Inhibition of p53 degradation

Details Inhibition of p53 degradation Inhibition of p53 degradation

2000-12-07

2004-12-14

Helms, Larry R. (Department: 1642)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Nucleoproteins, e.g., chromatin, chromosomal proteins,...

C514S002600

Reexamination Certificate

active

06831155

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to tumor suppression.
The p53 tumor suppressor gene encodes a nuclear phosphoprotein with cancer-inhibiting properties. The development of human cancer may involve inactivation of this suppressor function through various mechanisms, including gene deletions and point mutations. The p53 protein is a multi-functional transcription factor involved in the control of cell cycle progression, DNA integrity and survival of cells exposed to DNA-damaging agents. Loss of p53 activity predisposes cells exposed to the acquisition of oncogenic mutations.
P53 is critical in maintaining ordered proliferation, growth, and differentation of normal cells. Mutation in this gene may be one of the most significant genetic changes leading to the transformation of cells from normalcy to malignancy.
SUMMARY OF THE INVENTION
The invention provides compositions and methods for inhibiting degradation of p53, thereby enhancing p53-mediated tumor suppressor activity. Accordingly, the invention provides a polypeptide which contains the amino acid sequence of residues 92-112 of human p53 (PLSSSVPSQKTYQGSYGFRLG; SEQ ID NO:1). The polypeptide is substantially pure and inhibit degradation of endogenous p53 in a mammalian cell. The invention includes a polypeptide with the amino acid sequence PLSSSVPSQ KTYQGSYGFXLG (SEQ ID NO:2; where “X” is a positively-charged amino acid. For example, “X” is lysine (L) or histidine (H). p53-derived inhibitory polypeptides include a polypeptide which contains residues 1-113. of SEQ ID NO:3; a polypeptide which contains the proline-rich domain (PRD; residues 45-113 of SEQ ID NO:3) of p53; a polypeptide which does not contain the N-terminal acidic transactivation domain (TAD; residues 1-44 of SEQ ID NO:3 of p53; a polypeptide which does not contain a DNA binding domain (DBD; amino acids 144-290 of p53; a polypeptide which does not contain the oligomerization domain (OD; amino acids 319-363 of p53, and a polypeptide which does not contain residues 1-42 of SEQ ID NO:3.
The polypeptide is preferably at least 20 residues, more preferably at least 25 residues, more preferably at least 30 residues, more preferably at least 40 residues, and most preferably at least 50 residues in length. The polypeptide is less than the full-length of a naturally-occurring p53 polypeptide, i.e. the polypeptide is less than 393 amino acids in length.
The invention also includes peptide mimetics of p53 polypeptides. For example, the peptide mimetic contains the amino acid sequence of SEQ ID NO:1 or 2.
A method of inhibiting p53 degradation in a cell, e.g., a tumor cell or precancerous cell, is carried out by contacting the cell with a p53-derived inhibitory polypeptide described above. For example, the polypeptide contains the amino acid sequence of SEQ ID NO:1 or 2. Any cancer characterized by a p53 mutation or aberrant p53 expression is treated by the methods of the invention, e.g., squamous cell carcinoma, ovarian cancer, lung cancer (non-small cell and small cell carcinoma), pancreatic cancer, leukemias such as acute lymphoblastic leukemia, lymphomas, gliomas, and neuroblastomas. Cancers characterized by aberrant Mdm2 expression, e.g., overexpression or amplification, are also treated. Such cancers include sarcomas such as osteosarcoma as well as colon carcinoma, melanoma, choriocarcinoma, breast carcinoma, glioblastoma, neuroblastoma, and rhadomyosarcoma.
The methods are also useful to treat cancers in which p53 is degraded by human papillomavirus (HPV) E6 protein. For example, cervical cancers, in which HPV is often an etiological factor, are treated by administering a p53 inhibitory peptide to reduce E6-mediated degradation of endogeneous cellular p53.
In addition to administering p53-derived inhibitory polypeptides, DNA encoding such polypeptides are administered. For example, an isolated DNA encoding a polypeptide which contains the amino acid sequence of SEQ ID NO:1 or 2 is delivered to a target cell in a manner that allows uptake of the DNA by the cell and intracellular expression of the recombinant polypeptide in the cell.
Also within the invention is a degradation-resistant p53 polypeptide. A degradation-resistant polypeptide is refractory to Mdm2 or E6-mediated degradation but retains a tumor suppressor activity of p53, e.g., transcriptional activity or induction of apoptotic death of a cancer cell. For example, a degradation-resistant p53 mutant has a mutation in a degradation signal sequence of p53 such as a deletion (e.g., lacking the amino acid sequence of SEQ ID NO:1) or a point mutation (e.g., the amino acid at position 110 of p53 is substituted with an amino acid that is not positively charged). Preferably, the mutant contains an N-terminal transcriptional transactivation domain, e.g., amino acids 1-42 of SEQ ID NO:3 or amino acids 1-44 of SEQ ID NO:3. More preferably, the degradation-resistant p53 mutant retains at least 50%, more preferably at least 75%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 99%, and most preferably 100% of the tumor suppressive activity of a naturally-occurring p53 protein. Tumor suppressive activity is transcription activation, i.e., the ability to trans-activate transcription of a target gene such as Mdm2, or induction of apoptosis of tumor cells. A degradation-resistant p53 mutant polypeptide induces apoptosis in tumor cells.
The invention also includes an isolated DNA encoding a degradation-resistant p53 polypeptide and a method of inhibiting tumor growth by contacting a tumor cell with such a polypeptide or nucleic acid encoding it.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof and from the claims.


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