Sven Inerot

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. Int. J. Cancer 85:782–786, 2000.

Alterations in the regulatory pathway involving p16, pRb and cdk4 in human chondrosarcoma

The Gl 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 chondrosarcomas and in two chondrosarcoma cell lines. Both cell lines and one other 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 line and in three other samples pRb of expected size were detected in addition 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 tumor 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 heterozygous position in codon 148 was detected in one sample. The same base substitution was also found in two of the tissue samples. Finally, cytogenetic analysis of the samples with homozygously deleted CDKN2 revealed multiple structural abnormalities in all three cases. In conclusion, the p16/ pRb/cdk4 pathway may play an important role in the pathogenesis of some chondrosarcomas.

The helix-loop-helix transcription factors Id1 and Id3 have a functional role in control of cell division in human normal and neoplastic chondrocytes

The expression and localization of the helix‐loop‐helix transcription factors Id1 and Id3, as well as localization of the E12‐protein, were studied in cells isolated from human articular cartilage and chondrosarcoma. Serum withdrawal down‐regulated Id1 and Id3 expression in chondrocytes but not the Id1 expression in chondrosarcoma cells. Antisense oligonucleotides directed against Id1 and Id3 decreased BrdU labeling in both cell types. E12 was localized to the nucleus in chondrocytes and non‐confluent tumor cells and in confluent tumor cells, E12 had a cytoplasmic localization. This study suggests a functional role for Id1 and Id3 in the control of proliferation and differentiation of cartilage.

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.

Changes in p14ARF do not play a primary role in human chondrosarcoma tissues

The locus encoding the tumor suppressor p16 has been found to code for a second, different protein. This protein, p14ARF, has been shown to protect p53 from degradation. Like p16, its gene is often altered in different cancers. In this study, the first unique exon, exon 1β, of p14ARF, has been studied in 22 chondrosarcoma tissues using polymerase chain reaction, DNA sequencing and methylation‐specific polymerase chain reaction. One chondrosarcoma was found to have exon 1β homozygously deleted, but neither mutations nor methylations were found in any of the chondrosarcomas. This indicates that genetic changes of p14ARF are a rare event in chondrosarcoma.

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.