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The immortalization of neuronal precursor cells
EXPRESSION OF CELL ADHESION MOLECULES IN NORMAL AND PATHOLOGICAL TISSUES ROBERT BRACKENBURY, Department of Anatomy and Cell Biology, University of Cincinnati Medical Center, 231 Bethesda Ave., Cincinnati, OH 45267, U.S.A. Because cell-cell and cell-matrix interactions directly affect growth, differentiation and morphogenesis, abnormal changes in cell-cell interactions could have severe pathological consequences. It is now possible to investigate these alterations at the molecular level, due to progress over the last dozen years in the identification and characterization of several cell adhesion molecules, or CAMs. These molecules are integral membrane glycoproteins that mediate cell to cell binding. Two of these, the neural CAM, N-CAM, and the epithelial CAM, L-CAM, are expressed in early embryonic cells and in a wide variety of tissues during development. N-CAM and L-CAM appear to represent two different classes of adhesion molecules: N-CAM and some other adhesion molecules expressed in nerve tissue (the myelin-associated glycoprotein (MAG) and the myelin protein, PO ) do not require Ca and are built up of domains that are homologous to immunoglobulin gene super family proteins._ In contrast, L-CAM and some other CAMs (N-cadherln and P-cadherln) require Ca for blndlng and share some structural features that are distinct from the N-CAM family. •
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Specific antibodies and cDNA probes are available for many of these CAMs, and have been used to compare the amounts and forms of these molecules that are expressed in normal and pathological tissues. Such studies suggest that injury, oncogenic transformation, and genetic neurological disorders can be accompanied by striking changes in CAM expression that alter the adhesive or migratory behavior of cells•
THE
IMMORTALIZATION
OF NEURONAL
PRECURSOR
CELLS
K. F r e d e r i k s e n , D. L e v y , P - J Jat, N. V a l t z , G. A l m a z a n , R. M c K a y . D e p t s . B r a i n a n d C o g n i t i v e S c i e n c e , B i o l o g y , E 2 5 - 4 3 5 , M. I. T., Cambridge, MA 02139 T h e r e is a g r e a t d e a l of e v i d e n c e w h i c h s h o w s t h a t e a r l y e v e n t s in n e u r o n a l d i f f e r e n t i a t i o n a r e i m p o r t a n t in e s t a b l i s h i n g h i g h l y s p e c i f i c f e a t u r e s of a d u l t b r a i n o r g a n i z a t i o n . We have identif i e d a 200Kd. a n t i g e n p r e s e n t in p r e c u r s o r c e l l s in e a r l y d e v e l o p m e n t of t h e v e r t e b r a t e n e r v o u s s y s t e m . Using retroviruses expressing oncogenes we have established cell lines which express p200. The large T antigen from SV40 virus, the v-myc and neu o n c o g e n e s w e r e u s e d to g e n e r a t e c e l l l i n e s . The T antigen and v-myc induced lines have been most carefully studied. Two c l a s s e s ( p 2 0 0 + a n d p 2 0 0 - ) of T i n d u c e d c e l l l i n e s w e r e g e n erated. The p200- negative cell lines differentiate into cells w i t h n e u r o n a l m o r p h o l o g y a n d a n t i g e n i c i t y in c l o n a l c e l l c u l t u r e . P200+ cell lines differentiate into p200-. GFAP+ cells when they a r e g r o w n in c l o n a l c u l t u r e . When these cell lines are coc u l t u r e d in t h e p r e s e n c e of p r i m a r y c e l l s f r o m t h e e m b r y o n i c b r a i n t h e y c a n d i f f e r e n t i a t e i n t o f a t e s w h i c h t h e y do n o t a d o p t in clonal culture. In c o - c u l t u r e t h e p 2 0 0 - c e l l l i n e s c a n a d o p t a g l i a l as w e l l as n e u r o n a l fate, t h e p 2 0 0 + c e l l l i n e s c a n a c q u i r e a n e u r o n a l fate. These results suggest that we have immortalized functionally competent multipotential precursor cell states from the developing brain. They raise the exciting p o s s i b i l i t y of u s i n g f u r t h e r g e n e t i c a n d t r a n s p l a n t m a n i p u l a t i o n s to i d e n t i f y the m o l e c u l a r s i g n a l s w h i c h r e s t r i c t the f a t e of e a r l y c e l l s in the v e r t e b r a t e b r a i n .
z ÷
•
•
•
.
2 +
•
•
Specific antibodies and cDNA probes are available for many of these CAMs, and have been used to compare the amounts and forms of these molecules that are expressed in normal and pathological tissues. Such studies suggest that injury, oncogenic transformation, and genetic neurological disorders can be accompanied by striking changes in CAM expression that alter the adhesive or migratory behavior of cells•
THE
IMMORTALIZATION
OF NEURONAL
PRECURSOR
CELLS
K. F r e d e r i k s e n , D. L e v y , P - J Jat, N. V a l t z , G. A l m a z a n , R. M c K a y . D e p t s . B r a i n a n d C o g n i t i v e S c i e n c e , B i o l o g y , E 2 5 - 4 3 5 , M. I. T., Cambridge, MA 02139 T h e r e is a g r e a t d e a l of e v i d e n c e w h i c h s h o w s t h a t e a r l y e v e n t s in n e u r o n a l d i f f e r e n t i a t i o n a r e i m p o r t a n t in e s t a b l i s h i n g h i g h l y s p e c i f i c f e a t u r e s of a d u l t b r a i n o r g a n i z a t i o n . We have identif i e d a 200Kd. a n t i g e n p r e s e n t in p r e c u r s o r c e l l s in e a r l y d e v e l o p m e n t of t h e v e r t e b r a t e n e r v o u s s y s t e m . Using retroviruses expressing oncogenes we have established cell lines which express p200. The large T antigen from SV40 virus, the v-myc and neu o n c o g e n e s w e r e u s e d to g e n e r a t e c e l l l i n e s . The T antigen and v-myc induced lines have been most carefully studied. Two c l a s s e s ( p 2 0 0 + a n d p 2 0 0 - ) of T i n d u c e d c e l l l i n e s w e r e g e n erated. The p200- negative cell lines differentiate into cells w i t h n e u r o n a l m o r p h o l o g y a n d a n t i g e n i c i t y in c l o n a l c e l l c u l t u r e . P200+ cell lines differentiate into p200-. GFAP+ cells when they a r e g r o w n in c l o n a l c u l t u r e . When these cell lines are coc u l t u r e d in t h e p r e s e n c e of p r i m a r y c e l l s f r o m t h e e m b r y o n i c b r a i n t h e y c a n d i f f e r e n t i a t e i n t o f a t e s w h i c h t h e y do n o t a d o p t in clonal culture. In c o - c u l t u r e t h e p 2 0 0 - c e l l l i n e s c a n a d o p t a g l i a l as w e l l as n e u r o n a l fate, t h e p 2 0 0 + c e l l l i n e s c a n a c q u i r e a n e u r o n a l fate. These results suggest that we have immortalized functionally competent multipotential precursor cell states from the developing brain. They raise the exciting p o s s i b i l i t y of u s i n g f u r t h e r g e n e t i c a n d t r a n s p l a n t m a n i p u l a t i o n s to i d e n t i f y the m o l e c u l a r s i g n a l s w h i c h r e s t r i c t the f a t e of e a r l y c e l l s in the v e r t e b r a t e b r a i n .