U.S. patent application number 10/702720 was filed with the patent office on 2004-05-13 for chk1 and uses thereof.
This patent application is currently assigned to Millennium Pharmaceuticals, Inc.. Invention is credited to Jin, Shengfang, Shyjan, Andrew W., Williamson, Mark.
Application Number | 20040091948 10/702720 |
Document ID | / |
Family ID | 32069528 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040091948 |
Kind Code |
A1 |
Shyjan, Andrew W. ; et
al. |
May 13, 2004 |
Chk1 and uses thereof
Abstract
Increased expression of Chk1 is associated with drug resistance
of certain cells (e.g., cancer cells). The invention provides
methods for identifying drug resistant cells by measuring the
expression or activity of Chk1, methods for identifying modulators
of drug resistance, and methods for modulating drug resistance by
modulating the expression or activity of Chk1.
Inventors: |
Shyjan, Andrew W.; (Nahant,
MA) ; Williamson, Mark; (Saugus, MA) ; Jin,
Shengfang; (West Roxbury, MA) |
Correspondence
Address: |
MILLENNIUM PHARMACEUTICALS, INC.
40 Landsdowne Street
CAMBRIDGE
MA
02139
US
|
Assignee: |
Millennium Pharmaceuticals,
Inc.
|
Family ID: |
32069528 |
Appl. No.: |
10/702720 |
Filed: |
November 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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10702720 |
Nov 6, 2003 |
|
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09340264 |
Jun 30, 1999 |
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Current U.S.
Class: |
435/7.23 ;
435/6.18 |
Current CPC
Class: |
G01N 2333/9121 20130101;
G01N 33/573 20130101; C12Q 1/6886 20130101; G01N 33/5011 20130101;
G01N 2333/9108 20130101; C12Q 1/6809 20130101 |
Class at
Publication: |
435/007.23 ;
435/006 |
International
Class: |
C12Q 001/68; G01N
033/574 |
Claims
What is claimed is:
1. A method for determining whether a test compound modulates the
drug resistance of a cell, the method comprising: a) determining
the level of Chk1 expression in a cell in the presence of a test
compound; b) determining the level of Chk1 expression in the cell
in the absence of the test compound; and c) identifying the
compound as a modulator of drug resistance of the cell if the level
of expression of Chk1 in the cell in the presence of the test
compound differs from the level of expression of Chk1 in the cell
in the absence of the test compound.
2. The method of claim 1 wherein the Chk1 is encoded by an
endogenous gene.
3. A method for determining whether a test compound modulates the
drug resistance of a cell, the method comprising: a) incubating
Chk1 protein in the presence of a test compound; b) determining
whether the test compound binds to the Chk1 protein; c) selecting a
test compound which binds to the Chk1 protein; d) administering the
test compound selected in step c) to a non-human mammal having drug
resistant cells; e) determining whether the test compound alters
the drug resistance of the cells in the non-human mammal; and f)
identifying the test compound as a modulator of drug resistance of
the cell if the compound alters the drug resistance of the cells in
step e).
4. A method for determining whether a test cell has a
drug-resistant phenotype, the method comprising: a) measuring the
expression of Chk1 in the test cell; b) comparing the expression of
Chk1 measured in step a) to the expression of Chk1 in a control
cell not having a drug-resistant phenotype; and c) determining that
the test cell has a drug resistant phenotype if the expression of
Chk1 in the test cell is greater than the expression of Chk1 in the
control cell.
5. A method of determining whether a test cell has a drug-resistant
phenotype, the method comprising: a) measuring the activity of Chk1
in the test cell; b) comparing the activity of Chk1 measured in
step a) to the activity of Chk1 in a control cell not having a
drug-resistant phenotype; and c) determining that the test cell has
a drug resistant phenotype if the activity of Chk1 in the test cell
is greater than the activity of Chk1 in the control cell.
6. A method for determining whether a subject has or is at risk of
developing a drug resistant tumor, the method comprising: a)
measuring the expression of Chk1 mRNA in a biological sample
obtained from the subject; b) comparing the expression of Chk1 mRNA
measured in step a) to the expression of Chk1 mRNA in a biological
sample obtained from a control subject not having a drug resistant
tumor; and c) determining that the patient has or is at risk of
developing a drug resistant tumor if the expression of Chk1 mRNA in
the biological sample obtained from the patient is higher than the
expression of Chk1 mRNA in the biological sample obtained from the
control subject.
7. The method of claim 6, wherein step a) comprises the use of a
nucleic acid molecule that hybridizes to Chk1 mRNA.
8. A method for determining whether a subject has or is at risk of
developing a drug resistant tumor, the method comprising: a)
measuring the activity of Chk1 in a biological sample obtained from
the subject; b) comparing the activity of Chk1 measured in step a)
to the expression of Chk1 mRNA in a biological sample obtained from
a control subject not having a drug resistant tumor; and c)
determining that the patient has or is at risk of developing a drug
resistant tumor if the activity of Chk1 in the biological sample
obtained from the patient is higher than the activity of Chk1 in
the biological sample obtained from the control subject.
9. The method of claim 8, wherein step a) comprises the use of an
agent that binds to Chk1 protein.
10. A method for monitoring the effect of an anti-tumor treatment
on a patient, the method comprising: a) measuring the expression of
Chk1 in a tumor sample obtained from the patient; b) comparing the
expression of Chk1 measured in step a) to the expression of Chk1 in
a control sample of cells; and c) determining that the anti-tumor
treatment should be discontinued or modified if the expression of
Chk1 in the tumor sample is higher than the expression of Chk1 in
the control sample of cells.
11. The method of claim 10, wherein step a) comprises the use of a
nucleic acid molecule that hybridizes to Chk1 mRNA.
12. A method for monitoring the effect of an anti-tumor treatment
on a patient, the method comprising: a) measuring the activity of
Chk1 in a tumor sample obtained from the patient; b) comparing the
activity of Chk1 measured in step a) to the activity of Chk1 in a
control sample of cells; and c) determining that the anti-tumor
treatment should be discontinued or modified if the activity of
Chk1 in the tumor sample is higher than the activity of Chk1 in the
control sample of cells.
13. The method of claim 12, wherein step a) comprises the use of an
agent that binds to Chk1 protein.
14. A method for modulating the drug resistance of a cell, the
method comprising modulating Chk1 expression within the cell.
15. A method reducing the drug resistance of cell, the method
comprising contacting the cell with a molecule which reduces the
expression of Chk1 within the cell.
16. A method of increasing the effectiveness of a chemotherapeutic
compound in a patient suffering from a disorder associated with the
presence of drug-resistant neoplastic cells, the method comprising:
a) administering a chemotherapeutic compound to the patient; and b)
administering a compound with reduces Chk1 expression to the
patient.
17. A method of treating a mammal suspected of having a disorder
associated with the presence of drug-resistant cells, the method
comprising administering to the mammal a compound that reduces the
expression of Chk1 in the drug-resistant cells, the reduction be
sufficient to reduce the drug resistance of the drug resistant
cells.
18. A method for increasing the drug resistance of cell that has an
undesirably low level of Chk1 expression, the method comprising
exposing the cell to a compound that increases the expression of
Chk1.
19. A method for treating a drug resistant tumor in a patient, the
method comprising administering to said subject an amount of a Chk1
antagonist effective to reduce drug resistance of said tumor in the
patient.
20. The use of an inhibitor of Chk1 expression, or pharmaceutically
acceptable salt thereof, or a pharmaceutical composition containing
either entity, for the manufacture of a medicament for the
treatment of a drug resistant tumor in a patient.
Description
BACKGROUND OF THE INVENTION
[0001] The invention relates to chemotherapy and drug
resistance.
[0002] Cancer chemotherapy commonly involves the administration of
one or more cytotoxic or cytostatic drugs to a patient. The goal of
chemotherapy is to eradicate a substantially clonal population
(tumor) of transformed cells from the body of the individual, or to
suppress or to attenuate growth of the tumor. Tumors may occur in
solid or liquid form, the latter comprising a cell suspension in
blood or other body fluid. A secondary goal of chemotherapy is
stabilization (clinical management) of the afflicted individual's
health status. Although the tumor may initially respond to
chemotherapy, in many instances the initial chemotherapeutic
treatment regimen becomes less effective or ceases to impede tumor
growth. The selection pressure induced by chemotherapy promotes the
development of phenotypic changes that allow tumor cells to resist
the cytotoxic effects of a chemotherapeutic drug. Often, exposure
to one drug induces resistance to that drug as well as other drugs
to which the cells have not been exposed.
[0003] Cell cycle checkpoints are regulatory systems that control
the order and timing of certain events in the cell cycle. These
checkpoints are important for ensuring that cells divide properly.
For example, DNA damage leads to activation of a cell cycle
checkpoint regulatory system that arrests the cell cycle and
activates genes involved in repair of DNA damage. This system
prevents progression of the cell cycle until the DNA damage has
been repaired. Chk1, a kinase, is thought to be involved in the DNA
damage cell cycle checkpoint. Chk1 is thought to participate in the
phosphorylation of Cdc25 in response to DNA damage. Phosphorylation
of Cdc25 prevents activation of the Cdc2-cyclin B complex thereby
blocking mitotic entry.
SUMMARY OF THE INVENTION
[0004] The present invention concerns checkpoint kinase 1 (Chk1;
Genbank Accession No. AF016582; Sanchez et al. (1997) Science
277:1497). Applicants have found that expression of Chk1 is up
regulated in certain vinblastin resistant cancer cell lines and in
certain adromycin resistant cancer cell lines. Applicants have also
found that a ribozyme designed to decrease Chk1 expression can
increase drug sensitivity.
[0005] Chk1 nucleic acids and polypeptides are useful in diagnostic
methods related to identification of drug resistant cells (e.g.,
cancer cells). Chk1 nucleic acids and polypeptides are also useful
in screening methods directed to the identification of compounds
that can modulated (increase or decrease) the drug resistance of a
particular cell type or multiple cell types.
[0006] The invention includes a method for detecting the presence
of a Chk1 polypeptide in a sample, e.g., a biological sample. This
method features the steps of contacting the sample with a compound
which selectively binds to the polypeptide and then determining
whether the compound binds to a polypeptide in the sample. In some
cases, the compound which binds to the polypeptide is an
antibody.
[0007] The invention also features methods for detecting the
presence of a Chk1 nucleic acid molecule in a sample. This method
includes the steps of contacting the sample with a nucleic acid
probe or primer which selectively hybridizes to a Chk1 nucleic acid
molecule (e.g., an mRNA encoding Chk1); and then determining
whether the nucleic acid probe or primer binds to a nucleic acid
molecule in the sample.
[0008] Also within the invention are kits that include a compound
which selectively binds to a Chk1 polypeptide or nucleic acid and
instructions for use. Such kits can be used to determine whether
cells within a biological sample, e.g., a sample of patient cells,
are drug resistant.
[0009] The invention features methods for identifying a compound
which binds to a Chk1 polypeptide. These methods include the steps
of contacting a Chk1 polypeptide with a test compound and then
determining whether the polypeptide binds to the test compound. In
various embodiments of these methods, the binding of the test
compound to the Chk1 polypeptide is detected using an assay which
measures binding of the test compound to the polypeptide or using a
competition binding assay.
[0010] The invention also includes a method for modulating the
activity of a Chk1 polypeptide. This method includes the steps of
contacting the polypeptide or a cell expressing the polypeptide
with a compound which binds to the polypeptide in a sufficient
concentration to modulate the activity of the polypeptide.
[0011] In another aspect, the invention provides a method for
identifying a compound that modulates the activity of a Chk1
polypeptide (e.g., a Chk1 protein). In general, such methods entail
measuring a biological activity of the polypeptide in the presence
and absence of a test compound and identifying those compounds
which alter the activity of the polypeptide (e.g., alter the
ability of Chk1 to phosphorylated Cdc25). One such method includes
the steps of contacting the polypeptide with a test compound and
then determining the effect of the test compound on the activity of
the polypeptide to thereby identify a compound which modulates the
activity of the polypeptide.
[0012] The invention also features methods for identifying a
compound which modulates the expression of a Chk1 nucleic acid or a
Chk1 polypeptide by measuring the expression of the nucleic acid or
polypeptide in the presence and absence of a compound.
[0013] Other aspects of the invention are methods and compositions
relating to drug resistance. A "drug-resistant phenotype" refers to
a cellular phenotype which is associated with increased survival
(compared to a less drug-resistant cell) after exposure to a
particular dose of a drug, e.g., a chemotherapeutic drug, compared
to a cell that does not have this phenotype. A "drug-resistant
cell" refers to a cell that exhibits this phenotype. Drug
resistance can be characterized by lower intracellular
concentration of a drug compared to a non-resistant cell or a less
resistant cell as well as altered ability of a drug to affect its
target compared to a non-resistant cell or a less resistant cell.
Drug resistance is described in detail by Hochhauser and Harris
((1991) Brit. Med. Bull. 47:178-96); Simon and Schindler ((1994)
Proc. Nat'l Acad Sci USA 91: 3497-504); and Harris and Hochhauser
((1992) Acta Oncologica 31:205-213); Scotto et al. ((1986) Science
232: 751-55). Multi-drug resistance can be associated with, for
example, altered composition of plasma membrane phospholipids;
increased drug binding and intracellular accumulation; altered
expression or activity of plasma membrane or endomembrane channels,
transporters or translocators; altered rates of endocytosis and
associated alteration in targeting of endosomes; altered
exocytosis; altered intracellular ionic environments; altered
expression or activity of proteins involved in drug detoxification;
and altered expression or activity of proteins involved in DNA
repair or replication.
[0014] Also within the invention is a method of determining whether
a cell has a drug-resistant phenotype by measuring the expression
(or activity) of Chk1 in the cell and comparing this expression to
that in a control cell. Increased expression (or activity) of Chk1
in the cell compared to the control cell indicates that the cell
has a drug-resistant phenotype. In one embodiment of this method,
Chk1 expression is determined by measuring Chk1 protein (e.g.,
measuring Chk1 protein using an antibody directed against Chk1). In
another embodiment, Chk1 expression is measured by quantifying mRNA
encoding Chk1 or the copy number of the Chk1 gene. In another
embodiment Chk1 activity is measured using any assay which can
quantify a biological activity of Chk1.
[0015] The invention also includes a method for modulating the drug
resistance of a cell by modulating Chk1 expression or activity
within the cell. Thus, in one embodiment, the drug-resistance of a
cell is reduced by contacting the cell with a molecule (e.g., an
antisense nucleic acid molecule) that reduces the expression of
Chk1 within the cell.
[0016] Another aspect of the present invention is a method of
improving effectiveness of chemotherapy for a mammal having a
disorder associated with the presence of drug-resistant neoplastic
cells. In this method, a chemotherapeutic drug and a molecule that
reduces expression of Chk1 can be co-administered to a mammal.
Alternatively, the chemotherapeutic drug can be administered before
or after administration of the compound that reduces expression of
Chk1.
[0017] The invention also includes a method of identifying a
compound that modulates the drug resistance of a cell by first
contacting the cell with a test compound and then measuring and
comparing Chk1 expression in the cell exposed to the compound to
Chk1 expression in a control cell not exposed to the compound. The
compound is identified as modulator of drug resistance when the
level of Chk1 expression in the cell exposed to the compound
differs from the level of Chk1 expression in cells not exposed to
the compound. In one embodiment of this method, the cell has a
drug-resistant phenotype. In another embodiment, the cell is a
mammalian cell. This method may also include an optional step of
measuring the drug resistance of the cell in the presence of the
identified modulator of drug resistance. The Chk1 modulating
compounds that are identified in the foregoing methods are also
included within the invention.
[0018] The invention also features a method of treating a mammal
suspected of having a disorder associated with the presence of
drug-resistant cells. This method includes the steps of determining
whether a mammal has a disorder associated with the presence of
drug-resistant cells having increased Chk1 expression (e.g.,
drug-resistant cancer), and administering to the mammal a compound
that sufficiently reduces the expression of Chk1 so that the drug
resistance of the cells associated with the disorder is modulated
(i.e., reduced).
[0019] Another feature of the invention is a method for treating a
patient having a neoplastic disorder (e.g., cancer) by
administering to the patient a therapeutically effective amount of
a compound that decreases the expression of Chk1.
[0020] In the context of cancer treatment, the expression level of
Chk1 may be used to: 1) determine if a cancer can be treated by an
agent or combination of agents; 2) determine if a cancer is
responding to treatment with an agent or combination of agents; 3)
select an appropriate agent or combination of agents for treating a
cancer; 4) monitor the effectiveness of an ongoing treatment; and
5) identify new cancer treatments (either single agent or
combination of agents). In particular, Chk1 may be used as a marker
(surrogate and/or direct) to determine appropriate therapy, to
monitor clinical therapy and human trials of a drug being tested
for efficacy and in developing new agents and therapeutic
combinations.
[0021] Accordingly, the present invention provides methods for
determining whether an agent, e.g., a chemotherapeutic agent such
as vinblastin, will be effective in reducing the growth rate of
cancer cells comprising the steps of: a) obtaining a sample of
cancer cells; b) determining the level of expression in the cancer
cells of Chk1; and c) identifying that an agent will be effective
when Chk1 is not expressed or is expressed at relatively low level.
Alternatively, in step (c), an agent can be identified as being
relatively ineffective when to use to treat the cancer when Chk1 is
expressed or is expressed at relatively high level.
[0022] As used herein, an agent is said to reduce the rate of
growth of cancer cells when the agent can reduce at least 50%,
preferably at least 75%, most preferably at least 95% of the growth
of the cancer cells at a given concentration of the agent. Such
inhibition can further include a reduction in survivability and an
increase in the rate of death of the cancer cells. The amount of
agent used for this determination will vary based on the agent
selected. Typically, the amount will be a predefined therapeutic
amount.
[0023] As used herein, an agent is defined broadly as anything that
cancer cells can be exposed to in a therapeutic protocol. In the
context of the present invention, such agents include, but are not
limited to, chemotherapeutic agents, such as anti-metabolic agents,
e.g., Ara AC, 5-FU and methotrexate, antimitotic agents, e.g.,
taxol, vinblastine and vincristine, alkylating agents, e.g.,
melphanlan, BCNU and nitrogen mustard, Topoisomerase II inhibitors,
e.g., VW-26, topotecan and Bleomycin, strand-breaking agents, e.g.,
doxorubicin and DHAD, cross-linking agents, e.g., cisplatin and
CBDCA, radiation and ultraviolet light. A preferred agents is
doxorubicin.
[0024] The agents tested in the present methods can be a single
agent or a combination of agents. For example, the present methods
can be used to determine whether a single chemotherapeutic agent,
such as methotrexate, can be used to treat a cancer or whether a
combination of two or more agents can be used.
[0025] Cancer cells include, but are not limited to, carcinomas,
such as squamous cell carcinoma, basal cell carcinoma, sweat gland
carcinoma, sebaceous gland carcinoma, adenocarcinoma, papillary
carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary
carcinoma, undifferentiated carcinoma, bronchogenic carcinoma,
melanoma, renal cell carcinoma, hepatoma-liver cell carcinoma, bile
duct carcinoma, cholangiocarcinoma, papillary carcinoma,
transitional cell carcinoma, choriocarcinoma, semonoma, embryonal
carcinoma, mammary carcinomas, gastrointestinal carcinoma, colonic
carcinomas, bladder carcinoma, prostate carcinoma, and squamous
cell carcinoma of the neck and head region; sarcomas, such as
fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic
sarcoma, chordosarcoma, angiosarcoma, endotheliosarcoma,
lymphangiosarcoma, synoviosarcoma and mesotheliosarcoma; leukemias
and lymphomas such as granulocytic leukemia, monocytic leukemia,
lymphocytic leukemia, malignant lymphoma, plasmocytoma, reticulum
cell sarcoma, or Hodgkins disease; and tumors of the nervous system
including glioma, meningoma, medulloblastoma, schwannoma or
epidymoma.
[0026] The source of the cancer cells used in the methods of the
invention will be based on how the method of the present invention
is being used. For example, if the method is being used to
determine whether a patient's cancer can be treated with an agent,
or a combination of agents, then the preferred source of cancer
cells will be cancer cells obtained from a cancer biopsy from the
patient. Alternatively, cancer cells line of similar type to that
being treated can be assayed. For example if breast cancer is being
treated, then a breast cancer cell line can be used. If the method
is being used to monitor the effectiveness of a therapeutic
protocol, then a tissue sample from the patient being treated is
the preferred source. If the method is being used to identify new
therapeutic agents or combinations, then any cancer cells, e.g.,
cells of a cancer cell line, can be used.
[0027] A skilled artisan can readily select and obtain the
appropriate cancer cells that are used in the present method. For
cancer cell lines, sources such as The National Cancer Institute,
for the NCI-60 cells used in the examples, are preferred. For
cancer cells obtained from a patient, standard biopsy methods, such
as a needle biopsy, can be employed.
[0028] In the methods of the present invention, the level or amount
of expression of Chk1 is determined. As used herein, the level or
amount of expression refers to the absolute level of expression of
an mRNA encoded by the gene or the absolute level of expression of
the protein encoded by the gene (i.e., whether or not expression is
or is not occurring in the cancer cells).
[0029] As an alternative to making determinations based on the
absolute expression level of selected genes, determinations may be
based on the normalized expression levels. Expression levels are
normalized by correcting the absolute expression level of a
sensitivity or resistance gene by comparing its expression to the
expression of a gene that is not a sensitivity or resistance gene,
e.g., a housekeeping genes that is constitutively expressed.
Suitable genes for normalization include housekeeping genes such as
the actin gene. This normalization allows one to compare the
expression level in one sample, e.g., a patient sample, to another
sample, e.g., a non-cancer sample, or between samples from
different sources. Alternatively, the expression level can be
provided as a relative expression level. To determine a relative
expression level of a gene, the level of expression of the gene is
determined for 10 or more samples, preferably 50 or more samples,
prior to the determination of the expression level for the sample
in question. The mean expression level of each of the gene assayed
in the larger number of samples is determined and this is used as a
baseline expression level for the gene in question. The expression
level of the gene determined for the test sample (absolute level of
expression) is then divided by the mean expression value obtained
for that gene. This provides a relative expression level and aids
in identifying extreme cases of sensitivity or resistance.
Preferably, the samples used will be from similar tumors or from
non-cancerous cells of the same tissue origin as the tumor in
question. The choice of the cell source is dependent on the use of
the relative expression level data. For example, using tumors of
similar types for obtaining a mean expression score allows for the
identification of extreme cases of sensitivity or resistance. Using
expression found in normal tissues as a mean expression score aids
in validating whether the gene assayed is tumor specific (versus
normal cells).
[0030] Also within the invention is a method for increasing drug
resistance in a cell having an undesirably low level of Chk1
expression by administering a compound that increases the
expression of Chk1. Such methods are useful for the protection of
non-neoplastic cells during chemotherapy.
[0031] The invention features a method for determining whether a
test compound modulates the drug resistance of a cell, the method
including: a) determining the level of Chk1 expression (e.g., Chk1
encoded by an endogenous or heterologous gene) in a cell in the
presence of a test compound; b) determining the level of Chk1
expression in the cell in the absence of the test compound; and c)
identifying the compound as a modulator of drug resistance of the
cell if the level of expression of Chk1 in the cell in the presence
of the test compound differs from the level of expression of Chk1
in the cell in the absence of the test compound.
[0032] The invention features a method for determining whether a
test compound modulates the drug resistance of a cell, the method
including: a) determining the level of Chk1 activity in a cell in
the presence of a test compound; b) determining the level of Chk1
activity in the cell in the absence of the test compound; and c)
identifying the compound as a modulator of drug resistance of the
cell if the level of activity of Chk1 in the cell in the presence
of the test compound differs from the level of activity of Chk1 in
the cell in the absence of the test compound.
[0033] The invention also features a method for determining whether
a test compound modulates the drug resistance of a cell, the method
including: a) incubating Chk1 protein in the presence of a test
compound; b) determining whether the test compound binds to the
Chk1 protein; c) selecting a test compound which binds to the Chk1
protein; d) administering the test compound selected in step c) to
a non-human mammal having drug resistant cells; e) determining
whether the test compound alters the drug resistance of the cells
in the non-human mammal; and f) identifying the test compound as a
modulator of drug resistance of the cell if the compound alters the
drug resistance of the cells in step e).
[0034] The invention further features a method for determining
whether a test cell has a drug-resistant phenotype, the method
including: a) measuring the expression of Chk1 in the test cell; b)
comparing the expression of Chk1 measured in step a) to the
expression of Chk1 in a control cell not having a drug-resistant
phenotype; and c) determining that the test cell has a drug
resistant phenotype if the expression of Chk1 in the test cell is
greater than the expression of Chk1 in the control cell.
[0035] In another aspect the invention features a method of
determining whether a test cell has a drug-resistant phenotype, the
method including: a) measuring the activity of Chk1 in the test
cell; b) comparing the activity of Chk1 measured in step a) to the
activity of Chk1 in a control cell not having a drug-resistant
phenotype; and c) determining that the test cell has a drug
resistant phenotype if the activity of Chk1 in the test cell is
greater than the activity of Chk1 in the control cell.
[0036] In yet another aspect the invention features a method for
determining whether a subject has or is at risk of developing a
drug resistant tumor, the method including: a) measuring the
expression of Chk1 mRNA in a biological sample obtained from the
subject (using, e.g., a nucleic acid molecule that hybridizes to
Chk1 mRNA); b) comparing the expression of Chk1 mRNA measured in
step a) to the expression of Chk1 mRNA in a biological sample
obtained from a control subject not having a drug resistant tumor;
and c) determining that the patient has or is at risk of developing
a drug resistant tumor if the expression of Chk1 mRNA in the
biological sample obtained from the patient is higher than the
expression of Chk1 mRNA in the biological sample obtained from the
control subject.
[0037] In still another aspect the invention features a method for
determining whether a subject has or is at risk of developing a
drug resistant tumor, the method including: a) measuring the
activity of Chk1 in a biological sample obtained from the subject
(using, e.g., an agent that binds to Chk1 protein); b) comparing
the activity of Chk1 measured in step a) to the activity of Chk1 in
a biological sample obtained from a control subject not having a
drug resistant tumor; and c) determining that the patient has or is
at risk of developing a drug resistant tumor if the activity of
Chk1 in the biological sample obtained from the patient is higher
than the activity of Chk1 in the biological sample obtained from
the control subject.
[0038] The invention also features a method for monitoring the
effect of an anti-tumor treatment on a patient, the method
including: a) measuring the expression of Chk1 in a tumor sample
obtained from the patient (using, e.g., a nucleic acid molecule
that hybridizes to Chk1 mRNA); b) comparing the expression of Chk1
measured in step a) to the expression of Chk1 in a control sample
of cells; and c) determining that the anti-tumor treatment should
be discontinued or modified if the expression of Chk1 in the tumor
sample is higher than the expression of Chk1 in the control sample
of cells.
[0039] The invention also features a method for monitoring the
effect of an anti-tumor treatment on a patient, the method
including: a) measuring the activity of Chk1 in a tumor sample
obtained from the patient (using, e.g., an agent that binds to Chk1
protein); b) comparing the activity of Chk1 measured in step a) to
the activity of Chk1 in a control sample of cells; and c)
determining that the anti-tumor treatment should be discontinued or
modified if the activity of Chk1 in the tumor sample is higher than
the activity of Chk1 in the control sample of cells.
[0040] The invention further features a method for modulating the
drug resistance of a cell by modulating Chk1 expression within the
cell and a method for reducing the drug resistance of cell by
contacting the cell with a molecule which reduces the expression of
Chk1 within the cell.
[0041] The invention also features a method of increasing the
effectiveness of a chemotherapeutic compound in a patient suffering
from a disorder associated with the presence of drug-resistant
neoplastic cells, the method including: a) administering a
chemotherapeutic compound to the patient; and b) administering a
compound with reduces Chk1 expression to the patient.
[0042] The invention features a method of treating a mammal
suspected of having a disorder associated with the presence of
drug-resistant cells, the method including administering to the
mammal a compound that reduces the expression of Chk1 in the
drug-resistant cells, the reduction be sufficient to reduce the
drug resistance of the drug resistant cells and a method for
increasing the drug resistance of cell that has an undesirably low
level of Chk1 expression, the method including exposing the cell to
a compound that increases the expression of Chk1.
[0043] The invention also features a method for treating a drug
resistant tumor in a patient, the method comprising administering
to said subject an amount of a Chk1 antagonist effective to reduce
drug resistance of said tumor in the patient. In another aspect,
the invention features the use of an inhibitor of Chk1 expression,
or pharmaceutically acceptable salt thereof, or a pharmaceutical
composition containing either entity, for the manufacture of a
medicament for the treatment of a drug resistant tumor in a
patient.
[0044] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described
herein. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In the case of conflict, the present specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be limiting.
[0045] Other features and advantages of the invention will be
apparent from the detailed description and from the claims.
Although materials and methods similar or equivalent to those
described herein can be used in the practice or testing of the
invention, the preferred materials and methods are described
below.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 depicts the nucleotide sequence (SEQ ID NO:1) of a
cDNA encoding human Chk1 (GenBank Accession Number AF016582).
[0047] FIG. 2 depicts the predicted amino acid sequence (SEQ ID
NO:3) of human Chk1 (GenBank Accession Number AF016582).
[0048] FIG. 3 depicts the structure of a hammerhead ribozyme (3' to
5' strand) base-paired with a RNA that is to be cleaved (5' to 3'
strand).
[0049] FIG. 4 depicts a predicted secondary structure for Chk1
mRNA.
[0050] FIG. 5 depicts the results of an assay designed to measure
the drug resistance of 293EBNA cells transfected with a ribozyme
(Rz11; diamond), a mutant ribozyme (Rz11M; squares), or vector only
(empty; triangles).
DETAILED DESCRIPTION OF THE INVENTION
[0051] The nucleotide sequence of a cDNA encoding a human Chk1
protein (SEQ ID NO:1) and the predicted amino acid sequence of
human Chk1. protein (SEQ ID NO: 2) are shown in FIGS. 1 and 2
respectively.
[0052] The association between Chk1 expression and drug resistance
was discovered during a search for genes that are more highly
expressed in a drug resistant cell line than in the relatively drug
sensitive cell line from which the drug resistant cell line was
derived.
[0053] The studies described below in Example 1 demonstrate that
Chk1 is expressed at a higher level in certain cancers than in
no-cancerous cells. The studies in Example 2 demonstrate that Chk1
is expressed at a higher level in certain drug resistant cell lines
than in the less drug resistant cell lines from which the drug
resistant cell lines were derived. Example 3 describes the
preparation of purified human Chk1. The studies described in
Example 4 provide evidence that decreasing the expression of Chk1
renders cells more sensitive to doxorubicin.
[0054] Various aspects of the invention are described in further
detail in the following subsections.
[0055] I. Isolated Nucleic Acid Molecules
[0056] Isolated nucleic acid molecules that encode Chk1 proteins or
biologically active portions thereof, as well as nucleic acid
molecules sufficient for use as hybridization probes to identify
Chk1-encoding nucleic acids (e.g., Chk1 mRNA) and fragments for use
as PCR primers for the amplification or mutation of Chk1 nucleic
acid molecules, are useful in the methods of the invention. Various
methods for the preparation and use of Chk1 nucleic acid molecules
are described below.
[0057] As used herein, the term "nucleic acid molecule" is intended
to include DNA molecules (e.g., cDNA or genomic DNA) and RNA
molecules (e.g., mRNA) and analogs of the DNA or RNA generated
using nucleotide analogs. The nucleic acid molecule can be
single-stranded or double-stranded, but preferably is
double-stranded DNA.
[0058] An isolated nucleic acid molecule is one which is separated
from other nucleic acid molecules which are present in the natural
source of the nucleic acid. Preferably, an isolated nucleic acid is
free of sequences (preferably protein encoding sequences) which
naturally flank the nucleic acid (i.e., sequences located at the 5'
and 3' ends of the nucleic acid) in the genomic DNA of the organism
from which the nucleic acid is derived. An isolated Chk1 nucleic
acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1
kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the
nucleic acid is derived. Moreover, an isolated nucleic acid
molecule, such as a cDNA molecule, can be substantially free of
other cellular material, or culture medium when produced by
recombinant techniques, or substantially free of chemical
precursors or other chemicals when chemically synthesized.
[0059] A Chk1 nucleic acid molecule, e.g., a nucleic acid molecule
having the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or a
complement thereof, can be isolated using standard molecular
biology techniques and the sequence information provided herein.
Chk1 nucleic acid molecules can be isolated using standard
hybridization and cloning techniques (e.g., as described in
Sambrook et al., eds., Molecular Cloning: A Laboratory Manual. 2nd,
ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1989).
[0060] A Chk1 nucleic acid can be amplified using cDNA, mRNA or
genomic DNA as a template and appropriate oligonucleotide primers
according to standard PCR amplification techniques. The nucleic
acid so amplified can be cloned into an appropriate vector and
characterized by DNA sequence analysis. Furthermore,
oligonucleotides corresponding to Chk1 nucleotide sequences can be
prepared by standard synthetic techniques, e.g., using an automated
DNA synthesizer.
[0061] Useful Chk1 nucleic acid molecules can comprise only a
portion of a nucleic acid sequence encoding Chk1, for example, a
fragment which can be used as a probe or primer for identifying
and/or quatifying Chk1 mRNA in a biological sample. A probe or
primer can include at least about 12, 25, 50, 75, 100, 125, 150,
175, 200, 250, 300, 350 or 400 nucleotides and hybridizes, e.g.,
under stringent conditions, to a Chk1 mRNA, e.g, an mRNA comprising
the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.
[0062] Probes based on the human Chk1 nucleotide sequence can be
used to detect Chk1 transcripts or genomic sequences. The probe
comprises a label group attached thereto, e.g., a radioisotope, a
fluorescent compound, an enzyme, or an enzyme co-factor. Such
probes can be used as a part of a diagnostic test kit for
identifying cells or tissue which mis-express a Chk1 protein, such
as by measuring a level of a Chk1-encoding nucleic acid in a sample
of cells from a subject, e.g., detecting Chk1 mRNA levels or
determining whether a genomic Chk1 gene has been mutated, deleted,
or amplified.
[0063] A nucleic acid fragment encoding a "biologically active
portion of Chk1" can be prepared by isolating a portion of SEQ ID
NO:3 which encodes a polypeptide having a Chk1 biological activity,
expressing the encoded portion of Chk1 protein (e.g., by
recombinant expression in vitro) and assessing the activity of the
encoded portion of Chk1.
[0064] In addition to the probes and primers described above,
isolated nucleic acid molecules of at least 50, 100, 200, 300 ,
325, 350, 375, 400, 425, 450, 500, 550, 600, 650, 700, 800, 900,
1000, 1200, 1400, 1600, or 1800 nucleotides that hybridize under
stringent conditions to a nucleic acid molecule comprising the
nucleotide sequence, preferably the coding sequence of SEQ ID NO:1
or SEQ ID NO:3 are useful in the methods of the invention.
[0065] As used herein, the term "hybridizes under stringent
conditions" is intended to describe conditions for hybridization
and washing under which nucleotide sequences at least 60% (65%,
70%, preferably 75%) identical to each other typically remain
hybridized to each other. Such stringent conditions are known to
those skilled in the art and can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, New York (1989),
6.3.1-6.3.6. A preferred, non-limiting example of stringent
hybridization conditions are hybridization in x sodium
chloride/sodium citrate (SSC) at about 45.degree. C., followed by
one or more washes in 0.2.times.SSC, 0.1% SDS at 50 to 65.degree.
C.
[0066] Nucleic acid molecules encoding Chk1 proteins that contain
changes in amino acid residues that are not essential for activity
can be used in the methods of the invention. Such Chk1 proteins
differ in amino acid sequence from SEQ ID NO:2 yet retain
biological activity. For example, the isolated nucleic acid
molecule may include a nucleotide sequence encoding a protein that
includes an amino acid sequence that is at least about 45%
identical, 65%, 75%, 85%, 95%, or 98% identical to the amino acid
sequence of SEQ ID NO:2.
[0067] An isolated nucleic acid molecule encoding a Chk1 protein
having a sequence which differs from that of SEQ ID NO:2 can be
created by introducing one or more nucleotide substitutions,
additions or deletions into the nucleotide sequence of SEQ ID NO:3
such that one or more amino acid substitutions, additions or
deletions are introduced into the encoded protein. Mutations can be
introduced by standard techniques, such as site-directed
mutagenesis and PCR-mediated mutagenesis. Preferably, conservative
amino acid substitutions are made at one or more predicted
non-essential amino acid residues. A "conservative amino acid
substitution" is one in which the amino acid residue is replaced
with an amino acid residue having a similar side chain. Families of
amino acid residues having similar side chains have been defined in
the art. These families include amino acids with basic side chains
(e.g., lysine, arginine, histidine), acidic side chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine,
cysteine), nonpolar side chains (e.g., alanine, valine, leucine,
isoleucine, proline, phenylalanine, methionine, tryptophan),
beta-branched side chains (e.g., threonine, valine, isoleucine) and
aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,
histidine). Thus, a predicted nonessential amino acid residue in
Chk1 is preferably replaced with another amino acid residue from
the same side chain family. Alternatively, mutations can be
introduced randomly along all or part of a Chk1 coding sequence,
such as by saturation mutagenesis, and the resultant mutants can be
screened for Chk1 biological activity to identify mutants that
retain activity. Following mutagenesis, the encoded protein can be
expressed recombinantly and the activity of the protein can be
determined.
[0068] Antisense molecules, i.e., molecules which are complementary
to a sense nucleic acid encoding a protein, e.g., complementary to
the coding strand of a double-stranded cDNA molecule or
complementary to an mRNA sequence are useful in the methods of the
invention, e.g., for reducing expression of Chk1 to reduce the drug
resistance of a cell. The antisense nucleic acid can be
complementary to an entire Chk1 coding strand, or to only a portion
thereof, e.g., all or part of the protein coding region (or open
reading frame). An antisense nucleic acid molecule can be antisense
to a noncoding region of the coding strand of a nucleotide sequence
encoding Chk1. The noncoding regions ("5' and 3' untranslated
regions") are the 5' and 3' sequences which flank the coding region
and are not translated into amino acids.
[0069] An antisense oligonucleotide can be, for example, about 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, or 100 nucleotides in length.
An antisense Chk1 nucleic acid can be constructed using chemical
synthesis and enzymatic ligation reactions using procedures known
in the art. For example, an antisense nucleic acid (e.g., an
antisense oligonucleotide) can be chemically synthesized using
naturally occurring nucleotides or variously modified nucleotides
designed to increase the biological stability of the molecules or
to increase the physical stability of the duplex formed between the
antisense and sense nucleic acids, e.g., phosphorothioate
derivatives and acridine substituted nucleotides can be used.
Examples of modified nucleotides which can be used to generate the
antisense nucleic acid include 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridin- e,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiour- acil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and
2,6-diaminopurine. Alternatively, the antisense nucleic acid can be
produced biologically using an expression vector into which a
nucleic acid has been subcloned in an antisense orientation (i.e.,
RNA transcribed from the inserted nucleic acid will be of an
antisense orientation to a target nucleic acid of interest,
described further in the following subsection).
[0070] An antisense nucleic acid molecule is typically administered
to a subject or generated in situ such that they hybridize with or
bind to cellular mRNA and/or genomic DNA encoding a Chk1 protein to
thereby inhibit expression of the protein, e.g., by inhibiting
transcription and/or translation. The hybridization can be by
conventional nucleotide complementarity to form a stable duplex,
or, for example, in the case of an antisense nucleic acid molecule
which binds to DNA duplexes, through specific interactions in the
major groove of the double helix. An example of a route of
administration of antisense nucleic acid molecules of the invention
include direct injection at a tissue site. Alternatively, antisense
nucleic acid molecules can be modified to target selected cells and
then administered systemically. For example, for systemic
administration, antisense molecules can be modified such that they
specifically bind to receptors or antigens expressed on a selected
cell surface, e.g., by linking the antisense nucleic acid molecules
to peptides or antibodies which bind to cell surface receptors or
antigens. The antisense nucleic acid molecules can also be
delivered to cells using the vectors described herein. To achieve
sufficient intracellular concentrations of the antisense molecules,
vector constructs in which the antisense nucleic acid molecule is
placed under the control of a strong pol II or pol III promoter are
preferred.
[0071] An antisense nucleic acid molecule can be an
.alpha.-anomeric nucleic acid molecule. An .alpha.-anomeric nucleic
acid molecule forms specific double-stranded hybrids with
complementary RNA in which the strands run parallel to each other
(Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641). The
antisense nucleic acid molecule can also comprise a
2'-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.
15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987)
FEBS Lett. 215:327-330).
[0072] Ribozymes, which are catalytic RNA molecules with
ribonuclease activity which are capable of cleaving a
single-stranded nucleic acid, such as an mRNA, to which they have a
complementary region can be used in the methods of the invention.
Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff
and Gerlach (1988) Nature 334:585-591)) can be used to
catalytically cleave Chk1 mRNA transcripts to thereby inhibit
translation of Chk1 mRNA. A ribozyme having specificity for a
Chk1-encoding nucleic acid can be designed based upon the
nucleotide sequence of Chk1 (e.g., SEQ ID NO:1, SEQ ID NO:3). For
example, a derivative of a Tetrahymena L-19 IVS RNA can be
constructed in which the nucleotide sequence of the active site is
complementary to the nucleotide sequence to be cleaved in a
Chk1-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No. 4,987,071;
and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, Chk1 mRNA
can be used to select a catalytic RNA having a specific
ribonuclease activity from a pool of RNA molecules. See, e.g.,
Bartel and Szostak (1993) Science 261:1411-1418.
[0073] Other useful nucleic acid molecules are those which form
triple helical structures. For example, Chk1 gene expression can be
inhibited by targeting nucleotide sequences complementary to the
regulatory region of the Chk1 (e.g., the Chk1 promoter and/or
enhancers) to form triple helical structures that prevent
transcription of the Chk1 gene in target cells. See generally,
Helene (1991) Anticancer Drug Des. 6(6):569-84; Helene (1992) Ann.
N.Y. Acad. Sci. 660:27-36; and Maher (1992) Bioassays
14(12):807-15.
[0074] Nucleic acid molecules useful in the methods of the
invention can be modified at the base moiety, sugar moiety or
phosphate backbone to improve, e.g., the stability, hybridization,
or solubility of the molecule. For example, the deoxyribose
phosphate backbone of the nucleic acids can be modified to generate
peptide nucleic acids (see Hyrup et al. (1996) Bioorganic &
Medicinal Chemistry 4(1): 5-23). As used herein, the terms "peptide
nucleic acids" or "PNAs" refer to nucleic acid mimics, e.g., DNA
mimics, in which the deoxyribose phosphate backbone is replaced by
a pseudopeptide backbone and only the four natural nucleobases are
retained. The neutral backbone of PNAs has been shown to allow for
specific hybridization to DNA and RNA under conditions of low ionic
strength. The synthesis of PNA oligomers can be performed using
standard solid phase peptide synthesis protocols as described in
Hyrup et al. (1996) supra; Perry-O'Keefe et al. (1996) Proc. Natl.
Acad. Sci. USA 93: 14670-675.
[0075] PNAs of Chk1 can be used for therapeutic and diagnostic
applications. For example, PNAs can be used as antisense or
antigene agents for sequence-specific modulation of gene expression
by, e.g., inducing transcription or translation arrest or
inhibiting replication. PNAs of Chk1 can also be used, e.g., in the
analysis of single base pair mutations in a gene by, e.g., PNA
directed PCR clamping; as artificial restriction enzymes when used
in combination with other enzymes, e.g., S1 nucleases (Hyrup (1996)
supra; or as probes or primers for DNA sequence analysis and
hybridization (Hyrup (1996) supra; Perry-O'Keefe et al. (1996)
Proc. Natl. Acad. Sci. USA 93: 14670-675).
[0076] PNAs of Chk1 can be modified, e.g., to enhance their
stability or cellular uptake, by attaching lipophilic or other
helper groups to PNA, by the formation of PNA-DNA chimeras, or by
the use of liposomes or other techniques of drug delivery known in
the art. For example, PNA-DNA chimeras of Chk1 can be generated
which may combine the advantageous properties of PNA and DNA. Such
chimeras allow DNA recognition enzymes, e.g., RNAse H and DNA
polymerases, to interact with the DNA portion while the PNA portion
would provide high binding affinity and specificity. PNA-DNA
chimeras can be linked using linkers of appropriate lengths
selected in terms of base stacking, number of bonds between the
nucleobases, and orientation (Hyrup (1996) supra). The synthesis of
PNA-DNA chimeras can be performed as described in Hyrup (1996)
supra and Finn et al. (1996) Nucleic Acids Research 24(17):3357-63.
For example, a DNA chain can be synthesized on a solid support
using standard phosphoramidite coupling chemistry and modified
nucleoside analogs, e.g.,
5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can
be used as a linker between the PNA and the 5' end of DNA (Mag et
al. (1989) Nucleic Acid Res. 17:5973-88). PNA monomers are then
coupled in a stepwise manner to produce a chimeric molecule with a
5' PNA segment and a 3' DNA segment (Finn et al. (1996) Nucleic
Acids Research 24(17):3357-63). Alternatively, chimeric molecules
can be synthesized with a 5' DNA segment and a 3' PNA segment
(Peterser et al. (1975) Bioorganic Med. Chem. Lett.
5:1119-11124).
[0077] Useful oligonucleotide may include other appended groups
such as peptides (e.g., for targeting host cell receptors in vivo),
or agents facilitating transport across the cell membrane (see,
e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA
86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA
84:648-652; PCT Publication No. W088/09810) or the blood-brain
barrier (see, e.g., PCT Publication No. W089/10134). In addition,
oligonucleotides can be modified with hybridization-triggered
cleavage agents (See, e.g., Krol et al. (1988) Bio/Techniques
6:958-976) or intercalating agents (See, e.g., Zon (1988) Pharm.
Res. 5:539-549). To this end, the oligonucleotide may be conjugated
to another molecule, e.g., a peptide, hybridization triggered
cross-linking agent, transport agent, hybridization-triggered
cleavage agent, etc.
[0078] II. Isolated Chk1 Proteins and Anti-Chk1 Antibodies
[0079] Isolated Chk1 proteins, and biologically active portions
thereof, as well as polypeptide fragments suitable for use as
immunogens to raise anti-Chk1 antibodies are useful in the methods
of the invention. Methods for the preparation and use of these
molecules are described below. In general, Chk1 proteins can be
isolated from cells or tissue sources by an appropriate
purification scheme using standard protein purification techniques,
produced by recombinant DNA techniques, or synthesized chemically
using standard peptide synthesis techniques.
[0080] An "isolated" or "purified" protein or biologically active
portion thereof is substantially free of cellular material or other
contaminating proteins from the cell or tissue source from which
the Chk1 protein is derived, or substantially free from chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of Chk1 protein in which the protein is separated from
cellular components of the cells from which it is isolated or
recombinantly produced. Thus, Chk1 protein that is substantially
free of cellular material includes preparations of Chk1 protein
having less than about 30%, 20%, 10%, or 5% (by dry weight) of
non-Chk1 protein (also referred to herein as a "contaminating
protein"). When the Chk1 protein or biologically active portion
thereof is recombinantly produced, it is also preferably
substantially free of culture medium, i.e., culture medium
represents less than about 20%, 10%, or 5% of the volume of the
protein preparation. When Chk1 protein is produced by chemical
synthesis, it is preferably substantially free of chemical
precursors or other chemicals, i.e., it is separated from chemical
precursors or other chemicals which are involved in the synthesis
of the protein. Accordingly such preparations of Chk1 protein have
less than about 30%, 20%, 10%, 5% (by dry weight) of chemical
precursors or non-Chk1 chemicals.
[0081] Biologically active portions of.degree. a Chk1 protein
include peptides comprising amino acid sequences sufficiently
identical to or derived from the amino acid sequence of the Chk1
protein (e.g., the amino acid sequence shown in SEQ ID NO:2), which
include less amino acids than the full length Chk1 proteins, and
exhibit at least one activity of a Chk1 protein. Typically,
biologically active portions comprise a domain or motif with at
least one activity of the Chk1 protein. A biologically active
portion of a Chk1 protein can be a polypeptide which is, for
example, 10, 25, 50, 100, 200, 300, 400, or more amino acids in
length.
[0082] Moreover, other biologically active portions, in which other
regions of the protein are deleted, can be prepared by recombinant
techniques and evaluated for one or more of the functional
activities of a native Chk1 protein. A preferred Chk1 protein has
the amino acid sequence of SEQ ID NO:2. Other useful Chk1 proteins
are substantially identical to SEQ ID NO:2 and retain the
functional activity of the protein of SEQ ID NO:2 yet differ in
amino acid sequence due to natural allelic variation or
mutagenesis. Accordingly, a useful Chk1 protein is a protein which
includes an amino acid sequence at least about 45%, preferably 55%,
65%, 75%, 85%, 95%, or 99% identical to the amino acid sequence of
SEQ ID NO:2 and retains the functional activity of the Chk1
proteins of SEQ ID NO:2.
[0083] To determine the percent identity of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps can be introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino or nucleic acid sequence). The amino
acid residues or nucleotides at corresponding amino acid positions
or nucleotide positions are then compared. When a position in the
first sequence is occupied by the same amino acid residue or
nucleotide as the corresponding position in the second sequence,
then the molecules are identical at that position. The percent
identity between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % identity=# of
identical positions/total # of positions.times.100).
[0084] The determination of percent homology between two sequences
can be accomplished using a mathematical algorithm. A preferred,
non-limiting example of a mathematical algorithm utilized for the
comparison of two sequences is the algorithm of Karlin and Altschul
(1990) Proc. Nat'l Acad. Sci. USA 87:2264-2268, modified as in
Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5877.
Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul, et al. (1990) J. Mol. Biol. 215:403-410.
BLAST nucleotide searches can be performed with the NBLAST program,
score=100, wordlength=12 to obtain nucleotide sequences homologous
to Chk1 nucleic acid molecules of the invention. BLAST protein
searches can be performed with the XBLAST program, score=50,
wordlength=3 to obtain amino acid sequences homologous to Chk1
protein molecules of the invention. To obtain gapped alignments for
comparison purposes, Gapped BLAST can be utilized as described in
Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When
utilizing BLAST and Gapped BLAST programs, the default parameters
of the respective programs (e.g., XBLAST and NBLAST) can be used.
See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting
example of a mathematical algorithm utilized for the comparison of
sequences is the algorithm of Myers and Miller, CABIOS (1989). Such
an algorithm is incorporated into the ALIGN program (version 2.0)
which is part of the GCG sequence alignment software package. When
utilizing the ALIGN program for comparing amino acid sequences, a
PAM120 weight residue table, a gap length penalty of 12, and a gap
penalty of 4 can be used.
[0085] The percent identity between two sequences can be determined
using techniques similar to those described above, with or without
allowing gaps. In calculating percent identity, only exact matches
are counted.
[0086] Chk1 chimeric or fusion proteins are also useful in the
methods of the invention. As used herein, a Chk1 "chimeric protein"
or "fusion protein" comprises a Chk1 polypeptide operatively linked
to a non-Chk1 polypeptide. A "Chk1 polypeptide" refers to a
polypeptide having an amino acid sequence corresponding to Chk1,
whereas a "non-Chk1 polypeptide" refers to a polypeptide having an
amino acid sequence corresponding to a protein which is not
substantially identical to the Chk1 protein, e.g., a protein which
is different from the Chk1 protein and which is derived from the
same or a different organism. Within a Chk1 fusion protein the Chk1
polypeptide can correspond to all or a portion of a Chk1 protein,
preferably at least one biologically active portion of a Chk1
protein. Within the fusion protein, the term "operatively linked"
is intended to indicate that the Chk1 polypeptide and the non-Chk1
polypeptide are fused in-frame to each other. The non-Chk1
polypeptide can be fused to the N-terminus or C-terminus of the
Chk1 polypeptide.
[0087] One useful fusion protein is a GST-Chk1 fusion protein in
which the Chk1 sequences are fused to the C-terminus of the GST
sequences. Such fusion proteins can facilitate the purification of
recombinant Chk1.
[0088] Another useful Chk1 fusion protein is an Chk1-immunoglobulin
fusion protein in which all or part of Chk1 is fused to sequences
derived from a member of the immunoglobulin protein family.
Chk1-immunoglobulin fusion proteins of the invention can be used as
immunogens to produce anti-Chk1 antibodies in a subject, to purify
Chk1 ligands and in screening assays to identify molecules which
inhibit the interaction of Chk1 with a protein or nucleic acid
which binds Chk1.
[0089] A Chk1 chimeric or fusion protein can be produced by
standard recombinant DNA techniques. For example, DNA fragments
coding for the different polypeptide sequences are ligated together
in-frame in accordance with conventional techniques, for example by
employing blunt-ended or stagger-ended termini for ligation,
restriction enzyme digestion to provide for appropriate termini,
filling-in of cohesive ends as appropriate, alkaline phosphatase
treatment to avoid undesirable joining, and enzymatic ligation. The
fusion gene can be synthesized by conventional techniques including
automated DNA synthesizers. Alternatively, PCR amplification of
gene fragments can be carried out using anchor primers which give
rise to complementary overhangs between two consecutive gene
fragments which can subsequently be annealed and reamplified to
generate a chimeric gene sequence (see, e.g., Current Protocols in
Molecular Biology, Ausubel et al. eds., John Wiley & Sons:
1992). Moreover, many expression vectors are commercially available
that already encode a fusion moiety (e.g., a GST polypeptide). An
Chk1-encoding nucleic acid can be cloned into such an expression
vector such that the fusion moiety is linked in-frame to the Chk1
protein.
[0090] Variants of Chk1 protein which function as either Chk1
agonists (mimetics) or as Chk1 antagonists are useful in the
methods of the invention. Variants of the Chk1 protein can be
generated by mutagenesis, e.g., discrete point mutation or
truncation of the Chk1 protein. An agonist of the Chk1 protein can
retain substantially the same, or a subset, of the biological
activities of the naturally occurring form of the Chk1 protein. An
antagonist of the Chk1 protein can inhibit one or more of the
activities of the naturally-occurring form of the Chk1 protein by,
for example, competitively binding to polynucleotides or proteins
involved in Chk1 function. Thus, specific biological effects can be
elicited by treatment with a variant of limited function. Treatment
of a subject with a variant having a subset of the biological
activities of the naturally-occurring form of the protein can have
fewer side effects in a subject relative to treatment with the
naturally-occurring form of the Chk1 proteins.
[0091] Variants of the Chk1 protein which function as either Chk1
agonists (mimetics) or as Chk1 antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the Chk1 protein for Chk1 protein agonist or antagonist
activity. A library of Chk1 variants can be produced by, for
example, enzymatically ligating a mixture of synthetic
oligonucleotides into gene sequences such that a degenerate set of
potential Chk1 sequences is expressible as individual polypeptides,
or alternatively, as a set of larger fusion proteins (e.g., for
phage display) containing the set of Chk1 sequences therein. There
are a variety of methods which can be used to produce libraries of
potential Chk1 variants from a degenerate oligonucleotide sequence.
Chemical synthesis of a degenerate gene sequence can be performed
in an automatic DNA synthesizer, and the synthetic gene then
ligated into an appropriate expression vector. Use of a degenerate
set of genes allows for the provision, in one mixture, of all of
the sequences encoding the desired set of potential Chk1 sequences.
Methods for synthesizing degenerate oligonucleotides are known in
the art (see, e.g., Narang (1983) Tetrahedron 39:3; Itakura et al.
(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science
198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).
[0092] In addition, libraries of fragments of the Chk1 protein
coding sequence can be used to generate a variegated population of
Chk1 fragments for screening and subsequent selection of variants
of a Chk1 protein. For example, a library of coding sequence
fragments can be generated by treating a double stranded PCR
fragment of a Chk1 coding sequence with a nuclease under conditions
wherein nicking occurs only about once per molecule, denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA
which can include sense/antisense pairs from different nicked
products, removing single stranded portions from reformed duplexes
by treatment with S1 nuclease, and ligating the resulting fragment
library into an expression vector. By this method, an expression
library can be derived which encodes N-terminal and internal
fragments of various sizes of the Chk1 protein.
[0093] Several techniques are known in the art for screening gene
products of combinatorial libraries made by point mutations or
truncation, and for screening cDNA libraries for gene products
having a selected property. Such techniques are adaptable for rapid
screening of the gene libraries generated by the combinatorial
mutagenesis of Chk1 proteins. The most widely used techniques,
which are amenable to high through-put analysis, for screening
large gene libraries typically include cloning the gene library
into replicable expression vectors, transforming appropriate cells
with the resulting library of vectors, and expressing the
combinatorial genes under conditions in which detection of a
desired activity facilitates isolation of the vector encoding the
gene whose product was detected. Recursive ensemble mutagenesis
(REM), a technique which enhances the frequency of functional
mutants in the libraries, can be used in combination with the
screening assays to identify Chk1 variants (Arkin and Yourvan
(1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al.
(1993) Protein Engineering 6(3):327-331).
[0094] An isolated Chk1 protein, or a portion or fragment thereof,
can be used as an immunogen to generate antibodies that bind Chk1
using standard techniques for polyclonal and monoclonal antibody
preparation. The full-length Chk1 protein can be used or,
alternatively, the invention provides antigenic peptide fragments
of Chk1 for use as immunogens. The antigenic peptide of Chk1
comprises at least 8 (preferably 10, 15, 20, or 30) amino acid
residues of the amino acid sequence shown in SEQ ID NO:2 and
encompasses an epitope of Chk1 such that an antibody raised against
the peptide forms a specific immune complex with Chk1.
[0095] Preferred epitopes encompassed by the antigenic peptide are
regions of Chk1 that are located on the surface of the protein,
e.g., hydrophilic regions.
[0096] A Chk1 immunogen typically is used to prepare antibodies by
immunizing a suitable subject, (e.g., rabbit, goat, mouse or other
mammal) with the immunogen. An appropriate immunogenic preparation
can contain, for example, recombinantly expressed Chk1 protein or a
chemically synthesized Chk1 polypeptide. The preparation can
further include an adjuvant, such as Freund's complete or
incomplete adjuvant, or similar immunostimulatory agent.
Immunization of a suitable subject with an immunogenic Chk1
preparation induces a polyclonal anti-Chk1 antibody response.
[0097] Anti-Chk1 antibodies are useful in the methods of the
invention. The term antibody refers to immunoglobulin molecules and
immunologically active portions of immunoglobulin molecules, i.e.,
molecules that contain an antigen binding site which specifically
binds an antigen, such as Chk1. A molecule which specifically binds
to Chk1 is a molecule which binds Chk1, but does not substantially
bind other molecules in a sample, e.g., a biological sample, which
naturally contains Chk1. Examples of immunologically active
portions of immunoglobulin molecules include F(ab) and F(ab').sub.2
fragments which can be generated by treating the antibody with an
enzyme such as pepsin. The term monoclonal antibody or monoclonal
antibody composition refers to a population of antibody molecules
that contain only one species of an antigen binding site capable of
immunoreacting with a particular epitope of Chk1. A monoclonal
antibody composition thus typically displays a single binding
affinity for a particular Chk1 protein with which it
immunoreacts.
[0098] Polyclonal anti-Chk1 antibodies can be prepared as described
above by immunizing a suitable subject with a Chk1 immunogen. The
anti-Chk1 antibody titer in the immunized subject can be monitored
over time by standard techniques, such as with an enzyme linked
immunosorbent assay (ELISA) using immobilized Chk1. If desired, the
antibody molecules directed against Chk1 can be isolated from the
mammal (e.g., from the blood) and further purified by well-known
techniques, such as protein A chromatography to obtain the IgG
fraction. At an appropriate time after immunization, e.g., when the
anti-Chk1 antibody titers are highest, antibody-producing cells can
be obtained from the subject and used to prepare monoclonal
antibodies by standard techniques, such as the hybridoma technique
originally described by Kohler and Milstein (1975) Nature
256:495-497, the human B cell hybridoma technique (Kozbor et al.
(1983) Immunol Today 4:72), the EBV-hybridoma technique (Cole et
al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,
Inc., pp. 77-96) or trioma techniques. The technology for producing
various antibodies monoclonal antibody hybridomas is well known
(see generally Current Protocols in Immunology (1994) Coligan et
al. (eds.) John Wiley & Sons, Inc., New York, N.Y.). Briefly,
an immortal cell line (typically a myeloma) is fused to lymphocytes
(typically splenocytes) from a mammal immunized with a Chk1
immunogen as described above, and the culture supernatants of the
resulting hybridoma cells are screened to identify a hybridoma
producing a monoclonal antibody that binds Chk1.
[0099] Any of the many well known protocols used for fusing
lymphocytes and immortalized cell lines can be applied for the
purpose of generating an anti-Chk1 monoclonal antibody (see, e.g.,
Current Protocols in Immunology, supra; Galfre et al. (1977) Nature
266:55052; R. H. Kenneth, in Monoclonal Antibodies: A New Dimension
In Biological Analyses, Plenum Publishing Corp., New York, N.Y.
(1980); and Lerner (1981) Yale J. Biol. Med., 54:387-402. Moreover,
the ordinarily skilled worker will appreciate that there are many
variations of such methods which also would be useful. Typically,
the immortal cell line (e.g., a myeloma cell line) is derived from
the same mammalian species as the lymphocytes. For example, murine
hybridomas can be made by fusing lymphocytes from a mouse immunized
with an immunogenic preparation of the present invention with an
immortalized mouse cell line, e.g., a myeloma cell line that is
sensitive to culture medium containing hypoxanthine, aminopterin
and thymidine ("HAT medium"). Any of a number of myeloma cell lines
can be used as a fusion partner according to standard techniques,
e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma
lines. These myeloma lines are available from ATCC. Typically,
HAT-sensitive mouse myeloma cells are fused to mouse splenocytes
using polyethylene glycol ("PEG"). Hybridoma cells resulting from
the fusion are then selected using HAT medium, which kills unfused
and unproductively fused myeloma cells (unfused splenocytes die
after several days because they are not transformed). Hybridoma
cells producing a monoclonal antibody of the invention are detected
by screening the hybridoma culture supernatants for antibodies that
bind Chk1, e.g., using a standard ELISA assay.
[0100] Alternative to preparing monoclonal antibody-secreting
hybridomas, a monoclonal anti-Chk1 antibody can be identified and
isolated by screening a recombinant combinatorial immunoglobulin
library (e.g., an antibody phage display library) with Chk1 to
thereby isolate immunoglobulin library members that bind Chk1. Kits
for generating and screening phage display libraries are
commercially available (e.g., the Pharmacia Recombinant Phage
Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP
Phage Display Kit, Catalog No. 240612). Additionally, examples of
methods and reagents particularly amenable for use in generating
and screening antibody display library can be found in, for
example, U.S. Pat. No. 5,223,409; PCT Publication No. WO 92/18619;
PCT Publication No. WO 91/17271; PCT Publication WO 92/20791; PCT
Publication No. WO 92/15679; PCT Publication WO 93/01288; PCT
Publication No. WO 92/01047; PCT Publication No. WO 92/09690; PCT
Publication No. WO 90/02809; Fuchs et al. (1991) Bio/Technology
9:1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85;
Huse et al. (1989) Science 246:1275-1281; Griffiths et al. (1993)
EMBO J 12:725-734.
[0101] Additionally, recombinant anti-Chk1 antibodies, such as
chimeric and humanized monoclonal antibodies, comprising both human
and non-human portions, which can be made using standard
recombinant DNA techniques, are within the scope of the invention.
Such chimeric and humanized monoclonal antibodies can be produced
by recombinant DNA techniques known in the art, for example using
methods described in PCT Publication No. WO 87/02671; European
Patent Application 184,187; European Patent Application 171,496;
European Patent Application 173,494; PCT Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Canc. Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; and Shaw et al. (1988) J.
Natl. Cancer Inst. 80:1553-1559); Morrison, (1985) Science
229:1202-1207; Oi et al. (1986) Bio/Techniques 4:214; U.S. Pat. No.
5,225,539; Jones et al. (1986) Nature 321:552-525; Verhoeyan et al.
(1988) Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060.
[0102] An anti-Chk1 antibody (e.g., monoclonal antibody) can be
used to isolate Chk1 by standard techniques, such as affinity
chromatography or immunoprecipitation. An anti-Chk1 antibody can
facilitate the purification of natural Chk1 from cells and of
recombinantly produced Chk1 expressed in host cells. Moreover, an
anti-Chk1 antibody can be used to detect Chk1 protein (e.g., in a
cellular lysate or cell supernatant) in order to evaluate the
abundance and pattern of expression of the Chk1 protein. Anti-Chk1
antibodies can be used diagnostically to monitor protein levels in
tissue as part of a clinical testing procedure, e.g., to, for
example, determine the efficacy of a given treatment regimen.
Detection can be facilitated by coupling the antibody to a
detectable substance. Examples of detectable substances include
various enzymes, prosthetic groups, fluorescent materials,
luminescent materials, bioluminescent materials, and radioactive
materials. Examples of suitable enzymes include horseradish
peroxidase, alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; examples of bioluminescent materials include luciferase,
luciferin, and aequorin, and examples of suitable radioactive
material include .sup.125I, .sup.131I, .sup.35S or .sup.3H.
[0103] III. Recombinant Expression Vectors and Host Cells
[0104] Vectors, preferably expression vectors, containing a nucleic
acid encoding Chk1 (or a portion thereof) are useful in the methods
of the invention. A vector is a nucleic acid molecule capable of
transporting another nucleic acid to which it has been linked. One
type of vector is a "plasmid", which refers to a circular double
stranded DNA loop into which additional DNA segments can be
ligated. Another type of vector is a viral vector, wherein
additional DNA segments can be ligated into the viral genome.
Certain vectors are capable of autonomous replication in a host
cell into which they are introduced (e.g., bacterial vectors having
a bacterial origin of replication and episomal mammalian vectors).
Other vectors (e.g., non-episomal mammalian vectors) are integrated
into the genome of a host cell upon introduction into the host
cell, and thereby are replicated along with the host genome.
Moreover, certain vectors, expression vectors, are capable of
directing the expression of genes to which they are operatively
linked. In general, expression vectors of utility in recombinant
DNA techniques are often in the form of plasmids (vectors, e.g.,
viral vectors, replication defective retroviruses, adenoviruses and
adeno-associated viruses).
[0105] Useful recombinant expression vectors comprise a Chk1
nucleic acid in a form suitable for expression of the nucleic acid
in a host cell, which means that the recombinant expression vectors
include one or more regulatory sequences, selected on the basis of
the host cells to be used for expression, which is operatively
linked to the nucleic acid sequence to be expressed. Within a
recombinant expression vector, "operably linked" is intended to
mean that the nucleotide sequence of interest is linked to the
regulatory sequence(s) in a manner which allows for expression of
the nucleotide sequence (e.g., in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). The term "regulatory sequence"
is intended to include promoters, enhancers and other expression
control elements (e.g., polyadenylation signals). Such regulatory
sequences are described, for example, in Goeddel; Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1990). Regulatory sequences include those which direct
constitutive expression of a nucleotide sequence in many types of
host cell and those which direct expression of the nucleotide
sequence only in certain host cells (e.g., tissue-specific
regulatory sequences). It will be appreciated by those skilled in
the art that the design of the expression vector can depend on such
factors as the choice of the host cell to be transformed, the level
of expression of protein desired, etc. An expression vector can be
introduced into host cells to thereby produce proteins or peptides,
including fusion proteins or peptides, encoded by nucleic acids as
described herein (e.g., Chk1 proteins, mutant forms of Chk1, fusion
proteins, etc.).
[0106] The recombinant expression vectors of the invention can be
designed for expression of Chk1 in prokaryotic or eukaryotic cells,
e.g., bacterial cells such as E. coli, insect cells (using
baculovirus expression vectors) yeast cells or mammalian cells.
Suitable host cells are discussed further in Goeddel, Gene
Expression Technology: Methods in Enzyymology 185, Academic Press,
San Diego, Calif. (1990). Alternatively, the recombinant expression
vector can be transcribed and translated in vitro, for example
using T7 promoter regulatory sequences and T7 polymerase.
[0107] Expression of proteins in prokaryotes is most often carried
out in E. coli with vectors containing constitutive or inducible
promoters directing the expression of either fusion or non-fusion
proteins. Fusion vectors add a number of amino acids to a protein
encoded therein, usually to the amino terminus of the recombinant
protein. Such fusion vectors typically serve three purposes: 1) to
increase expression of recombinant protein; 2) to increase the
solubility of the recombinant protein; and 3) to aid in the
purification of the recombinant protein by acting as a ligand in
affinity purification. Often, in fusion expression vectors, a
proteolytic cleavage site is introduced at the junction of the
fusion moiety and the recombinant protein to enable separation of
the recombinant protein from the fusion moiety subsequent to
purification of the fusion protein. Such enzymes, and their cognate
recognition sequences, include Factor Xa, thrombin and
enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67:31-40),
pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,
Piscataway, N.J.) which fuse glutathione S-transferase (GST),
maltose E binding protein, or protein A, respectively, to the
target recombinant protein.
[0108] Examples of suitable inducible non-fusion E. coli expression
vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET
11d (Studier et al., Gene Expression Technology: Methods in
Enzymology 185, Academic Press, San Diego, Calif. (1990) 60-89).
Target gene expression from the pTrc vector relies on host RNA
polymerase transcription from a hybrid trp-lac fusion promoter.
Target gene expression from the pET 11d vector relies on
transcription from a T7 gn10-lac fusion promoter mediated by a
coexpressed viral RNA polymerase (T7 gn1). This viral polymerase is
supplied by host strains BL21(DE3) or HMS174(DE3) from a resident l
prophage harboring a T7 gnl gene under the transcriptional control
of the lacUV 5 promoter.
[0109] One strategy to maximize recombinant protein expression in
E. coli is to express the protein in a host bacteria with an
impaired capacity to proteolytically cleave the recombinant protein
(Gottesman, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. (1990) 119-128). Another strategy
is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an expression vector so that the individual codons
for each amino acid are those preferentially utilized in E. coli
(Wada et al. (1992) Nucleic Acids Res. 20:2111-2118). Such
alteration of nucleic acid sequences of the invention can be
carried out by standard DNA synthesis techniques.
[0110] An Chk1 expression vector is a yeast expression vector.
Examples of vectors for expression in yen be a S. cerivisae include
pYepSec1 (Baldari et al. (1987) EMBO J. 6:229-234), pMFa (Kurjan
and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al.
(1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,
CAalif., and picz (InVitrogen Corp, San Diego, Calif.).
[0111] Alternatively, Chk1 can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available for
expression of proteins in cultured insect cells (e.g., Sf 9 cells)
include the pAc series (Smith et al. (1983) Mol. Cell Biol.
3:2156-2165) and the pVL series (Lucklow and Summers (1989)
Virology 170:31-39).
[0112] An Chk1 nucleic acid can be expressed in mammalian cells
using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed (1987) Nature 329:840) and
pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When used in
mammalian cells, the expression vector's control functions are
often provided by viral regulatory elements. For example, commonly
used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression
systems for both prokaryotic and eukaryotic cells see chapters 16
and 17 of Sambrook et al. (supra).
[0113] In another embodiment, the recombinant mammalian expression
vector is capable of directing expression of the nucleic acid
preferentially in a particular cell type (e.g., tissue-specific
regulatory elements are used to express the nucleic acid).
Tissue-specific regulatory elements are known in the art.
Non-limiting examples of suitable tissue-specific promoters include
the albumin promoter (liver-specific; Pinkert et al. (1987) Genes
Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton
(1988) Adv. Immunol. 43:235-275), in particular promoters of T cell
receptors (Winoto and Baltimore (1989) EMBO J. 8:729-733) and
immunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen and
Baltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g.,
the neurofilament promoter; Byrne and Ruddle (1989) Proc. Natl.
Acad. Sci. USA 86:5473-5477), pancreas-specific promoters (Edlund
et al. (1985) Science 230:912-916), and mammary gland-specific
promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and
European Application Publication No. 264,166).
Developmentally-regulated promoters are also encompassed, for
example the murine hox promoters (Kessel and Gruss (1990) Science
249:374-379) and the .alpha.-fetoprotein promoter (Campes and
Tilghman (1989) Genes Dev. 3:537-546) Also useful in the methods of
the invention are recombinant expression vectors comprising an Chk1
nucleic acid molecule cloned into the expression vector in an
antisense orientation. That is, the DNA molecule is operatively
linked to a regulatory sequence in a manner which allows for
expression (by transcription of the DNA molecule) of an RNA
molecule which is antisense to Chk1 mRNA. Regulatory sequences
operatively linked to a nucleic acid cloned in the antisense
orientation can be chosen which direct the continuous expression of
the antisense RNA molecule in a variety of cell types, for instance
viral promoters and/or enhancers, or regulatory sequences can be
chosen which direct constitutive, tissue specific or cell type
specific expression of antisense RNA. The antisense expression
vector can be in the form of a recombinant plasmid, phagemid or
attenuated virus in which antisense nucleic acids are produced
under the control of a high efficiency regulatory region, the
activity of which can be determined by the cell type into which the
vector is introduced. For a discussion of the regulation of gene
expression using antisense genes See Weintraub et al.,
Reviews--Trends in Genetics, Vol. 1(1) 1986.
[0114] Host cells into which an Chk1 expression vector has been
introduced are useful in certain metods of the invention. The terms
"host cell" and "recombinant host cell" are used interchangeably
herein. It is understood that such terms refer not only to the
particular subject cell but to the progeny or potential progeny of
such a cell. Because certain modifications may occur in succeeding
generations due to either mutation or environmental influences,
such progeny may not, in fact, be identical to the parent cell, but
are still included within the scope of the term as used herein.
[0115] A host cell can be any prokaryotic or eukaryotic cell. For
example, Chk1 protein can be expressed in bacterial cells such as
E. coli, insect cells, yeast or mammalian cells (such as Chinese
hamster ovary cells (CHO) or COS cells). Other suitable host cells
are known to those skilled in the art.
[0116] Vector DNA can be introduced into prokaryotic or eukaryotic
cells via conventional transformation or transfection techniques.
As used herein, the terms "transformation" and "transfection" are
intended to refer to a variety of art-recognized techniques for
introducing foreign nucleic acid (e.g., DNA) into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE-dextran-mediated transfection, lipofection, or
electroporation. Suitable methods for transforming or transfecting
host cells can be found in Sambrook, et al. (supra), and other
laboratory manuals.
[0117] For stable transfection of mammalian cells, it is known
that, depending upon the expression vector and transfection
technique used, only a small fraction of cells may integrate the
foreign DNA into their genome. In order to identify and select
these integrants, a gene that encodes a selectable marker (e.g.,
resistance to antibiotics) is generally introduced into the host
cells along with the gene of interest. Preferred selectable markers
include those which confer resistance to drugs, such as G418,
hygromycin and methotrexate. Nucleic acid encoding a selectable
marker can be introduced into a host cell on the same vector as
that encoding Chk1 or can be introduced on a separate vector. Cells
stably transfected with the introduced nucleic acid can be
identified by drug selection (e.g., cells that have incorporated
the selectable marker gene will survive, while the other cells
die).
[0118] A prokaryotic or eukaryotic host cell in culture can be used
to produce (i.e., express) Chk1 protein, e.g., by culturing the
host cell (into which a recombinant expression vector encoding Chk1
has been introduced) in a suitable medium such that Chk1 protein is
produced. Chk1 protein can then be isolated from the medium or the
host cell.
[0119] Host cells which are capable of expressing Chk1 can also be
used to produce nonhuman transgenic animals. For example, in one
embodiment, a host cell of the invention is a fertilized oocyte or
an embryonic stem cell into which Chk1-coding sequences have been
introduced. Such host cells can then be used to create non-human
transgenic animals in which exogenous Chk1 sequences have been
introduced into their genome or homologous recombinant animals in
which endogenous Chk1 sequences have been altered. Such animals are
useful for studying the function and/or activity of Chk1 and for
identifying and/or evaluating modulators of Chk1 activity. As used
herein, a "transgenic animal" is a non-human animal, preferably a
mammal, more preferably a rodent such as a rat or mouse, in which
one or more of the cells of the animal includes a transgene. Other
examples of transgenic animals include non-human primates, sheep,
dogs, cows, goats, chickens, amphibians, etc. A transgene is
exogenous DNA which is integrated into the genome of a cell from
which a transgenic animal develops and which remains in the genome
of the mature animal, thereby directing the expression of an
encoded gene product in one or more cell types or tissues of the
transgenic animal. As used herein, an "homologous recombinant
animal" is a non-human animal, preferably a mammal, more preferably
a mouse, in which an endogenous Chk1 gene has been altered by
homologous recombination between the endogenous gene and an
exogenous DNA molecule introduced into a cell of the animal, e.g.,
an embryonic cell of the animal, prior to development of the
animal.
[0120] A transgenic animal can be created by introducing
Chk1-encoding nucleic acid into the male pronuclei of a fertilized
oocyte, e.g., by microinjection, retroviral infection, and allowing
the oocyte to develop in a pseudopregnant female foster animal. The
Chk1 cDNA sequence, e.g., that of SEQ ID NO:1 or SEQ ID NO:3 can be
introduced as a transgene into the genome of a non-human animal.
Alternatively, a nonhuman homologue of the human Chk1 gene, such as
a mouse Chk1 gene, can be isolated based on hybridization to the
human Chk1 cDNA and used as a transgene. Intronic sequences and
polyadenylation signals can also be included in the transgene to
increase the efficiency of expression of the transgene. A
tissue-specific regulatory sequence(s) can be operably linked to
the Chk1 transgene to direct expression of Chk1 protein to
particular cells. Methods for generating transgenic animals via
embryo manipulation and microinjection, particularly animals such
as mice, have become conventional in the art and are described, for
example, in U.S. Pat. Nos. 4,736,866 and 4,870,009, U.S. Pat. No.
4,873,191 and in Hogan, Manipulating the Mouse Embryo, (Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). Similar
methods are used for production of other transgenic animals. A
transgenic founder animal can be identified based upon the presence
of the Chk1 transgene in its genome and/or expression of Chk1 mRNA
in tissues or cells of the animals. A transgenic founder animal can
then be used to breed additional animals carrying the transgene.
Moreover, transgenic animals carrying a transgene encoding Chk1 can
further be bred to other transgenic animals carrying other
transgenes.
[0121] To create an homologous recombinant animal, a vector is
prepared which contains at least a portion of a Chk1 gene (e.g., a
human or a non-human homolog of the Chk1 gene, e.g., a murine Chk1
gene) into which a deletion, addition or substitution has been
introduced to thereby alter, e.g., functionally disrupt, the Chk1
gene. In a preferred embodiment, the vector is designed such that,
upon homologous recombination, the endogenous Chk1 gene is
functionally disrupted (i.e., no longer encodes a functional
protein; also referred to as a "knock out" vector). Alternatively,
the vector can be designed such that, upon homologous
recombination, the endogenous Chk1 gene is mutated or otherwise
altered but still encodes functional protein (e.g., the upstream
regulatory region can be altered to thereby alter the expression of
the endogenous Chk1 protein). In the homologous recombination
vector, the altered portion of the Chk1 gene is flanked at its 5'
and 3' ends by additional nucleic acid of the Chk1 gene to allow
for homologous recombination to occur between the exogenous Chk1
gene carried by the vector and an endogenous Chk1 gene in an
embryonic stem cell. The additional flanking Chk1 nucleic acid is
of sufficient length for successful homologous recombination with
the endogenous gene. Typically, several kilobases of flanking DNA
(both at the 5' and 3' ends) are included in the vector (see e.g.,
Thomas and Capecchi (1987) Cell 51:503 for a description of
homologous recombination vectors). The vector is introduced into an
embryonic stem cell line (e.g., by electroporation) and cells in
which the introduced Chk1 gene has homologously recombined with the
endogenous Chk1 gene are selected (see e.g., Li et al. (1992) Cell
69:915). The selected cells are then injected into a blastocyst of
an animal (e.g., a mouse) to form aggregation chimeras (see, e.g.,
Bradley in Teratocarcinomas and Embryonic Stem Cells: A Practical
Approach, Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A
chimeric embryo can then be implanted into a suitable
pseudopregnant female foster animal and the embryo brought to term.
Progeny harboring the homologously recombined DNA in their germ
cells can be used to breed animals in which all cells of the animal
contain the homologously recombined DNA by germline transmission of
the transgene. Methods for constructing homologous recombination
vectors and homologous recombinant animals are described further in
Bradley (1991) Current Opinion in Bio/Technology 2:823-829 and in
PCT Publication Nos. WO 90/11354, WO 91/01140, WO 92/0968, and WO
93/04169.
[0122] Transgenic non-human animals can be produced which contain
selected systems which allow for regulated expression of the
transgene. One example of such a system is the cre/loxP recombinase
system of bacteriophage P1. For a description of the cre/loxP
recombinase system, see, e.g., Lakso et al. (1992) Proc. Natl.
Acad. Sci. USA 89:6232-6236. Another example of a recombinase
system is the FLP recombinase system of Saccharomyces cerevisiae
(O'Gorman et al. (1991) Science 251:1351-1355. If a cre/loxP
recombinase system is used to regulate expression of the transgene,
animals containing transgenes encoding both the Cre recombinase and
a selected protein are required. Such animals can be provided
through the construction of "double" transgenic animals, e.g., by
mating two transgenic animals, one containing a transgene encoding
a selected protein and the other containing a transgene encoding a
recombinase.
[0123] Clones of the non-human transgenic animals described herein
can also be produced according to the methods described in Wilmut
et al. (1997) Nature 385:810-813 and PCT Publication Nos. WO
97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell,
from the transgenic animal can be isolated and induced to exit the
growth cycle and enter G.sub.o phase. The quiescent cell can then
be fused, e.g., through the use of electrical pulses, to an
enucleated oocyte from an animal of the same species from which the
quiescent cell is isolated. The reconstructed oocyte is then
cultured such that it develops to morula or blastocyte and then
transferred to pseudopregnant female foster animal. The offspring
borne of this female foster animal will be a clone of the animal
from which the cell, e.g., the somatic cell, is isolated.
[0124] IV. Pharmaceutical Compositions
[0125] Chk1 proteins, and anti-Chk1 antibodies, and modulators of
Chk1 expression or activity (also referred to herein as "active
compounds") can be incorporated into pharmaceutical compositions
suitable for administration. Such compositions typically comprise
the nucleic acid molecule, protein, or antibody and a
pharmaceutically acceptable carrier. As used herein the language
"pharmaceutically acceptable carrier" is intended to include any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the
like, compatible with pharmaceutical administration. The use of
such media and agents for pharmaceutically active substances is
well known in the art. Except insofar as any conventional media or
agent is incompatible with the active compound, use thereof in the
compositions is contemplated. Supplementary active compounds can
also be incorporated into the compositions.
[0126] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include parenteral, e.g.,
intravenous, intradermal, subcutaneous, oral (e.g., inhalation),
transdermal (topical), transmucosal, and rectal administration.
Solutions or suspensions used for parenteral, intradermal, or
subcutaneous application can include the following components: a
sterile diluent such as water for injection, saline solution, fixed
oils, polyethylene glycols, glycerine, propylene glycol or other
synthetic solvents; antibacterial agents such as benzyl alcohol or
methyl parabens; antioxidants such as ascorbic acid or sodium
bisulfite; chelating agents such as ethylenediaminetetraacetic
acid; buffers such as acetates, citrates or phosphates and agents
for the adjustment of tonicity such as sodium chloride or dextrose.
pH can be adjusted with acids or bases, such as hydrochloric acid
or sodium hydroxide. The parenteral preparation can be enclosed in
ampoules, disposable syringes or multiple dose vials made of glass
or plastic.
[0127] Pharmaceutical compositions suitable for injectable use
include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL (BASF; Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition must
be sterile and should be fluid to the extent that easy
syringability exists. It must be stable under the conditions of
manufacture and storage and must be preserved against the
contaminating action of microorganisms such as bacteria and fungi.
The carrier can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, and liquid polyetheylene glycol, and the like), and
suitable mixtures thereof. The proper fluidity can be maintained,
for example, by the use of a coating such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action of
microorganisms can be achieved by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars,
polyalcohols such as mannitol, sorbitol, sodium chloride in the
composition. Prolonged absorption of the injectable compositions
can be brought about by including in the composition an agent which
delays absorption, for example, aluminum monostearate and
gelatin.
[0128] Sterile injectable solutions can be prepared by
incorporating the active compound in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof.
[0129] Oral compositions generally include an inert diluent or an
edible carrier. They can be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Oral compositions can also be prepared using a fluid carrier for
use as a mouthwash, wherein the compound in the fluid carrier is
applied orally and swished and expectorated or swallowed.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition. The tablets,
pills, capsules, troches and the like can contain any of the
following ingredients, or compounds of a similar nature: a binder
such as microcrystalline cellulose, gum tragacanth or gelatin; an
excipient such as starch or lactose, a disintegrating agent such as
alginic acid, Primogel, or corn starch; a lubricant such as
magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a sweetening agent such as sucrose or saccharin; or a
flavoring agent such as peppermint, methyl salicylate, or orange
flavoring. For administration by inhalation, the compounds are
delivered in the form of an aerosol spray from pressured container
or dispenser which contains a suitable propellant, e.g., a gas such
as carbon dioxide, or a nebulizer.
[0130] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the active
compounds are formulated into ointments, salves, gels, or creams as
generally known in the art.
[0131] The compounds can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0132] In one embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes
targeted to infected cells with monoclonal antibodies to viral
antigens) can also be used as pharmaceutically acceptable carriers.
These can be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0133] It is especially advantageous to formulate oral or
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the subject to be treated; each unit containing a
predetermined quantity of active compound calculated to produce the
desired therapeutic effect in association with the required
pharmaceutical carrier. The specification for the dosage unit forms
of the invention are dictated by and directly dependent on the
unique characteristics of the active compound and the particular
therapeutic effect to be achieved, and the limitations inherent in
the art of compounding such an active compound for the treatment of
individuals.
[0134] Chk1 nucleic acid molecules of the invention can be inserted
into vectors and used as gene therapy vectors. Gene therapy vectors
can be delivered to a subject by, for example, intravenous
injection, local administration (see U.S. Pat. No. 5,328,470) or by
stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl.
Acad. Sci. USA 91:3054-3057). The pharmaceutical preparation of the
gene therapy vector can include the gene therapy vector in an
acceptable diluent, or can comprise a slow release matrix in which
the gene delivery vehicle is imbedded. Alternatively, where the
complete gene delivery vector can be produced intact from
recombinant cells, e.g. retroviral vectors, the pharmaceutical
preparation can include one or more cells which produce the gene
delivery system.
[0135] The pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
[0136] V. Uses and Methods of the Invention
[0137] The Chk1 nucleic acid molecules, proteins, protein
homologues, and antibodies described herein can be used in
screening assays, predictive medicine (e.g., diagnostic assays,
prognostic assays, monitoring clinical trials, and
pharmacogenomics), and methods of treatment (e.g., therapeutic
treatment methods and prophylactic treatment methods).
[0138] A. Screening Assays
[0139] The invention provides a method (also referred to herein as
a "screening assay") for identifying modulators, i.e., candidate or
test compounds or agents (e.g., peptides, peptidomimetics, small
molecules or other drugs) which bind to Chk1 proteins or have a
stimulatory or inhibitory effect on, for example, Chk1 expression
or Chk1 activity. Such identified compounds may be useful for the
modulation of drug resistance. In one embodiment, the invention
provides assays for screening candidate or test compounds which
bind to or modulate the activity of a Chk1 protein or polypeptide
or biologically active portion thereof. The test compounds of the
present invention can be obtained using any of the numerous
approaches in combinatorial library methods known in the art,
including: biological libraries; natural products libraries;
spatially addressable parallel solid phase or solution phase
libraries; synthetic library methods requiring deconvolution; the
`one-bead one-compound` library method; and synthetic library
methods using affinity chromatography selection. The biological
library approach is limited to peptide libraries, while the other
approaches are applicable to peptide, non-peptide oligomer or small
molecule libraries of compounds (Lam (1997) Anticancer Drug Des.
12:145).
[0140] Examples of methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. (1993) Proc.
Natl. Acad. Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl.
Acad. Sci. USA 91:11422; Zuckermann et al. (1994). J. Med. Chem.
37:2678; Cho et al. (1993) Science 261:1303; Carrell et al. (1994)
Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew.
Chem. Int. Ed. Engl. 33:2061; and Gallop et al. (1994) J. Med.
Chem. 37:1233.
[0141] Libraries of compounds may be presented in solution (e.g.,
Houghten (1992) Bio/Techniques 13:412-421), or on beads (Lam (1991)
Nature 354:82-84), chips (Fodor (1993) Nature 364:555-556),
bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos.
5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al. (1992)
Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and
Smith (1990) Science 249:386-390; Devlin (1990) Science
249:404-406; Cwirla et al. (1990) Proc. Natl. Acad. Sci.
87:6378-6382; and Felici (1991) J. Mol. Biol. 222:301-310).
[0142] In one embodiment, an assay is a cell-based assay in which a
cell which expresses a Chk1 protein, or a biologically active
portion thereof, is contacted with a test compound and the ability
of the test compound to bind to a Chk1 protein determined. The
cell, for example, can be a yeast cell or a cell of mammalian
origin. Determining the ability of the test compound to bind to the
Chk1 protein can be accomplished, for example, by coupling the test
compound with a radioisotope or enzymatic label such that binding
of the test compound to the Chk1 protein or biologically active
portion thereof can be determined by detecting the labeled compound
in a complex. For example, test compounds can be labeled with
.sup.125I, .sup.35S, .sup.14C, or .sup.3H, either directly or
indirectly, and the radioisotope detected by direct counting of
radioemmission or by scintillation counting. Alternatively, test
compounds can be enzymatically labeled with, for example,
horseradish peroxidase, alkaline phosphatase, or luciferase, and
the enzymatic label detected by determination of conversion of an
appropriate substrate to product. In a preferred embodiment, the
assay comprises contacting a cell which expresses a Chk1 protein,
or a biologically active portion thereof, with a known compound
which binds Chk1 to form an assay mixture, contacting the assay
mixture with a test compound, and determining the ability of the
test compound to interact with a Chk1 protein, wherein determining
the ability of the test compound to interact with a Chk1 protein
comprises determining the ability of the test compound to
preferentially bind to Chk1 or a biologically active portion
thereof as compared to the known compound.
[0143] In another embodiment, an assay is a cell-based assay
comprising contacting a cell expressing a Chk1 protein, or a
biologically active portion thereof, with a test compound and
determining the ability of the test compound to modulate (e.g.,
stimulate or inhibit) the activity of the Chk1 protein or
biologically active portion thereof. Determining the ability of the
test compound to modulate the activity of Chk1 or a biologically
active portion thereof can be accomplished, for example, by
determining the ability of the Chk1 protein to bind to or interact
with a Chk1 target molecule. As used herein, a "target molecule" is
a molecule with which a Chk1 protein binds or interacts in nature,
for example, a molecule in the nucleus or cytoplasm of a cell which
expresses a Chk1 protein. A Chk1 target molecule can be a non-Chk1
molecule or a Chk1 protein or polypeptide. The target, for example,
can be a second intracellular protein which has catalytic activity,
a protein which naturally binds to Chk1, or a protein which
facilitates the association of DNA with Chk1.
[0144] Determining the ability of the Chk1 protein to bind to or
interact with a Chk1 target molecule can be accomplished by one of
the methods described above for determining direct binding. In a
preferred embodiment, determining the ability of the Chk1 protein
to bind to or interact with a Chk1 target molecule can be
accomplished by determining the activity of the target molecule or
detecting a cellular response, for example, cell survival or cell
proliferation in the presence of a chemotherapeutic drug.
[0145] In yet another embodiment, an assay of the present invention
is a cell-free assay comprising contacting a Chk1 protein or
biologically active portion thereof with a test compound and
determining the ability of the test compound to bind to the Chk1
protein or biologically active portion thereof. Binding of the test
compound to the Chk1 protein can be determined either directly or
indirectly as described above. In a preferred embodiment, the assay
includes contacting the Chk1 protein or biologically active portion
thereof with a known compound which binds Chk1 to form an assay
mixture, contacting the assay mixture with a test compound, and
determining the ability of the test compound to interact with a
Chk1 protein, wherein determining the ability of the test compound
to interact with a Chk1 protein comprises determining the ability
of the test compound to preferentially bind to Chk1 or biologically
active portion thereof as compared to the known compound.
[0146] In another embodiment, an assay is a cell-free assay
comprising contacting Chk1 protein or biologically active portion
thereof with a test compound and determining the ability of the
test compound to modulate (e.g., stimulate or inhibit) the activity
of the Chk1 protein or biologically active portion thereof.
Determining the ability of the test compound to modulate the
activity of Chk1 can be accomplished, for example, by determining
the ability of the Chk1 protein to bind to a Chk1 target molecule
by one of the methods described above for determining direct
binding. In an alternative embodiment, determining the ability of
the test compound to modulate the activity of Chk1 can be
accomplished by determining the ability of the Chk1 protein further
modulate a Chk1 target molecule. For example, the
catalytic/enzymatic activity of the target molecule on an
appropriate substrate can be determined as previously
described.
[0147] In yet another embodiment, the cell-free assay comprises
contacting the Chk1 protein or biologically active portion thereof
with a known compound which binds Chk1 to form an assay mixture,
contacting the assay mixture with a test compound, and determining
the ability of the test compound to interact with a Chk1 protein,
wherein determining the ability of the test compound to interact
with a Chk1 protein comprises determining the ability of the Chk1
protein to preferentially bind to or modulate the activity of a
Chk1 target molecule.
[0148] The cell-free assays of the present invention are amenable
to use of both native and variant forms (e.g., peptide fragments
and fusion proteins) of Chk1. In the case of cell-free assays
comprising a hydrophobic form of Chk1, it may be desirable to
utilize a solubilizing agent such that the hydrophobic form of Chk1
is maintained in solution. Examples of such solubilizing agents
include non-ionic detergents such as n-octylglucoside,
n-dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide,
decanoyl-N-methylglucamide, Triton.RTM. X-100, Triton.RTM. X-114,
Thesit.RTM., Isotridecypoly(ethylene glycol ether)n,
3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),
3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane
sulfonate (CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane
sulfonate.
[0149] In more than one embodiment of the above assay methods of
the present invention, it may be desirable to immobilize either
Chk1 or its target molecule to facilitate separation of complexed
from uncomplexed forms of one or both of the proteins, as well as
to accommodate automation of the assay. Binding of a test compound
to Chk1, or interaction of Chk1 with a target molecule in the
presence and absence of a candidate compound, can be accomplished
in any vessel suitable for containing the reactants. Examples of
such vessels include microtitre plates, test tubes, and
micro-centrifuge tubes. In one embodiment, a fusion protein can be
provided which adds a domain that allows one or both of the
proteins to be bound to a matrix. For example,
glutathione-S-transferase/Chk1 fusion proteins or
glutathione-S-transfera- se/target fusion proteins can be adsorbed
onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.)
or glutathione derivatized microtitre plates, which are then
combined with the test compound or the test compound and either the
non-adsorbed target protein or Chk1 protein, and the mixture
incubated under conditions conducive to complex formation (e.g., at
physiological conditions for salt and pH). Following incubation,
the beads or microtitre plate wells are washed to remove any
unbound components, the matrix immobilized in the case of beads,
complex determined either directly or indirectly, for example, as
described above. Alternatively, the complexes can be dissociated
from the matrix, and the level of Chk1 binding or activity
determined using standard techniques.
[0150] Other techniques for immobilizing proteins on matrices can
also be used in the screening assays of the invention. For example,
either Chk1 or its target molecule can be immobilized utilizing
conjugation of biotin and streptavidin. Biotinylated Chk1 or target
molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)
using techniques well known in the art (e.g., biotinylation kit,
Pierce Chemicals; Rockford, Ill.), and immobilized in the wells of
streptavidin-coated 96 well plates (Pierce Chemical).
Alternatively, antibodies reactive with Chk1 or target molecules
but which do not interfere with binding of the Chk1 protein to its
target molecule can be derivatized to the wells of the plate, and
unbound target or Chk1 trapped in the wells by antibody
conjugation. Methods for detecting such complexes, in addition to
those described above for the GST-immobilized complexes, include
immunodetection of complexes using antibodies reactive with the
Chk1 or target molecule, as well as enzyme-linked assays which rely
on detecting an enzymatic activity associated with the Chk1 or
target molecule.
[0151] In another embodiment, modulators of Chk1 expression are
identified in a method in which a cell is contacted with a
candidate compound and the expression of Chk1 (mRNA or protein, or
the copy number of the Chk1 gene) in the cell is determined. The
level of expression of Chk1 in the presence of the candidate
compound is compared to the level of expression of Chk1 in the
absence of the candidate compound. The candidate compound can then
be identified as a modulator of Chk1 expression based on this
comparison. For example, when expression of Chk1 mRNA or protein is
greater (statistically significantly greater) in the presence of
the candidate compound than in its absence, the candidate compound
is identified as a stimulator of Chk1 mRNA or protein expression.
Alternatively, when expression of Chk1 mRNA or protein is less
(statistically significantly less) in the presence of the candidate
compound than in its absence, the candidate compound is identified
as an inhibitor of Chk1 mRNA or protein expression. The level of
Chk1 mRNA or protein expression in the cells, or the number of Chk1
gene copies per cell can be determined by methods described herein
for detecting Chk1 genomic DNA, mRNA, or protein.
[0152] Chk1 proteins can be used as "bait proteins" in a two-hybrid
assay or three hybrid assay (see, e.g., U.S. Pat. No. 5,283,317;
Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol.
Chem. 268:12046-12054; Bartel et al. (1993) Bio/Techniques
14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696; and
WO94/10300), to identify other proteins, which bind to or interact
with Chk1 ("Chk1-binding proteins" or "Chk1-bp") and modulate Chk1
activity.
[0153] The two-hybrid system is based on the modular nature of most
transcription factors, which consist of separable DNA-binding and
activation domains. Briefly, the assay utilizes two different DNA
constructs. In one construct, the gene that codes for Chk1 is fused
to a gene encoding the DNA binding domain of a known transcription
factor (e.g., GAL-4). In the other construct, a DNA sequence, from
a library of DNA sequences, that encodes an unidentified protein
("prey" or "sample") is fused to a gene that codes for the
activation domain of the known transcription factor. If the "bait"
and the "prey" proteins are able to interact, in vivo, forming an
Chk1-dependent complex, the DNA-binding and activation domains of
the transcription factor are brought into close proximity. This
proximity allows transcription of a reporter gene (e.g., LacZ)
which is operably linked to a transcriptional regulatory site
responsive to the transcription factor. Expression of the reporter
gene can be detected and cell colonies containing the functional
transcription factor can be isolated and used to obtain the cloned
gene which encodes the protein which interacts with Chk1.
[0154] This invention further pertains to novel agents identified
by the above-described screening assays and uses thereof for
treatments as described herein.
[0155] B. Predictive Medicine
[0156] The present invention also pertains to the field of
predictive medicine in which diagnostic assays, prognostic assays,
pharmacogenomics, and monitoring clinical trials are used for
prognostic (predictive) purposes to thereby treat an individual
prophylactically. Accordingly, one aspect of the present invention
relates to diagnostic assays for determining Chk1 protein and/or
nucleic acid expression as well as Chk1 activity, in the context of
a biological sample (e.g., blood, serum, cells, tissue) to thereby
determine whether an individual is afflicted with a disease or
disorder, or is at risk of developing a disorder, associated with
aberrant Chk1 expression or activity (e.g., altered drug
resistance). The invention also provides for prognostic (or
predictive) assays for determining whether an individual is at risk
of developing a disorder associated with Chk1 protein, nucleic acid
expression or activity (e.g., altered drug resistance). For
example, mutations in a Chk1 gene can be assayed in a biological
sample. Such assays can be used for prognostic or predictive
purpose to thereby prophylactically treat an individual prior to
the onset of a disorder characterized by or associated with Chk1
protein, nucleic acid expression or activity. For example, because
Chk1 is expressed at a higher level in drug resistant cells (e.g.,
the doxorubicin resistant cell lines A2780, U937, and HL60) than
non-drug resistant cell lines, higher than normal expression of
Chk1 can be used as an indicator of drug resistance.
[0157] Another aspect of the invention provides methods for
determining Chk1 protein, nucleic acid expression or Chk1 activity
in an individual to thereby select appropriate therapeutic or
prophylactic agents for that individual (referred to herein as
"pharmacogenomics").
[0158] Pharmacogenomics allows for the selection of agents (e.g.,
drugs) for therapeutic or prophylactic treatment of an individual
based on the genotype of the individual (e.g., the genotype of the
individual examined to determine the ability of the individual to
respond to a particular agent).
[0159] Yet another aspect of the invention pertains to monitoring
the influence of agents (e.g., drugs or other compounds) on the
expression or activity of Chk1 in clinical trials.
[0160] These and other agents are described in further detail in
the following sections.
[0161] 1. Diagnostic Assays
[0162] The invention provides a method of assessing expression,
especially undesirable expression, of a cellular Chk1 gene.
Undesirable (e.g., excessive) expression may indicate the presence,
persistence or reappearance of drug-resistant (e.g.,
vinblastin-resistant) tumor cells in an individual's tissue. More
generally, aberrant expression may indicate the occurrence of a
deleterious or disease-associated phenotype contributed to by
Chk1.
[0163] An exemplary method for detecting the presence or absence of
Chk1 in a biological sample involves obtaining a biological sample
(preferably from a body site implicated in a possible diagnosis of
diseased or malignant tissue) from a test subject and contacting
the biological sample with a compound or an agent capable of
detecting Chk1 protein or nucleic acid (e.g., mRNA, genomic DNA)
that encodes Chk1 protein such that the presence of Chk1 is
detected in the biological sample. The presence and/or relative
abundance of Chk1 indicates aberrant or undesirable expression of a
cellular Chk1 gene, and correlates with the occurrence in situ of
cells having a drug-resistant phenotype.
[0164] A preferred agent for detecting Chk1 mRNA or genomic DNA is
a labeled nucleic acid probe capable of hybridizing to Chk1 MRNA or
genomic DNA. The nucleic acid probe can be, for example, a
full-length Chk1 nucleic acid, such as the nucleic acid of SEQ ID
NO: 1 or 3, or a portion thereof, such as an oligonucleotide of at
least 15, 30, 50, 100, 250 or 500 nucleotides in length and
sufficient to specifically hybridize under stringent conditions to
Chk1 mRNA or genomic DNA. Other suitable probes for use in the
diagnostic assays of the invention are described herein.
[0165] A preferred agent for detecting Chk1 protein is an antibody
capable of binding to Chk1 protein, preferably an antibody with a
detectable label. Antibodies can be polyclonal, or more preferably,
monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or
F(ab').sub.2) can be used. The term "labeled", with regard to the
probe or antibody, is intended to encompass direct labeling of the
probe or antibody by coupling (i.e., physically linking) a
detectable substance to the probe or antibody, as well as indirect
labeling of the probe or antibody by reactivity with another
reagent that is directly labeled. Examples of indirect labeling
include detection of a primary antibody using a fluorescently
labeled secondary antibody and end-labeling of a DNA probe with
biotin such that it can be detected with fluorescently labeled
streptavidin. The term "biological sample" is intended to include
tissues, cells and biological fluids isolated from a subject, as
well as tissues, cells and fluids present within a subject. That
is, the detection method of the invention can be used to detect
Chk1 mRNA, protein, or genomic DNA in a biological sample in vitro
as well as in vivo. For example, in vitro techniques for detection
of Chk1 mRNA include Northern hybridizations and in situ
hybridizations. In vitro techniques for detection of Chk1 protein
include enzyme linked immunosorbent assays (ELISAs), Western blots,
immunoprecipitations and immunofluorescence. In vitro techniques
for detection of Chk1 genomic DNA include Southern
hybridizations.
[0166] In one embodiment, the biological sample contains protein
molecules from the test subject. Alternatively, the biological
sample can contain mRNA molecules from the test subject or genomic
DNA molecules from the test subject. A preferred biological sample
is a peripheral blood leukocyte sample isolated by conventional
means from a subject.
[0167] In another embodiment, the methods further involve obtaining
a control biological sample from a control subject, contacting the
control sample with a compound or agent capable of detecting Chk1
protein, mRNA, or genomic DNA, such that the presence of Chk1
protein, mRNA or genomic DNA is detected in the biological sample,
and comparing the presence of Chk1 protein, mRNA or genomic DNA in
the control sample with the presence of Chk1 protein, mRNA or
genomic DNA in the test sample.
[0168] The invention also encompasses kits for detecting the
presence of Chk1 in a biological sample (a test sample). Such kits
can be used to determine if a subject is suffering from or is at
increased risk of developing a disorder associated with aberrant
expression of Chk1 (e.g., the presence of a drug resistance
cancer). For example, the kit can comprise a labeled compound or
agent capable of detecting Chk1 protein or mRNA in a biological
sample and means for determining the amount of Chk1 in the sample
(e.g., an anti-Chk1 antibody or an oligonucleotide probe which
binds to DNA encoding Chk1, e.g., SEQ ID NO:1 or SEQ ID NO:3). Kits
may also include instruction for observing that the tested subject
is suffering from or is at risk of developing a disorder associated
with aberrant expression of Chk1 if the amount of Chk1 protein or
mRNA is above or below a normal level.
[0169] For antibody-based kits, the kit may comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to Chk1 protein; and, optionally, (2) a second, different
antibody which binds to Chk1 protein or the first antibody and is
conjugated to a detectable agent.
[0170] For oligonucleotide-based kits, the kit may comprise, for
example: (1) a oligonucleotide, e.g., a detectably labelled
oligonucleotide, which hybridizes to a Chk1 nucleic acid sequence
or (2) a pair of primers useful for amplifying a Chk1 nucleic acid
molecule;
[0171] The kit may also comprise, e.g., a buffering agent, a
preservative, or a protein stabilizing agent. The kit may also
comprise components necessary for detecting the detectable agent
(e.g., an enzyme or a substrate). The kit may also contain a
control sample or a series of control samples which can be assayed
and compared to the test sample contained. Each component of the
kit is usually enclosed within an individual container and all of
the various containers are within a single package along with
instructions for observing whether the tested subject is suffering
from or is at risk of developing a disorder associated with
aberrant expression of Chk1.
[0172] 2. Prognostic Assays
[0173] The methods described herein can furthermore be utilized as
diagnostic or prognostic assays to identify subjects having or at
risk of developing a disease or disorder associated with aberrant
Chk1 expression or activity. For example, the assays described
herein, such as the preceding diagnostic assays or the following
assays, can be utilized to identify a subject having or at risk of
developing a disorder associated with aberrant Chk1 protein,
nucleic acid expression or activity (eg., the presence of drug
resistant tumor cells). Alternatively, the prognostic assays can be
utilized to identify a subject having or at risk for developing
such a disease or disorder. Thus, the present invention provides a
method in which a test sample is obtained from a subject and Chk1
protein or nucleic acid (e.g., mRNA, genomic DNA) is detected,
wherein the presence or relative quantity of Chk1 protein or
nucleic acid is diagnostic for a subject having or at risk of
developing a disease or disorder associated with aberrant Chk1
expression or activity. As used herein, a "test sample" refers to a
biological sample obtained from a subject of interest. For example,
a test sample can be a biological fluid (e.g., serum), cell sample,
or tissue.
[0174] Furthermore, the prognostic assays described herein can be
used to determine whether a subject can be administered an agent
(e.g., an agonist, antagonist, peptidomimetic, protein, peptide,
nucleic acid, small molecule, or other drug candidate) to treat a
disease or disorder associated with aberrant Chk1 expression or
activity. Thus, if increased Chk1 expression is a cause of
increased drug resistance, such methods can be used to determine
whether a subject can be effectively treated with a specific agent
or class of agents (e.g., agents of a type which decrease Chk1
activity). Thus, the present invention provides methods for
determining whether a subject can be effectively treated with an
agent for a disorder associated with aberrant Chk1 expression or
activity in which a test sample is obtained and Chk1 protein or
nucleic acid is detected (e.g., wherein the presence or relative
quantity of Chk1 protein or nucleic acid is diagnostic for a
subject that can be administered the agent to treat a disorder
associated with aberrant Chk1 expression or activity). In some
embodiments, the foregoing methods provide information useful in
prognostication, staging and management of malignancies (tumors)
that are characterized by altered expression of Chk1 and thus by a
drug-resistance phenotype. The information more specifically
assists the clinician in designing chemotherapeutic or other
treatment regimes to eradicate such malignancies from the body of
an afflicted subject.
[0175] The methods of the invention can also be used to detect
genetic lesions (e.g., mutations or amplifications) in a Chk1 gene,
thereby determining if a subject with the altered gene is at risk
for a disorder characterized by aberrant cell proliferation and/or
differentiation. For example, genetic mutations, whether of
germline or somatic origin, may indicate whether the process of
developing drug resistance has been initiated or is likely to arise
in the tested cells. In preferred embodiments, the methods include
detecting, in a sample of cells from the subject, the presence or
absence of a genetic lesion characterized by at least one of an
alteration affecting the integrity of a gene encoding a
Chk1-protein, the mis-expression of the Chk1 gene, or the
amplification of a Chk1 gene. Preferably the sample of cells is
obtained from a body tissue suspected of comprising transformed
cells (e.g., cancer cells). Thus, the present method provides
information relevant to diagnosis of the presence of a tumor.
[0176] Genetic lesions can be detected, for example, by
ascertaining the existence of at least one of 1) a deletion of one
or more nucleotides from a Chk1 gene; 2) an addition of one or more
nucleotides to a Chk1 gene; 3) a substitution of one or more
nucleotides of a Chk1 gene, 4) a chromosomal rearrangement of a
Chk1 gene; 5) an alteration in the level of a messenger RNA
transcript of a Chk1 gene, 6) aberrant modification of a Chk1 gene,
such as of the methylation pattern of the genomic DNA, 7) the
presence of a non-wild type splicing pattern of a messenger RNA
transcript of a Chk1 gene, 8) a non-wild type level of a
Chk1-protein, 9) allelic loss of a Chk1 gene, 10) amplification of
a Chk1 gene, and 11) inappropriate post-translational modification
of a Chk1-protein. As described herein, there are a large number of
assay techniques known in the art which can be used for detecting
lesions in a Chk1 gene. A preferred biological sample is a biopsy
sample of tissue suspected of comprising transformed cells isolated
by conventional means from a subject.
[0177] In certain embodiments, detection of the lesion involves the
use of a probe/primer in a polymerase chain reaction (PCR) (see,
e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR
or RACE PCR, or, alternatively, in a ligation chain reaction (LCR)
(see, e.g., Landegran et al. (1988) Science 241:1077-1080; and
Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91:360-364), the
latter of which can be particularly useful for detecting point
mutations in the Chk1 gene (see Abravaya et al. (1995) Nucleic
Acids Res. 23:675-682). This method can include the steps of
collecting a sample of cells from a patient, isolating nucleic acid
(e.g., genomic, mRNA or both) from the cells of the sample,
contacting the nucleic acid sample with one or more primers which
specifically hybridize to a Chk1 gene under conditions such that
hybridization and amplification of the Chk1-gene (if present)
occurs, and detecting the presence or absence of an amplification
product, or detecting the size of the amplification product and
comparing the length to a control sample. It is anticipated that
PCR and/or LCR may be desirable to use as a preliminary
amplification step in conjunction with any of the techniques used
for detecting mutations described herein.
[0178] Alternative amplification methods include: self-sustained
sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci.
USA 87:1874-1878), transcriptional amplification system (Kwoh, et
al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta
Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), or any
other nucleic acid amplification method, followed by the detection
of the amplified molecules using techniques well known to those of
skill in the art. These detection schemes are especially useful for
the detection of nucleic acid molecules if such molecules are
present in very low numbers.
[0179] In an alternative embodiment, mutations in a Chk1 gene from
a sample cell can be identified by alterations in restriction
enzyme cleavage patterns. For example, sample and control DNA is
isolated, amplified (optionally), digested with one or more
restriction endonucleases, and fragment length sizes are determined
by gel electrophoresis and compared. Differences in fragment length
sizes between sample and control DNA indicates mutations in the
sample DNA. Moreover, the use of sequence specific ribozymes (see,
e.g., U.S. Pat. No. 5,498,531) can be used to score for the
presence of specific mutations by development or loss of a ribozyme
cleavage site.
[0180] In other embodiments, genetic mutations in Chk1 can be
identified by hybridizing a sample and control nucleic acids, e.g.,
DNA or RNA, to high density arrays containing hundreds or thousands
of oligonucleotides probes (Cronin et al. (1996) Human Mutation
7:244-255; Kozal et al. (1996) Nature Medicine 2:753-759). For
example, genetic mutations in Chk1 can be identified in
two-dimensional arrays containing light-generated DNA probes as
described in Cronin et al. supra. Briefly, a first hybridization
array of probes can be used to scan through long stretches of DNA
in a sample and control to identify base changes between the
sequences by making linear arrays of sequential overlapping probes.
This step allows the identification of point mutations. This step
is followed by a second hybridization array that allows the
characterization of specific mutations by using smaller,
specialized probe arrays complementary to all variants or mutations
detected. Each mutation array is composed of parallel probe sets,
one complementary to the wild-type gene and the other complementary
to the mutant gene.
[0181] In yet another embodiment, any of a variety of sequencing
reactions known in the art can be used to directly sequence the
Chk1 gene and detect mutations by comparing the sequence of the
sample Chk1 with the corresponding wild-type (control) sequence.
Additionally, sequencing of the DNA flanking the Chk1 can be used
to determine if the Chk1 gene has been amplified. Examples of
sequencing reactions include those based on techniques developed by
Maxim and Gilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or
Sanger ((1977) Proc. Natl. Acad. Sci. USA 74:5463). It is also
contemplated that any of a variety of automated sequencing
procedures can be utilized when performing the diagnostic assays
((1995) Bio/Techniques 19:448), including sequencing by mass
spectrometry (see, e.g., PCT Publication No. WO 94/16101; Cohen et
al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993)
Appl. Biochem. Biotechnol. 38:147-159).
[0182] Other methods for detecting mutations in the Chk1 gene
include methods in which protection from cleavage agents is used to
detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers
et al. (1985) Science 230:1242). In general, the art technique of
"mismatch cleavage" starts by providing heteroduplexes of formed by
hybridizing (labeled) RNA or DNA containing the wild-type Chk1
sequence with potentially mutant RNA or DNA obtained from a tissue
sample. The double-stranded duplexes are treated with an agent
which cleaves single-stranded regions of the duplex such as which
will exist due to basepair mismatches between the control and
sample strands. For instance, RNA/DNA duplexes can be treated with
RNase and DNA/DNA hybrids treated with S1 nuclease to enzymatically
digesting the mismatched regions. In other embodiments, either
DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or
osmium tetroxide and with piperidine in order to digest mismatched
regions. After digestion of the mismatched regions, the resulting
material is then separated by size on denaturing polyacrylamide
gels to determine the site of mutation. See, e.g., Cotton et al
(1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et al (1992)
Methods Enzymol. 217:286-295. In a preferred embodiment, the
control DNA or RNA can be labeled for detection.
[0183] In still another embodiment, the mismatch cleavage reaction
employs one or more proteins that recognize mismatched base pairs
in double-stranded DNA (so called "DNA mismatch repair" enzymes) in
defined systems for detecting and mapping point mutations in Chk1
cDNAs obtained from samples of cells. For example, the mutY enzyme
of E. coli cleaves A at G/A mismatches and the thymidine DNA
glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al.
(1994) Carcinogenesis 15:1657-1662). According to an exemplary
embodiment, a probe based on a Chk1 sequence, e.g., a wild-type
Chk1 sequence, is hybridized to a cDNA or other DNA product from a
test cell(s). The duplex is treated with a DNA mismatch repair
enzyme, and the cleavage products, if any, can be detected from
electrophoresis protocols or the like. See, e.g., U.S. Pat. No.
5,459,039.
[0184] In other embodiments, alterations in electrophoretic
mobility will be used to identify mutations in Chk1 genes. For
example, single strand conformation polymorphism (SSCP) may be used
to detect differences in electrophoretic mobility between mutant
and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad.
Sci USA: 86:2766, see also Cotton (1993) Mutat. Res. 285:125-144;
and Hayashi (1992) Genet Anal Tech Appl 9:73-79). Single-stranded
DNA fragments of sample and control Chk1 nucleic acids will be
denatured and allowed to renature. The secondary structure of
single-stranded nucleic acids varies according to sequence, the
resulting alteration in electrophoretic mobility enables the
detection of even a single base change. The DNA fragments may be
labeled or detected with labeled probes. The sensitivity of the
assay may be enhanced by using RNA (rather than DNA), in which the
secondary structure is more sensitive to a change in sequence. In a
preferred embodiment, the subject method utilizes heteroduplex
analysis to separate double stranded heteroduplex molecules on the
basis of changes in electrophoretic mobility (Keen et al. (1991)
Trends Genet 7:5).
[0185] In yet another embodiment, the movement of mutant or
wild-type fragments in polyacrylamide gels containing a gradient of
denaturant is assayed using denaturing gradient gel electrophoresis
(DGGE) (Myers et al. (1985) Nature 313:495). When DGGE is used as
the method of analysis, DNA will be modified to insure that it does
not completely denature, for example by adding a GC clamp of
approximately 40 bp of high-melting GC-rich DNA by PCR. In a
further embodiment, a temperature gradient is used in place of a
denaturing gradient to identify differences in the mobility of
control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem
265:12753).
[0186] Examples of other techniques for detecting point mutations
include, but are not limited to, selective oligonucleotide
hybridization, selective amplification, or selective primer
extension. For example, oligonucleotide primers may be prepared in
which the known mutation is placed centrally and then hybridized to
target DNA under conditions which permit hybridization only if a
perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki
et al. (1989) Proc. Natl Acad. Sci USA 86:6230). Such allele
specific oligonucleotides are hybridized to PCR amplified target
DNA or a number of different mutations when the oligonucleotides
are attached to the hybridizing membrane and hybridized with
labeled target DNA.
[0187] Alternatively, allele specific amplification technology
which depends on selective PCR amplification may be used in
conjunction with the instant invention. Oligonucleotides used as
primers for specific amplification may carry the mutation of
interest in the center of the molecule (so that amplification
depends on differential hybridization) (Gibbs et al. (1989) Nucleic
Acids Res. 17:2437-2448) or at the extreme 3' end of one primer
where, under appropriate conditions, mismatch can prevent, or
reduce polymerase extension (Prossner (1993) Tibtech 11:238). In
addition, it may be desirable to introduce a novel restriction site
in the region of the mutation to create cleavage-based detection
(Gasparini et al. (1992) Mol. Cell Probes 6:1). It is anticipated
that in certain embodiments amplification may also be performed
using Taq ligase for amplification (Barany (1991) Proc. Natl. Acad.
Sci USA 88:189). In such cases, ligation will occur only if there
is a perfect match at the 3' end of the 5' sequence making it
possible to detect the presence of a known mutation at a specific
site by looking for the presence or absence of amplification.
[0188] The methods described herein may be performed, for example,
by utilizing pre-packaged diagnostic kits comprising at least one
probe nucleic acid or antibody reagent described herein, which may
be conveniently used, e.g., in clinical settings to diagnose
patients exhibiting symptoms or family history of a disease or
illness involving a Chk1 gene.
[0189] Furthermore, any cell type or tissue, preferably biopsy
samples of tissue comprising or suspected of comprising transformed
cells, in which Chk1 is expressed may be utilized in the prognostic
assays described herein.
[0190] 3. Pharmacogenomics
[0191] Agents, or modulators which have a stimulatory or inhibitory
effect on Chk1 activity (e.g., Chk1 gene expression) as identified
by a screening assay described herein can be administered to
individuals to treat (prophylactically or therapeutically)
disorders (e.g., drug-resistance) associated with aberrant Chk1
activity. In conjunction with such treatment, the pharmacogenomics
(i.e., the study of the relationship between an individual's
genotype and that individual's response to a foreign compound or
drug) of the individual may be considered. Differences in
metabolism of therapeutics can lead to severe toxicity or
therapeutic failure by altering the relation between dose and blood
concentration of the pharmacologically active drug. Thus, the
pharmacogenomics of the individual permits the selection of
effective agents (e.g., drugs) for prophylactic or therapeutic
treatments based on a consideration of the individual's genotype.
Such pharmacogenomics can further be used to determine appropriate
dosages and therapeutic regimens. Accordingly, the activity of Chk1
protein, expression of Chk1 nucleic acid, or mutation content of
Chk1 genes in an individual can be determined to thereby select
appropriate agent(s) for therapeutic or prophylactic treatment of
the individual.
[0192] Pharmacogenomics deals with clinically significant
hereditary variations in the response to drugs due to altered drug
disposition and abnormal action in affected persons. See, e.g.,
Linder (1997) Clin. Chem. 43(2):254-266. In general, two types of
pharmacogenetic conditions can be differentiated. Genetic
conditions transmitted as a single factor altering the way drugs
act on the body (altered drug action) or genetic conditions
transmitted as single factors altering the way the body acts on
drugs (altered drug metabolism). These pharmacogenetic conditions
can occur either as rare defects or as polymorphisms. For example,
glucose-6-phosphate dehydrogenase deficiency (G6PD) is a common
inherited enzymopathy in which the main clinical complication is
haemolysis after ingestion of oxidant drugs (anti-malarials,
sulfonamides, analgesics, nitrofurans) and consumption of fava
beans.
[0193] As an illustrative embodiment, the activity of drug
metabolizing enzymes is a major determinant of both the intensity
and duration of drug action. The discovery of genetic polymorphisms
of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2)
and cytochrome P450 enzymes CYP2D6 and CYP2C19) has provided an
explanation as to why some patients do not obtain the expected drug
effects or show exaggerated drug response and serious toxicity
after taking the standard and safe dose of a drug. These
polymorphisms are expressed in two phenotypes in the population,
the extensive metabolizer (EM) and poor metabolizer (PM). The
prevalence of PM is different among different populations. For
example, the gene coding for CYP2D6 is highly polymorphic and
several mutations have been identified in PM, which all lead to the
absence of functional CYP2D6. Poor metabolizers of CYP2D6 and
CYP2C19 quite frequently experience exaggerated drug response and
side effects when they receive standard doses. If a metabolite is
the active therapeutic moiety, PM show no therapeutic response, as
demonstrated for the analgesic effect of codeine mediated by its
CYP2D6-formed metabolite morphine. The other extreme are the so
called ultra-rapid metabolizers who do not respond to standard
doses. Recently, the molecular basis of ultra-rapid metabolism has
been identified to be due to CYP2D6 gene amplification.
[0194] Thus, the activity of Chk1 protein, expression of Chk1
nucleic acid, or mutation content of Chk1 genes in an individual
can be determined to thereby select appropriate agent(s) for
therapeutic or prophylactic treatment of the individual. In
addition, pharmacogenetic studies can be used to apply genotyping
of polymorphic alleles encoding drug-metabolizing enzymes to the
identification of an individual's drug responsiveness phenotype.
This knowledge, when applied to dosing or drug selection, can avoid
adverse reactions or therapeutic failure and thus enhance
therapeutic or prophylactic efficiency when treating a subject with
a Chk1 modulator, such as a modulator identified by one of the
exemplary screening assays described herein.
[0195] 4. Monitoring of Effects During Clinical Trials
[0196] Monitoring the influence of agents (e.g., drugs, compounds)
on the expression or activity of Chk1 (e.g., the ability to
modulate the drug-resistant phenotype of a cell) can be applied not
only in basic drug screening, but also in clinical trials. For
example, the effectiveness of an agent determined by a screening
assay as described herein to decrease Chk1 gene expression, protein
levels, or downregulate Chk1 activity, can be monitored in clinical
trails of subjects exhibiting increased Chk1 gene expression,
protein levels, or upregulated Chk1 activity.
[0197] Alternatively, the effectiveness of an agent determined by a
screening assay to increase Chk1 gene expression, protein levels,
or upregulate Chk1 activity (e.g., to increase the drug resistance
of a non-cancerous cell), can be monitored in clinical trials of
compounds designed to increase Chk1 gene expression, protein
levels, or upregulate Chk1 activity. In such clinical trials, the
expression or activity of Chk1 and, preferably, other genes that
have been implicated in, for example, a cellular proliferation
disorder, can be used as a "read out" or markers of the drug
resistance of a particular cell.
[0198] For example, and not by way of limitation, genes, including
Chk1, that are modulated in cells by treatment with an agent (e.g.,
compound, drug or small molecule) which modulates Chk1 activity
(e.g., identified in a screening assay as described herein) can be
identified. Thus, to study the effect of agents on cellular
proliferation disorders, for example, in a clinical trial, cells
can be isolated and RNA prepared and analyzed for the levels of
expression of Chk1 and other genes implicated in the disorder. The
levels of gene expression (i.e., a gene expression pattern) can be
quantified by Northern blot analysis or RT-PCR, as described
herein, or alternatively by measuring the amount of protein
produced, by one of the methods as described herein, or by
measuring the levels of activity of Chk1 or other genes. In this
way, the gene expression pattern can serve as a marker, indicative
of the physiological response of the cells to the agent.
Accordingly, this response state may be determined before, and at
various points during, treatment of the individual with the
agent.
[0199] In a preferred embodiment, the present invention provides a
method for monitoring the effectiveness of treatment of a subject
with an agent (e.g., an agonist, antagonist, peptidomimetic,
protein, peptide, nucleic acid, small molecule, or other drug
candidate identified by the screening assays described herein)
comprising the steps of (i) obtaining a pre-administration sample
from a subject prior to administration of the agent; (ii) detecting
the level of expression of a Chk1 protein, mRNA, or genomic DNA in
the preadministration sample; (iii) obtaining one or more
post-administration samples from the subject; (iv) detecting the
level of expression or activity of the Chk1 protein, mRNA, or
genomic DNA in the post-administration samples; (v) comparing the
level of expression or activity of the Chk1 protein, mRNA, or
genomic DNA in the pre-administration sample with the Chk1 protein,
mRNA, or genomic DNA in the post administration sample or samples;
and (vi) altering the administration of the agent to the subject
accordingly. For example, increased administration of the agent may
be desirable to decrease the expression or activity of Chk1 to
higher levels than detected, i.e., to increase the effectiveness of
the agent.
[0200] C. Methods of Treatment
[0201] The present invention provides for both prophylactic and
therapeutic methods of treating a subject at risk of (or
susceptible to) a disorder or having a disorder associated with
aberrant Chk1 expression or activity. Such disorders include
cellular resistance to chemotherapeutic drugs.
[0202] 1. Prophylactic Methods
[0203] In one aspect, the invention provides a method for
preventing in a subject, a disease or condition associated with an
aberrant Chk1 expression or activity (e.g., the development of drug
resistance), by administering to the subject an agent which
modulates Chk1 expression or at least one Chk1 activity. Subjects
at risk for a condition which is caused or contributed to by
aberrant Chk1 expression or activity can be identified by, for
example, any or a combination of diagnostic or prognostic assays as
described herein. Administration of a prophylactic agent can occur
prior to the manifestation of symptoms characteristic of the Chk1
aberrancy, such that a disease or disorder is prevented or,
alternatively, delayed in its progression. For example,
administration of a prophylatic agent to a cancer patient may
prevent or delay the development of drug resistance in the
patient's cancer cells. Depending on the type of Chk1 aberrancy,
for example, a Chk1 agonist or Chk1 antagonist agent can be used
for treating the subject. The appropriate agent can be determined
based on screening assays described herein.
[0204] 2. Therapeutic Methods
[0205] Another aspect of the invention pertains to methods of
modulating Chk1 expression or activity for therapeutic purposes.
For example, the effectiveness of chemotherapy is "potentiated"
(enhanced) by restoring or improving vulnerability of the
transformed cells to the cytotoxic effects of a chemotherapeutic
drug that otherwise would be less effective by reducing the
expression of Chk1 in the cells. The modulatory method of the
invention involves contacting a cell with an agent that modulates
one or more of the activities of Chk1 protein activity associated
with the cell. An agent that modulates Chk1 protein activity can be
an agent as described herein, such as a nucleic acid or a protein,
a naturally-occurring cognate ligand of a Chk1 protein, a peptide,
a Chk1 peptidomimetic, or other small molecule. In one embodiment,
the agent stimulates one or more of the biological activities of
Chk1 protein. Examples of such stimulatory agents include active
Chk1 protein and a nucleic acid molecule encoding Chk1 that has
been introduced into the cell. In another embodiment, the agent
inhibits one or more of the biological activities of Chk1 protein.
Examples of such inhibitory agents include antisense Chk1 nucleic
acid molecules and anti-Chk1 antibodies. These modulatory methods
can be performed in vitro (e.g., by culturing the cell with the
agent) or, alternatively, in vivo (e.g, by administering the agent
to a subject). As such, the present invention provides methods of
treating an individual afflicted with a disease or disorder
characterized by aberrant expression or activity of a Chk1 protein
or nucleic acid molecule. In one embodiment, the method involves
administering an agent (e.g., an agent identified by a screening
assay described herein), or combination of agents that modulates
(e.g., upregulates or downregulates) Chk1 expression or activity.
In another embodiment, the method involves administering a Chk1
protein or nucleic acid molecule as therapy to compensate for
reduced or aberrant Chk1 expression or activity.
[0206] For example, in one embodiment, the method involves
administering the desired drug (e.g., cyclophosphamide) to an
individual afflicted with a drug-resistant cell population (a
tumor, e.g., a carcinoma, sarcoma, leukemia, lymphoma, or
lymphosarcoma), and coadministering an inhibitor of Chk1 expression
or activity. The administration and coadministration steps can be
carried out concurrently or in any order, and can be separated by a
time interval sufficient to allow uptake of either compound by the
cells to be eradicated. For example, an antisense pharmaceutical
composition (or a cocktail composition comprising an Chk1 antisense
oligonucleotide in combination with one or more other antisense
oligonucleotides) can be administered to the individual
sufficiently in advance of administration of the chemotherapeutic
drug to allow the antisense composition to permeate the
individual's tissues, especially tissue comprising the transformed
cells to be eradicated; to be internalized by transformed cells;
and to disrupt Chk1 gene expression and/or protein production.
[0207] Iinhibition of Chk1 activity is desirable in situations in
which Chk1 is abnormally upregulated and/or in which decreased Chk1
activity is likely to have a beneficial effect, e.g., in decreasing
the drug resistance of a cancer cell. Conversely, stimulation of
Chk1 activity is desirable in situations in which Chk1 is
abnormally downregulated and/or in which increased Chk1 activity is
likely to have a beneficial effect, e,g., in increasing the drug
resistance of a non-cancer cell.
[0208] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are hereby incorporated by
reference.
EXAMPLES
Example 1
Expression of Chk1 in Normal Cells and Cancer Cells
[0209] Nothern blot analysis was used to examine the expression of
Chk1 in various normal and cancerous tissues. This analysis
revealed that Chk1 is expressed at a considerably higher level in
breast carcinoma (eptithelial cells) than normal breast tissue. The
analysis also showed that Chk1 is expressed at a high level in
colon carcinoma (epithelial cells). In normal lung, Chk1 is
expressed at a lower level than in lung carcinoma, where it is
expressed at a high level in epithelial cells. The Northern
analysis also showed that Chk1 is expressed at a higher level in
prostate carcinoma (ductal epithelial cells) than in normal
prostate.
Example 2
Expression of Chk1 in Drug Resistant Cells Lines
[0210] A subtraction library was used to analyze differential
expression of genes in UCLA cells a resistant variants of UCLA
cells. This analysis revealed that Chk1 is expressed at a higher
level in the vinblastine resistant UCLA cells than in the
corresponding relatively vinblastine sensitive cells from which the
resistant cells were derived. Chk1 also proved to be upregulated in
a variety of relatively adromycin resistant cells lines compared to
the relatively adromycin resistant cells from which the cell lines
were derived.
Example 3
Preparation of Chk1 Proteins
[0211] Recombinant Chk1 can be produced in a variety of expression
systems. For example, the mature Chk1 peptide can be expressed as a
recombinant glutathione-S-transferase (GST) fusion protein in
insect cells and the fusion protein can be isolated and
characterized. For example, a gene encoding a GST-Chk1 fusion
protein can be created in the pGEX-2T vector (Promega). The
GST-Chk1 fusion gene can be removed from this vector and inserted
into the pFastBacl expression vector (Life Technologies, Inc.,
Bethesda, Md.). This vector permits expression of the fusion
protein in cultured insect cells (e.g., Sf9 cells). The cells
expressing the fusion protein are lysed and the fusion protein is
isolated using a Glutathione Sepharose 4B column (Pharmacia, Inc.
Piscataway, N.J.). After elution of the fusion protein from the
column, thrombin is used to cleave the GST polypeptide which is
then removed using a Glutathione Sepharose 4B column.
[0212] The Chk1 protein prepared as described above can be used to
generate antibodies directed against Chk1 and in in vitro screening
assays used to identify inhibitors of Chk1 activity.
[0213] Analysis of purified Chk1 prepared as described above
revealed that Chk1 is likely phosphorylated at one or more of the
following amino acid residues: Ser.sup.360, Ser.sup.376,
Ser.sup.406, and Thr.sup.403.
Example 4
Reduction in Chk1 Expression Using a Ribozyme
[0214] The following experiment indicates that 293 EBNA cells
transfected with a vector that expresses a hammerhead ribozyme
designed to selectively cleave Chk1 mRNA and thus reduce Chk1
expression are more sensitive to doxorubicin than control cells
that are not transfected with the ribozyme expression construct or
are transfected with a vector that expresses a mutant ribozyme.
[0215] In general, hammerhead ribozymes have the structure shown in
FIG. 3. The 5' to 3' strand is the RNA being cleaved, and the 3' to
5' strand is the ribozyme. The arrow indicates the location of the
cleavage.
[0216] Analysis of the predicted secondary structure of Chk1 mRNA
revealed several unpaired regions which might be cleaved by a
suitably designed hammerhead ribozyme. Several of these regions
were tested for accessibility using an RNaseH assay. Briefly, DNA
primers designed to basepair with various predicted unpaired
regions were incubated with in vitro transcribed, detectably
labeled Chk1 mRNA. The mRNA/primer mixture was exposed to RNAseH,
which cleaves the RNA strand of basepaired DNA/RNA hybrids. FIG. 4
depicts a predicted secondary structure of Chk1 mRNA with eight
potentially unpaired regions (2, 4, 6, 7, 11, 12, 13, and 14)
indicated. These regions include the one or more potential cleavage
sites (2: position 234; 4: position 282; 6: positions 309, 311, and
312; 7: position 333; 11: position 392; 12: position 411; 13:
positions 441 and 446; 14: positions 460 and 465). The RNAseH
analysis suggested that at least region 11 is unpaired. A triple
ribozyme designed to cleave this unpaired region was constructed
and inserted into and expression vector. A triple ribozyme is a
ribozyme which self-processes to release the ribozyme of interest.
An expression vector carrying the triple ribozyme construct
("Rz11") designed to cleave within region 11 at position 392 was
used to transiently transfect 293EBNA cells. As a control, 293EBNA
cells were also transfected with either an expression vector
lacking the triple ribozyme construct ("empty") or a an expression
vector carrying a mutant, inactive triple ribozyme construct
("Rz11M"). At 24 hr post-transfection the medium was changed. At 48
hr post-transfection the cultures were split and doxorubicin was
added to at concentrations up tp 2 micrograms/ml. Cytotoxicity
measurements were made at 72, 96, and 120 hr post-transfection
(corresponding to 24 hr, 48 hr, and 72 hr of drug treatment). The
transfected cells were then grown in the presence of various
concentrations of doxorubicin. As shown in FIG. 5, cells
transfected with the active triple ribozyme (Rz11) were more
sensitive to doxorubicin than the control cells (Rz11M and empty)
after 24 hr drug treatment. This result suggests that decreasing
the expression of Chk1 can lead to increased drug sensitivity.
[0217] Equivalents
[0218] 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.
Sequence CWU 0
0
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References