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Articles |
1
Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario M5G 1X5, Canada.
2
Department of Clinical Biochemistry, University of
Toronto, Toronto, Ontario M5G 1L5, Canada.
3
Division of Population Science, Fox Chase Cancer Center,
Philadelphia, PA.
a Address correspondence to this author, at Mount Sinai Hospital. Fax (416) 586-8628; e-mail epd{at}playfair.utoronto.ca
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4 ng/L; their changes in PSA content during the
menstrual cycle were studied in 7 informative cycles. We found that PSA
concentrations in serum are highest during the mid- to late follicular
phase, drop continuously with a half-life of 3–5 days between the late
follicular phase and midcycle, and reach a minimum during the mid- to
late luteal phase. PSA changes do not correlate with changes in
lutropin (LH), follitropin (FSH), or estradiol concentrations. However,
PSA peaks seem to follow the progesterone concentration peaks, with a
delay of 10–12 days. Sera of some volunteers were tested for their
ability to upregulate PSA protein and PSA mRNA in a tissue culture
system based on the T-47D breast carcinoma cell line. Only sera
obtained during the mid- to late luteal phase were able to upregulate
the PSA mRNA and protein. In stimulation experiments in vitro,
progesterone, but not LH, FSH, estradiol, human chorionic gonadotropin,
prolactin, or growth hormone, was able to upregulate PSA mRNA and
protein in the T-47D cell line. These data suggest that PSA is produced
in a cyclical manner during the menstrual cycle. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Prostate-specific antigen (PSA) is a 33-kDa serine protease produced at high concentrations by prostatic epithelial cells and secreted into the seminal plasma. PSA production in the prostate is regulated by androgens through the action of the androgen receptor (5). Recently, we have demonstrated that PSA is not a prostatic tissue-specific protein but is also expressed in the female breast and some other tissues in both males and females (6). Normal, benign, and malignant breast tissue produces PSA (7). Some, but not all, breast tumors produce PSA; our studies suggest that PSA is a favorable prognostic indicator in breast cancer (8). Previously we found a close association between PSA presence in breast tumors and the presence of both estrogen and progesterone receptors; this association was stronger between PSA and progesterone receptors (9). We have also found PSA in amniotic fluid at increasing concentrations between gestational weeks 11–22 (10). PSA concentrations were also greater in serum of pregnant women than in nonpregnant controls (10). We speculated that PSA is upregulated by placental steroids during pregnancy.
To investigate the mechanism of PSA gene regulation in the breast, we have developed a tissue culture system that reproduces in vitro the phenomenon of PSA production by breast cells. The steroid hormone receptor-positive breast carcinoma cell line T-47D does not produce detectable PSA when cultured in media lacking steroid hormones. Upon stimulation by steroid hormones, however, this cell line produces PSA in a dose-dependent manner. Strong positive regulators of PSA mRNA and protein expression in this system are androgens and progestins; estrogens not only do not induce upregulation but also partially block the effects of androgens and progestins (11)(12).
Regulation of the PSA gene by steroid hormones in the breast led us to speculate that this protein may be differentially expressed during the menstrual cycle. To investigate this possibility, we studied PSA concentrations in the serum of women during the menstrual cycle and correlated these with the concentrations of steroid and peptide hormones. In addition, we examined the ability of female serum during the menstrual cycle to induce PSA mRNA and protein expression in the tissue culture system. Our data support the view that the PSA protein is differentially expressed during the menstrual cycle, with the greatest concentrations occurring during the mid- to late follicular phase and the lowest being found during the mid- to late luteal phase. The peak in serum PSA concentration follows the peak in serum progesterone concentration but with a delay period of ~10–12 days. These data support the view that the PSA gene is regulated by corpus luteum steroids during the menstrual cycle.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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procedures
Tissue culture stimulation experiments.
The T-47D breast
carcinoma cell line was obtained from the American Type Culture
Collection. This cell line is positive for estrogen, progesterone,
androgen, and glucocorticoid receptors
(13)(14). T-47D cells were cultured in RPMI
media (Gibco BRL) supplemented with glutamine (200 mmol/L), bovine
insulin (10 mg/L), fetal bovine serum (100 g/L), antibiotics
(penicillin, streptomycin), and antimycotics (amphotericin B). The
cells were cultured to near confluency in plastic culture flasks and
then transferred to phenol red-free media containing charcoal-stripped
fetal bovine serum, 100 g/L, with antibiotics/antimycotics. Phenol
red-free media were used, given findings that phenol red has weak
estrogenic activity (15); charcoal-stripped fetal bovine
serum is devoid of any steroid hormones.
The T-47D cells were aliquoted into 24-well tissue culture plates (Corning no. 25820) and cultured to confluency with a change in media at 3 days. Stimulations were carried out with confluent cells in 1 mL of phenol red-free media containing charcoal-stripped fetal calf serum (100 g/L) and antibiotics/antimycotics. Stimulation was initiated by adding 1 mL of filter-sterilized serum sample and incubating for 24 h. Tissue culture supernatant (~150 µL) was removed for PSA protein analysis after 24 h. Slight modifications of this protocol were introduced as necessary. Appropriate multiple negative controls (no serum added) were included in each experiment. Positive controls consisted of T-47D cells stimulated with the synthetic progestin Norgestrel, which was found previously (11)(12) to induce PSA mRNA and PSA protein production by T-47D cells. Additional experiments were performed with progesterone at 0.11 µmol/L–10 pmol/L, prolactin at 5 µg/L–5 ng/L, growth hormone at 10 µg/L–10 ng/L, LH at 10 µg/L–10 ng/L, FSH at 10 µg/L–10 ng/L, and human chorionic gonadotropin (hCG) at 5–5000 IU/L (all concentrations are the final concentrations in the microtiter wells, and all the human hormones were purchased from Sigma Chemical Co.). The mRNA from the T-47D cells was extracted for PSA mRNA amplification as described below.
Measurement of PSA protein.
PSA protein was measured
with a highly sensitive time-resolved immunofluorometric procedure
described in detail elsewhere (16). This ultrasensitive
assay can measure PSA at 1–2 ng/L (and up to 10 000 ng/L) with a
precision of <10%. Further within-run precision data with a female
serum pool confirmed a CV of 8.5% at a PSA concentration of 4.9 ng/L
(n = 10). PSA measurements in all assays were performed in
duplicate or triplicate. PSA protein in all sera and tissue culture
supernatants was measured undiluted in 100-µL aliquots per assay.
Measurement of hormones in serum.
All serum samples
collected during the course of the menstrual cycle were analyzed for
concentrations of the hormones FSH, LH, estradiol, and progesterone.
FSH and LH were assayed with the Access® Immunoassay
Analyzer (Sanofi Diagnostics Pasteur). The lowest detectable
concentration of FSH and LH distinguishable from zero with 95%
confidence in these assays is 0.2 IU/L, and both assays exhibit total
imprecision <10% across the assay range. Estradiol was measured by a
solid-phase, chemiluminescence enzyme immunoassay system
(Immulite®; Diagnostic Products Corp.), which had a
detection limit of 0.044 nmol/L and a CV <10%. Progesterone was
measured with the Ciba-Corning ACS® progesterone
chemiluminescence immunoassay, with a minimum detectable concentration
of 0.35 nmol/L and CVs <10% within the measuring range.
Extraction of total RNA.
Total RNA from T-47D cells was
extracted with the commercial reagent, TRIZOLTM
(Gibco-BRL), according to the manufacturer's recommendations. The
quality and quantity of the extracted RNA were checked by
spectrophotometric measurements at 260 and 280 nm.
Reverse transcription.
To reverse-transcribe 1 µg of
total RNA, we used oligo dT primers and Superscript IITM
reverse transcriptase (Gibco BRL). Briefly, the RNA and 500 ng of oligo
dT primers were first denatured for 10 min at 70 °C, chilled on ice
for 1 min, and then incubated for 1 h at 42 °C in a 20-µL
reaction mixture containing 1x PCR buffer (Boehringer Mannheim; Tris
10 mmol/L, pH 8.3, plus KCl 50 mmol/L), 2.5 mmol/L MgCl2, 1
mmol/L deoxynucleoside triphosphates, 10 mmol/L dithiothrietol, and 200
units of Superscript II reverse transcriptase. The reaction was
terminated by heating for 15 min at 70 °C. Template RNA was digested
by incubation with RNase H for 20 min at 37 °C.
PCR procedure.
The two oligonucleotides we used to
amplify the cDNA of PSA by PCR were originally proposed by Deguchi et
al. (17) and have the following sequences:
PSA-1: 5'-TGC-GCA-AGT-TCA-CCC-TCA-3'
PSA-2: 5'-CCC-TCT-CCT-TAC-TTC-ATC-C-3'
These primers amplify a 754-bp fragment of PSA cDNA. Actin primers, which amplify a 372-bp fragment of actin cDNA, were used as controls; their sequences have been described previously (18).
In the PCR, 5 µL of cDNA was added to 45 µL of PCR mix containing 1x PCR buffer, 1.5 mmol/L MgCl2, 500 nmol of the PCR primers, 200 µmol/L of deoxynucleostide triphosphates, and 2 units of Taq DNA polymerase (Boehringer Mannheim). PCR was performed on a Perkin-Elmer 2400 thermal cycler for 30 cycles according to the following program: 94 °C for 30 s (5 min for the first cycle); 56 °C for 30 s; and 72 °C for 30 s (7 min for the last extension). Actin cDNA was amplified from 1 µL of the cDNA preparation under the same conditions used for PSA cDNA. From each PCR reaction 20 µL was electrophoresed on agarose gels (20 g/L) and visualized by ethidium bromide staining.
In other experiments, we incorporated digoxigenin-11-dUTP (Boehringer Mannheim), 0.7 µmol/L, into the PCR reaction mixture and detected the PCR product after Southern transfer to nylon membranes by probing with anti-digoxigenin antibodies conjugated to alkaline phosphatase; activity of the enzyme was detected with chemiluminescence. This method is ~500-fold more sensitive than ethidium bromide staining in detecting the PCR products.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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4 ng/L, we collected additional sera from two. These new
sera spanned two consecutive menstrual cycles and were collected at
least 2 months after the initial collection; thus, in total, we studied
7 informative menstrual cycles from three different patients. The days
of the menstrual cycle in each case were verified by analysis of
progesterone, estradiol, LH, and FSH.
In Fig. 1
, we present the serum PSA changes during the menstrual cycle of
one patient (3 cycles) and in Fig. 2
the data for the other two patients (3 cycles and 1 cycle). The
progesterone values are also presented; however, when we plotted the
PSA data along with the LH, FSH, or estradiol data, no recognizable
pattern or relationship was seen (data not shown). Figs. 1
and 2
show a
consistent pattern, in which PSA peaks during the mid- to late
follicular phase and reaches a minimum during the mid- to late luteal
phase. The difference between PSA and progesterone peaks is ~10–12
days.
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We further examined whether serum obtained during the menstrual cycle
could stimulate PSA production in the breast carcinoma cell line T-47D
by measuring the PSA mRNA and the PSA protein. We found that
nonstimulated T-47D cells do not produce PSA protein and do not express
PSA mRNA. Fig. 3
shows the results of stimulating the cells with serum obtained
during the menstrual cycle. The ability of the serum to induce PSA
production parallels the concentrations of progesterone present, the
greatest stimulation being achieved with the serum containing the most
progesterone (day 24 of the menstrual cycle).
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PSA mRNA can be detected in trace amounts in T-47D cells if the serum
used for stimulation is collected during the follicular phase of the
cycle. When the serum is collected during the luteal phase, PSA mRNA
expression is dramatically increased (Fig. 4
). These changes in PSA mRNA concentrations parallel the changes
in progesterone concentrations. PSA mRNA is undetectable in T-47D cells
that are not stimulated by serum (data not shown).
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We further stimulated T-47D cells with the glycoprotein hormones LH, FSH, and hCG and with prolactin and growth hormone. Despite the wide range of concentrations used, none induced PSA production. Progesterone was able to induce T-47D cells for PSA protein production and PSA mRNA expression at concentrations of 0.1-100 nmol/L (12).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The PSA concentration in female serum is very low and usually not measurable by commercial PSA assays. Using highly sensitive PSA procedures we developed (16), we demonstrated that many female sera have measurable PSA concentrations (20)(21). We do not know why some women have measurable serum PSA and some do not, but perhaps it is related to an ability of PSA to diffuse from the breast tissue into the general circulation. We have further demonstrated that much higher PSA concentrations can be found in female breast secretions. For comparison purposes, we here report the approximate concentrations (ng/L) of PSA in various human fluids: seminal plasma, 109; male serum, 1000–2000; normal breast discharge fluid, 5 x 106; milk of lactating women, 105; female serum, 2–4. That is, the difference between PSA concentrations in the prostatic or breast secretion (i.e., seminal plasma or breast discharge fluid) and the corresponding serum was ~106-fold. The physiological role of PSA in seminal plasma seems to have been established (22). The role of PSA in the breast and its secretions is still obscure. We found that breast cancer patients with PSA-producing tumors may have better prognosis than do patients with tumors that do not express PSA (8).
Because PSA expression is under the control of steroid hormones and
their receptors, we speculated that its concentration may change during
the menstrual cycle. Now we have demonstrated this, by assessing the
concentrations of serum PSA during the menstrual cycle of healthy women
and by examining the ability of serum from these women to stimulate PSA
production and PSA mRNA expression in a tissue culture system. We found
that serum PSA concentrations change widely during the menstrual cycle
and follow a specific pattern. This pattern was similar in the three
patients studied and was reproducible on repeated cycles from the same
patients (Figs. 1
and 2
). The increase in PSA concentration follows the
progesterone concentration increase but with a 10- to 12-day lag
period. On the basis of many recent indirect findings, we suggest that
the target tissue producing PSA is the female breast, but the
contribution of other steroid hormone-responsive tissues, e.g., the
endometrium, cannot be excluded (19). Recently, Clements
et al. measured PSA mRNA in endometrial tissue of normal cycling women
and found the highest amounts during the follicular phase and the
lowest during the late luteal phase (23), in general
accordance with the data reported here. They saw no association between
the changes of PSA and changes in the concentrations of LH, FSH, or
estradiol. Our tissue culture system with T-47D breast carcinoma cells
confirmed the following: (a) that progesterone can
upregulate PSA mRNA and protein production; (b) that
estradiol, LH, FSH, prolactin, growth hormone, and hCG do not mediate
PSA production in the T-47D cell line; (c) that sera
collected during the luteal phase of the cycle, but not sera collected
during the follicular phase or mid-cycle, have the ability to
upregulate PSA protein production and increase PSA mRNA expression in
the breast carcinoma cell line T-47D.
The data presented allow us to speculate that corpus luteum steroids stimulate target tissues capable of producing PSA (one of which is the breast) for PSA production and release into the mammary ducts. A fraction of this PSA diffuses into the general circulation and can be measured in the serum. Peak concentrations of PSA appear 10–12 days after the progesterone peak. Once the corpus luteum regresses, PSA concentration decreases, with an apparent half-life of ~3–5 days. Given these findings, we suggest that PSA is a protein regulated by corpus luteum steroids. Because this protein has serine protease activity, it will be interesting to find its biological role in the breast and its secretions and its possible physiological substrates (6).
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and progesterone (
) during menstrual
cycles of one volunteer: (A) in one menstrual cycle;
(B) in two additional consecutive menstrual cycles collected
at least 2 months after the sera of panel A.
