Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1998-05-12
2002-09-17
Ungar, Susan (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387300, C530S388100, C530S388200, C530S388800, C530S388850, C435S810000
Reexamination Certificate
active
06451979
ABSTRACT:
The present invention is directed to NBS-1 antibodies, and methods of using NBS-1 to suppress p53 directed growth.
BACKGROUND OF THE INVENTION
p53 is the most frequently mutated tumor suppressor protein identified to date in human cancers.(Levine A. J, et al.,
Nature
351:453-456 (1991); Hollstein M, et al,
Science
253:49-53 (1991)). The ability of p53 to negatively regulate cell growth is due, at least in part, to its ability to bind to specific DNA sequences and activate the transcription of target genes such as p21
Waf1
/Cip1 (Haffer R & Oren M.
Curr. Opin. Genet. Dev
. 5:84-90 (1995)). Recently, a gene was identified which encodes a protein having a deduced amino acid sequence showing varying levels of homology to certain p53 domains, for example, p53 residues implicated in sequence specific direct DNA binding are conserved in this protein (
Science
256:827-829 (1992)).
This gene, called NBS-1 (sometimes also called p73), maps to chromosome 1p36, a region which is frequently deleted in neuroblastomas (Versteeg R, et al.,
Eur J Cancer
31A:538-41 (1995)). See FIG.
1
A. Wild type NBS-1 mRNA is not detectable in these tumors. The protein shows 29% identity with p53 amino acids 1-45 (the transactivation domain) and 63% identity with p53 amino acids 113-290 (the DNA binding domain) and 38% identity with p53 amino acids 319-363 (the p53 oligomerization domain). (
FIGS. 1A and 1C
) Additionally, it has a sequence similar to p53's MDM2 binding domain of TFSDLW (SEQ ID NO:1), namely TFEDLW (SEQ ID NO:2). Although the homology between its N terminus and p53 is not as strong as the above-mentioned homology, there are also residues corresponding to amino acid residues in p53 that are frequently mutated which have been shown to be required for sequence specific DNA recognition by p53. Namely R175,G245, R248, R249, R273 and R282. There is no significant homology at the carboxy terminal (amino acid residues 364-393). Splice variants of NBS-1 at the carboxy terminus result in two different isoforms referred to as &agr; and &bgr; (see FIGS.
1
A and B). In addition, other isoforms have been identified.
Surprisingly, despite the homology between the two proteins in the core area where p53 binds to SV40 large T antigen, whereas p53 strongly interacts with SV40 large antigen, NBS-1 does not. For example, in the yeast 2-hybrid system, when p53 is used as the “bait” molecule strong interactions with p53 and SV40T large T antigen are shown (
FIG. 1D
) but not between NBS-1 and SV40 large T antigen.
In view of the strong correlation between mutations in p53, inactivation of p53 and cancer tumors, it would be important to have a means to supplement p53 function and/or replace p53 function.
Similarly, in view of the correlation between defects in 1p36 chromosomal deletions and neuroblastomas it would be useful to have means for detecting or monitoring the level of that NBS-1 gene product. We report that NBS-1 can activate the transcription of p53 responsive genes and can inhibit cell growth in a p53-like manner.
SUMMARY OF THE INVENTION
We have now discovered that NBS-1 can activate the transcription of p53 responsive genes and can inhibit cell growth in a p53-like manner.
For example, one can treat a subject having a p53 dependent tumor by determining the level of NBS-1 in the tumor cells, and comparing it to a corresponding non-malignant (normal) cell. If the level of NBS-1 in the tumor cell is not elevated, i.e., corresponding to or below the NBS-1 level in a normal cell, one can increase the level of NBS-1 in that cell. An alternative is to compare the NBS-1 level with the p53 level in the tumor cell. If the NBS-1 level is 10% or less, one can elevate the NBS-1 level. One preferred way of increasing NBS-1 levels is by transfecting the tumor cell with a vector containing a gene encoding NBS-1. Preferably, the NBS-1 gene is under the control of a powerful promoter. The NBS-1 then acts to reduce unchecked growth, for example, by negatively regulating a p53 response promoter.
REFERENCES:
patent: 5001225 (1991-03-01), Taylor
patent: WO 97/28186 (1997-08-01), None
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Jost Christine
Kaelin William
Dana-Farber Cancer Institute Inc.
Ungar Susan
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