Assays for identifying functional alterations in the p53...

Details Assays for identifying functional alterations in the p53... Assays for identifying functional alterations in the p53...

1999-10-13

2002-08-06

Park, Hankyel T. (Department: 1648)

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

Organic compounds

Carbohydrates or derivatives

C435S440000, C530S300000, C530S350000, C424S278100, C424S069000, C424S277100

Reexamination Certificate

active

06429298

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and genetics. More particularly, it concerns the various functions of the p53 tumor suppressors and methods for identifying useful mutants having altered p53 function.
2. Description of Related Art
p53, first thought to be an oncogene, is now the most widely recognized of the class of proteins known as tumor suppressors. Its complex involvement with gene transcription, genomic stability, chromosomal segregation, senescence, cell cycle regulation and apoptosis make it one of the most important regulatory molecules now known.
Mutations in p53 have been found in cells transformed by chemical carcinogenesis, ultraviolet radiation, and several viruses. The p53 gene is a frequent target of mutational inactivation in a wide variety of human tumors and is already documented to be the most frequently mutated gene in common human cancers. It is mutated in over 50% of human NSCLC (Hollstein et al., 1991) and in a wide spectrum of other tumors.
Although the specific molecular pathway(s) through which p53 responds to DNA damage are not known, it is well known that p53 binds to DNA in a sequence specific fashion. In addition, there is considerable evidence that p53 transactivates a number of important regulatory genes including p21
waf1
, a potent inhibitor or most cycling-dependent kinases. Other gene products upon which p53 has some effect are GADD45, Bax, Fas, TBP, RPA, XPB, XPD and CSB.
The number of p53 molecules in a cell is limited, estimated at between 10
3
and 10
4
per cell. This relatively small number of molecules suggests some kind of post-translational regulation. One type of regulation is thought to be a reversible serine phosphorylation by at least seven distinct kinases including cdc2, casein kinase II, DNA-dependent protein kinase I, a casein kinase I-like kinase, protein kinase C, mitogen-activated kinase and JNK1. Though the precise role these different kinases play in phosphorylating p53 is not completely understood, a better understanding of the mechanisms is evolving. For example, a mutation in the casein kinase II phosphorylation site at serine 392 can reduce the antiproliferative activity of p53. Sequence specific DNA binding to p21
waf1
is stimulated by phosphorylation of the serine at 315.
One of the primary goals for researchers in the p53 field has been to develop a cancer gene therapy that relies on the replacement of defective p53 genes with a wild-type p53 gene. Several clinical trials have been approved utilizing viral vectors to deliver the p53 gene and have shown notable success. Possible limitations on therapies include the amount of viral vector that is administered, duration of expression, as well as the amount of p53 being expressed in the cells. Repeated administrations also raise concerns about vector toxicity and immunogenicity.
Thus, despite the growing amount of information on p53 function, and mounting evidence of its utility in gene therapy for cancer and other hyperproliferative diseases, there remains a need for improved compositions and techniques to fully exploit p53's remarkable biological activity.
SUMMARY OF THE INVENTION
Thus, according to the present invention, there are provided methods for identifying various modified forms of the tumor suppressor p53. In a first embodiment, there is provided a method of identifying a thermostable p53 polypeptide comprising providing a population of polynucleotides encoding activated, mutated p53 polypeptides; transforming host cells lacking an endogenous p53 polypeptide with said population of polynucleotides and culturing said host cells at elevated temperatures and under other conditions permitting the expression of said mutated, activated p53 polypeptides; screening said mutated p53 polypeptides for p53 DNA binding activity; and comparing the measured DNA binding with the DNA binding of an activated p53 polypeptide produced at said elevated temperatures, wherein increased binding of said activated, mutated p53 polypeptide, as compared to an activated p53 polypeptide, identifies a thermostable p53 polypeptide.
The method may further comprise the step of generating said population by creating random mutations in polynucleotides encoding an activated p53 polypeptide. The random mutations may be created by chemical mutagenesis, PCR mutagenesis, RT hypermutagenesis or DNA shuffling. In a particular embodiment, the activated p53 polypeptide contains a truncation of a C-terminal portion of wild-type p53, for example the 30 C-terminal amino acid residues. Alternatively, the truncation may specifically encompass residue 360, or one or more point mutations when compared to wild-type p53. In another embodiment, the activated p53 molecule may contain an insertion as compared to wild-type p53, or a substitution in the C-terminus of wild-type p53. In yet another embodiment, the activated p53 polypeptide comprises an internal deletion.
The host cells may be bacterial cells, for example,
Escherichia coli
. Alternatively, the host cells may be eukaryotic cells, for example, yeast cells. The binding activity may be determined using a labeled target DNA, for example, where label is a radiolabel, a chemilluminescent label or a fluorescent label. The elevated temperature may be 37° C.
Also provided are thermostable p53 polypeptides comprising a first point mutation. Throughout the application, reference to a particular residues of p53 is made by reference to SEQ ID NO:2. The first point mutation may be Val
133
, Tyr
239
, Asp
268
, Val
336
, Pro
364
, Val
62
, Thr
116
Pro
166
, Th
270
, Ser
88
, Ile
157
, Val
344
, Gly
42
, Ser
268
, Lys
51
, Gly
326
, Glu
207
, Ser
212
, His
264
, Ala
203
, Leu
80
, Ala
30
, Lys
56
, Asn
106
, Arg
115
, Ser
277
, Met
344
, Gln
45
, Ala
102
, Ser
191
, Thr
322
, Ala
31
, Gly
49
, Thr
183
, Ile
264
or Val
346
. The polypeptide may further comprise a second point mutation. The combinations may be where said first point mutation is Gly
42
and said second point mutation is Ser
268
, where said first point mutation is Lys
51
and said second point mutation is Gly
326
, where said first point mutation is Leu
80
and said second point mutation is Ala
203
, where said first point mutation is Ser
277
and said second point mutation is Met
344
.
The polypeptide may further comprise a third point mutation. The combinations may be where said first point mutation is Tyr
239
, said second point mutation is Asp
269
, and said third point mutation is Val
336
; where said first point mutation is Val
62
, said second point mutation is Tyr
239
, and said third point mutation is Asp
268
; where said first point mutation is Asp
268
, said second point mutation is Val
336
, and said third point mutation is Pro
364
; where said first point mutation is Ser
88
, said second point mutation is Ile
157
, and said third point mutation is Val
344
; or where said first point mutation is Glu
207
, said second point mutation is Ser
212
, and said third point mutation is His
364
. The polypeptide may further comprise a fourth point mutation. The combinations may be where said first point mutation is Thr
116
, said second point mutation is Pro
166
, said third point mutation is Asp
268
, and said fourth point mutation is Thr
270
.
The polypeptide may further comprise a fifth point mutation. The combinations may be where said first point mutation is Val
133
, said second point mutation is Tyr
239
, said third point mutation is Asp
268
, said fourth point mutation is Val
336
, and said fifth point mutation is Pro
364
; where said first point mutation is Ala
30
, said second point mutation is Lys
56
, said third point mutation is Asn
106
, said fourth point mutation is Arg
115
, and said fifth point mutation is Ala
203
; where said first point mutation is Gln
45
, said second point mutation is Ala
102
, said third point mutation is Ser
191
, said fourth point mutation is Glu
207
, and said fifth point mutation is Thr
332
; or where said first point mutation is Ala
31
, said second po

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