SV40 large T antigen expression driven by col2a1 regulatory sequences immortalizes articular chondrocytes but does not allow stabilization of type II collagen expression.
Immortalization of chondrocytes by SV40 T Ag has often been reported to trigger the loss of expression of type II collagen, one of the main differentiation markers, although some immortalized chondrocyte lines maintaining a differentiated phenotype have also been described. Here, we show using transient cotransfections in differentiated chondrocytes that, in contrast to c-src, neither SV40 T Ag, nor c-myc, decreases col2a1 transcriptional activity. Then, we report the possibility of immortalizing rabbit articular chondrocytes by expression of SV40 T Ag controlled by the col2a1 promoter and enhancer (pCol2SV). This strategy allows one to select within a population of differentiated chondrocytes those which are able to maintain functional regulation of the col2a1 gene through long-term culture. In precrisis pCol2SV-transfected chondrocytes, all-trans-retinoic acid, a down-regulator of col2a1 expression, induced apoptosis, strongly suggesting the strict control of T Ag expression by col2a1 regulatory sequences. Some pCol2SV-transfected chondrocytes were definitively immortalized, after a short crisis period. However, type II collagen synthesis was restricted to a small proportion of cells, which went on to decrease with subculture, while the proportion of cells expressing T Ag was not affected. In these postcrisis cells, T Ag remained at least partially under the control of functional col2a1 regulatory elements as assessed by all-trans-retinoic acid down-regulation.
Steimberg
N
N
Ecole Pratique des Hautes Etudes, Laboratoire de Pharmacologie Cellulaire, Centre de Recherches Biomédicales des Cordeliers, 15 rue de l'Ecole de Médecine, Paris, 75006, France.
Viengchareun
S
S
Biehlmann
F
F
Guénal
I
I
Mignotte
B
B
Adolphe
M
M
Thenet
S
S
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000096:biosynthesis* / Q000235:genetics; D017209:Apoptosis / Q000187:drug effects; D002454:Cell Differentiation / Q000235:genetics; D002461:Cell Line, Transformed; D002472:Cell Transformation, Viral / Q000235:genetics; D019902:Chondrocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects; D004742:Enhancer Elements, Genetic; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000187:drug effects; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011817:Rabbits; D051381:Rats; D013539:Simian virus 40 / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology
D000952:Antigens, Polyomavirus Transforming; D014212:Tretinoin; D003094:Collagen