cyclin g1 has growth inhibitory activity linked to the arf ...cyclin g1 has growth inhibitory...

Cyclin G1 Has Growth Inhibitory Activity Linked to theARF-Mdm2-p53 and pRb Tumor Suppressor Pathways

Lili Zhao,1 Tina Samuels,1 Sarah Winckler,1 Chandrashekhar Korgaonkar,1 Van Tompkins,1

Mary C. Horne,1,2 and Dawn E. Quelle1,2

1Department of Pharmacology and 2The Molecular Biology Graduate Program, College of Medicine, The University ofIowa, Iowa City, IA

AbstractCyclin G1 is a p53-responsive gene that is induced in

alternative reading frame (ARF)-arrested cells, yet its

role in growth control is unclear. We tested its effects

on growth and involvement in the ARF-Mdm2-p53

tumor suppressor pathway. We show that cyclin G1

interacts with ARF, Mdm2, and p53 in vitro and in vivo.

At high levels, cyclin G1 induces a G1-phase arrest in

mammalian cells that coincides with p53 activation.

Conversely, lower levels of cyclin G1 lack intrinsic

growth inhibitory effects yet potentiate ARF-mediated

growth arrest. Notably, cyclin G1 is down-regulated by

Mdm2 through proteasome-mediated degradation.

These data suggest that cyclin G1 is a positive

feedback regulator of p53 whose expression is

restrained by Mdm2. Interestingly, growth inhibition by

cyclin G1 does not require p53 but instead exhibits

partial retinoblastoma protein (pRb) dependence. These

findings reveal that cyclin G1 has growth inhibitory

activity that is mechanistically linked to ARF-p53 and

pRb tumor suppressor pathways.

IntroductionInactivation of the p53 tumor suppressor gene is the most

frequent genetic event in human cancers (1). p53 is a

checkpoint regulator that maintains genomic stability in the

face of environmental and intracellular stresses, including

hypoxia, DNA damage, and oncogene activation (2). Normally,

p53 is a short-lived nuclear protein, but stress signals rapidly

stabilize and activate p53 through post-transcriptional mecha-

nisms, such as phosphorylation and acetylation (3). Activated

p53 suppresses growth by transactivating genes that trigger

growth arrest or apoptosis (4). Once cellular damage is

repaired, p53 must be down-regulated to allow normal cell

growth. This is primarily accomplished by Mdm2, a p53-

responsive gene product that acts in a negative autoregulatory

feedback loop to inactivate p53 (5). Mdm2 is an E3 ubiquitin

ligase that blocks p53 function through direct binding,

ubiquitination, and promotion of p53 nuclear export into

cytoplasmic proteasomes (5).

ARF is an alternative reading frame product derived from

the INK4a/ARF tumor suppressor locus on chromosome 9p21

(6, 7). It is the second most commonly inactivated gene in

human cancer (8), and it blocks cellular transformation in

response to activated oncogenes, such as Ras or Myc (9). As

with mice lacking p53, specific disruption of ARF results in

spontaneous tumor development (10, 11). p53 is the major

effector of ARF-mediated growth inhibition, and ARF activates

p53 by antagonizing Mdm2 (7, 9). Until recently, it was

generally accepted that ARF neutralized Mdm2 activity by

sequestering it within nucleoli, thereby allowing p53 to

accumulate in the nucleoplasm and induce expression of

growth inhibitory genes. However, recent studies showed that

ARF can inhibit growth without relocalizing endogenous

Mdm2 to nucleoli (12–14). Moreover, regions within the

amino terminus of ARF were identified that were dispensable

for Mdm2 binding and relocalization, but essential for its

activation of p53 and inhibition of growth (14). Those findings

suggested that other factors besides Mdm2 contribute to p53-

dependent growth suppression by ARF. Consistent with that

notion is the existence of multiple ARF signaling pathways. For

instance, once p53 is activated, both p21-dependent and p21-

independent pathways can contribute to the G1 and G2 arrest

elicited by ARF. p21Cip1 is a p53-responsive gene and potent

inhibitor of cyclin-dependent kinases (Cdks) that blocks

phosphorylation of the retinoblastoma tumor suppressor

protein, pRb (15). The consequent accumulation of active,

hypophosphorylated pRb arrests cells in G1 phase and prevents

S-phase entry. Although p21 is the primary downstream

effector of ARF-mediated cell cycle arrest, a p21-independent

pathway also exists that exerts a distinct biphasic growth arrest

(16, 17). In addition, ARF can induce a delayed G1-phase

growth arrest in cells lacking both p53 and Mdm2 (18).

Importantly, regulators of the p21- and p53/Mdm2-independent

pathways have yet to be identified.

We previously showed that cyclin G1 is induced by ARF in

both p21-positive and p21-negative cells (14, 17). Cyclin G1 is

a transcriptional target of p53 that contains two p53 binding

sites within its promoter, and its up-regulation coincides with

activation of p53 by various DNA-damaging agents (19–23).

Cyclin G1 is also induced by transforming growth factor h

Received 7/5/02; revised 11/1/02; accepted 12/13/02.The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.Grant support: D.E.Q. from the American Cancer Society (RSG-98-254-04-MGO) and NIH (RO1 CA90367), and by a grant from the NIH to M.C.H. (RO1GM56900).Requests for reprints: Dawn E. Quelle, Department of Pharmacology, College ofMedicine, The University of Iowa, 51 Newton Road, Iowa City, IA 52242. Phone:(319) 353-5749; Fax: (319) 335-8930. E-mail: [email protected] D 2003 American Association for Cancer Research.

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(TGF-h), BMP-4, p63, and p73, and is often detected in cells

and tissues lacking p53, indicating that it can be regulated

through p53-independent pathways (21–26). Notably, there is

no proven Cdk partner for cyclin G1. It has been found to

interact with pRb, cyclin G1-associated kinase (GAK), Cdk5,

and the regulatory BV and catalytic C subunits of protein

phosphatase 2A (PP2A) (27–32). The physiological impor-

tance of those associations has not been determined, although

there is suggestive new data concerning PP2A. Okamoto et al.

(32) recently reported that cyclin G1 recruits PP2A to

dephosphorylate Mdm2 and thereby regulate p53.

Apparently conflicting roles have been assigned to cyclin G1

in growth control. Some reports indicate that cyclin G1

promotes growth based on observations that its overexpression

enhances the growth of certain cancer cell lines, whereas

introduction of antisense constructs suppresses their growth

(29, 33–35). Conversely, it has been suggested that cyclin G1

may have growth inhibitory activities. This is based on

substantial yet largely correlative data showing that cyclin G1

expression is high in differentiated tissues and in G2-phase-

arrested hepatocytes, is induced by DNA-damaging agents, and

is up-regulated during TGF-h- or BMP4-mediated growth

arrest (23, 24, 36, 37). Moreover, it was recently shown that

cyclin G1�/� mouse embryo fibroblasts (MEFs) are partially

deficient in an irradiation-induced G2-M-phase checkpoint (38).

Others found that overexpression of cyclin G1 had no effect on

the growth properties of mouse fibroblasts, yet it sensitized

those cells to tumor necrosis factor a (TNFa)-mediated

apoptosis (19, 23).

The data presented here implicate cyclin G1 as a regulator

within the ARF-Mdm2-p53 and pRb tumor suppressor path-

ways. Moreover, our findings suggest that cyclin G1 has

intrinsic growth inhibitory activity that is dependent on the

magnitude of its expression. That observation may help to

explain, at least in part, why there are conflicting reports

concerning its role in growth control.

ResultsCyclin G1 Has Intrinsic Growth Inhibitory Activity

We previously demonstrated that cyclin G1 protein

expression is induced concomitantly with p53 activation in

ARF-arrested mouse fibroblasts (14, 17). Given that ARF can

inhibit growth independent of p53 and that cyclin G1 is

regulated by multiple transcription factors, we tested whether

up-regulation of cyclin G1 by ARF is p53 dependent. Cyclin

G1 protein levels were assayed following introduction of

ARF into cells expressing or lacking p53. Mouse NIH 3T3

fibroblasts (INK4a/ARF-null, wild-type p53) and triple-

knockout (tko) MEFs lacking p53, Mdm2, and ARF were

infected with retroviruses encoding mouse ARF or empty

vector control. ARF causes a rapid p53-dependent G1- and

G2-phase growth arrest in 3T3 cells versus a delayed G1-

phase block in tko cells (14, 18). Immunoblots showed

equivalent expression of ARF in both populations, yet cyclin

G1 expression and induction was only evident in NIH 3T3

cells (Fig. 1). For comparison, we also examined cyclin G1

expression in human Narf cells, a derivative of U2OS

osteosarcoma cells that express isopropyl-1-thio-h-D-galacto-

pyranoside (IPTG)-inducible ARF (39). Treatment with IPTG

for 2 days resulted in complete growth arrest (data not shown)

that coincided with induction of ARF, stabilization of p53, and

up-regulation of cyclin G1 (Fig. 1). These data indicate that

ARF-mediated induction of cyclin G1 is commonly observed

and requires p53.

The p53-dependent up-regulation of cyclin G1 in ARF-

arrested cells suggested that it might have intrinsic growth

inhibitory activity. This was supported by preliminary

experiments showing that ectopically expressed cyclin G1

induced a G1-phase arrest in Chinese hamster ovary (CHO)

and 293 cells.1 To further test that idea, green fluorescent

protein (GFP)-tagged cyclin G1 plasmids were expressed by

transient transfection in U2OS and NIH 3T3 cells. Cells

expressing GFP or GFP-cyclin G1 (GFP-G1) were collected

by fluorescence-activated cell sorting, and the DNA content

of each population was measured by staining with Hoescht

dye or propidium iodide (PI). Fig. 2 shows that cells

expressing high levels of GFP remained in cycle, similar to

GFP-negative cells isolated from the same populations. In

contrast, cells expressing GFP-G1 were dramatically arrested

in the G1 phase of the cell cycle. In experiments using live

U2OS cells stained with Hoescht, cells were simultaneously

stained with PI to identify dead cells within the population.

No increase in cell death was observed in the GFP-G1-

positive cells versus those expressing GFP (data not shown),

indicating that in this system, cyclin G1 does not initiate

apoptosis.

Cyclin G1 Potentiates ARF-Mediated Growth ArrestTo examine the significance of cyclin G1 up-regulation by

ARF, we tested its contribution to ARF-mediated growth arrest

in NIH 3T3 cells. Mouse fibroblasts were chosen for these

studies because we routinely achieve nearly complete intro-

duction of our genes of interest into the population via

retroviral-mediated infection (14, 17). As controls, retroviruses

encoding cyclin G1, empty vector, or mouse ARF were

individually transduced into the ARF-null cells. Western

blotting confirmed expression of the exogenous hemaglutinin

(HA)-tagged cyclin G1 (Fig. 3A), although levels achieved by

retroviral infection were approximately 5-fold lower than that

FIGURE 1. Cyclin G1 is induced by ARF in a p53-dependent manner.NIH 3T3 cells and tko MEFs were infected with vector (V ) or ARF (A )retroviruses, whereas Narf cells were treated with (+) or without (�) IPTG.Western blotting was performed to measure the expression of ARF, p53,and cyclin G1 in whole cell lysates (50 Ag per lane) from the indicated cells.

1S. Winckler and M.C. Horne, unpublished observations.

Cyclin G1 Activity Linked with p53 and pRb196



induced by ARF. Flow cytometric analyses revealed no

significant effect of cyclin G1 on 3T3 cell growth, similar to

vector control cells, whereas ARF induced a complete G1- and

G2-phase growth arrest (Fig. 3B and C).

The lack of growth arrest by cyclin G1 in these cells was

surprising given its ability to block U2OS and NIH 3T3 cell

growth in transfection experiments (Fig. 2). However, it was

consistent with earlier studies showing that cyclin G1 was

unable to initiate growth arrest or apoptosis when overex-

pressed in mouse fibroblasts (19). Interestingly, cell counts

taken 2 days after infection showed that cyclin G1 expressors

divided at a slightly faster rate (1.2-fold, P < 0.01) than vector

control cells (Table 1). This finding was in accordance with

studies showing that cyclin G1 modestly enhanced colony

formation of human diploid fibroblasts (29). It seems most

likely that an inability to achieve high levels of cyclin G1

expression in the retroviral system accounts for its lack of

growth inhibitory activity. Indeed, a correlation between growth

arrest and high-level expression of cyclin G1 was observed

(Fig. 3D). NIH 3T3 cells that were arrested, either by

transfection of GFP-G1 or introduction of ARF, expressed

high levels of tagged or endogenous cyclin G1, respectively

(Fig. 3D). Conversely, cells infected with cyclin G1 retroviruses

continued to proliferate and expressed relatively low levels of

exogenous cyclin G1. Thus, the magnitude of cyclin G1

expression correlated with its effects on cell growth.

Previous studies showed that cyclin G1 was unable to induce

apoptosis when overexpressed in NIH 3T3 cells, but it

potentiated cell death induced by TNFa (23). To test whether

exogenous cyclin G1 could enhance ARF-induced growth

arrest, NIH 3T3 cells were first infected with retroviruses

encoding empty vector or cyclin G1, followed by a second

round of infection with ARF retroviruses. Although this method

resulted in essentially complete infection with ARF (at least

96% of cells in each population expressed ARF, as determined

by immunofluorescence), reduced expression of ARF was

routinely achieved. Consequently, a less robust arrest and up-

regulation of p53 by ARF was observed (Fig. 3A and B). Under

these conditions, the addition of exogenous cyclin G1

consistently potentiated the G1-phase growth arrest exerted by

FIGURE 2. High levels of cyclin G1 induce G1-phase growth arrest.Representative histograms showing cell cycle distributions of sorted GFP-positive (+) and GFP-negative (�) U2OS and NIH 3T3 cells followingtransfection with GFP or GFP-cyclin G1 (GFP-G1 ) plasmids. Thepercentage of cells in S phase for each population is denoted.

FIGURE 3. Cyclin G1 enhancesARF-mediated growth arrest.A. Equivalent amounts of totalcellular protein from NIH 3T3 cellsinfected with the indicated retrovi-ruses were analyzed by Westernblotting for expression of ARF,cyclin G1 (HA-tagged form indicatedby asterisk ), p53, Mdm2, and B23(loading control). B. Cell cycledistributions of infected NIH 3T3cells from a representative experi-ment, in which V/A and G1/A repre-sent sequential infections of vectoror cyclin G1 plus ARF viruses.C. The relative percentage S phasefor cells expressing the indicatedviruses relative to vector control wascalculated from three independentexperiments. Asterisk, statisticallysignificant difference between G1/Aand V/A samples, as determined bya paired, two-tailed Student’s t testanalysis (P = 0.008); bars, SD.D. Cyclin G1 immunoblot assessingexpression levels in an equivalentnumber of NIH 3T3 cells infectedwith vector, ARF, or HA-cyclin G1retroviruses compared to NIH 3T3cells transfected with GFP or GFP-G1. Transfected ce l ls wereanalyzed (not sorted) by fluores-cence-activated cell sorting, and33% expressed GFP-G1. Asterisksindicate endogenous cyclin G1 (*),HA-cyclin G1 (**), and GFP-G1 (***).

Molecular Cancer Research 197



ARF (Fig. 3C). Although the effect was modest, it was highly

reproducible and statistically significant when subjected to a

Student’s t test analysis (P = 0.008). Identical results were

obtained in BrdUrd-incorporation assays, in which 40% of vec/

ARF-infected cells incorporated BrdUrd compared to only 30%

of G1/ARF-infected cells.

The above results indicated that cyclin G1 can contribute to

ARF-mediated growth arrest. It is notable that the enhanced

arrest of G1/ARF-expressing cells did not coincide with

increased p53 stabilization (Fig. 3A) or transcriptional

activation in reporter assays (negative data not shown). The

slight increase in Mdm2 expression observed in G1 + ARF-

versus Vec + ARF-infected cells was not reproducible in

multiple experiments (see Fig. 3A), and we have no evidence

for up-regulation of another p53 target, p21, in these cells (data

not shown). Therefore, the effects of exogenous cyclin G1

appeared to be independent of p53, as one would expect given

that its expression is normally downstream of p53.

Cyclin G1 Associates with ARF, Mdm2, and p53Previous studies showed that cyclin G1 is a nuclear protein,

ARF is nucleolar, and p53 and human Mdm2 (Hdm2) reside

within the nucleoplasm (22, 36, 40). Therefore, we tested

whether cyclin G1 might physically bind to those regulators.

In vitro binding assays were performed using glutathione

S-transferase (GST)-tagged cyclin G1 mixed with recombinant

ARF, p53, or Mdm2 produced in Sf9 insect cells (Fig. 4A).

GST-cyclin G2 and GST-PP2AC (the C subunit of PP2A) were

included for comparison. Cyclin G2 shares significant homol-

ogy with cyclin G1, and the BV and C subunits of PP2A are

known to associate with both G cyclins (24, 30–32). We found

that GST-cyclin G1 bound to ARF, p53, and Mdm2, although

the interaction with Mdm2 was reproducibly more efficient in

repeated assays (Fig. 4A and data not shown). Interestingly, p53

associated equally well with cyclin G2 and more strongly with

PP2AC than it did with cyclin G1. The interaction between

ARF and Mdm2 with cyclin G2 was weak in vitro . Likewise,

their ability to complex with PP2AC was limited but modestly

stronger given the relatively low levels of GST-PP2AC in the

reactions. Thus, cyclin G1 is able to associate independently

with ARF, p53, and Mdm2 in vitro , and p53 can also interact

with cyclin G2 and PP2AC.

To define the region(s) of cyclin G1 that interact with ARF,

p53, and Mdm2, similar in vitro binding studies were performed

with COOH-terminal deletion mutants of cyclin G1 fused with

GST. GST-G1 proteins containing NH2-terminal residues 1–24

or 1–57 of cyclin G1 were incapable of binding to either ARF,

p53, or Mdm2 (Table 2). Relatively weak binding was observed

between those proteins and residues 1–187 of cyclin G1, a

construct that contains the conserved cyclin box (24, 41). In

contrast, GST-G11–217 exhibited strong association with ARF

and optimal binding to p53 and Mdm2 when compared to the

binding ability of wild-type cyclin G11–294. Thus, residues

187–217 are required for efficient binding of ARF, p53, and

Mdm2. That region comprises the first three a helices of the so-

called ‘‘box repeat,’’ a COOH-terminal region of five a helices

containing structural similarity to the cyclin box (41).

To determine whether cyclin G1 formed complexes with

Mdm2, ARF, and p53 in vivo , immunoprecipitation (IP)-Western

blot analyses were performed from U2OS cells expressing

GFP-G1 with each of the individual proteins in U2OS cells

(Fig. 4B and C). GFP control did not associate with either

Hdm2, p53, or ARF. In contrast, GFP-G1 efficiently associated

with ectopically expressed Hdm2 and p53 (Fig. 4B). It also

coprecipitated with endogenous Hdm2 in p53 immunoprecipi-

tations from cells overexpressing p53, suggesting that a trimeric

complex between G1-Hdm2-p53 can be formed. By compar-

ison, cyclin G1 appeared to interact weakly with ARF relative

to the association between ARF and Hdm2 (Fig. 4C).

NIH 3T3 cells infected with ARF retroviruses were then used

to determine if endogenous cyclin G1, p53, and Mdm2 form

complexes in ARF-arrested cells. As expected from earlier

studies (39, 42), protein complexes between ARF, Mdm2, and

p53 were observed in the arrested cells (Fig. 4D). Two different

antibodies efficiently precipitated cyclin G1 and coprecipitated a

small amount of p53, yet failed to coprecipitate ARF or Mdm2.

Conversely, low levels of cyclin G1 were detectably precipitated

by antisera to Mdm2 and p53 compared to IgG control,

suggesting that a small percentage of cyclin G1 is associated

with those regulators during ARF-mediated growth arrest.

Cyclin G1 Is Relocalized by ARF and Hdm2Given that cyclin G1 forms complexes with Hdm2, ARF,

and p53 in vivo , we tested whether its localization was altered

by those proteins in U2OS cells (Fig. 5). As expected, GFP was

expressed in both the cytoplasm and nucleus, and its local-

ization was not altered by expression of ARF, p53, or Hdm2, or

vice versa . GFP-G1 was distributed throughout the entire

nucleus, including the nucleoli, when expressed alone in U2OS

cells. In contrast, GFP-G1 localization was altered by

exogenous ARF and Hdm2. GFP-G1 became exclusively

nucleolar in nearly 60% of transfected cells co-expressing

ARF, whereas Hdm2 retained GFP-G1 in the nucleoplasm in

65% of the population (Fig. 5). To assess the specificity of the

ARF effect, GFP-G1 was co-expressed with an ARF mutant,

D1-62, that localizes to the nucleoplasm and lacks growth

inhibitory activity (40, 43). GFP-G1 localization was unaffected

by D1-62. Importantly, expression levels were key determinants

of cyclin G1 localization because lower levels of ARF or Hdm2

were less efficient at relocalizing GFP-G1 (data not shown).

Overall, colocalization and/or relocalization of cyclin G1 with

p53, Hdm2, and ARF supports the notion that in vivo

complexes exist between those proteins.

Table 1. Low Levels of Exogenous Cyclin G1 ModestlyPromote Cell Growth in NIH 3T3 Fibroblasts

Retrovirus Relative Cell Number

Vector 1.0 F 0ARF 0.35 F 0.09Cyclin G1 1.21 F 0.02 (P < 0.01)

Note: Cells were infected with the indicated retroviruses and cell counts weretaken 2 days later. Each value represents the mean F SD from three independentexperiments. The probability (P) value, which indicates that cyclin G1-infectedsamples are statistically distinct from vector controls, was determined using apaired, two-tailed Student’s t test analysis.




Cyclin G1 Stabilizes and Activates p53The mechanism by which cyclin G1 potentiated ARF-

mediated growth inhibition appeared to be p53 independent.

However, the G1-phase arrest induced by high-level expression

of cyclin G1 correlated with an ability to bind Mdm2 and p53.

We hypothesized that an association between cyclin G1 with

Mdm2 or p53 might block the ability of Mdm2 to negatively

regulate p53, thereby fostering p53 activation. Consequently,

we measured the stability and transcriptional activity of p53 in

U2OS and NIH 3T3 cells expressing GFP-G1 (Fig. 6). GFP-

positive cells were collected by cell sorting from populations

transfected with GFP or GFP-G1 and analyzed by Western

blotting for expression of p53, Mdm2, and the respective GFP

proteins (Fig. 6A). In U2OS cells, GFP-ARF was included as a

positive control, and as anticipated, it stabilized p53 and led to

up-regulation of Mdm2 compared to GFP controls. Notably,

GFP-G1 also caused a 2- to 5-fold increase in p53 and Mdm2

levels, although the magnitude of up-regulation by cyclin G1

was consistently less than that achieved by ARF. A similar

result was observed in NIH 3T3 cells in which p53 and Mdm2

expression was enhanced by GFP-G1 compared to GFP-

positive and GFP-negative control cells (Fig. 6A, right panel).

The increased expression of p53 indicated that it was stabilized

by cyclin G1, whereas the up-regulation of Mdm2 suggested

that p53 was activated.

To directly measure the effects of cyclin G1 on p53 activity,

U2OS and NIH 3T3 stable cell lines expressing a p53 luciferase

reporter construct were generated and transiently transfected

with GFP, GFP-ARF, or GFP-G1. Luciferase assays revealed

that GFP-ARF and GFP-G1 activated p53 transcription, with an

average 5- to 8-fold increase above background levels observed

in GFP-expressing cells (Fig. 6B). Thus, cyclin G1 is a positive

regulator of p53. Because U2OS and NIH 3T3 cells lack the

INK4a/ARF gene, these results also demonstrate that cyclin G1

is able to activate p53 in the absence of ARF.

Cyclin G1 Function Does Not Require p53 Yet ShowsPartial Dependence on pRb

To test whether growth arrest mediated by cyclin G1

required p53 activity, GFP or GFP-G1 was expressed in mouse

and human cells lacking p53 (Fig. 7). As shown previously,

GFP expression alone had minimal effects on the cell cycle

distributions of each cell line tested, whereas p53-positive NIH

3T3 and U2OS were efficiently arrested by cyclin G1 (Fig. 7A).

Cyclin G1 also exhibited growth suppressive activity in murine

10-1 cells, an immortalized derivative of Balb 3T3 fibroblasts

that lacks p53 (44). In contrast, cyclin G1 failed to block

growth in p53-null Saos-2 osteosarcoma cells. Besides lacking

p53, Saos-2 cells carry a homozygous deletion of RB genes

(45), whereas 10-1 cells are pRb-positive.2

The above data suggested that growth inhibition by cyclin G1

was p53 independent but might require pRb. To test that idea

more directly, we established isogenic derivatives of U2OS cells

stably expressing the human papilloma viral proteins, E6 or E7,

FIGURE 4. Cyclin G1 interacts withMdm2, p53, and ARF in vitro and in vivo .A. Equal amounts of Sf9 lysates containingMdm2, p53, and ARF were mixed with theindicated GST fusion proteins, and Westernblots performed to measure binding. GSTfusion proteins were detected by Ponceau Sstaining (bottom panel ). B. GFP or GFP-G1was co-expressed with Hdm2 or p53 in U2OScells, and cell lysates were subjected to IP-Western blot analyses. Antibodies to GFP(G ), p53 (P ), and Hdm2 (H ) were used in theIPs. C. U2OS cells expressing GFP or GFP-G1 with ARF, or cells transfected with ARFand Hdm2, were analyzed by IP-Westernblotting, as described above. Antibodies toARF (A ) were used. D. Endogenous cyclinG1 complexes were examined in ARF-arrested NIH 3T3 cells treated with MG132for 3 h. Direct lysates (50 Ag per lane) fromvector (V )- and ARF (A)-infected cells, andimmunoprecipitated complexes (from 700 Agper IP) were analyzed by Western blotting.IPs were performed with the indicated anti-bodies, including pAb421 conjugated toSepharose (ap53 ) and two different anti-bodies to cyclin G1 (aG1-sc , Santa CruzBiotechnology, Santa Cruz, CA; aG1* ,polyclonal 1133).

2C. Korgaonkar and D.E. Quelle, unpublished observations.




or empty vector. The E6 protein targets p53 for degradation,

whereas E7 binds to and inactivates pRb (46–48). Cyclin G1

effectively arrested U2OS-vector and U2OS-E6 cells, support-

ing the notion that p53 is not required for cyclin G1-mediated

growth suppression (Fig. 7B). By comparison, cells expressing

E7 were only partially arrested by cyclin G1, consistent with the

data obtained in Saos-2 cells that suggested a role for pRb in

cyclin G1-induced growth arrest. Identical results were obtained

in BrdUrd incorporation assays using the same cell types

transfected with either GFP or GFP-G1 (data not shown).

Mdm2 Targets Cyclin G1 for Degradation in the 26SProteasome

During the course of our studies, we found it difficult to

achieve and maintain high levels of ectopically expressed cyclin

G1. This was evident in both transfection and infection

experiments in which exogenous cyclin G1 levels dramatically

decreased from 30 to 72 h post-expression (data not shown). As

such, we tested whether cyclin G1 was degraded by the 26S

proteasome. NIH 3T3 cells were infected with retroviruses

encoding vector or cyclin G1, and 2 days later, the populations

were treated with the proteasome inhibitor, MG132, for

increasing amounts of time. As shown in Fig. 8A, treatment

of vector control cells with MG132 resulted in the stabilization

of endogenous cyclin G1, p53, and Mdm2.

As a target of p53, the up-regulation of endogenous cyclin

G1 could be attributed to induction by stabilized p53 rather than

inhibition of proteasome-mediated degradation. Therefore, we

analyzed expression of HA- or GFP-tagged cyclin G1

constructs that lack p53 promoter sites. Both HA-cyclin G1

and GFP-G1 were markedly stabilized by MG132, indicating

that cyclin G1 is a target of the 26S proteasome (Fig. 8A and

B). Interestingly, the stabilization of exogenous HA-cyclin G1

was maximal after 1 h treatment with MG132, and this

coincided with accelerated stabilization and up-regulation of

endogenous p53 and Mdm2 compared to vector control cells

(Fig. 8A). Such data are consistent with the ability of cyclin G1

to stabilize and activate p53. However, the high levels of HA-

cyclin G1 obtained in MG132-treated cells, which are

comparable to the levels of endogenous cyclin G1 induced by

ARF, did not stabilize p53 as well as ARF. This indicates that

additional events besides up-regulation of cyclin G1 contribute

to ARF-mediated stabilization of p53.

Given that cyclin G1 interacts with Hdm2, an ubiquitin

ligase that targets p53 for degradation via the proteasome, we

assayed whether degradation of cyclin G1 was mediated by

Hdm2. Equivalent amounts of either p53, GFP-G1, or GFP

were co-expressed with increasing amounts of Hdm2 in U2OS

cells, and the expression of each protein was assessed by

Western blotting (Fig. 8C). As a control for Hdm2 activity and

to establish the validity of our assay, we showed that p53

expression was progressively reduced as Hdm2 expression was

increased. Moreover, p53 was not destabilized by an Hdm2

mutant (Hdm2.Ala466 –473) which is disrupted in the RING

domain required for ubiquitin ligase activity (49). Identical

results were obtained with GFP-G1, whereas GFP stability was

not affected by Hdm2 expression. These results strongly

suggested that cyclin G1, like p53, is a target of Hdm2-

mediated degradation.

DiscussionA key finding of this work was that cyclin G1 over-

expression caused a G1-phase growth arrest. Since its discovery

FIGURE 5. Cyclin G1 subnuclear distribution is distinctly altered byHdm2 and ARF. U2OS cells were transfected with GFP or GFP-G1constructs, with or without ARF, ARF mutant D1-62, Hdm2, or p53.Immunofluorescence was used to determine the localization of GFPand GFP-G1 proteins (green ) versus ARF, Hdm2, and p53 (TexasRed ). Relocalization of GFP-G1 by Hdm2 and ARF was quantifiedfrom three independent experiments in which 100 cells or more werecounted per condition.

Table 2. The COOH-Terminal ‘‘Box Repeat’’ Region of CyclinG1 Is Required for Efficient in Vitro Binding to ARF, Mdm2,and p53

Relative Binding

Cyclin G1 Fusion Protein ARF p53 Mdm2

GST-G11 – 24 � � �GST-G11 – 57 � � �GST-G11 – 187 ++ + +GST-G11 – 217 +++ ++++ ++++GST-G11 – 294(wt) ++++ ++++ ++++

Note: Relative in vitro binding efficiencies of various GST-cyclin G1 fusionproteins for recombinant ARF, p53, and Mdm2 were determined from at least twoexperiments.




in 1994 as a transcriptional target of p53 (19), cyclin G1 has

been described as either a positive or negative regulator of cell

growth. For instance, stable overexpression of cyclin G1 was

found to accelerate clonal expansion in primary fibroblasts (29),

whereas transient overexpression of cyclin G1 in cell lines and

primary hepatocytes induced apoptosis (36). Our finding that

low levels of cyclin G1 lack growth inhibitory activity (and

may even promote growth) while high levels suppress it may

help to reconcile data suggesting opposite roles for cyclin G1 in

growth control. One likely reason the growth inhibitory effects

of cyclin G1 have not been widely appreciated is that its

expression is markedly down-regulated by the proteasome in a

potentially Mdm2-dependent manner. As such, it is difficult to

achieve high levels of cyclin G1 except in transient assays.

Also, studies that relied on stable overexpression would

naturally exclude cells arrested by cyclin G1 (23, 29, 35).

A particularly novel observation was that growth inhibition

by cyclin G1 coincided with stabilization and activation of

p53. As depicted in the model in Fig. 9, this shows that cyclin

G1 can act in a positive regulatory feedback loop to activate

p53. The molecular mechanisms underlying p53 activation by

cyclin G1 are presently undefined, but we speculate that

Mdm2 function is somehow blocked. This could result from

cyclin G1 stoichiometrically limiting the binding between p53

and Mdm2, in keeping with the finding that both proteins

associate with the same COOH-terminal region of cyclin G1.

However, cyclin G1 overexpression actually enhanced the

detection of Mdm2-p53 complexes in vivo (Fig. 4 and Ref.

32), and binding studies revealed that p53 and cyclin G1

interact with distinct regions of Mdm2.3 Thus, Mdm2 might

FIGURE 7. Cyclin G1-mediated growth arrest does not require p53 butexhibits partial pRb dependence. A. The relative percentage S phase forsorted cells expressing GFP (black bar) or GFP-G1 (gray bar) comparedto GFP-negative cells within the same populations. Two independentexperiments were performed for each cell type. B. U2OS cell linesexpressing the human papilloma viral proteins, E6 or E7, or empty vector(Vec) were transfected with GFP (black bar) or GFP-G1 (gray bar). GFP-positive cells were collected by sorting and their DNA content measured byPI staining and flow cytometry. The mean percentage S phase for eachsample is shown from two independent experiments.

FIGURE 6. Cyclin G1 stabilizes and activates p53. A. U2OS and NIH3T3 cells were transfected with GFP, GFP-G1, or GFP-ARF plasmids, andGFP-positive cells (as well as GFP-negative cells from NIH 3T3 experi-ments) were collected by sorting. Cell lysates were electrophoresed onseparate gels and immunoblots performed to determine expression of p53and GFP (upper blots ) or Mdm2 (lower blots ), using Stat5 as the loadingcontrol for each membrane. B. U2OS and NIH 3T3 cells stably expressinga p53 luciferase reporter construct were similarly transfected, and therelative luciferase activity within GFP-G1- or GFP-ARF-expressing cellswas calculated compared to GFP controls. Data are representative ofthree independent experiments.

3L. Zhao and D.E. Quelle, unpublished observations.




bridge the interaction between cyclin G1 and p53 in vivo . As

such, introduction of cyclin G1 into Mdm2-p53 complexes

may partially disrupt Mdm2 conformation and cause reduced

p53 ubiquitination.

It is also conceivable that cyclin G1 promotes p53

activation via its association with PP2A. That interaction

causes the dephosphorylation of Mdm2 at S166 (32), an event

associated with the cytoplasmic relocalization of Mdm2 and

consequent stabilization of p53 in the nucleus (50). A notable

complication with that notion is that cyclin G1-PP2A

complexes also promote dephosphorylation of Mdm2 at

T216 (32). Dephosphorylation at that residue is thought to

result in p53 degradation, not stabilization, because cells

lacking cyclin G1 express elevated p53 and hyperphosphory-

lated Mdm2 at T216. Those results led Okamoto et al. (32) to

suggest that cyclin G1 can negatively regulate p53, although

given the opposing effects of cyclin G1-PP2A on Mdm2

phosphorylation, they also speculated that cyclin G1 could

activate p53. Unfortunately, the biological effects of cyclin G1

on p53 activity or cell growth were not tested in that study.

Because of its ability to activate p53, we were somewhat

surprised that growth inhibition by cyclin G1 did not require

p53. Several cell types lacking functional p53 were efficiently

arrested by cyclin G1. Although those results suggested that

p53 is not important for cyclin G1-mediated growth arrest,

they do not rule out the alternative possibility that cyclin G1

serves a role in p53 signaling. Indeed, a transient surge in

cyclin G1 expression following p53 stimulation might mimic

the high levels achieved in our transfection assays and amplify

p53 activity. Subsequent down-regulation of cyclin G1 by the

proteasome, presumably mediated by Mdm2, would then

reduce cyclin G1 expression and remove its contribution to

p53 signaling. Consistent with such a temporal response,

others showed that cyclin G1 expression precedes Mdm2 up-

regulation in response to DNA damage, and over time, cyclin

G1 levels decrease while Mdm2 expression increases (51, 52).

The circumstance or cellular context in which cyclin G1

expression is induced may also determine its impact on p53-

dependent events. There is evidence that cyclin G1 contributes

to DNA damage-induced checkpoints and G2-M-phase arrest

in primary fibroblasts and hepatocytes (36–38).

On the other hand, several lines of evidence support our

observation that cyclin G1 can function independent of

p53. First, cyclin G1 is expressed in cells and tissues

lacking p53 (21–25). Second, it can sensitize cells to

undergo apoptosis irrespective of p53 status (23), and its

ability to potentiate ARF-induced growth arrest did not

correspond with greater stabilization or activation of p53.

Third, if the sole function of cyclin G1 was to regulate

p53, cyclin G1-null animals would be expected to either

lack p53 function and be predisposed to cancer or possibly die

during embryogenesis due to unchecked p53 activity. Neither

FIGURE 8. Cyclin G1 degradation by the 26S proteasome is promoted by Hdm2. A. NIH 3T3 cells were infected with retroviruses encoding vector (V ) orHA-cyclin G1 (marked with an asterisk ), and treated with DMSO at time 0 or 50 AM MG132 for the indicated times. ARF-infected cells (A) were left untreated.Cyclin G1, p53, Mdm2, and B23 (loading control) expression was examined by immunoblotting. B. GFP-G1-transfected NIH 3T3 cells were treated for 6 hwith (+) or without (�) 20 AM MG132. Matched images using the same confocal settings are represented. Although not shown, GFP expression was notaltered by MG132 treatment. C. U2OS cells were transfected with the indicated plasmids, and the expression of p53, Hdm2, GFP, GFP-G1, and Stat5(loading control) was determined by Western blotting.




outcome was observed in mice lacking cyclin G1 (38). Indeed,

the lack of tumorigenesis or overt developmental defects in

cyclin G1-null mice suggests that it does not function as a tumor

suppressor or essential regulator of growth. Rather, it may

contribute to growth control in response to genotoxic stresses or

at particular times in development, and interactions with other

regulators, such as pRb and PP2A, may dictate its role. An

important point when considering cyclin G1 function, however,

is the possibility that cyclin G2 may have redundant or

compensatory functions. That idea is bolstered by findings that

cyclin G2 also inhibits cell growth (31), and it associates with

many of the same proteins, including p53, PP2A, Mdm2, and

ARF (data herein and Refs. 31, 32, and 52).

Our data showed that cyclin G1-mediated growth suppres-

sion was partially dependent on pRb. Cyclin G1 had no growth

inhibitory activity in RB-null Saos-2 cells and only partial

activity in U2OS cells expressing E7, an oncoprotein known to

cause the degradation and inactivation of pRb (46, 48, 53, 54).

Earlier work showed that cyclin G1 can associate with pRb, and

that the effects of cyclin G1 on growth in RKO colon cancer

cells were lost on inactivation of pRb (29). While the authors of

that study proposed that cyclin G1 promoted growth in a pRb-

dependent manner, we suggest that pRb may be required for

growth inhibition by cyclin G1. In fact, both ideas may be

correct. As mentioned earlier, the level of cyclin G1 expression

may determine its effects on growth. The noteworthy point of

agreement is that pRb may be essential for cyclin G1 action.

The pRb tumor suppressor protein and its closely related

family members, p107 and p130, are phosphoproteins that play

an essential role in controlling the G1-to-S phase transition (55).

When hypophosphorylated, the pRb proteins sequester E2F

transcription factors and block S-phase entry (56). Protein

phosphatase 1 (PP1) and PP2A represent two different classes

of serine/threonine phosphatases that have been implicated in a

number of biological processes, including phosphorylation of

the pRb proteins (57). Considerable evidence suggests that PP1

dephosphorylates pRb and activates pRb-dependent growth

arrest (58–60), whereas PP2A preferentially targets p107 (61,

62). However, there is a possibility that PP2A can also act on

pRb (63). Given its ability to associate with both PP2A and

pRb, it is conceivable that cyclin G1 induces G1-phase arrest by

promoting PP2A-mediated dephosphorylation of either pRb or

p107. A potential role for PP1 still cannot be excluded, nor can

the possibility that cyclin G1 binding to pRb directly blocks its

phosphorylation by Cdks. At this point, additional studies are

warranted to define the molecular basis of the functional

relationship between pRb and cyclin G1.

An important discovery during the course of these studies

was that cyclin G1 expression is limited by proteasomal

degradation. Moreover, Mdm2 overexpression accelerated that

process, whereas a RING finger mutant of Mdm2 that lacks

ubiquitin ligase activity failed to promote cyclin G1 degrada-

tion. Those findings are exciting because they represent the first

demonstration of cyclin G1 regulation by the proteasome, and

they suggest that Mdm2 normally controls cyclin G1 expression

via ubiquitination. In fact, preliminary data from a variety of in

vivo ubiquitination assays support that notion.4 The ability of

Mdm2 to regulate both p53 and cyclin G1 is striking,

particularly because cyclin G1 can activate p53. As such, this

work implies that Mdm2 can negatively regulate p53 function

by promoting cyclin G1 degradation, in addition to its more

direct effects on p53.

The significance of cyclin G1 relocalization to nucleoli in

cells overexpressing ARF is presently unclear. Binding studies

revealed relatively weak association between cyclin G1 and

ARF in vivo ; therefore, the relocalization of cyclin G1 to

nucleoli likely requires other factors. Although Hdm2 and p53

showed more efficient interaction with cyclin G1 in vivo , both

remained largely or completely nucleoplasmic in cells express-

ing cyclin G1 and ARF (data not shown), suggesting that they

were not responsible for directing cyclin G1 to nucleoli. It

remains to be determined whether the localization of cyclin G1

correlates with its effects on growth and ability to activate p53.

Various types of tumors, including osteosarcomas, and breast

and prostrate cancers, express high levels of cyclin G1 (22).

Because we found that high expression of cyclin G1 is growth

inhibitory, it is presumed that those tumor cells lack essential

regulators of G1 function, such as pRb. It is also possible that

mislocalization of cyclin G1 in cancer cells cancels its growth

inhibitory effects. Others showed that cyclin G1 failed to cluster

in discrete nuclear foci in response to DNA damage in

transformed cells, but did so in normal breast epithelial cells,

prompting them to postulate that ‘‘clustering’’ enabled cyclin

G1 to act as a p53 effector (22).

FIGURE 9. Model depicting the functional relationship between cyclinG1 with ARF, Mdm2, p53, and pRb. Cyclin G1 and Mdm2 are transcrip-tional targets of p53; ARF activates p53 and induces their expression byantagonizing Mdm2, an ubiquitin ligase and negative regulator of p53.Mdm2 may also down-regulate cyclin G1 activity because it enhancesproteasomal degradation of cyclin G1. At high levels of expression, cyclinG1 inhibits cell growth and stimulates p53 activity. We hypothesize thatcyclin G1 activates p53 by disrupting Mdm2 function (dashed line ),possibly by altering its phosphorylation status via PP2A. Once activated bycyclin G1, p53 would be expected to induce expression of p21, an inhibitorof Cdks that blocks growth by suppressing pRb phosphorylation.Consistent with that idea is our finding that cyclin G1 exhibits partialdependence on pRb to suppress growth. However, our observation thatgrowth inhibition by cyclin G1 does not require p53 is inconsistent with thatmodel. Consequently, a more direct functional link between cyclin G1 andpRb is likely, perhaps mediated through PP2A. Arrows, activating events;perpendicular bars, inhibitory processes.

4T. Samuels, S. Winckler, and M.C. Horne, unpublished observations.




This study was initiated to determine if cyclin G1 is a

regulator within the ARF signaling pathways. The data suggest

that it is, because it can contribute to ARF-mediated cell cycle

arrest and has intrinsic growth inhibitory activity. Notably, the

ability of cyclin G1 to potentiate growth arrest by ARF did not

involve activation of p53, suggesting that cyclin G1 functions

as a downstream effector of p53 in the p21-independent

pathway induced by ARF. We can conclude that cyclin G1 is

not a participant in the p53/Mdm2-independent pathway,

because it is not expressed in p53-null tko cells arrested by

ARF. At present, we have not addressed whether cyclin G1 is

required for ARF-induced arrest. That seems highly unlikely,

however, because ARF can inhibit growth in the absence of p21

or p53, demonstrating that it has multiple mechanisms of action

(16–18). Rather, cyclin G1 may affect the magnitude or

kinetics of growth suppression by ARF and p53.

In conclusion, these studies provide some explanation for

how cyclin G1 can exhibit differential effects on cell growth.

We propose that the levels and timing at which it is expressed

largely dictate its function, possibly by modulating the

regulators with which it associates. In agreement with that

idea, cyclin G1 affected both the accumulation and degradation

of p53 depending on whether it was in complexes with Mdm2/

ARF or Mdm2/PP2A, respectively (52). In that regard,

interesting parallels can be drawn with another p53 target,

p21, because low levels of p21 facilitate the assembly of

growth-promoting cyclin D/Cdk4 complexes while high levels

are redistributed among the Cdks and consequently inhibit

growth (15). Our current understanding of cyclin G1 suggests

that its different roles in growth control correlate, at least in part,

with differential regulation of the ARF-Mdm2-p53 pathway.

Materials and MethodsCell Culture and Protein Expression

NIH 3T3 fibroblasts and U2OS osteosarcoma cells (both

ARF-null, p53 and Mdm2 wild type) were grown in DMEM

containing 10% fetal bovine serum, 2 mM glutamine, and 100

Ag/ml of penicillin and streptomycin. Two p53-null cell lines,

Saos-2 and 10-1 (44), and primary MEFs lacking p53, Mdm2,

and ARF (kindly provided by Gerry Zambetti, St. Jude

Children’s Research Hospital) (18), were similarly maintained.

Narf6 cells (kindly provided by Gordon Peters, ICRF) were

treated with 1 Ag/ml IPTG for 2 days to induce ARF

expression. Cells were treated with 20–50 AM MG132

(Calbiochem, San Diego, CA) to inhibit the proteasome.

Retroviruses containing HA-tagged wild-type ARF or

murine cyclin G1 were produced and infected into NIH 3T3

cells, as described (6). For sequential infections, cells were first

infected with vector or cyclin G1 viruses for 6 h, followed by

4 ml of ARF retrovirus overnight. The next day, fresh medium

was added and cells were collected 24 h later. Plasmid DNAs

were transfected by a modified calcium phosphate precipitation

method (64). To select stable lines expressing human papilloma

virus E6 or E7 proteins, amphotropic viruses were first

collected from PA317 cells expressing either the empty pLXSN

retroviral vector, pLXSN-E6 or pLXSN-E7 (kindly provided by

Denise Galloway, Fred Hutchison Cancer Center). U20S cells

were then infected with 10 ml of each virus containing 8 Ag/ml

polybrene for 18 h, and selected in 0.6 mg/ml neomycin (G418)

for 2–3 weeks. Expression of E7 was confirmed by Western

blotting (Zymed, San Francisco, CA), whereas E6 expression

was confirmed by reduced p53 expression (measured by

Western blotting) and diminished ability of ARF to induce

growth arrest (data not shown).

DNA ConstructsExpression constructs containing HA-tagged ARF and its

mutant, D1-62, in pcDNA3.1, pSRa-MSV-tk-neo, or pEGFP,

have been described elsewhere (6, 65). DNA constructs for

GFP-tagged cyclin G1 were prepared by polymerase chain

reaction amplification of murine cyclin G1 cDNA (forward

p r ime r : 5 V-GCGAAGCTTGGATCCACCATGGTA-

GAAGTACTGACAACTGACTCTC-3V and reverse primer: 5V-GAGCCCGGGAATTCTTACAAATGGTCTCAG -

GAATCGTTGG-3V). A 950-bp BamHI-SmaI cyclin G1 cDNA

was subcloned into pEGFP-N1 (Clontech Laboratories, Inc.,

Palo Alto, CA) at BglII-SmaI sites. Cyclin G1 cDNA was

further amplified from pEGFP-cyclinG1 (forward primer:

5V-GCGAAGCTTGGATCCACCATGGTAGAAGTACTGA-

CAACTGACTCTC-3V and reverse primer: 5V-GCGGAATTCT-CAACTCGAGGTCGACTGACAAATGGTCTCAG-

GAATCGT-3V). Products were subcloned into pcDNA3.1, a

pBlueScript vector containing an HA epitope, and HA-cyclin

G1 was ligated into pSRa-MSV-tk-neo. GST expression

constructs for cyclin G1, cyclin G2, and PP2A/C have been

described (31). Mutants of cyclin G1 in the pGex vector were

generated by deletion of internal fragments from full-length

cyclin G1 and vector religation, including XbaI/XhoI (GST-

G11–24), BglII/XhoI (GST-G11–57), StuI/XhoI (GST-G11–187),

and SnaBI/XhoI (GST-G11–217).

Analyses for Growth ArrestThe DNA content of GFP-positive and GFP-negative cells

was determined by staining live cells with Hoescht dye 33342

(Sigma Chemical Co., St. Louis, MO), exactly as described

(31). Dead cells within the populations were identified by

staining with 5 Ag/ml PI for 5 min at room temperature. Cells

were sorted by an Epics 753 dual laser cytometer (Beckman

Coulter Corporation, Miami, FL). PI-positive cells and doublets

were excluded to ensure that only single viable cells were used

for analysis of DNA content and GFP expression. Alternatively,

1.5 � 105 GFP-positive and GFP-negative cells were sorted

from unstained populations, stained with PI, and analyzed for

DNA content using a FACScan (Becton Dickinson, San Jose,

CA) (6). For infected cells, DNA content was determined 48 h

post-infection by PI staining and FACScan analysis. Final cell

cycle distributions were determined using ModFit (Verity

Software House, Topsham, ME) or Watson Pragmatic (FlowJo,

Tree Star Inc., San Carlos, CA) software. Cell cycle progression

into S phase was also monitored by BrdUrd incorporation (14).

Protein Interaction AnalysesCells were lysed (1 � 107 cells/ml) for 1 h on ice in

NP40 buffer [50 mM Tris (pH 7.5), 120 mM NaCl, 1 mM

EDTA, 0.5% NP40] supplemented with 0.1 mM sodium

vanadate, 1 mM sodium fluoride, 5 Ag/ml leupeptin, and 30 AM




phenylmethylsulfonyl flouride. Lysates were sonicated (1 � 5 s

pulse) and clarified by centrifugation at 12,000 rpm for 10 min

at 4jC. Equivalent amounts of protein were immunoprecipi-

tated with protein A- or G-Sepharose at 4jC using antibodies to

mouse ARF (6), GFP (Abcam, Cambridge, UK), Mdm2 [2A10

or Ab-1 (Oncogene Research Products, Cambridge, MA)], p53

[DO-1 (Santa Cruz Biotechnology) or pAb421 conjugated to

Sepharose (generously provided by Ettore Appella from NIH)],

and cyclin G1 [SCH-46 (Santa Cruz Biotechnology) or

polyclonal 1133 (generated against human residues 11–25)5].

For in vitro interactions, insect cell lysates containing

recombinant ARF, p53, or Mdm2 were incubated with GST

fusion proteins on glutathione S-Sepharose (Amersham

Biosciences, Piscataway, NJ), as described (31). Protein

complexes, as well as lysates (50–100 Ag protein per lane),

were separated on denaturing gels and transferred onto

polyvinylidene difluoride membranes (Millipore, Bedford,

MA). Proteins were detected by enhanced chemiluminescence

(ECL, Amersham) according to the manufacturer’s specifica-

tions using the antibodies listed above, as well as antisera to

Stat5a (kindly provided by Fred Quelle, University of Iowa)

and nucleophosmin/B23 (Zymed).

p53 Reporter AssaysG418-resistant clones of U2OS and NIH 3T3 cells were

established that stably expressed the p53 luciferase reporter

construct, p53-luc (Stratagene, La Jolla, CA). Resulting reporter

lines were transfected with pEGFP, pEGFP-G1, or pEGFP-

ARF. Cells were collected 36–48 h later, lysed, and samples

measured in triplicate for luciferase activity (Promega Lucifer-

ase Assay System). The same populations were simultaneously

examined for GFP (transfected cells) or ARF-BrdUrd (infected

cells) immunofluorescence to determine the efficiency of

expression and to assess the growth arrest by ARF, respectively.

Relative luciferase activities were calculated by normalizing

luciferase readings to the percentage of cells expressing GFP

proteins or ARF.

Localization AssaysU20S cells (2.5 � 105) were seeded onto glass coverslips in

six-well dishes, fixed 36–48 h after transfection with 4%

paraformaldehyde, and permeabilized with 0.2% Triton X-100

for 15 min. Wild-type ARF and D1-62 were detected with

ARF antisera (6). Hdm2 was detected with SMP-14 (Santa

Cruz Biotechnology, 1:80 dilution), and p53 was detected with

1 Ag/ml DO-1 antibody (Santa Cruz Biotechnology). Secon-

dary antibodies were used as follows: biotinylated antirabbit at

1:500, biotinylated antimouse at 1:100, and Streptavidin Texas

Red (Amersham) at 1:200. Protein localization was analyzed

using Zeiss or Bio-Rad confocal microscopes. Z-sections were

performed to verify protein co-localization.

AcknowledgmentsThe authors thank Al Klingelhutz, Gerry Zambetti, Chuck Sherr, Fred Quelle,Karen Vousden, Ettore Appella, Denise Galloway, and Gordon Peters for

reagents. We also thank Jussara Hagen, Aruni S. Arachichige Don, and BrianHaugen for technical assistance. These studies were performed with assistancefrom the University of Iowa Flow Cytometry Facility, the Holden ComprehensiveCancer Center, and core facilities of the Diabetes and Endocrinology ResearchCenter at the University of Iowa.

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2003;1:195-206. Mol Cancer Res Lili Zhao, Tina Samuels, Sarah Winckler, et al. grant from the NIH to M.C.H. (RO1 GM56900).(RSG-98-254-04-MGO) and NIH (RO1 CA90367), and by a D.E.Q. from the American Cancer Society

1 1ARF-Mdm2-p53 and pRb Tumor Suppressor PathwaysCyclin G1 Has Growth Inhibitory Activity Linked to the

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