INHIBITION OF DNA MODULATION CAUSED BY MUTATED P53

INHIBITION DE LA MODULATION D'ADN DUE A LA PROTEINE P53 MUTANTE

English Abstract

The invention relates to a method for inhibiting DNA modulation caused by mut
p53, comprising the inhibition of mut p53 bonding on the DNA with strand
separating potential. The invention also relates to a system for identifying
substances suitable for said inhibition.

French Abstract

La présente invention concerne un procédé permettant d'inhiber la modulation d'ADN due à la protéine p53, lequel comprend l'opération consistant à inhiber la liaison de la protéine p53 mutante à l'ADN présentant un potentiel de séparation des brins. L'invention concerne en outre un système permettant l'identification de substances convenant pour une telle inhibition.

Claims

8
Claims
1. A method of inhibiting DNA modulation caused by mut p53,
comprising the inhibition of mut p53 bonding to DNA with
strand separating potential.
2. The method according to claim 1, wherein the modulation
is a firm complex of mut p53 and the DNA with strand
separating potential.
3. The method according to claim 1, wherein the modulation
is a strand separation of the DNA with strand separating
potential.
4. The method according to any claims 1 to 3 , wherein mut
p53 has a mutated core domain.
5. The method according to any of claims 1 to 3, wherein
mut p53 has a mutated C terminus.
6. The method according to any of claims 1 to 5, wherein
the DNA with strand separating potential comprises the
sequence AATATATTT or a variation thereof.
7. The method according to claim 6, wherein the variation
comprises the sequence AAAATATTT.
8. The method according to any of claims 1 to 7, wherein
the DNA with strand separating potential is present in
MAR. DNA .
9. The method according to any of claims 1 to 4 and 6 to 8,
wherein the inhibition takes place by a substance which
inhibits a mutated core domain of p53.
10. The method according to claim 9, wherein the substance
is the antibody PAb240.
11. The method according to any of claims 1 to 3 and 5 to 8,

wherein the inhibition takes place by a substance which
inhibits a mutated C terminus of p53.
12. The method according to claim 11, wherein the substance
is the antibody PAb421.
13. The method according to any of claims 1 to 8, wherein
the inhibition takes place by substances which inhibit a
mutated core domain and a mutated C terminus of p53.
14. The method according to claim 13, wherein the substances
are the antibodies PAb240 and PAb421.
15. The method according to any of claims 1 to 14, wherein
DNA modulation caused by mut p53 is inhibited in the
treatment of diseases.
16. The method according to claim 15, wherein the disease is
a tumoral disease.
17. A system for identifying substances which are suitable
for inhibiting DNA modulation caused by mut p53,
comprising mut p53 and a DNA with strand separating
potential.
18. The system according to claim 17, wherein the system
also comprises a substance whose inhibitory effect on
mut p53 bonding to a DNA with strand separating
potential is tested.
19. The system according to claim 17 or 18, wherein mut p53
has a mutated core domain.
20. The system according to claim 17 or 18, wherein mut p53
has a mutated C terminus.
21. The system according to any or claims 17 to 20, wherein
the DNA with strand separating potential is present in
MAR DNA.

22. The system according to any of claims 17 to 21, wherein
the DNA with strand separating potential comprises the
sequence AATATATTT or a variation thereof.
23. The system according to claim 22, wherein the variation
comprises the sequence AAAATATTT.

Description

CA 02318313 2000-07-13
Inhibition of DNA Modulation Caused by Mutated p53
The present invention relates to a method of inhibiting the
DNA modulation caused by mutated p53. The invention also
relates to a system for identifying substances suitable for
such an inhibition.
A protein referred to as p53 is present in cells. This
protein is a tumor suppressor which is activated in the case
of DNA damage. It then binds to promoters of target genes and
activates the transcription thereof. As a result, growth
stand-still of the cells and subsequent repair of DNA damage
and death of the cells, respectively, is achieved.
As has been shown, p53 is mutated in many tumors. In this
form, it often has no tumor-suppressor activity. It rather
presents itself even as a protein which has oncogenic
properties. Various experiments have been made to inhibit
mutated p53 (hereinafter referred to as mut p53) as regards
its oncogenic properties. However, these experiments did not
yield satisfactory results.
Therefore, it is the object of the present invention to
provide a product by means of which mut p53 can be
investigated and optionally inhibited as regards its
oncogenic properties.
According to the invention this is achieved by the subject
matters defined in the claims.
Thus, the subject matter of the present invention relates to
a method of inhibiting the DNA modulation caused by mut p53,
comprising the inhibition of mut p53 bonding to DNA with
strand separating potential.

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2
The present invention is based on the applicant's insight
that mut p53 but not wild-type p53 can cause modulation of
the DNA which has strand separating potential. He found that
such a DNA is wide-spread and frequently present in MAR DNA.
MAR ("matrix attachment region") DNA refers to regulatory
elements of higher order of chromatin by which chromatin is
divided into topologically independent "loops", which enables
independent spatial and temporal regulation of gene
expression and DNA replication. The applicant also found that
DNA with strand separating potential often comprises the
sequence AATATATTT or a variation thereof. In addition to
said sequence the DNA frequently comprises further regions
rich in AT as well. Moreover, he realized that the modulation
of the DNA may be different, e.g. a firm complex of mut p53
and the DNA or a strand separation thereof. He found that the
modulation is frequently a strand separation when the
indicated sequence and optionally the adjacent sequences have
an overall AT character. On the other hand, the modulation
often presents itself as a firm complex if no or only a weak
overall AT character is present in the sequences. Moreover,
the applicant found that the DNA modulation caused by mut p53
can be inhibited when mut p53 bonding to DNA with strand
separating potential is inhibited.
According to the invention these insights are used for a
method of inhibiting DNA modulation caused by mut p53. Such a
method comprises the inhibition of mut p53 bonding to DNA
with strand separating potential.
The expression "mut p53" comprises any p53 or a part thereof
which can bind to DNA with strand separating potential. In
particular, p53 may be one that has a mutated core domain
and/or a mutated C terminus. Examples of such p53 mutants are
Pro273 p53 and MethA p53. mut p53 can also be one that is
present together with another protein in a fusion protein.
The expression "DNA with strand separating potential"
comprises any DNA which can be modulated by mut p53. A
modulation can be different, e.g. a firm complex of mut p53

CA 02318313 2000-07-13
3
and a DNA with strand separating potential or a strand
separation thereof. In particular, the DNA with strand
separating potential be one that comprises one several
may or
copies of the sequence AATATATTT
or a variation
thereof.
Besides, so comprise further regions rich
the DNA in
can al
AT. A DNA found
with strand
separating
potential
is
preferably in MAR DNA.
The expression "bonding" comprises any kind and way by which
mut p53 can bind to DNA with strand separating potential. In
particular the bonding may be one where mut p53 binds
directly to the DNA. The bonding may also be one where mut
p53 binds indirectly, i.e. via other factors, such as
proteins, to the DNA.
The expression "inhibition" comprises any kind and way by
which mut p53 bonding to DNA with strand separating potential
can be inhibited. The kind of inhibition will depend on
whether mut p53 bonding to the DNA is direct or indirect. In
the case of indirect bonding, i.e. via further factors, such
as proteins, the inhibition may take place through substances
which inhibit said further factors. It is also possible to
add substances which inhibit mut p53. In the case of direct
bonding of mut p53 to the DNA, it seems to be useful to
employ substances inhibiting mut p53. Such substances may be
e.g. those which inhibit a mutated core domain of p53 and/or
a mutated C terminus of p53. Examples of such substances are
the antibodies PAb 240 and PAb 421.
According to the invention a system for identifying
substances is also provided which is suitable for inhibiting
DNA modulation caused by mut p53. Such a system comprises mut
p53 and a DNA with strand separating potential as well as
optionally a substance whose inhibitory effect on mut p53
bonding to a DNA with strand separating potential is tested.
As to individual components of the system the above
explanations apply correspondingly. Furthermore, reference is
made to the fact that a modulation of DNA and the inhibition
thereof, respectively, can be determined by common methods.

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For example, DNA strand separation and complex formation,
respectively, and the inhibitions thereof can be determined
by an EMSA ("Electrophoretic Mobility Shift Assay") test. For
this purpose, it is an obvious thing to incubate mut p53 with
labeled double-stranded DNA which has a strand separating
potential and to separate the mixture electrophoretically so
that the formation of single-stranded DNA and a complex
thereof, respectively, and the inhibitions thereof become
visible.
By means of the present invention it is possible to inhibit
the DNA modulation caused by mut p53. Such a modulation can
be a firm complex of mut p53 and a DNA with strand separating
potential or a DNA strand separation thereof. The modulation
of DNA plays a major part in many processes of the cell. For
example, the DNA strand separation is an essential step for
the expression of genes and the replication of DNA. The DNA
strand separation is subject to strong control. This control
is cancelled by mut p53. Thus, the way for the degeneration
of the cell, i.e. for the tumor formation, has been paved.
By means of the present invention it is possible to take
therapeutic steps in the case of diseases where mut p53
causes DNA modulation. Such diseases are in particular
tumoral diseases. Furthermore, the present invention
distinguishes itself in that it provides the possibility of
identifying substances suitable for the inhibition of DNA
modulation caused by mut p53, in particular in the case of
tumors. Such substances also represent a subject matter of
the present invention.

CA 02318313 2000-07-13
Brief description of the Drawings:
Fig. 1 shows the DNA strand separation caused by
mut p53 in the case of MARI DNA,
Fig. 2 shows the DNA strand separation caused by
mut p53 in the case of MARII DNA,
Fig. 3 shows the complex formation caused by mut
p53 in the case of MARS DNA,
Fig. 4 shows the complex formation caused by mut
p53 in the case of MAR7 and MAR8 DNAs, and
Fig. 5 shows the inhibition of the DNA strand
separation caused by mut p53 in the case of MARI DNA.
The invention is explained.by the below example.
Example: Modulation of DNA by mut p53 and the
Inhibition Thereof
The modulation of DNA is shown in the form of
a DNA strand separation and a complex formation,
respectively.
(a) Induction of DNA strand separation and a
complex formation, respectively, by mut p53
Two MAR regions in the form of
oligonucleotides were drafted from the 997 by
XbaI IgE MAR fragment which lies in the
enhancer region of the gene for the heavy
immunoglobulin chain. These were MARI, i.e.
the 3'-flanking region of the enhancer, and
MARII, i.e. the 5'-flanking region of the
enhancer. The oligonucleotides had the
following sequences:
MARI IgH enhancer 5'-
3'-flanking region
AGTGTCTTTAATTTCTAA-
TATATTTAGAAAACTGC

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G-3'
MARII IgH enhancer 5'-
5'-flanking region TTTTAACAATAATAAAT-
TAAGTTTAAAATATTT-
GCG
-3'
MARI has the above indicated sequence
AATATAATTT. MARII shows a variation of this
sequence, the overall AT character not being
modified.
Furthermore, oligonucleotides were drafted
which have variations of MARI such that the
overall AT character is reduced. These
oligonucleotides had the following sequences:
MARE: ACTATGCTT
MAR7: GCTCTCTTT
Furthermore, oligonucleotides were drafted
which have the sequence of MARI together with
the adjacent "GC clamps". The overall AT
character was reduced by the "GC clamps".
The oligonucleotides were synthesized, 32P-end
labeled and subjected to an annealing reaction
so that they were obtained in double-stranded
form. They were incubated with wild-type p53
and mut p53, e.g. MethA p53 and Pro 273 p53,
respectively, and subjected to an EMSA test.
For this purpose, the following steps were
taken:
Mixtures, which contained wild-type p53, MethA
p53 and Pro 273 p53, respectively, were pre-
incubated with 2 ~.g Poly dl:dC (non-specific
competitor) in 10 mM hepes, pH 7.8; 50 mM KCl;
1 mM EDTA; 5 mM MgClz; 10 % glycerin for 20
min.

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Following the pre-incubation, the labeled
oligonucleotides were added and the mixtures
were incubated at room temperature for 30 min.
Thereafter, the mixtures were subjected to 4 %
native polyacrylamide gel electrophoresis for
three hours before the gels were dried and
autoradiographed (cf. Figs. 1 - 4).
It showed that mut p53, e.g. MethA p53 and pro
273 p53, respectively, can cause a modulation
of DNA, e.g. strand separation and complex
formation, respectively, in the case of DNA
with strand separating potential.
(b) Inhibition by DNA strand separation caused by
mut p53
The steps as described under item (a) were
taken, with the exception that mixtures of
MethA p53 also contained the antibodies PAb
421 and PAb 240, respectively. The
oligonucleotides used were those which
contained MARI DNA ( cf . Fig . 5 ) .
It showed that by a product inhibiting the mut
p53 bonding to a DNA with strand separating
potential, such as antibodies PAb 421 and PAb
240, respectively, the mut p53-induced DNA
strand separation can be inhibited.