Group Title: Journal of Translational Medicine 2009, 7:8
Title: TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors
CITATION PDF VIEWER THUMBNAILS PAGE IMAGE ZOOMABLE
Full Citation
STANDARD VIEW MARC VIEW
Permanent Link: http://ufdc.ufl.edu/UF00100295/00001
 Material Information
Title: TRIP-Br2 promotes oncogenesis in nude mice and is frequently overexpressed in multiple human tumors
Series Title: Journal of Translational Medicine 2009, 7:8
Physical Description: Archival
Creator: Cheong JK
Gunaratnam L
Zang ZJ
Yang CM
Sun X
Nasr SL
Sim KG
Peh BK
Rashid SBA
Bonventre JV
Salto-Tellez M
Hsu SI
Publication Date: 39833
 Record Information
Bibliographic ID: UF00100295
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: Open Access: http://www.biomedcentral.com/info/about/openaccess/

Downloads

This item has the following downloads:

tripbr2 ( PDF )


Full Text




Journal of Translational Medicine lioved



Research

TRIP-Br2 promotes oncogenesis in nude mice and is frequently
overexpressed in multiple human tumors
Jit Kong Cheong1,2, Lakshman Gunaratnam1, Zhi Jiang Zang12,
Christopher M Yang2, Xiaoming Sun', Susan L Nasrl, Khe Guan Sim2,
Bee Keow Peh3, Suhaimi Bin Abdul Rashid3, Joseph V Bonventre1,
Manuel Salto-Tellez*3 and Stephen I Hsu*1,2,4


Address: 'Renal Division and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA,
2Department of Medicine, National University of Singapore and National University Hospital, 119074, Singapore, 3Department of Pathology,
National University of Singapore and National University Hospital, 119074, Singapore and 4Division of Nephrology, Hypertension and Renal
Transplantation, College of Medicine, University of Florida, 1600 SW Archer Road P.O. Box 100224, Gainesville, Florida 32610 USA
Email: Jit Kong Cheong jitkong.cheong@duke-nus.edu.sg; Lakshman Gunaratnam lgunaratnam@partners.org;
Zhi Jiang Zang Zhijiang.Zang@ medicine.ufl.edu; Christopher M Yang madscienc@yahoo.com; Xiaoming Sun xsun@partners.org;
Susan LNasr susan.l.nasr@gmail.com; Khe Guan Sim egypt09@gmail.com; Bee Keow Peh patbkp@nus.edu.sg; Suhaimi Bin
Abdul Rashid cmesar@nus.edu.sg; Joseph V Bonventre joseph_bonventre@hms.harvard.edu; Manuel Salto-Tellez* patmst@nus.edu.sg;
Stephen I Hsu* Stephen.Hsu@medicine.ufl.edu
* Corresponding authors



Published: 20 January 2009 Received: 15 May 2008
journal of Translational Medicine 2009, 7:8 doi: 10.1 186/1479-5876-7-8 Accepted: 20 January 2009
This article is available from: http://www.translational-medicine.com/content/7/1/8
2009 Cheong et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Members of the TRIP-Br/SERTAD family of mammalian transcriptional coregulators have recently been
implicated in E2F-mediated cell cycle progression and tumorigenesis. We, herein, focus on the detailed functional
characterization of the least understood member of the TRIP-Br/SERTAD protein family, TRIP-Br2 (SERTAD2).
Methods: Oncogenic potential of TRIP-Br2 was demonstrated by (I) inoculation of NIH3T3 fibroblasts, which were
engineered to stably overexpress ectopic TRIP-Br2, into athymic nude mice for tumor induction and (2) comprehensive
immunohistochemical high-throughput screening of TRIP-Br2 protein expression in multiple human tumor cell lines and
human tumor tissue microarrays (TMAs). Clinicopathologic analysis was conducted to assess the potential of TRIP-Br2
as a novel prognostic marker of human cancer. RNA interference of TRIP-Br2 expression in HCT-116 colorectal
carcinoma cells was performed to determine the potential of TRIP-Br2 as a novel chemotherapeutic drug target.
Results: Overexpression of TRIP-Br2 is sufficient to transform murine fibroblasts and promotes tumorigenesis in nude
mice. The transformed phenotype is characterized by deregulation of the E2F/DP-transcriptional pathway through
upregulation of the key E2F-responsive genes CYCLIN E, CYCLIN A2, CDC6 and DHFR. TRIP-Br2 is frequently
overexpressed in both cancer cell lines and multiple human tumors. Clinicopathologic correlation indicates that
overexpression of TRIP-Br2 in hepatocellular carcinoma is associated with a worse clinical outcome by Kaplan-Meier
survival analysis. Small interfering RNA-mediated (siRNA) knockdown of TRIP-Br2 was sufficient to inhibit cell-
autonomous growth of HCT-1 16 cells in vitro.
Conclusion: This study identifies TRIP-Br2 as a bona-fide protooncogene and supports the potential for TRIP-Br2 as a
novel prognostic marker and a chemotherapeutic drug target in human cancer.




Page 1 of 15
(page number not for citation purposes)







Journal of Translational Medicine 2009, 7:8


Background
Deregulation of E2F transcriptional activity due to altera-
tions in the p16INK4a/cyclin D/RB pathway is a hallmark of
many human cancers and more than half of all NCI-60
cell lines [1]. To date, the E2F family of proteins has been
shown to be involved in the regulation of genes whose
expression is pivotal for normal cell cycle progression and
numerous other cellular processes such as DNA repair,
programmed cell death and differentiation [2-4]. The
TRIP-Br/SERTAD (henceforth referred to as TRIP-Br) fam-
ily of novel mammalian transcriptional coregulators has
recently been shown to modulate E2F-dependent tran-
scriptional activities [5-7]. Family members include TRIP-
Brl/p34SEI-1/SERTAD1/SEI-1 (henceforth referred to as
TRIP-Brl), TRIP-Br2/SERTAD2/SEI-2 (henceforth referred
to as TRIP-Br2), TRIP-Br3/HEPP/CDCA4/SEI-3 (hence-
forth referred to as TRIP-Br3), RBT1 (Replication Protein
A Binding Transactivator 1)/SERTAD3 (henceforth
referred to as RBT1) and the recently-identified SERTAD4
[8]. In addition, the TRIP-Br homolog in Drosophila,
TARANIS (TARA), was identified in a screen for functional
partners of the homeotic loci and was shown to represent
a novel member of the trithorax group (trxG) of regula-
tory proteins [9].

Members of the TRIP-Br protein family possess three key
regions that we have previously coined TRIP-homology
domains (THD) [7]. THD1 contains a cyclin A-binding
motif (including a conserved nuclear localization signal,
KRK) at the amino terminal, followed by heptad repeats
that have been shown to be essential for protein-protein
interactions. THD2 consists of one or more PEST signals
rich in proline, serine and threonine residues, while
THD3 harbors a novel PHD zinc finger- and/or bromodo-
main-interacting motif and an acidic transactivation
domain at its carboxyl-terminus. The heptad repeats in
THD1 have been shown to be conserved in the TRIP-Br
family and were renamed as the SERTA (SEI-1, RBT1 and
TARA) domain [9]. It has been further shown that most of
the SERTA domain in TRIP-Brl consists of a cyclin-
dependent kinase 4 (CDK4)-binding site [10,11].

TRIP-Brl and RBT1 have recently been shown to be local-
ized in tandem within a 19ql3 amplicon frequently
found in human tumors, consistent with their putative
role as oncogenes that promote tumor growth [5]. Indeed,
cytogenetic studies have revealed a gain of chromosomal
region 19q13.1-13.2 in more than 30% of ovarian carci-
nomas [12,13] as well as a variety of other tumors includ-
ing pancreatic carcinomas [14] and lung cancers [15].
Although TRIP-Brl has been further demonstrated to be
amplified and overexpressed in several ovarian cancer cell
lines as well as in ovarian carcinomas [16], the association
of RBT1 amplification to human cancers remains elusive.
As a proof-of-principle that at least a subset of the TRIP-Br
gene family consists of novel protooncogenes that play


http://www.translational-medicine.com/content/7/1/8



important roles in cellular proliferation and human can-
cer, the knockdown of TRIP-Brl or RBT1 in cultured cell
lines has been shown to reduce cell growth and colony
formation [5,17,18]. Apart from their role as coactivators
in the stimulation of E2F-dependent transcription, the
corepressor function of TRIP-Br proteins has also been
described. Overexpression of TRIP-Brl has been found to
suppress CREB-mediated transcription and this suppres-
sion could be overcome by ectopic overexpression of CBP
[19]. In addition, TRIP-Br3 has been recently identified as
a novel E2F-responsive gene and as a repressor of E2F-
dependent transcriptional activation [6].

While most of the TRIP-Br family members have recently
been extensively characterized and shown to be involved
in a variety of important cellular processes including E2F-
mediated cell cycle progression, p53-dependent stress
response and cancer pathogenesis [6,7,9,11,18,20-22],
the physiological role of TRIP-Br2 in mammalian cells
remains poorly understood and its direct link to cancer
pathogenesis has not been established. We previously
reported that transcriptional downregulation of TRIP-Br2
in primary cell lines, achieved through DNA enzyme
knockdown or global knockout strategies, results in cellu-
lar proliferation arrest [17]. In the present study, we have
validated the oncogenic potential of TRIP-Br2. Overex-
pression of TRIP-Br2 resulted in the upregulation of E2F-
mediated transcription, the transformation of NIH3T3
fibroblasts and the promotion of tumor growth in ath-
ymic nude mice. We further performed high-throughput
expression profiling of TRIP-Br2 in comprehensive
human tumor tissue microarrays and showed that TRIP-
Br2 is frequently overexpressed in cancer.

Methods
Analysis of TRIP-Br2 gene structural organization,
prediction of TRIP-Br2 protein subcellular localization and
in silico profiling of TRIP-Br2 gene expression
The gene structural organization of human TRIP-Br2 was
analyzed by NCBI Entrez Gene, NCBI AceView and
BLAST/ClustalW http://www.ncbi.nlm.nih.gov/. The
PSORT II analysis software http://psort.nibb.ac.jp was
used to predict the subcellular localization of TRIP-Br2
proteins. The GNF SymAtlas v 1.2.4 (Novartis, http://
symatlas.gnf.org/SymAtlas/) human microarray database
was interrogated to determine the in silico gene expression
profiling of TRIP-Br2 across all human tissues. The NCBI
symbol SERTAD2 was used in the query of the GNF
SymAtlas database. The median (med) was calculated
based on expression of TRIP-Br2 across all human tissues;
med x 3: 3-fold more than the median; med x 10: 10-fold
more than the median. In silico TRIP-Br2 expression, (Z),
across all human tissues was scored via the following
scheme: +: (z) median; ++: median< () < med x 3, +++:
med x 3 < (X) < med x 10, ++++: med x 10 < (z).


Page 2 of 15
(page number not for citation purposes)







Journal of Translational Medicine 2009, 7:8


Cell culture and reagents
NIH3T3 mouse primary fibroblasts, WI38 human pri-
mary lung fibroblasts, U20S human osteosarcoma cells,
PC3 human prostate adenocarcinoma cells, 769-P human
renal adenocarcinoma cells, HCT-116 human colorectal
carcinoma cells, HepG2 human hepatocellular carcinoma
cells and MCF-7 human breast carcinoma cells were pur-
chased from American Type Culture Collection (Manas-
sas, VA). All cell lines were cultured in DMEM
supplemented with 10% FBS and maintained at 370C in
a 5% CO2 environment. Rabbit anti-TRIP-Br2 polyclonal
antibodies were generated as previously described [23]
and used in Western blot, immunocytochemical and
immunohistochemical analyses. All other antibodies used
in Western blot analyses were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). They include anti-
HA (sc-805), anti-cyclin E (sc-481) and anti-3-tubulin (sc-
5274). The use of expression plasmids pcDNA3.1 (Invit-
rogen, Carlsbad, CA) and pcDNA3.1-TRIP-Brl-HA have
been previously described [7]. The nucleotide sequence of
human TRIP-Br2 (hTRIP-Br2) was obtained from NCBI
PubMed (GenBank' accession no. BC101639) and used
as the template in the design of hTRIP-Br2-specific primers
for the construction of C-terminal HA-tagged hTRIP-Br2
expression plasmid (Additional File 1).

Generation of cells stably expressing TRIP-Br2
NIH3T3 fibroblasts were transfected with the empty vec-
tor pcDNA3.1 as a control or with the expression vectors
pcDNA3.1-TRIP-Brl-HA or pcDNA3.1-TRIP-Br2-HA
using FuGENE 6 Transfection Reagent (Roche Diagnostics
Co., Mannheim, Germany) in accordance with the manu-
facturer's instructions. Stable clones were selected using
Geneticin (Invitrogen, Carlsbad, CA) at a concentration of
750 ltg/ml. Expression levels of the carboxyl terminal HA-
tagged TRIP-Brl and TRIP-Br2 in each respective clone
were determined by Western blot analysis.

Serum deprivation, Bromodeoxyuridine (BrdU) labeling
and flow cytometric DNA content analysis
NIH3T3vector-only, NIH3T3TRIP-Brl-HA and NIH3T3TRIP-Br2-HA
fibroblasts were cultured in 96-well plates (for BrdU) or
100 mm culture dishes (for flow cytometry) in DMEM
supplemented with 0.2% FBS and were maintained for 72
h at 370C in a 5% CO2 environment. BrdU incorporation
was monitored using a cell proliferation/colorimetric
ELISA assay according to the manufacturer's instructions
(Boehringer Mannheim, Mannheim, Germany). Flow
cytometry was performed using a FACScan flow cytometer
(Becton Dickinson, Franklin Lakes, NJ) at a wavelength of
488 nm.

Soft agar colony formation and tumor induction assays
Soft agar assays were used to assess anchorage-independ-
ent growth of NIH3T3 cells as previously described [24].
For tumor induction assays, athymic nude mice (nu/nu)


http://www.translational-medicine.com/content/7/1/8



purchased from Charles River Laboratories, Inc. (Wilm-
ington, MA) were kept under SPF conditions and used
under protocol #06-231, which was approved by the Har-
vard Institutional Animal Care and Use Committee
(IACUC) and the Harvard Committee on Microbiological
Safety (COMS). 5 x 106 NIH3T3ector -only or NIH3T3TRIP-
Br2-HA fibroblasts were injected subcutaneously into 6-
week-old athymic nude mice (n = 4 for each group). On
day 13 post-injection, the mice were examined for tumor
formation. Tumor dimensions were measured every 2
days from day 13 until day 25 post-injection, at the end of
which time both groups were sacrificed and all tumors
were harvested for histological, immunohistochemical
and Western blot analyses. The experiment was repeated
by injection of new NIH3T3vector-only or NIH33T3TRIP-Br2-HA
clones into new groups of 6-week-old athymic nude mice
(n = 4). The penetrance of tumor induction from subcuta-
neous injection of NIH3T3vector-only or NIH33T3TRIP-Br2-HA
into these athymic nude mice was 0% and 100%, respec-
tively. Tumor ellipsoid volume was estimated using the
formulae previously described [25].

Semi-quantitative RT-PCR analyses
Total RNA was isolated from serum-deprived
NIH3T3ector-only, NIH3T3TRIP-Brl-HA and NIH3T3TRIP-Br2-HA
fibroblasts using the TRIZOL Reagent (Invitrogen,
Carlsbad, CA). Total RNA (3 Gig) was reverse transcribed
using the ABI High Capacity cDNA Archive Kit (Applied
Biosystems, Foster City, CA) according to the manufac-
turer's instructions. Polymerase Chain Reactions (PCR)
were performed on 1 gtl cDNA samples in the presence of
10 mM deoxyribonucleotide triphosphates (dNTPs) and
10 gtM of specific primer pairs in a total reaction volume
of 20 gtl. PCR was performed as follows: 20 cycles of dena-
turation (94C, 30 sec), annealing (51C, 30 sec) and
extension (72C, 1 minute) with a 2-minute initial dena-
turation step at 94 C and a 3-minute terminal polishing
step at 720C. The primer sequences used for RT-PCR are
available upon request.

Subcellular fractionation, denaturing SDS-PAGE and
Western blotting
Subcellular fractionation of the cells was performed using
the NE-PER Nuclear and Cytoplasmic Extraction Reagents
Kit (Pierce Biotechnology, Inc., Rockford, IL) according to
the manufacturer's instructions. Proteins from whole-cell
lysates were resolved using standard denaturing polyacry-
lamide gel electrophoresis and immunostained as
described previously [7].

Tissue microarray (TMA) construction,
immunohistochemistry and immunocytochemistry
Multiple TMA slides were obtained from the Department
of Pathology TMA Program at the National University of
Singapore, in compliance with Institutional Review Board
approval (IRB 05-017). These tumor TMAs were con-

Page 3 of 15
(page number not for citation purposes)







Journal of Translational Medicine 2009, 7:8


structed as previously described [26-29] and represented
samples from the following human tumor types that
occur in a broad range of organs: prostate carcinoma,
squamous cell lung carcinoma, lung adenocarcinoma,
breast carcinoma, gastrointestinal stromal tumor, ovarian
cystadenocarcinoma, colorectal carcinoma, basal cell car-
cinoma, renal cell carcinoma, osteosarcoma, hepatocellu-
lar carcinoma. Antigens were retrieved from the tissues
using a microwave histoprocessor (Milestone, Shelton,
CT) and DAKO pH 6.0 citrate buffer (DAKO, Via Real
Carpinteria, CA). Immunohistochemical staining was per-
formed on paraffin-embedded tissue sections using the
DAKO Envision kit (DAKO) and the rabbit anti-TRIP-Br2
antibody or its pre-immune serum control at a concentra-
tion of 1:300. Staining was visualized using a Leica DM
LB2 microscope. The intensity of TRIP-Br2 expression by
immunostaining in the tumor TMAs was scored inde-
pendently by three research pathologists in a double-
blinded manner. For immunocytochemistry, cells were
grown to 80% confluence on coverslips, washed three
times with PBS, fixed in pre-chilled 4% paraformaldehyde
for 20 minutes, and permeabilized in 0.1% Triton-X for
10 minutes. Primary immunostaining with rabbit anti-
TRIP-Br2 antibody (1:4000) was performed at room tem-
perature for 1 h. Pre-immune rabbit serum was used as a
negative control for the primary immunostaining of cells.
Secondary immunostaining with goat anti-rabbit-FITC
antibodies (sc-2012, Santa Cruz Biotechnology, Inc.,
Santa Cruz, CA) was performed at room temperature for 1
h, following 3 washes with PBS at the end of primary
immunostaining. Cellular DNA was subsequently coun-
terstained with DAPI. Staining was visualized and photo-
graphed using a Nikon Eclipse El000 fluorescence
microscope.

RNA interference of TRIP-Br2 expression
5 x 104 HCT-116 cells were plated in 12-well plates and
transfected with Cy3-labeled oligomer, scrambled siRNA
(negative control) or three different TRIP-Br2-specific siR-
NAs at the dose of 4 picomoles (pmol) or 40 pmol (in 1
ml of DMEM supplemented with 10% FBS) respectively
(TriFECTa'" kit, IDT, Coralville, IA) using Lipofactamine"T
Transfection Reagent (Invitrogen, Carlsbad, CA), in
accordance with the manufacturer's instructions. Twenty-
four hours post-transfection, these cells were cultured in
serum-free DMEM and maintained at 370C in a 5% CO2
environment for 72 h. HCT-116 cells that were not sub-
jected to transfection reagent treatment were included as
controls. Cells in colony forming assays were stained with
0.4% Giemsa stain as previously described [23]. The dye
in these cells was subsequently eluted with 1% SDS and
quantitated using a spectrophotometer at a wavelength of
595 nm. A standard curve was plotted using OD readings
taken from dye-eluted HCT-116 cells that were plated at
pre-determined cell densities.


http://www.translational-medicine.com/content/7/1/8



Statistical analysis
Survival curves for various patient cohorts were estimated
according to the method of Kaplan and Meier, and curves
were compared using the generalized Wilcoxon's test. The
log-rank test was used to assess the strength of association
between survival time and single variables corresponding
to factors thought to be prognostic for survival.

Results
TRIP-Br2, a novel proliferation marker, is highly expressed
in human lymphohematopoietic cell lineages
The TRIP-Br2 gene locus is approximately 22.3 kb long
and is localized at the poorly-characterized chromosome
2pl4 region of the human genome (position 64734550
bp to 64712250 bp; reverse strand) (Figure 1A). The pre-
cursor of TRIP-Br2 mRNA is approximately 5556 bp in
length and consists of two exons that are separated by a
long intron (Figure 1B). The intron encodes a splice donor
(GT) and a splice acceptor (AG) at either end, respectively.
The 297 bp-long 5' untranslated region (UTR) resides in
exon 1 of TRIP-Br2. It contains an in-frame stop signal
that is 6 bp prior to the 945 bp-long coding sequence of
TRIP-Br2, which is localized in exon 2. The 3' UTR of
TRIP-Br2 spans a region of approximately 4314 bp, fol-
lowed by a standard AATAAA polyadenylation signal. Due
to the lack of other splice donor-acceptor sites, transcrip-
tion of the human TRIP-Br2 gene is predicted to yield only
one mRNA transcript that encodes a 314 amino acid pro-
tein. We studied the primary protein sequence of human
TRIP-Br2 by BLAST/ClustalW analyses and found that
TRIP-Br2 is highly conserved in widely divergent species,
such as chimpanzee (99%), rhesus monkey (97%), rat
(86.9%), mouse (88.3%), chicken (81.4%) and zebrafish
(67.1%) (Figure 1C). Furthermore, based on the PSORT II
analysis, the subcellular localization of TRIP-Br2 protein
is predicted to be predominantly in the nucleus (69%),
with scant presence in the mitochondria (17%), in the
cytoplasm (4%), in the vacuoles (4%) or in vesicles of the
secretary system (4%).

In order to investigate the biological significance of TRIP-
Br2 in humans, we first performed in silico gene expression
profiling of TRIP-Br2 using a comprehensive web-based
human microarray database, GNF SymAtlas v 1.2.4
(Novartis, http://symatlas.gnf.org/SymAtlas/). As com-
pared to other tissues/cell types, TRIP-Br2 is highly
expressed in bone marrow, the thymus, the tonsil and
smooth muscle. It is also highly expressed in lymphohe-
matopoietic cell lineages, particularly in BDCA4+ den-
dritic cells, CD34+ cells (bone marrow hematopoietic
stem cells), CD71+ early erythroid cells, B lymphoblasts,
CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK
cells and CD33+ myeloid cells (Table 1). As these cell
types are highly proliferative, we postulated that TRIP-Br2
plays an important role in cellular proliferation and/or


Page 4 of 15
(page number not for citation purposes)











Journal of Translational Medicine 2009, 7:8










A
STRIP-Br2/SERTAD2 (H. sapiens)




Chromosome 2p14


0 20kb
II I


http://www.translational-medicine.com/content/7/1/8


SLC14 CEP8


2p113


TRIP-Br2f
SERTAD2


RABIA


TRIP-Br2SERTAD2 (H. sapiens)


Chromosome 2p14



I I -eUO,20
'UTR

f p"


s r3 (on the reverse strand)


3' UTR
3" UTR


00 1020074
00 1033-714
F0555 70
51578
00102 6 20
_00Ci1 -16- ci08


001020074
l07133 '/114
0 bb /0
515728
00100 920

-997959


(R. norvegious)
(Mr musculus)
(H, sapiens)
(P troglodytes)
(M mulatta)
(G. gallas)
(D. reno)

(R. norvegious)
(M. musculus)
(H sapiens)
(P. troglodytes)
(M. mulatta)
(G gallas)
(D. rerio)


001020074 (R. norvegious)
n1n n.,714 (M, musculus)
o055s70 (H. sapiens)
Jl 72.8 (P. troglodytes)
1 olOI3 920 (M. mulatta)
001 0oa'08 (G gallas)
97<)59 (D. rerio)


00101 UO 0 14 (R. noregious)
0010,33 14 (M musculus)
055570 (H. sapiens)
5 15 72 (P troglodytes)
ooo so )O (M. mulatta)
C 001 (C. rgallas)
99 b,9 (D. rerio)


0ni n: 7 4
0511i n:7 2B
07,5,70
515728
1C,1O1s 920
00109940
997959


(R. norvegious)
(M musculus)
(H sapiens)
(P. troglodytes)
(M. mulatta)
(G. gallas)
(D. rerio)


0olo0102 4 (R- norveglous)
-001 n_-7 14 (M. musculus)
05557(0 (H. sapiens)
51572S (P troglodytes)
0010o882 (M. mulatta)
oolo001026 (G gallas)
99-95 9 (D. rerio)


0010.00o74
001033"-14
05_ b0o
515721,
01 O *920
997959


(R. norvegious)
(M. musculus)
(H. sapiens)
(P troglodytes)
(M. mulatta)
(G. gallas)
(D, rerio)


ML hKGCGKRK FDnI EL0 EDL EGKEhVVp-- --SDAF RV YTL-QRQTI NT ISLNMKLYNItRFLTWEP
MT.r4[<,-;CGIKRK PrFi.HPO-L4I.-;I .K'.I V : ; ---- ,P RV YTI Q T PNI T T.MK.YNHRP I -'TRP
MLK.I -.l; -KRK 'I)i-FMHlIX l ,1:A K I VSP---(':I,1:.P- KVKSY'TI.Q1 QLT I *N i .SUMKL.YNHPRI. I I T[P
MLGKC PRKFDEHEDGLEGKIVSFP-- -C DGP V S Y T LQ Q T I F I SLMKLYJHR.PLT E
MLGKGc G;RKFDEHJEDGLE C IVSUP----CEDGPS .ISYTLQRQTI FN I SLMKLYNHRPLiTEP
ML3IKG KRK FDEHEG LE KVVSP ---TEGPS KV S YTLORT. T IFN I SLMKLYNMHRPLT E E
MI,.-;I;'-AKPK I f'r;:] -.''.:* ,;, INALA ;A-.A-A i', KV8 YI l.c-<2T I INPJM.N I ,I F YNtIPHAV VI-'


SLQ KTVL NNTILRRI QEEL KQEGSLRr TETSQrSD- SL YREAPAFTPL------
sLQPKTVLTTNM LTPRIQEELCKQEG S LRPA FTFSS PASN- SL SESQE PFA----------
SLTQKTVLI.MiNN MPRI QEELKQEGS4LRP 4FTFS SQ2PTT-EPS S7yP.EAPFAFSHL------
GtLQ VL~NNMLKRIQtELK<$E ILR 4tKFMt'TL b-TTb2LS.YREAKLAA 3 LP L-.....
LQOITVLZNNMJLRRIQEELIKQEG SLtRPMFTESQPTT-E ZpFDYREGEPAE sIL-----
sT.QITVTTh.iNNMhR flTQFF2T KQE r`pFrVFVTASQr AD-rPLDrFT R AQPArSHTrP- ...--
S I.KF:IRVI. I NNMI.RRI ( L21)FT. 'F RiNT.RP.PT, F'P F'P PSPPIP ]f PVIS FPRE1PQPA-F SV I..SMVAPP-


--ASpF=IFiCDLCrSTT---------p-lCLXTP3lIEG-DDTFrCTLQ-Vrifp7STRX=
- ---PAPHP FT.A-T ----- -P F.AC 1 .TPAS i., R oDNIDnT FC(:TTr .A- VH'PAAPT R, .S
--AM PH.-; HPCI, :1'.1I. ''I"---- ---PI ,A< : I PA: Ih, I.RI il )-I/lI J'l CT' A-M- OPTAPT 1 F .-! P
--ASPiSI1 IF(-,DLGSTT------- mFLEAtL TAAULLBED--D'TTFCTSTA-- M'TrAPTKLS P
--APFS SHIPDLGSTT------PLEACLTASLLED- DDITEC'TSQA-MQPTAE TKLS
-- A PR S T. IDT .V.TT ---- PT.PLST.PAST -----TFTPT'-VQH D-C4PT-.PP
I .'QASPPA. ;A; Vl..T9'-P -Sl, NPAPlA APP C T Al E,:l-NVC; .;L.c V s;PlP-IAPPA PTIH),S


EAL.S.3AEKDSZFSSAL.DE: IEELC .-F'- -- S-T TST E A--AAA..---E.. TS ------T -A-SET
Z LE EKDS FS L-DE EELCF ----TSMTTE HTAF---EGGTS-------- SES
PT ALLPF KDS F L'SALEE iA F LC' F .-- --T ST 1AAAAAT ---- D UVK WT, S--------- SEA
'AIPVlKurL) --JATlol--' C---- '9UTAA AAA---- -AJV-K A Ju ;.;---------_ CTEA
PT --S--EKS --ALETEc-----TT.AAAAAT----avK --------EA
PAT ,Q PV KDH; F'S PsA T, OF ETET, ESVnA--RDSFSSALDEIEELCFPSPELTATISAPGCTSSPLQLCEPPSLNRSGLDSKDRCSKP

SVQ K PF.-4PQ ----- F.RTODSRPFMFST.N-- TTT --- ---. TIF[.Tl.TT.DT) T.FADTT
VQKP! PK: .-- R.I] I R 'M .P l I"1"---- [- *| -. 4-1 ] ,1 I I,'Al
iQK ,iPQ- -- '.RAIt,; KIM .-;PCM- ~' IT 'rS-----Tc1--'ITD T] .' .1)' 1FADT
GTQKLDGPQ---ESRA DriS ]LMDSLrPGN- FE I TT S ----- TGELTDLTL DDI FAD I DT S
,3AQKFPDLeOQ O---ERTO'DD MS LP*OT-M I Fz I TW.S TIU-- ---TGErL T L TL3DDL I F AnI ET-
SVFPEPEGVA----- ESRTAES KLMEPLP GN--FMEEITTS-.---...TGFLTDELTLD'[DIL FADIDTS
Gi'PKL i-h ;. VP L.Ah",BHR;AV PNl'PP.TI' ,PPNS; L,Msla'S P.SA.-SMSOi/'LTIL/AI ,DL.L F 11 'AI b I.I 'l s

MYIDFDEPCTSASGTA- SK-ABP-VSADDLLKTLAPFS ---NQPVA'YPSQF KMLTELD-HIMEV
MIy') L-pl]'CITA-N;GT 'A-"- KMAP-V AuIL)] )L.K! I AP Y,--.NOT PVAEP 'Qp P-RKMI Sl ; IH IM V-;V
MYL'FLD-T.fSSrTIA-SKtiMAPt-VSAD)[LLTI Al-YS--f, VTlSQ-F MyDFDPC<-TSSSCGTA--S Ki4AP --VSADDLLXKTLArpYS- -SQ VASQP FKMDI-TEL HTMEV
MYDFDPCTSSS GTA-S ITMAP--LVS2ADDLL FTLA y S ----SQVVAPISgQI ERDLT-IET L.H INMEIV
MYD FDPCTsnAGAS- sKM4AF --VsADELL MYAE FF'DE)CTS S Si= AAAP SK% APhLJTMVTAD EL2L TFS Y =S GTflE AVS S -QP1 EKIImDLT E LDH IMEV


T.VCS 315
LVGS 309
LVGS 314

LVOS 314
T..VC.4 3 >1
jw- _3 C, 1


Figure I

Gene structural organization of human TRIP-Br2. (A) TRIP-Br2 is localized at chromosome 2pl4 of the human genome

(B) TRIP-Br2 consists of two exons that are separated by an intron encoding splice donor-acceptor (GT-AG) sequences at

either end. (C) Multiple sequence alignment of TRIP-Br2 proteins from widely divergent species by BLAST/ClustalW analyses.








Page 5 of 15

(page number not for citation purposes)








Journal of Translational Medicine 2009, 7:8


tumor progression. This is supported in part by our previ-
ous observation that ablation of TRIP-Br2 resulted in cel-
lular proliferation arrest in primary cells, which was
associated with downregulation of a subset of E2F-respon-
sive genes such as CYCLIN E [17].

TRIP-Br2 overexpression transforms murine fibroblasts by
upregulation of E2FIDP-mediated transcription
To validate the protooncogenic role of TRIP-Br2 in cell
cycle regulation and tumorigenesis, we stably overex-

Table I: TRIP-Br2 expression profiling in human tissues by
interrogation of the Novartis GNF SymAtlas vl.2.4 microarray
database


Human tissues


TRIP-Br2 gene expression


Bronchial epithelial cells
Lung
Whole brain
Bone marrow*
Thymus*
Lymph node
Tonsil*
Heart
Liver
Kidney
Skin
Pancreas
Skeletal muscle
Cardiac myocytes
Smooth muscle*
Placenta*
Prostate
Uterus
Ovary
Testis


Lymphohematopoietic cell lineages
BM-CD34+ cells* ++++
BM-CD71 + early erythroid cells* ++++
BM-CD 105+ endothelial cells* +++
BM-CD33+ myeloid cells* ++++
PB-CD56+ NK cells* ++++
PB-BDCA4+ dendritic cells* +++
PB-CD 14+ monocytes* ++++
PB-CD 19+ B cells* ++++
B lymphoblasts* ++++
CD4+ T cells* +++
CD8+ T cells* +++

The median (med) was calculated based on expression of TRIP-Br2
across all human tissues; med x 3: 3-fold more than the median; med
x 10: 10-fold more than the median. In silicon TRIP-Br2 expression, (Z),
across all human tissues was scored via the following scheme: +: (Z) <
median; ++: median < (Z) < med x 3, +++: med x 3 <(Z) < med x 10,
++++: med x 10 <(Z). TRIP-Br2 is found to be highly expressed in
human tissues such as bone marrow, thymus, tonsil and smooth
muscle. TRIP-Br2 is also highly expressed in the highly proliferative
lymphohematopoietic cell lineages. *Denotes high TRIP-Br2 expression
in these cells and tissues. BM: Bone marrow-derived; PB: Peripheral
blood-derived.


http://www.translational-medicine.com/content/7/1/8



pressed C-terminal HA-tagged-TRIP-Br2 in NIH3T3
fibroblasts (NIH3T3TR1P-Br2-HA; Figure 2A). Although TRIP-
Brl overexpression has been recently shown to transform
NIH3T3 fibroblasts, the underlying molecular mecha-
nism of cellular transformation by TRIP-Brl remains elu-
sive. Thus, we also stably overexpressed carboxyl-terminal
HA-tagged-TRIP-Brl in NIH3T3 fibroblasts (NIH3T3TRP-
Brl-HA) and investigated the mechanisms) by which TRIP-
Brl and TRIP-Br2 facilitate cellular transformation. Over-
expression of TRIP-Brl-HA or TRIP-Br2-HA in NIH3T3
fibroblasts conferred the ability to proliferate under low
serum concentrations, possibly by enhancing DNA syn-
thesis (Figure 2B). Flow cytometric DNA analysis revealed
significantly higher proportions of NIH3T3TRIP-Brl-HA and
NIH3T3TRIP-Br2-HA fibroblasts in S phase of the cell cycle as
compared to the NIH3T3vector-only control, despite serum
deprivation (Figure 2C). As TRIP-Br proteins have been
shown to regulate E2F/DP-mediated transcriptional activ-
ities [7], we screened these serum-deprived NIH3T3
fibroblasts for a panel of E2F-responsive cell cycle regula-
tors that govern cell cycle progression. Elevated levels of a
subset of these E2F-responsive cell cycle regulators com-
prised of CYCLINE (CCNE), CYCLING A2 (CCNA2), CDC6
and DHFR, were found in serum-deprived fibroblasts that
stably overexpress TRIP-Br proteins (Figure 2D, upper
panel). Notably, in serum-deprived NIH3T3 cells that sta-
bly overexpress TRIP-Brl-HA or TRIP-Br2-HA, we
observed a concomitant increase in cyclin E expression
(Figure 2D, lower panel). This is consistent with our previ-
ous observation that cyclin E was downregulated follow-
ing ablation of TRIP-Brl or TRIP-Br2. Hence, our data
suggest that CYCLIN E may be a TRIP-Brl- and TRIP-Br2-
coregulated gene.

TRIP-Br2 overexpression confers anchorage-independent
growth in soft agar and promotes tumor growth in athymic
nude mice
Next, we evaluated the oncogenic potential of TRIP-Br2 by
assessing anchorage-independent growth of these
NIH3T3TRIP-Br2-HA fibroblasts in soft agar (Figure 3A). As
many as 17.7% of the seeded NIH3T3TRIP-Br2-HA fibroblasts
formed colonies at 4 weeks post-plating, while
NIH3T3vector-only fibroblasts were incapable of anchorage-
independent growth in soft agar. PC3 cells and
NIH3T3TRIP-Brl-HA fibroblasts were used as positive con-
trols in this assay.

In addition, we validated the tumorigenic potential of
TRIP-Br2 by an in vivo tumor formation assay. One inocu-
lum (5 x 106 cells) of either NIH3T3vector-only or
NIH3T3TRIP-Br2-HA fibroblasts (from one representative
clone each) was injected subcutaneously into the lower
flanks of athymic nude mice (n = 4). This experiment was
repeated at least twice by subcutaneous injection of a dif-




Page 6 of 15
(page number not for citation purposes)








http://www.translational-medicine.com/content/7/1/8


A R1- V- V- R2- C 100
19 20 19 16 16 17 19 4 14 43 0.2% FBS, 72h
80
OR1-19
a 4 TRIP-Br2-HA 8 70
Non-specific *0 0R1-20
C 60
0 OR2-4
50









BrdU Assay, 5000 cells (0.2%FBS, 72 h)
1 I p o p M p0J | 5p IL 30











R pR2-00



ao o the R pc
20



Phases of cell cycle








B NIH3T3 NH3D Semi sRT-PCR (Serum deprivation)
pg0 V- R- R12- V1 RI- R-
005 1617 19 20 4 43 16 7 19 20 4 43






Cl CCNE CCNA2E




















(NIH3T3TRIP-Br2-HA), selected by G418, and analyzed by immunoblotting using an anti-HA antibody. Data was obtained from
three independent experiments that were performed in triplicates. (B) NIH3T3veoro-nlY, NIH3T3TRIP-Brl-HA and NIH3T3TRIP-Br2-
HA fibroblasts were cultured in 96-well plates in DMEM supplemented with 0.2% FBS and were maintained for 72 h at 37C in a
5% COz environment. BrdU incorporation was monitored using a cell proliferation/colorimetric ELISA assay. The fold increase
in BrdU incorporation of all clones was calculated relative to that of V 16, which was set arbitrarily to 1.0. The error bars rep-
resent the standard deviations of three independent experiments performed in triplicates. A Student's t-test was performed
and the respective p-values were indicated in the bar chart. (C) Upon serum deprivation, S phase cell counts were significantly
higher in the TRIP-Br-overexpressing NIH3T3 clones than the vector-only control. The results shown represent the mean +
SD for each independent RI (-19 and -20) and R2 clone (-4, 14, 43), compared to all V clones combined (-16, -17, -19), and
incorporate data from 3 independent experiments performed in triplicate. A Student's t-test was performed; *indicates p-value
< 0.001; **indicates p-value < 0.01 for the comparison of NIH3T3vector-onlyand NIH3T3TRIP-Bri-HAor NIH3T3TRIP-Br2-HA cells. (D)
Upper panel: Semi-quantitative RT-PCR analyses revealed up-regulation of CYCLIN E (CCNE), CYCLIN A2 (CCNA2), CDC6 and
DHFR in serum-deprived NIH3T3TRIP-Bri-HAand NIH3T3TRIP-Br2-HAfibroblasts. TS: Thymidylate synthase; 18srRNA was used as a
loading control. Data was obtained from three independent experiments that were performed in triplicates. Lower panel: West-
ern blot analyses showed an increase in cyclin E in serum-deprived NIH3T3TRIP-Brl-HAand NIH3T3TRIP-Br2-HA fibroblasts when
these cells were immunostained with anti-cyclin E antibodies. Data was obtained from three independent experiments that
were performed in triplicates. DHR
V16 V17 R2-4 R2-14 R2-43 Ri-19 Ri-20
NIH3T3'---- NIH3T3's'--' NIH3T3-r"- Western Blot (Serum deornvation)
Ri- R2-


















19 20Page 7 of 514 4
(page number not for citation purposes)
3-tubuhlno -



Figure 2
TRIP-Br-overexpressing-NIH3T3 fibroblasts proliferate in the absence of mitogenic stimulation as a result of
deregulation of the RBIE2FIDPI transcriptional pathway. V: Vector-only clones, R I TRIP Brl -HA-overexpressing
clones, R2: TRIP-Br2-HA-overexpressing clones. 3-tubulin was used as a loading control. (A) NIH3T3 fibroblasts were trans-
fected with pCDNA3.l vector (NlH3T3vector-only), pCDNA3. I -TRIP-BrI--A (NIH3T3TR1P-Br -HA) or pCDNA3. I TRIP Br2-HA
(NIH3T3TRIP-Br2-HA), selected by GA 18, and analyzed by immunoblotting using an anti-HA antibody. Data was obtained from
three independent experiments that were performed in triplicates. (B) NIH3T3ve.- anly, NIH3T3TR1P-Br -HA and NIH3T3TR1P-Br2
HA fibroblasts were cultured in 96-well plates in DMEM supplemented with 0.2% FBS and were maintained for 72 h at 37'C in a
5% CO2 environment. BrdU incorporation was monitored using a cell proliferationlcolorimetric ELISA assay. The fold increase
in BrdU incorporation of all clones was calculated relative to that of V 16, which was set arbitrarily to 1.0. The error bars rep-
resent the standard deviations of three independent experiments performed in triplicates. A Student's t-test was performed
and the respective p-values were indicated in the bar chart. (C) Upon serum deprivation, S phase cell counts were significantly
higher in the TRIP-Br-overexpressing NIH3T3 clones than the vector-only control. The results shown represent the mean +
SD for each independent R I (- 19 and -20) and R2 clone (-A, 14, 43), compared to all V clones combined (- 16, -17, -19), and
incorporate data from 3 independent experiments performed in triplicate. A Student's t-test was performed; *indicates p-value
< 0.00 1; **indicates p-value < 0.0 1 for the comparison of NlH 3T3vector-onyand NIH 3T3TRIP-Bri -HA or NIH 3T3TRIP-Br2-HA cells. (D)
Upper panel: Semi-quantitative RT-PCR analyses revealed up-regulation of CYCLIN E (CCNE), CYCLIN A2 (CCNA2), CDC6 and
DHFR in serum-deprived NIH3T3TRIP-BrI HA and NlH3T3TRIP-Br2-HA fibroblasts. TS: Thymidylate synthase; I8srRNA was used as a
loading control. Data was obtained from three independent experiments that were performed in triplicates. Lower panel. West-
ern blot analyses showed an increase in cyclin E in serum-deprived NIH3T3TR1P-Br -HA and NIH3T3TR1P-Br2HA fibroblasts when
these cells were immunostained with anti-cyclin E antibodies. Data was obtained from three independent experiments that
were performed in triplicates.




Page 7 of 15
(page number not for citation purposes)


Journal of Translational Medicine 2009, 7:8







Journal of Translational Medicine 2009, 7:8


ferent clone of either NIH3T3vector-only or NIH3T3TRIP-Br2-HA
fibroblasts into other groups of four athymic nude mice.
Data from two mice from a representative experiment are
shown in Figure 3B (Upper panel). All sites injected with
NIH3T3TRIP-Br2-HA fibroblasts developed a tumor, which
was typically ~0.7 cm3 (data derived from one tumor
induction assay, n = 4) at day 25 post-injection (Figure 2B,
lower panel). Tumors derived from NIH3T3TRIP-Br2-HA
fibroblasts were histologically fibrosarcomas (Figure 3C).
Western blot analyses of tumor extracts (Figure 3D, upper
panel) as well as HA-immunostaining of paraffin-embed-
ded tumor sections (Figure 3D, lower panel) indicated the
presence of the transgene product TRIP-Br2-HA.

TRIP-Br2 expression is dysregulated in many human
cancer cell lines
Given that overexpression of TRIP-Br2 alone was suffi-
cient to transform NIH3T3 fibroblasts, we hypothesized
that expression of TRIP-Br2 may be dysregulated and con-
tribute to oncogenesis in human cancer. We screened nor-
mal and cancer cell lines for TRIP-Br2 expression using
rabbit anti-TRIP-Br2 polyclonal antibodies and found
that TRIP-Br2 was overexpressed in human cancer cell
lines U2OS, PC3, 769-P, HCT-116, HepG and MCF-7
cells, but not in WI38 diploid fibroblasts (Figure 4A). The
higher molecular weight endogenous species of TRIP-Br2
observed in Figure 4A (and 4C below) are specific bands
that we have observed in only some human cancer cell
lines, associated with the use of the rabbit polyclonal anti-
TRIP-Br2 for immunoblot analysis [23].

We next sought to identify the cellular role(s) of TRIP-Br2
by investigating its localization in WI38 and U2OS cells.
Using rabbit anti-TRIP-Br2 polyclonal antibodies, we first
demonstrated by immunocytochemistry that TRIP-Br2
was predominantly localized to the nuclei of WI38 and
U20S cells (Figure 4B), with scant cytoplasmic expres-
sion. This is in agreement with our earlier PSORT II pre-
diction of TRIP-Br2 subcellular localization and a recent
observation made by Lai and coworkers [30]. As com-
pared to WI38 cells, TRIP-Br2 was clearly overexpressed in
the nuclei of U2OS cells. Our data from subcellular frac-
tionation analysis is consistent with this observation.
TRIP-Br2 was overexpressed and predominantly localized
to the nuclear fractions of U2OS cells as well as other
human cancer cell lines such as PC3, 769-P, HCT-116 and
HepG2 (Figure 4C), suggesting that TRIP-Br2 expression
and localization might be dysregulated in these cancer
cells.

TRIP-Br2 is aberrantly expressed in multiple human solid
tumors and its overexpression is associated with poor
prognosis in HCC
In order to address an oncogenic role for TRIP-Br2 in
human cancers, we assessed the immunohistochemical
expression of TRIP-Br2 by comparing normal and cancer


http://www.translational-medicine.com/content/7/1/8



tissue sections on microarrays that were constructed from
patient specimens of 10 different human tumor types. Tis-
sue microarray (TMA) is a high-throughput method for
the analysis of large numbers of formalin-fixed, paraffin-
embedded (FFPE) materials with minimum cost and
effort [31]. We found that TRIP-Br2 was overexpressed in
prostate carcinoma (50.8%), squamous cell lung carci-
noma (100%), lung adenocarcinoma (48.7%), ovarian
cystadenocarcinoma (73.1%), colorectal carcinoma
(64.9%), renal cell carcinoma (50%), osteosarcoma
(100%) and hepatocellular carcinoma (72.4%). Notably,
the frequency of TRIP-Br2 overexpression was lower in
breast carcinoma (25%), basal cell carcinoma (16.7%)
and gastrointestinal stromal tumor (15.6%). We also
observed minor variations of TRIP-Br2 overexpression
between different subtypes of ovarian carcinoma such as
serious, mucinous and endometroid ovarian cystadenocar-
cinoma (data not shown). A representative TRIP-Br2-
immunostained tumor specimen from each of the 10
tumor tissues and corresponding normal tissues exam-
ined by TMA are shown in Figure 5A and Additional File
2, respectively. The frequency of TRIP-Br2 upregulation in
these human cancers is summarized in Additional Table
S1 (see Additional File 1).

Next, we investigated the effect of TRIP-Br2 overexpres-
sion on the survival of hepatocellular carcinoma (HCC)
patients to determine whether TRIP-Br2 overexpression is
associated with poor prognosis. A patient cohort (n = 12)
with full survival data was divided into two groups, sur-
vival < 1 year (n = 8) and survival > 1 year (n = 4). These
two groups were subsequently scored as "TRIP-Br2 overex-
pressors" versus "TRIP-Br2 non-overexpressors" in the
corresponding tumor tissue biopsies represented on TMAs
(Figure 5A). A patient was scored as a "TRIP-Br2 overex-
pressor" if the intensity of TRIP-Br2 immunostaining in
tumor tissue was observed to be more intense than adja-
cent normal tissue. Six of eight HCC patients were TRIP-
Br2 overexpressors and were found to have survived for _
1 year, while three of four HCC patients were TRIP-Br2
non-overexpressors and were found to have survived for >
1 year. A survival analysis using the Kaplan Meier log rank
test was performed, which showed that the mean survival
of patients exhibiting tumor tissue TRIP-Br2 overexpres-
sion (9 months) was found to be significantly lower than
the survival of HCC patients without evidence of TRIP-Br2
overexpression (16 months) (p = 0.0452) (Figure 5B).
This observation is not only significant from a statistical
viewpoint, but also clinically in the context of a cancer
type with a particularly poor prognosis.

RNA interference of TRIP-Br2 expression inhibits cell-
autonomous growth of HCT-I 16 human colorectal cancer
cells
To validate the potential of TRIP-Br2 as a novel transcrip-
tion-based chemotherapeutic target for human cancers,

Page 8 of 15
(page number not for citation purposes)








Journal of Translational Medicine 2009, 7:8






A Coloni formaton (S

30 0%

250%

S2010% 14%

15,0%






0,0% -


ar Assay. 1000 cea 4 weAksl


PC3 V16 V17 V19 R2-4 R2-14 R243 R1-19 R1-20
NHST3"'O'"" NI3T3T3 'a" NIHI3T3 "'"-


http://www.translational-medicine.com/content/7/1/8





C H &E









Tumr
Sqi







__^ __ Tumr 2


D Cell lysates


R2. V. Tumors
A" Ir 17 10 1 1


ITRIP B2 HA


--- a- -tubu in

IHC: anti-HA


0 13 1 1t7 19 21 Z
Ofys po~r h~c, inlft


--fhr- riJf
...Nh., 7ri MNI:


Figure 3
Overexpression of TRIP-Br2-HA confers anchorage-independent growth on soft agar and induces tumors in
nude mice (nulnu). (A) Anchorage-independent growth of NIH3T3vector-only, NIH3T3TRIP-BrI-HA and NIH3T3TRIP-Br2-HAwas
assessed by colony formation in soft agar. The error bars represent the standard deviations of three independent experiments
performed in triplicates. PC3 cells were used as a positive control. V: Vector-only clones; RI: TRIP-Brl-HA-overexpressing
clones; R2: TRIP-Br2-HA-overexpressing clones. (B) Upper panel: Results of a representative experiment in which NIH3T3TRIP-
Br2-HA and NIH3T3vector-only fibroblasts were subcutaneously injected into the left and right flanks of nude mice, respectively.
Lower panel: Average tumor ellipsoid volume over 25 days post-subcutaneous injection was calculated, and the animals were
subsequently sacrificed. (C) Histological analyses of excised tumors indicated the presence of fibrosarcomas. (D) Western blot
(Upper panel) and immunohistochemical analyses (Lower panel) of excised tumors showed expression of TRIP-Br2-HA. Immu-
nopositive staining for TRIP-Br2-HA is represented by the brown color against the hematoxylin (blue) counterstain. Data was
obtained from three independent experiments that were performed in triplicates.




Page 9 of 15
(page number not for citation purposes)







Journal of Translational Medicine 2009, 7:8


we performed siRNA knockdown of TRIP-Br2 expression
in HCT-116 cells. Cy3-labeled oligomer transfection con-
trol (Cy3-O), scrambled siRNA non-specific control (Scr)
or TRIP-Br2-specific siRNAs (DS1, DS2 or DS3) were tran-
siently transfected into HCT-116 cells, respectively, at a
low dose of 4 pmol or a high dose of 40 pmol (in one ml
of DMEM supplemented with 10% FBS). Twenty-four
hours post-transfection, these cells were serum-deprived
for 72 h to investigate the role of TRIP-Br2 in cell-autono-
mous growth of HCT-116 cells. As shown in Figure 6A
(Left panel), specific knockdown of TRIP-Br2 expression in
HCT-116 cells (12-well plate) was only achieved by TRIP-
Br2-specific siRNAs, DS1 and DS2, at the higher dose of
40 pmol. There were no changes in the transcript levels of
other TRIP-Br gene family members upon treatment with
TRIP-Br2-specific siRNAs, DS1 and DS2, as assessed by
semi-quantitative RT-PCR (Figure 6A, right panel).Western
blot analyses further revealed that TRIP-Br2 protein
expression was significantly knocked down by TRIP-Br2-
specific siRNAs, DS1 and DS2 (Figure 6B). In addition,
colony forming assays (Figure 6C) and cell count analyses
(Figure 6D) showed that siRNA knockdown of TRIP-Br2
expression inhibited cell-autonomous growth of serum-
deprived HCT-116 cells.

Discussion
The TRIP-Br proteins represent a novel family of mamma-
lian transcriptional coregulators that recruit PHD zinc fin-
ger- and/or bromodomain-containing transcription
factors such as p300/CBP to the E2F/DP transcriptional
complexes in order to regulate E2F-mediated gene tran-
scription and cell cycle progression [7]. We recently
reported that ablation of TRIP-Brl or TRIP-Br2 expression
suppresses serum-inducible CYCLIN E expression. The
deficiency of either TRIP-Brl or TRIP-Br2 resulted in pro-
liferative block, indicating that these proteins may have
interdependent but not superimposable roles in the regu-
lation of serum-inducible cell cycle progression [17].
Although amplification of TRIP-Brl is commonly
detected in ovarian cancers [16] and overexpression of
TRIP-Brl has been shown to induce tumors in nude mice
[18], the role of its closely related family member, TRIP-
Br2, in cell cycle regulation and tumor progression has not
been elucidated.

With an increasing number of mRNA expression profiling
studies employing microarrays showing a positive correla-
tion between TRIP-Br2 overexpression and cellular prolif-
eration [32-37], we postulated that TRIP-Br2 plays an
important protooncogenic role in cell cycle regulation
and tumor progression. To validate its functions) in
growth and proliferation, we stably overexpressed TRIP-
Br2 in NIH3T3 fibroblasts and demonstrated that TRIP-
Br2 overexpression transformed these murine fibroblasts,
rendering them capable of proliferation under low serum
concentrations and of anchorage-independent growth in


http://www.translational-medicine.com/content/7/1/8



soft agar. We also demonstrated that overexpression of
TRIP-Br2 induced tumors in athymic nude mice (nu/nu).
Transformed cellular phenotypes were associated with
dysregulation of the E2F/DP-transcriptional pathway
through upregulation of a subset of key E2F-responsive
genes, such as CYCLIN E, CYCLIN A2, CDC6 and DHFR.
Furthermore, we have shown in our knockdown/knock-
out and overexpression studies that CYCLIN E is indeed a
TRIP-Br-coregulated gene. Ongoing microarray studies
will help us to identify other candidate TRIP-Br-coregu-
lated genes and to establish the mechanism by which
TRIP-Br proteins promote growth and tumor progression.

As overexpression of TRIP-Br2 resulted in the transforma-
tion of NIH3T3 fibroblasts, we hypothesized that TRIP-
Br2 expression is dysregulated in human cancer. We
found TRIP-Br2 to be overexpressed in many cancer cell
lines and observed its localization to the nucleus. We sub-
sequently showed that TRIP-Br2 was also overexpressed in
many human cancers, including prostate carcinoma,
squamous cell lung carcinoma, lung adenocarcinoma,
ovarian cystadenocarcinoma, colorectal carcinoma, renal
cell carcinoma, osteosarcoma and hepatocellular carci-
noma. Notably, we observed that the expression pattern
of TRIP-Br2 in these multiple human tumors in vivo
matched that observed in cultured cells originally derived
from these tumors. For instance, in both osteosarcoma tis-
sues and U2OS cells, TRIP-Br2 was overexpressed and
localized to the nucleus. No nuclear presence and little or
no cytoplasmic expression of TRIP-Br2 were observed in
normal prostate, lung, breast, gastric, ovary, colon, skin or
kidney sections (Additional File 2). These data demon-
strate that TRIP-Br2 is frequently and highly expressed in
tumors, but not in the corresponding normal tissues and
suggests that TRIP-Br2 expression and localization may be
dysregulated in tumors. We have also observed overex-
pression of TRIP-Br2 in the cytoplasm of a small subset of
these tumor specimens (data not shown), suggesting that
TRIP-Br2 may perform novel functions in the cytoplasm
and/or intracellular organelles to support oncogenesis in
these tumor subsets. Collectively, our data suggest that
TRIP-Br2 is a bona-fide protooncogene and that its overex-
pression may be associated with poor prognosis in human
cancers, as demonstrated in the case of hepatocellular car-
cinoma.

We envisage that the mechanism of overexpression of
TRIP-Br proteins may exist at the post-translational level
in human cancers and may involve dysregulation of pro-
tein turnover. Indeed, we have recently shown that muta-
tion of leucine residue 238 of the highly conserved
nuclear export signal (NES) motif of TRIP-Br2 led to the
nuclear entrapment of TRIP-Br2 and abolished it protein
turnover [38]. Ongoing high-throughput DNA sequenc-
ing of the corresponding human tumor samples identified
in our TMA immunoscreen will help us to identify novel

Page 10 of 15
(page number not for citation purposes)







http://www.translational-medicine.com/content/7/1/8


A


< TRIP-Br2



-i0i)- .0. 4 P-tubulin



B W138 U20S




C-


C 3S u2oS PC1 769-P ICT-11I HepG2
C N C N CN C N C N C N


-a i TRIP-Br2

o A Larmin B

j, en GAPDH



Figure 4
TRIP-Br2 expression is dysregulated in many human cancer cell lines. (A) Western blot analyses revealed overex-
pression of TRIP-Br2 in the human cancer cell lines U2OS, PC3, 769-P, HCT-1 16, HepG2 and MCF-7. Data was obtained from
three independent experiments that were performed in triplicates. (B) Immunocytochemical analyses showed that TRIP-Br2 is
found predominantly in both the nuclei of W138 and U20S cells. Cellular DNA was counterstained with DAPI (blue). (C) Sub-
cellular fractionation analyses revealed that TRIP-Br2 is overexpressed and preferentially localized to the nuclei of U2OS, PC3,
769-P, HCT- 16 and HepG2 cells. Data was obtained from three independent experiments that were performed in triplicates.



Page 11 of 15
(page number not for citation purposes)


Journal of Translational Medicine 2009, 7:8








http://www.translational-medicine.com/content/7/1/8


B Mortality
100 TRIP-Br2
overexpression
80 No TRIP-Br2
overexpression
60 -


40 1


20


0
0 5 10 15 20 25 30
Time(months)


Figure 5
TRIP-Br2 is overexpressed in multiple human solid tumors and associated with poor prognosis in hepatocellu-
lar carcinoma (HCC). (A) Multiple human tumor tissue arrays were immunostained with rabbit anti-TRIP-Br2 polyclonal
antibodies. I: Prostate carcinoma; 2: Squamous cell lung carcinoma; 3: Breast carcinoma; 4: Gastrointestinal stromal tumors,
GIST; 5: Renal cell carcinoma; 6: Ovarian carcinoma; 7: Colon carcinoma; 8: Basal cell carcinoma; 9: Hepatocellular carcinoma;
10: Osteosarcoma. The small insert represents 400x magnification of the tissue in each window (shown at I 00x magnification).
A scale is included in the small insert of window #1 (for all 400x magnified tissue specimens). Immunopositive staining for
hTRIP-Br2 is represented by the brown color against the hematoxylin (blue) counterstain. Data was obtained from three inde-
pendent experiments that were performed in triplicates. (B) TRIP-Br2 overexpression is associated with poor survival of HCC
patients (n = 12). The mean survival of patients with TRIP-Br2 overexpression (9 months) was significantly lower than that of
HCC patients without TRIP-Br2 overexpression (16 months). The p-value of this survival analysis was determined to be 0.0452
using the Kaplan Meier log rank test.




Page 12 of 15
(page number not for citation purposes)


Journal of Translational Medicine 2009, 7:8








http://www.translational-medicine.com/content/7/1/8


A TRlP-&2h C 5XiOV cells plated (OFBS. 72h1
NiRMA (4opmIn 1 2 3
$cr DS 1 DS DS3
Nol
skRNA (4pmol) -
Scr DS1I DS2 0S3
TRP-ar2 Scrambled ta
TrIP.&2 siRNA
ROT- ~W. '


18srRNA


TRIP-BF2
TRp-&3 g iRNA-D51

18srRNA TRiPBr2
ziRN A-1352


B TRIP-B2
-iRNA (4pmof)
NT Scr DS 0SO DS3Cy3.O 18000
1600e .
TRIP-Br2
S140000





NT Scr 0DS I DS2 S3Ry3-O


4Tr00m0










siRNA knockdown of TRIP-Br2 expression inhibits cell-autonomous growth of HCT-I 16 cells. 4 pmol or 40 pmol
-"





of C3-abeled oligomer control (C3-), scrambled siRNA (Scr) and TRP-r2-specific siRNAs (DS I, DS2 or DS3) were tran-
Traidment


Figure 6
siRNA knockdown of TRIP-Br2 expression inhibits cell-autonomous growth of HCT- 116 cells. 4 pmol or 40 pmol
of Cy3-labeled oligomer control (Cy3-O), scrambled siRNA (Scr) and TRIP-Br2-specific siRNAs (DS I, DS2 or DS3) were tran-
siently transfected into HCT-1 16 cells respectively. "Not transfected" (NT) samples were used as negative controls. The spe-
cificity of TRIP-Br2-specific siRNAs was assessed by semi-quantitative RT-PCR and Western blot analyses. Three independent
experiments were performed in triplicates. (A) Knockdown of TRIP-Br2 expression in HCT- 116 cells (12-well plate) was
achieved by TRIP-Br2-specific siRNAs, DSI and DS2, at the dose of 40 pmol. 18srRNA was used as a loading control. (B) TRIP-
Br2 protein expression was significantly knocked down by TRIP-Br2-specific siRNAs, DS I and DS2, at the dose of 40 pmol. B-
tubulin was used as a loading control. (C) Colony forming assays and (D) cell count analyses showed that siRNA knockdown of
TRIP-Br2 expression (DS- I or DS-2 at the dose of 40 pmol) inhibited cell-autonomous growth of serum-deprived HCT- 116
cells. The dashed line indicates the initial cell density plated. Data was obtained from three independent experiments that were
performed in triplicates.


Page 13 of 15
(page number not for citation purposes)


%W too WW "W


. ..lr


Journal of Translational Medicine 2009, 7:8








Journal of Translational Medicine 2009, 7:8


disease-inducing mutations in the coding sequence, and
the 5' and 3' regulatory regions of TRIP-Br2. We further
validated the potential of TRIP-Br2 as a novel transcrip-
tion-based chemotherapeutic target for human cancers by
demonstrating that siRNA knockdown of TRIP-Br2 inhib-
ited cell-autonomous growth of serum-deprived HCT- 116
cells. Notably, we have also shown that antagonism of the
TRIP-Br integrator function by synthetic decoy peptides,
which compete with TRIP-Br for binding to PHD zinc fin-
ger- and/or bromodomain-containing proteins, arrests
proliferation and induces caspase-3-independent sub-dip-
loidization in cancer cells in vitro [23].

In summary, we have identified TRIP-Br2 as a novel pro-
tooncogene that is aberrantly overexpressed in human
cancers. By making use of a comprehensive and high-
throughput tissue microarray technology, we were able to
advance rapidly from experimental validation of the pro-
tooncogenic role of TRIP-Br2 to identifying its value in
translational medicine for the potential treatment of a
wide variety of human cancers.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
All authors have read and approval the final manuscript.
JKC participated in study design, data acquisition, inter-
pretation and manuscript writing. LG participated in
study design and data interpretation. ZZ, CY, SLN, KGS,
JVB participated in data interpretation. XMS participated
in tissue culture-related work. SAR and BKP participated
in tissue microarray-related work. MST and SIH designed
the study and led the data interpretation and manuscript
writing.

Additional material


Additional file 1
Additional Materials. This file contains additional materials entitled 1)
"Construction of C-terminal HA-tagged hTRIP-Br2 expression plasmid"
(Additional Methods); 2) "Frequency of TRIP-Br2 overexpression in 10
Im .. . human cancers" (Additional Table Sl); 3) "TRIP-Br2 expres-
sion in multiple normal human tissues and benign tumors" (Figure Leg-
end for Additional Figure Sl).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1479-
5876-7-8-S1.doc]

Additional file 2
TRIP-Br2 expression in multiple normal human tissues and benign
tumors. The data presents the results of immunostaining of multiple nor-
mal or benign human tumor tissue arrays with rabbit anti-TRIP-Br2 pol-
yclonal antibodies.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1479-
5876-7-8-S2.tiff]


http://www.translational-medicine.com/content/7/1/8




Acknowledgements
We are grateful to Sushrut Waikar (Brigham and Women's Hospital) for
his kind assistance in the survival analysis of HCC patients. We thank Eileen
O'Leary (Brigham and Women's Hospital) for her technical support and
Antonis Zervos (University of Central Florida) for helpful discussions and
critical reading of the manuscript. This work was supported by SCS Grants
MN-05 & MN-77, awarded by the Singapore Cancer Syndicate, Agency for
Science, Technology and Research, Singapore, to M. Salto-Tellez and intra-
mural support from the Renal Division, Brigham and Women's Hospital to
S.I. Hsu.

References
I. Ikediobi ON, Davies H, Bignell G, Edkins S, Stevens C, O'Meara S,
Santarius T, Avis T, Barthorpe S, Brackenbury L, Buck G, Butler A,
Clements J, Cole J, Dicks E, Forbes S, Gray K, Halliday K, Harrison R,
Hills K, Hinton J, Hunter C, Jenkinson A, Jones D, Kosmidou V, Lugg
R, Menzies A, Mironenko T, Parker A, PerryJ, Raine K, Richardson D,
Shepherd R, Small A, Smith R, Solomon H, Stephens P, Teague J, Tofts
C, Varian J, Webb T, West S, Widaa S, Yates A, Reinhold W, Wein-
steinJN, Stratton MR, Futreal PA, Wooster R: Mutation analysis of
24 known cancer genes in the NCI-60 cell line set. Mol Cancer
Ther 2006, 5:2606-2612.
2. Guy CT, Zhou W, Kaufman S, Robinson MO: E2F-I blocks termi-
nal differentiation and causes proliferation in transgenic
megakaryocytes. Mol Cell Biol 1996, 16:685-693.
3. Lazzerini Denchi E, Helin K: E2F I is crucial for E2F-dependent
apoptosis. EMBO reports 2005, 6:661-668.
4. Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA,
Dynlacht BD: E2F integrates cell cycle progression with DNA
repair, replication, and G(2)/M checkpoints. Genes Dev 2002,
16:245-256.
5. Darwish H, Cho JM, Loignon M, Alaoui-Jamali MA: Overexpression
of SERTAD3, a putative oncogene located within the 19q 13
amplicon, induces E2F activity and promotes tumor growth.
Oncogene 2007.
6. Hayashi R, Goto Y, Ikeda R, Yokoyama KK, Yoshida K: CDCA4: a
nuclear factor induced by the E2F transcription factor family
that regulates E2F-dependent transcriptional activation and
cell proliferation. J Biol Chem 2006.
7. Hsu SI, Yang CM, Sim KG, Hentschel DM, O'Leary E, Bonventre JV:
TRIP-Br: a novel family of PHD zinc finger- and bromodo-
main-interacting proteins that regulate the transcriptional
activity of E2F- I/DP-I. EmboJ 2001, 20:2273-2285.
8. Bennetts JS, Fowles LF, Berkman JL, van Bueren KL, Richman JM,
Simpson F, Wicking C: Evolutionary conservation and marine
embryonic expression of the gene encoding the SERTA
domain-containing protein CDCA4 (HEPP). Gene 2006,
374:153-165.
9. Calgaro S, Boube M, Cribbs DL, Bourbon HM: The Drosophila
gene taranis encodes a novel trithorax group member
potentially linked to the cell cycle regulatory apparatus.
Genetics 2002, 160:547-560.
10. Li J, Melvin WS, Tsai MD, Muscarella P: The nuclear protein
p34SEI-l regulates the kinase activity of cyclin-dependent
kinase 4 in a concentration-dependent manner. Biochemistry
2004, 43:4394-4399.
II. Sugimoto M, Nakamura T, Ohtani N, Hampson L, Hampson IN, Shi-
mamoto A, Furuichi Y, Okumura K, Niwa S, Taya Y, Hara E: Regula-
tion of CDK4 activity by a novel CDK4-binding protein,
p34(SEI-1). Genes Dev 1999, 13:3027-3033.
12. Sham JS, Tang TC, Fang Y, Sun L, Qin LX, Wu QL, Xie D, Guan XY:
Recurrent chromosome alterations in primary ovarian car-
cinoma in Chinese women. Cancer Genet Cytogenet 2002,
133:39-44.
13. Thompson FH, Nelson MA, TrentJM, Guan XY, Liu Y, YangJM, Emer-
son J, Adair L, Wymer J, Balfour C, Massey K, Weinstein R, Alberts
DS, Taetle R: Amplification of 19q13. 1-q 3.2 sequences in
ovarian cancer. G-band, FISH, and molecular studies. Cancer
Genet Cytogenet 1996, 87:55-62.
14. Hoglund M, Gorunova L, Andren-Sandberg A, Dawiskiba S, Mitelman
F,Johansson B: Cytogenetic and fluorescence in situ hybridiza-
tion analyses of chromosome 19 aberrations in pancreatic
carcinomas: frequent loss of 19pl3.3 and gain of 19ql3.1-
13.2. Genes Chromosomes Cancer 1998, 21:8-16.


Page 14 of 15
(page number not for citation purposes)








Journal of Translational Medicine 2009, 7:8


15. Ried T, Petersen I, Holtgreve-Grez H, Speicher MR, Schrock E, du
Manoir S, Cremer T: Mapping of multiple DNA gains and losses
in primary small cell lung carcinomas by comparative
genomic hybridization. Cancer Res 1994, 54:1801-1806.
16. Tang TC, Sham JS, Xie D, Fang Y, Huo KK, Wu QL, Guan XY: Iden-
tification of a candidate oncogene SEI-I within a minimal
amplified region at 19q13.1 in ovarian cancer cell lines. Can-
cer res 2002, 62:7157-7161.
17. Sim KG, Cheong JK, Hsu SI: The TRIP-Br family of transcrip-
tional regulators is essential for the execution of cyclin E-
mediated cell cycle progression. Cell Cycle 2006, 5:11 I 1-1I 15.
18. Tang DJ, Hu L, Xie D, Wu QL, Fang Y, Zeng Y, Sham JS, Guan XY:
Oncogenic transformation by SEI-I is associated with chro-
mosomal instability. Cancer res 2005, 65:6504-6508.
19. Hirose T, Fujii R, Nakamura H, Aratani S, Fujita H, Nakazawa M,
Nakamura K, Nishioka K, Nakajima T: Regulation of CREB-medi-
ated transcription by association of CDK4 binding protein
p34SEI-l with CBP. IntJ MolMed 2003, I 1:705-712.
20. Gupta S, Takhar PP, Degenkolbe R, Koh CH, Zimmermann H, Yang
CM, Guan Sim K, Hsu SI, Bernard HU: The human papillomavirus
type I I and 16 E6 proteins modulate the cell-cycle regulator
and transcription cofactor TRIP-Brl. Virology 2003,
317:155-164.
21. Sim KG, Zang Z, Yang CM, BonventreJV, Hsu SI: TRIP-Br links E2F
to novel functions in the regulation of cyclin E expression
during cell cycle progression and in the maintenance of
genomic stability. Cell Cycle 2004, 3:1296-1304.
22. Watanabe-Fukunaga R, lida S, Shimizu Y, Nagata S, Fukunaga R: SEI
family of nuclear factors regulates p53-dependent transcrip-
tional activation. Genes Cells 2005, 10:851-860.
23. Zang ZJ, Sim KG, Cheong JK, Yang CM, Yap CS, Hsu SI: Exploiting
the TRIP-Br Family of Cell Cycle Regulatory Proteins as
Chemotherapeutic Drug Targets in Human Cancer. Cancer
Biol Ther 2007, 6:712-718.
24. Radkov SA, Kellam P, Boshoff C: The latent nuclear antigen of
Kaposi sarcoma-associated herpesvirus targets the retino-
blastoma-E2F pathway and with the oncogene Hras trans-
forms primary rat cells. Nat Med 2000, 6:1 121- 127.
25. Tomayko MM, Reynolds CP: Determination of subcutaneous
tumor size in athymic (nude) mice. Cancer Chemother Pharmacol
1989, 24:148-154.
26. Salto-Tellez M, Lee SC, Chiu LL, Lee CK, Yong MC, Koay ES: Micro-
satellite instability in colorectal cancer: considerations for
molecular diagnosis and high-throughput screening of archi-
val tissues. Clin Chem 2004, 50:1082-1086.
27. Salto-Tellez M, Nga ME, Han HC, Wong AS, Lee CK, Anuar D, Ng SS,
Ho M, Wee A, Chan YH, Soong R: Tissue microarrays character-
ise the clinical significance of a VEGF-A protein expression
signature in gastrointestinal stromal tumours. British j cancer
2007, 96:776-782.
28. Salto-Tellez M, Peh BK, Ito K, Tan SH, Chong PY, Han HC, Tada K,
Ong WY, Soong R, Voon DC, Ito Y: RUNX3 protein is overex-
pressed in human basal cell carcinomas. Oncogene 2006,
25:7646-7649.
29. Zhang D, Salto-Tellez M, Do E, Putti TC, Koay ES: Evaluation of
HER-2/neu oncogene status in breast tumors on tissue
microarrays. Human pathol 2003, 34:362-368.
30. Lai IL, Wang SY, Yao YL, Yang WM: Transcriptional and subcel-
lular regulation of the TRIP-Br family. Gene 2007, 388:102-109.
31. Kononen J, Bubendorf L, Kallioniemi A, Barlund M, Schraml P,
Leighton S, TorhorstJ, Mihatsch MJ, Sauter G, Kallioniemi OP: Tissue
microarrays for high-throughput molecular profiling of
tumor specimens. Nat med 1998, 4:844-847.
32. Albertini V, Jain A, Vignati S, Napoli S, Rinaldi A, Kwee I, Nur-e-Alam
M, Bergant J, Bertoni F, Carbone GM, Rohr J, Catapano CV: Novel
GC-rich DNA-binding compound produced by a genetically
engineered mutant of the mithramycin producer Strepto-
myces argillaceus exhibits improved transcriptional repres-
sor activity: implications for cancer therapy. Nucleic acids res
2006, 34:1721-1734.
33. Gentile M, Latonen L, Laiho M: Cell cycle arrest and apoptosis
provoked by UV radiation-induced DNA damage are tran-
scriptionally highly divergent responses. Nucleic acids res 2003,
31:4779-4790.
34. Klener P, Szynal M, Cleuter Y, Merimi M, Duvillier H, Lallemand F,
Bagnis C, Griebel P, Sotiriou C, BurnyA, Martiat P, Broeke A Van den:


http://www.translational-medicine.com/content/7/1/8




Insights into gene expression changes impacting B-cell trans-
formation: cross-species microarray analysis of bovine leuke-
mia virus tax-responsive genes in ovine B cells. J virol 2006,
80:1922-1938.
35. Kuchinskaya E, Heyman M, Grand6r D, Linderholm M, Soderhall S,
Zaritskey A, Nordgren A, Porwit-Macdonald A, Zueva E, Pawitan Y,
Corcoran M, Nordenskjold M, Blennow E: Children and adults
with acute lymphoblastic leukaemia have similar gene
expression profiles. European] Haematol 2005, 74:466-480.
36. Soo RA, Wu J, Aggarwal A, Tao Q, Hsieh W, Putti T, Tan KB, Low JS,
Lai YF, Mow B, Hsu S, Loh KS, Tan L, Tan P, Goh BC: Celecoxib
reduces microvessel density in patients treated with
nasopharyngeal carcinoma and induces changes in gene
expression. Ann Oncol 2006.
37. Turk R, Sterrenburg E, Wees CG van der, de Meijer EJ, de Menezes
RX, Groh S, Campbell KP, Noguchi S, van Ommen GJ, den Dunnen
JT, t Hoen PA: Common pathological mechanisms in mouse
models for muscular dystrophies. FASEBJ 2006, 20:127-129.
38. Cheong JK, Gunaratnam L, Hsu SI: CRMI-mediated nuclear
export is required for 26S proteasome-dependent degrada-
tion of the TRIP-Br2 protooncoprotein. J Biol Chem 2008.


Page 15 of 15
(page number not for citation purposes)


Publish with BioMed Central and every
scientist can read your work free of charge
"BioMed Central will be the most significant development for
disseminating the results of biomedical research in our lifetime.
Sir Paul Nurse, Cancer Research UK
Your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours you keep the copyright

Submit your manuscript here: BioMedcentral
http://ww .biomedcentral.com/info/publishingadv.asp




University of Florida Home Page
© 2004 - 2010 University of Florida George A. Smathers Libraries.
All rights reserved.

Acceptable Use, Copyright, and Disclaimer Statement
Last updated October 10, 2010 - - mvs