Note: Descriptions are shown in the official language in which they were submitted.
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DELIVERY OF AN AGENT TO THE DUCTAL EPITHELIUM IN THE PROPHYLACTIC AND
THERAPEUTIC TREATMENT OF CANCER
This application claims priority to U.S. patent
application serial number 08/510,623, which was filed on
August 3, 1995, and has been converted to U.S.
provisional patent application serial number not yet
known. The invention disclosed in this application was
made with government support under NIH planning grant P20
CA/ES66205, "Gene-mediated prevention of Cancer," and
grant NIH 1801 CA 57993, "Genetic and hormonal factors in
mammary carcinogenesis," awarded by the National
Institutes of Health, and with support of the American
Cancer Society under grant RD388, "Targeted disruption of
breast cells: a novel strategy for cancer prevention."
Therefore, the government has certain rights in this
invention.
Technical Field of the Invention
The present invention relates to the use of an
epithelium-destroying agent to destroy the epithelium of
an exocrine gland, particularly the mammary gland, in the
prophylactic and therapeutic treatment of disease, in
particular cancer.
Background of the Invention
Exocrine glands are glands that release a secretion
external to or at the surface of an organ by means of a
canal or duct. Examples of exocrine glands include,
among others, the mammary glands, prostate, liver, gall
bladder, pancreas, kidneys, sweat glands, and salivary
glands. Cancers of exocrine glands pose a major health
problem, frequently resulting in death. Currently,
' 35 cancers of the breast and prostate are among the leading
causes of death among women and men, respectively.
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The mature human breast comprises from six to nine
major ducts, which emanate from the nipple, serially
branch into ducts and terminate in lobuloalveolar
structures (Russo et al., Lab. Invest. 62(3): 244-278
(1990)). This branching network of ducts is composed of
epithelial cells in a supporting matrix of connective
tissue and endothelial cells.
Tissues removed from the human female breast during
surgery and autopsy have been examined in numerous
studies directed to the nature and site of origin of
neoplastic growth. Subgross sampling and histological
confirmation have enabled pathological characterization
of entire breasts, leading to the postulation of the
existence of four major possible sites of origin of
mammary carcinomas, namely ducts, terminal ducts,
ductules, and acini (Russo et al., supra). Ductal origin
is supported by the presence of more extensive epithelial
proliferations, which are presumed to be preneoplastic,
in surgically removed cancerous breasts as compared to
nonmalignant breasts removed during autopsies (Russo et
a 1. , s upra ) .
With a cumulative lifetime risk of a woman
developing breast cancer estimated to be 1 in 9, there is
an urgent need to develop therapeutic methods of
treatment that are more effective, less invasive and
accompanied by fewer side effects and prophylactic
methods of treatment that are more effective than
increased and intensified physical monitoring and far
less extreme than radical mastectomy. In spite of the
recent discovery of the heritable breast cancer
susceptibility loci, BRCA1 (Miki et al., Science 266: 66-
71 (1994)) and BRCA2, and other cancer susceptibility
loci, and the increasing ability of physicians to
identify women with elevated breast cancer risk, .
prophylactic methods are still currently limited to
physical monitoring and prophylactic mastectomy.
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In view of the above, it is an object of the present
invention to provide a method of locally treating an
exocrine gland prophylactically for disease. It is
another object of the present invention to provide a
method of locally treating an exocrine gland, in
particular the mammary gland, prophylactically for
cancer. Another object of the present invention is to
provide a method of locally treating an exocrine gland
therapeutically for disease. Yet another object of the
present invention is to provide a method of locally
treating a mammary gland therapeutically for cancer.
Still yet another object of the present invention is to
provide a method of locally treating a mammary gland both
therapeutically and prophylactically for cancer. These
and other objects and advantages, as well as additional
inventive features, will become apparent from the
detailed description provided herein.
Summary of the Invention
The present invention provides prophylactic and
therapeutic methods of treating the ductal epithelium of
an exocrine gland, in particular the mammary gland, for
disease, in particular cancer. The method comprises
contacting the ductal epithelium of the exocrine gland,
in particular the mammary gland, with an epithelial-
destroying agent. The ductal epithelium is preferably
contacted with the agent by ductal cannulation. The
epithelium-destroying agent is preferably a vector
comprising a thymidine kinase gene, which is used in
combination with ganciclovir (GCV), which can be
systemically administered. Another preferred epithelium-
destroying agent is a vector comprising a hypoxanthine
phosphoribosyl transferase (HPRT) gene, which is used in
combination with hypoxanthine aminopterin thymidine (HAT)
nucleotide, which can be systemically administered. Also
preferred as an epithelium-destroying agent is a vector
comprising a gene, which, upon transformation of a cell
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of the ductal epithelium and expression therein, induces
apoptosis or death of the transformed cell. A preferred
apoptosis-inducing gene is bclxs. Other preferred
epithelial-destroying agents include a cytolytic virus,
such as Vaccinia virus, and ethanol. The preceding
methods can additionally comprise contacting the ductal
epithelium with a cytokine or hematopoietic growth
factor, such as GM-CSF. Also provided by the present
invention is a method of treating the ductal epithelium
of a mammary gland both therapeutically and
prophylactically for cancer. The combined
therapeutic/prophylactic method comprises treating the
mammary gland therapeutically by surgery, radiation
and/or chemotherapy and contacting the ductal epithelium
of the mammary gland, either concomitantly or
subsequently, with an epithelium-destroying agent, which
does not specifically target cancerous cells. The
combined therapeutic/prophylactic method can additionally
comprise contacting the ductal epithelium with a cytokine
or hematopoietic growth factor, such as GM-CSF.
Brief Description of the Fi ures
Figure 1 is a graph of the ratio of cell no. in the
presence of GCV over cell no. in the absence of GCV (Cell
No. + GCV/Cell No. - GCV) versus viral multiplicity of
infection (viral MOI), which shows the effect of the
addition of 10 ~,i,g/ml GCV on NMU68 and RBA rat tumor cell
lines 6 hrs after transduction with adenoviral-Herpes
siircplex thymidine kinase (AdHS-tk) at titers of 0, 100,
500 and 1,000 moi. After the cells were maintained in
the presence of GCV for 3 days, they were counted using
trypan blue exclusion as a measure of cell viability and
cell numbers were normalized to the growth of cells in
the absence of ganciclovir (GCV).
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Detailed Descri tion of the Invention
The present invention is based on the observation
that the overwhelming majority of breast cancers arise
from epithelial cells, particularly those epithelial
5 cells which line the ducts of the mammary gland and are
collectively referred to as the ductal epithelium. The
present invention is also based on the exocrine nature of
the mammary gland. Given that the mammary gland is an
exocrine gland, it was further observed that the central
canal or duct could provide a means of directly accessing
the ductal epithelium for localized prophylactic and
therapeutic treatment of cancer. Based on these
observations, the prophylactic and therapeutic methods of
the present invention were developed.
Prophylactic Method
The prophylactic method of the present invention is
a method of treating the ductal epithelium of an exocrine
gland prophylactically for a disease that affects the
ductal epithelium of the exocrine gland. The method
comprises contacting, preferably by ductal cannulation,
the ductal epithelium of the exocrine gland with an
epithelium-destroying agent so as to destroy cells of the
ductal epithelium affected by the disease.
In one embodiment of the prophylactic method of the
present invention, the ductal epithelium of a mammary
gland is treated prophylactically for cancer so as to
inhibit the formation of cancer of ductal epithelial
origin. The method comprises contacting, preferably by
ductal cannulation, the ductal epithelium of the mammary
gland with an epithelium-destroying agent. The agent
preferably is a vector comprising a thymidine kinase
gene, such as a Herpes simplex thymidine kinase gene, and
ganciclovir, a vector comprising a HPRT gene and HAT
nucleotide, a cytolytic virus, such as a Vaccinia virus,
or ethanol. The method can additionally comprise
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contacting the ductal epithelium with a cytokine or
hematopoietic growth factor, such as GM-CSF.
In another embodiment of the prophylactic method of
the present invention, the ductal epithelium of an
exocrine gland, such as a mammary gland, is treated
prophylactically for cancer so as to inhibit the
formation of cancer of ductal epithelial origin. The
method comprises contacting, preferably by ductal
cannulation, the ductal epithelium of the exocrine gland
with an epithelium-destroying agent to destroy less than
all of the ductal epithelium so as to inhibit the
formation of cancer of ductal epithelial origin.
Preferably, up to about 70~, 80~, 85~, 90~ or 95~ of the
ductal epithelium is destroyed.
The epithelium-destroying agent is preferably a
vector comprising a thymidine kinase gene, such as that
from Herpes simplex, and ganciclovir, which can be
brought into contact with the ductal epithelium by any
suitable means, preferably by ductal cannulation or by
systemic administration, a vector comprising a HPRT gene
and HAT nucleotide, which can be brought into contact
with the ductal epithelium by any suitable means,
preferably by ductal cannulation or by systemic
administration, a vector comprising a gene, which upon
transformation of a cell of the ductal epithelium and
expression therein, induces apoptosis or death of the
transformed cell, such as bclxs, ethanol, or a cytolytic
virus, such as Uaccinia virus.
The above method can additionally comprise the
administration of a cytokine or hematopoietic growth
factor, such as GM-CSF. The GM-CSF can be brought into
contact with the ductal epithelium of the mammary gland
by any suitable means, such as by ductal cannulation of
GM-CSF or a vector comprising a gene encoding GM-CSF, in
which case the vector can be the same vector as the one
encoding the thymidine kinase, HPRT or apopotosis-
inducing gene, or it can be systemically administered.
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This embodiment of the prophylactic method can be
used to treat any exocrine gland. However, it is
particularly useful in the treatment of the mammary
gland.
The above-described prophylactic method of treating
a mammary gland is particularly useful in treating a
mammary gland in a mammal at risk for developing breast
cancer. The mammary gland can be characterized as one
that has never had a tumor, one that had a tumor
previously but the tumor is no longer detectable due to
other prior therapeutic treatment, or one that has an
incipient or occult tumor, preneoplasia or ductal
hyperplasia. Normally, hyperplasias and incipient and
occult tumors are not detectable by means of physical
examination or radiography. Accordingly, the
prophylactic method will find use in cases where there is
reason to take some prophylactic measures, such as when
there are known inherited factors predisposing to
cancers, where there are suspicious lesions present in a
breast with the potential for developing into a
malignancy, where there has been exposure to carcinogenic
agents in the environment, where age predisposes to a
cancer, where cancer of another gland, e.g., the mammary
gland of the contralateral breast, suggests a propensity
for developing cancer, or where there is a fear or
suspicion of metastasis.
Therapeutic Method
The therapeutic method of the present invention is a
method of treating the ductal epithelium of an exocrine
gland therapeutically for a disease that affects the
ductal epithelium of the exocrine gland. The method
comprises contacting, preferably by ductal cannulation,
the ductal epithelium of the exocrine gland with an
epithelium-destroying agent so as to destroy cells of the
ductal epithelium affected by the disease.
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In one embodiment of the therapeutic method of the
present invention, the ductal epithelium of a mammary
gland is locally treated therapeutically for cancer so as
to destroy cancerous and noncancerous cells of the ductal
epithelium and inhibit the spread of cancer. The method
comprises contacting, preferably by ductal cannulation,
the ductal epithelium of the mammary gland with an
epithelium-destroying agent, which need not, and
preferably does not, specifically target cancerous cells.
The agent preferably is a vector comprising a thymidine
kinase gene, such as a Herpes simplex thymidine kinase
gene, and ganciclovir, a vector comprising a HPRT gene
and HAT nucleotide, a cytolytic virus, such as a Vaccinia
virus, or ethanol. The method can additionally comprise
contacting the ductal epithelium with a cytokine or
hematopoietic growth factor, such as GM-CSF.
In the therapeutic method, the epithelial-destroying
agent should destroy all of the diseased or malignant
epithelium. In addition, the ductal epithelium
immediately surrounding the diseased/malignant epithelium
also preferably should be destroyed.
Combined Therapeutic/Prophylactic Method
The present invention also provides a method of
treating the ductal epithelium of a mammary gland both
therapeutically and prophylactically for cancer. The
method comprises treating the mammary gland
therapeutically with any given therapeutic method, such
as those currently known and used in the art. Examples
of such methods include surgical removal of the cancerous
tissue, radiation therapy and chemotherapy. The method
further comprises contacting, either concomitantly with
or subsequently to the therapeutic treatment, the ductal
epithelium of the mammary gland, e.g., by ductal
cannulation, with an epithelium-destroying agent, which
preferably does not specifically target cancerous cells,
so as to destroy any remaining cancerous cells and
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noncancerous cells and inhibit the spread of cancer. The
epithelium-destroying agent is preferably a vector
comprising a thymidine kinase gene, such as a Herpes
simplex thymidine kinase gene, combined with ganciclovir,
a vector comprising a HPRT gene combined with HAT
nucleotide, a cytolytic virus, such as a Uaccinia virus,
or ethanol. The method can additionally comprise
contacting the ductal epithelium with a cytokine or
hematopoietic growth factor, such as GM-CSF.
Alternative Embodiments of Prophylactic &
Therapeutic Methods
Although preferred embodiments have been described
above, the methods of the present invention can be used
to treat the ductal epithelium of any exocrine gland.
Examples of exocrine glands, other than the mammary
gland, which can be treated with the present inventive
methods include, among others, the prostate, liver, gall
bladder, pancreas, kidneys, sweat glands, and salivary
glands. The methods are especially useful in the
prophylactic and therapeutic treatment of the ductal
epithelium of mammary glands.
Similarly, the methods can be used to treat an
exocrine gland for any disease that affects the exocrine
ductal epithelium. The methods are particularly useful
in the treatment of cancer, including the stages of
hyperplasia, adenoma, carcinoma in situ, and carcinoma,
of ductal epithelial origin.
Any method can be used to destroy the ductal
epithelium. It is preferred, however, that the
destruction is limited to the ductal epithelium or a part
thereof .
Any method of contacting the ductal epithelium can
be used to effect local treatment. Preferably, ductal
cannulation is used. Although any duct or lobule can be
cannulated, it is preferred that the central canal or
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to
duct be cannulated. Ductal cannulation also enables
direct injection of a tumor mass, if desired.
Any epithelium-destroying agent can be used to
destroy the ductal epithelium of an exocrine gland. The
agent preferably should not destroy cells other than
cells of the ductal epithelium and preferably should not
result in side effects, the adversity of which outweigh
the benefits of destruction of the ductal epithelium. In
no event should the methods be used to destroy completely
the ductal epithelium of an exocrine gland in the
prophylactic/therapeutic treatment of a given disease,
wherein the complete destruction of the ductal
epithelium, in and of itself, would result in death of
the mammal so treated.
Examples of agents that can be used in the context
of the prophylactic and therapeutic methods of the
present invention include cytotoxic agents. Any
cytotoxic agent known in the art and suitable for
contacting the ductal epithelium of an exocrine gland of
a mammal can be used. In addition to ethanol and GCV
described above, other examples of cyotoxic agents and
their prodrugs include genistein, okadaic acid, 1-(3-D-
arabinofuranosyl-cytosine, arabinofuranosyl-5-aza-
cytosine, cisplatin, carboplatin, actinomycin D,
asparaginase, bis-chloro-ethyl-nitroso-urea, bleomycin,
chlorambucil, cyclohexyl-chloro-ethyl-nitroso-urea,
cytosine arabinoside, daunomycin, etoposide, hydroxyurea,
melphalan, mercaptopurine, mitomycin C, nitrogen mustard,
procarbazine, teniposide, thioguanine, thiotepa,
vincristine, 5-fluorouracil, 5-fluorocytosine,
adriamycin, cyclophosphamide, methotrexate, vinblastine,
doxorubicin, leucovorin, taxol, anti-estrogen agents such
as tamoxifen, intracellular antibodies against oncogenes,
the flavonol quercetin, Guan-mu-tong extract, retinoids
such as fenretinide, nontoxid retinoid analogues such as
N-(4-hydroxyphenyl)-retinamide (HPR), and monoterpenes
such as limonene, perillyl alcohol and sobrerol.
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Preferably, the agent is locally administered, especially
if administration of the agent is accompanied by toxic
side effects. Otherwise, the agent can be administered
by any suitable route, such as systemic administration.
A cytolytic virus also can be used as an agent. Any
cytolytic virus can be used as long as the organism
mounts a rapid immunological response to it such that the
virus cannot cause disease if it escapes the ductal
epithelium. Examples of cytolytic viruses include
Vaccinia viruses and Sindbis viruses, which can also be
used as vectors. Preferably, a Vaccinia virus is used.
Due to lack of mucosal immunity, Vaccinia infectious
particles enter and lyse the breast epithelial cells, yet
stromal immunity destroys the particles as soon as they
leave the ductile tree of the exocrine gland, thereby
preventing cytolysis beyond the ductal epithelium. The
advantages of Vaccinia administration are that it
eliminates the need for high titer virus, the need to
induce cell division in the breast, and the need to
administer a drug to effect cell death.
A vector comprising a suicide gene also can be used
as an agent, in conjunction with an agent that destroys
the ductal epithelium of an exocrine gland. The vector
comprising a suicide gene, upon transformation of a cell
of the ductal epithelium and expression therein, renders
the transformed cell sensitive to the epithelium-
destroying agent, increases the sensitivity of the
transformed cell to the agent, converts the agent from a
prodrug to an active drug, activates the conversion of
the agent from a prodrug to an active drug, enhances the
effect of the agent or, itself, produces a protein that
is cytotoxic. A preferred suicide gene for use in the
present inventive methods is the one described above,
. i.e., a thymidine kinase, such as the one from Herpes
simplex, which phosphorylates GCV, which, in turn,
inhibits DNA replication. Another example of a suicide
gene is cytosine deaminase, which is used in conjunction
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with 5-fluorocytosine. If the vector comprising the
suicide gene is administered locally to the ducts, the
cytotoxic agent or precursor can be administered ,
systemically, since only transfected cells will be
affected. In this regard, the bystander effect, i.e., ,
the death of neighboring uninfected cells, presumably due
to transfer of toxic byproducts through gap junctions
between cells in the same compartment, obviates the need
for every cell in the ductal epithelium, which is to be
destroyed, to be infected. However, sufficient time must
be allowed between contacting the ductal epithelium with
the suicide gene and the prodrug, for example, to achieve
efficient killing of the breast epithelial cells.
A vector comprising an apoptosis-inducing gene also
can be used as an agent that destroys the ductal
epithelium of an exocrine gland (Vaux, Cell 76: 777-779
(1994)). Examples of apoptosis-inducing genes include
ced genes, myc genes (overexpressed), the bclxs gene, the
bax gene, and the bak gene. The apoptosis-inducing gene
causes death of transfected cells, i.e., by inducing
programmed cell death. For example, the bclxs gene, bax
gene, or bak gene can be used to inhibit bc1-2 or bc1-xz,
leading to apoptosis. Where necessary, a vector
comprising an apoptosis-inducing gene can be used in
combination with an agent that inactivates apoptosis
inhibitors such as bc1-z, p35, IAP, NAIP, DAD1 and A20
proteins.
Suicide and apopotosis genes can be administered by
way of a viral vector, such as an adenoviral or
retroviral vector. Adenoviral vectors enable the
generation of high titer recombinant viruses (1011/m1) and
the efficient transduction of postmitotic cells because '
adenoviral DNA exists as an episome in the nucleus
(Verma, Molecular Medicine 1: 2-3 (1994)). -
According to one preferred embodiment, the gene can
be under the transcriptional regulation of a Rous sarcoma
viral promoter. Alternatively, it can be under the
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control of an epithelial tissue-specific or cell-specific
promoter.
Uptake of recombinant virus can be facilitated by
pretreatment or simultaneous treatment with polybrene or,
for example, in the case of a retrovirus, attachment of
the functional fragment of an antibody to the viral
particle.
In another embodiment, the apoptosis gene or suicide
gene can be present in a recombinant microorganism, which
will express the gene. One particularly preferred
microorganism for this purpose is the bacterium Listeria
monocytogenes.
Other methods known in the art for introduction of
raw DNA into cells can be used in the methods of the
present invention. Alternatively, liposomes, complexes
between polypeptide ligands for receptors on mammary
ductal epithelial cells, including complexes of
antibodies and functional fragments thereof, and plasmids
can be used (Mulligan, Science 260: 926-931 (1993)).
Epithelial cell-specific promoters, such as whey acidic
protein (wap), can be used to target expression of a
given gene, e.g., a suicide gene, in ductal epithelial
cells. Use can also be made of wild-type tumor
suppressor genes, such as p53 or Mcs-1 (rat), homeobox
genes expressed in normal cells but not in cancerous
cells, and the maspin gene.
Additionally, the ductal epithelium can be contacted
with an agent to effect the scavenging of epithelial
cells destroyed in accordance with the present invention,
e.g., a cytokine/growth factor. Suitable
cytokines/growth factors include GM-CSF, G-CSF, IL-2, IL-
4, IL-6, IL-7, hCG, TNF-a, INF-a and INF-y. Such factors
can be contacted with the ductal epithelium directly or
by expression of a vector comprising a gene encoding the
factor, in which case the vector can be the same one that
comprises a suicide gene, for example. The factors
stimulate a potent, long-lasting, and specific cell
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immunity, requiring both CD4 and CD8 cells. The immune
response is designed to scavenge destroyed ductal
epithelial cells by generating autoimmunity towards
epithelial cell antigens.
The ductal epithelium is preferably contacted with
the agent by introduction of the agent through the
central canal or duct of the exocrine ductal epithelium,
such as by ductal cannulation. However, in the case of
the mammary gland, for example, there are 6-9 major ducts
that emanate from the nipple and serially branch into
other ducts, terminating in lobulo-alveolar structures
(Russo et al. (1990), supra). Accordingly, in some
circumstances, such as those in which even more localized
treatment is necessary or desired, for example, by the
choice of anti-cancer agent, it may be preferable to
contact the ductal epithelium of the exocrine gland
through one of the other ducts or through a lobulo-
alveolar structure as opposed to the central canal or
duct. In this regard, ductal cannulation enables
intratumoral injection.
The methods of the present invention can be combined
with other methods of prophylactic and therapeutic
treatment in addition to those cited above, such as
methods that target destruction of cancer cells, e.g., by
targeting of cell-surface markers, receptor ligands,
e.g., ligands to gastrin-releasing peptide-like
receptors, tumor-associated antigens, e.g., the 57 kD
cytokeratin or the antigen recognized by the monoclonal
antibody GB24, the extracellular matrix glycoprotein
tamascin, antisense oncogenes such as c-fos, homeobox
genes that are expressed in cancer cells but not normal
cells, tumor-infiltrating lymphocytes that express
cytokines, RGD-containing peptides and proteins, which
are administered following surgery, lipophilic drug- -
containing liposomes to which are covalently conjugated
monoclonal antibodies for targeting to cancer cells, low
fat diet, moderate physical exercise and hormonal
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modulation. For prostate cancer, anti-testosterone
agents can be used as well as an inhibitor of cell
proliferation produced by prostatic stromal cells and C-
CAM, an epithelial cell adhesion molecule.
5 The following examples are presented to illustrate
the present invention, not to limit its scope. The
examples make use of the rat mammary tumor model, which
has been deemed an appropriate experimental model for
understanding breast cancer in humans (Sukumar et al.,
10 Mutation Res. 333 (1-2): 37-44 (1995); Russo et al.,
supra). In fact, 90-100 of female rats develop mammary
tumors in this model when they are administered the
carcinogen NMU at 55 days of age (Sukumar, Cancer Cells
4: 199-204 (1990) ) .
Examples
Example 1
This example demonstrates the successful delivery of
virus and other agents into the mammary ductile tree by
a
single injection through the teat.
ADV/CMV-(3-gal (from Dr. William Burns, Johns Hopkins
University) is an adenoviral 5 vector constructed with a
(3-galactosidase gene controlled by a cytomegaloviral
promoter. It was delivered into the mammary gland by
injection of a viral suspension in 20 ~.l of 0.2~ trypan
blue in Tris buffer through the teat of a rat. The
nipple was extruded, and the sphincter removed by
excising the nipple. In the rat, the muscle prevents
fluid from regurgitating into the breast and had to be
excised in order to visualize the ductal opening and
administer the agent. Trypan blue was used as a tracking
dye to ensure correct delivery to the ductile tree.
Injection about 30 days postpartum resulted in the
. mammary epithelial tree being clearly visible. Ethyl
alcohol (70~) was also successfully delivered by a single
injection through the teat.
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Example 2
This example demonstrates that adenovirus can
efficiently transduce human mammary epithelial cells in ,
vitro.
ADV/CMV-(3-gal was used to transduce HBL100 mammary
epithelial cells in vitro. The ~3-gal enzyme in this
construct contains a nuclear localization signal and
results in dense nuclear staining. HBL100 cells (102)
were plated in 24-well plates, and transduced with virus
at various doses and stained with X-gal 48 hrs later.
Essentially all cells were infected at a moi = 104.
This experiment also has been performed in human
mammary tumor cells MCF-7 (American Type Culture
Collection (ATCC), Rockville, MD), human mammary
epithelial cells MCF-l0A (ATCC), and two rat mammary
cancer cell lines, RBA (from Dr. Leonard Cohen) and 37-2
(from Dr. C. Marcelo Aldaz) with the same results. More
efficient adenoviral constructs have been used, thereby
achieving 100 infection at a moi = 103. These
experiments demonstrate successful infection by and
expression of adenovirus carrying the lacZ indicator
gene, which permits staining the cells blue by the
expression of the enzyme (3-galactosidase.
Example 3
This example demonstrates that infection with an
AdHS-tk construct followed by GCV treatment effectively
kills mammary tumor cells in vitro.
RBA and NMU68 are two rat mammary tumor cell lines
derived from a DMBA- and a NMU-induced tumor,
respectively [DMBA = dimethylbenz[aJanthracene, NMU = N'-
nitro N' -methylureaJ. Each cell line was plated at a '
density of 5 x 102 in 48-well plates (1.l cm) and allowed
to settle overnight. The next morning, they were '
transduced with AdHS-tk (Chen et al., PNAS(USA) 91: 3054-
57 (1994); obtained from S. Woo and E. Aguilar-Cordova,
Baylor College of Medicine, Houston, TX) at titers of 0,
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100, 500, and 1000 moi, and then, 6 hrs later, GCV (10
~g/ml) was added to the culture media. The cells were
maintained in the presence of GCV for 3 days and then
counted using trypan blue exclusion as a measure of cell
viability. The cell numbers were normalized to the
growth of cells in the absence of GCV. The results are
shown in Figure 1. More than 80~ of the cells of each
cell line were killed at a moi of 103.
Example 4
This example demonstrates the prophylactic effect of
the method of the present invention.
The mammary glands (6 on each side) of six virgin 50
day old Sprague Dawley rats were injected with AdHS-tk on
the left side and trypan blue on the right side or left
untreated. One rat remained completely untreated with
the virus or GCV and served as a positive control for
NMU-induced tumorigenesis. On the day of surgery, rats
were given an intramuscular injection of 5 Er.g estradiol
valerate and were anesthetized with an isofluorane/OZ
mixture. The nipples were cannulated with a 33 gauge
needle. Twenty ~,1 of AdHS-tk diluted in trypan blue
carrier (1 mM MgCl2, 20 ~tg/ml polybrene in 10~ glycerol,
0.4~ trypan blue in saline) at a concentration of 5 x 10'
particles/~.~,1 were injected into the duct. Carrier
control mammary glands received 20 ~,1 of trypan blue
carrier alone. An animal was considered treated when at
least three glands were successfully injected with trypan
blue and another three glands were successfully injected
with AdHS-tk. The remaining glands were left untreated.
Twelve hrs later, the rats were injected with 125 mg/kg
body weight GCV twice daily for three days. The rats
were then given a second intramuscular injection of 5 ~,g
- estradiol valerate and intraperitoneal injections of 100
~,~.g/kg body weight GCV once daily for three days. Five to
seven days after GCV treatment, the rats were given an
intravenous injection of NMU dissolved in 0.05 acetic
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18
acid (Ash Stevens, CO; 50 mg NMU/kg body weight) and were
subsequently monitored for general health and the
appearance of tumors at weekly intervals for 8 months.
The results were as follows:
Treatment No. of Tumors No. of Total ~ of Glands
Glands with Tumor
Untreated 5 12 41.7
Control
Trypan Blue 5 17 29.4
and
Ganciclovir
No injection; 5 20 25.0
Ganciclovir
AdHS-tk and 2* 35 5.7
Ganciclovir
(*) The two tumors (size < 5 mm) in this group were
detected during necropsy at the termination of the
experiment 8 months later. The difference between tumors
appearing in treated versus control glands was
significant by Chit analysis (p<0.01).
The above results show that the method of the
present invention inhibits the formation of cancer of
ductal epithelial origin in this rat model, in which NMU
induces the formation of mammary tumors in 90-100 of
female rats of similar age. Surprisingly and
unexpectedly, this prophylactic effect was achieved
without extensive destruction of the mammary glands.
These data demonstrate that selective destruction of
epithelial cells, e.g., key stem cells, can be sufficient -
to provide prophylactic protection against carcinogen-
induced tumor formation in the ductal epithelium of the
mammary gland.
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Example 5
This example demonstrates the efficient transfection
of mammary epithelial cells in vivo.
Lytic Vaccinia virus (106 Vaccinia-HA, which also
carries lacZ, in 20 ~.~,1 0.2$ trypan blue) was injected
into the mammary glands through the teat of 45 day old
virgin rats. Contralateral control glands were injected
with 0.2~ trypan blue. The glands were excised after 3
days. Frozen mammary gland sections were stained with X-
gal and counterstained with eosin. When Vaccinia-HA was
injected via the rat teat, it was able to infect the
epithelial cells. At 3 days post-infection, the X-gal
staining was confined primarily to the epithelial cells.
Exampl a 6
This example demonstrates the cytotoxicity of
Vaccinia/HA on HBL100 cells in vitro.
HBL100 cells (from ATCC, Rockville, MD) were plated
in DME:F12 medium (50~ Dulbecco's Modified Eagles Medium:
50~Ham's F12 Supplement) containing 10~ fetal bovine
serum and 10 E,i.g/ml insulin at a density of 5 x 104
cells/well and incubated at 37°C overnight. Vaccinia/HA,
at concentrations of 0 moi, 0.1 moi, or 1.0 moi, was
added to the culture medium, and the cells were incubated
at 37°C for at least 3 days. More than 90~ of the cells
were dead within 72 hrs of infection at 0.1 moi.
Exampl a 7
This example shows the death of rat mammary tumor
cells in culture by infection with Vaccinia/HA.
Cells of rat mammary cancer cell line RBA were
plated in growth medium at a density of 5 x 104 cells/well
and incubated at 37°C overnight. Vaccinia virus
engineered to express (3-galactosidase and hemagglutinin
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genes (Vaccinia/HA) was added to the culture medium at
concentrations of 0 moi, 0.1 moi, or 1.0 moi and
incubated at 37°C for at least 3 days. Up to 90~ of the
cells were lysed within 72 hours of injection at 1.0 moi.
5
Exarrcpl a 8
This example demonstrates the destruction of mammary
epithelium by transfection with Vaccinia/HA in vivo.
The mammary glands of 45-day old virgin rats were
10 injected through the teat with 1 x 10' particles of
Vaccinia/HA in 20 ~,~,1 0.2~ trypan blue (tracking dye) .
Contralateral control glands were injected with 0.2~
trypan blue. The glands were excised after 3 days, fixed
in chloroform: methanol: acetic acid and stained in iron-
15 hematoxylin. Branching structures of a whole-mounted
mammary gland injected with tracking dye alone were
visible up to the end buds and alveoli. Also visible as
brown bodies were the mammary lymph nodes. Examination
of a whole-mounted mammary gland of the same rat
20 receiving Vaccinia/HA in trypan blue on the contralateral
side revealed that only about 30~ of the ducts remained.
In addition, the lymph nodes were considerably enlarged,
denoting the mounting of an immune response to clear the
Vaccinia from the vicinity.
All publications, patents, and patent applications
cited herein are hereby incorporated by reference to the
same extent as if each individual document were
individually and specifically indicated to be
incorporated by reference and were set forth in its
entirety herein.
While this invention has been described with
emphasis upon preferred embodiments, it will be obvious
to those of ordinary skill in the art that the preferred -
embodiments may be varied. It is intended that the
invention may be practiced otherwise than as specifically
described herein. Accordingly, this invention includes
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21
all.modifications encompassed within the spirit and scope
of the appended claims.
