cathepsin-g and Leukemia

Here, we evaluated whether the overexpression of transcriptionally inactive ΔNp73 cooperates with PML/RARA fusion protein in the induction of an APL-leukemic phenotype, as well as its role in vitro in proliferation, myeloid differentiation, and drug-induced apoptosis. Using lentiviral gene transfer, we showed in vitro that ΔNp73 overexpression resulted in increased proliferation in murine bone marrow (BM) cells from hCG-PML/RARA transgenic mice and their wild-type (WT) counterpart, with no accumulation of cells at G2/M or S phases; instead, ΔNp73-expressing cells had a lower rate of induced apoptosis. Next, we evaluated the effect of ΔNp73 on stem-cell self-renewal and myeloid differentiation. Primary BM cells lentivirally infected with human ΔNp73 were not immortalized in culture and did not present significant changes in the percentage of CD11b. Finally, we assessed the impact of ΔNp73 on leukemogenesis or its possible cooperation with PML/RARA fusion protein in the induction of an APL-leukemic phenotype. After 120 days of follow-up, all transplanted mice were clinically healthy and, no evidence of leukemia/myelodysplasia was apparent. Taken together, our data suggest that ΔNp73 had no leukemic transformation capacity by itself and apparently did not cooperate with the PML/RARA fusion protein to induce a leukemic phenotype in a murine BM transplantation model. In addition, the forced expression of ΔNp73 in murine BM progenitors did not alter the ATRA-induced differentiation rate in vitro or induce aberrant cell proliferation, but exerted an important role in cell survival, providing resistance to drug-induced apoptosis.

Topics: Animals; Antimetabolites, Antineoplastic; Apoptosis; Bone Marrow Transplantation; Cathepsin G; Cell Differentiation; Cell Proliferation; Cell Self Renewal; Cell Transformation, Neoplastic; Cells, Cultured; Cytarabine; Gene Expression Regulation, Leukemic; Genetic Predisposition to Disease; Leukemia; Mice, Inbred NOD; Mice, SCID; Mice, Transgenic; Neoplastic Stem Cells; Phenotype; Promyelocytic Leukemia Protein; Retinoic Acid Receptor alpha; Signal Transduction; Time Factors; Transfection; Tumor Protein p73; Up-Regulation

The ectodomain of the human transferrin receptor (TfR) is released as soluble TfR into the blood by cleavage within a stalk. The major cleavage site is located C-terminally of Arg-100; alternative cleavage sites are also present. Since the cleavage process is still unclear, we looked for proteases involved in TfR ectodomain release. In the supernatant of U937 histiocytic cells we detected alternatively cleaved TfR (at Glu-110). In membrane fractions of these cells we identified two distinct proteolytic activities responsible for TfR cleavage within the stalk at either Val-108 or Lys-95. Both activities could be inhibited by serine protease inhibitors, but not by inhibitors of any other class of proteases. Protein purification yielded a 28 kDa protein that generated the Val-108 terminus. The protease activity could be ascribed to neutrophil elastase according to the substrate specificity determined by amino acid substitution analysis of synthetic peptides, an inhibitor profile, the size of the protease and the use of specific antibodies. The results of analogous experiments suggest that the second activity is represented by another serine protease, cathepsin G. Thus, membrane-associated forms of neutrophil elastase and cathepsin G may be involved in alternative TfR shedding in U937 cells.

Topics: Amino Acid Sequence; Biotin; Cathepsin G; Cathepsins; Cell Line; Cell-Free System; Cells, Cultured; Culture Media; Electrochemistry; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Humans; Immunohistochemistry; Leukemia; Leukocyte Elastase; Membranes; Molecular Sequence Data; Protein Conformation; Receptors, Transferrin; Serine Endopeptidases

An enzyme immunoassay has been developed for the quantitation of human polymorphonuclear leukocyte cathepsin G. The assay had a linear relationship over the range 0.23-4.7 nmol/l (6-125 ug/l) and a detection limit of 0.23 nmol/l (6 ug/l). Recovery in citrated plasma only occurred when the concentration was higher than 4.0 umol/l (110 mg/l). This effect could be overcome by diluting the plasma before adding the proteinase or by inactivating the proteinase before diluting it with plasma. The failure to detect cathepsin G in plasma was due to the plasma inhibitor alpha-2-macroglobulin masking the antigenic sites of the proteinase. Samples from several types of leukemia showed no detectable cathepsin G even when the total myeloid count was up to ten times the normal.

Topics: alpha-Macroglobulins; Cathepsin G; Cathepsins; Enzyme Activation; Humans; Immunoenzyme Techniques; Leukemia; Neoplasm Proteins; Neutrophils; Sensitivity and Specificity; Serine Endopeptidases

An antiserum to human cathepsin G has been raised in sheep and its reactivity with human tissues has been tested. The indirect immunoperoxidase staining sequence was employed and was applied to routinely processed paraffin sections. Mature granulocytes, especially those of the neutrophil variety, were intensely and consistently stained. Activity was not observed in other cell or tissue types. Many of the cells of acute and chronic myeloid leukemia were strongly stained, in contrast to those of acute lymphoblastic or chronic lymphocytic leukemias. The results of the technique are compared with those described with staining for muramidase (lysozyme), alpha 1-antitrypsin, leukocyte elastase, and naphthol-AS-D-chloroacetate esterase, and with certain monoclonal antisera directed against granulocyte determinants.

Topics: Cathepsin G; Cathepsins; Granulocytes; Humans; Immunoelectrophoresis; Immunoenzyme Techniques; Leukemia; Lymph Nodes; Serine Endopeptidases; Tissue Distribution