Oncogene (2011) 30, 434–444
& 2011 Macmillan Publishers Limited All rights reserved 0950-9232/11
www.nature.com/onc
ORIGINAL ARTICLE
The RNA-binding protein La contributes to cell proliferation and CCND1
expression
G Sommer1, J Dittmann1,2, J Kuehnert1, K Reumann2, PE Schwartz3, H Will2, BL Coulter4,
MT Smith4 and T Heise1,2
1
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA; 2HeinrichPette-Institut für Experimentelle Virologie und Immunologie, Hamburg, Germany; 3Protein Biotechnologies, Inc., Ramona, CA, USA
and 4Department of Pathology & Laboratory Medicine, Medical University of South Carolina, Charleston, SC, USA
The La protein is an essential RNA-binding protein
implicated in different aspects of RNA metabolism.
Herein, we report that small interfering (siRNA)mediated La depletion reduces cell proliferation of
different cell lines concomitant with a reduction in cyclin
D1 (CCND1) protein. To exclude off-target effects we
demonstrate that exogenous La expression in La-depleted
cells restores cell proliferation and CCND1 protein levels.
In contrast, proliferation of immortalized CCND1 knockout cells is not affected by La depletion, supporting a
functional coherence between La, CCND1 and proliferation. Furthermore, we document by reversible in vivo
crosslinking and ribonucleoprotein (RNP) immunoprecipitation an association of the La protein with CCND1
messengerRNA and that CCND1 internal ribosome entry
site (IRES)-dependent translation is modulated by La
protein level within the cell. In addition, we show elevated
La protein expression in cervical cancer tissue and its
correlation with aberrant CCND1 protein levels in
cervical tumor tissue lysates. In conclusion, this study
establishes a role of La in cell proliferation and CCND1
expression and demonstrates for the first time an overexpression of the RNA-binding protein La in solid tumors.
Oncogene (2011) 30, 434–444; doi:10.1038/onc.2010.425;
published online 20 September 2010
Keywords: La autoantigen; La protein; cyclin D1; CCND1;
cell proliferation; cervical cancer
Introduction
The predominant nuclear mammalian La protein is an
essential (Park et al., 2006) multifunctional RNAbinding protein involved in RNA processing (for
reviews, see Maraia and Intine, 2002; Wolin and
Cedervall, 2002) and translation (see below). Recent
work suggests that the La protein is aberrantly regulated
Correspondence: Dr T Heise, Department of Biochemistry and
Molecular Biology, Medical University of South Carolina, 173 Ashley
Avenue, Charleston, SC 29425, USA.
E-mail: [email protected]
Received 9 April 2010; revised 22 July 2010; accepted 10 August 2010;
published online 20 September 2010
in different types of cancer. It has been shown that the
La protein is elevated in BCR/ABL-transformed cells
from patients with chronic myeloid leukemia (Trotta
et al., 2003). In this study, the authors report that the La
protein promotes MDM2 protein expression through a
translational mechanism. Recently, La protein expression has been shown to be increased in different types of
tumor cell lines when compared with La protein levels of
normal cells (Al-Ejeh et al., 2007). Using murine glial
cells, Brenet et al. (2009) demonstrated that green
fluorescent protein (GFP)-tagged La protein shuttles in
an serine-threonine protein kinase (AKT)-dependent
manner to the cytoplasm, thereby supporting translation
of a subset of messengerRNAs (mRNAs). The authors
hypothesize that AKT-dependent shuttling of La to the
cytoplasm might contribute to the well-known oncogenic
effect of aberrant active AKT. Collectively, these studies
suggest an important role of the RNA-binding protein La
in cancer pathogenesis.
In addition to the well-described functions of the
RNA-binding protein La in the processing of RNA
polymerase III transcripts (for reviews, see Maraia,
2001; Wolin and Cedervall, 2002), the mammalian La
interacts with both cellular (McLaren et al., 1997;
Brenet et al., 2005) and viral mRNAs (Spangberg
et al., 2001; Ehlers et al., 2004), and many reports
suggest a role of La in regulation of cellular and viral
translation. For instance, human La supports internal
ribosome entry site (IRES)- and cap-dependent translation of viral mRNAs, such as hepatitis C virus, polio
virus (Ali et al., 2000; Costa-Mattioli et al., 2004; Pudi
et al., 2004), HIV TAR mRNA (Svitkin et al., 1994;
Chang et al., 1995), suppresses translation of the
hepatitis A virus (Cordes et al., 2008), and regulates
IRES- and cap-dependent translation of cellular
mRNAs (for example, BiP mRNA (Kim et al., 2001),
X-linked inhibitor of apoptosis protein (Holcik and
Korneluk, 2000), 50 TOP-mRNAs (Crosio et al., 2000)
and Mdm2 mRNA (Trotta et al., 2003)).
The primary objective of this study was to identify
novel cellular functions of the La protein in cancerous
cells. In our study, we show that the small interfering
(siRNA)-mediated reduction of La protein in different
cell lines correlates with both decrease in cell proliferation and downregulation of cyclin D1 (CCND1) protein.
La protein contributes to cell proliferation
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435
Our evidence that CCND1 knockout cells proliferate in
an La-independent manner and that La protein overexpression is accompanied by higher CCND1 protein
levels in cervical cancer patient samples supports our
hypothesis that the RNA-binding protein La contributes
to cell proliferation and CCND1 expression in cancer.
Results
The La protein is required for cell proliferation
Uncontrolled proliferation is a characteristic of most
cancerous cells. To test whether La affects cell proliferation, we first reduced La protein by transfecting LasiRNAs into the cervical adenocarcinoma-derived HeLa
cell line. After the second siRNA treatment, La protein
depletion was stable over the whole observation period
(24–96 h, Figure 1a). Next, we measured the proliferation
of La-depleted and control siRNA-treated cells over 4
days (Figure 1b). The similar cell number in both groups
4–5 h after plating indicated that La depletion did not
significantly affect cell re-attachment to culture dish (data
not shown). However, cell proliferation rate was significantly reduced in La-depleted HeLa cells (Figure 1c) and
calculation of doubling time of the cells revealed that
control cells doubled faster (B18.2 h) compared with
La-depleted cells (B39.5 h). To test whether La depletion
induced apoptosis, we analyzed caspase 3-mediated
cleavage of poly (ADP-ribose) polymerase (PARP) by
western blotting and observed no increase in PARP
cleavage (89 kDa, Figure 1d), no increase in the number
of dead cells by Annexin V and propidium iodide costaining after La depletion (Figure 1e) and no increase in
the sub-G1 peak during fluorescence-activated cell sorting
(FACS) analysis conducted at different time points after
La depletion (data not shown). Furthermore, cell cycle
analysis revealed no significant arrest of La-depleted cells
in any cell cycle phase (Supplementary Figure 1). We
conclude that the reduced proliferation rate of La-depleted
cancerous cells is not caused by enhanced cell death.
Figure 1 The La protein is required for proliferation of HeLa cells. (a) Western blot analysis shows that La is efficiently depleted by
La-specific siRNA over a period of 24–96 h after the second RNAi treatment. As control (con) cells were treated with luciferase-specific
siRNAs. a-Tubulin protein was used as a loading control. (b) Cell proliferation of La-depleted (La siRNA) and control-treated
(con siRNA) cells was measured over 4 days after the second RNAi treatment. Proliferation of La-depleted cells was reduced. (c) The
proliferation rate (slope of growth curve) of three independent experiments was calculated and plotted. The proliferation rate of Ladepleted cells (La siRNA) was statistically significant reduced by about 75% when compared with control-treated cells (con siRNA)
(P-value o0.05). (d) To test whether La-depletion induces programmed cell death, caspase-3 dependent cleavage of PARP was
visualized with western blotting. The PARP-specific antibody recognizes a 116-kDa fragment and an 89-kDa apoptosis-related
cleavage fragment. After La depletion, no difference in the amount of PARP cleavage products was observed indicating that caspase 3
pathways were not activated. GAPDH protein was used as a loading control. (e) To test whether the number of dead cells was different
in La-depleted and control-treated cells Annexin V and propidium iodide co-staining was conducted on day 2 after the second siRNA
treatment. Annexin V staining and propidium iodide incorporation was determined by FACS analysis. No increase in dead cells was
determined after La depletion.
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La protein contributes to cell proliferation
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Figure 2 CCND1 protein levels are strongly reduced in La-depleted HeLa cells. Cells were harvested 48 h after the second RNAi
treatment, and proteins were analyzed by western blotting. (a) CCND1 protein was dramatically reduced in La-depleted cells
(La siRNA), whereas only small changes were observed in p21 protein level. As a loading control a-Tubulin, HSC 70 or GAPDH were
used. (b) The CCND1 protein was restored by exogenous La (GFP-LaR) expression in La-depleted cells (compare lane 2 and 4).
As loading control, a-Tubulin was used. (c) RNA from La-depleted (La) and control (con) cells were reverse transcribed and CCND1
and GAPDH (for normalization) mRNA was measured with quantitative PCR. Three independent experiments were analyzed.
La-depletion did not reduce CCND1 mRNA level.
To test whether the phenotype observed in our
experiments was evoked by the lack of La protein and
not by La-siRNA off-target effects, we restored La
protein to cells in which the endogenous La protein was
depleted by siRNA treatment. For rescue experiments,
we introduced three silent point mutations into the LasiRNA binding site of a GFP-La expression plasmid
(GFP-LaR) from which a La-siRNA resistant GFP-La
mRNA can be transcribed. We transfected La-depleted
HeLa cells with GFP-La or GFP-LaR and observed that
GFP-LaR expression functionally complemented endogenous La protein and restored cell proliferation
(Supplementary Figure 2). These data support the
concept that La protein contributes to HeLa cell
proliferation.
Human La is required for CCND1 expression
To get insight into the overall effect on protein
expression in La-depleted cells we applied 2-D-gel
electrophoresis. About 6% of proteins are down
regulated and less than 1% were up regulated in Ladepleted cells (Supplementary Figure 3). As cell
proliferation of La-depleted cells was reduced, we
analyzed specifically the expression of proteins involved
in cell proliferation by western blotting. In La-depleted
HeLa cells, a significant reduction of the CCND1 was
observed (Figure 2a). In addition, we tested cyclin A,
cyclin B, cyclin E, cyclin-dependent kinase 1, cyclindependent kinase 2 and p27, and saw that only protein
levels of the cell cycle inhibitor, p21, were modestly
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reduced in La-depleted cells (Figure 2a). We found that
expression of Mdm2 and X-linked inhibitor of apoptosis
protein was not affected by La depletion, which suggests
that La-dependent translational regulation of those two
proteins is specific to cell types and growth conditions
(Figure 2a).
This finding suggests that La depletion does not
induce an overall reduction in protein expression, but
affects the expression of specific proteins and prompted
us to further investigate whether the reduction of
CCND1 protein expression was specifically evoked by
the lack of La protein. Therefore, we performed a rescue
experiment demonstrating that expression of the GFPLaR plasmid in La-depleted HeLa cells restored CCND1
protein levels by 66% (Figure 2b, lane 2 vs lane 4). The
experiment suggests that the La protein contributes to
CCND1 expression.
To elucidate the mechanism by which La affects
CCND1 expression, we compared CCND1 mRNA
levels of control-treated with La-depleted HeLa cells
by quantitative reverse transcriptase–PCR. No difference in CCND1 mRNA levels was observed (Figure 2c).
These data were confirmed by northern blot analysis
(data not shown). Next, we compared the half-life of
CCND1 protein in control- with that in La siRNAtreated cells and found that CCND1 half-life was
not shorter in La-depleted cells compared with control
cells (data not shown). These experiments suggest that
La protein regulates CCND1 expression post-transcriptionally.
La protein contributes to cell proliferation
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Figure 3 La protein is associated with CCND1 mRNA in living cells and regulates the CCND1 IRES element. To demonstrate an
association between La and CCND1 mRNA, HeLa cells were cross-linked to fix potential La:RNA interactions in living cells. Cell
lysates were prepared and used for La-specific IP. As control immunoglobulin isotype IgG 2a,k was applied (IgG con). After reversing
the crosslink, RNA was prepared from starting material, La and control IP pellets (P). RNA was analyzed by RT–PCR using CCND1
and hetero nuclear ribonucleoprotein (hnRNP)-E2 specific primer. (a) La-specific immunoprecipitation (IP). S ¼ IP supernatant
(b) RT–PCR analysis of La-associated RNA. La is specifically associated with CCND1 mRNA. M ¼ DNA marker. (c) Control-treated
and La-depleted cells were labeled with 35S-methionine for 20 min. Cell lysates were prepared and analyzed by SDS–polyacrylamide gel
electrophoresis and autoradiograph (Supplementary Figure 4). The lysates were used for CCND1- and tubulin-specific IP. IP pellets
were analyzed by SDS–polyacrylamide gel electrophoresis and autoradiography. CCND1 synthesis is strongly reduced in La-depleted
cells when compared with control-treated cells. (d) The CCND1 IRES plasmid pRCD1F (Shi et al., 2005; Jo et al., 2008) were
transfected into La-depleted and control-treated cells (in this experiments control siRNAs were a pool of random sequence obtained by
Dharmacon, Lafayette, CO, USA) and Renilla and Firefly luciferase activity was determined using the Dual-Luciferase Reporter
Assay (Promega). The Firefly and Renilla luciferase values were corrected by subtracting the backround luciferase values and the
Firefly:Renilla luciferase ratio was calculated and plotted. Four independent transfections were analyzed. (e) The CCND1 IRES
plasmid pRCD1F was co-transfected with GFP or GFP-La expression vectors. Cells were harvested 48 h later and luciferase expression
was determined using the Dual-Luciferase Reporter Assay. The Firefly and Renilla luciferase values were corrected by subtracting the
background luciferase values and the Firefly:Renilla luciferase ratio was calculated and plotted. Five independent transfections were
analyzed. Overexpression of La is able to stimulate CCND1 IRES-dependent translation.
Next, we were wondering whether we could recapitulate the defect in cell proliferation of La-depleted cells
by a direct siRNA-mediated knockdown of CCND1
expression. FACS analysis of the cell cycle phases
revealed that CCND1-depleted cells did not arrest in
the G1 or any other cell cycle phase (Supplementary
Figure 4) which, was analogous to our observations in
La-depleted cells (Supplementary Figure 1).
La associates with CCND1 mRNA
According to recent reports, La binds to cellular
mRNAs and effects its translation (Trotta et al., 2003).
As La depletion reduces CCND1 without changing
CCND1 mRNA level or protein half-life, we assumed
that La interacts with CCND1 mRNA in living cells to
regulate its expression. We prepared cell extracts from
in vivo cross-linked HeLa cells to purify La mRNA
complexes by immunoprecipitation (Figure 3a). After
reverse cross-linking, RNA preparation and reverse
transcriptase–PCR of the La-specific immunoprecipitated RNA, we conclude that CCND1 mRNA is
specifically associated with La in living cells
(Figure 3b). As specificity control, we show that
abundant hetero nuclear ribonucleoprotein (hnRNP)E2 mRNA was not immunoprecipitated with the
La-specific antibody (Figure 3b). Next, we analyzed
ongoing protein synthesis by 35-S methionine metabolic
labeling of La-depleted and control-treated cells. The
analysis of 35-S methionine protein lysates revealed that
La depletion did not cause an overall reduction in
protein synthesis (Supplementary Figure 5). Those
lysates were used for CCND1- and tubulin-specific
immunoprecipitation. As shown in Figure 3c, tubulin
protein was synthesized to similar degree in controltreated and La-depleted cells. However, CCND1 synthesis was massively reduced in La-depleted cells, suggesting
that CCND1 translation was specifically hampered.
Several reports suggest a role of La in IRESdependent translation (Belsham et al., 1995; Ali et al.,
2000; Holcik and Korneluk, 2000; Kim et al., 2001).
Hence, we tested whether La regulates the CCND1
IRES element (Shi et al., 2005; Jo et al., 2008). We
transfected the dual luciferase reporter construct
(pRCD1F, kind gift by Dr Gera in which the firefly
luciferase expression is dependent on the CCND1 IRES
element in La-depleted and control-treated cells) and
analyzed luciferase expression 48 h later. As shown
in Figure 3d, IRES-driven firefly luciferase expression
was reduced by about 40% in La-depleted cells. Next
we tested whether La overexpression stimulates the
CCND1 IRES (Figure 3e). In those experiments we
cotransfected the CCND1 IRES reporter with GFP or
GFP-La expression plasmids in HeLa cells and analyzed
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La protein contributes to cell proliferation
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Figure 4 La depletion reduces cell proliferation and CCND1 expression in different cell lines. After La-depletion cell proliferation of
(a) PC-3 and (b) NIH 3T3 cells were compared over 4 days after the second siRNA treatment. (c) The proliferation rate of PC-3 and
NIH 3T3 cells were statistically significant reduced after La-depletion (P-value o0.05). Three independent experiments were analyzed.
(d) Protein extracts from La-depleted (La) and control-treated (con) cells were analyzed by western blotting. In PC-3 and NIH 3T3
cells La depletion correlated with reduced CCND1 protein expression. GAPDH protein was used as a loading control.
Chemiluminescence signals were recorded using an ImageQuant ECL systems and quantified using ImageQuant TL software.
luciferase expression 48 h later. The calculation of the
firefly:renilla luciferase ratio revealed that exogenous La
protein is able to stimulate CCND1 IRES-dependent
translation by almost three fold. These data demonstrate that La is associated with CCND1 mRNA in
living cells and that La supports CCND1 IRESdependent translation.
La depletion effects proliferation and CCND1 expression
in different types of cells
Next we tested whether La protein contributes to cell
proliferation of other types of cell lines. The proliferation rate of La-depleted prostate cancer cell line PC-3
(Figure 4a) and immortalized mouse embryonic fibroblasts (NIH-3T3) (Figure 4b) was significantly reduced
by about 50% (Figure 4c). As shown for HeLa cells, an
increase in cell death was not detected in La-depleted
PC-3 cells by PARP analysis, Annexin V and propidium
iodide co-staining (data not shown). Next we tested
whether reduced proliferation rates were in coincident
with low CCND1 expression in La-depleted PC-3 and
NIH-3T3 cells, as shown for HeLa cells. Indeed La
depletion caused a drop in CCND1 protein levels in all
cell lines analyzed (Figure 4d). These results suggest that
the RNA-binding protein La is required for cell
proliferation and CCND1 expression in different types
of cells and is not restricted to a single cell line.
To support a functional link between La expression,
CCND1 expression and cell proliferation, we compared
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the proliferation rate of La-depleted and control-treated
immortalized mouse embryonic fibroblast (MEF) cells
originating from CCND1 knockout mice (MEFCCND1/
3T3 cells (Albanese et al., 1999; Wang et al., 2003)). In
contrast to all other tested cell lines, the proliferation
rate of La-depleted MEFCCND1/ was not reduced
(Figure 5a). By quantitative western blot analysis we
demonstrate that La protein expression was equally
reduced in MEFCCND1/ and NIH-3T3 control cells
(Figure 5b). These results support our notion that the La
protein affects cell proliferation by a CCND1-mediated
mechanism.
The La protein is overexpressed in cervical cancer
Our studies indicate that RNA-binding protein La can
regulate cell proliferation in different cancerous cell
lines. To determine whether the La protein is aberrantly
expressed in cancer tissue, we performed immunohistochemistry on cervical cancer tissue micro arrays (TMA)
(TMA CR481, Biomax, Rockville, MD, USA) containing 12 cases of cervical squamous cell carcinoma (SCC)
with 12 unmatched normal adjacent tissues, in a total of
24 patients for which 44 tissue cores were analyzed
(duplicated cores). The TMA was hybridized with highly
specific monoclonal La antibody (3B9) and counterstained with hematoxylin (Figures 6a, b, d and e). As a
control, hybridization with immunoglobulin isotype
IgG 2a,k and counterstaining with hematoxylin were
performed on a successive cut of the same TMA
La protein contributes to cell proliferation
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Figure 5 Cell proliferation of MEFCCND1/ cells is not affected by La-depletion. Cell proliferation of La-depleted (La) and controltreated (con) CCND1 knock out cells (MEFCCND1/) was compared over 4 days after the second siRNA treatment. (a) No significant
affect on cell proliferation of La-depleted MEFCCND1/ was observed. Three independent experiments were analyzed. (b) Protein
extracts from La-depleted (La) and control-treated (con) MEFCCND1/ cells and NIH-3T3 cells were analyzed by western blotting. La
protein was reduced in La-depleted MEFCCND1/ and NIH 3T3 cells to a similar extend. GAPDH protein was used as a loading
control. Chemiluminescence signals were recorded using an ImageQuant ECL systems and quantified using ImageQuant TL
software.
Figure 6 Nuclear expression of RNA binding La protein in cervical tumor and normal adjacent tissue. La-specific nuclear staining
was increased in cervical SCC tissue (a, b) compared with normal cervical tissue with chronic inflammation of cervical mucosa.
(d, e) Control staining with immunoglobulin isotype IgG 2a,k in SCC and normal tissue. (c, f) Scale bar ¼ 200 mm. Scoring of La
protein expression in TMA containing cervical SCC tumor and normal adjacent tissues. (g) The difference in La protein expression in
normal and tumor tissue was statistically significant (P-value o0.0001). The filled dots represent adenocarcinoma tissue samples.
Scoring represents that 80% (score 4), 50% (score 3), 25% (score 2) or o10% (score 1) of nuclei are positive for La-specific
staining.
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(Figures 6c and f). In SCC tissue, up to 80% of the
cells had La-specific brown staining of the nucleus
(Figures 6a and b). Compared with cancerous tissue, less
than 10% of the epithelial cells in adjacent normal
cervical tissue (which often has chronically inflamed
mucosa) were positive for nuclear La-specific staining
(Figures 6d and e). In addition, infiltrating cells within
the cervical mucosa also stained positive for La
(Figure 6e). Two pathologists performed the scoring of
the TMAs independently and the results were plotted as
shown in Figure 6g. Our data show, for the first time,
the expression of La in normal and cervical cancer tissue
and establishes the significant overexpression of the
RNA-binding protein La in cervical SCC tissue.
Elevated La expression correlates with elevated CCND1
expression in cervical cancer tissue lysates
Our work suggests that the La protein is overexpressed
in cervical cancer tissue and that it regulates CCND1
expression in different cell lines including cervical cancer
HeLa cells. Therefore, we investigated whether La
overexpression in cervical cancer tissue occurs coincidentally with an increase in CCND1 protein expression. We compared expression of La and CCND1 in
tissue lysates by quantitative western blotting. We
analyzed La and CCND1 expression in matched tissue
lysates prepared from tumor and adjacent normal
cervical tissue obtained from 12 patients (Figure 7).
After normalizing La and CCND1 protein against btubulin, we observed that of the 12 lysate sets, 7 tumor
samples had above-normal La protein expression
(Figure 7, high La in tumor, n ¼ 7 sets) and 5 tumor
samples had lower expression (Figure 7, low La in
tumor, n ¼ 5 sets). Interestingly, only in the seven tissue
lysates with elevated La protein did we determine a
strong increase in CCND1 protein levels. In contrast,
CCND1 levels were normal in tissue lysates with low La
protein. We conclude that our data support a model in
which La protein regulates cell proliferation and
contributes posttranscriptionally to enhanced CCND1
expression in different types of tumor cells.
Discussion
Here we present evidence that the cancer-associated
RNA-binding protein La is required for cell proliferation and CCND1 expression in cancer. In summary, our
data demonstrate the following: (I) La depletion causes
a defect in cell proliferation of different types of cell lines
(HeLa, PC-3, NIH-3T3); (II) La depletion led to a
decrease in CCND1 protein levels in different cell types,
which can be restored by exogenouse-La expression;
(III) La is associated with CCND1 mRNA in living cells
and activates CCND1-IRES; (IV) La protein is overexpressed in cervical cancer tissue; and (V) elevated La
levels are paralleled by increased CCND1 protein in
cervical tissue lysates.
Recently, a role was suggested for the RNA-binding
protein La in tumorigenesis: La protein is overexpressed
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Figure 7 Elevated La protein correlates with elevated CCND1
protein in cervical tumor tissue. Quantitative western blot analysis
for La and CCND1 protein in 12 tissue sets of matched normal and
tumor samples from cervical cancer patients. Seven tumor tissue
lysate sets (n ¼ 7) with high La expression in tumor samples
compared with matched normal tissue (high La) and five tumor
tissue lysate sets (n ¼ 5) with low or normal La expression in tumor
samples compared with matched normal tissue (low La) were
analyzed. Loading and quantification was normalized against
b-Tubulin protein levels. The dotted line represents the protein
expression of La and CCND1 in normal cervical tissue. Chemiluminescence signals were recorded using an ImageQuant ECL
systems and quantified using ImageQuant TL software.
in chronic myeloid leukemia cells and stimulates the
expression of Mdm2 (Trotta et al., 2003), a negative
regulator of the tumor suppressor p53. Brenet et al.
(2009) reported recently that in murine glial cells, the
GFP-tagged La protein is relocated to the cytoplasm
after phosphorylation by protein kinase AKT. Furthermore, Brenet et al. (2009) provided evidence that the
assortment of translational active La-associated
mRNAs changes upon AKT activation, suggesting that
La might contribute on the translational level to the
oncogenic effect of aberrant AKT activity in cancerous
cells. A recent report suggests that the La protein is
elevated in numerous cancer cell lines (Al-Ejeh et al.,
2007), but so far evidence is lacking to demonstrate that
the La protein is overexpressed in solid tumor tissue.
The highly significant overexpression of La in cervical
cancer (Figure 6), in chronic myeloid leukemia (Trotta
et al., 2003), and the AKT-dependent phosphorylation
of the RNA-binding protein La (Brenet et al., 2009)
might be a characteristic of various cancerous cells and
it is tempting to speculate that the La protein supports
tumorigenesis when aberrantly expressed.
Our attempt to illuminate cellular processes regulated
by the La protein revealed that cell proliferation of the
human cervical (HeLa) and prostate (PC-3) cancer cell
lines, and murine immortalized embryonic fibroblasts
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441
(NIH 3T3) is La-dependent (Figure 1 and 4). However,
the finding that proliferation of La-depleted MEFCCND1/
was not reduced (Figure 5a) suggests that CCND1
might mediate the effect of La on cell proliferation. Ladependent expression of the cell cycle regulatory protein
CCND1 in different cell lines (Figure 2a and 4d),
the CCND1 rescue experiments (Figure 2b), the La
dependent regulation of the CCND1 IRES element
(Figures 3d and e) and the finding that elevated La
expression in cervical cancer tissue lysates strongly
correlates with elevated CCND1 levels (Figure 7) are
further supportive for a model in which La contributes
to cell proliferation and CCND1 translation. CCND1
expression is upregulated in response to mitogens and
associates with cyclin-dependent kinases 4 or 6 (CDK4/
6) (Motokura et al., 1991; Fu et al., 2004; Knudsen
et al., 2006; Musgrove, 2006). The CCND1/CDK4/6
complex leads to phosphorylation of the retinoblastoma
protein, which normally exists in a hypophosphorylated
form associated with the transcription factor E2F. Upon
CCND1/CDK4/6-dependent phosphorylation, retinoblastoma protein releases the transcription factor E2F.
Subsequently, E2F activates a number of genes initiating the transition from the G1 into the S phase.
Importantly, the cell cycle regulator CCND1 is aberrantly expressed in different cancers (for review, see
Diehl, 2002; Fu et al., 2004). However, often in the
cervical cancer tissue and in the HeLa cervical cancer
cell line, CCND1/CDK4/6-dependent regulation is
nullified by the action of the human papilloma virus
protein E7 (Chellappan et al., 1992; McLaughlinDrubin and Munger, 2009). Hence, it is assumed that
cell proliferation in papilloma virus-infected cervical
cancer tissue/cells is mainly CCND1-independent and
contradicts our notion that La regulates cell proliferation through a CCND1-mediated mechanism. However,
CCND1 might contribute to cell proliferation by
another mechanism. It is well established that CCND1
fulfills CDK-independent functions and that CCND1
regulates a number of transcription factors or transcriptional regulatory proteins (for review, see Fu et al.,
2004). It is important to note that a comprehensive
analysis of gene expression data suggests that CCND1
counteracts the transcription factor C/EBPb (Lamb
et al., 2003), which regulates cell differentiation and cell
proliferation (for review, see Nerlov, 2007; Zahnow,
2009). C/EBPb is translated into two different isoforms:
LAP (C/EBPb, p44, p42) and LIP (p20) (Calkhoven
et al., 2000; Zahnow, 2009). The LAP:LIP ratio is
assumed to regulate cell proliferation through the LAPmediated repression of E2F target genes (Sebastian
et al., 2005). Lamb et al. (2003) suggested that CCND1
acts similar to LIP by counteracting LAP (C/EBPbeta)
functions. Hence, it is reasonable to speculate that
La-dependent CCND1 expression is an important mechanism which contributes to cell proliferation even in
human papilloma virus-infected cells. However, we also
have to assume that additional factors—for example not
yet identified La-regulated proteins (Supplementary
Figure 3)—in concert with CCND1 are required for La
to contribute to cell proliferation in different cell types.
Interestingly, in cervical tumor lysates elevated La
protein levels are observed along with an increase in
CCND1 protein levels (Figure 7). The finding that
CCND1 protein is elevated in those lysates is in agreement
with recent studies demonstrating elevated CCND1
expression in cervical cancer (Nichols et al., 1996; Cheung
et al., 2001; Carreras et al., 2007; Conesa-Zamora et al.,
2009). Although, in one study, CCND1 expression was
reduced in most of the analyzed samples, CCND1 was
elevated in invasive cancer and it correlates with lower
overall survival (Bae et al., 2001). Importantly, overexpression of CCND1 was not thought to be due to gene
amplification, suggesting posttranscriptional mechanism
in stimulating CCND1 expression (Muise-Helmericks
et al., 1998; Cheung et al., 2001; Shi et al., 2005; Inge
et al., 2008; Frost et al., 2009; Cowling, 2010).
It has been shown that the La protein regulates
translation of Mdm2 (Trotta et al., 2003) and IRESdependent translation of the X-linked inhibitor of
apoptosis (Holcik and Korneluk, 2000). Upregulation of
both proteins is expected to support cancer development
by repressing the expression of the tumor suppressor p53
and blocking apoptosis, respectively. In this study we
found for the first time that La contributes to the
expression of the cooperative oncogene CCND1 most
likely by regulating CCND1 IRES-dependent mRNA
translation (Figure 3). The CCND1 IRES element was
intensively studied in the past (Shi et al., 2005; Jo et al.,
2008). It will be interesting to dissect the molecular details
of La-dependent regulation of CCND1 IRES in the future
and the cellular conditions allowing La to activate the
CCND1 IRES. As an example, a change not only in total
La protein levels, as shown in this study, but also a
redistribution to the cytoplasm (Brenet et al., 2009) might
contribute to the ability of La to stimulate translation of
specific mRNAs in cancerous cells.
Our future work will focus on the dissection of the
molecular mechanism of La-dependent CCND1 regulation and cellular condition allowing La to stimulate
CCND1 translation. Our study clearly demonstrates
that La regulates the expression of CCND1 and cell
proliferation in different cell lines, suggesting a model in
which the La protein has an important role in cancer
pathology.
Materials and methods
Oligonucleotides
Please see Supplementary Materials.
DNA plasmids and mutagenesis
GFP-LaR was prepared by introducing three silent point
mutations in the La siRNA binding site of a GFP-La plasmid
(Horke et al., 2004) by PCR-based mutagenesis using
oligonucleotides La-M48S and La-M49AS. PCR products
were digested with DpnI and subsequently ligated using
the Rapid Ligation Kit (Roche, Germany) following the
manufacturer’s instructions. Plasmids were sequenced to
confirm mutagenesis.
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La protein contributes to cell proliferation
G Sommer et al
442
Cell culture and transfections
HeLa cells were purchased from ATCC, expanded and
aliquots were stored in liquid nitrogen. New HeLa cell cultures
were established every 2–3 months. MEFCCND1/ 3T3 cells
(Albanese et al., 1999; Wang et al., 2003) were kindly provided
by RG Pestell (Thomas Jefferson University, Philadelphia,
PA, USA). All cells were cultured in complete DMEM plus
10% fetal bovine serum. For siRNA treatment, 0.5 105
HeLa cells were plated per well (12-well plate). At 2 h before
transfection, complete DMEM was replaced by OPTI-MEM
(Invitrogen, Carlsbad, CA, USA) and siRNAs (final concentration 100 nM) were transfected using Oligofectamine (Invitrogen). For the second siRNA treatment, cells were replated
on day 5 after the first treatment and again transfected with
siRNA the following day as described. For siRNA treatment
of MEFCCND1/ and NIH 3T3 cells 1.0–2.0 106 cells were
transfected with 3 mg of La-specific or con-siRNA using a
nucleofector according to the manufacturer’s instructions
(AMAXA, Cologne, Germany, program A-023). For the
second siRNA treatment, cells were transfected on day 5 after
first treatment as described. Transfections of plasmid DNA
were performed using Fugene-6 reagent (Roche, Indianapolis,
IN, USA) according to the manufacturer’s instructions.
Proliferation assays, rescue experiments, FACS analysis and
Annexin V staining
For cell proliferation, the CyQuant Cell proliferation Assay
Kit (Invitrogen) was applied according to the manufacturer’s
instructions. At 2 days after the second siRNA treatment 4000
HeLa cells, 1250 MEFsCCND1/ cells, 5000 PC-3 cells or 1000
NIH 3T3 cells per well were plated in 96-well plates. Re-plating
efficiency was determined 4–5 h after plating and cell
proliferation was measured daily for 4 days.
For rescue experiments La-depleted and control-treated cells
were replated in 12 wells on day 2 after the first siRNA
treatment. GFP-La or GFP-LaR plasmid DNA was transfected on day 3 or 4. The next day, cells were sorted for GFP
signals with an Aria FACS cell sorter (BD Biosciences, San
Jose, CA, USA), and positive cells were cultured and replated
one day before the second siRNA treatment. For endpoint
analysis, on day 1 after the second treatment 0.25 105 cells
were transferred to 10-cm culture dishes and cultured for 3–4
days. Cells were stained with GIEMSA staining solution
(Merck, Whitehouse Station, NJ, USA) and counted. From
the same experiments, additional cells were harvested 48 h after
the second RNAi to analyze CCND1 protein expression in
GFP-La and GFP-LaR-expressing cells. Protein lysates were
prepared and analyzed for CCND1 by western blotting.
A number of dead cells were determined with the AnnexinV-FLUOS Staining Kit (Roche) according to the manufacturer’s instructions. FACS analysis was performed according
to standard protocols at the Flow Cytometry and Cell Sorting
facility (MUSC, Charleston, SC, USA). Caspase-3 dependent
processing of PARP was determined by western blot analysis.
Western blot analysis
Cells were lysed in cell lysis buffer (10 mM Tris, 10 mM KCl,
10 mM MgCl2, 0.2% Nonidet-P40, 1 mM EDTA, 420 mM NaCl,
1% protease inhibitor) and protein was analyzed by SDS–
polyacrylamide gel electrophoresis (PAGE). The antibodies
used and their dilutions are described under Supplementary
Materials. Secondary antibodies (1:20 000) were horseradish
peroxidase-conjugated (Dianova, Hamburg, Germany). Quantification was performed with an ImageQuant RT ECL
instrument and ImageQuant TL software (GE Healthcare,
Piscataway, NJ, USA).
Oncogene
In vivo crosslinking and immunoprecipitation
For reversible in vivo crosslinking we followed the protocol
(Niranjanakumari et al., 2002) with modifications (G Dreyfuss,
personal communication, University of Pennsylvania School
of Medicine) mentioned below. For formaldehyde fixation
the 1–2 106 trypsinized cells were washed twice with ice
cold 1 PBS, resuspended in 1 PBS containing 0.2% (v/v)
formaldehyde (Thermo Scientific, Pittsburgh, PA, USA, Methanol-free) and incubated at room temperature for 10 min with
slow mixing. The reaction was quenched by adding glycine (pH
7.0) to a final concentration of 0.25 M followed by incubation at
room temperature for 10 min with slow mixing.
For immunoprecipitation of La-associated RNA, cells were
harvested and lysed in ice-cold lysis buffer containing 20 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 10% glycerol,
1% NP-40, 1 Proteinease Inhibitor Mix (Roche), 50 mM
NaF, 1 mM Na3VO4, 1 mM DTT and 20 U rRNAsin (Promega,
Madison, WI, USA). The cell extract was sonicated and
clarified by centrifugation at 4 1C for 10 min. Cell extracts
were pre-cleared by incubation with Protein A-Sepharose
(GE Healthcare) in the presence of 20 U rRNAsin at 4 1C for
2 h followed by immunoprecipitation at 4 1C over night in
presence of Protein A-Sepharose (GE Healthcare), 20 U
rRNAsin and 2.5 mg of the following antibodies: mouse
monoclonal anti-La 3B9 (a kind gift from M Bachmann,
Technical University Dresden, Dresden, Germany) or purified
mouse immunoglobulin isotype IgG 2a,k (eBioscience, San
Diego, CA, USA).
For CCND1 and tubulin immunoprecipitation from 35Smethionine metabolic labeled protein lysates (Supplementary
Figure 5) the above protocol was used and monoclonal antiCCND1 and anti-a-tubulin were applied.
Total RNA isolation and quantitative RT–PCR
Total RNA was isolated using TriPure (Roche) following the
manufacturer’s instructions. Reverse transcription of RNA
was performed using the ThermoScript RT–PCR System
(Invitrogen). Quantitative PCR amplification was performed
on the BioRad iCycler-iQ system (BioRad, Hercules, CA,
USA) using the iQ-SYBR Green Supermix (BioRad).
Immunohistochemical staining
The cervical cancer TMAs with normal adjacent tissues (TMA
CR481) were purchased from Biomax. Staining was performed
according to standard protocols. Briefly, slides were baked for
2 h at 60 1C, deparaffinized in xylene, and stepwise rehydrated
in ethanol (100–50%) followed by ddH2O and 1 PBS washes.
Slides were boiled for 10 min in citrate-based antigen unmasking solution (Vector), washed with 1 PBS, treated with 0.3%
solution of H2O2 in ddH2O for 20 min and washed in 1 PBS.
Slides were blocked with horse serum, incubated with Laspecific antibody (1:200 dilution in horse serum), or mouse
isotype IgG 2a,k (eBioscience) at 4 1C overnight. Staining was
completed using ImmPress Reagent, anti-Mouse Ig Peroxidase, and ImmPACT DAB substrate (Vector). Tissue sections
were counterstained with Hematoxylin QS Nuclear Counterstain (Modifies Mayer’s Formula, Vector).
Cervical cancer tissue lysates
Please see Supplementary Materials.
Abbreviations
CCND1, cyclin D1; con, control; FACS, fluorescence-activated
cell sorting; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GFP, green fluorescent protein; MEF, mouse embryonic
fibroblasts; PARP, poly (ADP-ribose) polymerase; siRNA, small
La protein contributes to cell proliferation
G Sommer et al
443
interfering RNA; TMA, tissue micro array; XIAP, X-linked
inhibitor of apoptosis; IRES, internal ribosome entry site.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank Arne Düsedau (HPI) for sorting GFP-positive
cells and Rick Peppler for FACS analysis (MUSC). We
are grateful to R G Pestell (Thomas Jefferson University)
for providing the MEFCCND1/ cells, to M Bachmann
(Technical University Dresden, Germany) for providing La
antibodies, to Josef Gera for providing the CCND1-IRES
reporter plasmid pRCD1F, to D Watson (MUSC) for
experimental advice during this study, and D Fernandes
(MUSC) and J Schnellmann (MUSC) for critical reading of
the manuscript. This work was supported by the Deutsche
Forschungsgemeinschaft, HE 2814/3-2 (TH). The HeinrichPette-Institut (HPI) is financially supported by the
Bundesministerium für Gesundheit and Freie und Hansestadt
Hamburg.
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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)
Oncogene