1. 中文摘要
當細胞接受一些刺激因子，例如生長因子、賀爾蒙等等的刺激後，會經由
細胞內的訊息傳遞系統來調控基因的表現。在本研究計畫中，共分有三個分項
計畫：第一個分項計劃，是針對基因表現所需的轉錄調控因子，探討它們之間
的交互作用。我們發現 Sp1，C/EBPβ以及δ可以共同活化 interleukin-10 基因的
啟動區。在 p27Kip1 的基因調控方面，Sp1 也可以與維他命 D 的接受器交互作用。
此外，我們也證實 Sp1 可與 C/EBP 的家族蛋白共同參與 Mrp3 的基因調控。第
二個分項計劃，是針對發生在台灣的四種主要癌症，探討其新的訊息傳遞機制。
在膀胱癌的研究方面，由我們的結果顯示，經由 MSP 以及 RON 的訊息傳遞路
徑，可能是導致膀胱癌的一個重要成因。經 sodium butyrate 處理過後的結膓癌細
胞，可使其 FAK 以及 Src 的表現量降低，這可能歸因於 butyrate 可減低細胞的
增生。在肺癌的研究方面，在非小細胞型肺癌細胞中，我們得知 Stat-3 是屬於本
質上已呈活化狀，而 p53 的突變是與 IL-6 的分泌有關。在肝癌的研究方面，我
們證實大量表現 pre-S 的突變體可使細胞生長週期所需的基因表現增加而使細胞
克服休止期而進入正常的生長週期。第三個分項計畫，是針對病態生物學，來
探討導致抗細胞凋亡之新的訊息傳遞機制。在這一年中，我們已有一些斬獲，
包括 Fas 以及 Fas-L 之間的交互作用，caspase3 以及 p21WAF1 之間的結交作用，
藉由調降 FAK 之表現來阻抗 lithium 所引發之 ceramide 之作用。
在南台灣建立了核心研究設備是輔助本計劃成功的基礎。過去一年中，我
們已購置一些大型儀器，包括質譜分光計、共焦顯微鏡、螢光及化學冷光分析
儀，並把生物晶片與轉殖技術安置於核心研究設備中。結構生物學核心的品質
已獲提升並開始運作。
1. Executive Summary
Gene expression is regulated through intracellular signal transduction upon the
stimulation of growth factors, hormones and other stimulants. There are three
research subprojects in this project. In Sub-Project (I), we focused on the functional
interaction of transcription factors in gene expression. We found that Sp1 and C/EBP
β and δ cooperatively activate the promoter activity of interleukin-10 gene. Sp1
may also interact with vitamin D receptor in the gene regulation of p27Kip1. We also
demonstrated that Sp1 and C/EBP families may be cooperatively involved in gene
regulation of Mrp3. In Sub-Project (II), we focused on the novel mechanisms of
signal transduction of four major cancers in Taiwan. In the study of bladder cancer,
our results indicate that the signaling pathways mediated by MSP and RON may play
an important role in the formation of bladder cancer. Exposure of colon cancer cells
to sodium butyrate decreased the expression of both FAK and Src that might be
attributable to butyrate-reduced cellular proliferation. In the studies of lung cancer,
we focused that Stat3 is constitutively activated in non-small cell lung cancer
(NSCLC) cells, and secretion of IL6 is correlated with p53 mutation. In the studies of
hepatoma, we demonstrated that overexpression of pre-S mutant can overcome the
cell cycle arrest through up-regulation of gene expression in regulating cell cycle
progression. In Sub-Project (III), we focused on the novel signal transduction
mechanism that mediate anti-apoptotic effects in patho-biology. In the past year, we
made some progress in studies of signaling mechanisms regarding to interactions of
Fas/Fas-L, association of caspase3/p21WAF1, downregulation of FAK and
counteractions of lithium to ceramide.
The establishment of core laboratory facilities in Southern Taiwan is essential for
the success of the project. In the past year, we have deployed several large
instruments such as mass spectrometers, confocal microscope, fluorescense and
chemiluminescence analyzers together with biochips and transgenic technologies in
our core facilities. Structural Biology core was upgraded and started doing service
already.
2. General Description
The normal cellular function is under a sophisticated regulation network, so
called “signal transduction”, to support the integrity of the system. When cellular
growth control is abnormal, for example, the cell continuously grows until a tumor is
formed which may damage the neighboring tissue and cause the organism to die. In
addition, when a cell should go to apoptosis but does not, its presence may block the
function of the neighboring cells and the whole tissue. Thus, to continuously perform
normal cellular function, a cell needs to be cooperatively regulated by thousands of
signal transduction processes within itself. Furthermore, the signal transmission is
dynamic and cross-talk may occur within the cell. Therefore, it is also necessary for
scientists to work with cross-talk in the research field of signal transduction. We
proposed this project to integrate into a single research team all the intelligent
scientists working in this field in southern Taiwan. So far, our team has been
involved extensively in signal transduction and gene regulation research and has
provided major contributions to the field. Among them only two of the more
significant discoveries will be mentioned here. First, in the study of how c-Jun and
Sp1 work cooperatively in the activation of 12(S)-lipoxygenase expression, we
discovered a novel function of Sp1 as a carrier to bring the transcription factor c-Jun
to the GC-rich box-containing gene promoter. This is amongst the first few
discoveries of such a novel transcriptional factor function. Second, in the studies of
HBV-related hepato-carcinogenesis, we found that the mutated pre-S proteins of the
hepatitis B viral surface antigen are commonly present in liver tissues of chronic
hepatitis B viral infection, and the pre-S mutants may result in the down-regulation of
small HbsAg in endoplasmic reticulum (ER) resulting in ER stress. Through
intimate contact and intergration in this project, we will contribute to address, at the
molecular level, the tumorigenesis of the most important cancers in Taiwan. Also,
we will be able to provide knowledge about the regulation of transcriptional factors in
mediating gene expression and signal transduction in growth and apoptosis control.
We divided this proposal, into three sub-proposals; (I) functional interaction of
transcription factors in gene expression;
(II) novel mechanisms of signal
transduction of four cancers prevalent in Taiwan; and (III) studies of signal
transduction mechanisms that contribute to tumor cell survival.
3. Objectives
Specifically, our aims, which were actualized by three subprojects, were to:
1) Elucidate functional interactions of transcription factors in gene expression
regulation;
2) Study novel signal transduction mechanisms in four important cancers
prevalent in Taiwan; and
3) Elucidate novel signal transduction mechanisms that contribute to tumor
resistance.
4. Interface and Integration between Overall and Sub-Projects
The study of cellular signaling pathways and gene regulation is our main shaft in
this project. Instead of looking at
individual signaling pathways
(single dimensional studies), we
conducted our studies from a
multi-dimentional
prospective.
Through the study of “new
mechanism”, in search of “new
genes”, hopefully we will discover
“new functions”. In order to
improve the research infrastructure
in the NCKU medical research
center and form a technical
support base for the whole project, we established six core laboratories in Overall
project. They are (1) Mass Spectrometry (2) Microscopic Facility (3) Inducible
Gene Expression (4) Functional Genomics (5) Structural Biology and (6) Trangenic
Mice. The interface between Overall and Sub-Projects is indicated in the scheme.
5. Project
Dr. W.C.Chang is responsible for the project management.
In order to achieve
our goals, the following strategies were reinforced.
1) Integration ： There were frequent, informal intra-subproject interactions
among the PIs. A formal progress report meeting for each subprojects was
held once every 2~3 months. And the progress report for the whole project
was held once every 5~6 months. Through the informal and formal meetings,
we discussed about the technical help, insight sharing and discussion on
possible relationship with their own projects.
2) Quality control：In order to guarantee success and minimize unnecessary
waste of efforts, we have invited four distinguished scientists, three from
abroad and one local scientist to form an External Advisor Committee to
oversee our research progress annually. They will be responsible for critical
appraisal of our research directions and results, and give important
recommendations. The External Advisor Committee meeting of the first year
project is scheduled to be held on Feb. 15-16, 2003.
6. Describe in detail the approaches and methodologies to implement the
research works
CORE FACILITY Ⅰ: Proteomic Research Core Laboratory (PRCoL)
(Responsible Investigator: Pao-Chi Liao)
Objective:
To provide the following services:
(1) Protein identification (ID)
(2) Characterization of protein posttranslational modification (PTM)
(3) Protein MW measurement/confirmation
(4) Training courses for 2-D gel electrophoresis
Instrumentation:
Applied Biosystems DE-PRO MALDI-TOF mass spectrometer
Finnigan LCQ liquid chromatography-mass spectrometer (LC-MS)
Applied Biosystems QSTAR LC-MS with o-MALDI (funded by NSC)
Five sets 2-D gel electrophoresis (one set with multiple-gel capability)
Work completed in 2002 and plans for 2003
(1) Three mass spectrometers and 2-D gel electrophoresis sets have been acquired and
installed in 2002.
(2) Three full time operators have been employed for the operation of the core
laboratory in 2002 (two funded by MOE and one by NSC). Personnel training is
(3)
(4)
(5)
(6)
undergoing.
The core lab has provided five 2-D gel electrophoresis training courses in 2002.
“Protein MW measurement/confirmation” service has been available to users on
campus since December 1, 2002.
“Protein identification (ID)” service will be available in February of 2003.
“Characterization of protein posttranslational modification (PTM)” service will be
available by July of 2003.
CORE FACILITY II: Phosphoimage/Time-lapse video microscopy
(Responsible Investigator: Tzeng-Horng Leu)
Objective:
The main purpose of this core is to provide instrumentation support of (1)
phosphoimage analysis and (2) time-lapse video microscopy for researchers in the
MOE Program for Promoting Academic Excellence of Universities.
Facilities and Equipments:
(1) Phosphoimage analysis:
An FLA-5000 imaging system (Fujifilm) and a LAS-1000plus system (Fujifilm)
are purchased for analyzing images of radioisotope and fluorescent respectively.
The machines were set up in Dec. 2002 and are providing service now.
(2) Time-lapse video microscopy:
Leica AS MDW system is purchased and set up for live cell imaging. “All
components like camera, shutters, piezo z-positoner and monochromator are fully
integrated and optimized for light efficiency and acquisition speed” in this system.
Even fast cell dynamics can be recorded in 4D. This instrument will provide
recording of intracellular proteins/organelle translocation as well as long-time
observation of cellular movement. At this stage, the whole system, except CO2
incubator, is already set up and functioning.
The Core insures that these instruments are maintained in good working condition.
CORE FACILITY III: Multiple inducible gene expression cell model laboratory
(Responsible Investigator: Hsiao-Sheng Liu)
Objective:
The objectives of this core facility are to develop novel inducible systems and to
assist PIs in each subproject to utilize these systems to regulate the genes of interest.
Facility:
GenePulser XcellTM (BioRad) is an electroporator, which is extremely powerful for
DNA, RNA and protein delivery.
Accomplishments and service:
Because no personnel and budget are allocated to this core laboratory, therefore the
missions of this core laboratory are to make the plasmids of various inducible systems.
In addition, it functions as consultant center to help each laboratory to develop their
inducible systems. In this core facility, the following inducible systems are available
for use: 1. the lactose repressor system (Lac system), 2. the insect hormone
ecdysone-dependent expression system (Ecd system), 3. the tetracycline-dependent
expression system (Tet system). Furthermore, a Cre/lox P system has been used to
construct Tet inducible system. This system further simplifies the procedure of
cloning.
CORE FACILITY Ⅳ: DNA Microarray (Responsible Investigator: Li-Wha Wu)
Objectives:
There are several missions for this core facility. First, the core lab will provide all
the necessary equipment and facilities to assist all the PIs in the MOE program in
addition to those PI who also would like to use the microarray technology as their
research tools. Second, the coordinator will not only involve in setting up the
training courses from customized chip to sample preparation followed by
hybridization and washes of chips, but also adopt a super user list through which more
interested personnel will be trained to assist others. Third, the core lab will routinely
hold workshops to assist trainees on how to use the analysis software so that the data
obtained in each subproject can be transformed from meaningless signals into
meaningful data. Fourth, all the microarray data will be then integrated through
establishing databases using a server computer and maintenance program. As a
result, each principle investigator involved in this MOE will share their data with one
another and, if possible, the interested principle investigator can utilize these data for
further data mining.
Facilities:
Two PCR machines for high through amplification of cDNA fragments
Deep freezer for all the cDNA clone collection
Microarryer 06 for making customized chips on glass slides
Semi-automated hybridization and washing platform
Axon 4000B with scanning software (GenePix Pro 4.0)
Microarray analysis software (Two yearly licenses of Spotfire and one free yearly
license of GeneSpring 4.2)
One server computer with Oracle Database
Accomplishments and future direction:
Since all the proposed equipment have been purchased and set up, customized cDNA
chips spotted with 350 genes of interest have been produced through close
collaboration with the EGENOMIX company. The complete gene list of the
customized chip and all the updated information introducing this core facility have
been posted on the website (http://140.116.58.119/biochip/index.htm). Several PIs in
the MOE program are now in the phase of using these chips for their studies.
Furthermore, we have also routinely held workshops on the know-how of these
analysis softwares. As a part of MOE program, we will continue to achieve our
missions proposed in the objective. The ultimate goal of this core facility is to assist
all the PIs in achieving their excellence in the field of signal transduction and function
genomics. Ultimately, the overall research environment in the NCKU medical center
will be also accordingly upgraded.
CORE FACILITY Ⅴ: Structural Biology Core
A: Lab for NMR and Protein Expression
(Responsible Investigator: Woei-Jer Chuang)
Objective:
The main purpose of this core is to provide instrumentation support and service to
MOE investigators. The aims of this structural core lab are to:
(1) determine the 3D structures of proteins involving this project;
(2) produce large quantities of proteins for NMR studies; and
(3) model protein structure and analyze protein.
Facilities and Equipments:
Server: Sun Fire 6800
Workstation: SGI Octane Fuel
Softwares: SRS7, EMBOSS ,Artemis, InsightII, Xplor, CNS, Seqfold, Homology,
Modeller, Consensus, and Autodock
Fermentors: NBS Celligen (protein production in CHO cell)
NBS Bioflo 101 (mass protein production in Pichia pastoris)
NBS Bioflo 101 (mass protein production in E. coli)
The Core insures that these instruments are maintained in good working condition.
2002 Workplan Results
A number of important results have been achieved in the past year.
(1) The facility and equipments have been installed and the services were provided
starting from November, 2002.
(2) Two papers were published by using this core facility.
Liu et al. (2002) “Solution Structure of the DNA-Binding Domain of Interleukin
Enhancer Binding Factor 1 (FOXK1a) “, Proteins, 49, 543-553.
Chuang et al. (2002) “1H, 15N and 13C Resonance Assignments for the DNA-Binding
Domain of Myocyte Nuclear Factor (Foxk1)”, J. of Biomolecular NMR, 24, 75-76.
Future Outlook
This work will continue from the activities that have taken place in 2002 in the
respective working groups. This core provides instruction on the proper set up and use
of the equipment and will advise on the best procedures to accomplish the goal of
MOE.
B: Laboratory of Combinatorial Chemistry and Peptides Synthesis
(Responsible Investigator: Wai-Ming Kan)
Objective:
The main purpose of this core is to provide instrumentation support and service to
MOE investigators. The aims of this structural core lab are to:
(1) Visualization of peptides and small molecules by ab initio methods
(2) Peptide and Phosphopeptide Synthesis
(3) Combinatorial Peptide/Small Molecule Libraries Preparation—for the
identification of substrate selectivity, kinase recognition sequences, and enzyme
inhibitors or receptor ligands.
Facilities and Equipments:
Organic Synthesizers:
(1) EYELA solid phase organic synthesizer CCS-1200V
(2) EYELA ChemiStation Model PPW-2000
Workstation: SGI O2+
Softwares: Cerius2 C2•Visualizer; C2•DMol3 Interface; C2•DMol3-Molecular;
C2•Dynamics; C2•Minimizer; C2•MOPAC Interface and MOPAC
program; C2•Open Force Field
The Core insures that these instruments are maintained in good working condition.
2002 Workplan Results
The equipments were purchased and installed in Dec, 2002. Preliminary test runs
were successful. The Core insures that these instruments are maintained in good
working condition.
Future Outlook
A
pentadecamer
combinatorial
library
containing
M-A-X-X-X-X-Y-X-X-X-X-A-K-K-K for tyrosine kinase was planned for trial
synthesis in Febrary 2003. Custom combinatorial library synthesis and other
services will be available in April 2003.
CORE FACILITY Ⅵ: Core Laboratory for Construction of Transgenic Mice
(Responsible Investigator: Chao-Liang Wu)
Objective:
The main purpose of this core is to provide technical consultation and service to MOE
investigators. The aims of this structural core lab are:
(1) To generate transgenic mice with conventional pronuclear injection.
Tissue-specific transgene expression in the resulting mice is controlled by the
promoter of the transgene itself.
(2) To generate transgenic mice in an inducible expression manner.
(3) To provide embryo fibroblast cells with tetracycline or ecdysone inducible
modules.
(4) To construct gene targeting embryonic stem cells and mice.
Facilities and Equipments:
2 micromanipulators: one supported by NHRI and the other by MOE.
Several isoventilation cabinets: one supported by this project.
2002 Workplan Results
A transgenic animal facility has been set up in the Laboratory Animal Center, NCKU.
This facility already has the capability to serve not only the users from this program
project but also the scientific communities in Taiwan.
Our services include:
(1) Pronuclear microinjection for the generation of transgenic mice.
(2) Electroporation of targeting vectors into ES cells.
(3) Microinjection of ES cells to generate ES cell-derived chimerae.
(4) Cryopreservation and rederivation of murine embryos.
A detailed user manual has been posted at a web site, www.ncku.edu.tw/~animal/.
Future Outlook
This core facility will continue its work for consultation and service to accomplish the
goals of MOE.
Sub-Project ( Ⅰ ) Functional interaction of transcription factors in gene
expression (Principal Investigator: Wen-Chang Chang)
The ability of the core promoter to respond to activators and direct high levels of
transcription is dependent on the cooperative interaction between the transcription
factors. In this year, we focused our studies on the functional role of Sp1 and its
interaction with other transcription factors in the transcriptional regulation of cellular
genes including interleukin-10, p27Kip1 and multidrug-resistant protein 3. We found
that Sp1 cooperatively regulates the gene regulation of IL-10 with C/EBPβ and δ
in mouse macrophages. In our studies, we also provided new insight of a novel
molecular mechanism by which steroid hormones control the expression of
downstream target genes. We demonstrated that vitamin D receptor (VDR) may
interact with Sp1 and this complex may bind directly to DNA oligonucleotide
containing Sp1 consensus sequence. In the regulation of rat Mrp3 promoter, our
results suggested that Sp1 and C/EBP families may be cooperatively involved. The
underlying mechanism on the interaction of Sp1 with other transcription factors (e.g.
C/EBP and VDR) will be our next focus in this Sub-project.
I-1: Functional cooperation of Sp1 and C/EBPβand δ in
induced gene activation of interleukin-10 in
(Wen-Chang Chang)
BACKGROUND
lipopolysaccharidemouse macrophages
Interleukin-10 (IL-10) is one of induced cytokines following LPS stimulation. In
mouse macrophages, the Sp1 binding site residing at –89 to –78 bp of IL-10 promoter
was reported to be essential for its basal and LPS-induced expression (1 , 2). Although
Sp1 site is essential for activation of IL-10 gene, DNA binding activity of Sp1 is not
changed after LPS treatment. The immutability of Sp1-DNA binding activity is
insufficient to explain the mechanism of gene activation in mouse macrophages. In
this study, we further delineated the mechanism of promoter activation of IL-10
induced by LPS in mouse macrophages.
METHODS
Murine IL-10 promoter region (-553/+64 bp) was prepared by PCR amplification
of Raw 264.7 cell genomic DNA with specific primers. The DNA fragments were
inserted into a luciferase plasmid pGL3-Basic to form a reporter plasmid. The
mutated DNA fragments were also ligated into pGL3-Basic. Cells were tranfected
with plasmids by lipofection using Superfect. In the studies of SL2 cells, plasmids
were transfected by using calcium phosphate. Binding of nuclear transcription factors
to DNA promoter was analyzed by gel shift assay. And interaction between Sp1 and
C/EBP was analyzed by immunoprecipitation using anti-Sp1 antibodies-agarose
conjugate.
RESULTS
We identified that, in addition to Sp1, C/EBPβ and δ were also involved in
LPS-induced gene expression of IL-10. By transient transfection with 5’-deletion
mutants of IL-10 promoter, we found that there were two LPS-responsive elements in
promoter of mouse IL-10 gene. Analysis of these two regions by gel shift assay
suggested that Sp1 and C/EBP β and δ were found to these two regions
respectively. By site-directed mutagenesis, we found that disruption at both Sp1 and
C/EBP binding sites almost completely blocked the LPS response. By gel shift assay
and Western blotting, we found that the DNA binding complex and protein expression
of C/EBPβ and δ were increased by LPS treatment, but these results were not
found in Sp1. Overexpression of C/EBPβor C/EBPδ respectively activated the
promoter of IL-10 gene, and they were enhanced by LPS. Coimmunoprecipitation
experiments in intact cells indicated that LPS stimulated interaction between Sp1 and
C/EBPβ and δ induced by LPS cooperatively activated expression of IL-10 gene.
In the Sp1-deficient Drosophila Schneider SL2 cell system, we further confirmed the
functional cooperation of Sp1 and C/EBPβ and δ in the regulation of IL-10 gene
promoter.
DISCUSSION
In the present study, we conclude that transcription factors Sp1, C/EBPβ and
δ were all required for LPS-induced gene expression of IL-10 in mouse
macrophages. The increase of C/EBPβ and δ proteins by LPS treatment at least in
part explains the enhancement of IL-10 gene expression. In the overexpression of
C/EBPδ in RAW264.7 cells, LPS could enhance the luciferase activity of IL-10
promoter by about three folds, but there was just a little increase in overexpression of
C/EBP. These results indicate that both C/EBPβ and δ proteins might be modified
by LPS treatment to increase its transactivation activity on IL-10 gene promoter, and
C/EBPδ has a more important role upon LPS treatment. One of the interesting
discoveries of this research is the identification of protein-protein interaction between
Sp1 and C/EBPβ and δ protein in intact cells. By using coimmunoprecipitation
method, we found that Sp1 protein could interact with C/EBPβ and δ protein.
This is the first evidence to identify the physical interaction between Sp1 and C/EBP
β and δ, and LPS enhances these binding cernplexes. It is still not clear that
whether the increased physical interaction between Sp1 and C/EBP proteins stems
purely from the presence of more C/EBP proteins following LPS treatment or also
from changes in either Sp1 or C/EBP that allow stronger interaction. It has been
reported that transcription factors C/EBP family and Sp1 were identified to be
required, and cooperatively activate the promoter activity of several genes, but the
physical interaction between Sp1 and C/EBP protein by coimmunoprecipitation was
not proven. In the previous study of CYP2D5, Lee et al. (3) also found that glutamineand serine/threonine-rich domains of Sp1 are required for cooperating with C/EBPβ
in the presence of DNA. It is therefore interesting to study the direct interaction
between Sp1 and C/EBPβ and δ in our system.
REFERENCES
1. Brightbill, H. D., S. E. Plevy, R. L. Modlin, and S. T. Smale. (2000) A prominent
role for Sp1 during lipopolysaccharide-mediated induction of IL-10 promoter in
macrophages. J. Immunol. 164, 1940.
2.
3.
Tone, M., M. J. Powell, Y. Tone, and S. A. J. Thompson, and H. Waldmann.
(2000) IL-10 gene expression is controlled by the transcription factors Sp1 and
Sp3. j. Immunol. 165, 286.
Lee, Y. H., S. C. Williams, M. Baer, E. Sterneck, F.J. Gonzalez, and P.F. Johnson.
(1997) The ability of C/EBPβ but not C/EBPα to synergize with an Sp1
protein is specified by the leucine zipper and activation domain. Mol. Cell. Biol.
17, 2038.
I-2: Molecular mechanism of interaction between vitamin D receptor and Sp1 in
gene regulation (Wen-Chun Hung and Lea-Yea Chuang)
BACKGROUND
Recent studies demonstrate that steroid hormones may activate the expression
target genes in which the promoter regions lack steroid receptor response elements. It
is possible that steroid hormone/receptor complexes, instead of directly binding to
DNA, may interact with transcription factors to stimulate gene expression (1-3). In
this study, we examined the interaction between vitamin D receptor (VDR) and Sp1
transcription factor and studied the VDR/Sp1 complex in the control of p27Kip1
expression. Our study may explore a new molecular mechanism by which steroid
hormones regulate gene regulation under various physiological or pathological
circumstances.
METHODS
We used co-immunprecipitation and GST-pull down assays to study the
interaction between VDR and Sp1 in cell lines. To explore the binding domains
involved in this interaction, we constructed various VDR and Sp1 deletion mutants
and studied the binding between these mutants both in vitro and in cell lines. We also
investigated whether the VDR/Sp1 complex may bind to the promoter of p27Kip1 by
using double strand DNA oligonucleotide corresponding to the sequence –544 to -512
b.p. in p27Kip1 promoter which contains only Sp1 consensus sequence.
RESULTS
Our co-immunoprecipitation assays demonstrated that VDR indeed interacted
with Sp1 in cells. GST-pull down study also confirmed that VDR might physically
bind with Sp1 in vitro. We also investigated the binding domains of VDR and Sp1 by
using various deletion mutants. Our data suggested that the N-terminal (a.a. 1-283)
and C-terminal (a.a. 611-778) of Sp1 are needed for the binding of Sp1 to VDR. In
addition, we found that the interaction between VDR and Sp1 was significantly
enhanced after vitamin D3 stimulation. By using biotin-labeled double strand DNA
probe, we found that the VDR/Sp1 complex might bind to the oligonucleotide probe
contained only two Sp1 binding sites. Moreover, the binding activity was also
enhanced by vitamin D3. Taken together, these data support the notion that VDR and
Sp1 may interact with each other in vitro and in cells and the VDR/Sp1 complex may
bind to DNA via the Sp1 consensus sequence to regulate gene expression.
DISCUSSION
The classical idea of the genomic action of steroid hormone is that steroid
hormone may interact with its intracellular cognate receptor to activate the expression
of target genes in which the promoter regions contain hormone receptor response
element that can be bound by the hormone/receptor complex (4, 5). Conversely, a
gene will not be considered as a downstream target for steroid hormone if it lacks the
hormone receptor response element in its promoter. This study provides new insight
of a novel molecular mechanism by which steroid hormones control the expression of
downstream target genes. We demonstrate that VDR may interact with transcription
factor Sp1 and this complex may bind directly to DNA oligonucleotide containing
Sp1 consensus sequence. Therefore, results of our study will revise the idea of the
genomic action of steroid hormone that has been widely accepted for a long time.
VDR
VDR VDR
VDRE
Classical steroid signaling
SP1
Nonclassical steroid signaling
REFERENCES
1. Safe S. (2001) Transcriptional activation of genes by 17 beta-estradiol through
estrogen receptor-Sp1 interactions. Vitam. Horm. 62, 231-252.
2. Qin C, Nguyen T, Stewart J, Samudio I, Burghardt R, and Safe S. (2002) Estrogen
up-regulation of p53 gene expression in MCF-7 breast cancer cells is mediated by
calmodulin kinase-IV-dependent activation of a nuclear factor kappa B/CCAAT
-binding transcription factor-1 complex. Mol. Endocrinol. 16, 1793- 1809.
3. Inoue T, Kamiyama J, and Sakai T. (1999) Sp1 and NF-Y synergistically mediate
the effect of vitamin D3 in the p27Kip1 gene promoter that lacks vitamin D
response elements. J. Biol. Chem. 274, 32309-32317.
4. Khorasanizadeh S, and Rastinejad F. (2001) Nuclear-receptor interactions on
DNA- response elements. Trends Biochem. Sci. 26, 384-390.
5. Lemon BD, and Freedman LPA. (1999) Nuclear receptor cofactors as chromatin
remodelers. Curr. Opin. Cenet. Dev. 9, 499-504.
I-3: Transcriptional regulation of rat multidrug-resistant protein 3 (Mrp3)
(Jin-ding Huang)
BACKGROUND
MRP3, which transports bile salts, is localized in the basolaterol membrane of
hepatocytes and enterocytes. MRP3 may play a role in the enterohepatic circulation
of bile salts. In rats receiving common bile duct ligation, estrogen treatment, and
administratin of endotoxin as acquired cholestasis models, canalicular Mrp2 was
down-regulated by transcriptional and post-transcriptional mechanisms, and also
basolateral Mrp3 was induced as common bile duct ligation continues (1-3). It was
reported that MRP3 was up-regulated in a patient with Dubin-Johnson syndrome, an
autosomal recessive disease, is characterized by a MRP2 defect (4). MRP3 thus
appears to compensate for the impaired function of MRP2 in the liver and respond to
bile salts at the transcriptional levels.
METHODS
First, we investigated if Mrp3 gene expression can be detected in rat liver (H4IIE)
and intestine (IEC-18) cells, which served as target cells for transfection experiments.
Then, we cloned and analyzed the 5’-flanking region of rat Mrp3 gene. To
characterize the functional regions of the rat Mrp3 promoter involved in gene
expression, various deletions of the Mrp3 promoter were fused with the luciferase
reporter gene. Then to identify the region regulating basal promoter activity of the
rat Mrp3 gene, a series of deletion constructs were transiently transfected into H4IIE
and IEC-18 cells. Site-directed mutagenesis analysis and electrophoresis mobility
shift assay (EMSA) were used to examine the specific transcription factors binding.
In an alternative approach, we constructed the Sp1 variants to study the interaction
domain or functional group of Sp1, which interacts with other transcriptional factors.
The variants will be used in the future studies.
RESULTS
Multidrug resistance protein 3 (MRP3) is inducible under conditions of
extrahepatic cholestasis or MRP2 deficiency. Using a series of deletion mutants in
rat hepatoma and intestine cell line identified a basal transcription element
at –157/–106 bp, two negative response regions at –2723/–1128 and –530/–443 bp,
respectively, and a positive response region at –1128/–943. Functional analysis of
site-directed mutagenesis constructs demonstrated the region comprising Sp1 (3) or
Sp1 (4) is required for basal promoter activity. Gel mobility shift assays and DNA
supershift experiments suggest that the complexes formed with nuclear extracts
contain the Sp1 and/or Sp3 proteins.
Co-transfection of Mrp3 promoter (pWT-157)
constructs with Sp1 or Sp3 into Drosophila Schneider line 2 cells that lack Sp1/Sp3
could activate the Mrp3 promoter with dose dependence. Comparison of rat Mrp3
promoter activity between in H4IIE and in IEC-18 (intestine) cells, there are different
positive and negative response elements. It was indicated that other tissue specific
factors might contribute to this process. Further deletion, site-directed mutagenesis
and supershift assays also shows that C/EBPα and β may be involved in Mrp3 gene
regulation. These data suggest that Sp1 and C/EBP families may be cooperatively
involved in the regulation of rat Mrp3 promoter. In addition to promoter analyses,
fifteen Sp1 variants have been constructed. They are: S49A, T258A, S339A, T348A,
S353A, T419A, T419E, S439A, W457S, T476E, S503A, R590E, S713D, and S721A.
The variants are aiming the potential phosphorylation sites.
DISCUSSION
To date, three other Sp1-related proteins, Sp2, Sp3, and Sp4 have been identified.
Sp3, like Sp1, is expressed in various cells. Sp3 can serve not only as a
transcriptional activator but also can serve a repressor of Sp1-mediated transcription
depending on cellular and promoter contexts (5). The details of how these
transcription factors are involved in the Mrp3 gene activity of H4IIE and IEC-18 cells
may help to explain the different expression of Mrp3 in liver and intestine.
Moreover, how the interactions between Sp1, Sp3 and other transcription factors, like
CBF/NF-Y, FTF, nuclear receptor (PXR, FXR, and CAR), AP-1, and p53, provide
specificity in MRP family gene systems will be particularly interesting. Further
studies are required to elucidate these ideas.
REFERENCES
1. Lee, J., and Boyer, J. L. (2000) Molecular alterations in hepatocyte transport
mechanisms in acquired cholestatic liver disorders. Semin, Liver Dis. 20, 373-384.
2. Inokuchi, A., Hinoshita, E., Iwamoto, Y., Kohno, K., Kuwano, M., and Uchiumi, T.
(2001) Enhanced expression of the human multidrug resistance protein 3 by bile
salt in human enterocytes. J. Biol. Chem. 276, 46822-46829.
3. Ortiz, D. F., Li, S., Lyer, R., Zhang, X., Novikoff, P., and Arias, I. M. (1999)
MRP3, a new ATP-binding cassette protein localized to the canalicular domain of
the hepatocyte. Am. J. Physiol. 276, G1493-G1500.
4. Konig, J., Rost, D., Cui, Y. H., and Keppler, D. (1999) Characterization of the
human multidrug resistance protein isoform MRP3 localized to the basolateral
hepatocyte membrane. Hepatology 29, 1156-1163.
5. Suske, G. (1999) The Sp-family of transcritption factors. Gene 238, 291-300.
Sub-Project (II) Novel mechanisms of signal transduction of four major cancers
in Taiwan (Principal Investigators: Ih-Jen Su and Tzeng-Horng Leu)
There are several interesting observations in the first year. In the study of
bladder cancer, Drs. Chow and Liu have found that RON receptors are highly
overexpressed in the primary bladder tumors (60.7%). In addition, the macrophage
stimulating protein (MSP), one of the RON’s ligands, appeared in the urine of all the
tested bladder cancer patients (n=8). Thus the signaling pathways mediated by MSP
and RON may play an important role in the formation of bladder cancer. Studies
from Dr. Leu’s laboratory have indicated that the expression level of FAK and Src in
colorectal tumors is not only increased, but also in a parallel manner. Interestingly,
exposure of colon cancer cells to sodium butyrate, a histone deacetlylase inhibitor,
decreases the expression of both FAK and Src that might be attributable to
butyrate-reduced cellular proliferation. Since sodium butyrate is one of the
fermentation products derived from ingested dietary fibers in the intestines, this study
may provide a mechanism of cancer prevention mediated by dietary fibers. Studies
from the laboratories of Drs. Su and Lai have found that Stat3 is constitutively
activated in non-small cell lung cancer (NSCLC) cells. This is due to the autocrine
secretion of IL6 in these cells. Furthermore, the secretion of IL6 is correlated with
p53 mutation of tested NSCLC cells. Since Stat3 activation may have implication of
generation of malignant pleural effusion (MPE), tumor cell proliferation, and
resistance to chemotherapeutic drugs, the observation of p53 muation and IL6
production in the involvement of Stat3 activation may have clinical significance in the
treatment of MPE of NSLCL. Finally, Dr. Su have found that overexpression of
pre-S mutant can overcome the cell cycle arrest through up-regulation of gene
expression in regulating cell cycle progression (i.e. cyclin A, cyclin D1, CDK, and
PCNA). Further studies indicate that overexpression of pr-S mutant leads to
ER-stress and activation of GRP-78/79, Pi-ERK and JNK, which may participate in
the elevation of expression of genes as described above.
II-1:The novel mechanisms of tumor stroma and cellular oncogenes in
modulation of bladder carcinogenesis (Nan-Haw Chow and Hsiao-Sheng
Liu)
BACKGROUND
It is well known that extracellular matrix (ECM) of the stroma influences to a
great extent of the proliferation, differentiation, and morphogenesis of normal
epithelial cells, as well as the biological properties of carcinoma cells in vitro. The
aberration of ECM contents and its interaction with epithelial cells thus may play a
role in the epithelial carcinogenesis. As far as the ECM-derived growth factors are
concerned, scatter factor/hepatocyte growth factor (HGF), basic fibroblast growth
factor and vascular endothelial growth factors have been suggested to be the
examples in vivo [Willett et al., 1998]. We have demonstrated the importance of
HGF/c-met pathway in the progression of human bladder cancer (Cheng et al., 2002).
This study was designed to clarify the significance of macrophage stimulating protein
(MSP) and its cognate receptor RON signaling pathway, another member of c-met
receptor family, in bladder carcinogenesis.
METHODS
Several strategies were undertaken to test the potential relevance of MSP/RON
pathway in human bladder cancer. Immunoprecipitation of MSP was first performed
on human urine from 8 cases of bladder cancer and one case of xanthogranulomatous
nephritis. Then RT-PCR was screened for RNA expression of MSP in 10 uroepithelial
cell lines (E6, RT4, TSGH8301, TCCSUP, J82, T24, UB09, UB47, UB40, and UB37).
The expression of RON receptor was also assessed in uroepithelial cell lines by
western blotting. Since mutation of extracellular or kinase domain of RON was
reported in gastric and colon cancer cell lines, SSCP screening together with mutation
analysis of suspicious exon was carried out from exon 1 through exon 20. The
potential clinical relevance of receptor protein was estimated by
immunohistochemistry on a total of 56 primary tumors.
RESULTS
Immunoprecipitation found positive MSP expression in all bladder cancer urine
samples (n = 8), while the one case of xanthogranulomatous nephritis did not reveal
any detectable amount of MSP in the urine. RT-PCR analysis showed that MSP RNA
could be detected in E6, RT4, TCCSUP, J82, T24 and UB37 cells (5/10 cell lines
tested). As for RON receptor expression, western blot demonstrated the mature form
(140 kDa) in E6, RT4, TSGH8301, TCCSUP, and UB37 cells, with the highest level
observed in UB37 cells. The RON precursor (180 kDa) was found only in four cancer
cell lines, i.e. RT4, TSGH8301, TCCSUP, and UB37 cells. Interestingly, both UB47
and UB 40 cells revealed aberrant transcript of RON (165 kDa) reported to carry
constitutively active tyrosine phosphorylation of receptor in gastric and colonic cell
lines [Collesi et al., 1996; Chen et al., 2000]. Sequence analysis confirmed that both
UB47 and UB 40 cells contain 146 bp deletions (RON) at extracellular region
(2678 – 2824, lacking 49 amino acids). Immunohistochemistry reveal positive RON
expression in 34 of 56 primary bladder tumors (60.7%). There positive association of
receptor expression with non-papillary cancer (p = 0.0025) and multiple tumor (p =
0.05).
DISCUSSION
The current investigation supports that MSP/RON signaling pathway may play an
important role in the progression of human bladder cancer through autocrine/paracrine
mechanism. As a result, the signaling molecules could serve as potential targets for
cancer therapy in the future (Chow et al., 2003). The hypothesis, however, needs
additional experiments in support for its clinical implication. For this reason, we are
going to clarify the prognostic significance of RON expression in clinical cohort, the
signaling pathways of RON compared with wild-typed RON, and functional
relevance of RON in vivo. To deal with these objectives, we have made a lot of
efforts to construct RON plasmid, tyrosine phosphorylation assessment on
wild-typed and RONs (UB09, UB40 & UB37), the transfection of RON-negative
cell lines (J82 & UB09), and the RNAi experiments on high-RON cell lines (UB09,
UB40 & UB37). In the long run, microarray screening of inducible RON transfectant
effects and the animal study in vivo will be performed to elucidate the molecular
mechanisms of gene activation, and to confirm the clinical significance of
RON-targeting therapy in human bladder cancer.
REFERENCES
1. Willett, C. G., Wang, M. H., Emanuel, R. L., Graham, S. A., Smith, D. I., Shridhar,
V., Sugarbaker, D. J., and Sunday, M. E. (1998) Macrophage-stimulating protein
and its receptor in non-small-cell lung tumors: induction of receptor tyrosine
phosphorylation and cell migration. Am. J. Resp. Cell Mol. Biol. 18, 489-96.
2. Cheng, H.L., Trink, B., Tzai, T.S., Liu, H.S., Chan, S.H., Ho, C.L., Sidransky, D.,
Chow, N.H. (2002). Overexpression of c-met as a prognostic indicator for
transitional cell carcinoma of the urinary bladder. A comparison with p53 nuclear
accumulation. J Clin Oncol 20, 1544-1550.
3. Collesi C. Santoro MM. Gaudino G. Comoglio PM. (1996) A splicing variant of the
RON transcript induces constitutive tyrosine kinase activity and an invasive
phenotype. Mol Cell Biol 16, 5518-26.
4. Chen YQ. Zhou YQ. Angeloni D. Kurtz AL. Qiang XZ. Wang MH. (2000)
Overexpression and activation of the RON receptor tyrosine kinase in a panel of
human colorectal carcinoma cell lines. Exp Cell Res 261, 229-38.
5. Chow, N.H., Lin, Y.J., Cheng, H.L., Tzai, T.S., Ho, C.L., Chang, T.Y., Dai, Y.C.,
Liu, H.S. (2003) The significance of macrophage stimulating protein (MSP)/RON
signaling pathway in the progression of human bladder cancer. Proceedings of the
American Association for Cancer Research, 2003 (abstract).
Western bloting of uroepithelial cell lines
E6
RT4
8301 TCCSUP
J82
T24 UB09 UB47 UB40 UB37
Immunoprecipitation detection of MSP in human urine
HepG2
E6
701
704
512
580
MSP
II-2: Aberrant expression of signaling proteins in human colorectal tumors
(Tzeng-Horng Leu)
BACKGROUND
FAK and c-Src are two mutually interactive nonreceptor tyrosine kinases in
signal transduction. Overexpression of FAK and c-Src is observed in a variety of
human tumors. To investigate their role in the multistep colorectal carcinogensis
( Kinzler and Vogelstein, 1996) simultaneously, we analyzed their expression in 60
paired cancer-normal mucosa specimens from colorectal cancer patients. Compared
to normal mucosa, enhanced FAK and c-Src expression in tumor specimens (T/N > 2)
was observed in 48.3 % (29/60) and 68.3 % (41/60) tumor samples respectively while
no altered expression of p130Cas was detected. Interestingly, the expression levels of
both proteins are parallel in these tumors. Thus the coexpression of both FAK and
c-Src seems to be important for human colon cancer formation.
METHODS
To delineate the relationship between FAK and c-Src in human colon cancer, we
utilized the known anti-carcinogenic agents to analyze their expression in three
human colon cancer cell lines, i.e. Caco-2, SW480, and SW620. Since sodium
butyrate, an inhibitor of histone deacetylase (Riggs et al., 1977; Sealy and Chalkley,
1978), is able to regulate gene expression, we wondered whether signaling molecules
that modulate the expression of both proteins are sensitive to butyrate. Therefore,
we treated Caco-2, SW480, and SW620 with sodium butyrate and analyzed their
protein and mRNA expression. In addition, several other signaling molecules that
may contribute to colon tumor formation were also evaluated.
RESULTS
Butyrate treatment significantly reduces the growth rate of tested colon cancer
cells. Western immunoblot data revealed the expression of FAK, c-Src, p97Eps8, and
-catenin was reduced by butyrate treatment in a dose- and time-dependent manner.
Furthermore, the level of FAK Tyr-397 phosphorylation was also reduced in these
cells. By contrast, p21WAF1/CIP1 expression and histone acetylation were induced in
these cells as expected. RT-PCR analysis indicated that the mRNA level of either fak
or c-src was also decreased in response to butyrate.
DISCUSSION
The formation of colon cancer requires activation of oncogenes (i.e. K-Ras) and
inactivation of tumor suppressor genes (i.e. p53, APC) and several mismatch repairing
genes in a long period of time. However, in addition to these well-characterized
genetic markers, there were still ill defined alterations participated in this process.
Accumulated evidence demonstrated that both Src and FAK were either
overexpressed or activated in several human tumors including colon cancer.
However, its underlying mechanism is still elusive. In this study, we observe that
both FAK and Src are paralleled enhanced expression in human colon cancer. In
addition, pathways leading to co-express both proteins are butyrate sensitive.
Further analysis indicates that both protein and mRNA level of either fak or c-src in
colon cancer cells was reduced by butyrate. The butyrate-targeted molecules that
regulate the expression of c-Src and FAK remain to be solved.
REFERENCE
1. Kinzler, K.W., and Vogelstein B. (1996) Lessons from hereditary colorectal cancer.
Cell 87, 159-170.
2. Riggs, M.G., Whitaker, R.G., Neumann, J.R., Ingram, V.M. (1977) n-butyrate cause
histone modification in HeLa and Frienderythroleukaemia cells. Nature 268,
462-464.
3. Sealy, L., and Chalkley, R. (1978) The effect of sodium butyrate on histone
modification. Cell 14, 115-121.
II-3: Study of the pathogenesis of malignant pleural effusion associated lung
adenocarcinoma in Taiwan and Stat3 as a model gene (Wu-Chou Su and
Ming-Derg Lai)
BACKGROUND
Malignant pleural effusion (MPE) is a poor prognostic sign for patients with
non-small cell lung cancer (NSCLC). The generation of MPE is highly regulated by
vascular endothelial growth factor (VEGF). In some tumor cells, expression of VEGF
was found being regulated by activation of Stat3. In our clinical data, we found
elevated expression of IL-6 and VEGF in the pleural fluids of patients with MPE
associated lung adenocarcinoma. Furthermore, constitutively activated Stat3 was also
found in tumor tissues by immunohistochemical staining. Activation of Stat3 has been
shown to induce expression of a group of genes regulating cell cycle progression,
cellular proliferation and survival, such as cyclin D1, P21/WAF1, c-Myc and Bcl-xL.
Therefore, Stat3 activation may have implications of generation of MPE, tumor cells
proliferation, and resistance to chemotherapeutic drugs.
METHODS
Plasmids with EGFP (enhanced green fluorescent protein) or dn-Stat3
(dominant-negative Stat3) were introduced into PC14PE6/AS2 by a calcium
phosphate transfection procedure. Activation of Stat3 was analyzed by Western blot.
Cell survivals after treatment with drugs were measured by MTT colorimetric assay.
RESULTS
We have examined eight of lung cancer cell lines for Stat3 protein expression as
well as its tyrosine705 phosphorylation. The Stat3 in most of the cells is tyrosine705
phosphorylated, indicating it is in an activated state. The expression of Stat3, however,
is variable in those cells and does not correlate with its activation. When serum was
depleted from culture medium, the tyrosine705 phosphorylation of Stat3 had
decreased within 30 min but recovered spontaneously since 3 hours and to original
level at about 24 hours after manipulation. Further studies found the activation of
Stat3 in these NSCLC cells is regulated by autocrine IL-6 secretion. Since the
mutation of P53 gene has been related to autocrine secretion of IL-6, a direct DNA
sequencing on PC14PE6/AS2 cell was done by autosequencing method. We detected
mutations on exon 4 and 7 in the DNA of the cell.
To study the relationship between Stat3 activation and anti-cancer drug, we have
established a NIH-3T3 cell line (NIH3T3/S3C), which expresses constitutively
activated Stat3. Besides, PC14PE6/AS2 (with constitutively-active Stat3) was
transfected with Tet-controlled dominant-negative Stat3 plasmid. The AS2/dnStat3
cell line was subcloned and transfection of dnStat3 was verified by DNA sequencing.
With these two systems, the interaction between anti-cancer agents and activation of
Stat3 was explored. Cells with activated Stat3 are more resistant to anti-caner
agents— 5-FU, Cisplatin and Taxol. Several categories of anti-cancer agents have
been added to PC14PE6/AS2 cell to test their effects on Stat3 activation. To our
surprise, most of the agents enhanced activation of Stat3 at 3 hours, suggesting
activation of Stat3 is a protection mechanism of cells in response to stress. The
activation of Stat3 in AS2 cell declined to undetectable level at 24 hours after the
treatment by some agents but remained high by some other agents. Deactivation of
Stat3 at 24 hours was found to correlate well with anti-proliferative activities of
chemotherapeutic drugs.
The established AS2/dnStat3/EGFP cell will be injected into SCID or nude mice
to study the Stat3 function in vivo.
DISCUSSION
Our results show that autocrine of IL-6 in lung adenocarcinoma cells can active
Stat3 pathway through JAK2 and this may play an important role in lung
adenocarcinoma induced malignant pleural effusion. We have also demonstrated
intriguing interaction between activation of Stat3 and anti-cancer agents. The
established cell lines can be used for further biochemical studies and used to establish
animal models. The following figure summarizes our findings on how Stat3 is
activated and its implication of MPE:
P53 mutations
Other mechanisms
IL-6
Stat3-Y-P
VEGF
MPE
II-4:0ER Stree Signaling Role of pre-S Deletion Mutants in HBV-related
Hepatocarcinogenesis (Ih-Jen Su)
BACKGROUND
Hepatitis B virus ( HBV ) is a major etiologic agent in the development of
hepatocellular carcinoma ( HCC ). Epidemiologic studies have demonstrated an
approximately 100 fold increase in the relative risk of HCC among HBV carriers in
Taiwan.However, the exact mechanism of HBV-related hepatocarcinogenesis remains
to be explored.
We have previously identified two pre-S mutants from ground glass hepatocytes
at different replicative stages of chronic HBV replication. A mutant with deletion over
the pre-S2 region was of particular interest because the hepatocytes harboring pre-S2
mutant consistently cluster in groups, suggesting their potential growth advantage.
Furthermore, the pre-S2 mutant is prevalent in serum of patients at late replicative
phase ( 33% ) or in patients with HCC ( 65% ). The mutant pre-S proteins are
localized in endoplasmic reticulum ( ER ) and initiated ER stress signals ( Wang and
Su, paper submitted ). Whether the ER-stress signal is linked to the growth advantage
of GGHs leading to its clustering proliferation remains to be explored.
In this study, cell cycle regulation initiated by pre-S mutants in ER was studied
by flow cytometry. Microarray expression profile was used to identify the cellular
genes regulated by pre-S mutants.
METHODS
1. Pon-A inducible expression of pre-S mutants in Huh-7 cell line:
2. Microarray analysis of genes expressed by pre-S-induced ER stress:
3. Flow cytometry analysis of cell cycle regulation by mutant pre-S proteins:
4. Analysis of the expression of cell cycle regulators induced by pre-S-induced ER
stress.
RESULTS AND DISCUSSION
Pon-A-inducible expression of pre-S mutants led to the activation of ER-stress
signals GRP-78/94, PERK, and JNK. Microarray analysis of pre-S gene expression
revealed upregulation of several genes including cyclin A, cyclin D1, CDK, and
PCNA. Flow cytometry analysis of cell cycle revealed that pre-S2 mutant can
overcome the cell cycle arrest.. The pre-S-induced ER stress may therefore initiate
cell cycle progression through the upregulation of cyclin D1/CDK and cyclin A
FUTURE PLAN
1. To verify whether the cell cycle regulation by pre-S mutants is related to ER
stress.
2. To verify whether NFkB is involved in pre-S mutant-induced ER stress pathway.
3. To verify whether pre-S mutant will play a role in cell transformation by colony
formation and transgenic model.
REFERENCE
1. Pahl HL. (1999) Signal transduction from the endoplasmic reticulum to the cell
nucleus. Physiologic Review 79, 683-701.
2. Fan FY, Chen WC, Lu CC, Yao WJ, Wang HC, Chang TC, Lei HY, Su IJ. (2001)
Prevalence and significance of HBV pre-S mutants in serum and liver at different
replicative stages of chronic HBV infection. Hepatology 33, 277-286.
3. Wang HC, Lei HY, Su IJ. Molecular characterization of ground glass hepatocytes
in chronic HBV infection. ( submitted and revised )
4. Wang HC, Su IJ. Cyclin A upregulation and cell cycle regulation by a novel HBV
pre-S mutant-implication in HBV-related hepatocarcinogenesis. ( in preparation )
Sub-project (III)
Novel
signal
transduction
mechanisms
that
mediate
anti-apoptotic effects in patho-biology (Principal Investigator: Ming-Jer Tang)
Apoptosis and anti-apoptosis have become important issues for modern
biomedical science. Mechanisms that trigger apoptosis or anti-apoptosis in cell play
very important roles in morphogenesis during development or in patho-physiological
conditions, such as carcinogenesis or regeneration of specific organ. Apoptosis signals
may come from outside of the cell, work on cell membrane receptor, trigger
intracellular death machinery and finally degrade the integrity of cell structure. In the
past year, we made some progress in studies of signaling mechanisms regarding to
interactions of Fas/Fas-L, association of caspase 3/p21WAF1, downregulation of FAK
and counteractions of Lithium to ceramide. The death signaling pathways induced by
Fas/Fas-L interactions have been well documented. It is hypothesized that expression
of Fas-L in tumor cell may contribute to tumor escape from immune surveillance. Dr.
B. C. Yang demonstrates that the crosstalk between human glioma cells and
neutrophils through the Fas/FasL system results in enhanced tumor cell viability and
stimulation of cytokine production in neutrophils. In addition, the contact with human
glioma cells induces induction of IL-10 production in T cell lines. PKA-independent
pathway is involved in Fas-induced IL-10 production signaling. Caspase-3 is a
pro-apoptosis protein and p21/WAF1 is a cell cycle inhibitor. In cervical cancer,
activation of caspase-3 is accompanied by p21/WAF1 cleavage. The novel
mechanisms whereby capspase-3 activation induces cancer cell growth is examined
by Dr. C. Y. Chou. Focal adhesion complex proteins play very important roles in cell
adhesion, migration and survival. However, cells cultured on collagen gel exhibited
rapid downregulation of focal adhesion complex proteins. Dr. M. J. Tang
demonstrates that collagen gel-induced downregulation of focal adhesion proteins is
mediated by physical property of the gel and through 21 integrin, but not DDR1.
These findings may explain the mechanisms of downregulation of focal adhesion
proteins during the kidney development. Finally, Dr. Y. S. Lin works on the novel
mechanism by which Lithium serves to counteract ceramide-induced apoptosis in
immune cells and shows that Lithium confers protection from ceramide-induced
apoptosis via activation of MEK/ERK/Hsp70 and inhibition of mitochondrial
activation. Results of our studies should have impact in research field of cancer,
cancer immunology, and developmental biology.
III-1: Suppression of the Fas-mediated death signal in T cells upon contact with
Fas-L positive tumors (Bei-Chang Yang)
BACKGROUND
Fas/Fas-L system plays an important role in various immune functions. Recently,
many reports including findings of our group (1-3) demonstrated that Fas-L is
expressed on the surface membrane of tumors and may contribute to tumor escape
from immune surveillance. Nevertheless, using FasL+ cells for organ transplantation
or expression of FasL in transgenic animals caused severe tissue destruction, which
indicates a much subtler interaction of Fas and FasL in vivo than has previously been
perceived (4). Accumulating data indicate that the Fas signal may modulate gene
expression in addition to its well-known death-triggering capability. In the immune
system, responses to Fas activation vary with cell types and their differentiation
stages. Thus, the interplay between FasL+ tumors and immune cells deserves
reevaluation in a context without extensive apoptosis. This project is to reveal the Fas
signal in T cells/immune cells upon contact with the Fas-L molecule on tumor cells as
well as to screen potential agents to block the Fas-transduced death signal.
RESULTS AND DISCUSSION
During the period of the 2001-2002 we have completed two studies on the
responses of immune cells upon interaction of Fas and FasL.
1. W.S. Hor, W.L. Huang, Y.S. Lin and B.C. Yang, Crosstalk between tumor cells
and neutrophils through the Fas (APO-1, CD95)/FasL system: human glioma cells
enhance cell viability and stimulate cytokine production in neutrophils. J. Leuk.
Biol. (In press; 2003).
Many tumor cells are resistant to Fas-mediated killing, which has been
primarily used as a mechanism to evade immune attack. In this study, we found a new
action of Fas on tumors where activation of the Fas signal may force tumor cells to
produce survival factors for neutrophils. Human peripheral circulating neutrophils in
coculture with glioma cells showed significant delays in spontaneous apoptosis. IL-6
and IL-8 partially mediated the glioma cell-associated protective effect on neutrophils.
The Fas agonistic antibody CH-11 dose-dependently stimulated the expression of
IL-6 and IL-8 in glioma cells. Accordingly, blocking the Fas/FasL interaction reduced
IL-6 and IL-8 production in glioma cells and impaired their protective effect on
neutrophils. Coculture with glioma cells also affected the expression of cytokines in
neutrophils including IL-8, IFN- and TNF-to various extents. Collectively, our
results demonstrate bi-directional crosstalk between tumor and immune cells.
Although Fas activation alone cannot induce apoptosis in tumor cells, it may
potentially initiate an effective anti-tumor response through a circumvented
mechanism.
2. B.C. Yang, W.S. Hor, H.K. Lin, J.Y. Hwang, M.Y. Liu, and Y.J. Wang. Induction
of IL-10 in T cell lines upon contact with human glioma cells, that is mediated by
Fas signaling through a PKA-independent pathway. (J. Immunol. In revision)
Elevated expression of IL-10 has been frequently observed in tumor tissues and
tumor infiltrating cells that is implicated in tumorigenesis. We show here that the
transcription of IL-10 gene in Jurkat and Molt-4 T cell lines was up-regulated upon
contact with glioma cells without an induction of apoptosis in those T cells. The
glioma-associated IL-10 induction was suppressed by interrupting the engagement of
Fas and its ligand (Fas-L) with antagonistic antibody ZB4, reducing the Fas-L
expression of glioma cells using Fas-L-specific ribozyme, or preventing cell-to-cell
contact in a transwell culture system. Cross-linking of Fas with agonistic antibody
CH-11 triggered apoptosis and concomitantly enhanced the expression of IL-10 in
Jurkat cells. Activation of caspase-8, but not caspase-3 and caspase-9, was evident in
Jurkat cells in coculture with glioma cells. Moreover, caspase inhibitors Z-VAD and
Z-IETD inhibited the IL-10 induction in Jurkat cells treated by glioma cells or CH-11.
Direct activating protein kinase A (PKA) by forskolin in Jurkat cells also elevated the
expression of IL-10. However, KT5720, a selective PKA inhibitor, reduced neither
the anti-Fas-triggered nor the glioma-associated IL-10 induction. The cAMP-response
element binding proteins, CREB and ATF-1, in Jurkat cells were not further
phosphorylated in coculture with glioma cells or upon anti-Fas treatment. Moreover,
the application of KT5720 did not alter the glioma-associated IL-10 induction in
Jurkat cells further suggesting a PKA-independent pathway. In summary, our results
demonstrate a non-lethal crosstalk between tumor and immune cells through
Fas/Fas-L system leading to activation of Fas signal and IL-10 expression in T cells.
We hypothesize that such a mechanism would allow Fas-L expressing tumors to
down-regulate T cell-dependant antitumor immunity.
REFERENCES
1. B.C. Yang, Y.S. Wang, H.S. Liu, and S.J. Lin. (2000) Ras signaling is involved in
the expression of Fas-L in glioma. Lab. Invest. 8, 529-537.
2. C.C. Chio, Y.S. Wang, Y.L. Chen, S.J. Lin, and B.C Yang. (2001) Down-regulation
of Fas-L in glioma cells by ribozyme reduces cell apoptosis, tumor infiltrating
cells, and liver damage but accelerates tumor formation in nude mice. Br. J.
Cancer 85, 1185-1192.
3. Y.L. Chen, J.Y. Wang, H.S. Chen, and B.C. Yang. (2002) Granulocytes mediates
the Fas-L-associated apoptosis during lung metastasis of melanoma that
determines the metastatic behavior. Br. J. Cancer 87, 359-365.
4. P.R. Walker, P. Saas, and P.Y. Dietrich. (1988) Tumor expression of Fas ligand
(CD95L) and the consequences. Curr. Opin. Immunol. 10, 564-572.
III-2: Novel mechanisms of caspase-3 activation-induced cell growth in invasive
cervical carcinoma (Cheng-yang Chou)
BACKGROUND
Our preliminary results showed that both procaspase-3 and p21WAF1 were
over-expressed in cervical intraepithelial lesions (CIN) and in invasive cervical cancer.
Furthermore, 35 of 39 cervical cancer patients with tumor sizes > 3 cm3 showed
cleavage and activation of caspase 3 with cleavage of p21WAF1, whereas only one of
12 with a tumor size < 3 cm3 did. In contrast, none of the CIN specimens showed
evidence of p21WAF1 cleavage. In this study, we have examined the association of
p21WAF1 cleavage with apoptosis, proliferation, aneuploidy, the presence of HPV 16 or
18, and MMP-2 and –9 activation, and to identify the protein sequences of the p14
and p16 cleavage products and the enzymes responsible for the cleavage.
METHODS
We used immunohistochemistry to identify the expressions of procaspase 3 and
activated caspase 3, Ki-67 staining for cell proliferation and MMP 2 and 9 for the
potential of invasion and metastasis. We also used in-situ hybridization to detect HPV
16 or 18, and applied flowcytometry to assess DNA ploidy. The molecular identity of
p14 and p16 cleavage products will be analyzed by protein sequencing, and the
enzymes responsible for cleavage will be searched through available data bank.
RESULTS
Among the 64 cervical cancer patients recruited, 12 patients were
adenocarcinoma in histologic type and 52 were squamous cell carcinoma. Patients
with adenocarcinoma exhibited less procaspase and caspase 3 activation as compared
with patients with squamous cell carcinoma. There was no significant difference of
cell proliferation between patients with caspase 3/p21WAF1 cleavage and patients
without, suggesting the independence of p21WAF1 cleavage with cell proliferation. In
contrast, the presence of caspase 3/p21WAF1 cleavage was associated with apoptosis.
DISCUSSION
Protein sequencing of the p14 and p16 cleavage products and
immunohistochemical staining for MMPs are currently underway. An important
observation is to the phenomena of correlate caspase 3 activation/p21WAF1 cleavage to
the survival of these patients. In order to study the biologic significance of p21WAF1
cleavage products on cell functions such as apoptosis, proliferation, protein binding
with cyclin and PCNA, and cell cycle regulation, an in-vitro model with stable
transfection of p21WAF1 cleavage products in cervical cancer cells will be established.
The following figure summarizes our strategies to establish the in-vitro model.
Construction
insert
HindIII & XbaI
digestion
ligation
insert
PCR amplify
EcoRV & XbaI
digestion
pLacM
pTet-LacHyg
cotransfection
Cervical cancer cell
Hygromycin
selection
Stable expression cells
REFERENCES
1.
2.
3.
4.
5.
Asada, M., Yamada, T., Ichijo, H., Delia, D., Miyazono, K., Fukumuro, K., and
Mizutani, S. (1999) Apoptosis inhibitory activity of cytoplasmic p21
(Cip1/WAF1) in monocytic differentiation. EMBO J. 18, 1223-1234
Li, F., Ackermann, E. J., Bennett, C. F., Rothermel, A. L., Plescia, J., Tognin, S.,
Villa, A., Marchisio, P. C., and Altieri, D. C. (1999) Pleiotropic cell-division
defects and apoptosis induced by interference with survivin function. Nature
Cell Biol. 1, E199-E200
Waldman, T., Lengauer, C., Kinzler, K. W., and Vogelstein, B. (1996)
Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking
p21. Nature 381, 713-716
Pihan, G. A., Purohit, A., Wallace, J., Malhotra, R., Liotta, L. and Doxsey, S. J.
(2001) Centrosome defects can account for cellular and genetic changes that
characterize prostate cancer progression. Cancer Res. 61, 2212-2219
Zhang, Y., Fujita, N. and Tsuruo T. (1999) Caspase-mediated cleavage of
p21WAF1 converts cancer cells from growth arrest to undergoing apoptosis.
Oncogene 18, 1131-1138
III-3: Molecular mechanism of down-regulation of focal adhesion complex
proteins in kidney (Ming-Jer Tang)
BACKGROUND
Biochemical impacts exerted by extracellular matrix (ECM) on cell functions
have been studied intensively, whereas biophysical impacts exerted by threedimensional (3-D) ECM remains mostly unknown. It has been found collagen gel
overlay induces selective degradation of focal adhesion complex proteins (1, 2). In
this study, we examined whether collagen gel controls the expression of focal
adhesion complex proteins via its physical property, i.e. the rigidity. The physiological
significance of rigidity-regulated cell behavior was further explored in renal
regeneration and development.
METHODS
We used 3-D collagen gel as well as matrigel to establish that lowered
substratum rigidity affects focal adhesion complex proteins in cell lines and primary
culture of proximal tubule cells. To explore the signal pathways involved in collagen
gel-regulated focal adhesion protein expression, we employed cells harboring
collagen receptor DDR1 as well as FAK, either wild type or dominant negative.
Finally, we used 5/6 nephrectomized rat models to assess whether changes in kidney
rigidity, a result of tubulo-interstitial fibrosis, affected the expression of focal
adhesion complex proteins in kidney.
RESULTS
The collagen gel-induced down-regulation of focal adhesion complex proteins
was caused by reduction of protein synthesis and activation of proteases such as
calpain and was mediated by 21 integrin, but not DDR1. Freshly isolated renal
proximal tubule cells, initially exhibited little focal adhesion complex proteins like rat
kidney, re-expressed focal adhesion complex proteins in primary cultures when they
were cultured on collagen gel- or matrigel-coated dishes. Lowering substratum
rigidity by culturing cells on collagen gel or matrigel prevented the re-expression of
focal adhesion complex proteins in primary culture. Furthermore, in vivo studies
demonstrated that induction of tissue hardening by 5/6 nephrectomy resulted in
re-expression of focal adhesion complex proteins in kidney. Taken together, these data
indicate that the substratum rigidity determines expression of focal adhesion complex
proteins both in vitro and in vivo.
DISCUSSION
This study should facilitate our understanding of how substratum rigidity
controls cell behaviors through an important biomechanical regulatory mechanism.
We can speculate the flexible substrate may down-regulate the expression of focal
adhesion proteins and thereby hindrance the formation of focal adhesions as well as
the phosphorylation of FAK and paxillin, as observed in Pelham and Wang (3). Taken
together, our data provide a novel biomechanical signal mechanism that links the
substrate rigidity to the regulation of focal adhesions. The following figure
summarizes our findings on how mechanical property of collagen gel down-regulates
expression of focal adhesion proteins:
REFERENCES
1. Tang M.J., J.J. Hu, H.H. Lin, W.T. Chiu, and S.T. Jiang. (1998) Collagen gel
overlay induces apoptosis of polarized cells in cultures: disoriented cell death. Am.
J. Physiol. 275, C921-31.
2.
Wang Y.K., H.H. Lin, and M.J. Tang. (2001) Collagen gel overlay induces two
phases of apoptosis in MDCK cells. Am. J. Physiol. Cell Physiol. 280, C1440-8.
3.
Pelham R.J., and Y.L. Wang. (1997) Cell locomotion and focal adhesions are
regulated by substrate flexibility. Proc. Natl. Acad. Sci. USA 94, 13661-13665.
Geiger B. and A. Bershadsky. (2002) Exploring the neighborhood: adhesioncoupled cell mechanosensors. Cell 110, 139-142.
Yang-Kao Wang, Yao-Hsien Wang, Chau-Zen Wang, Junne-Ming Sung, Wen-Tai
Chiu, Shu-Han Lin, Yung-Hen Chang, and Ming-Jer Tang. (2002) Rigidity of
4.
5.
substratum controls expression of focal adhesion complex proteins. (Submitted to
J. Cell Biol.)
III-4:Lithium confers protection from ceramide-induced apoptosis via activation
of MEK/ERK/Hsp70 and inhibition of mitochondrial activation (Yee-Shin
Lin)
BACKGROUND
Apoptosis occurs not only by activation of pro-apoptotic signaling, but also by
suppression of survival pathways. Ceramide, a key mediator of apoptosis induced by
diverse stimuli, has been shown to inhibit both the PI 3-kinase/Akt and MAPK
pathways that may induce a turn-off mechanism of survival pathways (1-3). In this
study, the molecular mechanisms of apoptosis induced by ceramide was explored in
the immune cells. Furthermore, the effect of lithium, which has been shown to confer
protection against neuronal apoptosis, on ceramide-induced immune cell death was
investigated.
METHODS
Murine splenocytes and 10I T hybridoma cells were treated with ceramide in the
presence or absence of lithium. Cells were cultured with or without the addition of
various inhibitors, and examined by using propidium iodide staining and TUNEL
assay. Mitochondrial membrane potential was assessed by rhodamine 123 staining.
Protein expression, phosphorylation, and cleavage were analyzed by Western blotting.
RESULTS
Treatment of 10I T hybridoma cells with ceramide showed apoptotic
characteristics that were inhibited by lithium, but not by sodium and potassium.
Freshly isolated mouse splenocytes also underwent apoptosis when stimulated by
ceramide and this effect was blocked by lithium. Further investigation revealed that
lithium augmented MEK and ERK phosphorylation. The MEK inhibitor PD98059
reduced lithium-induced MEK/ERK activation and cell survival. Akt phosphorylation
was inhibited by ceramide and elevated by lithium, but lithium could not block
ceramide-mediated suppression of Akt. In search for the downstream target of ERK,
studies indicated that lithium enhanced Hsp70 expression and Hsp inhibitor
moderately abolished lithium-mediated protection of ceramide-induced apoptosis. Our
results indicated that lithium stimulated a survival pathway which involved activation
of MEK, ERK and Hsp70 (4).
During stress-induced apoptosis, the reduction of mitochondrial transmembrane
potential (MMP) results in the release of cytochrome c, which binds to Apaf-1 and
promotes apoptosome formation and caspase-9 activation. Once the initiator caspases
are activated, downstream effector caspases, such as caspases 3, 6, 7 are activated and
cell death occurs. In this study, the mitochondrial dysfunction and sequential caspase
activation induced by ceramide and the inhibitory effects of lithium were studied.
Results showed the activation of caspase-8 and MMP reduction, followed by the
increase in caspase-9 and caspase-3 activity and PARP degradation. Moreover,
ceramide-induced mitochondrial dysfunction and caspases 3, 8, 9 activation were
abolished by lithium treatment.
DISCUSSION
The caspase activation cascade following ceramide treatment remains further
dissection. Interestingly, activation of caspase-8, which is an initiator caspase in
Fas/TNFR-induced apoptotic pathways, is found in ceramide-induced 10I cell
apoptosis. Caspase-8 has been shown to play an essential role in
transcription-independent apoptosis triggered by p53 (5). Roles that p53 may play in
caspase activation and Hsp70 function will be further explored. The following
diagram summarizes our findings:
REFERENCES
1. Zhou H., S. A. Summers, M. J. Birnbaum, and R. N. Pittman. (1998) Inhibition of
Akt kinase by cell-permeable ceramide and its implications for ceramide-induced
apoptosis. J. Biol. Chem. 273, 16568-16575.
2. Widmann C., S. Gibson, and G. L. Johnson. (1998) Caspase-dependent cleavage
of signaling proteins during apoptosis. J. Biol. Chem. 273, 7141-7147.
3. Jan M.-S., H.-S. Liu, and Y.-S. Lin. (1999) Bad overexpression sensitizes
NIH/3T3 cells to undergo apoptosis which involves caspase activation and ERK
inactivation. Biochem. Biophys. Res. Commun. 264, 724-729.
4. Jan M.-S., L.-J. Hsu, C.-F. Lin, and Y.-S. Lin. Lithium confers protection from
ceramide-induced apoptosis via activation of MEK/ERK/Hsp70 and inhibition of
mitochondrial activation. (manuscript in preparation)
5. Ding H.-F., Y.-L. Lin, G. McGill, P. Juo, H. Zhu, J. Blenis, J. Yuan, and D. E.
Fisher. (2000) Essential role for caspase-8 in transcription-independent apoptosis
triggered by p53. J. Biol. Chem. 275, 38905-38911.
7. List of Research Results & Achievements
1) Tzeng-Horng Leu and Ming-Chei Maa. (2002) Tyr-863-phosphorylation enhances
focal adhesion kinase autophosphorylation at Tyr-397. Oncogene 21, 6992-7000.
2) Tzeng-Horng Leu and Ming-Chei Maa. (2003) Functional implication of the
interaction between EGF receptor and c-Src. Frontiers in Bioscience 8, s28-38.
3) Woei-Jer Chuang, I-Ju Yeh, Yu-Huei Hsieh, Pei-Phen Liu, Shu-Wan Chen, and
Wen-Yih Jeng. (2002) 1H, 15N and 13C resonance assignments for the
DNA-binding domain of myocyte nuclear factor (Foxk1). Journal of
Biomolecular NMR 24, 75-76.
4) Pei-Phen Liu, Yen-Chin Chen, Ching Li, Yu-Huei Hsieh, Shu-Wan Chen,
Shu-Huei Chen, Wen-Yih Jeng, and Woei-Jer Chuang. (2002) Solution structure of
the DNA-binding domain of interleukin enhancer binding factor 1 (Foxk1a)
Proteins: Structure, Function, and Genetics 49, 543-553.
5) Yi-Wen Liu, Hui-Ping Tseng, Ben-Kuen Chen, Lei-Chin Chen, and Wen-Chang
Chang. Functional cooperation of Sp1 and C/EBP β and δ in
lipopolysaccharide-induced gene activation of interleukin-10 in mouse
macrophages. (J. Immunol, submitted)
6) Hui-Ching Wang, Huan-Yao Lei , Nelson Fausto, and Ih-Jen Su. Ground Glass
Hepatocytes in Chronic Hepatitis B Virus Infection Contain Specific pre-S
Mutants Which May Activate Stress Signals and Confer Growth Advantage.
(Hepatology, submitted)
7) Lee TH, Chang HC, Chuang LY, and Hung WC. Involvement of of PKA and Sp1
in the induction of p27Kip1 by tamoxifen. (Biochemical Pharmacology,
submitted)
8) Hsiao-Chia Wu, Shwu-Jen Tzeng, Jau-Cheng Chiou, Ming-Derg Lai, and Jin-ding
Huang. Transcriptional Regulation of Rat Mrp3 Promoter by p53. (Biochemical
and Biophysical Research Communication, submitted)
9) Tsuey-Yu Chang, Wen-Jiuan Tsai, Chao-Kai Chou, Nan-Haw Chow, Tzeng-Horng
Leu, and Hsiao-Sheng Liu. Identifying the factors and signal pathways necessary
for anchorage-independent growth of Ha-ras oncogene- transformed NIH/3T3
cells. (Life Science, submitted)
10) Trei-Lien Hung, Fen-Fen Chen, Wu-Wei Lai, Ai-Li Hsiao, Wen-Tsung Huang,
Helen H.W. Chen, and Wu-Chou Su. Clinical evaluation of HER-2/neu protein in
malignant pleural effusion-associated lung adenocarcinoma and as a tumor marker
in pleural effusion diagnosis. (Clin Cancer Res, submitted)