leupeptins and Leukemia--Monocytic--Acute

Interleukin-1beta is a secreted protein that accumulates in the cytosol as an inactive precursor (pIL-1beta) before processing and release of biologically active protein. To understand the impact of this property on IL-1beta production, we examined the intracellular stability of pIL-1beta in lipopolysaccharide (LPS)-stimulated human monocytes. Precursor IL-1beta was degraded with a relatively short half-life of 2.5 h in the promonocytic cell line, THP-1, and in primary monocytes. MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) stabilized pIL-1beta levels in THP-1 cells, suggesting that degradation was proteasome-mediated, but this inhibitor was toxic for primary monocytes, causing release of pIL-1beta as well as the cytoplasmic enzyme, lactate dehydrogenase (LDH) into supernatants. In contrast, clasto-lactacystin beta-lactone, a specific inhibitor of the proteasome, caused a dose-dependent stabilization of intracellular pIL-1beta, and this led to a corresponding increase in mIL-1beta and pIL-1beta but not LDH release into culture supernatants. Therefore, by regulating intracellular levels of precursor IL-1beta, the proteasome plays an important and previously unrecognized role in controlling the amount of biologically active IL-1beta that is exported by activated monocytes.

Topics: Acrylates; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Half-Life; Humans; Interleukin-1; Lactones; Leucine; Leukemia, Monocytic, Acute; Leupeptins; Lipopolysaccharides; Monocytes; Multienzyme Complexes; Neoplasm Proteins; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

The ubiquitin-proteasome pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. We demonstrated that treatment of THP-1 human monocytic leukemia cells with Z-LLL-CHO, a reversible proteasome inhibitor, induced cell death through an apoptotic pathway. Apoptosis in THP-1 cells induced by Z-LLL-CHO involved a cytochrome c-dependent pathway, which included the release of mitochondrial cytochrome c, activation of caspase-9 and -3, and cleavage of Bcl-2 into a shortened 22-kDa fragment. Induction of apoptosis by protease inhibitor also was detected in U937 and TF-1 leukemia cell lines and cells obtained from acute myelogenous leukemia patients but not in normal human blood monocytes. Treatment of human blood monocytes with Z-LLL-CHO did not induce apoptosis or Bcl-2 cleavage in these cells that rarely proliferate. Interestingly, when THP-1 cells were induced to undergo monocytic differentiation by bryostatin 1, a naturally occurring protein kinase C activator, they were no longer susceptible to apoptosis induced by Z-LLL-CHO. Bryostatin 1-induced differentiation of THP-1 cells was associated with growth arrest, acquisition of adherent capacity, and expression of membrane markers characteristic of blood monocytes. Likewise, differentiated THP-1 cells were refractory to Z-LLL-CHO-induced cytochrome c release, caspase activation, and Bcl-2 cleavage. Resistance to Z-LLL-CHO-induced apoptosis in differentiated THP-1 cells was not due to cell cycle arrest. These findings show that the action of proteasome inhibitors is mediated primarily through a cytochrome c-dependent pathway and induces apoptosis in leukemic cells that are not differentiated.

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; Caspase 3; Caspase 9; Caspases; Cell Differentiation; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Enzyme Activation; Humans; Lactones; Leukemia, Monocytic, Acute; Leukemia, Promyelocytic, Acute; Leupeptins; Macrolides; Mitochondria; Monocytes; Multienzyme Complexes; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Ubiquitins