Proteasome proteolytic activity in hematopoietic cells from patients with chronic myeloid leukemia and multiple myeloma.
OBJECTIVE
The proteasome is a multicatalytic complex found in all eukaryotic cells; it is responsible for the degradation of key regulatory proteins associated with the cell cycle and apoptosis. In vitro, proteasome inhibitors can induce selective apoptosis in some malignant cell types as opposed to in their normal counterparts and first generation compounds are currently in clinical trials for the treatment of multiple myeloma. The objective of our study was to develop a method to extract and measure functional proteasome activity in primary human cells so that this method could then be used to determine whether patients might benefit from proteasome inhibitor therapy.


METHODS
Optimal proteasome extraction and assay conditions were established with myeloma and leukemic cell lines. These conditions were then applied to primary human cells from patients. Proteasome was extracted using lysis buffer and activity measured as turnover of a peptide fluorescent substrate.


RESULTS
Cells expressing bcr-abl showed significantly higher proteasome levels (372+/-16 AFU/1x10(6) cells/min) than did bcr-abl-negative cells (151+/- 8 AFU/1x10(6) cells/min) and were more sensitive to induction of apoptosis by proteasome inhibitor. Human myeloid leukemia cell lines showed higher levels of activity than those representing myeloma (eg HL-60 cells 947+/-25 AFU/1x10(6) cells/min; U266 177+/-6 AFU/1x10(6) cells/min). Primary cells from patients had similar levels of activity to those of the comparable cell line model.


CONCLUSIONS
This simple method measures functional proteasome activity in primary leukemic cells and demonstrates for the first time that this activity is higher in myeloid leukemia than in myeloma cells.
Magill
Laura
L
Department of Hematology, Queen's University Belfast, Belfast BT9 7AB, Ireland.
Lynas
John
J
Morris
Trenn C M
TC
Walker
Brian
B
Irvine
Alexandra E
AE
D017209:Apoptosis / Q000187:drug effects; D001854:Bone Marrow Cells / Q000201:enzymology; D045744:Cell Line, Tumor / Q000187:drug effects / Q000201:enzymology; D004492:Edetic Acid / Q000494:pharmacology; D016044:Fusion Proteins, bcr-abl / Q000032:analysis; D018922:HL-60 Cells / Q000187:drug effects / Q000201:enzymology; D006412:Hematopoietic Stem Cells / Q000201:enzymology*; D006801:Humans; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000201:enzymology* / Q000473:pathology; D009101:Multiple Myeloma / Q000201:enzymology* / Q000473:pathology; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013450:Sulfones / Q000494:pharmacology; D014162:Transfection
D009363:Neoplasm Proteins; D010436:Pepstatins; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D013450:Sulfones; C002010:4-(2-aminoethyl)benzenesulfonylfluoride; D004492:Edetic Acid; D016044:Fusion Proteins, bcr-abl; D046988:Proteasome Endopeptidase Complex; D007930:Leucine; C024974:E 64; C031375:pepstatin