cytochalasin-b and Leukemia--Myeloid--Acute

We have used laser-assisted confocal microscopy to evaluate the intracellular distribution of daunorubicin (DNR) in acute myeloid leukemia (AML) cell lines and fresh AML cells according to their differentiation phenotype. In KG1a, KG1, TF-1 and HEL cells, which express the early differentiation marker CD34, DNR was distributed in perinuclear vesicles which could be associated with the Golgi apparatus, as suggested by the distribution of fluorescent probes specific for intracellular organelles. In contrast, U937 and HL-60 cells, which display a more mature phenotype, exhibited nuclear and diffuse cytoplasmic DNR fluorescence. DNR sequestration was not correlated with P-glycoprotein (P-gp) or multidrug resistance protein expression. Furthermore, PSC833, a potent P-gp blocker, had little effect on drug sequestration in CD34+ AML cells. We also tested the effect of metabolic inhibitors, cytoskeleton inhibitors and carboxy-ionophores on DNR distribution in both CD34- and CD34+ AML cells. However, only non-specific metabolic inhibitors restored nucleic/cytoplasmic distribution in CD34+ cells. In these cells, the intracellular distribution of doxorubicin and idarubicin was very similar to that of DNR, while the distribution of methoxymorpholinyl-doxorubicin was nuclear and diffusely cytoplasmic. In fresh AML cells, DNR was also concentrated in the perinuclear region in CD34+ but not in CD34- cells. However, DNR sequestration was not observed in normal CD34+ cells. Finally, our results show that DNR is sequestered in organelles in CD34+ AML cells via an active mechanism which appears to be different from P-gp-mediated transport. Abnormal DNR distribution may account for the natural resistance of immature AML cells to anthracyclines.

Topics: Antibiotics, Antineoplastic; Antigens, CD34; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Blood Cells; Cell Nucleus; Colchicine; Cytochalasin B; Cytoplasm; Daunorubicin; Deoxyglucose; Fluorescent Antibody Technique, Indirect; Humans; Leukemia, Myeloid, Acute; Microscopy, Confocal; Monensin; Nigericin; Sodium Azide; Temperature; Tumor Cells, Cultured

The effect of human tumor necrosis factor (TNF) on early-passage HL-60 cells was studied. A transient phase of increased [3H]thymidine (TdR) incorporation was noted at 20-24 hr of exposure to TNF. This increase was disproportionate to the much slighter stimulation of the percentage of S-phase cells, which was measured by flow cytometry. Evidence for increased metabolic trapping of [3H]TdR following TNF treatment was apparent from whole cell uptake experiments. The salvage pathway enzyme TdR kinase was therefore measured and was found to be elevated comparably to [3H]TdR uptake. The mechanism of TNF regulation of TdR kinase was further investigated by a series of combination treatment experiments using other biologic factors and pharmacologic inhibitors of various intracellular steps. The response to TNF was not potentiated or reproduced by IL-1, IL-2, IL-3, IL-4, G-CSF, M-CSF, GM-CSF or alpha- or gamma-interferon. Blockers of early signal transduction steps, including H7, W7, sphingosine, and pertussis toxin, failed to inhibit TNF stimulation of [3H]TdR incorporation. mRNA synthesis inhibition with alpha-amanitin blocked this TNF effect, as did cAMP but not cGMP analogues. A sensitizing effect was noted with amiloride or cytochalasin B, characterized by greater relative increases of [3H]TdR incorporation and TdR kinase activity in response to TNF. In the presence of cytochalasin B, TNF treatment resulted in no change or slight decreases in the percentage of S-phase cells. Regulation of TdR kinase could thereby be dissociated from the usual cell cycle control. This study thus documents a unique example of stimulation of thymidine salvage pathway metabolism by a biologic factor, dissociable from overall cell cycle regulation.

Topics: Amiloride; Cell Cycle; Cell Division; Cell Line; Cytochalasin B; Humans; Leukemia, Myeloid, Acute; Thymidine; Thymidine Kinase; Tumor Necrosis Factor-alpha

Cytochalasin B and D enhanced vincristine (VCR) and daunomycin (DAU) accumulation in tumor cells, especially in VCR- and DAU-resistant cell lines. The effect of cytochalasin B, and to a lesser extent cytochalasin D, was almost equivalent to that observed for verapamil, a calcium channel blocker which has been reported to enhance drug accumulation in tumor cells. Cytochalasin B was most effective in VCR- and DAU-sensitive cells; however, the effect in resistant cells was less than that observed for verapamil, suggesting a different mode of action between these drugs in sensitive and resistant cells. Enhanced accumulation of VCR and DAU by cytochalasins was mediated by the inhibition of outward transport of VCR and DAU from tumor cells. Colchicine had no effect on VCR and DAU accumulation. Cytochalasins, especially cytochalasin D is a specific inhibitor of microfilament assembly in cells. These results indicate that the cellular microfilament system plays a prominent role in drug transport of tumor cells, and that an intact microtubular system is less involved.

Topics: Animals; Cell Line; Cell Survival; Colchicine; Cytochalasin B; Cytochalasin D; Cytochalasins; Daunorubicin; Drug Resistance; Humans; Leukemia; Leukemia P388; Leukemia, Myeloid, Acute; Mice; Verapamil; Vincristine

To identify the possible roles of Ca2+-related proteins, calmodulin and microfilaments in leukemic cells, we tested the effect of calmodulin antagonists and cytochalasins on proliferation and differentiation of human promyelocytic leukemic HL-60 cells. The growth of HL-60 was inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, and trifluoperazine dihydrochloride. In contrast, the 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]-induced differentiation of HL-60, as judged by plasma-membrane antigenic changes detected by monoclonal antibodies (OKM1, OKT9), nitroblue tetrazolium reduction, and induction of phagocytotic capacity, was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide or N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide, although phagocytosis was depressed by N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide or N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide. Trifluoperazine dihydrochloride also failed to inhibit the antigenic change induced by 1,25-(OH)2D3. Cytochalasins B and D, microfilament-disrupting agents, inhibited the cytoplasmic division and the growth of HL-60 but did not inhibit the 1,25-(OH)2D3-induced differentiation. These findings suggest that the calmodulin- and microfilament-dependent process may be involved in the proliferation of HL-60, but not in the differentiation induced by 1,25-(OH)2D3.

Topics: Calcitriol; Calmodulin; Cell Differentiation; Cell Division; Cell Line; Cytochalasin B; Cytochalasin D; Cytochalasins; Cytoskeleton; DNA Replication; Humans; Kinetics; Leukemia, Myeloid, Acute; Neoplasm Proteins; Phagocytosis; Structure-Activity Relationship; Sulfonamides