apaf-1 is a mediator of e2f-1-induced apoptosis
TRANSCRIPT
Apaf-1 Is a Mediator of E2 F-1-induced Apoptosis
Yu su ke Fu ru kawa§, Nor iko Nish im u ra , Yu taka Fu ru kawa, Masaaki Satoh +,
Hitosh i En d o+, Satsu ki Iwase# , Hisash i Yam ad a# , Mich io Matsu d a , Yasu h iko
Kan o*, an d Mitsu r u Nakam u r a
Division of Stem Cell Regu lat ion , Cen ter for Molecu lar Med icin e, an d
+Dep ar tm en t of Bioch em istr y, Jich i Med ica l Sch ool, Toch igi 32 9-04 98, Jap an ;
#Dep ar tm en t of In tern a l Med icin e, an d In stitu te of DNA Med icin e, Jikei
Un iver sity Sch ool of Med icin e, Tokyo 10 5-84 61, Jap an ; *Division of Med ica l
On cology, Toch igi Can cer Cen ter , Toch igi 32 0-08 34, Jap an .
Ru n n in g t it le: Ap af-1 is a m ed ia tor of E2 F-1-in d u ced ap op tosis
Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on July 30, 2002 as Manuscript M200805200 by guest on February 13, 2018
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Footn otes:
*Th is wor k was su p p or ted in p ar t b y Gran ts-in -Aid fr om th e Min istr y of
Ed u cat ion , Scien ce an d Cu ltu re of Jap an (to M. M.), an d gr an ts fr om th e
San kyo Fou n d ation of Life Scien ce, th e Ich iro Kan eh ar a Fou n d ation , an d th e
Jap an Leu kem ia Research Fu n d (to Y. F.).
§Cor resp on d en ce sh ou ld b e ad d ressed to: Yu su ke Fu ru kawa, M. D., Division of
Stem Cell Regu lat ion , Cen ter for Molecu lar Med icin e, Jich i Med ica l Sch ool,
33 11-1 Yaku sh iji, Min am ikawach i-m ach i, Toch igi 32 9-04 98, Jap an . Ph on e:
+8 1-28 5-58-7 400 ; Fax: +8 1-28 5-44-7 501 ; E-m ail: fu ru yu @jich i.ac.jp
1Th e abb reviat ion s u sed are: DMSO, d im eth ylsu lfoxid e; IPTG, isop r op yl-}-
D-th iogalactosid e; GFP, gr een flu or escen t p r otein ; ELISA, en zym e-lin ked
im m u n osor ben t assay; PI, p r op id iu m iod id e; CARD, casp ase-recr u itm en t
d om ain ; IAP, in h ib itor of ap op tosis.
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Abstract
E2 F-1 is cap able of p r om otin g b oth cell cycle p r ogr ession an d ap op tosis. Th e
la tter is im p or tan t for su p p ressin g u n towar d exp an sion of p r olifera tin g cells.
In th is stu d y, we in vest iga ted it s u n d er lyin g m ech an ism s. E2 F-1-in d u ced
ap op tosis was accom p an ied b y casp ase-9 activat ion , an d in h ib ited b y a
sp ecific in h ib itor of casp ase-9 in K562 su blin es over exp r essin g E2 F-1 . E2 F-
1 en h an ced th e exp ression of Ap af-1 with ou t th e cytosolic accu m u lat ion of
cytoch r om e c. Ap af-1-d eficien t m elan om a cell lin es were r esistan t to E2 F-1 ,
in d ica tin g th a t Ap af-1 is an essen tial elem en t of E2 F-1-m ed ia ted ap op tosis.
Fin a lly, we isola ted th e p r om oter r egion of th e Ap af-1 gen e, an d fou n d a
p u ta tive b in d in g site for E2 F. Ch r om at in im m u n op recip itat ion assay
r evealed th a t E2 F-1 b ou n d to Ap af-1 p r om oter u p on E2 F-1 over exp r ession ,
su ggestin g th a t Ap af-1 is u n d er tr an scr ip tion al r egu lat ion of E2 F-1 . Th ese
d ata d em on st ra te a n ovel m ech an ism of ap op tosis in wh ich an in cr ease in
Ap af-1 levels r esu lts in d irect activat ion of casp ase-9 with ou t m itoch on d r ial
d am age, lead in g to th e in it iat ion of a casp ase cascad e.
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Introduction
E2 F is a fam ily of tr an scr ip tion factor s th a t con tr ol G1/ S tr an sit ion of
eu karyot ic cells b y r egu lat in g th e exp ression of var iou s gr owth -r ela ted gen es
(1 ). Target gen es of th e E2 F fam ily in clu d e th ose en cod in g en zym es for DNA
syn th esis (DNA p olym erase |, th ym id in e kin ase, th ym id ylate syn th ase,
d ih yd r ofola te r ed u ctase, an d r ibon u cleotid e r ed u ctase), r egu lators of DNA
r ep licat ion (HsOrc1 , cd c6, MCM5 an d 6 , an d PCNA), an d com p on en ts of th e
cell cycle m ach in er y (cd c2, cd k2, cyclin s A, D1, D2, an d E, E2 F-1 an d -2 , p RB,
p 1 07, an d th e Myc an d Myb fam ilies) (2 ). Am on g E2 F fam ily m em bers, E2 F-1
is u n iq u e in it s ab ility to in d u ce ap op tosis, wh ich m ay p lay a r ole in th e
cellu lar h om eostasis of m u lticellu lar organ ism s (3 -5).
In r esp on se to m itogen ic st im u li, E2 F-1 is in d u ced in qu iescen t cells, an d
p r om otes b oth cell cycle p r ogr ession an d ap op tosis (6 ). Th e ap op tosis-
in d u cin g ability of E2 F-1 is im p or tan t for su p p ressin g u n towar d exp an sion of
p r olifera tin g cells, an d th u s, p r ovid es an in ter n a l d efen se m ech an ism again st
tu m or d evelop m en t . Th e im p or tan ce of E2 F-1-in d u ced ap op tosis u n d er
p h ysiologica l con d it ion s is clear ly d em on st ra ted b y sp on tan eou s
d evelop m en t of m u ltip le tu m ors in m ice lackin g E2 F-1 (7 , 8). In ad d ition , it
h as b een r ep or ted th a t d er egu la tion of E2 F-1 activity con tr ibu tes to en h an ced
p r olifera tion an d r esistan ce to cytotoxic d ru gs in h u m an m elan om a cells (9 ).
E2 F-1 is a lso in volved in th e acceler ated ap op tosis of h em atop oiet ic
p r ogen itor cells, wh ich is con sid er ed th e m ajor m ech an ism of b on e m arr ow
failu r e in m yelod ysp last ic syn d rom e (1 0). In ord er to clar ify th e m olecu lar
b asis of th ese d iseases, it is essen tia l to u n d erstan d th e p r ecise m ech an ism s of
E2 F-1-in d u ced ap op tosis.
Th ere are a few r ep or ts d ea lin g with th e m ech an ism s of E2F-1-m ed ia ted
ap op tosis. Fir st , Bates et a l. (1 1) r ep or ted th a t E2 F-1 in d u ces tr an scr ip tion al
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activat ion of ARF, wh ich stab ilizes p 5 3 b y sequ ester in g MDM2, a u b iqu itin
ligase for p 5 3. Th e stab iliza tion of p 5 3 r esu lts in facilita t ion of p 5 3-
d ep en d en t ap op tosis. Ob viou sly, th is is n ot th e sole m ech an ism of E2 F-1-
in d u ced ap op tosis, b ecau se E2 F-1 can tr igger cell d ea th in p 5 3-d eficien t
tu m ors in clu d in g m ost leu kem ia cell lin es (1 2). p 5 3 is a lso sh own to b e
d isp en sable for E2 F-1-m ed ia ted cell d ea th b y gen etic ap p r oach es (1 3).
Secon d , th e lack of NF-¥B activat ion h as b een d escr ib ed as a m ech an ism of
in cr eased sen sit ivity of E2 F-1-over exp ressin g cells to ap op totic st im u li
th r ou gh th e d ea th r ecep tor p a th ways (1 4). However , th is m ech an ism is n ot
ap p licab le to a ll cell typ es, as E2 F-1 can in d u ce ap op tosis in a d ea th
r ecep tor -in d ep en d en t m an n er in cer tain sett in gs. Most r ecen tly, th r ee
gr ou p s d em on st ra ted th e in volvem en t of th e p 5 3 h om ologu e p 7 3 in E2 F-1-
m ed ia ted ap op tosis (1 5-17 ). Alth ou gh p 7 3 is kn own to cau se ap op totic cell
d ea th , it s u n d er lyin g m ech an ism is st ill u n clear (1 8). In view of th e fact th a t
p 7 3 is a lso a tr an scr ip tion factor , it is p ossib le th a t oth er d irect m ed ia tor s act
d own str eam of p 7 3 in E2 F-in d u ced ap op tosis.
To exp lore th e m ech an ism s of E2 F-1-in d u ced ap op tosis, we establish ed
K562 su blin es wh ich can over exp r ess E2 F-1 con d it ion ally. Usin g th is system ,
we fou n d th a t E2 F-1 was cap able of activat in g casp ase-9 to in it iate th e casp ase
cascad e with ou t m itoch on d r ia l d am age. We fu r th er d em on st ra ted th a t th e
activat ion of casp ase-9 was cau sed b y u p -r egu lation of Ap af-1 , wh ich is a
d irect tr an scr ip tion al ta r get of E2 F-1 .
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Materials and Methods
Cell lines and ce ll culture . Th e h u m an im m atu re m yeloid cell lin e K562
was p r ovid ed b y Dr . Car m en B Lozzio (Un iversity of Ten n essee Med ica l
Cen ter ), an d m ain tain ed in RPMI1 640 m ed iu m su p p lem en ted with 10 % feta l
ca lf seru m . Hu m an m elan om a cell lin es, Malm e-3M an d SK-MEL-5 , were
p u r ch ased fr om ATCC (Manassas, VA). Sim ilar ly, G-361 an d Colo 67 9 were
obtain ed fr om RIKEN Cell Ban k (Tsu ku b a, Jap an ), an d MMAc was p r ovid ed b y
Dr . Hir osi Katayam a (Katayam a Der m atology Clin ic, Gu n m a, Jap an ).
Reagents. All ch em icals were p u r ch ased fr om Sigm a Ch em ical Co. (St. Lou is,
MO) u n less oth erwise stated . Cell-p er m eable flu or om eth ylketon e-
d er ivat ized p ep tid es cor r esp on d in g to sp ecific b in d in g sites for casp ases were
u sed as casp ase in h ib itors (R&D system s, Min n eap olis, MN). Stock solu tion s
were m ad e with d im eth ylsu lfoxid e (DMSO) a t 10 m M an d u sed a t a fin al
con cen t ra tion of 10 0 µM in gr owth m ed iu m (fin a l DMSO con cen tr a tion was
1%). Color im etr ic assay kit s for casp ase-8 an d -9 were a lso p u r ch ased fr om
R&D System s.
Establishment of K5 6 2 sublines which conditionally overexpress
E2 F-1 . K562 su blin es in wh ich fu ll-len gth E2 F-1 is in d u cib le b y th e ad d ition
of isop rop yl-}-D-th iogalactosid e (IPTG) were establish ed u sin g th e LacSwitch
m am m alian exp ression system (Str atagen e, La Jolla , CA). Br iefly, we fir st
tr an sfected th e eu karyot ic Lac r ep ressor -exp r essin g vector p 3 'SS in to th e
im m atu re h em atop oiet ic cell lin e K562 u sin g elect rop or a tion , an d establish ed
stab le tr an sform an ts b y selection with h ygrom ycin . Th e Lac-op era tor -
con ta in in g vector , p OPI3, car r yin g fu ll-len gth E2 F-1 cDNA was th en
tr an sfected in to th e p 3 'SS tr an sform an ts, an d E2 F-1-over exp ressin g clon es
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(r efer red to as Kp Ef1 in th is stu d y) were isola ted in th e p r esen ce of b oth
h ygrom ycin an d G418 . Usin g th e sam e p r oced u re, we establish ed K562
stab le tr an sform an ts wh ich con d ition ally over exp r ess fu ll-len gth E2 F-4 an d
E2 F-6, an d K562 su blin es car r yin g an em p ty p OPI3 vector .
Plasmids. An exp ression p lasm id for E2 F-1 was con str u cted b y in ser t in g
fu ll-len gth E2 F-1 cDNA (n t . 82 -140 8) in to th e Eco RI/ Ecl13 6 II sites of th e
p IRES2-EGFP b icistr on ic m am m alian exp ression vector (Clon tech , Pa lo Alto,
CA). Th e st ru ctu re of th e con st ru ct was ver ified b y r est r iction m ap p in g an d
d irect DNA sequ en cin g.
Isolation and analysis of the Apaf-1 promoter. We sear ch ed th e
h u m an gen om e p r oject d atab ase a t th e San ger Cen tr e (Hin xton , Cam br id ge,
UK), an d fou n d th a t th e 5 ' -u n tr an sla ted r egion of th e Ap af-1 gen e is in clu d ed
in th e h u m an ch r om osom e 12 clon e RP11-453 P16 (DDBJ/ EMBL/ Gen Ban k
accession n u m b er : AC0 261 12). Based on th e in form ation obtain ed , th e
p u ta tive p r om oter r egion of th e Ap af-1 gen e (n t . -11 36 to +3 4; see Fig. 7A for
th e sequ en ce) was am p lified b y PCR, an d in ser ted in to p CAT-basic vector
(Pr om ega, Mad ison , WI) to gen er ate a r ep or ter p lasm id . Pr om oter activity
was d eter m in ed b y tr an sien t tr an sfect ion in to Kp Ef1 cells an d CAT assays as
p r eviou sly r ep or ted (1 9). A sear ch for p u ta tive b in d in g sites of kn own
tr an scr ip tion factor s was p er form ed with th e TFSEARCH p r ogr am
(www.cb r c.jp / r esearch / d b / TFSEARCHJ.h tm l) b ased on th e TRANSFAC
d atab ase (2 0). To d efin e Cp G islan d s, th e r at io of obser ved / exp ected Cp G
was ca lcu la ted accord in g to th e followin g form u la: (Nu m ber of Cp G/ Nu m b er
of G x Nu m ber of C) x N, wh ere N is th e total n u m b er of n u cleotid es in th e
an alyzed sequ en ce (2 1).
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Transient transfection and fluorescence microscopy. Cells were
seed ed a t 5 x 10 5 cells/ m l on cover slip s in 60 -m m cu ltu re d ish es, an d
p lasm id s were tr an sfected with an Effecten e tr an sfect ion r eagen t (Qiagen ,
Valen cia , CA) accord in g to th e m an u factu r er 's in st ru ction s. After 72 h of
tr an sfect ion , cells on cover slip s were stain ed with An n exin -V-Cy3 (MBL,
Nagoya, Jap an ), an d exam in ed u n d er a flu or escen ce m icr oscop e u sin g sp ecific
filter sets to d istin gu ish gr een flu or escen t p r otein (GFP) an d An n exin -V-Cy3;
Olym p u s U-MWIBA for GFP an d U-MWG for Cy3 .
Flow cytometry. Th e cell cycle p r ofile was d eter m in ed b y stain in g DNA
with p r op id iu m iod id e (PI) in p r ep ara tion for flow cytom etr y with th e
FACScan / CellQu est system (Becton -Dickin son , San Jose, CA). Th e size of th e
su b-G1 fr act ion was ca lcu la ted as a p er cen tage with th e Mod FitLT 2 .0 p r ogr am
(Ver ity Software, Top sh am , ME). An n exin -V-p osit ive cells was d etected
accord in g to th e m an u factu r er 's p r otocol.
DNA fragmentation assay. DNA fr agm en ta tion was qu an t ita tively
m easu red b y d etect ion of cytosolic oligon u cleosom e-b ou n d DNA with th e Cell
Death Detect ion en zym e-lin ked im m u n osor ben t assay kit (ELISA) (Boeh r in ger
Man n h eim ) (2 2). Br iefly, cytosolic fr act ion (1 3 ,00 0g su p ern atan t) of th e
cells was u sed as an an t igen sou rce in a san d wich ELISA with a p r im ary an t i-
h iston e an t ibod y coated to m icr ot iter p lates an d a secon d ar y an t i-DNA an -
tibod y con ju gated with p er oxid ase. Dou ble absor ban ce a t 40 5 an d 49 5 n m
(A4 05n m / A49 5n m ) was m easu red again st su bst r ate solu tion as a b lan k. Th e
d ata a re exp ressed as fold -in cr ease over th e valu e of u n t rea ted
exp on en tia lly-growin g cells.
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Preparation of cytosolic prote ins. Cytosolic p r otein s were isola ted
accord in g to th e m eth od of Yan g et a l (2 3). Br iefly, cell p ellets were wash ed
on ce with ice-cold p h osp h ate-b u ffer ed sa lin e an d r esu sp en d ed in five
volu m es of b u ffer A (2 0 m M Hep es-KOH, p H 7 .5 , 10 m M KCl, 1 .5 m M MgCl2, 1
m M EDTA, 1m M EGTA, 1 m M d ith ioth r eitol, an d 0 .1 m M
p h en ylm eth ylsu lfon ylflu or id e) con ta in in g 25 0 m M su crose. Th e cells were
h om ogen ized with a Teflon h om ogen izer , an d cen tr ifu ged twice a t 75 0g for
10 m in a t 4°C. Th e su p ern atan ts were cen tr ifu ged a t 10 ,000g for 15 m in a t
4°C, an d th e r esu ltin g m itoch on d r ia p ellets were r esu sp en d ed in b u ffer A
con ta in in g 25 0 m M su crose. Th e su p ern atan ts of th e 10 ,000g sp in were
fu r th er cen tr ifu ged a t 10 0,00 0g for 1 h a t 4°C, an d th e r esu lt in g su p ern atan ts
were u sed as cytosol fr act ion s.
Confocal laser microscopy. Cells were collected on glass slid es u sin g a
Cytosp in cen tr ifu gator (Sh an d on Scien t ific, Ch esh ir e, En glan d ), an d fixed in
4% p ar afor m ald eh yd e in p h osp h ate-b u ffer ed sa lin e. In situ stain in g for
cytoch r om e c was car r ied ou t as p r eviou sly r ep or ted (2 4) u sin g an t i-
cytoch r om e c m on oclon al an t ibod y (6 H2.B4; Ph arm in gen , San Diego, CA) an d
Alexa 48 8-con ju gated an t ibod y to m ou se im m u n oglob u lin (Molecu lar Pr obes,
Eu gen e, OR) as p r im ary an d secon d ary an t ibod ies, r esp ectively. For co-
labelin g of m itoch on d r ia , we u sed two d iffer en t r ab bit p olyclon al an t ibod ies:
an t i-OPA1 an d an an t ibod y again st 62 -kDa m itoch on d r ia l p r otein (AB3 598 ;
Ch em icon In tern a tion al, Tem ecu la , CA). An ti-OPA1 was gen er ated b y
im m u n izin g r ab bit with th e GTPase d om ain of OPA1 (2 5), a d yn am in -related
p r otein localized a t th e in ter m em b ran e sp ace of m itoch on d r ia (Satoh , M. an d
En d o, H., m an u scr ip t in p r ep ara tion ). Goat an t ibod y to r ab bit
im m u n oglob u lin con ju gated with Cy3 (Am er sh am Ph arm acia Biotech ,
Piscataway, NJ) was u sed as a secon d ary an t ibod y. In situ stain in g for Bax
was p er form ed sim ilar ly u sin g an t i-Bax (clon e 3 ; Tr an sd u ction Labor ator ies)
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an d an t i-OPA1 an t ibod ies. Con focal m icr oscop ic an alysis was d on e as
p r eviou sly d escr ib ed (2 6).
Measurement of mitochondrial transmembrane potential.
Mitoch on d r ial tr an sm em b ran e p oten t ial was m easu red with a MitoCap tu r e
m itoch on d r ia l ap op tosis d etect ion kit (MBL, Nagoya, Jap an ). MitoCap tu r e is
a r h od am in e d er ivat ive, wh ich aggregates in m itoch on d r ia an d gen er ates a
b r igh t r ed flu or escen ce in in tact cells (2 7). Wh en th e m itoch on d r ia l
m em br an e p oten t ial is a ltered , MitoCap tu r e d ye r em ain s in th e cytop lasm
with ou t aggregation , givin g off a gr een flu or escen ce. Th e flu or escen t sign als
were d istin gu ish ed b y flow cytom etr y u sin g PI (FL-2) an d FITC (FL-1)
ch an n els.
Western blotting. Im m u n oblot t in g was car r ied ou t accord in g to th e
stan d ar d m eth od u sin g th e followin g an t ibod ies: an t i-E2F-1 (C-20; San ta Cr u z
Biotech n ology, San ta Cr u z, CA), an t i-}-actin (Ab -1; On cogen e Scien ce,
Un ion d ale, NY), an t i-casp ase-8 (B9 -2; Ph arm in gen ), an t i-casp ase-9 (6 808 6E;
Ph arm in gen ), an t i-cleaved casp ase-9 (# 950 1; Cell Sign allin g Tech n ology,
Bever ly, MA), an t i-casp ase-2 (clon e 35 ; Tr an sd u ction Labor ator ies, Lexin gton ,
KY), an t i-casp ase-6 (B9 3-4; Ph arm in gen ), an t i-casp ase-3 (clon e 19 ;
Tr an sd u ction Labor ator ies), an t i-cleaved casp ase-3 (# 966 1; Cell Sign allin g
Tech n ology), an t i-casp ase-7 (B9 4-1; Ph arm in gen ), an t i-Ap af-1 (NT; Millen iu m
Biotech n ology, Ram on a, CA), an t i-cytoch r om e c (7 H8.2C12 ; Ph arm in gen ),
an t i-p 5 3 (clon e 80 ; Tr an sd u ction Labor ator ies), an d an t i-Bax (clon e 3 ;
Tr an sd u ction Labor ator ies).
Northern blotting. We isola ted total cellu lar RNA u sin g cesiu m ch lor id e
u ltr acen tr ifu gation , an d p er form ed RNA b lot t in g as p r eviou sly d escr ib ed (1 9).
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Th e followin g cDNA fr agm en ts were gen er ated b y r ever se tr an scr ip tion -
p olym erase ch ain r eaction b ased on p u blish ed sequ en ces, an d u sed as p r obes
after p u r ificat ion (am p lified r egion s are sh own in p ar en th eses): Ap af-1 (n t .
20 99-3 609 ) (2 8), Nod -1 (n t . 13 9-60 0) (2 9), an d Sm ac (n t . 22 5-66 5) (3 0).
Chromatin immunoprecipitation assay. Ch r om at in im m u n op recip i-
ta tion assay was p er form ed accord in g to th e m eth od of Mor en o et a l. (3 1)
with m in or m od ificat ion s. Ap p roxim ately 5 x 10 7 cells were fixed in gr owth
m ed iu m with 1% form ald eh yd e a t r oom tem p er atu r e for 10 m in . After
wash in g with p h osp h ate-b u ffer ed sa lin e, cells were d isr u p ted in cell lysis
b u ffer (5 m M PIPES, p H 8 , 85 m M KCl, an d 0 .5 % NP-4 0) con ta in in g p r otease
in h ib itor com p lex (Roch e Diagn ost ics, Man n h eim , Ger m an y) on ice for 5 m in .
Th en , n u clei were collected b y m icr ocen tr ifu gat ion , r esu sp en d ed in n u clei
lysis b u ffer (5 0 m M Tr is HCl, p H 8 , 10 m M EDTA, an d 1% SDS) con ta in in g
p r otease in h ib itor com p lex, an d son ica ted on ice a t 10 secon d p u lses 6 t im es
to an average len gth of 50 0-10 00 b p . Sam p les were cen tr ifu ged a t 14 ,500
r p m for 10 m in , an d th e su p ern atan ts were stored a t -70 °C. Th e ch r om atin
su sp en sion s (1 00 µl each ; an equ iva len t of 5 x 10 6 cells) were ad d ed to IP
d ilu t ion b u ffer (1 67 m M NaCl, 16 .7 m M Tr is HCl, p H 8 , 1 .2 m M EDTA, 0 .0 1%
SDS, 1 .1 % Tr iton X-100, 20 µg/ m l sa lm on sp er m DNA, an d 50 µg/ m l yeast
tRNA) con ta in in g 5 µg of on e of th e followin g r ab bit p olyclon al an t ibod ies (a ll
p u r ch ased fr om San ta Cr u z Biotech n ology excep t an t i-c-kit ): an t i-E2F-1 (C-20),
an t i-E2F-2 (C-20), an t i-E2F-3 (C-18), an t i-E2F-4 (C-108 ), an t i-E2F-5 (E-19),
an d an t i-c-kit (On cogen e Research , Boston , MA). After in cu b at ion a t 4 °C for
2 h , th e m ixtu res were fu r th er r ocked with 20 µl of goat an t i-r ab bit IgG-
con ju gated m agn et ic b ead s (Milten yi Biotec, Au b u r n , CA) for 1 h , an d th e
im m u n e com p lexes were r ecover ed in a m agn et ic sep ara tor . Th e
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im m u n op recip itan ts were wash ed 3 t im es each with fou r d iffer en t b u ffers,
followed b y elu t ion with 50 m M NaHCO3 an d 1% SDS. Th e elu en ts were
h eat-t rea ted , eth an ol-p recip itated , d igested with p r otein ase K, ext racted with
p h en ol/ ch loroform , an d fin ally r esu sp en d ed in 30 µl of TE (p H 8). An
aliqu ot of 3 µl of th e fin al su sp en sion was su bjected to sem iqu an t ita tive PCR as
p r eviou sly d escr ib ed (3 2).
Results
Apoptosis is a relative ly late-onset event in E2 F-1 -overexpressing
cells
To in vest iga te th e m ech an ism s of E2 F-1-in d u ced ap op tosis, we establish ed
a K562 su blin e (Kp Ef1) in wh ich fu ll-len gth E2 F-1 is in d u cib le b y th e ad d ition
of IPTG u sin g th e LacSwitch m am m alian exp ression system . Up on ad d ition of
IPTG, E2 F-1 p r otein in cr eased m ore th an 5-fold after 6 h of cu ltu r e,
con firm in g th e in d u cib le over exp r ession of E2 F-1 (Fig. 1 , lower p an el) . Usin g
th is cell lin e, we fir st exam in ed th e effects of E2 F-1 on th e cell cycle p r ofile
an d ap op tosis b y flow cytom etr y. As a con tr ol, we sim u ltan eou sly an alyzed a
K562 stab le tr an sform an t car r yin g an em p ty vector (Mock), wh ich d id n ot
over exp r ess E2 F-1 in th e p r esen ce of IPTG (Fig. 1 , u p p er p an el) . At 12 h after
th e ad d ition of IPTG, a sign ifican t in cr ease in th e n u m b er of cells in S p h ase
was obser ved in an E2 F-1-over exp ressin g clon e b u t n ot in m ock-t ran sfected
cells, followed b y a r ela tive in cr ease in th e n u m b er of cells in G2/ M p h ase a t
24 h . After 72 h , ap op tosis, as ju d ged b y th e ap p earan ce of a su b-G1 fr act ion
in DNA h istogr am s, was r ead ily in d u ced in Kp Ef1 cells, wh ereas ap op tosis was
n ot obser ved in th e con tr ol (Fig. 1) an d K562 cells over exp r essin g eith er E2 F-4
or E2 F-6 (d a ta n ot sh own ). Th e in d u ct ion of ap op tosis b y E2 F-1 was
con firm ed b y oth er m eth od s in clu d in g DNA fr agm en ta tion ELISA an d
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An n exin -V stain in g (d a ta n ot sh own ). Th ese r esu lts su ggest th a t , am on g E2 F
fam ily m em bers, E2 F-1 sp ecifically in d u ces cell cycle p r ogr ession an d
ap op tosis sim u ltan eou sly b u t with d iffer en t kin etics, an d th e in d u ct ion of
ap op tosis is a r ela tively la te-on set even t.
Involvement of caspases-9 , -6 , and -3 in E2 F-1 -induced apoptosis
To d eter m in e th e p a th ways of apop totic sign al tr an sd u ction activated b y
E2 F-1, we exam in ed th e effects of sp ecific in h ib itors of casp ases on E2 F-1-
in d u ced ap op tosis. Kp Ef1 cells were p r e-tr ea ted with cell-p er m eable
casp ase-bin d in g p ep tid es, an d th en cu ltu red in th e p r esen ce of IPTG. Th e
p er cen tage of cells u n d ergoin g ap op tosis was m easu red b y ca lcu la tin g su b-G1
fr act ion s on DNA h istogr am s (Fig. 2A an d B) an d DNA fr agm en ta tion ELISA
(Fig. 2C) after 72 h . E2 F-1-in d u ced ap op tosis was sign ifican tly su p p ressed b y
a gen er al casp ase in h ib itor VAD as well as sp ecific in h ib itors for casp ases-9 , -6 ,
an d -3 .
To con firm th e activat ion of casp ases d u r in g E2 F-1-m ed ia ted ap op tosis,
we exam in ed th e exp ression an d cleavage of casp ases in E2 F-1-over exp ressin g
cells b y im m u n ob lottin g. As sh own in Fig. 3 , th e am ou n t of p r ocasp ase-9 (4 8
kDa) d ecr eased after 24 of cu ltu re with IPTG in Kp Ef1 cells with active
fr agm en ts of casp ase-9 (3 7 kDa/ 1 7 kDa) ap p ear in g after 48 h . We
sim u ltan eou sly m easu red th e activity of casp ase-9 u sin g color im etr ic assay
kit s, an d fou n d th a t it in d eed in cr eased in E2 F-1-over exp ressin g cells in
accord with th e p r ocessin g of p r ocasp ase-9 (d a ta n ot sh own ). With sim ilar
kin etics, p r ocasp ases-6 (3 4 kDa) an d -3 (3 2 kDa) were p r ocessed in to a la rge
active su bu n it (1 7 kDa) in th ese cells. No ap p ar en t effect on oth er in it ia tor
(casp ase-8 an d -2) an d execu t ion er (casp ase-7) casp ases was obser ved .
Th ese r esu lts su ggest th a t E2 F-1 activates casp ase-9 , wh ich in tu r n p r ocesses
casp ases-6 an d -3 to in it ia te a casp ase cascad e, lead in g to ap op totic cell d ea th .
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E2 F-1 -induced caspase-9 activation coincides with up-regulation of
Apaf-1
Th e la te on set of casp ase-9 activat ion in d icates th a t E2 F-1 is n ot d irectly
in volved in th e p r ocess b u t can in it ia te a casp ase cascad e th r ou gh
tr an scr ip tion al r egu lat ion of th e m olecu les th a t activate casp ase-9 . Th e
can d id ates for su ch m olecu les in clu d e CARD-con tain in g ad ap ter s, Ap af-1 (2 8)
an d Nod -1/ CARD4 (2 9, 33 ), an d an IAP-bin d in g factor Sm ac/ DIABLO (3 0, 34 ).
We scr een ed th e exp ression of th ese gen es in E2 F-1-over exp ressin g clon es b y
Nor th er n b lot t in g, an d fou n d th a t E2 F-1 in cr eased th e abu n d an ce of Ap af-1
m RNA after 24 h of cu ltu re (Fig. 4A). In ad d ition , Nod -1/ CARD4 m RNA was
tr an sien t ly u p -r egu lated in an E2 F-1-d ep en d en t m an n er , an d th e level of
Sm ac/ DIABLO tr an scr ip t in cr eased over t im e in b oth m ock-t ran sfected an d
E2 F-1-over exp ressin g cells. To ver ify th e in d u ction of Ap af-1 b y E2 F-1 , we
exam in ed th e exp r ession of Ap af-1 p r otein in E2 F-1-over exp ressin g clon es b y
Wester n b lot t in g. Ap af-1 p r otein was b ar ely d etectab le in u n t rea ted Kp Ef1
cells, b u t was r ead ily in d u ced 48 h after th e ad d ition of IPTG (Fig. 4B).
It h as b een sh own th a t Ap af-1 activates casp ase-9 b y in ter act in g with
p r ocasp ase-9 an d p r om otin g it s oligom er iza tion an d cleavage in th e p r esen ce
of d ATP an d cytoch r om e c (3 5 , 36 ). Wh en m itoch on d r ia a re d am aged b y
cer tain ap op totic st im u li su ch as an t ican cer d ru g tr eatm en t , cytoch r om e c is
r eleased fr om th e m itoch on d r ia in to th e cytosol, wh ere it tr igger s
oligom er iza tion of Ap af-1 an d activat ion of casp ase-9 (2 3 , 37 ). We
th erefor e exam in ed wh eth er E2 F-1 in d u ces cytop lasm ic tr an slocat ion of
cytoch r om e c in associat ion with th e in cr ease in Ap af-1 . Cytosolic p r otein s
were ser ia lly isola ted fr om E2 F-1-over exp ressin g cells, an d su bjected to
im m u n ob lottin g for th e evalu a tion of cytoch r om e c con ten t. As sh own in Fig.
4B, E2 F-1 d id n ot in cr ease th e am ou n t of cytoch r om e c in th e cytosol u p to 96
h of cu ltu re, wh ereas ad r iam ycin im m ed ia tely in d u ced a cytosolic
accu m u lat ion of cytoch r om e c in th ese cells. To corr obor ate th e r esu lt of
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im m u n ob lott in g, we exam in ed th e in tr acellu lar d istr ib u t ion of cytoch r om e c
u sin g con focal laser m icr oscop y. In u n t rea ted Kp Ef1 cells, cytoch r om e c was
stain ed in a p er in u clear p u n cta te p a ttern in d icat ive of in tr am itoch on d r ial
localizat ion (Fig. 4C, Con t rol). In tr am itoch on d r ia l localizat ion of
cytoch r om e c was con firm ed b y co-labellin g with m itoch od r ia l m arker s.
Du r in g ap op tosis tr igger ed b y ad r iam ycin , th e d istr ib u t ion of cytoch r om e c
b ecam e com p letely d iffu se, r eflectin g th e tr an slocat ion of cytoch r om e c in to
th e cytosol (Fig. 4C, ADR). In con tr ast , th e p a ttern of cytoch r om e c
d istr ib u t ion r em ain ed th e sam e as th a t of th e Con t rol in E2 F-1-over exp ressin g
Kp Ef1 cells u p to 48 h of E2 F-1 in d u ct ion (Fig. 4C, E2 F-1). Most if n ot a ll cells
r etain ed th e id en t ical p a ttern even after 72 h of cu ltu re wh en th ere was th e
in it ia l sign s of cell d ea th , in d icat in g th a t cytoch r om e c t r an sloca tion is n ot a
p r erequ isite for ap op tosis followin g E2 F-1 activat ion . Next , we m easu red
m itoch on d r ia l tr an sm em b ran e p oten t ial u sin g a r h od am in e-d er iva tive d ye in
ord er to assess m itoch on d r ia l d am age. As sh own in Fig. 4D, m itoch on d r ia l
tr an sm em b ran e p oten t ial was n ot sever ely affected d u r in g E2 F-1-in d u ced
ap op tosis, a lth ou gh it was lost in d ead cells a t d ay 3 . Th ese d ata su ggest th a t
th e E2 F-1-in d u ced u p -r egu lat ion of Ap af-1 is n ot a d irect r esu lt of
m itoch on d r ia l d am age or m assive cytosolic accu m u lat ion of cytoch r om e c. It
is h igh ly likely th a t Ap af-1 m ed ia tes E2 F-1-in d u ced ap op tosis as a
tr an scr ip tion al ta r get of E2 F-1 .
Fin a lly, we in vest iga ted wh y E2 F-1 d id n ot in d u ce cytop lasm ic
tr an slocat ion of cytoch r om e c in Kp Ef1 cells. Up on DNA d am age, p 5 3 is
stab ilized b y p h osp h or ylat ion an d in d u ces tr an scr ip tion al activat ion of Bax,
wh ich in tu r n acts on m itoch on d r ia l m em br an e to tr igger th e r elease of
cytoch r om e c (3 8). We th erefor e exam in ed th e exp ression of p 5 3 an d Bax in
Kp Ef1 cells. As sh own in Fig. 5A, p 5 3 was n ot d etectab le in th is cell lin e even
after th e in d u ct ion of E2 F-1 over exp r ession . We fou n d th a t th e lack of p 5 3
p r otein was a ttr ib u tab le to th e d efect of m RNA exp ression cau sed b y a p oin t
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m u tat ion of th e p 5 3 gen e (d a ta n ot sh own ). Con sisten t with th e absen ce of
p 5 3, th e am ou n t of Bax was n ot in cr eased b y E2 F-1 in Kp Ef1 cells (Fig. 5A).
Fu r th er m ore, we a lso d em on st ra ted th e absen ce of m itoch on d r ia l
tr an slocat ion of Bax u sin g con focal m icr oscop y. Bax was d etected in b oth
cytop lasm an d n u cleu s in u n t rea ted Kp Ef1 cells, wh ereas it accu m u lated in
th e cytop lasm an d b ecam e co-localized with m itoch on d r ia a fter ad r iam ycin
tr eatm en t (Fig. 5B). Th e ch an ge in Bax d istr ib u t ion was n ot obser ved after
E2 F-1 over exp r ession u p to 72 h , in d icat in g th a t Bax is n ot d irectly in volved in
cell d ea th cau sed b y E2 F-1 . Th ese d ata m ay exp lain wh y cytoch r om e c
r elease d ose n ot occu r followin g E2 F-1 over exp r ession in th is cell lin e.
Apaf-1 -defective ce lls are relative ly resistant to E2 F-1 -induced
apoptosis
To con firm th e in volvem en t of Ap af-1 in E2 F-1-in d u ced ap op tosis, we
exam in ed wh eth er E2 F-1 cou ld elicit ap op tosis in Ap af-1-d efect ive cells. For
th is p u r p ose, we fir st scr een ed th e exp ression of Ap af-1 in h u m an m elan om a
cell lin es, in som e of wh ich Ap af-1 is in act ivated d u e to h yp erm eth yla tion of
an en h an cer elem en t of th e Ap af-1 gen e (3 9), b y im m u n ob lottin g. Am on g 5
cell lin es exam in ed , th e Ap af-1 level was ver y low in G-361 an d b elow th e
d etect ion lim it in SK-MEL-5 (Fig. 6A). E2 F-1 was tr an sien tly over exp r essed in
th ese cell lin es b y tr an sfect in g a b icistr on ic vector th a t in d u ces sim u ltan eou s
exp ression of E2 F-1 an d GFP. At 72 h after tr an sfect ion , we d eter m in ed th e
p er cen tage of GFP-p osit ive cells u n d ergoin g ap op tosis b y flu or escen ce
m icr oscop y. Th e d ata fr om 3 in d ep en d en t exp er im en ts a re su m m ar ized in
Fig. 6B. Th e in d u cib ility of ap op tosis b y E2 F-1 was sign ifican tly lower in th e
two cell lin es with lit t le or n o exp ression of Ap af-1 th an in th e oth er th r ee
m elan om a cell lin es with in tact Ap af-1 exp ression . Th ere was n o d iffer en ce
in th e p r op or tion of ap op totic fr act ion am on g 5 cell lin es wh en an em p ty
p IRES2-EGFP vector was tr an sfected (Fig. 6B). Th ese r esu lts in d icate th a t
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E2 F-1-in d u ced ap op tosis is a t least in p ar t m ed ia ted th r ou gh Ap af-1-
d ep en d en t p a th ways.
E2 F-1 binds to a putative E2 F-binding site of Apaf-1 promoter
Fin a lly, we exam in ed wh eth er Ap af-1 is d irectly u n d er th e tr an scr ip tion al
r egu lat ion of E2 F-1 . To th is en d , we isola ted th e p u ta tive p r om oter r egion of
th e Ap af-1 gen e b y a d atab ase-or ien ted ap p r oach (Fig. 7A). Th e p r om oter
activity an d E2 F-1-r esp on siven ess of th e isola ted r egion were con firm ed b y
tr an sien t tr an sfect ion of p CAT-basic vector con ta in in g PCR-am p lified
sequ en ces b etween n t. -1136 an d +3 4 in to Kp Ef1 cells (d a ta n ot sh own ). Th is
r egion was fou n d to b e GC-r ich ; %(G+C) of th e en t ir e sequ en ce is 66 % an d th e
r at io of obser ved / exp ected Cp G is 0 .7 9 . In p ar ticu lar , th e sequ en ce b etween
n t. -31 6 an d -10 3 sh ows an ext rem ely h igh (G+C) con ten t (7 9%) with an
obser ved / exp ected Cp G r at io of 1 .2 1 , in d icat in g th a t th is r egion con stitu tes a
Cp G islan d or is p ar t of a la rge Cp G islan d . As a typ ical TATA m otif is n ot
p r esen t in th e Ap af-1 p r om oter , it is likely th a t m eth ylat ion of th e Cp G islan d
serves as th e m ajor m ech an ism of tr an scr ip tion al r egu lat ion of Ap af-1 . A
sear ch for p u ta tive b in d in g sites of kn own tr an scr ip tion factor s r evealed th a t
th ere is a con sen su s sequ en ce for E2 F b etween n t. -53 2 an d -52 6 (Fig. 7A). In
ad d ition , th ere are sever al GC b oxes, som e of wh ich are located arou n d th e
Cp G islan d , an d two p 5 3-b in d in g r egion s a t -76 5 to -73 9 an d -60 4 to -57 2.
We th en exam in ed wh eth er E2 F-1 b in d s to th e E2 F con sen su s site u p on th e
tr an scr ip t ion al activat ion of Ap af-1 cau sed b y E2 F-1 over exp r ession in v ivo
u sin g th e ch r om atin im m u n op recip itat ion assay. As sh own in Fig. 7B, E2 F-1
b ou n d to th e Ap af-1 p r om oter in Kp Ef1 cells 24 h after th e in d u ction , wh ereas
n o b in d in g of oth er E2 F fam ily p r otein s was obser ved excep t for a sm all
am ou n t of PCR p r od u ct d etected in E2 F-4 im m u n op recip itan ts fr om u n t rea ted
Kp Ef1 cells. Th ese r esu lts su ggest th a t E2 F-1 d irectly r egu lates tr an scr ip tion
of th e Ap af-1 gen e in a p osit ive m an n er a t least in E2 F-1-over exp ressin g cells.
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Discussion
In th is stu d y, we in vest iga ted th e m ech an ism s of E2 F-1-in d u ced ap op tosis
u sin g K562 su blin es wh ich can over exp r ess E2 F-1 con d it ion ally. Wh en E2 F-
1 was over exp r essed in th ese cells, ap op tosis was r ead ily in d u ced after 72 h ,
followin g a tr an sien t in cr ease in S p h ase a t 12 h . Th is con firm s th e n otion
th a t E2 F-1 p r om otes cell cycle p r ogr ession an d ap op tosis sim u ltan eou sly to
su p p ress u n towar d exp an sion of p r olifera tin g cells (3 -6 , 12 ).
We fou n d th a t E2 F-1 was cap able of activat in g casp ase-9 to in it ia te a
casp ase cascad e in th e absen ce of m itoch on d r ia l d am age. Th e activat ion of
casp ase-9 is u su ally tr igger ed b y th e r elease of cytoch r om e c fr om d am aged
m itoch on d r ia in r esp on se to cer tain ap op totic st im u li su ch as an t ican cer
d ru gs, u ltr aviolet r ad iat ion , an d seru m d ep r iva tion (4 0). Su b seq u en tly,
cytoch r om e c b in d s to Ap af-1 an d , in association with ad en in e n u cleotid es,
facilita tes a con form ation al ch an ge of Ap af-1 to exp ose it s CARD d om ain (3 5 ,
36 ). Ap af-1 oligom er izes th r ou gh th e exp osed CARD d om ain , an d r ecr u it s
p r ocasp ase-9 b y a h om op h ilic in ter act ion in volvin g CARDs, wh ich r esu lts in
ca ta lyt ic au toactiva tion of casp ase-9 . However , E2 F-1 activates casp ase-9
th r ou gh a d iffer en t p a th way; it b yp asses th e tr an slocat ion of cytoch r om e c
fr om m itoch on d r ia to cytosol, an d d irectly in d u ces au toactiva tion of
casp ase-9 via th e in cr ease in in tr acellu lar Ap af-1 levels. Th e in volvem en t of
oth er d irect activators of casp ase-9 , in clu d in g a CARD-con tain in g ad ap ter
Nod -1/ CARD4 (2 9, 33 ) an d an IAP-bin d in g factor Sm ac/ DIABLO (3 0, 34 ), is to
b e d eter m in ed b ecau se E2 F-1 a lso m od u la te th e exp ression of th ese m olecu les.
Up -r egu la tion of Apaf-1 is a lso obser ved d u r in g ap op tosis cau sed b y th e
ad en ovir al on cogen e E1 A, b u t it occu r s as a secon d ary even t of cytop lasm ic
accu m u lat ion of cytoch r om e c, an d th erefor e, is d ist in ct fr om th a t of E2 F-1
(4 1). Alth ou gh th e m ech an ism b y wh ich casp ase-9 is activated b y th e
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in cr ease in Ap af-1 is a t p r esen t u n clear , a h igh con cen t ra tion of Ap af-1 m ay
in cr ease th e p r obability of p h ysica l in ter act ion b etween p r ocasp ase-9
m olecu les even in th e absen ce of m itoch on d r ia l d am age, r esu lt in g in
oligom er iza tion an d su bsequ en t au toactiva tion of p r ocasp ase-9 . Th is
h yp oth esis is su bstan t iated b y stu d ies in wh ich forced exp ression of Ap af-1
solely p r om oted ap op tosis in h u m an m yeloid leu kem ia cell lin es (4 2 , 43 ).
Fin a lly, we p r ovid e evid en ce th a t Ap af-1 is a d irect tr an scr ip tion al ta r get of
E2 F-1 b y an alyzin g th e 5 ' -u n tr an sla ted r egion s of th e Ap af-1 gen e. Th is
fin d in g is con sisten t with th e r esu lts of a r ecen t effor t to id en t ify E2 F-
r esp on sive gen es b y global an a lysis of gen e exp ression u sin g h igh -d en sity
oligon u cleotid e ar r ays; Ap af-1 is on e of th e n ewly id en t ified E2 F-1-in d u cib le
gen es (4 4). In terest in gly, E2 F-2 an d E2F-3 d id n ot u p -r egu late Ap af-1 in th is
an alysis, wh ich is con sisten t with th e fact th a t E2 F-1 h as th e st ron gest ab ility
in p r om otin g ap op tosis am on g E2 F fam ily m em bers (5 ). However , th ere are
som e r ep or ts su ggestin g th a t oth er E2 F p r otein s, esp ecia lly E2 F-2 an d E2 F-4 ,
are a lso cap able of in d u cin g ap op tosis (4 5 , 46 ). Th e m ech an ism s u n d er lyin g
ap op tosis in d u ced b y E2 F-2 an d E2 F-4 m ay b e d iffer en t fr om th a t of E2 F-1 ,
b ecau se E2 F-2 an d E2 F-4 can n ot tr an sact iva te Ap af-1 (4 4 , 47 ). As E2 F-4 was
n ot able to in d u ce ap op tosis in ou r system (d a ta n ot sh own ), it is likely th a t
E2 F-4-m ed ia ted ap op tosis in volves p 5 3-d ep en d en t p a th ways.
Th e st ru ctu re of th e Ap af-1 p r om oter is ch ar acter ist ic; it lacks eith er a
typ ical TATA m otif or in it ia tor elem en ts, an d is d evoid of can on ica l b in d in g
sites for kn own tr an scr ip tion factor s excep t E2 F, Sp -1 , an d p 5 3. In stead , th e
Ap af-1 p r om oter sh ows an ext rem ely h igh (G+C) con ten t (> 60 %) with an
obser ved / exp ected Cp G r at io of gr ea ter th an 0 .8 , wh ich fu lfills th e
r eq u ir em en t of a Cp G islan d (2 1). Th ese featu r es su ggest th a t m eth ylat ion of
th e Cp G islan d serves as th e m ajor m ech an ism of tr an scr ip tion al r egu lat ion of
Ap af-1 . In d eed , Ap af-1 exp ression is d efective in ap p r oxim ately h alf of
m elan om a cell lin es, wh ich is r ever sed b y 5-aza-2 '-d eoxycyt id in e, an in h ib itor
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of m eth ylat ion (3 9). Th is im p lies th a t th e Ap af-1 gen e is silen ced b y
m eth ylat ion of th e Cp G islan d , an d th e m od u la tion of th e m eth ylat ion statu s
a lleviates th e r ep ression to in d u ce Ap af-1 tr an scr ip tion . In th is stu d y, we
d em on st ra ted th e b in d in g of E2 F-1 to th e Ap af-1 p r om oter in E2 F-1-
over exp r essin g cells u sin g th e ch r om atin im m u n op recip itat ion assay. Th is
r esu lt in d icates th a t E2 F-1 p ar ticip ates in th e d er ep r ession of th e Ap af-1 gen e
p r obably b y affect in g p r om oter m eth ylat ion . Th e m ech an ism s b y wh ich
E2 F-1 m od u la tes th e m eth ylat ion statu s of th e Ap af-1 p r om oter a re cu r r en t ly
u n d er in vest iga tion in ou r labor ator y.
Wh ile com p ilin g th is m an u scr ip t , th e a lm ost id en t ical obser vation was
p u blish ed b y Mor on i et a l (4 8). Mor eover , For tin et a l. (4 9) r ep or ted th a t
p 5 3 u p -r egu lates Ap af-1 as a d irect tr an scr ip tion al ta r get d u r in g n eu r on al cell
d ea th . Notably, th e form er stu d y clear ly d em on st ra ted th a t cytop lasm ic
tr an slocat ion of cytoch r om e c is accom p an ied b y E2F-1-in d u ced ap op tosis,
wh ich con tr ad icts ou r p r esen t fin d in g. Th is con tr ad ict ion m ay stem fr om th e
d iffer en ce in p 5 3 statu s of h ost cells u sed in each stu d y; p 5 3 exp ression is
d efective in ou r cell lin e, wh ereas Mor on i et a l. u sed p r im ary h u m an
fib rob lasts an d U2OS cells, b oth of wh ich p ossess n orm al p 53 . Alth ou gh ou r
fin d in g m ay b e u n iq u e to p 5 3-d eficien t cells, it in stead m akes two im p or tan t
p oin ts clear . Fir st , E2 F-1 can tr an sact iva te th e Ap af-1 gen e in th e absen ce of
p 5 3. Th is is clin ica lly im p or tan t b ecau se m ost can cer cells lost th e fu n ct ion
of p 5 3. Secon d , it is su ggested th a t th e E2 F-1/ Ap af-1 p a th way (gr owth -
associated in tr in sic r ou te) wor ks in p ar allel with th e p 5 3/ Bax/ cytoch rom e c
p ath way (DNA d am age-t r igger ed ext r in sic r ou te) in p r om otin g ap op totic cell
d ea th .
In con clu sion , th ese d ata d em on st ra te a n ovel m ech an ism of ap op tosis in
wh ich an in cr ease in Ap af-1 levels in d u ced b y E2 F-1 r esu lts in d irect
activat ion of casp ase-9 with ou t m itoch on d r ial d am age, lead in g to th e in i-
t iat ion of a casp ase cascad e. As b oth E2 F-1 an d Ap af-1 are fr eq u en t ly
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d er egu la ted in var iou s p a th ologic con d it ion s, th is fin d in g m ay con tr ibu te to a
b et ter u n d erstan d in g of th e p a th op h ysiology of m an y d iseases.
Ack n owled gm en ts - We th an k Drs. Tad ash i Mayu m i, Kiyosh i Kawakam i, an d
Keiko Iked a (Jich i Med ica l Sch ool) for h elp fu l d iscu ssion s. We are gr atefu l to
Ms. Yu kiko Fu ku d a-Kam iyosh ih ar a for excellen t tech n ica l assistan ce.
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Figure Legends
Fig. 1 E2 F-1 overexpression induces apoptosis in K5 6 2 ce lls.
A K562 su blin e, Kp Ef1, wh ich over exp r esses fu ll-len gth E2 F-1 in th e p r esen ce
of IPTG (E2 F-1) an d a m ock-t ran sfected con tr ol (Mock) were cu ltu red in th e
p r esen ce of 5 m M IPTG for 72 h , an d su bjected to im m u n ob lott in g for E2 F-1
exp ression an d cell cycle an alysis a t th e in d ica ted t im e p oin ts. Th e p osit ion s
of en d ogen ou s E2 F-1 (En d ) an d tr an sgen e (Tran s) a re sh own b y ar r ows. Th e
m olecu lar sizes a re sligh t ly d iffer en t b etween en d ogen ou s an d tr an sgen ic
E2 F-1 d u e to p h osp h or ylat ion an d acetylat ion . Data sh own are
r ep resen ta tive of m u ltip le in d ep en d en t exp er im en ts u sin g d iffer en t clon es
establish ed a t th e sam e tim e.
Fig. 2 Involvement of caspases-9 , -6 and -3 in E2 F-1 -induced
apoptosis.
(A) Kp Ef1 cells were seed ed a t 1 x 10 5 cells/ m l with var iou s casp ase in h ib itors
(1 00 µM fin al) or DMSO (solven t), an d cu ltu red in th e p r esen ce of 5 m M IPTG.
After 72 h , th e cells were h ar vested an d su bjected to flow cytom etr ic an alysis
for cell cycle p r ofile. Th e su b-G1 fr act ion was ca lcu la ted with th e Mod FitLT
2.0 p r ogr am , an d th e r esu lts of th r ee in d ep en d en t exp er im en ts a re
su m m ar ized in (B) (b ar =S. D.). (C) DNA fr agm en ta tion was qu an t ita tively
m easu red b y ELISA u sin g an t i-h iston e an d DNA an t ibod ies. Th e r esu lts a re
exp ressed as fold -in cr ease over th e valu e of u n t rea ted exp on en tia lly-growin g
cells. Data sh own are a su m m ar y of th r ee in d ep en d en t exp er im en ts (b ar =S.
D.). Con t rol: Kp Ef1 cells tr eated with DMSO alon e.
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Fig. 3 Activation of caspases-9 , -6 and -3 during E2 F-1 -induced
apoptosis.
Wh ole cell lysa tes were p r ep ared fr om Kp Ef1 cells a t given t im e p oin ts a fter
th e ad d ition of IPTG. Th e exp ression of casp ases was exam in ed b y
im m u n ob lott in g u sin g sp ecific an t ibod ies. Con t rol; Mock-tr an sfected K562
cells. Data sh own are r ep resen ta tive of m u ltip le in d ep en d en t exp er im en ts.
Fig. 4 E2 F-1 increases Apaf-1 leve ls without affecting mito-
chondrial transmembrane potential.
(A) Total cellu lar RNA was isola ted fr om Kp Ef1 an d Mock-tr an sfected K562
cells a t th e in d icated t im e p oin ts a fter th e ad d ition of IPTG, an d su bjected to
Nor th er n b lot t in g for Ap af-1 , Nod -1 , an d Sm ac m RNA exp ression . Eth id iu m
b rom id e-stain ed 28 s an d 18 s r RNAs are sh own as a load in g con tr ol. (B)
Kp Ef1 cells were cu ltu red in th e p r esen ce of IPTG for u p to 96 h . Wh ole cell
lysa tes an d cytosol fr act ion s were p r ep ared a t th e in d icated t im e p oin ts, an d
su bjected to im m u n ob lottin g for Ap af-1 an d cytoch r om e c, r esp ectively. }-
actin was u sed as a load in g con tr ol. ADR; Cytosol fr act ion isola ted fr om
Kp Ef1 cells tr eated with ad r iam ycin a t 1 µg/ m l for 24 h . (C) Kp Ef1 cells were
cu ltu red in th e absen ce (Con t rol) or p r esen ce of IPTG (E2F-1 ) for u p to 72 h ,
or tr eated with ad r iam ycin as above (ADR). Cells were d ou ble-stain ed with
an t i-cytoch r om e c an d an t i-m itoch on d r ia l p r otein an t ibod ies as d escr ib ed in
Mater ials an d Meth od s. ADR in corp ora ted in cellu lar DNA gave off a b r igh t
r ed flu or escen ce in th e n u clei. (D) Kp Ef1 cells were cu ltu red in th e absen ce
(Con trol) or p r esen ce of eith er IPTG (E2F-1 ) or ad r iam ycin (ADR), an d stain ed
with a MitoCap tu r e d ye to m easu re m itoch on d r ia l tr an sm em b ran e p oten t ial
a t th e in d icated t im e p oin ts. FL1 an d FL2 sign als in d icate m itoch on d r ia l
tr an sm em b ran e p oten t ial an d th e levels of ap op totic cells, r esp ectively. Data
sh own are r ep resen ta tive of m u ltip le in d ep en d en t exp er im en ts.
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Fig. 5 E2 F-1 was unable to up-regulate and activate Bax in p5 3 -
defective KpEf1 ce ll line.
(A) Wh ole cell lysa tes were isola ted fr om Kp Ef1 cells cu ltu red with IPTG a t
th e in d icated t im e p oin ts, an d su bjected to im m u n ob lot an a lysis for p 5 3 an d
Bax. A h u m an ep id er m oid carcin om a cell lin e A43 1 was u sed as a p osit ive
con tr ol for p 5 3 exp ression . Th e sam e sam p les were u sed for }-actin
im m u n ob lott in g to ver ify th e equ al load in g an d in tegr ity of sam p les. (B)
Kp Ef1 cells were cu ltu red in th e absen ce (Con t rol) or p r esen ce of IPTG (E2F-
1) for 72 h , or tr eated with ad r iam ycin for 16 h (ADR). Cells were d ou ble-
stain ed with an t i-Bax an d an t i-OPA1 an t ibod ies, an d an alyzed b y u sin g
con focal laser m icr oscop y.
Fig. 6 E2 F-1 failed to induce apoptosis in Apaf-1 -defective
melanoma ce ll lines.
(A) Th e exp ression of Ap af-1 p r otein was exam in ed in 5 h u m an m elan om a cell
lin es b y im m u n ob lott in g. Th e m em br an e was r ep rob ed with }-actin
an t ibod y to ver ify th e equ al load in g of sam p les. (B) E2 F-1 was over exp r essed
in m elan om a cell lin es b y tr an sfect in g a b icistr on ic vector th a t in d u ces
sim u ltan eou s exp ression of E2 F-1 an d GFP (E2F-1 ). As a con tr ol, cells were
tr an sfected with an em p ty p IRES2-EGFP vector (Vector ). After 72 h of
tr an sfect ion , th e p er cen tages of GFP-p osit ive cells u n d ergoin g ap op tosis were
d eter m in ed b y flu or escen ce m icr oscop y. Th e m ean s + S. D. (b ar ) of th r ee
in d ep en d en t exp er im en ts a re sh own . Aster isk in d icates p <0.0 5 b y Stu d en t 's
t-test .
Fig. 7 E2 F-1 binds to Apaf-1 promoter upon overexpression.
(A) Th e sequ en ce of th e 5 ' -u n tr an sla ted r egion of th e Ap af-1 gen e is sh own .
Posit ion +1 in d icates th e 5 ' en d of th e fu ll-len gth cDNA. Th e b ou n d ary of
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Exon 1 is in d ica ted b y a h or izon tal b ar . Pu ta tive b in d in g-sites of kn own
tr an scr ip tion factor s a re u n d er lin ed , an d a Cp G islan d is b oxed . Ar rows
corr esp on d to th e sequ en ces u sed for PCR p r im ers for ch r om atin
im m u n op recip itat ion assay. Th e n u cleotid e sequ en ce sh own h er e h as b een
d ep osited in th e DDBJ/ EMBL/ Gen Ban k d atab ase u n d er accession n u m b er
AB07 082 9. (B) Ch r om at in su sp en sion s were p r ep ared fr om Kp Ef1 cells
b efor e (T-0) an d after 24 h (T-24) of cu ltu re with IPTG, an d su bjected to
im m u n op recip itat ion with th e in d icated an t ibod ies. Th e r esu lt in g
p r ecip itan ts were su bjected to PCR u sin g a sp ecific p r im er p a ir cor r esp on d in g
to th e n u cleotid e p osit ion s of -61 8 to -59 7 an d -42 8 to -40 7 of th e Ap af-1
p r om oter . PCR was car r ied ou t in th e p r esen ce of [32P]d CTP, an d th e
am p lified p r od u cts were visu a lized b y au tor ad iogr ap h y after 8%
p olyacrylam id e gel elect rop h or esis. Th e r ep resen ta tive d ata of 30 PCR cycles
are sh own . No DNA: PCR was d on e with ou t DNA tem p lates. In p u t: Pr ior to
th e fir st wash , 20 0 µl of th e su p ern atan t was saved for each t im e p oin t , an d
u sed for PCR after p r otein ase tr eatm en t an d eth an ol-p recip itat ion .
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Con trolVAD
WEHDVDVAD
DEVDYVADVEIDIETD
LEHDAEVDLEED
0 10 2 0 30
Su b G 1 fr ac tion ( %)
Fig. 2B
DNA fr agme nta tio n ( A405 nm / A495 nm )
0 2 4 6 8 1 0
Con trolVAD
WEHDVDVAD
DEVDYVADVEIDIETD
LEHDAEVDLEED
Fig. 2C
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Fig. 4B
Apa f-1
}-Acti n
}-Acti n
Tim e af ter E2F-1 i nd ucti on
0 12 24 48 (h
0 12 24 48 72 96 (h
Cyto ch r om e c
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Fig. 4C
Cytoc hr om e c Mito ch on dr ia Merg e
Contro l
E2F-1(48 h)
E2F-1(72 h)
ADR
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p53
Bax
}-actin
KpEf1
0 6 12 24 48
+IPTG (h)
A431
Fig. 5A
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Bax Mito ch on d ri a Merge
Contro l
E2F-1(72 h)
ADR
Fig. 5B
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Fig. 6A
MMAc
SK-MEL-5
Mal me-3M
G-36 1
Colo 67 9
Ap opt oti c Cells (%)
0 10 20 30 40
*
*
VectorE2F-1
Fig. 6B
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Fig. 7A
Fig. 7B
-11 3 6 GCACAG CGGA CAG GAAGT AA C GGTTTAT AA C AAC GTGAA C GCTTCTGACG TGTAGCGGGA G CTTCAGAAA GCGTGGCCGA ATC TGCAG
-10 4 6 CTTAA CAAA T GGCTCGTTCC CAA ACCTTAG CCTCAAC TCC TTCTGA GCGA T AGG GGCATG CTACCAGCAC GGGGGGAAAT GAGAT ACAA
-95 6 AAAC GCCCGA AAG GGAAAAA C AAGG CTGGG CTGTTTCCTT CCTTTGGGGT CAA GACAGAG GCACAGAAG G CCCAGGT CTG GGAGG TTG -8 79 Lyf-1
-86 6 TCACCTCCGG CTGGATG TTG AGC CGGGTGG GAGCCCAAAC AGCAGG GGGC CCAC AGG AGG CCGGCCCAG G CAGCCTCGCG TCCACTTACC -81 2 c-E ts -8 04
-77 6 AG GCCAGGCC CAGG CACGTC CCCAG CGACA GCAGG CTCAG GCACGTTCGG GGGTCT GCCC AGCCCCCGCC TCCGCTGCTC CGGGCCACGG -7 65 p53 p53 -7 39 -7 20 Sp-1 Sp-1 -7 03
-68 6 G GGTCTTCCC GCCCTCGCTC CGCTTCCCGG GCTCCGCAG C AGGGGCTCCC TTGGGCCCCG ACTTCTTCCG GCTCTTCACC TCAG ACAT GT -6 85 NF-¥¥¥¥B -67 6 - 604 p53
-59 6 CTGGA GACCC TAGG ACGACA AG CCCAGG GC AGCTTCTTCA CCAGG GGGAG CAGGACGTGG CCGC CTTGGC GTTCGTGGGA ACCCTGGGCG p53 -57 2 -5 32 E2F -5 26
-50 6 GTGAC CGCGC CCCCTCACAG AC TTGGCACC GCCCAGAG CC CAGCCCCTTC CCTCTCCCCG GCATCCTCGT TGCTTCACTG AG TCTTTCAG -50 1 Sp-1 - 492 -4 68 Sp-1 -45 9
-41 6 CTGCCAGCTC CAT AGT TCCC CTA GGAG AGG TGGGCGGCGA CCTCAACCCA CAGCGCCTTC CACTGCGAT A TT GCTCCAAA T CCGAG GAAA
-32 6 TTCAA ACTCC CGGGCGCGCG CAGG CCGA CG GGACCCGAG G AG GAG GGGCA GGA CGAAGG G GTCGCGCGCG CCACGTCGGG CGCGCCGCC Ex on 1
-23 6 CTGCCCGAGT CCGGCAT TGG TGGGAAC GCG GCGCGTCCCT GAGGCTTA GC CACGCCCCGT CCGCGGGGTA GG CGGGCACT TCTACGCGCG -1 88 Sp-1 -17 8
-14 6 CGGGCAT GAG CCGTGGCAGG AGT GCGCGGC GGCAGCGGTG GCCGCCCCTT GGGGCTTGGG GTGTGTTTAT TTGCATA AGC GGGCGCGCGC +1-56 CGTCC GGGCT GGGTGGAT CC GGCGGGAT TT GACTGCTCCG CTGTCCAG AG G CGGAG AAGA AG AGG TA GCG AG TGGACGTG ACTGCTCTA -5 1 Sp-1 -4 4
+3 5 CCCGGGCAAA AGG GAT AGAA C CAGAG GTGG GGAGT CTGGG CAGTCGGCGA CCCGCGAAG A CTTGAG GTGC CGCA
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Satsuki Iwase, Hisashi Yamada, Michio Matsuda, Yasuhiko Kano and Mitsuru NakamuraYusuke Furukawa, Noriko Nishimura, Yutaka Furukawa, Masaaki Satoh, Hitoshi Endo,
Apaf-1 is a mediator of E2F-1-induced apoptosis
published online July 30, 2002J. Biol. Chem.
10.1074/jbc.M200805200Access the most updated version of this article at doi:
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