From the
Laboratory of Endocrine Cell Biology, National Research Laboratory
Program, Department of Internal Medicine,
Department of Pathology, Chungnam
National University School of Medicine, Daejeon 301-721 Korea,
Therapeutic Gene Group, Team 1, Samyang Genex
Biotech Research Institute, Yusung-Gu, Daejeon, the
**Department of Biochemistry, College of Natural
Sciences, Kyungpook National University, Daegu 702-701, Korea, the
Department of Radiation Oncology, Long
Island Jewish Medical Center, The Long Island Campus for Albert Einstein
College of Medicine, New Hyde Park, New York 11040
Received for publication, December 17, 2002 , and in revised form, April 24, 2003.
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Gadd45 is rapidly induced by agents that cause DNA damage including
UV, N-acetoxy-2-acetylaminofluorene, methylmethane sulfonate, and
H2O2 (6).
DNA damage-induced transcription of the Gadd45 gene is mediated by
both p53-dependent (7) and
p53-independent mechanisms (8).
The phenotype of Gadd45-deficient mice is similar to the phenotype of
p53-deficient mice, including genomic instability, sensitivity to
radiation-induced carcinogenesis, and a low frequency of exencephaly
(9). These findings suggest
that Gadd45 is one component of the p53 pathway that maintains genomic
stability.
Gadd45 proteins have also been implicated in DNA replication (10), DNA repair (10), G1 progression and G2/M checkpoint control (11, 12), apoptosis, and regulation of signal transduction (5, 13). Gadd45 proteins display a complex array of physical interactions with other proteins involved in these processes. It has been reported that Gadd45 proteins interact with proliferating cell nuclear antigen (10, 1417, 20) and with cyclin-dependent kinase inhibitor p21 (1720) in the nucleus. In addition, Gadd45 proteins activate the p38/Jun N-terminal kinase pathway by binding to MTK1/MEKK4 (5) in response to environmental stress. Gadd45 also interacts with Cdc2 and inhibits Cdc2-cyclin B1 kinase activity (21, 22). These findings led to the hypothesis that Gadd45 members play a critical role in negative growth control and apoptosis, but the detailed molecular mechanisms by which Gadd45 carries out this role remain to be elucidated.
This study used a yeast-two hybrid screen to identify novel proteins that
bind to CR6/Gadd45. CR6-interacting factor 1
(CRIF1)1 is a nuclear
protein identified in this screen. CRIF1 interacts with Gadd45
,
Gadd45
, and Gadd45
, and it is ubiquitously expressed in human
tissues. CRIF1 is exclusively localized to the nucleus and colocalizes with
Gadd45
. Gadd45 proteins and CRIF1 inhibit Cdc2-cyclin B1 kinase in an
additive manner, and CRIF1 inhibits histone H1 kinase activity associated with
immunoprecipitated Cdk2-cyclin E in the absence of Gadd45 proteins.
Overexpression of CRIF1 inhibits cell cycle progression at G1/S and
suppresses growth in serum-stimulated NIH3T3 cells. In addition, adrenal
adenoma and thyroid papillary cancer cells express CRIF1 at a very low level,
which is lower than in adjacent normal tissue. These findings suggest that
CRIF1 is a nuclear protein that modulates cell cycle progression and growth.
Altered expression of CRIF1 may also contribute to tumorigenesis in some cell
types.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The colon carcinoma cell line, RKO, the African green monkey cell line, COS-7, the mouse fibroblasts, NIH3T3, and the colon carcinoma cell line, HCT116, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, nonessential amino acids, 100 units/ml penicillin, and 100 µg/ml streptomycin in a humidified 5% CO2 and 95% air atmosphere at 37 °C.
PlasmidsPlasmid DNA was generated using standard cloning
procedures and constructs were verified by restriction enzyme analysis and DNA
sequencing. Plasmid pAS-Gadd45 encodes a fusion between the GAL4 DNA
binding domain and Gadd45
/CR6. pAS-Gadd45
was constructed by
inserting PCR-amplified human Gadd45
cDNA into the pAS21 vector
(Clontech Laboratories). Constructs for expression of GST fusion proteins were
constructed by cloning into pGEX-4T (Amersham Biosciences). Human
Gadd45
cDNA (supplied by Dr. A. J. Fornace Jr.) and PCR-amplified mouse
Gadd45
/MyD118 and human Gadd45
/CR6 cDNA were cloned into
pGEX-4T.
For mammalian expression, pFLAG-Gadd45, pFLAG-Gadd45
,
pFLAG-Gadd45
, and pFLAG-CRIF1 were constructed by inserting cDNA into
the EcoRI/XhoI sites of pCMV-Tag2 (Stratagene). pMyc-CRIF1
was constructed by inserting cDNA into the EcoRI/XhoI sites
of pCMV-Tag3 (Stratagene).
For mammalian two-hybrid assay, PCR-amplified Gadd45,
Gadd45
/MyD118, Gadd45
/CR6, and CRIF1 cDNA were cloned into
pCMV-BD and pCMV-AD (Stratagene).
Yeast Two-hybrid and cDNA Library ScreeningYeast strain
Y190 (Clontech Laboratories, Inc.) was transformed with pAS-Gadd45 and
a Matchmaker two-hybrid cDNA library from K-562 cells (Clontech Laboratories,
Inc.). Transformants were selected on SD medium lacking histidine, leucine,
and tryptophan and containing 45 mM 3-amino-1,2,4-triazole (Sigma).
After
-galactosidase filter assay, 72 blue colonies were obtained. To
determine the specificity of interaction, pACT2 plasmid DNA was rescued from
yeast, amplified in E. coli, HB101, and transformed into yeast Y190
with pAS-Gadd45
. One of the clones (His+ and
-gal+) was sequenced and further analyzed. Full-length cDNA
for CRIF1 was obtained from a human placenta cDNA library (Stratagene) as
described previously (23).
In Vitro Interaction ExperimentsIn vitro translated
35S-labeled CRIF1 was incubated for 20 min at room temperature with
glutathione-Sepharose (10 µl) preloaded with GST-Gadd45,
GST-Gadd45
, GST-Gadd45
, or GST-control in 250 µl of binding
buffer (20 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10%
glycerol, 1 mM dithiothreitol, 1 mM EDTA, 1
mM phenylmethanesulfonyl fluoride, 1 mM leupeptin, 1
mM pepstatin). Glutathione-Sepharose was washed extensively with
binding buffer, and bound proteins were eluted in 25 µl of Laemmli sample
buffer, boiled for 10 min, and analyzed by SDS-polyacrylamide gel
electrophoresis (10%) followed by autoradiography.
Mammalian Two-hybrid AssayPlasmids pCB-Gadd45 (for
GAL4 DBD-fusion) and pCA-CRIF1 (for NF-
B AD fusion) or pCB-CRIF1 and
pCA-Gadd45
(100 ng each) were co-transfected with 100 ng of pFR-Luc
reporter (Stratagene) into NIH-3T3 cells and grown in 24-well plates using
LipofectAMINE Plus (Invitrogen). Total plasmid DNA was adjusted to 400 ng/well
with vector DNA (pCMV-BD or pCMV-AD). 100 ng of pCMV-
-gal DNA (Clontech
Laboratories, Inc.) was used as internal control. Cells were lysed 24 h after
infection, and luciferase activity was measured by Berthold LB9507 luminometer
(Berthold). Luciferase activity was normalized to
-galactosidase
activity.
Northern Blot AnalysisThe human multiple tissue mRNA blots
(Clontech Laboratories, Inc.) containing 2 µg of poly(A)+ RNA
per lane were hybridized using QuickHyb solution (Stratagene) under stringent
conditions as recommended by the manufacturer. CRIF1 cDNA or DECAprobe
glyceraldehyde-3-phosphate dehydrogenase template (Ambion) was labeled with
[-32P]dATP with Strip-EZTM DNA labeling kit
(Ambion).
Immunoprecipitation and Western Blot AnalysisThe following
immunoprecipitation procedures were carried out at 4 °C using COS-7 cells
coexpressing tagged Gadd45 ,
, and
with/without
pFLAG-CRIF1 transfection. Cells were grown on 100-mm dishes, washed with
phosphate-buffered saline twice, and incubated for 30 min with radioimmune
precipitation lysis buffer containing protease inhibitors (20 µg/ml
leupeptin, 10 µg/ml pepstatin A, 10 µg/ml chymostatin, 2 µg/ml
aprotinin, 1 mM phenylmethylsulfonyl fluoride). The cell lysate was
collected and centrifuged at 10,000 x g for 10 min. The
supernatant was incubated with rocking for 2 h with 20 µl of protein A/G
beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and centrifuged for 15
min at 8,000 x g. The supernatant was removed and incubated
with primary antibody for 2 h with rocking. Protein A/G beads were added,
incubated for 12 h, and centrifuged at 10,000 x g.
Immunoprecipitates were collected and washed three times with radioimmune
precipitation buffer.
For Western blot analysis, cells were lysed at 4 °C for 20 min 10
mM KPO4, 1 mM EDTA, 5 mM EGTA, 10
mM MgCl2, 50 mM -glycerophosphate, 2
mM dithiothreitol, 1% Nonidet p-40, 1 mM Pefabloc (Roche
Applied Science), and 10 µg of each aprotinin and leupeptin per milliliter.
Total protein lysates were denatured by boiling in Laemmli sample buffer,
resolved on 7.515% SDS-polyacrylamide gels, and transferred to
polyvinylidene fluoride membranes. Membranes were blocked in PBS containing 5%
(w/v) nonfat dry milk and 0.1% Tween and incubated for 2 h with appropriate
antibodies. Blots were developed using horseradish peroxidase-linked
antirabbit secondary antibody and developed with a chemiluminescent detection
system (Phototope-HRP Western blot detection kit; New England Biolabs).
Kinase AssayExponentially growing RKO cells were lysed on
ice for 30 min in radioimmune precipitation buffer containing protease
inhibitors (20 µg/ml leupeptin, 10 µg/ml pepstatin A, 10 µg/ml
chymostatin, 2 µg/ml aprotinin, 1 mM phenylmethylsulfonyl
fluoride). One mg of cellular protein was immunoprecipitated with anti-Cdc2 or
Cdk2 antibodies. Immunoprecipitated Cdc2-cyclin B1 and Cdk2-cyclin E complexes
were incubated with recombinant Gadd45 and Gadd45
or CRIF1
proteins for 5 min. Histone H1 kinase assays were performed using 10 µg of
histone H1 (Upstate Biotechnology, Inc., Lake Placid, NY), 15 mM
MgCl2, 7 mM
-glycerol phosphate, 1.5 mM
EGTA, 0.25 mM sodium orthovanadate, 0.25 mM
dithiothreitol, and 10 µCi of [
-32P]ATP in 25 µl.
After 10 min at 37 °C, the reactions were transferred to numbered P81
paper and washed with 0.75% phosphoric acid and acetone. The activity in assay
squares was measured by scintillation counting (Hewlett-Packard).
Flow Cytometry AnalysisSamples were prepared for flow cytometry essentially as described previously (24). Briefly, cells were washed with 1x phosphate-buffered saline and then fixed with ice-cold 70% ethanol. Samples were washed with 1x phosphate-buffered saline and stained with propidium iodide 60 µg/ml (Sigma) containing RNase 100 µg/ml (Sigma) for 30 min at 37 °C. Cell cycle analysis was performed using a Becton Dickinson fluorescence-activated cell analyzer and Cell Quest version 1.2 software (Becton Dickinson Immunocytometry Systems, Mansfield, MA). At least 10,000 cells were analyzed per sample. Cell cycle distribution was quantified using the ModFit LT version 1.01 software (Verity Software House Inc., Topsham, ME).
Small Interfering RNA (siRNA) ExperimentsThe 21-nucleotide
siRNA were synthesized and purified using a Silencer siRNA
construction kit (Ambion Ltd.). The siRNA sequence targeting the human CRIF1
(GenBankTM accession number AF479749
[GenBank]
) corresponded to the coding region,
as showed in Fig. 6A.
The desalted DNA of the sense and antisense oligonucleotides targeting four
different regions on the human CRIF1 (Fig.
6A) were synthesized, and the eight nucleotides at the
3'-end of both oligonucleotides had the following sequence:
5'-CCTGTCTC-3', which is complementary to the T7 promoter. In
order to produce an efficient transcription template, the sense and antisense
oligonucleotides for each siRNA need to be converted to double-stranded DNA
with a T7 promoter in 37 °C. The sense and antisense siRNA transcripts
were transcribed for 2 h in separate reactions with T7 RNA polymerase. The
reactions were then mixed, and the combined reaction was incubated overnight
at 37 °C for double-stranded RNA. A single-strand specific ribonuclease
and DNase digestion were used to eliminate the 5'-overhanging leader
sequence and the DNA template, respectively. The resulting siRNA was recovered
from the mixture of nucleotides, enzymes, short oligomers, and salts in the
reaction by column purification. The cells were transiently transfected with
the CRIF1-siRNA duplex and the expression plasmids using LipofectAMINE Plus
(Invitrogen) according to the manufacturer's instructions. Briefly, the
plasmids were mixed with the Plus reagent and then incubated with
LipofectAMINE. The LipofectAMINE Plus DNA complex was added to the cells and
further incubated at 37 °C for 3 h. The control cells received the
LipofectAMINE Plus alone. The cell viability was detected using a trypan blue
dye exclusion test. After incubation, the medium was removed, and the cells
were given fresh medium and maintained for an additional 24 h. The
transfection efficiency was evaluated with pCMV-SPORT--gal (500 ng) and
pEGFP-CRIF1 (500 ng) in a six-well culture plate.
|
Immunofluorescence and ImmunohistochemistryCOS-7 cells were
transfected with pFLAG-Gadd45 and pMyc-CRIF1 and grown on uncoated
glass coverslips (Bellco Glass, Inc.). Cells were washed three times in PBS+
(PBS with 1 mM calcium and 1 mM magnesium). Cells were
fixed with 3.7% formaldehyde for 15 min at room temperature, washed briefly in
PBS+, and quenched with 50 mM NH4Cl in PBS for 5 min.
Cells were washed twice in PBS+ and incubated in blocking solution containing
3% bovine serum albumin in PBS+. For permeabilization, blocking and all
subsequent incubation steps included 0.1% Triton X-100. Anti-FLAG antibody
(Stratagene) and anti-c-Myc antibody (New England Biolabs, Inc., Beverly, MA)
were incubated with the cells overnight at 4 °C. Cells were washed five
times with PBS+ and incubated with secondary antibody conjugated with TRITC or
fluorescein isothiocyanate for 60 min at room temperature. The cells were
washed five times in PBS+, and fluorescence was visualized using a Nikon
Eclipse E400 microscope equipped with epifluorescence optics and a digital
camera; images were captured as Adobe Photoshop files.
Anti-CRIF1 antiserum was obtained from New Zealand White rabbits immunized with conjugated synthetic peptide AEEQKRREREQHIAEC corresponding to the midregion of CRIF1. The sera were titered against the GST-CRIF1 fusion protein by enzyme-linked immunosorbent assay, and the serum exhibiting the highest titer (1:25,600) was used in subsequent experiments. Thyroid gland and adrenal gland tissues were obtained from surgery patients. Tissue specimens were fixed in 10% formalin, embedded in paraffin, and sectioned and then stained with hematoxylin and eosin to evaluate histologic type. Samples used in this study were confirmed histologically as benign or malignant.
CRIF1 expression was analyzed by immunohistochemistry in two adrenal gland and seven thyroid cancer tissues. Samples were micro-waved for 10 min, and slides were incubated with anti-CRIF1 anti-serum (1:800) at room temperature for 1 h. Preimmune sera did not stain tissues at a 1:800 dilution. Slides were washed in phosphate-buffered saline and incubated with a linking solution containing biotinylated goat anti-rabbit IgG (Dako LSAB kit; Dako Laboratories) at room temperature for 30 min. Slides were sequentially incubated with streptavidin peroxidase at room temperature for 30 min and 3,3'-diaminobenzidine chromogen substrate solution (Dako Laboratories), counterstained with Meyer's hematoxylin and mounted. Three independent readers evaluated the slides. Two independent readers graded the slides in a blinded fashion. The slides were graded with respect to staining intensity using the following scale: 0 (no staining), +1 (weak), +2 (moderate), and +3 (high).
Other AssaysProtein concentration was determined using the Bradford method (Bio-Rad) with recrystallized bovine serum albumin as a standard.
Statistical SignificanceAll experiments were repeated at least three times with different batches of cells. Values shown are the mean ± S.E. of three or more experiments. Significance between experimental values was determined by two-way analysis of variance.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The CRIF1 cDNA had little 5'-untranslated sequence and a 669-bp open reading frame encoding a predicted polypeptide of 222 amino acids with a predicted molecular mass of 25 kDa (Fig. 1A). A polyadenylation signal (AATAA) is located 27 bp upstream of the poly(A) tail. A BLAST (26) search revealed that this cDNA encodes a novel protein with no homology to any proteins in the data base. The CRIF1 gene is located on chromosome 19 at 19p13.2. The CRIF1 gene has one exon of 351 bp and a second exon of 315 bp. Homology searches using BLAST identified the human and mouse genomic clones as BC004944 [GenBank] (similar to RIKEN cDNA 2310040G17) and NM026320 (mouse RIKEN cDNA 2310040G17), respectively. The amino acid sequence deduced from the CRIF1 cDNA isolated by two-hybrid screen is identical to BC004944 [GenBank] and has 90% homology with the mouse CRIF1 homologue NM026320 (Fig. 1B).
|
CRIF1 cDNA was in vitro translated, and the protein product had an apparent size of 28 kDa by SDS-PAGE (Fig. 2A). CRIF1 was also expressed transiently in COS-7 cells transfected with human CRIF1 cDNA cloned into pCMV-Tag2 (Stratagene). Cell lysates were analyzed by Western blot using anti-FLAG antibody (Stratagene), and an immunoreactive band was detected of the expected size (Fig. 2B).
|
CRIF1 mRNA was examined by Northern blot using mRNA from cultured cells and
human tissues (Clontech Laboratories). A CRIF1 mRNA of 0.7 kb was
detected in human cultured cells (data not shown). CRIF1 was highly expressed
in heart, thyroid, lymph node, trachea, and adrenal tissue and was expressed
at a low level in liver, skeletal muscle, kidney, pancreas, testis, ovary, and
stomach (Fig. 2C).
CRIF1 mRNA expression was similar to Gadd45
(data not shown).
Gadd45
is induced by DNA-damaging agents including methylmethane
sulfonate and UV (5). However,
CRIF1 mRNA was not induced by ionizing radiation, methylmethane sulfonate, or
UV in SW620 or MCF7 cells (data not shown).
Native CRIF1 was detected in human cells by Western blot analysis using anti-CRIF1 antiserum (Fig. 2D). The anti-CRIF1 antiserum specifically detects 28-kDa CRIF1 in anaplastic thyroid cancer cell lines NPA, FRO, and ARO and primary cultured smooth muscle cells and endothelial cells from umbilical vein (Fig. 2D). Immunoreactive CRIF1 protein was not detected in either the rat or mouse cells, such as the rat FRTL-5 thyroid cells and mouse NIH 3T3 cells (Fig. 2D).
Gadd45 proteins are localized predominantly in the nucleus
(5,
10,
17). CRIF1 has a putative
nuclear localization signal
(28) in its C-terminal region
(Fig. 1A), suggesting
that it might colocalize with Gadd45. The subcellular location of CRIF1 and
Gadd45 was studied using COS-7 cells transfected with pMyc-CRIF1 and
pFLAG-CR6. Myc and FLAG epitope tags were visualized by immunofluorescent
staining (Fig. 2E).
The results show that FLAG-CR6 protein and Myc-CRIF1 colocalize in the nucleus
(Fig. 2E).
Interaction between CRIF1 and Gadd45 Proteins in Vitro and in
VivoThe interactions between CRIF1 and Gadd45 proteins were
examined in vitro using recombinant GST-Gadd45, Gadd45
,
and Gadd45
immobilized on glutathione-agarose beads and
[35S]methionine-labeled in vitro translated CRIF1. In
vitro translated CRIF1 bound to GST-Gadd45
but did not bind to GST
(Fig. 3A, lane 5
versus lane 2). CRIF1 also binds to GST-Gadd45
and
GST-Gadd45
(Fig.
3A, lanes 3 and 4). In vitro
translated luciferase protein did not interact with GST or GST-Gadd45
proteins. As previously described
(10,
14,
16,
17,
27), in vitro
translated proliferating cell nuclear antigen and p21 bound to GST-Gadd45,
Gadd45
, and Gadd45
(data not shown).
|
CRIF1 interactions with Gadd45 proteins were also detected in mammalian
cells using a mammalian cell two-hybrid assay
(Fig. 3B).
Gadd45, Gadd45
, and Gadd45
were cloned into pCMV-BD
(Stratagene, La Jolla, CA) to create fusions with the DNA binding domain.
CRIF1 was cloned into pCMV-AD (Stratagene) to fuse with the NF-
B
activation domain. The luciferase reporter gene was activated when the CRIF1
fusion protein was co-expressed with Gadd45
, Gadd45
, or
Gadd45
fusion proteins (Fig.
3B). Similar results were obtained when CRIF1 was fused
with the DNA binding domain and Gadd45 proteins were fused with the activation
domain (data not shown). Co-expression of Gadd45
, Gadd45
, and
Gadd45
fusion constructs with vector pCMV-AD did not activate
luciferase expression. This result indicates that CRIF1 interacts specifically
with Gadd45 proteins.
In vivo interactions between CRIF1 and Gadd45 family proteins were
examined using COS-7 cells co-expressing FLAG-tagged Gadd45, -
,
and -
. CRIF1 was immunoprecipitated with anti-Myc and anti-FLAG
antibodies and the precipitates were blotted with anti-FLAG or anti-Myc
antibodies to detect tagged Gadd45 family proteins. As shown in
Fig. 3, CE,
tagged Gadd45 family proteins were present in the CRIF1 immunoprecipitates. In
addition, lysates from COS-7 cells expressing FLAG-tagged Gadd45
,
-
, and -
were immunoprecipitated with anti-FLAG antibody. CRIF1
was detected in the anti-FLAG immunoprecipitates
(Fig. 3F). These
results suggest that Gadd45 family proteins bind CRIF1 in vitro and
in vivo.
Inhibition of Cdc2 and Cdk2 Kinase and Regulation of Cell Cycle by
CRIF1Gadd45 proteins regulate G1/S and G2/M
checkpoints in several cell types
(11,
18,
21,
22). Gadd45 proteins bind Cdc2
kinase, which inhibits cellular proliferation
(11). Because these findings
suggest that CRIF1 interacts with Gadd45 proteins in vitro and in
vivo, Cdc2 kinase activity was examined in the presence or absence of
Gadd45 proteins and CRIF1. As shown in Fig.
4, recombinant Gadd45 and Gadd45
reduced histone H1
kinase activity in Cdc2-cyclin B1 immunoprecipitates to
60% of the
control (the presence of Cdc2-cyclin B1 was demonstrated by immunoblot; data
not shown). The addition of recombinant CRIF1 resulted in a progressive
inhibition of the enzymatic activity of the immunoprecipitated Cdc2-cyclin B1
complex. The simultaneous addition of recombinant Gadd45
or
Gadd45
and CRIF1 inhibited histone H1 kinase activity of Cdc2-cyclin B1
in an additive manner (Fig.
4A). In contrast, Gadd45 proteins do not inhibit
Cdk2-cyclin E histone H1 kinase activity, and Gadd45 proteins have no effect
on CRIF1-dependent inhibition of Cdk2-cyclin E histone kinase activity
(Fig. 4B).
|
Because CRIF1 interacts with Gadd45 and inhibits the activity of Cdc2-cyclin B1 and Cdk2-cyclin E complexes, it is possible that CRIF1 could also influence cell cycle progression. This idea was tested by overexpressing CRIF1 in NIH 3T3 cells and monitoring cell cycle progression by flow cytometry. LipofectAMINE Plus was used to reproducibly transfect NIH3T3 cells with pFLAG-CRIF1 with an efficiency of 3050% (Fig. 5A), eliminating the need for cotransfection with a selectable marker. Cell cycle progression was analyzed within 12 days after transfection. pFLAG-CRIF1 and pCMV-Tag2 (vector control) were transfected into NIH 3T3 cells, and CRIF1 expression was measured by immunoblotting with mouse anti-FLAG antibody. Cells transfected with pFLAG-CRIF1 expressed significantly more CRIF1 than pCMV-Tag2-transfected control cells (data not shown). Cells overexpressing CRIF1 (transfected with 4 µg of pFLAG-CRIF1) were more likely to be in G1 and less likely to be in S phase than control cells (Fig. 5A).
|
The growth rate was also examined in cells overexpressing pFLAG-CRIF1. NIH3T3 cells were cotransfected with pCMV-Tag2 or pFLAG-CRIF1 and pCMV-puro and grown for 5 days; pCMV-puro encodes a gene that confers puromycin resistance for selection purposes during cell growth. Cells transfected with pFLAG-CRIF1 grew more slowly than control cells transfected with pCMV-Tag2 (Fig. 5B). After 5 days of selection, the number of cells overexpressing pFLAG-CRIF1 was about half the number of control cells.
Effects of CRIF1-siRNA Duplexes on Rb Phosphorylation and Regulation of Cell CycleIn order to examine the role of endogenous CRIF1 in the regulation of cell proliferation and the cell cycle, 21-nucleotide duplexes of siRNA were used, and RNA interference studies in HCT116 cells were performed. This study selected four different target regions of human CRIF1 for the synthesis of the RNA duplexes as shown in Fig. 6A. The activities and specificity of the CRIF1-siRNAs was evaluated by the cotransfection of four different forms of siRNA duplexes with the expression plasmids, pFLAG-CRIF1 and pEGFP-CRIF1 (Fig. 6, B and D). As shown in Fig. 6B, two kinds of CRIF1-siRNAs, siRNA-263 and -379, effectively inhibited the exogenous FLAG-CRIF1 expression. However, siRNA-578 was not effective in inhibiting exogenous FLAG-CRIF1 expression. In similar experiments, siRNA-263 and siRNA-379 were effective in the reducing the green fluorescence protein fluorescence by inhibiting green fluorescence protein-tagged CRIF1 expression, as shown in Fig. 6D. However, siRNA-578 transfection did not alter the green fluorescence protein-CRIF1 fluorescence intensity compared with the control. These observations suggest that siRNA-263 and -379 are effective in inhibiting the exogenous CRIF1 expression. Therefore, the effects of siRNA-263 and -379 on the endogenous CRIF1 levels were investigated. The HCT116 cells transfected with CRIF1-siRNA-263 and -379 showed a very low level of endogenous CRIF1 compared with the nontransfected and siRNA-578-transfected cells (Fig. 6C). Collectively, these observations suggest that siRNA-263 and -379 are effective siRNA duplexes for the suppression of exogenous and endogenous CRIF1 expression.
During the G1 phase, the growth factor-initiated signals promoted the accumulation and assembly of the D-type cyclins (D1, D2, D3) with their cognate cyclin-dependent kinases (CDK4 and -6). The active holoenzyme promoted the G1 phase progression and S phase entry by virtue of its ability to phosphorylate the retinoblastoma protein Rb and titrate the CDK inhibitors p27Kip1 and p21Cip1. The initial phosphorylation of Rb and titration of the CDK inhibitors expedited the cyclin E-CDK2 kinase activation, thereby completing the Rb phosphorylation and inactivation prior to the initiation of DNA replication. As described above, the exogenous CRIF1 overexpression decreased the Cdk2-cyclin E kinase activity. This study observed the role of CRIF1 in the phosphorylation of Rb by the overexpression and down-regulation of CRIF1. As shown in Fig. 7A, CRIF1 expression in the HCT116 cells resulted a decrease in the phosphorylated forms of Rb with an increase in the CRIF1 expression level. The decrease in phosphorylated Rb level by CRIF1 overexpression was completely inhibited by CRIF1-siRNA-263 and -379 cotransfection but not by CRIF1-siRNA-578, which was unable to suppress CRIF1 expression (Fig. 7B). The level of Rb phosphorylation after transfection of the siRNA duplexes, siRNA-263, -379, and -578 was analyzed in order to observe the effect of the down-regulation of the endogenous CRIF1 on Rb phosphorylation. The HCT116 cells transfected with siRNA-263 and -379 duplexes showed an increase in Rb phosphorylation, but siRNA-578 did not alter the endogenous CRIF1 and phosphorylated Rb levels (Fig. 7C). These siRNA experiments suggest that modulating the intracellular level of CRIF1 results in the alteration of the Rb phosphorylation status, which is involved in regulating the G1/S phase of the cell cycle.
|
CRIF1 overexpression resulted in the accumulation of the G1 phase populations as shown in Fig. 5A. This study analyzed the cell cycle in the cells transfected with the CRIF1-siRNA duplexes to observe the roles of the endogenous CRIF1 in regulating the cell cycle. As shown in Fig. 7D, CRIF1 overexpression resulted in increased G1 phase populations, but the transfection of siRNA-263 and -379, which was highly effective in decreasing the endogenous CRIF1, resulted in a reduction of the G1 phase cell populations. However, transfecting the inefficient siRNA duplex, siRNA-578, did not change the G1 phase cell populations compared with the control. These observations suggest that the down-regulation of the endogenous CRIF1 promoted the cell cycle progression.
CRIF1 Expression in Endocrine TumorsAs shown in Fig. 2C, adrenal and thyroid gland tissue express CRIF1 at a high level. Therefore, the cellular distribution of CRIF1 in adrenal and thyroid glands was examined by immunohistochemistry in normal and cancerous tissue from patients who underwent surgery for nonfunctioning adrenal tumor or papillary thyroid cancer. Deparaffinized adrenal adenoma (n = 2) and thyroid (n = 7) papillary cancer tissue was sequentially incubated with anti-CRIF1 antiserum (1:800), biotinylated secondary antibody, streptavidin peroxidase, and 3,3'-diaminobenzidine substrate. High level expression of CRIF1 was expected in adrenal and thyroid tissue based on previous Northern blot results (Fig. 2C). CRIF1 immunoreactivity was mainly confined to the cortex of the adrenal gland (C) (Fig. 8). Expression was highest in the zona glomerulosa region of the cortex, and CRIF1 expression was very low in adrenal medulla (M). CRIF1 immunoreactivity was noted at cytoplasm and nucleus. However, the adenoma (A) in the adrenal cortex (C) expressed CRIF1 at a significantly lower level than the surrounding tissue (Fig. 8). In the thyroid gland, CRIF1 immunoreactivity was high within the nucleus in follicular epithelial cells (Fig. 8); however, papillary cancer cells within the follicular epithelium had much lower CRIF1 immunoreactivity than the surrounding cells as shown in Fig. 8. Immunohistochemical findings from three patients are shown in Fig. 8; in addition, CRIF1 immunoreactivity was lower in seven papillary thyroid cancers than in normal thyroid follicular epithelial cells. The DNA sequences of exon 1 and exon 2 in CRIF1 in thyroid papillary cancer tissues from seven patients did not carry detectable mutations (data not shown).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A partial human CRIF1 cDNA was isolated using a yeast two-hybrid screen
with Gadd45 as bait. CRIF1 genomic sequence, chromosomal location, and
a mouse CRIF1 homologue were identified by searching DNA databases. Evidence
presented here indicates that CRIF1 interacts with Gadd45
, -
, and
-
proteins in vitro and in vivo. In addition, CRIF1
colocalizes with Gadd45
in the nucleus. This result is consistent with
the possibility that CRIF1 and Gadd45 proteins co-regulate nuclear functions
such as cell cycle progression, replication, and transcription
(29).
The function of Gadd45 proteins has been implicated in activating a
G2/M checkpoint in response to genotoxic stress agents such as UV
radiation or methylmethane sulfonate. However, the function of Gadd45 proteins
in negative growth control is not fully understood. Recently, it was shown
that Gadd45 proteins ,
, and
interact with and inhibit
Cdc2cyclin B1 kinase. This study shows that CRIF1 suppresses the histone H1
kinase activity of Cdc2-cyclin B1 and Cdk2-cyclin E complexes in the presence
or absence of Gadd45 proteins. CRIF1 and Gadd45 inhibition was additive for
Cdc2-cyclin B1 kinase but not for Cdk2-cyclin E kinase. These findings suggest
that CRIF1 may modulate the G1/S and G2/M transition in
dividing cells. Overexpression of CRIF1 alters cell cycle progression in
NIH3T3 cells and increases the proportion of cells in G1; thus,
CRIF1 may participate in regulating the G1/S transition, which is
when cells commit to entering S phase and completing DNA replication and cell
division. Small interfering RNA experiments revealed that the down-regulation
of endogenous CRIF1 is related to the increase in Rb phosphorylation. This
observation suggests that CRIF1 has a constitutive inhibitory role in
G1 cell cycle regulation.
The tumor suppressor, p53, is a key protein that regulates G1 arrest in cells with DNA damage (30, 31). Genetic instability is one of the hallmarks of cancer, and cancer is often associated with aberrations in DNA repair or cell cycle checkpoint pathways (32). Proteins that regulate G1/S, such as ATM (ataxia telangiectasia-mutated) (33), Chk2 (checkpoint kinase 2) (34), BRCA1 (35), p53 (31), p16 (36), and Rb (36), are frequently altered in cancer cells. These major G1/S checkpoint transducers and effectors are tumor suppressors or protooncogenes, and loss of functional mutations in the genes encoding these proteins has been identified in many human malignancies.
CRIF1 expression is different in normal and cancer tissues. In normal tissue, thyroid and adrenal gland expressed the highest level of CRIF1 mRNA, and CRIF1 immunoreactivity is mainly localized in the nucleus by immunohistochemistry and in vitro immunofluorescence studies. However, histiocyte, osteoclast, terminally differentiated keratinocyte, and adrenal medulla expressed a very low or undetectable level of CRIF1 (data not shown), in contrast to high level CRIF1 expression in epithelial cells from thyroid follicle, colon (data not shown), and breast mammary duct (data not shown). It is interesting to note that some epithelial cell cancers in thyroid or breast expressed CRIF1 at a lower level than adjacent normal epithelial cells. Seven of seven papillary thyroid cancers expressed CRIF1 at a virtually undetectable level relative to normal epithelial cells and infiltrating T lymphocytes in the tumor, which expressed a high level of CRIF1. Reduced expression of CRIF1 was also observed in a benign adenoma from adrenal cortex. The mechanism by which CRIF1 expression is lowered in certain endocrine tumors was not evaluated in this study. No mutations that might account for altered expression of CRIF1 were found in exons 1 or 2 of CRIF1 in papillary thyroid cancer tissue.
In summary, the results presented here suggest that CRIF1 may negatively regulate cell growth by inhibiting the G1/S transition. Reduced expression of CRIF1 may occur in some cancers, and altered expression of CRIF1 may play a significant role in carcinogenesis.
![]() |
FOOTNOTES |
---|
* This work was supported by National Research Laboratory Program Grant
M1-0104-00-0014, Ministry of Science and Technology, Seoul, Korea and by
Department of Defense Breast Cancer Research Program Grant DAMD-17-02-1-0525
(to I. B.). The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
The first two authors contributed equally to this work.
¶ Present address: Neurogenex Inc., Sillimdong 56-1 Kwanakku, Seoul 151-744
Korea.
|| Present address: Dept. of Biological Science, Korea Advanced Institute of
Science and Technology, Daejeon, 305-721, Korea.
¶¶ To whom correspondence may be addressed: Dept. of Radiation Oncology, Long Island Jewish Medical Center, The Long Island Campus for Albert Einstein College of Medicine, 270-05 76th Ave., New Hyde Park, NY 11040. Tel.: 718-470-7456; Fax: 718-470-9756; E-mail: insoobae{at}hotmail.com.
|||| To whom co-correspondence should be addressed: Laboratory of Endocrine Cell Biology, Department of Internal Medicine, Chungnam National University College of Medicine, 640 Daesadong Chungku, Daejeon 301-721 Korea. Tel.: 82-42-220-7161; Fax: 82-42-257-5753; E-mail: minhos{at}cnu.ac.kr.
1 The abbreviations used are: CRIF1, CR6-interacting factor 1; GST,
glutathione S-transferase; PBS, phosphate-buffered saline; siRNA,
small interfering RNA; TRITC, tetramethylrhodamine isothiocyanate.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|