leupeptins and Leukemia-Lymphoma--Adult-T-Cell

We have recently shown constitutive IkappaB kinase (IKK) activation and aberrant p52 expression in adult T cell leukemia (ATL) cells that do not express human T cell leukemia virus type I (HTLV-I) Tax, but the mechanism of IKK activation in these cells has remained unknown. Here, we demonstrate distinct regulation of IKK activity in ATL and HTLV-I-transformed T cells in response to protein synthesis inhibition or arsenite treatment. Protein synthesis inhibition for 4 h by cycloheximide (CHX) barely affects IKK activity in Tax-positive HTLV-I-transformed cells, while it diminishes IKK activity in Tax-negative ATL cells. Treatment of ATL cells with a proteasome inhibitor MG132 prior to protein synthesis inhibition reverses the inhibitory effect of CHX, and MG132 alone greatly enhances IKK activity. In addition, treatment of HTLV-I-transformed cells with arsenite for 1 h results in down-regulation of IKK activity without affecting Tax expression, while 8 h of arsenite treatment does not impair IKK activity in ATL cells. These results indicate that a labile protein sensitive to proteasome-dependent degradation governs IKK activation in ATL cells, and suggest a molecular mechanism of IKK activation in ATL cells distinct from that in HTLV-I-transformed T cells.

Topics: Arsenites; Cell Line, Transformed; Cell Line, Tumor; Cell Transformation, Viral; Cycloheximide; Enzyme Inhibitors; Human T-lymphotropic virus 1; Humans; I-kappa B Kinase; Leukemia-Lymphoma, Adult T-Cell; Leupeptins; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Protein Synthesis Inhibitors; T-Lymphocytes

Cytokines, such as interleukin-2 (IL-2), activate intracellular signaling pathways via rapid tyrosine phosphorylation of their receptors, resulting in the activation of many genes involved in cell growth and survival. The deubiquitinating enzyme DUB-2 is induced in response to IL-2 but as yet its function has not been determined. The results of this study show that DUB-2 is expressed in human T-cell lymphotropic virus-I (HTLV-1)-transformed T cells that exhibit constitutive activation of the IL-2 JAK/STAT (signal transducers and activators of transcription) pathway, and when expressed in Ba/F3 cells DUB-2 markedly prolonged IL-2-induced STAT5 phosphorylation. Although DUB-2 did not enhance IL-2-mediated proliferation, when withdrawn from growth factor, cells expressing DUB-2 had sustained STAT5 phosphorylation and enhanced expression of IL-2-induced genes cis and c-myc. Moreover, DUB-2 expression markedly inhibited apoptosis induced by cytokine withdrawal allowing cells to survive. Taken together these data suggest that DUB-2 can enhance signaling through the JAK/STAT pathway, prolong lymphocyte survival, and, when constitutively expressed, may contribute to the activation of the JAK/STAT pathway observed in some transformed cells.

Topics: Apoptosis; Cell Line; Cell Line, Transformed; Cell Transformation, Viral; Cysteine Endopeptidases; DNA-Binding Proteins; Endopeptidases; Human T-lymphotropic virus 1; Humans; Immediate-Early Proteins; Interleukin-2; Leukemia-Lymphoma, Adult T-Cell; Leupeptins; Milk Proteins; Multienzyme Complexes; Phosphorylation; Proteasome Endopeptidase Complex; Signal Transduction; STAT5 Transcription Factor; T-Lymphocytes; Trans-Activators; Transcriptional Activation; Transfection; Ubiquitins

The MDM2 protein regulates the functional activity of the p53 tumor suppressor through direct physical association. Signals that control MDM2 expression are poorly understood but are likely to play an important role in the regulation of p53 activity. We show here that the half-life of MDM2 protein is shorter in proliferating than in quiescent peripheral blood mononuclear cells. We also demonstrate that MDM2 protein half-life is extended in some, but not all, p53 mutant human leukemic cell lines. In at least one of these p53 mutant lines, increased MDM2 protein stability is associated with higher amounts of MDM2 protein. Moreover, we demonstrate that MDM2 protein accumulates to a much greater extent in proteasome inhibitor-treated cells containing unstable MDM2 than in cells possessing stable MDM2. These results demonstrate that MDM2 expression is regulated by events that control the stability of the protein and suggest that the normal regulation of MDM2 turnover can be altered in tumor cell lines.

Topics: Burkitt Lymphoma; Cell Division; Cycloheximide; Cysteine Endopeptidases; Genes, p53; Half-Life; Humans; Jurkat Cells; Leukemia-Lymphoma, Adult T-Cell; Leukocytes, Mononuclear; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Nuclear Proteins; Protease Inhibitors; Proteasome Endopeptidase Complex; Protein Synthesis Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-mdm2; RNA, Messenger; RNA, Neoplasm; Tumor Cells, Cultured; Tumor Suppressor Protein p53

The endocytosis and intracellular metabolism of radiolabeled anti-CD3 MoAb 64.1 by the malignant human T cell line HPB-ALL were studied using biochemical, morphological, electrophoretic, and chromatographic techniques. Biosynthetically labeled [3H]64.1 and externally radioiodinated 125I-64.1 were similarly internalized and degraded by tumor cells, with approximately 70% of the initially bound radioactivity being released to the culture supernatant as trichloroacetic acid-soluble radioactivity in the first 24 hr of culture. Radiolabeled 64.1 was routed from the cell membrane to endosomes where initial proteolysis began and finally to lysosomes where terminal catabolism to single amino acids occurred. SDS-PAGE demonstrated four major intracellular metabolite species (46, 25, 15, and less than 10 kDa). Thin-layer chromatography demonstrated that greater than 95% of the trichloroacetic acid-soluble radioactivity in culture supernatants was 125I-monoiodotyrosine, indicating that proteases, not deiodinases, were of primary importance in catabolism of 125I-64.1. In the presence of inhibitors of lysosomal function (leupeptin, monensin, and ammonium chloride), 125I-64.1 degradation was impeded, causing prolonged retention of radioactivity in the lysosomal compartment of cells. However, although the pace of catabolism was markedly diminished by these agents, no major changes in the sizes of intermediate metabolites generated were observed. Our results suggest that judicious administration of lysosomal inhibitors (e.g. chloroquine, verapamil, monensin) may significantly enhance retention of radioimmunoconjugates by lymphoid malignancies, improving radioimmunoscintigraphic and radioimmunotherapeutic efforts.

Topics: Ammonium Chloride; Antibodies, Monoclonal; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Autoradiography; CD3 Complex; Endocytosis; Humans; In Vitro Techniques; Leukemia-Lymphoma, Adult T-Cell; Leupeptins; Molecular Weight; Monensin; Peptide Fragments; Receptors, Antigen, T-Cell; T-Lymphocytes; Tumor Cells, Cultured