Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR THE SCREENING OF CELL CYCLE
MODULATORS
Related Information
The contents of the patents, patent applications, and references cited
throughout
this specification are hereby incorporated by reference in their entireties.
Background of the Invention
Key transition points in the eukaryotic cell cycle are regulated by the
activity of
cyclin dependent kinases (cdks) (Nurse, P., (1990) Nature 344(6266):503-508
for
review see: Fisher, R.P. (1997) Curr Opin Genet Dev 7(1):32-38). Activity of
these
molecules is regulated by many mechanisms including phosphorylation and
protein-
protein interactions (Lew, et al., (1996) Curr Opin Cell Biol 8:795-804). For
example,
phosphorylation of Cdk by Cdk7 (on e.g., threonine residue 160) activates Cdk
activity
whereas phosphorylation by Mytl and Weel (on Thr-14 and Tyr-15) is inhibitory
(Gould, K.L., et al. (1989) Nature 42(6245):39-45; Krek, W., et al. (1991)
EMBOJ
10(2):305-16; Russell, P., et al., (1987) Cell 49(4):559-567). Moreover, Cdk
activity
can be regulated by the dephosphorylation of these residues by, for example, a
Cdc25
dual-specificity protein phosphatase which is a member of a group of highly
related dual
specificity phosphatases that promote cell cycle phase transitions
(Galaktionov, et al.,
(1991) Cell 67:1181-1194; Millar, J.B., et al., (1991) EMBOJ 10(13):4301-
4309).
Cdc25 phosphatases are expressed in all eukaryotes. A single form of Cdc25 is
expressed in yeast (Russell, P., et al. (1986) Cell 45(1):145-153) and two
forms are
present in Drosophila (Edgar, B.A., et al. (1989) Cell 57(1):177-187; Alphey,
L., et al.,
(1992) Cell 69, 977-988). In mammals, three forms of Cdc25 (A, B and C) are
present
and are encoded by three highly related but distinct genes (reviewed by
Draetta, et al.,
(1997) Biochem et Biophys Acta 1332:M53-M63). Recently a fourth form of Cdc25
was
reported in C. elegans raising the possibility that other forms may exist in
mammalian
cells (Ashcrot, N.R., et al., (1998) Gene 214:59-66). A crystal structure has
been
deduced for human Cdc25A (Fauman, E. B., et al., (1998) Cell 93, 617-625)
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Separate but perhaps over lapping functions have been assigned to each form of
Cdc25. Cdc25A has been proposed to mediate transition from G1 to S phase
(Jinno, S., et
al., (1994) EMBO Journal 13, 1549-1556). The role of Cdc25B has been cast both
at the
G1/S and G2M transitions (Sebastian et al. (1993) Proc Natl Acad Sci USA
90(8):3521-
3524; Galaktionov, K., et al., (1995) Genes & Development 9, 1046-1058;
Gabrielli,
B.G., et al., (1996) JCell Sci 109(Pt 5):1081-1093; Garner-Hamrick, et al.,
(1998) IntJ
Cancer 76:720-728) and Cdc25C is required for the onset of mitosis (Karlsson
C., et al.,
(1999) JCell Biol 146(3):573-584).
Based on gene disruption studies, Cdc25 phosphatases clearly play a role in
cell
cycle progression in yeast and Drosophila (Russell, P., et al. (1986) Cell
45(1):145-153;
Edgar, B.A., et al. (1989) Cell 57(1):177-187; Alphey, L., et al., (1992) Cell
69, 977
988). In mammalian cells, at least one report implicates Cdc25A in regulating
G1 arrest
due to DNA damage (Terada, Y., et al., (1995) Nature 376, 358-362). In
addition,
Cdc25A has been reported to play a role in regulating cell cycle progression
in response
to serum factors. For example, TGF(3 was proposed to cause G1 arrest in
certain cell
types by inhibiting Cdc25A expression (Iavarone, A., et al, (1999) Mol Cell
Biol 19,
916-922; Hoffman, L, et al., (1994) EMBO Journal 13, 4302-4310). Cdc25A has
also
been proposed to lie at the end of signal transduction pathways activated by
mitogens
(Galaktionov, K., et al., ( 1996) Nature 382, 511-517; Galaktionov, K., et
al., ( 1995)
Genes & Development 9:1046-1058).
In the mouse, Cdc25A and B are expressed in overlapping but distinct patterns
in
adult animals as well as in embryos (Wickramasinghe, D., et al., (1995)
Development
121, 2047-2056; Kakizuka, A., et al., (1992) Genes & Development 6, 578-590).
In
adult mice, Cdc25A is expressed most abundantly in testes and kidney and is
barely
detectable in lung and spleen (Wickramasinghe, D., et al., (1995) Development
121,
2047-2056). In the adult rat, Cdc25B is expressed most abundantly in spleen
and lung
but not detectable in kidney (Kakizuka, A., et al., ( 1992) Genes &
Development 6, 578-
590). In adult mice, Cdc25C is expressed most abundantly in thymus but was not
detected in lung or kidney (Nargi, J. L., et al., (1994) Immunogenetics 39, 99-
108).
Consistent with their proposed role as cell cycle regulators, over-expression
of
Cdc25A and B, but not C, has been reported in breast cancer (Galaktionov, K.,
et al.,
(1995) Genes & Development 9, 1046-1058), non-Hodgkin lymphomas (Hernandez,
S.,
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et al., (1998) Cancer Research 58, 1762-1767), in head and neck cancers
(Gasparotto,
D., et al., (1997) Cancer Research 57, 2366-2368), and in human gastric
carcinomas
(Kudo, Y., et al., (1997) Japanese Journal of Cancer Research 88, 947-952).
Moreover,
it has been reported that Cdc25A and B, but not C, can cooperate with the
activated ras
oncogene to immortalize mouse embryo fibroblasts (Galaktionov, et al., (1995)
Science
269:1575-1577).
The mechanism by which Cdc25 polypeptides regulate the cell cycle is still
incompletely understood. Moreover, current methods for identifying modulators
of
Cdc25 activity involve assays that are directed to screening for compounds
that alter
either, 1 ) Cdc25 phosphatase activity, or, 2) the ability of a Cdc25
polypeptide to cause
a phenotypic effect such as, e.g., apoptosis.
Examples of techniques for identifying modulators of Cdc25 phosphatase
activity are presented in, e.g., USPN 5,695,950, where potential inhibitor
compounds are
incubated with Cdc25 and a substrate, and a change in the ability of Cdc25 to
1 ~ dephosphorylate the substrate is taken as indicative of the test compound
as modulating
Cdc25 phosphatase activity. Similarly, in USPN 5,294,538, an assay is
presented for
screening anti-mitotic compounds using a Cdc25 phosphatase and test substrate,
e.g., p-
nitrophenyl, and Cdc25 phosphatase activity on the substrate in the presence
or absence
of a candidate compound is determined. And, in USPN 5,856,506 and USPN
5,700,821,
compounds for inhibiting the phosphatase activity of protein phosphatases such
as
Cdc25A and Cdc25B are presented.
Examples of techniques for assaying the ability of a Cdc25 polypeptide to
cause
a phenotypic effect such as, e.g., apoptosis, are presented in USPN 5,443,962,
where an
assay for identifying an inhibitor of Cdc25 phosphatase causing apoptosis is
scored
based on the ability of the inhibitor to cause the cell to proliferate.
Another phenotypic
assay is presented in USPN 5,861,249, where modulators of Cdc25 are identified
based
on their ability to alter Cdc25-induced apoptosis as compared to a control.
In summary, all of the foregoing assays are directed to screening compounds
that
alter, either phosphatase activity on a substrate, e.g., an artificial
substrate such as p-
nitrophenyl, or the ability of the modulator to alter a Cdc25-related change
of a
phenotypic effect such as, e.g., apoptosis.
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Accordingly, these assays have several limitations. The assays either look at
phosphatase activity in isolation of any downstream or biological effect, or,
are designed
to begin with cells that are severely compromised in their growth and yield a
qualitative
result. Thus, candidate compounds that may function in a phosphatase assay may
not
have been assayed for any downstream or biological efficacy. In addition,
potentially
useful compounds that are being assayed at levels or in a form that is
incompatible with
cell growth, may be erroneously dismissed as ineffective.
Accordingly, a need for improved assays for identifying modulators of Cdc25
exists.
Summary of the Invention
The invention solves the foregoing problems by providing a novel assay for
screening modulators of Cdc25 that relies on a robust cell culture system that
can
identify a candidate modulator by the appearance of a return or "rescue" of a
strong
reporter gene signal. The assay employs a catalytically active form of a Cdc25
that can
repress a selected promoter driving strong gene expression.
In parallel, a control is performed using a catalytically inactive form of
Cdc2~
that does not repress promoter activity but allows for a strong baseline
signal to be
established. This important control allows for determining if a particular
test compound
is inhibiting promoter activity independently of Cdc25. If the control signal
is
essentially unaffected, than the signal measured from the test reaction
containing the
catalytically active form of a Cdc25 can be accurately interpreted. Moreover,
candidate
modulators of Cdc25, e.g., inhibitors of Cdc25-mediated gene regulation, will
cause a
strong signal to appear.
Accordingly, the invention has several advantages which include, but are not
limited to, the following:
- provides an assay that measures the appearance of a strong signal from a
background of little or no signal;
- provides an assay that measures the appearance of a quantifiable signal and
not
a qualitative signal involving variable cell growth or other biological
effect;
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- provides an assay that contains a control that can accurately identify
compounds that are false positives (e.g., compounds that rescue the signal but
also
increase the signal in the test reaction) or false negatives (e.g., compounds
that produce
no signal but also lower the control signal, e.g., cytotoxic compounds) and
this insures
that inappropriate compounds are not further investigated and that candidate
compounds
are not erroneously dismissed;
- provides an assay that measures Cdc25-mediated gene regulation, e.g., at the
level of promoter control, and provides promoters that are mediated by Cdc25
(e.g., the
p21/WAF promoter; SV40 promoter) and control promoters that are not (e. g.,
the globin
promoter); and
- provides Cdc25A genomic and cDNA nucleic acids, polypeptides, and
transgenic animals having a Cdc25 gene disruption for extending determinations
made
using, e.g., the above assay for studies relating to Cdc25 autoregulation
and/or studies
requiring cells or an animal model with lowered or absent levels of a Cdc25
polypeptide.
Accordingly, in one aspect, the invention provides a method for identifying an
modulator of Cdc25 activity by providing a cell having a recombinant Cdc25
phosphatase gene where the expression of the gene alters the transcription of
a selected
gene. The method further includes contacting the test cell with a compound
under
conditions where the recombinant Cdc25 phosphatase gene is expressed and
alters the
transcription of a selected gene and determining the amount of transcription
of a selected
gene in the test cell as compared to the amount of transcription of the
selected gene in
the absence of the compound where a statistically significant change in the
amount of
transcription of the selected gene is indicative of the compound being a
modulator of
Cdc25-mediated transcription.
In one embodiment, the assay further includes comparing the change in
transcription measured in the above aspect in comparison with the
transcription
measured from a control test cell having a recombinant catalytically inactive
Cdc25
phosphatase under conditions where the catalytically inactive Cdc25
phosphatase is
expressed and does not substantially alter the transcription of the selected
gene and
determining the amount of transcription of the selected gene in the control
test cell in the
absence of the test compound as compared to the amount of transcription of the
selected
gene in the presence of the compound where a statistically significant change
in the
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amount of transcription of the selected gene is indicative of the compound as
a
modulator of transcription independent of Cdc25.
In one embodiment, the selected gene is a eukaryotic gene that contains, or is
operably linked, to a eukaryotic promoter element. In a related embodiment,
the
selected gene is p21/WAF, pGK, or Cdc25.
In a related embodiment, the selected gene contains a reporter gene,
preferably,
luciferase. In a related embodiment, the reporter gene is controlled by a
cellular
promoter such as the p21/WAF promoter, pGK promoter, or a Cdc25 promoter.
In another embodiment, the reporter gene is controlled by a viral promoter,
preferably the SV40 promoter.
In another embodiment, the recombinant Cdc25 phosphatase gene of the above
aspect encodes a mammalian Cdc25 phosphatase, preferably a mouse Cdc25
phosphatase, more preferably a human Cdc25 phosphatase. In a related
embodiment,
the Cdc25 phosphatase, preferably a human phosphatase, is selected from the
group
consisting of Cdc25A, Cdc25B, and Cdc25C.
In even another embodiment, the above aspect employs a test cell that is a
mammalian cell, preferably a murine cell, and more preferably a human cell.
In a yet another embodiment, the method includes determining transcription by
measuring reporter gene activity, preferably luciferase activity.
In preferred embodiment, a statistically significant increase in the amount of
transcription determined, indicates that the test compound is an inhibitor of
Cdc25
activity.
In another preferred embodiment, a statistically significant decrease in the
amount of transcription determined, indicates the test compound is an
activator of Cdc25
activity.
Other features and advantages of the invention will be apparent from the
following detailed description and claims.
Brief Description of the Drawings
Figure 1 shows the cDNA sequence and predicted amino acid sequence of murine
Cdc25A .
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Figure 2 shows in a schematic of the probes used for S 1 nuclease protection
analysis (Panel A). Panel B depicts an autoradiograph of an S 1 nuclease
protection
analysis. Genomic fragments were isolated and 5' end-labeled at the indicated
restriction
sites. These were hybridized to RNA isolated from 129sv mouse ES cells as
described
herein. The probe labeled at the NcoI site gave rise to a protected fragment
of 420
nucleotides (nt) (700 nt undigested) whereas the probe labeled at the NotI
site gave rise to
a product of 260 nt ( 1100 nt undigested). Probes hybridized to 50 pg yeast
tRNA did not
give rise to protections.
Figure 3 is a histogram of Cdc25A promoter activity. Mammalian 293 kidney
cells (Panel A) and H460 lung cells (Panel B) were transfected in triplicate
with the
indicated reporter construct. Shown is the average of each triplicate
transfection. Error
bars indicate the standard error of the mean and the Y axis scales have been
adjusted to
reflect the difference in transfection efficiency of the two cell lines.
Figure 4 is a histogram indicating the auto-regulatory activity of Cdc25A
phosphatase. Mammalian 293T cells were co-transfected with the indicated
Cdc25A
expression vector or an empty vector (pcDNA, Invitrogen) and 1.0 pg of the
indicated
luciferase (luc) reporter gene construct. Cells were harvested 48 hours later
and assayed
for luciferase activity. Error bars indicate the standard error of the mean;
for Cdc25A luc
n=8, for p21/WAF luc n=7 and for (3 Globin n=3. The lower panel depicts a
similar
analysis performed on the viral promoter SV40 and the eukaryotic promoter pGK.
Figure S shows an autoradiograph of Cdc25A RNA levels during the cell cycle.
RNA samples were isolated at the indicated times before or after release from
a cell
synchronization step (double thymidine block) and analyzed by Northern blot.
Replicate
blots were probed with a Cdc25A cDNA (Panel A) or ribosomal protein S 14 cDNA
(Panel B), quantitated using a phosphoimager, and values were normalized
(Panel C).
Figure 6 shows a schematic of two Cdc25A targeting vectors with the frequency
of homologous recombination achieved indicated. The conditional targeting
vector
contains the tNT cassette inserted into the NotI site (+260) in the
untranslated leader
sequence of the Cdc25A gene. This tNT cassette contains the coding sequences
of the
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_g_
tTA gene, a phosphoglycerate kinase gene promoter regulated neomycin (G418)
resistance gene and the Tet-o-7 tetracycline responsive promoter (Gossen, M.,
et al.,
(1992) Proc Natl Acad Sci USA 89(12):5547-5551). The Tet responsive promoter
is
fused (at +62) to the NotI site in the untranslated leader sequence of the
Cdc25A gene.
Also contained within the cassette are three polyadenylation sites and stop
codons in all
three reading frames. tTA driven, tetracycline dependent regulation of this
tet responsive
promoter can be demonstrated. The conventional targeting vector was
constructed by
removing a 2396 by XcmI fragment disrupting exons 1, 2 and 3 and replacing it
with a
PvuI-BsteII fragment containing the pGK regulated 6418 resistance gene derived
from
pPNT. After selection for 6418 resistance, EL1 ES cells electroporated with
these
vectors were expanded and analyzed by Southern blot for homologous
recombination.
Figure 7 shows a schematic of the genomic locus of murine Cdc25A and
targeting vector. Neomycin transferase (Neo') contained within the tNT
cassette and
inserted at the NotI site in exon l, was used for positive selection. The 1.3
kb 5' and 3.2
kb 3' homology regions of murine genomic sequences are represented by closed
boxes.
The P1 and P2 probes used in Southern blot analysis are indicated. A Southern
blot
analysis to identify heterozygous ES cell DNA is shown (Panel B) where BgIII
digests
probed with P1 detects a 7.Skb wild type band and 11.5 kb mutant band (Panel
B, Upper
Panel) and EcoRI digests probed with P2 detects a 6.5 and a 9.5 kb wild type
and mutant
band respectively (Panel B, Lower Panel).
Figure 8 shows photomicrographs of a histological analysis of Cdc25A
heterozygous intercross embryos (A-J). Embryos at E6.5, from a maternal
uterus, were
fixed in formaldehyde, sectioned serially and stained with hemotoxylin and
eosin. All
eight embryos (A-J) at E6.5 appeared healthy and displayed normal post-
implantation
development to the early primitive streak stage (K-T; 100X) embryos (R-T) were
disorganized and, in contrast to the adjacent normal embryos, had not
developed to the
late primitive streak stage (K-Q; 40X).
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Figure 9 shows photomicrographs of an in situ hybridization analysis of
heterozygous intercross embryos at E7.5 (A-F). Darkfield and partial
brightfield images
overlapped of adjacent sections probed with Cdc25A (C, E) and Cdc25B (D, F).
Control
sense probes for Cdc25A (A, A') and Cdc25B (B, B') are indicated. Digitized
darkfield
images of previous panel highlighting areas of hybridization to Cdc25A and
Cdc25B
probes as in panel I are shown in panels A'-F'. Arrows indicate position of
robust
Cdc25B hybridization to extra-embryonic mesodermal cells in D, D', F and F'.
Figure 10 shows the complete sequence of the murine Cdc2~A genomic locus.
Detailed Description of the Invention
In order to provide a clear and consistent understanding of the specification
and
claims, including the scope to be given such terms, the following definitions
are
provided.
Definitions
As used herein the term "Cdc25" is intended to include any art recognized cell
division control 25 gene and/or corresponding polypeptide expressed therefrom.
The
above term is also intended to refer to related Cdc25 genes and/or
polypeptides, e.g.,
Cdc25A, Cdc25B, and Cdc25C, unless indicated otherwise.
The term "Cdc25 activity" is intended to include any activity attributable to
the
Cdc25 polypeptide including direct catalytic activity, i.e., phosphatase
activity,
protein/protein interactions, and/or indirect activity such as,
transcriptional modulation.
The term "selected gene" is intended to include any eukaryotic gene or gene
capable of being transcriptionally regulated in a eukaryotic cell or cell
lysate, such as,
e.g., a cellular gene or a viral gene. Accordingly, the selected gene, unless
otherwise
noted, is typically operably linked to at least a minimal promoter and other
regulatory
elements, e.g., introns, termination sequences, polyadenylation sequences,
etc., necessary
for transcription of the gene. Accordingly, the selected gene may be a
cellular gene in a
natural context, e.g., encoded on a chromosome or encoded on, e.g., a plasmid.
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The term "transcription" is intended to include any measurable transcription
that
occurs when the genetic information of a DNA molecule is transferred to a
molecule of a
messenger RNA (mRNA). The transcription may take place in a cell or in a cell
lysate.
In addition, levels of transcription may be measured directly as a function
of, e.g., RNA
transcript production or indirectly as, e.g., a function of a resultant
polypeptide being
produced from the RNA transcript. The term is also intended to include an
indirect
determination of transcription by measuring, e.g., a modulation in polypeptide
occupancy
of a gene element, e.g., a promoter element, using, e.g., DNAse footprinting
or an
electrophoretic mobility shift assay (EMSA).
The term "statistically significant change" is intended to include any
reproducible
change in Cdc25 activity that is measurable with a minimum of statistical
significance.
Typically a change of 10%, more preferably 20%, and more preferably from 30% -
50% ,
and most preferably, 100%, 200%, 300%, or more, is considered a significant
change.
The term "eukaryotic promoter element" is intended to include any partial
promoter sequence which by itself is not capable of initiating normal
transcription but has
been determined to contribute to the activity of the overall activity of the
promoter.
The term "reporter gene" is intended to include any heterologous nucleotide
sequence that encodes a gene product that can be conveniently assayed and
includes, for
example, luciferase, chloramphenicol acetyltransferase, green fluorescent
protein, etc.
The term "cellular promoter" is intended to include a DNA sequence generally
described as the 5' region of a eukaryotic gene, located proximal to the start
codon. The
transcription of an adjacent genes) is initiated at the promoter region.
The term "viral promoter" is intended to include any promoter element
determined to regulate the transcription of a viral polypeptide, or, when
operably linked
to a heterologous gene, regulate the transcription of the heterologous gene in
a eukaryotic
cell or cell extract. Typical viral promoters intended to be encompassed by
the invention
include promoters derived from, e.g., SV40, adenovirus, CMV, herpesvirus, HIV,
papillomavirus, AAV, etc.
The term "cell'' is intended to include any eukaryotic cell such as yeast
cells, plant
cells, fungal cells, insect cells, e.g., Schneider and sF9 cells, mammalian
cells, e.g., HeLa
cells (human), NIH3T3 (murine), RK13 (rabbit) cells, embryonic stem cells
(e.g., D3 and
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J1), and cell types such as hematopoietic stem cells, myoblasts, hepatocytes,
lymphocytes, and epithelial cells.
The term "catalytically inactive Cdc25" is intended to include a Cdc25, such
as,
e.g., Cdc25A, that has reduced or absent levels of phosphatase activity as
compared to a
corresponding wild type Cdc25 polypeptide.
The term "test cell" is intended to include a eukaryotic cell having a
catalytically
active form of a Cdc25 polypeptide, e.g., a mammalian Cdc25A, B, or C.
The term "control test cell" is intended to include a eukaryotic cell having a
catalytically inactive form of a Cdc25 polypeptide, e.g., a mammalian Cdc25A,
B, or C
that has reduced or absent levels of phosphatase activity as compared to a
corresponding
wild type Cdc25 polypeptide.
Met/:ods and Compositions
The present invention will be described in detail as methods and nucleic
acids,
nucleic acid constructs, cells containing such nucleic acids, transgenic
animals, and cells
derived therefrom for screening compositions (including, e.g., small
molecules, peptides,
polypeptides, and genes) that modulate Cdc25 (e.g., mammalian Cdc25A, Cdc25B,
or
Cdc25C, or a combination thereof) transcription; Cdc25 activity (e.g.,
phosphatase
activity, protein/protein interactions); Cdc25-mediated gene regulation
(including, e.g.,
promoter repression), Cdc25-mediated cell cycle activity (e.g., apoptosis,
proliferation),
and Cdc25 pathways in general.
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of chemistry, molecular biology, microbiology,
recombinant
DNA technology, cell culture, and animal husbandry, which are within the skill
of the
art and are explained fully in the literature. See, e.g., Sambrook, Fritsch
and Maniatis,
Molecular Cloning: Cold Spring Harbor Laboratory Press (1989); DNA Cloning,
Vols.
1 and 2, (D.N. Glover, Ed. 1985); Oligonucleotide Synthesis (M.J. Gait, Ed.
1984);
Nucleic Acid Hybridization (B.D. Hames and S.J. Higgins, Eds. 1984); the
series
Methods In Enzymology (Academic Press, Inc.), particularly Vol. 1 ~4 and Vol.
155 (Wu
and Grossman, Eds.; Large-Scale Mammalian Cell Culture Technology, Lubiniecki,
A.,
Ed., Marcel Dekker, Pub., (1990); Molecular and Cell Biology of Yeasts,
Yarranton et
al., Ed., Van Nostrand Reinhold, Pub., (1989); Yeast Physiology and
Biotechnology,
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Walker, G., John Wiley & Sons, Pub., (1998); Baculovirus Expression Protocols,
Richardson, C., Ed., Humana Press, Pub., (1998); Methods in Plant Molecular
Biology:
A Laboratory Course Manual, Maliga, P., Ed., C.S.H.L. Press, Pub., (1995); and
Current Protocols in Molecular Biology, eds. Ausubel et al., John Wiley & Sons
( 1992)).
Other features of the invention will be apparent from the following examples
which should not be construed as limiting.
EXEMPLIFICATION
Throughout the examples, unless otherwise indicated, the following materials
and methods were used unless otherwise stated.
Materials and Methods
Cell Synchronization and Northern Blotting - Approximately 2 x 106 3T3 cells
were
synchronized at the G1/S boundary by double thymidine block protocol (Pagano,
M. (ed.)
Cell Cycle - Materials and Methods. New York: Springer-Verlag, 1990. Cells
were
harvested at 0, 3, 6, 9 and 12 hours post release. A fraction of the cells
were fixed,
propidium iodine stained and FACS analyzed to determine cell cycle stages.
Total RNA
from the remaining cells was isolated from 175cm2 flasks of synchronized 3T3
cells
utilizing RNAgents Total RNA Isolation System (Promega) according to the
manufacturer's protocol. Twenty micrograms of total RNA per time point and
S.Sug 0.5
- 9 kb RNA marker (New England Biolabs) were electrophoresed on 1.2%
agarose/formaldehyde gel and transferred to Nytran nylon membrane (Schleicher
and
Schuell) in lOX SSC. Prehybridization and hybridization in SX SSPE, lOX
Denhardt's,
50% formamide, 100 ~g/ml salmon sperm DNA and 2% SDS was carried out at
42°C
for 20 hours. The probe was a 1.7 kb C-terminal fragment of the murine Cdc2~A
cDNA
labeled with [alpha 32P] using random primer method (Stratagene). After a 20
hour
hybridization period, blots were washed in 2X SSC, 0.05% SDS at room
temperature. A
second wash at 50°C in O.1X SSC, 0.1% SDS followed. This blot was
reprobed with an
[alpha - 32P] labeled S 14 cDNA PCR product using the above hybridization and
washing
conditions, as a loading control.
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Luciferase Assay - Mammalian 293T cells grown in 6-well dishes at 1 x 10'
cells/well
were co-transfected with 1 ~g/well effector DNA (active and inactive Cdc25A)
and
1 pg/well reporter DNA (Cdc25A luciferase, p21 luciferase, and gamma globin)
using 3
~1/well FuGENE (Boehringer-Mannheim) in 100 ~1/well serum-free medium. Forty-
eight hours post-transfection, the cells were harvested and lysed using
Promega's
Luciferase Assay System. Cells were lysed in 250 p1 Cell Culture Lysis Reagent
and
stored at -80°C until needed for assaying. In a 96-well plate, 25 ~l
Luciferase Assay
Reagent (luciferin) was added to 25 ~l thawed cell extract. The plate was then
read
immediately by the luminometer.
SI Nuclease Analysis - An amount of 1.5 fmoles (approximately 9x106 cpm) of 5'
end
labeled probe was hybridized to 50 ~g total RNA isolated from 129sv mouse ES
cells
(51 °C, 80% formamide) and digested with 150 units of S 1 nuclease for
1 hr at 31 °C. The
products of the S 1 nuclease digestions were fractionated on a 6%
polyacrylamide gels
containing 8M urea and visualized by autoradiography.
Immunoblot Analysis - Extracts of 293T cells transfected with various Cdc25A
plasmids
were resolved by electrophoresis, transferred to nitrocellulose and probed
with rabbit
polyclonal anti-Human Cdc25A phosphatase (1 ~g/ml, Upstate Biotechnology). The
proteins were visualized using a donkey, anti-rabbit, secondary antibody
conjugated to
HRP and a chemiluminescence detection system (SuperSignal from Pierce).
EXAMPLE 1
CHARACTERIZATION OF THE MURINE Cdc25A GENE
In this example, the characterization of the genomic locus, cDNA sequence, and
amino acid sequence of murine Cdc25A, is described.
Sequence and Structure of the Mouse Cdc25A Gene
The complete genomic sequence of murine Cdc25A (Fig. 10) and the
transcription unit was determined by sequencing genomic DNA, cDNA, and by RNA
mapping. The coding sequence of Cdc25A (Fig. 1) was determined as being
expressed
from 18,314 by of genomic DNA comprising 15 exons.
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To make the foregoing determinations, genomic clones for DNA sequencing were
isolated by hybridization screening of shot gun libraries prepared from a P 1
vector
containing the entire Cdc25A locus using oligonucleotides derived from a
previously
published cDNA sequence (Wickramasinghe, D., et al., (1995) Development 121,
2047-
2056).
Sequencing of these clones revealed that the transcribed portion of the Cdc25
gene is encoded within 18,314 by of genomic DNA containing 15 exons, all of
which
contain coding regions. The exon structure of the Cdc25A gene is presented in
Table 1.
Table 1. Exon structure and coding capacity of the mouse Cdc25A gene.
Exon First Last Amino Genscan
NucleotideNucleotideAcids Predicted
1 1 589 1-57 Yes
2 1589 1662 57-83 Yes
3 2707 2749 83-97 No
4 3662 3698 97-109 Sub-optimal
5 4265 4366 110-144 No
6 5904 6008 145-179 Yes
7 7929 8054 180-222 Yes
8 8559 8633 223-247 Sub-optimal
9 8782 8950 248-303 Yes
10 11688 11786 304-336 Yes
11 13315 13377 337-357 No
12 13473 13571 356-390 Yes
13 14152 14282 391-434 Yes
14 16187 16298 434-471 Yes
16521 18335 472-516 Yes
Twelve of the fifteen exons where predictable using the Genscan gene
15 identification application (Burge, C., et al., (1997) JMoI Biol 268(1):78-
94)
(h~://bioweb.pasteur.fr/seqanal/interfaces/genscan-simple.html) (Table 1). No
coding
exons were predicted on the opposite strand. Intron size, exon size, and
splice junction
sequences are shown in Table 2.
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Table 2. Splicing junctions and intron structure of the mouse Cdc25A gene.
Exon 3' Splice Donor 5' Splice AcceptorExon Intron Size
Size (bp)
1 TGGGCAGgtgagagacc589 1009
2 ttggtttcagTGACTGT GATTCAGgtattgccat64 2544
3 tttctcaaagGTTTCTG AAGAAAAgtatgtattc43 912
4 ctcattacagCCTTGAA CCTACCTgtaagttctg37 566
actttgatagCAGAAGC AGAAAATgtttgctgag102 1537
tgttttgcagGAAGCAT TCGGATGgtacgtggtt105 1920
7 tgtctttcagCTATCTT TATGAAGgtacagtctg126 504
$ cttgctctagAATGATG AAACCTTgtaagttact75 148
9 tgaccctaagGCCGATC CGCCCAGgttagttacc169 2737
tttcttacagCTTCCAT CTCCAAGgtaactgcaa99 1528
11 ttttctaaagGTTTATC AGAAATTgtaagtcagg63 95
12 ctcttcatagATGGCAT CATCAAGgtttggattc99 580
13 gtctccacagGGTGCCG CTCGAATgtgagtacca131 1904
14 ttttcactagGTGCCGA GTGCCAGgtgagatgtc112 222
gttcctttagTCTCACT 1815
Exon and intron sequences are shown in upper- and lowercase letters,
respectively. The
5 splice acceptor and donor sequences are shown in boldface type.
A canonical polyadenylation site AATAAA (Wickens, M., et al., (1984) Science
226(4678):1045-1051) was identified 17,893 by down stream of the initiator
methionine
codon. The 3' most cluster of mouse ESTs present in the NCBI dbEST database
was
10 determined to overlap this site, consistent with this being the 3' end of
the gene. Previous
reports have proposed a 3' end further upstream. This conclusion likely
results from
false priming of a poly A track present in the transcribed region. The 3' UTR
contains
two copies of the mRNA destabilizing motif ATTTA at positions 16,811 and
16,939
(Wilson, T., et al. (1988) Nature 336(6197):396-399). Theses motifs are
conserved in the
15 corresponding sequence of the rat cDNA. The stability of Cdc25A mRNA in
mouse cells
was not determined however it is worth noting that in MCF7 cells, human Cdc25A
mRNA has a half life of approximately 2 hours.
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Based on the genomic sequence and the sequence of the Cdc25A cDNA cloned
from mouse kidney, the deduced amino acid sequence of murine Cdc25A was
determined
and is shown in Figure 1. This sequence differs at several positions form a
previously
published sequence that was based solely on cDNA sequence alone
(Wickramasinghe,
D., et al., (1995) Development 121, 2047-2056).
EXAMPLE 2
STRUCTURAL CHARACTERIZATION OF THE MURINE
Cdc25A PROMOTER
In this example, a characterization of the murine Cdc25A promoter is
presented.
Base on the sequence determinations made above, the structure of the murine
Cdc25A promoter could be determined. In particular, various key structural
motifs of the
Cdc25A promoter were identified. For example, the Cdc25A transcription
initiation site
was determined using S 1 nuclease protection analysis. Two genomic DNA probes
were
used for this analysis, one labeled at the NcoI site coincident with the
translation
initiation site and one labeled at the NotI site 152 nucleotides upstream
(Fig. 2A). As
shown in Figure 2B, the NotI probe yields a protected fragment of
approximately 260
nucleotides whereas the NcoI probe yields a product of approximately 420
nucleotides.
These results place the transcription initiation about 420 nucleotides 5' of
the translation
initiation site. This agrees roughly with the 5' end predicted based on the
published
sequence of the human Cdc25A cDNA (Galaktionov, K., et al., ( 1996) Nature
382, 511-
517) although the exact sequence in the vicinity of the start site is not well
conserved
between mouse and human. This region does not contain a consensus TATA box but
does contain the sequence AATTAA. This sequence is similar to degenerate TATA
elements shown to function, albeit very weakly, in yeast (Arndt et al., 1994,
Mol. Cell
Biol. 14:3719-3728). TATA independent transcription initiation can be directed
by
"initiator" (Inr) elements containing the consensus sequence PyPyAN(T/A)PyPy
(Javahery et al., (1994) Mol Cell Biol (14(1):116-127). A perfect match to
this consensus
is found at position -420 relative to the translation initiation site. Inr
sequence elements
are frequently associated with adjacent Spl binding sites (Faber et al., 1993,
J. Biol.
Chem. 268:9296-9301). Consistent with this, an Spl binding site is located at -
40
relative to the Cdc25A transcription initiation site and consensus Inr
element.
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Immediately adjacent to the putative Spl binding site is a binding site for
E2F that
has been reported to mediate cell cycle arrest by TGF (3 in keratinocytes
(Iavarone, A., et
al., (1999) Mol Cell Biol 19:916-922). In addition, it has been observed that
human
Cdc25A reporter gene activity is reduced 75% during cell cycle arrest induced
by
treatment with TGF (3 (Iaverone, A., et al. ( 1999) Mol Cell Biol 19:916-922).
In addition to the identification of the foregoing motifs, a GRAIL prediction
analysis (http://grail.lsd.ornl.gov/Grail-1.3/) was performed and it was
determined that
various CpG islands exist between nucleotides 852 and 1612, the region roughly
encompassing the transcription initiation site through the end of exon one.
EXAMPLE 3
FUNCTIONAL CHARACTERIZATION OF THE Cdc25A PROMOTER
In this example, a functional characterization of the murine Cdc25A promoter
is
presented.
To test for Cdc25A promoter function, a 1.3 Kb NotI Cdc25A genomic fragment
was fused (+260/-1040 relative to the transcription initiation site) to the
reporter gene
luciferase. It has been observed that Cdc25A is expressed over a wide range of
levels
depending on tissue type. For example, expression is very high in the kidney
but almost
undetectable in the lung. In order to determine if this 1.3 Kb fragment could
recapitulate
this expression pattern, transient transfection experiments were conducted.
The Cdc25A luciferase construct was transfected into mammalian 293 kidney
cells and NCI H460 lung cells in parallel with five other control constructs.
These
control constructs contained viral or house keeping gene promoters having
little cell type
specificity. As shown in Figure 3, the relative activity of the control
constructs differs
very little between the two cell types. However, although Cdc25A driven
luciferase
activity was detected in both cell types, it was at least ten times higher in
kidney cells
than lung cells relative to the panel of control reporter constructs.
These data suggest that cell type specific expression levels can be conferred
by
Cdc25A upstream sequences extending to approximately 1 Kb 5' of the
transcription
initiation site.
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In addition to tissue specific regulation of the murine Cdc25A promoter, the
autoregulation of the Cdc25A promoter was investigated. Accordingly, cells
were
cotransfected with the above-described mammalian luciferase reporter construct
driven
by the Cdc25A promoter and a plasmid encoding either a catalytically active
Cdc25A
phosphatase or a catalytically inactive phosphatase. Transcription from the
Cdc25A
promoter was repressed by over-expression of catalytically active but not
catalytically
inactive Cdc25A phosphatase.
Accordingly, it was concluded that the catalytically active Cdc25A phosphatase
can repress the transcriptional activity of the Cdc25A promoter. This result
may be
autoregulated by a mechanism of Cdc25A-mediated promoter suppression via
Cdc25A
phosphatase activity. This aspect of promoter suppression was further explored
as a
screening assay for modulators of Cdc25 phosphatase and these results are
described in
Example 4
EXAMPLE 4
ASSAY FOR SCREENING MODULATORS OF CDC25A REGULATION OF
CELLULAR AND VIRAL GENE TRANSCRIPTION
In this example, an assay for measuring Cdc25-mediated gene regulation an
identifying modulators thereof, is presented.
It has been observed that Cdc25A can affect the expression of genes containing
binding sites for the transcriptional repressor Cutl (Coqueret, O., et al.,
(1998) EMBO
Journal 17, 4680-4694). For example, Cdc25A can dephosphorylate Cutl thereby
increasing its affinity for binding sites in promoters such as the p21 /Waf
gene.
In addition, based on the functional characterization of the Cdc25A promoter
in
Example 3, it was discovered that the Cdc25A phosphatase is capable of
regulating the
Cdc25A promoter. Accordingly, an investigation of Cdc25A-mediated gene
regulation
of a number of different eukaryotic promoters was conducted in addition to
analysis of
Cdc25A autoregulation. In particular, Cdc25A was assayed for the ability to
regulate the
promoter of an important cell cycle gene, i.e., p21, the phosphoglycerate
kinase (pGK)
promoter, the promoter of a tissue restricted gene, i.e., (3-globin, and a
viral promoter
derived from SV40.
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Accordingly, mammalian cells were co-transfected with one of several reporter
constructs and a plasmid encoding either a catalytically active or
catalytically inactive
Cdc25 phosphatase. Following transfection, cells were harvested, and reporter
gene
activity as a function of luciferase activity was determined as described in
the materials
and methods subsection above.
As shown in Figure 4A, catalytically active Cdc25A was shown to repress p21
promoter expression in transient transfection experiments. Similarly, the
Cdc25A
promoter was inhibited by catalytically active Cdc25A but not catalytically
inactive
Cdc25A. Sequence analysis of the Cdc25A promoter revealed the presence of
consensus
Cutl binding sites at -625, -551 and -482 (Andres, V., et al., (1994) Genes
Dev 8(2):245-
257). This indicated that Cdc25A expression could repress its own expression
through a
feed-back mechanism. The effects of Cdc25A phosphatase expression on the
phosphoglycerate kinase (pGK) gene promoter and the (3-globin promoter (-108)
were
also analyzed (Figure 4A). The results show that the catalytically active but
not the
catalytically inactive form of Cdc25A was able to repress the pGK promoter but
not the
globin gene construct which does not contain Cutl binding sites.
In order to determine if Cdc25A could affect the gene transcription of other
promoters, a viral promoter derived from SV40 (simian virus 40) was tested. As
shown
in Fig 4 (lower panel), the catalytically active form of the Cdc25A
phosphatase, but not
the catalytically inactive form, was able to repress transcription of the SV40
promoter
and this was an extremely robust level of repression (42 fold repression).
To verified that the 293 cells were transfected with equivalent amounts of
constructs encoding Cdc25A, immunoblot analysis of Cdc25A polypeptide levels
using
polyclonal antisera was performed (Figure 4B).
Thus, these results show that Cdc25A expression can regulate gene expression
including Cdc25 expression via the phosphatase activity of the Cdc25
polypeptide. This
level of regulation is likely to be important in the regulation of cycling
cells especially,
e.g., in concert with other forms of regulation affecting, e.g., E2F activity
(Iaverone, et
al., (1999) Mol Cell Biol 19:916-922).
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These experiments further demonstrate the ability of Cdc25A to regulate not
only
different cellular promoters, such as its own promoter (but not all cellular
promoters, see,
e.g., the globin promoter), but also viral promoters, such as is exemplified
using the
SV40 promoter.
Accordingly, it will be appreciated that the assay has wide utility in
screening
modulators of Cdc25-mediated gene regulation. In particular, the viral
promoter SV40
may be used because of the unambiguous signal that can be assayed and because
an
inhibitor of Cdc25A will rescue signal output, i.e., reporter gene expression.
Because the
amount of Cdc25A repression of this promoter is 42-fold, even weak or partial
inhibitors
of Cdc25A activity can be readily assayed.
Moreover, the assay provides a control that can accurately identify compounds
that are false positives (e.g., compounds that rescue the signal but also
increase the
signal in the test reaction) or false negatives (e.g., compounds that produce
no signal but
also lower the control signal, e.g., cytotoxic compounds) and this insures
that
inappropriate compounds are not further investigated and that candidate
compounds or
not erroneously dismissed.
It will be further appreciated that any art recognized compound or library of
compounds containing, e.g., a test compound that is protein based,
carbohydrate based,
lipid based, nucleic acid based, natural organic based, synthetically derived
organic
based, or antibody based may be screened as a candidate compound that affects
Cdc25-
medated regulation of a promoter such as, e.g., the SV40 promoter.
Accordingly, any of
a number of art recognized high throughput assay techniques may be used in
conducting
the assay.
EXAMPLE 5
CHARACTERIZATION OF CELL CYCLE REGULATION OF CDC25A
EXPRESSION
In this example, the cell cycle regulation of Cdc25A is described.
To determine if murine Cdc25A levels changed during the cell cycle, Cdc25A
RNA levels were measured during the cell cycle of murine cells synchronized
with a
double thymidine block (Pagano, M. (ed.) Cell Cycle - Materials and Methods.
New
York: Springer-Verlag, 1995). Figure 5 shows that, relative to the level of
Cdc25A
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mRNA in cells arrested at G1/S (0 hours), Cdc25A mRNA levels peak at 9 hours
after
release corresponding roughly to the time when most cells are in G2 or M
phase. It was
also observed that mRNA levels begin to drop off as cells continue to finish
the first cell
cycle after release. These results are consistent with observations made of
human
Cdc25A protein levels using HeLa cells and NCI H460 cells. In addition, it has
been
observed that Cdc25A mRNA levels are cell cycle regulated in rat NRK cells
(Jinno, S.,
et al., (1994) EMBO Journal 13, 1549-1556).
Thus, consistent with rat and human cells, murine Cdc25A levels appear to be
modulated by factors affecting cell cycle progression. Moreover, these
findings indicate
that the mouse Cdc25A is expressed over a wide range, in a cell type specific
manner,
and its expression can be affected by check-point arrest. To explore the role
of Cdc25A
in vivo, transgenic animals were prepared as described in Example 7.
EXAMPLE 6
NOVEL VECTORS FOR CDC25A GENE TARGETING
In this example, the construction and recombination frequencies of gene
targeting
vectors constructed using the above-mentioned sequence information are
described.
The availability of genomic sequence from the Cdc25A locus facilitated the
construction of novel gene targeting vectors for disrupting the Cdc25A locus.
First, a
conventional targeting vector was constructed and designed to remove portions
of exon 1
and exon 2 resulting in disruption of the coding sequence (Figure 6).
Additionally, a
vector was constructed containing a cassette (tNT) designed to simultaneously
insert the
coding sequences of the tetracycline traps-activator (Gossen, M., et al.,
(1993) Trends
Biochem Sci 18(12):471-475), under control of the Cdc25A promoter and place
the
adjacent coding sequences of the Cdc25A gene under the control of the
tetracycline
response element tet-o-7 (Gossen, M., et al. (1993) Trends Biochem Sci
18(12):471-475)
(Figure 6). The second construct was expected to conditionally express Cdc25A
based
on the presence or absence of tetracycline. The embryonic cell line EL 1 was
electroporated with each of these constructs and placed under selection for
6418
resistance (negative selection was not used). Surprisingly, the construct
containing the
tNT insertion frequency was high (over 25% (14/54)) (Figure 6). Although
recombination between one arm of the conventional targeting vector and the
Cdc25A
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locus was detected, no homologous recombinants were identified among 210 6418
resistant clones (Figure 6).
These results may indicate that sequences within exons 1, 2, and 3 may
influence
homologous recombination frequency or are necessary for genetic stability of
the locus.
Accordingly, it was determined that relatively subtle differences in the
design of Cdc25
targeting vectors can have a significant impact on recombination frequencies.
EXAMPLE 7
PREPARATION AND ANALYSIS OF MICE LACKING A CDC25
PHOSPHATASE
In this example, the preparation and analysis of transgenic mice lacking a
Cdc25
polypeptide, is described.
Cyclin Dependent Kinase (CDK) activity controls cell division in eukaryotes
and
is positively regulated by CDC25, a family of dual specificity phosphatases.
In mice,
three Cdc25 genes, Cdc25A, B and C, have been identified and are expressed in
an
overlapping yet distinct manner during development (Sadhu, K., et al. ( 1990)
PNAS USA
87:5139-5143; Kakizuka, A., et al. (1992) Genes and Development 6:578-590;
Wickramasinghe, D., et al. (1995) Development 121:2047-2056; Wu, S., et al.
(1995)
Dev Biol 170:195-206). To examine the specific roles and potential redundancy
of these
genes during mouse development, Cdc25A deficient mice were generated. A lethal
phenotype is observed in Cdc25A deficient embryos in contrast to that of
Cdc25B
mutants that remain viable. These results indicate that Cdc25A is essential
and is not
redundant for early mouse development, in contrast to that of Cdc25B,
unequivocally
distinguishing the unique role played by these individual family members.
Targeting Strategy
The Cdc25A locus was targeted by site directed mutagenesis in embryonic stem
cells. Exon 1 was disrupted by insertion of a PGK-Neomycin' (PGK-NEO')
cassette
containing three polyadenylation signals and translation termination codons in
all three
reading frames. The targeting vector, which contained 1.3kb and 3.2kb of 5'
and 3'
homology regions respectively (Figure 7), was electroporated into Embryonic
Stem (ES)
cells and selected for resistance to 300~g/ml 6418. No negative selection was
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performed. Homologous recombination in these clones was identified by Southern
blot
analysis and 3 independent cell lines were expanded. These clones were
injected into
blastocysts and transferred to recipient mothers. Chimeric mice were
identified and
crossed with C57BL6 mice. Heterozygous offspring obtained from these crosses
were
healthy and fertile and have remained so through the 9-month duration of this
analysis.
Multiple litters of heterozygous intercross spring derived from the three
different cell
lines were genotyped. No homozygous mutant mice were identified although
heterozygous and wildtype offspring were represented (Table 3). These
observations
suggested that the homozygous mutant phenotype was associated with embryonic
lethality.
Table 3. Genotype analysis of Cdc25A heterozygous intercross offspring.
Wild type HeterozygousHomozygous Total
#2 5 11 0 16
#9 10 22 0 32
#36 32 59 0 91
Three independent lines (#2, # 9 # 36) of Cdc25A heterozygous intercrossed
offspring were
genotyped by Southern blot analysis using the 3'probe P2 described in Figure
7. In no case
were homozygous null offspring identified.
Phenotypic Analysis
In order to identify the developmental stage at which the embryonic lethality
occurs, various stages of embryos were dissected from heterozygous
intercrosses
ranging from E6.5 to E12.5. Examination of E6.5 embryos indicated that all
embryos
developed to the egg cylinder stage and appeared normal (n = 57). However, at
E7.5
disorganized embryos undergoing resorption were observed consistently in 25%
of the
embryos analyzed (n = 62). Embryonic death was observed in E8.5 and later
stages of
development at a similar ratio (n = 100). Histological examination of embryos
from
heterozygous intercrosses at E6.5 and E7.5 confirmed these observations
(Figure 2). In
addition, control crosses with wildtype and heterozygous mice do not display
embryonic
lethality at E7.5 in contrast to the heterozygous intercross embryos.
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These observations indicate that wildtype and heterozygous embryos do not
contribute to the embryonic lethal phenotype.
Wildtype embryos at E6.5 have developed to the egg cylinder stage (M. H.
Kaufman ( 1995) The Atlas of Mouse Development, Acad. Press. p 1-41 ). All
heterozygous intercross embryos develop to this stage and are
indistinguishable from
each other (Figure 8A-J). At E7.5 normal embryos develop to the late primitive
streak
stage. The presence of ectoderm, mesoderm and endoderm layers, in addition to
the
chorion, allantois, amnion and neural plate easily distinguish embryos at this
stage
(Figure 8K-Q). However, in contrast, we observed approximately 25% of the
embryos
were disorganized and did not display distinct embryonic layers or neural
plate
formation that resulted in early embryonic lethality.
In Sitzr Hybridization Analysis
To examine the embryonic lethality in greater detail, in-situ hybridization
analysis was carried out on E7.5 embryos (as described in J. Coligan et al.,
(1993) Cur.
Protocol Immunol (Whey ) Unit 12.8.1 - 21 ). Cdc25A RNA was detected
ubiquitously
in all embryos that appeared normal and had developed to the late primitive
streak stage
(Figure 2C, C'). Overall Cdc25A expression was observed in the endoderm,
mesoderm,
and ectoderm layers, the ectoplacental cone, extra-embryonic layers, and in
the allantois,
amnion and neural plate. An adjacent section of the same embryo hybridized to
a
cda25b probe revealed lower overall expression but specific localization of
Cdc25B
RNA to a subset of extra-embryonic mesodermal cells (Figure 8D, D'). In
contrast, the
disorganized embryos do not express Cdc25A (Figure 9E, E') in the ubiquitous
expression pattern observed in normal sibling embryos. However, Cdc25B RNA was
detected in an adjacent section of the same embryo (Figure 9F, F'). Although
the
embryonic tissue is disorganized the specific expression of Cdc25B is easily
distinguishable, while Cdc25A expression cannot be detected above background
levels.
These observations reveal that Cdc25A expression (wildtype or heterozygous) is
correlated with development of the embryo while the lack of Cdc25A expression
is
directly correlated with early embryonic lethality. Therefore, Cdc25A is
essential and is
not redundant for embryonic development.
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Since phenotypes have been identified in mice heterozygous for other cell
cycle
genes, we examined cell cycle profiles and DNA damage response in mice
heterozygous
for Cdc25A (A. Clarke et al. (1992) Nature 359: 328-330; L. Donehower et al.,
(1992)
Nature 356: 215-221; T. Jacks et a1.(1992) Nature 359: 295-300). Cell cycle
profiles,
by FACS analysis, of synchronized or DNA damaged heterozygous Mouse Embryo
Fibroblasts (MEFs) were indistinguishable from their wildtype counterparts.
Furthermore, Northern and immunoblot analysis of embryonic RNA and protein
respectively, reveals similar levels of Cdc25A in wildtype
and heterozygous embryos. cDNA expression arrays also indicate similar
expression
patterns in wildtype and heterozygous embryos and corroborate our previous
Northern
analysis data.
These results unequivocally demonstrate that Cdc25A is essential for early
embryonic development. At E7.5 overall developmental arrest is observed in
mutant
embryos suggesting that Cdc25A plays a critical role at this time of rapid
proliferation.
These results further distinguish the role of Cdc25A from that of Cdc25B.
Since
Cdc25B has been implicated in a role at G1/S, compensation for the lack of
Cdc25A
function by Cdc25B at G,/S would be predicted. However, the Cdc25A mutant
embryos
are not compensated for by Cdc25B, although it could be argued that the low
level of
Cdc25B expressed in these embryos is insufficient for compensation.
Furthermore, the
Cdc25A mutant phenotype is in sharp contrast to that of Cdc25B. Cdc25B is not
essential or is redundant in embryonic development. Cdc25B -/- mice develop
into adult
animals and do not display a mitotic phenotype.
These observations suggest that Cdc25A plays a central role in early
embryogenesis co-incident with tissue proliferation for subsequent development
and
differentiation. Zygotic expression of Cdc25A occurs most likely at the
blastocyst stage
of development (D. Wickramasinghe et al., (1995) Development 121:2047-2056).
Therefore, Cdc25A homozygous mutants probably survive preimplantation
development, due to maternally provided Cdc25A. Alternatively, Cdc25A is not
essential for these early embryonic divisions. Maternal support of early
development
has been documented extensively and survival of pre-implantation embryos is
not
unique in mouse knockout analysis (Telford, N., et al., (1990) Mol Reprod Dev
26:90-
100). For example, cyclin A2 -/- embryos express the protein in blastocysts.
The cyclin
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A2 is most likely derived from maternal stores and supports embryonic
development up
to E6.5 (Murphy, M. et al. (1997) Nature Genetics 15:83-86). In addition,
knockout
embryos of genes critical for cell division and DNA damage repair, such as
BRCA1,
BRCA2 and RAD51 display early embryonic lethal phenotypes ranging from E6.5-
8.5
(Gowen, L., et al. (1996) Nature Genetics 12:191-194).
The results presented here describe the pivotal role Cdc25A plays during mouse
development that is temporally coincident with rapid proliferation. Since
Cdc25A has
been implicated in numerous human cancers, these mutants extend a framework to
examine genetic interactions with other cell cycle regulators and tumor
suppressors in
creating a malignant state. In particular, these cells and resultant animals
provide
valuable tools for assaying Cdc25-mediated gene regulation.
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more
than
routine experimentation, many equivalents to the specific embodiments of the
invention
described herein. Such equivalents are intended to be encompassed by the
following
claims.
What is claimed:
