Alterations in the regulatory pathway involving p16, pRb and cdk4 in humanchondrosarcoma

The G1 regulatory pathway involving p16, pRb and cdk4 in the cell cycle has been investigated in human chondrosarcoma. The protein expression of p16, pRb and cdk4 was analyzed by Western blot in cultured cells from eight chon drosarcomas and in two chondrosarcoma cell Lines. Both cell lines and one o ther sample were negative for p16. Moreover, one of the cell lines was pRb- negative and showed a high expression of cdk4 as well. In the other cell li ne and in three other samples pRb of expected size were detected in additio n to a shorter form of the protein. To further investigate the reasons for down-regulation of the p16 protein, the p16-coding gene CDKN2 was analyzed by polymerase chain reaction (PCR, methyl-specific PCR (MSP) and sequencing in all tumor samples as well as in corresponding turner tissues from three of the samples. The p16-negative samples were all found to have homozygous deletion of CDKN2. Another sample showed partial gene methylation and a he terozygous position in codon 148 was detected in one sample. The same base substitution was also found in two of the tissue samples. Finally, cytogene tic analysis of the samples with homozygously deleted CBKN2 revealed multip le structural abnormalities in all three cases. In conclusion, the p16/pRb/ cdk4 pathway may play an important role in the pathogenesis of some chondro sarcomas. (C) 2001 Orthopaedic Research Society. Published by Elsevier Scie nce Ltd. All rights reserved.