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Department of Laboratory Medicine and Pathology, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Dr. R. Lloyd, Department of Laboratory Medicine and Pathology, 200 First Street, SW, Rochester, Minnesota 55905.
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Abstract |
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Immunocytochemical and RT-PCR analyses showed expression of all three D-type cyclins in the normal pituitary, with higher percentages of positive cells by immunocytochemistry in the nuclei of normal pituitaries (D1, 20–30%; D2, 50–60%; D3, 70–80%), compared with GH3 cells. In the normal pituitary, there were significantly higher levels of cyclins D2 and D3 in PRL, GH, LH, and TSH cells, compared with ACTH cells. Cyclin D1 protein was not detected in GH3 cells, while D2 was present in less than 1 percent and D3 in 10–15 percent of GH3 cells. There were low levels of cyclin D1 and D2 messenger RNA expression in GH3 cells, by RT-PCR.
When dissociated rat pituitary cells were cultured in the presence of basic fibroblast growth factor (5.6 nM) for 3 days, cyclin D2 was up-regulated 2-fold in normal PRL cells (control, 33 ± 1%; treated, 68 ± 2%). Similarly, bFGF treatment stimulated cyclin D3 expression 3-fold in GH3 cells (control, 15 ± 1%; treated, 44 ± 1%). Treatment of GH3 cells with 5-aza-2'-deoxycytidine, which induces gene demethylation, produced marked increases in cyclin D2 and D3 expression. Transfection of mouse cyclin D1 complementary DNA, driven by a cytomegalovirus promoter into GH3 cells, led to ectopic cyclin D1 expression; and there was a slight stimulation of cell proliferation and increased apoptosis in GH3 cells.
These results indicate that there is a differential expression of various D-type cyclins in different types of normal pituitary cells and between normal pituitary and GH3 cells. Growth factors, such as bFGF and demethylation increased D-type cyclin expression, whereas ectopic overexpression of cyclin D1 stimulates cell proliferation and increases apoptosis in GH3 pituitary tumor cells.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Rearrangement of the cyclin D1 gene has been reported in parathyroid adenomas and in B cell lymphomas, whereas gene amplification occurs in a subset of other malignancies, including carcinomas of breast, esophagus, head and neck, colon, liver, and lung (8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Human cyclin D1 (PRAD1) is involved in translocations observed in some parathyroid tumors (2, 3, 9). Cyclin D1 and D2 are also involved in the response to growth factor stimulation in a mouse macrophage cell line (27). Microinjection of cyclin D1 antibodies or antisense plasmids prevents passage of cells through G1, whereas overexpression of cyclin D1 shortens the G1 phase of the cell cycle (28, 29, 30). Similarly, overexpression of cyclin D2 and D3 shortens the G1 phase. The increase in cyclin D messenger RNA (mRNA) correlated with increased protein levels and preceded entry into S phase (28, 29, 30, 31, 32). D-type cyclins remain undefined in many respects, including subcellular localization and expression in many normal and neoplastic endocrine tissues such as the pituitary. Because the growth and differentiation of pituitary cells are regulated by various hormones and growth factors, such as transforming growth factor ß 1 (TGFß1) and bFGF, we analyzed the expression and regulation of D-type cyclins in normal and neoplastic rat pituitary cells to determine the possible role of D cyclins in pituitary tumor development. We observed that a differential distribution of D-type cyclins in normal and neoplastic pituitary exists and that D-type cyclins are regulated by growth factors, such as bFGF, in both normal and neoplastic pituitary.
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Materials and Methods |
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T3–1 was obtained from Dr. P. Mellon (University of
California, San Diego, La Jolla, CA). The GH-releasing hormone-CL1
(GHRH-CL1) cell line was developed in our laboratory from a GHRH
transgenic mouse pituitary tumor (33). TGFß1 from porcine platelet
was purchased from R&D Systems (Minneapolis, MN).
5-aza-2'-deoxycytidine was obtained from Sigma (St. Louis, MO).
Recombinant human bFGF was from Promega (Madison, WI). DMEM,
penicillin-streptomycin-fungizone, horse serum, FCS, HBSS, and RNA
TRIzol were purchased from Life Technologies (Grand Island, NY).
Cell culture
Normal rat anterior pituitaries were dissociated with 0.25%
trypsin, as previously described (34). Between 0.5 and 1 x
106 cells were obtained from each pituitary. Both normal
pituitary and the GH3 cell line were grown in DMEM
supplemented with 15% horse serum, 2.5% FCS, 1 ug/ml insulin, and 1%
antibiotics (100 U/ml penicillin, 100 ug/ml streptomycin, and 0.25
ug/ml fungizone). At the start of each experiment, 1 x
106 normal pituitary cells were plated in 35-mm dishes and
grown in DMEM for 2 days. The cells were then incubated in serum-free
DMEM without phenol red (Life Technologies), supplemented with 1
x ITS (insulin, 6.25 ug/ml; transferrin, 6.25 ug/ml; selenium, 6.25
ng/ml; BSA, 1.25 mg/ml; and linoleic acid, 5.35 ug/ml; Collaborative
Research, Bedford, MA). Additional supplements included 5
nM dexamethasone, 30 pM triiodothyronine
(Collaborative Research), and 1% antibiotics. Normal pituitary and
GH3 cells were treated with 5.6 nM bFGF and 1
nM TGFß1 for 3 days at 37 C in an atmosphere of 5%
CO2-95% air. These concentrations of bFGF and TGFß were
based on previous titration experiments to determine the optimal
concentrations in cultured pituitary cells (35). Aliquots of cells were
used to make cytospins (1 x 105 cells/slide), and the
remainder(4–5 x 106 cells/group) was used for RNA
extraction. Total RNA was extracted using a TRIzol reagent kit, as
recommended by the manufacturer (36).
Immunocytochemistry (ICC)
Dispersed cells were attached to
poly-L-lysine-coated glass slides by cytocentrifugation and
then fixed in 4% phosphate-buffered paraformaldehyde (pH 7.2) for 20
min. Antisera to rat pituitary hormones, PRL (used at a 1:4,000
dilution), GH (1:10,000), TSH (1:2,000), and LH (1:2,000) were obtained
from the National Pituitary Agency (Baltimore, MD). ACTH antiserum
(1:2,000) was purchased from Dako (Carpinteria, CA). The slides were
double-immunostained, as previously reported, using the
avidin-biotin-peroxidase and alkaline phosphatase kit (Vector,
Burligame, CA) methods (35). Monoclonal antibodies to cyclin D1, D2,
and D3 (Neomarkers, Fremont, CA) were used at a 1:500 dilution. Before
incubation with the cyclin D antibodies, the slides with pituitary
cells were microwaved for 5 min in 10 mM citric acid, pH
6.0. Immunostaining for cyclin was detected with
avidin-biotin-peroxidase conjugate. Detection of pituitary hormones was
accomplished with an avidin-biotin-alkaline phosphatase kit (Vector),
which was developed with nitroblue tetrazolium
chloride/5-bromo-4-chloro-3-indolyl phosphate. Negative control slides,
in which PBS was substituted for the primary antibodies, did not show
any staining. Positive cells were enumerated by counting a minimum of
1,000 cells per slide, and the results were expressed as the percentage
of each cell type determined by ICC.
Northern hybridization
The cyclin D complementary DNA (cDNA) clones used in this study
were obtained from Dr. C. J. Sherr (St. Judes, Memphis, TN). The
antisense RNA probe for Northern hybridization was generated in the
following manner. Cyclin D3 (pcN2.2-CYL3) was linearized with
HincII and transcribed with T3 RNA polymerase
for antisense probe. Transcription was carried out following the
manufacturer’s suggested protocols (Promega). Probes used for Northern
analysis were labeled with 32P-UTP (DuPont). RNA samples
(30 ug/lane) were electrophoresed on denaturing 1% agarose
formaldehyde gels. RNA was transferred to Nylon filters and baked for
1 h under vacuum at 80 C. Hybridizations with
32P-labeled riboprobe were performed according to
previously reported methods (37). Filters were washed at a final
stringency of 0.2 x SSC/0.1% SDS at 80 C for 2 h and
exposed to Kodak Omat-AR film (Eastman Kodak, Rochester, NY) with
intensifying screens at -70 C. The 0.24–9.5-kilobase (kb) RNA markers
were used as size standards to determine the size of each transcript.
To assess equivalent loading of RNA in the Northern blots, a
32P-labeled ß-actin oligonucleotide probe was used to
detect ß-actin mRNA. The amounts of cyclin D and ß-actin mRNAs were
quantitated by densitometry. Cyclin D3 mRNA level was expressed as
ratio relative to ß-actin.
GH3 cell synchronization
GH3 cells were synchronized in G1 phase by
incubating in DMEM medium containing aphidicolin (Sigma). After serum
stimulation for 20 h, aphidicolin was added to a final
concentration of 1 ug/ml. Incubation for 28 h led to the
accumulation of cells at the G1/S phase transition. After 28 h,
the medium containing aphidicolin was removed, and the cells were
rinsed twice with HBSS (15 min at 37 C, then 5 min at room
temperature). Fresh medium was added, and the cells progressed through
the cell cycle. The cells were harvested at different time periods, and
cell pellets were either stored at -70 C for RNA analysis or
resuspended for cell cycle analysis.
Cell cycle analysis
GH3 cells (5–10 x 106) were lysed
by resuspending in 0.2 ml of cold NIM buffer (0.01 M PBS,
pH 7.5, containing 1 mM CaCl2, 0.5
mM MgSO4, 0.2% BSA, and 0.6% Nonidet P-40).
The samples could be stored in this buffer at 4 C for up to 48 h
without significant degradation of the nuclei. The nuclei were purified
by centrifuging at 1500 x g through a 0.5-ml cushion
of NIM plus 5% BSA for 10 min at 4 C. The pelleted nuclei were
resuspended in 0.5 ml cold NIM, passed through a 25-gauge needle twice,
and filtered through 20 um nylon mesh to remove clumped nuclei.
Propidium iodide (PI) (50 mg/ml stock) was added to a final
concentration of 50 ug/ml. The nuclei were allowed to stain at least 30
min at 4 C before flow cytometric analysis. The fluorescence and size
of individual nuclei were measured with a flow cytometer (Becton,
Dickinson, CA). Analysis of computer-generated histograms, which
correlated fluorescence with DNA content, resulted in estimates of cell
cycle distribution. The G1 coefficient of variance for asynchronous
GH3 cell nuclei was approximate 3%, by this staining
protocol.
5-Aza-2'-deoxycytidine treatment
In some experiments, GH3 cells were treated with
different concentrations of 5-aza-2'-deoxycytidine (1, 5, and 10
µM) for 3 days, then the cells were harvested and cyclin
D expression was examined by ICC.
RT-PCR
First-strand cDNA was prepared from total RNA by using a
first-strand synthesis kit (Stratagene, La Jolla, CA), as previously
reported (35).
For semiquantitation of cyclin D1, D2, and D3 mRNA, the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control (38), which was coamplified with each cyclin in the same reactions (the sequences of primers and hybridization probes included: Cyclin D1: sense, 5-CGCCTTCCGTTTCTTACTTCA-3' (246–266); antisense, 5-AACTTCTCGGCAGTCAGGGGA-3' (476–496); internal probe, 5-CGCAGACCTCTAGCATCCAGGTGGCCACGA-3' (301–330) (39); Cyclin D2: sense, 5-CATTGAGCACATCCTACGCAA-3' (654–674); antisense, 5-CATTCACTTCCTCGTCCTGCT-3' (821–841); internal probe, 5-CGCAGATGGCTGCTCCCACGCTTCCAGTTGC-3' (784–814) (40); Cyclin D3: sense, 5-GCGTCCCCACCCGAAAGGCG-3' (351–370); antisense, 5-TAGAGCAGGCACCCAGGCCT-3' (718–737); internal probe, 5-CCAGTGCCTGCCGGTCACTGGGCAGAGAGA-3 (582–611) (40); and GAPDH: sense, 5-ATGGTGAAGGTCGGTGTGAACG-3' (72–93); antisense, 5-GTTGTCATGGATGACCTTGGCC-3' (545–566); internal probe, 5-CTTGCCGTGGGTAGAGTCATACTGGAACAT-3' (201–230) (41). PCR was performed in 100 ul final reaction vols containing 5 ul RT reaction product as template DNA, corresponding to cDNA synthesized from 500 ng total RNA, 1 x PCR buffer, 1.5 mM MgCl2, 0.2 mM of each deoxynucleotide, 300 ng for cyclins and 50 ng for GAPDH of each sense and antisense primer and 2.5 U Taq DNA polymerase (Promega). Programmable temperature cycling (Perkin-Elmer/Cetus 480, Norwalk, CT) was performed with the following cycle profile: 95 C for 5 min, followed by 30 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min. After the last cycle, the elongation step was extended by 10 min. To ensure coamplification within the linear range for both cyclin and GAPDH, cDNA titration was checked after 30 cycles, and the concentration of GAPDH primer in the reaction was adjusted to 50 ng/100 ul.
PCR product was analyzed by 2% agarose gel electrophoresis. 
X174
DNA digested with HaeIII was used as the molecular weight
standard. The separated PCR amplification products were transferred to
nylon membrane filters, and Southern hybridization with internal probes
that recognized regions within the amplified sequences was performed.
Hybridization was performed with 1 x 106 cpm/ml
33P dATP-labeled probe, and autoradiography was performed
at -70 C with Kodak Omat-AR film (Eastman Kodak) with intensifying
screens. Scanning densitometry of the autoradiogram was done with a
CS9000U densitometer (Shimadzu Corp., Tokyo, Japan). The results were
expressed, relative to the GAPDH internal control.
Analysis of various concentrations of cyclins and GAPDH cDNA was performed to ensure amplification in the linear portion of the curve. The linearity of densitometric analysis of the Southern hybridization products was determined using varying concentrations of PCR products and different exposure periods.
Generation of cyclin D1 plasmid and transfection
The 1.3-kb mouse cyclin D1 cDNA (pcBZ05.4-CYL1) that contains
the entire coding sequence was subcloned in its sense orientation into
the HindIII-XbaI sites of the pBK/cytomegalovirus
(pBK/CMV) plasmid (Stratagene). The cyclin D1 plasmid and control
plasmid (pBK/CMV) were transfected into GH3 cells using
lipofection. Approximately 0.5 x 106 GH3
cells in 2 ml DMEM were transfected with 6 ug plasmid DNA and 20 ug
lipofectin for 8 h, after which the cells were grown in fresh
complete DMEM for 2 days. Clones were subsequently selected in the
presence of 600 ug/ml G418 (Geneticin; Life Technologies) for 3 weeks.
Detection of cyclin D1 expression in GH3 cells was done by
ICC.
Detection of apoptosis
Apoptotic GH3 cells were detected in situ
with paraformaldehyde-fixed cytospin slides by 3'-end labeling of
genomic DNA with terminal deoxynucleotide transferase [TUNEL]
reaction from Boehringer Mannheim. Fluorescein-linked nucleotides,
incorporated into DNA breaks, were visualized by an alkaline
phosphatase detection system. In negative controls, terminal
deoxynucleotide transferase was omitted from reaction mixture, and some
samples were pretreated with deoxyribonuclease, which resulted in no
positive staining. The percentage of cells showing apoptosis was
determined by counting 3000 cells per slide.
Statistics analysis
Results represent a minimum of three independent experiments
using three or more replicates per treatment group. Statistical
analyses were done using the Student’s t test. Results were
expressed as the mean ± SE of the mean.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Immunostaining localized cyclin D1,
D2, and D3 proteins in normal pituitary. There was strong
immunoreactivity for all three D-type cyclins in the nuclei of normal
pituitaries. Double-staining with hormone and cyclin antibodies showed
a differential distribution of D cyclins in normal anterior pituitary
cells. Cyclin D1 constituted only a small percentage (3–5%) of
hormone-producing cells, and most of the positive immunoreactivity was
present in stromal cells, such as fibroblast and folliculo-stellate
cells, whereas D2 and D3 had higher levels in PRL, GH, LH, and TSH
cells, with significantly lower levels in ACTH cells (Figs. 2 and 3).
Cyclin D1 protein was not detected in GH3 cells by ICC. D2
was present in less than 1 percent and D3 between 10–15 percent of
GH3 cells.
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T3–1, expressed only cyclin D3 by RT-PCR, whereas both
cyclin D1 and D3 were detected in AtT 20 cells (Fig. 4). Fibroblast cells expressed all three
D-type cyclins assessed by both RT-PCR (Fig. 4) and ICC (data not
shown). These observations revealed differential expression of these
cyclins in different cell lines and suggested lineage-specific
differences.
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, 6, and 7).
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and 9). Northern hybridization with cyclin D3
RNA probe showed a 2.3-kb band, and the level of expression changed
during the cell cycle (Fig. 9). Cyclin D1 and D2 were not detected in
GH3 cells by Northern hybridization (data not shown).
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). Cyclin
D3 expression was up-regulated by bFGF (data not shown).
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). A 3-fold increase in the percentage
of immunoreactive PRL cells was also present with 5 µM
5-aza-2'-deoxycytidine (control, 3.3 ± 0.3%; treated, 10.3
± 0.9%; P < 0.01).
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). 3H-thymidine
incorporation demonstrated that GH3 cells transfected with
cyclin D1 transfectants had higher percentages of labeled cells,
compared with the transfectants with the control pBK/CMV vector
without insert (control pBK/CMV, 30.5 ± 0.7%; and cyclin D1
transfected cell, 35.3 ± 1.4%; P < 0.01 in
three independent experiments). Ectopic cyclin D1 expression
accelerated GH3 cell growth. There was no change in the
expression of cyclin D2 or D3 protein. We investigated whether ectopic
cyclin D1 expression induced apoptotic cell death and found that
ectopic cyclin D1 expression in GH3 cells increased the
number of cells undergoing apoptosis using the TUNEL in situ
staining method (Fig. 12) (control
pBK/CMV, 0.27 ± 0.03%; and cyclin D1 transfected cells,
0.44 ± 0.04%; P < 0.01 in three independent
experiments).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, GH3 cells, which secreted mainly PRL,
had very little cyclin D1 and D2 mRNA, compared with normal pituitary
PRL cells (which had abundant cyclin D2, as well as D3). Similarly,
other rat pituitary cell lines analyzed in these studies, including
GHRH-CL1, AtT20, and T3-1, all expressed mainly cyclin
D3, suggesting that cyclin D3 may have important roles in pituitary
cell proliferation in these neoplasms.
The role of various D cyclins in tumorigenesis has been investigated by various groups (7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19). Amplification of cyclin D1 (PRAD1) has been implicated in the pathogenesis of various tumors, including parathyroid adenomas (43) and breast carcinomas (15, 16, 22, 24). Interestingly, although cyclin D1 has been implicated in the development of various human malignancies, cyclins D2 and D3 have not been implicated in the development of human neoplasms. A recent study of mice with a disrupted cyclin D2 gene led to hypoplasia of the ovaries and testes. In this same study, some human ovarian and testicular tumors were found to have overexpression of cyclin D2 mRNA (44).
Our observation that bFGF, which is known to stimulate pituitary cell proliferation, up-regulated cyclin D2 in normal pituitary and cyclin D3 in GH3 cell, respectively, indicates that specific growth factors can regulate the cell cycle through the D cyclin proteins. It is possible that bFGF also stimulated cyclin D3 in normal pituitaries; however, the basal levels of cyclin D3 were very high, so slight increases could not be detected with the ICC assay.
Increased expression of cyclins D2 and D3, after demethylation induced by 5-aza-2'-deoxycytidine, suggests that methylation plays an important role in the expression of cyclin D in GH3 cells. Previous studies have shown that demethylation increased PRL and GH expressions in anterior pituitary cells (37, 45). More recent studies found that in some lymphoma cell lines, demethylation increased cyclin D2 expression (46), which is similar to our observations in pituitary cells.
The percentage of GH3 cells expressing immunoreactive PRL was relatively low but was increased 3-fold by 5-aza-2'-deoxycytidine treatment. The low levels of cells expressing PRL hormone may be related, in part, to the sensitivity of the assay, because a previous study detected more than a 4-fold increase in cells with PRL mRNA, compared with PRL protein (47). The failure of ICC to detect cyclin D3 in all GH3 cells may also be attributable to the lower sensitivity of the ICC assay, as well as to heterogeneity of the GH3 cells in expressing cyclin D3.
The experiments with synchronized GH3 cells suggest that expression of cyclin D3 mRNA and protein were induced in the G1 phase of the cell cycle. Cyclin D3 expression exhibited cell cycle periodicity. Expression of cyclin D3 peaked early in the G1 phase of the GH3 cell cycle after growth factor induction and varied only minimally throughout the remainder of the cell cycle. High concentrations of TGFß1 (1 nM) had no effect on the D-type cyclins in normal rat pituitary and GH3 cells but inhibited GH3 cell proliferation and prolonged the S phase of the cell cycle. Our previous studies indicated that TGFß1 had a biphasic effect on normal pituitary cell proliferation with inhibition at higher concentrations (1 nM) and stimulation at lower concentrations (0.1 pM) (35). The present results indicate that TGFß1 does not have a direct regulatory effect on the D cyclins. The mode of action of TGFß1 on cell cycle regulation in the pituitary suggests a complex interaction with various inhibitory and stimulatory proteins that are involved in cell cycle progression. TGFß1 inhibited growth of Mv1Lu epithelial cells in late G1 by preventing formation of active cyclin E-cdk2 complexes (3).
In the present study, transfected cyclin D1 accelerated GH3 cell cycle progression. Cyclin D1 overexpression in rodent fibroblasts led to a shortening of the fraction of cells in the G1 phase of the cell cycle, which is consistent with its role in enhancing G1-to-S progression (28, 29, 31). Previous studies with antisense cyclin D1 RNA expression from transfected cell lines inhibited the tumor growth and tumorigenicity in esophageal malignant cell lines (30). Ectopic cyclin D1 expression in GH3 cells did not induce endogenous cyclin D2 or change cyclin D3 expression. Our results indicate that G1-to-S phase transition does not require all three D-type cyclins and that the D cyclins have overlapping functions, because ectopic expression of cyclin D1 accelerated cell cycle progression. Moreover, ectopic cyclin D1 also induced apoptosis in GH3 cells, indicating that high levels of cyclin D1 in transfected GH3 cells had multiple effects. Proliferation and apoptosis may be regarded as related phenomena, with moderate ectopic expression of cyclin D1, resulting in growth stimulation; whereas overexpression in some cells may lead to apoptotic cell death. Transfection of cyclin D1 in neurons also induced apoptosis in vitro (48), which supports our observations in GH3 cells.
In summary, there are significant differences in D cyclin expressed in normal rat pituitary, compared with GH3 and other pituitary tumor cell lines. Our results indicate that during pituitary tumorigenesis, there are changes in the pattern of D-type cyclins expression and that ectopic overexpression of cyclin D1 by transfection experiments can stimulate both cell proliferation and apoptosis in GH3 cells.
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Footnotes |
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Received October 29, 1997.
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