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Liver-Specific pRB Loss Results in Ectopic Cell Cycle Entry

and Aberrant Ploidy

Christopher N. Mayhew,1Emily E. Bosco,

1Sejal R. Fox,

1Tomohisa Okaya,

2

Pheruza Tarapore,1Sandy J. Schwemberger,

2,3George F. Babcock,

2,3

Alex B. Lentsch,2Kenji Fukasawa,

1and Erik S. Knudsen

1

Departments of 1Cell Biology and 2Surgery, College of Medicine and 3Shriners Burns Institute, University of Cincinnati, Cincinnati, Ohio

Abstract

The liver exhibits an exquisitely controlled cell cycle,wherein hepatocytes are maintained in quiescence untilstimulated to proliferate. The retinoblastoma tumor sup-pressor, pRB, plays a central role in proliferative control byinhibiting inappropriate cell cycle entry. In many cases, livercancer arises due to aberrant cycles of proliferation, andcorrespondingly, pRB is functionally inactivated in themajority of hepatocellular carcinomas. Therefore, to deter-mine how pRB loss may provide conditions permissive forderegulated hepatocyte proliferation, we investigated theconsequence of somatic pRB inactivation in murine liver. Weshow that liver-specific pRB loss results in E2F target genederegulation and elevated cell cycle progression during post-natal growth. However, in adult livers, E2F targets arerepressed and hepatocytes become quiescent independent ofpRB, suggesting that other factors may compensate for pRBloss. Therefore, to probe the consequences of acute pRBinactivation in livers of adult mice, we gave adenoviral-Creby i.v. injection. We show that acute pRB loss is sufficient toelicit E2F target gene expression and cell cycle entry in adultliver, demonstrating a critical role for pRB in maintaininghepatocyte quiescence. Finally, we show that liver-specificpRB loss results in the development of nuclear pleomor-phism associated with elevated ploidy that is evident inadult mice harboring both acute and chronic pRB loss.Together, these results show the crucial role played by pRBin maintaining hepatocyte quiescence and ploidy in adultliver in vivo and underscore the critical importance ofdelineating the consequences of acute pRB loss in adultanimals. (Cancer Res 2005; 65(11): 4568-77)

Introduction

The retinoblastoma tumor suppressor (Rb) plays a critical role incellular proliferation control and inhibition of oncogenic transfor-mation (1, 2). Germ line mutations in the Rb gene predisposeindividuals to bilateral retinoblastoma aswell as osteosarcoma (3, 4).Somatic Rb inactivation contributes to the development of thesetumors as well as to tumors in a number of other organs(e.g., bladder, lung, breast, and prostate; refs. 5–8). Furthermore,inactivation of the Rb pathway, either by direct mutation of Rb orderegulation of upstream regulators of pRB, such as p16INK4a or

cyclin D1, is believed to be an obligatory step in sporadic tumorformation that renders cells hyperproliferative and unresponsive toantimitogenic signals (9).Rb encodes a nuclear phosphoprotein (pRB) that negatively

regulates G1-S phase cell cycle progression. It is believed that theantiproliferative function of pRB is attributable to its role as atranscriptional corepressor. In early G1, pRB is hypophosphorylatedand binds to members of the E2F family of transcription factorsand recruits corepressors to actively attenuate the transcription ofE2F-regulated genes (10). The E2F family of transcription factorsare critical regulators of numerous genes including thoseimportant for cell cycle progression (e.g., cyclin E and cyclin A)and DNA replication [e.g., proliferating cell nuclear antigen (PCNA),MCM5 , and MCM7]. In response to mitogenic signaling, theactivities of cyclin D-CDK4/6 and cyclin E-CDK2 are induced,resulting in phosphorylation of pRB and disruption of pRBassembled repressor complexes. Consequently, E2F-responsivegenes are subsequently expressed and cells progress into S phase(10, 11). Conversely, antimitogenic signaling results in diminishedcyclin-CDK activities, maintenance of pRB in its hypophosphory-lated/active form, and inhibition of cell cycle progression (12).As the Rb pathway is targeted at high frequency in human

cancer, there is considerable interest in understanding theconsequences of pRB loss. The biochemical effects of pRB losshave been studied extensively in cell culture model systems.Analysis of mouse embryonic fibroblasts from mice harboring germline Rb loss has shown that the expression of a number of pRB-E2Ftarget genes is deregulated (13, 14). Furthermore, Rb�/� MEFsexhibit deregulated cell cycle kinetics (13) and are unable to elicitcell cycle arrest following exposure to a variety of antimitogenicsignals (e.g., DNA damage and transforming growth factor-h;refs. 15–17). However, recent studies have suggested that Rbharbors additional activities that are associated with the mainte-nance of quiescence/senescence and are apparent only when Rb isinactivated acutely. These aspects of Rb function are masked whenpRB is chronically absent through functional compensation by thepRB-related pocket protein p107 (18).The complex consequences of pRB loss in cell culture are

mirrored by studies analyzing mice with targeted Rb deletion. Miceheterozygous for Rb are viable and develop pituitary and thyroidtumors early in life associated with loss of heterozygosity (19–21).Inactivation of both Rb alleles in mice results in unscheduled cellproliferation, apoptosis, and widespread developmental defects,leading to embryonic death by day 14.5 (20, 22, 23). Whereas manyof these events were suspected to be cell autonomous, it has sub-sequently been shown that many of the phenotypes arise due todefective extraembryonic development in Rb�/� embryos (24, 25).Additionally, in the Rb germ line–deficient animals, there isclear evidence for functional compensation by the other pocket

Requests for reprints: Erik S. Knudsen, Department of Cell Biology, Vontz Centerfor Molecular Studies, University of Cincinnati, Cincinnati, OH 45267-0521. Phone: 513-558-8885; Fax: 513-558-2445; E-mail: [email protected].

I2005 American Association for Cancer Research.

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proteins (26–28). To circumvent some of the difficulties associatedwith development and compensation, conditional models of pRBloss in specific organs have been generated (29–34). In thesemodels, a spectrum of phenotypes associated with both full andheterozygous inactivation of Rb has been described.The liver is an organ that is maintained in a quiescent state in

adults but harbors remarkable proliferative capacity regulated byincompletely understood pathways (35, 36). Following acute orchronic liver damage, quiescent hepatocytes enter the cell cycleand divide to restore the functional capacity of the liver. Precisecontrol over hepatocyte proliferation is critical for the suppressionof tumorigenesis in the liver, as chronic liver damage andcorresponding cycles of regeneration are known to fuel thedevelopment of hepatocellular carcinoma (37). There is clearevidence that CDK activities that function upstream (e.g., cyclin D)and downstream (e.g., cyclin E and cyclin A) of pRB are involved inregulating hepatocyte proliferation (38–40). Correspondingly, thepRB pathway is disrupted at high frequency in human hepatocel-lular carcinoma (41–43).Despite evidence that pRB plays a central role in the regulation

of hepatocyte proliferation and inhibition of hepatocellularcarcinoma, the biological consequences of pRB loss in the liverhave yet to be challenged. Therefore, here we investigated the effectof somatic pRB inactivation in murine liver. Liver-specific pRBablation results in E2F target gene deregulation and elevated cellcycle progression during post-natal growth. However, compensa-tion eventually occurs such that in adult livers, the expression ofE2F targets is repressed and hepatocytes become quiescentindependently of pRB. Furthermore, we show that acute pRBelimination in the livers of adult mice is sufficient to elicit E2Ftarget gene expression and cell cycle entry. Finally, we show thatliver-specific pRB loss results in rapid and dramatic pleomorphismwith nuclear enlargement and elevated ploidy. Together, theseresults reveal the critical role played by pRB in maintaininghepatocyte quiescence and ploidy in vivo and underscore thecritical importance of delineating the consequences of acute pRBloss in adult animals.

Materials and Methods

Mice. Mice harboring Rb alleles in which exon 19 is flanked by loxPsites (Rb f/f ; ref. 44) were obtained from the National Cancer Institute.

Albumin-Cre mice (45), hemizygous for the Alb-Cre transgene, were

purchased from The Jackson Laboratory (Bar Harbor, ME). To generateliver-specific Rb conditional knockouts, Rb f/f and Alb-Cre mice were

intercrossed to obtain mice homozygous for the floxed Rb locus and

hemizygous for Alb-Cre (Rb f/f ;Alb-Cre+). These mice were then interbred

with Rb f/f mice to produce Rb f/f and Rb f/f ;Alb-cre+ littermates at 1:1ratio. Rb f/f ;Alb-Cre+ mice were also intercrossed with nontransgenic

FVB/N mice to generate Rbwt/f and Rbwt/f ;Alb-Cre+ littermates at 1:1

ratio. Mice were housed in a pathogen-free animal facility under

standard 12-hour light/12-hour dark cycle with ad libitum water andchow. All of the experiments were conducted using the highest standards

for humane care in accordance with the NIH Guide for the Care and Use

of Laboratory Animals and were approved by the University of CincinnatiInstitutional Animal Care and Use Committee.

Mouse treatments and tissue harvesting. All experiments were done

using littermates. Bromodeoxyuridine (BrdUrd, 150 mg/kg, dissolved in

0.9% saline) was given by i.p. injection 1 hour before sacrifice. Livers wererapidly excised from euthanized mice, weighed, and the left lobe was fixed

in 10% buffered formalin for 16 hours for histologic analysis. Remaining

liver tissue was immediately frozen in liquid N2 and stored at �80jC until

use. Liver imprints (touch preparations) were prepared by excising the

liver and gently touching the sliced edge of the left lobe to a glass slide.This method allows single hepatocytes to attach in a nondisruptive

manner so that their nuclei remain intact. Fixation and staining of touch

preparations for centrosomes and centromeric DNA was then done as

described below. For adenoviral injections, male mice were anesthetizedwith pentobarbital (60 mg/kg, i.p.) before i.v. delivery of 1 � 109 plaque-

forming units (pfu) adenovirus via intrapenile injection. Adenovirus

expressing cre-recombinase (Ad-CMV-Cre) was purchased from the

University of Iowa Gene Transfer Core Facility (Iowa, IA). Controladenovirus expressing LacZ from the same promoter (Ad-CMV-LacZ)

was obtained from the Vector Core Laboratory, University of North

Carolina. Statistical analyses were done using unpaired two-tailed t test. Ps

< 0.05 were considered significant.PCR genotyping and detection of recombination. Genomic DNA was

isolated by standard phenol/chloroform extraction. Primers used for

detection of the Alb-Cre transgene were 5V-GCGGTCTGGCAGTAAAAAC-TATC-3V (sense) and 5V-GTGAAACAGCATTGCTGTCACTT-3V (antisense).

To analyze Rb genotype and to detect the presence of Cre-mediated

recombination, PCR analysis of Rb exon 19 was done (44, 46). Primers for

Rb18 (5V-GGCGTGTGCATCAATG-3V) and Rb19E (5V-CTCAAGAGCTCA-GACTCATGG-3V) yielded 283- and 235-bp products for the unrecombined-

floxed and wild-type alleles, respectively. Cre-mediated recombination was

detected using primers Rb18 and Rb212 (5V-GAAAGGAAAGTCAGGGA-CATTGGG-3V), yielding products of 746 and 260 bp for the unrecombined

and recombined-floxed Rb alleles respectively. Amplification was done

using 1 unit Taq polymerase (Promega, Madison, WI) and annealing

temperature of 58jC for 35 cycles. Total liver RNA was extracted with Trizol

(Invitrogen, Carlsbad, CA) and first-strand cDNA was synthesized from

1 Ag of RNA using the Superscript II reverse transcription-PCR (RT-PCR)

system (Invitrogen) following the manufacturer’s recommendations.

cDNA was subject to amplification using primers Rb exon 18 (5V-CCTTGAACCTGCTTGTCCTC-3V) and Rb exon 20 (5V-GAAGGCGTGCACA-GAGTGTA-3V) at 54jC for 35 cycles.

Preparation of liver nuclear extracts and Western blotting. Frozenliver was dissociated in buffer A [25 mmol/L Tris (pH 7.5), 50 mmol/L KCl,

2 mM MgCl2, 1 mmol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fluoride

(PMSF)] and incubated for 10 minutes on ice. Nuclei were then releasedusing a Dounce homogenizer and tight pestle. Following two washes in

buffer A, nuclei were extracted on ice for 10 minutes with radio-

immunoprecipitation assay buffer [150 mmol/L NaCl, 1.0% NP40,

0.5% deoxycholate, 0.1% SDS, 50 mmol/L Tris (pH 8.0), 50 mmol/L NaF,13 mg/mL h-glycerophosphate, 120 Ag/mL sodium vanadate supplemented

with 1 mmol/L PMSF and protease inhibitors]. Lysates were centrifuged at

13,000 rpm and total nuclear protein concentration determined by Bio-Rad

protein assay. Equal amounts of total nuclear protein were separated byPAGE and transferred to polyvinylidene difluoride membranes. Specific

proteins were detected by standard Western blotting procedures. pRB was

detected as previously described (47). Monoclonal antibodies to Brg1 (G-7),

MCM7 (141.2), and PCNA (PC10) were purchased from Santa CruzBiotechnology (Santa Cruz, CA). Monoclonal antibody to MCM5 (clone

33) was from BD Transduction Laboratories (San Diego, CA). Polyclonal

antibodies recognizing cyclin E (M-20), p107 (C-18), and p130 (C-20) werefrom Santa Cruz Biotechnology.

Bromodeoxyuridine labeling and immunohistochemistry. Sectionsfrom the left lobe were fixed in 10% neutral buffered formalin, paraffin

embedded, and cut into 5-Am sections. For immunohistochemical staining,sections were deparaffinized in xylenes and rehydrated through a graded

series of ethanol/water solutions. For analysis of cyclin E expression, liver

sections were initially boiled in Antigen Retrieval Solution (DakoCytoma-

tion, Cambridge, MA) using a microwave (5 minutes at 100% powerfollowed by 20 minutes at 30% power). Sections were then cooled to room

temperature before proceeding with staining. Endogenous peroxidase

activity was quenched by treatment in 3% H2O2 in methanol (DakoCyto-mation). Samples were blocked for 30 minutes in 5% goat serum in PBS.

Primary polyclonal anti–cyclin E antibody (Santa Cruz Biotechnology, M20)

was diluted 1:100 in blocking buffer and incubated for 1 hour at room

temperature. Following washing with PBS, biotinylated secondary antibody

Conditional Liver-Specific pRB Knockout

www.aacrjournals.org 4569 Cancer Res 2005; 65: (11). June 1, 2005



and streptavidin-peroxidase conjugate were applied according to theVectastain Elite avidin-biotin complex kit instructions (Vector Laboratories,

Burlingame, CA). Staining was developed in 3,3Vdiaminobenzidine solution

(Vector Laboratories) for 1 to 2 minutes and quenched in H2O. Slides were

counterstained with hematoxylin, dehydrated through graded ethanols andxylene, and mounted using permount (Sigma, St. Louis, MO). BrdUrd

incorporation was analyzed using a BrdUrd detection kit (Zymed, South San

Francisco, CA) exactly as recommended by the manufacturer. BrdUrd

incorporation was scored blind and at least 600 hepatocytes per sectionwere counted from several random fields. For histologic analysis, sections

were stained with H&E using standard techniques. TUNEL assay for

colorimetric apoptosis detection was done using the FragEL DNA

Fragmentation Detection Kit exactly as described by the manufacturer(Calbiochem, San Diego, CA).

Fluorescence-activated cell sorting analyses. Flow cytometry of

unfixed-propidium iodide (PI)–stained nuclei was done as previouslydescribed (48). Briefly, liver nuclei were prepared from frozen tissue as

described above. Unfixed nuclei were resuspended in 0.5 mg/mL PI, 0.1%

NP40, 40 Ag/mL RNase A, 0.1% sodium citrate in PBS. Following incubation

for 10 minutes at room temperature, samples were fluorescence-activatedcell sorting acquired. Flow Cytometry data was acquired on a Coulter Epics

XL (Beckman-Coulter, Miami, FL) with a 488-nm argon-ion laser.

Acquisition and ploidy analysis were done with System II software

(Beckman Coulter, Fullerton, CA). Doublet discrimination was done bygating on peak versus integral signals. The gates used to quantitate the

number of events at each ploidy level were set to correspond to 2-32N DNA

content (PI) and were not moved for all subsequent analysis. Histogramsand zebra plots were prepared using FlowJo software (Tree Star, Inc.,

Ashland, OR).

Centrosome staining. Imprints from freshly excised livers were fixed in

methanol for 20 minutes at �20jC. Cells were washed in PBS andpermeabilized with 1% NP40 in PBS for 5 minutes at room temperature.

Slides were blocked with 10% normal goat serum in PBS for 1 hour and

probed with mouse anti–g-tubulin antibody (49) for 1 hour at room

temperature. After washing in PBS, antibody-antigen complexes weredetected by incubation with rhodamine-conjugated goat anti-mouse

immunoglobulin G antibody for 1 hour at room temperature. Cells were

washed thrice in TBS and counterstained with 4V-6V-diamidino-2-phenyl-indole (DAPI) before mounting and visualization by fluorescence

microscopy.

Centromeric DNA staining. Freshly excised livers were gently pressed

onto glass slides. Liver cells were fixed using two changes of methanol/acetic acid (3:1) at �20jC for 20 minutes each. Cells were dried at room

temperature and stored at �20jC until use. For centromeric DNA staining,

fixed cells were dehydrated through graded ethanol solutions (70%, 80%,

and 95%) and incubated with 2 � SSC for 2 minutes at 73jC. Cells wereagain dehydrated through graded ethanol solutions and denatured in 70%

formamide/2� SSC for 5 minutes at 73jC. Cells were dehydrated again and

incubated with a Cy3-labeled pan-centromeric DNA probe exactly as

described by the manufacturer (Cambio, Cambridge, United Kingdom).Cells were counterstained with DAPI before mounting and visualized using

fluorescence microscopy.

Results

Somatic elimination of pRB in murine hepatocytes. Toinvestigate the action of pRB in liver, a model for tissue-specificinactivation of Rb was developed. In this model, mice transgenicfor Cre under the control of the albumin promoter (Alb-Cre) werecrossed to mice that harbor loxP sites flanking exon 19 of the Rbgene (Rbf/f ). Cre-mediated excision of the region flanked by theloxP sites results in truncation of the RB protein that is functionallyequivalent to a null allele (46, 50). Litters contained 50% control(Rbf/f ) and 50% experimental (Rbf/f ;Alb-Cre+) mice and littermateswere used for all analyses. Mice were born at the expected ratiosand histologic analysis of neonatal livers reflected no gross changes

in either the hepatic or hematopoietic cells in the liver (data notshown).We first evaluated whether Alb-Cre mediated recombination of

the Rb locus was specific to the liver. Consistent with a restrictedpattern of albumin promoter activity and Cre expression, PCRevaluation of recombination in DNA extracted from liver, spleen,lung, and kidney revealed recombination exclusively in the liver(Fig. 1A). In addition, recombination was not detected in DNAextracted from heart, skin, brain, thymus, bone marrow, ovary, orcolon (data not shown). We next determined the temporal extent ofrecombination in postnatal mice. Consistent with previous findings(51), recombination was incomplete in neonatal mice but wascomplete by 8 weeks of age (Fig. 1B , compare lanes 2 and 6). Todetermine the corresponding influence of recombination on the Rbtranscript, evaluation of Rb mRNA by RT-PCR was done andshowed predominance of the recombined message specific to thelivers of adult Rbf/f ;Alb-Cre+ mice (Fig. 1C). Lastly, to examine theinfluence of Cre-mediated recombination on pRB protein levels,pRB was immunoprecipitated from nuclear lysates derived fromRbf/f and Rbf/f ;Alb-Cre+ mice and immunoblotted for pRB. Theseresults confirmed a significant reduction in pRB levels in liversfrom Rbf/f ;Alb-Cre+ mice (Fig. 1D). Together, these data showeffective liver-specific ablation of pRB using Alb-Cre . Once

Figure 1. Liver-specific Cre expression results in pRB ablation in vivo . A, organspecificity of Cre-mediated recombination (RbDexon19) was detected by PCRanalysis of the floxed Rb locus using DNA extracted from the indicated organs of6-week-old mice. B, time course for Cre-mediated recombination was done byPCR using DNA extracted from livers of Rbf/f and Rbf/f ;Alb-Cre+ mice at theindicated times after birth. C, RT-PCR analysis of mRNA extracted from livers of16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice. D, pRB was immunoprecipitatedfrom nuclear lysates prepared from the livers of 8-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice using pRB polyclonal antisera. pRB was then detected byimmunoblotting using a pRB monoclonal antibody. Lysates were alsoimmunoblotted for Brg1 to ensure equal inputs into immunoprecipations.

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validated, this model system was used to assess the consequence ofsomatic pRB loss in the liver.pRB-E2F target genes are deregulated in livers of

Rbf/f ;Alb-Cre+ mice. Initially, the consequence of liver-specificpRB loss in 21-day-old mice was assessed. These mice were chosenbecause they are the youngest mice at which completerecombination was detected by PCR, and represent a point atwhich extramedullary hematopoiesis is complete (data notshown). Given the well-established role of pRB in repressing E2Factivity and inhibiting S-phase entry, the effect of pRB loss on theexpression of several pRB/E2F target genes was analyzed.Immunoblotting revealed elevated levels of cyclin E, MCM7,PCNA, and p107 proteins in nuclear lysates prepared from Rb f/

f ;Alb-Cre+ mice compared with littermate controls (Fig 2A ,compare lanes 5-8 with lanes 1-4). In contrast, levels of the pRB-related p130 pocket protein, which has not been shown to be

regulated by E2F, were unchanged in Rb f/f ;Alb-Cre+ mice.Furthermore, BrdUrd incorporation was f2-fold higher inhepatocytes from Rb f/f ;Alb-Cre+ mice than controls (Fig. 2Band C), demonstrating increased S-phase entry in pRB-deficienthepatocytes. Whereas the percentage of hepatocytes incorporat-ing BrdUrd was increased in Rb f/f ;Alb-Cre+ mice, there was nosignificant change in liver mass in these animals (Fig. 2D). Todelineate the basis for this finding, the apoptotic fraction in Rb f/f

and Rb f/f ;Alb-Cre+ livers was analyzed. TUNEL assays revealed noelevation in levels of apoptosis in Rb f/f ;Alb-Cre+ livers (data notshown), demonstrating that the lack of increased liver mass inthese animals was not due to removal of aberrantly proliferatinghepatocytes. Combined, these data suggest that the increase inthe number of hepatocytes in the S phase in pRB-deficient micewas not reflective of a gross deregulation of proliferation.pRB is dispensable for repression of pRB/E2F target genes

and inhibition of proliferation in adult mice. Adult hepatocyteshave a long half-life (f180 days in the mouse) and low basalproliferation rate; for example, mitosis is observed only in about 1in 20,000 hepatocytes at any given time (52). Given the observedderegulation of pRB/E2F targets and increased cell cycle progres-sion in young pRB-deficient mice, the consequences of prolongedpRB deficiency were assessed in adult mice. Interestingly, in thelivers of 16-week-old mice, cyclin E, MCM5, MCM7, and PCNAexpression was not detectable by immunoblot, even in mice lackingpRB (Fig. 3A , lanes 3 and 4). Comparable with young mice, therewas also no significant difference in the levels of apoptosis in thelivers of adult Rb f/f and Rb f/f ;Alb-Cre+ mice (data not shown).Additionally, BrdUrd was not incorporated (comparable withcontrol mice) in hepatocytes of adult pRB-deficient mice (data

Figure 3. pRB is dispensable for the down-regulation of E2F target geneexpression associated with hepatocyte aging. A, immunoblot analysis ofindicated pRB-E2F targets was done using nuclear lysates prepared from liversof 21-day-old and 16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice. B, liver weights of16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice are expressed as a percentage oftotal body weight. Columns, mean (n = 14 to 16 mice per group); bars, FSE.C, immunoblot analysis of pRB family members p107 and p130 was done usingnuclear lysates prepared from livers of 16-week-old Rbf/f and Rbf/f ;Alb-Cre+

mice. Brg1 served as loading control.

Figure 2. pRB-E2F target gene deregulation and ectopic S-phase entry inhepatocytes of 21-day-old pRB-deficient mice. A, immunoblot analysis ofindicated pRB-E2F targets was done using nuclear lysates prepared from liversof 21-day-old Rbf/f and Rbf/f ;Alb-Cre+ mice. B, representative BrdUrdimmunohistochemistry of liver sections prepared from 21-day-old Rbf/f and Rbf/f ;Alb-Cre+ mice. BrdUrd was administered 1 hour before sacrifice. Sectionswere counterstained with hematoxylin. Magnification, 20�. C, quantitation ofBrdUrd incorporation. Cells were scored as either BrdUrd positive (brown)or negative (blue ) and the percentage of BrdUrd-positive cells in each sectionwas calculated. Columns, means (n = 4); bars, FSE. D, liver weights of21-day-old Rbf/f and Rbf/f ;Alb-Cre+ mice are expressed as a percentage of totalbody weight. Columns, mean (n = 4); bars, FSE. Differences in liver weightwere not statistically different.





not shown). Consistent with these observations, the liver mass ofRb f/f ;Alb-Cre+ mice was not statistically different from Rb f/f mice(Fig. 3B). These data show that despite the deregulated geneexpression noted in young mice, pRB is not required for therepression of these genes or inhibition of proliferation andinduction of quiescence in adult mice.It is now clearly established that significant functional

redundancy exists between pRB and the closely related pocketproteins p107 and p130 (18, 26, 27). Furthermore, recent evidencehas suggested that functional compensation by p107 in pRB-deficient keratinocytes is critically dependent on the levels of p107protein (33). Therefore, given the observation that E2F target geneexpression and hepatocyte proliferation are repressed in both pRB-proficient and pRB-deficient adult livers, p107 and p130 proteinlevels were monitored in livers of adult Rb f/f and Rb f/f ;Alb-Cre+

mice. In 16-week-old Rb f/f ;Alb-Cre+ mice, the floxed Rb locusremains fully recombined (Fig. 1B), confirming that pRB is absentin these livers. Immunoblot analysis of p107 and p130 in livernuclear lysates prepared from 16-week-old mice showed signifi-cantly elevated levels of both of these proteins in Rb f/f ;Alb-Cre+

mice (Fig. 3C , compare lanes 1-2 with lanes 3-4). These datasuggest that with respect to the ability to repress E2F-directedtranscription and proliferation, p107 and p130 may coordinatelycompensate for pRB loss in the adult liver.Acute pRB ablation in adult liver deregulates E2F target

gene expression and promotes cell cycle entry. Althoughfunctional compensation by p107 and p130 may explain the lackof a proliferative phenotype in pRB-deficient livers of adult Rbf/f;Alb-Cre+ mice, our data do not rule out the possibility that pRB plays norole in maintaining the quiescence of adult mouse hepatocytes. Toaddress this question, recombinant adenovirus encoding Cre (Ad-Cre) was used to elicit acute pRB loss in livers of adult Rb f/f mice.We initially assessed the ability of Ad-Cre (1 � 109 pfu, delivered byi.v. injection) to facilitate recombination at the floxed Rb locus inliver tissue. PCR analysis showed the ability of Ad-Cre to mediatecomplete recombination of the floxed Rb locus within 3 days post-infection (Fig. 4A). We next determined whether acute pRB loss inthe liver was sufficient to deregulate the expression of E2F-responsive genes. Immunoblot analysis of the levels of several pRB-E2F targets that function in cell cycle control (p107, cyclin E, andcyclin A) and DNA replication (MCM5, MCM7, and PCNA) revealedelevated levels of each of these proteins in pRB-deficient livers(Fig. 4B). In contrast, acute pRB ablation did not influence levels ofp130 protein. These data show that acute pRB loss is sufficient toderegulate the expression of E2F target genes in the adult liver. Todetermine whether acute pRB loss was sufficient for cell cycle entryin adult hepatocytes in vivo , BrdUrd incorporation was analyzed. Asreported previously (53), infection with control (Ad-LacZ) adeno-virus led to a low level of hepatocyte DNA synthesis. However,compared with controls, Ad-Cre–injected mice displayed af4-foldincrease in the number of cells staining positive for BrdUrd (Fig. 4C).Nonetheless, in spite of the increased BrdUrd incorporation, nosignificant increase in liver mass was observed in Rb f/f miceinfected with Ad-Cre (Fig. 4D). Furthermore, TUNEL stainingshowed that acute pRB loss did not result in increased levels ofhepatocyte apoptosis (data not shown). Collectively, these datashow that acute pRB loss in adult liver is sufficient to deregulate E2Ftarget gene expression and to facilitate cell cycle entry but this doesnot result in loss of control over organ size.pRB controls hepatocyte ploidy. Together, the preceding data

show that pRB controls hepatocyte cell cycle entry, but that

compensation can occur with chronic loss of pRB. Whereas suchcompensation dampens some facets associated with pRB loss, (e.g.,maintenance of quiescence; ref. 18), clearly chronically deficientpRB-null cells harbor distinct phenotypes (13, 15). Therefore, toinvestigate possible deleterious consequences of pRB loss, histo-logic analysis of liver sections from 16-week-old Rb f/f ;Alb-Cre+

animals was done. Whereas no induction of hyperplastic prolifer-ation or tumorigenesis was observed, nuclear pleomorphism withdramatically enlarged nuclei was readily evident in pRB-deficientlivers by H&E staining (Fig. 5A). Such changes in nuclear size havebeen associated with increased hepatocyte ploidy. To determinewhether the large nuclei specifically arising in Rb f/f ;Alb-Cre+ micedid reflect elevated ploidy, in situ hybridization using a pan-centromeric DNA probe was done to monitor relative chromosome

Figure 4. Acute pRB ablation in livers of adult mice deregulates pRB-E2F targetgene expression and results in ectopic S-phase entry. A, 10- to 12-week-oldRbf/f mice were injected i.v. with 1 � 109 pfu of adenoviral (Ad-) Cre or Ad-LacZ(vector control). Ad-Cre–mediated recombination (RbDexon19) of the Rb locusin liver DNA was detected by PCR analysis. Livers were excised from mice3 days post-infection. B, immunoblot analysis of indicated proteins wasperformed using nuclear lysates prepared from livers of Rbf/f mice 6 days afterAd-Cre or Ad-LacZ injection. Brg1 served as loading control. C, quantitationof BrdUrd incorporation in livers of adult Rbf/f mice, 6 days after infection witheither Ad-LacZ or Ad-Cre. Hepatocytes were scored as either BrdUrd positive(brown ) or negative (blue ) and the percentage of BrdUrd-positive cells in eachsection was calculated. Columns, means (n = 3); bars, FSE. D, liver weights ofRbf/f mice 6 days following Ad-Cre or Ad-LacZ injection are expressed as apercentage of total body weight. Columns, means (n = 3); bars, FSE.

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number. As shown in Fig. 5B , the number of positively labeledcentromeres was dramatically elevated in the inherently largenuclei. These analyses indicate that pRB loss facilitates theacquisition of enhanced ploidy. To quantitatively assess ploidychanges throughout the liver, flow cytometry was used toconcurrently analyze liver nuclear size and DNA content. Analysisof DNA content (PI) in both 8- and 16-week-old animals showed ashift towards higher DNA levels in Rb f/f ;Alb-Cre+ mice (Fig. 6A , top).In addition, analysis of DNA content and nuclear size ( forwardscatter) showed that the populations with higher DNA content inRb f/f ;Alb-Cre+ mice were coincident with the larger nuclei(Fig. 6A , bottom ). Quantitative analysis of the changes inhepatocyte ploidy showed populations of nuclei with significantlyelevated 8- and 16N DNA content and significantly reduced nucleiwith 2N DNA content, in both 8- and 16-week-old Rb f/f ;Alb-Cre+

mice (Fig. 6B). Furthermore, in 16-week-old Rb f/f ;Alb-Cre+ animals,there was a small population of nuclei with 32N DNA.It has been shown that Rb , with haploinsufficiency, functions to

maintain genetic stability in mouse embryonic stem cells (54).Therefore, the influence of loss of a single Rb allele on hepatocyte

ploidy was determined by generating mice containing one wild-type and one floxed Rb allele, either with (Rbwt/f ;Alb-Cre+) orwithout (Rbwt/f ) Alb-Cre expression. Flow cytometric comparisonof nuclear DNA content and size in 12-week-old mice revealed nosignificant differences between Rbwt/f and Rbwt/Dexon19 mice (Fig. 6C).In contrast to loss of both Rb alleles, these data show that chronicloss of a single allele of Rb is insufficient to influence the ploidy ofadult hepatocytes. Furthermore, these findings also show that incontrast to previously reported data demonstrating the developmentof aneuploidy during prolonged high-level expression of Crerecombinase (55), long-term Cre expression in our animals is notassociated with ploidy changes. Together, the above data show thatpRB regulates hepatocyte ploidy in vivo , that a single Rb allele issufficient for this function, and that the changes in ploidy seen inpRB-deficient mice were specific to pRB loss and not due to Creexpression.We reasoned that changes in ploidy observed in Rb f/f ;Alb-Cre+

mice could be due to aberrant replication or some facet of mitoticderegulation. pRB loss has been associated with centrosomehyperamplification (29) that could fuel aberrant ploidy in specifictissues. Therefore, centrosome number was analyzed in hepato-cytes of Rb f/f and Rb f/f ;Alb-Cre+ animals. To quantitate centro-somes, liver touch preparations were stained for the centrosomemarker g-tubulin, counterstained with DAPI, and visualized byimmunofluorescence. Interestingly, in contrast to the effects of Cre-mediated pRB inactivation in mouse epidermis (29), we detectedno centrosome hyperamplification in pRB-deficient hepatocytes,even in those cells with significantly larger nuclei (Fig. 6D , left).Liver cells from both Rb f/f and Rb f/f ;Alb-Cre+ animals predomi-nantly contained a single centrosome (f85%) and all remainingcells contained two centrosomes (Fig. 6D , right). No cells wereidentified that contained more than two centrosomes, demon-strating that deregulation of centrosome duplication does notcontribute to the ploidy changes in pRB-deficient hepatocytes.Acute pRB loss results in rapid augmentation of hepatocyte

ploidy. Having shown that long-term pRB deficiency is associatedwith significant elevations in hepatocyte ploidy, the kinetics ofthese changes was assessed. To address this, Ad-Cre was used toacutely ablate pRB in vivo . Specifically, Rb f/f mice were injectedwith Ad-Cre or Ad-LacZ and sacrificed 6 days later, at which timelivers were excised and examined for changes in hepatocytenuclear size and ploidy. First, liver sections from adenovirus-infected mice were stained with DAPI to visualize nuclei.Numerous large nuclei were evident in sections specifically fromlivers of Ad-Cre–infected mice (Fig. 7A). Furthermore, flowcytometric analysis showed the presence of nuclei with elevatedDNA content and nuclear size in the livers of Ad-Cre–infected mice(Fig. 7B). Quantitation of these changes revealed significantlyincreased numbers of nuclei with 16- and 32N DNA content in Ad-Cre–infected mice (Fig. 7C). These data show that acute pRB lossresults in the rapid acquisition (within 6 days) of elevatedhepatocyte ploidy in vivo .

Discussion

The development of knockout mice has provided insight intothe roles of the retinoblastoma tumor suppressor in developmentand inhibition of tumorigenesis in mice (49). However, theembryonic lethality associated with germ line Rb deletion in micehas largely precluded study of the consequences of pRB lossin adult animals, a condition highly relevant to human cancer.

Figure 5. Increased nuclear size in pRB-deficient hepatocytes. A, histologicanalysis of formalin-fixed, paraffin-embedded sections of left liver lobe from16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice. Sections were stained with H&E.PV, portal vein. Top, magnification, 10�. Bottom has higher magnification viewsof boxed areas in top (magnification = 100�). B, centromeric DNA in touchpreparations from 16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice was identified byFISH analysis using a Cy3-01-labeled pan-centromeric DNA probe.Cells were counterstained with DAPI. Magnification, 60�.





Using an Rb conditional knockout, we show that pRB loss issufficient to deregulate E2F target gene expression and cell cyclecontrol in post-natal hepatocytes. However, despite this deregu-lated cell cycle progression, pRB loss is compensated for suchthat in adult mice pRB-deficient hepatocytes are biochemicallynormal and do not exhibit hyperplasia. Consistent withcompensation within the animal, the acute elimination of pRBin adult liver is sufficient to deregulate E2F target geneexpression and elicit cell cycle entry. As a consequence of pRBloss, both chronic and acute, there is a significant developmentof aberrant hepatocyte ploidy.

Studies using mice in which Rb is deleted in the germ linerevealed that pRB loss results in deregulated developmentassociated with extensive apoptosis and differentiation defectsin many tissues (20, 22, 23). Rb-null cells from these embryosundergo complete ectopic cell division followed by apoptotic celldeath (56). Furthermore, recent experiments showed thatconditional pRB loss in mouse epidermis is associated withepithelial cell proliferation defects and hyperplasia (29, 33). Incontrast, Rb-deficient cells in chimeric mice consisting of bothRb�/� and Rb+/+ cells survive but do not complete cytokinesis(56, 57), suggesting that these cells may undergo cell cycle arrest

Figure 6. Aberrant ploidy in pRB-deficienthepatocytes. A, nuclei were isolated from livers of8- and 16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice.DNA content (PI) and nuclear size (forwardscatter, FS ) were analyzed by flow cytometry.Data are from a single representative animalfrom each genotype and are expressed asDNA content versus cell number (top ) and nuclearsize versus DNA content (bottom ). B, quantitationof DNA content in isolated liver nuclei. Thepercent of nuclei with the indicated DNA content(2 to 32N) was calculated by scoring the number ofevents at each ploidy level from the cell numberversus DNA content histogram of each animal.Columns, means (n = 3); bars, FSE. Onlystatistically significant differences are indicated ongraphs. C, livers were resected from 12-week-oldRbwt/f and Rbwt/f ;Alb-Cre+ mice. Flow cytometricanalysis and quantitation of ploidy in isolatednuclei was done as described in (A ) and (B ).D, immunofluorescence analysis and quantitationof centrosome number in hepatocytes of16-week-old Rbf/f and Rbf/f ;Alb-Cre+ mice. Touchpreparations were stained with anti–g tubulinantibody to label centrosomes and counterstainedwith DAPI to label DNA. Left, representativeimages of cells from Rbf/f and Rbf/f ;Alb-Cre+ mice.Note that the large nuclei in the Rbf/f ;Alb-Cre+

section contains a single centrosome. Right, thenumber of centrosomes per cell was scored in atleast 150 cells per section. Columns, means[n = 2 (Rbf/f) or n = 3 (Rbf/f ;Alb-Cre+)];bars, FSE.

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before entering mitosis. Our analysis in the livers of postnatalmice similarly shows that liver-specific pRB knockout results in acell cycle defect, as evidenced by enhanced hepatocyte BrdUrdincorporation. However, the absence of evidence of mitotic figures(data not shown) or hyperplasia, coupled with the lack ofsignificantly increased liver weight suggests that although pRB-

deficient hepatocytes are able to ectopically enter the S phase,they do not progress through mitosis. Supporting this idea, theabsence of elevated levels of apoptosis in pRB-deficient hepato-cytes counters the possibility that organ mass is maintained bythe removal of ectopically dividing cells. Together, these datashow that in contrast to the epidermis, conditional pRBelimination in the liver, although sufficient to cause ectopic cellcycle entry, is not sufficient to result in aberrant cell division andhyperplasia in vivo .Acute ablation of pRB in the adult mouse liver results in

hepatocyte cell cycle entry and shows that a critical function ofpRB is the maintenance of quiescence in adult hepatocytes.However, it is also clear that in livers in which pRB is chronicallyabsent, the induction and maintenance of quiescence is largelyunaffected, demonstrating that other factors must compensate forpRB in this regard. We found that levels of the pRB-related p130protein were elevated in the livers of adult mice harboringchronic pRB loss. Importantly, such increases are specificallyassociated with conditions wherein there is evidence ofcompensation occurring in pRB-deficient livers (i.e., E2F targetgenes are not deregulated), strongly implicating a role for p130 incompensating for pRB in the liver. Although there is clearevidence that p130 and pRB have overlapping functions indevelopment (26), this is the first demonstration that p130 maybe involved in compensating for pRB loss in adult tissues.Additionally, levels of p107 protein were elevated in quiescentpRB-deficient adult livers, suggesting a compensatory role forp107. This idea is supported by a previous study showing thatp107 compensates for pRB loss in mouse epidermis by initiatingproliferative arrest in the prolonged absence of pRB (33). Themechanism through which p107 and p130 are up-regulated as aconsequence of pRB loss is unclear. It is well established thatp107 is an E2F target gene (58) and the levels of p107 RNA andprotein are up-regulated in pRB-deficient cells (14, 18, 59). Indeed,p107 levels were up-regulated during the deregulated cell cycleprogression in the pRB-deficient livers of young mice andfollowing acute pRB inactivation in adult mice. However, howthe levels of p107 remain elevated, whereas a number of otherE2F target genes (e.g., MCM7 and PCNA) are repressed inquiescence is unclear. Similarly, the basis for the up-regulation ofp130 is equally enigmatic. p130 is not an identified E2F targetgene and levels of p130 protein were not elevated in liversexhibiting deregulated proliferation following pRB loss, suggestingthe possibility that accumulation of this protein could be due tothe regulated stability of p130 protein. Thus, although pRB lossclearly deregulates cell cycle entry and target gene expression inpRB-deficient livers of young animals, the accumulation of p107/p130 through processes not completely understood can precludethese effects.Human cancer is primarily a sporadic disease arising in post-

mitotic tissues of adults. To mechanistically dissect whichfunction(s) of pRB are most relevant to its function as a tumorsuppressor, it is therefore critical to understand the biochemicalconsequences of acute, somatic pRB inactivation in adult tissues.Here we show that acute pRB ablation in liver results in E2F targetgene deregulation and ectopic cell cycle entry. Strikingly, we didnot detect any evidence for cell division or hypercellularity in liversfollowing acute pRB loss, suggesting that pRB loss alone is notsufficient to facilitate completion of mitosis. However, our dataclearly show that pRB is responsible for maintaining thequiescence of post-mitotic hepatocytes in mice. A formal

Figure 7. Rapidly increased nuclear size and ploidy following acute ablation ofpRB in livers of adult mice. A, sections of livers from 10- to 12-week-old Rbf/f

mice, 6 days after injection of Ad-Cre or Ad-LacZ, were stained with DAPI toidentify nuclei. Representative fields showing the presence of enlarged nucleifollowing Ad-Cre administration. B, nuclei were isolated from livers ofAd-Cre– or Ad-LacZ–injected mice. DNA content (PI) and nuclear size (forwardscatter, FS ) were analyzed by flow cytometry. Data are from a singlerepresentative animal from each genotype and are expressed as DNA contentversus cell number (top ) and nuclear size versus DNA content (bottom ).C, quantitation of DNA content in isolated liver nuclei. The percent of nuclei withthe indicated DNA content (2 to 32N) was calculated by scoring the number ofevents at each ploidy level from the cell number versus DNA content histogramof each animal. Columns, means (n = 3); bars, FSE. Statistically significantdifferences are indicated on graphs.





possibility is that the acute loss of pRB will lead to hyperplasiawith aging or that even in the adult compensation will occur withtime. Delineating these facets of pRB action will clearly forwardour understanding of the role of pRB loss on tumor etiology insporadic cancers.Whereas the loss of pRB does not lead to detectable

hyperplasia in the livers of adult mice, it does lead to a specifichistologic aberration within the liver, the appearance of nuclearpleomorphism. This is in fact so striking that the genotype ofmice can be determined by the histology of the liver. Here weshow that the likely cause of the pleomorphism is the enhancedploidy of pRB-deficient cells. We and others have found that pRBloss results in aberrant ploidy in cell culture (46, 60); however,although enlarged nuclei were detected in pRB-deficient hepato-cytes in chimeric animals (61), aberrant ploidy has not heretoforebeen shown in an animal model of pRB loss. The mechanismunderlying the change in ploidy is complicated in that two facetsof cellular biology cooperate to induce changes in nuclear ploidy.First, increased ploidy reflects the failure of nuclei with replicatedDNA to segregate their genetic material. This event is thenfollowed by subsequent cycles of replication to increase the ploidyabove 4N. As such, the generation of polyploid cells requires theuncoupling of DNA replication from mitosis (62). In the specificcase of hepatocytes, it has been recently shown that thegeneration of polyploidy is indeed due to a loosening of thenormally tight coupling of DNA replication and mitosis (63).Clearly, this coupling is further antagonized via the loss of pRB.Avni et al. recently showed that pRB blocks ectopic DNAsynthesis in fibroblasts by inhibiting the firing of specific originsof replication under some conditions (64). Thus, it is feasible thatthis function of pRB is transiently lost following pRB ablation inthe liver leading to endoreduplication and increased ploidy inhepatocytes the absence of cell division. An aspect of mitoticderegulation commonly manifested in tumors is centrosomehyperamplification. Centrosomes nucleate the microtubule arrayand as such play an important role in mitosis (65). Aberrantcentrosome numbers are found in a variety of tumors and havebeen shown in conditional knockout of Rb in the epidermis (29).

Here we failed to observe centrosome abnormalities associatedwith pRB loss, even in cells with the large (polyploid) nuclei,demonstrating that the development of aberrant ploidy is notassociated with centrosome hyperamplification.Together, our studies show that pRB in the liver has some

interesting biological consequences that may contribute to livertumorigenesis. Clearly, aberrant cycles of proliferation as occurduring chronic liver damage (e.g., hepatitis infection or alcohol-ism) fuels hepatocellular carcinoma (37). As such, loss of pRB andthe observed cell cycle deregulation in adult livers could representa progression event in liver tumorigenesis. Given that compensa-tion can occur, we speculate that the window of deregulated cellcycle progression associated with pRB loss is limited and thusprovides an explanation for the lack of a hyperplastic phenotypeassociated with chronic pRB loss. However, the manifestation ofincreased ploidy represents a durable characteristic of pRB loss.Although polyploid hepatocytes are a common feature of normalliver, their prevalence increases in a variety of pathophysiologicconditions (62, 66). Furthermore, it is believed that geneticinstability in these cells may provide a means to developaneuploidy and therefore contribute to tumorigenesis (62). Thus,these studies provide the basis for delineating the action of pRB inthe suppression of liver tumorigenesis and the maintenance ofappropriate liver function.

Acknowledgments

Received 11/24/2004; revised 3/16/2005; accepted 3/17/2005.Grant support: National Cancer Institute grant CA106471 (E.S. Knudsen) and

training grant T32 CA 59268 (C.N. Mayhew) and NIEHS Core grant E30-ES-06096 (E.S.Knudsen).

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

We thank Dr. David Witte (Department of Pathology, Cincinnati Childrens HospitalMedical Center) for evaluation of liver histology, Dr. Lique Coolen for assistance withmicroscopic image capture, the UC Comparative Pathology Core for histologicpreparations, Erin Williams for assistance with immunohistochemistry techniques,Dr. Jeff Albrecht (Division of Gastroenterology, Hennepin County Medical Center,Minneapolis) for helpful discussions, Drs. Karen Knudsen and Christin Petre forcritical reading of the article, and all the members of both of the Knudsen laboratoriesfor helpful comments.

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2005;65:4568-4577. Cancer Res Christopher N. Mayhew, Emily E. Bosco, Sejal R. Fox, et al. and Aberrant PloidyLiver-Specific pRB Loss Results in Ectopic Cell Cycle Entry

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