| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Department of Biomedical Sciences and Advanced Therapies, Section of Endocrinology, University of Ferrara, 44100 Ferrara, Italy
Address all correspondence and requests for reprints to: Ettore C. degli Uberti, M.D., Department of Biomedical Sciences and Advanced Therapies, Section of Endocrinology, University of Ferrara, Via Savonarola 9, 44100 Ferrara, Italy. E-mail: ti8{at}dns.unife.it
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
TGFß1 is an important regulator of human adrenal development, with marked effects on steroid-producing cell function, and the production of distinct TGFß subtypes has been suggested to be regulated by steroid hormones in several tissues. To address potential TGFß1 induction by DHT, quantitative PCR and enzyme-linked immunoadsorbent assay were performed in NCI-H295 cells treated with DHT (from 10-12–10-9 mol/L). DHT led to a significant dose-dependent increase in TGFß1 messenger ribonucleic acid expression and in biologically active TGFß1 protein levels in the conditioned media of NCI-H295 cells, demonstrating that androgen can induce TGFß1 expression and production. TGFß1 (10-7–10-6 mol/L) was capable of significantly reducing cell proliferation (P < 0.05) after 24 h of treatment, as assessed by measuring [3H]thymidine incorporation in NCI-H295 cells. The addition of TGFß1-neutralizing antibody to cell cultures treated with different DHT concentrations (10-9 and 10-10 mol/L) blocked the inhibitory effect of TGFß1 on adrenocortical cell proliferation.
These findings suggest that TGFß1 exerts an inhibitory action on adrenocortical cell proliferation. Therefore, it might be reasonable to suppose that DHT could also influence human adrenocortical cell growth by involving TGFß1.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
We previously demonstrated that the androgen receptor (AR) is expressed in human adrenal cortex and the cancer cell line, NCI-H295, and that dihydrotestosterone (DHT) significantly reduces human adrenocortical cell growth in vitro (7). The mechanism by which DHT exerts this inhibitory action on the adrenal cortex is still unknown. Recently, we have shown that TGFß1 and its receptor (type II) are expressed in human adrenocortical cells (8).
The present study was designed to investigate whether TGFß1 is involved in DHT action on human adrenocortical cells. In particular, we studied the effect of DHT on TGFß1 expression in NCI-H295 cells and the effect of TGFß1 on human adrenocortical cell growth by assessing the response of these cells to exogenous TGFß1 in terms of [3H]thymidine ([3H]thy) incorporation. Evidence is provided to demonstrate that DHT-induced growth inhibition could be mediated at least in part by TGFß1.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The human NCI-H295 adrenocortical cancer cell line was obtained from the American Type Culture Collection (Manassas, VA). Cells were maintained in RPMI 1640 medium without phenol red and with L-glutamine supplemented with 2% FCS and containing sodium selenite (1 ng/mL), insulin (5 µg/mL; Novo Nordisk, Princeton, NJ), transferrin (5 µg/mL), dexamethasone (10-8 mol/L), 17ß-estradiol (10-8 mol/L), 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 100 µg/mL amphotericin at 37 C in a humidified atmosphere of 5% CO2 and 95% air. To eliminate the influence of steroid hormones in the medium, cells were switched to RPMI 1640 medium supplemented with 2% dextran (0.1%)-coated charcoal (1%)-treated FCS (DCT-FCS), sodium selenite (1 ng/mL), insulin (5 µg/mL), transferrin (5 µg/mL), and antibiotics before incubation with hormones.
After overnight growth, subconfluent NCI-H295 cells were incubated in the absence (control untreated cells) or presence of DHT at the indicated concentrations in medium without steroids and supplemented with 2% DCT-FCS. DHT was added to the medium in alcohol, at a final ethanol concentration less than 0.1% in both the control and hormone-treated cultures. TGFß1 protein production was measured in conditioned media from culture flasks, and TGFß1 messenger ribonucleic acid (mRNA) expression was evaluated after total RNA extraction from cells harvested by trypsinization. Cell culture and chemical reagents were purchased from Mascia Brunelli (Milan, Italy) and Sigma (Milan, Italy), respectively, unless otherwise indicated.
Isolation of RNA
Total RNA was extracted from subconfluent NCI-H295 cells using the Ultraspec-II RNA Isolation System (Biotecx Laboratories, Inc., Houston, TX). To prevent DNA contamination, RNA was treated with ribonuclease-free deoxyribonuclease. The amount of total RNA was determined by optical density at 260 nm.
RT-PCR
Using a first strand complementary DNA (cDNA) synthesis kit (Roche, Mannheim, Germany), 1 µg total RNA was reverse transcribed according to the manufacturer’s protocol. RT mix in PCR tubes was covered with 50 µL light white mineral oil (Sigma-Aldrich Corp., Milan, Italy); the RT was carried out in the Minicycler (MJ Research, Inc., Watertown, MA) using a program with the following parameters: 10 min at 24 C, 60 min at 42 C, 5 min at 95 C, and 5 min at 4 C. After the reaction was completed, samples were stored at -20 C until the first PCR.
The complementary DNA (1 µL RT reaction) was then amplified by PCR in 30 cycles with 1 U Taq polymerase (Roche) in the conditions recommended by suppliers in a 25-µL reaction mixture. After initial denaturation at 95 C for 5 min, each cycle consisted of primer annealing at 63 C for 1 min, extension at 72 C for 1.5 min, denaturation at 95 C for 1 min, and final extension at 72 C for 10 min. Oligonucleotide primer sequences used for the amplification were: 1447–1470, 5'-CGGAGTTGTGCGGCAGTGGTTGA-3' (forward); and 1871–1894, 5'-GCGCCCGGGTTATGCTGGTTGTA-3' (reverse), located, respectively, in exons 3 and 7 of the human TGFß1 gene. These are expected to amplify a TGFß1 cDNA fragment 450 bp in size. PCR products were analyzed on a 2% agarose gel and visualized by ethidium bromide (ETB) staining.
To assure that no contamination occurred during the course of the RT-PCR procedure, two kinds of negative control were prepared. The first negative control was made by omitting the total RNA in the RT. The second was prepared by replacing the cDNA mix with water in the PCR reaction. The PCR was considered useful only if no band was observed in the negative control lanes on a 2% agarose gel.
For determination of TGFß1 mRNA, competitive quantitative RT-PCR was performed (9).
Construction of the target cDNA and competitive fragment for TGFß1
A 450-bp fragment of native TGFß1 cDNA (i.e. target) was obtained by PCR amplification of reverse transcribed total RNA from a human breast cancer cell line, MCF7, which is known to constitutively express TGFß1 (3). The PCR product was visualized by agarose gel electrophoresis and stained with ETB. The cDNA was extracted and purified from the gel with the Prep-A-Gene DNA purification kit (Bio-Rad Laboratories, Inc., Milan, Italy). By determination of the optical density we estimated the concentration (micrograms per µL) of the target cDNA; the absolute number of target cDNA molecules was then calculated (10) using the following formula: cDNA (molecules/µL) = [(cDNA (µg/µL) x Avogadro’s number)/mol wt (µg/mol)]. The mol wt of target cDNA was 270 g/mol, and Avogadro’s number was 6.023 x 1023 molecules/mol. The solution was aliquoted in serial dilutions and stored at -20 C.
To generate an internal standard, a previously described, low stringency PCR method was used (11). Briefly, RT-PCR was carried out at a lower annealing temperature (55 C) to decrease the stringency of priming. Lower stringency, indeed, allows more mismatches in primer sequences, yielding multiple products. As a competitor for the native TGFß1 message, a 200-bp fragment was selected and purified from agarose gel as previously described. Subsequently, the fragment was reamplified at the 60 C annealing temperature to determine its feasibility as a competitor for the native TGFß1 cDNA. The expected 200-bp fragment was again obtained and then used to compete with the native TGFß1 cDNA for the primers. The concentration of competitor fragment (CF) was estimated as described above.
Standard curve and competitive quantitative PCR
The standard curve for TGFß1 was constructed as previously
described by Krussel et al. (12). A constant amount of CF
(5 x 104 molecules) was coamplified with
declining amounts of target cDNA, obtained by serial dilutions. The
amounts of target cDNA that were added to each PCR are shown in Fig. 1
. PCR for TGFß1 was performed as
described above; the products were resolved on 2% agarose gel stained
with ETB and analyzed on the Fluor-S system (Bio-Rad Laboratories, Inc.). cDNA size calculation and UV densitometry
were carried out using Multi-Analyst software (Bio-Rad Laboratories, Inc.). The logarithmically transformed ratio of
the ETB densities of target cDNA to CF was calculated and plotted
against the log amount of initially added target cDNA in each PCR to
obtain the standard curves shown in Fig. 1. Simple regression analysis
showed a significant correlation (r2 = 0.9938;
P < 0.001) between these two parameters. The values
obtained from the regression line of the standard curve
(y = b + ax) allowed us to
measure the amount of cDNA TGFß1 transcripts in an unknown sample
when coamplified with 5 x 104 molecules of
CF. The ratio of the densities of the CF band (200 bp) and the target
cDNA band obtained from each sample (450 bp) was logarithmically
transformed and compared to the value obtained from the standard curve
[x = (y -
b)/a]. The reproducibility of this method
(quantitative PCR) was tested by repeating the assay using independent
control RNAs. The interassay coefficient of variation ranged from
2–3.7%. Quantitative PCR was performed on duplicate RT samples from
RNA extracted from control and DHT-treated
(10-12–10-8 mol/L)
NCI-H295 cells in seven independent experiments.
|
TGFß1 immunoassay was performed using the TGFß1 Emax Immunoassay System (Promega Corp., Madison, WI), which measures the level of biologically active TGFß1. Active TGFß1 was directly measured in cell culture media, and latent TGFß1 was indirectly determined after acid activation (0.1 N HCl) to convert any latent TGFß1 to the active form. All samples from conditioned media were assayed before and after acid activation to determine the amount of biologically active and total (latent plus biologically active) TGFß1, respectively. All samples from each experiment were processed in duplicate in the same assay. The lowest detection limit was 25 pg/mL TGFß1. Cross-reaction with TGFß2 and TGFß3 at 10 ng/mL was 5% or less. The interassay coefficient of variation for the TGFß1 method ranged from 6–11%. The intraassay coefficient of variation ranged from 3.2–5.1%. Results (nanograms per mL) were obtained by determining the mean value in culture medium collected from untreated control and DHT (10-11–10-8 mol/L)-treated cells in at least seven independent experiments.
DNA synthesis
The rate of [3H]thy incorporation was determined as previously described (13). NCI-H295 cells were plated in 24-multiwell plates (105 cells/well) and incubated for 24 h in a medium supplemented with DCT-FCS in the presence of [3H]thy (1.5 µCi/mL; 87 Ci/mmol) with or without TGFß1 at concentrations from 10-9–10-6 mol/L. In assays blocking the effects of DHT-induced TGFß1, NCI-H295 cells were treated with or without DHT (10-10 and 10-9 mol/L) and with or without 0.2 µg/mL TGFß1 neutralizing antibody (anti-TGFß1 pAB, Promega Corp.). A similar concentration of normal rabbit IgG was used as a negative control.
After incubation, the cells were washed three times with ice-cold PBS and twice with 10% ice-cold trichloroacetic acid (TCA). TCA-precipitable material was solubilized in 500 µL 0.2 mol/L sodium hydroxide and 0.1% SDS. Cell-associated radioactivity was then counted in a scintillation spectrometer. Results (counts per min/well) were obtained by determining the mean value of at least seven experiments in quadruplicate. The viability of NCI-H295 cells in control and treated cultures was evaluated by trypan blue staining, and the number of viable cells was always 85–95%.
Statistical analysis
Unless otherwise indicated, the values are expressed as the mean ± SE. A preliminary analysis was carried out to determine whether the datasets conformed to a normal distribution, and a computation of homogeneity of variance was performed using Bartlett’s test. Because of the inherent variability in the datasets and the skewed nature of the distribution of the results, all values were logarithmically transformed before statistical analysis. The results were compared within each group and between groups using ANOVA. If the F values were significant (P < 0.05), Student’s paired or unpaired t test was used to evaluate individual differences between means.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
After 24 h of treatment, DHT significantly (P
< 0.001) induced TGFß1 mRNA expression in the NCI-H295
adrenocortical cancer cell line at concentrations ranging from
10-12–10-8 mol/L, with a
12- to 14-fold increase compared with the control value and in a
dose-dependent fashion (Fig. 2). The time
course (Fig. 3) of TGFß1 gene induction
by DHT (10-9 mol/L) showed that TGFß1 mRNA
levels increased time-dependently. DHT caused a significant increase in
TGFß1 mRNA expression after 4 and 12 h (P <
0.05) and after 24 h (P < 0.01).
|
|
After 24-h incubation with DHT
(10-11–10-8 mol/L; Fig. 4), the levels of biologically active
TGFß1 in the culture medium of NCI-H295 cells were significantly
(P < 0.05) higher than those detected in the culture
medium of untreated control NCI-H295 cells (7.5 ± 0.6 ng/mL),
showing a 2-fold induction (15 ± 0.9 ng/mL) at
10-10–10-8 mol/L. On the
other hand, the latent form did not significantly change during DHT
treatment.
|
Data from seven individual experiments are expressed as the
percentage over the value in untreated control cells. As indicated in
Fig. 5, [3H]thy
incorporation in NCI-H295 cells was reduced by TGFß1 treatment after
24-h incubation, reaching statistical significance at
10-7 (P < 0.01) and
10-6 (P < 0.05) mol/L (mean
inhibition, 29.3 ± 8%).
|
To investigate whether TGFß1 is involved in the growth
inhibition induced by DHT, the action of TGFß1 was blocked using a
commercially available TGFß1-neutralizing antibody. This antibody is
identical to the one used for enzyme-linked immunosorbent assay. Figure 6 shows the results of seven independent
experiments. As expected, DHT treatment significantly (P >
0.05) reduced [3H]thy incorporation in NCI-H295
cells at both 10-10 and
10-9 mol/L by nearly 50%. The administration of
DHT together with a TGFß1-neutralizing antibody was associated with a
significant (P < 0.001) increase (2.2-fold) in
[3H]thy incorporation. When the antibody was
administered alone, [3H]thy incorporation
increased significantly (P < 0.01) by nearly 50%
compared with untreated control cells. Normal rabbit IgG did not
influence the rate of DNA synthesis in NCI-H295 cells (data not
shown).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
AR, which we found to be expressed in human adrenocortical tissues and cells (7), is a member of the steroid/thyroid receptor superfamily and, as such, is a transcription factor modulating gene expression in an androgen-dependent manner. Our results indicate that in a human adrenocortical cell line, DHT is capable of up-regulating both TGFß1 mRNA and protein, suggesting a possible regulatory mechanism at the transcriptional level. However, we cannot exclude that TGFß1 expression could be regulated posttranscriptionally, as suggested by Kim et al. (21), who demonstrated that the 5'-untranslated region of TGFß1 mRNA inhibits translation. Moreover, DHT could enhance TGFß1 transcription through an indirect pathway, as no androgen-responsive elements are present in the human TGFß1 gene promoter.
The demonstration that the majority of TGFß1 detected in conditioned media from DHT-treated NCI-H295 cells was biologically active, provides evidence that NCI-H295 cells are capable of activating TGFß1 protein in vitro. These results are in line with previous in vitro data showing that a significant fraction of TGFß1 secreted by cultured human cells is in the biologically active form (6).
We previously documented the presence of type II TGFß receptor in the human adrenocortical cancer cell line, NCI-H295, and that its expression can be enhanced by DHT treatment (8). This finding is consistent with the hypothesis of a TGFß1-mediated effect of DHT on adrenocortical gland proliferation and may provide the basis for an autocrine/paracrine role for TGFß1 in this model. In the present study we found that TGFß1 significantly reduced [3H]thy incorporation in NCI-H295 cells. As [3H]thy incorporation in the cells may be considered an indirect parameter of DNA synthesis, these results suggest that TGFß1 may exert an inhibitory action on adrenocortical cell DNA synthesis. The addition of TGFß1-neutralizing antibody to DHT-treated and untreated NCI-H295 cells produced a higher [3H]thy incorporation rate, indicating that the antibody counteracts the effect of both DHT-induced and constitutive TGFß1. This finding is in line with our data demonstrating that NCI-H295 cells constitutively produce TGFß1, which becomes a likely candidate for autocrine cell growth control. The demonstration that DHT enhances this putative autocrine feedback circuit could explain at least in part the inhibitory action of androgen on human adrenocortical cell proliferation. Furthermore, the normal human adrenal gland is known to express TGFß1, which inhibits human fetal adrenal growth (5). This provides the basis for the hypothesis that TGFß1 may exert autocrine/paracrine control on normal adrenocortical cell proliferation. Our experimental model cannot determine to what extent androgen could influence this system in the normal gland.
The demonstration of an antiproliferative action of TGFß1 on human adrenocortical cells is in accordance with previous evidence that this factor inhibits fetal and definitive adrenocortical cell proliferation (22, 23), possibly hampering the progression through the G1 phase of the cell cycle. Moreover, as recently described in the rat testis (24) and human endometrial epithelial cells (25), TGFß1 could reduce cell growth, triggering an apoptotic signal in adrenocortical cells. Szelei et al. (26) found that natural and synthetic androgens prevent MCF7 cells, stably transfected with a full human AR vector, from proliferating. The same effect was observed when these cells were cultured with serum-supplemented medium, suggesting that sex steroid target cells may be sensitive to a serum-borne component that conveys an inhibitory signal when cells enter the cell cycle. TGFß1 is highly represented in serum; therefore, we can argue that it could be an important component of the inhibitory complex influencing AR-positive cell proliferation. Besides, TGFß1 has been shown to inhibit epithelial cell growth in the absence of serum (27) in concordance with our data. This could suggest that TGFß1 does not require another serum-borne factor to be active. At the same time, up-regulation of collagen and matrix protein synthesis by the secreted TGFß1 could influence the proliferative rate of adrenocortical cells in vivo.
Recently, Boccuzzi et al. (28) showed by immunohistochemistry that adrenal carcinoma has very faint, if any, TGFß1 expression, whereas normal adrenal gland exhibits variable degrees of intensity of TGFß1 staining in the different layers. This would be in line with the idea that TGFß1 modulates adrenocortical cell proliferation and that the lack of TGFß1 expression might be correlated with more undifferentiated and highly proliferative pattern of neoplasia, whereas well differentiated and normally cycling cells express this cytokine at apparently detectable levels. By contrast, we found that the NCI-H295 cell line, derived from a human adrenocortical carcinoma, spontaneously produces quite high amounts of TGFß1, and that immunoneutralization of TGFß1 by a specific antibody is associated with greater [3H]thy incorporation in these cells. At present we are not able to exclude that other factors could influence the proliferation of adrenocortical cells in this model.
In conclusion, this study indicates that DHT treatment is associated with an increase in TGFß1 expression and production and that TGFß1 reduces the proliferation of the adrenocortical cancer cell line in vitro. We speculate that TGFß1 could account at least in part for the inhibitory effect of DHT on human adrenocortical cell growth that we have previously demonstrated (7).
|
Footnotes |
|---|
Received September 17, 1999.
Revised October 21, 1999.
Accepted October 26, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  L. S. Kirschner Emerging Treatment Strategies for Adrenocortical Carcinoma: A New Hope J. Clin. Endocrinol. Metab., January 1, 2006; 91(1): 14 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. P. Ng, B. Hinz, and M. A. Swartz Interstitial fluid flow induces myofibroblast differentiation and collagen alignment in vitro J. Cell Sci., October 15, 2005; 118(20): 4731 - 4739. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Zatelli, A. Luchin, D. Piccin, F. Tagliati, A. Bottoni, C. Vignali, M. Bondanelli, and E. C. degli Uberti Cyclooxygenase-2 Inhibitors Reverse Chemoresistance Phenotype in Medullary Thyroid Carcinoma by a Permeability Glycoprotein-Mediated Mechanism J. Clin. Endocrinol. Metab., October 1, 2005; 90(10): 5754 - 5760. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lafont, M. Laurent, H. Thibout, F. Lallemand, Y. Le Bouc, A. Atfi, and C. Martinerie The Expression of novH in Adrenocortical Cells Is Down-regulated by TGFbeta 1 through c-Jun in a Smad-independent Manner J. Biol. Chem., October 18, 2002; 277(43): 41220 - 41229. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Zatelli, F. Tagliati, J. E. Taylor, R. Rossi, M. D. Culler, and E. C. degli Uberti Somatostatin Receptor Subtypes 2 and 5 Differentially Affect Proliferation in Vitro of the Human Medullary Thyroid Carcinoma Cell Line TT J. Clin. Endocrinol. Metab., May 1, 2001; 86(5): 2161 - 2169. [Abstract] [Full Text]
|
|
|
|
|
|
J. E. Chipuk, S. C. Cornelius, N. J. Pultz, J. S. Jorgensen, M. J. Bonham, S.-J. Kim, and D. Danielpour The Androgen Receptor Represses Transforming Growth Factor-beta Signaling through Interaction with Smad3 J. Biol. Chem., January 4, 2002; 277(2): 1240 - 1248. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
