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10.1101/gad.12.19.2984Access the most recent version at doi:1998 12: 2984-2991Genes Dev.

Charles J. Sherr

p53 pathway−Tumor surveillance via the ARF

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PERSPECTIVE

Tumor surveillance via the ARF–p53pathway

Charles J. Sherr1

Howard Hughes Medical Inst itute, Department of Tumor Cell Biology, St . Jude Children’s Research Hospital,

Mem phis, Tennessee 38105 U SA

The retinoblastoma (Rb ) and p5 3 genes are not essential

for completion of the cell division cycle, but disruption

of their functions is central to the life history of most, if

not all, c ancer cell s (for review , see Weinberg 1995; Sherr

1996; Levine 1997). Surprisingly, Rb and p53 are them -

s elves regula t ed by t w o prot eins encoded by a s inglegenet ic locus, I N K 4 a / A R F , t h e pro du ct s o f w h i ch ,

p16 INK4a and p19ARF, are also potent tum or suppressors.

The role of p16INK4a a s a n inhibit or of cyclin D -depen-

dent kina s es ha s been a pprecia t ed s ince i t s dis covery

(Serrano et a l. 1993). N ow , emergin g evidence is provid-

i n g v a lu a bl e i n si gh t s i n t o t h e m o l ec ul a r c irc u it ry

t hrough w hich p19ARF modulates p53 activity as part of

a checkpoint response to oncogenic, hyperproliferative

signals.

Regulation of cell cycle progression by pRb andp53

D u ri n g m o s t o f G 1 pha s e of t he ma mma lia n cell cycle,Rb in it s hypophosphorylat ed form binds to several tran-

s cript ion fa ct ors of t he E2F fa mily, cons t ra ining t heir

activit y on som e promot ers and act ively repressing tran-

scription from others (see Dyson 1998). Phosphorylation

of Rb by cyclin-dependent kinases (cdks) in t he m id-to-

la t e G 1 phase of the cycle un tethers Rb from the E2Fs. In

turn, this enables the E2Fs to activate a series of target

genes , t he expres s ion of w hich is required for cells t o

enter S phase, thereby st imu lating proliferation (Fig. 1).

The cyclin D-dependent kinases cdk4 and cdk6 trigger

Rb phosphorylat ion, wh ich is likely com pleted by cyclin

E–cdk2 a s cells a pproa ch t he G 1-to-S phase transition.

Because induction and assembly of cyclin D-dependent

kina s es is dependent on mit ogenic s igna ling, ca ncella -t ion of R b’ s grow t h-s uppres s ive a ct ivit y is coupled t o

e xt r a ce ll u l ar s t i m u l i. B y i n h i bi t i n g c d k 4 a n d c d k6 , a

f a m i l y o f I N K 4 p r ot e i n s c a n p re v en t c el l s w i t h f u n c-

tiona l Rb from ent ering S phase. The prototypic m ember,

p16 INK4a (Serrano et al. 1993), is distinguished from its

close relatives (p15INK4b , p18INK4c , and p19INK4d) in i t s

role a s a pot ent t um or s uppress or. D is rupt ion of t he

p16 INK4a–c y cl in D 1 /c dk 4–R b pa t h w a y i s a c om m o n

e v en t i n h u m a n c a n c er , e i t h er r es u lt i n g f r o m l o ss o f

function of one of the t w o negative regulators (p16INK4a

or Rb) or from events leadin g to overexpression of one of

the t w o proto-oncogenes (cyclin D 1 or cdk4)(for review ,

see Weinberg 1995; Sherr 1996; Ruas and Peters 1998).

The p53 prot ein is a t ra ns cript ion fa ct or t ha t ca n in-

hibit cell cycle progres s ion or induce a popt os is in re-sponse to stress or DN A damage, and inact ivation of p53

attenuates both of these cellular responses (for review,

see Ko and P rives 1996; Levine 1997; G iacci a an d Kast an

1998). Elimina t ion of funct iona l p53 t hrough va rious

mecha nis ms is t he s ingle mos t comm on event in hum a n

cancer, occurring in ov er half of all tum ors (Hollstein et

al. 1994). The p53 protein is sho rt-lived and expressed at

very low levels in norma l cells but i t is s t a bil ized a nd

accumulates in cells that have sustained genotoxic dam-

age (Fig. 1). Am ong t he gene products induced by p53 is

t h e c d k i n h i bi t o r p 21C ip1/Waf1 , w h i c h c a n e ff ec t c el l

cycle a rrest (El-D eiry et a l . 1993; Ha rper et a l . 1993;

Xiong et al. 1993). Another key t arget is Mdm 2, wh ich

acts in a feedback loop to limit the action of p53 (Barak

et al. 1993; Wu et al. 1993), both by in hibiting its t rans-

a c t i v at i n g a c t i v i t y a n d b y c a t a l y z in g i t s d es t ru c t i on

(Ha upt et a l . 1997; Honda et a l . 1997; Kubbut a t et a l .

1997). Mutation of p53 compromises cell cycle arrest ,

a t t enua t es a popt os is induced by DNA da ma ge, predis -

poses cells to drug-induced gene amplification, affects

centrosome duplication, and rapidly leads to changes in

chromos ome num ber a nd ploidy (Ka s t a n et a l . 1991,

1992; Kuerbitz et al. 1992; Livingst one et al. 1992; Yin et

al. 1992; Clarke et al. 1993; Lowe a nd Ruley 1993; Low e

et al. 1993; Fukusawa et a l. 1996; Jacks and Weinberg

1996; H ermeking et al. 1997; P aulovich et al. 1997; G u-

alberto et al. 1998; Lanni and Jacks 1998). The resulting

genomic instabilit y greatly increases the probability th at

p53-null cells will evolve toward malignancy.

C o o p e r a t i o n b e t w e e n t h e R b a n d p 5 3 p a t h w a y s h a s

been amply demonstrated. Classic examples involve on-

coprot eins encoded by t he DNA t umor virus es , w hich

bot h ca ncel R b funct ion t o drive cells int o S pha s e a nd

neutralize p53 to prevent host cell suicide (for review,

see White 1996). Loss of function by Rb and related fam-

ily m embers can by pass p53-mediat ed G 1 arrest (Demers

et al. 1994; Slebos et al. 1994), but Rb loss induces E2F

a nd p53-dependent a popt os is (L ow e a nd R uley 1993;

How es et a l . 1994; M orgenbess er et a l . 1994; P a n a nd1E-MAIL [email protected]; FAX (901)495-2381.

2984 GENES & DEVELOPMENT 12:2984–2991 ©1998by Cold Spring Harbor Laboratory PressISSN 0890-9369/98 $5.00; www.genesdev.org











G riep 1994; Qin et al. 1994; Shan et a l. 1994; Sym onds et

a l . 1994; Wu a nd L evine 1994). I n s hort , mut a t iona l

e v en t s t h a t d i sa b l e t h e p 16INK4a–cyclin D 1/cdk4–Rb

pa t hw a y a nd enforce cell prolifera t ion a re count erba l-

anced by a p53-dependent apoptotic response that can

elimina t e incipient ca ncer cells . The a bil i t y of E2F t o

t r igger p53-dependent cell s uicide implies t ha t a bio-

chemica l connect ion l inks t heir funct ions . Ot her cellu-

lar oncogenes, such as m y c , also induce p53-dependent

a popt os is (Hermeking a nd Eick 1994; Wa gner et a l .

1994). Hence, p53 is not on ly act ivated by D N A dama ge,

but i t provides a n ‘ oncogene checkpoint ’ funct ion t ha t

gua rds cells a ga inst hyperprolifera t ive s igna ls (for re-

view , s ee V a n D yke 1994; Ja cks a nd Weinberg 1996;

White 1996; Levine 1997). This is t he sett ing in w hich

p19ARF plays a key role.

The I NK4a /ARF locus and tumorsuppression

T h e m a n n e r b y w h i c h a s i n g l e g e n e t i c l o c u s e n c o d e s

both p16INK4a and p19ARF is unprecedent ed in m a mm a ls

(Quelle et al. 1995) (Fig. 2). p16INK4a is encoded by three

closely link ed exons (designated 1␣, 2, and 3). An RNA

s eg m e n t a r is in g f ro m a n a l t er n at i v e f i rs t e xo n (1␤),

w hich m a ps 13–20 kb ups t rea m in t he huma n, mous e,

and rat genomes, is spliced to exon 2, yielding a ␤ t ra n-

s cript t ha t is a lm os t ident ica l in s ize t o t he ␣ transcript

tha t encodes p16INK4a (Duro et al. 1995; Mao et al. 1995;

Qu elle et al . 1995; Stone et al . 1995; Swa fford et al . 1997).

The init ia t or codon in exon 1␤ is not in fra me w it h s e-

quences encoding p16INK4a in exon 2, so the ␤ transcript

specifies a novel polypeptide. In the mouse, this 19-kD

protein consists of 65 amino acids encoded by exon 1␤,

and 105 am ino acids arising from t he alternative reading

frame (ARF) of exon 2 (Quelle et al. 1995). The h um an

protein terminates farther upstream in exon 2 and it has

a predicted m olecular mass of only 14 kD. Mouse p19ARF

a nd hum a n p14ARF are highly basic nuclear proteins that

induce G 1- a nd G 2-phase arrest w hen int roduced into a

variety of different cell ty pes (Quelle et a l. 1995; Stott et

al . 1998). I N K 4 a / A R F -null cells are susceptible t o ARF-

induced arrest , so th is activit y of p19ARF does not depend

upon p16INK4a.

M ut a t ions t ha t ina ct iva t e t he cdk inhibit ory funct ion

of p16INK4a occur frequent ly in a w ide s pect rum of hu-

ma n cancers (for review, see Ruas and Peters 1998). For

exa mple, cert a in ina ct iva t ing point mut a t ions impinge

on exon 1␣, s o m e o f w h i c h a r e i n h er it e d i n m e l a n om a

kindreds (Kamb et al. 1994a,b; G ruis et al. 1995). Al-

t h o u g h m a n y p o i n t m u t a t i o n s i n e x o n 2 o f I N K 4 a a re

a ls o predict ed t o a l t er p19ARF, t h o se t h a t h a ve b ee n

t e st e d e xp er im e n t a l l y h a v e b ee n f o un d t o i n a c t iv a t e

p16 INK4a w it hout a f fect ing t he a bil i t y of p19ARF t o i n -

duce cell cycle a rres t . M oreover, t h e a mino-t ermina l

moiety of ARF (amino acids 1–64), encoded entirely by

exon 1␤, is s uff icient t o induce cell cycle a rres t w hen

overexpressed (Quelle et al. 1997; Zh ang et al. 1998),

although tumor-specific point mutations in this domain

ha ve not been described (St one et a l . 1995; R ua s a n d

Peters 1998). Together, these data suggest that p16INK4a

i s d i s ru pt e d f r eq u e n t ly b y p oi n t m u t a t i o n s i n h u m a n

cancer, but p19ARF is not . How ever, t he common occur-

rence of homozygous deletions of I N K 4 a / A R F i n a w i d e

ra nge of huma n t um ors lea ves open t he poss ibil i t y t ha t

A RF plays an independent role as a tum or suppressor (see

below ).

Funct iona l a bla t ion of I N K 4 a / A R F i n m i c e by e li m i -

na t ion of exons 2 a nd 3 (Fig. 2) revea led t ha t derived

Figure 1. ARF checkpoint control. A RF re -

s pon d s t o p rol i fe ra t iv e s ign a ls t h a t a r e n or -

m a lly r eq u ir ed f or c e ll p rol i fe ra t ion . Wh e n

t h e s e s ig n a ls e x c e e d a c r i t ic a l t h r e s h old , t h e

ARF-dependent checkpoint (gray vert ical ba r-

rel) is act ivat ed, and ARF triggers a p53-depen-

dent response that induces growth arrest and/or apoptosis . Signals now know n to induce sig-

naling via the ARF–p53 pathway include Myc,

E1A, and E2F-1. In principle, ‘upstream’ onco-

proteins, such as products of muta ted Ras a lle-

les, const itut ively act iva ted receptors, or cyt o-

plasmic signal transducing oncoproteins, might

a l so t r ig ge r A R F a c t i vi t y v i a t h e c y cl i n D –

c d k 4–R b –E2F or M y c -d ep en d en t p a t h w a y s ,

b ot h of w h ic h a r e n or m a lly n e c e s s a r y f or S -

phase entry. In inhibit ing cyclin D-dependent

kinases, p16INK4a c a n d a m p e n t h e a c t iv i t y of

m it og e n ic s ig n a ls . E1 A is s h ow n t o w or k , a t

least in part , by canceling Rb function, a lthough its ability to inhibit p300 contributes to the response by interfering with m d m 2

expression. Again for simplicity , Myc an d E2F-1 are only sh ow n to act ivat e p53 via ARF. How ever, highly overexpressed levels of these

p rot e in s c a n a c t iv a t e p 5 3 in A RF -negat ive cells , a lbeit with an at tenuated eff iciency. ARF act ivat ion of p53 likely depends on

inact ivat ion of some Mdm2-specific function (implied by the unfilled box bracketing the la t ter two proteins). DNA damage signals

(ionizing and UV radiat ion, hypoxic stress, genotoxic drugs, etc.) access p53 through mult iple signaling pathways shown, again for

simplicity, as a s ingle DNA damage checkpoint (gray horizontal barrel) . Signals through the ARF and DNA damage pathways can

synergize in act ivat ing p53.

ARF tumor suppression

GENES & DEVELOPMENT 2985











nullizygous a nima ls w ere highly prone t o t um or devel-

opment (Serrano et al. 1996). Tumors arose early in life,

a nd t heir a ppea ra nce w a s a ccelera t ed by irra dia t ion of

new born mice or by t heir t rea t m ent w it h chemica l ca r-

cinogens. Intriguingly, m ouse embry o fibroblasts (MEFs)

explanted from the I N K 4 a / A R F knock-out mice did not

undergo replicative senescence in cult ure. Like many es-

t a blis hed mous e cell l ines , but unlike norma l prima ry

MEFs, they could be transformed by oncogenic r as alle-

les w it hout a requirement for s o-ca lled immort a liz ing

oncogenes such as m y c or adenovirus E1A . MEFs from

p5 3 -null mice exhibit s imila r propert ies (Ha rvey et a l .

1993), and p53-inactiv ating m utat ions are the m ost com -

mon single events in t he spontaneous conversion of MEF

strains into contin uously growin g cell lines (Harvey an d

Levine 1991). Results w ith bo th I N K 4 a / A R F -null or p5 3 -

n u l l M E Fs d i re ct l y c o n t ra s t w i t h t h o se o b t a in e d w i t h

norma l prima ry M EF s t ra ins , in w hich int roduct ion of

oncogenic ra s ins t ea d provokes a s t a t e of grow t h a rrest

resemblin g senescence, associated w ith accum ulation of

both p53 and p16INK4a (Serrano et al. 1997). Initially, it

w a s rea s oned t ha t t he phenot ype obs erved in I N K 4 a /

A RF -null m ice depended on th e loss of p16INK4a function

(Serrano et al. 1996). It follow ed tha t both p16INK4a a n d

p53 a ct ed a s det ermina nt s of cell s enes cence in M EFs ,

w it h t h e los s of eit her lea ding t o es t a blishment a nd im-

mort a liza t ion. R elea s e of t he s enes cence block by dis -

ruption of p16INK4a or p53 w ould be necessary for trans-

format ion of mouse fibroblasts by oncogenic ra s (Serrano

et al. 1997; for review , see Weinberg 1997). A persistent

a m b i gu i t y i s w h e t h er t h e se m i c e l a c k A RF function

completely. This is likely, because the targeting cassette

dis rupt ed t he mR N A polya denyla t ion s igna ls a s w ell a s

t h e I N K 4 a a n d A RF carboxy-terminal coding equences

(Fig. 2). How ever, the issue formal ly rem ains un resolved,

beca us e i t is conceiva ble t ha t a t runca t ed A R F prot ein

might somehow arise from undisrupted exon 1␤.

Surprisingly, w hen pure A RF -null m ice w ere crea t ed

t h a t l a c k e d o n l y t h e e x o n 1␤ sequences (Fig. 2), their

phenot ype w a s indis t inguis ha ble from t ha t a t t r ibut ed

previously to p16INK4a disruption (Kamijo et al. 1997).

I mport a nt ly, funct iona l p16INK4a was expressed in nor-

ma l t is s ues of A RF -null mice, in cultured MEFs, and in

cells from spontaneously a rising tum ors. Therefore, A RF

functions as a bona fide tumor suppressor, and the phe-

not ype init ia l ly a s cribed t o I NK4a los s is ins t ea d l ikelydue t o A R F ina ct iva t ion. I n t urn, t he phenot ypic cons e-

quences of p16INK4a los s in mice rema in uncert a in, a nd

cons t ruct ion of a pure I N K 4 a k n o ck o u t s t r a i n i s w a r-

ranted.

The ARF–p53 pathway

A cardinal feature of A RF -null M EFs is their capacity to

grow a s es t a blished cell l ines a nd t o be t ra ns formed by

oncogenic ra s genes alo ne (Kami jo et a l. 1997). Approxi-

mately 20% of spontaneously established fibroblast cell

lines derived from MEFs of wild-type mice undergo bi-

allelic A RF los s . M EF s t ra ins t ha t a re hemizygous for

A RF l o se t h e ir r em a i n i n g f u n ct i o n a l A RF a l l el e a n ds p o n t a n e o u s l y i m m o r t a l i z e a t a f a s t e r r a t e t h a n w i l d -

ty pe strains. In each ca se, established MEF cell lines that

lacked A RF preserved p53 function , w hereas those tha t

retained A RF ha d s us t a ined p53 mut a t ions . Thes e re-

sults suggested that p19ARF a nd p53 might funct ion in

t he s a me biochemica l pa t hw a y. Cons is t ent w it h t his hy-

pothesis, cells lacking a funct ional p5 3 gene are resistant

to p19ARF-induced cell cycle a rres t , implying t ha t p53

a ct s dow ns t rea m of A RF (Kam ijo et al . 1997). H ow ever,

A RF -null cells exhibit an in tact p53 checkpoint follow -

ing ionizing or UV irradiation, so p19ARF does not relay

signals to p53 in response to D N A dam age (Fig. 1). Loss

of p53 ca n occur in ca ncer cells t ha t a r is e in A RF -null

m ice, again indicating t hat ARF plays a more specialized

role in t um or s uppres sion t ha n p53, a nd t ha t s elect ion

a ga ins t p53 ca n furt her cont ribut e t o ma ligna ncy (Ka -

mijo et al. 1997).

Evidence s upport ing direct biochemica l int era ct ions

bet w een p19ARF a nd p53 is now in ha nd. Ect opic A R F

expression stabilizes p53 and induces p53-responsive

genes , M dm2 a m ong t hem . A R F ca n phys ica lly int era ct

w it h M dm2, a nd i t s binding blocks bot h M dm2-induced

p53 degradation and transactivational silencing (Kamijo

et a l . 1998; P om era nt z et a l . 1998; St ot t et a l . 1998;

Zhang et al. 1998). The interaction between Mdm2 and

p19ARF depends on t he ca rboxy-t ermina l h a lf of M dm 2

a nd on t he A R F a mino-t erminus (i . e. , t he a ct ive exon

1␤-coded segment) (Zhang et al. 1998). Because Mdm2

binds t o p53 t hrough i t s a mino-t ermina l doma in, A R F

ca n ent er int o t erna ry complexes w it h bot h M dm2 a nd

p53.

A lt hough h uma n p14ARF a ppea rs not t o int era ct w it h

p53 direct ly (P omera nt z et a l . 1998; St ot t et a l . 1998;

Z h a n g e t a l . 1 99 8), t h e re i s s o m e e vi d en c e t h a t t h e

mouse ARF protein can bind to p53 even in the absence

of Mdm2 (Kamijo et al. 1998). For example, in electro-

phoret ic m obili t y s hif t a s s a ys performed w it h purif ied,

a ct iva t ed p53 a nd a la beled oligonucleot ide cont a ining

t a n d em p5 3 c o n s en s u s D N A -b i n di n g s i t es f ro m t h e

Figure 2. The I N K 4 a / A R F locus. G enomic sequences encod-

ing p16INK4a are defined by com pletely f illed regions w ithin the

boxes designat ing exons 1␣, 2, and 3, whereas the segments of

exons 1␤ and 2 that encode ARF are defined by shaded areas.

Unfilled portions of the exons correspond to noncoding 5 Ј and 3Ј

regions. Splicing betw een the exons is indicat ed by the connect-

ing lines, and exons 1␣ a n d 1␤ are indicated to have separate

promoters (→). In the mouse genome, the alternative first exons

are separated by ∼13 kb of intervening sequences. Segments of

the genes t hat w ere disrupted by Serrano et a l . (1996) and Ka-

mijo et a l . (1997) are designated by horizontal lines below the

schematic.

Sherr

2986 GENES & DEVELOPMENT











p2 1 Cip1 promot er , a ddit ion of recombina nt p19ARF re-

t a r de d t h e m o b i l it y o f t h e p 53–o l ig on u c le ot i d e c o m -

plexes. In these assays, the otherwise latent DNA-bind-

ing capability of p53 needed to be activa ted by a ntibodies

directed to a carboxy-terminal p53 epitope, and p19ARF

w a s u n a b l e t o s u bs t i t u t e f or t h e a n t i b od y i n a c t i v a t i n g

D NA binding. Thes e obs erva t ions ra is e t he pos s ibil i t yt ha t int era ct ions bet w een p19ARF and p53 can occur on

chroma tin, alth ough there is no direct evidence that ARF

plays any physiologic role as a p53 coactivator.

ARF requires p53 to indu ce growt h arrest , but the di-

r ec t p h y si c a l i n t er a ct i o n s a m o n g p 19ARF, p53, a n d

Mdm2 in various binary and ternary complexes suggest

t ha t s ome p53 funct ions m a y reciproca lly depend on

ARF. Overexpression of p19ARF in A RF -null NIH-3T3

cells induced expression of a p2 1 Cip1 promot er-driven re-

port er gene in a ma nner t ha t depended on endogenous

p53. Paradoxically , ectopic overexpression of w ild-ty pe

p53 itself in A RF -null cells did not activa te th e reporter,

indica t ing t ha t s imple increa s es in t he a mount of p53

w ere insufficient to activat e transcription in this setting.p53-D ependent reporter gene expression w as restored

w h en s ub li m i n al a m o u nt s o f A RF expression vector

w ere reint roduced t oget her w it h increa s ing concent ra -

t ions of p5 3 , so p19ARF can provide some t ype of activat-

ing s igna l t ha t fa cil it a t es p53-dependent t ra ns cript ion

(Kamijo et al. 1998). In this respect, the functions of p53

and p19ARF are int erdependent.

Zha ng a nd colleagues (1998) reported that ARF accel-

era t ed M dm2 t urnover in HeL a cells cot ra nsfect ed w it h

vect ors encoding A R F a nd M dm2. They propos ed t ha t

d e s t a b i l i z a t i o n o f M d m 2 b y A R F w a s t h e m e c h a n i s m

underlying p53 accumulat ion. How ever, experiments by

ot hers ha ve yielded conflict ing res ult s . The idea t h a t

A R F des t a bil izes M dm2 s eems t o be a t odds w it h obs er-

v a t i o ns t h a t A RF a c t i v a t i o n i n M E Fs i n d uc es e n do g-

enous Mdm2 to accumulate in a p53-dependent manner

(de Stanchina et al. 1998; Kamijo et al. 1998; Zindy et al.

1998). Stot t and cow orkers (1998) confirm ed t hat in a

va riet y of cell t ypes cot ra nsfect ed w it h M dm 2 a nd p53,

int roduct ion of A R F overca me t he a bil i t y of M dm2 t o

induce p53 degradation. H ow ever, in t he presence or ab-

sence of exogenous p53, ARF caused Mdm2 to accumu-

late. M oreover, coexpression of t he E6 protein of hum an

papilloma virus 16, wh ich independently targets p53 for

degra da t ion, did not int erfere w it h t he a bil i t y of A R F t o

s t a bil ize cot ra ns fect ed M dm 2. M inima lly, i t s eems rea -

s o na b l e t o c o n cl u d e t h a t A R F c an a n t a g on i z e M d m 2

funct ion t hrough a m echa nism t ha t does not depend on

increased Mdm2 turnover.

How , then, does ARF stabilize p53? One possibility is

t ha t A R F int erferes w it h M dm 2’s a bil i t y t o t r igger p53

polyubiquit ina t ion. Support ing t his idea , M dm 2 s eems

t o induce t he a ppea ra nce of polyubiquit ina t ed forms of

p53, which are much less abundant in cells that overex-

press p19ARF (Pom erantz et a l. 1998). M dm2 and p19ARF

a ls o coloca lize in t he nucleoli of cells t ra ns fect ed w it h

both genes (Pomerantz et al. 1998). Because p53 degra-

dation depends upon its M dm2-m ediated nuclear export

(Roth et a l. 1998), ARF could conceivably retain M dm 2–

p53 com plexes in the n ucleolus, preventing t heir degra-

da t ion in t he cyt opla s m.

Fina lly, in cells la cking p53, A R F levels a re s ignif i-

cantly elevated (Quelle et al. 1995; Stott et al. 1998), but

reintroduction of w ild-ty pe p53 into p5 3 -null MEFs can

restore p19ARF t o norma l levels (Ka mijo et a l . 1998).

Simila rly, in huma n Sa os-2 os t eos a rcoma cells la ckingendogenous p53 funct ion, expres s ion of p14ARF w a s

dow n-regula t ed w hen cells w ere induced t o expres s ei-

ther tetracyclin e-regulated or t emperature-sensitive p53

(Stott et al. 1998). Therefore, not only can ARF stabilize

p53, but A R F expres sion is in t urn cont rolled by p53

t h ro u gh n eg at i v e f ee db a ck . Ag ai n , t h e u n de rl y in g

mecha nis m needs t o be cla r if ied. A mong t he pos sibil i-

t i es i s t h a t t h e A RF gene might be repressed by p53, or

the ARF protein could itself be a target of Mdm2-induced

turnover.

Oncogenic signals induceARFThe fa ct t ha t A R F-null M EFs grow a s es t a blis hed cell

l ines a nd ca n be t ra ns formed by oncogenic ra s m i m i c s

effect s induced by s o-ca lled imm ort a liz ing oncogenes ,

like m y c a nd E1A (Land et al. 1983; Ruley 1983). It there-

fore seems paradoxical th at m y c and E1A are also potent

inducers of apoptosis (Askew et a l. 1991; White et a l.

1991; Evan et al. 1992; Rao et al. 1992), a process aggra-

vated by depriving MEFs of serum survival factors (Evan

et a l . 1992; L ow e et a l . 1993). Thes e cont ra s t ing out -

comes of Myc and E1A action—extended life versus ac-

celerated death—can be reconciled by observations that

their overexpression provides a strong selective pressure

for event s t ha t dis ma nt le a popt ot ic s igna ling pa t hw a y s ,

w i t h A RF being a key target.

Overexpress ion of M yc, E1A , or E2F-1 in prima ry

MEFs rapidly induces A RF gene expression a nd leads to

p53-dependent apoptosis. How ever, A RF -null and p5 3 -

null MEFs resist t hese effects (de Stanchina et a l. 1998;

Zin dy et al. 1998). Simila rly, w ild-type or A RF hemizy-

gous M EFs t ha t s urvive M yc overexpres sion genera lly

s us t a in eit her p53 mut a t ion or A RF los s , but not bot h,

rapidly yielding established cell lines that tolerate supra-

phys iologic M yc levels even in t he a bs ence of s urviva l

factors (Zindy et a l. 1998). My c an d E1A can in duce p53

t h r ou g h b o t h A RF -dependent a nd A RF -independent

pathw ays, but m uch higher levels of oncoprotein expres-

s ion a re required t o a ct iva t e p53 w hen A RF is a bs ent .

Under the latter conditions, the p53 response is attenu-

ated a nd cells resistant to on cogene-induced killing rap-

idly emerge. R eint roduct ion of p19ARF int o s urviving

A RF -null cells expressing either Myc or E1A resensitizes

t h e m t o a po pt o s is , i n d i ca t i n g t h a t t h e a t t e n u a t i o n o f

dea t h is a direct cons equence of A RF los s a nd does not

res ult from ot her crypt ic m ut a t ions . Therefore, M yc,

E1A, an d E2F-1 trigger a p53-dependent oncogen e ch eck-

point gated by ARF (Fig. 1). Alth ough t he ARF–p53 path-

w a y is not es sent ia l for norma l prolifera t ion, t he check-

point could provide a fail-safe function during em bryonic

development . For example, in a m odel of the developing













murine lens, Rb deficiency triggers apoptosis in a p53-

dependent manner (Morgenbesser et al. 1994), but the

p ro c es s i s a t t e n ua t e d i n l en s es f ro m a n i m a l s l a c ki n g

I N K 4 a / A R F (Pomerantz et al. 1998).

One com ponent of t he E1A response involves its abil-

ity to activate p300, a coactivator required for p53-de-

pendent m d m 2 transcription (Thomas and White 1998).B ut t he a bil i t y of E1A t o induce A R F in M EFs is l ikely

media t ed by t he E2Fs , a s E1A mut a nt s t ha t bind p300

but do not int era ct w it h R b a re highly defect ive in t his

regard (de Stanchina et al. 1998) (Fig. 1). Conditional ex-

pression of E2F-1 in Saos-2 cells w as follow ed tem porally

by increased A RF mRNA and protein expression (Bates

et al. 1998). Cotransfection experiments indicated that

w ild-ty pe E2F-1 activat ed transcription from a m inim al

A RF promot er , w herea s a n E2F-1 mut a nt defect ive in

t ra ns a ct iva t ion w a s devoid of a ct ivit y . Des pit e t he fa ct

tha t My c also induces p19ARF to accum ulate very rapidly

(Zindy et al. 1998), it is presently unclear w hether M yc

a ct iva t es t he A RF promoter directly.

Coopera t ion bet w een m y c and oncogenic r as (Land etal. 1983; Ruley 1983) can be view ed to inv olve the ARF–

p53 pathw ay in directly. C ultured MEFs achieve replica-

t ive imm ort a li t y by ina ct iva t ing A RF or p5 3 , and by pro-

mot ing cell dea t h, oncogenes s uch a s E1A a nd m y c pro-

vide a strong selective pressure for disabling ARF or p53

function. Because enforced expression of p19ARF arrests

w ild-t ype M EFs but does not kil l t hem (Quelle et a l .

1995), ot her funct ions of M yc a nd E1A in a ddit ion t o

A RF induction are required for this process. The grow th

promoting properties of Myc and E1A are important be-

c a u s e w i t h o u t t h e m , t h e s e l e c t i o n f o r i m m o r t a l c e l l s

w ould l ikely not occur. This is even m ore obvious in

other cell ty pes in w hich transformat ion and tum origen-

esis strongly depend upon My c’s grow th promot ing func-

tions even in the absence of p5 3 (see, for example, Metz

et a l . 1995). I n t urn, M yc a nd E1A s eem t o ina ct iva t e

cellula r res pons es t ha t a re norma lly required for R a s -

media t ed inhibit ion of cell prolifera t ion, t hereby con-

verting ra s into a grow th-promot ing gene (Franza et al.

1986; Hicks et al. 1991; Hiraka w a et al. 1991; Lloyd et al.

1997; Serrano et al. 1997). The fact that oncogenic r as

a lone ca n t ra ns form M EFs la cking A RF or p5 3 argues

t ha t t heir ina ct iva t ion is key.

Because p19ARF addresses p53 through a path w ay t hat

is distinct from th ose activated by D NA dam age (Fig. 1),

induction of A RF by oncogenes may sensitize cells to the

effect s of genot oxic drugs t ha t a re us ed t o t rea t ca ncer.

Indeed, MEFs expressing E1A are significantly more sen-

s it ive t o kil l ing by a dria mycin t ha n t heir norma l coun-

terparts, w hereas E1A-expressing A RF -null M EFs no

longer manifest this synergy (de Stanchina et al. 1998).

The a bil i t y of A R F t o s ens e hy perprolifera t ive s t imuli

mus t be import a nt in t um or s urveilla nce, beca us e A RF

loss strongly predisposes to spontaneous cancer develop-

ment a nd a ccelera t es t he frequency of t um or induct ion

by irradiation or carcinogens (Serrano et al. 1996; Kamijo

et al. 1997). Indeed, in the absence of A RF emergence of

p5 3 -negative tumor cells that are resistant to DNA dam-

aging a gents should still occur (Fig. 1).

ARF in human cancer

M uch of t he experiment a l w ork on A RF t o da t e ha s in-

volved m urine systems. Senescence (and conv ersely, im -

mort a liza t ion) of hum a n cells is l ikely t o be s ubject t o

a ddit iona l a nd more s t r ingent cont rols , pa rt icula rly in

light of our longer life span. Whereas p53 and R b ina cti-vation can endow human fibroblasts with increased pro-

liferative potential, cells lacking these functions are not

immort a l , a nd chromos oma l t elomere s hort ening s oon

lim its con tin ued cell proliferati on (Bodnar et a l. 1998). In

cont ra s t , mous e chromos omes ha ve much longer t elo-

m e r es , a n d m i c e l a c ki n g t e l o m e ra s e a c t i v it y m u s t b e

bred t hrough ma ny genera t ions before t he delet erious

effects of t elomere shortening are ma nifest (Blasco et al.

1997; Lee et al. 1998).

D espit e funda ment a l dif ferences of t his t ype, A RF is

likely to funct ion as a tum or suppressor in hum ans. C er-

t a in ca ncers s uch a s mela noma s , bil ia ry t umors , non-

s ma ll cell lung ca rcinoma s , pa ncrea t ic , a nd esopha gea l

ca rcinoma s frequent ly s ust a in I N K 4 a point mut a t ions .Ot her tum or types, how ever, such as T- and B-cell acute

lymphobla s t ic leukemia s , bla dder a nd na s opha ryngea l

ca rcinoma s , mes ot helioma s , a na pla st ic a s t rocyt om a s ,

a nd gliobla s t oma mult iforme rout inely exhibit I N K 4 a /

A RF delet ions ra t her t ha n point mut a t ions (R ua s a nd

P et ers 1998). Whet her or not t hes e h omozygous dele-

t ions t a rget bot h A RF a n d I N K 4 a or A RF a lone, t heir

high frequency of occurrence strongly argues that A RF

l o ss c o n t ri b u t es s ig n if i ca n t l y t o h u m a n c a n ce r. Th i s

ma kes good sense. If p53 is directly targeted in >50% of

huma n ma ligna ncies, t hen p53-pos it ive t um ors ha ve

likely s ust a ined epis t a t ic m ut a t ions s uch a s M d m 2 a m -

plification or A RF loss. The concept that ARF monitors

prolifera t ive s igna ls ra t her t ha n DNA da ma ge helps t oexpand our understanding of p53 action and provides a

further rationale for A RF ina ct iva t ion t h rough chromo-

s oma l delet ion in ma ny forms of ca ncer.

Acknowledgments

I thank Mart ine F. Roussel, John C leveland, G erry Zam bett i ,

Tom Curran, A. Thomas Look, Suzy Baker, Peter McKinnon,

Scott W. Lowe, Ron D ePinho, Tyler Jacks, Manuel Serrano, and

Terry Van Dyke for s t imulat ing and helpful discussions, and

Dawn Quelle, Frederique Zindy, Takehiko Kamijo, Jason We-

ber, and Mangeng C heng for contributing crit ical data pert inent

to th e w ork from th e Sherr and Roussel laboratories. C.J.S . is aninvest igator of the Howard Hughes Medical Inst itute and also

acknow ledges support from the American Lebanese Syrian As-

sociated Charities (ALSAC)of St. Jude Children’s Research Hos-

pital.

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