calpain and hexamethylene-bisacetamide

As indicated by direct evidence, obtained by altering the cell-membrane permeability for Ca2+ in murine erythroleukaemia (MEL) cells, calpain is the triggering factor which connects fluctuations of the intracellular Ca2+ concentrations to the decay of protein kinase C (PKC), as well as to the kinetics of cell differentiation induced by hexamethylenebisacetamide. Cell exposure to verapamil caused a profound decrease in the rate of PKC down-regulation and a slower initial rate of accumulation of mature erythroid cells, whereas addition of the Ca2+ ionophore A23187 produced opposite effects. The high susceptibility of PKC-delta to calpain degradation, at concentrations of Ca2+ much lower than those required for degradation of the other PKC isoforms, may be explained by the finding that this kinase isoform is predominantly associated with the cell membrane. The different cellular localizations, as well as the different susceptibilities to calpain digestion, further support the hypothesis that in MEL cells the various PKC isoforms play distinct biological functions that are critical for the maintenance of the undifferentiated state of the cell and for its commitment to terminal erythroid differentiation.

Topics: Acetamides; Animals; Calcium; Calpain; Cell Differentiation; Cell Membrane; Cell Membrane Permeability; Down-Regulation; Enzyme Activation; Intracellular Fluid; Isoenzymes; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Protein Kinase C; Signal Transduction; Translocation, Genetic; Tumor Cells, Cultured

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca(2+)-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.

Topics: Acetamides; Antibodies; Antineoplastic Agents; Calcium; Calcium-Binding Proteins; Calpain; Cell Differentiation; Cell Line; Erythrocytes; Humans; Isoenzymes; Kinetics; Leukemia, Erythroblastic, Acute; Protein Kinase C; Tumor Cells, Cultured

Murine erythroleukemia cells contain a single type of calpain classified, on the basis of its calcium requirement, as a type I proteinase. The enzyme is practically inactive at concentrations of calcium below 10 microM and expresses maximal activity in the presence of 0.12-0.15 mM Ca2+. The affinity for Ca2+ cannot be reduced by exposure of the enzyme to conditions known to promote autoproteolysis of calpain. Expression of catalytic activity at lower concentrations of Ca2+, is promoted by the interaction with phospholipid vesicles or plasma membranes. Conditions that promote activation of calpain, induce also the self-inactivation of the enzyme. During terminal differentiation of murine erythroleukemia cells induced by HMBA, the intracellular level of calpain activity undergoes significative reduction. Similar decrease in calpain activity progressively occurs during the loss of sensitivity to HMBA as a result of density growth arrest.

Topics: Acetamides; Animals; Calcium; Calpain; Cell Differentiation; Enzyme Activation; Hemoglobins; Kinetics; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured