John A. Sokoloski

Induction of the differentiation of HL-60 promyelocytic leukemia cells by nonsteroidal anti-inflammatory agents in combination with low levels of vitamin D3

Previous experiments have shown that a variety of agents that interfere with the activity of the transcription factor NF-kB significantly enhanced the differentiation of HL-60 leukemia cells when combined with low levels of the monocytic/macrophagic differentiating agent vitamin D3. These include an antisense phosphorothioate oligonucleotide to the Rel A subunit of NF-kB, vitamin E and other antioxidants, and curcumin. Acetylsalicylic acid and other nonsteroidal anti-inflammatory agents represent another group of agents that have been reported to inhibit NF-kB at serum levels approximating those obtained during long-term therapy of chronic inflammatory states. To determine whether nonsteroidal anti-inflammatory agents also were capable of enhancing the differentiation of HL-60 leukemia cells produced by vitamin D3, we measured the effects of a variety of nonsteroidal anti-inflammatory agents on the maturation of HL-60 cells produced by low levels of vitamin D3. Acetylsalicylic acid by itself had no significant effect on the differentiation of HL-60 cells; however, this agent markedly increased the degree of differentiation produced by low levels of vitamin D3. Furthermore, a variety of other nonsteroidal anti-inflammatory agents of different chemical classes exhibited similar enhancements of the maturation of HL-60 cells when combined with vitamin D3. An analogous increase in the differentiation of HL-60 cells was also obtained by combination of several nonsteroidal anti-inflammatory agents with the granulocytic inducing agent, retinoic acid, but not with dimethylsulfoxide. The nonsteroidal anti-inflammatory agents also enhanced the differentiation of HL-60 cells when combined with vitamin D analogs which share the receptor binding properties of vitamin D3; however, a vitamin D analog which caused significant calcium mobilization, but was less effective in receptor binding than vitamin D3, did not induce the differentiation of HL-60 cells in the presence or absence of anti-inflammatory agents. The findings suggest that the nonsteroidal anti-inflammatory agents may have utility in the treatment of acute promyelocytic leukemia when used with the D vitamins or retinoic acid.

Induction of the differentiation of HL-60 and WEHI-3B D+ leukemia cells by lithium chloride.

The use of lithium chloride in manic-depressive patients and in patients receiving myelo-suppressive cancer chemotherapeutic agents is accompanied by a sustained leukocytosis due to an increase in granulocyte production. This property suggests that lithium chloride may have effects on hematopoietic differentiation. Treatment of cultured WEHI-3B D+ murine myelomonocytic and HL-60 human promyelocytic leukemia cells with millimolar concentrations of lithium chloride resulted in concentration-dependent increases in the number of differentiated myeloid cells, as determined by the ability of the cells to reduce nitroblue tetrazolium and by the binding of myeloid specific antibodies, and was associated with an inhibition of cellular proliferation. The effects of lithium chloride on growth and differentiation were antagonized by KCl, whereas NaCl had little effect. The induction of leukemic cell maturation by lithium chloride was markedly enhanced by the addition of low levels of retinoic acid. In contrast, other differentiation inducing agents (i.e. dimethyl sulfoxide and selenazofurin) had no effect on the degree of maturation induced by lithium. These findings suggest that the combination of lithium chloride and retinoic acid may have clinical utility in the treatment of leukemia through the induction of terminal differentiation.

Enhancement by antisense oligonucleotides to NF-kB of the differentiation of HL-60 promyelocytic leukemia cells induced by vitamin D3

We have demonstrated previously that a phosphorothioate antisense oligonucleotide to the p65 subunit of the inducible transcription factor NF-kappaB produced rapid changes in the expression of leukocyte integrin CD11b (Mo 1) and in the adhesion of dimethylsulfoxide (DMSO)-differentiated HL-60 cells stimulated by 12-O-tetradecanoylphorbol 13-acetate. We have also shown that a variety of agents which inhibit NF-kappaB, including vitamin E and related antioxidants, curcumin and several non-steroidal anti-inflammatory agents, significantly enhanced the differentiation of HL-60 leukemia cells when combined with low levels of 1,25-dihydroxyvitamin D3 (vitamin D3). To provide further evidence that interference with the activation of NF-kappaB affects the maturation of HL-60 leukemia cells by creating an environment conducive to terminal differentiation, we measured the effects of phosphorothioate antisense oligonucleotides to the various subunits of NF-kappaB on the differentiation of HL-60 cells produced by low levels of vitamin D3. When used alone these oligonucleotides had no significant effect on the differentiation of HL-60 cells. However, the antisense oligomer to the Rel A subunit of NF-kappaB markedly increased the extent of differentiation produced by low levels of vitamin D3. An enhancement of the differentiation of HL-60 cells induced by vitamin D3 was also obtained by several transcription factor decoys designed to mimic the consensus sequences of genes activated by Rel A. The findings provide additional support for the concept that inhibition of the activation of NF-kappaB may be involved in regulating the entry of promyelocytic leukemia cells into a differentiation pathway.

Effects of uridine on the growth and differentiation of HL-60 leukemia cells☆

HL-60 leukemia cells, induced to differentiate, activate a Na(+)-dependent nucleoside transport system, concomitant with a reduction in the nitrobenzylthioinosine (NBMPR)-sensitive facilitated transport of nucleosides. The consequence of these changes lead to the formation of intracellular pools of uridine. To examine the possible role of accumulated uridine in the commitment of HL-60 leukemia cells to undergo maturation, the effects of uridine on the growth and differentiation of HL-60 cells were monitored. Uridine at millimolar levels caused a concentration-dependent inhibition of cellular growth, resulting in the accumulation of cells in the G2/M phases of the cell cycle, phenomena that preceded the formation of differentiated cells. These effects of uridine were reduced by 10 microM NBMPR, an inhibitor of the facilitated transport of nucleosides. The effects of 24 mM uridine on growth and differentiation of HL-60 cells were also prevented by 5 mM inosine, and partially prevented by either 2 mM hypoxanthine or 20 microM adenosine. Pretreatment of HL-60 cells with 24 mM uridine for 6 days, followed by a 2 h exposure to TPA, resulted in the rapid attachment of cells to the tissue culture dish, and the extension of long processes. Although the concentrations of uridine required for the above effects are greater than those achieved during differentiation, these observations suggest that uridine may play a role in regulating the maturation process.

Induction of HL-60 leukemia cell differentiation by tetrahydrofolate inhibitors of de novo purine nucleotide biosynthesis.

Summary5,10-Dideazatetrahydrofolic acid (DDATHF) is a folate antimetabolite that shows activity against glycinamide ribonucleotide (GAR) transformylase, a folate-requiring enzyme in the de novo purine nucleotide biosynthetic pathway. Previous studies from our laboratory have shown that DDATHF is an effective inducer of the maturation of HL-60 promyelocytic leukemia. In solution, DDATHF is a mixture of two diastereomers due to an asymmetric configuration at carbon 6. Incubation of HL-60 cells with 1 μM of each diastereomer resulted in an inhibition of cellular proliferation after 48 h that preceded an increase in the number of differentiated myeloid cells, as determined by the ability of cells to reduce nitroblue tetrazolium (NBT) and by the binding of the myeloid-specific antibody Mo 1. Several analogs of DDATHF were also tested as inducers of the differentiation of HL-60 cells. With the exception of the 10-acetyl analog of 5-deazatetrahydrofolic acid, all compounds displayed similar activities as inducers of maturation. The finding that both stereoisomers of DDATHF, as well as the analogs tested, could selectively reduce intracellular purine nucleotide levels suggested that these compounds inhibited purine nucleotide biosynthesis de novo. This possibility was confirmed by the finding that hypoxanthine completely prevented the reduction of intracellular purine nucleotide levels, as well as the induction of differentiation and the inhibition of cellular growth, by these folate analogs. The results suggest that GAR transformylase is a target for a series of compounds whose structures resemble that of tetrahydrofolate and indicate that the inhibition of GAR transformylase by these compounds is sufficient to induce the maturation of HL-60 leukemia cells.

Regulation of the expression of enzymes involved in the replication of DNA in chemically-induced granulocytic differentiation of HL-60 leukemia cells

The expression of seven enzymes involved in the biosynthesis of DNA was measured in HL-60 promyelocytic leukemia cells treated with dimethylsulfoxide (DMSO) or all-trans retinoic acid (RA) to gain information on their role in the termination of proliferation in cells undergoing granulocytic differentiation. The steady-state levels of the mRNAs for topoisomerase I, topoisomerase II. DNA polymerase-alpha, thymidylate synthase, thymidine kinase and hypoxanthine-guanine phosphoribosyltransferase progressively declined from day 3 to day 7 of exposure to the polar solvent or the retinoid suggesting that the expression of these enzymes is coordinately regulated. In contrast, a pronounced difference between the two inducers of differentiation occurred in the expression of the mRNA of the M2 subunit of ribonucleotide reductase, with DMSO causing virtually complete inhibition of the expression of the M2 subunit of the enzyme from day 5 through day 7, with no change in the steady-state levels of the mRNA being produced by retinoic acid. Measurement of the enzymatic activities of two of these catalysts, thymidylate synthase and thymidine kinase, in cells exposed to the two inducers of maturation corroborated the findings at the level of the mRNAs, with corresponding decreases in the activity of these enzymes. The findings collectively demonstrate that the down-regulation of the expression of a relatively wide variety of enzymes involved in DNA replication occurs as late events in the granulocytic differentiation of HL-60 cells, ensuring that cellular replication cannot occur in terminally differentiated cells.

Induction of the differentiation of synchronized HL-60 leukemia cells by tiazofurin

Tiazofurin is an effective inducer of the maturation of HL-60 promyelocytic leukemia cells, as monitored by increased phagocytic ability and the capacity to reduce nitroblue tetrazolium (NBT). The antimetabolite acts as a potent inhibitor of IMP dehydrogenase, which results in a profound depression in the cellular levels of guanine nucleotides. Flow cytometric analysis of DNA histograms indicated that the commitment of HL-60 cells to differentiate when exposed to tiazofurin was preceded by a transient delay in the G1 phase of the cell cycle. HL-60 leukemia cells enriched in the various phases of the cell cycle by centrifugal elutriation were utilized to further characterize the relationship between the phase of the cell cycle and the commitment to enter a pathway of differentiation. Fractions composed mainly of G1 cells demonstrated an increased capacity to mature when exposed to tiazofurin, whereas fractions containing cells from the S and G2 + M phases of the cell cycle had a lower ability to enter a differentiation pathway. The findings suggest that the commitment of HL-60 cells to mature when exposed to tiazofurin is mediated during the G1 phase of the cell cycle.

Regulation of the expression of enzymes involved in the replication of DNA in chemically induced monocytic/macrophagic differentiation of HL-60 leukemia cells

The expression of a number of housekeeping enzymes of DNA biosynthesis was measured in HL-60 promyelocytic leukemia cells undergoing monocytic/macrophagic differentiation following treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1alpha,25-dihydroxyvitamin D3 (vitamin D3). Progressive decreases in the steady-state levels of the mRNAs for thymidylate synthase, topoisomerase II, and hypoxanthine guanine phosphoribosyltransferase occurred following exposure to TPA or vitamin D3. In contrast, the steady-state levels of the mRNAs for thymidine kinase, topoisomerase I, and DNA polymerase-alpha did not decrease until days 3-5 of treatment with vitamin D3 and then progressively declined thereafter. The mRNAs for thymidine kinase and topoisomerase I decreased slightly and the mRNA for DNA polymerase-alpha by 30-40%, and then remained constant between days 1 to 3 of treatment with the phorbol ester. The M2 subunit of ribonucleotide reductase exhibited an even greater difference, with no change in the steady-state concentration of mRNA over 3 days of exposure to TPA or vitamin D3. On days 5-7 of treatment with vitamin D3, essentially complete loss of the expression of the mRNA for the M2 subunit of ribonucleotide reductase occurred. Measurement of the enzymatic activities of thymidylate synthase and thymidine kinase in cells exposed to either of the inducers of maturation corroborated the findings at the level of the mRNAs, with corresponding decreases in the activity of these enzymes. The results indicate that the down-regulation of the expression of housekeeping enzymes of DNA replication occurs as late events in HL-60 cells undergoing monocytic/macrophagic differentiation, implying that the decreases in their gene expression are the result of the termination of proliferation rather than an initiating event in the cessation of DNA biosynthesis.

Differentiation of Malignant Cells as a Therapeutic Approach

The hematological malignancies may be considered to be diseases of altered maturation in which the rate of proliferation is increased relative to that of terminal differentiation. Many neoplastic cells, however, retain the capacity to mature to end-stage cells with a finite life-span. Differentiation to a nonmalignant state can be produced in many different malignant cell types by exposure to a variety of structurally diverse biological and chemical agents, including cytokines, solvents, hormones, vitamins, tumor promoters and cancer chemotherapeutic agents. The induced transformation to a terminally differentiated phenotype demonstrates the reversibility of the malignant state.