Induction of apoptosis in HL-60 cells by lithium.

It has been established that lithium, a preferred drug for the treatment of manic depression, affects the growth of HL-60 cells. Concentrations of lithium above 10 mM were shown to be cytotoxic to these cells. However, the mechanism by which lithium induces its cytotoxicity is still obscure. In this study, we examined whether programmed cell death was involved in lithium cytotoxicity. Our findings show that lithium induced apoptosis in a time- and concentration-dependent manner. Morphological features and DNA fragmentation revealed that with 10 mM lithium, apoptosis was maximal at 72 hours of treatment. Interestingly, other monovalent ions were unable to elicit DNA fragmentation, suggesting that this phenomenon is specific for lithium. Cell cycle studies revealed that lithium arrested the cells at the G2/M phase.

Effects of lithium on dimethyl sulfoxide induced differentiation of HL-60 promyelocytic leukemia cells.

The human promyelocytic leukemia cell line, HL-60, was used to investigate the effects of lithium on dimethyl sulfoxide (DMSO)-induced granulocytic differentiation of these cells. Dose-response studies showed an optimal increase of cellular proliferation when cells were incubated with 5 mM lithium for 5 days (127 +/- 5% of DMSO only treated cells). This enhancement in growth was preceded by significantly increased [methyl-3H]thymidine incorporation (143 +/- 4% of DMSO only treated controls) after 2 days. However, no significant changes in the ability of cells to reduce NBT could be detected irrespective of whether the cells were incubated with 1.25% (v/v) DMSO only, or with DMSO plus non-toxic concentrations (less than or equal to 10 mM) lithium. From the results obtained it would appear as if the arrest of growth induced by DMSO and the stimulation of proliferation effected by lithium occurs along independent pathways and that lithium exerts its mitogenic effect prior to the onset of terminal differentiation initiated by DMSO.

Protein synthesis in HL-60 cells treated with DMSO and hypoxanthine.

Short-term treatment of the HL-60 cells with DMSO and hypoxanthine, inducers of granulocytic differentiation, was reported to cause a rapid increase in protein synthesis. This effect was ascribed to the insertion of inosine in the wobble position of the tRNA anticodon and consequently increasing codon recognition potential. In this study we have re-investigated the effects of DMSO and/or hypoxanthine on protein synthesis. In contrast to their findings we were unable to demonstrate stimulated protein synthesis in either short- or long-term treatment with these agents. Polysome analysis under these conditions revealed that polysomes were disaggregated. Finally, the activity of tRNA-hypoxanthine ribosyltransferase, an enzyme responsible for the insertion of inosine in the anticodon, was also relatively low. Under these circumstances, we propose that tRNA modification is not essential in the regulation of protein synthesis.

The effects of lithium on the growth and phorbol ester (TPA) induced differentiation of two HL-60 sublines.

Two sublines of a human promyelocytic cell line, HL-60, were used to study the effect of lithium on TPA (12-o-tetradecanoylphorbol-13-acetate) induced macrophage-like differentiation. Although these sublines, HL-60 M and HL-60 JE, had different growth rates, both showed enhanced proliferation when treated with 5 mM lithium (128 +/- 2 and 141 +/- 1% in comparison to controls after 5 days of incubation, respectively). Treatment of the sublines with TPA for 72 h resulted in macrophage-like differentiation (assessed by cell adhesion) of about 90% at 10 nM TPA in HL-60 JE, whereas a maximum of 50% at 100 nM TPA was obtained in HL-60 M. Differentiation was also confirmed by non-specific esterase activity. However, incubation of both sublines with TPA and 5 mM lithium revealed that lithium has little or no effect on the macrophage-like differentiation of the HL-60 cell line.

Growth and morphological changes induced by lithium chloride treatment of HL-60 cells.

HL-60 cells were grown in culture and their proliferative behaviour and response to lithium were studied. Treatment of cells with lithium concentrations of up to 5 mM enhanced cell proliferation, however above 5 mM lithium, cell growth was inhibited. Cell viability remained above 90% with concentrations of lithium below 10 mM. With increasing concentrations of lithium cell death increased rapidly to about 70% after 3 days at 50 mM. DNA synthesis was also strongly inhibited by concentrations of lithium above 5 mM. At 50 mM lithium, [3H]-thymidine incorporation was 1%. Electron microscopy seems to indicate that the plasma membrane is the main target for lithium cytotoxicity, whilst lithium uptake studies suggest that cytotoxicity might be related to the accumulation of lithium within the cells.

Lithium enhances the proliferation of HL-60 promyelocytic leukemia cells.

The effects of lithium, an agent used in the treatment of manic depression and to attenuate myelosuppression during chemotherapy, on HL-60 promyelocytic leukemia cells were investigated. By monitoring cell growth at varying concentrations (0-50 mM), as well as by following cell proliferation over 8 days, it was established that lithium stimulates HL-60 proliferation within a very narrow concentration range. Enhancement of growth was optimal at 5 mM, whereas concentrations above 10 mM were toxic. Time course studies revealed that the greatest increase in cell number occurred after 5-6 days in the presence of lithium. This was preceded by DNA synthesis reaching a maximum after 1-2 days. Viability of the cells decreased gradually after 3 days with 5 mM, but not with 2.5 mM. We suggest that HL-60 cells are a suitable model to further investigate possible mitogenic and cytotoxic effects of lithium in vitro.

Control of cell-free protein synthesis by amino acids: effects on tRNA charging.

In order to resolve the observation that addition of glutamine and glutamate appears to be of particular importance in enhancing the activity of a cell-free protein synthesis system derived from rat liver (Manchester and Tyobeka, 1980), we have measured the KM of the aminoacyl-tRNA synthetases towards amino acids and the extent of aminoacylation of tRNA under the conditions of our earlier experiments. During incubation of the cell-free system in the presence of an amino acid mixture the extent of acylation to tRNA of 15 amino acids studied showed no clear change from initial time values. When incubation took place in the absence of added amino acids, however, the levels of glutamate and glutamine bound to their appropriate tRNAs dropped more rapidly and to lower levels than for other amino acids except tryptophan. The pronounced drop for these two amino acids does not seem to result from an abnormally high KM value for the synthetases towards the respective amino acids, nor an abnormally low Vmax, but probably from the fact that the amounts of glutamyl and glutaminyl-tRNA in the cell-free system are comparatively low.

Influence of individual amino acids on incorporation of [14C]leucine by rat liver ribosomes.

The report of Baliga et al. (1) that the nutritionally essential amino acids promote initiation of protein synthesis by isolated rat liver ribosomes has been reinvestigated. We were unable to observe any particular influence of essential amino acids on the incorporation of leucine into protein. We find instead that glutamine, glutamate, asparagine and aspartate appear to exert a greater influence than the essential amino acids on leucine incorporation by the ribosomes and suggest that the availability of amino acids in cell-free systems primarily affects elongation.