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We investigated the expression of the Chondromodulin-I (ChM-I) gene, a putative tumor suppressor gene in cartilaginous tumors, by quantitative RT-PCR in 15 chondrosarcomas (CSs). Eight CSs expressed the ChM-I gene at the level higher than those in articular cartilage (positive cases), whereas the expression of the ChM-I gene in the remaining seven CSs was lower than those in articular cartilage (negative cases). All of five peripheral CS were positive, and the ChM-I positive tumors shared expression profiles of cartilage-related genes with articular cartilage cells. On the other hand, all of four central CSs without extramedullary lesions were negative, and the ChM-I negative tumors expressed the parathyroid hormone-related peptide gene at the lower level and the COL10A1 genes at the higher level than articular cartilage cells. Neither the histological grade nor the rate of recurrence showed clear association with the level of ChM-I gene expression. These results suggested that the expression of ChM-I gene in CS has no direct role in tumorigenesis but rather reflects the site of tumor development and therefore precursor of tumor cells.

Mice lacking nuclear factor of activated T cell 1 (NFAT1) showed an abnormal proliferation of chondrocytes in articular cartilage and formed an extraosseous cartilaginous mass resembling a neoplastic lesion, suggesting that the NFAT1 gene is a tumor suppressor gene in cartilaginous neoplasms. Here we performed mutation analyses of the NFAT1 gene in human cartilaginous tumors including 30 chondrosarcomas and 15 enchondromas. Reverse transcription-polymerase chain reaction (PCR) analysis revealed the expression of the NFAT1 gene in 15/15 chondrosarcomas and 12/13 enchondromas. To find subtle alterations, the genomic structure of the NFAT1 gene was determined using human genome draft sequences, and a mutation analysis was performed using the exon-by-exon PCR-single-strand conformation polymorphism method. Two heterozygous missense mutations, A1557T (His446Leu) and C2859T (Pro880Leu), were found in eight tumor samples, but the same mutation was also present in the constitutional cells of corresponding patients. The incidence of the mutant alleles in the patient and control groups showed no significant difference, suggesting that these mutations are rare single nucleotide polymorphisms unrelated with tumorigenesis. These results suggest that the NFAT1 gene is not likely to be a tumor suppressor gene in human cartilaginous tumors.

Osteosarcoma is a malignant tumor with heterogeneous features both in histological and biological aspects. We have established three tumorigenic cell lines, MMOS1, MMOS2, and MMOS3, from three independent tumors that developed in nude mice after the inoculation of MMC2, an osteoblast-like cell line derived from p53-/- mice. Expression patterns of the osteoblast-related genes showed a marked difference between MMOS2 and the other two cell lines, and were correlated well with the features of the original tumors, ranging from an osteoblastic osteosarcoma (MMOS2) to tumors with scarce or no osteoid formation (MMOS1 and MMOS3). The properties of malignant cells also varied in the three cell lines. MMOS1, which was the most serum-dependent in vitro, developed markedly larger tumors in vivo than the other two cell lines. MMOS3 showed the fastest growth in low-serum conditions and produced the largest number of colonies in soft agar, but did not develop lung metastases, whereas MMOS1 and MMOS2 developed lung metastases with a frequency of 30 and 50%. These data suggest that the biological activities in vivo do not necessarily reflect those in vitro. Because the three tumorigenic cell lines share MMC2 as a common precursor, our data showed an example that the heterogeneity of osteosarcoma was created by genetic alterations that took place during the transformation process of each tumor.