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Cell Growth & Differentiation Vol. 10, 397-404, June 1999
© 1999 American Association for Cancer Research

Cyclin D1 Promotes Mitogen-independent Cell Cycle Progression in Hepatocytes1

Jeffrey H. Albrecht2 and Linda K. Hansen

Department of Medicine, Hennepin County Medical Center, Minneapolis, Minnesota 55415 [J. H. A.]; Minneapolis Medical Research Foundation, Minneapolis, Minnesota 55404 [J. H. A.]; and Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota 55455 [L. K. H.]


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cyclin D1 is widely believed to regulate progression through G1 phase of the cell cycle, and previous studies have shown that this protein is induced during hepatocyte proliferation in culture and in vivo. In this study, the role of cyclin D1 in the cell cycle of primary rat hepatocytes was further examined. Following epidermal growth factor stimulation, cyclin D1 was up-regulated at time points corresponding to the mitogen restriction point, and this was associated with enhanced cyclin D1-associated kinase activity. To test whether cyclin D1 expression was sufficient to promote mitogen-independent progression through the G1-S transition, we constructed a replication-defective adenovirus that overexpressed human cyclin D1. Transfection with the cyclin D1 vector but not a control vector resulted in hepatocyte DNA synthesis in the absence of growth factor that was similar to that seen in mitogen-treated cells. Furthermore, cyclin D1 transfection led to activation of downstream biochemical events, including cyclin A and proliferating cell nuclear antigen expression and cyclin E- and cyclin A-associated kinase activation. These results suggest that cyclin D1 expression is sufficient to promote progression of hepatocytes through the G1 restriction point.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Normal cell proliferation requires growth factors, which lead to orderly activation of regulatory proteins that control the transition through G1 phase of the cell cycle. After cells progress through the late G1 restriction point, they can proceed through the cell cycle, even in the absence of mitogens (1) . Understanding the biochemical basis of the G1 restriction point has been an important goal of cell cycle research. Numerous studies have implicated the retinoblastoma protein (Rb) and E2F transcription factors as key mediators of the restriction point (2 , 3) . Phosphorylation of Rb and related proteins during late G1 phase leads to release of E2F family members, which then regulate transcription of genes necessary for S-phase progression. Phosphorylation of Rb is carried out by cyclin/cdk3 complexes (3 , 4) . During G1 phase in most experimental systems, the D- and E-type cyclins are induced and bind to their respective cdk partners to form active Rb kinases (4 , 5) . In many cell types, cyclin D1 is the first cyclin to be up-regulated by growth factors during G1 phase. Cyclin D1 is thought to be a key intracellular mediator of extracellular signals, such as mitogens, that regulate proliferation (6 , 7) . Cyclin D1 complexes with cdk4 and cdk6, and after activation of the cdk by cyclin H/cdk7 ("cdk-activating kinase"), this complex is capable of phosphorylating Rb (2 , 8) . Thus, up-regulation of cyclin D1 appears to play a key role in the control of the G1 restriction point in many types of cells.

In the adult animal, hepatocytes are highly differentiated and perform numerous essential metabolic functions. In normal liver, they rarely undergo cell division, yet they retain a stem cell-like ability to proliferate in response to injuries that reduce functional hepatic mass, a feature that distinguishes them from other types of differentiated parenchymal cells (9, 10, 11) . The control of hepatocyte proliferation in the liver is complex and incompletely understood, but recent studies in the 70% PH model have offered a clearer picture of events that promote entry into the cell cycle (reviewed in Ref. 12 ). The transition from G0 to G1 phase of the cell cycle and progression through early G1 phase after PH appears to be mediated in part by changes in extracellular matrix and by cytokines including tumor necrosis factor-{alpha} and interleukin 6 (12, 13, 14, 15) . Progression of hepatocytes through late G1 phase in vivo is thought to require growth factors and involve activation of cyclin/cdk complexes, but the role of individual cyclins, cdks, and associated regulatory proteins has not been well studied (12) . Isolated primary hepatocytes in culture readily proliferate in response to mitogens such as EGF and have been used extensively to examine mechanisms of hepatocyte cell cycle control (12 , 16, 17, 18) .

Previous studies have shown that cyclin D1 is up-regulated during hepatocyte proliferation in culture and in the liver after PH (15 , 17 , 19, 20, 21, 22, 23, 24) . However, a functional role of cyclin D1 in hepatocytes has not been established. In this study, we further examined the regulation of cyclin/cdk holoenzymes and sought to determine whether expression of cyclin D1 was sufficient to trigger hepatocyte cell cycle progression in the absence of growth factor.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Cycle Progression and Cyclin/cdk Activation in Cultured Primary Rat Hepatocytes.
Primary rat hepatocytes were cultured on collagen film in the presence or absence of EGF and insulin. As demonstrated previously (16 , 17 , 20) , cultured hepatocytes demonstrated a long G1 interval, with DNA synthesis beginning at 52 h and peaking at 72 h in growth factor-stimulated cells (Fig. 1A)Citation . To determine the timing of the G1 restriction point in this system, we provided hepatocytes with medium supplemented with EGF and insulin, which was changed to growth factor-free medium at the intervals shown in Fig. 1BCitation , and DNA synthesis was assessed at 72 h. These growth factor-withdrawal experiments suggested that the restriction point occurred at {approx}40–44 h after plating, in close agreement with previous studies (16 , 17) .



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Fig. 1. DNA synthesis in cultured rat hepatocytes. A, time course of DNA synthesis. Hepatocytes were plated in the presence or absence of EGF/insulin as described in "Materials and Methods." DNA synthesis was determined by measuring [3H]thymidine uptake at the indicated time points. B, growth factor withdrawal experiments. Hepatocytes were plated in the presence of EGF/insulin for the indicated intervals, washed, and subsequently cultured in growth factor-free medium. DNA synthesis was determined at 72 h.

 
As demonstrated previously, induction of cyclin D1 protein in hepatocytes required growth factor and corresponded in time with passage through the G1 restriction point at 40–44 h (Fig. 2ACitation ; Refs. 17 and 20 ). Using a polyclonal antibody directed against rat cyclin E, we detected three isoforms at Mr 50,000–55,000, which likely represent alternatively spliced variants of this gene (25 , 26) . Cyclin E expression was detectable in the absence of growth factor, but up-regulation of the lower two forms of this protein occurred within 24 h of treatment with EGF (Fig. 2B)Citation . Consistent with its expression in the regenerating liver (20) , cdk4 expression was growth factor independent and did not vary during cell cycle progression. cdk2 protein was modestly up-regulated by EGF. Cyclin A was detected in EGF-treated hepatocytes at the G1-S interval and during S phase (52–72 h). Expression of the p21 cdk-inhibitory protein was induced by EGF, whereas p27 expression was growth factor independent and varied little as cells progressed through the cell cycle.



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Fig. 2. Expression of cell cycle proteins in primary hepatocytes. A, cyclin D1 expression. Hepatocytes cultured in the presence or absence of EGF/insulin were harvested at the indicated time points, and extracts were prepared for Western blot analysis, as described in "Materials and Methods." B, other cell cycle proteins. Western blot analysis was performed using the indicated antibodies.

 
The activity associated with cyclin/cdk complexes is shown in Fig. 3Citation . Immunoprecipitation/kinase assays demonstrated that induction of cyclin D1 protein was accompanied by an increase in associated Rb kinase activity. Similarly, kinase activity associated with cyclins E and A as well as cdk2 were up-regulated at the G1-S boundary (52 h) and during S phase (72 h). Thus, the regulation of G1- and S phase-associated cyclin/cdk complexes in primary hepatocytes was consistent with observations in other cell types and in the regenerating liver (22, 23, 24) .



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Fig. 3. Activation of cdks in primary hepatocytes. Extracts obtained from hepatocytes cultured in the presence or absence of EGF/insulin were subjected to immunoprecipitation/kinase assays, as described in "Materials and Methods."

 
Adenovirus-mediated Transfection of Cyclin D1 and Cell Cycle Progression.
Abundant data in other cell lines suggest that cyclin D1 plays a role in governing progression through the G1 restriction point (6 , 7) . Furthermore, in primary hepatocytes, cyclin D1 expression is completely growth factor dependent, and its up-regulation corresponds with the G1 restriction point (17 , 20) . We, therefore, sought to determine whether cyclin D1 expression was sufficient to promote progression of primary hepatocytes into S phase in the absence of growth factor. To test this, we constructed a replication-defective recombinant adenovirus containing the human cyclin D1 cDNA under the control of a constitutive promoter (ADV-D1). For control experiments, we used an identical adenoviral construct encoding ß-galactosidase (ADV-ßgal). Prior studies have shown that adenoviruses transfect hepatocytes with high efficiency, and under the conditions studied here, >80% of hepatocytes were transfected with ADV-ßgal (data not shown). We then examined whether transfection with ADV-D1 promoted cell cycle progression in hepatocytes.

Hepatocytes were transfected with ADV-D1 or ADV-ßgal for 2 h shortly after plating. Western blot analysis using a monoclonal antibody that preferentially recognizes human cyclin D1 (DCS-6) demonstrated that ADV-D1 led to high-level expression of this protein in transfected cells (Fig. 4)Citation . We then examined whether transfection with ADV-D1 led to up-regulation of cell-cycle proteins in a manner similar to growth factor stimulation. Cyclin E expression was modestly up-regulated by ADV-D1. Cyclin A and PCNA expression have been shown to be up-regulated during S phase in numerous cells, including hepatocytes (24 , 27 , 28) . At 72 h after plating, cells transfected with ADV-D1 (but not ADV-ßgal) demonstrated up-regulation of cyclin A and PCNA in the absence of growth factor, to levels similar to those seen after EGF stimulation. Transfection with ADV-D1 also led to induction of p21 protein expression at 72 h after plating.



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Fig. 4. Expression of cyclin D1 and cell cycle regulatory proteins in transfected hepatocytes. Hepatocytes were plated in the presence or absence of EGF/insulin and transfected with ADV-D1, ADV-ßgal, or no virus, as described in "Materials and Methods." Cells were harvested for Western blot analysis using the indicated antibodies. hu D1, monoclonal antibody to human cyclin D1 (DCS-6).

 
We then examined the activation of cyclin/cdk kinase complexes by ADV-D1 (Fig. 5A)Citation . The kinase activity associated with cyclin D1 was enhanced after ectopic expression of this protein. Furthermore, the kinase activities associated with cyclin E, cyclin A, and cdk2 were up-regulated at 72 h after plating in hepatocytes transfected with ADV-D1. Another function of the cyclin D1/cdk4 complex is to sequester cdk-inhibitory proteins such as p27, which then prevents inhibition of cyclin/cdk complexes acting downstream in the cell cycle (29) . To determine whether the transfected cyclin D1 may also act to sequester p27, we examined the association of p27 with cyclins D1 and E. As is shown in Fig. 5BCitation , the amount of p27 expressed in hepatocytes did not vary substantially after transfection with ADV-D1 or ADV-ßgal. The results of Western blot analysis using an antibody that recognizes both rodent and human cyclin D1 (Upstate Biotechnology, Inc., Lake Placid, NY) suggested that the level of cyclin D1 was slightly higher in EGF-treated cells than in ADV-D1-transfected cells under these conditions. Up-regulation of cyclin D1 by either EGF or by adenoviral transfection resulted in increased abundance of cyclin D1/p27 complexes, and this was associated with decreased abundance of cyclin E/p27 complexes. These results suggest that cyclin D1 expression resulting from ADV-D1 transfection may be capable of promoting hepatocyte cell cycle progression by forming an active Rb kinase and by binding cdk inhibitors.



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Fig. 5. Activation of cdks and complex formation in transfected hepatocytes. Hepatocytes were transfected with recombinant adenoviruses as described in Fig. 4Citation , and cells were harvested at the indicated time points. A, kinase activation. Extracts were subjected to immunoprecipitation/kinase assays using the indicated antibodies. B, p27-cyclin complex formation. In the top two panels, cellular lysates were subjected to Western blot with an antibody that recognizes human and rodent cyclin D1 (Upstate Biotechnology, Inc.) or an antibody to p27. In the bottom two panels, extracts were subjected to immunoprecipitation with antibodies to cyclin D1 or cyclin E, followed by Western blot of the precipitated proteins for p27.

 
Finally, we examined whether DNA synthesis was induced by cyclin D1 transfection. At 72 h after plating, hepatocytes transfected with ADV-D1 demonstrated increased DNA synthesis that was similar to EGF-treated cells (Fig. 6)Citation . Because of reports in other cell lines that transfection with cyclin D1 can lead to apoptosis, we examined hepatocytes at 72 and 96 h after plating, using an in situ DNA fragmentation assay. This did not demonstrate evidence of increased apoptosis in cells treated with ADV-D1 or ADV-ßgal (data not shown).



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Fig. 6. DNA synthesis in transfected hepatocytes. Hepatocytes were transfected as in Fig. 4Citation , and DNA synthesis was determined at 72 h, as described in "Materials and Methods."

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
This study was intended to address the hypothesis that induction of cyclin D1 is the critical growth factor-dependent step during late G1 in normal hepatocytes. Up-regulation of cyclin D1 is widely believed to play an important role during G1 phase in many types of cells (6 , 7) , and previous studies have suggested that this protein is involved in the regulation of hepatocyte proliferation. Cyclin D1 is induced during G1 in mitogen-stimulated hepatocytes in vitro and in rodent liver after PH and is up-regulated in regenerating human liver (20) . In models of impaired liver regeneration after PH, diminished hepatocyte proliferation is associated with decreased expression of cyclin D1 (15 , 19 , 30) . Although induction of cyclin D1 is thought to be the best marker of late G1 progression in hepatocytes (14 , 15 , 17 , 20) , a functional role of this protein has not been demonstrated. The most salient finding of this study is that expression of cyclin D1 in primary hepatocytes appears to be sufficient to trigger transition through the G1-S interval in a manner comparable to growth factor stimulation.

There is substantial evidence that cyclin D1 regulates progression through G1 in response to extracellular signals (6 , 7) . In numerous cell lines, increased synthesis of D-type cyclins (usually D1) occurs during early G1 phase and results in activation of cyclin D/cdk4 or cyclin D/cdk6 kinase complexes. Aside from phosphorylation of Rb and related proteins, induction of cyclin D1 appears to be involved in at least two other mechanisms of cell cycle control. As the abundance of cyclin D1/cdk4 increases during G1 phase progression, these complexes sequester cdk-inhibitory proteins, such as p21 and p27, thereby diminishing the concentration of "free" cdk-inhibitors capable of preventing activation of cyclin E/cdk2 and cyclin A/cdk2 holoenzymes (29) . Furthermore, cyclin D1 can directly regulate transcription factors in the absence of cdk4. Transcriptional activation by the estrogen receptor is enhanced by binding of cyclin D1 to this protein (31 , 32) . Alternatively, the growth-inhibitory transcription factor DMP1 is inactivated by binding to cyclin D1 (33) . At present, it is not clear whether all of these functions are necessary for cyclin D1 to promote progression through G1.

To further study the role of cyclin D1 in hepatocyte proliferation, we first examined the expression and activation of cyclins, cdks, and cdk inhibitors associated with late G1 and S phase. As demonstrated previously, primary rat hepatocytes progressed through a mitogen restriction point {approx}40–44 h after plating in the presence of growth factor, and induction of cyclin D1 protein corresponded temporally to the restriction point (16 , 17) . In agreement with the results of Loyer et al. (17) , cyclin D1 expression was growth factor dependent. Cyclin E protein was expressed in the absence of mitogen but was up-regulated after EGF stimulation, in contrast to previous results (17) . We further examined cyclin D1-, cyclin E-, cyclin A-, and cdk2-dependent kinases and found that these were activated at the G1-S boundary, as would be predicted by work in other systems. The cdk inhibitor p21 was induced by growth factors during G1 phase, consistent with its expression in regenerating liver after PH (22, 23, 24) and in other types of proliferating cells in culture (34 , 35) . In contrast, p27 expression did not vary substantially during the cell cycle and was not dependent upon growth factors. This is similar to the relatively static expression of p27 in regenerating liver after PH (22, 23, 24 , 36) .

There were two notable differences in the expression of G1-regulatory proteins in primary hepatocytes as compared to other types of cells in culture. (a) In many systems, cyclin E expression is growth factor dependent and is up-regulated after cyclin D1 induction during late G1 phase (7 , 25) . (b) In other cell lines, p27 expression diminishes as cells progress through G1 (7 , 37 , 38) . This decline is thought to allow activation of cyclin/cdk complexes during G1 phase by lowering the "threshold" of cdk inhibition (29) . Antisense inhibition of p27 allows mitogen-independent growth of BALB/c 3T3 cells, suggesting that the decline in p27 is an important determinant of progression through the restriction point in some cells (39) . Our finding that growth factor-mediated hepatocyte proliferation was not associated with diminished p27 expression suggests that up-regulation of cyclin D1 may be a particularly important growth factor-dependent event during late G1 in these cells. To further test this hypothesis, we examined whether high-efficiency transient transfection with a cyclin D1-encoding adenoviral vector would promote hepatocyte cell cycle progression in the absence of growth factor.

Adenoviruses efficiently transfect hepatocytes in culture and in vivo and have been used extensively to study the role of individual proteins in various biochemical pathways (40, 41, 42, 43) . In agreement with other studies, we found that ADV-ßgal transfected >80% of hepatocytes in culture. As expected, ADV-D1 led to abundant expression of human cyclin D1 that was functionally active, as demonstrated by its activation of Rb kinase activity. ADV-D1 but not the control vector stimulated entry into S phase, as evidenced by DNA synthesis, and the magnitude of this response was similar to EGF-treated cells under optimal conditions. Furthermore, ADV-D1 triggered "downstream" biochemical mediators of S phase, including cyclin A and PCNA expression and activation of cyclin A-, cyclin E-, and cdk2-associated kinase activity. These results suggest cyclin D1 expression is sufficient to induce progression through the G1-S interval in primary hepatocytes in a manner analogous to mitogen stimulation.

Previous studies have demonstrated diverse effects of cyclin D1 overexpression in cultured cells. Constitutive overexpression of cyclin D1 by permanent transfection in rodent fibroblasts led to a shortened G1 interval but did not promote mitogen-independent growth (44, 45, 46) . On the other hand, stable transfection of cyclin D1 was noted to cause growth inhibition of both human and mouse mammary epithelial cell lines and human fibroblasts (47, 48, 49) . Other studies have found that transient transfection or inducible overexpression of cyclin D1 caused apoptosis of rodent fibroblast and mammary epithelial cell lines grown under low-serum conditions (48 , 50 , 51) . In MCF7 breast cancer cells, inducible overexpression of cyclin D1 led to increased proliferation under low-serum conditions, but this was substantially less than the rate of proliferation induced by high-serum conditions (52) . Because other genetic alterations may enhance the proliferative effect of cyclin D1 overexpression (53 , 54) , perturbations in other cell cycle control genes may have enhanced the effect of cyclin D1 transfection in the MCF7 cells. A more recent study by Connell-Crowley et al. (55) indicates that microinjection of activated cyclin D1/cdk4 or cyclin E/cdk2 (but not cyclin A/cdk2) complexes led to proliferation of serum-starved WI38 fibroblasts. However, to our knowledge, previous studies have not shown that cyclin D1 expression is sufficient to induce DNA synthesis that is comparable to mitogen treatment and have not demonstrated induction of S phase-associated biochemical events in the absence of growth factor.

It has been previously demonstrated that hepatocytes undergo the transition from G0 to G1 during isolation and plating, as evidenced by expression of genes such as c-fos, c-jun, and c-myc (17 , 18 , 56) . Therefore, the finding that cyclin D1 transfection led to growth factor-independent cell cycle progression in hepatocytes (as opposed to other cell types in previous studies) may reflect the fact that mitogen-deprived hepatocytes are not in G0. Alternatively, in this study, we used a transient transfection system, whereas the permanent transfection techniques that were used in some of the prior studies could potentially lead to adaptive changes in the expression of other genes that inhibit growth. Here, ADV-D1 led to up-regulation of p21, suggesting that antiproliferative mechanisms may be triggered by cyclin D1 expression. Finally, it is possible that hepatocyte cell cycle control differs from previously studied cells. This latter possibility is supported by the finding that the expression of cell cycle control genes in proliferating hepatocytes and regenerating liver can differ from other systems used to study the cell cycle (17 , 19, 20, 21) . Furthermore, the remarkable capacity of differentiated adult hepatocytes to proliferate in vivo suggests that these cells may be highly responsive to certain mitogenic stimuli, such as cyclin D1 expression.

Previous studies in other cell types indicate that other G1-associated cyclins may also regulate the rate of progression through G1. Specifically, cyclins D2, D3, and E have been shown to accelerate progression through G1 in growth factor-stimulated cells (44 , 45 , 57 , 58) . This suggests that these cyclins may perform redundant functions, a possibility that is supported by the fact that cyclins D1 or D2 knockout mice show relatively mild phenotypic defects (59 , 60) . Alternatively, each G1-associated cyclin may demonstrate different substrate specificity when partnered with their corresponding cdks and may influence differentiation or other processes distinct from cell cycle progression (4, 5, 6, 7) . Therefore, further study is necessary to determine whether cyclin D1 is uniquely capable of promoting hepatocyte cell cycle progression or whether other G1-associated cyclins share this capability. Prior studies have suggested that cyclin D2 is not expressed in regenerating liver and that cyclin D3 expression in hepatocytes does not require growth factors (17 , 19) . In this study, we have chosen to focus on cyclin D1 because previous studies indicated that it may play a physiologically relevant role in liver regeneration.

In some cell types, cyclin E/cdk2 activation may be triggered by cyclin D1 expression, through E2F-mediated transcription of cyclin E (following phosphorylation of Rb and related proteins; Ref. 61) or as a result of p27 sequestration by cyclin D1 (29) . In this study, up-regulation of cyclin E occurred before that of cyclin D1 in hepatocytes, indicating that cyclin D1 expression is not a prerequisite for cyclin E induction in these cells. These data do suggest that induction of cyclin D1 by growth factor or adenoviral transfection was associated with increased binding of p27 to cyclin D1 and decreased binding of p27 to cyclin E. This is consistent with studies in other systems indicating that cyclin D1 may function to sequester p27, thereby promoting activation of cyclin E/cdk2 in late G1 (29) . The specific mechanisms by which cyclin D1 regulates downstream cyclin/cdk activity in hepatocytes requires further investigation.

The results of this study support the concept that cyclin D1 regulates progression of hepatocytes through late G1 in response to mitogens. Of note, related studies indicate that the control of hepatocyte proliferation by extracellular matrix may also be mediated by cyclin D1,4 further suggesting that this protein is a pivotal mediator of extracellular signals that regulate proliferation of these cells. Induction of cyclin D1, therefore, appears to be a "rate-limiting" step in hepatocyte proliferation. Previous publications indicate that p21 also regulates the rate of progression through G1 in hepatocytes. After PH, hepatocytes progress more rapidly through G1 in p21 -/- mice than in wild-type mice (24) . On the other hand, transgenic mice with forced hepatic overexpression of p21 demonstrate markedly impaired hepatocyte proliferation after PH (62) . Thus, functional data suggest that at least two components that control cyclin/cdk activity also regulate hepatocyte G1 progression: cyclin D1 promotes entry into S phase, whereas p21 delays the transition into S phase. Future studies examining the role of individual cell cycle control genes using knockout mice or gene targeting techniques will likely provide insight into the regulation of hepatocyte proliferation in vivo.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Culture.
Primary rat hepatocytes were harvested from 8–10-week-old male Lewis rat liver by collagenase perfusion followed by purification through a Percoll gradient (63 , 64) . Hepatocytes were placed at a density of 10,000 cells per cm2 in serum-free Williams E medium containing 20 milliunits/ml insulin, 5 nM dexamethasone, 20 mM sodium pyruvate, 50 µg/ml ascorbic acid, 100 units/ml penicillin/streptomycin, and 10 ng/ml EGF. Cells cultured without growth factors were provided the same medium excluding insulin and EGF. Nonadhesive Petri dishes were precoated with type I collagen (Vitrogen; Celtrix, Santa Clara, CA) that had been diluted in carbonate buffer (pH 9.4), yielding a density of 1 µg/cm2. Hepatocytes were maintained at 37°C in the presence of 5% CO2, and medium was changed daily, except in Fig. 1BCitation , where additional media changes were required for growth factor withdrawal studies. DNA synthesis was assessed by determining [3H]thymidine uptake essentially as described previously (20 , 64) . For Fig. 1ACitation , cells were labeled with 10 µCi/ml for 3 h before harvest, whereas for Figs. 1BCitation and 6Citation , cells were labeled for 18 h with 2 and 10 µCi/ml [3H]thymidine, respectively. Apoptosis assays were performed at 72 and 96 h using an immunohistochemical kit to detect DNA strand breaks (In Situ Cell Death Detection Kit; Boehringer Mannheim, Indianapolis, IN) according to the manufacturer’s instructions.

Protein Harvest and Western Blot Analysis.
At the indicated time points, hepatocytes were harvested as described previously (20) , washed in PBS, and lysed by Dounce homogenization and sonication. Cells were homogenized in a modified Tween 20 buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 0.1% Tween 20, 1 mM NaF, 7.5 mM EGTA, and 7.5 mM MgCl2] containing protease and phosphatase inhibitors (24 , 65) . Homogenates were clarified by centrifugation at 14,000 x g, and aliquots were frozen at -80°C for later use. Western blot analysis was performed as described (20 , 24) , using 10 µg of protein per lane. The following antibodies were used: monoclonal antimouse cyclin D1 (sc450) and anti-PNCA (sc56) and polyclonal antibodies to cyclin A (sc596), cyclin E (sc481), cdk2 (sc163), cdk4 (sc260), p21 (sc397G), and p27 (sc528) from Santa Cruz Biotechnology (Santa Cruz, CA). Additional antibodies included monoclonal antihuman cyclin D1 (DCS-6; Neomarkers, Fremont, CA), monoclonal anti-p27 (Transduction Laboratories, Lexington, KY), and polyclonal anti-cyclin D1 (Upstate Biotechnology, Inc.).

Immunoprecipitation and Kinase Assays.
Homogenates prepared as above were subject to immunoprecipitation followed by kinase assays, or Western blot was performed using the indicated antibodies, as described previously (24) . Control antibodies failed to show significant histone H1 or Rb kinase activity (data not shown). For cyclin D1-associated kinase assays involving transfected cells, a monoclonal antibody to cyclin D1 was used (DCS-11; Neomarkers). For cyclin A-associated kinase assays, a polyclonal antibody was used (a gift from Dr. Edward Leof, Mayo Clinic).

Preparation of Recombinant Adenovirus.
The recombinant replication-defective adenovirus was prepared using the methods outlined by Becker et al. (40) . In brief, the EcoRI-HindIII fragment of human cyclin D1 (a gift from Dr. Steven Reed, Scripps Institute; Ref. 66 ) was cloned into the corresponding restriction sites of pACCMVpLpA (a gift from Dr. Howard Towle, University of Minnesota), which contains the cytomegalovirus early promoter and the SV40 polyadenylation signal (40) . This plasmid was used, along with the pJM17 vector, to cotransfect 293 cells by calcium phosphate coprecipitation. Recombinant ADV-D1 adenovirus was isolated by plaque purification, and a single clone was used for large-scale amplification for use in later experiments. The identity of this clone was confirmed by Southern blot analysis demonstrating the presence of cyclin D1 cDNA. Viral titers were determined by a plaque-forming unit assay. Viral stock was stored at -20°C. ADV-ßgal, which is an identical construct containing the nuclear localizing variant of ß-galactosidase, was provided by Dr. Howard Towle, and large-scale amplification and titering of this virus was performed in an identical fashion.

Hepatocyte Transfection.
Hepatocytes were plated as outlined above for 3 h, followed by addition of ADV-D1 or ADV-ßgal in medium at a dose of 20 plaque-forming units per hepatocyte. After 2 h, the media containing adenovirus was removed and replaced with virus-free medium. Nontransfected cells in Figs. 4Citation 5Citation 6Citation underwent similar medium changes without transfection. Transfection efficiency was determined by ß-galactosidase histochemistry, as described previously (67) .


    Acknowledgments
 
We acknowledge excellent technical assistance from Brenda Rieland, Cory Ahonen, and Lisa Jungers. We appreciate the gifts of pACCMVpLpA, pJM17, and ADV-ßgal as well as assistance with adenoviral techniques from Drs. Howard Towle and Elizabeth Kaytor (University of Minnesota). We also acknowledge Drs. Steven Reed and Edward Leof for providing reagents used in these studies.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by NIH Grant DK-54921 and a grant from the American Liver Foundation (to J. H. A.) and by National Science Foundation Research Planning Grant MCB95-09600 and a grant from the Minnesota Medical Foundation (to L. K. H.). Back

2 To whom requests for reprints should be addressed, at Department of Medicine (865B), Hennepin County Medical Center, 701 Park Avenue, Minneapolis, MN 55415. Phone: (612) 347-8582; Fax: (612) 904-4299; E-mail: albre010{at}maroontc.umn.edu Back

3 The abbreviations used are: cdk, cyclin-dependent kinase; PH, partial hepatectomy; EGF, epidermal growth factor; PCNA, proliferating cell nuclear antigen. Back

4 L. K. Hansen and J. H. Albrecht. Regulation of hepatocyte cell cycle by type 1 collagen matrix: role of cyclin D1, manuscript in preparation. Back

Received for publication 11/ 6/98. Revision received 2/12/99. Accepted for publication 4/ 6/99.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cell Growth & Differentiation