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Max Planck Institute of Psychiatry (U.P., E.C., F.L., D.S., G.K.S.), 80804 Munich; and the Departments of Neurology, Technical University of Munich (T.A.), 81675 Munich, Germany; Centre National de la Recherche Scientifique (L.J., D.S.), 34094 Montpellier Cedex 05, France; and the Institute of Semeiotica Medica, University of Padua (C.P.), Padua, Italy
Address all correspondence and requests for reprints to: Uberto Pagotto, M.D., Department of Endocrinology, Max Planck Institute of Psychiatry, Kraepelinstrasse 10, 80804 Munich, Germany. E-mail: pagotto{at}mpipsykl.mpg.de
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the impact of Zac1 in pituitary cell proliferation, we ablated the endogenous Zac1 gene by antisense treatment in two murine cell types, AtT-20 and TtT/GF, that are representative of granular and agranular cell lineages, respectively. The decline in Zac1 protein levels under antisense treatment was accompanied by increased DNA synthesis in clonal corticotroph and folliculo-stellate cells, as demonstrated by enhanced [3H]thymidine incorporation (36% and 50%, respectively). Antisense oligonucleotides against Zac1 controlled cell proliferation in a dose-dependent way, and mutagenized antisense oligonucleotides were inert. Conclusively, our data provide the first evidence of a role for Zac1 in pituitary growth control.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We recently reported the isolation of a new gene from a mouse corticotroph library designated Zac1, which encodes a seven-zinc finger protein inducing concomitantly apoptosis and cell cycle arrest (14). Expression of Zac1 prevented the proliferation of tumor cells as measured by colony formation, growth rate, and cloning in soft agar, and precluded tumor formation in nude mice (14). Furthermore, Zac1 was demonstrated to induce the expression of pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PR1) in different cell lines (15).
Interestingly, Hamilton and colleagues independently identified a rat complementary DNA closely related to mouse Zac1 (90% homology) through its loss of expression in a rat model of epithelial ovary transformation. Accordingly, they named this gene LOT1 for "lost on transformation" (16). Moreover, the same researchers found a marked reduction of expression of the human LOT1 gene (85% homology to mouse Zac1) in human ovary cancer cell lines (17). Together, the functional properties of Zac1 and the loss of expression of its human counterpart suggest a growth-restraining function in differentiation and tumorigenesis.
To gain further insight into the role of Zac1 in the pituitary gland, we determined the regional and cellular expression pattern of Zac1 in the mouse pituitary using in situ hybridization (ISH). To identify the anterior pituitary cell type(s) that contains Zac1 transcripts, ISH was combined with immunohistochemistry (IHC) using antisera against anterior pituitary cell markers. Moreover, we investigated the effect of Zac1 on growth by inhibiting the endogenous Zac1 gene expression using antisense (AS) oligodeoxynucleotides (ODNs) in two murine clonal cells, representative of hormone-secreting or nonsecreting cells, corticotroph (AtT-20) and folliculo-stellate (FS)(TtT/GF) cells, respectively.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ISH
ISH was performed as previously described with minor
modifications (18). In brief, 8-µm pituitary sections were cut in a
cryostat, thaw-mounted onto sterile slides, postfixed in 4%
paraformaldehyde, and stored in 96% ethanol until use. Five different
ODNs (Pharmacia Biotech Benelux, Roosendaal, The
Netherlands) were selected for their ability to hybridize to the zinc
finger, the linker, the proline repeat, and the carboxyl-terminal
region of mouse Zac1-coding sequence (EMBL accession no. X95503) (14)
and were named accordingly to their location: ZF [nucleotides (nt)
238–282], L (nt 769–813), PR (nt 895–939), CT1 (nt 1156–1200), and
CT2 (nt 1311–1355). The sequences are presented in Table 1
. ODNs were 3'-end labeled with
[
-33P]deoxy-ATP (NEN, Cologne, Germany) by terminal
transferase (Boehringer Mannheim, Mannheim, Germany). After
hybridization and washing, sections were exposed to Betamax Hyperfilms
(Amersham International, Aylesbury, UK) for 14 days or dipped in Ilford
K5 photoemulsion (Ilford, Dreieich, Germany) and developed after
28 days. For a negative control, a 100-fold excess of nonlabeled ODNs
was added to the radioactive probe.
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Quantification of hybridization signals at the cellular level in
immunohistochemically identified pituitary cells
Slide preparations of three different colocalization experiments
for GH, PRL, ACTH, LH, TSH, FSH, and S-100 in mouse pituitary were
selected for signal quantification, and 10 different areas for each
experiment were scanned with a ProgRes 3012 Mikroscancamera (Imagine,
Munich, Germany) connected to a DMRD Leica Corp.
microscope (Leica Corp., Bensheim, Germany) in a
standardized manner at a resolution of 300 pixels/in. For
quantification of hybridization signals, the Optimas 5.2 computer
program (Optimas Corp., East Aspen, MT) was used. A threshold range for
gray values was set in brightfield images recognizing only
immunopositive structures, and their total area was automatically
determined by the program. This was followed by a determination of the
total area of silver grains inside these structures after overlaying
the corresponding darkfield image and setting a threshold range
recognizing only silver grains. For measurements of nonspecific
background, noncellular areas were manually outlined with the cursor,
and the total area of silver grains inside was determined. Two
different ratios were calculated: ratio 1, the sum of the total areas
of silver grains inside areas of immunostained structures (obtained in
all images of the same experiment for each kind of immunostaining)/the
sum of the total areas of immunostained or cellular structures; and
ratio 2, the sum of the total areas of silver grains inside selected
areas for background measurements/the sum of selected areas for
background measurements. Finally, ratio 2 was subtracted from ratio 1
to obtain background-free values.
Cell cultures
Cell culture materials and reagents were obtained from
Life Technologies (Karlsruhe, Germany), Falcon
(Heidelberg, Germany), Nunc (Wiesbaden, Germany), Seromed (Berlin,
Germany), Flow (Meckenheim, Germany), and Sigma Chemical Co. (St. Louis, MO). Stock cultures of AtT-20 and TtT/GF cells
were grown at 37 C and 5% CO2 in DMEM (pH 7.3)
supplemented with 10% (AtT-20) or 2% FCS (TtT/GF), 2 mM
glutamine, and antibiotics. Cells were seeded in 48-well plates for AS
experiments, in double slides for fluorescence experiments, or in
10-cm2 petri dishes for protein extraction. All experiments
were performed on cells in the midlog phase.
ODNs and transfection
The translation start region or its immediate vicinity of Zac1
messenger RNA (mRNA) were selected as a target to block Zac1 protein
translation. Three 20-mer AS ODNs (AS1, AS2, AS3) and three 20-mer
mutagenized (MUT) sequences (MUT1, MUT2, MUT3; phosphorothioates at 3'-
and 5'-terminals) with four mismatches maintaining the A, T, G, and C
contents were purchased from MWG-Biotech (Ebersberg, Germany). Their
sequences are shown in Table 1
. To visualize the ODN incorporation and
the intracellular distribution, the AS ODNs were 3'-labeled with
rhodamine (MWG-Biotech).
AtT-20 and TtT/GF cells were transfected by a recently reported procedure using polyethylenimine (PEI) (21). ODNs at various concentrations and PEI at 8 equivalents were first diluted in 150 mM NaCl and complexed by gently mixing the two solutions. After 10 min, the transfectant solution mixture was diluted in 450 µl DMEM without serum and applied to the cells for 2 h. Finally, the cells were rinsed and cultured with fresh DMEM supplemented with 10% (AtT-20) or 2% (TtT/GF) FCS for the studies described below. Control AtT-20 and TtT/GF were treated as described above with the ODNs omitted.
Determination of [3H]thymidine
incorporation
[3H]Thymidine incorporation was determined as
previously described with minor modifications (18). Sixteen hours after
transfection, the cells were incubated with 0.5 µCi/ml
[3H]thymidine for 3 h. The medium was then removed,
and the cells were precipitated with ice-cold 10% trichloroacetic acid
(1 h; 4 C) and washed with cold PBS. Finally, the DNA was hydrolyzed
overnight with 0.5 M NaOH-0.1% Triton X-100, and the
radioactivity was determined with a liquid scintillation counter.
Bromodeoxyuridine (BrdU) staining
BrdU staining was performed as previously described with minor
modifications (18) according to the instructions of the manufacturer
(5-Bromo-2'-Deoxy-Uridine Labeling and Detection Kit II, Boehringer
Mannheim). In brief, 16 h after transfection, the cells were
incubated with 10 µM BrdU for 45 min, medium was removed,
and cells were fixed in 70% ethanol for 30 min at -20 C. Mouse mAb
anti-BrdU (1:10) was applied, followed by antimouse Ig-alkaline
phosphatase (1:10).
Western blotting analysis
Western blotting was performed as described previously (14) with
minor modifications. In brief, AtT-20 and TtT/GF cells were harvested
in Tris-EDTA with 0.001% protease inhibitor cocktail (Sigma Chemical Co.) and 1 mM dithiothreitol and
disaggregated by passing through a 21-gauge needle. Total cellular
extracts (30 µg) were resolved on 8% SDS-PAGE gels and transferred
to nitrocellulose membrane. The blot was blocked in Tris-buffered
saline with 4% BSA and 0.05% Tween-20 for 1 h and incubated with
purified rabbit polyclonal against Zac1 (1:5000) (14) or rabbit
polyclonal against actin (1:200; Sigma Chemical Co.) for
quantification overnight at 4 C, followed by the second antibody
conjugated to horseradish peroxidase. Detection was performed using the
BM Chemiluminescence Western blotting kit (Boehringer Mannheim).
Statistics
Statistics for growth studies of AtT-20 and TtT/GF were
performed by ANOVA in combination with Scheffe’s test. Data are shown
as the mean ± SD. Each experimental condition was
performed in six replicates.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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At the regional level, very strong hybridization signals for Zac1 mRNA
were found in the anterior pituitary lobe, strong signals were found in
the intermediate lobe, and weak signals were seen in the posterior
lobe, as shown in Fig. 1a. Identical
results were obtained with ODN probes complementary to other parts of
the coding region of Zac1 (L, PR, C1, and C2), indicating that a single
class of transcripts corresponding to the mouse Zac1 gene were detected
(data not shown). Furthermore, the strong signals observed in the
anterior lobe were completely suppressed by competition with a 100-fold
excess of the corresponding unlabeled ODN (Fig. 1b), emphasizing the
specificity of the signal.
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). The number of silver grains per cell varied,
indicating that the cells differed in their content of Zac1 mRNA (Fig. 1c). This heterogeneity in Zac1 gene expression was consistently
observed in different tissue preparations and thus was unrelated to
variations in the preparation process. This finding was not confined to
the anterior lobe, but was also encountered in the intermediate lobe,
where the majority of cells contained Zac1 transcripts (Fig. 1e).
In the posterior lobe only scattered pituicytes expressed Zac1 mRNA at
low levels (Fig. 1f).
Colocalization of Zac1 mRNA and immunohistochemical markers for the
anterior pituitary cells
To identify pituitary cell populations expressing Zac1 mRNA, we
combined ISH for Zac1 with IHC for anterior pituitary hormones and
S-100.
All anterior pituitary cell types were found to contain Zac1
transcripts but at varying levels. Hybridization signals were most
intense in GH-producing (Fig. 2a) and
PRL-producing (Fig. 2b) cells; lower in LH-positive (Fig. 2c),
TSH-positive (Fig. 2d), and ACTH-positive (Fig. 2f) cells; and lowest
in FSH-producing cells (Fig. 2e). After GH- and PRL-secreting cells, FS
cells showed the highest hybridization signals for Zac1 transcripts
(Fig. 2g). The values of the quantified Zac1 mRNA levels in each
pituitary cell type are shown in Fig. 3
and are representative of one experiment. Similar results were obtained
in two other independent experiments.
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).
Briefly, three AS ODNs (AS1 to AS3) were used to investigate whether
the biological effect is reproducible with more than one AS ODN.
Accordingly, the effects of three different AS ODNs on the
proliferation of pituitary cells in midlog phase were compared with the
effects of three MUT ODN sequences (Table 1). As the outcome of a
transient knockout experiment will depend on the numbers of cells that
take up the AS ODN, we first tested the efficiency of our transfection
method. For this reason we used rhodamine-labeled ODNs and examined
their distribution in cultures exposed either to ODN alone or to
ODN/PEI complexes. With the latter condition in AtT-20 and TtT/GF
cells, we observed by fluorescence microscopy detectable signals in the
majority of the cells (Fig. 4), in
contrast to a complete absence of signal in cultures exposed solely to
rhodamine-coupled ODNs (data not shown). Counting at least 10 fields in
each of 3 different experiments, the highest transfection efficiency
(70%) was achieved for both cellular lineages when 1 µM
ODN was applied. Next, we tested the effects of all 3 AS on
[3H]thymidine incorporation in AtT-20 and TtT/GF cells.
We found that AS1 at a concentration of 1 µM ODN
increased [3H]thymidine incorporation by 36% in AtT-20
and by 50% in TtT/GF (Fig. 5, A and C),
whereas AS2 caused increases of 20% and 28%, respectively (Fig. 5, B
and D), compared with the MUT ODNs and the control. An increase in
thymidine uptake was also observed with 1 µM AS3 in both
AtT-20 and TtT/GF, but was not significant (data not shown). We then
performed a dose-response study using AS1 ODN concentrations between
0.3–1 µM with AtT-20 and between 0.3–1.5
µM with TtT/GF. AS1 ODN maximally stimulated
proliferation at 1.0 µM in AtT-20 and at 1.5
µM in TtT/GF cells. However, even at lower
concentrations, a significant effect was detectable. No significant
effect on proliferation was observed for MUT1 ODN at any of the
concentrations used (Fig. 6). BrdU
immunohistochemical staining of AtT-20 and TtT/GF cells confirmed that
the number of the nuclei incorporating BrdU was increased after
treatment with AS1 (1.0 µM) compared with MUT1 (1.0
µM) or PEI alone (Fig. 7).
Light microscopic inspection of the cells treated with the
above-mentioned AS ODN concentrations, either immediately after
transfection or 16 h before thymidine application, gave no
evidence of changes in cell morphology or reduced viability. Only at
ODN concentrations higher than 1.0 µM for AtT-20 and 1.5
µM for TtT/GF was a toxic effect noticed, and this could
be attributed to the transfectant vector PEI, as a similar decrease in
viability was seen with the equivalent PEI in controls. To examine
whether the increased DNA synthesis under AS ODNs was actually
accompanied by decreases in the levels of Zac1 protein in AtT-20 and
TtT/GF, cells were treated for 2 h with ODN/PEI, and cell extracts
were harvested for immunoblot analysis after 16 h. As shown in
Fig. 8, a clear reduction of the signal
for Zac1 protein was obtained after AS1 ODN treatment compared to that
after MUT1 ODN treatment. Densitometric measurements revealed a 62%
decrease for TtT/GF and 27% for AtT-20, indicating that the observed
decline in Zac1 protein levels correlates with the increase in DNA
synthesis under the same condition. The incomplete suppression of the
signal after AS ODN treatment was in accordance with our observation
that the majority, but not all, of the cells were transfected.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the past, the mitotic activity in the adult anterior pituitary gland has been thought to be low (22). This view has faced increasing challenge and has been highlighted in the recent work by Levy and colleagues (3), who concluded that, on the average, a pituitary cell enters mitosis (or apoptosis) once every 60–70 days, yielding a significant cellular turnover (>1.5%/day). Age, sex, and circadian differences (23, 24, 25) as well as exposure to hormones (26, 27, 28) have been reported to regulate the number of mitotic cells in anterior pituitary. Therefore, heterogeneous Zac1 mRNA levels in the anterior pituitary gland could reflect differences in the mitotic activity of individual subsets of cells.
In previous reports we have shown that Zac1 is able to induce the expression of PR1 in a panel of cell lines of various origin (15). Several peptidergic factors of hypothalamic origin are known to take part in the control of hormonal production and pituitary gland growth. Among these, PACAP acts as a hypophysiotropic factor, mainly through an activation of PR1 (for review, see Refs. 29, 30). Several studies demonstrated high expression of PR1 mRNA and protein in the anterior and intermediate lobes, in contrast to a very low expression in the posterior pituitary lobe (14, 31, 32). Moreover, using biotinylated ligands it was demonstrated that PACAP binds to all types of hormone-producing cells of the anterior pituitary gland, with the highest percentage binding to GH- and PRL-producing cells and the lowest to FSH-secreting cells (33). Our description of the regional and quantitative cellular distribution of Zac1 mRNA in the pituitary gland tightly correlates with the distribution of PR1 observed by others (14, 31, 32, 33). Thus, one may speculate that at the pituitary level Zac1 could take part in the mechanisms of action commonly attributed to PACAP, such as the ability to modify the paracrine milieu and to influence cell growth and differentiation, through the activation of PR1.
Together with the corticotroph lineage AtT-20, in which Zac1 was isolated, we found a constitutive expression of Zac1 protein in TtT/GF cells. This pituitary FS-like cell line represents a unique model to study the possible multifunctional roles of FS in regulating the extracellular environment of the anterior pituitary and in releasing paracrine factors that regulate the activity of other cell types (34).
In our previous report we demonstrated that an ectopic expression of Zac1 induces apoptosis and G1 arrest, resulting in a severe growth inhibition (14). As these studies relied on an enforced expression in a heterologous model, it remained of obvious interest to substantiate these findings in a model of physiological relevance. Based on the G1 arrest function of Zac1, we reasoned that the regulatory role of Zac1 on the cell cycle will be unmasked by an ablation of the endogenous gene, and we predicted an increased entry into the S phase under this condition. To this aim we used AS ODNs as a tool to block the expression of specific genes. The use of ODNs to modify gene expression is limited by their low cellular uptake and their rapid degradation by extracellular nucleases (35). Therefore, according to our previous experience (21), ODNs were complexed with PEI to protect them and to enhance their intracellular uptake (36). Moreover, ODNs were rendered resistant to nucleases by introducing phosphorothioate linkages. A number of recent reviews have suggested guidelines in interpreting antisense studies (37). A critical step using AS relates to the evaluation of specific gene inhibition (38). For this reason, we used mutagenized ODNs with four mismatches maintaining the same base composition as the antisense molecule. This approach represents a more stringent control than sense ODN complementary to the antisense sequence or ODN composed of a random mixture of all four nucleotides (39). In our experiments, AS1 ODN directed against Zac1 significantly stimulated DNA synthesis up to 36% and 50% in AtT20 and TtT/GF cells, respectively. Recent work indicates that ODNs may block proliferation through nonantisense mechanisms, which arise from cytotoxic ODN breakdown products. If such mechanisms are influencing our measurements, the anticipated effect would be opposite of those actually obtained, and the increases in DNA synthesis seen would therefore represent an underestimate. Furthermore, exposure to the transfection vector can be toxic, as observed in this study at high concentrations. Again, such a variable would lead to an underestimate of the actual effect and reinforces the view that our results represent a conservative estimate.
Together, these considerations strongly support a genuine mode of action of AS in our experiments, as made evident by the final net increase in [3H]thymidine incorporation, and the reproducibility of our results using two AS ODNs. Moreover, we could prove that AS treatment caused a decline in Zac1 protein levels giving further credit to a causal relation among an ablation of Zac1 mRNA, decreases in Zac1 protein, and concomitant increases in [3H]thymidine incorporation. Taking into account a transfection efficiency of 70%, the percentage of DNA synthesis increase after AS treatment was high notwithstanding the heterogeneity in the quantitative ODN uptake. In this view, the ODN rhodamine incorporation experiment also offered an explanation for the incomplete suppression of the signal for Zac1 protein as documented by immunoblot analysis.
In contrast, mouse primary pituitary cells seemed essentially refractory to transfection with ODN/PEI complexes, reaching an efficiency of transfection of not more than 5% of the cells transfected (data not shown). Thus, further studies are required to substantiate the role of Zac1 in vivo, and in this view, Zac null mutant mice will be valuable tools to follow up the present findings.
In conclusion, we provide new evidence for a role of Zac1 in pituitary proliferation, using AS treatment of pituitary cell lines. Moreover, the present analysis of Zac1 expression in each anterior pituitary gland cell population is paving the way to further studies aimed at investigation of Zac1 activity in human tumor samples. The elucidation of the interaction between epigenetic and genetic lesions in pituitary cell transformation and adenoma formation will provide important clues for the evaluation of tumor stage and advancement and for improvements in the therapeutical management.
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Acknowledgments |
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Footnotes |
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Received August 27, 1998.
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References |
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20/+ mice are predisposed to tumors
of the pituitary gland with a nearly complete penetrance. Oncogene 9:1021–1027[Medline]
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| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
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, AS1; 
and 
, MUT1. The mean ± SD are given
(n = 6). The experiments were repeated three times. Representative
results from one experiment are shown. *, P <
0.05; **, P < 0.01; ***, P <
0.001.
