Longer Staurosporine treatment times only minimally increase the degree of inhibition. The reduction of Akt Sp129 is also promptly induced, while the CK2 target site on Cdc37 seems to be more resistant to dephosphorylation, which is clearly observed only at higher concentrations and longer treatments. Similar results are observable in response to CX-5011. Inhibition of CK2 was also confirmed by analyzing the radioactive phosphorylation of endogenous proteins in treated cells. Figure 4 shows that several protein bands are less phosphorylated in S- and R-CEM cells treated with CX-4945 or CX-5011. To rule out the possibility of a non-specific effect, we treated cells with staurosporine, at concentrations which inhibit the majority of protein kinases but not CK2. This treatment, while inducing a high degree of cell death similarly to what caused by the CK2 inhibitors, it is almost ineffective on endogenous protein phosphorylations; this also suggests that, under the used conditions, CK2 is the major kinase responsible for the observed radioactivity, as expected for a highly expressed, pleiotropic and constitutively active enzyme. To ascertain if CK2 inhibition by CX-4945 and CX-5011 is effective in inducing cell death also in case of drug resistance, we treated cells with increasing concentrations of the compounds, and measured cell viability by the MTT method. Figures 5, 6, 7, 8 show representative results obtained under different time and assay conditions. We found that both 36338-96-2 inhibitors are able to induce appreciable cell death also in R cells, in a manner quite similar to S cells. In particular, the responsiveness of MDR cells indicates that these compounds are not substrate of the Pgp. Table 1 shows the DC50 values calculated from the MTT assays shown in Figure 5, 6, 7, 8; the 48 h assays were used for calculation, except for CEM cells in the presence of 10% FCS, where 24 h assays were considered. To understand if cell death induced by CX-4945 and CX-5011 in our cell lines was due to apoptosis, we evaluated the formation of nucleosomes in treated cells; the results, shown in Figure 9 for CEM cells, and confirmed for the other cell lines, indicated that apoptosis occurs to a similar degree in S and R cells, in response to these CK2 inhibitors. The results are also confir
