Sir2a-based therapeutic and prophylactic methods
This invention provides methods for treating and for inhibiting the onset of cancer in a subject comprising administering an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis. This invention also provides a related method for inducing the death of a cell. This invention further provides a method for decreasing the amount of damage to a subject's cells caused by physical stress comprising administering agent that increases the amount of Sir2α in the subject's cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject's cells. This invention further provides related methods for prolonging the life-span of a subject, decreasing the amount of damage to a cell caused by physical stress, and prolonging the life-span of a cell. Finally, this invention provides two articles of manufacture for performing the instant methods.
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This application claims the benefit of U.S. Provisional Application No. 60/298,506, filed Jun. 15, 2001, the contents of which are hereby incorporated by reference into this application.
BACKGROUND OF THE INVENTIONThe p53 tumor suppressor exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress (Levine, 1997; Prives and Hall, 1999; Vogelstein et al., 2000). Mutations within the p53 gene have been well documented in more than half of all human tumors (Hollstein et al., 1994). Accumulating evidence further indicates that, in the cells that retain wild-type p53, other defects in the p53 pathway also play an important role in tumorigenesis (Prives and Hall, 1999; Lohrum and Vousden, 1999). The molecular function of p53 that is required for tumor suppression involves its ability to act as a transcriptional factor in regulating downstream target gene expression (reviewed in Nakano and Vousden, 2001; Yu et al., 2001).
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 mainly post-translational modifications of p53, including phosphorylation and acetylation (reviewed in Appella and Anderson, 2000). 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). 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. This finding also indicates that acetylation may represent a general functional modification for non-histone proteins in vivo (Gu and Roeder, 1997).
Through the use of site-specific acetylated p53 antibodies, CBP/p300 mediated acetylation of p53 was further confirmed in vivo by a number of studies (Sakaguchi et al., 1998; Liu et al., 1999; Luo et al., 2000; Kobet et al., 2000; Ito et al., 2001). Significantly, the steady-state levels of acetylated p53 are stimulated in response to various types of stress, indicating the important role of p53 acetylation in stress response (reviewed in Ito et al., 2001).
By introducing a transcriptionally defective p53 mutant (p53Q25S26) into mice, it was found that the mutant mouse thymocytes and ES cells failed in undergoing 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 p53 acetylation in p53-dependent apoptotic response (Chao et al., 2000; Luo et al., 2000).
Furthermore, it has been found that oncogenic Ras as well as PML can upregulate the levels of acetylated p53 in normal primary fibroblasts, and also induce premature senescence in a p53-dependent manner (Pearson et al., 2000, Ferbeyre et al., 2000). p53 acetylation may also play a critical role in protein stabilization (Rodriguez et al., 2000; Nakamura et al., 2000; Ito et al., 2001). In addition, another independent study showed that acetylation, but not phosphorylation of the p53 C-terminus, may be required to induce metaphase chromosome fragility in the cell (Yu et al., 2000).
In contrast, much less is known about the role of deacetylation in modulating p53 function. The acetylation level of p53 is enhanced when the cells are treated with histone deacetylase (HDAC) inhibitors such as Trichostatin A (TSA). This observation led to the identification of a HDAC1 complex which is directly involved in p53 deacetylation and functional regulation (Luo et al., 2000; Juan et al., 2000).
PID/MTA2, a metastasis-associated protein 2, acts as an adaptor protein to enhance HDAC1-mediated deacetylation of p53, but this activity can be completely repressed by TSA (Luo et al., 2000). In addition, Mdm2, a negative regulator of p53, actively suppresses CBP/p300-mediated p53 acetylation, and this inhibitory effect can be abrogated by tumor suppressor p19ARF, suggesting that regulation of acetylation also plays a critical role in the p53-MDM2-p19ARF feed back loop (Ito et al., 2001; Kobet et al., 2000).
The yeast silent information regulator 2 (Sir2) protein belongs to a novel family of histone deacetylases (reviewed in Guarente, 2000; Shore, 2000). Sir2 activity is nicotinamide adenine dinucleotide (NAD)-dependent, but can not be inhibited by TSA (Imai et al., 2000; Landry et al., 2000a; Smith et al., 2000). The NAD-dependent deacetylase activity of Sir2 is essential for its functions, and this activity also connects its biological role with cellular metabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al., 2000; Smith et al., 2000). Recently, mammalian Sir2 homologs have been found to also contain the NAD-dependent histone deacetylase activity (Imai et al., 2000; Smith et al., 2000), further supporting the notion that the enzymatic activity is key to elucidating the molecular mechanism for its mediated functions (Min et al., 2001; Finnin et al., 2001).
Among Sir2 and its homolog proteins (HSTs) in yeast, Sir2 is the only protein exclusively localized in nuclei, whose activity is critical for both gene silencing and extension of yeast life-span (reviewed in Guarente, 2000). Based on protein sequence homology analysis, mouse Sir2α and its human ortholog SIRT1 (or human Sir2α) are the closest homologs to yeast Sir2 (Imai et al., 2000; Frye, 1999, 2000). However, their biological functions remain unclear.
SUMMARY OF THE INVENTIONThis invention provides a method for treating a subject afflicted with cancer comprising administering to the subject a therapeutically effective amount of an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby treating the subject.
This invention also provides a method for inhibiting the onset of cancer in a subject comprising administering to the subject a prophylactically effective amount of an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby inhibiting the onset of cancer in the subject.
This invention further provides a method for inducing the death of a cell comprising contacting the cell with an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby inducing the death of the cell.
This invention further provides a method for decreasing the amount of damage to a subject's cells caused by physical stress comprising administering to the subject a prophylactically effective amount of an agent that increases the amount of Sir2α in the subject's cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject's cells, thereby decreasing the amount of damage to the subject's cells.
This invention further provides a method for prolonging the life-span of a subject comprising administering to the subject a prophylactically effective amount of an agent that increases the amount of Sir2α in the subject's cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject's cells, thereby prolonging the subject's life-span.
This invention further provides a method for decreasing the amount of damage to a cell caused by physical stress comprising contacting the cell with an agent that increases the amount of Sir2α in the cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the cell, thereby decreasing the amount of damage to the cell.
This invention still further provides a method for prolonging the life-span of a cell comprising contacting the cell with an agent that increases the amount of Sir2α in the cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the cell, thereby prolonging the life-span of the cell.
Finally, this invention provides two articles of manufacture. The first article of manufacture comprises a packaging material and an agent contained therein that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, and a label indicating that the agent is used for treating a subject afflicted with cancer, inhibiting the onset of cancer in a subject, and/or inducing the death of a cell.
The second article of manufacture comprises a packaging material and an agent contained therein that increases the amount of Sir2α in a cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in a cell, and a label indicating that the agent is used for decreasing the amount of damage to a subject's cells caused by physical stress, prolonging the life-span of a subject, decreasing the amount of damage to a cell caused by physical stress, and/or prolonging the life-span of a cell.
“Anti-sense nucleic acid” shall mean any nucleic acid which, when introduced into a cell, specifically hybridizes to at least a portion of an mRNA in the cell encoding a protein (“target protein”) whose expression is to be inhibited, and thereby inhibits the target protein's expression.
“Catalytic nucleic acid” shall mean a nucleic acid that specifically recognizes a distinct substrate and catalyzes the chemical modification of this substrate.
“DNAzyme” shall mean a catalytic nucleic acid that is DNA or whose catalytic component is DNA, and which specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA. Each DNAzyme has a catalytic component (also referred to as a “catalytic domain”) and a target sequence-binding component consisting of two binding domains, one on either side of the catalytic domain.
“Inhibiting” the onset of a disorder shall mean either lessening the likelihood of the disorder's onset, or preventing the onset of the disorder entirely. In the preferred embodiment, inhibiting the onset of a disorder means preventing its onset entirely.
“Nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids thereof. The nucleic acid bases that form nucleic acid molecules can be the bases A, C, G, T and U, as well as derivatives thereof. Derivatives of these bases are well known in the art, and are exemplified in PCR Systems, Reagents and Consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J., USA).
“Ribozyme” shall mean a catalytic nucleic acid molecule which is RNA or whose catalytic component is RNA, and which specifically recognizes and cleaves a distinct target nucleic acid sequence, which can be either DNA or RNA. Each ribozyme has a catalytic component (also referred to as a “catalytic domain”) and a target sequence-binding component consisting of two binding domains, one on either side of the catalytic domain.
“Subject” shall mean any animal, such as a human, non-human primate, mouse, rat, guinea pig or rabbit.
“Treating” a disorder shall mean slowing, stopping or reversing the disorder's progression. In the preferred embodiment, treating a disorder means reversing the disorder's progression, ideally to the point of eliminating the disorder itself.
EMBODIMENTS OF THE INVENTIONThis invention provides a method for treating a subject afflicted with cancer comprising administering to the subject a therapeutically effective amount of an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby treating the subject.
This invention also provides a method for inhibiting the onset of cancer in a subject comprising administering to the subject a prophylactically effective amount of an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby inhibiting the onset of cancer in the subject. Such inhibition can be caused, for example, by altering the behavior of existing Sir2α or by decreasing Sir2α expression (e.g., via anti-Sir2α nucleic acids such as anti-sense and catalytic nucleic acids such as ribozymes and DNAzymes). Sir2α is exemplified by human Sir2α having GenBank accession number AF083106 and the mouse Sir2α having GenBank accession number AF214646.
In the preferred embodiment of these methods, the subject is a human. The agent used in the instant methods can be any agent that inhibits p53 deacylation, such as vitamin B3 or nicotinamide. In a further embodiment, the instant methods further comprise administering to the subject an agent that enhances p53-dependent apoptosis via a mechanism other than Sir2α inhibition, such as Trichostatin A or Etoposide.
This invention further provides a method for inducing the death of a cell comprising contacting the cell with an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby inducing the death of the cell.
In the preferred embodiment of this method, the cell is a human cell. The agent used in the instant methods can be any agent that inhibits p53 deacylation, such as vitamin B3 or nicotinamide. In a further embodiment, this method further comprises contacting the cell with an agent that enhances p53-dependent apoptosis via a mechanism other than Sir2α inhibition, such as Trichostatin A or Etoposide.
This invention further provides a method for decreasing the amount of damage to a subject's cells caused by physical stress comprising administering to the subject a prophylactically effective amount of an agent that increases the amount of Sir2α in the subject's cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject's cells, thereby decreasing the amount of damage to the subject's cells.
The damage to the subject's cells can be any type of cellular damage including, for example, DNA damage and membrane damage. Physical stress includes, without limitation, ultraviolet radiation and oxidation. In this method, the agent can be administered prior to, concurrently with or subsequent to the occurrence of the physical stress.
This invention further provides a method for prolonging the life-span of a subject comprising administering to the subject a prophylactically effective amount of an agent that increases the amount of Sir2α in the subject's cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject's cells, thereby prolonging the subject's life-span.
In the preferred embodiment of the instant methods, the subject is human. The agent that increases the amount of Sir2α and/or the ability of Sir2α to inhibit p53-dependent apoptosis can be, for example, a Sir2α-encoding nucleic acid.
This invention further provides a method for decreasing the amount of damage to a cell caused by physical stress comprising contacting the cell with an agent that increases the amount of Sir2α in the cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the cell, thereby decreasing the amount of damage to the cell.
The damage to the cell can be any type of cellular damage including, for example, DNA damage and membrane damage. Physical stress includes, without limitation, ultraviolet radiation and oxidation. In this method, the agent can be contacted with the cell prior to, concurrently with or subsequent to the occurrence of the physical stress.
This invention still further provides a method for prolonging the life-span of a cell comprising contacting the cell with an agent that increases the amount of Sir2α in the cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the cell, thereby prolonging the life-span of the cell.
In the preferred embodiment of the instant methods, the cell is a human cell. The agent that increases the amount of Sir2α and/or the ability of Sir2α to inhibit p53-dependent apoptosis can be, for example, a Sir2α-encoding nucleic acid.
In this invention, administering agents can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, nasally, via implant, transmucosally, transdermally, intramuscularly, and subcutaneously. The following delivery systems, which employ a number of routinely used pharmaceutical carriers, are only representative of the many embodiments envisioned for administering the instant compositions.
Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprolactones and PLGA's). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprolactone.
Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc).
Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid).
Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids), and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.
Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, xanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine), preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid), anti-caking agents, coating agents, and chelating agents (e.g., EDTA).
Finally, this invention provides two articles of manufacture. The first article of manufacture comprises a packaging material and an agent contained therein that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, and a label indicating that the agent is used for treating a subject afflicted with cancer, inhibiting the onset of cancer in a subject, and/or inducing the death of a cell.
The second article of manufacture comprises a packaging material and an agent contained therein that increases the amount of Sir2α in a cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in a cell, and a label indicating that the agent is used for decreasing the amount of damage to a subject's cells caused by physical stress, prolonging the life-span of a subject, decreasing the amount of damage to a cell caused by physical stress, and/or prolonging the life-span of a cell.
This invention is illustrated in the Experimental Details section that follows. This section is set forth to aid in an understanding of the invention but is not intended to, and should not be construed to, limit in any way the invention as set forth in the claims which follow thereafter.
Experimental Details IntroductionSince homologues of Sir2 have been identified in almost all organisms examined including bacteria, which has no histone proteins (reviewed in Guarente, 2000; Frye, 1999; 2000), it is likely that Sir2 also targets non-histone proteins for functional regulation (Muth et al., 2001). Our preliminary results uncovered a novel activity in protein fractions from mammalian nuclear extract that could effectively deacetylate p53 in the presence of TSA. In support of the notion that mammalian Sir2α targets p53 for functional regulation, we present evidence that mouse Sir2α as well as human SIRT1 can directly bind p53 both in vitro and in vivo, and promotes cell survival under stress by specifically repressing p53-dependent apoptotic response.
Synopsis of Experimental FindingsThe NAD-dependent histone deacetylation of Sir2 connects cellular metabolism with gene silencing as well as aging in yeast. Here, we show that mammalian Sir2α physically interacts with p53 and attenuates p53-mediated functions. Nicotinamide (Vitamin B3) inhibits an NAD-dependent p53 deacetylation induced by Sir2α, and also enhances the p53 acetylation levels in vivo. Furthermore, Sir2α represses p53-dependent apoptosis in response to DNA damage and oxidative stress, whereas expression of a Sir2α point-mutant increases the sensitivity of cells in the stress response. Thus, our findings implicate a novel p53 regulatory pathway mediated by mammalian Sir2α. These results have significant implications regarding an important role for Sir2α in modulating the sensitivity of cells in p53-dependent apoptotic response and the possible effect in cancer therapy.
A. ResultsMammalian Sir2α Interacts with P53 Both In Vitro and in Vivo
Since mouse Sir2α shares a highly conserved region at the C-terminus with human SIRT1 (
Furthermore, we tested whether Sir2α directly interacts with p53 in vitro. As shown in
Deacetylation of p53 by Mammalian Sir2α
In order to test whether p53 could be specifically deacetylated by mammalian Sir2α in vitro, the mouse Sir2α protein was expressed with the N-terminal Flag epitope in cells and purified to near homogeneity on the M2-agarose affinity column (lane 3,
To establish the role for mammalian Sir2α in deacetylating p53 in cells, we used an acetylated p53-specific antibody to monitor the steady-state levels of acetylated p53 in vivo (Luo et al., 2000). As indicated in
Inhibition of Sir2α-Mediated p53 Deacetylation by Nicotinamide
To further elucidate the in vivo effect by endogenous Sir2α, we tried to identify an inhibitor for Sir2α-mediated deacetylase activity on p53. Deacetylation of acetyl-lysine by Sir2α 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). Although the molecular mechanism of Sir2 mediated NAD-dependent deacetylation needs to be detailed, it was proposed that formation of an enzyme-ADP-ribose intermediate through NAD hydrolysis is 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). We thus tested whether nicotinamide is able to inhibit the deacetylase activity of Sir2α on acetylated p53 in vitro.
Similar reactions as described above (
Furthermore, we examined the effect of Sir2α expression on endogenous levels of acetylated p53. Mouse embryonic fibroblast (MEF) cells, which express the wild type of p53, were infected with either a pBabe retrovirus empty vector or a pBabe retrovirus containing Sir2α, and cultured for a week under pharmacological selection. We first examined the protein levels of p53 activation in response to DNA damage in these cells by western blot analysis. Similar protein levels of p53 activation were induced in both types of cells after etoposide treatment for 6 hrs (lanes 3, 4 vs. lanes 1, 2, lower panel,
Maximum Induction of p53 Acetylation Levels in Normal Cells Requires Inhibition of Endogenous Sir2α Activity
After we found that nicotinamide has a strong inhibitory effect on Sir2α mediated deacetylation in vivo (
As indicated in
Repression of p53-Mediated Functions by Mammalian Sir2α Requires its Deacetylase Activity
To determine the functional consequence of mammalian Sir2α-mediated deacetylation of p53, we tested its effect on p53-mediated transcriptional activation. A mammalian p53 expression vector (CMV-p53), alone or in combination with different amounts of mouse Sir2α expressing vector (CMV-Sir2α) 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
To further test the biological role of mammalian Sir2α, we examined its modulation on p53-dependent apoptosis. p53 null cells (H1299) were transfected with p53 alone or cotransfected with p53 and Sir2α. The transfected cells were fixed, stained for p53, and analyzed for apoptotic cells (SubG1) (Luo et al., 2000). As indicated in
The Role of Mammalian Sir2α in Stress-Induced Apoptotic Response
Our data have indicated that mammalian Sir2α can deacetylate p53 both in vitro and in vivo (
Since stimulation of p53 acetylation as well as p53-dependent apoptosis have also been implicated in many other types of stress response (reviewed in Ito et al., 2001), we examined the role of mammalian Sir2α in the oxidative stress response. Recent studies have indicated that oxidative stress-induced cell death is p53-dependent (Yin et al., 1998; Migliaccio et al., 1999). We chose early-passage normal human fibroblast (NHF) IMR-90 cells for this study since it has been demonstrated that p53-dependent apoptosis can be strongly induced by hydrogen peroxide treatment in these cells (Chen et al., 2000). IMR-90 cells were infected with either a pBabe retrovirus empty vector or a pBabe retrovirus containing Sir2α, and cultured for a week under pharmacological selection. By immunofluorescence staining, we found that p53 in these infected cells was induced significantly after hydrogen peroxide treatment, along with Sir2α localized in the nuclei detected by immunostaining with specific antibodies (
Taken together, these results suggest that mammalian Sir2α promotes cell survival under stress by inhibiting p53-dependent apoptosis.
Mammalian Sir2α has No Effect on p53-Independent Cell Death Induced by Anti-Fas.
In order to determine the specificity of mammalian Sir2α-mediated protection of cells from apoptosis, we examined whether Sir2α has any effect on p53-independent, Fas-mediated apoptosis. The MEF (p53−/−) cells were first infected with either a pBabe retrovirus empty vector or a pBabe retrovirus containing Sir2α, 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 (SubG1). As shown in
Expression of a Sir2α Point-Mutant Increases the Sensitivity of Cells in the Stress Response
To further demonstrate that endogenous Sir2α regulates endogenous p53 under normal conditions, we introduced a Sir2α point-mutant (hSIRT1-363Y), which is functionally-defective in p53 deacetylation (
Taken together, these results suggest that endogenous Sir2α is critically involved in deacetylating p53 as well as regulating p53-mediated biological functions under physiological conditions.
B. DiscussionThe present data reveal the existence of a novel p53 regulatory pathway that is controlled by mammalian Sir2α. Sir2α is involved in gene silencing and extension of life span in yeast and C. elegans (reviewed in Guarente, 2000; Shore, 2000; Kaeberlein et al., 1999; Tissenbaun and Guarente, 2001). Although the mammalian homolog has also been shown to contain a unique histone deacetylase activity (Imai et al., 2000; Smith et al., 2000), its biological function remains to be elucidated. We show here (i) that p53 strongly binds to mouse Sir2α as well as its human ortholog hSIRT1 both in vitro and in vivo, (ii) that p53 is a substrate for the NAD-dependent deacetylase of mammalian Sir2α, (iii) that the Sir2α-mediated deacetylation antagonizes p53-dependent transcriptional activation and apoptosis, (iv) that the Sir2α-mediated deacetylation of p53 is inhibited by nicotinamide both in vitro and in vivo, (v) that Sir2α specifically inhibits p53-dependent apoptosis in response to DNA damage as well as oxidative stress, but not the p53-independent, Fas-mediated cell death, and (vi) expression of a Sir2α point mutant increases the sensitivity of the cells in response to stress. These results are especially relevant to the multiple regulatory pathways of p53 in vivo and, since the acetylation levels of p53 are stimulated in response to various types of stress, to the role of mammalian Sir2α in stress response.
The Sir2α-Mediated Pathway is Critical for Cells Under Stress
Our study implicates a novel, Mdm2-independent, negative regulatory pathway for p53, which further supports the views that there are multiple pathways in cells for tight regulation of p53 function (Prives and Hall, 1999; Appella and Anderson, 2000). In normal cells, Mdm2 is the major negative regulator for p53, and Mdm2-mediated repression appears sufficient to downregulate p53 activity. Interestingly, while no obvious effect by Sir2α expression was observed in cells at normal conditions, Sir2α becomes critical in protecting cells from apoptosis when cells were either treated by DNA damage or under oxidative stress (
In this regard, p53 is often found as latent 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 events contribute to p53 stabilization and activation by preventing binding with Mdm2 (reviewed in Appella and Anderson, 2000; Shieh et al., 1997). Mdm2 itself is also phosphorylated by ATM during DNA damage response, and this modification attenuates its inhibitory potential on p53 (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 occupy the same sites responsible for Mdm2-mediated ubiquitination (Rodriguez et al., 2000; Nakamura et al., 2000), and the highly acetylated p53 can not be effectively degraded by Mdm2 without deacetylation (Ito et al., 2001). Thus, in contrast to unstressed cells, the main p53 negative regulatory pathway mediated by Mdm2 is severely blocked at several levels in response to DNA damage (Maya et al., 2001). Under these circumstances, the Sir2α-mediated regulation may become a major factor in controlling p53 activity, making it possible for cells to adjust the p53 activity for DNA repair before committing to apoptosis.
Attenuation of p53-Mediated Transactivation by Sir2α
Earlier studies indicated that p53-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 p53-mediated apoptosis (reviewed in Prives and Hall, 1999). However, there is now growing evidence showing that p53 can effectively induce apoptosis by activating pro-apoptotic genes in vivo (reviewed in Nakano and Vousden, 2001; Yu et al., 2001). Thus, tight regulation 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 Sir2α is essential for its mediated functions (reviewed in Gurante, 2000). Reversible acetylation was originally identified in histones, however, accumulating evidence indicates that transcriptional factors are also functional targets of acetylation (reviewed in Sterner and Berger, 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 Berger, 2000; Kuo and Allis, 1998). Interestingly, 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), Sir2 may also be targeted to specific endogenous genes for transcriptional regulation in yeast.
In fact, there are at least seven different Sir2 homologs present in mammalian cells, but only mouse Sir2α and human SIRT1 are truly orthologs to yeast Sir2 based on the amino acid sequence homology and protein structure similarity (Frye, 1999, 2000; Imai et al., 2000). In addition, mouse SIR2L2 and SIR2L3 (or Human SIRT2 and SIRT3), are cytoplasmic proteins (Yang et al., 2000; Perrod et al., 2001). We have found that neither human SIRT5 nor SIRT6 binds to, or has any effect in deacetylating p53 in vivo (
Our results suggest that mammalian Sir2α inhibits p53-mediated apoptosis through attenuation of the transcriptional activation potential of p53. Our study also predicts that other cellular factors may use a similar mechanism to recruit Sir2 family proteins for TSA-insensitive transcriptional regulation in mammalian cells.
Novel Implications for Cancer Therapy
Inactivation of p53 functions has been well documented as a common mechanism for tumorigenesis (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). Based on our results that the maximum induction of p53 acetylation in normal cells requires both types of deacetylase inhibitors in addition to DNA damage, there are at least three different p53-negative regulatory pathways in mammalian cells. Interestingly, inhibitors for HDAC-mediated deacetylases, including sodium butyrate, TSA, SAHA and others, have been also proposed as antitumor drugs (reviewed in Marks, et al., 2001; Butler et al., 2000; Yoshida et al., 1995). Thus, we envision as one embodiment of this invention propose that the combining DNA damage drugs, HDAC-mediated deacetylase inhibitors, and Sir2α-medicated deacetylase inhibitors, in cancer therapy for maximally activating p53.
In contrast to PID/HDAC1-mediated p53 regulation (Luo et al., 2000), our results have shown that mammalian Sir2α-mediated effect on p53 is NAD-dependent, indicating that this type of regulation is closely linked to cellular metabolism (reviewed in Guarente 2000; Campisi, 2000). In fact, null mutants of NPT1, a gene that functions in NAD synthesis, show phenotypes similar to that of Sir2 mutants in silencing (Smith et al., 2000) and in life extension in response to caloric restriction in yeast (Lin et al., 2000). Thus, metabolic rate may play a role in Sir2α-mediated regulation of p53 function and, perhaps, modulate the sensitivity of cells in p53-dependent apoptotic response.
C. Experimental ProceduresPlasmids and Antibodies
To construct Sir2α expression constructs, the full-length cDNA was subcloned from pET28a-Sir2α (Imai et al., 2000) into pcDNA3 or pBabepuro vector. Site-directed mutation was generated in the plasmid pRS305—Sir2α using the Gene Edit system (Promega). To construct the human SIRT1 expression construct, DNA sequences corresponding to the full-length hSIRT1 (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 Sir2α antibody that can recognize both human and mouse Sir2α, we made a polyclonal antibody against the highly conserved C-terminus of Sir2α. DNA sequences corresponding to this region (480-737) were amplified by PCR and subcloned into pGEX-2T (Pharmacia). α-Sir2α antisera were raised in rabbits against the purified GST-Sir2α(480-737) fusion protein (Covance), and further affinity-purified on both protein-A and antigen columns. By western blot analysis and immunofluorescent staining, this antibody can detect both mouse Sir2α and human SIRT1 proteins.
In Vitro p53 Deacetylation Assay
The Flag-tagged Sir2α 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). The proteins were purified under a very high stringency condition (300 mM NaCl 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 p53 (2.5 μg) was incubated with purified Sir2α (10 ng) at 30° C. for 1 hr either in the presence of 50 μM NAD or as indicated. The reactions were performed in a buffer containing 50 mM Tris-HCl (pH 9.0), 50 mM NaCl, 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.
Virus Infection and Stress Response
All MEF cells were maintained in DMEM medium supplemented with 10% fetal bovine serum, and the IMR-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). After one-week selection, the cells were either frozen for stock or immediately used for further analysis. About 500,000 MEF cells were plated on a 10-cm dish 24 hr before treatment. The cells were then exposed to etoposide (20 μm) 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 (SubG1) according to DNA content. In case of the Fas-mediated apoptosis assay, the cells were treated with actinomycin D (0.25 μg/ml) and Fas antibody (100 ng/ml) as previously described (Di Cristofano et al., 1999). In the case of oxidative stress response, the IMR-90 cells were treated with H2O2 (50 to 200 μM) for 24 hrs.
Detecting Acetylation Levels of p53 in Cells
The cells (human lung carcinoma cell lines H460 (wild-type p53) and H1299 (p53-null), human colon carcinoma HCT116 (wild-type p53), mouse embryonal carcinoma cell line F9 (wild-type p53), 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 μM) and or other drugs (0.5 μM of TSA, 5 mM of nicotinamide, and 50 μM of ALLN) as indicated for 6 hr. After treatment, the cells were harvested for western blot analysis. The rabbit polyclonal antibody specific for p300-mediated acetylated p53-[α-p53(Ac)-C] was raised and purified against the acetylated human p53 C-terminal peptide [p53 (Ac)-C: H-S55GQSTSRH55LMF-OH (5=acetylated Lysine)] as described before (Luo et al., 2000). In the case of cotransfection assays testing for p53 acetylation levels, H1299 cells were transfected with 5 μg of CMV-p53 plasmid DNA, 5 μg of CMV-p300 plasmid DNA, and 10 μg of pcDNA3—Sir2α plasmid DNA as indicated. 24 hr after the transfection, the cells were lysed in a Flag-lysis buffer (50 mM Tris, 137 mM NaCl, 10 mM NaF, 1 mM EDTA, 1% Triton X-100 and 0.2% Sarkosyl, 1 mM DTT, 10% glycerol, pH 7.8) with fresh proteinase inhibitors, 10 μM TSA and 5 mM nicotinamide (Sigma). The cell extracts were resolved by either 8% or 4-20% SDS-PAGE gels (Novex) and analyzed by western blot with α-p53(Ac)-C and α-p53(DO-1).
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Claims
1. A method for treating a subject afflicted with cancer comprising administering to the subject a therapeutically effective amount of an agent that inhibits the ability of Sir2α to inhibit p53-dependent apoptosis, thereby treating the subject.
2. (canceled)
3. The method of claim 1, wherein the subject is a human.
4-5. (canceled)
6. The method of claim 1, further comprising administering to the subject an agent that enhances p53-dependent apoptosis via a mechanism other than Sir2α inhibition.
7-15. (canceled)
16. A method for decreasing the amount of damage to a subject's cells caused by physical stress comprising administering to the subject a prophylactically effective amount of an agent that increases the amount of Sir2α in the subject's cells and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the subject's cells, thereby decreasing the amount of damage to the subject's cells.
17. The method of claim 16, wherein the damage is selected from a group consisting of DNA damage and membrane damage.
18. The method of claim 16, wherein the physical stress is selected from the group consisting of ultraviolet radiation and oxidation.
19. The method of claim 16, wherein the agent is administered prior to and/or concurrently with the occurrence of the physical stress.
20. The method of claim 16, wherein the agent is administered subsequent to the occurrence of physical stress.
21-23. (canceled)
24. A method for decreasing the amount of damage to a cell caused by physical stress comprising contacting the cell with an agent that increases the amount of Sir2α in the cell and/or the ability of Sir2α to inhibit p53-dependent apoptosis in the cell, thereby decreasing the amount of damage to the cell.
25. The method of claim 24, wherein the damage is selected from the group consisting of DNA damage and membrane damage.
26. The method of claim 24, wherein the physical stress is selected from the group consisting of ultraviolet radiation and oxidation.
27. The method of claim 24, wherein the cell is contacted with the agent prior to and/or concurrently with the occurrence of the physical stress.
28. The method of claim 24, wherein the cell is contacted with an agent subsequent to the occurrences of the physical stress.
29. (canceled)
30. The method of claim 24, wherein the cell is a human cell.
31. The method of claim 24, wherein the agent is a Sir2α-encoding nucleic acid.
32-33. (canceled)
Type: Application
Filed: Jul 9, 2007
Publication Date: May 22, 2008
Applicant: The Trustees of Colubia University (New York, NY)
Inventors: Wei Gu (New York, NY), Jian Yuan Luo (New York, NY)
Application Number: 11/825,944
International Classification: A61K 31/70 (20060101); C12N 5/08 (20060101); A61P 35/00 (20060101);