[CANCER RESEARCH 56, 860-865, February 15, 1996]
MATERIALS AND METHODS
Mammaglobin, a Mammary-specific
Member of the Uteroglobin Gene Family, Is
Overexpressed in Human Breast Cancer1 2
Mark A. Watson and Timothy P. Fleming3
Departments of Pathology [M. A. W.J, Ophthalmology and Visual Sciences IT. P. F.J,
and Genetics [T. P. F.J, Washington University School of Medicine, St. Louis, Missouri
63110
ABSTRACT
In this report, we describe a novel cDNA isolated from a
primary human breast adenocarcinoma and differentially
expressed in several breast carcinoma cell lines. The protein
encoded by this cDNA, which we have named mammaglobin, is
homologous to a family of secreted proteins that includes rat
prostatic steroid-binding protein subunit C3, human Clara cell 10-
kilodalton protein, and rabbit uteroglobin. Expression of the
mammaglobin gene is restricted to the adult mammary gland.
More significantly, in an analysis of 35 breast tumor biopsies,
mammaglobin mRNA levels were increased at least 10-fold
relative to normal breast tissue in 23% of cases. The breast-
specific expression of this potentially secreted protein and its
frequent overexpression in primary human breast tumors suggest
that mammaglobin may be a novel marker for the management of
breast cancer.
INTRODUCTION
The evolution of breast cancer is accompanied by multiple genetic
changes that result in qualitative and quantitative alterations in
individual gene expression (1). Our hypothesis is that many of these
quantitative genetic changes manifest themselves as alterations in the
cellular complement of novel transcribed mRNAs. We believe that the
identification of these mRNAs, if sufficiently characterized, could
provide clinically useful information for patient management and
prognosis while enhancing our understanding of breast cancer
pathogenesis. Using a “shotgun” approach that uses modifications of
the differential display PCR technique (2), we have previously
identified several PCR fragments or DESTs 4 from human breast tumor
biopsies (3). These PCR fragments correspond to mRNA transcripts
that are under- or overexpressed in breast tumors relative to patient-
matched normal breast tissue.
In this report, we describe the isolation of a novel full-length cDNA
corresponding to one of these previously isolated DEST sequences.
The protein encoded by this cDNA, which we have named
mammaglobin, is homologous to several secreted epithelial proteins
including hCCIO protein (4), rUg (5), and rPSC3 (6). We demonstrate
that expression of the mammaglobin gene is restricted to the adult
mammary gland, and that compared to normal breast tissue, 23% (8 of
35) of primary human breast tumors overexpress mammaglobin
mRNA. The breast-specific expression of this potentially secreted
protein and its frequent dysregulation in primary mammary tumors
suggest that mammaglobin may be an important new marker for the
management of breast cancer.
1Received 10/11/95; accepted 12/6/95.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
2 This work was supported in part by American Cancer Society IRG Grant No.
ACS-IRG36-37.
3 To whom requests for reprints should be addressed, at Department of
Ophthalmology, Box 80%, Washington University School of Medicine, 660 South Euclid
Avenue, St. Louis, MO 63110.
4 The abbreviations used are: DEST, differential expressed sequence tag;
hCCIO, human Clara cell 10-kD protein; rUg, rabbit uteroglobin; rPSC3, rat prostatic
steroidbinding protein subunit C3; RACE, rapid amplification of cDNA ends; RT-PCR,
reverse transcription-PCR; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; kD,
kilodalton.
Cell Culture and Tissues. Cell lines were obtained from American
Type Culture Collection and grown in DMEM supplemented with 10% FCS.
Tissue biopsy specimens and accompanying pathological reports were obtained
from the Human Cooperative Tissue Network (7).
Isolation of Mammaglobin cDNA. Total cellular RNA from the cell
line MDA-MB415 was isolated using the standard guanidinium isothiocyanate
method. This RNA was used in the RACE PCR procedure using the 5'-
Amplifinder Race kit (Clonetech) and following the manufacturer’s protocol.
The two downstream, mammaglobin-specific, nested oligonucleotides used for
RACE PCR were D2R (5'-ATA AGA AAG AGA AGG TGT GG-3') and D2Rb
(5'-AAT CCG TAG TTG GTT TCT CAC C-3'). An upstream mammaglobin-
specific control oligonucleotide was also used according to the manufacturer’s
recommendations [D2F (5'-CTT TCT GCA AGA CCT TTG GC-3')].
Amplification steps were performed with Vent DNA polymerase (New England
Biolabs) in a volume of 50 /a1 containing 1 x polymerase buffer [10 mM KC1,
20 mM Tris • HC1 (pH 8.8), 10 mM (NH4)2S04, 2 mM MgS04, 0.1% Triton X-
100], 400 /am dNTPs, 2 units Vent polymerase, and 0.2 /am each appropriate
primer. Amplification was performed at 94°C for 45 s, 55°C for 1 min, and
72°C for 1 min over 40 cycles. A single, 400-bp amplified RACE product was
obtained, digested with EcoRI, and ligated into the EcoRl and Smal sites of the
plasmid vector pGEM7Z (Promega). All sequencing was performed on both
strands using a Taq DNA polymerase thermal cycle sequencing kit according to
the manufacturer’s protocol (Promega) and mammaglobin sequence-specific
primers.
RNA Isolation, RT-PCR, and Northern Analysis. All indicated total
cellular RNA samples were isolated using the standard guanidinium
isothiocyanate method and treated with RNase-free DNase (Promega). For RT-
PCR analysis, 1 /Ag of indicated total RNA was reverse transcribed with
oligo(dT2j) and Superscript II reverse transcriptase (GIBCO-BRL) in a volume
of 20 /a1 containing 1X reverse transcriptase buffer [50 mM Tris • HC1 (pH
8.3), 75 mM KC1, 3 mM MgCl2] provided by the manufacturer, 10 mM DTT,
200 /am dNTPs, 100 ng oligo(dT21), and 200 units reverse transcriptase.
Reactions were performed at 45°C for 60 min. 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') and D2102 (5'-CAG CGG CTT CCT
TGA TCC TTG-3') or the GAPDH primers FI (5'-CCA CCC ATG GCA AAT
TCC ATG GCA-3') and R1 (5'-TCT AGA CGG CAG GTC AGG TCC ACC-
3'). Reactions were performed in a 50-/xl volume containing 1X polymerase
buffer provided by the manufacturer, 2 mM MgCl2, 200 /am dNTPs, 0.5 /am each
primer, and 2 units Taq DNA polymerase (Perkin Elmer Cetus). Amplification
occurred for 40 cycles at 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min.
For Northern analysis, 20 /Ag of total RNA were analyzed as described
previously (3) using the full-length mammaglobin cDNA probe. Integrity, equal
loading, and uniform transfer of each RNA sample were assessed by ethidium
bromide staining and visualization of 28S and 18S ribosomal bands. In no case
did the intensity of ribosomal bands differ by more than 50% between tumors,
tumor cell lines, or tumor/normal tissue pairs.
RESULTS
Isolation of the Full-length Mammaglobin cDNA. We previously
isolated a DEST PCR fragment, the corresponding mRNA of which
was abundant in the cell line MDA-MB415 (3). We therefore elected
to use the RACE PCR technique (8) with this cell line to isolate the
corresponding full-length cDNA. Sequence from the 403-bp fragment
isolated by this technique was combined with a sequence obtained
previously from the corresponding DEST sequence
[CANCER RESEARCH 56, 860-865, February 15, 1996]

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9 1 2 3 4 5
G A 8 7 6
GG T C G C T C C C C A A G A A AG 5 4
AC GC C TC TT AT CT GC AC CG GA TG AC CC AC GC GC C
63 7 8 9 9 10
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C A AT A T C A G C A C G G C T C C TG
TC CC MeG AG
L TG
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M TC
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A TG
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T TC
l TAs
yr la y er
2 Cys ro eu eu lu sn al ie er 3 ys hr ie n
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C C GT T A A G T A G C C C G T A G GA
CA
P AA
G VaG CT
S AG
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AspC
ro in l er ys hr
4 lu yr ys lu eu eu in lu he ie5 Asp
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A G AC A A G A G G T A G T T C A C AC
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sp lu r eu er sn al lu al he
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T T TA T A C C G A T T A C C T A T AT
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495

TTA ATA AAT TGA TGG CA 31

Fig. 1. Human mammaglobin sequence. Nucleotide sequence of the cDNA and encoded protein are numbered respectively. Open bars, extent of the previously isolated DEST PCR
sequence. Closed bars, sequence obtained from the RACE PCR clone. The putative signal peptide sequence of the mammaglobin protein and poly(A) addition site at the 3' end of the
cDNA are shown in bold.
[CANCER RESEARCH 56, 860-865, February 15, 1996]

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 MAMMAGLOBIN EXPRESSION IN BREAST CANCER

ruo -21 MKLAITLALVTLALLCS PAS AG ICPRFAHVIENLLLGTPS S - - YETSLKEF

linn nI n n II mi mi n i i in
hCCIO -21

Fig. 2. Mammaglobin homology with other gene

family members. Amino acid sequences of human
mammaglobin (ZiAfAAf), rPSC3, hCCIO, and rUG
are shown. Identities between hMAM and rPSC3 or
hMAM and hCCIO are shown in bold and double
lines. Identities between hMAM and rUG only are
shown in bold and single lines. Numbering for
hCCIO and rUG is based on the known site of signal
peptide cleavage.

MKLAVTLTLVTLALCCSSASAEICPSFQRVIETLLMDTPSS - - YEAAMELF
linn nI n n n INI INI I I i in
hMaM oi MKLLMVLMLAALSQHCY-A-GSGCPLLENVISKTINPQVSKTEYKELLQEF
mm I mi II mi INI i nil mm n I n
rPSC3 oi MKLVFLFLLVTIPICCY-
ASGSGCSILDEVIRGTINSTVTLHDYMKLVKPY

rUG 29 EPDDTMKDAGMQMKKVLDSLPQT- -TRENIMKLTEKIVKSPLCN

II II II II III I I II II INI
hCCIO 29 spdqdmreagaqlkklvdtlpqk--presiiklmekiaqsslcn
II II INI II III I I II II IIINIII
hMaM 50 IDDNATTNAIDELKECF - -LNQTDETLSNVEVFMQLIYDSSLCDLF
II II II IIINI II llllll INI INIII II IIII II
rPSC3 51 VQDHFTEKAVKQFKQCF- -
LDQTDKTLENVGVMMEAIFNSESCQQPS

to deduce the full-length 503-bp cDNA sequence now designated as amino acid identity (58% homology including conservative
mammaglobin (Fig. 1). Because these sequence data were obtained substitutions) with rPSC3 (Fig. 2). This protein is one of three subunits
from a tumor cell line by PCR amplification, it was conceivable that that forms a tetrameric structure, constituting the major secretory
mutations may have been introduced into this cDNA clone. To exclude protein in the rat ventral prostate (6, 11). Using manual alignment
this possibility, two specific primers based on the MDA-MB415 methods with other sequences that had less significant BLAST scores
mammaglobin cDNA sequence were used to amplify the with both mammaglobin and rPSC3 protein sequences, we identified
mammaglobin cDNA from normal human breast tissue. Cloning and other homologies with hCCIO (4) and rUg (Ref. 5; Fig. 2). These
sequencing of the mammaglobin cDNA obtained from human breast sequences, depending on species, were 26% identical or 40%
tissue revealed no sequence discrepancies with the first MDA-MB415 homologous including conservative amino acid substitutions. In
clone. This demonstrated that the cDNA is not mutated in this cell line, particular, a number of amino acids were perfectly conserved among
nor were mutations introduced during the RACE procedure. The full- all proteins, including Cys-3 and Cys-69, which are known to play a
length mammaglobin sequence is displayed in Fig. 1. Within this 503- role in disulfide bond formation between uteroglobin subunits and
bp cDNA is a 279-bp open reading frame that encodes a polypeptide Tyr-21, which is required for progesterone binding to the uteroglobin
of 93 amino acids and a predicted molecular mass of 10.5 kD (Fig. 2). dimer (5). These homologies suggest that mammaglobin is a novel
The first 19 residues of this sequence also predict a hydro- phobic member of a small family of proteins that are secreted by epithelial
peptide signal sequence. The initial methionine of the open reading cells (12, 13).
frame contains a near-perfect Kozak consensus sequence, and 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 dT 19AC oligonucleotide priming site of the
original DEST sequence. On the basis of these data, we conclude that
we have isolated the full-length mammaglobin cDNA.
Mammaglobin Is a Novel Member of the Uteroglobin Gene
Family. Using the mammaglobin cDNA sequence to query Genbank
with the BLAST search algorithm (9, 10), no obvious DNA sequence
homologies were identified. However, using the amino acid sequence
of the putative translation product, mammaglobin exhibited 42%
 Mammaglobin Expression Is Restricted to the Mammary
Gland. rPSC3 is expressed in rat ventral prostate (6), whereas
expression of hCCIO and rUg has been demonstrated in numerous
tissues including lung, uterus, prostate, and breast (5, 13). Because of
the sequence homology between mammaglobin and these proteins, we
sought to compare their patterns of tissue-specific expression. As
shown in Fig. 3A, an ~500-bp mammaglobin message was easily
detected in tumor specimen 2410 (the tissue from which the DEST
corresponding to the mammaglobin cDNA was isolated) and to a much
less extent in normal human breast tissue. However, expression of
mammaglobin was undetectable in human uterus and lung, two sites of
uteroglobin expression, as well as ovary and placenta. To broaden the
scope and increase the sensitivity of our study, we used RT-PCR
analysis in several other adult human tissues to assay for
mammaglobin gene expression. As shown in Fig. 3£, RT-PCR analysis
detected mammaglobin mRNA in both tumor 2410 and normal breast
tissue. Of 15 other tissues surveyed, however, no mammaglobin
expression was seen. This included tissues that normally express
rPSC3 and uteroglobin (lung, uterus, prostate), hormonally responsive
and steroidogenic tissues (ovary, testis, placenta), and other epithelial
organs (colon, bladder). Although a band similar in size to the
mammaglobin PCR product was detected in testis, this band originated
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from the GAPDH RT-PCR reaction. Therefore, it most likely
represents a nonspecific product resulting from GAPDH
oligonucleotide priming and not an authentic mammaglobin PCR
product. Detection of GAPDH message (Fig. 3B) and epidermal
growth factor receptor message (data not shown) in all reactions
demonstrated that absence of expression was not due to degraded RNA
or other trivial explanations. Therefore, to the extent of tissues
examined and the sensitivity of the RT-PCR assay used, mammaglobin
is a mammary-specific gene.
Mammaglobin Expression in Breast Carcinoma Cell Lines.
Although we previously demonstrated that the DEST corresponding to
the mammaglobin cDNA was abundant in the cell line MDA- MB415
(3), we sought to expand this analysis to multiple carcinoma cell lines
and nontumorigenic immortalized breast cell lines using the full-length
mammaglobin cDNA. As shown in Fig. 4, mammaglobin expression
could not be detected in primary breast myoepithelial cells (hMMC),
primary breast stromal cells (HA2403, HA2407), the immortalized
breast cell line B5/589 (14), or an immortalized “normal” luminal
ductal breast cell line (MCF10A) (15). On the other hand,
mammaglobin expression was easily detected in the tumor cell lines
MDA-MB415 and MDA-MB361 and to a lesser extent in lines
 MAMMAGLOBIN EXPRESSION IN BREAST CANCER

A
GAPDH
hMAM

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u o
in

2 X X 2 C m 2 2 2 2 2 2 2 U X
1 ;1 1 1 1 /1 i 1 1 J
Fig. 3. Mammaglobin expression in adult human tissues.
A, Northern blot analysis of mammaglobin expression in
indicated tissues. Arrowhead, 500-bp mammaglobin message.
1 1 1 1 1 1 1 1 1
B, RT-PCR analysis of mammaglobin expression in indicated
tissues. PCR amplification products of GAPDH and
mammaglobin are indicated. Note that the faint band in the
testis lane does not correspond to an authentic mammaglobin
PCR product (see text). PBL, peripheral blood leukocytes;
Breast CA, breast carcinoma.

Fig. 4. Mammaglobin expression in tumor cell lines. Northern blot analysis

of mammaglobin gene expression in indicated mammary and nonmammary
(LnCAP, E2, HeLa) immortalized and tumor cell lines. hMMC, primary human
mammary myoepithelial cells.

BT474, MDA-MB175, and MDA-MB468. In cell lines (and tumor mammaglobin gene predicts a 3-kb unspliced mammaglobin
tissues, see below) that demonstrated elevated mammaglobin message,5 we believe that this 3-kb transcript represents unprocessed
expression, a second ~3-kb transcript was detected in addition to the nuclear mRNA that is detectable in tissues and cell lines with a high
0.5-kb mammaglobin message. Because low-stringency Southern Blot
analysis with a mammaglobin genomic probe has failed to reveal other
human mammaglobin-related genes and sequence analysis of the

5 Unpublished data.
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 MAMMAGLOBINEXPRESSION
EXPRESSIONININBREAST
BREASTCANCER
CANCER
MAMMAGLOBIN

A mRNA was again detected in normal breast tissue and tumor 2410.
IIIIIIIIIIIIIIIIII1 Mammaglobin message was also abundant in seven other tumors. In
<
& < < < four of these, expression was at least 10-fold higher than patient-
O C9 O o O
© matched normal breast tissue. In all, 23% (8 of 35) of tumors
lo < CO
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examined overexpressed mammaglobin mRNA. These data suggest
r- CO CO CO
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<
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that overexpression of mammaglobin is not unique to a single tumor
specimen and is, in fact.

iiiiiIlllIii

level of mammaglobin transcription. Overall, these results

demonstrate that mammaglobin gene expression defines a unique
B <
o
to CO CM CO r- CM CM
oooo
CDCDCDCD

Fig. 5. Mammaglobin expression in primary human

tumors. A, Northern blot analysis of mammaglobin
gene expression in normal breast and indicated
tumor biopsy specimens. B, Northern blot analysis
of mammaglobin expression in patient-matched
pairs of breast tumor biopsies (G4) and surrounding
normal breast tissue. Note that exposure times differ
between A and B so that mRNA levels cannot be
compared directly between these two experiments.

phenotype to a subset of breast carcinoma cell lines and that, in

culture, expression is absent in many of the normal cellular
constituents that compose the mammary gland in vivo.
Overexpression of Mammaglobin in Primary Breast Carcinoma.
To determine the significance of mammaglobin expression in vivo and
its frequency of dysregulation in primary breast tumors, we next
examined a panel of 35 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. 5, the 500-bp mammaglobin
 relatively frequent among primary breast tumors. Review of clinical
and pathological data available for each tumor (Table 1) revealed no
clear relation between mammaglobin expression and tumor grade,
stage, histological classification, or hormone receptor status.
DISCUSSION
We have described previously the use of a modified differential
display PCR technique to identify sequences (DESTs) that are
differentially expressed between breast carcinoma and normal breast
tissue in vivo (3). Using this technique, we are interested in isolating
genes that contribute to novel pathways in breast cancer progression
and that may serve as useful prognostic markers for patient
management. In this report, we have identified a novel cDNA that
encodes a mammary-specific protein with homologies to uteroglobin
and a prostatic steroid-binding protein. This protein, mammaglobin, is
overexpressed in 23% of primary tumors examined thus far.
The amino acid sequence homology between mammaglobin,
rPSC3, and uteroglobin suggests that these proteins constitute a gene
family. Rat prostatic steroid binding protein (prostatein) is the major
secretory protein in the rat ventral prostate (11). It binds steroid
hormones, and its synthesis is stimulated by testosterone. The mature
protein is a tetramer composed of two different dimeric subunits,
C3/C1 and C3/C2, each linked by disulfide bonding. The C7, C2, and
C3 genes all encode 8-12-kD peptides and are thought to have arisen
from gene duplication, but whereas the Cl and C2 genes show strong
homology to each other, they are much less similar to the C3 gene
(16). Correspondingly, mammaglobin shows no sequence homology
with the Cl or C2 peptides. The transcriptional regulation of the rPSC3
gene has been studied extensively (17). It is of interest that recent
reports have demonstrated the rPSC3 promoter fused to SV40 T
antigen produces both prostatic and mammary carcinomas in
transgenic mice (18). However, the true biological function of
prostatein and its individual subunits is unknown, and no
corresponding human genes have been identified.
The hCCIO gene is the human homologue of the rUg gene (4, 5,
13). 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. The precise pattern of tissue
expression, however, is species specific and related to a diverse set of
regulatory elements in the rabbit and human genes. Unlike rat
prostatein, uteroglobin is a homodimeric protein coupled by two
disulfide linkages at the conserved residues Cys-2 and Cys-69.
Uteroglobin gene transcription is regulated by tissue-specific responses
to steroid hormones, and transcriptional control of this gene has been
studied extensively both in vitro and in transgenic animal models (19).
rUg is capable of binding progesterone and structurally related steroids
and is a substrate for transglutaminases. Furthermore, several
investigators have demonstrated that rUg inhibits phospholipase A 2
activity and may interfere with the immune and inflammatory activity
of several cell types. The hCCIO protein and corresponding mouse and
rat

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homologues may have similar functional properties as rUg but are less suggested for other epithelial specific genes (23). Finally, because
well characterized (13). In all cases, the true biological function of mammaglobin is a potentially secreted protein, its presence may be
these proteins is also unknown. detectable in serum of patients whose tumor overexpresses this gene
Unlike hCCIO or rPSC3, mammaglobin has been isolated based on product. As such, mammaglobin may be equally or more clinically
its relative abundance in breast tumor tissue. As an initial attempt to useful than other solid tumor markers used for managing patients with
determine its relevance to human breast cancer, we examined the breast cancer (22).
expression of mammaglobin in several breast tumor biopsies. Among
35 tumor specimens examined, 23% overexpressed mammaglobin REFERENCES
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identified both estrogen receptor-positive and estrogen receptor-
negative tumors that express mammaglobin. It is possible, therefore,
that mammaglobin expression independently defines a subclass of
tumor cell phenotypes. Mammaglobin is expressed in early,
noninvasive ductal carcinoma in situ, as well as in late stage invasive
disease. We have not yet investigated whether mammaglobin is
overexpressed in benign breast disease, but this information should
assist in defining the relationship between breast cancer progression
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utility of measuring mammaglobin expression in breast cancer
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demonstrates several properties of a clinically useful breast tumor
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overexpressed in breast carcinoma, mammaglobin is a breast-specific
protein, the overexpression of which may reflect a more cell-specific
alteration of the mammary epithelium rather than representing 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, as is
currently the case for the estrogen and progesterone receptors (22).
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nodes or peripheral leukocytes 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 also be
useful for detecting occult breast cancer metastases, as has been
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EXPRESSION IN and uteroglobin-like
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 AA American Association
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Cancer Research
The Journal of Cancer Research (1916-1930) | The American Journal of Cancer (1931-1940)

Mammaglobin, a Mammary-specific Member of the Uteroglobin

Gene Family, Is Overexpressed in Human Breast Cancer
Mark A. Watson and Timothy P.
Fleming Cancer Res 1996;56:860-865.

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