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CA 02519161 2002-07-08
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SIR2 ACTIVITY
CLAIM OF PRIORITY
This application claims priority under 35 USC ~ 119(e) to U.S. Patent
Application Serial
No. 60/303,370, filed on July 6, 2001, and U.S. Patent Application Serial No.
60/303,456, also
filed on July 6, 2001, the entire contents of which are hereby incorporated by
reference.
GOVERNMENT SUPPORT
The invention was supported, in whole or in part, by a grant RO1 CA78461 to
RAW;
NHhBl/NIFI Fellowship to SKD K08 HL04463. The U.S. Government has certain
rights in the
invention.
BACKGROUND
Regulation of the cell cycle is important in homeostasis of both cells and
organisms (e.g.,
mammalian cells or mammals). Disruptions in the normal regulation of the cell
cycle can occur,
for example, in tumors which proliferate uncontrollably, in response to DNA
damage (e.g.,
ionizing radiation) to the cell or organism, and under conditions of stress
(e.g., oxidative stress)
in the cell or organism.
The p53 tumor suppressor protein exerts anti-proliferative effects, including
growth
arrest, apoptosis, and cell senescence, in response to various types of
stress, e.g., DNA damage
(Levine, 1997; Giaccia and Kastan, 1998; Drives and Hall, 1999; Oren, 1999;
Vogelstein et al.,
2000). Inactivation ofp53 function appears to be critical to tumorigenesis
(Hollstein et al.,
1999). Mutations in the p53 gene have been shown in more than half of all
human tumors
(Hollstein et al., 1994). Accumulating evidence further indicates that, in the
cells that retain
wild-type pS3, other defects in the p53 pathway also play an important role in
tumorigenesis
(Drives and Hall, 1999; Lohrum and Vousden, 1999; Vousden, 2000). The
molecular.fixnction of
p53 that is required for tumor suppression involves its ability to act as a
transcriptional factor in
regulating endogenous gene expression. A number of genes which are critically
involved in
either cell growth arrest or apoptosis have been identified as p53 direct
targets, including
p21CIP1/WAF1, Mdm2, GADD45, Cyclin GS 14-3-3F, Noxa, p53AIP1, PUMA, and others
CA 02519161 2002-07-08
,. '"°"' WO 03/004621 PCT/US02/21461
(Nakano and Vousden, 2001; Yu et al., 2001; Oda et al., 2000a, 2000b; El-
Deriry et al., 1993;
Wu et al., 1993; Barak et al., 1993; Kastan et al., 1992; Olcamoto and Beach,
1994).
p53 is a short-lived protein whose activity is maintained at low levels in
normal cells.
Tight regulation of p53 is essential for its effect on tumorigenesis as well
as maintaining normal
cell growth. The precise mechanism by which p53 is activated by cellular
stress is not
completely understood. It is generally thought to involve primarily post-
translational
modifications of p53, including phosphorylation and acetylation (reviewed in
Appella and
Anderson, 2000; Giaccia and Kastan, 1998). Early studies demonstrated that
CBP/p300, a
histone acetyl-transferase (HAT), acts as a coactivator of p53 and potentiates
its transcriptional
activity as well as biological function in vivo (Gu et al., 1997; Lill et al.,
1997; Avantaggiati et
al., 1997). Genetic studies have also revealed that p300 mutations are present
in several types of
tumors, and that mutations of CBP in human Rubinstein-Taybi syndrome as well
as CBP
knockout mice lead to higher risk of tumorigenesis, further supporting an
important role for this
interaction in the tumor suppressor pathway (reviewed in Goodman and Smolik,
2000; Gile et
al., 1998; Kung et al., 2000; Gayther et al., 2000). Significantly, the
observation of functional
synergism between p53 and CBP/p300 together with its intrinsic HAT activity
led to the
discovery of a novel FAT (Transcriptional factor acetyl-transferase) activity
of CBP/p300 on p53
which suggests that acetylation represents a general functional modification
for non-histone
proteins in vivo (Gu and Roeder, 1997) which has been shown for other
transcriptional factors
(reviewed in Kouzarides, 2000; Sterner and Berger, 2000; Muth et al., 2001).
p53 is specifically acetylated at multiple lysine residues (Lys 370, 371, 372,
381, 382) of
the C-terminal regulatory domain by CBP/p300. The acetylation of p53 can
dramatically
stimulate its sequence-specific DNA binding activity, perhaps as a result of
an acetylation-
induced conformational change (Gu and Roeder, 1997; Sakaguchi et al., 1998;
Liu et al., 1999).
By developing site-specific acetylated pS3 antibodies, CBP/p300 mediated
acetylation of p53
was confirmed in vivo by a number of studies (reviewed in Chao et al., 2000;
Ito et al., 2001). In
addition, pS3 can be acetylated at Lys320 by another H.AT cofactor, PCAF,
although the in vivo
functional consequence needs to be further elucidated (Sakaguchi et al., 1998;
Liu et al., 1999;
Liu et al., 2000). Steady state levels of acetylated p53 are stimulated in
response to various
types of stress (reviewed in Ito et al., 2001).
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Recently, by introducing a transcription defective pS3 mutant (pS3Q2SS26) into
mice, it
was found that the mutant mouse thymocytes and ES cells failed to undergo DNA
damage-
induced apoptosis (Chao et al., 2000; Jimenez et al., 2000). Interestingly,
this mutant protein
was phosphorylated normally at the N-terminus in response to DNA damage but
could not be
acetylated at the C-terminus (Chao et al., 2000), supporting a critical role
of pS3 acetylation in
transactivation as well as pS3-dependent apoptotic response (Chao et al.,
2000; Luo et al., 2000).
Furthermore, it has been found that oncogenic Ras and PML upregulate
acetylated pS3 in normal
primary fibroblasts, and induce premature senescence in a p53-dependent manner
(Pearson et al.,
2000; Ferbeyre et al., 2000). Additionally acetylation, not phosphorylation of
the pS3 C-
terminus, may be required to induce metaphase chromosome fragility in the cell
(Yu et al.,
2000). Thus, CBP/p300-dependent acetylation of pS3 may be a critical event iii
pS3-mediated
transcriptional activation, apoptosis, senescence, and chromosome fragility
In contrast, much less is known about the role of deacetylation in modulating
pS3
function. Under normal conditions, the proportion of acetylated pS3 in cells
remains low. This
may reflect the action of strong deacetylase activities in vivo. The
acetylation level of pS3 is
enhanced when the cells are treated with histone deacetylase (HDAC) inhibitors
such as
Trichostatin A (TSA).. These observations led to identification of a HDAC1
complex which is
directly involved in pS3 deacetylation and functional regulation (Luo et al.,
2000; Juan et al.,
2000). PID/MTA2, a component of the HDAC1 complex, acts as an adaptor protein
to enhance
HDAC1-mediated deacetylation of pS3 which is repressed by TSA (Luo et al.,
2000). In
addition, Mdm2, a negative regulator of pS3, actively suppresses CBP/p300-
mediated pS3
acetylation, and this inhibitory effect can be abrogated by tumor suppressor
pl9ARR,
Acetylation may have a critical role in the pS3-MDM2 pl9ARF feed back loop
(Ito et al., 2001;
Kobet et al., 2000).
The Silent Information Regulator (SIIZ) family of genes represents a highly
conserved
group of genes present in the genomes of organisms ranging from archaebacteria
to a variety of
eukaryotes (Frye, 2000). The encoded SIR proteins are involved in diverse
processes from
regulation of gene silencing to DNA repair. The proteins encoded by members of
the SIRZ gene
family show high sequence conservation in a 2S0 amino acid core domain. A
well=characterized
gene in this family is S. cerevisiae SIR2, which is involved in silencing HM
loci that contain
information specifying yeast mating type, telomere position effects and cell
aging (Guarente,
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1999; Ka.eberlein et a1.,1999; Shore, 2000). The yeast Sir2 protein belongs to
a family ~f
histone deacetylases (reviewed in Guarente, 2000; Shore, 2000). The Sir2
homolog, CobB, in
Salnaonella typhiryZUrium, functions as an NAD (nicotinamide adenine
dinucleotide)-dependent
ADP-ribosyl transferase (Tsang and Escalante-Semerena,1998).
The Sir2 protein is a deacetylase which uses NAD as a cofactor (Imai et al.,
2000;
Moazed, 2001; Smith et al., 2000; Tanner et al., 2000; Tanny and Moazed,
2001). Unlike other
deacetylases, many of which are involved in gene silencing, Sir2 is
insensitive to histone
deacetylase inhibitors like trichostatin A (TSA) (Imai et al., 2000; Landry et
al., 2000a; Smith et
al., 2000).
Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NAD hydrolysis,
producing
nicotinamide and a novel acetyl-ADP ribose compound (1-O-acetyl-ADP-ribose)
(Tanner et al.,
2000; Landry et al., 2000b; Tanny and Moazed, 2001). The NAD-dependent
deacetylase
activity of Sir2 is essential for its functions which can connect its
biological role with cellular
metabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al., 2000;
Smith et al.,'2000).
Mammalian Sir2 homologs have NAD-dependent histone deacetylase activity (Imai
et al., 2000;
Smith et al., 2000). Most information about Sir'Z mediated functions comes
from the studies in
yeast (Gaxtenberg, 2000; Gottschling, 2000).
Among Sir2 and its homolog proteins (HSTs) in yeast, Sir2 is the only protein
localized
in nuclei, which is critical for both gene silencing and extension of yeast
life-span (reviewed in
Guarente, 2000). Based on protein sequence homology analysis, mouse Sir2a
and,its human
ox~tholog SIRTl (or human Sir2a or hSir2) are the closest homologs to yeast
Sir2 (Imai et al.,
2000; Frye, 1999, 2000) and both exhibit nuclear localization (Figure 7C).
Homologues of Sir2
have been identified in almost all organisms examined including bacteria,
which has no histone
pxoteins (reviewed in Gray and Ekstrom, 2001; Frye, 1999; 2000; Brachmann et
al., 1995). For
this reason it is likely that Sir2 also targets non-histone proteins for
functional regulation (Muth
et al., 2001).
The S. cerevisiae Sir2 is involved in DNA damage responses (Martin et al.,
1999;
McAinsh et al., 1999; Mills et al., 1999). In mammalian cells, one of the
primary mediators of
the DNA damage response is the p53 protein (Levine, 1997; Oren, 1999;
Vogelstein et al.,
2000). Following DNA damage, the p53 protein is protected from rapid
degradation and
acquires transcription-activating functions, these changes being achieved
largely through post-
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translational modifications (Abraham et al., 2000; Canman et al., 1998; Chehab
et al., 1y99;
Sakaguchi et al., 1998; Shieh et ad., 2000; Siliciano et al., 1997).
Transcriptional activation of
p53 protein in turn upregulates promoters of a number of genes including
p21WAF1 (eI-Deiry et
al., 1993) that promotes cell cycle exit or death-inducing proteins lilce PIDD
(Lin et al., 2000).
The p53 protein is phosphorylated in response to DNA damage (Siliciano et al.,
1997).
There are at Ieast 13 different residues both at the N and C terminal portions
of p53 protein that
are phosphorylated by various kinases (Appella and Anderson, 2000). For
example, the ATM
and ATR proteins phosphorylate p53 at residue SerlS (Khanna et al., 1998;
Siliciano et al.,
1997; Tibbetts et al., 1999) and Chk1/2 kinases at residue Ser20 (Chehab et
al., 1999; Shieh et
al., 2000).
Modification of SerlS is importazxt for the functional activation of the p53
protein.
Phosphorylation of SerlS may increase the affinity of the p300 acetylase for
p53 (Dumaz and
Meek,1999; Lambert et al., 1998).
p53 is acetylated in vitro by p300 at Lys 370-372, 381 and 382 (Gu and Roeder,
1997).
In response to DNA damage, p53 is also acetylated in viva at Lys 373 and Lys
382 (Abraham et
al., 2000; Sakaguchi et al., 1998). Other factors that can affect acetylation
of p53 include
MDM2 protein, which is involved in the negative regulation of p53 (Oren, 1999)
and can
suppress acetylation of p53 protein by p300 (Ito et al., 2001; Kobet et al.,
2000). While
acetylation by p300 and deacetylation by the TSA-sensitive HDAC1 complex (Luo
et al., 2000)
have been shown to be important in regulation of p53 protein activity, the
remaining factors
responsible for its regulation as a transcription factor remain elusive.
Analogs of NAD that inhibit endogenous ADP-ribosylases reduce induction of p21
WAF1
in response to DNA damage and overcome p53-dependent senescence (Vaziri et
al., 1997). In
addition, p53 protein can bind to the NAD-dependent poly-ADP-ribose
polymerase.
The S1R complex in Saccharomyces cerevisiae was originally identified through
its
involvement in the maintenance of chromatin silencing at telomeres and at
mating type loci. It is
composed of four components, Sirlp, Sir2p, Sir3p, and Sir4p, that normally
reside at yeast
telomeres. In response to DNA damage, the STR complexes relocate to the site
of double-
stranded breaks where they participate in the repair of the lesions by non-
homologous end
joining. This ANA damage response is dependent on the function of the
MEC1/RAD9 DNA
checkpoint pathway MEClis a homolog of the ATM protein that coordinates the
DNA damage
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response in mammalian cells, in part by triggering the cascade of events that
lead to the
stabilization of the pS3 protein (Canman et al., 199$). Another major function
of Sir2, gene
silencing, is closely tied to the regulation of lifespan in S. cerevisiae
(Guarente, 1999).
Double-strand breaks in the genome of mammals invoke a cascade of signaling
events
that ultimately cause phosphorylation and subsequent stabilization of pS3
protein. In addition,
these strand breaks lead to activation of p53 protein as a transcription
factor. This activation may
be due largely to its acetylation (Gu and Roeder, 1997; Sakaguchi et al.,
I998). The resulting
stabilized, activated pS3 protein contributes to the upregulation of cyclin-
dependent kinase
inhibitors such as p21WAF1 and hence to the cytostatic effects ofp53.
Alternatively, depending
on the cellular background or degree of damage, the apoptotic effects of pS3
may predominate
through its ability to induce expression of pro-apoptotic proteins such as
PIDD (Lin et al., 2000).
These various phenomena indicate that specific components of the machinery
that monitors the
integrity of the genome are clearly able to alert pS3 to the presence of
genetic damage, leading to
its functional activation. Conversely, in the event that damage has been
successfully repaired,
signals must be conveyed to pS3 in order to deactivate it. Thus, a cell cycle
advance that has
been halted by p53 to enable repair to proceed should be relieved following
completion of repair,
enabling the cell to return to its active growth state. For this reason, the
inactivation of pS3
becomes as important physiologically as its activation.
In light of this information, modulators of Sir2 and/or p53 activity would be
useful in
modulating various cellular processes including, e.g., repair of DNA damage,
apoptosis,
oncogenesis, gene silencing and senescence, inter alia.
SUMMARY
In one aspect, the present invention relates to methods and compositions
employing pS3
and Sir2 proteins. Cellular and organismal processes are regulated by
modulating the activity of
Sir2 and/or pS3. In some cases the regulated processes control a program of
regulated aging
and/or metabolism of a cell or an organism. Compounds that regulate the
activity of Sir2 and/or
pS3 can be identified, for example, by a method described herein.
As used herein, the term "Sir2" refers to a protein that is at least 25%
identical to the 250
amino acid conserved Sir2 core catalytic domain, amino acids 258451 of SEQ ID
NO. 12. A
6
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Sir2 protein can be for example, at least 30, 40, 50, 60, 70, 80, 85, 90, 95,
99% identical to amino
acids 258-451 of SEQ ID NO. 12. For example, the Sir2 protein is human SIRT1,
GenBank
Accession No: AF083106. There are at least seven different Sir2 homologs
present in
mammalian cells (Frye, 1999, 2000; Imai et ad., 2000; Gray and El~strom,
2001). The mouse
Sir2a and human SIRTl, are preferred Sir2 proteins.
Sir2 can be a protein (e.g., SEQ m NOS. 8, 10, 12, 14, 16 or 18) or a fragment
of the
protein capable of deacetylating a substrate in the presence or NAD and/or an
NAD analog
and/or a fragment capable of binding to a target protein, e.g., a
transcription factor. Such
functions can be evaluated by a method described herein. A Sir2 fragment can
include a
"domain" which is a structurally stable folded unit of the full-length
protein. The Sir2 protein
can be encoded by the nucleic acid sequence of SEQ >D NOS. 7, 9, 11, 13, 15 or
17. In a
preferred embodiment, the Sir2 is a human Sir2. A model of the three-
dimensional structure of a
Sir2 protein has been determined (see, e.g., Bedalov et al. (2001), Min et al.
(2001), Finnin et al.,
(2001)) and provides guidance for identifying domains of Sir2. '
A "full length" Sir2 protein refers to a protein that has at least the length
of a naturally
occurring Sir2 protein. A "full length" Sir2 protein or a fragment thereof can
also include other
sequences, e.g., a purification tag., or other attached compounds, e.g., an
attached fluorophore, or
cofactor.
The invention includes sequences and variants that include one or more
substitutions,
e.g., between one and six substitutions, e.g., with respect to a naturally-
occurring protein.
Whether or not a particular substitution will be tolerated can be determined
by a method
described herein. One or more or all substitutions may be conservative. A
"conservative amino
acid substitution" is one in which the amino acid residue is replaced with an
amino acid residue
having a similar side chain. Families of amino acid residues having similar
side chains have
been defined in the art. These families include amino acids with basic side
chains (e.g., lysine,
arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid),
uncharged polar side
chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic
side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine).
The terms "identical" or percent "identity," in the context of two or more
nucleic acids or
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polypeptide sequences, refer to two or more sequences or subsequences that are
the same or have
a specified percentage of amino acid residues or nucleotides that are the sane
(i.e., about 50%
identity, preferably 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%,
96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., the C.
elegans proteins
provided herein), when compared and aligned for maximum correspondence over a
comparison
window or designated region) as measured using a sequence comparison
methodology such as
BLAST or BLAST 2.0 with default parameters described below, or by manual
alignment and
visual inspection. Such sequences are then said to be "substantially
identical." This definition
also refers to, or may be applied to, the complement of a test nucleic acid
sequence. The
definition also includes sequences that have deletions and/or additions, as
well as those that have
substitutions. As described below, the preferred algorithms can account for
gaps and the like.
Preferably, identity exists over a region that is at least about 25 amino
acids or nucleotides in
length, or more preferably over a region that is at least 50 or 100 amino
acids or nucleotides in
length.
The pS3 polypeptide can have greater than or equal to 25%, 50%, 75%, 80%, 90%
overall identity or greater than or equal to 30%, 50%, 75%, 80%, 90% overall
similarity to SEQ
lD NO. 3. Preferably, the Sir2 or p53 polypeptide is a human protein (e.g., as
described herein),
although it may also be desirable to analyze Sir2 or p53 polypeptides isolated
from other
organisms such as yeast, worms, flies, fish, reptiles, birds, mammals
(especially rodents), and
primates using the methods of the invention.
In one aspect, the invention features a method of screening a compound. The
method
includes providing a reaction mixture including Sir2, a transcription factor,
and the compound,
and determining if the compound modulates Sir2 interaction with, e.g.,
binding, of the
transcription factor. Deterniining if the compound modulates Sir2binding may
be accomplished
by methods known in the art, including comparing the binding of Sir2 to the
transcription factor
at a first concentration of the compound and at a second concentration of the
compound. In a
further embodiment, either of the first or second concentration of the
compound may be zero,
e.g., as a reference or control.
In a further embodiment, the reaction mixture also includes a Sir2 cofactor,
such as NAD
or an NAD analog.
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In a further embodiment, the transcription factor is p53 or a Sir-2 binding
fragment
thereof. The transcription factor, e.g., pS3, or fragment thereof may be
acetylated or labeled. In
a preferred embodiment, the transcription factor is an acetylated pS3
fragment, and the fragment
includes lysine 382.
In a further embodiment, the Sir2 included in the reaction mixture is a Sir2
variant, e.g., a
variant that has reduced deacetylase activity, such as the H363Y mutation. The
Sir2 may be
human, e.g., human SIRT1. Alternatively, the Sir2 may be marine, e.g., Sir2a.
Tn one
embodiment of the inventions, the Sir2 is exogenous and expressed from a
heterologous nucleic
acid. Additionally, in a further embodiment, the transcription factor may be
exogenous and
expressed from a heterologous nucleic acid.
The method of screening can be used to identify compounds that modulate, e.g.,
increase
or decrease, cell growth, modulate, e.g., slow or speed, aging, modulate,
e.g., increase or
decrease, lifespan, modulate cellular metabolism, e.g., by increasing or
decreasing a metabolic
function or rate.
In another aspect, the invention features a method of screening a compound by
providing
a reaction mixture comprising Sir2, a transcription factor, and the compound,
and determining if
the compound modulates Sir2-mediated deacetylation of the transcription
factor. The step of
determining if the compound modulates Sir2-mediated deacetylation of the
transcription factor
may be performed by methods lrnown in the art, including comparing the binding
of Sir2 to the
transcription factor at a first concentration of the compound and at a second
concentration of the
compound. In a further embodiment, either of the first or second concentration
of the compound
may be zero, e.g., as a reference or control. In a further embodiment, the
reaction mixture also
includes a Six2 cofactor, such as NAD or an NAD analog.
In a further embodiment, the transcription factor is p53 or a Sir-2 binding
fragment
thereof. The pS3 or fragment thereof may be acetylated or labeled. Tn a
preferred embodiment,
the transcription factor is an acetylated p53 fragment, and the fragment
includes lysine 382.
In a further embodiment, the Sir2 included in the reaction mixture is a Sir2
variant that
has reduced deacetylase activity, such as the H363Y mutation. The Sir2 may be
human, e.g.,
human SIRT1. Alternatively, the Sir2 may be marine, e.g., Sir2a. In one
embodiment of the
inventions, the Sir2 is exogenous and expressed from a heterologous nucleic
acid. Additionally,
9
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in a further embodiment, the transcription factor may be exogenous and
expressed from a
heterologous nucleic acid.
The method of screening can be used to identify compounds that modulate, e.g.,
increase
or decrease, cell growth, modulate, e.g., slow or speed, aging, modulate,
e.g., increase or
decrease, lifespan, modulate cellular metabolism, e.g., by increasing or
decreasing a metabolic
function or rate.
The present invention also relates to a method of screening a compound by
providing a
compound that interacts with Sir2, e.g., a compound that binds Sir2;
contacting the compound
with a cell or a system; and determining if the compound modulates
transcription of a p53-
regulated gene. Determining if the compound modulates transcription of a p53-
regulated gene
may be by any of the methods known in the art, including comparing the
modulation of
transcription of a p53-regulated gene at a first concentration of the compound
and at a second
concentration of the compound. In a further embodiment, either of the first or
second
concentration of the compound may be zero, e.g., as a reference or control.
In a related aspect, the invention features a method of evaluating a compound,
the method
comprising: contacting Sir2 or a transcription factor, e.g., p53, with a test
compound; evaluating
an interaction between the test compound and the Sir2 or the transcription
factor, e.g., pS3;
contacting a cell or organism that produces the Sir2 or transcription factor
polypeptide with the
test compound; and evaluating the effect of the test compound on the rate of
aging on the cell or
organism. The interaction can, for example, be a physical interaction, e.g., a
direct binding
interaction, a covalent change in one or both of the test compound or the Sir2
or transcription
factor, a change in location of the test compound (e.g., a change in
subcellular localization), or a
functional interaction (e.g., an alteration in activity, stability, structure,
or activity of the
polypeptide).
In some embodiments, the method is repeated one or more times such that, e.g.,
a library
of test compounds can be evaluated. In an related embodiment, the evaluating
of the interaction
with the test compound and the Sir2 or the transcription factor, e.g., p53, is
repeated, and the
evaluating of the rate of aging is selectively used for compounds for which an
interaction is
1o
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detected. Possible test compounds include, e.g., small organic molecules,
peptides, antibodies,
and nucleic acid molecules.
In some embodiments, the interaction between the test compound and the Sir2 or
transcription factor, e.g., p53, is evaluated in vitro, e.g., using an
isolated polypeptide. The Sir2
or transcription factor, e.g., p53, polypeptide can be in solution (e.g., in a
micelle) or bound to a
solid support, e.g., a column, agarose beads, a plastic well or dish, or a
chip (e.g., a microarray).
Similarly, the test compound can be in solution or bound to a solid support.
In other embodiments, the interaction between the test compound and the Sir2
or
transcription factor, e.g., p53, is evaluated using a cell-based assay. For
example, the cell can be
a yeast cell, an invertebrate cell (e.g., a fly cell), or a vertebrate cell
(e.g., a Xenopus oocyte or a
mammalian cell, e.g., a mouse or human cell). In preferred embodiments, the
cell-based assay
measures the activity of the Sir2 or transcription factor, e.g., p53,
polypeptide.
In preferred embodiments, the effect of the test compound on the rate of aging
of a cell or
animal is evaluated only if an interaction between the test compound and the
Sir2 or transcription
factor, e.g., p53, is observed.
In some embodiments, the cell is a transgenic cell, e.g., a cell having a
transgene. In
some embodiments, the transgene encodes _a protein that is normally exogenous
to the transgenic
cell. In some embodiments, the transgene encodes a human protein, e.g., a
human Sir2 or
transcription factor, e.g., p53, polypeptide. In some embodiments, the
transgene is linked to a
heterologous promoter. In other embodiments, the transgene is linked to its
native promoter. In
some embodiments, the cell is isolated from an organism that has been
contacted with the test
compound. In other embodiments, the cell is contacted directly with the test
compound.
In other embodiments, the rate of aging of an organism, e.g., an invertebrate
(e.g., a
worm or a fly) or a vertebrate (e.g., a rodent, e.g., a mouse) is determined.
The rate of aging of
an organism can be determined by a variety of methods, e.g., by one or more of
a) assessing the
life span of the cell or the organism; (b) assessing the presence or abundance
of a gene transcript
or gene product in the cell or organism that has a biological age-dependent
expression pattern;
(c) evaluating resistance of the cell or organism to stress, e.g., genotoxic
stress (e.g., etopicide,
L1V irradition, exposure to a mutagen, and so forth) or oxidative stress; (d)
evaluating one or
more metabolic parameters of the cell or organism; (e) evaluating the
proliferative capacity of
the cell or a set of cells present in the organism; (fj evaluating physical
appearance or behavior
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of the cell or organism, and (g) assessing the presence or absence of a gene
transcript or gene
product in the cell or orgauism that has a p53-regulation-dependent expression
pattern. In one
example, evaluating the rate of aging includes directly measuring the average
life span of a group
of animals (e.g., a group of genetically matched animals) and comparing the
resulting average to
the average life span of a control group of animals (e.g., a group of animals
that did not receive
the test compound but are genetically matched to the group of animals that did
receive the test
compound). Alternatively, the rate of aging of an organism can be determined
by measuring an
age-related parameter. Examples of age-related parameters include: appearance,
e.g.; visible
signs of age; the expression of one or more genes or proteins (e.g., genes or
proteins that have an
. age-related expression pattern); resistance to oxidative stress; metabolic
parameters (e.g., protein
synthesis or degradation, ubiquinone biosynthesis, cholesterol biosynthesis,
ATP levels, glucose
metabolism, nucleic acid metabolism, ribosomal translation rates, etc.); and
cellular proliferation
(e.g., of retinal cells, bone cells, white blood cells, etc.). Tn some
embodiments, the organism is a
transgenic animal. The transgenic animal can include a transgene that encodes,
e.g., a copy of a
Sir2 or transcription factor protein, e.g., a p53 protein, e.g., the Sir2 or
transcription factor, e.g., a
p53 polypeptide that was evaluated for an interaction with the test compound.
In some
embodiments, the transgene encodes a protein that is normally exogenous to the
transgenic
animal. For example, the transgene can encode a human protein, e.g., a human
Sir2 or
transcription factor, e.g., p53, polypeptide. In some embodiments, the
transgene is linked to a
heterologous promoter. In other embodiments, the transgene is linked to its
native promoter. In
some embodiments, the transgenic animal further comprises a genetic
alteration, e.g.; a point
mutation, insertion, or deficiency, in a gene encoding an endogenous Sir2 or
transcription factor,
e.g., p53, protein, such that the expression or activity of the endogenous
Sir2 or transcription
factor protein is reduced or eliminated.
In some embodiments, the organism is on a calorically rich diet, while in
other
embodiments the organism is on a calorically restricted diet.
In some embodiments, a portion of the organism's life, e.g., at least 20%,
30%, 40%,
50%, 60%, 70%, 80%, 90%, or more, of the expected life span of the organism,
has elapsed prior
to the organism being contacted with the test compound.
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In another aspect, the invention features a method of evaluating a protein,
comprising:
identifying or selecting a candidate protein, wherein the candidate protein is
a Sir2 or
transcription factor, e.g. p53, polypeptide; altering the sequence, expression
or activity of the
candidate protein in a cell or in one or more cells of an organism; and
determining whetlier the
alteration has an effect on the interaction, e.g., binding, of Sir2 with a
transcription factor, e.g.
p53, or on the deacetylation of transcription factor, e.g. p53.
In some embodiments, the candidate protein is identified by amplification of
the gene or
a portion thereof encoding the candidate protein, e.g., using a method
described herein, e.g., PCR
amplification or tlne screening of a nucleic acid library In preferred
embodiments, the candidate
protein is identified by searching a database, e.g., searching a sequence
database for protein
sequences homologous to Sir2 or a transcription factor, e.g., p53.
In preferred embodiments, the candidate protein is a human protein. In other'
embodiments, the candidate protein is a mammalian protein, e.g., a mouse
protein. In other
embodiments, the protein is a vertebrate protein, e.g., a fish, bird or
reptile protein, or an
invertebrate protein, e.g., a worm or insect protein. In still other
embodiments, the protein is a
eukaryotic protein, e.g., yeast protein.
In another aspect, the invention features method of evaluating a protein, the
method
comprising a) identifying or selecting a candidate protein, wherein the
candidate protein is Sir2
or a transcription factor, e.g., p53; b) identifying one or more polymorphisms
in a gene, e.g., one
or more SNPs that encodes the candidate protein; and c) assessing
correspondence between the
presence of one or more of the polymorphisms and an interaction, e.g.,
binding, of Sir2 with the
transcription factor, e.g., p53, or with the deacetylation of the
transcription factor, e.g., p53. The
polymorphisms can be naturally occurring or laboratory induced. In one
embodiment, the
organism is an invertebrate, e.g., a fly or nematode; in another embodiment
the organism is a
mammal, e.g., a rodent or human. A variety of statistical and genetic methods
can be used to
assess correspondence between a polymorphism and longevity. Such correlative
methods
include determination of linkage disequilibrium, LOD scores, and the like.
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In another aspect, the invention features a method of modulating cell growth
in an
animal, e.g., a mammal, by modulating the Sir2-mediated deacetylation of a
transcription factor
in the animal.
In one embodiment, the method includes modulating cell growth by increasing
acetylation of p53. In a futther embodiment, the method includes inactivating
Sir2, e.g., by the
use of antisense, RNAi, antibodies, intrabodies, NAD depletion, a dominant
negative mutant of
Sir2, or by the addition of Sir2 cofactor-analogs, e.g., N.AD analogs such as
those described in
Vaziri et al. (1997) or nicotinamide. In a further embodiment, the method
includes introducing a
deacetylation-resistant form of pS3. In still another embodiment, the
invention is a method for
treating a mammal, e.g., a mammal having a disease characterized by unwanted
cell
proliferation, e.g., cancer, accelerated senescence-related disorders,
inflammatory and
autoimmune disorders, Alzheimer's disease, and aging-related disorders.
Tn another embodiment, the method includes modulating cell growth by
decreasing
acetylation of p53. In a further embodiment, the method includes increasing
NAD
concentrations. In a further embodiment, the method includes increasing Sir2
concentrations, e.g.
by addition of purified Sir2, by expression of Sir2 from heterologous genes,
or by increasing the
expression of endogenous Sir2, or by the addition of Sir2 cofactor-analogs,
e.g., NAD analogs
such as those described in Vaziri et aI. (I99?).
The present invention also relates to a method of modulating the growth of a
cell in vivo
or ira vitro by modulating the Sir2-mediated deacetylation of a transcription
factor in the cell.
In one embodiment, the method includes modulating the growth of a cell by
increasing
acetylation of p53, thereby decreasing cell growth. In a further embodiment,
the method
includes inactivating Sir2, e.g., by the use of antisense, RNAi, antibodies,
intrabodies, NAD
depletion, a dominant negative mutant of Sir'L, or nicotinamide, or decreasing
Sir2 activity by the
addition of Sir2 cofactor-analogs, e.g., NAD analogs such as those described
in Vaziri et al.
(1997). In a further embodiment, the method includes introducing a
deacetylation-resistant form
of p53.
In one embodiment, the method includes modulating the growth of a cell by
decreasing
acetylation of p53, thereby increasing cell growth. In a further embodiment,
the method includes
increasing NAD concentrations. In a further embodiment, the method includes
increasing Sir2
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concentrations, e.g. by addition of purified Sir2, by expression of Sir2 from
heterologous genes,
or by increasing the expression of endogenous Sir2, or by the addition of Sir2
cofactor-analogs,
e.g., NAD analogs such as those described in Vaziri et aI. (1997).
In one aspect the invention features a method of directing Sir2 to a
transcription factor
binding site, e.g., a p53 binding site, and thereby modifying the acetylation
status of the binding
site on histone or DNA. The method includes providing a Sir2-transcription
factor complex
under conditions such that the transcription factor targets Sir2 to the
transcription factor binding
site, allowing the Sir 2 to modify the acetylation status of histories and DNA
at the transcription
factor binding site.
In a preferred embodiment, the method is performed in vivo or in vitro, e.g.,
in an animal
or in a cell.
In a preferred embodiment, the Sir2-transcription factor complex is provided
at a
different stage of development of the cell or animal or at a greater
concentration than occurs
naturally
Tn a preferred embodiment, the Sir2 or transcription factor or both is
increased, e.g., by
supplying exogenous Sir2 and/or transcription factor, e.g., p53, by supplying
an exogenous
nucleic acid encoding Sir2 or transcription factor, e.g., p53, or by inducing
endogenous
production of Sir2 or a transcription factor, e.g., p53.
In one embodiment, the present invention relates to a method of evaluating a
compound,
e.g., a potential modulator of Sir2 or transcription factor, e.g., p53
activity, comprising the steps
of contacting the transcription factor, e.g., p53, Sir2, and NAD or an NAD
analog with the
compound; evaluating an interaction between the compound and one or more of
the transcription
factor, e.g., p53, Sir2, and a cofactor such as NAD or an NAD analog;
contacting the compound
with a cell or organism having transcription factor, e.g., p53 or Sir2
activity; and evaluating the
rate of aging of the cell or organism. In a preferred embodiment, evaluating
the rate of aging
comprises one or more of:
a) assessing the life span of the cell or organism;
b) assessing the presence or absence of a gene transcript or gene product in
the cell
or organism that has a biological age-dependent expression pattern;
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c) evaluating resistance of the cell or organism to stress;
d) evaluating one or more metabolic parameters of the cell or organism;
e) evaluating the proliferative capacity of the cell or a set of cells present
in the
organism;
f) evaluating physical appearance, behavior, or other characteristic of the
cell or
organism; and
g) assessing the presence or absence of a gene transcript or gene product in
the cell
or organism that has a pS3-regulation-dependent expression pattern.
The details of one or more embodiments of the invention are set forth in the
accompanying drawings and the description below. Other features, objects, and
advantages of
the invention will be apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
Figure 1. Interactions between pS3 and mammalian Sir2a both in vitro and in
vivo.
(A) is an autoradiograph demonstrating direct interactions of Sir2a with GST
pS3. The
GST pS3 full length protein (GST pS3) (lane 1), the N-terminus of pS3 protein
(1-73) (lane 2),
the middle part of pS3 (100-290) (lane 3), the C-terminus of pS3 (290-393)
(lane 4), and GST
alone (lane ~ were used in GST pull-down assay with ifa vitro translated 35S-
labeled full length
mouse Sir2a. (B) is two western blots demonstrating pS3 interactions with
Sir2a in H1299 cells.
Western blot analyses of the indicated whole cell extract (WCE) (lanes 1, 3,
5, 7), or the pS3
immunoprecipitates with M2 antibody (TP/Flag-pS3) prepared from the
transfected H1299 cells
(lane 6, 8), or the Sir2a immunoprecipitates (IP/Flag-Sir2a) with M2 antibody
prepared from the
transfected H1299 cells (lanes 2, 4) with either anti-pS3 monoclonal antibody
(DO-1) (lanes 1-
4), or anti-Sir2a polyclonal antibody (lanes S-8). The cells were either
transfected with pS3
(lanes 3, 4) or Sir2a (lanes 7, 8) alone, or cotransfected with pS3 and Sir2a
(lanes 1, 2, S, 6). (C)
is a schematic representation of the lugh homology regions between mouse Sir2a
and human
SIRT1 (hSIRTl). The core domain represents the very conserved enzymatic domain
among all
Sir2 family proteins (Frye, 1999, 2000). (D) is a western blot demonstrating
pS3 interactions
with human SIRT1 in H1299 cells. Western blot analyses of the indicated whole
cell extract
(WCE) (lanes 1, 3) or the Flag-hSIRTI immunoprecipitates with M2 antibody
(IP/hSIRTl)
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(lanes 2, 4) prepared from either the hSIRTl and p53 cotransfectedH1299 cells
(lanes 1, 2) or the
p53 alone transfected cells (lanes 3, 4) with anti-p53 monoclonal antibody (DO-
1).
Figure 2. P53 interacts with mammalian Sir2a (mouse Six2a and hSIRTl) in
normal
cells.
(A) is two western blots demonstrating the interaction between p53 and hSIRTl
in H460
cells. (B) is two western blots demonstrating the interaction between pS3 and
Sir2a in F9 cells.
(C) The interaction between p53 and hSIRTl un HCT116 cells either at the
normal condition
(lanes, 1-3) or after DNA damage treatment by etoposide (lanes, 4-6). Western
blot analyses of
the indicated whole cell extract (WCE) (lanes 1, 4), or immunoprecipitates
with anti-Sir2a
antibody (IP/anti-Sir2a) (lanes 2, 5) prepared from different cell extracts,
or control
iinmunoprecipitates with pre-inununoserum from the same extracts (lanes 3, 6),
with anti-p53
monoclonal antibodies (DO-I for human p53, 42I for mouse p53), or anti-Sir2a
antibody.
Figure 3. TSA-insensitive deacetylation of p53 by mammalian Sir2a.
(A) Colloidal blue staining of a SDS-PAGE gel containing protein Marker (lane
I), a
control eluate from M2 loaded with untransfected cell extract (lane 2), and
100 ng of the highly
purified Flag-tagged Sir2a recombinant protein (lane 3). (B) Deacetylation of
p53 by Sir2a, 2.5
pg of 14C-labeled acetylated p53 (lane 1) was incubated with either the
control eludate (lane 4),
the purified 10 ng of Sir2a (lanes 2 and 3), or the same amount of Sir2a in
the presence of 500
nM TSA (lane 5) for 60 min at 30EC. NAD (50 Vim) was also added in each
reaction except lane
2. The proteins were analyzed by resolution on SDS-PAGE and autoradiography
(upper) or
Coomassie blue staining (lower). (C). Reduction of the steady-state levels of
acetylated p53 by
both mouse Sir2a and human SIRTI expression. Western blot analysis of H1299
cell extracts
from the cells cotransfected with p53 and p300 (lane 1), or in combination
with Sir2a (lane 2), or
in combination with hSIRTl (lane 4), or in combination with Sir2aH355A (lane
3), in
combination with hSIRTS (lane 5), or in combination with PARP (lane 6) by
acetylated pS3-
specific antibody (upper) or DO-1 for total pS3 (lower). (D) Deacetylation of
p53 by Sir2a izi
the presence of TSA. Western blot analysis of acetylated p53 levels in H1299
cells cotransfected
with p53 and p300 (lanes 1, 3), or cotransfected with p53, p300 and Sir2a
(lanes 2, 4) by
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acetylated p53-specific antibody (upper) or OF-1 for total p53 {lower). Cells
were either not
treated (lanes 1, 2) or treated with 500 nM TSA (lanes 3, 4).
Figure 4. Abrogation of mammalian Sir2a mediated deacetylation of p53 by
nicotinamide.
(A) Sir2a-mediated deacetylation of p53 is inhibited by nicotinamide. 2.5 :g
of 14C-
labeled acetylated p53 (lane 1) was incubated with 10 ng of purified Sir2a and
50 p,M NAD
alone (lane 2), or in the presence of either 5mM of nicotinamide (lane 3) or 3
mM of 3-AB (3-
aminobenzamide) (lane 4) fox 60 min at 30EC. The proteins were analyzed by
resolution on
SDS-PAGE and autoradiography (upper) or Coomassie blue staining (lower). (B)
The Sir2a-
mediated deacetylation of endogenous p53 was abrogated in the presence of
nicotinamide. Cell
extracts from the mock-infected MEF p53 (+/+) cell (lanes 1-2, 5-6), or the
pBabe-Sir2ainfected
cells (lanes 3-4, 7-8), either untreated (lanes 1, 3, 5, 7), or treated with
etoposide and TSA (lane
2, 4), or in combination with nicotinamide (lanes 6, 8) for 6 hr were analyzed
by Western blot
with acetylated p53-specific antibody (upper) or DO-1 for total p53 (lower).
(C) Synergistic
induction of p53 acetylation levels by TSA and nicotinamide during DNA damage
response.
Western blot analysis of cell extracts from the H460 cells treated with
etoposide alone (lane 2),
or in combination with TSA (lane 3), or TSA and nicotinamide (lane 4), or TSA
and 3-AB (lane
5) for 6 hr by acetylated p53-specific antibody (upper) or DO-1 for total p53
(lower). The cell
extracts from untreated cells (lane 1), or treated with a proteasome inhibitor
LLNL (50 :M) were
also included (lane 6).
Figure 5. Bar graphs illustrating repression of p53-mediated transcriptional
activation by
mammalian Sir2a.
(A), (B) MEF (p53-/-) cells were transiently transfected with 10 ng of CMV p53
alone, or
in combination with indicated Sir2a constructs together with either the PG13-
Luc reporter
construct (A), or a control reporter construct (TK-Luc) (B) by calcium
phosphate precipitation
essentially as previously described (Luo et al., 2000). (C), {D) MEF (p53-!-)
cells were
transiently transfected with 10 ng of CMV p53 alone, or in combination with
S:g of either CMV
Sir2a, or CMV hSIRTl, or CMV hSIRTS (C), or CMV Sir2aH355A as indicated (D)
together
with the PG13-Luc reporter construct. All transfections were done in duplicate
and .
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representative experiments depict the average of three experiments with
standard deviations
indicated.
Figure 6. Inhibition of pS3-dependent apoptosis by Sir2a.
(A) H1299 cells were transfected with p53 alone, or cotransfected with p53 and
Sir2a, or
cotransfected with pS3 and Sir2aH3SSA. After transfection, the cells were
fixed, stained for pS3
by FTTC-conjugated a-p53 antibody, and analyzed by flow cymtometry for
apoptotic cells
(subGl) according to DNA content (PI staining). (B) The experiments were
repeated at least
three times; this bar graph depicts the average of three experiments with
standard deviations
indicated.
Figure 7. Inhibition of p53-dependent apoptotic response to stress by
mammalian Sir2a.
(A) Repression of the apoptotic response to DNA damage by Sir2a. Both mock
infected
cells and p/babe-Six2a infected MEF pS3(+/+) cells were either not treated (1
and 2) or treated
with either 20 E.LM etoposide. The cells were analyzed by flow cytometry for
apoptotic cells
(subG1) according to DNA content (PI staining). (B) Similar results were
obtained for three
times, and this bar graph of representative data depicts the average of three
experiments with
standard deviations indicated (B).
Figure 8. Co-precipitation of hSir2 and pS3 pxotein.
(A) Immunoprecipitation of hSir2 with a C-terminal polyclonal rabbit antibody
followed
by immunoblotting with the same antibody revealed the existence of a 120Kd
protein in normal
BJ fibroblasts (left panel), and increased levels in these cells expressing
the wild type (middle
panel) and HY mutant (right panel) of hSir2. (B) Immunofluorescence analysis
of hSir2
indicated the existence of a nuclear protein with a punctuate staining
pattern. (C) Nuclear
lysates from H1299 cells ectopically expressing pS3 and hSir2 were
precipitated with the anti-
hSir2 antibody The blot was probed the anti-hSir2 antibody and a polyclonal
sheep anti-pS3
antibody (bottom panel). (D) p53 protein was immunoprecipitated with the Do-1
anti-pS3
antibody from lysates of non-irradiated and irradiated (6Gy) BJT cells
(expressing ~telomerase)
that had been stably infected with pYESir2wt and pYESir2HY mutant vectors. The
blot was
probed with anti-hSir2 antibody and rabbit anti-p53 polyclonal antibodies
(CMl+SC6243).
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Figure 9. Effect of hSir2 expression on p53 acetylation ih vitro.
The deacetylation activity of mSir2 on the human p53 C-terminal peptide
(residues 368-
386) di-acetylated at positions 373 and 382. (A, B) HPLC chromatograms of
products of
deacetylation assays with mSir2 and the indicated concentrations of NAD. Peaks
1 and 2
correspond to the monomeric and dimeric forms of the p53 peptide, respectively
Peak 3
corresponds to the singly deacetylated monomer identified by mass spectroscopy
(C-F) Amino-
terminal Edman sequencing of peaks 1 and 3. Chromatograms of positions 373 and
382 are
shown. Peaks of acetyl-lysine (AcK) and simple lysine (K) are indicated in
each panel. Small
peaks of lysine in panels C, D and F are due to residual fractions of previous
lysines at positions
372 and 381.
Figure 10. hSir2 effects on p53 acetylation irc vivo.
(A) Reconstitution of the acetylation and deacetylation cascade in immortal
human
epithelial H1299 cells by transient co-transfection of the indicated genes.
After co-transfection of
the mentioned constructs, the cellular lysates were analyzed by Western blot
analysis, using Ab-I
to detect K382 p53, DO-1 for total p53 or ~ actin for loading control. Lane 3,
co-transfection of
CMVwtp53 and p300 generates acetylated p53 at K382, lane 4, co-transfection of
the acetylation
mutant K382R of p53 with p300. Lane 5, Same as 4 but with co-transfected wild
type hSir2.
Lanes 7-8, co-transfection of the acetylation mutant K320R with or without
wild type hSir2.
Lane 9, Co-transfection of CMVwtp53, CMVp300 and wild type hSir2.
(B) BJ cells expressing telomerase (BJT), were stably infected with either a
wild type
hSir2 or a mutant hSir2HY virus. The hSir2-expressing mass cultures were subj
ected to 6Gy of
ionizing radiation in presence of low concentrations of TSA (0. lmg/ml) and
the p53 acetylation
was measured at indicated time points by immunoblotting with Ab-1 that
recognizes specifically
the deacetylated K382 p53 protein. The blots were subsequently probed with
anti-p53, anti p21,
anti-~ actin and anti-hSir2 antibodies. Time (hrs) post 6 Gy of irradiation is
shown inside the
brackets.
(C) Deacetylation of p53 in vivo in MCF7 cells. Four-fold ectopic expression
of wild
type hSir2 or hSir2HY mutant in MCF7 cells radiated with 6Gy of ionizing
radiation and its
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effect on p53 acetylation at K382: The blot was probed for acetylation with Ab-
1 and reprobed
with other antibodies as in (B). Times shown are post irradiation in hours.
Figure 11. hSir2 expression and its influence on p53 activity
(A) is a bar graph depicting transcriptional activity of p53 protein, as
measured in H1299
cells by co-transfection p53 with a p21 WAFT promoter-luciferase construct
(p2lPluc).
Transcriptional activity of p53 protein was measured upon ectopic expression
of wild type hSir2,
hSir2HY (B) is a bar graph illustrating results from control SV40-Luciferase
transfections with
CMVp53 and increasing amounts of wild type hSir2 in to H1299 cells and
luciferase activity was
measured and expressed as Relative Light Unit (%RLII). (C) Is an immunoblot
demonstrating
levels of p21 WAFT in MCF73L cells expressing wt hSir2 or hSir2HY protein in
response to 6Gy
of ionizing radiation. The blot was probed with Dol for detection of p53 and
~i actin for loading
control.
Figure 12. Effects of hSir2 on p53-dependent apoptosis and radiosensitivity
(A) is a bar graph illustrating ectopic expression of hSir2wt and its
influence on p53-
dependent apoptosis in HI299 cells. H1299 cells were transfected with a wild
type p53
expression construct to induce p53-dependent apoptosis. Annexin V positive and
propidium
iodide negative cells were measured.
(B) is a line graph comparison of gamma-ray survival. Dose-response curves are
shown
for different types of BT cells treated with ionizing radiation while growing
exponentially and
asynchronously Twelve days after radiation the colonies were counted and
survival calculated as
described previously (Dhar et al., 2000). The ataxia-telangiectasia (A-T) cell
line was used a
positive control to indicate radiosensitivity in an exponentially growing
population.
Figures 13A and 13B. The coding nucleic acid (SEQ ID NO. 2) and deduced amino
acid
(SEQ ID NO. 3) of human p53.
Figure 14. The nucleic acid (SEQ 1D NO. 4) sequence of human p53 (GenBanlc
Accession No: K03199).
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Figures 15A, B, C and D. The nucleic acid (SEQ ID NO. S) and deduced amino
acid
sequence (SEQ D7 NO. 6) ofmouse Sir2.
Figures 16A, B and C. The nucleic acid (SEQ ID NO. 7) and deduced amino acid
sequence (SEQ ID NO. 8) of mouse Sir2 GenBanlc Accession No: AF214646.
Figures 17A and B. The nucleic acid (SEQ ID NO. 9) and deduced amino acid
sequence
(SEQ IrD NO. 10) of human Sir2 SIRT2 GenBanl~ Accession No: AF083107.
Figures 18A, B and C. The nucleic acid (SEQ ID NO. 11) and deduced amino acid
sequence (SEQ 117 NO. 12) ofhuman Sir2 SIRTl GenBank Accession No: AF083106.
Figure 19. The nucleic acid (SEQ ll~ NO. 13) and deduced amino acid sequence
(SEQ
I17 NO. 14) of human Sir2 SIRT3 GenBank Accession No: AF083108.
Figures 20A and B. The nucleic acid (SEQ ID NO. 15) and deduced amino acid
sequence (SEQ ID NO. 16) of human Sir2 SIRT4 GenBank Accession No: AF083109.
Figures 21A and B. The nucleic acid (SEQ DJ NO. 17) and deduced amino acid
sequence (SEQ m NO. 18) of human Sir2 SIRTS GenBank Accession No: AF083110.
DETAILED DESCRIPTION
As described below, hSir2 directly binds the human p53 protein both in vitro
and in vivo
and can deacetylate p53, e.g., at the K382 residue of p53. A functional
consequence of this
deacetylation is an attenuation of the p53 protein's activity, e.g., as a
transcription factor
operating at a cellular promoter, e.g., the p21WAF1 promoter. In another
cellular Context, in
which the DNA damage response leads to apoptosis, hSir2 activity attenuates
the pS3-dependent
apoptotic response. Hence, hSir2 can negatively regulate a program of cellular
death.
Sir2 proteins can also deacetylate~histones. For example, Sir2 can deacetylate
lysines 9
or 14 of histone H3. Histone deacetylation alters local chromatin structure
and consequently can
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regulate the transcription of a gene in that vicinity Sir2 proteins can bind
to a number of other
proteins, termed "Sir2-binding par(ners." For example, hSlRT1 binds to p53. In
many instances
the Sir-2 binding partners are transcription factors, e.g., proteins that
recognize specific DNA
sites. Interaction between Sir2 and Sir2-binding partners delivers Sir2 to
specific regions of a
genome and can result in local modification of substrates, e.g., histories and
transcription factors
localized to the specific region. Accordingly, cellular processes can be
regulated by compounds
that alter (e.g., enhance or diminish) the ability of a Sir2 protein to
interact with a Sir2-binding
partner or that alter that ability of a Sir2 protein to modify a substrate.
While not wishing to be
bound by theory, a Sir2-transcription factor complex may be directed to a
region of DNA with a
transcription factor binding site; once there, Sir2 may alter the acetylation
status of the region,
e.g., by deacetylating histories, non-histone proteins, and/or DNA. This would
locally raise the
concentration of Sir2 and may potentially result in the Sir2-mediated
silencing of genes located
at or near transcription-factor binding sites. Certain organismal programs
such as aging or
metabolism and disorders such as cancer can be controlled using such
compounds.
While not wishing to be bound by theory, in mammalian cells, signals
indicating the
successful completion of DNA repair may be relayed via hSir2 to acetylated
proteins like p53
that have been charged with the task of imposing a growth arrest following DNA
damage. These
signals enable hSir2 to reverse part or all of the damage-induced activation
of p53 as a
transcription factor by deacetylating the K382 residue of p53. By doing so,
hSir2 reduces the
likelihood of subsequent apoptosis and, at the same time, makes it possible
for cells to re-enter
the active cell cycle, enabling them to return to the physiological state that
they enjoyed prior to
sustaining damage to their genomes.
Inactivation of the p53 signaling pathway is involved in the pathogenesis of
most if not
all human tumors (Hollstein et al., 1994; Lohrum and Vousden, 1999). In about
half of these
tumors, mutation of the p53 gene itself suffices to derail function. In some
of the remaining
tumors, loss of p14'~, which acts to down-regulate p53 protein levels, has
been implicated
(Lohrum and Vousden, 1999; Prives and Hall, 1999). The present invention is
related to the
discovery of a novel mode by which an incipient cancer cell attenuate at least
some p53
functions via modulation of the activity of hSir2, which, like the other two
genetic strategies,
may result in the inactivation of both the cytostatic and pro-apoptotic
functions of p53.
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The invention is thus based in part on the discovery of the existence of a p53
regulatory
pathway that is regulated by mammalian Sir2a. Sir2a is involved in gene
silencing and
extension of life span in yeast and C. elegarzs (reviewed in Guarente, 2000;
Shore, 2000;
Kaeberlein et al., 1999; Tissenbaun and Guarente, 2001). p53 binds to mouse
Sir2a as well as
its human ortholog hSIRTl both in vitro and in vivo. p53 is a substrate for
the NAD-dependent
deacetylase activity of mammalian Sir2a. Sir2a-mediated deacetylation
antagonizes p53-
dependent transcriptional activation and apoptosis. Sir2a-mediated
deacetylation of p53 is
inhibited by nicotinamide both in vitro and in vivo. Sir2oc specifically
inhibits p53-dependent
apoptosis in response to DNA damage and/or oxidative stress, but not p53-
independent, Fas-
mediated cell death. Accordingly, compounds that alter (e.g., decrease or
enhance) the
interaction between Sir2 and p53 can be used to regulate processes downstream
of p53, e.g.,
apoptosis. Such compounds may alter the catalytic activity of Sir2 for a
substrate such as p53 or
may alter the interaction between Sir2 and p53.
The present invention relates to the discovery that p53 is a binding partner
of mammalian
Sir2a, which physically binds to p53 both in vitro and in vivo. In some cases,
p53 is ~.lso a
substrate of Sir2. Sir2a specifically represses p53-mediated functions
including p53-dependent
apoptotic response to stress.
p53 can be, for example, the mature protein (e.g., SEQ ID NO. 3) or a fragment
thereof.
The p53 protein can be encoded by the nucleic acid sequence of SEQ ID NOS. 2
and/or 4). In a
preferred embodiment, p53 is the human p53. Deacetylation of p53 can be
mediated by
Sir2,e.g., in combination with a cofactor, such as NAD and/or an NAD analog.
The phrase "deacetylating p53" refers to the removal of one or more acetyl
groups (e.g.,
CH3C02-) from p53 that is acetylated on at least one amino acid residue. In a
preferred
embodiment, p53 is deacetylated at a lysine of p53 selected from the group
consisting of lysine
370, lysine 371, lysine 372, lysine 381 and lysine 382 of SEQ ID NO. 3. p53
can be
deacetylated in the presence or absence of DNA damage or oxidative cellular
stress. The DNA
damage can be caused by, for example, ionizing radiation (e.g., 6 Gy of
ionizing radiation), or a
tumor or some other uncontrolled cell proliferation. p53 is deacetylated in
the presence of DNA
damage or oxidative stress by combining p53, Sir2, NAD and/or an NAD analog.
Sir2 can be the mature protein (e.g., SEQ ?D NOS. 8, 10, 12, 14, 16 or 18) or
a fragment
of the mature protein capable of deacetylating p53 in the presence or NAD
and/or an NAD
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analog. The Sir2 protein can be encoded by the nucleic acid sequence of SEQ m
NOS. 7, 9, 11,
13, 15 or 17). In a preferred embodiment, the Sir2 is human Sir2.
In one embodiment, the invention is a method of deacetylatir~g p53 comprising
the step of
combining Sir2 and NAD and/or an NAD analog with p53. The combination can be
performed
in the presence or the absence of cells. Such combinations can be in tissue
culture (e.g., BJT
cells, MCF-7 cells) or in an organism (e.g., a mammal, e.g., as a human).
Combination of p53,
Sir2 and NAD and/or an NAD analog can be any placement of p53, Sir 2 and NAD
or a NAD
analog in sufficient proximity to cause Sir2 to deacetylate p53 that is
acetylated on at least one
amino acid residue, which deacetylation by Sir2 requires the presence of NAD
and/or an NAD
analog.
"1VAD" refers to nicotinamide adenine dinucleotide. An "NAD analog" as used
herein
refers to a compound (e.g., a synthetic or naturally occurring chemical, drug,
protein, peptide,
small organic molecule) which possesses structural similarity to component
groups of NAD
(e.g., adenine, ribose and phosphate groups) or functional similarity (e.g.,
deacetylates p53 in the
presence of Sir2). For example, an NAD analog can be 3-aminobenzamide or 1,3-
dihydroisoquinoline (H. Vazixi et al., EMBO J. 16:6018-6033 (1997), the entire
teachings of
which are hereby incorporated by reference).
"p53 activity" refers to one or more activity of p53, e.g., p-53 mediated
apoptosis, cell
cycle arrest, and/or senescence,
"Modulating p53 activity" refers to increasing or decreasing p53 activity,
e.g., p-53
mediated apoptosis, cell cycle arrest, and/or senescence, e.g. by altering the
acetylation or
phosphorylation status of p53.
"Acetylation status" refers to the presence or absence of one or more acetyl
groups (e.g.,
CH3C0z~ at one or more lysine (K) residues, e.g., K370, K371, K372, K381,
and/or K382 of
SEQ m NO. 3. "Altering the acetylation status" refers to adding or removing
one or more acetyl
groups (e.g., CH3C02'~ at one or more lysine (K) residues, e.g., K370, K371,
K372, K381, and/or .
K382 of SEQ ID NO. 3, e.g., by modulating Sir2 activity
Similarly, "phosphorylation status" refers to the presence or absence of one
or more
phosphate groups (P03~ at one or more residues, e.g., serine 15 and/or serine
20 of SEQ m NO.
3. "Altering the phosphorylation status" refers to adding or removing one or
more phosphate
groups (P03~) at one or more residues, e.g., serine 15 and/or serine 20 of SEQ
Ba NO. 3.
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"Sir2 activity" refers to one or more activity of Sir2, e.g., deacetylation of
p53 or histone
proteins.
"Modulating Sir2 activity" refers to increasing or decreasing one or more
activity of Sir2,
e.g., deacetylation of p53 or histone proteins, e.g., by altering the binding
affinity of Sir2 and
p52, introducing exogenous Szr2 (e.g., by expressing or adding purified
recombinant Sir2),
increasing or decreasing levels of NAD and/or an NAD analog (e.g., 3-
aminobenzamide, 1,3-
dihydroxyisoquinoline), and/or increasing or decreasing levels of a Sir2
inhibitor, e.g.,
nicotinamide and/or a nicotinamide analog. Additionally or alternatively,
modulating Sir2
activity can be accomplished by expressing, e.g. by transfection, a dominant
negative gene of
Sir2 (e.g., SirH~. The dominant negative gene can, for example, reduce the
activity of
endogenous Sir2 on p53 deacetylation thereby modulatiizg the activity of Sir2.
A "nicotinamide analog" as used herein refers to a compound (e.g., a synthetic
or
naturally occurring chemical, drug, protehl, peptide, small organic molecule)
which possesses
structural similarity to component groups of nicotinamide or functional
similarity (e.g., reduces
Sir2 deacetylation activity of p53).
The Sir2a-mediated pathway is critical for cells under stress
It is believed that there are multiple pathways in cells for regulation of p53
function
(Drives and Hall, 1999; Giaccia and Kastan, 1998; Ashcroft et al., 2000). In
normal cells, Mdm2
is the major negative regulator for p53, and Mdm2-mediated repression appears
sufficient to
downregulate pS3 activity. Sir2 regulation of p53 may be an Mdm2-independent,
negative
regulatory pathway for p53. Interestingly, while no obvious effect by Sir2a
expression was
observed in cells at normal conditions, Sir2a became critical in protecting
cells from apoptosis
when cells were either treated by DNA damage or under oxidative stxess (Figure
7). Thus,
Sir2a-mediated pathway can be critical for cell survival when the p53 negative-
control mediated
by Mdm2 is severely attenuated in response to DNA damage or other types of
stress.
p53 is often found in latent or inactive forms and the levels of p53 protein
are very low in
unstressed cells, mainly due to the tight regulation by Mdm2 through
functional inhibition and
protein degradation mechanisms (reviewed in Freedman et al., 1999). However,
in response to
DNA damage, p53 is phosphorylated at multiple sites at the N-terminus; these
phosphorylation
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events contribute to pS3 stabilization and activation by preventing Mdm2
binding to pS3
(reviewed in Appella and Anderson, 2000; Giaccia and Kastan,1998; Shieh et
al., 1997, 2000;
Unger et al., 1999; Hirao et al., 2000). Mdm2 itself is also phosphorylated by
ATM during DNA
damage response, and this modification attenuates its inhibitory potential on
pS3 (Maya et al.,.
2001). Furthermore, while p53 is strongly stabilized and highly acetylated in
stressed cells,
acetylation of the C-terminal multiple lysine sites may occur at the same
sites responsible fox
Mdm2-mediated ubiquitination (Rodriguez et aL, 2000; Nakamura et al., 2000),
and the highly
acetylated pS3 may not be effectively degraded by Mdm2 without deacetylation
(Ito et al.,
2001). Thus, in contrast to unstressed cells, the main pS3 negative regulatory
pathway mediated
by Mdm2 is blocked at several levels in response to DNA damage (Maya et al.,
2001). Under
these circumstances, Sir2a-mediated regulation may become a major factor in
controlling pS3
activity, making it possible for cells to adjust pS3 activity to allow time
fox DNA repair before
committing to apoptosis.
In oncogene-induced premature senescence of cells, the pS3 negative regulatory
pathway
controlled by Mdm2 may be blocked (reviewed in Sherr and Weber, 2000;
Sharpless and
Depinho, 1999; Serrano et al., 1997). However, in contrast to DNA damage
response, the
Mdm2-mediated pathway is abrogated by induction of p 14A~ (or mouse p 19A~) in
these cells
(Honda and Yasuda, 1999; Weber et al., 1999; Tao et al., 1999a, 1999b; Zhang
et al., 1998;
Pomerantz et al., 1998). Furthermore, when primary fibroblasts undergo
senescence, a
progressive increase of the pS3 acetylation levels was observed in serially
passaged cells
(Pearson et al., 2000). Oncogeiuc Ras and PML induced pS3-dependent premature
senescence,
and upregulated the pS3 acetylation levels in both mouse and human normal
fibroblasts (Pearson
et al., 2000; Ferbeyre et al., 2000). Thus, mammalian Sir2a-mediated
regulation may also play
an important role in oncogene-induced premature senescence.
Attenuation of p53-mediated transactivatiton by Sir2a
Earlier studies indicated that pS3-mediated transcriptional activation is
sufficient and also
absolutely required for its effect on cell growth arrest, while both
transactivation-dependent and -
independent pathways are involved in pS3-mediated apoptosis (reviewed in
Prives and Hall,
1999; Vousden, 2000). p53 may be effective to induce apoptosis by activating
pro-apoptotic
genes in vivo (reviewed in Nalcano and Vousden, 2001; Yu et al., 2001). Thus,
tight regulation
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,WO 03/004621 PCT/US02121461
of p53-mediated transactivation is critical for its effect on both cell growth
and apoptosis (Chao
et al., 2000; Jimenez et al., 2000).
Recent studies indicate that the intrinsic histone deacetylase activity of
Sir2a is essential
for its mediated functions (reviewed in Gurante, 2000). Reversible acetylation
was originally
identified in histories (reviewed in Cheung et al., 2000; Wolffe et al.,
2000); however,
accumulating evidence indicates that transcriptional factors are also
functional targets of
acetylation (reviewed in Serner and Bergen 2000; Kouzarides, 2000). Thus, the
transcriptional
attenuation mediated by histone deacetylases may act through the effects on
both histone and
non-histone transcriptional factors (Sterner and Bergen 2000; Kuo and
Allis,1998). Microarray
surveys for transcriptional effects of Sir2 in yeast revealed that Sir2
appears to repress amino
acid biosynthesis genes, which are not located at traditional "silenced" loci
(Bernstein et al.,
2000). Thus, in addition to silencing (repression) at telomeres, mating type
loci and ribosomal
DNA (reviewed in Guarente, 2000; Shore, 2000), Six2 may also be targeted to
specific
endogenous genes for transcriptional regulation in yeast.
In contrast to the yeast counterpart Sir2, the mouse Sir2a protein does not
colocalize with
nucleoli, telomeres or centromeres by co-immunofluorescence assay, indicating
that this protein
is not associated with the most highly tandemly repeated DNA in the mouse
genome, The
immunostaining pattern of human SILZT1 as well as mouse Sir2a indicates that
mammalian
Sir2a is, similar to HDAC1, broadly localized in the nucleus, further
supporting the notion that
mammalian Sir2a may be recruited to specific target genes for transcriptional
regulation in vivo.
Mammalian Sir2a may inhibit pS3-mediated functions by attenuation of the
transcriptional activation potential of pS3. Since deacetylation of pS3 is
critical, but may not be
the only function mediated by this Sir2a-p53 interaction, additional functions
mediated by Sir2a,
such as histone deacetylation, may also contribute to this regulation, As one
theory, not meant to
be limiting, pS3 and Sir2a may strongly interact to deacetylate pS3 and
possibly recruit the p53-
Sir2a complex to the target promoter. The subsequent transcription repression
may act both
through decreasing p53 transactivation capability and through Sir2a-mediated
llistone
deacetylation at the target promoter region. In contrast to I~DAC1-mediated
effect, this
transcriptional regulation is not affected by TSA treatment. Other cellular
factors may use a
similar mechanism to recruit Sir2a for TSA-insensitive transcriptional
regulation in mammalian
cells.
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Novel implications for cancer therapy
Inactivation of p53 functions has been well documented as a common mechanism
for
tumorigenesis (Hollstein et al., 1999; Vogelstein et al., 2000). Many cancer
therapy drugs have
been designed based on either reactivating p53 functions or inactivating p53
negative regulators.
Since p53 is strongly activated in response to DNA damage, mainly through
attenuation of the
Mdm2-mediated negative regulatory pathway (Maya et al., 2001), many DNA damage-
inducing
drugs such as etoposide are very effective antitumor drugs in cancer therapy
(reviewed in
Chresta and Hickman, 1996; Lutzker and Levine, 1996). Maximum induction of p53
acetylation
in normal cells, however, requires both types of deacetylase inhibitors in
addition to DNA
damage, and there may be at least three different p53 negative regulatory
pathways in
mammalian cells. Inhibitors for HDAC-mediated deacetylases, including sodium
butyrate, TSA,
SARA and others, have been also proposed as antitumor drugs (Butler et al.,
2000; Finnin et al.,
1999; Taunton et al., 1996; Yosliida et al., 1995; Buckley et al., 1996).
Combining DNA
damage drugs, HDAC-mediated deacetylase inhibitors, and Sir2a-mediated
deacetylase
inhibitors, may have synergistic effects in cancer therapy for maximally
activating pS3.
In contrast to PID/HDAC1-mediated p53 regulation (Luo et al., 2000), the
invention
shows that mammalian Sir2a-mediated effect on p53 is NAD-dependent, indicating
that this type
of regulation is closely linked to cellular metabolism (reviewed Guarente
2000; Alfred, 2000;
Campisi, 2000; Min et al., 2001). In fact, null mutants of NPT1, a gene that
functions in NAD
synthesis, show phenotypes similar to that of Sir2 mutants in gene silencing
(Smith et al., 2000)
and in life extension in xesponse to caloric restriction in yeast (Lin et al.,
2000). Thus, metabolic
rate may play a role in Six2a-mediated regulation of p53 function and,
perhaps, modulate the
sensitivity of cells in p53-dependent apoptotic response.
In yet another embodiment, the invention is a method of modulating p53-
mediated
apoptosis by modulating Sir2 activity Sir2 activity can be modulated as
described' herein (e.g.,
overexpressing Sir2, transfecting a cell with a dominant negative regulating
gene). An increase
in Sir2 activity (e.g., by overexpressing Sir2) can result in a decrease in
p53-mediated apoptosis.
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A decrease in Sir2 activity (e.g., transfecting a cell with a dominant
negative gene) can result in
an increase in pS3-mediated apoptosis.
In still another embodiment, the invention is a method of screening for a
compound(e.g.,
a small organic or inorganic molecule) which modulates (e.g., increases or
decreases) SirZ-
mediated deacetylation of p53. In the method, SirZ, p53, NAD and/or an NAD
analog, and the
compound to be tested are combined, the SirZ-mediated deacetylation of pS3 is
measured and
compared to the Sir2-mediated deacetylation of pS3 measured in the absence of
the compound.
An increase in the Sir2-mediated deacetylation of pS3 in the presence of the
compound being
tested compared to the Sir2-mediated deacetylation of p53 in the absence of
the compound
indicates that the compound increases Sir2 deacetylation of pS3. Likewise, a
decrease in the
SirZ-mediated deacetylation of p53 in the presence of the compound being
tested compared to
the SirZ-mediated deacetylation ofpS3 in the absence of the compound indicates
that the
compound decreases deacetylation of pS3 by SirZ. As used herein, "SirZ-
mediated
deacetylation" refers to the NAD-dependent removal of acetyl groups which
requires SirZ.
In another embodiment, the present invention relates to a method of screening
a
compound by providing an in vitro test mixture comprising a transcription
factor or a fragment
thereof, Sir2, and a Sir2 cofactor with the compound, evaluating an activity
of a component of
the test mixture in the presence of the compound, and comparing the activity
in the presence of
the compound to a reference obtained in the absence of the compound.
In another embodiment, the present invention relates to a method of screening
a
compound that is a potential NAD analog by providing an in vitro test mixture
comprising a
transcription factor or a fragment thereof, SirZ, and the compound, evaluating
an activity of a
component of the test mixture in the presence of the compound, and comparing
the activity in the
presence of the compound to a reference obtained in the absence of the
compound,
In one embodiment the SirZ is human, e.g., human SIRTl. In another embodiment,
the
Sir2 is marine, e.g., marine Sir2a.
In one embodiment the Sir2 cofactor is NAD or an NAD analog.
In another embodiment the transcription factor is pS3 or a fragment thereof,
and it may be
acetylated and/or labeled.
In a fiuuther embodiment, the evaluated activity is Sir2 activity, e.g.,
deacetylation of a
protein, e.g., deacetylation of a histone protein, and/or deacetylation of the
transcription factor,
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e.g., deacetylation of p53. The Sir2 activity may also be binding of a
protein, e.g., binding of a
histone protein and/or binding of the transcription factor, e.g.,. binding of
p53. The Sir2 activity
may be evaluated by detecting production of nicotinamide.
In a further embodiment, the evaluated activity is p53 activity The p53
activity may be
evaluated by detecting cell cycle arrest, apoptosis, senescence, and/or a
change in the levels of
transcription or translation products of a gene regulated by p53. Methods fox
detecting such
changes and genes regulated by p53 are known in the art and include those
methods and genes
disclosed in U.S. Pat. No. 6,171,789, which is incorporated herein by
reference in its entirety.
In one embodiment, the test mixture is provided in a cell-free system.
In another embodiment, the test mixture is provided in a cell-based system,
wherein one
of the components is exogenous. The term "exogenous" refers to a component
that is either
added directly, or expressed from a heterologous DNA source, such as
transfeeted DNA. Many
methods axe known in the art for expression of heterologous or exogenous gene
products.
In a further embodiment, the evaluated activity is an effect on the rate of
aging of a cell or
organism. Such an effect may be evaluated by contacting the compound with a
cell or organism
having p53 or Sir2 activity, e.g., endogenous or exogenous p53 or Sir2
activity; and evaluating
the rate of aging of the cell or organism. The rate of aging may be evaluated
by several methods,
including:
a) assessing the life span of the cell or organism;
b) assessing the presence or absence of a gene transcript or gene product in
the cell
or organism that has a biological age-dependent expression pattern;
c) evaluating resistance of the cell or organism to stress;
d) evaluating one or more metabolic parameters of the cell or organism;
e) evaluating the proliferative capacity of the cell or a set of cells present
in the
organism;
fj evaluating physical appearance, behavior, or other characteristic of the
cell or
organism; and
(g) assessing the presence or absence of a gene transcript or gene product in
the cell
or organism that has a p53-regulation-dependent expression pattern.
The compounds identified by the methods of the invention can be used, for
example, to
treat cancer (e.g., a compound which decreases Sir2-mediated deacetylation of
p53) or prevent
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p53-mediated apoptosis (e.g., acompound which increases Sift-mediated
deacetylation of pS3).
The compounds can be used in methods oftreating a cell or an organism, e.g., a
cell or organism
that has been exposed to DNA-damaging ionizing radiation, by modulating Sir2
activity in the
cell. In the method of treating cancer in a mammal, Sir2 activity can be
reduced. In a preferred
embodiment, Sir2 activity is reduced by nicotinamide or a nicotinamide analog.
In yet another embodiment, the invention is a method of screening for analogs
of NAD.
In the method, Sir2, p53 and a compound to be tested as an analog of NAD
(e.g., a small organic
or inorganic molecule) are combined. Deacetylation of the p53 by the Sir2 is
measured and
compared to the measured deacetylation of p53 by Sir2 in the presence of NAD.
A compound
which, fox example, promotes Sir2-mediated deacetylation of p53 when combined
with Sir2 and
p53, is an NAD analog and can be used in place of NAD, for example, as a
cofactor with Sir2 to
prevent ox decrease p53-mediated apoptosis.
In a further embodiment, the invention is a method of treating cancer in a
mammal
comprising the step of modulating Sir2 activity in tumor cells to cause an
increase in p53
activity The Sir2 activity can be modulated as described herein (e.g.,
overexpression of Sir2,
transfection of a cell with a dominant negative regulatory gene, or
nicotinamide or a
nicotinamide analog).
In another embodiment, the invention includes a method of treating a cell that
has been
exposed to ionizing radiation, the method comprising modulating Sir2 activity
in the cell. In a
particular embodiment, in a cell which has undergone DNA damage or oxidative
stress, Sir2
activity can be modulated to reduce Sir2 activity (e.g., by transfecting a
cell with a dominant
negative regulatory gene, or by addition or expression of nicotinamide or a
nicotinamide analog)
which can result in the arrest of the growth cycle of the cell, allowing the
cell to repair at least a
portion of the DNA damage caused by the ionizing radiation. Once the cell has
repaired a
portion of the DNA damage, the reduction in Sir2 activity can be removed and
the cell cycle of
the cell resumed.
In still another embodiment, the invention includes an isolated protein
complex of Sir2
and acetylated p53. p53 can also be phosphorylated (e.g., on one or both of
serine 15 or serine
20 of SEQ m NO. 3).
The compounds or NAD analogs identified by the methods of the invention can be
used
in the treatment of diseases or conditions such as cancer, or following DNA
damage or oxidative
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stress. The compounds or NAD analogs can be administered alone or as mixtures
with
conventional excipients, such as pharmaceutically, or physiologically,
acceptable organic, or
inorganic carrier substances such as water, salt solutions (e.g., Ringer's
solution), alcohols, oils
and gelatins. Such preparations can be sterilized and, if desired, mixed with
lubricants,
preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing
osmotic pressure,
buffers, coloring, and/or aromatic substances and the Like which do not
deleteriously react with
the NAD analogs or compounds identified by the methods of the invention.
The dosage and frequency (single or multiple doses) of the compound or NAD
analog
administered to a mammal can vary depending upon a variety of factors,
including the duration
of DNA damage, oxidative stress or cancer condition.
In some embodiments of the present invention, the rate of aging of a cell,
e.g., a yeast
cell, invertebrate cell (e.g., fly cell), or vertebrate cell (e.g., mammalian
cell, e.g., human or
mouse cell) is determined. For example, the rate of aging of the cell can be
evaluated by
measuring the expression of one or more genes or proteins (e.g., genes or
proteins that have an
age-related expression pattern), by measuring the cell's resistance to stress,
e.g., genotoxic stress
or oxidative stress, by measuring one or more metabolic parameters (e.g.,
protein synthesis or
degradation, ubiquinone biosynthesis, cholesterol biosynthesis, ATP levels
within the cell,
glucose metabolism, nucleic acid metabolism, ribosomal translation rates,
etc.), by measuring
cellular proliferation, or any combination of measurements thereof.
In other embodiments, the rate of aging of an organism, e.g., an invertebrate
(e.g., a worm
or a fly) or a vertebrate (e.g., a rodent, e.g., a mouse) is determined. The
rate of aging of an
organism can be determined by directly measuring the average life span of a
group of animals
(e.g., a group of genetically matched animals) and comparing the resulting
average to the
average life span of a control group of animals (e.g., a group of animals that
did not receive the
test compound but are genetically matched to the group of animals that did
receive the test
compound). Alternatively, the rate of aging of an organism can be determined
visually, e.g., by
looking for visible signs of age (e.g., physical appearance or behavior), by
measuring the
expression of one or more genes or proteins (e.g., genes or proteins that have
an age-related
expression pattern), by measuring the cell's resistance to genotoxic (e.g.,
caused by exposure to
etoposide, W irradiation, mutagens, etc.) or oxidative stress, by measuring
one or.more
metabolic parameters (e.g., protein synthesis or degradation, ubiquinone
biosynthesis, cholesterol
33
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WO 03/004621 PCT/US02/21461
biosy~.ithesis, ATP levels, glucose metabolism, nucleic acid metabolism,
ribosomal translation
rates, etc.), by measuring cellular proliferation (e.g., of retinal cells,
bone cells, white blood cells,
etc.), or any combination of measurements thereof. In one embodiment, the
visual assessment is
for evidence of apoptosis, e.g., nuclear fragmentation.
All animals typically go through a period of growth and maturation followed by
a period
of progressive and irreversible physiological decline ending in death. The
length of time from
birth to death is known as the life span of an orgaiusm, and each organism has
a characteristic
average life span. Aging is a physical manifestation of the charges underlying
the passage of
time as measured by percent of average life span.
In some cases, characteristics of aging can be quite obvious. For example,
characteristics
of older humans include skin wrinkling, graying of the hair, baldness, and
cataracts, as well as
hypermelanosis, osteoporosis, cerebral cortical atrophy, lymphoid depletion,
thymic atrophy,
increased incidence of diabetes type II, atherosclerosis, cancer, and heart
disease. Nehlin et al.
(2000), Annals NY Acad Sci 980:176-79. Other aspects of mammalian aging
include weight
loss, lordokyphosis (hunchback spine), absence of vigor, lymphoid atrophy,
decreased bone
density, dermal thickening and subcutaneous adipose tissue, decreased ability
to tolerate stress.
(including heat or cold, wounding, anesthesia, and hematopoietic precursor
cell ablation), liver
pathology, atrophy of intestinal villi, skin ulceration, amyloid deposits, and
joint diseases. Tyner
et al. (2002), Nature 415:45-53.
Careful observation reveals characteristics of aging in other eukaryotes,
including
invertebrates. For example, characteristics of aging in the model organism C.
elegans include
slow movement, flaccidity, yolk accumulation, intestinal autofluorescence
(lipofuscin), loss of
ability to eat food or dispel waste, necrotic cavities in tissues, and germ
cell appearance.
Those skilled in the art will recognize that the aging process is also
manifested at the
cellular level, as well as in mitochondria. Cellular aging is manifested in
loss of doubling
capacity, increased levels of apoptosis, changes in differentiated phenotype,
and changes in
metabolism, e.g., decreased levels of protein synthesis and turnover.
Given the programmed nature of cellular and organismal aging, it is possible
to evaluate
the "biological age" of a cell or organism by means of phenotypic
characteristics that are
correlated with aging. For example, biological age can be deduced from
patterns of gene
expression, resistance to stress (e.g., oxidative or genotoxic stress), rate
of cellular proliferation,
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WO 03/004621 PCT/US02/21461
and the metabolic characteristics of cells (e.g., rates of protein synthesis
and turnover,
mitochondrial function, ubiquinone biosynthesis, cholesterol biosynthesis, ATP
levels within the
cell, levels of a Krebs cycle intermediate in the cell, glucose metabolism,
nucleic acid
metabolism, ribosomal translation rates, etc.). As used herein, "biological
age" is a measure of
the age of a cell or organism based upon the molecular characteristics of the
cell or organism.
Biological age is distinct from "temporal age," which refers to the age of a
cell or organism as
measured by days, months, and years.
Described below are exemplary methods for identifying compounds that can
reduce the
rate of aging of an organism and thereby slow or ameliorate the pathologies
associated with
increased temporal age. Activation of p53 may lead to cell cycle arrest or to
apoptosis; Sir2 can
suppress this effect by deacetylating p53. Accordingly, the expression or
activity of p53 and/or
Sir2 gene products in an organism can be a determinant of the rate of aging
and life span of the
organism. Reduction in the level and/or activity of such gene products would
reduce the rate of
aging and may ameliorate (at least temporarily) the symptoms of aging. A
variety of techniques
may be utilized to inhibit the expression, synthesis, or activity of such
target genes and/or
proteins. Such molecules may include, but are not limited to small organic
molecules, peptides,
antibodies, antisense, ribozyme molecules, triple helix molecules, and the
like.
The following assays provide methods (also referred to herein as "evaluating a
compound" or "screening a compound") for identifying modulators, i.e.,
candidate or test
compounds (e.g., peptides, peptidomimetics, small molecules or other drugs)
which modulate
Sir2 or p53 activity, e.g., have a stimulatory or inhibitory effect on, for
example, Sir2 or p53
expression or activity, or have a stimulatory or inhibitory effect on, for
example, the expression
or activity of a Sir2 or p53 substrate. Such compounds can be agonists or
antagonists of Sir2 or
p53 function. These assays may be performed in animals, e.g., mammals, in
organs, in cells, in
cell extracts, e.g., purified or unpurified nuclear extracts, intracellular
extracts, in purified
preparations, in cell-free systems, in cell fractions enriched for certain
components, e.g.,
organelles or compounds, or in other systems known iii the art. Given the
teachings herein and
the state of the art, a person of ordinary skill in the art would be able to
choose an appropriate
system and assay for practicing the methods of the present invention.
CA 02519161 2002-07-08
WO 03/004621 PCT/US02/21461
Some exemplary screening assays for assessing activity or function include one
or more
of the following features:
- use of a transgenic cell, e.g., with a transgene encoding Sir2 or pS3 or a
mutant thereof;
- use of a mammalian cell that expresses Sir2 or pS3;
- detection of binding of a labeled compound to Sir2 or a transcription factor
where the
compound is, for example, a peptide, protein, antibody or small organic
molecule; e.g., the
compound interferes with or disrupts an interaction between Sir2 and a
transcription factor
- use of proximity assays that detect interaction between Sir2 and a
transcription factor
(e.g., pS3), or fia.gments thereof, for example, fluorescence proximity
assays..
- use of a two hybrid assay to detect interaction between Sir2 and a
transcription factor
(e.g., pS3) or fragments thereof. In some instances, the two hybrid assay can
be evaluated in the
presence of a test compound, e.g., to determine if the test compound disrupts
or interferes with
an interaction. Two hybrid assays can, for example, be conducted using yeast
or bacterial
systems.
- use of radio-labelled substrates, e.g. 355, 3H,14C, e.g., to determine
acetylation status,
metabolic status, rate ofprotein synthesis, irater alia.
- use of antibodies specific for certain acetylated or de-acetylated forms of
the substrate.
One embodiment herein accordingly comprises methods for the identification of
small molecule
drug candidates from large libraries of compounds that appear to have
therapeutic activity to
affect metabolic maintenance and/or to reverse or prevent cell death and thus
exhibits potential
therapeutic utility, such as the ability to enhance longevity Small organic
molecules and
peptides having effective inhibitory activity may be designed de novo,
identified through assays
or screens, or obtained by a combination of the two techniques. Non-protein
drug design may be
carried out using computer graphic modeling to design non-peptide, organic
molecules able to
bind to pS3 or Sir2. The use of nuclear magnetic resonance (Nl~) data for
modeling is also
known in the art, as described by Lam et al., Science 263: 380,1994, using
information from x-
ray crystal structure studies of pS3 or Sir2, such as that described in Min,
J. et al., Cell 105:269-
279, 2001.
Small molecules may also be developed by generating a library of molecules,
selecting
for those molecules which act as Iigands for a specified target, (using
protein functional assays,
for example), and identifying the selected ligands. See, e.g., Kohl et al.,
Science 260: 1934,
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1993. Techniques for constructing and screening combinatorial libraries of
small molecules or
oligomerie biomolecules to identify those that specifically bind to a given
receptor protein are
Irnown. Suitable oligomers include peptides, oligonucleotides, carbohydrates,
nonoligonucleotides (e.g., phosphorothioate oligonucleotides; see Chem. and
Engineering News,
page 20, 7 Feb. 1994) and nonpeptide polymers (see, e.g., "peptoids" of Simon
et al., Proc. Natl.
Acad. Sci. USA 89 9367, 1992). See also U.S. Pat. No. 5,270,170 to Schatz;
Scott and Smith,
Science 249: 386-390, 1990; Devlin et al., Science 249: 404-406, 1990;
Edgington,
BIOlTechnology, l l : 285, 1993. Libraries may be synthesized in solution on
solid supports, or
expressed on the surface of bacteriophage viruses (phage display libraries).
Known screening methods may be used by those spilled in the art to screen
combinatorial
libraries to identify active molecules. For example, an increase (or decrease)
in p53 or Sir2
activity due to contact with an agonist or antagonist can be monitored.
Tn one embodiment, assays for screening candidate or test compounds that are
substrates
of a Sir2 or p53 protein or polypeptide or biologically active portion thereof
are provided. In
another embodiment, assays for screening candidate or test compounds which
bind to or
modulate the' activity of a Sir2 or pS3 protein or polypeptide or biologically
active portion
thereof, e.g., modulate the ability of SirZ or p53 to interact with a ligand,
are provided. In still
another embodiment, assays for screening candidate or test compounds for the
ability to bind to
or modulate the activity of a Sir2 or p53 protein or polypeptide and to also
alter the rate of aging
of a cell or an organism are provided.
Examples of methods for the synthesis of molecular libraries can be found in
the art, for
example in: De~tt et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6909, 1993; Erb.
et al., Proc. Natl.
Acad. Sei. USA 91: 11422,1994; Zuckermann et al., J. Med. Chem. 37: 2678,
1994; Cho et al.,
Science 261 : 1303, 1993; Carrell et al., Angew. Chem. /nt. Ed. Engl. 33:
2059, 1994; Carell et
al., Angew. Chem. /nt. Ed. Engl. 33: 2061, 1994; and in Gallop et al., J. Med
Chem. 37:1233,
1994. '
Libraries of compounds may be presented in solution (e.g., Houghten,
Biotechniques 13:
412-421, 1992), or on beads (Lam, Nature 354: 82-84, 1991), chips (Fodor,
Nature 364: 555-
556, 1993), bacteria (Ladner U.S. P.N. 5,223,409), spores (Ladner U.S. PN.
'409), plasmids (Cull
et al., Proc Natl Acad Sci USA 89: 1865-1869, 1992) or on phage (Scott and
Smith" Science 249:
37
CA 02519161 2002-07-08
WO 03/004621 PCT/US02/21461
386-390, 1990); (Devlin, Science 249: 404-406, 1990); (Cwirla et al., Proc.
Natl. Acad. Sci
U.S.A. 87: 6378-6382, I990); (Felici, J. Mol. Biot. 222: 30I-310, 1991);
(Ladner supra.).
The compounds tested as modulators of Sir2 or p53 can be any small chemical
compound, or a biological entity, such as a protein, e.g., an antibody, a
sugar, a nucleic acid, e.g.,
an antisense oligonucleotide or a ribozyme, or a lipid. .Alternatively,
modulators can be
genetically altered versions of Sift or p53. Typically, test compounds will be
small chemical
molecules and peptides, or antibodies, antisense molecules, or ribozymes.
Essentially any
chemical compound can be used as a potential modulator or ligand in the assays
of the invention,
although most often compounds that can be dissolved in aqueous or organic
(especially DMSO-
based) solutions are used. The assays are designed to screen large chemical
libraries by
automating the assay steps and providing compounds from any convenient source
to assays,
which are typically run in parallel (e.g., in microtiter formats on microtiter
plates in robotic
assays), It will be appreciated that there are many suppliers of chemical
compounds, including
Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO),
Fluka
Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
In one preferred embodiment, high throughput screening methods known to one of
ordinary skill in the art involve providing a combinatorial chemical or
peptide library containing
a large number of potential therapeutic compounds (potential modulator or
ligand compounds).
Such "combinatorial chemical libraries" or "ligand libraries" are then
screened in one or more
assays, as described herein, to identify those library members (particular
chemical species or
subclasses) that display a desired characteristic activity: The compounds thus
identified can
serve as conventional "lead compounds" or can themselves be used as potential
or actual
therapeutics.
A combinatorial chemical library is a collection of diverse chemical compounds
generated by either chemical synthesis or biological synthesis, by combining a
number of
chemical "building blocks" such as reagents. For example, a linear
combinatorial chemical
library such as a polypeptide library is formed by combining a set of chemical
building blocks
(amino acids) in everypossible way for a given compound length (i.e., the
number of amino
acids in a polypepfiide compound). Millzons of chemical compounds can be
synthesized through
such combinatorial mixing of chemical building blocks. Moreover, a
combinatorial library can
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WO 03/004621 PCT/USOZ/21461
be designed to sample a family of compounds based on a parental compound,
e.g., based on the
chemical structure of NAD or nicotinamide.
Preparation and screening of combinatorial chemical libraries is well known to
those of
skill in the art. Such combinatorial chemical libraries include, but are not
limited to, peptide
libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res.
37:487-493 (1991) and ,
Houghton et al., Nature 354:84-88 (1991)). Other chemistries for generating
chemical diversity
libraries can also be used. Such chemistries include, but are not limited to:
peptoids (e.g., PCT
Publication No. WO 91119735), encoded peptides (e.g., PCT Publication No. WO
93/20242),
random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines
(e.g., U.S.
Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and
dipeptides (Hobbs et
al., Proc. Nat. Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides
(Hagihara et al.,
J. Amen. Chem. Soc. 114:6568 (1992)), nonpeptidal peptidomimetics with glucose
scaffolding
(Hirschmann et al., J. Amer. Chem. Soc. 114:9217-9218 (1992)), analogous
organic syntheses of
small compound libraries (Chen et ad., J. Amer. Clzem. Soc. 116:2661 (1994)),
oligocarbamates
(Cho et al., Science 261:1303 (1993)), and/or peptidyl phosphonates (Campbell
et al., J. Org.
Chem. 59:658 (1994)), nucleic acid libraries (see Ausubel, Berger and
Sambrook, all supra),
peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody
libraries (see, e.g.,
Vaughn et al., Nature Biotechnology, I4(3):309-3I4 (1996) and
PCT/CTS96/10287), carbohydrate
libraries (see, e.g., Liang et al., Science, 274:1520-1522 (1996) and U.S.
Patent 5,593,853), small
organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18,
page 33 (1993);
isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S.
Patent 5,549,974;
pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S.
Patent
5,506,337; benzodiazepines, 5,288,514, and the Iike).
Devices for the preparation of combiliatorial libraries are commercially
available (see,
e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin,
Woburn,
MA, 433AApplied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford,
MA). In
addition, numerous combinatorial libraries are themselves commercially
available (see, e.g.,
ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, M0,
ChemStar, Ltd,
Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD,
etc.).
In one embodiment, the invention provides solid phase based in vitro assays in
a high
throughput format, e.g., where each assay includes a cell or tissue expressing
Sir2 and/or p53. In
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CA 02519161 2002-07-08
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a high throughput assays, it is possible to screen up to several thousand
different modulators or
ligands in a single day In particular, each well of a microtiter plate can be
used to run a separate
assay against a selected potential modulator, or, if concentration or
incubation time effects are to
be observed, every 5-10 wells can test a single modulator. Thus, a single
standard microtiter
plate can assay about 96 modulators. If 1536 well plates are used, then a
single plate can easily
assay from about 100- about 1500 different compounds. It is possible to assay
many plates per
day; assay screens for up to about 6,000, 20,000, 50,000, or 100,000 or more
different
compounds are possible using the integrated systems of the invention.
Candidate Sir2- or p53-interacting molecules encompass many chemical classes.
They
can be organic molecules, preferably small organic compounds having molecular
weights of 50
to 2,500 Daltons. The candidate molecules comprise functional groups necessary
for structural
interaction with proteins, particularly hydrogen bonding, for example,
carbonyl, hydroxyl, and
carboxyl groups. The candidate molecules can comprise cyclic carbon or
heterocyclic structures
and aromatic or polyaromatic structures substituted with the above groups. In
one embodiment,
the candidate molecules are structurally andlor chemically related to NAD or
to nicotinamide.
Other techniques are known in the art for screening synthesized molecules to
select those
with the desired activity, and for labeling the members of the library so that
selected active
molecules may be identified, as in U.S. PN. 5,283,173 to Fields et al., (use
of genetically altered
Saccharomyces cerevisiae to screen peptides for interactions). As used herein,
"combinatorial
library' refers to collections of diverse oligomeric biomolecules of differing
sequence, which can
be screened simultaneously for activity as a ligand for a particular target.
Combinatorial libraries
may also be referred to as "shape libraries", i.e., a population of randomized
fragments that are
potential ligands. The shape of a molecule refers to those features of a
molecule that govern its
interactions with other molecules, including Van der Waals, hydrophobic,
electrostatic and
dynamic.
Nucleic acid molecules may also act as ligands for receptor proteins. See,
e.g.,
Edgington, BIOlTechnology 11: 285, 1993. U.S. P.N. 5,270,163 to Gold and Tuerk
describes a
method for identifying nucleic acid ligands for a given target molecule by
selecting from a
library of RNA molecules with randomized sequences those molecules that bind
specifically to
the target molecule. A method for the in vitro selection of RNA molecules
immunologically
cross-reactive with a specific peptide is disclosed in Tsai et a1, Proc.
lVatl. Acad. Sci. USA 89:
CA 02519161 2002-07-08
WO 03/004621 PCT/US02/21461
8864, (1992); and Tsai et al. Immunology 150:1137, (1993). In the method, an
antiserum raised
against a peptide is used to select RNA molecules from a library of RNA
molecules; selected
RNA molecules and the peptide compete for antibody binding, indicating that
the RNA epitope
functions as a specific inhibitor of the antibody-antigen interaction.
Antibodies that are both specific for a target gene protein and that interfere
with its
activity may be used to inhibit target gene function. Such antibodies may be
generated using
standard techniques, against the proteins themselves or against peptides
corresponding to
portions of the proteins. Such antibodies include but are not limited to
polyclonal, monoclonal,
Fab fragments, single chain antibodies, chimeric antibodies, and the like.
Where fragments of
the antibody are used, the smallest inhibitory fragment which binds to the
target protein's binding
domain is preferred. For example, peptides having an amino acid sequence
corresponding to the
domain of the variable region of the antibody that binds to the target gene
protein may be used.
Such peptides may be synthesized chemically or produced via recombinant DNA
technology
using methods well known in the art (e.g., see Sambrook et al., Eds.,
Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, (1989), or
Ausubel, F. M. et
aZ., eds. Current Protocols in Molecular Biology (1994).
Alternatively, single chain neutralizing antibodies that bind to intracellular
target gene
epitopes may also be administered. Such single chain antibodies may be
administered, for
example, by expressing nucleotide sequences encoding single-chain antibodies
within the target
cell population by utilizing, for example, techniques such as those described
in Marasco et al.,
Pt-oc. Natl. Acad. Sci. USA 90: 7889-7893 (1993).
Also encompassed are assays for cellular proteins that interact with Six2 or
p53. Any
method suitable for detecting protein-protein interactions may be used. The
traditional methods
that may be used include, for example, co-immunoprecipitation, crosslinlcing,
and co-purification
through gradients or chromatographic columns. For these assays, Sir2 or p53
can be a full-
length protein or an active fragment. Additional methods include those methods
that allow for
the simultaneous identification of genes that encode proteins that interact
with Sir2 or p53.
These methods include, for example, probing expression libraries using a
labeled Sir2 or p53
protein, Sir2 or p53 fragment, or Sir2 or p53 fusion protein.
One method to detect protein-protein interaction in vivo is the two-hybrid
system, see, for
example, Chien et al., Proc. Natl. Acad. Sci, USA 88: 9578-9582 (1991). In
brief, the two-hybrid
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system utilizes plasmids constructed to encode two hybrid proteins: one
plasmid comprises the
nucleotides encoding the DNA binding domain of a transcriptional activator
protein fused to the
Sir2 or p53 nucleotide sequence encoding the Sir2 or p53 polypeptide, and the
other plasmid
comprises the nucleotides encoding the transcriptional activator protein's
activation domain
fused to a cDNA encoding an unknown protein that has been recombined into the
plasmid from a
cDNA library The DNA binding domain fusion plasmid and the cDNA fusion protein
library
plasmids are transformed into a strain of yeast that contains a reporter gene,
for example lacZ,
whose regulatory region contains the activator's binding site. Either hybrid
protein alone cannot
activate translation of the reporter gene because it is lacking either the DNA
binding domain or
the activator domain. Tnteraction of the two hybrid proteins, however,
reconstitutes a functional
activator protein and results in activation of the reporter gene that is
detected by an assay for the
reporter gene product. The colonies that reconstitute activator activity are
purified and the
library plasmids responsible for reporter gene activity are isolated and
sequenced. The DNA
sequence is then used to identify the protein encoded by the library plasmid.
Macromolecules that interact with Sir2 or p53 are referred to as Sir2 or p53
binding
partners. Sir2 or p53 binding partners are likely to be involved in the
regulation of Sir2 or p53
function. Therefore, it is possible to identify compounds that interfere with
the interaction
between Sir2 or p53 and its binding partners. The basic principle of assay
systems used to
identify compounds that interfere with the interaction of Sir2 or p53 and a
binding partner is to
prepare a reaction mixture containing Sir2 or p53 or a Sir2 or p53 fragment
and the binding
parhier under conditions that allow complex formation. The reaction mixture is
prepared in the
presence or absence of the test compound to test for inhibitory activity The
test compound may
be added prior to or subsequent to Sir2/ or p53/binding partner complex
formation. The
formation of a complex in a control but not with the test compound confirms
that the test
compound interferes with complex formation. The assay can be conducted either
in the solid
phase or in the liquid phase.
In another embodiment, an assay is a cell-based assay comprising contacting a
cell
expressing Sir2 or p53 with a test compound and determining the ability of the
test compound to
modulate (e.g. stimulate or inhibit) the activity of Sir2 or p53. A preferred
activity is the
deacetylation function of Sir2 on p53; a further preferred activity is the
ability of p53 to cause
ERU cycle arrest or apoptosis. Determining the ability of the test compound to
modulate the
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activity of Sir2 or p53 can be accomplished, for example, by determining the
ability of Sir2 or
pS3 to bind to or interact with the test molecule, or by determining the
ability of the test
molecule to stimulate or inhibit the activity of Sir2 or pS3. CeII-based
systems can be used to
identify compounds that inhibit Sir2 or p53, Such cells can be recombinant or
non-recombinant,
such as cell lines that express the Sir2 or p53 gene. Preferred systems are
mammalian or yeast
cells that express Sir2 or pS3. 1n utilizing such systems, cells are exposed
to compounds
suspected of ameliorating body weight disorders or increasing lifespan. After
exposure, the cells
are assayed, for example, for expression of the Sir2 or p53 gene or activity
of the Sir2 or p53
protein. .Alternatively, the cells are assayed for phenotypes such as those
resembling body
weight disorders or lifespan extension. The cells may also be assayed for the
inhibition of the
deacetylation function of Sir2 on p53, or the apoptotic or cytostatic function
of p53.
Another preferred cell fox a cell-based assay comprises a yeast cell
transformed with a
vector comprising the Sir2 or p53 gene. One use for a yeast cell expressing
Sir2 or p53 is to
mutagenize the yeast and screen for yeast that will survive only when the Sir2
or p53 polypeptide
is functioning normally Synthetic lethal screens are described in Holtzman et
al. (1993), J. Cell
Bio. 122: 635-644. The yeast that require Sir2 or p53 function for survival
can then be used to
screen test compounds for those that inhibit Sir2 or p53 activity Test
compounds that results in a
decrease in yeast survival are likely inlubitors of Sir2 or p53 in this
system.
In yet another embodiment, an assay is a cell-free assay in which Sir2 or p53
protein or
biologically active portion thereof is contacted with a test compound and the
ability of the test
compound to bind to the Sir2 or p53 protein or biologically active portion
thereof is determined.
Binding of the test compound to the Sir2 or p53 protein can be determined
either directly or
indirectly as described above. In a preferred embodiment, the assay includes
contacting the Sir2
or pS3 protein or biologically active portion thereof with a known compound
which binds Sir2 or
p53 to form an assay mixture, contacting the assay mixture with a test
compound, and
determining the ability of the test compound to interact with an Sir2 or p53
protein, wherein
deterniining the ability of the test compound to interact with an Sir2 or p53
protein comprises
determining the ability of the test compound to preferentially bind to Sir2 or
p53 or a
biologically active portion thereof as compared to the known compound.
In yet another embodiment, an assay is a cell-free system in which Sir2
protein or
biologically active portion thereof is contacted with p53 protein or
biologically active portion
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WO 03/004621 PCT1US02/21461
thereof, to form a mixture comprising a detectable amount bound p53:Sir
complex. And a test
compound is contacted with the mixture, and the ability of the compound to
effect the stability or
formation of the p53:Sir2 complex is determined. Interaction of the test
compound with he
p53:Sir2 complex may be determined directly or by methods known in the art. Iu
a preferred
embodiment, the method comprises contacting p53 with Sir2 to form a mixture
comprising the
p53:Sir2 complex, further contacting the mixture with a compound to be tested,
and evaluating
the binding kinetics of p53:Sir2 complex both in the presence and the absence
of the test
compound to directly bind the p53:Sir2 complex is evaluated. The cell-free
assays are amenable
to use of both soluble and/or membrane-bound forms of proteins. In the case of
cell-free assays
in which a membrane-bound form of a protein is used it may be desirable to
utilize a solubilizing
agent such that the membrane-bound form of the protein is maintained in
solution. Examples of
such solubilizing agents include non-ionic detergents such as n-
octylglucoside, n-
dodecylglucoside, n-dodecylinaltoside, octanoyl-N-methylglucamide, decanoyl-N-
methylglucamide, Triton X-100, Triton X-114, Thesit, Isotridecypoly(ethylene
glycol ether)n, 3-
[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), 3-[(3-
cholamidopropyl)dimethylammonio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-
dodecyl,N,N-dimethyl-3-amino-1-propane sulfonate.
In more than one embodiment of the above assay methods, it rnay be desirable
to
immobilize either Sir2 or p53 or its target molecule to facilitate separation
of complexed from
uncomplexed forms of one or both of the proteins, as well as to accommodate
automation of the
assay Binding of a test compound to an Sir2 or p53 protein, or interaction of
an Sir2 or p53
protein with a target molecule in the presence and absence of a candidate
compound, can be
accomplished in any vessel suitable for containing the reactants. Examples of
such vessels
include microtiter plates, test tubes, and micro-centrifuge tubes. in one
embodiment, a fusion
protein can be provided which adds a domain that allows one or both of the
proteins to be bound
to a matrix. For example, glutathione-S-transferase/Sir2 or /p53 fusion
proteins or glutathione-S-
firansferase/target fusion proteins can be adsorbed onto glutathione sepharose
beads (Sigma
Chemical, St. Louis, Mo.) or glutathione derivatized microtitre plates, which
are then combined
with the test compound or the test compound and either the non-adsorbed target
protein or Sir2
or p53 protein, and the mixture incubated under conditions conducive to
complex formation
(e.g., at physiological conditions for salt and pI-~. Following incubation,
the beads or microtiter
44
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~'VO 03/004621 PCT/US02/21461
plate wells are washed to remove any unbound components, the matrix
immobilized in the case
of beads, complex determined either directly or indirectly, fox example, as
described above.
Alternatively, the complexes can be dissociated from the matrix, and the level
of Sir2 or p53
binding or activity determined using standard techniques.
Other techniques for immobilizing proteins on matrices can also be used in the
screening
assays of the invention. For example, either a Six2 or p53 protein or a Sir2
or p53 target molecule
can be immobilized utilizing conjugation of biotin and streptavidin.
Biotinylated Sir2 or p53
protein or target molecules can be prepared from biotin-NHS (N-hydroxy-
succinzmide) using
techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals,
Rockford, Ill.), and
immobilized in the wells of streptavidin-coated 96 well plates (Pierce
Chemical). Alternatively,
antibodies reactive with Sir2 or p53 protein or target molecules but which do
not interfere with
binding of the Sir2 or p53 protein to its target molecule can be derivatized
to the wells of the
plate, and unbound target Sir2 or p53 protein trapped in the wells by antibody
conjugation.
Methods for detecting such complexes, in addition to those described above for
the GST
immobilized complexes, include immunodetection of complexes using antibodies
reactive with
the Sir2 or p53 protein or target molecule, as well as enzyme-linked assays
which rely on
detecting an enzymatic activity associated with the Sir2 or p53 protein or
target molecule.
In addition to cell-based and in vitro assay systems, non-human organisms,
e.g.,
transgenic non-human organisms, can also be used. A transgenic organism is one
in which a
heterologous DNA sequence is chromosomally integrated into the germ cells of
the animal. A
transgenic organism will also have the transgene integrated into the
chromosomes of its somatic
cells. Organisms of any species, including, but not limited to: yeast, worms,
flies, fish, reptiles,
birds, mammals (e.g., mice, rats, rabbits, guinea pigs, pigs, micro-pigs, and
goats), and non-
human primates (e.g., baboons, monkeys, chimpanzees) may be used in the
methods of the
invention.
Accordingly, in another embodiment, the invention features a method of
identifying a
compound that alters the rate of aging of a cell or an organism, comprising:
contacting a Sir2 or
p53 polypeptide with a test compound; evaluating an interaction between the
test compound and
the Sir2 or pS3 polypeptide; and further evaluating the effect of the test
compound on the rate of
aging of a cell or organism.
CA 02519161 2002-07-08
WO 03/004621 PCT/US02/21461
The interaction between a test compound and the Sir2 or p53 polypeptide can be
performed by any of the methods described herein, e.g., using cell-based
assays or cell-free in
vitro assays. Weather the interaction between the test compound and the Sir2
or p53 polypeptide
is evaluated prior to the evaluation of the effect of the text compound on the
rate of aging of a
cell or organism is not critical to the method. However, it is preferable to
evaluate the interaction
between the test compound and Sir2 or p53 polypeptide first, so that test
compounds that do not
interact with the Sir2 or p53 polypeptide do not have to be tested for their
effect upon the rate of
aging. It can also be preferable to use an assay for evaluating the
interaction between the test
compound and the Sir2 or p53 polypeptide that can be adapted for high
throughput screening,
thus malting it possible to screen one or more libraries of test compounds,
Possible test
compounds include, e.g., small organic molecules, peptides, antibodies, and
nucleic acid
molecules, as described above.
The rate of aging of an organism can be determined using methods known in the
art. For
example, the rate of aging of an organism can be determined by directly
measuring the life span
of the organism. Preferably, a statistical measure, e.g., an average or median
value, of the life
span of a group of animals, e.g., a group of genetically matched animals, will
be determined and
the resulting statistical value compared to an equivalent statistical value,
e.g, an average of
median value, of the life span of a control group of animals, e.g., a group of
animals that did not
receive the test compound but are genetically matched to the group of animals
that did receive
the test compound. Such methods are suitable for organisms that have a short
life span, such as
worms or flies. See, for example, Rogina, et al. (2000), Science 290:2137-40,
Direct
measurement of life span can also be preformed with other organisms such as
rodents, as
discussed, fox example, in Weindruch et al. (1986), .Tournal of Nutrition
116(4):641-S4. Those
skilled in the art will recognize that there are many ways of measuring the
statistical difference
(e.g., using the Student's T test) between two sets of data, any of which may
be suitable for the
methods of the invention.
To reduce the time that it takes to measure a change in the rate of aging
using data an the
life span of the organisms treated with the test compound, various
modifications or treatments of
the organisms can be implemented. For example, animals fed on a calorically
rich diet tend to
live shorter lives, thus reducing the time that needs to elapse to determine
when the average life
span of the test group of animals has exceeded the average life span of the
control group of
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WO 03/004621 PCT/US02/21461
animals. Alternatively, the test compound can be administered to test animals
that have already
lived for SO%, 60%, 70%, 80%, 90%, or more of their expected life span. Thus,
the test
compound can be administered to an adult organism, or even an old adult
organism. Other
possibilities include the use of genetically modified organisms. For example,
the organisms
could harbor mutations (e.g., a Hype~l~inetic~ or Shalcers mutation in
Drosophila, or a mutation in
a silent information regulator gene (e.g., Sir2), or a catalase or superoxide
dismutase gene) or
transgenes (e.g., encoding a transporter protein (e.g., a carboxylate
transport protein such as
IND~ or a protein involved in insulin signaling and metabolic regulation
(e.g., IG.F-1)) that
reduce their average life span. See Rogina et al. (1997), PYOC. Natl. Acad.
Sci., USA 94:6303-6;
Rogina and Helfand (2000), Biogerontology 1:163-9; and Guarente and Kenyon
(2000), NatuYe
408:255-62. Those skilled in the art will understand that it may also be
desirable to practice the
methods of the invention using organisms that are long-lived, such as
calorically restricted
animals, or animals carrying mutations or transgenes that increase their life
span.
A proxy for rate of aging of a cell or an organism can be determined using
biomarkers
that are indicative of the biological age of the organism (i.e., age-related
parameters). Using
biomarkers for determining biological age can greatly facilitate screens for
compounds that alter
the rate of aging, as they bypass the requirement of waiting for the animal to
die in order to
determine the rate of aging. Biomarkers suitable for use in the present
invention include, but are
not limited to, levels of protein modification, e. g., accumulation of
glycosylated proteins, rates or
levels of protein turnover, levels or composition of T cell populations,
protein activity, physical
characteristics, macular degeneration, and/or increased copper and zinc
concentrations i.n
neuronal tissues. The expression of genes whose regulation is biological age-
dependent is a
particularly preferred biomarker fox use in the methods of the invention.
Numerous genes are
known to be expressed in a biological age-dependent manner. In Drosophila, for
example, such
genes include wingless and engrailed. See Rogina and Helfand (1997),
Mechanisms of
Development 63:89-97. In mice, the expression of the ras oncogene is elevated
in older animals.
See Hass et al. (1993), Mutat. Res. 295(4-6):281-9. Similarly, in rodents and
worms, genes that
are differentially expressed in young and old organisms have been identified
by transcriptional
profiling using microarrays. See, e.g., Lee et al. (1999), Science 285:1390-
93; WO 01/12851;
and Hill et al. (2000), Science 290:809-812. For example, Hill et al. (2000)
Science 90:809
discloses genes whose transcripts are up-regulated in nematodes that are at 2
weeks in
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WO 03/004621 PCT/US02/21461
development. Examples of such genes include the genes described in cluster
(4,1):69 of Hill,
supra. Any gene whose regulation is biological age-dependent is suitable for
the methods of the
invention. Preferably, more than one gene is analyzed so as to improve the
accuracy of the
determination. Analysis of gene expression can be performed by any technique
known in the art,
including Northern, in-situ hybridization, quantitative PCR, and
transcriptional profiling using
microarrays. Methods of determining biological age based on gene expression
patterns are
described in WO 01/12851.
Metabolic parameters can also be used to evaluate the xate of aging of a cell
or organism.
For example, the rate of protein synthesis and degradation decreases in
biologically aged cells,
and the levels proteins having advanced glycosylation end product
modifications increases. See,
Lambert and Merry (2000), Exp. Gerontol 3S(S):583-94; and WO 01/79842. In
addition,
animals that harbor mutations conferring longer life span (and thus a reduced
rate of aging) can
show defects in ubiquinone biosynthesis, mitochondria) biogenesis, glucose
metabolism, nucleic
acid metabolism, ribosomal translation rates, and cholesterol biosynthesis.
See, for example,
WO 98/17823 and WO 99/10482. Thus, by measuring any of these parameters or
some
combination thereof, it is possible to indirectly evaluate the rate of aging
of a cell or an organism.
Methods of analyzing protein synthesis, degradation, and modification with
advanced
glycosylation end products are known in the art, as described in Lambert and
Merry (2000), Exp.
Gerontol 3S(S):583-94 and WO 01/79842. Similarly, methods of analyzing
ubiquinone
biosynthesis, mitochondria) biogenesis, and glucose metabolism are known in
the art (see, e.g.,.
Marbois et al. J. Biol. Chem. 271:299S; Proft et al. EMBO J. 14:6116; and WO
98/17823), as are
methods of analyzing nucleic acid metabolism, ribosomal translation rates, and
cholesterol
biosynthesis (see, e.g., WO 99/10482).
Cellular proliferation is another parameter that can be used to evaluate the
biological age
of a cell or organism. Cells from biologically aged organisms demonstrate
reduced proli~erative
capacity as compared to the cells of a corresponding younger organism. See Li
et al. ( 1997),
Invest. Ophthalmol. 38(1):100-7; and Wolf and Pendergrass (1999), J Gerontol.
A Biol. Sci. Med.
Sci. S4(11):BS02-17. It will be understood by one skilled in the art that
there are many methods
for evaluating the proliferative capacity of cells that are suitable for use
in the methods of the
invention. For example, cells can be labeled in vitro (or in vivo) with BrdU
to determine the
percent of dividing cells or evaluated using a colony forming assay, as
described in Li et al.
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WO 03/004621 PCT/US02/21461
(1997), supra. Cells suitable for the analysis of proliferative capacity
include cells grown in
tissue culture, cells isolated from an animal that has been treated with a
test compound, cells that
are part of a live animal, or cells that are part of a tissue section obtained
from an animal. With
respect to cells present in an animal or tissue section thereof, preferable
cells include lens
epithelial cells, osteoblasts, osteoclasts, and lymphoid cells.
Basically, any biomarker that is altered in a biological age-dependent manner
has the
potential to be used to evaluate the effect of a test compound upon the rate
of aging of a cell or
an organism. Thus, additional biomarkers include visual appearance, resistance
to oxidative
stress, cellular transformation (the ability to adopt a transformed (i.e.,
cancerous or malignant)
phenotype), or DNA methylation (e.g., of a ras oncogene). See, for example,
Finkel and
Holbrook (2000), Nature 408:239-47; Kari et al. (1999), JNuty: Health Aging
3(2):92-101; and
Hass et al. (1993), Mutat. Res. 295(4-6):281-9.
A cell used in the methods of the invention can be from a stable cell line or
a primary
culture obtained from an organism, e.g., a organism treated with the test
compound.
A transgenic cell or animal used in the methods of the invention can include a
transgene
that encodes, e.g., a copy of a Sir2 or p53 protein, e.g., the Sir2 or p53
polypeptide that was
evaluated for an interaction with the test compound. The transgene can encode
a protein that is
normally exogenous to the transgenic cell or animal, including a human
protein, e.g., a human
Sir2 or p53 polypeptide. The transgene can be linked to a heterologous or a
native promoter.
Transgenic Organisms
This disclosure further relates to a method of producing transgenic animals,
e.g., mice or
flies. In one embodiment, the transgenic animal is engineered to express,
overexpress or
ectopically express Sir2 or p53, which method comprises the introduction of
several copies of a
segment comprising at least the polynucleotide sequence encoding SEQ ID NO. 2
with a suitable
promoter into the cells of an embryo at an early stage. Techniques laiown in
the art may be used
to introduce the Sir2 or p53 transgene into animals to produce the founder
line of animals. Such
techniques include, but are not limited to: pronuclear microinjection (U.S.
P.N. 4,873,191);
retrovirus mediated gene transfer into germ lines (Van der Putten et al.,
Proc. Natl. Acad. Sci.
USA 82: 6148-6152, 1985; gene targeting in embryonic stem cells (Thompson et
al., Cell 56:
3I3-321, 1989; electroporation of embryos (Lo, Mol. Cell Biol. 3: 1803-1814,
1983; and sperm-
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mediated gene transfer (Lavitrano, et ad., Cell 57: 717-723, 1989; etc. For a
review of such
techniques, see Gordon, Ihtl. Rev. Cytod. 115: 171-229, 1989.
Gene targeting by homologous recombination in embryonic stem cells to produce
a
trausgenic animal with a mutation in the Sir2 or p53 gene ("knock-out"
mutation) can also be
performed . In such so-called "knock-out" animals, there is inactivation of
the Sir2 or p53 gene
or altered gene expression, such that the animals can be useful to study the
function of the Sir2 or
p53 gene, thus providing animals models of human disease, which are otherwise
not readily
available through spontaneous, chemical or irradiation mutagenesis.
A particularly useful transgenic animal in one in which the Sir2 or p53
homolog has been
disrupted or knocked out.
Transgenic animals such as mice, for example, may be used as test substrates
for the
identification of drugs, pharmaceuticals, therapies and interventions that can
be used for the
ameliorating or slowing the effects of aging.
Accordingly, the invention features a transgenic organism that contains a
transgene
encoding a Sir2 or p53 polypeptide. In preferred embodiments, the Sir2 or p53
r polypeptide is a
human Sir2 or p53 polypeptide. The Sir2 or p53 polypeptide can be exogenous to
(i.e., not
naturally present in) the transgenic organism.
The transgenic organism can be a yeast cell, an insect, e.g., a worm or a fly,
a fish, a
reptile, a bird, or a mammal, e.g., a rodent.
The transgenic organism can furhher comprise a genetic alteration, e.g., a
point mutation,
insertion, or deficiency, in an endogenous gene. The endogenous gene harboring
the genetic
alteration can be a gene involved in the regulation of life span, e.g., a gene
in the insulin
signaling pathway, a gene encoding a Sir2 or transcription factor protein, or
both. In cases where
the genetically altered gene is a Sir2 or transcription factor, e.g., p53,
polypeptide, it is preferable
that the expression or activity of the endogenous Sir2 or transcription
factor, e.g., p53, protein is
reduced or eliminated.
Therapeutic Uses
In another embodiment, the invention features a method of altering the
expression or
activity of a Sir2 or p53 polypeptide, comprising administering to a cell or
an organism a
compound that increases or decreases the expression or activity of the Sir2 or
p53 polypeptide in
an amount effective to increase or decrease the activity of the Sir2 or p53
polypeptide.
so
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The Sir2 or p53 polypeptide can also be a yeast, invertebrate (e.g., worm or
fly), or
vertebrate (e.g.; fish, reptile, bird, or mammal (e.g., mouse)) protein. .
The cell to which the compound is administered can be an invertebrate cell,
e.g., a worm
cell or a fly cell, or a vertebrate cell, e.g., a fisli cell (e.g., zebrafish
cell), a bird cell (e.g., chicken
cell), a reptile cell (e.g., amphibian cell, e.g., Xenopus cell), or a
mammalian cell (e.g., mouse or
human cell). Similarly, the organism to which the compound is administered can
be an
invertebrate, e.g., a worm or a fly, or a vertebrate, e.g., a fish (e.g.,
zebrafish), a bird (e.g.,
chicken), a reptile (e.g., amphibian, e.g., Xenopus), or a mammal (e.g.,
rodent or a human).
When the organism is a human, it is preferred that the human is not obese or
diabetic.
The compound that is administered to the cell or organism can be an agonist
that
increases the expression or activity of the Sir2 or p53 polypeptide or an
antagonist.that decreases
the expression or activity of the Sir2 or p53 polypeptide. Whether agonist or
antagonist, the
compound can be a small organic compound, an antibody, a polypeptide, or a
nucleic acid
molecule.
The agonist or antagonist can alter the concentration of metabolites, e.g.,
Krebs Cycle
intermediates, e.g., succinate, citrate, or a-keto-glutarate, within the cell
or within one or more
cells of the organism. Such action is expected to alter the cell's or the
organism's resistance to
oxidative stress. For example, an antagonist could increase the cell's or the
organism's
resistance to oxidative stress. In addition, the agonist or antagonist can
alter one or more aging-
related parameters, e.g., the expression of one or more genes or proteins
(e.g., genes or proteins
that have an age-related expression pattern), or the value of one or more
metabolic parameters
(e.g., one or more metabolic parameters that reflect the rate of aging of the
cell or organism). ,
the agonist or antagonist alters the rate of aging of the cell or organism.
Ideally, the compound reduces, e.g., partially reduces, the expression of the
Sir2 or p53
polypeptide. For example, anti-sense RNA, or ribozymes can be used to reduce
the expression
of the Sir2 or p53 polypeptide. Double-stranded inhibitory RNA is particularly
useful as it can
be used to selectively reduce the expression of one allele of a gene and not
the other, thereby
achieving an approximate 50% reduction in the expression of the Sir2 or p53
polypeptide. See
Garrus et al. (200I), Cell 107(1):55-65.
In one embodiment, treatment of aging comprises modulating the expression of a
Sir2 or
p53 polypeptide. A cell or subject can be treated with a compound that
modulates the expression
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,WO 03/004621 PCT/US02/21461
of a Sir2 or p53 gene. These compounds can be nucleic acid molecules
substantially
complementary to a Sir2 or p53 gene. Such approaches include oligonucleotide-
based therapies
such as antisense, ribozymes, and triple helices .
Oligonucleotides may be designed to reduce or inhibit mutant target gene
activity.
Techniques for the production and use of such molecules are well known to
those of ordinary
skill in the art. Antisense RNA and DNA molecules act to directly block the
translation of
mRNA by hybridizing to targeted mRNA and preventing protein translation. With
respect to
antisense DNA, oligodeoxyribonucleotides derived from the translation
initiation site, e.g.,
between the -10 and +10 regions of the target gene nucleotide sequence of
interest, are preferred.
Antisense oligonucleotides are preferably 10 to 50 nucleotides in length, and
more preferably 15
to 30 nucleotides in length. An antisense compound is an antisense molecule
corresponding to
the entire Sir2 or p53 mRNA or a fragment thereof.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific
cleavage of
RNA. The mechanism of ribozyme action involves sequence specific hybridization
of the
nibozyme molecule to complementary target RNA, followed by an endonucleolytic
cleavage.
The composition of ribozyme molecules includes one or more sequences
complementary to the
target gene mRNA, and includes the well known catalytic sequence responsible
for mRNA
cleavage disclosed, for example, in U.S. P.N. 5,093,246. Within the scope of
this disclosure are
engineered hammerhead motif ribozyme molecules that specifically and
efficiently catalyze
endonucleolytic cleavage of RNA sequences encoding target gene proteins.
Specific ribozyme
cleavage sites within any potential RNA target are initially identified by
scanning the molecule
of interest for ribozyme cleavage sites that include the sequences GUA, GUU,
and GUC. Once
identified, short RNA sequences of between 15 and 20 ribonucleotides
corresponding to the
region of the target gene containing the cleavage site may be evaluated for
predicted structural
features, such as secondary structure, that may render the oligonucleotide
sequence unsuitable.
The suitability of candidate sequences may also be evaluated by testing their
accessibility to
hybridization with complementary oligonucleotides, using ribonuclease
protection assays.
Nucleic acid molecules used in triple helix formation for the inhibition of
transcription
should be single stranded and composed of deoxyribonucleotides. The base
composition of these
oligonucleotides are designed to promote triple helix formation via Hoogsteen
base pairing rules,
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which generally require sizeable stretches of either purines or pyrimidines to
be present on one
strand of a duplex. Nucleotide sequences may be pyrimidine-based, which will
result in TAT
and CGC triplets across the three associated strands of the resulting triple
helix. The pyrimidine-
rich molecules provide base complementarity to a purine-rich region of a
single strand of the
duplex in a parallel orientation to that strand. In addition, nucleic acid
molecules may be chosen
that are purine-rich, for example, containing a stretch of G residues. These
molecules will form a
triple helix with a DNA duplex that is rich in GC pairs, in which the majority
of the purine
residues are located on a single strand of the targeted duplex, resulting in
GGC triplets across the
three strands in the triplex.
Alternatively, the potential sequences targeted for triple helix formation may
be increased
by creating a "switchback" nucleic acid molecule. Switchback molecules are
synthesized in an
alternating S'-3', 3'-5' manner, such that they base pair with first one
strand of a duplex and then
the other, eliminating the necessity for a sizeable stretch of either purines
or pyrimidines to be
present on one strand of a duplex.
The antisense, ribozyme, and/or triple helix molecules described herein may
reduce or
inhibit the transcription (triple helix) and/or translation (antisense,
ribozyme) of mRNA produced
by both normal and mutant target gene alleles. If it is desired to retain
substantially normal
levels of target gene activity, nucleic acid molecules that encode and express
target gene
polypeptides exhibiting normal activity may be introduced into cells .via gene
therapy methods
that do not contain sequences susceptible to whatever antisense, ribozyme, or
triple helix
treatments are being utilized. Alternatively, it may be preferable to
coadminister normal target
gene protein into the cell or tissue in order to maintain the requisite level
of cellular or tissue
target gene activity.
Antisense RNA and DNA, ribozyme, and triple helix molecules may be prepared by
any
method known in the art for the synthesis of DNA and RNA molecules. These
include
techniques fox chemically synthesizing oligodeoxyribonucleotides and
oligon'bonucleotides, for
example solid phase phosphoramidite chemical synthesis. Alternatively, RNA
molecules may be
generated by in vitro and in vivo transcription of DNA sequences encoding the
antisense RNA
molecule. Such DNA sequences may be incorporated into a wide variety of
vectors that
incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase
promoters.
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Alternatively, antisense cDNA constructs that synthesize antisense RNA
constitutively or
inducibly, depending on the promoter used, can be introduced stably into cell
lines. Various
well-lcnown modifications to the DNA molecules may be introduced as a means of
increasing
intracellular stability and half life. Possible modifications include but are
not limited~to the
addition of flanking sequences of ribonucleotides or deoxyribonucleotides of
the 5' andlor 3' ends
of the molecule or the use of phosphorothioate or f O-methyl rather than
phosphodiesterase
linkages within the oligodeoxyribonucleotide backbone.
Modulators of Sir2 or p53 expression can be identified by a method wherein a
cell is
contacted with a candidate compound and the expression of Sir2 or p53 mRNA or
protein in the
cell is determined. The level of expression of Sir2 or p53 mRNA or protein in
the presence of the
candidate compound is compared to the level of expression of mRNA or protein
in the absence
of the candidate compound. The candidate compound can then be identified as a
modulator of
Sir2 or p53 expression based on this comparison. For example, when expression
of Sir2 or p53
mRNA or protein is greater in the presence of the candidate compound than in
its absence, the
candidate compound is identified as a stimulator of Sir2 or p53 mRNA or
protein expression.
Alternatively, when expression of Sir2 or p53 mRNA or protein is less in the
presence of the
candidate compound than in its absence, the candidate compound is identified
as an inhibitor of
Sir2 or p53 mRNA or protein expression. The level of Sir2 or p53 mRNA or
protein expression
in the cells can be determined by methods described herein for detecting Sir2
or p53 mRNA or
protein.
Delivery of antisense, triplex agents, ribozymes, and the like can be achieved
using a
recombinant expression vector such as a chimeric virus or a colloidal
dispersion system or by
injection. Useful virus vectors include adenovirus, herpes virus, vaccinia,
and/or RNA virus such
as a retrovirus. The retrovirus can be a derivative of a marine or avian
retrovirus such as
Moloney marine leukemia virus or Rous sarcoma virus. All of these vectors can
transfer or
incorporate a gene for a selectable marker so that transduced cells can be
identified and
generated. The specific nucleotide sequences that can be inserted into the
retroviral genome to
allow target specific delivery of the retroviral vector containing an
antisense oligonucleotide can
be determined by one of skill in the art.
Another delivery system for polynucleotides is a colloidal dispersion system.
Colloidal
dispersion systems include macxomolecular complexes, nanocapsules,
microspheres, beads, and
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lipid-based systems including oil-in-water emulsions, micelles, mixed micelles
and liposomes.
A preferred colloidal delivery system is a liposome, an artificial membrane
vesicle useful as in
vivo or in vitro delivery vehicles. The composition of a liposome is usually a
combination of
phospholipids, usually in combination with steroids, particularly cholesterol.
The Sir2 or p53 gene may also be underexpressed.
Methods whereby the level of Sir2 or p53 gene activity may be increased to
levels
wherein disease symptoms are ameliorated also include increasing the level of
gene activity, for
example by either increasing the level of Sir2 or p53 gene present or by
increasing the level of
gene product which is present.
For example, a target gene protein, at a level sufficient to ameliorate
metabolic imbalance
symptoms, may be administered to a patient exhibiting such symptoms. One of
skill in the art
will readily know how to determine the concentration of effective, non-toxic
doses of the normal
target gene protein. Additionally, RNA sequences encoding target gene protein
may be directly
administered to a patient exhibiting disease symptoms, at a concentration
sufficient to produce a
level of target gene protein such that the disease symptoms are ameliorated.
Administration may
be by a method effective to achieve intracellular administration of compounds,
such as, for
example, liposome administration. The RNA molecules may be produced, for
example, by
recombinant techniques such as those described above.
Further, patients may be treated by gene replacement therapy. One or more
copies of a
normal target gene, or a portion of the gene that directs the production of a
normal target gene
protein with target gene function, may be inserted into cells using vectors
that include, but are
not limited to adenovirus, adenoma-associated virus, and retrovirus vectors,
in addition to other
particles that introduce DNA into cells, such as liposomes. Additionally,
techniques such as
those described above xnay be utilized for the introduction of normal target
gene sequences into
human cells.
Cells, preferably autologous cells, containing and expressing normal target
gene
sequences may then be introduced or reintroduced into the patient at positions
which allow for
the amelioration of metabolic disease symptoms. Such cell replacement
techniques may be
preferred, for example, when the target gene product is a secreted,
extracellular gene product.
In instances where the target gene protein is extracellular, or is a
transmembrane protein,
any of the administration techniques described, below wluch are appropriate
for peptide
CA 02519161 2002-07-08
WO 03/004621 PCT/US02/21461
administration may be utilized to effectively administer inhibitory target
gene antibodies to their
site of action.
The identified compounds that inhibit target gene expression, synthesis and/or
activity
can be administered to a patient at therapeutically effective doses to treat
or ameliorate or delay
the symptoms of aging. A therapeutically effective dose refers to that amount
of the compound
sufficient to result in amelioration or delay of symptoms of aging.
Toxicity and therapeutic efficacy of such compounds can be determined by
standard
pharmaceutical procedures in cell cultures or experimental animals, e.g., for
determining the
LD50 (the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective
in 50% of the population). The dose ratio between toxic and therapeutic
effects is the therapeutic
index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit
large
therapeutic indices are preferred. While compounds that exhibit toxic side
effects may be used,
care should be taken to design a delivery system that targets such compounds
to the site of
affected tissue in order to minimize potential damage to uninfected cells and,
thereby, reduce
side effects. The data obtained from the cell culture assays and animal
studies can be used in
formulating a range of dosage for use in humans. The dosage of such compounds
lies preferably
within a range of circulating concentrations that include the ED50 with little
or no toxicity. The
dosage may vary within this range depending upon the dosage form employed and
the route of
administration utilized. For any compound used in the method of the invention,
the
therapeutically effective dose can be estimated initially from cell culture
assays. A dose may be
formulated in animal models to achieve a circulating plasma concentration
range that includes
the IC50 (i.e., the concentration of the test compound which achieves a half
maximal inhibition
of symptoms) as determined in cell culture. Such information can be used to
more accurately
determine useful doses in humans. Levels in plasma may be measured, for
example, by high
performance liquid chromatography.
Pharmaceutical compositions may be formulated in conventional manner using one
or
more physiologically acceptable carriers or excipients. Thus, the compounds
and their
physiologically acceptable salts and solvates may be formulated for
administration by inhalation
or insufflation (either through the mouth or the nose) or oral, buccal,
parenteral or rectal
administration.
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WO 03/004621 PCT/US02/21461
For oral administration, the pharmaceutical compositions may take the form of,
fox
example, tablets or capsules prepared by conventional means with
pharmaceutically acceptable
excipients such as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline
cellulose or calcium
hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g.,
potato starch or sodium starch glycolate); or wetting agents (e.g., sodium
lauryl sulphate). The
tablets may be coated by methods well known in the art. Liquid preparations
for oral
administration may take the form of, for example, solutions, syrups, or
suspensions, or they may
be presented as a dry product for constitution with water or other suitable
vehicle before use.
Such liquid preparations may be prepared by conventional means with
pharmaceutically
acceptable additives such as suspending agents (e.g., sorbitol syrup,
cellulose derivatives or
hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-
aqueous vehicles
(e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils);
and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also
contain buffer
salts, flavoring, coloring, and sv~eetening agents as appropriate.
Preparations fox oral administration may be suitably formulated to give
controlled release
of the active compound. For buccal administration the compositions may take
the form of tablets
or lozenges formulated in conventional manner. For administration by
inhalation, the
compounds for use according to the present invention are conveniently
delivered in the form of
an aerosol spray presentation from pressurized packs or a nebuliser, with the
use of a suitable
propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane,
carbon dioxide or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be
determined by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g.
gelatin for use in an inhaler or insufflator may be formulated containing a
powder mix of the
compound and a suitable powder base such as lactose or starch. The compounds
may be
formulated for parenteral administration by injection, e.g., by bolus
injection or continuous
infusion. Formulations for injection may be presented in unit dosage form,
e.g., in ampoules or
in mufti-dose containers, with an added preservative. The compositions may
take such forms as
suspensions, solutions, or emulsions in oily or aqueous vehicles, and may
contain formulatory
agents such as suspending, stabilizing, and/or dispersing agents.
Alternatively, the active
ingredient may be in powder form for constitution with a suitable vehicle,
e.g., sterile pyrogen-
57
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free water, before use. The compounds may also be formulated in rectal
compositions such as
suppositories or retention enemas, e.g., containing conventional suppository
bases such as cocoa
butter or other glycerides. In addition to the formulations described
previously, the compounds
may also be formulated as a depot preparation. Such long acting formulations
may be
administered by implantation (for example subcutaneously or intramuscularly)
or by
intramuscular injection. Thus, for example, the compounds may be formulated
with suitable
polymeric or hydrophobic materials (for example as an emulsion in an
acceptable oil) or ion
exchange resins, or as sparingly soluble derivatives, for example, as a
sparingly soluble salt.
All references cited herein are incorporated by reference in their entirety.
The invention
is illustrated by the following non-limiting examples.
Materials and Methods
Plasmids and antibodies
To construct mSir2a expression constructs, the full-length cDNA was subcloned
from
pET28a-Sir2a (Itnai et al., 2000) into pcDNA3 or pBabepuro vector. Site-
directed mutation was
generated in the plasmid pRS305-Sir2a using the Gene Edit system (Pmmega). To
construct the
human SIRTI expression construct, DNA sequences corresponding to the full-
length hSIRTl
(Frye, 1999) were amplified by PCR from Marathon-Ready Hela cDNA (Clontech),
and initially
subcloned into pcDNA3.1/V5-His-Topo vector (Invitrogen), and then subcloned
with a Flag-tag
into a pCIN4 vector for expression (Gu et al., 1999). To prepare the Sir2a
antibody that can
recognize both human and mouse Sir2a, a polyclonal antibody against the highly
conserved C-
terminus of Sir2a was generated. DNA sequences corresponding to this region
(480-737) were
amplified by PCR and subcloned into pGEX-2T (Pharmacia). a-Sir2a antisera was
raised in
rabbits against the purified GST-Sir2a (480-737) fusion protein. (Covance),
and fiwther affinity-
purified on both protein-A and antigen columns. By Western blot analysis and
immunofluorescent staining, this antibody can defect both mouse Sir2a and
human S1RTI
proteins.
To construct hSir2 expression constructs, BamHI/SnaBI fragment ofhS7R2SIRT1
cDNA
was inserted into pBabe-Y-Puro. The resulting plasmid was designated pYESir2-
puro. Similarly
a BamHI/SnaBI fragment of hSir2 that was mutated at residue 363 from Histidine
(H~ to
Tyrosine (Y) by site-directed mutagenesis (Stratagene) was used to create the
retroviral vector
pYESirZHY. pBabe-hTERT-hygro contained an EcoRT/Sall fragment of hTERT cloned
into
58
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EcoRl/SaII site of pBabe-Hygro. pCMVwtp53, pCMVK382R and pCMVK320R were a gift
from Dr. E. Appella ~.
Cell culture and derivation of cell lines
All cells were grown in presence of 20% OZ and 5% COZ at 37 ° C in
humidified
chambers. Human diploid fibroblast BJ cells, human epithelial breast carcinoma
cell line MCF7
and H1299 human epithelial carcinoma cell Iines were grown in DME +10% FCS.
PBS(-/-)
(phosphate buffered saline) without magnesium or calcium was used for washing
cells and other
applications described herein.
Amphotrophic viruses were produced by transient co-transfection of pCL-pCL-
Ampho
with the LTR containing pBabe vectors (Morgenstern and Land, 1990), pYESir2 or
pYESir2HY
in to 293T cell line using Fugene6 (Roche). Three days post transfection
supernatants were
collected and filtered with 0.4 micron filters. Primary BJ cells or MCF7 cells
were infected with
retrovirus containing media in presence of 8 mg/ml of polybrene ovenught and
48 hours later
cells were selected in puxomycin at 1 mg/mI.
Following selection and during the experimentation all the mass cultures were
maintained
in presence of puromycin. These selected BJ cells were subsequently infected
and selected with
a pBabe-hTERT virus carrying the hygromycin resistance gene (200mg/ml). The
resulting cells
were: BJT (carrying pYE-Puro backbone and pBabe-hTERT-hygro), BJThSir'2wt
(carrying
pYESir2 wild type hSir2 and pBabe-hTERT hygro) and BJThSir2HY (pYESir2HY
mutant hSir2
and pBabe-hTERT-hygro). MCF7 cells were transfected with the vector p2lP-Iuc
(Vaziri et al.,
1997) and pCMVneo, clones were selected in 500 mglml of 6418 and the clone
designated
MCF73L was selected that was able to upregulate the p21WAF1 promoter-
luciferase in response
to treatment with 6 Gy of ionizing radiation. MCF7 cells or MCF73L were
infected with the
same viruses as described before to yield the following cell lines: MCF73LP
(carrying pBabe Y-
puro backbone), MCF73L-hSir2wt and MCF73L-hSir2HY. Cells were kept under
appropriate
selection throughout experiments.
ha vitYO p53 deacetylation Assay
The Flag-tagged SirZa-expressing cells were established and expanded in DMEM
medium, and cell extracts were prepared essentially as previously described
(Luo et al., 2000;
Gu et al., 1999; Ito et al., 1999). The proteins were purified under a very
high stringency
59
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~'O 03/004621 PCT/US02121461
condition (300 mM NaCI and 0.5 % NP-40). The eluted proteins were resolved by
a SDS-PAGE
gel and analyzed by colloidal blue staining (Novex). Acetylated GST-p53 was
prepared by p53
acetylation assay as previously described (Gu and Roeder, 1997) and further
purified on
glutathione-Sepharose (Luo et al., 2000). The 14C-labeled acetylated pS3 (2.5
p,g) was incubated
with purified Sir2a (10 ng) at 30 °C for 1 hr either in the presence of
50 ,uM NAD or as
indicated. The reactions were performed in a buffer containing 50 mM Tris-HCl
(pH 9.0),
SOmM NaCI, 4 mM MgCl2, 0.5 mM DTT, 0.2 mM PMSF, 0.02% NP-40 and 5% glycerol.
The
reactions were resolved on SDS-PAGE and analyzed by Coomassie blue staining
and
autoradiography.
Immunoprecipitation and Immunofluorescence
H1299 cells transiently expressing p53 and hSir2 were lysed using the NP40
buffer and
lysates described above and immunoprecipitated with lul of anti-hSir2 antibody
overnight.
Protein G-sepharose beads (50 ml) were added to the lysates and rotated at
4°C for 3hrs. The
immune complexes were collected, washed 3 times, and resolved using the Nupage
gradient 4-
12% Bis-Tris MOPS (3-N-morpholino propane sulfonic acid) protein geI (Novex)
in the
presence of provided anti-oxidant (Novex).
The gels used were transferred to nitrocellulose and probed with anti-p53
antibody (pAb7
sheep anti human polyclonal antibody, Oncogene Science), signal detected using
a goat anti-
sheep HRP secondary antibody. The membranes were subsequently washed and
reprobed with
anti-hSir2 antibody.
For immunoprecipitation in BJ cells, lmg of protein per reaction were
incubated with lul
of Ab-6(anit-p53 monoclonal, Oncogene Science) and immunoprecipitation was
performed as
described above except that the time of incubation in primary antibody was
2hrs and 4 times
higher concentrations of protease inhibitors were used, due to the observed
high instability of
p53 protein in BJ cells. Immune complexes were resolved as previously
described using the
Novex system (Tnvitrogen) and membranes were exposed to a mix of polyclonal
antibodies at
1:1000 dilution (SC6432, polyclonal rabbit and CM1, polyclonal rabbit). A
secondary goat anti-
rabbit HRP was used at 1:30,000 concentration for detection. Membranes were
subsequently
blocked again and re-probed with anti-hSir2 antibody.
GO
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WO 03/004621 PCT/US02/21461
Immunofluorescence of U20S and BJ cells was undertaken by fixing the cells m
microchamber slides (LabTek) in 70% Ethanol and subsequent staining with anti-
hSir2 antibody
at 1:500 dilution. A secondary goat anti-rabbit FITC antibody at 0.5 ug/ml was
used for
detection of signal.
GST pull-down assay and co-immunoprecipitation assay ,
GST fusion proteins were expressed in E. coli, extracted with buffer BC500 (20
mM
Tris-HCI, pH 8.0, 0.5 mM EDTA, 20% glycerol, 1mM DTT and 0.5 mM PMSF)
containing 50
mM KCI and 1% NP-40, and purified on glutathione-sepharose (Pharmacia). 35S-
labeled Sir2a
was in vitro translated by a TNT kit (Promega) using pcDNA3-Sir2a as a
template. 5 ~,l Of 35S-
labeled Sir2a were incubated at 4°C for 60 min with each of the
different immobilized GST
fusion proteins in BC200 buffer containing 200 mM KCI and 0.2% NP-40. Beads
were then
washed five times in 0.5 ml of the same buffer. Bound proteins were eluted
with an equal
volume of SDS sample buffer, resolved by SDS-PAGE, and analyzed by Coomassie
blue
staining and autoradiography.
The co-immunoprecipitation assay was performed essentially as described
previously
(Luo et ad., 2000). Cells were extracted with lysis buffer (25mM HEPES-KOH, pH
8.0, 150 mM
KCl, 2mM EDTA, 1mM DTT, 1mM PMSF, l0,ug/ml aprotinin, l0,ug/ml leupeptin,
l,ug/ml
pepstatin A, 20 mM NaF, 0.1% NP-40). After centrifugation, the supernatants
were incubated
with M2 beads (Sigma) for 4 hr at 4°C. The M2 beads were washed five
times with 0.5 ml lysis
buffer, after which the associated proteins were eluted with Flag peptides to
avoid the cross-
reaction from the mouse IgG in western blot analysis. In the case of the co-
immunoprecipitation
in normal cells, 50 million cells were extracted in the same lysis buffer. The
supernatants were
incubated with 20 ,ug a-Sir2a antibody or pre-immune antiserum from the same
rabbit and 40 ~.1
protein A/G plus-agarose (Santa Cruz) for overnight. The agarose beads were
washed five times
with 0.5 ml of lysis buffer, after which the associated proteins were eluted
with BC1000 (20 mM
Tris-HC1, pH 8.0, 0.5 mM EDTA, 20% glycerol, lxnM DTT and 0.5 mM PMSF)
containing 1 M
NaCI, 1% NP-40, 0.5% Deoxycholic Acid. The eluted proteins were resolved on 8%
SDS
PAGE and Western blot with a-Sir2a antibody and a-p53 (DO-1) for human cells
and a-p53
(421) for mouse cell.
G1
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''~O 03/004621 PCT/US02/21461
Immunoblot analysis
For detection of acetylated forms of p53 in BJ cells and MCF7 cells, equal
numbers of
cells were plated 24 hrs before the experiment. l.5xI0~ BJ cells or 107 MCF7
cells exponentially
growing phase in 150 cm2 dishes were exposed to 6Gy of ionizing radiation
(137C~esium gamma
source at dose rate of 1 Gy/min). At the appropriate time point, cells were
washed and harvested
by trypsinization and subsequent neutralization with 10% serum. After washing
the cells once in
PBS(-/-), cell pellets were frozen on dry ice instantly at the appropriate
time point. Once all
time points were collected, cell pellets were all lysed on ice at once by
adding 0.5% NP40, I50
mM NaCI (in the presence of complete protease inhibitor mix, Roche), for 30
minutes and
vortexing. Cell lysates were prepared by centrifugation for 20 minutes at
4°C. Protein content
of lysates were measured using Lowry based assay (BioRad DC protein assay).
Protein (300
mg) was resolved on gradient 4-20% criterion Tris-HC gels (Biorad),
transferred to
nitrocellulose and blocked in I O% skim milk.
The resulting membrane was incubated overnight in 1:400 dilution of Ab-1
(Oncogene
Science, peptide based rabbit polyclonal anti K382 p53). This membrane was
then washed twice
in PBS(-l-) containing 0.05%Tween 20 for 15 minutes. Secondary Goat anti-
rabbit antibody
conjugated to HRP (Pierce) was used at a concentration of 1:30,000 for lhr in
1% Milk.
Membrane was subsequently washed twice for 30 minutes total time.
The membrane was incubated with Supersignal west femto maximum substrate
(Pierce)
for 2 minutes and exposed to X-GMAT sensitive film (Kodak) for up to 30
minutes. The
membrane was subsequently blotted with a monoclonal p2I WAFI antibody (F5,
Santa Cruz
Biotech), p53 antibody (SC6243, polyclonal rabbit, Santa Cruz) (Ab-6, Oncogene
Science),
anti-hSir2 (polyclonal rabbit). ~3-actin was used (Abeam) for loading control.
96715 is an anti-
acetyl H3 Lys9 was a monoclonal antibody (Cell Signaling).
Virus infection and stress response
All MEF cells were maintained in DMEM medium supplemented with 10% fetal
bovine
serum, and the 11VIR-90 cells were maintained in Eagle's minimal essential
medium
supplemented with 10% fetal bovine serum and non-essential amino acids. The
virus infection
and selection were essentially as described previously (Ferbeyre et al.,
2000). A$er one-week
selection, the cells were either frozen for stock or immediately used fox
further analysis. About
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WO 03/004621 PCT/US02/21461
500,000 MEF cells were plated on a 10-cm dish 24 hr before treatment. The
cells were then
exposed to etoposide (20 Vim) for 12 hr. After treatment, the cells were
washed with PBS and
fed with normal medium. Another 36 hrs later, the cells were stained with PI
and analyzed by
flow cytometric analysis for apoptotic cells {SubGl) according to DNA content.
In case of the
Fas-mediated apoptosis assay, the cells were treated with actinomycin D (0.25
p.g/ml) and Fas
antibody (100 ng/ml) as previously described (Di Cristofano et al., 1999). In
the case of
oxidative stress response, the 1MR-90 cells were treated with H202 (200 ~.M)
for 24 hrs.
Luciferase and apoptosis assays
H1299 cells were transfected using the Fugene6 protocols (Roche) with
pCMVwtp53 in
presence or absence of pCMVp300 and 5 ~g of p2IP-Luc (containing a 2.4kb
fragment of p21
linked to luciferase gene) as previously described (Vaziri et al.,1997). All
experiments were
performed in triplicates.
Apoptosis was measured at approximately 48 hrs post transfection using the
annexin V
antigen and propidium iodide exclusion (Clontech laboratories).
Radiation survival curves of BJ cells were performed as described previously
(Dhar et
al., 2000; Vaziri et al., 1999).
FRCS analysis for apoptosis assay
Both adherent and floating cells were combined and washed in cold PBS. For
SubGl/FACs analysis, cells were fixed in methanol for 2 hr at -20 °C,
rehydrated in PBS for 1 hr
at 4 °C, and then reacted with the primary antibody (DO-1) for 30 min
at room temperature.
Cells were washed twice in PBS and incubated with a goat anti-mouse FITC-
conjugated
secondary antibody for 30 min at room temperature. Following incubation, cells
were washed in
PBS and treated with RNase A (50 ,ug/ml) for 30 min at room temperature.
Propidum iodide (PI:
2.5 ,ug/ml) was added to the cells, and samples were then analyzed in a
FACSCalibur (BD). A
region defining high FITC fluorescence was determined, and the cells falling
into this region
were collected separately. The PI staining was recorded simultaneously in the
red channel.
Immunofluorescence Assay
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1'V0 03/004621 PCT/US02/21461
Immunofluorescence was performed essentially as the standard protocol (Guo et
al.,
2000). After fixation, cells were exposed to two primary antibodies: p53
monoclonal antibody
DO-1 (Santa Cruz) and a-Sir2a for 1 hr at room temperature. The cells were
washed three times
with 1% BSA plus 0.2% Tween-20 in PBS and then treated with two secondary
antibodies [a
goat anti-rabbit IgG conjugated to Alexa S68 (Molecular Probes), and anti-
mouse IgG-FITC
(Santa-Cruz)]. DAPI was used for counter-staining to identify nuclei. The
cells were further
washed four times. Images were acquired from a Nikon Eclipse E600 fluorescent
microscope
(Hamamatsu Photonics).
Detecting acetylation levels of p53 in cells
The cells (human lung carcinoma cell lines H460 (wild-type pS3) and H1299 (pS3-
null),
human colon carcinoma HCT116 (wild-type p53), mouse embryonal carcinoma cell
line F9
(wild-type pS3), mouse embryonic fibroblast MEFs or others) were maintained in
DMEM
medium supplemented with 10% fetal bovine serum. For DNA damage response,
about 1
million cells were plated on a 10-cm dish 24 hr before treatment. The cells
were then exposed to
etoposide (20~.M) and or other drugs (0.5 ~,M of TSA, 5 mM of nicotinamide,
and 50 ~,M of
LLNL) as indicated for 6 hr.
After treatment, the cells were harvested for Western blot analysis. The
rabbit polyclonal
antibody specific for p300-mediated acetylated pS3 [a-pS3(Ac)-C] was raised
and purified
against the acetylated human p53 C-terminal peptide [p53 (Ac)-C: H-
SSSGQSTSRHSSLMF-
OH SEQ. I)7 No:l (5 = acetylated Lysine)] as described before (Luo et al.,
2000).
This antibody recognizes the p300-mediated acetylated forms of both human and
mouse
pS3. In the case of cotransfection assays testing for p53 acetylation levels,
H1299 cells were
transfected with 5 ~.g of CMV-p53 plasmid DNA, S ~,g of CMV-p300 plasmid DNA,
and 10 p.g
of pcDNA2-Sir2a plasmid DNA as indicated. 24 hr after the transfection, the
cells were lysed in
a Flag-lysis buffer (SO mM Tris, 137 mM NaCI, 10 mM NaF, 1mM EDTA, 1% Triton X-
100
and 0.2% Sarkosyl, 1 mM DTT, 10% glycerol, pH 7.8) with fresh proteinase
inhibitors, 10 ~,M
TSA and SmM nicotinamide (Sigma). The cell extracts were resolved by either 8%
or 4-20%
SDS-PAGE gels (Novex) and analyzed by Western blot with a-p53 (Ac)-C and a-p53
(DO-1).
Deacetylation assay of the p53 C-terminal peptide
64
CA 02519161 2002-07-08
' The human p53 C-terminal peptide (residues 368-385+Cys;
HLKSK(AcK)GQSTSRHK(AcK)LMFKC); (SEQ ID N0. 24) di-acetylated at positions 373
and
382 was synthesized and purified with HPLC. Deacetylation assays of this
peptide by Sir2 and
analyses of the reaction products were performed as described previously (Tmai
et al., 2000).
EXAMPLES
Example 1. Mammalian Sir2a interacts with p53 both i~a vitro and i~a vivo.
Mouse Sir2a interacts with p53. The p53 protein can be divided into three
distinct
functional domains (Gu and Roeder,1997): an amino-terminus that contains the
transcriptional
activation domain (NT: residues 1-73), a central core that contains the
sequence-specific DNA-
binding domain (M: residues I00-300), and the multifunctional carboxyl-
terminus (CT:
residues 300-393). The GST-pS3 fusion proteins containing each domain as well
as the full-
length protein were expressed in bacteria and purified to near homogeneity on
gluthathione-
agrose beads. As shown in Figure IA, 35S-labeled ira vitYO translated Sir2oc
strongly bound to
immobilized GST-p53 but not to immobilized GST alone (lane 1 vs. 6). Sir2a was
tightly bound
to the C-terminal domain of p53 (GST-p53CT) (lane 4, Figure IA), also bound to
the central
DNA-binding domain (GST-p53M), but showed no binding to the N-terminal domain
of p53
(GST-p53NT) (lane 3 vs. 2, Figure 1A).
To test for the interactions between p53 and Sir2a in cells, extracts from
transiently-
transfected p53-null cells (H1299) were immunoprecipitated with anti-Flag
monoclonal antibody
(M2). As shown in Figure 1B, p53 was detected in the immunoprecipitate
obtained from H1299
cells cotransfected with constructs encoding Flag-Sir2a and p53 (lane 2), but
not from cells
transfected with the p53 construct alone (lane 4). Conversely, Sir2a was
detected in the
immunoprecipitates obtained from H1299 cells cotransfected with constructs
encoding Sir2a and
Flag-p53 (lane 6, Figure 1B), but not from cells transfected with the Sir2a
construct alone (lane
8, Figure IB). p53 interacts similarly with human SIRT1 (hSlRT1) (Figure 1C,
D), the human
ortholog of mouse Sir2a (Frye, 1999; 2000), showing that p53 and mammalian
Sir2a interact.
Since mouse Sir2a shares a highly conserved region at the C-terminus with
human
SIRT1 (Figure 1C), but not with any other mammalian Sir2 homologs (Frye, 1999;
2000), a
polyclonal antibody against the C-terminus (amino acid 480-737) of mouse Sir2a
was
developed. Anti-Sir2a antisera (a-Sir2a) was raised in rabbits against the
purified GST-
GS
CA 02519161 2002-07-08
CVO 03/004621 PCT/US02/21461
Sir2a(480-737) fusion protein. As shown in Western blots, this antibody can
detect both mouse
Sir2a and human STRTl proteins, but not other human Sir2 homologs (Figure 2A,
B).
p53 interaction with Sir2a or hSIRTI in normal cells without overexpression
was studied
employing this antibody. Cell extracts from human (H460) and mouse cells (F9),
which express
wild-type pS3 proteins, were immunoprecipitated with a-Sir2a, or with the pre-
immune serum.
Western blot analysis revealed that this antibody immunoprecipitated both
Sir2a and hSIRTI
(lower panels, Figure 2A, 2B). Human and mouse p53 were detected in the
respective a-Sir2a
immunoprecipitations from cell extracts, but not in the control
immunoprecipitations with the
preimmune serum, showing that p53 interacts with mammalian Sir2a in normal
cells. In contrast
to abrogation of the Mdm2-p53 interaction by DNA damage as previously reported
(Shieh et al.,
1.997), this interaction was stronger in cells after DNA damage treatment
(Figure 2C), which
shows mammalian Sir2a is involved in regulating p53 during the DNA-damage
response. Thus,
p53 interacts with mammalian Sir2a both in vitf-o and in vivo.
Example 2. Deacetylation of p53 by mammalian Sir2a
p53 was deacetylated by mammalian Sir2a in vitro. Mouse Sir2a protein was
expressed
with the N-terminal Flag epitope in cells and purified to near homogeneity on
the M2-agrose
affinity column (lane 3, Figure 3A to determine). The GST-p53 fusion protein
was acetylated by
p300 in the presence of [14C]-acetyl-CoA, and the acetylated p53 protein was
purified on the
GST affinity column. These highly purified recombinant proteins were used in
this assay in
order to avoid possible contamination by either inhibitory factors or other
types of deacetylases.
As shown in Figure 3B,14C-labeled acetylated p53 was efficiently deacetylated
by
purified Sir2a (lane 3), but not by a control eluate (lane 4). NAD is required
for Sir2a-mediated
dea.cetylation of p53 (lane 2 vs. 3, Figure 3B). Further, the deacetylase
inhibitor TSA, which
significantly abrogates HDAC1-mediated deacetylase activity on p53 (Luo et
al., 2000), had no
apparent effect on Sir2a-mediated p53 deacetylation (lane 5, Figure 3B). These
results show
that Sir2a can strongly deacetylate p53 in vitro, and that this activity
depends on NAD.
A role for mammalian Sir2a in deacetylating p53 in cells was established using
acetylated p53-specific antibody to monitor the steady-state levels of
acetylated p53 in vivo (Luo
et al., 2000). As shown in Figure 3C, a high level of acetylated p53 was
detected in the cells
cotransfected with p300 and p53 (lane 1). However, p53 acetylation levels were
significantly
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abolished by expression of either Sir2a or hSIRTl (lanes 2, 4). In contrast, a
Sir2a mutant
(Sir2aH3S5A) containing a point mutation at the highly conserved core domain
causing
defective histone deacetylase activity in vitf~o had almost no effect (lane 3
vs. 2, Figure 3 C).
Furthermore, neither S1RT5, another human Sir2 homolog, nor poly (ADP-ribose)
polymerase
(PAltP), whose activity is also NAD-dependent (reviewed in Vaziri et al.,
1997), had any
significant effect on p53 acetylation (lanes 5, 6, Figure 3C). hi addition, in
contrast to HDAC-
mediated deacetylation of p53 (Luo et al., 2000) Sir2a still strongly
deacetylated p53 in the
presence of TSA (lane 4 vs. 3, Fig. 3D) even though the steady state level of
acetylated p53 was
elevated when the cells were treated with TSA (lane 3 vs. 1, Fig. 3D). Thus,
mammalian Sir2a
has robust TSA-independent p53 deacetylation activity. .
Example 3. Inhibition of Sir2a-mediated p53 deacetylation by nicotinamide
Sir2a-mediated deacetylase activity of p53 can be inhibited. Deaeetylation of
acetyl-
lysine by Sir2a is tightly coupled to NAD hydrolysis, producing nicotinamide
and a novel
acetyl-ADP-ribose compound (1-O-acetyl-ADPribose) (Landry et al., 2000b;
Tanner et al.,
2000; Tanny and Moazed, 2001). The formation of an enzyme-ADP-ribose
intemediate through
NAD hydrolysis may be critical for this chemical reaction (Landry et al.,
2000b). Since
nicotinamide is the first product from hydrolysis of the pyridinium-N-
glycosidic bond of NAD, it
may function as an inhibitor for its deacetylase activity (Landry et al.,
2000b). Nicotinamide is
able to inhibit the deacetylase activity of Sir2a on acetylated p53 in vitro.
Similar reactions as described above (Figure 3B), were set up by incubating
labeled p53
substrate, recombinant Sir2a and NAD (50 ~Z11~ alone, or in combination with
nicotinamide
(SmM). As shown in Figure 4A, '4C-labeled acetylated p53 was efficiently
deacetylated by
Sir2a (lane 2) however, the deacetylation activity was completely inhibited in
the presence of
nicotinamide (lane 3 vs. lane 2 Figure 4A). As a negative control, 3-AB (3-
aminobenzamide), a
strong inhibitor of PARP which is involved in another type of NAD-dependent
protein
modification (Va.ziri et al., 1997), showed no significaalt effect on Sir2a
mediated deacetylation
(lane 4 vs. 3, Figure 4A).
To further investigate the role of mammalian Sir2cx-mediated regulation iJn
vivo, the
effect of Sir2a expression on p53 acetylation levels during the DNA damage
response was
determined. Mouse embryonic fibroblast (1VIEF) cells, which express the wild
type of p53, were
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infected with either a pBabe-puro retrovirus empty vector or a pBabe-puro
retrovirus containing
Sir2a, and cultured for a week under pharmacological selection. The protein
levels of p53
activation in response to DNA damage in these cells was determined by Western
blot analysis.
Similar protein levels of p53 activation were induced in the pBabe vector
infected cells and
pBabe-Sir2a infected cells after etoposide treatment far 6 hrs (lanes 3, 4 vs.
lanes l, 2, lower
panel, Figure 4B).
rn the mock-infected cells, the acetylation level of p53 was significantly
enhanced by
DNA damage (lane 2 vs. lane 1, Upper panel, Figure 4B). However, DNA damage
treatment
failed to stimulate the p53 acetylation in the pBabe-Sir2a infected cells even
in the presence of
TSA (lane 4 vs. lane 2, Upper panel, Figure 4B), showing that Sir2a expression
results in
deacetylation of endogenous p53. This Sir2a-mediated effect was completely
abrogated by
nicotinamide treatment (lane 8 vs. lane 6, Figure 4B). Thus, Sir2a mediated
deacetylation of p53
can be inhibited by nicotinamide both ifa vltYO and in vivo.
Example 4. Maximum induction of p53 acetylation levels in normal cells
requires
inhibition of endogenous Sir2a activity
Endogenous Sir2a in the regulation of p53 acetylation levels in normal cells
during the
DNA damage response was determined.
As shown in Figure 4C, after the wild-type p53 containing human lung carcinoma
cells
(H460) were treated by etoposide, acetylation of p53 was induced (lane 2 vs.
lane 1). No
significant p53 acetylation was detected in the cells treated with a
proteasome inhibitor LLNL
(lane 6, Figure 4C), indicating that the observed stimulation of p53
acetylation is induced by
DNA damage, not through p53 stabilization.
p53 can be deacetylated by a Pm/MTA2/HDAC1 complex, whose activity is
completely
abrogated in the presence of TSA (Luo et al., 2000). The mild enhancement of
the acetylation
level of p53 by TSA during DNA damage response may be due mainly to its
inhibitory effect on
endogenous HDAC1-mediated deacetylase activity (lane 3 vs. lane 2, Figure 4C).
A super
induction of p53 acetylation was showed when the cells were treated with both
TSA and
nicotinamide (lane 4 vs. lane 3, Figuxe 4C). In contrast, 3-AB treatment had
no effect on the
level of p53 acetylation (lane 5 vs. lane 3, Figure 4C), indicating that PARP-
mediated poly-ADP
ribosylation has no effect on p53 acetylation. Similar results were also
observed in other cell
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types including either mouse cells (MEFs, F9) or human cells (BL2, HCT116).
Thus, maximum
induction of pS3 acetylation requires inhibitors for both types of
deacetylases (HDACl and
Sir2a), and endogenous Sir2a plays a major role in the regulation of the p53
acetylation levels
induced by DNA damage.
Example 5. Repression of p53-mediated functions by mammalian Sir2a requires
its
deacetylase activity
The functional consequence of mammalian Sir2a-mediated deacetylation of p53
was
determined by testing its effect on p53-mediated transcriptional activation. A
mammalian pS3
expression vector (CMV-p53), alone or in combination with different amounts of
mouse Sir2a
expressing vector (CMV-Sir2a), was cotransfected into MEF (p53-~-) cells along
with a reporter
construct containing synthetic p53 binding sites placed upstream of the
luciferase gene (PG13-
Luc).
As shown in Figure SA, Sir2a strongly repressed p53-mediated transactivation
in a dose-
dependent manner (up to 21 fold), but had no significant effect on the
transcriptional activity of
the control reporter construct (TK-Luc) (Figure 5B), which has no p53 binding
site at the
promoter region. Also, expression of human SIRT1 showed a similar effect on
the p53 target
promoter (Figure SC). Neither the SifZaH3SSA mutant or SIRTS, both of which
are defective in
p53 deacetylation (Figure 3C), had any effect on the p53-mediated
transactivation (Figure SC,
D). Thus, mammalian Sir2a specifically represses p53-dependent
transactivation, and that this
repression requires its deacetylase activity.
The modulation of Sir2 on p53-dependent apoptosis was determined. p53 null
cells
(H1299) were transfected with p53 alone or cotransfected with p53 and Sir2a.
The transfected
cells were fixed, stained for pS3, and analyzed for apoptotic cells (SubGl)
(Luo et al., 2000). As
indicated in Figure 6A, overexpression of p53 alone induced significant
apoptosis (32.3%
SubGl). However, co-transfection ofp53 with Sir2a significantly reduced the
level of apoptosis
(16.4% SubGl), while the mutant Sir2aH355A was impaired in this effect (29.5%
SubGl)
(Figure 6A, B). Thus, mammalian Sir2a is involved in the regulation of both
p53 mediated
transcriptional activation and p53-dependent apoptosis, and deacetylase
activity is required for
these Sir2cc-mediated effects on p53.
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Example 6. The role of mammalian Sir2a in stress induced apoptotic response
Mammalian Sir2a can deacetylate p53 both in vitro and in vivo (Figure 3).
Sir2a can
block the induction of endogenous p53 acetylation levels by DNA damage (Figure
4B, 4C). To
elucidate the physiological signif cance for this Sir2a mediated regulation,
the effect on DNA
damage-induced apoptotic response was determined.
MEF (p53*~~ cells as described above (Figure 4B), were infected with either a
pBabe-
puro retrovirus empty vector or a pBabe-puro retrovirus containing Sir2a.
After the DNA
damage treatment by etoposide, the cells were stained with PT and analyzed by
flow cytometric
analysis for apoptotic cells (SubGl) according to DNA content. As shown in
Figure 7A, the
cells mock infected with the pBabe-vector, were susceptible to etoposide-
induced cell death,
with about 48% of the cells apoptotic, after exposure to 20 ~,M of etoposide
(3 vs. 1, Figure 7A).
In contrast, the pBabe-Sir2a infected MEF (p53+~~ cells were more resistant to
apoptosis
induced by the same dose of etoposide, with only 16.4% apoptotic cells (4 vs.
3, Figure 7A).
Since no significant apoptosis was detected in MEF (p53-~') cells by the same
treatment, the
induced apoptosis observed in MEF (p53+~~ cells is totally p53-dependent.
Thus, Sir2a
significantly inlxibits p53-dependent apoptosis in response to DNA damage.
The role of mammalian Sir2a in the oxidative stress response was determined.
Recent
studies have indicated that oxidative stress-induced cell death is p53-
dependent (Yin et al., 1998;
Migliaccio et al., 1999). Early-passage normal human fibroblast (NHF) nYIR-90
cells were
employed for this study since p53-dependent apoptosis can be induced by
hydrogen peroxide
treatment in these cells (Chen et al., 2000).
llVIR-90 cells were infected with either a pBabe-puro retrovirus empty vector
or a pBabe-
puro retrovinzs containing Sir2a, and cultured for a week under
pharmacological selection. By
immunofluorescence staining, p53, in these infected cells, was induced
significantly after
hydrogen peroxide treatment, along with Sir2a localized in the nuclei detected
by
immunostaining with specific antibodies (Figure 7C). Sir2a expression
significantly promotes
cell survival under oxidative stress. As indicated in Figure 7D, the cells
mock infected with the
pBabe-vector, were susceptible to H202-induced cell death, with more than 80%
of the cells
being killed after 24 hr exposure to 200 ~,M H202 (II vs. >]. Tn contrast, the
pBabe-Sir2a
infected cells were much more resistant to death by the same dose of H202,
with about 70% of
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the cells surviving after 24 hr of H202 treatment (IV vs. III, Figure 7D).
Mammalian Sir2a
promotes cell survival under stress by inhibiting p53-dependent apoptosis.
Example 7. Mammalian Sir2a has no effect on p53-independent cell death induced
by anti-Fas
The specificity of mammalian Sir2a-mediated protection of cells from apoptosis
was
examined by determining whether Sir2a has any effect of p53-independent, Fas-
mediated
apoptosis. The MEF (p53'~-) cells were first infected with either a pBabe-puro
retrovirus empty
vector or a pBabe-puro retrovirus containing Sir2a, then cultured for a week
under
pharmacological selection. After the treatment by anti-Fas (100 ng/ml) for 24
hrs, the cells were
harvested and further analyzed for apoptotic cells (SubGl).
Cells mock infected with the pBabe vector, were susceptible to anti-Fas
induced cell
death, with about 31.7% of the cells becoming apoptotic. However, in contrast
to the strong
protection of p53-dependent apoptosis by Sir2a during DNA damage response in
the MEF
(p53~~~ cells (Figure 7A, B), Sir2a expression had no significant effect on
Fas-mediated
apoptosis in the MEF (p53'~') cells. Thus, mammalian Sir2cx regulates p53-
mediated apoptosis.
Mammalian Sir2a has no effect on the Fas mediated apoptosis. (A) Both mock
infected
cells and pBabe-Sir2a infected MEF p53(-/-) cells were either not treated (1
and 2) or treated
with 100 ng/ml Fas antibody in presence of actinomycin D (0.25 ~.g/mI) (3 and
4). The cells
were analyzed for apoptotic cells (subGl) according to DNA content (PI
staining). The
representative results depict the average of three experiments with standard
deviations indicated.
Example 8. Physical interaction of hSir2 with p53
p53 protein is acetylated in response to DNA damage and the acetylation
contributed to
the functional activation of p53 as a transcription factor (Abraham et al.,
2000; Sakaguchi et al.,
1998). Sir2 is a deacetylase of p53, thereby modulating functioning of p53 as
a transcription
factor:
In order to study the functional interaction between p53 and hSir2, a full
length human
hSir2SIRTl cDNA clone (obtained from the IMAGE consortium (Frye, 1999)) was
introduced
into a pBabe-based retroviral expression vector which also carries puromycin
resistance gene as
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a selectable marker. The resulting construct was termed pYESirwt. A retroviral
construct
bearing a derived, mutant allele of Sir2 and termed pYESirHY was constructed
and used in
parallel as control. This mutant allele encodes an amino acid substitution at
residue 363, at
which site the normally present histidine is replaced by tyrosine. This H to Y
substitution results
in an alteration of the highly conserved catalytic site of the hSir2 protein
and subsequent
neutralization of its deacetylase activity. These vector constructs were used
to transduce the
hSIR2SIRT1 gene both by transfection and retroviral infection.
A polyclonal rabbit antibody that specifically recognizes the C-terminal
portion of hSir2
was developed and its specificity validated by immunoprecipitation and Western
blotting (Figure
8A). Both the endogenous and the ectopically expressed hSir2 proteins were
detected as protein
species of 120 Kilodalton (Kd) rather than as SOKd polypeptide predicted from
the known
primary sequence of hSIR2STRTI (Figure 8A). Localization of hSir2 protein by
immunofluorescence using the hSir2 antibody showed a punctate nuclear staining
pattern (Figure
8B).
The physical interactions between hSir2 and p53 were evaluated by co-
transfecting the
pYESir2wt plasmid and a vector expressing wt p53 under the control of the
cytomegalovirus
promoter (pCMV-wtp53) transiently into H1299 human non-small cell lung
carcinoma cells
(Brower et al., 1986) which have a homozygous deletion of the p53 gene and
produce no p53
mRNA or protein (Mitsudomi et aL, 1992). Cell lysates were subsequently mixed
with the rabbit
anti-hSir2 antibody and resulting immune complexes were collected by protein G
and analyzed
by SDS-PAGE electrophoresis and immunoblotting. The immunoblot was probed with
a sheep
anti-p53 antibody (Figure 8C) and reprobed it subsequently with an anti-hSir2
antibody (top
panel) to verify presence of hSir2 in the complex. As indicated in Figure 8C,
immunoprecipitation of hSir2 resulted in co-precipitation of p53.
W the reciprocal experiment, lysates of BJT cells, human fibroblasts into
which the
telomerase gene has been introduced, were examined. In addition, these cells
express either the
wild type hSir2 vector or the hSir2HY mutant. Two cell populations were
created by infection
of mass cultures of BJT cells with the respective vectors and subsequent
selection in puromycin.
The anti-p53 antibody was employed to immunoprecipitate complexes and
subsequently probe
the resulting immunoblot with either polyclonal anti-p53 antibodies or an anti-
hSir2 antibody.
These immunoblots demonstrated a physical interaction between hSir2 and p53
proteins (Figure
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8D). Formation of these complexes was unaffected by the H to Y mutation
introduced into the
hSir2 catalytic site (Figure 8D), Furthermore, radiation used to increase the
levels of p53 protein
in BJ cells had no effect on the levels of p53:hSir2 complexes. Comparison of
the
immunoprecipitated p53 to total input pS3 resulted in an estimate of
approximately 1% of the
cells complement of p53 protein was present in physical complexes with hSir2.
Example 9. Deacetylation of p53 by hSir2 iii vitro
Since liSir2 forms physical complexes with p53, the ability of Sir2 to
deacetylate human
p53 in vitro was evaluated. Since adequate quantities of bacterially produced
hSir2 were not
available, bacterially expressed mouse SIRZ (mSir2a) enzyme was used in in
vitro assays (Imai
et al., 2000).. A 20 residue-long oligopeptide that contains the sequence
corresponding to
residues 368-386+Cys of the human p53 protein was used as a substrate in these
reactions.
Lysine residues corresponding to residues 373 and 382 of the p53 protein were
synthesized in
acetylated form in this oligopeptide substrate. These two residues of p53 are
known.to be
acetylated by p300 (Gu and Roeder, 1997) following y or W irradiation (Liu et
al., 1999;
Sakaguchi et ad., 1998) with acetylation of lysine residue 382 being favored
in response to
ionizing radiation in vivo (Abraham et al., 2000). This p53 oligopeptide
serves as an excellent.
surrogate p53 substrate in vitro for acetylation studies (Gu and Roeder,
1997).
The deacetylase activity of hSir2 utilizes NAD as a co-factor (Imai et al.,
2000; Moazed,
2001; Smith et al., 2000; Tanner et al., 2000; Tanny et al., 1999). In the
absence of added NAD,
incubation of mSir2 with p53 oligopeptide gave rise to a single prominent peak
(peak 1) and a
small, minor peak (peak 2) upon high pressure liquid chromatography (HPLC),
corresponding to
the monomeric and dimeric forms of the peptide, respectively (Figure 9A).
However, incubation
in the presence of 1rnM NAD produced a singly deacetylated species as the
major product (peak
3, Figure 9B). Edman sequencing of this singly deacetylated species revealed
that mSir2
preferentially deacetylated the residue corresponding to Lys 382 ofpS3 (Figure
9, C-F), having
relatively weak effect on Lys 373. Thus, the acetylated p53 peptide acted as a
substrate for hSir2
and indicated that the de-acetylation of pS3 at Lys 382 by mammalian Sir2 is
specific and not the
result of an indiscriminate deacetylase function.
Example 10. Deacetylation of p53 by hSir2 in vivo
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The ability of hSir2 to deacetylate intact p53 protein in vivo was evaluated.
To produce
acetylated p53 an vivo, the p53 expression plasmid was co-transfected with one
expressing p300.
This protocol leads to acetylation ofp53 in the absence of exposure to DNA-
damaging agents
(Luo et al., 2000). The ability of hSir2 to deacetylate the p53 protein at its
K382 residue in
H1299 cells that lack endogenous p53 gene was determined. The levels of
acetylation of p53 at
Lys382 were monitored by using a rabbit polyclonal antibody, termed Ab-1,
which had been
raised against the acetylated K382 of p53 protein. The specificity of the Ab-I
antibody has been
demonstrated (Sakaguchi et al., 1998).
Co-transfection of plasmids expressing wild-type p53 and p300 into H1299 cells
showed
that p53 protein is readily acetylated at K382, as detected by probing the
immunoblot with the
Ab-1 antibody (Figure 10A, lane 3). Recognition of this acetylated form of p53
by the Ab-1
antibody was specific, since a mutant p53 protein that was expressed in a
parallel culture of
H1299 cells and carries an arginine rather than a lysine at residue 382 was
not recognized by the
Ab-1 antibody, despite ectopic expression of the p300 acetylase. (Figure 10A,
lane 6).
Co-transfection of the hSir2-expression plasmid with the p53- and p300-
expressing
plasmids substantially decreased the acetylated p53 that could be detected by
the Ab-1 antibody.
(Figure 10A, Iane 5). The residual level of acetylated p53 could be further
reduced by increasing
the amount of co-transfected hSir2 expression plasmid. Thus, hSir2 can
deacetylate p53 protein
at the Lys382 residue ire vivo.
The hSir2HY vector, which expresses the mutant-catalytically inactive hSir2,
was
introduced into these H1299 cells. The mouse equivalent of this hSir2HY mutant
lacks 95% of
its deacetylase activity (Imai et al., 2000). The hSir2HY mutant failed to
deacetylate wt p53
efficiently, indicating that the catalytic activity of the introduced wild
type hSir2 gene product
was required for specific deacetylation of p53 Lys 382 (Figure 10A, lane 9).
The lysine 320 residue of p53 is also acetylated by PCAF in response to DNA
damage
(Sakaguchi et al., 1998). Whether the state of acetylation of residue 320
affected the ability of
hSir2 to deacetylate residue 382 was determined. A mutant p53 allele that
specifies a lysine-to-
arginine substitution at residue 320 was expressed, This amino acid
substitution did not affect
the ability of hSir2 to deacetylate the K382 residue in H1299 cells,
indicating that the action of
hSir2 on the acetylated K382 residue is independent of the state of
acetylation of the K320
residue (Figure 10A, lanes 7, 8).
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As a measure of the substrate specificity of hSir2, the effects of hSifz on
histone
acetylation, specifically the acetylated residue lysine 9 of bi.stone H3, were
determined using cell
nuclei from the above experiments. H3 Lys9 acetylation was monitored through
the use of the
967IS monoclonal antibody. The 9671 S antibody specifically recognizes histone
H3 that is
acetylated at this position.
Neither wildtype hSir2 nor the catalytically inactive hSir2HY altered the
acetylation of
histone H3 at this position (Figure 10A, bottom). Thus, de-acetylation of pS3
Lys382 in vivo
reflects a defined substrate specificity of hSir2 and not a non-specific
consequence of its over-
expression.
Example I1. liSir2 and p53 acetylation in primary and tumor cell lines
Acetylation of lysine residue 382 of p53 accompanies and mediates the
functional
activation of pS3 as a transcription factor following exposure of a cell to
ionizing radiation
(Sakaguchi et al., 1998). To determine whether hSir2 could antagonize and
reverse this
activation of p53, by its deacetylase function, either wildtype hSir2 or the
mutant form specified
by the hSir2HY vector was expressed in BJT human fibroblast cells. Ectopic
expression of the
telomerase enzyme in these BJT cells, undertaken to extend their lifespan, had
no effect on either
their activation of p53 protein ox their responses to DNA damage (Vaziri et
al., 1999).
Tn order to facilitate detection of i~a vivo acetylated p53 protein, BJT cells
were expressed
to 6Gy of ionizing radiation in the presence of low trichostatin A (TSA)
concentrations. While
not directly inhibiting hSir2 catalytic activity (Imai et al., 2000), TSA
appears to increase the
stability of acetylated pS3 protein (Sakaguchi et al., 1998), perhaps by
inhibiting nori-hSir2
deacetylases that also recognize the acetylated p53 K382 residue. The
resulting immunoblot was
probed with the polyclonal rabbit antiserum (Ab-1 ) which specifically
recognizes the acetylated
K382 form of p53.
Following 6 Gy of ionizing radiation, a I.5-2 fold increase in the level of
acetylated pS3
protein was observed, as indicated by the levels of p53 protein recognized by
the Ab-1 antiserum
(Figure 10B). A four-fold increase in hSir2 levels, achieved through ectopic
expression of hSir2,
resulted in the reversal of the radiation-induced increase in acetylated K382
pS3 protein (Figure
l OB). In contrast, expression of the catalytically inactive hSirHY protein at
comparable levels
increased the radiation-induced levels of p53 acetylated at residue K382
(Figure 10B) suggesting
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that the hSir2HY mutant may act in a dominant negative fashion in BJT cells. A
re-probing of
this immunoblot with a polyclonal anti-p53 antibody showed normal
stabilization ofp53 in
control cells in response to DNA damage and at most, slightly reduced levels
of stabilization in
fihe presence of ectopically expressed wild type hSir2 (Figure 10B). Hence,
while hSir2 is able to
reverse the radiation-induced acetylation of p53 in these cells, it has only
minimal effects on the
metabolic stabilization of p53 induced by exposure to radiation.
A similar phenomenon was observed in MCF-7 human breast carcinoma line cells,
which
have retained an apparently intact p53-dependent checkpoint in response to
ionizing radiation.
Irradiation of these cells led to a three-fold increase in acetylated p53
levels, while a four-fold
ectopic expression of wild type hSir2 in irradiated MCF-7 cells led to
deacetylation of pS3
protein (Figure l OC). Tn contrast to BJT cells, no significant change in the
stability of total pS3
protein was observed. However, MCF-7 cells expressing the liSirHY mutant
showed a level of
radiation-induced acetylation that was comparable to control irradiated cells
(Figure Z OC). Thus,
hSir2 is able to reverse the radiation-induced acetylation in both BJT and MCF-
7 cells,
suggesting that hSir2 acts as an antagoiust of p53 function in vivo.
The differences observed in deacetylation activities of hSir2HY in MCF7 and
BJT cells
may reflect the ability of hSir2HY to act as a dominant-negative allele in BJT
cells. BJT cells do
express significantly lower levels of endogenous hSir2 when compared with MCF7
cells. These
lower levels of hSir2 in BJT cells may enable hSir2HY to form inhibitory
complexes with
endogenous wild type hSir2 or with other proteins required for its function.
In tlus context,
evidence in yeast suggests that H363Y mutant does indeed act as a potent
dominant-negative
(Tanny et al., 1999).
Example 12. Effects of hSir2 on the transcriptional activity of p53 protein
The effects of hSir2 on the transcriptional activity of p53 were determined by
co-
transfecting H1299 cells transiently with a p53 expression plasmid and a
reporter construct in
which the promoter of the p21WAF1 gene (el-Deiry et al., 1993), a known target
of
transcriptional activity by p53, is able to drive expression of a luciferase
reporter gene (Vaziri et
al., 1997). As indicated in Figure 11A, luciferase activity increased in
response to increasing
amount of co-transfected wtp53 expression vector. Conversely, tha
transcriptional activity of
p53 protein was suppressed by co-expression of wild type hSir2 in a dose-
dependent fashion.
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The catalytically inactive hSir2HY mutant had no effect on pS3 transcriptional
activity (Figure
1 1A). The specificity of hSir2 in affecting promoter activity was determined
using a
constitutively active SV40 promoter linked to the luciferase gene. Expression
of this control
construct was not affected by increasing amounts of hSir2 expression vector at
any level (Figure
11B).
The above observations were confirmed in a more physiologic context using a
subline of
MCF-7L cells. The subline of MCF-7 cells was stably transfected with a p21
WAF1 promoter-
reporter construct. In addition, these cells were infected stably with
retroviral vector constructs
expressing either the wild type hSir2 or the mutant hSir2HY. These cells were
expressed to 6 Gy
of ionizing radiation and subsequently measured total pS3 and p21WAF1 protein
levels (Figure
11 C).
pS3 protein levels increased normally in all cell populations in response to
irradiation of
these cells. However, the levels of p21 WAFT protein were reduced in cells
expressing wild type
hSir2 (Figure 11C). Moreover, MCF-7L cells expressing the mutant hSir2HY
protein had a
higher level of p21 WAF 1 when compared with the irradiated controls and with
the wild type
hSir2-overexpressing cells (Figure 11C) showing that the hSir2HY mutant may
act in a
dominant-negative fashion in these cells. Thus, hSir2 can antagonize the
transcriptional
activities of p53 that enable it to exert cytostatic effects via
transcriptional activation of the
p2lWAFl gene.
Example 13. Inhibition of p53-dependent apoptosis by hSir2
hSir2 can antagonize the ability of pS3 to act in a cytostatic fashion through
induction of
p21 WAF1 synthesis. The ability of hSir2 to blunt the pro-apoptotic functions
of p53 was
determined. Restoration of wild-type pS3 function in H1299 cells, achieved via
introduction of a
wt pS3-expressing vector, induces apoptosis, as W dicated by the expression of
the cell surface
annexin V antigen (Figure 12A). Co-transfection of a p300 vector with the pS3
gene increased
this p53-dependent apoptosis (Figure 12A). This apoptotic response was
abolished in a dose-
dependent manner in cells co-transfected with increasing amounts of the wt
hSir2 expression
plasmid (Figure 12A). Hence, hSir2 antagonizes both the cytostatic effects of
pS3 (as mediated
by p21WAF1) and its pro-apoptotic effects.
77
CA 02519161 2002-07-08
~7V0 03/004b21 PCT/US02/21461
Example 14. Effects of mutant hSir2HY on radiosensitivity of human fibroblasts
In contrast to the behavior of many other marine or human cell lines, human
fibroblasts
become relatively radioresistant upon inactivation of p53 function (Tsang et
al., 1995). This
behavior suggested an additional test of the ability of hSir2 to antagonize
p53 function, which
depended on measuring the long-term survival of human BJT fibroblasts cells
following
exposure to various doses of low-level ionizing radiation.
Ectopic expression of wild type hSir2 in these cells led to a greater long-
term.survival
(Figure 12B, triangles), while expression of the mutant hSir2HY in BJT cells
led to a
radiosensitive phenotype (Figure 12B, diamonds) consistent with hSir2HY
constructs acting in a
dominant-negative fashion in BJT cells. A positive control cell line derived
from an individual
with ataxia telangiecstasia (AT) was highly radiosensitive (Figure 12B,
circles). The central role
of pS3 in these various responses was also shown in the behavior of a subline
of BJT fibroblasts
that express a dominant-negative form of p53 and also have acquired a measure
of
radioresistance (Figure 12B, open square). Thus, wt hSir2 antagonizes p53
activity while the
hSir2HY mutant potentiates its activity.
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AlI patents, patent applications, and published references cited herein are
hereby
incorporated by reference.
Anumber of embodiments of the invention have been described. Nevertheless, it
will be
understood that various modifications may be made without departing from the
spirit and scope
of the invention. Accordingly, other embodiments are within the scope of the
following claims.
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