| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ARTICLES |
Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medical Sciences, Kyushu University (Y.N., T.Y., Y.-M.M., K.O., I.I., M.S., M.N., C.M., T.O., K.G., R.T., H.N.), 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan; CREST (Core Research for Evolutional Science and Technology) (T.T., K.G., R.T., H.N.) and Division of Internal Medicine, National Kokura Hospital (Y.N.), Harugaoka 10-1, Kokura-Minami-Ku 802-0803, Japan; Division of Internal Medicine, Kyushu-Rosai Hospital (M.H.), Kuzuhara-Takamatsu 1–3-1, Kokura-Minami-Ku 800-0296, Japan; and Division of Gynecology, Kyushu-Rosai Hospital (Y.K.), Kuzuhara-Takamatsu 1-3-1, Kokura-Minami-Ku 800-0296, Japan
Address all correspondence and requests for reprints to: Hajime Nawata, M.D., Ph.D., Department of Medicine and Bioregulatory Science (Third Department of Internal Medicine), Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan. E-mail: nawata{at}mailserver.med.kyushu-u.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxylated steroids, dehydroepiandrosterone, androstenedione,
or estradiol was observed. The aromatase activity of KGN was relatively
high and was further stimulated by (Bu)2cAMP or FSH. These
findings showed a pattern similar to that of steroidogenesis in human
granulosa cells, thus allowing analysis of naturally occurring
steroidogenesis in human granulosa cells. Fas-mediated apoptosis of KGN
was also observed, which mimicked the physiological regulation of
apoptosis in normal human granulosa cells. Based on these findings,
this cell line is considered to be a very useful model for
understanding the regulation of steroidogenesis, cell growth, and
apoptosis in human granulosa cells. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
During the last 2 decades, although several animal ovarian granulosa cell lines, mostly immortalized by oncogenes and oncoviruses have been reported (12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25), only five human granulosa cell lines have been established (26, 27, 28, 29, 30). These human granulosa cell lines include 1) lines established by the long-term culture of human granulosa cell tumor cell (26, 27), 2) human granulosa-lutein cells immortalized with SV40 large T antigen (28), 3) a line established by a forced introduction of the human papillomavirus gene to the primary human granulosa cells (29), and 4) a line most recently established by triplet transfection of primary human granulosa cells with the SV-40 DNA, Ha-ras oncogene, and a temperature-sensitive (ts) mutant of the p53 tumor suppresser gene (30). Although these human cell lines are useful and offer some promise of further discoveries in this field, none was reported to express functional FSH receptor.
In the present study we established a new line of human granulosa-like cells from a tumor specimen enucleated from a patient who showed a local recurrence of a granulosa cell tumor after menopause. We consider this cell line, KGN, to be very unique and useful, because it maintains most physiological activities, including the expression of functional FSH receptor, as well as the same pattern of steroidogenesis and Fas-mediated apoptosis as those observed in normal granulosa cells.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
, A and B). In December 1993, the tumor
recurred in the pelvic region. A portion of the granulosa tumor tissue
obtained at the time of reoperation in January 1994 was used as the
source of the cell culture.
|
In vitro cell morphology and growth characteristics
The morphological appearance of the KGN cells in a monolayer
culture was observed by phase difference microscopy
(Nikon, Tokyo, Japan). The rate of cellular proliferation
was measured for monolayer cultures of KGN in a logarithmic growth
phase at a starting concentration of 2 x
105 cells/dish in 60-mm petri dishes (Falcon
3002, Becton Dickinson and Co., Rutherford, NJ). The
population doubling time was determined by cell counting at 24-h
intervals for 5 days while changing the medium (DMEM/Ham’s F-12 with
10% FCS) every other day. The determinations were carried out with
three dishes for each experiment.
Immunohistochemical staining of cytochrome P450arom
The KGN cells were plated on cover glass and fixed with 4%
paraformaldehyde at 4 C for 1 h. After treatment with 0.2% Triton
X-100 for 2 min, the cells were incubated with 0.3%
H2O2 in methanol for 30 min
at room temperature to avoid nonspecific endogenous peroxidase
reaction. Next, the cells were preincubated with 2% skim milk in PBS
(pH7.5) for 1 h, and then incubated with diluted rabbit antiserum
raised against human cytochrome P450arom (32) (1:500 in
PBS) at 4 C for overnight. Control KGN cells were also incubated with
normal (preimmune) rabbit serum (1:500 in PBS). After the cells were
washed with PBS three times, the antibody-antigen complexes were
detected by the streptavidin-biothin-peroxidase method using a
Histofine kit (Nichirei, Tokyo, Japan). The specific staining was
identified by the presence of brown reaction procedures.
Chromosome analysis
The chromosomes were examined in exponentially growing KGN
cells (passage 20) in an in vitro culture. The karyotype was
analyzed by standard trypsin G-banding and was described according to
the ISCN (33).
Measurement of the steroid content in medium secreted from KGN
KGN cells were inoculated on culture dishes (Falcon 3002) and
cultured for 3 days in a medium containing 10% FCS. After 3 days, when
the cells reached confluence, they were transferred to medium
containing 10% dextran-treated charcoal-treated FCS (DCS) and then
further cultured for 6–72 h in the presence or absence of
10-5–10-2
M (Bu)2cAMP (Wako Pure Chemical Industries Ltd., Tokyo, Japan) or
10-10–10-6
M phorbol 12-myristate 13-acetate (TPA; Wako Pure Chemical Industries Ltd.). The steroid contents of progesterone,
17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione
secreted into the culture medium were assayed using the respective
commercial RIA kits (Diagnostic Products, Los Angeles,
CA). Pregnenolone, 17-hydroxypregnenolone, estrone
(E1), and estradiol (E2)
were measured by SRL Co. Ltd. (Tokyo, Japan) using the respective RIA
systems (34). The antibody against
17-hydroxypregnenolone cross-reacts 1.0% with pregnenolone, 0.5%
with 17-hydroxyprogesterone, 0.2% with 16-pregnenolone, and
0.4% with 20-progesterone and 20ß-progesterone. The antibody
against the 17-hydroxyprogesterone cross-reacts 0.6% with
progesterone, 2.1% with 11-deoxycortisol, 3.2% with
17-hydroxypregnenolone, and 3.8% with 17-hydroxypregnenolone
sulfate.
Measurement of cAMP content in KGN cells
We measured the content of cAMP in KGN cells under the
stimulation of human FSH (hFSH; Sigma, St. Louis, MO) by
RIA using a commercially available kit (Yamasa Syoyu Co. Ltd., Chiba,
Japan) (35). The biological potency of hFSH was about 7000
IU/mg. Cultured KGN cells at confluent state in petri dishes (Falcon
3002, Becton Dickinson and Co., Inc., Franklin Lakes, NJ)
were transferred to the medium containing 1.0% (wt/vol) BSA
(Sigma) and 0.5 mM 3-isobutyl-1-methylxanthine
(Sigma). The cells were further cultured for 3 h in
the presence or absence of 0.5–500 ng/ml hFSH or
10-6 M
forskolin (Sigma). For the measurement of intracellular
cAMP, cells were washed twice with PBS, lysed with 3 ml 0.1
M HCl, scrapped into Eppendorf tubes using a
rubber policeman, and sonicated three times for 20 sec each time on ice
with sonicater (Cell disruptor-185, SmithKline Co.). The cell
debris was precipitated by centrifugation for 10 min at 4 C, and cAMP
in the supernatant was measured.
Aromatase assay
The aromatase activity of KGN was determined by measuring the
amount of [3H]H2O
released upon the conversion of
[1ß-3H]androstenedione to estrone by a
modification of the method of Ackerman et al.
(36). KGN cells were plated on a petri dish (Falcon 3001)
in culture medium with 10% FCS. At confluence, the culture medium was
replaced with DMEM/Ham’s F-12 containing 10% DCS and incubated for
another 12 h in the presence or absence of
10-5–10-2
M (Bu)2cAMP (Wako Pure Chemical Industries Ltd.),
10-10–10-6
M TPA(Wako Pure Chemical Industries Ltd.), 0.5–500 ng/ml hFSH (Sigma), 5–5000 mIU/ml
human menopausal gonadotropin (hMG; Teikoku Zouki Co. Ltd., Tokyo,
Japan), 0.1–100 IU/ml hCG (Teikoku Zouki Co. Ltd.), or
10-8–10-5
M dexamethasone (Sigma). After
treatment, the cells were further incubated with 12.5
nM
[1ß-3H]androstenedione (NEN Life Science Products, Boston, MA; SA, 27.5 Ci/mmol) for 12 h.
After incubation, the medium (2.0 ml) was transferred to tubes
containing 1.0 ml ice-cold 30% (wt/vol) trichloroacetic acid and then
centrifuged to remove precipitated protein. The cells were harvested
using 0.25% trypsin-1 mM EDTA to determine the
protein concentration. The following protocol for the extraction of the
medium to measure the amount of
[3H]H2O was performed
exactly as previously described (37). Finally, the amount
of radioactivity in the
[3H]H2O was corrected by
subtracting the blank values from each sample. The cell protein content
was determined using a micro bicinichoninic acid kit (Pierce Chemical Co., Rockford, IL) after the cells were dissolved in
1.0 N NaOH. The aromatase activity was expressed
as picomoles per mg cell protein. As a control, the aromatase
activities of human granulosa cells (obtained from in vitro
fertilization programs), human fibroblast, and HOS cell (human
osteoblast-like cell line) (37) were measured in the same
manner.
To ensure that KGN cells can produce estrogens, they were incubated with 10-5 M androstenedione for 72 h in the presence or absence of 10-3 M (Bu)2cAMP, and the contents of E1 and E2 secreted into the culture medium were measured by RIAs as described above.
FSH binding assay
To determine whether KGN cells have functional FSH receptor, an
[125I]FSH binding study was performed in the
manner described by Li et al. (38). In brief,
KGN cells were inoculated on 12-well culture plates (Falcon) and
cultured for 2 days in a medium containing 10% FCS. After 2 days, when
the cells reached confluence, they were washed twice with binding
buffer [DMEM/Ham’s F-12 medium containing 0.5% (wt/vol) BSA] and
then incubated with [125I]hFSH (SA, 155
TBq/mmol; concentration, 787 kBq/ml; NEN Life Sciences Products) in the presence of different concentrations of cold
hFSH (Sigma) at 37 C for 1 h in the binding buffer
and lysed with 1 M NaOH. The amount of bound
radioactivity was counted using a 
-counter. Specific binding was
defined as that remaining after subtraction of nonspecific binding from
the total amount of [125I]FSH binding. Protein
concentrations of the cell lysate were determined using a commercial
protein assay kit (micro bicinchoninic acid kit, Pierce Chemical Co.).
Effects of interferon- and anti-Fas antibody on cultured KGN
cells
To determine the utility of the KGN cell line for an experiment
on apoptosis, Fas-mediated apoptosis was tested as an example. KGN
cells were inoculated on culture dishes (Falcon 3001) at a
concentration of 1 x 105 cells/dish. After
incubating for 24 h, the cells were transferred to the medium
containing 10% DCS. Then, the cells were preincubated for 24 h in
the presence of 100 IU/ml interferon- (Shionogi Co. Ltd., Osaka,
Japan) (39, 40). Next, monoclonal antihuman Fas antibody,
CH-11 (Medical and Biological Laboratories Co. Ltd., Nagoya, Japan),
the active-form antihuman Fas antibody (IgM-type antibody derived from
mouse) that stimulates post-Fas signaling by binding to Fas
(41), was added to the cell culture at 1 µg/ml. At the
same time, 1 µg/ml mouse IgM (Medical and Biological Laboratories Co.
Ltd.) was added to the control culture. All cells on the culture dishes
were collected 24 and 48 h after the addition of CH-11. Dead cells
were counted by the trypan blue dye exclusion method, and the ratio of
dead cells was compared with the control value. DNA fragmentation was
examined by 1.8% agarose-TBE gel electrophoresis of the DNA
samples extracted from the collected cells following a previously
described protocol (42).
Statistics
All experiments were carried out more than three times with
triplicate plates per point. All values represent the mean ±
SD. A one-factor ANOVA was used for statistical evaluation.
P < 0.05 was considered to indicate statistical
significance.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) and changed to an
epithelial cell-like shape in the high cell density state (Fig. 1D).
They had an estimated doubling time of about 46.4 h in
vitro.
Karyotype analysis
The chromosome counting of 50 metaphase KGN cells revealed the
modal peak to be 45. G-Banded karyotype analyses of 10 KGN cells
exhibiting 45 chromosomes revealed all to be an abnormal karyotype of
45,XX, 7q-, -22. Namely, the 7q deletion and monosomy 22 were observed
(data not shown).
Immunohistochemical staining of KGN cells with antibody against
cytochrome P450arom
To show the clonality of this cell line as a steroidogenic cell,
we immunohistochemically stained the cells with normal rabbit serum or
immune rabbit serum against human cytochrome P450arom. As shown in Fig. 2, compared with the cells stained with
normal rabbit serum as a control (Fig. 2A), most of the KGN cells were
specifically stained with antiserum against human cytochrome P450arom,
especially in the perinuclear region, although the intensities were
somewhat different among the cells (Fig. 2, B and C). These results
indicate that a large percentage of KGN cells are steroidogenic cells,
which really expresses cytochrome P450arom.
|
indicates the levels of the various
steroids in the medium secreted from 106 KGN
cells for 24 h in the presence or absence of
10-4 M
(Bu)2cAMP. KGN cells showed detectable levels
of basal secretion of pregnenolone and progesterone in the medium, both
of which significantly increased after stimulation with
10-4 M
(Bu)2cAMP. On the other hand, the basal
concentrations of 17-hydroxypregnenolone, 17-hydroxyprogesterone,
androstenedione, dehydroepiandrosterone, and estradiol in the medium
were either very low or undetectable, and none of the above steroids
showed any significant change after the stimulation with
10-4 M
(Bu)2cAMP for 24 h.
|
and 5). A
significant and time-dependent increase in the secretion of
progesterone compared with basal secretion was noted during stimulation
with 10-3 M
(Bu)2cAMP for 24–72 h (Fig. 4). Further, a
dose-dependent increase in the secretion of progesterone was observed
during stimulation with
10-5–10-2 M
(Bu)2cAMP for 24 and 48 h (Fig. 5). The
effect of the C kinase stimulator, TPA, was also tested (Fig. 6). Although
10-3 M
(Bu)2cAMP caused a dramatic increase in the
secretion of progesterone compared with the basal secretion (0.26
± 0.08 vs. 8.50 ± 1.83 ng/106
cells·48 h; mean ± SD; n = 3;
P < 0.01), the coincubation with TPA
(10-8–10-6
M) significantly suppressed the
(Bu)2cAMP-stimulated secretion of progesterone
(coincubation with 10-8 M
TPA, 3.24 ± 1.03; 10-7
M TPA, 0.45 ± 0.21;
10-6 M TPA, 0.77 ±
0.34 ng/106 cells·48 h).
|
|
|
). Whereas, the treatment of KGN with
10-10–10-6
M TPA in the presence or absence of
10-3 M
(Bu)2cAMP caused no significant change in the
aromatase activity (data not shown). Treatment of KGN cells with
5–5000 mIU/ml hMG (Fig. 8A) and 0.5–500
ng/ml hFSH (Fig. 9) caused a
dose-dependent and significant increase in aromatase activity. The
minimum effective doses for the stimulation of aromatase activity were
50 mIU/ml hMG (1.83 ± 0.35 pmol/mg protein; control, 0.86 ±
0.14 pmol/mg protein; mean ± SD; n = 3;
P < 0.05) and 50 ng/ml hFSH (2.35 ± 0.47 pmol/mg
protein; control, 0.96 ± 0.15 pmol/mg protein; n = 3;
P < 0.01). On the other hand, no significant changes
in aromatase activity were noted during stimulation with 0.1–100 IU/ml
hCG (Fig. 8B). A slight, but significant, increase in aromatase
activity was observed during treatment with
10-8–10-5
M dexamethasone for 12 h in a dose-dependent
manner (data not shown). The maximum stimulation was observed with
10-6
M dexamethasone (1.71 ± 0.19 pmol/mg
protein; control, 0.92 ± 0.16 pmol/mg protein; n = 3;
P < 0.01). These results suggest that aromatase
activity in KGN cells is responsive to FSH partly through the protein
kinase A-mediated activation and is also under the regulation of
glucocorticoid.
|
|
|
, significant amounts of
E1 (103.7 ± 6.5 pg/106 cells)
and E2 (170.7 ± 3.5 pmol/106 cells) were
produced and secreted in the culture medium, and significant increases
in the levels of E1 (11.2-fold) and
E2 (23.8-fold) were noted during stimulation with
10-3 M
(Bu)2cAMP.
|
). The minimal effective dose of hFSH
was 50 ng/ml (P < 0.05 vs. control).
However, the magnitude of the stimulation of intracellular cAMP
production by hFSH was much smaller than that by forskolin (1
µM forskolin; 683 ± 36
pmol/106 cells).
|
Effect of interferon- and active-form anti-Fas antibody (CH-11)
on cultured KGN cells
To determine whether KGN cells exemplify the Fas- mediated
apoptotic phenomenon observed in human granulosa cells, KGN cells
were exposed to active-form antihuman Fas antibody (CH-11)
(40) for 24 or 48 h after pretreatment with 100 IU/ml
interferon-. Interferon- pretreatment was used to facilitate the
Fas-induced apoptotic mechanism partly by the induction of Fas antigen
(39, 40). Although the pretreatment with only
interferon- did not cause any significant change in the ratio of the
number of dead cells to that of the total cells, the addition of CH-11
to the interferon--pretreated cells caused a dramatic increase in
the ratio (Fig. 10A). DNA fragmentation
was recognized by electrophoresis in the DNA specimens extracted from
the KGN cells cultured in the presence of CH-11 in a time-dependent
manner (Fig. 10B).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
As for the mechanism of steroidogenesis in ovarian tissues, a two-cell,
two-gonadotropin theory has been proposed, which assumes that ovarian
estrogen synthesis may require both thecal and granulosa cells. It is
assumed that minor androgens mainly produced in thecal cells under the
control of LH are transported to granulosa cells and then are converted
to estrogen under the control of FSH (49, 50). Most of the
enzymatic or immunohistochemical findings of animal or human granulosa
cells correlate with this theory, as the activity and expression of
P450arom were present in normal granulosa cells, whereas those of
P450C17, a single enzyme mediating both 17-hydroxylase and
17,20-lyase activities (51) were either very low or
undetectable in the cells (6, 52). However, several
investigators have reported the presence of both 17-hydroxylase
activity and estrogen production in human granulosa cells (2, 3, 4, 26). The inconsistency of such studies may be due to the
difference in the culture condition or to the stage of follicular
development at which the experiments were performed, as a
development-related difference in steroidogenesis of human granulosa
cell has been suggested (10, 11).
In our KGN cells, based on the steroid concentrations secreted for
24 h, basal and cAMP-stimulated production of pregnenolone and
progesterone was observed, whereas very low or undetectable levels of
17-hydroxylated steroids and androgens were produced either before
or after treatment with (Bu)2cAMP. A slight
detection of 17-hydroxypregnenolone or 17-hydroxyprogesterone in
the medium is probably due to the slight cross-reactivities of
respective antibodies with other steroids in the RIAs, as no
significant increase in these steroids was observed after stimulation
with cAMP for 24 h. In addition, P450C17 transcript roughly
assessed by RT-PCR was undetectable before and after treatment with
cAMP for 24 h (data not shown). However, it is not completely
denied that even such an undetectable level of P450c17 expression may
still contribute to some detection of 17-hydroxylated steroids.
Because of the absence or low level formation of androstenedione, no
detectable formation of estradiol was observed in the KGN cells
incubated in 10% DCS medium despite the presence of aromatase
activity. However, when the cells were exogenously provided with enough
androstenedione, detectable amounts of E1 and
E2 were produced and secreted by KGN, indicating
the real capacity of KGN cells to produce estrogens. All of these
findings together indicate the mechanism of steroidogenesis in KGN to
be quite similar to that in normal granulosa cells (6, 50, 52), regarding the concept of two-cell, two-gonadotropin theory.
Furthermore, the suppressive effect of TPA (relatively high dose) on
cAMP-induced progesterone production in KGN mimicked the pattern
observed in normal granulosa cells (53, 54).
In the previously established granulosa cell lines, little has been reported on the aromatase activity and its regulation, except a report by Rainey et al. (29) demonstrating the enhanced expression of P450arom messenger RNA by forskolin in a transformed human granulosa cell line (HGL5). This is probably because aromatase activity has proven difficult to maintain in long-term cultures of granulosa cells. In our study the basal aromatase activity of KGN cells was easily measurable and was relatively high compared with those of other aromatase-expressing cells such as fibroblast and osteoblast (37). Although the aromatase activity of KGN cells was about 50–100 times lower than that of primary cultured human granulosa cells measured by us or reported by Steinkampf et al. (8), it was highly inducible by treatment with (Bu)2cAMP or FSH, but was only slightly inducible by treatment with glucocorticoid. These patterns of regulation regarding the aromatase activity in KGN cells were quite similar to those observed in normal human granulosa cells (5, 8), suggesting granulosa cell-specific promoter usage of the human P450arom gene (55, 56). Actually, the usage of granulosa cell-specific promoters, consisting of either exon 1c or 1d (or called promoter II) of the human aromatase gene, in KGN cells was confirmed by RT-PCR analysis (data not shown). Thus, the continued expression of P450arom in this cell line is also very useful for the study of regulation of P450arom in human granulosa cells.
The marked increase in aromatase activity caused by treatment with hFSH or hMG suggests that this cell line expresses functional FSH receptor. The concomitant increase in the levels of intracellular cAMP by hFSH treatment further supports this finding. Indeed, specific [125I]FSH binding to KGN cells was observed by binding assays, and the estimated binding capacity and Kd were almost similar to those noted in the ovarian cancer of sex cord-stromal origin (57). The expression of the FSH receptor on KGN cells and its responsiveness to FSH are worthy of note, as no clear response to FSH has been demonstrated in any of the previously established human granulosa cell lines (26, 27, 28, 29, 30). As most of the previous human cell lines have been established from normal granulosa cells by the forced introduction of a part of oncogenes or viral genes (26, 27, 28, 29, 30), it has been speculated that the native FSH receptor might be lost upon cell transformation (1). In contrast, our cell line, KGN was established by long-term culture of cells from an originally transformed human granulosa cell tumor, probably resulting in the preservation of physiologically more normal conditions. In a rat granulosa cell line, to overcome the absence of native FSH receptor, a stable transformant cell line expressing FSH receptor has been obtained by the forced introduction of FSH receptor gene (21). In contrast, treatment with hCG caused no change in the aromatase activity of KGN cells. The responsive pattern of aromatase activity to gonadotropin in human granulosa cells has been reported to be different depending on the developmental stage of the follicle; in granulosa cells from immature follicles, treatment with FSH, but not LH, increased aromatase activity, whereas in mature granulosa cells, both treatments markedly stimulated aromatase activity (9). These findings suggest that the developmental stage of KGN cells may be close to that of immature granulosa cells.
In recent years the involvement of apoptosis in granulosa cells
regarding the process of follicular atresia has been established, and
several mechanisms of the apoptosis have been proposed
(1). As one such mechanism, the Fas-Fas ligand system has
been reported to be important for the apoptosis of granulosa cells
using a primary culture system (39, 40). However, a
molecular biological analysis using the primary culture system is
usually limited, because relatively large populations of uniform cells
are required for such analyses. In this respect it is also very
meaningful that the Fas-mediated mechanism of apoptosis could be
reproduced in our KGN cells by exposure to interferon- and the
active form of Fas antibody. This fact enables us to perform a detailed
analysis of the mechanism using our newly established cell line and may
also provide insight for a new therapeutic approach to the granulosa
cell tumor.
In conclusion, we established a new ovarian granulosa-like tumor cell line, KGN, from a granulosa cell tumor. The KGN cells had steroidogenic activities similar to those of normal granulosa cells and expressed functional FSH receptor. The relatively high level of aromatase activity of KGN cells will greatly help in the study of P450arom gene regulation as well as in the study of the aromatase inhibitor as an anticancer drug. In addition, it is highly probable that the Fas-mediated apoptotic mechanism in normal granulosa cells is maintained in KGN cells. This cell line is therefore expected to allow us to study various aspects of the physiological regulation of human granulosa cells in the future.
|
Acknowledgments |
|---|
Received May 2, 2000.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-hydroxylase/17,20-lyase) in cultured human granulosa
cells. J Clin Endocrinol Metab 63:202–207- subunit promoter/simian virus
T-antigen fusion gene: characterization of ovarian tumors and
establishment of gonadotropin-responsive granulosa cell lines. Mol
Endocrinol 9:616–627-hydroxypregnenolone and dehydroepiandrosterone. Clin Endocrinol
(Tokyo) 26:309–314
-hydroxylase cytochrome P450cDNA in nonsteroidogenic
(Cos1) cells. Science 234:1258–1261-hydroxylase and side-chain-cleavage cytochromes P-450 in the human
ovary. J Reprod Fertil 85:163–169This article has been cited by other articles:
 |
|
 |
|
  P. Pierre, P. Froment, D. Negre, C. Rame, V. Barateau, C. Chabrolle, P. Lecomte, and J. Dupont Role of adiponectin receptors, AdipoR1 and AdipoR2, in the steroidogenesis of the human granulosa tumor cell line, KGN Hum. Reprod., November 1, 2009; 24(11): 2890 - 2901. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Komagata, M. Nakajima, S. Takagi, T. Mohri, T. Taniya, and T. Yokoi Human CYP24 Catalyzing the Inactivation of Calcitriol Is Post-Transcriptionally Regulated by miR-125b Mol. Pharmacol., October 1, 2009; 76(4): 702 - 709. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Wang, P. K Nicholls, P. G Stanton, C. A Harrison, M. Sarraj, R. B Gilchrist, J. K Findlay, and P. G Farnworth Extra-ovarian expression and activity of growth differentiation factor 9 J. Endocrinol., September 1, 2009; 202(3): 419 - 430. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Dipietromaria, B. A. Benayoun, A.-L. Todeschini, I. Rivals, C. Bazin, and R. A. Veitia Towards a functional classification of pathogenic FOXL2 mutations using transactivation reporter systems Hum. Mol. Genet., September 1, 2009; 18(17): 3324 - 3333. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Wang, R. Li, and Y. Hu The Alternative Noncoding Exons 1 of Aromatase (Cyp19) Gene Modulate Gene Expression in a Posttranscriptional Manner Endocrinology, July 1, 2009; 150(7): 3301 - 3307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Bilandzic, S. Chu, P. G. Farnworth, C. Harrison, P. Nicholls, Y. Wang, R. M. Escalona, P. J. Fuller, J. K. Findlay, and K. L. Stenvers Loss of Betaglycan Contributes to the Malignant Properties of Human Granulosa Tumor Cells Mol. Endocrinol., April 1, 2009; 23(4): 539 - 548. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Benayoun, F. Batista, J. Auer, A. Dipietromaria, D. L'Hote, E. De Baere, and R. A. Veitia Positive and negative feedback regulates the transcription factor FOXL2 in response to cell stress: evidence for a regulatory imbalance induced by disease-causing mutations Hum. Mol. Genet., February 15, 2009; 18(4): 632 - 644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Aghajanova, A. Hamilton, J. Kwintkiewicz, K.C. Vo, and L.C. Giudice Steroidogenic Enzyme and Key Decidualization Marker Dysregulation in Endometrial Stromal Cells from Women with Versus Without Endometriosis Biol Reprod, January 1, 2009; 80(1): 105 - 114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kyronlahti, M. Ramo, M. Tamminen, L. Unkila-Kallio, R. Butzow, A. Leminen, M. Nemer, N. Rahman, I. Huhtaniemi, M. Heikinheimo, et al. GATA-4 Regulates Bcl-2 Expression in Ovarian Granulosa Cell Tumors Endocrinology, November 1, 2008; 149(11): 5635 - 5642. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Benayoun, S. Caburet, A. Dipietromaria, M. Bailly-Bechet, F. Batista, M. Fellous, D. Vaiman, and R. A. Veitia The identification and characterization of a FOXL2 response element provides insights into the pathogenesis of mutant alleles Hum. Mol. Genet., October 15, 2008; 17(20): 3118 - 3127. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Moumne, A. Dipietromaria, F. Batista, A. Kocer, M. Fellous, E. Pailhoux, and R. A. Veitia Differential aggregation and functional impairment induced by polyalanine expansions in FOXL2, a transcription factor involved in cranio-facial and ovarian development Hum. Mol. Genet., April 1, 2008; 17(7): 1010 - 1019. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Chand, G. T. Ooi, C. A. Harrison, A. N. Shelling, and D. M. Robertson Functional analysis of the human inhibin {alpha} subunit variant A257T and its potential role in premature ovarian failure Hum. Reprod., December 1, 2007; 22(12): 3241 - 3248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Gao, C. De Geyter, K. Kossowska, and H. Zhang FSH stimulates the expression of the ADAMTS-16 protease in mature human ovarian follicles Mol. Hum. Reprod., July 1, 2007; 13(7): 465 - 471. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Chabrolle, L. Tosca, and J. Dupont Regulation of adiponectin and its receptors in rat ovary by human chorionic gonadotrophin treatment and potential involvement of adiponectin in granulosa cell steroidogenesis Reproduction, April 1, 2007; 133(4): 719 - 731. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lu, A. Amleh, J. Sun, X. Jin, S. D. McCullough, R. Baer, D. Ren, R. Li, and Y. Hu Ubiquitination and Proteasome-Mediated Degradation of BRCA1 and BARD1 during Steroidogenesis in Human Ovarian Granulosa Cells Mol. Endocrinol., March 1, 2007; 21(3): 651 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Batista, D. Vaiman, J. Dausset, M. Fellous, and R. A. Veitia Potential targets of FOXL2, a transcription factor involved in craniofacial and follicular development, identified by transcriptomics PNAS, February 27, 2007; 104(9): 3330 - 3335. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sugawara, N. Sakuragi, and H. Minakami CREM confers cAMP responsiveness in human steroidogenic acute regulatory protein expression in NCI-H295R cells rather than SF-1/Ad4BP. J. Endocrinol., October 1, 2006; 191(1): 327 - 337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. N. Parakh, J. A. Hernandez, J. C. Grammer, J. Weck, M. Hunzicker-Dunn, A. J. Zeleznik, and J. H. Nilson Follicle-stimulating hormone/cAMP regulation of aromatase gene expression requires beta-catenin PNAS, August 15, 2006; 103(33): 12435 - 12440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Pannetier, S. Fabre, F. Batista, A. Kocer, L. Renault, G. Jolivet, B. Mandon-Pepin, C. Cotinot, R. Veitia, and E. Pailhoux FOXL2 activates P450 aromatase gene transcription: towards a better characterization of the early steps of mammalian ovarian development. J. Mol. Endocrinol., June 1, 2006; 36(3): 399 - 413. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Miyoshi, F. Otsuka, J. Suzuki, M. Takeda, K. Inagaki, Y. Kano, H. Otani, Y. Mimura, T. Ogura, and H. Makino Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells Biol Reprod, June 1, 2006; 74(6): 1073 - 1082. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Tsuchiya, M. Nakajima, S. Takagi, M. Katoh, W. Zheng, C. R Jefcoate, and T. Yokoi Binding of Steroidogenic Factor-1 to the Regulatory Region Might Not Be Critical for Transcriptional Regulation of the Human CYP1B1 Gene. J. Biochem., March 1, 2006; 139(3): 527 - 534. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Watanabe, M. Noda, and S. Nakajin Effect of epidermal growth factor and prostaglandin on the expression of aromatase (CYP19) in human adrenocortical carcinoma cell line NCI-H295R cells J. Endocrinol., January 1, 2006; 188(1): 59 - 68. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. E. Harris, A. L. Chand, I. M. Winship, K. Gersak, Y. Nishi, T. Yanase, H. Nawata, and A. N. Shelling INHA promoter polymorphisms are associated with premature ovarian failure Mol. Hum. Reprod., November 1, 2005; 11(11): 779 - 784. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xia, Y. Sidis, A. Mukherjee, T. A. Samad, G. Brenner, C. J. Woolf, H. Y. Lin, and A. Schneyer Localization and Action of Dragon (Repulsive Guidance Molecule b), a Novel Bone Morphogenetic Protein Coreceptor, throughout the Reproductive Axis Endocrinology, August 1, 2005; 146(8): 3614 - 3621. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Fan, T. Yanase, H. Morinaga, Y.-M. Mu, M. Nomura, T. Okabe, K. Goto, N. Harada, and H. Nawata Activation of Peroxisome Proliferator-Activated Receptor-{gamma} and Retinoid X Receptor Inhibits Aromatase Transcription via Nuclear Factor-{kappa}B Endocrinology, January 1, 2005; 146(1): 85 - 92. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Wu, S. Ghosh, Y. Nishi, T. Yanase, H. Nawata, and Y. Hu The Orphan Nuclear Receptors NURR1 and NGFI-B Modulate Aromatase Gene Expression in Ovarian Granulosa Cells: A Possible Mechanism for Repression of Aromatase Expression upon Luteinizing Hormone Surge Endocrinology, January 1, 2005; 146(1): 237 - 246. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ohno, N. Araki, T. Yanase, H. Nawata, and M. Iida A Novel Nonradioactive Method for Measuring Aromatase Activity Using a Human Ovarian Granulosa-Like Tumor Cell Line and an Estrone ELISA Toxicol. Sci., December 1, 2004; 82(2): 443 - 450. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Steinmetz, H. A. Wagoner, P. Zeng, J. R. Hammond, T. S. Hannon, J. L. Meyers, and O. H. Pescovitz Mechanisms Regulating the Constitutive Activation of the Extracellular Signal-Regulated Kinase (ERK) Signaling Pathway in Ovarian Cancer and the Effect of Ribonucleic Acid Interference for ERK1/2 on Cancer Cell Proliferation Mol. Endocrinol., October 1, 2004; 18(10): 2570 - 2582. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chu, Y. Nishi, T. Yanase, H. Nawata, and P. J. Fuller Transrepression of Estrogen Receptor {beta} Signaling by Nuclear Factor-{kappa}B in Ovarian Granulosa Cells Mol. Endocrinol., August 1, 2004; 18(8): 1919 - 1928. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Morinaga, T. Yanase, M. Nomura, T. Okabe, K. Goto, N. Harada, and H. Nawata A Benzimidazole Fungicide, Benomyl, and Its Metabolite, Carbendazim, Induce Aromatase Activity in a Human Ovarian Granulose-Like Tumor Cell Line (KGN) Endocrinology, April 1, 2004; 145(4): 1860 - 1869. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Fan, T. Yanase, Y. Wu, H. Kawate, M. Saitoh, K. Oba, M. Nomura, T. Okabe, K. Goto, J. Yanagisawa, et al. Protein Kinase A Potentiates Adrenal 4 Binding Protein/Steroidogenic Factor 1 Transactivation by Reintegrating the Subcellular Dynamic Interactions of the Nuclear Receptor with Its Cofactors, General Control Nonderepressed-5/Transformation/ Transcription Domain-Associated Protein, and Suppressor, Dosage-Sensitive Sex Reversal-1: a Laser Confocal Imaging Study in Living KGN Cells Mol. Endocrinol., January 1, 2004; 18(1): 127 - 141. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Sugawara, H. Shimizu, N. Hoshi, A. Nakajima, and S. Fujimoto Steroidogenic Acute Regulatory Protein-binding Protein Cloned by a Yeast Two-hybrid System J. Biol. Chem., October 24, 2003; 278(43): 42487 - 42494. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Mukasa, M. Nomura, T. Tanaka, K. Tanaka, Y. Nishi, T. Okabe, K. Goto, T. Yanase, and H. Nawata Activin Signaling through Type IB Activin Receptor Stimulates Aromatase Activity in the Ovarian Granulosa Cell-Like Human Granulosa (KGN) Cells Endocrinology, April 1, 2003; 144(4): 1603 - 1611. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Tajima, K. Hosokawa, Y. Yoshida, A. Dantes, R. Sasson, F. Kotsuji, and A. Amsterdam Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes Mol. Hum. Reprod., January 1, 2002; 8(1): 48 - 57. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. P. Risbridger, J. F. Schmitt, and D. M. Robertson Activins and Inhibins in Endocrine and Other Tumors Endocr. Rev., December 1, 2001; 22(6): 836 - 858. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Sugawara, S. Abe, N. Sakuragi, Y. Fujimoto, E. Nomura, K. Fujieda, M. Saito, and S. Fujimoto RIP 140 Modulates Transcription of the Steroidogenic Acute Regulatory Protein Gene through Interactions with Both SF-1 and DAX-1 Endocrinology, August 1, 2001; 142(8): 3570 - 3577. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y.-M. Mu, T. Yanase, Y. Nishi, A. Tanaka, M. Saito, C.-H. Jin, C. Mukasa, T. Okabe, M. Nomura, K. Goto, et al. Saturated FFAs, Palmitic Acid and Stearic Acid, Induce Apoptosis in Human Granulosa Cells Endocrinology, August 1, 2001; 142(8): 3590 - 3597. [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 |
) and
48 h (
). Progesterone content was measured by RIA. Each value
indicates the mean ± SD of three experiments, with
triplicate plates per point. *, P < 0.01; **,
P < 0.001 [vs. control (absence of
(Bu)2cAMP)]. a, P < 0.05; b,
P < 0.01; c, P < 0.001
(vs. corresponding values).
