dihydropyridines and Leukemia--Erythroblastic--Acute

Dexniguldipine hydrochloride (DNIG) is a potent antineoplastic agent with well-documented anti-(protein kinase C) activity and an ability to reverse multidrug resistance. Given the importance of protein kinase C (PKC) activity in proliferation and differentiation, we examined the effect of DNIG on several parameters of Friend erythroleukemia cell (FELC) activity. Particular attention was paid to proliferation, hexamethylene-bisacetamide-(HMBA)-induced differentiation, nuclear localization of protein kinase C, and nuclear protein phosphorylation. P-glycoprotein expression was also followed as an indicator of changes in multidrug resistance. At 2.5 microM, DNIG caused a significant decrease in the rate of FELC proliferation, while maintaining a cellular viability of greater than 80%, whether exposure to the drug was continuous over 96 h or took the form of a 6-h pulse/chase. DNA synthesis was decreased in cells exposed to DNIG for 20 h. Flow cytometry showed a marked increase in the percentage of cells in S phase of the cell cycle. Phosphorylation studies revealed decreased phosphorylation of two nuclear proteins (80 kDa and 47 kDa) following a 4-h exposure to the drug. HMBA-induced differentiation was significantly inhibited with continuous exposure to DNIG, and this effect appears to be a pre-commitment one, as 6-h pulse/chase exposures also resulted in inhibition of differentiation. Cells induced to differentiate with HMBA also demonstrated a decrease in the quantity of the 80-kDa phosphoprotein. Western blotting revealed that, even in the face of decreased phosphorylation, exposure to this PKC inhibitor resulted in an increase in the amount of nuclear PKC alpha. Finally, levels of P-glycoprotein were decreased in the presence of this drug. Our work identifies several effects of the PKC inhibitor DNIG on FELC and suggests several roles for PKC in regulating FELC proliferation and differentiation. Additionally, these results suggest that this PKC inhibitor may increase the effect of other chemotherapeutic drugs, particularly S-phase-specific ones, by increasing the length of S phase and decreasing multidrug resistance. The possibility of combination therapy with DNIG and other antineoplastic agents should be investigated further in light of these findings.

Topics: Acetamides; Animals; Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Cycle; Cell Differentiation; Dihydropyridines; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Mice, Inbred DBA; Nuclear Proteins; Phosphorylation; Protein Kinase C; Tumor Cells, Cultured

We studied the restoration of doxorubicin accumulation and sensitivity by verapamil and quinine in a variant of the human erythroleukemia cell line K562 selected for resistance to doxorubicin and presenting a multidrug-resistance (MDR) phenotype. Verapamil was able to completely restore doxorubicin accumulation in the resistant cells to the level obtained in sensitive cells, but only partially reversed doxorubicin resistance. Quinine, in contrast, had a relatively weak effect on doxorubicin accumulation but was able to completely restore doxorubicin sensitivity in the resistant cells. In addition, verapamil was able to decrease azidopine binding to P-glycoprotein, whereas quinine was not. Quinine also modified the intracellular tolerance to doxorubicin, which suggests that it is able to modify drug distribution within the cells. Confocal microscopy revealed that verapamil and quinine were able to restore nuclear fluorescence staining of doxorubicin in resistant cells; since this was obtained for quinine without significant increase of doxorubicin accumulation, this observation confirms that quinine acts principally on doxorubicin redistribution within the cells, allowing the drug to reach its nuclear targets. When used in association, verapamil and quinine reversed doxorubicin resistance in a synergistic fashion. We conclude that verapamil and quinine do not share the same targets for reversal of MDR in this cell line; whereas verapamil directly interferes with P-glycoprotein and mainly governs drug accumulation, quinine has essentially intracellular targets involved in drug redistribution from sequestration compartments.

Topics: Affinity Labels; ATP Binding Cassette Transporter, Subfamily B, Member 1; Azides; Dihydropyridines; Doxorubicin; Drug Resistance; Drug Screening Assays, Antitumor; Drug Synergism; Humans; Kinetics; Leukemia, Erythroblastic, Acute; Microscopy, Confocal; Microscopy, Fluorescence; Phenotype; Quinine; Subcellular Fractions; Tumor Cells, Cultured; Verapamil

Calcium entry, via a dihydropyridine-sensitive pathway, is required for differentiation in murine erythroleukemia cells (MELC). Calcium channel currents have been identified physiologically in some non-excitable cells, but little is known regarding the structure of these channels. We show that a truncated form of the alpha 1 subunit of the cardiac voltage-gated calcium channel (dihydropyridine receptor, DHPR) is expressed in MELC. This MELC calcium channel lacks the first four transmembrane segments of the DHPR (IS1 to IS4). A MELC calcium channel/cardiac DHPR chimera, co-expressed with the alpha 2 and beta subunits of the DHPR, forms a functional calcium channel in Xenopus oocytes.

Topics: Acetamides; Alternative Splicing; Amino Acid Sequence; Animals; Base Sequence; Blotting, Western; Calcium Channels; Calcium Channels, L-Type; Cell Differentiation; Cloning, Molecular; Dihydropyridines; DNA, Complementary; Leukemia, Erythroblastic, Acute; Mice; Molecular Sequence Data; Muscle Proteins; RNA, Messenger; Sequence Homology, Amino Acid; Tumor Cells, Cultured; Xenopus laevis

Dexniguldipine-HCl (DNIG)--a prospective clinical modulator of p170-glycoprotein (pgp170)-mediated multidrug resistance (MDR)--was evaluated in a drug-accumulation assay in MDR murine leukemia cell strain F4-6RADR expressing pgp170. The compound elevated low accumulation of either doxorubicin (DOX), daunorubicin (DNR), or mitoxantrone (MITO) in resistant F4-6RADR cells to the very levels observed in drug-sensitive F4-6 precursor cells. In parallel with the increase in DNR content (F4-6RADR, solvent: 303 +/- 27 pmol/mg protein; DNIG (3.3 mumol/l): 1,067 +/- 174 pmol/mg protein; F4-6P, solvent: 948 +/- 110 pmol/mg protein; n = 8-9, SEM), the amount of DNR tightly bound to the acid precipitate pellet obtained from F4-6RADR (i.e., protein, DNA, RNA) increased 3.9-times to the levels observed in sensitive F4-6 cells. The main pyridine metabolite of DNIG displayed similar activity. Concentration-response analysis revealed that DNIG and R,S-verapamil (VER) induced 100% reversal of the DNR accumulation shortage associated with the MDR phenotype but DNIG was 8 times more potent than VER (50% inhibitory concentration (IC50), 0.73 vs 5.4 mumol/l). In keeping with the accumulation assay, DNIG was about 10 times more potent than VER in sensitizing F4-6RADR cells to the cytostatic and cytotoxic effects of DNR in proliferation assays. In conclusion, DNIG is a potent in vitro modulator, improving (a) the accumulation of anthracycline-like cytostatics, (b) drug access to cellular binding sites, and (c) the cytostatic action of DNR in F4-6RADR leukemia cells of the MDR phenotype.

Topics: Animals; Cell Division; Daunorubicin; Dihydropyridines; Dose-Response Relationship, Drug; Drug Resistance; Friend murine leukemia virus; Leukemia, Erythroblastic, Acute; Mice; Tumor Cells, Cultured; Verapamil