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WO2002064790A2 - Proteine jfy induisant l'apoptose rapide - Google Patents

Proteine jfy induisant l'apoptose rapide Download PDF

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Publication number
WO2002064790A2
WO2002064790A2 PCT/US2001/047455 US0147455W WO02064790A2 WO 2002064790 A2 WO2002064790 A2 WO 2002064790A2 US 0147455 W US0147455 W US 0147455W WO 02064790 A2 WO02064790 A2 WO 02064790A2
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jfyl
seq
expression
isolated
purified
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PCT/US2001/047455
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WO2002064790A3 (fr
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Jian Yu
Kenneth W. Kinzler
Bert Vogelstein
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The Johns Hopkins University
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Priority to CA002432111A priority Critical patent/CA2432111A1/fr
Priority to EP01270126A priority patent/EP1343884A2/fr
Priority to JP2002565103A priority patent/JP2004530422A/ja
Priority to US10/450,436 priority patent/US20040077832A1/en
Publication of WO2002064790A2 publication Critical patent/WO2002064790A2/fr
Publication of WO2002064790A3 publication Critical patent/WO2002064790A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/20—Screening for compounds of potential therapeutic value cell-free systems

Definitions

  • JFY1 Protein induces rapid apoptosis
  • Inactivation of the growth-controlling functions of p53 appears to be critical to the genesis of most human cancers (Hollstein et al., 1999; Hussain and Harris, 1999).
  • the p53 protein controls rumor growth by inhibiting cell cycle progression and by stimulating apoptosis (Lane, 1999; Levine, 1997; Oren, 1999; Prives and Hall, 1999). It has been shown that the inhibition of cell cycle progression by p53 is in large part due to its ability to transcriptionally activate genes that directly control cyclin- dependent kinase activity (reviewed in (El-Deiry, 1998)).
  • p53 induces p2 j CiPi/WAFi ⁇ wn
  • the apoptosis stimulated by p53 involves disruption of mitochondrial membrane potential, accumulation of reactive oxygen species, stimulation of caspase 9 activity and subsequent activation of a caspase cascade (Li et al., 1999; Polyak et al., 1997; Schuler et al, 2000; Soengas et al., 1999).
  • an isolated and purified JFYl protein having the sequence shown in SEQ ID NO: 1 or 2 is provided.
  • an isolated and purified JFYl polynucleotide comprises a coding sequence having the sequence shown in SEQ NO: 3 or 4.
  • an isolated and purified JFYl BS1 or BS2 nucleic acid is provided. It has the sequence shown in SEQ ID NO: 5, 6, or ' 27.
  • a method of inducing apoptosis in cancer cells is provided.
  • a nucleic acid comprising a JFYl coding sequence is supplied to cancer cells. JFYl is thereby expressed and induces apoptosis in said cancer cells.
  • a method of screening drugs for those which can induce apoptosis is provided.
  • a test compound is contacted with a cell comprising a mutant p53 and no wild-type p53. JFYl expression is detected in the cell.
  • a test compound which increases JFYl expression is a candidate drug for treating cancer.
  • a method of screening drugs for those which can induce apoptosis is provided.
  • a test compound is contacted with a cell comprising a mutant p53 and a JFYl-BS2-reporter gene construct.
  • the cell comprises no wild-type p53. Reporter gene expression is detected.
  • a test compound which increases reporter gene expression is a candidate drug for treating cancer.
  • a method for diagnosing cancer cells is provided.
  • An expression product of JFYl is assayed in a biological sample suspected of being neoplastic.
  • the amount of the expression product in the biological sample is compared to the amount of the expression product in a control sample which is not neoplastic.
  • the biological sample is identified as neoplastic if the amount of the expression product in the biological sample is significantly less than the amount in the control sample.
  • a method to aid in determining prognosis of a cancer patient is provided.
  • An expression product of JFYl is assayed in a tumor sample.
  • the amount of the expression product in the tumor sample is compared to amount of the expression product in a control sample which is not neoplastic.
  • the biological sample is identified as having a negative prognostic indicator if the amount of the expression product in the tumor sample is significantly less than the amount in the control sample.
  • the present invention provides the art with a new gene and protein which are important in mediating p53 induced apoptosis in cancer cells.
  • FIG. 1A to 1C Induction of JFYl by ⁇ 53 in CRC cells.
  • Figure 1A Northern blot analyses of RNA samples prepared from p53 -inducible DLD1 cells at the indicated time points are shown. The JFYl gene was induced as early as 3 hours after doxycycline removal, similar to that of ⁇ 21, while the B AX and Noxa genes were not induced as robustly. p53AIPl transcripts were not detectable under these conditions. A GAPDH probe was used as a loading control.
  • RNA from the indicated colorectal cancer cells lines infected with adenovirus expressing wt p53 (W) and mutant ⁇ 53R75H (M) for 17 hours were analyzed by Northern blotting.
  • Figure 1C RNA from the indicated colorectal cancer cells lines treated with adriamycin (Adr) or 5-Fluorouracil (5-FU) for 24 hours was analyzed by Northern blotting. RNA from untreated cells ("Un”) was used as a control.
  • FIG. 2 A to 2B The JFYl protein contains a BH3 domain.
  • Figure 2 A Alignment of the predicted amino acids of human (SEQ ID NO: 1) and mouse (SEQ ID NO:2) JFYl reveals 90% identity. The identical residues are colored blue and non-conserved residues are colored red. The residues comprising AA128 -165 were predicated to form an ⁇ -helix by the Chou-Fasman method. The middle third of the ⁇ -helix corresponding to the BH3 (AA141-149) domain is completely identical in both human and mouse JFYl.
  • Figure 2B Alignment of BH3 domains of JFYl with other Bcl-2 family members. (SEQ ID NO.7-17) conserveed residues (contained in more than three members of the eleven shown) are colored blue, whereas the non- conserved residues are colored red.
  • FIG. 3A to 3D p53 activates the JFYl promoter
  • Figure 3A The two potential p53 binding sites (BSl and BS2; SEQ ID NOs: 5 and 6) within 300 bp of the putative transcription start site are indicated.
  • the predicted open reading frame (ORF) starts at the indicated ATG. Fragl and Frag2 were used in reporter constructs.
  • Figure 3B The indicated fragments were cloned into pBVLuc and cotransfected into HI 299 cells together with a wt (wt) or mutant (R175H) p53 expression construct (Baker et al., 1990). The ratio of luciferase activity in the presence of wt p53 compared to that in the presence of mutant p53 is plotted on the ordinate. All experiments were performed in triplicate with a ⁇ -galactosidase reporter included in the transfection mix for normalization, with means and one standard deviation indicated by the bars and brackets, respectively.
  • FIG. 4A to 4C JFYl encodes a mitochondrial protein that interacts with Bcl-2 and BCI-XL.
  • PTK and P C MV refer to the Herpes Virus thymidine kinase promoter and CMV promoter, respectively.
  • Hyg hygromycin-B-phosphotransferase gene, conferring resistance to Hygromycin B.
  • TRE tetracycline responsive elements
  • tTA Tet activator
  • Pm CMv minimal CMV promoter. This system is activated by removal of Doxycycline (Dox).
  • FIG. 4B HA-tagged JFY 1 constructs were transfected into 911 cells and visualized by indirect immunofluorescence (green). Mit ⁇ Tracker Red dye was used to visualize mitochondria. JFY1- ⁇ BH3 encodes a tagged JFYl protein with a 15 amino acid deletion and is therefore missing the BH3 domain.
  • Figure 4C Different pairs of expression constructs were transfected into 911 cells and total lysates were immunoprecipitated with a rabbit anti-HA antibody, then analyzed by western blotting with the indicated antibodies. The lanes labeled "total lysate" contain -25% of the amount of lysate represented in the lanes containing immunoprecipitates.
  • JFYl potently suppresses the growth of human tumor cells.
  • the indicated cell lines were transfected with constructs encoding JFYl, JFY1- ⁇ BH3, or the empty vector.
  • Cells were harvested 24 hours after transfection and equal cell numbers serially diluted inT25 flasks and grown under selection in hygromycin B for 17 days. Only the highest density flasks are shown. There was no observable difference in colony formation between transfection with JFY1- ⁇ BH3 and that with the empty vector, while the number of colonies obtained after transfection with the JFYl expression vector was reduced by more than 1000-fold.
  • FIG. 6A An expression vector containing separate cassettes for GFP and JFYl (see Fig. 4A) was used to establish inducible clones of DLD1 cells. Representative results are shown for cells that were maintained in the uninduced state (Off) or after induction by removal of doxycycline from the medium for 12 hours (On). The same fields are shown in the first two columns as viewed under phase contrast (Phase) or fluorescence microscopy (GFP) for the clones that inducibly expresses both GFP and JFYl (JFYl) or GFP alone (Vector).
  • Phase contrast Phase contrast
  • GFP fluorescence microscopy
  • the third column shows nuclei of the same cell cultures harvested immediately after microscopy and stained with Hoechst 33528. Apoptotic cells stained with this dye have characteristic condensed chromatin and fragmented nuclei. Virtually all JFYl -induced cells were apoptotic by 12 hours.
  • Figure 6B The indicated clones were grown in the presence (Off) or absence (On) of doxycycline for 10 days, then stained with crystal violet. Two different flasks, containing either two million or two thousand cells at the start of the experiment, are shown to illustrate the profound effect of JFYl induction.
  • Figure 6C DLD1 cells inducibly expressing JFYl were harvested at the indicated times following doxycycline withdrawal.
  • Polynucleotides provided by the present invention include those which are very closely related to SEQ ID NO:3 or 4, including any which encode the same amino acid sequence as shown in SEQ ID NO: 1 or 2. Also included are those which are polymorphic variants of JFYl as shown, as well as those which are naturally occurring JFYl mutants and species homologues. Polynucleotide variants typically contain 1, 2, or 3 base pair substitutions, deletions or insertions. Polymorphic protein variants typically contain 1 amino acid substitution, typically a conservative substitution.
  • polynucleotides are considered homologues if they achieve at least 90 % identity. Preferably they are at least 91 %, 93 %, 95 %, 97 %, or even 99 % identical. Percent identity between a putative JFYl polypeptide variant or mutant or homologue can be determined using the Blast2 alignment program. Default settings can be used in comparing the putative sequence to the amino acid sequence of SEQ ID NO: 1 or 2 .
  • Polynucleotides preferably comprise at least 730 nucleotides in length of JFYl coding sequence or at least 1640 nucleotides of total JFYl transcript or genomic sequence. Any naturally occurring variants of the JFYl sequence that may occur in human tissues and which has apoptosis inducing activity are within the scope of this invention. Thus, reference herein to either the nucleotide or amino acid sequence of JFYl includes reference to naturally occurring variants of these sequences. Nonnaturally occurring variants which differ by as much as four amino acids and retain biological function are also included here. Preferably the changes are conservative amino acid changes, i.e., changes of similarly charged or uncharged amino acids.
  • JFYl minor amino acid variations from the natural amino acid sequence of JFYl are contemplated as being encompassed by the term JFYl ; in particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains.
  • Polynucleotide sequences according to the present invention can be isolated away from other sequences to which they are naturally adjacent in chromosome 19q. Thus they can be isolated away from all or some other human 19q sequences. In a particularly preferred embodiment they are isolated away from all other 19q sequences.
  • the polynucleotides can include a vector for replicating and/or expressing the coding sequence of JFYl .
  • the vector may contain a regulatory sequence which permits control, for example by an inducer or repressor, of expression of JFYl sequences.
  • the vectors are formed by recombinant in vitro techniques. Vectors can be replicated and maintained in suitable host cells as are known in the art. Pure cultures of the host cells are preferred.
  • Suitable regulatory sequences are known in the art and any such sequence can be used without limitation.
  • the polynucleotide can be joined to another coding sequence, for example, one which encodes an easily assayable epitope or enzyme activity.
  • Such polynucleotides will produce fusion proteins having the properties of both JFYl and the fusion partner. Fusion proteins can contain all or a part of JFYl and all or a part of a second protein.
  • Polynucleotides according to the invention also can be used as primers or probes. Such polynucleotides can be at least 15, 18, 10, or 25 nucleotides in length. They can be double or single stranded. Preferably for use they will be single stranded or denatured. Probes and primers can be labeled using, for example, radiolabels, fluorescent moieties, restriction endonuclease sites, specific hybridization sequences, etc. These can be synthesized according to any technique known in the art for making oligonucleotides. Primer pairs are typically used in tandem and can be packaged together. In one particular embodiment, the primers and/or probes are used to monitor expression of JFYl as discussed below. Primer pairs of the invention employ at least one primer which is substantially complementary to nucleotides 1-235 of SEQ ID NO: 1 or its complement. Substantial complementarity means that the primer will hybridize and initiate template-based extension during amplification.
  • Polypeptides containing at least 9, 10, 12, 14, 16, or 18 contiguous amino acids of SEQ ID NO: 1 or 2 can be used inter alia to make antibodies. Such polypeptides can be used alone or conjugated or fused to other proteins as immunogens to induce specific binding antibodies to JFYl in an inoculated animal, such as a mouse, rabbit or goat. Thus polyclonal or monoclonal preparations of JFYl -specific binding antibodies are also provided. Methods for making and screening for such antibodies are well known in the art and can be used by the skilled artisan without recourse to undue experimentation.
  • the control sequences permit binding of p53 which upregulates JFYl expression.
  • Two such binding sequences were located although one appears to be more active than the other. Either or both of these can be used for coordinately expressing a reporter or other gene sequence with JFYl.
  • the binding sequences can be used with the endogenous coding sequence or with other sequences to exert p53 control.
  • Suitable reporter genes are known in the art, and any can be used including but not limited to Green Fluorescent Protein, ⁇ galactosidase, and alkaline phosphatase.
  • the binding sequence can be used singly, or in tandem arrays.
  • a polynucleotide may comprise at least two or at least four copies of the binding sequence. Isolated and purified polynucleotides containing the binding sequences are purified away from other genetic sequences located on chromosome 19q.
  • JFYl or polynucleotides encoding JFYl can be used to treat cancers or other diseases characterized by unwanted cellular proliferation.
  • the polynucleotide can be administered directly to the tumor or to the body cavity containing the rumor.
  • the polynucleotide can be administered in a virus or in a viral vector.
  • the polynucleotide can be administered in a liposome or other gene delivery particle or formulation. In some situations, the polynucleotide can be delivered by particle bombardment.
  • Those of skill in the art will recognize and be able to match the appropriate delivery method and vehicle for the particular type of tumor or other disease.
  • JFYl expression can be monitored, either directly or using a reporter gene under the control of a BSl (SEQ ID NO:5) and/or BS2 (SEQ ID NO:6 or 27) sequence.
  • a compound or composition which is able to increase JFYl expression (or surrogate reporter expression) is identified as a candidate for treating cancer or other disease involving cellular proliferation.
  • Monitoring expression can be done by any means known in the art, including measuring a particular protein immunologically or by activity, or by measuring a particular mRNA species. Techniques for measuring expression are well known in the art and any can be used as is convenient. Similar screening techniques can be set up for cell-free systems in which JFYl expression is monitored, either directly or by surrogate.
  • p53 can be used diagnostically and prognostically for detection and prediction of cancer disease severity, so can JFYl .
  • a biological sample can be assayed for the amount of an expression product of JFYl .
  • a significantly lower amount in the biological sample than in a control sample identifies a neoplastic sample.
  • Control samples can be obtained from the same individual as the biological sample or it can be obtained from a normal healthy individual. Preferably the control sample will be obtained from the same tissue type as the test sample. If a bonafide tumor sample is tested for expression of JFYl then a prognosis can be determined. Lower or absent amounts of JFYl expression products are a negative prognostic indicator, as is lowered expression of p53 in cancer cells.
  • CRC cell line DLD1 undergoes apoptosis -18 hours following expression of exogenous p53 under the control of a doxycycline-regulated promoter. Moreover, these cells are committed to apoptosis after only 9 hours of p53 exposure, as addition of doxycycline after this period does not diminish apoptosis (Yu et al., 1999).
  • the candidate genes should exhibit biochemical and physiologic properties that suggest they can directly stimulate apoptosis through a mitochondrial pathway.
  • DLD1 cells inducibly expressing p53 were studied using the Serial Analysis of Gene Expression (SAGE) technique (Velculescu et al., 1995; Yu et al., 1999).
  • SAGE Serial Analysis of Gene Expression
  • JFYl The JFYl gene was discovered through a SAGE tag that matched to ESTs (Expressed Sequence Tags) but to no known genes.
  • the SAGE data indicated that JFYl was induced over ten-fold in DLD1 cells following p53 expression for 9 hours.
  • Northern blotting showed that JFYl was induced as soon as 3 hours following doxycycline withdrawal, just as was p21 apl/W ⁇ Fl (Fig. 1 A).
  • JFYl expression was maximal by 6 hours, well before the 9-hour "commitment point" for apoptosis determined previously (Yu et al., 1999).
  • JFYl mRNA expression was found to be induced in HCT116 and SW48 cells following treatment with 5-FU (5-fluorouracil), the mainstay of treatment for CRC, as well as by the DNA-damaging agent adriamycin (Fig. 1C).
  • HCT116 and SW48 cells contain wt p53 genes, and the results in Fig. 1C demonstrate that endogenous levels of ⁇ 53 were sufficient to induce JFYl.
  • the apoptosis following 5- FU treatment is totally dependent on intact ⁇ 53 (Bunz et al., 1999).
  • HCT116 cells in which the p53 genes had been disrupted by targeted homologous recombination (Bunz et al., 1998), we found that the transcriptional induction of JFYl by 5-FU was also entirely dependent on p53 (Fig. 1C).
  • JFYl which is only related to the Bcl-2 family through its BH3 domain, may affect pore formation when complexed with other Bcl-2 family members or with other mitochondrial proteins. Expression of high levels of JFYl is sufficient for apoptosis, but it is not known whether expression of this gene is necessary for apoptosis. Additionally, JFYl was expressed, albeit at very low levels, in all normal human tissues analyzed. Targeted deletions of JFYl in human somatic and mouse ES cells, facilitated by the sequence data provided in Fig. 2, should provide answers to these questions in the future. Finally, the fact that JFYl expression led to a very rapid and profound apoptosis suggests that it should be considered as a substitute for p53 in cancer gene therapy.
  • the mouse homolog of JFYl was identified through searches of mouse EST and genomic databases.
  • the deduced murine gene contains four exons corresponding to the four coding exons of the human homolog, and the corresponding coding exons were of identical length in the two species.
  • the human and murine genes were 91% and 90% identical at the amino acid and nucleotide levels, respectively (Fig. 2A).
  • Example 2 JFYl encodes a BH3 domain-containing mitochondrial protein that interacts with Bcl-2 and Bcl-X L
  • BH3 domains are one of the four Bcl-2 homology domains present in Bcl- 2 family of proteins (Chittenden et al., 1995). Several of the pro-apoptotic members of this family contain the BH3 domain but not the BH1, 2, or 4 domains and reside at least partially in mitochondria (reviewed in (Korsmeyer, 1999; Reed, 1997)).
  • BH3 domains are essential for their pro-apoptotic activities and for their ability to heterodimerize with other Bcl-2 family members (Wang et al., 1998; Wang et al., 1996; Zha et al., 1997).
  • a GenBank entry (Accession U82987) corresponding to a partial JFYl cDNA sequence carried the intriguing annotation of "Human Bcl-2 binding component 3". The basis for this annotation was not specified and the amino acid sequence included with this entry was out of frame with respect to the major protein we predicted to be encoded by the JFYl gene.
  • Fig. 4A To determine the subcellular localization of human JFYl, we constructed an expression vector encoding the full length JFYl protein with an amino-terminal hemaglutanin (HA) tag (Fig. 4A). This vector was expressed in 911 cells, which have a flat morphology that facilitates subcellular localization studies. Indirect immunofluorescence with an anti-HA antibody showed punctate perinuclear staining in all transfected cells (Fig. 4B). Comparison of this localization with that of a dye that labeled mitochondrial membranes (MitoTracker Red) indicated complete co- localization (Fig. 4A).
  • a dye that labeled mitochondrial membranes Mitsubishi Red
  • the BH3 domain was not required for this localization, as the protein generated from another JFYl expression vector, JFY1- ⁇ BH3, (identical except for the deletion of the BH3 domain), was also found exclusively in mitochondria (Fig. 4B). This lack of dependence on BH3 for mitochondrial localization is consistent with data on other BH3 -containing proteins, though it distinguished JFYl from Noxa, in which the BH3 domain was required (Oda et al., 2000a).
  • the human colorectal cancer cell lines DLD-1, HCTl 16, SW48, SW480 and the human lung cancer cell line H1299 were obtained from ATCC.
  • HCT 116 cells with a targeted deletion of ⁇ 53 has been previously described (Bunz et al., 1998). All lines were maintained in McCoy's 5 A media (Life Technologies) supplemented with 10% fetal bovine serum (HyClone), 100 units/ml of penicillin and 100 ug/ml of streptomycin at 37°C.
  • the retinal epithelial cell line 911 was kindly provided by A. J. Van der Eb of the University of Leiden and maintained as described (Fallaux et al., 1996).
  • Chemotherapeutic agents were used at concentrations of 0.2 ug ml (adriamycin) and 50 ug/ml (5-FU) and cells were treated for 24 hours. Transfections were performed with Fugene M 6 (Boehringer Mannheim) according to the manufacturer's instructions.
  • JFYl expression plasmids The HA-tagged, full length JFYl expression vector pHAHA-JFYl was constructed by cloning RT-PCR products into the pCEP4 vector (Invitrogen). Variants of this vector containing JFYl with the BH3 domain deleted, or the alternatively spliced form of JFYl, were constructed similarly. Sequences for the primers and details of vector construction are available from authors upon request. In all cases, inserts of multiple individual clones were completely sequenced and the ones that were free of mutation were subsequently used for experiments. The Bcl-2 expression vector was described previously (Pietenpol et al., 1994) and the V5- tagged Bel -X expression vector was purchased from Invitrogen.
  • Promoter-containing fragments were amplified from human genomic DNA of HCTl 16 cells and cloned into the pBVLuc luciferase reporter vector containing a minimal promoter (He et al., 1998). To test presumptive p53-binding sites, the following oligo pairs containing two copies of wildtype or mutant binding sites were used: 5'- CTAGGCTCCTTGCCTTGGGCTAGGCCACACTCTCCTTGCCTTGGGCTAGGC
  • oligonucleotide pairs were concatamerized and cloned into the Nhe I site of pBVLuc.
  • Transfections of 911 cells were performed in 12-well plates using 0.2 ug luciferase reporter plasmid, 0.2 ug pCMN ⁇ and 0.8 ug pCEP4 encoding either wt p53 or mutant p53R175H.
  • the ⁇ -galactosidase reporter pCMN ⁇ Promega was included to control for transfection efficiency. Luciferase and ⁇ -galactosidase activities were assessed 24-48 hours following transfection with reagents from Promega and IC ⁇ Pharmaceuticals, respectively.
  • DLD1-TET cells are DLD1 derivatives containing a constitutively expressed tet activator (Gossen and Bujard, 1992; Yu et al., 1999).
  • Immunoprecipitation was performed essentially as described (Chan et al., 1999) with the following modifications. 911 cells were seeded in T75 flasks 18 hours prior to transfection with 5 ug of each of two expression constructs (10 ug total) and harvested 20 hours after transfection. The cell suspension was sonicated for 15 seconds in a total volume of 1 ml and incubated with 30 ul protein A:protein G beads (4:1, Boehringer Mannheim) for one hour at 4°C. The supernatants collected after centrifugation ("total lysates") were subsequently used for immunoprecipitation with rabbit antibody against HA (sc-805, Santa Cruz).
  • 911 cells were seeded on glass chamber slides ( ⁇ alge ⁇ unc, Lab-Tek 177372) and transfected with JFYl expression constructs. Twenty hours later, Mito Tracker Red (0.5 uM, Molecular Probes) was added to the medium and the cells were incubated at 37°C for an additional 20 minutes. Cells were fixed with 4% paraformaldehyde in PBS, permeablized with cold acetone and blocked with 100% goat serum for 1 hour at room temperature. After three washes in PBST (PBS with 0.05% Tween-20), slides were incubated with anti-HA antibody (12CA, Boehringer Mannheim) diluted 1 :200 with 50% goat serum in PBST at 4°C overnight.
  • Mito Tracker Red 0.5 uM, Molecular Probes
  • the medium was replaced with fresh growth media with or without doxycycline and cells were allowed to grow for 10 days, and then stained with crystal violet.
  • crystal violet To determine the fraction of apoptotic cells, all cells (attached and floating) were collected and stained with Hoechst 33258 as described (Waldman et al., 1996). Cells with characteristic condensed chromatin and fragmented nuclei were scored as apoptotic.
  • the Bcl-2 protein family arbiters of cell survival, Science 257, 1322-1326.
  • Cipl is a potent inhibitor of Gl cyclin-dependent kinases, Cell 75, 805-16.
  • Noxa a BH3-only member of the Bcl-2 family and candidate mediator of ⁇ 53-induced apoptosis, Science 255, 1053-8.
  • ⁇ 53AIPl a potential mediator of p53 -dependent apoptosis, and its regulation by Ser-46-phosphorylated p53, Cell 102, 849-62.
  • Drosophila p53 is a structural and functional homolog of the tumor suppressor p53, Cell 707, 91- 101.
  • Bcl-2 proteins regulators of apoptosis or of mitochondrial homeostasis?, Nat Cell Biol 7, E209-E216.
  • BID a novel BH3 domain-only death agonist, Genes Dev 10, 2859-69.
  • p21 is a universal inhibitor of cyclin kinases, Nature 366, 701-704.

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Abstract

Selon l'invention, la caractérisation globale des gènes exprimés immédiatement après l'expression de p53 a permis d'identifier un gène appelé JFY1. La protéine codée par JFY1 s'est avérée être exclusivement mitochondriale et se lier à Bcl-2 et Bcl-XL par le biais d'un domaine BH3. L'expression exogène de JFY1 a résulté en une apoptose extrêmement rapide et profonde survenant bien avant celle résultant de l'expression exogène de p53. Sur la base de ses seuls motifs d'expression, de la dépendance à p-53 et des propriétés biochimiques, JFY1 est probablement un médiateur direct de l'apoptose associée à p53.
PCT/US2001/047455 2000-12-19 2001-12-12 Proteine jfy induisant l'apoptose rapide WO2002064790A2 (fr)

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CA002432111A CA2432111A1 (fr) 2000-12-19 2001-12-12 Proteine jfy induisant l'apoptose rapide
EP01270126A EP1343884A2 (fr) 2000-12-19 2001-12-12 Proteine jfy induisant l'apoptose rapide
JP2002565103A JP2004530422A (ja) 2000-12-19 2001-12-12 急速なアポトーシスを誘導するｊｆｙ１蛋白質
US10/450,436 US20040077832A1 (en) 2001-12-12 2001-12-12 Jfy1protein induces rapid apoptosis

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US25632800P 2000-12-19 2000-12-19
US60/256,328 2000-12-19

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EP1343884A2 (fr) 2003-09-17
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CA2432111A1 (fr) 2002-08-22

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