Evidence That Cyclin D1 Mediates Both Growth and Proliferation Downstream of TOR in Hepatocytes*

Signaling through the target of rapamycin is required for increased protein synthesis, cell growth, and proliferation in response to growth factors. However, the downstream mediators of these responses, and the elements linking growth and proliferation, have not been fully elucidated. Rapamycin inhibits hepatocyte proliferation in culture and liver regenerationin vivo. In cultured rat hepatocytes, rapamycin prevented the up-regulation of cyclin D1 as well as proteins acting downstream in the cell cycle. Transfection with cyclin D1 or E2F2, but not cyclin E or activated Akt, overcame the rapamycin-mediated cell cycle arrest. Rapamycin also inhibited the induction of global protein synthesis after growth factor stimulation, and cyclin D1 overcame this inhibition. Rapamycin inhibited hepatocyte proliferation and cyclin D1 expression in the mouse liver after 70% partial hepatectomy. In rapamycin-treated mice, transfection with cyclin D1 induced hepatocyte proliferation, increased hepatocyte cell size, and promoted growth of the liver. These results suggest that cyclin D1 is a key mediator of increased protein synthesis, cell growth, and proliferation downstream of target of rapamycin in mitogen-stimulated hepatocytes.

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][4][5][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 cyclindependent kinases (cdks), which control discrete phases of proliferation (see Refs. 5,12,and 13). Mitogenic signals activate complexes that control G 1 phase, consisting of the D-type cyclins and cdk4 and cdk6, which are followed by activation of cyclin E/cdk2 in late G 1 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 G 1 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 D⅐cdk4 -6 and/or cyclin E⅐cdk2, and thus these G 1 cyclin⅐cdk complexes are crucial targets of extracellular signals that control proliferation.
Inhibition of TOR signaling leads to down-regulation of G 1 cyclin⅐cdk complexes, through mechanisms that differ between cell types (9). In yeast, rapamycin inhibits proliferation through down-regulation of the G 1 cyclin CLN3 (14). In mammalian cells, several studies have shown that rapamycin inhibits the expression of cyclin D1 (9), which regulates G 1 progression in a variety of cells including hepatocytes (1,5,12,(15)(16)(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 Dtype 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 Ͼ50% 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 cyclin⅐cdk complexes) show gigantism and enhanced organ size (25)(26)(27)(28)(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 previ-ous 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.

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 ϫ 10 9 plaque-forming units via tail vein injection of the indicated adenoviruses. Rapamycin (Calbiochem) was diluted in Me 2 SO at a concentration of 17.5 g/l. This was further diluted into 250 l of saline immediately before intraperitoneal injection. Control mice received an equivalent amount of Me 2 SO 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 [ 3 H]thymidine as outlined elsewhere (16,17). Protein synthesis was assessed by [ 3 H]leucine incorporation using techniques described elsewhere (32,33). In brief, hepatocytes were cultured at a density of 10 4 /cm 2 in 35-mm dishes and [ 3 H]leucine (ICN Biochemicals, 1 mCi/ml) was added at a final concentration of 2 Ci/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).
Northern Blot Analysis-Total RNA was isolated from hepatocytes in 4 M GIT lysis buffer, followed by Northern blot analysis (10 g/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.

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 G 1 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 G 1 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 G 1 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 mitogendependent 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 G 1 mitogen restriction point.
In some but not all experimental systems, cyclin D1 is downregulated 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.
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 G 1 regulatory protein in cultured hepatocytes.
Cyclin D1 Promotes Global Protein Synthesis in Rapamycintreated 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 Ͼ3-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)(4)(5)(6), these studies suggest that cyclin D1 may induce growth downstream of TOR in hepatocytes.
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 G 1 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 Ϯ 9.1% of hepatocytes were positive by BrdUrd immunohistochemistry, as compared with 3.0 Ϯ 2.9% in the rapamycintreated mice (p Ͻ 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.
Cyclin D1 Promotes Hepatocyte Proliferation and Growth in the Livers of Rapamycin-treated Mice-Our results in primary  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.
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 BrdUrdpositive hepatocytes (14.0 Ϯ 5.3% versus 0% for control-transfected mice, p Ͻ 0.004). Furthermore, transfection with cyclin D1 led to substantial liver growth, increasing relative liver size by more than 40% (p Ͻ 0.007).
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). 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)(4)(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 G 1 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 G 1 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 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.

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. 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 upregulating 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 smallsize 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)(16)(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.