Connective Tissue Oncology Society

2001 CTOS Annual Meeting Posters— Medical Oncology

THE MUTATION ANALYSIS OF NFAT1 IN CHONDROSARCOMA
Tomoki Aoyama¹, Satoshi Nagayama³, Takeshi Okamoto¹, Taisuke Hosaka¹, Takeharu Nakamata¹, Koichi Nishijo¹, Tomitaka Nakayama², Takashi Nakamura², junya Toguchida¹
¹Institiute for Frontier Medical Science Kyoto Univeresity, ²Department of Orthopaedics Kyoto University, ³Department of Surg. Surgical Basic Science Kyoto Univeresity

OBJECTIVE: NFAT(Nuclear factor of activated T cell) is a family of transcription factor regulating gene expression in the immune system. In NFAT1(-/-) mice, however, ectopic proliferation of cartilageous tissues were observed, some of which resembled with chondrosarcomas (Ranger, et al. 2000). These results suggest that in addition to the role in the immune system, NFAT1 is an important regulator in chondrocyte proliferation/differentiation, and may have a role as a tumor suppressor gene in chondrosarcomas. From this standpoint, we investigated the mutation analysis of the NFAT1 gene in human chondrosarcomas.

METHODS: From the cDNA sequence and GeneBank human genome information, we were able to determine the genomic structure of NFAT1 gene, which consisted of 10 exons. Fifteen sets of PCR primers were designed to amplify the entire coding sequences including intron sequences at exon-intron boundaries. Initial screening was performed by PCR-SSCP in 26 cases of chodrosarcomas, and samples showing abnormal bands were further investigated by direct sequence. The expressions of NFAT1 in tumor samples were analyzed by RT-PCR in 15 of 26 cases. The expressions of NFAT1 was also analyzed in 10 cases of osteosarcoma, 23 cases of soft tissue sarcomas, and 4 cases of lipomas. Cultured cell lines of osteosarcomas and mesenchymal stem cell (MSC) were also analyzed.

RESULTS: We found single base change in the coding sequences at five sites, of which three, C373G(Val51Val), C1015G(Pro265Pro), C1723A(Ile501Ile) did not alter the coding amino acid. In other two sites, A1557T (His446Leu), C2859T(Pro880Leu), identical base changes were present in patientsÕ normal somatic cells, denying the possibilities of somatic mutations. Therefore, all of five base changes seemed to be single nucleotide polymorphisms, and not disease-relating mutations. The expression of NFAT1 was observed in all of examined chondrosarcomas (15/15). In addition, NFAT1 were also expressed in9/10 osteosarcomas, 5/5 synovial sarcomas, 3/5 leimyosarcomas, 2/3 myxoid liposarcomas, 1/3 well differentiated liposarcomas, 1/3 MFH and 1/4 lipomas. We also found the expresison of NFAT1 in most of osteosarcoma cel lines(6/7), indicating that messages found in primary samples were not merely derived from infiltrateing lymphocytes. The expression of NFAT1 was detected in undifferentiated MSC, and retained after the induction of chondrogenesis or osteogenesis, but disappeared after the induction of adipogenesis.

CONCLUSION: The results in this study suggest that the NFAT1 gene is unlikely to be a tumor suppressor in chondrosarcoma. Because the expression was detected in various mesenchymal tumors and also MSC, the NFAT1 may play a role in proliferation and/or differentiation of mesenchymal cells. Down-regulation of the expression after adipogenic indution in MSC, and lower expression in tumors derived from adipose tissues suggest that one of the roles of NFAT1 in mesenchymal tissues is the inhibition of adipogenesis.