Patent Application
20080193488
The invention relates to a screening assay for the identification of agents that inhibit the interaction of p53 with p53 inhibitory polypeptides and including p53 inhibitory polypeptide variants that are inactive with respect to the inhibition of p53 activity.
Tumour suppressor genes encode proteins which function to inhibit cell growth or division and are therefore crucially 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. Arguably, the tumour suppressor gene which has been the subject of the most intense research is p53.
The p53 gene 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. The identification of the ASPP family of proteins as specific regulators of p53 revealed a novel mechanism by which the apoptotic function of p53 is regulated1 (see WO02/12325).
Whereas the apoptotic function of p53 is stimulated by ASPP1 and ASPP22, the related iASPP inhibits p53-dependent apoptosis 3. Regulation of p53 by the ASPP family members is evolutionarily conserved from worm to human2 3 and is disclosed in currently unpublished PCT application PCT/GB04/003492 which is incorporated by reference. Moreover, the involvement of the ASPP family in tumour development is reflected by the observation that the expression of ASPP1 and ASPP2 is often reduced while iASPP is increased in a large percentage of tumours examined2 3-5. An inverse relationship between ASPP2 expression and clinical outcome of B-cell lymphomas was also observed6.
The mechanistic basis by which ASPP1 and ASPP2 are activators of p53 while iASPP is an inhibitor of p53 remains unclear.
All ASPP family members bind p53 through their C-terminus. Furthermore, the most homologous region among the ASPP family members is located within its C-terminus and it carries the signature sequences of this family of proteins; Ankryin repeats, SH3 domain and Proline rich region containing Protein (ASPP). ASPP1 and ASPP2 belong to a unique class of SH3 domain containing proteins in which one of the critical proline contact residues in the SH3 domain of ASPP1 and ASPP2 is changed from Tyr to Leu. Based on the analysis of the co-crystal structure of the DNA binding domain of p53 and the SH3 domain of ASPP2 it was shown that this change allows ASPP2 to have higher binding affinity to the DNA binding domain of p53.
Moreover, p53 contains a proline rich sequence with five PXXP motifs which is known to be required for p53 to induce apoptosis but not cell cycle arrest7-9. The proline rich region of p53 is required for p53-dependent transactivation of target genes such as PIG3 but not p21waf1 or mdm27,10. Similarly, ASPP1 and ASPP2 selectively enhance the ability of p53 to transactivate pro-apoptotic genes such as Bax and PIG3 but not p21waf1 or mdm22.
Additionally, the most common polymorphism of p53 specifically found in human is located within the proline rich region of p53 at codon 72. In humans, the naturally occurring amino acid is either Proline (Pro) or Arginine (Arg). However the majority of vertebrates has Proline in the corresponding residue11. Extensive studies have been carried out in the last decade to investigate the link between the expression of p53 polymorphic variants at codon 72 (p53Pro72 and p53Arg72) and cancer susceptibility. However, the outcome has been disappointing because of a lack of understanding of how p53Pro72 and p53Arg72 function in vivo12-17. Interestingly, the proline at residue 72 of p53 is part of a PXXP motif which is known to be critical in contacting the Tyr residue of the SH3 domain containing protein.
In our co-pending application PCT/GB04/004341, currently unpublished, which is incorporated by reference, we disclose, amongst other things, methods to screen for agents that modulate the interaction of iASPP with p53 and the preferential binding of iASPP and ASPP1/2 for the p53 polymorphic variant p53Pro72. A conserved Tyr and Leu in the SH3 domain of iASPP and ASPP2 determines their distinct binding preference to the proline-rich region and DNA binding domain of p53 respectively. The proline-rich region of p53 is required for the ASPP family members to regulate p53-mediated apoptosis. Importantly, the ASPP family members, particularly iASPP, bind and regulate the activities of p53Pro72 more efficiently than that of p53Arg72. Endogenous iASPP level dictates the activities of codon 72 polymorphic p53 and over-expression of iASPP occurs more frequently in tumours homozygous for p53Pro72 than for p53Arg72. Hence escape from the negative regulation of iASPP is one of the mechanisms by which p53Arg72 activates apoptosis more efficiently than p53Pro72.
We describe a further screening method that allows the identification of agents that inhibit the interaction of iASPP polypeptides that comprise the SH3 domain of iASPP with the proline rich domain of p53. We also describe variant iASPP polypeptides which show reduced binding to p53, in particular p53Pro72.
According to an aspect of the invention there is provided an isolated nucleic acid molecule as represented by the nucleic acid sequence in
In a preferred embodiment of the invention said nucleic acid molecule comprises the nucleic acid sequence represented in
Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, N.Y., 1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (Allows Sequences that Share at Least 90% Identity to Hybridize)
In a preferred embodiment of the invention said nucleic acid molecules are part of an expression vector, preferably an expression vector adapted for eukaryotic gene expression.
Typically said adaptation includes, by example and not by way of limitation, the provision of transcription control sequences (promoter sequences) which mediate cell/tissue specific expression. These promoter sequences may be cell/tissue specific, inducible or constitutive.
“Promoter” is an art recognised term and, for the sake of clarity, includes the following features which are provided by example only, and not by way of limitation. 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 (polypeptides) which have been shown to bind specifically to enhancer elements. The binding/activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of physiological/environmental cues which include, by example and not by way of limitation, intermediary metabolites (eg glucose, lipids), environmental effectors (eg light, heat,).
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.
Adaptations which facilitate the expression of vector encoded genes include the provision of transcription termination/polyadenylation sequences. This also includes the provision of internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bi-cistronic or multi-cistronic expression cassettes. Expression control sequences also include so-called Locus Control Regions (LCRs). These are regulatory elements which confer position-independent, copy number-dependent expression to linked genes when assayed as transgenic constructs. LCRs include regulatory elements that insulate transgenes from the silencing effects of adjacent heterochromatin, Grosveld et al., Cell (1987), 51: 975-985.
There is a significant amount of published literature with respect to expression vector construction and recombinant DNA techniques in general. Please see, Sambrook et al (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour, N.Y. and references therein; Marston, F (1987) DNA Cloning Techniques: A Practical Approach Vol III IRL Press, Oxford UK; DNA Cloning: F M Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).
The use of viruses or “viral vectors” as therapeutic agents is well known in the art. Additionally, 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, picornoviridiae, herpesveridiae, poxyiridae, 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.
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 a 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 WO00/22137 and WO00/22136.
It has been demonstrated that viruses which are attenuated for replication are also useful in gene therapy. For example the adenovirus dl1520 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. The present invention may also be used in combination with the administration of such vectors to minimize the pre-existing or induced humoral immune response to such vectors.
It may be valuable in some instances to utilize or design vectors to achieve introduction of the exogenous transgene in a particular cell type. Certain vectors exhibit a natural tropism for certain tissue types. For example, vectors derived from the genus herpesviridiae have been shown to have preferential infection of neuronal cells. Examples of recombinantly modified herpesviridiae vectors are disclosed in U.S. Pat. No. 5,328,688 issued Jul. 12, 1994. Cell type specificity or cell type targeting may also be achieved in vectors derived from viruses having characteristically broad infectivity's by the modification of the viral envelope proteins. For example, cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fibre coding sequences to achieve expression of modified knob and fibre domains having specific interaction with unique cell surface receptors. Examples of such modifications are described in Wickham, et al (1997) J. Virol 71(11):8221-8229 (incorporation of RGD peptides into adenoviral fiber proteins); Amberg, et al. (1997) Virology 227:239-244 (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Harris and Lemoine (1996) TIG 12(10):400-405; Stevenson, et al. (1997) J. Virol. 71(6):4782-4790; Michael, et al. (1995) Gene Therapy 2:660-668 (incorporation of gastrin releasing peptide fragment into adenovirus fiber protein); and Ohno, et al. (1997) Nature Biotechnology 15:763-767 (incorporation of Protein A-IgG binding domain into Sindbis virus). Other methods of cell specific targeting have been achieved by the conjugation of antibodies or antibody fragments to the envelope proteins (see, e.g. Michael, et al. (1993) J. Biol. Chem 268:6866-6869, Watkins, et al. (1997) Gene Therapy 4:1004-1012; Douglas, et al. (1996) Nature Biotechnology 14: 1574-1578. Alternatively, particularly moieties may be conjugated to the viral surface to achieve targeting (See, e.g. Nilson, et al. (1996) Gene Therapy 3:280-286 (conjugation of EGF to retroviral proteins)). Additionally, the virally encoded therapeutic transgene also be under control of a tissue specific promoter region allowing expression of the transgene preferentially in particular cell types.
According to a further aspect of the invention there is provided an isolated variant polypeptide comprising an amino acid sequence wherein said polypeptide is modified by the deletion or substitution of at least amino acid residue tyrosine 814 of the amino acid sequence represented 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 modifications 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 variant 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 preferred embodiment of the invention said tyrosine amino acid residue is substituted with a leucine amino acid residue.
Preferably said variant polypeptide is modified only at amino acid residue tyrosine 814. Preferably said variant polypeptide is a tyrosine for a leucine substitution.
According to a further aspect of the invention there is provided the use of a modified nucleic acid or modified polypeptide according the invention as a pharmaceutical.
According to a further aspect of the invention there is provide a pharmaceutical composition comprising a modified nucleic acid or modified polypeptide according to the invention.
When administered, the nucleic acids/polypeptides of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary immune potentiating agents such as adjuvants and cytokines and optionally other therapeutic agents, such as chemotherapeutic agents.
The nucleic acids/polypeptides the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
The compositions of the invention are administered in effective amounts. An “effective amount” is that amount of a composition that alone, or together with further doses, produces the desired response. In the case of treating a particular disease, such as cancer, the desired response is inhibiting the progression of the disease. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods.
Such amounts will depend, of course, on the particular condition being treated, the severity of the condition, the individual patient parameters including age, physical condition, size and weight, the duration of the treatment, the nature of concurrent therapy (if any), the specific route of administration and like factors within the knowledge and expertise of the health practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the individual components or combinations thereof be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art, however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons.
The pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of nucleic acid/polypeptided for producing the desired response in a unit of weight or volume suitable for administration to a patient. The response can, for example, be measured by determining regression of a tumour, decrease of disease symptoms, modulation of apoptosis, etc.
The doses of nucleic acid administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
In general, doses of nucleic acids of between 1 ng and 0.1 mg generally will be formulated and administered according to standard procedures. Other protocols for the administration of compositions will be known to one of ordinary skill in the art, in which the dose amount, schedule of injections, sites of injections, mode of administration (e.g., intra-tumoral) and the like vary from the foregoing. Administration of compositions to mammals other than humans, (e.g. for testing purposes or veterinary therapeutic purposes) is carried out under substantially the same conditions as described above. A subject, as used herein, is a mammal, preferably a human, and including a non-human primate, cow, horse, pig, sheep, goat, dog, cat or rodent.
When administered, the pharmaceutical preparations of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
Compositions may be combined, if desired, with a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as syrup, elixir or an emulsion.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of nucleic acids, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.
In a further preferred embodiment of the invention said composition further comprises at least one further therapeutic agent. Preferably said agent is a chemotherapeutic agent.
Preferably said agent is selected from the group consisting of: cisplatin; carboplatin; cyclosphosphamide; melphalan; carmusline; methotrexate; 5-fluorouracil; cytarabine; mercaptopurine; daunorubicin; doxorubicin; epirubicin; vinblastine; vincristine; dactinomycin; mitomycin C; taxol; L-asparaginase; G-CSF; etoposide; colchicine; derferoxamine mesylate; and camptothecin.
According to an aspect of the invention there is provided a screening method for the identification of an antagonist that inhibits the interaction of a p53 inhibitor polypeptide with a p53 polypeptide comprising the steps of:
In a preferred method of the invention said p53 inhibitor polypeptide comprises a part of the amino acid sequence as represented in
In a further preferred method of the invention said p53 polypeptide comprises a part of the amino acid sequence represented in
In a preferred method of the invention said p53 polypeptide comprise an arginine amino acid residue at position 72 of the amino acid sequence represented in
In a further 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 part thereof. Preferably said antibody or binding part is a monoclonal antibody.
Preferably, said antibody interferes with the binding of said p53 inhibitor polypeptide with p53 at tyrosine 814 and amino acid residues 62-91 of p53.
In a preferred method of the invention said antibody fragment is a single chain antibody variable region fragment or a domain antibody fragment.
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 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 complimentarity 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 iASPP and p53 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 induce apoptosis. 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. Alternatively, 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: 398-412; 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 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.
In a preferred method of the invention said method further comprises a step wherein said agent is tested for activity with respect to a second different p53 polypeptide variant, preferably said p53 variant is modified by substitution of an amino acid residue encoded by codon 72 of the nucleic acid sequence as represented in
In a preferred method of the invention said p53 variant varies at codon 72 wherein said codon encodes an arginine or proline amino acid residue.
According to a further aspect of the invention there is provided a nucleic acid molecule as represented by the nucleic acid sequence in
In a preferred embodiment of the invention said peptide fragment is at least 8 amino acid residues in length.
In a further preferred embodiment of the invention said peptide fragment is between about 9 amino acid residues and 18 amino acid residues in length.
In still further preferred embodiment of the invention said peptide fragment is between about 18 amino acid residues and 32 amino acid residues in length.
According to a further aspect of the invention there is provided a peptide fragment encoded by a nucleic acid according to the invention.
According to a still further aspect of the invention there is provided an immunogenic composition comprising a nucleic acid or peptide according to the invention. Preferably said composition further comprises an adjuvant or carrier.
An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells. Examples of adjuvants include, by example only, Freunds adjuvant, muramyl dipeptides, liposomes. The term carrier is construed in the following manner. A carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter. Some antigens are not intrinsically immunogenic (i.e. not immunogenic in their own right) yet may be capable of generating antibody responses when associated with a foreign protein molecule such as keyhole-limpet haemocyanin or tetanus toxoid. Such antigens contain B-cell epitopes but no T cell epitopes. The protein moiety of such a conjugate (the “carrier” protein) provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen. Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.
According to a further aspect of the invention there is provided a method for preparing a hybridoma cell-line producing monoclonal antibodies comprising the steps of:
Preferably, said immunocompetent mammal is a rodent, for example a mouse, rat or hamster.
According to a further aspect of the invention there is provided a hybridoma cell-line obtainable by the method according to the invention.
According to a still further aspect of the invention there is provided an antibody obtained from the hybridoma cell-line according to the invention.
According to a further aspect of the invention there is provided a method for the treatment of an animal which would benefit from a stimulation of apoptosis comprising administering a nucleic acid molecule or polypeptide or composition according to the invention
According to a further aspect of the invention there is provided a method for the immunisation of an animal comprising administering a nucleic acid or peptide or immunogenic composition according to the invention.
In a preferred method of the invention said animal is a human.
In a further preferred method of the invention said treatment or immunisation is the treatment of cancer or vaccination against, cancer. Preferably, said cancer is breast cancer.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with reference to the following Figures:
Table 1 and Table 2 illustrate mRNA expression of iASPP in human breast-tumor samples (DCIS, grade1-2-3) expressing either wild type or mutant p53. Up arrows represent overexpression of iASPP mRNA in comparison with their matched normal samples. Table 2 shows the percentage of tumour samples homozygous for p53Pro72 (PP) or p53Arg72 (RR) overexpressing iASPP with either wild type or mutant p53. “n” represents the number of samples in each category.
Cells were grown in DMEM supplemented with 10% FCS. DO-1 is a mouse anti-p53 antibody. The V5 epitope is recognised by the mouse monoclonal antibody V5. CD20Leu is an FITC conjugated monoclonal antibody specific for the cell surface marker CD20 (Becton Dickinson). The mouse and rabbit antibodies to ASPP1 and ASPP2 were described previously (Rabbit anti-ASPP1 antibody pAb ASPP1.88, Rabbit anti-ASPP2 antibody pAb DX77 Rabbit anti-iASPP antibody pAb iASPP.18, mouse monoclonal anti-ASPP1 antibody LX011, mouse monoclonal anti-ASPP2 antibody DX54.10, mouse monoclonal anti-iASPP antibody miASPP49.3)2,28,29.
ASPP1, ASPP2, ASPP2L1113Y, iASPP and iASPPY814L expression plasmid were tagged with the V5 epitope. For transient expression of p53 the pCB6 plasmid was used, whereas the p53Arg72 and p53Pro72 used for the in vitro binding assays were cloned in pSP65 vector. p53Δpro was cloned in pcDNA3 vector.
Saos-2 or H1299 cells (5×105) were plated 24 hours prior to transfection in 6 cm dishes. All transactivation assays contained 1 μg of reporter plasmid. 50 ng of p53Pro72 or p53Arg72, 4 μg of ASPP1 or ASPP2, 500 ng of iASPP expression plasmids were used as indicated. In
Cells (106) were plated 24-48 hours prior to transfection in 10 cm plates. All cells were transfected with 2 μg of CD20 expressing plasmid as a transfection marker. Transfection consisted of 1 μg of human p53Pro72 or p53Arg72, 2 μg of p53□pro, 10 μg of ASPP1, ASPP2 or ASPP2L1113Y, 1 μg of iASPP or iASPPY814L and 8 μg of pSuper plasmid containing RNAi of iASPP as indicated. 36 hours after the transfection, both attached and floating cells were harvested and analysed as previously described15.
For western blotting, cells were lysed in either Nonidet P-40 (NP40) lysis buffer or luciferase reporter lysis buffer (Promega). Between 15-50 μg of protein extract was loaded on SDS-PAGE gels. For immunoprecipitation, cells were lysed in NP40 lysis buffer and pre-cleared with protein G beads for 1 hour at 4° C. The protein concentration was determined and then 1-2 mg of the extract was incubated with antibody pre-bound to protein G beads for 4 hours or overnight at 4° C. The beads were washed twice in NP40 lysis buffer and twice in NET buffer. The IP beads were mixed with 5× sample buffer and loaded onto an SDS-polyacrylamide gel. The gels were transferred (wet) to Protran nitrocellulose membranes, and the resulting blots were incubated first with primary antibody and subsequently with the appropriate secondary HRP conjugated antibody (Dako). The blot was exposed to hyperfilm following the use of ECL substrate solution (Amersham Life Science). The expression of endogenous ASPP1, ASPP2, iASPP, p53Pro72 and p53Arg72 were detected by using antibodies specific to these proteins derived from different species from that used in IP,
p53Pro72, p53Arg72, p53Δpro, ASPP1, ASPP2, ASPP2L1113Y, iASPP and iASPPY814L were in vitro translated and labeled with [35S]methionine using the TNT T7 and TNT sp6 Quick coupled transcription-translation systems (Promega). The lysates containing indicated proteins were incubated at 30° C. for 1 h. The anti-V5 antibody immobilized on protein G-agarose beads was added to the binding reaction mixtures and incubated on a rotating wheel at 4° C. for 16 h. The beads were then washed with PBS. The bound proteins were released in SDS gel sample buffer and analyzed by SDS-10% polyacrylamide gel electrophoresis. Results were visualized by autoradiography.
Construction and Transfection of siRNA of iASPP
Oligonucleotides (19 bp) derived from iASPP were ligated into pSuper expression plasmids as described previously30. The plasmids containing correct 19 bp oligonucleotides of iASPP were confirmed by sequencing. The sequences of iASPP sense and antisense oligonucleotides used in this study are as follow (lowercase indicates the vector sequence from pSuper; upper case indicates the target sequence for the RNAi):
Sense (S) and Antisense (A) oligos for iASPP
For transfection, 1×106 H1299 or Saos2 cells were plated into 10 cm dishes. Cells were transfected with 2.5 μg of pMACS H-2KK alongside either pSuper or pSuper-si-RNA iASPP (10 μg). 48 h after transfection, cells expressing the pMACS H-2KK plasmid were separated using the MACS system (Miltenyi Biotec) according to the manufacturer's instructions. This gave rise to two populations of cells: H-2KK expressing (transfected) cells and non-expressing (non-transfected cells). Both cell populations were lysed with RIPA buffer on ice for 30 minutes followed by centrifugation at 20 000 g for 30 minutes at 4° C.
The breast cancers were all ductal carcinomas of no special type. The presence of an adequate proportion of tumour tissue was confirmed histologically prior to analysis. Codon 72 single nucleotide polymorphism was performed as described previously31. Mutations in p53 were analysed by single strand conformational polymorphism (SSCP) and sequencing as described. Expression of the ASPP family members was performed using TaqMan PCR. The primer sequences are as follows.
iASPP
Alignment of the SH3 domains of the ASPP family members with those SH3 from other proteins reveals the presence of Leu instead of the highly conserved Tyr at residues 1075 and 1113 for ASPP1 and ASPP2 respectively. Interestingly, the corresponding residue in iASPP is Tyr (Y814) (
To investigate whether the amino acid sequence difference found in the SH3 domain of ASPP2 (L1113) and iASPP (Y814) was one of the determining factors for the binding specificity of iASPP versus ASPP2 to the proline rich region of p53, we used oligonucleotide-directed mutagenesis to introduce an amino acid interchange between iASPP and ASPP2. Y814 of iASPP was changed to Leu while Leu 1113 of ASPP2 was changed to Tyr and the efficiency with which ASPP2 and iASPP bind to p53 and p53Δpro was compared to ASPP2/L1113Y and iASPP/Y814L in in vitro assays. Interestingly, the amount of p53 in complex with iASPP/Y814L was less than that in complex with iASPP, even though slightly more iASPP/Y814L was immunoprecipitated. Under the same conditions, similar amounts of p53Δpro were co-immunoprecipitated with iASPP and iASPP/Y814L (
The biological consequences of the interaction between the proline rich region of p53 and the ASPP family of proteins were tested using p53Pro72 and p53Δpro. Consistent with our previous observations, expression of ASPP1 or ASPP2 stimulated the transactivating activity of p53Pro72 on the human promoters of Bax but not mdm2. However, no stimulatory effects on p53Δpro were observed even though similar amounts of ASPP1 and ASPP2 were expressed (
To further demonstrate the importance of binding to the proline rich region of p53 in the iASPP-dependent inhibition of the apoptotic function of p53Pro72, we used flow cytometry to test the ability of iASPP/Y814L to inhibit p53-dependent apoptosis. Consistent with results from the binding assays, iASPP/Y814L has almost completely lost its ability to inhibit apoptosis induced by p53Pro72 (
The proline rich region of p53 spans residues 62-917. Interestingly, the proline residue at position 72 is part of the PXXP motif present in p53, implying that Pro72 is one of the critical amino acids in p53 which contacts iASPP. A computer based three-dimensional structure modelling study was carried out based on the existing co-crystal structure of ASPP2/p53 (PDB code 1YCS). Initially iASPP was modelled based on ASPP2 and docked to p53 based on the complex structure 1YCS. After minimization, the iASPP-p53 complex was investigated to explore the differences between the binding interface of the DNA-binding region of p53 and ASPP2 versus iASPP. To investigate which part of the proline rich region of p53 would bind to the ASPP family; both sequential and structural studies were performed. A region was identified that contains both an arginine and the proline at codon 72. This poly-proline helix was modelled and docked into ASPP2 and iASPP based on the structure of Grb2-SOS complex (1AZE). The structural investigations showed that, as expected from the analysis of other SH3-poly pro complexes the Y814 of iASPP is an important contact residue to the prolines. When this is mutated to a Leu as in ASPP2, a large gap develops and the hydrophobic/aromatic contacts are lost (
To formally test this hypothesis, we used in vitro translated p53Arg72, p53Pro72, ASPP1, ASPP2 and iASPP. As shown in
Since Y814 of iASPP could potentially contact the proline at residue 72 of p53, the ability of iASPP/Y814L and ASPP2/L11113Y to bind to p53Pro72 and p53Arg72 was also tested. Consistent with previous findings, there was a clear reduction in the binding ability of iASPP/Y814L to p53Pro72 but not to p53Arg72 (
Having established that the ASPP family members, particularly iASPP, selectively interact with the two codon 72 p53 polymorphic variants, we next investigated whether the two polymorphic variants of p53 are subject to differential functional regulation by the ASPP family of proteins. In Saos-2 cells, the transactivating activity of p53Pro72 on the promoters of Bax and PIG3 is similar to or greater than p53Arg72 when expressed alone (
The ability of endogenous ASPP family of proteins in regulating the activities of the two polymorphic p53 variants in vivo was first tested by examining the expression levels of ASPP1, ASPP2 and iASPP in H1299 and Saos-2 cells. Although the levels of ASPP1 and ASPP2 were similar in both cell lines, H1299 cells express 3-5 folds more iASPP than Saos-2 cells (
The more efficient binding of iASPP to pS3Pro72 than to p53Arg72, implies that p53Arg72 is less sensitive to the inhibitory effects of iASPP, a mechanism which potentially explains the relatively greater apoptosis-inducing activity of p53Arg72 in H1299 cells. The ability of iASPP to influence the activities of p53Pro72 and p53Arg72 was further tested using RNA interference to reduce the expression of endogenous iASPP. In Saos-2 cells, iASPP RNAi stimulated the transactivating activity of p53Arg72 and pS3Pro72 on the Bax promoter by 1 and 2 fold respectively (
To further investigate the biological and potentially clinical importance of our findings, we examined the expression levels of iASPP in a panel of matched normal and human breast carcinomas homozygous for p53Arg72 (n=62) or p53Pro72 (n=16) (table 1) using TaqMan (real-time) RT-PCR. Detailed analysis revealed that the frequency of over-expression of iASPP is significantly higher in cases with wild type p53 which are homozygous for p53Pro72 than in those homozygous for p53Arg72 in the germ-line (table 2). Whereas iASPP was over expressed in 90% of the tumours homozygous for p53Pro72 (14/15), only 32% (15/47) of the tumours homozygous for p53Arg72 over-expressed iASPP mRNA (table 1, table 2 and
Here we show that the proline rich region is the second binding site of the ASPP family members to p53. This initial observation led us to delineate a novel mechanism by which the ASPP family members regulate the apoptotic function of p53. We provide evidence that the proline rich region of p53 binds to iASPP more efficiently than to ASPP1 (data not shown) and ASPP2. This finding provides molecular insights into the mechanistic basis by which iASPP inhibit, while ASPP1 and ASPP2 stimulate, the apoptotic function of p53. The failure of iASPP/Y814L to bind the proline rich region of p53 and to inhibit the apoptotic function of p53 demonstrates for the first time that iASPP predominantly inhibits the apoptotic function of p53 through its ability to selectively bind the proline rich region of p53. In contrast previous studies showed that many proteins can stimulate the activities of p53 through their ability to interact with the proline rich region of p53. Binding to the proline rich region and enhance the acetylation of p53 is one of the mechanism by which p300 stimulates the transactivation function of p5318.The binding of corepressor mSin3a protein to the proline rich region of p53 can also increased the stability and transrepression function of p5319, one of the property of p53 which is closely linked to its apoptotic function20. Additionally IKbΔ can bind to the proline rich region of p53 and enhance p53 mediated apoptosis. This interaction of IkbΔ requires the phosphorylation of p53 at Ser4621. We do not yet know the precise mechanism by which the proline rich region of p53 is involved in activating the full apoptotic function of p53. However the results shown in this study suggested that binding to the proline rich region of p53 and preventing other activators such as p300, Sin3A and IKbΔ from binding to the same region of p53 is perhaps one of the mechanisms by which iASPP inhibits the apoptotic function of p53.
In the absence of any co-crystal structure information between the proline rich region of p53 and the ASPP family members, it remains unclear whether the binding of iASPP to the proline rich region of p53 could change the protein conformation of p53 and alter the ability of p53 to bind DNA, ASPP1, ASPP2 and other interacting partners of p53. Nonetheless, the findings shown here provide an attractive possibility that binding to iASPP and abrogating its anti-apoptotic function is one of the reasons why the proline rich region sequence of p53 is required for its full apoptotic function. Future structural studies of p53 containing the proline rich region sequence are needed to provide a detailed molecular explanation of these questions. The failure of ASPP1 and ASPP2 to stimulate the apoptotic function of the proline rich region deleted p53 mutant, p53Δpro, as well as p53Arg72 suggested that counteracting the inhibitory activity of iASPP is one of the main mechanisms by which ASPP1 and ASPP2 stimulate the apoptotic function of p53 (
The findings reported here also reveal a novel insight into the mechanism by which the apoptosis function of the p53 polymorphic variants, p53Pro72 and p53Arg72, is regulated. The regulation of p53 by ASPP family proteins is evolutionarily conserved and the most conserved member of the ASPP family and the only ASPP family member present in C.elegans3 is iASPP. Hence it is interesting to note that iASPP has a higher binding affinity to p53Pro72 than to p53Arg72. The polymorphism of p53 at codon 72 only exists in human and p53Arg72 is human specific. Moreover, the frequency of the allele encoding p53Pro72 varies among different ethnic populations. The number of individuals homozygous for p53Pro72 is closely linked to latitude and is much higher in the black populations living near the equator, suggesting that p53Pro72 is selected in an environment with high levels of UV light22,23. A molecular explanation of this selection may be that the ASPP family of proteins, iASPP in particular, selectively regulate the apoptotic function of p53Pro72. In response to various stress signals, two different pathways are involved in regulating the apoptotic function of p53Pro72 or p53Arg72. It was previously shown that the p53Arg72 preferentially localizes to the mitochondria24. Here, we have identified an alternative pathway by which p53Arg72 is more active than p53Pro72 in induction of apoptosis and has, therefore, evolved in humans. The insensitivity of p53Arg72 to inhibition by iASPP implies that the most efficient way to inactivate the apoptotic function of p53Arg72 in human tumourigenesis is by intragenic mutation. Consistent with this hypothesis and previous publication, the percentage of tumours expressing mutant p53 that are homozygous for p53Arg72 is much higher than those with p53Pro72 (42% and 6% respectively) in the panel of breast tumours examined25. Being a more potent inhibitor of p73, there is also a selective advantage to mutate p53Arg72 in tumours26. In contrast, inactivation of p53Pro72 can occur by reduction in expression of ASPP1, ASPP2 or over-expression of iASPP, in addition to mutation in p53 itself. Therefore the ASPP family of proteins provided another level of regulation of p53Pro72. As a result, p53Pro72 is less prone to mutation than p53Arg72 in normal cells in response to signals that induce the apoptotic function of p53. This may be why the percentage of p53Pro72 homozygous carriers is highest in the ethnic populations that have evolved in an environment consistently exposed to high dose of p53 inducing agents such as UV radiation. Nevertheless, homozygosity for p53pro72 does not necessarily mean protection against p53 mutation in other types of cancer because the expression levels of the ASPP family proteins vary dramatically among different tissues (data not shown)27. Only when the expression levels of the ASPP family members are taken into consideration, can clear conclusions be drawn on association of expression of polymorphic p53 variants with cancer susceptibility. The results shown here suggest the possibility of improved strategies to treat cancer according to their p53 polymorphism and ASPP expression patterns.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0514538.8 | Jul 2005 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2006/006850 | 7/13/2006 | WO | 00 | 2/20/2008 |
