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(Received for publication, September 26, 1994; and in revised form, November 17, 1994) From the
Prostate-specific antigen (PSA) is widely used as a tumor marker
of prostatic adenocarcinoma. We recently found that 30% of breast
tumors produce PSA and that PSA is a favorable prognostic marker in
female breast cancer. We measured immunoreactive PSA in cytosolic
extracts of normal breast tissue from eight women receiving no
medication and one woman who was receiving the progestin-containing
oral contraceptive Brevicon. None of the eight cytosolic extracts of
normal breast tissue contained appreciable amounts of immunoreactive
PSA. However, left and right breast tissues from the woman receiving
Brevicon contained high levels of PSA. This immunoreactive species was
shown to have a molecular weight identical to that of seminal PSA.
Furthermore, reverse transcription of RNA and polymerase chain reaction
amplification produced a 571-base pair cDNA that hybridized to a
labeled cDNA PSA probe. Upon sequencing, the cDNA polymerase chain
reaction product was found to have 100% homology with cDNA from
prostatic tissue. PSA production by breast carcinoma cell lines was
achieved after in vitro stimulation with norethindrone and
ethinylestradiol. Our data suggest that PSA can no longer be regarded
as a specific prostatic protein because it is produced by breast tumors
with good prognosis and by normal breast tissue after steroid hormone
stimulation.
Oral contraceptives are among the most widely used drugs, but
the genes that are regulated by these agents are currently unknown. No
genes have as yet been found which are specifically regulated by oral
contraceptives in the female breast. We have recently found that about
30% of female breast tumors overexpress prostate-specific antigen
(PSA)(
After
electrophoresis the PSA cDNA PCR products were transferred onto Hybond
nylon membranes (Amersham) and hybridized with a PSA cDNA probe (kindly
provided by Dr. Jose Moreno). Southern blotting, probe radiolabeling,
hybridization, and autoradiography were performed by standard
techniques(10) . A second PCR amplification was performed on
the remaining PCR reaction to provide sufficient material for
subsequent analysis. Finally, the fragments were purified by ion
exchange columns (Qiagen, Dusseldorf, Germany) according to the
instructions of the manufacturer.
We have prepared cytosolic extracts from 18 normal breast
tissues removed from nine women (left and right breasts) during
cosmetic breast reduction surgery. PSA immunoreactivity was measured in
these extracts using a highly specific and sensitive immunofluorometric
technique (6) and by two widely used commercial PSA assays.
Breast extracts from eight of the nine women were found to contain
<0.03 ng of PSA/mg of total protein and were considered negative for
PSA. Two breast extracts from the same woman (left and right breasts)
had a relatively high concentrations of PSA (0.11 and 1.53 ng/mg). None
of the eight PSA-negative women was receiving oral contraceptives or
other medications. The woman with PSA-positive breasts was receiving
only one medication, Brevicon. The PSA-positive and -negative results
in the breast extracts by the immunofluorometric procedure were
verified by using two widely used commercial PSA methods: the IMx from
Abbott Laboratories and the Tandem-E kit from Hybritech Inc.
Additionally, the highly positive breast extract was serially diluted
in female serum from 2- to 32-fold and analyzed by immunofluorometry
and the IMx assay. Excellent agreement between results was obtained.
The serum from the patient on oral contraceptives and sera from another
10 women on oral contraceptives were analyzed for PSA and found to
contain <0.02 µg/l PSA. The highly positive breast extract
was subjected to HPLC (Fig. 1), and fractions were analyzed by
two immunofluorometric procedures that measure either total PSA (free
PSA plus PSA bound to
Figure 1:
HPLC with a gel
filtration column. Details of the method are given in (6) .
Each HPLC fraction (0.5 ml) was analyzed with an assay that measures
free and
Figure 2:
Western blot analysis. Samples were
electrophoresed on 8-16% gradient polyacrylamide minigels under
reducing conditions, electrotransferred to nitrocellulose membranes,
and probed with a rabbit polyclonal anti-PSA antibody. Detection was
achieved by using a horseradish peroxidase-conjugated goat anti-rabbit
antibody and chemiluminescence. Lane 1, molecular mass
markers. Lane 2, purified seminal PSA dissolved in bovine
serum albumin; the PSA band appears at 33 kDa (just above the marker at
31 kDa). Lane 3, supernatant from a prostatic carcinoma cell
line (LNCaP) producing PSA. Lane 4, PSA-positive normal breast
extract from the woman receiving Brevicon, containing a band at 33 kDa. Lane 5, another normal breast extract tested negative for PSA
by the immunofluorometric procedure. Lane 6, an amniotic fluid
tested for comparison; the identity of the band at 28 kDa, present in
PSA-positive and PSA-negative normal breast extracts and in amniotic
fluids, is unknown(16) .
To study the oral contraceptive-induced PSA
production further, we cultured T-47D and MCF-7 breast carcinoma cell
lines in the absence of any steroid hormones or in the presence of
norethindrone or ethinylestradiol at various concentrations (Fig. 3). No PSA was detected in the tissue culture supernatants
in the absence of steroid hormones after 11 days of confluent cultures.
Ethinylestradiol stimulated low levels of PSA production at
concentrations
Figure 3:
Production of PSA by the breast carcinoma
cell line MCF-7. Cells were grown to confluence and then stimulated
with varying concentrations of either norethindrone (1) or
ethinylestradiol (2) at the final concentrations indicated, in
the absence of fetal calf serum from the culture medium. PSA was
measured in the culture supernatant 10 days poststimulation. No PSA was
detected in cell cultures grown identically but either nonstimulated or
stimulated with the solvent alone (ethyl alcohol). Norethindrone
stimulates PSA production at concentrations as low as 10
Two RNA
samples isolated from normal human breast tissue obtained at reduction
mammoplasty were analyzed for PSA gene expression by PCR. Southern blot
hybridization of the PCR products with a PSA cDNA probe detected a PSA
band of the expected size (571 bp) in the sample obtained from the
woman who was receiving oral contraceptives. The other sample, obtained
from a woman not receiving any medication, was PSA-negative (Fig. 4). To determine whether the PSA mRNA detected in breast
tissue is identical to the mRNA present in prostate tissue, we
performed DNA sequence analysis on the PSA cDNA fragment isolated from
breast tissue and compared it with the published PSA cDNA sequence.
Partial results are shown in Fig. 5. DNA sequence analysis of
the breast tissue PSA cDNA fragment showed 100% identity with the PSA
cDNA sequence data from normal prostate tissue recently obtained by
Monne and Croce (
Figure 4:
Top panel, reverse transcription-PCR of
RNA isolated from normal breast tissue and detected by Southern
blotting and hybridization of the filter with a radiolabeled PSA cDNA
probe. A PSA hybridization band of 571 bp is detected in lane
2, but the band is absent in lane 1. Bottom
panel, ethidium bromide-stained agarose gel of
Figure 5:
Representative chromatogram of a
60-nucleotide region of the PSA cDNA sequence from normal breast
tissue. Total RNA was isolated from a breast tissue specimen obtained
at reduction mammoplasty from a woman receiving Brevicon. RNA was
reversed transcribed to cDNA, amplified by PCR, purified, and directly
sequenced. We obtained a 100% identity between the PSA cDNA sequence
from breast tissue (bottom) and the PSA cDNA published
sequence (top).
PSA is a serine protease found at very high concentrations in
sperm. The molecular mass of the glycosylated and nonglycosylated PSA
is 28.430 and 26.079 kDa, respectively(12) . However, with
polyacrylamide gel electrophoresis, PSA runs as a 32-33-kDa
protein. It has been suggested that PSA is involved in semen
liquefaction postejaculation. PSA is considered a highly specific
biochemical marker of the prostate gland and is currently used for
prostate cancer diagnosis, population screening, and postsurgical
monitoring of patients with prostate cancer(2, 3) .
Recently, we have demonstrated that PSA is produced by 30% of breast
tumors and provided evidence that this marker may be a new favorable
prognostic indicator of the disease(1) . Patients whose tumors
produce PSA live longer and relapse less frequently in comparison with
patients with PSA-negative tumors. In this paper we have
examined if PSA could also be produced by normal breasts either under
physiological conditions or under conditions of stimulation by
exogenously administered steroid hormones. Eight patients who received
no medication had breast PSA levels below 0.03 ng/mg protein and were
considered negative for PSA. One patient who was receiving a
progestin-containing oral contraceptive was found to have breast PSA
levels of 0.11 and 1.53 ng/mgf total protein (left and right breast,
respectively). This immunoreactive PSA molecule was measurable by the
immunofluorometric assay (6) as well as by commercial PSA
assays that are used widely for prostate cancer diagnosis. We have
verified, using HPLC (Fig. 1), that the immunoreactive PSA
species in breast is present in two molecular forms: as a 33-kDa
protein, corresponding to the seminal form of prostatic PSA; and as a
100-kDa protein, corresponding to PSA complexed with
We have further molecularly characterized the presence of
PSA in the normal breast at the mRNA level. For this analysis, we
extracted total RNA from the PSA-positive and a PSA-negative breast
tissue and amplified it, after reverse transcription, with specific
primers derived from the known PSA gene sequence. We found that
amplification occurred only with RNA from the PSA-positive breast
tissue. The PCR product was hybridized with a specific cDNA probe for
PSA and revealed the expected 571-bp fragment (Fig. 4).
Furthermore, sequencing of the PCR product derived from breast tissue
has shown that the sequence was identical to the sequence of the cDNA
for PSA derived from prostatic tissue. No mutations were identified (Fig. 5). The data presented support the notion that the PSA
gene is expressed in normal breast tissue under conditions of
stimulation by steroid hormones. To reproduce the phenomenon in
vitro we cultured T-47D and MCF-7 cells, two breast cancer cell
lines that are both positive for steroid hormone receptors. Tissue
culture supernatants from the nonstimulated cell lines contained no
detectable PSA. However, upon stimulation with norethindrone or
ethinylestradiol, the two components of the contraceptive used by the
patient whose breast was positive for PSA, the cell lines produced PSA.
Notably, norethindrone was active at concentrations as low as
10 The physiological
relevance of our finding is currently unknown. However, it has recently
been suggested (13, 14) that PSA, a serine protease,
is a new potential growth factor regulator, enzymatically digesting
insulin growth factor-binding protein-3 to release insulin growth
factor-I or enzymatically activating latent human transforming growth
factor-
Volume 270,
Number 12,
Issue of March 24, 1995 pp. 6615-6618
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
)(1) , a protein that was thought to be
produced exclusively by the epithelial cells of the prostate gland (2, 3) . PSA overexpression in breast cancer is
associated with the presence of steroid hormone receptors(4) .
The mechanism of such overexpression in breast cancer is currently
obscure. It has been hypothesized that either ovarian or adrenal
steroids or tumor-derived molecules derepress the PSA gene through the
steroid hormone receptors or that the PSA gene is constitutively
overexpressed because of poststeroid hormone-receptor complex defects,
loss of physiological repressors, or because of defects in the hormone
response elements. We have observed a significant advantage in both
overall and disease-free survival of breast cancer patients who are
PSA-positive. (
)As PSA was found to be associated with more
benign breast tumors, we speculated that it could also be expressed by
normal breasts either under physiological circumstances or after
steroid hormone stimulation. In this paper we demonstrate that the
breasts of one woman who was receiving a progestin-based oral
contraceptive contained high levels of PSA. This protein was absent
from the breasts of eight other women who were not receiving any
medication. These data strongly suggest that PSA is not a
prostate-specific protein and that it can be produced by the female
breast either in cases of malignancy or after stimulation by steroidal
compounds. The biological role of PSA in either normal or malignant
breast is currently under investigation.
Patients
We have studied nine women who
underwent breast reduction surgery for cosmetic reasons. Normal breast
tissue was immediately frozen on dry ice after resection and was stored
at -70 °C until extraction. Eight of the nine women were not
receiving any medication. One woman was receiving Brevicon, a highly
prescribed oral contraceptive containing 1 mg of norethindrone (a
progestin) and 0.035 mg of ethinylestradiol/tablet. Breast tissue was
obtained from both the left and right breasts for each patient.Preparation of Cytosolic Extracts
Breast tissue
cytosolic extracts were prepared as described previously(1) .Measurements
PSA in the cytosolic breast extracts
was measured with a highly sensitive and specific immunofluorometric
technique described in detail elsewhere(6) . PSA was also
measured in the cytosolic breast extracts with commercially available
kits: the Hybritech Tandem-E PSA kit (Hybritech Inc., San Diego) and
the IMx automated PSA method (Abbott Laboratories, Chicago). PSA in the
tissue was expressed as ng/mg of total protein.High Performance Liquid Chromatography (HPLC)
HPLC
was performed as described previously(6) .Western Blot Analysis
All necessary reagents and
equipment for Western blot analysis were purchased from Novex (San
Diego). The manufacturer's protocols were followed throughout.
Samples for Western blot analysis were electrophoresed under reducing
conditions on 8-16% gradient polyacrylamide minigels, and
proteins were transferred to Hybond-ECL nitrocellulose membranes
(Amersham Corp.). Membranes were blocked for 1 h and processed further
as recommended by the manufacturer of the ECL-Western blot detection
kit (Amersham Corp.). Briefly, the membrane was probed with a
polyclonal anti-PSA antibody (from Medix Biotech, Foster City, CA, 1
mg/ml, diluted 2,000-fold in the blocking buffer), washed, and reacted
with a horseradish peroxidase-conjugated goat anti-rabbit antibody. The
enzyme activity on the membrane was revealed by chemiluminescence,
captured on x-ray film. Biotinylated molecular weight markers were
visualized by reacting with a streptavidin-horseradish peroxidase
conjugate added simultaneously with the goat anti-rabbit antibody.Tissue Culture
The T-47D and MCF-7 breast
carcinoma cell lines and the LNCaP prostate carcinoma cell line were
obtained from the American Type Culture Collection (Rockville, MD).
These cell lines were cultured as described previously(7) .
Stimulation experiments with steroids, obtained from Sigma, were
performed as described previously (7) . PSA was measured in the
supernatants of the tissue cultures with the immunofluorometric
procedure(6) .RNA Extraction
Total RNA extraction was performed
using the RNazol B method (Tel-Test Inc., Friendswood, TX) following
the manufacturer's instructions.cDNA Synthesis
One microgram of total RNA was used
for the synthesis of the first strand of cDNA using the SuperScript II
reverse transcriptase (Life Technologies, Inc.). Briefly, RNA,
oligo(dT)
primers (100 ng), and random hexamer
primers (100 ng) were first denatured for 5 min at 70 °C, chilled
on ice for 1 min, and then incubated for 1 h at 37 °C in 20 µl
of a reaction mixture containing 1 
first stand buffer, 250
µM deoxynucleoside triphosphate mix (Boehringer Mannheim),
10 mM dithiothreitol, and 200 units of SuperScript II reverse
transcriptase.Oligonucleotide Primers
Oligonucleotides were
synthesized by the Jefferson Cancer Institute Nucleic Acid Facility
(Philadelphia) and used without further purification. The PSA
oligonucleotide sequences used as primers were: PR5,
5`-AGCCCCAAGCTTACCACCT-3`; PR6, 5`-CAGACTGCCCTGCCACGA-3`; PR2 and PR3
sequences have been published previously(8) . Previously
published actin PCR primer sequences were used to rule out degraded
RNA(9) . Amplification yielded a 571-base pair (bp) PSA cDNA
fragment with PR5 (from exon 1) and PR2 (from exon 4) oligonucleotide
primers and a 154-bp cDNA fragment with actin oligonucleotide primers.PCR Procedure and Southern Blot Analysis
One
microliter of cDNA was added to 39 µl of PCR mix containing 1
PCR buffer (Boehringer Mannheim), a 18 pM concentration of each primer, 250 µM deoxynucleoside
triphosphate mix, and 1.25 units of Taq DNA polymerase
(Boehringer Mannheim). PCR was performed with primers PR5 and PR2 for
30 cycles according to the following program: 94 °C for 1 min (2
min for the first cycle), 56 °C for 1 min, and 72 °C for 2 min
(5 min for the last extension). Actin fragments were amplified from 1
µl of the cDNA preparation under the same conditions used for PSA
PCR. Twenty microliters of each PCR reaction was electrophoresed in 2%
agarose gel and visualized by ethidium bromide staining.Sequencing
An automated 373A DNA sequencer
(Applied Biosystems) and dye terminator kits from the same manufacturer
were used for direct sequencing of the PSA cDNA fragments by the
dideoxynucleotide chain termination method using fluorescent labels.
The coding and noncoding strands of each fragment were sequenced with
primers generating overlapping sequence data. Sequences were
reassembled and analyzed using the SAP program(11) .
-antichymotrypsin) or
specifically the PSA--antichymotrypsin (ACT)
complex(6) . More than 80% of the total PSA in normal breast
was in the free, 33-kDa form; a small proportion was present as
ACT
PSA complex (100 kDa). Another minor species, containing PSA
and ACT, was also detected (660 kDa), but its identity is unknown. The
presence of PSA in the highly positive breast extract was further
confirmed by Western blot analysis (Fig. 2). The 33-kDa form of
PSA, shown here to be present in normal breasts stimulated by oral
contraceptives, is similar to the form of PSA found in breast
tumors(1) . In male serum, the majority of PSA is present as
ACTPSA complex with a molecular mass of 100 kDa (data not
shown)(6) .
-antichymotrypsin-bound PSA (ACTPSA)
(
) or an assay that measures only ACTPSA (
). The
response of the latter assay is in arbitrary fluorescence units since
no ACTPSA standard exists. Panel A, injection of
purified seminal PSA, which elutes at fraction 39 corresponding to a
molecular mass of 33 kDa. No ACTPSA is detected. Panel
B, injection of a breast extract from the woman receiving the oral
contraceptive Brevicon. The PSA assay detects two peaks: one at
fraction 39 (free PSA, major peak) and one at fraction 30 (100-kDa
minor peak). The latter peak is ACTPSA as confirmed by the
ACTPSA assay. The identity of the minor peak at fraction 21 (650
kDa) is unknown. These data confirm that more than 80% of the breast
tissue PSA is in the free, 33-kDa form. The HPLC column was calibrated
with molecular mass standards eluting at fraction 21 (660 kDa), 28 (160
kDa), 37 (44 kDa), 42 (17 kDa), and 49 (1.4
kDa).
10
M. Norethindrone was
effective in mediating intense PSA gene expression at concentrations as
low as 10
M. Other progestins were also
effective in mediating PSA gene expression (data not shown). The
identity of PSA in the tissue culture supernatants was characterized
further by HPLC and Western blot analysis (data not shown).
M.
)(GenBank accession number U17040). No
mutations were identified in the 571-bp fragment that was sequenced.
-actin reverse
transcription-PCR products. Lane 1 represents RNA isolated
from breast tissue negative for PSA protein immunoreactivity. Lane
2 represents RNA isolated from the breast tissue of the woman
receiving Brevicon. This breast tissue was positive for PSA protein
immunoreactivity.
-antichymotrypsin. The latter form, which is
predominantly found in the serum of prostate cancer patients, was
present in the breast extract at relatively low levels (<10% of
total PSA); the major form was comprised of the 33-kDa protein (Fig. 1B). We have further characterized PSA in the
positive breast extract using Western blot analysis. This data have
shown that the PSA-positive breast extract, but not a PSA-negative
breast extract, contained an immunoreactive band with a molecular mass
of 33 kDa (Fig. 2). PSA was not elevated in the serum of
patients receiving oral contraceptives. The additional band on Western
blots, at 28 kDa, present in PSA-positive and PSA-negative breast
extracts and in amniotic fluid, may represent a PSA isoform or a PSA
fragment.M (Fig. 3). and proteolytically modulating cell adhesion
receptors(15) . Our previous data on PSA gene expression in
breast cancer that has good prognosis (1, 5) and the
demonstration here that the PSA gene is regulated by oral
contraceptives in normal breast suggest that this enzyme, until
recently thought to be associated only with male prostatic tissue, may
have important, previously unrecognized extraprostatic functions
related to breast and other tissue growth and possibly breast cancer.
This suggestion is further supported by our finding of the PSA presence
in amniotic fluid during gestational weeks 13-21 (16) and
in the milk of lactating women(5) . The role of PSA in
embryonal life and its involvement in growth factor regulation are
currently under investigation.
)-antichymotrypsin.))
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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