Tumour suppressor gene

Details Tumour suppressor gene Tumour suppressor gene

1999-02-25

2001-10-23

Ungar, Susan (Department: 1642)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S320100, C435S253300, C435S069100

Reexamination Certificate

active

06307036

ABSTRACT:

BACKGROUND TO THE INVENTION
Mutations of the p53 tumour suppressor gone have been observed in a number of different tumour types isolated from human cancer patients. The central role of the p53 protein in protecting cells against genotoxic damage and the high prevalence (>50%) of p53 mutations in cancer identifies this protein as an important target in the clinical diagnosis of cancer as well as in the development of more effective anticancer treatments.
Recently, mutations have been observed in partial p53 gene sequences isolated from canine cancer patients. The identification of tumour-associated canine p53 mutations indicates that the p53 protein may play an equally important role in the suppression of cancer in the dog. Characterisation of the canine p53 protein may lad to the generation of clinically relevant diagnostic reagents useful in the classification and treatment of p53-associated cancers.
Certain canine breeds display an increased disposition to cancer. In addition, there is evidence for familial-associated canine cancers. The recent identification of a p53 germ line mutation in the dog strongly suggests that inherited p53 mutations may predispose certain canine families to cancer. The identification of these canine families would be beneficial in screening for healthy individuals suitable for vital functions in the community (eg guide dogs for the blind, dogs in law enforcement). The development of a genetic screen for individual carriers of germ line p53 mutations would benefit from information on the canine p53 cDNA and full length protein. The current information describes for the first time the isolation of the complete canine p53 cDNA sequence and expression of the full length canine p53 protein. Additionally the specification describes a method of screening for healthy individuals.
STATEMENTS OF INVENTION
The present invention provides full length canine p53 protein, cDNA and RNA sequences, degenerate primers and immunological reagents based on the full length protein.
Among the uses of the present invention are the following:
1. Degenerate primers (SEQ ID NO:5; SEQ ID NO:6) have the potential to be used in the isolation of a number of different p53 cDNA from higher vertebrate organisms.
2. Canine p53 cDNA (SEQ ID NO:1) sequence can be used;
in the isolation of the complete canine p53 gene sequence
to generate probes for cytogenetic screening of canine turnouts
to identify tumour-associated mutations of the canine p53 gene
to identify individual carriers of germ line p53 gene mutations and determine breed disposition to p53-dependent cancers
to generate tumour-identified mutants for study
to study p53-dependent activities in canine normal and tumour cells
to express p53 protein within in vitro expression systems (eg rabbit reticulocyte lysate)
to express p53 protein in prokaryotic and eukaryotic (eg baculovirus-mediated) in vivo expression systems
to determine the efficacy of anti-cancer therapies based on p53 genetic status
3. Canine p53 RNA sequence (SEQ ID NO:4) information can be used to generate probes to assess changes in the stability or expression of p53 in different cell types. Antisense probes can also be designed to interfere with canine p53 RNA expression in vivo.
4. Canine p53 protein can be used as an antigen to generate monoclonal antibodies tat are directed to both conformation-specific epitopes and primary epitopes. Such antibodies would be useful in the clinical identification of canine cancers and in the analysis of p53 protein status. Canine p53-specific antibodies may also be useful in the analysis of a canine immune response to cancer. A diagnostic assay to detect circulating antibodies in canine cancer patients that are directed towards the canine p53 protein could be developed.
In accordance with the above, the present invention provides an isolated DNA coding for the canine p53 protein. Preferably the protein has the amino acid sequence (SEQ ID NO:2) as set forth in
FIG. 4
of the accompanying drawings, or is a modified form of said protein which is fictionally equivalent or associated with a predisposition to a cancer.
The present invention also provides an isolated DNA which comprises the nucleotide sequence as set forth in
FIG. 2
(SEQ ID NO:1) of the accompanying drawings or a corresponding RNA. Further, the present invention provides an isolated DNA which comprises an allelic variant of the nucleotide sequence set forth in
FIG. 2
(SEQ ID NO:1) of the accompanying drawings or a corresponding RNA. Additionally The present invention provides an isolated nucleic acid which is a DNA comprising a mutated form of the nucleotide sequence set forth in
FIG. 2
(SEQ ID NO:1) of the accompanying drawings and associated with a predisposition to a cancer. The mutation may be an insertion or deletion mutation, a nonsense mutation or a missense mutation.
In addition the present invention provides the following:
A. Oligonucleotide primers having nucleotide sequences (SEQ ID NO:6) as set forth in
FIG. 1
of the accompanying drawings,
B. A replicative cloning vector which comprises an isolated DNA of the invention and a replicon operative in a host cell for said vector
C. An expression vector which comprises an isolated DNA of the invention wherein the coding sequence for the canine p53 protein or modified form thereof is operably-linked to a promoter sequence capable of directing expression of said coding sequence in host cells for said vector.
D. Host cells transformed with a vector of the invention.
E. A method of producing a canine p53 protein or modified form thereof which comprises culturing host cells of the invention under conditions suitable for production of said protein and recovering said protein.
F. A method of producing a canine p53 proteus or modified from thereof in a cell-free system under conditions suitable for production of a protein which is characterised by the radiograph shown in
FIG. 5
of the accompanying drawings.
G. A preparation of canine p53 protein substantially free of other canine proteins and having the amino acid sequence (SEQ ID NO:2) set forth in
FIG. 4
of the accompanying drawing.
H. A reparation of a protein substantially free of other proteins, said protein being a mutated canine p53 protein obtainable by expression of a mutated form of the nucleotide sequence (SEQ ID NO:1) set forth in
FIG. 2
of the accompanying drawings.
I. An antibody capable of specifically binding to a protein of the invention.
J. An antibody of the invention which is a monoclonal antibody.
K. A preparation of a polypeptide substantially free of other proteins, said polypeptide being an antigenic fragment of a protein of the invention and which is suitable for use as an immunogen to obtain an antibody of the invention.
L. A kit for detecting mutations in the canine p53 gene resulting in cancer comprising at least one oligonucleotide primer specific for the canine p53 gene and instructions relating to detecting mutations in the canine p53 gene.
M. A kit for detecting mutations in the canine p53 gene resulting in susceptibility to cancer comprising at least one allele-specific oligonucleotide probe for the p53 gene and instructions relating to detecting mutations in the canine p53 gene.
N. A kit for detecting circulating antibodies linked with development of canine cancer involving wild type or mutant p53 and instructions relating to detecting p53 protein.
O. A kit for immunocytochemical detection of p53 abnormalities associated with canine cancer and instructions relating to the detection of p53 protein.
The invention also provides a method of identifying individual carriers of germ line p53 gene mutations, the method comprising the steps of obtaining a sample from the individual to be screened, isolating generic DNA from said sample preferably by using a reverse transcriptase polymerase chain reaction (RT-PCR), sequencing the sample and comparing it to the nucleotide sequence (SEQ ID NO:1) as set forth in
FIG. 2
of the accompanying drawing, or a modified form thereof which is functionally equivalent or associated with

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