Patent Application
20070225240
The invention relates to an screening assay for the identification of agents which modulate the activity of polypeptides which affect the apoptotic activity of the tumour suppressor protein p53 and including gene therapy vectors comprising p53.
Tumour suppressor genes encode proteins which function to inhibit cell growth or division and are therefore important with respect to maintaining proliferation, growth and differentiation of normal cells. Mutations in tumour suppressor genes result in abnormal cell-cycle progression whereby the normal cell-cycle check points which arrest the cell-cycle, when, for example, DNA is damaged, are ignored and damaged cells divide uncontrollably. The products of tumour suppressor genes function in all parts of the cell (e.g. cell surface, cytoplasm, nucleus) to prevent the passage of damaged cells through the cell-cycle (i.e. G1, S, G2, M and cytokinesis). Arguably the tumour suppressor gene which has been the subject of the most intense research is p53. p53 encodes a protein which functions as a transcription factor and is a key regulator of the cell division cycle. It was discovered as a protein shown to bind with affinity to the SV40 large T antigen. The p53 gene encodes a 393 amino acid polypeptide with a molecular weight of 53 kDa.
We have described a family of proteins in WO02/12325 which function to enhance the apoptotic activity of p53. ASPP1 and ASPP2 selectively interact with p53 to enhance the apoptotic function of p53 at p53 responsive promoters to promote apoptosis in vivo. We herein describe the interaction of ASPP family members with the oncogene Ras. ASPP 1 and 2 are also phophoproteins.
Ras oncogenes are frequently activated by mutation or over expression in many human tumours. For example, approximately 95% of pancreatic tumours contain so called K-Ras mutations. Ras oncogenes are believed to exert their effect by over-riding the normal cell-cycle control mechanisms by activating protein kinases (e.g. Raf, Mek, Erk kinase pathways) which regulate the function of cell-cycle cyclins which promote the proliferation of eukaryotic cells.
In its inactive state Ras is bound to GDP. The activation of Ras by growth factors results in exchange of GDP for GTP and a consequent change in the conformation of Ras to an activated form. In vitro, Ras has an intrinsic GTPase activity which becomes active when growth factor stimulation is removed and returns Ras to its GDP bound state. Ras is also a post-translationally modified protein and it is this modification which facilitates the localisation of Ras to the cell membrane and allows Ras to receive growth factor signals. The post-translational modification is farnesylation which results in the alkylation of cysteine residues in a conserved motif “CAAX”. Ras has three CAAX motifs located in the C-terminus of the protein and it has been shown that inhibition of the farnesylation reaction of Ras blocks its processing and thereby inactivates the protein. The farnesylation reaction has been a target for the rational design of agents which inhibit the reaction thereby preventing the localisation of Ras at its site of action, the inner cell membrane. However, the farnesylation reaction is more complicated that was first seemed. For example, H Ras is exclusively modified by farnsyltransferase whereas K-Ras and N-Ras can also be modified by gerangylgerangyltransferase. This has meant that there is a continuing need to identify new targets which can modulate Ras activity or oncogenic Ras activity, either directly or indirectly,
We have identified ASPP 1/2 as Ras binding targets. The binding domain in ASPP1/2 is the amino terminus of the protein. We also show that Ras activates ASPP through the MAPK and Raf CX pathway and that dephosphorylation by phosphatase action is an important factor regulating ASPP action. The interaction of these protein factors with ASPP provides an opportunity to identify agents which enhance or inhibit the action of ASPP on p53 mediated apoptosis.
According to an aspect of the invention there is provided method for the identification of agents which modulate the interaction of the proto-oncogene/oncogene Ras with the p53 binding protein family ASPP, either directly or indirectly.
According to an aspect of the invention there is provided a screening method for the identification of agents which modulate the interaction of a first polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
In a preferred method of the invention said first polypeptide is represented by the amino acid sequence as shown in
A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations which may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and asparatic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan.
In addition, the invention features polypeptide sequences having at least 75% identity with the polypeptide sequence as hereindisclosed, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99% identity with the amino acid sequence illustrated herein.
In a further preferred method of the invention said first polypeptide comprises the amino acid sequence +1 to +120 of the sequence shown in
In a further preferred method of the invention said second polypeptide is represented by the amino acid sequence shown in
In a preferred method of the invention said second polypeptide is modified at amino acid residue 12. Preferably said modification is the substitution of amino acid 12 for amino acid valine. Preferably said second polypeptide is K-RasV12. Alternatively said polypeptide is H-RasV12.
In a yet further preferred method of the invention said second polypeptide is modified at amino acid residue 17. Preferably said modification is substitution of serine for asparagine at amino acid residue 17.
In a further preferred method of the invention said first and second polypeptides are expressed by a cell.
In a preferred method of the invention said cell is a cell transfected with at least one nucleic acid molecule(s) which encodes said first and/or second polypeptides.
Preferably the expression of said nucleic acid molecule(s) is regulatable.
In a preferred method of the invention said cell is a cancer cell.
In a yet further preferred method of the invention said cell is part of a transgenic animal wherein the genome of said animal has been modified to include nucleic acid molecules which encode first and second polypeptides. Preferably said nucleic acid molecules are expressed in a specific cell/tissue.
In a yet still further preferred method of the invention said preparation includes at least one chemotherapeutic agent.
According to an aspect of the invention there is provided a screening method for the identification of agents which modulate the phosphorylation of a first polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
According to a further aspect of the invention there is provided a screening method for the identification of agents which modulate the phosphorylation state of a first polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
In a preferred method of the invention said agent is a polypeptide.
In a preferred method of the invention said polypeptide is an antibody, or active binding fragment thereof.
Preferably said antibody or binding fragment is a monoclonal antibody.
Antibodies or immunoglobulins (Ig) are a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) (low molecular weight) chain (κ or λ), and one pair of heavy (H) chains (γ, α, μ, δ and ε), all four linked together by disulphide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are non-variable or constant. The L chains consist of two domains. The carboxy-terminal domain is essentially identical among L chains of a given type and is referred to as the “constant” (C) region. The amino terminal domain varies from L chain to L chain and contributes to the binding site of the antibody. Because of its variability, it is referred to as the “variable” (V) region. The variable region contains complementarity determining regions or CDR's which form an antigen binding pocket. The binding pockets comprise H and L variable regions which contribute to antigen recognition. It is possible to create single variable regions, so called single chain antibody variable region fragments (scFv's). If a hybridoma exists for a specific monoclonal antibody it is well within the knowledge of the skilled person to isolate scFv's from mRNA extracted from said hybridoma via RT PCR. Alternatively, phage display screening can be undertaken to identify clones expressing scFv's.
Alternatively said fragments are “domain antibody fragments”. Domain antibodies are the smallest binding part of an antibody (approximately 13 kDa). Examples of this technology is disclosed in U.S. Pat. No. 6,248,516, U.S. Pat. No. 6,291,158, U.S. Pat. No. 6,127, 197 and EP0368684 which are all incorporated by reference in their entirety.
In a preferred method of the invention said antibody fragment is a single chain antibody variable region fragment.
In a further preferred embodiment of the invention said antibody is a humanised or chimeric antibody.
A chimeric antibody is produced by recombinant methods to contain the variable region of an antibody with an invariant or constant region of a human antibody. A humanised antibody is produced by recombinant methods to combine the complementarity determining regions (CDRs) of an antibody with both the constant (C) regions and the framework regions from the variable (V) regions of a human antibody.
Chimeric antibodies are recombinant antibodies in which all of the V-regions of a mouse or rat antibody are combined with human antibody C-regions. Humanised antibodies are recombinant hybrid antibodies which fuse the complimentarity determining regions from a rodent antibody V-region with the framework regions from the human antibody V-regions. The C-regions from the human antibody are also used. The complimentarily determining regions (CDRs) are the regions within the N-terminal domain of both the heavy and light chain of the antibody to where the majority of the variation of the V-region is restricted. These regions form loops at the surface of the antibody molecule. These loops provide the binding surface between the antibody and antigen.
Antibodies from non-human animals provoke an immune response to the foreign antibody and its removal from the circulation. Both chimeric and humanised antibodies have reduced antigenicity when injected to a human subject because there is a reduced amount of rodent (i.e. foreign) antibody within the recombinant hybrid antibody, while the human antibody regions do not elicit an immune response. This results in a weaker immune response and a decrease in the clearance of the antibody. This is clearly desirable when using therapeutic antibodies in the treatment of human diseases. Humanised antibodies are designed to have less “foreign” antibody regions and are therefore thought to be less immunogenic than chimeric antibodies.
In a further preferred method of the invention said agent is a peptide, preferably a modified peptide.
It will be apparent to one skilled in the art that modification to the amino acid sequence of peptides which modulate the interaction of ASPP family members with polypeptides involved in regulating ASPP activity could enhance the binding and/or stability of the peptide with respect to its target sequence. In addition, modification of the peptide may also increase the in vivo stability of the peptide thereby reducing the effective amount of peptide necessary to inhibit an interaction. This would advantageously reduce undesirable side effects which may result in vivo. Modifications include, by example and not by way of limitation, acetylation and amidation.
In a preferred method of the invention said peptide is acetylated. Preferably said acetylation is to the amino terminus of said peptide.
In a further preferred method of the invention said peptide is amidated. Preferably said amidation is to the carboxyl-terminus of said peptide.
In a further preferred method of the invention said peptide is modified by both acetylation and amidation.
Alternatively, or preferably, said modification includes the use of modified amino acids in the production of recombinant or synthetic forms of peptides. It will be apparent to one skilled in the art that modified amino acids include, by way of example and not by way of limitation, 4-hydroxyproline, 5-hydroxylysine, N6-acetyllysine, N6-methyllysine, N6,N6-dimethyllysine, N6,N6,N6-trimethyllysine, cyclohexyalanine, D-amino acids, ornithine. Other modifications include amino acids with a C2, C3 or C4 alkyl R group optionally substituted by 1, 2 or 3 substituents selected from halo (e.g. F, Br, I), hydroxy or C1-C4 alkoxy.
Altematively, peptides could be modified by, for example, cyclisation. Cyclisation is known in the art, (see Scott et al Chem Biol (2001), 8:801-815; Gellerman et al J. Peptide Res (2001), 57: 277-291; Dutta et al J. Peptide Res (2000), 8: 398412; Ngoka and Gross J Amer Soc Mass Spec (1999), 10:360-363.
In a preferred method of the invention peptides according to the invention are modified by cyclisation.
In a further preferred method of the invention said agent is an aptamer.
Nucleic acids have both linear sequence structure and a three dimensional structure which in part is determined by the linear sequence and also the environment in which these molecules are located. Conventional therapeutic molecules are small molecules, for example, peptides, polypeptides, or antibodies, which bind target molecules to produce an agonistic or antagonistic effects. It has become apparent that nucleic acid molecules also have potential with respect to providing agents with the requisite binding properties which may have therapeutic utility. These nucleic acid molecules are typically referred to as aptamers. Aptamers are small, usually stabilised, nucleic acid molecules, which comprise a binding domain for a target molecule. A screening method to identify aptamers is described in U.S. Pat. No. 5,270,163 which is incorporated by reference. Aptamers are typically oligonucleotides which may be single stranded oligodeoxynucleotides, oligoribonucleotides, or modified oligodeoxynucleotide or oligoribonucleotides.
The term “modified” encompasses nucleotides with a covalently modified base and/or sugar. For example, modified nucleotides include nucleotides having sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3′ position and other than a phosphate group at the 5′ position. Thus modified nucleotides may also include 2′ substituted sugars such as 2′-O-methyl-; 2-O-alkyl; 2-O-allyl; 2′-S-alkyl; 2′-S-allyl; 2′-fluoro-; 2′-halo or 2; azido-ribose, carbocyclic sugar analogues a-anomeric sugars; epimeric sugars such as arabinose, xyloses or lyxoses, pyranose sugars, furanose sugars, and sedoheptulose.
Modified nucleotides are known in the art and include, by example and not by way of limitation, alkylated purines and/or pyrimidines; acylated purines and/or pyrimidines; or other heterocycles. These classes of pyrimidines and purines are known in the art and include, pseudoisocytosine; N4, N4-ethanocytosine; 8-hydroxy-N6-methyladenine; 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil; 5-fluorouracil; 5-bromouracil; 5-carboxymethylaminomethyl-2-thiouracil; 5-carboxymethylaminomethyl uracil; dihydrouracil; inosine; N6-isopentyl-adenine; 1-methyladenine; 1-methylpseudouracil; 1-methylguanine; 2,2-dimethylguanine; 2-methyladenine; 2-methylguanine; 3-methylcytosine; 5-methylcytosine; N6-methyladenine; 7-methylguanine; 5-methylaminomethyl uracil; 5-methoxy amino methyl-2-thiouracil; β-D-mannosylqueosine; 5-methoxycarbonylmethyluracil; 5-methoxyuracil; 2-methylthio-N6-isopentenyladenine; uracil-5-oxyacetic acid methyl ester; psueouracil; 2-thiocytosine; 5-methyl-2 thiouracil, 2-thiouracil; 4-thiouracil; 5-methyluracil; N-uracil-5-oxyacetic acid methylester; uracil 5-oxyacetic acid; queosine; 2-thiocytosine; 5-propyluracil; 5-propylcytosine; 5-ethyluracil; 5-ethylcytosine; 5-butyluracil; 5-pentyluracil; 5-pentylcytosine; and 2,6,-diaminopurine; methylpsuedouracil; 1-methylguanine; 1-methylcytosine.
The aptamers of the invention are synthesised using conventional phosphodiester linked nucleotides and synthesised using standard solid or solution phase synthesis techniques which are known in the art. Linkages between nucleotides may use alternative linking molecules. For example, linking groups of the formula P(O)S, (thioate); P(S)S, (dithioate); P(O)NR′2; P(O)R′; P(O)OR6; CO; or CONR′2 wherein R is H (or a salt) or alkyl (1-12C) and R6 is alkyl (1-9C) is joined to adjacent nucleotides through —O— or —S—. The binding of aptamers to a target polypeptide is readily tested by assays hereindisclosed.
According to a further aspect of the invention there is provided an antibody, or active binding fragment thereof, wherein said antibody or fragment specifically binds a polypeptide encoded by a nucleic acid molecule selected from the group consisting of:
In a preferred embodiment of the invention said antibody is a monoclonal antibody.
In a preferred embodiment of the invention said antibody fragment is a single chain antibody fragment or a domain antibody.
In a preferred embodiment of the invention said phosphorylated epitope comprises amino acid residue 671 of the amino acid sequence as shown in
In an alternative preferred embodiment of the invention said phosphorylated epitope comprises amino acid residue 698 of the amino acid sequence shown in
In a further preferred embodiment of the invention said phosphorylated epitope comprises amino acid residue 746 of the amino acid sequence as shown in
In a yet preferred embodiment of the invention said phosphorylated epitope comprises amino acid residue 827 of the amino acid sequence shown in
According to a further aspect of the invention there is provided a cell transfected with at least one nucleic acid molecule wherein the genome of said cell is modified to include at least one copy of a nucleic acid molecule encoding a polypeptide selected from the group consisting of:
According to a further aspect of the invention there is provided a cell transfected with at least one nucleic acid molecule wherein the genome of said cell is modified to include at least one copy of a nucleic acid molecule encoding a polypeptide selected from the group consisting of:
According to a yet further aspect of the invention there is provided a cell transfected with at least one nucleic acid molecule wherein the genome of said cell is modified to include at least one copy of a nucleic acid molecule encoding a polypeptide selected from the group consisting of:
In a preferred embodiment of the invention said cell further comprises a nucleic acid molecule which includes a reporter gene to monitor the activity of said pro-apoptotic polypeptide(s).
In a preferred embodiment of the invention said cell is a cancer cell.
According to a yet further aspect of the invention there is provided a non-human transgenic animal comprising at least one cell according to the invention.
In a preferred embodiment of the invention said non-human animal is a non-human primate.
In a further preferred embodiment of the invention said transgenic animal is a rodent, preferably a mouse, rat or hamster.
In an alternative preferred embodiment of the invention said transgenic animal is a pig.
It is well within the knowledge of the skilled person to successfully generate both a heterozygous and homozygous transgenic animals with intergation of a desired nucleic acid encoding a particular gene or combination of genes using modified embryonic stem cells. These same steps can be easily applicable to species other than for example, mice. For example, Ware et al teaches an embryonic stem cell culture condition amenable for such animals as cattle, pigs and sheep (Society for the Study of Reproduction, 38:241 (1988)). In addition, this reference illustrates that the state of the art with respect to generation of transgenic species, other than mice, using modified embryonic stem cells is a well developed methodology.
According to an aspect of the invention there is provided a combined preparation of p53 and ASPP1 and/or ASPP2.
According to a further aspect of the invention there is provided a combined preparation comprising a nucleic acid molecule which encodes a p53 polypeptide, or sequence variant thereof, and at least one nucleic acid molecule which encodes at least one polypeptide, or sequence variant thereof, as represented by the amino acid sequences shown in
In a preferred embodiment of the invention there is provided a nucleic acid molecule which encodes both a p53 polypeptide and at least one polypeptide as represented by the amino acid sequences shown in
In a preferred embodiment of the invention said nucleic acid molecule is part of a vector. Preferably said nucleic acid molecules are operably linked to at least one promoter sequence which controls expression of said nucleic acid molecules. “Tromoter” is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only. Enhancer elements are cis acting nucleic acid sequences often found 5′ to the transcription initiation site of a gene (enhancers can also be found 3′ to a gene sequence or even located in intronic sequences). Enhancers function to increase the rate of transcription of the gene to which the enhancer is linked. Enhancer activity is responsive to trans acting transcription factors which have been shown to bind specifically to enhancer elements.
Promoter elements also include so called TATA box and RNA polymerase initiation selection sequences which function to select a site of transcription initiation. These sequences also bind polypeptides which function, inter alia, to facilitate transcription initiation selection by RNA polymerase.
Adaptations also include the provision of selectable markers and autonomous replication sequences which facilitate the maintenance of said vector in either the eukaryotic cell or prokaryotic host. Vectors which are maintained autonomously are referred to as episomal vectors. Episomal vectors are desirable since these molecules can incorporate large DNA fragments (30-50 kb DNA). Episomal vectors of this type are described in WO98/07876.
A number of viruses are commonly used as vectors for the delivery of exogenous genes. Commonly employed vectors include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picomoviridiae, herpesveridiae, poxviridae, adenoviridiae, or picornnaviridiae. Chimeric vectors may also be employed which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al.(1997) Nature Biotechnology 15:866-870). Such viral vectors may be wild-type or may be modified by recombinant DNA techniques to be replication deficient, conditionally replicating or replication competent.
Preferred vectors are derived from the adenoviral, adeno-associated viral and retroviral genomes. In the most preferred practice of the invention, the vectors are derived from the human adenovirus genome. Particularly preferred vectors are derived from the human adenovirus serotypes 2 or 5. The replicative capacity of such vectors may be attenuated (to the point of being considered “replication deficient”) by modifications or deletions in the E1a and/or E1b coding regions. Other modifications to the viral genome to achieve particular expression characteristics or permit repeat administration or lower immune response are preferred. Most preferred are human adenoviral type 5 vectors containing a DNA sequence encoding the p53 tumor suppressor gene. In the most preferred practice of the invention as exemplified herein, the vector is replication deficient vector adenoviral vector encoding the p53 tumor suppressor gene A/C/N/53 as described in Gregory, et al., U.S. Pat. No. 5,932,210 issued Aug. 3, 1999 (the entire teaching of which is herein incorporated by reference).
Alternatively, the viral vectors may be conditionally replicating or replication competent. Conditionally replicating viral vectors are used to achieve selective expression in particular cell types while avoiding untoward broad spectrum infection. Examples of conditionally replicating vectors are described in Pennisi, E. (1996) Science 274:342-343; Russell, and S. J. (1994) Eur. J. of Cancer 30A(8):1165-1171. Additional examples of selectively replicating vectors include those vectors wherein an gene essential for replication of the virus is under control of a promoter which is active only in a particular cell type or cell state such that in the absence of expression of such gene, the virus will not replicate. Examples of such vectors are described in Henderson, et al., U.S. Pat. No. 5,698,443 issued Dec. 16, 1997 and Henderson, et al., U.S. Pat. No. 5,871,726 issued Feb. 16, 1999 the entire teachings of which are herein incorporated by reference.
Additionally, the viral genome may be modified to include inducible promoters which achieve replication or expression only under certain conditions. Examples of inducible promoters are known in the scientific literature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; Iida, et al. (1996) J. Virol. 70(9):6054-6059; Hwang, et al.(1997) J. Virol 71(9):7128-7131; Lee, et al. (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al.(1997) J. Biol. Chem 272(46); 29364-29371.
The viruses may also be designed to be selectively replicating viruses. Particularly preferred selectively replicating viruses are described in Ramachandra, et al. PCT International Publication No. WO00/22137, International Application No. PCT/US99/21452 published Apr. 20, 2000 and Howe, J., PCT International Publication No. WO0022136, International Application No. PCT/US99/21451 published Apr. 20, 2000. A particularly preferred selectively replicating recombinant adenovirus is the virus d101/07/309 as more fully described in Howe, J.
It has been demonstrated that viruses which are attenuated for replication are also useful in the therapeutic arena. For example the adenovirus d11520 containing a specific deletion in the E1b55K gene (Barker and Berk (1987) Virology 156: 107) has been used with therapeutic effect in human beings. Such vectors are also described in McCormick (U.S. Pat. No. 5,677,178 issued Oct. 14, 1997) and McCormick, U.S. Pat. No 5,846,945 issued Dec. 8, 1998.
According to a further aspect of the invention there is provided a method to treat a condition which would benefit from an increase in apoptosis comprising administering a preparation comprising a first nucleic acid molecule comprising a nucleic acid sequence which encodes a p53 polypeptide, or sequence variant thereof, and administering a second preparation comprising a second nucleic acid molecule comprising a nucleic acid sequence which encodes a polypeptide, or sequence variant thereof, as represented by the amino acid sequence as shown in
According to a further aspect of the invention there is provided a method to treat a condition which would benefit from a stimulation of apoptosis comprising administering a combined preparation according to the invention.
In a preferred method of the invention said condition is cancer.
The preparations of the invention can be typically be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route, as an oral or nasal spray or via inhalation.
An embodiment of the invention will now be described by example only and with reference to the following Figures:
a is the ASPP1 nucleic acid sequence;
a is the H-Ras wild-type nucleic acid sequence;
a is the MAPK nucleic acid sequence;
a is the PKA nucleic acid sequence;
a is the phosphatase 1 nucleic acid sequence;
Methods
Cell culture, Antibodies and Plasmids
Cells were grown in DMEM plus 10% FCS. The mouse monoclonal DO-1 is specific to p53. The V5 epitope is recognized by the mouse monoclonal anti-V5 antibody (Invitrogen) and the haemagglutinin epitope is recognized by the anti-HA antibody (mAb) (Covance). The mouse monoclonal PC-10 is specific to the PCNA protein. CD20Leu is a fluorescein isothiocyanate-conjugated monoclonal antibody specific for the cell surface marker CD20 (Becton Dickinson). The mouse and rabbit antibodies to ASPP1 and ASPP2 were described previously 12 (rabbit anti-ASPP1 polyclonal antibody ASPP1.88, rabbit anti-ASPP2 polyclonal antibody ASPP2/77, mouse monoclonal anti-ASPP2 antibody DX54.10). The mouse monoclonal antibodies mAbASPP1.54.1 and mAbASPP1.54.2 were raised against an amino-terminal fragment of ASPP1 that has high homology to ASPP2. mAbASPP1.54.1 recognizes ASPP1 and cross-reacts with ASPP2 whereas mAbASPP1.54.2 does not cross-react with ASPP2 (data not shown). The mouse monoclonal antibody used against Ras for Western Blots was RO2120 (BD Transduction Laboratories) and to detect Ras in immunoprecipitation the rat monoclonal antibody 238 was used (Santa Cruz). The rabbit polyclonal C-12 (Santa Cruz) was used to detect Raf-1. Mcl-1 was detected by a rabbit polyclonal antibody (Santa Cruz). The deletion mutants of ASPP1 were tagged with the V5-epitope as were full length ASPP1 and ASPP2. H-RasV12 and K-RasV12 were tagged with the haemagglutinin epitope. All expression plasmids used in this study were driven by the cytomegalovirus immediate-early promoter, with the exception of the Ras and RafCX plasmids which were driven by the EF1a promoter. The ASPP1 and ASPP2 mutants were constructed using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). Using the peptide CPAPSpPGLDY (representing residues 824-832) with the serine phosphorylated as an immunogen, a rabbit polyclonal antibody NGH.S4 which specifically recognizes phosphorylated ASPP2 at amino acid 827 was raised. An affinity column was made by cross-linking the phospho-peptide to epoxy-sepharose-6B (Amersham Pharmacia Biotech) according to the manufacturer's instructions. Serum from the final bleed was clarified by centrifugation and filtration through 0.45 μm filter and was supplemented with 1× TTBS (0.5 M NaCl, 20 mM Tris [pH 8.0], 0.1% Tween-20). This was passed over the affinity column and washed with TTBS until the flowthrough had an OD280 nm <0.01. The antibody was then eluted with 0.2M glycine (pH2.8) and neutralized with Tris-HCl (pH 8.0).
RNAi of H-Ras and K-Ras
The mammalian expression vector pSUPER was used for expression of RNAi. In the case of K-Ras RNAi the gene-specific insert specifies a 19-nucleotide sequence corresponding to nucleotides 25-43 downstream of the transcription start site (gttggagctggtggcgtag) of K-Ras, which is separated by a 9-nucleotide noncomplementary spacer (ttcaagaga) from the reverse complement of the same 19-nucleotide sequence. This vector was referred to as K-Ras RNAi. The H-Ras RNAi construct was cloned into the same pSUPER vector with a 20-nucleotide insert corresponding to the nucleotides 299-316 downstream of the transcription start site (tcaaacgggtgaaggactc). These sequences were inserted into the pSUPER.2 backbone after digestion with BglII and HindIII and transformed into TOP10 One ShotTM supercompetent cells (Invitrogen) according to the manufacturer's instructions. Upon ligation, the BglII site is destroyed, allowing for selection of positive clones.
Transactivation Assay
Saos2 cells (7×105) were plated 24 hours prior to transfection in 6-cm-diameter dishes. All transactivation assays contained 1 μg of reporter plasmid. Fifty nanograms of p53, 1 μg of p63© and p73<, 4 μg of ASPP1 or ASPP2 or H-Ras RNAi or K-Ras RNAi, and 1.5 μg of H-rasV12 or K-RasV12 or RafCX expression plasmids were used as indicated. Cells were lysed in reporter lysis buffer 16 to 24 hours after transfection and assayed using the luciferase assay kit (Promega). The fold increase of p53 and ASPP by HRasV12/K-RasV12/RafCX was determined by the activity of p53 and ASPP in combination with RasV12/RafCX divided by the activity of p53 and ASPP alone.
Flow Cytometry
For FACS analysis, 106 cells were plated 24-48 hours prior to transfection in 10-cm diameter plates. All cells were transfected with 2 μg of a plasmid expressing CD20 as a transfection marker, 10 μg ASPP1 or ASPP2, 9 μg H-Ras RNAi or K-Ras RNAi, 6 μg E6, 15 μg anti-sense ASPP1 or anti-sense ASPP2 as indicated. Following transfection, some cells were subsequently treated with 3.5 μg/μl cisplatin for 24 hours. Both attached and floating cells were harvested using 4 mM EDTA/PBS and stained with FITC-conjugated anti-CD20 antibody. The cells were then fixed and stained with propidium iodide. The DNA content of all the cells expressing CD20 was analyzed using a flow cytometry (Becton Dickinson) as described in Hsieh et al.
Protein Biochemistry
For Western blotting, cells were lysed in either NP40 lysis buffer (1% Nonidet P40, 50 mM Tris [pH 8.0], 150 mM NaCl, and 1 mM EDTA), RIPA buffer (150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 50 Mm Tris-HCl [pH 8.0]) or luciferase lysis buffer. Between 35 and 150 μg of extract was mixed with 6× sample buffer and loaded on SDS-PAGE gels. For immunoprecipitation, cells were lysed in Ras IP buffer (20 mM Trish-HCl [pH 7.5], 1 mM EDTA, 1000 mM KCl, 5 mM MgCl2, 10% v/v glycerol, 1% v/v Triton X-100, 0.05% v/v 2-Mercaptoethanol and protease and phosphatase inhibitors) and the protein concentration determined.
Protein Purification
Both recombinant Ras and the N-terminus of ASPP1 were purified from BL21 bacteria. Protein expression was induced by IPTG (0.2 mM) for 3 hours and the cells were sonicated and then lysed. The lysate was spun at 10000 g and the supernatant recuperated. The recombinant proteins were purified by Glutathionine Sepharose→4B beads (Pharmacia Biotech AB, Sweden) as described by manufacturer in the case of Ras or in the case of N-terminus ASPP1 by nickel columns (Qiagen). The GST tag of Ras was cleaved off with bovine thrombin (Sigma) at a concentration of 5 units/ml and the thrombin removed by P-aminobenzamidine-agarose beads (Sigma). The GST-tagged ASPP2 fragment (693-1128) was purified by Glutathionine Sepharose→4B beads (Pharmacia Biotech AB, Sweden). The protein was concentrated and resupsended in 1× kinase buffer (50 mM Tris-HCl pH 7.5, 270 mM sucrose, 0.1 mM EGTA and 0.1% mercaptoethanol).
Ras GDP/GTP Loading
2.5 μg of recombinant protein purified from Escherichia coli was incubated in a total volume of 320 μl assay buffer containing 2 μCi [3H] GDP or [3H] GTP in a water bath for 10 minutes at 30° C. An aliquot of each sample had its tritium content measured to check the equal loading of GDP and GTP.
In Vitro Immunoprecipitation
1.5 μg of recombinant loaded Ras and 1.5 μg of recombinant ASPP1 fragment (1-308) were mixed together in 500 μl PBS. The lysate was pre-cleared with G-beads for 30 minutes. V5 antibody pre-attached to G-beads was added to the recombinant proteins and incubated for 1 hour at 4° C. The beads were washed three times with NET with 1% NP40 buffer.
In Vivo Immunoprecipitation
1-4 mg of lysate was pre-cleared with protein G beads for 30 minutes at 4° C. and subsequently incubated with antibody pre-bound to protein G beads for 2-16 hours at 4° C. The beads were washed three times with NP40 buffer. The immunoprecipitation beads were mixed with 6× sample buffer and loaded on SDS-PAGE gels. The gels were wet-transferred onto Protran nitrocellulose membrane and the resulting blots were blocked and incubated first in primary antibody and then with the appropriate secondary horseradish peroxidase-conjugated antibody (Dako). The blot was exposed to hyperfilm following the use of enhanced chemiluminescence substrate solution (Amersham Life Science).
In Vitro Phosphorylation Assay.
2 μg of GST-ASPP2 (693-1128) expressed in E. coli was incubated in 30 μl of reaction mixture containing 50 mM Tris-HCl, pH 7.5, 0.1% 2-mercaptoethanol, 1 μM microcystine and 100 μM [©-32P]ATP (10000 c.p.m./pmol) at 30° C. for 30 minutes. The reactions were terminated as described in Sapkota et al. and analyzed by chromatorgraphy on a C18 column.
Protein Analysis by Cycloheximide Experiments
Cells were either transfected with the appropriate expression plasmids or treated with 20 ng/ml EGF and 20 μM UO126 as indicated, the day prior to cycloheximide treatment. Cycloheximide was added to cells at a final concentration of 50 μg/ml in the appropriate medium. Cells were harvested and lysed at the time points indicated.
The Amino Terminus of ASPP1 and ASPP2 is Necessary for its Full Activity
p53 is the most common tumour suppressor protein found mutated in cancers with more than half of all human cancers carrying p53 mutations. p53 is activated by stress signals such as DNA damage and its activation can lead to one of two responses: cell cycle arrest or apoptosis. It has recently been shown that a novel family of tumour suppressor proteins, known as the ASPP family, can interact with p53 and specifically enhance p53-induced apoptosis but not cell-cycle arrest. ASPP1 and ASPP2 enhance the DNA binding and transactivation function of p53 on the promoters of pro-apoptotic genes only in vivo. Mutant ASPP1 and ASPP2 missing the first 150 amino acids are not fully functional, suggesting that the domain is essential for full activity of the ASPP proteins. This amino-terminal domain that is required for ASPP activity contains a putative Ras-association domain (see
Oncogenic H-rasV12 and K-rasV12 Increases ASPP2 Transactivation Activity
It has previously been reported that ASPP2 can increase p53 transactivation on pro-apoptotic genes and we wanted to see whether ras would have any effect on ASPP2 activity. We have shown, using transactivation assays, that both oncogenic H-rasV12 and K-rasV12 can increase ASPP2 activity 2-3 fold (see
Oncogenic Ras Can Activate Endogenous ASPP1, ASPP2 and P53 to Transactivate a Pro-apoptotic Reporter
U2OS cells were used to look at the effect of oncogenic ras on endogenous ASPP2 and p53.
Endogenous H-ras is Necessary for Full ASPP2 Activity
H-ras and K-ras RNAi constructs were made in both the pSUPPPRESSOR and pSUPER vectors. Only the constructs in the pSUPER vector were able to reduce ras levels specifically as shown in
Ras Activates ASPP2 Via its Raf-MAPK Pathway
Ras is a GTPase which is upstream of many effector pathways, the most well-known of which is the Raf-MAPK pathway. As it was unclear as to whether ras activated ASPP2 directly or via one of its pathways, we looked to see whether an activated form of Raf had any effect on ASPP2 activity.
The C-term of ASPP2 is Phosphorylated in Vitro by MAPK and Raf CX is Unable to Activate a Phosphorylation Mutant of ASPP2
The purified C-terminus of ASPP2 was used as a substrate to screen for kinases in an in vitro assay. An array of kinases were added to the purified C-terminus of ASPP2 and the phosphorylation status of the protein was analysed using P32 as a phosphorylation marker. MAPK1, PKA, p38SAPK and p90rsk were all found to be able to phosphorylate ASPP2 (
ASPP2 phosphorylation mutants were then constructed to see whether this site is phosphorylated in vivo. A serine was replaced by an alanine residue at the MAPK phosphorylation site identified by the in vitro phosphorylation assay (mutant 3), at another MAPK putative site (mutant 1) and at a PKA putative site that was shown to be phosphorylated in vitro (mutant 2) as shown in
The ASPP2 mutants were compared to wild type ASPP2 in a transactivation assay with p53 using bax-luciferase as a reporter (
H-ras Binds to ASPP1 in its Amino-terminus
As ASPP1 and ASPP2 contain a putative ras-association domain in their amino-terminus, we investigated whether ASPP was able to bind ras. We used different fragments of ASPP1 to see what parts of the protein bound to ras (
Activated Endogenous Ras Binds ASPP1 and ASPP2
EGF ligand activates EGFR, which leads to the recruitment of a guanine nucleotide exchange factor (GNEF). Once recruited to the plasma membrane and in close proximity to Ras, the GNEF leads to the exchange of GDP- to GTP-associated ras, making ras active and able to activate its downstream effectors. Thus EGF is able to activate endogenous ras in a physiological manner. An ASPP2 inducible clone was used to see whether endogenous, wild-type ras bound ASPP2 with different activity after stimulation with EGF. An immunoprecipitation was performed with endogenous ras, with and without ASPP2 and ras induction.
An immunoprecipitation was performed to determine whether endogenous ras is able to bind to endogenous ASPP1 and ASPP2.
The Amino-terminus of ASPP1 Binds ras.GTP More Efficiently than ras.GDP
In order to determine whether the interaction of ras and ASPP1/2 is direct, as we would expect if ASPP contains an active ras-association domain, we performed an in vitro binding assay with purified ras and amino-terminus of ASPP1. As suggested with the EGF-dependent binding of ras to ASPP, we speculated that ras in its GTP form could bind the amino-terminus of ASPP1 more efficiently than ras in its GDP form. We therefore purified ras and loaded it with either GDP or GTP (
Oncogenic ras Co-localizes with ASPP2 But not ASPP1 and Changes the ASPP1 Cellular Localization.
U2OS cells co-transfected with oncogenic H-rasV12 and ASPP2 clearly show co-localization at the plasma membrane of the cells (
ASPP1 and ASPP2 Interact with Activated Ras in Vitro and in Vivo
Residues 1-89 of ASPP1 and ASPP2 share sequence homology with the Ras association domain of RBL2 and AF6, suggesting that ASPP1 and ASPP2 may bind Ras (
The ability of ASPP1 to interact with Ras was confirmed by the observation that the purified ASPP1 fragment (1-308), containing the Ras-association domain (RAD), interacted with purified Ras in vitro. The ASPP1 amino-terminus bound Ras-GTP with a 4-fold higher affinity than Ras-GDP (
More Ras was detected in the ASPP1 and ASPP2 immunoprecipates derived from serum- and EGF-stimulated cell lysates than in those unstimulated (
ASPP1 and ASPP2 have been shown to synergize with p53 to induce the transcription of pro-apoptotic genes and this activity requires intact ASPP1 and ASPP1/2. Therefore, to test the biological significance of the Ras/ASPP interactions, oncogenic H-RasV12, which is constitutively in its GTP-bound form, was co-transfected with ASPP2 and p53 in Saos2 cells as indicated. The results shown in
In contrast, wild-type Ras had very little effect on the transactivation function of p53. The levels of Ras bound to GTP or GDP is tightly regulated in cells and increasing the amount of total Ras by transfection does not necessarily lead to increased levels of Ras-GTP in cells, which could explain why wild-type H-Ras had no effect on ASPP2 activity. Both H-RasV12 and K-RasV12 could stimulate the transactivation function of p53 to the same extent, namely 2.5 fold, and this was mediated by ASPP1 and ASPP2 (
Endogenous Ras is Required for the Full Activity of ASPP1 and ASPP2
The requirement of endogenous Ras to stimulate the activity of p53 via ASPP1 and ASPP2 was tested using RNA interference (RNAi). H-Ras RNAi and K-Ras RNAi were cloned into a PSUPER vector and their ability to specifically reduce the expression of H-Ras or K-Ras was confirmed (data not shown). The results in
All these results demonstrated that endogenous H-Ras and K-Ras are necessary for the full activities of ASPP1 and ASPP2.
Ser827 of ASPP2 is Phosphorylated by MAPK in Vitro and in Vivo
One of the most studied downstream pathways of Ras signalling is the Raf-MAPK pathway which acts as a phosphorylation cascade. To investigate whether the Ras-Raf-MAPK pathway stimulates p53 via ASPP1 and ASPP2, an activated form of Raf known as Raf CX, was used in a transactivation assay with a bax-luciferase reporter (
Phosphorylation of ASPP1 and ASPP2 by MAPK is Required for K-rasV12 and RafCX to Stimulate the Activity of ASP-P1 and ASPP2
Knowing that endogenous Ras is necessary for full ASPP2 activity and that Ras can activate ASPP1 and ASPP2 via the Raf-MAPK pathway, we investigated the requirement of endogenous MAPK activity for ASPP2 to stimulate the transactivation function of p53. A known MEK inhibitor, UO126, was used to test its effect on the transactivation function of p53 via ASPP2. Interestingly, the presence of UO126 had a small effect on the transactivation function of p53 alone but it significantly inhibited the transactivation function of p53 when ASPP2 was present (
In conditions where the cells were starved of serum, these mutants had identical activity to wild-type ASPP1 or ASPP2 in their ability to enhance the transactivation function of p53 (data not shown). However, whereas KrasV12 or activated Raf CX were able to stimulate wild-type ASPP2 and ASPP2 (S698A) by 2.5 fold, they were unable to increase the activity of mutant ASPP2 (S827) (data not shown and
Ras-Raf-MAPK Pathway Enhances the Stability of ASPP1 and ASPP2
We have consistently observed that ASPP1 and ASPP2 protein levels were increased when co-transfected with oncogenic Ras. Similarly, we have noticed a decrease in ASPP2 protein levels when cells were treated with the MAPK inhibitor UO126. Thus we hypothesised that Ras, via the Ras-MAPK pathway, could enhance the activity of ASPP1 or ASPP2 by increasing their protein levels.
The ASPP2 protein levels in the presence or absence of transfected oncogenic Ras were measured after cycloheximide addition (
| Number | Date | Country | Kind |
|---|---|---|---|
| 0328106.0 | Dec 2003 | GB | national |
| 0404242.0 | Feb 2004 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB04/03899 | 9/13/2004 | WO | 1/16/2007 |
| Number | Date | Country | |
|---|---|---|---|
| 60554852 | Mar 2004 | US |
