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Department of Obstetrics and Gynecology (T.S., A.H., K.K., M.O., I.K., T.N.), Shinshu University School of Medicine, Matsumoto 390-8621, Japan; Department of Gynecology and Obstetrics (S.F.), Faculty of Medicine, Kyoto University, Kyoto 606-8507, Japan; and Department of Organ Regeneration (T.N.), Shinshu University Graduate School of Medicine, Matsumoto 390-8621, Japan
Address all correspondence and requests for reprints to: Toshio Nikaido, Ph.D., Department of Obstetrics and Gynecology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto 390-8621, Japan. E-mail: tnikaido{at}hsp.md.shinshu-u.ac.jp
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Introduction |
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Advances in cell cycle research have revealed that cell proliferation is regulated by the interaction between cyclins/cyclin-dependent kinases (cdks) and tumor suppressor gene products such as the retinoblastoma gene product (pRb) and cdk inhibitors (10, 11, 12). The cyclin/cdk complexes inactivate pRb, leading to cell cycle progression. Conversely, the cyclin/cdk complexes are inactivated by cdk inhibitors, resulting in growth arrest (12). p27Kip1 (p27) is a cdk inhibitor that functions as a tumor suppressor, because 1) p27 associates mainly with the cyclin E/cdk2 complex, and inhibits pRB phosphorylation via the cyclin E/cdk2 complex (13), 2) overexpression of p27 blocks cells from entering the S phase (13, 14), and 3) mice lacking p27 exhibit multiorgan hyperplasia (15, 16, 17). We previously reported that immunohistochemical expression of p27 protein in the normal endometrium is observed not in the proliferative phase but in the secretory phase and that MPA treatment induces p27 expression in endometrial hyperplasia. Therefore, p27 has been suggested to be involved in growth suppression by progestins in endometrial glandular cells (18).
In the present study, to further clarify the involvement of p27 in the progestin-mediated growth inhibition of normal and malignant endometrial epithelia, the effect of progestins on the expressions of p27 and other cell cycle-related molecules was examined using cultured normal endometrial glandular cells and endometrial carcinoma cells in vitro. Growth suppression by p27 was confirmed by a transfection experiment using a p27 expression vector. The actual function of p27 as a cdk inhibitor was examined by immunoprecipitation for the complex formation between p27 and cdk2/cyclin E. In addition, the molecular mechanism for elevation of p27 expression by progestins, either transcriptional or posttranslational, was also investigated.
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Materials and Methods |
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Cell culture
1) Endometrial carcinoma cells. Two established endometrial
cancer cell lines Ishikawa (PR-positive) and KLE (PR-negative) were
studied. Ishikawa cells (clone No. 3-H-4) were kindly provided by Dr.
Nishida at Tsukuba University. KLE cells were purchased from
ATCC (Manassas, VA). Cells were cultured in DMEM
supplemented with 15% FCS (Life Technologies, Inc., Grand
Island, NY) and antibiotics, at 37 C in a humidified atmosphere of 5%
CO2. Expression of PR in Ishikawa cells and its
absence in KLE cells were confirmed by immunohistochemistry and Western
blotting.
2) Normal endometrial glandular cells. Human endometria were obtained at hysterectomy from premenopausal women with regular menstrual cycles, aged 36–47 yr, who underwent hysterectomy for nonendometrial abnormalities such as uterine leiomyoma and cervical dysplasia. This study was approved by the Ethical Committee at Shinshu University School of Medicine, and informed consent was obtained from every patient. A portion of each endometrial specimen obtained was histologically examined and dated according to the criteria of Noyes et al. (19). In the present study, endometrial tissues obtained in the late-proliferative phase were used for all experiments except for p27 protein degradation assay, and those in the early secretory phase (d 7–17) for p27 protein degradation assay.
Normal endometrial glands were isolated and glandular cells were cultured as described previously (20, 21, 22) with some modifications. In brief, endometrial tissues were washed with PBS and minced into small pieces of less than 1 mm3. The tissues were then incubated in DMEM containing 0.25% collagenase (Wako, Osaka, Japan) and 0.005% deoxynuclease (Sigma) for 60–90 min at 37 C with continuous shaking. The resulting cell suspension was filtered through a 250-µm sieve to remove mucus and undigested tissues. The filtered cell suspension was then filtered through a 37-µm sieve. Intact glands were recovered by backwashing of mesh. Glands were then dispersed into single epithelial cells by incubation with trypsin (0.025%), EDTA (0.01%, Sigma), and deoxynuclease (0.005%) at 37 C for 5–10 min with occasional pipetting. The cell suspension was centrifuged for 3 min at 3000 rpm and resuspended in Ham’s F12 (phenol red-free, Life Technologies, Inc.) supplemented with 0.1% or 15% dextran charcoal-filtered FCS. Expressions of ER and PR were confirmed by immunohistochemistry and Western blotting.
[3H]-thymidine incorporation assay
To evaluate the growth inhibition of normal endometrial
glandular cells by progesterone, a
[3H]-thymidine incorporation assay was
performed. In brief, 5 x 103 isolated
endometrial glandular cells (obtained on d 9–12 of the menstrual
cycle) were dispersed onto type IV collagen-coated 96-well plates
(IWAKI, Chiba, Japan). The cells were cultured with Ham’s F12 + 15%
charcoal filtered FCS for 2 d after dispersion. The medium was
then changed to Ham’s F12 + 0.1% FCS for 24 h. After serum
starvation, E2 alone (10-8 M,
10-6 M) and E2
(10-6 M) + progesterone (P4
10-8 M), as well as 10 µl of
[3H]-thymidine solution (0.05 µCi/µl,
Amersham Pharmacia Biotech, Buckinghamshire, UK), were
added to the medium and incubated at 37 C for 24, 48, and 72 h.
After incubation, media were aspirated and cells were harvested using a
trypsin/EDTA solution, and their radioactivity was measured with a ß
scintillation counter. The results are indicated by the relative ratio
against the control. Eight wells were used for each treatment, and
experiments were repeated twice. Statistical analysis of the control
and MPA-treated groups was done by Scheffé’s test. A tied
P-value of less than 0.05 was considered significant.
MTT assay
To evaluate the growth inhibition of endometrial carcinoma
cells by progestins, an
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was performed. Endometrial carcinoma cells
(Ishikawa and KLE) were dispersed in 96-well plates at a density of
2 x 103 per well in 100 µl of DMEM with 0.5%
charcoal-filtered FCS. MPA was added at concentrations of 1 x
10-6 M and 1 x
10-8 M every day from the second day
of culture, and the cells were refed with fresh culture medium every
3 d. After 14 d of culture, growth inhibition was determined
by the MTT (Sigma) method. After changing the medium, 10
µl of MTT (5 mg/ml PBS) solution was added to each well. After
incubation of 4 h, the medium was aspirated, and formazan crystals
were dissolved with 50 µl of DMSO by shaking. The absorbance was
measured at 550 nm with a microplate reader (Bio-Rad Laboratories, Inc., Richmond, CA). Eight wells were used for
each treatment, and experiments were repeated twice. Growth inhibition
of each MPA concentration was described as the relative ratio of
absorbance to control groups. Statistical analysis between control and
MPA-treated groups was done by Scheffé’s test. A tied
P-value of less than 0.05 was considered significant.
Western blotting
Expressions of the p27 protein and other cell-cycle related
molecules in cultured normal and malignant endometrial cells after
progestin treatment were examined by Western blotting. Normal
endometrial glandular cells (1 x 106) were
dispersed in type IV collagen-coated 6-cm dishes and incubated with
Ham’s F12 + 15% FCS for 24 h. The medium was then changed to
Ham’s F12 + 0.1% dextran charcoal-filtered FCS. P4 was added daily at
a concentration of 10-8 and
10-6 M and incubated for 7 d.
The medium was changed every 3 d. Endometrial carcinoma cells (KLE
and Ishikawa, 1 x 105) were also dispersed
in a 6-cm dish with DMEM and 15% FCS for 24 h. The medium was
then changed to DMEM + 0.1% charcoal-filtered FCS. MPA was added daily
at concentrations of 10-8 and
10-6 M for 14 d. The medium was
changed every 3 d. After the cell culture, cells were collected
with trypsin/EDTA solution. Collected cells were homogenized and lysed
in 0.5 ml of a cell lysis buffer consisting of 50 mM
Tris-HCl (pH 8.0), 0.25 M NaCl, 0.5% NP-40, 1
mM phenylmethylsulfonyl fluoride (Sigma), 1
µg/ml aprotinin (Roche Molecular Biochemicals Co.,
Indianapolis, IN), 1 µg/ml leupeptin (Roche Molecular Biochemicals), and 20 µg/ml
N-tosyl-L-phenylalanine chloromethyl
ketone (Roche Molecular Biochemicals). The lysates
were centrifuged at 13,000 x g for 20 min at 4 C, and
the supernatants were stored at -80 C. Extracts equivalent to 50 µg
of total protein were separated by SDS-polyacrylamide gels (10%
acrylamide). The proteins were then transferred to supported
nitrocellulose membranes (Amersham Pharmacia Biotech) with
a plate electrode apparatus (Semi Dry Blotter II, Ken En Tec,
Copenhagen, Denmark) for 90 min. Filters were incubated with antibodies
against cyclin D1 (DCS-6, Progen, Heidelberg, Germany), and against
cyclin E (HE1), cdk4 (C-22), cdk2 (M2), p27 (C-19), and proliferating
cell nuclear antigen (a growth marker), all of which were purchased
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). An
antibody for ß-actin (AC-15, Biomakor, Rehovot, Israel) was used
as the internal standard. Then, the filters were incubated in
peroxidase-conjugated antimouse or rabbit IgG and the bound
antibody was detected with an enhanced chemiluminescence system
(Amersham Pharmacia Biotech). The bands were
quantitatively analyzed by densitometry.
Immunoprecipitation
Complex formation between p27 and cyclin E/cdk2 was examined by
immunoprecipitation. The isolated endometrial glandular cells and
Ishikawa cells were cultured as described in Western
blotting for 7 d with or without P4 and MPA treatment,
respectively. The cell lysates obtained from these four samples were
subject to immunoprecipitation. Briefly, 50 µg of the lysate was
immunoprecipitated with 2 µl of an anti-cdk2 antibody (Santa Cruz Biotechnology, Inc.) for 60 min at 4 C. The
cdk2-precipitates were collected for 1 h on 20 µl of protein G
plus-agarose (Oncogene, Cambridge, MA). After washing with a lysis
buffer twice, precipitates were resuspended in a Laemmli SDS sample
buffer and resolved by SDS-PAGE. The immunoprecipitated protein
complexes were resolved and probed for immunoblotting to detect
associated proteins using antibodies against p27 and cyclin E
(Santa Cruz Biotechnology, Inc.).
Northern blotting
Total RNA from the normal endometrium obtained at the
proliferative phase (two cases) and the secretory phase (two cases), as
well as Ishikawa cells cultured with MPA at concentrations of
10-8 M and
10-6 M for 14 d, were extracted
using Isogene (Wako). Fifteen micrograms of total RNA were
electrophoresed on a denaturing 1.0% agarose formaldehyde gel and
transferred to Hybond-N membranes (Amersham Pharmacia Biotech). The membranes were prehybridized at 42 C for 4 h
in 50% formamide, 5x saline-sodium phosphate-EDTA, 5x Denhardt’s
solution, 1% SDS, and 100 µg/ml denatured herring sperm DNA. For
probes, genomic DNA obtained from the normal endometrium was used as a
template, and [32P
]-deoxy-CTP (Amersham Pharmacia Biotech) was labeled by PCR using the template and
primers specific for p27 (23). After prehybridization,
filters were hybridized overnight at 42 C and washed with 0.1 x
SCC and 0.1% SDS firstly for 30 min at 42 C, then for 20 min at 55 C.
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an
internal control. The bands were analyzed using a MacBas system
(Fuji Photo Film Co., Ltd. Film, Tokyo, Japan).
p27 protein degradation assay
Endometrial glandular cells (obtained on d 14–17, 5 x
105) were dispersed on type IV collagen-coated
6-cm dishes. The cells were cultured for 2 d with Ham’s F12 +
15% FCS. The cells were washed twice with PBS and the medium was
changed to Ham’s F12 + 0.1% FCS. Cycloheximide (Sigma),
a protein synthesis inhibitor, at a concentration of 20
µM and P4 at a concentration of
10-6 M was then added to the medium.
Endometrial carcinoma cells (Ishikawa, 1 x
105) were also dispersed on 6-cm dishes with DMEM
+ 15% FCS. The cells were washed by PBS, and cycloheximide (20
µM) and MPA (10-6 M)
were also added. Both types of cells were harvested after 6, 12, and
24 h of incubation at 37 C, and the amount of p27 protein with or
without P4 or MPA was compared by Western blotting.
DNA transfection
To confirm the growth suppressive function of p27, the p27
expression vector was transfected to normal endometrial glandular
cells, and the effect on the expression of cell cycle-related molecules
was assessed by Western blotting. In brief, 1 µg of the expression
vector plasmid pcDNA3-p27 (a kind gift from Dr. Nakanishi at Nagoya
City University) was transfected to the normal glandular cells cultured
in 6 cm dishes with Ham’s F12 + 15% FCS, using the Effectene
Transfection Reagent (QIAGEN, Hilden, Germany).
Seventy-two hours after transfection, the expression of cell
cycle-related molecules in the p27-transfected cells was evaluated
using Western blotting. In addition, the effects of p27 overexpression
on growth suppression were also evaluated by
[3H]-thymidine uptake assay. In brief, 0.1 µg
of pcDNA-p27 or vector were transfected to the normal glangular cells
cultured in 96-well plate with 10 µl of
[3H]-thymidine solution using the Effectene
system. Seventy-two hours after transfection,
[3H]-thymidine uptake was counted.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The difference in
the uptake between the control and E2 (at 10-6
M)-treated group reached a significant difference with
relative ratio 1.45 ± 0.20 (P = 0.01) after
72 h of incubation. The estrogen-induced
[3H]-thymidine uptake was suppressed by the
addition of P4 at 10-8 M
after 48 and 72 h of incubation. Interestingly, however, the
[3H]-thymidine uptake slightly increased with
the addition of P4 after 24 h of incubation.
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). Relative ratio of
growth inhibition obtained by MTT assay was 0.92 ± 0.15 at
10-8 M and 0.62 ± 0.07 at
10-6 M of MPA. The difference
between the MPA (10-6 M)-treated
group and control was significant (P = 0.009). In KLE
cells which did not express PR, the growth suppression due to MPA
treatment was negligible (Fig. 2b).
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). The expression of cyclin D1, cyclin
E, cdk4 and proliferating cell nuclear antigen decreased in P4-treated
cells. The expression of cdk2 showed a slight decrease when P4 was
added at 10-6 M.
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). The expression of cyclin D1 slightly decreased in
MPA-treated cells (relative ratio; 1: 0.68: 0.75). The expressions of
cyclin E, cdk4 and cdk2 did not show any marked changes due to MPA
treatment. In KLE cells, none of the factors examined in the present
study showed significant differences among the control and MPA-treated
groups (data not shown).
Complex formation between p27 and cyclin E/cdk2
The protein extracted from the normal endometrial glandular cells
and Ishikawa cells was first immunoprecipitated by cdk2 and resolved
with anti-p27 or anti-cyclin E antibodies. In the normal glandular
cells, P4 treatment increased cdk2-bound p27 protein and slightly
decreased cdk2-bound cyclin E (Fig. 4a).
In Ishikawa cells, MPA treatment also increased cdk2-bound p27 protein
(Fig. 4b). The amount of cdk2-bound cyclin E did not show any apparent
changes in Ishikawa cells due to MPA treatment (Fig. 4b).
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). In Ishikawa cells, the expression of
p27 mRNA did not show any marked increase due to the addition of MPA
(relative p27 mRNA/GAPDH ratio of 10-8
M and 10-6
M against the control was 0.89 and 0.69,
respectively) (Fig. 5b).
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). In Ishikawa cells, a
larger amount of p27 protein was also observed in MPA-treated groups
(Fig. 6b).
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). The
[3H]-thymidine uptake of the p27-transfected
cells decreased by 57% (relative ratio; 0.43±0.35) compared with the
vector-transfected controls (P = 0.012) (Fig. 7b),
suggesting the possible involvement of p27 in the growth suppression of
endometrial glandular cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrated that the growth suppression of endometrial glandular cells by P4 treatment is associated with elevated expression of p27 protein, as well as reduced expression of cyclin D1, cyclin E, and cdk4. This is consistent with our previous report on the human endometrium in vivo, which showed elevated expression of p27 and decreased expression of cyclins/cdks in the secretory phase (18, 26). In addition, the complex formation of p27 with cdk2 and cyclin E, as indicated by immunoprecipitation, supports the functional involvement of p27. Moreover, the transfection experiment showed that forced p27 expression decreased [3H]-thymidine uptake in glandular cells. Thus, P4 suppresses the growth of normal endometrial glandular cells, possibly via the up-regulation of p27 protein, which actually functions as a cdk inhibitor. Recent reports revealed that growth inhibition by differentiation-inducing agents is often associated with the elevated expression of cdk inhibitors such as p27 and p21WAF/CIP1 (27, 28). The arrest of cell growth by vitamin D derivatives is associated with cell differentiation, as well as increased p27 expression (29, 30). In our study, however, P4 treatment not only increased p27 expression but also decreased the expression of cyclins and cdks; the down-regulation mechanism remains to be clarified. As for another important tumor suppressor p21WAF/CIP1 (p21), our research group previously reported that the expression of p21 in endometrial glands is very focal and weak in its staining intensity (31). The limited topological distribution of p21 suggests the possible involvement p21 in the apoptosis rather than the growth suppression.
In endometrial carcinoma cells, MPA treatment showed growth suppression of PR-positive Ishikawa cells in a dose-dependent fashion. This is consistent with a previous report (32). MPA is a potent differentiation inducer and has antitumor activities; it has been used to treat advanced or recurrent endometrial carcinoma with a response rate of 30–35% (2, 3). Endometrial carcinomas with PR showed a higher response rate of 80% (4, 5). The growth inhibitory mechanism of progestins on PR-positive endometrial carcinoma cells has been explained with respect to metabolizing enzymes (33), or transforming growth factors (34, 35). However, the intracellular mechanism of the growth inhibition has not fully been clarified. The present study demonstrated that growth suppression of PR-positive Ishikawa cells induced by MPA treatment was also associated with elevated expression of the p27 protein. In contrast, MPA treatment did not increase the p27 expression along with no growth suppression in PR-negative KLE cells. We previously observed the increased expression of the p27 protein in patients with endometrial hyperplasia after MPA treatment (18). In addition, the immunoprecipitation experiment showed that MPA treatment induces an increase in cdk2-bound p27 protein. These findings strongly suggest the functional involvement of p27 in the growth suppression of Ishikawa cells.
In endometrial carcinoma cells, however, the growth suppression rate due to treatment with MPA was at most approximately 38% when MPA was added at 10-6 M. Growth-suppressive effect was minimal when MPA was added at 10-8 M, although the increase of p27 expression was evident, suggesting that the p27 protein induced by 10-8 M MPA is insufficient to suppress the potent growth of Ishikawa cells. This is contrast to normal endometrial glandular cells. Down-regulation of cyclins and cdks by progestins was observed in normal endometrial glandular cells, but not in Ishikawa cells. The weaker suppression of carcinoma cells by progestins may be due to the persistent overexpression of cyclins and cdks, which does not seem to be under hormonal control. Thus, overexpressed cyclins and cdks, possibly because of genetic changes acquired during the pathogenetic process, may override the growth inhibitory function of p27, even if the p27 protein increased due to MPA treatment in Ishikawa cells.
To analyze further the mechanism of up-regulation of the p27 protein by progestins, we studied the change in p27 mRNA expression, as well as the degradation of intracellular p27 protein. The results obtained by Northern blot analysis revealed that the amount of p27 protein is not controlled at transcriptional level both in the normal and malignant endometrial cells. In addition, p27 protein degradation experiments using cycloheximide indicated that the amount of p27 protein increased with progestins. These data suggest that the intracellular p27 protein level is regulated mainly by a posttranslational mechanism. Several lines of evidence also support the posttranslational control of p27 (36, 37, 38). Recent reports showed that phosphorylation of threonine 187, or lysines of the p27 protein, is required for the ubiquitination and subsequent digestion by proteinase of this molecule (39, 40, 41). Although the exact target molecule is unknown, progestins may suppress the ubiquitin-proteasome proteolytic pathway.
In conclusion, the p27 protein is an important regulator that is functionally involved in progestin-induced growth suppression of normal and malignant endometrial epithelial cells. In addition, p27 is up-regulated by progestins possibly via posttranslational mechanisms.
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Acknowledgments |
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Footnotes |
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Abbreviations: cdks, Cyclins/cyclin-dependent kinases; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MPA, medroxyprogesterone acetate; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; P4, progesterone; pRb, retinoblastoma gene product.
Received February 14, 2001.
Accepted for publication June 21, 2001.
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