PMC:3940921 / 15544-17985 JSONTXT

{"project":"2_test","denotations":[{"id":"24590311-18181098-79065524","span":{"begin":229,"end":231},"obj":"18181098"},{"id":"24590311-16778216-79065525","span":{"begin":233,"end":235},"obj":"16778216"},{"id":"24590311-17024115-79065526","span":{"begin":237,"end":239},"obj":"17024115"},{"id":"24590311-19671918-79065527","span":{"begin":1102,"end":1104},"obj":"19671918"}],"text":"The presence of immunoproteasome subunits increases sensitivity to carfilzomib\nWe, along with others, have reported that the sensitivity of tumor cells to PIs may be associated with differential expression of proteasome subunits.28, 29, 30 In this study, we found that in contrast to the proteasome profiles observed for CML cell lines and primary samples above, immunoproteasome subunits made little or no contribution to the overall proteasome activity in K562 cells (Supplementary Table 2). In this regard, the K562 cell line is more similar to a nonhematopoietic cell line. We demonstrate that although carfilzomib is a potent inhibitor of the β5 CT-L subunit in K562 cells (IC50=6.68 nM; Supplementary Table 2), the IC50 value for carfilzomib in reducing the proliferation of this cell line was \u003efivefold higher than the corresponding values for the other CML cell lines. We therefore sought to investigate whether the difference in sensitivity we observed with K562 cells was related to immunoproteasome expression. A549 lung carcinoma cell line, known to express low amounts of immunoproteasome,26 was included as a comparison. K562 and A549 cells were cultured in the presence of 100 U/ml IFN-γ for 72 h to induce immunoproteasome expression, before treatment with carfilzomib (1–1000 nM continuous exposure for 24 h). IFN-γ treatment resulted in an increase in the expression of LMP7 by fourfold in K562 cells and 10-fold in A549 cells (Figures 5a and b); IFN-α treatment produced similar results (Supplementary Figure 4C). The increase in LMP7 expression corresponded with a significantly increased sensitivity to carfilzomib (IC50 value reduced from 608 nM to 179 nM in K562 cells and 92 nM to 10 nM in A549 cells, P⩽0.01; Figures 5c and d). Conversely, we looked at the effect of LMP7 knockdown on the sensitivity of cells to carfilzomib. KCL22S and LAMA84S cells were treated with siRNA targeted against LMP7 before treatment with carfilzomib (1–1000 nM continuous exposure for 24 h). Downregulation of LMP7 (Supplementary Figure 4) was associated with a significant decrease in sensitivity to carfilzomib; the IC50 value for KCL22S cells increased from 5 nM to 16 nM (P=0.01) and LAMA84S cells increased from 7 nM to 20 nM (P=0.04). These results demonstrate that the presence of both the β5 and LMP7 CT-L subunits are required for optimal effect with carfilzomib and pretreatment with IFN-γ can sensitize cells to carfilzomib."}