cytochalasin-d and Leukemia--Myeloid--Acute

Acquired mutations truncating the C-terminal domain of the granulocyte colony-stimulating factor receptor (G-CSF-R) are found in about 20% of severe congenital neutropenia (SCN) patients, with this cohort of patients predisposed to acute myeloid leukemia (AML). In myeloid cells, such mutations act in a dominant-negative manner leading to hyperproliferation and lack of differentiation in response to G-CSF. However, why these truncated receptors are dominant in function over wild-type receptors has remained unclear. We report that ligand-induced internalization of truncated G-CSF-R is severely impaired compared with the wild-type receptor, which results in sustained activation of STAT proteins. Strikingly, in cells coexpressing both truncated and wild-type forms, the truncated receptors acted dominantly with regard to both internalization and sustained activation. Site-directed mutagenesis of the C-terminus showed that receptor tyrosines in this region were dispensable for internalization, whereas a di-leucine-containing motif in Box B3 played some role. However, loss of the di-leucine motif was not the critical determinant of the sustained activation status of truncated receptors. These data suggest that defective internalization, leading to extended receptor activation, is a major cause of the dominant hyperproliferative effect of truncated G-CSF receptors, which is only partially due to the loss of a di-leucine motif present in the Box B3 region of the full-length receptor.

Topics: Animals; Cytochalasin D; DNA Mutational Analysis; DNA-Binding Proteins; Granulocyte Colony-Stimulating Factor; Humans; Leucine; Leukemia, Myeloid, Acute; Mice; Milk Proteins; Mutagenesis, Site-Directed; Mutation; Neutropenia; Receptors, Granulocyte Colony-Stimulating Factor; STAT1 Transcription Factor; STAT3 Transcription Factor; STAT5 Transcription Factor; Structure-Activity Relationship; Trans-Activators; Tumor Cells, Cultured; Tyrosine

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