Enhanced Ca2(+)-dependent proteolysis associated with Adriamycin-resistant HL-60 cells

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Pharmacology

Background:

• Adriamycin-resistant HL-60 (HL-60/ADR) cells exhibit high levels of M-kinase, a fragment of protein kinase C.
• M-kinase activity leads to the in vitro generation of pp130 in these resistant cells.

Purpose of the Study:

• To investigate the presence and activity of proteases in Adriamycin-resistant HL-60 cells compared to wild-type cells.
• To characterize the role of calcium-activated proteases in the phenotype of multidrug resistance.

Main Methods:

• Analysis of cell extracts from HL-60/ADR and wild-type HL-60 cells.
• In vitro assays to detect protease activity and protein generation.
• Use of protease inhibitors, specifically leupeptin, to block enzymatic activity.
• Metabolic labeling and in vivo studies to assess phosphoprotein levels.

Main Results:

• A Ca2(+)-activated protease was identified in HL-60/ADR cell extracts, absent in wild-type cells.
• This protease generated 26 kDa and 86 kDa phosphoproteins (pp26, pp86) in vitro and reduced pp130 levels.
• Leupeptin inhibited the formation of pp26 and pp86 and the reduction of pp130.
• Elevated phosphorylation of pp26 was observed in resistant cells both in vitro and in vivo.

Conclusions:

• Ca2(+)-dependent protease activity is a distinguishing characteristic of this multidrug-resistant cell line.
• This protease activity contributes to the altered phosphoprotein profile observed in resistant cells.
• The findings suggest a potential role for this protease in the mechanisms of multidrug resistance.