- Email: [email protected]
Identification from a subtracted CDNA library of novel genes associated with differentiation of cytotrophoblast in vitro
Abstracts
ENDOTHELINS-INDUCED INOSITOL PHOSPHATE RELEASE IN SMOOTH MUSCLE OF STEM VILLI VESSELS OF HUMAN TERM PLACENTA : ARE ETA RECEPTORS INVOLVED ? F. Mondon, F. Doualla-Bell Kotto Maka, F. Ferr6. INSERM U. 361, Maternit6 Baudelocque, Paris, France. Specific and very high affinity binding sites for [125I]ET-1 have been previously characterized on membranes of smooth muscle cells of stem villi vessels. Our results suggest the presence of distinct endothelin subtype receptors (1). In the present study we examined in explants of stem villi vessels (deprived of endothelial layer) the relationship between endothelin receptors and phosphoinositide breakdown. ET-1, ET-3 and sarafotoxin S6b but not the ETB receptor selective agonist : sarafotoxin $6c induced dose-dependent increases in inositol phosphate production. In contrast preincubation of explants with increasing concentrations of the ETA receptor antagonist BQ-123 (D-Trp-D-Asp-Pro-D-Val-Leu) resulted in a dosedependent inhibition of ET-1 (10-6M) induced inositol phosphate release. This inhibition was complete at 10"4M BQ-123. These data demonstrated that in smooth muscle of stem villi vessels only the ETA subtype receptors were coupled with the phospholipase C system. (1) C. Robaut et al., Placenta, 1991, 12, 55-67. This study was supported by MGEN and INSERM.
IDENTIFICATION FROM A SUBTRACTED CDNA LIBRARY OF NOVEL GENES ASSOCIATED WITH DIFFERENTIATION OF CYTOTROPHOBLAST IN VITRO. D.W.Morrish, E.Linetsky, D.Bhardwaj, M.C.Paterson, R.Godbout, Departments of Medicine, University of Alberta, and Molecular Oncology Program, Cross Cancer Institute, Edmonton, Alberta Canada We have shown that the growth factors EGF, CSF-1, and GM-CSF are inducers, and TGI~ 1 is an inhibitor, of cytotrophoblast differentiation in vitro. To identify genetic regulators of this process, we have constructed a subtractive cDNA library between freshly isolated cytotrophoblast ("-" pool) and cytotrophoblast stimulated by 10 ng/ml rhEGF to differentiate into syncytium ("+" pool; harvested at 24,48,72 h of EGF exposure), cDNA was synthesized from the "-" and "+" pools and subtraction performed by the deletion cloning method. The resultant DNA hybrids were inserted into the Lambda ZAPII vector and amplified. The library was differentiallyscreened 3 times with cDNA probes from the "-" and "+" pools and ultimately 94 subtracted clones were isolated. Amino acid sequencing to date has identified a-subunit of ATP synthase, fibronectin, and three novel clones. One of these may have partial homology to tyrosine kinase. Characterization of these clones is ongoing. We conclude that differentiating cytotrophoblast expresses a variety of known and potentially novel genes. This method permits further identification of factors that may regulate trophoblast differentiation.
A.53
ENDOTHELINS-INDUCED INOSITOL PHOSPHATE RELEASE IN SMOOTH MUSCLE OF STEM VILLI VESSELS OF HUMAN TERM PLACENTA : ARE ETA RECEPTORS INVOLVED ? F. Mondon, F. Doualla-Bell Kotto Maka, F. Ferr6. INSERM U. 361, Maternit6 Baudelocque, Paris, France. Specific and very high affinity binding sites for [125I]ET-1 have been previously characterized on membranes of smooth muscle cells of stem villi vessels. Our results suggest the presence of distinct endothelin subtype receptors (1). In the present study we examined in explants of stem villi vessels (deprived of endothelial layer) the relationship between endothelin receptors and phosphoinositide breakdown. ET-1, ET-3 and sarafotoxin S6b but not the ETB receptor selective agonist : sarafotoxin $6c induced dose-dependent increases in inositol phosphate production. In contrast preincubation of explants with increasing concentrations of the ETA receptor antagonist BQ-123 (D-Trp-D-Asp-Pro-D-Val-Leu) resulted in a dosedependent inhibition of ET-1 (10-6M) induced inositol phosphate release. This inhibition was complete at 10"4M BQ-123. These data demonstrated that in smooth muscle of stem villi vessels only the ETA subtype receptors were coupled with the phospholipase C system. (1) C. Robaut et al., Placenta, 1991, 12, 55-67. This study was supported by MGEN and INSERM.
IDENTIFICATION FROM A SUBTRACTED CDNA LIBRARY OF NOVEL GENES ASSOCIATED WITH DIFFERENTIATION OF CYTOTROPHOBLAST IN VITRO. D.W.Morrish, E.Linetsky, D.Bhardwaj, M.C.Paterson, R.Godbout, Departments of Medicine, University of Alberta, and Molecular Oncology Program, Cross Cancer Institute, Edmonton, Alberta Canada We have shown that the growth factors EGF, CSF-1, and GM-CSF are inducers, and TGI~ 1 is an inhibitor, of cytotrophoblast differentiation in vitro. To identify genetic regulators of this process, we have constructed a subtractive cDNA library between freshly isolated cytotrophoblast ("-" pool) and cytotrophoblast stimulated by 10 ng/ml rhEGF to differentiate into syncytium ("+" pool; harvested at 24,48,72 h of EGF exposure), cDNA was synthesized from the "-" and "+" pools and subtraction performed by the deletion cloning method. The resultant DNA hybrids were inserted into the Lambda ZAPII vector and amplified. The library was differentiallyscreened 3 times with cDNA probes from the "-" and "+" pools and ultimately 94 subtracted clones were isolated. Amino acid sequencing to date has identified a-subunit of ATP synthase, fibronectin, and three novel clones. One of these may have partial homology to tyrosine kinase. Characterization of these clones is ongoing. We conclude that differentiating cytotrophoblast expresses a variety of known and potentially novel genes. This method permits further identification of factors that may regulate trophoblast differentiation.
A.53