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Proliferation and early expression of soluble cytokeratin in differentiating murine F9 embryonal carcinoma (EC) cells
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Detection of DNA synthesis of early mammalian cleavage stages with Anti-bromodeoxyuridine.
PROLIFERATION AND EARLY EXPRESSION OF SOLUBLE CYTOKERATIN IN DIFFERENTIATING MURINE F9 EMBRYONAL CARCINOMA (EC) CELLS *A. Laasonen, #P. Kurki, and #E. Lehtonen *Helsinki Univ. Central Hosp., Dept. of Radiother. & Oncol.; #Univ. of Helsinki, Dept. of Pathol., SF00290 Helsinki, Finland Flow cytometry (FCM), immunofluorescence microscopy (IF) and immunoblotUng (IB) were used to study changes in proliferation and dillerentation of F9 EC cells upon treatment with retinoic acid (RA) and dibutyryl cyclic AMP (dbc-AMP). Proliferation was monitored by bromodeoxyuridine labeling, and cytokeratin (CK)' was .used as a marker for differentiation. The cell cycle time (Tc) of untreated F9 cells was 12 hr. After 3 and 5 days of RA/dbcAMP-treatment, the Tc had prolonged to 4 hr and 12 hr, respectively, due to lengthening of G1 and S phases. The change in the proliferation rate of differentiating cells was not homogeneous. A small fraction of cells, possibly noncycling cells, stayed in G1 phase at 3 days after treatment. By FCM, CK was found already after 2 days of treatment in whole cells but not in detergent-extracted cells. This result and the IF- and IB-findings suggest that CK is initially in a soluble, nonfilamentous form, and becomes later organized into cytoplasmic filaments thai resist detergent extraction. We conclude that CK gene(s) (coding for an Mr-52 000 polypeptide) become expressed at an early phase of Fg cell differentiation, and typical CK filaments appear somewhat later.
G. te Kronnie 1, M.L. 5oerjan 2 and 5. Talsma 1. Dept. of (l)Experimental Animal Morphology and Cell Biology (2) Genetics. Agricultural University Wageningen NL. In vitro point labeling of early mammalian cleavage stages offers a possibility to detect DNA synthesis of individual b l a s t o m e r e s at chosen time intervals upon fertilization. We used 4h bromodeoxyuridine 5rdUrd incorporation in vitro followed by anti-SrdUrd monoclonal antibody incubation of nucleus spread preparations which allows rapid evaluation of DNA synthesis of nuclei. Moreover the localization of the anti-BrdUrd reaction product on the nuclei gives information on the phases within the DNA-S(synthetic) phase. Our studies with BrdUrd point labeling of early mammalian cleavage stages shows (1) Asynchrony in DNA synthetic activity between the two blastomeres of mouse 2Cell embryos occurs. We report minimal values of 2Cell G2÷M ( 3h and OCell G2÷M ( 3 h . (2) Both mouse ans pig show asynchrony between blastomeres which becomes more apparent in later cleavage stages. The relevance of blastomere asynchrony with respect to early differentiation in inner- and outer-cells will be discussed.
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A N E W M E M B E R OF I M M U N O G L O B U L I N S U P E R F A M I L Y W I T H H O M O L O G Y TO B - C H A I N OF C L A S S II A N T I G E N A N D TO V D O M A I N IS E X P R E S S E D IN T E R A T O C A R C I N O M A STEM CELLS. T. M i y a u c h i , T. K a n e k u r a , M, Q z a w ~ and T. M u r a m a t s u . J a p a n I m m u n o r e s e a r c h Lab., T a k a s a k i 370, J a p a n and Dept. of Biochem. and D e r m a t o l . , Fac. of Med., K a g o s h i m a Univ., K a g o s h i m a 890, Japan. As a c e l l - s u r f a c e g l y c o p r o t e i n exp r e s s e d in t e r a t o c a r c i n o m a s t e m cells, we i d e n t i f i e d a n e w m e m b e r of i m m u n o globulin (Ig) s u p e r f a m i l y , EI2. S t r u c ture of EI2 w a s e l u c i d a t e d f r o m the s t r u c t u r e of the c D N A clones. EI2 h a d one I g - l i k e d o m a i n in a m o l e c u l e . The I g - l i k e d o m a i n w a s u n i q u e : it h a d h o m o l o g y w i t h b o t h B - c h a i n of M H C class II a n t i g e n a n d V d o m a i n of Ig. EI2 R N A w a s a l s o d e t e c t e d in 9 d a y m o u s e e m b r y o s a n d s e v e r a l o r g a n s of a d u l t mice, such as the testis. We p r e v i o u s l y d e s c r i b e d a n o t h e r m e m b e r of Ig s u p e r f a m i l y (Gp70) e x p r e s s e d in teratocarcinoma s t e m cells (J. Biol0 Chem., 263, 3059, 1988). T h e r e f o r e , we p r o p o s e that c e l l - s u r f a c e r e c o g n i t i o n b e t w e e n m e m b e r s of Ig s u p e r f a m i l y is i m p o r t a n t in e a r l y e m b r y o g e n e s i s just as in the case of l y m p h o i d cell i n t e r a c t i o n s and n e r v e cell i n t e r a c t i o n s .
Presence of actin during "in vivo" growth of Embryoid Bodies derived from OTT6050 Teratocarc~. Monzo,M. ,de Anta, J.M~. ,Barnadas,A. ,Ruano~D. Asc itic form of Teratocarcincrna (TC )shows structures resembling to early mouse embryos called Embryoid BodiesfEB).Morphologically • EB show an external endoderma! cell layer which sorrounds an amount of Embryona! Carcinoma cells that can be divided into two types, simple and cystic EB, depending on the presence or absence of internal cavities. However, an aspect which has not studied in depth is the "in vivo'EB growth dynamics. In this work, we have isolated a morphologically homogeneus EB population characterized by showing a few EC cells. We have injected intraperitonea!!y these F/3 into isogenic 129 ter/Sv mice and we have observed their morphology throughout different days of evolution by Scanning Electron Microscopy.We have verified that injected EB turn into more complex EB, which show a great amount of EC cells, that they are able to form cavities, to ~buddlng" and to generate a great diversity of morphological EB types. At the same time, using a monoc!ona! antibody anti-actln and by immunof !uorescence techniques we have studied the presence of actin during'in vivo'growth and evolution of EB.
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Detection of DNA synthesis of early mammalian cleavage stages with Anti-bromodeoxyuridine.
PROLIFERATION AND EARLY EXPRESSION OF SOLUBLE CYTOKERATIN IN DIFFERENTIATING MURINE F9 EMBRYONAL CARCINOMA (EC) CELLS *A. Laasonen, #P. Kurki, and #E. Lehtonen *Helsinki Univ. Central Hosp., Dept. of Radiother. & Oncol.; #Univ. of Helsinki, Dept. of Pathol., SF00290 Helsinki, Finland Flow cytometry (FCM), immunofluorescence microscopy (IF) and immunoblotUng (IB) were used to study changes in proliferation and dillerentation of F9 EC cells upon treatment with retinoic acid (RA) and dibutyryl cyclic AMP (dbc-AMP). Proliferation was monitored by bromodeoxyuridine labeling, and cytokeratin (CK)' was .used as a marker for differentiation. The cell cycle time (Tc) of untreated F9 cells was 12 hr. After 3 and 5 days of RA/dbcAMP-treatment, the Tc had prolonged to 4 hr and 12 hr, respectively, due to lengthening of G1 and S phases. The change in the proliferation rate of differentiating cells was not homogeneous. A small fraction of cells, possibly noncycling cells, stayed in G1 phase at 3 days after treatment. By FCM, CK was found already after 2 days of treatment in whole cells but not in detergent-extracted cells. This result and the IF- and IB-findings suggest that CK is initially in a soluble, nonfilamentous form, and becomes later organized into cytoplasmic filaments thai resist detergent extraction. We conclude that CK gene(s) (coding for an Mr-52 000 polypeptide) become expressed at an early phase of Fg cell differentiation, and typical CK filaments appear somewhat later.
G. te Kronnie 1, M.L. 5oerjan 2 and 5. Talsma 1. Dept. of (l)Experimental Animal Morphology and Cell Biology (2) Genetics. Agricultural University Wageningen NL. In vitro point labeling of early mammalian cleavage stages offers a possibility to detect DNA synthesis of individual b l a s t o m e r e s at chosen time intervals upon fertilization. We used 4h bromodeoxyuridine 5rdUrd incorporation in vitro followed by anti-SrdUrd monoclonal antibody incubation of nucleus spread preparations which allows rapid evaluation of DNA synthesis of nuclei. Moreover the localization of the anti-BrdUrd reaction product on the nuclei gives information on the phases within the DNA-S(synthetic) phase. Our studies with BrdUrd point labeling of early mammalian cleavage stages shows (1) Asynchrony in DNA synthetic activity between the two blastomeres of mouse 2Cell embryos occurs. We report minimal values of 2Cell G2÷M ( 3h and OCell G2÷M ( 3 h . (2) Both mouse ans pig show asynchrony between blastomeres which becomes more apparent in later cleavage stages. The relevance of blastomere asynchrony with respect to early differentiation in inner- and outer-cells will be discussed.
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A N E W M E M B E R OF I M M U N O G L O B U L I N S U P E R F A M I L Y W I T H H O M O L O G Y TO B - C H A I N OF C L A S S II A N T I G E N A N D TO V D O M A I N IS E X P R E S S E D IN T E R A T O C A R C I N O M A STEM CELLS. T. M i y a u c h i , T. K a n e k u r a , M, Q z a w ~ and T. M u r a m a t s u . J a p a n I m m u n o r e s e a r c h Lab., T a k a s a k i 370, J a p a n and Dept. of Biochem. and D e r m a t o l . , Fac. of Med., K a g o s h i m a Univ., K a g o s h i m a 890, Japan. As a c e l l - s u r f a c e g l y c o p r o t e i n exp r e s s e d in t e r a t o c a r c i n o m a s t e m cells, we i d e n t i f i e d a n e w m e m b e r of i m m u n o globulin (Ig) s u p e r f a m i l y , EI2. S t r u c ture of EI2 w a s e l u c i d a t e d f r o m the s t r u c t u r e of the c D N A clones. EI2 h a d one I g - l i k e d o m a i n in a m o l e c u l e . The I g - l i k e d o m a i n w a s u n i q u e : it h a d h o m o l o g y w i t h b o t h B - c h a i n of M H C class II a n t i g e n a n d V d o m a i n of Ig. EI2 R N A w a s a l s o d e t e c t e d in 9 d a y m o u s e e m b r y o s a n d s e v e r a l o r g a n s of a d u l t mice, such as the testis. We p r e v i o u s l y d e s c r i b e d a n o t h e r m e m b e r of Ig s u p e r f a m i l y (Gp70) e x p r e s s e d in teratocarcinoma s t e m cells (J. Biol0 Chem., 263, 3059, 1988). T h e r e f o r e , we p r o p o s e that c e l l - s u r f a c e r e c o g n i t i o n b e t w e e n m e m b e r s of Ig s u p e r f a m i l y is i m p o r t a n t in e a r l y e m b r y o g e n e s i s just as in the case of l y m p h o i d cell i n t e r a c t i o n s and n e r v e cell i n t e r a c t i o n s .
Presence of actin during "in vivo" growth of Embryoid Bodies derived from OTT6050 Teratocarc~. Monzo,M. ,de Anta, J.M~. ,Barnadas,A. ,Ruano~D. Asc itic form of Teratocarcincrna (TC )shows structures resembling to early mouse embryos called Embryoid BodiesfEB).Morphologically • EB show an external endoderma! cell layer which sorrounds an amount of Embryona! Carcinoma cells that can be divided into two types, simple and cystic EB, depending on the presence or absence of internal cavities. However, an aspect which has not studied in depth is the "in vivo'EB growth dynamics. In this work, we have isolated a morphologically homogeneus EB population characterized by showing a few EC cells. We have injected intraperitonea!!y these F/3 into isogenic 129 ter/Sv mice and we have observed their morphology throughout different days of evolution by Scanning Electron Microscopy.We have verified that injected EB turn into more complex EB, which show a great amount of EC cells, that they are able to form cavities, to ~buddlng" and to generate a great diversity of morphological EB types. At the same time, using a monoc!ona! antibody anti-actln and by immunof !uorescence techniques we have studied the presence of actin during'in vivo'growth and evolution of EB.
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