NVP-BKM120 PI3K inhibitor were designated as the nuclear fraction.

m for 5 min in an Eppendorf centrifuge were designated as the nuclear fraction. Full length cDNA of MIZ 1 was cloned into an eukaryotic NVP-BKM120 PI3K inhibitor expression vector, pEAK12. The neuroblastoma cells indicated were transfected with the pEAK/MIZ 1 construct by electroporation using an XCell electroporator . To examine NVP-BKM120 PI3K inhibitor MIZ 1 protein expression by Western blot analysis and 2 D gel analysis, the cells were harvested at 24 h after transfection. The 2 D gel electrophoresis was done according to the ReadyPrep�?2 D Starter Kit and PROTEAN® IEF cell instruction manuals. Briefly, cell extracts for 2 D gel electrophoresis were made in the 2 D sample buffer. An 11 cm, pH 3.
0 10, immobilized pH gradient strip was re hydrated directly with 200 l ReadyPrep rehydration/sample SKI-606 buffer, which included 50 g cell extract at room temperature, overnight.
The re hydrated IPG strips were then placed on a PROTEAN IEF cell and the first dimension electrophoresis was performed using the rapid voltage ramping program. After the first dimension electrophoresis, the IPG strips were equilibrated consecutively with Equilibration Buffer I and with Equilibration Buffer II containing iodoacetamide. The IPG strips were then SKI-606 placed on 4 20% Criterion pre cast gels and the second dimension electrophoresis was performed using a Criterion Cell. All neuroblastoma cell lines to date are derived from unfavorable neuroblastomas.
To examine the effect of Hsp90 inhibition on growth of unfavorable neuroblastoma cells, the four cell lines IMR5, CHP134, SY5Y and SKNAS were used. IMR5 and CHP134 are MYCN amplified neuroblastoma cell lines and express high levels of MYCN.
SY5Y and SKNAS are non MYCN amplified cell lines and express high levels of MYC. 17 DMAG was used as a model agent for Hsp90 inhibitors because of its water solubility and potency. As shown in Fig. 1, 17 DMAG inhibited growth of the four neuroblastoma cell lines in dose dependent fashions after two days of the treatment. Among the cell lines, CHP134 was most sensitive to 17 DMAG treatments, whereas SKNAS was least sensitive to the treatments. In addition, there was a biphasic growth inhibitory effect of Hsp90 inhibition for SKNAS, SY5Y and IMR5.
In these three cell lines, 17 DMAG showed similar growth inhibitory effects between the concentrations of 0.63 and 2.5 M, and its effect was further enhanced up to 10 M according to the dose.
Based on these results, subsequent assays were done using 17 DMAG at the dose of 5 M for all neuroblastoma cell lines. It has been shown that inhibition of Hsp90 leads to the down regulation of known oncoproteins, including AKT, ERBB2, BRAF and BCR ABL. Nonetheless, whether or not Hsp90 inhibition can affect MYC and MYCN stability has not been well documented. In this study, we examined whether the growth suppressive effect of Hsp90 inhibition on the neuroblastoma cells was associated with MYCN and MYC destabilization in these cells. As shown in Fig. 2A, treatment of these cell lines with 17 DMAG resulted in a clear decrease in MYCN or MYC expression as early as day 1 of the treatment. Early time course studies showed that the effect of the drug treatment on MYCN and MYC stability varied among the cell lines examined. The drug treatment was most effective against MYCN and MYC in IMR5 and SY5Y, respectively.