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INTRODUCTION |
In the normal adult liver, hepatocytes are highly differentiated
and rarely replicate, yet they retain a remarkable proliferative capacity. In response to acute injuries that result in decreased functional cell mass, parenchymal hepatocytes promptly enter the cell
cycle and can restore liver mass and function within days. Compensatory
hepatocyte proliferation is an important adaptive response in patients
with liver disease, and experimental models of liver regeneration
present a unique opportunity to study physiologic cell growth,
proliferation, and organ size homeostasis (1, 2). Hepatocyte
proliferation in vivo is controlled by an array of
extracellular stimuli including growth factors, hormones, inflammatory cytokines, nutrients, and extracellular matrix. Whereas intracellular mediators of hepatocyte cell cycle progression have been extensively studied, the signaling pathways controlling growth of these cells have
not been as clearly characterized.
Although the terms are sometimes used interchangeably, cell growth and
proliferation represent distinct cellular processes. Cell growth refers
to an increase in cell mass as a result of enhanced synthesis of
proteins and other macromolecules (reviewed in Refs. 3-6).
Proliferation refers to the orderly progression through the cell cycle
resulting in cell division. Under many circumstances, mitogens
coordinately regulate cell growth and proliferation, so that each
generation of cells is the appropriate size. Substantial insight into
the regulation of cell growth and proliferation has been gained from
yeast systems, which frequently utilize pathways analogous to mammalian
cells. In the yeast systems, cell proliferation is dependent upon
proper cell growth; inhibition of growth regulatory genes can arrest
proliferation, whereas inhibition of cell cycle genes does not
necessarily prevent cell growth (3, 4). In yeast and mammalian cells,
growth is dependent on the protein synthetic machinery, which must be
substantially up-regulated during mitogenesis to allow for normal cell
division (6, 7). The marked induction of protein synthesis following
mitogenic stimulation is mediated through several mechanisms, including ribosome biogenesis, increased translational rates, and enhanced uptake
of amino acids. Numerous studies have demonstrated that the increased
rate of protein synthesis necessary for cell growth is mediated by
pathways involving phosphoinositide 3-kinase, target of
rapamycin (TOR),1 and S6
kinases. In particular, TOR appears to play a pivotal role in
integrating signals from mitogens and nutrients that regulate cell
growth (for review, see Refs. 8-11). Specific inhibition of TOR with
rapamycin (sirolimus), a drug that is used for immunosuppression, impairs protein synthesis, cell growth, and proliferation in response to mitogens.
The primary intracellular mediators of cell cycle progression are
protein kinase complexes consisting of cyclins and
cyclin-dependent kinases (cdks), which control discrete
phases of proliferation (see Refs. 5, 12, and 13). Mitogenic signals
activate complexes that control G1 phase, consisting of the
D-type cyclins and cdk4 and cdk6, which are followed by
activation of cyclin E/cdk2 in late G1 phase. Cyclin
D/cdk4-6 and cyclin E/cdk2 cooperate to phosphorylate and inactivate
the retinoblastoma (Rb) protein and derepress E2F-mediated
transcription, which then promotes entry of cells into S phase. After
cells in culture progress beyond a critical point in late
G1 phase (called the restriction point), they no longer
require growth factors to complete the cell cycle. The biochemical
basis of the restriction point appears be inactivation of Rb by cyclin
D·cdk4-6 and/or cyclin E·cdk2, and thus these G1 cyclin·cdk complexes are crucial targets of
extracellular signals that control proliferation.
Inhibition of TOR signaling leads to down-regulation of G1
cyclin·cdk complexes, through mechanisms that differ between cell types (9). In yeast, rapamycin inhibits proliferation through down-regulation of the G1 cyclin CLN3 (14). In mammalian
cells, several studies have shown that rapamycin inhibits the
expression of cyclin D1 (9), which regulates G1 progression
in a variety of cells including hepatocytes (1, 5, 12, 15-17). This suggests that in higher eukaryotes, as well as yeast, cell cycle control proteins are "downstream" of growth control proteins (3, 4). However, recent studies have suggested a more complicated arrangement, because cell cycle control proteins can also regulate growth. For example, overexpression of D-type cyclins leads
to enhanced organ growth in Drosophila,
Arabidoposis, and mice (18-24). We have recently found that
transient transfection of hepatocytes with cyclin D1 leads to robust
proliferation and a >50% increase in liver mass within 6 days (15).
Conversely, cyclin D1 knockout mice are smaller than wild-type mice,
and mice with homozygous deletion of the p27 gene (which inhibits
cyclin·cdk complexes) show gigantism and enhanced organ size
(25-29). These findings suggest that cyclin D1 is capable of inducing
growth, and by inference, enhanced biosynthesis of proteins. Thus, in
addition to its well defined role in cell cycle progression, cyclin D1
may also play a role in the regulation of cell growth.
In the current study, we further explored the relationship between TOR,
cyclin D1, growth, and cell cycle progression in hepatocytes. Prior
studies have suggested that cyclin D1 is a key target of TOR in growth
factor-stimulated cells. However, rapamycin inhibits the expression of
many proteins (8-11), and previous studies have not determined whether
expression of cyclin D1 is sufficient to promote cell cycle progression
in the setting of TOR inhibition. Furthermore, although genetic studies
suggest that cyclin D1 induces cell growth, there is relatively little
direct evidence in mammalian systems, and previous studies have not
examined whether cyclin D1 is capable of inducing overall protein
synthesis in rapamycin-treated cells. Our results suggest that cyclin
D1 mediates overall protein synthesis, growth, and proliferation
downstream of TOR in hepatocytes.
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EXPERIMENTAL PROCEDURES |
Animals--
All animal studies were performed in accordance
with IACUC approval and NIH guidelines. Male BALB/c mice (Harlan
Sprague-Dawley) were provided laboratory chow and water ad
libitum. At 8 weeks of age, PH was performed as previously
described (30). Adenovirus-mediated transfection was performed as
previously described (15, 31). In brief, mice were injected with 5 × 109 plaque-forming units via tail vein injection of the
indicated adenoviruses. Rapamycin (Calbiochem) was diluted in
Me2SO at a concentration of 17.5 µg/µl. This was
further diluted into 250 µl of saline immediately before
intraperitoneal injection. Control mice received an equivalent amount
of Me2SO in saline. Rapamycin was administered at a dose of
1.5 mg/kg/day beginning 2 h prior to PH or adenovirus injection
and daily after this. Two hours before harvest, animals were injected
with bromodeoxyuridine (50 mg/kg) by intraperitoneal injection. Liver
harvest, biopsy for immunohistochemistry, and tissue homogenization was
performed as described elsewhere (30, 31).
Hepatocyte Culture--
Primary rat hepatocytes were harvested
from 8-10-week-old male Lewis rats (Harlan Sprague-Dawley) by
collagenase perfusion followed by purification through a Percoll
gradient (16, 17). These were plated on collagen film in Williams E
medium as previously described, in the presence or absence of EGF (10 ng/ml) and insulin (20 milliunits/ml) as indicated (16, 17). Rapamycin
was diluted in methanol as provided by the manufacturer (Cell Signaling
Technologies, Inc.) and used at a final concentration of 10 nM. Control samples were treated with an equivalent amount
of vehicle (methanol). Hepatocytes were transfected with adenoviruses
as previously described (16, 31). Media and additives were changed
daily. Cells harvest for protein and RNA isolation was performed as
previously described (16, 17). DNA synthesis was determined by
[3H]thymidine as outlined elsewhere (16, 17). Protein
synthesis was assessed by [3H]leucine incorporation using
techniques described elsewhere (32, 33). In brief, hepatocytes were
cultured at a density of 104/cm2 in 35-mm
dishes and [3H]leucine (ICN Biochemicals, 1 mCi/ml) was
added at a final concentration of 2 µCi/ml 68 h after plating
under the conditions indicated in Fig. 4. After 4 h, cells were
washed twice with cold phosphate-buffered saline, lysed in 10% cold
trichloroacetic acid, incubated at 95 °C for 15 min, and placed on
ice for 30 min. The acid-insoluble material was collected by vacuum
filtration onto Whatman GF/C glass fiber filters. Retained
radioactivity was determined by scintillation counting on a 1450 Microbeta counter (EG & G Wallac).
Adenoviruses--
Recombinant adenoviruses encoding human cyclin
D1 and cyclin E, along with the control virus encoding
-galactosidase, were described elsewhere (16, 31). Adenoviruses
encoding human E2F2 and constitutively active Akt were provided by Drs.
J. Nevins and K. Walsh, respectively (34, 35).
Western Blot Analysis--
Liver tissue and hepatocytes were
homogenized in lysis buffer as previously described (30). Western blots
were performed as reported elsewhere, using commercially available
antibodies (15, 30, 31). Additional antibodies include human cyclin E
(NeoMarkers, Fremont, CA) and rat cyclin E (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); phospho-S6 (Ser-235/236), total S6, phospho-Akt (Ser-473), and eIF-4E binding protein 1 (4E-BP1, Cell Signaling Technologies, Inc.); and eukaryotic initiation factor 4E (eIF-4E, BD
Transduction Labs). Previous studies have shown that the relative migration of 4E-BP1 bands corresponds with different phosphorylated forms of this protein (36).
Northern Blot Analysis--
Total RNA was isolated from
hepatocytes in 4 M GIT lysis buffer, followed by Northern
blot analysis (10 µg/lane) using a cDNA probe to mouse cyclin D1
as outlined previously (16, 17).
FACS Analysis and Microscopy--
Primary mouse hepatocytes from
adult male BALB/c mice (7-8 weeks old) were isolated from mice treated
as indicated by collagenase perfusion followed by purification through
a Ficoll gradient as previously described (16, 17). Freshly isolated
hepatocytes were subjected to forward area light scattering using a
FACSCaliber scanner (BD Pharmingen). Mean cell size and coefficient of
variation were determined using CellQuest Pro program (BD Pharmingen)
as recommended by the manufacturer. Cells were also air-dried on microscope slides and stained with hematoxylin and eosin for microscopy.
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RESULTS |
Rapamycin Inhibits Proliferation and Cyclin D1 Expression in
Cultured Hepatocytes--
Prior studies have demonstrated that
rapamycin inhibits cell cycle progression in response to mitogens in
numerous types of cells in culture, including rat hepatocytes (9, 37,
38). Primary rat hepatocytes in culture proliferate readily in the presence of mitogens. They demonstrate a relatively long G1
phase of about 48 h, and peak DNA synthesis (representing S phase)
occurs at about 72 h (16, 39). Previous studies have shown that
these cells progress through the mitogen restriction point at 40-44 h,
which corresponds in time with the induction of cyclin D1 in late
G1 phase (16, 39).
In Fig. 1A, we confirmed that
rapamycin inhibited cell cycle progression (as measured by DNA
synthesis) in cultured rat hepatocytes stimulated with EGF and insulin.
If rapamycin was provided before the restriction point (e.g.
at 24 h), cell cycle progression was inhibited, whereas addition
of rapamycin after the restriction point (i.e. at 48 h)
did not substantially reduce DNA synthesis at 72 h (Fig.
1B). This indicates that cells that have traversed the
restriction point were no longer sensitive to the rapamycin-mediated cell cycle arrest. Because induction of cyclin D1 corresponds in time
with progression of hepatocytes through the G1 restriction point (16, 39), this suggests the possibility that expression of this
protein may make cells resistant to the effects of rapamycin. To
confirm that rapamycin inhibited downstream effects of TOR, we examined
the phosphorylation of S6 at Ser-235/236 (which are rapamycin-sensitive
sites (38)) using a phosphospecific antibody. As predicted, rapamycin
added during the entire course of the experiment inhibited S6
phosphorylation at 48 and 72 h (Fig. 1C). As shown in
other systems (40), rapamycin also diminished the expression of total
S6 protein. Somewhat surprisingly, mitogen treatment did not appear to
substantially affect S6 phosphorylation at these sites in primary
hepatocytes. Studies of mitogen-dependent signaling
frequently examine phosphorylation events that occur within a shorter
time frame, because signal transduction kinases are often transiently
induced after growth factor treatment (41). In Fig. 1D,
cells were treated with EGF and insulin for 30-60 min before harvest.
This again demonstrated that mitogen treatment had little effect on S6
phosphorylation, whereas rapamycin was inhibitory. This suggests that
S6 phosphorylation at these sites (and possibly TOR activity) is
regulated by other factors in cultured hepatocytes, such as the
nutrient supply (11). These results indicate that TOR activity is
necessary for S6 phosphorylation and progression of hepatocytes through
the G1 mitogen restriction point.

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Fig. 1.
Rapamycin inhibits DNA synthesis and S6
phosphorylation in hepatocytes. A, primary rat
hepatocytes were plated in the presence or absence of EGF (10 ng/ml)
and insulin (20 milliunits/ml) and DNA synthesis was assessed by
[3H]thymidine uptake at 24-72 h as described under
"Experimental Procedures." Rapamycin (10 nM) or vehicle
were added to the indicated samples for the entire experiment. Results
are expressed as a percentage of the [3H]thymidine uptake
measured at 72 h in EGF/insulin-stimulated cells, and represent
the mean ± S.D. of six separate samples. B,
rapamycin was added to the culture medium for the indicated time frames
and DNA synthesis was determined at 72 h. C,
hepatocytes were cultured as in A and harvested at 48 and
72 h for Western blot using antibodies to
Ser-235/236-phosphorylated and total S6. D, hepatocytes
were cultured in the absence of growth factors for 44 h, followed
by the addition of EGF/insulin. Rapamycin or vehicle were added to the
indicated samples 15 min before the growth factors. Cells were
harvested after 30 or 60 min for Western blot analysis using the
indicated antibodies.
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In some but not all experimental systems, cyclin D1 is down-regulated
by rapamycin treatment (9). Consistent with these findings, rapamycin
significantly inhibited the expression of cyclin D1 in hepatocytes
(Fig. 2A). In addition, the
expression of proteins acting downstream in the cell cycle, such as
cyclin E and proliferating cell nuclear antigen, were also
substantially inhibited. The p21 cdk-inhibitor protein, which is
up-regulated by mitogens and cyclin D1 expression in hepatocytes (15,
16), was similarly inhibited by rapamycin. The expression of cyclin D1
mRNA was not affected by rapamycin treatment (Fig. 2B),
indicating that diminished expression of this protein was because of
impaired translation and/or enhanced proteolysis, as has been
demonstrated in other systems (9). These studies indicate that cyclin
D1 is a target of TOR signaling in hepatocytes.

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Fig. 2.
Inhibition of cyclin D1 and cell cycle
proteins by rapamycin. A, hepatocytes were
cultured under the indicated conditions as described in Fig.
1A and harvested at 72 h for Western blot analysis
using the indicated antibodies. B, total RNA was
harvested from hepatocytes at 72 h for Northern blot analysis of
cyclin D1. The bottom panel shows the 18 S ribosomal RNA
band as visualized by ethidium bromide staining.
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Cyclin D1 and E2F2 Overcome the Rapamycin-induced Cell Cycle Arrest
in Hepatocytes--
Prior studies have suggested that down-regulation
of cyclin D1 is an important component of the antiproliferative effect
of rapamycin, but because this agent inhibits the expression of many proteins, it is possible that other factors mediate this effect. To
test whether cyclin D1 was the pivotal cell cycle target of rapamycin,
we transfected the cells with a recombinant adenovirus encoding cyclin
D1 (ADV-D1). Recombinant adenoviruses transfect hepatocytes in culture
and in vivo with a high efficiency (16, 42). Transfection of
rapamycin-treated hepatocytes with ADV-D1 promoted DNA synthesis to
levels seen in untreated mitogen-stimulated cells and induced the
expression of downstream cell cycle proteins such as cyclin E,
proliferating cell nuclear antigen, and p21 (Fig.
3, A and B).
Transfection with E2F2, which mediates the transition into S phase
downstream of cyclin D1 (13), similarly promoted cell cycle progression
in rapamycin-treated cells. On the other hand, transfection with
activated Akt, which can act upstream of TOR (8-11), was ineffective.
Similarly, cyclin E did not induce cell cycle progression in the
setting of rapamycin treatment. These data suggest that cyclin D1 is
the key rapamycin-sensitive G1 regulatory protein in
cultured hepatocytes.

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Fig. 3.
Cyclin D1 and E2F2, but not cyclin E or
activated Akt, overcome rapamycin-mediated cell cycle arrest.
Rapamycin-treated hepatocytes were cultured under the conditions
described in Fig. 1A, transfected with recombinant ADV as described
under "Experimental Procedures," and harvested at 72 h.
A and B, hepatocytes were transfected with
cyclin D1, human E2F2, or the control vector followed by measurement of
DNA synthesis and Western blot analysis, respectively. The cyclin E
antibody used in B was directed against the rat protein.
C and D, hepatocytes were transfected with cyclin
D1, human cyclin E, constitutively active Akt, or the control vector,
and DNA synthesis assays and Western blot analysis were performed. The
cyclin E antibody used in D was directed against the human
protein.
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Cyclin D1 Promotes Global Protein Synthesis in Rapamycin-treated
Cells--
A major downstream effect of TOR is control of the protein
translation apparatus through several mechanisms, including S6 and
4E-BP1 phosphorylation and transcription of ribosomal RNA (8-11).
Previous studies have shown that inhibition of TOR with rapamycin
substantially diminishes the mitogen-stimulated induction of protein
synthesis in several types of cells (8-11, 32). In hepatocytes,
mitogen treatment promoted a >3-fold increase in protein synthesis
(Fig. 4); similar to studies in other
cells, rapamycin inhibited this response by 75% (32). Transfection of
rapamycin-treated hepatocytes with cyclin D1 restored overall protein
synthesis to levels similar to untreated mitogen-stimulated cells.
These results suggest that the rapamycin-mediated inhibition of
specific cell cycle proteins (Fig. 3) and global protein synthesis (Fig. 4) were because of diminished expression of cyclin D1. Because increased protein synthesis is a key component of cell growth (3-6),
these studies suggest that cyclin D1 may induce growth downstream of
TOR in hepatocytes.

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Fig. 4.
Cyclin D1 restores the induction of protein
synthesis by mitogens in rapamycin-treated hepatocytes.
Hepatocytes were cultured and transfected with the indicated
adenoviruses as described in the legend to Fig. 3, and harvested
following a 4-h pulse with [3H]leucine. Protein synthesis
was measured as outlined under "Experimental Procedures." Results
are expressed as the percentage of the [3H]leucine uptake
seen in EGF/insulin-stimulated cells in the absence of rapamycin and
are the average + S.D. of four different experiments.
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Rapamycin Inhibits Liver Regeneration and Cyclin D1 Expression in
Vivo--
Previous reports have shown that rapamycin inhibits
hepatocyte proliferation and liver growth in rats following growth
stimulation by PH (36, 38, 43). In the PH model, a large
population of hepatocytes progress through the cell cycle in a
relatively synchronous manner, with peak DNA synthesis (S phase)
occurring at 36-42 h in BALB/c mice (1, 2). Previous studies have shown that cyclin D1 is markedly induced at time points corresponding to late G1 and S phase after PH (but not after sham
surgery) in rodents (44-46). To determine whether cyclin D1 is a
target of rapamycin-sensitive pathways in hepatocytes
in vivo, mice were injected with 1.5 mg/kg/day of rapamycin
beginning 2 h before PH, and livers were harvested at 42 h.
As demonstrated in previous studies (36, 38, 43), rapamycin markedly
inhibited hepatocyte DNA synthesis after PH (Fig.
5). In vehicle-treated mice, 20.8 ± 9.1% of hepatocytes were positive by BrdUrd immunohistochemistry, as
compared with 3.0 ± 2.9% in the rapamycin-treated mice
(p < 0.005). The expression of cyclin D1, as well as
proteins acting downstream in the cell cycle, was substantially
diminished. Rapamycin selectively inhibited protein expression, because
other proteins (e.g. p27, actin, and eIF-4E) were unaffected
by this treatment. As recently shown (38), rapamycin inhibited the
appearance of the phosphorylated species of S6 after PH. A recent study
showed that 4E-BP1 phosphorylation is rapamycin-insensitive in
regenerating rat liver (36). Consistent with these findings, rapamycin
did not markedly diminish the appearance of the highly phosphorylated (slower migrating) species of 4E-BP1 in mouse liver after PH, but did
slightly increase the abundance of the unphosphorylated (faster-migrating) species (Fig. 5C). These data indicate
that cyclin D1 is a target of TOR signaling in the regenerating liver after PH, and that diminished expression of this protein corresponds with impaired liver regeneration.

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Fig. 5.
Rapamycin inhibits cyclin D1 expression and
hepatocyte proliferation after PH in vivo. Male
BALB/c mice were treated with rapamycin (1.5 mg/kg/day by
intraperitoneal injection) or vehicle (Me2SO) beginning
2 h before PH followed by harvest of the livers at 42 h as
described under "Experimental Procedures." Two hours before
harvest, the mice were given an intraperitoneal injection BrdUrd (50 mg/kg). A, hepatocyte DNA synthesis was determined by
BrdUrd immunohistochemistry 42 h after PH in Me2SO or
rapamycin-treated mice. The percentage of BrdUrd-positive hepatocyte
nuclei is expressed as the mean ± S.D. of 5 animals.
B, liver extracts were subjected to Western blot
analysis of cell cycle proteins. Specimens of normal liver and liver
harvested 42 h after PH (without other treatment) are also shown.
C, Western blot analysis was performed using antibodies
to Ser-235/236-phosphorylated and total S6, eIF-4E, and 4E-BP1.
Specimens from two separate Me2SO- and rapamycin-treated
animals are shown.
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Cyclin D1 Promotes Hepatocyte Proliferation and Growth in the
Livers of Rapamycin-treated Mice--
Our results in primary
hepatocytes indicated that expression of cyclin D1 was sufficient to
restore the induction of both cell cycle progression and protein
synthesis in the setting of rapamycin treatment. However, prior studies
have shown that primary hepatocytes demonstrate patterns of metabolism
and gene expression that can be distinct from hepatocytes in the liver
(1, 2, 39). To examine whether cyclin D1 promoted hepatocyte
proliferation and growth in vivo, we transfected
rapamycin-treated mice with this protein using the adenoviral system.
Previous studies have shown that intravenously injected adenoviruses
transfect hepatocytes in vivo with high efficiency (42), and
numerous studies have used this system to study the effect of
single-gene expression in the liver. As previously shown in normal mice
(15), transfection with ADV-D1 led to expression of cyclin D1 in the
livers of rapamycin-treated mice and promoted the expression of
proteins that act downstream in the cell cycle, including cyclin A,
proliferating cell nuclear antigen, and p21 (Fig.
6). In the rapamycin-treated mice, cyclin D1 also triggered hepatocyte DNA synthesis as measured by the number of
BrdUrd-positive hepatocytes (14.0 ± 5.3% versus 0%
for control-transfected mice, p < 0.004). Furthermore,
transfection with cyclin D1 led to substantial liver growth, increasing
relative liver size by more than 40% (p < 0.007).

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Fig. 6.
Cyclin D1 induces hepatocyte proliferation
and liver growth in vivo in rapamycin-treated
mice. Mice were treated with rapamycin (1.5 mg/kg/day) and
transfected by intravenous injection with the cyclin D1 or control
adenovirus as described under "Experimental Procedures."
A, hepatocyte DNA synthesis was determined by BrdUrd
immunohistochemistry in liver biopsies obtained 1 day after
transfection. B, Western blot analysis was performed on
liver lysates obtained at 1 day. C, liver mass (as a
percentage of body mass) was determined at 6 days.
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The increased liver size seen after cyclin D1 transfection suggests
that this protein induces hepatocyte growth as well as proliferation.
However, it is possible that other effects (e.g. edema or
accumulation of other cells) could contribute to the increased liver
mass, although these effects were not observed on routine histologic
examination of liver sections (data not shown). To examine whether
cyclin D1 promoted the growth of individual hepatocytes, we isolated
purified primary hepatocytes from the livers of normal and
rapamycin-treated mice 3 days after transfection with ADV-D1 or the
control vector. By forward angle light scattering, which quantitates
cell size, cyclin D1 induced substantial growth of individual
hepatocytes, even in the setting of TOR inhibition (Fig.
7). The increase in cell size was also
evident subjectively on microscopic examination. The observed increase
in cell size suggests that persistent expression of cyclin D1 in
vivo may induce hepatocyte growth to a greater degree than
proliferation, because a proportional increase in growth and
proliferation would be expected to result in normal-sized cells (3,
5).

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Fig. 7.
Cyclin D1 induces growth of hepatocytes
in vivo. Normal or rapamycin-treated mice were
transfected with ADV-D1 or the control adenovirus as described in the
legend to Fig. 6, and hepatocytes were isolated by collagenase
perfusion at 3 days as described under "Experimental Procedures."
Hepatocytes were purified by Percoll gradient centrifugation.
A, hepatocytes from untreated or rapamycin-treated mice
transfected with the indicated adenoviruses were analyzed by forward
angle light scattering measurements on a FACS scanner. The curves for
the untransfected and control-transfected hepatocytes are nearly
superimposed. B, mean cell size and coefficient of
variation was determined by FACS analysis. C, isolated
hepatocytes were subjected to hematoxylin and eosin staining and
microscopy.
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DISCUSSION |
Previous studies have shown that inhibition of TOR with rapamycin
prevents cell cycle progression, the induction of global protein
synthesis, and cell growth in mitogen-stimulated cells (8-11).
Interestingly, the mechanism(s) by which rapamycin inhibits proliferation varies between cell types. In several systems, cyclin D1
expression is markedly down-regulated by rapamycin, and therefore this
protein has been presumed to be a key cell cycle target of this agent.
However, to our knowledge, previous studies have not confirmed that
cyclin D1 expression is sufficient to overcome the rapamycin-mediated
cell cycle arrest. Furthermore, prior studies have not documented that
cyclin D1 promotes growth or overall protein synthesis in the setting
of rapamycin treatment. Our results suggest that cyclin D1 is a key
mediator of both proliferation and growth downstream of TOR in hepatocytes.
Under normal circumstances, growth and proliferation are stimulated in
a similar fashion following mitogen treatment, so that the cell
accumulates sufficient mass to ensure adequate distribution of cellular
contents to the daughter cells (3, 4). In the absence of sufficient
growth, subsequent generations of cells would become progressively
smaller, which would eventually compromise viability (5). Although the
pathways that promote growth and proliferation share several key
elements (including TOR), the downstream effectors of cell growth are
poorly understood. An important component of cell growth is induction
of ribosomes and the protein translation apparatus, which is necessary
to accommodate the required increase in protein synthesis (6, 7).
Compared with the current knowledge of the cell cycle machinery, less
is known about the mechanisms that regulate cell growth, ribosome biogenesis, and increased protein synthesis in response to mitogenic stimuli. Furthermore, the mechanisms that link growth and overall protein synthesis to the cell cycle are poorly understood (3-5).
Proteins involved in the regulation of both growth and proliferation
include Ras, phosphoinositide 3-kinase, TOR, Myc, and according to
recent studies cyclin D1 (3, 4). As recently proposed (3), these
proteins probably do not represent a linear pathway, but rather "a
web of interacting proteins" that have distinct effects. For example,
when overexpressed in the liver, Myc produces growth but not
proliferation (47). Furthermore, the effect of each protein
is highly dependent on the context. Overexpression of cyclin D in
developing Drosophila increases both growth and cell cycle
progression in proliferating cells, but only growth in post-mitotic
cells (24). In plants, D-type cyclins coordinately induce
growth and proliferation, so that cells maintain a normal size but the
plant size is increased (22). In several strains of transgenic mice,
targeted overexpression of cyclin D1 leads to enhanced proliferation
and organ growth, suggesting that this protein can promote both
processes in mammalian systems. However, in each of these studies, the
target cells were also subjected to normal growth signals during the
process of development and/or adult life. Thus, it is not clear whether
stimulation of other pathways by mitogenic stimuli is required for the
proliferative or growth-promoting effect of cyclin D1 in these systems.
To examine this further, we chose a population of cells that is
normally quiescent in the adult animal (hepatocytes), and inhibited a
key mediator of both growth and proliferation (TOR). Our results
suggest that in quiescent cells and in the presence of rapamycin,
cyclin D1 induced growth and proliferation in vivo.
Furthermore, cyclin D1 restored maximal protein synthesis rates in
rapamycin-treated hepatocytes in culture. We believe that this is the
most direct evidence to date that induction of cyclin D1 alone is
capable of promoting both growth and proliferation in a mammalian system.
In addition to playing a pivotal role in promoting normal cell cycle
progression, cyclin D1 is an important oncogene that is overexpressed
in many human tumors (5, 12). Constitutive expression of cyclin D1 is
likely to contribute to malignant transformation by reducing the
dependence on extracellular signals that normally control
proliferation, that is, it diminishes the requirement for mitogens in
the transition through the G1 restriction point (5, 12).
Phosphorylation of Rb and related proteins by cyclin D1/cdk4-6 appears
to be a key event during progression through the restriction point.
However, cyclin D1 has several other effects that are independent of Rb
or cdks (48). The mechanisms by which cyclin D1 may trigger cell growth
are poorly understood. In Drosophila, cyclin D/cdk4 appears
to promote cell growth independently of Rb (24). Recent studies have
suggested that G1 cyclin-cdk complexes enhance the
transcription of ribosomal RNA by RNA polymerases I and III (49, 50),
and thus may contribute to ribosome biogenesis downstream of mitogens.
However, neither this nor prior studies have clearly determined how
cyclin D1 promotes the protein translational apparatus and cell growth,
and further investigation is needed to identify the mechanisms and
downstream mediators involved (3, 4). The induction of cell growth by
cyclin D1 may play an important role in both tissue development and the
physiologic response to growth factors. Furthermore, cell growth is an
important component of the neoplastic process, and the ability to
promote both growth and proliferation may underlie the oncogenic effect
of cyclin D1 (5).
The current data build on prior studies examining the involvement of
TOR signaling in hepatocyte proliferation. Several groups have shown
that rapamycin inhibits S6K1 activation, S6 phosphorylation, DNA
synthesis, and restoration of liver mass after PH (36, 38, 43). In the
adult liver, increased S6 phosphorylation is thought to play an
important role in up-regulating ribosomal protein synthesis observed in
this model (51, 52). Interestingly, fetal hepatocytes in
late gestation demonstrate a high rate of proliferation that is not
inhibited by rapamycin or dependent on S6 phosphorylation (38). The
fetal hepatocytes express significant amounts of cyclin D1 and appear
to proliferate in a mitogen-independent manner (53, 54). The current
studies suggest that expression of cyclin D1, which is induced in fetal
hepatocytes through pathways distinct from those observed in adult
cells (53, 54), promotes rapamycin resistance. The effect of rapamycin
treatment on cyclin D1 expression in our studies contrasts with the
effect seen in conditional S6 knockout mice, which also show impaired
liver regeneration after PH (52). In the S6 knockout livers, cyclin D1
expression and associated kinase activity are normally up-regulated
after PH, but cyclin E expression, hepatocyte proliferation, and liver
growth are substantially inhibited. These results indicate that S6
deletion induces a cell cycle and growth checkpoint that acts
downstream of cyclin D1/cdk4 and is distinct from that induced by
rapamycin. Further study is required to clarify the role of downstream
mediators of TOR and translational regulatory elements in liver regeneration.
The results shown here may have implications for the clinical use of
TOR inhibitors. Although the dose of rapamycin in these studies exceeds
that used in the setting of human liver transplantation, this drug
might inhibit liver growth in small-size allografts that normally adapt
to the size of the recipient (55). Rapamycin analogs are also being
tested as antineoplastic agents because of their inhibitory effects on
growth and proliferation (9). The studies outlined here suggest that
persistent expression of cyclin D1 causes cells to be resistant to
rapamycin. Tumors with constitutive overexpression of cyclin D1 may
therefore be insensitive to the growth and cell cycle inhibitory
effects of rapamycin analogs.
One interpretation of our data is that cyclin D1 is the single relevant
downstream effector of TOR signaling during mitogenesis in regard to
both growth and proliferation. However, our system has several
limitations, and it is highly likely that a more refined model will
arise from further studies. It is important to point out that
transfection of cyclin D1 does not necessarily induce expression of
this protein equivalent to that seen in normal or malignant cells.
Thus, it is possible that when cyclin D1 is expressed in a more
physiologic manner, other proteins are required to induce growth and
proliferation. Furthermore, we have limited our studies to standard
parameters of growth and proliferation (DNA and protein synthesis, cell
and organ size, and cell cycle protein expression), and a more detailed
analysis may reveal that cyclin D1 does not overcome all of the effects
of TOR inhibition. Finally, the effects of cyclin D1 expression are
likely to be dependent on the cell type and thus it is not clear
whether these results can be generalized to other systems; hepatocytes
appear to be highly responsive to this protein (1, 15-17). However,
our data support prior conclusions that cyclin D1 is a key downstream
target of TOR. Furthermore, these studies indicate that cyclin D1
promotes both hepatocyte growth and proliferation in the setting of TOR inhibition.