AU755775B2 - Inhibition of DNA modulation caused by mutated p53 - Google Patents
Inhibition of DNA modulation caused by mutated p53 Download PDFInfo
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- AU755775B2 AU755775B2 AU29200/99A AU2920099A AU755775B2 AU 755775 B2 AU755775 B2 AU 755775B2 AU 29200/99 A AU29200/99 A AU 29200/99A AU 2920099 A AU2920099 A AU 2920099A AU 755775 B2 AU755775 B2 AU 755775B2
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4746—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used p53
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- General Engineering & Computer Science (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Peptides Or Proteins (AREA)
Description
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.
.o Therefore, it would be desirable to provide a product by means of which mut p53 can be investigated and optionally inhibited as regards its oncogenic properties.
The subject matter of the present invention relates to a method of inhibiting DNA modulation, wherein the binding of mut p53 having a mutated core domain and/or a mutated C Sterminus to the sequence AATATATTT or a variation thereof of a DNA with strand separating potential is inhibited.
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. Moieover, 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, wherein the binding of mut p53 having a mutated core domain and/or a mutated C terminus to the sequence AATATATTT or a variation thereof of a DNA with strand separating potential is inhibited.
The expression "mut p53" comprises any p53 or a part thereof which can bind to DNA with strand separating potential. In Sparticular, 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.
PAWPDOCS\CRNJSPEC[\7508S90.d.24/6/02 2a 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 and a DNA with strand separating potential or a strand separation thereof. In particular, the DNA with strand separating potential may be one that comprises one or several copies of the sequence AATATATTT or a variation thereof.
Besides, the DNA can also comprise further regions rich in AT. A DNA with strand separating potential is found 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 also provided is a system for identifying substances for inhibiting DNA modulation caused by mut 53, comprising mut 53 having a mutated core domain and/or a mutated DNA with strand separating potential consisting of the sequence AATATATTT or a variation thereof.
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.
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.
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 MAR6- and MAR7-DNAs, Fig. 4 shows the complex formation caused by mut p53 in the case of MAR1 and MAR5 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.
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 bp 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 3'-flanking region AGTGTCTTTAATTTCTAA
PTATATTTAGAAAACTGC
G-3' MARII IgH enhancer 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: MAR6: 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, 2 P-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 preincubated with 2 /ig Poly dl:dC (non-specific competitor) in 10 mM hepes, pH 7.8; 50 mM KC1; 1 mM EDTA; 5 mM MgC1,; 10 glycerin for min.
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.
Inhibition by DNA strand separation caused. by mut p53 The steps as described under item 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. 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 S99- strand separation can be inhibited.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers or steps but not the exclusion of any other integer or group of integers or steps.
P:\WPDOCS\CRN\SPECI\7508890.pe.doc- 11/07/02 7a The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of the common general knowledge in Australia.
*3
Claims (6)
- 2. The method according to claim i, wherein the modulation is a firm complex of mut p53 and the DNA with strand separating potential. o.0 3. The method according to claim 1, wherein the modulation is a strand separation of the DNA with strand S15 separating potential. e.:
- 4. The method according to anyone of claims 1 to 3, wherein the variation comprises the sequence AAAATATTT. 20 5. The method according to anyone of claims 1 to 4, eeo* wherein the DNA with strand separating potential is S"present in MAR DNA.
- 6. The method according to anyone of claims 1 to wherein the inhibition takes place by substances which inhibit a mutated core domain and/or a mutated C terminus of p53.
- 7. The method according to claim 6, wherein the substances are the antibodies PAb240 and PAb421.
- 8. A system for identifying substances for inhibiting DNA modulation caused by mut p53, comprising mut p53 having a mutated core domain and/or a mutated C terminus and a DNA with strand separating potential consisting of the Tsequence AATATATTT or AAAATATTT. I 9
- 9. The system according to claim 8, wherein the system also comprises a substance whose inhibitory effect on mut p53 binding to the DNA with strand separating potential is tested. Methods of inhibiting DNA modulation or systems for identifying substances which are suitable for inhibiting the DNA modulation, substantially as hereinbefore described with reference to the Example and the accompanying drawings. DATED this 29th day of October, 2002 WOLFGANG DEPPERT 15 By his Patent Attorneys DAVIES COLLISON CAVE
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE19802792A DE19802792A1 (en) | 1998-01-26 | 1998-01-26 | Inhibiting DNA modulation caused by p53 mutant, useful for treatment of cancer |
| DE19802792 | 1998-01-26 | ||
| PCT/DE1999/000221 WO1999037803A2 (en) | 1998-01-26 | 1999-01-22 | INHIBITION OF DNA MODULATION CAUSED BY MUTATED p53 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2920099A AU2920099A (en) | 1999-08-09 |
| AU755775B2 true AU755775B2 (en) | 2002-12-19 |
Family
ID=7855657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU29200/99A Ceased AU755775B2 (en) | 1998-01-26 | 1999-01-22 | Inhibition of DNA modulation caused by mutated p53 |
Country Status (13)
| Country | Link |
|---|---|
| EP (1) | EP1049810A2 (en) |
| JP (1) | JP2002507389A (en) |
| KR (1) | KR100413929B1 (en) |
| CN (1) | CN1289373A (en) |
| AU (1) | AU755775B2 (en) |
| BR (1) | BR9907725A (en) |
| CA (1) | CA2318313A1 (en) |
| DE (2) | DE19802792A1 (en) |
| NO (1) | NO20003762D0 (en) |
| PL (1) | PL342399A1 (en) |
| RU (1) | RU2235786C2 (en) |
| SK (1) | SK11052000A3 (en) |
| WO (1) | WO1999037803A2 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100348113C (en) * | 2005-05-14 | 2007-11-14 | 章建庆 | Method for preparing edible indica rice grass carp |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994012202A1 (en) * | 1992-11-26 | 1994-06-09 | University Of Dundee | ACTIVATION OF p53 PROTEIN |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB9521544D0 (en) * | 1995-10-20 | 1995-12-20 | Univ Dundee | Activation of P53 protein and therapeutic applications thereof |
-
1998
- 1998-01-26 DE DE19802792A patent/DE19802792A1/en not_active Withdrawn
-
1999
- 1999-01-22 PL PL99342399A patent/PL342399A1/en not_active Application Discontinuation
- 1999-01-22 DE DE19980080T patent/DE19980080D2/en not_active Ceased
- 1999-01-22 CN CN99802374A patent/CN1289373A/en active Pending
- 1999-01-22 KR KR10-2000-7008117A patent/KR100413929B1/en not_active IP Right Cessation
- 1999-01-22 CA CA002318313A patent/CA2318313A1/en not_active Abandoned
- 1999-01-22 BR BR9907725-6A patent/BR9907725A/en not_active IP Right Cessation
- 1999-01-22 SK SK1105-2000A patent/SK11052000A3/en unknown
- 1999-01-22 JP JP2000528710A patent/JP2002507389A/en active Pending
- 1999-01-22 RU RU2000121564/13A patent/RU2235786C2/en not_active IP Right Cessation
- 1999-01-22 AU AU29200/99A patent/AU755775B2/en not_active Ceased
- 1999-01-22 EP EP99910089A patent/EP1049810A2/en not_active Withdrawn
- 1999-01-22 WO PCT/DE1999/000221 patent/WO1999037803A2/en not_active Application Discontinuation
-
2000
- 2000-07-21 NO NO20003762A patent/NO20003762D0/en not_active Application Discontinuation
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1994012202A1 (en) * | 1992-11-26 | 1994-06-09 | University Of Dundee | ACTIVATION OF p53 PROTEIN |
| AU5533194A (en) * | 1992-11-26 | 1994-06-22 | University Of Dundee, The | Activation of p53 protein |
Non-Patent Citations (1)
| Title |
|---|
| ONCOGENE, 12, P.1941-1952 (1996) * |
Also Published As
| Publication number | Publication date |
|---|---|
| DE19980080D2 (en) | 2001-03-29 |
| CA2318313A1 (en) | 1999-07-29 |
| JP2002507389A (en) | 2002-03-12 |
| NO20003762L (en) | 2000-07-21 |
| WO1999037803A3 (en) | 1999-10-14 |
| NO20003762D0 (en) | 2000-07-21 |
| PL342399A1 (en) | 2001-06-04 |
| KR100413929B1 (en) | 2004-01-07 |
| AU2920099A (en) | 1999-08-09 |
| CN1289373A (en) | 2001-03-28 |
| EP1049810A2 (en) | 2000-11-08 |
| WO1999037803A2 (en) | 1999-07-29 |
| RU2235786C2 (en) | 2004-09-10 |
| WO1999037803A9 (en) | 1999-11-18 |
| DE19802792A1 (en) | 1999-07-29 |
| BR9907725A (en) | 2001-09-04 |
| KR20010040407A (en) | 2001-05-15 |
| SK11052000A3 (en) | 2001-05-10 |
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| FGA | Letters patent sealed or granted (standard patent) |