WO2005073404A2 - Functional element of siah-1b responsive p53 - Google Patents

Abstract

A functional element of siah-lb that directly interacts with p53 is discloses. Also disclosed are methods of screening compounds useful in the treatment, prevention or management of cancer or a neurodegenerative disorder based on the interaction between p53 and the functional element, and methods of treating, preventing or managing cancer or a neurodegenerative disorder using the compounds so screened.

Description

FUNCTIONAL ELEMENT OF SIAH-lb RESPONSIVE P53

1. FILED OF THE INVENTION This invention relates to the functional element in siah-lb promotor, which directly interacts with the tumor suppressor p53. Methods of screening agents useful in treating, preventing and managing cancer or a neurodegenerative disorder are also encompassed.

2. BACKGROUND OF THE INVENTION The Drosophila SL A is a protein required for neuronal differentiation of the R7 photoreceptor cells in the eye of Drosophila melanogastex (1). It acts downstream of the Sevenless tyrosine kinase receptor to degrade the transcriptional repressor Tramtra (2, 3). The Drosophila SINA protein and its mammalian Siah homologues are phylogenetically conserved E3 ligases, enzymes involved in ubiquitination and proteasome-mediated degradation of protein substrates. This function is due to their N-terminal RING-finger domain, which recruits ubiquitin conjugating E2 enzymes and promotes the ligation of ubiquitin to the substrate (4). The two functional human genes siah-1 and siah-2 are responsible for degradation of Kid, BOB/OBF1, synaptophysin, synphilin, TGF Beta-induced early gene and TRAF2 (5-10). Siah-1, through the binding and downmodulation of Numb, is a positive regulator of Notch activity (11). Although it has been reported that human siah-1 and its mouse homologous siah-lb are induced by p53 during apoptosis and tumor reversion, it is unclear whether siah genes are directly interacting with p53. Therefore, determining whether siah genes are direct target of interaction by p53 is important in understanding the mechanism of tumor reversion and/or cell apoptosis, thereby allowing the screening and designing of an effective anti-tumor agents. In addition, since the relevance of tumor reversion or cell apoptosis to a neurodegenerative disorder such as Alzheimer's disease has been implicated, an effective agents for treating, preventing or managing a neurogenerative disorder can also be screened or designed using that piece of information.

3. SUMMARY OF THE INVENTION In one embodiment, this invention is directed to a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb. In another embodiment, this invention encompasses a method of identifying a compound that reduces or inhibits cell apoptosis comprising: (a) contacting the compound with a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb and p53; (b) determining whether binding between the DNA fragment and p53 is decreased; and if the binding between the DNA fragment and p53 is decreased, then, (c) contacting the compound with a cell; and (d) determining whether cell apoptosis is reduced as compared to a cell that has not been contacted with the compound. In another embodiment, this invention encompasses a method of treating, preventing or managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of the compound identified using the screening method above. The neurodegenerative disorders that can be treated, prevented or managed using the methods of this invention include, but are not limited to, Alzheimer's disease, Huntington's disease and Parkinson's disease. In another embodiment, this invention encompasses a method of identifying a compound that promotes cell apoptosis comprising: (a) contacting the compound with a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb and 53; (b) determining whether binding between the DNA fragment and p53 is decreased; and if the binding between the DNA fragment and p53 is decreased, then, (c) contacting the compound with a cell; and (d) determining whether cell apoptosis is increased as compared to a cell that has not been contacted with the compound. In another embodiment, this invention encompasses a method of treating, preventing or managing cancer comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective arriount of the compound identified using the screening method described above. hi another embodiment, this invention encompasses a method of diagnosing a cause of a disease or disorder associated with reduced Siah-1 expression comprising: (a) obtaining sequence information of2155 to 2103 nucleotides downstream from translational start site of siah-lb obtained from a patient; (b) obtaining sequence information of 2155 to 2103 nucleotides downstream from translational start site of siah-lb obtained from a normal subject; and (c) comparing the sequences obtained from steps (a) and (b), wherein if t ie sequence obtained from step (a) is not identical to the sequence obtained from step (b), the cause is a defect in binding of p53 to siah-lb or activation of siah-lb by ρ53.

4. BRIEF DESCRIPTION OF FIGURES Fig. 1 illustrates activation of p53 induces increased siah-lb mRNA levels. Schematic alignment of siah-1 a and siah-lb mRNAs. Exons are represented by bars and the CDS as large rectangles. Homologous sequences are colored gray. White segments identify sequences specific to each mRNA. The relative positions of the siah-la and siah-lb probes (black bars) used for Northern blot analysis are also indicated. (B) Northern blots of mouse lriRNA hybridized with the siah-1 CDS (probe CDS), the siah-la probe (probe a) or the siah-lb probe (probe b). (C) Northern blot analysis of mRNA isolated from LTR6 cells after shifting to 32°C for 1, 2, 4 and 6 hr. LTR6 cells at 38°C are used as negative controls. (D) Northern blot of mRNA extracted from NIH3T3 cells after 16 hr of incubation with 15 nM Actinomycin D (ActD); untreated cells (0 hr) were used as control. (C and D) Blots were hybridized with a siah-lb specific probe. p21 and cyclin Gl were used as positive controls for p53 activation. GAPDH was used as control for equal loading. Fig. 2 illustrates DNA derived from the siah-lb gene confers transcriptional activation by p53. Promoter activity was measured in luciferase assays with reporter plasmids comprising the region nt -2613/- 1694 of siah-lb or its three mutants (siah-lb mutl, siah-lb mut2 and siah-lb Δ), following transient transfection into HI 299 cells. The different constructs are schematically represented on the left panel and their corresponding promoter activity is shown on the right panel. A p21 promoter fragment containing the distal p53RE was used as positive control and empty pGL3 vector as negative control. Luciferase activity is reported as fold activation, representing the ratio of the values standardized by protein concentration measured in presence or absence of p53. Values shown are mean +/- SD (n^). Fig. 3 illustrates that p53 binds to the siah-lb promoter in vitro : (A) EMSA was performed with a probe corresponding to the p53RE of the siah-lb promoter (nt -2160/-2098). This probe was incubated with recombinant p53 protein (lanes 2, 3, 4, 6 and 7) in absence (lanes 1, 2, 4 and 6) or in presence (lanes 3, 5 and 7) of the PAb421 antibody. In lanes 6 and 7, the probe contained a double mutation in the p53 consensus site (siah-lb mut3). A pool of mouse IgGs was used as control for the PAb421 antibody specificity (lane 4) ; and (B) in vitro DNase I footprinting experiment with labeled probes designed to have the p53RE placed in their center. Probes containing the p53RE of siah-lb (lanes 1, 2 and 3) or its mutants (lanes 4, 5 and 6 for siah-lb mutl and lanes 7, 8 and 9 for siah-lb mut2) were incubated with either 0.5 (lanes 2, 5 and 8) or 1 μg of purified p53 (lanes 3, 6 and 9) or with an irrelevant protein (lane 10) before digestion with DNase I. The positions of the p53 binding sites were identified by running DNA sequencing reactions next to the footprinting experiment (left panel). Fig. 4 illustrates that p53 binds to siah-lb chromatin in vivo. Chromatin immunoprecipitation (ChIP) was performed following p53 activation in vivo, using antibodies against clathrin heavy chain (ip ctrl) or p53 (ip p53). PCR was performed with gene-specific primers. Total lysate was used as a control for PCR amplification (input). p53 binding was tested using siah-lb specific primers; p21 primers and β2 microglobulin primers were used as positive and negative controls, respectively. (A) Results of ChIP . analysis done on LTR6 cells, activated by temperature shift to 32 °C for 6 hr; Ml and LTR6 cells maintained at 38°C served as negative controls. (B) Western blots analysis of p53 in NIH3T3 cells activated with 15 nM Actinomycin D (ActD) for 16 or 24 hr. Untreated cells were used as negative control (0 hr). α-tubulin served as an equal loading control. (C) Results of ChIP analysis done onNIH3T3 cells treated as in (B). Fig. 5 illustrates Transcriptional activation of siah-lb by p53 in NIH3T3. To confirm the results obtained in the HI 299 human cell line, we performed luciferase assay in another cellular environment. (A) Luciferase activity of the fragment nt -2613/-1694 of the siah-lb promoter and of the three mutants (siah-lb mutl, siah-lb mut2 and siah-lb Δ) in NIH3T3 cells. Empty pGL3 vector is used as negative control. The activity is reported as fold of activation, representing the ratio of the values, standardized by protein concentration and measured in the presence or absence of p53. (B) Promoter activity of siah-lb and p21 was performed in luciferase assay using 75 or 30 ng of the p53 pCMV construct transiently transfected in NTH3T3 cells. Luciferase expression is represented in arbitrary units. Data are calculated on the mean values with +/- SD (n=3). Fig. 6 illustrates that p53 binds to the siah-lb promoter in vivo. To confirm the results obtained with the baculovirus-produced p53, we tested in gel shift assay the p53 protein produced by INT. EMSA was performed with a probe containing the p53 responsive element of the siah-lb promoter (nt -2160/-2098) (lanes 1, 2, 3, 4, 7 and 8) was incubated with the p53 produced by INT (lanes 2, 3, 4, 5 and 6) in absence (lanes 1, 2, 4, 5 and 7) or in presence (lanes 3, 6 and 8) of the PAb421 antibody. The effect of the double mutation in the p53 consensus sites (siah-lb mut3) is shown in lanes 5 and 6. To verify that no protein of the INT reaction mix is involved, we tested the reticulocyte lysate alone (INT control, lanes 7 and 8). A pool of mouse IgGs was used as control of the antibody specificity (lane 4). The supershift and a non specific gel retardation observed without p53 (INT control) are indicated by an arrow.

5. DETAILED DESCRIPTION OF THE INVENTION We cloned the siah-1 gene, one of the human homologues of SIN A, and showed its overexpression in the epithelium of the small intestine, a well- established example of physiological programmed cell death (12). Moreover, in our studies of tumor reversion we found siah-1 overexpressed at the mRNA level (12), as well as at the protein level (13, 14), in cells with a suppressed malignant phenotype. In a three-dimensional basement membrane reconstituted in matrigel, Siah-1 is able to reorganize MCF7 cells in structures similar to those observed with normal breast cells (14, 15). Cells overexpressing Siah-1 showed an increase in apoptosis and gave rise to significantly fewer tumors than the parental cells when injected into scid/scid mice (13, 14). We demonstrated that cells transfected with the cyclin-dependent kinase inhibitor p21 exhibit high levels of Siah-1 (16) and that Siah-1 has common downstream effectors with p21 and the tumor suppressor p53 (13). Moreover, Siah-1 acts in a complex with Skpl, Ebl, SIP (Siah interacting protein), and adenomatous polyposis coli protein (pAPC) to facilitate, in a p53 -dependent manner, the degradation of beta-catenin thus inducing apoptosis and inhibiting cell proliferation and transformation (17, 18). Pwl/Peg3 is another p53-inducible gene product that cooperates with Siah-1 in promoting cell death (19) whereas BAG-1, an anti-apoptotic protein, antagonizes ' the effect of Siah-1 on apoptosis (20). The human siah-1 gene has two murine homologues, siah-la and siah-lb, which are widely expressed in various tissues of the embryo and adult (21). The RNA sequences of siah-la and siah-lb are 95% homologues with each other, 90% with their human counterpart and 72% with Drosophila SINA. Siah-la drives the degradation of c-myb, a proto-oncogene involved in cellular proliferation and apoptosis (22), and is necessary for progression past metaphase during meiosis I of spermatogenesis (23). We also identified siah-lb by cDNA differential display as a gene induced by p53 in murine Ml myeloid leukemia cells (24). This suggests a function for Siah-lb in apoptosis, as observed for its human homologue. Gene expression analysis revealed that human siah-1 transcription was significantly correlated to the dosage of p53 (25). p53 is a homotetrameric transcription factor that can activate or repress the transcription of a series of genes controlling cell cycle progression, apoptosis, DNA repair, and other types of stress response. These genes include p21, MDM2, cyclin G, BAX, noxa, puma, TSAP6 (26-32) and many others. The p53 protein is normally short-lived and present at low levels, but in response to stress it accumulates in the nucleus where it binds to specific DNA sequences within chromatin (33). The consensus p53 binding site is composed of two 10 bases half sites, each conforming to the sequence 5'-PuPuPuC(A/T)(T/A)GPyPyPy-3' (where Pu and Py represent purines and pyrimidines, respectively) and separated by a spacer of 0-13 bp (34-37). Based on this consensus sequence, a computer algorithm developed to identify p53 binding sites in the human genome disclosed siah-1 as a possible downstream target of p53 (38). In the present study, we show that the siah-lb promoter contains a functional p53 responsive element (RE), which is able to bind p53 in vitro and in vivo. Activation of p53 in different cell systems leads to a significant increase in siah-lb transcription. Thus, the siah-lb gene is a direct transcriptional target of p53. In the present study, we show that the mouse siah-lb gene, shown previously to be upregulated during p53-mediated apoptosis (24), contains a functional p53RE within its second intron. This p53RE enables the transactivation of the siah-lb gene by p53 in response to anti-cancer agents such as ActD. Through the use of suitable molecular tools, which discern between the different highly homologous mouse siah-1 transcripts, we show that siah-lb mRNA is increased as a, result of p53 activation, thus resolving previous ambiguities stemming from the use of non-specific probes (12, 16, 21, 24, 40). Basal levels of siah-lb mRNA are also present before p53 activation, suggesting that the protein has a role also in non-stressed cells. Moreover, siah-lb expression can also be induced by p53-independent mechanisms (13), although expression is maximal when p53 is activated. A recent report, utilizing siah- lb-null mouse embryo fibroblasts, has concluded that Siah-lb is not involved in the p53 pathway (40). However, it is possible that in such cells p53 activates multiple downstream effectors. The role of Siah-lb could thus be substituted by proteins encoded by one or more of the other p53 -responsive genes that are transactivated simultaneously with siah-lb. Moreover, the apparent discrepancy between our data and those of Bowtell and co-workers (40) may also be due to different experimental conditions; it is conceivable that the contribution of Siah-lb to the p53 response may vary as a function of the cellular context. In this regard, it is noteworthy that the effect of p53 itself is also greatly cell context-dependent; thus, activation of the same temperature sensitive p53 mutant can induce growth arrest, apoptosis or differentiation in a context-dependent manner (47). The pattern and extent of induction of different p53 target genes is greatly dependent on the concentration of active p53 protein within a given cell (48). In this regard, it is of note that the siah-lb gene seems to require higher levels of p53 activity for maximum activation as compared to the p21 gene (Fig. 1C), and this is also seen when the corresponding p53RE's are compared in luciferase assays (see Supplementary Fig. 5B). This is most likely due to differences in the affinity of p53 for the corresponding p53RE's, as seen also with other p53 target genes (49). This is also suggested by the fact that while for p21 10 minutes of crosslinking were sufficient to obtain an optimal PCR signal in the ChIP analysis, 15 minutes were required in the case of siah-lb (data not shown). The identified p53RE is functional, despite its unusual structure. So far, no functional p53RE with such a long spacer between the two half sites has been described. It is possible that the sequence of the spacer also has a role in enabling such an unusual p53RE to function properly. The footprinting analysis suggests that the spacer is not directly involved in interactions with p53; nevertheless, this region may be recognized in vivo by other proteins required for optimal p53- induced transcription of siah-lb. It is also noteworthy that each of the two half sites seems to function even on its own, when the other half site is mutant and incapable of binding p53; this is seen both in DNA binding analysis (Fig. 3B) and in functional assays (Fig. 2). Several studies have proposed a role for the Siah proteins in p53-mediated responses (11, 13, 19, 50-52). It is also noteworthy that siah-lb knockout mice are not viable, underlying the importance of this gene in embryonic development. The exact way in which Siah contributes to p53- mediated apoptosis or cell cycle arrest is still not clearly defined; one possibility is that this may have to do with the proposed role of Siah-1 in mediating p53- dependent degradation of β-catenin in response to DNA damage (51). By establishing a direct link between p53 and transcriptional regulation of the siah-lb gene, our study further supports the importance of the latter in the p53 response. 5.1 Methods of Treatment. Prevention or Management i one embodiment, this invention encompasses methods of treating, preventing and/or managing cancer comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of a compound identified using certain methods of this invention. As used herein, and unless otherwise specified, the terms "treat," "treating" and "treatment" refer to the eradication or amelioration of a disease or condition, or of one or more symptoms associated with the disease or condition. In certain embodiments, the terms refer to minimizing the spread or worsening of the disease or condition resulting from the administration of one or more prophylactic or therapeutic agents to a subject with such a disease or condition. As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or condition, or of a symptom thereof. As used herein, and unless otherwise specified, the terms "manage,"

"managing" and "management" refer to preventing or slowing the progression, spread or worsening of a disease or condition. Often, the beneficial effects that a subject derives from a prophylactic or therapeutic agent do not result in a cure of the disease or condition. As used herein and unless otherwise indicated, the term "managing" encompasses preventing the recurrence of cancer in a patient who had suffered from cancer, lengthening the time a patient who had suffered from cancer remains in remission, preventing the occurrence of cancer in patients at risk of suffering from cancer (e.g., patients who had been exposed to high amounts of radiation or carcinogenic materials, such as asbestos; patients infected with viruses associated with the occurrence of cancer, such as, but not limited to, HIN and Kaposi's sarcoma-associated herpes virus; and patients with genetic predispositions to cancer, such as those suffering from Downs syndrome), and preventing the occurrence of malignant cancer in patients suffering from pre-malignant or non- malignant cancers. As used herein, and unless otherwise specified, a "therapeutically effective amount" of a compound is an amount sufficient to provide a therapeutic benefit in the treatment or management of a disease or condition, or to delay or minimize one or more symptoms associated with the disease or condition. A therapeutically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment or management of the disease or condition. The term "therapeutically effective amount" can encompass an amount that improves overall therapy, reduces or avoids symptoms or causes of disease or condition, or enhances the therapeutic efficacy of another therapeutic agent. As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or condition, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with other agents, which provides a prophylactic benefit in the prevention of the disease. The tenn "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent. As used herein, the term "cancer" includes, but is not limited to, solid tumors and blood born tumors. The term "cancer" refers to disease of skin tissues, organs, blood, and vessels, including, but not limited to, cancers of the bladder, bone or blood, brain, breast, cervix, chest, colon, endrometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testis, throat, and uterus. Specific cancers include, but are not limited to, advanced malignancy, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastases, glioblastoma multiforms, glioblastoma, brain stem glioma, poor prognosis malignant brain tumor, malignant glioma, recurrent malignant giolma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, Dukes C & D colorectal cancer, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, karotype acute myeloblastic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous T-Cell lymphoma, cutaneous B- Cell lymphoma, diffuse large B-Cell lymphoma, low grade follicular lymphoma, localized or metastatic melanoma (of any kind, including, but not limited to, ocular), peritoneal carcinoma, papillary serous carcinoma, gynecologic sarcoma, soft tissue sarcoma, scelroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressive, hormone refractory prostate cancer, resected high-risk soft tissue sarcoma, unrescectable hepatocellular carcinoma, Waldenstrom's macroglobulinemia, smoldering myeloma, indolent myeloma, fallopian tube cancer, androgen independent prostate cancer, androgen dependent stage IN non-metastatic prostate cancer, hormone- insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, and leiomyoma. hi a specific embodiment, the cancer is metastatic. In another embodiment, the cancer is refractory or resistance to chemotherapy or radiation. In some embodiments of this invention, compounds of this invention can be administered, sequentially or simultaneously, with another anticancer agent. The administration may be via same route or different routes. Examples of anti- cancer agents include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; celecoxib; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexonnaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; iproplatin; irinotecan; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; taxotere; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; and zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-l,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; arnrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein- 1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-aletbine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole;

CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorlns; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cyto statin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; doxorubicin; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflomithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imatinib (e.g., Gleevec®), imiquimod; iirimunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxanfrone; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim;Erbitux, human chorionic gonadotrophin; monophosphoryl lipid

A+myobacterium cell wall sk; mopidamol; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline;

N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; oblimersen

(Genasense®); O""benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rohitukine; romurtide; roquinimex; rubiginone Bl; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinostatin stimalamer. The magnitude of a prophylactic or therapeutic dose of each active ingredient in the treatment, prevention and/or management of cancer will typically vary with the specific active ingredients, the severity and type of cancer, and the route of administration. The dose, and perhaps the dose frequency, may also vary according to age, body weight, response, and the past medical history of the patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors by following, for example, dosages reported in the literature and recommended in the Physician 's Desk Reference® (54th ed., 2000). In one embodiment, the patient is an animal. In another embodiment, the patient is a mammal, specifically, a human. In another embodiment, this invention encompasses methods of treating, preventing and managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention and/or management a therapeutically or prophylactically effective amount of a compound identified certain methods of this invention. Examples of neurodegenerative disorders that can be treated using methods of this invention include, but are not limited to, amylotrophic lateral sclerosis, Alzheimer's disease, Huntington's disease and Parkinson's disease. In one embodiment, the neurodegenerative disorder that can be treated, prevented and/or managed using methods of this invention is Alzheimer's disease. In one embodiment, the patient is a mammal, in particular, a human. In some embodiments, compound of this invention may be administered, sequentially or simultaneously, with another agent useful in treating a neurodegenerative disorder. The administration may be via same route or different routes. Examples of other agents include, but are not limited to: acridine derivatives such as tacrine; amantadine; dopamine depleting agents such as tetrabenazine and reserpine; dopamine receptor antagonists such as bromocriptine and pergolide; metabolic precursors of dopamine such as levodopa; inhibitors of acetylcholinesterase (AChE) such as physostigmine; inhibitors of L-amino acid decarboxylase such as benserazide and carbidopa; muscarinic receptor antagonists such as benzotropine mesylate, diphenhydramine hydorchloride and trihexyphenidyl; precursors of acetylcholine synthesis such as choline chloride and phosphatidyl choline (lecithin); and selegiline. The magnitude of a prophylactic or therapeutic dose of each active ingredient in the treatment, prevention and/or management of a neurodegenerative disorder will typically vary with the specific active ingredients, the severity and type of disorder, and the route of administration. The dose, and perhaps the dose frequency, may also vary according to age, body weight, response, and the past medical history of the patient. Suitable dosing regimens can be readily selected by those skilled in the art with due consideration of such factors by following, for example, dosages reported in the literature and recommended in the Physician's

Desk Reference® (54th ed., 2000).

6. EXAMPLES This invention can be further illustrated by the following, non-limiting examples. 6.1 Materials and Methods 6.1.1 Cell Culture and p53 Activation The Ml murine myeloid leukemia cell line and its derivative LTR6 clone, expressing a temperature sensitive p53, have been described (24, 39). In mouse NIH3T3 cells p53 was activated by incubation with 15 nM Actinomycin D

(Sigma) for 16-24 hr. Human p53 null HI 299 cells were grown according to the

ATCC recommendations. 6.1.2 Cloning and Mutagenesis The siah-lb promoter region (nt -2613/-1694) was cloned into the pGL3- enhancer vector (Promega). The siah-lb p53 binding element was mutated (CATG to TATA) either within the first (siah-lb mutl) or within the second half site (siah-lb mut2), using the QuikChange Multi SiteDirected Mutagenesis kit (Stratagene). The p53RE (nt -2160/-2098) was deleted in the siah-lb Δ with the following primers: Forward 5'- CTAAAATGGGTCTCAAGACCTCCCCTGAGA-3' and Reverse 5'- TCTCAGGGGAGGTCTTGAGACCCATTTTAG-3'. The distal region of the p21 promoter (nt -7615/-7354 from ATG) containing the p53RE was cloned into the pGL3 enhancer vector using the primers Forward 5'- CTAGGTACCCCAGAGGATACCTTGCAAGGCTGCA-3' and Reverse 5'- TATAGATCTTCTCTGTCTCCATTCATGCTCCTCC-3'. The mouse p53 coding sequence (CDS) was cloned in a pCMN vector. 6.1.3 Proteins and Antibodies The human p53 protein was purified from insect cells infected with an appropriate recombinant baculovirus. The following antibodies were used: anti- p53 FL-393 (Santa Cruz Biotechnologies) and PAb421 (Oncogene); anti-clathrin heavy chain H-300 (Santa Cruz Biotechnologies); anti alpha-tubulin B-5-1-2

(Sigma). 6.1.4 Northern Blot Analysis Northern blots were performed as described (24). The following probes were used: mouse p21 nt 111/340 (ace. n. NM_007669), cyclin Gl nt 202/443 (ace. n. BC005534) and human GAPDH (Clontech). The probe CDS corresponds to the region nt 251/896 of the human siah-1 cDNA (ace. n. U63295). A specific probe for siah-la (probe a) was designed from the 3' UTR (nt 1676/1934) of siah- la mRNA (ace. n. Z19579) while the specific probe for siah-lb (probe b) corresponds to nt 1/230 in the 5' UTR of the mRNA (ace. n. Z19580). 6.1.5 Rapid Amplification of cDNA Ends (RACEVPCR The RACE-PCR was performed using the Marathon cDNA Amplification kit (Clontech) following the manufacturer's instructions. Briefly, mRNA derived from mouse cells was subjected to reverse transcription using a modified oligo d(T) primer. The resulting cDNAs were ligated to an adaptor and used as templates in 5' or 3' RACE PCR using the adaptor specific primer with siah-1 primers designed to amplify both siah-la and siah-lb cDNA. siah-1 Forward: 5'-CCCCTTGTGAGTCAACACATAGTGCTGC-3'; siah-1 Reverse: 5'- TGGGGCGACAGTTGCTACAAACAAG-3'. For the 5' amplification we performed a nested PCR using an internal adaptor specific primer and the reverse siah-lb primer 5'-

AGACTCGCCAAGTCATTGTTGGATGC-3 ' . 6.1.6 Luciferase Assays 120,000 cells were transfected with 300 ng of the different siah-lb promoter constructs (see Cloning and Mutagenesis) and 300 ng of pCMN vector, either empty or expressing mouse p53. Transfection was performed using LipofectAMIΝE PLUS reagent (Life Technologies Inc.) according to the manufacturer's recommendations. 24 hr after transfection the cells were washed and cell extracts prepared using a reporter lysis reagent (25 mM Tris, 8 mM MgCl₂, 1 mM DTT, 1% Triton X-100 and 15% glycerol). Following normalization of each extract for protein content, luciferase activity was measured using the Victor Luminometer (Perkin Elmer) after addition of 20 nmol Luciferin (Roche) and 100 nmol ATP (Sigma). 6.1.7 Electrophoretic Mobility Shift Assay (EMSA Synthetic oligonucleotides containing the p53RE of siah-lb, either wild type

(5'TCTCAAGACATGTCCAGACCTCCCCTGATCACATTCAAAAGGGTCTC AAGACATGTCCAGACC-3') or double mutant (5'-

TCTCAAGATATATCCAGACCTCCCCTGATCACATTCAAAAGGGTCTCA AGATATATCCAGACC-3 ') were radiolabeled at their 5' end using T4

Polynucleotide Kinase (Biolabs) and [γ- PJATP (Amersham Biosciences). The complementary oligonucleotides were then annealed and purified on a polyacrylamide gel. The probes were incubated for 20 min at room temperature (RT) with 18 ng of recombinant p53 in a buffer containing 20 mM Tris HCl pH 7.5, 50 mM KCl, 5 mM MgCl₂, 1 mM DTT, 0.5 mM EDTA, 0.5 mg/ml BSA; then 20 ng of DΝA competitor poly dl-dC (Amersham) was added to a final volume of 30 μl. To induce a supershift, 300 ng of p53 -specific PAb421 antibody were included in the mixture and the incubation was continued for 20 min at RT. The reaction products were analyzed by electrophoresis on a 4% non-denaturing polyacrylamide gel. 6.1.8 In vifro DNase I footprinting The radiolabeled forward primer 5'-CCATGGAGCCACCTCAGCTC-3' and the reverse primer 5'-ACTAGTAATGAGTTTCCTCTCCTACATGAA-3' were used in PCR reactions with 25 ng DNA of the following constructs: siah-lb, siah-lb mutl or siah-lb mut2. The 410 bp labeled PCR product containing the p53RE in its center was used in DNase I footprinting experiments following the Sure Core Footprinting protocol (Promega) with minor modifications. For each reaction, 500 ng or 1 μg of p53 or 1 μg of irrelevant protein were mixed with 500 ng of PAb421 antibody. After 30 min of preincubation on ice, 10 ng (30000 c.p.m.) of 5 '-end-labeled DNA was added and incubation carried on for another 30 min on ice. After adjusting the concentration to 5 mM CaCl₂ and 10 mM MgCl_2; samples were digested with DNase I (0.15 U) for 1 min at RT and resolved on a sequencing gel. DNA sequencing reactions were done using the Sequenase Version 2.0 DNA Sequencing Kit (USB). 6.1.9 Chromatin immunoprecipitation and Western blot analysis Experiments were performed with the Chromatin Immunoprecipitation

(ChIP) assay kit (Upstate biotechnologies) following the manufacturer's instructions. Samples were immunoprecipitated with antibodies against clathrin heavy chain or against p53 (FL-393). PCR analysis of the isolated DNA fragments employed the following primer pairs: siah-lb Forward 5'- CCATTGTGTGCATCTTCCTGAGCCC-3'; Reverse 5'-

GAACTAACCTAGCACTAGTAATGAG-3', β2 microglobulin (β2m) Forward 5'-GCTCTGAAGATTCATTTGAACCTGC-3'; Reverse 5'- ATCCAAGTAATGAGAGTACAGAGG-3', p21 Forward 5'-

CCAGAGGATACCTTGCAAGGC-3'; Reverse . 5'-

TCTCTGTCTCCATTCATGCTCCTCC-3'. Specificity of the primers was checked by Blast analysis using the Ensembl mouse genomic database. PCR reactions with the different primers gave rise to a single specific product of the expected size. The linear range for each primer pair was determined empirically, using increasing amounts of LTR6 genomic DNA. PCR products were resolved on 2% agarose gels. Whole cell extracts were generated using standard conditions. Extracts containing 20 μg total protein were subjected to Western blot analysis using the FL-393 and B 5-1-2 antibodies. 6.2 Results 6.2.1 p53-dependent induction of siah-lb mRNA To confirm and extend previous studies showing that siah-lb is activated during p53 -induced apoptosis, we used the informations available in the NCBI mRNA database to analyze the sequences of siah-la and siah-lb, whose alignment is schematically shown in Fig. 1A. This analysis confirmed that the siah-1 coding sequence (CDS) probe used previously (23) recognizes both siah-la and siah-lb.

In Northern blot analysis this probe hybridizes to two transcripts, corresponding to sizes of 2.3 and 1.9 kb, respectively (Fig. IB), i order to study more specifically siah-lb transcription, we designed a probe in the 5' UTR (ace. n. Z19580) of its mRNA (nt 1/230) (Fig. 1A). the same manner we designed a probe specific for siah-la (ace. n. Z19579) in the 3'UTR of its mRNA (nt 1676/1934) (Fig. 1A). On mRNA from mouse cells the siah-la probe (probe a) recognizes the 2.3 kb band, while the siah-lb probe (probe b) recognizes only the 1.9 kb band (Fig. IB). These results disagree with a previous suggestion (40) that the band at 2.3 kb includes both siah-la and siah-lb transcripts. According to the published sequences of siah-lb mRNA (ace. n. Z19580 and ace. n. BC052887) the expected size of the siah-lb transcript is 1.7 kb. As our siah-lb specific probe recognizes a band of bigger size, we performed RACE PCR experiments to determine more precisely the 5' and 3' ends of siah-lb mRNA. The 3' RACE PCR confirmed the published sequences, but the preliminary 5' RACE analysis revealed a possible alternative splicing of the siah-lb first exon. The two different first exons described by Delia et al. (21) or sequenced by the I.M.A.G.E consortium (ace. n. BC052887) each represent 1/7 of the clones analyzed by us. The remaining 5/7 of the clones were found to contain a first exon of 231 bp, which so far has not been described (ace. n. AY495086). Transcripts containing this longer exon are expected to have a size of 1.9 kb, confirming the results of our Northern analysis. To confirm that siah-lb is activated in the process of p53 -induced apoptosis, we used the LTR6 clone derived from Ml mouse myeloid leukemia cells. LTR6 cells are stably transfected with a temperature-sensitive p53 mutant (vail 35) that gains wild type function upon shifting the temperature from 38 to 32°C, leading cells into programmed cell death (39). As expected, Northern blot analysis confirmed the transcriptional activation of mouse p21 and mouse cyclin Gl, both of which are well-established p53 target genes, upon shift of LTR6 cells to 32°C (Fig. 1C). Importantly, a parallel albeit more modest increase in siah-lb mRNA was also observed under the same conditions (Fig. 1C), consistent with the conclusion that siah-lb is ap53-inducible gene. Activated p53 can trigger different effector pathways, depending on cell type and mode of activation. To assess the generality of p53 -mediated siah-lb induction, we therefore subjected another cell line, NIH3T3, to treatment with the transcription inhibitor Actinomycin D, a well-documented activator of the p53 pathway (41, 42) . As seen in Fig. ID, this also resulted in a marked increase in siah-lb mRNA. 6.2.2 Identification of a putative p53 consensus site in the siah-lb gene To determine whether the siah-lb gene is a direct transcriptional target of p53, 20 kb of the region of murine chromosome X containing the siah-lb locus (nt -18621/+1379 relative to the initiator ATG) were analyzed using the Patch software (http://www.gene- regulation. com/c ibin/pub/pro grams/patch/bin/patch, cgi . A putative p53 consensus binding site was identified in the intronic region comprising nt -2155/- 2103, between exon 2 and exon 3. This DNA element consists of two identical half sites: 5'-AGACATGTCC-3'. The distance between the two half sites is unusual: while the consensus specifies a spacer of 0-13 bp (33), the two half sites found in siah-lb are 33 bp apart. Moreover, from the analysis of many confirmed p53 target sites, it seems that the majority of the physiologically relevant ones have no spacer at all (34, 38). It was therefore of particular interest to test the interaction of p53 with this putative binding site. 6.2.3 Functionality of the p53 responsive element in the siah-lb gene The region nt -2613/-1694 of the siah-lb gene, containing the putative p53RE located at nt -2155/-2103, was subcloned into the pGL3-enhancer vector and tested for p53-dependent transcriptional activity in a luciferase assay. The different constructs represented on the left panel of Fig. 2 were transiently transfected into p53-null H1299 lung adenocarcinoma cells with or without a plasmid driving the expression of the mouse p53 protein. Luciferase activity in the presence of p53 was normalized for the activity in the absence of p53, to obtain a fold of activation. While the empty luciferase vector (negative control) was not affected by the presence of p53, inclusion of siah-lb sequences led to an 8 fold increase in luciferase activity when p53 was present (Fig. 2). To verify that the positive effect of p53 on the transcriptional activity of the reporter plasmid is mediated by the consensus site identified within the siah-lb gene, we mutated nucleotides within this site that are known to be necessary for direct binding of p53. Mutation of CATG to TATA in the first half site (siah-lb mutl, Fig. 2) led to a two-fold reduction in the activating effect of p53, and a similar mutation in the second half site (siah-lb mut2) resulted in a 3 -fold reduction. Deletion of the entire putative p53RE (64 bp) (siah-lb Δ) abrogated almost completely the effect of p53. Similar results were obtained in mouse NIH3T3 cells transfected with the same set of reporter plasmids (Fig. 5 A, provided as supporting information on the PNAS web site). Moreover, the extent of activation was p53 dose-dependent (Fig. 5B, provided as supporting information on the PNAS web site). These observations imply that p53 -responsiveness of the reporter plasmid is indeed due to the presence of the putative intronic p53 binding site identified by us in the siah-lb gene. 6.2.4 p53 binds to the siah-lb promoter in vitro Electrophoretic mobility shift assays were employed to analyze the binding and affinity of p53 to the p53RE identified in the siah-lb gene. A 63 bp radiolabeled probe including this element was incubated with purified baculovirus-derived p53 protein, in presence of the p53 -specific monoclonal antibody PAb421, which stimulates the formation of p53-DNA complexes by imparting a conformational change in p53 (43, 44). As seen in Fig. 3 A, no lanes marked such incubation resulted in specific gel retardation (lane 3). A similar effect was seen when in vitro translated p53 was used in the reaction (Lane 3 of Fig. 6, provided as supporting information on the PNAS web site). The specificity of the binding was verified by competition with an excess of unlabeled DNA containing a p53 consensus site derived from the p21 gene (data not shown). In contrast, no shift was observed when the p53 consensus binding site was mutated (siah-lb mut3, Fig. 3A lanes 6 and 7). Comparison with the positions of molecular weight markers, run on the same gel, suggests that the supershifting complex has an approximate size of 850 kDa, corresponding to a p53 tetramer plus four antibody molecules and the probe (45). Thus, despite the non-conventional nature of the consensus sequence, the mode of binding of p53 appears to be similar to that occurring with more typical p53 binding sites. The use of short double stranded oligonucleotide probes for p53

' bandshift assays has been questioned recently (46). Therefore, we also performed DNAse I footprinting studies using a 410 bp labeled probe corresponding to nt - 2290/- 1880 of the siah-lb gene, centered around the p53RE. As shown in Fig. 3B, p53 protected the two half sites from DNase I cleavage but did not protect the spacer DNA between the two half sites: the latter may be due to the relatively long distance between the two half sites. A similar footprint was obtained in the absence of antibody (data not shown). Remarkably, mutation of either of the two half sites from CATG to TATA abrogated p53 -mediated protection of that particular half site, while not affecting the protection of the other one, whose sequence remained wild type (Fig. 3B, no lanes marked lanes 4, 5 and 6 for siah- lb mutl and lanes 7, 8 and 9 for siah-lb mut2). These results demonstrate that p53 can bind to either one or both of the consensus half sites found in the siah-lb gene. In conclusion, EMSA and DNase I footprinting demonstrate that p53 is able to associate in vitro with the p53 consensus located at nt -2155/-2112 of the siah- lb gene. 6.2.5 p53 associates with siah-lb chromatin in vivo To determine if p53 could bind the intronic siah-lb p53RE in vivo, we perfonned chromatin immunoprecipitation (ChIP) experiments. The LTR6 cell system was used for this purpose, under the same conditions where endogenous siah-lb transcription is efficiently activated by p53 (see Fig. 1C). Ml and LTR6 cells were maintained at 38°C or 32°C for 6 hr and treated with formaldehyde to generate covalently cross-linked DNA-protein complexes within the cells. Cross- linked chromatin was then immunoprecipitated with antibodies against clathrin heavy chain for negative control (ip ctrl) or against p53 (ip p53). PCR amplification was then performed on the immunoprecipitated DNA as well as on total input DNA after fragmentation (input). Primers specific for siah-lb, corresponding to positions -2208/-2184 (forward) and -1891/-1967 (reverse) were designed in the genomic sequence containing the p53RE, thus avoiding cross- reactivity with the other siah homologues. As seen in Fig. 4 A, siah-lb and p21 chromatin were specifically immunoprecipitated with anti-p53 antibodies only from LTR6 cells maintained at 32°C; hence, like the corresponding elements in the p21 gene promoter, the p53RE within the siah-lb intron is indeed occupied by wild type p53 within living cells. In order to rule out the possibility that the in vivo binding of p53 to the siah-lb genomic DNA was due to a p53 overexpression artefact, we induced activation of endogenous p53 in NIH3T3 cells by treatment with Actinomycin D, which led to a substantial increase in the steady state levels of p53 (Fig. 4B). When such Actinomycin D (ActD) treated NIH3T3 cells were used as the starting material for ChIP analysis, clear association was observed between p53 and siah- lb chromatin, as well as with p21 chromatin (Fig. 4C). Interestingly, p53 binding to the siah-lb chromatin remained maximal after 24 hr of ActD treatment, whereas binding to the p21 promoter region decreased between 16 and 24 hours of treatment. Thus, both transfected and endogenous ρ53 are found specifically associated with the siah-lb gene in vivo, further confirming that this is a bonafide direct p53 target gene.

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Claims

CLAIMSWhat is claimed is:

1. A DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah- 1 b .

2. A method of identifying a compound that reduces or inhibits cell apoptosis comprising:

(a) contacting the compound with a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb and p53;

(b) determining whether binding between the DNA fragment and p53 is decreased; and if the binding between the DNA fragment and p53 is decreased, then,

(c) contacting the compound with a cell; and (d) determining whether cell apoptosis is reduced as compared to a cell that has not been contacted with the compound.

3. A method of treating, preventing or managing a neurodegenerative disorder comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of the compound identified using the method of claim 2.

4. The method of claim 3, wherein the neurodegenerative disorder is Alzheimer's disease, Huntington's disease or Parkinson's disease.

5. A method of identifying a compound that promotes cell apoptosis comprising:

(a) contacting the compound with a DNA fragment comprising 2155 to 2103 nucleotides downstream from translational start site of siah-lb and p53; (b) determining whether binding between the DNA fragment and p53 is decreased; and if the binding between the DNA fragment and p53 is decreased, then,

(c) contacting the compound with a cell; and (d) determining whether cell apoptosis is increased as compared to a cell that has not been contacted with the compound.

6. A method of treating, preventing or managing cancer comprising administering to a patient in need of such treatment, prevention or management a therapeutically or prophylactically effective amount of the compound identified using the method of claim 5.