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Differentially expressed genes in human trophoblast cells with different degrees of invasiveness
Abstracts:
R.T.C.
and T.G.W.M.S.
Canada
A.15
1996
DifferentiallyExpressedGenes in Human Jrophoblast Cells with Different Degrees of Invasiveness.G. Huch, H.-P. Hohn,
H.-W. Denker; Institute for Anatomie, Universrtyof Essen, MedicalSchool,D-45122Essen,Germany. The majorityof molecularcontrol mechanismsof trophoblast invasivenessare unknown so far. We have analyzed gene expressionof normal trophoblast cells and of JAr malignant trophoblast cells with differing invasive potential by DDRTPCR (differential-display-RT-PCR). In JArcells,high invasivenesswas inducedwith phorbol-12-my ristoyl-13-acetatewhilefor low invasivenesscellswere treated with dibutyryl-CAMP (Placenta14, 1993, A44). Normaltrophoblast cells were isolated from first trimester (highly invasive) and term placentae(“non”-invasive),Accordingto DDRT-PCR, majordifferences’inmRNA-patternswere seen betweennormal and malignanttrophoblast cells, between early and term trophoblast cells, but not between JAr cells with different treatments DifferentiallygeneratedPCR-productsof normaltrophoblast were reamplified, cloned into the vector pCRII, and sequenced. Data base searches identifiedonly a few of the cloned fragments. One of them isolated from first trimester trophoblastshowed 100% homologyto j31-integrinwhich has been suggestedon the protein levelto be involvedin directing trophoblastinvasion(Damskyet al., 1994, Development120, 3657). This result demonstratesthat DDRT-PCRis a reliable tool to detect changesin gene expressionas relatedto regulation of trophoblastdifferentiation/invasiveness. Since RNA probes for cloned PCR-fragmentsusuallyrepresenting3’-regions(mostlyuntranslated)did not give reliableresultsin in-situ-hybridizations we are constructinga cDNAlibrary from first trimester human placentae.This and another one from term placentais being screenedfor the respectivecoding regionsas a basisfor the constructionof appropriateprobes. Supported
by Dr:Mildred-Scheel-Stiftung
Growth hormone (GH) treatment increases circulating concentrations of glucose, fatty acids and IGF-1. We hypothesised that maternal GH treatment may enhance fetal and placental growth. Twelve chronically catheterised pregnant sheep were treated with GH O.lmg/Kg SCbd for 10d from 125f0.3d (meanfSE), while 11 controls received saline. GH increased placental capacity for simple diffusion, measured by steady state 14C-urea clearance. Maternal and fetal blood urea fell, but there were no other changes in fetal or placental metabolism. Fetal and placental weights were similar in GH and control groups at 134f0.3d (fetus 4.3f0.2 vs 4.3+0.3Kg, placenta 441f36 vs 411*42g). These data suggest placental diffusion capacity does not limit fetal growth under these circumstances.
Control
Maternal GH
DO D5 (1nhnn7p=O.O03)
57:4+3i3 REE Blood Urea (r~M&4=0.002)’
4:1&0:4 Fetal Blood Urea (mM~pex~001)
Control
~!-itrol
4:4&0:4
DIO
’
2.3x1.3
3.w.5
2.m.3 4.w.5
H.Hodgess, A.K.Hadjantonskis, J.Bliek, M.de Meulemeester, A.Westerfeld, P.Little, and MMannens. Departments of Clinical Chemistry, Free University Hospital, Amsterdam, Human Genetics, Univ. of Amsterdam, and Biochemistry, Imperial College London. Mice deficient for the Mash-2 gene coding for the lineage-specific transcription factor of the basic helix-loop-helix (bHLH) family die at 10 days post coitum due to placental failure as a consequence of deficient spongiotrophoblast formation. By screening of chromosome 1l-specific cosmids with a degenerate FCR probe, the human Mash2 (HASHI) was isolated. By pulsed field gel electrophoresis, HASH2 was found to map to chromosome 11~15.5 proximal to and in close vicinity of IGF.2 in the syntenic human chromosome region of mouse chromosome 7. This region harbours a cluster of imprinted genes (Mash2, Ins-2, IGF-2, and H19). In the bHLH-region, the human gene was 92.7% and 98% homologous to mouse MASH2 at the DNA and peptide levels, respectively. HASH2 expression was studied by non-radioactive RNA in situ hybridization of early human trophoblast cells of normal and androgenetic (molar) origin. in early human placentae, HASH2 expression was found in extravillus trophoblast cells only. Chorionic villas trophoblast, placental stromal cells as well as maternal placental bed giant cells were negative. In trophoblast cells of androgenetic origin, a differential distribution pattern was observed. Extravillus trophoblast cells with malignant potential (invasive moles) were positive, while extravillus trophoblast cells from complete moles without malignant potential were negative. These expression data suggest, although indirectly, that HASH2 is genetically imprinted with preferential activation of the maternal aliele.
fiir Krebsforschung
Maternal Growth Hormone Treatment Increases Placental Diffusion Capacity but not Fetal or Placental Growth in Sheep. J.E.Harding, P.C.Evans and P.D.Gluckman, Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of Auckland, Auckland, New Zealand.
Days of Treatment rf-Urea Clearance
Human MASH2 (HASH2) Maps lo Chromosome11~15 and is Expressed in Extravillus Trophoblast. :,Oudeians, M.Alders, J.Postmus, Lvan Wijk,
‘E.%8i4 . . 3.0k0.6 3.6kO.5
. .s :*Ef
Effect of hyposmotic cell swelling on [Ca2+li in human placental cytotrophoblast cells in culture. F.H.M.M. van de
S.L.Greenwood and C.P.Siblev, School of Biological Sciences,University of Manchester, Manchester, U.K.
Put,
In the present study we have examined the effect of hyposmotic solutions on [Ca”], in cultured multinucleate cvtotrophoblast cells.
z ’
Figure
“.5w
tune
hnl
0
10
20
30
1. Changesin [Ca2’], were monitored in cellsloaded
with the fluorescent Ca” indicator fitra-2 and bathed in isosmotic (iso; 55mM NaCl HEPES buffer with mannitol; 283mOsm KgH,O-‘) or hyposmotic (hypo; 55mM NaCl HEPES buffer; 138mOsm KgH,O”) solutions. (A) Hyposmotic solutions (+1.2mM Ca*‘) caused a 20% increase in relative cell volume (--; reflected as a fall in fura- fluorescencemeasured at the isosbesticwavelength) which was consistently (n=5) accompanied by a transient rise in [Ca*‘], (-; arrow). (B) In the absenceof extracellular Ca*’ (+lmM EGTA), hyposmotic solutions again causedan increasein cell volume (--;n=3) which was associatedwith a much smaller rise in [Ca2’li (-; arrow). Thus, cytotrophoblast cell swelling in response to hyposmotic solutions
is accompanied
by an increase
in [Ca”],.
R.T.C.
and T.G.W.M.S.
Canada
A.15
1996
DifferentiallyExpressedGenes in Human Jrophoblast Cells with Different Degrees of Invasiveness.G. Huch, H.-P. Hohn,
H.-W. Denker; Institute for Anatomie, Universrtyof Essen, MedicalSchool,D-45122Essen,Germany. The majorityof molecularcontrol mechanismsof trophoblast invasivenessare unknown so far. We have analyzed gene expressionof normal trophoblast cells and of JAr malignant trophoblast cells with differing invasive potential by DDRTPCR (differential-display-RT-PCR). In JArcells,high invasivenesswas inducedwith phorbol-12-my ristoyl-13-acetatewhilefor low invasivenesscellswere treated with dibutyryl-CAMP (Placenta14, 1993, A44). Normaltrophoblast cells were isolated from first trimester (highly invasive) and term placentae(“non”-invasive),Accordingto DDRT-PCR, majordifferences’inmRNA-patternswere seen betweennormal and malignanttrophoblast cells, between early and term trophoblast cells, but not between JAr cells with different treatments DifferentiallygeneratedPCR-productsof normaltrophoblast were reamplified, cloned into the vector pCRII, and sequenced. Data base searches identifiedonly a few of the cloned fragments. One of them isolated from first trimester trophoblastshowed 100% homologyto j31-integrinwhich has been suggestedon the protein levelto be involvedin directing trophoblastinvasion(Damskyet al., 1994, Development120, 3657). This result demonstratesthat DDRT-PCRis a reliable tool to detect changesin gene expressionas relatedto regulation of trophoblastdifferentiation/invasiveness. Since RNA probes for cloned PCR-fragmentsusuallyrepresenting3’-regions(mostlyuntranslated)did not give reliableresultsin in-situ-hybridizations we are constructinga cDNAlibrary from first trimester human placentae.This and another one from term placentais being screenedfor the respectivecoding regionsas a basisfor the constructionof appropriateprobes. Supported
by Dr:Mildred-Scheel-Stiftung
Growth hormone (GH) treatment increases circulating concentrations of glucose, fatty acids and IGF-1. We hypothesised that maternal GH treatment may enhance fetal and placental growth. Twelve chronically catheterised pregnant sheep were treated with GH O.lmg/Kg SCbd for 10d from 125f0.3d (meanfSE), while 11 controls received saline. GH increased placental capacity for simple diffusion, measured by steady state 14C-urea clearance. Maternal and fetal blood urea fell, but there were no other changes in fetal or placental metabolism. Fetal and placental weights were similar in GH and control groups at 134f0.3d (fetus 4.3f0.2 vs 4.3+0.3Kg, placenta 441f36 vs 411*42g). These data suggest placental diffusion capacity does not limit fetal growth under these circumstances.
Control
Maternal GH
DO D5 (1nhnn7p=O.O03)
57:4+3i3 REE Blood Urea (r~M&4=0.002)’
4:1&0:4 Fetal Blood Urea (mM~pex~001)
Control
~!-itrol
4:4&0:4
DIO
’
2.3x1.3
3.w.5
2.m.3 4.w.5
H.Hodgess, A.K.Hadjantonskis, J.Bliek, M.de Meulemeester, A.Westerfeld, P.Little, and MMannens. Departments of Clinical Chemistry, Free University Hospital, Amsterdam, Human Genetics, Univ. of Amsterdam, and Biochemistry, Imperial College London. Mice deficient for the Mash-2 gene coding for the lineage-specific transcription factor of the basic helix-loop-helix (bHLH) family die at 10 days post coitum due to placental failure as a consequence of deficient spongiotrophoblast formation. By screening of chromosome 1l-specific cosmids with a degenerate FCR probe, the human Mash2 (HASHI) was isolated. By pulsed field gel electrophoresis, HASH2 was found to map to chromosome 11~15.5 proximal to and in close vicinity of IGF.2 in the syntenic human chromosome region of mouse chromosome 7. This region harbours a cluster of imprinted genes (Mash2, Ins-2, IGF-2, and H19). In the bHLH-region, the human gene was 92.7% and 98% homologous to mouse MASH2 at the DNA and peptide levels, respectively. HASH2 expression was studied by non-radioactive RNA in situ hybridization of early human trophoblast cells of normal and androgenetic (molar) origin. in early human placentae, HASH2 expression was found in extravillus trophoblast cells only. Chorionic villas trophoblast, placental stromal cells as well as maternal placental bed giant cells were negative. In trophoblast cells of androgenetic origin, a differential distribution pattern was observed. Extravillus trophoblast cells with malignant potential (invasive moles) were positive, while extravillus trophoblast cells from complete moles without malignant potential were negative. These expression data suggest, although indirectly, that HASH2 is genetically imprinted with preferential activation of the maternal aliele.
fiir Krebsforschung
Maternal Growth Hormone Treatment Increases Placental Diffusion Capacity but not Fetal or Placental Growth in Sheep. J.E.Harding, P.C.Evans and P.D.Gluckman, Research Centre for Developmental Medicine and Biology, Department of Paediatrics, University of Auckland, Auckland, New Zealand.
Days of Treatment rf-Urea Clearance
Human MASH2 (HASH2) Maps lo Chromosome11~15 and is Expressed in Extravillus Trophoblast. :,Oudeians, M.Alders, J.Postmus, Lvan Wijk,
‘E.%8i4 . . 3.0k0.6 3.6kO.5
. .s :*Ef
Effect of hyposmotic cell swelling on [Ca2+li in human placental cytotrophoblast cells in culture. F.H.M.M. van de
S.L.Greenwood and C.P.Siblev, School of Biological Sciences,University of Manchester, Manchester, U.K.
Put,
In the present study we have examined the effect of hyposmotic solutions on [Ca”], in cultured multinucleate cvtotrophoblast cells.
z ’
Figure
“.5w
tune
hnl
0
10
20
30
1. Changesin [Ca2’], were monitored in cellsloaded
with the fluorescent Ca” indicator fitra-2 and bathed in isosmotic (iso; 55mM NaCl HEPES buffer with mannitol; 283mOsm KgH,O-‘) or hyposmotic (hypo; 55mM NaCl HEPES buffer; 138mOsm KgH,O”) solutions. (A) Hyposmotic solutions (+1.2mM Ca*‘) caused a 20% increase in relative cell volume (--; reflected as a fall in fura- fluorescencemeasured at the isosbesticwavelength) which was consistently (n=5) accompanied by a transient rise in [Ca*‘], (-; arrow). (B) In the absenceof extracellular Ca*’ (+lmM EGTA), hyposmotic solutions again causedan increasein cell volume (--;n=3) which was associatedwith a much smaller rise in [Ca2’li (-; arrow). Thus, cytotrophoblast cell swelling in response to hyposmotic solutions
is accompanied
by an increase
in [Ca”],.