The present invention relates to a combination comprising a plurality
of polynucleotide probes that are modulated in response to EGF and
which are associated with breast cancer, and which may be used in
their entirety or in part as to diagnose, to stage, to treat, or
to monitor the treatment of a subject with a breast cancer.
What is claimed is:
1. A combination comprising a plurality of cDNAs, wherein the plurality
of cDNAs consists of SEQ ID NOs: 1-16 or their complements.
2. The combination of claim 1, wherein the cDNAs are immobilized
on a substrate.
3. A high throughput method for detecting differential expression
of one or more cDNAs in a sample containing nucleic acids, the method
comprising: (a) hybridizing the substrate of claim 2 with nucleic
acids of the sample, thereby forming one or more hybridization complexes;
(b) detecting the hybridization complexes; and (c) comparing the
hybridization complexes with those of a standard, wherein differences
between the standard and sample hybridization complexes indicate
differential expression of cDNAs in the sample.
4. The method of claim 3, where in the nucleic acids of the sample
are amplified prior to hybridization.
5. A high throughput method of screening a plurality of molecules
or compounds to identify a ligand which specifically binds a cDNA,
the method comprising: (a) combining the combination of claim 1
with the plurality of molecules or compounds under conditions to
allow specific binding; and (b) detecting specific binding between
each cDNA and at least one molecule or compound, thereby identifying
a ligand that specifically binds to each cDNA.
6. The method of claim 5 wherein the plurality of molecules or
compounds are selected from DNA molecules, RNA molecules, peptide
nucleic acid molecules, mimetics, peptides, transcription factors,
repressors, and regulatory proteins.
FIELD OF THE INVENTION
The present invention relates to a composition comprising a plurality
of polynucleotide probes which may be used in detecting expression
of genes modulated in response to EGF, and which are associated
with breast cancer.
BACKGROUND OF THE INVENTION
Intercellular communication is essential for the development and
survival of multicellular organisms. Communication is achieved through
the secretion of proteins by signaling cells and the internalization
of these proteins by target cells. Growth factors are secreted proteins
that mediate communication between signaling and target cells. The
secreted growth factors bind to specific receptors on the surfaces
of target cells, and bound receptors trigger second messenger signal
transduction pathways. These signal transduction pathways elicit
specific cellular responses in the target cells. Such responses
can include the modulation of gene expression and the stimulation
or inhibition of cell division, cell differentiation, and cell motility.
Epidermal growth factor (EGF) is a member of a broad class of polypeptide
growth factors that generally act as mitogens in diverse cell types
to stimulate wound healing, bone synthesis and remodeling, extracellular
matrix synthesis, and proliferation of epithelial, epidermal, and
connective tissues. In addition, EGF produces non-mitogenic effects
in certain tissues. The EGF receptor (EGFR), and its stimulation
by EGF, has been linked with a number of cell proliferative disorders
or malignancies. These include skin hyperplasia, erythroblastosis,
and fibrosarcoma in animals; and in humans, benign hyperplasia of
the skin, mammary carcinoma, glioblastoma, and hepatic carcinoma.
Other epithelial carcinomas associated with EGFR activity include
prostatic hyperplasia/cancer, renal carcinoma, bladder cancer, laryngeal
cancer, esophageal tumors, stomach cancer, colon carcinoma, ovarian
adenomas, and lung cancer (Khazaie, K. et al. (1993) Cancer and
Metastasis Rev. 12:255-274).
The relationship of EGFR expression to human mammary carcinoma
has been particularly well studied. (See Khazaie et al., supra,
and references cited therein for a review of this area.) Overexpression
of EGFR, particularly coupled with down-regulation of the estrogen
receptor (ER), has been a marker of poor prognosis in breast cancer
patients. In addition, EGFR expression in breast tumor metastases
is frequently elevated relative to the primary tumor, suggesting
that EGFR is involved in tumor progression and metastasis. This
is supported by accumulating evidence that EGF has pleiotropic effects
on cell motility, chemotaxis, secretion and differentiation; cell
functions related to metastatic potential. For example, EGF has
been found to influence the expression and organization of integrins,
a family of receptors known to function in cell attachment to the
extracelluar matrix during metastasis (Nicolson, G. L. (1984) Expl.
Cell Res. 150:3-22; Schirrmacher, V (1985) Adv. Cancer Res. 43:1-73).
EGFR may influence cell-cell adhesion by affecting changes in the
phosphorylation of certain proteins involved in the process, such
as .beta.-atenin, fodrin, spectrin, and tubulin (Khazaie et al.,
supra). EGF has also been shown to affect the production and release
of various proteinases involved in cell invasion of the extracelluar
matrix, such as metalloproteinases, aminopeptidases, serine proteases,
cysteine proteases and aspartic proteinases, as well as proteinase
inhibitors such as plasminogen activator inhibitor (PAI-1) and tissue
inhibitors of metalloproteases (TIMP).
In addition to the various proteins indicated above that are affected
by EGF activity, the EGF signal transduction pathway itself involves
the recruitment and activation of a variety of molecules including
phospholipase C, phosphoinositol-3 kinase, MAP kinase, raf kinase,
and a GTPase-activating protein (GAP). The expression of these and
other molecules effected by EGF activity may be useful for the prediction
or monitoring of cell proliferative disorders, pre-malignant conditions,
or the presence and progression of malignant diseases in which EGF
Array technology can provide a simple way to explore the expression
of a single polymorphic gene or a large number of related or unrelated
genes. When the expression of a single gene is explored, arrays
are employed to detect the expression of specific gene variants.
For example, a p53 tumor suppressor gene array is used to determine
whether individuals are carrying mutations that predispose them
to cancer. The array has over tens of thousands of DNA probes to
analyze more than 400 distinct mutations of p53.
DNA-based array technology is especially relevant for the rapid
screening of expression of a large number of genes. There is a growing
awareness that gene expression is affected in a global fashion.
A genetic predisposition, disease or therapeutic treatment may affect,
directly or indirectly, the expression of a large number of genes.
In some cases the interactions may be expected, such as where the
genes are part of the same signaling pathway. In other cases, the
interactions may be totally unexpected, such as when the genes participate
in separate signaling pathways. Therefore, DNA-based arrays can
be used to investigate how genetic predisposition, disease, or therapeutic
treatment affects the expression of a large number of genes.
The potential application of gene expression profiling to breast
cancer is particularly relevant to improving diagnosis and prognosis
of this disease. The mortality rate for breast cancer approaches
10% of all deaths in females between the ages of 45-54 (K. Gish
(1999) AWIS Magazine, 28:7-10). However the survival rate based
on early diagnosis of localized breast cancer is extremely high
(97%), compared with the advanced stage of the disease in which
the tumor has spread beyond the breast (22%). Current procedures
for clinical breast examination are, however, lacking in sensitivity
and specificity, and efforts are underway in other laboratories
to develop comprehensive gene expression profiles for breast cancer
that may be used in conjunction with conventional screening methods
to improve diagnosis and prognosis of this disease (Gish, supra).
It would be advantageous to prepare DNA-based arrays that can be
used for monitoring expression of a large number of genes associated
with cell proliferative disorders and with pre-malignant and malignant
conditions. The present invention provides for a composition comprising
a plurality of polynucleotide probes for use in detecting changes
in expression of a large number of genes encoding proteins associated
with EGFR expression and activity. Such a microarray can be employed
for diagnosis and monitoring of the treatment of any disease or
precondition where EGFR activation is involved, in particular, breast
The present invention provides a combination comprising a plurality
of cDNAs, wherein each of the cDNAs comprises at least a fragment
of a polynucleotide sequence or a complement thereof whose expression
is modulated by EGF and is associated with breast cancer and which
are selected from SEQ ID NOs: 1-16 as presented in the Sequence
Listing. In one aspect, the combination is immobilized on a substrate.
The invention also provides a high throughput method to detect
differential expression of one or more of the cDNAs of the combination.
The method comprises hybridizing the substrate comprising the combination
with the nucleic acids of a sample, thereby forming one or more
hybridization complexes, detecting the hybridization complexes,
and comparing the hybridization complexes with those of a standard,
wherein differences in the size and signal intensity of each hybridization
complex indicates differential expression of nucleic acids in the
The invention further provides a high throughput method of screening
a library or a plurality of molecules or compounds to identify a
ligand. The method comprises combining the substrate comprising
the combination with a library or a plurality of molecules or compounds
under conditions to allow specific binding and detecting specific
binding, thereby identifying a ligand. The library or a plurality
of molecules or compounds are selected from DNA molecules, RNA molecules,
peptide nucleic acid molecules, mimetics, peptides, transcription
factors, repressors, and other regulatory proteins.
The invention still further provides an isolated cDNA selected
from SEQ ID NOs: 12-16 as presented in the Sequence Listing. The
invention still further provides a pharmaceutical composition comprising
the cDNA and a suitable pharmaceutical carrier. The invention also
provides a vector comprising the cDNA, a host cell comprising the
vector, and a method for producing a protein comprising culturing
the host cell under conditions for the expression of a protein and
recovering the protein from the host cell culture. The invention
additionally provides a method for purifying a ligand, the method
comprising combining a cDNA of the invention with a sample under
conditions which allow specific binding, recovering the bound cDNA,
and separating the cDNA from the ligand, thereby obtaining purified
The present invention provides a purified protein encoded and produced
by a cDNA of the invention. The invention also provides a high-throughput
method for using a protein to screen a library or a plurality of
molecules or compounds to identify a ligand. The method comprises
combining the protein or a portion thereof with the library or a
plurality of molecules or compounds under conditions to allow specific
binding and detecting specific binding, thereby identifying a ligand
which specifically binds the protein. A library or a plurality of
molecules or compounds are selected from DNA molecules, RNA molecules,
peptide nucleic acid molecules, mimetics, peptides, proteins, agonists,
antagonists, antibodies or their fragments, immunoglobulins, inhibitors,
drug compounds, and pharmaceutical agents. The invention further
provides for using a protein to purify a ligand. The method comprises
combining the protein or a portion thereof with a sample under conditions
to allow specific binding, recovering the bound protein, and separating
the protein from the ligand, thereby obtaining purified ligand.
The invention yet still further provides a method for using the
protein to produce an antibody. The method comprises immunizing
an animal with the protein or an antigenically-effective epitope
under conditions to elicit an antibody response, isolating animal
antibodies, and screening the isolated antibodies with the protein
to identify an antibody which specifically binds the protein. The
invention yet still further provides a method for using the protein
to purify antibodies which bind specifically to the protein.
DESCRIPTION OF THE SEQUENCE LISTING AND TABLES
A portion of the disclosure of this patent document contains material
which is subject to copyright protection. The copyright owner has
no objection to the facsimile reproduction by anyone of the patent
document or the patent disclosure, as it appears in the Patent and
Trademark Office patent file or records, but otherwise reserves
all copyright rights whatsoever.
The Sequence Listing is a compilation of nucleic acid sequences
obtained by sequencing clone inserts (isolates) of different cDNAs.
Each sequence is identified by a sequence identification number
(SEQ ID NO) and by the clone number from which it was obtained.
Table 1 lists genes differentially expressed in human BT20 breast
carcinoma cells and in primary breast carcinoma tissue. In each
page, the table contains (by column): 1) SEQ ID NO: as shown in
the Sequence Listing; 2) the Genbank ID and; 3) gene description
to which the Incyte sequence was annotated; 4-10) the differential
expression of the gene in each of seven breast carcinoma tissue
samples, and 5) the maximal differential expression of the gene
measured during the time course of the experiment for BT20 cells.
DESCRIPTION OF THE INVENTION
"Array" refers to an ordered arrangement of at least
two cDNAs on a substrate. At least one of the cDNAs represents a
control or standard sequence, and the other, a cDNA of diagnostic
interest. The arrangement of from about two to about 40,000 cDNAs
on the substrate assures that the size and signal intensity of each
labeled hybridization complex formed between a cDNA and a sample
nucleic acid is individually distinguishable.
The "complement" of a nucleic acid molecule of the Sequence
Listing refers to a cDNA which is completely complementary over
the full length of the sequence and which will hybridize to the
nucleic acid molecule under conditions of high stringency.
A "combination" comprises at least two and up to 16 sequences
selected from the group consisting of SEQ ID NOs: 1-16 as presented
in the Sequence Listing.
"cDNA" refers to a chain of nucleotides, an isolated
polynucleotide, nucleic acid molecule, or any fragment or complement
thereof. It may have originated recombinantly or synthetically,
be double-stranded or single-stranded, coding and/or noncoding,
and purified or combined with carbohydrate, lipids, protein or inorganic
elements or substances. Preferably, the cDNA is from about 4000
to about 5000 nucleotides.
The phrase "cDNA encoding a protein" refers to a nucleic
acid sequence that closely aligns with sequences which encode conserved
regions, motifs or domains that were identified by employing analyses
well known in the art. These analyses include BLAST (Basic Local
Alignment Search Tool; Altschul (1993) J Mol Evol 36: 290-300; Altschul
et al. (1990) J Mol Biol 215:403410) which provides identity within
the conserved region. Brenner et al. (1998; Proc Natl Acad Sci 95:6073-6078)
who analyzed BLAST for its ability to identify structural homologs
by sequence identity found 30% identity is a reliable threshold
for sequence alignments of at least 150 residues and 40% is a reasonable
threshold for alignments of at least 70 residues (Brenner et al.,
page 6076, column 2).
"Derivative" refers to a cDNA or a protein that has been
subjected to a chemical modification. Derivatization of a cDNA can
involve substitution of a nontraditional base such as queosine or
of an analog such as hypoxanthine. These substitutions are well
known in the art. Derivatization of a protein involves the replacement
of a hydrogen by an acetyl, acyl, alkyl, amino, formyl, or morpholino
group. Derivative molecules retain the biological activities of
the naturally occurring molecules but may confer advantages such
as longer lifespan or enhanced activity.
"Differential expression" refers to an increased, upregulated
or present, or decreased, downregulated or absent, gene expression
as detected by the absence, presence, or at least two-fold changes
in the amount of transcribed messenger RNA or translated protein
in a sample.
"Fragment" refers to a chain of consecutive nucleotides
from about 200 to about 700 base pairs in length. Fragments may
be used in PCR or hybridization technologies to identify related
nucleic acid molecules and in binding assays to screen for a ligand.
Nucleic acids and their ligands identified in this manner are useful
as therapeutics to regulate replication, transcription or translation.
A "hybridization complex" is formed between a cDNA and
a nucleic acid of a sample when the purines of one molecule hydrogen
bond with the pyrimidines of the complementary molecule, e.g., 5'-A-G-T-C-3'
base pairs with 3'-T-C-A-G-5'. The degree of complementarity and
the use of nucleotide analogs affect the efficiency and stringency
of hybridization reactions.
"Ligand" refers to any agent, molecule, or compound which
will bind specifically to a complementary site on a cDNA molecule
or polynucleotide, or to an epitope or a protein. Such ligands stabilize
or modulate the activity of polynucleotides or proteins and may
be composed of inorganic or organic substances including nucleic
acids, proteins, carbohydrates, fats, and lipids.
"Oligonucleotide" refers a single stranded molecule from
about 18 to about 60 nucleotides in length which may be used in
hybridization or amplification technologies or in regulation of
replication, transcription or translation. Substantially equivalent
terms are amplimer, primer, and oligomer.
"Portion" refers to any part of a protein used for any
purpose; but especially, to an epitope for the screening of ligands
or for the production of antibodies.
"Post-translational modification" of a protein can involve
lipidation, glycosylation, phosphorylation, acetylation, racemization,
proteolytic cleavage, and the like. These processes may occur synthetically
or biochemically. Biochemical modifications will vary by cellular
location, cell type, pH, enzymatic milieu, and the like.
"Probe" refers to a cDNA that hybridizes to at least
one nucleic acid molecule in a sample. Where targets are single
stranded, probes are complementary single strands. Probes can be
labeled with reporter molecules for use in hybridization reactions
including Southern, northern, in situ, dot blot, array, and like
technologies or in screening assays.
"Protein" refers to a polypeptide or any portion thereof.
A "portion" of a protein retains at least one biological
or antigenic characteristic of a native protein. An "oligopeptide"
is an amino acid sequence from about five residues to about 15 residues
that is used as part of a fusion protein to produce an antibody.
"Purified" refers to any molecule or compound that is
separated from its natural environment and is from about 60% free
to about 90% free from other components with which it is naturally
"Sample" is used in its broadest sense as containing
nucleic acids, proteins, antibodies, and the like. A sample may
comprise a bodily fluid; the soluble fraction of a cell preparation,
or an aliquot of media in which cells were grown; a chromosome,
an organelle, or membrane isolated or extracted from a cell; genomic
DNA, RNA, or cDNA in solution or bound to a substrate; a cell; a
tissue; a tissue print; a fingerprint, buccal cells, skin, or hair;
and the like.
"Specific binding" refers to a special and precise interaction
between two molecules which is dependent upon their structure, particularly
their molecular side groups. For example, the intercalation of a
regulatory protein into the major groove of a DNA molecule, the
hydrogen bonding along the backbone between two single stranded
nucleic acids, or the binding between an epitope of a protein and
an agonist, antagonist, or antibody.
"Similarity" as applied to sequences, refers to the quantification
(usually percentage) of nucleotide or residue matches between at
least two sequences aligned using a standardized algorithm such
as Smith-Waterman alignment (Smith and Waterman (1981) J Mol Biol
147:195-197) or BLAST2 (Altschul et al. (1997) Nucleic Acids Res
25:3389-3402). BLAST2 may be used in a standardized and reproducible
way to insert gaps in one of the sequences in order to optimize
alignment and to achieve a more meaningful comparison between them.
"Substrate" refers to any rigid or semi-rigid support
to which cDNAs or proteins are bound and includes membranes, filters,
chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels,
capillaries or other tubing, plates, polymers, and microparticles
with a variety of surface forms including wells, trenches, pins,
channels and pores.
"Variant" refers to molecules that are recognized variations
of a cDNA or a protein encoded by the cDNA. Splice variants may
be determined by BLAST score, wherein the score is at least 100,
and most preferably at least 400. Allelic variants have a high percent
identity to the cDNAs and may differ by about three bases per hundred
bases. "Single nucleotide polymorphism" (SNP) refers to
a change in a single base as a result of a substitution, insertion
or deletion. The change may be conservative (purine for purine)
or non-conservative (purine to pyrimidine) and may or may not result
in a change in an encoded amino acid.
The present invention provides a combination comprising a plurality
of polynucleotide probes, comprising at least a fragment of a gene
whose transcript is modulated in response to EGF. Preferably, the
plurality of probes comprise at least a fragment of one or more
of the sequences, SEQ ID NOs: 1-16, presented in the Sequence Listing;
and they are arranged on a substrate, preferably a microarray.
The microarray can be used for large scale genetic or gene expression
analysis of a large number of targets. The microarray can also be
used in the diagnosis of diseases and in the monitoring of treatments
where altered expression of genes is associated with a cell proliferative
disorder, in particular, breast cancer. Further, the microarray
can be employed to investigate an individual's predisposition to
a disease, in particular, breast cancer.
In a preferred embodiment, the combination provides the expression
of those probes selected from SEQ ID NOs: 1-16 which are associated
with breast cancer
When the composition of the invention is employed as hybridizable
elements in a microarray, the elements are organized in an ordered
fashion so that each element is present at a specified location
on the substrate. Because the elements are at specified locations
on the substrate, the hybridization patterns and intensities, which
together create a unique expression profile, can be interpreted
in terms of expression levels of particular genes and can be correlated
with a particular metabolic process, condition, disorder, disease,
stage of disease, or treatment.
The combination comprising a plurality of cDNAs can also be used
to identify or purify a molecule or compound which specifically
binds to at least one of the cDNAs. These molecules may be identified
from a sample or in high throughput mode from a library of mRNAs,
cDNAs, genomic fragments, and the like. Typically, samples or libraries
will include targets of diagnostic or therapeutic interest. If nucleic
acids in a particular sample enhance the hybridization background,
it may be advantageous to remove these nucleic acids. One method
for removing additional nucleic acids is by hybridizing the sample
with immobilized probes and washing away those nucleic acids that
do not form hybridization complexes. At a later point, hybridization
complexes can be dissociated, thereby releasing the purified targets.
cDNAs and Their Uses
cDNAs can be prepared by a variety of synthetic or enzymatic methods
well known in the art. cDNAs can be synthesized, in whole or in
part, using chemical methods well known in the art (Caruthers et
al. (1980) Nucleic Acids Symp. Ser. (7)215-233). Alternatively,
cDNAs can be produced enzymatically or recombinantly, by in vitro
or in vivo transcription.
Nucleotide analogs can be incorporated into cDNAs by methods well
known in the art. The only requirement is that the incorporated
analog must base pair with native purines or pyrimidines. For example,
2, 6-diaminopurine can substitute for adenine and form stronger
bonds with thymidine than those between adenine and thymidine. A
weaker pair is formed when hypoxanthine is substituted for guanine
and base pairs with cytosine. Additionally, cDNAs can include nucleotides
that have been derivatized chemically or enzymatically.
cDNAs can be synthesized on a substrate. Synthesis on the surface
of a substrate may be accomplished using a chemical coupling procedure
and a piezoelectric printing apparatus as described by Baldeschweiler
et al. (PCT publication WO95/251116). Alternatively, the cDNAs can
be synthesized on a substrate surface using a self-addressable electronic
device that controls when reagents are added as described by Heller
et al. (U.S. Pat. No. 5,605,662). cDNAs can be synthesized directly
on a substrate by sequentially dispensing reagents for their synthesis
on the substrate surface or by dispensing preformed DNA fragments
to the substrate surface. Typical dispensers include a micropipette
delivering solution to the substrate with a robotic system to control
the position of the micropipette with respect to the substrate.
There can be a multiplicity of dispensers so that reagents can be
delivered to the reaction regions efficiently.
cDNAs can be immobilized on a substrate by covalent means such
as by chemical bonding procedures or UV irradiation. In one method,
a cDNA is bound to a glass surface which has been modified to contain
epoxide or aldehyde groups. In another method, a cDNA is placed
on a polylysine coated surface and UV cross-linked to it as described
by Shalon et al. (WO95/35505). In yet another method, a cDNA is
actively transported from a solution to a given position on a substrate
by electrical means (Heller, supra). cDNAs do not have to be directly
bound to the substrate, but rather can be bound to the substrate
through a linker group. The linker groups are typically about 6
to 50 atoms long to provide exposure of the attached cDNA. Preferred
linker groups include ethylene glycol oligomers, diamines, diacids
and the like. Reactive groups on the substrate surface react with
a terminal group of the linker to bind the linker to the substrate.
The other terminus of the linker is then bound to the cDNA. Alternatively,
polynucleotides, plasmids or cells can be arranged on a filter.
In the latter case, cells are lysed, proteins and cellular components
degraded, and the DNA is coupled to the filter by UV cross-linking.
The cDNAs may be used for a variety of purposes. For example, the
combination of the invention may be used on an array. The array,
in turn, can be used in high-throughput methods for detecting a
related polynucleotide in a sample, screening a plurality of molecules
or compounds to identify a ligand, diagnosing a breast cancer, or
inhibiting or inactivating a therapeutically relevant gene related
to the cDNA.
When the cDNAs of the invention are employed on a microarray, the
cDNAs are arranged in an ordered fashion so that each cDNA is present
at a specified location. Because the cDNAs are at specified locations
on the substrate, the hybridization patterns and intensities, which
together create a unique expression profile, can be interpreted
in terms of expression levels of particular genes and can be correlated
with a particular metabolic process, condition, disorder, disease,
stage of disease, or treatment.
The cDNAs or fragments or complements thereof may be used in various
hybridization technologies. The cDNAs may be labeled using a variety
of reporter molecules by either PCR, recombinant, or enzymatic techniques.
For example, a commercially available vector containing the cDNA
is transcribed in the presence of an appropriate polymerase, such
as T7 or SP6 polymerase, and at least one labeled nucleotide. Commercial
kits are available for labeling and cleanup of such cDNAs. Radioactive
(Amersham Pharmacia Biotech (APB), Piscataway N.J.), fluorescent
(Operon Technologies, Alameda Calif.), and chemiluminescent labeling
(Promega, Madison Wis.) are well known in the art.
A cDNA may represent the complete coding region of an mRNA or be
designed or derived from unique regions of the mRNA or genomic molecule,
an intron, a 3' untranslated region, or from a conserved motif.
The cDNA is at least 18 contiguous nucleotides in length and is
usually single stranded. Such a cDNA may be used under hybridization
conditions that allow binding only to an identical sequence, a naturally
occurring molecule encoding the same protein, or an allelic variant.
Discovery of related human and mammalian sequences may also be accomplished
using a pool of degenerate cDNAs and appropriate hybridization conditions.
Generally, a cDNA for use in Southern or northern hybridizations
may be from about 400 to about 6000 nucleotides long. Such cDNAs
have high binding specificity in solution-based or substrate-based
hybridizations. An oligonucleotide, a fragment of the cDNA, may
be used to detect a polynucleotide in a sample using PCR.
The stringency of hybridization is determined by G+C content of
the cDNA, salt concentration, and temperature. In particular, stringency
is increased by reducing the concentration of salt or raising the
hybridization temperature. In solutions used for some membrane based
hybridizations, addition of an organic solvent such as formamide
allows the reaction to occur at a lower temperature. Hybridization
may be performed with buffers, such as 5.times. saline sodium citrate
(SSC) with 1% sodium dodecyl sulfate (SDS) at 60.degree. C., that
permit the formation of a hybridization complex between nucleic
acid sequences that contain some mismatches. Subsequent washes are
performed with buffers such as 0.2.times.SSC with 0.1% SDS at either
45.degree. C. (medium stringency) or 65.degree.-68.degree. C. (high
stringency). At high stringency, hybridization complexes will remain
stable only where the nucleic acid molecules are completely complementary.
In some membrane-based hybridizations, preferably 35% or most preferably
50%, formamide may be added to the hybridization solution to reduce
the temperature at which hybridization is performed. Background
signals may be reduced by the use of detergents such as Sarkosyl
or Triton X-100 (Sigma Aldrich, St. Louis Mo.) and a blocking agent
such as denatured salmon sperm DNA. Selection of components and
conditions for hybridization are well known to those skilled in
the art and are reviewed in Ausubel et al. (1997, Short Protocols
in Molecular Biology, John Wiley & Sons, New York N.Y., Units
2.8-2.11, 3.18-3.19 and 4-64.9).
Dot-blot, slot-blot, low density and high density arrays are prepared
and analyzed using methods known in the art. cDNAs from about 18
consecutive nucleotides to about 5000 consecutive nucleotides in
length are contemplated by the invention and used in array technologies.
The preferred number of cDNAs on an array is at least about 100,000,
a more preferred number is at least about 40,000, an even more preferred
number is at least about 10,000, and a most preferred number is
at least about 600 to about 800. The array may be used to monitor
the expression level of large numbers of genes simultaneously and
to identify genetic variants, mutations, and SNPs. Such information
may be used to determine gene function; to understand the genetic
basis of a disorder; to diagnose a disorder; and to develop and
monitor the activities of therapeutic agents being used to control
or cure a disorder. (See, e.g., U.S. Pat. No. 5,474,796; WO95/11995;
WO95/35505; U.S. Pat. No. 5,605,662; and U.S. Pat. No. 5,958,342.)
Screening and Purification Assays
A cDNA may be used to screen a library or a plurality of molecules
or compounds for a ligand which specifically binds the cDNA. Ligands
may be DNA molecules, RNA molecules, peptide nucleic acid molecules,
peptides, proteins such as transcription factors, promoters, enhancers,
repressors, and other proteins that regulate replication, transcription,
or translation of the polynucleotide in the biological system. The
assay involves combining the cDNA or a fragment thereof with the
molecules or compounds under conditions that allow specific binding
and detecting the bound cDNA to identify at least one ligand that
specifically binds the cDNA.
In one embodiment, the cDNA may be incubated with a library of
isolated and purified molecules or compounds and binding activity
determined by methods such as a gel-retardation assay (U.S Pat.
No. 6,010,849) or a reticulocyte lysate transcriptional assay. In
another embodiment, the cDNA may be incubated with nuclear extracts
from biopsied and/or cultured cells and tissues. Specific binding
between the cDNA and a molecule or compound in the nuclear extract
is initially determined by gel shift assay. Protein binding may
be confirmed by raising antibodies against the protein and adding
the antibodies to the gel-retardation assay where specific binding
will cause a supershift in the assay.
In another embodiment, the cDNA may be used to purify a molecule
or compound using affinity chromatography methods well known in
the art. In one embodiment, the cDNA is chemically reacted with
cyanogen bromide groups on a polymeric resin or gel. Then a sample
is passed over and reacts with or binds to the cDNA. The molecule
or compound which is bound to the cDNA may be released from the
cDNA by increasing the salt concentration of the flow-through medium
The cDNA may be used to purify a ligand from a sample. A method
for using a cDNA to purify a ligand would involve combining the
cDNA or a fragment thereof with a sample under conditions to allow
specific binding, recovering the bound cDNA, and using an appropriate
agent to separate the cDNA from the purified ligand.
Protein Production and Uses
The full length cDNAs or fragment thereof may be used to produce
purified proteins using recombinant DNA technologies described herein
and taught in Ausubel et al. (supra; Units 16.1-16.62). One of the
advantages of producing proteins by these procedures is the ability
to obtain highly-enriched sources of the proteins thereby simplifying
The proteins may contain amino acid substitutions, deletions or
insertions made on the basis of similarity in polarity, charge,
solubility, hydrophobicity, hydrophilicity, and/or the amphipathic
nature of the residues involved. Such substitutions may be conservative
in nature when the substituted residue has structural or chemical
properties similar to the original residue (e.g., replacement of
leucine with isoleucine or valine) or they may be nonconservative
when the replacement residue is radically different (e.g., a glycine
replaced by a tryptophan). Computer programs included in LASERGENE
software (DNASTAR, Madison Wis.), MACVECTOR software (Genetics Computer
Group, Madison Wis.) and RasMol software (www.umass.edu/microbio/rasmol)
may be used to help determine which and how many amino acid residues
in a particular portion of the protein may be substituted, inserted,
or deleted without abolishing biological or immunological activity.
Expression of Encoded Proteins
Expression of a particular cDNA may be accomplished by cloning
the cDNA into a vector and transforming this vector into a host
cell. The cloning vector used for the construction of cDNA libraries
in the LIFESEQ databases may also be used for expression. Such vectors
usually contain a promoter and a polylinker useful for cloning,
priming, and transcription. An exemplary vector may also contain
the promoter for .beta.-galactosidase, an amino-terminal methionine
and the subsequent seven amino acid residues of .beta.-galactosidase.
The vector may be transformed into competent E. coli cells. Induction
of the isolated bacterial strain with isopropylthiogalactoside (IPTG)
using standard methods will produce a fusion protein that contains
an N terminal methionine, the first seven residues of .beta.-galactosidase,
about 15 residues of linker, and the protein encoded by the cDNA.
The cDNA may be shuttled into other vectors known to be useful
for expression of protein in specific hosts. Oligonucleotides containing
cloning sites and fragments of DNA sufficient to hybridize to stretches
at both ends of the cDNA may be chemically synthesized by standard
methods. These primers may then be used to amplify the desired fragments
by PCR. The fragments may be digested with appropriate restriction
enzymes under standard conditions and isolated using gel electrophoresis.
Alternatively, similar fragments are produced by digestion of the
cDNA with appropriate restriction enzymes and filled in with chemically
synthesized oligonucleotides. Fragments of the coding sequence from
more than one gene may be ligated together and expressed.
Signal sequences that dictate secretion of soluble proteins are
particularly desirable as component parts of a recombinant sequence.
For example, a chimeric protein may be expressed that includes one
or more additional purification-facilitating domains. Such domains
include, but are not limited to, metal-chelating domains that allow
purification on immobilized metals, protein A domains that allow
purification on immobilized immunoglobulin, and the domain utilized
in the FLAGS extension/affinity purification system (Immunex, Seattle
Wash.). The inclusion of a cleavable-linker sequence such as ENTEROKINASEMAX
(Invitrogen, San Diego Calif.) between the protein and the purification
domain may also be used to recover the protein.
Suitable host cells may include, but are not limited to, mammalian
cells such as Chinese Hamster Ovary (CHO) and human 293 cells, insect
cells such as Sf9 cells, plant cells such as Nicotiana tabacum,
yeast cells such as Saccharomyces cerevisiae, and bacteria such
as E. coli. For each of these cell systems, a useful vector may
also include an origin of replication and one or two selectable
markers to allow selection in bacteria as well as in a transformed
eukaryotic host. Vectors for use in eukaryotic host cells may require
the addition of 3' poly(A) tail if the cDNA lacks poly(A).
Additionally, the vector may contain promoters or enhancers that
increase gene expression. Many promoters are known and used in the
art. Most promoters are host specific and exemplary promoters includes
SV40 promoters for CHO cells; T7 promoters for bacterial hosts;
viral promoters and enhancers for plant cells; and PGH promoters
for yeast. Adenoviral vectors with the rous sarcoma virus enhancer
or retroviral vectors with long terminal repeat promoters may be
used to drive protein expression in mammalian cell lines. Once homogeneous
cultures of recombinant cells are obtained, large quantities of
secreted soluble protein may be recovered from the conditioned medium
and analyzed using chromatographic methods well known in the art.
An alternative method for the production of large amounts of secreted
protein involves the transformation of mammalian embryos and the
recovery of the recombinant protein from milk produced by transgenic
cows, goats, sheep, and the like.
In addition to recombinant production, proteins or portions thereof
may be produced manually, using solid-phase techniques (Stewart
et al. (1969) Solid-Phase Peptide Synthesis, W H Freeman, San Francisco
Calif.; Merrifield (1963) J Am Chem Soc 5:2149-2154), or using machines
such as the ABI 431A peptide synthesizer (Applied Biosystems, Foster
City Calif.). Proteins produced by any of the above methods may
be used as pharmaceutical compositions to treat disorders associated
with null or inadequate expression of the genomic sequence.
Screening and Purification Assays
A protein or a portion thereof encoded by the cDNA may be used
to screen a library or a plurality of molecules or compounds for
a ligand with specific binding affinity or to purify a molecule
or compound from a sample. The protein or portion thereof employed
in such screening may be free in solution, affixed to an abiotic
or biotic substrate, or located intracellularly. For example, viable
or fixed prokaryotic host cells that are stably transformed with
recombinant nucleic acids that have expressed and positioned a protein
on their cell surface can be used in screening assays. The cells
are screened against a library or a plurality of ligands and the
specificity of binding or formation of complexes between the expressed
protein and the ligand may be measured. The ligands may be DNA,
RNA, or PNA molecules, agonists, antagonists, antibodies, immunoglobulins,
inhibitors, peptides, pharmaceutical agents, proteins, drugs, or
any other test molecule or compound that specifically binds the
protein. An exemplary assay involves combining the mammalian protein
or a portion thereof with the molecules or compounds under conditions
that allow specific binding and detecting the bound protein to identify
at least one ligand that specifically binds the protein.
This invention also contemplates the use of competitive drug screening
assays in which neutralizing antibodies capable of binding the protein
specifically compete with a test compound capable of binding to
the protein or oligopeptide or fragment thereof. One method for
high throughput screening using very small assay volumes and very
small amounts of test compound is described in U.S Pat. No. 5,876,946.
Molecules or compounds identified by screening may be used in a
model system to evaluate their toxicity, diagnostic, or therapeutic
The protein may be used to purify a ligand from a sample. A method
for using a protein to purify a ligand would involve combining the
protein or a portion thereof with a sample under conditions to allow
specific binding, recovering the bound protein, and using an appropriate
chaotropic agent to separate the protein from the purified ligand.
Production of Antibodies
A protein encoded by a cDNA of the invention may be used to produce
specific antibodies. Antibodies may be produced using an oligopeptide
or a portion of the protein with inherent immunological activity.
Methods for producing antibodies include: 1) injecting an animal,
usually goats, rabbits, or mice, with the protein, or an antigenically-effective
portion or an oligopeptide thereof, to induce an immune response;
2) engineering hybridomas to produce monoclonal antibodies; 3) inducing
in vivo production in the lymphocyte population; or 4) screening
libraries of recombinant immunoglobulins. Recombinant immunoglobulins
may be produced as taught in U.S Pat. No. 4,816,567.
Antibodies produced using the proteins of the invention are useful
for the diagnosis of prepathologic disorders as well as the diagnosis
of chronic or acute diseases characterized by abnormalities in the
expression, amount, or distribution of the protein. A variety of
protocols for competitive binding or immunoradiometric assays using
either polyclonal or monoclonal antibodies specific for proteins
are well known in the art. Immunoassays typically involve the formation
of complexes between a protein and its specific binding molecule
or compound and the measurement of complex formation. Immunoassays
may employ a two-site, monoclonal-based assay that utilizes monoclonal
antibodies reactive to two noninterfering epitopes on a specific
protein or a competitive binding assay (Pound (1998) Immunochemical
Protocols, Humana Press, Totowa N.J.).
Immunoassay procedures may be used to quantify expression of the
protein in cell cultures, in subjects with a particular disorder
or in model animal systems under various conditions. Increased or
decreased production of proteins as monitored by immunoassay may
contribute to knowledge of the cellular activities associated with
developmental pathways, engineered conditions or diseases, or treatment
efficacy. The quantity of a given protein in a given tissue may
be determined by performing immunoassays on freeze-thawed detergent
extracts of biological samples and comparing the slope of the binding
curves to binding curves generated by purified protein.
Labeling of Molecules for Assay
A wide variety of reporter molecules and conjugation techniques
are known by those skilled in the art and may be used in various
cDNA, polynucleotide, protein, peptide or antibody assays. Synthesis
of labeled molecules may be achieved using commercial kits for incorporation
of a labeled nucleotide such as .sup.32 P-dCTP, Cy3-dCTP or Cy5-dCTP
or amino acid such as .sup.35 S-methionine. Polynucleotides, cDNAs,
proteins, or antibodies may be directly labeled with a reporter
molecule by chemical conjugation to amines, thiols and other groups
present in the molecules using reagents such as BIODIPY or FITC
(Molecular Probes, Eugene Oreg.).
The proteins and antibodies may be labeled for purposes of assay
by joining them, either covalently or noncovalently, with a reporter
molecule that provides for a detectable signal. A wide variety of
labels and conjugation techniques are known and have been reported
in the scientific and patent literature including, but not limited
to U.S Pat. No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437;
4,275,149; and 4,366,241.
The cDNAs, or fragments thereof, may be used to detect and quantify
differential gene expression; absence, presence, or excess expression
of mRNAs; or to monitor mRNA levels during therapeutic intervention
associated with breast cancer. These cDNAs can also be utilized
as markers of treatment efficacy against breast cancer over a period
ranging from several days to months. The diagnostic assay may use
hybridization or amplification technology to compare gene expression
in a biological sample from a patient to standard samples in order
to detect altered gene expression. Qualitative or quantitative methods
for this comparison are well known in the art.
For example, the cDNA may be labeled by standard methods and added
to a biological sample from a patient under conditions for hybridization
complex formation. After an incubation period, the sample is washed
and the amount of label (or signal) associated with hybridization
complexes is quantified and compared with a standard value. If the
amount of label in the patient sample is significantly altered in
comparison to the standard value, then the presence of the associated
condition, disease or disorder is indicated.
In order to provide a basis for the diagnosis of a condition, disease
or disorder associated with gene expression, a normal or standard
expression profile is established. This may be accomplished by combining
a biological sample taken from normal subjects, either animal or
human, with a probe under conditions for hybridization or amplification.
Standard hybridization may be quantified by comparing the values
obtained using normal subjects with values from an experiment in
which a known amount of a substantially purified target sequence
is used. Standard values obtained in this manner may be compared
with values obtained from samples from patients who are symptomatic
for a particular condition, disease, or disorder. Deviation from
standard values toward those associated with a particular condition
is used to diagnose that condition.
Such assays may also be used to evaluate the efficacy of a particular
therapeutic treatment regimen in animal studies and in clinical
trial or to monitor the treatment of an individual patient. Once
the presence of a condition is established and a treatment protocol
is initiated, diagnostic assays may be repeated on a regular basis
to determine if the level of expression in the patient begins to
approximate that which is observed in a normal subject. The results
obtained from successive assays may be used to show the efficacy
of treatment over a period ranging from several days to months.
Gene Expression Profiles
A gene expression profile comprises a plurality of cDNAs and a
plurality of detectable hybridization complexes, wherein each complex
is formed by hybridization of one or more probes to one or more
complementary sequences in a sample. The cDNAs of the invention
are used as elements on a microarray to analyze gene expression
profiles. In one embodiment, the microarray is used to monitor the
progression of disease. Researchers can assess and catalog the differences
in gene expression between healthy and diseased tissues or cells.
By analyzing changes in patterns of gene expression, disease can
be diagnosed at earlier stages before the patient is symptomatic.
The invention can be used to formulate a prognosis and to design
a treatment regimen. The invention can also be used to monitor the
efficacy of treatment. For treatments with known side effects, the
microarray is employed to improve the treatment regimen. A dosage
is established that causes a change in genetic expression patterns
indicative of successful treatment. Expression patterns associated
with the onset of undesirable side effects are avoided. This approach
may be more sensitive and rapid than waiting for the patient to
show inadequate improvement, or to manifest side effects, before
altering the course of treatment.
In another embodiment, animal models which mimic a human disease
can be used to characterize expression profiles associated with
a particular condition, disorder or disease; or treatment of the
condition, disorder or disease. Novel treatment regimens may be
tested in these animal models using microarrays to establish and
then follow expression profiles over time. In addition, microarrays
may be used with cell cultures or tissues removed from animal models
to rapidly screen large numbers of candidate drug molecules, looking
for ones that produce an expression profile similar to those of
known therapeutic drugs, with the expectation that molecules with
the same expression profile will likely have similar therapeutic
effects. Thus, the invention provides the means to rapidly determine
the molecular mode of action of a drug.
Assavs Using Antibodies
Antibodies directed against epitopes on a protein encoded by a
cDNA of the invention may be used in assays to quantify the amount
of protein found in a particular human cell. Such assays include
methods utilizing the antibody and a label to detect expression
level under normal or disease conditions. The antibodies may be
used with or without modification, and labeled by joining them,
either covalently or noncovalently, with a labeling moiety.
Protocols for detecting and measuring protein expression using
either polyclonal or monoclonal antibodies are well known in the
art. Examples include ELISA, RIA, and fluorescent activated cell
sorting (FACS). Such immunoassays typically involve the formation
of complexes between the protein and its specific antibody and the
measurement of such complexes. These and other assays are described
in Pound (supra). The method may employ a two-site, monoclonal-based
immunoassay utilizing monoclonal antibodies reactive to two non-interfering
epitopes, or a competitive binding assay. (See, e.g., Coligan et
al. (1997) Current Protocols in Immunology, Wiley-Interscience,
New York N.Y.; Pound, supra)
The cDNAs and fragments thereof can be used in gene therapy. cDNAs
can be delivered ex vivo to target cells, such as cells of bone
marrow. Once stable integration and transcription and or translation
are confirmed, the bone marrow may be reintroduced into the subject.
Expression of the protein encoded by the cDNA may correct a disorder
associated with mutation of a normal sequence, reduction or loss
of an endogenous target protein, or overexpression of an endogenous
or mutant protein. Alternatively, cDNAs may be delivered in vivo
using vectors such as retrovirus, adenovirus, adeno-associated virus,
herpes simplex virus, and bacterial plasmids. Non-viral methods
of gene delivery include cationic liposomes, polylysine conjugates,
artificial viral envelopes, and direct injection of DNA (Anderson
(1998) Nature 392:25-30; Dachs et al. (1997) Oncol Res 9:313-325;
Chu et al. (1998) J Mol Med 76(3-4):184-192; Weiss et al. (1999)
Cell Mol Life Sci 55(3):334-358; Agrawal (1996) Antisense Therapeutics,
Humana Press, Totowa N.J.; and August et al. (1997) Gene Therapy
(Advances in Pharmacology. Vol. 40), Academic Press, San Diego Calif.).
In addition, expression of a particular protein can be regulated
through the specific binding of a fragment of a cDNA to a genomic
sequence or an mRNA which encodes the protein or directs its transcription
or translation. The cDNA can be modified or derivatized to any RNA-like
or DNA-like material including peptide nucleic acids, branched nucleic
acids, and the like. These sequences can be produced biologically
by transforming an appropriate host cell with a vector containing
the sequence of interest.
Molecules which regulate the activity of the cDNA or encoded protein
are useful as therapeutics for breast cancer. Such molecules include
agonists which increase the expression or activity of the polynucleotide
or encoded protein, respectively; or antagonists which decrease
expression or activity of the polynucleotide or encoded protein,
respectively. In one aspect, an antibody which specifically binds
the protein may be used directly as an antagonist or indirectly
as a delivery mechanism for bringing a pharmaceutical agent to cells
or tissues which express the protein.
Additionally, any of the proteins, or their ligands, or complementary
nucleic acid sequences may be administered as pharmaceutical compositions
or in combination with other appropriate therapeutic agents. Selection
of the appropriate agents for use in combination therapy may be
made by one of ordinary skill in the art, according to conventional
pharmaceutical principles. The combination of therapeutic agents
may act synergistically to affect the treatment or prevention of
the conditions and disorders associated with EGF regulation. Using
this approach, one may be able to achieve therapeutic efficacy with
lower dosages of each agent, thus reducing the potential for adverse
side effects. Further, the therapeutic agents may be combined with
pharmaceutically-acceptable carriers including excipients and auxiliaries
which facilitate processing of the active compounds into preparations
which can be used pharmaceutically. Further details on techniques
for formulation and administration used by doctors and pharmacists
may be found in the latest edition of Remington's Pharmaceutical
Sciences (Maack Publishing, Easton Pa.).
Animal models may be used as bioassays where they exhibit a phenotypic
response similar to that of humans and where exposure conditions
are relevant to human exposures. Mammals are the most common models,
and most infectious agent, cancer, drug, and toxicity studies are
performed on rodents such as rats or mice because of low cost, availability,
lifespan, reproductive potential, and abundant reference literature.
Inbred and outbred rodent strains provide a convenient model for
investigation of the physiological consequences of underexpression
or overexpression of genes of interest and for the development of
methods for diagnosis and treatment of diseases. A mammal inbred
to overexpress a particular gene (for example, secreted in milk)
may also serve as a convenient source of the protein expressed by
Transgenic Animal Models
Transgenic rodents that overexpress or underexpress a gene of interest
may be inbred and used to model human diseases or to test therapeutic
or toxic agents. (See, e.g., U.S Pat. No. 5,175,383 and U.S Pat.
No. 5,767,337.) In some cases, the introduced gene may be activated
at a specific time in a specific tissue type during fetal or postnatal
development. Expression of the transgene is monitored by analysis
of phenotype, of tissue-specific mRNA expression, or of serum and
tissue protein levels in transgenic animals before, during, and
after challenge with experimental drug therapies.
Embryonic Stem Cells
Embryonic (ES) stem cells isolated from rodent embryos retain the
potential to form embryonic tissues. When ES cells such as the mouse
129/SvJ cell line are placed in a blastocyst from the C57BL/6 mouse
strain, they resume normal development and contribute to tissues
of the live-born animal. ES cells are preferred for use in the creation
of experimental knockout and knockin animals. The method for this
process is well known in the art and the steps are: the cDNA is
introduced into a vector, the vector is transformed into ES cells,
transformed cells are identified and microinjected into mouse cell
blastocysts, blastocysts are surgically transferred to pseudopregnant
dams. The resulting chimeric progeny are genotyped and bred to produce
heterozygous or homozygous strains.
In gene knockout analysis, a region of a gene is enzymatically
modified to include a non-natural intervening sequence such as the
neomycin phosphotransferase gene (neo; Capecchi (1989) Science 244:1288-1292).
The modified gene is transformed into cultured ES cells and integrates
into the endogenous genome by homologous recombination. The inserted
sequence disrupts transcription and translation of the endogenous
ES cells can be used to create knockin humanized animals or transgenic
animal models of human diseases. With knockin technology, a region
of a human gene is injected into animal ES cells, and the human
sequence integrates into the animal cell genome. Transgenic progeny
or inbred lines are studied and treated with potential pharmaceutical
agents to obtain information on the progression and treatment of
the analogous human condition.
As described herein, the uses of the cDNAs, provided in the Sequence
Listing of this application, and their encoded proteins are exemplary
of known techniques and are not intended to reflect any limitation
on their use in any technique that would be known to the person
of average skill in the art. Furthermore, the cDNAs provided in
this application may be used in molecular biology techniques that
have not yet been developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known to the
person of ordinary skill in the art, e.g., the triplet genetic code,
specific base pair interactions, and the like. Likewise, reference
to a method may include combining more than one method for obtaining
or assembling full length cDNA sequences that will be known to those
skilled in the art. It is also to be understood that this invention
is not limited to the particular methodology, protocols, and reagents
described, as these may vary. It is also understood that the terminology
used herein is for the purpose of describing particular embodiments
only, and is not intended to limit the scope of the present invention
which will be limited only by the appended claims. The examples
below are provided to illustrate the subject invention and are not
included for the purpose of limiting the invention.
For purposes of example, the preparation and sequencing of the
breast tissue cDNA library (BRSTNOT07), from which Incyte Clone
1297817 was identified is described. Preparation and sequencing
of cDNAs in libraries in the LIFESEQ database (Incyte Genomics)
have varied over time, and the gradual changes involved use of kits,
plasmids, and machinery available at the particular time the library
was made and analyzed.
I cDNA Library Preparation
The BRSTNOT07 cDNA library was constructed from diseased breast
tissue removed from a 43-year-old Caucasian female during unilateral
extended simple mastectomy. Pathology indicated mildly proliferative
fibrocystic changes with epithelial hyperplasia, papillomatosis,
and duct ectasia. Pathology for the matched tumor tissue indicated
invasive grade 4, nuclear grade 3 mammary adenocarcinoma with extensive
The frozen tissue was homogenized and lysed in a guanidinium isothiocyanate
solution using a POLYTRON homogenizer (PT-3000, Brinkmann Instruments,
Westbury, N.J.). The lysate was centrifuged over a 5.7 M CsCl cushion
using a SW28 rotor in an L8-70M ultracentrifuge (Beckman Instruments,
Fullerton, Calif.) for 18 hours at 25,000 rpm at ambient temperature.
The RNA was extracted with acid phenol pH 4.7, precipitated using
0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in
RNAse-free water, and treated with DNase at 37.degree. C. RNA extraction
and precipitation was repeated as before. The mRNA was then isolated
using the OLIGOTEX kit (Qiagen, Inc., Chatsworth, Calif.) and used
to construct the cDNA library.
The mRNA was handled according to the recommended protocols in
the SUPERSCRIPT plasmid System (Life Technologies). The cDNA were
fractionated on a SEPHAROSE CL4B column (Amersham Pharmacia Biotech,
Piscataway N.J.), and those cDNAs exceeding 400 bp were ligated
into pSPORT I. The plasmid was subsequently transformed into DH5.alpha.
competent cells (Life Technologies).
II Isolation and Sequencing of cDNA Clones
Plasmid DNA was released from the cells and purified using the
REAL Prep 96 plasmid kit (Qiagen). This kit enabled the simultaneous
purification of 96 samples in a 96-well block using multi-channel
reagent dispensers. The recommended protocol was employed except
for the following changes: 1) the bacteria were cultured in 1 ml
of sterile Terrific Broth (Life Technologies) with carbenicillin
at 25 mg/l and glycerol at 0.4%; 2) after inoculation, the cells
were cultured for 19 hours and lysed with 0.3 ml of lysis buffer;
and 3) following isopropanol precipitation, the DNA pellet was resuspended
in 0.1 ml of distilled water. After the last step in the protocol,
samples were transferred to a 96-well block for storage at 4.degree.
The cDNAs were prepared using a MICROLAB 2200 system (Hamilton,
Reno Nev.) in combination with DNA ENGINE thermal cyclers (PTC200;
M J Research, Waltham Mass.). The cDNAs were sequenced by the method
of Sanger and Coulson (1975; J. Mol. Biol. 94:441f) using ABI PRISM
377 DNA sequencing systems (PE Biosystems). Most of the sequences
were sequenced using standard ABI protocols and kits (PE Biosystems)
at solution volumes of 0.25.times.-1.0.times.. In the alternative,
some of the sequences were sequenced using solutions and dyes from
Amersham Pharmacia Biotech.
III Homology Searching of cDNA Clones and Their Deduced Proteins
As used herein, "homology" refers to sequence similarity
between a reference sequence and at least a fragment of a newly
sequenced clone insert, and can refer to either a nucleic acid or
amino acid sequence. The GenBank databases which contain previously
identified and annotated sequences, were searched for regions of
homology using BLAST (Altschul (1993 and 1990) supra).
BLAST involves first finding similar segments between the query
sequence and a database sequence, then evaluating the statistical
significance of any matches that are found and finally reporting
only those matches that satisfy a user-selectable threshold of significance.
BLAST produces alignments of both nucleotide and amino acid sequences
to determine sequence similarity. The fundamental unit of the BLAST
algorithm output is the High scoring Segment Pair (HSP). An HSP
consists of two sequence fragments of arbitrary, but equal lengths,
whose alignment is locally maximal and for which the alignment score
meets or exceeds a threshold or cutoff score set by the user.
The basis of the search is the product score, which is defined
The product score takes into account both the degree of identity
between two sequences and the length of the sequence match as reflected
in the BLAST score. The BLAST score is calculated by scoring +5
for every base that matches in an HSP and -4 for every mismatch.
For example, with a product score of 40, the match will be exact
within a 1% to 2% error, and, with a product score of 70, the match
will be exact. Homologous molecules are usually identified by selecting
those which show product scores between 15 and 40, although lower
scores may identify related molecules. The P-value for any given
HSP is a function of its expected frequency of occurrence and the
number of HSPs observed against the same database sequence with
scores at least as high. Percent sequence identity is found in a
comparison of two or more amino acid or nucleic acid sequences.
Percent identity can be determined electronically, e.g., by using
the MEGALIGN program (DNASTAR). The percentage similarity between
two amino acid sequences, e.g., sequence A and sequence B, is calculated
by dividing the length of sequence A, minus the number of gap residues
in sequence A, minus the number of gap residues in sequence B, into
the sum of the residue matches between sequence A and sequence B,
times one hundred. Gaps of low or of no homology between the two
amino acid sequences are not included in determining percentage
Sequences with conserved protein motifs may also be searched using
the BLOCKS search program. This program analyses sequence information
contained in the Swiss-Prot Database and PROSITE and is useful for
determining the classification of uncharacterized proteins translated
from genomic or cDNA sequences (Bairoch, supra; Attwood, supra).
PROSITE is a useful source for identifying functional or structural
domains that are not detected using motifs due to extreme sequence
divergence. Using weight matrices, these domains are calibrated
against the SWISS-PROT database to obtain a measure of the chance
distribution of the matches.
The PRINTS database can be searched using the BLIMPS search program
to obtain protein family "fingerprints". The PRINTS database
complements the PROSITE database by exploiting groups of conserved
motifs within sequence alignments to build characteristic signatures
of different protein families. For both BLOCKS and PRINTS analyses,
the cutoff scores for local similarity were: >1300=strong, 1000-1300=suggestive;
for global similarity were: p<exp-3; and for strength (degree
of correlation) were: >1300=strong, 1000-1300=weak.
IV Extension of cDNA Clones
Some of the nucleic acid sequences of SEQ ID NO: 1-16 were produced
by extension of an appropriate fragment of the molecule using oligonucleotide
primers designed from this fragment. One primer was synthesized
to initiate 5' extension of the known fragment, and the other primer,
to initiate 3' extension of the known fragment. The initial primers
were designed using OLIGO 4.06 software (National Biosciences),
or another appropriate program, to be about 22 to 30 nucleotides
in length, to have a GC content of about 50% or more, and to anneal
to the target sequence at temperatures of about 68.degree. C. to
about 72.degree. C. Any stretch of nucleotides which would result
in hairpin structures and primer- primer dimerizations was avoided.
Selected human cDNA libraries were used to extend the sequence.
If more than one extension was necessary or desired, additional
or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well
known in the art. PCR was performed in 96-well plates using the
DNA Engine thermal cycler (MJ Research, Inc.). The reaction mix
contained DNA template, 200 nmol of each primer, reaction buffer
containing Mg.sup.2+, (NH.sub.4).sub.2 SO.sub.4, and .beta.-mercaptoethanol,
Taq DNA polymerase (Amersham Pharmacia Biotech), ELONGASE enzyme
(Life Technologies), and Pfu DNA polymerase (Stratagene), with the
following parameters for primer pair PCI A and PCI B: Step 1: 94.degree.
C., 3 min; Step 2: 94.degree. C., 15 sec; Step 3: 60.degree. C.
1 min; Step 4: 68.degree. C., 2 min; Step 5: Steps 2, 3, and 4 repeated
20 times; Step 6: 68.degree. C., 5 min; Step 7: storage at 4.degree.
C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step 1: 94.degree. C., 3 min; Step 2: 94.degree.
C., 15 sec; Step 3: 57.degree. C., 1 min; Step 4: 68.degree. C.,
2 min; Step 5: Step 2, 3, and 4 repeated 20 times; Step 6: 68.degree.
C., 5 min; Step 7: storage at 4.degree. C.
The concentration of DNA in each well was determined by dispensing
100 .mu.l PICOGREEN quantitation reagent (0.25% (v/v) PICOGREEN;
Molecular Probes, Eugene Oreg.) dissolved in 1.times. TE and 0.5
.mu.l of undiluted PCR product into each well of an opaque fluorimeter
plate (Corning Costar, Acton Mass.), allowing the DNA to bind to
the reagent. The plate was scanned in a Fluoroskan II (Labsystems
Oy, Helsinki, Finland) to measure the fluorescence of the sample
and to quantify the concentration of DNA. A 5 .mu.l to 10 .mu.l
aliquot of the reaction mixture was analyzed by electrophoresis
on a 1% agarose mini-gel to determine which reactions were successful
in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred
to 384-well plates, digested with CviJI cholera virus endonuclease
(Molecular Biology Research, Madison Wis.), and sonicated or sheared
prior to religation into pUC 18 vector (Amersham Pharmacia Biotech).
For shotgun sequencing, the digested nucleotides were separated
on low concentration (0.6 to 0.8%) agarose gels, fragments were
excised, and agar digested with Agar ACE (Promega). Extended clones
were religated using T4 ligase (New England Biolabs, Beverly Mass.)
into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu
DNA polymerase (Stratagene) to fill-in restriction site overhangs,
and transfected into competent E. coli cells. Transformed cells
were selected on antibiotic-containing media, individual colonies
were picked and cultured overnight at 37.degree. C. in 384-well
plates in LB/2.times. carb liquid media.
The cells were lysed, and DNA was amplified by PCR using Taq DNA
polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene)
with the following parameters: Step 1: 94.degree. C., 3 min; Step
2: 94.degree. C., 15 sec; Step 3: 60.degree. C., 1 min; Step 4:
72.degree. C., 2 min; Step 5: Step 2, 3, and 4 repeated 29 times;
Step 6: 72.degree. C., 5 min; Step 7: storage at 4.degree. C. DNA
was quantifie by PICOGREEN reagent (Molecular Probes) as described
above. Samples with low DNA recoveries were reamplified using the
same conditions as described above. Samples were diluted with 20%
dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC energy
transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham
Pharmacia Biotech) or the ABI PRISM BIGDYE Terminator cycle sequencing
ready reaction kit (PE Biosystems).
V Propagation and Maintenance of Cultured Cells
The human breast carcinoma cell line (BT-20) was purchased from
ATCC (Manassus, Va.); and primary mammary epithelial cells (HMEC),
from Clonetics (San Diego, Calif.). The cells were propagated in
media according to the supplier's recommendations.
VI Experimental Treatment of Cultured Cells
Cells were plated in culture dishes and grown at 37.degree. C.
in 5% CO.sub.2 till 80% confluent. The cells were then grown for
48 hours in the presence of 1% fetal bovine serum (FBS), following
which the spent media was removed and replaced with either media
alone or media containing 50 ng/ml EGF (R and D system, Minneapolis,
Minn.). Control and EGF-stimulated cells were lysed at different
time points (e.g. 4, 8, 12, 24, 36, and 48 hours for the BT20 experiment)
VII Preparation of mRNA
Following the experimental treatments described above, total RNA
was extracted from cell samples using the TRIZOL reagent (Life Technologies)
extraction protocol based on the supplier's recommendations, and
the mRNA purified using the OLIGOTEX kit (Qiagen) described above.
The mRNA from four non-diseased breast tissue samples, prepared
from three female patients ages 3242 and a pooled tissue sample
from two donors ages 43 and 58, were obtained from BioChain Institute
(San Leandro Calif.). mRNA from seven ductal carcinoma primary tumors,
prepared from six female patients, ages 46-56, and one pool of 18
donors, ages 40-72, was also obtained from the same source.
VIII Labeling of Probes and Hybridization Analyses
Target nucleic acids were amplified from bacterial vectors by thirty
cycles of PCR using primers complementary to vector sequences flanking
the insert. Amplified target nucleic acids were purified using SEPHACRYL-400
beads (Amersham Pharmacia Biotech). Purified target nucleic acids
were robotically arrayed onto a glass microscope slide (Corning
Science Products, Corning N.Y.). The slide was previously coated
with 0.05% aminopropyl silane (Sigma-Aldrich, St. Louis Mo.) and
cured at 110.degree. C. The arrayed glass slide (microarray) was
exposed to UV irradiation in a STRATALINKER V-crosslinker (Stratagene).
In an alternative method, a mixture of target nucleic acids, a
restriction digest of genomic DNA, is fractionated by electrophoresis
through an 0.7% agarose gel in 1.times.TAE [Tris-acetate-ethylenediamine
tetraacetic acid (EDTA)] running buffer and transferred to a nylon
membrane by capillary transfer using 20.times. saline sodium citrate
(SSC). Alternatively, targets are individually ligated to a vector
and inserted into bacterial host cells to form a library. Target
nucleic acids are arranged on substrate by one of the following
methods. In the first method, bacterial cells containing individual
clones are robotically picked and arranged on a nylon membrane.
The membrane is placed on bacterial growth medium, LB agar containing
carbenicillin, and incubated at 37.degree. C. for 16 hours. Bacterial
colonies are denatured, neutralized, and digested with proteinase
K. Nylon membranes are exposed to UV irradiation in a STRATALINKER
UV-crosslinker (Stratagene) to cross-link DNA to the membrane.
Each mRNA sample was reverse transcribed using MMLV reverse transcriptase
in the presence of dCTP-Cy3 or dCTP-Cy5 (Amersham Pharmacia Biotech)
according to standard protocol. After incubation at 37.degree. C.,
the reaction was stopped with 0.5 M sodium hydroxide, and RNA was
degraded at 85.degree. C. The probes were then purified using Chroma
Spin 30 gel filtration spin columns (Clontech, Palo Alto, Calif.)
and ethanol precipitation.
Competitive hybridization was performed using RNA from (1) Control
untreated versus EGF treated cells or (2) Normal breast versus tumor
The hybridization mixture, containing 0.2 mg of each of Cy3 and
Cy5 labeled cDNA probes as starting material, was heated to 65 .degree.
C., and added to the microarray surface. The array was covered with
a coverslip and incubated at 60 .degree. C. The microarrays were
washed at 45 .degree. C. in high stringency buffer (1.times.SSC
and 0.1% SDS) followed by low stringency washes (0.1.times.SSC)
A laser microscope was used to detect the fluorescence-labeled
probed. Excitation wavelengths were 488 nm for Cy3 and 632 nm for
Cy5. Each array was scanned twice, one scan per fluorophore. The
emission maxima was 565 nm for Cy3 and 650 nm for Cy5. The emitted
light was split into two photomultiplier tube detectors based on
wavelength. The output of the photomultiplier tube was digitized
and displayed as an image, where the signal intensity was represented
using a linear color transformation, with red representing a high
signal and blue a low signal. The fluorescence signal for each element
was integrated to obtain a numerical value corresponding to the
signal intensity using GEMTOOLS gene expression analysis software
IX Data Analysis and Results
Data analysis using the GEMTOOLS gene expression analysis software
(Incyte Genomics) was performed to identify those genes which exhibited
a 2-fold or more change in expression in response to EGF and displayed
a signal intensity of over 300. The sequences found in the Sequence
Listing were selected because they showed at least a 2-fold change
in expression in response to EGF treatment of a human breast tumor
cell line, BT-20, and were also differentiatially expressed in primary
breast carcinoma tissue samples. Comparisons of expression among
these cells and tissues allowed the identification of genes potentially
useful in diagnosing a breast cancer differentially expressed in
BT20 cells and breast carcinoma tissue, but not in HMEC cells).
Table 1 lists genes differentially regulated by EGF treatment at
least 2-fold in BT20 breast carcinoma cells that are also differentially
regulated at least 2-fold in at least four of seven breast carcinoma
tissue samples (BC). Column 1 lists the SEQ ID NO, column 2 the
Genbank ID, and column 3 the description of the gene by BLAST, where
identified. Sequences not identified by BLAST are indicated as "Incyte
unique". Columns 4-10 list the differential expression of the
gene in seven breast carcinoma tissue samples (BC1-BC7), and column
11 lists the maximal differential expression of the gene during
the time course of the experiment for BT20 cells (BT20). Positive
values indicate upregulation of the gene, and negative (-) values
indicate downregulation. This comparison shows that genes differentially
regulated in vitro by EGF treatment of BT20 cells are also differentially
regulated in vivo in breast carcinoma. These genes may be useful
in diagnosing and monitoring the progression of breast cancer and
the response to treatment.
X Expression of the Encoded Protein
Expression and purification of a protein encoded by a cDNA of the
invention is achieved using bacterial or virus-based expression
systems. For expression in bacteria, cDNA is subcloned into a vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into bacterial hosts, such as BL21(DE3). Antibiotic
resistant bacteria express the protein upon induction with IPTG.
Expression in eukaryotic cells is achieved by infecting Spodoptera
frugiperda (Sf9) insect cells with recombinant baculovirus, Autographica
californica nuclear polyhedrosis virus. The polyhedrin gene of baculovirus
is replaced with the cDNA by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid intermediates.
Viral infectivity is maintained and the strong polyhedrin promoter
drives high levels of transcription.
For ease of purification, the protein is synthesized as a fusion
protein with glutathione-S-transferase (GST; APB) or a similar alternative
such as FLAG. The fusion protein is purified on immobilized glutathione
under conditions that maintain protein activity and antigenicity.
After purification, the GST moiety is proteolytically cleaved from
the protein with thrombin. A fusion protein with FLAG, an 8-amino
acid peptide, is purified using commercially available monoclonal
and polyclonal anti-FLAG antibodies (Eastman Kodak, Rochester N.Y.).
XI Production of Specific Antibodies
A denatured protein from a reverse phase HPLC separation is obtained
in quantities up to 75 mg. This denatured protein is used to immunize
mice or rabbits following standard protocols. About 100 .mu.g is
used to immunize a mouse, while up to 1 mg is used to immunize a
rabbit. The denatured protein is radioiodinated and incubated with
murine B-cell hybridomas to screen for monoclonal antibodies. About
20 mg of protein is sufficient for labeling and screening several
In another approach, the amino acid sequence translated from a
cDNA of the invention is analyzed using PROTEAN software (DNASTAR)
to determine regions of high antigenicity, essentially antigenically-effective
epitopes of the protein. The optimal sequences for immunization
are usually at the C-terminus, the N-terminus, and those intervening,
hydrophilic regions of the protein that are likely to be exposed
to the external environment when the protein is in its natural conformation.
Typically, oligopeptides about 15 residues in length are synthesized
using an ABI 431 peptide synthesizer (Applied Biosystems) using
Fmoc-chemistry and then coupled to keyhole limpet hemocyanin (KLH;
Sigma Aldrich) by reaction with M-maleimidobenzoyl-N-hydroxysuccinimide
ester. If necessary, a cysteine may be introduced at the N-terminus
of the peptide to permit coupling to KLH. Rabbits are immunized
with the oligopeptide-KLH complex in complete Freund's adjuvant.
The resulting antisera are tested for antipeptide activity by binding
the peptide to plastic, blocking with 1% BSA, reacting with rabbit
antisera, washing, and reacting with radioiodinated goat anti-rabbit
Hybridomas are prepared and screened using standard techniques.
Hybridomas of interest are detected by screening with radioiodinated
protein to identify those fusions producing a monoclonal antibody
specific for the protein. In a typical protocol, wells of 96 well
plates (FAST, Becton-Dickinson, Palo Alto Calif.) are coated with
affinity-purified, specific rabbit-anti-mouse (or suitable anti-species
Ig) antibodies at 10 mg/ml. The coated wells are blocked with 1%
BSA and washed and exposed to supernatants from hybridomas. After
incubation, the wells are exposed to radiolabeled protein at 1 mg/ml.
Clones producing antibodies bind a quantity of labeled protein that
is detectable above background.
Such clones are expanded and subjected to 2 cycles of cloning at
1 cell/3 wells. Cloned hybridomas are injected into pristane-treated
mice to produce ascites, and monoclonal antibody is purified from
the ascitic fluid by affinity chromatography on protein A (APB).
Monoclonal antibodies with affinities of at least 10.sup.8 M.sup.-1,
preferably 10.sup.9 to 10.sup.10 M.sup.-1 or stronger, are made
by procedures well known in the art.
XII Purification of Naturally Occurring Protein Using Specific
Naturally occurring or recombinant protein is substantially purified
by immunoaffinity chromatography using antibodies specific for the
protein. An immunoaffinity column is constructed by covalently coupling
the antibody to CNBr-activated SEPHAROSE resin (APB). Media containing
the protein is passed over the immunoaffinity column, and the column
is washed using high ionic strength buffers in the presence of detergent
to allow preferential absorbance of the protein. After coupling,
the protein is eluted from the column using a buffer of pH 2-3 or
a high concentration of urea or thiocyanate ion to disrupt antibody/protein
binding, and the protein is collected.