Note: Descriptions are shown in the official language in which they were submitted.
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REGULATION OF APOPTOSIS AND IN VITRO MODEL FOR STUDIES THEREOF
BACKGROUND OF THE INYENTION
The field of this invention is the area of ~ul,lo~i~ (plog~ led cell death) and methods for the study
of the regulation thereof. Spel~ifir~lly, the present invention provides an in vitro system for the analysis of
apoptosis and specific regulators of the apoptotic pathway.
Apoptosis is a distinct form of cell death controlled by an internally encoded suicide prograrn [reviewed
by Steller, H. ~1995) Science 267, 1445-1449; White, E. (1996) Gene d~ Dev. 10, 1-151. Morphologic changes
aCcociatpd with apoptosis include con~1Pnca-ion of nucleoplasm and cytopla m, blebbing of cytoplasmic
es, and rl,.~ ion of the cell into apoptotic bodies that are rapidly phagocytosed by neighboring
cells [Kerr, J. (1971) J. Pathol. 105, 13-20; Wyllie et al. (1980) Int. ~ev. Cytol. 68, 251-305]. Bjorhrmir.~a.
markers of ~yO~)lOSiS include DNA rl~g.. ,l; ~ion into nucleosomal r~a~lu.,nls lWyllie, A. (1980) Nature 284,
555-556], activation of the interleukin Ib collvellillg enzyme (ICE)-farnily of ~ tcases [Schlegel et al., 1996;
Duan et al. (1996) J. Biol. Chem. 271, 1621-1625; Wang et al. (1996) EMBO J. 15, 1012-1020], and cleavage
of substrates of the ICE-family of proteases, including poly(ADP-ribose) polymerase (PARP) [Tewari et al.
(1995) Cell 81, 801-809; Nicholson et al. (1995) Nature 376, 37-43] sterol regulatory element binding proteins
(SREBPs) lWang et al. (1995) J. Biol. Chem. 270, 18044-18050; Wang et al. 1996, supra], nuclear lamin
[Lazebnik et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9042-9046], and the Ul ~ccoriqt~(l 70 kDa protein
[Cacciola-Rosen et al. (1994) J. Biol. Chem. 269, 30757-30760].
The cell suicide program is illustrated by genetic studies in the nenlato~e Caenorhabditis elegans
- [Hengartner and Horvitz (1994) Philos. Trans. P. Soc. London Ser. B 345, 243-246]. Two genes involved in
the control of prograrnmed cell death in C. elegans have been well chd~a~ iL~d. One gene (ced-9) encodes
a protein that prevents cells from ulldGrgoillg apoptosis [Ileng~ et al. (1992) Natl~re 356, 494-499], and
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the ced-3 gene encodes a protease required for ini~i~tinn of a~,u~lo~is [Yuan and Horvitz (1990) Dev. Biol. 138,
33-41~ .
The bc1-2 family of genes are m~nm~ ll co~ e.~ of ced-9 [Hengartner and Horvitz (1994) Cell
76, 665-676] . Over-t;A,u~ ioll of bc1-2 coding ~c.~ prevents m:lmm~ n eells from ul~d.,. ~ohlg apoptosis
in response to a variety of stimuli [reviewed by Reed, 1. C. (1994) J. Cell Biol. 124, 1-6]. The BCL-2 protein
is located primarily on the outer ~ ,s of mito. ~ M~-n~gh~l et al. (1992) J. hrist. Cytochem. 40,
1819-1825;Krajewskietal.(19933CancerRes.53,4701-4714;deJongetal.(1994)CancerRes.54,256-260].
The presenee of BCL-2 on the mito~hon~lri~ surface is eorrelated with a bloek in the release of eytoehrome e
in response to triggers of apoptosis in eells whieh do not express the BCL-2 protein on the mi~r ehonrlrial surfaee
[Yang et al . (1997) Science 275, 1129-1132] . Holoeytoehrome e, but not apo~;ylo~Lru.. ,e c, triggers activation
of CPP32 and the ap~Lolic eascade. Without wishing to be bound by theory, it is believed that the Be1-2
protein inhibits ~u~lo~is by pl~ tillg release of holoeyto~hlol"c e from the mitochondrial "~ b-d~le and also
prevents depo~-i7~tion of the mitorh~ n~lrj~ llb~ c.
The CED-3 protein is a cysteine protease related to the ICE-family of proteases in m~nm~ cells
[Yuan et al. (1993) Cell 75, 641-652]. The closest m~mm~ n homolog of CED-3 is CPP32 ~Fernandes-
Alnernri et al. (1994) J. Biol. Chem. 269, 30761-30764], whieh cleaves PARP and SREBPs in eells undergoing
apoptosis [Tewari et al. (1995) supra; Nieholson et al. (1995) supra; Wang et al. (1996) supra]. CPP32,
which is also called caspase-3, is closely related to CED-3 in tenns of arnino aeid sequenee identity and
substrate crerifirity [Xue and Horvitz (1995) Nah~re 377, 248-251]. Like CED-3 in C. elegans, CPP32
20 norrnally e~ists in the eytosolie fraetion as an inaaive pl~cul~ol, that pl~,Cul~ùl iS activated proteolytically in
cells u..d~.goillg apoptosis [Schlegel et al. (1996) J. Biol. Chem. 271, 1841-lX44, 1996; Wang et al. (1996)
supra]. Further evidenee for the l~,~Ui~ for aetive CPP32 in aL'o~: c is that a ~ ~e~ide aldehyde
inhibitor that ~ ,;r~ 1y inhibits CPP32 aetivity blocks the ability of eytosol from apoptotic eells to induee
~optosic-like ehanges in normal nuclei in vitro. [Nicholson et al. (1995) supra].
Triggering of ~osis by activated CPP32 is part of the highly regulated I~IF.~ for ini~ ion of
apoptosis; careful regulation of this pathway is necessary to prevent u-lw~lled eell death. CPP32 is activated
by multiple proteolytic cleavages of its 32 kDa pl~ UI~Ol form, &~ g the 17111 kDa or 20111 kDa active
form [Nicholson et al. (1995) supra; Wang et al. (1995) supra]. CPP32 is activated by eleavage at aspartic acid
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residues, a hq~lr~qrk of ICE-like plotedses [Tho,lll,c,l~y et al. (1992) Nature 356, 768-774], and a cascade of
ICE-like proteolytic cleavages leading to a~,ulno~is has been proposed [Tewari et al. ~1995) supra; Wang et al.
(1996) supra]. Activated CPP32 from HeLa cell extracts cleaves the CPP32 p.c~ul~or [Wang et al. (1996)
supra], in-lirqting that CPP32 can be activated through autocatalysis. Autocatalytic cleavage is probably
S responsible for active enzyme when the CPP32 ~ ,u~ol is expressed in large quantity in bacteria [Xue and
Horvit,q (1995) supra]. Recently, another ICE-family protease has been i~l ntifiPd that may be lui~unsible for
cleaving the CPP32 pl~ ul:~ol into the 20/11 kDa active form. This enzyme has been purified from hamster
liver eAtracts and j~Pntifipd as the hamster homolog of Mch2a [Liu et al. (1996) J. Biol. Chem. 271, 13371-
13376; Fernandes-Alnemri et al. (1995) Cancer ~es. 55, 2737-2742]. Autocatalysis and the protease cascade
10 may provide the signal amplifirqti~n necessary for rapid and irreversible apoptosis, but the intracellular factors
that trigger this ~tnrlifirq~inn have yet to be identif~d.
There have been several previous reports of cell-free a~lo~i~ systems that induce apoptotic changes
in the added nuclei [T q7Pbnik et al. (1993) J. Cell Biol. 123, 7-22; Nc~.l,leycr et al. (1994) Cell 79, 353-364;
Eeari et al. (1995) EMBO. J. 14, 5201-5208; Martin et al. (1995) EMBO J. 14, 5191-5200]. These systems
15 require cytosol from cells that are a1ready ul~d~ ,uhlg apoptosis in vivo; thus, they cannot be used to detect
triggering factors.
There is a need in the art for in vitro methods for the analysis of COIlllJUulldS and biological factors
which trigger or accelerate apoptosis or which interfere with the in~ ticn of a~Ju~Jto~;s, as well as those which
can increase the apoptotic effect of rhPmnthpr~q~reutic agents in cancers, especially those eAI/l.,ssillg oncogenic
20 bc1-2. This need is met by the present invention, which allows the study of a~ iS and regulators thereof
in a cell-free system in which the analysis is not complicated by previous in-i..rtinn of the apoptotic pathway
in the cells used to prepare the test extracts.
SUMMARY OF THE INVENTION
The present invention provides an in vitro system and methods for the analysis of the regulation of
25 ~~roptocic and for the i-i~ntifi~q~inn of activators and h~ of the d~ pt()lic pathway; the present system is
improved over prior art systems for the study of apoptosis in that the prior art systems d- ~.. n~iPd on cell free
e~ctracts prepared from olgani~ in which the apoptosis pathway had already been induced. Thus, the present
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system and methods perrnit freedom from the potential i.~ fe,.,,.ce of apoptosis-inducing factors or other
conAitif~nc on which prior art systems have relied.
As e~PmplifiPd herein, the present invention provides an in vitro system for analysis of apoptosis and
its regulation, where the test system includes a 100,000 x g ~ 7--~ of HeLa cells from sllcrPncir?n culture
(S-100). In its first aspect, the HeLa S-100, to which chqllPngp cvJll~Joullds are added, is assayed for CPP32
proteolytic activity using r~lio!~llPlPcl poly(~ .,oc;..~ Airhosrh~t-p-ribose polymerase (PARP) and radiolabeled
sterol regulatory binding protein 2 (SREBP-2) and sodium dodecyl sulfate polyacrylamide gel ele.,~luphol.,i,ls
(SDS-PAGE) and ~l ~trrP~ graphy. The radiolabeled PARP and SREBP-2 can be prepared by in vitro
translation in the presence of 35s-m-pthi~ninp as described in Example 3 herein. In a second aspect, the HeLa
10 S-100, to which challenge Culll~vun~]a are added, is assayed for DNA fra~lrpntiltion activity, by hlcuba~ g the
treated S-100 with hamster liver cell nuclei and then extracting the genomic DNA and analyzing by agarose gel
elc~LIul~horesis. The specific proteolytic activity is q~c~h ~ 1 by the addition of dATP or dADP (at a
con..~...ldtion from about 0.1 to about 2 mM, ~l~,ruldbly about 1 mM. DNA lln~ e activity is similarly
~1. y..nfL ...t on the presence of dATP. It has been d~ l. rl~d that cytochrome c is required in the cell-free
15 extract for the dATP-~ r~ ~l activation of the apoptotic pathway, especially for the activation of the apoptosis
marker protease.
The present invention provides a cell-free system which d-ll)lirqtPs the features of the apoptotic
program, inr.lllAine the activation of CPP32 and DNA r.~ ion Apoptosis in this system is initiated by
the presence of soluble l;yloulllulllc c and dATP at snfflri~pnt con~ tlalions~ This system allows the
20 frPrti--nq~ion and pllrifir~in,n of the bioçhem:-q1 c~lll~vl.cllla that trigger the activation of the apoptotic l~lutcascs
and DNA fri~ement.~ion
The present invention further provides a method for ide.llirying antagonists of dATP in the cyîosol of
a~ r ~ n~ -deficient cells, such as T cells from persons with severe cu.nl,illed irnmnnrlA~PfiriPnçy
dATP levels in ~Pn~sinp ~l~O~ Qc-deficient cells are elevated in co-~ ;.cvll to those of normal cellsl and
25 without wishing to be bound by any particularly, this is believed tû co.lllil,ule to the a,~ lOIna of the deficiency.
TAPntifir~ion of antagonists of the initi~ ~ion of apoptosis can lead to L~ 5 for the amelioration of the
clinical state of deficient individuals.
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The present invention also provides methods for j-~Pntifirqtion of co.l.~uundD which trigger apoptosis
even where the bd-2 oncogene protein is present. The bc1-2 oncc",_..c is q~gori tpd with lesiDt~lce to
chemotherapy in human cancer, and compounds which cause CPP32 protease and DNA rl~ r~ ion nuclease-
activation in bc1-2 oncogene extracts can be id~ntifi~d in the cell free assays of the present invention where the
S S-100 extract is prepared from BCL-2 ~A~ sillg cells.
litionqlly, the present invention allows the idPntifirqtion of compounds which effectively increase
the apoptotic response to dATP and/or cytochrome c, including those which increase dATP levels in treated
cells and those which promote release of iylu~ LIulllc c from mitn~ ;aMIl.,.lll,l~~s. Such compounds can
be used to increase the err~~ -css of chPm- thl a~uLiC agents which act by inducing ap-)~JIoDis.
BRIEF DESCRIPTION OF THE DRAWINGS
~igures lA-lD illustrate dATP--~P.pen~nt activation of CPP32 and DNA fi~ t.~ion in vitro.
Aliquots ~10 ~1) of HeLa cell S-100 (50 ~g) were ;I~-ul~ t~ d alone (larle 1), in the presence of 1 mM ATP (lane
2), or in the presence of 1 mM dATP (lane 3) at 30~C for 1 hr in a final volume of 20 ~1 of buffer A. Fig.
lA, samples were subjected to SDS-PAGE and llal-Df~ ,d to a nitrocellulose filter, probed with a mt)norlon~l
anti-CPP32 antibody, and the antigen/antibody complex was visualized by the ECL method. The filter was
exposed to Kodak X-OMAT AR X-ray film for 1 min. In Fig. lB, an aliquot of 10 ~1 of in vitro trqn~ P-l,
35S-labeled PARP was added to each reaction. After S min, the samples were Dul~_~,lud to SDS-PAGE and
r~l..,d to a nitrocellulose filter. The filter was exposed to film for 2 hr at room Ir~ . In Fig. lC,
a S ~1 aliquot of in vitro 11 ' ~i, 35S-labeled SREBP-2 was added to each reaction. After incubation at 30~C
20 for 30 min, the samples were subjected to SDS-PAGE, the gel was dried and exposed to film for 2 hr at room
te.~ ul~i. In Fig. lD, a 50 ~1 aliquot of HeLa cell S-100 (250 ~Lg) was i..cub ~ d with 6 ~I Hamster Liver
nuclei in the absence (lane 1), or presence of 1 mM ATP (lane 2) or dATP (lane 3) for 2 hr at 37~C in a final
volume of 60 ~1 of buffer A. DNA was isolated as df ~ ' ;bed in the Examples and size-sepqrqtr~ by agarose
gel ele~;~,uphoresis (2% agarose), and the DNA was visualized by ethidium bromide staining.
Figures 2A-2B illustrate the nucleotide specifi~ity for in vitro activation of CPP32 and DNA
fragmPnt~ti~n Fig. 2A shows the results of in~nbqting a 10 ~1 aliquot of HeLa S-100 (50 ~.~g) was i,.,~
with a 3 ~1 aliquot of in vitro ~lansl..~, 35S-labeled CPP32 at 30~C for 1 hr in a final volume of 20 ~1 in the
presence of 1 mM ' ' nucleotide. The samples were subjected to SDS-PAGE and ll~uDÇ~ ,dto a
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nitrocP~ lose filter. The filter was exposed to film for 16 hours at room le ,.I.f..,n..~. In Fig. 2B, a 50 ~1
aliquot of HeLa S-100 ~250 ~Lg) was inrob~ted with each aliquot of 6 ~I hamster liver nuclei at 37~C for 2 hr
in the presence of 1 rnM int~ nucleotide. The DNA was isolated, analyzed by 2% agarose gel
elecllol,horesis and visualized by ethidium bromide staining.
S Figure 3 illustrates fra~rion~ti~-n and l~c.,.. ~ llion of dATP--lepen-lent activation of CPP32 by
phosrhocelll-lose cl~.ull.dtugraphy. HeLa cell S-100 was subjected to rhocphoc~ lose chromatography and the
column flow through and bound material were collected as described in the Examples. Aliquots (10 ,ul) of HeLa
S-100 (50 ~cg) (lanes 1 and 2), pl~o~ ncell-llose flow through fraction (PC-FT) (lanes 3 and 4),
ph- .cphoce~ lose bound fraction (PC-B) (lanes S and 6), and the rnixture of phosphocellulose flow through and
10bound material (lanes 7 and 8) were inr.~lb.~t~d with aliquots of 3 ~1 in vitro tr~ncl~d, 35S-labeled CPP32 at
30~C for I hr in the absence (lanes 1,3,5,7) or presence (lanes 2,4,6,8) of I mM dATP. The sarnples were
subjected to SDS-PAGE, ~ r~...,d to a nitrocellulose filter, and the filter was exposed to film for 16 hours
at room tl ..~ "~;.
Figure 4 shows the results of Mono S column purificatiûn of Apaf-2. The Apaf-2 activity that bound
15to the ph~-crhocelllllose column was purified through the Mono S colurnn as dc~-;filcd in Exarnple 7. Fig. 4A
shows the results of inrub~ tion of 1 ~LI aliquots of Mono S colurnn fractions with aliquots of 10 ~1
ph~ c~llulose flow through fraction and 3 ~1 of in vitro translated, 35S-labeled CPP32 at 30~C for 1 hr in
the presence of 1 mM dATP in a final volume of 20 ~1 of buffer A. Sarnples were subjected to SDS-PAGE,
.lcd to a nitroc~thllose filter, and the filter was exposed to film for 16 hours at room t~.U~.dlUl~. In
20Fig. 4B, aliquots (30 ~1) of the Mono-S fractions were subjected to 15% SDS-PAGE and the proteins were
visualized by silver staining.
Figure 5 provides the absol~lioll spectrum of Apaf-2. An aliquot of 1 ml of Apaf-2 purified through
the Mono S colurnn was subjected to absorption spectrum scaoniog using a CARY 219 ~.e~ u~holu,l,~t~
Absorption s~e~;l,u,ll was recorded between 330 nm and 600 nm at a scaoning speed of 1 nm/sec.
25Figure 6 ~eml - - that cytochrome c proteins from bovine heart aod rat liver have Apaf-2 activity.
Aliquots of 0.2 ~g of Apaf-2 purified through the Mono S colurnn (lanes 1, 2), cytochrome c from bovine heart
(lanes 3, 4), and rat liver (lanes 5, 6) were inr~lb~ with ali~uots of 10 ~I phosphoc.~llulose flowthrough
fraction aod 3 ~1 in vitro translated, 35S-labeled CPP32 at 30~C for I hr in a final volume of 20 ~I buffer A
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in the absence (lanes 1,3,5) or presence of (lanes 2, 4, 6) I mM dATP. Samples were ~ul);ccl~d to SDS-PAGE
and Llal~ cd to a nitrocellulose filter wbich was then exposed to a film for 16 hr at room ~.u~Jeldlul~.
Figures 7A-7D d~ o~ immnn- ~1epletion of cytodnv~ c from HeLa S- 100 and rc~CO~ n of
dATP~ A. ~-1 activation of CPP32, DNA rr~ l;on and nuclear morphological change using purified
S cytochrome c. Cylucluul--c c present in the HeLa cell S-lO0 was immnn~xlepleted as described in the Example
lO. In Fig. 7A, 10 ~l aliquots of HeLa S-lO0 (50 ~g) (lanes I and 2), or lO ~1 aliquots of HeLa S-100
imm11n-~rpleted of cytochrome c (lanes 3 and 4), or 10 ~1 of HeLa S-100 i~"..~ leted of cytochrome c
s~ k.~ d with 0.2 ~g Apaf-2 purified through the Mono S column (H) (lanes S and 6), bovine heart
cytochrome c (B) (lanes 7 and 8), or rat liver ~;ylu~,hlullle c (R) (lanes 9 and lO), were i.~ ul,~ l with aliquots
of 3 ~l in vitro translated, 35S-labeled CPP32 in the absence (lanes 1, 3, 5, 7, 9) or presence (lanes 2, 4, 6, 8,
10) of 1 mM dATP at 30~C for 1 hr in a final reaction volume of 20 ~1 of buffer A. Samples were subjected
to SDS-PAGE and ll~u.s~ ,d to a nitrocellulose filter which were then exposed to film for 16 hr at room
. In Fig. 7B, HeLa S-100 (50 ~g) imm~n(~ ed of eylo~hl~ll.e c were ,~c,~ .1l d with the
in~ d amount of Apaf-2 (purified through Mono S column step) in a CPP32 cleavage reaction as de;,~-ibed
in Fig. 7A. The cleaved products (p20) were quqntifi~ d in a Fuji-1000 phospho.. ulagel machine and plotted
in co...,~ on with that t,~ by HeLa S-100. In Fig. 7C, aliquots of 50 ~1 of HeLa S-100 (250 ~g) (lanes
1 and 2), or 50 ~1 of HeLa S-100 -~p!: ~ of ~,ylol_hlullle c (lanes 3 and 4), or 50 ~l of HeLa S-lO0
im~mmnfleF' ~ ~ of cytochrome c .u,~ d with I ~g apaf-2 purified through the Mono S column (H) (lanes
5 and 6), bovine heart cytochrome c (B) (lanes 7 and 8), or rat liver cytochrome c (R) (lanes 9 and 10), were
i _~ ~ with aliquots of 6 ~I hamster liver nuclei in the absence (lanes 1, 3, 5, 7, 9) or presence (lanes 2,
4, 6, 8, lO) of 1 mM dATP at 37~C for 2 hr in a final reaction volume of 60 ~1. The DNA were isolated as
~s~ -ed in the F~ nples~ analyzed on 2% agarose gel el~l.o~v-~,;.is, and the DNA was visualized by
ethidium bromide staining. In Fig. 7D, DNA L.~ ion assays were carried out as in Panel C using HeLa
S-lO0 cd~h: ' of L;yLu~,hl~lllc c alone (a,b) or su~ d with Apaf-2 purified through Mono S
column step (c,d) in the absence (a,c) or presence of 1 mM dATP (b,d). After 2 hr inr1~h~ion at 37~C, an
aliquot of each reaction (30 ~1) was stained with 4',6'-d;~ --2-phenylindole (DAPI), and observed under
a nuu,c~-;cl.ce uu ;-u;,cù~e with a UV-2A co,.ll.i.l~lion filter.
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Figure 8 illustrates dATP and cytoehrome c-~leprn~1rnt activation of CPP32 in S- 100 cytosol
prep~ra~innc (imml1n~l~lPpleted of cytochrome c) from human elllhl~ollic kidney 293 eells and human monoblastic
U937 cells. CPP32 activation reactions were earried out as described in Figure 7 except 25 ~g of S-100 was
used in each reaction. 1 mM of dATP was present in lanes 2, 4, 6, 8, 10, and 12. Lanes I and 2, S-100
S fraction from 293 eells; Lanes 3-4, S-100 fraction from 293 eells immnn~depleted of eytoehrome c; lanes 5 and
6, S-100 fraction from 293 eells i~ o~pleted of cytochrome c supp1emrntrd with 0.2 ~4g of Apaf-2 purified
through the Mono S eolumn step; Lanes 7 and 8, S-100 fraetion from U937 eells; Lanes 8-9, S-100 fraction
from U937 cells immnnodep~ n, of cylu~lllull.e c; lanes 11,12, S-100 fraetiûn from U937 eells
imm ~n<)depleted of cytochrome c auppl~ d with 0.2 ,ug of Apaf-2 purified through Mono S column step.
Figure 9 df~ ion of dATp-depen~1pr~t activation of CPP32 with S-cytosol and
purified Apaf-2. On day 0, Hela cells were set up at 5 x 105 cells per 100 mrn dish in medium A as described
he.._;lll)elow. On day 2, cells were harvested, eollected by centrifugation (1000 g, 10 min, 4~C). After washed
onee with ice-cold PBS, the eell pellet was au~c ~ d in 5 volumes of ice-cold buffer A c~ntilining 250 mM
sucrose. The cells were disrupted by dounring 3 times in a 5 ml Wheaton douncer with a pestle polished with
sand paper. After microcPnt~ g~ )n for S min at 4~C, the au~ lL~l~ were further centrifuged at 105 x g
for 30 min in a table top UIL~ f.;r~g~ (Rerkm~n I~laLlU~llC.-L~, Fullerton, CA). The resulting S~
were decign~ as S-cytûsol. Aliquots of S-cytosol (50 ~g) alone (lanes 1 and 2), or suppl~m~nled with 0.2
~g Apaf-2 purified through the Mono S column (lanes 3 and 4), were inrub ~ ~ with aliquots of 3 ~l in vitro
translated, 35S-labeled CPP32 in the absenee (lanes 1, 3) or presence (lanes 2, 4) of 1 mM dATP at 30~C for
1 hr in a final reaction volume of 20 ~1 of buffer A. Samples were subjected to SDS-PAGE and transferred
to a nitroce1~ ose filter whieh were then exposed to film for 16 hr at room t~ly~d~Ul~;.
Figures 10A-lOB shows increased relea_e of cytoehrome c to the cytosol upon apoptotic ctimll~ n
HeLa cells were treated as d~ c~1 in Figure 9. On day 2, sLdu,us~ol;~le at a ~mal concelllldtiull of I ~M was
added to the medium as inr1: 1 After i~ ul. -~ .. at 37~C for 6 hr, the eells were ha~ ,1 and S-eytosols
were prepared as deseribed in Figure 9. In Fig. 10A, a 50 ~4g aliquot of ~eLa cell S-100 as in Figures 1-7
(lane 1), or S-cytosol from HeLa eells (lane 2), or S-cytosol from HeLa cells treated with st~uloa~ûlinc for 6
hr. In lane 4, aliquot of 0.2 ,ug of Apaf-2 purified through Monû S column step. Proteins were s~u~ted using
15 % SDS-PAGE, lla"ar~ ,d to a nitrocellulose filter, and probed with a m~ rlon:l1 anti-cytochrome c antibody
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and the antigen/antibody complex was visualized by the ECL method as dc~ ibcd herein. Kodak X-OMAT
AR X-ray film was exposed for 15 seconds. The arrow denotes the position of cytochrome c; X denotes protein
bands cross-reacting with this antibody. In Fig 10B, aliquots co.llh;nillg 4.5 ~g of S-cytosol from HeLa cells
(-staurosporine) or HeLa cells treated with 1 ~LM ~laUlU~JVlillc for 6 hr (+sla.l~ospuli..e) were inr.ub~ed with
10 ~1 aliquots of in vitro translated, 35S-labeled PARP for 30 min at 30~C in a volume of 20 ~1 of buffer A.
Samples were then subjected to 12% SDS-PAGE, transferred to a nitrocellulose filter, and film was exposed
for 4 hr at room t~ lalul~.
DETAILED DESCRIPTION OF THE INVENTION
Apoptosis, or cell death, is a natural ph~.-.. -- "~ n Modlllqti~n of normal ~ol~lo~is or a.;li~ldlioll of
10 the apoptotic pathway in cells in which apoptosis is inhibited due to the t;A~ sioll of uncog~ s~ for example,
can lead to longer and e .ll~ ,red life and/or improved medical ll~a~lu~ methods, for exarnple, in cancer
patients.
The present invention provides a method for the identifir~ion of inducers and/or h~hibilol~ of apoptosis
in a cell-free system colu~ ing 100,000 X g ~u~ of cell cytosol (S-100) prepared from actively growing
cells and c~ ui.. g the inactive CPP32 and nuclease pl~,CUl:~UI~. Desirably, the S-100 is prepared from
m~nmqliqn cells, for example, HeLa cells. Activation of the apoptosis marker protease CPP32 and the marker
nuclease are triggered in this system in the presence of dATP and soluble cytochrome c in a 100,000 x g cytosol
MoAifir~ n of the assay preparation con~litinng allows the id~ntifirqtion of co~ ùu~lds, proteins
or co.,.l,osilions which can ,.,I,~ le either for the dATP or the soluble cytochrome c or for both. Initiation
20 of the apoptotic pathway is detected by the proteolytic cleavage of SREBPs or PARP by the CPP32 protease
which is activated at an early step of the apoptotic pathway. Triggering of the a~ ulic pathway can also be
detected via the activation of the nuclease. Active CPP32 protease and active apoptotic DNA fragm~.nt.~ion
nuclease are marker enzymes of the apoptotic pathway. In this system soluble ~lochloll,e c and dATP trigger
activation of the marker enzymes for apoptosis. It is understood that analogs of dATP and dADP function in
25 triggering the apoptotic activation as well.
Col..~uu..d~ or proteins which inhibit the initi~ n of the apoptotic pathway are detected by their
p,~ ion of the activation of the CPP32 protease or the marker nuclease in the presence of cytochrome c and
dATP, con~itil nc which norrnally activate the pathway.
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Compounds or proteins which cl,ullt,_la.;t the apoptosis-inhibiting activity of the bc1-2 gene product (or
of other oncogene products) can be i~ntifird by their ability to allow the activation of the marker enzymes of
the ~u~lOliC pathway even in the presence of dATP and cytochrome c in S-100 extracts of cells e~ h-g bcl-
2 or similar ollcogenes. Compositions i-~l ntifi~ d in the present assay system can be then used to increase the
S activity of rhrm- thf, .l~le.llic agents used in the llcallll.,lll of cancers and other hyperplastic disorders, especially
in cells ~A~ si--g oncogenic bc1-2 or other oncogenes which decrease apoptosis.
Activation of CPP32 and DNA rl~ icn are two well ch~ ,te.i~,d biochemical markers of
apoptosis and its initi~ti~ n With the goal of producing an in vitro system that duplicates apoptosis, we prepared
100,000 x g cytosolic s 'l~ (S-100) from sllcpPn~ion cultures of HeLa cells. The activation of CPP32
is the result of cleavage of its 32 kDa ~.~u-~or into the 20 kDa NH2-terminal fragment and 11 kDa COOH-
terminal fragment [Nicholson et al. (1995) supra], thus the activation of CPP32 in the HeLa cell S-100 was
monitored by Western blot analysis using a monoclonal antibody against the 20 kDa fragment of CPP32 (Figure
lA). The enzymatic activity of CPP32 was assayed by ll~,aS~llillg the cleavage of two 35S-labeled sul~7l~dt~s,
PARP (Figure lB) and SREBP-2 (Figure lC). DNA fr~gm~n~rtion was assayed by i--- ~IbJ~ E; the HeLa cell
S-100 with nuclei isolated from hamster liver followed by genomic DNA extraction and analysis by agarose gel
el~llo~ho.~,;,is. We found that deoxy.~lrn--cinr-5-ll;~ G~l)h 1r (dATP) markedly accel~l~ted the activation of
CPP32 in the HeLa cell S-100. As shown in Figs. lA-lB, no activation of CPP32 was observed when the
HeLa cell S-100 was ;~ . d in the presence or absence of 1 mM ATP at 30~C for 1 hr (Figure lA, lanes
1 and 2). However, in the presence of 1 mM dATP, most of the CPP32 in the HeLa cell S-100 was activated
(Figure lA, lane 3). The activated extracts readily cleaved PARP into 85 kDa and 24 kDa L.. 6~ ll1s (Figure
lB, lane 3) and SREBP-2 into 55 kDa and 70 kDa L..6lllcll~s (Figure lC, lane 3). The sizes of the cleaved
products of PARP and SREBP-2 were the same as observed in cells ullder6ohlg al~optosis [Kr~lFrn~nn et al.
(1993) Cancer Res. 53, 3976-3985; Wang et al. (1996) supra]. It is likely that this cleavage was the result of
the activation of CPP32, and related enzymes such as SCA2/Mch3, which are known to cleave PARP and
SREBPs at these positions [Fernandes-Alnemri et a, . (1995) Cancer Res. 55, 6045-6052; Pai et al . (1996) Proc.
N~tl. Acad. Sci. USA 93, 5437-5442]. HeLa cell S-100 e~tract in the presence of dATP induced DNA
r.~g,.. " -~;0,- when inrub~tPd with hamster liver nuclei (Fig. lD, lane 3). Such rl,.t,.,.. ~n ~ion did not occur
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with HeLa S-100 in ehe presence or absence of ATP, co.. r.. ;.-e the ~~4uile~ for dATP (Figure ID, lanes
1 and 2).
To test the mlcleoti~le ~p~PCifirity for activation of CPP32 and DNA fr~gm~nt~tion, HeLa cell S-100
was in~llh~t~d with in vitro tr~ncl~t~d, 35S-labeled CPP32 in the presence of 1 mM of various nucleotides
(Figure 2A). Cleavage occurred only in the presence of dATP or dADP (Fig. 2A). CTP, dCTP, GTP, dGTP,
UTP, dTTP, ADP, AMP, dAMP, ~ nr~inP, deoxy~lPn~-sin~ and cAMP did not replace dATP. Identical
ml~lPQti(le .I~e~ ;r.~ y was observed in the DNA fr~mPnt.q~ion assay (Fig. 2B).
To isolate the protein~s) required for the a~,liva~iull of CPP32, HeLa S-100 was loaded onto a
phosrhoc~Plhllose column, and the flow-through and bound fractions were collected. Neither fraction alone
supported dATP-cleFPn-l~nt activation of CPP32 (Fig. 3, lanes 3-6). When the flowthrough and bound fractions
were mixed, CPP32-aL;Li~ i.lg activity was restored ~lanes 7-8). This eA~ lll indicates that there are
multiple factors, which can be s~p~-~tPd by the phosph-lc~lhllose chromatography, cc...l.;l,.l~;..~ to dATP-
dPrPn~ nt activation of CPP32. The factor(s) that flow through the ph~rhoce~ ose column are cle~ign~tPd
apoptotic protease acli~ralillg factor-l (Apaf-l) and the factor that bound to the column is clesien~t~d apoptotic
15 protease activating factor-2 (Apaf-2). It is ~mdersrood that "Apaf-l" may l.,~,~sclll more than one protein or
it may l~ clll a colll~ tiun of protein(s) and other factors.
Apaf-2 activity was assayed by l~.,ollll,illillg with Apaf-l after pllliflc~lion by the following steps.
First, the Apaf-2 fraction was s.ll~je.,ted to 50% ~ .. sulfate ~l.,.,il,il~iul,. All of the activity l~ .ed
in the s~ ". ~ while most of the protein prLç;pi~ l (Table 1). The ~ .l was loaded onto a phenyl-
20 --r~ ose (hydrophobic ;l .~ ~iu. ) colurnn and t_e activity was eluted with 1 M - ~ l,u~ill. sulfate. The eluate
was passed through a gel filtration colurnn; active fractions were subjected to se~ ;sil Mono Q (anion
PY-'h~r~g~) and Mono S (cation t~h---gi)LIllL~ a~ugraphy. The Apaf-2 activity flowed through the Mono Q
column, and the flow through was directly loaded onto the Mono S column. Bound Apaf-2 activity was then
eluted with a 100-300 rnM NaCI linear salt gradient. The fractions from the Mono S column were collected
and assayed. As shown in Figure 4A, the Apaf-2 activity eluted from the Mono S column at alJIJlL Aillldtl,ly 120
mM NaCI (fractions 2-4). The active fractions were analyzed by SDS-PAGE (Fig. 4B). A protein of apparent
m- lecnl~r mass of 15 kDa was co-eluted with the activity. No other proteins were detected by silver staining
in the active Apaf-2 fractions.
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Table I s.~ s the results of a complete l~ulir~,atiOn of Apaf-2 starting with the S-100 fraction
from 20-liters of HeLa cells (348.5 mg protein). The Apaf-2 protein was purified more than 2000-fold with
an overall recovery of 152% activity. The > 100% recovery indicates the elimination of inhibitory activities
during the purification.
Purified Apaf-2 had a noticeable pink color, and it showed absorbance peaks at 415, 520 and 549 nm,
a spectrum shared by reduced cytochrome c LMargoliash and Walasek (1967) Meth. Enzymol. X, 339-348].
Identity of Apaf-2 with cytochrome c was confirmed by comparison of amino acid seq~ n~fcgcnt~ l from
tryptic peptides isolated from the 15 kDa Apaf-2 with known cytochrome c amino acid sequence inr~Jlll~alion.
All those se~lu....~ec show 100% identity with portions of the reported sc~u~llce of human cytochrome c (Table
10 Il).
To confirm that cytochrome c has Apaf-2 activity, purified bovine heart and rat liver cytochrome c
(from a cuuuu~ ;ial source) were tested for Apaf-2 activity. As shown in Figure 6, cytochrome c from both
sources initiated dATP-depf nflPnt activation of CPP32 as efficiently as Apaf-2 (lanes 3-6).
To rule out the possibility that the Apaf-2 activity is due to a minor co~ g protein that co-
purified with ~,ylo~ uuuc c, an il....... ~ d~ hlio~ 1 was carried out using a monoclonal antibody
against rat cytochrome c. This m-)nl rlon~l antibody cross-reacts with purified Apaf-2. As shown in Figures
7A-7D, HeLa cell S-100 depleted of cytochrome c using the m--~o~lon~l anti-cytochrome c antibody lost the
dATP-~ ~J. ,~ acliv.llivn of CPP32 and the ability to induce DNA r~..c" .. ~ ion in the added nuclei (Figure
7A and 7C, lanes 3 and 4). Adding back either the purified Apaf-2 from HeLa cells or the cc",.u.~..;ial
20 ~to~luu.llc c from bovine heart or rat liver to the imrnnnn~iepleted extracts restored the dATP-depçn~ nt
~ dlioll of cpp32 and DNA rl .~ ;nn (Figure 7A and 7C~ lanes S-10)~ The ..c~ ion of cytochrome
c ~ tioll of CPP32 was evident with the addition of 0.01 ~g (33 nM) of purified cytochrome c
to the cytochrome c-depleted e~tracts (Figure 7B). Addition of 0.3 ~g of cytochrome c l~co~ d more than
100% of control activity, in~ tin~ that the cytochrome c in the cytosol is not at saturation level (Figure 7B).
The dATP and cyto~,luull.c c-d~f .\A~ .. l activation of CPP32 and DNA frAgmPnt~tion was accuu.p~i~d by the
morphological change in the co-i~ b~id nuclei that is ~,h~ t~ ic of apoptosis (Figure 7D).
To investigate whether the dATP and cytochrome c-dependent activation of CPP32 is a general
phc~.uul.,,wll, cytosols were prepared from human e...l~.yonic kidney 293 ceils and human monoblastic iellk~mi~
... . . . .. .
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WO 98102579 PCT/US97112090
U937 cells. As shown in Figure 8, S-100 fractions from both cell types co~ d a dATP--l..pf ~. ..1 CPP32
activating activity (Lanes 1, 2 and 7, 8). ~ 'h~ ,letion of cytochrome c from these cytosols resulted in the
loss of CPP32 activating activity (lanes 3, 4 and 9, 10) and addition of purified cytochrome c restored the
activity (lane 5, 6 and 11, 12).
Human cytochrome c is encoded by a single copy nuclear gene ~Evans and Scarpulla (1988) Proc. Natl.
Acad. Sci. USA 85, 9625-9629] which is translated on cytopl~rnic libosvllles as apocytochrome c. The heme
group of cytochrome c is attached to apocyLocl,~ul.le c upon its translocation into mito~h~-n~ria; holocytochrome
c is a soluble protein located in the hllellllelllbl~ulc space of mi~orhon-lria [Gonzales and Neupert (1990) J.
Bioenergetics & Bi~ ru~.c5 22, 753-768]. The presence of cytochrome c in the cytosolic fraction can
therefore be the result of ruptured outer ~ilo~h~nl1rial ll.~ I,I~,c by hypotonic shock during its prep~r~iom
To test this hypothesis, cytosol from HeLa cells was prepared in the presence of 250 mM sucrose to protect
mitochnn~ri~l integrity. The cells were broken gently by dollnring in a sand paper polished piston [Hayakawa
et al. (1993) Mol. Cell. Biochem. 119, 95-103]. Cytosol prepared this way (~cign~t~d S-cytosol) c~ ;,.Pd
little cytochrome c as COIll~ ,d to the cytosol used in the previous ~A~,Ihlle~ (Figure lOA, lanes 1 and 2).
As shown in Figure 9, S-cytosol was incapable of initiating the dATP-dependent activation of CPP32 (lanes 1
and 2) unless purified cytochrome c was added (lanes 3 and 4).
The ,~i~uh~ for uyluchlu~l~e c in the ~rop~otic program in vitro indicates there is increased release
of cytochrome c to the cytosol in cells ul~d~,.boing apoplo~is. HeLa cells were treated with s~ .u~porillc.
Sl~l,us~line is a broad-spectrum inhibitor of protein kinases, and it has been found to be a potent apoptosis
inducer in a variety of cell types [Rueggs and Burgess (1989) Trends Pharmacol. Sci. 10, 218-220; l~robson
et al. (1993) Natl re 361, 365-36; Wang et al. (1996) supra]. Cytosol was prepared from ~ uHIlc-treated
cells using sucrose c.~ ;.;"i.~e buffer, and the cells were dounced by the sand paper polished piston. As shown
in Figure IOB, DLa~lu~l~ulil~e ~ L of HeLa cells resulted in activation of the londogent~us CPP32 as
detected by the cleavage of PARP. S-cytosol from ~ u~vlillc treated HeLa cells c~ fd markedly elevated
cytochrome c as cOI~ d to that from non-treated cells (Figure lOA, lanes 2 and 3). The same phenûlll~,non
was also observed in human monoblastic U937 cells. Arabinosylcytosine, etoposide and mitoxantrone HCI also
- act to initiate apoptosis.
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W O 98/02579 - PCTrUS97112~90
The present invention provides an in vitro system that faithfully duplicates the two best characterized
bio~ .";.~l markers of apoploDis, i.e. DNA r.~,..~ on into nucleosomal r ..6-l.cllls and the activalion of the
ICE-related apoptotic protease CPP32. This in vitro system allowed us to fractionate and begin to isolate the
required co-~ ,nents. One required protein factor was purified to homogeneity and irlPnlifif-d as the human
C~lu~ u~ C.
The present dATP- and cyto~;l..~l,..c c-dependent in vitro d~ ODis system ~ sel.ls a general apoptotic
program. Identical results were obtained from cytosols of HeLa cells, human embryonic kidney 293 cells, and
human monoblastic U937 cells.
There have been several previous reports of cell-free a~u~)lOSiS systerns based on extracts from
10 hormone-treated Xenopus eggs [N~ ,l et al. (1994) supra], double Dyll~hlu~ d mitotic chicken hlop~--m:l
cells (T ~bnik et al. (1993) supral, or extracts from Fas, UV irradiated and ceramide treated cells lEeari et
al. (1995) supra; Martin et al. (1995) supra]. Our system differs from the previously reported systems in that
it uses extracts from normally growing cells which have not been induced to undergo apoptosis. This allows
apoptosis to be initiated in vitro. Because it uses only soluble comron~n~C, the system is amenable to
fr~rtion~til~n and l~c~ n
In vitro ~O~)loD;s in our system was initiated by the addition of dATP. Although the finding that dATP
plays a critical role for inili~-ion of aroptosiC in vitro was empirical, dATP has long been implicated in cell
death. The best known case is the inherited ll. r~-: -l y of ~ ~o~;.lr ~ c~ (ADA), which results in severe
co,l.l,il,ed immnnn-lcfi. :~ .I. y (SCID). In ADA patients, there is an a~ rms~1 qt~cl-m~ tirm of dATP up to mM
~0 level in their Iymphocytes and death of CD8~~W tr~nCi~inn~ and CD4-CD8 double-positive thymocytes by an
l e~ [Cohen et al. (1978) Proc. Natl. Acad. Sci. USA 75, 472-476; Goday, A. et al. (lg85)
Bio~nl. Pharm. 34, 3561-3569; B~ Y~ iDte and Cohen (1995) Proc. Natl. Acad. Sci. USA 92, 8373-8377].
It has also been reported that de~"-y ' ~ td~lllenl of cultured chick embryonic D~ .. lir neurons results
in the ~rc-lmlll ltion of dATP and death through apoptosis ~Wakade et al. (1995) J. Biol. Chem. 270, 17986-
17992]. N~ ,onal cell death was ~ y~llt~d by an l.ll~lFo~ c kinase inhibitor, sll~g~s~ing that dATP
~cnmlll ~tion was the cause of cell death [Wakade et al. (1995) supral. Our finding that dATP can initiate the
activation of CPP32 and DNA fi~..F..u f ;on provides a .II ~ lic explanation for the dATP m~ tPd cell
14
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W O 98/02579 PCTrUS97/12090
toxicity. dADP can s ~ for dATP. In cells treated with an ADA inhibitor, dADP also ~rcumlllqt~c7
although to a lesser extent than dATP [Goday et al. (1985) supra].
The fractionation of the factors necessary for dATP-d~ activation of CPP32 resulted in the
i~orltifir~io~ of soluble cytochrome c'as one of the nc.,ess~y CO~ JOJl~ for apoptosis in vitro. It is unlikely
5 that cytochrome c mimics the function of another protein, because cytochrome c is the only protein with Apaf-2
activity purified from the S-100 fraction. The lc4ui~ t for cytochrome c was cnnfirmPd by the depletion
and .~co~ ion r~
Cytochrome c is an essential colllyulle~ll of the mitorhf-n~1rial lI,i,y;ldtUly chain. It is a soluble protein
which is localized in the i,,l~ ",. ~hlall~, space and is loosely attached to the surface of the inner mitochondrial
10 ll~,lll,.,u~e [Gonzales and Neupert (1990) supra]. Cytochrome c is translated by cytoplasmic ribosomes and
follows a unique pathway into ",;I<~h~ ia which does not require the signal se~u---re, electro-chemical
potential, and general protein tr~nC~oc ~tinn ~ ..,hill~,.y [Mayer et al. (1995) J. Biol. Chem. 270, 12390-12397).
Mito~hon~iria have been implicated in apoptosis since the discovery that the bc1-2 farnily of proteins
are located in the outer mitorhontlri~l lu.,.llbl~lc ~Monaghan et al. (1992) supra; Krajewski et al. (1993) supra;
15 de Jong et al. (1994) supra]. In vitro apoptosis in Xenopus egg extracts requires a dense organelle fraction
enriched in l..;tv~J.-.. hia [Nc~ . et al. (1994) supra]. The present inventors have shown that purified
mits)~h~-n~lria from hamster heart can ~uyplc~ nl cytosol immlln- ~PplPt~d of cytochrome c, or cytosol prepared
in the presence of sucrose to support CPP32 activating reaction. However, a potential ~.llll.,.ll against the
involvement of l"iLo~ l,o~ in apoptosis comes from a report that ~oyLosis and bc1-2 protection of apoptosis
20 are normal in cells lacking ",;lo~ Ariq DNA [Jqrobsnn et al. (1993) supra] . None of the known ",i~orl~
r~ c, such as ATP profl~r~ion electron transfer, o~cidative phosy~lylation, generation of reactive o~ygen
species and Ca~+ uptake, appear to account for its involvement in apoptosis [Jacobson et al. (1993) supra;
HG-~ ~h~lY et al. (1993) Cell 75, 241-251; Nc....l~"~.,. et al. (1994) supra]. That cytochrome c is a nece~.D~uy
celllyo~ l of cellular apoptotic program indicates that mitorhon-1ria are involved in clyuylO5~iS by releasing
25 cytoclllul.,~ c. Because cyl-xll,u..lc c is encoded by a nuclear gene and tr~ cloc~tirn of apocytochrome c into
orl~ a does not require I~ bl~lC potential and general protein trpnclo~Dtion l.la~Li.l.,ly [Evans and
Scarpulla (1988) supra; Mayer et al. (1995) supra], it can be totally funr~inn~' in dyOytOSiS in cells lacking
mitorhontlrjal DNA. Cr~nci~tpnt with this model, the cells undergoing apoptosis induced by S~alllO~ululC
CA 02260766 1999-01-12
W 098/02579 PCT~US97/12090
showed il~clcdscd cytosolic ~,yloC]l~u~l~C c. Release of cytochrome c into the cytosol provides a target for
regulation of apoptosis, possibly by the bc1-2 family of proteins.
The biochernical merhqni~m of cytochrome c function in the activation of CPP32 remains to be
d~ t~ ,.,i..?l 7'he ~U.;r~ cn and cha~ io~- of Apaf-1, which is at least one other component required
S for the CPP32 activation reaction, will provide further llnrl.. :~lh.~(~i"~ of the early events in apoptosis.
Monoclonal or polyclonal qn~ibo~iPs~ preferably mono~ l~)n~l, crecihr~ ly reacting with a target protein
can be made by methods known in the rt. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Labo~aloli~s; Goding (1986) Monoclonal Antibodies: Principles and Practice, 2d
ed., Acadernic Press, New York; and Ausubel et al. (1987) supra.
St. ndard l~ s for cloning, DNA isolation, amplification and purification, for enzymatic reactions
involving DNA ligase, DNA polymerase, restriction ~n~on~lelelcPC and the like, nd various separation
t~ rhni~nP.c are those known and co~r~ nly employed by those skilled in the art. A number of standard
t: ' iqllPc are ~escnhed in Sambrook et al. (1g89) Molecular Cloning, Second Edition, Cold Spring Harbor
Labo.aloly Press, Plainview, New York; Maniatis et al. (1982) Molecular Cloning, Cold Spring Harbor
Lal)olduly Press, Pl, inview, New York; Wu (ed.) (1993) Meth. Enzymol. 218, Part l; Wu (ed.) (1979) Meth
Enzymol. 68; Wu et al. (eds.) (1983) Meth. Enymol. 100 and 101; Crus~ nd Moldave (eds.) Meth.
Enymol. 65; Miller (ed.) (1972) Exp~,i,..~,.ls in Molecular Genetics, Cold Spring Harbor Ldhcr-dl~Jly, Cold
Spring Harbor, New York; Old and Primrose (1981) Principles of Gene Manipulation, University of California
Press, Berkeley; Schleif and Wensink (1982) Practical Methods in Molecular Biology; Glover (ed.) (1985~ DNA
Cloning Vol. I .nd Il, IRL Press, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic Acid Hybridization,
IRL Press, Oxford, UK; and Setlow and Hnllq~n-l~r (1979) Genetic Engineering: Principles and Met~ods, Vols.
14, Plenum Press, New York. Ab~l.,vialiOl1s and ~omPnrlqhlre~ where employed, are deemed standard in the
field and cc""~ ly used in professional journals such as those cited herein. All .~ ,..ces cited herein are
incol~u~ d ~y ,~,f~ ce in their entirety.
16
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WO 98/02579 - PCT/US97/12090
EXAMPLES
Example 1. General Methods and Materials
Nucleotide Ll;pi~n~h"~ . were purchased from Pharmacia (Piscataway, NJ). ADP, dADP, AMP,
dAMP, ~ nnSin~ and deoxyal~nnginp were from ICN BiomP~lic:~lc, Inc. (Costa Mesa, CA). Pepstatin A,
5 I~ hl, N-acetyl-leucyl-leucyl-norleucine (ALLN) were obtained from Boehringer h/l~nnhpim Corporation
(Indianapolis, IN). Pl~c.lyl~ l-sulfonyl fluoride (PMSF), Imidazole, cAMP, aprotinin, bovine heart
cytochrome c and rat liver cytochrome c were purchased from Sigma l'hPm~ Co. (St. Louis, MO). 35S-
.... ~I.io.~ was purchased from ~m.orsll~m Corporation (Arlington Heights, IL). Molecular weight standards
for SDS-PAGE and gel-filtration ulll~Jllldography were obtained from Bio-Rad Laboratories (Hercules, CA).
cDNA clones of human SREBP-2 and hamster CPP32 were described in Wang et al . (1995) and protein
con.,e..lldlion was d~lr~ d by the Bradford method [Bradford, M.M. (1976) Anal. Biochem. 72, 248-254].
Silver staining was carried out using a Silver Stain Plus kit from Bio-Rad Laboratories (Hercules, CA).
Plasmids were purified using a Me~;~l.,~) kit (Qiagen, Chatsworth, CA).
Example 2. Pr~-p~ra~iorl of S-100 Fractions from HeLa cells, 293 cells and U937 cells
Human HeLa S3 cells were grown as described [Wang et al. (lg93) J. Biol. Chem. 268, 14497-14504].
The cells (5 x 1051ml) were ha.~,eDted by ~. ~Ir;r ~ on at 1,800 x g for 10 min at 4~C. After washed once
with ice-cold pho~l.h~P buffered saline (PBS), the cell pellet was suspPn-lPd in S volumes of ice-cold buffer A
r20 mM Hepes-KOH, pH 7.5, 10 rnM KCI, 1.5 mM MgCI2, I mM sodium EDTA, I mM sodium EGTA, 1
mM dithiothreitol (DTT) and 0.1 mM PMSF~ s"rF' ~ with protease i.~il,ilu.~ (5 ,u/ml pepstatin A, 10
,uglrnl h ~ ;.., 2 ~gml aprotinin, and 25 ~g/rnl ALLN). After holding on ice for 15 rnin, the cells were
.LDI~. ~ by ~iountitlg 15 times in a 100 ml Kontes douncer with the B pestle (Kontes Glass Co., Vineland, NJ).
The nuclei were centrifuged at 1000 x g for 10 rnin at 4~C. The Du~c...d~ was further centrifuged at 105 X
g for 1 hr in a R~- L ~ Sw 28 rotor. The resulting ~ (S- 100 fraction) was stored at -80~C and used
25 for the in vitro apoptosis assay and the starting rnaterial for the purification of Apaf-2.
293 cells were set up at 5 x 105 cells per 100 mm dish in medium A [Dulbecco's mnrlifiPd Eagle's
medium (DMEM) s~MIPm~nt~ with 10% (v/v) heat-i..a~;lival~d fetal calf serum, 100 U/ml penicillin and 100
~g/rnl streptomycin sulfate]. After inrllb~rinn for 48 hr at 37~C in a 5% CO2 incubator, the cells were
CA 02260766 1999-01-12
WO 98/02579 PCTJUS97112090
harvested, collected by centrifugation (1000 g, 10 min, 4~C). U937 cells were set up at 5 x 105 cell/ml in
medium B [RPMI 1640 medium supp'- .d with 10% fetal calf serum, 100 U/rnl penirillin and 100 ~g/ml
streptomycin sulfate]. After incubation for 48 hr in a 5% C0~ incubator, the cells were collected by
centrifugation (1000 g, 10 min, 4~C). The cell pellets of 239 cell and U937 cell were washed once with ice-
cold PBS and ~ .c~ rd in 5 volumes of ice-cold buffer A ~ le .l.. lrd with protease inhibitors. Aher
holding on ice for 15 min, the cells were broken by passing 15 times through a G22 needle. After
ce.~l~iru~;dlion in a miclu~ ruge for 5 min at 4~C, the ~ were further centrifuged at 105 x g for
30 min in a table top ul~ ce..lliruge (Re~kn~~;, Illallul~ , Fullerton, CA). The resulting sl~pprn~-t~t tC were
used for the in vitro apoptosis assay.
Example 3. In vitro Tr-~-ncloti~n of CPP32, SREBP, and PARP
A PCR rl~lll~,.ll encoding amino acids 29-277 of harnster CPP32 [Wang et al. (1996) supra] was
cloned into Ndel and BarnHI sites of pET 15b vector (Novagen, Madison, WI). The resulting fusion protein
of six hictiflinP5 with hamster CPP32 (arnino acids 29-277) was translated in a TNT T7 Il~l~ ion/translation
kit (Promega, Madison, Wl) in the presence of 35S-m~thit ninr- according to the mP~~llf~tnrer's instructions.
The translated protein was passed through a 1 ml nickel affinity column (Qiagen, Chat~ , CA) equilibrated
with buffer A. After washing the column with 10 ml of buffer A, the translated CPP32 was eluted with buffer
A c~-,u;~ g 250 mM imi-l~71~'~. Human SREBP-2 was translated in a TNT SP6 lla.lsclil)tion/translation kit
as d~ 5e~ ;l.ed lWang et al. (1995) rHua et al. (1993) Proc. Natl. Acad. Sc~. USA 90, 11603-11607] . Full length
human PARP cDNA [Cherney et al. (1987) Proc. Natl. Acad. Sci. USA 84, 8370-B374] was cloned into Smal
and EcoRI sites of pBK-CMV vector (Stratagene, La Jolla, CA) and I -'~ted in a TNT T7
.t;.~ n kit [Promega, Madison, Wll. The translated SREBP-2 and PARP (200 ~1 each) were
purified by passing each translation mixture through a 10-ml Seph,~ G-25 gel-filtration column equilibrated
with buffer A. The translated proteins c~.J..~ d within the exclusion volume of the column were collected.
2~
Example 4. Western Blot Analysis
A ll,ol~oclonal antibody against human CPP32 was purchased from Trncdllrtion Laboratories and a
m~-n~rlon~l antibody against cytochrome c (7H8.2C12) was obtained as de5~. ;l.cd previously [Jc~ on and
CA 02260766 1999-01-12
WO 98/02~79 - PCTIUS9711Z090
Johnson (1991) Proc. N~tl. Acod. Sci. USA 88, 4428-4432]. Monoclonal antibody specific for cytochrome c
is available from ph~rrningf n l~ .oblot analysis was p~l ~ull~ed with horseradish peroxidase-conjugated anti-
mouse immunoglobulin G using the F.nh~nred l~hf mil~l.,.;nf sc- ..re (ECL) Western Blotting Detection reagents
(~mfr~hsm Corporation, Arlington Heights, IL).
Example 5. Assay for dATP-~IJ- .~ 1 Activation of CPP32 Protease
CPP32 was trqncl ~ed and purified as ~ srrihed above. Aliquot of 3 ~1 of the in vitro translated CPP32
was inr~lbqted with the in~ d protein fraction, mlrleoti~lrc, and I n~ additional MgC12 at 30~C for 1 hour
in a final volume of 20 ~1 of buffer A. At the end of the inrllh~ion, 7 ~41 of 4x SDS sarnple buffer was added
10 to each reaction. After boiling for 3 min, each sample was subjected to a 15% SDS-polyacrylamide gel
electrophoresis (SDS-PAG~). The gel was ~ d to a nitrocellulose filter which was sllhseqllf ntly exposed
to a Kodak X-OMAT AR X-ray film (Eastman Kodak, Rochester, NY) for 16 hr at room t~ l~u~e.
Exarnple 6. Purification of Apaf-2 from HeLa S-100
All purification steps were carried out at 4~C. All the chlu~dtography steps except the
phnal.hocf lhllose colurnn were carried out using an q-ltf-mq~ir fast protein liquid cl-ru ~c~raphy (FPLC) station
(Ph~llal~ia, Piscala~,.~/, NJ).
85 m~ of HeLa S-100 was applied to a phncrh~-cellulose column (40 ml bed volume) equilibrated with
Buffer A. The column was washed with 3 column volumes of buffer A and eluted with 2 column volumes of
buffer A c4~-lh;.. ;llg 0.5 M NaCl.. A.. ,.. ~ ,. sulfate (50%) was added directly to the ph~ hl~c~ sf 0.5
M eluate. After rotating at 4~C for 1 h, the _ixture was cfnr~ çd at 15,000 rpm for 15 min in a JA 20
rotor (P~erlrmqn In~llu~ ts, Fullerton, CA). The ~ was directly applied to a 10 ~ phenyl-agarose
colurnn [phenyl-sepharose, Pha~ a ia, Piscataway, NJl equilibrated with buffer A cu l;.; ;..g 50% ~
sulfate. The column was washed with two bed volumes of buffer A c~ i. g 50% ~ sulfate and
25 eluted with buffer A ~o~ g 1 M -~ sulfate. The eluate was loaded onto a Superdex-200 gel
filtration column (Phs-mqriq. Pisca~a~.a~, NJ) (300 ml) equilibrated with buffer A and eluted with the same
buffer. Fractions of 10 ml were collected and assayed for Apaf-2 activity. The active fractions from the gel-
filtration colum~n were pooled and loaded onto an anion eYrhqnge Mono Q 5/5 column and a cation eYrh~ nge
19
CA 02260766 1999-01-12
WO 98/02579 PCTIUS97/lZ090
Mono S 5/5 column corlllf~ d together. The columns were pre-equilibrated with Buffer A. After loading, the
columns were ~ CO~ f~ ~ ,i and the Mono S column was washed with S rnl of buffer A c-. l;.;..i..g 0 1 M NaCl
and the Apaf-2 activity was eluted from the column with a 20 ml 0.1-0.3 M linear NaCI gradient. Fractions
of 1 ml were co!lPc~ed
s
Exarnple 7. Preparation of Hamster Liver Nuclei
Livers from 4 male Golden Syrian hamsters (Sasco) were rinsed with ice-cold pho~hale-buffered saline
(PBS) and holllo6~ ,d in 0.25 glml of buffer B (10 mM Hepes-KOH, pH 7.6, 2.4 M sucrose, 15 mM KCI,
2 mM sodium EDTA, 0.15 mM ~l,llhlc, 0.15 mM ~rPnni-iine~ 0.5 mM DTT, 0.5 mM PMSF) by three
10 strokes of a motor-driven homogenizer. The ho...ng~ ~rs were centrifuged through a 10-ml cushion of buffer
B at 25,000 rpm for 1 h in a SW 28 rotor at 4~C. The nuclei pellet was resl-~pPndPd in buffer C (10 rnM
PIPES, pH 7.4, 80 mM KCI, 20 mM NaCI, 5 mM sodium EGTA, 250 rnM sucrose, and 1 mM Dl~) at 8.5
x 107 nuclei/ml and stored at -80~C in multiple aliquots.
Example 8. DNA r,~ ion Assay
Aliquots of 50 ~l HeLa cell S-100 and 6~1 hamster liver nuclei were infrlh~Pd at 37~C for 2 h with
1 mM ~litinn~l MgCl2 in the absence or presence of 1 rnM indicated nucleotide. After incubation, an aliquot
of 500 ~ul buffer D (100 rnM Tris-HCI, pH 8.5, 5 mM EDTA, 0.2 M NaCI, 0.2% wlv SDS, and 0.2 mg/rnl
~ r --. K) was added to each reaction and inrllb~pd at 37~C overnight. NaCI was then added to a final
20 f~ ;on of 1.5 M, and the nuclear debris was spun down for 15 tnin in a llliwu~e~llliruge at room
t~ ci. The DNA in the s-~ .t was pl~ d with an equal volume of 100% (vlv) ethanol. The
DNA p~ ) was washed once with 70% ethanol and l~ Afd in 40 ,ul of buffer E t....l;;~.;.lglO rnM
Tris-HCI, pH 7.5, 1 mM sodium EDTA, and 200 ~glrnl DNAse-free RNase A (Wollhillg~ull Bie~ f~l
Corporation, Freehold, NJ). After i... ~Ib~l;nn at 37~C for 2 hr, the DNA was loaded onto a 2% agarose gel
and ele~,l.u~hu~sis was con~ t~d at 50 V for 2 hr in 0.5 x TBE buffer (1 x TBE buffer contains 90 mM Tris-
borate/2 rnM EDTA). The gel was stained with 2 ~glml ethidium bromide for 15 min, df~ d with water
for 1 hr, and the DNA was visualized using UV light.
. . .
CA 02260766 1999-01-12
W O 98/02579 PCT~US97/12~90
Example 9. Tmmllno~pletj(m of Cytochrome c from HeLa S-100
An anti-cytochrome c mnn~ n~l antibody (6H2. B4) which l~,C(~ the native form of cytochrome
c was described previously [Jemmerson et al. (1991) Eur. J. Immunol. 21, 143-151]. An aliquot of 100 ~1
(0.7 mg/ml of IgG 2A) of this antibody was in~uh7~Pd with a 1:1 mixture of 50 ~1 protein A and protein G
S agarose beads ~ d in 200 ~1 of PBS (Santa Cruz) ai 4~C for 3 hr. The beads were collected by
centrifllg~tion for 15 min in a mi~;lu~ -iru~ at 4~C. After removal of the sllrPrn ~f~rlt, the beads were washed
once with 1 ml of buffer A and inr~ ; with 1.5 ml S-100 fractions for 5 hr in a rotator at 4~C. The beads
were sllbs~qu~ntly pelleted by centrifugation for 15 min in a n~icrocentrifuge at 4~C. The su~ was used
as S-100 immlmt (lepleted of cytochrome c.
The foregoing examples and disclosure are provided for illustrative puIposes, and they are not intended
to limit the scope of the invention as provided herein. Any variations in the e~ nrlifi. d c~,l")osilions and
methods which occur to the skilled artisan are intended to fall within the scope of the present invention.
Ta~le I. Purification of Apaf-2 from l~eLa cells
S-100 was prepared from 20-liters of HeLa cells in spinner culture as d~i,.;.i~ed in the E~amples. An aliquot
of each fraction was dialyzed against buffer A and the Apaf-2 activity was assayed by ~t;.,~,..ll i~li--g with 35S-
labeled CPP32 at four conc~ ions of protein. The results were quantified by phosphorim~in~.
Step Fraction Protein Specific Total Purification Recovery
Activity Activity
mg units/mg unit -fold %
1 S-100 348.5
2 Phosphocellulose 104 126.6 13166 1 100
3 50% Ammonium- 23.8 833.3 19824 6.6 150
Sulfate Precipitation
4 Phenyl-Sepharose 0.473 42145 19934 333 151
5 Superdex-200 0.460 43367 19950 343 152
6 Mono Q/Mono-S 0.076 263150 20000 2079 152
a Protein concentrations of various fractions were determined by the Bradford method.
35 b One unit of activity is defined as the cleavage of 1 % of the input substrate in 60 min.
CA 02260766 1999-01-12
W 098/02579 PCTrUSg7112090
~able ll. Sequences of tryptic peptides from the 1 5-kDa Apaf-2: comparison withhuman cytochrome c
Sequences were obtained from Edman degradation performed on the HPLC-purified
tryptic (Lys-C) peptides generated from the SDS-PAGE purified 15 kDa Apaf-2. Thesequence of human cytochrome c was reported by Evans and Scarpulla, 1988, supra.o The * denotes a residue in Apaf-2 that could not be assigned based on peptide sequence
analysis. Numbers in parentheses denote the arnino acid position in the cDNA sequence
of human cytochrome c.
Tryptic peptide
1 . EERADLIAY (89-96) (SEQ ID NO: 1 )
2. TGPNLHGLFGR (28-38) (SEQ ID NO:2)
3. TGQAPGYSYTAANK (40-53) (SEQ ID NO:3)
4. YIPGTK (74-79) (SEQ ID NO:4)
5. *II*GEDTLMEYL (56-68) (SEQ ID NO:5)
6. IFIMK (9-13) (SEQ ID NO:6)
7. TGPNL (28-32) (SEW ID NO:7)
CA 02260766 l999-0l-l2
W O 98/02579 PCTrUS97/12090
SEQUENCE LISTING
(1) GENERAL INFORMATION:
~i) APPLICANT: EMORY UNIVERSITY
~ii) TITLE OF INVENTION: Regulation of Apoptosis and In Vitro
Model for Studies Thereof
(iii) NUMBER OF SEQUENCES: 7
~iv) CORRESPONDENCE ADDRESS:
~A) ADDRESSEE: Greenlee, Winner and Sullivan, P.C.
(B) STREET: 5370 Manhattan Circle, Suite 201
(C) CITY: Boulder
(D) STATE: Colorado
(E) COUNTRY: US
(F) ZIP: 80303
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #l.o, Version #1.30
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: WO
(B) FILING DATE: 11-JUL-1997
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/021,268
(B) FILING DATE: 12-JUL-1996
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Ferber, Donna M.
(B) REGISTRATION NUMBER: 33,878
(C) REFERENCE/DOCKET NUMBER: 45-96 WO
~ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (303) 499-8080
(B) TELEFAX: (303) 499-8089
(2) INFORMATION FOR SEQ ID NO:1:
Q~N~ CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: amino acid
(C) sTR~Nn~nNR-~s single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Glu Glu Arg Ala Asp Leu Ile Ala Tyr
1 5
23
CA 02260766 l999-0l-l2
WO 98/02579 PCT/US97/12~90
~2~ INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 11 amino acids
B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Thr Gly Pro Asn Leu His Gly Leu Phe Gly Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 14 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: sinyle
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Thr Gly Gln Ala Pro Gly Tyr Ser Tyr Thr Ala Ala Asn Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
~B) TYPE: amino acid
(C) STR~N~ N~ S: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Tyr Ile Pro Gly Thr Lys
1 5
24
CA 02260766 1999-01-12
W O 98/02579 PCT~US97/lZU90
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 13 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(ix) FEATURE:
(A) NAME/KEY: Modified-site
(B) LOCATION: 1..13
(D) OTHER INFORMATION: /note= "X at positions 1 and 4 were
not identified in analysis of amino acid sequence.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Xaa Ile Ile Xaa Gly Glu Asp Thr Leu Met Glu Tyr Leu
1 5 lO
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ile Phe Ile Met Lys
1 5
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LEN¢TH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: not relevant
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Thr Gly Pro Asn Leu
1 5
