U.S. patent application number 10/522043 was filed with the patent office on 2006-05-11 for polypeptide.
Invention is credited to Patricia Kuwabara, Xin Lu, David Selwood.
Application Number | 20060100143 10/522043 |
Document ID | / |
Family ID | 32071253 |
Filed Date | 2006-05-11 |
United States Patent
Application |
20060100143 |
Kind Code |
A1 |
Lu; Xin ; et al. |
May 11, 2006 |
Polypeptide
Abstract
The invention relates to a polypeptide, or part thereof, which
inhibits the apoptotic activity of the tumour suppressor protein
p53 and including screening methods to identify agents which
interfere with the activity of said polypeptide.
Inventors: |
Lu; Xin; (London, GB)
; Kuwabara; Patricia; (Bristol, GB) ; Selwood;
David; (London, GB) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 SW SALMON STREET
SUITE 1600
PORTLAND
OR
97204
US
|
Family ID: |
32071253 |
Appl. No.: |
10/522043 |
Filed: |
October 3, 2003 |
PCT Filed: |
October 3, 2003 |
PCT NO: |
PCT/GB03/04296 |
371 Date: |
October 13, 2005 |
Current U.S.
Class: |
424/85.1 ;
435/320.1; 435/325; 435/6.13; 435/69.1; 514/18.9; 514/19.3;
530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/193 20130101;
A61K 38/193 20130101; A61K 38/1709 20130101; A61P 35/00 20180101;
A61K 38/1709 20130101; A61K 38/50 20130101; C07K 14/4747 20130101;
A61K 2300/00 20130101; A61K 38/50 20130101; A61K 2300/00 20130101;
A61K 2300/00 20130101 |
Class at
Publication: |
514/012 ;
530/350; 435/006; 435/069.1; 435/320.1; 435/325; 536/023.5 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/82 20060101 C07K014/82; C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 7, 2002 |
GB |
0223193.4 |
Mar 19, 2003 |
GB |
0306261.9 |
Claims
1. An isolated nucleic acid molecule which encodes a polypeptide,
or sequence variant thereof, wherein said polypeptide is a fragment
of the polypeptide sequence represented in SEQ ID NO: 8 or 9,
wherein the fragment is: a polypeptide fragment consisting of amino
acid residues from about residue 128-224 of the amino acid sequence
presented in SEQ ID NO: 8 or 9; or a polypeptide fragment
consisting of amino acid residues from about residue 128-224 of the
amino acid sequence presented in SEQ ID NO: 8 or 9 wherein said
sequence has been modified by addition, deletion or substitution of
at least one amino acid residue, wherein the polypeptide inhibits
the apoptoic activity of p53.
2. The nucleic acid molecule according to claim 1, wherein said
molecule encodes a fragment consisting of amino acid residues from
about residue 128-224 of the sequence represented in SEQ ID NO:
8.
3. The nucleic acid molecule according to claim 2, wherein said
molecule is isolated from a human.
4. The nucleic acid molecule according to claim 1, wherein said
molecule encodes a fragment consisting of amino acid residues from
about residue 128-224 of the sequence represented in SEQ ID NO:
9.
5. The nucleic acid molecule according to claim 4, wherein said
molecule is isolated from a nematode.
6. The nucleic acid molecule according to claim 5, wherein said
nematode is of the genus Caenorhabditis spp.
7. (canceled)
8. The nucleic acid molecule according to claim 1, wherein said
nucleic acid molecule is a cDNA or genomic DNA.
9. (canceled)
10. A polypeptide fragment or sequence variant thereof, encoded by
the nucleic acid molecule according to claim 1.
11. A vector comprising the nucleic acid according to claim 1.
12. The vector according to claim 11, wherein said vector is an
expression vector.
13. A cell tranformed or transfected with the nucleic acid molecule
according to claim 1.
14. A pharmaceutical composition comprising the nucleic acid
according to claim 1.
15. A pharmaceutical composition comprising the polypeptide
according to claim 10.
16. (canceled)
17. A transgenic non-human animal comprising the nucleic acid
molecule according to claim 1.
18. (canceled)
19. A screening method to identify agents which inhibit the binding
of a polypeptide, or fragment thereof, to p53 comprising: forming a
preparation comprising the polypeptide of claim 10; and a p53
polypeptide, or a fragment thereof consisting of the binding
site(s) for the polypeptide of claim 10; providing at least one
agent to be tested; and determining the activity of the agent with
respect to the binding of the polypeptide of claim 10 to the p53
polypeptide, or a fragment thereof consisting of the binding
site(s) for the polypeptide of claim 10.
20. The method according to claim 19, wherein said agent is a
polypeptide or a peptide.
21. (canceled)
22. The method according to claim 20, wherein said polypeptide is
an antibody or binding part thereof.
23. The method according to claim 22, wherein said antibody is a
monoclonal antibody.
24. The method according to claim 22, wherein said fragment is an
Fab fragment.
25. The method according to claim 24, wherein said Fab fragment is
an F(ab').sub.2 fragment, an Fab fragment, an Fv fragment, or CDR3
regions.
26. The method according to claim 23, wherein said antibody is
humanised.
27. The method according to claim 23, wherein said antibody is a
chimeric antibody.
28. An isolated nucleic acid molecule, wherein said molecule is
isolated from a nematode worm and hybridises to the nucleic acid
sequence shown in SEQ ID NO: 9, wherein said nucleic acid molecule
encodes an inhibitor of p53 and inhibits the apoptotic activity of
p53.
29. The nucleic acid molecule according to claim 28, wherein said
molecule hybridises under stringent hybridisation conditions.
30. (canceled)
31. An isolated polypeptide comprising the amino acid sequence as
represented in SEQ ID NO: 9 or a variant polypeptide which
polypeptide is modified by addition, deletion or substitution of at
least one amino acid residue and is an inhibitor of p53.
32. A method of treatment of an animal, comprising administering an
effective amount of the polypeptide according to claim 10, wherein
said effective amount induces the apoptotic activity of p53.
33. A method of treatment of an animal comprising administering an
effective amount of a nucleic acid molecule according to claim 1,
wherein said effective amount induces the apoptotic activity of
p53.
34. The method according to claim 32, wherein said treatment is of
cancer.
35. The polypeptide of claim 10, wherein the polypeptide is a
peptide comprising the amino acid sequence DGPEETD (SEQ ID NO: 2);
GPEETD (SEQ ID NO: 2); TTLSDG (SEQ ID NO: 3); AEFGDE (amino acids
294-299 of SEQ ID NO: 8); or PRNYFG (SEQ ID NO: 4).
36. The peptide according to claim 35, wherein said peptide is at
least 6 amino acid residues.
37. The peptide according to claim 35, wherein said peptide is at
least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino
acid residues.
38. The peptide according to claim 35, wherein said peptide is at
least 20; 30; 40; 50; 60; 70; 80; 90; or 100 amino acid
residues.
39. The peptide according to claim 35, wherein the peptide consists
of the amino acid sequence DGPEETD (SEQ ID NO: 2); GPEETD (SEQ ID
NO: 1); TTLSDG (SEQ ID NO: 3); AEFGDE (amino acids 294-299 of SEQ
ID NO: 8); or PRNYFG (SEQ ID NO: 4).
40. (canceled)
41. The peptide according to claim 35, wherein the peptide further
comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 arginine
residues.
42. (canceled)
43. A pharmaceutical composition comprising the peptide of claim
35.
44. The pharmaceutical composition according to claim 43, wherein
said composition further includes a carrier, diluent or
excipient.
45. The pharmaceutical composition of claim 35 further comprising
at least one anti-cancer agent.
46. The pharmaceutical composition according to claim 45 wherein
said anticancer agent is 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; or
camptothecin.
47. (canceled)
48. (canceled)
49. A complex comprising the peptide according to claim 35 and an
antibody, or binding part thereof.
50. The complex according to claim 49, wherein said antibody or
binding part is a cell specific antibody.
51. The complex according to claim 49 wherein said antibody is a
cancer cell specific antibody.
52. A method of treatment of an animal that would benefit from the
induction of apoptosis, comprising administering an effective
amount of the peptide of claim 35 to the animal.
53. (canceled)
54. The method according to claim 52, wherein said treatment is
cancer treatment.
Description
[0001] The invention relates to a polypeptide, or part thereof,
which inhibits the apoptotic activity of the tumour suppressor
protein p53; screening methods to identify agents which interfere
with the activity of said polypeptide and agents with said
activity.
[0002] Apoptosis, or programmed cell death, is a process by which
multi-cellular organisms regulate cell number and differentiation.
The process is regulated by factors which either induce or prevent
apoptosis. Inducers of apoptosis include Bcl-2 family members,
caspase family members and their associated factors Apaf-1 and
Fadd. Caspases are synthesised as proenzymes which become activated
after proteolytic cleavage. The active caspase then induces many of
the morphological and biochemical changes associated with
apoptosis. Mitochondria play a pivotal role in the activation
process through the release of pro-apoptotic factors such as
cytochrome c, AIF and Diablo. The release from mitochondria is
controlled by the Bcl-2 family of proteins; (e.g. Bcl-2 and Bcl-xl
inhibit release; Bax and Bak induce release).
[0003] WO9953051 discloses a cytokine dependent protein p21 which
has pro-apoptotic activity. p21 is expressed in a cytokine
dependent manner in myeloid/erthyroid cells. These cells are
dependent on IL-3 for growth and in the absence of IL-3 the
translation of p21 is induced resulting in apoptosis and cell
death. p21 is a cytoplasmic protein which translocates to the outer
mitochondrial membrane to induce pro-apoptotic activities.
[0004] Tumour suppressor proteins also have pro-apoptotic
activities.
[0005] 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).
[0006] 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 in 1978 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. Genes regulated by the transcriptional activity of p53
contain a p53 recognition sequence in their 5' regions. These genes
are activated when the cellular levels of p53 are elevated due to,
for example DNA damage. Examples of genes which respond to p53
include, mdm2, Bax and PIG-3. Bax and PIG-3 are involved in one of
the most important functions of p53, the induction of
apoptosis.
[0007] In our co-pending application WO02/12325 we disclose a
family of proteins, referred to as ASPP, as specific activators of
p53 and revealed a mechanism by which wild type p53 is tolerated in
tumours, such as human breast carcinomas. We also disclose an
inhibitor of ASPP family members referred to as iASPP. iASPP is an
oncogene and is the most conserved member of the ASPP family. iASPP
is the only ASPP-like protein found in C. elegans. Similar to human
iASPP, the C. elegans homologue functions as a key inhibitor of
p53. These findings indicate that regulation of p53 function by
members of the ASPP family has been evolutionarily conserved across
phyla.
[0008] The C. elegans iASPP is capable of substituting for human
iASPP in all of the assays performed in human cells. Moreover,
reciprocal substitution studies reveal that the apoptotic function
of C. elegans p53 is enhanced or inhibited by human ASPP and iASPP,
respectively. Using RNAi we further demonstrate that iASPP is a key
inhibitor of p53 mediated apoptosis in C. elegans. All of these
observations show that the regulation of p53 by ASPP family members
is evolutionarily conserved. Control of p53 activity plays a
pivotal role in development and tumourigenesis. Hence, inhibiting
the oncogenic function of iASPP could provide an important new
strategy for treating tumours expressing wild type p53. Sequence
comparision between C. elegans and human iASPP reveals a conserved
domain between the nematode and human sequence which likely
explains the functional conservation between the proteins.
[0009] According to an aspect of the invention there is provided an
isolated nucleic acid molecule which encodes a polypeptide, or
sequence variant thereof, wherein said polypeptide is a fragment of
the polypeptide sequence represented in FIG. 1a or 1b, which
fragment is selected from the group consisting of: [0010] i) a
polypeptide fragment consisting of amino acid residues from about
residue 128-224 of the amino acid sequence presented in FIG. 1a or
1b; [0011] ii) a polypeptide fragment consisting of amino acid
residues from about 128-244 of the amino acid sequence presented in
FIG. 1a or 1b wherein said sequence has been modified by addition,
deletion or substitution of at least one amino acid residue; and
[0012] iii) a polypeptide as defined in (i) and (ii) wherein said
polypeptide substantially retains the biological activity of the
polypeptide represented in FIG. 1a or 1b.
[0013] In a preferred embodiment of the invention said nucleic acid
molecule encodes a polypeptide fragment consisting of amino acid
residues from about 128-224 of the sequence represented in FIG. 1a.
Preferably said nucleic acid molecule is isolated from a human.
[0014] In an alternative preferred embodiment of the invention said
nucleic acid molecule encodes a polypeptide fragment consisting of
amino acid residues from about 128-224 of the sequence represented
in FIG. 1b. Preferably said nucleic acid molecule is isolated from
a nematode. Preferably said nematode is of the genus Caenorhabditis
spp.
[0015] In a preferred embodiment of the invention said nucleic acid
molecule encodes a polypeptide, or sequence variant thereof, which
polypeptide inhibits the activity of a polypeptide represented by
the amino acid sequence represented in FIG. 1a or 1b.
[0016] In a preferred embodiment of the invention said nucleic acid
molecule is a cDNA.
[0017] In an alternative preferred embodiment of the invention said
nucleic acid molecule is genomic DNA.
[0018] According to a further aspect of the invention there is
provided a polypeptide fragment or sequence variant thereof,
encoded by a nucleic acid molecule according to the invention.
[0019] It will be apparent that fragments which are sequence
variants may retain the biological activity of the full length
polypeptide or alternatively have antagonistic activity by
competing for binding sites in p53. In general, the specificity of
polypeptides according to the invention with respect to binding to
p53 is shown by binding equilibrium constants. Polypeptides which
are capable of selectively binding p53 preferably have binding
equilibrium constants of at least about 10.sup.7 M.sup.-1, more
preferably at least about 10.sup.8 M.sup.-1, and most preferably at
least about 10.sup.9 M.sup.-1.
[0020] A sequence variant, i.e. a fragment polypeptide and
reference 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) phenylalaine, tyrosine and
tryptophan.
[0021] A functionally equivalent polypeptide according to the
invention is a variant wherein one or more amino acid residues are
substituted with conserved or non-conserved amino acid residues, or
one in which one or more amino acid residues includes a substituent
group. Conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile;
interchange of the hydroxl residues Ser and Thr; exchange of the
acidic residues Asp and Glu; substitution between amide residues
Asn and Gln; exchange of the basic residues Lys and Arg; and
replacements among aromatic residues Phe and Tyr.
[0022] In addition, the invention features polypeptide sequences
having at least 75% identity with the polypeptide sequences 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 sequences illustrated herein.
[0023] As mentioned above, the invention also provides, in certain
embodiments, "dominant negative" polypeptides derived from the
polypeptides hereindisclosed. A dominant negative polypeptide is an
inactive variant of a protein, which, by interacting with the
cellular machinery, displaces an active protein from its
interaction with the cellular machinery or competes with the active
protein, thereby reducing the effect of the active protein. For
example, a dominant negative receptor which binds a ligand but does
not transmit a signal in response to binding of the ligand can
reduce the biological effect of expression of the ligand. Likewise,
a dominant negative catalytically-inactive kinase which interacts
normally with target proteins but does not phosphorylate the target
proteins can reduce phosphorylation of the target proteins in
response to a cellular signal. Similarly, a dominant negative
transcription factor which binds to another transcription factor or
to a promoter site in the control region of a gene but does not
increase gene transcription can reduce the effect of a normal
transcription factor by occupying promoter binding sites without
increasing transcription.
[0024] The end result of the expression of a dominant negative
polypeptide in a cell is a reduction in function of active
proteins. One of ordinary skill in the art can assess the potential
for a dominant negative variant of a protein, and using standard
mutagenesis techniques to create one or more dominant negative
variant polypeptides. For example, given the teachings contained
herein of iASPP polypeptides, one of ordinary skill in the art can
modify the sequence of iASPP polypeptides by site-specific
mutagenesis, scanning mutagenesis, partial gene deletion or
truncation, and the like. See, e.g., U.S. Pat. No. 5,580,723 and
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled
artisan then can test the population of mutagenized polypeptides
for diminution in a selected activity (e.g., p53 binding,
modulation of apoptosis) and/or for retention of such an activity.
Other similar methods for creating and testing dominant negative
variants of a protein will be apparent to one of ordinary skill in
the art.
[0025] According to a further aspect of the invention there is
provided a vector comprising a nucleic acid according to the
invention.
[0026] In a further preferred method of the invention said vector
is an expression vector conventionally adapted for gene
expression.
[0027] 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.
[0028] 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
and is therefore position independent). 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 environmental cues which include, by example and not
by way of limitation, intermediary metabolites (eg glucose,
lipids), environmental effectors (eg light, heat,).
[0029] Promoter elements also include so called TATA box and RNA
polymerase initiation selection (RIS) 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.
[0030] Adaptations also include the provision of selectable markers
and autonomous replication sequences which both 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.
[0031] 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 bicistronic
or multi-cistronic expression cassettes.
[0032] 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 in mice. LCRs include
regulatory elements that insulate transgenes from the silencing
effects of adjacent heterochromatin, Grosveld et al., Cell (1987),
51: 975-985.
[0033] These adaptations are well known in the art. 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).
[0034] According to a further aspect of the invention there is
provided a cell transformed or transfected with a nucleic acid
molecule or vector according to the invention.
[0035] Preferably, said host cells are eukaryotic cells, for
example, insect cells such as cells from a species Spodoptera
frugiperda using a baculovirus expression system. This expression
system is favoured in the instance where post-translational
modification of the polypeptide is required. Host cells and cell
lines, can be prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO
cells, COS cells, yeast expression systems and recombinant
baculovirus expression in insect cells). Especially useful are
mammalian cells such as human, mouse, hamster, pig, goat, primate,
etc. They may be of a wide variety of tissue types, and include
primary cells and cell lines. Specific examples include
keratinocytes, peripheral blood leukocytes, fibroblasts, bone
marrow stem cells and embryonic stem cells. The expression vectors
require that the pertinent sequence, i.e., those nucleic acids
described above, be operably linked to a promoter.
[0036] According to a further aspect of the invention the invention
there is provided a polypeptide according to the invention for use
as a pharmaceutical.
[0037] According to a further aspect of the invention there is
provided a nucleic acid according to the invention for use as a
pharmaceutical.
[0038] In a preferred embodiment of the invention said
pharmaceutical further comprises a a diluent, carrier or
excipient.
[0039] When administered, the therapeutic compositions 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.
[0040] The therapeutics of 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. When antibodies are used therapeutically, a preferred
route of administration is by pulmonary aerosol. Techniques for
preparing aerosol delivery systems containing antibodies are well
known to those of skill in the art. Generally, such systems should
utilize components which will not significantly impair the
biological properties of the antibodies, such as the paratope
binding capacity (see, for example, Sciarra and Cutie, "Aerosols,"
in Remington's Pharmaceutical Sciences, 18th edition, 1990, pp
1694-1712; incorporated by reference). Those of skill in the art
can readily determine the various parameters and conditions for
producing antibody aerosols without resort to undue
experimentation. When using antisense preparations of the
invention, slow intravenous administration is preferred.
[0041] 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 or can be monitored according to
diagnostic methods of the invention discussed herein.
[0042] 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.
[0043] The pharmaceutical compositions used in the foregoing
methods preferably are sterile and contain an effective amount of
dominant negative iASPP or nucleic acid encoding a dominant
negative iASPP, 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 the signal
transduction inhibited by the dominant negative iASPP-1,
composition via a reporter system as described herein, by measuring
downstream effects such as gene expression, or by measuring the
physiological effects of the iASPP composition, such as regression
of a tumour, decrease of disease symptoms, modulation of apoptosis,
etc.
[0044] The doses of dominant negative iASPP polypeptide or 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.
[0045] In general, doses of dominant negative iASPP are formulated
and administered in doses between 1 ng and about 500 mg, and
between 10 ng and 100 mg, according to any standard procedure in
the art. Where nucleic acids encoding dominant negative iASPP are
employed, doses of between 1 ng and 0.1 mg generally will be
formulated and administered according to standard procedures. Other
protocols for the administration of iASPP 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 iASPP 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.
[0046] 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.
[0047] iASPP 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.
[0048] 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.
[0049] The pharmaceutical compositions also may contain,
optionally, suitable preservatives, such as: benzalkonium chloride;
chlorobutanol; parabens and thimerosal.
[0050] 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.
[0051] 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 a syrup, elixir or an emulsion.
[0052] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous or non-aqueous preparation
of iASPP polypeptides or 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.
[0053] According to a further aspect of the invention there is
provided a transgenic non-human animal comprising a nucleic acid
according to the invention.
[0054] The invention also includes transgenic non-human animals. As
used herein, "transgenic non-human animals" includes non-human
animals having one or more exogenous nucleic acid molecules
incorporated in germ line cells and/or somatic cells. Thus the
transgenic animal include "knockout" animals having a homozygous or
heterozygous gene disruption by homologous recombination, animals
having episomal or chromosomally incorporated expression vectors,
etc. Knockout animals can be prepared by homologous recombination
using embryonic stem cells as is well known in the art. The
recombination can be facilitated by the cre/lox system or other
recombinase systems known to one of ordinary skill in the art. In
certain embodiments, the recombinase system itself is expressed
conditionally, for example, in certain tissues or cell types, at
certain embryonic or post-embryonic developmental stages, inducibly
by the addition of a compound which increases or decreases
expression, and the like. In general, the conditional expression
vectors used in such systems use a variety of promoters which
confer the desired gene expression pattern (e.g., temporal or
spatial). Conditional promoters also can be operably linked to
iASPP family nucleic acid molecules to increase expression of these
nucleic acid molecules in a regulated or conditional manner.
Trans-acting negative regulators of iASPP activity or expression
also can be operably linked to a conditional promoter as described
above. Such trans-acting regulators include antisense nucleic acids
molecules, nucleic acid molecules which encode dominant negative
molecules, ribozyme molecules specific for iASPP nucleic acids, and
the like. The transgenic non-human animals are useful in
experiments directed toward testing biochemical or physiological
effects of diagnostics or therapeutics for conditions characterized
by increased or decrease iASPP expression. Other uses will be
apparent to one of ordinary skill in the art.
[0055] According to a further aspect of the invention there is
provided the use of the polypeptide, or fragment thereof, in a
screening method for the identification of agents which inhibit the
binding of said polypeptide to p53.
[0056] According to a further aspect of the invention there is
provided a screening method to identify agents which inhibit the
binding of a polypeptide or fragment thereof to p53 comprising:
[0057] i) forming a preparation comprising [0058] a) a polypeptide
according to the invention; and [0059] b) a p53 polypeptide, or a
fragment thereof consisting of the binding site(s) for the
polypeptide in (a); [0060] ii) providing at least one agent to be
tested; and [0061] iii) determining the activity of the agent with
respect to the binding of the polypeptide in (a) to the polypeptide
in (b).
[0062] In a preferred method of the invention said agent is a
polypeptide, preferably a peptide.
[0063] In preferred method of the invention said peptide comprises
an amino acid sequence selected from the group consisting of:
GPEETD; DGPEETD; TTLSDG; AEFGDE; or PRNYFG.
[0064] In a preferred method of the invention said peptide is at
least 6 amino acid residues in length. Preferaby the length of said
peptide is selected from the group consisting of: at least 7 amino
acid residues; 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
amino acid residues in length. Alternatively the length of said
peptide is at least 20 amino acid residues; 30; 40; 50; 60; 70; 80;
90; or 100 amino acid residues in length.
[0065] In a further preferred method of the invention said peptide
consists of an amino acid sequence consisting of: GPEETD; DGPEETD;
TTLSDG; AEFGDE; or PRNYFG.
[0066] It will be apparent to one skilled in the art that
modification to the amino acid sequence of peptides agents 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 p53 binding
of iASPP. 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. 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,
N.sup.6-acetyllysine, N.sup.6-methyllysine,
N.sup.6,N.sup.6-dimethyllysine,
N.sup.6,N.sup.6,N.sup.6-trimethyllysine, cyclohexyalanine, D-amino
acids, ornithine. Other modifications include amino acids with a
C.sub.2, C.sub.3 or C.sub.4 alkyl R group optionally substituted by
1, 2 or 3 substituents selected from halo (eg F, Br, I), hydroxy or
C.sub.1-C.sub.4 alkoxy.
[0067] It will also be apparent to one skilled in the art that
peptides which retain p53 binding activity could be modified by
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.
[0068] In a further preferred method of the invention said
antagonist is an antibody or antibody binding part. Preferably said
antibody is a monoclonal antibody or binding part thereof.
[0069] Antibodies, also known as immunoglobulins, are protein
molecules which usually have specificity for foreign molecules
(antigens). 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 (.kappa. or
.lamda.), and one pair of heavy (H) chains (.gamma., .alpha., .mu.,
.delta. and .epsilon.), 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.
[0070] 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.
[0071] The H chains of Ig molecules are of several classes,
.alpha., .mu., .sigma., .alpha., and .gamma. (of which there are
several sub-classes). An assembled Ig molecule consisting of one or
more units of two identical H and L chains, derives its name from
the H chain that it possesses. Thus, there are five Ig isotypes:
IgA, IgM, IgD, IgE and IgG (with four sub-classes based on the
differences in the `constant` regions of the H chains, i.e., IgG1,
IgG2, IgG3 and IgG4). Further detail regarding antibody structure
and their various functions can be found in, Using Antibodies: A
laboratory manual, Cold Spring Harbour Laboratory Press.
[0072] In a preferred embodiment of the invention said fragment is
a Fab fragment.
[0073] In a further preferred embodiment of the invention said
antibody is selected from the group consisting of: F(ab').sub.2,
Fab, Fv and Fd fragments; and antibodies comprising CDR3
regions.
[0074] Preferably said fragments are single chain antibody variable
regions (scFV's) or domain antibodies. If a hybidoma 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. 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.
[0075] A modified antibody, or variant antibody, and reference
antibody, 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. Most highly preferred are variants which show enhanced
biological activity.
[0076] Preferably said antibody is a humanised or chimeric
antibody.
[0077] 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.
[0078] 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.
[0079] 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.
[0080] 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.
[0081] According to a further aspect of the invention there is
provided an isolated nucleic acid molecule wherein said molecule is
isolated from a nematode worm which nucleic acid molecule
hybridises a nucleic acid sequence as represented by FIG 1b.
[0082] In a preferred embodiment of the invention said nucleic acid
molecule hybridises under stringent hybridisation condtions.
Preferably said nematode worm is of the genus Caenorhabditis
spp.
[0083] According to a further aspect of the invention there is
provided an isolated polypeptide comprising the amino acid as
represented in FIG. 1b which polyeptide is modified by addition,
deletion or substitution of at least one amino acid residue.
[0084] According to a further aspect of the invention there is
provided a method of treatment of an animal comprising
administering an effective amount of a polypeptide or nucleic acid
or vector according to the invention wherein said effective amount
induces the apopoptic activity of p53.
[0085] In a preferred method of the invention said treatment is of
cancer.
[0086] According to an aspect of the invention there is provided a
peptide comprising an amino acid sequence selected from the group
consisting of: GPEETD; DGPEETD; TTLSDG; AEFGDE; or PRNYFG.
[0087] In a preferred embodiment of the invention said peptide is
at least 6 amino acid residues in length. Preferably the length of
said peptide is selected from the group consisting of: at least 7
amino acid residues; 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20 amino acid residues in length. Alternatively the length of
said peptide is at least 20 amino acid residues; 30; 40; 50; 60;
70; 80; 90; or 100 amino acid residues in length.
[0088] In a further preferred embodiment of the invention said
peptide consists of an amino acid sequence consisting of: GPEETD;
DGPEETD; TTLSDG; AEFGDE; or PRNYFG.
[0089] According to a further aspect of the invention there is
provided a pharmaceutical composition comprising at least one
peptide according to the invention and at least one anti-cancer
agent
[0090] In a preferred embodiment of the invention said said
anticancer 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.
[0091] According to a yet further preferred aspect of the invention
there is provided a complex comprising a peptide according to the
invention and at least one antibody, or active binding part
thereof.
[0092] In a preferred embodiment of the invention said antibody or
fragment is a cell specific antibody. Preferably said cell is a
cancer cell.
[0093] A complex of a peptide according to the invention and an
antibody which specifically binds a cell, preferably a cancer cell,
enables the targeting of said peptide to cell. Typically, the
complex would be internalised releasing the peptide intracellularly
to deliver the peptide to its target. Means to form a complex of
antibody and peptide are known in the art and include the use of,
for example bi-functional crosslinking agents, these may be
hetero-bifunctional or homo-bifunctional. The crosslinkers may be
reducible thereby facilitating release of said peptide(s). Cancer
specific cell markers are known in the art. For example, tumour
rejection antigen precursors. These include by example and not by
way of limitation the the MAGE, BAGE, GAGE and DAGE families of
tumour rejection antigens, see Schulz et al Proc Natl Acad Sci USA,
1991, 88, pp991-993 which is incorporated by reference.
[0094] According to an aspect of the invention there is provided a
method of treatment of an animal, preferably a human, wherein said
animal would benefit from the induction of apoptosis comprising
administering an effective amount of a peptide according to the
invention.
[0095] According to a further aspect of the invention there is
provided a method of treatment of an animal, preferably a human,
wherein said animal would benefit from the induction of apoptosis
comprising administering an effective amount of a composition
according to the invention
[0096] In a preferred method of the invention said treatment is
cancer treatment.
[0097] An embodiment of the invention will now be described by
example only and with reference to the following figures:
[0098] FIG. 1a is the nucleic acid sequence of human iASPP; FIG. 1b
is the C. elegans nucleic acid sequence of iASPP;
[0099] FIG. 2a is the amino acid sequence of human iASPP; FIG. 2b
is the C. elegans amino acid sequence of iASPP;
[0100] FIG. 3 illustrates that FITC labelled peptide (3a) DGPEETD
and (3b) TTLSDG can penetrate cells;
[0101] FIG. 4 illustrates the stimulation of the Bax promoter by
p53 after incubation with various peptides, in particular
DGPEETD;
[0102] FIG. 5 illustrates the stimulation of p53 transactivation in
a human tumour cell line U2SO after UV damage of DNA in the
presence of peptide DGPEETD;
[0103] FIG. 6 illustrates various experiments showing the
interaction of C. elegans iASPP and p53;
[0104] FIG. 7 illustrates a homology comparison between C. elegans
iASPP and human iASPP; and
[0105] FIG. 8 illustrates further experiments showing the
interaction of C. elegans iASPP and p53;
[0106] FIG. 9 illustrates the effect of RNAi on C. elegans iASPP
expression;
[0107] FIG. 10 illustrates p53 contact sites in iASPP;
[0108] FIG. 11 Influence of peptides on transactivation level of
Bax-Luc reporter. Cells were transfected with Bax reporter gene and
exposed to UV (20J/m2). Peptides were added for 24 hours in medium
at two different concentrations as indicated;
[0109] FIG. 12 illustrates the influence of peptides on Pig3
reporter activity for 12 hours. Transactivated cells were grown
with medium containing Arg-tagged peptides at two concentrations.
The left bar graph represents the transactivation activity at a
concentration of 25 uM and the second is with a concentration of 50
uM. Yap peptide is used as a control peptide here instead of p9.
Except for the control, cells were treated with cisplatin (3
ug/ml);
[0110] FIG. 13 illustrates the effects of various chemotherapeutic
drugs on p53 induced apoptosis;
[0111] FIG. 14a illustrates the effects of antisense or si-RNA of
ASPP1, ASPP2 or iASPP, in combination with the activity of the
anti-cancer agent doxorubicin; FIG. 14b illustrates the effects of
antisense or si-RNA of ASPP1, ASPP2 or iASPP in combination with
the activity of the anti-cancer agent cisplatin;
[0112] FIG. 15 illustrates the activity of inhibitory peptides in
combination with cisplatin on ASPP1, ASPP2 and iASPP.
MATERIALS AND METHODS
Cell Culture, Antibodies and Plasmids
[0113] Saos-2, MCF-7, and U2OS cells were grown in DMEM
supplemented with 10% FCS, 100 IU/ml penicillin-streptomycin and 2
mM glutamine. Anti-p53 antibodies DO-1 and DO-13 are monoclonal
antibodies while CM-1 is a rabbit polyclonal antibody specific to
p53. The V5 and 9E10 epitopes are recognised by the mouse
monoclonal antibodies V5 and 9E10 respectively. The mouse
monoclonal PC-10 is specific to the PCNA protein. 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.sup.1. Mouse and rabbit
antibody to iASPP (peptide RLQPALPPEAQSVPELEE) was produced as
described by Harlow and Lane .sup.13. All expression plasmids used
in this study were driven by the CMV immediate early promoter.
ASPP1, iASPP and Ce-iASPP are tagged with V5 epitope while Ce-p53
is tagged with 9E10 epitope.
DNA Transfection
[0114] The transfection mix included the DNA of interest in
1.times.HBS buffer (280 mM NaCl, 10 mM KCl, 1.4 mM
Na.sub.2HPO.sub.4.2H.sub.2O, 12 mM Glucose, 39 mM HEPES, adjusted
to pH 6.9-7.3) precipitated with 2.5 M CaCl.sub.2. The transfection
mix was added dropwise to the cells and washed off after 6 hours
with DMEM. 16-24 hours following the wash, cells were lysed either
in Reporter lysis buffer (Promega) for Luciferase assays and
western blots or in NP40 lysis buffer for western or
immunoprecipitation procedures.
Transactivation Assays
[0115] For transcriptional assay, 5.times.10.sup.5 Saos-2 cells
were plated 24 hr prior to transfection in 6 cm dishes. Various
combinations of plasmid DNA were transfected using the following
amounts. All transfection assays contain 1 .mu.g of reporter
plasmid. 50 ng of wild type human p53, 100 ng of plasmid expressing
C. elegans p53, 4 .mu.g of ASPP2, or 8 .mu.g of ASPP1, 5 .mu.g of
human iASPP or 7.5 .mu.g of Ce-iASPP were used as indicated. After
transfection, the cells were lysed in Reporter Lysis Buffer
(Promega, WI, USA) 16-24 hr post-wash and assayed using the
Luciferase Assay kit (Promega, WI, USA). The fold activation of a
particular reporter was determined by the activity of the
transfected plasmid above the activity of vector alone. The fold
increase of p53 transactivation activity by ASPP was obtained by
the activity p53 in combination with ASPP divided by the activity
of p53 alone on the promoters used in each assay.
Cell Transformation Assay
[0116] Rat Embryo Fibroblasts (REFs) obtained from Biowhittaker
were grown in DMEM to 50% confluence in a 90 mm dish. Cells were
then transformed as described previously .sup.14. Briefly, 2 .mu.g
of EJ ras 6.6, 2 .mu.g of pCE (E1A), 5 .mu.g of pCB6-16E, 5 .mu.g
of wild type human p53, 1 .mu.g or 5 .mu.g of human or C. elegans
iASPP were transfected into the REFS as indicated. All the cells
were transfected with the same amount of plasmid DNA expressing neo
gene. The transfected cells were then selected with 400 .mu.g/ml of
G418 and the morphologically transformed colonies were scored after
3-4 weeks after transfection.
Flow Cytometry
[0117] For FACS analysis, 10.sup.6 Saos-2 cells were plated 24-48 h
prior to transfection in 10 cm plates. The cells were then
transfected with 2 .mu.g of a plasmid expressing CD20. CD20
expression was used as a transfection marker. The transfections
consisted of 1 .mu.g of human p53 or 4 .mu.g of Ce-p53, 10 .mu.g of
ASPP1 and ASPP2, 2 .mu.g of Bax, 15 .mu.g of anti-sense iASPP, 7.5
.mu.g-10 .mu.g of human iASPP or 7.5 .mu.g of Ce-iASPP plasmid as
indicated. 36 hours after the transfection, both attached and
floating cells were harvested using 4 mM EDTA/PBS and stained with
FITC conjugated anti-CD20 antibody CD20Leu. For each experiment,
one dish of cells was transfected with the control vector only
without CD20. These cells were later stained with antibody CD20Leu
under the same conditions as those co-transfected with CD20 plasmid
to serve as a negative control. The cells lacking expression of
CD20 plasmid were used to set the base line to allow the gating of
the CD20 positive (and hence transfected) cells. After staining
with the antibody CD20Leu, the cells were fixed and stained with
propidium iodide. The DNA content of all the cells expressing CD20
was analysed using the flow cytometer (Becton Dickson) as
described.sup.15.
Protein Biochemistry
[0118] For western blotting, cells grown in monolayers were washed
with 1.times.PBS and lysed in either NP40 lysis buffer (1% Nonidet
P40, 50 mM Tris pH8.0, 150 mM NaCl, 1 mM EDTA pH 8.0) or luciferase
reporter lysis buffer. The protein concentration of the cell
extracts was determined against a standard curve using the BioRad
protein assay system (BioRad). Between 15-100 .mu.g of extract was
mixed with 5.times.sample buffer and loaded on SDS-PAGE gels. The
gels were wet transferred on to Protran nitrocellulose membrane and
the resulting blots blocked in 10% reconstituted milk powder in
1.times.PBS. Subsequently the blots were incubated with primary
antibody prepared either in tissue culture medium or as undiluted
hybridoma supernatant and incubated with the appropriate secondary
HRP conjugated antibody (Dako). In between each stage the blot was
washed with repeat changes of TBST (10 mM Tris pH 8.0, 150 mM NaCl,
0.5% Tween 20). The blot was exposed to hyperfiln following the use
of ECL substrate solution (Amersham Life Science).
[0119] For immunoprecipitation cells were lysed in NP40 lysis
buffer on ice for 30 minutes and pre-cleared with protein G beads
for 1 hour at 4 C. The protein concentration was determined and
1000 .mu.g of the extract was incubated with antibody pre-bound to
protein G beads for 4 hours at 4 C. The beads were washed twice in
NP40 lysis buffer and twice in NET buffer (50 mM Tris pH 8.0, 150
mM NaCl, 1 mM EDTA pH 8.0). The IP beads were mixed with
5.times.sample buffer and loaded onto a SDS-PAGE gel.
In Vitro Translation and In Vitro Immunoprecipitation
[0120] ASPP family members and p53 were in vitro translated and
labelled with .sup.35S--Methionine using the TNT T7 Quick coupled
Transcription/Translation System (Promega). For the experiments
shown in FIG. 1E, 15, 30 and 45 .mu.l of in vitro translated
lysates of iASPP were added in addition to p53 and ASPP2. Rabbit
anti-p53 antibody CM1 was used to detect the presence of unlabelled
p53.
[0121] For FIG. 6A, 5-10 .mu.l of human iASPP lysate (unlabelled)
was incubated with 15 .mu.l of the lysate containing the in vitro
translated Ce-p53. The mixture of proteins was allowed to
co-translate at 30 C for 1 hr. 200 .mu.l of phosphate-buffered
saline (PBS) was then added to the mixture of proteins and
incubated at 4 C for a further hour on a rotating wheel. The
anti-V5 antibody irmnobilised on protein G agarose beads was added
to the binding reactions and incubated on a rotating wheel at 4 C
for 16 hours. The beads were then washed with PBS. The bound
proteins were released in SDS gel sample buffer and analysed by 10%
SDS-polyacrylamide gel electrophoresis (PAGE). Ce-p53 was detected
by autoradiography and Human iASPP was detected by anti-V5 antibody
following a western blot. For the rest of the figures, the proteins
were labelled with .sup.35S -Methionine and immunoprecipitated as
above using the indicated antibodies. Results were visualised using
autoradiography.
[0122] In vivo Labelling of Cells with .sup.35S -Methionine and
.sup.35S -Cysteine
[0123] U20S cells in the absence or presence of transfected
plasmids (as indicated in FIG. 1A and 2C) were washed with PBS and
then incubated with 250 .mu.ci/ml of .sup.35S--methionine and 250
.mu.ci of 35-cysteine in DMEM lacking both methionine and cysteine
for 2 hours at 37C. Cells were then washed with PBS before
harvesting. For FIG. 2C, twenty four hours after transfection the
cells were in vivo labelled with .sup.35S-methionine and
.sup.35S-cysteine for 2hrs. Cells expressing CD20 (transfected
cells) were stained with FITC-conjugated anti-CD20 antibody. A
biotin conjugated anti-FITC antibody was then added to the cell
pellet, and after the incubation, the cells were mixed with
streptavidin conjugated magnetic beads to isolate the CD20
expressing cells. The cells were then lysed with NP40 lysis buffer
and the proteins immunoprecipitated with mouse anti iASPP antibody.
The immunoprecipitates were washed with NET buffer and resolved by
SDS-PAGE and autoradiography.
Cell Fixation
[0124] Monolayers of cells were grown in 30 mm dishes and washed
with 1.times.PBS. Cells were fixed with 1 ml of 4%
para-formaldehyde for 15 minutes and then washed in 1.times.PBS. 1
ml of 0.2% Triton-X100 in 1.times.PBS was used to permeabilise the
cells for 2 minutes and this was washed off with three washes of
1.times.PBS. Primary antibody was prepared in tissue culture medium
at the appropriate concentration and added to the dishes for 3
hours. The dishes were washed with 1.times.PBS and the secondary
antibody of either anti-rabbit TRITC (Tetramethyl rhodamine
isothiocyanate) or anti-mouse FITC (Fluorescein isothiocyanate)
prepared in tissue culture medium at the manufacturers recommended
dilution (Sigma, UK) and added to the dishes for one hour. The
cells were washed in 1.times.PBS and left to air dry. Citifluor
shielding agent (Citifluor, UK) was applied as a drop to the
surface of the cells and a cover slip placed on top. A drop of
immersion oil on top of the cover slip allowed the immunocomplexes
to be visualised using a Zeiss Axiophot fluorescence microscope.
Antibodies 9E10 and V5 were used to detect the expression of
epitope tagged Ce-p53 (9E10), human iASPP (V5) and Ce-iASPP (V5)
respectively. Human p53 was detected by DO.1 antibody.
Cloning Ce-p53 and Ce-iASPP cDNA
[0125] cDNAs carrying the complete coding regions of Ce-ape-1
(iASPP) and Ce-cep-1 (p53) were generated by RT-PCR using the
Promega Access kit, cloned into the vector pCR4-TOPO (Invitrogen)
and sequenced. C. elegans p53 and iASPP were then subcloned into a
mammalian expression vector pcDNA3 in frame with the epitope of
9E10 and V5 respectively. The full-length Ce-ape-1 is predicted to
be trans-spliced to SL1 (Y. Kohara, unpublished).
RNA Interference (RNAi) and Cell Corpse Assays
[0126] RNAi was performed by feeding or microinjection using
established procedures (Fire et al., 1998; Timmons and Fire, 1998)
In order to eliminate Ce-iASPP and Ce-p53 activity by RNAi, N2
animals were first subjected to Ce-iASPP RNAi feeding.
Subsequently, 20 F1 animals were removed from the feeding plate,
injected with Ce-p53 dsRNA and returned to independent Ce- iASPP
dsRNA feeding plates. The F2 progeny of animals fed Ce-iASPP (+/-
injection of Ce-p53 dsRNA) were stained with SYTO12 and apoptotic
corpses scored as described12. Hence, because all animals have been
subjected to Ce-iASSP feeding RNAi, any differences in the mean
number of apoptotic cell corpses detected in the two populations
are likely to result from Ce-p53 dsRNA injection.
EXAMPLES
iASPP is the Most Conserved Member of the ASPP Family
[0127] Sequence analysis indicates that the C. elegans p53 gene,
cep-1, is a distant member of the p53 family, however, the residues
critical for ASPP and DNA binding activity appear to be
conserved.sup.3,4 Hence, we searched the C. elegans genome for an
ASPP homologue and found that F46F3.4 is the only C. elegans gene
encoding a protein with significant sequence homology to all three
members of the ASPP family. The gene corresponding to F46F3.4 has
been named ape-1 (for apoptotic enhancer) based on the mutant
phenotype produced by ape-1(RNAi) (see below); however, the protein
product will henceforth be referred to as Ce-iASPP. Ce-iASPP
consists of 769 amino acids; sequence comparisons reveal that the
C-terminus of Ce-iASPP is the region most conserved with other ASPP
members (FIG. 7). It was previously shown that the C-terminus of
ASPP2 interacts with p53. Moreover, all but one (6 out of 7)
residue involved in this interaction are conserved in both iASPP
and Ce-iASPP. Taken together these results suggested that Ce-iASPP
might interact with both human and C. elegans p53. This was tested
in vitro by co-immunoprecipitation. As shown in FIG. 8B, Ce-iASPP
interacts with both human and Ce-p53. The interaction between
Ce-p53 and Ce-iASPP was further confirmed by reciprocal
immunoprecipitation (FIG. 8B, right panel).
[0128] Ce-iASPP contains the hallmark ankyrin repeats and SH3
domain found in other ASPP family members and is expressed in the
cytoplasm and nucleus of human cells (FIG. 8C). The expression of
human iASPP is predominantly nuclear but cytoplasmic staining is
also detectable. Both human and C. elegans p53 are primarily
expressed in the nucleus of transfected human cells (FIG. 8C).
Since ASPP and iASPP can positively and negatively regulate the
apoptotic function of p53, respectively, we tested the effect of
Ce-iASPP on the activities of p53. When Ce-iASPP was co-expressed
with human p53 in mammalian cells, it produced a small reduction in
the transactivation and apoptotic function of p53, presumably by
inhibiting endogenous ASPP function. However, in the presence of
ASPP1 or ASPP2, co-expression of Ce-iASPP prevented ASPP1 or ASPP2
from stimulating the transactivation and apoptotic function of p53
to the same extent as human iASPP (FIG. 8D). Furthermore, the
ability of human and C. elegans iASPP to inhibit apoptosis is
p53-dependent since they both failed to inhibit Bax induced
apoptosis under the same conditions (FIG. 8E). The ability of iASPP
to inhibit the activity of p53 is not due to the reduced expression
of p53 (FIG. 8F). Like human iASPP, the Ce-iASPP also has oncogenic
activity. The expression of Ce-iASPP enhanced the transforming
activity of ras and E1A. Moreover, expression of Ce-iASPP inhibited
the suppressor function of wild type human p53 (FIG. 8G). These
results demonstrate that Ce-iASPP is more likely to be an
orthologue of human iASPP than of ASPP. Also, the ability of
Ce-iASPP to inhibit p53 suggests that the regulation of p53
apoptotic function by the ASPP family of proteins is evolutionarily
conserved.
The Regulation of p53 by ASPP Family of Proteins is Evolutionarily
Conserved
[0129] It was unclear whether Ce-p53 could induce apoptosis in
mammalian cells because of the limited sequence similarity between
human and C. elegans p53, although most of the Ce-p53 residues that
contact ASPP are conserved. If Ce-iASPP inhibits the activities of
human p53 in a manner similar to that of human iASPP, this argues
that the regulation of p53 by the ASPP family has been
evolutionarily conserved. This further suggests that the activities
of Ce-p53 could be subject to regulation by the ASPP family of
proteins. To address these issues, Ce-p53 was tested for its
ability to interact in vitro with members of the human ASPP family
by co-immunoprecipitation. As shown in FIG. 6A, Ce-p53 interacts
with ASPP2 and iASPP. The expression of Ce-p53 induced apoptosis in
human cells with an efficiency similar to human p53. Remarkably,
expression of human ASPP, ASPP2 in particular, significantly
enhanced the ability of Ce-p53 to induce apoptosis to an extent
similar to human p53 and the expression of human iASPP also
inhibited the apoptotic function of Ce-p53 (FIG. 6B). In addition,
both human and C. elegans iASPP inhibited Ce-p53 induced apoptosis
to the same extent (FIG. 6C). The ability of Ce-p53 to
transactivate p53 target gene promoters, such as Bax-luc, was also
tested and was found to be much lower than that of human p53.
Interestingly, co-expression of ASPP2 and Ce-p53 resulted in a very
small but detectable increase in the transactivation function of
Ce-p53, indicating that the human ASPP family could regulate Ce-p53
in a manner similar to that of human p53 (FIG. 6D). The small
increase in the transactivation function of Ce-p53 by ASPP2 is not
observed on the mdm2 promoter. All of these results suggest that
the residues conserved between human and C. elegans p53 are crucial
and sufficient for the apoptotic function of p53 and for p53 to be
regulated by the ASPP family.
iASPP is an Evolutionarily Conserved Inhibitor of p53 In Vivo
[0130] Like human p53, one of the most important functions of C.
elegans p53 is its ability to induce apoptosis in germ cells in
response to DNA damage .sup.3,4. Knowing that co-expression of
human or C. elegans iASPP can inhibit the apoptotic function of p53
in mammalian cell lines, we hypothesised that expression of
Ce-iASPP might similarly protect C. elegans germ cells from death
by apoptosis. This question was addressed in vivo using RNA
mediated interference (RNAi) .sup.5. Depletion of endogenous
Ce-iASPP increased the number of germ cells undergoing apoptosis,
indicating that the normal function of Ce-iASPP is to inhibit
apoptosis (FIG. 9A, lanes 1 and 5, 6). The enhancement of germ cell
apoptosis caused by depletion of Ce-iASPP was not detected when
RNAi was performed in a mutant lacking the C. elegans CED-3
caspase, indicating that the core apoptotic machinery is involved
in this process (FIG. 9A, lanes 3 and 4) .sup.6. We also obtained
additional support for the hypothesis that the primary role of
Ce-iASPP is to inhibit the pro-apoptotic activity of Ce-p53, which
is normally stimulated in response to genotoxic stress .sup.3,4.
First, it was found that the increase in the number of C. elegans
germ cells undergoing apoptosis after depletion of Ce-iASPP was
abrogated by simultaneously depleting Ce-p53 by RNAi (FIG. 9A,
compare lanes 5, 6 and 8). Moreover, the depletion of both Ce-iASPP
and Ce-p53 by RNAi did not completely eliminate apoptosis, but
instead returned the number of germ cells undergoing apoptosis to
wild-type physiological levels. Second, the increase in the number
of apoptotic germ cell corpses detected after wild type worms were
exposed to 100-Gy IR was no greater than that observed after
depletion of Ce-iASSP by RNAi in the presence or absence of
exposure to 100-Gy IR (FIG. 9A, lanes 2 and 7). These results
clearly demonstrate that iASPP is an important inhibitor of p53
function in C. elegans, although we can not exclude the possibility
that a genetic knockout might reveal that Ce-iASPP has additional
activities. Since the regulation of p53 by the ASPP family is
highly conserved, it is likely that iASPP is also a key inhibitor
of p53 in other organisms including humans.
[0131] We show here that iASPP is the most phylogenetically
conserved inhibitor of p53, so far identified, and also the most
evolutionarily conserved member of the ASPP family. Remarkably, the
ability of ASPP family members to regulate the apoptotic function
of p53 has been conserved between C. elegans and human. This argues
that the apoptotic function of p53 is likely to be more conserved
than its ability to induce cell cycle arrest; this agrees with
recent observations showing that ectopic expression of both C.
elegans and Drosophila p53 induces apoptosis but not cell cycle
arrest .sup.3,4,7,8. In C. elegans p53-mediated apoptosis appears
to play an important role in maintaining the fidelity of germ
cells, which might have incurred DNA damage .sup.3,4.
Interestingly, the most important tumour suppressor function of p53
is also linked to its ability to induce apoptosis. Therefore, ASPP
family members, the evolutionarily conserved regulators of p53,
must play a critical role in tumourigenesis.
The Oncoprotein iASPP
[0132] iASPP shares more sequence similarity with the N-terminally
truncated ASPP2 mutant 53BP2 than the full-length ASPP. Expression
of iASPP inhibited the apoptotic function of p53. Like 53BP2, the
most profound effect of iASPP on the apoptotic function of p53 is
mediated through its ability to act as a competitor of ASPP.
However, in C. elegans, iASPP is the only gene that has homology to
the human ASPP family. Thus iASPP directly inhibits the apoptotic
function of p53 in C. elegans. A similar mechanism might also apply
in mammalian cells. In agreement with this, iASPP antisense RNA
induced a 3 to 5-fold increase in apoptotic cells in U2OS and MCF7
cells. In this latter model, ASPP could stimulate the apoptotic
function of p53 by removing the negative effects that iASPP imposed
on p53. The failure of iASPP antisense RNA to produce a significant
increase of apoptosis in cisplatin treated U2OS and MCF7 cells
might be due to the fact that cisplatin stimulates the apoptotic
function of p53 by increasing the activities of ASPP. This, in
turn, might explain why the expression of ASPP antisense RNA
generated a profound inhibitory effect on apoptosis induced by
cisplatin. It is also under this condition that the anti-apoptotic
function of iASPP is most pronounced. Thus the apoptotic function
of p53 is negatively regulated by iASPP and positively regulated by
ASPP. The competition between these two opposing signals could
determine the apoptotic state of p53 and ultimately cell fate.
Regardless of whether iASPP acts as a dominant negative regulator
of ASPP or a direct inhibitor of p53, a competition between iASPP
and ASPP for binding to p53 is critical for the apoptotic function
of p53. Consistent with this model, a change in the percentage of
p53 complexed with ASPP2 was seen in response to DNA damage. It is
likely that the percentage of p53 complexed with iASPP and ASPP
would be regulated by signals that induce death or survival.
[0133] Being an inhibitor of p53, iASPP enhanced the transforming
activities of oncogenes such as ras plus E7 or E1A of human
papilloma virus and adenovirus but not ras plus mutant p53. This is
particularly interesting since E7 and E1A are known to induce
p53-dependent apoptosis .sup.9. While E7 and E1A can bind and
inactivate the tumour suppressor function of Rb, their oncogenic
function is largely reduced due to their ability to activate
p53-dependent apoptosis. Proteins that can inhibit apoptosis
induced by E7 and E1A would enhance the oncogenic function of E7
and E1A. Therefore, like the dominant negative p53 mutants, iASPP
was able to stimulate the oncogenic function of E7 and E1A by
inhibiting the apoptotic function of p53. It is important to point
out that iASPP was not as active as mutant p53, p53H175 or p53L173
in co-operating with ras to transform REFs under the experimental
conditions described here. Part of the reason was due to the low
expression level of iASPP in the assay (data not shown). However,
the differences in the transforming activities of iASPP and mutant
p53 might also be caused by other known activities of mutant p53,
which are independent of its ability to act as a simple dominant
negative inhibitor of p53. Nevertheless the ability to confer
cellular resistance to the cytotoxic effects of UV and cisplatin
suggested that the overexpression of iASPP would be selected for in
human tumours expressing wild type p53. Consistent with this, iASPP
expression is increased in human breast carcinomas expressing wild
type p53. The majority of tumours (7 out of 8) expressing high
levels of iASPP also express wild type p53 and normal levels of
ASPP, indicating that iASPP is an inhibitor of ASPP in vivo. Our
previous study showed that the expression levels of ASPP1 and ASPP2
were down regulated in 60% of human breast tumours express wild
type p53. Taken together, the abnormal expression of ASPP family
members would account for almost 80% of human breast carcinomas
examined. The ASPP family members are encoded by three different
genes located on different chromosomes (data not shown). We do not
know why the frequency of human breast carcinomas showing down
regulation of ASPP is much higher than those showing increased
expression of iASPP. However, it is possible that the expression
pattern of the ASPP family members varies in different types of
human tumours. The percentage of tumours with altered ASPP
expression could also differ in various tumour types. Nevertheless,
inhibiting the oncogenic function of iASPP could provide an
important new strategy to treat tumours expressing wild type
p53.
Evolutionarily Conserved Regulation of p53 by the ASPP Family
[0134] Sequence comparison reveals that there is 38% identity
between the human and C. elegans iASPP amino acid sequences; within
the ankyrin repeats and SH3 domain, the homology is as high as 78%
(residues 154-227 of human iASPP and 557-630 of Ce-iASPP, 55/74
residues are similar). Most of the iASPP residues contacting p53
are conserved. The structural conservation between human and C.
elegans iASPP is reflected by their ability to regulate p53
function in human cells. Like human iASPP, C. elegans iASPP
interacts with and inhibits the transactivation and apoptotic
function of human p53 in cell lines. The conservation of ASPP/p53
regulation is further demonstrated in a study of C. elegans p53 in
human cells. It is interesting and important to point out that the
sequence homology between human and C. elegans p53 is very limited
(13.7% identity at protein level). The highest level of p53
homology between the two species is around 50% in a very limited
region (residues 9/18 residues are similar). However, many of the
ASPP2 contact residues identified from a crystal structure.sup.10
are conserved between human and C. elegans p53 (5 out of 8 residues
are conserved). The ability of C. elegans p53 to interact with
human ASPP family members in vitro highlights the importance of
these conserved residues.
[0135] Remarkably, C. elegans p53 induces apoptosis very
effectively in human cells. Similar to human p53, the apoptotic
function of C. elegans p53 is positively and negatively regulated
by the human ASPP and iASPP, respectively. These results
demonstrate for the first time that the apoptotic function of p53
is conserved despite the limited sequence homology between human
and C. elegans p53. The few key residues conserved between human
and C. elegans p53 are sufficient for ASPP family members to
regulate the apoptotic function of p53 both in vitro and in
vivo.
[0136] The C. elegans p53 showed little ability to transactivate
human p53 target genes such as Bax and PIG3 in comparison to human
p53 (data not shown). Even though Bax is frequently a p53 target
gene during apoptosis, it is not the only target gene that is
required for p53 induced apoptosis. p53 is known to transactivate
over 20 pro-apoptotic genes none of which has so far proved to be
indispensable in p53 induced apoptosis. There are over 4000
putative p53 target genes in the human genome and many of them are
pro-apoptotic genes .sup.11. It is possible that Ce-p53 might
transactivate some of these other human p53 target genes as
effectively as human p53. Unfortunately none of the ones we tested
so far belong to this category. Although transactivation of the
human Bax-luc reporter in human cells by Ce-p53 is not very strong,
it is important to point out that the co-expression of ASPP
stimulated the transactivation function of Ce-p53 only on the
promoters of Bax but not on mdm2. This pattern of ASPP action is
similar to that seen with human p53. Alternatively, Ce-p53 might
induce apoptosis independent of its transcriptional activity; human
p53 is known to induce apoptosis through both transcriptional
dependent and independent pathways .sup.12. Regardless of how
Ce-p53 induces apoptosis in human cells, the most remarkable and
important fact is that the human ASPP family of proteins regulate
the apoptotic function of both Ce-p53 and human p53 in a similar
way. Ce-iASPP is also able to completely replace human iASPP in all
the assays performed in human cells. These results argue strongly
that the regulation of p53 function by ASPP family is conserved
from worm to human. The link between p53 and the ASPP family
suggests that the regulation of p53 by the ASPP family should be
the future target in developing new strategies for cancer therapy.
Tumours expressing wild type p53 can be sensitised to treatments by
enhancing the activity of ASPP and removing the activity of
iASPP.
Chemotherapy Drugs Appear to Modify ASPP Family Protein
Function
[0137] As 50% of human tumours maintain wild-type p53, one approach
to treating cancer might be to reactivate p53 in tumour cells to
induce apoptosis. Indeed, chemotherapy and radiotherapy agents
generate DNA damage, which could activate functional p53 in tumour
cells. However, the pathways affected by these drugs differ and
tumour cells can develop protective strategies to enable survival.
As mentioned previously, ASPP1 and ASPP2 specifically enhance the
apoptotic function of p53 by stimulating the DNA binding and
transactivation function of p53 on the promoters of proapoptotic
genes (Samuels-Lev et al., 2001), while iASPP is a very conserved
inhibitor of p53 (Bergamaschi et al., 2003). Taken together, this
suggests that the function of the ASPP family of proteins may be
affected by chemotherapy agents, which in turn vary the subsequent
p53 apoptotic response, and thus play a role in the effectiveness
of drugs used in cancer treatment.
[0138] Specifically, experiments were carried out to investigate
the hypothesis that chemotherapy drugs could be made more effective
in cell lines undergoing DNA damage by modulating the activity of
the ASPPs.
[0139] Chemotherapy agents were used to investigate whether the
apoptosis induced in cell lines correlated directly with p53
expression levels. U-2OS and MCF7 cells were transfected with the
Pig3 promoter (Pig3-luc 17 mer) and treated with various
chemotherapy drugs (doxorubicin, cisplatin, etoposide, colchicine,
deferoxamine mesylate (DFO), daunorubicin, camptothecin and
5'fluorouracil).
[0140] Luciferase levels were assayed 24 hours later, FIG. 13. The
results illustrate that drug-induced apoptosis, as measured by
luciferase, did not correlate directly with p53 protein levels (for
example, compare doxorubicin and cisplatin responses) indicating
that p53 alone is not responsible for the activation of
proapoptotic genes. It is possible that the ASPP proteins respond
differently to the various chemotherapy drugs to give the
discrepancy observed between Pig3 activation and p53 protein
expression level.
[0141] To test whether the ASPPs play a role in the process, short
interfering RNA (si-RNA) (Elbashir et al., 2001) and antisense RNA
were used to knock down ASPP gene expression. Sequences to generate
si-RNA were cloned into pSuper plasmid (Brummelkamp et al., 2002).
Since responses of proapoptotic genes differed depending on the
drug used, both doxorubicin and cisplatin were tested in U-2OS
cells. Pig3-luciferase was transfected together with either
antisense or si-iASPP (FIGS. 14A and 14B). The results show that
antisense ASPP1 or antisense ASPP2 both reduce the induction of
Pig3-luciferase by doxorubicin (FIG. 14A) but antisense or si-RNA
for iASPP caused no reduction. A si-RNA for p53 prevented the
doxorubicin response (FIG. 14A). In contrast, antisense ASPP1 or
antisense ASPP2 had little effect on the cisplatin induction of the
Pig3 promoter but the si-RNA for iASPP caused a significant
increase. Again siRNA for p53 prevented the cisplatin response.
[0142] These data could be interpreted to indicate that endogenous
p53 activity was induced by doxorubicin cooperating with ASPP1 and
ASPP2 in U-2OS cells but in response to cisplatin endogenous p53
activity was being restrained by iASPP. These differential effects
might be due to the modulatory effects of the drugs on ASPP or
iASPP activity.
The Disruption of iASPP/p53 Binding can Enhance Apoptosis and Could
Potentially be Used in Cancer Therapy
[0143] Inhibiting the p53/iASPP interaction with synthetic
molecules should lead to p53-mediated apoptosis in p53-positive
stressed cells.
[0144] It is known that p53 binds to the C terminal part of iASPP;
specific contact amino acids are indicated in FIG. 10. Three
different peptides containing particular iASPP sequences were
designed to inhibit the iASPP/p53 binding and test the effects on
proapoptotic genes, FIG. 10. The three peptides were linked to a
Tat sequence and to FITC.
[0145] The peptides were initially tested for their effects on p53
transactivation of the Bax promoter using UVradiation to activate
p53, FIG. 11. The luciferase activity of the Bax promoter was only
slightly affected by the control peptide, which should not affect
iASPP & p53 interaction. However, addition of peptide 3, 6 or 7
caused about a two fold increase in the reporter activity, using 50
.mu.M peptide. Some peptides were less active at 100 .mu.M.
[0146] The study was continued with the Pig3-luciferase reporter.
The Pig3 gene is transactivated by p53 in a different way to Bax.
Whereas Bax is transactivated by a conventional transcription
domain (El-Deiry et al., 1992) (Bourdon et al., 1997), p53 binds to
the Pig3 promoter via repeat sequences (TGYCC)n, where Y=C or T
(Polyak et al., 1997). The number of repeats is polymorphic within
populations (Contente et al., 2002). Two reporters containing 10 or
17 repeats were used in case there was a difference in response
between them. Other variables to consider are time and
concentration of peptide and the location of the peptides in the
cells since p53 and iASPP are mostly nuclear proteins.
[0147] A second generation of peptides was used for this
experiment, which are identical to the previous ones except for the
tag. These peptides were tagged with nine arginine residues, which
are thought to give the peptides better penetration into cells
(Lindsay, 2002). In this case a peptide 7 from the ASPP2 sequence
was included (see FIG. 10) and a different control peptide (Yap)
was used. Cells were treated with cisplatin and peptides were
applied for 12 hours at two different concentrations (25 uM and 50
uM) and the results are shown in FIG. 15. Despite some variability
in the results the P3-iASPP or P7-iASPP peptides showed a
substantial increase in the reporter activity over the response
seen with control Yap peptide and cisplatin treatment, suggesting
that apoptosis induced during chemotherapy could be enhanced
through the use of iASPP peptide inhibitors. TABLE-US-00001 TABLE 1
Human Mouse C.El. Drosophila Fugu I Fugu II Fugu III Fugu IV Human
(352) X X X X X X X X Mouse (260) 93.7 X X X X X X X C.El. (769)
20.4 38.8 X X X X X X Drosophila (1071) 43.2 40.0 32.4 X X X X X
Fugu I (260) 51.9 45.0 48.1 55.4 X X X X Fugu II (252) 54.8 54.8
51.6 58.7 X X X X Fugu III (144) 54.2 53.5 54.2 64.6 X X X X Fugu
IV (132) 51.5 50.8 55.3 62.9 X X X X
[0148]
Sequence CWU 1
1
9 1 6 PRT Artificial sequence p53 inhibitor peptide 1 Gly Pro Glu
Glu Thr Asp 1 5 2 7 PRT Artificial Sequence p53 inhibitor peptide 2
Asp Gly Pro Glu Glu Thr Asp 1 5 3 6 PRT Artificial Sequence p53
inhibitor peptide 3 Thr Thr Leu Ser Asp Gly 1 5 4 6 PRT Artificial
Sequence p53 inhibitor peptide 4 Pro Arg Asn Tyr Phe Gly 1 5 5 18
PRT Artificial Sequence p53 inhibitor peptide 5 Arg Leu Gln Pro Ala
Leu Pro Pro Glu Ala Gln Ser Val Pro Glu Leu 1 5 10 15 Glu Glu 6
2156 DNA Homo sapiens 6 gcggccgcgt cgacccggcg ttcagacgcg ggcagctacc
ggcgctcgct gggctccgcg 60 gggccgtcgg gcactttgcc tcgcagctgg
cagcccgtca gccgcatccc catgcccccc 120 tccagccccc agccccgcgg
ggccccgcgc cagcgtccca tccccctcag catgatcttc 180 aagctgcaga
acgccttctg ggagcacggg gccagccgcg ccatgctccc tgggtccccc 240
ctcttcaccc gagcaccccc gcctaagctg cagccccaac cacaaccaca gccccagcca
300 caatcacaac cacagcccca gctgccccaa cagccccaga cccaacccca
aacccctacc 360 ccagcctccc acatccgcat ccccaacaga catggccccc
tgtgaacgaa ggacccccca 420 aaccccccac cgagctggag cctgagccgg
agatagaggg gctgctgaca ccagtgctgg 480 aggctggcga tgtggatgaa
ggaccctgta gcaaggcctc tcagccccac gaggctgcag 540 ccagcactgc
caccggaggc acagtcggtg cccgagctgg aggaggtggc acgggtgttg 600
gcggaaattc cccggcccct caaacgcagg ggctccatgg agcaggcccc tgctgtggcc
660 ctgcccccta cccacaagaa acagtaccag cagatcatca gccgcctctt
ccatcgtcat 720 ggggggccag ggcccggggg gcggagccag agctgtcccc
catcactgag ggatctgagg 780 ccagggcagg gccccctgct cctgccccac
cagctcccat tccaccgccc ggccccgtcc 840 cagagcagcc caccagagca
gccgcagagc atggagatgc gctctgtgct gcggaaggcg 900 ggctccccgc
gcaaggcccg ccgcgcgcgc ctcaaccctc tggtgctcct cctggacgcg 960
gcgctgaccg gggagctgga ggtggtgcag caggcggtga aggagatgaa cgacccgagc
1020 cagcccaacg aggagggcat cactgccttg cacaacgcca tctgcggcgc
caactactct 1080 atcgtggatt tcctcatcac cgcgggtgcc aatgtcaact
cccccgacag ccacggctgg 1140 acacccttgc actgcgcggc gtcgtgcaac
gacacagtca tctgcatggc gctggtgcag 1200 cacggcgctg caatcttcgc
caccacgctc agcgacggcg ccaccgcctt cgagaagtgc 1260 gacccttacc
gcgagggtta tgctgactgc gccacctacc tggcagacgt cgagcagagt 1320
atggggctga tgaacagcgg ggcagtgtac gctctctggg actacagcgc cgagttcggg
1380 gacgagctgt ccttccgcga gggcgagtcg gtcaccgtgc tgcggaggga
cgggccggag 1440 gagaccgact ggtggtgggc cgcgctgcac ggccaggagg
gctacgtgcc gcggaactac 1500 ttcgggctgt tccccagggt gaagcctcaa
aggagtaaag tctagcagga tagaaggagg 1560 tttctgaggc tgacagaaac
aagcattcct gccttccctc cagacctctc cctctgtttt 1620 ttgctgcctt
tatctgcacc cctcaccctg ctggtggtgg tccttgccac cggttctctg 1680
ttctcctgga agtccaggga agaaggaggg ccccagcctt aaatttagta atctgcctta
1740 gccttgggag gtctgggaag ggctggaaat cactggggac aggaaaccac
ttccttttgc 1800 caaatcagat cccgtccaaa gtgcctccca tgcctaccac
catcatcaca tcccccagca 1860 agccagccac ctgcccagcc gggcctggga
tgggccacca caccactgga tattcctggg 1920 agtcactgct gacaccatct
ctcccagcag tcttggggtc tgggtgggaa acattggtct 1980 ctaccaggat
ccctgcccca cctctcccca attaagtgcc ttcacacagc actggtttaa 2040
tgtttataaa caaaatagag aaactggttt aatgtttata aacaaaatag agaaactttc
2100 gcttataaat aaaagtagtt tgcacagaaa tgaaaaaaaa aaaaaaaaaa aaaaaa
2156 7 2310 DNA Caenorhabditis elegans 7 atggtcacga ccagtagcgg
agggggtata gggtacccgg caaacaacgg tgtcacacag 60 gtgtctctga
ttcactcgtc ggattctgta cgaactgttt caactgcccc aatataccgt 120
ccgacgtcat caatggcatc tacgatggct cataaatctt cgacggctcc gttcatctcc
180 gcaaatcaac gaatgtcaaa accgccggtt cgggtggtcg ctcaaccacc
accaccacat 240 ccacaagcat tgtcccaaca gtatcaccag cagaatccga
tgatgatgta ttccgcacca 300 aatacacgac cacacgttat tccgacaatg
caagtgcaac cgacaatggc cgctcaaatt 360 aaacgaaata atcctgttaa
tgcacagttt cagaaccctt ctgaaatgat cgccgattac 420 ggtgtaaaac
cgcagtcagt agaaatggtg caaagagttc gagctgttcg aagacaagtc 480
gccgacgagg agaccgaact gcgaagactc agagagcttg aacacgaaac ggcacagctt
540 caaaataaga attatggaag agaaagagag ttgaatgtgc aaggatccat
gctgaaagaa 600 gctcaattag agttgagaaa tgcttcaatg agggcgcaat
ctttaaacaa gcatttggaa 660 gaaatgtacc ggagaagaca aactgcagca
gcggcagcgc tcgtggaaca acgaaaaatg 720 cagcaacatc agattcttct
agcccgagct gcaaatcaag tatccacaca agaagttata 780 agacctcgtg
cttctgtcga accattccaa gttaataata cccaacagca acaaccatca 840
cctcaaatga tgaaatcaga agaattttcg gagaaaagag atttgaatgg acaaactggc
900 agttatgatg ctatcgatgg atcaggagat catcaaaaaa taccgacgga
gccatcgtac 960 ttggcaccat gtaaagaaaa ccagcaaaaa tactcggagt
taagtaaaat ggcatctacg 1020 gatcctcatt caaatcacag ttcaccatca
acttcttcgc agaaagctcc gacgttgatc 1080 acattttctc caccaagttt
tgaacagaaa atcaactcgt ctacaatgac tcgggattct 1140 ccgttcgttg
agcgtccaac atcgtttggt gatagtctag acgaatcacg actgagaagt 1200
ggaaagactg atttggtatc acttcgatca gattccctga aagctacgaa acgtcgttct
1260 tgggctgctt ccgaaggtac ttcaatgtca gaggcagaga tgattcatag
gcttcttgat 1320 gaacaacgtc gtgggagatc acattttatt ccacaattgc
caacatcaca agaagaacca 1380 tcggcaataa catcagaaac atatgccgaa
gaagttgtca attcagaatc gaaacaagtt 1440 gctacaagtt cggattccac
taataatctt gaattgccaa ccgaacaaat ggtattaggt 1500 agtgatacca
caacagaaga agatgcaagt tcgtgttcaa cacgttctga tgatggacag 1560
aatcttgaaa tggaagttgc gattgaaaga agaactgtta aaggcatttt gagaagacct
1620 aatgaaaaga tgaacaaagg tcgcattgaa tttgacccat tagcactctt
gctcgatgct 1680 gctttagaag gagaactcga tttagtgaga agcagtgcct
caaagctaac agatgtctca 1740 caggccaatg atgaagggat tacggcgttg
cacaatgcga tttgtgctgg acactatgag 1800 attgtaagat ttttgatcga
gaacgacgct gatgtgaatg ctcaagattc cgatggttgg 1860 actccacttc
attgtgcagc ttcctgtaat aaccttccaa tggttagaca acttgtggaa 1920
ggaggaggat gcgttctcgc ttcgacacta tctgatatgg aaacacctgt ggagaagtgt
1980 gaagaagatg aagatggtta tgatggatgt ttgaagtatc tttccgcagc
ccataactca 2040 acgggatcaa ttaatactgg aaaagtttac gctgcttatg
gatatgaggc ggcatttgaa 2100 gatgagctca gttttgatgc aggagatgaa
ttgacggtta ttgagaaaga taaagtcgat 2160 aaaaattggt ggacatgtga
gaagaacaat ggagagaagg gacaagtacc aagaacatat 2220 ttggcgttgt
acccatcgtt aaaatacaga aagaagctca actttgtgat gttcgatctt 2280
ccattggaat cgaacaacaa tgtcgaataa 2310 8 350 PRT Homo sapiens 8 Met
Trp Met Lys Asp Pro Val Ala Arg Pro Leu Ser Pro Thr Arg Leu 1 5 10
15 Gln Pro Ala Leu Pro Pro Glu Ala Gln Ser Val Pro Glu Leu Glu Glu
20 25 30 Val Ala Arg Val Leu Ala Glu Ile Pro Arg Pro Leu Lys Arg
Arg Gly 35 40 45 Ser Met Glu Gln Ala Pro Ala Val Ala Leu Pro Pro
Thr His Lys Lys 50 55 60 Gln Tyr Gln Gln Ile Ile Ser Arg Leu Phe
His Arg His Gly Gly Pro 65 70 75 80 Gly Pro Gly Gly Arg Ser Gln Ser
Cys Pro Pro Ser Leu Arg Asp Leu 85 90 95 Arg Pro Gly Gln Gly Pro
Leu Leu Leu Pro His Gln Leu Pro Phe His 100 105 110 Arg Pro Ala Pro
Ser Gln Ser Ser Pro Pro Glu Gln Pro Gln Ser Met 115 120 125 Glu Met
Arg Ser Val Leu Arg Lys Ala Gly Ser Pro Arg Lys Ala Arg 130 135 140
Arg Ala Arg Leu Asn Pro Leu Val Leu Leu Leu Asp Ala Ala Leu Thr 145
150 155 160 Gly Glu Leu Glu Val Val Gln Gln Ala Val Lys Glu Met Asn
Asp Pro 165 170 175 Ser Gln Pro Asn Glu Glu Gly Ile Thr Ala Leu His
Asn Ala Ile Cys 180 185 190 Gly Ala Asn Tyr Ser Ile Val Asp Phe Leu
Ile Thr Ala Gly Ala Asn 195 200 205 Val Asn Ser Pro Asp Ser His Gly
Trp Thr Pro Leu His Cys Ala Ala 210 215 220 Ser Cys Asn Asp Thr Val
Ile Cys Met Ala Leu Val Gln His Gly Ala 225 230 235 240 Ala Ile Phe
Ala Thr Thr Leu Ser Asp Gly Ala Thr Ala Phe Glu Lys 245 250 255 Cys
Asp Pro Tyr Arg Glu Gly Tyr Ala Asp Cys Ala Thr Tyr Leu Ala 260 265
270 Asp Val Glu Gln Ser Met Gly Leu Met Asn Ser Gly Ala Val Tyr Ala
275 280 285 Leu Trp Asp Tyr Ser Ala Glu Phe Gly Asp Glu Leu Ser Phe
Arg Glu 290 295 300 Gly Glu Ser Val Thr Val Leu Arg Arg Asp Gly Pro
Glu Glu Thr Asp 305 310 315 320 Trp Trp Trp Ala Ala Leu His Gly Gln
Glu Gly Tyr Val Pro Arg Asn 325 330 335 Tyr Phe Gly Leu Phe Pro Arg
Val Lys Pro Gln Arg Ser Lys 340 345 350 9 769 PRT Caenorhabditis
elegans 9 Met Val Thr Thr Ser Ser Gly Gly Gly Ile Gly Tyr Pro Ala
Asn Asn 1 5 10 15 Gly Val Thr Gln Val Ser Leu Ile His Ser Ser Asp
Ser Val Arg Thr 20 25 30 Val Ser Thr Ala Pro Ile Tyr Arg Pro Thr
Ser Ser Met Ala Ser Thr 35 40 45 Met Ala His Lys Ser Ser Thr Ala
Pro Phe Ile Ser Ala Asn Gln Arg 50 55 60 Met Ser Lys Pro Pro Val
Arg Val Val Ala Gln Pro Pro Pro Pro His 65 70 75 80 Pro Gln Ala Leu
Ser Gln Gln Tyr His Gln Gln Asn Pro Met Met Met 85 90 95 Tyr Ser
Ala Pro Asn Thr Arg Pro His Val Ile Pro Thr Met Gln Val 100 105 110
Gln Pro Thr Met Ala Ala Gln Ile Lys Arg Asn Asn Pro Val Asn Ala 115
120 125 Gln Phe Gln Asn Pro Ser Glu Met Ile Ala Asp Tyr Gly Val Lys
Pro 130 135 140 Gln Ser Val Glu Met Val Gln Arg Val Arg Ala Val Arg
Arg Gln Val 145 150 155 160 Ala Asp Glu Glu Thr Glu Leu Arg Arg Leu
Arg Glu Leu Glu His Glu 165 170 175 Thr Ala Gln Leu Gln Asn Lys Asn
Tyr Gly Arg Glu Arg Glu Leu Asn 180 185 190 Val Gln Gly Ser Met Leu
Lys Glu Ala Gln Leu Glu Leu Arg Asn Ala 195 200 205 Ser Met Arg Ala
Gln Ser Leu Asn Lys His Leu Glu Glu Met Tyr Arg 210 215 220 Arg Arg
Gln Thr Ala Ala Ala Ala Ala Leu Val Glu Gln Arg Lys Met 225 230 235
240 Gln Gln His Gln Ile Leu Leu Ala Arg Ala Ala Asn Gln Val Ser Thr
245 250 255 Gln Glu Val Ile Arg Pro Arg Ala Ser Val Glu Pro Phe Gln
Val Asn 260 265 270 Asn Thr Gln Gln Gln Gln Pro Ser Pro Gln Met Met
Lys Ser Glu Glu 275 280 285 Phe Ser Glu Lys Arg Asp Leu Asn Gly Gln
Thr Gly Ser Tyr Asp Ala 290 295 300 Ile Asp Gly Ser Gly Asp His Gln
Lys Ile Pro Thr Glu Pro Ser Tyr 305 310 315 320 Leu Ala Pro Cys Lys
Glu Asn Gln Gln Lys Tyr Ser Glu Leu Ser Lys 325 330 335 Met Ala Ser
Thr Asp Pro His Ser Asn His Ser Ser Pro Ser Thr Ser 340 345 350 Ser
Gln Lys Ala Pro Thr Leu Ile Thr Phe Ser Pro Pro Ser Phe Glu 355 360
365 Gln Lys Ile Asn Ser Ser Thr Met Thr Arg Asp Ser Pro Phe Val Glu
370 375 380 Arg Pro Thr Ser Phe Gly Asp Ser Leu Asp Glu Ser Arg Leu
Arg Ser 385 390 395 400 Gly Lys Thr Asp Leu Val Ser Leu Arg Ser Asp
Ser Leu Lys Ala Thr 405 410 415 Lys Arg Arg Ser Trp Ala Ala Ser Glu
Gly Thr Ser Met Ser Glu Ala 420 425 430 Glu Met Ile His Arg Leu Leu
Asp Glu Gln Arg Arg Gly Arg Ser His 435 440 445 Phe Ile Pro Gln Leu
Pro Thr Ser Gln Glu Glu Pro Ser Ala Ile Thr 450 455 460 Ser Glu Thr
Tyr Ala Glu Glu Val Val Asn Ser Glu Ser Lys Gln Val 465 470 475 480
Ala Thr Ser Ser Asp Ser Thr Asn Asn Leu Glu Leu Pro Thr Glu Gln 485
490 495 Met Val Leu Gly Ser Asp Thr Thr Thr Glu Glu Asp Ala Ser Ser
Cys 500 505 510 Ser Thr Arg Ser Asp Asp Gly Gln Asn Leu Glu Met Glu
Val Ala Ile 515 520 525 Glu Arg Arg Thr Val Lys Gly Ile Leu Arg Arg
Pro Asn Glu Lys Met 530 535 540 Asn Lys Gly Arg Ile Glu Phe Asp Pro
Leu Ala Leu Leu Leu Asp Ala 545 550 555 560 Ala Leu Glu Gly Glu Leu
Asp Leu Val Arg Ser Ser Ala Ser Lys Leu 565 570 575 Thr Asp Val Ser
Gln Ala Asn Asp Glu Gly Ile Thr Ala Leu His Asn 580 585 590 Ala Ile
Cys Ala Gly His Tyr Glu Ile Val Arg Phe Leu Ile Glu Asn 595 600 605
Asp Ala Asp Val Asn Ala Gln Asp Ser Asp Gly Trp Thr Pro Leu His 610
615 620 Cys Ala Ala Ser Cys Asn Asn Leu Pro Met Val Arg Gln Leu Val
Glu 625 630 635 640 Gly Gly Gly Cys Val Leu Ala Ser Thr Leu Ser Asp
Met Glu Thr Pro 645 650 655 Val Glu Lys Cys Glu Glu Asp Glu Asp Gly
Tyr Asp Gly Cys Leu Lys 660 665 670 Tyr Leu Ser Ala Ala His Asn Ser
Thr Gly Ser Ile Asn Thr Gly Lys 675 680 685 Val Tyr Ala Ala Tyr Gly
Tyr Glu Ala Ala Phe Glu Asp Glu Leu Ser 690 695 700 Phe Asp Ala Gly
Asp Glu Leu Thr Val Ile Glu Lys Asp Lys Val Asp 705 710 715 720 Lys
Asn Trp Trp Thr Cys Glu Lys Asn Asn Gly Glu Lys Gly Gln Val 725 730
735 Pro Arg Thr Tyr Leu Ala Leu Tyr Pro Ser Leu Lys Tyr Arg Lys Lys
740 745 750 Leu Asn Phe Val Met Phe Asp Leu Pro Leu Glu Ser Asn Asn
Asn Val 755 760 765 Glu
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