PTEN is known to be the most highly mutated tumor suppressor gene after p53 [10]. It plays an important role in regulating proliferation, migration, survival, cell invasion and tumor angiogenesis [11, 12]. Freeman et al. [13] reported that loss of PTEN was a common occurrence in osteosarcoma. It was further demonstrated that PTEN can control p53 half-life independent via a currently unknown mechanism [14]. In addition, mutations of tumor-suppressor retinoblastoma gene (Rb) in osteosarcoma are associated with a poor prognosis [15]. However, none of these

alterations can characteristically reflect the biologic nature or clinical features of all osteosarcomas. IDH1 is a cytosolic NADP-dependent isocitrate dehydrogenase. It catalyzes decarboxylation VS-4718 concentration of isocitrate into alpha-ketoglutarate [16]. Shechter et al. [17] described that the activity of AUY-922 chemical structure IDH1 is coordinately regulated through the cholesterol and fatty acid biosynthetic pathways, suggesting that IDH1 provides the cytosolic NADPH required by these pathways. Memon et al. [18] found that expression of IDH1 was downregulated in a poorly differentiated bladder cancer cell line compared with a well-differentiated bladder cancer cell line. Tissue biopsies of late-stage bladder cancers also showed IDH1 downregulation compared with early-stage bladder cancers. Yan et al. [19] described

that mutations of NADP (+)-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur in a majority of Tideglusib several types of malignant gliomas. Interestingly, Parsons et al. [20] found that IDH1 mutations in human glioblastoma had a very high frequency of p53 mutation. Mutation of the IDH1 gene was also strongly correlated with a normal cytogenetic status [21]. IDH1 appears to function as a tumor suppressor that, when mutationally inactivated, contributes to tumorigenesis [21, 22]. But, there is no study on the expression of IDH1 in osteosarcoma. As to the previous study PIK3C2G on IDH1 and p53, we are also curious intensively about the correlation between IDH1 and p53. So, we developed a study to characterize the expression and significance

of IDH1 and p53 in osteosarcoma cell lines (MG63 and U2OS) as well as in clinical patient biopsies. Methods Cell lines and cell culture The human osteosarcoma (OS) cell lines MG63 and U2OS (obtained from ATCC through LGC Promochem, Wesel, Germany) were cultured in RPMI 1640 media (Sigma, USA) with 10% fetal bovine serum (Amresco, USA) and antibiotics. Cells were cultured according to standard techniques in cell culture flasks in a humidified incubator in 5% CO2 atmosphere. Immunocytochemistry Cell lines were grown on coverslips treated with the appropriate growth media in 24 well cluster plates. Cells were fixed in 2% formaldehyde in 0.1 mol/L phosphate-buffered saline (PBS, pH 7.4) for 20 min at room temperature and subsequently washed three times in PBS.