673

Expression of Cyclin E in Placentas with Hydropic

Change and Gestational Trophoblastic Diseases
Implications for the Malignant Transformation of Trophoblasts

Young Tae Kim, M.D.1 BACKGROUND. Although much is known about the morphologic, cytogenetic, and
Nam Hoon Cho, M.D.2 clinical characters of gestational trophoblastic diseases, little information has
Jae Hung Ko, Ph.D.3 appeared concerning the parameters related to their persistence or neoplastic
Woo Ick Yang, M.D.2 transformation. Cell cycle alterations in tumor tissue were examined in this study
Jae Wook Kim, M.D.1 in light of obvious changes in the clinical behavior of malignant cells. There is an
Eun Kyoung Choi, M.D.1 increasing body of evidence suggesting that the abnormal expression of cyclins is
Seung Ho Lee, M.D.4 considered one of the most important events in malignant transformation of
various human cancers. Among these cell cycle regulators, the role of cyclin E in
1
Department of Obstetrics and Gynecology, Yonsei the neoplastic transformation of trophoblast populations has been poorly defined.
University College of Medicine, Seoul, Korea. METHODS. Using formalin fixed, paraffin embedded trophoblastic tissues, the
2
Department of Pathology,Yonsei University Col- authors investigated the expression of cyclin E by immunohistochemistry in pla-
lege of Medicine, Seoul, Korea. centas with hydropic change and gestational trophoblastic diseases. The speci-
3 mens examined included tissue from 29 patients with complete hydatidiform
Department of Biology, Yonsei University, Seoul,
Korea. mole, 18 patients with partial hydatidiform mole, and 6 patients with choriocar-
4
cinoma after term pregnancy or abortion. The authors also studied four cases of
Department of Obstetrics and Gynecology, Sam-
hydropic abortion.
sung Jeil Hospital, Seoul, Korea.
RESULTS. The cyclin E indexes (CEI) were as follows: 25.7% ⫾ 6.2% for hydropic
change, 35.3% ⫾ 12.7% for triploid partial moles, 42.2% ⫾ 13.1% for diploid/
tetraploid complete moles, and 63.6% ⫾ 9.5% for choriocarcinomas. There was a
significant difference in CEI between placentas with hydropic change and partial
mole (P ⫽ 0.04) and placentas with hydropic change and complete mole (P
⫽ 0.003). Choriocarcinomas had significantly higher cyclin E expression compared
with placentas, partial moles, and complete moles, respectively. A significant
correlation between the expression of cyclin E and S-phase fraction was observed
in gestational trophoblastic diseases (rank correlation coefficient ⫽ 0.45, P ⬍ 0.05).
The relation between cyclin E expression and proliferation was abrogated in
placentas with hydropic change, suggesting that cyclin E up-regulation represents
a genuine aberration.
CONCLUSIONS. The results of this study were consistent with the concept that
cyclin E overaccumulation may play an important role in the uncontrolled prolif-
eration and neoplastic transformation of trophoblasts. Cancer 2000;89:673–9.
© 2000 American Cancer Society.

The authors thank Seung Ho Lee, M.D., for his KEYWORDS: cyclin E, cell cycle, hydatidiform moles, gestational trophoblastic
assistance in preparing this work.
diseases.
Address for reprints: Jae Wook Kim, M.D., Depart-
ment of Obstetrics and Gynecology, Yonsei Univer-
sity College of Medicine, C.P.O. Box 8044, Seoul
120-752, Korea.
G estational trophoblastic disease encompasses a heterogeneous
group of diseases including complete and partial hydatidiform
moles, invasive moles, choriocarcinoma, and placental site tropho-
Received January 31, 2000; accepted April 6, blastic tumors. Hydatidiform moles are not truly malignant, in con-
2000. trast to choriocarcinomas. The significance of hydatidiform moles,

© 2000 American Cancer Society

674 CANCER August 1, 2000 / Volume 89 / Number 3
however, lies in the risk of subsequent choriocarcino-
ma.1,2 Although much is known about the morpho-
logic character and genetic composition of complete
and partial hydatidiform moles, there has been little
information concerning the factors related to persis-
tence or neoplastic transformation of trophoblasts.3– 6
By contrast, choriocarcinoma is a malignant tumor of
cytotrophoblast and syncytiotrophoblast that can
arise in any type of gestation including hydatidiform
mole, normal term pregnancy, spontaneous abortion,
and ectopic pregnancy.
Cell cycle progression is controlled by different
groups of highly conserved proteins consisting of cy-
clin dependent kinases (CDKs) and sets of activating
and inhibitory proteins.8 –13 Cyclins are proteins that
bind and activate various CDKs at different stages of
the cell cycle, a given cyclin–CDK complex being es-
sential for transition through a specific stage in the
cycle. Recently, there has been a growing recognition
of new cyclins, particularly the G1 cyclins, D and E,
which regulate cell cycle transition from G1 into S-
phase.14 –17 D-type and E-type cyclins sequentially ac-
tivate CDK4 and -6 and CDK2 in the G1-S transition,
causing phosphorylation and inactivation of the reti-
noblastoma protein (pRB) and initiation of DNA rep-
lication. Cyclin E drives cell proliferation by phosphor-
ylating pRB. Hyperphosphorylated pRB then releases
transcription factors of the E2F family to cause DNA
synthesis. Cyclin E is both a regulator and a target of
the E2F transcription family in an autoregulatory loop
that is essential for progression from the G1- to the
S-phase of the cell cycle.16,17 Recent evidence suggests
that cyclin E is one of the key regulators in the cell
cycle machinery and furthermore, because the over-
expression of cyclin E shortens the G1-phase and pro-
longs the S-phase, cells blocked in G1 can be forced
into DNA duplication by aberrantly expressed cyclin
E.18,19 Owing to a crucial role for cyclins in cell cycle
progression, these proteins may be potential targets in
tumorigenesis.
Neoplastic transformation is characterized by de-
regulation of the cell cycle. Although p53 is still the
most important cell cycle regulator in human malig-
nancies, there is an increasing body of evidence indi-
cating that aberrant expression of cyclins is consid-
ered to be one of the most important events in the
tumorigenesis of various human cancers.8,10 –13 This
abnormal expression, especially that of cyclin E, has
been reported in a number of human cancers, includ-
ing breast carcinoma, endometrial carcinoma, and
malignant lymphoma.20 –24 However, there have been
very few reports on cyclin E in human placentas and
fewer on gestational trophoblastic disease.25,26
In our previous report,27 we reported that cyclin E
might play a leading role in neoplastic transformation
of the uterine cervix, whereas cyclin D was down-
regulated, especially when associated with human
papillomavirus.
In this study, we investigated the expression of
cyclin E with immunohistochemistry in well docu-
mented cases of placentas with hydropic change and
53 cases of gestational trophoblastic diseases. We also
examined the potential link between the malignant
transformation of trophoblasts and the expression of
cyclin E.
MATERIALS AND METHODS
Sample Composition
Tissue samples fixed with 10% neutral buffered forma-
lin and embedded in paraffin were selected from 57
patients treated at Yonsei University Medical Center.
These included the tissue from 29 patients with com-
plete hydatidiform mole, 18 cases of partial hydatidi-
form mole, and 6 cases of choriocarcinoma after term
pregnancy or abortion. We also studied four cases of
hydropic abortion. In this study, no patients had been
treated with chemotherapy before tissue sampling.
The selected cases were obtained from uterine curet-
tage and blocks from hysterectomy specimens. The
cases of gestational trophoblastic disease were diag-
nosed and classified according to well established his-
topathologic criteria and flow cytometric studies of
DNA content.28 –30 Four-micrometer-thick sections
were cut from a paraffin block for immunohistochem-
ical stains of cyclin E. One section was stained with
hematoxylin and eosin and examined microscopically
to confirm the histologic diagnosis of each lesion.
Subsequently, 4 50-␮m sections were cut and col-
lected into microcentrifuge tubes for DNA flow cytom-
etry.
Immunohistochemical Studies
A modification of the immunohistochemical staining
for cyclin E was used as described elsewhere.27,31 Par-
affin sections for immunohistochemical staining were
mounted on slides coated with poly-L-lysine. Briefly,
slides were deparaffinized and rehydrated. They were
immersed in 10 mM sodium citrate, pH 6.0, and
heated twice in a microwave oven for 5 minutes each
time (700 Watts). The slides then were sequentially
placed in running cold water for 15 minutes. Subse-
quently, a circle was marked on all sections by Dako
Co. (Carpinteria, CA) pencil and blocked with 3% hy-
drogen peroxide for 10 minutes. Sections were incu-
bated with normal goat serum for 30 minutes and then
sequentially incubated with primary antibodies in-
cluding cyclin E (1:40; Pharmingen, San Diego, CA),
overnight at 4 °C, respectively. Sections were addition-
 Expression of Cyclin E in Gestational Trophoblastic Diseases/Kim et al. 675

FIGURE 1. Distribution of cyclin E expression among placentas with hydropic

change (placenta), partial moles (PM), complete moles (CM), and choriocarci- FIGURE 2. Scatter diagram shows the distribution of cyclin E and S-phase
noma (CCA) (short horizontal bar indicates mean) is shown. fraction in gestational trophoblastic diseases (rank correlation coefficient
⫽ 0.45, P ⬍ 0.05).

TABLE 1
Comparison of Cyclin E Indexes
for 15 minutes to obtain the nuclear pellet. The iso-
Placentas PM CM CCA lated nuclei were sequentially treated with trypsin (T-
9253; Sigma) and ribonuclease A (R-4875; Sigma) and
Placentas
stained with propidium iodide (p-5264; Sigma) for at
PM *
CM * least 2 hours. Up to 20,000 nuclei were analyzed on a
CCA * * * FACScan flow cytometer (Becton-Dickinson, Sunny-
vale, CA). Cell cycle evaluation of the DNA histograms
PM: partial moles; CM: complete moles; CCA: choriocarcinoma. including DNA index, ploidy, and S-phase fraction was
* Significant difference.
performed with a Modfit computerized software pro-
gram (Verity Software, Inc., Topsham, ME). Samples
were interpreted as DNA diploid when a single peak
ally incubated at room temperature for 30 minutes
on the DNA histogram occupied the diploid position
with biotinylated goat anti-mouse immunoglobulin G,
with a DNA index of 1.0. Triploidy DNA content was
and for another 30 minutes at room temperature with
defined by an additional G0/G1 peak with a DNA index
ABC reagent (Dako). Aminoethylcarbazole was used as
of 1.5 (range, 1.3–1.6). Tetraploidy DNA content was
chromogen with hematoxylin counterstain. Only nu-
defined when the peak in the G2/M region represented
clear staining was regarded as a positive reaction. The
greater than 20% of the cells and was followed by a
cyclin E index (CEI) was expressed as a percentage of
corresponding G2/M peak with a DNA index between
positive cells per 1000 trophoblastic cells by using grid
1.8 and 2.2. Aneuploidy was defined by any additional
counting.
G0/G1 peak not falling into the range for discrete mul-
tiples of haploid DNA content. The accepted value of
Flow Cytometric Analysis
the coefficient variation for the G0/G1 peak was less
Nuclear suspensions were prepared from the paraffin
than 8%.
embedded tissue blocks by the method of Hedley et
Statistical analysis was performed by analysis of
al.32 Representative paraffin blocks were chosen with a
variance test, Student t test, and Spearman rank cor-
component of decidual tissue to serve as an internal
relation test. Differences were considered significant
diploid reference for the DNA histograms. Fifty-mi-
when the probability of error was below 5% (P ⬍ 0.05).
crometer-thick sections were obtained and deparaf-
finized with Histo-Clear (National Diagnostics, Inc.,
Sommerville, NJ), rehydrated with progressively de- RESULTS
creasing ethanol concentrations (100, 95, and 70%) for The staining patterns were found to be heterogeneous
10 minutes each, and then washed 2 times in distilled in the different groups of trophoblastic tissue studied.
water. The sections then were digested in 2 mL of 5% The CEI was as follows: 25.7% ⫾ 6.2% for hydropic
pepsin (Sigma Chemical Co., St. Louis, MO) for 90 –120 change; 35.3% ⫾ 12.7% for partial moles; 42.2%
minutes at 37 °C, filtered, and centrifuged at 3000 rpm ⫾ 13.1% for complete moles; and 63.6% ⫾ 9.5% for
676 CANCER August 1, 2000 / Volume 89 / Number 3
FIGURE 3. Immunostaining for cyclin E in hydropic abortion is shown. Cyclin E reactivity is primarily restricted to the nuclei of cytotrophoblasts. (A) Cyclin E
immunostaining in complete mole (original magnification ⫻100). (B) Cyclin E is strongly expressed and localized to the syncytiotrophoblasts. (original magnification
⫻200).
choriocarcinomas (Fig. 1). There was a significant dif-
ference in the CEI between placentas with hydropic
change and partial mole (P ⫽ 0.04) and placentas with
hydropic change and complete mole (P ⫽ 0.003). Cho-
riocarcinomas had a significantly higher cyclin E ex-
pression compared with placentas, partial moles, and
complete moles. Although there was a difference in
staining between partial and complete moles, this dif-
ference did not reach statistical significance (P ⫽ 0.08)
(Table 1). A statistically significant correlation be-
tween the expression of cyclin E and S-phase fraction
was observed (rank correlation coefficient ⫽ 0.45, P
⬍ 0.05) as shown in Figure 2.
Figure 3A shows the immunohistochemical stain-
ing for cyclin E in placenta with hydropic change.
Immunostaining with anti– cyclin E antibody was
prominent in the nuclei of villous cytotrophoblasts,
whereas immunoreactivity for cyclin E was barely ob-
served in the nuclei of syncytiotrophoblasts. Con-
versely, the patterns of immunostaining for cyclin E
were quite different in complete hydatidiform mole.
As could be observed, cyclin E was strongly expressed
and localized to the nuclei of the syncytiotrophoblasts
(Fig. 3B)
Figure 4A illustrates the immunohistochemical
staining for cyclin E in partial hydatidiform mole. No
immunoreactivity for cyclin E was observed in any
cells of swollen villi, whereas immunostaining with
anti– cyclin E antibody was pronounced in nonmolar
villi showing strong expression of cyclin E in covering
syncytiotrophoblasts as well as cytotrophoblasts.
Figure 4B represents the expression of cyclin E in
choriocarcinoma. Immunostaining with anti– cyclin E
antibody was found in both syncytiotrophoblastic and
cytotrophoblastic cells.
DISCUSSION
Cell machine regulation of cell cycle progression and
cessation is quite complex and not fully understood in
 Expression of Cyclin E in Gestational Trophoblastic Diseases/Kim et al. 677

FIGURE 4. Immunohistochemical staining of cyclin E in partial hydatidiform mole is shown. (A) Cyclin E immunostaining in choriocarcinoma (original magnification
⫻200). (B) Immunostaining with anti– cyclin E antibody is shown in both cytotrophoblastic and syncytiotrophoblastic cells (original magnification ⫻100).

mammalian cells. Recent studies have demonstrated
new cell cycle regulators, particularly the G1 cyclins
and CDK inhibitors, which govern cell cycle progres-
sion from G1- to S-phase.8-13,33–35 Cyclin E is a late G1
cyclin that is required for entry into S-phase along
with its catalytic partner. The gene for cyclin E is
located on human chromosome 19q12 and translates
cyclin E, which is composed of 395 amino acids.36
Cyclin E is considered to play an important role in
the neoplastic transformation of various human tu-
mors.20,23,24 It has been reported that overaccumula-
tion of cyclin E reflects amplification of the gene,37,38
although other mutations have been detected. How-
ever, cyclin E is in part modified by a posttranscrip-
tional mechanism,20 so that up-regulated protein ex-
pression can occur without mutations in the cyclin E
gene. We reported that cyclin E might play a leading
role in the neoplastic transformation of the uterine
cervix, whereas cyclin D1 was down-regulated.27
The main purpose of this study was to evaluate a
potential link between neoplastic transformation of
trophoblasts and the expression of cyclin E. Chorio-
carcinomas had a significantly higher cyclin E expres-
sion compared with placentas with hydropic change
and hydatidiform moles, respectively. There was also a
significant difference in the expression of cyclin E
between placentas with hydropic change and partial
mole and between placentas with hydropic change
and complete mole. The observed difference in the
expression of cyclin E in each type of trophoblastic
disease seems to relate to the differing degree of neo-
plastic transformation of the trophoblasts. Moreover,
the expression of cyclin E in this series was linked with
proliferation. A significant correlation between the ex-
pression of cyclin E and S-phase fraction was observed
in gestational trophoblastic disease (rank correlation
coefficient ⫽ 0.45, P ⬍ 0.05), but this correlation
seems ambiguous for placentas with hydropic change,
suggesting that cyclin E up-regulation represents a
genuine aberration. Although some investigators have
reported that the cell cycle phase specific expression
of cyclin E was disturbed in breast tumor cells,39,40
other reports have shown a connection between cyclin
E expression and proliferation as well as a cell cycle
678 CANCER August 1, 2000 / Volume 89 / Number 3
phase specific expression of the cyclin E protein eval-
uated by immunohistochemistry or flow cytom-
etry.20,41 Therefore, it can be assumed that cyclin E
protein expression in neoplastic trophoblasts in-
creases over the course of malignant transformation of
the trophoblasts.
In the current study of trophoblastic tissues, cy-
clin E immunoreactivity in placentas with hydropic
change was demonstrated mainly in the cytotropho-
blasts, whereas immunostaining for cyclin E was
barely observed in the nuclei of syncytiotrophoblasts.
This finding was in agreement with the previously
published study by Bamberger et al.25 They reported
that cyclin E was strongly expressed in the nuclei of
cytotrophoblast and intermediate trophoblasts but
not in the terminally differentiated, nondividing syn-
cytiotrophoblast. This observation suggests that cell
type specificity might be involved in the expression of
cyclin E in the human trophoblast populations.
Conversely, we found that cyclin E was expressed
in both cytotrophoblastic and syncytiotrophoblastic
cells of partial mole, complete mole, and choriocarci-
noma. Trophoblast proliferation is a tightly controlled
situation, and one of the keys to its regulation might
appertain to the balance between the positive cell
cycle factors, such as cyclin E and the negative cell
cycle inhibitors. Deregulation of the cell cycle can
cause uncontrolled cell division, resulting in the de-
velopment or progression of human tumors. This dys-
topic expression of cyclin E in the trophoblastic ele-
ments suggests that cyclin E protein expression may
be deregulated in gestational trophoblastic disease.
In summary, the current study demonstrated that
the cellular levels of cyclin E expression were signifi-
cantly increased in choriocarcinoma trophoblasts
compared with those in nonmalignant trophoblasts.
This change may play a role in uncontrolled prolifer-
ation and neoplastic transformation. To our knowl-
edge, this is the first report to describe the expression
of cyclin E in relation to gestational trophoblastic dis-
ease, to demonstrate the increase in cyclin E expres-
sion in the malignant transformation of trophoblasts,
and to reveal the dystopic expression of cyclin E in
gestational trophoblastic disease. However, additional
molecular studies to identify the amplification of the
cyclin E gene or the source of cyclin E protein over-
accumulation will be necessary to clarify these find-
ings.
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