SV40 large T antigen expression driven by col2a1 regulatory sequences immortalizes articular chondrocytes but does not allow stabilization of type IIcollagen expression

Immortalization of chondrocytes by SV40 T Ag has often been reported to tri gger the loss of expression of type II collagen, one of the main differenti ation markers, although some immortalized chondrocyte lines maintaining a d ifferentiated phenotype have also been described. Here, we show using trans ient cotransfections in differentiated chondrocytes that, in contrast to c- src, neither SV40 T Ag, nor c-myc, decreases col2a1 transcriptional activit y. Then, we report the possibility of immortalizing rabbit articular chondr ocytes by expression of SV40 T Ag controlled by the col2a1 promoter and enh ancer (pCol2SV). This strategy allows one to select within a population of differentiated chondrocytes those which are able to maintain functional reg ulation of the col2a1 gene through long-term culture. In precrisis pCol2SV- transfected chondrocytes, all-trans-retinoic acid, a down-regulator of col2 a1 expression, induced apoptosis, strongly suggesting the strict control of T Ag expression by col2al regulatory sequences. Some pCol2SV-transfected c hondrocytes were definitively immortalized, after a short crisis period. Ho wever, type II collagen synthesis was restricted to a small proportion of c ells, which went on to decrease with subculture, while the proportion of ce lls expressing T Ag was not affected. In these postcrisis cells, T Ag remai ned at least partially under the control of functional col2al regulatory el ements as assessed by all-trans-retinoic acid down-regulation, (C) 1999 Aca demic Press.