed to be frompatient leukemia cells in the aggressive phase, which aretransformed from cells in the chronic phase. Our preliminary
results using allele-specific PCR showed that this mutation
was present in the patient blast of the aggressive phase but
not occurred during the in vitro culture process. Thus, KRAS
mutation of this patient is a late event during his LGL
leukemia progression.
The frequency of KRAS mutation in LGL leukemia
remained to be determined previously. The mechanism of thecytokine-independent growth of LGL clinical samples has
not been disclosed fully. Thus, the clinical significance of ourobservation is not clear enough at the moment and the future
analysis of LGL leukemia samples is warranted. Recently,
the comprehensive dynamical and structural analysis of a
network model of this disease has revealed several key
components important for the leukemogenesis and the valueof importance of Ras as the network component in T-LGL
leukemia has been shown.4,15)
In the present study, KRAS G12V as well G12A induced
ERK1 and ERK2 activation and anchorage-independent growth
in NIH3T3 cells and the growth advantage of mutated KRAStransfected MOTN-1 cells in IL-2 depleted medium was clearlyshown. However, KRAS G12A mutation has been reported in
IL-2-dependent KHYG-1 cell lines.16) Ras-overexpressed factordependent FDC-P1 cells exhibit resistance to apoptotic cell
death by cytokine depletion,17) and RAS mutation induces
increased anti-apoptosis in some models.18) Thus, RAS mutationis necessary but not always sufficient for cytokine-independent
growth. Other factors such as c-myc cannot be ruled out toconfer IL-2 independent growth to PLT-2 cells.
Taken together, we found that KRAS G12A mutation isimportant for IL-2- independent proliferation, which occurred
during disease progression of the patient from whom these twocell lines were established.
Reference
1) Gilliland DG. Semin Hematol, 2002; 39: 6-11.
2) Matsuo Y. Leuk Res, 2002; 26: 873-879.
3) Chan G. Cancer Metastasis Rev, 2008; 27: 179-192.
4) Zhang R. Proc Natl Acad Sci USA, 2008; 105: 16308-16313.
5) Vamosi G. Proc Natl Acad Sci USA, 2004; 101: 1108211087.
6) Epling-Burnette PK. Oncogene, 2004; 23: 9220-9229.
7) Ulku AS. Cancer Treat Res, 2003; 115: 189-208.
8) Zhang J. Blood, 2009; 113: 1304-1314.
9) Guerrero S. Cancer Res, 2000; 60: 6750-6756.
10) Deschoolmeester V. Br J Cancer, 2010; 103: 1627-1636.
11) Seeburg PH. Nature, 1984; 312: 71-75.
12) Davidsson J. Leukemia, 2010; 24: 924-931.
13) Kiessling MK. Blood, 2011; 117: 2433-2440.
14) Ricci C. Clin Cancer Res, 2010; 16: 2246-2256.
15) Saadatpour A. PLoS Comput Biol, 2011; 7: e1002267.
16) Choi YL. Leuk Res, 2005; 29: 943-949.
17) McGlynn AP. Leuk Res, 2000; 24: 47-54.
18) Shinjyo T. Tohoku J Exp Med, 2008; 216: 25-34.
Author address

E-Mail:mizuani.naoki@e.mbox.nagoya-u.ac.jp

Changes of Matrix Metalloproteinases (MMPs) Expression in Triglyceride TreatedMacrophages.
Jaewon Lim1, Yoonjung Cho1, Byung Chul Jung1, Hyun-Kyung Kim1, 2, Yeo Wool Kang1,
Ki-Jong Rhee1, Tae Ue Kim1 and Yoon Suk Kim1
1

Department of Biomedical Laboratory Science, College of Health Sciences, Yonsei University, Wonju 220-710, Korea
Department of Biomedical Laboratory Science, College of Natural Science, Gimcheon University, Gimcheon 740-704, Korea

2Introduction
Triglyceride (TG) is known to be associated with
inflammatory disease including atherosclerosis. Recently, it
has been reported that triglyceride is an independent risk
factor in atherogenesis. TG has been proposed to induce cell
death of macrophage or foam cell, resulting in increased
inflammatory reactions in plagues. Moreover, it has beenreported that matrix metalloproteinases (MMPs) have an
important role in a variety of inflammatory reactions. MMPs
are zinc-dependent endopeptidases which digest extracellularmatrix (ECM) in normal processes. In addition, dysregulationof MMPs leads to destruction of connective tissue andvarious pathological inflammatory conditions. Depending on
their substrate specificity and domain structure, MMPs are
divided into six subclasses including collagenases,
gelatinases, stromelyisins, matrilysins, membrane-type
MMPs and others. Under physiological conditions,expression of MMPs is tightly regulated on mRNA level by avariety of growth factors, cytokines, chemokines and contact
to the ECM. In this study, changes in expression of MMPs
were investigated during monocyte differentiation into
macrophages, and when the macrophages ingest TG after
differentiation.

Total RNA was extracted from cultured THP-1
cells and PMA-differentiated THP-1 cells by using TRIzol
reagent according to the manufacturer’s instructions. cDNA
was synthesized by reverse transcription with 2 µg total RNA,
0.25 µg of random hexamer (Invitrogen) and 200 unit of
Murine Molony Leukemia Virus Reverse Transcriptase(MMLV-RT; Invitrogen) for 10min at 25°C, 50min at 37°C
and 15min at 70°C. Diluted reverse-transcribed cDNA wasused for subsequent 20 µl PCR amplification. PCR
amplification using Prime Taq premix PCR kit (Genet Bio,
Chungnam, Korea) was performed in a thermocycler(Applied Biosystems, New York, NY, USA) for 25 ~ 40cycles using MMPs primers for MMP-1, MMP-8, MMP-3,
MMP-10, MMP-12, MMP-19, MMP-2, MMP-9 and MMP-7.
The levels of gene expression were normalized against GAPDH. PCR products were electrophoresed on 2.0% (w/v)
agarose gels containing 0.5 µg/ml ethidium bromide (Et-Br),and the sizes of the products determined by comparison to
100 bp DNA ladder marker (Intron, Gyeonggi, Korea). Gelimages were taken using Gel-Doc (Bio-Rad, Hercules, CA,
USA). The intensity of each band amplified by RT-PCR was
analyzed using Image Lab (version 2.0, Bio-Rad) andnormalized to GAP-DH in corresponding samples.

Materials and Methods

Results

Inhibitors
Phorbol 12-myristate 13-acetate (PMA) was
purchased from Sigma-Aldrich (St. Louis, MO, USA). TG
was provided by B. Braun Melsungen AG (Melsungen,Germany). TRIzol for RNA isolation were obtained from
Invitrogen (Carlsbad, CA, USA). Specific inhibitors ofclassical PKC (RO-31-7549), MEK1 (PD 98059), PI3-K (Ly294002), p38 MAPK (SB 203580) and cRAF-1 kinase (GW5074) were purchased from Calbiochem (Darmstadt,
Germany). Specific inhibitor of NF-κB (BAY 11-7085) was
purchased from Enzo life science (New York, NY, USA).
Specific inhibitor of PLC (U 73122) was purchased from
Cayman (Ann Arbor, MI, USA). Dimethyl sulfoxide(DMSO) was obtained from Sigma-Aldrich (St. Louis, MO,
USA).

Increased expression of MMPs during PMA induced
differentiation of THP-1 cells
PMA, which promotes differentiation of monocytes,
is known to stimulate secretion of MMP-1, -3, -7, -9, -12 and-14 in macrophages. Almost all MMPs showed increased
expression during differentiation of monocyte into
macrophage except for MMP-2 (Fig 1). However, theinitiation of expression was different for each MMP.

Cell culture and treatments
The human monocytoid THP-1 leukemia cell line
was cultured in RPMI medium 1640 supplemented with 2
mM L-glutamine, 100 U/ml penicillin, 100 µg/ml
streptomycin and 10% fetal bovine serum (FBS) at 37℃
under 5% CO2. THP-1 cells were seeded at a density of 1 ×
106 cells/well in 6-well plates and treated with the indicatedconcentrations of PMA to induce differentiation into
macrophages. Differentiated macrophages were incubatedwith the indicated concentrations of TG.
mRNA extraction and semi-quantitativetranscriptase PCR (RT-PCR)

reverseFig 1. Time-dependent expression of MMPs during PMA
induced differentiation of THP-1 cells.

PKC is not involved in PMA-induced increase of MMPs
expression
The THP-1 cell lines were treated with PMA which
is PKC activator to induce differentiation into macrophages.
To elucidate the mechanism by which PMA treatment affects
expression of MMPs, we examined the PKC signaling
pathway associated with PMA-stimulated induction of MMPs
(Fig 2). Interestingly, expression pattern of MMPs was notchanged by RO-31-7549 (an inhibitor of classic PKC). The
results showed that PKC was not associated with PMAdependent increased mRNA expression of MMPs.

TG induced reduction of MMPs expression in PMA
differentiated THP-1 macrophage cells
To investigate the modulation of MMPs expression
by accumulation of TG in differentiated THP-1 cells, THP-1
cells were treated with 200 nM PMA for 48 h to induce
differentiation into macrophages and then treated with
1mg/ml TG for the last 24 h. TG treatment of differentiatedmacrophages resulted in inhibition of expression of most
MMPs except for MMP-1, -10, and -2. The most dramatic
decreased expression was observed for MMP-8, -12 and -9.
However, expression of MMP-2 increased slightly. The
results showed that TG was decreased expression of most
MMPs in differentiated THP-1 cells.

Discussion

Fig 2. MEK1-mediated up-regulation of MMPs during
PMA-induced differentiation of THP-1 cells.
PMA-dependent increased mRNA expression of MMPs
was mediated by MEK-1
To elucidate the mechanism by which PMA
treatment affects expression of MMPs, we examined thevarious signaling pathway molecules associated with PMAstimulated induction of MMPs. Treatment with GW 5074 (aninhibitor of cRAF-1 kinase) or U 73122 (an inhibitor of PLC)
did not influence PMA-mediated expression of MMPs. In
contrast, treatment with PD 98059 (an inhibitor of MEK-1)
almost completely blocked PMA-mediated expression of
MMPs except for MMP-2. PD 98059 had slight negative
effects on PMA-mediated expression of MMP-19, -9 and -7(Fig 2). These results suggested that the signal transduction
molecule, MEK-1, was involved in the increased expressionof MMPs during macrophage differentiation.

Fig 3. TG induced reduction of MMPs expression in PMA
differentiated THP-1 macrophage cells.

MMPs was known to be expressed only in low
level in peripheral blood monocytes. When monocytes
contact with the ECM, expression level of MMPs is activatedand extracellular secretion of MMPs is increased. Thesephenomenon increased degradation of ECM, therefore play aimportant role of cell migration in tissue. In this study, we
showed that expression level of most MMPs increased during
differentiation of monocyte into macrophage, except for
MMP-2. These results suggest that increased expression levelof MMPs during macrophage differentiation helps
differentiated monocyte invasion in tissue of inflammatorysite. In addition, MEK-1 inhibtor decreased the rising of
expression level of MMPs. These results showed that thesignal transduction molecule, MEK-1, mediated expressionof MMPs during macrophage differentiation. Therefore,
These results suggest that developments of selective
inhibitory drugs of MEK-1 would be helpful in effect of
reduce inflammation response by cell migration ofmacrophage. TG treatment in differentiated THP-1 cell
reduced expression of several MMPs. Independent of MMPs
subtypes, TG accumulation changed expression level of
MMPs in differentiated THP-1 cells. In particular, expression
level of MMP-8, -12 and -9 was dramatically decreased.
These results show that TG accumulation in differentiatedmacrophage reduced expression of MMPs. One of thesuggestion is that reduction of MMPs maybe leads topartially blocked cell migration of macrophage ininflammatory site and associated weakening of inflammation
response. Another suggestion is that these reponses increased
plaque formation and inflammation response by decreased
escaping of TG uptaked macrophage in atherosclerotic
regions. However, more study should be investigated toprove these hypotheses. These results provide basic
information for studying MMPs expression during
macrophage differentiation and foundation for studies of roleof MMP in triglyceride mediated inflammatory diseases.
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and macrophages and its relationship to atherosclerotic
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