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Papers
Expression of c-myc and c-fms oncogenes introphoblastic cells in
hydatidiform mole andnormal human placenta
A N Y Cheung, G Srivastava, S Pittaluga, T K Man, H Ngan, R J
Collins
AbstractAims:To compare the expression ofc-incand c-fins
proto-oncogenes in the pla-centa and hydatidiform mole.Methods:
Twelve hydatidiform moles andsix induced abortion cases were
coilected.c-inyc and c-fins proto-oncogene expres-sion was analysed
by northern blothybridisation and
immunohistochemicalstaining.Results: The results of northern
blothybridisation analysis showed that c-finswas expressed more
strongly in hydatidi-form moles compared with normalplacenta of
similar gestational age.Moreover, c-fJns mRNA
concentrationsincreased with more advanced gestationalage in moles
but not in normal placentas.c-nyc expression was very low in
hydati-diform moles and normal placentas. Bothoncogenes, however,
had no direct corre-lation with the clinical course ofthe
molarpregnancies.Conclusion: The difference in c-finsexpression
between hydatidiform molesand normal placentas suggests that
c-finsmay have a role in the development ofmolar pregnancies.
(7 Clin Pathol 1993;46:204-207)
Department ofPathology, UniversityofHong Kong, QueenMary
Hospital, HongKongA N Y CheungG SrivastavaS PittalugaT K ManR J
CollinsDepartment ofObstetrics andGynaecologyH NganCorrespondence
to:Dr Annie CheungAccepted for publication7 August 1992
The trophoblast of early placenta can pro-liferate rapidly and
infiltrate the decidua andmyometrium at the placental site.'
Expressionof certain cellular proto-oncogenes may beassociated with
rapid cell growth in these earlytrophoblast. A strong correlation
betweenc-nyc transcript concentrations and tropho-blastic
proliferation has been reported.2 It hasalso been suggested that
colony stimulatingfactor-l (CSF-1), a glycoprotein growth
factorrequired for the proliferation and differentia-tion of
mononuclear phagocytic cells, mayhave a role in placental
development andfunction under hormonal influence.3 The
c-finsproto-oncogene product is related to the recep-tor for the
CSF-1,4 but in situ hybridisationstudies indicate that c-fins mRNA
is associatedwith trophoblast in human placenta.5The pathogenesis
of hydatidiform moles has
remained a mystery for decades. The difference
between physiological oncogene expression inearly placenta and
that seen in molar pregnan-cies is unknown. It would also be useful
toascertain whether there is a particular patternof oncogene
expression in moles which willlater progress into invasive moles or
chor-iocarcinoma. This study aimed to investigatethe expression of
c-myc and c-fins proto-oncogenes in hydatidiform moles comparedwith
that in normal placenta.
MethodsTwelve clinically and ultrasonographically sus-pected
cases of hydatidiform mole and sixlegally induced abortions were
suction evac-uated. Fresh chorionic villi and molar vesicleswere
selected and snap frozen in liquid nitro-gen and stored at -
70°C.The rest of the tissuewas fixed in formalin for histological
exam-ination.Human HL-60 promyelocytic leukaemia cell
line6 and human HUT78T cell lymphoma cellline were maintained in
logarithmic growthphase; total RNA was extracted and used
aspositive controls for c-mc and c-fins expres-sion,
respectively.The probes used were c-myc-exonIII (1-8
kilobase insert) from Oncor (Gaithersburg,USA) and c-fins cDNA
clone pCfins 104 inPUC12 (1-23 kilobase insert) from AmericanType
Culture Collection (Maryland, USA).The cDNA probes were labelled by
an oligo-labelling procedure (Bresatec, Australia).
Total cellular RNA was extracted by theguanidine hydrochloride
procedure and unde-graded RNA was obtained from eight molesand six
abortion cases.7 Haematoxylin andeosin stained sections of each
frozen tissueblock were examined to exclude contaminationby decidua
and to ensure roughly equal num-bers of molar villi in the tissue
block. Aliquotsof total RNA (20 ,pg) from each sample weredenatured
and subjected to electrophoresis on1-0% agarose gels containing l
lM formalde-hyde. Equivalent amounts ofRNA were loadedinto each
lane as judged by ethidium bromidestaining. RNA was then
transferred to nylonmembranes (Gene Screen Plus, NEN, DuPont,
Boston, Massachusettes, USA). Themembranes were prehybridised in a
solutioncontaining 50% formamide, 1M sodium chlo-
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c-myc and c-fins expression in trophoblastic cells
A Controls
28S-* -42kb
18S-
Ba,
Normal placenta
(Gestational age)
5 8 9 10 16 (weeks)
281kb
181
A=iI aI
Hydatidiform moles
(Gestational age)
6 9 10 12 16 (weeks)
|v-28S.a a rRNA
Figure 1 Representative autoradiogram of northern blot
hybridised with 32P-labelledc-fins cDNA probe. Total RNA was
isolatedfrom the normal placentas at five, eight,nine, I0 and 16
weeks ofpregnancy as well as from hydatidiform moles at six, nine,
10,12, 16 weeks'gestational age. The transcripts of c-fins was
detected in the appropriaterange of 4-2 kilobases. The positions of
18S and 28S ribosomal RNA are indicated. Theresults of the probing
of '2P-labelled 28S ribosomal RNA is shown in the lower panel
B.
Signals were quantitated by densitometricscanning of the
autoradiograms.Frozen sections (6 pm) of the stored frozen
samples were cut, air-dried, and fixed in 4%paraformaldehyde.
The sections were stainedwith mouse monoclonal antibody to
c-myconcoprotein and rabbit polyclonal antibody toc-fins
oncoprotein (Cambridge Research Bio-chemials, England)9 and
monoclonal mouseantibody to human Ki-67 antigen (Dako,Copenhagen,
Denmark). The alkaline-phos-phatase anti-alkaline-phosphatase
method, asdescribed by Cordell, was used.'0 Negativecontrols were
used by omitting the primaryantibody in each case. Positive
controls wereobtained by cytospin of the human HL-60 andHUT-78 cell
lines as well as by including alymphoma case with a high
proliferative rate.
ride, 10% dextran sulphate, 1% sodiumdodecyl sulphate (SDS),
0-2% polyvinylpyrro-lidone, 0-2% ficoll, 0-2% bovine serum
albu-min, 50 mM TRIS-HCI (pH 7-5), 0-1%sodium pyrophosphate, 01 mM
EDTA, anddenatured salmon sperm DNA (200 ,ug/ml) at42°C for
overnight. The membranes werehybridised to the denatured
32P-oligolabelledc-nyc and c-fins DNA probes (0-5-2 ng/ml) inthe
same solution for 24 hours. The final washof the membranes was in a
solution containing0 1 x SSC (30 mM sodium chloride, 3 mMsodium
citrate), 1% SDS, 0-1% sodium pyro-phosphate, and 0 1 mM EDTA at
65°C for 60minutes. The membranes were also strippedand reprobed
with 28S ribosomal RNA oligo-meric probe to ensure that equal
amounts ofRNA were loaded in each lane and that thetransfer was
quantitative.8 Autoradiographywas carried out using X-OMAT AR x-ray
film(Kodak) with intensifying screens at -70°C.
Figure 2 Relative changesin c-fins RNA expressionin (A) normal
placentasand (B) hydatidiformmoles. The arbitraryscanner units
representdensitometric readings fromthe five normal placentasand
the five hydatidiformmoles at the statedgestational age.
c
L-
a)
cJ
cJ
C.
a,
C.,
.0
C0
C
0
zEc"
25 A Normal placenta
20
1.5-
1-0 _
0-5
0l5 8 9 10 16
ResultsHistological examination showed that all themoles
collected were complete moles. Therewere no detectable differences
in the accumu-lation of macrophages in the moles and pla-centas
examined. The gestational ages of thehydatidiform moles thus
selected for analysisranged from six to 16 weeks while the
abortioncases ranged from five to 16 weeks. In the casesstudied two
hydatidiform moles had persis-tently raised serum concentrations of
humanchorionic gonadotrophin (hCG) during followup and single agent
chemotherapy was given.The results of northern blot
hybridisation
analysis showed that higher mRNA concentra-tions of c-fins were
found in hydatidiformmoles compared with those in normal placentaof
comparable gestational age (fig 1). More-over, c-fins expression
increased with moreadvanced gestational age in moles, but innormal
placentas the c-fms concentrationreduced with increased gestational
age. Den-sitometric measurements of the relative c-fmisRNA
expression in normal placentas andhydatidiform moles is shown in
fig 2. Immuno-histochemical staining carried out with theanti-c-fis
antibody clearly localised c-fms anti-gen expression to the
syncytiotrophoblast withoccasional positive staining in the
Hofbauercells in the molar villi (fig 3). There was nodetectable
accumulation ofmacrophages in thesections. No correlation between
c-fms expres-sion and serum concentrations ofhCG duringfollow up
and subsequent chemotherapy in themolar pregnancies studied could
be demon-strated.
Contrary to previous reports, c-myc expres-sion was very low in
the cases of moles andnormal placentas studied except in one case
ofinduced abortion at five weeks' gestation (fig4). However, the
total RNA extracted from theHL60 cell line showed high
concentrations ofc-myc mRNA. The Ki67 indices were less than5% in
all the placentas and molar pregnanciesstudied.
6 9 10 12Gestational age (weeks)
llff_ Discussion16 Using northern blot and in situ
hybridisation,
Pfeifer-Ohlsson et al, have demonstrated the
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Cheung, Srivastava, Pittaluga, Man, Ngan, Collins
Figure 3 Immunohistochemical staining with anti-fins antibody in
hydatidiform mole to localise c-fins antigenexpression in
syncytiotrophoblast.
expression ofhuman c-myc proto-oncoearly human villous
cytotrophoblastpeak expression at five weeks after corand a deline
by the end of the first trinpregnancy related to differentiation
ofphoblasts into syncytiotrophoblasts.'low expression of c-myc in
the norncentas analysed in our study correspon(advanced gestational
age of the studiedfive to 16 weeks. On the other hand,
....7WM,t
Figure 4 Representative northern blot hybridised with 32p
labelled c-myc cDN.,Total RNA was isolated from normal placentas at
five, eight, nine, 10, and 16pregnancy andfrom hydatidiform moles
at six, nine, 10, 12, and 16 weeks' ges,age. The transcript of
c-myc was detected in the range of 2-4 kilobases in the plafive
weeks' gestation. The human HL-60 leukaemia cell line RNA was used
aspositive control. The positions of 18S and 28S ribosomal RNA are
indicated. T)of the probing of '2P-labelled 28S ribosomal RNA is
shown in the lower panel E
tgenes in expression of c-myc in the moles we studied iswith a
contrary to the findings of previous studies.
iception Sarkar et al have demonstrated c-myc and c-rasaester of
proto-oncogene expression by in situ hybrid-cytotro- isation in
cytotrophoblasts of six hydatidiform2 The moles.'2 The gestational
age of the molesnal pla- studied was not specified. Yokoyama et al
haveds to the also reported the expression of c-myc, c-fims,cases-
and c-sis proto-oncogenes in one mole at thethe low 10th week of
gestation by northern blot analy-
sis and in situ hybridisation."'While such low c-myc expression
in our
study may have been related to the com-paratively advanced
gestational age of themoles studied-six to 16 weeks-it may
beimportant to the pathogenesis of hydatidiformmole: this has
always been controversial.Park'3A attributed the primary
disturbance toabnormalities in the trophoblast, resultingindirectly
in death of the fetus, but Hertig andEdmonds proposed early death
of the embryoas the initiating process.'4 Fox also proposedthat
moles were simply a form of abortion andnot neoplastic. 5 Suresh et
al, from theircomparative study of silver stained
nucleolarorganiser region counts on complete andincomplete
hydatidiform moles as well as onmissed abortions, supported the
latter view.'6Flow cytometric studies also suggested that
thecorrelation of the clinical course with pro-liferative index was
not significant while thecorrelation with ploidy was
positive.'7Thus the
S. low c-myc expression in the moles we studiedfavours the
hypothesis that most hydatidiform
A probe. moles are a form of abortion due to abnormalweeks of
conceptions, rather than a true neoplasia astational c-myc is
usually high in actively proliferatingicenta at cells. This is
further supported by the low:e results growth fraction determined
by anti-Ki673. antibody.
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c-myc and c-fins expression in trophoblastic cells
Pregnancy induces a 1000-fold increase inthe murine uterine
CSF-1 concentration,which appeared to be regulated by
chorionicgonadotrophin,'8 and the synergistic action ofthe female
sex hormones, oestradiol and pro-gesterone.' c-fins expression in
murine pla-centas rises steadily throughout gestation toplateau on
the 18th day.'8 Moreover, theexistence of the CSF-1 receptor and
the c-finsgene product in human choriocarcinoma andteratocarcinoma
cell lines has been demon-strated in previous studies.3 19 20
Simultaneousexpression of a-subunits of hCG was alsoobserved in the
teratocarcinoma. The c-finstranscript has been localised to the
syncytio-trophoblast of hydatidiform moles and chor-iocarcinoma,
where hCG is produced.'3Hence, c-fins may have a role in
placentaldevelopment and may be related to hCGmetabolism. In our
study the highest c-finsexpression found in the normal placentas
wasat the 5th week of gestation; there was a steadydecline in its
expression in placentas of moreadvanced pregnancies. On the other
hand, aparallel increase in c-fins mRNA concentrationwith advancing
gestational age of moles wasobserved. However, there is no
demonstrablerelation between the hCG activity at diagnosisand the
intensity of c-fins expression in thisstudy. The patients with
hydatidiform moleswere followed up with an estimation of
hCGactivities and two of them received treatmentfor persistently
high hCG concentrations. Bothoncogenes did not seem to have a
direct effecton the clinical course of molar pregnancies.This
difference in the intensity of c-fins expres-sion between normal
placentas and molessuggests that c-fins may have a role in
thedevelopment of hydatidiform moles.
This study was supported by grants from the Committee onResearch
and Conference Grant (335/046/0035) and theMedical Faculty Fund
(362/030/3410).
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