DKK-1 is a candidate gene for tumor suppressor in glioma and considered as a serologic and prognostic biomarker.In our recent study of 12 human glioma cell lines, GW3965 manufacturer we found that the supernatant fluid and lysate of 9 cell lines had high level of DKK-1 protein and the other 3 had

very low level or non-detectable DKK-1 protein (Zhou et al, unpublished data). The high level of DKK-1 protein in most glioma cell lines suggested that DKK-1 may play an important role in glioma and attracted our intention to further study this DKK-1′s function in glioma. In this study we constructed a eukaryotic expression Selleckchem Barasertib vector of human DKK-1(pcDNA3.1-DKK-1) and stably transfected the vector into the glioma cell line SHG44, which had no expression of DKK-1 under normal growth condition. We found that elevated expression of DKK-1 increased the sensitivity of SHG44 cells to the anti-cancer drug BCNU in vitro. Materials and methods Construction of expression vector The 816-base pair human DKK-1 cDNA was amplified from the RNA of human placenta tissue using reverse transcription polymerase chain reaction (RT-PCR). The sequence of sense primer was 5′-CTA GCTAGC ACATGATGGCT CTGG-3′ (NHe I enzyme digestion site was indicated as underline) and antisense primer was 5′-G GAATTC GTGTCTCTGACAAGTGTG-3′ (EcoR I enzyme digestion site was indicated

as underline). The PCR reaction (10 μl) contained 1 μl cDNA, l μl 10 × buffer (MgCl2), 0.4 mM dNTPs, 1umol primer, 1U TaqDNA

Polymerase. After denaturation at 95°C for 5 min, PCR was performed for 35 cycles (30 s at 95°C, Ro 61-8048 30 s at 50°C and 30 s at 72°C) and extended at 72°C for 5 min. The linear NHeI-EcoRI fragment containing the DKK-1 cDNA was subcloned into pcDNA3.1 (Invitrogen Company), which yielded pcDNA3.1-DKK-1 by T4 ligase (TaKaRa Company). The insertion of DDK-1 in pcDNA3.1 was confirmed by PCR, restriction enzyme digestion analysis (NHeI and EcoRI) and DNA sequencing. Cell culture The human glioma cell line SHG44 was established by our lab in 1984 and has been widely used in China. It was originally obtained from a patient with grade II-III astrocytoma (according to World Health Organization). Cells were cultured in Exoribonuclease RPMI1640 medium (Giboc Company) supplemented with 10% fetal bovine serum, 100 IU/ml penicillin and 100 μg/ml streptomycin. Cells were cultured at 37°C in a humidified atmosphere containing 5% carbon dioxide. The culture medium was changed every 48 h. Determination of the optimal concentration of G418 G418 is an aminoglycoside and is commonly used as a selective agent for the bacterial neo r/kan r genes. The optimal concentration of G418 for selection of resistance was determined by the following procedure. SHG44 cells were plated at the same concentration of 5 × 104/well, in 24-well plates containing 2 ml culture medium per well.