A purified and isolated DNA sequence and the encoded mammary-specific
protein, mammaglobin, are disclosed. Also disclosed are methods
for the detecting breast cancer based upon the overexpression and
secretion of mammaglobin by breast cancer cells. The methods detect
and/or quantitate the presence of mammaglobin or the mRNA encoding
What is claimed is:
1. An isolated and purified polypeptide comprising an epitope of
mammaglobin, wherein said epitope comprises at least five contiguous
amino acids of SEQ ID NO: 2.
2. The polypeptide of claim 1 which comprises SEQ ID NO: 2.
3. The polypeptide of claim 1 which comprises a sequence of 93
amino acids, said sequence being a conservatively substituted derivative
of SEQ ID NO: 2, wherein said polypeptide is antigenic.
4. An isolated and purified antibody which is specific for an epitope
of mammaglobin comprising at least five contiguous amino acids of
SEQ ID NO: 2.
5. The antibody of claim 4 which is generated against a polypeptide
comprising SEQ ID NO: 2.
6. The antibody of claim 4 which is generated against a polypeptide
comprising a sequence of 93 amino acids, said sequence being a conservatively
substituted derivative of SEQ ID NO: 2.
7. The antibody of claim 4, wherein said epitope is secreted by
a breast cancer neoplasia cell.
BACKGROUND OF THE INVENTION
(1) Field of the Invention
This invention relates generally to the field of breast cancer
pathogenesis and, more particularly, to a cDNA sequence and encoded
mammary-specific protein for use in detecting and treating breast
(2) Description of the Related Art
Breast cancer is one of the most common and potentially lethal
of cancers. Although early diagnosis and treatment can reduce morbidity
and mortality related to the disease, the positive predictive value
of mammography has been estimated to be only about 25% (Hall et
al., N Engl J Med 327: 319-328, 1992 which is incorporated by reference).
It would, therefore, be desirable to have a means for detecting
the cancer earlier than the cancer can be detected using mammography
and a genetic or biochemical marker might be able to provide such
means to complement and increase the predictive value of mammography.
(Hayes, Hematol Oncol Clin N Am 8: 485, 1994 which is incorporated
The development of breast cancer is accompanied by a number of
genetic changes (For review see Porter-Jordan, Hematol Oncol Clin
N Am 8: 73, 1994 which is incorporated by reference). Such changes
include gross chromosomal alterations and loss of genetic markers
(Devilee et al, Biochim Biophys Acta 1198: 113, 1994; Callahan et
al, J Cell Biochem Suppl 17: 167, 1993 which are incorporated by
reference). The progression of breast neoplasia has also been shown
to result in qualitative and quantitative changes in expression
of previously identified genes that encode growth factors and their
receptors (Zajchowski et al., Cancer Res 48: 7041, 1988 which is
incorporated by reference), structural proteins (Trask et al., Proc
Natl Acad Sci 87: 2319, 1990 which is incorporated by reference),
second messenger proteins (Ohuchi et al., Cancer Res 26: 2511, 1986
which is incorporated by reference), and transcription factors (Harris,
Adv Cancer Res 59: 69: 1992 which is incorporated by reference).
These changes in gene expression could potentially form the basis
for developing a breast cancer marker, although the precise role
of these gene changes in the pathogenesis of breast carcinoma in
patient biopsy samples is not well understood.
In addition to providing a genetic or biochemical marker for breast
cancer for early detection of the disease, it would also be desirable
to have a tumor marker that might provide an estimation of prognosis,
a means for selection and evaluation of therapy and a means for
the targeting of therapy. Although a number of tissue markers have
been identified, none are sufficiently sensitive or tumor specific
to be ideally suited for diagnosis or for screening the general
population. (Id.). Thus, there remains a continuing need for a breast
cancer marker such as a gene along with its expressed protein that
can be used to specifically and selectively identify the appearance
and pathogenic development of breast cancer in a patient.
Using a modified differential display polymerase chain reaction
technique to isolate differentially expressed sequence tags from
mammary carcinoma, several sequence fragments were isolated that
were uniquely expressed in neoplastic mammary epithelial tissue
as compared to normal tissue controls (Watson and Fleming, Cancer
Res 54: 4598-4602, 1994 which is incorporated by reference). The
discovery of one of these sequence tags identified as DEST002 has
led to the discovery and isolation of the novel full length cDNA
and encoded protein now referenced as mammaglobin. The cDNA and
protein are both new.
SUMMARY OF THE INVENTION
Briefly, therefore, the present invention is directed to the identification
of novel genes whose expression is increased in breast cancer and
to the isolating of cDNA's from the mRNA's of these genes. Accordingly,
applicants have succeeded in discovering a novel cDNA and the encoded
mammary-specific secretory protein, mammaglobin. The cDNA is in
purified and isolated form and identified as SEQ ID NO: 1 and the
encoded protein, mammaglobin is in purified and isolated form and
identified as SEQ ID NO: 2.
Mammaglobin is overexpressed in 27% of stage I primary breast cancer
tumors. This suggests that dysregulation of the mammaglobin gene
occurs early and frequently in breast cancer. The discovery of mammaglobin
and its cDNA, therefore, provide the basis for the development of
novel methods and compositions for the detection and treatment of
breast neoplastic disease in humans and other mammals.
Thus, the present invention is also directed to novel methods for
detecting the presence of breast neoplasia cells in a sample. In
one embodiment cDNA encoding mammaglobin or a derivative of said
cDNA is used to detect the presence of mammaglobin mRNA in a sample.
The method comprises the steps of: (a) providing a polynucleotide
containing a nucleotide sequence having the sequence of SEQ ID NO:
1 or a derivative thereof, (b) incubating the nucleotide sequence
with the sample under conditions in which the sequence can hybridize
with mRNA from breast neoplasia cells, and (c) detecting the existence
of a DNA-RNA hybridization complex.
Another aspect the present invention provides for a kit for detecting
the presence of breast neoplasia cells in a sample. The kit comprises
a polynucleotide containing a nucleotide sequence having the sequence
of SEQ ID NO: 1 or a derivative thereof packaged in a container.
In another embodiment of the present invention, mammaglobin or
a derivative thereof is used to detect the presence of cDNA that
is reverse transcribed from mammaglobin mRNA in a sample. The method
comprises the steps of: (a) producing a cDNA from mRNA using the
reverse transcription method in a sample obtained from a patient,
(b) providing two oligomers which are primers for the polymerase
chain reaction method and which flank or lie within a cDNA encoding
mammaglobin, and (c) amplifying the cDNA encoding mammaglobin by
the polymerase chain reaction method. The two oligomers comprise
SEQ ID NO: 3 and SEQ ID NO: 4.
Another embodiment to the present invention provides a kit for
detection of the presence of breast neoplasia cells in a sample.
The kit comprises two oligomers which are primers for the polymerase
chain reaction method and which are flank or lie within a cDNA encoding
mammaglobin packaged in a container. The two oligomers comprise
SEQ ID NO: 3 and SEQ ID NO: 4.
In another embodiment of the present invention, the presence of
the mammaglobin expressed by a tumor cell is detected in a sample
using specific antibodies to the protein, mammaglobin. The specific
antibodies can be polyclonal or monoclonal antibodies.
Among the several advantages found to be achieved by the present
invention, therefore, may be noted the provision of a nucleotide
sequence and encoded amino acid sequence that can serve as markers
for breast cancer cells; the provision of methods for early detection
of the presence of breast neoplasia cells; the provision of means
for detecting breast cancer that can complement mammography and
increase the predictive value; and the provision of methods that
can provide an estimation of prognosis; and the provision of markers
that will allow the targeting of therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the strategy used to isolate the full length
mammaglobin cDNA using the Rapid amplification of cDNA Ends (RACE)
Polymerase Chain Reaction (PCR) technique and subsequent subcloning
into vectors pGEM7Z and pCEV27.
FIG. 2 illustrates the human cDNA sequence of SEQ ID NO: 1 (nucleotides
numbered above) and the amino acid sequence of the encoded the mammary-specific
protein, mammaglobin (SEQ ID NO: 2)(amino acids numbered below),
the solid bar illustrating the 403 bp fragment (SEQ ID NO: 5) isolated
by the RACE PCR method and the open bar indicating the 206 bp DEST002
sequence (SEQ ID NO: 6);
FIG. 3 illustrates the amino acid sequence of the mammary-specific
protein, mammaglobin (hMAM), (SEQ ID NO: 2) compared to rat prostatic
steroid binding protein subunit C3 (rPSC3)(SEQ ID NO: 7) and human
clara cell 10 kD protein (hCC10)(SEQ ID NO: 8) with identities marked
by bold letters and double lines and structurally similar amino
acids marked by single lines;
FIG. 4 illustrates (A) the Northern blot analysis of hybridization
of the human cDNA sequence encoding the mammary-specific protein,
mammaglobin (hMAM), to mRNA expressed by tissues from breast neoplasia,
normal breast and other adult tissues and (B) the analysis of RT/PCR
amplified samples of tissues from breast neoplasia, normal breast
and other adult tissues;
FIG. 5 illustrates the translation of the mammary-specific cDNA
sequence in an in vitro rabbit reticulocyte lysate assay system;
FIG. 6 illustrates Northern blot hybridization with the cDNA encoding
mammaglobin detecting mRNA in tumor 2410, in tumors from three of
eight other patients (shown in bold), and to a lesser extent, in
normal breast tissue (shown in italics) comparing in two cases,
mammaglobin expression in tumor tissue and patient matched normal
DESCRIPTION OF THE PREFERRED EMBODIMENTS
One aspect of the present invention is based upon the identification
and sequencing of the cDNA identified as SEQ ID NO: 1 which encodes
a mammary-specific secretory protein, mammaglobin, identified by
SEQ ID NO: 2 (FIG. 2). As described below, the full length mammaglobin
cDNA was isolated starting from tumor cell mRNA that was reverse
transcribed, amplified using the technique of PCR and subcloned
into expression vectors. In addition, the protein, mammaglobin,
encoded by the cDNA was identified and characterized.
Using the anonymous sequence tag previously designated DEST002,
it was demonstrated that the corresponding gene product, which was
up until now unknown but herein identified as mammaglobin, is particularly
abundant in the breast cancer tumor cell line MDA-MB415. To isolate
the full length mammaglobin cDNA, the mRNA was reverse transcribed
from this cell line and cloned using the RACE PCR technique (Edwards
et al. Nucleic Acids Research 19: 5227-32, 1991 which is incorporated
by reference). This technique is based upon the strategy of ligation
of single-stranded oligodeoxyribonucleotide to the 3' end of single-stranded
cDNA. The method by which the mammaglobin cDNA was isolated is represented
schematically in FIG. 1. The full length 503 bp cDNA sequence (SEQ
ID NO: 1) was deduced from the sequence information obtained from
the 403 bp fragment (SEQ ID NO: 5) (FIG. 2) isolated by this technique
along with sequence information previously obtained from the corresponding
DEST sequence (DEST002, SEQ ID NO: 6) in our earlier study (Watson
and Fleming, supra) (FIG. 2). The full length mammaglobin cDNA and
the encoded polypeptide is shown in FIG. 2. Within the 503 bp cDNA
is a 279 bp open reading frame which encodes a polypeptide of 93
amino acids and predicted molecular mass of 10.5 kD (FIG. 2). The
first 19 residues of this open reading frame also predict a hydrophobic
peptide signal sequence. The initial methionine of the open reading
frame contains a near-perfect Kozak consensus sequence (Kozak, Cell
22: 7-8, 1980 which is incorporated by reference). The 60 bp upstream
of this sequence contain no other in-frame methionines or translational
stops. The 3' untranslated sequence of the cDNA constitutes 163
bp and contains a polyadenylation signal, AATAAA, 12 bp upstream
of the priming site of the original DEST002 sequence. These data
indicate that the full length mammaglobin cDNA has been isolated.
A search for DNA sequences similar to the mammaglobin cDNA sequence
in Genbank using the BLAST algorithm (Benson et al., Nucl Acid Res21:
2963-2965, 1993; Altschul et al, J Mol Biol 215: 403-410, 1990 which
are incorporated by reference), identified no obvious DNA sequence
homologies. Thus, mammaglobin cDNA is believed to be a novel, heretofore
unknown DNA sequence.
A search of other polypeptides for sequences related to mammaglobin
revealed an amino-acid sequence homology between mammaglobin and
other polypeptides. Mammaglobin exhibited 42% amino acid identity
(58% including conservative substitutions) with rat prostatic steroid
binding protein (prostatein) subunit C3 (rPSC3) (FIG. 3) (SEQ ID
NO: 7). Rat prostatic steroid binding protein is a major secretory
protein in the rat ventral prostate consisting of a tetrameric protein
composed of two different dimeric subunits; C3/C1 and C3/C2 (Parker
et al., Ann N Y Acad Sci 438: 115-124; Parker et al., J Steroid
Biochem 20: 67-71, 1984 which are incorporated by reference). The
C1, C2, and C3 genes all encode approximately 6 kD secretory proteins
and are thought to have arisen from gene duplication, but while
the C1 and C2 genes show strong homology to each other, they are
much less similar to the C3 gene. Correspondingly, mammaglobin shows
no sequence homology with the C1 or C2 proteins.
As noted above, prostatic steroid binding protein (prostatein)
is the major secretory protein in the rat ventral prostate and its
expression is regulated by androgenic steroids (Parker et al, Ann
N Y Acad Sci 438: 115-24, 1984; Parker et al, J Steroid Biochem
20: 67-71, 1984 which are incorporated by reference). Another protein,
human estramustin-binding protein (hEMBP) has been reported to be
expressed in human prostate, human breast cancer and human malignant
melanoma. (Bjork et al, Cancer Res 42: 1935-1942, 1982; Bjork et
al, Anticancer Res 11: 1173-82, 1991 which are incorporated by reference).
Human estramustin-binding protein is immunochemically similar to
rat estramustin-binding protein, which has been postulated to be
identical to rat steroid-binding protein, prostatein. As noted above,
the amino acid sequence of mammaglobin exhibited 42% amino acid
identity and 58% homology including conservative substitutions with
the C3 subunit of prostatein. Thus it is possible that mammaglobin
could be in some way related to hEMBP. However, while both prostatein
and hEMBP are detected in the prostate gland, mammaglobin mRNA is
completely absent in this tissue. Hence, mammaglobin is neither
the same protein nor a subunit of hEMBP and, furthermore, the sequence
of hEMBP has not been determined so that it is not known whether
there is even any similarity of mammaglobin with some fragment or
subunit of hEMBP.
Although recent reports have demonstrated the rPSC3 promoter fused
to SV40 T antigen produces both prostatic and mammary carcinomas
in transgenic mice (Maroulakou et al., Proc Nat Acad Sci U.S. 91:11236-11240,
1994; Sandmoller et al, Oncogene 9: 2805-2815, 1994 which are incorporated
by reference), the true biological function of this protein is unknown.
Furthermore, notwithstanding the hypothesized relationship of rat
prostatic steroid binding protein to human EMBP, no human polypeptide
or human gene corresponding to rPSC3 has been identified. Thus,
mammaglobin and the cDNA encoding mammaglobin represent novel sequences
Using manual alignment with other sequences that had less significant
BLAST scores with both mammaglobin and rPSC3 protein sequences,
we identified other homologies with human clara cell 10 kD protein
(hCC10) (SEQ ID NO: 8) (Peri et al, J Clin Invest 92:2099-2109,
1993 which is incorporated by reference) (FIG. 3) and, in addition,
with rabbit and mouse uteroglobin proteins (Miele et al., Endocrine
Rev 8: 474-90, 1987; Cato and Beato, Anticancer Res 5: 65-72, 1985;
Miele et al., J Endocrinol Invest 17: 679-692, 1994 which are incorporated
by reference). These homologies, depending on species, were 26%
identity or 40% including conservative substitutions. In particular,
a number of amino acids were perfectly conserved among all proteins,
including Cys-3 and Cys-69 which are known to play a role in disulfide
bond formation between uteroglobin subunits (see below). These homologies
suggest that mammaglobin is a novel member of a small family of
proteins that are secreted by epithelial cells (Miele et al, 1994,
The hCC10 gene is the human homologue of rabbit and mouse uteroglobin
genes (Peri et al, J Clin Invest 92: 2099-2109, 1993 which is incorporated
by reference). Uteroglobin was originally characterized as a secretory
protein in rabbit uterus, but has since been found in other epithelial
organs including lung, breast and prostate. Unlike rat prostatein,
uteroglobin is a homodimeric protein coupled by two disulfide linkages
at the conserved residues Cys-2 and Cys-69 (Miele et al, 1994, supra).
Although uteroglobin gene transcription is regulated by steroid
hormones, the ability of the protein itself to bind progesterone
or other steroid hormones is controversial and again, the true biological
function of this protein is unknown (Miele et al., 1994, supra).
Mammaglobin expression is restricted to the mammary gland. This
is in contrast to the observation that rPSC3 is expressed in rat
ventral prostate (Parker et al., Ann N Y Acad Sci 438: 115-1124,
1984), and the expression of hCC10/uteroglobin in numerous tissues
including lung, uterus, prostate, and breast (Miele et al., 1987,
supra; Cato and Beato, supra; Miele et al., 1994 supra). Because
of the sequence homology between mammaglobin and these proteins,
we determined the pattern of tissue specific expression. The 500
bp mammaglobin message was easily detected in tumor specimen 2410
(the tissue from which this original sequence tag was isolated)
and to a much less extent in normal human breast tissue (FIG. 4A).
The mammaglobin message could not be detected in the immortalized
breast epithelial cell line B5-589. Expression of mammaglobin was
also undetectable in human uterus and lung, two sites of uteroglobin
Amplification using RT/PCR detected mammaglobin mRNA in both tumor
2410 and normal breast tissue, but not in 15 other tissues surveyed,
including tissues that normally express rPSC3 and uteroglobin (lung,
uterus, prostate), hormonally responsive and steroidogenic tissues
(ovary, testis, placenta), and other secretory epithelial organs
(colon) (FIG. 4B). Therefore, the expression of mammaglobin mRNA
is relatively specific for mammary tissue.
Based on the studies in this report, mammaglobin is a relatively
mammary-specific protein. Two other genes known to be overexpressed
in breast carcinoma are erb-B and cyclin D (Jardines et al, Patholology
61: 268-282, 1994; Keyomars and Pardee, Proc Nat Acad Sci U.S. 90:
1112-1116, 1993 which is incorporated by reference). Unlike the
overexpression of erb-B or cyclin D, the overexpression of mammaglobin
may reflect a more specific alteration of the mammary epithelial
cell rather than a general increased growth potential or mitotic
rate. As such, appearance of mammaglobin gene dysregulation may
have more specific import for the therapeutic vulnerability or clinical
course of a tumor.
Mammaglobin expression could not be detected in normal lymph nodes
or peripheral lymphocytes at the level of sensitivity afforded by
a single step RT/PCR assay. This suggests that analysis of mammaglobin
transcripts in peripheral lymph nodes may be useful for detecting
occult breast cancer metastases, as has been suggested for other
epithelial specific genes (Schoenfeld et al., Cancer Res 54: 2986-90
which is incorporated by reference).
To demonstrate that the mammaglobin cDNA encoded a translatable
protein, the cDNA clone was used in an In vitro translation assay.
FIG. 5 shows the protein product from a rabbit reticulocyte lysate
programmed with the mammaglobin cDNA. An approximately 6 kD protein
is generated using the mammaglobin cDNA. The apparent molecular
weight is smaller than that predicted from conceptual translation
of the open reading frame, but this finding is commonly observed
with rabbit and human uteroglobin translation products as well.
Although we detected overexpression of mammaglobin RNA in one tumor
specimen (i.e. 2410), it was not clear at what frequency this overexpression
is seen in other breast carcinomas. We therefore examined a panel
of fifteen, stage I primary breast carcinomas of differing histological
types by Northern blot hybridization with the mammaglobin cDNA probe.
Because of potential variability in expression due to environment
influences (e.g. patient hormonal status), we also sought to compare
tumor specimens directly with patient-matched normal breast tissues
samples, although this was not possible in many cases. As shown
in FIG. 6, the 500 bp mammaglobin mRNA was again detected in normal
breast tissue and tumor 2410. Mammaglobin was also detected in three
other tumors, two of which demonstrated little or no expression
in patient-matched normal tissue. In all, 4 of 15 (27%) of tumors
examined overexpressed mammaglobin mRNA. These data suggest that
overexpression of mammaglobin is not unique to a single tumor specimen
and is, in fact, relatively frequent among primary breast tumors.
Furthermore, the fact that all tumors examined were stage I suggests
that this dysregulation occurs relatively early in the progression
of breast neoplasia.
Because Applicants believe mammaglobin is likely to be a secreted
protein, its presence would be expected to be detectable in sera
from patients whose tumor overexpresses this gene product. As such,
mammaglobin is likely to be as clinically useful as prostate specific
antigen (PSA) and other solid tumor markers for managing patients
with breast cancer (Tumor markers in diagnostic pathology, Clin
Lab Med 10: 1-250, 1990 which is incorporated by reference).
We determined the prevalence of mammaglobin as a tumor marker in
the general population of breast cancer tumors by examining the
expression of mammaglobin in several primary breast carcinomas.
Although the number of specimens examined in this study was small,
27% of tumors evaluated overexpressed mammaglobin mRNA. This percentage
is comparable to the prevalence of other genetic alterations such
as erb-B amplification and p53 mutation (Slamon et al. Sci 244:
707-712, 1989; Thor et al, J Nat'l Cancer Inst 84: 845-855, 1992
which are incorporated by reference). Furthermore, because we have
restricted our analysis to stage I tumors, overexpression of mammaglobin
would actually be more prevalent than any other genetic alteration
reported in this subgroup of tumors (Alllerd et al, J Nat'l Cancer
Inst 85: 200-206, 1993 which is incorporated by reference).
The identification of mammaglobin as a breast cancer marker provides
the basis for another aspect of the present invention, which involves
methods for detecting the presence of breast cancer in a patient.
The term "detection" as used herein in the context of
detection of breast neoplastic disease is intended to be a comprising
aspect of the determining of the presence of breast cancer in a
patient, the distinguishing of breast cancer from other diseases,
the estimation of prognosis in terms of probable outcome of the
disease and prospect for recovery, the monitoring of the disease
status or the recurrence of the disease, the determining of a preferred
therapeutic regimen for the patient and the targeting of antitumor
The method for detecting breast cancer comprises hybridizing a
polynucleotide to mRNA from breast neoplasia cells. The polynucleotide
comprises SEQ ID NO: 1 or a derivative of SEQ ID NO: 1. By derived
from a nucleotide sequence it is meant that the derived nucleotide
sequence is substantially the same as the sequence from which it
is derived in that the derived nucleotide sequence has sufficient
sequence complementarity to the sequence from which it is derived
to hybridize to mRNA from breast neoplasia cells under the same
stringency conditions that the sequence from which it is derived
hybridizes to the mRNA from breast neoplasia cells.
The derived nucleotide sequence is not necessarily physically derived
from the nucleotide sequence, but may be generated in any manner
including for example, chemical synthesis or DNA replication or
reverse transcription or transcription.
To detect the presence of mRNA encoding mammaglobin in a detection
system for breast cancer, a sample is obtained from a patient. The
sample can be a tissue biopsy sample or a sample of blood, plasma,
serum or the like. The sample may be treated to extract the nucleic
acids contained therein. The resulting nucleic acid from the sample
is subjected to gel electrophoresis or other size separation techniques.
Detection involves contacting the nucleic acids and in particular
the mRNA of the sample with a DNA sequence serving as a probe to
form hybrid duplexes. The term "probe" refers to a structure
comprised of a polynucleotide which forms a hybrid structure with
a target sequence, due to complementarity of probe sequence with
a sequence in the target region.
Detection of the resulting duplex is usually accomplished by the
use of labeled probes. Alternatively, the probe may be unlabeled,
but may be detectable by specific binding with a ligand which is
labeled, either directly or indirectly. Suitable labels and methods
for labeling probes and ligands are known in the art, and include,
for example, radioactive labels which may be incorporated by known
methods (e.g., nick translation or kinasing), biotin, fluorescent
groups, chemiluminescent groups (e.g., dioxetanes, particularly
triggered dioxetanes), enzymes, antibodies, and the like.
When using the cDNA encoding mammaloglobin or a derivative thereof
as a probe, high stringency conditions can be used in order to prevent
false positives. When using sequences derived from mammaglobin,
less stringent conditions can be used. The stringency of hybridization
is determined by a number of factors during hybridization and during
the washing procedure, including temperature, ionic strength, length
of time and concentration of formamide. These factors are outlined
in, for example, Sambrook et al. (Molecular Cloning: A Laboratory
Manual, 2d ed., 1989).
In order to increase the sensitivity of the detection in a sample
of mRNA encoding mammaglobin, the technique of reverse transcription/polymerization
chain reaction (RT/PCR) can be used to amplify cDNA transcribed
from mRNA encoding mammaglobin. The method of RT/PCR is well known
in the art (for example, see Watson and Fleming, supra).
The RT/PCR method can be performed as follows. Total cellular RNA
is isolated by, for example, the standard guanidium isothiocyanate
method and the total RNA is reverse transcribed. The reverse transcription
method involves synthesis of DNA on a template of RNA using a reverse
transcriptase enzyme and a 3' end primer. Typically, the primer
contains an oligo(dT) sequence. The cDNA thus produced is then amplified
using the PCR method and mammaglobin specific primers. (Belyavsky
et al, Nucl Acid Res 17: 2919-2932, 1989; Krug and Berger, Methods
in Enzymology, Academic Press, N.Y., Vol.152, pp. 316-325, 1987
which are incorporated by reference)
The polymerase chain reaction method is performed using two oligonucleotide
primers that are complementary to the two flanking regions of the
DNA segment to be amplified. The upstream and down stream primers
are typically from 20 to 30 base pairs in length and hybridize to
the flanking regions for replication of the nucleotide sequence.
The polymerization is catalyzed by a DNA-polymerase in the presence
of deoxynucleotide triphosphates or nucleotide analogs to produce
double-stranded DNA molecules. The double strands are then separated
by any denaturing method including physical, chemical or enzymatic.
Commonly, the method of physical denaturation is used involving
heating the nucleic acid, typically to temperatures from about 80.degree.
C. to 105.degree. C. for times ranging from about 1 to 10 minutes.
The process is repeated for the desired number of cycles.
The primers are selected to be substantially complementary to the
strand of cDNA being amplified. Therefore, the primers need not
reflect the exact sequence of the template, but must be sufficiently
complementary to selectively hybridize with the strand being amplified.
Following amplification, the PCR product is then detected by ethidium
bromide staining (Sambrook, et al., 1989, supra).
In another embodiment of the present invention, the mammaglobin
cDNA sequence or derivative thereof can be used to characterize
any alteration of the mammaglobin gene (i.e. gene rearrangement,
gene amplification, or gene deletion) in a specimen from a breast-cancer
patient. This provides a method whereby patient specimens or samples,
which do not contain intact mRNA, can still be examined for changes
in gene structure.
In one application of this technique, the mammaglobin cDNA sequence
or derivative thereof is hybridized to patient genomic DNA that
had been isolated from a patient's tumor, normal tissue, or lymphocytes
and digested with one or more restriction endonucleases. Using the
Southern blot protocol, which is well known in the art, this assay
determines whether a patient or a patient's breast tumor has a mammaglobin-gene,
which was deleted, rearranged, or amplified. Detection of these
changes can then provide important information useful for predicting
prognosis and for patient management.
In a second application of this technique, one or more pairs of
oligonucleotide primers based on the mammaglobin cDNA sequence or
derivative thereof could be used in the polymerase chain reaction
to amplify segments of the mammaglobin gene from a patient sample.
Analysis of the resulting PCR products indicate whether a particular
segment of the mammaglobin gene is deleted or rearranged. Such information
is useful for prognosis and patient management.
The present invention further provides for methods to detect the
presence of the polypeptide, mammaglobin, in a sample obtained from
a patient. Any method known in the art for detecting proteins can
be used. Such methods include, but are not limited to immunodiffusion,
immunoelectrophoresis, immunochemical methods, binder-ligand assays,
immunohistochemical techniques, agglutination and complement assays.
(for example see Basic and Clinical Immunology, Sites and Terr,
eds., Appleton & Lange, Norwalk, Conn. pp 217-262, 1991 which
is incorporated by reference). Preferred are binder-ligand immunoassay
methods including reacting antibodies with an epitope or epitopes
of mammaglobin and competitively displacing a labeled mammaglobin
protein or derivative thereof.
As used herein, a derivative of mammaglobin is intended to refer
to a polypeptide containing amino acids or modified amino acids
in which the polypeptide derivative cross-reacts with mammaglobin.
By cross-reaction it is meant that an antibody reacts with an antigen
other than the one that induced its formation.
Numerous competitive and non-competitive protein binding immunoassays
are well known in the art. Antibodies employed in such assays may
be unlabeled, for example as used in agglutination tests, or labeled
for use a wide variety of assay methods. Labels that can be used
include radionuclides, enzymes, fluorescers, chemiluminescers, enzyme
substrates or co-factors, enzyme inhibitors, particles, dyes and
the like for use in radioimmunoassay (RIA), enzyme immunoassays,
e.g., enzyme-linked immunosorbent assay (ELISA), fluorescent immunoassays
and the like.
Polyclonal or monoclonal antibodies to mammaglobin or an epitope
thereof can be made for use in immunoassays by any of a number of
methods known in the art. By epitope reference is made to an antigenic
determinant of a polypeptide. An epitope could comprise 3 amino
acids in a spacial conformation which is unique to the epitope.
Generally an epitope conists of at least 5 such amino acids. Methods
of determining the spatial conformation of amino acids are known
in the art, and include, for example, x-ray crystallography and
2 dimensional nuclear magnetic resonance.
One approach for preparing antibodies to a protein is the selection
and preparation of an amino acid sequence of all or part of the
protein, chemically synthesizing the sequence and injecting it into
an appropriate animal, usually a rabbit or a mouse.
Methods for preparation of mammaglobin or an epitope thereof include,
but are not limited to chemical synthesis, recombinant DNA techniques
or isolation from biological samples. Chemical synthesis of a peptide
can be performed, for example, by the classical Merrifeld method
of solid phase peptide synthesis (Merrifeld, J Am Chem Soc 85: 2149,
1963 which is incorporated by reference) or the FMOC strategy on
a Rapid Automated Multiple Peptide Synthesis system (DuPont Company,
Wilmington, Del.) (Caprino and Han, J Org Chem 37: 3404, 1972 which
is incorporated by reference).
Polyclonal antibodies can be prepared by immunizing rabbits by
injecting antigen into the popliteal lymph nodes followed by subsequent
boosts at two week intervals with intraperitoneal injection of antigen.
The animals are bled and sera assayed against purified mammaglobin
protein usually by ELISA. Monoclonal antibodies can be prepared
after the method of Milstein and Kohler by fusing splenocytes from
immunized mice with continuously replicating tumor cells such as
myeloma or lymphoma cells. (Milstein and Kohler Nature 256: 495-497,
1975; Gulfre and Milstein, Methods in Enzymology: Immunochemical
Techniques 73: 1-46, Langone and Banatis eds., Academic Press, 1981
which are incorporated by reference). The hybridoma cells so formed
are then cloned by limiting dilution methods and supernates assayed
for antibody production by ELISA or RIA.
The unique ability of antibodies to recognize and specifically
bind to target antigens expressed by a tumor cell provides an approach
for the treatment of cancer. (For review see LoBuglio and Saleh,
Am J Med Sci 304: 214-224, 1992; Bagshawe, Adv Pharmacol 24: 99-121,
1993 which are incorporated by reference). Thus, another aspect
of the present invention provides for a method for preventing the
onset and treating breast cancer in an animal based upon the use
of antibodies to mammaglobin, which has been discovered to be overexpressed
by breast cancer cells. Specific antibodies to mammaglobin, either
polyclonal or monoclonal, are produced by any method known in the
art. For example, murine or human monoclonal antibodies can be produced
by hybridoma technology. Alternatively, mammaglobin, or an immunologically
active fragment thereof, or an anti-idiotypic antibody, or fragment
thereof can be administered to an animal to elicit the production
of antibodies capable of recognizing the mammaglobin-expressing
The antibodies so produced or fragments thereof are labeled with
one or more oncolytic substances such as radionuclides, toxins,
or cytotoxic drugs and administered to a patient suspected of having
breast cancer. The binding of the labeled antibody to the mammaglobin
being overexpressed by the breast cancer cell will cause the death
of the cancer cell.
Any of a variety of oncolytic substances known in the art can be
used to produce such labeled antibodies. For example, immunotoxins
can be made by coupling plant and bacterial toxins to antibodies.
Such toxins include, for example, ricin, diphtheria toxin and Pseudomonas
exotoxin A. Drug-antibody conjugates can also be made in which chemotherapeutic
agents are linked to the antibody. Chemotherapeutic agents suitable
for such use include, for example, tomoxifen, doxorubicin, methotrexate,
chlorambucil, Vinca alkaloids, and mitomycin. In addition, radioimmunoconjugates
can be made in which a radionuclide is stably linked to the antibody.
Radionuclides suitable for making radioimmunoconjugates include,
for example, .beta.-emmitters such as .sup.131 I, .sup.188 Re, .sup.186
Re, .sup.67 Cu, .sup.90 Y and .sup.47 Sc; .alpha.-emitters such
as .sup.211 At, .sup.212 Bi and .sup.212 Pb; auger electron emitters
such as .sup.125 I and .sup.77 Br; and fissionable nuclides such
as .sup.10 B.
Preferred embodiments of the invention are described in the following
examples. Other embodiments within the scope of the claims herein
will be apparent to one skilled in the art from consideration of
the specification or practice of the invention as disclosed herein.
It is intended that the specification, together with the examples,
be considered exemplary only, with the scope and spirit of the invention
being indicated by the claims which follow the examples.
In the examples below, cell lines were obtained from American Type
Culture Collection and grown in Dulbecco's minimal essential medium
supplemented with 10% fetal calf serum. Tissue biopsy specimens
were obtained from the Human Cooperative Tissue Network (LiVolsi
et al, Cancer 71: 1391-1394, 1993 which is incorporated by reference).
This example illustrates the isolation of mammaglobin cDNA.
Total cellular RNA from the cell line MDA-MB415 was isolated using
the standard guanidinium isothiocyanate method. (Belyavsky et al,
supra). This RNA was used in the RACE PCR procedure employing the
Amplifinder kit (Clonetech) and following the manufacturer's protocol.
The synthesis of first strand cDNA was performed in a standard
reaction containing 1 .mu.g RNA, 10 .mu.M specific mammaglobin primer
D2R (5'-ATA AGA AAG AGA AGG TGT GG-3')(SEQ ID NO: 4), 4 .mu.l of
5.times. RT buffer (250 mM TrisCl pH8.3, 375 mM Kcl, 15 mM MgCl.sub.2),
2 .mu.l of 100 mM DTT, 1 .mu.l of 10 mM dNTPs and 200 units of Superscript.TM.
II reverse transciptase (Gibco/BRL) in a reaction volume 20 .mu.l.
The reaction proceeded for 1 hour at 45.degree. C. and was terminated
by incubating at 95.degree. C. for 5 minutes. RNA was hydrolyzed
with 400 .mu.M NaOH at 65.degree. C. for 30 minutes and neutralized
with 400 .mu.M acetic acid. Reaction was then added to 3 volumes
of 6M NaI and 10 .mu.l of treated glass beads. Beads were washed
three times with 80% EtOH and nucleic acid was eluted from the beads
in 45 .mu.l of water. Nucleic acid was then precipitated and resuspended
in 10 .mu.l of water. The purifed first strand cDNA was ligated
to the manufacturer's provided anchor oligonucleotide (SEQ ID NO:
9, 5'-CAC GAA TTC ACT ATC GAT TCT GGA ACC TTC AGA GG-3'), using
T4 RNA ligase at 27.degree. for 20 hours. One tenth of a ligation
reaction was used for PCR amplification in a 50 .mu.l reaction containing
1 .mu.M manufacturer's anchor primer (SEQ ID NO: 10, 5'-CTG GTT
CGG CCC ACC TCT GAA GGT TCC AGA ATC GAT AG-3'), 1 .mu.M mammaglobin
specific primer D2Rb (SEQ ID NO: 11, 5'-AAT CCG TAG TTG GTT TCT
CAC C-3'), 200 .mu.M dNTPs, 5 units of Vent.TM. DNA polymerase,
and 1.times. polymerase buffer (10 mM Kcl, 20 mM TrisCl, 10 mM (NH.sub.4).sub.2
SO.sub.4, 2 mM MgSO.sub.4, 0.1% Triton X-100). The reaction was
incubated at 94.degree. for 2 minutes and then 94.degree. for 45
seconds, 50.degree. for 1 minute, and 72.degree. for 90 seconds
for a total of 40 times.
The two downstream mammaglobin-specific nested oligonucleotides
were D2R (SEQ ID NO: 4) and D2Rb (SEQ ID NO: 11). An upstream mammaglobin-specific
control oligonucleotide was also used as per the manufacturer's
recommendations, D2F (5'-CTT TCT GCA AGA CCT TTG GC-3') (SEQ ID
NO: 12). All PCR amplifications were performed with Vent DNA polymerase
(New England Biolabs). The amplified RACE product was digested with
EcoRI and ligated into the EcoRI and SmaI sites of the plasmid vector
All sequencing was performed using the Taq DNA polymerase thermal
cycle sequencing kit as per the manufacture's protocol (Promega).
Briefly the procedure used is as follows.
10 pmol of sequence specific oligonucleotide was end labeled with
10 pmol of .sup.32 P-.gamma. ATP (3,000 Ci/mmol and 10 mCi/ml) using
T4 polynucleotide kinase in a 10 .mu.l reaction for 30 minutes at
37.degree. C. A polymerization reaction containing 100 ng of plasmid
template, 1.5 pmol of labeled sequencing primer, and 5 units of
sequencing grade Taq polymerase was created in 17 .mu.l of the manufacturer's
provided sequencing buffer. This reaction was aliquoted to a set
of four reaction tubes containing manufacturer's provided mix of
deoxynucleotides and either dideoxy-A, C, G, or T. The set of four
tubes were incubated at 95.degree. C. for 2 minutes and then, 94.degree.
C. for 45 seconds, 45.degree. C. for 30 seconds, and 72.degree.
C. for 1 minute for 30 times. After reactions were completed, 3
.mu.l of 80% formamide/bromphenol blue dye was added to each tube.
Samples were heated to 70.degree. C. for 2 minutes and loaded on
a 6% acrylamide/7.5M urea sequencing gel and run for 2-4 hours and
60 W constant power. The gel was dried and then exposed to Kodak
XAR5 Xray film for 2 to 24 hours.
The sequence thus obtained was a 403 bp fragment (SEQ ID NO: 5)
as shown in FIG. 2, solid bar. In earlier work the DEST002 Tag sequence
was isolated (Watson and Fleming, supra). This sequence was a 206
bp fragment (SEQ ID NO: 6) as shown in FIG. 2, open bar. Combining
the information from these two sequences allowed the full-length
503 bp cDNA of mammaglobin to be deduced. (FIG. 2).
This example demonstrates that mammaglobin expression is restricted
to mammary gland tumor cells and to a lesser extent normal mammary
Total cellular RNA samples were isolated using the standard guanidinium
isothiocyanate method and treated with RNase-free DNase (Promega).
For RT/PCR analysis, 1 .mu.g of indicated total RNA was reverse
transcribed with oligo dT.sub.21 (SEQ ID NO:13) and Superscript
II reverse transcriptase (Gibco/BRL) according to the manufacture's
Two hundred ng of oligo dT.sub.21 (SEQ ID NO: 13) and 1 .mu.g of
total RNA were incubated at 65.degree. C. for 5 minutes in a 10
.mu.l volume. Sample was chilled on ice and added to it were 4 .mu.l
of 5.times. RT buffer (250 mM TrisCl pH8.3, 375 mM Kcl, 15 mM MgCl.sub.2),
2 .mu.l of 100 mM DTT, 1 .mu.l of 10 mM dNTPs and 200 units of Superscript.TM.
II reverse transcriptase (Gibco/BRL). The reaction proceeded for
1 hour at 45.degree. C. and was terminated by incubating at 95.degree.
C. for 5 minutes.
One tenth of each RT reaction was subject to PCR analysis using
the mammaglobin specific primers D2R (5'-ATA AGA AAG AGA AGG TGT
GG-3') (SEQ ID NO: 4) and d2102 (5'-CAG CGG CTT CCT TGA TCC TTG-3')
(SEQ ID NO:3) and standard reaction conditions for 40 cycles at
94.degree..times.30 sec./55.degree..times.1 min./72.degree..times.1
For Northern analysis, 20 .mu.g of total RNA was analyzed as previously
described (Watson and Fleming, supra) using the full length mammaglobin
cDNA probe. Integrity and equal loading of each RNA sample was assessed
by ethidium bromide staining.
As shown in FIG. 4A, the 500 bp mammaglobin message is easily detected
in tumor specimen 2410 (the tissue from which this original DEST
was isolated) and to a much less extent in normal human breast tissue
but not in the immortalized breast epithelial cell line B5-589,
or in human lung, placenta, uterus and ovary (FIG. 4A). Following
amplification using RT/PCR analysis, mammaglobin expression was
still not detected in 15 tissues surveyed (FIG. 4B). Detection of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) message (FIG. 4B)
and EGF receptor message (data not shown) in these reactions demonstrated
that absence of expression was not due to degraded RNA or other
trivial explanations. Thus the expression of mammaglobin mRNA is
relatively specific for mammary tissue.
This example demonstrates that the mammaglobin cDNA encodes a translatable
nucleotide sequence which results in protein product of appropriately
predicted molecular mass. In vitro translations were performed using
the TNT.TM. rabbit reticulocyte translation kit with T7 RNA polymerase
(Promega) and .sup.35 S-Methionine (>1000 Ci/mmol; 10 mCi/ml,
Amersham) according to the manufacturer's protocol.
To 25 .mu.l of TNT.TM. rabbit reticulocyte lystae was added 2 .mu.l
of manufacturer's prepared reaction buffer, T7 RNA polymerase, 20
.mu.M amino acid mixture minus methionine, 40 .mu.Ci.sup.35 S-methionine
(1,000 Ci/mmol and 10 mCi/ml), 40 units ribonuclease inhibitor,
1 .mu.g of mammaglobin/pGEM7 plasmid, and sufficient DEPC treated
water to create a final reaction volume of 50 .mu.l. This reaction
was incubated at 30.degree. C. for 60 minutes. 5 .mu.l of this reaction
was removed into 20 .mu.l of SDS gel buffer, boiled for 2 minutes,
and loaded on a 17.5% SDS-polyacrylamide gel.
Rabbit reticulocyte lysate programmed with mammaglobin cDNA produced
a 6 kD protein while that programmed with no cDNA did not produce
any protein product.
This example illustrates the prevalence of overexpression of mammaglobin
in primary breast carcinoma.
To determine the frequency of mammaglobin overexpression in breast
carcinomas, we examined a panel of fifteen, stage I primary breast
carcinomas of differing histological types using Northern blot hybridization
with the mammaglobin cDNA probe. Patient-matched normal breast tissues
samples were also compared in tissues from two patients (FIG. 6).
The 500 bp mammaglobin mRNA was detected in normal breast tissue
and tumor 2410 and in three other tumors, two of which when tested
demonstrated little or no expression in patient-matched normal tissue
(BO15 v. BO16; BO22 v. BO23) (FIG. 6). In all, 4 of 15 (27%) of
tumors examined overexpressed mammaglobin mRNA. These data indicate
that overexpression of mammaglobin is not unique to a single tumor
specimen and is, in fact, relatively frequent among primary breast
tumors. Furthermore, the fact that all tumors examined were stage
I suggests that this dysregulation occurs relatively early in the
progression of breast neoplasia.
In view of the above, it will be seen that the several advantages
of the invention are achieved and other advantageous results attained.
As various changes could be made in the above methods and compositions
without departing from the scope of the invention, it is intended
that all matter contained in the above description and shown in
the accompanying drawings shall be interpreted as illustrative and
not in a limiting sense.