Oncogene (2006) 25, 4207–4216
& 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00
www.nature.com/onc
ORIGINAL ARTICLE
Expression of GCIP in transgenic mice decreases susceptibility to chemical
hepatocarcinogenesis
W Ma1,2,3,4, X Xia5, LJ Stafford1,2,3, C Yu1,2,3, F Wang1,2,3, G LeSage5 and M Liu1,2,3
1
Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, Houston, TX, USA; 2Center
for Cancer Biology and Nutrition, Texas A&M University System Health Science Center, Houston, TX, USA; 3Department of
Molecular and Cellular Medicine, Texas A&M University System Health Science Center, Houston, TX, USA; 4The Graduate School
of Biomedical Sciences, The University of Texas-Houston Health Science Center, Houston, TX, USA and 5Department of Internal
Medicine, The University of Texas-Houston Health Science Center, Houston, TX, USA
Transcription factors with helix–loop–helix (HLH) motif
play critical roles in controlling the expression of genes
involved in lineage commitment, cell fate determination,
proliferation, and tumorigenesis. To examine whether
the newly identified HLH protein GCIP/CCNDBP1
modulates cell fate determination and plays a role in
hepatocyte growth, proliferation, and hepatocarcinogenesis, we generated transgenic mice with human GCIP
gene driven by a liver-specific albumin promoter. We
demonstrated that in GCIP transgenic mice, the overall
liver growth and regeneration occurred normally after
liver injury induced by carbon tetrachloride (CCl4). In the
diethylnitrosamine (DEN)-induced mouse hepatocarcinogenesis, we demonstrated that overexpression of GCIP in
mouse liver suppressed DEN-induced hepatocarcinogenesis at an early stage of tumor development. The number
of hepatic adenomas at 24 weeks was significantly lower
or not detected in GCIP transgenic male mice compared
to the control mice under the same treatment. Although
GCIP has little inhibition on the number of hepatic tumors
at later stages (40 weeks), hepatocellular tumors in GCIP
transgenic mice are smaller and well-differentiated
compared to the poorly differentiated tumors in wildtype mice. Furthermore, we demonstrate that GCIP functions as a transcriptional suppressor, regulates the
expression of cyclin D1, and inhibits anchorage-independent cell growth and colony formation in HepG2 cells,
suggesting a significant role of GCIP in tumor initiation
and development.
Oncogene (2006) 25, 4207–4216. doi:10.1038/sj.onc.1209450;
published online 27 February 2006
Keywords: GCIP; CCNDBP1; DIP1/HHM; transgenic
mouse;
hepatocarcinogenesis;
diethylnitrosamine;
HLH-Zipper proteins
Correspondence: Dr M Liu, Alkek Institute of Biosciences and
Technology, Texas A&M University System Health Science Center,
2121 W Holcombe Blvd, Houston, TX 77030, USA.
E-mail: [email protected]
Received 28 September 2005; revised 27 December 2005; accepted 27
December 2005; published online 27 February 2006
Introduction
Liver cancer is the third most common causes for cancer
death in the world. In 2005, a total of 15 420 estimated
deaths by liver/intrahepatic bile duct cancer are expected
in the US, including 10 330 male cases and 5090 female
cases (Jemal et al., 2005). Although lots of research has
been performed to study the mechanism of hepatocarcinogenesis, few of them are related to the early stage of
liver tumor development. Helix–loop–helix (HLH)
proteins are key regulators of cell growth and differentiation in embryonic and adult tissues. They have
been demonstrated to play critical roles in regulation of
gene expression, cell cycle control, cell lineage commitment and numerous developmental processes (Blackwell
et al., 1990; Kreider et al., 1992; Zebedee and Hara,
2001). From the yeast to humans, over 240 HLH
proteins have been identified to date (Massari and
Murre, 2000). Based upon the presence or absence of
DNA-binding domain and other additional functional
domains, HLH proteins can be classified into five major
classes, basic HLH (bHLH) proteins, basic HLH
Per-AhR-Arnt-Sim (bHLH-PAS) proteins, basic HLH
leucine zipper (bHLH-LZ) proteins, dominant-negative
HLH (dnHLH) proteins, and HLH leucine zipper
(HLH-LZ) proteins. In multicellular organisms, bHLH
proteins are required for many important developmental
processes, including neurogenesis (Lee et al., 1995; Ma
et al., 1996; Sun et al., 2001), myogenesis (Nabeshima
et al., 1993; Molkentin et al., 1995; Spicer et al., 1996),
hematopoiesis (Kreider et al., 1992; Zhao and Aplan,
1999; Barndt et al., 2000), and pancreatic development
(Jensen et al., 2000; Pin et al., 2001). On the basis of
tissue distribution and dimerization capabilities, bHLH
protein can be further divided into two subgroups. One
subgroup also known as the E proteins and includes
E12, E47, E2-5, E2-2, HEB, and Daughterless. These
proteins are ubiquitously expressed in many tissues and
capable of forming either homo- or heterodimers
(Zhuang et al., 1992; Shirakata and Paterson, 1995).
The DNA-binding specificity of E proteins is limited to
the E-box site, which have the consensus sequence
CANNTG (Cordle et al., 1991; Voronova and Lee,
1994). The other subgroup bHLH proteins show a
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4208
tissue-restricted pattern of expression and regulate
tissue-specific cell growth and differentiation. Examples
include the myogenic bHLH family of proteins (MyoD,
Myogenin, Myf-5, MRF4), the cardiogenic family of
proteins (eHand and dHand), the neurogenic family of
proteins (Neurogenin, NeuroD, Mash-1, Mash-2, Hes,
NSCL), and the haemopoietic family of bHLH proteins
(SCL/TAL-1, Lyl-1, and ABF-1) (Murre et al., 1989;
Lassar et al., 1991; Hu et al., 1992; Kee et al., 2000).
With few exceptions, they are incapable of forming
homodimers and preferentially heterodimerize with the
E proteins. The heterodimers can bind both canonical
(CANNTG) and noncanonical (CACNAG) E-box sites
(Gaubatz et al., 1994). Dominant-negative HLH proteins, including Id proteins (Id1, Id2, Id3, Id4) and emc,
are small proteins lack a basic region. Owing to a lack
of the basic region, the Id proteins appear to function
as dominant-negative regulators of bHLH proteins
through the formation of ‘nonfunctional’ Id/bHLH
heterodimers, thus quench the activity of the E proteins,
inhibit cell differentiation and regulate tumorigenesis
(Benezra et al., 1990; Sun et al., 1991; Peppelenbosch
et al., 1995; Yokota and Mori, 2002; Perk et al., 2005).
Mice deficient in the Id family of proteins have profound
defects in neurogenesis, angiogenesis, vascularization of tumor xenografts, impaired immune responses,
and tumorigenesis (Lyden et al., 1999; Rivera et al.,
2000; Rivera and Murre, 2001; Ruzinova and Benezra,
2003; Sikder et al., 2003).
GCIP (CCNDBP1) is a HLH leucine zipper (HLHLZ) proteins without basic DNA-binding region, which
was identified and characterized in our lab and others to
interact with cyclinD1 (DIP1) and as a human homologue of MAID protein (HHM) (Hwang et al., 1997;
Terai et al., 2000; Xia et al., 2000; Yao et al., 2000).
GCIP gene is localized on chromosome 15q15, a region
frequently deleted or undergoes loss of heterozygosity
(LOH) in different tumors, including colorectum,
breast, lung, and bladder tumor (Natrajan et al.,
2003). Previous data demonstrate that GCIP is expressed specifically in foci of the 2-acetylaminofluorene/
partial hepatectomy (AAF/PH) model, which is used to
monitor the differentiation of rat hepatic stem cells into
hepatocytes, suggesting GCIP may be involved in the
hepatic stem cell development and differentiation
processes (Terai et al., 2000). GCIP was also showed
to regulate the transcription of hepatocyte nuclear
factor 4 (HNF4), which has E-box sequences in its
promoters, thus suggesting that GCIP might be involved
in the liver growth and differentiation (Terai et al., 2000;
Takami et al., 2005). High-level expression of GCIP was
found from the very early stages of hepatocarcinogenesis, suggesting that GCIP might be involved in
hepatocarcinogenesis (Takami et al., 2005).
In this study, we have generated GCIP transgenic
mice in the liver and characterized the effects of GCIP in
liver regeneration after injury and in hepatocarcinogenesis induced by diethylnitrosamine (DEN). Our data
demonstrate that in GCIP transgenic mice, the
overall liver growth, cell proliferation, and regeneration
occurred normally compared to the wild-type mice after
Oncogene
liver injury induced by carbon tetrachloride (CCl4).
In the mouse model of hepatocarcinogenesis induced by
DEN, we observed that overexpression of GCIP in
transgenic mice suppressed the number of chemicalinduced hepatocarcinogenesis at the early stage of tumor
development. At the late stage, hepatocellular tumors
in GCIP transgenic mice are smaller and welldifferentiated compared to the poorly differentiated
tumors in wild-type mice. We further demonstrated that
GCIP functions as a general transcriptional suppressor.
Overexpression of GCIP inhibited anchorage-independent cell growth and colony formation whereas downregulation of GCIP promotes colony formation in
HepG2 cells. These data suggest that GCIP may
function as an important negative regulator in tumor
initiation and progression but does not alter the
proliferation of normal hepatocytes following liver
injury. These findings suggest GCIP maybe an attractive
target for prevention of hepatocellular carcinoma.
Results
Generation of transgenic mice overexpressing GCIP under
the control of the albumin promoter
The full-length human GCIP was cloned from a human
bone marrow cDNA library (clontech) (Xia et al., 2000).
Human GCIP cDNA fragment (1.1 kb) containing the
entire coding region was inserted into the EcoRI site
of an albumin promoter-driven expression vector
(Figure 1a) and a chicken insulator is inserted at
30 -end of the transgene to ensure the expression of
target gene. Two GCIP transgenic lines were developed
and utilized for characterization. Transcription of
human GCIP was identified by Northern blot analysis
of liver mRNA using a 32P-labeled human GCIP cDNA
probe. Expected transcripts of the human GCIP
transgene were found only in GCIP transgenic mice
but not in control wild-type mice (Figure 1b), whereas
transcript of the mouse GCIP were found in both
transgenic mice lines and control wild-type mice. The
expression of GCIP mRNA in transgenic mice was
approximately two to five folds higher than that of the
endogenous murine GCIP gene. RT–PCR with primers
specific to human GCIP also showed the presence of
human GCIP mRNA in the liver of both Tg1 and Tg2
GCIP transgenic mice, with higher levels of expression
in Tg1 GCIP transgenic animals, which is consistent
with our results obtained from Northern blot analysis
(Figure 1b and c). Furthermore, we examined the
expression of GCIP protein in transgenic mice using
specific anti-GCIP antibody in Western blot analysis. As
shown in Figure 1d, GCIP protein was at about two- to
fourfold higher in transgenic mice than the endogenous
mouse GCIP protein (Figure 1d).
All GCIP transgenic mice appear to develop and
reproduce normally. The GCIP transgenic mice showed
no significant difference in liver weight relative to total
body weight in comparison to age-matched controls
in first 12 months (data not shown). Examination of
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4209
Figure 1 Generation and characterization of the GCIP-transgenic
mice. (a) Schematic representation of GCIP transgene construct.
The 1.1 kb cDNA of human GCIP was ligated downstream of
albumin promoter for overexpression in the hepatocytes. A chicken
insulator is inserted at 30 -end of the transgene to ensure the
expression of target gene. The pronucleus of FVB mice fertilized
eggs were injected with GCIP transgene construct and eggs were
transferred to the oviduct of pseudopregnant female mice.
(b) Northern blot analyses of RNAs from adult liver tissues of
GCIP transgenic and wild-type (WT) mice were performed with the
32
P-labeled human GCIP cDNA probe and mouse GCIP cDNA
probe, respectively. GAPDH cDNA probe was used as a control to
equal loading of total RNA. (c) RT–PCR with primers specific to
the human GCIP and murine GCIP were preformed to show the
presence of human GCIP mRNA in liver of both Tg1 and Tg2 of
GCIP-transgenic mice, with higher levels of expression in Tg1GCIP transgenic animals. RT–PCR with GAPDH-specific primer
was used as control. (d) Western blot analysis of GCIP protein in
the liver of transgenic mice and WT mice with anti-GCIP rabbit
polyclonal antibody. Western blot analysis of b-actin was served as
a loading control.
multiple livers from Tg1 and Tg2 lines of transgenic
GCIP mice and wild-type mice revealed no significantly
difference in liver structure and hepatocyte morphology.
Normal liver regeneration and hepatocyte proliferation in
GCIP transgenic mice after acute CCl4 treatment
The liver has a remarkable capacity to regenerate after
injury. To determine the effect of GCIP overexpression
on hepatocyte proliferation and regenerative capability,
we induced liver injury and regeneration on 7-week-old
male wild-type and GCIP transgenic mice by performing
a single intraperitoneal injection of the liver toxin CCl4,
which is known to cause hepatic injury by forming
trichloromethyl free radical and altering the hepatocyte
membrane permeability (Recknagel et al., 1989).
Histologically, GCIP transgenic mice and wild-type
mice showed similar extent of hepatocellular injury at
each time point (from 38 to 72 h) following acute
CCl4 exposure (Figure 2a), demonstrating little
or no difference in liver regeneration and heptocyte
proliferation.
Figure 2 Expression of GCIP in transgenic mice does not affect
histology, liver injury, and cell proliferation in livers following
intraperitoneal injection of CCl4. (a) Histological analysis of wild-type
(WT) and GCIP-transgenic mice (Tg) after acute CCl4 treatment,
respectively. Liver tissues were fixed, paraffin-embedded, then stained
by hematoxylin and eosin (H&E) as described in Materials and
methods. (b) Serum enzyme levels (ALT) show similar liver damage
responses for WT and GCIP-transgenic mice. Plasma ALT activity was
measured using the GP-Transaminase kit as described. No significant
difference between GCIP transgenic and WT mice at different time
points was observed (Po0.01) after acute CCl4 treatment. (c) Kinetics
of DNA synthesis and cell proliferation in GCIP transgenic and WT
mice. Cellular proliferation was measured by BrdU incorporation as
the percentage of the number of BrdU positive hepatocytes in total
hepatocytes by four or five visual fields per animal. All results are based
on analysis of 3–5 animals per time point and a minimum of 3000 cells
per animal was counted. No significant difference was detected in
hepatocyte DNA synthesis between GCIP transgenic and WT mice at
different time points after acute CCl4 treatment.
Oncogene
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4210
Alanine transaminase (ALT), is used as a specific
maker for hepatocyte injury (Wroblewski, 1959). To
determine the potential role of GCIP in hepatocyte
injury and regeneration, we examined whether overexpression of GCIP affects ALT level in transgenic
GCIP mice and in wild-type control mice. As shown in
Figure 2b, in CCl4-induced liver injury, ALT levels
increased and reached peak at 24 h in both wild-type and
GCIP transgenic mice. There was no significant
difference at ALT levels between wild-type and GCIP
transgenic mice up to 72 h post-CCl4 injection
(Figure 2b), indicating little effect of GCIP on the
injury responses of the liver induced by CCl4 injection.
To further examine whether overexpression of GCIP
in transgenic mice affect cell proliferation during injury
responses, we examined BrdU incorporation in the
CCl4-injured liver and evaluated hepatocyte DNA
synthesis and cell proliferation after CCl4 injection. As
shown in Figure 2c, both GCIP transgenic and wild-type
mice showed similar patterns for BrdU incorporation
with peak incorporation occurring at 48 h post-CCl4
injection, suggesting GCIP has little effect on DNA
synthesis and cell proliferation after CCl4 injection in
the transgenic liver. Together, these data support the
idea that GCIP transgenic mice in the liver showed an
unaltered liver damage and hepatocyte proliferation
after acute CCl4 treatment.
GCIP overexpression suppressed diethylnitrosamineinduced hepatocarcinogenesis in early stage of liver
tumor development
To elucidate the role of GCIP in hepatocarcinogenesis,
DEN was administered to GCIP transgenic mice to
induce liver tumorigenesis. As female mice are known to
be resistant to hepatocarcinogenesis in experimental
mouse models, including those employing chemical
carcinogenesis, only male mice were used and analysed
in our experiments (Diwan and Meier, 1976; Moore
et al., 1981; Vesselinovitch, 1987; Lee et al., 1998;
Nakatani et al., 2001). In this commonly used neonatal
DEN-induced hepatocarcinogenesis model, a single dose
of 10 mg/kg DEN was intraperitoneally injected into
14-day-old wild-type and GCIP transgenic mice. The
mice were killed 4, 6 or 10 months later. DEN-induced
microfoci could be readily observed under light microscope in wild-type liver by 4 months. The most common
foci were basophilic cell type, followed by clear cell type
and the mixed type containing both types of cells.
Hepatocyte dysplasia was apparent with increased
nuclear/cytoplasm ratio, pleomorphism, vacuolization,
and clear cell change. In general, while variations
existed, there were no apparent differences in the
histological changes between the wild-type and GCIP
transgenic livers. However, there were significant
differences in the number of foci and the size of foci
between the GCIP transgenic mice and the wild-type
mice. The number of hepatic adenomas at 24 weeks was
significantly lower or not detected in GCIP transgenic
male mice compared to the control mice under the same
treatment by 24 weeks (Figure 3a–d). Only 7.8% of
Oncogene
GCIP transgenic males versus 38.5% of wild-type males
developed liver adenomas by 24 weeks (Figure 3a–d,
Table 1). However, after40 weeks, 85.7% of GCIP
transgenic male mice developed liver tumors while 100%
of the wild-type male mice developed liver tumor after
DEN-treatment (Figure 3e–h, Table 1). The tumors in
GCIP transgenic mice at 40 weeks are smaller and welldifferentiated (Figure 3g and h) compared to those
found in wild-type mice (Figure 3e and f). These data
were consistent with the microfoci development at the
early stage in the wild-types of mice and suggested that
tumor development in the GCIP transgenic mice was
delayed.
GCIP suppresses the tumorigenesis in HepG2 cells
To examine the effect of GCIP in tumorigenesis, we
examined how GCIP regulated anchorage-independent
cell growth through soft agar assays. HepG2 cells were
stably transfected with pCMV-Tag2B control vector,
pCMV-Tag2B-GCIP, and pU6-GCIPsiRNA plasmid,
respectively. The transfected cells were analysed and
compared in soft agar assays for tumorigenesis. As
shown in Figure 4, overexpression of GCIP inhibited
HepG2 cell colony formation on soft agar (Figure 4a
and b), whereas downregulation of GCIP by specific
siRNA significantly increased the colony formation
of HepG2 cells (Figure 4a and b), suggesting that
GCIP can function as a potential suppressor gene
for anchorage-independent cell growth and colony
formation.
GCIP functions as a transcriptional repressor through
TSA/NaB insensitive pathways
GCIP contains a putative HLH domain, an Asp/Glurich acidic domain, and a leucine-zip domain. The
distinct domain structures suggest that GCIP could be a
potential transcriptional regulator. To further determine
the role of GCIP in general transcriptional regulation,
we used L8G5-luc reporter, a luciferase reporter gene
controlled by eight copies of the binding site for LexA
immediately adjacent to five copies of the binding site
for GAL4 (Hollenberg et al., 1995) (Figure 5a). LexAVP16 fusion coactivator was used as positive control to
Table 1 Incidence of liver tumors in GCIP transgenic mice and
wild-type mice treated with diethylnitrosamine (DEN)
Mouse incidence of liver tumor (weeks)
Genotype
WT
TG
24 (weeks)
40 (weeks)
5/13 (38.5%)
1/13 (7.7%)
8/8 (100%)
6/7 (85.7%)
The number of mice per group possessing at least one liver adenoma/
tumor, relative to the total number of mice in that group. The numbers
in parentheses represent the percentage of liver tumor occurrence rate.
Diethylnitrosamine was administered as a single intraperitoneal
injection of 10 mg/kg body weight at 14 days of age. Incidence of
liver tumors in GCIP transgenic mice is significantly lower than that in
wild-type mice at 24 weeks based on t-test (Po0.05). Abbreviations:
WT, wild-type mice; TG, GCIP transgenic mice.
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4211
Figure 4 GCIP regulates tumorigenesis in HepG2 cells in colony
formation assays. (a) Overexpression of GCIP suppresses anchorage-independent colony formation of HepG2 cells while downregulation of GCIP by siRNA promotes colony formation. HepG2
cells were stably transfected with empty pCMV-Tag2B, pCMVTag2B-GCIP, pU6-GCIPsiRNA, plated in soft agar, and assayed
for colony formation after 3 weeks. (b) The effect of GCIP on
tumorigenesis was counted as the number of colonies formed on
soft agar assays. Overexpression of GCIP inhibited HepG2 colony
formation (column 2) while downregulation of GCIP by siRNA
promoted colony formation (column 3).
of the class I and class II HDACs, which are sensitive to
the inhibition by TSA and NaB.
Figure 3 Comparison of liver tumors arising in GCIP-transgenic
and wild-type (WT) mice treated with DEN. (a–b) Histology of WT
male mouse livers at 24-weeks after DEN-treatment shows
adenomas in five out of 13 WT mice. (c–d) No obvious adenoma
was observed in GCIP transgenic mice (only one small adenoma
was found in GCIP transgenic mice). (e–f) Tumors of WT male
mice at 40 weeks’ old, showing poorly differentiated tumors and
tumor necrosis (e) found in eight out of eight WT mice. (g–h) Liver
adenomas found in GCIP transgenic mice at 40 weeks, showing
smaller and well-differentiated tumors in six out of seven GCIPtransgenic mice. Tumors were labeled by the cycle lines.
activated reporter to high levels of transcription
(Figure 5a). By fusing GCIP to the Gal4 DNA-binding
domain, the Gal4-GCIP fusion protein was able to
efficiently reduce the transactivation of L8G5-luc
reporter activated by LexA-VP16. This Gal4-GCIPinduced inhibition was not affected when cells were
incubated with either of the two HDAC inhibitors,
trichostatin A (TSA) and sodium butyrate (NaB)
(Figure 5a). Both TSA and NaB are well-known HDAC
inhibitors and were able to strongly promote transcriptional activation at indicated concentration. In the
presence of TSA or NaB, cotransfection of Gal4-GCIP
still strongly inhibited VP16-induced transcriptional
activation in the L8G5-luciferase assays, suggesting that
GCIP induced transcriptional inhibition is independent
GCIP inhibits the expression of cyclin D1
To understand the potential mechanism of GCIP
inhibition of cell proliferation and tumorigenesis, we
examined how GCIP affects the expression of cyclin D1,
a key molecule in cell proliferation and tumorigenesis.
First, we investigated how GCIP regulates the transcriptional activation of cyclin D1 by transfecting HepG2
cells with the pCMV-Tag2B vector, pCMV-Tag2BGCIP plasmid, or pU6-GCIPsiRNA plasmids, together
with the 1745 cyclin D1 promoter-luciferase reporter.
This cylcin D1 reporter is the orginal fragment of cyclin
D1 50 sequence cloned from the PRAD1 break point
(Motokura and Arnold, 1993). Cell extract were assayed
for luciferase or b-galactosidase activities 48 h later. As
shown in Figure 5b, overexpression of GCIP can
markedly inhibit cyclin D1 transcriptional activity while
decreasing GCIP expression by specific siRNA in the
cells can alleviate the inhibitory effect of cyclin
D1 transcription (Figure 5b). Based on the inhibitory
effect of GCIP on the transcriptional activity of cyclin
D1 promoter in the cell, we further examined whether
GCIP could inhibit the expression of cyclin D1 protein
in the cells. Cells were transfected with pCMV-Tag2B
vector, pCMV-Tag2B-GCIP, or pU6-GCIPsiRNA,
Oncogene
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4212
Figure 5 GCIP functions as a transcription repressor and regulates cyclin D1 expression. (a) Regulation of transcriptional activities
by the expression of Gal4-GCIP. Cos-7 cells were transiently cotransfected with L8G5-luc reporter, LexA-VP16, Gal4-GCIP, or Gal4vector control as indicated. Gal4-GCIP can efficiently suppress L8G5-luc transcription activated by LexA-VP16. TSA or NaB has little
or no effect on the GCIP-induced transcriptional repression. (b) GCIP inhibits transcriptional activation of 1745 cyclin D1 promoter.
HepG2 cells were plated and transiently transfected with pCMV-Tag2B vector, pCMV-Tag2B-GCIP plasmid, or pU6-GCIPsiRNA,
together with the 1745 cyclin D1-luciferase reporter. Overexpression of GCIP significantly inhibited the transcriptional activity of
cyclinD1 promoter while decreasing GCIP expression can upregulate cyclin D1 transcription. (c) Inhibition of cyclin D1 protein
expression by GCIP. HepG2 cells were plated and transiently transfected with pCMV-Tag2B vector, pCMV-Tag2B-GCIP, and
pU6-GCIPsiRNA plasmid, respectively. Cell extracts were assayed for the expression of GCIP, cyclin D1, and b-actin with specific
antibodies using Western blot analysis 72 h later. Results showed that overexpression of GCIP markedly inhibited cyclinD1 expression
while decreasing GCIP expression by siRNA significantly increased the expression of cyclin D1 protein in the cells. (d) RT–PCR with
primers specific to the murine cyclin D1 were preformed to show the expression of cyclin D1 mRNA in liver of both 24 weeks and 40
weeks old GCIP transgenic mice and wild-type (WT) mice treated with DEN, with lower levels of cylin D1 expression in 24 weeks old
GCIP transgenic mice. RT–PCR with GAPDH-specific primer was used as control.
respectively. Total proteins were extracted and separated by SDS-PAGE. Western blot analysis was
performed to examine the expression levels of the GCIP,
cyclin D1, and actin. As shown in Figure 5c, GCIP
protein was decreased in the cells transfected with pU6GCIPsiRNA, confirming that pU6-GCIPsiRNA could
efficiently decrease GCIP expression level (Figure 5c).
Down-regulation of GCIP greatly increased the expression level of cyclinD1 protein in the cells transfected
with pU6-GCIPsiRNA while overexpression of GCIP
decreased the expression of cyclin D1 in cells transfected
with pCMV-Tag2B-GCIP (Figure 5c), suggesting that
GCIP is able to inhibit tumorigenesis by regulating the
expression and function of cyclin D1 in the cell.
To further examine the in vivo effect of GCIP on
cyclin D1 expression, RT–PCR analysis was performed
to check the expression of cyclin D1 in liver of both 24
weeks and 40 weeks GCIP transgenic mice and wildtype mice that were treated with DEN. The results
Oncogene
showed that lower levels of cylin D1 expression was
present in 24 weeks’ old GCIP transgenic mice
compared with wild-type mice (Figure 5d). However,
there is little difference between the expression levels of
cyclin D1 in 40 weeks old GCIP transgenic and wildtype mice (Figure 5d), suggesting that cyclin D1 plays a
key role in the effect of GCIP in tumor suppression.
Discussion
In the previous study, our group reported that GCIP
was expressed highly in terminal differentiated tissues,
including heart, muscle, peripheral blood leukocytes,
and brain. Furthermore, in cells transfected with GCIP,
phosphorylation of retinoblastoma (Rb) protein by
cyclin D-dependent protein kinase was reduced and
E2F1-mediated transcription activity was inhibited,
suggesting GCIP could play a role in cell proliferation
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4213
and differentiation (Xia et al., 2000). Previous data from
Thorgeirsson’s group had shown that GCIP/HHM is
expressed specifically in the foci of the 2-acetylaminofluorene/partial hepatectomy (AAF/PH) model and is
likely to be involved in hepatic stem cell (hepatic oval
cell) proliferation and differentiation (Terai et al., 2000).
GCIP/HHM was also demonstrated to regulate the
transcriptional activation of hepatocyte nuclear factor 4
(HNF4) in reporter assays, suggesting a role of GCIP/
HHM in liver growth and differentiation (Terai et al.,
2000). Mouse GCIP, which is named as Maid (Maternal
Id-like molecule), was isolated from a subtraction
cDNA library enriched for maternal transcripts that
are still present in the mouse 2 cell stage embryo (Hwang
et al., 1997). However, Maid is just one of the shorter
splicing isoform of the mouse GCIP, the full-length
mouse GCIP isoform has extra 47 amino acids at its
amino terminus and shares an overall amino acid
sequence identity of 79.2% homology with GCIP.
Interesting, human GCIP also has multiple different
splicing isoform. Totally 14 splice patterns (SP) from the
Alternative Splicing Database (ASD) are shown for
human GCIP. Five of them were valuated at high
confidence level. Alternative splicing is an important
mechanism to generate protein diversity and to determine the binding properties, intracellular localization,
enzymatic activity, protein stability and posttranslational modifications of proteins in a tissue-specific or
development-specific way (Sierralta and Mendoza, 2004;
Cooper, 2005; Lee and Wang, 2005; Stamm et al., 2005).
Splice site mutations of tumor suppressor genes often
create truncated proteins as classical nonsense mutations. Over the last two decades, many studies have
reported cancer-specific alternative splicing in the
absence of genomic mutations (Venables, 2004). The
ubiquitous expression of GCIP and multiple different
splicing isoforms of GCIP suggest that GCIP may have
multiple functions and involve in multiple different
processes.
In this study, we have generated the GCIP transgenic
mice by subcloning the human GCIP into a vector
containing the albumin promoter to evaluate the in vivo
function of GCIP in liver proliferation, differentiation,
and tumorigenesis. No abnormal development was
detected in GCIP transgenic mice both before and after
birth. Cell proliferation during liver regeneration in
response to CCl4 induced liver injury was normal in
GCIP transgenic mice compared with the wild-type
mice. Although GCIP may affect E2F-1 and cyclinD1
activities in our in vitro assays, data obtained from the
GCIP transgenic mice are similar to the overexpression
of E2F-1 and cyclin D1 in mice liver (Conner et al.,
2000; Ledda-Columbano et al., 2002), in which no
obvious changes was observed in hepatic regenerative
growth and proliferation after partial hepatectomy
(Conner et al., 2000) and mitogenic stimuli (LeddaColumbano et al., 2002), respectively.
Liver regeneration following liver injury is known to
depend on two processes. The first one is through
hepatocyte division and usually happens after acute liver
injury. If this process is impaired, liver repair depends
on the recruitment of hepatic oval cells. Hepatic oval
cells are able to proliferate and differentiate into
hepatocytes. Oval cell proliferation is commonly seen
in the preneoplastic stages of liver carcinogens and is
believed to be the source of hepatocarcinogenesis after
the exposure to hepatocarcinogens (Knight et al., 2000;
Roskams et al., 2003; Theise et al., 2003; Lemmer et al.,
2004). GCIP is expressed specifically in the foci of the
2-acetylaminofluorene/partial hepatectomy (AAF/PH)
model, indicating that it is more likely to be involved in
hepatic oval cell proliferation and differentiation but not
hepatocyte proliferation (Terai et al., 2000). The
involvement of GCIP in hepatic oval cell proliferation
and differentiation may partly explain why there exists
no difference in liver injury and hepatocyte proliferation between GCIP transgenic and wild-type mice after
CCl4-treatment, while hepatic carcinogenesis after
DEN-treatment was attenuated in the GCIP transgenic
mice compared to wild-type mice.
It is known that early hepatocellular carcinoma
(HCC) is usually a well-differentiated carcinoma and
then gradually changes into a moderately differentiated
and poorly differentiated phenotype during tumor
progression (Sugihara et al., 1992). GCIP expression
was also detected in human liver tumor samples with
immunostaining by specific anti GCIP antibody. GCIP
protein was stained in 23 of 32 adenomatous hyperplasia
(AH) samples (72%), 19 of 28 well-differentiated HCC
samples (68%), and 9 of 18 poorly moderately
differentiated HCC samples (50%). Expression of GCIP
was high in the adenomatous hyperplasia (AH) and
well-differentiated HCC samples but relatively low in
the poorly differentiated HCC samples, suggesting that
the decrease of GCIP might be a prerequisite event for
further tumor progression (Takami et al., 2005). In our
present study, we demonstrated that overexpression of
GCIP in mouse liver decreased susceptibility to DEN
induced hepatocarcinogenesis in transgenic mice in the
early stage of tumorigenesis. After given a single
intraperitoneal injection of DEN, 38.5% of wild-type
males developed liver tumors by 24 weeks, however,
only 7.8% of transgenic males developed liver tumors at
the same stage (Table 1). These results raised the
possibility that GCIP may have a tumor suppressor
function in the early stage of the hepatocarcinogenesis.
Although GCIP has little effect on the number of liver
tumors in the later stage of tumor development (86% in
GCIP transgenic mice versus 100% in WT mice), tumors
in GCIP transgenic mice are smaller and well-differentiated compared to the tumors found in wild-type
mice, suggesting an important inhibitory role of GCIP
in liver tumor initiation and progression processes.
In our investigation of the potential mechanisms of
GCIP in tumorigenesis and transcription regulation, we
found that GCIP could function as a transcription
repressor independent of the class I and class II HDAC
proteins. The D-type cyclins (cyclins D1, D2, and D3)
are key molecules in cell cycle progression, cell
proliferation and tumorigenesis. The cyclin D proteins
complex with cdk4 and cdk6 and thereby regulate
transition from the G1 phase into the S phase by
Oncogene
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4214
phosphorylation and inactivation of the retinoblastoma
protein (pRb) tumor suppressor. Amplification and/or
overexpression of the cyclin D1 (bcl1, PRAD1, and
CCND1) gene has been found in 11–13% of human
hepatomas (Nishida et al., 1994, 1997; Suzui et al., 2002)
and other human cancers, such as in 30–40% of human
breast cancers (Kenny et al., 1999; Pestell et al., 1999).
Overexpression of cyclin D1 is sufficient to initiate
hepatocarcinogenesis in transgenic mice (Deane et al.,
2001, 2004). Thus, cyclin D1 can function as an
oncogene in the liver and is a potential target for
hepatoma prevention and therapy. Our finding that
GCIP suppresses the liver tumorigenesis by significantly
down-regulating the transcriptional activity and protein
expression of cyclin D1 suggest a novel mechanism for
GCIP as a potential tumor suppressor gene in human
cancers. Future studies are underway in our laboratory
to help us understand the function of GCIP as a tumor
suppressor and a regulator in cell proliferation and
differentiation processes.
Materials and methods
Generation of GCIP transgenic mice
1.1 kb human GCIP cDNA containing the entire coding region
for the full-length human GCIP was inserted into the EcoRI
site of an albumin promoter-driven expression vector
(Figure 1a). A chicken insulator is inserted at 30 -end of
transgene to ensure the expression of target gene. This
transgene was used to generate transgenic mice by injecting
GCIP transgene into the pronucleus of FVB mice fertilized
eggs, then eggs were transferred to the oviduct of pseudopregnant female FVB mouse. Transgenic mice were identified
by Southern blot analysis of EcoRI digested tail genomic DNA
using a 32P-labeled 1.1 kb GCIP cDNA probe. To confirm
Southern results, PCR analysis of mouse tail DNA was
performed using oligonucleotide primers designed from the
human GCIP gene. Transgenic mice and control mice used in
this study were propagated by breeding with GCIP heterozygote transgenic males with wild-type FVB females. Mice
were maintained in 12-h light/12-h dark cycles with free access
to food and water. All animal studies were performed in
accordance with the guidelines of the Institutional Animal Use
and Care Committee of the Institute of Biosciences and
Technology, Texas A&M University System Health Science
Center.
GCIPsiRNA plasmid construction
For stable expression of GCIP siRNAs within cells, the U6
promoter was cloned in front of GCIP gene-specific targeting
sequence (19 nt sequences GACTCAATGAGGCAGCTGT
from GCIP cDNA separated by a 9 nt spacer from the reverse
complement of the same sequence) and five thymidines (T5) as
termination signal.
Experimental liver injury by administration of CCl4
All mice were 7-week-old males and kept under 12-h light/12-h
dark lighting cycles at 231C. Three to five 7-week-old male
mice were used in each experimental group. A single dose of
2.0 ml/kg of body weight (2:5 v/v in mineral oil) was
administered by intraperitoneal injection for CCl4-induced
liver damage study. After the mice were weighed, anesthetized
and exsanguinated, livers were excised for analysis.
Oncogene
ALT analyses
Blood was drawn from anesthetized mice through heart
puncture and the serum was separated by centrifugation for
20 min. Blood plasma levels of ALT (aminotransferase)
activity were immediately measured using the GP-Transaminase kit (No. 505-P, Sigma, St Louis, MO, USA) in per
time point after CCl4 injection.
Hepatocyte DNA synthesis assays
Hepatocyte DNA synthesis was measured by bromodeoxyuridine (BrdU) incorporation. Mice were given intraperitoneally injections of 20 mg/ml BrdU (Boehringer, Mannheim,
Indianapolis, IN, USA) in a dose of 50 mg/g body weight 2 h
before killing. The liver samples were collected, fixed and
paraffin-embedded at per time point after CCl4 treatment as
described in the text. Sections, 5-mm, were cut for immunohistochemistry (IHC). An ABC staining kit was purchased
from Santa Cruz Biotechnology. Immunohistochemical staining follows the manufacturer’s protocol from Santa Cruz
Biotechnology, Santa Cruz, CA, USA. Briefly, anti-BrdU
primary antibody (Sigma, no. 2531) was used at 1:500 in block
serum. BrdU incorporation in fixed liver sections was
visualized with an anti-BrdU monoclonal antibody and an
alkaline phosphatase-conjugated secondary antibody. Positively staining hepatocytes nuclei and unstained cells were
counted under a Nikon Digital camera Dxm1200 microscope,
and BrdU incorporation was expressed as the percentage of
the number of labeled hepatocytes in four or five visual fields
per animal (magnification 100).
Diethylnitrosamine-induced liver tumor and histological
procedures
At 14 days of age, mice were given a single intraperitoneal
injection of DEN (Sigma Chemical Co.) in a dose of 10 mg/kg
of body weight. At 21 days of age, mice were separated by sex
and their transgenicity was determined.
At 24 and 40 weeks of age, animals were killed and analysed.
Representative sections from each lobe of the liver and visible
tumors were fixed, sectioned, stained with hematoxylin and
eosin (H&E), and examined for the presence of adenomas.
Statistical analysis of tumor incidence was carried out using
the Student’s t-test.
Liver tissues were fixed overnight in Histochoice Tissue
Fixative MB (no. H120–4L, Amresco, Solon, OH, USA),
dehydrated through a series of ethanol treatments, and
embedded in paraffin according to standard procedure.
Sections were prepared and stained with H&E.
Northern blot analyses
Total RNA was isolated from liver using TRIZOL reagent
(Invitrogen, Carlsbad, CA, USA) and separated on 1%
agarose-formaldehyde gel. RNA was transferred by capillary
blotting overnight onto Hybond þ membrane (AmershamPharmacia Biotech, Piscataway, NJ, USA) in 10 SSC.
GCIP probe was labeled with [a-32P]dCTP (Amersham) using
the Random Primers DNA Labeling system (Invitrogen),
and hybridized according to the manufacturer’s protocol.
Prehybridization and hybridization were done in 10 ml Rapid
Hyb buffer (Amersham) at 681C for 1 h and overnight,
respectively. The blot was washed two times in 2 SSC with
0.1% SDS at room temperature for 30 min, followed by two
washes in 0.1 SSC with 0.1% SDS at 681C for 30 min. We
stripped the GCIP probe with 0.1% SDS and reprobed the
blot with b-actin probe using the same protocol. Blots were
exposed 3 h to overnight on BioRad Phosphorimager screen.
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4215
Cell culture and luciferase reporter assays
The HepG2 cells were obtained from ATCC (Rockville, MD,
USA) and maintained in a 371C incubator with 5% CO2
humidified air in Dulbecco’s minimal modified Eagle’s medium
(DMEM) supplemented with 10% (v/v) fetal bovine serum.
At 48 h after transfection of the plasmids, the cells in a
24-well plate were lysed and harvested in 200 ml reporter lysis
buffer (Promega), and cell lysates were assayed for luciferase
activities. The luciferase assay was carried out using luciferase
assay kit (Promega) and Packard Topcount Scintillation
Counter. Extracts were also assayed for b-galactosidase
activity with the Galacto-Light Plust b-Galactosidase
Reporter Gene Assay Systems system (Tropix). Each extract
was assayed three times, and the mean relative light unit
(RLU) was corrected by values obtained from an extract
prepared from nontransfected cells. The relative luciferase
activity was calculated as RLU/b-galactosidase.
Soft agar assay
Human liver HepG2 cells were cultured in DMEM containing
10% fetal calf serum and transfected by pCMV-Tag2B vector,
the pCMV-Tag2B-GCIP plasmid, and the siRNA plasmidspecific for GCIP. Transfected cells will be selected with G418
for 7 days. After selection, cells will be analysed for colony
growth by suspending in 0.3% agarose medium containing
DMEM þ 5% FBS and layered onto a 2.5-ml bed of 0.6%
agarose in a 35-mm dish with grids. Plates will be incubated 3
weeks, and the number of colonies >100 mm will be counted.
Statistical analysis
Difference between wild-type and GCIP-transgenic animals in
the liver tumor incidence was examined for statistical
significance using the t-test.
Acknowledgements
This work was supported partially from NIH Grants
(5R01HL064792 and 1R01CA106479). We would like to
thank members of the Liu laboratory and members of the
Center for Cancer Biology and Nutrition for their comments
and discussion.
References
Barndt RJ, Dai M, Zhuang Y. (2000). Mol Cell Biol 20:
6677–6685.
Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H.
(1990). Cell 61: 49–59.
Blackwell TK, Kretzner L, Blackwood EM, Eisenman RN,
Weintraub H. (1990). Science 250: 1149–1151.
Conner EA, Lemmer ER, Omori M, Wirth PJ, Factor VM,
Thorgeirsson SS. (2000). Oncogene 19: 5054–5062.
Cooper TA. (2005). Cell 120: 1–2.
Cordle SR, Henderson E, Masuoka H, Weil PA, Stein R.
(1991). Mol Cell Biol 11: 1734–1738.
Deane NG, Lee H, Hamaamen J, Ruley A, Washington MK,
LaFleur B et al. (2004). Cancer Res 64: 1315–1322.
Deane NG, Parker MA, Aramandla R, Diehl L, Lee WJ,
Washington MK et al. (2001). Cancer Res 61: 5389–5395.
Diwan BA, Meier H. (1976). Cancer Lett 1: 249–253.
Gaubatz S, Meichle A, Eilers M. (1994). Mol Cell Biol 14:
3853–3862.
Hollenberg SM, Sternglanz R, Cheng PF, Weintraub H.
(1995). Mol Cell Biol 15: 3813–3822.
Hu JS, Olson EN, Kingston RE. (1992). Mol Cell Biol 12:
1031–1042.
Hwang SY, Oh B, Fuchtbauer A, Fuchtbauer EM, Johnson
KR, Solter D et al. (1997). Dev Dyn 209: 217–226.
Jemal A, Murray T, Ward E, Samuels A, Tiwari RC, Ghafoor
A et al. (2005). CA Cancer J Clin 55: 10–30.
Jensen J, Pedersen EE, Galante P, Hald J, Heller RS, Ishibashi
M et al. (2000). Nat Genet 24: 36–44.
Kee BL, Quong MW, Murre C. (2000). Immunol Rev 175:
138–149.
Kenny FS, Hui R, Musgrove EA, Gee JM, Blamey RW,
Nicholson RI et al. (1999). Clin Cancer Res 5: 2069–2076.
Knight B, Yeoh GC, Husk KL, Ly T, Abraham LJ, Yu C et al.
(2000). J Exp Med 192: 1809–1818.
Kreider BL, Benezra R, Rovera G, Kadesch T. (1992). Science
255: 1700–1702.
Lassar AB, Davis RL, Wright WE, Kadesch T, Murre C,
Voronova A et al. (1991). Cell 66: 305–315.
Ledda-Columbano GM, Pibiri M, Concas D, Cossu C,
Tripodi M, Columbano A. (2002). Hepatology 36:
1098–1105.
Lee C, Wang Q. (2005). Brief Bioinform 6: 23–33.
Lee GH, Ooasa T, Osanai M. (1998). Cancer Res 58:
1665–1669.
Lee JE, Hollenberg SM, Snider L, Turner DL, Lipnick N,
Weintraub H. (1995). Science 268: 836–844.
Lemmer ER, Vessey CJ, Gelderblom WC, Shephard EG, Van
Schalkwyk DJ, Van Wijk RA et al. (2004). Carcinogenesis
25: 1257–1264.
Lyden D, Young AZ, Zagzag D, Yan W, Gerald W, O’Reilly
R et al. (1999). Nature 401: 670–677.
Ma Q, Kintner C, Anderson DJ. (1996). Cell 87: 43–52.
Massari ME, Murre C. (2000). Mol Cell Biol 20: 429–440.
Molkentin JD, Black BL, Martin JF, Olson EN. (1995). Cell
83: 1125–1136.
Moore MR, Drinkwater NR, Miller EC, Miller JA, Pitot HC.
(1981). Cancer Res 41: 1585–1593.
Motokura T, Arnold A. (1993). Genes Chromosomes Cancer 7:
89–95.
Murre C, McCaw PS, Vaessin H, Caudy M, Jan LY, Jan YN
et al. (1989). Cell 58: 537–544.
Nabeshima Y, Hanaoka K, Hayasaka M, Esumi E, Li S,
Nonaka I. (1993). Nature 364: 532–535.
Nakatani T, Roy G, Fujimoto N, Asahara T, Ito A. (2001).
Jpn J Cancer Res 92: 249–256.
Natrajan R, Louhelainen J, Williams S, Laye J, Knowles MA.
(2003). Cancer Res 63: 7657–7662.
Nishida N, Fukuda Y, Ishizaki K, Nakao K. (1997). Histol
Histopathol 12: 1019–1025.
Nishida N, Fukuda Y, Komeda T, Kita R, Sando T,
Furukawa M et al. (1994). Cancer Res 54: 3107–3110.
Peppelenbosch MP, Qiu RG, de Vries-Smits AM, Tertoolen
LG, de Laat SW, McCormick F et al. (1995). Cell 81: 849–856.
Perk J, Iavarone A, Benezra R. (2005). Nat Rev Cancer 5:
603–614.
Pestell RG, Albanese C, Reutens AT, Segall JE, Lee RJ,
Arnold A. (1999). Endocr Rev 20: 501–534.
Pin CL, Rukstalis JM, Johnson C, Konieczny SF. (2001).
J Cell Biol 155: 519–530.
Recknagel RO, Glende Jr EA, Dolak JA, Waller RL. (1989).
Pharmacol Ther 43: 139–154.
Rivera R, Murre C. (2001). Oncogene 20: 8308–8316.
Rivera RR, Johns CP, Quan J, Johnson RS, Murre C. (2000).
Immunity 12: 17–26.
Oncogene
GCIP suppresses hepatocarcinogenesis in transgenic mouse
W Ma et al
4216
Roskams TA, Libbrecht L, Desmet VJ. (2003). Semin Liver
Dis 23: 385–396.
Ruzinova MB, Benezra R. (2003). Trends Cell Biol 13:
410–418.
Shirakata M, Paterson BM. (1995). EMBO J 14: 1766–1772.
Sierralta J, Mendoza C. (2004). Brain Res Brain Res Rev 47:
105–115.
Sikder H, Huso DL, Zhang H, Wang B, Ryu B, Hwang ST
et al. (2003). Cancer Cell 4: 291–299.
Spicer DB, Rhee J, Cheung WL, Lassar AB. (1996). Science
272: 1476–1480.
Stamm S, Ben-Ari S, Rafalska I, Tang Y, Zhang Z, Toiber D
et al. (2005). Gene 344: 1–20.
Sugihara S, Nakashima O, Kojiro M, Majima Y, Tanaka M,
Tanikawa K. (1992). Cancer 70: 1488–1492.
Sun XH, Copeland NG, Jenkins NA, Baltimore D. (1991).
Mol Cell Biol 11: 5603–5611.
Sun Y, Nadal-Vicens M, Misono S, Lin MZ, Zubiaga A, Hua
X et al. (2001). Cell 104: 365–376.
Suzui M, Masuda M, Lim JT, Albanese C, Pestell RG,
Weinstein IB. (2002). Cancer Res 62: 3997–4006.
Oncogene
Takami T, Terai S, Yokoyama Y, Tanimoto H, Tajima K,
Uchida K et al. (2005). Gastroenterology 128: 1369–1380.
Terai S, Aoki H, Ashida K, Thorgeirsson SS. (2000).
Hepatology 32: 357–366.
Theise ND, Yao JL, Harada K, Hytiroglou P, Portmann B,
Thung SN et al. (2003). Histopathology 43: 263–271.
Venables JP. (2004). Cancer Res 64: 7647–7654.
Vesselinovitch SD. (1987). Toxicol Pathol 15: 221–228.
Voronova AF, Lee F. (1994). Proc Natl Acad Sci USA 91:
5952–5956.
Wroblewski F. (1959). Am J Med 27: 911–923.
Xia C, Bao Z, Tabassam F, Ma W, Qiu M, Hua S et al. (2000).
J Biol Chem 275: 20942–20948.
Yao Y, Doki Y, Jiang W, Imoto M, Venkatraj VS, Warburton
D et al. (2000). Exp Cell Res 257: 22–32.
Yokota Y, Mori S. (2002). J Cell Physiol 190: 21–28.
Zebedee Z, Hara E. (2001). Oncogene 20: 8317–8325.
Zhao XF, Aplan PD. (1999). J Biol Chem 274: 1388–1393.
Zhuang Y, Kim CG, Bartelmez S, Cheng P, Groudine M,
Weintraub H. (1992). Proc Natl Acad Sci USA 89:
12132–12136.