U.S. patent application number 09/940766 was filed with the patent office on 2002-10-03 for tumor suppressor waf1.
Invention is credited to Kinzler, Kenneth W., Vogelstein, Bert.
Application Number | 20020142442 09/940766 |
Document ID | / |
Family ID | 22531967 |
Filed Date | 2002-10-03 |
United States Patent
Application |
20020142442 |
Kind Code |
A1 |
Vogelstein, Bert ; et
al. |
October 3, 2002 |
Tumor suppressor WAF1
Abstract
A human gene, WAF1, has been identified which is induced by
wild-type but not mutant p53 in human brain tumor cells. The gene
is located on chromosome 6p21.2 and directs the synthesis of an
18.1 kd protein. Introduction of WAF1 cDNA suppresses growth of
human brain and colon tumor cells. The WAF1 gene and protein are
useful inter alia for diagnosis and treatment of human tumors.
Inventors: |
Vogelstein, Bert;
(Baltimore, MD) ; Kinzler, Kenneth W.; (Baltimore,
MD) |
Correspondence
Address: |
BANNER & WITCOFF
1001 G STREET N W
SUITE 1100
WASHINGTON
DC
20001
US
|
Family ID: |
22531967 |
Appl. No.: |
09/940766 |
Filed: |
August 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09940766 |
Aug 29, 2001 |
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08456297 |
Jun 1, 1995 |
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08456297 |
Jun 1, 1995 |
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08149829 |
Nov 10, 1993 |
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Current U.S.
Class: |
435/226 ;
514/17.7; 514/19.3; 530/388.26; 536/23.2 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 15/1135 20130101; C12N 2310/53 20130101; C12N 2310/15
20130101; C07K 14/4703 20130101; A61K 38/00 20130101 |
Class at
Publication: |
435/226 ; 514/12;
530/388.26; 536/23.2 |
International
Class: |
A61K 038/17; C07H
021/04; C12N 009/64; C07K 016/40 |
Goverment Interests
[0001] This invention was made with support from NIH grant CA 0907
and CA 43460. The U.S. government therefore retains some rights in
the invention.
Claims
1. An isolated and purified subchromosomal DNA molecule which
encodes WAF1 protein as shown in SEQ ID NO: 2, said DNA molecule
containing three exons of 168 bp, 450 bp and 1600 bp, wherein the
sequence of said exons is shown in SEQ ID NO: 1.
2. The DNA molecule of claim 1 which is not more than 90 kb.
3. The DNA molecule of claim 1 which is a P1 clone.
4. The DNA molecule of claim 1 which contains at least two
intervening sequences.
5. The DNA molecule of claim 1 which contains no intervening
sequences.
6. An isolated and purified WAF1 protein having a sequence as shown
in SEQ ID NO: 2.
7. An antibody which is specifically reactive with human WAF1
protein.
8. A method of suppressing growth of tumor cells, comprising the
step of: administering to said cells a WAF1 protein having a
sequence as shown in SEQ ID NO: 2.
9. A method of suppressing growth of tumor cells, comprising the
step of: administering to said cells a DNA molecule which causes
said cells to express WAF1, said DNA molecule having a sequence as
shown in SEQ ID NO: 1.
10. A method for screening potential therapeutic agents for the
ability to suppress the growth of tumor cells by activating the
expression of WAF1, comprising the steps of: incubating a potential
therapeutic agent with a cell which contains a WAF1 reporter
construct, said reporter construct comprising a WAF1 transcription
regulatory region covalently linked in a cis configuration to a
gene encoding an assayable product; measuring the production of the
assayable product, a potential therapeutic agent which increases
the production by the cell of the assayable product being an agent
which will suppress the growth of tumor cells by activating the
expression of WAF1.
11. The method of claim 10 wherein the WAF1 transcription
regulatory region comprises about 2.4 kb upstream from WAF1
transcriptional start site.
12. The method of claim 10 wherein the WAF1 transcription
regulatory region comprises the sequence of SEQ ID NO: 3.
13. A method for diagnosing cancer, comprising the steps of:
testing a tissue to determine if the tissue expresses less WAF1
than normal tissue.
14. The method of claim 13 wherein the step of testing utilizes an
antibody which is specifically reactive with WAF1 protein.
15. The method of claim 13 wherein the step of testing utilizes a
nucleic acid probe which specifically hybridizes to a WAF1 mRNA,
said probe having a sequence selected from SEQ ID NO: 1.
16. A method for diagnosing cancer, comprising the steps of:
testing a tissue to determine if DNA in said tissue contains a
mutant WAF1 gene.
17. The method of claim 16 wherein DNA of the tissue is compared to
DNA of a normal tissue to determine whether the WAF1 gene is
mutant.
18. A WAF1 reporter construct, said reporter construct comprising a
WAF1 transcription regulatory region covalently linked in a cis
configuration to a gene encoding an assayable product.
19. The reporter construct of claim 18 wherein the WAF1
transcription regulatory region comprises about 2.4 kb upstream
from WAF1 transcriptional start site.
20. The reporter construct of claim 18 wherein the WAF1
transcription regulatory region comprises the sequence of SEQ ID
NO: 3.
21. An antisense WAF1 construct comprising: a. a transcriptional
promoter; b. a transcriptional terminator; c. a DNA segment
comprising one or more segments of the WAF1 gene, said gene segment
located between said promoter and said terminator, said DNA segment
being inverted with respect to said promoter and said terminator,
whereby RNA produced by transcription of the DNA segment is
complementary to a corresponding segment of WAF1 RNA produced by
human cells.
22. The antisense WAF1 construct of claim 21 wherein said
transcriptional promoter is inducible.
23. A WAF1 antisense oligonucleotide comprising: at least ten
nucleotides complementary to WAF1 mRNA.
24. The WAF1 antisense oligonucleotide of claim 23 which comprises
at least about twenty nucleotides complementary to WAF1 mRNA.
25. The WAF1 antisense oligonucleotide of claim 23 which contains
one or more modified nucleotide analogs.
26. The WAF1 antisense oligonucleotide of claim 23 which is a
circular molecule.
27. A method for promoting the proliferation of cells, comprising
the step of: administering a WAF1 antisense oligonucleotide
comprising at least ten nucleotides complementary to WAF1 mRNA to
said cells to inhibit the expression of WAF1.
28. A method for promoting the proliferation of cells, comprising
the step of: administering a WAF1 triplex-forming oligonucleotide
comprising at least ten nucleotides complementary to WAF1 gene to
said cells to inhibit the expression of a WAF1 gene.
29. A method for promoting growth of cells, comprising the step of:
administering to said cells to inhibit the expression of WAF1, an
antisense WAF1 construct comprising: a. a transcriptional promoter;
b. a transcriptional terminator; c. a DNA segment comprising one or
more segments of the WAF1 gene, said gene segment located between
said promoter and said terminator, said DNA segment being inverted
with respect to said promoter and said terminator, whereby RNA
produced by transcription of the DNA segment is complementary to a
corresponding segment of WAF1 RNA produced by human cells.
30. The method of claim 29 wherein said transcriptional promoter is
inducible.
31. A method for assessing susceptibility to cancers, comprising
the step of: testing a tissue selected from the group consisting of
blood, chorionic villi, amniotic fluid, and a blastomere of a
preimplantation embryo, to determine if DNA in said tissue contains
a mutant WAF1 gene.
Description
TECHNICAL FIELD
[0002] The invention relates to the fields of diagnosis and therapy
of cancers. More particularly, the invention relates to a protein
which can suppress tumor cell growth.
BACKGROUND OF TIE INVENTION
[0003] Inactivation of p53 is a common event in the development of
human neoplasia (Hollstein et al. (1991) Science 253, 49-53). A
variety of mechanisms can lead to such functional inactivation,
including p53 point mutations of deletions of p53 (Baker et al.
(1989) Science 244, 217-221; Wolf, D., and Rotter, V. (1985) Proc.
Natl. Acad. Sci. USA 82, 790-794), and interaction with oncogenic
viral or cellular proteins (Mietz et al. (1992) EMBO J. 11,
5013-5020; Momand et al. (1992) Cell 69, 1237-1245). Wild-type p53
has been shown to be a suppressor of tumor cell growth (for reviews
see Mercer, W. E. (1992) Crit. Rev. Eucar. Gene Exp. 2, 251-263;
Oren, M. (1992) FASEB J. 6, 3169-3176; Lane, D. P. (1992) Nature
358, 15-16; Perry, M. E., and Levine, A. J. (1993) Curr. Opin. in
Genet. and Devel. 3, 50-54). Inactivation of p53 by any of the
above mechanisms thereby leads to a selective growth advantage,
generally observed as tumor progression.
[0004] The mechanism underlying p53 growth suppression is still
undefined. Several biochemical features of p53 have been
elucidated, and at least two of these are currently of much
interest. First, p53 has been shown to transcriptionally suppress a
variety of promoters containing TATA-elements (Ginsberg et al.
(1991) Proc. Natl. Acad. Sci. USA 88, 9979-9983; Santhanam et al.
(1991) Proc. Natl. Acad. Sci. USA 88, 7605-7609; Kley et al. (1992)
Nucl. Acids Res. 20, 4083-4087; Mack et al. (1993) Nature 363,
281-283). This suppression is apparently sequence independent, and
may involve p53 binding to the TATA-binding protein (TBP) or to
other transcription factors (Seto et al. (1992) Proc. Natl. Acad.
Sci. USA 89, 12028-12032; Truant et al. (1993) J. Biol. Chem. 268,
2284-2287; Ragimov et al. (1993) Oncogene 8, 1183-1193; Martin et
al. (1993) J. Biol. Chem. 268, 13062-13067; Liu et al. (1993) Mol.
and Cell. Biol. 13, 3291-3300). Second, p53 can bind to DNA in a
sequence-specific manner (Kern et al. (1991) Science 252,
1707-1711). A 20 bp consensus binding site, consisting of two
copies of the 10 bp sequence 5'-RRRCWWGYYY-3', separated by up to
13 bp, has been identified (El-Deiry et al. (1992) Nature Genet. 1,
45-49; Funk et al. (1992) Mol. Cell. Biol. 12, 2866-2871). Both
copies of the 10 bp sequence are required for efficient binding by
p53. p53 contains a strong transcriptional activation sequence near
its amino terminus (Fields, S., and Jang, S. K. (1990) Science 249,
1046-1049; Raycroft et al. (1990) Science 249, 1049-1051), and can
stimulate the expression of genes downstream of its binding site.
Such stimulation has been demonstrated in both mammalian (Kern et
al. (1992) Science 256, 827-830; Funk et al. (1992) Mol. Cell.
Biol. 12, 2866-2871; Zambetti et al. (1992) Gen. and Devel. 6,
1143-1152) and yeast cells (Scharer, E., and Iggo, R. (1992) Nucl.
Acids Res. 20, 1539-1545; Kern et al. (1992) Science 256, 827-830)
as well as in an in vitro system (Farmer et al. (1992) Nature 358,
83-86).
[0005] The sequence-specific transcriptional activation by p53 has
led to the hypothesis that p53-induced genes may mediate its
biological role as a tumor suppressor (Pietenpol et al. (1993) Cell
(submitted)). To date, several genes containing p53-binding sites
have been identified. These include muscle creatine kinase (M C K,
Weintraub et al. (1991) Proc. Natl. Acad. Sci. USA 88, 4570-4574;
Zambetti et al. (1992) Gen. and Devel. 6, 1143-1152), GADD45
(Kastan et al. (1992) Cell 71, 587-597), MDM2 et al. (1993) EMBO
12, 461-468; Wu et al. (1993) Genes and Devel. 7, 1126-1132), and a
GLN retroviral element (Zauberman et al. (1993) EMBO J. 12,
2799-2808). Each of these genes contains a 20 bp sequence with high
homology to the p53 consensus binding site (Prives, C., and
Manfredi, J. J. (1993) Gen. and Devel. 7, 529-534). The p53-binding
sites in GADD45 and MDM2 are located within introns, the MCK site
is 3 kb upstream of the transcription start site, and the GLN
element is located within an LTR. The relationship of any of these
genes to suppression of cell growth by p53 remains unclear. It has
been suggested that MDM2 may be a feedback regulator of p53 action,
by being transcriptionally induced (Barak et al. (1993) EMBO 12,
461-468; Wu et al. (1993) Genes and Devel. 7, 1126-1132), then
inhibiting p53 function (Momand et al. (1992) Cell 69, 1237-1245;
Oliner et al. (1993) Nature 362, 857-860; Wu et al. (1993) Genes
and Devel. 7, 1126-1132). In this regard, MDM2 functions as an
oncogene rather than as a tumor suppressor gene (Fakharzadeh et al.
(1991) EMBO J. 10, 1565-1569; Finlay, C. A. (1993) Mol. and Cell.
Biol. 13, 301-306).
[0006] There is a need in the art for elucidation of the pathway by
which p53 exerts its tumor suppressive effects. There is also a
need in the art for new diagnostic and therapeutic tools for
evaluating and ameliorating human cancers.
SUMMARY OF THE INVENTION
[0007] It is an object of the invention to provide DNA molecules
useful for diagnosing and treating human tumors.
[0008] It is another object of the invention to provide proteins
useful for treating human tumors and for raising diagnostically
useful antibodies.
[0009] It is still another object of the invention to provide
antibodies which are useful for diagnosing human cancer.
[0010] It is yet another object of the invention to provide methods
of suppressing growth of tumor cells.
[0011] It is an object of the invention to provide a method for
screening potential therapeutic agents for treating cancer.
[0012] It is another object of the invention to provide methods for
diagnosing cancer.
[0013] It is yet another object of the invention to provide a
reporter construct, useful for screening potential antineoplastic
agents.
[0014] It is an additional object of the invention to provide an
antisense construct for inhibiting expression of a tumor suppressor
gene.
[0015] It is still another object of the invention to provide
antisense oligonucleotides for inhibiting expression of a tumor
suppressor gene.
[0016] It is yet another object of the invention to provide methods
for promoting growth of cells in which a tumor suppressor gene's
expression is inhibited.
[0017] It is another object of the invention to provide a method
for assessing susceptibility to cancers.
[0018] These and other objects of the invention are provided by one
or more of the embodiments described below. In one embodiment of
the invention an isolated and purified subchromosomal DNA molecule
is provided. The molecule encodes WAF1 protein as shown in SEQ ID
NO: 2, and contains three exons of 168 bp, 450 bp and 1600 bp. The
sequence of said exons is shown in SEQ ID NO: 1.
[0019] In another embodiment of the invention an isolated and
purified WAF1 protein is provided. The protein has a sequence as
shown in SEQ ID NO: 2.
[0020] In yet another embodiment of the invention an antibody is
provided. The antibody is specifically reactive with human WAF1
protein.
[0021] In still another embodiment of the invention a method of
suppressing growth of tumor cells is provided. The method comprises
administration of a WAF1 protein having a sequence as shown in SEQ
ID NO: 2 to said cells.
[0022] In an additional embodiment of the invention a method of
suppressing growth of tumor cells is provided. The method comprises
administration to said cells of a DNA molecule which causes said
cells to express WAF1, said DNA molecule having a sequence as shown
in SEQ ID NO: 1.
[0023] According to another embodiment of the invention a method
for screening potential therapeutic agents for the ability to
suppress the growth of tumor cells by activating the expression of
WAF1 is provided. The method comprises incubation of a potential
therapeutic agent with a cell which contains a WAF1 reporter
construct, said reporter construct comprising a WAF1 transcription
regulatory region covalently linked in a cis configuration to a
gene encoding an assayable product. Further, the method comprises
measurement of the production of the assayable product. A potential
therapeutic agent is identified as useful if it increases the
production by the cell of the assayable product.
[0024] In still another embodiment of the invention a method for
diagnosing cancer is provided. The method comprises testing a
tissue to determine if the tissue expresses less WAF1 than normal
tissue.
[0025] In another embodiment of the invention a method for
diagnosing cancer is provided. The method comprises testing a
tissue to determine if DNA in said tissue contains a mutant WAF1
gene.
[0026] In still another embodiment of the invention a WAF1 reporter
construct is provided. The reporter construct comprises a WAF1
transcription regulatory region covalently linked in a cis
configuration to a gene encoding an assayable product.
[0027] In another embodiment of the invention an antisense WAF1
construct is provided. The construct comprises: a transcriptional
promoter; a transcriptional terminator; and a DNA segment
comprising one or more segments of the WAF1 gene, said gene segment
located between said promoter and said terminator, said DNA segment
being inverted with respect to said promoter and said terminator,
whereby RNA produced by transcription of the DNA segment is
complementary to a corresponding segment of WAF1 RNA produced by
human cells.
[0028] In another embodiment of the invention a WAF1 antisense
oligonucleotide is provided. The oligonucleotide comprises at least
ten nucleotides complementary to a sequence present in WAF1
mRNA.
[0029] In yet another embodiment of the invention a triplex
oligonucleotide is provided. The oligonucleotide comprises at least
ten nucleotides complementary to a sequence present in a WAF1
gene.
[0030] In still another embodiment of the invention a method is
provided for promoting growth of cells. The method comprises:
administering a WAF1 antisense or triplex-forming oligonucleotide
comprising at least ten nucleotides complementary to WAF1 mRNA or
WAF1 gene, respectively, to said cells to inhibit the expression of
WAF1. In an alternative method an antisense WAF1 construct is
administered to said cells to inhibit the expression of WAF1. The
construct comprises:
[0031] a. a transcriptional promoter;
[0032] b. a transcriptional terminator;
[0033] c. a DNA segment comprising one or more segments of the WAF1
gene, said gene segment located between said promoter and said
terminator, said DNA segment being inverted with respect to said
promoter and said terminator, whereby RNA produced by transcription
of the DNA segment is complementary to a corresponding segment of
WAF1 RNA produced by human cells.
[0034] In still another embodiment of the invention a method is
provided for assessing susceptibility to cancers. The method
comprises testing a tissue selected from the group consisting of
blood, chorionic villi, amniotic fluid, and a blastomere of a
preimplantation embryo, to determine if DNA in said tissue contains
a mutant WAF1 gene.
[0035] Thus the subject invention provides the art with useful
means for diagnosing and treating cancers in humans and other
animals. Moreover, it opens new avenues for the design and
screening of additional anti-neoplastic therapeutic agents which
operate by means of a new mechanism as detailed below. Conversely,
the subject invention provides a new approach for promoting the
proliferation of cells when large numbers of such cells are
desired.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 shows p53-dependent transactivation in GM and DEL
cell lines. GM cells (containing an inducible wild-type p53 gene)
or DEL cells (containing an inducible mutant p53 gene) were
transfected with reporter plasmids as indicated, and luciferase
activity was measured after 18 hours of incubation either in the
absence or presence of dexamethasone as shown. Wild-type p53
expression plasmid was co-transfected with PG13-Luc into DEL cells
as indicated in the two right-most lanes.
[0037] FIG. 2 shows the time course of induction of p53, WAF1, and
MDM2 transcripts in GM cells treated with dexamethasone. A Northern
blot was prepared using 10 .mu.g of total RNA isolated from GM
cells treated with dexamethasone for 0, 4, 6, 8, or 16 hours, and
probed with p53 cDNA (top panel), WAF1 cDNA (middle panel), or MDM2
cDNA (lower panel). The endogenous mutant (mut) and induced
wild-type (wt) p53 mRNA species are indicated with arrows.
[0038] FIG. 3 shows CDNA and predicted amino acid sequence of human
WAF1. The predicted translation begins at nt 76 and ends at nt 567.
The nucleotide sequence of human WAF1 has been deposited with
GenBank; accession number VO3106.
[0039] FIG. 4 shows Southern blot analysis of WAF1 in human and
mouse cells. Four .mu.g of human genomic DNA (lanes 1 and 2) or 8
.mu.g of mouse genomic DNA (lanes 3 and 4) were digested with EcoRI
(lanes 1 and 3) or BamHI (lanes 2 and 4). Following transfer, the
blot was probed with a radioactively labeled WAF1 cDNA fragment
encompassing WAF1 nt 1 to 1004.
[0040] FIG. 5 shows chromosomal localization of the gene encoding
WAF1. FIG. 5A shows partial metaphase chromosomes after FISH with
the biotin-labeled genomic WAF1 probe (arrow indicates chromosome
6). FIG. 5B shows identical G-banded metaphase chromosomes as in
FIG. 5A, documenting the localization of the fluorescent signal to
6p21.2. FIG. 5C shows an idiogram of chromosome 6 (arrow indicates
6p21.2).
[0041] FIG. 6 shows that WAF1 is induced in the presence of
transcriptionally active wild-type p53. A Northern blot was
prepared from 10 .mu.g of total RNA from GM cells in the presence
or absence of dexamethasone for 16 hours (lanes 1 and 2
respectively), DEL cells treated with dexamethasone (lane 3), or
SW480 cells infected with either Ad-gal (lane 4) or Ad-p53 (lane 5)
for 16 hours. The blot was probed with WAF1 DNA, GADD45 DNA, MDM2
DNA, or TGF-beta DNA, as indicated. An ethidium bromide stain of
the RNA, prior to Northern transfer, is shown in the lower-most
panel. The expression of wild-type p53 in the various cells is
indicated at the top of the figure.
[0042] FIG. 7 shows the sequence of the second exon of mouse WAF1.
FIG. 7A shows the predicted amino acid sequence of mouse WAF1 shown
above the nt sequence. FIG. 7B shows comparison of the predicted
amino acid sequences between human and mouse WAF1. Identical amino
acids are indicated by a line between human and mouse amino acids,
whereas similar amino acids are indicated by a dot.
[0043] FIG. 8 shows that WAF1 induction by p53 is conserved in rat
and mouse. A Northern blot was prepared using total RNA from GM
cells, either untreated (lane 1) or treated for 6 hours with
dexamethasone (lane 2), REF 112 cells grown either at 37.degree. C.
(uninduced; lane 3) or 31.degree. C. (lane 4), or MCO1 cells
infected with either Ad-gal (lane 5) or Ad-p53 (lane 6). The RNA
was hybridized with radioactive probes made from human WAF1 cDNA
(FIG. 7A) or mouse WAF1 DNA (FIG. 7B). An ethidium bromide stain of
the RNA, prior to transfer, is shown in the lowest panel.
[0044] FIG. 9 shows that WAF1 suppresses the growth of human tumor
cells. The human brain tumor line (DEL; FIG. 9) or the human colon
tumor line (SW480; FIG. 9) were transfected with the pCFP4 vector,
or vectors encoding sense WAF1, antisense WAF1, mutant WAF1, or
wild-type p53, as indicated. The photographs show low power views
of the transfected flasks following 17 days of hygromycin
selection. Below each photograph, the fraction of colonies (%) in
each flask compared to the vector transfected cells is indicated
(means of three flasks.+-.standard deviation). The vector
transfectants contained an average of 310 and 850 colonies in rows
A and B, respectively.
[0045] FIG. 10 shows schematic representation of WAF1 transcription
regulatory region. Diagram shows the promoter-reporter constructs
(FIG. 10A), and partial DNA sequence of the WAF1 upstream
regulatory region (FIG. 10B), including promoter and upstream p53
binding sequences. Small letters in the latter represent deviations
from the p53 consensus binding sequence. The TATA-element and Sp1
recognition sequences within the WAF1 promoter are surrounded by
boxes. The Sac I site, used for making the DM-Luc construct devoid
of the p53 binding site, occurred at the 3'-end of the sequence
shown (g is the 1st nt of the Sac I recognition site).
[0046] FIG. 11 shows activation of WAF1-promoter by wild-type p53.
GM or DEL cells were transfected with either the WWP-Luc or DM-Luc
reporters (FIG. 10), and luciferase activity was measured after
incubation with or without dexamethasone for 14 hours.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0047] One of the major goals of tumor biology is to understand the
biochemical nature of the pathways leading to growth suppression.
For p53, this understanding has recently been enhanced by finding
that p53, a nuclear phosphoprotein, binds to DNA in a
sequence-specific manner, and activates transcription from such
sequences. A variety of experiments has suggested the hypothesis
that genes whose expression is activated by p53 are likely to be
mediators of p53 action (Pietenpol et al. (1993) Cell (submitted)).
It is a discovery of the applicants that WAF1 is such a gene. WAF1
gene expression is induced by p53, and this induction is observed
in cell lines from human, mouse, and rat. The data indicate that
not only are the coding sequence and exon structure of WAF1
conserved, but also its regulation by p53. This is consistent with
the fact that p53 tumor suppressive function is conserved between
rodents and humans.
[0048] The activation of a gene following wild-type p53 expression
could be indirect, a result of induction by a second gene directly
controlled by p53. In the case of WAF1, the p53 induction is likely
to be direct, as at least one strong binding site exists within its
transcription regulatory region. The binding site also functions in
a p53-dependent manner in yeast. Moreover, the entire WAF1 promoter
region is unambiguously under p53 control in mammalian cells (FIG.
11). Finally, WAF1 mimics the growth suppression of p53 when
introduced into three different cell lines. There are a few other
genes whose expression is increased as a result of p53 expression,
but none of them has been shown to meet the numerous criteria
described here for a direct mediator of p53 action.
[0049] DNA molecules according to the present invention are
isolated and purified from other chromosomal genes. They may be
either genomic sequences or cDNA sequences, i.e., they may or may
not contain intervening sequences. A genomic clone of about 90 kb
has been isolated which encodes the whole WAF1 gene. The WAF1 mRNA
has been found to be approximately 2.1 kb. The WAF1 gene contains
three exons of 168, 450 and 1600 bp. The sequence of the exons is
shown in SEQ ID NO: 1.
[0050] Now provided with the sequence of WAF1, one of ordinary
skill in the art can readily obtain the 18.1 kd WAF1 protein. It
can be expressed in bacteria, yeast, or other convenient cell.
Portions of it can be synthesized and linked to a carrier protein
for immunization of laboratory animals to raise antibodies
specifically immunoreactive with WAF1 protein. The antibodies can
be used to purify the WAF1 protein from natural or recombinant
sources. Such antibodies can be polyclonal or monoclonal, as is
convenient for the particular application.
[0051] As described herein, WAF1 protein has a growth-suppressing
effect on tumor cells. Thus its administration to tumor cells may
be desirable to effect such growth suppression. Other cells which
are involved in proliferative diseases may also be targeted for
WAF1-mediated growth suppression. Such proliferative diseases
include psoriasis, polyps, warts, and inflammatory diseases. WAF1
protein may be administered in suitable formulations to tumor
cells. It may be microinjected, or simply supplied externally to
tumor cells. It may be encapsulated, e.g., in liposomes. If
WAF1-encoding DNA is administered to the tumor cells then the cells
can express their own WAF1 protein for growth suppression. Such DNA
can be genomic or cDNA, as described above. Other cells involved in
proliferative diseases may be treated similarly.
[0052] According to another aspect of the invention WAF1 reporter
constructs are provided. They are recombinant DNA molecules which
contain a WAF1 transcription regulatory region covalently linked in
a cis configuration to a reporter gene. Many suitable reporter
genes are known in the art, including, but not limited to
.beta.-galactosidase, luciferase, chloramphenicol acetyl
transferase, neomycin phosphotransferase. If expression of the
reporter gene is increased in the presence of a test compound, then
one can assume that the test compound will function similarly to
increase expression of WAF1 when it is located downstream from its
own transcription regulatory region, as it is in vivo. Since
increased expression of WAF1 is shown herein to have a growth
suppressing effect on tumor cells, it can be assumed that the test
compound which enhances the expression of the reporter construct
will similarly have a growth suppressive effect in vivo. The
transcription regulatory region of WAF1 which is sensitive to the
presence of wild-type p53 is located within about 2.4 kb of the
WAF1 transcriptional start site. The region includes the p53
binding site shown in SEQ ID NO:3. If the reporter construct is in
a cell, the cell can be incubated with the test compounds and the
effect on the expression of the reporter gene can be monitored and
measured. Alternatively, the reporter construct may be employed in
vitro in cell-free transcription and optionally translation
systems.
[0053] WAF1 is shown herein to be regulated by wild-type but not
mutant p53. Therefore, one can use the expression of WAF1 as a
marker for the expression of wild-type p53. Diminished WAF1
expression, relative to normal tissues, can indicate cancer, just
as diminished wild-type p53 expression or presence of mutated p53
expression can be indicative of cancer. Assays for WAF1 expression
can be used in addition to, or in place of, assays for wild-type
p53 directly. Tissues which are suitable for comparison purposes to
provide a normal control are typically adjacent, morphologically
normal tissues. Tests for the presence or amount of WAF1 expression
can employ either antibodies specific for WAF1 protein, nucleic
acid probes of at least about 10 nucleotides complementary to all
or a portion of the sequence of SEQ ID NO:1, or other tests known
in the art. Similarly, DNA of a tumor tissue can be tested to
determine whether it contains mutations. WAF1 mutations would be
expected to confer a neoplastic phenotype on cells, as do p53
mutations. Mutations can be determined by determining the sequence
of the genes in the tissue being tested, and comparing that
sequence to that disclosed in SEQ ID NO: 1. Such mutations may
arise in the germline or in somatic tissues. If the mutations arise
in somatic tissues, then they will not be found in other tissues of
the same individual. If the mutations arise in the germline, they
will be found in all tissues of the body, and will, like germline
p53 mutations, indicate a susceptibility to cancers. Tissues
suitable for testing for germline mutations include blood,
chorionic villi, amniotic fluid, and blastomeres if preimplantation
fertilized embryos.
[0054] Antisense WAF1 constructs contain a transcriptional promoter
and a transcriptional terminator (polyadenylylation signal), with a
DNA segment between them. The DNA segment comprises one or more
segments of the WAF1 gene, but that segment(s) is in an inverted
orientation in the construct, compared to the orientation in the
human genome. Transcription from the transcriptional promoter of
the construct produces an (antisense) RNA molecule which is
complementary to WAF1 RNA which is produced from the WAF1 promoter
in normal human cells. The promoter used to make the antisense RNA
molecule can be an inducible promoter which can be regulated by
certain prescribed stimuli. For example, a metallothionein promoter
or a hormone responsive promoter can be advantageously used. Other
promoters and terminators can be used as is convenient in the
particular application.
[0055] The antisense WAF1 constructs of the present invention can
be used in one type of cell to produce antisense RNA which is then
applied to other cells by techniques known in the art.
Alternatively, the WAF1 constructs can be administered to the
ultimate target cells in which regulation of WAF1 is desired.
Suitable means for introducing DNA constructs into cells are known
in the art. Administration of antisense constructs may be by
transfection, transformation, electroporation, fusion, etc., as is
known in the art. Inhibition of WAF1 expression causes cells to
proliferate and prevents cell death. This can be particularly
useful in situations where growing large numbers of certain cells
in culture is desirable, such as in the case of culturing epidermal
cells for transplantation. Alternatively, administration to certain
cells of the body may be desirable, such as immune cells or cells
of the gastrointestinal tract.
[0056] WAF1 antisense oligonucleotides are also provided for the
same purpose as the antisense constructs, discussed above. The
oligonucleotides are at least ten nucleotides and may be twenty or
thirty nucleotides in length. They may consist of normal
nucleotides or nucleotide analogs or mixtures of the two. Analogs
include methylphosphonates, aminoalkylphosphonates,
phosphorothioates, phosphorodithioates, substituted or
unsubstituted phosphoramidates. The antisense oligonucleotides are
typically linear, single-stranded molecules which are complementary
to the natural WAF1 mRNA made by human cells, though circular
molecules can also be utilized. These can be administered to cells
in liposomes, or naked, for uptake by the cells by passive or
receptor-mediated transport. It is often desirable that the
antisense oligonucleotide be designed to be complementary to the 5'
end of the mRNA, in particular to the translation start site.
However, other portions of mRNA molecules have been found to be
amenable to antisense inhibition, and may be used in the practice
of the present invention. It is also desirable to avoid portions of
the mRNA as target for the antisense oligonucleotides which have
secondary structures which involve hydrogen bonding with other
portions of the molecule. For example, it is desirable to avoid
regions which appear to be involved in formation of stems of
stem-loop structures.
[0057] Some suitable oligonucleotide sequences which may be used
are: 5'-
1 GGTTCTGACATGGCGCCTCC-3'; 5'-CCCAGCCGGTTCTGACATGG-3';
5'-ATGCAGCCCGCCATTAGCGC-3'; 5'-GTCATGCTGGTCTGCCGCCG-3';
5'-GTGGGCGGATTAGGGCTTCC-3'; 5'-TAAATAGTATTTCATAAAAT-3'.
[0058] These correspond to nucleotides number 67-86, 74-93,
181-200, 499-518, 560-579, 803-822, of the WAF1 cDNA shown in SEQ D
NO: 1.
[0059] The expression of WAF1 may also be inhibited by interference
with transcription, by adding oligonucleotides or modified
oligonucleotides than can form triple-stranded structures
(triplexes) by complexing with a segment of the WAF1 gene.
EXAMPLE 1
[0060] This example demonstrates an experimental gene expression
system which is sensitive to and specific for the presence of
wild-type p53.
[0061] As a first step towards the isolation of p53-regulated
genes, we determined optimal cell culture conditions under which an
exogenous wild-type p53 protein could activate transcription
through specific DNA binding. A reporter plasmid containing a p53
DNA-binding site upstream of a basal promoter (Kern et al. (1992)
Science 256, 827-830) linked to a luciferase reporter gene
(PG13-Luc) was cloned and cotransfected into SW480 colon cancer
cells with either a human wild-type p53 expression plasmid (p53-wt)
or a mutant p53 expression plasmid (p53-273). High luciferase
activity was observed only when wild-type p53 was present (data not
shown). No luciferase activity was detected if the reporter plasmid
contained mutant p53 binding sites (MG15-Luc), regardless of
whether or not wild-type p53 was present. This validated reporter
was then used in a p53-inducible system.
[0062] The glioblastoma cell line GM contains endogenous mutant p53
(Ullrich et al. (1992) Oncogene 7, 1635-1643) and
dexamethasone-inducible exogenous human wild-type p53 (Mercer et
al. (1990) Proc. Natl. Acad. Sci. USA 87, 6166-6170). The related
line DEL expresses the same endogenous mutant p53 and a
dexamethasone-inducible exogenous mutant p53 (Lin et al. (1992)
Natl. Acad. Sci. USA 89, 9210-9214). Both cell lines were
transfected with either PG13-Luc or MG15-Luc and incubated in the
presence or absence of dexamethasone. FIG. 1 shows that
dexamethasone-induced wild-type p53 (GM) but not mutant p53 (DEL)
expression activated transcription of the luciferase reporter gene
linked to a p53 binding site. No luciferase activity was observed
when the p53 binding site was mutant (MG15-Luc), or when the p53
protein was mutant (GM without dexamethasone or DEL with or without
dexamethasone). Transfection of wild-type p53 into DEL cells
activated the PG13-Luc reporter with or without dexamethasone (FIG.
1), confirming that the failure of expression of luciferase
reporter gene in this cell line was due to the absence of wild-type
p53. These experiments demonstrated that reporter gene expression
in these two cell lines was both sensitive and specific to the
presence of wild-type p53.
[0063] Methods:
[0064] The SW480 colon cancer cell line was maintained in culture
as previously described (Baker et al. (1990) Science 249, 912-915).
GM4723 (GM cells) and del4A (DEL cells) lines were passaged in
Eagle's minimal essential media and log phase cells were induced
with dexamethasone as previously described (Mercer et al. (1990)
Proc. Natl. Acad. Sci. USA 87, 6166-6170). PG13-Luc and MG15-Luc
plasmids were cloned by inserting the Hind III/EcoR I fragments
containing wild-type or mutant p53 binding elements (PG13-CAT and
MG15-CAT; Kern et al. (1992) Science 256, 827-830) into the Hind
III/EcoRI sites of pBluescript II SK+ (pBS; Stratagene, La Jolla,
Calif.). PG13 contains 13 copies of a p53 binding site, while MG15
contains 15 copies of a subtly mutated p53 binding site. The 200 bp
EcoR I/BamH I fragment containing the polyoma promoter (from
pBEL.Py; Munholland et al. (1992) EMBO J. 11, 177-184) was cloned
into pBS constructs containing either PG13 or MG15. A 2.6 kb Sac I
luciferase cassette (or a 3 kb beta-galactosidase cassette, see
below) without promoter elements, was then cloned downstream to
create either PG13-Luc or MG15-Luc.
[0065] Transfected cells were washed twice with 4 ml Dulbecco's PBS
per T-25 flask. The cells were lysed with 0.3 ml (per T-25) of
1.times. CCLR buffer (Promega, Madison, Wis.) for 10 minutes at
room temperature. After a 5 second spin to pellet large debris, 10
.mu.l of supernatant was added to 90 .mu.l of reconstituted
Luciferase Assay Reagent (Promega). Light emission was detected by
scintillation counting.
EXAMPLE 2
[0066] This example demonstrates the isolation of a wild-type p53
activated fragment (WAF1) by subtractive hybridization.
[0067] Based on the reporter gene experiments, we chose to use
subtractive hybridization to identify endogenous genes regulated by
p53 in GM cells. In order to determine the optimal time to isolate
RNA enriched for p53-induced genes, Northern blot analysis was
performed, using RNA isolated from GM cells at various intervals
following dexamethasone induction. FIG. 2 shows that under the
logarithmic growth conditions used, the exogenous wild-type p53
mRNA was detectable by 4 hours after induction and remained
elevated for at least 16 hours in GM cells upon dexamethasone
induction. A p53-induced cDNA library was therefore prepared from
GM cells treated with dexamethasone for 6 hours.
[0068] Eighty percent of the clones obtained carried inserts,
generally of 1.5- to 2.0-kb in length. A total of 120,000 clones
were screened by hybridization to a subtracted p53-induced cDNA
probe. This probe was made from cDNA of dexamethasone-induced GM
cells after subtraction with an excess of dexamethasone-induced DEL
RNA. Control experiments showed that the subtraction procedure
used, involving chemical crosslinking (Hampson et al. (1992) Nucl.
Acids Res. 20, 2899) provided an enrichment of over 100-fold for
cDNA sequences not present in the RNA used for subtraction (data
not shown). Following hybridization to the subtracted probe, the
clones were rehybridized to a probe made from RNA of
dexamethasone-induced DEL cells. A total of 99 clones
differentially hybridized to the subtracted probe on the initial
screen and forty-five of these reproducibly displayed differential
hybridization when re-tested.
[0069] Hybridization probes were prepared from these clones and
used in Northern blots containing RNA isolated from dexamethasone
treated or untreated GM cells. Of the 45 clones, 28 were found to
be highly induced upon dexamethasone treatment. The other 27 clones
were less induced by dexamethasone, and were not studied further.
Hybridization, sequencing and restriction endonuclease analysis
indicated that all of the 28 highly induced cDNA clones were
derived from a single 2.1 kb mRNA. The gene encoding this message
was named WAF1 (wild-type p53 activated fragment #1).
Rehybridization to the cDNA library revealed that WAF1 cDNA was
present at a frequency of 0.4% following dexamethasone
induction.
[0070] Methods:
[0071] 3.5 .mu.g poly A+RNA obtained from GM cells induced with
dexamethasone for 6 hours was isolated using oligo-dT cellulose
(Clontech, Palo Alto, Calif.) according to the supplier's
recommendations, from total RNA prepared by CsCl gradient
ultracentrifugation of guanidine isothiocyanate lysed cells, as
described (Davis, L. G., et. al. (1986) Elsevier Science Pub. Co.,
Inc.). The poly A+RNA was used to make an oligo-dT primed cDNA
library with the SuperScript Choice System (BRL Research Products
Life Technologies, Grand Island, N.Y.). A total of 100 ng of cDNA,
comprising the 1.5 to 5 kb fraction, was ligated to lambda Ziplox
EcoR I arms (Gibco BRL Life Technologies, Inc., Gaithersburg, Md.),
and phage clones were obtained following infection of E. coli
strain Y109OZL. Phage clones were screened by hybridization of
colony lifts to either subtracted or unsubtracted cDNA probes
prepared as described below. Excision of pZLI plasmid clones was
carried out by phage infection of the excision strain DH1OB-Zip
(Elledge et al. (1991) Proc. Natl. Acad. Sci. USA 88,
1731-1735).
[0072] Unsubtracted cDNA probes were prepared from 2 .mu.g poly
A+RNA "driver" using oligo-dT as primer and MMLV Super-Script II as
described (Hampson et al., (1992) Nucl. Acids Res. 20, 2899),
except that following alkaline hydrolysis with NaOH and
neutralization with HCl, the cDNA was isopropanol precipitated in
the presence of 0.17 M sodium perchlorate, washed with 70% ethanol,
vacuum dried and resuspended in 10 .mu.l of water (Kinzler et al.
(1989) Nucl. Acids Res. 17, 3645-3653). Twenty ng of unsubtracted
cDNA was then labelled with random primers using Sequenase as
described (Hampson et al. (1992) Nucl. Acids Res. 20, 2899).
Subtracted cDNA probes were prepared after a 22 hour hybridization
of 500 ng "target" cDNA to 10 .mu.g poly A+"driver" RNA, chemical
crosslinking with 2,5-diaziridinyl-1,4-benzoquinone(generously
provided by John Butler), and labelling as described (Hampson et
al. (1992) Nucl. Acids Res. 20, 2899).
EXAMPLE 3
[0073] This example demonstrates the structure of the WAF1
gene.
[0074] Eighteen of the 28 WAF1 clones appeared to contain near
full-length cDNA, predicted to be 2.1 kb on the basis of Northern
blot analysis (FIG. 2). DNA sequencing revealed that most of the
clones contained the same 5'-end. Because the cDNA library used was
not amplified, this likely represented the 5'-end of the
transcript. The WAF1 cDNA sequence is shown in FIG. 3. (SEQ ID NO:
1) The first ATG codon occurred at nucleotide 77, and an in-frame
termination codon occurred at nucleotide 570, predicting a
translation product of 18.1 kd. In vitro transcription and
translation of WAF1 cDNA clones produced a protein of the expected
size (not shown). Analysis of the amino-acid sequence of WAF1
protein revealed a cysteine-rich region
C(X).sub.4C(X).sub.15,C(X).sub.6C between amino acids 13 and 41
with the potential for zinc-binding (Berg, Science 232:485-487
(1986)), and a basic region between amino acids 140 and 163
containing two potential bipartite nuclear localization signals
(Robbins et al., Cell 64:615-623 (1991)) near the C-terminus (SEQ
ID NO: 2) No significant homologies at the amino-acid level were
found to known proteins (NBRF-PIR release #35.0). Southern blot
analysis showed that WAF1 was probably a single copy gene, with no
close relatives in the human genome (FIG. 4).
[0075] Methods:
[0076] Northern blot analysis was performed as previously described
(El-Deiry et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3470-3474)
except that Quickhyb (Stratagene, La Jolla, Calif.) was used as the
hybridization solution.
EXAMPLE 4
[0077] This example demonstrates that WAF1 is localized to
chromosome 6, band p21.2 of the human genome.
[0078] To identify the chromosomal location of the WAF1 gene, a
human genomic P1 clone (P1-WAF1) containing WAF1 sequences was
obtained (as described below). The clone contained about 90 kb of
DNA, and partial sequencing revealed that the WAF1 gene consisted
of three exons of 68, 450, and 1600 bp (exons 1, 2, and 3
respectively). The translation initiation signal was contained in
exon 2, a relatively long coding exon (Sterner et al (1993) Mol.
and Cell. Biol. 13, 2677-2687). The P1-WAF1 clone was labelled with
biotin and hybridized to metaphase chromosomes as previously
described (Meltzer et al. (1992) Nature Genet. 1, 24-28). A total
of 18 metaphase cells were examined, and each had at least one
"double" fluorescent signal (i.e., signals on each of 2 chromatids)
on the middle of the short arm of chromosome 6 (FIG. 5). In 15/18
cells, double signals were observed on both chromosome 6 homologs.
Only chromosomes in which both chromatids displayed a signal were
included for analysis, making the background hybridization close to
zero. The same cells subjected to FISH had been previously G-banded
using Trypsin-Giemsa and photographed to allow direct comparison of
the results. The results demonstrated that sequences hybridizing to
WAF1 DNA fragment were localized to 6p21.2.
[0079] Methods:
[0080] A screen of human genomic P1 clones for WAF1 was performed
using the primers 5'-CTTTCTAGGAGGGAGACAC-3' and
5'-GTTCCGCTGCTAATCAAAG-3' from WAF1 exon 3 for PCR (Genome Systems,
Inc., St. Louis, Mo.). The PCR was performed using the Bind-Aid kit
(USB) in a 25 .mu.l reaction containing 2.5 .mu.l 10.times. USB PCR
buffer, 2 .mu.l 2.5 mM each dNTP (dATP, dCTP, dGTP, and dTTP), 0.5
.mu.l Bind-Aid (0.5 .mu.g/.mu.l SSB), 0.5 .mu.l each primer (350
ng/.mu.l), 10 ng DNA template, and 2 Units AmpliTaq (Pekin Elmer
Cetus). Amplification was carried out for 35 cycles (following the
profile: 95.degree. C. for 30 seconds, 57.5.degree. C. for 1
minute, and 70.degree. C. for 1 minute), yielding a 99 bp PCR
product. The P1 clone obtained (P1-WAF1) was labelled with biotin
and hybridized to metaphase chromosomes as previously described
(Meltzer et al. (1992) Nature Genet. 1, 24-28). Eighteen metaphase
nuclei were examined for WAF1 localization.
EXAMPLE 4
[0081] This example demonstrates that (1) WAF1 is induced in more
than one cell type following wild-type p53 expression; (ii) WAF1 is
highly conserved among species; and (i) WAF1 is induced by p53 in
other species.
[0082] FIG. 6 illustrates the expression of WAF1 in GM cells
following dexamethasone treatment for 16 hours (lane 2), compared
to either uninduced GM cells (lane 1) or dexamethasone treated DEL
cells containing induced mutant p53 (lane 3). Controls for the
experiment included two other genes known to be induced by p53,
MDM2 and GADD45, as well as an unrelated gene, TGF-beta. Both MDM2
and GADD45 were induced in the GM cells when wild-type p53 was
present, but much less so than WAF1
[0083] To examine the induction of WAF1 by wild-type p53 in a
different cell line, a wild-type p53 construct in an adenoviral
vector (Ad-p53) was used to infect human SW490 colon cancer cells.
That Ad-p53 produced transcriptionally active p53 was demonstrated
by assaying an SW480 cell line carrying a stably integrated
reporter responsive to wild-type but not mutant p53 as described
below. SW480 cells were infected with either Ad-p53 or Ad-gal (a
control adenoviral vector producing beta-galactosidase instead of
p53) for 16 hours and RNA used for Northern blot analysis. FIG. 6
shows that WAF1 was highly induced in SW480 cells infected with
Ad-p53 (lane 5), but not those infected with Ad-gal (lane 4).
[0084] We next assessed the evolutionary conservation of WAF1. "Zoo
blots" revealed that single copy sequences from mouse cells
hybridized to the human WAF1 clone (FIG. 4). Attempts to clone a
mouse WAF1 cDNA from a mouse adult brain cDNA library were
unsuccessful. Therefore, we obtained a mouse genomic clone
containing the WAF1 gene as described below. The nucleotide and
predicted amino acid sequence of the mouse WAF1 (mWAF1) second exon
is shown in FIG. 7A. The mouse and human WAF1 second exon sequences
were 76% identical and 80% similar at the amino acid level (FIG.
7B). A stretch of 26 amino acids (human aa 21-56) was almost
perfectly conserved, as was the zinc finger-like motif between aa
13 and 41 in human WAF1 (H(X).sub.4C(X).sub.15C(X).sub.6C in the
mouse). The positions of introns surrounding exon 2 in the WAF1
gene were identical in both human and mouse (not shown).
[0085] To determine whether rodent WAF1 gene expression was induced
by wild-type p53, two experimental systems were used. The first
consisted of rat embryo fibroblasts containing a stably integrated
murine temperature-sensitive mutant p53 (REF-112 cells; Michalovitz
et al. (1990) Cell 62, 671-680). These cells were transfected with
the PG13-Luc reporter and incubated either at 37.degree. C. (mutant
p53 conformation), or 31.degree. C. (wild-type p53 conformation)
for 24 hours. No measurable increase in luciferase activity was
observed at 37.degree. C., but luciferase activity increased
1000-fold at 31.degree. C., confirming the presence of
transcriptionally active murine wild-type p53 at the latter
temperature. RNA was then prepared from REF-112 cells incubated for
14 hours either at 37.degree. C. or 31.degree. C. FIG. 8 shows that
expression of WAF1 mRNA was detected at 31.degree. C. but not at
37.degree. C., demonstrating that the WAF1 gene is conserved in
rat, and that the gene is inducible by the murine p53 at the
wild-type permissive temperature.
[0086] Second, the murine fibrosarcoma cell line MCO1 (Halevy et
al. (1991) Oncogene 6, 1593-1600), which lacks p53 due to a splice
site mutation and a deletion, was infected with either Ad-p53 or
Ad-gal. At 22-hours following adenoviral infection, RNA was
prepared and used in Northern blot analysis. FIG. 8 shows that
mWAF1 was highly induced in MCO1 cells infected with Ad-p53, but
not in cells infected with Ad-gal. Thus, WAF1 induction by p53 was
conserved in both rat and mouse cells.
[0087] The fact that WAF1 was (i) induced in more than one human
cell type following wild-type p53 expression; (ii) highly conserved
among species; and (iii) induced by p53 in other species, suggests
that WAF1 is important for p53 function.
[0088] Methods:
[0089] The MDM2 probe was made from a 1.6 kb cDNA fragment (Oliner,
J. D. et al. (1993) Nature 362, 857-860), and the GADD45 probe was
generously provided by A. Fornace (Kastan et al. (1992) Cell 71,
587-597). Probes were made by oligo-labelling DNA fragments
isolated from agarose gels (Feinberg, A. P. and Vogelstein, B.
(1983) Anal. Biochem. 132, 6-13).
[0090] A mouse WAF1 (mWAF1) genomic clone was isolated by screening
1.times.10.sup.6 clones of a mouse genomic DNA library in Lambda
Fix II (Stratagene), using the human WAF1 cDNA as a probe. One
hybridizing clone was obtained. An 11 kb Hind III fragment
containing the second exon of mWAF1 was subcloned into the Hind III
site of pBS. An 0.3 kb Pst I fragment from this clone (containing
part of mWAF1 exon 2) was used to probe the Northern blot in FIG.
8.
[0091] The cDNA for p53 was obtained as a BamH I fragment from the
p53-wt vector (Baker et al. (1990) Science 249, 912-915; Kern et
al. (1992) Science 256, 827-830) and cloned into the BamH I site of
pMV10 (Wilkinson, G. W. G., and Akrigg, A. (1992) Nucl. Acids Res.
20, 2233-2239). The Hind III fragment of pMV10-p53-wt was then
subcloned into the Hind III site of the pMV60 vector (Wilkinson, G.
W. G., and Akrigg, A. (1992) Nucl. Acids Res. 20, 2233-2239) to
make the vector pMV60-p53-wt. The plasmids pMV60-p53-wt and pJM17
(Wilkinson, G. W. G., and Akrigg, A. (1992) Nucl. Acids Res. 20,
2233-2239) were co-transfected into 293 cells. Recombinants were
plaque purified and tested for production of transcriptionally
active p53 by infection of the SW480-IAB3 cell line. A plaque
purified recombinant (Ad-p53) induced beta-galactosidase activity
in infected SW480-IAB3 cells. The beta-galactosidase producing
defective adenovirus (Ad-gal) was obtained from plaque purified
recombinants following co-transfection of 293 cells with pMV35 and
pJM17. Both Ad-p53 and Ad-gal were further purified by CsCl
banding.
[0092] The SW480-IAB3 cell line was obtained following
co-transfection of SW480 cells with plasmids PG13-Gal and
pCMV-Neo-Bam (Baker et al. (1990) Science 249, 912-915), and
selection with genetecin. Individual clones were isolated by
limiting dilution and tested for the presence of stably integrated
intact reporter by transfection with either plasmid p53-wt or
p53-143 (Kern et al. (1992) Science 256, 827-830) followed 24 hours
later by in-situ X-gal staining. The SW480-IAB3 was chosen for
passaging because no beta-galactosidase activity was detectable
unless wild-type p53 was present in the cells. The cells were
maintained in Leibovitz L15 medium supplemented with 10% fetal
bovine serum and 0.5 mg/ml genetecin. REF-112 and MCO1 cells were
obtained through the generosity of Moshe Oren. For transfection
experiments, 1.5.times.10.sup.6 cells were plated in 25-cm.sup.2
tissue culture flasks 24 hours before transfection. A total of 5
.mu.g of CsCl banded DNA and 25 .mu.g Lipofectin (Bethesda Research
Laboratories, Gaithersburg, Md.) were used for transfections. For
growth inhibition experiments (FIG. 9), hygromycin (0.25 mg/ml)
selection began 24 hours after transfection.
EXAMPLE 5
[0093] This example demonstrates that WAF1 suppresses tumor cell
growth.
[0094] If WAF1 played a role in mediating the tumor growth
inhibition of p53, one might expect it to have a growth suppressive
role of its own. To address this possibility, mammalian expression
vectors containing p53 cDNA or WAF1 cDNA in either the sense
(pC-WAF1-S) or antisense pC-WAF1-AS) orientation were constructed.
The vectors each contained a gene conferring hygromycin resistance
in addition to the cDNA. The vectors were transfected into SW480
cells, previously shown to be inhibited by wild-type p53 expression
(Baker et al. (1990) Science 249, 912-915). Following transfection,
cells were grown in the presence of hygromycin and the number of
colonies was scored after 2-3 weeks.
[0095] The data in FIG. 9 show that introduction of WAF1 sense cDNA
expression vectors resulted in substantial growth suppression, as
seen by a 10-20 fold decrease in the number of hygromycin-resistant
colonies. This growth suppression was similar to, but not as
complete as, that observed with p53 (FIG. 9). Introduction of the
WAF1 antisense cDNA expression vector, or the vector devoid of WAF1
sequences, resulted in a similar number of clones. The few small
clones which did appear after transfection of the WAF1 sense cDNA
expression vector grew at a slow rate and could not be passaged.
Similar results were obtained in four separate experiments, each
with triplicate transfections, using different preparations of
plasmid DNA. We additionally used the brain tumor cell lines GM and
DEL in similar experiments, and found that their growth was also
suppressed by the introduction of wild-type WAF1 (FIG. 9 and data
not shown). As an additional control, we constructed a WAF1 mutant
(pC-WAF1-ES), with a stop codon at nt 222. Introduction of
pC-WAF1-ES into either SW480 or DEL cells did not result in
significant growth suppression (FIG. 9).
[0096] Methods
[0097] pC-WAF1-S (sense) and pC-WAF1-AS (antisense) expression
plasmids were prepared by cloning the full-length WAF1 cDNA as a
Not I fragment from cDNA library clone #33 into the Not I site of
pCEP4 (Invitrogen). The pC-WAF1-ES mutant vector was similarly
obtained from a PCR generated cDNA insert, containing a G to A
transition at nt 222, resulting in a stop codon instead of Trp at
amino acid 49.
EXAMPLE 6
[0098] This example demonstrates that p53 activates the WAF1
promoter.
[0099] Having demonstrated that WAF1 expression is induced by
wild-type p53, we attempted to determine whether this resulted from
a direct interaction of p53 with regulatory elements in WAF1. To
search for sequences transcriptionally responsive to p53, we used
the 90 kb genomic clone P1-WAF1 in a yeast enhancer trap system. In
this system, yeast cells auxotrophic for histidine were transformed
with a plasmid library constructed by insertion of random fragments
of P1-WAF1 upstream of a truncated GAL1 promoter regulating
histidine reporter gene expression. Clones were selected for
histidine prototropy in the presence of human p53 expression. Three
libraries were constructed, using Alu I, Hae III, or Sau 3AI
fragments of P1-WAF1. Through the screening of 1.6.times.10.sup.5
transformants, 22 wild-type p53-dependent, histidine prototrophs
were obtained. No histidine prototropy was observed if yeast
expressed mutant instead of wild-type p53. All but one of the 22
clones were found to contain either of two sequence elements, both
matching the previously defined p53 binding site consensus. Mapping
of the two elements revealed that one of them was located 2.4 kb
upstream of WAF1 coding sequences (FIG. 10). Thus, a p53-binding
site was present upstream of WAF1, and this element, when placed in
an artificial system with a standard promoter, could stimulate
expression of a reporter gene in the presence of wild-type p53.
[0100] To determine whether the natural promoter elements of WAF1
could mediate p53-dependent transcriptional activation, a 2.4 kb
genomic fragment, with its 3'-end at nt 11 of WAF1 cDNA, was cloned
upstream of a promoterless luciferase reporter gene. A partial
sequence of the WAF1 promoter, and a map of this clone, is shown in
FIG. 10. This promoter was G:C rich and contained a TATA-element 43
nt upstream of the putative transcription start site. Two Sp1
binding sites were located at nt -50 and -104, and there was a
sequence weakly matching the p53 binding site consensus at
nt-75.
[0101] FIG. 11 shows that the WAF1 promoter construct WWP-Luc
activated expression of luciferase only in the presence of
wild-type p53. In the absence of wild-type p53 (GM cells without
dexamethasone or DEL with or without dexamethasone), expression of
this reporter was less than 2% of levels observed in the presence
of wild-type p53. When the 2 kb upstream p53 binding site was
deleted (DM-Luc), the majority of the luciferase activity was
abolished, though the residual activity was still wild-type
p53-dependent. This observation suggests the presence of a second
(weaker) p53 response element within the WAF1 promoter, perhaps at
nt -75 (FIG. 10). The same pattern of reporter activation was
observed following co-transfection of WWP-Luc or DM-Luc with the
wild-type p53 expression plasmid in SW480 cells. There was a
200-fold increase in luciferase activity with wild-type p53
compared to mutant p53 (273.sup.His) transfection (data not shown).
Similar to the GM cell results, luciferase activity decreased by
approximately 80% when the upstream p53 response element was absent
(DM-Luc construct, FIG. 11).
[0102] Methods
[0103] The P1-WAF1 clone was digested to completion with Hae III,
Alu I, or Sau 3AI, subcloned into the plasmid pBM947 and used to
identify p53 binding sites by genetic selection in yeast (Wilson et
al. (1991) Science 252, 1296-1300; T. Tokino et al., unpublished
data). A total of 530,000 clones were obtained in E. coli, and the
DNA from these clones was used to transfect S. cerevisiae cells
containing a p53 expression vector and a HIS3 gene under the
control of p53 binding sequences (Nigro et al. (1992) Mol. Cell.
Biol. 12, 1357-1365; Kern et al. (1992) Science 256, 827-830; T.
Tokino and S. Thiagalingam, unpublished data). A total of 160,000
yeast clones were assayed for histidine prototropy. Selection in
the absence of histidine allowed the isolation of clones containing
a p53 binding sequence; transcriptional activation by p53 resulted
in HIS3 production and subsequent survival of the yeast
transformants. DNA was isolated from such clones and tested for
induction of histidine prototropy in yeast strains with or without
human p53 expression vectors.
[0104] WWP-Luc and DM-Luc plasmids were cloned by inserting the 2.6
kb BamH I luciferase cassette (from PG13-Luc) into the Xho I sites
of pWWP and pDM. The 2.4 kb WAF1 promoter region was obtained as a
Hind III cassette by PCR amplifications using a P1-WAF1 subclone as
template and the primers 5'-CCACAAGCTTCTGACTTCGGCAG-3' and
5'-CCCAGGAACAAGCTTGGGCAGCAG- -3'. This cassette was cloned into the
Hind III site of pBC KS+ (Stratagene) to yield plasmid pWWP
containing the endogenous WAF1 promoter including the upstream p53
binding element near one end and WAF1 nt 11 at the other end (FIG.
10). The plasmid pDM, which lacks the p53 binding element 2.4 kb
upstream of WAF1, was obtained by digesting pWWP with Sac I, and
recloning the deleted fragment after circularization.
[0105] SEQ ID NO: 1
[0106] See Nucleotide Sequence of FIG. 3.
[0107] SEQ ID NO: 2
[0108] See Amino Acid Sequence of FIG. 3.
[0109] SEQ ID NO: 3
[0110] GAACATGTCCCAACATGTTG
Sequence CWU 0
0
* * * * *