Molecular tools for differentiating normal breast tissue and cells
from cancerous breast tissue and cells are provided. The tools are
derived from a novel tumor suppressor gene which encodes a protein
referred to hereinafter as the "EDG1" protein. One tool
is an isolated polynucleotide which encodes the EDG1 protein. The
other tool is an antibody which is immunospecific for the EDG1 protein.
Methods of detecting cancerous cells which employ the antibody and
polynucleotide are also provided. Methods for decreasing proliferation
of breast cancer cells, prostate cancer cells, testicular cancer
cells, and ovarian cancer cells are also provided. Such method comprises
increasing levels of the EDG1 protein in such cells.
What is claimed is:
1. An isolated polynucleotide which is 1080 nucleotides in length,
wherein the nucleic acid sequence of said isolated polynucleotide
is set forth in SEQ ID NO:1 or is the complete complement thereof.
2. An isolated polynucleotide which is 1080 nucleotides in length
and encodes a full-length EDG1 protein, wherein the amino acid sequence
of said full-length EDG1 protein is set forth in SEQ ID NO.2.
3. The isolated polynucleotide of claim 2, wherein said polynucleotide
is incorporated into an expression vector, a viral genome, or a
liposome, or is fused with polynucleotide which encodes a protein
tag or an amino acid tag for stabilizing the EDG1 protein or for
simplifying purification of the EDG1 protein, or is operably linked
with a promoter which drives expression of the EDG1 protein in a
Breast cancer is a significant health problem for women in the
United States and throughout the world. Despite recent advances
in detection and treatment of the disease, breast cancer remains
the second leading cause of cancer-related deaths in women. Management
of the disease currently relies on a combination of early diagnosis
through routine breast screening procedures and aggressive treatment.
Such treatment may include surgery, radiotherapy, chemotherapy,
hormone therapy or combinations of these therapies.
Ninety-five percent of all breast tumors, at least initially, are
dependent on estrogens for growth. Estrogens are steroid hormones
that are essential for normal sexual development and functioning
of female reproductive organs. Estrogens are also important for
growth, differentiation, and functioning of the testis, epididymis
and prostate in males. Estrogens also have important non-reproductive
effects on bones and the heart. Estrogens comprise a group of natural
and synthetic substances. Natural estrogens include estradiol (i.e.,
17-.beta.-estradiol or E2), estrone and estriol. Estrogens are sometimes
given therapeutically in the form of a conjugate, such as for example,
ethinyl estradiol, conjugated estrogens or diethylstilbestrol.
Tissues in the body that are responsive to estrogens are called
"estrogen-sensitive" or "estrogen-responsive"
tissues and include cells of the urogenital tract, cardiovascular
system and skeletal system. The cells that comprise estrogen-sensitive
tissues contain estrogen receptors (ER). ER can be of the .alpha.
type or .beta. type. Estrogens enter cells and bind to ER in the
cytoplasm of such cells and an estrogen-ER complex is formed. Herein,
a molecule such as estrogen that binds to a receptor is generally
called a "ligand." Herein, a receptor such as ER that
has formed a complex with a ligand is called a "liganded"
Once the estrogen ligand binds to ER, the estrogen-ER complex migrates
to the nucleus of the cell and binds to specific sequences of DNA
within the cellular genome called "estrogen response elements."
Such estrogen response elements are located in the promoters of
specific genes in the cell nucleus.
Binding of the estrogen-ER complex to estrogen-responsive elements
causes activation or suppression of the transcription of the specific
genes (Beato, et al., 1995, Cell, 83:851-7.; Katzenellenbogen, et
al., 1995, J Steroid Biochem Mol Biol, 53:387-93.; Tsai and O'Malley,
1994, Annu Rev Biochem, 63:451-86.). The activation or suppression
of specific gene transcription is one type of molecular and/or cellular
response that can result from formation of a ligand-receptor complex.
When such a response occurs, the receptor is said to have been "activated."
Estrogen-ER complexes, therefore, act as transcription factors
to regulate the expression of these genes. When a ligand binds to
a receptor and a molecular and/or cellular response (e.g., transcriptional
regulation of genes) occurs, such ligands are referred to as "agonists"
and the response produced is called "agonism." Herein,
therefore, the term agonist refers to ligands, such as estrogen,
that produce the molecular and/or cellular responses.
Estrogens and ER play significant roles in certain human cancers,
breast cancer being one specific example. Cells in female breast
tissue normally contain ER. Interaction of estrogens with ER in
breast cells normally causes the breasts to grow at puberty and
again during pregnancy. Since breast cells normally contain ER,
it is not surprising that cells comprising tumors of the breast
also contain ER. Ninety-five percent of all breast tumors, at least
initially, have ER and are dependent on estrogens for growth. In
such breast tumor cells, estrogens acting via the ER, dramatically
escalate proliferative and metastatic activity (Osborne, et al.,
1980, Cancer, 46:2884-8.).
Treatment of such ER-positive breast tumors comprises administration
to the individual with the tumor, compounds such as tamoxifen (TOT).
TOT can also administered to individuals who may be at high risk
for developing breast tumors in the future, for the purpose of prevention
of such tumors. Chemically, tamoxifen is one of a number of compounds
referred to as triphenyethylene derivatives. Tamoxifen is a mainstay
of breast cancer treatment and inhibits the proliferation promoting
effect of estrogens (Katzenellenbogen, et al., 1995, J Steroid Biochem
Mol Biol, 53:387-93.; Osborne, et al., 1980, Cancer, 46:2884-8.;
Jordan and Murphy, 1990, Endocr Rev, 11:578-610.). Like estrogens,
TOT binds to ER and, therefore, is also an ER ligand. Unlike estrogen
binding to ER, however, TOT binding to ER does not result in production
of significant molecular and/or cellular responses. The changes
in gene expression resulting from TOT binding to ER are significantly
less in magnitude than those resulting from estrogen binding to
ER. Such decreased responses are referred to as "partial agonism."
Ligands such as TOT, that result in partial agonism, are referred
to as "partial agonists."
Of significance is that binding of ER by TOT prevents estrogens
from producing their effect on ER (i.e., the partial agonist precludes
effects of the agonist). Since estrogens are prevented from producing
a molecular and/or cellular response through the ER, the response
produced in the presence of both estrogens and TOT will be partial
agonism, rather than agonism. Such partial agonism is the basis
by which TOT impairs breast tumor growth (i.e., by blocking the
agonist effects of estrogens).
With regard to TOT, while it is effective in preventing proliferation
of ER-positive breast tumor cells (i.e., cells that contain ER)
in the early stages of breast cancer treatment, such ER-positive
tumor cells invariably develop resistance to TOT. That is, after
a time (e.g., 5 years), TOT is no longer effective in preventing
estrogen stimulation of tumor proliferation and, in fact, causes
stimulation of proliferation of ER-positive tumor cells.
The high mortality observed in breast cancer patients indicates
that additional methods and tools for diagnosing and treating breast
cancer are needed. Methods and tools for differentiating between
normal breast tissue and cells and cancerous breast tissue and cells
are desirable. Additional methods and tools for reducing or inhibiting
the growth or proliferation of breast cancer cells are also desirable.
SUMMARY OF THE INVENTION
The present invention provides tools and methods for differentiating
normal breast tissue and cells from cancerous breast tissue and
cells. The tools are derived from a novel tumor suppressor gene
designated as Estrogen Downregulated Gene (EDG1) that is down-regulated
by estrogen in mammary epithelial cells. EDG1 encodes a protein
referred to hereinafter as the "EDG1" protein (SEQ. ID.
NO.2). In one aspect the tool is an isolated polynucleotide which
encodes the EDG1 protein. In one embodiment, the isolated polynucleotide
comprises the nucleotide sequence of SEQ ID NO.1. The present invention
also relates to fragments of the isolated polynucleotide that can
be used as probes or primers for identifying cells that are or are
not expressing EDG1.
In another aspect, the tool is a monoclonal antibody which is immunospecific
for the EDG1 protein. The antibody may further comprise a detectable
label, such as a fluorescent label, a chemiluminescent label, a
radiolabel or an enzyme. Also encompassed are hybridoma cells and
cell lines that produce such antibody. In another aspect, the tool
is a polyclonal sera, antibodies of which bind immunologically to
the EDG1 protein.
In another aspect, the present invention provides a method of detecting
cancerous cells in an hormone responsive tissue test sample. Preferably,
the sample is a prostate tissue, ovarian tissue, testes tissue,
uterine tissue, cervical tissue or, more preferably a breast tissue
sample. In one embodiment, the method comprises contacting the sample
or a protein extract therefrom with at least one antibody to the
EDG1 protein under conditions wherein antibody binding to the EDG1
protein occurs; and assaying for the presence or absence of a complex
between the antibody and a protein in the sample, wherein a decrease
in the level of the antigen-antibody complex, as compared to the
levels found in a sample of control cells, indicates that the sample
comprises cancerous cells. Preferably, the assay is an immunocytochemical
assay which permits determination of the intracellular location
of the antigen-antibody complexes. In another embodiment, the method
comprises assaying for the presence of EDG1 transcript in the sample,
wherein a decrease in the level of the EDG1 transcript in the sample,
as compared to the level of the EDG1 transcript in a control sample,
denotes that the test sample comprises cancerous cells.
The present invention also relates to the protein encoded by EDG1
and biologically active or immunologically reactive fragments thereof.
In one embodiment the EDG1 protein has the amino acid sequence of
SEQ ID NO.2. In one embodiment the EDG1 protein fragment has the
amino acid sequence of SEQ ID NO.3.
The present invention also provides a method for decreasing proliferation
of breast cancer cells, prostate cancer cells, testicular cancer
cells, and ovarian cancer cells. Such method comprises increasing
levels of the EDG1 protein in such cells. In one embodiment, the
cells are contacted with the EDG1 protein or a biologically active
equivalent or fragment thereof under conditions permitting uptake
of the protein or fragment. In another embodiment, the cells are
contacted with (i) a nucleic acid encoding the EDG1 protein, and
(ii) a promoter active in the cancer cell, wherein the promoter
is operably linked to the region encoding the EDG1 protein, under
conditions permitting the uptake of the nucleic acid by the cancer
cell. The cancer cell may be derived from an endocrine tissue such
as breast, ovary, prostate or testes tissue.
The present invention also provides a method for inhibiting the
transcriptional activity of estrogen-liganded ER.alpha. in cancer
cells. Such method comprises increasing the levels of the EDG1 protein
in such cells.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. shows the nucleotide sequence, SEQ ID NO.1, of a human
EDG1 cDNA and the predicted amino acid sequence, SEQ ID NO.2, of
the EDG1 protein.
FIG. 2. Functional interaction of EDG1 with ER.alpha.. a, in vitro
translated and [.sup.35 S]methionine-labeled Estrogen Receptor .alpha.
(ER.alpha.) was incubated with GST alone or GST-EDG1 bound to Sepharose
in the presence of vehicle, 10.sup.-6 M Estradiol (E.sub.2) or 10.sup.-6
M trans-hydroxytamoxifen (TOT). Bound protein was eluted and analyzed
by 12.5% SDS-polyacrylamide gel electrophoresis. "Input"
is an input lane and represents in vitro translated product added
in the samples. No interaction of in vitro translated products was
observed with GST alone. The autoradiograph is representative of
3 separate experiments. b, CHO cells were transfected with expression
vectors for activators (5 ng)/reporter constructs (2 .mu.g)--(ER.alpha.)/(ERE).sub.2
-TATA-CAT or Progesterone Receptor .beta. (PR .beta.)/MMTV-CAT or
retinoic acid receptor (RAR)/DR5-CAT or Gal4-VP16/G5-E1b-CAT. MCF7
cells were transfected with (ERE).sub.2 -pS2-CAT. The cells were
cotransfected with cmv5 control expression vector or increasing
concentration of an expression vector for EDG1 (cmv5-EDG1) as indicated.
MCF7 cells, which expresses high endogenous ER, were transfected
with (ERE).sub.2 -pS2-CAT reporter vector. CHO and MCF7 cells were
also transfected with a .beta.-galactosidase internal control reporter
to correct for transfection efficiency. Cells were then treated
for 24 h with 10.sup.-8 M estradiol (E.sub.2), 10.sup.-8 R5020,
or 10.sup.-8 all trans retinoic acid. Values are the means.+-.S.E.
from three separate experiments. c, CHO cells were transfected with
the 100 ng of pEGFP-C3-EDG1 vector or pEGFP-C3-PCMT (PCMT is a known
non-nuclear protein). For fluorescence images a fluorescein filter
was used. (Original images at 400.times.total magnification).
FIG. 3. EDG1 expression and intracellular localization in normal
breast and breast cancer tissue and epithelial cells. a, Total RNA
was collected from untreated MCF7 cells (-), and cells treated for
24 h with 10.sup.-9 M 17.beta.-Estradiol (E.sub.2), 10.sup.-6 M
all trans retinoic acid (RA), or 5 mM hexamethylene bisacetanide
(HMBA). Total RNA was also collected from different breast epithelial
cell lines. The blot was probed with random primer-labeled EDG1
cDNA. To control for RNA loading the same blot was reprobed with
36B4. The autoradiographs are representative of three separate experiments.
b, Sections obtained from breast tumor and adjacent normal breast
tissue of 3 patients were stained for endogenous EDG1 using the
EDG1 (peptide 152-171) polyclonal rabbit antibody and the goat,
anti-rabbit Alexa 488 secondary antibody. c, EDG1 expression in
human tissues. Master human normal blots (Invitrogen) containing
mRNA from different tissues was probed with random primer-labeled
EDG1 cDNA. To control for RNA loading the same blot was reprobed
with actin. EDG1 and .beta.-actin mRNA levels were quantified using
densitometry. EDG1 mRNA levels were normalized to .beta.-actin levels
and expressed relative to EDG1 expression in the lung.
FIG. 4. Regulation of EDG1 intracellular localization and relevance
in breast cell growth. a, MCF10A and MCF7 cells were stained for
endogenous EDG1 b, MCF7 cells weaned out of phenol-red free- and
fall serum-containing medium for 2 weeks and treated with 10.sup.-9
M E.sub.2 or 20 ng/ml EGF for the indicated time periods. Cells
were then stained for endogenous EDG1. c, MCF7 and MCF10A were infected
with control, EDG1, or antisense EDG1 retrovirus in the presence
or absence of tetracycline or 10.sup.-8 M Estradiol (E.sub.2). Five
days after infection, cells were stained for EDG1 expression and
cell number was determined using the CellTiter 96 Aqueous One Solution
Proliferation Assay. Values for cell number are expressed relative
to the absorbance in control cells grown in the presence of tetracycline
(which is set at 1). Values are the means.+-.S.E. from two separate
experiments with triplicate wells for each group. d, MCF7 cells
were infected with control, EDG1 or EDG1 .sub.AS retrovirus in the
presence of 3 .mu.g/ml tetracycline. Twenty-four hours later cells
were given fresh media tetracycline. Four days later cells were
detached and plated for anchorage independent growth. Values are
expressed as the number of colonies formed per number of cells plated.times.100.
Values are expressed relative to the number of colonies/cells plated
for cells infected with control retroviruses grown in the presence
of tetracycline (which is set at 1). Values are the means.+-.S.E.
from two separate experiments with triplicate wells for each group.
e, MCF10A and MDA-MD-231 cells were infected with retroviruses and
plated for proliferation or immunostaining as described in (d).
In a, b, c, and e, cells were stained for endogenous EDG1 using
the EDG1 (peptide 152-171) polyclonal rabbit antibody and the goat,
anti-rabbit Alexa 488 secondary antibody. Cells were viewed under
a fluorescence microscope at 200.times.magnification. MCF10A cells
were also stained with nile red to examine lipid vacoule formation.
FIG. 5. Functional interaction of EDG1 with binding proteins for
components of the extracellular matrix and regulation of anchorage
independent growth. a, in vitro translated and [.sup.35 S]methionine-labeled
Integrin .beta.4 Receptor or Laminin Binding Protein were incubated
with GST alone or GST-EDG1 bound to Sepharose. Bound protein was
eluted and analyzed by 12.5% SDS-polyacrylamide gel electrophoresis.
The numbers at the right indicate molecular size markers in kilodaltons.
"Input" is an input lane and represent 10% of total in
vitro translated products added in the samples. The autoradiograph
is representative of 3 separate experiments. b, MCF7 infected with
control, EDG1 or EDG1.sub.As retroviruses were immunostained using
67LR IgG monoclonal mouse antibody and goat, anti-mouse Alexa 594
secondary antibody and EDG1 polyclonal rabbit IgG antibody and goat,
anti-rabbit Alexa 488 secondary antibody. c, MCF7 cells were transfected
with 100 ng of pEGFP-EDG1, pRFP-67LR, pEGFP-C3, pRFP-C1, or pEGFP-hPMC2
as indicated. pRFP-C1 and pEGFP-C3 are control fluorescent protein
vectors without a cDNA insert and pEGFP-hPMC2 is an unrelated nuclear
protein. The cells were observed under the microscope 24 h later.
For fluorescence images rhodamine or fluorescein filters were used.
Original images are at 400.times. total magnification.
DETAILED DESCRIPTION OF THE INVENTION
As used herein the following terms have the following meanings:
"Antibody" means a protein molecule that binds to, cross
reacts with, or is immunoreactive with a specific antigen or immunogen.
The binding reaction between an antibody and its antigen is specific
in that the antibody binds only to an amino acid sequence present
within the specific protein (i.e., an epitope). An anti-EDG1 antibody
means an antibody molecule that binds to one or more epitopes of
the EDG1 protein.
"Biological sample" means a sample of mammalian cells.
These cells may be part of a tissue or organ sample obtained, for
example, by biopsy, or they may be individual cells, for example,
blood cells or cells grown in tissue culture.
"Cancer cell" or "cancerous cell" means a cell
in or from a carcinoma.
"Breast cancer" means any of various carcinomas of the
breast or mammary tissue.
"cDNA" means a DNA prepared using messenger RNA (mRNA)
as template. The advantage of using a cDNA, as opposed to genomic
DNA or DNA polymerized from a genomic, non- or partially-processed
RNA template, is that the cDNA primarily contains coding sequences
of the corresponding protein.
"Expression" means the production of a protein or a gene
transcript (i.e. mRNA) in a cell.
"Hormone responsive tissue" as used herein refers to
tissues that are normally responsive to estrogens or androgens.
Hormone responsive tissues include the mammary glands, testes, prostate,
uterus and cervix. A tissue which is normally responsive to estrogens
or androgens may lose its responsiveness to the hormone. Thus, "hormone
responsive tissue" is a broad term as used herein and encompasses
both hormone-sensitive and hormone insensitive tissues.
"Estrogen-receptor positive" as used herein refers to
a cell that comprises estrogen receptor and is responsive to estrogen
and to agents that bind to the estrogen receptor, such as tamoxifen.
"Estrogen-receptor negative" as used herein refers to
a cell that is normally responsive to estrogen, such as a mammary
epithelial cell, but that contains little to no estrogen receptor.
As a result, the estrogen receptor negative cell is estrogen-insensitive
and refractory to treatment with tamoxifen.
"Label" means to incorporate into a compound a substance
that is readily detected. Such substances include radioactive substances
and fluorescent dyes, for example.
"Native" means the nucleic acid of a non-mutated gene
or peptide sequence encoded by such a gene as found in a phenotypically
"Neoplasia" means the process resulting in the formation
and growth of an abnormal tissue that grows by cellular proliferation
more rapidly than normal, and continues to grow after the stimuli
that initiated the new growth ceases.
"Normal cell" means a non-cancerous cell.
"Proliferation" means growth and reproduction, i.e.,
division of cells
"Tumor" refers to a spontaneous, new growth of tissue
in the body that forms an abnormal mass. Tumors are comprised of
cells and such cells are known as tumor cells. Tumors and cells
derived from tumors can be either benign or malignant. Cells that
are malignant have a variety of properties that benign cells and
non-tumor cells do not have. Malignant cells invade, grow and destroy
adjacent tissue, metastasize, and usually grow more rapidly than
benign tumor cells. "Neoplasm" is essentially synonymous
"Tumor suppressor gene" refers to a gene whose expression
within a tumor cell suppresses the ability of such cells to grow
spontaneously and form an abnormal mass.
All publications and other references mentioned herein are incorporated
by reference in their entirety.
The present invention provides a protein referred to hereinafter
as EDG1 protein and functional equivalents thereof The EDG1 protein
is encoded by the tumor suppressor gene designated Estrogen Downregulated
Intracellular Localization of EDG1 Protein
EDG1 is localized in different intracellular compartments in normal
breast and breast cancer epithelial cells. (See FIG. 4) In normal
mammary epithelial cells, represented by the cell line MCF10A, EDG1
is localized primarily in the nucleus. In contrast, lower and more
diffuse cytoplasmic staining occurs in MCF7 cells, which are representative
of estrogen receptor positive breast cancer epithelial cell (FIG.
4A). Interestingly, EDG1 localized primarily to the nucleus when
MCF7 cells were weaned out of their maintenance medium containing
phenol red and full serum to phenol red-free media containing charcoal-stripped
serum (FIG. 4B). Estradiol, (E.sub.2) and epidermal growth factor
(EGF) affect the levels and/or intracellular localization of EDG1
protein in breast cancer cells. A slight decrease in EDG1 expression
is evident 12 h and 16 h after E.sub.2 treatment. (FIG. 4B) After
treatment with either Epidermal Growth Factor (EGF) or E.sub.2,
a decrease in the nuclear localization of EDG1 was evident in MCF7
cells (FIG. 4B). Estrogen- and EGF-induced nuclear export of EDG1
is inhibited by antiestrogen ICI182,780 and Mitogen-Activated Protein
Kinase Kinase (MAPKK) inhibitor PD098,059 respectively (data not
shown). It was observed that EGF, not E.sub.2, induces EDG1 nuclear
export in MCF10A cells which expresses very low levels of estrogen
receptor (ER) (data not shown).
Interaction of EDG1 Protein with Other Cellular Proteins
EDG1 interacting proteins were identified using the yeast two-hybrid
system and the interactions were verified in vitro using GST pull-down
assays (FIG. 5A). The strongest interactors from the yeast two hybrid
screenings are two proteins involved in cell adhesion--the 67 kD
laminin receptor (67LR) and the integrin .beta.4 interactor protein
(p27/BBP). Stronger interaction of EDG1 with p27/BBP, when compared
to its interaction with 67LR, was observed in GST-pull down assays.
Both 67LR and p27/BBP have been proposed to be part of the structural
link between extracellular matrix proteins and the cytoskeleton
(Biffo S, Sanvito F, Costa S, Preve L, Pignatelli R, Spinardi L,
Marchisio PC (1997) Isolation of a novel beta4 integrin-binding
protein (p27(BBP)) highly expressed in epithelial cells. J. Biol.
Chem. 272: 30314-30321; Ardini E, Tagliabue E, Magnifico A, Buto
S, Castronovo V, Colnaghi MI, Menard S. (1997) Co-regulation and
physical association of the 67-kDa monomeric laminin receptor and
the alpha6beta4 integrin. J. Biol. Chem. 272: 2342-2345). The functional
relatedness of these two proteins supports the biological relevance
of the interaction of EDG1 with these proteins.
To determine the functional consequence of the interaction of EDG1
with 67LR, the expression of 67LR was examined in cells infected
with control or EDG1 retroviruses. 67LR is a cell surface-associated
protein that interacts specifically and directly with laminin. Increased
cell surface expression of 67LR is associated with increased invasiveness
and less differentiated phenotypes of several types of human malignancies
(Sanvito F, Vivoli F, Gambini S, Santambrogio G, Catena M, Viale
E, Veglia F, Donadini A, Biffo S, Marchisio pc. (2000) Expression
of a highly conserved protein, p27bbp, during the progression of
human colorectal cancer. Cancer Res. 60: 510-516; Cress AE, Rabinovitz
I, Zhu W, Nagle RB. (1995) The alpha 6 beta 1 and alpha 6 beta 4
integrins in human prostate cancer progression. Cancer Metastasis
Rev. 14: 219-228.). Increased cell surface expression of 67LR is
seen in breast cancer cells treated with estrogen and progesterone
(Castronovo V, Taraboletts G, Liotta LA, Sobel M (1989) Modulation
of Laminin receptor Expression by Estrogen and Progestins in Human
Breast Cancer Cell Lines, J. Nat. Cancer Instit. 81: 781-788.).
The role of 67LR in breast cancer is not limited to invasion because
breast cancer cells undergoing proliferation express increased cell
surface 67LR. While 67LR shows primary membrane localization in
control cells, cytoplasmic staining was also evident (FIG. 5B).
Cells infected with EDG1 retroviruses show an overall decrease in
67LR expression, especially in the membrane. Cells that were treated
with estradiol, which induces EDG1 nuclear export, show increased
67LR staining. 67LR has been proposed to originate from a 37 kDa
precursor ribosomal protein (37LRP) that can be localized to the
nucleus and the cytoplasm (Ardini E, Posole G, Tagliabue E, Magnifico
A, Castronovo V, Sobel ME, Colnaghi MI, Menard S. (1998) the 67-kDa
laminin receptor originated from a ribosomal protein that acquired
a dual function during evloution, Mol. Biol. Evol. 15:1017-1025).
Acylation of 37LRP leads to the formation of the 67LR dimer and
the acquisition of laminin binding capacity. Thus it is possible
that EDG1 interferes with 67LR processing. Because the antibody
utilized in these experiments does not detect 37LRP, an expression
vector wherein 67LR cDNA was cloned in frame with Red Fluorescent
Protein (RFP) (pRFP-67LR) was used to explore this possibility.
In cells transfected with pRFP-67LR, red fluorescence can be localized
throughout the cell (FIG. 4C). Interestingly, when RFP-67LR was
coexpressed with green fluorescence protein-tagged EDG1 the localization
of red fluorescence was more limited, showing localization primarily
in the nucleus. The change in localization was specific to cells
expresssing EDG1. No change in red fluorescence localization was
evident in cells cotransfected with control GFP expression vector
(i.e. no EDG1 cDNA) or with an unrelated protein that we have previously
reported to be primarily nuclear (GFP-hPMC2, Montano MM, Wittman,
BM, Bianco NR (2000) Identification and Characterization of a Novel
Factor that Regulates Quinone Reductase Gene Transcriptional Activity,
J. Biol. Chem. 275: 34306-34313). The data support an effect of
EDG1 on 67LR processing.
Structure of EDG1 Protein
In one embodiment, the EDG1 protein is 359 amino acids in length
and comprises the amino acid sequence, SEQ ID NO.2, shown in FIG.
1. The EDG1 protein has a nuclear localization signal spanning residues
150-177. DNAStar analyses predict that the EDG1 protein consists
mostly of alpha helices interspersed with turns and coils. EDG1
protein is a highly hydrophilic and highly charged, with a large
proportion of the amino acids surface exposed. Many of the alpha
helices are amphipathic (i.e. negatively or positively charged).
Positive charges come from runs of triple arginine or lysines and
negative charges come from triple runs of aspartate or glutamate.
These indicate the potential for electrostatic interactions coming
about from protein-protein or protein-nucleic acid contacts.
EDG1 Protein Functional Equivalents
The present invention also encompasses functional equivalents of
the EDG1 protein that may vary structurally from the EDG1 protein
(SEQ. ID. NO.2), but have equivalent function. Such functional equivalents
are immunologically cross reactive or biologically active equivalents
of the EDG1 protein which comprises SEQ ID NO.2. Such functional
equivalents have an altered sequence in which one or more of the
amino acids in the corresponding reference sequence is substituted
or in which one or more amino acids are deleted from or added to
the corresponding reference sequence.
While it is possible to have nonconservative amino acid substitutions,
it is preferred that, except for the substitutions that are made
to replace cysteine, the substitutions be conservative amino acid
substitutions, in which the substituted amino acid has similar structural
or chemical properties with the corresponding amino acid in the
reference sequence. By way of example, conservative amino acid substitutions
involve substitution of one aliphatic or hydrophobic amino acids,
e.g., alanine, valine, leucine and isoleucine, with another; substitution
of one hydroxyl-containing amino acid, e.g., serine and threonine,
with another; substitution of one acidic residue, e.g., glutamic
acid or aspartic acid, with another; replacement of one amide-containing
residue, e.g., asparagine and glutamine, with another; replacement
of one aromatic residue, e.g., phenylalanine and tyrosine, with
another; replacement of one basic residue, e.g., lysine, arginine
and histidine, with another; and replacement of one small amino
acid, e.g., alanine, serine, threonine, methionine, and glycine,
Preferably, the deletions and additions are located at the amino
terminus, the carboxy terminus, or both, of SEQ ID NO.2. As a result
of the alterations, the EDG1 functional equivalent has an amino
acid sequence which is at least 90% identical, preferably at least
95% identical, more preferably at least 97% identical to SEQ ID
NO.2 Sequences which are at least 90% identical have no more than
1 alteration, i.e., any combination of deletions, additions or substitutions,
per 10 amino acids of the reference sequence. Percent identity is
determined by comparing the amino acid sequence of the variant with
the reference sequence using MEGALIGN project in the DNA STAR program.
In making such changes, the hydropathic index of amino acids may
be considered. The importance of the hydropathic amino acid index
in conferring interactive biologic function on a protein is generally
understood in the art (Kyte & Doolittle, (1982) A simple method
for displaying the hydropathic character of a protein. J. Mol. Biol.
157:105-132). It is accepted that the relative hydropathic character
of the amino acid contributes to the secondary structure of the
resultant protein, which in turn defines the interaction of the
protein with other molecules, for example, enzymes, substrates,
receptors, DNA, antibodies, antigens, and the like.
It is known in the art that certain amino acids may be substituted
by other amino acids having a similar hydropathic index or score
and still result in a protein with similar biological activity,
i.e., still obtain a biological functionally equivalent protein.
It is also understood in the art that the substitution of like amino
acids can be made effectively on the basis of hydrophilicity. U.S.
Pat. No. 4,554,101, incorporated herein by reference, states that
the greatest local average hydrophilicity of a protein, as governed
by the hydrophilicity of its adjacent amino acids, correlates with
a biological property of the protein.
It is understood that an amino acid can be substituted for another
having a similar hydrophilicity value and still obtain a biologically
active and immunologically cross-reactive protein. As outlined above,
amino acid substitutions are generally based on the relative similarity
of the amino acid side-chain substituents, for example, their hydrophobicity,
hydrophilicity, charge, size, and the like.
The immunologically cross-reactive EDG1 variants immunologically
bind to one or more of the antibodies that are raised using the
EDG1 protein as an immunogen. The biologically active EDG1 variants
inhibit or reduce estrogen-bound estrogen receptor transcriptional
activity in MCF7 cells and proliferation of normal mammary epithelial
cells or cancerous mammary epithelial cells.
While it is difficult to predict the exact effect of the substitution,
deletion or insertion in advance of doing so, for example, when
modifying an immune epitope on the EDG1 protein, one skilled in
the art will appreciate that the effect will be evaluated by routine
screening assays. For example, a change in the immunological character
of the EDG1 protein, such as affinity for a given antibody, is measured
by a competitive-type immunoassay. Modifications of protein properties
such as redox or thermal stability, hydrophobicity, susceptibility
to proteolytic degradation, or the tendency to aggregate with carriers
or into multimers may be assayed by methods well known to one of
skill in the art.
The present invention also encompasses fusion proteins comprising
the EDG1 protein or a functional equivalent thereof and a tag, i.e.,
a second protein or one or more amino acids, preferably from about
2 to 65 amino acids, more preferably from about 34 to about 62 amino
acids, which are added to the amino terminus of, the carboxy terminus
of, or any point within the amino acid sequence of the EDG1 protein,
or a variant of such protein. Typically, such additions are made
to stabilize the resulting fusion protein or to simplify purification
of an expressed recombinant form of the corresponding EDG1 protein
or variant of such protein. Such tags are known in the art. Representative
examples of such tags include sequences which encode a series of
histidine residues, the epitope tag FLAG, the Herpes simplex glycoprotein
D, beta-galactosidase, maltose binding protein, or glutathione S-transferase.
The EDG1 protein and functional equivalents thereof may be produced
by conventional peptide synthesizers. The EDG1 proteins and functional
equivalents thereof may also be produced using cell-free translation
systems and RNA molecules derived from DNA constructs that encode
the EDG1 proteins. Alternatively, EDG1 proteins and functional equivalents
thereof are made by transfecting host cells with expression vectors
that comprise a DNA sequence that encodes the respective EDG1 protein
or functional equivalents and then inducing expression of the protein
in the host cells. For recombinant production, recombinant constructs
comprising a sequence which encodes the EDG1 protein or functional
equivalents thereof are introduced into host cells by conventional
methods such as calcium phosphate transfection, DEAE-dextran mediated
transfection, transvection, microinjection, cationic lipid-mediated
transfection, electroporation, transduction, scrape loading, ballistic
introduction or infection.
The EDG1 protein and functional equivalents thereof may be expressed
in suitable host cells, such as for example, mammalian cells, yeast,
bacteria, or other cells under the control of appropriate promoters
using conventional techniques. Following transformation of the suitable
host strain and growth of the host strain to an appropriate cell
density, the cells are harvested by centrifugation, disrupted by
physical or chemical means, and the resulting crude extract retained
for further purification of the EDG1 protein.
Conventional procedures for isolating recombinant proteins from
transformed host cells, such as isolation by initial extraction
from cell pellets or from cell culture medium, followed by salting-out,
and one or more chromatography steps, including aqueous ion exchange
chromatography, size exclusion chromatography steps, and high performance
liquid chromatography (HPLC), and affinity chromatography may be
used to isolate recombinant EDG1 protein.
EDG1 Polypeptides and Oligopeptides
The present invention also encompasses oligopeptides or polypeptides,
referred to hereinafter collectively as "EDG1 polypeptides,"
that are less than 359 amino acids in length and comprise a consecutive
sequence in SEQ ID NO.2. In one aspect, the EDG1 polypeptides are
immunologically cross-reactive with the EDG1 protein. Such polypeptides
can be used to prepare antibodies that form antigen-antibody complexes
with the EDG1 protein. In one embodiment, the EDG1 polypeptide comprises
amino acids 152-171 of SEQ ID NO.2. In other words, the EDG1 polypeptide
comprises the hydrophilic region, KHRRRPSKKKRHWKPYYKL SEQ ID NO.3.
In another aspect, the EDG1 polypeptide has the biological activity
of the native EDG1 protein, i.e., the EDG1 polypeptide has the ability
to reduce or inhibit proliferation of a non-cancerous or cancerous
mammary epithelial cell.
The present invention provides isolated polynucleotides which encode
the EDG1 protein or a functional equivalent thereof. The EDG1 polynucleotides
may be single-stranded or double stranded. Such polynucleotides
may be DNA or RNA molecules In one embodiment the isolated polynucleotide
comprises the EDG1 cDNA sequence, SEQ ID NO.1, shown in FIG. 1.
Sequence analysis of the EDG1 cDNA clone indicates an open reading
frame of 1077 bp (359 amino acids) encoding a 40 kDa protein. The
EDG1 polynucleotides are useful for preparing EDG1 proteins.
In vivo, EDG1 acts as a tumor suppressor gene. Database searches
indicate that EDG1 can be localized to chromosome arm 17q. EDG1
mRNA expression is prevalent in normal mammary epithelial cells
and in other human hormone responsive tissues such as the ovary
and prostate, and testes (FIG. 2C). Expression of EDG1 mRNA is low
in breast cancer epithelial cells. Estradiol or E.sub.2 which induces
breast cancer cell growth, has an inhibitory effect on EDG1 mRNA
expression in breast cancer cells. Conversely, hexamethylene-bis-acetamide
(HMBA), which is known to be an inducer of differentiation and apoptosis,
upregulates EDG1 mRNA expression in breast cancer cells (FIG. 3A).
The present invention also encompasses isolated polynucleotides
whose sequence is the complement of the EDG1 cDNA sequence, SEQ
ID NO.1, and polynucleotides that hybridize under stringent conditions,
preferably under highly stringent conditions, to the open reading
frame sequence of the EDG1 cDNA sequence, SEQ ID NO.1, or the complement
thereof. Such hybridization conditions are based on the melting
temperature, Tm, of the nucleic acid binding complex or probe, as
described in Berger and Kimmel (1987) Guide to Molecular Cloning
Techniques, Methods in Enzymology, vol 152, Academic Press. The
term "stringent conditions," as used herein, is the "stringency"
which occurs within a range from about Tm-5 (5.degree. below the
melting temperature of the probe) to about 20.degree. C. below Tm.
As used herein, "highly stringent" conditions employ at
least 0.2.times.SSC buffer and at least 65.degree. C. As recognized
in the art, stringency conditions can be attained by varying a number
of factors such as the length and nature, i.e., DNA or RNA, of the
probe; the length and nature of the target sequence, the concentration
of the salts and other components, such as formamide, dextran sulfate,
and polyethylene glycol, of the hybridization solution. All of these
factors may be varied to generate conditions of stringency which
are equivalent to the conditions listed above.
Variations in the above conditions may be accomplished through
the inclusion and/or substitution of alternate blocking reagents
used to suppress background in hybridization experiments. Typical
blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured
salmon sperm DNA, and commercially available proprietary formulations.
The inclusion of specific blocking reagents may require modification
of the hybridization conditions described above, due to problems
The present invention also relates to polynucleotide encoding a
protein having a sequence that is at least 85%, preferably at least
90%, more preferably at least 95%, most preferably at least 97%
identical to the amino acid sequences depicted in FIG. 1 and set
forth in SEQ ID NO. 2, provided that such protein is an immunologically
cross-reactive or biologically reactive equivalent of the EDG1 protein.
Such sequences include allelic variants, species variants and other
amino acid sequence variants (e.g., including "muteins"
or "mutant proteins"), whether naturally-occurring or
Polynucleotides that encode the EDG1 protein and sequences which
are the complements thereof are useful tools for designing hybridization
probes for screening tissue samples, particularly tissues from patients
known to have or suspected of having breast cancer, and for isolating
and identifying cDNA clones and genomic clones encoding the EDG1
genes or allelic forms thereof. Such hybridization techniques are
known to those of skill in the art. SEQ ID NO. 1, and sequences
which are the complement thereof are also useful for designing primers
for polymerase chain reaction (PCR), a technique useful for obtaining
large quantities of cDNA molecules that encode the EDG1 proteins.
Also encompassed by the present invention, are single stranded
polynucleotides, hereinafter referred to as antisense polynucleotides,
having sequences which are complementary to the DNA and RNA sequences
which encode the EDG1 protein. The term complementary as used herein
refers to the natural binding of the polynucleotides, through hydrogen
bond formation between complementary nucleotide bases, under permissive
salt and temperature conditions by base pairing.
The present invention also provides primers which can be used in
PCR to obtain the EDG1 poylnucleotides from cDNA libraries, for
screening tissue samples, or for diagnostic purposes. The present
invention also encompasses oligonucleotides that are used as primers
in polymerase chain reaction (PCR) technologies, reverse transcriptase-PCR
(RT-PCR) for example, to amplify transcripts of the genes which
encode the EDG1 proteins or portions of such transcripts. Preferably,
the primers comprise 12-50 nucleotides, more preferably 15-30 nucleotides.
Preferably, the primers have a G+C content of 40% or greater. Such
oligonucleotides are at least 98% complementary with a portion of
the DNA strand, i.e., the sense strand, which encodes the respective
EDG1 or a portion of its corresponding antisense strand. Preferably,
the primer has at least 99% complementarity, more preferably 100%
complementarity, with such sense strand or its corresponding antisense
strand. Primers which have 100% complementarity with the antisense
strand of a double-stranded DNA molecule which encodes a EDG1 protein
have a sequence which is identical to a sequence contained within
the sense strand. The identity of primers which are 15 nucleotides
in length and have full complementarity with a portion of the antisense
strand of a double-stranded DNA molecule which encodes the EDG1
protein and is determined using the nucleotide sequence, SEQ ID
NO:1, shown in FIG. 1.
Such primers for PCR comprise a pair of set of primers. One primer
of the pair is called the "forward primer" and is located
at the left end of the sequence to be amplified. The second primer
of is called the "reverse primer" and is located at the
right end of the sequence to be amplified. The forward primer hybridizes
to the opposite strand of the template (the DNA to be amplified)
than does the reverse primers. Selection of forward and reverse
primers, for the purpose of amplifying a sequence of DNA by PCR,
is well known to one skilled in the art.
The present invention also encompasses oligonucleotides that are
useful as hybridization probes for detecting transcripts of the
genes which encode the EDG1 protein Preferably, such oligonucleotides
comprise at least 200 nucleotides. Such hybridization probes have
a sequence which is at least 90% complementary with a contiguous
sequence contained within the sense strand or antisense strand of
a double stranded DNA molecule which encodes the EDG1 protein. Such
hybridization probes bind to the sense strand or antisense under
stringent conditions, preferably under highly stringent conditions.
The probes are used in Northern assays to detect transcripts of
EDG1 homologous genes and in Southern assays to detect EDG1 homologous
genes. The identity of probes which are 200 nucleotides in length
and have full complementarity with a portion of the sense or antisense
strand of a double-stranded DNA molecule which encodes the EDG1
protein is determined using the nucleotide sequence, SEQ ID NO:1,
shown in FIG. 1.
The present invention also encompasses isolated polynucleotides
which are alleles of the genes which encode the EDG1 proteins. As
used herein, an allele or allelic sequence is an alternative form
of the gene which may result from one or more mutations in the sequences
which encode the EDG1 proteins. Such mutations typically arise from
natural addition, deletion of substitution of nucleotides in the
open reading frame sequences Any gene may have none, one, or several
allelic forms. Such alleles are identified using conventional techniques,
such as for example screening libraries with probes having sequences
identical to or complementary with one or more EDG1 polynucleotides.
The present invention also encompasses altered polynucleotides
which encode the EDG1 protein or a functional equivalent of the
EDG1 protein. Such alterations include deletions, additions, or
substitutions. Such alterations may produce silent changes and result
in an EDG1 protein having the same amino acid sequence as the EDG1
protein encoded by the unaltered polynucleotide. Such alterations
may produce a nucleotide sequence possessing non-naturally occurring
codons. For example, codons preferred by a particular prokaryotic
or eucaryotic host may be incorporated into the nucleotide sequences
shown in FIG. 1 to increase the rate of expression of the proteins
encoded by such sequences. Such alterations may also introduce new
restriction sites into the sequence or result in the production
of a EDG1 protein variant. Typically, such alterations are accomplished
using site-directed mutagenesis.
Synthesis of Polynucleotides Encoding EDG1 Proteins or Variants
Polynucleotides comprising sequences encoding a EDG1 protein or
a functional equivalent thereof may be synthesized in whole or in
part using chemical methods. Polynucleotides which encode an EDG1
protein, particularly alleles of the genes which encode an EDG1
protein, may be obtained by screening a genomic library or cDNA
library with a probe comprising sequences identical or complementary
to the sequences shown in FIG. 1 or with antibodies immunospecific
for an EDG1 protein to identify clones containing such polynucleotide.
The probes are used in Northern blot or colony hybridization assays
under high stringency conditions. Alternatively, polynucleotides
encoding EDG1 proteins may be made using polymerase chain reaction
(PCR) technology and primers which bind specifically to sequences
which are known to encode a EDG1 protein.
The present invention also provides antibodies that are immunospecific
for the EDG1 protein. As used herein the term immunospecific means
the antibody binds with greater affinity to an EDG1 protein than
other proteins that are found in normal breast cells.
The term "antibody" encompasses monoclonal antibodies,
polyclonal antibodies, multispecific antibodies (e.g., bispecific
antibodies), and antibody fragments, so long as they exhibit the
desired biological activity. "Antibody fragments" comprise
a portion of a full length antibody, generally the antigen binding
or variable region thereof. Examples of antibody fragments include
Fab, Fab', F(ab').sub.2, and Fv fragments.
Antibodies raised against EDG1 are produced by immunizing a host
animal with an EDG1 protein or an antigenic fragment thereof. Suitable
host animals for injection of the protein immunogen include, but
are not limited to, rabbits, mice, rats, goats, and guinea pigs.
Various adjuvants may be used to increase the immunological response
in the host animal. The adjuvant used depends, at least in part,
on the host species. For example, guinea pig albumin is commonly
used as a carrier for immunizations in guinea pigs. Such animals
produce heterogenous populations of antibody molecules, which are
referred to as polyclonal antibodies and which may be derived from
the sera of the immunized animals.
The term "monoclonal antibody" as used herein refers
to an antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the population
are identical except for possible naturally-occurring mutations
that may be present in minor amounts. Monoclonal antibodies are
highly specific, being directed against a single antigenic site.
Furthermore, in contrast to conventional (polyclonal) antibody preparations,
which typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed against
a single determinant on the antigen.
The monoclonal antibodies to be used in accordance with the present
invention may be made by the hybridoma method, first described by
Kohler et al., Nature 256: 495 (1975), in which case the hybridoma
cell lines that are obtained secrete the monoclonal antibodies during
growth. In order to grow the hybridoma cell lines and obtain the
secreted antibodies, the hybridoma cell lines may be grown in cell
culture and culture medium containing the monoclonal antibodies
collected. Alternatively, the hybridoma cell lines may be injected
into, and grown within, the peritoneal cavity of live animals, preferably
mice. As the hybridoma cell lines grow within the peritoneal cavity
of the animal, the monoclonal antibodies are secreted. This peritoneal
fluid, called "ascites," is collected using a syringe
to obtain the monoclonal antibodies. Such antibodies may be of any
immunoglobulin class including IgG, IgM, IgE, Iga, IgD and any class
Antibody preparations may be isolated or purified. An "isolated"
antibody is one which has been identified and separated and/or recovered
from a component of its natural environment. Contaminant components
of its natural environment are materials which would interfere with
diagnostic or therapeutic uses for the antibody, and may include
enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.
In preferred embodiments, the antibody may be purified (1) to greater
than 95% by weight of antibody as determined by the Lowry method,
and most preferably more than 99% by weight, (2) to a degree sufficient
to obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using Coomassie
blue or, preferably, silver stain. Isolated antibody includes the
antibody in situ within recombinant cells since at least one component
of the antibody's natural environment will not be present. Ordinarily,
however, isolated antibody will be prepared by at least one purification
Antibodies immunospecific for EDG1 are useful diagnostic markers
detecting cancerous epithelial cells in a tissue selected from breast
tissue, ovarian tissue, testicular tissue, prostate tissue, uterine
tissue and cervical tissue. In accordance with the present invention,
it has been shown that cancerous mammary epithelial cells have lower
levels of EDG1 protein than non-cancerous mammary epithelial cells.
The diagnostic method comprises the steps of contacting a sample
of test cells or a protein extract thereof with immunospecific anti-EDG1
antibodies and assaying for the formation of a complex between the
antibodies and a protein in the sample. Because EDG1 protein localizes
to the nucleus in non-cancerous mammary epithelial cells and, if
present, to the cytoplasm of cancerous mammary epithelial cells,
it is preferred that the assay be an immunocytochemical assay. The
cells may be fixed or premeablized to permit interaction between
the antibody and intracellular proteins. Interactions between antibodies
and a protein or peptide in the sample are detected by radiometric,
colorimetric, or fluorometric means. Detection of the antigen-antibody
complex may be accomplished by addition of a secondary antibody
that is coupled to a detectable tag, such as for example, an enzyme,
fluorophore, or chromophore. Formation of low levels of complex
in the test cell as compared to the normal cells indicates that
the test cell is cancerous.
Cancer Detection Methods Employing EDG1 Polynucleotides
The EDG1 polynucleotides or fragments are also useful for detecting,
defining the borders of, or grading mammary epithelial cell carcinomas
in patients known to have or suspected of having a mammary epithelial
cell carcinoma. The EDG1 polynucleotides or fragments may also be
used to detect cancerous cells in prostate, testicular, and ovarian
tissue. In accordance with the present invention, it has been determined
that mammary epithelial cell lines derived from mammalian tissues
obtained from individuals with breast cancer have lower levels of
EDG1 mRNA than mammary epithelial cells derived from normal mammary
tissues. In accordance with the present invention, it has also been
determined that cells derived from prostate tissue, testicular tissue,
and ovarian tissue contain relatively high levels of EDG1 transcript.
Thus, the polynucleotides of the present invention may be used
as probes in Northern analysis to identify tissues which have comparatively
lower and higher levels of EDG1 mRNA. In such procedures total RNA
or mRNA is obtained from the cells that are know to be or suspected
of being cancerous and from non-cancerous cells, i.e. breast epithelial
cells, testicular epithelial cells, prostate cells, or ovarian cells,
preferably from the same patient, and then assayed using the EDG1-designed
probe. In general, the non-cancerous cells will be obtained from
tissues near but outside the border of the expected carcinoma.
In one example, the coding sequence is radioactively labeled with
.sup.32 P or digoxigenin, and then hybridized in solution to RNA
that is isolated from test cells, e.g., mammary epithelial cells
suspected of being cancerous, and separated by size using gel electrophoresis
and blotted to nitrocellulose paper. After hybridization and washing
of the nitrocellulose paper, hybridization of the EDG1 probe to
RNA on the nitrocellulose, as revealed by autoradiography, indicates
expression of the EDG1 mRNA. Decreased levels of EDG1 mRNA expression
in the test cells as compared to levels of EDG1 mRNA present in
normal epithelial cells derived from the same type of tissue indicates
that the test cells are cancerous.
In another embodiment of the present invention, EDG1 probes, labeled
as described above, are used to hybridize directly to test cells,
e.g. mammary epithelial cells or tissues suspected of being cancerous,
and to normal cells derived from the same type of tissue, i.e. control
cells. The cells or tissues are fixed before hybridization, using
procedures well known to those skilled in the art. Hybridization
is performed under conditions similar to those described above.
Detection of hybridization, by autoradiography for example, indicates
the presence of EDG1 transcripts within the cells or tissues. A
reduced level of EDG1 transcripts in the test tissues or cells as
compared to control cells indicates that the test cells are cancerous.
Similarly, EDG1-designed primers may be used in RT-PCR to quantify
the amount of EDG1 mRNA in the test tissues and cells. Examples
of such primers include, but are not limited to (for EDG1)
rt1: cagtgtgatttctagagc, SEQ ID NO. 4, and rt2: agagcagaactactcaag,
SEQ ID NO. 5.
Alternatively, EDG1-designed primers may be used to analyze tissue
sections from human patients by an RT in situ-PCR hybridization
protocol as described Nuovo et al (1994) in Am J. Pathol., 144,
659-666, which is specifically incorporated herein by reference.
Cancer Detection Methods Employing Anti-EDG1 Antibodies
Anti-EDG1 antibodies have a diagnostic use, since simple immunochemical
staining of tissue sections, cells, and protein extracts derived
from mammary, prostate, testicular, and ovarian tissues can be used
to estimate the portion of cells expressing the EDG1 protein. Such
a test based on the use of anti-EDG1 antibodies and other standard
secondary techniques of visualization will be useful in cancer diagnosis,
particularly cancer diagnosis of breast tissue. Such a test of tumor
suppressor gene expression might also be useful to the scientific
In a diagnostic method of the present invention, the anti-EDG1
antibodies are used to determine the extent to which EDG1 protein
is present in a tissue sample obtained from an individual known
to have or suspected of having carcinoma, particularly breast carcinoma.
This can be determined using known techniques. Comparison of results
obtained from the tissue sample with results obtained from an appropriate
control (e.g., cells or tissue of the same type known to have normal
EDG1 levels) is carried out. Decreased EDG1 levels are indicative
of an increased probability of abnormal cell proliferation or oncogenesis
or of the actual occurrence of abnormal proliferation or oncogenesis.
It is contemplated that the levels of EDG1 in cancerous cells will
be at least 50% less than the level of EDG1 protein in non-cancerous
cells, more preferably the levels will be less than 30% of normal
levels, most preferably EDG1 will not be expressed. In accordance
with the present invention, it has been shown that cells derived
from more advanced carcinomas will have lower levels of EDG1 than
cells derived from less advanced carcinomas. Thus, the levels of
EDG1 in the test cells can be used as a prognostic marker of the
The sample may be untreated, or subjected to precipitation; fractionation,
separation, or purification before combining with the anti-EDG1
protein antibody. In those cases where proteins are extracted from
the sample, it is preferred that isolated proteins from the sample
be attached to a substrate such as a column, plastic dish, matrix,
or membrane, preferably nitrocellulose. For isolated protein, the
preferred detection method employs an enzyme-linked immunosorbent
assay (ELISA) or a Western immunoblot procedure.
Formation of the complex is indicative of the presence of the EDG1
protein in the test sample. Thus, the method is used to determine
whether there is a decrease or increase in the levels of the EDG1
protein in a test sample as compared to levels of the EDG1 protein
in a control sample and to quantify the amount of the EDG1 protein
in the test sample. Deviation between control and test values establishes
the parameters for diagnosing the disease.
In accordance with the present invention, it has been determined
that EDG1 protein is primarily localized in the nucleus of normal
mammary epithelial cells. It has also been determined that EDG1
protein, if present, localizes predominantly in the cytoplasm in
cancerous mammary epithelial cells and that the extent of cytoplasmic
localization correlates with the stage of the cancer, i.e., more
EDG1 protein localizes in cytoplasm of cells derived from advanced
carcinomas. Thus, it is preferred that the antibody-based detection
methods employ cell tissue sections, since the information obtained
from such samples permit not only detection of cancerous cells,
but also an assessment of the grade of the tumor that is detected.
Methods of Inhibiting Proliferation of Cancer Cells
The EDG1 polynucleotides and proteins may also be used to block
the growth or decrease the proliferation of hormone responsive cancer
cells derived from breast tissue, prostate tissue, ovarian tissue,
uterine tissue and testicular tissue. The polypeptides may be used
to decrease proliferation of both hormone sensitive and hormone
insensitive cancer cells that are derived from these tissues, including
estrogen receptor positive and estrogen receptor negative breast
cancer cells. The EDG1 polynucleotides and proteins may be used
to block proliferation of these cancer cells in vitro or in vivo.
The EDG1 polynucleotides and proteins may also be used to reduce
or inhibit proliferation of colon cancer cells. The method involves
increasing the levels of the EDG1 protein in the cancerous cells.
Inhibiting Proliferation with EDG1 Polynucleotides and Oligonucleotides
In one embodiment, polynucleotides encoding the EDG1 protein or
a functional equivalent thereof are introduced into such cells to
permit expression or overexpression of the EDG1 protein. Viral or
plasmid vectors may be used to deliver the polynucleotide to the
Levels of EDG1 may be increased in cancer cells by introducing
a DNA fragment comprising an EDG1 polynucleotide and a promoter
into the cell and expressing the EDG1 protein. Preferably, the promoter,
which is operably linked to the EDG1 polynucleotide is a tissue
specific promoter. The DNA fragment may be incorporated into a viral
vector or into a liposome which, preferably, further comprises a
molecule which targets the liposome to the cancer cell. Alternatively,
levels of EDG1 are increased in the target cancer cell by delivering
EDG1 into the cell via a liposome.
Examples of known viral vectors are recombinant viruses which are
generally based on several virus classes including poxviruses, herpesviruses,
adenoviruses, parvoviruses and retroviruses. Such recombinant viruses
generally comprise an exogenous gene under control of a promoter
which is able to cause expression of the exogenous gene in vector-infected
host cells. Recombinant viruses which can be used to transfect cells
are mentioned and cited for example in a review by Mackett, Smith
and Moss (1994) J Virol 49(3): 857-864.
Preferably, the virus vector is a defective adenovirus which has
the exogenous gene inserted into its genome. The term "defective
adenovirus" refers to an adenovirus incapable of autonomously
replicating in the target cell. Generally, the genome of the defective
adenovirus lacks the sequences necessary for the replication of
the virus in the infected cell. Such sequences are partially or,
preferably, completely, removed from the genome. To be able to infect
target cells, the defective virus must contain sufficient sequences
from the original genome to permit encapsulation of the viral particles
during in vitro preparation of the construct.
Preferably, the adenovirus is of a serotype which is not pathogenic
for man. Such serotypes include type 2 and 5 adenoviruses (Ad 2
or Ad 5). In the case of the Ad 5 adenoviruses, the sequences necessary
for the replication are the E1A and E1B regions. Methods for preparing
adenovirus vectors are described in U.S. Pat. No. 5,932,210, which
issued in August, 1999 to Gregory et al., U.S. Pat. No. 5,985,846
which issued in November, 1999 to Kochanek et al, and U.S. Pat.
No. 6,033,908 which issued in March, 2000, to Bout et al.
More preferably, the virus vector is an immunologically inert adenovirus.
As used herein the term "immunologically inert" means
the viral vector does not encode viral proteins that activate cellular
and humoral host immune responses. Methods for preparing immunologically
inert adenoviruses are described in Parks et al., Proc Natl Acad
Sci USA 1996; 93(24) 13565-70; Leiber, A. et al., J. Virol. 1996;
70(12) 8944-60; Hardy s., et al, J Virol. 1997, 71(3): 1842-9; and
Morsy et al, Proc. Natl. Acad. Sci. USA 1998. 95: 7866-71, all of
which are specifically incorporated herein by reference. Such methods
involve Cre-loxP recombination. In vitro, Cre-loxP recombination
is particularly adaptable to preparation of recombinant adenovirus
and offers a method for removing unwanted viral nucleotide sequences.
Replication deficient recombinant adenovirus lacks the E1 coding
sequences necessary for viral replication. This function is provided
by 293 cells, a human embryonic kidney cell line transformed by
adenovirus type. First generation adenoviruses are generated by
co-transfecting 293 cells with a helper virus and a shuttle plasmid
containing the foreign gene of interest. This results in the packaging
of virus that replicates both the foreign gene and numerous viral
proteins. More recently, 293 cells expressing Cre recombinase, and
helper virus containing essential viral sequences and with a packaging
signal flanked by loxP sites, have been developed (See Parks et
al.) In this system, the helper virus supplies all of the necessary
signals for replication and packaging in trans, but is not packaged
due to excision of essential sequences flanked by loxP. When 293-Cre
cells are co-transfected with this helper virus, and a shuttle plasmid
(pRP1001) containing the packaging signal, nonsense "filler
DNA", and the foreign gene, only an adenovirus containing filler
DNA and the foreign gene is packaged (LoxAv). This results in a
viral recombinant that retains the ability to infect target cells
and synthesize the foreign gene, but does not produce viral proteins,
for targeting cancer cells.
Methods for targeting vectors to cancer cells are described in
Nakanishi T, Tamai I, Takaki A, Tsuji A. (2000) Cancer cell-targeted
drug delivery utilizing oligopeptide transport activity. Int. J.
Cancer. 88: 274-280, and Poul M A, Becerril B, Nielsen U B, Morisson
P, Marks J D. (2000) Selection of tumor-specific internalizing human
antibodies from phage libraries. J. Mol. Biol. 301: 1149-1161, both
of which are incorporated herein in their entirety. Methods for
delivering isolated oligonucleotides and polynucleotides to cells,
including the nucleus of cells, are described in Lebedeva I, Benimetskaya
L, Stein C A, Vilenchik M. (2000) Cellular delivery of antisense
oligonucleotides. Eur. J. Pharm. Biopharm. 50: 101-119. Review.,
and Fisher K D, Ulbrich K, Subr V, Ward C M, Mautner V, Blakey D,
Seymour L W. (2000) A versatile system for receptor-mediated gene
delivery permits increased entry of DNA into target cells, enhanced
delivery to the nucleus and elevated rates of transgene expression.
Gene. Ther. 7: 1337-1343.
In a further embodiment an expression construct comprising the
polynucleotide may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and
an inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation
of closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat (1991) Targeting of liposomes
to hepatocytes. Targeted Diagn. Ther 4: 87-103). Also contemplated
are lipofectamine-DNA complexes.
Inhibiting Proliferation of Cancer Cells with EDG1 Protein and
Biologically Active Equivalents Thereof
Proliferation of cancer cells, particularly breast cancer cells,
may also be accomplished introducing an EDG1 protein or a biologically
active oligonucleotide or polynucleotide derived therefrom into
the cancer cell. A variety of methods exist for introducing proteins
and polypeptides into cells. Such methods include, but are not limited
to, "protein transduction" or "protein therapy"
as described in publications by Nagahara et al. (Nagahara, et al.,
1998, Nat Med. 4: 1449-52.) and in publications from the laboratory
of Dowdy (Nagahara, et al., 1998, Nat Med. 4: 1449-52.; Schwarze,
et al., 1999, Science, 285:1569-72.; Vocero-Akbani, et al., 2000,
Methods Enzymol, 322:508-21; Ho, et al., 2001, Cancer Res, 61:474-7.;
Vocero-Akbani, et al., 2001, Methods Enzymol, 332:36-49; Snyder
and Dowdy, 2001, Curr Opin Mol Ther, 3:147-52.; Becker-Hapak, et
al., 2001, Methods, 24:247-56.), publications which are incorporated
herein by reference.
In one embodiment an eleven amino acid sequence, the "protein
transduction domain" (PTD), from the human immunodeficiency
virus TAT protein (Green and Loewenstein, 1988, Cell, 55:1179-88.;
Frankel and Pabo, 1988, Cell, 55:1189-93.) is fused to the wild-type
EDG1 protein. The purified protein is then put in contact with the
surface of cells and the cells take up the wild-type EDG1 protein
which functions to inhibit or suppress growth of that cell. In the
case where it is desired to introduce the wild-type EDG1 protein
containing the fused PTD into cells comprising a tumor in a human
or animal, the protein is administered to the human by a variety
of methods. Preferably, the protein is administered by injection
(e.g., intravenously) or by inhalation in an aerosol.
EDG1 proteins that contain the fused PTD are preferably made by
fusing the DNA sequence encoding the EDG1 protein or a functional
equivalent thereof with the DNA sequence encoding the PTD. The resulting
EDG1-PTD fusion gene is preferably incorporated into a vector, for
example a plasmid or viral vector, that facilitates introduction
of the fusion gene into a organism and expression of the gene at
high levels in the organism such that large amounts of the fusion
protein are made therein. One such organism in which the vector
containing the fusion gene can be expressed is a bacterium, preferably
Escherichia coli. Other organisms are also commonly used by those
skilled in the art. After the fusion protein is expressed at a high
level in any of these organisms, the fusion protein is purified
from the organism using protein purification techniques well known
to those skilled in the art.
The present invention also provides a method for inhibiting the
transcriptional activity of estrogen-liganded ER.alpha. in cells,
particularly in breast cancer cells. Such method comprises increasing
levels of the EDG1 protein in such cells.