Changes of the p16 gene but not the p53 gene in human chondrosarcoma tissues.

The role of two important tumour suppressor genes, p16 and p53, was evaluated in cartilaginous tumour tissues. Genomic DNA from 22 chondrosarcomas, 5 benign chondroid tumours, 1 sample of reactive proliferative cartilage and 2 samples of normal cartilage were analysed using polymerase chain reaction, single strand conformational polymorphism, DNA sequencing and methylation-specific polymerase chain reaction. The p16 gene was found to be partly methylated in 5 high-grade chondrosarcomas and homozygously deleted in 1 chondrosarcoma. Moreover, a polymorphism was detected in 3 malignant tumours, but not in benign tumours or normal cartilage. Analysis of the p53 gene revealed an unchanged structure in all samples. These findings show a role for p16, but not p53, in chondrosarcoma.

Partial deletions of the CDKN2 and MTS2 putative tumor suppressor genes in a myxoid chondrosarcoma.

Cytogenetic abnormalities of chromosome 9 (9p21) have been reported in a large number of tumors that include malignant melanomas, gliomas, lung cancers and leukemias. These aberrations on 9p have been previously shown to involve the loss of the interferon gene cluster and the gene for methylthioadenosine phosphorylase (MTAP), both of which have been mapped to the 9p21 region. Recently, two putative tumor suppressor gene(s) CDKN2 and MTS2, have been mapped to the 9p21 region, and have been shown to be deleted in a large number of hematopoietic and solid malignancies. In this study we report a cytogenetic and a detailed molecular analysis of a myxoid chondrosarcoma cell line 105KC and its clonal derivatives 105AJ, 105AJ1.1, 105AJ3.1, and 105AJ5.1. Specifically, we have demonstrated chromosome 9p21 related abnormalities by cytogenetic analysis, the associated loss of the interferon gene cluster, and the loss of the immunoreactive MTAP protein and activity. In addition, we have also shown the presence of deletions involving the CDKN2 and the MTS2 putative tumor suppressor genes in these chondrosarcoma cell lines. The above studies were extended to other chondrosarcoma cell lines and primary tumors, where similar deletions of the CDKN2 and MTS2 genes were found to be present (unpublished data). This suggests a potential role for the involvement of the CDKN2 and MTS2 putative tumor suppressor genes in the development of chondrosarcomas.

Heterogeneity of keratan sulfate substituted on human chondrocytic large proteoglycans.

Newly synthesized 35S-labeled chondrocytic keratan sulfate chains were generated by chondrocytes of human chondrosarcoma cell line 105KC and were analyzed for heterogeneity of regional substitution, hydrodynamic size, and charge density. After isolation of the high density large chondrocytic proteoglycans and sequential digestions with chondroitinase ABC, L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin, and alpha-chymotrypsin, followed by Superose 6 chromatography, two populations of keratan sulfate-containing proteoglycan fragments were identified and pooled separately. Keratan sulfate chains from each of the regions were compared after release by Pronase digestion, and differences in substitution patterns were observed; keratan sulfate chains of greater polydispersity, as well as a population of larger hydrodynamic size, were present in only one of the two regions. Alkaline/borohydride treatment confirmed both the existence of a population of uniquely large keratan sulfate chains and its restriction to a single region of proteoglycan fragments. In addition to heterogeneity of hydrodynamic size, the keratan sulfate chains exhibited regional heterogeneity of charge density and hence, of sulfation patterns. Analysis by Mono Q chromatography identified distinct groups of keratan sulfate that segregated by charge density and whose proportionate composition differed between the proteoglycan regions. Furthermore, the most highly charged species were unique to a single region and encompassed the chains of larger hydrodynamic size. This suggests that there may be regional heterogeneity of keratan sulfate chains substituted along a single class of proteoglycans and identifies a novel population of large, highly sulfated chondrocytic keratan sulfate chains.

Synthesis of chondrocytic keratan sulphate-containing proteoglycans by human chondrosarcoma cells in long-term cell culture.

Keratan sulphate is an integral component of the large aggregating proteoglycans of mature human articular cartilage. The keratan sulphate content of chondrocytic proteoglycans increases during maturation, and it is a useful marker of mature-type chondrocytic proteoglycans. Ordinarily, in cell culture, chondrocytes from non-neoplastic tissues dedifferentiate, diminish or cease to synthesize aggregating proteoglycans with the same amount of keratan sulphate as those formed in vivo, and do not maintain their in vivo phenotype. In tissue culture, this down-regulation of synthesis of keratan sulphate is irreversible. The study of the metabolism of mature human chondrocytes has been hampered by the absence of stable models. We report a cell-line, 105KC, derived from a human chondrosarcoma, that has maintained a stable proteoglycan phenotype during more than three years of culture. Analysis with immunofluorescence suggested that 105KC cells continued to synthesize keratan sulphate in long-term culture. Biochemical analysis demonstrated that 105KC cells maintained the production of chondrocytic large-aggregating proteoglycans and that keratan sulphate composed 13 per cent of their glycosaminoglycan content. To our knowledge, 105KC represents the first model to have maintained the post-fetal chondrocytic proteoglycan phenotype in stable culture. This study documents the feasibility of the development of mature chondrocytic cell-lines and sheds light on the biological characteristics of chondrosarcomas.

The effects of long term monolayer culture on the proteoglycan phenotype of a clonal population of mature human malignant chondrocytes.

Articular cartilage chondrocytes maintain biosynthetic heterogeneity in cell culture, but undergo irreversible dedifferentiation of their proteoglycan phenotype, as defined by keratan sulfate content. A recently described cell line of malignant human chondrocytes, 105KC, was the first to maintain a differentiated keratan sulfate-proteoglycan phenotype in long-term culture. A clone of 105KC, labeled KC2H3, is currently described and represents a distinct and metabolically more homogeneous population of mature chondrocytes than 105KC. KC2H3 cells universally express keratan sulfate biosynthesis, as defined by indirect immunofluorescence. In addition, KC2H3 expresses a more mature proteoglycan phenotype than 105KC, as demonstrated by the keratan sulfate content: 24% of glycosaminoglycan content of the aggregating proteoglycans of KC2H3 versus 13% for 105KC. Further reported are the effects of long term monolayer culture on the proteoglycan phenotype expressed by KC2H3. After more than 16 months in continuous monolayer, KC2H3 cells remained morphologically indistinguishable from those maintained in suspension alternating with monolayer. In addition, the proteoglycan phenotype remained mature, without a tendency towards dedifferentiation. The flattened morphology adopted by chondrocytes while in monolayer has been considered a stimulus of dedifferentiation; the present study is the first to examine the direct effects of physical state on a homogeneous and stable population of chondrocytes.