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Original Studies |
Neuroendocrine Unit, Departments of Neurosurgery (B.S.), and Neuropathology (E.T.H.W.), Massachusetts General Hospital, and Harvard Medical School, Boston, Massachusetts 02114
Address all correspondence and requests for reprints to: Anne Klibanski, M.D., Neuroendocrine Unit, Bulfinch 457, Massachusetts General Hospital, Boston, Massachusetts 02114. E-mail: aklibanski{at}partners.org
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Abstract |
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We also investigated tumor expression of follistatin messenger ribonucleic acid, an activin-binding protein with two isoforms of different potencies. In contrast to normal pituitary tissue, only four tumors expressed both follistatin isoforms, and three tumors expressed only the less potent form. Tumors in which activin induced antiproliferative responses showed diminished or no follistatin messenger ribonucleic acid expression compared to normal pituitary. These data indicate that activin has an antiproliferative effect in a subgroup of human pituitary tumors.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Growth inhibitory effects of activin have been shown in a number of normal and neoplastic human cell types, including tumor cell lines such as a prostate cancer-derived line, LNCaP (7, 8, 9, 10, 11, 12, 13, 14). Activin-induced cellular growth arrest is accompanied by altered expression and activity of cell cycle nuclear factors. Activin up-regulates the mitotic inhibitor p21WAF1/cip1 and suppresses cyclin D2, resulting in hypophosphorylation and activation of the tumor suppressor Rb protein (7, 12, 14, 15). Activin-induced growth arrest has also been reported in several pituitary cell lines, such as mouse corticotroph AtT20 cells (16) and rat somatotroph GH3 cells (17). Although activin increases the number of FSH-secreting cells in primary rat pituitary cultures, an increase in cellular proliferation has not been demonstrated (18). In contrast, in several other cell types, such as ovarian cells, activin may stimulate cell proliferation (19, 20, 21). Therefore, activin effects on cell proliferation seem to be tissue specific.
The biological actions of activin may be influenced in part by follistatin (FS). FS is an activin-binding single chain glycoprotein that was first isolated from ovarian follicular fluid and exists in two isoforms with different potencies in neutralizing the effects of activin. The two isoforms of FS are generated by alternative splicing, containing 288 (FS288) and 315 (FS315) amino acids, respectively. Recently, it has been shown that FS expression is decreased in human pituitary adenomas (22). There is also evidence for differential cellular expression and affinity for activin of the two FS isoforms. FS288, bound to the cell surface, is 8–10 times more potent in neutralizing activin than the secreted FS315 (23, 24). Differential expression of these two isoforms may mediate activin effects in a number of established human cell lines. For example, the androgen-responsive prostate cancer cell line LNCaP demonstrates a clear antiproliferative response to activin (8, 9, 10, 11), whereas the androgen-resistant human prostate cancer cell line PC3 is refractory to growth arrest by activin (9, 11). It has been reported that activin effects in these cell lines may be due to differences in FS isoform expression (11, 25). In pituitary tissue, previous studies detected only FS315 messenger ribonucleic acid (mRNA) transcripts in normal rat pituitary gland (26). Although in human gonadotroph adenomas FS mRNA levels have been reported to be decreased compared to those in normal pituitary tissue (22), expression of different FS mRNA isoforms has not been investigated or linked to proliferation (22, 27).
Therefore, we investigated whether activin can affect cell proliferation in clinically nonfunctioning human pituitary adenomas. In addition, we determined the regulation of the cell cycle nuclear factor p21WAF1/cip1 by activin as well as FS isoform expression in these tumors.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Tumor tissue from 16 clinically nonfunctioning pituitary macroadenomas was placed in 0.9% saline immediately after transsphenoidal surgery. Patients were classified as having clinically nonfunctioning tumors based on the presence of a macroadenoma without a diagnosis of acromegaly or Cushing’s syndrome and serum PRL levels of less than 100 ng/mL. A portion of each adenoma was fixed in formalin and included in paraffin for histological examination. Another fragment was snap-frozen in liquid nitrogen before total RNA was extracted and subjected to RT. A third fragment was used for proliferation studies.
Activin effects on proliferation were studied in primary cell culture obtained by enzymatically dispersing the tumor fragment as previously described (28). The pituitary adenomas and LNCaP cells were maintained in DMEM supplemented with penicillin/streptomycin, 10% nonessential amino acids, and insulin/transferrin/selenium with or without 10% FBS at 37 C and 90% relative humidity in 5% CO2 air. At the end of each pituitary tumor primary cell culture, we examined the morphology of the cultured cells to exclude any fibroblast contamination. In parallel experiments we also cultured tumor primary cells on poly-D-lysine-covered slides and performed immunostaining for cytokeratin, an epithelial-origin cell marker, and for vimentin, a component of the intermediary filaments found in fibroblasts and in the folliculostellate cells in the pituitary gland (29). We used normal human HS 27 fibroblasts, cultured and stained under the same conditions, as controls. In this determination we could confirm that the number of cells that stained positively for vimentin is minimal. Therefore, in these short term primary cultures we could exclude the possibility that fibroblast contamination could influence the pituitary tumor cell proliferation data after each experiment.
Activin treatment of cultured cells
Approximately 1 x 106 primary cultured cells/well were aliquoted into 6- or 12-well plates for [3H]thymidine incorporation or cell cycle nuclear factor regulation studies, respectively. Each experiment was performed in duplicate or triplicate, depending on the sample availability. Cells were incubated in medium with 10% FBS overnight. To study cell proliferation, fresh medium with 10% FBS, 10 µCi/mL [3H]thymidine, and recombinant human activin A (National Hormone and Pituitary Program, Torrance, CA) to a final concentration of 0, 1, or 10 ng/mL was added to corresponding wells for 24 h. To study the effect of activin on p21WAF1/cip1 expression, culture medium was changed to serum-free medium immediately before the experiments, and cells were treated with 0, 1, 10, and 100 ng/mL activin for 4 h in duplicate experiments. Total RNA was extracted from three well pools for each activin dose.
LNCaP cells were cultured in 100-mm dishes. In time-course experiments we used 50 ng/mL activin treatment and harvested cells at 0, 2, 4, 8, 16, and 24 h (for RNA extraction) and at 0, 4, 8, and 24 h (for protein extraction). The dose response to activin was studied by adding activin to medium in concentrations of 0, 1, 10, and 100 ng/mL. Total RNA was extracted after 4 h, and proteins were extracted after treatment for 24 h.
Tritiated thymidine incorporation
Quantification of DNA synthesis by [3H]thymidine incorporation as an indirect measurement of proliferation was performed as previously reported (8). After lysis in buffer containing 1% Triton X-100, 10% glycerol, 2 mmol/L ethylenediamine tetraacetate, 2 mmol/L dithiothreitol, and 25 mmol/L Tris-phosphate (pH 7.8), tumor genomic DNA containing incorporated [3H]thymidine was precipitated onto glass-fiber scintillation filters using 10% trichloroacetic acid. Radioactivity was quantitated by scintillation counting of filters for 1 min in 10 mL Scintiverse BD liquid scintillation counting liquid (Fisher Scientific, Pittsburgh, PA).
RT-PCR
Total RNA was extracted as previously described (30). Each pituitary tumor fragment and five postmortem pituitary specimens from control subjects (Harvard Brain Bank, McLean Hospital, Belmont MA) were homogenized in guanidinium isothiocyanate reagent using 1 mL for 100 mg tissue. Activin-treated cultured cells were dispersed in 500 µL guanidinium isothiocyanate and extracted, and the RNA was precipitated with isopropanol, washed once in 70% ethanol, air-dried, and dissolved in water. All RNA samples were stored at -80 C until used. Genomic DNA contamination was removed by incubation with RQ1-DNase (Promega Corp., Madison, WI) for 30 min at 37 C, followed by heat inactivation of the enzyme. The concentration of total RNA was measured by OD at 260 nm.
RT of RNA extracted from tissue fragments was performed on 1 µg total RNA in a 20-µL reaction containing 50 mmol/L Tris-HCl (pH 8.3), 5 mmol/L KCl, 5 mmol/L MgCl2, 5 mmol/L dithiothreitol, 0.25 mmol/L spermadine, 200 µmol/L deoxy (d)-NTPs, 1 µg oligo(dT)15, and 12 U AMV reverse transcriptase (Promega Corp.). Reactions were carried out in an MJ thermocycler (MJ Research, Inc., Watertown, MA) at 42 C for 15 min and at 99 C for 5 min. Control reactions were carried out in the absence of reverse transcriptase.
All PCR amplifications used approximately 40 ng first strand
complementary DNA from a single RT reaction. PCR was performed in 50
mmol/L KCl, 10 mmol/L Tris-HCl (pH 9.0), 3.5 mmol/L
MgCl2, 0.1% Triton X-100, 40 µmol/L dNTPs, and
1.25 U Taq polymerase (Promega Corp.) in a
final volume of 50 µL. PCR primers (12.5 pmol) were used for each
reaction, and amplifications were carried out in an MJ thermocycler
(MJ Research, Inc.). A control tube containing the PCR
reaction mixture with no template was included to assess extraneous
contamination. All PCR reactions started with 5 min at 94 C, followed
by 30 cycles (1 min at 94 C, 1 min at optimal annealing temperature,
and 1 min and 15 s at 72 C) with a final step of 10 min at 72 C
and were maintained thereafter at 4 C. PCR products were visualized by
incorporation of [
-32P]dCTP (100
nCi/reaction) in the amplification reactions, which were resolved by
6% nondenaturing Tris/borate/EDTA electrophoresis
buffer/polyacrylamide gel electrophoresis (Protogel, National
Diagnostics, Atlanta, GA) and autoradiography onto Kodak
X-Omat film (Eastman Kodak Co., Rochester, NY) for 2–48
h. Images were digitally scanned, and signals were quantitated using
IPLabGel software (Signal Analytics, Vienna, VA). The amounts of
amplification products were compared between samples in correlation
with the equal amount of glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and cyclophylin A products, as described for comparative RT-PCR
(31, 32, 33).
All PCR primers were designed using Oligo software (National Diagnostics, Plymouth, MN) and were cross-referenced against GenBank sequences to ensure specificity. We used the following sets of primers, described in 5'-3' sequence and with the expected product size in parentheses [activin receptor (Act-R)]: Pit-1, TTC TAC TCT CTT GTG GGA ATG and CTG CCA TCA CTC CAT AGG TTG (232 bp); GAPDH, GAG CCA CAT CGC TCA GAC and TTC TCC ATG GTG GTG AAG (340 bp); Act-R type I (Act-RI), GAG ATC GTG GGC ACC CAA GGG and AGC TGG GAG AGG GTC TTC TTG (530 bp); Act-RII, GCA AAA TGA ATA CGA AGT CTA and GCA CCC TCT AAT ACC TCT GGA (435 bp); Act-RIIB, CAA CTT CTG CAA CGA GCG CTT and GCG CCC CCG AGC CTT GAT CTC (283 bp); FS, GGC CGG TGT TCC CTC TGT GAT and CTC CTC TTC CTC GGT GTC TTC; p21, GGC CCA GTG GAC AGC GAG CAG and CGG CGT TTG GAG TGG TAG AAA (401 bp); and cyclophilin A, CAT GGT CAA CCC CAC CGT GTT CTT and TAG ATG GAC TTG CCA CCA GTG CCAT (255 bp). FS was amplified using a primer set that allowed detection of the known alternate splice, with the forward primer located in exon 5 and the reverse primer located in exon 6. Using this primer set, FS288 gives a PCR product of 452 bp, and FS315 gives a PCR product of 188 bp.
Protein expression by Western blotting
The LNCaP cells were lysed for 10 min on ice using 1 mL lysis buffer [25 mmol/L Tris-HCl, 250 mmol/L NaCl, 2 mmol/L ethylenediamine tetraacetate, 1% Triton X-100, and 5% protease inhibitor mixture (Sigma, St. Louis, MO)] for one 100-mm tissue culture dish. The homogenates were centrifuged at 10,000 x g for 15 min at 4 C. The protein concentration in each sample was determined by Bradford assay (Sigma), and 100 µg proteins were separated by 12.5% SDS-PAGE. The resolved proteins were electroblotted onto polyvinylidene difluoride filters (MSI, Marlborough, MA) in transfer buffer (192 mmol/L glycine, 20% methanol, and 25 mmol/L Tris-HCE). To reduce nonspecific binding, the filters were incubated in a blocking buffer [20 mmol/L Tris (pH 7.6), 137 mmol/L Nace, 10% nonfat milk, and 0.1% Tween-20] at 4 C overnight. Filters were then incubated with specific primary antibodies at the indicated dilutions for 45 min at room temperature and rinsed in wash buffer (Tris-buffered saline, pH 7.6, and 0.1% Tween-20) four times for 10 min each time. The primary antibodies used were anti-p21 (goat polyclonal; 1:3,000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and antiactin (mouse monoclonal; 1:2,000 dilution; Roche Molecular Biochemicals, Indianapolis, IN). The secondary antibodies used were antigoat (1:10,000) or antimouse (1:5,000) IgG conjugated with horseradish peroxidase (Roche Molecular Biochemicals) in a 30-min incubation at room temperature. Western blots were visualized by enhanced chemiluminescence (Roche Molecular Biochemicals).
Statistical analysis
Because of the high degree of variability in raw counts between tumor cultures, the results of the [3H]thymidine incorporation experiments were expressed as a percentage of the results for the control wells (activin dose, 0 ng/mL). Because the data were nonparametric, Kruskal-Wallis one-way ANOVA was used to examine the effect of increasing activin dose on [3H]thymidine incorporation. Post-hoc analysis was undertaken using Dunnett’s method, comparing results with both the activin doses to the control values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clinical characteristics of the 16 patients, tumor
immunohistochemistry, and RT-PCR results for Pit-1 are summarized in
Table 1
. The ages of the patients ranged
from 37–86 yr (mean, 57 yr). All tumors were positive for -subunit
immunostaining, whereas immunostaining for 1 or more glycoprotein
hormone subunits was positive in 12 tumors. Screening for the
presence of Pit-1 expression by RT-PCR showed 1 positive tumor that was
also TSHß positive by immunohistochemistry. The normal pituitary
samples used as control were all positive for Pit-1 mRNA. These data
indicate that these surgical specimens were not contaminated with
significant numbers of normal pituitary cells.
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Activin induced a dose-dependent decrease in
[3H]thymidine incorporation in five tumors
(Table 2). The range of percent
suppression at the 10 ng/mL activin dose was 15–65%. No tumor
demonstrated a statistically significant increase in
[3H]thymidine incorporation in response to
activin. Activin effects on tumor cell proliferation did not correlate
with either clinical tumor characteristics or the results of
immunohistochemistry analysis. Figure 1A
shows the inhibition of [3H]thymidine
incorporation with activin at doses of 1 and 10 ng/mL as a percentage
of the control value (P = 0.02 and 0.005, respectively)
in a representative tumor (no. 7). Similar results were observed in
LNCaP cells treated with same doses of activin (Fig. 1B).
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As shown in Table 2
, most pituitary adenomas expressed Act-RII
mRNA (15 of 16), Act-RIIB mRNA (15 of 16), and Act-RIB mRNA (15 of 16).
In normal pituitary tissue both forms of FS (FS288 and FS315) mRNA were
expressed (Fig. 2). In contrast, only 4
tumors expressed both FS isoforms, and in 3 tumors the long form of FS,
FS315, was expressed alone. The intermediary band (
300 bp) in lane 6
is a nonspecific PCR product. The 5 tumors that exhibited an
antiproliferative response to activin showed no expression or decreased
levels of FS315 mRNA (lanes 3, 7, 8, 13, and 16) compared to normal
pituitary tissue.
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To investigate the mechanism of activin antiproliferative effects,
we first used a known activin-responsive tumor cell line, LNCaP, to
establish the optimal experimental time course and treatment dose. In a
24-h time-course treatment of these cells with 50 ng/mL activin,
up-regulation of p21WAF1/cip1 basal expression
was shown by comparative RT-PCR (Fig. 3a)
and Western blotting (Fig. 3b). Steady state levels of mRNA increased
as early as 2 h. Protein levels increased by 4 h, but there
was no further increase thereafter up to 24 h. Increasing
doses of activin caused an up-regulation of
p21WAF1/cip1 mRNA levels, with induction observed
at activin concentrations as low as 1 ng/mL (Fig. 3c). The
p21WAF1/cip1 protein levels also showed a dose
response to activin (Fig. 3d) with an increase observed with 1 ng/mL
activin and maximal induction achieved at a dose of 10 ng/mL.
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Because p21WAF1/cip1 was up-regulated
by activin in LNCaP cells, we performed similar studies in nine
primary cultures of pituitary adenomas. The three tumors showing
an antiproliferative response to activin (no. 8, 13, and 16)
demonstrated a dose-dependent increase in
p21WAF1/cip1 mRNA levels in response to 4 h
of activin treatment. The increases in
p21WAF1/cip1 mRNA steady state levels were 306%,
394%, and 297%, respectively, with 100 ng/mL activin treatment
compared by scanning densitometry to the control, after normalization
with cyclophilin A expression. In contrast, activin failed to increase
p21WAF1/cip1 expression in four tumors (no. 10,
11, 14, and 15) that showed no proliferative inhibition with activin.
In two tumors (no. 9 and 12), although we did not see a decrease in
proliferation in our study, p21WAF1/cip1 mRNA was
up-regulated by activin in a comparative RT-PCR assay. In Fig. 4, it can be seen that
p21WAF1/cip1 expression with activin treatment in
two representative tumors as RT-PCR products of
p21WAF1/cip1 correlated with equal amounts of the
coamplified cyclophilin A products (upper panel) and the
ratio calculated from scanning densitometry, comparing activin-treated
cells to controls (lower panel). Tumor 16 showed
an inhibition of proliferation and dose-dependent
p21WAF1/cip1 up-regulation with activin treatment
(Fig. 4a), whereas in tumor 15 proliferation and
p21WAF1/cip1 expression were unchanged by activin
(Fig. 4b).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One mechanism for the antiproliferative effect of activin shown in other human tumor cell lines involves up-regulation of p21WAF1/cip1, a cyclin-dependent kinase inhibitor (12). We chose to use a proven activin-responsive human tumor cell line, LNCaP cells, as a model to investigate the experimental time and dose course of cell cycle nuclear factor regulation. LNCaP cells exhibit an activin-induced reduction in [3H]thymidine incorporation and cell number (8, 11), similar to what we observed in primary pituitary tumor cell culture. In LNCaP cells, activin caused a prompt, dose-dependent increase in p21WAF1/cip1 gene transcription, and Western blotting confirmed up-regulation of p21WAF1/cip1 protein in response to activin. This effect in LNCaP cells is consistent with that in other cell lines, which have been reported to show increased levels of p21WAF1/cip1 in response to activin and transforming growth factor-ß. For example, in the hepatoma cell line HepG2, the activin-induced antiproliferative effects were suggested to be mediated by increased p53-dependent p21WAF1/cip1 gene expression, resulting in hypophosphorylation of the Rb protein (12). Our studies revealed that in pituitary adenomas in which activin caused an antiproliferative effect, p21WAF1/cip1 expression was up-regulated. In four tumors in which thymidine incorporation was unchanged by activin treatment, p21WAF1/cip1 remained at steady state levels. However, p21WAF1/cip1 gene expression was also increased in two tumors in which thymidine incorporation was not significantly altered by activin treatment. Therefore, although p21WAF1/cip1 may be involved in activin-mediated growth arrest, up-regulation of this protein alone is not sufficient for this growth inhibitory effects in pituitary tumors. The heterogeneity of such responses in human pituitary adenomas in vitro has been well documented, in contrast to the uniformity of results using well established homogenous cell lines.
Activin receptor mRNA expression, detected by an extremely sensitive RT-PCR technique, was highly prevalent in the group of pituitary tumors we studied, consistent with previously reported data (36). However, no correlation was found between the presence of activin receptors and activin-mediated inhibition of cell growth. Although the presence of activin receptors is not sufficient to cause growth arrest by activin, we cannot exclude the following possibilities. Because activin receptor mRNA expression was not quantitatively examined, it is not known whether there are differences in activin receptor expression levels between activin-responsive and nonresponsive tumors. Also, it is not known whether the lack of activin responsiveness in some tumors is due to the presence of mutations of these receptors. However, in a recent study we found that somatic point mutations within intracellular kinase regions of type I/type II activin receptors are rare in human pituitary tumors (37). Nonetheless, expression of truncated forms of activin receptors in pituitary tumors may interfere with normal receptor function (36). Therefore, further functional studies of these activin receptor forms are needed. Other potential tumor-specific defects in activin signaling pathways include alterations in the expression or function of Smad proteins, crucial mediators for signal transduction from the cell surface receptor to the nucleus (38). Other somatic mutations, although rarely found in pituitary tumors, or growth factors may also be involved in pituitary tumorigenesis (39).
We have demonstrated that activin has an antiproliferative effect in a subset of clinically nonfunctioning human pituitary tumors in primary culture in vitro. The expression of the cyclin-dependent kinase inhibitor p21WAF1/cip1 is up-regulated by activin in the cells; thus, this cell cycle regulator is a potential factor in mediating activin antiproliferative effects. However, the up-regulation of p21WAF1/cip1 was also observed in some tumors that had no response to activin in cell proliferation, indicating that other factors are also required for this antiproliferative effect. Although the extent of the antiproliferative response is variable between individual tumors, no tumor demonstrated a proliferative response to activin.
Our results suggest that defects in activin-mediated signal transduction pathways and dysregulation of modulating peptides may be involved in abnormal activin-mediated growth control in pituitary tumors.
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Acknowledgments |
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Footnotes |
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2 Recipient of a Health Research Council of New Zealand Overseas
Postdoctoral Fellowship 1996–1997 and the Odlin Research Fellowship
from the Royal Australasian College of Physicians 1997–1998.
Received June 11, 1999.
Revised August 19, 1999.
Accepted November 24, 1999.
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