calcimycin and hexamethylene-bisacetamide

The goal of the present study was to determine whether partial restoration of the differentiation-inducing capacity of the PKC activator bryostatin 1 by the calcium ionophore A23187 is accompanied by enhancement of apoptosis in ara-C-pretreated human leukemia cells. When HL-60 cells were exposed to ara-C (10 or 100 microM;6 h) followed by bryostatin 1 alone (10 nM; 24 h), no increase in apoptosis was noted. In contrast, subsequent exposure of ara-C-pretreated cells to A23187 (250 nM; 24 h) increased apoptosis by approximately 100%. When ara-C-pretreated cells were incubated with A23187 and bryostatin 1, no further potentiation of cell death (compared to cells exposed to A23187 alone) was observed. Nevertheless, the combination of bryostatin 1 and A23187 substantially increased inhibition of clonogenicity in cells preincubated with ara-C (e.g., by > or = 2 logs). This effect was associated with morphological and functional evidence (i.e., plastic adherence) of enhanced leukemic cell maturation. The differentiating capacity of the combination of bryostatin 1 and A23187 was significantly weaker than that of the phorbol diester, PMA (10 nM), and unaccompanied (at 24 h) by induction of the cyclin-dependent kinase inhibitors (CDKIs) p21WAF1/CIP1 and p27KIP1. However, the extent of apoptosis was comparable in cells exposed to ara-C followed by PMA or bryostatin 1 + A23187, suggesting that differentiation per se is not solely responsible for enhancement of cell death in ara-C-pretreated cells. Coadministration of bryostatin 1 and the organotellurium compound AS101, which mimics the actions of A23187 in some systems, after ara-C also led to enhanced antiproliferative effects which were unaccompanied by an increase in apoptosis. Finally, exposure of cells to ara-C followed by other differentiation-inducing agents, including dimethylsulfoxide and sodium butyrate also resulted in increases in cell death in this cell line. These findings indicate that the inability of bryostatin 1 to potentiate apoptosis in ara-C-pretreated HL-60 cells may involve factors other than an inadequate differentiation stimulus. They also suggest that loss of leukemic self-renewal capacity following exposure to cytotoxic and differentiation-inducing agents may involve mechanisms other than, or in addition to, potentiation of apoptosis, particularly cellular maturation.

Topics: Acetamides; Antineoplastic Agents; Apoptosis; Bryostatins; Calcimycin; Calcium; Cell Differentiation; Cytarabine; DNA Fragmentation; HL-60 Cells; Humans; Lactones; Macrolides; Tetradecanoylphorbol Acetate

A high-mobility group 1 (HMG1) protein type isolated from murine erythroleukaemia (MEL) cells promotes acceleration of the differentiation process when added to a MEL cell culture together with the inducer hexamethylene bisacetamide. We now provide direct evidence that the presence of HMG1 protein in the extracellular medium is essential for terminal erythroid differentiation. An extracellular function for HMG1 protein in MEL cell is further supported by a demonstration that this protein is released from MEL cells exposed to the chemical inducer and that the addition of an anti-(HMG1 protein) monoclonal antibody to the cell culture inhibits the differentiation process almost completely. The release of HMG1 protein from MEL cells is modulated by compounds affecting cell calcium homoeostasis, such as a calcium ionophore or verapamil. In fact, in the presence of the ionophore an increased rate of differentiation is accompanied by an enhanced extracellular release of HMG1 protein, whereas in the presence of verapamil both phenomena are significantly decreased.

Topics: Acetamides; Animals; Antineoplastic Agents; Calcimycin; Cell Differentiation; High Mobility Group Proteins; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured

The role of the plasma membrane potential (delta psi p) in the commitment to differentiation of murine erythroleukemia (MEL) cells has been studied by analyzing the ionic basis and the time course of this potential in the absence or the presence of different types of inducers. delta psi p was determined by measuring the distribution of tetraphenylphosphonium (TPP+) across the plasma membrane and displayed a 22-hour depolarization phase (from -28 to +5 mV) triggered by factors contained in foetal calf serum (FCS) and followed by a nearly symmetrical repolarization phase. After measuring the electrochemical equilibrium potential of Na+, K+, and Cl-, the relative contribution of these ions to delta psi p was evaluated by means of ion substitution experiments and by the addition of ion flux inhibitors (tetrodotoxin [TTX], 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate [SITS]) and ionophores (Valinomycin, A23187). The Na+ contribution to delta psi p appeared negligible, the potential being essentially generated by K+ and Cl- fluxes. When evaluated by a new mathematical approach, the effects of Valinomycin and A23187 at different times of incubation provided evidence that both the depolarization and the repolarization phase were due to variations of the K+ permeability across the plasma membrane (PK) mediated by Ca2+-activated K+ channels. All the inducers tested (dimethylsulfoxide [DMSO], hexamethylen-bis-acetamide [HMBA], diazepam), although they did not modify the ionic basis of delta psi p, strongly attenuated the depolarization rate of this potential. This attenuation was not brought about when the inducers were added to noninducible MEL cell clonal sublines. Cell commitment occurred only during the depolarization phase and increased proportionally to the attenuation of this phase up to a threshold beyond which the further increase of the attenuation was associated with the inhibition of commitment. The major role of the inducers apparently consisted of the stabilization of the Ca2+-activated K+ channels, suggesting that a properly modulated delta psi p depolarization through these channels is primarily involved in the signal generation for MEL cell commitment to differentiation.

Topics: 4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; Acetamides; Animals; Calcimycin; Calcium; Cell Differentiation; Cell Membrane Permeability; Chlorides; Diazepam; Dimethyl Sulfoxide; Electrochemistry; Ion Channels; Leukemia, Erythroblastic, Acute; Mathematics; Membrane Potentials; Mice; Potassium; Sodium; Tetraethylammonium Compounds; Tetrodotoxin; Time Factors; Valinomycin