Magnesium deprivation inhibits the expression of differentiation‐related phenotypes in human promyelocytic leukemia HL‐60 cells

Abstract

The role of magnesium ions in the differentiation of human promyelocytic leukemia HL-60 cells was investigated. When HL-60 extracellular magnesium was deficient (less than 0.01 mM), the total intracellular magnesium content and [3H] leucine incorporation rates decreased to 61 and 28%, respectively, on day 3. When the cells were treated with various inducers (100 nM 1 alpha, 25 dihydroxyitamine D3 (1,25(OH)2D3), 100 nM beta-all-trans retinoic acid (RA), 20 nM 12-o-tetradecanoyl phorbol-13-acetate (TPA), 1.25% dimethylsulfoxide (DMSO) and 30 nM aclacinomycin (AcM] in magnesium-deficient medium, the expression of differentiation-related phenotypes (nitroblue tetrazolium (NBT) reducing ability, nonspecific esterase (NSE) activity and monoclonal antibody, OKM1 binding activity) was almost completely inhibited. After a 2-day treatment with 100 nM 1,25(OH)2D3 in magnesium-deficient medium, the expression of differentiation-related phenotypes was restored by further incubation in the absence of inducer in standard magnesium medium (0.4 mM). These results suggested that magnesium deprivation inhibited the expression of HL-60 differentiation-related phenotypes but not their commitment to differentiation. These phenotypes were expressed without inducer in standard magnesium medium after a 2-day simultaneous treatment with 1,25(OH)2D3 and cyclohexamide (protein synthesis inhibitor) in magnesium-deficient medium, but not after simultaneous pretreatment with 1,25(OH)2D3 and alpha-amanitin (RNA synthesis inhibitor). Thus, it was suggested that the magnesium-requiring step in HL-60 cell differentiation is in protein but not mRNA synthesis. This conclusion is supported by the findings that changes in c-myc and c-fms mRNA levels in HL-60 cells treated with 100 nM 1,25(OH)2D3 in magnesium-deficient medium and those in standard magnesium medium were the same. In addition, dibutyryl cyclic adenosine monophosphate (dbc AMP) could restore expression of differentiation-related phenotypes inhibited by magnesium deprivation but not those inhibited by cyclohexamide, even though magnesium deprivation inhibited protein synthesis as much as did cyclohexamide. This suggests that magnesium-requiring step in HL-60 cell differentiation is different from that inhibited by cyclohexamide.

Effect of Uremic Serum on 1,25-Dihydroxyvitamin D₃-lnduced Differentiation of Human Promyelocytic Leukemia Cell, HL-60

“…However, as HL-60 cell proliferation is not coupled to its differentiation as we [3,22] and others [29,30] have previously demonstrated, the proliferationsuppressive effect of uremic serum could be neglected in assessing the effect of I,25-(OH)2D3 on cell differentiation. Although the Ca and Mg levels in the culture medium are known to affect the l,25-(OH)2Dr induced HL-60 cell dif ferentiation [31,32], we did not detect a significant dif ference in those levels between the uremics and the controls. Aluminium is known to cause l,25-(OH)2D3 resistance at a post-receptor step with a compensatory rise in the 1,25-(OH)2D3 receptor levels in target tissues [33].…”

Magnesium in cell proliferation and differentiation

“…Inasmuch as re-introduction of Mg in the culture medium enabled DMSO-treated cells to differentiate, the authors also concluded that Mg deprivation could compromise appearance of the differentiated phenotype but not of cell commitment to differentiation. Such conclusion can be tentatively reconciled with the well known inhibition of protein synthesis by Mg deprivation (54). Clearly, one might wonder whether such conditions are specific enough to probe the possible role of Mg in differentiation.…”

Evidence of intracellular and trans‐acting differentiation‐inducing activity in human promyelocytic leukemia HL‐60 cells: Its possible involvement in process of cell differentiation from a commitment step to a phenotype‐expression step

“…Since we previously demonstrated that HL-60 cells could become committed to differentiation and yet not express the differentiation-related phenotypes in magnesium-deficient medium (Okazaki et al, 1987), the results obtained here suggested that int-DIA was present in 1,25(0H)2D3-treated HL-60 cells and could accumu- late in differentiation-committed phenotype-nonexpressing cells to a greater degree than it could in differentiation-committed phenotype-expressing cells.…”

“…In the previous report, we showed that HL-60 cells treated in magnesium-deficient medium with optimal concentrations of various inducers including 1,25- (OH)2D3, RA, TPA, and actinomycin D were committed to differentiate but did not express the differentiationrelated phenotypes (Okazaki et al, 1987). Using this system, it has become possible to divide the process of HL-60 cell differentiation into at least two steps, namely differentiation-committed phenotype-nonexpressing and phenotype-expressing steps.…”

“…In the previous report, we demonstrated that HL-60 cells treated with 1,25(OH)2D3 in the magnesium-deficient medium were committed to differentiate but did not express the differentiation-related phenotypes, and that the transcription of a certain kind of mRNA might have been indispensable for this commitment (Okazaki et al, 1987). Thus, it was possible to assume that the differentiation signal were blocked and accumulated in differentiation-committed phenotype-nonexpressing cells.…”