PMC:3940921 / 12746-15542 JSONTXT

{"project":"2_test","denotations":[{"id":"24590311-19671918-79065517","span":{"begin":228,"end":230},"obj":"19671918"},{"id":"24590311-17616698-79065518","span":{"begin":232,"end":234},"obj":"17616698"},{"id":"24590311-19157685-79065519","span":{"begin":824,"end":826},"obj":"19157685"},{"id":"24590311-17616698-79065520","span":{"begin":1163,"end":1165},"obj":"17616698"},{"id":"24590311-18181098-79065521","span":{"begin":1167,"end":1169},"obj":"18181098"},{"id":"24590311-19671918-79065522","span":{"begin":1928,"end":1930},"obj":"19671918"},{"id":"24590311-16778216-79065523","span":{"begin":2167,"end":2169},"obj":"16778216"}],"text":"Proteasome profiles and subunit selectivity of carfilzomib in CML\nCarfilzomib has been demonstrated to primarily target CT-L subunits of both the constitutive proteasome (β5) and immunoproteasome (LMP7) in MM and lymphoma cells.26, 27 To evaluate the inhibitory effect of carfilzomib in CML cells, we quantified the levels of constitutive proteasome and immunoproteasome subunits in primary CML cells, normal mononuclear cells and imatinib-sensitive and -resistant CML cell lines (Supplementary Table 2). In primary cells, the amount of active proteasome subunits per total protein concentration was significantly higher in CML cells compared with normal donors (24.05±1.38 ng/μg vs 16.69±2.86 ng/μg, P=0.001), consistent with our previous observations that Bcr-Abl activity is associated with increased proteasome activity.21 We found that immunoproteasome subunits were the major constituent of total proteasome in primary cells (59.1–65.4%) and Ba/F3 cell lines (61.1–70.2%), and comprised 16.3–30.1% of total proteasome in KCL22-S and -R and LAMA84-S and -R CML cell lines (Table 1). This is in agreement with previous reports on cells of hematopoietic origin27, 28 and confirms that the immunoproteasome is a substantive component of total proteasome in myeloid as well as lymphoid cells. The amount of CT-L subunits relative to total cellular protein was similar across all cell lines and primary CML cells (6.29±1.04 ng/μg). LMP7 was the predominant form of CT-L subunit expressed in normal mononuclear cells (83.8%), CML mononuclear cells (76.6%) and Ba/F3 cell lines (52.6–65.8%), and accounted for a significant proportion of CT-L activity in human CML cell lines (36.6–51.2%) (Figure 4a). In keeping with previous observations, we found that primary CML cells and KCL22-S and -R and LAMA84-S and -R predominantly express the immunoproteasome subunit LMP7 (12–25.3%) relative to MECL1 (2.2–15.2%) and LMP2 (1.2–18.6%).26 The contribution of individual proteasome subunits in Ba/F3 cell lines differs quite markedly from both primary cells and human CML cell lines. We have previously demonstrated that the proteasome subunit profiles are cell type dependent29 and this difference may reflect the different lineage origin and species of the cell lines.\nTo examine the effect of carfilzomib on individual proteasome activities, cell lines were pulsed with increasing concentrations of carfilzomib (1–1000 nM) for 1 h and analyzed using the ProCISE assay. Whereas there was a dose-dependent inhibition of all proteasome activities (Figures 4b–d), carfilzomib displayed preferential inhibitory activity against CT-L subunits, with IC50 values of ⩽8.32 nM for the β5 subunit and ⩽16.55 nM for LMP7. IC50 values for T-L and C-L subunits were at least threefold higher (Supplementary Table 3)."}