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. 2007 Mar 13;110(1):267–277. doi: 10.1182/blood-2006-03-013128

Figure 1.

Figure 1

NPI-0052 inhibits the proteolytic activities of the 20S proteasome in leukemia cell lines. (A) NPI-0052 inhibits the chymotrypsin-like activity of the 20S proteasome in Jurkat, K562, and ML-1 cells. Cells were incubated for 1 hour in the presence (black bars) or absence (white bars) of 1 μM NPI-0052. The chymotrypsin-like activity of the 20S proteasome of leukemia cells was determined by measurement of fluorescence generated from the cleavage of the fluorogenic substrate suc-LLVY-amc. Release of fluorescence (amc) was measured using a spectrofluorometer using an excitation of 380nm and an emission of 460nm. Proteasome activity was evaluated in relative fluorescence units (RFU). (B) Measurement of caspase-like activity in Jurkat, K562, and ML-1 cells treated with NPI-0052. Caspase-like activity of the 20S proteasome detected with fluorogenic peptide z-LLE-amc. (C) Effects on the trypsin-like activity by NPI-0052 in Jurkat, K562, and ML-1 cells. Trypsin-like activity of 20S proteasome measured with boc-LRR-amc fluorogenic substrate. For panels A, B, and C, the data presented are from 3 independent experiments with similar results, mean ± SD (P < .05, significantly different from control). (D) NPI-0052–induced accumulation of proteasomal substrate, p27. Lysates from Jurkat cells treated with NPI-0052 for 3 hours and 6 hours were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted for p27 and actin. (E) Comparison between NPI-0052 and bortezomib inhibition of proteolytic activities of the 20S proteasomes. 1 μM NPI-0052 (black bars) inhibits the caspase-like activity (P = .019) and chymotrypsin-like activity more effectively (P = .005) than 1 μM bortezomib (white bars) in Jurkat cells. The RFU for control was 58081.5 (data not shown). Dose response curves for NPI-0052 (black rhombus) and bortezomib (white squares), measuring the chymotrypsin-, caspase-, and trypsin-like activities (P < .05 for 200 nM dose). The data presented are the mean ± SD from 3 independent experiments performed in triplicate with similar results.