U.S. patent application number 12/903706 was filed with the patent office on 2011-03-03 for dna damaging agents in combination with tyrosine kinase inhibitors.
This patent application is currently assigned to ARCH DEVELOPMENT CORPORATION. Invention is credited to Donald KUFE, Ralph R. WEICHSELBAUM.
Application Number | 20110053240 12/903706 |
Document ID | / |
Family ID | 26887731 |
Filed Date | 2011-03-03 |
United States Patent
Application |
20110053240 |
Kind Code |
A1 |
KUFE; Donald ; et
al. |
March 3, 2011 |
DNA DAMAGING AGENTS IN COMBINATION WITH TYROSINE KINASE
INHIBITORS
Abstract
The present invention relates to the signalling pathways
connecting DNA damage, such as that induced by ionizing radiation
or alkylating agents, and phosphorylation by tyrosine kinases.
Inventors: |
KUFE; Donald; (Wellesley,
MA) ; WEICHSELBAUM; Ralph R.; (Chicago, IL) |
Assignee: |
ARCH DEVELOPMENT
CORPORATION
Chicago
IL
DANA-FARBER CANCER INSTITUTE
Boston
MA
|
Family ID: |
26887731 |
Appl. No.: |
12/903706 |
Filed: |
October 13, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11381311 |
May 2, 2006 |
7838512 |
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12903706 |
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10375684 |
Feb 24, 2003 |
7070968 |
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11381311 |
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08309315 |
Aug 19, 1994 |
6524832 |
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10375684 |
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08192107 |
Feb 4, 1994 |
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08309315 |
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Current U.S.
Class: |
435/173.1 ;
435/375 |
Current CPC
Class: |
A61K 31/40 20130101;
A61K 31/70 20130101; A61K 31/7072 20130101; A61K 31/70 20130101;
A61K 31/38 20130101; A61K 2300/00 20130101; A61K 31/41 20130101;
A61K 2300/00 20130101; A61K 31/35 20130101; A61K 31/704 20130101;
A61K 31/495 20130101; A61N 5/10 20130101; A61K 31/40 20130101; A61K
31/70 20130101; A61K 31/7048 20130101; A61K 41/0023 20130101; A61K
41/17 20200101; A61K 31/70 20130101; A61K 31/40 20130101; A61K
31/365 20130101; A61K 31/70 20130101 |
Class at
Publication: |
435/173.1 ;
435/375 |
International
Class: |
C12N 13/00 20060101
C12N013/00; C12N 5/00 20060101 C12N005/00 |
Goverment Interests
[0002] The U.S. Government owns rights in the present disclosure
pursuant to grant number CA19589 from the National Cancer
Institute.
Claims
1. A process of selectively activating the lyn family of Src-like
tyrosine kinases in a cell that expresses the lyn gene, the process
comprising exposing the cell to an effective activating dose of
ionizing radiation.
2. The process of claim 1, wherein the lyn family of Src-like
tyrosine kinases comprises p56/p53.sup.lyn.
3. A process of stimulating tyrosine phosphorylation of Src-like
tyrosine kinase substrates comprising exposing a cell that
expresses the lyn gene to an effective activating dose of ionizing
radiation.
4. The process of claim 3, wherein the substrate is phospholipase
C-.gamma.1, phospholipase C-.gamma.2, mitogen-activated protein
kinase GTPase activating protein, phosphatidylinositol 3-kinase,
enolase or p34.sup.cdc2.
5. A process of altering the response of a cell that expresses the
lyn gene to ionizing radiation comprising inhibiting the activity
of Src-like tyrosine kinase.
6. The process of claim 5, wherein the Src-like tyrosine kinase is
p56/p53.sup.lyn.
7.-21. (canceled)
Description
[0001] The present application is a Divisional of copending U.S.
application Ser. No. 11/381,311, filed May 2, 2006, which is a
Continuation of U.S. application Ser. No. 10/375,684, filed Feb.
24, 2003, now U.S. Pat. No. 7,070,968, which is a Continuation of
U.S. application Ser. No. 08/309,315, filed Sep. 19, 1994, now U.S.
Pat. No. 6,524,832, which is a Continuation-in-Part of U.S.
application Ser. No. 08/192,107, filed Feb. 4, 1994, which is
abandoned. The disclosures set forth in the referenced applications
are incorporated herein by reference in their entireties.
BACKGROUND
[0003] 1. Field of the Disclosure
[0004] The present disclosure relates generally to the field of
biochemical pathways. More particularly, it concerns the pathways
connecting DNA damage and phosphorylation by tyrosine kinases.
[0005] 2. Description of the Related Art
[0006] Current treatment methods for cancer, including radiation
therapy alone, surgery and chemotherapy, are known to have limited
effectiveness. Cancer mortality rates will therefore remain high
well into the 21st century. The rational development of new cancer
treatment methods will depend on an understanding of the biology of
the cancer cell at the molecular level. Certain cancer treatment
methods, including radiation therapy, involve damaging the DNA of
the cancer cell. The cellular response to DNA damage includes
activation of DNA repair, cell cycle arrest and lethality (Hall,
1988). The signaling events responsible for the regulation of these
events, however, remain unclear. Several checkpoints in cell cycle
progression control growth in response to diverse positive and
negative regulatory signals (Lau & Pardee, 1982). Ionizing
radiation, for example, slows growth by inducing delays in
G.sub.1/S and G.sub.2 phases of the cell cycle. The available
evidence suggests that G.sub.2 arrest in necessary for repair of
DNA damage before entry into mitosis (Steinman et al., 1991;
Weinert & Hartwell, 1988). Genetic studies in Saccharomyces
cerevisiae have demonstrated that the RAD9 protein controls G.sub.2
arrest induced by DNA damage (Schiestl et al., 1989; Murray, 1989).
Mutants of the rad9 locus are unable to delay entry into mitosis
following exposure to genotoxic agents and thereby replicate
damaged DNA. Although the mammalian homolog of rad9 remains
unidentified, other studies in various eukaryotic cells have
demonstrated that entry into mitosis is regulated by a 34 kD
serine/threonine protein kinase, designated p34.sup.cdc2 (Nurse,
1990; Pines & Hunter, 1989; Russell & Nurse, 1987).
[0007] Recent studies have shown that exposure of eukaryotic cells
to ionizing radiation is associated with induction of certain early
response genes that code for transcription factors. Members of the
jun/fos and early growth response (EGR) gene families are activated
by ionizing radiation (Sherman et al., 1990; Datta et al., 1992a).
Expression and DNA binding of the nuclear factor kB (NF-kB) are
also induced in irradiated cells (Brach et al., 1991; Uckun et al.,
1992a). Other studies have shown that levels of the tumor
suppressor p53 protein increase during X-ray-induced arrest of
cells in G1 phase (Kastan et al., 1991; 1992). The activation of
these transcription factors presumably represents transduction of
early nuclear signals to longer term changes in gene expression
that constitute the response to irradiation. Ionizing radiation
also induces protein kinase C (PKC) and protein tyrosine kinase
activities (Hallahan et al., 1990; Uckun et al., 1993). However,
the specific kinases responsible for these activities and their
substrates require further study.
[0008] Mitomycin C (MMC) is an antitumor antibiotic isolated from
Streptomyces caespitosus that covalently binds to DNA (Tomasz et
al., 1988). This agent induces both monofunctional and bifunctional
DNA lesions (Carrano et al., 1979). Other studies have demonstrated
that MMC stimulates the formation of hydroxyl radicals (Dusre et
al., 1989). Although the precise mechanism of action of this agent
is unclear, MMC-induced cytotoxicity has been attributed to DNA
alkylation and the formation of interstrand cross-links (Carrano et
al., 1979; Dusre et al., 1989; Tomasz et al., 1988). Treatment of
mammalian cells with MMC is associated with inhibition of DNA
synthesis and induction of sister-chromatid exchange (Carrano et
al., 1979). Previous work has demonstrated that MMC also enhances
transcription of HIV-1 and collagenase promoter constructs
transfected into HeLa cells (Stein et al., 1989). These studies
indicated that AP-1 is involved in MMC-induced activation of the
collagenase enhancer. However, little is known about the effects of
this agent on other signaling events.
[0009] Protein tyrosine phosphorylation contributes to the
regulation of cell growth and differentiation. Protein tyrosine
kinases can be divided into receptor-type and nonreceptor-type
(Src-like) kinases (Cantley et al., 1991; Hanks et al., 1988; Bonni
et al., 1993; Larner et al., 1993; Ruff-Jamison et al., 1993).
Several protein tyrosine kinases have been purified from the
cytosolic fractions of various tissues (Nakamura et al., 1988; Wong
& Goldberg, 1984; Zioncheck et al., 1986).
[0010] The Src-like kinases, which can associate with receptors at
the plasma membrane, induce rapid tyrosine phosphorylation and/or
activation of effectors such as phospholipase C-.gamma.1
(PLC.gamma.1) (Carter et al., 1991), PLC.gamma.2 (Hempel et al.,
1992), mitogen-activated protein (MAP) kinase (Casillas et al.,
1991), GTPase activating protein (GAP) (Gold et al., 1992a) and
phosphatidylinositol 3-kinase (PI3-K) (Gold et al., 1992b). Recent
studies have demonstrated an increase in tyrosine phosphorylation
following irradiation of B-lymphocyte precursors (Uckun et al.,
1993). Studies of p59.sup.fyn, p56/p53.sup.lyn, p55.sup.blk and
p56.sup.lck activity demonstrated that these Src-family tyrosine
kinases were not responsible for radiation-induced tyrosine
phosphorylation (Uckun et al., 1992a). These findings suggested
that other protein tyrosine kinases, perhaps of the receptor-type,
are involved in the response of cells to ionizing radiation.
[0011] Varying the environmental conditions following exposure to
ionizing radiation or DNA damaging agents can influence the
proportion of cells that survive a given dose due to the expression
or repair of potentially lethal damage (PLD). The damage is
potentially lethal because while under normal circumstances it
causes cell death, manipulation of the post-irradiation environment
can modify the cell response. Studies show that cell survival can
be increased if the cells are arrested in the cell cycle for a
protracted period of time following radiation exposure, allowing
repair of DNA damage. (Hall, 1988). Thus, PLD is repaired and the
fraction of cells surviving a given dose of x-rays is increased if
conditions are suboptimal for growth, such that cells do not have
to undergo mitosis while their chromosomes are damaged.
[0012] For some diseases, e.g., cancer, ionizing radiation is
useful as a therapy. Methods to enhance the effects of radiation,
thereby reducing the necessary dose, would greatly benefit cancer
patients. Therefore, methods and compositions were sought to
enhance radiation effects by increasing the sensitivity of cells to
damage from ionizing radiation and DNA damaging agents such as
alkylating compounds. Cells that are irradiated or treated with DNA
damaging agents halt in the cell cycle at G.sub.2, so that an
inventory of chromosome damage can be taken and repair initiated
and completed before mitosis is initiated. By blocking the stress
or survival response in these cells, they undergo mitosis with
damaged DNA, express the mutations, and are at a greater risk of
dying.
SUMMARY OF THE INVENTION
[0013] In one aspect, the present invention provides a process of
selectively activating the lyn family of Src-like tyrosine kinases
in a cell that expresses the lyn gene. In accordance with that
process, the cell is exposed to an effective activating dose of
ionizing radiation. In a preferred embodiment, p56/p53.sup.lyn is
activated.
[0014] In another aspect, the present invention provides a process
of stimulating tyrosine phosphorylation of Src-like tyrosine kinase
substrates. In such a process, a cell that expresses the lyn gene
is exposed to an effective activating dose of ionizing radiation.
Exemplary Src-like tyrosine kinase substrates include phospholipase
C-.gamma.1, phospholipase C-.gamma.2, mitogen-activated protein
kinase, GTPase activating protein, phosphatidylinositol 3-kinase
and enolase.
[0015] In another aspect, the present invention provides a process
of inhibiting ionizing radiation induced tyrosine phosphorylation
in a cell that expresses the lyn gene comprising inhibiting the
activity of p56/p53.sup.lyn. In a preferred embodiment,
p56/p53.sup.lyn activity is inhibited by exposing the cell to an
effective inhibitory amount of herbimycin A or genistein.
[0016] The present invention also provides a process of stimulating
the activity of p56/p53.sup.lyn with out the concomitant
stimulation of other Src-like tyrosine kinases by treating cells
with genotoxic alkylating agents. In such a process, a cell that
expresses the lyn gene is exposed to an effective activating dose
of an alkylating agent, for example, mitomycin C. Subsequent to
this exposure, the cells are treated with a tyrosine kinase
inhibitor that inhibits the activity of p56/p53.sup.lyn, preventing
phosphorylation on p34.sup.cdc2 on tyrosine, effectively allowing
the cells to progress past G.sub.2 arrest.
[0017] The G.sub.2 phase is the point in the cell cycle used DNA
repair following damage from ionizing radiation or alkylating
agents. Other DNA damaging agents also are able to cause cell
arrest in the G.sub.2 phase. By preventing delays in G.sub.2, cells
will enter mitosis before the DNA is repaired and therefore the
daughter cells will likely die. By lengthening the G.sub.2 period,
cells undergo repair and survival following exposure to a DNA
damaging agent increases.
[0018] DNA damaging agents or factors are defined herein as any
chemical compound or treatment method that induces DNA damage when
applied to a cell. Such agents and factors include ionizing
radiation and waves that induce DNA damage, such as, T-irradiation,
X-rays, UV-irradiation, microwaves, electronic emissions, and the
like. A variety of chemical compounds, also described as
"chemotherapeutic agents", function to induce DNA damage, all of
which are intended to be of use in the combined treatment methods
disclosed herein. Chemotherapeutic agents contemplated to be of
use, include, e.g., alkylating agents such as mitomycin C,
adozelesin, cis-platinum, and nitrogen mustard. The invention also
encompasses the use of a combination of one or more DNA damaging
agents, whether ionizing radiation-based or actual compounds, with
one or more tyrosine kinase inhibitors.
[0019] To kill a cell in accordance with the present invention, one
would generally contact the cell with a DNA damaging agent and a
tyrosine kinase inhibitor in a combined amount effective to kill
the cell. The term "in a combined amount effective to kill the
cell" means that the amount of the DNA damaging agent and inhibitor
are sufficient so that, when combined within the cell, cell death
is induced. Although not required in all embodiments, the combined
effective amount of the two agents will preferably be an amount
that induces more cell death than the use of either element alone,
and even one that induces synergistic cell death in comparison to
the effects observed using either agent alone. A number of in vitro
parameters may be used to determine the effect produced by the
compositions and methods of the present invention. These parameters
include, for example, the observation of net cell numbers before
and after exposure to the compositions described herein.
[0020] Similarly, a "therapeutically effective amount" is an amount
of a DNA damaging agent and tyrosine kinase inhibitor that, when
administered to an animal in combination, is effective to kill
cells within the animal. This is particularly evidenced by the
killing of cancer cells within an animal or human subject that has
a tumor. "Therapeutically effective combinations" are thus
generally combined amounts of DNA damaging agents and tyrosine
kinase inhibitors that function to kill more cells than either
element alone and that reduce the tumor burden.
[0021] The present invention generally provides novel strategies
for the improvement of chemotherapeutic intervention. It is
proposed that the combination of a DNA damaging agent and a
tyrosine kinase inhibitor will lead to synergistic cancer cell
killing effects over and above the actions of the individual DNA
damaging component.
[0022] In certain embodiments, a process of enhancing cell death is
provided, which comprises the steps of first treating cells or
tumor tissue with a DNA damaging agent, such as ionizing radiation
or an alkylating agent, followed by contacting the cells or tumors
with a protein kinase inhibitor, preferably a tyrosine kinase
inhibitor. Examples of alkylating agents are mitomycin C,
adozelesin, nitrogen mustard, cis-platinum. Exemplary tyrosine
kinase inhibitors are genistein or herbimycin.
[0023] DNA damaging agents or factors are defined herein as any
chemical compound or treatment method that induces DNA damage when
applied to a cell. Such agents and factors include radiation and
waves that induce DNA damage, such as, .gamma.-irradiation, X-rays,
UV-irradiation, microwaves, electronic emissions, and the like. A
variety of chemical compounds, which may be described as
"chemotherapeutic agents", also function to induce DNA damage, all
of which are intended to be of use in the combined treatment
methods disclosed herein. Chemotherapeutic agents contemplated to
be of use, include, e.g., mitomycin C (MMC), adozelesin,
cis-platinum, nitrogen mustard, 5-fluorouracil (5FU), etoposide
(VP-16), camptothecin, actinomycin-D, cisplatin (CDDP).
[0024] The invention provides, in certain embodiments, methods and
compositions for killing a cell or cells, such as a malignant cell
or cells, by contacting or exposing a cell or population of cells
to one or more DNA damaging agents and one or more tyrosine kinase
inhibitors in a combined amount effective to kill the cell(s).
Cells that may be killed using the invention include, e.g.,
undesirable but benign cells, such as benign prostate hyperplasia
cells or over-active thyroid cells; cells relating to autoimmune
diseases, such as B cells that produce antibodies involved in
arthritis, lupus, myasthenia gravis, squamous metaplasia,
dysplasia, and the like. Although generally applicable to killing
all undesirable cells, the invention has a particular utility in
killing malignant cells. "Malignant cells" are defined as cells
that have lost the ability to control the cell division cycle, as
leads to a "transformed" or "cancerous" phenotype.
[0025] To kill cells, such as malignant cells, using the methods
and compositions of the present invention, one would generally
contact a "target" cell with at least one DNA damaging agent and at
least one tyrosine kinase inhibitor in a combined amount effective
to kill the cell. This process may involve contacting the cells
with the DNA damaging agent(s) or factor(s) and the tyrosine kinase
inhibitor at the sate time. This may be achieved by contacting the
cell with a single composition or pharmacological formulation that
includes both agents, or by contacting the cell with two distinct
compositions or formulations, at the same time, wherein one
composition includes the DNA damaging agent and the other
composition includes the tyrosine kinase inhibitor.
[0026] Naturally, it is also envisioned that the target cell may be
first exposed to the DNA damaging agent(s) and then contacted with
a tyrosine kinase inhibitor, or vice versa. In such embodiments,
one would generally ensure that sufficient time elapses, so that
the two agents would still be able to exert an advantageously
combined effect on the cell. In such instances, it is contemplated
that one would contact the cell with both agents within about 12
hours of each other, and more preferably within about 6 hours of
each other, with a delay time of only about 4 hours being most
preferred. These times are readily ascertained by the skilled
artisan.
[0027] The terms "contacted" and "exposed", when applied to a cell,
are used herein to describe the process by which a tyrosine kinase
inhibitor, such as genistein or herbimycin A, and a DNA damaging
agent or factor are delivered to a target cell or are placed in
direct juxtaposition with the target cell. To achieve cell killing,
both agents are delivered to a cell in a combined amount effective
to kill the cell, i.e., to induce programmed cell death or
apoptosis. The terms, "killing", "programmed cell death" and
"apoptosis" are used interchangeably in the present text to
describe a series of intracellular events that lead to target cell
death.
[0028] Tyrosine kinases participate in diverse signalling pathways,
and also participate in signal transduction in smooth muscle.
"Genistein", as used herein, refers to the compound described in
the Merck Index (7th Edition, 1960, p 474), or a derivative or
analogue thereof that functions as a tyrosine kinase inhibitor. The
ability of a genistein analogue to inhibit a tyrosine kinase, may
be readily determined by methods known to those of skill in the
art, as described, for example in Akiyama et al. (1987),
incorporated herein by reference. Also encompassed are
genistein-like compounds that form an active tyrosine kinase
inhibitor upon ingestion.
[0029] The present invention also provides advantageous methods for
treating cancer that, generally, comprise administering to an
animal or human patient with cancer a therapeutically effective
combination of a DNA damaging agent and a tyrosine kinase
inhibitor. Chemical DNA damaging agents and/or inhibitors may be
administered to the animal, often in close contact to the tumor, in
the form of a pharmaceutically acceptable composition. Direct
intralesional injection is contemplated, as are other parenteral
routes of administration, such as intravenous, percutaneous,
endoscopic, or subcutaneous injection.
[0030] In terms of contact with a DNA damaging agent, this may be
achieved by irradiating the localized tumor site with ionizing
radiation such as X-rays, UV-light, .gamma.-rays or even
microwaves. Alternatively, the tumor cells may be contacted with
the DNA damaging agent by administering to the animal a
therapeutically effective amount of a pharmaceutical composition
comprising a DNA damaging compound, such as mitomycin C,
adozelesin, cis-platinum, and nitrogen mustard. A chemical DNA
damaging agent may be prepared and used as a combined therapeutic
composition, or kit, by combining it with a tyrosine kinase
inhibitor, as described above.
[0031] Other embodiments of the invention concern compositions,
including pharmaceutical formulations, comprising a DNA damaging
agent in combination with a tyrosine kinase inhibitor. These
compositions may be formulated for in vivo administration by
dispersion in a pharmacologically acceptable solution or buffer.
Suitable pharmacologically acceptable solutions include neutral
saline solutions buffered with phosphate, lactate, Tris, and the
like. Preferred pharmaceutical compositions of the invention are
those that include, within a pharmacologically acceptable solution
or buffer, mitomycin C in combination with genistein or herbimycin
A.
[0032] Still further embodiments of the present invention are kits
for use in killing cells, such as malignant cells, as may be
formulated into therapeutic kits for use in cancer treatment. The
kits of the invention will generally comprise, in suitable
container means, a pharmaceutical formulation of a DNA damaging
agent and a pharmaceutical formulation of a tyrosine kinase
inhibitor. These agents may be present within a single container,
or these components may be provided in distinct or separate
container means.
[0033] The components of the kit are preferably provided as a
liquid solution, or as a dried powder. When the components are
provided in a liquid solution, the liquid solution is an aqueous
solution, with a sterile aqueous solution being particularly
preferred. When reagents or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0034] Although kits have been described as part of this invention,
it should be noted that the use of ionizing radiation to create DNA
damage is an important aspect of the invention not specifically
provided in kit form.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0036] FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D. Activation of
Src-like tyrosine kinases by mitomycin C (MMC). HL-60 cells were
exposed to 10.sup.-5 M MMC and harvested at 1 h. Cell lysates were
subjected to immunoprecipitation with pre-immune rabbit serum
(PIRS) (FIG. 1A); anti-Fyn antibodies (FIG. 1B); anti-Lyn
antibodies (FIG. 1C); and anti-Src antibodies (FIG. 1D).
Phosphorylation reactions were performed in the presence of
[.gamma..sup.32P]ATP for 10 min at 30.degree. C. Phosphorylated
protein was analyzed by 10% SDS-PAGE and autoradiography.
[0037] FIG. 2A, FIG. 2B and FIG. 2C. Activation of p56/p53.sup.lyn
kinase by MMC. In FIG. 2A, HL-60 cells were exposed to the
indicated concentrations of MMC for 1 h. In FIG. 2B, cells were
exposed to 10.sup.-5 M MMC for the indicated times. Anti-Lyn
immunoprecipitates were incubated with [.gamma.-.sup.32P]ATP and
enolase. Phosphorylated protein was analyzed by SDS-PAGE and
autoradiography. In FIG. 2C, anti-Lyn immunoprecipitates were
analyzed by immunoblotting with anti-Lyn.
[0038] FIG. 3A and FIG. 3B. Tyrosine phosphorylation of
p56/p53.sup.lyn in MMC-treated cells. HL-60 cells were treated with
MMC for 1 h. Cell lysates were immunoprecipitated with anti-Lyn and
the immunoprecipitates were subjected to immunoblotting with
anti-P-Tyr (FIG. 3A) or anti-Lyn (FIG. 3B).
[0039] FIG. 4A, FIG. 4B and FIG. 4C. MMC-induced p56/p53.sup.lyn
activation is sensitive to tyrosine kinase inhibitors and is not a
direct effect. In FIG. 4A, cells were treated with 10.sup.-5 M
herbimycin A (H) or genistein (G) for 1 h and then MMC for an
additional 1 h. In FIG. 4B, cells were treated with
5.times.10.sup.-5 M H7 for 1 h and then MMC for 1 h. Anti-Lyn
immunoprecipitates were analyzed for phosphorylation of
p56/p53.sup.lyn and enolase. In FIG. 4C, cells were treated with
MMC for 1 h. Anti-Lyn immunoprecipitates were analyzed for
phosphorylation of p56/p53.sup.lyn and enolase. Lysates from
untreated HL-60 cells were immunoprecipitated with anti-Lyn. MMC
(10.sup.-5 M) was added to the kinase reaction and incubated for 15
min. The reaction was analyzed for phosphorylation of
p56/p53.sup.lyn and enolase.
[0040] FIG. 5A, FIG. 5B and FIG. 5C. Other alkylating agents active
p56/p53.sup.lyn. HL-60 cells were treated with 2.times.10.sup.-6 M
adozelesin (FIG. 5A), 10.sup.-5 M nitrogen mustard (FIG. 5B) and
10.sup.-5 M cis-platinum (FIG. 5C) for 1 h. Anti-Lyn
immunoprecipitates were analyzed for phosphorylation of
p56/p53.sup.lyn and enolase.
[0041] FIG. 6A and FIG. 6B. Association of p56/p53.sup.lyn and
p34.sup.cdc2. HL-60 cells were treated with 10.sup.-5 M MMC for 1
h. In FIG. 6A, cell lysates were incubated with GST or GST-Lyn
proteins immobilized on beads. The resulting complexes were
separated by SDS-PAGE and analyzed by immunoblotting with anti-cdc2
antibody. In FIG. 6B, lysates from control (labeled HL-60) and
MMC-treated cells were subjected to immunoprecipitation with
anti-cdc2. The immune complexes were assayed for in vitro kinase
activity by incubation with [.gamma.-.sup.32P]ATP. One aliquot of
the kinase reaction was analyzed by SDS-PAGE and autoradiography.
The other aliquot was washed to remove free ATP and boiled in SDS
buffer to disrupt complexes. A secondary immunoprecipitation was
then performed with anti-Lyn. The anti-Lyn immunoprecipitates were
separated by SDS-PAGE and analyzed by autoradiography.
[0042] FIG. 7A and FIG. 7B. Effects of MMC treatment on tyrosine
phosphorylation of p34.sup.cdc2. HL-60 cells were exposed to MMC
for 1 h. In FIG. 7A, cell lysates were subjected to
immunoprecipitation with anti-cdc2. The immunoprecipitates were
analyzed by SDS-PAGE and immunoblotting with anti-P-Tyr. In FIG.
7B, cell lysates were subjected to immunoprecipitation with
anti-cdc2 and immunoblot analysis with anti-cdc2.
[0043] FIG. 8. Phosphorylation of cdc2 peptides by p56/p53.sup.lyn.
HL-60 cells were treated with MMC for 1 h. Cell lysates were
subjected to immunoprecipitation with anti-Lyn. The
immunoprecipitates were assayed for phosphorylation of either a
cdc2 (IEKIGEGTYGVVYK) or mutated cdc2 (mcdc2; Y-15 to F-15)
peptide. The results represent the mean.+-.S.D. of two independent
studies each performed in duplicate and are normalized to control
phosphorylation of the cdc2 peptide. Control cells (cross hatch);
MMC-treated cells (stripes).
[0044] FIG. 9A, FIG. 9B and FIG. 9C. Activation of Src-like protein
tyrosine kinases by ionizing radiation. HL-60 cells were exposed to
200 cGy ionizing radiation and harvested at 15 min or 2 hours. In
FIG. 9A, Cell lysates were subjected to immunoprecipitation with
anti-Fyn antibodies; in FIG. 9B, cell lysates were subjected to
immunoprecipitation with anti-Lyn antibodies; and in FIG. 9C, cell
lysates were subjected to immunoprecipitation with anti-Lck
antibodies. Autophosphorylation reactions were performed by adding
[.gamma.-.sup.32P]ATP for 10 min at 30.degree. C. Phosphorylated
protein was analyzed by 10% SDS-PAGE and autoradiography.
[0045] FIG. 10A and FIG. 10B. Activation of p53/56.sup.lyn kinase
by ionizing radiation. HL-60 cells were exposed to 200 cGy ionizing
radiation for 5 min, 15 min, 30 min, 6 hours, 12 hours, or 24
hours. Cell lysates were subjected to immunoprecipitation with
anti-Lyn. In FIG. 10A, the immunoprecipitates were analyzed in
autophosphorylation reactions. In FIG. 10B, enolase phosphorylation
assays are shown. Samples were separated in 10% SDS-PAGE gels and
analyzed by autoradiography. The fold increase of Enolase
phosphorylation, increased as measured by scintillation counting of
the excised bands, is indicated at the bottom.
[0046] FIG. 11. Different doses of ionizing radiation induce
activation of p53/p56.sup.lyn. HL-60 cells were exposed to the
indicated doses of ionizing radiation and then harvested at 12 h.
Soluble proteins were subjected to immunoprecipitation with
anti-Lyn. The immunoprecipitates were analyzed for phosphorylation
of p56/p53.sup.lyn and enolase. The fold increase in enolase
phosphorylation is indicated at the bottom.
[0047] FIG. 12A and FIG. 12B. Effects of H.sub.2O.sub.2, NAC and
protein tyrosine kinase inhibitors on activation of
p56/p53.sup.lyn. In FIG. 12A, HL-60 cells were either treated with
H.sub.2O.sub.2 for the indicated times or pretreated with 30 mM NAC
for 1 h, irradiated (200 cGy) and harvested at 12 h. In FIG. 12B,
HL-60 cells were treated with 10 .mu.M herbimycin (H) or 10 .mu.M
genistein (G) for 1 h, irradiated (200 cGy) and then harvested at
12 h. Cell lysates were immunoprecipitated with anti-Lyn and the
immunoprecipitates were analyzed for phosphorylation of
p56/p53.sup.lyn and enolase.
[0048] FIG. 13A and FIG. 13B. Ionizing radiation exposure induces
tyrosine phosphorylation of a 34 kD substrate. HL-60 cells were
exposed to 200 cGy ionizing radiation and harvested at the
indicated times. In FIG. 13A, soluble proteins were subjected to
immunoblot (IB) analysis with anti-P-Tyr; and in FIG. 13B soluble
proteins were subjected to immunoblot (IB) analysis with
anti-p34.sup.cdc2 antibodies. The arrow indicates the position of
34 kD signals.
[0049] FIG. 14A and FIG. 14B. Different doses of ionizing radiation
induce tyrosine phosphorylation of the 34 kD protein. HL-60 cells
were exposed to the indicated doses of ionizing radiation and then
harvested at 5 min. In FIG. 14A, soluble proteins were subjected to
immunoblot (IB) analysis with anti-P-Tyr; and in FIG. 14B, soluble
proteins were subjected to immunoblot (IB) analysis with
anti-p34.sup.cdc2 antibodies. The arrows indicate the position of
the 34 kD signals.
[0050] FIG. 15A and FIG. 15B. Ionizing radiation induces tyrosine
phosphorylation of p34.sup.cdc2. HL-60 cells were exposed to 50 cGy
ionizing radiation and harvested at 5 min. Cell lysates from
control and irradiated cells were subjected to immunoprecipitation
(IP) with p34.sup.cdc2 antiserum and protein A-Sepharose. In FIG.
15A, the immunoprecipitates were subjected to immunoblot (IB)
analysis with anti-P-Tyr antibodies; and in FIG. 15B, the
immunoprecipitates were subjected to immunoblot (IB) analysis with
anti-p34.sup.cdc2 antibodies.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0051] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example I
Alkylating Agents Activate the Lyn Tyrosine Kinase and Promote
Tyrosine Phosphorylation of p34.sup.cdc2
A. Materials and Methods
[0052] Cell culture. HL-60 cells were grown in RPMI-1640 medium
containing 15% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin,
2 mM L-glutamine, 1 mM sodium pyruvate and 1 mM non-essential amino
acids. Cells were treated with MMC (Sigma Chemical Co., St. Louis,
Mo.), adozelesin (Sigma), cis-platinum (Sigma), nitrogen mustard
(Sigma), genistein (GIBCO/BRL, Gaithersburg, Md.), herbimycin A
(GIBCO/BRL) and H-7 (Seikagaku America Inc., Rockville, Md.). Cell
viability was determined by trypan blue exclusion.
[0053] Immune complex kinase assays. Cells (2-3.times.10.sup.7)
were washed twice with ice cold phosphate buffered saline (PBS) and
lysed in 2 ml of lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 1%
NP-40, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 1
mM DTT and 10 mg/ml of leupeptin and aprotinin). After incubation
on ice for 30 min, insoluble material was removed by centrifugation
at 14000 rpm for 10 min at 4.degree. C. Soluble proteins were
precleared by incubating with 5 mg/ml rabbit-anti-mouse IgG for 1 h
at 4.degree. C. and then for an additional 30 min after addition of
protein A-sepharose.
[0054] The supernatant fraction was incubated with pre-immune
rabbit serum, anti-Fyn, anti-Lyn, anti-Src (UBI, Lake Placid, N.Y.)
or anti-cdc2 (sc-54, Santa Cruz Biotechnology, Santa Cruz, Calif.)
antibodies for 1 h at 4.degree. C. followed by 30 min after
addition of protein A-sepharose. The immune complexes were washed
three times with lysis buffer and once with kinase buffer (20 mM
HEPES, pH 7.0, 10 mM MnCl.sub.2 and 10 mM MgCl.sub.2) and
resuspended in 30 ml of kinase buffer containing 1 mCi/ml
[.gamma.-.sup.32P]ATP (3000 Ci/mmol; NEN, Boston, Mass.) with and
without 5-8 mg of acid-treated enolase (Sigma). The reaction was
incubated for 15 min at 30.degree. C. and terminated by the
addition of 2.times.SDS sample buffer. The proteins were separated
in 10% SDS-polyacrylamide gels and analyzed by autoradiography.
Radioactive bands were excised from certain gels and quantitated by
scintillation counting.
[0055] Immune complexes were also resuspended in 30 ml kinase
buffer containing 1 mCi/ml [.gamma.-.sup.32P] ATP and either 100 mM
cdc2 peptide (amino acids 7 to 20; IEKIGEGTYGVVYK; SEQ ID NO:1) or
100 mM mutated cdc2 peptide with Phe-15 substituted for Tyr-15
(IEKIGEGTFGVVYK; SEQ ID NO:2). The reactions were incubated for 15
min at 30.degree. C. and terminated by spotting on P81
phosphocellulose discs (GIBCO/BRL). The discs were washed twice
with 1% phosphoric acid and twice with water before analysis by
liquid scintillation counting.
[0056] Immunoblot analysis. Immune complexes bound to protein
A-sepharose were prepared as for the autophosphorylation assays.
Proteins were separated in 10% SDS-polyacrylamide gels and
transferred to nitrocellulose paper. The residual binding sites
were blocked by incubating the filters in 5% dry milk in PBST
(PBS/0.05% Tween-20) for 1 h at room temperature. The blots were
subsequently incubated with anti-cdc2 or anti-phosphotyrosine
(anti-P-Tyr; MAb 4G10, UBI). After washing twice with PBST, the
filters were incubated for 1 h at room temperature with anti-mouse
IgG (whole molecule) peroxidase conjugate (Sigma) in 5% milk/PBST.
The filters were then washed and the antigen-antibody complexes
visualized by the ECL detection system (Amersham, Arlington
Heights, Ill.).
[0057] Coimmunoprecipitation. Immunoprecipitations were performed
with anti-p34.sup.cdc2 at 5 mg/ml cell lysate. Immune complexes
were collected on protein A-Sepharose beads (Pharmacia), washed
three times with lysis buffer and twice with kinase buffer,
resuspended in kinase buffer and then incubated for 10 min at
30.degree. C. in the presence of 1 mCi/ml [.gamma.-.sup.32P] ATP.
One aliquot of the kinase reaction was subjected to SDS-PAGE and
autoradiography. The other aliquot was washed in lysis buffer to
remove free ATP and then boiled in 20 mM Tris-HCl, pH 8.0
containing 0.5% SDS and 1 mM DTT to disrupt protein-protein
interaction. After dilution to 0.1% SDS, a secondary
immunoprecipitation was then performed by adding anti-Lyn antibody
and protein A-Sepharose beads. The anti-Lyn immunoprecipitates were
then subjected to SDS-PAGE and autoradiography.
[0058] Fusion protein binding assays. The plasmid encoding a
glutathione S-transferase (GST)-Lyn (amino acids 1 to 243) fusion
protein was obtained from T. Pawson, Toronto, Canada and
transfected into E. coli DH5a (Pleiman et al., 1993). The fusion
protein was induced with IPTG, purified by affinity chromatography
using glutathione-Sepharose beads (Pharmacia) and equilibrated in
lysis buffer. HL-60 cell lysates were incubated with 50 mg
immobilized GST or GST-Lyn for 2 h at 4.degree. C. The protein
complexes were washed three times with lysis buffer and boiled for
5 min in SDS sample buffer. The complexes were then separated in
10% SDS-PAGE and subjected to silver staining or immunoblot
analysis with anti-cdc2.
B. Results
[0059] Previous studies have demonstrated that HL-60 cells express
the p59.sup.fyn, p56/p53.sup.lyn and pp60.sup.c-src tyrosine
kinases (Barnekow & Gessler, 1986; Gee et al., 1986; Katagiri
et al., 1991). In this example, the inventors have shown that
certain of these tyrosine kinases are activated during treatment of
HL-60 cells with MMC.
[0060] Immunoprecipitates from control and MMC-treated cells were
assayed for autophosphorylation. There was no detectable kinase
activity in precipitates obtained with pre-immune rabbit serum
(FIG. 1A). Other studies with an anti-Fyn antibody demonstrated
that autophosphorylation of p59.sup.fyn is decreased at 1 h of MMC
treatment (FIG. 1B). Similar results were obtained at multiple time
points through 6 h of MMC exposure. In contrast, immunoprecipitates
with anti-Lyn demonstrated an increase in p56/p53.sup.lyn activity
as a result of MMC exposure (FIG. 1C). The finding that anti-Src
immunoprecipitates also exhibited a decrease in pp60.sup.c-src
activity in MMC-treated cells (FIG. 1D) suggests that MMC exposure
is associated with selective activation of p56/p53.sup.lyn.
[0061] Activation of p56/p53.sup.lyn was confirmed at different
concentrations of MMC and by assaying for phosphorylation of the
substrate protein enolase. Increases in p56/p53.sup.lyn activity
were found at 10.sup.-8 and 10.sup.-7 M MMC, while more pronounced
stimulation of this kinase was apparent at 10.sup.-6 and 10.sup.-5
M (FIG. 2A). The results further demonstrate that p56/p53.sup.lyn
activity is rapidly induced in MMC-treated cells. Increases in
MMC-induced phosphorylation of p56/p53.sup.lyn and enolase were
first detectable at 30 min (4.2-fold increase for enolase) and
persisted through at least 12 h (4.1-fold for enolase) of drug
exposure (FIG. 2B). The induction of p56/p53.sup.lyn activity was
not related to cell death since viability as determined by trypan
blue exclusion was >90% at 12 h of MMC treatment.
[0062] Immunoblot analysis was also performed to determine whether
the increases in p56/p53.sup.lyn activity were due to a greater
abundance in protein. The results demonstrate similar levels of
p56/p53.sup.lyn protein (FIG. 2C). These findings supported a rapid
and prolonged activation of p56/p53.sup.lyn in response to MMC
treatment.
[0063] In order to confirm that activation of p56/p53.sup.lyn is
associated with tyrosine phosphorylation, the anti-Lyn immune
complexes were assayed by immunoblotting with anti-P-Tyr. The
results demonstrate an increase in tyrosine phosphorylation of
p56/p53.sup.lyn from MMC-treated as compared to control cells (FIG.
3A). Analysis of the anti-Lyn immunoprecipitates by immunoblotting
with anti-Lyn confirmed the presence of similar levels of protein
after MMC treatment (FIG. 3B).
[0064] The involvement of tyrosine phosphorylation was further
supported by the demonstration that pretreatment of cells with the
tyrosine kinase inhibitors, genistein (Akiyama et al., 1987) and
herbimycin A (Uehara et al., 1989) completely blocks the
stimulation of p56/p53.sup.lyn activity associated with MMC
treatment (FIG. 4A). In contrast, pretreatment with the
isoquinoline sulfonamide inhibitor of serine/threonine protein
kinases, H-7 (Hidaka et al., 1984), had no detectable effect on the
MMC-induced activity (FIG. 4B). These effects of MMC on induction
of p56/p53.sup.lyn could be related to direct interaction of this
agent with Lyn kinase. However, incubation of anti-Lyn immune
complexes in the presence of MMC was associated with a decrease in
kinase activity (FIG. 4C). Taken together, these findings indicated
that MMC induces the tyrosine kinase activity of p56/p53.sup.lyn by
an indirect mechanism.
[0065] The available evidence indicates that MMC acts as a
monofunctional and bifunctional alkylating agent (Carrano et al.,
1979). Consequently, adozelesin, another monofunctional but
structurally distinct alkylating agent (Bhuyan et al., 1992; Hurley
et al., 1984), was investigated. The results demonstrate that
treatment of HL-60 cells with adozelesin is similarly associated
with stimulation of p56/p53.sup.lyn and enolase phosphorylation
(FIG. 5A). Other studies were performed with agents that also
induce the formation of DNA cross-links. Nitrogen mustard, an agent
that forms monoadducts and DNA interstrand cross-links (Ewig &
Khon, 1977; Hartley et al., 1992), was effective in inducing
p56/p53.sup.lyn activity (FIG. 5B). Moreover, treatment of cells
with cis-platinum, an agent that forms intrastrand cross-links
(Sherman & Lippard, 1987), was associated with stimulation of
the p56/p53.sup.lyn kinase (FIG. 5C). These findings indicated that
the response of cells to diverse alkylating-type agents induces
activation of p56/p53.sup.lyn.
[0066] In order to examine the significance of p56/p53.sup.lyn
activation, the association of this kinase with specific
intracellular proteins that undergo tyrosine phosphorylation in
MMC-treated cells was investigated. This issue was initially
addressed using a GST-Lyn fusion protein to identify molecules
which interact with p56/p53.sup.lyn. Lysates from MMC-treated cells
were incubated with immobilized GST or GST-Lyn. Analysis of the
adsorbates by SDS-PAGE and staining demonstrated the presence of a
34 kD protein.
[0067] The inventors assayed the adsorbates for reactivity with
anti-cdc2. The results indicate that p34.sup.cdc2 associates with
the GST-Lyn fusion protein and not the GST control (FIG. 6A). The
potential interaction between p56/p53.sup.lyn and p34.sup.cdc2 was
further examined in coimmunoprecipitation studies. Lysates of
control and MMC-treated cells were subjected to immunoprecipitation
with anti-cdc2 and the immunoprecipitates were assayed for
autophosphorylation (FIG. 6B). One aliquot of the in vitro kinase
reaction was assayed by SDS-PAGE and autoradiography. While
immunoprecipitates from MMC-treated cells exhibited phosphorylation
of 53-56 kD proteins, there was little if any of this activity in
control cells (FIG. 6B). In order to determine whether the
anti-cdc2 immunoprecipitates contain p56/p53.sup.lyn, the other
aliquot of the in vitro kinase reaction was treated to disrupt
protein complexes and then subjected to immunoprecipitation with
anti-Lyn. The results demonstrate increased levels of
autophosphorylated p56/p53.sup.lyn when assaying MMC-treated as
compared to control cells (FIG. 6B).
[0068] The finding that MMC exposure induces an interaction between
p56/p53.sup.lyn and p34.sup.cdc2 prompted further studies to
determine whether p34.sup.cdc2 exhibits increased tyrosine
phosphorylation in MMC-treated cells. Immunoprecipitation of
p34.sup.cdc2 and then immunoblotting of the precipitates with
anti-P-Tyr demonstrated an increase in reactivity as a result of
MMC treatment (FIG. 7A). Reprobing the filter with the anti-cdc2
antibody demonstrated similar levels of p34.sup.cdc2 protein (FIG.
7B). Since these findings indicated that MMC treatment is
associated with increased tyrosine phosphorylation of p34.sup.cdc2,
other studies were performed to determine whether p56/p53.sup.lyn
can phosphorylate p34.sup.cdc2 in vitro.
[0069] In order to study a potential phosphorylation site for
Src-like kinases located at Tyr-15 of p34.sup.cdc2, synthetic
peptides were prepared with sequences derived from amino acids 7 to
20 of p34.sup.cdc2 and another with substitution at Tyr-15 with
Phe-15. While anti-Lyn immune complexes from control cells
phosphorylated the cdc2 peptide, similar complexes from MMC-treated
cells exhibited nearly a 2-fold stimulation in this activity (FIG.
8). In contrast, there was little phosphorylation of the mutated
cdc2 peptide with anti-Lyn complexes from control or MMC-treated
cells (FIG. 8). These findings indicated that p56/p53.sup.lyn
phosphorylates the Tyr-15 site of p34.sup.cdc2.
[0070] The present results demonstrate that treatment of HL-60
cells with MMC is associated with selective activation of the
p56/p53.sup.lyn tyrosine kinase. These findings are not limited to
HL-60 cells since other cell lines, for example U-937 myeloid
leukemia cells, also respond to this agent with increases in
p56/p53.sup.lyn activity.
[0071] The lyn gene encodes two forms of the tyrosine kinase,
p56.sup.lyn and p53.sup.lyn, due to alternate mRNA splicing
(Yamanashi et al., 1987; Yamanashi et al., 1989). As a member of
the Src-like family of tyrosine kinases, p56/p53.sup.lyn is related
to pp60.sup.c-src and p59.sup.fyn (Cantley et al., 1991). However,
only p56/p53.sup.lyn was activated in MMC-treated cells. These
kinases are often associated with cell surface receptors at the
interface between the cell membrane and cytoplasm. Studies of
p56/p53.sup.lyn in B cells have demonstrated an association with
the B-cell antigen receptor (Pleiman et al., 1993; Yamanashi et
al., 1992). Engagement of the B-cell antigen receptor induces
activation of p56/p53.sup.lyn, as well as other Src-like kinases,
and tyrosine phosphorylation of substrates that include PLCg2, MAP
kinase and GAP (Pleiman et al., 1993). Other studies have shown
that p56/p53.sup.lyn associates with the 85 kDa a-subunit of PI 3-K
and induces PI 3-K activity (Yamanashi et al., 1992). Thus,
p56/p53.sup.lyn is capable of associating with and phosphorylating
diverse downstream effector molecules.
[0072] Although the cellular effects of alkylating agents such as
MMC are generally attributed to DNA damage, their action may be
related to alkylation of RNA or protein. The demonstration that MMC
treatment of intact cells is associated with activation of
p56/p53.sup.lyn raised the possibility that this effect might be
due to direct alteration of Lyn protein. p56/p53.sup.lyn activity
was however decreased in vitro by incubation of anti-Lyn immune
complexes with MMC. In order to address the possibility that
MMC-induced activation of p56/p53.sup.lyn is related to formation
of DNA lesions, another agent, adozelesin, was used that covalently
binds to the N-3 of adenine within the minor groove of DNA (Bhuyan
et al., 1992; Hurley et al., 1984). Adozelesin also induces
p56/p53.sup.lyn activity.
[0073] HL-60 cells also respond similarly to other alkylating
agents, such as nitrogen mustard which reacts predominantly with
guanines by alkylation of their N-7 positions or forms DNA
interstrand cross-links (Ewig & Khon, 1977; Hartley et al.,
1992). Moreover, p56/p53.sup.lyn activity was stimulated by
cis-platinum which induces intrastrand cross-links (Sherman &
Lippard, 1987). Thus, structurally distinct agents that damage DNA
by diverse mechanisms are capable of inducing p56/p53.sup.lyn
activity. Recent studies have demonstrated that treatment of HeLa
cells with ultraviolet (UV) irradiation is associated with
increases in the catalytic activity of c-Src and c-Fyn, but not
that of c-Yes (Devary et al., 1992). Taken together with the
absence of detectable pp60.sup.c-src or p59.sup.fyn activation in
MMC-treated HL-60 cells, these results suggest that induction of
these tyrosine kinases may be cell-type or agent specific.
[0074] The p34.sup.cdc2 serine/threonine protein kinase controls
entry of cells into mitosis (Nurse, 1990; Pines & Hunter,
1990). This kinase is regulated by networks of kinases and
phosphatases that appear to respond to the state of DNA
replication. Activation of p34.sup.cdc2 involves association with
cyclin B and posttranslational modifications of the
p34.sup.cdc2/cyclin B complex (Norbury & Nurse, 1992).
Phosphorylation of p34.sup.cdc2 on Thr-161 is required for
activation (Atherton-Fessler et al., 1993; Desai et al., 1992;
Solomon et al., 1992), while Tyr-15 phosphorylation results in
inhibition of both p34.sup.cdc2 activity and entry of cells into
mitosis (Gould & Nurse, 1989; Gould et al., 1990).
[0075] Studies have demonstrated that treatment of mammalian cells
with alkylating and other DNA-damaging agents is associated with
G.sub.2 arrest (Konopa, 1988; Lau & Pardee, 1982; Tobey, 1975).
However, the precise mechanisms responsible for this effect have
remained unclear. Exposure of cells to ionizing radiation is
associated with rapid inhibition of p34.sup.cdc2 activity and
G.sub.2 arrest (Lock & Ross, 1990). Other studies have
demonstrated that arrest of nitrogen mustard-treated cells at
G.sub.2 is temporally related to formation of DNA cross-links and
p34.sup.cdc2 inhibition (O'Connor et al., 1992). In the present
studies, it is demonstrated that MMC treatment results in rapid
tyrosine phosphorylation of p34.sup.cdc2. Similar findings have
been obtained in cells treated with ionizing radiation (see
following Examples). This modification of p34.sup.cdc2 is
associated with loss of kinase activity as determined by assaying
anti-cdc2 immunoprecipitates for phosphorylation of H1 histone.
Thus, the phosphorylation of p34.sup.cdc2 on tyrosine appears to
represent in part the response of mammalian cells to DNA damage and
may contribute to G.sub.2 arrest by inhibition of p34.sup.cdc2
activity.
[0076] The available evidence indicates that the p107.sup.wee1
dual-specificity kinase is responsible for phosphorylation of
p34.sup.cdc2 on Tyr-15 (Featherstone & Russell, 1991; Parker et
al., 1991; Parker et al., 1992). While p107.sup.wee1 appears to
control p34.sup.cdc2 activity to ensure completion of S-phase,
other studies suggest that p107.sup.wee is not required for the
DNA-damage-dependent mitotic checkpoint. In this context, normal
mitotic arrest has been observed after irradiation of
Schizosaccharomyces pombe cells with a defective or missing wee1
gene (Barbet & Carr, 1993). Other studies have shown that
p34.sup.cdc2 is phosphorylated on tyrosine in yeast wee1 minus
mutants (Gould et al., 1990). The present results in mammalian
cells suggest that regulation of p34.sup.cdc2 following exposure to
alkylating agents involves activation of p56/p53.sup.lyn. The
association of p56/p53.sup.lyn and p34.sup.cdc2 in MMC-treated
cells, as well as the finding that p56/p53.sup.lyn can
phosphorylate the Tyr-15 site of p34.sup.cdc2 in vitro, support the
possibility that p56/p53.sup.lyn contributes to signaling from the
mitotic checkpoint that monitors for alkylating agent-induced
damage.
Example II
Ionizing Radiation Activates the Lyn Tyrosine Kinase and Promotes
Tyrosine Phosphorylation of p34.sup.cdc2
[0077] Treatment of human HL-60 myeloid leukemia cells with
ionizing radiation is associated with activation of the Lyn
tyrosine kinase. The lyn gene encodes two forms of this kinase,
p56.sup.lyn and p53.sup.lyn, as a result of alternate splicing
(Yamanashi et al., 1987; 1989). Both p56/p53.sup.lyn, but not
certain other Src-related kinases, are activated in irradiated
HL-60 cells. Activation of p56/p53.sup.lyn represents a signaling
pathway distinct from those involved in X-ray-induced early
response gene expression.
[0078] HL-60 myeloid leukemia cells were grown in RPMI-1640 medium
containing 15% heat-inactivated fetal bovine serum (FBS)
supplemented with 100 units/ml penicillin, 100 mg/ml streptomycin,
2 mM L-glutamine, 1 mM sodium pyruvate and 1 mM non-essential amino
acids. Cells in logarithmic growth phase were suspended in complete
RPMI-1640 medium with 0.5% FBS 18 hours prior to irradiation.
[0079] Irradiation was performed at room temperature using a
Gammacell 1000 (Atomic Energy of Canada, Ottawa) under aerobic
conditions with a .sup.137Cs source emitting at a fixed dose rate
of 13.3 Gy/min as determined by dosimetry. HL-60 cells were also
treated with 50 mM H.sub.2O.sub.2 (Sigma Chemical Co., St. Louis,
Mo.), 30 mM N-acetyl cysteine (NAC; Sigma), 10 .mu.M genistein
(GIBCO/BRL, Gaithersburg, Md.) or 10 .mu.M herbimycin A
(GIBCO/BRL).
[0080] Cells (2-3.times.10.sup.7) were washed twice with ice cold
phosphate buffered saline (PBS) and lysed in 2 ml of lysis buffer
(20 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 1 mM sodium vanadate, 1
mM phenylmethylsulfonyl fluoride, 1 mM DTT, and 10 mg/ml of
leupeptin and aprotinin). After incubation on ice for 30 min,
insoluble material was removed by centrifugation at 1400 rpm for 10
min at 4.degree. C. Soluble proteins were precleared by incubation
with 5 mg/ml rabbit anti-mouse IgG for 1 hour at 4.degree. C. and
then addition of protein A sepharose for 30 min.
[0081] The supernatants were incubated with 2.5 .mu.l of anti-human
Fyn, 2 .mu.l of anti-human Lyn, 3 .mu.l of anti-human Lyk
(N-terminal) or 3 .mu.l of anti-Src antibody (UBI, Lake Placid,
N.Y.) for 1 hour at 4.degree. C. followed by 30 min with protein
A-sepharose. The immune complexes were washed three times with
lysis buffer, once with kinase buffer (20 mM HEPES, pH 7.0, 10 mM
MnCl.sub.2 and 10 mM MgCl.sub.2) and resuspended in 30 .mu.l of
kinase buffer containing 1 mCi/ml [.gamma.-.sup.32P]ATP (3000
Ci/mmol; NEN, Boston, Mass.). The reaction was incubated for 10 min
at 30.degree. C. and terminated by the addition of 2.times.SDS
sample buffer. The proteins were resolved in 10% SDS-polyacrylamide
gels, dried and analyzed by autoradiography.
[0082] Immune complexes as prepared for autophosphorylation assays
were washed three times with lysis buffer and once with kinase
buffer. The beads were resuspended in 30 .mu.l of kinase buffer
containing 1 mCi/ml [.gamma.-.sup.32p]ATP and 3-5 mg of acid
treated enolase (Sigma). The reaction was incubated for 10 min at
30.degree. C. and terminated by the addition of 2.times.SDS sample
buffer. The proteins were resolved by 10% SDS-PAGE. Equal loading
of the enolase was determined by staining with Coomassie blue. The
gels were then destained and analyzed by autoradiography.
Radioactive bands were also excised from the gel and quantitated by
scintillation counting.
[0083] Previous studies have demonstrated that p59.sup.fyn and
p56/p53.sup.lyn are expressed in HL-60 cells (Katagiri et al.,
1991). Using autophosphorylation assays, the present inventors
herein show that irradiation of HL-60 cells with 200 cGy was
associated with little if any change in p59.sup.fyn activity at 15
min and 12 hours (FIG. 9A). A more detailed analysis between those
time points revealed similar findings. In contrast, p56/p53.sup.lyn
activity was increased at both 15 min and 12 hours after
irradiation as compared to that in untreated cells (FIG. 9B).
Studies of p56.sup.lck demonstrated little detectable activity in
HL-60 cells before or after exposure to ionizing radiation (FIG.
9C). These findings show that p56/p53.sup.lyn is selectively
activated in HL-60 cells by ionizing radiation. This conclusion is
further supported by the absence of an increase in c-Src activity
following irradiation.
[0084] HL-60 cells were also irradiated with 200 cGy and
immunoprecipitates assayed for both p56/p53.sup.lyn
autophosphorylation and enolase (a substrate protein)
phosphorylation. Irradiation was associated with an increase in
p56/p53.sup.lyn autophosphorylation at 5 min that persisted through
12 hours (FIG. 10A). However, assays at 24 hours after X-ray
treatment revealed declines in p56/p53.sup.lyn signals (FIG.
10A).
[0085] Similar findings were obtained when using enolase as the
substrate. While stimulation of p56/p53.sup.lyn autophosphorylation
was less apparent under these conditions, increases in enolase
phosphorylation were clearly detectable when comparing anti-Lyn
immunoprecipitates from control and irradiated HL-60 cells (FIG.
10B). This increase in activity was rapid and sustained for at
least 12 hours (FIG. 10B). Quantitation of .sup.32P-incorporation
into enolase by scintillation counting demonstrated X-ray-induced
increases in p56/p53.sup.lyn activity of approximately 3-fold at 15
min to 12 hours (FIG. 10B). As observed in autophosphorylation
studies, enolase phosphorylation was also decreased at 24 hours
(FIG. 10B).
[0086] Similar studies were performed at different doses of
ionizing radiation (FIG. 11). Treatment with 25 cGy had little if
any effect on phosphorylation of p56/p53.sup.lyn or enolase. Doses
of 50 cGy, however, were associated with increases in
p56/p53.sup.lyn activity (FIG. 11). Moreover, on the basis of
enolase phosphorylation there was an apparent dose-dependent
stimulation of this kinase (FIG. 11).
[0087] The cellular effects of ionizing radiation are believed to
be related to direct interaction of X-rays with DNA or through the
formation of reactive oxygen intermediates (ROIs) which damage DNA
and cell membranes (Hall, 1988). While the role of different
classes of ROIs in activation of the Src-like kinases is unclear,
recent studies have demonstrated that H.sub.2O.sub.2 and diamide,
which oxidize free sulfhydryl groups in cells, activate p56.sup.lck
in T cells (Nakamura et al., 1993).
[0088] HL-60 cells were either treated with H.sub.2O.sub.2 for the
indicated times or pretreated with 30 mM NAC for 1 hour, irradiated
(200 cGy) and harvested at 12 hours. Irradiated HL-60 cells treated
with H.sub.2O.sub.2 did not show a detectable increase in
phosphorylation of p56/p53.sup.lyn or enolase (FIG. 12A). Cells
were also treated with the antioxidant NAC (Roederer et al., 1990;
Staal et al., 1990), an agent that abrogates oxidative stress by
scavenging certain ROIs and increasing intracellular glutathione
levels (Aruoma et al., 1989; Burgunder et al., 1989). NAC had
little effect on X-ray-induced p56/p53.sup.lyn activity (FIG. 12A),
while this agent completely blocks induction of c-jun and EGR-1
gene expression in irradiated HL-60 cells (Datta et al., 1992b;
1993).
[0089] HL-60 cells were treated with 10 .mu.M herbimycin (H) or 10
.mu.M genistein (G) for 1 hour, irradiated (200 cGy) and then
harvested at 12 hours. Cell lysates were immunoprecipitated with
anti-Lyn and the immunoprecipitates were analyzed for
phosphorylation of p56/p53.sup.lyn and enolase. In marked contrast,
the tyrosine kinase inhibitors, herbimycin and genistein inhibited
X-ray-induced p56/p53.sup.lyn activity (FIG. 12B).
[0090] Previous work has demonstrated that both ionizing radiation
and H.sub.2O.sub.2 are potent inducers of c-jun gene transcription
(Datta et al., 1992b). These two agents have also been used to
support the role of ROIs in targeting CC(A/T).sub.6GG sequences to
mediate activation of the EGR-1 gene (Datta et al., 1993). The
finding that such induction of early response gene transcription is
inhibited by NAC further supports the role of some of these
intermediates in X-ray-induced nuclear signaling mechanisms.
[0091] The present invention provides for the activation of
p56/p53.sup.lyn as a distinct cellular response to ionizing
radiation and not to H.sub.2O.sub.2-induced oxidative stress. These
findings contrast work by others which suggested that Src-like
tyrosine kinases, including p56/p53.sup.lyn, are not responsible
for signaling in irradiated B cells (Uckun et al., 1992a). The
demonstration that ionizing radiation, and not H.sub.2O.sub.2,
induces p56/p53.sup.lyn activity by an NAC-insensitive mechanism
therefore indicates that activation of this tyrosine kinase is
independent from those signals responsible for X-ray-induced early
response gene expression.
[0092] The finding in B cells that p56/p53.sup.lyn is functionally
associated with the cell surface (Yamanashi et al., 1992) suggests
that activation of this kinase by ionizing radiation may be
generated near the plasma membrane rather than in the nucleus.
Indeed, the available evidence supports the involvement of
receptor-mediated signaling in the activation of p56/p53.sup.lyn
(Yamanashi et al., 1992; Pleiman et al., 1993). Src-like proteins
may be activated through dephosphorylation by tyrosine phosphatases
(Mustalin & Altman, 1990; Cantley et al., 1991; Hartwell &
Weinart, 1989) and potentially other mechanisms (Cantley et al.,
1991; Hartwell & Weinart, 1989).
[0093] In regard to the effect of ionizing radiation on the
phosphorylation of p34.sup.cdc2 on tyrosine, HL-60 cells were grown
in RPMI 1640 medium containing 15% heat-inactivated total bovine
serum supplemented with 100 units/ml penicillin, 100 .mu.g/ml
streptomycin and 2 mM L-glutamine. Exponentially growing cells were
suspended in serum free media 18 h prior to irradiation.
Irradiation was performed at room temperature using a Gammacell
1000 (Atomic Energy of Canada, Ottawa) with a .sup.137Cs source
emitting at a fixed dose rate of 13.3 Gy/min as determined by
dosimetry.
[0094] Cells were washed twice with ice cold phosphate buffered
saline and lysed in buffer A (10 mM Tris, pH 7.4, 1 mM EGTA, 1 mM
EDTA, 50 mM NaCl, 5 mM .beta.-glycerophosphate, 1% Triton X-100,
0.5% NP-40, 1 mM sodium vanadate, 1 mM DTT, 1 mM
phenylmethylsulfonyl fluoride and 10 .mu.g/ml of leupeptin and
aprotinin). Insoluble material was removed by centrifugation at
14000 rpm for 5 min at 4.degree. C. Protein concentration was
determined by Coomassie Blue staining using BSA as standard.
[0095] Soluble proteins (50 .mu.g) were separated by
electrophoresis in 10% SDS-polyacrylamide gels and then transferred
to nitrocellulose paper. The residual binding sites were blocked by
incubating the filter in 5% dry milk in PEST (PBS/0.05% Tween 20)
for 1 h at room temperature. The filters were then incubated for 1
h with either mouse anti-phosphotyrosine (anti-P-Tyr; 4G10)
monoclonal antibody (4G10, UBI, Lake Placid, N.Y.) or a mouse
anti-p34.sup.cdc2 monoclonal antibody which is unreactive with
other cyclin-dependent kinases (sc-54; Santa Cruz Biotechnology,
Santa Cruz, Calif.). After washing twice with PEST, the blots were
incubated with anti-mouse or anti-rabbit IgG peroxidase conjugate
(Sigma Chemical Co., St. Louis, Mo.). The antigen-antibody
complexes were visualized by chemiluminescence (ECL detection
system, Amersham, Arlington Heights, Ill.).
[0096] Immunoprecipitations were performed with anti-P-Tyr or
anti-p34.sup.cdc2 at 5 .mu.g/ml cell lysate. Immune complexes were
collected with protein A-Sepharose (Pharmacia) and
immunoprecipitates were analyzed by 10% SDS-PAGE. After transfer to
nitrocellulose and blocking, immunoblot analysis was performed with
either anti-p34.sup.cdc2 or anti-P-Tyr and detected with the
appropriate HRP-conjugated second antibody using the ECL
system.
[0097] HL-60 cells were exposed to 200 cGy ionizing radiation and
monitored for proteins with increased levels of phosphotyrosine.
Using an anti-P-Tyr antibody in immunoblot analyses, reactivity
with a protein of approximately 34 kD was increased at 1 min after
ionizing radiation treatment (FIG. 13A). Similar findings were
obtained at 5 and 10 min, while reactivity was decreased at 15 min
(FIG. 13A). The filters were washed and reprobed with an
anti-p34.sup.cdc2 antibody. The anti-P-Tyr and anti-p34.sup.cdc2
signals were superimposable. Moreover, there was little detectable
change in p34.sup.cdc2 protein levels following exposure to
ionizing radiation (FIG. 13B). Similar findings were obtained with
doses of ionizing radiation from 50 to 500 cGy (FIG. 14A). The
finding that the signals obtained with the anti-p34.sup.cdc2
antibody (FIG. 14B) were also superimposable over those found with
anti-P-Tyr suggested that p34.sup.cdc2 may undergo phosphorylation
on tyrosine following ionizing radiation treatment.
[0098] Extracts of irradiated cells were subjected to
immunoprecipitation with anti-p34.sup.cdc2. The immunoprecipitates
were then monitored by immunoblotting with anti-P-Tyr. The signal
for p34.sup.cdc2 was increased in irradiated as compared to control
cells (FIG. 15A). While this result further supported increased
tyrosine phosphorylation of p34.sup.cdc2, the filter was washed and
reprobed with anti-p34.sup.cdc2 to assay for levels of p34.sup.cdc2
protein. The finding that the anti-p34.sup.cdc2 signals were
similar in control and irradiated cells (FIG. 15B) indicated that
p34.sup.cdc2 undergoes increased phosphorylation on tyrosine
following ionizing radiation exposure.
[0099] Activation of p34.sup.cdc2 requires association with cyclin
B (Pines & Hunter, 1989; Russel & Nurse, 1987) and certain
posttranslational modifications. In Schizosaccharomyces pombe, the
p34.sup.cdc2/cyclin B complex is inactivated by phosphorylation of
p34.sup.cdc2 on tyrosine 15 by Weel (Featherstone & Russell,
1991; Parker et al., 1991; 1992; Gould & Nurse, 1989).
Dephosphorylation of p34.sup.cdc2 on Tyr-15 by the cdc25 gene
product is necessary for activation of p34.sup.cdc2 and entry into
mitosis (Gould et al., 1989; Enoch & Nurse, 1990). The weel and
cdc25 gene products thus determine the timing of entry into mitosis
by a series of phosphorylations and dephosphorylations of
p34.sup.cdc2. Other work in S. pombe has demonstrated that mitotic
checkpoints monitor DNA synthesis and the presence of DNA damage
(Al-Khodairy & Carr, 1992; Rowley et al., 1992; Lock &
Ross, 1990). The DNA damage checkpoint evidently regulates
p34.sup.cdc2 by mechanisms distinct from those induced by the
replication checkpoint (Rowley et al., 1992; Lock & Ross,
1990). Other studies have demonstrated that p34.sup.cdc2 kinase
activity is decreased when CHO cells are exposed to 8 Gy ionizing
radiation (Uckun et al., 1992b).
[0100] The present invention discloses activation of Src-like
tyrosine kinases and phosphorylation of tyrosine kinase substrates,
such as p34.sup.cdc2, as a rapid response to ionizing radiation.
Inhibition of the radiation-induced activation of those tyrosine
kinases prevents or inhibits substrate phosphorylation. Because the
function of those substrates depends on their state of
phosphorylation, inhibition of phosphorylation alters the function
of those substrates. To the extent that substrate function is
responsible for all or part of the cascade of changes associated
with radiation, altering substrate function by inhibition of
phosphorylation alters the cells response to radiation. Thus, the
present invention contemplates a process to alter the response of
cell to radiation, the process comprising inhibiting tyrosine
kinase activity. In a preferred embodiment, the tyrosine kinase in
a Src-like tyrosine kinase of the lyn family.
Example III
DNA Damaging Agents
[0101] Following radiation exposure, many single strand breaks are
produced in DNA, but these are readily repaired using the opposite
strand of DNA as a template. X-ray energy deposition on DNA may
lead not only to strand breakage but to base damage. The breakage
may result in incorrect rejoining in pre-replication chromosomes in
the G.sub.1 phase, leading to chromosomal aberrations, or if the
radiation is given late in S or G.sub.2, chromatid aberrations will
result.
[0102] The skilled artisan in directed to "Remington's
Pharmaceutical Sciences" 15th Edition, chapter 33, in particular
pages 624-652. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biologics standards.
[0103] A variety of other DNA damaging agents may be used with the
tyrosine kinase inhibitors, as provided by this invention. This
includes agents that directly crosslink DNA, agents that
intercalate into DNA, and agents that lead to chromosomal and
mitotic aberrations by affecting nucleic acid synthesis.
[0104] Agents that induce DNA alkylation, such as mitomycin C, may
be used. Mitomycin C is an extremely toxic antitumor antibiotic
that is cell cycle phase-nonspecific. It is almost always given
intravenously, at a dose of 20 mg/meter.sup.2, either in a single
dose or given in 10 separate doses of 2 mg/meter.sup.2 each given
over 12 days. It has been used clinically against a variety of
adenocarcinomas (stomach, pancreas, colon, breast) as well as
certain head and neck tumors.
[0105] Another option is to employ cisplatin, which has also been
widely used to treat cancer, with efficacious doses used in
clinical applications of 20 mg/m.sup.2 for 5 days every three weeks
for a total of three courses. Cisplatin is not absorbed orally and
must therefore be delivered via injection intravenously,
subcutaneously, intratumorally or intraperitoneally.
[0106] Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis, and chromosomal segregation.
Examples of these compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in clinical setting for the treatment of neoplasms
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals
for adriamycin, to 35-50 mg/m2 for etoposide, intravenously or
double the intravenous dose orally.
[0107] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors, and subunits also lead to DNA damage. As such a
number of nucleic acid precursors have been developed. Particularly
useful are agents that have undergone extensive testing and are
readily available. As such, agents such as 5-fluorouracil (5-FU),
are preferentially used by neoplastic tissue, making this agent
particularly useful for targeting to neoplastic cells. Although
quite toxic, 5-FU, is applicable in a wide range of carriers,
including topical, however intravenous administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
Example IV
Tyrosine Kinase Inhibitors
[0108] Tyrosine protein kinase activities are known to be
associated with oncogene products of the retroviral src gene
family, and also with several cellular growth factor receptors such
as that for epidermal growth factor (EGF). Activation of protein
tyrosine phosphorylation by p56/p53.sup.lyn in the present studies
demonstrates that the lyn protein is associated with the cell cycle
regulatory protein p34.sup.cdc2, contributing to mitotic arrest. If
this association is blocked, such as by use of protein tyrosine
kinase inhibitors such as genistein or herbimycin A, the cells are
unable to arrest in the G.sub.2 phase, forcing cell cycle traverse
and expression of potentially lethal damage. Thus, the combined use
of DNA damaging agents such as ionizing radiation or alkylating
agents with tyrosine kinase inhibitors is a novel approach to
enhancing cell killing.
[0109] Genistein, a natural isoflavonoid phytoestrogen, has been
reported to exhibit specific inhibitory activity against tyrosine
kinases of EGF receptor, pp60.sup.v-src and pp110.sup.gag-fes. It
has been generally shown to block a number of EGF dependent
phenomena, including both receptor autophosphorylation and histone
phosphorylation.
[0110] Herbimycin A has also been shown to inhibit the
autophosphorylation of EGF-stimulated receptors in intact cells in
a time and dose dependent manner. Herbimycin A both decreases the
receptor quantity and the EGF-stimulated receptor kinase
activity.
[0111] Other tyrosine kinase inhibitors may also be used, for
example, those isolated from natural sources. One such compound is
erbstatin (Umezawa and Imoto M, 1991; Sugata et al., 1993) and its
analogues, e.g., RG 14921 (Hsu et al., 1992). Lavendustin A from
Streptomyces griseolavendus (Onoda et al., 1989), which is about 50
times more inhibitory than erbstatin, and analogues thereof, are
also contemplated for use as protein-tyrosine kinase inhibitors
(Smyth et al., 1993b). Piceatannol
(3,4,3',5'-tetrahydroxy-trans-stilbene; Geahlen and McLaughlin,
1989) and polyhydroxylated stilbene analogues thereof (Thakkar et
al., 1993) may also be used.
[0112] Further natural tyrosine kinase inhibitors that may be used
are emodin (3-methyl-1,6,8-trihydroxyanthraquinone), an inhibitor
from the Chinese medicinal plant Polygonum cuspidatum (Jayasuriya
et al., 1992; Chan et al., 1993); desmal
(8-formyl-2,5,7-trihydroxy-6-methylflavanone), isolated from the
plant Desmos chinensis (Kakeya et al., 1993); the chlorosulfolipid,
malhamensilpin A, isolated from the cultured chrysophyte
Poterioochromonas malhamensis (Chen et al., 1994); flavonoids
obtained from Koelreuteria henryi (Abou-Shoer et al., 1991);
fetuin, a natural tyrosine kinase inhibitor of the insulin receptor
(Rauth et al., 1992).
[0113] Another group of compounds known to be tyrosine kinase
inhibitors are the tyrphostins, which are low molecular weight
synthetic inhibitors (Gazit et al., 1989). The tyrphostins AG17,
AG18, T23 and T47 have been shown to inhibit pancreatic cancer cell
growth in vitro (Gillespie et al., 1993). Tyrphostins have also
been shown to have antiproliferative effects on human squamous cell
carcinoma in vitro and in vivo (Yoneda et al., 1991). RG-13022 and
RG-14620 were found to suppress cancer cell proliferation in vitro
and tumor growth in nude mice. Another active tyrphostin is AG879
(Ohmichi et al., 1993).
[0114] Various chemical compounds may also be used in combination
with DNA damaging agents, such as ionizing radiation, as have been
described in the literature for use alone. One example is RG50864
(Merkel et al., 1993). Further examples are the indole substituted
2,2'-dithiobis(1-methyl-N-phenyl-1H-indole-3-carboxamides,
especially the 5-substituted derivative, as described by Rewcastle
et al. (1994).
(Z)-alpha-[(3,5-dichlorophenyl)methylene]-3-pyridylacetonitrile (RG
14620) is another active tyrosine kinase inhibitor that may be used
in a topical or intravenous form (Khetarpal et al., 1994).
[0115] BE-23372M,
(E)-3-(3,4-dihydroxybenzylidene)-5-(3,4-dihydroxyphenyl)-2(3H)-furanone,
is also a tyrosine kinase inhibitor (Tanaka et al., 1994a). This
may be synthesized from 3-(3,4-dimethoxybenzoyl)propionic acid and
veratraldehyde or 3,4-diacetoxy-benzaldehyde, as described by
Tanaka et al. (1994b). BE-23372M may also be isolated from the
culture broth of a Rhizoctonia solani fungus (strain F23372) using
acetone and then purified by solvent extraction and column
chromatography (Okabe et al., 1994).
[0116] Further tyrosine kinase inhibitors that may be used include
4,5-Dianilinophthalimide, which has, alone, been shown to have in
vivo antitumor activity (Buchdunger et al., 1994). Hydroxylated
2-(5'-salicyl)naphthalenes form another group of inhibitors that
could be used in the present invention, and may be prepared as
described by Smyth et al. (1993a).
Example V
Treatment Protocols
[0117] 1) Patients exhibiting neoplastic disease are treated with a
protein kinase inhibitor, for example genistein, at a concentration
of between 1 and 100 .mu.M, or herbimycin A at a concentration of
between about 1 and 100 .mu.M, for 6 hours prior to exposure to a
DNA damaging agent. [0118] 2) Patients are exposed to ionizing
radiation (2 gy/day for up to 35 days), or an approximate a total
dosage of 700 gy. [0119] 3) As an alternative to ionizing radiation
exposure, patients are treated with a single intravenous dose of
mitomycin C at a dose of 20 mg/m.sup.2.
[0120] It is contemplated that mitomycin C treatment in combination
with tyrosine protein kinase inhibitors will be effective against
cancer of the stomach, pancreas, oral cavity, breast and
head/neck.
[0121] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the composition, methods and in the
steps or in the sequence of steps of the method described herein
without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
2114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 1Ile Glu Lys Ile Gly Glu Gly Thr Tyr Gly Val Val
Tyr Lys1 5 10214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 2Ile Glu Lys Ile Gly Glu Gly Thr Phe Gly
Val Val Tyr Lys1 5 10
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