Cell cycle genes in chondrosarcoma. Involvement of the Id1 and p16 pathways

Abstract: Chondrosarcoma is a malignant, cartilaginous tumour arising from bone. The wide variationin morphology, behaviour and clinical outcome raise the question whether chondrosarcomasare several different but closely related tumours rather than a single entity. On the cellularlevel, little is known about the molecular events responsible for the development andprogression of chondrosarcoma. The aim of this thesis was to investigate if the tumoursuppressor p16 and the helix-loop-helix (HLH) transcription factor Id1 and their regulatorypathways in the G1 phase of the cell cycle were affected in chondrosarcoma.Cultured cells and tissue from 34 human chondrosarcomas were investigated for geneticdefects by polymerase chain reaction (PCR) based methods and for changes in expressionpatterns of p16, p14ARF, p53, pRb, cdk4, Id1, Id3, E12 and MIDA1.While a structurally unchanged p53-gene was revealed in all samples analysed and only onehomozygous deletion of the p14ARF gene was found, the p16 gene showed homozygousdeletion, methylation and sequence deviations in preferable high-grade tumour tissue fromnine of 22 chondrosarcomas and in three of eight cultures of chondrosarcoma cells as well asin two chondrosarcoma cell lines. The protein expression of p16, pRb and cdk4 was analysedby Western blot in the cultured cells and several changes in the expression was detected incells from four of the eight tumours and in both cell lines. The expression and localisation ofId1 and Id3, as well as localisation of the E12-protein in cultured cells were studied byribonuclease protection assay, Western blot and immunohistochemistry. Id1 demonstrated astrong expression in chondrosarcoma cells also after serum withdrawal from the culturemedia, in contrast to normal chondrocytes where the expression was down-regulated.Moreover, antisense oligonucleotides directed against Id1 and Id3 decreased the mitoticactivity as measured by BrdU-labelling in both cell types. E12 had a nuclear localisation inchondrocytes and non-confluent tumour cells but in confluent tumour cells, E12 was found inthe cytoplasm. A previously unreported splicing form of MIDA1, a non-HLH protein bindingto Id1, was also found. The expression of MIDA1, Id1 and p16 studied by real-time PCRshowed that the expression levels of MIDA1 were rather constant between samples, while theexpressions of Id1 and p16 varied. The lowest levels of p16 expression were found inchondrosarcomas with worse prognosis, suggesting a possible role for p16 expression as aprognostic factor.The results from this thesis show that important genes in the G1 regulation of the cell cycleare changed in chondrosarcoma. Different defects in tumours with the same histological gradesupport the theory that chondrosarcomas may be several closely related tumours.