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Immortalization of human chondrocytes by temperature sensitive SV40 large T antigen—phenotypic characterization of several clones
Osteoarthritis and Cartilage (1993) 1, 1-84 © 1993 The Osteoarthritis Research Society
1063 4584/93f010001 -$08.00]0
OSTEOARTHRITIS and CARTILAGE R E G U L A T I O N OF CHONDROCYTE DIVISION AND D I F F E R E N T I A T I O N The p r o t e i n k i n a s e inhibitor HA1004 e n h a n c e s TGFfl-dependent r e - e x p r e s s i o n of the c h o n d r o c y t e p h e n o p t y p e u n d e r s e r u m - f r e e c o n d i t i o n s PAUL D. BENYA AND SILVIA R. PADILLA
University of Southern California, Orthopaedic Hospital, Los Angeles, CA, U.S.A. The aim of this s t u d y was to identify p a r t s of t h e s i g n a l i n g p a t h w a y used by TGFfl to e n h a n c e e x p r e s s i o n of t h e c h o n d r o c y t e d i f f e r e n t i a t e d phenotype. We h a v e p r e v i o u s l y shown t h a t r e t i n o i c acid (RA)-modulated r a b b i t c h o n d r o c y t e s , w h i c h no l o n g e r synthesize t y p e II collagen, c a n be r e l e a s e d from RA t r e a t m e n t in t h e a b s e n c e of serum w i t h o u t r e g a i n i n g type II synthesis. However, TGFfl s t i m u l a t e s re-expression of type II synthesis, a n d at low TGFfl doses this re-expression is e n h a n c e d by t h e m i c r o f i l a m e n t modifying d r u g dihydroc y t o c h a l a s i n B (DHCB). Because of t h e p u t a t i v e k i n a s e d o m a i n of t h e type II TGFfl receptor, and the p o t e n t i a l for a k i n a s e cascade, we e v a l u a t e d t h e effect of t h e p r o t e i n k i n a s e i n h i b i t o r HA1004 on TGFfl d e p e n d e n t c h o n d r o c y t e re-expression in this system. R a b b i t a r t i c u l a r c h o n d r o c y t e s were t r e a t e d w i t h 1 pg/ml RA d u r i n g p r i m a r y c u l t u r e and t r a n s f e r r e d to s e c o n d a r y c u l t u r e u n d e r the same conditions. RA was removed from confluent c u l t u r e s a n d cells were maint a i n e d serum-free in m e d i u m s u p p l e m e n t e d with insulin, t r a n s f e r r i n , s e l e n i u m and BSA linoleic acid. A f t e r 1 week, t r e a t m e n t with 0.3 or 5 ng/ml T G F f l l was i n i t i a t e d in t h e presence or a b s e n c e of DHCB and HA1004. C o l l a g e n p h e n o t y p e was d e t e r m i n e d by 2D C N B r
• peptide m a p p i n g of t r i t i a t e d b i o s y n t h e s i z e d collagen; PG s y n t h e s i s was d e t e r m i n e d as pronase-soluble. CTAB-precipitable cpm. A low level of re-expression was i n d u c e d by 0.3 ng/ml TGFfl a l o n e on d a y 1 of t r e a t m e n t . A d d i t i o n of HA1004 (10 ]IM caused a d o u b l i n g in this r e s p o n s e w h e t h e r m e a s u r e d by t y p e II c o l l a g e n re-expression o r PG synthesis. In the l a t t e r case a s i m i l a r s t i m u l a t i o n was caused by DHCB, and t h e c o m b i n a t i o n of HA1004 a n d DHC8 was additive. A t 5 ng/ml TGFfl, HA10004 was no l o n g e r s t i m u l a t o r y , in c o n t r a s t to DHCB. HA1004 was less effective at 40 a n d 120 gM, w h e r e it would be e x p e c t e d to i n h i b i t P K A and PKC, respectively. The e n h a n c e m e n t of TGFfl-dependent re-expression by HA1004 suggests t h a t TGFfl s i g n a l i n g is s u p p r e s s i b l e by a n u n k n o w n kinase. HA1004 a p p e a r s to remove this inhibition. HA1004 c l e a r l y does not i n h i b i t a n y p a r t of t h e expected TGFfl k i n a s e cascade. Its a c t i o n occurs a t doses lower t h a n those t h a t effectively i n h i b i t t h e m a j o r k i n a s e t r a n s d u c t i o n systems, and its a c t i o n a p p e a r s d i s t i n c t from t h a t of DHCB. Given the m u l t i p l e levels of t r a n s c r i p t i o n t h a t a r e expected to p a r t i c i p a t e in TGFfl d e p e n d e n t re-expression, it is not possible to d e t e r m i n e an e x a c t l o c a t i o n for the effects of HA1004 at this time.
I m m o r t a l i z a t i o n of h u m a n c h o n d r o c y t e s by t e m p e r a t u r e s e n s i t i v e SV40 large T a n t i g e n - - p h e n o t y p i c c h a r a c t e r i z a t i o n of s e v e r a l clones B. BENOIT*, A. TACHET DES COMBES*, G. VERBRUGGENt, S. DEMIGNOT*, A.-M. MALFAIRt, S. THENET*, E. VEYSt AND M. ADOLPHE*
*Laboratoire de Pharmacologie Cellulaire, EPHE, Paris, France ¢ UZ-Rijkuniversiteit, Gent, Belgium The d e v e l o p m e n t of an i m m o r t a l i z e d h u m a n a r t i c u l a r c h o n d r o c y t e cell line would be a v a l u a b l e tool for p h a r m a c o - t o x i c o l o g i c a l studies. T r a n s f e c t i o n of h u m a n c h o n d r o c y t e s o r i g i n a t e d from seven donors was undert a k e n with s e v e r a l oncogenes. A f t e r a period of l a t e n c y of 6-9 m o n t h s i m m o r t a l i z e d clones emerged from cells t r a n s f e c t e d by t e m p e r a t u r e sensitive SV40 large T antigen. Thirty-two clones from t h r e e different donors h a v e been i s o l a t e d a n d expanded. I n d i r e c t immunofluorescence study showed positive s t a i n i n g for l a r g e T a n t i g e n and t y p e II a n d / o r type I c o l l a g e n for some clones. This i m m u n o l a b e l i n g p e r m i t t e d the s e l e c t i o n of clones for f u r t h e r c h a r a c t e r i z a t i o n . Cell g r o w t h was studied at b o t h permissive (33°C) and n o n p e r m i s s i v e t e m p e r a t u r e (39°C). G r o w t h curves showed t h a t t h e
g r o w t h r a t e of all t h e clones studied is h i g h e r at 39°C -~han at 33°C. C o l l a g e n p h e n o t y p e expression was s t u d i e d by SDS-PAGE a n a l y s i s of t h e i n t a c t c h a i n s of c o l l a g e n after t r i t i a t e d p r o l i n e i n c o r p o r a t i o n . The p a t t e r n o b t a i n e d showed t h a t some of t h e clones s t u d i e d synthesized t y p e III a n d t y p e I collagens. However, it is n o t possible to infer from SDS-PAGE w h e t h e r t h e r e is t y p e II and type I t r i m e r c o l l a g e n synthesis. F o r some clones the p a t t e r n a t 39°C differs from t h e p a t t e r n at 33°C. Two-dimensional e l e c t r o p h o r e s i s a n d N o r t h e r n blot a n a l y s i s should p e r m i t to e l u c i d a t e the t y p e of c o l l a g e n synthesized in o r d e r to select a few cell lines p o t e n t i a l l y i n t e r e s t i n g for p h a r m a c o - t o x i c o l o g i c a l studies and to c h a r a c t e r i z e them more deeply.
1063 4584/93f010001 -$08.00]0
OSTEOARTHRITIS and CARTILAGE R E G U L A T I O N OF CHONDROCYTE DIVISION AND D I F F E R E N T I A T I O N The p r o t e i n k i n a s e inhibitor HA1004 e n h a n c e s TGFfl-dependent r e - e x p r e s s i o n of the c h o n d r o c y t e p h e n o p t y p e u n d e r s e r u m - f r e e c o n d i t i o n s PAUL D. BENYA AND SILVIA R. PADILLA
University of Southern California, Orthopaedic Hospital, Los Angeles, CA, U.S.A. The aim of this s t u d y was to identify p a r t s of t h e s i g n a l i n g p a t h w a y used by TGFfl to e n h a n c e e x p r e s s i o n of t h e c h o n d r o c y t e d i f f e r e n t i a t e d phenotype. We h a v e p r e v i o u s l y shown t h a t r e t i n o i c acid (RA)-modulated r a b b i t c h o n d r o c y t e s , w h i c h no l o n g e r synthesize t y p e II collagen, c a n be r e l e a s e d from RA t r e a t m e n t in t h e a b s e n c e of serum w i t h o u t r e g a i n i n g type II synthesis. However, TGFfl s t i m u l a t e s re-expression of type II synthesis, a n d at low TGFfl doses this re-expression is e n h a n c e d by t h e m i c r o f i l a m e n t modifying d r u g dihydroc y t o c h a l a s i n B (DHCB). Because of t h e p u t a t i v e k i n a s e d o m a i n of t h e type II TGFfl receptor, and the p o t e n t i a l for a k i n a s e cascade, we e v a l u a t e d t h e effect of t h e p r o t e i n k i n a s e i n h i b i t o r HA1004 on TGFfl d e p e n d e n t c h o n d r o c y t e re-expression in this system. R a b b i t a r t i c u l a r c h o n d r o c y t e s were t r e a t e d w i t h 1 pg/ml RA d u r i n g p r i m a r y c u l t u r e and t r a n s f e r r e d to s e c o n d a r y c u l t u r e u n d e r the same conditions. RA was removed from confluent c u l t u r e s a n d cells were maint a i n e d serum-free in m e d i u m s u p p l e m e n t e d with insulin, t r a n s f e r r i n , s e l e n i u m and BSA linoleic acid. A f t e r 1 week, t r e a t m e n t with 0.3 or 5 ng/ml T G F f l l was i n i t i a t e d in t h e presence or a b s e n c e of DHCB and HA1004. C o l l a g e n p h e n o t y p e was d e t e r m i n e d by 2D C N B r
• peptide m a p p i n g of t r i t i a t e d b i o s y n t h e s i z e d collagen; PG s y n t h e s i s was d e t e r m i n e d as pronase-soluble. CTAB-precipitable cpm. A low level of re-expression was i n d u c e d by 0.3 ng/ml TGFfl a l o n e on d a y 1 of t r e a t m e n t . A d d i t i o n of HA1004 (10 ]IM caused a d o u b l i n g in this r e s p o n s e w h e t h e r m e a s u r e d by t y p e II c o l l a g e n re-expression o r PG synthesis. In the l a t t e r case a s i m i l a r s t i m u l a t i o n was caused by DHCB, and t h e c o m b i n a t i o n of HA1004 a n d DHC8 was additive. A t 5 ng/ml TGFfl, HA10004 was no l o n g e r s t i m u l a t o r y , in c o n t r a s t to DHCB. HA1004 was less effective at 40 a n d 120 gM, w h e r e it would be e x p e c t e d to i n h i b i t P K A and PKC, respectively. The e n h a n c e m e n t of TGFfl-dependent re-expression by HA1004 suggests t h a t TGFfl s i g n a l i n g is s u p p r e s s i b l e by a n u n k n o w n kinase. HA1004 a p p e a r s to remove this inhibition. HA1004 c l e a r l y does not i n h i b i t a n y p a r t of t h e expected TGFfl k i n a s e cascade. Its a c t i o n occurs a t doses lower t h a n those t h a t effectively i n h i b i t t h e m a j o r k i n a s e t r a n s d u c t i o n systems, and its a c t i o n a p p e a r s d i s t i n c t from t h a t of DHCB. Given the m u l t i p l e levels of t r a n s c r i p t i o n t h a t a r e expected to p a r t i c i p a t e in TGFfl d e p e n d e n t re-expression, it is not possible to d e t e r m i n e an e x a c t l o c a t i o n for the effects of HA1004 at this time.
I m m o r t a l i z a t i o n of h u m a n c h o n d r o c y t e s by t e m p e r a t u r e s e n s i t i v e SV40 large T a n t i g e n - - p h e n o t y p i c c h a r a c t e r i z a t i o n of s e v e r a l clones B. BENOIT*, A. TACHET DES COMBES*, G. VERBRUGGENt, S. DEMIGNOT*, A.-M. MALFAIRt, S. THENET*, E. VEYSt AND M. ADOLPHE*
*Laboratoire de Pharmacologie Cellulaire, EPHE, Paris, France ¢ UZ-Rijkuniversiteit, Gent, Belgium The d e v e l o p m e n t of an i m m o r t a l i z e d h u m a n a r t i c u l a r c h o n d r o c y t e cell line would be a v a l u a b l e tool for p h a r m a c o - t o x i c o l o g i c a l studies. T r a n s f e c t i o n of h u m a n c h o n d r o c y t e s o r i g i n a t e d from seven donors was undert a k e n with s e v e r a l oncogenes. A f t e r a period of l a t e n c y of 6-9 m o n t h s i m m o r t a l i z e d clones emerged from cells t r a n s f e c t e d by t e m p e r a t u r e sensitive SV40 large T antigen. Thirty-two clones from t h r e e different donors h a v e been i s o l a t e d a n d expanded. I n d i r e c t immunofluorescence study showed positive s t a i n i n g for l a r g e T a n t i g e n and t y p e II a n d / o r type I c o l l a g e n for some clones. This i m m u n o l a b e l i n g p e r m i t t e d the s e l e c t i o n of clones for f u r t h e r c h a r a c t e r i z a t i o n . Cell g r o w t h was studied at b o t h permissive (33°C) and n o n p e r m i s s i v e t e m p e r a t u r e (39°C). G r o w t h curves showed t h a t t h e
g r o w t h r a t e of all t h e clones studied is h i g h e r at 39°C -~han at 33°C. C o l l a g e n p h e n o t y p e expression was s t u d i e d by SDS-PAGE a n a l y s i s of t h e i n t a c t c h a i n s of c o l l a g e n after t r i t i a t e d p r o l i n e i n c o r p o r a t i o n . The p a t t e r n o b t a i n e d showed t h a t some of t h e clones s t u d i e d synthesized t y p e III a n d t y p e I collagens. However, it is n o t possible to infer from SDS-PAGE w h e t h e r t h e r e is t y p e II and type I t r i m e r c o l l a g e n synthesis. F o r some clones the p a t t e r n a t 39°C differs from t h e p a t t e r n at 33°C. Two-dimensional e l e c t r o p h o r e s i s a n d N o r t h e r n blot a n a l y s i s should p e r m i t to e l u c i d a t e the t y p e of c o l l a g e n synthesized in o r d e r to select a few cell lines p o t e n t i a l l y i n t e r e s t i n g for p h a r m a c o - t o x i c o l o g i c a l studies and to c h a r a c t e r i z e them more deeply.